International Review of Cytology: A Survey of Cell Biology, Volume 112

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME112

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN DEAN BOK GARY G. BORISY PIET BORST BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO BERNDT EHRNGER CHARLES J. FLICKINGER NICHOLAS GILLHAM M. NELLY GOLARZ D E BOURNE YUKlO HIRAMOTO Y UKINORI HIROTA MARK HOGARTH K. KUROSUMI ARNOLD MITTELMAN KEITH E. MOSTOV

AUDREY MUGGLETON-HARRIS DONALD G. MURPHY ANDREAS ODSCHE MURIEL J . ORD VLADIMIR R. PANTIC W. J . PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL L. EVANS ROTH JOAN SMITH-SONNEBORN WILFRED STEIN RALPH M. STEINMAN HEWSON SWIFT K . TANAKA DENNIS L. TAYLOR TADASHI UTAKOJI ALEXANDER YUDIN

INTERNATIONAL

Review of Cytology A

S U R V E Y OF C E L L

BIOLOGY

Editor-in-Chief

G. H. BOURNE

St. George's University School of Medicine St. George's, Grenada West Indies

Editors

K. W. JEON Department of Zoology University of Tennessee Knoxville, Tennessee

M. FRIEDLANDER Jules Stein Eye Institute UCLA School of Medicine Los Angeles, California

VOLUME112

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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0 1988 BY ACADEMICPRESS. INC.

ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING. OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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NUMBER:52-5203

Contents Prolactins of Pregnancy and Their Cellular Source LINDA OGREN 1. Introduction 11. Placental Lactogens Ill. Decidual Prolactin

AND

FRANKTALAMANTES I 4

......................................................... ............................................

41

50 51 52

..................... References ..........................................................................................

Membrane Oligosaccharides: Structure and Function during Differentiation PAULL. MANN

I. Introduction ... .................................................................. The Aging Cell Surface ......... .................... Developmental Phenomena .................................................................... Immune Regulation .... ...................................... Neoplastic Regulation ................................................................ odulation and IMR-90 Cellular Senescence Cell-Surface Oligosacc References ................................................................

11. 111. IV. V. VI.

67 71 78

92

Endosperm Development in Maize RICHARD v . KOWLES A N D RONALD L. PHILLIPS

I. Introduction ........................ Early Development ............................................................................... Microscopic Characterization of Endosperm Cells Cellular and Nuclear Activity ................................................................. Evidence for Endoreduplicati Differences in Nuclear DNA .................................. Biological Significance of DN Further Directions ................................................................................ References ................................ ...............

11. 111. IV. V. VI. VII. VIII.

V

97 97 101 I04 1 I4 I22 125 131 I33

vi

CONTENTS

Ameboid Movement and Related Phenomena W. STOCKEM A N D W. KLOFQCKA

........................................ ........................................................................................ Phenomena ............................................................ Organization of the Microfilament System ................................................ stem ................................. Function of the Microfil Concluding Remarks ... .............................................................. Summary ..................................................................... References ................ ....................................................

I. Introduction 111.

IV. V. VI. VII.

137 139 140 149 156 175 177 I79

The Role of Hepatocytes and Sinusoidal Cells in the Pathogenesis of Viral Hepatitis PATRICIA S. LATHAM

............................................................... ................

11. Role of Liver Architecture in the Pathogenesis of Viral Hepatitis 111. Role of Liver-Derived Cells in The Pathogenesis of Viral Hepatitis

185

185

............... 196

IV. Role of Interferon and the Immune Response in the Viral Pathogenesis of Hepatitis ......................................................... V. Genetic and Age-Dependent Determinants of Susceptibility ............................................................... in Viral Hepatitis ....... VI. Conclusion ....................................................... ........ References ................. ................................................................

213 216 219 220

“Leaky” Cells of Glandular Epithelia S. S. ROTHMAN A N D T. MELESE I. Introduction ............................................................. 11. Paracellular versus Transcellular Movement ...................

Ill. IV. V. VI.

The Experimental System ..................................................... Other Evidence of Transcellular Transport and Permeabilit Nature of the Paracellular Path .......................................... Concluding Remarks ... ................................................................... References ....................................................................

INDEX .........................................................................................................

243

245

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. I12

Prolactins of Pregnancy and Their Cellular Source LINDAOGRENAND FRANK TALAMANTES Department of Biology, University of California, Santa Cruz, Santa Cruz, California 95064

I. Introduction

The placentas of numerous species produce hormones that are structurally and functionally similar to the pituitary hormones prolactin (PRL) and growth hormone (GH), which are 20-25K molecular weight proteins that regulate various processes including mammary gland differentiation, steroidogenesis, somatic growth, and intermediary metabolism. The most extensively studied placental PRL-like hormones are the placental lactogens (PLs). Historically, hormones called PLs were identified in the fetal component of placentas from various species on the basis of their ability to mimic the actions of pituitary PRL in various bioassays and radioreceptor assays. Subsequently, substances with PRL-like activity were purified from placentas and characterized biochemically. Although each of these hormones is called a PL because it possesses activity in assays for PRL-like activity, the PLs differ from one another in size and primary function, and some species produce more than one hormone that is a PL. Table I summarizes the nomenclature used to describe PLs in various species. In addition to PLs, the fetal component of the rat, mouse, and bovine placenta has recently been reported to produce other molecules that have amino acid sequence homology to pituitary PRL; their functions are currently unknown. In the mouse these substances include proliferin and proliferin-relatedprotein. In the rat they include PRL-like protein A. This PRL-like protein of the bovine placenta has not been named, but its cDNA has been designated PRL-related cDNA I. The maternal component of the placenta, the decidua, also produces PRL-like substances in some species. The primate decidua secretes a molecule that appears to be structurally identical to pituitary PRL. The rat decidua produces a substance designated decidual luteotropin, which has PRL-like biological activity but is not identical to rat pituitary PRL. In this review we will discuss the biochemistry, the mechanism and regulation of secretion, and the functions of these hormones. The literature from several areas of research has been summarized in tabular I Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

TABLE I NOMENCLATURE A N D SOMEBIOCHEMICAL PROPERTIES OF PLs" Percentage sequence homology* Hormone (synonyms)

Molecular weight

Number of amino acids

PRL

GH

Number of cysteines

p1

Human

hPL (hCS, chorionic PRL)

22,279'

191

67

%

4

4.6-6.2

Rhesus monkey

rhPL (rhCS)

-20,000-22,5Wd

?

7

?

4

?

Baboon

baboon PL

-20,000-25,000'

?

?

?

?

?

Mouse

mPL-I complex (midpregnancy) lactogen) mPL-I1 (mPL)

5

?

31

4

6.6-7.0

?

?

4.5

Species

References

rd .-

Rat

29,000-42,000d

1 94

21,812'

191

51

Li er al. (1973); Belleville er al. (1975); Chattejee er al. (1977); Cooke er a / . (1981) Shome and Friesen (1971); Vinik el al. (1973) Josimovich er a / . (1973) Colosi ef a / . (1987a,b)

Colosi er al. (1982); Jackson er a / . ( 1986) Robertson e r a / . (1982)

34

Hamster

W

4

6.0-6.4

9

> >

6

? 8.3-8.8 6.8-9.0

-25.000d

,

Sheep

haPL-I haPL-I1 (haPL) OPL (OCS)

-20,000-23.000d

?

?

Bovine

bPL (bCS)

-30,000-34,000d

?

?

?

?

4.8-6.3

Goat

CPL

-20,000-25,00Od

?

?

?

?

?

-35,000‘

?

1

Robertson and Friesen (1975): Robertson ef 01. (1982); Duckworth er a / . (1986a) Southard cf u / . (1987) Southard ef a / . (1986) Hurley ef a / . (1975, 1977a.b): Marta1 and Djiane (1975); Chan el a / . (1976. 1986): Reddy and Watkins (1978a) Murthy cf al. (1982); Eakle er a / . (1982); Arima and Bremel (1983); Byatt er nl. (1986) BeCka ef al. (1977)

Abbreviations: PL, placental lactogen; PRL, prolactin; GH, growth hormone: PI,isoelectric point; CS. chorionic somatomammotropin. Percentage homology figures include conservative amino acid replacements. ‘ Molecular weight obtained from amino acid sequence. Apparent molecular weight determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. ‘ Apparent molecular weight determined by gel exclusion chromatography. I T h e term rPL (without the designation “I” or “11”) has been used in two different contexts to refer to PLs from the rat. The term was originally used to designate rPL-I1 (Robertson and Friesen, 1975). More recently, it has been used by several authors to designate placental substances that are detected by assays for PRL-like activity and presumably reflects a combination of rPL-I and rPL-I1 (see Section 11,C.l). a

4

LINDA OGREN AND FRANK TALAMANTES

form to conserve space. Reference citations for literature appearing in the tables are included in the tables rather than in the text. 11. Placental Lactogens

Placental lactogens have been identified in the placentas of a large number of primates, rodents, and artiodactyls (for a complete list see Talamantes, 1975a,b; Kelly et al., 1976; Forsyth, 1986). A PL does not appear to be produced by the pig, cat, dog, horse, zebra, bat, armadillo, ferret, several shrews, and several rhinoceroses (Forsyth, 1986). The existence of a PL in the rabbit is controversial. Purification of the hormone has been reported (Bolander and Fellows, 1976), but its presence in placental tissue has not been confirmed by bioassay or radioreceptor assay (Talamantes, 1975a,b; Kelly et al., 1976). A. BIOCHEMICAL CHARACTERIZATION PLs have been purified from the placentas of the human (e.g., Josimovich and MacLaren, 1962; Cohen et al., 1964; Friesen, 1965a,b; Hunt et a1.,1981), rhesus monkey (Shome and Friesen, 1971; Vinik et al., 1973), mouse (mPL-I: Colosi et al., 1987a; mPL-11: Colosi et al., 1982), rat (rPL-11: Robertson and Friesen, 1975), hamster (haPL-11: Southard et al., 1986), sheep (e.g., Hurley et al., 1975, 1977a; Marta1 and Djiane, 1975; Chan et al., 1976), and bovine (Murthy et al., 1982; Eakle et al., 1982; Arima and Bremel, 1983). Placental lactogens from the baboon (Josimovich et al., 1973) and goat (BeEka et al., 1977) have been partially characterized. Some properties of these hormones are listed in Table I. Structurally, PLs can be divided into two general groups: (1) PLs that have structures that are highly similar to those of the PRL and GH from the same species, and (2) PLs that have structures that appear to differ somewhat from those of GH and PRL. Placental lactogens in the first group are single-chain polypeptides having molecular weights ranging from about 20,000 to 25,000. This group of proteins is characterized by the presence of either two or three intrachain disulfide bonds that are in positions analogous to those of GH and PRL. Both GH and PRL contain a small disulfide loop in the carboxy terminal region of the molecule and a large loop enclosing about 110 amino acid residues. In addition, PRL contains a third small disulfide loop at the amino terminus. Structural similarity to PRL and GH has been best demonstrated for hPL, mPL-11, and rPL-11, which are the only PLs whose complete amino acid sequences are known. As shown in Table I, each of

PROLACTINS OF PREGNANCY

5

these hormones shows significant sequence homology to both PRL and GH from the same species (hPL: Bewley et al., 1972; Cooke et al., 1981; mPL-11: Jackson et al., 1986; rPL-11: Duckworth et al., 1986a). The other PLs of this group are haPL-11, oPL, rhPL, baboon PL, and probably cPL. With the exception of cPL, each of these PLs has been shown to be related immunologically to the homologous PRL and/or GH and to other PLs of this group; the immunological properties of cPL have not been reported (haPL-11: Southard and Talamantes, 1987; oPL: Hurley et al., 1975; rhPL: Shome and Friesen, 1971; Vinik et al., 1973; baboon PL: Josimovich et al., 1973). The amino acid compositions of oPL (Hurley et al., 1977a,b; Chan et al., 1986) and rhPL (Shome and Friesen, 1971) are similar to those of other members of the PRL-GH family. Although the PLs in this group are structurally related to both PRL and GH, some of the PLs are more PRL- than GH-like structurally, while others are more GH- than PRL-like. Analysis of the primary structures of hPL, rPL-11, and mPL-I1 has revealed that the rodent PL-11s share more sequence homology with the homologous PRL than with the homologous GH, while the converse is true for hPL (Table I). Since data on the biological activities and amino acid sequences of PLs are still limited to very few species, it is not known how accurately the degree of sequence homology between a PL and the PRL and GH from the same species predicts the primary functions of the PL. In the case of mPL-11, the relationship between greater sequence homology to mPRL and biological activity appears to be a good one, since all of the known functions of mPL-I1 are PRL-like and the hormone does not bind to mGH receptors in several tissues (Haro and Talamantes, 1985, and unpublished observations). In the case of hPL, however, the high degree of amino acid sequence homology with hGH suggests that the GH activity of hPL should be very high, when in fact, it is relatively low in several different bioassays (Sherwood et al., 1980), demonstrating that seemingly minor differences in amino acid sequence between a PL and GH or PRL can result in significant differences in the activity of the molecules. [The specific differences in primary structure between hGH and hPL that may account for the differences in their bioactivity have been discussed by Nicoll et al. (1986).] Since amino acid sequence data are not available for the other PLs. the number of cysteine residues obtained from amino acid composition analysis has been used by some investigators as a criterion for comparing PLs with the homologous GH and PRL. Based on their cysteine content, hPL, rhPL, mPL-11, and rPL-I1 are similar to GHs, whereas oPL is similar to PRL (Table I). The conclusion suggested by these data on mPL-I1 and rPL-I1 differs from that based on the analysis of the hormones’ amino acid sequences, which raises questions about the

6

LINDA OGREN AND FRANK TALAMANTES

suitability of this method for assessing the structural relatedness of PLs, GHs, and PRLs. The other structural group of PLs includes bPL, mPL-I, haPL-I, and rPL-I. Bovine PL is a single-chain polypeptide having a molecular weight of 30,000 to 34,000 (Eakle et al., 1982; Murthy et al., 1982; Arima and Bremel, 1983). Although complete amino acid sequence data for bPL have not been reported, preliminary studies indicate that it shares about 45% amino acid sequence homology with bPRL (Schuler and Hurley , 1985).The large difference in molecular weight between bPL and bPRL or bGH is due to the presence of additional amino acids in the polypeptide chain of bPL (Schuler and Hurley, 1985). Despite its larger size, bPL appears to share at least some structural features with oPL since it crossreacts with antiserum to oPL (Murthy et al., 1982). Mouse PL-I consists of two complexes of proteins that have been classified by molecular weight and their behavior during purification; they have been designated mPL-I (29-32K) and mPL-I (36.5-42K) (Colosi et al., 1987a). Mouse PL-I (29-32K) comprises three proteins with apparent molecular weights of 29,000, 30,500, and 32,000; the two larger molecular weight components are glycosylated. Mouse PL-I (36.5-42K) is composed of 5 glycoproteins. The protein moiety of mPL-I is a single-chain, 194-amino acid polypeptide (Colosi et al., 1987b). It differs structurally from mPRL, mGH, and mPL-I1 in that it does not contain the large disulfide loop that is characteristic of the family (Colosi et al., 1987a). Very little is known about the structures of rPL-1 and haPL-I. Neither hormone crossreacts with antiserum to the PL-I1 from the same species (rPL-I: Robertson et al., 1982; haPL-I: Southard et al., 1987). The fact that the molecular weights of rPL-I and haPL-I are similar to that of mPL-I and the gestational maternal serum profile of rPL-I is similar to that of mPL-I (Section II,C,2) suggests that these hormones may be analogous to mPL-I. Variant forms of PL that differ with respect to molecular weight, amino acid composition, and net surface charge have been described for PLs from both structural groups. The most extensively examined of these variants are the high molecular weight forms of hPL and haPL-11. These PLs differ from the other PLs that have been characterized in that they exist to a significant extent in the placenta and circulation as high molecular weight forms. The large molecular weight forms of hPL consist of dimers and higher oligomers of the monomeric hormone. About 25% of dimeric hPL is composed of noncovalently-associated monomers, and the remainder consists of two molecules of monomeric hPL that are joined in antiparallel configuration by a disulfide bond between Cys-182 of one monomer and Cys-189 of the other (Schneider et al., 1977, 1979). Higher

PROLACTINS OF PREGNANCY

7

oligomers are non-covalently-associated aggregates of monomer (Schneider et al., 1975a; Cox et al., 1979). The large molecular weight forms of hPL are biologically active (Schneider et al., 1977) and account for about 55 and 10% of the total hPL in the placenta during the first and third trimesters, respectively (Schneider et al., 1975a,b; Calvert et al., 1985). They are also present in serum throughout pregnancy, but account for less than 10% of the total hPL activity (Schneider et al., 1975a; Calvert et al., 1985). haPL-I1 exists in both the placenta and circulation almost entirely as high-molecular-weight forms that are maintained by both disulfide linkages and noncovalent interactions (Southard et al., 1986, 1987). The high-molecular-weight forms of haPL-I1 in the blood differ from those in the placenta. In maternal plasma, haPL-I1 is present primarily as 600K, 210K, and monomeric forms, whereas placental haPL-I1 is very heterogeneous, with molecular weight forms ranging from monomer to >1500K (Southard er al., 1987; J. Southard and F. Talamantes, unpublished observations). It is not known whether these forms are composed of several molecules of monomeric haPL-I1 or whether they consist of molecules of haPL-I1 linked to other proteins. The high-molecular-weight forms of haPL-I1 are probably biologically active since they show activity in radioreceptor assays for PRL-like activity (Southard et al., 1986). The reason hPL and haPL-I1 exist to a significant extent as high molecular weight forms, whereas other PLs do not, is not known. It is possible that in the native proteins, the disulfide linkages of hPL and haPL-I1 are oriented in a configuration that increases their likelihood of forming interchain disulfide bonds with other molecules. Variants of oPL (Chan et al., 1986) and rhPL (Shome and Friesen, 1971) having slightly different amino acid compositions, and charge isoforms of hPL (Belleville et al., 1975; Chattejee et al., 1977), mPL-I1 (Southard et al., 1986), haPL-I1 (Southard et al., 1986),rPL-I1 (Robertson et al., 1982), oPL (Marta1 and Djiane, 1975; Chan et al., 1976; Hurley et al., 1977a; Reddy and Watkins, 1978a; Southard et al., 1986), and bPL (Arima and Bremel, 1983; Byatt et al., 1986) have also been reported. The structural basis for differences in the net surface charge of the PLs is not understood. The existence of forms of rhPL and oPL that differ with respect to amino acid composition could be due to genetic variation, but this question has not been examined experimentally. Structure-function relationships of PLs have not been examined extensively. Studies carried out on hPL suggest that biological and immunological activity are conferred by the amino terminus, and the carboxy terminus is involved in maintaining the conformation of the molecule (Burstein et al., 1978; Russell et al., 1979). The disulfide bonds

8

LINDA OGREN AND FRANK TALAMANTES

of hPL confer stability to the secondary and tertiary structures of the molecule (Aloj et al., 1972). B. SECRETION 1. Site of Production Information about the cell types in the placenta that contain PLs is available for the human, mouse, rat, sheep, and bovine. In all cases, PL-containing cells originate in the fetal component of the placenta. In the human placenta, mRNA for hPL has been localized to the syncytial layer by in situ hybridization (McWilliams and Boime, 1980; Boime et al., 1982; Hoshina et al., 1982a, 1985), indicating that the hormone is synthesized in the syncytium. In situ hybridization studies on mRNAs for other PLs have not been carried out. Information about the cell types that produce PLs in ruminants and rodents has been obtained largely by immunohistochemical staining methods, whereby cells containing the hormone are detected by staining with an antiserum to the hormone. With these methods it is not possible to determine whether a cell stains for the hormone because the hormone is synthesized there or because the cell is a target of the hormone. In the sheep and bovine, oPL and bPL have been localized largely to the binucleate cells of the chorion (Marta1 et al., 1977; Reddy and Watkins, 1978b; Watkins and Reddy, 1980; Wooding, 1981; Verstegen et al., 1985; Duello et al., 1986), and within the binucleate cells of the sheep placenta, oPL has been localized to secretion granules (Wooding, 1981; Lee et al., 1986; Rice and Thorburn, 1986a). The presence of oPL in secretion granules suggests that these hormones are synthesized within the binucleate cells. In the mouse, mPL-I1 has been localized to the giant cells of the chorioallantoic and choriovitelline placentas and to basophilic cytotrophoblasts (Hall and Talamantes, 1984). Less information is available about the cells in the mouse and rat placenta that contain mPL-I, rPL-I, and rPL-11. Data obtained from culturing mouse (Soares et al., 1983)and rat (Soares et al., 1985)placental explants indicate that each of these hormones is secreted by both the chorioallantoic and choriovitelline placentas, probably by giant cells, 2. Synthesis and Mechanism of Secretion Most of the work that has been carried out on the synthesis and mechanism of secretion of PLs has focused on hPL. Two genes, HCS-A and HCS-B, code for hPL, and both are expressed under normal conditions. Their products differ in one amino acid, which is in the signal sequence (Barrera-SaldaAa et al., 1983). HCS-A and HCS-B are linked

PROLACTINS OF PREGNANCY

9

to three other genes of the GH-PL gene family and are located at band q22-24 of chromosome 17 (Harper et al., 1982). The structure of these genes has been reviewed by Barsch et al. (1983) and Parks ( 1984). hPL is synthesized as a prehormone containing a 25-amino acid signal peptide (Sherwood et al., 1979) which is cleaved by microsomal membrane peptidases (Boime et al., 1975, 1980; Cox et al., 1976; Szczesna and Boime, 1976; Strauss et al., 1980). Studies examining the kinetics of hPL synthesis and release in vitro have suggested that the mature hormone exists in two different pools in the tissue: a stable storage pool and a readily releasable pool, with newly synthesized hormone being preferentially channeled into the latter (Suwa and Friesen, 1969; Golander et al., 1978a). The physical basis of this compartmentalization is not understood. A recent study suggests that hPL may be packaged into small secretory granules (Fujimoto et al., 1986), but whether the hormone in these granules represents the storage pool suggested by kinetic experiments is not known. The kinetics of PL secretion have also been examined in mice (Basch and Talamantes, 1986). As is the case with hPL, newly synthesized mPL-I1 is rapidly released. In contrast to the secretion kinetics of hPL, however, there does not appear to be a significant storage pool of mPL-I1 in the mouse placenta. The mechanism by which PLs are released from the cell is not well understood. Evidence obtained from humans and sheep suggests that the processes regulating basal and secretagogue-stimulated PL release differ. Basal hPL and oPL release are not calcium dependent (Choy and Watkins, 1976; Handwerger et al., 1981a; Rice and Thorburn, 1986b),and hPL release is in fact stimulated by the absence of extracellular calcium and by agents that inhibit the influx of calcium or interfere with formation of a calcium-calmodulin complex (Choy and Watkins, 1976; Handwerger et al., 1981a; Handwerger and Siegel, 1983; Zeitler et al., 1983; Hochberg et al., 1984). In contrast, secretagogue-stimulated release of both hormones requires the presence of extracellular calcium (Zeitler et al., 1986; Rice and Thorburn, 1986b). The manner in which PLs are delivered to the maternal blood differs between species. In the human placenta, hPL is released from the syncytial layer of the trophoblast directly into the maternal blood in the intervillous space. In contrast, delivery of PL to the maternal blood in the sheep and bovine appears to depend on the continued movement of PL-containing binucleate cells from the fetal to the maternal component of the placenta (Steven et al., 1978; Wooding, 1981; Duello et al., 1986). PL is synthesized and packaged into secretion granules in the binucleate cells of the chorion. These cells then migrate across the fetal-maternal

10

LINDA OGREN AND FRANK TALAMANTES

junction and fuse to form a syncytium which is closely associated with the maternal circulation. C. GESTATIONAL PROFILES A N D METABOLISM 1, Assays for Measuring PL Concentrations

PL concentrations in various biological fluids were originally estimated with various bioassays and radioreceptor assays for PRL-like, and in some cases, GH-like activity. Although many of these assays have been valuable in identifying tissues containing PLs, their usefulness in determining the absolute concentrations of PLs in tissues is limited by the fact that these assays detect other hormones in addition to PLs. Consequently, as pure preparations of PLs have become available, highly specific radioimmunoassays (RIAs) for the hormones have been developed. At the present time RIAs are available for hPL, rhPL, mPL-I, mPL-11, rPL-11, haPL-11, oPL, and bPL (see references in Section II,C,2). Concentrations of rPL-I and cPL have only been estimated by bioassay and radioreceptor assay. In some cases, the specificity of bioassays and radioreceptor assays for PLs from the rat and goat have been improved by ( I ) neutralizing the activities of PRL-like hormones that are not of interest by treating samples with specific antisera (e.g., Tonkowicz et al., 1983), ( 2 ) using RIAs to measure the concentrations of PRL-like hormones that are not of interest and then “subtracting” these values from the total PRL-like activity measured by radioreceptor assay or bioassay (e.g., Hayden et al., 1980), or (3) in the case of samples containing rPL-I and rPL-11, by separating the two hormones chromatographically prior to assay (Soares et al., 1985). However, in many other instances the total PRL-like activity of samples has been reported, which has created confusion, particularly in the rat PL literature. In these experiments, no attempt was made to distinguish between rPL-I and rPL-I1 and investigators have referred to the species being measured as “rPL.” In the present discussion, references to the “rPL” literature have been omitted when similar experiments utilizing specific RIAs have been reported. When the “rPL” literature has been cited, we have designated the activity “total PRL-like activity” or “total placental lactogenic activity” to indicate that all PRL-like activity in the placenta or blood was measured. 2 . Gestational Projles of PLs The gestational profiles of PLs in the maternal blood of several species are shown in Figs. 1-3. These profiles differ between species with respect to ( I ) their overall pattern, (2) the absolute concentration of PL that is

PROLACTINS OF PREGNANCY

Mid-gestation

Term (-38 weeks)

FIG. I . Gestational profiles of PI, in the maternal circulation of the human (top) and rhesus monkey (bottom). Hormone concentrations were measured by RIA. Kedrawn from Berle (1974) (hPL), Walsh ('1 rrl. (1977:)) (rhPL). and Novy i ~ fctl. (1981) (rhPL).

present, (3) the stage of gestation when the hormone first appears in the circulation, and (4) the relative concentrations of hormone in the maternal and fetal circulations. The gestational profiles of PLs in the maternal circulation fall into two general patterns. The first includes the serum profiles of rat and mouse PL-I, which are characterized by a very large peak during or slightly after midpregnancy , with hormone concentrations falling to very low to undetectable values several days later (Fig. 2). In contrast to this general pattern, the gestational profiles of the other PLs are characterized by the continued presence of the hormones in the blood from the time they first appear until parturition. In some species of this group, the PL concentration of the maternal blood increases gradually throughout pregnancy and

12

LINDA OGREN AND FRANK TALAMANTES

P I

b c

5.0

c

C

a, -

._ 9

3

a, 0

7

2.5

a .c

.

E

m

I

Y

J

Mid-gestation

a L

Term (-16 days) 800

8.0

I

? A

E 400 &

-

<m 4.0

-

C

-

I

Y

-

L a

i a

E

E

Mid-gestation

?

Term (-19 days) 900

3.0

A

h

m

I

c

a, m

._ 3

450

1.5 1

P

a:

-

. -

a;

a 0 -

l l

E

m

5

-

-J

a;

Mid-gestation

Term (-22 days)

FIG. 2. Gestational profiles of PL-I and PL-11 in the maternal circulation of the hamster (top). mouse (middle), and rat (bottom). The concentrations of haPL-11, mPL-I, mPL-11, and rPL-II were measured by RIA. The concentrations of haPL-I and rPL-I were estimated by radioreceptor assay as PRL-like activity not due to PRL or PL-11. Redrawn from Southard el d.(1987) (haPL-I, haPL-II), Colosi er a / . (1986) (mPL-I), Robertson and Friesen (1981) (rPL-11)- and Robertson el ul. (1982) (rPL-I). Data for mPL-I1 from D. Bravo and F. Talamantes, unpublished.

PROLACTINS OF PREGNANCY

13

in others it levels off or drops toward the end of pregnancy. The rodent PL-11s appear in the maternal blood somewhat later in pregnancy than do the other PLs; the serum profile of haPL-I1 is particularly striking in this respect. The PL-I1 profile shown for the mouse in Fig. 2 is that of the Swiss Webster strain. Balb/c mice show a similar profile, whereas the PL-I1 profile of C3H mice levels off during the final week of gestation (Soares and Talamantes, 1982, 1983, 1985). The species differences in the maximum PL concentrations of the maternal blood are quite marked, with values ranging from a few nanograms per milliliter of serum in the bovine to micrograms per milliliter of serum in the primates, sheep, mouse, and hamster. It is interesting to note that major differences in maximum PL concentrations exist even between species from the same order; for example, compare PL (PL-11) concentrations between the sheep and bovine (Fig. 3) or between the hamster and mouse or rat (Fig. 2). Of the species that have been examined thus far, it appears that the ruminants differ from both the primates and rodents in the distribution of PL between the maternal and fetal circulations. In the sheep, the oPL concentration of the fetal blood exceeds that of the maternal blood for the first half of pregnancy (Chan et al., 1978a; Gluckman et al., 1979), and in the bovine, the fetal serum PL concentration is about 5 to 10 times higher than the PL concentration of the maternal blood throughout gestation (Beckers et al., 1982; Duello et al., 1986). In contrast, the PL (PL-11) concentration of the fetal blood is less than 1% that of the maternal blood at term in the human (Kaplan and Grumbach, 1965) and rhesus monkey (Novy et al., 1981) and less than 10% that of the maternal blood during the last third of pregnancy in the mouse (D. Bravo and F. Talamantes, unpublished observations). It is not known whether the differences in gestational PL profiles between species are associated with differences in the primary functions of these hormones. This question cannot be answered until more information is obtained about the biological activities of the PLs in homologous systems (see Section 11,E).The species differences in the distribution of PL between the fetal and maternal compartments are particularly interesting because they suggest that the role of PL in the fetus may be relatively more important in those species in which fetal PL concentrations are high than in species in which the fetal blood contains a very low concentration of the hormone. 3. Placental Lactogens in Amniotic and Allantoic Fluid Human PL (Grumbach et al., 1968; Berle, 1974; Nielsen and Schioler, 1981) and mPL-I1 (D. Bravo and F. Talamantes, unpublished observa-

14

LINDA OGREN AND FRANK TALAMANTES

Bovine

C 1 a I]

0.5 '

Mid-gestationO

'

Term Z (-40 weeks)

Sheep e

E

-4

1.5 -

1

a

I

//

300

-c

I

-

0,

1

%

I

I

Mid-gestation

I

I

Term (-21 weeks)

F I G . 3. Gestational profiles of PLs in the maternal circulation of the bovine (top), sheep (middle), and goat (bottom). The concentrations of bPL and oPL were measured by RIA. The concentration of cPL was estimated by radioreceptor assay as PRL-like activity not due to PRL. Redrawn from Beckers et a / . (1982) (bPL), Handwerger PI ul. (1977) (oPL). and Hayden et ul. (1980) (cPL).

tions) have also been detected in the amniotic fluid, their concentrations in this compartment being significantly higher than those of fetal serum. Ovine PL (Chan et al., 1978a; Handwerger et al., 1977) and bPL (Beckers et al., 1982) are present in both amniotic and allantoic fluid. The role of PLs in these compartments is unknown. The source of amniotic fluid hPL and oPL appears to be direct secretion into the amnion, since very little hormone is transferred from either the maternal or fetal circulation into the amniotic fluid (Kaplan et al., 1968; Reddy and Watkins, 1983).

15

PROLACTINS OF PREGNANCY TABLE I1 HALF-TIME OF DISAPPEARANCE OF PLS FROM Rapid phase (minutes)

(,<,,

PL hPL rhPL mPL-II rPL-I rPL-I1 OPL

11.3 20 0.2 19.5 31 I .2 6 29.1

THE

phase (minutes)

MATERNAL CIRCULATION

ill? Slow

48.5 18. I -b 1063

-h -b -

References Kaplan et al. (1968) Belanger et al. (1971) Pirion et a / . (1988) Kelly er a / . (1975) Glaser et al. (1985) Kelly el al. (1975) Glaser et a / . (1985) Handwerger et al. (1977)

" The disappearance curves for rhPL were multiexponcntial but a half-life value was calculated only for the rapid phase. " The disappearance curves did not show a slow phase. ' A slow phase oldisappearance ofoPL was present in some hut not all animals. When present. the of the \low phase was about 40 minutes.

4. Circadian Variation Circadian variation in the PL concentration of the maternal blood has not been noted in any of the species that have been examined (hPL: Pavlou et al., 1972; Ayala et al., 1984; mPL-11: Soares and Talamantes, 1984; oPL: Chan et al., 1978a; Taylor et al., 1980; cPL: Hayden et al., 1980).

5. Metabolism The clearance of PLs from the maternal circulation has been investigated in the human, rhesus monkey, sheep, mouse, and rat (Table 11). In each of these species except the rat, the disappearance of PL has been reported to show biexponential kinetics. The disappearance of rPL-I1 has been reported to follow first-order kinetics. The data shown in Table I1 suggest that the clearance rates of the 20,000-25,000 molecular weight PLs are roughly inversely related to body mass. Factors other than body mass also influence the rate of disappearance of the hormones from the circulation, however, since a 4-fold difference in the half-life of the rapid phase of the disappearance curves remained between hPL and mPL-I1 when these data were expressed as a function of metabolic body size (Piiion et a/., 1988). The much slower half-time of disappearance of rPL-I compared with that of rPL-I1 is most likely due to differences in the size and structure of the hormones. The mechanisms by which PLs are cleared from the maternal circulation have not been investigated except in

16

LINDA OGREN AND FRANK TALAMANTES

humans, where removal of hPL is known to occur by mechanisms that do not involve excretion of the intact hormone into urine (Kaplan et a l . , 1968; Grumbach et al., 1968).

D. REGULATION OF SECRETION Although the regulation of secretion of PLs has been the subject of intensive investigation since hPL was first purified in the early 1960s, a good understanding of what controls the concentrations of these hormones in the maternal and fetal blood has still not been achieved for any species. One difficulty in studying the regulation of secretion of the PLs stems from the fact that it is not clear whether all of the PLs are regulated by similar mechanisms. This is probably unlikely, in fact, given that the structures and major functions of the hormones differ between species. However, in discussing the regulation of secretion of these hormones, some investigators have unfortunately ignored species distinctions and drawn conclusions about the control of a generic PL. These conclusions have frequently been based on data obtained in the human, in which most of the work has been carried out. A second difficulty in understanding the regulation of PLs arises from the fact that the mechanisms through which putative regulators act have rarely been examined. Thus, most of the work in this field describes changes in the concentration of a PL in the blood or in incubation medium after treatment with a putative regulator. In the case of in uiuo studies, changes in the PL concentration of the maternal or fetal blood could arise from changes in any of the following parameters: the metabolic clearance rate of PL, the number of PLproducing cells, the rate of PL synthesis by each cell, and the rate of release of PL by the cells. Furthermore, the effects of putative regulators in uiuo could be due to direct action of the regulator on PL-producing cells or they could be mediated by other factors produced by other tissues or other placental cell types. Although data from in vitro studies are easier to interpret in that direct action of a putative regulator on the placenta can be assumed, many of these studies suffer from the drawback that no attempt was made to demonstrate that the putative regulator affected PL secretion specifically, and consequently, generalized effects of the regulator on cellular protein synthesis and tissue viability cannot be ruled out. One other deficiency of the literature in this field is the absence of information about gestational changes in the regulation of the PLs. Although gestational changes in the effects of several putative regulators have been reported for oPL (Taylor et af.,1983a,b)and hPL (Belleville et

PROLACTINS OF PREGNANCY

17

al., 1978), a systematic examination of this question has not been undertaken. The following discussion of the literature on the regulation of PLs is organized by putative regulator. We have attempted to point out features of PL regulation that appear to be common to several species, but such generalities are difficult to formulate at the present time because only limited data are available.

I . Placental Mass and the Number of PL-Producing Cells The absolute concentration of hPL (Spellacy et al., 1978), mPL-I1 (Soares and Talamantes, 1983), rPL-I1 (Robertson and Friesen, 1981), oPL (Handwerger et al., 1977; Taylor et al., 1980; Butler et al., 1981),and cPL (Hayden et al., 1979, 1980; Forsyth et al., 1985) in the maternal blood is influenced by the number of conceptuses, with PL concentrations being higher in animals carrying more fetuses. The effect of litter size is probably due primarily to the increase in placental mass that accompanies multiple fetuses. This conclusion is supported by data collected in sheep, where restricting the growth of the placenta was accompanied by a reduction in the oPL concentration of the maternal plasma without changing the production rate of oPL per unit mass of tissue (Falconer et al., 1985). Changes in the number of PL-producing cells during gestation appear to contribute significantly to determining the gestational PL profile of the human and probably the sheep. In the human, several lines of evidence suggest that the gestational increase in the hPL concentration of the maternal blood results from an increase in the production rate of the hormone rather than from a decrease in the rate at which it is cleared from the circulation. First, the metabolic clearance rate of hPL does not differ between men and pregnant and nonpregnant women (Kaplan et al., 1968), indicating that pregnancy-specific factors do not affect the rate of hPL elimination from the circulation. Second, translation of polysomes or total placental poly(A)-mRNA from third trimester placentas yielded more hPL than did translation of polysomes or poly(A)-mRNA from first trimester tissue (Boime and Boguslawski, 1974a,b; Boime et al., 1976; Chattejee et al., 1976; Hubert et al., 1981), indicating that the ability of the placenta to synthesize hPL increases as gestation advances. This increase in synthetic capacity results from increases in both the total amount of hPL mRNA in the placenta and the fraction of poly(A)-mRNA that is hPL mRNA (Hubert et al., 1981; McWilliams et al., 1977); translational efficiency does not change significantly between the first and third trimesters (Boime and Boguslawski, 1974a,b; Boime et al., 1976;

18

LINDA OGREN AND FRANK TALAMANTES

Chatterjee et al., 1976; Hubert et al., 1981). The concentration of hPL mRNA in the syncytial layer remains relatively constant during pregnancy (Hoshina et al., 1982b) while the proportion of syncytial tissue in the trophoblast (Pierce and Midgely, 1963; Hoshina et al., 1982b) and the mass of the trophoblast (Braunstein et al., 1980) increase as pregnancy advances, which suggests that the gestational increase in the amount of hPL mRNA in the placenta results primarily from an increase in the mass of the syncytium. In sheep the number of binucleate cells in the placenta appears to play a role in determining the serum profile of oPL during the first half and again at the end of pregnancy. As shown in Fig. 3, the oPL concentration of the maternal blood increases until about day 120 of gestation. The percentage of binucleate cells in the trophectoderm remains relatively constant until approximately the last two weeks of pregnancy (Wooding, 1982),and fetal cotyledonary mass increases until about day 85 (Marta1and Djiane, 1977), suggesting that the total number of binucleate cells increases until about day 85, parallelling the gestational serum oPL profile. At the end of pregnancy, the number of binucleate cells decreases (Wooding, 1982), as does the oPL concentration of both the fetal and maternal blood. Unfortunately, information about gestational changes in the number of PL-producing cells is not available for other species, and consequently, it remains to be determined whether regulation of the gestational maternal serum PL profile by the number of PL-producing cells is a common feature of PL production. It should be noted that the gestational maternal serum PL profiles of several species have been examined for correlations with gestational changes in placental mass, the assumption apparently being that an increase in placental mass is accompanied by an increase in the number of PL-producing cells. Although this relationship appears to be valid in the human (as mentioned earlier), it has not been verified in other species, and these types of data should be interpreted cautiously. 2. Fetus There is evidence that the fetus is important for normal development of the gestational serum PL profile of the mother in the rhesus monkey, rat, and sheep. In the rhesus, a stimulatory effect of the fetus on the rhPL concentration of the maternal blood appears to result from its stimulation of normal placental growth and development (Belanger et al., 1971; Walsh et al., 1977b). A similar situation appears to occur in the case of fetal stimulation of rPL-I levels in the rat, where fetectomy in early pregnancy results in failure of giant cell development (Tonkowicz et af., 1983). The rat fetus also regulates the rPL-I1 concentration of the maternal serum during the second half of pregnancy but in this case the mechanism, which

PROLACTINS OF PREGNANCY

19

is unknown, appears to be more specific than a general effect on placental growth (Robertson et al., 1984a). In the sheep, examination of fetal control of the maternal serum oPL concentration has focused primarily on regulation of the preparturitional decline in oPL concentration. Hypophysectomy of fetal sheep prevented this decline (Taylor et al., 1983a), but fetal adrenalectomy (Wintour et al., 1982) and treatment of fetuses with adrenocorticotropin or dexamethasone (Taylor et al., 1983b; Lowe et al., 1984) did not. The fetal sheep pituitary does not appear to regulate the concentration of oPL in the maternal blood earlier in pregnancy, however (Taylor et al., 1983a,b). 3. Genetic Factors Recent reports have described the occurrence of normal pregnancies in several women having low to undetectable serum and placental concentrations of hPL (Nielsen et al., 1979; Sideri et al., 1983). The low concentration or complete absence of the hormone was due to partial or complete deletion of the hPL genes, with hPL concentration being approximately one-fourth of normal in an individual who had only one of the four hPL genes (Wurzel et al., 1982; Parks et al., 1985). The correlation between blood hormone concentration and the number of remaining hPL genes in this woman indicates that a compensatory increase in the expression of the remaining hPL gene did not occur and further suggests that at any stage of gestation, the maximum production rate of hPL is regulated largely by the number of hPL genes that is present (Parks et al., 1985). In mice, the genotype of the feto-placental unit plays an important role in determining both the absolute concentration and the gestational pattern of mPL-I1 in the maternal serum (Soares and Talamantes, 1983). As discussed in Section II,C,2, the gestational profile of mPL-I1 differs between mouse strains. When animals from two different strains were mated, the absolute concentration of mPL-I1 in the maternal serum was greater than that of females bred to males of the same strain, and the gestational maternal serum mPL-I1 profile was characteristic of the male strain. These observations suggest that in mice, the genotype of the feto-placental unit is more important than the maternal environment in regulating the maternal serum gestational mPL-I1 profile. 4. Metabolic Factors

The studies examining the regulation of secretion of PLs by the diet and various metabolic factors are summarized in Table 111. The rationale for these studies is based largely on suggestions that one of the functions of PLs during pregnancy is to bring about changes in maternal intermediary

REGULATIONOF

PL hPL

Putative regulator Fasting

Protein-rich food

hPL

TABLE Ill PLs BY DIETAND METABOLITES

Experimental design" (endpoint) Fasting up to 90 hours first half of pregnancy ([hPL] of maternal blood) Ingestion of protein-rich meal ? glucose, third trimester ([hPL] of maternal blood) Infusion or oral glucose, second and third trimesters ([hPL] of maternal blood)

Result

t No effect No effect/ -1

Glucose (hypoglycemia)

Insulin infusion, second and third trimesters ([hPL] of maternal blood)

No effect/ t

Glucose

Long-term incubation of third trimester explants or cells in various concentrations of glucose ([hPL] of medium) Alter triglyceride and/or fatty acid levels of blood by drug treatment, third trimester [ (hPL] of maternal blood)

No effectloptima1 glucose concentration exists for hPL release in vitro

Tnglycerides, free fatty acids

No effect

References Kim and Felig (1971); Tyson er a/. (1971a) Tyson er al. (1971b)

Spellacy ef a [ . (1966, 1971); Samaan et al. (1966); Grumbach et al. (1%8): Burt er al. (1970); Ajabor and Yen (1972): Gaspard et al. (1974): Surmaczynska ef al. (1974); KUN er al. (1975) Gaspard et al. (1974); Spellacy et al. (1966, 1971); Sarnaan et al. (1966); Grumbach et al. (1968); Ajabor and Yen (1972) Belleville et al. (1978): Zeitler er al. (1983)

Moms et al. (1974); Gaspard et al. (1975, 1977)

rhPL

Fasting

Protein deprivation Glucose (hypoglycemia)

!2

mPL-I1

Fasting

rat total PRL-like activity

Protein deprivation

OPL

Fasting

Glucose (hyperglycemia) Glucose (hypoglycemia)

a

Fasting up to 72 hr, third trimester ([rhPL] of maternal blood) Low protein diet, entire pregnancy ([rhPL] of maternal blood) Insulin infusion to mother or fetus, third trimester ([rhPL] of maternal, fetal blood) Fasting up to 48 hr, second half of pregnancy ([mPL-I11of maternal blood) Low protein diet, second and third trimesters (total PRLlike activity of maternal blood and placenta) Fasting up to 72 hr, third trimester ([oPL] of maternal, fetal blood) Glucose infusion of fetus, third trimester ([oPL] of maternal, fetal blood) Insulin infusion of ewe or fetus, third trimester ([oPL] of maternal, fetal blood)

No effect

Friesen ef al. (1972); Novy ef al. (1981)

No effect

Novy et al. (1981)

N o effect

Chez er al. (1970).

t

Fielder et al. (1987)

1

Wunderlich ef al. (1979); Pilistine and Munro (1984)

t

Brinsmead er al. (1981)

No effect

Brinsmead er a / . (1981)

No effect

Brinsmead et al. (1981)

The terms “short-term” and “long-term” designate treatments that lasted less than 24 hours and more than 24 hours, respectively

22

LINDA OGREN AND FRANK TALAMANTES

metabolism that ensure the availability of adequate nutrients for the fetus (Section II,E,2). The model predicts that PL secretion should increase when nutrient availability is low (e.g., during fasting and between meals) and decrease when nutrient availability is high (e.g., after eating). Effects of prolonged fasting on maternal and fetal serum PL concentrations have been examined in the human, mouse, sheep, and rhesus monkey. In the human and mouse, fasting was followed by an increase in the hPL and mPL-I1 concentrations of the maternal serum, respectively, and in the sheep, fasting resulted in an increase in the oPL concentrations of both the maternal and fetal blood. Fasting did not affect the PL concentration of the maternal blood in the rhesus monkey, however, which suggests that an increase in the maternal serum PL concentration may not be a universal response to starvation. The factors that stimulate the increase in the PL concentration of the blood in response to fasting are not known, nor is it known whether the stimulus originates in the mother or fetus or both. Although it is well established that the concentrations of numerous hormones and metabolites in the blood are affected by fasting, none of these substances has been examined for an effect on PL secretion under conditions designed to mimic conditions of prolonged starvation. The effects of changes in the composition of the diet on circulating PL concentrations have received little attention. Protein deprivation did not affect the maternal serum PL concentration of the rhesus monkey, but it did result in a decrease in the total PRL-like activity of the serum and placenta of rats. Although it is likely that the major PRL-like species in the latter study was rPL-11, interpretation of these data is complicated by the fact that the contribution of each of the PRL-like hormones to the total PRL-like activity was not assessed (see Section II,C,l). A number of studies have examined the effects of short-term changes in the serum concentrations of glucose and lipids on PL concentrations. In humans, altering the free fatty acid and triglyceride concentrations of the maternal blood did not affect the hPL concentration of the maternal blood. Data on the effects of changes in glucose concentration on PL concentrations in the human, rhesus monkey, and sheep are contradictory, with investigators reporting ( I ) increases in serum PL concentrations in response to insulin-induced hypoglycemia, (2) decreases in serum PL concentrations after glucose infusion, and (3) no effect of either treatment. The data from studies in which insulin was used in humans to induce hypoglycemia are difficult to interpret because insulin itself has been reported to stimulate (Hochberg et al., 1983; Perlman et al., 1985; Bhaumick et al., 1987) and to have no effect (Suwa and Friesen, 1969; Zeitler et al., 1983) on hPL secretion in vitro. In many of the in vivo studies in which a change in the glucose concentration of the blood was followed by change in the serum hPL concentration, a causal relationship

PROLACTINS OF PREGNANCY

23

was difficult to establish because the time course of the hPL response was not consistent with that of the change in glucose concentration (see, for example, Burt et al., 1970; Gaspard et al., 1974). In addition, the magnitude of the hPL response to a change in glucose concentration has generally been small, and in one case it was attributed to the change in blood volume that occurred after injection of a hypertonic glucose solution (Spellacy et al., 1971; Gaspard et al., 1974). Thus, it appears unlikely that short-term changes in the glucose or lipid concentrations of the blood play a significant role in regulating PL secretion in humans, and the few studies that have been carried out in the rhesus monkey and sheep suggest that the same may hold true in these species as well. 5. Pituitary The influence of the maternal pituitary on the maternal serum PL concentration differs significantly among species. In rhesus monkeys (Walsh et al., 1977b) and goats (Buttle et al., 1979; Hayden et al., 1980), maternal hypophysectomy during the second half of gestation did not affect the PL concentration of the maternal blood. In contrast, hfpophysectomy of mice (Day et al., 1986) and rats (Robertson et al., 1984b; Voogt et al., 1985) resulted in a large increase in the maternal serum PL-I1 concentration. The response of mice to hypophysectomy was rapid, a significant increase in serum mPL-I1 concentration occurring within 12 hours after surgery on day 12 of pregnancy (Day et al., 1986). The response of rPL-I1 to hypophysectomy was much slower, with maternal serum rPL-I1 concentrations increasing about 6 days after surgery on day 12 (Robertson et al., 1984b).The difference in the time course of the PL-I1 response between the two species suggests that there may be some species differences in the mechanisms that mediate the response, although an increase in the half-life of the hormone in the circulation has been implicated as a causal factor in both species (Glaser et al., 1985; Pirion et al., 1988). It is not known whether the pituitary also regulates the secretion rates of mPL-I1 and rPL-11. Recent studies suggest that GH is the pituitary factor that suppresses mPL-I1 concentrations in mice (Kishi et al., 1988). Preliminary data suggest that the inhibitory effect of the pituitary on mPL-I1 levels is not direct but is mediated by the ovaries and/or adrenals (K. Kishi, G. Thordarson, and F. Talamantes, unpublished observations). 6. Ovary, Adrenal Gland, and Steroid Hormones Our understanding of the regulation of PL concentrations by the ovary, adrenal gland, and the steroids produced by these tissues is very fragmentary. Studies carried out in the human on the effects of progestogens and

24

LINDA OGREN AND FRANK TALAMANTES

estrogens are contradictory, but most have shown no effect of either group of steroids on hPL secretion (Table IV). An interesting exception is the work reported by Belleville et al. (1978). In this study, progesterone stimulated hPL secretion by placental explants from the first trimester of pregnancy but inhibited hPL secretion by tissue from the third. If confirmed, these data demonstrate a clear difference between stages of pregnancy in the response of hPL to a single regulator. Studies examining effects of glucocorticoids have suggested that these hormones do not affect hPL secretion, although a recent report has identified binding regions for the glucocorticoid receptor in the hPL gene (Eliard et al., 1985).

In the mouse and rat, the regulation of PL concentrations by the ovary and by progesterone has been difficult to examine because ovarian progesterone is required for the maintenance of pregnancy. Thus, it is difficult to separate effects of ovariectomy or progesterone treatment that are specific to PL-I1 secretion from effects of these treatments on the viability of the feto-placental unit. Despite this limitation, it has been possible to show that ovariectomy during the second half of pregnancy causes an increase in the PL-I1 concentration of the maternal blood in both species, but the mechanism by which the ovaries lower PL-I1 concentrations has not been determined. In the rat, progesterone alone or in combination with estradiol did not affect the maternal serum rPL-I1 concentration of ovariectomized animals, whereas estrogens lowered the serum rPL-I1 concentration of animals that had been subjected to both ovariectomy and fetectomy. Effects of ovariectomy in the rat have also been examined in one study that made use of a nonspecific assay for placental lactogenic activity which detected both rPL-I and rPL-I1 (Tonkowicz and Voogt, 1984; also see Section II,C,l). In this study, ovariectomy prior to midpregnancy resulted in a large decrease in the placental lactogenic activity of the maternal serum and the activity was partially restored by progesterone and estradiol. The marked difference between these data and those showing an increase in the rPL-I1 concentration of the maternal serum after ovariectomy (Robertson et al., 1984b) is probably due to the fact that Tonkowicz and Voogt (1984) performed ovariectomy at an earlier stage of pregnancy, which disrupted normal placental development. In both mice and rats, treatment of placental explants or cells from midpregnancy with progesterone in vitro inhibited PL-I1 secretion. It is noteworthy that in the rat placenta, progesterone treatment was effective only when applied to a mixed population of labyrinth cells that was undergoing differentiation into cell types that secrete PL-11; it had no effect on PL production by mature trophoblast giant cells. These data

TABLE IV REGULATION OF PLs BY THE OVARY,ADRENAL GLAND,AND STEROIDS PL

Putative Regulator

Experimental Design" (endpoint)

hPL

Estrogens

Short-term treatment in uiuo, third trimester ([hPL] of maternal blood) Long-term incubation of cells or explants from first and third trimesters ([hPL] of medium) Short- and long-term incubation of cells or explants from first and third trimesters ([hPL] of medium) Long-term treatment in uiuo, third trimester ([hPL] of maternal blood) Short- and long-term incubation of third trimester explants ([hPL] of medium) Ovariectomy, second half of pregnancy ([mPL-111 of maternal blood) Long-term incubation of explants from midpregnancy ([mPL-111 of medium) Long-term incubation of explants from midpregnancy ([mPL-I11 of medium)

Progestogens

Glucocorticoids

Glucocorticoids

mPL-I1

Ovary

Progesterone

Estradiol

Result

References

No effect

N o effect/

Niven et a / . (1974)

t

N o effect/ f /

Belleville et a / . (1978); Zeitler et a / . (1983)

1

Suwa and Friesen (1969); Belleville et d. (1978); Wilson el d. (1980); Zeitler el al. (1983)

No effect

Lange and Anthonsen (1980); Ylikorkala et a / . (1974)

No effect

Suwa and Friesen (1969); Wilson and Jawad (1982)

t

Soares and Talamantes (1985)

1

Soares and Talamantes (1985)

No effect

Soares and Talamantes (1985)

(continued)

TABLE IV (Continued) Putative Regulator

PL rPL-I1

Ovary and adrenal gland Adrenal gland

Progesterone and estrogens

Estrogens

Progesterone

OPL

a

Progesterone

Experimental Design” (endpoint) Ovariectomy 2 adrenalectomy, second half of pregnancy ([rPL-Ill of maternal blood) Adrenalectomy. second half of pregnancy ([rPL-111 of maternal blood) Long-term treatment of ovariectomized rats with progesterone 2 estrogens, second half of pregnancy ([rPL-Ill of maternal blood) Long-term treatment of ovariectomized-fetectomized rats with estrogen, second half of pregnancy ([rPL-11] of maternal blood) Long-term incubation of cells from midpregnancy (total PRL-like activity, probably PL-11, of medium) Inhibit progesterone synthesis in uiuo, late pregnancy ( [oPL] of maternal blood)

Result

References

t

Robertson er a/.(1984b)

N o effect

Robertson er a/.(l984b)

No effect

Robertson et a / . (1984b)

Robertson er a/.(1984a)

Soares and Glasser (1987)

No effect

Taylor e f al. (1982)

The terms “short-term” and “long-term” designate treatments that lasted less than 24 hours and more than 24 hours, respectively.

PROLACTINS OF PREGNANCY

27

suggest that progesterone may inhibit the differentiation of PL-producing cells rather than PL production per se (Soares and Glasser, 1987). Only one study has examined the regulation of PL concentrations by steroids in the sheep, and these data indicated no effect of short-term inhibition of progesterone synthesis on the maternal serum of oPL concentration. These data do not rule out the possibility that progesterone or other steroids affect oPL secretion over the long term, however. 7. Neurotransmitters and Neuropeptides In most cases, the substances in this category that have been examined as putative regulators of PL secretion were chosen because they are known to regulate the secretion of GH and/or PRL, the hypothesis being that structurally similar hormones may be regulated by similar factors. As shown in Table V, this hypothesis has generally not been substantiated in the case of PLs from the human and ruminants, where somatostatin, thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), p-endorphin, acetylcholine, and p-adrenergics have not affected PL concentrations either in vivo or in vitro. Studies examining the effects of dopamine and dopamine agonists on PL concentrations have yielded conflicting data. Treatment with dopamine or its agonists in vivo has generally produced no change in the concentration of PLs in the blood, while treatment in vitro has resulted in a decrease in bPL and hPL secretion. The reliability of the data from the in vitro studies has been questioned by a report that dopamine alters the activity of PLs in some assays (Thordarson and Forsyth, 1984). The significance of the increase in the oPL concentration of the sheep placenta in response to dopamine treatment is not known. In the mouse, preliminary data from our laboratory suggest that GH-releasing hormone (GH-RH) and GnRH stimulate the secretion of mPL-I1 in vitro. Although it is not known whether GnRH and GH-RH are present in the mouse placenta, GnRH (Tan and Rousseau, 1982) and a GH-RH-like molecule (Baird et a f . , 1985) have been identified in the human and rat placenta, respectively. If similar substances are present in the mouse placenta, the stimulation of mPL-I1 secretion by these neuropeptides in vitro may have physiological significance. 8. Arginine Treatment of sheep (Handwerger et al., 1978; Grandis and Handwerger, 1983) and women (Prieto et al., 1976) with large concentrations of arginine results in an increase in the PL concentration of the maternal serum. In the sheep, the effect appears to be mediated by ornithine, which is a metabolite of arginine in the urea cycle (Handwerger et al., 1981b).

TABLE V REGULATION OF PLs BY NEUROTRANSMITTERS A N D NEUROPEPTIDES PL hPL

Putative regulator" Dopamine and its agonists

Somatostatin

TRH

hPL

GnRH

Acetylcholine

p- Adrenergic agonists

OPL

Dopamine and its agonists

Experimental designb (endpoint) Short-term incubation of third trimester explants ([hPL] of medium) Long-term treatment in uiuo, first trimester ([hPL] of maternal blood) Short-term incubation of third trimester explants ([hPL] of medium) Short-term treatment in uiuo, 8-22 weeks of gestation ([hPL] of maternal blood) Short-term incubation of third trimester explants ([hPL] of medium) Long-term incubation of explants, all stages of pregnancy ([hPL] of medium) Short-term incubation or perfusion of third trimester tissue ([hPL] of medium) Long-term incubation of third trimester explants ([hPL] of medium) Short-term incubation of third trimester explants ([hPL] of medium) Short- and long-term treatment of fetus in uiuo, second half of gestation ( [oPL] of maternal, fetal blood)

Result

1

References Macaron et al. (1978b)

No effect

Ylikorkala et al. (1980); Tolino et al. (1985)

No effect

Macaron et al. (1978a.b)

N o effect

Hershman et a/. (1973)

No effect

Hershman et al. (1973)

No effect

Khodr and Siler-Khodr (1978); Siler-Khodr et a/. (1986)

No effect

Welsch (1979)

.1

Belleville et a/. (1974, 1978)

No effect

Shu-rong ei al. (1982)

No effect

Lowe et a/. (1979); MartiHenneberg et a / . (1981); Taylor ei a/. (1983a)

OPL

Dopamine and its agonists

TRH

Somatostatin

P-Endorphin h) W

CPL

Dopamine agonist

bPL

Dopamine

mPL-I1

GH-RH

GnRH

TRH

a

Long-term treatment of ewes in uiuo, second half of pregnancy ( [oPL] of maternal blood, fetal blood) Long-term treatment of ewes in uivo. second half of pregnancy ([oPL] of placenta) Long-term treatment of fetus in uiuo,second half of gestation ([oPL] of maternal, fetal blood) Long-term treatment of fetus in uiuo, second half of gestation ( [oPL] of maternal, fetal blood) Short-term treatment of fetus in uiuo, second half of gestation ( [oPL] of fetal blood) Long-term treatment of mother in uiuo, week 8 -+ term ( [cPL] of maternal blood) Short-term incubation of explants from mid and late pregnancy ([bPL] of medium) Short-term incubation of cells from midpregnancy ([mPL-I11 of medium) Short-term incubation of cells from midpregnancy ([mPL-11] of medium) Short- and long-term incubation of cells from midpregnancy ([mPL-111 of medium)

No effectlmarginal J

T

Martal and Lacroix (1978); Schams er al. (1984); Lowe al. (1979)

et

Martal and Lacroix (1978)

No effect

Taylor cr al. (1983a)

No effect

Taylor et al. (1983a)

No effect

Gluckman er al. (1980)

No effect

Forsyth

el

al. (1985)

.1

Forsyth and Hayden (1980)

T

G. Thordarson and F. Talamantes (unpublished)

t

G. Thordarson and F. Talamantes (unpublished)

No effect

G. Thordarson and F. Talamantes (unpublished)

Abbreviations: TRH, thyrotropin-releasing hormone; GnRH. gonadotropin-releasing hormone; GH-RH, growth hormone-releasing hormone. The terms “short-term” and “long-term” designate treatments that lasted less than 24 hours and more than 24 hours, respectively.

30

LINDA OGREN AND FRANK TALAMANTES

Arginine has also been reported to stimulate the secretion of bPL in vitro (Forsyth and Hayden, 1980).These data probably have little physiological significance because very high concentrations of arginine were necessary to stimulate a PL response, but they are of interest because arginine also stimulates the secretion of GH, illustrating that PLs and GHs in the human and in ruminants are responsive to some of the same factors. 9. Serum Factors Human serum was recently reported to contain one or more substances that stimulate the secretion of hPL in vitro (Barrett et al., 1986). The substance is present in the sera of men and nonpregnant women and its concentration increases during pregnancy. Partial characterization of the substance indicated that it is a protein having an apparent molecular weight of 3 1,000. The substance appears to be a specific secretagogue of hPL since it did not stimulate the secretion of several pituitary hormones, decidual PRL, or other placental proteins. 10. High-Density Lipoproteins (HDL)

HDL were recently reported to stimulate the release of hPL from placental explants in vitro (Handwerger et al., 1987). The PL-stimulating activity of HDL was caused by the apolipoproteins AI, AH, and CI, and was specific for the release of hPL. The extent to which HDL regulate hPL secretion during pregnancy remains to be determined. 11. Second Messengers Second messenger systems that regulate PL secretion have been studied most extensively in the human. The secretion of hPL is stimulated by arachidonic acid (Handwerger et al., 1981c; Zeitler et al., 1983, 1986; Zeitler and Handwerger, 1985) and diacylglycerols (Harman et al., 1986a). The stimulation by diacylglycerols is accompanied by an increase in protein kinase C activity (Harman et al., 1986a) and the stimulation by arachidonic acid is accompanied by increases in calcium mobilization (Zeitler et al., 1986) and phosphoinositide hydrolysis to yield diacylglycerols and inositol phosphates (Zeitler and Handwerger, 1985). The latter reaction probably results from activation of phospholipase C by arachidonic acid (Zeitler and Handwerger, 1985). Together these data suggest that hPL secretion is regulated by phospholipase C-stimulated phosphoinositide hydrolysis. Arachidonic acid and activation of phospholipase C also appear to play a role in regulating the secretion of oPL (Huyler et al., 1985; Rice and Thorburn, 1986b), but the details of the mechanism have not been worked out. The role of CAMP in regulating PL release has been examined in the

PROLACTINS OF PREGNANCY

31

human placenta and remains unresolved. The majority of studies, utilizing designs in which effects of cAMP were assessed after exposure periods of several hours, reported no effect of CAMP on hPL release (Handwerger et af., 1973; Belleville et af., 1974; Zeitler et al., 1983; Winikoff and Braunstein, 1985). Exceptions include a report by Hochberg et af. (1987), who noted inhibition of hPL secretion by cells from term placenta after treatment with dibutyryl CAMP, and another by Welsh (1979), who noted stimulation of hPL release after perfusion of placentas with dibutyryl cAMP in some but not all experiments. Recently Harman et af. (1987) reported that treatment of term placental cells with drugs that increase intracellular cAMP levels stimulated the release of hPL for a short period of time, after which the rate of hPL release declined. The latter observation may explain the failure of some previous investigators, using longer term incubations, to demonstrate stimulation of hPL release by CAMP.

E.

BIOLOGICAL

ACTIVITIES

Because many of the PLs are structurally very similar to PRL and GH, most of the work carried out on the biological activities of the PLs has examined the PRL- and GH-like effects of these hormones in various tissues. The most widely studied activities of the PLs include the regulation of mammary gland secretory differentiation and ovarian steroidogenesis, the stimulation of growth, and the regulation of intermediary metabolism. Unfortunately, much of the work examining the biological activities of the PLs has been carried out in heterologous rather than homologous systems, where a heterologous system is one in which the effects of a PL from one species are studied in tissue from another. This has come about for two reasons. In the case of hPL, experimentation in humans has obviously been limited by ethical considerations, even though the hormone itself can be obtained readily. In the case of PLs from other species, the availability of adequate amounts of highly pure hormone has been the limiting factor. Interpreting data collected in a heterologous system is difficult because PLs may show activities in tissues from other species that they do not show in tissues from their own species. This results because of the structural relationships that exist between PLs, PRLs, and GHs from different species. Thus, a heterologous PL may bind to receptors that normally bind the animal’s own PRL or GH; the homologous PL may not contain a similar binding region and consequently will not mimic the activity of the heterologous PL. The converse of this argument is also true, and the absence of biological activity of a heterologous PL in a given tissue should not be taken as proof against a

32

LINDA OGREN AND FRANK TALAMANTES

role for the homologous PL in the same tissue. In this context it is important to note that calling a hormone a “placental lactogen” does not mean that it is known to stimulate lactogenesis in the mammary gland of the homologous species, nor does it imply that regulation of mammary gland function is the primary function of the hormone in all species. Most of the PLs were originally designated as such because they mimicked actions of PRLs in heterologous bioassays and radioreceptor assays. As discussed in Section II,A, PLs from some species share more amino acid sequence homology with the PRL than the GH from the same species, whereas the converse is true for other PLs. A similar relationship appears to exist for the biological activities of the hormones, in that PLs from some species primarily regulate functions that are influenced by GH and other PLs primarily affect processes regulated by PRL, although it should be noted that the amount of amino acid sequence homology between a PL and the PRL and GH from the same species does not always predict whether the PL is functionally more PRL- or GH-like (see Section 11,A). The fact that some PLs are functionally more GH- than PRL-like and others are more PRL- than GH-like indicates that even when biological activity experiments have been carried out in the homologous species, extrapolating findings from one species to the next is difficult, especially when the species are not closely related. In the following discussion of the biological activities of the PLs, data from homologous systems are emphasized. Some information obtained from heteroiogous systems is also included because in many cases only a few studies have been carried out in the homologous system. 1. Regulation of Mammary Gland Secretory Differentiation

Secretory differentiation of the mammary gland during pregnancy encompasses several processes, including ductal and lobuloalveolar growth and acquisition of the ability to synthesize specific milk constituents. Numerous endpoints have been used to assess the extent of these processes. For example, mammary gland growth has been estimated by changes in the DNA content and the DNA polymerase a activity of the gland and by uptake of radiolabeled thymidine. The RNA content, RNA-to-DNA ratio, and the rate of RNA synthesis have been used to assess changes in total mammary gland protein synthesis. The appearance of secretion in lobuloalveolar structures and the concentrations of casein and a-lactalbumin have been used as indices of the ability of the gland to secrete specific milk constituents (reviewed by Thordarson and Talamantes, 1987). As summarized in Table VI, hPL, mPL-I, mPL-11, and oPL have all been shown, in the homologous species, to stimulate at least one of the

33

PROLACTINS OF PREGNANCY

EFFECTSOF PLs PL hPL

mPL-I mPL-I1

OPL bPL

ON THE

TABLE VI MAMMARYGLAND:STUDIES CARRIED OUT I N THE HOMOLOGOUS SPECIES

Result Stimulated DNA synthesis in benign breast tumors maintained in athymic mice and in vitro Stimulated growth of ductal epithelium in vitro; did NOT maintain already established secretion in vitro Stimulated synthesis of a-lactalbumin in vitro Stimulated synthesis of a-lactalbumin and secretion, judged by histological criteria, in vitro Stimulated synthesis of a-lactalbumin in v i m Did NOT stimulate synthesis of casein, a-lactalbumin, or lipid in vitro

References Welsch and McManus (1977); McManus et al. (1978)

Prop (1968)

Colosi

el ul.

(1987a)

Colosi et a / . (1982); Thordarson et a / . (1986)

I. Forsyth (personal communication) Byatt and Bremel (1986)

endpoints associated with secretory differentiation of the mammary gland. So far, the only effects of the mouse and ovine PLs on the mammary gland that have been examined are those dealing with their ability to stimulate the secretion of specific milk constituents; effects on the growth of lobuloalveolar structures have not been assessed. The effects of hPL on the human mammary gland have been examined with respect to both cell proliferation and milk secretion. These data suggest a role for hPL in stimulating mammary epithelial cell proliferation during pregnancy and less involvement of the hormone in regulating mammary gland secretory activity, although more investigation is required before a role for hPL in regulating the secretion of specific milk constituents can be ruled out. In contrast to the PLs listed in Table VI that have been shown to regulate mammary gland function in the homologous species, bPL has recently been reported to have no effect on the secretion of several specific milk constituents by the bovine mammary gland; effects on lobuloalveolar growth were not assessed. Since bPL has been shown to stimulate secretion of specific milk constituents in mammary tissue from other species (Buttle and Forsyth, 1976; Byatt and Bremel, 1986) and to be active in heterologous mammary gland radioreceptor assays for

34

LINDA OGREN AND FRANK TALAMANTES

PRL-like activity (Murthy et al., 1982; Eakle et al., 1982; Arima and Bremel, 1983), these data in the homologous species, if confirmed, lend support to the argument that conclusions obtained in heterologous systems about the biological activities of PLs may not in fact be valid in the homologous system. Effects of pure preparations of other PLs on secretory differentiation of mammary tissue have not been studied in homologous species. There is evidence that placentas of the rat, goat, and rhesus monkey produce substances that are lactogenic in the homologous species (reviewed by Thordarson and Talamantes, 1987), and haPL-I1 (Southard et al., 1986) and rPL-I and -11 have been reported to show activity in heterologous mammary gland radioreceptor and bioassays for PRL-like activity (Kelly et al., 1975; Robertson and Friesen, 1975, 1981; Glaser et al., 1984).

2 . Regulation of Maternal Intermediary Metabolism A number of changes in maternal intermediary metabolism occur during human pregnancy including (1) an increase in the insulin response to a glucose load, (2) resistance to the effects of insulin in some tissues, (3) a decrease in glucose tolerance, and (4)an increase in lipid mobilization (Kalkhoff et al., 1978; Freinkel, 1985). The net effect of these changes is sparing of maternal glucose for utilization by thee fetus. The involvement of hPL in bringing about these adaptations was suggested shortly after the hormone was purified (Grumbach et al., 1968), and since that time a large number of studies, in both homologous and heterologous systems, have been carried out to examine effects of hPL on each of these endpoints. Studies carried out in humans on effects of hPL on insulin secretion under basal conditions have generally reported no effect of the hormone on fasting insulin levels in normal men and nonpregnant women (Beck and Daughaday, 1967;Josimovich and Mintz, 1968; Kalkhoff et al., 1969). In hypopituitary dwarfs, hPL was reported to increase the fasting insulin concentration of the blood (Grumbach et al., 1968). The apparent discrepancy between these data may be due partly to the difference in the serum GH concentration between the subjects. In heterologous systems, hPL treatment did not affect the basal insulin concentration of the blood of rhesus monkeys (Beck, 1970) or dogs (Burt et al., 1966), but it did stimulate insulin secretion in vitro by pancreatic tissue from the mouse and rat (Laube et al., 1972; Nielsen, 1982; Nielsen et al., 1986). It is not known whether the response of the mouse and rat pancrease to hPL reflects species differences or the fact that the experiments were carried out in vitro, where actions of hPL on other aspects of intermediary metabolism do not come into play. Effects of hPL treatment on the insulin response to a glucose load in

PROLACTINS OF PREGNANCY

35

humans have been inconsistent, with some investigators reporting an increase in the insulin response to glucose after hPL administration (Beck and Daughaday, 1967; Grumbach et al., 1968; Samaan et al., 1968) and others reporting no effect on the insulin response (Josimovich and Mintz, 1968; Kalkhoff et al., 1969). The differences between these data have not been explained. In heterologous systems, hPL administration did not affect the insulin response to glucose in rhesus monkeys (Beck, 1970) but it enhanced the insulin response to glucose in hypophysectomized rats (Martin and Friesen, 1969; Malaisse et al., 1969; Bennett et al., 1976). Enhancement of insulin resistance after hPL treatment has been reported in heterologous systems (rhesus monkey: Beck, 1970; rat: Lostroh, 1974). In these studies an increase in insulin resistance was defined as the occurrence of a smaller-than-expected decline in blood glucose concentration in response to insulin administration, but the sensitivity of individual tissues to insulin was not examined. Other investigators have reported that the sensitivity of the rat liver to insulin decreased after hPL treatment (Sladek, 1975), whereas that of rat adipose tissue increased (Leake and Burt, 1969). Studies examining effects of hPL on glucose tolerance in humans have produced conflicting data. A reduction in glucose tolerance was defined as an abnormal elevation in the glucose concentration of the blood following administration of a glucose load. In some cases hPL treatment reduced carbohydrate tolerance (Beck and Daughaday, 1967; Grumbach et al., 1968), whereas in others, the hormone was without effect ( Josimovich and Mintz, 1968; Samaan et al., 1968; Kalkhoff et al., 1969). The differences between these data have not been resolved. A large number of studies have been carried out on the effects of hPL on lipid metabolism. Human PL has generally been reported to stimulate lipolysis in human adipose tissue when administered either in vivo (Grumbach et al., 1966; Berle, 1973) or in vitro (Williams and Coltart, 1978) and to increase the sensitivity of human adipose tissue to other lipolytic stimuli such as theophylline (Williams and Coltart, 1978), although no effect of the hormone on basal lipolysis has been noted (Beck and Daughaday, 1967; Berle et al., 1974; Recio et al., 1979). In addition to its effects on iipoiysis, hPL has been reported to stimulate other aspects of adipocyte metabolism in humans including glucose uptake and oxidation and glycogen synthesis; these actions of hPL mimic those of insulin (Recio et al., 1979). The fact that hPL appears to stimulate basal and theophylline-stimulated lipolysis as well as glucose uptake and utilization suggests that the overall effect of hPL on human adipose tissue metabolism is to increase the basal activity of the tissue. In periods of fasting (for example, between meals) the increase in basal and hormone-stimulated

36

LINDA OGREN AND FRANK TALAMANTES

lipolysis provides fatty acids that can be used as an energy source by the mother, sparing glucose for utilization by the fetus. In the fed state, the increase in glucose uptake and utilization by adipose tissue ensures the availability of triglycerides as an energy source in subsequent periods of fasting. In many heterologous systems, including the rat, rabbit, dog, and Cercopithecus monkey, the effects of hPL on adipose tissue metabolism have been similar to those observed in human tissue (Friesen, 1965a; Burt et al., 1966; Riggi et al., 1966; Turtle and Kipnis, 1967; Genazzani et al., 1969, 1970; Felber et al., 1972; Sirek et al., 1972; Fredholm and Person, 1973; Strange and Swyer, 1974; Recio et al., 1979). In the mouse, hPL did not stimulate lipolysis in tissue from pregnant animals (Fielder and Talamantes, 1987). Although many of the data cited above are consistent with a role for hPL in regulating some of the metabolic adaptations that occur during the second half of pregnancy in humans, questions still remain about the physiological significance of many of these observations. In many cases, and particularly when considering the .effects of hPL on lipolysis, the concentrations of hormone that have been required to elicit a response have been orders of magnitude higher than those present in the blood of pregnant women. The reason for this is not known, but there are !everal possible explanations. One is that the activity of hPL on these endpoints is truly marginal, that any activity of the hormone in these systems merely reflects the fact that it is a low potency “analog” of hGH. An alternative explanation is that many effects of hPL on maternal intermediary metabolism have not been examined under experimental conditions that mimic the hormonal milieu of pregnancy, and consequently, the possibility remains that much lower doses of hPL might be active when the system has been appropriately “primed” by other hormones. Similarly, since the metabolic adaptations of pregnancy occur over a long period of time, it is possible that providing long-term exposure to the hormone would be a more appropriate model in which to study biological activity. As discussed in Section II,D,4, short-term changes in the glucose and fatty acid concentrations of the maternal blood have not affected the maternal serum hPL concentration in a consistent manner. These observations argue against a role for hPL in regulating glucose and fatty acid concentrations of the maternal blood over the short term-during the period between meals, for example. It is more likely that hPL acts over the long term, in conjunction with other hormones, to alter the set point and response time of the system to render it more sensitive to hormones that respond to short-term changes in nutrient availability. Effects of oPL and mPL-I1 on maternal intermediary metabolism have

PROLACTINS OF PREGNANCY

37

also been reported. The effects of treatment of pregnant and nonpregnant sheep with partially pure preparations of oPL have been examined in two studies. In one, oPL administration was followed by decreases in the circulating concentrations of free fatty acids, glucose, and amino nitrogen of fasted ewes (Handwerger et al., 1976), whereas oPL increased the circulating concentrations of free fatty acids, glucose, and urea and fatty acid turnover in fed ewes in the other (Thordarson et al., 1987). Short-term treatment of ewes with antiserum to oPL did not affect circulating concentrations of free fatty acids, lactate, or hydroxybutyrate or alter glucose utilization by fed, pregnant ewes (Waters et al., 1985). The latter data suggest that oPL is not an important regulator of maternal intermediary metabolism over the short term in ewes. Long-term effects of the hormone warrant additional investigation. In the mouse, mPL-I1 did not stimulate lipolysis in vitro in adipose tissue from pregnant mice during the second half of pregnancy (Fielder and Talamantes, 1987).

3. Regulation of Fetal Growth and Metabolism Evidence from studies carried out in the homologous species suggests that hPL and oPL are involved in regulating the metabolism and growth of the fetus. Human PL has been reported to stimulate amino acid uptake, incorporation of ['Hlthymidine, and IGF-I production by myoblasts and fibroblasts of human fetuses from the first half of gestation (Hill et al., 1985, 1986). In human fetal hepatocytes, the hormone stimulated IGF-I production and the incorporation of ['Hlthymidine (Strain et al., 1987). The effect of hPL on thymidine incorporation was mediated at least partly by IGF-I, but the stimulation of amino acid uptake was thought to occur independently of hPL's stimulation of IGF-I secretion (Hill et al., 1986; Strain et al., 1987). In the fetal sheep, oPL has been reported to stimulate glycogen synthesis by the liver (Freemark and Handwerger, 1986). In both species, the concentrations of hPL and oPL that were active were in the physiological range. The homologous GHs had little or no activity in ovine hepatocytes and human myoblasts and fibroblasts (Hill et al., 1985; Freemark and Handwerger, 1986), although GHs regulate these functions in postnatal animals. These data suggest that hPL and oPL play roles in regulating fetal metabolism and growth that are filled by GH after birth. It is interesting to note that hGH was as effective as hPL in human fetal hepatocytes in vitro (Strain et al., 1987), suggesting that both hormones regulate fetal hepatocyte function in utero. A specific receptor for oPL has been identified in the fetal ovine liver. Although this receptor also binds oGH and oPRL, its affinity for these hormones is much lower than its affinity for oPL (Freemark and Hand-

38

LINDA OGREN AND FRANK TALAMANTES

werger, 1986; Freemark et al., 1986, 1987). The fetal ovine liver does not appear to contain a high-affinity GH receptor (Freemark et al., 1986), which accounts for the failure of oGH to stimulate glycogen synthesis in this tissue. The demonstration of a receptor specific for oPL in the fetal ovine liver is significant in that it discounts the widely held assumption that PLs act exclusively by binding to receptors for either PRL or GH. The presence of binding sites for hPL in human fetal liver and skeletal muscle has been described in a preliminary report (Hill et al., 1987). In addition to the studies just discussed, a number of reports have examined effects of oPL and hPL on tissues of the fetal rat. In this system, oPL has been shown to inhibit glucagon-induced glycogenolysis in the liver (Freemark and Handwerger, 1985)and to stimulate amino acid uptake by skeletal muscle (Freemark and Handwerger, 1983), ornithine decarboxylase activity (Hurley et al., 1980) and glycogen synthesis (Freemark and Handwerger, 1984a,b) in the liver, and the production of IGF-I by fibroblasts (Adams et al., 1983). In contrast, hPL did not affect glycogen synthesis or amino acid uptake in the same experiments (Freemark and Handwerger, 1983, 1984a) nor did it affect the production of IGF-I and -11 by fetal rat hepatocytes (Richman et al., 1985). The suitability of the fetal rat model for studying effects of heterologous PLs on fetal tissues is difficult to evaluate. On one hand, the effects of oPL on hepatic glycogen synthesis in the model were similar to those of oPL in tissue from the homologous species. On the other hand, data on the effects of hPL in the model could be used as evidence against a role for the hormone in regulating fetal growth and metabolism, although studies in homologous systems clearly suggest the hormone affects growthassociated endpoints, albeit at an earlier stage of gestation. The situation is further complicated by observations that oPL was effective in stimulating amino acid transport, ornithine decarboxylase activity, and glycogen synthesis in fetal rat tissues when rat GH and rat PRL were not, or were effective only at much higher concentrations (Hurley et al., 1980; Freemark and Handwerger, 1983, 1984a). In these instances, did oPL mimic the action of an rPL? Placental lactogens have also been identified in the blood of the fetal mouse, rhesus monkey, and bovine (Section II,C,2), but no information is available about the roles of these hormones in this compartment. In the mouse, mPL-I1 is present in the fetal circulation by day 14 of gestation, and until day 16, it is the only PRL-like hormone that has been detected in this compartment (D. Bravo and F. Talamantes, unpublished observations), which suggests that prior to day 16, mP-I1 may affect functions in the fetus that are regulated by PRL and GH after birth. Binding sites for mPL-I1 have been identified in the fetal mouse liver on day 17 of

PROLACTINS OF PREGNANCY

39

gestation. Preliminary data indicate that these sites also bind mouse PRL (mPRL) but not mouse GH (Harigaya et al., 1988). Since the relative affinity of these binding sites for mPL-I1 and mPRL has not yet been determined, it is not known whether these sites represent a specific PL-I1 receptor, as found in the fetal ovine liver (Freemark et al., 1986), or whether mPL-I1 acts via a receptor that also binds mPRL with high affinity. In addition to whatever direct role PLs play in regulating fetal growth and metabolism, the hormones also regulate fetal growth indirectly to the extent that they bring about adaptations in the metabolism of the mother that ensure the availability of an adequate supply of nutrients for the fetus (see Section II,E,2). 4. Regulation of Steroidogenesis The role of PLs in regulating steroidogenesis has been examined in the mouse, rat, human, and sheep. These investigations have focused on the effects of PLs on the production of progesterone, which is required for the maintenance of pregnancy. It is well established in both mice and rats that substances secreted by the conceptus are important in regulating progesterone production by the ovaries during the second half of pregnancy (Ray et al., 1955; Matthies, 1967; Cohen and Gala, 1969; Linkie and Niswender, 1973; McCormack, 1974; Critser et al., 1980, 1982). These substances have not been identified, but it has been suggested that the PLs may be responsible for at least some of this luteotropic activity (Gibori et al., 1977; Ochiai and Rothchild, 1981, Tabarelli et al., 1982; Glaser et al., 1985). So far, the evidence supporting this suggestion is circumstantial. In both species, the appearance of luteotropic bioactivity in the serum and placenta coincides with the appearance of PL-I in the blood (mouse: Critser et al., 1980, 1982; Colosi et al., 1986; rat: Ray et al., 1955; Matthies, 1967; Linkie and Niswender, 1973; Robertson and Friesen, 1981). In the rat, preliminary biochemical characterization of the luteotropic activity of placental extracts from midpregnant animals indicated that its molecular weight was similar to that of rPL-I (Linkie and Niswender, 1973; Robertson et al., 1982). In addition, the luteotropic substances in placental extracts and serum of midpregnant rats have been shown to play a PRL-like role in the ovary (Gibori and Richards, 1978; Gibori et al., 1979; Glaser et al., 1984), and PL-I and PL-I1 from both rats and mice have been reported to bind to PRL receptors of various tissues of the homologous species (Glaser et al., 1984; Colosi et al., 1987a). Effects of pure preparations of PL-I and PL-I1 from the mouse and rat on steroidogenesis have not been examined. A partially pure preparation of mPL-I that did not contain

40

LINDA OGREN AND FRANK TALAMANTES

mPL-I1 was not active in a bioassay for LH-like activity (Colosi et al., 1987a). In the human and sheep the evidence suggests that PLs do not influence progesterone production by the ovary. For example, treatment of women with hPL did not prolong the luteal phase of the menstrual cycle (Stock et al., 1971) and receptors for hPL could not be detected in luteal membranes obtained at parturition (Goldsmith et al., 1978), which argues against a luteotropic role for hPL during both early and late pregnancy, respectively. In the sheep, reducing the oPL concentration of the maternal blood with antiserum did not affect the maternal plasma progesterone concentration (Waters et al., 1985),and short-term administration of oPL in vivo did not affect the basal secretion of progesterone by the ovaries or inhibit normal or prostaglandin-induced luteol ysis in cycling animals (Marta1et al., 1979; Schramm et al., 1984). Ovine PL also had no effect on progesterone production by the sheep ovary in uitro (Rodgers et al., 1983). It is important to note that effects of long-term oPL treatment on ovarian steroidogenesis in sheep have not been examined, and until they are, a luteotropic role for the hormone cannot be ruled out. In this context it is interesting to note that binding of oPL to oGH receptors in the sheep ovary has been reported (Chan et al., 1978b); the role of oGH in the ovary is not understood. Ovine PL did not bind to oPRL receptors in the sheep ovary (Chan et al., 1978b), which suggests that the hormone probably does not play a PRL-like role in this gland. In contrast to the marked absence of luteotropic effects of hPL and oPL in the homologous species, both hormones show luteotropic activity in nonpregnant rodents (hPL: Josimovich et al., 1963; Florini et al., 1966; Crisp, 1977; Talamantes et al., 1977; oPL: Chan et al., 1980; De la Llosa-Hermier et al., 1983). This activity is probably due to structural similarity between the rodent PRLs and human and ovine PLs, and again illustrates the need to evaluate biological activities of PLs in the homologous species. In both the human and sheep, the placenta is the major source of circulating progesterone by the second half of pregnancy (Heap and Flint, 1984). It would be interesting to examine the effects of hPL and oPL on steroidogenesis by this tissue.

5 . Placental Lactogen Receptors Until the recent discovery of specific receptors for oPL that have low affinity for oGH and oPRL in the fetal ovine liver (Freemark and Handwerger, 1986; Freemark et al., 1986, 1987), it was generally assumed that PLs act exclusively by binding to receptors for GH and/or PRL. In this scheme, the relative affinity of a PL for PRL versus GH receptors determines whether the PL has PRL- or GH-like functions in a tissue, and

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the importance of a PL to the regulation of a process is proportional to its affinity for the GH or PRL receptor and its concentration. At the present time it is not known whether the existence of specific PL receptors is associated with post-receptor-binding events that are not shared by GH and PRL, or whether the activities mediated by specific PL receptors are essentially the same as those mediated by GH or PRL receptors. In either case, the existence of specific PL receptors provides an additional mechanism by which tissue responsiveness to hormones from the GHPRL-PL family can be regulated. 6 . Relevance of the PLs

Recent reports describing the occurrence of normal pregnancies in women having low to undetectable concentrations of hPL in the blood and placenta due to the absence of the genes for hPL (see Section 11,D,3), have raised questions about the physiological importance of this hormone (Nielsen et al., 1979; Wurzel et al., 1982; Sideri et al., 1983; Parks et al., 1985). Although these data clearly indicate that in healthy women, hPL itself is not required for the maintenance of pregnancy, there is some evidence that the hGH gene, a variant hPL gene (HCS-L),or a hybrid of the two was expressed in the placentas of these individuals (Frankenne et al., 1986), which suggests that these PL-like molecules could have performed functions carried out by hPL in normal women. Although circulating concentrations of hGH and hPRL were normal in women lacking hPL, it is possible that the sensitivity of target tissues to these hormones was enhanced, also compensating for the absence of hPL. Until more information is obtained about endocrine parameters in PL-deficient women, and about the response of these individuals to stresses such as starvation, it is difficult to determine whether or not there is an absolute requirement for a PL-like hormone in human pregnancy.

111. Decidual Prolactin

A. IDENTIFICATION A N D BIOCHEMICAL CHARACTERIZATION In the early 1970s several laboratories reported the presence of substances in amniotic fluid from the human and rhesus monkey that had PRL-like activity in bioassays and RIAs (Friesen et al., 1972; Tyson et al., 1972; Parke, 1973; Josimovich et al., 1974). Preliminary biochemical characterization of this activity in human amniotic fluid indicated that its molecular size, estimated by polyacrylamide gel electrophoresis, and its net charge and isoelectric point were very similar to those of a human pituitary PRL standard (Ben-David et al., 1973). The material

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was subsequently purified by several laboratories (Ben-David and Chrambach, 1974; Hwang et al., 1974; Rathnam et al., 1977). The purified material was indistinguishable immunologically from a human pituitary PRL standard (Hwang et al., 1974; Rathnam et al., 1977), and the sequence of its 25 N-terminal amino acid residues was identical to that of human pituitary PRL (Hwang et af.,1974), which suggested that the PRL in the amniotic fluid of humans was very similar, if not identical, to pituitary PRL. The discovery of PRL in the amniotic fluid prompted investigation into the source of the hormone. Although several early studies suggested that the PRL was from the fetal (Fang and Kim, 1975) or maternal pituitary (Josimovich et al., 1974; Schenker et al., 1975), later work demonstrated that it is synthesized by the decidua (Section III,E,l), and the hormone was consequently designated decidual PRL. The amino acid sequence of decidual PRL was obtained from sequence analysis of cDNAs prepared from human decidual poly(A)-mRNA (Takahashi et al., 1984). These data demonstrated that the amino acid sequence of decidual PRL is identical to that of human pituitary PRL. Recent studies have also reported the presence of a glycosylated form of PRL in the decidua and amniotic fluid (Heffner et al.. 1986; Lee and Markoff, 1986). PRL from the amniotic fluid of rhesus monkeys has not been purified. By analogy to human decidual PRL, it is assumed to be identical to rhesus pituitary PRL.

B. SECRETION 1. Site of Production Immunohistochemical methods have been used to demonstrate that decidual PRL is present predominantly in decidualized stromal cells of the endometrium in humans (Frame et al., 1979a; Markoff et al., 1983a; Braverman et al., 1984; Kaurna and Shapiro, 1986; McRae et al., 1986). When considered in conjunction with data demonstrating the presence of mRNA for PRL in the decidua (Clements et al., 1983; Takahashi et al., 1984; Suganuma et al., 1986), these data suggest that PRL is synthesized by these cells. Localization of decidual PRL in the rhesus monkey has not been reported. 2 . Mechanism of Secretion Decidual PRL does not appear to be packaged into classical secretion granules (Handwerger et al., 1984b; Handwerger and Capel, 1985), and

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there is evidence suggesting that very little of the hormone is stored in the cells after it is synthesized (Markoff et al., 1983a; Handwerger et al., 1981d, 1984a; Maslar and Ansbacher, 1986). The process by which PRL is released from decidual cells requires the presence of calcium (Markoff et al., 1982; Richards et al., 1982), but unlike the release of pituitary PRL, which is stimulated by higher than normal calcium concentrations, the release of decidual PRL is not affected by raising the calcium concentration above normal (Markoff et al., 1982). Studies examining the transfer of PRL from the maternal to the fetal surface of the amnion-chorion-decidua have demonstrated the choriodecidual contact is required for this process (McCoshen et al., 1982a; McCoshen and Barc, 1985). No transfer of radiolabeled PRL occurred when the hormone was applied to either the maternal or fetal side of isolated amniochorion (Schenker et al., 1979). Interestingly, the PRL that is transferred from the decidua to the fetal side of the amniochorion must originate within the decidual cells that adhere to the amniochorion; transfer of radiolabeled PRL from the maternal to the fetal side did not occur if the hormone was merely incubated with amniochorion with adherent decidua (Riddick and Maslar, 1981). C. GESTATIONAL PROFILES AND METABOLISM

In the human, PRL can be detected for the first time in decidualized stromal cells of the endometrium on about days 23-25 of an ideal menstrual cycle whether or not conception has occurred (Maslar and Riddick, 1979; Kauma and Shapiro, 1986). The PRL concentration of the amniotic fluid remains low ( 4 0ng/ml) until about week 14 of pregnancy and then increases rapidly to very high values by about week 20 (-4000 ng/ml). After about week 28, the PRL concentration of the amniotic fluid decreases until about week 34 and then remains relatively constant until parturition (-500 ng/ml) (Kletzky et al., 1985). The gestational profile of PRL concentrations in the amniotic fluid of the rhesus monkey differs from that of humans. In the rhesus, the PRL concentration of the amniotic fluid does not decline markedly during the last trimester of pregnancy. Maximal concentrations are similar in the two species (Friesen et al., 1972). The half-life of PRL in the amniotic fluid of humans is 4.2 hours (Tyson, 1982). Significant amounts of decidual PRL do not appear to be secreted into the maternal blood since serum PRL concentrations are very low in hypophysectomized women (Riddick et al., 1979) and rhesus monkeys (Walsh et al., 1977b) and in women treated with inhibitors of pituitary

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PRL secretion (Bigazzi et al., 1979a; Lehtovirta and Ranta, 1981; Miyakawa et al., 1982). There is no transfer of PRL from the amniotic fluid to the maternal circulation (Josimovich et al., 1974). D. REGULATION OF SECRETION

1. The Number of PRL-Producing Cells During the menstrual cycle in humans, the appearance of PRL in the endometrium coincides with the appearance of decidualized stromal cells, and the ability of endometrium from nonpregnant women to secrete PRL in vitro is correlated with the extent of decidualization (Maslar and Riddick, 1979; Maslar et al., 1980; Daly et al., 1981; Ying et al., 1985). These observations suggest that a major determinant of the rate of decidual PRL production during the luteal phase of the menstrual cycle is the number of stromal cells that have undergone decidualization. After implantation the ability of the human decidua to secrete PRL in vitro increases significantly (Maslar et al., 1980), and during the last trimester of gestation, it declines with a pattern similar to the gestational profile of the PRL concentration of the amniotic fluid (Rosenberg et al., 1980). Whether these changes in the PRL secretory ability of the decidua are due to changes in the number of PRL-producing cells or to changes in the rate of PRL synthesis by individual decidual cells is not known.

2. Progesterone and Estrogens Progesterone has been reported to induce both decidualization and PRL secretion in endometrium from nonpregnant women in vitro (Daly et al., 1983a; Maslar and Ansbacher, 1986; Maslar et al., 1986). In this system estradiol antagonized the effect of progesterone on PRL secretion (Daly et al., 1983a). Although these data suggest that both steroids are involved in regulating decidual PRL production during the luteal phase of the menstrual cycle, it is not clear whether the stimulation of PRL secretion by progesterone is direct or whether it is secondary to the induction of decidualization. Effects of progesterone and estrogens on decidual PRL secretion have also been examined in tissue from late gestation, but the findings have been contradictory. Several investigators have reported no effect of short- or long-term treatment of decidual cell and explant cultures with progesterone, estradiol, or estriol (Richards et al., 1982; Markoff et al., 1983a). Others noted that estradiol stimulated PRL secretion by decidual explants in long-term culture, while progesterone alone did not affect PRL production, although it antagonized the effect of estradiol

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(Rosenberg and Bhatnagar, 1984). Finally, effects of these steroids on decidual PRL production have been reported to depend on whether the tissue was obtained from labor versus nonlabor pregnancies (Daly et al., 1983b). In these studies, labor-exposed decidua did not respond to either progesterone or estradiol in uitro, whereas PRL secretion was stimulated by progesterone and estradiol in nonlabor tissue. 3. Osmolarity Effects of changes in the osmotic environment of the decidua on PRL secretion have been examined because of suggestions that a major function of the hormone is regulating the ionic composition and volume of the amniotic fluid (Section III,E,2). The data from these studies are somewhat contradictory. In one case (Markoff et al., 1982), PRL secretion by decidual explants from the end of gestation was not affected when the osmolality of the incubation medium was varied between 224 and 336 mOsm/kg. In other studies, increasing the extracellular osmolality from 252 to 457 mOsm/kg resulted in an increase in PRL secretion by term-decidual explants (Andersen et al., 1982, 1984). The differences between these data may be due to differences in the length of incubation between studies; tissues were incubated four times longer in the experiments that showed stimulation of PRL secretion in a hyperosmotic environment.

4. Local Factors Several laboratories have reported the production of substances by the feto-placental unit that regulate the production of decidual PRL. One of these factors is a placental product that stimulates both the synthesis and release of decidual PRL (Handwerger et al., 1983; Markoff et al., 1983a). It has not been purified but is known to be a heat-stable protein having a molecular weight of greater than 10,000. The other factors that have been reported are secreted by the decidua and inhibit decidual PRL secretion. Markoff et al. (1983b) described a heat-labile protein in the 38,000-45,000 molecular weight range that inhibited the release but not the synthesis of decidual PRL. Recently, Daly et al. (1986a,b) reported the secretion by decidua of a 10,000-35,000 molecular weight substance that inhibited decidual PRL synthesis. This substance was not characterized and the possibility that the inhibitory factor was PRL itself, thus demonstrating autofeedback, could not be eliminated.

5 . Serum Factors Stimulation of decidual PRL secretion in vitro by human and fetal calf sera has been described in several reports (Lucian0 et al., 1980; Krug et al., 1983). Partial characterization of the active substance in fetal calf

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serum indicated that it was heat-stable and had a molecular weight greater than 12,000 (Lucian0 et al., 1980). Since effects of serum on the synthesis and release of total decidual proteins were not evaluated in these studies, it is not known whether the serum factors were specific regulators of decidual PRL production or whether their effects on PRL production were secondary to improved tissue viability and enhanced total cellular protein synthesis. The absence of an effect of fetal calf serum on decidual PRL production has also been noted (Handwerger et al., 1983). 6. Dopamine and TRH Since decidual PRL is structurally identical to pituitary PRL, the first studies examining the regulation of decidual PRL secretion focused on effects of dopamine and TRH, which inhibit and stimulate pituitary PRL secretion, respectively. Neither dopamine (and its agonists) nor TRH affected decidual PRL secretion under numerous experimental conditions (Bigazzi et a f . , 1979a,b; Golander et al., 1979b; Ben-Jonathan and Munsick, 1980; Lehtovirta and Ranta, 1981; Liu et al., 1981; Miyakawa et a f . , 1982; Richards et a f . , 1982; Lee and Markoff, 1986). 7. Second Messengers The secretion of PRL by decidual cells and explants has been reported to be inhibited by CAMP (Handwerger et al., 1980), arachidonic acid (Handwerger et a f . ,1981d),and compounds that activate protein kinase C (Harman et al., 1986b). An integrated scheme for the actions of these compounds in regulating decidual PRL secretion has not been proposed, however.

E. BIOLOGICAL ACTIVITIES 1. Source of Amniotic Fluid PRL Several lines of evidence suggest that the decidua is the major source of the PRL in amniotic fluid. First, in humans the gestational profiles of PRL in the maternal and fetal blood and amniotic fluid differ markedly, and the PRL concentration of the amniotic fluid is not correlated with the PRL concentration of either the maternal or fetal blood (Tyson et al., 1972; Schenker et al., 1975; Biswas, 1976; Soria et al., 1977; Clements et al., 1977; Furuhashi et a f . , 1983; Freeman et al., 1984; Kletzky et al., 1985). These observations and the fact that the PRL concentration of the amniotic fluid is much higher than that of the maternal blood in the human (see, for example, Kletzky et al., 1985) argue against passage of PRL from the maternal circulation into amniotic fluid along a concentration gradi-

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ent. This has been confirmed in the rhesus monkey, where only a small amount of radiolabeled human PRL appeared in amniotic fluid after its injection into the maternal or fetal circulation (Friesen et al., 1972; Josimovich et al., 1974). Second, the PRL concentration of the amniotic fluid was normal in hypophysectomized women (Riddick, et al., 1979) and rhesus monkeys (Walsh et al., 1977b) and in women treated with drugs that inhibit PRL secretion by the pituitary (Bigazzi et al., 1979a; Lehtovirta and Ranta, 1981; Miyakawa et al., 1982). In the latter studies, the PRL concentration of both the maternal and fetal blood was very low, demonstrating that amniotic fluid PRL levels are maintained despite very low concentrations of pituitary PRL. Third, the PRL concentration of the amniotic fluid was very low in tuba1 pregnancies (Rosenberg, 1984), which suggests that close proximity of the decidua to the gestational sac is required for the appearance of PRL in the amniotic fluid. Fourth, the gestational profile of the PRL concentration of the decidua is very similar to the gestational profile of PRL in the amniotic fluid, and the capacity of the decidua to secrete PRL in vitro parallels the amniotic fluid PRL profile (Rosenberg et al., 1980; Fukumatsu et al., 1984; Lucian0 and Varner, 1984). Finally, the decidua secretes PRL in virro that is indistinguishable from pituitary and amniotic PRL fluid by its behavior in gel exclusion chromatography and polyacrylamide gel electrophoresis, whereas the placenta, amnion, and chorion do not secrete significant amounts of PRL under the same conditions (Riddick and Kusmik, 1977; Golander et al., 1978b, 1979a; Riddick et al., 1978; Healy et al., 1979; Frame et al., 1979b; Bigazzi et al., 1979b; Kubota et al., 1981; Liu et al., 1981; Krug el al., 1983). In addition, poly(A)-mRNA from decidua-chorion hybridizes with cDNA for human pituitary PRL, whereas poly(A)-mRNA from the amnion and trophoblast do not (Clements et al., 1983), which indicates that the PRL gene is expressed in the decidua. These data and the fact that newly synthesized PRL appeared on the fetal side of amnionchorion-decidua when radiolabeled amino acid was added to the decidual side in vitro (McCoshen and Barc, 1985) suggest that within the conceptus, the decidua is the tissue that secretes PRL.

2. Amniotic Fluid and Fetal Hydromineral Balance Of all the studies performed on possible functions of decidual PRL, its role in regulating the volume and osmolarity of the amniotic fluid has probably received the most attention. In the rhesus monkey, raising the concentration of PRL in the amniotic fluid by injecting ovine PRL was

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followed by a decrease in amniotic fluid volume that appeared to result primarily from the transfer of fluid to the maternal compartment (Josimovich et al., 1977a). The hormone also protected the fetus from large changes in extracellular fluid volume that otherwise followed changes in amniotic fluid osmolarity (Josimovich et al., 1977a). In the sheep, injection of ovine PRL into the amniotic fluid prevented the increase in the osmolarity of the amniotic fluid that followed infusion of hypertonic mannitol into the maternal circulation (Ross et al., 1983). Although the physiological relevance of the latter experiment is questionable since the PRL concentration of the amniotic fluid of sheep is extremely low (<2 ns/ml) (Ross et al., 1983), this study and that performed in rhesus monkeys nevertheless suggest that PRL in the amniotic fluid compartment may be involved in regulating amniotic fluid and fetal extracellular fluid volumes. The mechanisms by which this occurs are not well understood, however, due partly to limited understanding of the regulation of these processes in general. The total volume of the amniotic fluid is determined by the net exchange of fluid between the fetal, maternal, and amniotic fluid compartments (reviewed by Seeds, 1980). Of these possible sites of PRL action on amniotic fluid volume, only the effects of PRL on water movement between the amniotic fluid and maternal compartments have been considered in any detail. In experiments using isolated human amnion from late gestation, human and ovine PRL decreased the movement of water from the fetal to the maternal side of the membrane when applied to the fetal side (Leontic and Tyson, 1977a; Leontic et al., 1979). The effect was due to a decrease in the diffusion of water and was mediated by the epithelial layer of the amnion; the bulk flow of water in response to an osmotic gradient was not affected (Leontic et al., 1979; Raabe and McCoshen, 1986). The physiological importance of these observations is not clear, however, since the chorion, and not the amnion, has been reported to determine the permeability of the intact amniochorion to water (Lloyd et ul., 1969). When the effect of PRL on water movement was measured across membrane composed of amnion, chorion, and decidua, which is the membrane as it exists naturally, the hormone did not affect water movement when applied to the fetal (amniotic) side, but it reduced the permeability of the membrane to the transfer of water from the maternal to the fetal side when applied to the maternal (decidual) side (Tyson e? a / . , 1984), the net effect being an increase in the movement of water from the amniotic fluid to the maternal compartment. The relative importance of PRL’s regulation of the water permeability of the amniochorion to the determination of amniotic fluid volume is not known.

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Receptors for PRL have been reported in human chorion laeve (Herington et al., 1980; McWey et al., 1982), which suggests that the effects of PRL on the water permeability of the intact amniochorion may be mediated at least partly by the chorion. The uptake of radiolabeled human PRL by the light cells of the amnion has also been reported (McCoshen et al., 1982b), but despite numerous attempts, it has not been possible to demonstrate the presence of receptors for PRL in this tissue by biochemical methods (see, for example, McWey et al., 1982; Raabe and McCoshen, 1986). Effects of PRL on water and mineral transport by fetal tissues have been examined only briefly. Ovine PRL did not affect the permeability of fetal rhesus monkey skin to water (Leontic and Tyson, 1977b), but the presence of PRL receptors in the fetal rhesus monkey lung has been reported (Josimovich et al., 1977b). The latter observation is of interest because the fetal lung appears to be an important area of fluid exchange between the fetus and amniotic fluid (reviewed by Seeds, 1980). In the adult rat, ovine PRL has been reported to stimulate hydromineral transport across the small intestine (Mainoya, 1975), suggesting that PRL’s actions on the fetal gut should also be examined. 3 . Other Activities PRL has been reported to stimulate the activity of estrogen sulfatase in human decidual cells obtained prior to the initiation of labor (Braverman and Gurpide, 1986) and to reduce the production of prostaglandin E, by amnion-chorion-decidua during late gestation (Tyson et al., 1985). Both of these observations suggest that decidual PRL may be involved in regulating processes associated with the initiation of parturition. A role for amniotic fluid PRL in regulating surfactant production by the fetal lung has also received some attention, but the data from these studies have not been conclusive. Treatment of fetal rabbits with ovine PRL has been reported to stimulate pulmonary surfactant production (Hamosh and Hamosh, 1977), and a significant correlation has been reported between amniotic fluid PRL concentration and several parameters of fetal lung maturation in rhesus monkeys (Johnson et al., 1985). Although these observations are consistent with the involvement of amniotic fluid PRL in regulating fetal lung maturation, other data, showing no correlation between amniotic fluid PRL concentrations and fetal lung surfactant levels in human fetuses (for example, Hatjis et al., 1981), might argue against such a role for the hormone. Examination of this question is complicated by the fact that the fetal lung is also affected by PRL produced by the fetal pituitary, and consequently, the presence or absence of a correlation between the PRL concentration of the

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amniotic fluid and parameters of fetal lung maturation is difficult to interpret.

IV. Decidual Luteotropin The presence of substances in the rat decidua that possess PRL-like luteotropic activity was originally suggested by observations that ovarian progesterone secretion was maintained in pregnant or pseudopregnant animals when PRL secretion was suppressed if decidual tissue was present whereas progesterone secretion declined rapidly in PRL-inhibited animals if decidual tissue was absent (Gibori et al., 1974; Basuray and Gibori, 1980). The effects of decidual tissue on ovarian function were subsequently investigated in detail. These studies demonstrated that the active substance in the decidua are released into the systemic circulation (Castracane and Rothchild, 1976) and that they have numerous PRL-like functions in the ovary. These substances ( I ) act with intraluteal estradiol to regulate progesterone synthesis (Gibori et al., 1981), (2) maintain luteal cell estrogen (Basuray et al., 1983) and LH (Gibori et al., 1984, 1985) receptor concentrations, (3) maintain LH-responsive adenylyl cyclase activity (Gibori et al., 1984), (4) enhance human chorionic gonadotropin (hCG)-stimulated estradiol and testosterone secretion by the corpus luteum (Gibori et ul., 1985), ( 5 ) inhibit basal estradiol and testosterone secretion and inhibit hCG-stimulated estradiol secretion by ovarian follicles (Gibori et al., 1985). Recently, decidual extracts were reported to bind to PRL receptors in membrane fractions of the rat corpus luteum (Jayatilak et al., 1985) and rat decidua (Jayatilak and Gibori, 1986). The latter observation suggests that these substances may have autocrine or paracrine roles in addition to their activities in the ovary. The PRL-like activity of rat decidual tissue has been designated decidual luteotropin, but at the present time it is not clear whether all of the activities described above are due to a single substance or whether more than one molecule with PRL-like biological activity is present in the decidua. Fractionation of decidual extracts by gel exclusion chromatography showed a major peak of PRL-like activity, measured by the rat ovary radioreceptor assay, in the 20,000-25,000 molecular weight range, as well as several minor peaks of larger molecular weight (Jayatilak er al., 1985). The decidual luteotropin was heat- and trypsin-labile, indicating that it is a protein (Jayatilak et al., 1985), and it did not cross-react with antiserum to rat PRL, which suggests that the structure of decidual luteotropin differs from that of pituitary PRL (Gibori et al., 1974).

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The secretion of decidual luteotropin has been examined briefly using the rat ovary radioreceptor assay for PRL-like activity to estimate its concentration. The decidual luteotropin concentration of the decidua was reported to be low on day 6 of pseudopregnancy, to increase to maximal levels on day 9, and then to decline by day 14 (Jayatilak and Gibori, 1986). The gestational profile of decidual luteotropin in pregnant animals has not been reported. The secretion of decidual luteotropin was stimulated by progesterone in an in vivo model (Jayatilak et al., 1984). Synthesis of decidual luteotropin by decidual explants and cells in vitro has been reported (Herz et al., 1986).

V. Other PRL-like Molecules of the Placenta

The fetal component of the mouse, bovine, and rat placenta produces other molecules, in addition to PL-I and PL-11, that are structurally similar to PRL. Two of these molecules have been identified in the mouse. They are proliferin (Linzer et al., 1985) and proliferin-related protein (Linzer and Nathans, 1985). Proliferin is a glycoprotein containing 224 amino acids, 29 of which are in the signal sequence (Linzer et al., 1985; Lee and Nathans, 1987). The preprotein shares 40% sequence homology with mPL-11, including conservative replacements (Jackson et al., 1986). Proliferin-related protein contains 244 amino acids, 30 of which form the signal peptide (Linzer and Nathans, 1985). The prehormone shares 37-45% amino acid sequence homology with proliferin, mouse PRL, and mPL-11, and 25% sequence homology with mouse GH, including conservative substitutions (Linzer and Nathans, 1985; Jackson et al., 1986). Proliferin and mRNA for proliferin have been localized to the giant cells of the trophoblast (Lee et al., 1988). By day 8 of pregnancy, mRNA for proliferin is present, and its levels increase significantly by day 10 and then decline slowly until day 18 (Linzer et al., 1985). The gestational profile of proliferin in maternal serum is similar to that of proliferin mRNA in the placenta (Lee et al., 1988). The protein is also present in the amniotic fluid, where its gestational profile is similar to that of proliferin in the maternal blood (Lee et al., 1988). The gestational profile of mRNA for proliferin-related protein is similar to that of proliferin mRNA, except that the levels do not peak until day 12 (Linzer and Nathans, 1985). Secretion of proliferin-related protein by placental explants in vitro has been described (Linzer and Nathans, 1985), but the distribution of the protein in the maternal, fetal, and amniotic fluid compartments during pregnancy is not known. The functions of proliferin and proliferin-related protein during pregnancy are not known, although it has been suggested that

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these molecules may be involved in regulating placental and/or fetal growth since high concentrations of proliferin have been observed during periods of growth in murine cell lines (Linzer and Nathans, 1984; Linzer et al., 1985). The rat placenta has been reported to contain mRNA for a molecule designated PRL-like protein A (Duckworth et al., 1986b). Sequence analysis of the cDNA for PRL-like protein A indicates that it contains 227 amino acids, 31 of which form the signal peptide. The preprotein shares 43 and 45% amino acid sequence homology with rat PRL and rPL-11, respectively, including conservative replacements. mRNA for PRL-like protein A appears in the placenta on day 14 of the pregnancy and its levels then increase to a peak on day 18. The functions of the protein and whether it is secreted by the placenta in vivo are not known. A PRL-related mRNA that is distinct from bPL mRNA has been described in the bovine placenta (Schuler and Hurley, 1987). The corresponding cDNA was designated PRL-related cDNA I (PRC-I). The predicted protein shares 39% amino acid sequence homology with bPRL and 17% homology with bGH. The secretion patterns and function of the protein predicted by PRC-I are unknown. ACKNOWLEDGMENTS We thank Dr. Jonathan Southard and Dr. Gudmundur Thordarson for their comments and suggestions, Gulla GIslad6ttir for drawing the illustrations, and Dotty Hollinger for assistance in preparing the manuscript. This work was funded by NSF Grant DCB 8602865 and NIH Grants HD14966 and RR08132 to Dr. F. Talamantes.

REFERENCES Adams, S. O., Nissley, S. P., Handwerger, S., and Rechler, M. M. (1983). Nature (London) 302, 150-153. Ajabor, L. N., and Yen, S. S. C. (1972). Am. J . Obsrer. Gynecol. 1l2, 908-911. Aloj, S. M., Edelbach, H., Handwerger, S., and Shenvood, L. M. (1972). Endocrinology 91, 728-737. Andersen, J. R., Borggaard, B., 0rnvold, K., and Knoth, M. (1982). Acra Endocrinol. (Copenhagen) 100, 623-629. Andersen, J. R., Borggaard, B., Schroeder, E., Olsen, E. B., Stimpel, H., and Nyholm, H. C. (1984). Acra Endocrinol. (Copenhagen) 106, 405-410. Arima, Y . , and Brernel, R. D. (1983). Endocrinology 113, 2186-2194. Ayala, A. R . , Bustos, H., and Aguilar, R. M. (1984). Inr. J . Gynaecol. Obsrer. 22, 173-176. Baird, A., Wehrenberg, W. B., Bohlen, P., and Ling, N. (1985). Endocrinology 117, 1598-1601. Barrera-Saldafia, H. A., Seeburg, P. H., and Saunders, G. F. (1983). J . Biol. Chem. 258, 3787-3793.

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Talamantes, F. (1975b). Gen. Comp. Endocrinol. 27, 115-121. Talamantes, F., Guzman, R., and Lopez, J. (1977). J. Endocrinol. 75, 333-334. Tan, L., and Rousseau, P. (1982). Biochem. Biophys. Res. Commun. 109, 1061-1071. Taylor, M. J., Jenkin, G., Robinson, J. S., Thorburn, G. D., Friesen, H., and Chan, J. S. D. (1980). J. Endocrinol. 85, 27-34. Taylor, M. J., Jenkin, H., Robinson, J. S., and Thorburn, G. D. (1982). J. Endocrinol. 95, 275-279. Taylor, M. J., McMillen, I. C., Jenkin, G., Robinson, J. S., and Thorburn, G. D. (1983a). J. Dev. Physiol. 5 , 251-258. Taylor, M. J., Robinson, J. S., Jenkin, G., and Thorburn, G. D. (1983b). J . Endocrinol. 98, 197-200. Thordarson, G., and Forsyth, 1. A. (1984). J . Reprod. Fertil. 72, 261-267. Thordarson, G., and Talamantes, F. (1987). In "The Mammary Gland: Development, Regulation, and Function" (M. C. Neville and C. Daniel, eds.), pp. 459-498. Plenum, New York. Thordarson, G., Villalobos, R., Colosi, P., Southard, J., Ogren, L., and Talamantes, F. (1986). J . Endocrinol. 109, 263-274. Thordarson, G., and McDowell, G. H., Smith, S. V., Iley, S., and Forsyth, I. A. (1987). J. Endocrinol. 113, 277-283. Tolino, A., Tedeschi, A., and Montemagno, U. (1985). Biol. Res. Pregnancy Perinatol. 6 , 70-72. Tonkowicz, P. A,, and Voogt, J. L. (1984). Endocrinology 114, 254-259. Tonkowicz, P., Robertson, M., and Voogt, J. (1983). Biol. Reprod. 28, 707-716. Turtle, J. R., and Kipnis, D. M. (1967). Biochim. Biophys. Acta 144, 583-593. Tyson, J. E. (1982). Semin. Perinatol. 6 , 216-228. Tyson, J. E., Austin, K. L., and Farinholt, J. W. (1971a). Am. J. Obster. Gynecol. 109, 1080- 1082. Tyson, J. E., Jones, G. S., Huth, J., and Thomas, P. (1971b). Am. J. Obsrer. Gynecol. 110, 934-942. Tyson, J. E., Hwang, P., Guyda, H., and Friesen, H. G. (1972). A m . J . Obsrer. Gynecol. 113, 14-20. Tyson, J. E., Mowat, G. S., and McCoshen, J. A. (1984). Am. J . Obster. Gynecol. 148, 296-300. Tyson, J. E., McCoshen, J. A., and Dubin, N. H. (1985). Am. J . Obsrer. Gynecol. 151, 1032- 1038. Verstegen, J., Fellmann, D., and Beckers, J. F. (1985). Acta Endocrinol. (Copenhagen)109, 403-410. Vinik, A. I., Kaplan, S. L., and Grumbach, M. M. (1973). Endocrinology 92, 1051-1064. Voogt, J. L., Pakrasi, P. L., Johnson, D. C., and Dey, S. K. (1985). J . Endocrinol. 107, 121- 126. Walsh, S . W., Wolf, R. C., Meyer, R. K., Aubert, M. L., and Friesen, H. G. (1977a). Endocrinology 100, 851-855. Walsh, S. W., Meyer, R. K., Wolf, R. C., and Friesen, H. G. (1977b). Endocrinology 100, 845-850. Waters, M. J., Oddy, V. H., McCloghry, C. E., Gluckman, P. D., Duplock, R., Owens, P. C., and Brinsrnead, M. W. (1985). J. Endocrinol. 106, 377-386. Watkins, W. B., and Reddy, S. (1980). J. Reprod. Ferril. 58, 411-414. Welsch, C. W., and McManus, M. J. (1977). Cancer Res. 37, 2257-2261. Welsch, F. (1979). Res. Commun. Chem. Pathol. Pharmacol. 24, 211-222. Williams, C., and Coltart, T. M. (1978). Br. J . Obsret. Gynaecol. 85, 43-46.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. I I2

Membrane Oligosaccharides: Structure and Function during Differentiation PAULL. MANN Department of Anutomy, and Center for Non-Invasive Diagnosis, School of Medicine, University of New Mexico, Albuquerque, New Mexico 87/31

I. Introduction

In vitro cellular senescence as a model for aging has been the subject of intensive research by basic scientists for over three decades. Within the context of the total problem the cellular model represents the link between the organismic and the molecular levels. Aside from the obvious experimental importance of such models, they should provide insight into testable hypotheses, ultimately leading to detailed chemical mechanisms to describe the cellular events. This information can then be applied to the organismic level and to the molecular level for further, more detailed investigation. The chemical mechanism provides a logical basis from which predictive intervention strategies can be designed and tested. In the case of the senescence model, these strategies would take the form of modification of the onset or severity of the senescence event itself. In this organization the model becomes a interactive part of the whole process, not just the observational endpoint. These interactions in turn lead to perturbations of the process and to more detailed mechanistic descriptions of the original biological events. This intervention potential has not been realized in aging research. The diversity of potential mechanisms requires the development of a restricted view of the overall problem. This article will develop the concept that cell-surface oligosaccharide structures are directly involved in the control and maintenance of cellular differentiative capacity. An attempt has been made to present information from the literature to support this hypothesis. The influence of cell-surface oligosaccharides will be explored from the perspective of the aging model, developmental systems, immune regulation, and growth-transformed cell systems. These areas have been chosen specifically to demonstrate that these carbohydrate moieties have a general function in growth control. As can be seen from the breadth of these topics, the intent is to present a rationale for the development of testable hypotheses for cellular aging within the context of general growth/differentiation regula67 Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

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tion. These overviews will necessarily focus on a narrow study area, namely, cell-surface oligosaccharides and differentiation. The term ofigosaccharide is meant to be a general designation, and could refer to material attached to glycopeptides, glycolipids, or which is an integral part of glycosarninoglycans (GAG) or proteoglycans (PG). In the last section a rationale will be developed for the hypothesis that cell-surface oligosaccharide structures have a controlling influence over the biological behavior of the senescence model. 11. The Aging Cell Surface

There have been a number of reviews relating the structure and function of the cytoplasmic membrane in the aging cell model (Phillips and Cristofalo, 1983; Kay, 1984; Kelley and Vogel, 1984; Rifiind et al., 1985; Cristofalo, 1986). Little will be said about the earlier, very valuable, work done to document morphological changes related to the development of cellular senescence (for example, see Mitsui and Schneider, 1976a-c). Population doubling level (PDL)-dependent compositional and microviscosity changes in membranes were not observed in earlier work (Polgar et al., 1978) with WI-38 fibroblasts. More recently, using lateral diffusion in the murine dorsal-root ganglion neurons as a model, Horie et al. (1986) demonstrated an age-related (developmental stage) change in mobility. These changes were also associated with altered cell function. These results draw a clear functional distinction between senescent and developmental systems, and have tended to isolate the cellular senescence model from the general perspective of developmental biology. The proposed hypothesis presented here will try to draw attention to possible areas of similar functionality among aging, development, maturation processes, and, finally, aberrant growth control. The erythrocyte model has been a popular model in aging as it relates to the clinical management of the aging patient and also as it relates to the appropriate storage of blood products. The erythrocyte model has been explored in a number of studies in an attempt to correlate cell turnover with physical changes on the cell surface. Significant reductions in D-galactose (Gal) and/or 2-acetamido-2-deoxy-~-galactosyl (GalNAc) residues with aging has been reported (Gattegno et al., 1981). These authors reported similar findings for in vivo aging of red cells (Gattegno et al., 1982). Gattegno et al. (1983) also showed that there were quantitative differences between young and old cell surfaces with respect to the number of sialic acid residues. Using an anodic electrophoretic mobility determination on human peripheral blood leukocytes, it was observed

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that the cells from older individuals had higher densities of the carbonyl groups, attributed to sialic acid residues (Gomariz-Zilber et al., 1985). In a series of articles, Kay has examined what she refers to as a senescent cell differentiation antigen (for a review see Kay, 1985). This surface antigen appears to be an altered band-3 molecule. Band 3 is the major anion transport membrane complex. In senescent cells and/or damaged cell populations a proteolytic product of band 3 is found on the cell surface that appears to direct the cell's turnover. In studies on hematopoietic cell differentiation in the chicken, Kline et al. (1984) and Nelson er al. (1984) have found distinct transitions of surface markers from the fetal (CFA) to the adult (CAA) phenotype. Many of the differences appear to be related to either glycosylation or oligosaccharide termination variations. Thus, even in this small sampling of studies, there is a developing theme of involvement of the cell-surface oligosaccharide in developmental transitions, of functional importance in the mature process, and perhaps even the existence of a specific signal for clearance from the circulation at the end of its useful lifespan. These studies which relate function and structure are very important in the ultimate unifying description of a testable hypothesis for cellular aging. A discussion of functionally related changes in the cell surface and their potential relationship to the aging process, including work on the effect of developmental, maturation, and aging processes on the immune system, follows. MacDonald et al. (1981) described the development of Lyt-1+,2and Lyt-1+,2' subsets of T cells in the congenically athymic mouse as a function of aging. Subsequent studies (Maryanski et al., 1981) showed that these Thy-I/Lyt-2 cells are the precursors of the cytolytic lymphocyte. Dumont et al. (1985) have found that the Lyt-2' cells can be assigned to functional subsets which either do or do not bear the marker 9F3 (a specific monoclonal antibody reactivity). Murine strains bearing the genotype for lymphoproliferation and autoimmunity induction (lpr) exhibit a marked increase in the percentage of cells bearing both Lyt-2' and 9F3 with increasing age. These cells have a concomitant decrease in their phytohemagglutinin (PHA) and concanavalin A (Con A) reactivity, suggesting a carbohydrate alteration on this specific cellular subset. This tentative correlation between oligosaccharide and functional activity is discussed further in the section on oligosaccharides and immune regulation. The immune function of the elderly human subject was investigated by Antonaci er al. (1985). These workers showed that T cell immunoregulation was compromised in both the helper and suppressor compartments to the same extent. The effect could be transferred to young cell populations by culture supernatants, suggesting the production of a suppressive factor. Hallgren et al. (1983) in a study of T cell-surface

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markers and aging showed the presence of a less differentiated thymocyte in the circulation of the older donor. Later these same workers (Hallgren et al., 1985) suggested that this primitive T cell expression in the aged donor was the result of cell-surface modulation of the various T cell markers. These studies suggest that there is a type of reversion to developmentally primitive markers on the cells of the aged donor. These rather sketchy and diverse results suggest that many of the observed deficits in the functioning of the immune response in the aging subject can be traced to changes in the cell surface, and perhaps to the oligosaccharide compositional or structural changes. Aizawa et al. (1979, 1980a-d) described in a series of papers a carbohydrate-based alteration in the cell surface of aging human diploid fibroblasts. Their assay consisted of a Con A-mediated red blood cell adsorption onto the fibroblasts. They showed that the effect was a generalized phenomenon with respect to various tissues and cell types, and correlated with various age-related disease situations. These workers used the Con A at 100 pg/ml, thus probably supersaturating the surfaces of their system. At these concentrations it is possible that the cross linking properties of the multivalent Con A produced a microcrystallite on the surface of the cell. This would not affect their data (except in a positive sense, that is, the rosetting function of these cells would be more avid), but this type of assay could not be used for quantitation of the effect or characterization of the binding epitope. Wever et al. (1980) showed that the treatment of human fibroblasts with heparin over a period of time resulted in an increase in the surface expression of heparin sulfate and hyaluronic acid. Studies by Vogel et al. (1981) showed a slight alteration in the secretion of GAGS with Phase I11 (senescent) fibroblasts but no quantitative changes related to age. Matuoka and Mitsui (1981) found that heparin sulfate accumulated on the cells of advancing PDL in the absence of an elevated synthesis level. Confluent cultures showed a PDL-dependent increased total GAG level. Later, the same workers (Mitsui et al., 1985) showed that these heparin sulfate changes directly affected the older cells’ ability to replicate. Moley and Engelhardt (1981) demonstrated that there were no significant changes in the major peptide antigenic structures present on sensecent and presenescent cells using a protein A/specific antisera adsorption assay. These data suggest that any surface oligosaccharide PDL-dependent changes were not related to major peptide transcriptional changes. The apparent anomaly in these data (demonstrating PDL-dependent changes in cell-surface moieties in the absence of cell-surface protein changes) could be explained by the following hypothesis: Although the surface glycopeptides are the same, modifications in their oligosaccharide side

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chains (either in structure or conformation) result in an altered presentation and control potential. It might also be related to the sensitivity of the protein A assay. De Vos and van Gansen (1982) showed that there was no direct correlation between cytoskeletal changes and the induction of the sensecence, suggesting that the morphological “flattening” of the senescent cell was surface dependent. This is significant, as the morphological changes which occur very late in the cell’s life span appear to be secondary to the actual induction event. Lee et al. (1982) showed that a high-affinity low-density lipid (LDL) receptor was inducible in aging fibroblasts by withdrawal of the lipoproteins from the culture medium. They also showed that the total number of LDL receptors decreased with advancing PDL. These data present a picture of an aging cell surface which is both less inducible and less suppressible by feedback because of some surface alteration. HMG-CoA reductase, a cytoplasmic enzyme system which both regulates cholesterol synthesis and contributes to the synthesis of dolichol, follows a similar pattern. Oka (1985) showed that senescent fibroblasts showed a marked decreased in their ability to either synthesize or secrete fibronectin. A study by Blondal et al. (1985) showed that senescent cells bound more Con A than their younger counterparts but at the same time incorporated less mannose into their surfaces. These studies are in disagreement with those of Aizawa et al. (1979, 1980a-d). Mitsui et al., (1985) found cell-surface charge anomalies in the Werner’s syndrome skin fibroblast model. The increase of negative charge with advancing age in the normal model or rapid aging model (Werner’s) was associated with an increase in heparin sulfate. GAG preparations from the older cells could inhibit growth of the younger cells. Taken together, even this fragmentary information suggests that cell-surface changes are correlated with senescence. These changes occur in the absence of major cell-surface protein antigenic changes, and appear to be related to oligosaccharide structure. 111. Developmental Phenomena

The cell surface is a prime clearinghouse for information. The boundary conditions representing the cell surface imply that information must undergo physical changes (transduction) before being of functional use to the cell. The exchange of information at the boundary is a bidirectional activity and has certain physical restraints. These include diffusioncontrolled access to the surface and structural and conformational regulation of interaction with the boundary (binding). In developmental systems, the time scales for both growth and differentiation are remark-

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ably compressed as compared to maturation processes. This makes developmental systems a very useful area for the investigation of oligosaccharide control over growth/differentiation. Signal diffusion can be used as a natural control mechanism in this context; however, for most cellular activites the assumption is that there must be a mechanism which is functional at the cell surface to actively control access. In mature models this function has been largely attributed to glycopeptide-receptor interaction. In some developmental models either the specific glycopeptide or the cell-surface receptor has not yet developed. In this case the signaling process is more generic in nature. Since cellular differentiation and growth control are required throughout the cell’s lifespan regardless of the status of glycopeptide/receptor-specificcontrols, it must be assumed that a less signal specific, more generic control mechanism exists. Using photobleaching techniques to determine diffusion coefficients, Gall and Edelman (1981) showed that if reversible modulation of cell surfaces represents a specific signal it must involve a very few distinct receptors and could not be a general property of cell-surface receptor systems. This work suggests that the basal level of control does not rest with the specific receptor. Because the developmental systems have an inherent simplicity, they have also been used extensively for biochemical studies. This is a complex area and is beyond the scope of this article; the following example, however, makes an interesting point. A great deal of attention has been paid to the biochemical pathways involved in signal transduction, primarily from the perspective of protein kinase C activity. Nishizuka (1984, 1986) has described a pathway whereby this kinase enzyme was activated by diacylglycerol present in the cell membrane. This signal system is controlled by inositol phospholipid turnover loops, and poses a possible point of interaction for the phorbol ester promoters in the malignant transformation of growth control. The phorbols can irreversibly substitute for the diacylglycerol and are not regulated, thereby supplying a constant activation signal. Under normal growth/differentiation control the cell-surface receptor-ligand complexing event “triggers” phospholipase C action on phosphatidyl inositol 4,5-bisdiphosphate to yield the diacylglycerol and inositol 1,4,S-triphosphate (Berridge, 1984), which enters the cytoplasm and releases calcium from intracellular stores. A second messenger, inositol 1,3,4,5-tetrabisphosphate, also appears in stimulated cells (Rossier et al., 1986). This represents the current thinking on intracellular events subsequent to the cell-surface event. Many of these reactions are controlled by feedback loops, but seldom is any mention made of feedback (interactive) signals at the cell-surface level. The unstated assumption is that the receptor-driven

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signal is an “on” switch with no coherent method for controlling either access of the signal to the receptor or the dynamics of the binding event itself. We will attempt to develop a rationale for such a control over the cell-surface events. Developmental models, especially those involved with evolutionarily primitive organisms, offer the clarity of simple, temporally intense responses. A theoretical approach to growth control in the plant cell-wall system has recently been developed (Ricard and Noat, 1986) that utilizes cell-surface charge density as a driver of growth. A hypercycle is hypothesized, involving charge display followed by neutralization through the deposition of new neutral carbohydrates. This growth model is highly dependent on local pH and divalent cation concentration, thus providing a possible link with the mammalian models. More importantly, it offers a surface-interactive model which expresses control over growth and perhaps also control of differentiative properties of the system. The evolutionarily primitive systems are important models for our purposes because they should be more dependent on a “simpler” form of control system. Dictyostelium responds uniquely to exogenous signals. Roos et al. (1977), as well as later studies by Schapp and van Driel(1985), have shown a cyclic behavior of cyclic AMP and adenylcyclase activation. Pulsed addition of cAMP resulted in pulsed activation. The stimulation of postaggregation differentiation via cAMP activation is a surface phenomenon. Specific surface carbohydrate ligands were shown to direct differentiation-specific adhesion mechanisms in Dictyostelium by Bozzaro ( 1985). Dictyostelium surface oligosaccharide types and characteristics were directly related to the developmental stage of the slime mold (Ivatt rt al., 1981). Links between the behavior of the primitive slime molds and more advanced species have shown a continuity of general control type (Barondes et al., 1981). Intracellular “lectins” from both the cellular slime molds and chicken were found to act as induction signals. These inductive signals are largely unidirectional, but in some instances bidirectional effects have been observed in the differentiation of some of these surface entities into structural/conformational forms. In the sea urchin developmental model, Bolanowski et al. (1984) showed that fatty-acid acylation of glycopeptides is developmentally regulated, as it is in higher species. Specific oligosaccharides have also been associated with distinct differentiation stages of the trypanosome (Engel and Parodi, 1985). We can also find examples of cell-surface oligosaccharides involved in the differentiation and growth control of mammalian models. Skutelsky and Bayer (1983) showed specific cell-surface determinants involved in the specific differentiation pathways. Erythroid stem cells have high

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levels of galactose-containing determinants, whereas the lymphoid precursor cells lacked this specific marker. These data may suggest that a specific cellular differentiation resulted in a specific oligosaccharide change on the cell surface. This can be viewed as the cell altering its own ability to interact with its environment in a specific manner. In other studies, Codogno and Aubery (1983) showed, using a chick developmental model, that during early embyronic growth there is a marked increase in surface sialic acid residues due to both increases in sialylation and the conversion to 0-linked oligosaccharides. The changes in sialic acid, however, did not account for the biological changes noted in cell adhesion, which resulted from cellular differentiation. These data suggest a complex bidirectional control over differentiation function involving both the cell surface and the cell’s environment. The central role of the cell surface in interactive control mechanisms is suggested by the work of Chizzonite and Zak (1981) and Frank and Rich (1983). In the developmental model of the rat heart, these workers have shown that there is a direct correlation between the development of the cell-surface glycocalyx and a pattern of functional sensitivity to calcium ion concentration changes. This suggests that the cell surface has a role in maintaining and modulating calcium ion fluxes, which in turn suggests a generic control of the glycocalyx over numerous cell reactions. Tunicamycin, at dolichol synthesis-inhibitory concentrations, prevented the topological orientation and physical fusion of embryonic quail myocytes during differentiation of the muscle, thus suggesting a spatial recognition role for the cell-surface carbohydrates. When nonfusing myoblast cells were placed in mitogen-depleted medium they reinitiated their differentiation and expressed a number of “muscle-dependent” genes (Olson et al., 1983). These changes have been shown to parallel the induction of specific transferases, and thus form a significant link between gene induction and oligosaccharide differentiation (Grant et al., 1986). The direct link between the cell-surface oligosaccharide expression of a model and its differentiation status was extended in several models. These models provided an additional perspective of the interaction potential of the surface oligosaccharides with surrounding tissues. Miyauchi et al. (1982) traced the expression of specific cell-surface carbohydrate determinants as a function of development and found distinct expression patterns suggesting a primary role for carbohydrates in differentiation. The migration of intestinal epithelial cells is under differentiative control in conjunction with positional signals as they proceed from crypt to villus locations (Wilson et al. 1984). Superimposed on these morphological positions are biochemical gradients of various transferases and acceptors, indicating that changes in carbohydrate are again “driving” differentiation. Exogenous addition of dolichyl phos-

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phates has been shown to affect the differentiation of bone marrow hematopoietic progenitors (Shimamura et al., (19851, again showing an interactive potential for the system. Cell-surface oligosaccharide alterations have been correlated with resistance to differentiation signals of some variant cell clones, much like the development of drug resistance (Felsted et ul., 1985). These data reiterate the potential of the cell surface oligosaccharide structure as a bidirectional controller of differentiation. Glycophorin A, an exclusive erythrocyte sialoglycopeptide has only 1 N-linked and 15 0-linked oligosaccharides which show a differentiationdependent modulation with maturity (Gahmberg et al., 1984). Malignant transformation also shows changes in the level of 0-linked material, suggesting that molecular heterogeneity is an expression of diversity, perhaps in the form of skewed distributions of oligosaccharide side chains on otherwise normal protein. Taken together, these data illustrate the truly interactive status for these molecules with the ability to discriminate certain signals. There have been a number of studies on neural development which indicate another level of oligosaccharide control over differentiation. In a neural cell line model a surface glycopeptide was shown to go through developmental stages directly related to its glycosylation (Rougon et al., 1982). Leon et al. (1984) have shown using the chick dorsal-root ganglion model that a specific monosialoganglioside facilitates the differentiation and redifferentiation of the cells in culture. In a glaucoma model, Beauchamp et al. (1985) demonstrated that cell surface and extracellular complex carbohydrates and specific glycoconjugates were responsible for the initiation\of differentiative signals. Landreth et al. (1985) showed that the binding and differentiative properties of nerve growth factor could be blocked by wheat germ agglutinin, thus indicating that a specific carbohydrate moiety was involved in the binding and therefore the signal transduction. Sobue et al. (1986) used two cAMP analogs to induce a specific cell surface gal-cerebroside in Schwann cells. These induced cell-surface changes resulted in morphological and differentiative changes. This effect was independent of extracellular cAMP concentrations, indicating that proliferative and differentiative signals were separate. Addition of purified gangliosides from brain tissue to neuroblastoma cultures has been shown to affect differentiation responses in the cells (Leskawa and Hogan, 19851, indicating the endogenous and exogenous signal potential. Different pathways appear to have separate signals. This work has been confirmed and extended in the dorsal-root ganglion system, where it was found that not only was the differentiative stage of the cell determined by the cell-surface oligosaccharides (the lactosamine series in this case), but the signal transference was affected in a similar

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manner (Dodd et al., 1984; Jessell and Dodd, 1985; Dodd and Jessell, 1985).

The cell surface has also been shown, in numerous models, to exercise a controlling influence over various general growth parameters. McAuslan et al. (1980) characterized a variant endothelial cell line which secreted a 3-fold increase in fibronectin, much of it depositing directly onto its luminal surface. This was correlated with an increased binding of Con A. When the cell was induced to migrate, the fibronectin was found to redistribute and the cells demonstrated the ability to grow over or under other cells in culture. Normal endothelial cells have most of the fibronectin positioned on the basolateral surface and are confined in their migration behavior. These data suggest that a number of differentiative functions of the cell may be not only moderated by the cell surface oligosaccharide structures, but also absolutely defined by these residues. This type of cell-surface interaction appears to have general biological significance in that it has been shown that surface extracts from confluent endothelial cells can inhibit the growth of endothelial cells (Heimark and Schwartz, 1985). This effect is tissue specific. These same cell surface GAGS have been implicated in all phases of regulation (Klebe et ul., 1986). Heparin sulfate was shown to bind endogeneous proliferative factors, was reversible by platelet-derived growth factor (PDGF) addition, and was modulated by the presence of serum. Thus, glycosylated cell products are not only implicated in signal transduction and modulation but also in general biological reactions which are necessary for tissue architecture. In a study of cell-surface carbohydrate structures during differentiation, Sat0 et al. (1986) have shown that there are diverse and highly specific mechanisms for regulating the appearance and disappearance of developmentally important carbohydrate markers. This expression of carbohydrate markers appears again in the neoplasia model, in which reexpression of some of these markers represents the major tumorspecific membrane markers (Feizi, 1985). This in turn forms a natural link between normal and malignant growth/differentiation control. The growth-transformed models are a very important part of our discussion of the senescence model and will be discussed in Section V.

IV. Immune Regulation Regulation of the synthesis of the complex oligosaccharide side chains of the glycopeptides has been studied extensively. Ivatt (1981) described the primitive sequential production mechanism for their synthesis. As will be seen later, specific controls have subsequently been found for the branching points of oligosaccharides which account for the majority of

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their diversity. Crumpton (1982) suggested two mechanisms for oligosaccharide alterations in malignant transformation: The glycopeptide could be glycosylated differently, or entirely new glycopeptides could be produced. Both mechanisms are operative, but recently more and more information tends to implicate the oligosaccharide itself as the focus in altered regulation. The pertinent question is, how is the information managed? Schachter et al. (1982) developed a rationale for the structural diversity of oligosaccharides which involved two enzymes functional at the same site to induce branching, as well as the concept that specific carbohydrate substitutions may “fix” a certain structure. The linearity of the glycosylation process itself also acts as a powerful control mechanism. This strongly suggests that diversity in oligosaccharides rests primarily with their conformational, three-dimensional spatial interactions. Thus, a structural component can be found directing development and maturation, while at the same time be implicated in the aberrant transformed growth processes. The “difference” may be as simple as the conformation of the oligosaccharide which changes the signaling potential of the molecule. Immune regulation and some of its control mechanisms may give us some insights into this process. The expression of cell-surface gangliosides have been shown to be directly related to the phenotypic state of the cell. Stallcup et al. (1984a,b) have shown that cell-surface determinants have the physical ability to modulate recognition and/or regulation reactions. A major histocompatibility complex (MHC) class I, non-H2, substance was extracted from lymphocytes which could inhibit the growth of intact lymphoid cells, either normal or transformed. This cell-surface molecular modulation effect appears to be generic, in that a sponge surface “lectin” has been described which increases the growth potential of a murine lymphoma line (Diehl-Seifert et al., 1985). This galactose-specific material interacts with a family of cell-surface glycopeptides (170, 140, and 88 kDa). Robb (1985) presents data which indicates that the interleukin-2 (IL-2) receptor system occurs in both high- and low-affinity forms. Studies using cDNA probes suggest that the protein component is not sufficient for the high-affinity interaction, therefore suggesting another type of auxiliary interaction, perhaps involving ligand or local cell-surface modification of binding. Treatment of T cells with antisera to the T-3 marker produces shedding of the T-3 antigen and an increased expression of IL-2. This is specific to an accessory cell interaction, again presenting the possibility that even these highly specific interactions are modulatable by general cell-surface interactions (Schwab et al., 1985). Chen et al. (1986) have shown that there is a separation and specificity in certain signals. In this instance, lectin-stimulated T cells were differentiated into cytotoxic cells only after the addition of either a- or y-interferon. This second interaction

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was mediated by a specific cell-surface receptor, possibly indicating that there is also a temporal sequence involved in the enunciation of certain specific signals. T-6 is a thymocyte marker with a heterogeneous differentiation stage-related presentation which shows marked similarity to the murine thymic leukemia antigen. It is associated with pz-microglobulin, and is a class I MHC, non-HLA A or B, antigen (van Agthoven and Terhorst, 1982; Lerch et al., 1983). The T-6 presentation is reminiscent of Hallgren’s work, described earlier, where redifferentiation of T cells in the aging subjects was found. These phenomena could be related to oligosaccharide modification and they are discussed below. Childs et al. (1983), studying the leukocyte common antigen (T200), found that both 0- and N-linked oligosaccharides were more susceptible to enzyme attack, which suggests that it is this site which changes during differentiation reactions. The cytotoxic T cell determinant has been shown to be associated with T200 as well as two other glycopeptides (140 and 85 kDa), and the carbohydrate moiety is essential for activity (Lefrancois et al., 1985, 1986). These glycans appear to be 0-linked. Their expression is modulated by stimuli such as IL-2 and Con A. An N-acetylgalactose (GalNAc) residue is probably involved in the binding epitope. Another T cell surface antigen, Thy-1, shows a direct link between the differentiation status of the cell and its N-linked oligosaccharide (Morrison et al., 1986). Using a B-lymphocyte model to study response to different surface stimuli, DeFranco et al. (1982) showed that just as T cell growth and differentiation are under complex surface control, the B cell compartment also shows signs of complex and highly specific control. Specific cell-surface antigenic systems have been described for a number of lymphoid models (Clark and Ledbetter, 1986). In this study, a Blymphocyte activation model, two cell-surface glycopeptide complexes have been described whose control appears to be affinity modulated. Although this is a recently emerging area of active research, it appears plausible at this stage to hypothesize that cell-surface oligosaccharide structures modulate both normal and transformed cell growth/differentiation patterns in the regulation of the immune system. V. Neoplastic Regulation

Neoplastic growth regulation plays an important part in the overall discussion of cellular senescence because the neoplastic model presumably lacks the regulatory mechanism that allows the normal cell to senesce. In the preceding sections the relationship between cell-surface oligosaccharide structures and control over developmental and matura-

MEMBRANE OLIGOSACCHARIDES

79

tion processes has been stressed. The identity and structural characteristics of some neoplastic cell-surface antigenic determinants have been studied (Feizi, 1984; Thorpe and Feizi 1984; Feizi et al., 1984; Tatteroo et al., 1984; Tabilio et al., 1984). N-Acetyllactosamine units (SSEA-I) were found to markedly change during embryonic life, maturation processes, and malignant transformations. Phorbol ester (TPA) or dimethyl sulfoxide (DMSO) treatment of HL-60 cells stimulates a differentiation response correlated with a masking of specific carbohydrates by N-acetylneuraminic acid (NANA). SSEA-I is expressed on all cell lineages as a generic differentiation antigen; this brings into question some of the classification criteria for malignancy using monoclonal antibodies, as these tend to react with the SSEA-I antigenic structure and may not differentiate between altered forms (conformational?) associated with malignant transformation. Mechanistic studies for the possible role of carbohydrate in differentiation have shown that stimulant/promoter type substances, such as diethylstilbestrol in the chick-oviduct differentiation model, cause an enormous increase in dolichyl phosphate-linked oligosaccharide synthesis (Hayes and Lucas, 1983). Cell-surface oligosaccharides have been implicated in differentiation for a number of years. In early studies with the HL-60 system (Nakayasu et al., 1980) it was shown that tunicamycin, a potent dolichol synthesis inhibitor, could induce the differentiation of HL-60s into the phagocytic pathway. Forced differentiation of HL-60s with DMSO or TPA results in specific changes in the oligosaccharides, including outer chain elongation (SSEA-I antigens). These results indicate that myeloid differentiation pathways may require specific oligosaccharide signals (Mizoguchi et al., 1984a,b). Treatment of HL-60 with exogenous specific gangliosides has been shown to modulate growth and monocytic differentiation (Nojiri et al., 1986). Using 3T3-SV40, 3T3, and 3T3-SV40 revertant combinations, Mugnal et af. (1984) showed that the revertant had certain cell-surface characteristics in common with the parent line while others were unique. Transformed cells appear to be amenable to differentiative changes by surface modulation. Kiguchi et al. (1986) have analyzed the cell-surface glycosphinolipids (GSL) and gangliosides (GM3) of a human T cell leukemia model. TPA activation results in a suppressor phenotype, which in turn could be related to changes in GSL and GM3. A disialoganglioside (GD3) was shown to have a different expression which was either related to an in situ modification of its expression, or to the utilization of a novel synthesis pathway. Winterbourne and Mora (1981) showed in a clonedmouse solid-tumor model that, although there was no difference in the level of heparin sulfate between the various clones, there were differences in the number of 0-sulfate residues found in oligosaccharides of higher molecular weight. These data suggest a possible altered interaction

80

PAUL L. MANN

potential between some tumor clones and their immediate environment. In the B-16 metastatic lung mouse model, Irimura et al. (1981) demonstrated that inhibition of glycosylation mechanisms with tunicamycin prevented normally metastatic variants from adhering to endothelial cells and also reduced their growth. The Thomsen-Friedenreich antigen (T-antigen), a p( 1-3) Gal/GalNAc residue has also been associated with T cell differentiation and malignant transformation. Newman and Delia (1983) studied the appearance of peanut agglutinin (PNA, fi (1-3) GaUGalNAc nominal specificity) binding on acute lymphoblastic leukemias (T-ALL) and found about 25% positive cells, indicating a thymocyte origin. After treatment with TPA the p(1-3) Gal/GalNAc residues were masked by NANA, implying a differentiation step. Bresalier et al. (1984) showed that primary colon tumors secreted a mucin with p( 1+3) Gal/GalNAc specificity and that the metastatic counterparts of these tumors lost this specificity, suggesting still another differentiation step. Orntoft et al. (1985) found in a survey study that the T-antigen was widespread among colorectal malignancies. Yuan et al. (1986) found that the expression of T-antigen was detection system dependent, in that the results were dependent on the use of specific reagents (PNA and monoclonal or polyclonal antibodies). This indicates that there may be conformational differences not yet described which are related to differentiation and/or malignant transformation. The results of most of the experiments discussed above are directly related to the use of reagents (antibodies/lectins) with specific binding properties. The general concept of binding is that it is a complex physical process comprising both primary structural and conformational components. Thus, three reagents with the same nominal structural specificity may have very different aggregate binding properties. By the same token, the same structural entity on a cell surface may have very different interaction potentials depending on its spatial disposition and that of its nearest neighbors. Thus, simple binding of a reagent and an unknown cell-surface epitope is not sufficient information as to the epitope’s identity. Either extensive affinity information or independent structural verification of the epitope is required.

VI. Cell Surface Oligosaccharide Modulation and IMR-90 Cellular Senescence In the preceding sections an overview of experimental evidence to support the hypothesis that cell-surface oligosaccharide structures have a

MEMBRANE OLIGOSACCHARIDES

81

major influence over a variety of cell differentiative functions including cellular senescence was presented. This has been rather sketchy and at times disjointed, primarily because of the number and diversity of fields involved. However, several general conclusions can be drawn from these studies: (1) oligosaccharides, their appearance and disappearance have a major influence over developmental processes; (2) cell surface oligosaccharide alterations, mainly structural epitopic changes, have been found to influence differentiative processes in the adult; (3) in some instances these influences appear to be truly interactive, in that the purported signaling is bidirectional; and (4)these same oligosaccharide structures appear to be missing or altered under aberrant cellular grow.th control conditions. This last point is crucial to our discussion of differentiative control mechanisms involved with cellular senescence. It draws an obvious distinction between “normal” and “aberrant” behavior, and any theoretical discussion of cellular senescence must consider both. The general reference to interactive influences made above is also important because it draws a natural distinction between the traditional signal transduction systems (glycopeptide-receptor), and the oligosaccharide conformational signals. The glycopeptide-receptor systems accept specific information from the external environment and, through a transduction system, pass that information on to the cytoplasm. There is a subsequent reaction to this information, but it may be temporally and spatially quite remote from the original transduction event. The oligosaccharide interactions are hypothesized to be more generic than those of the specific glycopeptide receptor model. “Generic” in this context is meant to signify certain minimal functions required for biological existence, and can be contrasted with the presumed highly specific signaling capacity of the glycoprotein receptor. Although a distinction (nonspecific versus specific) is being made, it is mainly for discussion purposes as the major logical source for these oligosaccharide structures is the cell-surface glycopeptides themselves. The distinction arises from evolutionary considerations. It has always been necessary to process information relative to cell contacts (initially as chemical boundary conditions) but, for instance, the development of insulin control over glucose levels in the circulation and tissues is probably a relatively recent requirement of organismic development. The assumption is that signal specificity arose from nonspecificity placed under some type of “pressure,” and that the nonspecific activity is still functional. This is of course pure speculation at this point, but it provides a very useful operational basis from which to design experiments and test predictions from hypotheses. This type of analysis also provides a rationale for viewing the cell’s ability to exist in its environment. The logical drawback to “specific” (glycopeptide recep-

82

PAUL L. MANN

tor) control over all phases of growth is the enormous, unmanageable number of specific signals required. A generic system would be, by necessity, much simpler. If there were a natural connection between the two alternative control systems via, for instance, conformation-induced changes in the affinity of the glycopeptide receptor sites caused by their own oligosaccharide side-chain interactions, then a functional continuity would be possible. As a concept this would require the cell surface to have the ability to functionally restrict its own oligosaccharide/environment interactions through bidirectional interactions. The major intrinsic drawback to the concept of the oligosaccharide as a modulator of functioddifferentiation is the molecule’s lack of the primary and secondary structural “stiffness” inherent in the amide linkage. Thus, in the case of oligosaccharides we must place more emphasis on molecular conformational information. In fact, with nine major monosaccharide units available for use in mammalian species and a freely rotating carbon-carbon bond (with its apparently infinite capacity to form branching structures), the oligosaccharide has more diversity potential than the peptides. This diversity is regulated at the enzyme level (Roseman, 1970; Hill et al., 1977; Waechter and Lennarz, 1976) by feedback control over substrate and through specific substitutions (Schachter et al., 1979; Baenziger and Kornfeld, 1974; Tai et al., 1975). Even with these physical restrictions at the production level there remains an enormous diversity, especially when one considers the cell surface with each glycopeptide having 12-20 oligosaccharide side chains, each with 5-15 monosaccharide units. Most of this diversity will be expressed as conformational diversity, and could be further restricted on a surface by association into packing structures mediated by divalent cations. Hoekstra and Duzgunes (1986), in a study on the RCA-Imediated agglutination and fusion of vesicles, found that calcium regulated the steric orientation of the carbohydrate head groups, therefore mediating the physical properties of the approaching vesicles and their consequent fusion potential. A galactose-specific sponge lectin undergoes a conformational change in the presence of calcium to specifically expose galactose residues, which in turn mediate cell-substrate adhesion (DiehlSeifert et al., 1985). Divalent cations have been shown to be permissive for lectidligand interactions (Carver and Brisson, 1984; Borrebaeck and Mattiasson, 1980). These data suggest that oligosaccharides mediate a more interactive reaction than simple binding. The binding event can be seen to depend on some other permissive event (perhaps calcium-dependent conformational change?). The result of the binding event itself induces calcium binding (selfregulation), and either lowers or raises the activation energy for a specific interaction.

MEMBRANE OLIGOSACCHARIDES

83

In order to address some of these speculations, our model system had to have the capacity to determine oligosaccharide conformational interactions within the realm of growth control in general and cellular senescence specifically. Working with the IMR-90 model, we have investigated the identity and functional interactive potential of the cell-surface oligosaccharides during the “senescence process.” Lectins were used for the initial studies because of their structural specificity for defined oligosaccharide groups. It was important to establish biological relevance while at the same time developing an analytical capability. Fluorescently conjugated lectins were first used to visualize the ligands within the context of the whole cell (Mann et al., 1987a). Simple binding studies showed that the IMR-90 cells had three lectin specificities: concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA120). The nominal carbohydrate specificites are: Con A, mannosyl; WGA, N-acetylglucosyl; RCA-120, galactosyl residues. In order to establish the identity of the actual binding epitope, detailed competition analyses of the binding events are required. The simple binding curves for these lectins on young (PDL ~ O S ) , midlife (PDL ~ O S ) ,and old (PDL 45s) IMR-90 are similar in shape. When the same number of cells from early and advanced PDLs are plated at approximately 30% culture-surface confluency, and a concentration of fluorescently conjugated lectin (FITC-lectin), determined to produce approximately 8 0 4 5 % binding-site saturation for optimal sensitivity, is incubated with the cells, a quantitative decrease in uptake of Con A (-22%), WGA (-27%), and RCA-120 (-33%) specificities is observed in the advanced PDL cells. The advanced PDL cells also appear to develop a “new” specificity, Dolichos biJorus agglutinin (DBA), which has a nominal specificity for N-acetylgalactosyl residues. This basic finding is in agreement with the work of Aizawa. Cellular heterogeneity has been a hallmark of many of the morphological descriptions of cellular senescence; thus, we undertook to investigate the contribution of this type of heterogeneity to this observation. Using photometric readings to quantitate the amount of fluorescence associated with individual cells, it was possible to compare the labeling patterns of individual cells. N o heterogeneity was apparent in the PDL-dependent quantitative difference in lectin uptake. When 80-pm’ areas of membrane were quantitated, similar results were seen. Thus these quantitative differences appear to be generic effects of all the cells within the population, dependent only on the PDL of the culture. Kinetic experiments designed to investigate the temporal heterogeneity of the observation also indicated that the individual lectin-dependent quantitative changes were a general population property. This is in spite of the fact that an enormous amount of morphological heterogeneity is present,

84

PAUL L. MANN

which is especially significant in the morphologically senescent populations (in our experience with IMR-90 this occurs at PDL45-47). The morphological events associated with cellular senescence occur over a very short period of time (within the last few PDLs) compared with the entire life span. When a kinetic study was done on the lectin uptake it was found that the quantitative changes occurred much earlier in the cells’ life span (for instance Con A changes become significant at PDL 3 3 , and that the onset of the changes is lectin dependent. RCA-120 is the first to show quantitative differences and reaches its quantitative maximum at PDL 39. It should be emphasized that these quantitative changes precede the morphological events. The use of the fluorescein isothiocyanate ( F I T 0 conjugated lectins also permitted capping and endocytosis experiments (Mann et af., 1987b). By using the photometer it was possible to determine the membrane clearance rates for the complexed material. The finding that the lectinlligand conjugates cap and endocytose suggests that the ligand is part of an integral membrane protein. The clearance rates again show a lectin dependency. In the case of the Con A complex there appears to be two distinct classes of membrane complex, one rapidly cleared (50% clearance time = 50 minutes) and the other cleared more slowly (50% clearance time = 340 minutes). Furthermore, the highmobility class is completely absent from the advanced PDL cells. The disappearance of this functional class of cell-surface oligosaccharides accounts for >85% of the quantitative PDL-dependent difference. The RCA-120 mobility studies show that both young and old populations have the same rate of clearance (mobility), thus suggesting that the PDLdependent difference is primarily related to the number of binding sites. The WGA data are complex and indicate that there are both numerical and functional changes in the clearance (mobility) behavior of this complex. In order to study these cell-surface PDL-dependent changes in analytical detail we developed an assay which could be used for ligand competition assessment (Mann et af.,1988a). This entailed the application of a biotinylated lectin (in the case of the competition analysis, mixed directly with graded amounts of either specific or nonspecific synthetic carbohydrate), and avidin and biotinylated alkaline phosphatase in separate steps. The multistep nature of the assay allows the competition for the original binding event to be carried out in the absence of the assay reporter molecule (biotinylated enzyme). This assay was also extraordinarily sensitive because of the added amplification of the multiple steps. The assay is capable of a full oligosaccharide analysis on as few as 100 cells. This assay showed that only three of the lectins (from a screening assessment of seven lectins which tests the major specificities) reacted

85

MEMBRANE OLIGOSACCHARIDES

with the IMR-90 cell surface, in agreement with the FITC-lectin experiments. Figure I shows the simple binding curves of PDL 30 and PDL 45 IMR-90 cells incubated with graded concentrations of Con A in the biotinylated lectin/avidin/biotinylated enzyme assay. It is clear that the nonsenescent and senescent cell populations differ only in their quantitative uptake of the lectin. Similar results were seen with both WGA and RCA-120. These data are in agreement with that of Aizawa as discussed above. Because of the analytical capability of the assay system we are able to investigate the nature of the quantitative differences seen in Fig. 1 . In order to do this we have chosen a constant concentration of lectin (approximately 85% of the saturation concentration) and added graded concentrations of synthetic specific carbohydrate to the lectin incubation mixture. This competition data can then be analyzed by a series of microprocessor-aided analyses (LIGAND and ISIS-12) to derive Scatchard plots from the competition data. This, in turn, provides calculated apparent association constants (K,, in units of liters/mole), and binding capacities (R,,),which are the basis for the analytical comparison between lectin-binding events on the cell surface. The crucial assumptions for these analyses are that the exogenous synthetic ligand is identical to the cell-surface determinanj, and that the reaction has gone to equilibrium. The first assumption is justified, in this case, by our independent knowledge of the lectin specificities and the use of multiple synthetic ligands to assure detailed knowledge of the binding event. If we assume that the binding epitope of Con A is a rnannosyl residue of a particular conformation, there should be a relationship between the results of

I C

m 0 v 0 0

I

-2

4

0 L O G L E C T I N CONC. (M)

-1

1

FIG. I . Simple binding of concanavalin A to IMR-90 cells; open figures represent PDL 3 I cells and closed figures PDL 42 cells.

86

PAUL L. MANN

-5

-4 CARBO. CONC.

-3

-2

(LOG MI FIG. 2. A Scatchard analysis of mannosyl residues on PDL 30 IMR-90 cells. Concanavalin A was competed for by a-methyl-d-mannopyranoside at various concentrations.

affinity determinations for a group of ligands that have a range of binding affinities for the lectin by separate analysis. Under equilibrium conditions, the calculated affinity for a variety of ligands should increase until the affinity matches the unknown epitope, then there should be an apparent leveling off of the calculated affinity. This value is the “true” unknown affinity. Figure 2 shows a typical analysis for Con A binding to IMR-90 cells at PDL 30. The numerical analysis accounts for 90% of the total lectin bound within two distinct binding site classes, with apparent equilibrium binding constants of 1.44 X lo6 and 3000 liters/mol (Mann et al., 1988b). Figure 3 shows a similar analysis for PDL 45 cells, and in this case only one binding site class is predicted with a binding constant of 5000 liters/mol. The total number of binding sites is very similar in both cases, leading us to the conclusion that there has been a quantitative loss of the high-affinity binding class as the IMR-90 cells advance through their lifespan. This oligosaccharide change occurs temporally before the morphological manifestations of senescence, therefore making it a candidate for being a causal factor. These mathematical determinations are representative of the K values for what we assume are “families” or classes of binding sites. The remarkable feature is that we observe any class distinction at.all. We would assume from these results that there are distinct distributions of binding affinities superimposed on the general diversity of the oligosaccharides displayed on the cell surface. This type of speculation which relies on the lectin interaction will require confirmation. We are currently undertaking the purification and characterization of these purported distinct affinity classes. The affinity information

87

MEMBRANE OLIGOSACCHARIDES

LL

\

m

-5

-4 CARBO. CONC.

-3

-2

MI FIG. 3. A Scatchard analysis of mannosyl residues on PDL 42 IMR-90 cells. Concanavalin A was competed for by 0-methyl-d-mannopyranosideat various concentrations. (LOG

generated by the biological experiments and lectin-affinity columns will be used to design specific separation protocols. Nuclear magnetic resonance (NMR) and mass spectrographic techniques will be used to independently establish the structural and conformational homogeneity of the oligosaccharides. In spite of the current gaps in our information there are intriguing correlations between the biological and analytical data. The highly mobile Con A class correlates with the high-affinity Con A class, in that they both disappear as a function of advancing PDL. The kinetic experiments also suggest that these classes are the same. Similar affinity changes occur with WGA and RCA-120. The RCA-120 class of binding affinities appears to follow a single distribution pattern and shows concomitant changes in the median affinity and binding capacity with advancing PDL. The results with the WGA show a complex series of changes involving both affinity class distributions (KJ and numbers of binding capacities ( R J . The most puzzling feature of these findings, as mentioned above, is the apparent level of functional restriction of these oligosaccharides on the cell surface. Given the enormous diversity of structures discussed above, why are there such “tightly” distributed affinity classes? Carver and Brisson (1984) have documented distinct “allowable” states of interaction of the Con A molecule itself, attributed to a specific diversity restriction in the oligosaccharides. These states are defined by the probability of interaction and are related to the activation energy required for interaction at any given conformation. The same type of physical description is possible for the oligosaccharide structures on the cell

88

PAUL L. MANN

surface. If we assume that these experimental data are indicative of a coherent organization, then we can calculate the Gibb’s free energy for the cell surface oligosaccharides in early and late PDL cultures. When this is done a difference of as much as 8-11 kcal exists between the populations, with respect to the Con A binding site class distribution alone, This, in its simplest interpretation, indicates a much higher activation-energy barrier to be overcome for any subsequent reaction in the late PDL population. This prediction, of course, makes several highly speculative assumptions. These include the concepts that there is coherence between the oligosaccharide specificites, that they have some coupling function with other receptor systems, and that the cell surface has the capacity to behave as a functional (coupled) unit. Do these changes in cell-surface oligosaccharide structure have any other experimental correlates besides those described above for cellular senescence? When the IMR-90 cells are plated at different densities a cell concentration-dependent regulation of the cell surface oligosaccharide structures is apparent (Mann et af., 1987b). This provides further support for the potential biological significance of the oligosaccharide structures. The K values, which may have control over a significant amount of potential binding energy, are maintained on the cell surface independent of the number of members in the population as long as the population density is appropriate for growth. However, when the extent of the oligosaccharide affinity class stabilization was investigated it was observed that this was stable only when the plated cell density was 3080% of the total surface area. This is the general surface confluency range which defines optimal growth conditions. Above 80% confluency , contact-induced growth inhibition begins to slow the growth of “normal” cells and below 20-30% surface confluency, cells show extende.d cell cycle times. Figure 4 shows the incorporation of tritiated thymidine into newly synthesized DNA as a function of cell concentration. This is a typical growth curve for normal cells, showing a pronounced lag phase at low-density and high-density inhibition of growth. Table I shows the effect of growth control on the oligosaccharide structures at the cell surface. The oligosaccharides down-regulate at cell concentrations at which high and low density growth inhibition occur. Thus, the observations concerning the oligosaccharide cell-surface structures appear to have functional correlates with general growth control phenomena as well as cellular senescence. These results are best compared with data from experiments with growth-transformed cell lines. In particular, we have used a transplantable canine glioma line (Mann et al., 1988b). The canine glioma cell line was developed by Salcman et af. (1982). This cell line was chosen as a model for this work because of its adherent growth character-

MEMBRANE OLICOSACCHARIDES 6T

89

I

0 0 0

if

. I

X

r a

U

a: 0

I-

I

5

4

6

L O G CELL CONC.

FIG.4. Cell-growth curve for PDL 30 IMR-90 cells. Uptake of tritiated thymidine into newly synthesized DNA as a function of cell number in culture.

istics, and the fact that its size is similar to that of the IMR-90. It shows typical transformed-cell growth characteristics. In general, the number and specificities of the lectins that bind to both normal and growthtransformed cells are similar. Table I1 presents data from the lectin screen on this cell line. The shapes of the simple binding curves are also similar to those of the IMR-90 cells. However, the analytical data, K,, and R,, values are markedly dissimilar. The transformed cells generally lack the TABLE 1 THEEFFECTOF CELLCONCENTRATION ON GROWTH CHARACTERISTICS A N D SURFACE REGULATION Cell concentration ( X IO'/mI) I 5 10

so 100

500 1000

Surface confluency (%)

DNA synthesis"

Surface regulation

< 10

-

Down" Downh Downb UP UP UP Down'

< 10 < 10 20 30 70 >85

t

+ + ?

Uptake of tritiated thymidine into acid-insoluble precipitate as a function of cell number. Combined regulation of Con A , RCA-120, and WGA classes. These are complex and at least partially independent variables. Combined regulation of Con A, RCA-120. and WGA classes. This pattern of general down-regulation is not the same as the low-density pattern. "

90

PAUL L. MANN TABLE I1 LECTINBINDING TO CANINE GLIOMA CELLS“

Lectin

Nominal specificity

OD (405 nM)

WGA Con A RCA- 120 UEA-I SBA PNA DBA ECA

GlcNAc a-d-Man P-d-Gal I-FUC a,P-GalNAc Gal(PI+3)GalNAc P-d-GalNAc Gal(pl+4)GlcNAc

0.77 5 0.008 1.16 t 0.003 2.20 5 0.005 0.55 2 0.02 0.42 t 0.003 0.72 0.002 0.48 t 0.003 1.34 5 0.006

*

“ Canine glioma cells cultured at 1 x 1OS/ml, labeled with lectin at 5 pg/ml for 90 minutes, and the optical density of PNP read after 120 minutes incubation. Note the increased “nonspecific” uptake relative to normal IMR-90 control cells.

two-site configuration and have a K value similar to the low-affinity class of the IMR-90 cells (in the case of Con A binding). For instance, the K for the Con A class is 3000 liters/mol. The last major difference involves the plating properties of the transformed cells. Unlike the regulated cells, the oligosaccharide affinity display of the transformed cells does not appear to be dependent on cell density. Cells can be plated at densities as low as 5 x IO’/ml (
MEMBRANE OLIGOSACCHARIDES

91

of the oligosaccharide surface. The major advantage of our model is that it will be possible to make predictions and test various hypotheses. There are two major types of theoretical analysis for cellular senescence (Aufderheide. 1984): stochastic and programmed senescence. The stochastic theories suggest that senescence is the result of accumulated errors or damage which eventually become lethal. Pereira-Smith and Smith (1983) suggest that immortal growth-transformed cell behavior is recessive to mortal behavior, thus giving the primary role to programmed senescence with stochastic events providing the potential “escape” route. Both general types are probabilistic in nature. It is important to establish some causal relationship between the cell-surface oligosaccharide analysis shown here and the functional biology of the cell. This was done above in the form of correlates. In very recent experiments we made the prediction that if one could modulate the oligosaccharide display on IMR-90 cells at a point in time before the senescence-related phenotype change (PDL 35-37), then it might be possible to affect the kinetics of the onset of the senescence event. We tried a number of regimens which are known to affect carbohydrate synthesis. Swainsonine ( a ,P-indolizidine l-a, 2-a, 8-P triol), an amannosidase I1 inhibitor (Elbein, 1984; Schwarz and Datema, 1984), when used at concentrations which did not significantly affect the proliferation rate of the IMR-90 cells ( I p M ) , caused a substantial delay in the onset of senescence (from PDL 45-47 to PDL 60). The cell-surface oligosaccharide display changes associated with senescence as described above were also proportionally delayed. These studies are now being confirmed and extended to other types of modulation which are potentially more biological in nature. The rationale behind this type of intervention experiment involves a number of assumptions, namely that the oligosaccharide structures referred to here are responsible for growth regulation, that there is an “appropriate” structure, and that this structure is modulated at the cytoplasmic level. A range of modulants are currently being developed which broaden this perspective to include the potential modulation of genetic mechanisms through the use of differentiation agents. This intervention approach must also be tested and verified with the nonregulated neoplastic models. Table 111 shows some preliminary results on the effect of swainsonine in p M concentrations on the generation times of the canine glioma system (Mann et al., 1988b). A cycle of 7 days with the drug and 7 days without, repeated four times, commits the cells to a slowing of growth rate (from 19 to 23 hours for cell-generation times) and an up-regulation of the RCA- 120 binding. These modest results, we hope, indicate the potential utility of the concept. The major point is that with the development of the analytical

92

PAUL L. MANN TABLE 111 THEEFFECTOF SWAINSONINE MODULATION ON CANINE GLIOMA CELLSO

RCA-120 affinity class

K,*

Treatment cycle

T8

0

18.7 2 1.57

6412 k 996

1

18.2 2 2.1

* 1.8

2

25.4

3

22.8 2 3.5

4

22.6

f

1.0

7006 f3074

18660 f I386 17131 f5898 22523 *686

R2 6.36E-M 2 1 .2E-M 6.08E-" f I . IE-M 3 .87E-04 f I .3E-05 1 .79E-M f3SE-O5 2.18E-" f 4 S E -OJ

Tanovar -

-0.255 -6.723 -3.030 -9.456

" Canine glioma cells were treated with swainsonine ( I p M )for I week, then for I week without the drug (treatment cycle), their generation time (T,) and surface oligosaccharide composition analyzed. The RCA-I20 class showed significant (Tand analysis of variance) changes after the second drug cycle. These cells also showed some contact-induced growth inhibition and acted as better targets in cytotoxicity assays (see Mann, el a / . , 1988b).

model, we have the opportunity to study these processes in detail and develop both experimental and theoretical approaches. The results of our study could be viewed as support for either general group of aging theory, but the major significance is that it appears possible to modulate the morphological onset of senescence. Therefore it should be possible to study the major predictions of these and other theories. Regardless of the outcome, this approach then becomes an interactive component between theory and experiment and will ultimately result in a functional understanding of the basic process of senescence. If, as it appears possible, cellular senescence is integrated as a component of more general biological control over growth/differentiation processes, then the results will have even broader significance.

REFERENCES Aizawa, S., and Kurimoto, F. (1979). Exp. Gerontol. 14, 311-321. Aizawa, S., Mitsui, Y . , and Kurimoto, F. (1980a). Exp. Cell Res. 125, 287-296. Aizawa, S., Mitsui, Y . , Kurimoto, F., and Matsuoka, K. (1980b). Exp. Cell Res. 125, 297-303. Aizawa, S., Mitsui, Y . , Kurimoto, F., and Matsuoka, K. (1980~).Mech. Ageing Dev. 13, 297-306. Aizawa, S . , Mitsui, Y . , Kurimoto, F., and Nomura, K. (1980d). Exp. Cell Res. 127, 143-157.

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INTERNAIIONAL REVIEW OF CYTOLOGY. VOL. I12

Endosperm Development in Maize RICHARDv. KOWLESAND RONALDL. PHILLIPS Biology Depmrttnent, Saint Muty’s College, Winonu, Minnesota 55987 and Agronomy and Plant Genetics Depurtment, Uniuersity of Minnesota, S t . Puul, Minnesota 55108

I. Introduction Interest in Zea mays L. (maize) endosperm development is generated by both its importance in agriculture and by the opportunity it affords to study developmental mechanisms. The endosperm of maize is a large storage organ that constitutes 80-90% of the mature kernel dry weight. Approximately 380 billion pounds of endosperm are produced in the United States every year. The mature maize kernel is the result of an integrated developmental process involving both the embryo and the endosperm. Understanding the development of the endosperm as it relates to overall kernel maturation is of great interest. Pertinent information in this regard may eventually lead to useful manipulations to gain increased yields and improved quality. Biotechnological alterations will undoubtedly require a close integration of molecular and cytological information. The objective of this review is to present the genetic and molecular aspects of maize endosperm development. 11. Early Development

Botanical descriptions of kernel development in maize, along with other cereals, are numerous. Early reports about the origin of the endosperm via the double fertilization process and the subsequent events of kernel development were made by True (1893), Sargant (1900), Poindexter (1903), Miller (1919), and Weatherwax (1919, 1923, 1930). More complete accounts of the morphological aspects of the endosperm and its development were reported by Lampe (1931), Randolph (1936), Brink and Cooper (1947a,b), and Kiesselbach (1949). A great amount of detail, concerning the progression of cytological events in the developing endosperm has been provided by Duvick (1951, 1955, 1963). The maize endosperm is generally described as having a triploid origin; however, this condition rapidly changes in much of the tissue following 97 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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fertilization. The initial ploidy level of the endosperm is 3X, because of double fertilization in which two polar nuclei of the central cell of the embryo sac fuse with one of the two sperm nuclei of the pollen grain. Within 2-4 hours, the triploid nucleus undergoes rapid synchronous divisions that ensue for several days, resulting in a syncytial tissue (Fig. 1). Cell walls begin to develop at about 3 days following fertilization. By the fourth or fifth day after fertilization, the endosperm is completely cellular and uninucleate. Both karyokinesis and cytokinesis cease to occur in the central region of the tissue by about 12 days after pollination (dap), while mitotic activity persists for the longest time in the peripheral areas. The outermost region of cells cytologically behaves much like meristematic tissue, generating additional cells by mitosis toward the interior of the endosperm. Cells of the interior region and their nuclei, however, dramatically increase in size during endosperm development. A steady increase in the mean nuclear size can be readily noted, although a large amount of variation in this regard still exists. The development of endosperm tissue in the kernel proceeds at a tremendously fast rate (Fig. 2). The changes in the size of the entire endosperm body that occur between 8 and 12 dap are particularly rapid. The rapid growth at this stage is due to an increase both in cell number and cell size. Thereafter, increases in cell size, not cell number, are observed in the cells of the central region. In the inbred A188 during one growing season, the mean nuclear volume was 53,000 pm’ at 18 dap. This volume constitutes a 34-fold increase compared to the mean nuclear volume at 4 dap (Table I). Size comparisons among endosperm, maize root-tip, and chicken erythrocyte nuclei are illustrated in Fig. 3. The rapidly growing endosperm gradually replaces the nucellus and ultimately compresses any remaining nucellar cells to the outer edge of the kernel cavity. Usually by 12 dap, the endosperm has completely filled the kernel cavity. At about this time, or possibly a little later, the outermost layer of cells of the endosperm differentiates into the aleurone. Much research has been focused upon the unique characteristics of this single layer of cells and its development. Sass (1977) describes the morphology of the endosperm and the aleurone. The haploid antipodal cells of the embryo sac also increase in number by mitotic division concomitantly with early endosperm growth. Approximately 24-48 antipodals can be found making up a loosely formed body of cells, some of which may be multinucleate (Randolph, 1936; Cooper, 1937; Diboll and Larson, 1966). Contrary to earlier assumptions that antipodal cells degenerate during the initial stages of endosperm development, Weatherwax (19261, Randolph (1936), and Cooper (1937) all reported persistence of antipodal cells in the late stages of kernel

FIG. 1 . Synchronous cell division in endosperm during the first 2 days of development following pollination. (Courtesy of S. Hake.)

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FIG. 2. Development of the endosperm as illustrated by kernel cross sections. At 4 dap, the endosperm is the small structure at the base of the kernel while the bulk of the kernel initially consists of nucellar tissue and surrounding carpels; at 8 dap, the endosperm is easily discerned as the body in the lower part of the kernel; during the period of 9-12 dap, the endosperm compresses the nucellus toward the outer edge of the kernel: by 15 dap, the volume of the kernel is mostly endosperm; the embryo can also be observed in the 24-dap kernel.

development in many instances. Brink and Cooper (1944) postulated that antipodal activity is essential for very early endosperm development in Hordeum jubatum x Secale cereale hybrid seeds. Evidence for a similar role in the maize kernel has not been reported. Progressions of cellular changes occurring in the developing endosperm have been clearly described by Duvick (1951, 1955, 1963). Combining his observations with the research of other workers, Duvick was able to present an overall cytological view of endosperm development. Mitotic

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101

TABLE I MEAN VOLUME OF CENTRALLY LOCATED NUCLEIDURING ENDOSPERM DEVELOPMENT Days after pollination

Number of nuclei

Mean nuclear volume (pm ’)

4 6 8 10 12 14

41 47 37 42 39 41 41 29 29 20

1560 1394 2193 2828 22572 29708 41 II6 53394 37765 38376

16

18 20 22

activity stops first in the extreme basal region, and then in the centrally located cells. Thereafter, the cessation of mitotic activity progresses from the silk-scar region of the kernel near the apex downward toward the base and outward toward the periphery. This progression has been described as occurring in a wavelike manner until all cell division, except for some activity in the aleurone, is terminated at about 20-25 dap. Cell enlargement follows a similar pattern; that is, a general advance from the basal cells to the central region and then to the periphery simultaneously with that occurring from the upper region to the base. Duvick reported similar overlapping wavelike patterns for nuclear size increases and for starch grain synthesis. Protein body enlargement was also observed as beginning in the centrally located cells and spreading towards peripheral cells; however, the rate of thier enlargement becomes progressively greater in the cells toward the outer regions of the developing endosperm. This difference in rate of synthesis eventually results in larger protein bodies among the peripheral cells than in the central cells. It appears that most facets of cell differentiation originate in certain regions and gradually spread to other parts of the endosperm. Such waves are interesting, but also perplexing from the standpoint of the mechanisms that control such developmental patterns. 111. Microscopic Characterization of Endosperm Cells

Cross-section preparations of the endosperm show the overall structure consisting of irregularly shaped cells with prominent nuclei and nucleoli.

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FIG. 3. A maize root-tip cell and its nucleus (top) is compared with a 22-dap endosperm nucleus (bottom) against a background of chicken erythrocyte nuclei in which several are marked by arrows (original microscopic magnification, x 200).

Squash preparations and macerations reveal endosperm cells to be extremely fragile throughout their development. The cell walls are very thin, and they are easily disrupted when making standard squashes. Such cytological techniques usually result in microscopic preparations consist-

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16 dap A188 Endosperm

ii

Mean Diameter is 32.1 pm

J

0

3

z

U

0 K W

rn

fz 11-15 16-20 21-25 26-30 31-35 36-40 41-45 46-50

51

DIAMETER OF NUCLEI (pm) FIG. 4. Distribution of nuclear diameters in the endosperm of the A188 inbred at 16 dap.

ing of naked nuclei along with some scattered cell wall fragments and cytoplasmic debris. Occasionally, remnants of cytoplasm will remain attached to the nuclei. Most centrally located cells are in interphase by about 12 dap since mitotic activity has stopped in this region. The cells and their nuclei continue to enlarge, reaching peak sizes at approximately 16 dap. The mean diameter of the nuclei at 16 dap is 32 pm (Fig. 4). A cytological survey of 148 inbred lines at 14 and 18 dap did not show any appreciable differences in nuclear morphology among them (Kowles and LaPorte, unpublished). A few lines, however, did display some mitotic activity in the central regions at 14 dap. Such activity, however, is very infrequent in most of the lines studied. Several other lines appeared to have nuclei with a more condensed chromatin than the usual interphase nuclei. Nonetheless, it appears that different lines generally follow a comparable development with regard to nuclear enlargement and morphology. Cell preparations treated with acridine orange and viewed with fluorescence microscopy simultaneously demonstrate the location of RNA (red) and DNA (yellow/green). These cytological techniques have proven to be ideal for the demonstration of a maximum of three nucleolar organizer regions per nucleus, a reticulum of chromatin strands embedded within the nucleolus, and a cytoplasm containing vast amounts of RNA (Kowles and Springer, unpublished). Observations of centrally located tissue at 12 dap by electron microscopy confirm the thin-walled structure of endosperm cells. The cytoplasm contains a typical array of cellular organelles, inclusions, and membranous structures. No cell

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structure out of the ordinary has been observed in several series of electron micrographs (Kowles and Springer, unpublished). IV. Cellular and Nuclear Activity A. CELLDIVISION

The rapid change in overall endosperm size between 8 and 12 dap is due to both an increase in cell number and, to some extent, nuclear and cell enlargement. Subsequent to this period, changes in the central region of the endosperm are principally due to large increases in nuclear and cell size. Mitotic activity reaches a peak in the developing endosperm between 8 and 10 dap (Phillips et al., 1983, 1985; Kowles and Phillips, 1985). The mitotic index at this peak time is approximately 10% (Table 11). Mitoses are nearly absent in the centrally located endosperm cells when development reaches 12-14 dap in most of the 148 strains and hybrids cytologically studied thus far (Kowles and Laporte, unpublished). A few mitoses can be observed in the extreme peripheral tissue until about 20 dap. Several investigators have calculated the number of nuclei per endosperm at the peak points of development. Reddy and Daynard (1983) reported a maximum of 176,000 nuclei/endosperm at 18 dap for the LGl hybrid. Endosperm from other hybrids were composed of considerably fewer nuclei. Jones et al. (1985) modified the methods of calculating the nuclear number and reported 880,000 nuclei/endosperm from greenhouseTABLE I1 MITOTIC INDICES OF CENTRALLY LOCATED CELLS IN DEVELOPING ENDOSPERM AT 4 TO 24 DAYSAFTER POLLINATION Days after pollination

Total nuclei observed

Number of mitotic nuclei

4 6 8 10 12 14 16

970 445 433 617 429 413 33 1 714 430 352 259

13 22 46 59 4

18

20 22 24

1

0 0 0 0 0

Mitotic index (%) 1.3 4.9 10.6 9.6 0.9 0.2 0.0 0.0 0.0 0.0 0.0

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ENDOSPERM DEVELOPMENT IN MAIZE

grown plants and 720,000 nuclei/endosperm from field-grown plants. The strain used by the latter researchers was a single cross hybrid A619 x W64A. Both research groups showed a general decrease in nuclear number after 20-22 dap. Duvick (1951) had earlier speculated that this decline was probably due to an eventual disintegration of the nuclei.

B. DNA AMPLIFICATION A number of reports since 1950 have characterized the nuclei of the endosperm. Duncan and Ross (1950) provided information on the endosperm with an emphasis upon nuclear size, chromosome knobs, nucleoli, and chromatin strandedness. Increases in DNA content per nucleus were suggested based upon observed increases in nuclear size. Similar observations have been made for maize endosperm grown under in vitro conditions (Straus, 1954). Several reports indicated increased DNA contents ranging from 6C to 384C (Swift, 1950; Punnett, 1953; Tschermak-Woess and Enzenberg-Kunz, 1965). None of these investigations, however, provided definitive information with regard to the extent of DNA changes that take place on a temporal basis during development of the endosperm. Phillips et al. (1983, 1985) and Kowles and Phillips (1985) have described the cytogenetic patterns of maize-endosperm growth and have assessed the changes of DNA levels of endosperm nuclei during development. A dramatic surge in nuclear size and DNA content per nucleus normally occurs at about 10-12 dap following the cessation of mitotic activity (Fig. 5 ) ., Both nuclear size and DNA content peak at 16-18 dap. Nuclear volume and DNA content means at each period of development I

1

30

4

6

8

10 12 14 16 18 20 22

Days Post-Pollination

FIG. 5. The mitotic index (A),DNA content (0). and nuclear volume (0) for centrally located endosperm nuclei in A188 maize.

106

RICHARD V. KOWLES AND RONALD L. PHILLIPS

are significantly correlated ( r = 0.92; p < 0.001). In A188, the strain most intensively studied by cytophotometry thus far, the DNA content per nucleus reaches a maximum mean level of 90-IOOC. DNA content of interphase nuclei measured before the amplification activity average between 4 and 5.6C. At least some of these nuclei are presumably in the S or G , phase of the nuclear cycle, which accounts for the mean DNA content being greater than 3C. Some of these nuclei may have already begun DNA amplification with an absence of cell division. Within any particular endosperm region, DNA amplification appears to occur among nuclei with little synchrony (Kowles, unpublished). Figure 6 illustrates the distribution of nuclear DNA content in C values for a group of endosperm samples taken at various developmental stages. The distribution does not show much clustering of values, nor does it reveal any pronounced peaks. DNA content measurements of individual nuclei for particular postpollination periods show similar nonclustered dispersions. These results are based upon a small number of measurements, however, and larger studies should be more informative. The overall rate of DNA amplification per nucleus during the 10- to 16-dap period of endosperm development in the A188 strain is striking. Assuming that repeated rounds of DNA replication result in an exponential increase, each round of replication requires about 22-24 hours. The last round of replication, including the gap period during this period of amplification, therefore requires an overall linear DNA synthesis of 916 pm/second/nucleus. This rate of synthesis, in turn, requires an overall incorporation of 2.75 X lo6 base pairs/second/nucleus. DNA content has also been measured in dividing nuclei at all stages of 5

192c -

tt

7

d 6

z5 u 4 0

a 3 : 2

I

2 1

1

= o DNA CONTENT/NUCLEUS (C values)

FIG. 6. Distribution of centrally located endosperm nuclei from all postpollination periods grouped according to their DNA content in C values.

107

ENDOSPERM DEVELOPMENT IN MAIZE

mitosis and in interphase within different regions of the endosperm; that is, the peripheral and central regions, and in tissue between these two regions (Kowles and Ploense, unpublished). No statistically significant differences in DNA content were noted among these regions relative to nuclei measured in prophase and metaphase. Nonsignificant differences were also found for nuclear figures in anaphase and telophase among these regions. As expected, interphase nuclei showed a highly significant difference for nuclear DNA content among different endosperm regions (Table 111). This statistically significant deviation among regions occurs even though a great amount of heterogeneity exists among the interphase nuclei within each region. The ratio of DNA per nucleus between prophase-plus-metaphase figures compared to anaphase-plus-telophase figures was 2.28 : 1. This ratio deviates slightly from that expected under these conditions, that is, 2.0: 1. Whether some nuclei undergo mitotic activity following a certain degree of DNA amplification has not been established by definitive tests. Uptake experiments using tritiated thymidine in endosperm tissue later than 12 dap reveal centrally located nuclei with silver grains directly over the chromatin. In the same autoradiographs, some nuclei are relatively free of silver grains. Assuming that these unlabeled cells are viable, one TABLE 111 DNA CONTENT/NUCLEUS COMPARED AMONG PERIPHERAL, CENTRAL, AND INTERMEDIATE REGIONS OF THE ENDOSPERM Number of nuclei measured

Mean DNA/nucleus in arbitrary units

Interphase Peripheral Intermediate Central

9 12 24

1949 2019 41 I 8

6.41"

Prophase /metaphase Peripheral Intermediate Central

21 II 20

1783 1942 2007

0.34

Ariaphase/ telophase Peripheral Intermediate Central

18 16 6

77 I 929 177

2.30

Nuclear stage by position in the endosperm

"

0.01 level of significance.

Calculated F value

108

RICHARD V. KOWLES AND RONALD L. PHILLIPS

can conclude the existence of a G stage. Taken together, cytological observations and uptake experiments indicate a cell cycle devoid of a mitotic stage, but with synthesis and gap stages. Observations of the large DNA increases per nucleus prompt consideration of genomic balance and the ploidy barrier within endosperm tissue. Various ploidy imbalances have been suggested for endosperm failure in kernel development, such as a deviation from a 2 : 3 : 2 ratio of maternal tissue, endosperm, and embryo, respectively (Muntzing, 1930). Another explanation has attributed endosperm failure to a deviation from a 3 : 2 ratio of endosperm to embryo (Watkin, 1932). Nishiyama and Inomata (1966) inferred that a 2 : I ratio between the maternal and the paternal genomes might be essential. Sarkar and Coe (1971) proposed that the endosperm needs to be either triploid or triploid multiples for normal development. Lin (1984) used an indeterminate gametophyte mutant (is) to gain various ploidy level combinations. He concluded that endosperm failure was due to factors within the endosperm tissue itself, rather than embryo or maternal tissue factors. Lin also concluded that the 2 : I ratio of maternal to paternal chromosomes may be important. Assuming that the maternal and paternal genomes of the inital triploid nucleus undergo DNA amplification at the same rate, the DNA increases first reported by Phillips et al. (1983) are not counter to a genomic balance requirement suggested by Nishiyama and Inomata (1966) and Lin (1984). C. NUCLEAR HETEROGENEITY The nuclei of the endosperm cells are of uniform size only during the very early stages of development. As the endosperm develops, the nuclei become increasingly variable in size. Distribution patterns of nuclear size for different periods following pollination are displayed in Fig. 7. Distribution patterns of DNA content per nucleus in the inbred A188 for different periods after pollination follow a similar pattern (Fig. 8). Some nuclei at 16 and 18 dap have DNA levels surpassing 200C. The largest nucleus measured so far was 690C. The distributions in Fig. 8 do not show any of the extremely large nuclei at the earlier dates after pollination. These observations further support the temporal nature of the DNA amplification events; nevertheless, the ultimate result is a tissue evidently characterized by a high level of variation. Without doubt, the endosperm is not a uniform mass of cells. Wilson (1978) has also pointed out that the endosperm does not develop uniformly. He notes that the tissue differs from top to bottom during development and also at maturity. Starch synthesis, protein body forma-

I09

ENDOSPERM DEVELOPMENT IN MAIZE

100 50 1

bp

100

u

50

n

50

Y

days PP

-

8

-

1

1

8

I

I

I

I

I

I

0 .-; 100

c

.o

c

.--c 0

100 50

100

50

L

16

i

22

Nuclear Volume (x

l o 4 pm3)

FIG. 7. Distribution patterns of nuclear volume of centrally located endosperm nuclei in A188 for each period from 8 to 22 dap.

tion, and carbon products from photosynthesis begin near the apex and proceed downward during the kernel-filling stages. Variation extends further than that noted from one region of the endosperm to another; that is, a high degree of heterogeneity also exists within very small areas of the tissue. In many instances, fluorescence cytophotometry measurement of DNA content has shown much greater than 2-fold differences among nuclei all taken from the central region of the endosperm. These results cannot be due simply to the measurement of nuclei at different points within the S stage of the cell cycle. Extensive heterogeneity of nuclear DNA content can also be demonstrated statistically with the coefficient of variation (CV); that is, standard deviation/ mean x 100%. The average CV for DNA content of centrally located endosperm nuclei for early developmental periods in 17 different strains is 61.6. The average CV for these same strains at the later periods of development is 69.5 . A large amount of heterogeneity seems to exist

110

RICHARD V. KOWLES AND RONALD L. PHILLIPS

0

50

100

150

200

250

DNA/Nucleus (C) FIG. 8. Distribution patterns of DNA content of centrally located endosperm nuclei in A188 for each period from 8 to 22 dap.

throughout the DNA amplification period of endosperm development. As a comparison, the same cytophotometric techniques yield an average CV of only 25.1 for four different tests of DNA content in root-tip mitotic figures. The average for a series of CV tests with Galfus erythrocytes (supposedly all 2C) is only 7.1 (Kowles, unpublished). Generally, a gradient of sizes can be noted from the smaller, more peripheral, nuclei to the larger centrally located nuclei. Still, some regions within the endosperm are seen to have very large nuclei in juxtaposition to the smaller peripheral nuclei. This is especially the case in the region contiguous to the embryo. Serial sections of about 60-75 pm in thickness have been obtained with a cryostat microtome, and the technique has allowed for analyses of nuclear heterogeneity (Kowles, unpublished). All of the nuclei in an entire cross-section can be tracked and measured for DNA content. Again, this sectioning method coupled with Feulgen cytophotometry reveals rather striking nuclear differences within relatively small regions. The term "nucleotype" has been used to describe plant nuclei (Bennett, 1973). The definition refers to the condition of the nucleus, which may affect the phenotype independently of the informational

ENDOSPERM DEVELOPMENT IN MAIZE

111

content of the DNA. In other words, not only do plant nuclei differ with regard to their genetic activity, they also vary in their physical properties, including size and mass. Bennett further notes that, in general, a negative correlation exists between the number of genomes and the life expectancy of cells. The high DNA content of maize endosperm nuclei and the ephemeral nature of this tissue certainly support that correlation. In addition to differences in size and mass, cytological observations of endosperm nuclei reveal other differences relative to their general morphology and degree of chromatin condensation. Different nucleotypes are quite evident in this tissue. Variation in gene expression within the developing endosperm is also of paramount interest. Chourey et al. (1986) have reported the possible presence of two distinct cell types in the developing endosperm with regard to sucrose synthetase expression. Two different sucrose synthetase enzymes are synthesized in maize tissue; these enzymes are called SS-1 and SS-2. Chourey et al. presented evidence that both SS-1 and SS-2 are present in endosperm tissue and in seedling shoots. Both enzymes are tetramers and are similar to each other such that random polymerization occurs between the polypeptides of the two enzymes, forming isozymes. In seedling shoots, random polymerization into tetramers results in five different isozymes, as expected. In the endosperm, however, only the homomers of the individual SS-1 and SS-2 types are identified that is, heteropolymers are not found. The SS-1 and SS-2 enzymes appear to be separated as the endosperm tissue develops, either spatially or temporally. Studies of variation among endosperm cells at the level of gene expression is barely beginning. D. ABERRANT CHROMOSOME BEHAVIOR I N THE ENDOSPERM Collins (1913) noted that aleurone color will sometimes display a mosaic appearance, that is, colored spots varying in size from nearly the entire kernel surface to as little as the dimensions of a single cell. A mosaic texture of the endosperm was also observed, but on a much rarer basis. Interestingly, some of the mosaic endosperm kernels for texture (waxy) were also mosaic for aleurone color with the two characters exactly coincidental. East (191 3) also found occasional kernels that showed a half-and-half condition relative to color. Several explanations were suggested to account for the irregularly mosaic kernels described by Collins and the half-and-half kernels by East. Emerson ( 1915) offered arguments which reduced the possibilities to either the occurrence of somatic mutation or the somatic segregation of characters in the heterozygous condition. Later, Emerson (1918) noted

112

RICHARD V . KOWLES AND RONALD L. PHILLIPS

that such anomalous endosperm development occurred mostly when the aleurone character in question was known to be heterozygous and then only when the dominant factor enters the cross from the male parent and the recessive allele from the female parent. For example, an anomalously colored aleurone would occasionally occur from the aaA condition, but not the AAa or M A conditions. Based upon genetic evidence, Emerson (1921) concluded that such aberrant kernels were possibly due to chromosome nondisjunction. Emerson set up crosses in which the female parent contributed linked recessive aleurone and endosperm characters, and the male parent provided the linked corresponding dominant character in each case; for example, in some tests Emerson used the characters C and c (colored and noncolored) and Wx and wx (waxy and nonwaxy). It was shown that when aberrant kernels occurred, the recessive aleurone character was almost always underlaid by the recessive endosperm character. Since the two characters are genetically linked, the breeding tests strongly supported aberrant chromosome behavior rather than somatic mutation, which would require two rare events occurring almost simultaneously. Weatherwax (1923) reported that two types of nuclear division occurred in the developing endosperm of maize, which he called meitotic division (mitotic) and direct division (amitotic). He claimed that the first type occurs in the early stages of endosperm development, and that the second type occurs in the later developmental stages. Direct division, or amitosis, is taken to mean that the chromatin does not undergo chromosome condensation; rather, the interphase nucleus simply divides into two nuclei by mass division. Fisk (1927), however, disputed many of these conclusions, pointing out that no cytological evidence had been offered to support the contentions of Weatherwax. Fisk did not observe any evidence of amitosis; instead, he found the nuclei divided in a typically mitotic manner. In addition, our own extensive cytological work with maize endosperm has not revealed any indications of amitotic division. Fisk also could not find any direct evidence of nondisjunction to support Emerson’s genetic data indicating aberrant chromosome behavior. This is not surprising, however, considering the infrequency of nondisjunction and the difficulty in cytologically discerning the event. Recessive gene expression in heterozygous endosperm tissue was also studied by Jones (1935). The frequency of mosaic events in kernel development varied greatly, dependent upon the family. At first, Jones attributed endosperm mosaicism more to deletions than nondisjunction, because he often observed the separation of linked genes. Subsequent studies, however, led Jones (1936a, 1937a,b) to include nondisjunction (somatic segregation) as a possibility. This latter conclusion was based

ENDOSPERM DEVELOPMENT IN MAIZE

113

upon the occurrence of twin spots, which could be explained by a different number of alleles in adjacent cells having visible effects. The atypical growths were considered to be the result of either a removal or a concentration of certain substances in cells which, in turn, was brought about by unequal mitoses. Although somatic segregation was deemed to be the more common occurrence underlying changes in endosperm expression, Jones also implicated mutation (1936b), random chromosome translocations (1938), and somatic crossover (1940) as playing roles. The high frequency in which these spontaneous changes were observed is very striking in some of the work by Jones (1941). Jones attributed a more numerous occurrence of chromosomal aberrations at mitotic division in the endosperm as a possible explanation. In 1944, Jones reported additional evidence for the breakage and relocation of chromosomal parts in endosperm cells. Through the series of studies by Jones and the earlier workers, evidence was collected that supported anomalous endosperm growth due to nondisjunction, deletions, somatic mutation, and translocations.

E. CLONAL DEVELOPMENT Phenotypic modifications occurring in tissues have been helpful in understanding their mode of development. Such studies are based upon the rationale that each well-defined tissue sector represents a clone of cells, all of which are derived from a single modified cell. These modifications occur spontaneously, but they can also be induced in order to facilitate such studies. Clonal technique has been used with great advantage by Steffensen (l968), who employed X-irradiation to investigate the modes of development of the maize plant. Coe and Neuffer (1978) and Johri and Coe (1983) used similar methods to determine the destinies of embryo cells as they proliferate into the plant body. McClintock (1951, 1965, 1978) analyzed clones of modified cells in the maize endosperm to reveal certain patterns of its development. In some of her classic work concerning endosperm development, McClintock used a method that included the alleles nonwaxy (Wx) and waxy ( w x ) and the activator (Ac) regulatory system. Wx is the dominant allele that allows for the production of amylose, resulting in a dark blue color when I-KI solution is applied. When homozygous, the recessive wx allele produces an endosperm having only amylopectin rather than additional amylose. Instead of a blue color, amylopectin takes on the red-brown color of the I-KI solution which, in turn, can be further removed with hot water. In this way, McClintock could differentiate the active cells ( W x ) from the inactive cells (wx) relative to amylose produc-

114

RICHARD V. KOWLES AND RONALD L. PHILLIPS

tion. The action of the Wx gene conveniently came under the control of the Ac system which is capable of modifying Wx expression during endosperm development. The clones resulting from wx changing to the Wx expression were made evident in the mature endosperm with the application of the I-KI staining solution. McClintock used these methods to interpret the origins of cells in the development of the endosperm. In general, the initial cell divisions establish left and right halves of the kernel. Subsequent cell proliferation occurs in a spherical manner resulting in a pattern of cones and V-like sectors. Although the basic theme of endosperm development can be illustrated in this way, McClintock has cautioned that many variations on this theme have been encountered and need further study.

V. Evidence for Endoreduplication

A. CYTOLOGICAL ASPECTS DNA increases in the nuclei of developing endosperm prompt a consideration of the mode by which this increase occurs. The possibilities include polyploidy (an increase in sets of chromosomes), nuclear fusion, or endoreduplication (polyteny). Polytene chromosome structures presumably arise as a result of polyploidization without interruption by chromatin condensation. Polytenization may take the form of underreplication which includes most, but not all, of the genome; on the other hand, preferential amplification may take place in which only a relatively small part of the genome is increased Many examples of variation in nuclear DNA content in plant cells have been reported. Several comprehensive reviews of endopolyploidy and polytenization in plants are available (Nagl, 1978, 1982; D’Amato, 1984; Nagl et al., 1985; Brodsky and Uryvaeva, 1985). Duncan and Ross (1950) reported the occurrence of endomitotic events leading to polyteny. They based their conclusion upon observations of a maximum of three nucleoli, a constant heterochromatic knob number, approximately 30 chromosome-like bodies in older endosperm tissue, and a large amount of multistrandedness. The cytological studies of Phillips et al. (1983, 1985) and Kowles and Phillips (1985) concur with those of Duncan and Ross. In addition, three nucleolar organizer regions (NOR) are always present in nuclei having only one large nucleolus. Further support is provided by in situ hybridization in which a 17/26 S rRNA probe was hybridized to endosperm nuclei (Wang and Phillips, unpublished; Kowles and Phillips, 1985). Figure 9A shows the typical three

ENDOSPERM DEVELOPMENT IN MAIZE

1 I5

clusters of silver grains associated with the nucleolus, each of the clusters over an NOR. Figure 9B displays the results of in situ hybridization (Kowles, unpublished) using an RNA probe generated from a 185-base pair repeated DNA segment located in knob heterochromatin (Peacock et al., 1981; Dennis and Peacock, 1984). Hybridization of this probe with the inbred A188 usually shows 12 substantial silver grain clusters over the nucelus. Such results are in agreement with expectations since A188 has four knobs of significant size and the tissue is mostly triploid, although highly polytenized, after about 12 dap. Once again, it needs to be pointed out that the endosperm tissue is not homogeneous. Several reports have indicated the occurrence of polyploidy in endosperm nuclei. Punnett (1953) observed some hexaploid cells in 8-dap endosperm from greenhouse-grown plants. Stephen (1973) provided data that revealed 17% of the endosperm nuclei to be hexaploid in tissue of 9-12 dap. He also suggested that some nuclei may have been of a higher ploidy level. Lin (1975, 1977) has shown that 95% of the endosperm nuclei are triploid in 6- to 10-dap tissue. The other 5% of the nuclei were mostly hexaploid, but a few were also 9X or 12X. Lin's fine photomicrographs clearly provide evidence that the endosperm tissue is not uniformly triploid. Kowles and Phillips (1985) have also observed occasional polyploid nuclei. Polyploidy beyond the triploid condition seems to arise in a few nuclei, especially in the younger, more actively dividing, endosperm tissue.

B. PROPORTION OF GENOMEAMPLIFIED A number of tests have been conducted in efforts to determine whether preferential amplification or underreplication occurs as a result of the DNA increase in nuclei of the developing endosperm (Phillips et al., 1985; Kowles et al., 1986). Slot-blot nitrocellulose hybridization assays have been carried out with DNA extracted from total endosperm tissue over a range of postpollination dates with several "P-labeled DNA probes nick translated by the technique of Rigby et al., (1977), specifically, 17/26 S rDNA, zein. waxy, sucrose synthetase, and total DNA from 2n leaf tissue. The results obtained from these tests with the 17/26 S rDNA and the 2n-leaf DNA probes are presented in Fig. 10. Nitrocellulose hybridization differences could not be detected among the varying stages of development with any of the probes used thus far, at least within the level of resolution of this technique. Another analysis, devised by Roninson (1983), was used in continued attempts to detect whether differential DNA amplification occurs in the endosperm. The experimental approach of the Roninson assay is outlined

116

RICHARD V . KOWLES AND RONALD L. PHILLIPS

FIG. 9. (A) In situ hybridization of labeled ribosomal RNA to an endosperm nucleus. The three clusters of silver grains denoted by arrows are associated with the lightly stained nucleolus (original microscopic magnification, x 150). (B) In situ hybridization of an endosperm nucleus with labeled RNA generated from a 185-bp repeated DNA segment located in knob heterochromatin. The dark areas are dense clusters of silver grains indicative of a large amount of hybridization (original microscopic magnification, X250).

in Fig. 11A. A DNA segment needs to be repeated at least 50 times per genomic equivalent to rehybridize under the conditions of the technique. The resulting bands following renaturation in the gel consist of repeated or amplified DNA segments of specific length relative to the particular restriction enzyme used in the test. An advantage of the Roninson assay is that repetitive DNA sequences dispersed in the genome cannot hybridize with each other, because they most likely occur on differently sized restriction fragments. An autoradiograph of a gel is presented in Fig. 11B which compares 2n-leaf DNA with 17-dap total endosperm DNA digested with Hind111 (Phillips et al., 1985). The 2n-leaf DNA had a higher specific activity than the 17-dap endosperm DNA, causing the difference in overall intensity. In this regard, it can be noted that the relative differences in the intensities of bands between the two lanes are similar throughout the gel. In addition, no bands appear in the endosperm DNA that are not present in the 2n-leaf DNA. Differential DNA amplification is not detected in this experiment; however, the resolution of the test is not

ENDOSPERM DEVELOPMENT IN MAIZE

117

FIG. 9. (continued)

definitive enough to completely eliminate the possibility of low-level amplification of some DNA sequences. Still another test has been performed to determine whether heterochromatin and euchromatin amplify proportionately. Fluorescence cytophotometry was employed with the fluorochromes Hoechst-33342, chromomycin A3, and propidium iodide. Hoechst-33342 is AT-rich DNA sensitive, chromomycin A3 is GC-rich DNA sensitive, and propidium iodide is not differentially sensitive to either AT-rich or GC-rich DNA. Heterochromatin in maize consists of AT-rich DNA segments; this is also supported by fluorescence techniques. The application of quinacrine followed by fluorescence microscopy results in Q-bright heterochromatin

118

A

RICHARD V. KOWLES AND RONALD L. PHILLIPS

Probe 9kb rDNA

5.0

1.0

0.2 ug.

2N D17

C. T. FIG. 10. (A) DNA slot-blot hybridization in which DNA extractions from leaves ( 2 n ) , endosperm DNA at 17 dap, and calf thymus were probed with a 9-kb rDNA probe representing the 17/26 S ribosomal RNA genes in maize. The DNA probe was made radioactive by I2Pnick translation. DNA amounts applied to the slots were 5.0, I .O, and 0.2 pg with replicates for each type of DNA; (B) DNA slot-blot hybridization in which DNA extractions from leaves (2n) and endosperm at different stages of development ( 1 I , 17, and 23 dap) were probed with 2n DNA from the entire maize genome. The 2n DNA was made radioactive by '*P nick translation. DNA amounts applied to the slots were 1 .O, 0.2, and 0.04 pg with replicates for each type of DNA. [Fig. 10B is from Kowles et al. (1986). Used with permission.].

and Q-dull euchromatin (Kowles and Springer, unpublished). Q-bright regions, in turn, tend to consist of AT-rich DNA segments (Jalal et al., 1974). In this analysis, fluorescence was measured from nuclei selected for specific sizes. Table IV presents data which show no significant difference between small and large nuclei relative to the mean fluorescence per unit of nuclear area (Kowles and Ploense, unpublished). The same observation holds true for three different tests with Hoechst-33342 (AT sensitive) and three different tests with chromomycin A3 (GC sensitive). Only the propidium iodide test showed a significant difference between nuclei of different sizes; however, the 1-test probability in this case was only slightly below the 5% level of significance. Since a strong positive correlation exists between the mean volume and the mean DNA

ENDOSPERM DEVELOPMENT IN MAIZE

B

119

Probe 2N DNA 1.0

0.2

.04 ug.

215

Dl1 017

023 FIG. 10. (continued)

content of nuclei for each postpollination period, it is tempting to conclude that both heterochromatin and euchromatin of endosperm nuclei are being amplified. If any preferential amplification or underreplication is taking place in the developing endosperm, it must not be very widespread across the genome nor at a very high level. Such conclusions, however, are offered tentatively. The experiments such as slot-blot hybridizations and Roninson assays previously described all rely upon the situation in which total DNA is extracted from bulk endosperm tissue. The possibility that a certain proportion of nuclei behaves in a different manner during development cannot be excluded without additional tests. Subtle differences

120

RICHARD V. KOWLES AND RONALD L. PHILLIPS

I . Digest genomic DNA with a restriction enzyme that generates a 5’ overhang 2. Remove about 100 bases in 3’ direction with Exo 111 3. Fill in with Klenow, 32P-dXTP to original size 4. Mix labeled probe with unlabeled genomic DNA digested with the same restriction enzyme 5 . Electrophorese on an agarose gel 6. Denature DNA in the gel 7. Renature the DNA in the gel 8. Digest single-stranded DNA with SI 9. Repeat the previous three steps 10. Dry gel and expose for autoradiograph y

FIG. I I . Roninson analysis for detecting preferential amplification of unknown repeated sequences. (A) Experimental method, (B) Comparison of 2n leaf DNA and endosperm DNA at 17 dap.

TABLE IV FLUORESCENCE CYTOPHOTOMETRIC RESULTSWITH THREEFLUOROCHROMES FOR NUCLEIOF DIFFERENT SIZES

Experiment 1

2 3 4

Endosperm tissue daP

Area of nucleus (pm')

13 13 16 16 16 16 14 14

I23 314 314 I964 314 1964 123 804 314 1964 314 1964 123 3 14

5

16

6

16 16 16

7

13

13 Significant at the 5% level.

Fluorochrome Hoechst-33342 Hoechst-33342 Hoechst-33342 Chromomycin A3 Chromomycin A3 Chromomycin A3 Propidium iodide

N

Mean fluorescence/unit area

Standard error

59 60 43 25 20 50 16 5 30 10 I5 30 60 60

22.5 21.7 56.7 64.8 54.0 54.8 53.4 62.0 13.3 11.1 8.6 8.5 29.4 22.8

1.03 0.69 2.98 4.39 3.79 2.57 4.60 7.02 0.87 0.95 1.oo 0.47 2.41 1.69

t Value

1.245 1.554 0.170 0.895 1.359 0.165 2.247"

122

RICHARD V. KOWLES AND RONALD L. PHILLIPS

among individual nuclei could be obscured in analyses that use total DNA extracts. It seems sensible to consider this possibility and to further test it in view of the vast amount of heterogeneity observed in these nuclei. In situ hybridization is a means by which differential DNA amplification might be tested with greater resolution. Such experiments would be especially useful if DNA content and specific gene numbers could be determined and correlated for each individual nucleus. Many DNA to DNA and RNA to DNA in situ hybridization experiments have been conducted with a variety of probes applied over a range of postpollination dates. As previously discussed, RNA probes generated from 17/26 S rDNA and a 185-bp repeated DNA segment from knob heterochromatin cause distinct silver grain clusters over endosperm nuclei (Kowles and Phillips, 1985; Kowles et al., 1986; Kowles and Lorang, unpublished). These results are indicative of a 3X chromosome number. In sitii hybridization used in conjunction with cytophotometry may ultimately give definitive information concerning nuclear structure within the endosperm.

VI. Differences in Nuclear DNA Content among Strains A. INBREDS

AND

ENDOSPERM MUTANTS

Mean DNA content per centrally located nucleus during endosperm development has been determined in a number of different strains, including inbreds, endosperm mutants in various backgrounds, and F, crosses (Kowles and Phillips, 1985; Phillips et al., 1985). In all of the strains studied, statistically significant differences resulted for the mean DNA levels among different days after pollination, in spite of the large variation of DNA content among the nuclei within each postpollination period. Table V lists the maximum mean DNA levels per nucleus for a selected group of inbreds and endosperm mutants in various backgrounds. The overall patterns of DNA amplification during development among the strains are comparable, differing only in the timing of the large DNA increases and the peak levels of DNA ultimately attained. All of the strains studied thus far have an initially low DNA level, rapidly increasing levels during development to a peak around 14-18 dap, followed by a gradual reduction in nuclear DNA levels. A large range exists for the peak levels of DNA among strains. An F test of the peak DNA levels among all strains shows highly significant differences. Other measurements indicate that the level of DNA amplification may be independent of the 2C level of DNA in the strain. Table VI groups data

ENDOSPERM DEVELOPMENT IN MAIZE

123

TABLE V M A X I M U MEAN-DNA M LEVELS/NUCLEUS REACHED DURING ENDOSPERM DEVELOPMENT FOR I2 STRAINS (EXPRESSED AS C VALUES) Strains

dap

A188 B37 L289 Wilbur's Knobless Flint Zapalote Chico Black Mexican Sweet Endosperm mutants A 188 o p a y ~ e - 2 A619 opuqrre-2 837 opaque-2 837 j l ~ u r y - 2 B37 waxv B37 ~ u g a r y - 2

14 19

Peak DNA level

15

17 19 16 13 18

17 19 19 19

93 2 141 2 155 2 I47 2 112 2 147 ?

2

SE

9 18

17 II 10

22

114 2 24 153 2 25 178 2 19 162 t 16 134 +. 14 802 9

of peak DNA levels in the endosperm with the DNA levels found in 2C anaphase figures for the same strains. The latter data were obtained by Feulgen cytophotometric measurements of root-tip mitotic cells (Kowles and Ploense, unpublished). These data show a correlation of -0.61, although the corresponding probability is only 20%. The 2C DNA levels are very similar to those obtained by other workers (Laurie and Bennett, 1985; Baer and Schrader, 1985a). Differences in 2C DNA values among strains of maize is not unusual. Laurie and Bennett reported a 37% difference between the strains with the lowest and highest 2C DNA levels TABLE V1 PEAK LEVELSOF MEANDNAKENTRALLY LOCATED ENDOSPERM NUCLEUS A N D MEAN2c DNA LEVELS I N ROOTTIPSOF T H E SAMESTRAINS"

Strain A188 Zapalote Chico B37 Wilbur's Knobless Flint Black Mexican Sweet

DNA/endosperrn nucleus (pg)

DNA/ZC anaphase

230.9 276.0 349.7 363. I 366.3

6.20 6.50 6.46 5.30 5.23

(Pi$

'' D N A measurements are based upon 2.62 pg of DNA/chicken erythrocyte as an internal reference.

124

RICHARD V. KOWLES AND RONALD L. PHILLIPS

as measured with a scanning microdensitometer. Baer and Schrader (1985a) measured nuclei from young fully expanded leaves with flow cytometry and found the variation to be about 15% among the strains in their studies. The data reported in Table VI amount to a 24% variation between the low (5.23 pg) and high (6.50 pg) DNA-containing strains. In most cases, the endosperm mutation strains measured thus far possess a normal DNA content per nucleus. Preliminary data indicated that sugary-2 may have lower DNA peak levels (Kowles, unpublished; Glover, personal communication). This latter endosperm trait also reduces seed size (Glover et al., 1975). To the contrary, some of the strains with an endosperm mutation contained nuclei with higher mean DNA contents. For example, opaque-2 in a B37 background, opaque-2 in an A619 background, andfloury-2 in a B37 background averaged peak DNA levels of 178C, 153C, and 162C, respectively. These DNA levels are almost twice that of A188, the line initially and most intensively studied. The A188 strain may prove to be one of the lowest in this regard. B. F, Crosses Limited investigation of nuclear DNA content in the endosperm of F,, F,, and reciprocal crosses has been carried out thus far. The peak DNA level resulting from the F, cross of Zapalote Chico and Wilbur’s Knobless Flint was similar to the lower of the two parents; that is, Zapalote Chico. From several other F, reciprocal crosses, the endosperm had lower DNA levels per nucleus than either of the parents. Neither heterosis nor dosage effect appears to exist relative to DNA content per endosperm nucleus, at least in these few preliminary F, tests. Groszmann and Sprague (1948), however, reported marked heterosis for endosperm weight in one direction of a reciprocal cross, but not in the other direction. They attributed these results to the action of specific genes rather than chromosomal dosage effects. Baer and Schrader (1985b) reported little or no heterosis in DNA concentration of leaf blades (and relative cell volume). As a result of their breeding experiments, they concluded that DNA concentration in this tissue is due to polygenic inheritance. In contrast, Palacios (1982) showed heterotic effects of nuclear DNA content in root-tip meristematic tissue when perennial teosinte and maize (Gaspe) were crossed. In this study, teosinte was reported to have 31.6 arbitrary units (AU) of DNA, maize 23.2 AU units and the F, hybrid 41.4 AU. Interesting results also have been reported by Chandlee and Scandalios (1981) relative to gene expression. They have concluded that only maternal genes express prior to 6-8 dap, at least for the enzymes investigated in their study. More studies of this kind need to be applied to the maize endosperm before

ENDOSPERM DEVELOPMENT IN MAIZE

125

sound conclusions can be made about hybrids and nuclear DNA content in the tissue. C. DEFECTIVE KERNEL MUTANTS Defective kernel mutants (dek)in maize have long been recognized and studied from genetic standpoints (Jones, 1920; Demerec, 1923; Mangelsdorf, 1926; Eyster, 1931; Brink and Cooper, 1947a,b). Lowe and Nelson (1946) also researched the development of a similar defective kernel called miniature seed. Phenotypically, these kernels have a reduced endosperm which, in most cases, is due to a single recessive gene. In a few cases the endosperm appears normal, but the kernel does not have an embryo (called germless). Recent investigations of dek mutants have been reported by Manzocchi et al. (1980a,b), Neuffer and Sheridan (1980), Sheridan and Neuffer (1980), and Clark and Sheridan (1986). In a large study, Sheridan and Neuffer induced 855 recessive dek mutants by ethylmethane sulfonate treatment. Emerging from both the earlier reports and the recent studies is information that four types of dek mutants exist: ( I ) those that affect the endosperm and the embryo resulting in a nonviable embryo, (2) those that affect the endosperm and the embryo resulting in a viable embryo, (3) those that affect only the endosperm, and (4) those that affect only the embryo. Several of the dek mutants generated by Sheridan and Neuffer have been cytologically investigated (Kowles et al., 1986; Kowles and McMullen, unpublished). Most of these mutants have relatively small nuclei at 14-18 dap compared with normal kernels on segregating ears. Only 2 of 12 dek mutant lines studied thus far have comparable DNA content per nucleus with those of normal kernels at 16-18 dap. The other 10 dek mutants have from 1.4- to 13.8-fold less DNA per nucleus than the normal kernels on segregating ears. In addition, cytological preparations show dek cells with a much reduced level of storage products, especially starch granules. These preliminary studies, however, do not give information relative to cause and effect. Still, the dek mutants could prove extremely useful in addressing fundamental questions about endosperm development (Carlson and Rice, 1974; Scandalios, 1982; Kowles et al., 1986).

VII. Biological Significance of DNA Amplification in the Endosperm An association exists between DNA amplification and endosperm development, although at this time it is difficult to be more definitive

126

RICHARD V. KOWLES AND RONALD L. PHILLIPS

4

6

8

10 12 14 16 18 20 22 24

DAY8 POST-POLLINATION

FIG. 12. DNA content per nucleus for centrally located A188 endosperm tissue over four consecutive growing seasons: 1980 (O), 1981 (m), 1982 ( O ) ,and 1983 (A).A slightly earlier increase and peak in DNA content occurred in 1983 than in the other three growing seasons, all of which began rapid DNA increases at about 10 dap.

about the nature of that association. Supporting this contention, nonetheless, are data presented in Fig. 12 that show DNA amplification patterns to be very similar over four consecutive growing seasons (Kowles and Phillips, 1985). Further support is offered in Fig. 13, in which the general patterns for DNA amplification are comparable among different strains (Kowles et al., 1986). As previously discussed, the timing of the DNA increases will vary slightly, and the maximum peak levels of DNA per nucleus show a large range. Still, the consistent patterns observed regardless of varying environmental conditions and genetic backgrounds are noteworthy. Several roles could possibly exist for the marked DNA amplification in the endosperm. One possibility already considered is the presence of a DNA salvage pathway, that is, the production of storage DNA for subsequent degradation into nucleosides (or nucleotides) and transport to the developing embryo. Figure 14 shows that measurements of DNA per nucleus during endosperm development among 18 different lines average a reduction of DNA beginning at about 18-20 dap (Kowles and Phillips, 1985; Phillips ef al., 1985). The timing of this DNA reduction coincides well with the period in which the embryo begins an extensive enlargement (Randolph, 1936). A preliminary experiment failed to show a DNA salvage and mobilization system between 10 and 22 dap (Kowles et al., 1986). An in vitro ear culture system was used to carry out a ['Hlthy-

I27

ENDOSPERM DEVELOPMENT IN MAIZE A

A188

3

Wilbur F l i n t

F- Z a p a l o t e

9

X

Wilbur

DAYS POST-POLLINATION

FIG. 13. Patterns of DNA amplification during the postpollination period in several strains. endosperm mutants, and endosperm from an F, cross. The f12 837 strain may not have been sampled late enough to show the decline in DNA per nucleus observed in the other strains. [From Kowles et ul. (1986). Used with permission.]

midine pulse/chase experiment in which samples were taken every 24 hours over a period of 12 days. The tissue samples were subjected to autoradiography (Fig. 15). Silver grain numbers over endosperm nuclei rapidly increased, reached a peak, and remained somewhat constant as one would expect with a cessation of cell division in that tissue. The embryo, which was growing very little at the time of the pulse, did not show silver grains above background throughout the experiment. These results need to be taken cautiously since the experiment only tests a 10-day period fairly early in development. Such a reserve mobilization system could still be operative much later in kernel development, or even during germination. In general terms, there are compelling reasons for considering increased gene expression and product formation to be the result of increased DNA content. In Fig. 16A and B, the DNA amplification pattern has been superimposed upon the levels of two enzymes involved in carbohydrate synthesis as assayed by Tsai et al. (1970). These are only

128

RICHARD V . KOWLES AND RONALD L. PHILLIPS

6-7

8-9

10-11 12-13 14-15 16-17 18-19 20-

DAYS AFTER POLLINATION 14. Distribution of DNA content per nucleus during maize endosperm development (6 to over 20 dap). The data represent mean values of 18 maize lines. FIG.

two of many examples of the close association that exists between DNA amplification and the synthesis of substances in the developing endosperm. Numerous investigations have shown that the 10- to 12-dap period, or slightly later, is the point in the development of the endosperm in which many cellular activities are greatly enhanced, therefore, coinciding with DNA amplification. Such enhancement has been noted for dry weight (Brink and Cooper, 1947b; Groszmann and Sprague, 1948), fresh weight (Hadzi-Taskovic Sukalovic, 1986), protein granule appearance and enlargement (Duvick, 1961, 1963; Khoo and Wolf, 1970), total proteins (Ingle et al., 1965; DiFonzo et al., 1977, 1979), zein mRNA (Marks et al., 1983), and zein accumulation (Wilson, 1978; Soave et al., 1981; Lee and Tsai, 1984). Numerous enzyme assays have resulted in a similar temporal pattern as shown by Tsai and Nelson (1968), Tsai et al. (1970), Burr and Nelson (1973), and Baba et al. (1982). RNase, however, shows a rapid increase slightly later than 12 dap (Ingle et al., 1965; Wilson, 1978, 1980; Tsai, 1979). A comparable pattern is seen for free amino acids (Crawford and Rendig, 1982; Dierks-Ventling, 1983; Arruda and DaSilva, 1983) and for cytokinin levels (Miller, 1967). Even the effectiveness of tissue culturing of the endosperm is greatest at about 12 dap (Straus and LaRue, 1954; Tamaoki and Ullstrup, 1958; Bhojwani, 1984). The 10- to 12-dap period is certainly a critical time, followed by very prolific activity relative to the development of the endosperm in maize. Observations of defective kernel (dek) mutants with a reduced endo-

129

ENDOSPERM DEVELOPMENT IN MAIZE

110

2

4

1

100

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90

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sperm are also of considerable interest (Neuffer and Sheridan, 1980; Sheridan and Neuffer, 1980). These dek mutants initially appear normal and cannot be distinguished from normal kernels on segregating ears until about 10-15 dap. This timing closely corresponds to the period in which prolific DNA amplification normally occurs. Also, preliminary measurements have shown that DNA amplification is greatly reduced in the dek endosperms. Existence of a group of mutations for this characteristic should facilitate the study of DNA amplification, although separating cause from response will be difficult. One particular dek mutant has one-half as much DNA per nucleus, but twice as much nucleolar material than normal kernels on the same segregating ear (Kowles et al., 1986). The ratio of the nuclear area to the nucleolar area in the defective kernels average 1.8 : 1, while the same ratio in normal kernels on segregating ears averages 9.5 : 1. These and other ratios which highlight this dek mutant as an exceptional line are summarized in Table VII (Kowles and McMullen, unpublished). Phillips and Wang (1981) and Phillips et ul. (1983) have shown that a strong correlation exists between nuclear volume and nucleolar volume in normal strains

130

RICHARD V. KOWLES AND RONALD L. PHILLIPS

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(Fig. 17A). They have also shown through the application of in situ hybridization with an rRNA probe that a similarly strong correlation exists between the silver grain number per nucleolus and nucleolar volume (Fig. 17B). These observations indicate that the copies of rDNA increase with the nuclear and nucleolar volumes. The size relationships of the dek mutant described drastically differ from the typical situation. Not known, however, is whether the rDNA segments of the genome in this particular dek mutant have undergone a disproportionate amplification, or, on the other hand, whether the reduced amount of DNA in the dek mutant compensates by increased expression.

131

ENDOSPERM DEVELOPMENT IN MAIZE

TABLE VII COMPARISON OF NUCLEARA N D NUCLEOLAR SIZESI N DEFECTIVE KERNEL MUTANTS, ONEOF WHICHHASA N ABNORMALLY LARGENUCLEOLUS

Stock number 3409 3412

3409 3412 3409 3412

Kern e I s from segregating ear

Nuclei (areay

Normal dek Normal dek (Large nucleolus) Normal Normal dek drk (Large nucleolus)

Nucleoli (area)”

N

Mean

SE

Mean

SE

Nucleus to Nucleolus ratio

17 19 14 20

114.4 18.1 155.9 54.9

13.04 1.33 10.66 7.86

15.2 3.4 16.4 29.8

1.90 0.34 1.04 5.32

7.5: 1 5.3: 1 9.5: 1 1.8: I

17 14 19 20

114.4 155.9 18. I 54.9

13.04 10.66 1.33 7.86

15.2 16.4 3.4 29.8

1.90 1.04 0.34 5.32

‘ Arbitrary units.

A final speculation focuses upon the translocation of amino acids and carbohydrates to the developing embryo by way of the scutellum. These activities are occurring during embryo growth, kernel germination, and early seedling growth. Much of the translocation of storage products requires the degradation of cell walls and a dissolution process. One might assume that dissolution is aided by a structural arrangement of very thin, fragile cell walls in the endosperm. This morphology of the maize endosperm is easily confirmed by microtome techniques with both light and electron microscopy (Kowles and Springer, unpublished). Mathematically, a tissue composed of large cells has lesser cell wall surface area than a tissue composed of small compact cells and might more easily break down. Generally, large cells tend to have large nuclei (Bennett, 1973; Cavalier-Smith, 1978; Nagl, 1982); however, one might question whether large cells require large nuclei. Extensive DNA amplification might provide both a multiplicity of genes necessary for product synthesis and cell enlargement, and, as a consequence, a minimum of cell wall material in this ephemeral tissue.

VIII. Further Directions The endosperm of Zea mays is a unique tissue. Some early researchers have called it a formless mass of tissue and even a “monster.” Such designations were made partly because the endosperm tissue is the direct

132

RICHARD V . KOWLES AND RONALD L. PHlLLlPS

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VOLUME ( ~ 1 0 0 0r3) FIG. 17. (A) Relationship between nuclear volume and nucleolar volume (closed circles) and between nuclear volume and the number of silver grains from in siru hybridization with '*'l-labeled RNA (open circles), (B) relationship between nucleolar volume and silver grains in the hybrid Wf9x 8 3 7 self-pollinated.

result of fertilization, but it does not thereafter pass through a complete life history to result in another organism (Sargant, 1900). The endosperm has also been described as the embryo's "sister-and-a-half" because of the initial triploid nucleus being composed of two polar nuclei (genetically identical to the egg) and one sperm nucleus (Weatherwax, 1923). The tissue greatly increases the number of genomes per cell, develops rapidly, and lives for a relatively short time. These characteristics are not unique for cells with a short life expectancy. Bennett (1973) has noted a general correlation between a large number of genomes per cell and short-lived cells in other plant tissues such as root-cap cells, tapetal cells, and antipodals. Interest in the biology of the maize endosperm is prompted by several considerations. The system is obviously important from an agricultural

ENDOSPERM DEVELOPMENT IN MAIZE

133

standpoint. Pertinent information regarding the genetic and molecular mechanism of endosperm development could conceivably lead to new ideas on protein synthesis, and even hybrid vigor. In addition, the endosperm tissue may be excellent material for the elucidation of basic biological concepts, especially those being pursued by developmental biologists. The aleurone has already been shown to be a worthy genetic tool in classical, molecular, and developmental investigations (Coe and Neuffer, 1977; Coe, 1978). Research efforts in maize endosperm development have thus far resulted in more questions than answers. Many questions still relate to DNA amplification since it is not known how the process is controlled, how many genes are involved in the control, and whether maternal-paternal dosage effects play a role. Well-designed studies are needed to determine whether a relationship exists between DNA amplification and yield, and, if so, the specific nature of that relationship. Functions have not yet been definitively related to the vast DNA amplification of the endosperm. The maize endosperm is a very heterogeneous tissue. The question of how this heterogeneity is related to gene expression during endosperm development, if at all, is worth further attention. Growth and development of the endosperm and the embryo undoubtedly require precisely timed expressions of many genes. As in all biological systems, understanding the mechanisms underlying the orchestration of genes involved in development is ambitious research. Optimistically, however, genetic and molecular tools are now available to conduct investigations that will accumulate critical information about the development of this important tissue. ACKNOWLEDGMENTS We thank Sarah Hake for the use of an unpublished photomicrograph, Shin Enomoto and Michael McMullen for help in performing the slot-blot hybridization tests, and Clare Korte for critically reading the manuscript. Parts of the work described in this review were supported by the National Science Foundation, the U.S.D.A. Competitive Grants Program, the McKnight Foundation, and Pioneer Hi Bred International, Inc.

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Palacios, 1. G. (1982). Maize Genet. Coop. News Lett. 56, 102-104. Peacock, W. J., Dennis, E. S., Rhoades, M. M., and Pryor, A. J. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 4490-4494. Phillips, R. L., and Wang, A. S. (1981). Maize Genet. Coop. News Lett. 55, 89-91. Phillips, R. L., Wang, A. S., and Kowles, R. V. (1983). Stadler Genet. Symp. 15, 105-1 18. Phillips, R. L., Kowles, R. V., McMullen, M. D., Enomoto, S, and Rubenstein, I. (1985). In “Plant Genetics” (M. Freeling, ed.), pp. 739-754. Liss, New York. Poindexter, C. C. (1903). Ohio Nat. 4, 3-9. Punnett, H. H. (1953). J . Hered. 44,257-259. Randolph, L. F. (1936). J . Agric. Res. 53, 881-916. Reddy, V. M., and Daynard, T. B. (1983). Maydica 28, 339-355. Rigby, P. W. J., Dieckman, M., Rhodes, C., and Berg, P. (1977). J . Mol. B i d . 113,237-25 I . Roninson, I. B. (1983). Nucleic Acids Res. 11, 5413-5431. Sargant, E. (1900). Ann Bot. 14, 689-712. Sarkar, K. R., and Coe, E. H., Jr. (1971). Crop Sci. 11, 539-542. Sass, J. E. (1977) I n “Corn and Corn Improvement” (G. F. Sprague, ed.), pp. 98-99. American Society of Agronomy, Madison, Wisconsin. Scandalios, J. G . (1982). Annu. Rev. Genet. 16, 85-1 12. Sheridan, W. F., and Neuffer, M. G. (1980). Genetics 95, 945-960. Soave, C., Tardani, L., DiFonzo, N., and Salamini, F. (1981). Cell 27, 403-410. Steffensen, D. M. (1968). A m . J. Bot. 55, 354-369. Stephen, J. (1973). Sci. Cult. 39, 323-324. Straus, J. (1954). A m . J. Bot. 41, 833-839. Straus, J., and LaRue, C. D. (1954). A m . J. Bot. 41, 687-694. Swift, H. (1950). Genetics 36, 643-654. Tamaoki, T., and Ullstrup, A. P. (1958). Bull. Torrey Bot. Club 85, 260-272. True, R. H . (1893). Bot. Gaz. 18, 212-227. Tsai. C. Y. (1979). Maydica 24, 129-140. Tsai, C. Y., and Nelson, 0. E. (1968). Plant Physiol. 43, 103-112. Tsai, C. Y., Salamini, F., and Nelson, 0. E. (1970). Plant Physiol. 46, 299-306. Tschermak-Woess, E., and Enzenberg-Kunz, U. (1965). Plantn 64, 149-169. Watkin, A. E. (1932). J . Genet. 25, 125-162. Weatherwax, P. (1919). Bull. Torrey Bot. Club 46, 73-102, plus plates 6 and 7. Weatherwax, P. (1923). “The Story of the Maize Plant.” Univ. of Chicago Press, Chicago. Weathervrsix, P. (1926). Bull. Torrey Bot. Club 53, 381-384. Weatherwax, P. (1930). Am. J . Bot. 17, 371-380. Wilson, C. (1978). In “Maize Breeding and Genetics” (D. B. Walden, ed.), pp. 405-419. Wiley, New York. Wilson, C. (1980). Plant Physiol. 66, 119-125.

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. II2

Ameboid Movement and Related Phenomenal w.STOCKEM* A N D w.K L O P O C K A t *Institute of Cytology, University of Bonn, 0-5300 Bonn I, Federal Republic of Germany und ?Department of Cell Biology, Nencki Institute of Experimental Biology, PL-02093 Warsaw 22, Poland

I. Introduction

A. GENERAL CONSIDERATIONS Recent progress in cell biology research has revealed both the dynamic and stabilizing function of an extended system of different types of filaments commonly defined by the term cytoskeleton. Some topical reviews summarized and discussed the present knowledge on microfilaments, microtubules, and intermediate filaments as the major morphological components of this system in a large variety of cells (Weihing, 1979; Geiger, 1983; Jockusch, 1983; Porter et al., 1983; Grain, 1986). In the meantime, numerous cytoskeletal proteins were identified and localized by antibodies against them (Groschel-Stewart, 1980; GroschelStewart and Drenckhahn, 1982). In general these proteins are involved in different cellular phenomena, such as (1) generation of motive force for cytoplasmic streaming and cell locomotion, (2) changes in cell shape and cell surface morphology, (3) maintenance of cell body stability, (4) determination of the rheological properties of the cytoplasmic matrix, ( 5 ) movement of intracellular organelles and particles, (6) cytotic membrane flow processes, (7) mitotic or cytokinetic activities, and (8) cellular wound healing. Unlike tissue cells, the giant free-living amebas, as typical representatives of primitive motile systems, seem to possess a less complex cytoskeleton chiefly composed of actin-containing microfilaments and some other filamentous elements (Taylor, 1977; Grebecki, 1982; Stockem et af.,1982; Paulin-Levasseur and Gicquaud, 1984; Christiani et al., 1986). Experimental data were gained indicating that controlled G-F-actin transformation and F-F-actin aggregation cycles can explain morphodynamic and rheological changes in the protoplasm of amebas during active 'In memoriam to Prof. Dr. Robert D. Allen and his famous work on ameboid movement, and dedicated to Prof. Dr. Karl E. Wohlfarth-Bottermann on the occasion of his sixt y-fifth birthday. 137 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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locomotion (Pollard and Ito, 1970; Jeon and Jeon, 1975; Taylor et al., 1973, 1977; Hellewell and Taylor, 1979). Thus, the internal face of the plasma membrane functions as a site for the anchorage of polymerized actomyosin and plays an important role in transducing contractile activities of the cortical filament system into hydraulic pressure and cytoplasmic streaming (Wehland ef al., 1979). Since numerous new data were obtained by different laboratories during the last years, the following review attempts to summarize the present knowledge on the spatial arrangement, ultrastructural organization, and function of the contractile apparatus in Amoeba proteus as a highly limited portion of the field of cell motility.

FEASIBILITIES B. TECHNICAL In a series of physiological, biochemical, immunocytochemical, and ultrastructural investigations, the phenomenon of ameboid movement was studied by a large number of experimental cell biologists (WohlfarthBottermann, 1964; Wolpert, 1965; Jahn and Bovee, 1969; Allen and Allen, 1978; Grebecki, 1982). According to the different techniques employed, four periods can be distinguished. During the first period (prior to 1960), the various motile activities (sol-gel transformation, protoplasmic streaming, ameboid movement, formation and retraction of pseudopodia) and accompanying phenomena (organelle transport, membrane dynamics during locomotion and cytotic activities, reaction to chemical and physical stimuli) were described by conventional light microscopy (Grebecki, 1964). The second period (1960-1975) used the electron microscope and different techniques such as epoxy-resin embedding, thin sectioning, and negative straining to identify and analyze cell structures (thin and thick filaments) probably participating in the generation of motive force for ameboid movement (Komnick et al., 1973). In the third period (19751980), cell scientists returned to improved light microscopic methods (immunocytochemistry, fluorescent-analog cytochemistry, videoenhanced contrast microscopy) in order to demonstrate the cellular organization and dynamics of microfilaments and other cytoskeletal elements in amebas and a large variety of ameboid cells (Taylor and Wang, 1980; Allen et al., 1981; Kukulies and Stockem, 1986). Currently, the fourth period uses new electron microscopic and biochemical techniques (cryotechniques, protein analysis) to understand the molecular organization of contractile proteins and the mechanism of chemomechanical energy transformation into controlled movement phenomena (Heuser and Kirschner, 1980).

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11. Theories

In the course of about 150 years of research on the mechanism of ameboid movement, the most important progress was achieved by concepts interpreting this phenomenon on the basis of cytoplasmic contractility (Schulze, 1875; Hyman, 1917; Pantin, 1923; Mast, 1926; 1932.) The classical concept of Mast (1926) postulated that endoplasmic streaming follows the hydrostatic pressure gradient created by contraction of an ectoplasmic cylinder. This concept was later developed in a more uncompromising manner by the tail-contraction theory (Goldacre and Lorch, 1950; Goldacre, 1956, 1961, 1964; Marsland, 1956; Rinaldi and Jahn, 1963; Jahn, 1964; Rinaldi et al., 1975a) in which the site of motive-force generation within amebas was limited to the ectoplasm of the posterior region. The generalized cortical contraction theory (Grebecki, 1979, 1982) refers to the peripheral distribution of F-actin in the form of a cortical layer at the cytoplasmic face of the cell membrane (Korohoda and Stockem, 1975; Stockem et al., 1978; 1983a,b; Grebecka and Hrebenda, 1979; Wehland et al., 1979; Gawlitta et al., 1980a; Condeelis, 1981) and is based on the classical hydrodynamic concept of Mast (1926); however, this theory differs in a characteristic way from the tail-contraction hypothesis because it suggests that in locomoting amebas the cortical cylinder can contract in any cell body region isotonically or isometrically, depending on t h e mode of cell contact (Grebecki, 1984). According to the recently proposed solation-contraction coupling hypothesis (Hellewell and Taylor; 1979; Taylor et al., 1979; Condeelis, 1981; Taylor and Fechheimer, 1982), the ectoplasmic gel cylinder acts in its most rigid form as an antagonist to contraction, i.e., contraction can occur only in regions of increasing or decreasing gel structure. Solation of the gel structure is suggested to be a consequence of the dissociation of actin-binding proteins from actin and/or by the action of proteins that sever long actin filaments into shorter fragments. Solation-contraction processes in the tail or in retracting pseudopodia of the cell seem to be initiated by an increase in free Ca” and accompanied by the release of a fraction of cytoskeletal and regulative proteins into the soluble endoplasmic pool. This highly solated endoplasm is then pushed forward along the hydraulic pressure gradient, i.e., the combination of contractile and cytoskeletal functions of the ectoplasmic gel is responsible for ameboid movement. In contrast to the above-mentioned theories, the frontal zone contrac-

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tion hypothesis (Allen, 1961, 1973) denies the active contraction of a cortical microfilament system and the participation of a hydrostatic pressure gradient in the generation and transmission of motive force for ameboid movement. In place of that, this concept locates the site of contraction just behind the tip of advancing pseudopodia, i.e., within the loop where streaming endoplasm is converted into the ecotoplasmic cylinder. The viscoelastic character of the moving endoplasm should enable the transmission of tension, and contraction in the front of amebas should pull cytoplasm forward against the stationary ectoplasmic cylinder. Consequently, the withdrawal of the posterior region is explained by a passive shortening of the cell body and not by localized active contraction. 111. Phenomena

A. EXTERNAL SHAPEA N D MOTILEACTIVITY Amoeba proteus grown under normal culture conditions shows various motile activities and cell shapes (Figs. 1 and 21). Active locomotion is always combined with the expression of a distinct polarity (Figs. la, band 21, c-e), whereas resting cells show a rather apolar organization (2I,a, b, and f-h). Recently, Grebecki and Grebecka (1978) suggested a new terminology based mainly on the external morphology, mode of cell contact to the substratum, and other physiological parameters in order to classify the different morphodynamic types of amebas. Migrating monopodia1 (Fig. 21,c) and polypodial (Fig. 21,d and e) cells with a strictly bipolar and multipolar orientation are called “orthotactic” and “polytactic,” respectively, whereas resting cells with an apolar orientation are called “heterotactic” (Fig. 21, a, b, and f-h). In orthotactic cells protoplasmic streaming originates at the rear end (uroid) and is directed toward the front zone (advancing pseudopodium, Fig. 3a). The most striking phenomena of migration in polytactic amebas (Fig. l a and b) are changes in cell shape caused by the continuous formation and retraction of pseudopodia. Cinematographic studies (Stockem et al., 1969; Grebecka and Grebecki, 1975) demonstrate a permanent decrease in contour at the uroid or at retracting pseudopodia (striated areas), and a permanent increase in contour at the front or at extending pseudopodia (black areas). The uroid and front regions are always separated by an intermediate zone of more or less constant outline. During locomotion the cell surface exhibits alternate unfolding and refolding in that the uroid and retracting pseudopodia are wrinkled (Fig.

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a

FIG. 1. Drawings obtained from normal locomoting A . profeus to demonstrate changes of shape in two different cells (a,b). Each sequence extends over a time period of 20 minutes and comprises 10 consecutive stages (1-10). The stages show two outlines of the same cell, which differ by I minute: the outline containing white and hatched areas represents the previous time stage, whereas the white and black areas compose the stage attained 1 minute later. A. Av, autophagic vacuoles; Ac, actin cortex; Ca, food cavity; Cv, contractile vacuole; DIK, differential interference contrast; E, endosome; Ec, ectoplasm; En, endoplasm; F, front; Fc, filament cortex; Fv, food vacuole; Gp, granuloplasm; Hp, hyaloplasm; IF, intermediate filaments; M, mitochondria; Mc, myosin cortex; MI, mucous layer; Mt, microtubules; N, nucleus; Pm, plasma membrane; U, uroid.

3c), whereas advancing pseudopodia of polytactic cells (Fig. 3b) and the front region of orthotactic amebas (Fig. 3a) are smooth. Morphometric measurements have shown that comparable volumes in retracting cell areas are surrounded by more than twice the membrane

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area than in advancing ones. The simultaneous existence of areas with different folding degrees is a necessary precondition for the motility of amebas and indicates the contractile activity of the microfilament system beneath the plasma membrane (see Sections IV and V). OF PSEUDOPODIUM FORMATION B. CONTROL

Under normal culture conditions the external shape of A . proteus is always correlated with definite electrical membrane potentials (PDs: Batueva, 1965a,b; Bingley, 1966; Braatz-Schade et al., 1973; BraatzSchade and Haberey, 1975). Measurements carried out on eight different morphodynamic types in normal Chalkley medium at pH 6.5 (Fig. 21, and IV) reveal maximal PDs of up to -75 mV in rapidly moving orthotactic cells (Fig. 2Ic and IV), whereas polytactic amebas (Fig. 2Id, e, IV) with a lower rate of locomotion show average PDs of -44 and -50 mV, respectively. Heterotactic specimens with no locomotor activity (Fig. 2Ia,b, f-h, and IV) range between -32 and -8 mV, and occasionally resting cells can be found with positive PDs between +9 and +24 mV. Corresponding morphodynamic forms of A . proteus can be induced by

I Shape

i I

I

pn-value

6.0

-18

- 5 22

-21t7

-73 211

-4529

-M'-9

2

8

--_

*23* 6

-20'- 3 -442 7

*la'- 7

(m)

FIG. 2. Correlation of cell shape, number of pseudopodia, and membrane potential in A . proreus. I (a-h): Eight different morphodynamic types: I1 (2.0-9.5): Chalkley media with eight different pH values; I11 (-5-+ 16/- 15): shape-dependent membrane potentials in Chalkley media of different pH (see 11); IV (-8-+9/ -22): shape-dependent membrane potentials in Chalkley medium of constant pH 6.5. From Braatz-Schade and Haberey (1975).

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controlled changes of the H' ion concentration in the external medium (Fig. 211 and 111). Except for minute variations at extreme acid or alkaline pH, the PD values measured in such cells correspond exactly to the values in respective forms from normal cultures. In addition, these results clearly point to a correlation between the pH of the external medium and the number of pseudopodia, which increases with decreasing H' concentration (Fig. 21 and 11). Possible binding sites for the adsorption and accumulation of protons and other cations are acid mucopolysaccharides with negative neoinositol-bound phosphate polymers (Allen et al., 1974). There are strong indications that, besides H', extracellular Ca" plays a predominant role in controlling the physiological state of the plasma membrane (Brandt and Freeman, 1967; Brandt and Hendil, 1972; Kukulies et al., 1986). Accordingly, high concentrations of external Ca" have a stabilizing effect on the membrane (Gingell, 1972), whereas low concentrations and the simultaneous presence of other cations can induce cellular activities such as endocytotic membrane internalization (Marshall and Nachmias, 1965; Hendil, 1972; Braatz-Schade and Haberey, 1975; Josefsson, 1975; Stockem, 1977; Prusch, 1981; see also Section V,A,5). It is interesting that different amebas from the same culture exhibit remarkable differences in the degree of externally bound Ca" and, hence, in the physiological state of the plasma membrane (Kukulies et al., 1986). This finding may explain why a reliable and reproducible ultrastructural and physiological analysis of this organism has been impeded in many respects. C. INTERNALORGANIZATION

In normal locomoting amebas the streaming sollike endoplasm is transported within a cylinder which consists of rigid, gellike ectoplasm (Fig. 3a). At the light microscopic level, endoplasm and ectoplasm exhibit a similar structural organization, i.e., they consist of hyaline groundplasm (Stockem, 1969) containing various granular components such as the nucleus, contractile vacuole, mitochondria, vacuolar apparatus, dictyosomes, and endoplasmic reticulum (Fig. 3e). Cells experimentally treated with drugs and anesthetics, high hydrostatic pressure, or high temperatures often exhibit a distinct separation of hyaline groundplasm (hyaloplasm) in the cell periphery and granular cell components (granuloplasm) in the central cell region (Fig. 3d; Korohoda and Stockem, 1975). Similar hyaline zones are also present in migrating amebas at the tip of advancing pseudopodia and at the periphery of the intermediate and posterior cell body region (Fig. 3a). It was shown that they contain pure

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FIG. 3. General morphology of A. proteus. (a) Orthotactic cell during normal locomotion. (b,c) Extending (b) and retracting pseudopodium (c) of a polytactic cell showing a smooth and highly folded surface, respectively. (d) Heat-shocked cell with distinct separation of hyaloplasm and granuloplasm. (el Characteristic structure of the cytoplasm in a control cell. Arrows show cytoplasmic streaming direction. DIK. Scales: a = 50 p m : b,c, = 20 pm; d,e, = 10 p m . Abbreviations as in Fig. I . (a) From Stockem et a / . (1982); (b,c) from Stockem e f a / . (1969).

groundplasm (Korohoda and Stockem, 1975), which is not extruded from the granuloplasm by syneresis (Marsland, 1956; Allen, 1968, 1973), but by the detachment of a cortical microfilament layer from the plasma membrane during active contraction (Stockem et al., 1978; Stockem and Hoffman, 1986).

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D. MOVEMENTPHENOMENA Normal locomotion of A . proteus is accompanied by characteristic changes in the position of the plasma membrane, the ectoplasmic gel cylinder, and the granular constituents of the central endoplasm. The rate and moving direction of these compartments depend on the polarity of the cell, the speed of migration, and the zone of cell contact to the substratum.

1 . Movement of the Plasma Membrane Treatment of the cell surface with different marker techniques (Griffin and Allen, 1960; Wolpert and O’Neil, 1962; Czarska and Grebecki, 1966; Wolpert and Gingell, 1968) produced evidence that the plasma membrane is a structure with a long-time constancy in surface area, following exactly the movement and shape transformations of amebas. Czarska and Grebecki (1966) advanced this view and demonstrated the importance of the continuous unfolding and refolding processes at the cell surface for the formation and retraction, respectively, of pseudopodia. This hypothesis was later confirmed by more extensive cinematographic and electron microscopic studies (Haberey et al., 1969; Stockem et al., 1969; Haberey, 1971; Stockem, 1972). Accordingly, the dynamic behavior of the plasma membrane during active locomotion of A . proteus can be characterized in detail as follows. The entire membrane slides forward along the surface of the cell body (Fig. 4G, a-c), and the sliding direction is always the same as that of endoplasmic flow. In lateral and dorsal cell areas the plasma membrane moves with approximately the same speed as the tip of advancing pseudopodia, whereas the movement velocity of the membrane at the ventral side of the cell is distinctly less. The difference in speed is probably due to local and temporary contacts of the cell surface to the substratum. Although the majority of marker particles externally attached to the plasma membrane flow forward in the direction of endoplasmic streaming (anterograde transport), some of them are transported backward (Fig. 4H, a-c; retrograde transport; Grebecki, 1985, 1986). Both movements exist within the same region, and particles moving in opposite directions may cross each other at a distance of less than 1 pm (Fig. 41,a-d). A transition from forward to backward transport occurs immediately behind the front, where endoplasm is transformed into new ectoplasm. As a rule, backward transport is always slower than forward transport and further decreases in velocity when the particles approach cell regions adhering to the substratum. Retrograde transport of particles is interpreted as the backward flow ’

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FIG. 4. (A-F) Schematic drawing to demonstrate the movement patterns of the ectoplasmic gel cylinder in side view (A, C, E) and corresponding top view (B, D, F). Straight arrows show movement direction of the ectoplasmic gel cylinder; striated arrows show endoplasmic streaming direction; arrowheads denote retraction or extension of pseudopodia; dotted areas denote outline regions of cell contact. G-I: Light micrographs showing the movement patterns of the ectoplasmic gel cylinder and the plasma membrane in living A . proleus; DIK. G (a-c): Forward movement of latex beads attached to the cell surface (white arrowheads) independent of the backward movement of ectoplasmic granules (black arrowheads) in the frontal part of a monotactic cell at intervals of 4 seconds; H (a-c): Unisonous backward movement of a latex particle at the cell surface (white arrowheads) and of granules in the ectoplasm (black arrowheads) of an extending lateral pseudopodium at intervals of 2 seconds; 1 (a-d): Bidirectional movement of two latex particles at the surface of a moving cell demonstrating anterograde (black arrowheads) and retrograde transport (white arrowheads) at intervals of 2 seconds; White arrows show direction of endoplasmic streaming. Scales G-I = 10pm. ((3-1) From Grebecki (1986).

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of membrane components externally linked to the marker and internally linked to the cortical microfilament layer. Anterograde transport is considered to reflect the general forward flow of membrane components which are not anchored to the cortical filament layer. In general, the bidirectional movement pattern of particles attached to the cell surface indicates the high fluidity of the plasma membrane and the recirculation of membrane components between the tail and the front of locomoting amebas. 2. Movement of the Ecto- and Endoplasm The ectoplasmic cylinder of moving A . proreus (Grebecki, 1984, 1985) continuously retracts from each distal and free cell region toward the actual site of adhesion (Fig. 4A, B, G, and H). In cells frontally attached to the substratum the ectoplasm moves forward (Fig. 4C and D), whereas in amebas adhering at the tail ectoplasm moves backward, thus producing the characteristic fountain-streaming phenomenon (Fig. 4E and F). In cells without any contact to the substratum, the whole peripheral ectoplasmic layer is retracted toward the geometric center, which coincides with the posterior uroid region. The retraction velocity increases from the site of adhesion to any free distal body region in a linear way, and the retraction of the ectoplasmic layer is independent from the endoplasmic flow, i.e., a pseudopodium may be withdrawn as a whole even when endoplasmic streaming is directed forward (Fig. 4B and F). Endoplasmic particles transported to the front cannot become frontal again without recirculation through the cell interior. The rate of endoplasmic flow depends on the degree of the hydrostatic pressure gradient along the cell body and is related to the speed of cell locomotion. The velocity profiles are correlated with the rheological properties of the streaming endoplasm, and microscopic measurements (Allen, 1961; Allen et al., 1963, 1966; Francis and Allen, 1971) show that the axial endoplasm behaves as a thixotropic non-Newtonian fluid. Moreover, a gradient of decreasing viscoelasticity in the endoplasm between the uroid and the front (Wang et al., 1982) is probably induced by a decreasing gradient of free Ca” in the same regions (Nuccitelli et al., 1977). E. MEMBRANE TURNOVER During normal locomotion of A . proreus, membrane turnover occurs as the result of slow, permanent endocytosis at the uroid and simultaneous exocytosis at the front (Fig. 5 ; Stockem, 1972). Morphometric studies indicate that about 0.15% of the total cell membrane is internalized per

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exocytotic vacuole

primary lysosomes

endoplasmic reticulum

secondary lysosome

recycled lysosomal vesicles preexisting lysosome secondary endosomes primary endosome primary endosome endocytotic channel

UROlD

FIG.5. Schematic drawing to demonstrate the membrane turnover in normal locomoting A . proteus. For description, see Section M E .

minute and recycled during these processes, i.e., migrating cells renew their cell surface once every 12 hours. This rate is too low to play any role in the generation of motive force for ameboid movement (Stockem, 1973). The fate of internalized material in amebas is similar to the concept of cytoplasmic digestion as proposed by de Duve (1963) for other cells. Primary endosomes fuse directly or after division into smaller secondary endosomes with so-called preexisting lysosomes (Thoenes et al., 1970) or with primary lysosomes which both contain digestive enzymes (Hausmann and Stockem, 1972). Simultaneously, small vesicles with digested substrate are pinched off from secondary lysosomes and seem to fuse

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with other cytoplasmic compartments such as the endoplasmic reticulum. The indigestible lysosomal content is then extruded by defecation, and the membrane of the defecation vacuole, which differs from the plasma membrane because it lacks a mucous layer, is recycled back into the cytoplasm by endocytosis (Stockem, 1972; Hausmann and Stockem, 1972). According to different investigations, membrane regeneration is accomplished by exocytotic vacuoles arising from the distal region of the dictyosomes and fusing with the plasma membrane, probably in frontal cell regions (Stockem, 1969; Wise and Flickinger, 1970). Microfilaments play an important role in generating motive force for cytotic membrane flow processes (see Section V,A,5).

IV. Organization of the Microfilament System Most attempts to explain the phenomenon of active locomotion in large amebas were more or less unsuccessful because morphological and physiological data necessary to understand the mechanism of motive force generation were lacking. In particular, these data must explain the nature, spatial arrangement, ultrastructural organization, and function of the contractile system in A. proteus. A. NATURE OF

THE

CONTRACTILE SYSTEM

Investigations on cells glycerol extracted according to the technique of Hoffman-Berling (1956) provided the first evidence for the actomyosin nature of the contractile system in A. proteus (Simard-Duquesne and Couillard, 1962; Schafer-Danneel, 1967). Upon addition of ATP and inorganic ions, the glycerinated cell models specifically react with a distinct volume contraction. Simultaneously, the contractile effect of adenosine triphosphate (ATP) proved to be morphologically manifested in the condensation of a network of thin and thick filaments (Rinaldi et al., 1975a; Opas, 1976). Hence, it was concluded that the contractile system in A. proteus is equivalent to the actomyosin system of muscle and a large variety of nonmuscle cells (Komnick et al., 1973).

ARRANGEMENT OF B. SPATIAL

THE

CONTRACTILE SYSTEM

The spatial arrangement of cytoplasmic actin and myosin in normal locomoting A. proteus was recently demonstrated by indirect immunofluorescence microscopy (Stockem et al., 1983b).According to this study

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FIG. 6. lmmunocytochemical localization of the actomyosin system in A . proteus. (a) Conventional light transmission micrograph of an orthotactic cell after methanol fixation at -20°C. (b) Corresponding fluorescence micrograph of the cell shown in (a) after staining with actin antibodies. The arrowheads point to hyaline pseudopodia in the posterior cell body region. (c) Fluorescence micrograph of an orthotactic cell after methanol fixation at -20°C and staining with myosin-antibodies. Scales = I 0 0 pm. Abbreviations as in Fig. I . From Stockem et a!. (1983b).

both proteins are distinctly concentrated in a cortical layer immediately beneath the plasma membrane (Fig. 6). In cell areas where a hyaloplasmic sheet borders the plasma membrane the actomyosin layer delineates the central granuloplasm from the peripheral hyaloplasm (Fig. 6a and b, arrowheads). The distributions of actin and myosin within the cortical layer are not uniform, i.e., the amount and density of actin increase from the advancing front to the intermediate cell region and decrease again toward the uroid (Fig. 6b), whereas the myosin content gradually

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increases from the front to the rear end (Fig. 6c). A considerable amount of actin and myosin is also found around the cell nucleus and the contractile vacuole. It is important to mention that distinct fibrillike differentiations in both the endoplasm and ectoplasm of locomoting A . proteus are completely lacking. C. ULTRASTRUCTURAL ORGANIZATION OF THE CONTRACTILE SYSTEM A large number of investigations on A . proteus delivered morphological evidence that the internal face of the plasma membrane functions as a privileged site for the display and anchorage of polymerized actomyosin (Comly, 1973; Korn and Wright, 1973; Pollard and Korn, 1973; Korohoda and Stockem, 1975; Rinaldi et al., 1975a; Taylor et al., 1976b; Hauser, 1978; Grebecka and Hrebenda, 1979; Condeelis, 1981; Stockem et al., 1982). Based on the experience that the actomyosin system of giant amebas is difficult to stabilize during electron microscopic preparations (Szamier et al., 1975; Allen and Allen, 1978), improved fixation and embedding techniques were applied recently in order to obtain optimally preserved specimens (Stockem et al., 1982, 1984). According to electron micrographs taken from serial sections of whole cells (Figs. 7a, 8a and b) and used to reconstruct the ultrastructural organization of the actomyosin system in schematic drawings (Fig. 7b), the contractile system of A . proteus is represented by a continuous cortical layer along the entire cell surface (Fig. 7b, broken lines). The layer varies in thickness and density depending on the cell region: in the front region it is very thin with an average thickness of 0.1-0.2 pm and mostly in direct contact with the plasma membrane (Figs. 7b and 8a); in the intermediate and posterior regions the layer normally increases in density and thickness to 0.5 pm or more and often loses contact with the plasma membrane, thus separating the peripheral hyaloplasm from the central granuloplasm (Figs. 7b-d and 8b). Higher magnifications show that the contractile system is constructed mainly of two components: thin filaments with a diameter of 6 nm representing actin (Fig. 8c and d, arrows) and thick filaments with a diameter of 10-30 nm representing myosin (Fig. 8d, arrowheads). The thin filaments are linked with the plasma membrane by regularly arranged crossbridges of 5-10 nm thickness and 20-30 nm length, whereas the thick filaments are connected to the thin filaments by crossbridges measuring 60 nm between the two filament types and 30 nm in diameter (Fig. 8e). In contrast to thin filaments, which are a general constituent of the contractile system, the display of thick filaments is largely restricted to the intermediate and posterior region (Fig. 7b, thick broken lines). The

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1

FIG.7. Electron microscopic localization of the actomyosin system in A. proieus. (a) Electron micrograph of an orthotactic cell after GA fixation to demonstrate the homogeneous hyaloplasm and particulate granuloplasm, which contains the entire mass of membranesurrounded cell structures. (b) Drawing prepared at high magnification to demonstrate the course of the actomyosin system in the same section as shown in (a). The actomyosin system is a continuous layer separating the hyaloplasm (white) and the granuloplasm (dotted). At the anterior end it consists of actin filaments (thin broken lines) and at the posterior end of actin and myosin filaments (thick broken lines). (c) DIK micrograph showing the clear separation of hyaloplasm and granuloplasm in the uroid region of a living cell. (d) Higher magnification of the uroid region marked by arrowhead 2 in (b). For arrowhead 1 in (b) and 3 in (d), see higher magnifications in Fig. 8a and b, respectively. Scales a-c = 50 pm, d = 10 pm. Abbreviations as in Fig. I . From Stockem et a / . (1982).

number of thick filaments increases toward the uroid and their abundance within this region is responsible for the high contrast of the contractile system in survey micrographs (Figs. 7d and 8b). Both thin and thick filaments reveal a different degree of order with respect to the spatial

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FIG. 8. Ultrastructure of the actomyosin system in A. proteus. (a) Membrane-attached microfilament layer in the anterior cell body region (indicated by arrowhead 1 in Fig. 7b). The filament layer consists of actin filaments. (b) Membrane-detached microfilament layer in the posterior cell body region (indicated by arrowhead 2 and 3 in Fig. 7b and d, respectively). The filament layer consists of actin and myosin filaments. (c,d) Tangential sections through the microfilament layer in the anterior (c) and posterior cell body region (d). The anterior layer is composed of actin filaments, the posterior layer of actin (arrows) and myosin filaments (arrowheads). (e) Single actin filament (arrows) connected with the plasma membrane by numerous cross bridges and with a myosin filament (arrowheads) by globular structures. Scales a, b = I pm; c, d = 0.5 pm; e = 0.2 pm. Abbreviations as in Fig. I . (a, b, e) From Stockem et a / . (1982); (c, d) from Wehland et a/. (1979).

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orientation (Fig. 8c-e); in the front region thin filaments run mostly parallel to the plasma membrane and longitudinal axis of the cell (Figs. 8e and IN), whereas in the intermediate (Fig. 8c) and uroid regions (Figs. 8d and 181) thin and thick filaments form a netlike structure without any predominant pattern of the two filament types.

D. PHYSIOLOGICAL POLARITY OF THE CONTRACTILE SYSTEM The physiological polarity of the contractile system in A . proteus was investigated by fluorescent-analog cytochemistry (FAC; Taylor and Wang, 1978; Kreis and Birchmeier, 1982; Kukulies and Stockem, 1986). The new high-resolution technique for light microscopic studies on living cells combines fluorescence microscopy, microinjection, biochemistry, video technology, and digital image processing. Fluorescent G-actin was successfully used for the in vivo labeling of F-actin in different cell types (Wang and Taylor, 1980). After microinjection into normal locomoting amebas the fluorescent G-actin copolymerizes with endogenous unlabeled G-actin to F-actin, thus giving rise to a selective staining of organized microfilaments (Gawlitta et al., 1980a; Taylor, et al., 1980; Stockem et al., 1983a; Hoffmann et al., 1984). The time interval of 10-20 minutes between microinjection and the first appearance of a distinct fluorescent signal within the cortex at the cell periphery indicates a rather high rate of actin exchange between the cytoplasmic matrix and the microfilament system. (Fig. 9). Gray value scanning measurements across the cortical microfilament system of moving orthotactic specimens regularly reveal a four to five times higher fluorescence in the intermediate cell body and anterior uroid region as compared to the advancing front and the rear end (Fig. 9a and b). The distinctly increased fluorescent signal within this intermediate cortical segment is due to a rather high local density of fluorochrome molecules, thus indicating a strongly condensed state of the microfilament system during active contraction. On the other hand, comparatively weak fluorescence of the cortex within the front and the lower uroid region points to more relaxed and reduced microfilament systems at the two poles. Independent of the actual cell shape and cytoplasmic streaming pattern, the localized contractile activity of the filament cortex within the intermediate cell body region is maintained even over long periods (Fig. 9c). Once the incorporation of labeled G-actin into the filament cortex is accomplished, the distribution of the residual fluorescent protein is no longer uniform. The granuloplasm inside the cortex clearly exceeds the peripheral hyaloplasm in the amount of fluorescent actin (Fig. 9). This

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Fic;. 9. Fluorescent-analog cytochemistry of normal locomoting A . proreus. Light transmission (a) and fluorescence micrographs (b, c) of a living cell after microinjection and incorporation of 1AF-actin into the microfilament system. The increased fluorescence intensity in the intermediate cell body region indicates the distinct condensation of actin filaments within this segment. Time, minutes after microinjection of IAF-actin; arrows show endoplasniic streaming direction. Scales = 100 prn. Abbreviations as in Fig. I . From Stockem ef crl. (1983a).

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distribution differs from the observed actin content of corresponding areas in experimentally treated amebas (Section V,A) and indicates varying polymerization degrees of nonorganized actin within the cytoplasmic matrix (Stockem et al., 1983a; Hoffmann et al., 1984). In polytactic amebas with a multidirectional polarity the spatial organization and physiological state of the contractile system are much more complicated than in orthotactic specimens. Large pseudopodia formed when the direction of locomotion changes exhibit a corresponding microfilament pattern, as compared to complete orthotactic cells. Moreover, frame-by-frame sequences provide clear evidence for the existence of a hydraulic pressure mechanism, because active contraction of the tubular filament cortex becomes directly visible by a gradual increase in the fluorescence intensity and a simultaneous decrease in the radial diameter of the system around the endoplasmic channel (Stockem et al., 1983a). The question of whether single pseudopodia represent ‘‘functional units which move independently of one another” (Allen, 1968; Allen and Allen, 1978) or whether entire polytactic amebas “act as organized units” (Mast, 1932; Klopocka and Grebecki, 1980) is discussed again later (see Section V,B,l).

V. Function of the Microfilament System As the most important structure in large amebas to generate motive force for pseudopodium formation and active locomotion, the cortical microfilament system can be analyzed with respect to its functional significance by chemical (Jennings, 1904; Edwards, 1923; Jeon and Bell, 1965) and physical stimulation (Hyman, 1917; Goldacre, 1952) or inhibition. In the following sections, some recent results are described by which the influence of different agents (Hoffmann et al., 1984; Stockem and Hoffmann, 1986), light-shade differences (Grebecki, 1980, 1981), and externally applied pressure (Allen et al., 1971; Rinaldi et al., 1975b)on the morphology and function of the contractile system in A . proteus were demonstrated. INVESTIGATIONS A. CHEMICAL

In a first series of experiments (Stockem et al., 1978, 1983a; Wehland et al., 1979; Gawlitta et al., 1980a,b, 1981; Hoffmann et al., 1984; Stockem and Hoffman, 1986) 88 different agents were used (via external application or microinjection) for a functional analysis of the contractile system in A . proteus (see list of chemical agents in Table I). The different reaction

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Microinjected substances

Acetylcholine, Acridine Orange, asparagine. atropine

Acetylthiocholine iodide, Acridine Orange, actin, actinomycin D., ammonium molybdate, ATP Bovine serum albumin Caffeine, caprylic acid, chlorpromazine, chlortetracycline, cocaine, collagenase, cysteine. cytochalasin B

Benzamide, butyrylcholine Caffeine, chloral hydrate, chloroform. pchloromercuribenzoic acid, chlorpromazine, cocainehydrochloride, colchicine, collagenase, cysteine, cytochalasin B D-600, dibucaine, digitonin, dinitrophenol (DNP) Ether, ethylenediamine. EDTA, EGTA, ephedrine hydrochloride. eserine Heparin, 8-hydroxyquinoline iso-OMPA Lysolecithin Monoiodoacetic acid, morphine N-Ethylmaleimide (NEM), noradrenaline Ouabain Papain, phalloidin, phloretin, phloridzin, phospholipase, philocarpine, poly-I-lysine (MG: 500,000, MG: 6000-9000), polymyxin-B-sulfate. procaine hydrochloride, prostaglandin F?., puromycin, putrescine Quinine RNase. Ruthenium Red Serotonin, spermidine, sperrnine, sucrose Theobrornine. theophylline, thiourea, Trypan Red, trypsin Urea, urethane

D-600, DMSO, DNase I, DNP, D?O EDTA, EGTA, eserine, ethanol, ethylenediamine Ferritin, fluphenazin, fragrnin Heparin, histamine, HMM, H?Oz1 hyaluronidase iso-OMPA L ysolecithin Morphine NEM Ouabain Papain, PCMB, pepsin, phalloidin, phloretin, phloridzin, phospholipase, pH graded Chalkley medium from 3-1 1 , poly-L-glutamic acid, poly-Llysine, polymyxin B, procaine hydrochloride, putrescine Quinine RNase, Ro 20-1724, Ruthenium Red Sodium monovanadate, spermidine, spermine, sucrose Theophylline, thiourea, trypsin

Vitamin K ,

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patterns which were induced by the agents depend mainly on two phenomena, i.e., on the localized or general contraction or relaxation of the cortical microfilament system. Some of the most interesting reponses obtained by chemical stimulation can be summarized as follows (Fig. 101, A-F). 1. General Contraction of the Filament Cortex

Substances such as phalloidin, ouabain, procaine, N-ethylmaleimide (NEM), puromycin, dinitrophenol (DNP), and others cause general contraction and immobilization in conjunction with spherulation of amebas by destroying the morphological and physiological polarity of the cortical microfilament system (Figs. 101, A and 1la-d). Frequently, during contraction the filament cortex loses contact with the plasma membrane, thus initiating characteristic morphogenetic changes: hyaloplasm is squeezed through the narrow filamentous meshwork and enters the cell periphery (Fig. I la-h). The experimentally induced separation of hyaloplasm and granuloplasm is comparable to the situation in normal locomoting A . proteus (Figs. 3a, 7b-d, 8b, and 9a-c) and other species of ameba, such as Vanella, Hyalodiscus, or Thecamoeba (Haberey, 1973; Haberey and Hiilsmann, 1973) in which a permanent display of hyaline and granular cytoplasmic areas is due to the regular morphological appearance. After the formation of large hyaline areas by chemical induction, a second, smaller, fluorescent actin layer is usually displayed beneath the plasma membrane; this layer is sometimes connected with the detached original layer by fibrils (Fig. 1 le-h). The fibrillar structures can originate from discrete patches (Fig. llh) which seem to function as generation sites where further polymerization of actin starts until a new continuous layer has developed (Fig. 1 li; see also Hoffmann et al., 1984).This clearly demonstrates that some of the cortex-organizing factors are bound to the internal face of the plasma membrane and are concentrated within distinct areas. So far, it is still unknown whether the same factors also control the contact between the plasma membrane and the filament cortex. The existence of such a control mechanism is demonstrated by the fact that general contraction of the cortical microfilament system and immobilization of A . proteus can also occur without any distinct separation of hyaloplasm and granuloplasm (Fig. 101, B). Substances such as Acridine Orange, PCMB, Ruthenium Red, and others cause destruction of the morphological and functional polarity without detachment of the filament cortex from the internal face of the plasma membrane (see also Sections V,A,3, and 5).

I

...

FIG. 10. I: Localized or general contraction and relaxation of the actomyosin system in A . proreus upon chemical stimulation with altogether 88 different substances. (A) General

contraction of the detached microfilament system with granuloplasm/ hyaloplasm separation. (B) General contraction of the microfilament system without detachment from the plasma membrane. (C) Localized relaxation of the microfilament system at the front and localized contraction at the uroid. (D) General relaxation of the microfilament system and cell flattening. (E) General relaxation and destruction of the microfilament system and cell death. (F) Cytokinetic activity upon localized contraction of the microfilament system in the intermediate cell body region. 11. Locally induced pseudopodium (B) and uroid formation (A) by external application of substances with relaxing and contractile influence. respectively. Arrows show endoplasmic streaming direction. White areas show hyaloplasm; dotted, granuloplasm; black, filament cortex. From Hoffrnann (unpublished).

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FIG. 11. Fluorescent-analog cytochemistry of experimentally treated A. proreus. Light transmission (a, b) and corresponding fluorescence micrographs (c, d) of a cell 30 minutes after microinjection of IAF-actin to demonstrate the influence of subsequently microinjected 1 mM phalloidin. The filament cortex (Fc) detaches from the plasma membrane and causes separation of hyaloplasm and granuloplasm. Light transmission (e, g) and corresponding fluorescence micrographs (f, h, i) of two different cells (e, f and g-i) 30 minutes after

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2 . General Relaxation of the Filament Cortex Anesthetics such as urethane, benzamide, ether, chloral hydrate, chloroform, ethanol, and others initiate immobilization and relaxation of A . proteus (Hiilsmann et al., 1976; Korohoda and Stockem, 1975) by completely destroying the integrity of the microfilament system (Fig. 101, D and E). In some cases the relaxed state is reached over an initial phase of general contraction. As shown by externally applied Ruthenium Red (Stockem et al., 1983a) any ordered locomotor and cytoplasmic streaming activity first stops ; the intermediate region of the microfilament system is then no longer predominant in motive force generation. The cortex at the front and uroid also starts to contract until the cells have completely rounded. Thereafter, the filament cortex is slowly destroyed, with the exception of a small actin sheath around the pulsating vacuole. Simultaneous with the equal distribution of actin the cytoplasm relaxes, a result clearly visible by cell body flattening (Fig. 101, D; see also Hiilsmann et al., 1976); upon complete destruction of the filament cortex most cells die by membrane disruption (Fig. 101, E). It follows that a membraneattached microfilament system is important not only for the generation of motive force for cytoplasmic streaming and cell locomotion, but also for the maintenance of cell body stability. 3. Localized Contraction and Relaxation of the Filament Cortex Substances such as the actin-binding proteins DNase I (Wehland et al., 1979) and fragmin (Gawalitta et al., 1980b) can cause progressive relaxation of the front and simultaneous contraction of the intermediate and uroid region (Fig. 101, C) by destroying the balanced equilibrium between organized actin present in the filament cortex in a high polymer, insoluble state, and disorganized actin distributed in the cytoplasmic matrix in a low polymer, soluble state. DNase I binds rapidly, specifically, and very strongly to G-actin, blocking its ability to polymerize (Mannherz et al., 1975). In addition, the protein depolymerizes F-actin slowly and also binds G-actin complexed with profilin (Harris and Weeds, 1978). Fragmin is an actin-modulating (AM) protein from Physarum polycephalitm and also a powerful inhibitor of actin polymerization when added to G-actin (Hasegawa et al., 1980; Hinssen, 1981). Microinjected DNase I and microinjection of IAF-actin to demonstrate the influence of externally applied 5 mM DNP. The original filament cortex (Fc,) detaches from the plasma membrane and a new one (Fc?) is formed. The formation of the new filament cortex starts from discrete patches at the internal face of the plasma membrane (arrowheads) which are often connected with the original filament cortex by fibrils (arrows). Time, minutes-seconds after application of phalloidin and DNP. Scales = 100 pm. Abbreviations as in Fig. I . From Hoffman el d. ( 1984).

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fragmin transform the normal polytactic cell morphology of A . proreus (Fig. 12a) into an orthotactic one (Fig. 12b) characterized by a strictly bipolar organization, i.e., a flattened advancing front showing clear suppression of lateral pseudopod formation, and a contractile uroid undergoing a continuous decrease in volume (Fig. 101, C ) . Under the

FIG. 12. Influence of microinjected fragmin (a, b) and DNase I (c-f) on the cell shape and microfilament organization of A . proreus. Fragmin-induced transformation of a polytactic cell (a) into a bipolar cell model by permanent relaxation of the front and permanent contraction of the uroid (b). Arrows show endoplasmic streaming direction; time, minutesseconds after application of fragmin. DNase I-induced gradual destruction of the filament cortex as shown by comparing the front (c, e ) and uroid region (d, f ) of a control (c, d) and microinjected cell (e, f ). Scales a, b = 100 pm; c-f = I pm. Abbreviations as in Fig. 1. (a, b) From Gawlitta et a / . (1980b); (c-f) from Wehland et a / . (1979).

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assumption that the anterior part of a locomoting ameba is one of the most favored regions for the formation of a new microfilament layer beneath the plasma membrane, a slow destabilization, and consequently a gradual relaxation, of the front should be caused by the two proteins upon a significant reduction of the polymerizable actin pool. Since DNase I and, especially, fragmin cause only a long-term depolymerization of preexisting microfilaments and no inhibition of actin-myosin interaction, the intermediate and posterior cell regions are able to perform continuous contraction for 10-15 minutes until ultrastructural studies show a distinct destruction of the actomyosin system (Fig. 12c-f). These results suggest that a controlled reversible equilibrium between soluble and polymerized forms of actin is a necessary requirement for ameboid movement. In strong support of this, the first biochemical study of A . proteus revealed the existence of ameba actin (MW 44K) and ameba profilin (MW 22K) as well as a cytoplasmic kinase (Sonobe et al., 1985, 1986). The phosphorylation of ameba actin with this kinase in the presence of ameba profilin prevents the polymerization of G-actin to F-actin and keeps G-actin in the monomeric state. It seems probable that the two proteins are essential factors of a control system for sol-gel transformation cycles during ameboid movement and related phenomena. A localized chemotactic response of the microfilament system is also inducible in any cell body region by applying substances with a positive or negative chemotactic influence using a micropipette (Fig. 1011, A and B). As shown by other authors for narcotics (Korohoda, 1977; KaliszNowak, 1978) and hydra extracts (Jeon and Bell, 1965), all substances with a positive chemotactic influence cause relaxation of discrete cell surface areas and local pseudopodium formation (Fig. 1011, B) by detachment of the microfilament layer from the plasma membrane (Fig. 14a,b), Further pseudopodium elongation is always a discontinuous process brought about by the successive reformation, detachment, and destruction of several cortex generations (Fig. 14c and d; see also Sections V,A,5. and V,B,I). On the other hand, all substances with a negative chemotactic influence cause constriction of discrete cell surface areas and local uroid formation by contraction of the microfilament layer (Fig. 1011, A). In a series of experiments using nucleated and enucleated fragments of A . proteus, Korohoda (1977) locally applied different narcotics in a micropipette and concluded from these results that in the intact ameba the nucleus secretes a relaxing substance which controls cell body polarity by pseudopodium formation and retraction. 4. Induced Cytokinetic Activities Spermine has a specific effect on actin. Isolated G-actin from muscle and other cells incubated with spermine polymerizes to F-actin (Oriol-

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Audit, 1978). When spermine is microinjected into normal locomoting A . proteus (Fig. 13a) a distinct cytokinetic activity is induced (Fig. 101, F ) while such cells form one, or even two, cleavage furrows within the intermediate cell region (Fig. 13b). After 4- 10 minutes the constrictions are completed, and the resulting nucleated cell parts show normal streaming and locomotion (Fig. 13c, arrow), whereas the enucleated cell parts remain stationary and later degenerate (Fig. 13c, asterisk). The intracellular distribution of actin during induced cytokinesis as followed by fluorescent-analog cytochemistry shows the formation of a ringlike structure within the cleavage furrow (Fig. 13d-h, arrowheads), which disappears immediately after cytokinesis is completed (Fig. 13i). At the same time, the distinct actin caps polymerized at the two adjacent poles of the dividing cell parts (Fig. 13 d-h, asterisks) are also destroyed (Fig. 13i). Electron microscopy of cells fixed during spermine-induced cytokinesis reveals numerous well-aligned actin and myosin filaments as a specialized manifestation of the cell cortex in the developing cleavage furrow (Fig. 13 k-m). These results resemble similar observations in dividing eggs (Schroeder, 1975; Wang and Taylor, 1979) and demonstrate that cycles of actin and myosin polymerization and depolymerization as well as the crosslinking of preexisting microfilaments represent a basic mechanism in the generation of motive force not only for normal locomotion but also for cytokinesis of A . proteus.

5 . Induced Endocytotic Activities During pinocytosis induced by the external application of different polycations (Chapman-Andresen, 1962; Josefsson, 1975; Stockem, 1977), A . proteus attains a spherical shape and produces numerous short hyaline pseudopodia at the cell periphery (Fig. 14e). Most pseudopodia contain channellike membrane invaginations (arrow) which undergo vesiculation

FIG. 13. Influence of microinjected spermine (25 mM) on the cell shape and microfilament organization of A . proreus. a-c: Spermine-induced local contraction of the filament cortex in the intermediate cell body region (b. arrowheads) and formation of a nucleated (c, arrow) and enucleated (c, asterisk) cell fragment by cytokinesis. Time distance a-c = 6 minutes. d-i: Fluorescence micrographs of a cell 30 minutes after microinjection of IAF-actin and subsequent injection of spermine to show the formation of a contractile microfilament ring in the cleavage furrow (arrowheads) and actin caps at the two cell poles (asterisks). Time distance d-i, 4 minutes. k-m: Electron micrographs demonstrating the accumulation of actin (I) and myosin filaments (k asterisks, m) in the cleavage furrow a few minutes after microinjection of 25 mM spermine and GA fixation; (k) low and (I, m) high magnification. Arrow shows endoplasmic streaming direction. Scales a-i = 100 p n : k = 10 p m , 1, m = 0.2 Km. From Gawlitta et ul. (1981).

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FIG. 14. Pseudopodium formation and pinocytosis in A . proteus. Schematic drawings demonstrating the activity of the filament cortex during normal pseudopodium formation (a-d) and induced pinocytosis (a’-d’). The numbers I , 2, and 3 indicate cortex generations of different age. DIK (e) and fluorescence micrographs (f, g) demonstrating the formation of a pinocytotic channel (arrow) and of an endosome as well as the position of the microfilament system after staining with actin antibodies. Fc,, Fc2, Fc, show cortex generations of different ages. Scale = 50 pm. Abbreviations as in Fig. I . (a-d’) From Koster (unpublished); (f, g) from Stockem et al. (1983b).

and thus formation of endosomes (E). Staining of pinocytosing amebas with myosin and actin antibodies (Fig. 14f and g) reveals a distinct actomyosin layer at the internal face of the plasma membrane. In comparison to normal locomoting cells the layer is thicker and shows a uniform diameter along the entire cell, including the endocytotic channels and vacuoles. At the tip of hyaline pseudopodia the membrane-associated layer (Fig. 14f, Fc,) often splits into second (Fc,) and third sheathes

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(Fc,), which connect the channel with the internal pseudopodial surface. In pinocytosing specimens with a distinct separation of hyalo- and granuloplasm, the lowest sheath normally borders these two regions. Cinematographic investigations (Klein and Stockem, 1979) have shown that pinocytotic channel formation is the result of a special mode of pseudopodium formation. As described in Section V,A,3 (see Fig. 14 a-d), channel formation starts by active contraction of the microfilament system within a limited region of the cell surface (Fig. 14a’ and b’). After the formation of a short invagination by traction forces, the layer partially detaches from the plasma membrane, with the exception of the basic channel region (Fig. 14c’), i.e., a small pseudopodium is formed by the efflux of hyaloplasm. Comparable to the situation in normal pseudopodium formation, the channel elongates by the successive polymerization of a new microfilament layer after detachment of the previous one (Fig. 14d’; see also Koster, 1980; Taylor et al., 1980). A distinct phagocytotic activity can also be induced in A . proteus by applying both natural food organisms (Mast and Root, 1916; Jeon and Jeon, 1983; Prusch and Minck, 1985) and certain substances such as prostaglandins (Stockem et al., 1983a) or small peptides (R. D. Prusch, personal communication). During the first phase of phagocytosis, fluorescently labeled actin shows a uniform intracellular distribution comparable to the situation in relaxed cells. When capturing is initiated by the extension of large pseudopodia, however, considerable amounts of actin are concentrated at the two pseudopodial tips (Fig. 15a-d) near the existing connection between the food cavity (Ca) and the external medium (arrows). Immediately after fusion of the pseudopodia and formation of a food vacuole (Fig. 15e), however, the local fluorescence disappears, and a distinct filament cortex encircling the vacuole is formed (Fig. 15f). Whereas the diameter of the food vacuole slowly decreases, the filament cortex increases in thickness and density as the consequence of active contraction. These results are in good agreement with electron microscopic observations of Jeon and Jeon (1983), who found that the food vacuole membrane is in close contact with a prominent network of microfilaments, and that-comparable to the process of pinocytosismicrofilaments also play an important role in the generation of mechanical forces for phagocytosis.

B. PHYSICAL INVESTIGATIONS I . Influence of Light-Shade Differences Lateral illumination of A . proteus causes migration away from the source of light (Davenport, 1897; Mast, 1910), a method used to produce

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FIG. 15. Fluorescent-analog cytochemistry of experimentally treated A. proleus. a-f Fluorescence micrographs of a cell 30 minutes after microinjection of IAF-actin to demonstrate the induction of phagocytosis by externally applied 0.5 mg/ml prostaglandin. During formation of a food cavity the actin accumulates at the tip of two pseudopodia (a-d, arrow). After fusion of the pseudopodial tips and formation of a food vacuole (e), a filament cortex forms around the vacuolar membrane (f). Time, minutes-seconds after induction of phagocytosis. Scale = 100 p m . Abbreviations as in Fig. I . From Stockem et a / . (1983a).

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the orthotactic morphodynamic type (Wohlfarth-Bottermann and Stockem. 1966; Haberey and Stockem, 1971) and to study the influence of light-shade differences on the polarity and motile activity of large amebas (Schaeffer, 1917; Mast, 1932; Grebecki, 1980, 1981; Grebecki and Klopocka, 1981; Grebecki et al, 1981; Klopocka, 1982). Investigations on the photophobic response of A . proteus (Mast, 1932; Grebecki, 1980) showed that it is possible to induce local contraction or relaxation in any cell body region. Discrete illumination at the tip immediately stops the further elongation of an advancing pseudopodium, but has no influence on retracting regions. Polytactic amebas are incapable of moving across a luminous stripe intersecting a shaded background, because the contact with light rapidly induces a one-sided formation of lateral pseudopodia and, consequently, a change in the direction of cell locomotion parallel to the stripe (Fig. 16a-d). The motor and cell body polarity of amebas is also modified by the application of shade to different cytoplasmic regions during migration in the bright field. A complete reversal of polarity occurs when shade is applied to the uroid, which is then transformed into a new advancing front, whereas the former anterior region becomes the actual tail of the ameba (Fig. 16e-h). When the tip segment of a single pseudopodium of a polytactic ameba is constantly stimulated by shade, the cell changes its morphodynamic shape gradually and becomes strictly orthotactic. Polytactic specimens can also become orthotactic by coming into contact with straight or curved thin stripes of shade along which they permanently move (Fig. l6i-r). During such a locomotion the tip of the leading pseudopodium penetrates periodically into the illuminated surrounding field, quickly stops, and then resumes the previous direction. Therefore, oriented locomotion is controlled by the effect of light-shade differences, i.e., endoplasm always flows toward the cell region kept in shade because the cortical activity of any locally shaded part of ameba is less efficient than that of regions which are exposed to higher light intensity. More detailed information on the reactions of polytactic amebas to localized photic stimulation was gained in cinematographic studies by inducing lateral pseudopodia with transversally applied stripes of shade in three different (anterior, intermediate, and posterior) cell body regions (Fig. 16s, A-F). As a rule, pseudopodia in the anterior part of the cell are always formed more rapidly as compared to the intermediate and posterior regions. This finding also points to functional differences in the physiological state of the three regions of A . proteus (see also Wehland et al., 1979; Wang er a/., 1982). Moreover, the further development of such pseudopodia produced clear evidence for a cell region-dependent determination of the actual moving direction as measured by the deviation

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angle between the former and the new longitudinal axis: the deviation angle distinctly increases in value from the front (0-45") over the intermediate (45-90") to the uroid region (90" or more). When amebas have a choice between a small or wide deviation angle (Klopocka and Grebecki, 1982), the cells usually take advantage of the first possibility and refuse the second one (Fig. 16t). This result agrees with the observations of Sayers et al., (1979) on freely migrating amebas in which new pseudopodia are normally formed at an angle of about 35", as compared to the main axis of the cell body. Moreover, the fact that the formation of a new front always precedes the contraction of an old one clearly points to the essential role of localized relaxations of the cortical layer for the control of ameboid movement (Grebecki and Klopocka, 1981). Quantitative investigations measuring the relative changes of movement velocity at the uroid and front under stimulation revealed that light-induced contraction of any cell body region-with the exception of the front-results in a distinct acceleration of cell locomotion (Fig. 16u,A I-V). However, the rates of changes in velocity differ at the uroid and front in that the accelerating effect on the uroid gradually decreases from 134 to 104 when more anterior body regions are exposed to light, whereas the accelerating effect on the front distinctly increases from 115 to 152 under corresponding conditions. On the other hand, shade-induced relaxation of any cell body region generally retards the velocity of cell locomotion (Fig. 16u,B I-V). The effects are weaker, however, and, apparently, less regular than after local application of light, that is, only the reduction of light at the front increases the rate of uroidal retraction and frontal extension to 140 and 148, respectively. All observations on the behavior of amebas stimulated by light-shade differences are in good agreement with the chemical data and the postulation that motive force for protoplasmic streaming is generated by FIG. 16. Influence of light-shade differences on the motile activity and cell shape of A . (a-d) Modification of the moving direction as a consequence of the contact of a polytactic cell with an illuminated stripe of light. (e-h) Reversal of motor polarity of a polytactic cell induced by shading the uroid region. (i-r) Transformation of a polytactic cell into an orthotactic one by forcing the ameba to move along a stripe of shade. [s (A-F)] Schematic demonstration of the relationship between the direction of endoplasmic streaming and the mode of pseudopodium formation in different cell body regions. (t) Behavior of a cell migrating along a branched stripe of shade to demonstrate the preferred formation of pseudopodia at low angles between the axis of the pseudopodium and the cell body. [u (A,B)] Relative velocities of uroidal contraction and frontal extension measured after local application of light (A) and shade (B) in different cell body regions. Arrows show endoplasmic streaming direction. Abbreviations as in Fig. I . (a-s, u) From Grebecki (1980); (t) from Klopocka and Grebecki (1982).

proteus.

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the contraction of a cortical microfilament layer in the intermediate and posterior regions, whereas the anterior region is in a more relaxed state and controls the velocity and direction of movement. Moreover, these investigations also demonstrate that orthotactic and polytactic amebas represent organized units in which pseudopodium formation and retraction occur, not independently of one another, but in a highly coordinated manner (see Section IV,D).

2. Influence of Externally Applied Pressure Gradients In another series of experiments, externally applied positive and negative pressures have been used both as a direct test for the hydraulic pressure hypothesis (Mast, 1932; Allen et al., 1971; Rinaldi et al., 1975b; Grebecka, 1980) and as an attempt to measure the magnitude of the cytoplasmic pressure gradient (Allen and Roslansky, 1959; Kamiya, 1964; Kanno, 1964). It was demonstrated by the adaption of a double-chamber technique to amebas (Kamiya, 1964) and the imposition of a low positive pressure to the front of cells kept in agar capillaries (Rinaldi et al., 1975b) that small differences in hydrostatic pressure can cause the mass transport of endoplasm. The motive force for endoplasmic motion was found to range from 0.5 to 15 mm H,O column pressure. High negative pressure of 35 cm HzO, however, locally applied with a glass micropipette to different regions of the cell surface, does not cause reversal of streaming (Allen et al., 1971). The alternative application of positive and negative pressure induces oscillations in the velocity of flow only near the orifice of the capillary. Further, when one pseudopodium of a polytactic ameba is sucked into a narrow capillary with less than - 10 mm H,O, only some pseudopodia outside the pipette respond to the externally applied pressure, whereas others extend and retract independently of the stimulus (Rinaldi et al., 1975b). Such experiments were interpreted to mean that streaming cannot result from a positive pressure gradient generated along the longitudinal axis of the ameba (Allen et al., 1971; Allen, 1973; Allen and Allen, 1978). On the contrary, observations obtained in sucking experiments by introducing capillaries into the flowing mass of endoplasm are consistent with the classical concept of the hydraulic pressure hypothesis. A low negative pressure (below 5 cm H,O) applied directly to the interior of the tail region of A . proteus induces the reversal of streaming and the maintenance of this streaming direction even after the release of suction (Grebecka, 1980). Since a negative pressure locally applied to the outer surface of amebas by suction does not prevent pseudopodium extension even when the force is 10 times higher than normally required for endoplasmic streaming, the

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question was raised (Rinaldi et al., 1975b; Winet, 1975) whether the externally applied negative pressure is really transmitted to the cell interior. The distinct folding of the cell surface region kept inside the pipette as well as the slight influence of alternative positive-negative pressures on the endoplasmic flow suggest rather that the application of negative pressure to the outer surface of the cell locally induces a strong contraction of the cell cortex (Grebecka. 1980). The contraction of the sucked part inside the pipette interferes with contractions taking place in other free, withdrawing pseudopodia, and thus, interferes with the escape behavior of the ameba. For such reasons, these experiments seem inconclusive as tests of the different theories of ameboid movement. On the other hand, locally applied pressure is one of the factors capable of influencing the polarity by modifying the structural and physiological intracellular gradient of a moving A . proterrs. C. CELLMODELS Glycerol-extracted specimens have been used in different studies as cell models to analyze the contractile properties of A . proleus and to test the different theories of ameboid movement (Hoffmann-Berling, 1956; Simard-Duquesne and Couillard, 1962; Schafer-Danneel, 1967; Rinaldi et al., 1975a; Opas and Rinaldi, 1976; Kuroda and Sonobe, 1981; Sonobe et ul., 1985). Upon application of a reactivation solution containing Mg-ATP at concentrations of 0.5 or 1 mM, the glycerinated models exhibit calcium-regulated reactions such as active contraction and cytoplasmic streaming but not organized ameboid movement (Sonobe et al., 1985). A necessary precondition for successful reactivation is a free calcium concentration of about M , a range also physiologically necessary for muscle contraction (Ebashi and Endo, 1968) and found to induce streaming in naked ameba cytoplasm (see Section V,D). The inhibition of motile activities was observed in glycerol-extracted cell models at lower or M Ca”, a distinct separation of higher calcium concentrations; at hyaloplasm and granuloplasm took place without any cytoplasmic M Ca ’+ , no reaction was induced with the streaming, and at exception of a contraction around the nucleus. AMP and ADP at a 0.5 mM concentration also failed to elicit a contraction of the models (Rinaldi et al., 1975a; Kuroda and Sonobe, 1981). and reactivation of the glycerinated amebas was inhibited by cytochalasin B or N-ethylmaleimide (Kuroda and Sonobe, 1981). Since the degree of Brownian movement of particles in glycerinated models is higher at 10 than at lo-” M Ca”, it was suggested that the deficiency of calcium induces the contractile gel

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state of the cytoplasm, whereas excess calcium causes the relaxed sol state. More recently, Sonobe et al. (1986) have shown the existence of some regulatory proteins which seem to control the rheological properties of the cytoplasm and, through that, active locomotion. The electron miscroscopic investigation of glycerol-extracted cell models of A . proteus demonstrated a rather reliable preservation of the ultrastructure of the cytoplasm (Rinaldi and Hrebenda, 1975; Rinaldi et al., 1975a; Sonobe et al., 1985; Sonobe and Kuroda, 1986). The thin filaments observed in such models were identified as actin by HMM binding (Ishikawa et al., 1969; Comly, 1973; Sonobe et al., 1985), whereas the morphological feature of the thick filaments (Hinssen et al., 1978) points to the myosin nature of this filament type. The reactivation of ameba models is accompanied by the assembly of thick filaments into more or less ordered aggregates and of thin filaments laterally connected to thicker bundles and fibrils (see also Section V,A,4.).

D. ISOLATED CYTOPLASM The first demonstration of a Ca2+ion requirement for active contraction in ameboid cells results from investigations on cytoplasm mechanically isolated from single specimens of Chaos carolinensis (Taylor et al., 1973, 1976a; Taylor, 1976). These experiments demonstrate that contractions of the isolated cytoplasm are elicited at free calcium ion concentrations above 7.0 x lo-’ M in the presence of Mg-ATP as the energy source. Physiological media can successfully control contractility in isolated ameba cytoplasm. In a stabilization solution which lacks ATP and exogenous calcium, isolated cytoplasm is motionless?highly viscoelastic, and exhibits a strain birefringence (especially when stretched) for up to 15 minutes. However, stabilized cytoplasm treated with relaxation solution which contains 1 mM disodium ATP gradually loses its birefringence and viscoelasticity. At the ultrastructural level, the relaxation solution causes dissociation of actomyosin fibrils into individual myosin aggregates and actin filaments. A decrease in cytoplasmic birefringence and contractility was also observed after incubation in stabilization solutions containing higher concentrations of [ethylenebis (oxoethylene nitrilo)] tetraacetic acid (EGTA) (Condeelis et al., 1976). On the other hand, calcium concentrations just below the threshold concentration of 1.0 x lo-’ M stabilized actin and myosin filaments in bundles. These results indicate that extremely low divalent cation concentrations cause a decrease in optical anisotropy , the number of visible fibrils, and the contractility of isolated cytoplasm of ameba. It is suggested that the viscoelastic proper-

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ties of stabilized cytoplasm are due to crossbridges between the actin and myosin filaments and that these connections are broken at extremely low divalent cation concentrations and under relaxation conditions. The fact that isolated cytoplasm can cycle repeatedly through stabilized, contracted, and relaxed states by manipulating the exogenous free calcium and ATP concentrations suggests that the rheological events occurring in the intact ameba during locomotion may be similar. The explanation for cyclic rheological changes in the intact cell would be, by analogy, a control mechanism exercised by endogenous free calcium ions, regulative proteins, and ATP on the interaction of actin and myosin.

VI. Concluding Remarks New methodological feasibilities provided the basis for numerous investigations on the three-dimensional organization of the cytoskeleton in a large variety of tissue cells by combining the replica technique with detergent extraction, rapid freezing, freeze drying, or critical-point drying (Heuser and Kirschner, 1980; Schliwa et al., 1982). According to a recent study (Christiani er al., 1986)on the microfilament system of A . proteus as obtained with the same techniques, this system shows a similar spatial and ultrastructural organization (Fig. 17) when correlated with results of the immunocytochemical (Section IV,B), fluorescent-analog cytochemical (Section IV,D) and conventional electron microscopic investigations (Section IV,C). Moreover, the study delivered clear evidence that at least three further types of filaments exist within ecto- and endoplasm of normal amebas: filaments measuring 2-4, 10-12, and 24-26 nm (Fig. 17a and c). PaulinLevasseur and Gicquaud (1984) already described the existence of single 10-nm filaments by negative staining in isolated cytoplasm of A . proreus. In addition to the observations of these authors it is now obvious that the 10-nm filaments run separately or slightly aggregated, especially through the ectoplasm in a loose network (Fig. 17c). The 10-nm filaments can be locally associated with another filament type of 24-26 nm thickness, which is hollow and resembles a microtubule (Fig. 17a). Although Preston (1985) recently succeeded in demonstrating an abundance of microtubules in the cytoskeleton of motile Acanthamoeba castellanii, microtubules in A . proteus were so far only detected during mitosis in the mitotic apparatus (Gromov, 1985). The 24- to 26-nm filaments described in the present article are more rare than the 5- to 7-nm and 10- to 12-nm filaments and seem to originate from MTOCs (Fig. 17a). Within such regions, the 24- to 26-nm filaments are sporadically interconnected with

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FIG. 17. Ultrastructure of the cytoskeleton in A . profeus after Triton X extraction and PtlC replication. (a) Survey electron micrograph demonstrating microtubules in surface view and cross-fraction (arrowheads) as well as other filamentous structures in the ectoplasm. (b) Internal face of the plasma membrane showing the three-dimensional organization of the cortical microfilament system. Arrows point to single thick filaments. (c) Microtubules and intermediate filaments in the ectoplasm connected by 2- to 4-nm-thick crossbridges (arrowheads). Scales = I p m . Abbreviations as in Fig. I . From Christiani et nl. ( 1986).

the 10- to 12-nm filaments by another filament type measuring 2-4 nm in diameter (Fig. 17c). Such superthin filaments were also described by Paulin-Levasseur and Gicquaud ( I 984) and can be compared to bridging filaments of a corresponding thickness observed in tissue culture cells (Schliwa and van Blerkom, 1981). So far, no data are available about the chemical nature of the 2- to 4-, 10- to 12-, and 24- to 26-nm filaments in A . proteus, because sufficient

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amounts of cells are difficult to obtain by classical cultivation methods. In addition, normally grown A . proterrs are carnivorous and always contain numerous ingested cells within food vacuoles (bacteria, flagellates, and ciliates) which impair a reliable protein analysis. It is therefore necessary to develop an axenic culture method for A . proteus for the isolation of cytoskeletal proteins and the production of specific antibodies against them (Sonobe el al., 1986). VII. Summary Recent results gathered by normal light microscopy, immunocytochemistry, fluorescent-analog cytochemistry, and electron microscopy have allowed an improved interpretation of ameboid movement and related phenomena. 1 . The contractile system responsible in Amoeba proteus for the generation of motive force for protoplasmic streaming and a large variety of dynamic activities is represented mainly by a thin cortical filament layer at the cytoplasmic face of the cell membrane (Fig. 181). During normal locomotion this layer exhibits a distinct structural and physiological polarity with three different zones: a zone of reformation at the front (A), a zone of contraction in the intermediate cell region (B), and a zone of destruction at the uroid (C). 2. Two types of filaments participate in the formation of the cortical layer: (1) randomly distributed thin (actin) filaments exhibiting a parallel orientation in the anterior (Fc,) and a disordered arrangement in the intermediate and posterior cell region (Fc2; see also Fig. 17b), and (2) thick (myosin) filaments in close association with F-actin and mostly restricted to the intermediate and posterior cell region (Fc?). 3. The internal hydraulic pressure generated by localized active contraction of the cortical layer is transmitted to the endoplasm via the cell membrane and converted into directed streaming by a gel-sol gradient of decreasing viscosity between the uroid and the front. Calcium ions, ATP, and regulative proteins (profilin and a kinase) play an essential role in controlling both the interaction of actin and myosin and the sol-gel state of the cytoplasmic matrix. 4. Any alterations externally induced in the polarity of the cortical filament system by chemical or physical stimulation and inhibition cause immobilization of the amebas (Fig. 1811) with characteristic changes in (1) cell shape (spherulation and cell flattening), (2) membrane dynamics (cytotic and cytokinetic activities), and (3) cytoplasmic organization (hyalogranuloplasmic separation).

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a

b

C

d

e

FIG. 18. Schematic drawings to summarize the results on the organization of the microfilament system in normal locomoting (I) and resting A. proteus (11) as well as during pseudopodium formation (111). (A, B, C) Anterior, intermediate, and posterior segment of the microfilament system. (Fc,, Fc2) Ultrastructural organization of the microfilament system in the anterior and posterior cell body region. 1, Solated cytoplasm; 2, gelated cytoplasm; 3, newly formed microfilament system; 4, contracted microfilament system; 5 , destructing microfilament system; arrows show endoplasmic streaming direction. For description, see Section VII. Abbreviations as in Fig. I .

5 . The formation of new pseudopodia, an important event for the conversion of endoplasmic streaming into ordered ameboid movement, is initiated by a local relaxation of the ectoplasmic cylinder. This relaxation includes (1) separation of the cortical layer from the plasma membrane (Fig. 18111, a + b, c + d), (2) reformation of a new layer at the

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pseudopodial tip (Fig. 18111, b -+ c, d + e), (3) destruction of the old layer at the hyalogranuloplasmic border (Fig. 18111, c,e), and (4) alternate solation (Fig. 18111, b and d) and gelation (Fig. 18111, c and e) of the hyaloplasm between the layer and the plasma membrane. The retraction of pseudopodia is accomplished by a local contraction of the cortical layer in conjunction with a simultaneous gel-sol transformation of the ectoplasmic cylinder. 6. The expression of a rather complex cytoskeleton which is composed not only of microfilaments and associated proteins, but also of intermediate- and microtubularlike structures has to be considered in future discussions concerning the mechanisms of ameboid movement.

ACKNOWLEDGMENTS The investigations summarized in this review were partly supported by a grant of Deutsche Forschungsgemeinschaft (Stockem 126/4-2) and a scholarship of the Alexander von Humboldt-Stiftung for Dr. A. Klopocka. The authors wish to thank Dipl. Biol. M. Christofidou for reading the manuscript.

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Paulin-Levasseur, M. P., and Gicquaud, C. (1984). Eur. J. Cell Biol. 34,60-63. Pollard, T. D., and Ito, S. (1970). J . Cell Biol. 46,267-289. Pollard, T. D., and Korn, E. D. (1973). J . Biol. Chem. 248, 448-450. Porter, K., Beckerle, M., and McNiven, M. (1983). Mod. Cell Biol. 2, 259-302. Preston, T. M. (1985). Cell Biol. lnt. R e p . 9, 307-314. Prusch, R. D. (1981). I n “Membrane Physiology of Invertebrates” (R. B. Podesta and S. F. Timmers, eds.), pp. 19-36. Dekker, New York. Prusch, R. D., and Minck, D. R. (1985). Cell Tissue Res. 242, 557-564. Rinaldi, R. A., and Hrebenda, B. (1975). J . Cell Biol. 66, 193-198. Rinaldi, R. A., and Jahn, T. L. (1963). J . Protozool. 10, 344-357. Rinaldi, R. A., Opas, R., and Hrebenda, B. (1975a). J . Protozool. 22, 286-292. Rinaldi, R. A,, Korohoda, W., and Wohlfarth-Bottermann, K. E. (1975b). Acta Protozool. 14, 363-369. Sayers, Z., Roberts, A. M., and Bannister, L. H. (1979). Acta Protozool. 18, 313-325. Schaeffer, A . A. (1917). Biol. Bull. 32, 45-74. Schafer-Danneel, S. (1967). 2. Zellforsch. 78, 441-462. Schliwa, M., and van Blerkom, J. (1981). J . Cell B i d . 90, 222-235. Schliwa, M., Pryzwansky, K. B., and van Blerkom, J. (1982). Philos. Truns. R. Soc. London Ser. B 299, 199-205. Schroeder, T. E. (1975). I n “Molecules and Movement” (S. Inouk and R. E. Stephens, eds.), pp. 305-332. Raven, New York. Schulze, F. E. (1875). Arch. Mikrosk. Anat. 11, 329-353. Shard-Duquesne, N., and Couillard, P. (1962). Exp. Cell Res. 28, 85-91. Sonobe, S., and Kuroda, K. (1986). Protoplasma 130, 41-50. Sonobe, S., Hatano, S., and Kuroda, K. (1985). I n “Cell Motility” (H. Ishikawa, S. Hatano, and H. Sato, eds.), Vol. 2, pp. 271-282. Univ. of Tokyo Press, Tokyo. Sonobe, S., Takahashi, S., Hatano, S., and Kuroda, K. (1986). J . Biol. Chem. 261, 14837- 14843. Stockem, W. (1969). Histochernie 18, 217-240. Stockem, W. (1972). Acta Protozool. 11, 83-93. Stockem, W. (1973). Z . Zellforsch. 136, 433-446. Stockem, W. (1977). In “Mammalian Cell Membranes” ( G . A. Jamieson and D. M. Robinson, eds.), Vol. 5 , pp. 151-195. Butterworths, London. Stockem, W., and Hoffman, H. U. (1986). Acta Protozool. 25, 245-254. Stockem, W., Wohlfarth-Bottermann, K. E., and Haberey, M. (1969). Cytobiologie 1, 37-57. Stockem, W., Weber, K., and Wehland, J. (1978). Cytobiologie 18, 114-131. Stockem, W., Hoffman, H. U . , and Gawlitta, W. (1982). Cell Tissue Res. 221, 505-519. Stockem, W., Hoffman, H. U., and Gruber, B. (1983a). Cell Tissue Res. 232, 79-96. Stockem, W., Naib-Majani, W., Wohlfarth-Bottermann, K. E., Osborn, M., and Weber, K. (1983b). Eur. J . Cell Biol. 29, 171-178. Stockem, W., Naib-Majani, W., and Wohlfarth-Bottermann, K. E. (1984). Cell B i d . l n t . R e p . 8, 207-213. Szamier, P. M., Pollard, T. D., and Fujiwara, K. (1975). J. Cell Biol. 67, 424a. Taylor, D. L. (1976). In “Cell Motility” (T. D. Pollard, J. Rosenbaum, and R. Goldman, eds.), pp. 797-821. Cold Spring Harbor Lab. Press, Cold Spring Harbor, New York. Taylor, D. L. (1977). Exp. Cell Res. 105, 413-426. Taylor, D. L., and Fechheimer, M. (1982). Philos. Trans. R. Soc. London Ser. B 299, 185-197. Taylor, D. L., and Wang, Y. L. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 857-861.

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Taylor, D. L., and Wang, Y. L. (1980). Nature (London) 284, 405-410. Taylor, D. L., Condeelis, J . S., Moore, P. L., and Allen, R. D. (1973). J.Cell Biol. 59, 378-394. Taylor, D. L., Moore, P. L., Condeelis, J . S., and Allen, R. D. (1976a). Exp. Cell Res. 101, 127- 133. Taylor, D. L., Rhodes, J. A., and Hammond, S. A. (1976b). J. CellBiol. 70, 123-143. Taylor, D. L., Condeelis, J. S., and Rhodes, J. A. (1977). Prog. Clin. B i d . Res. 17,581-598. Taylor, D. L., Hellewell, S. B., Virgin, H. W., and Heiple, J. (1979). I n “Cell Motility” (S. Hatano, H. Ishikawa, and H. Sato, eds.) pp. 363-377. Univ. Press of Tokyo, Tokyo. Taylor, D. L., Wang, Y. L., and Heiple, J. M. (1980). J. Cell B i d . 86, 590-598. Thoenes, W.. Langer, K. H., Heifer, U., and Romen, W. (1970). Virchow Arch. Abt. B . Zellpathol. 5, 124-143. Wang, Y. L.. and Taylor, D. L. (1979). J . Cell Biol. 82, 672-679. Wang, Y. L., and Taylor, D. L. (1980). J. Hisrochem. Cytochem. 28, 1198-1206. Wang, Y. L., Lanni, F., McNeil, P. L., Ware, B. R., and Taylor, D. L. (1982). Proc. Nail. Acad. Sci. U . S . A . 79, 4660-4664. Wehland, J . , Weber, K., Gawlitta, W., and Stockem, W. (1979). Cell Tissue Res. 199, 353-372. Weihing, R. R. (1979). Math. Achiev. Exp. Pathol. 8, 42-109. Winet, H. (1975). I n “Molecules and Cell Movement” (S. lnouC and R. E. Stephens, eds.), p. 257. Raven, New York. Wise, G. F., and Flickinger, C. J. (1970). Exp. Cell Res. 61, 13-23. Wohlfarth-Bottermann. K. E. (1964). I n t . Rev. Cytol. 16, 61-131. Wohlfarth-Bottermann, K. E., and Stockem, W. (1966). Z . Zellforsch. 73, 444-474. Wolpert, L. (1965). S y m p . Soc. Gen. Microbiol. 15, 270-293. Wolpert, L., and Gingell, D. (1968). Symp. SOC. Exp. B i d . 22, 169-198. Wolpert, L., and O’Neil, C. H. (1962). Nature (London) 196, 1261-1266.

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INTEKNATIONAL KEVIEW OF CYTOLOGY. VOL. 112

The Role of Hepatocytes and Sinusoidal Cells in the Pathogenesis of Viral HepaGtis PATRICIAS. LATHAM University o j Maryland Hospital and Baltimore Veteran’s Administrution Hospital, Baltimore, Maryland 21201

I. Introduction Knowledge of the pathogenesis of human viral hepatitis due to types A, B, non-A, and non-B virus continues to be limited to descriptions of liver pathology and the evolution of serum and tissue viral markers, where available. Progress in delineating the pathogenesis of human hepatitis is limited by the long and variable incubation period for the viruses, the limited availability of appropriate animal models, and the lack of liver tissue cultures supportive to viral replication. The recent identification of animal models for the hepadnaviruses, which share many features in common with hepatitis B, promises to advance our understanding of the human disease (Summers and Mason, 1982). There is already available, however, a burgeoning experience with the pathogenesis of other viral models of hepatitis in common laboratory animals. This review will present an overview of that literature, concentrating on the early interactions of viruses with sinusoidal and parenchymal cells of the liver in the acute phase of hepatitis.

11. Role of Liver Architecture in the Pathogenesis of Viral Hepatitis A. NORMALLIVERARCHITECTURE The liver is uniquely disposed in the circulation to play a major role in the host response to many viral infections. The sinusoidal capillary network of the liver is interposed between the gut and the systemic circulation, and the reservoir capacity of this capillary system at any one time is nearly one-third of the circulating volume. The blood enters via branches of the veinous-portal and hepatic-arterial circulations, which are contained, along with lymphatics, in fibrous tracts. These tracts branch 185 Copyright 0 198X by Acddemic Press. Inc All right? of reproduction in d n y form reserved

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into ever finer subdivisions until the blood flows out of the vessels into the sinusoidal capillary network in the parenchyma. The blood percolates along these capillary channels to exit at terminal venules of the hepatic vein, which in turn drain into the inferior vena cava. The capillary sinusoids circulate between rows of hepatocytes which are aligned in plates one to two cells in thickness. The architectural design is such that each hepatocyte has at least one complete surface exposed to capillary flow beneath a sinusoidal lining of endothelial cells (refer to Figs. 1-4). 1 . Sinusoidal Cells

There are four types of sinusoidal cells of the liver, the so-called nonparenchymal cells: the endothelial lining cells, the fixed tissue macrophages, the lipocytes, and the neuroendocrine-like cells. In one study, the distribution of these cell types in the nonparenchymal cell population is reported to be 48% endothelial cells, 39% Kupffer cells, and 13% lipocytes (Widmann et al., 1972). The entire population of sinusoidal cells makes up approximately 30% of the total number of cells in the liver. The endothelial cells which line the sinusoidal channels of the liver are unlike those elsewhere in that they have numerous fenestrae through their cytoplasm, like a sieve plate, and they have no electron-dense basement membrane beneath them (refer to Figs. 2-4). Under normal physiological conditions, these pores admit particles and molecules with a diameter of approximately 100 nm or less (Fahimi, 1982). The structure of the endothelial cells and their relationship to the underlying hepatocytes provide unique access of serum contents to a relatively undisturbed layer between these cells and the underlying parenchymal cells. This layer, referred to as Disse’s space, contains relatively few strands of reticulin and numerous microvilli of the hepatocytes. Morphologically these endothelial cells are actively pinocytotic and contain lysosomes. It is presumed that they have functions similar to endothelial cells in other sites (Fahimi, 1982). Interspersed with the endothelial cells and projecting into the sinusoidal space, and occasionally into Disse’s space, are numerous reticuloendothelial cells, the Kupffer cells, which represent the largest organ population of fixed tissue macrophages in the body (Walker, 1976) (refer to Figs. 2B-4). The Kupffer cells are actively pinocytotic and phagocytic and are surrounded by a microenvironment, described as the “fuzzy coat,” which many be important in their ability to bind and adequately phagocytose various particles (Jones and Summerfield, 1982). The Kupffer cells are able to remove as much as 90% of the particulate matter entering the circulation, including viruses (Mims, 1957, 1964). The macrophages also have important accessory and effector roles in the immune response.

FIG. 1. Normal hepatocellular architecture is illustrated in this photomicrograph of a lobule from a normal human liver biopsy. The portal and periportal areas, or zone 1, are at the right, and the pericentral area, or zone 3, is at the left. Note the plates of hepatocytes 1-2 cells in thickness and the intervening sinusoidal spaces. x 110.

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FIG. 2. (A) This scanning electron photomicrograph was obtained after fixation perfusion of mouse liver. It demonstrates a sinusoidal space lined by an endothelial cell (E). Note the nuclear area of the endothelial cell beneath the letter E, and the sieve platelike fenestrations through its surface (arrow). Disses’ space beneath the endothelial cell is filled with the microvilli of two hepatocytes (H) which are seen there. The bile canaliculus of these hepatocytes is seen in the lower left of the photo. x8240. (B) This scanning electron photomicrograph obtained after fixation perfusion of mouse liver shows the body of an endothelial cell (E) lining the sinusoidal space with two hepatocytes beneath it (H). A Kupffer cell (K) is sitting above the endothelial cell within the sinusoidal space. X 11,250.

FIG. 3. This transmission electron photomicrograph of perfusion-fixed mouse liver demonstrates a sinusoidal space lined by an endothelial cell (E) and containing a portion of a Kupffer cell (K) in which a large debris-containing lysosome is evident. Hepatocytes surround the sinusoidal space (H). Note the gaps in the endothelial lining (arrows) caused by the fenestrae of the endothelial cell. The hepatocyte microvilli fill Disses’ space beneath the endothelial lining cell. A bile canaliculus is evident at the border between two hepatocytes just to the left of the endothelial cell

FIG.4. This transmission electron photomicrograph obtained after perfusion tixation of mouse liver demonstrates the features of a Kupffer cell filling the sinusoidal space. Note that the cell is separated from the surrounding hepatocytes by a fenestrated endothelial cell sinusoidal lining. The Kupffer cell is filled with many lysosomes containg debris. x 15,960.

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The other nonparenchymal (NP) cells in the sinusoids include the lipocytes (fat-storing cells) and the Pit cells (cells containing neuroendocrine-like granules). The lipocytes can be identified by their content of lipid-containing vacuoles rich in vitamin A. These cells are believed to serve as storage sites for that vitamin; they are also believed to play a role in fibrogenesis (Fahimi, 1982). Lipocytes are not actively pinocytotic or phagocytic, and they are not known to have a role in the pathogenesis of viral infections. The Pit cells are difficult to identify under ordinary circumstances and their physiologic function in the liver is not known.

2. Hepatocytes Hepatocytes, or parenchymal cells, of the liver contribute approximately 70% to the total number of cells in the organ. The hepatocyte is an active metabolic cell with numerous functions: synthesis, detoxification, storage, and excretion. The parallel rows and plates of hepatocytes and their interspersed sinusoids radiate out from the incoming blood flow at the portal tracts to the terminal venules in units of liver tissue which are functionally referred to as acini and histologically referred to as lobules (refer to Fig. 1). The area of the lobule adjacent to the portal tract is referred to as periportal, or as functional zone 1. The area surrounding the terminal venule is referred to as centrilobular, or as functional zone 3. The area of the lobule between these is referred to as midzonal, or as functional zone 2. The cell surface facing the sinusoids is markedly amplified by the presence of numerous microvilli projecting into Disse’s space (refer to Figs. 2 and 3). The hepatocytes are arranged in rows with a distinctive polarity relative to the sinusoidal surface. The polarity of the plasma membrane in the hepatocyte is established by the presence of tight junctions which divide the plasma membrane of the hepatocyte into a basolateral surface with exposure to the sinusoid, and an apical, or canalicular surface. The apposition of canalicular surfaces between pairs of hepatocytes along a plate of cells creates a luminal channel called the canaliculus. Bile formed by the hepatocytes flows through the canaliculi countercurrent to the circulation into ever-larger channels which are eventually lined by epithelial cells of the bile ducts. The bile ducts are supported by the same fibrous septas which contain branches of the portal vein and hepatic artery, the so-called “portal tracts.” In addition to fibroblasts, the connective tissue of the portal tracts usually contains a few lymphocytes and macrophages. The liver is a common site of viral clearance and replication within the body. Hepatotrophic viruses home to the liver and from that site mount a systemic viremia associated with lethal cellular destruction in the liver

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itself. Alternatively, the liver may fail to effectively clear virus from the circulation, or it may enhance the circulatory viremia; this allows the virus to overwhelm host defenses and to arrive at its true target site of replication and injury. In either case, the hepatocyte may serve as a potential target cell for viral replication in the liver, but the virus can reach the liver only by passing through the gauntlet of sinusoidal cells which reside in the liver capillary network.

B.

PATHOLOGY OF

LIVERARCHITECTURE I N ACUTE VIRAL HEPATITIS

Heterogeneity in zonal characteristics and hepatocellular function in the liver are known to be present and to contribute to a characteristic pathology due to some etiologies, or stages, of hepatitis. Hepatitis due to ischemia, for example, is associated with centrilobular hepatocellular necrosis and collapsed sinusoids, which characteristically occur without inflammatory response. Hepatitis due to direct hepatotoxins is typified by centrilobular hepatocellular macrovesicular fatty change and hepatocellular necrosis, also without much inflammatory response. Hepatitis due to most viral etiologies, however, is initially characterized by diffuse, but focal, cytopathic effects. A tendency for these early lesions to appear in periportal areas after intravenous inoculation of virus has been attributed to the fact that the periportal Kupffer cells have the initial opportunity to clear the bulk of the virus from the incoming circulation. Kupffer cells have been noted to engulf leukocytes and viruses and the following types of morphological changes have been described: nuclear inclusions, as seen after infection by mouse hepatitis virus (MHV) (Miyai et al., 1962), early and selective necrosis and lysis, as seen after infection with frog virus 3 (FV3) (Kirn et ul., 1982a), acidophilic Coucilman bodies, as seen after infection with yellow fever virus (YFV) (Mogensen, 1979), and hyperplasia due to proliferation and/or recruitment of monocytes, as seen in infection by MHV (Boss and Jones, 1963; Jones and Cohen, 1963). Focal zones of hepatocellular necrosis appear next, as described in infection with ectromelia (Mims, 1964). These necrotic foci may be associated with an inflammatory reaction presumably due to the stimulation of an immune response, or there may be no inflammatory reaction, where presumably there has been no immune response elicited. These necrotic foci may remain small with eventual recovery of normal tissue as local regulatory factors control the spread of virus, or a progressive increase in the size of the foci may ensue, with or without an inflammatory response, until a fulminant, lethal hepatitis results in death of the animal. Figure 5 illustrates such a course of infection after subcutaneous inoculation of susceptible 3- to 4-week-old mice with Punta Tor0 virus of

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the Bunyavirus family (Latham, unpublished observations). When very large doses of virus are presented to the liver, they may overwhelm the initial restriction to Kupffer cells and infect hepatocytes directly; this results in a rapidly progressive, diffuse hepatocellular necrosis, as described after the introduction of large concentrations of ectromelia (Mims, 1964), or Rift Valley fever (RVF), another virus of the Bunyavirus family (Mims, 1957).

111. Role of Liver-Derived Cells in the Pathogenesis of

Viral Hepatitis A. ISOLATION A N D CULTURE OF LIVER-DERIVED CELLS In considering virus-liver interactions in producing hepatitis, it is important to remember that much of what we understand of pathogenesis is derived from in vivo and in vitro studies involving peritoneal macrophages or macrophages from other sites. It is only in the last 10-15 years or so that methods have been developed to study virus-liver cell interactions directly in vitro. It is now possible to isolate, in a relatively homogeneous population, each of the liver-derived cells that would appear in a mixed-cell suspension of liver-hepatocytes, Kupffer cells, endothelial cells, and lipocytes. The standard technique employed today involves an in vivo double perfusion of the liver via the portal vein with calcium-free and then collagenase-containing solutions to separate the liver-derived cells from the underlying superstructure and from one another (Knook et al., 1976, 1977; Sleyster et al., 1977; Seglen, 1976). The liver is then removed in toto and the cells are gently teased away from the capsule with subsequent separation of individual cell types by differential centrifugation and elutriation, with or without the use of pronase to cause a differential lysis of hepatocytes, and with preservation of nonparenchymal cells. The resultant populations of liver-derived cells can each be retrieved at approximately 90% or better purity, and greater than 90% viability. The primary cultures of these cells do not divide in routine culture, but can survive for periods ranging from several days to several weeks, depending on the media, substrate, and hormonal supplementation. I . Role of Kupffer Cells a. Characteristic Features of Kupffer Cells. The study of isolated Kupffer cells reveals that these macrophages have many similarities, but also some important dissimilarities from other macrophages and monocytes (refer to Table I) (LePay et al., 1985). It is important to remember

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TABLE I MACROPHAGE CHARACTERISTICS EXPRESSED BY KUPFFERC E L L S Fixed-tissue macrophage Active phagocytosis la expression necessary for antigen presentation in the immune response Deficient in generating reactive oxygen intermediates Peroxidase and esterase positive Fc and C3b receptors Capable of limited proliferation

this fact in considering the role of Kupffer cells in various models of viral hepatitis, since this role is often inferred from in vitro studies of virus-macrophage interaction using macrophages from other sources. The Kupffer cell has some features in common with activated macrophages, including enhanced phagocytic capability and the ability to function as both an accessory cell and an antigen-presenting cell in the immune response (LePay et a / . , 1985; Rogoff and Lipsky, 1980; Richman et al., 1979). The Kupffer cell does not have the capacity to spontaneously lyse tumor cells, or cells containing intracellular organisms, but it can, like other macrophages, be activated to do so (Diamond, 1982). The Kupffer cell is also unable to generate reactive oxygen metabolites, as most other macrophages are able to do (LePay et al., 1985; Shiratori et a/., 1984; Richman et ul., 1979). Kupffer cells can be induced to proliferate in vitro by the action of colony-stimulating factor, a lymphokine (Decker et a / . , 1985). It is likely that Kupffer cells also divide at a low level in vivo to maintain a steady state of tissue macrophages in the liver sinusoids. However, the large number of macrophages seen in the inflammation of an acute viral infection is more likely due to monocytic recruitment from the circulation than Kupffer cell proliferation (Decker et al., 1985).

b. Clearance of Circulating Virus by Kupffer Cells. Kupffer cells are uniquely situated to play a particularly pivotal role in the pathogenesis of systemic and hepatic viral infections, since they reside in the sinusoids of the first major capillary network between that which is absorbed from the gastrointestinal tract and entry into the systemic circulation. Kupffer cells represent the largest population of phagocytic macrophages in the circulation and as such, they are largely responsible for the clearance of particulate matter (Jones and Summerfield, 1982). As particulate matter, viral particles are cleared predominantly by Kupffer cells, with an efficiency which is directly proportional to their increasing size (Mims, 1964). The clearance of RVF virus, for example, may be as great as 50-150 times the saturating inoculum (Mims, 1956a,b). A variable resid-

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ual of virus, which is referred to as the “uncleared tail,” remains in the circulation after passage through the system (Mims, 1964). The “uncleared tail” is composed of virus which is not as readily phagocytosed due to its association with red blood cells, or perhaps an element of reversible release from phagocytic vacuoles. In a nonspecific manner, macrophage phagocytosis of virus may be enhanced or inhibited by factors which increase or decrease the phagocytic activity of these cells, for example, Bacillus Calmette-Guerin or endotoxin lipopolysaccharide may stimulate phagocytotic activity and increase viral uptake (Potterfield, 1985; Hirsch et a f . , 1970; Gledhill, 1959), but thorotrast (Mims, 1964), antimacrophage serum (Mogensen, 1979), carbon blockade, methyl palmitate, or silica (Zisman et al., 1970; Mogensen, 1978) may inhibit it and decrease or prevent viral uptake. The viral uptake itself may also inhibit uptake of additional virus, as has been described for FV3 (Gut, unpublished observations, described in Kirn et al., 1982a). In a more specific manner, viral uptake may be enhanced by receptors for the virus which reside on this cell, as has been suggested for vesicular stomatitis virus (VSV) (Brunner et af., 1960). Viral uptake may also be specifically enhanced by antibody-dependent endocytosis (ADE) (Porterfield, 1985), where the antibody associated with the virus binds to the cell via the Fc receptor in the case of IgG antibody, or by the complement receptor in the case of IgM-viral-complement complexes. The virus-antibody complex which is so bound to the cell may then be incorporated in phagocytic vacuoles for later internalization. If the macrophage is permissive to replication of a particular virus, antibody-enhanced uptake of the virus will increase viral replication; if, however, the macrophage is nonpermissive to viral replication, such uptake will tend to further decrease the virus available to the permissive target cell. Once exposed to the virus, the Kupffer cell has the potential to be a permissive or nonpermissive host cell to viral replication. The Kupffer cell-virus interaction may also result in secondary extrinsic effects on surrounding cells, or affect the subsequent interaction of the virus with surrounding cells (Stohlman et al., 1982); in case of the Kupffer cell, the surrounding cells are endothelial cells and hepatocytes. The interplay of these direct viral effects and host responses in the liver may result in several possible outcomes including recovery, persistent hepatitis, or fulminant liver failure and death, depending on a delicate balance of variables. c. Kupffer Cell-Virus Interactions. The interactions of Kupffer cells with viruses able to produce a lethal hepatitis may be nonparticipatory , passive without injury to the host macrophage, passive with injury to the host macrophage, nonpermissive, or permissive to viral replication. This

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classification represents a modification of that presented or reviewed by Mims (1964), Sabesin and Koff (1974a,b), Morahan and Morse (1979), and Mogensen (1978) (refer to Fig. 6 and Table 11). In reviewing the synopsis of potential Kupffer cell-virus interactions, however, it is important to remember that most examples are drawn from studies using macrophages derived from sites other than the liver. The few reports which utilize Kupffer cells directly for in vitro investigation are still relatively few, including studies of vaccinia (Keller et ul., 1985; Kirn et al., 1980), FV3 (Kirn et al., 1980, 1982a), and MHV (Pereira et al., 1984a). i. Nonparticipatory Kupffer cell-virus interaction. A nonparticipatory interaction of Kupffer cells with a virus is one in which the virus is not effectively engulfed, or cleared, by the Kupffer cells from the circulation. The virus does not, therefore, enter the Kupffer cell and has essentially no interaction with that cell. Such a nonparticipatory interaction with macrophages has been described for low doses of poliovirus-I in mice. However, it has also been observed that when large doses of poliovirus-I are introduced, some virus can be cleared from the circu-

cellu

v w

FIG. 6. Kupffer cells-virus interactions.

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INTRINSIC

TABLE 11 RESPONSE OF KUPFFER CELLS TO VIRUSES

Virus

Response

Poliovirus- 1 Rift Valley fever Frog virus 3 Cytomegalovirus Herpes Simplex virus Vaccinia Ectromelia Mouse hepatitis virus Flaviviruses Influenza

Nonparticipatory Passive Nonpermissive

Permissive

lation to later appear in the bile, presumably by the action of macrophages (Mims, 1964). It remains uncertain whether the clearance of poliovirus in the liver is due to Kupffer cell uptake, since it may be possible for the virus-to bypass that cell and enter directly into hepatocytes through the endothelial fenestrae lining the sinusoids (Mims, 1964) refer to Figs. 2 and 3). ii. Passive Kupffer cell-viral interuction. A passive interaction of macrophage and virus is one in which the macrophage may phagocytose the virus and internalize it, but will not permit any phase of viral replication. The virus may be rereleased without altering its infectivity and without injury to the Kupffer cell. In this role, the Kupffer cell may modulate the course of a hepatotrophic virus by concentrating its delivery to the adjacent hepatocyte, but it does not play a key role in enhancing or restricting viral replication and spread. The macrophage is reported to play such a role in infection by the R V F virus (Mims, 1957). When mice are experimentally inoculated intravenously with a large dose of RVF virus, the virus is cleared via the liver, presumably by the Kupffer cells, with an efficiency of 50-150 times the saturating dose (Mims, 1956a,b). The mice die after 6 hours with fulminant hepatic necrosis. Since the time interval from inoculation of R V F to death of the mouse is insufficient for more than one sequence of viral replication, that replication must have occurred in hepatocytes to have resulted in such fulminant liver cell necrosis; replication of RVF in Kupffer cells, therefore, cannot be an essential part of the pathogenesis of this virus (Mims, 1964). A passive interaction of macrophage with virus may also occur in which the viral protein and lysosomal degradation products of the virus are toxic to the macrophage; liver macrophages appear to react to FV3 in such a

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20 I

manner. Frog virus 3 is able to produce a lethal hepatitis in mice and rats despite the fact that this virus is unable to replicate at body temperature (Kirn et al., 1982a). In infection by FV3, Kupffer cells take up the virus by pinocytosis, phagocytosis, and membrane fusion (Gendrault et a/., 1981; Kirn et al., 1982a). The viral proteins are subsequently toxic to macrophages without evidence for viral replication; in fact, the inoculation of viral proteins alone produces a similar response. The toxic proteins of FV3 result in an inhibition of RNA and protein synthesis in the macrophage with complete sinusoidal cell necrosis, including adjacent endothelial cells, which have a similar viral uptake and subsequent necrosis in vitro (Kirn et al., 1982b; Gut et al., 1984). Within 3 hours after intravenous administration of the virus, incomplete viral proteins appear in hepatocytes (Kirn et al., 1982a; Gut et al., 1984). Since FV3 viral proteins have not been demonstrated to cause hepatocytolysis (Gut et al., 1984), the hepatic necrosis following FV3 infection must be related to an additional factor. There is evidence to suggest that this additional factor is endotoxin which is endogenously absorbed from the gut (Kirn et al., 1982a; Gut et a / . , 1984) (refer to Section 111,D).Concurrent infection of FV3 and a hepatotrophic virus such as vaccinia can also produce a lethal hepatitis in a mouse normally resistant to vaccinia (Steffan and Kirn, 1979). iii, Nonpermissive infection of Kupffer cells with virus. In nonpermissive infections of Kupffer cells, the macrophages internalize the virus, but little or no infectious progeny are subsequently released. The macrophage/Kupffer cell in this case serves to restrict the spread of virus to other cells, as was described for the hepatitis due to cytomegalovirus (CMV) (Selgrade and Osborn, 1974; Mims and Gould, 1978) and Herpes Simplex virus (HSV) (Mogensen, 1978). Macrophages are also nonpermissive to infection by vaccinia; however, the ability of the virus to form complete virions, or to undergo an incomplete cycle of replication is more variable, dependent on the source and treatment of the macrophage (Morahan et al., 1979). For example, Kupffer cells isolated from human (Kirn et al., 1980) or rat liver (Keller et a/., 1983, and resident peritoneal macrophages (Silverstein, 1974) from mice are nonpermissive to vaccinia virus, resulting in an abortive cycle of replication (Keller et a/., 1985; Kirn et a / . , 1980). In contrast, elicited peritoneal macrophages from mice are permissive to vaccinia replication, unless the macrophages are obtained from preimmunized mice (Koszinowski et a / . , 1975). In infection of susceptible mice by CMV in vivo, immunofluorescence studies of the liver show that Kupffer cells are the cells first infected when low-virulence viral strains are introduced, with effective restriction of viral replication to a relatively few foci having little inflammatory

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reaction. On the other hand, when mice are infected with high-virulence strains of CMV, hepatocytes rather than macrophages show the first evidence of infection, suggesting that Kupffer cells are only infected secondarily (Mims and Gould, 1978). When peritoneal macrophages from these mice are infected in vitro, the macrophages show a low efficiency of infection, wherein a large inoculum of virus is required to produce a relatively small number of infectious progeny (Mims and Gould, 1978; Selgrade and Osborn, 1974). Although unstimulated peritoneal exudate cells (PEC)/macrophages are more readily infected than elicited or activated PEC in vitro (Mims and Gould, 1978), there is no difference in the minimal ability of macrophages to replicate this virus when they are derived from animals of differing genetic susceptibility, or when viral strains of variable virulence are studied (Selgrade and Osborn, 1974). When susceptible cells are exposed to virus in the presence of macrophages, however, a decrease in infectious foci is noted which is inversely proportional to the virulence of the viral strain (Mims and Gould, 1978). These data demonstrate that the ability of macrophages to sequester CMV exceeds their ability to replicate it in vitro, giving them an ultimately protective role in the course of CMV infection in vivo. It is likely that animals susceptible to CMV have macrophages which are less able to restrict infection. The in vivo histological findings in liver support a protective role for the Kupffer cells (Selgrade and Osborn, 1974). Susceptible strains infected with CMV in vivo evidence many more foci of infection with active inflammatory infiltration, compared to the few foci with little inflammation seen in resistant animals. Conditions which diminish the ability of macrophages to restrict the spread of CMV, such as treatment with silica, enhance the severity of infection (Selgrade and Osborn, 1974). The action of macrophages alone, however, is not sufficient to result in the survival of the animal. This is evidenced by the finding that CMV-infected athymic nude mice have an improved control of early infection, presumably related to their relatively active macrophages, but an increase in the final mortality, presumably related to their loss of cell-mediated immunity (Starr and Allison, 1977). The interaction of macrophages with HSV is very similar to that described for CMV; resistance in uiuo is positively correlated to restriction of infection by macrophages in vitro (Mogensen, 1977a,b, 1978; Stevens and Cook, 1971; Johnson, 1964). Macrophages support a nonpermissive viral infection (Stevens and Cook, 1971) with a protective effect in vivo which is lost on pretreatment with macrophage toxins such as silica (Mogensen, 1977a, 1978; Morahan et al., 1977; Schlabach et al., 1979; Zisman et al., 1970) or antimacrophage serum (Mogensen, 1979; Zisman et al., 1970), but which can be enhanced by pretreatment with

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reticuloendothelial system (RES) stimulators such as Corynebacterium parvum or pyran (Morahan et ul., 1977). The nonpermissive infection of macrophages by HSV is related to an abortive infection of these cells in which complete virions are not formed and virus-destructive products, such as lysosomal hydrolases, are released (Stevens and Cook, 1971). Those virions which are identified in these cells fail to survive. The abortive infection is specific for macrophages and associated with the death of these cells. There are no data at present on the specific interaction of Kupffer cells with herpes virus. There is one in uitro study of peripheral monocytes, however, which suggests that activated tissue macrophages may be able to support the viral replication which is ordinarily restricted in the unstimulated circulating monocytes (Daniels et al., 1978). This latter report describes an HSV infection of isolated human blood monocytes as an abortive one, associated with the formation of viral membranes containing no DNA. When these cells are infected with HSV after 7 days in culture, however, a permissive infection results. In this study, the investigators demonstrate an increase in acid phosphatase and lysosomes over time in the cultured monocytes characteristic of more mature tissue macrophages. The findings of this study suggest that activated tissue macrophages may differ from unstimulated monocytes in their response to some viruses, supporting replication of a virus which would normally be restricted in the unstimulated cell. This report underscores the importance of taking into account differences in virus-macrophage interactions dependent on the source and activity of the macrophage involved. A difference between Kupffer cell- and circulating monocytevirus interactions may be of particular importance in the natural pathogenesis of viral infection in the liver, sincemonocytes arerecruited to this site during infection andlor inflammation (Tanner el al., 1982). In contrast to studies with CMV suggesting that activated macrophages are better able to resist infection than unstimulated ones, studies with HSV demonstrate that elicited or activated macrophages are better able to replicate the virus than unstimulated ones, regardless of the underlying virulence of the viral strains (Hirsch et al., 1970; Lopez and Dudas, 1979; Frank rt a / . , 1978; Kirchner et al., 1978a,b). When the viral interactions with unstimulated macrophages are compared between susceptible and resistant mouse strains, however, macrophages from resistant mice are observed to restrict viral infection more effectively (Lopez and Dudas, 1979). The ability of activated macrophages to modulate in vivo infections by HSV is further supported by the studies of Kirchner et al. (1978a), who demonstrated that spleen cells (macrophages and lymphocytes) can

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support growth of HSV only if stimulated by endotoxin, which is known to activate macrophages and lymphocytes. When spleen cultures are derived from C3H/HeJ mice with a genetic resistance to the effects of endotoxin, the cells do not support viral growth in uitro, and these animals are more resistant to HSV infection in vivo (Kirchner et al., 1978a). In these experiments, T lymphocytes do not appear to play a direct role in the interaction of spleen cultures with HSV, since stimulation of these cells by concanavalin A (Con A) or phytohemagglutinin does not influence the course of the infection. Neither do T lymphocytes appear to be crucial to the course of in uiuo hepatitis, since thymectomy or antithymocyte serum does not influence resistance to this virus in uiuo (Schlabach et al., 1979). However, it is likely that T lymphocytes do play some role in the course of the hepatitis, since a persistent viremia is more frequently seen in uiuo when lymphocyte action is compromised by antilymphocyte serum (ALS), or after HSV infection of athymic nude mice (Mogensen, 1978; Zisman et al., 1970). iv. Permissive infection of Kupffer Cells with Virus. A permissive infection of macrophages, resulting in replication of the virus, is another common interaction of these cells with viruses causing hepatitis, such as ectromelia (Roberts, 1963; Mims, 1964), flaviviruses (Goodman and Koprowski, 1962), or mouse hepatitis virus (MHV) (Shif and Bang, 1970; Bang, 1978). The interaction of the macrophage with influenza virus is also an intrinsically permissive one, but the infection will be nonpermissive in macrophages from a resistant animal in the presence of interferon (Lindenmann et al., 1978) (refer to Section IV). The relative efficiency of macrophages, and particularly Kupffer cells, to serve as host cells to viral replication determines the concentration and timing of viral delivery to the hepatocytes, thereby influencing the rate of onset and severity of the resultant hepatitis. The route of infection, of course, particularly affects the outcome of viral hepatidites in which viral pathogenesis is mediated by replication in macrophages. In extrapolating the course of natural infection from experimental models, it is well to remember which route of inoculation best reporduces that which would be seen in nature. After subcutaneous injection, the virus is first accumulated in peripheral macrophages which deliver it, and possibly amplify its presentation, to regional lymph nodes. In lymph nodes, the virus is better able to incite an immune response, and macrophages there can host the early cycle(s) of viral replication with ultimate delivery of the viremia into the circulation. In the circulation, the virus is cleared predominantly by Kupffer cells which, if not overwhelmed, mediate viral replication before releasing the virus to the surrounding target cells, the hepatocytes. After intraperitoneal injection,

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peritoneal macrophages and recruited monocytes accumulate the virus, amplify it by replication, and deliver it to regional lymph nodes with a subsequent course as described for subcutaneous injection. After intravenous inoculation, virus is delivered directly to circulating monocytes and the reticuloendothelial cells of the liver and spleen. Virus in the circulation may be associated with circulating platelets, monocytes, and lymphocytes, although it is less effectively cleared when bound in this way. Differences in routes of inoculation may have profound effects on the outcome of viral hepatitis. Early in vivo studies with ectromelia in mice demonstrate that mice resistant to subcutaneous ectromelia can be made susceptible when the virus is injected intraperitoneally (Mims, 19641, despite the fact that the antibody response to ectromelia is the same in both groups of mice. The difference in survival of these two groups of mice following ectromelia infection may be explained by differences in the response of cells in contact with the virus following its absorption, differences in the rate of effective immune response, and sequestration of virus at a site which evades protective host responses. In vivo viral infection due to macrophage-permissive viruses affect hepatocytes in the liver by first replicating in Kupffer cells. Positive immunofluorescence of viral antigens is first detected in these sinusoidal cells after infection of mice by ectromelia (Mims, 1964, 1972) or MHV (Boss and Jones, 1963; Jones and Summerfield, 1982; Taguchi et al., 1976). The focal nature of the initial hepatitis in vivo supports the idea that the infection has spread from the Kupffer cells, since a more diffuse infection of hepatocytes would be expected to have a more diffuse or zonal sort of injury (Mims, 1964; Miyai et al., 1962; Boss and Jones, 1963; Sabesin and Koff, 1974a,b). The focal nature of the lesions in the liver caused by ectromelia may reflect the fact that only a fraction, approximately 25%, of the Kupffer cells are at first infected, even after massive doses of virus (Mims, 1964, 1972). It is possible to bypass the initial “gate-keeping” function of the Kupffer cell to produce hepatitis even in resistant animals by introducing the virus through the bile ducts rather than the circulation (Mims, 1964, 1972), or by introducing an inoculum of virus which can overwhelm the inital phagocytic clearance by the Kupffer cells, allowing the virus to arrive at and replicate in the susceptible hepatocytes (Mims, 1964). In vitro studies indicate that only macrophages from susceptible mice allow ectromelia (Roberts, 1963) or MHV (Shif and Bang, 1970; Bang, 1960; Stohlman et al., 1982) to replicate, despite the fact that macrophages from both susceptible and resistant mice have a similar uptake of virus (Krystniak and Dupuy, 1981). In fact, the in vitro macrophage response to a number of MHV viral strains can accurately predict the in

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vivo response of the donor mouse to these viruses (Taguchi et ul., 1976, 1981). It is of particular interest that an intermediate, semisusceptible response of macrophages in vitro is associated with a persistent infection in donor mice (Virelizer and Allison, 1976). Persistent infection of MHV in vivo is associated with low viral titers and a depression of delayed hypersensitivity, which may be the result of low-grade viral replication in macrophages and a resultant suppression of the immune response (LePrevost et al., 1975b). Studies using isolated Kupffer cells specifically as the macrophage population for MHV infection in vitro show no differences in resultant viral titers or cytopathic effects of this virus when cells are derived from resistant or susceptible donor mice; however, the replication in Kupffer cells from resistant mice is delayed by 24-36 hours, perhaps allowing time for an adequate immune response of the host (Pereira et al., 1984b). It is also noteworthy that one viral strain of MHV, MHV-3, does not show any correlation of in vitro macrophage replication and in vivo outcome, indicating that additional factors must be involved in determining the course of hepatitis after viral infection (Taguchi et al., 1981). The studies described above indicate that a key factor in the pathogenesis of macrophage-permissive viruses is their ability to replicate in macrophages; therefore, factors which inhibit viral uptake in macrophages can diminish the severity of hepatitis in susceptible animals. Thorotrast administration in ectromelia (Mims, 1964), or silica administration in MHV (Schindler et al., 1984) renders susceptible mice more resistant by compromising the function of macrophages; in the case of MHV, no viral replication can be detected in liver or spleen after such treatments (Schindler et al., 1984). On the other hand, when silica is intraperitoneally administered to mice which are normally resistant to MHV, having macrophages which do not support viral replication, the mice become more susceptible (Taguchi et al., 1980;Tamura et al., 1979). The activity of the macrophage also plays a modulating role in susceptibility to viral infection. Factors which increase activity of the macrophage tend to increase resistance to macrophage-permissive viruses. Previous infection of a virus with MHV, for example, is shown to activate macrophages and to make them more resistant to future infections with MHV (Taguchi et al., 1980). However, the increased viral resistance induced by activation of macrophages is not the sole determinant of outcome in vivo, as indicated by the observation that mice treated with glucan to activate macrophages have diminished hepatitis and prolonged survival (DiLuzio et al., 1982), but no decrease in ultimate mortality (Williams and DiLuzio, 1980). The activation of macrophages in

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vivo obviously has many more consequences than merely affecting the interaction of that cell with virus; it also alters macrophage participation in several responses induced by viral infection, including immune responses. It is also possible that the agent used to activate the macrophage has multiple modulating effects on the pathogenesis of the viral-host interaction. Corynebacterium parvum, for example, when used to activate macrophages in mice susceptible to MHV, is protective when virus is introduced at high dose, but not when MHV is introduced at low dose, an effect which is not related to natural killer activity or interferon response (Schindler et al., 1981). Endotoxin is another macrophage-activating agent with multiple effects. Endotoxin is found to increase the survival of ectromelia-susceptible mice when administered at high concentrations, but not at low concentrations (Gledhill, 1959).This difference in outcome may be caused by a shift in the balance of in vivo effects which this agent has, including effects on the macrophage-virus interaction, as well as effects on the immune response; macrophages are necessary to the integrity of both humoral and cell-mediated immune response (refer to Section IV). The immune response of the infected host is, of course, ultimately crucial to the survival of the animal and recovery from the viral infection. If the virus is able to rapidly spread to involve enough tissue in critical organs before an effective immune response can be mounted, the host animal will die. In fact, this may well be the case in mice susceptible to infection by ectromelia, in which the immune response is observed to occur I day later than a similar response seen in surviving resistant animals (Sabesin and Koff, 1974a,b). Mims describes the interaction of virus and host response well when he states “In an infectious process where the outcome depends on the race between growth in the liver and lymphoid tissue on the one hand and the immune response on the other, a delayed initiation of growth in these target organs may be of crucial importance” (Mims, 1964). The balance of virulence in the virus and defenses, including immunity, in the host determines the ultimate outcome of the infectious process in vivo (Mims, 1972).

d. Role of Extrinsic Effects in Kupffer Cell-Virus Interactions. The extrinsic effects of macrophages and Kupffer cells upon surrounding cells and hepatocytes are another important consideration in the pathogenesis of viral infections which involve macrophages (Stohlman et al., 1982; Morahan and Morse, 1979; refer to Table 111). These secondary effects of macrophage-virus interaction upon surrounding cells and tissue are of several different types: nonspecific, direct without the involvement of

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PATRICIA S. LATHAM TABLE 111 EXTRINSIC EFFECTS OF KUPFFERCELLS-VIRUS

INTERACTIONS

Nonspecific Release of lysosomal enzymes Direct extrinsic effects Synthesis and release of interferon Synthesis and release of tumor necrotic factor Direct inhibition of viral target cell protein synthesis Indirect extrinsic effects Synthesis and release of procoagulant

additional mediating factors, or indirect, in which a sequence of events and intermediate factors are involved, and specific immune effects which are discussed in Section IV. i. Nonspecific extrinsic effects of Kupffer cell-virus Interaction. There are several nonspecific effects of virus-Kupffer cell/macrophage interaction. One nonspecific effect of macrophage-virus interaction may be to preclude the ability of this cell to serve in its usual capacity of host defense against other possible toxins. In the case of Kupffer cells, endotoxin absorbed from the colon is implicated in the pathogenetic mechanism of at least one form of viral hepatitis due to FV3 (Kirn et al., 1982a; Gut et al., 1984). The FV3-induced loss of Kupffer cells as a hepatocellular barrier leaves the liver cells especially vulnerable to a variety of toxins. Another nonspecific effect of virus-macrophage interaction is the toxic effect of released lysosomal enzymes which may be increased in these cells by stimulation/activation and released from them by injury. Kupffer cells, like other macrophages, contain large numbers of lysosomes filled with proteases and hydrolases for the killing of phagocytosed living pathogens, such as bacteria and fungi, and for the denaturation of proteins to remove the detritus of cellular and tissue debris. The release of such factors may be a contributing factor to tissue injury, but it is not the only factor, since lysis of these same cells by FV3 in a previously colectomized animal results in no liver parenchymal cell injury (Kirn et al., 1982a; Gut et al., 1984). Macrophages may also be stimulated to produce activated oxygen species which can result in tissue injury. Although Kupffer cells do not produce reactive oxygen intermediates, recruited monocytes do (LePay et al., 1985; Tanner et al., 1982). ii. Direct extrinsic effects of Kupffer cell-virus interaction. Kupffer cells and macrophages may also be stimulated/activated by the viral infection to have direct effects upon the interaction of virus with surrounding cells. A complete enumeration of possible macrophage extrinsic effects to viral infection is presented in the review by Morahan

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and Morse (1979). However, known direct extrinsic effects of macrophage-virus interaction are antiviral and include the synthesis and release of interferon to inhibit viral replication, the recognition of virus-containing cells with subsequent synthesis and release of tumor necrotic factor (TNF) for lysis of the target cell, and an effect of direct cell contact whose mechanism is not yet clear, and may be a macrophage-induced inhibition of viral protein synthesis in the target cell (Morahan and Morse, 1979). iii. Indirect extrinsic effects of Kupffer cell-virus interaction. Other extrinsic effects of virus-macrophage interaction may result from the synthesis and release of macrophage factors which do not directly affect virus-cell interactions, but rather affect the survival and function of the liver tissue. One such factor is procoagulant, which is released from MHV-infected macrophages (Levy et al., 1982; Dindzans et al., 1985). Procoagulant can induce the formation of fibrin clots and thrombosis. Thrombosis in the liver can result in ischemia and necrosis of liver tissue which might, in turn, be a significant cause of the hepatitis seen in response to MHV (Levy et al., 1982; Dindzans et al., 1985). Levy et a f . (1982) demonstrate that the in vitro ability of peripheral monocytes to respond to viral infection with procoagulant release correlates with the in vivo MHV susceptibility of the mouse from which the cells were derived, and requires the cooperation of T lymphocytes. The increase in synthesis and release of procoagulant by the monocytes is associated with a decrease in synthesis and release of plasminogen (Dindzans et al., 1985). In addition, the activated macrophage and tissue injury can also activate complement which can bind to the C3b receptors of macrophages to further enhance their stimulated response. The complement itself can also cause membrane injury through immune and nonimmune mechanisms. 2. Role of Endothelial Cell-Virus Interactions Endothelial cells of the liver are presently the subject of a limited number of studies investigating their response to viral infection (Kirn et al., 1982b; Pereira et al., 1984b). Rat and mouse endothelial cells are seen to take up virus predominantly by pinocytosis and membrane fusion, to support viral replication under appropriate culture conditions, and to produce interferon in response to infection by vaccinia and FV3 (Kirn et al., 1982b). A permissive infection of liver endothelial cells in vitro is also known to occur after infection with MHV (Pereira et al., 1984b). Since endothelial cells predominate among the nonparenchymal cells in the sinusoid, the observation that they are able to synthesize interferon may have an influential impact on the course of hepatitis in vivo, but there are no investigations yet available. In infection by MHV in vitro, mouse

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endothelial cells show a similar response to the virus as seen for Kupffer cells in vitro; they are able to replicate the virus to the same extent when derived from susceptible or resistant animals, but with a 24- to 36-hour delay when derived from mice which are resistant (Pereira et a f . , 1984b). Endothelial cells may also play an important role in hepatitis by determining access of hepatotrophic cells to the parenchyma. The fenestrae in endothelial cells are approximately 100 nm, and may be dynamic in size (Kirn et al., 1982b; Fahimi, 1982). The size of the fenestrae approximates that of many viruses. It is possible that conditions which may dilate or close these fenestrae can affect the course of viral infection in the liver.

C. HEPATOCYTES I N VIRAL PATHOGENESIS OF HEPATITIS 1. Intrinsic Hepatocellular- Virus Interactions

a. Viral Hepatotropism. Viruses may infect hepatocytes in particular for one of two reasons. First, the virus may be accumulated in the liver by the action of Kupffer cell clearance with or without replication of the virus, infecting hepatocytes as adjacent susceptible cells. A second reason that the virus might favor replication in hepatocytes is that it is uniquely hepatotrophic, as shown by an attraction of the viral molecule to the microenvironment of the hepatocytes, or by the action of specific receptors for the virus on the plasma membrane of the cell. There is no example as yet of a virus having a particular predilection for the microenvironment surrounding the hepatocyte, although this is likely to be an important factor in the binding characteristics of any substance to this cell. In intact hepatocytes, it is expected that viruses most commonly enter the cell at the sinusoidal, or basolateral, plasma membrane, since most viruses arrive at the hepatocyte by the vascular route, and most viral particles are larger than the approximately 100 nm size admissable to the semipermeable tight junction between these cells (Schneeberger and Lynch, 1984). It is not known at this time if any viruses must bud from one surface or another of hepatocytes in the course of hepatitis. It is likely that some viruses are normally released at the apical surface of hepatocytes, such as hepatitis A and other viruses which are transmitted by the fecal-oral route (Mims, 1964). There is a precedent in viral morphogenesis for an enveloped virus to bud selectively from one surface of a cell rather than another (Rodriquez-Boulan et a l . , 1983). Secretory and absorptive cells, in particular, have polarity of transcellular flow and have apical and basolateral plasma membranes which express differences in

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their biochemical composition, and which are segregated from one another by the tight junction as a plasma membrane barrier (Schneeberger and Lynch, 1984). Influenza virus is described to bud uniquely from the apical surface, and VSV to bud from the basolateral surface of MadenDarby canine kidney (MDCK) cell in vitro (Rodriquez-Boulan et al., 1983). b. Viral Hepatocellular Replication. In addition to the hepatotrophic inclination of the virus and the modulating role of sinusoidal cells in hepatitis, the relative intrinsic ability of hepatocytes themselves to serve as a site of viral replication is an important determinant of outcome. Most viruses causing lethal hepatitis do replicate in hepatocytes, with the noteworthy exception of FV3 (Kirn, et al., 1982b; Kirn et al., 1983) (refer to Section II,B,I ,c,ii). The yield of infectious viral progeny from hepatocytes for some viruses may be increased when these cells are in an active metabolic or regenerative cycle. Rat virus, for example, is one virus described to have an affinity for hepatocytes in mitosis; thus, the virus undergoes increased replication and produces more severe disease in neonatal animals than in adults (Margolis et al., 1968). Viruses with enhanced replication in metabolically active tissue also replicate more effectively in liver tissue which is actively mitotoic. In adult animals, such a mitotically active liver can be produced by the regenerative pulse seen after 50-75% partial hepatectomy; HSV is one such virus described to increase its yield of viral progeny in the regenerative cycle after partial hepatectomy in rats (Ruffolo et d.,1966). When HSV is introduced into rats treated in this manner, it is able to induce susceptibility in an otherwise resistant animal. The yield of infectious viral progeny from hepatocytes for some viruses may also be decreased when these cells cannot support a complete cycle of viral replication. Inefficient viral replication in hepatocytes may occur in such a way as to release large quantities of incomplete virions which are able to compete with complete virions for cellular uptake, thereby decreasing the production of virus and decreasing the severity of hepatitis. The defective particles may also act to bind available humoral antibody or cytoeffector sites, thereby preventing them from acting against complete virions. In infection with RVF virus in rats, the production of defective virions is found to correlate with an improved survival when very high concentrations of virus are introduced (Mims, 1964). In infection of mice by influenza A, the production of defective viral particles is associated with the resistance of a genetic strain to that viral infection, even though the yield of infectious progeny in the two strains of mice is the same (Dimmock et al., 1986).

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Isolated hepatocytes in culture were recently reported as a useful model system to explore primary interactions of these cells with virus (Arnheiter, 1980). Using such a model system, a correlation is described between the susceptibility of mice in uiuo to hepatotrophic influenza or HSV-1 and the ability of the virus to replicate and produce cytopathic effects in isolated hepatocytes (Arnheiter, 1980). However, the correlation of in uitro hepatocellular replication and in uivo susceptibility is not uniform and may vary for each strain of virus (Arnheiter et al., 1980), and for the strains of resistant or susceptible mice (Arnheiter et al., 1980; Amheiter, 1982). The in uiuo outcome of infection in some resistant (A2G) and susceptible (C57BL/6 or CBA) mouse strains, for example, is found to correlate with the ability of influenza A to replicate in isolated hepatocytes from these strains (Arnheiter et al., 1980). The interaction of these hepatocytes with influenza A is specific for the virus involved, since there is no direct correlation of that response to those seen with other hepatotrophic viruses, such as herpes and VSV virus (Arnheiter et al., 1980) or MHV (Taguchi et al., 1983). 2. Extrinsic Effects of Hepatocellular- Virus Interactions

In addition to the ability of the virus to replicate in hepatocytes, these cells may also influence the course of hepatitis by their ability to synthesize interferon and to respond to its antiviral effects (Arnheiter et al., 1980). Hepatocytes are known to produce interferon spontaneously (in uitro,) and in response to viral infection (Arnheiter et al., 1980). No differences have yet been described in the quantity or quality of interferon produced by hepatocytes from resistant or susceptible animals; however, differences have been noted in the sensitivity of the host cells to respond to the antiviral effects of the interferon that is produced (Arnheiter et al., 1980). The interferon sensitivity of the host cell is dictated by the phenotypic expression of a single gene, the Mx gene, and is specific for each infecting virus.

D. NONVIRAL FACTORS I N THE PATHOGENESIS OF VIRAL HEPATITIS Additional factors may have other important modulating influences on antiviral response, such as endotoxin lipopolysaccharide (LPS). LPS may have practical significance as an important effector in the immune response of naturally occurring infections, since it can activate B lymphocytes and macrophages and it can endogenously enter the systemic circulation from the reservoir of gram-negative organisms which usually colonize the colon. In fact, it is probable that low levels of endotoxin are routinely absorbed and cleared from the portal circulation by the Kupffer cells of the liver (Nolan and Camera, 1982). However, endotoxin may also

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have a direct role in the pathogenesis of viral hepatitis, since it is demonstrated to cause direct liver injury to isolated perfused liver (Nolan and Camera, 1982). The idea that endotoxin-induced liver injury may be significant in the pathogenesis of hepatitis related to viral infections is suggested by the observation that thymectomy-enhanced susceptibility to ectromelia is not seen in “low pathogen mice” or in mice fed antibiotics, which would tend to cleanse the gut of the bacterial endotoxin source (Subrahmanyan, 1968). Endotoxin is also suggested to play a role in the hepatitis related to FV3 infection in rats. FV3 does not replicate in vivo during infection, although its proteins are toxic and result in the lysis of sinusoidal cells. The release of viral proteins and/or the release of injurious agents from necrotic sinusoidal cells cannot alone result in the hepatocellular necrosis which ensues, however, since no hepatitis occurs when endotoxin is removed by colectomy, or neutralized with polymixin B, prior to infection of rats with FV3 (Gut et al., 1984; Kirn et al., 1982a). IV. Role of Interferon and the Immune Response in the Viral Pathogenesis of Hepatitis A. THEROLE OF MACHROPHAGE-T LYMPHOCYTE INTERACTIONS

Specific extrinsic effects of macrophages on virus-target cell interaction are the so-called “immune effects” (see Table IV). The macrophage plays a pivotal role in cell-mediated immunity and in many of the pathogenetic models which we now understand for viruses causing TABLE IV I N IMMUNE ROLEOF KUPFFERCELLS/MACROPHAGES RESPONSETO VIRUS Effects of viral-activated macrophages on immune response Remove virus antigen-antibody complexes Clearance Antibody-dependent complement cytolysis Antigen presentation to the immune system Macrophage release of monokines, interluekin I Leukocyte-activating factor (LAF) Mitogenic protein Effects of the immune response on the macrophage T Lymphocyte release of lymphokines, y-interferon Macrophage-activating factor (MAF) Migration-inhibition factor (MIF) Also interleukin 2

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hepatitis. When viruses are introduced intraperitoneally , subcutaneously, or intravenously, then peritoneal macrophages, Langerhans cells, and Kupffer cells, respectively, are the tissue macrophages which interact with the virus, along with monocytes which are recruited from the circulation. The interaction with virus activates the macrophage and, for the most part, it is the activated macrophage which interacts with lymphocytes to effect an appropriate immune response to viral infection (Morahan and Morse, 1979). Circulating antigen-antibody complexes of viral proteins can be bound to macrophages through Fc receptors, or through C3b receptors with the help of complement, to stimulate enhanced phagocytosis by machrophages and, in turn, to stimulate an activation of the cell (Diamond, 1982; Porterfield, 1985). If the activated macrophage expresses HLA locus antigens (Ir or Ia antigens), as do Kupffer cells, it may then recognize the antigen as foreign, such that it will ingest and degrade the antigen, and “present” the antigen to lymphocytes (Richman et al., 1979; Diamond, 1982). A soluble factor released from macrophages, interleukin 1 (IL-1, also known as leukocyteactivating factor, or mitogenic protein), stimulates a mitogenic response in B lymphocytes and in helper T lymphocytes (Diamond, 1982; Roitt et al., 1985). Activated macrophages also induce T lymphocytes to secrete the lymphokines macrophage-activating factor (MAF ) and macrophageinhibiting factor (MIF) (Roitt et al., 1985; Diamond, 1982). These factors activate the macrophage and inhibit migration of macrophages, respectively. MIF acts to increase the local population of macrophages at the site of inflammation. MAF acts to further activate macrophages to become cytocidal to virally infected cells. An additional factor, interleukin-2 (IL-2) is also released by T lymphocytes in the presence of macrophages (Roitt et al., 1985). The release of IL-2 by T lymphocytes stimulates regulatory T lymphocyte subsets and variably increases the activity of natural killer (NK) cells (Pereira et al, 1984a; Welsh et al., 1986),which are also enhanced by the stimulus of interferon. A rough, but inconsistent, correlation does exist between the appearance of interferon and the activity of NK cells after viral infection (Stohlman et af., 1983). Although a direct correlation between interferon production and NK cells has been described in response to HSV (Armerding, and Rossiter, 1981) and CMV (Bancroft et af., 1981), no correlation has been found in response to influenza (Leung et al., 1981)or MHV (Schindler et al., 1982). It is probable that NK cells are important effectors of immune response in the pathogenesis of some viruses, but an insufficient number of studies are yet available which explore the role of these cells in viral hepatitis (Pereira et al., 1984a). In macrophage-nonpermissive infections, it is probable that T lymphocytes cooperate with macrophages to confine infection to defined foci in

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the liver by the release of MIF, macrophage chemotactic factor, and y-interferon, as has been described for HSV (Ianello and Mogeuseu, 1985). Although such action may not be crucial to the development of a lethal hepatitis, its absence may allow the virus to seed itself more effectively into the circulation with a resultant increased incidence in other sequelae of infection, such as meningitis and encephalitis (Zisman et al., 1970). The cell-mediated T lymphocyte reaction is also necessary to the integrity of resistance and to recovery in macrophage-permissive viral infections. Thymectomy or antithymocyte serum can make resistant mice susceptible without affecting antibody, interferon, or intrinsic macrophage response to a virus such as ectromelia (Blanden, 1970) or MHV (Virelizier, 198 1). Macrophages are necessary cofactors for T lymphocyte cell-mediated immunity, as indicated by spleen transfer (Blanden, 1971a,b) and irradiation experiments (Dupuy et al., 1975, 1984), which indicate that both cells are necessary for antiviral effects. Dupuy et al., 1975, 1984) demonstrate that lymphocytes are most important in the host-virus immune response by their effects on macrophages, since macrophages alone can sustain resistance to MHV after irradiation of the animal, or after antilymphocyte serum, if the macrophages are “preimmunized” (Dupuy et al., 1975, 1984). Dindzans et al. (1985) underscore the importance of T lymphocyte effects in the viral response of macrophages when they provide direct evidence that there are differences in the responses of peritoneal macrophages and T lymphocyte-exposed macrophages to MHV infection in vitro (Weiser and Bang, 1976, 1977). In one study, Weiser and Bang (1976) demonstrated that normally resistant macrophages from resistant mice in vitro can be made susceptible to MHV after exposure to the fluid, and presumably lymphokines, of allogeneic mixed lymphocytes. In a later study, these same investigators demonstrate that the fluids released from Con A-stimulated spleen cells contain factors which can confer MHV resistance to otherwise susceptible macrophages in vitro and mice in vivo (Weiser and Bang, 1977). In this latter study, lymphokines are suggested to be the mediating factor able to confer resistance on the susceptible cells. Interferon production, however, is not entirely ruled out (Weiser and Bang, 1977). Levy et al. (1985) add further support to the hypothesis that macrophage-T lymphocyte interactions are crucial to survival of MHV viral infection by demonstrating that cultures of spleen cells from susceptible mice, in contrast to resistant mice, do not proliferate, release IL-1 or IL-2, or enhance IL-2-T lymphocyte receptors in response to MHV (Levy et ul., 1985). Other examples of the need for an integrated lymphocyte-macrophage response to viral infection in vivo are demonstrated in the studies of age-dependent susceptibility, discussed in Section V,B.

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Humoral immune response may also directly influence macrophagevirus interaction by enhancing antibody-dependent complement cytolysis (ADCC) and by antibody-dependent enhancement (ADE) of viral uptake. In ADE, virus-antibody complexes are more effectively adherent to macrophages through Fc receptors and are internalized more easily to effect enhanced replication of virus (Porterfield, 1985).

B. THEROLE OF INTERFERON Another antiviral factor produced in the course of the immune response is interferon, Interferon synthesis is critical to the survival and recovery of most, if not all, viral infections since antibody to interferon can increase the severity and mortality of infection (Virelizer and Gresser, 1978). There is no uniform correlation, however, between the amount of interferon produced and the resistance to infection. MHV induces an interferon production which has a rough direct correlation to the virulence of the viral strain (Taguchi and Giddell, 1985); however, low levels of interferon can still be seen to have an adequate antiviral effect for some strains of MHV. Proietti et al. (1986) were able to demonstrate an antiviral effect of mouse peritoneal machrophages in vitro for VSV which is consistent with interferon synthesis, and which is abolished by the presence of antiinterferon, despite the fact that no interferon is measurable in the cultures of these cells. The sensitivity of the host to the antiviral effects of the interferon induced may be specific to the infecting virus, as indicated by studies with influenza (Arnheiter et al., 1980, 1982). In the case of influenza A infection, the antiviral effect of interferon in the host is determined by a single gene, the “Mx gene,” which is present in mice resistant to influenza infection (Arnheiter, 1980). The effect of the interferon on the course of the viral infection is specific to the infecting virus, as indicated by the observation that isolated hepatocytes from influenza-resistant mice successfully respond to interferon in vitro to prevent viral infection, but the interferon response of these cells only partially protects them from HSV and confers negligible protection from VSV (Arnheiter et al., 1980, 1982; Haller et al., 1979). V. Genetic and Age-Dependent Determinants of Susceptibility in Viral Hepatitis A. GENETICSUSCEPTIBILITY Susceptibility of an animal species to hepatitis is dependent on both the specific strain of the inciting virus and the specific strain of the animal species. In vivo resistance to a virus is the phenotypic expression of a

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gene, or genes, which may influence the response of cells involved in the host-virus interaction. Genetic influences which have been described to alter the response of cells to the viruses producing hepatitis include the efficiency of viral permissive or nonpermissive replication, cytolysis and release of virus from cells, response of a cell to interferon, and the ability of a macrophage to respond with activation to viral infection. Genetic resistance of mice to HSV in vivo has been found to correlate with the ability of macrophages in uitro to support only a nonpermissive viral replication (Mogensen, 1977b). Mogensen et al. (1977) determined that the ability of the macrophage to restrict the spread of virus to other cells was inherited as an X-linked dominant trait in mice. Arnheiter later determined that the gene was also expressed in isolated hepatocytes from HSV-resistant strains of mice (Arnheiter, 1980). Genetic resistance of mice to macrophage-permissive viral infections in vivo is found to correlate with the ability of macrophages to prevent viral replication in vitro, even though these cells have a similar adsorption and uptake of the virus (Krystniak and Dupuy, 1981). Only macrophages from susceptible mice permit replication of MHV (Roberts, 1963; Shif and Bang, 1970; Bang, 1978; Stohlman et al., 1982) or the flaviviruses (Mogensen, 1979). The ability of macrophages to resist viral replication of MHV is inherited as an autosomal dominant trait (Bang, 1978), correlates with in vivo susceptibility, and is viral specific (Arnheiter et al., 1980). Lamontagne and Dupuy also found expression of MHV genetic viral resistance at the cellular level (Lamontagne and Dupuy, 1984); however, their model system of mouse fibroblasts was found to resist viral release and cell lysis rather than viral replication. The studies of Dupuy et al. (1984) further suggest that genetic susceptibility to virus may be expressed not only by the response of the macrophage itself to virus, but also by deficiencies of macrophage immune interactions with T lymphocytes. Dupuy et al. (1984) demonstrated that resistance or susceptibility of mice to MHV depends on both splenic lymphocytes and macrophages when these two cell types are studied in radiation chimeras. Another mode of genetic resistance to hepatitis in mice is expressed by the sensitivity of potential virus-host cells to the antiviral effects of interferon, as represented by the response of liver-derived cells to infection with influenza virus. Influenza infectivity of hepatocytes is controlled by a single gene, the Mx gene, which determines the sensitivity of hepatocytes to antiviral interferon effects (Arnheiter et al., 1980; Haller et al., 1979). Cells from strains of animals containing this gene respond to endogenous or exogenous interferon to restrict viral infection, while cells from susceptible animals synthesize an equivalent amount of interferon, but cannot respond to it with restriction of viral spread.

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Genetic resistance to FV3 appears to depend on yet another model of genetic resistance in rats related to the ability of lymphocytes and macrophages to become activated, as they normally do in the course of immune response to infection (Kirn et al., 1982; Gut et al., 1984). The FV3 virus cannot replicate in vivo, and resistance is not correlated to any difference in the response of macrophages or hepatocytes to this virus. However, C3HlHeJ mice are resistant to endotoxin activation of B lymphocytes and macrophages, and it is the presence of endotoxin and the response to it which appear to best correlate with genetic susceptibility in this animal model. Although the pathogenesis of hepatitis due to HSV is very different from that due to FV3, resistance of C3H/HeJ mice to HSV viral infection may also be related to a failure of macrophage activation (Kirchner et al., 1978a). In this model system, isolated spleen cells from C3H/HeJ mice fail to respond to endotoxin with the enhanced viral replication seen in other strains.

B. AGE-DEPENDENT SUSCEPTIBILITY Age-dependent susceptibility in viral hepatitis may be due to several factors, including maturation of macrophages and/or other cells involved in the immune response. Maturation of macrophages is considered important in age-dependent susceptibility, since in vivo macrophages in liver and spleen, and in vitro macrophages from susceptible weanling mice produce a greater yield of viral progeny than macrophages from resistant mice infected with the same multiplicity of MHV (Taguchi et al., 1979; Galliliy et al., 1967) or CMV (Mims and Gould, 1978; Selgrade and Osborn, 1974). Transfer of peritoneal macrophages from adult resistant mice to susceptible weanling mice enhances the resistance of the weanling mice exposed to MHV (Levy-Leblond and Dupuy, 1977; Taguchi et al., 1979) or CMV (Selgrade and Osborn, 1974). In HSV infection, susceptibility of newborn mice in vivo is also positively correlated to maturation of macrophages (Johnson, 1964; Stevens and Cook, 1971). Macrophages from both newborns and adult mice are equally well infected in vitro, but macrophages from resistant animals do not spread the infection as readily to other cells (Johnson, 1964). As in CMV infection, transfer of adult peritoneal macrophages to newborns can provide protection (Mogensen et al., 1978; Hirsch et al., 1970). The macrophages used in these transfer studies are most effective if they are stimulated, in which state they also more effectively limit viral spread in vitro (Mogensen et al., 1978; Hirsch et al., 1970). Hirsch et al. (1970) demonstrated that macrophages from immature mice in vitro cannot respond to activation with more effective viral restriction or destruction, and neither do they respond with in-

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creased phagocytosis or production of interferon, as do more mature cells (Hirsch et al., 1970). The relevance of these findings in experimental hepatitis to human disease is suggested by the observation that HSV-2 in humans shows a predilection for more severe infections in neonates and premature infants (Mogensen, 1979). Factors interrelated with macrophage-virus interactions are also suggested to influence age-dependent susceptibility, such as the effectiveness of systemic viral transport from entry site to target site, or the effectiveness of the immune response. In age-dependent susceptibility of mice to ectromelia, the lack of an age-related difference in replication of virus at peripheral sites is suggested as evidence that the difference in outcome of viral infection may be related to the action of Kupffer cells specifically, or to the immune response (Sabesin and Koff, 1974a,b; Subrahmanyan, 1968). This hypothesis is supported by the knowledge that the immune response and the action of T lymphocytes are still immature in mice younger than 3-4 weeks of age (Sherr et al. 1981; Yang and Skinses, 1973). The hypothesis is also supported by studies which show that the transfer of adult resistant spleen cells, adherent macrophages, and T lymphocytes to susceptible weanling mice can protect these mice from infection by MHV (Tardieu et al., 1979); transfer of preimmunized T lymphocytes or antibody alone, however, cannot provide complete protection (LePrevost et al., 1975a). Interferon production or sensitivity to its effects may be additional factors in age-dependent susceptibility, as suggested by the observation that weanling mice susceptible to MHV produce less interferon than adult mice that are resistant to this virus (Taguchi et al., 1979).

VI. Conclusion The complex interplay of factors operative in the pathogenesis of viral hepatitis may be broadly organized into two catagories of viral-host responses. The first viral-host response involves the ability of the virus to adsorb, infect, and replicate in the host tissues with which it comes in contact, dependent on its route of inoculation. In the case of hepatitis, these cells always include hepatocytes, endothelial cells, and lymphoreticular cells which reside in liver, circulation, and extrahepatic lymphoid tissue. The second interaction involves the ability of the host to impede, contain, and finally free itself of the infecting virus. This article has focused on those viral-host responses which involve liver-derived cells directly; it should be appreciated, however, that many of the viral-host responses may effectively occur before the virus ever arrives at the liver.

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The lymphoreticular cells in lymph nodes and circulation may have important interactions with the virus before it enters liver tissue. Intrinsic and extrinsic virus-T lymphocyte-macrophage interactions can result in variable nonspecific host responses such as interferon production, natural killer cell activation, and macrophage activation, as well as specific responses of humoral and cell-mediated immunity. These host-virus interactions may be important determinants in the outcome of viral infection in the liver; in fact, the hepatitis may be the result in part of factors, such as endotoxin, complement, or the effects of ischemia, which are independent of direct viral effects on the hepatocytes themselves. In summary, the outcome of viral hepatitis depends on a number of variables which are richly intertwined. An increased understanding of the action and interaction of these variables on the course of hepatitis is essential to future investigations leading to prevention and cure.

ACKNOWLEDGMENTS The author was supported in part by grants from NIH (R01 A121 844-01) and the Veteran’s Administration Merit Review.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. I I2

“Leaky” Cells of Glandular Epithelia S. S. ROTHMAN”AND T. M E L E S E ~ *University of California, San Francisco, San Francisco, California 94143 tColumbia University, New York, New York 10032

I. Introduction Epithelial cells form an imperfect, partially selective barrier to the exchange of matter between environment and organism. In addition to the plasma membrane of these cells, matter may enter or leave the organism through spaces between them; these spaces are known as inter- or paracellular spaces. The question of the relative permeability of these two potential pathways to the passage of matter has been a subject of continuing interest. It has been considered most extensively for flattened, membranous epithelial layers, such as the intestinal epithelium, gallbladder, and urinary bladder, as well as for the tubular system of the kidney. Glandular epithelia have received relatively little attention in this regard. In this review we focus on the experimental work done on one such epithelial gland, the pancreas, and its permeability to polar nonelectrolytes of substantial size. 11. Paracellular versus Transcellular Movement

A. GENERAL

Electron microscopic images of thin transverse sections through epithelial tissues demonstrate that the membranes of the surface cells of the epithelial layer are not directly apposed or fused to each other laterally, but separated by a space through which material can potentially pass. However, visual observation of the intercellular space at the cell’s apex also shows that it is narrow, and appears to be filled with material. This suggested that the movement of matter via these junctions might be severely limited, if it occured at all. This observation, coupled with the fact that the surface area available for the movement of material between cells is only of the order of 0.01-1% of that available for passage through cells, depending upon the particular anatomical arrangement, gave rise to the idea that epithelial junctions were “tight” or impermeable. It seemed that the dominant, if not the sole, means available to broach the epithelial barrier was across the cell. 225 Copyright 0 1988 by Academic P r e s . Inc. All rights of reproduction in any form re\erved.

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In the 1970s, this perception underwent a major shift. Now, the movement of material, particularly ions and water, across at least certain epithelia, is thought to occur in great part through leaky paracellular conduits (Diamond, 1974; Wright and Pietras, 1974; Powell, 1981), because these junctions present a much less severe barrier than the cell membrane, with a much higher permeability constant to compensate for the minimal surface area. Frompter and Diamond (1972) scanned the surface of the gallbladder epithelium with a discrete flow of electrical current and found that areas of low resistance outlined the array of cells. That is, the junctions between cells were low-resistance shunts-leaky , not tight. Because current is carried in biological systems by ions, it seemed that at least the major ions, sodium and chloride, must pass through the paracellular shunt in order to carry the current. If such passage was accounted for by simple diffusion, alone or in combination with hydrodynamic flow, then other substances of the same or smaller size, most significantly water, would seemingly travel the same route. Various epithelia have been classified as being either tight or leakyleaky, if substances (water and ions carrying current) were thought to move readily through junctions between cells, or tight, if not (Augustus et al., 1977). If a substantial electrical potential can be maintained across an epithelium, then paracellular shunting could be minimal (low conductance), whereas if transmural potentials are small or absent, high conductance through paracellular shunts could be assumed. Thus, whether or not a particular epithelial tissue is leaky, in electrical terms or to various substances otherwise, has come to be associated with the nature of the paracellular path. Does a particular epithelium have a leaky intercellular junction? However cellular permeability might vary, the relative leakiness of the epithelial surface is predominantly a function of intercellular, not cellular, permeability. It is the realization that this does not seem to be a satisfactory generalization, applicable to all epithelia, that forms the basis of the discussion that follows. At least two glandular epithelia appear to be leaky as the result of transcellular, not paracellular, processes. B. “THE LEAKIEST EPITHELIUM STUDIED So FAR” Jansen et al. (1979) and Bonting et al. (1980) reported that the pancreatic epithelium was permeable-apparently uniquely so-to water-soluble nonelectrolytes of substantial size, in particular, sucrose, mannitol, and inulin. These groups reported further that epithelial permeability to sucrose and inulin increased when protein secretion by the gland

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was stimulated. As a result of these observations, these workers stated that the pancreas was “the leakiest epithelium studied so far.” In spite of the fact that permeability increased in response to increased, presumably membrane-associated, cellular activity (namely, protein secretion), they concluded that the leakiness to large polar nonelectrolytes was between cells, and that the increased leakiness seen when the gland was stimulated was the result of an increase in the permeability of paracellular shunts. This interpretation followed the view that the leakiness of an epithelium is, in general, a function of paracellular permeability, but was, in addition, based on knowledge that the large polar nonelectrolytes mannitol, sucrose, and inulin, that crossed this epithelium do not enter a variety of cells significantly, if at all. These and other similar large, water-soluble molecules were thought unable to cross biological membranes in general-because they are not sufficiently soluble in lipid solutions, the cells lack special “facilitated” (selective) processes to transport them, and they are too large to permeate by way of the small pores or channels buried in the membrane. For these reasons, these substances have been used, by convention, to demarcate noncellular spaces in many tissues. Thus, if the epithelial layer was permeable to such substances, paracellular passage could be assumed. Indeed, this permeability seemed to provide evidence that epithelial leakiness was paracellular ! Despite this reasonable conclusion, however, the leakiness of this particular epithelium appears to be expressed by way of its cells, not by paracellular shunts. This is suggested by certain observations of Jansen et al. (1979, 1980) and Bonting et al. (1980), as well as our own (Melese and Rothman, 1983a-c) using the same experimental system, the pancreatic epithelium of the rabbit in organ culture. 111. The Experimental System

Most of the observations that we shall discuss were made using the rabbit pancreas in short-term organ culture (Rothman, 1964, 1966; Rothman and Brooks, 1965). The rabbit pancreas, unlike the pancreas of most mammals, is very thin and can be studied whole in vitro without the necessity of vascular perfusion. The pancreatic duct is cannulated, then the whole gland is removed from the animal, mounted in a chamber, bathed by a physiological salt solution (often enriched with amino acids and glucose), and gassed continuously with oxygen and carbon dioxide. The gland secretes water, salts, and proteins (primarily digestive enzymes) at rates comparable to those found in situ, and among available in uitru preparations of this tissue is uniquely responsive to stimulants of

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protein secretion. Ductal fluid is collected undiluted by means of the indwelling catheter. The volume of medium bathing the gland varies somewhat in the different studies that we shall discuss, but in all of them is very large relative to the rate of fluid secretion-from about 500 to 3000 times the volume of material secreted over a period of an hour or so. Therefore, the amount of a given material extracted from the bathing medium (to be secreted) represents a small percentage of that present at the outset, and for this reason we can assume, as an approximation, that the concentration of a given substance added to the bathing medium remains unchanged during the course of study (up to several hours). OF SUBSTANCES ACROSSSECRETORY EPITHELIA A. THEDISTRIBUTION

In a simple equilibrating system one cannot determine the permeability of a barrier epithelium to a particular substance in terms of that substance’s relative concentration on the two sides of the barrier when the system is at the equilibrium or steady state. Of course, at the steady state in such a system, all substances to which the barrier is passively permeable, i.e., substances with a reflexion coefficient of less than 1.0, presumably will be present in equal concentrations on both sides (questions of charge aside), regardless of their relative permeabilities. In a secretory system such as the pancreas in organ culture, however, disequilibria may exist as steady-state phenomena even for passively distributed substances, and for this reason, the trandcis ratio of concentrations across the epithelium at the steady state can provide an estimate of the barrier’s permeability to the substance. The existence of steadystate disequilibria across this epithelial surface is due to the flow of fluid through the duct system. Material that crosses the epithelium is swept down and out of the ductal conduits by fluid flow; thus the attainment of an equilibrium state across the epithelium can be prevented because the ductal compartment is not static. Fluid flow continually regenerates the transepithelial gradient. As a result, disequilibria exist not only on the way to an equilibrium state, but as steady-state phenomena. The concentration of a substance in fluid collected from the secretory duct (trans), even one to which the gland is quite permeable, may be less than 100% of that in the bathing medium (cis) at the steady state. If the fluid flow was great enough, even a freely permeable substance might be present at a very low concentration in fluid collected from the duct system. The deviation from 100% (degree of disequilibration) at the steady state is a measure of both the relative permeability of the barrier to the substance and the rate at which material is removed from the locus of its

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transepithelial transport (a function of the rate of fluid movement). Differences in the relative concentrations (trandcis) of different substances at the steady state become solely indices of permeability, whether flow is kept constant or is equivalent from circumstance to circumstance (for different substances under study). The latter is the case for the rabbit pancreas in uitro. Under these conditions, for trans values well below equilibrium (say of the order of 10% or less of the cis concentration), the extent of disequilibrium at the steady state is roughly proportional to the difference in the permeability of the epithelium to different substances. Equilibration being an exponential function, as values approach equilibrium (equal concentrations on both sides of the epithelial surface) the cis/trans ratio underestimates permeability.

B. THEDISTRIBUTION OF SUBSTANCES ACROSS PANCREATIC EPITHELIUM

THE

Urea, the small organic molecule (60 Da), to which biological membranes are often freely permeable, is present at the steady state in secreted fluid at approximately 90% of it‘s concentration in the cis compartment (Jansen et al., 1979). Thus, the system is close to equilibrium for urea at the steady state. From this we can conclude that the pancreas is quite permeable to urea, and that fluid flow is not sufficiently great to substantially reduce its concentration in ductal fluid. For the larger six-carbon alcoholic-sugar mannitol(l82 Da), permeability is somewhat lower and the steady state is displaced further from equilibrium, with a trans/& concentration ratio of about 60% (Fig. 1) (Jansen et al., 1979; Melese and Rothman, 1983a). Permeability to sucrose (342 Da) is lower yet, with steady-state values of about 8-12% achieved within about 30 minutes, depending upon the particular experimental circumstance (Jansen et al., 1979; Bonting et al., 1980; Melese and Rothman, 1983~).Finally, the polysaccharide inulin (-5-7 kDA) attains a steady state in about 45 minutes, at a trans/cis ratio of about 2-6% (Jansen et al., 1979; Melese and Rothman, 1983~). Although these values suggest, at least at first glance, a particularly leaky epithelial surface, without knowing the true thickness of the diffusion barrier and the ratio of cell or tissue volume to effective surface area, an accurate comparison of permeabilities between different tissues is not possible. Moreover, the rate of equilibration may reflect geometric features of the particular experimental system specifically the size of the volume into which the material equilibrates (the size of the trans compartment). Because the whole pancreas in organ culture provides a

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Time (minutes) FIG. I . The concentration of inositol (diamond) and mannitol (square) in ductal fluid before and after the addition of a cholinergic stimulant [ I mgl100 ml bath fluid acetyl-Pmethylcholine chloride (MCh)]. Values are expressed as percentages of the bath concentration (100%). The appearance of labeled inositol and mannitol was followed in samples of secretion collected from the cannulated duct of the rabbit pancreas in organ culture in the continuous presence of the labeled sugars in the bathing medium. The steady-state concentration of inositol under unstimulated conditions was approximately one-tenth that of mannitol in this group of experiments. Cholinergic stimulation increased inositol concentration some 6-fold, but mannitol concentration was unchanged. Data are mean values +. S E (n = 4 for inositol and 3 for mannitol). Reprinted from Melese and Rothman (1983a), with permission.

very large ratio of surface area to trans-compartment volume, a rapid rate of equilibration is to be expected even in the presence of a substantial permeability restriction. The tissue weight is about 1 g and the volume into which material equilibrates (the volume of the duct system) is approximately 0.1 ml, or a tissue-to-trans compartment volume ratio of about 10. In comparison, a 10-mg wet weight (about 0.5 cm’) piece of ileum in an Ussing chamber, bathed symmetrically by about 10-15 ml of fluid, has a tissue-to-trans compartment volume ratio of about lo-’. Thus, the pancreas in culture would equilibrate transepithelially at approximately lo4times the rate of a piece of ileum in such an Ussing chamber for a substance to which both tissues are equally permeable. If we use steady-state unidirectional (cis-to-trans) fluxes, estimated from the initial or maximal rate of transport during equilibration, in order to compare permeability, the size of the trans compartment can be ignored. When this is done, the maximal rate of inulin transport across the

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23 1

“leaky” ileum of the rabbit is about 0.007% (of medium concentration)/ 10 minutes/0.5 cm’ tissue area (Goetze and Rothman, 1978), whereas the equivalent rate of transport across the pancreas is about 2%/10 minutes/g wet weight (Melese and Rothman, 1983~).Equating for tissue volume, and assuming equal diffusion length and tissue volume/surface area ratio, this reflects an approximately 3-fold permeability difference, that is, the pancreas is about three times more permeable to inulin than the ileum. In the presence of a protein secretion stimulant, this value is increased, by about 3- to 5-fold, to a difference in permeability of about one order of magnitude. Whatever the degree of leakiness may be, movement via paracellular shunts may be consistent with the observations we have discussed to this point, namely, permeability decreases with increasing molecular size for substances thought not to enter cells. Such shunts may just happen to be particularly leaky in the pancreas. Indeed, this leakiness may be hard to imagine as a property of cells that must retain a variety of disequilibrium steady states, for large and small molecules alike, in order to function (how could such a leaky cell membrane effectively maintain disequilibrium steady states?). Nevertheless, whatever preconceptions we might hold, a variety of additional observations do not fit a model of passive paracellular passage. For example, if a paracellular shunt accounts for the presence of mannitol in ductal fluid, then other molecules of similar size and physical properties should be present at about the same concentration, having followed the same passive paracellular pathway to enter the duct. If a structurally analogous molecule is also taken up by cells, such as those that are metabolically active (e.g., the hexose and hexitol glucose and inositol), then its concentration is ductal fluid should be higher than mannitol, reflecting the sum of both paracellular and transcellular fluxes. But this is not the case. The steady-state concentration of the metabolizable inositol-almost identical to mannitol in terms of size, shape, and charge-in ductal fluid is not only lower then mannitol, but has a trans/cis ratio of at most, 20% of mannitol’s (in our hands 6-12% for inositol as compared to 54% on the average for mannitol) (Melese and Rothman, 1983a) (Fig. I ) . That is, the epithelium appears about 5-10 times more permeable to mannitol than to the almost identical inositol, even though inositol enters cells and, hence, can also move transcellularly . Similarly, the glucose analog, 3-O-methylglucose, is found in secretion at a trans/cis ratio of about 8%, which is only 15% of that for mannitol (Melese and Rothman, 1983a). If these three substances (mannitol, inositol, and 3-0-methylglucose) travel through a paracellular shunt, then the lowest permeability value

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sets the upper limit for movement of six-carbon monosaccharides by this route, assuming that the paracellular path is passive (that is, it is unable to distinguish between these similar substances). If this is the case, then the much higher permeability to mannitol must reflect cellular, and not paracellular, permeability. To explain the lower permeability values for inositol and the glucose derivative relative to mannitol, we have to propose that specific cellular processes restrict their transport; in particular, their chemical transformation or utilization within the cell, or specific cellular permeability and transport limitations (that perhaps involve intermediary cellular compartments), or both, that do not exist for the nonmetabolizable mannitol. We noted that stimulants of protein secretion increase the permeability of the pancreatic epithelium. In the presence of a cholinergic stimulant, the concentration of inositol and 3-O-methylglucose, as well as sucrose and inulin, in ductal fluid is increased some 3- to 4-fold or more (Fig. 1) (Melese and Rothman, 1983a-c). Mannitol concentration, however, is unaffected. Why does mannitol concentration not increase as well? The fact that its concentration in ductal fluid is much higher in the first instance (in the absence of the stimulant) suggests an explanation. Perhaps the higher value indicates that mannitol concentration in the trans (ductal) compartment is maximal, i.e., as close as possible to equilibrium for the particular system (given the effect of flow) in the unstimulated state, and for this reason cannot increase when epithelial permeability increases. If this is the case, however, then why is the concentration of urea in ductal fluid at the steady state, also seemingly as close to equilibrium as possible, substantially higher than mannitol (-90 versus -60%) ( Jansen et al., 1979)? Shouldn’t they be identical? The difference might be explained if the equilibrium were not between ductal contents and the medium, but between ductal and cell contents. In this case, the difference could be due to differences in the relative ease of entrance of mannitol versus urea into the cell across its basolateral surface. Such a difference might lead to different intracellular concentrations for the two substances at the steady state, which in turn would lead to different concentrations in ductal fluid, even if both were at equilibrium across the apical cell surface.

C. TISSUEUPTAKE If mannitol is passively distributed across cells, rather than between them, then of course it should be found in them. Using albumin space, 16% as reported by Swanson and Solomon (1973), as the standard for interstitial space in this tissue, tissue uptake of mannitol(62%) (Jansen et

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al., 1979; Melese and Rothman, 1983a) clearly includes a substantial cellular component. We can use this value to test the notion of an equilibrium distribution between cell and duct contents for mannitol. If we correct for mannitol’s presence in interstitial space (assuming equilibrium), its cellular concentration (per unit volume) would be approximately 56% (of medium concentration). This is essentially indistinguishable from its concentration in ductal fluid at the steady state [54% in our hands (1983a) and 60% in the study of Jansen et al. (1979)l. Thus, cellular and ductal concentrations indeed appear to be almost the same (i.e., close to equilibrium relative to each other, even in the absence of a stimulant). Similarly, the uptake of both sucrose and inulin also indicates a cellular component. Tissue sucrose concentration averaged about 64%, depending upon the experimental situation, yielding a cellular concentration 48% of medium concentration (Melese and Rothman, 1983~).Tissue inulin concentration averaged about 41%, yielding a cellular concentration 25% of medium concentration on the average (Melese and Rothman, 1983~). The concentration of sucrose in secretion varied from about 8 to 12%, and inulin was 2 to 6% (Melese and Rothman, 1983~).Thus, unlike mannitol, both inulin and sucrose are apparently secreted in the presence of a substantial concentration gradient from cell to duct. The concentration ratio (cell to duct) for sucrose in two different experimental settings was 4.1 and 4.5, and the ratio for inulin was 5.0. In the presence of a cholinergic stimulant that increases the concentration of sucrose and inulin in secretion, the concentration ratio decreased to values that approximate the equilibrium state between cellular and ductal content [1.4 and 0.94 for sucrose and inulin, respectively, now similar to the value for mannitol (0.96-1.07) (Melese and Rothman, 1983c)l. This suggests that the permeability change produced by the cholinergic agonist occurs at the membrane separating cell from ductal contents, the apical cell membrane. For substances at equilibrium across this membrane prior to stimulation (e.g., mannitol), concentration is unaltered by stimulation. In contrast, when a gradient from cell to duct is present prior to stimulation, the concentration of the substance in ductal fluid increases and the equilibrium state between cell and duct is more closely approached subsequent to stimulation. Thus, tissue uptake data support the view that transepithelial passage of these substances is in great part cellular, and that cellular events are responsible for the increase in permeability seen with cholinergic stimulation. Moreover, this increase in permeability appears to be the result of an increase in the permeability of the apical cell membrane. This latter conclusion makes considerable sense in that, however one views protein secretion as occurring (vesicular or membrane transport processes or

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some combination thereof ), changes in the permeability properties of the apical membrane of the cell with stimulation of protein secretion (as occurs with cholinergic agonists) would not be suprising.

IV. Other Evidence of Transcellular Transport and Permeability Changes A. APICAL CELLMEMBRANE

There is a variety of additional experimental evidence that supports these conclusions, some of which follows: 1. Although the concentration of inositol in ductal fluid increases in the presence of a cholinergic stimulant, its transepithelial transit time does not decrease (Melese and Rothman, 1983a). If the increase in concentration were due to an increase in the permeability of a passive paracellular pathway, then transit time should be reduced. This is because P = x / k r , where P is permeability, x is the thickness of the epithelium, r is transit time, and k is a geometric constant (Melese and Rothman, 1983a).Transit time should be reduced in proportion to the increase in concentration (in ductal fluid) when the system is far from equilibrium (when P is proportional to the change in concentration), or greater than proportionately when the system is close to equilibrium (when the change in concentration underestimates P ) . If a proportional relationship between concentration and permeability applies, as it more or less does for inositol, then transit time should have been reduced by some 70-80%, from approximately 6 to 1-2 minutes, for the observed increase in concentration. As noted, no change was observed. 2. No change in the permeability of the basolateral membrane to inositol occurs with stimulation, that is, the initial rate of inositol uptake into tissue across the basolateral surface remains unchanged (Melese and Rothman, 1983a). Given that the effect is transcellular, this indicates, by exclusion, that the increased inositol concentration in ductal fluid is due to events at the apical cell membrane. 3. The concentration of phosphate ion in ductal fluid increases some 3to 4-fold in the presence of a cholinergic stimulant when phosphate is in the medium bathing the gland, but increases equally when it is absent from the medium (Melese and Rothman, 1983b)(Fig. 2). In the latter case, only a cellular source of phosphate is available, and the equivalent increase in concentration in both situations suggests increased phosphate exit from the cell across the apical membrane in both cases.

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Time (min) FIG.2. Phosphate (inorganic) output in ductal fluid in the presence (open bars) and absence (closed bars) of phosphate in the medium. “Unstimulated” refers to the 15-minute periodjust prior to the addition of MCh ( 1 mg/100 ml bath fluid) (15-60 minutes). Samples of secretion were collected from the cannulated duct of the rabbit pancreas in organ culture. Cholinergic stimulation increased phosphate output maximally by about 3-fold in this particular group of experiments. Phosphate output, in both unstimulated and stimulated states, was approximately the same whether or not phosphate was present in the medium bathing the gland. Values are means k SE, n = 5-6 for all data. *p < 0.05, * * p < 0.025, * * * p < 0.01, ****p < 0.001 versus “unstimulated.” Reprinted from Melese and Rothman (1983b). with permission.

4. Removal of [‘‘C]sucrose (or [“Clinulin) from the medium bathing the gland should, of course, lead to a decline in the concentration of the isotope in ductal fluid as well, over time reaching a new lower steady-state concentration related to the concentration of the substance remaining in the medium after the change of fluid. If this reequilibration is by means of a passive paracellular shunt, the decline should occur over a period of time, and be of a form, that, in essence, mirrors the original equilibration when isotope is first added to the medium. This is because the rates of both equilibration and reequilibration are functions of the phenomenological constants governing the particular process, and of course these would not change merely because we removed, rather than added, the substance whose equilibrium was being studied. That is, reequilibration would be described by an exponential function with the same constants as the initial

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equilibration, and should take approximately the same time, 30-45 minutes, to reach the new steady state. For a simple three-compartment system, the net flux of a substance is Y = A,(l-e?I') + Az(l-e-'2'), where A , and A, are the concentrations of the substance at the steady state in two recipient compartments in series with each other, and k, and k, are the rate constants for the filling of each compartment. Similarly, during reequilibration, - Y = A,(l-e-kl') - A2(1-e-'2'). In the latter case, for reequilibration, because the two exponentials are related in a subtractive fashion, the time would be reduced; the reduction would be significant if both expressions (exponentials 1 and 2) are of comparable magnitude. Thus, by 30-45 minutes after removal of the isotope from the bathing medium, we would expect a new, lower steady-state concentration to have been established in ductal fluid. The concentration should be reduced, relative to the prior steady state, in direct proportion to the decrease in medium concentration. As a result, we would expect a trans/cis ratio identical to that seen prior to removal of the isotope (expressed as percentages, 8-12% for sucrose and 2-6% for inulin). When this experiment was performed (and the isotope removed after the tissue was labeled for 2 hours) (Melese and Rothman, 1983c), reequilibration took far longer than the initial period of equilibration; even 2 hours after its removal, the concentration of isotope in ductal fluid had only fallen by some 80-85% of the >97% required to reestablish the original trandcis ratio and a proper steady state (Fig. 3). At 2 hours, the concentration of sucrose in ductal fluid was equal to its concentration in the medium (100%) and inulin was present at 70% of its medium concentration, not 8- 14 and 2-6%, respectively, the expected steadystate values (Figs. 3 and 4). Thus, despite a rapid initial decline, secreted fluid only slowly reequilibrated with the new, much lower, medium concentration. 5 . If stimulants act by increasing the permeability of a paracellular shunt, then their addition during reequilibration should lead to a more rapid reduction in the substance's concentration in secreted fluid and hasten attainment of the new steady state. For example, for sucrose, the stimulant should hasten reduction in the trans/cis ratio from -2 to 20, the approximate range of ratios immediately after the isotope was removed, to 0.5, the ratio seen in the continuing presence of the isotope when the stimulant was added. When a stimulant was added in this situation, however, the concentration of the substance in ductal fluid not only did not fall more rapidly, it increased (Figs. 3 and 5 ) (Melese and Rothman, 1983~).Indeed, for

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Time ( m i d FIG. 3. Concentration of labeled sucrose in ductal fluid in the presence of labeled sucrose in the bathing medium and after the pancreas was transferred to a sucrose-free medium (arrow), both in the presence (solid triangles) and absence (open triangles) of the hormone cholecystokinin (CCK) (6.0 Ivy dog unitdl00 ml of bath fluid). Although there was initially a roughly exponential decrease in labeled sucrose concentration in ductal fluid after the transfer, reequilibration did not occur at a rate comparable to the initial equilibration (dotted line is the inverse of the initial equilibration), but was protracted, only achieving concentration parity with the bathing medium after 2 hours in the absence of sucrose. In the presence of CCK, the decline in labeled sucrose in ductal fluid was not accelerated, as expected if the permeability of a passive paracellular shunt had been increased, but was further truncated to the point that the concentration remained elevated above medium concentration over time [2.6 times medium concentration and ductal concentration (in the absence of the stimulant)] at the end of the experiment. Concentration of labeled sucrose in the bath immediately after transfer of the organ to a sucrose-free medium and at the end of the experiment is shown (open squares). Data are means 2 SE, n = 24 (18 unstimulated after bath change and 6 stimulated),p < 0.001. CCK at 270 minutes versus control (-CCK). Reprinted from Melese and Rothman (1983c), with permission.

sucrose, the increase was proportionately identical (4- to 5-fold) to that observed in the continued presence of labeled sucrose in the medium (Fig. 5). Thus, sucrose concentration in ductal fluid had risen to values four t o five times the sucrose concentration in the bathing medium, not only not falling more sharply, but giving the illusion of uphill transport from medium to duct (if we apply movement through passive paracellular shunts as our model). To explain these observations, we must propose a retained (parallel) source of label within the tissue (presumably within cells) that reequilibrates slowly, release from which is enhanced by the stimulant, whatever the concentration of isotope in the medium might be. 6. Although the concentration of sucrose in fluid collected from the pancreatic duct increased greatly with cholinergic stimulation when the sucrose concentration in the medium bathing the gland was either 2 or 25

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"

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FIG. 4. Concentration of labeled inulin in ductal fluid in the presence of labeled inulin in the bathing medium and after the pancreas was transferred to an inulin-free medium (arrow). The concentration of inulin 2 hours after its removal from the inulin containing medium was 70% of the concurrent remaining medium concentration (open squares), not 2-6% as predicted. Despite the initial falloff in inulin concentration in ductal fluid after the gland was transferred to an inulin-free medium, reequilibration occurred much more slowly than the initial period of equilibration. The minimal rate of reequilibration expected if it occurred through a passive paracellular shunt is shown by the dotted line. Data are means 2 SE, n = 6. Reprinted from Melese and Rothman (1983c), with permission.

mM, an increase was not seen when sucrose concentration was 100 mM (Bonting et al., 1980). This suggests that transport is in some fashion

concentration limited, even for the presumably passively distributed sucrose. If transepithelial transport were by means of a passive paracellular conduit, then as long as sucrose dissolved fully in water, as it does at 100 mM, a proportional increase should be seen. 7. The addition of 2,4,6-triaminopyrimidine, a substance thought to block paracellular channels, had no effect on transepithelial transit of Na', other electrolytes, water, sucrose, or mannitol (Jansen et al., 1980). This suggests that either the substance was not effective in blocking these particular channels, or that passage did not occur by this route. 8. The blocking agent did, however, attenuate the increase in permeability due to stimulation with a cholinergic drug, although it did not diminish the increase in permeability seen when a gastrointestinal hormone, cholecystokinin (CCK), was used to stimulate secretion instead (Jansen et al., 1980). If we apply a paracellular model, and assume that the agent blocks channels, then we must propose that it is effective only in the presence of the cholinergic drug, but not in the unstimulated state or in the presence of another stimulant that increases permeability (CCK).

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Time ( m i d FIG. 5 . Effect of MCh ( I mg/100 ml bath fluid) (bar) on the concentration of sucrose in ductal fluid 2 hours after labeled sucrose had been removed from the bathing medium (see Fig. 3). Sucrose reached a peak concentration in ductal fluid five times the prestimulus value and five times its concurrent concentration in the bathing medium. The concentration of labeled sucrose in the bath immediately before addition of the stimulant and at the end of the experiment is shown (open squares). Data are expressed as means t S.E.; n = 6 . p < 0.01, without MCh (120 minutes) versus with MCh (140 minutes). Reprinted from Melese and Rothman (1983c), with permission.

B. SODIUM PERMEABILITY A N D THE SODIUM GRADIENT A consideration of sodium permeability may be helpful in putting the notion of leaky cell membranes into the context of a functioning cell. As we have already noted, the gland is quite active in the in vitro system used in these studies. It secretes water and electrolytes at rates comparable to those seen in situ, maintains a 4- to 6-fold HC0,- concentration gradient from medium to duct that is comparable to that seen in situ, and is quite responsive to stimulants of protein secretion (Rothman, 1964, 1966; Rothman and Brooks, 1965; Ridderstap and Bonting, 1969). This of course suggests that the tissue is functioning normally, at least in the sense that the cells of the gland do osmotic work, carry out active ion transport, and are responsive to stimulants in vitro. Another characteristic of this tissue in vitro that suggests more or less normal function is its ability to maintain ion gradients between cell and medium, notably a sodium gradient (Swanson and Solomon, 1973; Rossier and Rothman, 1975). The sodium (-potassium) gradient, perhaps the premiere example of a disequilibrium steady state essential for cell function, requires energy for its maintenance, and in turn provides a potential energy gradient for other cellular processes. For the rabbit pancreas in vitro, cellular sodium concentration is maintained at approximately 25 mM when bathed by a medium containing sodium at 143 mM. This sodium gradient is maintained in spite of the substantial leakiness

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of the cell membrane to this ion. Pancreatic tissue equilibrates with *”a in about 1-3 minutes, i.e., the cellular sodium pool exchanges completely within this interval (Rossier and Rothman, 1975). Although this rapid rate may in part reflect an acceleration in the equilibration of labeled sodium due to diffusional exchange of labeled for unlabeled material, it is nevertheless very rapid in comparison to the rate of sodium equilibration seen for many cells (Stein, 1967). In spite of this substantial permeability, pancreatic secretory cells are capable of maintaining the ion gradients necessary to carry out the cell’s physiological functions. Moreover, even though the pancreatic epithelium is quite permeable to large substances such as sucrose and inulin, it is nonetheless proportionally less so than to the much smaller sodium ion. That is, the cell membrane’s leakiness to large molecules must be viewed in relative terms. Its leakiness to large molecules does not compromise the cell’s ability to maintain ion gradients and function normally any more than its substantial permeability to small ones does. Indeed, the large difference in permeability between sodium (and other small molecules) and inulin (and other large substances) indicates that channels permeable to large substances occupy only a minor portion of the total channel cross-section (“total pore area”), and thus only account for a small fraction of transmembrane ion fluxes and conductance. C. MAIN DUCTOF THE RABBITSALIVARY GLANDS Nonetheless, the fact that the secretory cells of the pancreas appear very permeable to both large and small molecules alike suggests that the cell membrane is, in a general sense, leaky. A similar conclusion was drawn by Augustus et al. (1977) about the permeability properties of the cells of the main duct of the rabbit salivary gland (Augustus et al., 1978). They noted that two criteria are widely applied to distinguish between, and to describe, tight and leaky epithelia-the magnitude of the transmural resistance (R) (or the conductance, G) and the size of the electrical potential across the epithelium (p.d.).Tight epithelia are characterized by a high p . d . and a high R; the opposite is true for leaky epithelia. A direct proportionality can be demonstrated among a variety of epithelial tissues for these two parameters (R andpd.) (Fig. 6). Of course, such a relationship is predicted, indeed must be observed, if a simple passive paracellular electrical shunt is the primary conductive pathway. In part on this basis, the transmural electrical properties (tight or leaky) of epithelia have come to be viewed as being expressive of the nature of the paracellular pathway. The tighter the paracellular junction, the higher the resistance and greater the transmural potential difference, and vice versa.

“LEAKY” CELLS OF GLANDULAR EPITHELIA

24 1

Augustus et al. (1977, 1978) observed, however, that the salivary duct epithelium was able to display transmural potentials as great as those of the “tightest” epithelia (over 100 mV), and yet simultaneously have a remarkably low transmural resistance, equivalent to that of the “leakiest” epithelia (as low as 10 (n cm-2)(Fig. 6). Moreover, they observed an inverse, not a direct, relationship between p . d . and R. They drew the conclusion that their observations were not explicable in terms of passive paracellular electrical shunts, but required “cellular explanations,” presumably being accounted for by the more complex electrical circuitry of the cell. As to the notion of leaky cells they conclude: “Interestingly, the resistance of cell membranes can be as low as 10 ohms cm-*-we found no reason to ascribe this low resistance to an extracellular pathway” (Augustus et al., 1977). V. Nature of the Paracellular Path

A. PARACELLULAR ROUTE In our discussion to this point we have assumed that a paracellular route is “simple” and “passive.” That is, transepithelial movement by

d~,,(~’*z cm) FIG. 6. Relationship between transepithelial potential (e,,) and the square root of resistance in various epithelia (solid line): (1) rabbit proximal tubule, (2) rabbit ileum, (3) rabbit gallbladder, (4) human gallbladder, ( 5 ) rat early distal tubule, (6) rabbit colon, (7) lizard gastric mucosa, (8) rat late distal tubule, (9) dog late distal tubule, (10) rabbit collecting duct, ( I I ) hamster collecting duct. (For citations to these values see Augustus e? a / . . 1977.) The dashed line gives values for the main duct of the rabbit salivary glands in symmetrical solutions of varying chloride concentration, as specified. Note that a substantial transmural potential can be developed across the salivary duct epithelium even though the resistance is quite low. Reprinted from Augustus e? a / . (1977), with permission.

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means of such a route can be described in terms of a three-compartment (medium/paracellular space/ductal space), simple diffusion, hydraulic flow model. It is this model that does not fit many of the observations discussed above. This view, however, makes two assumptions about the paracellular environment that we should consider. The first is that the paracellular space can be properly thought of as a single compartment, and therefore that nonelectrolytes traveling through it would not be “sequestered” in other (more slowly equilibrating) subcompartments within that space, as they might within the cell. If such subcompartmentalization existed and displayed the necessary properties, it might account for some of the results. The second assumption is that the substances in the medium and in the paracellular space are roughly at equilibrium with each other, and that equilibration between these two compartments occurs very rapidly, relative to transepithelial movement. If this were not the case, then the uptake of substances into the cell from the paracellular space might reduce the concentration of a particular substance within that space, and lead to a lower concentration of this particular substance in ductal fluid, than for another similar substance that is not accumulated by the cell (i.e., whose concentration in the paracellular space is not reduced). Such an effect might explain the large difference between mannitol and inositol concentration in ductal fluid without invoking transcellular transport, at least if one ignores the tissue uptake data. Inositol would be accumulated by the cell, whereas mannitol would not be. If such a difference accounted for the results, then we should be able to restore the concentration of the substance in the paracellular channel to equal that in the medium simply by raising medium concentration to saturate cellular uptake processes. We performed this experiment with inositol, but were unable to increase its trans/cis ratio to levels even approaching those seen for mannitol, as should have been possible if this explanation applied (Melese and Rothman, 1983a).

B. VESICLES Although the membranes of the pancreatic secretory cell are permeable to large substances, the mechanism of “permeation” might involve the formation of vesicles from the basolateral membrane of the cell-specifically, the uptake of substances into the cell in vesicles formed from the basolateral cell membrane (endocytosis), the transcellular movement of these vesicles, and the eventual release of vesicle contents into the duct as a result of fusion of the vesicle membrane with the apical cell

“LEAKY” CELLS OF GLANDULAR EPITHELIA

243

membrane (exocytosis). That is, the membranes are only permeable to these substance in this “indirect” sense. If, however, transepithelial transport is due to the trapping of substances (such as the nonelectrolytes mannitol, sucrose, and inulin) within forming vesicles, given no reason on purely physical grounds to expect that one or the other should bind differentially to vesicle membranes, their concentrations in tissue and ductal fluid, relative to that in the medium, should be identical. As we have discussed, however, large differences are observed. For example, a 5- to 10-fold difference in concentration is seen between the physically “identical” inositol and mannitol, with mannitol being much greater. To account for concentrations of such substances in secreted fluid of the order of 50% or more of that in the medium, as was seen in a variety of situations for several of the substances studied, would require that the transcellular vesicle system carry approximately half the bulk of secreted material. Similarly, if the tissue uptake of these various substances were vesicular, then such vesicles would be expected to occupy a large fraction of cell volume, given tissue uptake values. In the case of mannitol, for example, we would have to conclude that more than half the cell volume, a volume about equal to all intracellular membrane-enclosed spaces in this cell, is occupied by such vesicles. VI. Concluding Remarks

The rabbit pancreas was found to be quite permeable to relatively large polar nonelectrolytes, such as mannitol, sucrose, and inulin. Indeed, in terms of this criterion it may be the leakiest epithelium thus far identified. In this article we have reviewed the evidence for this permeability, as well as the unexpected finding that it is expressed across the cells of the gland, not between them. Both the extent of the nonspecific permeability and the fact that it is expressed across cells give a substantially different picture of epithelial leakiness than have studies of planar, predominantly nonglandular, epithelial surfaces. The nonelectrolyte permeability of the rabbit pancreas appears to be due to the passage of substances directly through the cell’s membranes and is not the result of special vesicle transport processes. Permeability is increased several-fold when protein secretion is stimulated. This increase, traced to the apical surface of the secretory cell, indicates a reorganization of the cell membrane at this site that is maintained in the presence of the stimulating hormone. Despite the seemingly analogous results obtained for the cells lining the

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major duct of the rabbit salivary glands, conclusions regarding the extent to which the permeability characteristics of the rabbit pancreas reflect a more general characteristic of glandular epithelia must await results of similar studies on other glands.

REFERENCES Augustus, J., Bijman, J., Van Os, C. H., and Slegers, J. F. G. (1977). Nature (London) 268, 657-658. Augustus, J., Bijman, J., and Van Os, C. H. (1978). J . Membr. B i d . 43, 203-226. Bonting, S. L., DePont, J. J. H. H. M., Fleuren-Jakobs, A. M. M., and Jansen, J . W. C. M. (1980). J. Physiol. (London) 309, 547-555. Diamond, J. M. (1974). Fed. Proc. Fed. Am. SOC.Exp. Biol. 33, 2220-2224. Frompter, E., and Diamond, J. M. (1972). Nature (London) New Biol. 235, 9-13. Goetze, H., and Rothman, S. S. (1978). Biochim. Biophys. Acta 512, 214-220. Jansen, J. W. C. M., De Pont, J. J. H. H. M., and Bonting, S. L. (1979). Biochim. Biophys. Act0 55, 95-109. Jansen, J. W. C. M., Fleuren-Jakobs, A. M. M., De Pont, J. J. H. H. M., and Bonting, S. L. (1980). Biochim. Biophys. Acra 598, 115-126. Melese, T., and Rothman, S. S. (1983a). Biochim. Biophys. Acta 763, 212-219. Melese, T., and Rothman, S. S. (1983b). Am J . Physiol. 245, C121-Cl24. Melese, T., and Rothman, S. S. (1983~).Proc. Natl. Acad. Sci. U.S.A. 80, 4870-4874. Powell, D. W. (1981). Am. J . Physiol. 241, G275-G288. Ridderstap, A. S., and Bonting, S. L. (1969). Am. J. Physiol. 217, 1721-1727. Rossier, M., and Rothman, S. S. (1975). A m . J. Physiol. 228, 1199-1205. Rothman, S. S. (1964). Nature (London) 204, 84-85. Rothman, S. S. (1966). A m . J . Physiol. 211, 777-780. Rothman, S. S., and Brooks, F. P. (1965). Am. J. Physiol. 208, 1171-1176. Swanson, C. H., and Solomon, A. K. (1973). J. Gen. Physiol. 62, 407-429. Stein, W. D. (1967). “The Movement of Molecules across Cell Membranes.” Academic Press, New York. Wright, E. M., and Pietras, R. J. (1974). J . Membr. Biol. 17, 293-312.

Index

A

Acanrhamoeba cnstellanii, ameboid movement and, 175 Acetylcholine, placental lactogens and, 27, 28 N-Acetylgalactosamine, membrane oligosaccharides and, 79 N-Acetylgalactose, membrane oligosaccharides and, 78 N-Acetylgalactosyl, membrane oligosaccharides and, 83 N-Acetylglucosyl, membrane oligosaccharides and, 83 N-Acetylneuraminic acid, membrane oligosaccharides and, 79, 80 Acid phosphatase, hepatitis and, 203 Acidity, ameboid movement and, 143 Acini, hepatitis and, 192 Acridine Orange ameboid movement and, I58 endosperm, maize and, 103 Actin, ameboid movement and, 137, 177 microfilament system function, 158, 161, 163, 165-167, 174, 175 microfilament system organization, 149-151, 154-156 theories, 139 Actin-modulating protein, ameboid movement and, 161 Actomyosin, ameboid movement and, 138 microfilament system function, 159, 163, 166, 174 microfilament system organization, 149-151, 153 Acute lymphoblastic leukemias, membrane oligosaccharides and, 80

245

Acylation, membrane oligosaccharides and, 73 Adenyl cyclase, decidual luteotropin and, 50 Adhesion ameboid movement and, 147 membrane oligosaccharides and, 73, 74 Adipocytes, placental lactogens and, 35 Adipose tissue, placental lactogens and, 35-37 Adrenal gland, placental lactogens and, 23, 26 B-Adrenergics, placental lactogens and, 27, 28 Adrenocorticotropin, placental lactogens and, 19 Adsorption ameboid movement and, 143 hepatitis and, 217 membrane oligosaccharides and, 70 Age-dependent susceptibility, hepatitis and, 215, 218, 219 Agglutination, membrane oligosaccharides and, 82 Aging, membrane oligosaccharides and, 67 cell surface, 68-71 immune regulation, 78 senescence, 92 Albumin, glandular epithelia and, 232 Alcohol, glandular epithelia and, 229 Aleurone, endosperm, maize and, 133 activity, 111, 112 early development, 98, 101 Alkaline phosphatase, membrane oligosaccharides and, 84, 85 Alkalinity, ameboid movement and, 143

246

INDEX

Allantoic fluid, placental lactogens and, 13, Antigens hepatitis and, 197, 214 14 Ameboid movement, 137, 138, 175-179 membrane oligosaccharides and microfilament system function, 156 aging cell surface, 69, 71 cell models, 173, 174 immune regulation, 77, 78 chemical investigations, 156-167 neoplastic regulation, 79, 80 isolated cytoplasm, 174, 175 Antiinterferon, hepatitis and, 2 16 physical investigations, 167- 173 Antilymphocyte serum, hepatitis and, 204, microfilament system organization, 149 215 nature, 149 Antimacrophage serum, hepatitis and, 198, physiological polarity, 154-156 202 spatial arrangement, 149-151 Antithymocyte serum, hepatitis and, 203 ultrastructural organization, 151-154 Apical cells, glandular epithelia and phenomena, 140-142 experimental system. 232. 233 internal organization, 143- 147 transcellular transport. 234-238 membrane turnover, 147-149 Apolipoproteins, placental lactogens and, pseudopodium formation, 142, 143 30 theories, 139, 140 Arachidonic acid Amino acids decidual prolactin and, 46 decidual prolactin and, 42, 47 placental lactogens and, 30 endosperm, maize and, 131 Arginine, placental lactogens and, 27, 30 placental lactogens and ATP, ameboid movement and, 149, 174, biochemical characterization, 4-7 175, 177 biological activities, 32, 37, 38 Autofeedback, decidual prolactin and, 45 secretion, 8, 9 Autoradiography, endosperm, maize and, prolactins and, I , 51, 52 116, 127 Amitosis, endosperm, maize and, 112 Avidin, membrane oligosaccharides and, Amniochorion, decidual prolactin and, 43, 84, 85 48, 49 Amnion, decidual prolactin and, 43, 47-49 Amniotic fluid decidual prolactin and, 41-50 B placental lactogens and, 13, 14 prolactins and, 51 B cells, membrane oligosaccharides and, Amoeba proteus, see Ameboid movement 78 Amylopectin, endosperm, maize and, B lymphocytes, membrane I I3 oligosaccharides and, 78 Amylose, endosperm, maize and, I13 Bacteria, hepatitis and, 208, 213 Anaphase, endosperm, maize and, 107 Basal cells, endosperm, maize and, 101 Antibodies Basement membranes, hepatitis and, 186 ameboid movement and, 137, 166, 177 Benzamide, ameboid movement and, 161 hepatitis and Bile, hepatitis and, 192, 200, 205 immune response, 214, 215 Bioassays interferon, 216 decidual prolactin and, 41 liver-derived cells, 198, 205, 21 I placental lactogens and, 10, 32, 34 susceptibility, 219 prolactins and, 4 Antibody-dependent complement cytolysis, Bladder, glandular epithelia and. 225 hepatitis and, 216 Bone marrow, membrane oligosaccharides Antibody-dependent endocytosis, hepatitis and, 75 and, 198, 216 Bunyavirus, hepatitis and, 196

247

INDEX

C Calcium ameboid movement and, 177 microfilament system function, 173-175

phenomena. 143, 147 theories, 139 decidual prolactin and, 43 membrane oligosaccharides and, 72, 74. 82

placental lactogens and, 9, 30 Calmodulin, placental lactogens and, 9 Canaliculus, hepatitis and, 192 Canine glioma cells, membrane oligosaccharides and, 88, 90-92 Carbohydrates endosperm, maize and, 127, 131 membrane oligosaccharides and, 67 aging cell surface, 69, 70 developmental phenomena, 73-76 immune regulation, 77, 78 neoplastic regulation, 79 senescence, 82. 83, 85, 91 placental lactogens and, 35 Carbon blockade, hepatitis and, 198 Carbonyl groups, membrane oligosaccharides and, 69 Casein, placental lactogens and, 32 cDNA decidual prolactin and, 42, 47 membrane oligosaccharides and. 77 prolactins and, I , 52 Cell-mediated immunity, hepatitis and, 202, 213, 215, 220 Chaos ccirolinrnsis, ameboid movement and, 174 Chloral hydrate, ameboid movement and, 161 Chloride, glandular epithelia and. 226 Chloroform, ameboid movement and, 161 Cholecystokinin. glandular epithelia and, 238 Cholinergic stimulants, glandular epithelia and experimental system. 232, 233 transcellular transport, 234, 237, 238 Chorion decidual prolactin and, 43, 47-49 placental lactogens and, 8, 9

Chromatin, endosperm, maize and activity, 105, 107, I I I endoreduplication, 114 microscopic characterization, 103 Chromomycin A3, endosperm, maize and, 117, 119

Chromosomes endosperm, maize and activity, 105, 108, I 1 1-1 13 endoreduplication, 122 nuclear DNA content, 124 placental lactogens and, 9 Circadian variation, placental lactogens and, 15 Cleavage ameboid movement and, 165 placental lactogens and, 9 Clones endosperm, maize and, 113, 114 membrane oligosaccharides and, 75, 79, 80

Colectomy, hepatitis and, 213 Collagenase, hepatitis and, 196 Colon hepatitis and, 208, 212 membrane oligosaccharides and, 80 Colony-stimulating factor, hepatitis and, I97

Concanavalin A hepatitis and, 203, 215 membrane oligosaccharides and aging cell surface, 69, 70 developmental phenomena, 76 immune regulation, 78 senescence, 83-89 Corpus luteum, decidual luteotropin and, 50

Corynebacrerium paruum, hepatitis and, 203, 207

Cyclic AMP decidual prolactin and, 46 membrane oligosaccharides and, 73, 75 placental lactogens and, 30, 31 Cysteine, placental lactogens and, 5 Cytochalasin B, ameboid movement and, 173

C ytokinesis ameboid movement and. 137, 163, 165 endosperm, maize and, 98 Cytokinin, endosperm, maize and, 128

248

INDEX

Cytolysis hepatitis and, 216, 217 membrane oligosaccharides and, 69 Cytomegalovirus, hepatitis and immune response, 214 liver-derived cells, 201, 202, 204 susceptibility, 2 18 Cytophotometry, endosperm, maize and activity, 106, 109, 110 endoreduplication, 117, 121, 122 nuclear DNA content, 123 Cytoskeleton, ameboid movement and, 137-139, 175 Cytotoxicity, membrane oligosaccharides and, 77, 78

D Decidual luteotropin, I , 50, 51 Decidual prolactin biological activities, 46-50 gestation, 43, 44 identification, 41, 42 metabolism, 43, 44 regulation, 44-46 secretion, 42, 43 Degradation, endosperm, maize and, 126, 131 Deletion endosperm, maize and, 113 placental lactogens and, 19 Dexamethasone, placental lactogens and, 19 Diac ylgl ycerol membrane oligosaccharides and, 72 placental lactogens and, 30 Dictyosomes, ameboid movement and, 149 Dicryosreliurn, membrane oligosaccharides and, 73 Diet, placental lactogens and, 19-22 Diethylstilbestrol, membrane oligosaccharides and, 79 Differentiation, membrane oligosaccharides and, see Membrane oligosaccharides Dimethyl sulfoxide, membrane oligosaccharides and, 79 Dinitrophenol, ameboid movement and, 158

Disease, membrane oligosaccharides and, 70

Disse’s space, hepatitis and, 186, 192 DNA endosperm, maize and activity, 105-1 1 1 amplification, 125-121, 133 endoreduplication, 114-120, 122 microscopic characterization, 103 nuclear DNA content, 122-125 hepatitis and, 203 membrane oligosaccharides and, 88, 89 placental lactogens and, 32 DNA polymerase, placental lactogens and, 32 DNase I, ameboid movement and, 161, 163 Dolichol, membrane oligosaccharides and, 74, 79 Dolichyl phosphate, membrane oligosaccharides and, 74, 75 Dopamine decidual prolactin and, 46 placental lactogens and, 27, 28

E Ectoplasm, ameboid movement and, 175, 178, 179 microfilament system organization, 151

phenomena, 143, 145, 1479 theories, 139, 140179 Ectromelia, hepatitis and immune response, 215 liver architecture, 193, 196 liver-derived cells, 204-207, 213 susceptibility, 219 Electron microscopy ameboid movement and, 138, 175, 177 microfilament system function, 165, 167, 174 microfilament system organization, I51 phenomena, 145 endosperm, maize and, 103, 104, 131 glandular epithelia and, 225 Electrophoresis decidual prolactin and, 41, 47 membrane oligosaccharides and, 68 Encephalitis, hepatitis and, 215

INDEX Endocytosis ameboid movement and microfilament system function, 165- 167 phenomena, 143, 147, 149 membrane oligosaccharides and, 84 Endometrium, decidual prolactin and, 42-44 Endoplasm, ameboid movement and, 175, 177, 178 microfilament system function, 172, 173 microfilament system organization, 151, 156 phenomena, 143, 145, 147 theories, 139, 140 Endoplasmic reticulum, ameboid movement and, 143, 149 Endoreduplication, endosperm, maize and cytological aspects, 114, 115 genome amplification, 115-122 &Endorphin, placental lactogens and, 27, 29 Endosomes, ameboid movement and, 148, I66 Endosperm, maize and, 97, 131-133 activity aberrant chromosome behavior, I 1 1-1 13 cell division, 104, 105 clonal development, 113, I14 DNA amplification, 105-108 nuclear heterogeneity, 108-1 1 1 DNA amplification, 125-131 early development, 97-101 endoreduplication cytological aspects, 114, 115 genome amplification, 115-122 microscopic characterization, 101-104 nuclear DNA content defective kernel mutants, 125 F, crosses, 124, 125 inbreds, 122-124 mutants, 122-124 Endothelial cells hepatitis and, 219 liver architecture, 186 liver-derived cells, 196, 198, 200, 201, 209, 210 membrane oligosaccharides and, 76, 80 Endotoxin, hepatitis and, 220

249

liver-derived cells, 198, 201, 203, 207, 208, 212, 213 susceptibility, 218 Enzymes endosperm, maize and activity, 111 DNA amplification, 127, 128 endoreduplication, 116 nuclear DNA content, 124 hepatitis and, 208 membrane oligosaccharides and, 71, 77, 78, 82, 85 Epithelia, glandular, see Glandular epithelia Epithelial cells decidual prolactin and, 48 hepatitis and, 192 membrane oligosaccharides and, 74 placental lactogens and. 33 Epitopes, membrane oligosaccharides and aging cell surface, 70 immune regulation, 78 neoplastic regulation, 80, 81 senescence, 83, 85, 86 Erythrocytes endosperm, maize and, 98, I10 membrane oligosaccharides and, 68, 75 Erythroid stem cells, membrane oligosaccharides and, 73 Estradiol decidual luteotropin and, 50 decidual prolactin and, 44 placental lactogens and, 24, 25 Estriol, decidual prolactin and, 44 Estrogen sulfatase, decidual prolactin and, 49 Estrogens decidual luteotropin and, 50 decidual prolactin and, 44 placental lactogens and, 24-26 Ethanol, ameboid movement and, 161 Ether, ameboid movement and, 161 N-Ethylmaleimide, ameboid movement and, 158, 173 Ethylmethane sulfonate, endosperm, maize and, 125 Euchromatin, endosperm, maize and, 117-1 19 Exocytosis, ameboid movement and, 147, I49

250

INDEX

F Fasting, placental lactogens and biological activities, 35, 36 regulation, 20-22 Fatty acids membrane oligosaccharides and, 73 placental lactogens and biological activities, 36, 37 regulation, 21, 22 Feedback, membrane oligosaccharides and, 71, 72 Fertilization, endosperm, maize and, 97. 98, 132 Fetus decidual prolactin and, 45-50 placental lactogens and biological activities, 36-39 gestational profiles, I I , 13 regulation, 18, 19 secretion, 8, 9 prolactins and, I , 52 Fibrils, ameboid movement and, 158, 174 Fibrin, hepatitis and, 208 Fibroblasts hepatitis and, 192, 217 membrane oligosaccharides and, 68, 70, 71 placental lactogens and, 37 Fibrogenesis, hepatitis and, 192 Fibronectin membrane oligosaccharides and, 71, 76 Filaments, ameboid movement and, 137, 138, 175-177 microfilament system function, 174 microfilament system organization, 149, 151, 152, 154, 156 Flaviviruses, hepatitis and, 204, 217 Flow cytometry, endosperm, maize and, 124 Fluorescence ameboid movement and, 154, 158, 167 endosperm, maize and, 109, 117, 118, 121 membrane oligosaccharides and, 83, 85 Fluorescent-analog cytochemistry. ameboid movement and, 154, 155, 160, 165, 168, 175, 177 Fractionation, decidual luteotropin and, 50

Fragmin, ameboid movement and, 161-163 Frog virus 3, hepatitis and liver architecture, 193 liver-derived cells, 198, 200, 201, 208, 209, 21 I , 213 susceptibility, 2 18 Fungi, hepatitis and, 208 Fusion ameboid movement and, 149, 167 endosperm, maize and, 98, I14 glandular epithelia and, 225 hepatitis and, 201, 209 membrane oligosaccharides and, 82 placental lactogens and, 10

G Galactose, membrane oligosaccharides and, 74, 17, 82 Galactosyl, membrane oligosaccharides and, 83 Gallbladder, glandular epithelia and, 225, 226 Callus, endosperm, maize and, I10 Gangliosides, membrane oligosaccharides and, 75, 17, 79 Genotype membrane oligosaccharides and, 69 placental lactogens and, 19 Glandular epithelia, leaky cells of, 225, 243, 244 experimental system, 227, 228 distribution, 228, 229 pancreas, 229-232 tissue uptake, 232-234 movement, 225-227 paracellular path route, 241, 242 vesicles, 242, 243 transcellular transport apical cell membrane, 234-238 rabbit salivary glands, 240, 241 sodium permebility, 239, 240 Glaucoma, membrane oligosaccharides and, 75 Glucagon, placental lactogens and, 38 Glucocorticoids, placental lactogens and, 24. 25

INDEX Glucose glandular epithelia and, 231, 232 membrdne oligosaccharides and. 81 placental lactogens and biological activities, 34-37 regulation, 20-23 Glycans, membrane oligosaccharides and. 78 Glycerol. ameboid movement and, 149, 174, 1173 Glycocalyx. membrane oligosaccharides and, 74 Glycogen, placental lactogens and. 35, 37, 38 Glycogenolysis, placental lactogens and, 38 Glycolipids, membrane oligosaccharides and, 68 Glycopeptides, membrane oligosaccharides and, 68 aging cell surface. 70 developmental phenomena, 72, 73, 75 immune regulation, 76-78 senescence, 81, 82 Glycophorin, membrane oligosaccharides and, 75 GI ycoprotein placental lactogens and, 6 prolactins and, 51 Glycosarninoglycans, membrdne oligosaccharides and, 68, 70, 71, 76 Glycosphingolipids. membrane oligosaccharides and, 79 Glycosylation decidual prolactin and, 42 placental lactogens and, 6 Gonadotropin-releasing hormone, placental lactogens and, 27-29 Growth hormone placental lactogens and biochemical characterization, 4-6 biological activities, 31, 32, 34, 36-41 gestational profiles, 10 regulation. 27, 30 secretion, 9 prolactins and, I , 51, 52 Growth hormone-releasing hormone. placental lactogens and, 27-29

25 I H

Hematopoietic cells, membrane oligosaccharides and, 69, 75 Hepadnaviruses, hepatitis and, 185 Heparin, membrane oligosaccharides and, 70 Heparin sulfate, membrane oligosaccharides and, 70, 71, 76, 79 Hepatectomy, hepatitis and, 21 I Hepatitis, 185, 219 immune response, 213-216 interferon, 216 liver architecture normal, 185-193 pathology, 193- 196 liver-derived cells endothelial cells, 209, 210 hepatocytes, 2 10-2 12 Kupffer cells, 196-209 nonviral factors, 212, 213 susceptibility age-dependent, 218, 219 genetic, 216-218 Hepatocytes hepatitis and, 220 interferon, 216 liver architecture, 186, 187, 192. 193. 196 liver-derived cells, 196, 198, 200-202, 204, 205, 207 pathogenesis, 2 10-2 I3 susceptibility, 217, 218 placental lactogens and, 37 Hepatocytolysis, hepatitis and, 201 Hepatotropism, hepatitis and, 210-212 Herpes simplex virus, hepatitis and immune response, 214, 215 interferon, 216 liver-derived cells, 201, 203, 204, 211, 212 susceptibility, 217, 218 Heterochromatin, endosperm, maize and, 114, 115, 117, I I9 Heterogeneity endosperm, maize and, 133 activity, 107-1 1 I endoreduplication, 122 hepatitis and, 193

252

INDEX

membrane oligosaccharides and, 75, 78. 83 placental lactogens and, 7 Heterosis, endosperm, maize and, 124 High-density lipoproteins, placental lactogens and, 30 Homogeneity endosperm, maize and, 1 I5 hepatitis and, 196 membrane oligosaccharides and, 87 Homology placental lactogens and biochemical characterization, 5 , 6 biological activities, 31-34, 37-40 gestational profiles, 13 prolactins and, 1, 51, 52 Hormones glandular epithelia and, 238, 243 hepatitis and, 196 Human chorionic gonadotropin, decidual prolactin and, 50 Hyaluronic acid, membrane oligosaccharides and, 70 Hybridization decidual prolactin and, 47 endosperm, maize and, 133 DNA amplification, 130 early development, 100 endoreduplication, 114-1 16, 119, 122 microscopic characterization, 104, I05 nuclear DNA content, 125 placental lactogens and, 8, 41 Hydrolases, hepatitis and, 207 Hydrolysis, placental lactogens and, 30 Hydromineral balance, decidual prolactin and, 47-49 Hydroxybutyrate, placental lactogens and, 37 Hyperplasia, hepatitis and, 193 Hypoglycemia, placental lactogens and, 22 H ypophy sectom y decidual prolactin and, 43, 47 placental lactogens and, 19, 23, 35

I

Ileum, glandular epithelia and, 230, 231 Immune regulation, membrane oligosaccharides and, 67, 76-78

Immune response hepatitis and liver architecture, 186, 193 liver-derived cells, 197, 204-207, 212 pathogenesis, 213-216 susceptibility, 218, 219 membrane oligosaccharides and, 70 Immunofluorescence ameboid movement and, 149 hepatitis and, 201, 205 Immunoglobulins, hepatitis and, 198 Incubation, hepatitis and, 185 Inflammation, hepatitis and immune response, 214 liver architecture, 193 liver-derived cells, 197, 201, 202, 204 Influenza virus, hepatitis and immune response, 214, 216 liver-derived cells, 204, 211, 212 Inositol, glandular epithelia and experimental system, 231, 232 paracellular path, 242, 243 transcellular transport, 234 Inositol phosphates, placental lactogens and, 30 Insulin membrane oligosaccharides and, 81 placental lactogens and, 22, 34, 35 Insulin growth factors, placental lactogens and, 37 Interferon hepatitis and, 220 liver-derived cells, 204, 207, 209, 212 pathogenesis, 2 14-2 16 susceptibility, 217, 219 membrane oligosaccharides and, 77 Interleukin-I, hepatitis and, 214, 215 Interleukin-2 hepatitis and, 214, 215 membrane oligosaccharides and, 77, 78 Internalization, hepatitis and, 198, 200, 201, 216 Interphase, endosperm, maize and, 103, 107, 112 Inulin, glandular epithelia and experimental system, 229-233 movement, 226, 227 transcellular transport, 235, 240 Irradiation, hepatitis and, 215 Ischemia, hepatitis and, 193, 209, 220

INDEX

K

253

Lipocytes, hepatitis and, 186, 192, 196 Lipolysis, placental lactogens and, 35-37 Karyokinesis, endosperm, maize and, 98 Lipopolysaccharides, hepatitis and, 198, Kidney, glandular epithelia and, 225 212 Kupffer cells, hepatitis and Liver, see also Hepatitis clearance, 197, 198 placental lactogens and, 35, 37-40 extrinsic effects, 207-209 Localization features, 196, 197 ameboid movement and, 137 immune response, 213, 214 microfilament system function, 158, interaction, 198-207 159, 161, 171 liver architecture, 186, 190, 191, 193, 196 theories, 140 liver-derived cells, 210, 212 decidual prolactin and, 42 susceptibility, 219 placental lactogens and, 8 prolactins and, 51 Lungs, decidual prolactin and, 49, 50 L Luteolysis, placental lactogens and, 40 Luteotropic activity c~-Lactalbumin,placental lactogens and, 32 decidual luteotropin and, 50 Lactate, placental lactogens and, 37 placental lactogens and, 39, 40 Langerhans cells, hepatitis and. 214 Lymph nodes, hepatitis and, 204, 222 Lectin, membrane oligosaccharides and Lymphocytes developmental phenomena, 73 hepatitis and, 220 immune regulation, 77 immune response, 214, 215 senescence, 82-85, 87, 89, 90 liver architecture, 192 Lesions, hepatitis and, 193 liver-derived cells, 203-205, 212 Leukemia, membrane oligosaccharides susceptibility, 2 17-2 19 and, 78, 79 membrane oligosaccharides and, 69, 77 Lymphoid cells, membrane Leukocyte common antigen. membrane oligosaccharides and, 78 oligosaccharides and, 74, 77 Leukocytes Lymphoid tissues, hepatitis and, 207, 220 hepatitis and, 193 Lymphokines, hepatitis and, 197, 214, 215 membrane oligosaccharides and, 68 Lymphoproliferation, membrane LH oligosaccharides and, 69 decidual luteotropin and, 50 Lymphoreticular cells, hepatitis and, 200 placental lactogens and, 40 Lysis, hepatitis and Ligands, membrane oligosaccharides and liver architecture, 193 developmental phenomena, 73 liver-derived cells, 196, 197, 208, 209, immune regulation, 77 213 senescence, 82-86 susceptibility, 217 Light, ameboid movement and, 167-172 Lysosomal hydrolases, hepatitis and, 203 Light microscopy Lysosomes ameboid movement and, 138, 143, 154, ameboid movement and, 148, 149 177 hepatitis and, 186, 200, 203, 208 endosperm, maize and, 131 Lipids glandular epithelia and, 227 hepatitis and, 192 M placental lactogens and biological activities, 34, 35 Macrophage-activating factor, hepatitis regulation, 22. 23 and, 214

254

INDEX

Macrophage-inhibiting factor, hepatitis and, 214, 215 Macrophages, hepatitis and, 219, 220 liver architecture, 186, 192 liver-derived cells, 196-209, 212 pathogenesis, 213-215 susceptibility, 217-219 Madin-Darby canine kidney cells, hepatitis and, 21 1 Maize, see Endosperm, maize and Major histocompatibility complex, membrane oligosaccharides and, 77, 78 Malignancy, membrane oligosaccharides and, 79, 80 Mammary gland placental lactogens and, 3 1-34 prolactins and, 1 Mannitol decidual prolactin and, 48 glandular epithelia and, 243 experimental system, 229, 231-233 movement, 226, 227 paracellular path, 242, 243 transcellular transport, 238 Mannose, membrane oligosaccharides and, 71 Mannosyl, membrane oligosaccharides and, 83, 85 Maternal intermediary metabolism, placental lactogens and, 35-37 Membrane oligosaccharides, 67, 68 aging cell surface, 68-71 developmental phenomena, 71-76 immune regulation, 76-78 IMR-90 cellular senescence, 80-92 neoplastic regulation, 78-80 Meningitis, hepatitis and, 215 Menstrual cycle, placental lactogens and, 40, 43, 44 Metaphase, endosperm, maize and, 107 Methyl palmitate, hepatitis and, 198 3-O-Methylglucose, glandular epithelia and, 23 1, 232 Microfilaments, ameboid movement and, 137, 138, 175, 179 function, 156 cell models, 173, 174 chemical investigations, 156-167 isolated cytoplasm, 174, 175

physical investigations, 167-173 organization, 149 nature, 149 physiological polarity, 154-156 spatial arrangement, 149-151 ultrastructural organization, 15 1-154 phenomena, 142, 144, 147, 149 theories, 140 Microtubules, ameboid movement and, 137, 175, 179 Microvilli, hepatitis and, 186, 192 Milk, placental lactogens and, 32, 33 Mitochondria, ameboid movement and, 143 Mitogens hepatitis and, 214 membrane oligosaccharides and, 74 Mitosis ameboid movement and, 137, 175 endosperm, maize and activity, 104, 105, 107, 108, 110, 112, 113 early development, 98 microscopic characterization, 103 hepatitis and, 21 1 Monoclonal antibodies, membrane oligosaccharides and, 79 Monoc ytes hepatitis and immune response, 214 liver architecture, 193 liver-derived cells, 196, 197, 203-205, 208, 209 membrane oligosaccharides and, 79 Monosaccharides, glandular epithelia and, 232 Morphology ameboid movement and, 137 microfilament system function, 156, 158, 162, 174 microfilament system organization, 149, 151 phenomena, 140 endosperm, maize and activity, 1 I I DNA amplification, 131 early development, 97, 98 microscopic characterization, 103 hepatitis and, 186, 193 membrane oligosaccharides and

255

INDEX aging cell surface. 68, 71 developmental phenomena, 74, 75 senescence, 83, 84, 86, 92 Mosaicism, endosperm, maize and, I12 Mouse hepatitis virus immune response, 214, 215 interferon, 216 liver architecture, 193 liver-derived cells, 204-207, 209, 212 susceptibility, 217-219 mRNA decidual prolactin and. 42, 47 placental lactogens and, 8. 17, 18 prolactins and, 5 I Mucopolysaccharides, ameboid movement and, 143 Mutation, endosperm, maize and activity, 108, 1 11-1 13 DNA amplification, 128-131 nuclear DNA content, 122-125 Myoblast cells membrane ohgosaccharides and, 74 placental lactogens and, 37 Myosin, ameboid movement and, 177 microfilament system function, 163, 165, 166, 174, 175 microfilament system organization, 149- IS I

N Narcotics, ameboid movement and, 163 Natural killer cells, hepatitis and, 207, 214, 220 Necrosis, hepatitis and liver architecture, 193, 196 liver-derived cells, 200, 201, 209, 213 Neoplasia, membrane oligosaccharides and, 76. 91 Neoplastic regulation. membrane oligosaccharides and. 78-80 Nerve growth factor, membrane oligosaccharides and, 75 Neuropeptides, placental lactogens and, 27 Neurotransmitters, placental lactogens and, 27 Nitrocellulose, endosperm, maize and, 1 IS Nitrogen, placental lactogens and, 37

Nonparenchymal cells, hepatitis and, 186, 192, 196, 209 Nuclear magnetic resonance, membrane oligosaccharides and, 87 Nucleolar organizer regions, endosperm, maize and, I14 Nucleosides, endosperm, maize and, 126 Nucleotype, endosperm, maize and, 110, Ill

0 Oligosaccharides, membrane, see Membrane oligosaccharides Ornithine, placental lactogens and, 27 Ornithine decarboxylase, placental lactogens and, 38 Osmolarity, decidual prolactin and, 45, 47, 48 Osmosis, glandular epithelia and, 239 Ouabain, ameboid movement and, 158 Ovariectomy, placental lactogens and, 24 Ovary decidual luteotropin and, 50, 51 placental lactogens and biological activities, 31, 39, 40 regulation, 23-26 Oxidation, placental lactogens and, 35 Oxygen glandular epithelia and, 227 hepatitis and, 197, 208

P Pancreas, glandular epithelia and, 225, 243 experimental system, 227-232 movement, 226, 227 paracellular path, 242 transcellular transport, 237, 239, 240 Pancrease, placental lactogens and, 34 Parenchymal cell. hepatitis and, 185, 186, 208, 210 Peanut agglutinin, membrane oligosaccharides and, 80 Peptidases, placental lactogens and. 9 Peptides ameboid movement and, 167 membrane oligosaccharides and, 70, 82

256

INDEX

placental lactogens and, 9 prolactins and, 51, 52 Peritoneal exudate cells, hepatitis and, 201 Permeability, glandular epithelia and, 225, 243, 244 experimental system, 228-234 movement, 226, 227 paracellular path, 242 transcellular transport, 234, 236, 238, 240 PH ameboid movement and, 143 membrane oligosaccharides and, 73 Phagocytosis ameboid movement and, 167 hepatitis and immune response, 214 liver architecture, 186, 192 liver-derived cells, 197, 198, 200, 201, 205, 208 susceptibility, 219 membrane oligosaccharides and, 79 Phalloidin, ameboid movement and, 158 Phenotype endosperm, maize and, 110, 113 hepatitis and, 212, 216 membrane oligosaccharides and, 69, 77, 79, 91 Phorbol esters, membrane oligosaccharides and, 72, 79, 80 Phosphate ameboid movement and, 143 glandular epithelia and, 234 Phospholipase C, placental lactogens and, 30 Phospholipids, membrane oligosaccharides and, 72 Phosphorylation, ameboid movement and, 163 Photosynthesis, endosperm, maize and, 109 Physarum polycephalum, ameboid movement and, 161 Ph ytohemagglutinin hepatitis and, 203 membrane oligosaccharides and, 69 Pinocytosis ameboid movement and, 165-167 hepatitis and, 186, 192, 201, 209 Pit cells, hepatitis and, 192

Pituitary decidual luteotropin and, 50 decidual prolactin and, 41-43, 47, 49 placental lactogens and, 19, 23, 30 prolactins and, 1 Placenta, see also Placental lactogens decidual prolactin and, 45, 47 prolactin-like molecules of, 5 I , 52 prolactins and, I , 5 I , 52 Placental lactogens, I , 4 biochemical characterization, 4-8 biological activities, 3 I , 32 fetal growth, 37-39 mammary gland secretory differentiation, 32-34 maternal intermediary metabolism, 34-37 receptors, 40, 41 relevance, 41 steroidogenesis, 39, 40 concentrations, 10 gestational profiles, 10-13 allantoic fluid, 13, 14 amniotic fluid, 13, 14 circadian variation, I5 metabolism, 15, 16 regulation, 16, 17 adrenal gland, 23 arginine, 27, 30 fetus, 18, 19 genetic factors, 19 high density lipoproteins, 30 metabolic factors, 19-23 neuropeptides, 27-29 neurotransmitters, 27-29 ovary, 23 pituitary, 23 placental mass, 17, 18 second messengers, 30, 31 serum factors, 30 steroid hormones, 23-27 secretion, 8-10 Plasma, placental lactogens and, 7, 40 Plasma membrane ameboid movement and, 138, 178, 179 microfilament system function, 158, 163, 166, 167 microfilament system organization, 150, 151, 154

phenomena, 142-147, 149

INDEX glandular epithelia and, 225 hepatitis and, 192, 210 Plasminogen, hepatitis and, 209 Platelet-derived growth factor, membrane oligosaccharides and, 76 Platelets, hepatitis and, 205 Ploidy, endosperm, maize and, 98, 108 Polarity ameboid movement and, 177 microfilament system function, 158, 163, 169, 173 microfilament system organization, 154- I56 phenomena, 140, 145 hepatitis and, 192, 210 Poliovirus, hepatitis and, 199, 200 Pollination, endosperm, maize and activity, 104. 108 DNA amplification, 128 early development, 98, 99 nuclear DNA content, 122 Polymerization ameboid movement and, 138 microfilament system function, 158, 161, 163, 165, 167 microfilament system organization, 151, 156 endosperm, maize and, 1 I 1 Polymixin, hepatitis and, 213 Polypeptides endosperm, maize and, 1 1 1 placental lactogens and, 4, 6 Polyploidy, endosperm, maize and, 114, 1 15

Polysaccharides, glandular epithelia and, 229 Polysomes, placental lactogens and, 17 Polyteny, endosperm, maize and, 114, 1 15

Population doubling level, membrane oligosaccharides and aging cell surface, 68, 70, 71 senescence, 83-88, 90, 91 Portal vein. hepatitis and, 192, 196 Pregnancy decidual luteotropin and, 50, 51 dccidual prolactin. ,wi’Decidual prolactin placental lactogens and, see Placental lactogens

257

PRL-like molecules of placenta and, 5 1, 52 prolactins and, 1-4 Prehormones placental lactogens and, 9 prolactins and, 51 Procaine, ameboid movement and, 158 Procoagulant, hepatitis and, 209 Profilin, ameboid movement and, 161, 163 Progesterone decidual luteotropin and, 50, 5 1 placental lactogens and biological activities, 39, 40, 44,45 regulation, 24-27 Progestogens, placental lactogens and, 23, 24 Prolactin, I , 51, 52 decidual luteotropin and, 50, 51 decidual prolactin and, 41 -49 placental lactogens and biochemical characterization, 4-6 biological activities, 31, 32, 34, 38-41 gestational profiles, 10 regulation, 27, 30 Prolactin-like molecules of placenta, 51, 52 Prolactins, 1-4, see also specific prolactin Proliferation endosperm, maize and, 113, 114 hepatitis and, 193, 197, 215 membrane oligosaccharides and, 75, 76, 91 Proliferin, I , 51, 52 Pronase, hepatitis and, 196 Prophase, endosperm, maize and, 107 Propidium iodide, endosperm, maize and, 1 I8 Prostaglandins ameboid movement and, 167 decidual prolactin and, 49 placental lactogens and, 40 Proteases, hepatitis and, 205 Protein ameboid movement and, 137, 138, 177, 179 microfilament system function, 161, 163, 174 microfilament system organization, 150 theories, 139 decidual prolactin and, 45, 46

258

INDEX

endosperm, maize and, 101, 108, 128, 133 glandular epithelia and experimental system, 227, 228, 23 1-234 movement, 226 transcellular transport, 239 hepatitis and immune response, 214 liver-derived cells, 200, 201, 208, 209, 213 membrane oligosaccharides and, 70, 71, 75, 84 placental lactogens and biochemical characterization, 6, 7 biological activities, 32 regulation, 16, 20-22, 30 prolactins and, I. 51, 52 Protein kinase C decidual prolactin and, 46 membrane oligosaccharides and, 72 placental lactogens and, 30 Proteoglycans, membrane oligosaccharides and, 68 Proteolysis, membrane oligosaccharides and, 69 Protoplasm, ameboid movement and, 140, 171, 177 Pseudopodia, ameboid movement and, 178, 179 microfilament system function, 156, 162, 163, 165-167, 169, 172, 173 microfilament system organization, 156 phenomena, 140-143, 145, 147 theories, 139, 140 Pseudopregnancy , decidual luteotropin and, 51 Punta Toro virus, hepatitis and, 193. I95 Purification decidual prolactin and, 42, 45 placental lactogens and, 4, 6, 34 Puromycin, ameboid movement and, I58

Q Quinacrine, endosperm, maize and, I17

R Rabbit, glandular epithelia and, 227, 229, 231, 240, 241, 243, 244 Radioimmunoassays decidual prolactin and, 41 placental lactogens and, 10 Radioreceptor assays decidual luteotropin and, 50, 5 I placental lactogens and, 4 biochemical characterization, 7 biological activities, 32-34 gestational profiles, 10 prolactins and, I Rat virus, hepatitis and, 21 1 Replication endosperm, maize and, 106 hepatitis and, 185 immune response, 216 liver architecture, 192, 193 liver-derived cells, 198, 200-202, 204-206, 209-212 susceptibility, 217-219 Reticuloendothelial cells, hepatitis and, 186, 203, 205 Reticulum, hepatitis and, 186 Ricinus commrrnis agglutinin, membrane oligosaccharides and, 83-85, 87, 91 Rift Valley fever virus, hepatitis and, 196, 197, 200, 21 1 RNA endosperm, maize and, 103, 115, 122 hepatitis and, 201 placental lactogens and, 32 rRNA, endosperm, maize and, 114, 130

S Salivary glands, glandular epithelia and, 240, 241. 244 Schwann cells, membrane oligosaccharides and, 75 Secale cereale, endosperm, maize and, 100 Second messengers decidual prolactin and, 46 membrane oligosaccharides and, 72 placental lactogens and, 30, 31 Senescence, membrane oligosacrharides and, 67

259

INDEX aging cell surface, 69, 71 developmental phenomena, 76 IMR-90. 80-92 neoplastic regulation, 78 Serum factors, placental lactogens and, 30 Sialic acid, membrane oligosaccharides and, 68, 69, 74 Sialoglycopeptides, membrane oligosaccharides and, 75 Silica, hepatitis and, 198, 202, 206 Sinusoidal cells, hepatitis and, 185 liver architecture, 185, 186, 188, 189, 192, 193 liver-derived cells, 197, 200, 201, 205, 209-211. 213 Sodium, glandular epithelia and. 226. 239. 240 Somatostatin, placental lactogens and, 27-29 Spermine, ameboid movement and, 163, I65 Starch, endosperm, maize and, 101, 108 Starvation, placental lactogens and, 22, 41 Steroidogenesis placental lactogens and, 31, 39, 40 prolactins and, I Steroids decidual prolactin and, 44, 45 placental lactogens and, 23, 24, 27 Stromal cells, decidual prolactin and, 42-44 Sucrose endosperm, maize and. I 1 1 , I15 glandular epithelia and, 243 experimental system, 229, 232, 233 movement, 226, 227 transcellular transport, 235-238, 240 Sugar. glandular epithelia and, 229 Surfactant, decidual prolactin and, 49 Swainsonine. membrane oligosaccharides and, 91, 92 Synctium, placental lactogens and, 10, 18 Syneresis, ameboid movement and, 144

T T cells. membrane oligosaccharides and aging cell surface, 69. 70 immune regulation, 77, 78

neoplastic regulation, 79, 80 T lymphocytes, hepatitis and, 220 immune response, 214, 215 liver-derived cells, 203, 209 susceptibility, 217, 219 Telophase, endosperm, maize and, 107 Temperature ameboid movement and, 143 hepatitis and, 201 Testosterone, decidual luteotropin and, 50 Theophylline, placental lactogens and, 35 Thorotrast, hepatitis and, 198, 206 Thrombosis, hepatitis and, 209 Thymectomy, hepatitis and, 203, 213, 215 Thymidine endosperm, maize and, 107, 126, 127 membrane oligosaccharides and, 88 placental lactogens and, 32, 37 Thymocytes, membrane oligosaccharides and, 70, 78, 80 Thyrotropin-releasing hormone decidual prolactin and, 46 placental lactogens and, 27-29 Translocation, endosperm, maize and, I13 Transmural resistance, glandular epithelia and, 240, 241 Triglycerides, placental lactogens and, 20, 22, 36 Trypsin, decidual luteotropin and, 50 Tumor necrotic factor, hepatitis and, 209 Tumors hepatitis and, 197 membrane oligosaccharides and, 76, 79, 80 Tunicamycin, membrane oligosaccharides and, 74, 79, 80

U

Underreplication, endosperm, maize and, 114, 115, 119 Urea, glandular epithelia and. 229, 232 Urethane, ameboid movement and, 161 Uroid, ameboid movement and, 177 microfilament system function, 161-163, 171 microfilament system organization, 150. I52 phenomena, 147, 148

INDEX V Vaccinia, hepatitis and, 199, 201, 209 Vesicles, glandular epithelia and, 242 Vesicular stomatitis virus, hepatitis and, 198, 21 1, 212, 216 Viral hepatitis, see Hepatitis Viremia, hepatitis and, 192, 193, 204 Vitamin A, hepatitis and, 192

Wheat germ agglutinin, membrane oligosaccharides and, 75, 83-85, 87

Y Yellow fever virus, hepatitis and, 193

W Z

Werner’s syndrome, membrane oligosaccharides and, 71

Zea mays L., see Endosperm, maize and