The Plasma Membrane of Uterine Epithelial Cells: Structure and Histochemistry

The Plasma Membrane of Uterine Epithelial Cells: Structure and Histochemistry CHRISTOPHER

R.

MURPHY

With 25 Figures

)! SEMPER ~

GUSTAV FISCHER VERLAG· STUTTGART· NEW YORK· 1993

Ph.D. CHRISTOPHER R. MURPHY, B. Sc. (Hons.) Department of Histology and Embryology The University of Sydney Sydney, New South Wales 2006 (Australia)

Acknowledgements

I am grateful to my collaborators past and present who contributed to the author's studies reported here. Several of my present students contributed micrographs: Ms Karen Luxford, fig. 1-3, 6, 7,24 and 25; Miss Bronwyn Jones, fig. 4, 5, 18 and 19; Ms Margot Hosie, fig. 16 and , 17; Miss Y.-L. Shion, fig. 22 and 23. Mr Roland Smith prepared the micrographs for publication and his photographic expertise is greatly appreciated.

Die Deutsche Bibliothek - CIP-Einheitsaufnahme Murphy, Christopher: The 'plasma membrane of uterine epithelial cells : structure and histochemistry / Chnstopher R. Murphy. - Stuttgart; Jena ; New York: G. Fischer, 1993 (Progress in histochemistry and cytochemistry; Vol. 27, No.3) ISBN 3~437-11514-6

NE: GT Library of Congress Card-No. 88-20469

Published jointly by: Gustav Fischer VerlaglVCH Publishers 220 East 23rd Street, Suite 909, New York, New York 10010 Gustav Fischer Verlag Wollgrasweg 49, 70599 Stuttgart © Gustav Fischer Verlag· Stuttgart· Jena· New York· 1993 AIle Rechte vorbehalten Gesamtherstellung: Laupp & Gobel, NehrenlTiibingen Printed in Germany

Contents 1 2 3 3.1 3.2

4 4.1 4.2

4.3 5 5.1

5.2 5.3 6 7 7.1

7.2 8 9

Opening remarks . . . . . . . . . . . . . . . . . . . . . . Introduction: the role of uterine epithelial cells . . . . . . Membrane structure: conventional morphological studies Effects of ovarian hormones . . . . . . . . . The attachment picture . . . . . . . . . . . . Membrane structure: freeze-fracture studies. Intramembranous particles . . . . . . . . . . Lipids . The tight junction and the lateral plasma membrane. Histochemistry of the glycocalyx and related cell surface molecules Enzymes and determinants . . The glycocalyx . The glycocalyx in attachment . . . . . . . . . The basal plasma membrane and basal lamina Biochemical approaches. . . . . . . . . . . . Cell culture, glycans and uterine fluid proteins Isolation of the plasma membrane . . . . The membrane skeleton and cytoskeleton Concluding remarks .

1 1 3 3 5 12 14 17 21 24 24

References . .

50

Subject index

67

27 34 34 39 39

43 44 48

1 Opening remarks Some plasma membranes are clearly more dynamic than others. That is, while we may expect that in some instances membranes act to mostly separate different biological activities, in others, the structure of the membrane is a crucial contributor to the success of that biological activity. Among the latter we would have to number the plasma membrane of uterine epithelial cells since this membrane is the initial site of contact between maternal and embryonic cells at the time of attachment and implantation of the blastocyst and we might reasonably expect the structure and dynamics of this plasma membrane to playa crucial role in the success or otherwise of these events. Its central role in such vital biological phenomena also suggests that studying this plasma membrane may not only throw light on these events but may also contribute to our understanding of membrane phenomena at a more general level.

2 Introduction: The role of uterine epithelial cells There are many intriguing aspects of attachment and implantation but some are particularly revealing in the present discussion of the plasma membrane of uterine epithelial cells. It has been appreciated for many years that while blastocysts can attach to a multitude of sites outside the uterus they can only attach in the uterus during a very narrow range of times and under very specific hormonal conditions. FAWCETT et al. (1947), FAWCETT (1950), NICHOLAS (1942, 1950), and KIRBY (1963, 1965) all transferred blastocysts to a variety of extra-uterine sites including the anterior chamber of the eye, the abdominal cavity, kidney, intestine and testis. These workers found that while the transferred blastocysts displayed varying degrees of differentiation of the inner cell mass, the trophoblast continued to proliferate for some days. It is also well established that blastocysts can attach to a variety of artificial sites including culture dishes with or without special additives and without the need for hormonal conditioning of any kind (GLENISTER 1965; SHERMAN and SALOMON 1975; SHERMAN and WUDL 1976; GLASS et al. 1979; FARACH et al. 1987; GLASSER et al. 1991). Moreover, even bIastocysts such as in the pig which are not invasive inside the uterus, display invasive behaviour outside this organ (SAMUEL 1971; SAMUEL and PERRY 1972). Other tissues also behave in a highly atypical fashion inside the uterus. Microorganisms are unable to invade non-receptive uterine epithelial cells (WIRA and MERRITT 1977; PARR and PARR 1985) and highly invasive tumors that have little trouble invading numerous other sites, are only able to invade the uterus when the hormonal conditioning is like that prevailing at normal blastocyst attachment (SHORT and YOSHINAGA 1967; WILSON and POTTS 1970). Thus it is something of an enigma that the only site in which blastocysts cannot attach ubiquitously is the natural tissue of attachment - the uterus. This enigma becomes less so, perhaps, when we appreciate that while bIastocysts may be able to attach almost any-

2 . Ch. R. Murphy

where, they can only develop and be born as new individuals from the uterus. So the fastidiousness of the uterus can be seen in evolutionary terms at least, as a device to ensure that the energy the individual expends in pregnancy results in the best chance of a viable successor. However, while evolution may provide a rationale for what at first seems enigmatic, it .tells us little about the cell biology of uterine fastidiousness. COWELL (1969) provided what is probably the first indication that the barrier to ubiquitous intrauterine attachment and implantation resides in the epithelial cells lining the uterus with the finding that mouse blastocysts could implant at any stage provided that the epithelium was damaged. Conversely, with an intact epithelium, implantation only occurred when the uterus was appropriately conditioned with ovarian hormones (COWELL 1969). Similarly, the aforementioned tumors behaved in like fashion being able to invade either when the hormonal conditioning was appropriate or regardless of hormonal status if the epithelium was already breached (SHORT and Y OSHINAGA 1967). The barrier function of the uterus ., thus seems to reside in the epithelial cells for both blastocysts and other invasive tissues and the barrier can be breached when these cells become appropriately conditioned with progesterone and oestrogen. Roles other than that of barrier which are fulfilled by the uterine epithelium have been considered by MARTIN (1980). That ovarian hormonal conditioning is essential for implantation has been known for some time and in rodents at least, the requirements have been very precisely worked out (FINN and MARTIN 1972, 1974; FINN and PORTER 1975; FINN 1977; PSYCHOYOS 1961, 1963, 1973, 1986). If the barrier to attachment and implantation resides in the epithelial cells, then it is eminently reasonable to assume that the plasma membrane of these cells plays a crucial role since this membrane and its apical portion in particular, is the first site of contact between embryonic and maternal cells in a wide variety of species (ENDERS and SCHLAFKE 1969; SCHLAFKE and ENDERS 1975). This is remarkable because the mechanism of implantation differs widely among species. It varies from the different types of invasive implantation where the epithelium is breached leading to haemo-chorial and endothelio-chorial placentae to the non invasive types resulting in epithelio-chorial placentae. Regardless however, of the mechanism of implantation, the process always seems to start with attachment of the plasma membrane of the trophoblast to the apical plasma membrane of uterine epithelial cells (ENDERS 1972; BJORKMAN 1973; SCHLAFKE and ENDERS 1975). Thus attachment, unlike the process of implantation, appears to be a general phenomenon. DENKER(1990) has highlighted the unusual nature of attachment in that the normally non-adhesive apical plasmalemma of a polarised epithelial cell .becomes adhesive and in an insightful suggestion has proposed that uterine epithelial cells lose polarity as early pregnancy proceeds. On the plasma membrane, MURPHY (1992) has drawn attention to structural changes in different regions of the membrane. Given both the centrality and unique nature of attachment, it is likely that generalities in the response of the plasma membrane of uterine epithelial cells at this time exist across species boundaries.

Plasma membrane of uterine epithelial cells . 3

3 Membrane structure: Conventional morphological studies One of the most enduring models in molecular biology must surely be that of · GORTER and GRENDEL (1925) who proposed that the plasma membrane of erythrocytes consisted of two oriented layers of lipids. This model was extended by DANIELL! and · DAVSON (1935) to include protein molecules on the outer surfaces and contributed to the development of the model of ROBERTSON (1964) based on electron microscopical studies. Early electron microscopy of membranes has been summarized by ELBERS (1964) who emphasized differences in the trilaminar appearance obtained with that technique and while the genesis in thin section preparations of the trilaminar image is unclear (LUFTIG et al. 1977), it is seen in most if not all plasma membranes including that of uterine epithelial cells.

!

3.1 Effects of ovarian hormones Among the first to study the plasma membrane of uterine epithelial cells was NILSSON (1958 a, b, 1959 a, b) in a series of papers using routine embedding and thin sectioning techniques for electron microscopy. NILSSON (1958 a) compared cells from oestrous and control ovariectomised mice and noted that the microvilli on the plasma membrane of cells from oestrous animals were some three fold longer than those on controls and that the lateral plasma membrane was much more folded than in controls. Cells in oestrous animals were also taller than controls which suggested additional membrane was present as well. NILSSON (1958 b, 1959 a) injected ovariectomised mice with oestrogen and reported a massive increase in both length and frequency of apical microvilli thus confirming the effect of oestrogen on microvillar development and he further suggested that the depletion of lipid droplets seen at oestrus might have contributed to the increased membrane at that stage. Structurally, NILSSON (1958 b) observed that at four hours and twenty hours after exogenous oestrogen injection to ovariectomised mice, the width between the of the apical plasma membrane was changed. In controls, the distance between dense layers was 7 nm but following oestrogen treatment, it was narrowed to 4 nm. The lateral plasma membranes were also affected with the distance between adjacent cells narrowing from 26nm to 16nm. NILSSON (1958b) also noted that oestrogen treatment caused deposition of a dumenal substance> on the apical tips of the microvilli which was not seen in controls. NILSSON (1959 b) confirmed and quantified the changes in width between of the apical plasma membrane and demonstrated that oestrogen resulted in increases in both number and height of apical microvilli as well as a twenty fold increase in luminal surface membrane overall. These findings on the effect of oestrogen on the apical plasma membrane have largely been confirmed by more recent work, but NILSSON'S findings on the changes in width •between dense layers does not appear to have been taken up by later workers, possibly

4 . Ch. R. Murphy

because of the difficulties in extrapolating from the earlier model of membranes (ROBERTSON 1964; LUFTIG et al. 1977) to more current ideas on membrane structure. Even so, an investigation into such a fundamental structural change using presently available histochemical methods may repay future study. In the rat, the influence of hormones on plasma membrane outline has also been established. LJUNGKVIST (1971 b) injected ovariectomised rats with progesterone and found that microvilli increased in length and number and that numerous pinocytotic invaginations were frequent as compared with control ovariectomised animals (LJUNGKVIST 1971 a). In addition, large apical protrusions of the plasma membrane as well as smaller irregular protrusions and numerous large apical vesicles became apparent with progesterone. LJUNGKVIST (1971 b) also commented that this in the luminal epithelial cells was widespread and consistent as compared with the patchy and irregular response seen in glandular epithelial cells. LJUNGKVIST (1971 c) found that oestrogen dramatically increased the length of apical microvilli from 0.3 ~m in control ovariectomised rats to 1 ~m or longer in those treated with oestrogen and also that the microvilli became more numerous. Longitudinal striations - now established to be actin microfilaments (LUXFORD and MURPHY 1992 a) - also developed in the microvilli under oestrogen treatment as did an on their tips which was much more extensive than in untreated rats (LJUNGKVIST 1971 a). The lateral plasma membrane in these studies was seen to be affected by oestrogen but not progesterone with the former hormone producing a much more folded and interdigitated membrane (LJUNGKVIST 1971 c). Studies on changes in the structure of the plasma membrane of uterine epithelial cells in species other than rats and mice have also been carried out. While usually not as extensive, there is little doubt that ovarian hormones affect the plasma membrane in ways similar to those described above, in all species studied so far ranging from rabbits (HENZYL et al. 1968; DAVIES and HOFFMAN 1975; MOTTA and ANDREWS 1976) and guinea pigs, (BoURGOS and WISLOCKI 1958; HEDLUND and NILSSON 1971; ALKHALAF et al. 1987) to the roe deer (AITKEN 1974), pigs (KEYS and KING 1989, 1992; BLAIR et al. 1991), sheep (MURRAY 1992), cows (GUILLOMOT and GUAY 1982), seals (BOSHIER 1979), as well as non human primates (BRENNER et al. 1983) and the human being GOHANNISSON and NILSSON 1972; FERENCZY et al. 1972; FERENCZY 1977; MARTEL et al. 1987; DEHOU et al. 1987; MURPHY et al. 1987; WYNN 1989). While generalisations always have a degree of inaccuracy, it would seem that oestrogen which results in' numerous long, regular microvilli, has a more dramatic effect on membrane outline than progesterone, at least when acting alone. Several other hormones and hormone-anologues have also been found to alter cell . surface arrangement in uterine epithelial cells. Thus ANDERSON et al. (1975) found diethylstilbestrol and the inhibitor, nafoxidine, resulted in massive formation of microvilli within 72 hours of injection into rats and they speculated on increased absorption across the apical plasma membrane. NILSSON et al. (1980) reported that levonorgestrol

Plasma membrane of uterine epithelial cells . 5

retard~d development of both cilia and microvilli in women. NAYEL et al. (1987) and PAL and GUHA (1990) studied the effects of differertt oral contraceptives on the surface of rat and human epithelial cells and reported both increases and reductions in microvillar length and frequency depending on the contraceptive and its length of use. FEDELE et al. (1987) found deciliation of some ciliated cells and retardation of microvilli with a combined progestagen-oestrogen contraceptive (SBH 209 AE) in women and FERENCZY (1977) also noted the suppressive effects of the synthetic progestin, provera, on microvilli and cilia in women. Similarly, MARTEL et al. (1989) found that the progesterone antagonist, RU 486, retarded microvillar changes as well as development of the large apical protrusions. STEIN and KRAMER (1989) studied the effects of exogenous gonadotropic hormones (FSH, LH) superimposed on the normal hormonal levels and reported exceptionally long and numerous microvilli which lacked a glycocalyx on luminal epithelial cells. These unusual microvilli no doubt resulted from the elevated oestrogen levels present. The widely used superovulatory drug, clomiphene citrate, can also alter the plasma membrane of uterine epithelial cells. Although not all workers have documented clear effects with clomiphene (FEDELE et al. 1988), BIRKENFELD et al. (1988) reported increased secretory protrusions of the apical membrane with this drug. HOSIE and MURPHY (1992) recently found that clomiphene citrate given to ovariectomised rats could produce structures such as branched microvilli and bare areas of plasma membrane which are not commonly associated with either progesterone or oestrogen action. That various hormone derived drugs produce effects on the plasma membrane of uterine epithelial cells is not perhaps surprising. Understanding the origin and variety of these effects requires much more work however, but is of undoubted importance since many have useful fertility or contraceptive effects.

3.2 The attachment picture

During early pregnancy and the period of blastocyst attachment, the hormones progesterone and oestrogen act together to the uterus to receive the blastocyst. As already mentioned, in rats the requirements have been clearly worked out in an elegant series of experiments (PSYCHOYOS 1973, 1986). Compared with their individual actions, the hormones acting together have dramatically different effects on the appearance of the plasma membrane of uterine epithelial cells. In rats and mice the long, thin, regular microvilli characteristic of oestrus and the first two days of pregnancy are gradually replaced by shorter, less regular microvilli and other irregular protrusions. By day 4 of pregnancy the large protrusions are also in evidence amoung the shorter microvilli. The apical plasma membrane then gradually loses most microvilli until by days 5 to 6 of pregnancy - when the blastocyst can attach - it only bears different forms of highly irregular projections scarcely worthy of the naine microvilli. Concomitantly, and by day 6 of pregnancy, the uterine lumen closes down so that the irregular projec-

6 . Ch. R. Murphy

Plasma membrane of uterine epithelial cells ' 7

tions interdigitate with each other, or where a blastocyst is. present, with similar projections on the surface of the blastocyst. This phenomenon of uterine closure is incompletely understood: there can be little doubt that it is important in normal attachment but it probably does not precipitate it (MARTIN et al. 1970). Later on day 6, the apical plasma membranes of opposing uterine epithelial cells or of blastocyst and uterine epithelium come to lie almost parallel along the area of contact by which time most types of apical projection are lost. The blastocyst then penetrates the epithelium by 'displacing the epithelial cells «displacement> implantation in rats and mice - SCHLAFKE and ENDERS 1975) and over the following days of pregnancy, lost uterine epithelial cells are gradually replaced and the plasma membrane comes to bear more or less regular microvilli again. These changes in the apical plasma membrane during attachment have attracted the attention of many investigators who have divided them into different phases using different terminologies. The earlier stage has been termed the first phase of attachment (POTTS 1968), the first stage of closure (POLLARD and FINN 1972), the apposition stage (ENDERS and SCHLAFKE 1969), the preattachment phase (MAYER et al. 1967; NILSSON 1966 a), implantation stage 1 (ENDERS and SCHLAFKE 1967), or the early attachment stage (TACHI et al. 1970). The later stages where microvilli are remodelled and extensive close contact is established has been referred to as the second phase of attachment (POTTS 1968), the second stage of closure (POLLARD and FINN 1972), the late attachment stage (TACHI et al. 1970), the adhesion stage (ENDERS 1976), implantation stage 2 (ENDERs and SCHLAFKE 1967) or the attachment stage (NILSSON 1966 a). The nomenclature of NILSSON (1966 a), LJUNGKVIST and NILSSON (1971) and LJUNGKVIST (1972 a) referring to the entire process as the thus distinguishing it from actual penetration of the epithelium, and the plasma membrane changes as the probably represents the most accepted terminology and is used here. The attachment picture is essential if blastocysts are to begin implantation successfully. LJUNGKVIST and NILSSON (1971) and LJUNGKVIST (1972 a) performed a carefully timed series of experiments with both hormone injections to ovariectomised virgin rats and normally pregnant rats in which attachment had been delayed by ovariectomy on day 4 of pregnancy. They found that in either case, blastocysts were only able to begin Fig. 1, 2 and 3. Thin section electron micrographs of the apical plasma membrane of rat uterine luminal epithelial cells showing the appearance under different hormQnal conditions and the remarkable transformation undergone by the membrane during early pregnancy. Fig. 1, on day 1 of pregnancy (or at oestrus) under oestrogen influence, the membrane displays numerous long thin, regular microvilli with a glycocalyx and microfilaments are also evident in the core of the microvilli. Fig. 2, on day 3 of pregnancy, during early progesterone secretion, microvilli become shorter and somewhat less regular. Fig. 3, on day 6 of pregnancy under the influence of both progesterone and oestrogen the receptive appearance is evident (see text). The membrane is thrown into numerous irregular projections and microvilli are no longer evident. All x 50,000.

8 . Ch. R. Murphy

implantation when an attachment picture was present. Moreover, since this picture can be induced in virgin ovariectomised rats in the absence of blastocysts, it must be predominantly a maternal response not requiring a blastocyst for its occurrence (LJUNGKVIST 1972 a; MURPHY and ROGERS 1981). This view is further strengthened by the observation that the apical plasma membrane undergoes the same sequence of changes whether it is apposed to a blastocyst or to uterine epithelial cells from the other side of the uterus (POTTS 1968; TACHI et al. 1970). In all cases, the hormonal requirements for pregnancy worked out by PSYCHOYOS (1973) were the same ones necessary for the attachment picture. Adding to the significance of the attachment picture are the experiments of LJUNGKVIST (1972 b) who showed that amounts of oestrogen which were too high to allow implantation when acting on a progesterone primed uterus also prevented development of the attachment picture. The attachment picture of the apical plasma membrane of uterine epithelial cells thus represents a morphological sign of an uterus that is receptive for the onset of implantation. (Fig. 1 to 5 show the membrane changes in both transmission and scanning electron microscopy). In species other than rats and mice, morphological changes also take place in the apical plasma membrane during early pregnancy although they have not been as well studied in general as in the former two species. HEDLUND and NILSSON (1971) and HEDLUND et al. (1972) reported an attachment reaction in the hamster and guinea-pig and different types of membrane reorganization in the rabbit and mink. LARSEN (1962) showed micrographs of irregular microvilli and cytoplasmic processes in rabbit uterine epithelial cells at attachment and SEGALEN et al. (1982) also reported increasingly irregular microvilli in the rabbit as pregnancy progressed. ENDERS and SCHLAFKE (1969) found membrane reorganization as well as the formation of different types of membrane junctions in the ferret, armadillo and bat and ENDERS and SCHLAFKE (1972) showed that punctate desmosomes were formed at regions of close apposition between blastocyst and uterine epithelial cell membranes where microvilli had been lost in the ferret. During early pregnancy in marsupials, ENDERS and ENDERS (1969) found areas of flattened membrane with shared desmosomes between uterine epithelial and trophoblast cells in the opossum, Philander opossum: in the marsupial bandicoot, Isoodon macrourus, PADYKULA and TAYLOR (1982) showed irregular microvilli on the surface of maternal epithelial homokaryons and the marsupial mouse (Sminthopsis crassicaudata) loses regular microvilli on epithelial cells, the apex of which bulge into the uterine lumen, as the time of attachment approaches (ROBERTS, C. T., personal communication). In the roe deer, AITKEN et al. (1973) and AITKEN (1975) found increases in, and remodelling of, microvilli and the appearance of large apical membrane smooth-surfaced protrusions. In the horse, ALLEN et al. (1973) and ENDERS and Lru (1991) de-. Fig. 4 and 5. Scanning electron micrographs showing surface views of the transformation of the apical plasma membrane during early pregnancy. Regular microvilli on day 1 are seen in fig. 4 and the flattened, irregular cell surface of day 6 is seen in fig. 5. - Both x 15,900.

Plasma membrane of uterine epithelial cells . 9

10· Ch. R. Murphy

scribed loss of microvilli and remodelling of the apical cell surface. PARKENING (1976) showed that microvilli, while maintaining their integrity during early implantation in the golden hamster, were still remodelled so becoming more irregular and BLANKENSHIP et al. (1990) have recently reported that attachment in the chinese hamster is quite similar to that in the rat with extensive remodelling of microvilli into irregular protrusions of the apical plasma membrane. W ATHES and WOODING (1980) noted flattening of epithelial microvilli and GUILLOMOT and GUAY (1982) reported the presence of additionallarge irregular protrusions the surface of which were depleted of microvilli, on the apical membrane of cow epithelial cells during the early pregnancy period. In another ruminant, the sheep, GUILLOMOT et al. (1981) found loss of microvilli and formation of large rounded cytoplasmic protrusions of the apical plasma membrane as pregnancy progressed. WOODING and MORGAN (1989) have recently noted that uterine epithelial cell microvilli in ruminants are completely displaced during attachment. DANTZER (1985) working on crossbred pigs, reported loss of microvilli on the epithelial surface and conversion of that surface into a flattened, protruding profile in areas close to the trophoblast and KEYS and KING (1990) as well as BLAIR et al. (1991) also noted loss of microvilli and conversion of those remaining to a short profile as well as apical epithelial protrusion in pigs as pregnancy progressed towards the time of attachment. Further along the evolutionary pathway, in an non-human primate, the rhesus monkey, RENIUS et al. (1973) and ENDERS et al.(1983) found that blastocyst and epithelium come to share membrane junctions orginally formed between epithelial cells and that loss of microvilli as well as other types of flattened membrane areas characterised uterine epithelial cell surfaces at attachment. Seeking to emphasize the generality of attachment picture membrane changes, NILSSON (1962) compared mouse and man and upon finding irregular projections during the luteal phase concluded that perhaps similar morphological changes were a requirement for successful attachment in a variety of species. MARTEL et al. (1981; 1987; 1991), ROGERS and MURPHY (1989) and MURPHY et al. ,(1987; 1990) have also pointed out similarities between the apical plasmalemmas of, rodents and man both in their individual responses to hormones and during the period of attachment especially with regard to the increasing irregularity of the cell surface. Of particular interest in this context, are the large apical protrusions. They were described by LJUNGKVIST (1971 b) and BERGSTROM (1974) as , by PSYCHOYOS and MANDON (1971) as and by NILSSON (1974b) as . These structures which measure several micrometres across, are comparatively lacking in cellular organelles and have an undulating irregular surface the membrane of which is devoid of microvilli (Fig.6 and 7). They were originally thought to be involved in Fig. 6 and 7. The characteristic pinopods of the apical plasma membrane are seen in these micrographs. Fig. 6, scanning electron micrograph showing three pinopods at low power on day 4 of pregnancy. Notice the
Plasma membrane of uterine epithelial cells . 11

12 . Ch. R. Murphy

apocrine secretion (NILSSON 1972) and are known to be progesterone-dependant (LJUNGKVIST 1971 b; HAMMER et al. 1978; MURPHY and ROGERS 1981; MARTEL et al. 1989). ENDERS and NELSON (1973) and PARR and PARR (1974) have shown that these structures are in fact endocytotic in rodents at least, and they are now appropriately referred to as . PARR and her colleagues in a meticulous series of experiments (PARR and PARR 1977,1989; PARR et al. 1978; PARR 1980,1983) have also emphasised the combined exocytotic-endocytotic activity of the uterine epithelium in which the entire plasmalemma is engaged, as well as the involvement of the pinopods. The pinopods however, are particularly illustrative of the commonality across species of some morphological aspects of the plasma membrane because they or similar structures are found in a wide variety of species during the receptive period for blastocyst attachment (GIvEN and ENDERS 1989; PSYCHOYOS and MARTEL 1985; MARTEL et al. 1991; and previous references). There is thus good agreement in rodents and laboratory animals in particular but also in many other species, that dramatic changes in membrane structure as seen in conventional electron microscopy accompany attachment. But how is this reflected in more fundamental plasma membrane molecular organization?

4 Membrane structure: Freeze fracture studies The evolution of ideas on membrane structure have been greatly influenced by a single morphological method of tissue preparation and the unique view {t provides of biological membranes: the freeze-fracture technique. As summarized by SINGER (1971), the situation to that stage seemed largely ad-hoc with various workers adding features to the then prevailing models of membrane structure based on little evidence. SINGER (1971) however, pointed out that the situation was not really so amorphous and he proposed a model of membrane structure based on thermodynamic criteria which argued that, like previous models, the lipids were organised in an oriented bilayer, but that many membrane proteins - the integral membrane proteins - were intercalated into the bilayer and that some of them may pass through the bilayer from extracellular to cytoplasmic surfaces. This model was subsequently modified to take account of new degrees of fluidity of some components and appeared as the Fluid Mosaic Model of membrane structure (SINGER and NICOLSON 1972). With several subsequent alterations of degree rather than substance, this model is generally agreed to be the best interpretation of the organization of lipids, proteins and carbohydrates that is available, especially where plasma membranes are concerned (SINGER 1992). The introduction by STEERE (1957) of a method of frozen biological specimen preparation for electron microscopy, along with improvements in technology and interpretation (MOOR et al. 1961; BRANTON 1966; PINTO DA SILVA and BRANTON 1970; BRANTON 1971), allowed the freeze-fracture technique to establish that biological membranes

Plasma membrane of uterine epithelial cells . 13

could indeed split along the hydrophobic inner plane and that this region was occupied by at least some proteins represented by approximately 8 nm intramembranous particles (IMP). The technique thus provided a visual confirmation of some essential features of the Fluid Mosaic Model of SINGER and NICOLSON (1972) and contributed much to establishing the credibility of this model. Although limited to date by the inability to identify the chemical nature of many features seen so clearly in replicas (but see PINTO DA SILVA 1984; SEVERS 1991), the freeze-fracture method remains the only way to visualize the interior hydrophobic region of biological membranes.

Fig. 8. Freeze-fracture electron micrograph showing apical plasma membrane of rat uterine epithelial cell on day 1 of pregnancy. Round profiles of cross-fractured microvilli (m) are evident at this time and IMP are randomly dispersed and well separated from each other. - x 100,000.

14 . Ch. R. Murphy

4.1 Intramembranous particles Among the first freeze-fracture observations on the plasma membrane of uterine epithelial cells were those of MURPHY et al. (1979) who studied cells from rats which had been ovariectomised and then injected with exogenous ovarian hormones. They found that whereas oestrogen alone had no eff~ct on IMP density, progesterone caused elevated levels of IMP in the apical plasma membrane above control levels. The two hormones acting together however, as they do during early pregnancy, were seen to produce a density of IMP of 3800/[lm2 of apical plasma membrane (two and a half times that seen in controls) and also resulted in the appearance of linear and other arrays of IMP not seen in any other treatment group. MURPHY et al. (1982 a) extended these studies into the apical plasma membrane in rats during normal pregnancy and found an increase in IMP density from 2350/[lm2 on day 1 of pregnancy to 4000/[lm 2 on day 6 with an intermediate density between these times. The high density at day 6 around the time of attachment, was also accompanied by the appearance of rods and rows of IMP in various arrays with were not seen at other stages of pregnancy. These studies also confirmed, with the laterally extended views of membranes so obtained, the changes in membrane contours seen in the aforementioned thin section electron microscope work: they showed that whereas early in pregnancy, well-rounded cross-fractured profiles of microvilli were visible, later, on day 6, numerous profiles of cross-fractured irregular protrusions were apparent in the hydrophobic plane of the apical membrane (Fig. 8 to 11). Freeze-fracture studies have also been conducted on IMP in the apical plasma membrane of other species. In the amphibian salamander where uterine ultrastructure does not seem to be influenced by hormones and the uterine epithelium is not specialised for reproductive functions (GREVEN and ROBENEK 1980), little difference in IMP density between pregnant and non-pregnant animals could be found (GREVEN and ROBENEK 1982), but in other mammals, JOHNSON et al. (1988) in the pig and WINTERHAGER and DENKER (1990) in the rabbit, have also reported an increase in apical plasma membrane IMP during early pregnancy and the period of attachment. A particularly interesting finding in rats treated with the receptivity-inducing hormone regime (PSYCHOYOS 1973) as well as those at day 6 was the appearance of reflexive gap junctions (MURPHY et al. 1982a, b) (Fig. 10). These enigmatic structures are gap junctions formed between physically touching projections of different regions of the plasma membrane of the same cell - in this case the apical plasma membrane of uterine

Fig. 9. Apical plasma membrane in freeze fracture view on day 6 of pregnancy. The membrane lacks the characteristic profiles of cross-fractured microvilli and instead the irregular nature of the membrane is seen in the form of irregular areas of cross fractured cytoplasm (cyt). IMP are noticeably more dense than on day 1 (fig. 8) and some IMP form rods and rows. - x 100,000.

Plasma membrane of uterine epithelial cells . 15

16 . Ch. R. Murphy

Fig. 10 and 11. Freeze-fracture electron micrographs showing various arrangements of IMP present during the attachment period. In fig. 10 two gap junctions of the apical membrane are seen (arrows) and rods of IMP (arrows) are seen in fig. 11. lu = lumen. - Both x 130,000.

Plasma membrane of uterine epithelial cells . 17

epithelial cells - and have also been reported in ovarian decidual cells (HERR and HEIDGER 1978). It is far from clear why a cell which already has cytoplasmic continuity between projections from the same surface should also need gap junctions between them and while there is no evidence that these junctions are actually communicating, their appearance exclusively at the time of attachment suggests an important role in this phenomenon. Together with the large increase in IMP, it emphasizes the remarkable molecular changes occurring in the membrane. The chemical nature and function of IMP has long been an area of debate. There have been suggestions that some IMP are lipidic in nature although these particles are generally found in pure lipid mixtures and have a different morphology from the more usual IMP seen in biological membranes (VERKLEIJ 1984). Most experiments seem to confirm that IMP are protein, or at least protein complexed with lipid in biological systems (BRANTON 1971; SPETH et al. 1972; YAMANAKA and DEAMER 1976). In uterine epithelial cells, MURPHY and SWIFT (1983) were able to remove IMP in the apical plasma membrane with pronase treatment and concluded that over half of these IMP contained protein with the remainder probably too deeply embedded in the bilayer to be accessible to J?!0!..e~~..c!~ge~tion. The density of IMP have generally been thought to reflect metabolic activity of biological membranes and some may function as membrane transport proteins (BRANTON and DEAMER 1972; McNUTT 1977; BROWN et al. 1978; PERANZI et al. 1991). The higher number of IMP seen at attachment would make this membrane an membrane compared with IMP density seen in other membranes (BRANTON and DEAMER 1972; McNUTT 1977) which is consistent with suggestions that the apical plasma membrane plays an important role in regulation of the luminal fluid composition for attachment (NILSSON and LUNDKVIST 1979; MURPHY et al1982 a). IMP may have other functions: some IMP in nucleated cell membranes including the apical plasma membrane of uterine epithelial cells, have carbohydrates and receptors attached to them extracellularly (PINTO DA SILVA and MARTINEZ-POLOMO 1974; TORPIER and CAPRON 1980; MAURER and MUHLETHALER 1981; MURPHY and BRADBURY 1984). The increased density of IMP, especially the aggregates, seen at attachment in the apical plasma membrane could reflect a changed presentation or clustering of carbohydrates or other receptors projecting into the lumen and which serve as recognition sites for the blastocyst. In this context, the expression of novel carbohydrate epitopes at attachment (d. Section 5.2) is particularly interesting. 4.2 Lipids Freeze-fracture remains a predominantly morphological technique. However, in combination with various reagents which produce ultrastructurally identifiable lesions on replicas, freeze-fracture can be used to localize particular lipid molecules in membranes and some of these reagents have been applied to the plasma membrane of uterine epithelial cells.

18 . Ch. R. Murphy

Polymyxin B (PXB) is a cationic polypeptide antibiotic which binds to anionic phospholipids ip rpembranes and produces recognizable lesions in freeze-fracture preparations (LOUNATMA and NANNINGA 1976; HARTMANN et al. 1978; BEARER and FRIEND 1980; SIXL and GALLA 1981). Since it is a peptide, PXB cannot readily be applied to fixed cells and most of its use to date has been on isolated or naturally ocurring freefloating cells (SEVERS and ROBENEK 1983). Moreover the lesions produced by PXB are strikingly pleiomorphic which adds to the interpretational difficulties of this reagent. It has heven so produced some interesting results in uterine tissues. MURPHY and MARTIN (1984) injected PXB into the uterine lumen of anaesthetised rats in oestrus and early pregnancy. Circular lesions of 35 nm diameter were the most common lesion seen in this study and they could only be found on the apical plasma membrane with lateral plasma membranes always unlabelled. The apical plasma membranes of all cells seen by MURPHY and MARTIN (1984) displayed lesions although in different densities and it was concluded by these authors that anionic phospholipids varied in density both between cells and between different regions of the plasma membrane of the same cell. WINTERHAGER and DENKER (1990) used a similar in vivo injection technique in rabbits and also found a variety of PXB lesions in the plasma membrane of uterine epithelial cells but also that some cells were completely unlabelled. The heterogeneous response to PXB was diminished however in the plasma membrane of cells adjacent to implanting blastocysts although no overall change in density was conspicuous, and the reduced heterogeneity may in part reflect the fusion of membranes that occurs in rabbit epithelial cells at implantation. Notwithstanding the technical and interpretational difficulties, it does seem that anionic phospholipids can be localized in the plasma membrane of uterine epithelial cells with PXB and that these lipids undergo changes during early pregnancy and in different regions of the membrane. An important lipid in many mammalian cell membranes is cholesterol and this molecule can also be localized using freeze-fracture cytochemical methods. In this case ,the reagents which complex with cholesterol and form ultrastructurally identifiable lesions are the saponins, tomatin and digitonin and the polyene antibiotic filipin (ELIAS et al. 1978, 1979; SEVERS and ROBENEK 1983). Unlike polymyxin B these reagents are relatively easy to use and can be applied to fixed tissues: indeed it is generally preferable to use fixed tissues because of the powerful detergent properties of these reagents. The membrane deformations produced are relatively uniform in size and appearance and are easily recognized even in low densities in replicas of fractured membranes. In the case of tomatin and digitonin, lesions appear as tubular ridges or scallops of approximately 50 nm width and 250 nm length and filipin appears as circular protuberances (or depressions depending on the fracture face) of 25 nm diameter. The mechanism of action of these reagents is not well understood but lesion formation seems to involve intercalation of the cytochemical into the bilayer and its interaction with cholesterol as well as probable clustering and phase separation of complexes of the two molecules (MEYER 1981; SEVERS and ROBENEK 1983; SINOWATZ et al. 1989). Related to the mechanism of

Plasma membrane of uterine epithelial cells . 19

action, the reliability of the reagents in detecting cholesterol has been questioned and absence of lesion cannot be taken to indicate lack of cholesterol especially where clusters of IMP or other protein is present (SEVERS and ROBENEK 1983; BEHNKE et al. 1984). The proportion of cholesterol usually detected is also unclear because upon artificial rearrangement of lipids in the plasma membrane of uterine epithelial cells at least, many more filipin lesions appear than before perturbation (MURPHY and DWARTE 1987 b). Nevertheless, especially when used to confirm each other, digitonin and filipin are generally thought to give an indication of cholesterol in biological membranes (SEVERS and ROBENEK 1983; SIMONS and SEVERS 1986; SINOWATZ et al. 1989) and may be particularly useful in indicating changes in expression of this lipid. In uterine epithelial cells, MURPHY and MARTIN (1985) used digitonin to follow cholesterol changes in the plasma membrane of rats during early pregnancy. The lateral plasma membrane was seen to have a constant frequency and distribution of digitonincholesterol complexes indicating a comparatively unchanging content of the sterol during early pregnancy, at least as could be determined with this method. The frequency of the tubular-scalloped lesions in the apical plasma membrane however, increased from occupying 7% of membrane area on day 1 of pregnancy to 98% on day 6 of pregnancy the time of attachment, and new types of digitonin-cholesterol lesions became apparent indicating phase-separation of lipids (MEYER 1981). Extending these studies, MURPHY and DWARTE (1987 a) used filipin to see if this cytochemical could confirm the changes in cholesterol (Fig. 12 and 13). They found that lesions increased from 36% on day 1 to occupy 96% of apical membrane area on day 6 and that again, the lateral plasma membrane was apparently unchanged. WINTERHAGER and DENKER (1990) similarly found a large increase in cholesterol detected with filipin in the apical plasma membrane of uterine epithelial cells in the implantation chamber of rabbits as compared with nonimplantation regions and pseudopregnant animals. These authors did not study days 1 or 2 so changes over the period of early pregnancy could not be detected but they found that lateral plasma membranes also displayed a higher and more uniform density of filipin lesions in the implantation chamber and this may reflect the increased coupling of these epithelial cells to each other and the membrane fusion they undergo in the rabbit (WINTERHAGER et at. 1988;WINTERHAGER and DENKER 1990). It thus appears that in the two mammalian species studied, rats and rabbits, an increase in cholesterol in the apical plasma membrane is seen at the time of attachment. This is likely brought about in rats by the large apical vesicles which appear under progesterone stimulation (LJUNGKVIST 1971 b; MURPHY and ROGERS 1981), have been shown to fuse with the apical plasma membrane (PARR 1982) and which have cholesterol-rich membranes with both digitonin and filipin cytochemistry (MURPHY and MARTIN 1987; MURPHY and DWARTE 1987 a). The reproductive function of this increase in cholesterol is not clear. Cholesterol is generally thought to increase ordering of membrane lipids and so decrease membrane fluidity but depending on the phase transitions of lipids present, an increase in fluidity is also possible (YEAGLE 1985; LEWIS and

20 . Ch. R. Murphy

Plasma membrane of uterine epithelial cells . 21

McELHANEY 1992). Cholesterol can also alter the function of other membrane molecules including enzymes and carbohydrates (YEAGLE 1985) and is known to be required to maintain the clustering of some receptors (ROTHBERG et al.1990). In view of the clustered IMP reported above, perhaps its increase in the plasma membrane of uterine epithelial cells at the time of attachment contributes to the expression of an altered cell surface of clustered receptors and novel carbohydrates (see section 5.2) which the blastocyst can recognise.

4.3 The tight junction and the lateral plasma membrane Unlike the apical and basal portions of a plasma membrane which face different external and internal environments respectively, the lateral plasma membrane of epithelial cells face the relatively constant environment of another epithelial cell and are generally similar to each other. This is also true of the uterine epithelium notwithstanding the reproductive specialisation of these epithelial cells. Several workers have commented on the interdigitation of lateral plasma membranes of uterine epithelial cells and that this interdigitation apparently increases during early pregnancy or under the influence of ovarian hormones, particularly progesterone, in some species (LARSEN 1962; LJUNGKVIST 1971 b; BLANKENSHIP et al. 1990; KEYS and KING 1990). It may be a reflection of reorganization in the epithelial sheet rather than a phenomenon of particular reproductive significance. Gap junctions are also components of the lateral membrane but are not apparently common in uterine epithelial cells and the aforementioned freeze-fracture studies have not widely discussed them. MURPHY et al. (1981) reported small gap junctions among the tight junctional strands of ovariectomised rat uterine epithelial cells but noted their absence in more physiological states. MURPHY (unpublished observations) also found small and infrequent gap junctions between epithelial cells of pregnant rats but could find few differences during early pregnancy, a finding similarly reported for pregnant rabbits by WINTERHAGER and KUHNEL (1982). More recently however, WINTERHAGER et al. (1988) have found significant formation of gap junctions in rabbit epithelial cells immediately adjacent to the blastocyst and have concluded that this increased coupling of epithelial cells may be a precondition of the symplasm formation which occurs in that species.

Fig. 12 and 13. Freeze-fracture micrographs following treatment of tissue with filipin to reveal cholesterol. Donut-shaped lesions produced by filipin and indicating cholesterol are evident and are much more numerous in the membrane on day 6 (fig. 13) than on day 1 (fig. 12). Below the irregular apical membrane on day 6, numerous vesicles with membranes crowded with filipin lesions are also seen. - Both x 65,000.

22 . Ch. R. Murphy

By far the most interest in the lateral plasma membrane of uterine epithelial cells has been directed to the tight junctions. These are the apical-most in the junctional complex and were originally described in a thin-section study of several epithelia by FARQUHAR and PALADE (1963) who termed them zonulae occludentes (occluding junctions) and surmised their role in preventing or reducing paracellular movement of molecules. Because of its capacity to expose laterally extended regions of plasma membrane, freezefracture is uniquely suited for the study of tight junctions and has been extensively employed to examine the rows of closely-packed IMP which appear as strands in replicas and are the freeze-fracture appearance of these junctions (CLAUDE and GOODENOUGH 1973; STAEHELIN 1974; CEREIJIDO 1992). In uterine epithelial cells, tight junctions were reported on by MURPHY et al. (1981) who studied the capacity of ovarian hormones injected into ovariectomised rats to generate different patterns of tight-junctional organization. Oestrogen produced a tight junction consisting largely of strands running parallel to the apical surface and with few vertical connections between them but progesterone resulted in a much more complex pattern with many interconnections and a depth down the lateral membrane some two to three times greater than with oestrogen. Progesterone and oestrogen together produced effects not discernably different from progesterone alone. During early pregnancy, MURPHY et al. (1982 c) found on day 1 that junctions consisted of the parallel arrangement of strands and that by the time of blastocyst attachment in rats the junctions had become much deeper and had the interconnected appearance consisting of arrays of branching, anastomosing ridges (Fig. 14 and 15). An increase in depth and geometrical complexity of tight junctions has similarly been described during early pregnancy in rabbits by WINTERHAGER and KUHNEL (1982) who also noted formation of isolated strands of macular tight junctions below the main complex and JOHNSON et al. (1988) reported an increase in complexity but not depth around the time of implantation in pigs which they too concluded was progesterone-induced. In women, MURPHY et al. (1982 d; 1992) have found that tight junctions are more geometrically complex earlier than later in the menstrual cycle. It is thus remarkable that in all the species where sufficient evidence exists, it points to a progesterone-induced increase in tight junctional complexity. Tight junctions are well-known as regulators of paracellular flow with more strands and particularly a more complex geometry, generally indicating decreased paracellular permeability (CLAUDE and GOODENOUGH 1973; GONZALEZMARISCAL 1992). It would seem likely that the increasing complexity seen during early

Fig. 14 and 15. Freeze-fracture micrographs of tight junctions on the lateral plasma membrane of rat uterine epithelial cells. On day 1 of pregnancy (fig. 14) most junctional strands run parallel to the apical membrane. By day6 (fig. 15) a much more complex and interconnecting pattern of strands is evident and the junction extends much further down the lateral membrane. a = apical plasma membrane. - Both x 75,000.

Plasma membrane of uterine epithelial cells . 23

24 . Ch. R. Murphy

pregnancy is a reflection of the need to preserve the luminal contents specially developed by epithelial secretion for blastocyst development, from dilution or loss by unwanted flow into or out of the lumen (MURPHY et al. 1982 c). Freeze-fracture studies thus extend those based on thin-section electron microscopy and in particular establish an increase in apical plasma membrane IMP and lateral membrane tight junctions. An increase in apical cholesterol is also seen in the species studied. The commonality across species of these changes is especially impressive given the different types of implantation they display with pigs even having a non-invasive implantation. The similarity in plasma membrane response would seem to be further evidence of the generality of attachment membrane changes.

5 Histochemistry of the glycocalyx and related cell surface molecules Carbohydrates of the glycocalyx and related molecules on the cell surface are likely to be involved in the very first contact between blastocyst and uterine epithelium. Consequently these_J:D21~cules have/received considerable attention although no comprehensive appreciation ort:h~i~;ole in attachment yet exists. Even so, numerous carbohydrates, enzymes, receptors and other determinants have been identified on the surface of uterine epithelial cells employing a variety of histochemical and cytochemical approaches to specifically localize them either as components of, or associated with, the plasma membrane. It is to these molecules that we now turn attention.

5.1 Enzymes and determinants

The likely role of various enzymes of the plasma membrane of uterine epithelial cells in modifying the function of that membrane has attracted the attention of numerous workers. CHRISTIE (1966, 1967) studied a variety of enzymes in several different species (rat, rabbit, guinea pig, cat) using the catalytic activity of the enzymes in light microscopic approaches to histochemical identification. Precise localization is naturally difficult with these techniques but several dehyrogenases and alkaline phosphatase showed apical distribution and in a related study CHRISTIE (1968) suggested that the enzymes may be involved in lipid metabolism and membrane transport phenomena. BOSHIER (1969) also employed light microscopical enzyme histochemical methods to localize alkaline phosphatase activity in the sheep, but in this study localization to the apical plasma membrane of uterine cells was achieved. It was also found that enzyme activity increased during early pregnancy and the period of blastocyst attachment and BOSHIER (1969) suggested that the increased activity indicated increases in carbohydrate metabolism in the apical plasma membrane. HALL (1971) using both lead and calcium visualization procedures was also able to specifically localize enzyme activity to the plasma

Plasma membrane of uterine epithelial cells . 25

membrane. In this case the enzyme, 5-nucleotidase, was found on the free (apical) surface where it was thought likely to be involved in nucleotide absorption. LIf:SER and WILLE (1975) used both light and electron microscopic techniques to study alkaline phosphatase activity in cow uterine epithelial cells. These authors achieved an unequivocal localization of delicate lead phosphate deposits, indicating enzyme ativity, all along the apical microvillar membrane and found moreover that the activity of this enzyme was greatest at the time of blastocyst attachment. COLVILLE (1968) and SAWARAGI and WYNN (1969) using electron microscope histochemistry have also reported changes in alkaline phosphatase as well as adenosine triphosphatase on the apical microvilli during the menstrual cycle in women. Using ovariectomy-induced delay of implantation in the mouse, NILSSON and LUNDKVIST (1979) examined alkaline phosphatase and glucose-6-phosphatase activities and found both to be negative with electron microscopical cytochemistry. Following oestrogen injection to initiate blastocyst attachment however, alkaline phosphatase activity over the apical microvilli and pinopods became strongly positive and increased molecular transport across the apical plasma membrane was judged to be occurring at this time in response to oestrogen action. Several proteinases have also been studied in uterine epithelial cells of the cat and rabbit. DENKER (1977) and DENKER et al. (1978) extensively examined a range of glycosidases and proteolytic enzymes and found activities of an amino acid arylamidase and endopeptidase at the surface of the uterine epithelial cells. The proteinases appeared to have optimum activities in the slightly acid pH range and DENKER et al. (1978) were unable to ascribe a definite physiological function to these uterine surface proteases although they could have a role, along with similar molecules on the trophoblast, in dissolution of trophoblast coverings. In further extensive studies in the rabbit, CLASSEN-LINKE et al. (1987) and CLASSEN-LINKE and DENKER (1990) used both light and electron microscopical methods to study a range of enzymes on the surface of uterine epithelial cells. Alkaline phosphatase, aminopeptidase M, gamma glutamyl transferase and dipeptidyl peptidase IV were all found to have maximal activity on the apical plasma membrane at day 5 of pregnancy and this activity declined by day 8 - the day of implantation in the rabbit. CLASSEN-LINKE and DENKER (1990) point out that while there is no reason to connect the four enzymes directly with attachment, their concomitant decline at the same time is another indicator of the profound changes this plasma membrane undergoes at attachment. These authors further suggest that the decline in enzymes may also indicate a loss of polarity by the apical plasma membrane because two other proteins they studied, desmoplakin I and II, which are normally located in the subapical lateral plasma membrane domain, became more evenly distributed around the cell surface as the apical enzyme activity declined. Other enzymes have been found on, or associated with, the surface of uterine epithelial cells. MANCARELLA et al. (1981) studied a leucine ~-naphthylamidase in pigs. It had a molecular weight of 480 kDa by electrophoresis and several lines of evidence, including histochemical, led these workers to conclude that the enzyme was a component of the

26 . Ch. R. Murphy

microvillar membrane of uterine epithelial cells and that in addition, it underwent a protease-induced cleavage resulting in release of at least some of the enzyme into the uterine luminal fluid. ISHIKAWA et al. (1984) localized an NAD(P)H oxidase to the microvillar membrane of rat cells using an electron microscopic cerium method. This enzyme which generates hydrogen peroxide might also be involved in lysis of blastocyst coverings. More recently PARR et al. (1988) and MARSHBURN et al. (1990) showed micrographs suggesting that activity of different prostaglandin synthases might be located, among other cellular regions, in the apical surface of mouse uterine epithelial cells. Several other molecules have also been localized to the surface of uterine epithelial cells using histochemical methods. LAMPELO et al. (1986) produced a monoclonal antibody which localized an antigen on rabbit uterine epithelial cells. Although the antibody cross-reacted with antigens in several tissues at high concentrations, it specifically localized the antigen to the apical plasma membrane of pregnant animals at lower concentrations. THOR et al. (1987) and OSTEEN et al. (1990) have also localized another so-far unidentified antigen to the surface of human uterine epithelial cells during postovulatory periods with a monoclonal antibody method. Among more clearly identified antigens, are histocompatibility complex antigens (I a) localized by TABIBZADEH et al. (1987) with peroxidase-labelled monoclonal antibodies in the electron microscope to the apical microvilli of human uterine epithelial cells and thought to be involved in antigen presentation by these authors. Perhaps these molecules could present antigen to the blastocyst? Employing an affinity purified antibody, SVALANDER et al. (1990) studied the expression of cell-cell adhesion molecule 105 in uterine epithelial cells during the oestrous cycle, delayed implantation and normal pregnancy. The cell-CAM 105 molecule is a membrane integrated cell surface glycoprotein and was localized to the apical plasma membrane at several different stages studied. It is down-regulated by oestrogen at the time of implantation and so is unlikely to directly participate in adhesion, but SVALANDER et al. (1990) speculate that the disappearance of cell-CAM 105 at the time of attachment may render the apical plasma membrane more easily penetrated by the blastocyst. Interestingly, and consistent with the suggestion that cell-CAM 105 is lost from the apical plasma membrane in receptive uterine epithelial cells, cell-CAM 105 has also been found on the surface of cultured rat uterine epithelial cells at states concluded to be compatible with oestrogen-dominance and so non-receptive for attachment (JULIAN et al. 1992). Several fu~osylated carbohydrate deter~i~ants also containing N-acetyl-glucosamine were followed with a panel of fluoresceinated monoclonal antibodies by KIMBER et al. (1988) and KIMBER and LINDENBERG (1990) and were localized to the surface of uterine epithelial cells. One determinant, lacto-N-fucopentaose I, was clearly demonstrated on the apical plasma membrane of mouse uterine epithelial cells and is induced by oestrogen acting on a progesterone primed uterus. Since LINDENBERG et al. (1988) had earlier shown that blocking the determinant prevents attachment of blastocysts to uterine monolayers, it was concluded that this apical

Plasma membrane of uterine epithelial cells . 27

plasma membrane carbohydrate determinant was clearly involved in interactions between blastocyst and uterine surface (see also section 7.2). Several ABO(H) blood group related carbohydrate antigens have also recently been localized to the surface of human uterine epithelial cells with fluoresceinated monoclonal antibodies by RAVN et al. (1992) who noted changes during the menstrual cycle. At the opposite surface of uterine epithelial cells, NISHIDA et al. (1991) localized laminin, collagen type IV and a fibronectin receptor predominantly to the basal surface of cells in rats. No prominent changes in distribution of the first two of these molecules was noted during the cycle, but the antibody for fibronectin receptor which showed strongest fluorescence at the basal cell surface and much weaker labelling at lateral surfaces during pro-oestrous, declined at other stages of the cycle. Also on the basal and lateral (but not apical) plasma membrane, are major histocompatibility complex class I (MHC I) molecules, which were localized by VAN EIJKEREN (1991) with an immunogold procedure and thought by these workers to contribute to the maintenance of polarization of the human uterine epithelial cells they studied. Another group of mostly basolateral molecules - heparan sulphate proteoglycans (HSPG) - were followed recently by POTTER and MORRIS (1992) in mice with a monoclonal antibody against syndecan which is a cell surface HSPG. Peroxidase staining indicating localization of this molecule changed from basolateral to predominantly basal as the oestrous cycle progressed from metoestrus to oestrus and as early pregnancy progressed to the attachment period it became difficult to detect any basal or lateral staining. Very little apical staining could be detected at any stage suggesting that this HSPG may not have a direct role in attachment of the blastocyst (see also section 7.1) although POTTER and MORRIS (1992) point out that the changes they detected in syndecan on the surface of uterine epithelial cells emphasize the dramatic turnover in that cell surface during early pregnancy.

5.2 The glycocalyx

Of all the surface molecules studied histochemically, carbohydrates of the glycocalyx have received the most attention. BENNETT (1963) coined the term glycocalyx (meaning on microvilli in mice under the influence of oestrogen. These early micrographs however, leave no doubt that a glycocalyx was present and highlight the role of hormones in contributing to this structure. LJUNGKVIST (1971 b, c) also noted changes caused in a similar coat material on the tips of microvilli in rats and his micrographs

28 . Ch. R. Murphy

show that both progesterone and oestrogen produced different effects on this structure with oestrogen again more dominant. In the ferret during normal pregnancy, ENDERS and SCHLAFKE (1972) also employing routine staining of thin sections for electron microscopy, found that the glycocalyx of uterine epithelial cells was selectively removed at sites of adhesion with the blastocyst. These early observations and developing awareness of the possible role of the glycocalyx in cell adhesion in general, as well as suggestions on uterine adhesiveness (NILSSON 1966 b), then led to numerous studies on, in particular, changes in cell surface charge produced by alterations in surface carbohydrates. Thus, ENDERS and SCHLAFKE (1974) used ruthenium red and colloidal thorium dioxide to study surface carbohydrates in rats and while they suggested a role for these molecules in attachment of blastocysts, they could not demonstrate any specific change in thickness or surface charge. The histochemical methods used however, unequivocally demonstrated a thick glycocalyx of complex chemical composition which was much thicker on the uterine epithelial cell surface than on the blastocyst. Also using cationic ferritin and colloidal iron hydroxide, ENDERS et al. (1980) were again unable to demonstrate either a generalised change in surface carbohydrates or a localised change at the attachment site between blastocyst and epithelium in rats. GUILLOMOT et al. (1982) similarly could not find alterations to the glycocalyx of uterine epithelial cells in sheep using several cationic markers and WORDINGER and AMSLER (1980) could likewise see no histochemical changes in the uterine glycocalyx in pigs although NILSSON (1974 a) had in the meantime been able to show an increase in some ruthenium red stainable surface components in the rat. There appears nonetheless to be an emerging view that in some species at least, the epithelial glycocalyx is indeed reduced in either or both of thickness and negative charge as the attachment period approaches. HEWITT et al. (1979) employed cationic ferritin with electron microscopy to show that anionic sites progressively disappeared from uterine epithelial cell surfaces from day 1 to day 6 of pregnancy in rats. These authors concluded that the generalised decrease they observed must be under maternal hormonal control and not dependant on the presence of a blastocyst because the changes also occurred in pseudopregnant animals where there were no blastocysts. MURPHY and ROGERS (1981) came to a similar conclusion based on studies on ovariectomised rats given hormonal replacement treatment, where a replacement regime compatible with attachment also showed a reduction in the extent of the glycocalyx and in negatively charged sites on uterine epithelial cells when compared with regimes that were not compatible with attachment. In addition to these reports, others have appeared which indicate one or more types of alterations to charged cell surface molecules. SALAZARFig. 16 and 17. Thin section micrographs of rat uterine epithelial cells treated with cationic ferritin to label the negatively charged surface coat. Labelling is seen as electron dense and is much reduced on day 6 (fig. 17) as compared with the heavy labelling on day 1 (fig. 16). Sections are not counter-stained. - Both x 75,000.

Plasma membrane of uterine epithelial cells

29

J

\'

30 . Ch. R. Murphy

RUBIO et al. (1980) working on rats, CHAVEZ and ANDERSON (1985) on mice, ANDERSON and HOFFMAN (1984)onrabbits, DANTZER(1985) and BLAIR et al. (1991) working with crossbred pigs, JANSEN et al. (1985) and NAVOT et al. (1989) working with timed human endometrial biopsies, as well as ANDERSON et al. (1990) who studied changes in cationic ferritin and alcian blue binding in monkeys, have all shown decreases in thickness as well as changes in the staining of one or more reagents that indicate reduced surface negativity of uterine epithelial cells. The reduction in surface charg~ is generally held to result from loss of sialic acids and this is strongly supported by the work of CHAVEZ (1990)with Limulus polyphemus lectin the binding of which indicates sialic acid and which was found to decrease on the surface of mouse uterine epithelial cells at attachment. MORRIS and POTTER (1984) used a different approach with positively charged beads to show that both blastocyst and uterine epithelial cell surfaces were negatively charged although the latter surface was found to have four times the negative charge which nonetheless, was reduced at the time of attachment. HOSIE and MURPHY (1989) also employed ruthenium red and cationic ferritin stains for negatively charged groups and similarly found a generalised decrease in these molecules from day 1 to day 6 of pregnancy in the rat (Fig. 16 and 17). These authors however, further reported that the reduced cell surface charge on the day 6 apical plasma membrane could be restored at that time by treating tissues with proteolytic enzymes and they suggested that the reduction in surface charge could result from masking of charged surface carbohydrates by re-arrangements in their sequence as well as reduction in their number. Clearly though, this finding is also consistent with a turnover of surface carbohydrates and the likely addition of new molecules which could mask preexisting carbohydrates as suggested by these authors. Thus there appears to be a reduction in both negativity and amount of glycocalyx in several species at the time of attachment. That the amount is reduced can be seen directly. JONES and MURPHY (unpublished) used high resolution, field emission gun scanning electron microscopy (FEGSEM) to study the glycocalyx of uterine epithelial cells in rats. This technique allows direct visualization of the native glycocalyx without the need for histochemical staining and clearly revealed fine radiating filamentous attachments on the regular microvilli of the day 1 cell surface. It also showed that by day 6 of pregnancy, the time of implantation in rats, these filaments were completely absent from the smooth membrane of the irregular surface projections then present (Fig. 18, 19). The reduction in glycocalyx which apparently accompanies the reduced negative surface charge on uterine epithelial cells is of particular interest because it is also known Fig. 18 and 19. High resolution (field emission gun) scanning electron micrographs showing the glycocalyx of rat uterine epithelial cells. On day 1 (fig. 18) fine filamentous elements of glycocalyx can be seen radiating from the microvilli, but these are absent from the protrusions of irregular membrane on day 6 (fig. 19). - Both x 70,000.

Plasma membrane of uterine epithelial cells' 31

32 . Ch. R. Murphy

that there are reductions in both amount and surface negativity of blastocysts as well at the time of attachment to uterine epithelium; a finding that has been confirmed in a few species with both histochemical staining and more direct physical measurements of surface charge (CLEMETSON et al. 1970; NILSSON et al. 1973; JENKINSON and SEARLE 1977; NILSSON and HJERTEN 1982). It seems unlikely however that mere reduction in surface charge is sufficient to bring about attachment by itself (although it may be necessary) because even with the reductions described above, the apposing cell surfaces of blastocyst and uterine epithelium are still quite strongly negatively charged. Moreover, oestrogen can reduce surface charge by itself and attachment will not occur with this treatment (MURPHY and ROGERS 1981). Rather, if the glycocalyx is involved other mechanisms must exist and several predominantly non-charged carbohydrates are known to change on the surface of uterine epithelial cells during early pregnancy. Surface carbohydrates can also be studied by means of of lectins. These large molecules are carbohydrate binding proteins, more frequently of plant than animal origin, which bind non-covalently to a variety of carbohydrates depending on their specificity and which can be conveniently labelled with different markers thus enabling histochemicallocalization of the target sugars. Lectins per se have been the subject of n~merous excellent reviews and so do not need to be revisited here (LIS and SHARON 1973; NICOLSON 1974; BROWN and HUNT 1978; BARONDES 1984; GABIUSl991). Adding to the usefulness and interest of lectins in the present context is the finding that when injected into the uterine lumen during early pregnancy, they can prevent implantation (HICKS and GUZMAN-GONZALEZ 1979; Wu and Gu 1981), presumably by binding to carbohydrates of either or both of blastocyst and uterine epithelial cell surfaces and preventing the assumed carbohydrate interactions occurring there. Batteries of lectins coupled with fluorescein or electron dense markers have been used by several workers to study generalised carbohydrate changes throughout uterine und related tissues and alterations to cell surface carbohydrates in particular. Thus, LEE et al. (1983) used twenty different leetins to describe patterns of change in reproductive tissues with uterine epithelium displaying the most pronounced modifications. At the light microscopic level these authors found terminal galactose sugars were most prominent in pregnant mice uteri. LEE and DAMJANOV (1985) used similar methods with fluorescein labelling to study human endometrium with a battery of lectins only one of which - Bauhinia purpurea (BPA) that localizes N-acetyl-galactosamine - reacted exclusively with uterine epithelial cells. Similar studies have also examined endometrial epithelia of mice and humans and have detected numerous reproductive cycle-related changes in carbohydrates as well as those associated with neoplastic transformation of uterine epithelial cells (Wu et al. 1983; BYCHKOV and TOTO 1986; KUPRYJANCZYK 1989; AOKI et al. 1989, 1990) and changes induced by antibodies (WHYTE et al. 1987). Carbohydrates specifically associated with the plasma membrane of the epithelial cells have also been localized, and while the usual complement appears to be present (including a-D-galactose, a-L-fucose and other fucosylated sugars, B-D-galactose,

Plasma membrane of uterine epithelial cells . 33

sialic acids, N-acetyl-glucosamine, N-acetyl-galactosamine, a-D-mannose, a-D-glucose, ~- D-glucose, and numerous undetermined oligosaccharides) only certain sugars have so far been shown to alter in distribution or amount during the attachment period. BUKERS et al. (1990) used seven fluoresceinated leetins to study carbohydrates in the plasma membrane and other regions of uterine epithelial cells in rabbits. Increases in Nacetyl-glucosamine in the apical plasma membrane region was found in pregnant animals whereas staining for fucose sugars was only weak at all stages examined. Lectins which bound D-galactose and N -acetyl-galactosamine gave the strongest reactions at most stages of early pregnancy however, and differences in binding between the plasma membranes of mesometrial and anti-mesometrial rabbit epithelial cells were also reported. THIE et al. (1986) with an autoradiographic technique also showed that Nacetyl-glucosamine was more strongly localized by wheat germ agglutinin (WGA) in the rabbit microvillous border towards the end of the preimplantation period. WHYTE and ROBSON (1984) and WHYTE and ALLEN (1985) used fluoresceinated leetins to show that L-fucose and N-acetylglucosamine were associated with the onset of implantation receptivity by uterine epithelial cells in pigs, sheep and especially horses. Fucose-containing molecules have similarly been implicated in attachment by KIMBER et al. (1988) and KIMBER and LINDENBERG (1990) using fluoresceinated monoclonal antibodies. Studies with electron dense markers at the electron microscopic level which provides more precise localizations, have also demonstrated the existence of, and changes in, carbohydrates in the uterine glycocalyx. Thus, increases in surface fucose of rat uterine epithelial cells in a ferritin-lectin study was described by MURPHY and TURNER (1991). N-acetyl-glucosamine sugars localized with wheat germ agglutinin (WGA) labelled with ferritin or colloidal gold have also been shown to be present on cultured uterine epithelial cells (VALDIZAN et al. 1992) and to increase on the surface of rabbit (ANDERSON et al. 1986) and rat uteri (MURPHY and TURNER 1991) at the time of attachment. Nacetyl-galactosamine studied with the lectin, soy bean agglutinin (SBA) likewise increases on the epithelial plasma membrane by the time of attachment in rats (MURPHY and TURNER 1991). Another sugar strongly implicated in attachment membrane changes is D-galactose which has been studied in some detail using the lectin Ricinus communis I (RCA-I). This sugar increases on mouse uterine epithelial cell surfaces (CHAVEZ and ANDERSON 1985; CHAVEZ 1986, 1990) as well as on the surface of uterine epithelial cells in rabbits (ANDERSON et al. 1986; ANDERSON 1989) and monkeys (ANDERSON et al. 1990) and is also involved in surface interactions in other systems (ANDERSON 1989). MURPHY and TURNER (1991) have recently shown, using an avidin-ferritin-Iectin technique, that at least four different carbohydrate molecules increase by day 6 of pregnancy on rat uterine epithelial cells (see above). One of the lectins used is of particular interest. This lectin, Phytolacca americana, does not bind to monosaccharides like many others and cannot be inhibited by them. Instead, it reveals oligosaccharides of N-acetyl-glucosamine and similar sugars and these molecules were found by MURPHY and TURNER (1991) to be virtually absent on day 1 of pregnancy but to increase many-

34 . Ch. R. Murphy

fold by day 6 - the time of implantation in rats (Fig. 20 and 21). Moreover, STEIN and MURPHY (unpublished) have also found with Jihe same lectin that these or similar oligosaccharides significantly increase on the surface of rat blastocysts between the morning and evening of day 5 of pregnancy. Thus the sugars, or variants of them, most commonly implicated in attachmentrelated phenomena in the plasma membrane of uterine epithelial cells appear to be Lfucose, D-galactose and N-acetyl-glucosamine. 5.3 The glycocalyx in attachment Most postulated recognition systems between carbohydrates at attachment have involved glycosyltransferase or similar molecules acting as bridges between plasma membranes of blastocyst and uterine epthelial cells (ENDERS and SCHLAFKE 1974; MURPHY and ROGERS 1981; ANDERSON et al. 1990; LINDENBERG et al. 1990) along lines originally suggested by ROSEMAN (1970). Perhaps however, other models should be considered in addition. Increases in carbohydrates and our finding that common oligoccharides increase on both uterine epithelial and blastocyst cell surfaces around implantation (above) when taken with the observation that in other systems carbohydrates can interact specifically without the need of protein intermediaries (EGGENS et al. 1989; GABIUS, 1991) suggests that homophilic interactions between carbohydrates could contribute to attachment of blastocysts to the plasma membrane of uterine epithelial cells as well. The reductions of some carbohydrates and the masking or reductions in charged carbohydrates already described, could facilitate this type of interaction by reducing mass and surface charge in the glycocalyx sufficently to permit the more specific homophilic carbohydrate binding to occur.

6 The basal plasma membrane and basal lamina The basal plasma membrane and its associated basal lamina have in general received less attention than other regions of the plasma membrane of uterine epithelial cells. This is probably because this segment is not directly concerned with the very first interaction with the blastocyst and nor does it apparently display the spectacular changes in outline seen in the more readily observable apical plasma membrane. Nevertheless, the basal lamina remains for some time even after the epithelial cells are removed in those species Fig.20 and 21. Thin section electron micrographs of rat uterine epithelial cells with the apical plasma membrane stained for carbohydrates using the biotinylated lectin-avidin-ferritin method. The ferritin label is seen as electron-dense on the cell surface. The lectin staining shown here is Phytolacca americana which displays a large increase from day 1 of pregnancy (fig. 20) to day 6 of pregnancy (fig. 21). Sections are not counter-stained. - Both x 75,000.

Plasma membrane of uterine epithelial cells . 35

20

36 . Ch. R. Murphy

with invasive implantation and even in species without this form of implantation, changes have been described in this surface of uterine epithelial cells which faces the internal environment of the body. The terminology of basal lamina and the related basement membrane can be confusing with some workers using the two terms almost synonomously. Here, and following FAWCETT (1986), the term will be used to denote the thin continuous layer seen underlying all epithelia in electron microscopy and consisting of an electron lucent layer or plus the electron dense layer of predominantly collagen type IV called the . The term denotes a much thicker and less consistent layer and originated in descriptions based on light microscopy: it generally includes the basal lamina as well as components of the extracellular matrix which are not arranged in a continuous layer. The major interest in the basal portion of uterine epithelial cells has been in how the blastocyst penetrates it in those species with invasive implantation. SCHLAFKE and ENDERS (1975) found that in laboratory animals, the trophoblast paused at the residual lamina densa which remained intact even though the overlying epithelial cells which largely produced it had been removed. It was originally thought that this residual lamina densa was primarily penetrated by either the trophoblast or uterine epithelial cells and more likely the former because of the well known phagocytic activity of the trophoblast during implantation and its degradative capacity when proliferating in ectopic sites (SCHLAFKE and ENDERS 1975; FAWCETT 1950; KIRBY 1963; SALOMON and SHERMAN 1975; GLASS et al. 1983; LOPATA et al. 1992). Despite these speculations, it has been shown in rats and mice, that whenever cell processes are found penetrating through the residual lamina densa they are processes of the underlying decidual cells and not those of the trophoblast and it now seems that the decidual cells are responsible for the initial physical penetration at least (SCHLAFKE et al. 1985; BLANKENSHIP and GIVEN 1992). As pointed out by SCHLAFKE et al. (1985) however, these observations do not eliminate a role for uterine epithelial cells in lamina densa breakdown because the relationship between lamina densa and epithelial cells must be modified since epithelial cells are lost some distance from the trophoblast (WELSH and ENDERS 1983). Moreover, in some areas the lamina densa is absent between degenerating uterine epithelial cells and decidual cells and no penetrating decidual cell processes can be seen in these regions (SCHLAFKE et al. 1985). Uterine epithelial cells are also known to be able to degrade artificial substrates in vitro (WELSH and ENDERS 1989) and human (ROBERTS et al. 1988) and horse (ENDERS and Lw 1991) uterine epithelial cells can extend processes of their basal plasma membrane through the lamina densa towards the underlying stromal cells as has been similarly found both in vitro and in vivo in the rabbit where the epithelial cells form a symplasm (HOHN and DENKER 1990; MARX et al. 1990). It may also be that since uterine epithelial cells largely produce and maintain the lamina densa, they secrete a biochemically different structure by the time of implantation which facilitates penetration of the blastocyst even though it is morphologically similar to the structure seen earlier in pregnancy (although see SHION and MURPHY (unpublished) below).

Plasma membrane of uterine epithelial cells . 37

Of particular interest in the present context, changes in the appearance of the basal plasma membrane and its attendant lamina densa have been reported during early pregnancy. LARSEN (1962) described an increasingly complex folding of the basal plasma membrane and lamina densa in rabbits as pregnancy progressed that was not seen in cells from oestrous animals which had a relatively smooth and softly undulating basal surface. GUILLOMOT et al. (1981) also noted a highly folded basal plasma membrane in sheep during pregnancy and the irregular folds were thought to penetrate the lamina densa and establish contact with the underlying decidual cells. These authors did not study the basal surface in non pregnant sheep so progressive changes could not be discerned. In pigs, KEYS and KING (1989, 1990, 1992) described an increasing irregularity in the basal plasma membrane and lamina densa in pregnant animals as compared with those in oestrus and also found that pregnant animals as well as those treated with oestrogen displayed long whorls of lamina densa which were separated from the membrane. They attributed these changes, as well as changes in the smoothness of the lateral plasma membrane which they also observed to redistribution of cells associated with the reorganization of the epithelial sheet. MARX et al. (1990) showed that at the time of symplasm formation in uterine epithelial cells in pregnant rabbits, the lamina lucida becomes thin or missing and the lamina densa becomes thicker although more diffuse as well as being absent in some areas. It was also reported by MARX et al. (1990) that numerous membrane-bound branching basal folds of uterine epithelial cells penetrated the lamina densa at this time in rabbits. In mice during early pregnancy, BLANKENSHIP and GIVEN (1992) found that the usually linear appearance of the basal surface of uterine epithelial cells was changed as pregnancy progressed and became more tortuous in outline. These workers also noted that the lamina densa frequently became diffuse in outline and variable in thickness at this time. In an effort to appreciate the changing nature of the basal plasma membrane and basal lamina over the full period of early pregnancy up to the time of attachment in the rat, SHION and MURPHY (unpublished) recently compared cells from day 1 and day 6 pregnant animals as well as those treated with various regimes of exogenous ovarian hormones. The basal plasma membrane on day 1 was seen to be relatively smooth in outline with the attendant lamina densa averaging 13 nm in thickness and with frequent thickenings between the two which resembled immature hemidesmosomes. By day 6 of pregnancy however, this study found that the immature hemidesmosomes were no longer visible but that the basal plasma membrane and lamina densa had become highly folded. The lamina densa was found on day 6 to have significantly increased in thickness to 23 nm although it still ran parallel to the basal plasma membrane in all regions observed (Fig. 22 and 23). SHION and MURPHY (unpublished) also found oestrogen to have little effect on the basal surface of uterine epithelial cells, but that progesterone was responsible for most of the increase in basal folding and thickness of the lamina densa. Thus the dynamism of the plasma membrane of uterine eptihelial cells is also seen in, perhaps less spectacular, but nonetheless noteworthy changes in its basal portion.

38 . Ch. R. Murphy

Plasma membrane of uterine epithelial cells . 39

7 Biochemical approaches The small contribution uterine epithelial cells make to the overall composition of the uterus belies their importance in attachment. Most estimates put this contribution at around 5% of uterine weight and the proportion of the epithelial cells occupied by plasma membrane is of course, only a tiny fraction of the cell. As a consequence, the plasma membrane is relatively inaccessible to many biochemical methods which require larger amounts of pure preparations of membranes than can readily be extracted. Indeed, to answer many of the questions about changes in membrane composition posed by the above-mentioned structural studies, we would ideally like to isolate and analyse apical plasma membrane and distinguish its composition from that of the remaining lateral and basal plasma membrane. Thus considerably more is known about the plasma membrane of uterine epithelial cells. at the structural level than about its biochemical composition. Cell culture, analysis of uterine fluid proteins, and other biochemical approaches in combination with immunological and histochemical methods have recently however, begun to give us some appreciation of the sorts of molecular changes that may accompany structural differentiation.

7.1 Cell culture, glycans and uterine fluid proteins Using a cell culture system which allows uterine epithelial cells to polarize by employing a matrix coated permeable surface, GLASSER et al. (1988) were able to obtain differentiated cells which formed functional tight junctions as seen in thin section electron microscopy at least. The protein, ovomorulin, was found to be restricted to the lateral cell surfaces and these polarized cultured cells displayed different secretory profiles on their apical and basolateral secretory compartments. Using the same culture system, CARSON et al. (1988) found that the degree of polarization of uterine epithelial cells influenced their ability to secrete glycoconjugates including proteoglycans especially at the apical surface. Moreover, these authors pointed out that since uterine epithelial cells face a lumen and since at least some of the secreted proteoglycans were expressed at the apical cell surface (TANG et al. 1987; MORRIS et al. 1988 a), it was unlikely that the proteoglycans performed a purely structural role (as they might be expected to do at the basal surface). Rather, they were likely involved in adhesion between blastocyst and uterine epithelium at attachment. APLIN (1991) in commenting on glycans as markers of human endometrial differentiation has also suggested that Fig. 22 and 23. Thin section micrographs showing the basal plasma membrane and lamina densa of rat uterine epithelial cells. On day 1 (fig. 22) the basal cell surface is comparatively smooth in outline. By day 6 (fig. 23) both plasma membrane and lamina densa have become highly folded and the lamina densa is thicker than on day 1. - Both x 45,000.

40 . Ch. R. Murphy

these molecules may be involved in molecular interactions between embryo and uterine epithelial cells at attachment. Since in addition, proteoglycans are capable of rapid turnover, they provide a ready means of change in expression of molecules of the cell surface of uterine epithelial cells (CARSON et al. 1990). Rapid changes in proteoglycan may well accompany the structural membrane changes particularly remodelling of microvilli as already discussed, although the apical plasma membrane of these cultured cells can not yet be made to respond to hormones in the same coordinated fashion that uterine epithelial cells do in vivo. APLIN et al. (1988), MORRIS et al. (1988 b), BABIARZ and HATHAWAY (1988) and DUTT and CARSON (1990) also studied glycan assembly, cell surface expression and hormone induction of glycans. Oestrogen appears to have the principal responsibility for the synthesis of these molecules and a distinct cell surface subset of lactosaminoglycans was identified by DUTT and CARSON (1990) using electrophoretic and gel filtration chromatographic methods. DUTT et al. (1987) have found that lactosaminoglycans are located on the surface of uterine epithelial cells and these glycans are clearly involved in adhesion of uterine epithelial cells to each other in vitro. Whether they are also involved in attachment of blastocysts to uterine epithelial cells is less clear although fucosylated lacto-pentasaccharides have been shown to exist on the apical surfaces of mouse uterine epithelial cells (KIMBER et al. 1988) and DUTT et al. (1988) also demonstrated that primary uterine epithelial explants synthesize oligosaccharides containing lactosaminoglycans. These glycans may be involved in attachment by virtue of their likely redistribution over apical and basolateral cell surfaces in response to hormone action during early pregnancy (DUTT and CARSON 1990). On the other hand, heparan sulphate proteoglycans which are also associated with the plasma membrane (see above), have been more directly implicated in adhesion between blastocyst and uterine epithelium by CARSON et al. (1990). WILSON et al. (1990) using scintillation counting of tritiated bound heparin, have added weight to this suggestion by showing that the cultured mouse uterine epithelial cells they studied expressed heparan sulphate binding sites on their apical and basal surfaces. Although more of these binding sites were found basally, their expression on the apical plasma membrane suggests a role in recognition of the blastocyst which has been shown to express heparan sulphate (FARACH et al. 1987; CARSON et al. 1992). This increase in uterine epithelial HSPG receptors could result from de novo synthesis of receptors, or release of bound uterine HSPG (MORRIS et al. 1988 b; MORRIS and POTTER 1990). Perhaps uterine HSPG could also be displaced by the blastocyst-expressed HSPG which may have a higher affinity for the uterine receptor. Thus the roles of the large glycan molecules of the plasma membrane of uterine epithelial cells are likely to be several and varied. Analyses of uterine luminal fluids have also provided some insight into the sorts of molecular changes that may take place in the plasma membrane of uterine epithelial cells even if only because of their proximity to the membrane. Moreover, injected antibodies to uterine fluid proteins can inhibit attachment in the rat thus indicating the importance

Plasma membrane of uterine epithelial cells . 41

of these proteins (YING and GREEP 1972) and HEGELE-HERTUNG and BEIR (1985) have highlighted the close proximity in which secreted fluidal proteins may be held to the apical plasma membrane. Additionally, the distinction between a secreted molecule which enters the luminal fluid and one which remains attached to the cell surface may often be difficult to draw as recently found for lactosaminoglycans by DUTT and CARSON (1990). Some findings on fluidal proteins and related molecules are included here for these reasons. GORE-LANGTON and SURANI (1975), FISHEL (1979, 1980) and QUARMBY and KORACH (1986) have studied changes in mice and SURANI (1975, 1976), SHALGI et al. (1977) and KOMM et al. (1986) have examined uterine flushings from rats and identified hormone induced changes in profile usually employing electrophoretic methods and radiolabelling with 1 D or 2 D analysis of separated proteins. In these studies, progesterone alone did not appear to induce synthesis of many specific proteins, but strong temporal correlations were apparent in protein profiles at the time of implantation when the two hormones are acting together (SURANI 1977). Oestrogen on the other hand induces synthesis of several specific proteins which can be identified electrophoretically (AITKEN 1977) and in particular a 11 0 kDa protein thought to be produced by uterine epithelial cells (KurvANEN and DESOMBRE 1985). TENG et al. (1986) have also purified and identified a 70 kDa protein using electrophoretic and chromatographic techniques. There was no indication that this 70 kDa protein was associated with the cell surface but it comprised 10-20% of total fluidal protein, was glycosylated and was clearly induced by oestrogen. TAKATA and TERAYAMA (1979) found moreover that progesterone could arrest oestrogen induced synthesis and secretion of sulphated glycoproteins in rat uterine luminal epithelial cells. TAKEDA et al. (1988) have identified and characterised an oestrogen induced protein of 97 kDa molecular weight they termed USP-1 which is synthesized by uterine epithelial cells and at least some of which appears to be associated with the apical surface of these cells. WANG and BROOKS (1986) and HORVAT et al. (1992) have also recently identified several proteins in the 115 to 130 kDa molecular weight range largely induced by oestrogen and NIEDER and MACON (1987) using radiolabelling and electrophoretic techniques found several specific molecules they identified with the maternal recognition of pregnancy in uterine epithelial cells. In commenting on functions of uterine fluidal proteins especially in the pig, ROBERTS and BAZER (1988) identified several proteins with differing molecular weights and having roles as diverse as antiviral and immunoprotective effects and, in the cat, MURRAY et al. (1986) have suggested that an oestrogen-induced uterine protein they identified by radioimmunoassay may coat the apical surface of uterine epithelial cells in preparation for attachment. More recently, Lru and GODKIN (1992) identified a retinol-binding protein of 23 kDa in uterine fluid which also was immunolocalised to the apical surface of bovine uterine epithelial cells and was considered to be involved in vitamin transport between maternal and foetal cells. It is thus clear that ovarian hormones induce changes in protein profile in uterine

42 . Ch. R. Murphy

luminal fluid just as they induce structural changes in the plasma membrane of uterine epithelial cells. While oestrogen appears to have a major role in many studies, as it does in much structural differentiation, the effect of progesterone in the production of uteroglobin in rabbits (BEIER 1982), uteroferrin in pigs (ROBERTS and BAZER 1980) and a 46 kDa protein in sheep (SALOMONSEN et al. 1987) is well known. Although some of these many luminal proteins appear to be associated with the plasma membrane, which ones and in what capacity, is not clear at present. Several other molecules have also been identified as components of, or associated with, the plasma membrane of uterine epithelial cells employing various biochemical and histochemical methods. APLIN and SElF (1985, 1987) and SErF and APLIN (1990) raised panels of monoclonal antibodies against fragments of human uterine epithelial cells in culture. Using immunofluorescence microscopy these authors were able to localize several antigens to the surface of uterine epithelial cells although some of the antibodies also reacted with antigens on cells not of epithelial origin. Two of the antibodies so produced however (CC 25 and F 51) both recognized cell surface antigens and also displayed a changing expression during the menstrual cycle so indicating their usefulness in investigating changes during that cycle. The nature of the antigens so localized is not at present clear. Monoclonal antibodies were also used by Fox et al. (1981) to localize an early embryonic antigen (SSEA-1) on several reproductive epithelia including uterine epithelial cells. The antigen recognized in these studies is thought to be a carbohydrate determinant of a glycolipid and Fox et al. (1981) suggest that it is important in interactions between blastocyst and uterus. Glycolipids have also rec~ived attention in their own right. Using thin layer chromatography, ZHU et al. (1990) identified the major glycosphingolipids and plotted changes in these molecules during the menstrual cycle. These workers did not specifically identify glycolipids of the plasma membrane of uterine epithelial cells since they worked with pooled tissues, but GD 3 content of total endometrium was found to decline by 70% during pregnancy whereas GM 3 expression increased at the same time. ZHU et al. (1990) point out however that since glycolipids are instrumental in regulating membrane physiology they may be implicated in terms of altered cell contacts for foetal-maternal interactions. Also on lipid changes, MOULTON and KOENIG (1986 a, 1986 b) have found that phosphorylation of membrane lipids is a likely transducer of message transmission since transmethylation of phospholipids increases during early pregnancy in the plasma membrane of uterine epithelial cells. Such methylation could act to enhance availability of membrane receptors via alterations to membrane fluidity (MOULTON and KOENIG 1986 a). An additional biochemical indicator of the importance of lipids in membrane events was provided by the studies of VILAR-RoJAS et al. (1982) who found that intraluminal administration of phospholipases A z and C inhibited implantation in the rat and that this was accompanied by a reduction in the glycocalyx on the apical plasma membrane of uterine epithelial cells caused by the phospholipases.

Plasma membrane of uterine epithelial cells . 43

7.2 Isolation of th~ plasma membrane Direct attempts to isolate and analyse the apical plasma membrane of uterine epithelial cells have been few, have met with dificulties and have not always yielded unequivocal results. MULLINS et al. (1980) isolated a plasma membrane fraction from pig uterine epithelium at different stages during the oestrous cycle and early pregnancy and found that their preparation was 10-50 times enriched in typical marker enzymes (alkaline phosphatase, leucine aminopeptidase) of the plasma membrane but that contamination with cytoplasmic fractions was still likely. The plasma membrane fraction did not however display any major alterations in protein makeup as determinded by autoradiography of 2 D-gel electrophoresis although a shift to lower isoelectric points of two polypeptides was noted by these autors. RICKETTS et al. (1984) cultured isolated rabbit endometrial cells and following radioiodination of surface proteins, homogenized lysates of the cells were run on 1 D and 2 D gel electrophoresis. Comparisons were made between receptive and non-receptive cell surface profiles and distinctive changes in two proteins in the 40 kDa molecular weight range from uterine epithelial cells could be determined, although no functional significance could be directly attributed to these proteins and RICKETTS et al. (1984) commented that proteins of this size are smaller than that usually associated with adhesive functions between cells. LAMPELO et al. (1985) also isolated plasma membrane fractions of rabbit endometrial cells and electrophoretically analysed the results. Three proteins ranging from 38 kDa to 84 kDa could be associated with the receptive phase for implantation but it was not possible in this study to resolve whether the identified proteins were from epithelial or stromal plasma membranes. In the most comprehensive attempt so far to isolate plasma membrane proteins, ANDERSON et al. (1986) developed an intra-luminal detergent solublization procedure for the apical plasma membrane of rabbit uterine epithelial cells down to the tight junction. These authors ran the solublized fractions on a discontinuous polyacrylamide gel electrophoresis system with both coomassie and silver staining. Transferred proteins also underwent lectin staining on Western blots and membrane proteins from oestrous and pseudopregnant animals as well as those from membranes from implantation and inter-implantation sites were analysed. Several apical plasma membrane proteins across a wide rang of molecular weights were identified with those of 26, 80-86 and 145 kDa being most conspicuous. Three proteins of 24 kDa, 42 kDa and 58 kDa could be associated with the acquisition of receptivity by uterine epithelial cells in the rabbits and at least two of these were further shown to be new glycosylated proteins by lectin binding to the Western blots. HOFFMAN et al. (1990) used a modified detergent extraction method together with a purfication and immunohistochemical technique to further study the 42 kDa protein. Specific localization to the apical plasma membrane of uterine epithelial cells and not other sites was achieved for this protein which is glycosylated and contains galactose and glucosamine residues at least by lectin binding (see also the

44 . Ch. R. Murphy

section on the glycocalyx). HOFFMAN et al. (1990) suggested that the 42 kDa protein, whose function is currently unknown, would be a useful marker of uterine receptivity in the rabbit. . '

T~e finding ~y AN~ER~ON et al. (1986) that s~me of .thi'~tage-s~ecif~cprot~ins acqUlred by utenne epIthelIal cells are glycosylated IS espeClalJ:y mterestmg uythe lIght of the histochemical studies on carbohydrates already discussed (section 5)~and work involving a different biochemical approach by LINDENBERG et al. (1988). TMese authors used several oligosaccharides from milk in competitive inhibition experiments between blastocysts and isolated cultured endometrial monolayers. One compound, lacto-:Nfucopentaose I, produced a significant reduction in blastocyst attachment to, and oUk growth on, these monolayers. Monoclonal antibodies raised by these authors against the fucopentaose compound localized it to the surface of the cultured cells so suggesting involvement of fucosylated oligosaccharides on uterine epithelial cell surfaces in the adhesion process. VALDIZAN et al. (1992) employing WGA-affinity chromatography and electrophoresis have also implicated oligosaccarides in the apical plasma membrane of cultured mouse uterine epithelial cells in the acquisition of receptivity in that species. In this case however, the sugar is N-acetyl-glucosamine which is attached to a 200 kDa protein and these authors suggest that a drastic reduction in this sugar and others is likely to be neccessary to allow the blastocysts to attach to the otherwise heavily glycosylated apical plasma membrane. Thus, conclusions based on biochemical evidence are in good agreement with the histochemical studies examined earlier. 8 The membrane skeleton and cytoskeleton The cytoskeleton of epithelial cells is a diffuse organelle consisting of three principle elements: microtubules, intermediate filaments and microfilaments together with the associated connecting molecules. The membrane skeleton is usually considered to be one component of the cytoskeleton which is restricted to the zone immediately underlying the lipid bilayer and which consists mostly of actin microfilaments and associated proteins (WEATHERBEE 1981; GEIGER 1983; CARROWAY and CAROTHERS-CARROWAY 1989; REPASKY and GREGORIO 1992). The large changes to epithelial cell surface contours already discussed along with the alterations and additions to the many plasma membrane molecules we have examined would seem to provide unequivocal evidence that the membrane skeleton and cytoskeleton of uterine epithelial cells must undergo major alterations especially in the light of the known involvement of these organelles in a wide variety of cellular and cell surface phenomena in other systems (WEATHERBEE 1981; CARRAWAY and CAROTHERSCARRAWAY 1989). It would thus also be reasonable to assume that considerable attention would have been devoted to the cytoskeleton. The latter is not the case and few

Plasma membrane of uterine epithelial cells . 45

workers have so far examined the cytoskeleton of uterine epithelial cells although the possible involvement of microfibrillar elements in stabilizing uterine microvilli has been mentioned in passing (LAWN 1973; FINN and PORTER 1975). Indeed in a recent review of the cytoskeleton in some reproductive epithelia, uterine luminal epithelial cells were not mentioned and there was little to report on glandular epithelial cells (WEINTRAUB 1990). In view of this paucity of information, this section will include studies on both the cytoskeleton and membrane skeleton of uterine epithelial cells although attention will centre on membrane-associated structures which might add to our understanding of the plasma membrane and the remarkable changes it undergoes, and which are the focus of this review. Interest in the cytoskeleton of uterine epithelial cells though limited has to some extent been in the utility, especially of intermediate filament proteins, as indicator molecules in neoplastic canges and has grown out of the surgical need for differential diagnosis of tumors (WEINTRAUB 1990). Thus MOLL et al. (1983) identified different cytokeratin proteins in extracts of normal and neoplastic human epithelial cells and DABBS et al. (1986) in an immunocytochemical study identified keratin and vimentin during the proliferative phase of the menstrual cycle and reported that whereas keratin was found throughout the cytoplasm of the epithelial cells, vimentin had a more localized distribution. KHONG et al. (1986) could only localise cytokeratin in human glandular epithelial cells and found no staining for vimentin. VIALE et al. (1988) similarly followed these two cytoskeletal proteins in epithelial tumors of the ovary and glandular epithelial cells. WONODIREKSO et al. (1992) also using immunofluorescence, recently reported a reduction in different cytokeratins in human uterine luminal epithelial cells in response to a long-term progestin hormonal contraceptive agent. The influence of ovarian hormones on cytoskeletal elements, in this case microtubules, was studied more directly with high-resolution electron microscopy by SZEGO et al. (1988) who described a remarkably rapid influence, measured in seconds, on microtubule depolymerization and concomittent growth of microvilli in uterine epithelial cells of oestrogen-treated rats. These authors emphasized the general lability of these cytoskeletal elements to oestrogen in the ovariectomised animals they studied and did not examine early pregnancy. During early pregnancy and referring specifically to the membrane alterations occurring then, LUNAM and MURPHY (1983) used colchicine to show that inhibition of microtubules could produce changes in the apical plasma membrane of uterine epithelial cells which somewhat resembled the transformation which occurs on days 5 to 6 in rats and so suggested the involvement of microtubules in the process. LUXFORD and MURPHY (1989) used immunofluorescence microscopy of actin microfilaments to demonstrate that these structures were less apically concentrated as the period of attachment and membrane changes advanced. HOCHFELD et al. (1990) also used immunofluorescence to illustrate that while cytokeratin was seen in an unchanging distribution in rabbit uterine epithelial cells during early pregnancy, vimentin distribution which was basolateral early in pregnancy was also found apically as implantation proceeded.

46 .

Ch.

R.

Murphy

In the only studies to date to specifically relate membrane skeletal elements to membrane structure, LUXFORD and MURPHY (1992a,:1992 b) used myosin decoration of actin microfilaments and electron microscopic histochemistry to follow the relationship between actin and apical membrane contours in rat uterine epithelial cells (Fig. 24 and 25). On day 1 of pregnancy these workers found that a prominent terminal web was visible with branches extending microfilaments .into the regular microvilli and other filaments were associated with the membrane in a less organized fashion. As pregnancy progressed however, and microvilli gradually became less regular, the terminal web progressively disassociated into disconnected bundles of filaments until by day 6 a terminal web was no longer present. At this time of uterine receptivity in rats, the irregular projections of the apical plasma membrane contained either isolated bundles or meshworked microfilaments with few lateral filaments connecting them and the large apical protrusions or pinopods contained a dense microfilament meshwork. LUXFORD and MURPHY (1992 a) also found that as the terminal web was lost, cellular vesicles and other organelles which previously were excluded from the submembranous cytoplasm, presumably by the dense web of filaments, were able to approach the apical plasma membrane. In a continuation of these studies, LUXFORD and MURPHY (1992 b) showed that oestrogen and progesterone have quite different effects on the actin microfilaments but that the two hormones acting together in the receptivity-producing combination (PSYCHOYOS 1973) produced a pattern of microfilament organization in ovariectomised rats indistinguishable from that described above on day 6. Thus the loss of terminal web is under maternal ovarian hormonal control. This finding of a loss of the terminal web in uterine epithelial cells during early pregnancy is truly remarkable and may well be unparalleled in epithelial cells. It may also have considerable significance for the dramatic change seen in the apical plasma membrane at attachment. Disassociation of microfilaments could clearly contribute to the loss of microvilli which seems to be essential for receptivity. Moreover, as the terminal web is lost, the apical vesicles previously excluded from the region which then approach the membrane, could add new molecules to the membrane and alter the expression of others. As already mentioned (Section 4.2) these vesicles fuse with the apical ?lasma membrane around the receptive phase for attachment and have cholesterol-rich membranes which probably contribute to the known large increase in cholesterol in the apical plasma membrane at this time (PARR 1982; MURPHY and MARTIN

Fig. 24 and 25. Thin section electron micrographs of rat uterine epithelial cells showing the apical membrane and apical cytoplasmic region. The tissue has been processed to reveal the apical actincontaining membrane skeleton/cytoskeleton elements with the myosin subfragment 1 decoration method following detergent permeablisation. Fig. 24 from day 1 pregnant tissue shows a regular terminal web into which actin microfilaments of the microvilli descend. By day 6 (fig.25) the terminal web has been completely lost and the irregular membrane projections contain areas of meshworked actin-microfilaments. - Fig. 24 x 36,000: fig. 25 x 40,000.

Plasma membrane of uterine epithelial cells . 47

48 . Ch. R. Murphy

1987; MURPHY and DWARTE 1987 a). Cholesterol is a powerful modulator of many membrane functions (YEAGLE 1985) and a large increase in this molecule, promoted by membrane skeleton alterations, could facilitate expression of a new or altered apical cell surface including the carbohydrates discussed earlier.

9 Concluding remarks It is clear that in addition to the well-recognised changes in contours during early pregnancy, the plasma membrane of uterine epithelial cells also undergoes alterations in much more basic molecular structure. These changes in the apical plasma membrane in particular are truly remarkable and may well be unparalleled in regularity and magnitude. Thus the picture presented here of the plasma membrane is of a highly dynamic structure whose changes are largely controlled by ovarian hormones both during the reproductive cycle and in preparation for attachment with the blastocyst. The remarkable events in preparation f<;>r attachment include the replacement of regular microvill with increasingly irregular surface projections accompanied by, and perhaps promoted by, progressive dissociation of the apical terminal web. This loss of the terminal web appears to facilitate delivery of new molecules into the apical plasma membrane including cholesterol. New protein species also appear as witnessed by freeze-fracture, biochemical and histochemical approaches and new carbohydrate epitopes are found and are accompanied by loss of others and an apparent reduction in surface charge. The expression and clustering of these new molecules as well as pre-existing ones could also be influenced by the increase in cholesterol and we have seen that homophilic interaction between carbohydrates could contribute to attachment. As we have also seen, it has been proposed that uterine epithelial cells lose polarity by the time of attachment. As far as the plasma membrane is involved, the loss of some surface enzyme activities and the terminal web could be seen as indicators of reduced polarity, but as has also been discussed, tight junctions actually increase in complexity quite dramatically by attachment and these structures are usually considered a sine qua non of polarization in epithelia. Perhaps the most interesting question is: why is it apparently essential for the plasma membrane of uterine epithelial cells to undergo such a remarkable transformation to allow attachment? Why not just express the correct molecules - like other cells do when undergoing cell-cell interactions? Perhaps the global alterations seen in the membrane are reflections of the much more unusual requirements necessary to allow genetically dissimilar tissues to adhere and for an epithelium to be invaded from its apical side. Perhaps more importantly, the plasma membrane contribution to attachment lies as much in the stereoscopic presentation of molecules as in the nature of those molecules per se, and the transformation is a device to ensure that presentation.

Plasma membrane of uterine epithelial cells . 49

This review has concentrated on the structural and histochemical changes in the plasma membrane of uterine epithelial cells. While the mode of implantation differs between species, attachment appears to be a common beginning about which we have much to learn in many species. Such studies will undoubtedly add to our appreciation of the changes of the different molecular components in this plasma membrane and should eventually explain aspects of the uterine barrier function to attachment and implantation. At a more general level however, further studies on this unusually changeable membrane should tell us much about fundamental membrane molecular structure and dynamics.

50 . Ch. R. Murphy

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