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Morphological variations in gap junctions of ovarian granulosa cells
TISSUE Puhlihd
& CELL
1975 7 (2) 389-405
h_v Lonprnan
DAVID
Group
Ltd. Prinrrd
F. ALBERTINI,
in Gwnt
Britain
DQN W. FAWCETT
and PATRICIA
J. OLDS
MORPHOLOGICAL VARIATIONS IN GAP JUNCTIONS OF OVARIAN GRANULOSA CELLS ABSTRACT. Gtanulosa cells in ovarian follicles of tat, mouse, rabbit and hamster were studied by lanthanum tracer and freeze-fracture techniques. Abundant gap junctions exhibited striking intraspecific variation in size and pattern of particle aggregation. The smaller gap junctions showed close packing of the inttamembtanous A face particles. In large gap junctions, tanging up to 6 p in diameter, particles were packed in rectilinear arrays separated by a labytinthine network of particle-free ‘aisles’. Small clusters of particles in a particle-poor circumferential zone suggested enlargement of junctions by peripheral accretion. Linear inttamembtanous structures, resembling those of occluding junctions, occasionally bounded large gap junctions. Spherical inttacytoplasmic structures limited by gap junctional membranes were shown by tracer studies to arise by invagination of the cell surface. These were intrepteted as a means of disposal of junctions by interiotization.
1973). Such junctions have also been observed on the boundaries between coupled cells in embryonic tissues, and in cell cultures (Johnson and Sheridan, 1971; Pinto da Silva and Gilula, 1972; Revel et al., 1973). Freeze-cleaving now permits a more detailed examination of the structure of gap junctions and other membrane specializations. Using this technique, Friend and Gilula (1972) described variations in the intramenbranous differentiation at gap junctions in several mammalian organs with respect to their number, distribution and size. Staehelin (1972) has described several variants of the gap junction in rat intestinal epithelium. Interest in the intercellular junctions of ovarian tissue stems from the belief that the specializations for cell communication may ultimately be found to play a significant role in differentiation and in the integration of cellular responses to hormonal stimulation. To date the basis for this speculation is that in the rabbit ovary, gap junctions are not present in primary follicles but appear in the early antral follicle and continue to increase in number and size as the follicle enlarges Albertini and Anderson, 1974). A similar progressive evolution of these junctions has been observed in the present study in young
Introduction cell contacts between granulosa cells have been identified in various mammalian species by several investigators studying the ultrastructure of ovarian follicles in thin sections (Byskov, 1969; Espey and Stutts, 1971; Motta et al., 1971; Zamboni, 1974). In these studies the predominant junctional specialization has been variously described as a ‘close junction’ or a ‘tight’ or ‘occluding’ Junction. Recent work employing lanthanum as an electron-opaque tracer for delineating extracellular spaces (Anderson, 1971; Merk ef al., 1973) and freeze-fracturing studies (Fletcher, 1973 ; Albertini and Anderson, 1974) have provided convincing evidence that these cell contacts are, for the most part, gap junctions or nexuses. This type of junction is believed to be the morphological basis for electrical and metabolic coupling of cells in many normal epithelial tissues (Furshpan and Potter, I968 ; Bennett, 1973 : McNutt and Weinstein, CLOSE
Department of Anatomy and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 021 IS. Received 6 Fcbtusty
1975. 389
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rats. It has also been reported that the number of such junctions is responsive to hormonal stimulation (Merk et al., 1972). In order to describe more completely the morphology of cell contacts in ovarian tissue and to clarify the types of membrane specialization present, we have studied the follicles of mice, rats, rabbits, and hamsters by electron microscopy of thin sections, by freeze fracturing and by use of lanthanum as a probe of the extracellular space. Materials and Methods Thirty-day rats and sexually mature mice, hamsters and Dutch Belted rabbits were used in this study. Follicles or pieces of follicles were dissected quickly from whole ovaries in air or in 2% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer (Karnovsky, 1965). Paraformaldehyde was omitted from the fixative used for rabbit tissue. Follicular tissue was fixed 15-25 min in the aldehyde solution, trimmed and washed in buffer, equilibrated with 30%) glycerol in buffer for l-2 hr at room temperature, frozen on paper discs in the liquid phase of Freon 22 and stored in liquid nitrogen. Freeze-fracturing and platinumcarbon shadowing were done in a Balzer apparatus (Balzers, Lichtenstein) operating at a stage temperature of - 115°C. Replicas were subsequently freed from tissue in Clorox, mounted on 300-mesh uncoated copper grids and examined with a Phillips 200 or 300 electron microscope. For the preparation of thin sections, tissue were fixed for 1 hr in the same fixatives used for freeze-cleaving. Both the aldehyde fixative and buffer washes contained 2%
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lanthanum nitrate. After a I-hr incubation in 0.03 N NaOH, tissues were post-fixed in 17; 0~04, dehydrated in a graded series of ethanols and embedded in an EponAraldite mixture (Anderson and Ellis, 196.5). Thin sections were cut on a Porter-Blum MT-2 ultramicrotome, collected on 300mesh grids, and stained lightly with uranyl acetate and a mixture of lead salts (Sato, 1968) before viewing in an electron microscope. Freeze-fracturing cleaves the plasma membrane through its hydrophobic interior, exposing two complementary internal aspects of the membrane (Branton, 1966; Pinto da Silva and Branton, 1970). The outwardly directed inner half membrane or inner leaflet, rich in membrane-intercalated particles, is conventionally termed the A-face. The inwardly directed outer half membrane or outer leaflet, which contains relatively few particles, is referred to as the B-face. This terminology will be used throughout this paper. Observations The lining of the developing mammalian follicle is a stratified epithelium composed of multiple layers of granulosa cells resting upon a thick basal lamina that separates it from the surrounding theta folliculi (Fig. 1). In early antral follicles, the granulosa cells are closely packed and in contact over a large part of their surface, as in most other types of epithelium. As follicular development progresses, however, the epithelium becomes much more loosely organized. Conspicuous intercellular spaces appear and the polygonal cells gradually become stellate
Fig. 1. Early antral follicle from a macaque ovary showing the eccentric antral cavity and the granulosa cells forming a compact multilayered epithelium. Epon section. x 600. Fig. 2. In more advanced follicles the eranulosa cells near the basal lamina remain in close contact, but those in the remainder of the epithelium become very loosely organized and irregularly stellate in shape with large intercellular spaces. Thus they come to resemble mesenchyme instead of typical epithelium. The appearance of an area of cell contact such as that at the arrows is seen in a freeze-fractured specimen in Fig. 5. x 960.
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as the area of cell-to-cell contact is diminished. As a result of these changes, the granulosa layer comes to resemble mesenchyme rather than a typical epithelium (Fig. 2). Study of electron micrographs confirms that gap junctions continue to be prevalent despite the looser organization of the tissue. Freeze-fracture preparations show many gap junctions with closely packed particles on the A-face and a corresponding pattern of small pits on the B-face (Figs. 3, 4). These junctions are remarkable not only for their number but also for the variation in their packing pattern and for their very large size. Whereas the average diameter of gap junctions in other epithelia may be of the order of 0.5 p, the gap junctions of granulosa cells range in size from under 1 p to as much as 6 ,Uin diameter. Those less than 1 p in diameter found on appositional areas of similar size tend to be irregular in outline and are composed of particles and complementary pits in close hexagonal array. In studying replicas of fracture planes that cross the interface between cells, one not infrequently encounters rows of 3-5 gap junctions spaced at rather regular intervals and separated by lenticular profiles of extracellular spaces where the cell membranes diverge and then come together again (Figs. 3, 4). This suggests that there is a gap
Fig. 3. Freeze-fracture mouse ovary. Where the junctions of varying size. diverge around lenticular
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junction wherever the cell surfaces are in apposition; while between these areas of contact, there is a system of interfacial canals-a topographic relationship not entirely unlike that of the prickle-cells or keratinocytes of the epidermis except that in stratified squamous epithelium, the end-on contacts of the short cell processes are maintained mainly by desmosomes instead of gap junctions. If this interpretation is correct, then a surprising proportion of the contact surface of granulosa cells is occupied by gap junctions. The larger junctions, over 1 p in diameter, are more regular in outline and usually round or oval. The planar area of junctional specialization is often elevated some distance above the remainder of the replica suggesting that it covers the end of a short, broad process that abuts a similar process of the neighboring cell (Fig. 5). Another feature of interest in these large junctions is a marked tendency for the particles to be clustered in rectilinear arrays separated by narrow particle-free aisles (Figs. 6, 7). The particle arrays are generally composed of 3-5 rows of particles 12-25 to a row. The particles in adjacent rows may be in register or less precisely ordered (Figs. 6, 7). For reasons that are not clear, when both A- and B-face are available for comparison, the pits on the
preparation membranes Between the intercellular
of the interface between two granulosa cells in are in contact they are specialized to form gap four gap junctions shown here, the membranes spaces. x 26,000.
Fig. 4. Another example of successive gap junctions and intervening intercellular spaces, from hamster ovary. The A-face of the junctions show closely packed intramembranous particles. The B-face exhibits a corresponding pattern of shallow pits. Preparations of this kind give the impression that gap junctions occupy a large portion of the total area of cell contact. x 53,000. Fig. 5. A very large gap junction covering a slightly elevated region of cell contact between granulosa cells of rabbit ovary. Notice the complex pattern of particle aggregates separated by particle-free aisles. An area such as that enclosed in the rectangle is shown at higher magnification in Fig. 6. x 34,000. Fig. 6. A portion of the periphery of a large gap junction on a rabbit granulosa cell. Notice the relatively sparse population of intramembranous particles in the unspecialized membrane; the sharp boundary of the junction; and the labyrinthine pattern of particle-free paths between rectilinear arrays of particles. x 230,000.
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B-face are more precisely ordered than are the particles on the A-face (Figs. 16, 19). Peripheral to the large gap junctions, there is often a zone of fairly uniform width in which the concentration of A-face particles is very much lower than that found in the unspecialized areas of membrane generally (Fig. 7). Scattered in this particle-poor satellite aggregates of peripheral zone, particles of varying size are seen. The relationship of these to the main junction creates the impression that the junctions may grow by accretion of these new clusters of particles at their perimeter. Such a recruitment of particles from surrounding unspecialized areas of membrane would be consistent with the current concept of a fluid lipid bilayer permitting lateral movement of particles. This membrane intercalated impression is strengthened by the observation that in young animals in which gap junctions are just appearing or in immature animals injected with pregnant mare’s serum gonadotropin, it is not uncommon to see areas in which there are several discrete clusters of particles a short distance apart in a configuration suggesting that they may be coalescing to form a gap junction. Of less common occurrence are isolated meandering strands or ridges on the A-face like those found at the zonulae occludentes of columnar epithelia. These are of limited length, end blindly and show no consistent pattern. They are termed ‘focal tight junctions’ in the literature and have been reported in regenerating liver and various other rapidly renewing epithelia (Yee, 1973). A line of membrane fusion of such limited extent could not subserve the function of occluding or sealing the intercellular cleft and at present their significance is quite obscure. Occasionally clusters of particles of what appear to be developing gap junctions are associated with these strands or ridges (Fig. 8). Not infrequently in rabbit granulosa cells, a large and otherwise typical gap junction is bounded by a long more or less continuous strand around its periphery (Figs. IO, 1I). Similar ridges on the A-face and grooves on the B-face may extend across the gap junction dividing it into polygonal areas of various size and shape (Fig. 12). Rarely such intramembranous strands may completely circumscribe an area that contains no particle aggregates (Fig. 9).
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The very large gap junctions of rabbit granulosa cells (Fig. 5) often present a very flat surface suggesting that this specialization may confer a certain degree of stiffness upon the membrane. This does not seem to be invariably true, however, for in thin sections of lanthanum-stained hamster granulosa cells, very extensive gap junctions can be identified in regions where the boundary between cells is elaborately corrugated and interdigitated (Fig. 13). Such areas when sectioned perpendicular to the membranes show a 30 8, dense line of lanthanum that has permeated the junction, but where the plane of section is parallel or tangential to the surface, the junctional subunits can be seen in negative image outlined by lanthanum (Fig. 13). In all species studies, the granulosa cells were found to have processes that project from one cell deep into the neighboring cell, thus confirming earlier studies based upon thin sections (Espey and Stutts, 1972; Merk et al., 1972). In sections of tissue in which lanthanum is used as an electron-opaque probe of the extracellular space, these interdigitating processes appear as circular profiles clearly outlined by the tracer, thus verifying their continuity with the cell surface (Figs. 14, 15). Similar profiles that are not infiltrated by lanthanum can also be identified. These evidently represent processes that have constricted at their base and detached from the cell surface becoming spherical cytoplasmic inclusions bounded by a double membrane. These were first described as ‘sphaerae occlusae’ (Espey and Stutts, 1972) on the erroneous assumption that they were bounded by tight or occluding junctions. When these processes and inclusions are studied by freeze-cleaving, their membrane specializations are found to be typical gap junctions (Albertini and Anderson, 1974). This is found to be true in the mouse and rat, as well as in the rabbit. Furthermore, the junctional specializations of the processes and spherical inclusions exhibit the same diversity of particle patterns as is seen among the gap junctions on the cell surface-close hexagonal packing: parallel rectilinear ranks of particles separated by particle-free aisles (Figs. 17, 19); reticular arrangements of closely packed particles outlining small rounded or polygonal areas devoid of particles (Fig. 20): and intermediates between these latter two (Fig. 18).
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Fig. 7. A peripheral portion of a large gap junction from rabbit granulosa cell illustrating the rectilinear particle arrays, and intervening ‘aisles’. Notice also the particle-poor zone that often surrounds gap junctions. Several satellite aggregates of particles in this zone suggest that the large gap junctions may grow by accretion of such aggregations to their periphery. x 123,000. Fig. 8. Specimen from hamster granulosa cell. Several aggregations of gap junctional particles partially connected by rows of particles and short ridges or strands. x 141,000. Fig. 9. An strands on the epithelia. The is not known.
area of rabbit granulosa cell membrane exhibiting intramembranous A-face resembling those found in tight or occluding junctions of other significance of these intramembranous differentiations in follicular cells x 250,000.
Figs. 10 and 11. Portions of the periphery of a very large gap junction on a rabbit granulosa cell. The junction is surrounded by an extensive but discontinuous intramembranous ridge resembling one of the elements that comprise tight junctions in simple columnar epithelia. x 65,000. Fig. 12. A region of rabbit granulosa cell membrane in which tight junctional elements circumscribe rounded or polygonal units of both gap junctional and non-junctional membrane. x 97,000. Fig. 13. A thin unstained section of hamster granulosa cells showing a very extensive gap junction on an irregularly corrugated area of the cell boundary. Where the plane of section is normal to the cell membranes, a dense 30 A line of lanthanum can be seen permeating the gap junction. Where the plane of section is parallel to the membranes, a lattice of hexagonally packed particles can be seen in negative image outlined by the lanthanum. x 102,500. This is more evident at higher magnification in the two insets. x 175,000. Fig. 14. A thin unstained section of rabbit granulosa cell from a tissue sample immersed in lanthanum. The two circular profiles outlined by lanthanum are sections of cell processes of an adjacent cell. These deep invaginations are bounded by gap junctions in which one can sometimes discern parallel lines of lanthanum corresponding to the particle-free ‘aisles’ seen in freeze-cleaving preparations. x 33,000. Fig. 15. Three lanthanum outlined processes are shown here projecting lutein cell from the sixth day of pregnancy. x 24,500.
into mouse
Fig. 16. A small gap junction of a rabbit granulosa cell showing both A-face particles and B-face pits. This junction exhibits an uncommonly uniform hexagonal lattice. For reasons that are not understood, the pits often seem more highly ordered than the particles. x 250,000. Fig. 17. A gap junction from hamster of particle arrays. x 80,000.
granulosa
cell showing a parallel arrangement 1*’
Fig. 18. A gap junction of a rat granulosa cell lining an invagination occupied by a process of an adjoining cell. The pattern of junctional particles is similar to many seen on planar portions of the cell surface. x 100,000. Fig. 19. A path of cleavage through the cytoplasm of a granulosa cell encountering a deeply invaginated cell process. Parallel arrays of pits are seen on the B-face and a corresponding pattern of particles in a smaller exposure of the A-face. x 77,000. Fig. 20. A fracture through granulosa cell cytoplasm. The round concave membrane in the center is an interiorized gap junction. Its shape suggests that is is part of a hollow spherical inclusion formed by invagination and constriction of a large gap junction formerly on the surface of the cell. x 75,000.
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Discussion This study has confirmed that the commonest type of junctional specialization of the granulosa cells is the nexus or gap junction (Merk et al., 1973; Fletcher, 1973; Albertini and Anderson, 1974). It leaves unexplained the large number of these junctions found in each cell, their exceptional size (up to 6 y), the variability in the arrangement of their intramembranous particles, and their physiological significance in development and maturation of the follicle. Some discussion of these gaps in our knowledge may be useful as a stimulus to further investigation. Gap junctions are believed to play an important role in cell-to-cell communication by providing low resistance pathways for diffusion of ions (Furshpan and Potter, 1968). The occurrence of such junctions between granulosa cells might therefore be advantageous for coordinating the synthetic activities of cells throughout the follicular epithelium. Electrical coupling of the granulosa cells to each other and to the oocyte has yet to be demonstrated. It has been suggested however that these cells may be responsible for maintaining the meiotic arrest of the oocyte or for initiating its maturation. Such an effect could conceivably be mediated by junctions permitting flow of ions between processes of cells in the corona radiata and those of the oocyte where the two interdigitate in the zona pellucida. There is abundant evidence that only a few, very small gap junctions are sufficient to maintain electrical coupling of epithelial cells and little is gained by a great increase in the area of the low resistance pathways. Thus it is difficult to account for the abundance and the large size of the junctional specializations of granulosa cells on the basis of electrical coupling alone. It has been demonstrated that small molecules can also diffuse from cell to cell via gap junctions (Lowenstein, 1966; Johnson and Sheridan, 1971; Sheridan, 1971) and this may result in metabolic coupling of cells (Gilula et al.., 1972). Although ovarian follicles grow to a large size, they remain avascular and the diffusion distance from the capillaries of the theta to the cumulus oophorus is considerable. However, there would seem to be no need for
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transcellular diffusion of blood-borne nutrients or hormones for there is no permeability barrier in the ovary comparable to the blood-testis barrier. Electron-opaque tracers of large molecular size rapidly diffuse from the blood throughout the follicles via the extracellular spaces (Anderson, 1972). Nevertheless, as previously suggested (Merk et al., 1973) gap junctions might provide for uniform dissemination of intermediary metabolites, second messengers or other regulatory molecules throughout the population of granulosa cells-insuring a uniform metabolic response to circulating hormone. For such metabolic coupling a larger area of junctional membrane might be advantageous (Sheridan, 1974). Other consequences of the extensive gap junctions between granulosa cells must be considered. It was emphasized above that as follicular growth progresses, intercellular spaces become more prominent throughout the epithelium. The area of contact between the cells is thus reduced until the epithelium takes on the appearance of a stellate reticulum with the cells adhering via short processes. The abundance of gap junctions in freeze-cleaved preparations and their large size suggests that the greater part of the contact surface of adjacent granulosa cells may be specialized in this manner. It is important to bear in mind that, in addition to their function in communication, gap junctions are also sites of very tenacious adherence of cells. Whatever advantage may accrue to granulosa cells by having large areas of specialization for communication would seem to be gained at the expense of mobility. This would seem to pose a problem in that cells with extensive gap junctions could not easily change their position relative to their neighbors to allow for enlargement of the antrum or growth of the follicle as a whole. It is evident, however, that the cellular relationships do continually change during follicular growth and as ovulation approaches, a sizeable mass of cumulus cells around the oocyte is cast off from its attachments to the rest of the epithelium. In the thinning of the follicular wall and formation of the stigma, there is a marked attenuation of the epithelium that must involve either extensive passive displacement or active migration of the cells. It is not uncommon at this stage to find individual
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follicular cells detached from the epithelium exhibiting amoeboid shape or rounded up and floating free in the follicular fluid (Parr, 1974; Motta and DiDio, 1974). Consideration of these developmental events raises the interesting question as to how the cells free themselves from their firm attachments at gap junctions in order to participate in these dynamic morphogenetic processes. This question directs attention to the deficiency in our knowledge of the life span and fate of junctional specializations. Once formed, can they be dismantled in situ? We know of no morphological evidence suggestive of disassembly of gap junctions in situ. It is known, however, that when epithelial cells are experimentally dissociated in calcium-free solutions, desmosomes separate and the half desmosomes are then interiorized and can be identified in the walls of endocytotic vacuoles in the cytoplasm (Berry and Friend, 1961; Overton, 1968). It is assumed that they are then broken down by the lysosomal system. If interiorization and lysosomal degradation are the only mechanism by which cells can free themselves of attachment at gap junctions, this may provide an explanation for the very frequent occurrence of deep invagination of junctional areas of granulosa cell membranes into neighboring cells and the abscission of these invaginations to form spherical cytoplasmic inclusions bounded by two-cell membranes that exhibit typical gap junctional specialization. These so-called ‘annular gap junctions’ or ‘sphaera occlusa’ were interpreted by Espey and Stutts (1972) as a mechanism for exchange of cytoplasm between granulosa cells. While it cannot be denied that incorporation of a small amount of cytoplasm of the adjacent cell does occur when these structures are formed, it seems to us unlikely that an ‘exchange of cytoplasm’ is their principal function. More plausible is the suggestion that interiorization may represent a means of removing immobilizing junctions from the surface (Merk et al., 1973) to permit movement of the cells in the course of the continual internal reorganization of the follicular epithelium. If this interpretation is valid, then one would expect to find interiorized junctions in greatest abundance in those periods of follicular development and luteal transformation which involve the greatest amount of cell movement.
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There is experimental evidence that this is true. The number of surface nexuses and interiorized nexuses in Graafian follicles of young rabbits has been studied before, and after, injection of human chorionic gonadotropin (Bjersing and Cajander, 1974). Following administration of the hormone, the granulosa cells became much more loosely organized. Associated with this change there was a significant decline in surface nexuses and a doubling of the number of ‘annular junctions’. Thus it seems likely that the formation of these structures is a means of interiorizing and disposing of junctional membrane. Gap junctions are maintained in large numbers throughout follicular growth despite the apparent loss of many by interiorization. Therefore we assume that as cells change position in the epithelium, new junctions are formed rapidly in newly established areas of cell contact. This continual turnover of the cell surface may account for some of the diversity in structure of gap junctions and suggests that the growing follicle may be unusually favorable material for study of the initiation and growth of gap junctions. A considerable number of the junctions examined in this study were encircled by membrane containing relatively few granules. The particle-poor region observed around gap junctions has been referred to by other authors as a ‘zone of particle exclusion’. Small clusters of particles often observed in this zone are probably destined to traverse it and to fuse with the main junctional aggregation of particles thus contributing to its growth. In ingenious experiments on reconstitution of gap junctions among reaggregating hepatoma cells, Johnson et al. (1974) found the earliest indications of nascent gap junctions were smooth, flattened areas of membrane called ‘formation plaques’ surrounding a few 90- 110 8, particles. At later times, there was an aggregation of smaller particles within the plaque into tightly adherent groups indistinguishable from small gap junctions. These rapidly enlarged by addition of individual particles and by fusion of the smaller aggregates. These findings strengthen the interpretation put forward in the present study that the satellite aggregations of particles observed in the clear zone around the large granulosa cell junctions are probably clusters of par-
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titles being recruited in the growth of the junction. If it be assumed that junctional particles arise in unspecialized areas of membrane and then aggregate at the site of a new junction, then ‘zone of particle depletion’ might be a term more appropriate for this clear zone than ‘zone of particle exclusion’. The observations reported here have demonstrated in the gap junctions of granuloss cells patterns of particle packing that are rarely seen in such junctions in other organs. The degree of variability in pattern is quite unusual, some showing rectilinear particle arrays a few rows wide separated by particlefree aisles; others consisting of loosely and irregularly packed particles, and still others exhibiting a very close precisely hexagonal packing. The possible functional correlates of these variations in pattern remain obscure. However, it is not unreasonable to speculate that they may represent different stages in development or different functional states of the junctions or both. This latter possibility becomes more credible in the light of recent experimental studies which have shown that when the gap junctions of gastric epithelium or those in the septa of crayfish axons are exposed to conditions which result in electrical uncoupling, the junctional particles become closely packed in a regular hexagonal lattice and when coupling is restored. they become more loosely and irregularly spaced (Perrachia and Dulhunty, 1974). Our thinking about the significance of gap junctions has been dominated to date by their correlation with electrical coupling. There is some danger that acceptance of all such particle aggregates on the A-face as pathways for cell-to-cell communication may close our minds to other possible interpretations. Aggregation of intramembranous particles has been reported at sites of secretory particle discharge (Satir et a/., 1973): after exposure of erythrocytes to changes of pH and temperature (Pinto da Silva, 1972): in immune reactions (Scott and Marchesi, 1972); and after physiological activation of spermatozoa (Friend and Fawcett, 1973 ; Koehler and Gaddum-Rosse, 1975). Indeed, closely packed hexagonal arrays of particles resembling gap-junctions have been reported in membranes at the free surface of transitional epithelium
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(Staehelin et al., 1972) and on the ectoderm of hydra where there can be no cell-to-cell communication (Wood, 1974). There is broad acceptance of the concept that the sites of binding of antibodies, lectins, hormones and drugs to the cell surface may be oligosaccharide moieties of glycoprotein or glycolipid constituents of the membranes. Incontestable evidence for this interpretation is difficult to obtain, but freezefracturing studies of myoneural junctions reveal distinctive, irregular rows of I I@ 140 8, intramembranous particles at the tops of the junctional folds in the postsynaptic membrane (Rash and Ellisman. 1974)-sites where immunohistochemical methods also localize acetylcholine receptor complexes (Daniels and Vogel, 1974). Thus in this case the receptors appear to he associated with intramembranous particles visible by freeze cleaving. Freed from the concept that membrane particle organization must necessarily be related to intercellular communication. one can think of other possible roles for the aggregations of particles found in the membranes of granulosa and lutein cells. For example, glycoprotein hormones of the pituitary are known to bind to the membranes of their target organs and to exert their effect via activation of adenylcyclase and the intracellular accumulation of the second messenger cyclic AMP (Butcher et al.. 1972). The fate of the bound hormone after it has exerted its effect is unclear but continuous modulation of target organ function would seem to require either a mechanism for dissociation of the hormone from its binding site or a means of local degradation of the hormone receptor complex, together with continual replacement of the receptor population in the membrane. In a foregoing section of this discussion, we expressed perplexity over the exceptional size and number of gap junctions in the granulosa cells--a profusion of particle aggregates that would seem to be far in excess of the needs for functional integration. We now offer for consideration the proposition that not all particle aggregations on the A-face are concerned with cell communication. Evidence for fluidity of the cell membrane and movement of protein particles within it is now compelling. It gonadotropic hormone receptors are indeed exposed portions of membrane intercalated
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particles or projecting oligosaccharides, it is conceivable that the particles representing occupied receptors may move within the plane of the membrane assembling to form large particle aggregates. These then may be interiorized by invagination and degraded within the cytoplasm while new receptors are added to the cell surface. Although such a disposal mechanism for occupied receptors may seem very far fetched, it offers an alternative explanation for the spherical vesicles bounded by gap junctional membrane that are so common in the cytoplasm
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of these cells. Though highly conjectural, this suggestion may have some value in keeping before investigating the possibility that not all of the varied patterns of particle aggregation seen in membranes may be concerned with the single function of cell communication. Acknowledgement
Supported by a grant from the Human Life Foundation.
References ALBERTINI, D. F. and ANDERSON, E. 1974. The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. J. Cell Biof., 63, 234-250. ANDERSON, E. 1971. Intercellular junctions in the differentiating Graafian follicle of the mouse. Amt. Rec., 169, 473a. ANDERSON, W. 1972. Permeability of ovarian blood vessels and follicles of juvenile rats. Microvasc. Res., 4, 348-373. ANDERSON, W. A. and ELLIS,R. A. 1965. Ultrastructure of Trypanosoma lewisi; flagellum, microtubules, and the kinetoplast. J. Protozool., 12, 483-499. BENNETT, M. V. L. 1973. Function of electrotonic junctions in embryonic and adult tissues. Fedn Proc. Fedn. Am. Sots exp. Biol., 32, 65-15. BERRY, M. N. and FRIEND, D. S. 1969. High yield preparation of isolated rat liver parenchymal cells. J. Cell Biol., 43, 506. BJERSING, L. and CAJANDER, S. 1974. Ovulation and the mechanism of follicle rupture. IV. Ultrastructure of membrane granulosa of rabbit Graafian follicles prior to induced ovulation. Cell Tiss. Res., 153, I-14. BRANTON, D. 1966. Fracture faces of frozen membranes. Proc. mtn. Acad. Sci., U.S.A., 55, 1048-1056. BUTCHER, R. W., ROBINSON, G. A. and SUTHERLAND, E. W. 1972. Cyclic AMP and hormone action. In: Biochemical Actions of Hormones (ed. G. Litwack) Vol. 2, pp. 21-54, Academic Press, New York. BYSKOV,A. G. S. 1969. Ultrastructural studies on the preovulatory follicle in the mouse ovary. 2. ZeNforsch. Mikrosk. Amt., 100, 285-299. DANIEL& M. P. and VOGEL, 2. 1974. Immunoperoxidase staining of a-bungarotoxin bound to acetylcholine receptors in mouse motor endplates. J. Cell Biol., 63, 76a. ESPEY, L. L. and STUTTS, R. H. 1972. Exchange of cytoplasm between cells of the membrana granulosa in rabbit ovarian follicles. Biol. Reprod., 6, 168-175. FLETCHER, W. H. 1973. Diversity of intercellular contacts in the rat ovary. J. CeN Biol., 59, 10la. FRIEND, D. S. and GILULA, N. B. 1972. Variations in tight and gap junctions in mammalian tissues. J. CeN Biol., 53, 759-776. FRIEND, D. S. and FAWCETT, D. W. 1974. Membrane differentiations in freeze-fractured mammalian sperm. J. Cell Biol., 63, 641-664. FURSHPAN, E. J. and POTTER, D. D. 1968. Low resistance junctions between cells in embryos and tissue culture. In Current Topics in Developmental Biology (eds. A. A. Moscona and A. Monroy), Vol. 3, pp. 95-127. Academic Press, New York. GILULA, N. B., REEVES,0. R. and STEINBACH,A. 1972. Metabolic coupling, ionic coupling and cell contacts. Nature, Lond., 235, 262-265. JOHNSON, R. G. and SHERIDAN, J. D. 1971. Junctions between cancer cells in culture: ultrastructure and permeability. S&m-c, N. Y., 174, 717-719.
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JOHNSON, R. G., HAMMER, M., SHERIDAN, J. D. and REVEL, J. P. 1974. Gap junction formation between reaggregated Novikoff hepatoma cells. Proc. ncrtn. Acad. Sci., U.S.A., 71, 4536-4540. KARNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27, 137A-138A. KOFHLFII, J. K. and GADDUM-ROSSE, D. 1975. Media induced alterations of the membrane associated particles of the guinea-pig sperm tail. J. Ultrastrucf. Res. (in press). LowEi%srE]N, W. R. 1966. Permeability of membrane junctions. Ann. N. Y. Acad. Sci., 137, 441-472. McNurr, N. S. and WEINSTEIN,R. S. 1973. Membrane ultrastructure at mammalian intercellular junctions. In Prugwss in Biophysics and Molecular Biology (eds. J. Butler and E. Noble), Vol. 26, pp. 45 107. Pergamon Press, Oxford. MERK, F. B., BOTTICELLI,C. R. and ALBRIGHT, J. T. 1972. An intercellular response to estrogen by granulosa ceils in the rat ovary: an electron microscope study. Endocrinology, 90, 992-1007. MURK, F. B., ALBRIGHT, J. T. and BOTTICELL~C. R. 1973. The fine structure of granulosa cell nexuses In rat ovarian follicles. Anal. Rec., 175, 107-125. MOT~A. P., TAKEVA, S. and NESCI, E. 1971. Etude ultrastructurale et histochemique des rapports entre les cellules folliculaires et I’ovocyte pendent le development du follicule ovarien chez les mammafreres. Ac,fa Amt., 80, 537-562. MOTTA. P. and DIDIO, L. J. A. 1974. Microfilaments in granulosa cells during follicular development and transformation in corpus luteum in the rabbit ovary. J. Suhmicr. CyroL, 6, 15-27. OVERTON, J. 1968. The fate of desmosomes in trypanized tissue. J. exp. Zoo/., 168, 203-214. Paaa, I. L. 1974. Histological examination of the ovarian follicular wall prior to ovulation. Biol. Reprod.. 11. 483-503. P~RRAWIA, C. and DULHUNTY, A. F. 1974. Gap junctions: structural changes associated with changes ,n permeability. J. Cell Biol., 63, 263a. PI?~TODA SIL~A, P. 1972. Translational mobility of the membrane intercalated particles of human erythrocyte ghosts. pH-dependent, reversible aggregation. J. Cell Biol., 53, 727-787. PINTO DA SILVA. P. and GILULA, N. B. 1972. Gap junctions in normal and transformed fibroblasts in culture. Expl Cell Ras., 71, 383-401. PINIO DA SILLA. P. and BRANTON, D. 1970. Membrane splitting in freeze-etching. Covelently bound ferritin as a membrane marker. J. CeN Biol., 45, 598-605. RASH, J. E. and ELLISMAN, M. H. 1974. Studies of excitable membranes. 1. Macromolecular specializatlont of the neuromuscular junction and the nonjunctional sarcolemma. J. Cc/l Biol., 63, 567-586. R~:vFL.,J. P.. YIP, P. and CHANG, L. L. 1973. Cell junctions in the early chick embryos--a freeze etch study. DcvI Biol., 35, 302-3 17. SATIR, B., SVHOOLEY,C. and SATIR, P. 1973. Membrane fusion in a model system. Mucocyst secretion in Tetrahymena. J. Cclll Biol., 56. 153-l 76. Sa-ro, 7‘. 1968. A modified method for lead staining of thin section. J. Elec,. Mk. (Jopcrn), 17, 158-159. S(XI?I, R. E. and MARCHESI, V. T. 1972. Structural changes in membranes of transformed lymphocytes demonstrated by freeze-etching. Cell Immunol., 3, 301-317. SH~WD~N. J. D. 1971. Dye movement and low resistance junctions between reaggregated embryonic cells. Dcvi. Biol.
. 26 3627-636.
SHFRID~N. J. D. 1974. Functional evaluation of low resistance junctions: influence of cell shape and size. Anz. Zoo/., 13. I 119-l 128. SIAEHLI~, L. A. 1972. Three types of gap junctions interconnecting intestinal epithelial cells visualized by freeze-etching. Proc. mtn. Acad. Sci., U.S.A., 69, 1318-1321. STAFHFLIN, L. A.. CHLAPOWSKI, F. J. and BONNEVILLE, M. A. 1972. Lumenal plasma membrane of the urinary bladder. I. Three dimensional reconstruction from freeze-etch images. J. CeN Biol., 53. 73-91. WOOD. R. L. 1974. A closely packed array of membrane intercalated particles at the free surface of Hydrtr. J. Cell Biol., 62, 556-560. YEI. A. G. 1973. Studies on the origin and distribution
Sciences, Harvard University. ZAMBONI. L. 1974. Fine morphology IO. 125~149.
of intercellular
junctions.
of the follicle wall and follicle cell-oocyte
Thesis. Division of Medical association.
Biol.
Rc~pwd..
& CELL
1975 7 (2) 389-405
h_v Lonprnan
DAVID
Group
Ltd. Prinrrd
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in Gwnt
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and PATRICIA
J. OLDS
MORPHOLOGICAL VARIATIONS IN GAP JUNCTIONS OF OVARIAN GRANULOSA CELLS ABSTRACT. Gtanulosa cells in ovarian follicles of tat, mouse, rabbit and hamster were studied by lanthanum tracer and freeze-fracture techniques. Abundant gap junctions exhibited striking intraspecific variation in size and pattern of particle aggregation. The smaller gap junctions showed close packing of the inttamembtanous A face particles. In large gap junctions, tanging up to 6 p in diameter, particles were packed in rectilinear arrays separated by a labytinthine network of particle-free ‘aisles’. Small clusters of particles in a particle-poor circumferential zone suggested enlargement of junctions by peripheral accretion. Linear inttamembtanous structures, resembling those of occluding junctions, occasionally bounded large gap junctions. Spherical inttacytoplasmic structures limited by gap junctional membranes were shown by tracer studies to arise by invagination of the cell surface. These were intrepteted as a means of disposal of junctions by interiotization.
1973). Such junctions have also been observed on the boundaries between coupled cells in embryonic tissues, and in cell cultures (Johnson and Sheridan, 1971; Pinto da Silva and Gilula, 1972; Revel et al., 1973). Freeze-cleaving now permits a more detailed examination of the structure of gap junctions and other membrane specializations. Using this technique, Friend and Gilula (1972) described variations in the intramenbranous differentiation at gap junctions in several mammalian organs with respect to their number, distribution and size. Staehelin (1972) has described several variants of the gap junction in rat intestinal epithelium. Interest in the intercellular junctions of ovarian tissue stems from the belief that the specializations for cell communication may ultimately be found to play a significant role in differentiation and in the integration of cellular responses to hormonal stimulation. To date the basis for this speculation is that in the rabbit ovary, gap junctions are not present in primary follicles but appear in the early antral follicle and continue to increase in number and size as the follicle enlarges Albertini and Anderson, 1974). A similar progressive evolution of these junctions has been observed in the present study in young
Introduction cell contacts between granulosa cells have been identified in various mammalian species by several investigators studying the ultrastructure of ovarian follicles in thin sections (Byskov, 1969; Espey and Stutts, 1971; Motta et al., 1971; Zamboni, 1974). In these studies the predominant junctional specialization has been variously described as a ‘close junction’ or a ‘tight’ or ‘occluding’ Junction. Recent work employing lanthanum as an electron-opaque tracer for delineating extracellular spaces (Anderson, 1971; Merk ef al., 1973) and freeze-fracturing studies (Fletcher, 1973 ; Albertini and Anderson, 1974) have provided convincing evidence that these cell contacts are, for the most part, gap junctions or nexuses. This type of junction is believed to be the morphological basis for electrical and metabolic coupling of cells in many normal epithelial tissues (Furshpan and Potter, I968 ; Bennett, 1973 : McNutt and Weinstein, CLOSE
Department of Anatomy and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 021 IS. Received 6 Fcbtusty
1975. 389
ALBERTINI,
rats. It has also been reported that the number of such junctions is responsive to hormonal stimulation (Merk et al., 1972). In order to describe more completely the morphology of cell contacts in ovarian tissue and to clarify the types of membrane specialization present, we have studied the follicles of mice, rats, rabbits, and hamsters by electron microscopy of thin sections, by freeze fracturing and by use of lanthanum as a probe of the extracellular space. Materials and Methods Thirty-day rats and sexually mature mice, hamsters and Dutch Belted rabbits were used in this study. Follicles or pieces of follicles were dissected quickly from whole ovaries in air or in 2% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer (Karnovsky, 1965). Paraformaldehyde was omitted from the fixative used for rabbit tissue. Follicular tissue was fixed 15-25 min in the aldehyde solution, trimmed and washed in buffer, equilibrated with 30%) glycerol in buffer for l-2 hr at room temperature, frozen on paper discs in the liquid phase of Freon 22 and stored in liquid nitrogen. Freeze-fracturing and platinumcarbon shadowing were done in a Balzer apparatus (Balzers, Lichtenstein) operating at a stage temperature of - 115°C. Replicas were subsequently freed from tissue in Clorox, mounted on 300-mesh uncoated copper grids and examined with a Phillips 200 or 300 electron microscope. For the preparation of thin sections, tissue were fixed for 1 hr in the same fixatives used for freeze-cleaving. Both the aldehyde fixative and buffer washes contained 2%
FAWCETT
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lanthanum nitrate. After a I-hr incubation in 0.03 N NaOH, tissues were post-fixed in 17; 0~04, dehydrated in a graded series of ethanols and embedded in an EponAraldite mixture (Anderson and Ellis, 196.5). Thin sections were cut on a Porter-Blum MT-2 ultramicrotome, collected on 300mesh grids, and stained lightly with uranyl acetate and a mixture of lead salts (Sato, 1968) before viewing in an electron microscope. Freeze-fracturing cleaves the plasma membrane through its hydrophobic interior, exposing two complementary internal aspects of the membrane (Branton, 1966; Pinto da Silva and Branton, 1970). The outwardly directed inner half membrane or inner leaflet, rich in membrane-intercalated particles, is conventionally termed the A-face. The inwardly directed outer half membrane or outer leaflet, which contains relatively few particles, is referred to as the B-face. This terminology will be used throughout this paper. Observations The lining of the developing mammalian follicle is a stratified epithelium composed of multiple layers of granulosa cells resting upon a thick basal lamina that separates it from the surrounding theta folliculi (Fig. 1). In early antral follicles, the granulosa cells are closely packed and in contact over a large part of their surface, as in most other types of epithelium. As follicular development progresses, however, the epithelium becomes much more loosely organized. Conspicuous intercellular spaces appear and the polygonal cells gradually become stellate
Fig. 1. Early antral follicle from a macaque ovary showing the eccentric antral cavity and the granulosa cells forming a compact multilayered epithelium. Epon section. x 600. Fig. 2. In more advanced follicles the eranulosa cells near the basal lamina remain in close contact, but those in the remainder of the epithelium become very loosely organized and irregularly stellate in shape with large intercellular spaces. Thus they come to resemble mesenchyme instead of typical epithelium. The appearance of an area of cell contact such as that at the arrows is seen in a freeze-fractured specimen in Fig. 5. x 960.
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as the area of cell-to-cell contact is diminished. As a result of these changes, the granulosa layer comes to resemble mesenchyme rather than a typical epithelium (Fig. 2). Study of electron micrographs confirms that gap junctions continue to be prevalent despite the looser organization of the tissue. Freeze-fracture preparations show many gap junctions with closely packed particles on the A-face and a corresponding pattern of small pits on the B-face (Figs. 3, 4). These junctions are remarkable not only for their number but also for the variation in their packing pattern and for their very large size. Whereas the average diameter of gap junctions in other epithelia may be of the order of 0.5 p, the gap junctions of granulosa cells range in size from under 1 p to as much as 6 ,Uin diameter. Those less than 1 p in diameter found on appositional areas of similar size tend to be irregular in outline and are composed of particles and complementary pits in close hexagonal array. In studying replicas of fracture planes that cross the interface between cells, one not infrequently encounters rows of 3-5 gap junctions spaced at rather regular intervals and separated by lenticular profiles of extracellular spaces where the cell membranes diverge and then come together again (Figs. 3, 4). This suggests that there is a gap
Fig. 3. Freeze-fracture mouse ovary. Where the junctions of varying size. diverge around lenticular
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junction wherever the cell surfaces are in apposition; while between these areas of contact, there is a system of interfacial canals-a topographic relationship not entirely unlike that of the prickle-cells or keratinocytes of the epidermis except that in stratified squamous epithelium, the end-on contacts of the short cell processes are maintained mainly by desmosomes instead of gap junctions. If this interpretation is correct, then a surprising proportion of the contact surface of granulosa cells is occupied by gap junctions. The larger junctions, over 1 p in diameter, are more regular in outline and usually round or oval. The planar area of junctional specialization is often elevated some distance above the remainder of the replica suggesting that it covers the end of a short, broad process that abuts a similar process of the neighboring cell (Fig. 5). Another feature of interest in these large junctions is a marked tendency for the particles to be clustered in rectilinear arrays separated by narrow particle-free aisles (Figs. 6, 7). The particle arrays are generally composed of 3-5 rows of particles 12-25 to a row. The particles in adjacent rows may be in register or less precisely ordered (Figs. 6, 7). For reasons that are not clear, when both A- and B-face are available for comparison, the pits on the
preparation membranes Between the intercellular
of the interface between two granulosa cells in are in contact they are specialized to form gap four gap junctions shown here, the membranes spaces. x 26,000.
Fig. 4. Another example of successive gap junctions and intervening intercellular spaces, from hamster ovary. The A-face of the junctions show closely packed intramembranous particles. The B-face exhibits a corresponding pattern of shallow pits. Preparations of this kind give the impression that gap junctions occupy a large portion of the total area of cell contact. x 53,000. Fig. 5. A very large gap junction covering a slightly elevated region of cell contact between granulosa cells of rabbit ovary. Notice the complex pattern of particle aggregates separated by particle-free aisles. An area such as that enclosed in the rectangle is shown at higher magnification in Fig. 6. x 34,000. Fig. 6. A portion of the periphery of a large gap junction on a rabbit granulosa cell. Notice the relatively sparse population of intramembranous particles in the unspecialized membrane; the sharp boundary of the junction; and the labyrinthine pattern of particle-free paths between rectilinear arrays of particles. x 230,000.
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OVARIAN
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B-face are more precisely ordered than are the particles on the A-face (Figs. 16, 19). Peripheral to the large gap junctions, there is often a zone of fairly uniform width in which the concentration of A-face particles is very much lower than that found in the unspecialized areas of membrane generally (Fig. 7). Scattered in this particle-poor satellite aggregates of peripheral zone, particles of varying size are seen. The relationship of these to the main junction creates the impression that the junctions may grow by accretion of these new clusters of particles at their perimeter. Such a recruitment of particles from surrounding unspecialized areas of membrane would be consistent with the current concept of a fluid lipid bilayer permitting lateral movement of particles. This membrane intercalated impression is strengthened by the observation that in young animals in which gap junctions are just appearing or in immature animals injected with pregnant mare’s serum gonadotropin, it is not uncommon to see areas in which there are several discrete clusters of particles a short distance apart in a configuration suggesting that they may be coalescing to form a gap junction. Of less common occurrence are isolated meandering strands or ridges on the A-face like those found at the zonulae occludentes of columnar epithelia. These are of limited length, end blindly and show no consistent pattern. They are termed ‘focal tight junctions’ in the literature and have been reported in regenerating liver and various other rapidly renewing epithelia (Yee, 1973). A line of membrane fusion of such limited extent could not subserve the function of occluding or sealing the intercellular cleft and at present their significance is quite obscure. Occasionally clusters of particles of what appear to be developing gap junctions are associated with these strands or ridges (Fig. 8). Not infrequently in rabbit granulosa cells, a large and otherwise typical gap junction is bounded by a long more or less continuous strand around its periphery (Figs. IO, 1I). Similar ridges on the A-face and grooves on the B-face may extend across the gap junction dividing it into polygonal areas of various size and shape (Fig. 12). Rarely such intramembranous strands may completely circumscribe an area that contains no particle aggregates (Fig. 9).
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The very large gap junctions of rabbit granulosa cells (Fig. 5) often present a very flat surface suggesting that this specialization may confer a certain degree of stiffness upon the membrane. This does not seem to be invariably true, however, for in thin sections of lanthanum-stained hamster granulosa cells, very extensive gap junctions can be identified in regions where the boundary between cells is elaborately corrugated and interdigitated (Fig. 13). Such areas when sectioned perpendicular to the membranes show a 30 8, dense line of lanthanum that has permeated the junction, but where the plane of section is parallel or tangential to the surface, the junctional subunits can be seen in negative image outlined by lanthanum (Fig. 13). In all species studies, the granulosa cells were found to have processes that project from one cell deep into the neighboring cell, thus confirming earlier studies based upon thin sections (Espey and Stutts, 1972; Merk et al., 1972). In sections of tissue in which lanthanum is used as an electron-opaque probe of the extracellular space, these interdigitating processes appear as circular profiles clearly outlined by the tracer, thus verifying their continuity with the cell surface (Figs. 14, 15). Similar profiles that are not infiltrated by lanthanum can also be identified. These evidently represent processes that have constricted at their base and detached from the cell surface becoming spherical cytoplasmic inclusions bounded by a double membrane. These were first described as ‘sphaerae occlusae’ (Espey and Stutts, 1972) on the erroneous assumption that they were bounded by tight or occluding junctions. When these processes and inclusions are studied by freeze-cleaving, their membrane specializations are found to be typical gap junctions (Albertini and Anderson, 1974). This is found to be true in the mouse and rat, as well as in the rabbit. Furthermore, the junctional specializations of the processes and spherical inclusions exhibit the same diversity of particle patterns as is seen among the gap junctions on the cell surface-close hexagonal packing: parallel rectilinear ranks of particles separated by particle-free aisles (Figs. 17, 19); reticular arrangements of closely packed particles outlining small rounded or polygonal areas devoid of particles (Fig. 20): and intermediates between these latter two (Fig. 18).
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Fig. 7. A peripheral portion of a large gap junction from rabbit granulosa cell illustrating the rectilinear particle arrays, and intervening ‘aisles’. Notice also the particle-poor zone that often surrounds gap junctions. Several satellite aggregates of particles in this zone suggest that the large gap junctions may grow by accretion of such aggregations to their periphery. x 123,000. Fig. 8. Specimen from hamster granulosa cell. Several aggregations of gap junctional particles partially connected by rows of particles and short ridges or strands. x 141,000. Fig. 9. An strands on the epithelia. The is not known.
area of rabbit granulosa cell membrane exhibiting intramembranous A-face resembling those found in tight or occluding junctions of other significance of these intramembranous differentiations in follicular cells x 250,000.
Figs. 10 and 11. Portions of the periphery of a very large gap junction on a rabbit granulosa cell. The junction is surrounded by an extensive but discontinuous intramembranous ridge resembling one of the elements that comprise tight junctions in simple columnar epithelia. x 65,000. Fig. 12. A region of rabbit granulosa cell membrane in which tight junctional elements circumscribe rounded or polygonal units of both gap junctional and non-junctional membrane. x 97,000. Fig. 13. A thin unstained section of hamster granulosa cells showing a very extensive gap junction on an irregularly corrugated area of the cell boundary. Where the plane of section is normal to the cell membranes, a dense 30 A line of lanthanum can be seen permeating the gap junction. Where the plane of section is parallel to the membranes, a lattice of hexagonally packed particles can be seen in negative image outlined by the lanthanum. x 102,500. This is more evident at higher magnification in the two insets. x 175,000. Fig. 14. A thin unstained section of rabbit granulosa cell from a tissue sample immersed in lanthanum. The two circular profiles outlined by lanthanum are sections of cell processes of an adjacent cell. These deep invaginations are bounded by gap junctions in which one can sometimes discern parallel lines of lanthanum corresponding to the particle-free ‘aisles’ seen in freeze-cleaving preparations. x 33,000. Fig. 15. Three lanthanum outlined processes are shown here projecting lutein cell from the sixth day of pregnancy. x 24,500.
into mouse
Fig. 16. A small gap junction of a rabbit granulosa cell showing both A-face particles and B-face pits. This junction exhibits an uncommonly uniform hexagonal lattice. For reasons that are not understood, the pits often seem more highly ordered than the particles. x 250,000. Fig. 17. A gap junction from hamster of particle arrays. x 80,000.
granulosa
cell showing a parallel arrangement 1*’
Fig. 18. A gap junction of a rat granulosa cell lining an invagination occupied by a process of an adjoining cell. The pattern of junctional particles is similar to many seen on planar portions of the cell surface. x 100,000. Fig. 19. A path of cleavage through the cytoplasm of a granulosa cell encountering a deeply invaginated cell process. Parallel arrays of pits are seen on the B-face and a corresponding pattern of particles in a smaller exposure of the A-face. x 77,000. Fig. 20. A fracture through granulosa cell cytoplasm. The round concave membrane in the center is an interiorized gap junction. Its shape suggests that is is part of a hollow spherical inclusion formed by invagination and constriction of a large gap junction formerly on the surface of the cell. x 75,000.
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OVARIAN
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Discussion This study has confirmed that the commonest type of junctional specialization of the granulosa cells is the nexus or gap junction (Merk et al., 1973; Fletcher, 1973; Albertini and Anderson, 1974). It leaves unexplained the large number of these junctions found in each cell, their exceptional size (up to 6 y), the variability in the arrangement of their intramembranous particles, and their physiological significance in development and maturation of the follicle. Some discussion of these gaps in our knowledge may be useful as a stimulus to further investigation. Gap junctions are believed to play an important role in cell-to-cell communication by providing low resistance pathways for diffusion of ions (Furshpan and Potter, 1968). The occurrence of such junctions between granulosa cells might therefore be advantageous for coordinating the synthetic activities of cells throughout the follicular epithelium. Electrical coupling of the granulosa cells to each other and to the oocyte has yet to be demonstrated. It has been suggested however that these cells may be responsible for maintaining the meiotic arrest of the oocyte or for initiating its maturation. Such an effect could conceivably be mediated by junctions permitting flow of ions between processes of cells in the corona radiata and those of the oocyte where the two interdigitate in the zona pellucida. There is abundant evidence that only a few, very small gap junctions are sufficient to maintain electrical coupling of epithelial cells and little is gained by a great increase in the area of the low resistance pathways. Thus it is difficult to account for the abundance and the large size of the junctional specializations of granulosa cells on the basis of electrical coupling alone. It has been demonstrated that small molecules can also diffuse from cell to cell via gap junctions (Lowenstein, 1966; Johnson and Sheridan, 1971; Sheridan, 1971) and this may result in metabolic coupling of cells (Gilula et al.., 1972). Although ovarian follicles grow to a large size, they remain avascular and the diffusion distance from the capillaries of the theta to the cumulus oophorus is considerable. However, there would seem to be no need for
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transcellular diffusion of blood-borne nutrients or hormones for there is no permeability barrier in the ovary comparable to the blood-testis barrier. Electron-opaque tracers of large molecular size rapidly diffuse from the blood throughout the follicles via the extracellular spaces (Anderson, 1972). Nevertheless, as previously suggested (Merk et al., 1973) gap junctions might provide for uniform dissemination of intermediary metabolites, second messengers or other regulatory molecules throughout the population of granulosa cells-insuring a uniform metabolic response to circulating hormone. For such metabolic coupling a larger area of junctional membrane might be advantageous (Sheridan, 1974). Other consequences of the extensive gap junctions between granulosa cells must be considered. It was emphasized above that as follicular growth progresses, intercellular spaces become more prominent throughout the epithelium. The area of contact between the cells is thus reduced until the epithelium takes on the appearance of a stellate reticulum with the cells adhering via short processes. The abundance of gap junctions in freeze-cleaved preparations and their large size suggests that the greater part of the contact surface of adjacent granulosa cells may be specialized in this manner. It is important to bear in mind that, in addition to their function in communication, gap junctions are also sites of very tenacious adherence of cells. Whatever advantage may accrue to granulosa cells by having large areas of specialization for communication would seem to be gained at the expense of mobility. This would seem to pose a problem in that cells with extensive gap junctions could not easily change their position relative to their neighbors to allow for enlargement of the antrum or growth of the follicle as a whole. It is evident, however, that the cellular relationships do continually change during follicular growth and as ovulation approaches, a sizeable mass of cumulus cells around the oocyte is cast off from its attachments to the rest of the epithelium. In the thinning of the follicular wall and formation of the stigma, there is a marked attenuation of the epithelium that must involve either extensive passive displacement or active migration of the cells. It is not uncommon at this stage to find individual
402
follicular cells detached from the epithelium exhibiting amoeboid shape or rounded up and floating free in the follicular fluid (Parr, 1974; Motta and DiDio, 1974). Consideration of these developmental events raises the interesting question as to how the cells free themselves from their firm attachments at gap junctions in order to participate in these dynamic morphogenetic processes. This question directs attention to the deficiency in our knowledge of the life span and fate of junctional specializations. Once formed, can they be dismantled in situ? We know of no morphological evidence suggestive of disassembly of gap junctions in situ. It is known, however, that when epithelial cells are experimentally dissociated in calcium-free solutions, desmosomes separate and the half desmosomes are then interiorized and can be identified in the walls of endocytotic vacuoles in the cytoplasm (Berry and Friend, 1961; Overton, 1968). It is assumed that they are then broken down by the lysosomal system. If interiorization and lysosomal degradation are the only mechanism by which cells can free themselves of attachment at gap junctions, this may provide an explanation for the very frequent occurrence of deep invagination of junctional areas of granulosa cell membranes into neighboring cells and the abscission of these invaginations to form spherical cytoplasmic inclusions bounded by two-cell membranes that exhibit typical gap junctional specialization. These so-called ‘annular gap junctions’ or ‘sphaera occlusa’ were interpreted by Espey and Stutts (1972) as a mechanism for exchange of cytoplasm between granulosa cells. While it cannot be denied that incorporation of a small amount of cytoplasm of the adjacent cell does occur when these structures are formed, it seems to us unlikely that an ‘exchange of cytoplasm’ is their principal function. More plausible is the suggestion that interiorization may represent a means of removing immobilizing junctions from the surface (Merk et al., 1973) to permit movement of the cells in the course of the continual internal reorganization of the follicular epithelium. If this interpretation is valid, then one would expect to find interiorized junctions in greatest abundance in those periods of follicular development and luteal transformation which involve the greatest amount of cell movement.
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There is experimental evidence that this is true. The number of surface nexuses and interiorized nexuses in Graafian follicles of young rabbits has been studied before, and after, injection of human chorionic gonadotropin (Bjersing and Cajander, 1974). Following administration of the hormone, the granulosa cells became much more loosely organized. Associated with this change there was a significant decline in surface nexuses and a doubling of the number of ‘annular junctions’. Thus it seems likely that the formation of these structures is a means of interiorizing and disposing of junctional membrane. Gap junctions are maintained in large numbers throughout follicular growth despite the apparent loss of many by interiorization. Therefore we assume that as cells change position in the epithelium, new junctions are formed rapidly in newly established areas of cell contact. This continual turnover of the cell surface may account for some of the diversity in structure of gap junctions and suggests that the growing follicle may be unusually favorable material for study of the initiation and growth of gap junctions. A considerable number of the junctions examined in this study were encircled by membrane containing relatively few granules. The particle-poor region observed around gap junctions has been referred to by other authors as a ‘zone of particle exclusion’. Small clusters of particles often observed in this zone are probably destined to traverse it and to fuse with the main junctional aggregation of particles thus contributing to its growth. In ingenious experiments on reconstitution of gap junctions among reaggregating hepatoma cells, Johnson et al. (1974) found the earliest indications of nascent gap junctions were smooth, flattened areas of membrane called ‘formation plaques’ surrounding a few 90- 110 8, particles. At later times, there was an aggregation of smaller particles within the plaque into tightly adherent groups indistinguishable from small gap junctions. These rapidly enlarged by addition of individual particles and by fusion of the smaller aggregates. These findings strengthen the interpretation put forward in the present study that the satellite aggregations of particles observed in the clear zone around the large granulosa cell junctions are probably clusters of par-
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OVARIAN
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titles being recruited in the growth of the junction. If it be assumed that junctional particles arise in unspecialized areas of membrane and then aggregate at the site of a new junction, then ‘zone of particle depletion’ might be a term more appropriate for this clear zone than ‘zone of particle exclusion’. The observations reported here have demonstrated in the gap junctions of granuloss cells patterns of particle packing that are rarely seen in such junctions in other organs. The degree of variability in pattern is quite unusual, some showing rectilinear particle arrays a few rows wide separated by particlefree aisles; others consisting of loosely and irregularly packed particles, and still others exhibiting a very close precisely hexagonal packing. The possible functional correlates of these variations in pattern remain obscure. However, it is not unreasonable to speculate that they may represent different stages in development or different functional states of the junctions or both. This latter possibility becomes more credible in the light of recent experimental studies which have shown that when the gap junctions of gastric epithelium or those in the septa of crayfish axons are exposed to conditions which result in electrical uncoupling, the junctional particles become closely packed in a regular hexagonal lattice and when coupling is restored. they become more loosely and irregularly spaced (Perrachia and Dulhunty, 1974). Our thinking about the significance of gap junctions has been dominated to date by their correlation with electrical coupling. There is some danger that acceptance of all such particle aggregates on the A-face as pathways for cell-to-cell communication may close our minds to other possible interpretations. Aggregation of intramembranous particles has been reported at sites of secretory particle discharge (Satir et a/., 1973): after exposure of erythrocytes to changes of pH and temperature (Pinto da Silva, 1972): in immune reactions (Scott and Marchesi, 1972); and after physiological activation of spermatozoa (Friend and Fawcett, 1973 ; Koehler and Gaddum-Rosse, 1975). Indeed, closely packed hexagonal arrays of particles resembling gap-junctions have been reported in membranes at the free surface of transitional epithelium
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(Staehelin et al., 1972) and on the ectoderm of hydra where there can be no cell-to-cell communication (Wood, 1974). There is broad acceptance of the concept that the sites of binding of antibodies, lectins, hormones and drugs to the cell surface may be oligosaccharide moieties of glycoprotein or glycolipid constituents of the membranes. Incontestable evidence for this interpretation is difficult to obtain, but freezefracturing studies of myoneural junctions reveal distinctive, irregular rows of I I@ 140 8, intramembranous particles at the tops of the junctional folds in the postsynaptic membrane (Rash and Ellisman. 1974)-sites where immunohistochemical methods also localize acetylcholine receptor complexes (Daniels and Vogel, 1974). Thus in this case the receptors appear to he associated with intramembranous particles visible by freeze cleaving. Freed from the concept that membrane particle organization must necessarily be related to intercellular communication. one can think of other possible roles for the aggregations of particles found in the membranes of granulosa and lutein cells. For example, glycoprotein hormones of the pituitary are known to bind to the membranes of their target organs and to exert their effect via activation of adenylcyclase and the intracellular accumulation of the second messenger cyclic AMP (Butcher et al.. 1972). The fate of the bound hormone after it has exerted its effect is unclear but continuous modulation of target organ function would seem to require either a mechanism for dissociation of the hormone from its binding site or a means of local degradation of the hormone receptor complex, together with continual replacement of the receptor population in the membrane. In a foregoing section of this discussion, we expressed perplexity over the exceptional size and number of gap junctions in the granulosa cells--a profusion of particle aggregates that would seem to be far in excess of the needs for functional integration. We now offer for consideration the proposition that not all particle aggregations on the A-face are concerned with cell communication. Evidence for fluidity of the cell membrane and movement of protein particles within it is now compelling. It gonadotropic hormone receptors are indeed exposed portions of membrane intercalated
404
ALBERTINI,
particles or projecting oligosaccharides, it is conceivable that the particles representing occupied receptors may move within the plane of the membrane assembling to form large particle aggregates. These then may be interiorized by invagination and degraded within the cytoplasm while new receptors are added to the cell surface. Although such a disposal mechanism for occupied receptors may seem very far fetched, it offers an alternative explanation for the spherical vesicles bounded by gap junctional membrane that are so common in the cytoplasm
FAWCETT
AND
OLDS
of these cells. Though highly conjectural, this suggestion may have some value in keeping before investigating the possibility that not all of the varied patterns of particle aggregation seen in membranes may be concerned with the single function of cell communication. Acknowledgement
Supported by a grant from the Human Life Foundation.
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of the follicle wall and follicle cell-oocyte
Thesis. Division of Medical association.
Biol.
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