Human Reproduction vol.7 no.3 pp 391 -398, 1992

Structure, distribution and composition of the extracellular matrix of human oocytes and cumulus masses

Pramilia Dandekar1, Judith Aggeler2 and P.Talbot3 'Department of Obstetrics, Gynecology and Reproductive Medicine, M1489, University of California, School of Medicine, San Francisco, CA 94143, 2Veteran's Administration Medical Center, 4150 Clement Street No. 151E, San Francisco, CA 94121 and 'Department of Biology, University of California, Riverside, CA 92521, USA 3 To whom correspondence should be addressed

The structure, distribution and composition of the extracellular matrix present around the human oocyte and in the cumulus was examined following fixation in the presence of ruthenium red. An extracellular matrix comprising granules and filaments is present in the cumulus layer, in the corona radiata, in the outer pores of the zona pellucida and in the perivitelline space surrounding unfertilized oocytes. In replicate samples, the extracellular matrix comprised filaments which were mostly very long, occasionally crossconnected by shorter filaments, and usually decorated with numerous small granules. Enzymatic digestion with affinitypurified trypsin or Streptomyces hyaluronidase removes the granules and filaments, respectively, at all levels of the oocyte-cumulus complex. These results are interpreted to mean that protein and hyaluronic acid are present in all extracellular compartments of the human oocyte-cumulus complex. The significance of this distribution of hyaluronic acid with respect to the role of sperm hyaluronidase in fertilization is discussed. Key words: human oocyte cumulus complex/hyaluronic acid/ extracellular matrix/fertilization/hyaluronidase

Introduction Freshly ovulated mammalian oocytes are surrounded by a zona pellucida, corona radiata, and cumulus layer which together comprise the oocyte-cumulus complex. Since these vestments usually remain around the oocyte until fertilization is complete, a fertilizing spermatozoon must swim through several thick coats before reaching the perivitelline space adjacent to the oolemma. At each level of penetration, the sperm passes through an extracellular matrix. These matrices are of interest because they may, to varying degrees, present an obstacle to incoming spermatozoa (Talbot, 1985) and because they may alter sperm motility (Tesarik et al., 1990) and induce the acrosome reaction (Lenz et al., 1983; Tesarik, 1985; Meizel and Turner, 1986; Tesarik and Kopecny, 1986; Wassarman, 1988; Siiteri etai, 1988). One extracellular matrix of the oocyte, the zona pellucida, is comprised of three glycoproteins, ZP1, ZP2 and ZP3 which have been well characterized (reviewed by Wassarman, 1988). This © Oxford University Press

matrix is probably the most difficult for spermatozoa to penetrate. The matrix between the cumulus cells and the corona radiata cells has been less throughly examined, although it is known to contain hylauronic acid (Eppig, 1979; Ball et al., 1982; Talbot and DiCarlantonio, 1984a,b; Kan, 1990) and several glycoproteins (Cherr et al., 1990; Virji et al., 1990). At one time, it was thought that spermatozoa had to release hyaluronidase during the acrosome reaction to penetrate the hyaluronic acid-rich matrix of the cumulus and corona radiata. However, recent work suggests that these hyaluronic acid-rich matrices present little physical resistance to incoming spermatozoa (Talbot et al., 1985). This extracellular matrix and its relationship with spermatozoa needs further characterization. The perivitelline space around unfertilized oocytes has been shown in opossums (Talbot and DiCarlantonio, 1984c) and pigs (Kopecny et al., 1984) to contain a matrix comprised of granules and filaments following processing in the presence of ruthenium red. Apart from these studies, little is known about the extracellular matrix in the perivitelline space surrounding unfertilized mammalian oocytes. The purpose of the present investigation was to examine the ultrastructure, composition, and distribution of the extracellular matrix around unfertilized human oocytes and in cumulus masses. Because these matrices are rich in carbohydrate, they do not fix well for ultrastructural examination using conventional techniques. However, we have shown previously that the matrix of hamster and mouse oocyte—cumulus complexes (Talbot, 1984; Talbot and DiCarlantonio, 1984b) and of the opossum perivitelline space (Talbot and DiCarlantonio, 1984c) can be stabilized for ultrastructural examination if fixation is done in the presence of the polycation ruthenium red. Fixation with this protocol yields an extracellular matrix comprised of granules and filaments which have been shown, using enzymatic treatment, to represent protein and hyaluronic acid, respectively. In this study, we have examined the structure, composition and distribution of the extracellular matrix stabilized by ruthenium red in human oocytes and cumulus masses. In addition, we have made replicas of the matrix between cumulus cells for high resolution analysis. The replica technique has previously been used to study extracellular matrices in other systems, and it provides a better overview of matrix components than does conventional thin sectioning (see e.g. Furthmayr and Madri, 1982; Aggeler, 1988). Materials and methods Collection and handling of human oocytes and cumulus masses Human oocytes which failed to fertilize in vitro, as well as pieces of cumulus masses which were separated from oocyte—cumulus 391

P.Dandekar, J.Aggeler and P.Talbot

392

Extracellular matrix of the human oocyte and cumulus

complexes at the time of the follicular aspiration were obtained from an in-vitro fertilization programme. Details of the procedure have been described previously (Dandekar et al., 1990). Oocytes and cumulus masses were washed 4—6 times with cacodylate buffer and processed with ruthenium red (Talbot and DiCarlantonio, 1984c). Enzyme treatments Some human oocytes and cumulus masses were placed in 200 y\ droplets of Streptomyces hyaluronidase (50—100 IU/ml) (Calbiochem) or trypsin (10-20 IU/ml) (Worthington TRL-3) at room temperature and observed using a dissecting microscope. When evidence of an enzyme effect became apparent (e.g. the zona pellucida was partially hydrolysed in trypsin), the incubation was terminated by replacing the enzyme solution with 3 % glutaraldehyde and 0.5 % ruthenium red in 0.1 M cacodylate buffer. Additional details of this method are given in Talbot and DiCarlantonio (1984c). Electron microscopy All samples that were processed for thin sectioning were fixed for - 1.5 h in 3% glutaraldehyde plus 0.5% ruthenium red in 0.1 M calodylate buffer, pH 7.4. Oocytes and cumulus masses were washed three times in cacodylate buffer containing 0.5% ruthenium red, then post-fixed for 1 - 3 h in 1 % osmium tetroxide in 0.1 M cacodylate buffer containing 0.5 % ruthenium red. After several washes in cacodylate buffer, oocytes and cumulus masses were dehydrated in a graded series of ethanol and infiltrated and embedded in Spurr's plastic. Additional detail on the preparation of samples is given in Talbot (1985) and Talbot and DiCarlantonio (1984b). Replicas of some samples were made for examination by transmission electron microscopy. Cumulus cells that were fixed, as described above, were critical point dried from bone dry CO2, mounted in a Balzers Freeze-etch Apparatus and rotary shadowed with 5 nm of Pt—C at an angle of 25°, followed by carbon at 90° to stabilize the replica. Cleaned replicas were mounted on grids and examined in a Phillips 400 transmission electron microscope. Some cumulus samples were fixed for scanning electron microscopy in the presence of ruthenium red, using the above protocol (Talbot, 1984). After osmium treatment, samples were dehydrated in ethanol, critical point dried out of CO2, sputtered coated with 200 A of gold and examined with a Phillips 500 scanning electron microscope. Results General appearance of extracellular matrix in human oocytes and cumulus masses When human oocytes and cumulus masses were fixed in the presence of ruthenium red and examined in thin sections using

transmission electron microscopy, an extracellular matrix comprised of granules and filaments was observed in the cumulus layer (Figures 1 and 2), the corona radiata (not shown), the pores of the zona pellucida (not shown) and the perivitelline space (Figure 3). The matrix is structurally similar in all locations. Individual granules measure —42-48 nM in diameter. Both granules and filaments appeared to associate with the plasma membrane of cumulus cells (Figure 2); corona radiata cells (not shown) and the oocyte (Figure 3). Some clusters of expanded cumulus cells were fixed in the presence of ruthenium red, dehydrated, critical point dried and coated with platinum/carbon. The platinum/carbon replicas were examined using transmission electron microscopy (Figure 4). This technique gave a better high-resolution overview of the elements in the matrix. Many individual filaments were found to be very long, a feature that was not appreciated in thin sections, which reveal only a short segment of each filament. Long filaments often appeared to be interconnected by shorter filaments. Occasionally, short segments of filaments were observed which were devoid of granules; however, most filaments were covered along their entire length with small granules. Each granule, in general, touched or overlapped adjacent granules. Effects of enzymes on the granules and filaments of the extracellular matrix In the hyaluronidase experiments, the control samples of cumulus layer (Figures 5 and 7), corona radiata (not shown) and perivitelline space (Figure 9) had an abundance of granules and filaments in the extracellular matrix. In contrast, the number of filaments in the extracellular matrix of the cumulus layer (Figures 6 and 8), corona radiata (not shown) and perivitelline space (Figure 11) was greatly reduced in samples that were treated for 2 - 5 min with hyaluronidase prior to processing in ruthenium red for electron microscopy. In both control (Figures 5, 7 and 9) and experimental (Figures 6, 8 and 11) samples the abundance of granules in the extracellular matrix was similar at all levels of the oocyte—cumulus complex. The granules in hyaluronidasetreated samples (Figures 6, 8 and 11) were more aggregated than in controls, making their diameters difficult to measure accurately. In control samples, numerous granules and filaments were present in the matrix of the perivitelline space (Figure 9), corona radiata (not shown) and cumulus layer (Figure 12). Brief treatment with trypsin caused a significant reduction in the number of granules present in the perivitelline space (Figure 10) and cumulus layer (Figures 13 and 14). Moreover, the diameter of the granules which remained after trypsin treatment was significantly smaller than the diameter of control granules. In trypsin-treated samples, numerous filaments were present in the extracellular matrix at all levels of the oocyte-cumulus corn-

Figs 1—4. All samples were fixed for electron microscopy in the presence of ruthenium red. Fig. 1. Human cumulus not exposed to spermatozoa. The extracellular spaces contain a matrix comprised of granules (G) andfilaments(unlabelled arrowheads), x 15 100. Fig. 2. Human cumulus cell not exposed to spermatozoa and showing granules andfilamentsattached to the cell's plasma membrane (arrowheads). x69 500. Fig. 3. Perivitelline space (PVS) surrounding a human oocyte which failed to fertilize. The PVS contains an extracellular matrix (ECM) comprised of granules (G) and filaments (F). This matrix is indistinguishable structurally from that found in the cumulus layer, corona radiata and pores of the zona pellucida. Some of the granules and filaments attach to the oocyte microvilli (M). ZP = zona pellucida, C = cortex of oocyte. x64 000. Fig. 4. Replica of the granule/filament matrix found between cumulus cells. This sample was not exposed to spermatozoa. Mostfilamentsare quite long. Shortfilamentsappear to interconnect adjacentfilaments.The filaments are covered with numerous small granules; occasionally, short regions offilamentslack granules (arrowheads). x67 025. 393

P.Dandekar, J.Aggeler and P.Talbot

'7

394

Extracellular matrix of the human oocyte and cumulus

plex (Figures 10, 13, 14). Some filaments in trypsin-treated samples attached to the plasma membrane of cumulus cells (Figure 14) and the oocyte (Figure 10), as was also the case in controls (not shown). Discussion The main findings to emerge from this study are that the human oocyte—cumulus complex contains an extracellular matrix in the cumulus layer, corona radiata, outer pores of the zona pellucida and perivitelline space which is comprised of granules and filaments when fixed in the presence of ruthenium red. Based on enzymatic digestion, the granules appear to contain protein, while the filaments are comprised primarily of hyaluronic acid. This extracellular matrix is similar, ultrastructurally, to that found in hamster, mouse, opossum, pig extracellular matrix and hydromine rodent oocyte-cumulus complexes which have been fixed in the presence of ruthenium red (Talbot and DiCarlantonio, 1984b,c; Kopecny et al., 1984; McGregor et al., 1989). The best method for preserving the structure of the extracellular matrix in the mammalian oocyte—cumulus complex has been controversial (Phillips et al., 1990). We previously showed that ruthenium red processing for transmission electron microscopy results in some shrinkage of the matrix (Talbot and DiCarlantonio, 1984a), probably due to removal of water. In fact, the extracellular matrix of hamster oocyte-cumulus complexes shrinks during dehydration even if ruthenium red has not been used (Talbot and DiCarlantonio, 1984a). Cryofixation followed by cryosubstitution could be expected to give excellent structural preservation of the extracellular matrix. However, the technique is subject to fixation artefacts, in particular, ice crystal damage (Phillips et al., 1990), especially in very hydrated tissues, and is useful only for fixing the outer 10—20 pm of a sample (Humbel and Miiller, 1984). It would be very difficult to obtain satisfactory fixation of the perivitelline matrix using this method, as it would normally be too deep in the oocyte—cumulus complex to freeze properly, and any treatment used to remove the cumulus and corona radiata would also destabilize it. While not entirely free of artefacts, fixation of the mammalian oocyte and cumulus matrix in the presence of ruthenium red does provide certain advantages over other methods. Ruthenium red processing gives clear, useful information on the distribution of the extracellular matrix. Because it appears as a granule/filament matrix when fixed this way, it has also been possible to enzymatically analyse its granular and filamentous components. These analyses have shown that the extracellular matrix found in the cumulus layer, corona radiata, outer pores of the zona pellucida and perivitelline space are not only structurally similar, but at all locations, the granules

are comprised of protein and the filaments of hyaluronic acid. Ruthenium red processing is, in fact, the only method thus far that has been used successfully to demonstrate structurally the perivitelline extracellular matrix. In addition to the factors already mentioned, ruthenium red processing is easy to use and reproducible. New information on the structure of the granule/filament matrix was obtained by examining replicas by scanning electron microscopy. Replicas show the presence of very long, thin filaments which are sensitive to hyaluronidase, as would be expected in a matrix rich in hyaluronic acid. The length of these filaments was not appreciated in conventional thin sections, where they appear in any one section to be very short. Moreover, the replicas also reveal that granules were associated with almost all filaments. In well-preserved cryofixed extracellular matrix at the periphery of the oocyte-cumulus complex, Phillips et al. (1990) also observed filaments. The granules seen in the extracellular matrix of oocyte-cumulus complexes following fixation in the presence of ruthenium red are known by enzymatic extraction to contain protein, and may represent the glycoproteins described by others (Cherr et al, 1990; Virji et al., 1990). The glycoproteins of the extracellular matrix are probably more affected by fixation and processing than the hyaluronic acid molecules, and may not be globular in unfixed material. The granule/filament matrix observed probably originates from the cumulus and corona radiata cells. These cells have been shown by others to synthesize hyaluronic acid (Ball et al., 1982; Eppig, 1979). Moreover, the corona radiata cells send processes through the zona pellucida to the oocyte surface (Szollozi, 1967). Secretion of extracellular matrix from the tips of these processes could account for the presence of the granule/filament matrix in the perivitelline space. However, the cumulus and corona radiata may not be the exclusive source of the granule/filament matrix. Kan (1990) has provided evidence in hamsters that a matrix component is secreted by the oocyte. Moreover, in humans, Tesarik and Kopecny (1986) found that both late preovulatory oocytes and cumulus cells synthesize and secrete proteoglycans. The relative abundance of the granule/filament matrix in the perivitelline space varies among species. The matrix is sparse in hamsters and mice (Dandekar and Talbot, 1992) and was not found in the perivitelline space of hydromyine rodents (McGregor et al., 1989), where it may also be difficult to demonstrate. In contrast, the perivitelline space around human, opossum and pig oocytes has a rich matrix which is readily demonstrated by ruthenium red processing (this study; Talbot and DiCarlantonio, 1984b,c; Talbot, 1985). Even human oocytes which had been incubated with spermatozoa and, presumably, been exposed to

Fig. 5. Control human cumulus not exposed to spermatozoa or test enzymes. The granule/filament matrix between cumulus cells is intact and well preserved. x67 800. Fig. 6. Human cumulus not exposed to spermatozoa but treated for 2 - 5 min with hyaluronidase before fixing for electron microscopy. The granules of the extracellular matrix are well preserved, but filaments are rarely observed. The granules appear more aggregated than in the untreated control (Fig. 5). x90 900. Fig. 7. Replica of control human cumulus which was not exposed to spermatozoa or test enzymes. The granule/filament matrix is well preserved. X43 400. Fig. 8. Replica of a human cumulus which was not exposed to spermatozoa but was treated for 5 min with hyaluronidase before fixation for electron microscopy. Fewer filaments are present. Granules (G) are still abundant but are aggregated. X43 900.

395

P.Dandekar, J.Aggeler and P.Talbot

11

Fig. 9. Perivitelline space surrounding an oocyte which did not undergo fertilization. This oocyte was not exposed to test enzymes. The granule/filament matrix of the PVS is well defined. x51 200. Fig. 10. Perivitelline space (PVS) surrounding an oocyte which did not undergo fertilization. This oocyte was treated with trypsin for 2 - 3 min before fixation for electron microscopy. Filaments (F) are still very abundant in the PVS; however, granules (unlabelled arrowhead) are rare and those that are present are smaller in diameter than in untreated controls. ZP = zona pellucida, C = cortex of oocyte. x53 300. Fig. 11. Perivitelline space (PVS) surrounding an oocyte which did not undergo in-vitro fertilization. This oocyte was treated with hyaluronidase for 10 min before fixation for electron microscopy. The granules (arrowheads) are still abundant in the PVS; however, filaments are lacking. x52 900. 396

Extracellular matrix of the human oocvte and cumulus

\

Fig. 12. Scanning electron micrograph of the extracellular matrix in a cumulus which was not exposed to spermatozoa. The matrix comprises granules and filaments. X2800. Fig. 13. Scanning electron micrograph of cumulus similar to that in Fig. 12 except that this cumulus was treated with trypsin for 2—3 min before fixation for electron microscopy. A rich network of filaments is still present, but many of the granules have been removed by trypsin treatment. X28OO. Fig. 14. Thin section of a cumulus sample not exposed to spermatozoa but treated with trypsin for 10 min before fixation for electron microscopy. Filaments are abundant and well preserved but granules (arrowheads) are rare and reduced in diameter. x83 700.

some hyaluronidase from dead spermatozoa, retained a fair amount of extracellular matrix in the perivitelline space. The demonstration that hyaluronic acid exists at all levels of the mammalian oocvte—cumulus complex should be kept in mind when interpreting the role of the acrosomal hyaluronidase in fertilization. While it is possible that hyaluronidase facilitates penetration of the cumulus and corona radiata, several lines of evidence suggest this is not the case. First, in mice (Wassarman, 1988) and in other mammals (e.g. Cherr et al, 1986; O'Rand and Fisher, 1987; Cross et al., 1988), the zona pellucida contains an acrosome reaction-inducing factor (ZP3) suggesting that the reaction normally occurs on the zona pellucida surface. Secondly, the extracellular matrix between cumulus cells and corona radiata cells presents little physical resistance to incoming motile cells which lack hyaluronidase (Talbot et al., 1985) and, finally, motile hamster spermatozoa have been seen to penetrate to the zona pellucida surface without initiating an acrosome reaction (Cherr et al., 1986; Corselli and Talbot, 1987). When taken together, these observations suggest that acrosomal hyaluronidase is not needed for penetration of the cumulus and corona radiata. It seems probable that the same would be true for penetration of the zona.

While some hyaluronic acid-containing matrix does penetrate into the pores of the zona, the main frame-work of the zona would be a more formidable barrier to spermatozoa. Hyaluronic acid in the perivitelline space however, may be very important in fertilization, not because it provides a significant barrier to sperm penetration of the perivitelline space, but because it may inhibit gamete membrane fusion. The presence of hyaluronic acid on cell surfaces in other systems has been shown to block cell fusion (Vollet and Roth, 1974; Kujawa and Tepperman, 1983; Orkin etai, 1985). In our micrographs, the granule/filament matrix appeared to attach directly to the oolemma. It may be necessary for spermatozoa to remove this thin coating of hyaluronic acid to gain sufficient proximity to the oocyte to fuse. It should also be noted that hyaluronic acid coats the surfaces of cumulus cells, and this coating could prevent premature fusion of spermatozoa with an inappropriate cell type. Acrosomal hyaluronidase could gain access to the perivitelline space by diffusing across the zona pellucida following the acrosome reaction or, more likely, it may be carried into the perivitelline space with the acrosome-reacted spermatozoa. Several investigators have shown that spermatozoa 397

P.Dandekar, J.Aggeler and P.Talbot

retain a significant complement of hyaluronidase following the normal acrosome reaction (Brown, 1975; Morton, 1976; Harrison, 1988). The function of sperm hyaluronidase in mammalian fertilization is not currently understood. However, its substrate, hyaluronic acid, is now known to be present at all levels of the oocyte-cumulus complex and this may be helpful in future analyses of acrosomal enzyme function. Acknowledgements We thank Dr Mary Martin and Dr Robert H.Glass for providing resources for this project through the Human In Vitro Fertilization Program at the University of California, San Francisco, and for their helpful suggestions regarding the manuscript. We also thank David Demers, Vanessa Hsieh and Jocelyn Wu for their assistance in preparing the plates. This research is supported in part by a grant from the Academic Senate.

References Aggeler,J. (1988) Three-dimensional organization of the extracellular matrix secreted by cultured rat smooth muscle cells. In Vitro Cell. Dev. Biol, 24, 633-638. Ball,G.D., Bellin.M.E., Ax,R.L. and First,N.L. (1982) Glycosaminoglycans in bovine cumulus-oocyte complexes: Morphology and chemistry. Mol. Cell. Endocrinoi, 28, 113-122. Brown,C.R. (1975) Distribution of hyaluronidase in the ram spermatozoon. J. Reprod. FertiL, 45, 537-539. Cherr.G.N., Lambert,H., Meizel.S. and Katz.D.F. (1986) In vitro studies of the golden hamster sperm acrosome reaction: Completion on the zona pellucida and induction by homologous soluble zona pellucidae. Dev. Biol., 114, 119-131. Cherr,G.N., Yudin.A.I. and Katz,D.F. (1990) Organization of the hamster cumulus extracellular matrix: A hyaluronate-glycoprotein gel which modulates sperm access to the oocyte. Dev. Growth Differ., 32, 353-365. Corselli.J. and Talbot,P. (1987) In vitro penetration of hamster oocytecumulus complexes using physiological numbers of sperm. Dev. Biol., 122, 227-242. Cross,N.L., Morales,P., Overstreet,J.W. and Hansen.F.W. (1988) Induction of acrosome reactions by human zona pellucida. Biol. Reprod., 38, 235-244. Dandekar.P. and Talbot,P. (1992) The perivitelline space of mammalian oocytes: extracellular matrix of unfertilized oocytes and the formation of a cortical granule envelope following fertilization, in press. Dandekar,P., Martin.M.C. and Glass,R.G. (1990) Polypronuclear embryos after in vitro fertilization. Fertil. Steril., 53, 510-514. Eppig.J.J. (1979) FSH stimulates hyaluronic acid synthesis by oocytecumulus cell complexes from mouse preovulatory follicles. Nature, 281, 483-484. Furthmayr.H. and Madri.J.A. (1982) Rotary shadowing of connective tissue macromolecules. Collagen Relat. Res., 2, 349-363. Harrison,R.A.P. (1988) Hyaluronidase in ram semen: quantitative determination, and isolation of multiple forms. Biochem. J., 252, 865-874. Humbel,B. and Miiller,M. (1984) Freeze substitution and low temperature embedding. In Csanady,A., Ro!ich,P. and Szabo,O. (eds), Proceedings 8th European Congress of Electron Microscopy, Budapest, 3, pp. 1789-1798. Kan,F.W.K. (1990) High-resolution localization of hyaluronic acid in the golden hamster oocyte-cumulus complex by use of a hyaluronidasegold complex. Anat. Res., 228, 370-383. Kopecny,V., Flechon,J.E.. Motlik.J. and Pivko.J. (1984) Glycosaminoglycan synthesis by pig oocytes during meiotic 398 maturation: Fine structure autoradiography and cytochemistry.

Abstract, 8th European Congress on Electron Microscopy, Budapest. Kujawa,M.J. and Tepperman,K. (1983) Culturing chick muscle cells on glycosaminoglycan substrates: attachment and differentiation. Dev. Biol, 99, 277-286. Lenz.R.W., Bellin.M.E. and Ax,R.L. (1983) Rabbit spermatozoa undergo an acrosome reaction in the presence of gycosaminoglycans. Gamete Res., 8, 11-19. McGregor.L., Flaherty.S.P. and Breed,W.G. (1989) Structure of the zona pellucida and cumulus oophorus in three species of native Australian rodents. Gamete Res., 23, 279-287. Meizel,S. and Turner,K.O. (1986) Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm. J. Exp. Zool., 237, 137-139. Morton,D.B. (1976) Lysosomal enzymes in mammalian spermatozoa. In Dingle,J.D. and Dean,R.T. (eds), Lysosomes in Biology and Pathology, Vol. 5. Elsevier/North-Holland, Amsterdam, pp. 203-255. O'Rand,M.G. and Fisher,S.J. (1987) Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae. Dev. Biol., 119, 551-559. Orkin.R.W., Knudson.W. and Toole,B.P. (1985) Loss of hyaluronatedependent coat during myoblast fusion. Dev. Biol., 107, 527-530. Phillips.D.M., Zacharopoulos.V.R. and Perotti.M.E. (1990) Structure of the cumulus oophorus at the time of fertilization. Cell Tissue Res., 261, 249-259. Siiteri,J., Dandekar.P. and Meizel,S. (1988) Human sperm acrosomeinitiating activity associated with the human cumulus oophorus and murine granulosa cells. J. Exp. Zool., 246, 71 — 80. Szollosi,D. (1967) Development of cortical granules and the cortical reaction in rat and hamster eggs. Anat. Rec, 159, 431—446. Talbot,P. (1984) Hyaluronidase dissolves a component of the hamster zona pellucida. J. Exp. Zool., 229, 309—316. Talbot.P. (1985) Sperm penetration through oocyte investment in mammals. Am. J. Anat., 174, 331—346. Talbot.P. and DiCarlantonio.G. (1984a) The architecture of the hamster oocyte-cumulus complex. Gamete Res., 9, 261—272. Talbot,P. and DiCarlantonio,G. (1984b) The oocyte-cumulus complex: Ultrastructure of the extracellular matrix in hamster and mice. Gamete Res., 10, 127-142. Talbot.P. and DiCarlantonio.G. (1984c) Ultrastructure of opossum oocyte investing coats and their sensitivity to trypsin and hyaluronidase. Dev. Biol, 103, 159-167. Talbot.P., DiCarlantonio.G., Zao,P., Penkala.J. and Haimo.L.T. (1985) Motile cells lacking hyaluronidase can penetrate the hamster oocyte cumulus complex. Dev. Biol, 108, 387-398. Tesarik,J. (1985) Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid, and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro. Reprod. Fertil, 74, 383-388. Tesarik.J. and Kopecny,V. (1986) Late preovulatory synthesis of proteoglycans by the human oocyte and cumulus cells and their secretion into the oocyte—cumulus-complex extracellular matrices. Histochemistry, 85, 523-528. Tesarik,J., Testart,J., Leca.G. and Nome.F. (1990) Reversible inhibition of fertility in mice by passive immunization with anticumulus oophorus antibodies. Biol. Reprod., 43, 385—391. Virji.N., Phillips.D.M. and Dunbar.B.S. (1990) Identification of extracellular proteins in the rat cumulus oophorus. Mol. Reprod. Dev., 25, 339-344. Vollet.J.J. and Roth.L.E. (1974) Cell fusion by nascent membrane induction and divalent cation treatment. Cryobiologie, 9, 249—262. Wassarman,P.M. (1988) Zona pellucida glycoproteins. Annu. Rev. Biochem., 57, 415-442. Received on August 19, 1991; accepted on October 30, 1991

Structure, distribution and composition of the extracellular matrix of human oocytes and cumulus masses.

The structure, distribution and composition of the extracellular matrix present around the human oocyte and in the cumulus was examined following fixa...
4MB Sizes 0 Downloads 0 Views