The oocyte-cumulus complex: Ultrastructure of the extracellular components in hamsters and mice

To enhance preservation of the extracellular materials, we have fixed hamster and mouse oocyte cumulus complexes (OCC) for transmission electron microscopy in the presence of ruthenium red. Ruthenium red had four effects on the extracellular components of the freshly ovulated hamster OCC. It interacted with the surface of cumulus and corona radiata cells; it stabilized the extracellular matrix (ECM) that was comprised of granules and filaments; it produced moderate electron density and good structural definition in the zona pellucida, and it revealed occasional smalls granular depsits on the oolemma. The ECM observed between cells of the cumulus and corona radiata layers extended into the outer one third of the zona pellucida. The granule and filament matrix was removed from the cumulus layer, corona radiata, and pores of the zona pellucida by brief treatment with hyaluronidase. The extracellular components of oviducal OCC from hamsters and mice appeared similar to OCC removed from follicles of the hamster shortly before ovulation. However, oviducal OCC did show increased aggregation of granules in the ECM. In most cases where females had been mated and oocytes were fertilized, the extracellular components appeared similar to those seen in fresh OCC. Exceptions were noted in some oocytes that lacked cumulus and corona radiata cells. In these instances, the zona pellucida generally lacked the granule/filament matrix. After fertilization numerous small electrondense granules were noted in the perivitelline space. These were presumed to originate in the cortical granules and formed a new investing layer around the zygote. Our data suggest that the OCC becomes more difficult for a sperm to penetrate as it approaches the oocyte. The significance of these results is discussed with respect to sperm traffic in the OCC and the cortical reaction.     

Motile cells lacking hyaluronidase can penetrate the hamster oocyte cumulus complex

Sperm hyaluronidase is thought to assist in penetration of the extracellular matrix (ECM) between the cumulus and corona radiata cells surrounding mammalian oocytes. The question was asked: Can motile cells which lack hyaluronidase penetrate the hamster oocyte cumulus complex (OCC)? Sea urchin (Strongylocentrotus purpuratus) and frog (Rana catesbeiana) sperm and the unicellular, biflagellated, green alga Chlamydomonas reinhardtii were extracted and found to contain no hyaluronidase activity. Moreover, none of these cells was able to disperse the cumulus cells of hamster OCC, nor did they affect the ultrastructure of the ECM between cells. Fresh hamster OCC were challenged with suspensions of each cell type. Frog and sea urchin sperm penetrated to the zona pellucida surface in less than 5 min. A cell wall-less mutant of Chlamydomonas also penetrated to the zona surface but required longer than 5 min. Wild-type Chlamydomonas penetrated only halfway to the zona, perhaps because its cell wall adhered to the ECM between the cumulus cells and retarded its movement. The motility of the frog and sea urchin sperm was not affected by the ECM of the OCC. Frog sperm exhibited slow lethargic motility yet had no difficulty penetrating to the zona; this indicates that hyperactivated motility is not required for penetration of the ECM. None of the challenge cells penetrated the zona pellucida, although the frog sperm did compress the weave of the zona. These data show that motile cells which lack the enzyme hyaluronidase can readily penetrate the ECM of the hamster cumulus and corona radiata and suggest that the significance of hyaluronidase in fertilization should be reevaluated.     

In vitro penetration of hamster oocyte-cumulus complexes using physiological numbers of sperm

We have compared the ability of uncapacitated, capacitated acrosome intact, and acrosome-reacted hamster sperm to penetrate the cumulus and corona radiata of fresh hamster oocyte-cumulus complexes (OCC) in vitro. This was done using physiological numbers (1-20) of sperm so that cumulus and corona radiata cells did not disperse during challenge. Uncapacitated sperm did not penetrate to the zona pellucida surface; most (74%) uncapacitated sperm bound to cumulus cells at the periphery of the OCC. Capacitated acrosome-intact sperm penetrated to the zona pellucida surface; a significant percentage of these sperm arrived at the zona pellucida without showing evidence of initiating an acrosome reaction. Most capacitated acrosome-reacted sperm did not enter the extracellular matrix between cumulus and corona radiata cells; those which did penetrated to the zona surface with difficulty, if at all. These results suggest that the changes which occur in the sperm surface during capacitation are more important than the acrosome reaction in enabling hamster sperm to penetrate the cumulus and corona radiata. The effects of gold sodium thiomalate (GST) and polyphloretin phosphate (PPP) (inhibitors of hyaluronidase) on penetration of the OCC by capacitated sperm were also examined. Both synthetic inhibitors blocked sperm penetration to the zona pellucida, but the effective concentrations of inhibitors were far in excess of what was needed to block hyaluronidase activity. Reasons for concluding that the action of these inhibitors is nonspecific are discussed. These data show that hamster sperm with intact acrosomes can penetrate the cumulus and corona radiata cell layers of fresh OCC in vitro and support the hypothesis that the acrosome reaction occurs on the zona pellucida surface.     

Ultrastructure of opossum oocyte investing coats and their sensitivity to trypsin and hyaluronidase

Ovulated opossum oocytes are surrounded by a zona pellucida, but not by cumulus cells. Opossum sperm carry at least four acrosomal hydrolases (hyaluronidase, acrosin, N-acetylhexosaminidase, and arylsulfatase); the functions of these enzymes in opossum fertilization are uncertain. To identify possible substrates for these hydrolases, the ultrastructure of opossum oocytes was examined after fixation in the presence of ruthenium red which stabilizes extracellular matrices. This oocyte is unusual in having a wide perivitelline space containing a highly structured extracellular matrix (ECM). The ECM is comprised of granules and filaments, and it resembles matrices known to contain hyaluronic acid in other systems. Hydrolases, known to be present in opossum acrosomes, were tested for their effect on the ultrastructure of the zona pellucida and matrix of the perivitelline space. Trypsin dissolved the zona pellucida and decreased the size of the granules in the perivitelline space. Streptomyces hyaluronidase, which specifically attacks hyaluronic acid, removed only matrix filaments. Arylsulfatase, N-acetylhexosaminidase, and beta-glucuronidase did not affect the zona pellucida or ECM in our assay. These observations are consistent with the ideas that (1) opossum sperm must penetrate two oocyte investments, the zona pellucida and ECM of the perivitelline space; (2) the ECM contains hyaluronic acid (filaments) and protein (granules); (3) opossum sperm acrosin may function in penetration of the zona pellucida and ECM; and (4) opossum sperm hyaluronidase may function in penetration of the ECM by degrading hyaluronic acid (filaments). Dissolution of the granules and filaments from oocyte microvilli is probably necessary to permit close apposition and fusion of the sperm and oocyte membranes. The evolutionary significance of these results is discussed.     

Architecture of the hamster oocyte-cumulus complex

The structure of the hamster oocyte-cumulus complex (OCC) has been analyzed to help understand how mammalian sperm penetrate the investing coats of the oocyte. At ovulation, oocytes of most mammalian species are surrounded by a zona pellucida, corona radiata, and cumulus layer. Cells of the cumulus and corona radiata are separated by an extracellular matrix (ECM) that contains hyaluronic acid. In hamsters, the diameter of mature follicular OCCs is 0.61 ± 0.12 mm, whereas in freshly ovulated OCCs in culture medium it is 0.78 ± 0.15 mm. This indicates the OCC expands at or after ovulation. The corona radiata is 1–4 cell layers deep, and corona radiata cells are closely packed (center-to-center distances between adjacent cells in follicular OCCs averaged 14 ± 3 μm). The cumulus layer is 5–8 cell layers deep, and intercellular spaces are much larger (center-to-center distances between adjacent cumulus cells in follicular OCCs averaged 50 ± 20 μm). An ovulated OCC has an approximate volume of 248 × 10⁶ μm³ and was estimated to contain approximately 5,700 cells. Studies with stretched OCCs show that ink particles can readily penetrate the extracellular spaces of the cumulus and corona radiata layers and interact with the ECM, staining it black. At the periphery of the OCC, the ECM appeared discontinuous and formed “cords” containing cumulus cells, whereas closer to the corona radiata the matrix completely filled intercellular spaces. Ink penetrated the corona radiata, but the ECM was difficult to visualize because of the close packing of cells in this layer. Our observations on unfixed OCCs show that cell packing becomes more dense proceeding from the periphery of the OCC to the zona pellucida and that the ECM at the periphery differs from the matrix deeper in the OCC. These data suggest that an incoming sperm would find penetration of the investing coats to increase in difficulty (that is physical resistance would increase) as it got closer to the oocyte. We have also examined the effects of processing for microscopy on the structure of the OCC. Both standard fixation procedures and fixation in the presence of ruthenium red caused condensation of the ECM, especially in the cumulus layer, and thus produced a significant decrease in the diameter of the OCC.     

An in vitro technique to study penetration of hamster oocyte-cumulus complexes by using physiological numbers of sperm

We have developed an inexpensive in vitro system for studying cumulus penetration and fertilization by using physiological numbers of sperm. This system simulates conditions believed to exist in vivo more closely than any in current usage. In this system, 1–100 hamster sperm are used to challenge fresh hamster oocyte-cumulus complexes (OCC). Only fresh (nonoviducal) OCC are used, as they present the most stringent challenge to sperm. Because sperm numbers are low, OCC do not disperse, and sperm can be studied microscopically during penetration of the cumulus oophorus and corona radiata. These conditions permit microscopic assessment of the sperm acrosome. Video tapes of experiments allow easy review and analysis of experiments. Results obtained employing this technique show that, in vitro, (1) capacitated, acrosome-intact hamster sperm can penetrate the extracellular matrix between cumulus cells and bind to the zona pellucida; (2) the “figure-eight motility” characteristic of hyperactivated hamster sperm swimming in culture medium is suppressed when sperm swim in the extracellular matrix between cumulus cells; and (3) fertilization occurs in capillary tubes when low numbers of sperm are used. The in vitro system that we have described will be useful in analyzing the mechanisms used by sperm to penetrate the cumulus and corona radiata and to clarify the role of the acrosomal enzymes in fertilization.     

The extracellular matrix of porcine mature oocytes: Origin,composition and presumptive roles

The extracellular matrix (ECM) of porcine mature oocytes was revealed by transmission electron microscopy (TEM) after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS) and on the surface of the zona pellucida (ZP), it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells) or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine – precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA) bound to proteoglycans – for various times (with or without chase) and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose) and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI) and metaphase II (MII) and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of ³H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted ³H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a favourable factor for sperm penetration.     

Sperm penetration through oocyte investments in mammals

Literature on the interactions between eutherian gametes is reviewed. The oocyte cumulus complex of the female is surrounded by a zona pellucida, corona radiata, and cumulus layer. Sperm undergo an acrosome reaction before penetrating the zona pellucida. The morphological consequences of this reaction and its possible role(s) in penetration of the oocyte cumulus complex are considered. The acrosomal enzyme, hyaluronidase, has been thought to aid sperm in penetrating the cumulus layer and corona radiata. Several recent investigations, including one that shows that motile cells lacking hyaluronidase can penetrate to the zona surface, do not support this idea. Other possible roles of this enzyme in fertilization are discussed. The development of in vitro fertilization systems that employ physiological numbers (1-100) of sperm will be valuable in studying the mechanisms used by sperm to penetrate the oocyte cumulus complex.     

A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg

A typical mammalian egg is surrounded by an outer layer of about 3,000 cumulus cells embedded in an extracellular matrix rich in hyaluronic acid. A current, widely proposed model is that the fertilizing sperm, while it is acrosome intact, passes through the cumulus cell layer and binds to the egg zona pellucida. This current model lacks a well- supported explanation for how sperm penetrate the cumulus layer. We report that the sperm protein PH-20 has a hyaluronidase activity and is present on the plasma membrane of mouse and human sperm. Brief treatment with purified, recombinant PH-20 can release all the cumulus cells surrounding mouse eggs. Acrosome intact mouse sperm incubated with anti-PH-20 antibodies can not pass through the cumulus layer and thus can not reach the zona pellucida. These results, indicating that PH-20 enables acrosome intact sperm to penetrate the cumulus barrier, reveal a mechanism for cumulus penetration, and thus provide the missing element in the current model.     

Cellular associations in the rat oocyte-cumulus cell complex: Morphology and ovulatory changes

Cumulus-oocyte complexes were isolated from the follicles of proestrous rats or from the oviducal ampullae of estrous rats. The zona pellucida of some complexes was dissolved before fixation. The follicular cumulus cells were seen to be held together mainly by long processes, which often extended over a distance of several cells. Large numbers of straight processes from the corona radiata cells, passing to the oocyte, surface, were seen in the space formerly occupied by the zona pellucida. Oocyte microvilli were uniformly short; none traversed the zona. The postovulatory complexes were covered by amorphous extracellular material which also filled the spaces between the cells. By lysis of this material with hyaluronidase the cumulus cells were detached. The surfaces of these cells were covered with blebs. By testing the ability of hyaluronidase to remove the corona cells from the zona pellucida of complexes isolated around the time of ovulation, it was found that the completion of retraction of the corona cells processes occurred in the oviduct, immediately after ovulation. It is suggested that the oviducal environment may influence the final step of the withdrawal of the corona cells' projections from the zona pellucida.     

Structure of the cumulus matrix and zona pellucida in the golden hamster: A new view of sperm interaction with oocyte-associated extracellular matrices

Summary Hamster oocyte-cumulus complexes (OCC), with and without sperm, were structurally analyzed by light- and electron microscopy using freeze substitution. This method has yielded a clear picture of the extracellular oocyte investments, the cumulus cell matrix and the zona pellucida. The cumulus matrix has an overall homogeneous fibrillar structure which appears to attach to cumulus cells at their filopodial extensions. The matrix also extends into the outer regions of the zona pellucida. The zona pellucida has a distinct porous configuration throughout its entire structure. During gamete interaction experiments, capacitated hamster sperm with ultrastructurally intact acrosomes were found throughout the matrix. Sperm had dramatic effects on the matrix, resulting in compression and stretching. Sperm found on the zona pellucida had initiated or completed the acrosome reaction. During the initial stages of the acrosome reaction, the matrix was in contact with the sperm. At later stages of the acrosome reaction, there was a complete loss of matrix material in regions near the sperm.     

The dual functions of GPI-anchored PH-20: hyaluronidase and intracellular signaling

The ovulated mammalian oocyte is surrounded by the "cumulus ECM", composed of cells embedded in an extracellular matrix that is rich in hyaluronic acid (HA). The cumulus ECM is a viscoelastic gel that sperm must traverse prior to fertilization. Mammalian sperm have a GPI-anchored hyaluronidase which is known as PH-20 and also as SPAM 1. PH-20 is located on the sperm surface, and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. PH-20 appears to be a multifunctional protein; it is a hyaluronidase, a receptor for HA-induced cell signaling, and a receptor for the zona pellucida surrounding the oocyte. The zona pellucida recognition function of PH-20 was discovered first. This function is ascribed to the inner acrosomal membrane PH-20, which appears to differ biochemically from the PH-20 on the sperm surface. Later, when bee venom hyaluronidase was cloned, a marked cDNA sequence homology with PH-20 was recognized, and it is now apparent that PH-20 is the hyaluronidase of mammalian sperm. PH-20 is unique among the hyaluronidases in that it has enzyme activity at both acid and neutral pH, and these activities appear to involve two different domains in the protein. The neutral enzyme activity of plasma membrane PH-20 is responsible for local degradation of the cumulus ECM during sperm penetration. Plasma membrane PH-20 mediates HA-induced sperm signaling via a HA binding domain that is separate from the hyaluronidase domains. This signaling is associated with an increase in intracellular calcium and as a consequence, the responsiveness of sperm to induction of the acrosome reaction by the zona pellucida is increased. There is extensive evidence that GPI-anchored proteins are involved in signal transduction initiated by a diverse group of cell surface receptors. GPI-anchored proteins involved in signaling are often associated with signaling proteins bound to the cytoplasmic leaflet of the plasma membrane, typically Src family, non-receptor protein tyrosine kinases. PH-20 appears to initiate intracellular signaling by aggregating in the plasma membrane, and a 92-kDa protein may be the cell signaling molecule linked to PH-20.     

Quantitative and qualitative cytochemical analysis of changes in polysaccharides-proteins in rat ovulable and atretic ovarian follicles during the estrous cycle

Summary The glycosaminoglycan (periodic acid — Schiff, PAS) and hyaluronic acid (alcian blue) content of the membrana granulosa, zona pellucida and antrum of rat ovarian follicles was analyzed qualitatively and quantitatively during the estrous cycle in three types of follicles: ovulable, early atretic and late atretic. The qualitative analysis consisted of the conjunctive localization of PAS-reactive, fluorescent granules within the membrana granulosa. The quantitative analysis consisted of microdensitometric measurements of PAS and alcian blue staining within the zona pellucida and antrum of the ovulable and atretic follicles. For the localization of PAS granules within the granulosa cells, ovaries were removed on the day of proestrus, fixed in 6% paraformaldehyde, embedded in methacrylate and sectioned. Following the examination of the cells for fluorescence, the same section was stained with PAS and lead-hematoxylin. In ovulable follicles there was no fluorescence in the membrana granulosa while PAS granules occurred exclusively within the cells of the cumulus and corona radiata. In late atretic follicles, fluorescent-PAS reactive granules were located in the granulosa cells at the periphery of the follicle. During early atresia no fluorescence and very few PAS granules were observed in the granulosa cells. Since fluorescence is a marker for some lysosomes, these observations suggest that the PAS granules in the ovulable follicles may not be a type of lysosome. The amount of stain in the zona pellucida and antrum of the three follicular types was quantified using a scanning and integrating microdensitometer. On all days of the estrous cycle, PAS intensity was higher in the zona pellucida than in the antrum of the three follicular types. PAS staining in the respective antra was the same on all days of the estrous cycle. Intrafollicular PAS staining in the zonae pellucidae differed during the cycle. With respect to the zonae pellucidae, staining intensity in the three follicles was identical on estrus. On diestrus-1, staining intensity was the same in the ovulable and early atretic follicles and less in the late atretic follicle. By diestrus-2 and on proestrus, PAS intensity was highest in the zona pellucida of the ovulable follicle and less in the zona pellucida of both types of atretic follicle. In contrast to this pattern of staining, alcian blue staining intensity was identical in the zona pellucida of all follicles throughout the cycle. There was no difference in intra-antral alcian blue staining intensity on estrus and diestrus-2. On diestrus-1 and proestrus, staining intensity was greater in the antrum of the late atretic follicle than in the antra of the other follicular types. These studies indicate that glycosaminoglycan content is greater in the zona pellucida of the ovulable follicle of the rat on the last two days preceding ovulation than in the zona pellucida of either the early or late atretic follicles. In contrast, hyaluronic acid content remains constant in the zona pellucida of the three follicular types throughout the estrous cycle. These studies also give the first indication that, in the rat, the localization of PAS granules exclusively in the cumulus oophorus and corona radiata may be used to identify ovulable follicles.This work was supported by a research grant from the National Institute of Child Health and Human Development, HD-12684     

Structure of the zona pellucida and cumulus oophorus in three species of native Australian rodents

The sperm head of many Australian hydromyine rodents has three curved hooks projecting from its anterior margin; the structure of the hooks has been characterized, but their function is unknown. In this study, we have investigated whether the hooks might have evolved to assist sperm penetration through more formidable egg vestments, particularly the zona pellucida. Cumulus-oocyte complexes were obtained from two species that possess a three-hooked sperm head (Pseudomys australis and P. nanus) and one species that does not (Notomys alexis) and examined by light and electron microscopy. After fixation in the presence of ruthenium red, the zona pellucida was found to consist of a fibrillar meshwork, but there were no interspecific structural differences. A corona radiata was absent, and the cumulus extracellular matrix was composed of filaments and electron-dense granules in each species. Measurements of the zona thickness in freshly ovulated, unfixed oocytes revealed that it was thinnest (7.8 μm) in P. australis. Which has a three-hooked sperm head, and thickest (11.4 μm) in N. alexis, the species in which the ventral hooks are absent. Hence, no correlation was found between the thickness of the zona pellucida or the structure of the cumulus-oocyte complex, and the presence of three hooks on the sperm head. We conclude, therefore, that it is unlikely that the evolution of the three-hooked sperm head is an adaptation for penetration of increased barriers around the oocyte.     

Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg

The function of glycosylphosphatidylinositol-anchored sperm hyaluronidase PH-20 in fertilization has long been believed to enable acrosome-intact sperm to pass through the layer of cumulus cells and reach the egg zona pellucida. In this study, we have produced mice carrying a null mutation in the PH-20 gene using homologous recombination. Despite the absence of sperm PH-20, the mutant male mice were still fertile. In vitro fertilization assays showed that mouse sperm lacking PH-20 possess a reduced ability to disperse cumulus cells from the cumulus mass, resulting in delayed fertilization solely at the early stages after insemination. Moreover, SDS-PAGE of sperm extracts and subsequent Western blot analysis revealed the presence of other hyaluronidase(s), except PH-20, presumably within the acrosome of mouse sperm. These data provide evidence that PH-20 is not essential for fertilization, at least in the mouse, suggesting that the other hyaluronidase(s) may play an important role in sperm penetration through the cumulus cell layer and/or the egg zona pellucida, possibly in cooperation with PH-20, although the importance of sperm motility cannot be neglected.     

Perivitelline space of mammalian oocytes: extracellular matrix of unfertilized oocytes and formation of a cortical granule envelope following fertilization.

Extracellular matrices (ECM) present around unfertilized and fertilized mammalian oocytes were studied ultrastructurally in samples prepared in the presence of ruthenium red to facilitate stabilization of extracellular materials. Unfertilized mouse, hamster, and human oocytes have an ECM comprising granules and filaments in their perivitelline spaces (PVS). This matrix is more abundant in the human than in hamsters and mice. The granule/filament matrix appears identical to the matrix seen between cumulus and corona radiata cells following ruthenium red processing and previously shown to comprise protein and hyaluronic acid. By including ruthenium red during fixation, it is possible to demonstrate the existence of cortical granule exudate in the PVS of fertilized oocytes from hamsters, mice, and humans. Much of the cortical granule exudate is trapped in the PVS and forms a new coat around the fertilized oocyte. This material is particulate when stained with ruthenium red and appears to be uniformly dispersed around the entire oocyte surface. We refer to this new coat as the cortical granule envelope. This envelope is observed in the PVS of all developmental stages up to and including blastocysts in all three species. Following hatching of mouse and hamster blastocysts, the cortical granule envelope is no longer present. Possible functions of this envelope are discussed.     

In vitro studies of the golden hamster sperm acrosome reaction: completion on the zona pellucida and induction by homologous soluble zonae pellucidae

We have studied the occurrence of the golden hamster sperm acrosome reaction (AR) in vitro during interaction with the oocyte investments: the cumulus cell matrix and the zona pellucida. Hamster sperm were capacitated in a defined medium that does not induce the AR. These spermatozoa were allowed to interact with the ovum vestments, the events of which were recorded using high-speed videomicrography. Frame-by-frame analysis revealed that sperm did not complete the AR in the cumulus cell matrix, but did so on the zona pellucida. Furthermore, a higher percentage of sperm completed the AR on the zona pellucida of cumulus-invested than on cumulus-free eggs. We also investigated the effect of solubilized hamster and mouse zonae pellucidae on the hamster sperm AR. Addition of solubilized hamster zonae to capacitated sperm elicited the AR within 15 min. Solubilized mouse zonae were significantly less effective, indicating that the zona-induced AR in hamster sperm may be species specific. These results suggest that the hamster zona pellucida is an inducer of the AR in the intact or soluble form, and that the majority of spermatozoa traverse the cumulus cell matrix without completing the AR in our in vitro system.     

Ultrastructural evidence for an association between an oviductal glycoprotein and the zona pellucida of the golden hamster egg

The ultrastructural localization of an oviductal glycoprotein, designated ZP-0 in golden hamster oviductal eggs, was investigated by immunolabeling methods using a monoclonal antibody (C11E8). Immunofluorescence staining showed that C11E8 specifically reacted with the zona pellucida of the oviductal egg but not the ovarian egg. In an immunoelectron microscopic study applying the protein-A gold technique, gold particles were distributed throughout the zona pellucida of the oviductal eggs and were also associated with the perivitelline matrix. Structures within the eggs and cumulus cells did not react with C11E8. Quantitative evaluations of the degree of labeling demonstrated that a large number of gold particles was bound to the zone pellucida, especially in the middle layer. Moreover, in bovine testicular hyaluronidase-treated eggs the density of labeling decreased only in the outer third of the zona pellucida. These results show that ZP-0 to the was associated with the zona pellucida and perivitelline matrix of the golden hamster egg after ovulation and suggest that there are topographical differences in the binding activity of ZP-0 to the zona pellucida. In addition, the decrease in labeling density of ZP-O induced by hyaluronidase appears to be related to changes in the properties of the outer layer of the zona pellucida.     

Sperm penetration through cumulus mass and zona pellucida

Mammalian fertilization requires sperm to penetrate the cumulus mass and egg zona pellucida prior to fusion with the egg. Although sperm penetration through these physical barriers is essential, the molecular mechanism has not yet been completely elucidated. In addition to sperm motility, hyaluronan-hydrolyzing and proteolytic enzymes of sperm have been suggested to participate in the penetration events. Here we focus on the functional roles of hyaluronidase and protease in sperm passage through the cumulus mass and zona pellucida.     

Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm

The glycosaminoglycans (GAGs) hyaluronic acid and heparin were added (10 micrograms and 100 micrograms/ml to golden hamster sperm suspensions previously incubated for 4.5 h under capacitating conditions. After additions, sperm were incubated for 5-15 min and acrosome reactions (AR) assayed in motile sperm by phase contrast microscopy. Hyaluronic acid and heparin significantly stimulated AR over control levels. Hyaluronic acid did not stimulate AR 15 min after addition to sperm previously incubated for only 2.5 h. Pre-incubation of hyaluronic acid with streptomyces hyaluronidase destroyed the ability of that GAG to stimulate the AR. These results indicate that GAGs (at least one of which, hyaluronic acid, is present in the oocyte cumulus oophorous) can rapidly stimulate the acrosome reaction in motile previously capacitated hamster sperm.