Hyaluronidase dissolves a component in the hamster zona pellucida

Mammalian sperm must pass between cumulus cells and corona radiata cells before reaching the surface of the zona pellucida which surrounds the oocyte. The cumulus and corona radiata cells are separated from each other by an extracellular matrix (ECM) containing hyaluronic acid. The structure of this ECM and of the zona pellucida was investigated in the hamster oocyte-cumulus complex (OCC) using transmission electron microscopy (TEM) following processing in ruthenium red. When fixed in the presence of ruthenium red, the ECM of the OCC and the zona pellucida were well preserved and highly structured. The ECM between corona radiata cells was comprised of a network of granules and filaments which resembled hyaluronic acid containing matrices described in other systems. The outer one-third to one-half of the zona pellucida was porous; the ECM of the corona radiata extended into these pores. Bovine testicular hyaluronidase, Streptomyces hyaluronidase, and hamster sperm extracts containing hyaluronidase each dispersed the cumulus cells and most of the corona radiata cells. TEM examination revealed that brief (5-10 min) hyaluronidase treatment of OCCs removed the matrix filaments and caused clumping of the granules in both the corona radiata and zona pellucida. Longer hyaluronidase treatments (15-30 min) removed both filaments and granules. Our observations are consistent with the ideas that: 1) the ECM between corona radiata cells contains hyaluronic acid, and 2) hyaluronic acid is present in the outer one-third to one-half of the zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Mechanistic studies of the plasma membrane block to polyspermy in mouse eggs

The mechanisms responsible for the plasma membrane associated block to polyspermy in mouse eggs were studied. Reinsemination experiments using zona-free eggs indicated that, after fertilization, the egg plasma membrane is altered such that sperm binding to the egg plasma membrane is blocked, except in the region of the second polar body. Activation of the egg with either ethanol or strontium chloride did not result in a block to polyspermic penetration, as artificially activated eggs displayed identical penetration levels as to nonactivated control eggs. The penetrability of activated eggs was not altered by the presence or absence of the zona pellucida during activation. Lectin staining for egg cortical granule material indicated that activation did cause cortical granule exocytosis; however, activated eggs remained penetrable. These data support the following conclusions: (1) an alteration in the ability of the egg plasma membrane to allow sperm adherence accounts for the block to polyspermy; (2) establishment of the plasma membrane block to polyspermy is sperm dependent, since artificial egg activation does not result in a block response; (3) the contents of the egg's cortical granules do not play a role in the establishment of the plasmalemma block response. © 1993 Wiley-Liss, Inc.     

Preventing polyspermy in mammalian eggs—Contributions of the membrane block and other mechanisms

The egg's blocks to polyspermy (fertilization of an egg by more than one sperm) were originally identified in marine and aquatic species with external fertilization, but polyspermy matters in mammalian reproduction too. Embryonic triploidy is a noteworthy event associated with pregnancy complications and loss. Polyspermy is a major cause of triploidy with up to 80% of triploid conceptuses being the result of dispermic fertilization. The mammalian female reproductive tract regulates the number of sperm that reach the site of fertilization, but mammals also utilize egg‐based blocks to polyspermy. The egg‐based blocks occur on the mammalian egg coat (the zona pellucida) and the egg plasma membrane, with apparent variation between different mammalian species regarding the extent to which one or both are used. The zona pellucida block to polyspermy has some similarities to the slow block in water‐dwelling species, but the mammalian membrane block to polyspermy differs substantially from the fast electrical block that has been characterized in marine and aquatic species. This review discusses what is known about the incidence of polyspermy in mammals and about the mammalian membrane block to polyspermy, as well as notes some lesser‐characterized potential mechanisms contributing to polyspermy prevention in mammals.     

A cell surface block to polyspermy occurs in golden hamster eggs

We have examined the frequency and fate of supernumerary sperm in the perivitelline space (PVS) of in vitro fertilized hamster eggs to determine if there is a cell surface block to polyspermy. The zona pellucida block to polyspermy is very effective since only one sperm penetrated the zona pellucida in 72.8% of the 876 fertilized eggs examined. Of the polypenetrated eggs, 41.6% had a supernumerary sperm within the PVS. The proportion of polypenetrated eggs with PVS sperm did not change when the duration of coincubation was increased from 3 to 6 hr. PVS sperm were found in 67% of the inseminations. From these data we conclude that there is a cell surface block to polyspermy in the hamster. To investigate the mechanism of the cell surface block, we used the Hoechst-transfer technique (R. Hinkley, B. Wright, and J. Lynn, 1986, Dev. Biol. 118, 148-154) to monitor sperm-egg fusion. We first demonstrated that dye transfer from zona pellucida-free eggs to sperm only occurred when fusion was possible, i.e., in the presence of calcium, and that dye was transferred to all fused sperm. When cumulus-free, zona-intact eggs were preloaded with Hoechst dye and viewed 3 hr postinsemination, three classes of eggs with supernumerary sperm in the PVS were observed: eggs with only Hoechst-positive sperm (62%), eggs with only Hoechst-negative sperm (27%), and eggs with both a Hoechst-positive and a Hoechst-negative sperm (11%). Because of the limited time resolution of the Hoechst-transfer technique, the cell surface block could operate by preventing sperm fusion (Hoechst-negative), by the failure of the eggs to incorporate fused sperm (Hoechst-positive), and/or by the "unfusing" of fused sperm (Hoechst-positive and Hoechst-negative). We are unable at this time to differentiate between these mechanisms.     

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.     

Specialised plasma membranes in the preovulatory follicle op the fowl

Summary In the hen's follicle processes from the zona granulosa cells extend into the zona radiata. The terminal plasma membrane of these processes has a total thickness of around 160 Å and consists of five layers. Small granules spaced at regular intervals are attached to the cytoplasmic aspect of the inner layer by short stalks. In the 2 mm and 7 mm follicles the plasma membrane of the ovum facing the specialised terminal membranes has a striated appearance and shows a regular arrangement of granules attached by stalks to both its inner and outer aspects. The terminal and striated membranes are separated by an interval although there are areas of closer contact. In the 15 mm and 35 mm pre-ovulatory follicles the plasma membrane round the whole surface of the ovum is now typical striated membrane with bristles and attached granules. No explanation can be given at present of the function of the terminal membranes of the granulosa processes. They may indicate some change in the permeability permitting the intercellular diffusion of particles. It is suggested that the striated ovum plasma membrane is associated with the adsorption and transport of substances into the ovum for yolk synthesis.     

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.     

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.     

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.     

Monospermy and polyspermy after partial zona dissection of reinseminated human oocytes

Partial zona dissection (PZD), a zona drilling method that uses mechanical force to open the zona pcllucida while the oocyte is shrunken in a sucrose solution, was applied to 121 unfertilized 1-day-old mature human oocytes prior to reinsemination. The 115 surviving oocytes were divided into three groups in which the duration between sucrose addition and reinsemination was varied: I) Less than 20 minutes, II) 21 to 45 minutes, and III) longer than 45 minutes. There was a trend toward a reduced fertilization and polyspermy rate as the time between sucrose exposure and insemination in sucrose-free medium increased. Moreover, there was a statistically significant reduction in the number of oocytes penetrated by more than four sperm in group III (0/41) versus group I (7/34), and in group III, parthcnogcnctic development was observed. The incidence of polyspermy was also increased in oocytes manipulated more than 25 hours after retrieval compared with those manipulated 21-24 hours after recovery, supporting the idea that aged oocytes have a reduced ability to block polyspermy. Oocyte contraction in sucrose occurred in three different patterns: spherical, pear-shaped, and crenated. Both the fertilization and polyspermy rates were significantly higher in the crenated group. These results indicate that changes resembling activation occur following sucrose exposure and that sucrose activation can be used to reduce the risk of polyspermic fertilization in zona drilling procedures. In addition, the pattern of shrinkage in sucrose can be used as an indicator of oocyte receptivity to sperm penetration.     

The interaction of living boar sperm and sperm plasma membrane vesicles with the porcine zona pellucida

Enzymological, morphological, and immunological methods were used to characterize further the interaction of noncapacitated boar spermatozoa with the porcine zona pellucida. Transmission electron microscopy showed that sperm usually bind to the zona over the head region of the cell. Only the plasma membrane is involved in this binding. Bound sperm will undergo the acrosome reaction when treated with calcium and the ionophore A23187. The ability of intact sperm to bind to porcine eggs in vitro and the ability of sperm plasma membrane vesicles to absorb univalent antibody to the sperm binding site for the zona were used to determine the effects of various physical, chemical, and enzymological treatments on the sperm binding sites. These sites were resistant to a number of enzymes including proteases and polysaccharidases, but were inactivated by heat and trichloroacetic acid. Binding sites on the zona were inactivated by extracts from small quantities of sperm. Binding was also blocked by Fab antibody to whole zonae absorbed to other swine tissue and by similarly absorbed Fab antibody to sperm plasma membranes. These data provide further support for the presence of zona recognition sites on the plasma membrane of noncapacitated boar sperm. The binding sites on the sperm plasma membrane do not appear to be peripheral membrane proteins nor major constituents of a surface glycocalyx.     

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.     

Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10^?5 to 10^?3M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10^?3M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the egg's surface, which give rise to cortical projections in the presence of SBTI.     

Inhibition of boar sperm hyaluronidase activity by tannic acid reduces polyspermy during in vitro fertilization of porcine oocytes

The present study was conducted to examine the effects of three polyphenols (tannic acid, apigenin and quercetin) on hyaluronidase activity and in vitro fertilization (IVF) parameters. Among them, tannic acid showed by far the strongest potency for blocking hyaluronidase activity extracted from preincubated boar sperm, causing a dose-dependent inhibition over the range of 2-10 microg/ml. When cumulus-intact and cumulus-free oocytes were inseminated in IVF medium containing tannic acid, the penetration and the polyspermy rates were significantly decreased in the presence of 10 microg/ml tannic acid compared with those in the absence of tannic acid, and the addition of 5 microg/ml tannic acid significantly reduced the polyspermy rate (p < 0.05) compared with that of the control while maintaining the high penetration rate. However, apigenin and quercetin had no effect on the rate of polyspermy. Interestingly, the incidence of polyspermy was significantly reduced in oocytes inseminated with sperm pretreated with 5 microg/ml tannic acid (p < 0.05), although the pretreatment of oocytes had no effect against the polyspermy after insemination with untreated sperm. Treatment with tannic acid caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization, nor a reduction of the proteolytic activity of acrosomal contents or the number of zona-bound spermatozoa. These data suggest that an appropriate concentration of tannic acid prevents polyspermy through the inhibition of sperm hyaluronidase activity during IVF of porcine oocytes.     

Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.     

The block to sperm penetration in zonal-free mouse eggs

The rate of sperm penetration and the number of sperm penetrating zona-free mouse eggs were found to be dependent on sperm concentration. At the lowest sperm concentrations examined (10² cells/ml, sperm-egg ratios of approximately 1:1), most eggs were penetrated (75%), and polyspermy was low (19%) following 3 hr of incubation. The number of sperm penetrating the egg was logarithmically related to sperm concentration. All eggs showed a delay of at least 20 min between insemination and penetration, and penetration was complete in approximately 2 hr at 10⁴ sperm/ml; this penetration block was attributed to egg-related changes. The existence and timing of the egg plasma membrane block to polyspermy were evaluated by reinsemination experiments. In this approach, the block was triggered in zona-free eggs with a low concentration of capacitated epididymal sperm at time 0, and the eggs were subsequently challenged with high sperm concentrations. The presence or absence of a block was inferred from the degree of polyspermy observed in these eggs after 3 hr of incubation. Adjusting for sperm concentration-dependent delays between insemination and sperm penetration, a blocking time of approximately 40 min was obtained.     

Effects of the porcine oviduct-specific glycoprotein on fertilization, polyspermy, and embryonic development in vitro

This study evaluated the effects of porcine oviduct-specific glycoprotein (pOSP) on in vitro fertilization (IVF), polyspermy, and development to blastocyst. Experiment 1 evaluated the effects of various concentrations (0-100 microgram/ml) of purified pOSP on fertilization parameters, including penetration, polyspermy, male pronuclear formation, and mean number of sperm penetrated per oocyte. Experiment 2 examined the ability of an anti-pOSP immunoglobulin G to inhibit the observed effects of pOSP on fertilization parameters. Experiments 3 and 4 examined various concentrations of pOSP (0-100 microgram/ml) on zona pellucida solubility and sperm binding, respectively. Lastly, experiment 5 assessed the effects of various concentrations of pOSP (0-100 microgram/ml) on the in vitro embryo cleavage rate and development to blastocyst. Pig oocytes matured and fertilized in vitro were used for all experiments. An effect of treatment (P < 0.05) was detected for pOSP on penetration, polyspermy, and mean number of sperm per oocyte. Concentrations for pOSP of 0-50 microgram/ml had no effect on sperm penetration rates; however, compared with the control, 100 microgram/ml significantly decreased the penetration rate (74% vs. 41%). Addition of 10-100 microgram/ml significantly reduced the polyspermy rate compared with the control (61% vs. 24-29%). The decrease in polyspermy achieved by addition of pOSP during preincubation and IVF was blocked with a specific antibody to pOSP. No effect of treatment was observed on zona digestion time relative to the control; however, the number of sperm bound to the zona pellucida was significantly decreased by treatment (P < 0.05). Compared with the control, all concentrations of pOSP examined reduced the number of sperm bound per oocyte (45 vs. 19-34). A treatment effect (P < 0.05) was observed for pOSP on embryo development to blastocyst but not on cleavage rates. Addition of pOSP during preincubation and fertilization significantly increased postcleavage development to blastocyst, but a synergistic stimulation on development was not detected when pOSP was included during in vitro culture. These results indicate that exposure to pOSP before and during fertilization reduces the incidence of polyspermy in pig oocytes, reduces the number of bound sperm, and increases postcleavage development to blastocyst.     

Membrane depolarization facilitates sperm entry, large fertilization cone formation, and prolonged current responses in sea urchin oocytes

Depolarization of the sea urchin egg's membrane is required for two processes during fertilization: the entry of the fertilizing sperm and the block to polyspermy which prevents the entry of supernumerary sperm. In an immature sea urchin oocyte, the depolarization is very small in response to the attachment of a sperm. The purpose of this study was to determine whether the depolarization evoked by sperm attaching to an oocyte can facilitate sperm entry or induce the block to polyspermy. Individual oocytes of the sea urchin with diameters which ranged from 86 to 102% that of the average diameter for mature eggs from the same female were examined. The oocytes have a membrane potential of -73 +/- 6 mV (SD, n = 80) and a very low input resistance compared to that of mature eggs. Single sperm, following attachment to an oocyte, elicit a brief, small depolarization with a maximum amplitude of 8 +/- 1.4 mV (SE, n = 15), frequently followed by the formation of tiny filament-like fertilization cones, but the sperm fail to enter. If oocytes are voltage-clamped at membrane potentials more negative than -20 mV, following attachment of the sperm small transient inward currents occur, similar filament-like cones form, and the sperm do not enter. When many sperm attach to an oocyte which is not voltage clamped, the depolarizations sum to create a large depolarization with an amplitude of 60 to 80 mV, which shifts the oocyte's membrane potential to a value between -10 and +5 mV; more positive values are not attained. At such membrane potentials, whether the potential is maintained by the summed depolarizations of many attached sperm or by voltage clamp, large fertilization cones form, the sperm enter, and the oocytes can become highly polyspermic. In oocytes voltage clamped at +20 mV, however, both sperm entry and fertilization cone formation are suppressed. Therefore, both types of voltage-dependence for sperm entry are present in oocytes, although the depolarization caused by a single sperm is not large enough to permit its entry, nor is the depolarization caused by many sperm sufficient to prevent the entry of supernumerary sperm.     

Molecular basis of fertilization in the mouse

Recent studies of mouse fertilization have identified two complementary gamete receptors that mediate sperm-egg binding. Sperm surface β1,4-galactosyltransferase (GalTase) binds to specific oligosaccharides of the egg coat (zona pellucida) glycoprotein ZP3. Evidence suggests that these same molecules may stimulate the acrosome reaction in sperm. After the acrosome reaction, it is thought that sperm remain adherent to the zona by binding another glycoprotein, ZP2. The acrosome-reacted sperm releases hydrolytic enzymes, including acrosin and N-acetylglucosaminidase, enabling it to penetrate the zona pellucida. After the penetrating sperm binds to the egg membrane and activates development, N-acetylglucosaminidase is exocytosed from egg cortical granules and, as part of the zona block to polyspermy, globally removes the sperm GalTase binding site from ZP3 oligosaccharides.