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.     

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.     

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.     

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.     

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.     

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)     

Use of salt-stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization

Zonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4-cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x ) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41). In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt-stored zonae, zonae-free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt-stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona-free hamster ova that were penetrated by sperm cells from the same sample. In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt-stored zonae, zona-free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy-six of 80 salt-stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different. The salt-stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt-stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.     

Organization of the Hamster Cumulus Extracellular Matrix: A Hyaluronate-Glycoprotein Gel which Modulates Sperm Access to the Oocyte

The mammalian oocyte-cumulus complex contains an extracellular matrix rich in hyaluronate. Recently, the microstructure of the hamster cumulus extracellular matrix was described (52). In the present work, we investigated the organization of this matrix. We employed freeze-substitution methodologies to investigate ultrastructural effects of various treatments, including sperm enzymes, on the matrix. Protease treatment resulted in disruption with a loss of the fibrillar structures and some expansion; in contrast, hyaluronidase treatment completely solubilized the matrix. EDTA extraction revealed that the fibrils are composed of fine filaments. A discrete region of the matrix immediately surrounding the oocyte, the corona radiata, was resistant to EDTA disruption. We found that hyaluronate is an ubiquitous constituent of the microstructural elements of this extracellular matrix. The matrix exhibits a carbohydrate:protein ratio of approximately 2:1. SDS-PAGE revealed that glycosylated polypeptides are bound to the matrix. The lectins LCA and WGA had differing affinities for these polypeptides, and bound ubiquitously to the intact matrix. The present data suggest that glycoprotein-hyaluronate interaction is critical for maintenance of the cumulus extracellular matrix microstructure and for its physical properties.     

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.     

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.     

Release of hyaluronidase and beta-N-acetylhexosaminidase during in vitro incubation of hamster sperm

Previous studies have shown that hamster sperm release a significant amount of hyaluronidase before and independently of the normal acrosome reaction. In this study, we have used improved methods for in vitro incubation to investigate the time course of the release of hyaluronidase and hexosaminidase from hamster sperm. When hamster sperm are incubated in medium which allows capacitation, 34 to 47% of the total mechanically extractable hyaluronidase and 34 to 51% of beta-N-acetylhexosaminidase are released into solution prior to and independently of the normal acrosome reaction (ARx). An additional 40 to 50% of the hyaluronidase and 34 to 51% of the hexosaminidase are released at the time of the normal ARx. Control experiments indicate that the early release is not due to the presence of dead sperm in culture and that the normal ARx is required for the second release. Increasing amounts of TCA-precipitated bovine serum albumin in the culture medium stimulated the early (1 hr) release of both enzymes. The data are consistent with the ideas that a significant amount of both enzymes is released from the sperm surface by 1 hr of incubation and that about the same amount of each enzyme is released during the normal ARx. Hyaluronidase and hexosaminidase release at the time of the acrosome reaction was measured for the first time using hamster sperm. The biphasic release of these enzymes may indicate that they have a dual function in fertilization and may help explain how sperm can penetrate the cumulus and corona radiata without undergoing an acrosome reaction.     

Kinematics of hamster sperm during penetration of the cumulus cell matrix

During capacitation, mammalian spermatozoa gain the ability to penetrate the cumulus cell matrix (CCM). The role of hyperactivated motility for this capacity is uncertain. In the present study, hamster sperm were observed during penetration and progression through the CCM, and flagellar beat patterns were quantitated by characterization of the underlying flagellar bends. Small numbers of sperm were added to cumulus masses slightly compressed on a slide (150 μm depth), and penetration was videorecorded using interference contrast optics. During penetration of the cumulus surface, sperm did not generate the large flagellar bends and asymmetric beats that are hallmarks of hyperactivation in low viscosity media. Instead, they entered slowly using high-frequency, low-amplitude sinusoidal flagellar motions. Within the CCM, sperm continued to move slowly, and they exhibited three distinct patterns of motility. The first was sinusoidal, produced by alternating, propagated bends: principal bends (PB) moved the head away from the beat midline, with the convex edge of the head leading, and reverse bends (RB) had the opposite curvature. The second pattern was asymmetric and sinusoidal: an extreme RB developed in the distal flagellum, was propagated distally, and was followed by a PB of less curvature. The third motility pattern was a hatchet-like stroke of the sperm head which resulted when an extreme, nonpropagated PB developed slowly in the proximal midpiece, and was released rapidly. In this mode there were no reverse bends, and sperm did not progress. There were subpopulations of capacitating sperm in free-swimming medium which had these same bend types and motility patterns, suggesting that qualitative flagellar movement may not change during CCM penetration. Sperm velocity in the CCM was not strongly correlated with flagellar beat kinematics, suggesting local heterogeneity in cumulus mechanical resistance and/or differences in interaction of the matrix with the surfaces of individual sperm. An effective viscosity of the cumulus near its border was estimated to be of the order of 1–4 P.     

Hyperactivation enhances mouse sperm capacity for penetrating viscoelastic media.

A movement pattern known as hyperactivation has been observed among sperm recovered from the periovulatory oviduct of several species. In culture medium, hyperactivated sperm swim in a pattern that is far less progressive than that of freshly ejaculated sperm. In the oviduct, sperm encounter highly viscoelastic substances, such as mucus and the cumulus matrix. We have previously reported that hyperactivated hamster sperm become more progressive in vitro when the viscosity of medium is increased. In the present study, we tested the effect of increasing the viscosity and viscoelasticity of the medium on the swimming progressiveness of mouse sperm. Caudal epididymal sperm were incubated in a medium that produced hyperactivated motility in 60 min. Swimming velocities of sperm incubated for 60 min were compared with those of fresh sperm after addition of one of the following to culture medium: solutions of 1.8% methylcellulose (high viscosity), 1.8% long chain polyacrylamide (high viscoelasticity), or culture medium alone (low viscosity). In culture medium, hyperactivated sperm had significantly lower mean straight-line velocities than fresh sperm (p = 0.004); this difference disappeared in methylcellulose (p = 0.085) and was reversed in polyacrylamide (p = 0.004). This and other velocity measurements indicated that hyperactivated mouse sperm penetrate viscoelastic media more efficiently than fresh sperm and therefore may be more efficient at penetrating oviductal mucus and cumulus matrix in vivo.     

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.     

Immunoinhibition of human fertilization in vitro by antibodies to the cumulus oophorus intercellular matrix

Rabbit polyvalent antiserum raised against the solubilized cumulus matrix was a powerful inhibitor of human fertilization in vitro, affecting sperm-zona pellucida interaction. Both sperm binding to, and penetration of, the zona pellucida were severely impaired by the anti-cumulus matrix antiserum, whereas no effects of this antiserum on cumulus matrix solubilization or penetration of zona-free human eggs were evident. Moreover, the anti-cumulus antiserum partly neutralized the acrosome reaction-inducing activity of the cumulus matrix. These results warrant further research into the functional specificity of different cumulus matrix components and into the effects of the respective antibodies on reproductive function as a possible lead to a new approach to contraceptive vaccine development.     

Hyaluronidase activity of macaque sperm assessed by an in vitro cumulus penetration assay

A model system consisting of cynomolgus macaque sperm and ovulated hamster ova-cumulus complexes (OCCs) was utilized to study the role of the sperm protein PH-20 in cumulus penetration. The hyaluronidase activity of solubilized macaque sperm PH-20 was evaluated using an ELISA-like microplate assay prior to and following the addition of the hyaluronidase inhibitors heparin (0–100 μg/ml) and apigenin (250 μM), as well as the Ig fraction of a polyclonal antibody raised against purified recombinant macaque PH-20 (R10; 10–400 μg/ml). Sperm motility following exposure to enzyme inhibitors was evaluated using computer-aided sperm motility analysis. Macaque sperm were labeled with the permeant fluorescent nuclear dye, Hoechst 33342, and were coincubated with ovulated hamster OCCs for 30 min at 37°C. The addition of heparin, apigenin, or R10 antibody to solubilized sperm extracts resulted in a linear dose-dependent decrease in hyaluronidase activity (P < .01). In the heterologous cumulus penetration assay, fluorescently labeled macaque sperm that were pretreated with heparin (1–100 μg/ml), apigenin (250 μM), or R10 antibody (Ig fraction, 10–400 μg/ml) demonstrated a dose-dependent decrease in the ability to penetrate hamster OCCs (P < 0.01), in the absence of effects on sperm motility. In the homologous assay, experiments using macaque OCCs and fluorescently labeled macaque sperm confirmed that the same concentrations of heparin and R10 antibody similarly suppressed spermatozoal cumulus penetration (P < .01). These results suggest that macaque sperm PH-20-derived hyaluronidase participates in cumulus penetration in this species, and that this model system is useful for further studies into primate gamete interaction. Mol. Reprod. Dev. 46:392–400, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated in vitro,in relation to the timing of the acrosome reaction

Hamster spermatozoa were tested for their ability to penetrate the intact cumulus matrix at low sperm:egg ratios (approximately 3:1). Uncapacitated spermatozoa attached to the surface of the cumulus and could not penetrate. Spermatozoa capacitated in vitro began to be able to penetrate after about 2 hr of preincubation, coincidentally with the first appearance of hyperactivation and spontaneous acrosome reactions. In all, 628 in vitro incubated spermatozoa were evaluated on and in cumuli: 270 could penetrate, but only ten of these were judged to have intact, “unmodified” acrosomes. Almost all spermatozoa capable of penetrating showed optically “modified” and swelling acrosomal caps, and this confirms previous observations on cumulus penetration in vivo. Penetration appeared limited to a phase in capacitation prior to completion of the acrosome reaction, as spermatozoa that had lost the acrosomal cap penetrated poorly and showed reduced viability. Penetration of the cumulus was inhibited by the hyaluronidase inhibitor sodium aurothiomalate. Cumulus penetrating ability could result either from a change in surface properties of the sperm at capacitation, which renders them less “sticky” to the matrix, or from release or activation of a “cumulus lysin.” We conclude that the ability to enter the cumulus matrix coincides with physiological changes in spermatozoa that occur during a terminal phase of capacitation preceding complete loss of the acrosomal cap, and that initiation of this process in vivo must precede sperm-zona contact.     

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators

Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.     

CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization

Ca²⁺-dependent mechanisms are critical for successful completion of fertilization. Here, we demonstrate that CRISP1, a sperm protein involved in mammalian fertilization, is also present in the female gamete and capable of modulating key sperm Ca²⁺ channels. Specifically, we show that CRISP1 is expressed by the cumulus cells that surround the egg and that fertilization of cumulus–oocyte complexes from CRISP1 knockout females is impaired because of a failure of sperm to penetrate the cumulus. We provide evidence that CRISP1 stimulates sperm orientation by modulating sperm hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, patch clamping of sperm revealed that CRISP1 has the ability to regulate CatSper, the principal sperm Ca²⁺ channel involved in hyperactivation and essential for fertility. Given the critical role of Ca²⁺ for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orientation for successful penetration of the cumulus during fertilization.