Modulation of fertilization by the vitellus after interaction with peptides released by hamster sperm-zona pellucida contact

Two to three minutes after hamster sperm make contact with and adhere to the surface of homologous zonae pellucidae in vitro, the first of several sets of peptides (S1 peptides) is released into the supernatant. This release occurs whether the zonae have or have not been mechanically separated from the vitellus (cellular part of the egg). Presence of the S1 peptides is detected by means of a sperm-egg assay, the premature binding assay. This assay is based on the ability of an aliquot of the medium, in which sperm are interacting with the zona surface, to induce early binding, upon addition of the aliquot to a second drop of interacting gametes. To determine if the vitellus affected the 2-min S1 peptides the ultrafiltrates of the supernatants containing them, released through sperm-egg and sperm-zona interactions, were fractionated on Biogel P-6 and their elution profiles were compared using the premature binding assay. The sperm-egg ultrafiltrates were resolved into two main domains of activity, while those of the sperm-zona formed three. The ultrafiltrates collected 2 min after the interaction of sperm with eggs or with isolated zonae were compared for their abilities to inhibit the penetration of the zona pellucida, a previously demonstrated capacity of the 2-min sperm-egg S1 peptides. The ultrafiltrate containing the sperm-zona peptides, except at a very low level, failed to inhibit penetration significantly. However, when the sperm-zona ultrafiltrate was preincubated with eggs then the resulting supernatant inhibited penetration in a dose-related manner, and the three-domain elution profile, characteristic of the sperm-zona ultrafiltrate, was converted to the egg-like two-domain profile. Taken together these data suggest that the 2-min S1 peptides consist of several subpopulations, at least one of which interacts with the vitellus. The resulting solution then acquires the ability to inhibit penetration of the egg by the sperm in a dose-related manner. Taken together these data indicate that by interacting with at least one of the components of the 2-min peptides, the vitellus is involved in regulating sperm-zona interactions.     

Role of the vitellus in the block to polyspermy in golden hamster eggs

The block to polyspermy in golden hamster eggs is believed to operate only at the zona pellucida. However, changes in the egg vitellus also prevent further entry of capacitated sperm. When zona-free hamster eggs spontaneously activated in vitro, and in vivo fertilized eggs at pronuclear stage were inseminated with capacitated human sperm, penetration did not occur. In the case of a homologous system using hamster sperm and in vivo fertilized hamster eggs, slight attachment of sperm was observed but no penetration. The cortical granules were found to be released in spontaneously activated and in fertilized eggs as observed by phase contrast microscopy. These observations suggest that the egg vitellus plays a role in the block to poiyspermy in addition to that of the zona block.     

Surface interactions between mammalian sperm and egg: variation of spermatozoa concentration as a probe for the study of binding in vitro.

The pre-penetration binding interactions between gametes of the golden hamster were investigated in vitro. Binding between capacitated spermatozoa and the surface of eggs, that is the zonae pellucidae with intact vitelli, as a function of the concentration of spermatozoa, followed a sigmoidal curve. This was in sharp contrast to the linear binding obtained with mechanically isolated zonae pellucidae (zonae lacking vitelli). Penetration of eggs as a function of the concentration of spermatozoa paralleled the binding curve that occurred between gametes. The binding curve obtained with uncapacitated spermatozoa and eggs was not sigmoidal but was linear after a slight lag and parallel to the curve obtained with uncapacitated spermatozoa and isolated zonae pellucidae. Taken together these results support previous work which implicated a vitelline factor in the binding reaction between the surfaces of eggs and capacitated spermatozoa. By scoring binding at one minute intervals it was possible to relate the rapid uninterrupted binding that occurs between capacitated spermatozoa and isolated zonae pellucidae with the equally rapid but transient and vitellus-influenced binding that occurs between gametes. It was concluded that the vitelline factor acts by preventing most of the early type of binding that occurs between spermatozoa and isolated zonae pellucidae and not by terminating the early, rapid, initial binding as previously postulated. Thus, this early binding never occurs between most of the gametes that finally bind 30 to 40 minutes later and, therefore, does not play a role in the establishment of the late binding step which leads to penetration.     

Hamster zonae pellucidae cannot induce physiological acrosome reactions in chemically capacitated hamster spermatozoa in the absence of albumin

Cauda epididymal hamster spermatozoa were capacitated with D-penicillamine in a chemically defined (protein-free) medium (= "chemical" capacitation). Hamster zonae pellucidae were incapable of inducing functional acrosome reactions in chemically capacitated hamster sperm in a protein-free medium during sperm-egg coincubation. The culture medium used throughout incubation was a modified Tyrode's solution containing 10 mM sodium lactate, 100 microM sodium pyruvate and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm motility was maintained in all media with PHE (20 microM penicillamine, 100 microM hypotaurine, and 1.0 microM epinephrine). Additional D-penicillamine (125 or 500 microM) or 3 mg/ml bovine serum albumin (high control: TALP-PVA) was used to capacitate sperm during preincubation at 1-2 X 10(6) sperm/ml for 4.0 h at 37 degrees C in 5% CO2 in air. Sperm were then coincubated (2 X 10(4) sperm/ml) with cumulus-free hamster eggs in TALP-PVA or TLP-PVA +/- additional D-penicillamine (total: 500 or 125 microM) for 1.5 or 6.0 h. Percent egg penetration was used as the definitive index of sperm capacitation and functional acrosome reactions. Chemically capacitated sperm did not penetrate eggs (0.0 +/- 0.0%) in the absence of albumin during 1.5 h of sperm-egg coincubation. When sperm were chemically capacitated with 125 microM or 500 microM D-penicillamine, then coincubated with eggs for 6.0 h in the absence of albumin, only 18.8 +/- 28.6% and 23.7 +/- 29.7%, respectively, of eggs were penetrated. Significantly (p less than 0.05) more eggs (67.7 +/- 22.4%) were penetrated when coincubated with chemically capacitated sperm for 1.5 h in medium containing albumin. These results demonstrate that zonae pellucidae of hamster eggs require the presence of albumin to efficiently induce functional acrosome reactions in sperm that are chemically capacitated with D-penicillamine.     

Time and process of sperm penetration into cumulus-free mouse eggs fertilized in vitro

Sperm penetration through the zona pellucida and fusion of the sperm head with the vitellus were observed continuously and filmed under phase optics in cumulus-free living mouse eggs inseminated in vitro with capacitated epididymal sperm. Most spermatozoa penetrated the zona pellucida, traversed the perivitelline space, and fused with the vitellus at an angle nearly perpendicular to the surface. The mean duration required for sperm to penetrate the zona pellucida was 20 minutes with a range of 15–26 minutes. Sperm traversed the perivitelline space in less than one second. The initial contact of sperm with the vitellus generally took place at the tip of the sperm head. When the tip of the sperm head contacted the vitellus there was an immediate reduction in the rate of flagellation, followed by the gradual sinking of the sperm head into the vitellus.     

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.     

Evidence suggesting a role for sperm metalloendoprotease activity in penetration of zona-free hamster eggs by human sperm

It has been reported that metalloendoprotease (MEP) activity is involved in somatic cell membrane fusion events and in the sea urchin sperm acrosome reaction (AR). MEP activity also has been demonstrated in human and other mammalian sperm. The present study was concerned with investigating whether a human sperm MEP is important in membrane events necessary for sperm egg fusion. Ejaculated human sperm were washed, capacitated in vitro, and preincubated with the competitive MEP inhibitors phosphoramidon (50 microM) or CBZ-L-phenylalanine (1 mM), with 100 microM diethylenetriaminepentaacetic acid (DTPA), a heavy metal chelator, or as controls, with the appropriate solvents. The AR was initiated in vitro with preovulatory human follicular fluid and the sperm washed to dilute inhibitors and then coincubated with zona-free golden hamster eggs (zonae and cumuli removed with trypsin and hyaluronidase, respectively). Eggs were washed after 0.5 h, and the number of sperm remaining bound was counted. After 2.5 h further incubation, the eggs were stained with acetolacmoid or acetoorcein and penetration was assayed by counting the number of decondensed sperm heads per egg (penetration index) and the percent of penetrated eggs. The inhibitor treatments did not decrease the percentage of penetrated eggs (range 80-90%), but a significant reduction in the penetration index was observed. Phosphoramidon reduced the penetration index by 45%, CBZ-L-phenylalanine by 57%, and DTPA by 56%. None of the inhibitors decreased the penetration index or the percentage of penetrated eggs when added directly to suspensions of acrosome-reacted sperm and zona-free eggs at the diluted levels that would have been present after washing inhibitor-treated sperm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scanning electron microscope study of in vitro prepenetration gamete interactions

Hamster and mouse capacitated spermatozoa were interacted in vitro with hamster and mouse eggs in homologous and heterologous combinations. Also, fertilized and trypsin treated unfertilized hamster eggs, and unfertilized rat eggs were made to interact with capacitated hamster spermatozoa. The surface of the zona pellucida was then examined with the scanning electron microscope. It was found that sperm attachment, followed by sperm binding and penetration through the zona pellucida, was observed only when homologous gamete combinations were used. Binding of the spermatozoa to the zona was evidenced by the lytic effect of the acrosomal enzymes on the zona substance. When fertilized eggs and trypsin-treated unfertilized hamster eggs were mixed with capacitated hamster spermatozoa as well as in the heterologous gamete combinations, we found that the spermatozoa were able to establish attachment but not binding. Under these conditions the outer surface of the zona pellucida was never found to have penetration tracks made by the spermatozoa. Failure of heterologous spermatozoa to cross the foreign zona pellucida is believed to be associated with the inability of the foreign spermatozoa to establish binding and to the inability of their acrosomal enzymes to digest the zona. A similar mechanism is believed to work in zona-reacted and in trypsin-treated hamster eggs inseminated in vitro with homologous spermatozoa.     

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.     

Detection of a soluble acrosome reaction-inducing factor, different from serum albumin, associated with the ovulated egg-cumulus complex.

Soluble extracts of the ovulated hamster egg-cumulus complex (ECC) were tested on capacitated sperm for activity in inducing the physiological acrosome reaction (AR). Evidence for occurrence of the physiological AR included enhanced sperm penetration of intact homologous zonae pellucidae as well as induction of AR in nonattached and in zona-bound sperm following a brief coincubation with test compound. Since hamster serum albumin, a major protein of hamster body fluids, also induces spontaneous ARs under certain conditions, it was used as one of the comparators for the acrosome reaction inducing factor (ARIF; Westrick et al., Biol Reprod 32 [Suppl 1]. 213, 1985) activity in the ECC. Sperm exposure to concentrations of the soluble ECC extract ranging from 0.04 to 0.2 mg protein/ml significantly increased penetration of salt-stored zonae by 36%, mean numbers of penetrating sperm by 90%, ARs in nonattached sperm by 65%, and ARs in zona-bound sperm by 102%. Hamster serum albumin added after completion of capacitation had no significant effect on these parameters. We conclude that 1) the ovulated ECC contains a soluble ARIF that augments zona-induced ARs and sperm penetration and 2) the ARIF is not serum albumin.     

Nature and fate of the factors released during early contact interactions between hamster sperm and egg prior to fertilization in vitro

Initial attempts have been made to characterize three factors (Hartmann and Hutchison, 1977, J. Cell Physiol., 93, 41) which are released in vitro at 2, 31, and 50 min after capacitated hamster sperm make contact with but prior to penetration of the zona pellucida. A fourth factor is known to be released at 20–25 min but in the experiments described no effort was made toward its characterization. The assay for the factors is based upon their ability to induce early binding between gametes. Because the release of each of the factors occurs at a different time they can be harvested by sampling the supernatant at the appropriate time. The short-lived activity of these factors, which normally disappears soon after their release, was stabilized by buffering the medium to pH 7.0–7.4 with Tris or TES and removing the cells. Under these conditions, the factors released at 31 and 50 min were stable when incubated at 37°C in the absence of cells for at least 60 min, but the activity of the factor released at 2 min was erratic under similar incubation conditions. The factors released at 31 and 50 min passed unimpeded through filters with molecular weight cutoffs of 2000, and both eluted off Bio-Gel P-2 columns as single peaks of activity in regions corresponding to molecular weights of at least 1800 (void volume) and approximately 1400, respectively. The 2-min factor passed unimpeded through a filter capable of excluding molecules of molecular weight larger than 5000, but 50–86% of the activity was recovered after passing through a filter with a molecular weight cutoff at 2000. The release of the 2- and 31-min factors was inhibited 48 and 74%, respectively, by macromolecular trypsin inhibitors at concentrations which also blocked penetration of the egg by the sperm; these inhibitors had little or no effect on the release of the 50-min factor. The activity of ultrafiltered and pH-stabilized 31- and 50-min factor was destroyed by the proteases subtilisin and leucine aminopeptidase but was unaffected by trypsin or glycosidases. The disappearance of the 31- and 50- min factors after their release was investigated by incubating each factor with each of the cell types present in the drop and it was found that activity only declined in the presence of the eggs. Taken together, these results provide evidence that (1) at least two populations of peptides are released in a time-dependent manner when capacitated sperm make contact with the zona pellucida and (2) these peptides may disappear from the supernatant through an egg-mediated mechanism.     

Zona-induced acrosome reaction of hamster spermatozoa

It is well established that the zonae pellucidae of mature unfertilized eggs have the ability to induce the acrosome reaction of capacitated spermatozoa. To determine if this capacity of the zona is species-specific, hamster spermatozoa were allowed to attach to the zonae of homologous and heterologous eggs and examined for the acrosome reaction. The zonae of eggs from six different species were tested and the zona of hamster egg was found to have the strongest capacity to induce the acrosome reaction of hamster spermatozoa, followed by human and rat zonae. The zonae and mouse, guinea pig, and domestic fowl eggs were incapable of inducing the acrosome reaction of hamster spermatozoa. The acrosome reaction-inducing ability of the hamster zona was found to increase during maturation in the ovary. The zona of mature unfertilized hamster eggs maintained their acrosome reaction-inducing ability even after aldehyde fixation or storage in a highly concentrated solution of ammonium sulfate.     

An investigation of the latency period between sperm oolemmal adhesion and oocyte penetration

In clinical studies of the ability of capacitated human sperm to penetrate zona-free hamster eggs, we have previously observed that the ratio of oolemmal adherent to penetrating sperm varied between men. Sperm incorporation did not occur immediately following gamete adhesion and not all adherent sperm penetrated the egg. To further investigate this phenomenon, comparisons were made of the kinetics of gamete adhesion, membrane fusion, and sperm incorporation of capacitated mouse and human spermatozoa by zona-free hamster eggs and of mouse sperm by zona-free mouse and hamster eggs. Eggs were inseminated with either capacitated human or mouse sperm or combinations of both, washed out of sperm suspension after initial gamete adherence, and further incubated in sperm-free medium. Gamete membrane fusion was judged by dye transfer of Hoechst 33342 and sperm entry of the cortical ooplasm by observation of expanded sperm heads within acridine orange stained eggs. Oolemmal adherent mouse and human sperm fused with and penetrated zona-free hamster eggs at different times whether eggs were inseminated in parallel or with combinations of sperm of both species. Oolemmal adherent mouse sperm penetrated zona-free hamster eggs prior to their penetration of zona-free mouse eggs. Ultrastructural studies of zona-free human eggs inseminated with human sperm confirmed prior observations with hamster eggs that only acrosome-reacted human sperm adhere to the oolemma. These results have lead us to postulate that sperm entry into the egg may occur through a "zipper" mechanism involving the ligation of local gamete receptors similar to the incorporation of target particles by phagocytes and suggest that not all oolemmal adherent human sperm are capable of being incorporated although they have undergone an acrosome reaction.     

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.     

Time requirement for capacitation of boar spermatozoa assessed by their ability to penetrate the zona-free hamster egg.

Boar spermatozoa were preincubated for various times in the isolated uterus and oviduct from a maturing gilt and used to inseminate zona-free hamster eggs. The proportions of eggs penetrated and activated were increased, and the interval between insemination and sperm penetration was shortened when the spermatozoa were preincubated for 4--5.5 h instead of 2--.5 h. Overall penetration rates were higher and sperm penetration occurred about 1 h earlier when the eggs were inseminated with spermatozoa preincubated in the uterus than in the oviduct. It is concluded that the change in ability of boar spermatozoa to penetrate zona-free hamster eggs is due to capcitation which requires 4--4.5 h and 5--5.5 h of preincubation in the isolated uterus and oviduct, respectively.     

Properties of the first and second factors released after hamster sperm make contact with the zona pellucida prior to fertilization in vitro

An investigation was made as to the nature of two of the factors, termed S1, released within the first 30 minutes after contact is made between capacitated hamster sperm and the zona pellucida in vitro. Previous studies showed that these S1 factors were detected two and 20 to 25 minutes after the gametes were combined and that, based on filtration studies, the former possessed a molecular weight of less than 5,000 daltons. The present results show that the quantity of the 20–25-minute S1 factor released into the supernatant increased linearly as a function of the sperm concentration. This factor passed unimpeded through a filter with a 5,000 molecular weight cutoff but only 42% of the activity traversed a filter with a cutoff of 2,000 daltons. The two-minute S1 factor, in the virtual total absence of cells, was stable for 10 to 15 minutes, but lost significant activity upon longer incubation. Under the same conditions, the 20–25-minute factor lost approximately 25% of its activity within 15 minutes, but remained stable at this level for at least 45 minutes of incubation. Both S1 factors were not affected by a mixture of glycosidases, but were inactivated by subtilisin, trypsin, and leucine aminopeptidase which was contaminated with endopeptidases. The activity of the two-minute S1 factor appeared more susceptible to the action of the proteases than that of the 20–25-minute S1 factor. In contrast to previous results obtained with the two-minute S1 factor, the release of the 20–25-minute S1 factor was not inhibited by the inclusion of soybean trypsin inhibitor a t concentrations which are known to inhibit penetration of the zona by the sperm. The results suggest that the two- and 20–25-minute S1 factors are peptides which are not identical.     

Sperm tail entry into the mouse egg in vitro

Cumulus-free mouse eggs were placed on microscope slides and inseminated with capacitated mouse spermatozoa. Fertilization could then be observed through the phase contrast microscope and recorded by time-lapse cinematography. Following the penetration of the fertilizing spermatozoon through the zona pellucida and the fusion of the sperm head with the vitelline membrane, the entire sperm tail gradually entered the vitellus. The time required for tail incorporation into the vitellus as measured in 49 eggs varied from 3 h 3 min to 5 h 49 min, with a mean time of 4 h 23 min. When tail incorporation began, the greater part of the flagellum was still outside the zona pellucida; occasionally it slipped into the perivitelline space, but generally it remained outside the zona and shortened by degrees as incorporation proceeded. The motility of the fertilizing spermatozoon declined abruptly very soon after fusion of the sperm head with the vitellus and remained at a very low level during the 3–6 h required for tail incorporation. Sperm motility, therefore, does not appear to be the main determinant in tail incorporation and the primary mechanism responsible for it remains unclear. As the sperm tail slowly entered the vitellus, the second meiotic division was completed with concomitant extrusion of the second polar body. Key stages in second polar body formation were correlated with events in tail incorporation. Differences between fertilization in vitro and in vivo are discussed.     

A guanine nucleotide-binding regulatory protein in human sperm mediates acrosomal exocytosis induced by the human zona pellucida.

Guanine nucleotide-binding regulatory proteins play key intermediary roles in regulating zona pellucida-mediated acrosomal exocytosis in mouse and bull sperm. Since human sperm possess a Gi-like protein and undergo the acrosome reaction in response to the human zona pellucida, we investigated whether this G protein plays a regulatory role in this exocytotic process. Zonae pellucidae isolated from eggs that had been inseminated but had shown no signs of fertilization after retrieval for in vitro fertilization and embryo transfer were pooled into groups of greater than or equal to 50 in order to reduce variability in biological responses due to the possible presence of ZP that had undergone modifications associated with the polyspermy block. Acid-solubilized zonae pellucidae were incubated with capacitated sperm, and the sperm then assessed for the acrosome reaction using both the P. sativum agglutinin and chlortetracycline fluorescence assays; both assays gave similar results. Sperm incubated with solubilized zonae pellucidae at a final concentration of 2, 4, or 6 ZP/microliter underwent acrosomal exocytosis to a similar extent as compared with A-23187. Sperm were incubated with 1 microgram/ml pertussis toxin during capacitation to functionally inactivate the Gi-like protein. Pertussis toxin treatment of sperm did not affect sperm motility and the ability of the cells to bind to structurally intact zonae pellucidae. Pertussis toxin, however, completely inhibited the percentage acrosome reactions induced by solubilized zonae pellucidae. By contrast, the A-23187-induced acrosome reaction was insensitive to PT treatment. Pertussis toxin inhibition of the zona pellucida-induced acrosome reaction occurred in a concentration-dependent manner with maximal effects observed at 100 ng/ml PT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro

The question of whether the acrosome reaction, which leads to fertilization, occurs in intact sperm bound to the zona pellucida of the egg or in intact sperm before contact with the egg, was addressed by assessing the effect of 3-quinuclidinyl benzilate (QNB) on the two types of acrosome reaction. QNB is a specific inhibitor of the fertilization of zona-intact mouse eggs by mouse sperm. Mouse spermatozoa in suspension underwent acrosome reactions at a low rate, which could be accelerated by addition of 5 μM divalent cation ionophore A23187; the occurrence of such acrosome reactions was not inhibited by QNB. The rate at which acrosome reactions occurred in sperm bound to the zona pellucida of cumulus-free eggs, bound to isolated zonae, or exposed to acid-solubilized zona components, was greatly accelerated relative to that observed in the absence of zonae. These acrosome reactions were strongly inhibited by QNB at concentrations which inhibit the fertilization of zona-intact mouse eggs in vitro. These data suggest that the zona pellucida can induce acrosome reactions in mouse spermatozoa and that these acrosome reactions are the ones which lead to the fertilization of zona-intact eggs. In contrast, the acrosome rection in sperm which are not in contact with the zona is not associated with fertilization of zona-intact eggs.     

Structural and functional relationships between mouse and hamster zona pellucida glycoproteins

The hamster egg's extracellular coat, or zona pellucida, consists of three glycoproteins, designated hZP1, hZP2, and hZP3, that exhibit extensive heterogeneity on SDS-PAGE. hZP1 is a relatively minor component of hamster zonae pellucidae, as compared with hZP2 and hZP3. In the presence of reducing agents, hZP1, 200,000 apparent Mr, migrates on SDS-PAGE with an apparent Mr of 103,000. This suggests that hZP1, like mouse ZP1, is composed of two polypeptides held together by intermolecular disulfides. When purified hamster ZP glycoproteins were tested at relatively low concentrations in an in vitro competition assay, employing either hamster or mouse gametes, only hZP3 (56,000 apparent Mr) exhibited sperm receptor activity (i.e., inhibited binding of sperm to eggs). Thus, apparently hZP3 is the hamster counterpart of mouse ZP3, the mouse egg receptor for sperm. Furthermore, at relatively high concentrations, solubilized hamster egg ZP preparations induced both hamster and mouse sperm to undergo the acrosome reaction in vitro. hZP3 is encoded by a relatively abundant ovarian mRNA that is detected by a mouse ZP3 cDNA probe and is the same size, about 1.5 kb, as mRNA encoding the mouse sperm receptor, ZP3 (83,000 apparent Mr). Like mouse ZP2, hZP2 undergoes limited proteolysis following artificial activation of hamster eggs in vitro. Results of in vitro assays employing intact eggs and isolated zonae pellucidae demonstrate that hamster eggs possess a ZP2-proteinase which has a substrate specificity similar to that of the mouse enzyme. These observations are discussed in terms of structural and functional relationships that may exist between hamster and mouse zona pellucida glycoproteins.