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.     

Sperm entry into zona-free oocytes in the hamster oviduct: Implications for the mechanisms of acrosome reaction induction

The possible importance of the zona pellucida for induction of the acrosome reaction (AR) and establishment of sperm/egg associations in the fallopian tube was investigated by instilling zona-free eggs into the oviductal ampulla of hamsters that had been inseminated with epididymal spermatozoa 6–7 hours previously. The eggs were recovered only 60–90 minutes later because of increasing difficulty with time of collecting zona-free eggs from the oviduct. In the zona-free group, 41 (4%) of 1,101 transferred eggs were recovered, of which 20% contained spermatozoa with decondensing nuclei (mean 4.4/egg). A similar (22%) fertilization rate (mean 3.2 spermatozoa/egg) was found among intact (control) eggs recovered after instillation into the contralateral oviduct. Mammalian spermatozoa are not incorporated even into zona-free eggs before AR occurs. These results thus demonstrate that an AR in functional hamster spermatozoa in vivo and establishment of sperm/egg associations in vivo require no interaction with the zona pellucida nor with other products of ovulation.     

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)     

A tale of two cells: endocannabinoid-signaling regulates functions of neurons and sperm

Sea urchin and human sperm contain receptors for neurotransmitters and psychoactive drugs, including cannabinoid receptors (CNRs). Anandamide, arachidonoylethanolamide (AEA), is a lipid-signal molecule that is an endogenous agonist for CNRs. AEA is enyzmatically released from membrane phospholipids when neurons are stimulated. Retrograde AEA signals from depolarized postsynaptic neurons inhibit neurotransmitter release at synapses in mammalian brain. Analogous processes regulate sperm functions during fertilization in sea urchins. AEA and (-)delta9tetrahydrocannabinol [(-)delta9THC], the major psychoactive constituent of marijuana, inhibit fertilization by blocking acrosomal exocytosis/acrosome reactions (AR) stimulated by egg jelly. The acrosome is a Golgi-derived secretory granule in sperm analogous to synaptic vesicles in neurons. AEA and (-)delta9THC do not block ionophore-induced AR, suggesting that they inhibit AR by modulating signal transduction event(s) before opening of ion channels. Unfertilized sea urchin eggs have enzymes required to release AEA from membrane phospholipids. These results indicate that sea urchin eggs may release AEA after activation by the fertilizing sperm. Released AEA may then react with CNRs in nearby sperm to block AR, thereby helping to prevent polyspermy. AEA is present in human seminal plasma, midcycle oviductal fluid, and follicular fluid. Sperm are sequentially exposed to these fluids as they move from the vagina to the site of fertilization in the oviduct. R-methanandamide (AM-356), a metabolically stable AEA analog, and (-)delta9THC modulate capacitation and fertilizing potential of human sperm in vitro. These findings suggest that AEA signaling directly affects sperm functions required for fertilization and provide additional evidence for common signaling processes in neurons and sperm.     

Penetration of zona-free hamster eggs by liposome-treated sperm from the bull, ram, stallion, and boar

Spermatozoa from each of four rams, four stallions, and three boars (six semen samples) were treated with dilauroylphosphatidylcholine (PC12) liposomes and compared with control bull sperm to induce the acrosome reaction (AR) and study possible penetration of the sperm into zona-free hamster eggs. Diluted sperm were incubated with several concentrations of PC12 for 7 min at 39 degrees C prior to insemination of the hamster eggs in vitro. The sperm from the bull were diluted to 10(6) cells/ml, as previously studied. Sperm from the ram, stallion, and boar were diluted to 6 X 10(6) and 20 X 10(6) cells/ml. After addition to the eggs, the sperm concentration was reduced by 75 percent. Inseminated eggs were incubated with sperm for 3 h at 39 degrees C prior to being fixed, stained, and observed for sperm penetration. At an initial concentration of 6 X 10(6) cells/ml, bull sperm treated with 36.7 microM PC12 achieved an egg penetration rate of 92%, whereas under nearly identical conditions stallion spermatozoa achieved only 54% egg penetration. Under similar conditions, ram spermatozoa failed to penetrate eggs, but when the initial sperm concentration was increased to 20 X 10(6) cells/ml, sperm incubated with 51.1 microM PC12 achieved 52% egg penetration. Boar spermatozoa treated with PC12 at either sperm concentration failed to exhibit an AR or penetrate hamster eggs. In general, as PC12 concentration increased the percentage of sperm with an AR increased and sperm motility decreased. It is concluded that 1) PC12 liposomes are effective in inducing the AR in sperm from the bull, ram, and stallion, but under conditions tested are ineffective with boar sperm;(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: I. A fertility assay for fresh semen

Fresh sperm from five bulls having nonreturn rates ranging from 48% to 77% were treated with 15.7, 21.0, 26.2, 31.5, 36.7, and 42.0 μM dilauroylphosphatidylcholine (PC12) to induce the sperm acrosome reaction (AR). Treated sperm were incubated 3 hr with zona-free hamster eggs at 39°C prior to fixation. The eggs were then stained and examined for sperm penetration. Differences in the percentages of motile sperm and of sperm exhibiting an AR among bulls were small when compared on a within-liposome-concentration basis. Increasing the PC12 concentration from 15.7 μM to 42.0 μM increased the percentage of sperm exhibiting an AR for all bulls. At the lowest lipid concentration (15.7 μM), the percentage of eggs penetrated by sperm from the five bulls was 6% to 36%, with 0% in controls. When sperm were incubated with increasing lipid concentrations, the egg penetration rate increased to over 80%, and the total number of sperm increased to over 100 per 36 eggs in each treatment for every bull. These penetration rates decreased at the highest lipid concentration. A correlation between the PC12 concentration maximizing egg penetration and the nonreturn rate of ?.63 was found. The correlation between the PC12 concentration maximizing the total number of penetrated sperm per treatment and the bull nonreturn rate was ?.96. It was concluded that PC 12 liposomes induce the AR in bull spermatozoa, which enables them to penetrate zona-free hamster eggs. High fertility bulls required less lipid to induce the AR than did lower fertility bulls. Consequently, this assay of fresh semen could provide a laboratory method to estimate the fertility of a bull.     

Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis

At fertilization, spermatozoa bind to the zona pellucida (ZP1, ZP2, ZP3) surrounding ovulated mouse eggs, undergo acrosome exocytosis and penetrate the zona matrix before gamete fusion. Following fertilization, ZP2 is proteolytically cleaved and sperm no longer bind to embryos. We assessed Acr3-EGFP sperm binding to wild-type and huZP2 rescue eggs in which human ZP2 replaces mouse ZP2 but remains uncleaved after fertilization. The observed de novo binding of Acr3-EGFP sperm to embryos derived from huZP2 rescue mice supports a ;zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for at least 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

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.     

Remodeling of the actin cytoskeleton during mammalian sperm capacitation and acrosome reaction

The sperm acrosome reaction and penetration of the egg follow zona pellucida binding only if the sperm has previously undergone the poorly understood maturation process known as capacitation. We demonstrate here that in vitro capacitation of bull, ram, mouse, and human sperm was accompanied by a time-dependent increase in actin polymerization. Induction of the acrosome reaction in capacitated cells initiated fast F-actin breakdown. Incubation of sperm in media lacking BSA or methyl-beta-cyclodextrin, Ca(2+), or NaHCO(3), components that are all required for capacitation, prevented actin polymerization as well as capacitation, as assessed by the ability of the cells to undergo the acrosome reaction. Inhibition of F-actin formation by cytochalasin D blocked sperm capacitation and reduced the in vitro fertilization rate of metaphase II-arrested mouse eggs. It has been suggested that protein tyrosine phosphorylation may represent an important regulatory pathway that is associated with sperm capacitation. We show here that factors known to stimulate sperm protein tyrosine phosphorylation (i.e., NaHCO(3), cAMP, epidermal growth factor, H(2)O(2), and sodium vanadate) were able to enhance actin polymerization, whereas inhibition of tyrosine kinases prevented F-actin formation. These data suggest that actin polymerization may represent an important regulatory pathway in with sperm capacitation, whereas F-actin breakdown occurs before the acrosome reaction.     

Acrosome reaction in the cumulus oophorus revisited: involvement of a novel sperm-released factor NYD-SP8

Fertilization is a process involving multiple steps that lead to the final fusion of one sperm and the oocyte to form the zygote. One of the steps, acrosome reaction (AR), is an exocytosis process, during which the outer acrosome membrane fuses with the inner sperm membrane, leading to the release of acrosome enzymes that facilitate sperm penetration of the egg investments. Though AR has been investigated for decades, the initial steps of AR in vivo, however, remain largely unknown. A well elucidated model holds the view that AR occurs on the surface of the zona pellucida (ZP), which is triggered by binding of sperm with one of the ZP glycosylated protein, ZP3. However, this model fails to explain the large number of ‘falsely’ acrosome-reacted sperms found within the cumulus layer in many species examined. With the emerging evidence of cross-talk between sperm and cumulus cells, the potential significance of AR in the cumulus oophorus, the outer layer of the egg, has been gradually revealed. Here we review the acrosome status within the cumulus layer, the cross-talk between sperm and cumulus cells with the involvement of a novel sperm-released factor, NYD-SP8, and re-evaluate the importance and physiological significance of the AR in the cumulus in fertilization.     

Comparison of the penetrability of the egg vestments in follicular oocytes, unfertilized and fertilized ova of the rabbit

Mixed populations of rabbit ovulated eggs and follicular oocytes, one labelled with a fluorescent marker, were transferred to the same tubal ampulla of an inseminated recipient female and were then recovered 3 hr later. There was no significant difference in the number spermatozoa penetrating to the perivitelline space or within the substance of the zona pellucida of follicular oocytes (immature or atretic) and mature ovulated ova. In contrast to mature ovulated ova, however, none of the spermatozoa reaching the perivitelline space of vesicular (dictyate) oocytes had attached to or penetrated the vitelline surface to enter the ooplasm.The same approach involving transfer of nonpenetrated eggs together with eggs penetrated previously in a donor female, demonstrated that prior entry of spermatozoa does not reduce the penetrability or receptivity of the rabbit zona pellucida to subsequent spermatozoa.These experiments indicate: (a) that the penetrability of the granulosa cell investment and/or zona pellucida of the rabbit follicular oocyte does not change from the time of antrum formation until the point at which follicular atresia ensures; (b) that between the time of initial LH stimulation and ovulation important changes mediating the onset of the fertizability of the dictyate oocyte of the rabbit probably occur at the vitelline surface; and (c) that in neither a qualitative nor quantitative sense has the demonstrably greater resistance of the rabbit zona pellucida to proteolysis following fertilization any physiological significance for sperm penetration.     

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.     

Effect of dilauroylphosphatidylcholine liposomes on motility, induction of the acrosome reaction, and subsequent egg penetration of ram epididymal sperm

The effects of dilauroylphosphatidylcholine (PC12) on ram epididymal sperm motility, acrosome reaction (AR) induction, plasma membrane permeability, mitochondrial function, and sperm penetration into zona-free hamster eggs were determined. PC12 (50 microM) induced cell motility in caput and cauda sperm, as measured by subjective estimation and automated motility analysis. Motion parameters of treated caput sperm approached those of control ejaculated sperm. Flow cytometric analysis revealed that membrane permeability to propidium iodide and mitochondrial uptake of rhodamine 123 changed during epididymal transit. PC12 induced the AR in sperm from all epididymal regions relative to control incubated sperm (caput 17% vs. control 8%; corpus 29% vs. control 13%; proximal cauda 48% vs. control 4%; distal cauda 51% vs. control 9%). After PC12 treatment, egg penetration by sperm was increased for sperm from the corpus (corpus 7% vs. control 0%) and cauda (proximal 48% vs. control 0%; distal 51% vs. control 0%), but not for caput sperm (caput 0% vs. control 0%). These studies establish that some sperm in each region of the epididymis possess the capacity for movement and the AR. Caput sperm, however, were unique in that they could not penetrate eggs. Additional maturational changes must occur in the caput and/or corpus epididymidis before penetration capacity can be expressed.     

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

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

Zona drilling increases the penetrability of rat oocytes matured in vitro

Immature rat follicular oocytes were cultured either with cumulus cells intact (CI) or cumulus-free (CF) in bovine serum albumin (BSA)- or serum-supplemented medium under conditions in which meiotic maturation occurs spontaneously. After 12 h of culture to permit in vitro maturation (IVM), the cumulus cells were stripped from the CI group. Control oocytes recovered 2-4 h after ovulation from oviducts of pregnant mare's serum gonadotropin (PMSG)-treated rats were similarly stripped of cumulus cells. Half the oocytes in each group had holes "drilled" in their zonae pellucidae by topical application of acid Tyrode's solution with a micropipette to enable bypass of the zona barrier to penetration. They were cultured for a further 14-16 h with epididymal sperm and then were assessed for sperm penetration and pronuclear formation. In a preliminary study using various concentration of sperm, 50,000 sperm/ml was identified as an appropriate concentration and was used in all subsequent experiments. For oocytes matured in serum-supplemented medium, penetration rates of non-drilled oocytes-expressed as a percentage of oocytes exposed to sperm for CF, CI, and ovulated oocytes were 10%, 34%, and 80%, respectively (p less than 0.01). Drilling significantly increased the penetration rates of both IVM groups (CF: 40%, CI: 77%) but not of ovulated oocytes (78%). Forty-one percent of non-drilled CF oocytes failed to form normal pronuclei after penetration. This was significantly higher than either the CI (0%) or ovulated (1%) groups (p less than 0.001). Drilling increased the incidence of failure to form normal pronuclei in penetrated oocytes of the CF group (64%) but not of the CI or ovulated groups.2z=     

Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster

Using a semi-chemically defined medium, the requirement of extracellular Ca²⁺ for survival, capacitation, and acrosome reaction of spermatozoa as well as various stages of fertilization in the hamster was studied. A Ca²⁺-deficient environment is unfavorable for long-term survival of spermatozoa. Sperm capacitation may occur in Ca²⁺-deficient media, but not as efficiently as in normal media. The acrosome reaction definitely requires extracellular Ca²⁺. Other processes or phenomena that require extracellular Ca²⁺ are initiation and maintenance of hyperactivated motility of spermatozoa, penetration of acrosome-reacted spermatozoa into the zona pellucida, fusion of the spermatozoa with eggs, and the development of pronuclear eggs into two-cell embryos. Extracellular Ca²⁺ is apparently unnecessary for the attachment of spermatozoa to the zona and egg surfaces, decondensation of the sperm nucleus, and the development of sperm and egg pronuclei within the egg. These results were compared with data obtained in other species such as the sea urchin, mouse, rat and guinea pig.     

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.     

Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.