Protein serine and threonine phosphorylation,hyperactivation and acrosome reaction in in vitro capacitated hamster spermatozoa

Monoclonal antibodies against phosphoserine and phosphothreonine were used in the present study to investigate the changes in serine and threonine phosphorylation respectectively during capacitation of hamster spermatozoa. Immunoblot analysis of hamster spermatozoa capacitated in TALP, a medium that supports capacitation, showed that a set of four proteins of molecular weight 56, 63, 66, and 100 kDa was phosphorylated both at the serine and threonine residues. In addition, five other proteins of molecular weight 32, 39, 45, 53, and 61 kDa were phosphorylated specifically at the threonine residues. Of these nine proteins, the 100 kDa protein showed a time dependent or capacitation-dependent decrease in intensity which coincided with the percentage acrosome-reacted spermatozoa. In contrast, the 49 and 63 kDa threonine phosphorylated proteins showed increased phosphorylation coinciding with capacitation. H8 (a serine and threonine kinase inhibitor) had a transient effect on the phosphorylation of these two phosphothreonine proteins but inhibited acrosome reaction substantially all through the treatment period. Okadaic acid (OA) (a serine and threonine protein phosphatase inhibitor) inhibited hyperactivation but had no effect on acrosome reaction. In fact, OA stimulated acrosome reaction. Finally the immunofluorescence studies indicated localization of the serine phosphorylated proteins in tail as well as in head of the capacitated hamster spermatozoa whereas the threonine phosphorylated proteins were localized mostly in the tail of the spermatozoa. The findings of the present study suggest that serine/threonine phosphorylation and the enzymes responsible for regulating the level of phosphorylation play an important role in capacitation and capacitation-associated events namely hyperactivation and acrosome reaction. However, further studies are needed in order to establish the exact role of these proteins in capacitation of spermatozoa.     

Capacitation,acrosome reaction,and egg penetration by canine spermatozoa in a simple defined medium

A defined medium (CCM) is described in which washed ejaculated canine spermatozoa can be induced to undergo capacitation and the acrosome reaction, and to penetrate corona-free eggs in vitro. The composition of the medium is similar to other Krebs-Ringer's bicarbonate buffered media except for the absence of magnesium and adjustments in the concentration of NaHCO₃, glucose, and albumin. The concentration of NaCI is adjusted to maintain the osmolality at approximately 300 mOsm, and the pH is 7.8 under 5% CO₂ in air. This pH was found more favorable for the occurrence of the acrosome reaction than the lower pH (7.4) of media with more usual bicarbonate concentrations. Calcium ions are essential not only for the occurrence of the acrosome reaction of canine spermatozoa, but also for their motility. Potassium ions are apparently necessary for the process of sperm-egg fusion following zona penetration. When CCM was compared with two other media, it allowed the best combination of good rates for acrosome reaction (46%), motility (83%), and zona penetration (71%). Sperm-egg fusion also occurred. The requirements for capacitation of canine spermatozoa are compared with those of some other species.     

Observations on the acrosome reaction of human sperm in vitro

Human sperm were incubated in vitro in serum or the defined medium TMPA and were periodically assessed for acrosome reactions using two new methods of assay. The first method, FITC-RCA labeling, was previously shown to be valid for estimating the percentage of normal acrosome reactions of human sperm. The second method, a triple staining technique, is shown in this study to give results comparable to those obtained with FITC-RCA labeling. The percentage of acrosome-reacted sperm was determined at 0, 2.5, 5, and 7 hr of incubation. In both media, some sperm had reacted by 2.5 hr; a maximum percentage of reactions occurred between 5 and 7 hr. The maximum percentage never exceeded 20–25%, which represents only one-third of the live sperm, ie, those potentially able to undergo normal acrosome reactions. It will be important in future studies to determine if this low-peak percentage is due to the fact that: (1) Commonly used culture media are suboptimal or (2) only about 25% of the sperm in a human ejaculate are capable of undergoing normal acrosome reactions.     

Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids in the acrosome reaction

The effects of lipids on the survival, acrosome reaction, and fertilizing capacity of guinea pig spermatozoa were studied by incubating the spermatozoa in media containing various concentrations of the lipids. Lipids tested were: phosphatidyl-choline (PC), -ethanolamine (PE), -inositol (PI), -serine (PS), sphingomyelin (S), cholesterol (C), lysophosphatidyl-choline (LC), -ethanolamine (LE), -inositol (LI), -serine (LS), and glyceryl monooleate (M). When spermatozoa were incubated in a regular medium (containing 2 mM Ca²⁺) with M, the majority underwent the acrosome reaction within 1 hour. None of the other lipids were as effective as M, and some were totally ineffective under the same conditions. However, when spermatozoa were preincubated in Ca²⁺-free medium containing LC, LE, or LI, they gained the ability to undergo the acrosome reaction. One hour of preincubation in Ca²⁺-free medium with LC, LE, or LI was enough to render the vast majority of spermatozoa capable of undergoing the acrosome reaction in response to Ca²⁺. The optimum concentrations for LC, LE, and LI were approximately 85 μg/ml, 210 μg/ml, and 140 μg/ml, respectively. Spermatozoa that had undergone the acrosome reaction by pretreatment with LC, LE, or LI remained actively motile and were capable of fertilizing eggs. LS was totally ineffective in rendering the spermatozoa capable of undergoing the acrosome reaction, and in fact it inhibited the acrosome reaction by itself and also inhibited the LC-, LE-, or LI-mediated acrosome reaction. LS did not prevent acrosome-reacted spermatozoa from penetrating the zona pellucida, but did prevent sperm-egg fusion. Based on these findings, it is suggested that lysophospholipids are intricately involved in the sperm acrosome reaction and perhaps in sperm-egg fusion.     

Effect of the human cumulus oophorus on movement characteristics of human capacitated spermatozoa

The effect of human cumulus oophorus on movement characteristics of human spermatozoa previously incubated in vitro under capacitating conditions was studied using automated digital image analysis. When spermatozoa were incubated for a short time with whole cumuli, most of those that penetrated the cumulus intercellular matrix were characterized by a linear movement with small amplitudes of lateral head displacement, but with elevated values of beat cross frequency. Short (5 min) incubation with solubilized cumulus intercellular matrix of spermatozoa preincubated in capacitating conditions (6 h) significantly reduced the percentage of spermatozoa showing the 'hyperactivated' type of motility characterized by high curvilinear velocity, low progressive velocity and elevated values of lateral head displacement. Moreover, a subpopulation of spermatozoa with very high values of progressive velocity and beat cross frequency and with reduced amplitudes of lateral head displacement appeared in these conditions. This cumulus-related motility pattern was not seen in fresh spermatozoa or in those incubated in the absence of cumulus material. Changes in the sperm movement characteristics similar to those observed in the presence of the solubilized cumulus matrix could also be induced by some of its h.p.l.c. fractions. These results show that the intercellular matrix of the human cumulus oophorus exerts a specific effect on human sperm motility, probably acting preferentially on the 'hyperactivated' sperm subpopulation.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W-7

The effect of the calmodulin antagonist W-7 on the capacitation and the acrosome reaction of guinea pig spermatozoa was examined. The characteristic features of the acrosome reaction induced by W-7 were the dependence on the composition and pH of the medium and on the presence of sodium bicarbonate. The most effective concentration of W-7 for inducing the acrosome reaction was approximately 5 μM, which is far less than the Kd for calmodulin. Moreover, W-7 enhanced the ability of spermatozoa to acquire capacitation in a Ca²⁺-free medium. The spermatozoa induced to undergo the acrosome reaction by W-7 were capable of penetrating the zona-free hamster eggs. W-5, which has a lower affinity for calmodulin than W-7, also induced the acrosome reaction in the same manner as W-7. These results suggest that the naphthalenesulfonamide derivatives W-7 and W-5 can induce the acrosome reaction in guinea pig spermatozoa via capacitation in a pH-dependent, Ca²⁺-calmodulin-independent manner.     

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.     

Solubilized zona pellucida proteins and progesterone induce calcium influx and the acrosome reaction in capacitated dog spermatozoa.

Spermatozoa from the sperm-rich fractions of the semen of 6 beagle dogs were capacitated and the effect of both zona pellucida (ZP) proteins and progesterone on calcium flux and the acrosome reaction measured. Sperm calcium flux was determined using the dual wavelength calcium probe indo-1/AM (6 microM) in a flow cytometric assay (one ejaculate from each dog examined; n = 6). No calcium flux was observed in the negative control treatments (RPMI medium or DMSO). Both heat-solubilized bitch ZP proteins and progesterone caused a similar response characterized by a gradual but marked influx of calcium ions which was sustained over 2 min. Acrosomal status was assessed by indirect immunofluorescence using a specific monoclonal antibody following 1 hr incubation for each treatment (four ejaculates from each dog examined; n = 24). The level of acrosomal exocytosis was very high for samples treated with ZP proteins (70.3 +/- 2.1%) and progesterone (84.6 +/- 1.5%) and was significantly different from the respective controls (P < 0.001). Interestingly the patterns of calcium flux in response to both ZP proteins and progesterone were in contrast to the situation in other species studied to date raising the possibility that the mechanism for triggering the acrosome reaction may be different in dog spermatozoa. In addition the high degree of progesterone-induced acrosomal exocytosis compared to other species raises the probability that the majority of dog spermatozoa are already undergoing the acrosome reaction before they reach the egg ZP.     

Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm

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

Procedures for obtaining high percentages of viable in vitro capacitated hamster sperm

Suspensions of nearly 100% viable golden hamster sperm were prepared by passing washed cauda epididymal sperm through a column of 0.25–0.3 mm glass beads. Incubations of these viable sperm under in vitro capacitation conditions in volumes of 0.1 or 1 ml (2–2.5 × 10⁶/ml) resulted in 85–92% viable sperm after four hours and 45 minutes of incubation. More than 70% of these sperm were judged to have been capacitated after four hours and 45 minutes of incubation on the basis of their having undergone acrosome reactions and the presence of high numbers of sperm exhibiting the activated motility characteristic of capacitated hamster sperm. Thus, for the first time, procedures are available that will yield large numbers of viable capacitated sperm for biochemical analysis and that will also allow other studies of hamster sperm capacitation with minimum interference from molecules released from dead sperm.     

Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa

The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 M W dextran sulfate fertilized more bovine oocytes than control sperm (P <0.05), sperm treated with 500,000 M W dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.     

Production,characterization, and use of ionophore-induced,calcium-dependent acrosome reaction in ram spermatozoa

A system has been developed for inducing a calcium-dependent acrosome reaction in ram spermatozoa in vitro using the calcium ionophore A23187. The resultant reaction is accompanied by release of the acrosomal enzymes hyaluronidase and acrosin, but there is no release of the cytoplasmic enzyme glucose 6-phosphate isomerase. In any given cell, the visible acrosome reaction apparently takes place rapidly, but there is a variable delay before the reaction occurs. Under optimum conditions, about 90% of treated spermatozoa show an acrosome reaction within one hour. Preincubation of the spermatozoa with the proteinase inhibitors p-amino-benzamidine or p-nitrophenylguanidinobenzoate allows two stages of the reaction to be distinguished ultrastructurally, a membrane fusion stage followed by a dispersal of the acrosomal matrix. In the presence of the inhibitors, the first stage is delayed but is completed within 1 hour, whereas the second remains largely incomplete. In the presence of calcium, ionophore concentrations which induce an acrosome reaction abolish sperm motility rapidly and completely. However, by adding serum albumin shortly after addition of ionophore, motility can be preserved while the acrosome reaction occurs as usual; the motility pattern observed under these conditions is of the “whip-lash” or “activated” type. Although the motile ionophore-treated spermatozoa were unsuccessful at penetrating normal mature sheep oocytes in vitro, they were able to penetrate zona-free oocytes, after which swelling and decondensation of the sperm head took place.     

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa

Calmodulin has been postulated as a mediator in the calcium-dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM-PL) after in vitro capacitation 2) by 0.003% Triton-X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM-PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome-reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.     

Evidence of new antigens in the mouse cumulus oophorus during preovulatory cumulus expansion

The effects of an antiserum (anti-COC) against ovulated mouse cumulus-oocyte complexes (COC) on in vitro fertilization of mouse oocytes were studied. Preincubation of ovulated COC with various concentrations of anti-COC led to dose-dependent impairment of fertilization rates as well as to a decrease in the number of spermatozoa attached to the zona pellucida. Anti-COC was used to probe Western blots of cumulus proteins. These cumuli were obtained from 2 experimental groups of mice corresponding to 2 different maturational stages (preovulatory immature COC or preovulatory mature COC). Two antigens (70 and 80 kDa) present in cumulus intercellular matrix from mature COC were only found as traces in matrix from immature COC. In addition, the protein pattern of the cumulus intercellular matrix was different from that of cumulus cells, whatever the COC maturational stage. These results indicate the appearance of new proteins in the cumulus oophorus during preovulatory expansion and are consistent with the contraceptive action of anti-COC. © 1993 Wiley-Liss, Inc.     

Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS-2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell-conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO₂ in air. Viability and the occurrence of true acrosome reactions were assessed using a triple-stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS-2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS-2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS-2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley-Liss, Inc.     

Capacitation and the acrosome reaction in squirrel monkey (Saimiri sciureus) spermatozoa evaluated by the chlortetracycline fluorescence assay

Capacitation and the acrosome reaction in squirrel monkey seminal spermatozoa diluted in Tyrode's medium (TALP) and TC-199 were monitored by a chlortetracycline (CTC) fluorescence assay. Four CTC patterns, similar to those found in human sperm, were readily characterized by fluorescent staining on the heads of the spermatozoa. The appearance of the capacitated (CP) pattern was dependent on the concentration of the bovine serum albumin. Acrosomal loss was observed in a maximum of 15% of the sperm in the populations studied here. Calcium ionophore A23187 (5 μM to 20μM) induced acrosomal loss in 60–70% of capacitated spermatozoa. However in freshly ejaculated sperm incubated under capacitating conditions or in spermatozoa incubated in Ca^{+ +}-free medium, A23187 failed to induce acrosomal loss. Furthermore, spermatozoa incubated in the presence of seminal plasma or spermatozoa obtained following a 1-hour “swim-up” procedure showed an identical timecourse of appearance of the CP pattern, indicating the lack of effect of seminal plasma on capacitation in the squirrel monkey.     

Inhibition of domestic cat spermatozoa acrosome reaction and zona pellucida penetration by tyrosine kinase inhibitors

Spermatozoa from teratospermic domestic cats (>60% morphologically abnormal spermatozoa per ejaculate) consistently exhibit lower levels of oocyte penetration in vitro than their normospermic (<40% abnormal spermatozoa per ejaculate) counterparts. This could be caused by structural abnormalities or intracellular defects resulting in disruption of normal cellular functions. Spermatozoa from teratospermic cats also are compromised in the ability to capacitate and undergo the acrosome reaction (AR) in vitro. Further, we recently identified two tyrosine phosphorylated proteins (95- and 160-kDa) localized over the acrosome region in domestic cat spermatozoa. Phosphorylation of these proteins is reduced in teratospermic compared with normospermic ejaculates. To begin to understand the relationship between tyrosine phosphorylation and sperm function, we examined the effects of two protein tyrosine kinase inhibitors (tyrphostin RG-50864 and genistein) on (1) sperm motility; (2) protein tyrosine phosphorylation; (3) the ionophore A23187-induced AR; (4) the spontaneous and zona pellucida (ZP)–induced AR, and (5) the ability of spermatozoa from normospermic cats to penetrate conspecific ZP-intact oocytes. Over a wide range of concentrations, neither inhibitor affected sperm percentage motility during incubation (P > 0.05). Preincubation with either inhibitor reduced tyrosine phosphorylation of both (95- and 160-kDa) sperm proteins. Although both inhibitors blocked the ZP-induced AR, neither influenced the spontaneous AR nor the A23187-induced AR, suggesting that tyrosine phosphorylation may be involved in physiologic AR. No differences (P > 0.05) were observed in the ability of control or inhibitor-treated spermatozoa to bind to or penetrate the outer ZP layer. However, percentages of oocytes with treated spermatozoa in the inner ZP (tyrphostin, 8.7%; genistein, 20.4%) and perivitelline space (tyrphostin, 0%; genistein, 2.3%) were less (P < 0.001) than untreated controls (inner ZP, 62.7%; perivitelline space, 10.2%). These results (1) demonstrate that ZP-induced acrosomal exocytosis in domestic cat spermatozoa is regulated via a tyrosine kinase–dependent pathway and (2) suggest that defects in these signaling pathways may represent one of the causes for compromised sperm function in teratospermic males. Mol. Reprod. Dev. 49:48–57, 1998. © 1998 Wiley-Liss, Inc.