Xenoestrogenic compounds promote capacitation and an acrosome reaction in porcine sperm

There is growing evidence that endocrine disruptors bind to hormone receptors; since these receptors are present on the sperm membrane, sperm are potentially a useful model for examining estrogenic activities of endocrine disruptors. The objective of the present study was to compare the effects of two xenoestrogenic compounds (genistein and 4-tert-octylphenol) to those of two steroids (estrogen and progesterone) and heparin on in vitro capacitation and the acrosome reaction in a porcine sperm model. Porcine sperm were incubated with various concentrations (0.001-100 μM) of each chemical for 15 or 30 min, and then capacitation and the acrosome reaction were assessed using chlortetracycline. Estrogen and progesterone were considerably more potent than the other chemicals in stimulating capacitation. Estrogen stimulated sperm capacitation at all tested concentrations after 15 min of incubation (P < 0.05), whereas progesterone stimulated sperm capacitation at all tested concentrations after 15 and 30 min (P < 0.05). The effect of genistein on sperm capacitation was comparable with that of estrogen, and it was the most potent in stimulating the acrosome reaction. Genistein stimulated the acrosome reaction at all tested concentrations after 30 min (P < 0.05). However, 4-tert-octylphenol had the least effect on capacitation and the acrosome reaction. In summary, since all chemicals studied effectively altered capacitation and the acrosome reaction, it was concluded that porcine sperm could be a useful model for in vitro screening of potential endocrine disruptors. It was noteworthy that concurrent comparisons to steroids increased the ability to determine estrogenic characteristics of the tested chemicals.     

The N-terminal of a heparin-binding sperm membrane mitogen possess lectin-like sequence

Glycosaminoglycans like heparin and heparin sulfate in follicular fluid induce changes in the intracellular environment during the spermatozoal functional maturation. We previously reported the isolation, purification and partial characterization of a heparin binding sperm membrane protein (HBSM). In the present study, the amino acids analysis provided evidence of a single sequence, which suggest the homogeneity of the purified HBSM. Fourteen amino acids--(1)A D T I V A V E L D T Y P N(14)--correspond to the amino terminal sequence of Concanavalin A (Con A) and contain 45.2% carbohydrate by weight. HBSM possess mitogenic property on lymphocytes with comparable magnitude to the well-known mitogen; Con A, inducing 83% radiolabel thymidine incorporation in growing lymphocytes. Unlike Con A, there was no agglutination of cell by HBSM upto 5 ng/ml concentration. Interestingly, we found that heparin and chondroitin sulfate-conjugated HBSM inhibit the proliferative activity. Similar effect was also found with an in-house isolate sulfated glycans; G-I (28% sulfate). In contrast, there was no inhibition by the desulfated form; G-ID. Altogether, our data suggest that the mechanism of cell proliferative pathway may be different for HBSM and Con A.     

Binding of a murine proteinase inhibitor to the acrosome region of the human sperm head

Proteinase inhibitors are present in the various glands, tissues, and secretions of the male reproductive tract. Some of these inhibitors bind to the acrosomal region of the sperm, and their release during in vitro or in utero incubation suggests that they may play a role in capacitation. In the mouse, the binding site for a trypsin-acrosin inhibitor, the acceptor, has been implicated in capacitation, zona binding, and the acrosome reaction. This presentation demonstrates that a component, molecular weight ?T20,000, on the human sperm head may recognize the murine inhibitor. Furthermore, the acrosome reaction can be induced in capacitated human sperm by immunoaggregation of bound murine inhibitor. The data indicate that the proteinase inhibitor binding site on the human sperm head may, as with a similar site on murine sperm, play a role in the early events of fertilization. © 1993 Wiley-Liss, Inc.     

Differential involvement of rat sperm choline glycerophospholipids and sphingomyelin in capacitation and the acrosomal reaction

Rat spermatozoa main lipid classes and their fatty acids were studied to assess their possible changes in capacitation and the acrosomal reaction (AR), induced in vitro. Capacitation-associated protein tyrosine phosphorylation, and the efflux of 30% of the total cholesterol from gametes to the medium, took place concomitantly with the release of a similar percentage, i.e., a larger amount, of the total phospholipid, mostly after hydrolysis of the major choline glycerophospholipids (CGP). Main medium lipid metabolites after capacitation were lyso-CGP and polyenoic fatty acids typical of CGP (22:4n-9, 22:5n-6), as free fatty acids (FFA). The AR, induced by a calcium ionophore, resulted in further phospholipid loss, but the produced metabolites remained in the gametes. CGP decrease in AR accounted for some additional FFA and lyso-CGP, but mostly for (22:5n-6-rich) diglycerides. Hydrolysis of sphingomyelins (SM) to ceramides also occurred, mostly affecting species with very long chain polyenoic fatty acids. Quantitatively, CGP and SM were the lipid classes decreasing the most after capacitation and AR, respectively. The massive cholesterol and phospholipid loss from the gametes during capacitation is thus associated with protein phosphorylation, a function that has been located to the sperm tail. The lipid metabolites produced during AR, by accumulating in the gamete heads, could be implicated in sperm–oocyte interactions.     

Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatoza by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization. © Wiley-Liss, Inc.     

Formation and dynamic alterations of horizontal microdomains in sperm membranes during progesterone-induced acrosome reaction

A peptidomics approach was applied to determine the peptides in the larval central nervous system of the grey flesh fly, Neobellieria bullata. Fractions obtained by high performance liquid chromatography were analysed by MALDI-TOF and ESI-Q-TOF mass spectrometry. This provided biochemical evidence for the presence of 18 neuropeptides, 11 of which were novel Neobellieria peptides. Most prominently present were the FMRFamide-related peptides: 7 FMRFamides, 1 FIRFamide, and Neb-myosuppressin. The three putative capa-gene products Neb-pyrokinin and the periviscerokinins Neb-PVK-1 and -2 were detected, as well as another pyrokinin. This Neb-PK-2 was also present in the ring gland along with corazonin, Neb-myosuppressin, and Neb-AKH-GK, an intermediate processing product of the adipokinetic hormone. Furthermore, the central nervous system contained Neb-LFamide, proctolin, and FDFHTVamide, designated as Neb-TVamide. With this study, we considerably increased our knowledge of the neuropeptidome of the pest fly N. bullata, which is an important insect model for physiological research.     

ABCG2 is expressed in late spermatogenesis and is associated with the acrosome

An increasingly exploited strategy for the isolation of stem cells is based on the increased efflux of Hoechst 33342 lipophilic dye mediated by ABCG2, an ATP-binding cassette transporter which is highly expressed in various stem cells. We found ABCG2 expression to be present at later stages of spermatogenesis. Western blot analysis using an anti-ABCG2 antibody revealed expression of a 72 kDa band in mature sperm obtained from mice, rats, bulls or humans. Immunocytochemistry studies revealed acrosomal staining pattern of ABCG2 in spermatozoa. Experiments using the Hoechst 33342 ABCG2 substrate and the ABCG2-specific inhibitor FTC demonstrated efflux activity of ABCG2 in mature sperm. Incubation of sperm in capacitating medium in the presence of the ABCG2-inhibitor FTC resulted in decreased cholesterol depletion compared to sperm incubated in the absence of FTC. Our results demonstrate that ABCG2 is expressed at the acrosome in mature sperm. ABCG2 may thus serve to mediate cholesterol removal.     

Enhancement of acrosome reaction and subzonal insemination of a single spermatozoon in mouse eggs.

The acrosome reaction in mouse spermatozoa was induced by various means. These were 1) varying incubation time in T6 medium, 2) incubation in T6 medium with added A23187, 3) incubation in T6 medium with added dbcGMP and imidazole, 4) exposure to an electric field, and 5) a combination of incubation in a medium with dbcGMP and imidazole and electroporation. The mean percentages of acrosome-free spermatozoa obtained by these various methods and assessed on the basis of both Bryan's stain and immunolocalization by FITC-labeled monoclonal antibodies increased by steps from 36% to 67%, 73%, 86%, and 92%. Individual spermatozoa from the various treatments were afterwards microinjected under the zona pellucida of a mouse oocyte. The fertilization rate for eggs microinjected with a spermatozoon treated with A23187, dbcGMP, and imidazole, by electroporation and by a combination of the last two methods also increased by steps from 17% to 34%, 36%, and 70%, respectively. Ninety-five percent of the fertilized oocytes reached the early blastocyst stage, thirty-eight percent of these blastocysts implanted in pseudopregnant mice, and twenty-eight percent developed to term. These results indicated the varying degrees of success of different ways of inducing acrosomal loss in spermatozoa and their subsequent success rates in fertilization and further in vitro and in vivo development.     

Signal transduction pathways that regulate sperm capacitation and the acrosome reaction

Ejaculated spermatozoa must undergo physiological priming as they traverse the female reproductive tract before they can bind to the egg’s extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fertilize the egg. The preparatory changes are the net result of a series of biochemical and functional modifications collectively referred to as capacitation. Accumulated evidence suggests that the event that initiates capacitation is the efflux of cholesterol from the sperm plasma membrane (PM). The efflux increases permeability and fluidity of the sperm PM and causes influx of Ca²⁺ ions that starts a signaling cascade and result in sperm capacitation. The binding of capacitated spermatozoa to ZP further elevates intrasperm Ca²⁺ and starts a new signaling cascade which open up Ca²⁺ channels in the sperm PM and outer acrosomal membrane (OAM) and cause the sperm to undergo acrosomal exocytosis. The hydrolytic action of the acrosomal enzymes released at the site of sperm-egg (zona) binding, along with the hyperactivated beat pattern of the bound spermatozoon, are important factors in directing the sperm to penetrate the ZP and fertilize the egg. The role of Ca²⁺-signaling in sperm capacitation and induction of the acrosome reaction (acrosomal exocytosis) has been of wide interest. However, the precise mechanism(s) of its action remains elusive. In this article, we intend to highlight data from this and other laboratories on Ca²⁺ signaling cascades that regulate sperm functions.     

"In vitro" capacitation and subsequent acrosome reaction are related to changes in the expression and location of midpiece actin and mitofusin-2 in boar spermatozoa

The induction of “in vitro” capacitation (IVC) and subsequent, progesterone-induced “in vitro” acrosome reaction (IVAR) was concomitant with an increase in actin polymerization, also showing an increase in actin presence at the apical area of the midpiece. The presence of mitofusin-2, a protein involved in the regulation of the coordinated mitochondrial function, expanded from midpiece to the principal piece after IVC and IVAR. All of these results indicate that the increase of boar sperm mitochondrial activity during IVC and the first minutes of IVAR is concomitant with changes in the expression and location of both actin and mitofusin-2. Our results suggest that both actin and mitofusin-2 play important roles in the modulation of boar sperm mitochondrial function, both by originating changes in the protein membrane environment and by changes in the mitochondrial structure itself.     

Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba2+, and has a PK+/PNa+ selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization.     

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A₂, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A₂ activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.     

Subzonal insemination with a single spermatozoon using manipulation assisted sperm adhesion onto the ooplasmic membrane in mouse ova.

A simple and successful method of microinjection of a single spermatozoon under the zona pellucida of a mouse oocyte has been developed. A characteristic of this method is that the tip of the sperm injection needle pierces the zona pellucida without touching the ooplasmic membrane. All the ova (277) used for this series of experiments had normal morphology after the injection procedure. Spermatozoa preincubated in culture medium for capacitation and those treated with ionophore A23187 for induction of acrosome reaction were used. In combination with some of these injections, a manipulation assisting the adhesion of the sperm head onto the ooplasmic membrane was employed. The fertilization rate (67.3%) of the ova injected with the ionophore-treated sperm using the sperm-adhesion treatment was significantly higher (P less than 0.005) than that obtained by the injection of the preincubated sperm without applying the adhesion treatment (23.6%). All three of the recipients that received the 24 fertilized ova became pregnant and gave birth to 11 offspring (45.8%). The inseminations performed with the sperm-adhesion treatment using the immotile sperm from the preincubated population and/or those from the ionophore-treated population did not result in fertilization in any case. These results suggest that the fertilization rate of subzonal insemination with motile ionophore-treated sperm can be improved by applying the sperm-adhesion treatment and that sperm motility might be involved in the establishment of fertilization, even after the adhesion of the sperm head with the mouse ovum membrane.     

Effect of reactive oxygen species on capacitation and associated protein tyrosine phosphorylation in buffalo (Bubalus bubalis) spermatozoa

In the present study, the effect of two particular reactive oxygen species (ROS), superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) on buffalo (Bubalus bubalis) sperm capacitation and associated protein tyrosine phosphorylation was studied. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50 x 10(6)/mL and incubated at 38.5 degrees C for 6h with or without heparin (10(g/mL; a positive control), or xanthine (X; 0.5mM)-xanthine oxidase (XO; 0.05 U/mL)-catalase (C; 2100 U/mL) system that generates O(2)(-) or NADPH (5mM) that stimulates the endogenous O(2)(-) production or H(2)O(2) (50 microM). The specific effect of O(2)(-), H(2)O(2) and NADPH on buffalo sperm capacitation and protein tyrosine phosphorylation was assessed by the addition of superoxide dismutase (SOD), catalase and diphenylene iodonium (DPI), respectively, to the incubation medium. Each of X+XO+C system, NADPH and H(2)O(2) induced a significantly higher percentage (P<0.05) of capacitation in buffalo spermatozoa compared to control. However, DPI inhibited this NADPH-induced capacitation and protein tyrosine phosphorylation and suggested for existence of an oxidase in buffalo spermatozoa. Using immunoblotting technique, at least seven tyrosine-phosphorylated proteins (20, 32, 38, 45, 49, 78 and 95 kDa) were detected in capacitated buffalo spermatozoa. Out of these, the tyrosine phosphorylation of p95 was induced extensively by both O(2)(-) as well as exogenous source of H(2)O(2) and using specific activators and inhibitors of signaling pathways, it was found this induction was regulated through a cAMP-dependent PKA pathway. Further, immunofluorescent localization study revealed that these ROS-induced tyrosine-phosphorylated proteins are mostly distributed in the midpiece and principal piece regions of the flagellum of capacitated spermatozoa and suggested for increased molecular activity in flagellum during capacitation. Thus, the study revealed that both O(2)(-) and H(2)O(2) promote capacitation and associated protein tyrosine phosphorylation in buffalo spermatozoa and unlike human and bovine, a different subset of sperm proteins were tyrosine-phosphorylated during heparin- and ROS-induced capacitation and regulation of these ROS-induced processes were mediated through a cAMP/PKA signaling pathway.     

Effect of hen's egg yolk on capacitation and acrosome reaction of diluted canine spermatozoa

The aim of this study was to investigate the influence of progesterone, cholesterol and calcium (Ca²⁺) in an egg-yolk-containing extender on capacitation and acrosome reactions (AR) of diluted canine spermatozoa during 4 days of cooled-storage. For this purpose, we first investigated the effect of supplementation of a Tris–citrate–fructose buffer (TCF) with progesterone in a final concentration of 0.1, 0.2 and 1.0 μg progesterone/ml TCF-diluted semen. We then compared the effects of TCF and the same buffer-containing 20% egg yolk (TCF–EY). In egg yolks and the TCF–EY, progesterone was measured by enzyme immunoassay, cholesterol by enzymatic colorimetry and Ca²⁺ by flame atomic absorption spectrophotometry. For both experiments, ejaculates from eight dogs were used. For the comparison of diluents, one ejaculate was divided and one half diluted with TCF, the other with TCF–EY. One half of each TCF- and TCF–EY-diluted sample was evaluated immediately (D1), the other after storage for 4 days at +4 °C (D4). In diluted semen, motility and viability were measured by a computer assisted sperm analyzer (CASA; Sperm Vision, Minitüb, Germany), capacitation and AR were evaluated with a modified chlortetracycline assay (CTC) and the AR additionally by flow cytometry. Results: Supplementation of progesterone revealed, that between D1 and D4, total and progressive motility decreased with all progesterone concentrations, while viability as well as percentage of capacitated and acrosome reacted spermatozoa stayed constant. Progesterone-, cholesterol- and Ca²⁺ concentrations in egg yolks were 524.8 ± 131.4 ng/g, 13.9 ± 2.03 mg/g and 1.27 ± 0.17 mg/g, respectively. In the TCF–EY-diluent, the respective values were 210.9 ng/g, 2.52 mg/g and 1.1 mg/g. In TCF–semen, at D1, motility and viability were significantly higher than in TCF–EY-samples (p < 0.05), however at D4, no significant differences were detectable. Further, in TCF–semen, percentages of spermatozoa with intact membranes decreased significantly (p < 0.05) and capacitated spermatozoa increased (p < 0.05), which was not seen in TCF–EY-samples. In all samples, low percentages of AR were detected and after 4 days, the highest value of AR in TCF–EY-samples was 5.3% on average, as detected by flow cytometry. We therefore conclude that progesterone from egg yolk in routine extenders does not substantially influence semen longevity or AR of canine semen during cold-storage for 4 days. In contrary, egg yolk seems to prevent a significant increase in capacitated spermatozoa.     

Implication of cAMP during porcine sperm capacitation and protein tyrosine phosphorylation

Second messengers are involved in sperm fertilizing potential, as both motility and the acrosome reaction are influenced by cAMP. Moreover, the activity of cyclic nucleotides is implicated in the appearance of tyrosine phosphorylated sperm proteins, which is associated with capacitation in the mammalian spermatozoa. Nevertheless, the involvement of the cAMP/protein kinase A (PK-A) pathway during pig sperm capacitation may be different from that observed in other mammals. The objective of the present study was to clarify the cAMP/PK-A pathway during the capacitation of porcine spermatozoa and to evaluate this impact on the p32 sperm tyrosine phosphoprotein appearance. The presence of p32 was assessed after incubating fresh pig sperm with IBMX/db-cAMP, H-89, a PK-A inhibitor or bistyrphostin, a tyrosine kinase inhibitor, in capacitating (CM) or non-capacitating conditions (NCM) by immunoblotting SDS-extracted and separated sperm proteins using an anti-phosphotyrosine antibody. When pig spermatozoa were incubated in CM supplemented with H-89 (50 microM) or bistyrphostin (1.2 microM), capacitation decreased significantly (P < 0.001). The p32 sperm tyrosine phosphoprotein, previously shown to be associated with capacitation of porcine sperm though not necessarily an end point of this phenomenon, was not modulated by IBMX/db-cAMP (100 microM/1 mM), H-89 (50 microM) nor bistyrphostin (1.2 microM). Our results indicate, therefore, that pig sperm are regulated somewhat differently than as described for other mammals, because although the cAMP/PK-A and tyrosine kinase pathways are involved in capacitation, they do not influence the appearance of p32.     

Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm.

Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place. Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2, 2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 microM, and with an EC50 of 100 microM. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium. In contrast, addition of 500 microM dibutyryl cAMP (dbcAMP), alone or together with 100 microM 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium. These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium. When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX. The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation.     

Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.