Evidence for sequential deployment of secretory enzymes during the normal acrosome reaction of guinea pig sperm in vitro

Experiments were conducted to determine if acrosomal enzymes are released simultaneously or in sequence during the normal acrosome reaction. Epididymal guinea pig sperm were incubated in a chemically defined, calcium-containing medium which supports normal acrosome reactions within 4–5 hours at 37°C. The sperm suspensions were monitored for motility, normal acrosome reactions, and false acrosome reactions during in vitro incubation. At specified time intervals, the sperm were separated from the incubation medium by centrifugation, and the distribution of dipeptidyl peptidase (DPP II) and acrosin activity was determined by biochemically assaying the hydrolysis of trialanine and N-benzoyl-L-arginine ethyl ester (BAEE), respectively. When calcium was present, there was a significant increase in DPP II activity in the supernatants by 1 hour of incubation and a slight decline at later time points. This release was not correlated with false or normal acrosome reactions (loss of the acrosomal cap) monitored by phase-contrast microscopy but probably represents a very early stage in the normal acrosome reaction. This early stage is difficult to detect at the light microscope level because sperm are still in rouleaux and because membrane fusion is not directly observable. In contrast, acrosin activity, which was assayed in the same supernatants, increased at later times when sperm were observed to have completed normal acrosome reactions. The ultrastructural distribution of DPP II was determined in sperm pellets collected during in vitro incubation by using the DPP II substrate lysyl-alanyl-4-methoxy-2-naphthyamide. In freshly isolated cauda epidiymal sperm, reaction product is confined to the light-staining area in the dorsal bulge of the acrosome. However, by 1 hour of incubation, the light-staining area of many sperm was partially or completely dispersed, while other regions of the acrosome were unchanged. Our data are consistent with the conclusions that DPP II is a highly soluble component of the guinea pig sperm acrosome and that its release occurs during the initial phase of the acrosome reaction while sperm are still in rouleaux. Structural changes in the acrosome associated with DPP II release were detectable by electron microscopy but not by light microscopy. Acrosin, which is less soluble than DPP II, is released at a later time during the acrosome reaction. Both DPP II and acrosin appear to be partially inhibited following their release from sperm. A complete understanding of the sequential release and extracellular activities of the acrosomal enzymes will be necessary to fully define their functions in fertilization.     

Acrosomal status and motility of guinea pig spermatozoa during in vitro penetration of the cumulus oophorus

Previous studies have suggested that both acrosome-intact and acrosome-reacted guinea pig sperm are capable of binding to the zona pellucida of cumulus-free oocytes, but the acrosomal status of guinea pig sperm during penetration of the cumulus has not been reported. We made video recordings of the interaction between capacitated guinea pig sperm and cumulus-invested guinea pig oocytes. The videotapes were analysed to identify sperm with hyperactivated motility and to classify the acrosomal status of sperm during penetration of the cumulus and after binding to the zona pellucida. The resolution of the video recordings was not sufficient to recognise sperm with swollen acrosomes. However, sperm that had completed the acrosome reaction were easily identified. Acrosome-reacted sperm were found adherent to the outer boundary of the cumulus, but were never observed to penetrate the cumulus. The percentage of acrosome-intact, hyperactivated sperm was higher in the cumulus oophorus than in culture medium, suggesting that changes in motility were elicited in response to contact with the cumulus. Fully acrosome-reacted sperm were found adherent to the zona pellucida, and solubilised guinea pig zona pellucida was capable of inducing acrosome reactions in capacitated guinea pig sperm. Acrosome-intact sperm were also observed on the zona, but they were not tightly bound and did not have hyperactivated motility, suggesting that these sperm were not functionally capacitated. Our observations demonstrate that guinea pig sperm penetrate the cumulus matrix in an acrosome-intact state. Although we did not observe sperm undergoing the acrosome reaction, our observations and experimental data suggest that the acrosome reaction of guinea pig sperm is completed on or near the surface of the zona pellucida.     

猪精子体外获能与顶体反应的超微结构研究

用４种方法,检测了猪精子体外获得的效果。结果证明：高离子浓度的前培养液和猪镦泡液,具有促进获能过程的作用,实验还获得了获能后顶体反尖的一些重要的形态学变化资料,包括质膜的膨胀、断裂、顶体膨胀、顶体外膜内陷或原位局部囊泡化,质膜再全部丢失。顶体内膜直到与卵母细胞质膜融合,才发生可见的变化。受精过程无论体内或体外,都容易发生多精入卵,体外受精则更甚。在精子穿过卵丘细胞之间时,一方面开始进行顶体反应,另     

THE IMPORTANCE OF HYDROLYTIC ENZYMES TO AN EXOCYTOTIC EVENT, THE MAMMALIAN SPERM ACROSOME REACTION

The mammalian sperm acrosome reaction is a unique form of exocytosis, which includes the loss of the involved membranes. Other laboratories have suggested the involvement of hydrolytic enzymes in somatic cell exocytosis and membrane fusion, and in the invertebrate sperm acrosome reaction, but there is no general agreement on such an involvement. Although reference was made to such work in this review, the focus of the review was on the evidence (summarized below) that supports or fails to support the importance of certain hydrolytic enzymes to the mammalian sperm acrosome reaction. Because the events of capacitation, the prerequisite for the mammalian acrosome reaction, and of the acrosome reaction itself are not fully understood or identified, it is not yet always possible to determine whether the role of a particular enzyme is in a very late step of capacitation or part of the acrosome reaction. (1) The results of studies utilizing inhibitors of trypsin-like enzymes suggest that such an enzyme has a role in the membrane events of the golden hamster sperm acrosome reaction. The enzyme involved may be acrosin, but it is possible that some as yet unidentified trypsin-like enzyme on the sperm surface may play a role in addition to or instead of acrosin. Results obtained by others with guinea pig, ram and mouse spermatozoa suggest that a trypsin-like enzyme is not involved in the membrane events of the acrosome reaction, but only in the loss of acrosomal matrix. Such results, which conflict with those of the hamster study, may have been due to species differences or the presence of fusion-promoting phospholipase-A or lipids contaminating the incubation media components, and in one case to the possibly damaging effects of the high level of calcium ionophore used. The role of the trypsin-like enzyme in the membrane events of the hamster sperm acrosome reaction may be to activate a putative prophospholipase and/or to hydrolyse an outer acrosomal or plasma membrane protein, thus promoting fusion. A possible role of the enzyme in the vesiculation step rather than the fusion step of the acrosome reaction cannot be ruled out at present. (2) Experiments utilizing inhibitors of phospholipase-A₂, as well as the fusogenic lysophospholipid and cis-unsaturated fatty acid hydrolysis products that would result from such enzyme activity, suggests that a sperm phospholipase-A₂ is involved in the golden hamster sperm acrosome reaction. Inhibitor and LPC addition studies in guinea pig spermatozoa have led others to the same conclusion. The fact that partially purified serum albumin is important in so many capacitation media may be explained by its contamination with phospholipase-A and/or phospholipids. Serum albumin may also play a role, at least in part, by its removal of inhibitory products released by the action of phospholipase-A₂ in the membrane. The demonstration of phospholipase-A₂ activity associated with the acrosome reaction vesicles and/or the soluble component of the acrosome of hamster spermatozoa, and the fact that exogenous phospholipase A₂ can stimulate acrosome reactions in hamster and guinea pig spermatozoa, also support a role for the sperm enzyme. The actual site or the sites of the enzyme in the sperm head are not yet known. The enzyme may be on the plasma membrane as well as, or instead of, in the acrosomal membranes or matrix. A substrate for the phospholipase may be phosphatidylcholine produced by phospholipid methylation. It is possible that more than one type of ‘fusogen’ is released by phospholipase activity (LPC and/or cis-unsaturated fatty acids, which have different roles in membrane fusion and/or vesiculation. In addition to acting as a potential ‘fusogen’, arachidonic acid released by sperm phospholipase-A₂ probably serves as precursor for cyclo-oxygenase or lipoxygenase pathway metabolites, such as prostaglandins and HETES, which might also play a role in the acrosome reaction. Although much evidence points to a role for phospholipase-A₂, phospholipase-C found in spermatozoa could also have a role in the acrosome reaction, perhaps by stimulating events leading to calcium gating, as suggested for this enzyme in somatic secretory cells. (3) A Mg²⁺-ATPase H⁺-pump is present in the acrosome of the golden hamster spermatozoon. Inhibition of this pump by certain inhibitors of ATPases (but not by those that only inhibit mitochondrial function) leads to an acrosome reaction only in capacitated spermatozoa and only in the presence of external K⁺. The enzyme is also inhibited by low levels of calcium, and such inhibition, combined with increased outer membrane permeability to H⁺ and K⁺, and possibly plasma membrane permeability to H⁺ (perhaps by the formation of channels), may be part of capacitation and/or the acrosome reaction. The pH of the hamster sperm acrosome has been shown to become more alkaline during capacitation, and such a change may result in the activation of hydrolytic enzymes in the acrosome or perhaps in a change in membrane permeability to Ca²⁺. A similar Mg²⁺-ATPase has not been found in isolated boar sperm head membranes. However, that conflicting result could have been due to the use of noncapacitated boar spermatozoa for the preparation of the membranes or to protease modification of the boar sperm enzyme during assay. (4) Inhibition of Na⁺, K⁺-ATPase inhibits the acrosome reaction of golden hamster spermatozoa, and the activity of this enzyme increases relatively early during capacitation. A late influx of K⁺ is important for the acrosome reaction. However, this late influx may not be due to Na⁺, K⁺-ATPase, but instead may be due to a K⁺ permeability increase (possibly via newly formed channels) in the membranes during capacitation. It is suggested in this review that Na⁺, K⁺-ATPase has a role early in capacitation rather than directly in the acrosome reaction (although such a role cannot yet be completely ruled out). One possible role for the enzyme in capacitation might be to stimulate glycolysis (which appears to be essential for capacitation and/or the acrosome reaction of hamster and mouse spermatozoa). The function of the influx of K⁺ just before the acrosome reaction is probably to stimulate, directly or indirectly, the H⁺-efflux required for the increase in intraacrosomal pH occurring during capacitation. Direct stimulation of the acrosome reaction by a change in membrane potential resulting directly from K⁺-influx is not a likely explanation for the hamster results. However, the importance of an earlier membrane potential change, due to increased Na⁺, K⁺-ATPase during capacitation, and/or of later membrane potential changes resulting from the pH change, cannot be ruled out. Although K⁺ is required for the hamster acrosome reaction, other workers have reported that K+ inhibits guinea pig sperm capacitation. However, the experimental procedures used in the guinea pig sperm studies raise some questions about the interpretation of those inhibition results. (5) Ca²⁺-influx is known to be required for the acrosome reaction. Others have suggested that increased Ca²⁺-influx due to inhibition or stimulation of sperm membrane calcium transport ATPases are involved in the acrosome reaction. There is as yet no direct or indirect biochemical evidence that inhibition or stimulation of such enzymatic activity is involved in the acrosome reaction, and further studies are needed on those questions. (6) I suggest that the hydrolytic enzymes important to the hamster sperm acrosome reaction will also prove important for the acrosome reaction of all other eutherian mammals.     

Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae

The binding of mammalian spermatozoa to the egg's extracellular coat, the zona pellucida, is a complex process which culminates in species-specific penetration of the sperm to the egg plasma membrane. To investigate where on the spermatozoon's surface the zona binding sites are located, whole rabbit zonae were labeled with FITC, heat solubilized and used to observe the surface binding patterns on live spermatozoa. Before the acrosome reaction the zona binding sites are located either over the entire head as well as the middle piece or alternatively in patches along the apical ridge of the head. After the acrosome reaction there is a 29% loss of fluorescence and the zona binding sites are present in the posterior aspect of the acrosomal region, the anterior postacrosomal region and the middle piece. These results demonstrate the presence of zona binding sites after the acrosome reaction which would account for the sperm's ability to remain bound to the zona after the acrosome reaction. Further, we report for the first time that solubilized rabbit zonae pellucidae will induce the acrosome reaction in in vitro capacitated rabbit sperm whereas solubilized pig zonae pellucidae will not. Since rabbit sperm bind pig zonae, the induction and specificity of the physiological acrosome reaction must reside in the affinity of the binding rather than the binding itself.     

Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm

Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.     

Redistribution of mouse sperm surface galactosyltransferase after the acrosome reaction

Gamete recognition in the mouse is mediated by galactosyltransferase (GalTase) on the sperm surface, which binds to its appropriate glycoside substrate in the egg zona pellucida (Lopez, L. C., E. M. Bayna, D. Litoff, N. L. Shaper, J. H. Shaper, and B. D. Shur, 1985, J. Cell Biol., 101:1501-1510). GalTase has been localized by indirect immunofluorescence to the dorsal surface of the anterior sperm head overlying the intact acrosome. Sperm binding to the zona pellucida triggers induction of the acrosome reaction, an exocytotic event that results in vesiculation and release of the outer acrosomal and overlying plasma membranes. Consequently, we examined the fate of sperm surface GalTase after the acrosome reaction. Contrary to our expectations, surface GalTase is not lost during the acrosome reaction despite the loss of its membrane domain. Rather, double-label indirect immunofluorescence assays show that GalTase is redistributed to the lateral surface of the sperm, coincident with the acrosome reaction. This apparent redistribution of GalTase was confirmed by direct enzymatic assays, which show that 90% of sperm GalTase activity is retained during the acrosome reaction. No GalTase activity is detectable on plasma membrane vesicles released during the acrosome reaction. In contrast, removal of plasma membranes by nitrogen cavitation releases GalTase activity from the sperm surface, showing that GalTase redistribution requires a physiological acrosome reaction. The selective redistribution of GalTase to a new membrane domain from one that is lost during the acrosome reaction suggests that GalTase is repositioned for some additional function after initial sperm-zona binding.     

Gossypol-induced inhibition of guinea pig sperm capacitation in vitro

The effect of gossypol acetate at various concentrations (10(-6) to 10(-4) M) on guinea pig sperm forward progressive movement, capacitation, and the acrosome reaction was explored in vitro. We found that 10(-4) M gossypol completely abolished the forward progressive motility of the sperm, and that this inhibition of motility was proportional to the concentration of gossypol used. Also, a dose-dependent decrease in acrosome reactions occurred with concentrations of the agent as low as 5.0 X 10(-6) M. However, we observed that such prevention of the acrosome reaction apparently happens at the capacitation stage rather than during the acrosome reaction itself. Inhibition of capacitation by gossypol was reversible--once the spermatozoa were capacitated in gossypol-free medium, the compound did not block the reaction.     

On the role of glucose in capacitation and acrosomal reaction of guinea pig sperm

In order to determine whether metabolizable sugars delayed capacitation of guinea pig spermatozoa, these cells were pre-incubated in Tyrode's pyruvate lactate glucose medium (T-PLG) or Tyrode's glucose solution (T-G). They were then transferred to minimal culture medium containing pyruvate and lactate (MCM-PL) and the occurrence of acrosomal reactions (AR) was determined by light microscopic observations of wet mount aliquots. The percentage of acrosomal reactions was quantitated in fixed samples and occurrence of a true AR was confirmed by electron microscopy. Activated acrosome-reacted spermatozoa were observed within 5 min when cells were transferred to MCM-PL solution, after preincubating them for 60–120 min either in T-PLG or T-G media. By 15 min in MCM-PL the percentage of acrosome-reacted spermatozoa reached values similar to those obtained in cells pre-incubated from the beginning in MCM-PL medium (P > 0.05 in both) but significantly different from T-PLG and T-G controls (P < 0.0005 in both). The acrosomal reaction was external calcium dependent and independent of the Tyrode's media pH ranging from 7.2 to 8.0. The results obtained suggested that capacitation occurred in T-PLG and that it was not delayed by glucose; the results also suggested that capacitation could occur within a short time with glucose as the only exogenous substrate, but that the acrosome reaction could have been arrested by a glucose metabolite. Data are presented which suggest that intracellular levels of glucose-6-phosphate (as 2-deoxyglucose-6-phosphate)could play a key role in the expression of the acrosome reaction in sperm already able to perform it. A new hypothesis is suggested for the development of the fertilizing potential of guinea pig sperm when in the female genital tract.     

Immunocytochemical alterations in the intra-acrosomal antigen MN7 during epididymal maturation of guinea pig spermatozoa

We have previously shown that a 90-kDa intra-acrosomal antigen, MN7, is restricted to the anterior acrosomal region of mouse, rat, and hamster spermatozoa. The present study has examined the localization and the behavior of MN7 during sperm maturation in the epididymis of the guinea pig by immunoelectron microscopy. MN7 showed not only a specific localization in the apical segment of the guinea pig sperm acrosome, but also a distinct alteration during maturation, as follows. MN7 was exclusively found both at the dorsal matrix and on the outer acrosome membrane (OAM)/matrix-associated materials in the apical segment. MN7 was initially distributed throughout the electron-lucent dorsal matrix in immature sperm but, during maturation, became more restricted to the spherical bodies within the electron-lucent area. MN7 on OAM/matrix-associated materials was first distributed along the ventral margin and the small area posterior to the dorsal matrix but, during maturation, disappeared from the ventral margin and became restricted to the dorsal region. These results indicate that MN7 is a good tool for studying the stepwise maturation of epididymal spermatozoa.     

Aggregation of beta-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction

beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.     

F-actin in guinea pig spermatozoa: its role in calmodulin translocation during acrosome reaction.

The presence of actin has been determined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques, actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDS-PAGE and Western blots immunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa. Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosomal region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility.     

Effect of Ca2+ channel antagonists on the acrosome reaction of guinea pig and golden hamster spermatozoa

The effects of Ca²⁺ channel antagonists on the motility and acrosome reaction of guinea pig spermatozoa were examined by incubating the spermatozoa continuously in Ca²⁺-containing capacitating media with 10^?6 M to 10^?4 M antagonist. Antagonists tested were four voltage-gated Ca²⁺ channel antagonists (verapamil, nifedipine, nimodipine, and FR–34235) and two ligand-gated channel antagonists (NaNO₂ and Na-nitroprusside). None of these antagonists could block the acrosome reaction. Instead, three antagonists (verapamil, nimodipine, and FR-34235, each at 10^?4 M) accelerated the onset of the acrosome reaction with a subsequent decrease in sperm motility. Nifedipine and Na-nitroprusside at the same concentration caused a complete loss of sperm motility by 4 hr of incubation with no substantial effect on the rate of acrosome reaction. The detrimental effect of antagonists on the motility of spermatozoa appears to be due to a direct, Ca²⁺-independent, membrane-perturbing action of the reagents. The acrosome reaction was not inhibited when guinea pig spermatozoa were precapacitated in Ca²⁺-free medium (with a low concentration of lysolecithin) in the continuous presence of antagonists. An acceleration of the onset of the acrosome reaction by verapamil (10^?4 M) was also demonstrated in the golden hamster. These results may be interpreted as indicating that the entry of extracellular Ca²⁺ into spermatozoa, which triggers the acrosome reaction of guinea pig and hamster spermatozoa, is not mediated by Ca²⁺ channels. This is in marked contrast with the case reported in invertebrate spermatozoa. Possible mechanisms by which some of the antagonists stimulate the acrosome reaction and affect the motility of mammalian spermatozoa are discussed.     

Localization of the Rho GTPases and some Rho effector proteins in the sperm of several mammalian species

The acrosome reaction is a fundamental event in the biology of the sperm and is a prerequisite to fertilization of the egg. Members of the Rho family of GTPases and their effectors are present in the cytoplasm and/or plasma membrane overlying the acrosome of porcine sperm. We have implicated the Rho family of GTPases and the Rho-activated kinase, ROCK-1, in mediating the zona-pellucida-induced acrosome reaction. Others have implicated the Rho GTPase in regulating the ionophore-induced acrosome reaction in the sperm of several mammalian species as well as in motility of bovine sperm. In this study, the localization of the Rho GTPases (RhoA, RhoB, Rac1 and Cdc42) as well as the effectors RhoGDI, PI(4)P5K and ROCK-1, was determined in boar, human, rat, ram, bull and elephant sperm. The four GTPases were each present in the sperm head of all species examined. RhoGDI was expressed in the head and tail of sperm from all species except pig, where it was present only in the head. PI(4)P5K was expressed in both head and tail of sperm from all species, but expression was typically weaker in the tail. Finally, ROCK-1 was expressed in the heads and tails of all sperm except that of the boar, where it was present only in the acrosomal region. These observations taken together suggest that the expression of Rho GTPases in sperm has been conserved throughout mammalian evolution, most likely due to the role of these GTPases in regulating acrosomal exocytosis.     

Capacitation is associated with tyrosine phosphorylation and tyrosine kinase-like activity of pig sperm proteins

Capacitation represents the final maturational steps that render mammalian sperm competent to fertilize, either in vivo or in vitro. Capacitation is defined as a series of events that enables sperm to bind the oocyte and undergo the acrosome reaction in response to the zona pellucida. Although the molecular mechanisms involved are not fully understood, sperm protein phosphorylation is associated with capacitation. The hypothesis of this study is that protein tyrosine phosphorylation and kinase activity mediate capacitation of porcine sperm. Fresh sperm were incubated in noncapacitating or capacitating media for various times. Proteins were extracted with SDS, subjected to SDS-PAGE, and immunoblotted with an antiphosphotyrosine antibody. An M(r) 32 000 tyrosine-phosphorylated protein (designated as p32) appeared only when the sperm were incubated in capacitating medium and concomitant with capacitation as assessed by the ionophore-induced acrosome reaction. The p32 was soluble in Triton X-100. Fractionation of sperm proteins with Triton X-114 demonstrated that after capacitation, this tyrosine phosphoprotein is located in both the cytosol and the membrane. Enzyme renaturation of sperm proteins was conducted in gels with or without either poly glu:tyr (a tyrosine kinase substrate) or kemptide (a protein kinase A substrate). An M(r) 32 000 enzyme with kinase behavior was observed in all gels but was preferentially phosphorylated on tyrosine, as assessed by phosphorimagery and by thin layer chromotography to identify the phosphoamino acids. Indirect immunolocalization showed that the phosphotyrosine residues redistribute to the acrosome during capacitation, which is an appropriate location for a protein involved in the acquisition of fertility.     

Enzyme activity in anuran spermatozoa upon induction of the acrosome reaction.

At the time of sperm-egg fusion in Discoglossus pictus, a large amount of electron-dense material of an unknown nature is liberated from the sperm. In the present work we studied this material in D. pictus sperm, using an assay utilising strips of autoradiographic film as a gelatin substrate for proteolytic enzymes. Upon treatment with A23187, D. pictus sperm produced a large halo on the gelatin substrate, indicating the presence of enzymes released by the sperm at the time of the acrosome reaction. In contrast, Xenopus laevis sperm did not produce halos upon treatment with A23187. The use of protease inhibitors such as TLCK, leupeptin, chymostatin, SBTI and EACA strongly suggests that the D. pictus whole acrosome contains trypsin and chymotrypsin activity while an SBTI-sensitive activity is absent in a small portion of the acrosome, possibly the anteriormost region. Furthermore, the material released at the acrosome reaction also contains an EACA-inhibited activity, indicating the presence of plasminogen activator. We conclude that D. pictus sperm release proteolytic enzyme(s) that may act at the egg surface at the time of gamete fusion.     

Isolation of the peri-acrosomal plasma membrane protein D40 enriched fraction from guinea pig spermatozoa using a monoclonal antibody

Membrane fragments were obtained from guinea pig spermatozoa by mechanical shearing. A membrane-enriched fraction was separated from other cellular debris, mainly sperm nuclei and tails, by centrifugation on 20% Ficoll 70 solution. Peri-acrosomal plasma membrane protein, D40, enriched fraction was separated from this membrane preparation using a mouse monoclonal antibody to D40 attached to magnetic beads. Enrichment of D40 antigen in this fraction was demonstrated by western blotting. The method provides a preparative route to a membrane, the constituents of which play an important role in sperm recognition of the zona pellucida and the acrosome reaction. Some constituents of the peri-acrosomal plasma membrane over the equatorial segment of the acrosome may also play a role in sperm docking with the oocyte plasma membrane and fusion of the two cells.     

Evidence suggesting the importance of fatty acids and the fatty acid moieties of sperm membrane phospholipids in the acrosome reaction of guinea pig spermatozoa

When guinea pig spermatozoa were preincubated 1 hr in Ca2+-free medium containing dilysocardiolipin (100--125 micrograms/ml) then exposed to Ca2+, the majority underwent an immediate acrosome reaction. Monolysocardiolipin was much less effective and the native cardiolipin was totally ineffective. Some fatty acids added to the medium could also render the spermatozoa capable of undergoing their acrosome reactions, arachidonic acid in methyl ester form being most potent. It is known that sperm membrane contains phospholipase A which cleaves membrane phospholipids into lysophospholipids and fatty acids. Most lysophospholipids are known to be potent acrosome reaction-promoting agents. As some forms of fatty acids are also potent acrosome reaction-promoting agents, both products of membrane phospholipid hydrolysis by phospholipase A (i.e., both fatty acids and lysophospholipids) may work synergistically to effect the conversion of stable sperm membranes to a fusion competent state capable of engaging in the acrosome reaction.     

Distribution, crypticity, stability, and localization of α-L-fucosidase of mouse cauda epididymal sperm

Sperm‐associated and semen‐specific isoforms of α‐L ‐fucosidase are thought to function in fertilization in numerous organisms. Here, we report the localization, distribution, crypticity, and stability of this enzyme in mouse cauda epididymal sperm and cauda fluid. Western analysis revealed that the sperm‐associated α‐L ‐fucosidase is present as two isoforms (Mr ～49 and 56 kDa), whereas the cauda fluid α‐L ‐fucosidase shows a single band at 50 kDa. α‐L ‐Fucosidase activity was detected using the fluorogenic substrate 4‐MU‐FUC. Of the total α‐L ‐fucosidase activity recovered in the cauda epididymal contents, 74% was found in the cell‐free cauda fluid and about 7% was found in sperm cells. During capacitation or permeabilization, cryptic intracellular stores of soluble enzyme were released to the supernatant, while leaving bound enzyme concentrated within the small volume of sperm. Moreover, membrane‐associated enzyme activity was still detectable in acrosome‐reacted cells. Immunofluorescence studies support the presence of α‐L ‐fucosidase (originally localizing at the acrosomal area) at the equatorial segment after the acrosome reaction. α‐L ‐Fucosidase activity of both cauda fluid and sperm at 37°C, 5% CO₂ was relatively stable and detectable up to 72 hr. The stability and appearance of mouse sperm‐associated α‐L ‐fucosidase in the equatorial segment after the acrosome reaction suggest that α‐L ‐fucosidase may be involved in sperm–egg interaction. Mol. Reprod. Dev. 79: 208–217, 2012. © 2011 Wiley Periodicals, Inc.     

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.