ACROSOMAL DISRUPTION IN SPERM : Freeze-Fracture of Altered Membranes

"Capacitation" is a physiological event which alters sperm to permit rapid penetration through oocyte investments and fusion between gametes. Acrosomal "reaction," the physiological release of acrosomal contents, occurs after this facilitating process. In this study, acrosomal "disruption" of guinea pig and rat sperm was achieved in vitro by incubating sperm together with the follicular contents of superovulated mice. The samples contained both "reacted" and "disrupted" sperm. Thin sections of affected sperm revealed rupture and vesiculation of the plasma membrane overlying the acrosome, as well as loss of both the outer acrosomal membrane and the acrosomal content. Freeze-fracture revealed disintegration of the characteristic geometric patterns in regions of the acrosomal and plasma membranes thus disrupted and major modifications in particle distribution in the sperm tail. In the guinea pig, strands of 6–8-nm particles, usually confined to the plasma membrane of the midpiece, which overlies mitochondria, also appeared in the principal piece. Likewise, in rat sperm, bands of similarly small particles formed acute angles throughout the membrane of the principal piece. Compared with the membranes of control preparations, these membrane alterations are apparently a direct consequence of incubation with ovarian follicular contents.     

Electron microscopical studies of membrane injuries in blue fox spermatozoa subjected to the process of freezing and thawing

Disintegration of blue fox sperm membranes is studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In unfrozen spermatozoa studied by SEM, the plasmalemma and the acrosome appeared to be intact, except for a few cases of disruption of the former structure at the anterior part of the head. In semen frozen in 0.5-ml plastic straws by use of N2 vapor after dilution with Tris-fructose-citric acid with 8 vol % glycerol and 20 vol % egg yolk and thawed at 70 degrees C for 8 sec, the spermatozoa displayed different degrees of membrane damage. These alterations could be classified into three main categories of which the first included only minor changes in the plasmalemma, but vesiculation and disintegration of the outer part of the acrosomal membrane. In the second category (also the most frequent one) the outer part of the acrosomal membrane was extensively vesiculated, and the plasmalemma was discharged proximal to the equatorial segment. Extensive loss of plasmalemma and complete absence of the outer part of the acrosomal membrane characterized the last category of membrane damage. The functional implications of the three categories of membrane alterations are discussed.     

Localization of intracellular calcium during the acrosome reaction in ram spermatozoa

Calcium was identified by a pyroantimonate-osmium fixation technique in ram spermatozoa undergoing a spontaneous acrosome reaction induced by incubation of diluted semen at 39°C. Intracellular calcium was only detected in diluted spermatozoa and increased in amount and distribution over 4 hr At 4 hr, the majority of the spermatozoa displayed ultrastructural evidence of an acrosome reaction. Calcium was initially evident on the outer acrosomal membrane in multiparticulate clusters, which were seen to be located on scalloped crests of acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly. As vesiculation advanced, calcium particles became associated with the periphery of the vesicles attached in the region of the fusion between the two membranes, but were never seen inside the vesicles. The equatorial segment was not labelled until much later in the reaction, at which time calcium particles were also evident on the nuclear membrane; vesiculation of the equatorial segment was also noted at this time. Dense labelling of the postacrosomal dense lamina was seen in all incubated spermatozoa. At the anterior margin of this structure the labelling was seen to be in a “sawtooth” arrangement. The disposition of the calcium both temporally and spatially is discussed in relation to its possible mechanisms in bringing about membrane fusion. © 1995 Wiley-Liss, Inc.     

Acrosome reaction in spermatozoa from hagfish (Agnatha) Eptatretus burgeri and Eptatretus stouti: acrosomal exocytosis and identification of filamentous actin

Spermatozoa of the hagfishes Eptatretus burgeri and Eptatretus stouti, caught in the sea near Japan and North America, respectively, were found to undergo the acrosome reaction, which resulted in the formation of an acrosomal process with a filamentous core. The acrosomal region of spermatozoa of E. stouti exhibited immunofluorescent labeling using an actin antibody. The midpiece also labeled with the antibody. The acrosomal region showed a similar labeling pattern when sperm were probed with tetramethylrhodamine isothyocyanate (TRITC)-phalloidin; the midpiece did not label. Following induction of the acrosome reaction with the calcium (Ca2+) ionophore ionomycin, TRITC-phalloidin labeling was more intense in the acrosomal region, suggesting that the polymerization of actin occurs during formation of the acrosomal process, as seen in many invertebrates. The potential for sperm to undergo acrosomal exocytosis was already acquired by late spermatids. During acrosomal exocytosis, the outer acrosomal membrane and the overlying plasma membrane disappeared and were replaced by an array of vesicles; these resembled an early stage of the acrosome reaction in spermatozoa of higher vertebrates in which no formation of an acrosomal process occurs. It is phylogenetically interesting that such phenomena occur in spermatozoa of hagfish, a primitive vertebrate positioning between invertebrates and high vertebrates.     

Gamete membrane fusion in hamster spermatozoa with reacted equatorial segment

Mammalian spermatozoa must undergo many changes to be able to fertilize the oocyte. One of these changes, the acrosome reaction, has been established as a requisite for gamete membrane fusion to occur; it consists of the fusion and vesiculation of the sperm plasma membrane with the outer acrosomal membrane of the principal segment of the acrosome. Reaction of the equatorial segment has occasionally been observed. The objective of the present work was to determine whether the presence of the sperm plasma membrane over the equatorial segment is necessary for gamete membrane fusion to occur. Golden hamster spermatozoa were capacitated in vitro in TAPL 10K, and the maximum possible percentage of acrosome reaction was determined at 82.79% + 1.69% SD (P = 0.27; r = 0.21). Ultrastructural studies showed that 93.6% of the reacted spermatozoa in this population had their principal and equatorial segments reacted. The fertilizing ability of these spermatozoa was assayed using zona-free hamster oocytes. The percentage of fertilized ova obtained was 98.8% (308/312). Ultrastructural studies snowed the presence of spermatozoa with reacted equatorial segment inside the cytoplasm of immature oocytes. The evidence presented in this work demonstrates that the plasma membrane of spermatozoa with reacted equatorial segment retains its ability to fuse with the oocyte.     

Immunocytological characterization of the outer acrosomal membrane (OAM) during acrosome reaction in boar

In order to study the acrosome reaction in boar, spermatozoa were incubated in a calcium-containing medium in the presence of the calcium ionophore A23187. The time course of the acrosome reaction was assessed by phase-contrast microscopy and correlated with the movement characteristics of the spermatozoa determined by means of multiple-exposure photography (MEP). Different stages of the acrosome reaction could be observed by indirect immunofluorescence using an antibody fraction raised in rabbits against the isolated outer acrosomal membrane (OAM). At the start of the acrosome reaction, a bright fluorescence located exclusively at the acrosomal cap of the sperm head could be observed, whereas after 60-120 min, the fluorescence vanished, indicating the complete loss of the OAM. However, to gain more insight into the stages of the plasma membrane and OAM during the acrosome reaction, immunoelectron-microscopical studies were performed using anti-OAM antibodies detected by the protein-A gold method. Ultrathin sections and total preparations in combination with transmission electron microscopy (TEM) confirmed, that boar spermatozoa start their acrosome reaction by a vesiculation of the plasma membrane, thus exposing the heavily labelled OAM, which is then lost as sheets or large vesicles. The newly exposed inner acrosomal membrane did not show any labelling with gold, thereby indicating clear differences in the antigenicity of both acrosomal membranes.     

Immunocytological characteriziation of the outer acrosomal membrane (OAM) during acrosome reaction in boar

Summary In order to study the acrosome reaction in boar, spermatozoa were incubated in a calcium-containing medium in the presence of the calcium ionophore A23187. The time course of the acrosome reaction was assessed by phasecontrast microscopy and correlated with the movement characteristics of the spermatozoa determined by means of multiple-exposure photography (MEP). Different stages of the acrosome reaction could be observed by indirect immunofluorescence using an antibody fraction raised in rabbits against the isolated outer acrosomal membrane (OAM). At the start of the acrosome reaction, a bright fluorescence located exclusively at the acrosomal cap of the sperm head could be observed, whereas after 60–120 min, the fluorescence vanished, indicating the complete loss of the OAM. However, to gain more insight into the stages of the plasma membrane and OAM during the acrosome reaction, immunoelectron-microscopical studies were performed using anti-OAM antibodies detected by the protein-A gold method. Ultrathin sections and total preparations in combination with transmission electron microscopy (TEM) confirmed, that boar spermatozoa start their acrosome reaction by a vesiculation of the plasma membrane, thus exposing the heavily labelled OAM, which is then lost as sheets or large vesicles. The newly exposed inner acrosomal membrane did not show any labelling with gold, thereby indicating clear differences in the antigenicity of both acrosomal membranes.     

Proteoglycan from bovine follicular fluid enhances an acrosome reaction in bovine spermatozoa

Bovine epididymal and ejaculated spermatozoa were incubated for 22 and 9.5 h respectively, in a chemically defined medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. Addition of bovine follicular proteoglycan or chondroitin sulfates ABC significantly increased the incidence of acrosome reaction. The stimulatory effects of the proteoglycan or chondroitin sulfates were negated by exposure to the enzyme chondroitinase ABC. Viability of spermatozoa was not affected by the various experimental treatments. Transmission electron microscopy of spermatozoa showed that vesiculation had occurred between the plasma and outer acrosomal membrane. These results suggest that proteoglycan present in follicular fluid at the time of ovulation may promote the acrosome reaction which precedes the ability of sperm to fertilize an ovum.     

pH and protease control of acrosomal content stasis and release during the guinea pig sperm acrosome reaction

The purpose of this study was to examine how trypsin inhibitors affect the guinea pig sperm acrosome reaction in vitro. Using spermatozoa pretreated with lysophosphatidyl choline, we found that both naturally occurring high molecular weight and the smaller synthetic trypsin inhibitor p-aminobenzamidine (PAB) delayed the onset of the acrosome reaction as monitored by light microscopy. Examination with electron microscopy revealed that acrosomal matrix dispersal rather than membrane fusion was affected. Despite the morphologic delay in acrosomal content release, PAB unexpectedly permitted 96% of soluble acrosomal antigen to be released into the supernatant. In addition, total acrosin release in the presence of PAB was 74% of control, with the vast majority as latent rather than active enzyme. A morphologically intact but membrane-free target of acrosomal matrix (AM), which is sensitive to trypsin inhibitor, was partially purified using Triton-x-100 at pH 5.2. AM remained morphologically stable at pH 5.2; however, shift up to pH 7 resulted in rapid dissolution within several minutes as monitored by light and electron microscopy and light scattering. Trypsin inhibitor prevented dispersion of AM at pH 7. The results suggest that, during the acrosome reaction, one distinct region of the acrosomal contents disperses after membrane vesiculation in a pH and trypsin inhibitor-insensitive fashion while a pH sensitive trypsin-like activity (acrosin?) disperses another discrete region of acrosomal matrix.     

Localization of a syntaxin isoform, syntaxin 2, to the acrosomal region of rodent spermatozoa

The acrosome reaction includes a membrane fusion event that is a prerequisite for sperm penetration through the zona pellucida and subsequent fertilization. Since SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have been shown to be key players in membrane fusion during regulated exocytosis in nerve terminals and secretory cells, and since the acrosome reaction has some features in common with regulated exocytosis, we hypothesized that SNARE proteins might also regulate acrosomal exocytosis. RT-PCR analysis demonstrated the expression of SNARE proteins, three isoforms of syntaxin 2 (2A, 2B, and 2C) and syntaxin 4A, in rat testes. Immunoblot analysis with anti-syntaxin 2 antibody showed that the protein was expressed in rodent spermatozoa, and that it was associated with membrane components of spermatozoa prepared by sucrose density gradient centrifugation. Confocal laser scanning microscopy with double immunolabeling revealed that syntaxin 2 was colocalized with acrin 1, a 90 kDa acrosomal protein, over the acrosomal region of spermatozoa but was not associated with the posterior half of head or tail. Localization of syntaxin 2 over the acrosomal region was supported by the finding that it was shed from sperm heads during an acrosome reaction induced by calcium ionophore A23187 in vitro. In view of the putative role of syntaxin proteins in other membrane fusion systems, these data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa.     

Sperm surface changes during the acrosome reaction as observed by freeze-fracture

The mammalian acrosome reaction is an exocytotic process that can be analyzed by the technique of freeze-fracture; only sperm cells capacitated in vitro or treated to elicit the acrosome reaction in vitro have been studied, and all pictures published are from material fixed before freezing. All the authors point out the appearance of particle-free areas in the plasma membrane of the acrosomal region during capacitation and before any fusion. This is interpreted as an increase in membrane fluidity as suggested by studies on membrane lipid composition in guinea-pig sperm. We have recently described the induced acrosome reaction in ram spermatozoa. Fusion starts at the limit of the anterior and equatorial segments and progresses forward in the anterior segment along ramified paths, resulting in a fenestration gradient of the acrosomal cap. Fusion propagation may be controlled by fluidity increase in the plasma membrane of the anterior segment, and it is probably inhibited in the equatorial segment by the ordered structure of the acrosomal membrane.     

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.     

Rab3A triggers the acrosome reaction in permeabilized human spermatozoa

The acrosome reaction is a regulated exocytotic process leading to a massive fusion between the outer acrosomal membrane and the cell membrane. In spite of the great amount of information available related to the acrosome reaction in several species, there is a remarkable paucity about the role of monomeric guanosine triphosphatases (GTPases) of the Rab family-well-established participants in exocytosis in other cell types-in the acrosome reaction. Western blot and immunofluorescence analysis indicate that Rab3A is present in human spermatozoa and localizes to the acrosomal region in the sperm head. One difficulty in studying the role of proteins in intact cells is the fact that they are unable to cross the cell membrane. Therefore, we established a working model of streptolysin O-permeabilized human spermatozoa. Permeabilized spermatozoa were able to respond in a regulated way to different stimuli, such as G protein activators and calcium. An acrosomal reaction was also triggered by a Rab3A peptide corresponding to the effector region. More important, recombinant Rab3A protein in the GTP-bound form caused acrosome exocytosis. The same protein loaded with GDP or Rab11 in the GTP-bound form was inactive. Also, recombinant GDI (GDP dissociation inhibitor)-a protein that releases Rab proteins from membrane-inhibited a GTPgammaS-stimulated acrosome reaction. Our results indicate that 1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human spermatozoa.     

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.     

Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.     

The acrosomal region of the spermatozoon of Ciona intestinalis: Its relationship with the binding to the vitelline coat of the egg

This paper describes a study of the apical region of the spermatozoon of Ciona intestinalis before and during its binding to the vitelline coat of the egg. A combination of the techniques of thin sectioning, negative staining, and freeze fracture has revealed that in the apical-most region, where a small acrosomal vesicle lies on the flat tip of the nucleus, there is a cap-like region almost completely free of particles on the P face of the plasma membrane. The particle-free area is surrounded by two circlets of orderly arranged particles. Upon binding to the vitelline coat the particles of the distal circlet show a partial displacement, while the particles of the apical circlet remain unaltered. The relationship between the apical circlet and the binding process is discussed. The final step of the acrosome reaction, which occurs in only a few of the bound spermatozoa, consists in the fusion of the plasma membrane with the acrosomal membrane, in the dehiscence of the acrosomal contents and finally in the formation of membrane tubules.     

Protein tyrosine phosphorylation of a heparin-binding sperm membrane mitogen (HBSM) is associated with capacitation and acrosome reaction

Protein tyrosine phosphorylation in spermatozoa is associated with epididymal maturation and though to be central for attainment of a capacitated state and expression of hyperactivated motility. Heparin, the most highly sulfated glycosaminoglycans, was also the most potent at stimulating the acrosomal reaction in bovine epididymal spermatozoa. Studies using radiolabeled inorganic phosphate showed 11-fold increase (32)Pi incorporation in heparin-binding sperm membrane protein (HBSM) during spermatozoal capacitation, and the phosphorylation occurs at the tyrosine residue. Epididymal spermatozoa were induced to undergo capacitation and acrosome reaction by 70% when the cells were incubated in BWW medium supplemented with heparin. The spermatozoa pre-treated with anti-HBSM antibody showed 46% reduction in the hyperactivated motility and lowers the acrosome reaction. This was confirms by measuring the hydrolysis of benzoyl-l-arginine ethyl ether (BAEE) by the acrosomal enzyme; acrosin. The preliminary finding suggests that HBSM may play an important role in the sperm capacitation and acrosome reaction.     

Relationship between calcium binding sites and membrane fusion during the acrosome reaction induced by ionophore in ram spermatozoa

Ram spermatozoa treated with the ionophore A23187, to induce the acrosome reaction, were fixed in pyroantimonate-osmium in order to demonstrate calcium at an ultrastructural level. Prior to vesiculation, granules of precipitate occurred at points of contact between plasma and outer acrosomal membranes, suggesting a discrete role for calcium during membrane fusion. The earliest stages of vesiculation always occurred in the region just anterior to the equatorial segment and it was this same site where a marked concentration of precipitate, indicating calcium binding sites, could be demonstrated.     

Simple histochemical stain for acrosomes on sperm from several species

The acrosome reaction is an exocytotic process that enables a sperm to penetrate the zona pellucida and fertilize an egg. The process involves the fenestration and vesiculation of the sperm plasma membrane and outer acrosomal membrane releasing the acro somal contents. Many different methods have been devel oped to detect the acrosomal status of sperm. These techniques are sometimes complicated, costly, and can be used on only a few species. The aim of this study was to develop an efficient and inexpensive method to assess the acrosomal status of sperm from a variety of species. We prepared and fixed sperm from humans, cattle, swine, rabbits, guinea pigs, and mice and stained them with Coomassie G250. The acrosomes were stained intensely blue in color. Following capacitation, some sperm were incubated for 1 hr with 10 microM calcium ionophore A23187 to induce the acrosome reaction. They were also stained with Coomassie G-250. Ionophore-treated sperm lacked Coomassie staining over the acrosomal region. Differential interference contrast (DIC), bright field microscopy or Pisum sativum agglutinin staining confirmed that the acrosomes of sperm from these species were reacted in response to calcium ionophore treatment and the acrosome reaction frequencies matched results with Coomassie staining. These results demonstrate that the acrosomal status of mammalian sperm from several species can be determined easily and reliably using this simple Coomassie Blue G-250 staining method.     

Surface alterations during the capacitation of mammalian spermatozoa

Capacitation is the process by which mammalian sperm acquire the ability to undergo the acrosome reaction which, in turn, is a prerequisite for sperm-egg fusion and penetration. Until recently, it was thought that capacitation involved subtle physiological and chemical changes which had no morphological counterparts even at the electron microscopic level. However, it has now been shown by a number of investigators that material associated with the plasma membrane surface is either lost or extensively redistributed during in vitro or in vivo capacitation. We have made use of lectins and antibodies as probes of the sperm surface during capacitation and the acrosome reaction. Concanavalin A (Con A), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) have been used in conjunction with fluorescent tags (FITC) and ultrastructural markers (ferritin, hemocyanin) to study the surface of golden hamster, guinea pig, mouse and human spermatozoa. Con A and WGA label the plasma membrane overlying the acrosomal region quite uniformly on these species. After capacitation there is a specific loss (or masking) of lectin binding sites over the acrosomal region of the sperm head in all species examined. Antibodies prepared against sperm and specific antibodies to a cell surface protein (fibronectin) were also tagged with fluorescent or ultrastructural markers and used to label the surfaces of sperm before and after capacitation. These probes also indicate a specific loss of surface associated material over the acrosomal surface after capacitation. These results are consistent with the notion that there is a general removal of surface components during capacitation and that this denuding of the surface is a prerequisite for the following membrane fusion events involved in the acrosome reaction and sperm-egg fusion.