大熊猫精子获能和顶体反应过程中钙分布变化规律的研究

应用焦锑酸钾原位定位法对大熊猫精子获能和顶体反应过程中进行钙定位研究,发现未获能精子的 Ｃａ２＋主要结合于顶体前区和赤道段质膜外侧和顶体内膜内侧（核膜侧）;随着获能的进行,Ｃａ２＋进入精子内部并主要结合于顶体区质膜内侧和顶体外膜外侧;顶体反应的精子,Ｃａ２＋结合于顶体内膜外侧、顶体后区质膜外侧和分散存在于释放的顶体内容物中,有些顶体反应精子的顶体内膜外侧结合的Ｃａ２＋特别丰富。精子尾部的Ｃａ２＋主要分布于中段线粒体内,且其内所含Ｃａ２＋含量随着获能和顶体反应而增加。另外尾部致密纤维和轴丝处也有少量Ｃａ２＋分布。     

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.     

Signaling pathways in sperm capacitation and acrosome reaction.

The binding to the egg's zona pellucida stimulates the spermatozoon to undergo acrosome reaction, a process which enables the sperm to penetrate the egg. Prior to this binding, the spermatozoa underago in the female reproductive tract a series of biochemical transformations, collectively called capacitation. The first event in capacitation is cholesterol efflux leading to the elevation of intracellular calcium and bicarbonate to activate adenylyl cyclase (AC) to produce cyclic-AMP, which activates protein kinase A (PKA) to indirectly phosphorylate certain proteins on tyrosine. During capacitation, there is also an increase in protein tyrosine phosphorylation dependent actin polymerization and in the membrane-bound phospholipase C (PLC). Sperm binding to zona-pellucida causes further activation of cAMP/PKA and protein kinase C (PKC), respectively. PKC opens a calcium channel in the plasma membrane. PKA together with inositol-trisphosphate activate calcium channels in the outer acrosomal membrane, which leads to an increase in cytosolic calcium. The depletion of calcium in the acrosome will activate a store-operated calcium entry mechanism in the plasma membrane, leading to a higher increase in cytosolic calcium, resulting in F-actin dispersion which enable the outer acrosomal and the plasma membrane to come into contact and fuse completing the acrosomal reaction.     

Evidence for the capacitation-associated membrane priming of mouse spermatozoa

An important feature of male fertility is the physiological priming of mammalian spermatozoa by a multifaceted process referred to as capacitation. It is a prerequisite event before spermatozoa can bind to the egg's extracellular coat, the zona pellucida, and undergo a signal transduction cascade. The net result is the fusion of the plasma membrane (PM) and underlying outer acrosomal membrane at multiple sites and the release of acrosomal contents (i.e., glycohydrolases, proteinases, etc.) at the site of sperm-zona binding. In this study, we have used an indirect immunofluorescence (IIF) assay and other staining approaches to examine capacitation-associated membrane priming of mouse spermatozoa. For IIF studies, we used affinity-purified antibodies against two glycohydrolases that cross-reacted with the acrosomal enzymes only when the uncapacitated spermatozoa were permeabilized. Incubation of spermatozoa in a medium that favors in vitro capacitation induced membrane priming that allowed the antibodies to cross-react with the acrosomal enzymes in capacitating acrosome-intact spermatozoa without permeabilization, as revealed by the appearance of several distinct fluorescent patterns, including an initial immunopositive lining over the acrosome cap to an intense immunopositive reaction throughout the acrosome. These early immunopositive patterns were followed by the appearance of intense fluorescent spots (droplets) that seem to establish contact with the PM in a time-dependent manner. Inclusion of calmodulin, a 17-kDa Ca(2+)-binding protein which promotes capacitation, in the incubation medium did not alter the overall rate of capacitation; however, its presence accelerated the initial stages of membrane priming. The potential similarities between sperm capacitation and early events of Ca(2+)-triggered membrane fusion among eukaryotes and among various stations of the secretory and endocytotic pathways are discussed.     

Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association

The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.     

Calmodulin signals capacitation and triggers the agonist-induced acrosome reaction in mouse spermatozoa

Capacitated acrosome-intact spermatozoa interact with specific sugar residues on neoglycoproteins (ngps) or solubilized zona pellucida (ZP), the egg's extracellular glycocalyx, prior to the initiation of a signal transduction cascade that results in the fenestration and fusion of the sperm plasma membrane and the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents (i.e., induction of the acrosome reaction (AR)). The AR releases acrosomal contents at the site of sperm-zona binding and is thought to be a prerequisite event that allows spermatozoa to penetrate the ZP and fertilize the egg. Since Ca(2+)/calmodulin (CaM) plays a significant role in several cell signaling pathways and membrane fusion events, we have used a pharmacological approach to examine the role of CaM, a calcium-binding protein, in sperm capacitation and agonist-induced AR. Inclusion of CaM antagonists (calmodulin binding domain, calmidazolium, compound 48/80, ophiobolin A, W5, W7, and W13), either in in vitro capacitation medium or after sperm capacitation blocked the npg-/ZP-induced AR. Purified CaM largely reversed the AR blocking effects of antagonists during capacitation. Our results demonstrate that CaM plays an important role in priming (i.e., capacitation) of mouse spermatozoa as well as in the agonist-induced AR. These data allow us to propose that CaM regulates these events by modulating sperm membrane component(s).     

Influence of a capacitation period on human sperm acrosome loss.

The present study investigates whether a 5 hour capacitation period modifies the ability of human spermatozoa to undergo induced acrosomal loss. Human sperm acrosomal loss was induced by treatment with either the calcium ionophore A23187, low concentrations of the phospholipid dilauroylphosphatidylcholine (PC12), or 2 hours incubation in conditioned medium prepared from human cumulus cells (CM/CC). The use of a dual staining method (FITC-ConA and Hoechst 33258) for simultaneous assessment of acrosomal status and viability demonstrated that induction of acrosomal loss with calcium ionophore was not dependent on a capacitation period. A short (5 hour) incubation period was not sufficient to induce acrosomal loss with CM/CC above spontaneous acrosome reaction rates in medium alone. A significant capacitation-dependent increase (P < 0.05) in acrosomal loss was observed when human spermatozoa were incubated with PC12. Induction of acrosomal loss of capacitated human spermatozoa with PC12 therefore provides a simple assay for the simultaneous assessment of human sperm capacitation and the acrosome reaction in vitro.     

Processes controlling sperm-egg fusion

Sperm interaction with the egg envelopes triggers the acrosome reaction. Indeed, sperm-egg fusion is accomplished by the fusion of the acrosomal process (or of the exposed inner acrosomal membrane in mammals) with the egg plasma membrane. Fusion must be preceded by the establishment of molecular contact between the two membranes. It is suggested that, as in the case of artificial phospholipid membranes, the two major obstacles to the establishment of molecular contact are electrostatic repulsion and the hydration barrier. It is argued that morphology of the acrosome is such as to favour the overcoming of such barriers. By analogy with the conditions governing fusion of artificial phospholipid membranes and cell fusion, it is proposed that the following processes play a role in sperm-egg fusion. The large calcium uptake accompanying the acrosome reaction may help fusion either through the known effect of calcium on fusion of phospholipid membranes or by shielding the surface charges of the acrosomal process. Fusogenic proteins at the surface of the acrosomal process are likely to play a role in the fusion of the acrosomal process with the egg plasma membrane. The activation of phospholipases in conjunction with the acrosome reaction may also be instrumental in sperm-egg fusion through the transient production of lysophosphatides. Clearance or translocation of intramembraneous proteins in the egg plasma membrane at the site of contact with the acrosomal process may also be required for fusion. Lastly it is suggested that a translocation or a conformational change of some proteins of the egg plasma membrane, which is required for fusion, may be induced by the depolarization of the egg plasma membrane that follows molecular contact with the acrosomal process.     

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.     

Is sperm capacitation analogous to early phases of Ca2+‐triggered membrane fusion in somatic cells and viruses?

An important feature of male fertility is the physiological priming of spermatozoa by a multifaceted process collectively referred to as capacitation. The end point of this evasive process is the hyperactivated spermatozoa capable of binding to terminal sugar residues on the egg's extracellular coat, the zona pellucida (ZP), and undergoing acrosomal exocytosis (i.e., induction of the acrosome reaction). The hydrolytic action of acrosomal enzymes released at the site of zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoa, are important factors that regulate the penetration of ZP and fertilization of the egg. Despite many advances in identifying sperm components that promote capacitation, the mechanism underlying the calcium-triggered process remains elusive. The purpose of this review article is to focus on new advances that have enhanced our understanding of in vivo/in vitro capacitation, a prerequisite event resulting from a dramatic modification and reorganization of the sperm membrane molecules. Special emphasis has been laid on accumulating evidence suggesting potential similarities between the sperm capacitation and early phases of calcium-triggered membrane fusion (i.e., tethering and docking) during secretory and endocytotic pathways among eukaryotes.     

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.     

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.     

The role of F-actin cytoskeleton-associated gelsolin in the guinea pig capacitation and acrosome reaction

The acrosomal reaction (AR) is a regulated sperm exocytotic process that involves fusion of the plasma membrane (PM) with the outer acrosomal membrane (OAM). Our group has described F-actin cytoskeletons associated to these membranes. It has been proposed that in regulated exocytosis, a cortical cytoskeleton acts as a barrier that obstructs membrane fusion, and must be disassembled for exocytosis to occur. Actin-severing proteins from the gelsolin family have been considered to break this barrier. The present study attempted to determine if gelsolin has a function in guinea pig sperm capacitation and AR. By indirect immunofluorescence (IIF), gelsolin was detected in the apical and postacrosomal regions of the head and in the flagellum in both capacitated and non-capacitated guinea pig spermatozoa. By Western blotting, gelsolin was detected in isolated PM and OAM of non-capacitated spermatozoa. Gelsolin and actin were detected in a mixture of PM-OAM obtained by sonication, and both proteins were absent in membranes of capacitated spermatozoa. Inhibition of three different pathways of PIP2 hydrolysis during capacitation did not cancel gelsolin loss from membranes. Gelsolin was detected by Western blotting associated to membrane cytoskeletons obtained after phalloidin F-actin stabilization and Triton-X treatment; additionally, by immunoprecipitation, it was shown that gelsolin is associated with actin. By electron microscopy we observed that skeletons disassemble during capacitation, but phalloidin prevents disassembly. A three-dimensional skeleton was observed that apparently joins PM with OAM. Exogenous gelsolin stimulates AR assayed in a permeabilized spermatozoa model. Results suggest that gelsolin disassembles F-actin cytoskeletons during capacitation, promoting AR.     

Changes of Con A Receptor Sites on Mammalian Sperms during Capacitation and Acrosome Reaction

ＣｈａｎｇｅｓｏｆＣｏｎＡＲｅｃｅｐｔｏｒＳｉｔｅｓｏｎＭａｍｍａｌｉａｎＳｐｅｒｍｓｄｕｒｉｎｇＣａｐａｃｉｔａｔｉｏｎａｎｄＡｃｒｏｓｏｍｅＲｅａｃｔｉｏｎＤＵＡＮＣｈｏｎｇ－ｗｅｎ（段崇文）,ＣＨＥＮＤａ－ｙｕａｎ（陈大元）（ＳｔａｔｅＫｅｙＬ...     

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.     

Differential release of soluble and matrix components: evidence for intermediate states of secretion during spontaneous acrosomal exocytosis in mouse sperm

Although its exact role in fertilization is unknown, the acrosome is a very important, exocytotic organelle overlying the anterior aspect of sperm from many species. Structurally and functionally, the acrosome can be considered to consist of soluble and particulate compartments. One component of the particulate acrosomal matrix is the zona pellucida-binding protein sp56. Our demonstration that this protein is within the acrosomal matrix and not on the sperm plasma membrane has led us to reexamine the events of acrosomal exocytosis and the role of the sperm acrosomal matrix in the fertilization process. To visualize the soluble compartment, we have utilized sperm from transgenic mice that carry soluble green fluorescent protein (GFP) in their acrosomes and, as a means to assess the exposure of acrosomal matrix components, we have tested the ability of these sperm to bind beads coated with antibodies to sp56. The loss of GFP from the acrosomes and the binding of the beads by the sperm undergoing capacitation serve as indicators of distinct stages of acrosomal exocytosis, allowing us to define intermediates of acrosomal exocytosis that occur during the course of sperm capacitation. These experiments demonstrate that the exposure and release of acrosomal proteins during spontaneous acrosomal exocytosis is not synchronous but is regulated during capacitation. Furthermore, acrosomal exocytosis under these conditions required calcium in the medium. On the basis of these findings, we propose an alternative model for acrosomal exocytosis that considers a role for these intermediates of exocytosis during capacitation and sperm-ZP interactions.     

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.     

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.     

Equine sperm-oocyte interaction: the role of sperm surface hyaluronidase.

The plasma membrane over the sperm head of several mammalian species has been shown to express a glycerolphosphatidylinositol-linked hyaluronidase known as PH-20. This protein has been associated with the sperm's interaction with the oocyte cumulus matrix and zona pellucida. The characteristics of PH-20 in equine sperm have not been clearly defined. In this study, ejaculated gel-free semen from five stallions and epididymal sperm from isolated epididymis from 10 stallions was used to characterize the PH-20 activity in equine sperm. Affinity purified anti-equine PH-20 polyclonal antibody was used to immunodetect sperm surface-associated PH-20 and immunolabel whole sperm. The intracellular calcium indicator, Fluo-3, was used to assess sperm intracellular calcium. Stallion sperm express a surface-associated hyaluronidase localized to the posterior sperm head region in ejaculated sperm. Following in vitro capacitation and acrosomal exocytosis, the inner acrosomal membrane (IAM) displays intense hyaluronidase fluorescence suggesting that the IAM and hyaluronidase plays a significant role in zona penetration by sperm. Sperm incubated in hyaluronan (HA)-containing capacitation medium display an elevated intracellular calcium concentration (P<0.01) that is associated with translocation of PH-20 antigenic sites on the sperm surface in addition to increases in protein tyrosine phosphorylation. Caput- and cauda-derived sperm display developmentally unique PH-20 immunofluorescence expression patterns. These data suggest that the differential expression of PH-20 in ejaculated and epididymal sperm could be involved in cumulus penetration, sperm-egg recognition, and oolemmal fusion in this species.     

Cholesterol efflux promotes acrosome reaction in goat spermatozoa

Cholesterol efflux and membrane destabilization play an important role in sperm capacitation and membrane fusion in the acrosome reaction (AR). In this study we establish the effect of cholesterol removal from spermatozoa on acrosomal responsiveness. Mature goat spermatozoa were incubated in BSA-free medium in the presence of beta-cyclodextrin (betaCD) as cholesterol acceptor. After incubation with 8 mM betaCD, 50-60% of cholesterol was released from sperm membranes with no loss in the phospholipid content, and 35% of AR was induced. However, when 30% of cholesterol was lost, this moderate cholesterol decrease was unable to initiate AR. Cholesterol desorption was very rapid, following an exponential kinetics with a half-time of around 10 min, which is in contrast with the slow sigmoidal kinetics of acrosomal responsiveness: around 2 h was required for maximal AR. Our results suggest that cholesterol efflux has a direct influence on the onset of the AR, that is, merely removing cholesterol would trigger the AR.