Difference in the Manner of Association of Acrosome-Intact and Acrosome-Reacted Hamster Spermatozoa with Egg Microvilli as revealed by Scanning Electron Microscopy

Scanning electron microscopy was employed to examine the manner of association between in vitro capacitated spermatozoa and zona-free eggs of the hamster. Spermatozoa with intact acrosomes, which were unable to fuse with eggs, were seen in general associated with egg microvilli in the region of the acrosomal cap. Acrosome-reacting spermatozoa were seen associated with egg microvilli with the dissociating acrosomal caps. Acrosome-reacted spermatozoa, which were able to fuse with eggs, generally associated with egg microvilli by the equatorial segment and the anterior portion of the postacrosomal region. It is inferred that the completion of the acrosome reaction signals changes in the plasma membrane over the equatorial segment of the acrosome and the anterior area of the postacrosomal region which give it a greater affinity to and fusibility with the oolemma.     

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.     

Immunolocalization of heat shock protein 70 (Hsp 70) in boar spermatozoa and its role during fertilization

The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P < 0.05) increase in the proportion of unfixed cells showing the fluorescent signal has been recorded. No differences in Hsp70 amount between fresh, capacitated, and reacted semen were observed by Western blot. The presence of anti-Hsp70 antibody in the fertilization medium significantly reduced, in a concentration-dependent manner, the fertilization rate of both zona-intact and zona-free oocytes. The overall data demonstrate that Hsp70 is present on boar sperm with a dynamic redistribution as the sperm undergoes capacitation and acrosome reaction and suggest an important role of this protein during porcine gamete interaction.     

Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis

To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.     

Actin polymerization in the equatorial and postacrosomal regions of guinea pig spermatozoa during the acrosome reaction is regulated by G proteins

The acrosome reaction (AR) is an exocytotic process of spermatozoa, and an absolute requirement for fertilization. During AR, actin polymerization is necessary in the equatorial and postacrosomal regions of guinea pig sperm for spermatozoa incorporation deep into the egg cytoplasm, but not for plasma membrane (PM) fusion nor the early steps of egg activation. To identify the mechanisms involved in this sperm actin polymerization, we searched for the protein members, known to be involved in a highly conserved model, that may apply to any cellular process in which de novo actin polymerization occurs from G protein activation. WASP, Arp 2/3, profilins I and II, and Cdc42, RhoA and RhoB GTPases were localized by indirect immunofluorescence (IIF) in guinea pig spermatozoa and their presence corroborated by Western blotting. WASP and profilin II were translocated to the postacrosomal region (Arp2/3 already were there) in long-term capacitated and acrosome-reacted spermatozoa, at the same time as actin polymerization occurred. These events were inhibited by GDP-beta-S and promoted by lysophosphatidic acid (LPA) and GTP-gamma-S, a small GTPase inhibitor and two activators, respectively. By immunoprecipitation, Cdc42-WASp association was identified in capacitated but not in noncapacitated gametes. Polymerized actin in the postacrosomal region is apparently anchored both to the postacrosomal perinuclear theca region and the overlying PM. Results suggest that GTPases are involved in sperm actin polymerization, in the postacrosomal region and the mechanism for polymerization might fit a previously proposed model (Mullins, 2000: Curr Opin Cell Biol 12:91-96).     

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.     

A new procedure for rapidly scoring acrosome reactions of human sperm

Because the acrosome of human sperm is too small to be directly visualized by phase-contrast microscopy, acrosome reactions (that is loss of the acrosome) are generally not evaluated in studies of human sperm capacitation and fertilization. Nevertheless, it would be useful in such studies to have a technique for easily identifying and quantitating acrosome-reacted sperm. In this paper, we describe a method for labeling the human sperm acrosome with fluorescein-conjugated Ricinus communis agglutinin-60 (FITC-RCA); we show that in sperm without acrosomal caps, FITC-RCA labeling occurs either not at all or only in the equatorial segment of the acrosome. To determine if the absence of FITC-RCA labeling in the acrosomal cap region gives a reliable estimate of acrosome reactions, washed sperm or sperm incubated in a capacitating medium (BWW) were divided into two groups, which were then fixed for FITC-RCA labeling or transmission electron microscopy. Counts of acrosome reactions made by each method were similar, and we observed an increase in the percentage of reactions following incubation in BWW. We conclude that the FITC-TCA labeling technique is a reliable method for accurately scoring the percentage of acrosome-reacted human sperm.     

Role of monoclonal antibody J-23 in inducing acrosome reactions in capacitated mouse spermatozoa.

A monoclonal antibody (J-23) to the 15 kDa component on the sperm head, the acceptor, which functions in zona binding, was shown to induce the acrosome reaction in capacitated cells, but not in fresh cells. The antibody recognized its epitope in the acrosomal cap region of fresh spermatozoa and in the equatorial region on washed and capacitated spermatozoa. However, equatorial expression did not depend on the acrosome reaction, since washing fresh spermatozoa increased the percentage with equatorial fluorescence, but did not increase the percentage with reacted acrosomes. The data indicate that the acrosome reaction can be induced in capacitated spermatozoa in the absence of zona glycoproteins.     

Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon

Experiments to bind fluorescein-conjugated Arachis hypogea (peanut) agglutinin (FITC-PNA) to washed human spermatozoa demonstrated that this lectin binds to the acrosome region in air-dried preparations. Since there was no binding when labelling was performed in suspension, and comparable labelling to that seen in air-dried preparations was seen when spermatozoa treated with saponin (to lyse the plasma membrane) were labelled in suspension, the lectin must bind to an intracellular structure, probably the outer acrosomal membrane. This was confirmed by ultrastructural localization of colloidal gold-conjugated lectin in saponin-treated spermatozoa. Treatment of spermatozoa with the detergent Nonidet P-40 caused a marked change in the binding pattern: more spermatozoa showed binding in the equatorial segment of the acrosome with no binding in the anterior cap region. A comparable, less marked, change was seen when spermatozoa were incubated overnight under conditions known to support the capacitation and spontaneous acrosome reactions. Treatment with the calcium ionophore A23187 for 1 h to induce acrosome reactions artificially in uncapacitated spermatozoa resulted in the appearance of patchy acrosome fluorescence. From these experiments it is concluded that PNA binds specifically to the outer acrosomal membrane, and that FITC-PNA-labelling may be used to monitor the human sperm acrosome reaction.     

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.     

An investigation using lectins of glycocomponents of mouse spermatozoa during capacitation and sperm-zona binding

Ten different lectins conjugated to fluorescein isothiocyanate (FITC) were used to study the distribution of surface carbohydrates on mouse spermatozoa, and to monitor the possible changes of their distribution during capacitation in vitro and sperm-egg interaction. Most of the lectins gave a restricted pattern of binding to fixed or unfixed epididymal spermatozoa. Binding was highly specific because no staining occurred in the presence of appropriate monosaccharides. Binding of UEA I, DBA and Con A was unaffected by the type of fixative used, but it was influenced by mild centrifugation. While unwashed spermatozoa showed binding mainly over the acrosomal cap and equatorial or postacrosomal regions, spermatozoa washed by mild centrifugation showed a change in the staining of the equatorial segment. Binding of 5 different lectins to spermatozoa did not change during capacitation in vitro. In contrast, capacitated spermatozoa bound to the zona pellucida exhibited a UEA I binding pattern which was strikingly different from that of the capacitated but unbound spermatozoa. We conclude that glycocomponents of specific regions of mouse spermatozoa do not change dramatically during capacitation, but do alter significantly during binding to the zona pellucida.     

Effects of angiotensin II on the acrosome reaction in equine spermatozoa

Angiotensin II is a hormone with a wide array of physiological effects that exerts its effect via interaction with two major subtypes of receptor. The results of this study show that angiotensin II (from 1 to 100 nmol l(-1)) initiates acrosomal exocytosis in equine spermatozoa that have undergone capacitation in vitro in a TALP-TEST (Tyrode's albumin lactate pyruvate; 188.7 mmol TES l(-1), 84.8 mmol Tris l(-1)) buffer with cAMP. The acrosome reaction and sperm viability were assessed with fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) and Hoechst 33258, respectively. The initiation of the acrosome reaction by angiotensin II was strongly inhibited by losartan, a specific angiotensin II type 1 receptor antagonist. Although angiotensin II as well as progesterone both initiated the acrosome reaction in equine spermatozoa, there was no synergistic effect when both agonists were added simultaneously. Initiation of acrosomal exocytosis by angiotensin II was accompanied by a rapid and transient calcium influx that was assessed in capacitated spermatozoa loaded with Fura-2AM. In addition, the angiotensin II-mediated calcium influx was inhibited when spermatozoa were preincubated with losartan. Western blotting with an antibody against angiotensin II type 1 receptor detected a major sperm protein of 60 kDa. Indirect immunofluorescence of non-capacitated spermatozoa with the angiotensin II type 1 receptor antibody revealed labelling in the midpiece and tail. In capacitated spermatozoa, the angiotensin II type 1 receptor was localized mainly over the anterior region of the sperm head, the equatorial segment and occasionally on the postacrosomal region in addition to the sperm tail. In conclusion, this study demonstrated the ability of angiotensin II to stimulate the acrosome reaction in capacitated equine spermatozoa. This effect is mediated via the angiotensin II type 1 receptor and is accompanied by an increase in intracellular calcium.     

Localization of rabbit sperm acrosin during the acrosome reaction induced by immobilized zona matrix

To better understand the loss of the acrosomal cap on the surface of the zona pellucida and the function of the equatorial-postacrosomal region after the acrosome reaction, we have constructed an in vitro system using heat-solubilized zonae pellucidae dried onto a coverslip and incubated with capacitated spermatozoa. This system allows good optical resolution of spermatozoonzona interaction. Induction of the acrosome reaction by zonae on coverslips (30%) is comparable to the induction of the reaction reported previously for rabbit spermatozoa using solubilized zonae in solution. Antiserum to rabbit proacrosin, antiserum to a porcine 49-kDa proacrosin fragment, and antiserum to a porcine 14-kDa C-terminal acrosin fragment were utilized to monitor the acrosome reaction. Rabbit proacrosin/acrosin is not present on the surface of live, acrosome-intact, swimming spermatozoa. After contact with zona, the acrosome reaction begins and proacrosin/acrosin becomes available to bind antibody, first as a crescent in the apical region and then more posteriorly until the entire anterior acrosome is labeled. Proacrosin/acrosin remains on the equatorial and postacrosomal regions of acrosome-reacted spermatozoa and also remains associated with the acrosomal cap even after the spermatozoon is no longer associated with it. Further studies using zona-coated coverslips should lead to a more detailed understanding of the mechanism of zona penetration.     

Purified and refolded recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B expressed in Escherichia coli binds to spermatozoa

Bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B (bmZPB), excluding the N:-terminal signal sequence and the C:-terminus transmembrane-like domain, has been expressed in Escherichia coli as polyhistidine fusion protein. A requirement of 4 M urea to maintain the purified protein in soluble state rendered it unsuitable for biological studies. Purification of refolded r-bmZPB without urea and devoid of lower molecular weight fragments was achieved by following an alternate methodology that involved purification of inclusion bodies to homogeneity and solubilization in the presence of a low concentration of chaotropic agent (2 M urea) and high pH (pH 12). The solubilized protein was refolded in the presence of oxidized and reduced glutathione. The circular dichroism spectra revealed the presence of both alpha helical and beta sheet components in the secondary structure of the refolded r-bmZPB. The binding of the refolded r-bmZPB to the spermatozoa was evaluated by an indirect immunofluorescence assay and also by direct binding of the biotinylated r-bmZPB. The binding was restricted to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. In the acrosome-reacted spermatozoa a shift in the binding pattern of r-bmZPB was observed and it bound to the equatorial segment, postacrosomal domain, and midpiece region. Binding of biotinylated r-bmZPB was inhibited by cold r-bmZPB as well as by monoclonal and polyclonal antibodies generated against r-bmZPB. These results suggest that nonglycosylated bmZPB binds to capacitated as well as acrosome-reacted spermatozoa in a nonhuman primate and may have a functional role during fertilization.     

Effects of the purified IGF-I complex on the capacitation and acrosome reaction of rabbit spermatozoa

A protein complex containing IGF-I, purified from rabbit seminal plasma, was used to investigate its effects on the capacitation and acrosome reaction of rabbit spermatozoa. Uncapacitated sperm (Pattern F), capacitated sperm (Pattern B), and acrosome-reacted sperm (Pattern AR) were determined by CTC staining, and the results were validated by PSA-staining. The addition of the IGF-I complex to the capacitative medium directed the spermatozoa to spontaneous acrosome reaction. On the other hand, IGF-I complex, added to capacitated spermatozoa, acted as inducer of the acrosome reaction. Results of IVF experiments showed high rates of fertilization with capacitated spermatozoa, acrosome-reacted by either A23187 or IGF I complex, whereas significantly lower rates were obtained with spermatozoa capacitated in the presence of IGF-I complex.     

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O- linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and - reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.     

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.     

Post-testicular development of a novel membrane substructure within the equatorial segment of ram,bull, boar,and goat spermatozoa as viewed by atomic force microscopy

Atomic force microscopy has been used to investigate changes in the plasma membrane overlying the head region of mammalian spermatozoa (bull, boar, ram, goat, stallion, mouse, and monkey) during post-testicular development, after ejaculation, and after exocytosis of the acrosomal vesicle. On ejaculated ram, bull, boar, and goat spermatozoa the postacrosomal plasma membrane has a more irregular surface than that covering the acrosome. The equatorial segment, by contrast, is relatively smooth except for an unusual semicircular substructure within it that has a coarse uneven appearance. This substructure (referred to as the equatorial subsegment) is situated adjacent to the boundary between the postacrosomal region and the equatorial segment itself and seems to be confined to the order Artiodactyla as it has not been observed on stallion, mouse, or monkey spermatozoa. The equatorial subsegment develops during epididymal maturation, and following induction of the acrosome reaction with Ca(2+) ionophore A23187, its topography changes from a finely ridged appearance to that resembling truncated papillae. A monoclonal antibody to the equatorial subsegment binds only to permeabilized spermatozoa, suggesting that the subsegment is related to the underlying perinuclear theca that surrounds the sperm nucleus. A role for the equatorial subsegment in mediating fusion with the oolemma at fertilization is discussed.     

Recent advances in sperm maturation in the human epididymis

As spermatozoa move through the human epididymis they encounter a varied environment with respect to the proteins with which they come into contact. In the proximal epididymis sperm are subjected to the action of enzymes and exposure to proteins involved in membrane modification. In the middle region another set of proteins and enzymes predominates; those associated with sterol transport could modify the sperm membrane to permit the uptake of GPI-anchored zona binding proteins P34H and CD52. More distally sperm encounter increasing activities of lytic enzymes, proteins involved in both zona binding and oocyte fusion, the major maturation antigen CD52, antimicrobial activity and decapacitation factors, that help them to survive before ejaculation. Adherence of the proteins to different domains (e.g. anterior acrosome or equatorial acrosomal segment) may depend on the nature of the protein, the lipid composition of the particular membrane and the ionic environment in the epididymal lumen. The eventual location on a capacitated sperm (acrosomal membrane) or acrosome-reacted sperm (equatorial region) may dictate their role in, for example, zona-binding (P34H) or oocyte-binding (gp20). Both proteins and membranes may be modified during epididymal transit by the enzymes which may add to or remove carbo-hydrates and peptides from the sperm surface.