Acrosome reaction in sperm of the toad,bufo bufo japonicus

The acrosome in the sperm of the toad, Bufo bufo japonicus, consists of a membrane-limited acrosomal cap and a fibrous perforatorium. When sperm are incubated with the oviducal pars recta extract (PRE) for 30–60 min, the outer acrosomal membrane fuses with the overlying plasma membrane at several points with concomitant loss of the contents of the acrosomal cap. The inner acrosomal membrane thus exposed fuses with the plasma membrane at the caudal end of the acrosomal region. This PRE-induced acrosome reaction is completely inhibited by soybean trypsin inhibitor. Sperm found in the innermost jelly layer of inseminated eggs possess an intact acrosome, but those either passing through the vitelline coat or localizing in the perivitelline space are acrosome-reacted in the same manner as when treated with PRE. These observations, combined with recent evidence showing involvement of the pars recta substance in fertilization, indicate that the acrosome reaction occurring in a fertilizing sperm at or near the surface of the vitelline coat is a response to a substance that is derived from the pars recta and deposited in the vitelline coat.     

东方扁虾精子的超微结构

利用电镜研究了东方扁虾（Ｔｈｅｎｕｓ ｏｒｉｅｎｔａｌｉｓ）精子的形态和结构。精子由核、膜复合物区和顶体区３部分组成。核内含非浓缩的染色质、微管及细纤维丝,外被核膜;５～６条辐射臂自核部位伸出,臂内充满微管。膜复合物区位于核与顶体之间,由许多膜片层结构及其衍生的囊泡共同组成。顶体区由顶体囊和围顶体物质组成,顶体结构复杂,由顶体帽、内顶体物质和外顶体物质等构成;围顶体物质呈细颗粒状,主要分布于顶体囊     

Localization of boar sperm proacrosin during spermatogenesis and during sperm maturation in the epididymis.

The localization of proacrosin was determined by using colloidal gold labeling and electron microscopy of boar germ cells during spermiogenesis to post-ejaculation. Proacrosin was first localized in round spermatids during the Golgi phase of spermiogenesis; it was associated with the electron-dense granule, or acrosomal granule that was conspicuous within the acrosome. It remained within the acrosomal granule during the cap and acrosome phases of spermiogenesis. At these stages, there was no apparent association of the proacrosin molecule with the acrosomal membranes. During the maturation phase of spermiogenesis, proacrosin was seen to become dispersed into all regions of the acrosome except the equatorial segment. When sperm from different segments of the epididymis and ejaculated sperm were examined, localization was observed throughout the acrosome except for the equatorial segment. Here proacrosin appeared to be localized on both the inner and outer acrosomal membranes as well as with the acrosomal matrix, although further studies are required to verify the membrane localization. No labeling was seen on the plasma membrane. These data suggest that the synthesis and movement of proacrosin to sites in the acrosome are controlled by an as yet unknown process. The absence of proacrosin on the plasma membrane of mature ejaculated sperm makes it unlikely that this enzyme plays a role in sperm-zona adhesion prior to capacitation.     

Ultrastructural localization of proacrosin and acrosin in ram spermatozoa

Proacrosin and acrosin were localized immunocytochemically at the electron microscope level in ram spermatozoa undergoing an ionophore-induced acrosome reaction. Antigenicity was preserved after fixation with 0.5% w/v ethyl-(dimethylaminopropyl)-carbodimide, and an antibody preparation was used that reacted with all major forms of ram acrosin. All stages of the acrosome reaction could be observed in a single preparation. At the earliest stage, labeling was observed throughout the acrosomal contents, which were just beginning to disperse. As dispersal proceeded, labeling diminished, being associated only with visible remnants of the acrosomal matrix. By the time the acrosome had emptied, almost no labeling could be detected on the inner acrosomal membrane. The relationship between matrix dispersal and proacrosin activation was studied in isolated ram sperm heads. While proacrosin was prevented from activating, the acrosomal matrix remained compact; but as activation proceeded, the matrix decondensed and dispersed in close parallel. By the time proacrosin activation was complete, the acrosomal contents had almost entirely disappeared. We conclude that proacrosin is distributed throughout the acrosomal contents as an intrinsic constituent of the acrosomal matrix. During the acrosome reaction, proacrosin activation occurs, resulting directly in decondensation of the matrix. All the contents of the acrosome including acrosin disperse and, by the time the acrosome is empty and the acrosomal cap is lost, only occasional traces of acrosin remain on the inner acrosomal membrane. Since the acrosomal cap is normally lost during the earliest stages of zona penetration, acrosin's role in fertilization is unclear: it does not appear to be a zona lysin bound to the inner acrosomal membrane.     

Egg jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope

The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+). Fluorescein isothiocyanate-labeled DBA bound specifically to the acrosomal cap of the intact sperm in the presence of a Ca(2+)-chelating agent, EDTA, suggesting that binding of DBA to the native receptor for the egg jelly substance on the acrosomal region took the place of the egg jelly substance-induced acrosome reaction. In contrast, the sperm that had been acrosome reacted by DBA treatment did not bind to the vitelline envelope of the egg whose jelly layers were removed. Subsequent addition of jelly extract caused the sperm binding to vitelline envelope, indicating that the egg jelly of the newt contains substances that are involved in not only inducing the acrosome reaction but also binding to the vitelline envelope. This is the first demonstration of the involvement of egg jelly substance in the binding of acrosome-reacted sperm to the vitelline envelope.     

PH‐20 but not acrosin is involved in sperm penetration of the macaque zona pellucida

In this study, we investigated the functions of PH‐20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm‐zona binding in other species. Anti‐macaque PH‐20 IgG, anti‐pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD‐46, which is also located on the inner acrosomal membrane, but has no known function in sperm‐zona pellucida interaction. After labeling with anti‐acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH‐20 and CD‐46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti‐acrosin IgG nor anti‐CD‐46 IgG affected sperm penetration of the zona at concentrations up to 300 μg/ml, but zona penetration was blocked completely when anti‐PH‐20 IgG (100 μg/ml) was present during sperm‐oocyte interaction. Ultrastructural observations of oocytes incubated with anti‐PH‐20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti‐PH‐20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm‐zona binding, rather than primary sperm‐zona binding or the zona‐induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida. Mol. Reprod. Dev. 53:350–362, 1999. © 1999 Wiley‐Liss, Inc.     

锯缘青蟹精子超微结构的研究

利用光镜和电镜观察了锯缘青蟹成熟精子的形态和超微结构。精子呈陀螺形，无鞭毛，在较宽的一端环生着１０余辐射臂。精子由球状的顶体、核杯以及核衍生的辐射臂三部分组成。顶体包括顶体管和顶体囊，后者包绕在顶体管的中央管周围，并可分为头帽带，内层和外层区。顶体被杯状的核包裹，仅头帽露于精子表面。成熟的精子中，位于核杯和顶体管之间的核膜出现局部断续或消失，中心粒和一些胞器出现的核杯腔中。     

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.     

THE ACROSOME REACTION IN MYTILUS EDULIS : I. Fine Structure of the Intact Acrosome

The intact acrosome of the Mytilus edulis spermatozoon consists of a conical vesicle, the basal side of which is deeply invaginated so that the whole vesicle forms a sheath around a very slender axial rod, about 2.7 µ long, inserted in a tube passing through the nucleus. The annular base of the acrosomal vesical is filled with a homogeneous substance; the outer wall of the vesicle is lined with a somewhat irregular layer of a particulate substance interspersed with very fine tubular elements, and its lumen is nearly filled by a strand of material which extends from the inner tip of the invagination to the apex of the acrosome. The lumen of the invagination appears empty except for the rod and a delicate sleeve-like structure which surrounds it. The plasma membrane of the sperm cell lies in immediate contact with the acrosomal membrane over its whole outer surface. In its general organization, this molluscan acrosome shows a rather close homology with that of the annelid Hydroides.     

The subacrosomal granule and its evolution during spermiogenesis in a lizard

Summary Some aspects of spermiogenesis have been studied in the testis of the teiid lizard Cnemidophorus lemniscatus lemniscatus by electron microscopy. Shortly after the acrosomal vesicle is lodged in a nuclear concavity of the spermatid, a dense granule differentiates in the center of the subacrosomal space. It is cone-shaped and shows a longitudinal striation. Its base applies to the acrosomal membrane and, through this, to the acrosomal granule. Its rounded vertex causes a depression of the nuclear membranes which, initially juxtaposed, separates at this point to form a vesicle. The granule develops and becomes a rod when spermiogenesis is advanced and the subacrosomal space has taken the form of a secondary cap. The rod is cylindrical, retains its original striation and has a convex acrosomal end. It encloses the vesicle formed by the nuclear envelope in its base and follows the apex of the nucleus. Meanwhile, the acrosomal granule loses its identity and the acrosomal cap is filled with a dense substance, in which a fringe of translucent material differentiates. This fringe lies in the dorsal and apical margins of the acrosome and is incompletely divided by longitudinal crests of the dense acrosomal substance. A projection of the Sertoli cell forms an accessory cap which envelops the acrosome and is in turn covered by the cytoplasm of the spermatid, constituting an intricate association. Two reflex membranes underlie the plasmalemma in the outer surface of the projection of the Sertoli cell. They are continuous with one another at their ends and with the cell membrane in the edge of pores. In the peripheral cytoplasm of the spermatid facing the accessory cap, numerous microtubules run longitudinally. By means of thin membranes some are interconnected or connected with the plasmalemma, from which they seem to originate.This research forms part of project N. 31.26.S1-0244 supported by the Consejo Nacional de Investigaciones Científicas y Tecnológicas     

Characterization and expression pattern of p53 during spermatogenesis in the Chinese mitten crab Eriocheir sinensis

p53, as a “Guardian of the Genome”, plays an important role in cell cycle arrest, apoptosis, DNA repair and inhibition of angiogenesis in different tissues including testis. p53 gene and its protein perform many essential roles for mammalian spermatogenesis. To explore its functions during spermatogenesis in Eriocheir sinensis, we have cloned and sequenced the cDNA (1,218 bp) of p53 from the testis by degenerating primer PCR and rapid-amplification of cDNA ends. The protein alignment of p53 shows the conserved DNA binding domain, dimerization site and zinc binding site consisted of the predicted structures. Phylogenetic analysis revealed that p53 was more closer to Marsupenaeus japonicus and Tigriopus japonicus than other examined species. Tissue expression analysis of p53 mRNA showed p53 was distinctly expressed in accessory sexual gland, muscle, gill, heart, hepatopancreas and testis. In situ hybridization revealed that the p53 mRNA was weakly distributed around the nucleus, but stronger in the invaginated acrosomal tubule at the early stage. At the middle stage, p53 mRNA signal was increased than the early stage and the signal displayed dot-like pattern on the surface of cup-like nucleus. The signal on acrosomal cap is stronger than on the acrosomal tubule, despite acrosomal tubule signal was also distinct. At the late stage, the signal was still mainly located in acrosomal cap and acrosomal tubule. Sporadic signal were found surrounding the cup-like nucleus, but they were very weak. In the mature sperm, the signal was dramatically decreased. Even though the signal on cup-like nucleus and acrosomal tubule were distinct, they were weaker than those in middle stage. Based on these results, we concluded that p53 may play an important role in formation of acrosome biogenesis and nuclear shaping during spermiogenesis of E. sinensis.     

Sialyl glycoprotein distribution on the plasma membrane of ejaculated ram spermatozoa

Localization of sialyl residues on unfixed ejaculated ram sperm membrane using the direct covalent probes of either ferritin hydrazide or latex hydrazide revealed a unique regional distribution on the plasmalemma covering the sperm head only. Three different labelling zones were identified based on the intensity and the nature of the sialyl glycoconjugates: a patchy-like zone which included the plasma membrane overlaying the post-nuclear cap and the convex side of the apical body of the acrosome; highly ordered heavily labelled zones including the plasmalemma adjacent to the concave apical body of the acrosome and to the posterior part of the equatorial acrosomal segment; a paucity-labelling zone which included the plasma membrane underlying the principal acrosomal region and the anterior part of the equatorial acrosomal segment. The possible physiological role of the highly ordered labelled zones is discussed.     

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.     

Video microscopic analysis of ionophore induced acrosome reactions of lobster (Homarus americanus) sperm

Sperm from the American lobster (Homarus americanus) are normally nonmotile. However, during fertilization, the sperm undergo a calcium-dependent acrosome reaction that propels them forward about 18 μMm. The reaction occurs in two phases, eversion and ejection, which take place too quickly to permit analysis by direct observation. The purposes of this study were to examine the structural changes occurring in sperm during the normal acrosome reaction and to determine the rate of the reaction using video microscopy. The reaction was induced in vitro by ionophore A23187 and recorded using a video system attached to a Nikon Nomarski interference microscope. Videotapes were played back frame by frame (30 frames/sec), and images of reactions from 10 sperm were analyzed. The acrosome reaction, including the eversion of the acrosomal vesicle and ejection of the subacrosomal material and nucleus, can be divided into 4 steps: (1) expansion of the apical cap followed by expansion of the remainder of the acrosomal cylinder; expansion of the cylinder begins at its apical end and proceeds toward its base, (2) eversion of the apical half of the acrosomal vesicle and initial contraction of the apical cap, (3) eversion of the basal half of the acrosomal vesicle, continued contraction of the apical cap, and ejection of the subacrosomal material and nucleus, and (4) final contraction of the apical cap and ejection of the acrosomal filament. During steps 2, 3, and 4, the mean forward movement of sperm is 12.7, 3.9, and 1.1 μMm, respectively. Although the time required to complete the reaction ranged from 0.66 to 5.16 s, most sperm reacted in less than 3. s, and these sperm were considered to have typical rates. For sperm that reacted in less than 3 s, both step 1 and step 4 take about 0.2 s and show little variation among sperm. the time required to complete steps 2 and 3 averaged 0.63 and 0.37 s, respectively. Forward movement of the sperm during the acrosome reaction is caused by eversion of the inner and outer acrosomal material and contraction of the apical cap. The protein(s) responsible for this contraction is not yet known. © 1993 Wiley-Liss, Inc.     

ROLE OF THE GAMETE MEMBRANES IN FERTILIZATION IN SACCOGLOSSUS KOWALEVSKII (ENTEROPNEUSTA) : II. Zygote Formation by Gamete Membrane Fusion

An earlier paper showed that in Saccoglossus the acrosomal tubule makes contact with the egg plasma membrane. The present paper includes evidence that the sperm and egg plasma membranes fuse to establish the single continuous zygote membrane which, consequently, is a mosaic. Contrary to the general hypothesis of Tyler, pinocytosis or phagocytosis plays no role in zygote formation. Contact between the gametes is actually between two newly exposed surfaces: in the spermatozoon, the surface was formerly the interior of the acrosomal vesicle; in the egg, it was membrane previously covered by the egg envelopes. The concept that all the events of fertilization are mediated by a fertilizin-antifertilizin reaction seems an oversimplification of events actually observed: rather, the evidence indicates that a series of specific biochemical interactions probably would be involved. Gamete membrane fusion permits sperm periacrosomal material to meet the egg cytoplasm; if an activating substance exists in the spermatozoon it probably is periacrosomal rather than acrosomal in origin. The contents of the acrosome are expended in the process of delivering the sperm plasma membrane to the egg plasma membrane. After these membranes coalesce, the sperm nucleus and other internal sperm structures move into the egg cytoplasm.     

The Acrosome Reaction in Ciona intestinalis (Ascidia, Tunicata)

An acrosome reaction occurs by fusion between the acrosomal outer membrane and the plasmalemma enclosing the acrosome in Ciona intestinalis spermatozoa. The fusion seems to proceed along the peripheral margin of the acrosome, which causes vesiculation. The membrane bound vesicle formed by this process is probably shed by the sperm. The acrosomal inner membrane is exposed and becomes a part of the plasmalemma enclosing the anterior region of the sperm head. During this process, any acrosomal substance might be released through the opening formed by membrane fusion. The acrosome reaction most likely occurs in C. intestinalis spermatozoa, via vesiculation, in fundamentally the same way as observed in mammalian spermatozoa.     

Fine structure of the unistellate sperm of the shrimp, Sicyonia ingentis (Natantia)

Sperm of the prawn Sicyonia ingentis were studied cytochemically and ultrastructurally. Striking cytological differences were noted between these natantian sperm and previously studied reptantian sperm. In general, the S. ingentis sperm are composed of a spherical main body that is partially encompassed by a morphologically diverse cap region, from which extends a single appendage or spike. The main body houses an uncondensed, Feulgen-positive nuclear region that is partially surrounded by a cytoplasmic band. A single layer of small, 600 Å, vesicles lines the periphery of the cytoplasmic band. Large membranous vesicles extend from the inner surface of the cytoplasmic band into the nuclear region. The nucleus is separated from the cap or acrosomal complex by a dense plate and a highly organized crystalline lattice, which is composed of geometric squares that are approximately 350 Å in dimension. The cap region also contains convoluted membrane pouches; a central granular core; spherical bodies; an electron-dense, saucer-shaped plate; and a large anterior granule. The convoluted membrane pouches and anterior granule are periodic acid-Schiff (PAS) positive. The anterior granule also demonstrates RNAase-stable red fluorescence with acridine orange staining. A spiralled spike, approximately 6 μm long, extends from the anterior end of the cap. The cap and spike are bound by a double membrane, which results from the fusion of the plasma membrane and the convoluted pouch membrane. The sperm's acrosome is thought to be composed of the two PAS-positive cap components and the spike.     

Biochemical characterization of the PH-20 protein on the plasma membrane and inner acrosomal membrane of cynomolgus macaque spermatozoa

Preparations of sperm membranes (plasma membranes and outer acrosomal membranes) and denuded sperm heads were isolated from macaque sperm, and the PH-20 proteins present were characterized by Western blotting, hyaluronic acid substrate gel analysis, and a microplate assay for hyaluronidase activity. Because we have shown previously that PH-20 is located on the plasma membrane and not on the outer acrosomal membrane, the PH-20 in the membrane preparations was presumed to be plasma membrane PH-20 (PM-PH-20). PM-PH-20 had an apparent molecular weight of 64 kDa and the optimum pH for its hyaluronidase activity was 6.5. The PH-20 associated with denuded sperm heads was localized by immunogold label to the persistent inner acrosomal membrane (IAM) and was presumed to be IAM-PH-20, which included a major 64 kDa form and a minor 53 kDa form. The 53 kDa form was not detected in extracts of denuded sperm heads from acrosome intact sperm that were boiled in nonreducing sample buffer, but was present in extracts of sperm heads from acrosome reacted sperm and in the soluble material released during the acrosome reaction, whether or not the samples were boiled. Substrate gel analysis showed that the hyaluronidase activity of the 53 kDa form of PH-20 was greatest at acid pH, and this activity was probably responsible for the broader and lower optimum pH of IAM hyaluronidase activity. When hypotonic treatment was used to disrupt the sperm acrosome and release the acrosomal contents, less than 0.05% of the total hyaluronidase activity was released. The PH-20 protein released by hypotonic treatment was the 64 kDa form and not the 53 kDa form, suggesting that its source might be the disrupted plasma membranes. Our experiments suggest that the soluble form of hyaluronidase, which is released at the time of the acrosome reaction, is derived from the IAM. This soluble hyaluronidase is composed of both the 64 kDa form and 53 kDa form of PH-20. The 53 kDa form appears to be processed from the 64 kDa form at the time of the acrosome reaction. Mol. Reprod. Dev. 48:356–366, 1997. © 1997 Wiley-Liss, Inc.     

Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy

Actin was localized in testicular spermatids and in ionophore-treated ejaculated sperm of boar by use of a monoclonal anti-actin antibody labeled with colloidal gold. With the on-grid postembedding immunostaining of Lowicryl K4M sections, actin was identified in the subacrosomal region of differentiating spermatids, in the microfilaments of the surrounding Sertoli cells, and in the myoid cells of the tubular wall. Ejaculated sperm, labeled with the preembedding method, showed actin between the plasma membrane and the outer acrosomal membrane of the equatorial segment. Indirect immunofluorescence was positive in the equatorial segment and in the acrosomal cap of intact sperm, whereas reacted sperm at the anterior head region retained fluorescence only in the inner acrosomal membrane. Rhodamine-phalloidin failed to stain intact and reacted sperm. The distribution of actin in sperm head membranes (inner acrosomal membrane, membranes of the equatorial segment), which are retained after the acrosome reaction, is discussed.     

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.