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.     

A reevaluation of the fertilizin hypothesis of sperm agglutination and the description of a novel form of sperm adhesion

The classical isoagglutination of sea urchin sperm by egg jelly is not an agglutination of cells, as proposed by the fertilizin-antifertilizin hypothesis. Sperm motility is required to obtain the isoagglutination of Strongylocentrotus purpuratus sperm, and the sperm do not adhere to each other in the isoagglutination clusters, which cannot be fixed for microscopy and which disperse rapidly into individual cells when sperm motility is inhibited. These observations suggest that isoagglutination is the swarming of freely moving sperm to a common focus and is quite distinct from the agglutination of sperm by known crosslinking agents (antibodies or lectins).A previously unrecognized form of sperm agglutination is described which follows induction of an acrosome reaction by egg jelly, ammonia, or the ionophore A23187 in a suspension of sea urchin or sand dollar sperm. The sperm form rosettes of up to 100 cells in which the newly extended acrosomal processes adhere to each other. Rosettes can form containing sperm of different species, in which the acrosomal processes adhere without species preference.As observed by transmission electron microscopy, the acrosomal process of Lytechinus pictus sperm consists of an acrosomal tubule covered by a sheath of extracellular material. Rosette formation results from attachment between the extracellular materials of adjacent sperm.Less frequently, the acrosomal process of one sperm adheres to the midpiece of another by fusion of the acrosomal tubule and midpiece plasma membranes.     

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     

Differential release of guinea pig sperm acrosomal components during exocytosis

The contents of the sperm acrosome are compartmentalized at the biochemical and morphological levels. Biochemically, the acrosome can be considered to be comprised of two compartments: one consisting of readily soluble proteins and one containing a particulate acrosomal matrix. To test the hypothesis that compartmentalization affects the release of acrosomal components during the course of secretion in guinea pig sperm, we examined the relationship between the presence of specific proteins and acrosomal status and monitored the recovery of acrosomal constituents in the medium surrounding sperm induced to undergo exocytosis with the ionophore A23187. Cysteine-rich secretory protein 2 (CRISP-2), a soluble component of the acrosome, was rapidly lost from the acrosome soon after ionophore treatment. However, acrosomal matrix components remained associated with the sperm for longer periods. AM67, a matrix component and the guinea pig orthologue of the mouse sperm zona pellucida-binding protein sp56, was released at a slower rate than was CRISP-2 but at a faster rate than were two other matrix proteins, AM50 and proacrosin. Coincident with their release from the sperm, AM50 and proacrosin were posttranslationally modified, probably by proteolysis. The release of proacrosin from the matrix appears associated with the conversion of this protein to the enzymatically active acrosin protease. These results provide strong support for the hypothesis that compartmentalization plays a significant role in regulating the release of proteins during the course of acrosomal exocytosis. Acrosomal matrix proteins remain associated with the sperm for prolonged periods of time following the induction of acrosomal exocytosis, suggesting that transitional acrosomal intermediates may have significant functions in the fertilization process.     

A new procedure for determining acrosomal status of very small numbers of human sperm

The acrosome of human sperm cannot be easily distinguished by light microscopy. Although several techniques are now available to label the acrosomal region of human sperm and report acrosomal status, they generally require large numbers of sperm. We describe here a new procedure in which sperm are collected and treated on small-pore filters. The acrosomal region is then labeled using fluoresceinated lectin. The main advantage of this method is that it enables the study of the acrosomal status of sperm in samples with very low sperm concentration.     

Phagocytosis of sperm heads lacking the acrosomal process by unfertilized starfish oocytes

In sperm of the starfish Asterina pectinifera, the acrosomal process and the flagellum were mechanically separated from the sperm head with a disperser. The sperm head fraction was then used to examine the direct interaction between the sperm head and the egg surface. Sperm heads lacking the acrosomal process and the flagellum did not fertilize oocytes, even after removal of the vitelline coat. Transmission electron microscopy showed that each denuded oocyte engulfed the sperm head without gamete membrane fusion. The sperm-engulfing response, similar to phagocytosis, was induced without the mediation of the acrosomal process. The present results suggest that the process of sperm incorporation consists of two independent events, acrosomal process-egg surface fusion and the phagocytotic movement of the egg surface.     

Evaluation of rabbit sperm acrosomal integrity and fertilizing ability by use of vital stains.

Three staining procedures to detect sperm acrosome integrity were compared via electron microscopy. Stains were applied to epididymal, freshly ejaculated, in vivo capacitated, and sonicated sperm cells in addition to spermatozoa displaying sequentially removed plasma and outer and inner acrosomal membranes. Sequential membrane removal procedures resulted in removal of plasma membranes from 73% of all sperm cells, removal of plasma and outer acrosomal membranes from 74% of all sperm cells, and removal of plasma and outer and inner acrosomal membranes from 87% of all sperm cells as determined by electron microscopy. Live/dead staining results were not statistically different from subjective microscopic motility evaluations (P less than 0.005) for epididymal, sonicated, freshly ejaculated, and in vivo capacitated sperm samples. All three stains assessed were similarly capable of detecting the acrosome status of freshly ejaculated and of sonicated spermatozoa compared to data obtained by electron microscopy (P = 0.010). However, only the Bryan-Akruk stain afforded data that were closely correlated with data obtained via electron microscopy for all sperm types assessed; the latter included in vivo capacitated spermatozoa and sperm cells rendered free of plasma membranes. Results confirmed an earlier report by successfully effecting sequential removal of rabbit acrosomal membranes and documented use of the Bryan-Akruk acrosomal stain for evaluation of sperm cell populations for fertilizing ability. These findings should prove useful in further investigations of mechanisms involved in achievement of fertilizing ability by rabbit spermatozoa.     

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.     

Zonadhesin assembly into the hamster sperm acrosomal matrix occurs by distinct targeting strategies during spermiogenesis and maturation in the epididymis

Zonadhesin is the only sperm protein known to bind in a species-specific manner to the zona pellucida. The zonadhesin precursor is a mosaic protein with a predicted transmembrane segment and large extracellular region composed of cell adhesion, mucin, and tandem von Willebrand D domains. Because the precursor possesses a predicted transmembrane segment and localizes to the anterior head, the mature protein was presumed to be a sperm surface zona pellucida-binding protein. In this study of hamster spermatozoa, we demonstrate that zonadhesin does not localize to the sperm surface but is instead a constituent of the acrosomal matrix. Immunoelectron microscopy revealed that distinct targeting pathways during spermiogenesis and sperm maturation in the epididymis result in trafficking of zonadhesin to the acrosomal matrix. In round spermatids, zonadhesin localized specifically to the acrosomal membrane, where it appeared to be evenly distributed between the outer and inner membrane domains. Subsequent redistribution of zonadhesin resulted in its elimination from the inner acrosomal membrane and restriction to the outer acrosomal membrane of the apical and principal segments and the contents of the posterior acrosome. During sperm maturation in the epididymis, zonadhesin dissociated from the outer acrosomal membrane and became incorporated into the forming acrosomal matrix. These data suggest an important structural role for zonadhesin in assembly of the acrosomal matrix and further support the view that the species specificity of zona pellucida adhesion is mediated by egg-binding proteins contained within the acrosome rather than on the periacrosomal plasma membrane.     

Pisum sativum agglutinin used as an acrosomal stain of porcine and caprine sperm

Fluorescein isothiocyanate-labelled pisum sativum agglutinin (FITC-PSA) was evaluated as an acrosomal stain for porcine and caprine sperm that had previously been stained with Hoechst 33258 to assess cell viability. The FITC-PSA procedure was as accurate as other procedures in assessing acrosomal presence or absence on either fresh or liquid-stored porcine sperm. Approximately half of the incubated porcine sperm with acrosomal loss maintained membrane impermeability to the Hoechst 33258; these were potentially viable acrosome-reacted sperm. The FITC-PSA procedure was significantly correlated with the assessment of acrosomal status of cryopreserved caprine sperm by transmission electron microscopy (TEM). However, TEM results indicated a higher percentage of caprine sperm with acrosomal loss. Ability to penetrate zona-free hamster ova was not highly correlated with the percentage of viable acrosome-reacted porcine or caprine sperm. The FITC-PSA procedure provides an estimate of viable acrosome reactions and may be a useful tool in the evaluation of sperm fertility.     

Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies

An important question in mammalian gamete physiology concerns how capacitation and the occurrence of acrosome reactions in motile sperm relate to fertility. Evaluation of these relationships has been restricted by practical limitations because rapid, quantitative assays are unavailable. We have developed a rapid, reproducible assay for the evaluation of acrosomal status utilizing monoclonal antibodies specific to antigens localized in the acrosomal cap region of the sperm head. Mice were immunized with human ejaculated sperm preparations and the resultant hybridomas producing antisperm antibody were selected by solid-phase radioimmunoassay and indirect immunofluorescence (IIF). Two monoclonal antibodies (HS-19, HS-21) recognized target antigens restricted to the acrosomal cap by IIF, and 87 +/- 8.5% of the sperm in fresh ejaculates from 10 different sperm donors showed positive cap fluorescence with these reagents. Loss of HS-21 binding as measured by IIF was correlated with disappearance of the acrosomal cap as observed directly by transmission electron microscopy. Acrosomal disappearance, artificially induced in vitro using the calcium ionophore A23187, also resulted in a loss of HS-21 binding. The induction of acrosomal loss by ionophore was dependent upon extracellular calcium. The data presented suggest that specific monoclonal antibodies can be used for the rapid evaluation of acrosomal status in mammalian sperm.     

Assembly of the fluorescent acrosomal matrix and its fate in fertilization in the water strider,Aquarius remigis

Animal sperm show remarkable diversity in both morphology and molecular composition. Here we provide the first report of intense intrinsic fluorescence in an animal sperm. The sperm from a semi‐aquatic insect, the water strider, Aquarius remigis, contains an intrinsically fluorescent molecule with properties consistent with those of flavin adenine dinucleotide (FAD), which appears first in the acrosomal vesicle of round spermatids and persists in the acrosome throughout spermiogenesis. Fluorescence recovery after photobleaching reveals that the fluorescent molecule exhibits unrestricted mobility in the acrosomal vesicle of round spermatids but is completely immobile in the acrosome of mature sperm. Fluorescence polarization microscopy shows a net alignment of the fluorescent molecules in the acrosome of the mature sperm but not in the acrosomal vesicle of round spermatids. These results suggest that acrosomal molecules are rearranged in the elongating acrosome and FAD is incorporated into the acrosomal matrix during its formation. Further, we followed the fate of the acrosomal matrix in fertilization utilizing the intrinsic fluorescence. The fluorescent acrosomal matrix was observed inside the fertilized egg and remained structurally intact even after gastrulation started. This observation suggests that FAD is not released from the acrosomal matrix during the fertilization process or early development and supports an idea that FAD is involved in the formation of the acrosomal matrix. The intrinsic fluorescence of the A. remigis acrosome will be a useful marker for following spermatogenesis and fertilization. J. Cell. Physiol. 226: 999–1006, 2011. © 2010 Wiley‐Liss, Inc.     

In vitro release of house fly,Musca domestica l. (Diptera : Muscidae), acrosomal material after treatments with secretion of female accessory gland and micropyle cap substance

Mature house fly, Musca domestica L. (Diptera : Muscidae), sperm were treated with female accessory gland secretion and micropyle cap substance to determine their effectiveness in eliciting an acrosome response. Scanning and transmission electron microscopy revealed that release of acrosomal material was achieved by treatment of the sperm with a combination of accessory gland secretion and micropyle cap substance but not by gland secretion alone. The gland secretion was utilized to dissolve the cap substance from mature ovarian eggs, and this combined solution was applied to sperm removed from the spermathecae of mated females. The acrosomes of several of the sperm were completely lacking, while others showed a partial effect of the treatment, i.e. extensive acrosomal membrane fragmentation and vesiculation. Most sperm that reacted to the treatment lacked a plasma membrane around the acrosome, suggesting that this membrane, along with the acrosomal membrane, is involved in formation of the vesicles alongside the acrosomal cavity.     

Acrosomal status in fresh and capacitated human ejaculated sperm

The acrosomal status of human sperm was evaluated by immunofluorescence utilizing a specific monoclonal antibody that recognizes target antigen(s) localized in the acrosomal cap region. Spontaneous acrosomal loss was first examined in sperm preparations used for successful in vitro fertilization of human eggs. In these sperm populations, less than 20% of the sperm underwent degenerative or spontaneous acrosomal loss following 24 h of incubation. The correlation of acrosomal loss with changes in motility and viability suggested that sperm senescence was not necessarily coupled to acrosomal loss. Chemical induction of acrosomal loss by calcium ionophore A23187 and lysophosphatidylcholine (LPC) was characterized. Maximal ionophore induction (10 microM A23187 in media containing calcium) was observed in cells exposed to capacitating conditions in vitro; sperm exposed to noncapacitating conditions did not readily acquire the ability to respond to ionophore. The reaction induced by ionophore was slow (60 min), and at least 30% of the cells were always resistant to induction. In contrast, LPC induced rapid, synchronous acrosomal loss in either freshly ejaculated or capacitated sperm in the presence or absence of extracellular calcium, suggesting that this loss was not a physiologic reaction. These studies may provide a basis for evaluating capacitation and ultimately fertility potential in the human male.     

Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa

Polyclonal antisera directed against conserved and subtype-specific peptide sequences of the alpha-subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to characterize the nature of mammalian sperm G proteins and to determine whether their localization was consistent with their proposed roles in mediating ZP3-induced acrosomal exocytosis. Mouse and guinea pig sperm exhibit positive immunofluorescence in the acrosomal region using an antiserum directed against a peptide region common to all alpha-subunits of G proteins (G alpha). The immunofluorescence disappears after sperm have undergone the acrosome reaction, suggesting that the immunoreactive material is associated with the plasma membrane/outer acrosomal membrane region overlying the acrosome. The presence of G proteins in this region is confirmed by the presence of a Mr 41,000 substrate for pertussis toxin (PT)-catalyzed [32P]ADP-ribosylation in purified plasma membrane/outer acrosomal membrane hybrid vesicles obtained from acrosome-reacted guinea pig sperm. Immunoprecipitation and polyacrylamide gel electrophoresis of PT-catalyzed [32P]ADP-ribosylated protein(s) using anti-peptide antisera generated against sequences unique to Gi alpha 1, Gi alpha 2, and Gi alpha 3 confirm the existence of all three Gi subtypes in mouse sperm extracts. Indirect immunofluorescence using an antiserum directed against a peptide region present in Gz alpha, a PT-insensitive G protein, demonstrates positive immunoreactivity in the postacrosomal/lateral face region of the mouse sperm head. This immunoreactivity is retained during acrosomal exocytosis in response to solubilized ZP and then disappears subsequent to this exocytotic event. These data demonstrate that Gi protein alpha-subunits are present in the acrosomal region of mammalian sperm, consistent with their postulated role in regulating ZP3-mediated acrosomal exocytosis, and that PT-insensitive Gz alpha is found in a region of the sperm head distinct from that of the Gi alpha subunits.     

The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo

Adult female golden hamsters were induced to superovulate. When they were mated several hours prior to ovulation or artificially inseminated about the time of ovulation, nearly 100% of their eggs were subsequently fertilized monospermically. During the progression of fertilization when the eggs were still surrounded by compact cumulus oophorus, the contents of the ampullary region of the oviducts were collected and spermatozoa moving in the ampullary fluid, within the cumulus and on/in the zonae pellucidae of unfertilized eggs, were examined by light and electron microscopy to evaluate the status of their acrosomal caps. Most spermatozoa swimming in the ampullary fluid had apparently intact acrosomal caps, while the vast majority moving within the cumulus had distinctly modified acrosomal caps. Most spermatozoa that had passed through the cumulus and reached the zona surfaces had remnants of their acrosomal caps (“acrosomal ghosts”). When the ghosts were present around the sperm heads on the zona, the heads pivoted about a point roughly corresponding to the places where the ghosts were located. The ghosts seemed to firmly attach to the zona surfaces, then were split open by the sperm heads and left behind as the sperm heads advanced into the zona. A few spermatozoa on the zona surfaces had no acrosomal ghosts (at least not detectable by light microscopy). In this case, the sperm head pivoted about either the inner acrosomal membrane or the equatorial segment of the acrosome. In no instance were spermatozoa with intact acrosomal caps found on zona surfaces. We infer from these observations that most spermatozoa in vivo initiate their acrosome reactions while they are advancing through the cumulus. When they arrive at the zona surfaces, acrosomal ghosts are generally present on the sperm heads. These ghosts appear to hold sperm heads to zona surfaces as well as to restrict the direction of advancement of sperm head through the zona. In a minority of cases, ghostless spermatozoa reach the zona surfaces. As these spermatozoa appear to be able to penetrate the zona successfully, structures other than the acrosomal ghost (ie, the inner acrosomal membrane and the plasma membrane over the equatorial segment of the acrosome) may also attach to zona surfaces before spermatozoa penetrate into the zona.     

Acrosomal reaction of Thyone sperm. II. The kinetics and possible mechanism of acrosomal process elongation

Thyone sperm were induced to undergo the acrosomal reaction with a calcium ionophore A23187 in sea water containing 50 mM excess CaCl2, and the extension of the acrosomal process was recorded with high- resolution, differential interference contrast video microscopy at 60 fields/sec. The length of the acrosomal process was measured at 0.25-s intervals on nine sperm. When the data were plotted as (length)2 vs. time, the points fell exactly on a straight line except for the initial and very final stages of elongation. Cytochalasin B alters the rate of elongation of the acrosomal process in a dose-dependent way, inhibiting the elongation completely at high concentrations (20 micrograms/ml). However, no inhibition was observed unless excess Ca++ was added to sea water. The concentration of actin in the periacrosomal cup of the unreacted sperm is as high as 160 mg/ml; we calculate this concentration from the number and lengths of the actin filaments in a fully reacted sperm, and the volume of the periacrosomal cup in the unreacted sperm. These results are consistent with the hypothesis proposed earlier that monomers add to the ends of the actin filaments situated at the tip of the growing acrosomal process (the preferred end for monomer addition), and that the rate of elongation of the process is limited by diffusion of monomers from the sperm head (periacrosomal cup) to the tip of the elongating process. During the extension of the acrosomal process, a few blebs distributed along its lengths move out with the process. These blebs maintain a constant distance from the tip of the growing process. At maximum length, the straight acrosomal process slackens into a bow, and numerous new blebs appear. A few seconds later, the process suddenly straightens out again and sometimes actually contracts. The behavior of the blebs indicates that membrane is inserted at the base of the growing acrosomal process, and that membrane assembly and water uptake must be coupled to actin assembly during elongation. We discuss how the dynamic balance of forces seems to determine the shape of the growing acrosomal process, and how actin assembly may be controlled during the acrosomal reaction.     

Morphological identification of sperm receptors above egg microvilli in the polychaete, Neanthes japonica

A fine-structural study of sperm-egg interactions in the polychaete Neanthes japonica was carried out. Unfertilized eggs are surrounded by a chorion 0.6-0.7 micrometers thick. Oocyte microvilli are inserted into the inner layer of the chorion. The outer layers of the chorion are opened just above the tips of the microvilli, where a membrane vesicle (microvillus tip vesicle, about 0.2 micrometers in diameter) plugs the chorion's opening. During fertilization, the acrosomal process of the sperm fuses with an egg microvillus within 1 min of insemination. All the microvillus tip vesicles disappear from the chorion surface within 5 min of insemination. When eggs, which are prefixed with glutaraldehyde, are inseminated, numerous sperm undergoing the acrosome reactions attach to the eggs. In the majority of these sperm, the tip of acrosomal process which is coated with the acrosomal content, adhere to a microvillus tip vesicle. These findings suggest that the microvillus tip vesicle serves as a sperm receptor, which induces the acrosome reactions and adhere to the sperm acrosomal process. The adhesion of the acrosomal process to the microvillus tip vesicle seems to be a prerequisite event for its fusion with the microvillus.     

A study of factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arbacia punctulata.

Several factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arbacia punctulata, have been investigated quantitatively using a simple substrate film technique to monitor extension of the acrosomal process by electron microscopy. Verification of typical acrosomal process formation has been accomplished using thin sections. Sperm were found to undergo the acrosomal reaction in artificial sea water in the absence of egg jelly coat at pH values above 9.6. In the presence of egg jelly a high percentage of sperm react at pH 8.6. At this pH, the fraction of sperm that undergo the acrosomal reaction is directly proportional to the concentration of egg jelly. The Ca²⁺ ionophore A23187 induces the acrosomal reaction in the absence of egg jelly at pH 8.6. The proportion of sperm that react is dependent on the concentration of ionophore and on the concentration of Ca²⁺ in the medium. Pretreatment of sperm with low levels of La³⁺ ion, which is known to be a Ca²⁺ ion antagonist, results in inhibition of egg jelly induction of the acrosomal reaction. These findings suggest that there are marked similarities between the acrosomal reaction in sea urchin sperm and membrane fusion dependent secretory processes in other cell types.     

Assessing acrosomal status of bovine sperm using fluoresceinated lectins

Binding of 12 lectins to bull sperm was analyzed to select a lectin that bound preferentially to the acrosomal region. Peanut agglutinin (PNA) and Pisum sativum agglutinin (PSA) were suitably specific for intracellular, acrosome-associated glycoconjugates. Peanut agglutinin exhibited almost no detectable binding to sperm surface receptors, but intense binding to the area of the acrosome anterior to the equatorial segment. In contrast, PSA bound intensely to anterior and equatorial acrosomal regions, and weakly to the other regions of the sperm. Acrosomal labeling by both lectins decreased when sperm were induced to acrosome-react with calcium ionophore. To determine if these lectins could be used to assess acrosomal status, we compared the percentage of acrosome-reacted sperm that were detected by staining with naphthol yellow and erythrosin B with the percentage that were detected by lectin labeling. The incidence of reacted sperm detected by PSA labeling was not significantly different from that detected by naphthol yellow/ erythrosin B (P = 0.46). The incidence of reacted sperm detected by PNA was correlated with the incidence detected by naphthol yellow/erythrosin B, but was significantly lower (P = 0.003). We conclude that labeling permeabilized sperm with fluoresceinated PSA can serve as a rapid assay for acrosomal status.