Evaluation of acrosomal status of bovine spermatozoa using concanavalin a lectin

We report here that fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) specifically labels the acrosomal region of acrosome-reacted bovine spermatozoa. This labeling is found to be useful in evaluating the acrosome status of bovine spermatozoa. When fresh bovine spermatozoa that had been fixed with 4% formaldehyde, smeared on glass slides and then air-dried were stained by FITC-ConA, weak fluorescence was observed on the acrosomal region, although almost all the spermatozoa appeared to be acrosome-intact. However, when fresh sperm suspensions were incubated with FITC-ConA and then mounted on glass slides, no fluorescence was observed on the acrosomal region. Therefore, in the ensuing experiments, both the fixation and the FITC-ConA staining of spermatozoa were done in suspension. When ethanol-treated spermatozoa, whose outer membrane may be permeabilized, were stained with FITC-ConA, the fluorescence was extensively observed on the inner acrosomal region. This fluorescence was inhibited in the presence of 0.2 M D-mannose, a competitive sugar, suggesting that FITC-ConA binds specifically to glycocomponents on the inner acrosomal membrane. We next tried to stain fresh or frozen-thawed spermatozoa from 3 different bulls that had been treated with the calcium ionophore A23187, which is known to induce acrosome reaction of bovine spermatozoa, with FITC-ConA. A significant correlation between the percentage of ConA-labeled spermatozoa and that of rose bengal stained negative ones at various time points during A23187 incubation was achieved. Furthermore, suitability of dual staining to distinguish between physiological acrosome reaction (acrosome-lost and live) and degenerative acrosomal loss (acrosome-lost and dead) using FITC-ConA and Hoechst bis-benzimide 33258 (H258) supravital stain was also confirmed. From these results, it was concluded that the FITC-ConA labeling procedure is a feasible and reliable method for the assessment of physiological acrosome reaction of bovine spermatozoa.     

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.     

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.     

Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody

A monoclonal antibody generated against hamster epididymal spermatozoa and recognizing an antigen within the acrosome was used in conjunction with FITC-antimouse immunoglobulin as a marker of the human acrosome during sperm development, capacitation, and the acrosome reaction. The specificity of binding of the monoclonal antibody was assessed using immunolocalization by epi-fluorescence and electron microscopy. Immunofluorescence revealed that antibody bound over the entire anterior acrosome in hamster and human spermatozoa. Ultrastructural localization indicated that antigen was predominantly present on the inner face of the outer acrosomal membrane and within the acrosomal content. Qualitative specificity was studied using a highly purified preparation of hamster acrosomes in an enzyme-linked immunosorbent assay. Since the antibody rapidly visualized human acrosomes, it was used to detect abnormal acrosome morphology of mature spermatozoa and to mark spermatids present in the ejaculate. During incubation in capacitating medium, changes in the immunofluorescence of live or methanol fixed spermatozoa were correlated with incubation interval and the ability of spermatozoa to fuse with zona-free hamster oocytes. Spermatozoa bound to zona-free hamster oocytes displayed no fluorescence, confirming that acrosome loss occurred before spermatozoa attached to the vitellus.     

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

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

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

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

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Immunodetection of acrosin during the acrosome reaction of hamster, guinea-pig and human spermatozoa.

Mammalian sperm acrosomes contain a trypsin-like protease called acrosin which causes limited and specific hydrolysis of the extracellular matrix of the mammalian egg, the zona pellucida. Acrosin was localized on hamster, guinea-pig and human sperm using monoclonal and polyclonal antibodies to human acrosin labelled with colloidal gold. This was visualized directly with transmission electron microscopy, and with light and scanning microscopy after silver enhancement of the colloidal gold probe. Four distinct labelling patterns were found during capacitation and the acrosome reaction in hamster and guinea-pig spermatozoa, and three patterns were found in human spermatozoa. In the hamster, acrosin was not detected on the inner acrosomal surface after the completion of the acrosome reaction, thus correlating with the observation that hamster spermatozoa lose the ability to penetrate the zona after the acrosome reaction. With guinea-pig and human spermatozoa, acrosin was still detected after the completion of the acrosome reaction, thus correlating with the observation that acrosome reacted guinea-pig spermatozoa bind to and penetrate the zona pellucida.     

Binding of zona binding inhibitory factor-1 (ZIF-1) from human follicular fluid on spermatozoa

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.     

Assessment of acrosome-reacted boar spermatozoa using monoclonal antibody GB 24 and propidium iodide

Fluorescein-labeled GB 24, a mouse monoclonal antibody, was evaluated as an acrosomal dye for boar spermatozoa that had previously been stained with propidium iodide (PI) to assess sperm viability. A specific sperm-staining pattern with fluorescein-labeled GB 24 was shown to be associated with acrosome reaction on freshly ejaculated sperm when fixed with acetone or induced with ionophore A 23187, whereas the presence of PI staining was typical of dying spermatozoa. The GB 24-PI procedure was as accurate as the glutaraldehyde method in assessing acrosomal presence or absence on freshly ejaculated spermatozoa when spontaneous or A 23187-induced acrosomal reactions were considered. Approximately half of A 23187-induced spermatozoa with acrosomal loss did not exhibit a PI fluorescence; these were potentially viable acrosome-reacted spermatozoa. On semen diluted in a boar sperm-specific diluent (BTS-A) and stored, percentages of spermatozoa with nonintact acrosome from glutaraldehyde and GB 24-PI were not significantly different. Conversely, data from GB 24-PI was significantly lower than those from glutaraldehyde when semen were undiluted. This suggested that spermatozoa with reacted acrosome gradually lost their ability to bind with GB 24. Providing unequivocal and rapid scoring of acrosome-reacted spermatozoa, the GB 24-PI procedure may be a valuable tool in the evaluation of the acrosomal status of porcine fresh spermatozoa.     

Soybean trypsin inhibitor as a probe for the acrosome reaction in motile cynomolgus macaque sperm

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to acrosome react with calcium ionophore A23187 in the presence of SBTI and were fixed for ultrastructural observation. Transmission electron microscopy (TEM) revealed secondary labelling of anti-SBTI-IgG with colloidal gold in association with the acrosomal matrix and fused membranes of sperm undergoing the acrosome reaction, but gold labelling was not observed on acrosome-intact sperm. When SBTI was conjugated with the fluorochrome Alexa 488, labelled (acrosome-reacted) sperm showed bright fluorescence that ranged from a patchy or punctate appearance to solid labelling over the region of the acrosomal cap. Following treatment with ionophore, the percentages of total acrosome-reacted sperm (motile and non-motile) as assessed with Alexa-SBTI, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and TEM were 54.6%, 51.6% and 61.5%, respectively. Measures of acrosomal status with FITC-PSA and Alexa-SBTI were highly correlated (r = 0.94; n = 3). Macaque zonae pellucidae were co-incubated with activated sperm for 1 min and then rinsed in medium containing Alexa-SBTI and immediately observed with epifluorescence microscopy. The mean percentage of Alexa-SBTI-labelled (acrosome-reacted) motile sperm bound to the zona was 45.7 +/- 14 (range: 22-80.4%; n = 4). Fewer than 1% of the motile sperm in suspension surrounding the zonae were acrosome-reacted. Alexa-SBTI had no effect on sperm motility, survival, or zona binding capability.     

Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation.

Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin-enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids.     

A Simple Staining Procedure for Detecting the true Acrosome Reaction in Buffalo (Bubalus bubalis) Spermatozoa

A simple dual staining procedure for detecting the true acrosome reaction in dried smears of buffalo spermatozoa is described. Trypan blue is used first to differentiate live from dead spermatozoa and the dried smears which have been prepared are stained with Giemsa for acrosome evaluation. Four categories of spermatozoa were recognized: A) live, intact acrosome (acrosome pink, postnuclear cap clear); B) dead, intact acrosome (acrosome pink, postnuclear cap blue); C) live, detached acrosome (acrosome clear, postnuclear cap clear); and D) dead, detached acrosome (acrosome clear, postnuclear cap blue). The procedure is simple, rapid and convenient for assessing true acrosome reaction in buffalo spermatozoa. Simultaneous assessment of sperm viability and its acrosomal status in dried smears makes this procedure attractive because the true acrosome reaction can be studied thoroughly at a later state after the incubation period.     

Lysophosphatidylcholine disrupts the acrosome of tammar wallaby (macropus eugenii) spermatozoa

The acrosomal status of wallaby spermatozoa was evaluated by light and electron microscopy after incubation in 1–100 μM lysophosphatidylcholine (LPC) for up to 120 min. Treatment with 1 and 10 μM LPC for 120 min did not lead to acrosomal loss, or detectable alteration to the acrosome, as detected by Bryan's staining and light microscopy. Incubation with 25 μM LPC had little effect on acrosomal loss, however statistically significant changes (P < 0.05) in the acrosomal matrix (altered) were detected after 10-min incubation by light microscopy. Around 50% of acrosomes were altered after 20-min incubation in 50 μM LPC (P < 0.001), and 40% of spermatozoa had lost their acrosome after 60-min incubation (P < 0.001). Treatment with 75 and 100 μM LPC led to rapid acrosomal loss from around 50% of spermatozoa within 10 min (P < 0.001), and by 60 min acrosomal loss was 70–80%. LPC, like the diacylglycerol DiC₈ (1,2-di-octanoyl-sn-glycerol), is thus an effective agent to induce loss of the relatively stable wallaby sperm acrosome, and it also induces changes within the acrosomal matrix. Ultrastructure of the LPC-treated spermatozoa revealed that the plasma membrane and the acrosomal membranes were disrupted in a manner similar to that seen after detergent treatment (Triton X-100). There was no evidence of point fusion between the plasma membrane overlying the acrosome and the outer acrosomal membrane. The plasma membrane was the first structure to disappear from the spermatozoa. The acrosomal membranes and matrix showed increasing disruption with time and LPC concentration. Wallaby spermatozoa incubated with LPC at concentrations that induced significant acrosomal loss also underwent a rapid decline in motility that suggested that acrosomal loss may be due to cell damage, rather than a physiological AR. This study concluded that LPC-induced acrosomal loss from tammar wallaby spermatozoa is due to its action as a natural detergent and not as a phosphoinositide pathway intermediate. The study further demonstrates the unusual stability of the marsupial acrosomal membranes. © 1993 Wiley-Liss, Inc.     

An investigation of capacitation and the acrosome reaction in dog spermatozoa using a dual fluorescent staining technique

The processes of capacitation and acrosomal exocytosis of dog spermatozoa in vitro have yet to be fully investigated. Firstly, we investigated the effectiveness of a technique for staining dog spermatozoa with the fluorescent labels Hoechst 33258 and chlortetracycline. A modified fluorescence microscopy staining method was shown to be effective for the assessment of both viability and functional status in this species. Secondly, the presence of the ionophore A23187 in culture medium was shown to promote capacitation and the acrosome reaction of dog spermatozoa. We have therefore established that this dual fluorescent staining method can be used for monitoring these events in the dog, and it may be useful in future studies of optimal in vitro culture conditions.     

C-kit receptor and its possible function in human spermatozoa

The presence and role of the c-kit proto-oncogene protein was investigated in the mature sperm of the human. A polyclonal antibody against the c-kit peptide was used to perform immunohistochemical (IHC) staining, electron microscopy (EM) studies, and Western blot analysis. The acrosomal region of fresh sperm specifically stained with the antibody. No acrosomal staining or staining limited to the equatorial region was noted in the acrosome-reacted (AR) sperm. EM studies demonstrated immunogold label on the plasma membrane (PM) of the acrosome, and confirmed the lack of binding following the acrosome reaction. A 150 kDa band was detected by Western blot analysis. This protein was released from the sperm surface during sperm capacitation and the acrosome reaction. Antibody against the c-kit receptor significantly inhibited the acrosome reaction and increased sperm agglutination, but did not significantly inhibit sperm motility. These results suggest that the c-kit receptor protein is present in mature human sperm and is released during capacitation and/or the acrosome reaction. The assessment of the c-kit receptor may also be a useful assay for sperm function in male infertility.     

Rab3A triggers the acrosome reaction in permeabilized human spermatozoa

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

Rat caltrin protein modulates the acrosomal exocytosis during sperm capacitation

Caltrin is a small and basic protein of the seminal vesicle secretion that inhibits sperm calcium uptake. The influence of rat caltrin on sperm physiological processes related to fertilizing competence was studied by examining its effect on 1) spontaneous acrosomal exocytosis, 2) protein tyrosine phosphorylation, and 3) sperm-egg interaction. Results show that the presence of caltrin during in vitro capacitation both reduced the rate of spontaneous acrosomal exocytosis without altering the pattern of protein tyrosine phosphorylation, and enhanced the sperm ability to bind to the zona pellucida (ZP). The significantly higher proportion of sperm with intact acrosome observed in the presence of caltrin was accompanied by a strong inhibition in the acrosomal hyaluronidase release. Enhancement of sperm-ZP binding was evident by the increase in the percentage of eggs with bound spermatozoa as well as in the number of bound sperm per egg. Similar results were obtained when the assays were performed using spermatozoa preincubated with caltrin and then washed to remove the unbound protein, indicating that the sperm-bound caltrin was the one involved in both acrosomal exocytosis inhibition and sperm-ZP binding enhancement. Caltrin bound to the sperm head was partially released during the acrosomal exocytosis induced by Ca-ionophore A23187. Indirect immunofluorescence and immunoelectron microscopy studies revealed that caltrin molecules distributed on the dorsal sperm surface disappeared after ionophore exposure, whereas those on the ventral region remained in this localization after the treatment. The present data suggest that rat caltrin molecules bound to the sperm head during ejaculation prevent the occurrence of the spontaneous acrosomal exocytosis along the female reproductive tract. Consequently, more competent spermatozoa with intact and functional acrosome would be available in the oviduct to participate in fertilization.     

Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.     

The equatorial subsegment in mammalian spermatozoa is enriched in tyrosine phosphorylated proteins

The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.