Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.     

Distribution of endogenous and exogenous 5′-nucleotidase on bovine spermatozoa

A polyclonal rabbit antibody against 5-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.     

Localisation of low molecular weight sperm antigens during capacitation and acrosome reaction

FITC-labelled sperm-specific antibodies against hamster spermatozoa were utilized as probes in acrosome reaction assays. An indirect immunofluorescence test demonstrated the localisation of two sperm proteins of 19 kDa and 23 kDa on the anterior acrosomal cap region of washed cauda epididymal sperm. These proteins were not detected in reacted acrosome or on immature or immotile sperm. Antisperm agglutinating antibodies specific to these two low molecular weight sperm antigens could be useful probes for evaluating the acrosomal status of mammalian spermatozoa.     

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.     

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.     

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.     

Capacitation and acrosome reaction in buffalo bull spermatozoa assessed by chlortetracycline and Pisum sativum agglutinin fluorescence assay

Studies on buffalo sperm capacitation have been limited because of the non-availability of a direct assay system. We describe two methods for detecting the acrosomal status of buffalo spermatozoa, namely chlortetracycline (CTC) fluorescence assay and Pisum sativum agglutinin (FITC-PSA) stain. We also test them under various treatment regimens and simultaneously standardize and calibrate them with transmission electron microscopy. An initial comparison of three physiological media, such as Krebs-Ringer bicarbonate buffer, Tyrode solution and Brackett & Oliphant medium (having different calcium concentrations and osmolality) used for studying the capacitation of buffalo spermatozoa and assessed by CTC, FITC-PSA, Giemsa stain and TEM, revealed Brackett & Oliphant medium to be marginally better than the other two media. When stained with chlortetracycline, three distinct fluorescent patterns were visible in buffalo spermatozoa under capacitating conditions. These were 'F' with fluorescence in the post acrosomal region characteristic of uncapacitated acrosome-intact cells; 'B' with fluorescence on the anterior portion of the sperm head and a dark band in the post-acrosomal region, characteristic of capacitated, acrosome intact cells and 'AR' with a fluorescent band on the posterior portion of the head, characteristic of acrosome-reacted cells. The FITC-PSA intensely labels the acrosomal region of acrosome intact buffalo sperm. Acrosome reacted sperms had diminished acrosomal labelling by both the probes used. Buffalo spermatozoa was not capacitated when calcium was either omitted from the medium or chelated with EGTA. In the presence of Ca2+ ionophore, A23187, 68% at 4 h and 85% at 8 h completed the acrosome reaction. Time course studies revealed a 4 h incubation period at 1.71 mM Ca2+ concentration to be necessary before transformation of 'F' to 'B' cells could take place. Spontaneous acrosome reaction induced at 6 and 8 h incubation of buffalo spermatozoa in KRB medium resulted in conversion of 'B' cells to 'AR' cells while 'F' cells remained unchanged. A simultaneous evaluation of acrosome intact and acrosome-reacted cells using FITC-PSA, Giemsa and TEM gave results similar to examination by CTC stain. Both the assays are rapid, reproducible, reliable and they detect an increase or decrease in physiological acrosome reactions. They thus can be used to study effects of calcium and prove to be good monitoring systems to identify buffalo sperm capacitation and acrosome reaction in individual buffalo bulls for fertility studies.     

First evidence of prothymosin alpha localization in the acrosome of mammalian male gametes

Prothymosin α (PTMA) is a highly acidic intrinsically unstructured protein. Its expression in male gonads is evolutionary conserved; in rat testis it is specifically localized in the cytoplasm of post‐meiotic germ cells, in association with the developing acrosome system. In the present paper we investigated on PTMA localization inside the head of mammalian spermatozoa (SPZ). We chose a confocal approach to ascertain whether PTMA is expressed in the acrosome or in the perinuclear theca, two regions that are tightly linked and partially overlapped in the mature haploid cells. The obtained results showed that PTMA is specifically localized in the acrosome of rat epididymal SPZ; the same experimental approach evidenced, for the first time, PTMA presence in human ejaculated SPZ. A Western blot analysis on protein extracts from human sperm head fractions confirmed the confocal data and demonstrated that the peptide is specifically associated with the inner acrosomal membrane fraction. Finally, when the acrosome reaction was induced in vitro by progesterone treatment on both rat and human sperm, PTMA signal was retained in the apical region of reacted SPZ. In conclusion, this study confirms the conservation of PTMA distribution in vertebrate male gametes and strongly supports a role for this polypeptide in their physiology. J. Cell. Physiol. 228: 1629–1637, 2013. © 2013 Wiley Periodicals, Inc.     

Use of atomic force microscopy for morphological and morphometric analyses of acrosome intact and acrosome-reacted human sperm

The objective of this study was to use atomic force microscopy (AFM), with submicron resolution, for morphophologic and morphometric analyses of acrosome intact and acrosome-reacted human sperm heads. A mixed population of acrosome intact and reacted sperm was produced by treating capacitated sperm with A23187, which induced the acrosome reaction in approximately 50% of total sperm population. This A23187-treated sperm suspension was then plated onto a coverslip and acrosome reacted sperm were preidentified by their specific staining with rhodamine-conjugated Concanavalin A. The sperm coverslip was then air-dried and scanned by a Nanoscope IIIa atomic force microscope, using the contact mode. Top and side view images processed through the illuminate mode revealed three dimensional sperm head contour, with the highest point situated in the head posterior in both acrosome intact and acrosome reacted sperm. Maximum height, length, and width measured in 50 acrosome intact and 50 acrosome-reacted sperm were the same in both populations. However, head length at half maximum height was significantly decreased in acrosome reacted sperm (2.99 +/- 0.24 microm vs. 3.56 +/- 0.32 microm of acrosome intact sperm), due to the sudden change of the height contour from the maximum peak to the anterior tip of acrosome-reacted sperm. Our results described here can therefore be used to differentiate acrosome intact and reacted sperm from each other. This would allow future studies on subcellular changes, related to the acrosome reaction, at the submicron resolution level under more physiological conditions, since AFM does not require fixing or staining of the samples.     

Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae

The binding of mammalian spermatozoa to the egg's extracellular coat, the zona pellucida, is a complex process which culminates in species-specific penetration of the sperm to the egg plasma membrane. To investigate where on the spermatozoon's surface the zona binding sites are located, whole rabbit zonae were labeled with FITC, heat solubilized and used to observe the surface binding patterns on live spermatozoa. Before the acrosome reaction the zona binding sites are located either over the entire head as well as the middle piece or alternatively in patches along the apical ridge of the head. After the acrosome reaction there is a 29% loss of fluorescence and the zona binding sites are present in the posterior aspect of the acrosomal region, the anterior postacrosomal region and the middle piece. These results demonstrate the presence of zona binding sites after the acrosome reaction which would account for the sperm's ability to remain bound to the zona after the acrosome reaction. Further, we report for the first time that solubilized rabbit zonae pellucidae will induce the acrosome reaction in in vitro capacitated rabbit sperm whereas solubilized pig zonae pellucidae will not. Since rabbit sperm bind pig zonae, the induction and specificity of the physiological acrosome reaction must reside in the affinity of the binding rather than the binding itself.     

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.     

Seminal vesicle secretion of African catfish, its composition, its behaviour in water and saline solutions and its influence on gamete fertilizability

The seminal vesicle secretion (SVS) of the African catfish, Clarias gariepinus, was investigated by analytical and experimental methods. SVS consists mainly of proteins and glycoproteins which are responsible for its viscous and sticky nature. The secretion contains also high activities of acid phosphatase, alkaline phosphatase, and proteases. These catabolic enzymes do not have functions in autolysis or liquefaction of SVS but are considered to eliminate aging spermatozoa from the proximal portions of seminal vesicle and from the spermatic duct. SVS of the African catfish is unstable in the environment relevant for natural spawning. When SVS was mixed with water, seminal plasma or different types of saline solutions its protein coagulated forming fibrous or granular particles of variable size within a few seconds. Pure SVS completely inhibited the motility as the sticky secretion hindered spermatozoa in free swimming. SVS had also a negative effect on sperm fertility, egg fertility, and sperm egg contact, as the fertilization was drastically suppressed in the presence of SVS. Basing on our analytical and experimental results we exclude that SVS has functions in stabilizing the viability of spermatozoa stored in the spermatic ducts or is an energy resource of spermatozoa. It also does not improve or stabilize the fertilization process and has no functions in adhering the eggs to substrates or in covering the eggs for mechanical protection or antibacterial defense. A function of SVS in the male and female communication during the prenuptial spawning behaviour is discussed.     

Effect of Spermine-NONOate on acrosome reaction and associated protein tyrosine phosphorylation in Murrah buffalo (Bubalus bubalis) spermatozoa

The present study was conducted to know the role of Nitric Oxide (NO) on the acrosome reaction (AR) in Murrah buffalo (Bubalus bubalis) spermatozoa. Ejaculated buffalo spermatozoa were washed, suspended in sp-TALP media containing 6 mg BSA/mL and cell concentration was adjusted to 50×10(6) cells/mL. The cells were incubated for 6h in the absence or presence of heparin (10 μg/mL) to induce capacitation. Fully capacitated spermatozoa were incubated in presence of 100 μg/mL Lysophosphatidyl choline (LPC, T1) or 100 μM Spermine-NONOate (T2) or 100 mM L-NAME (T3) or 100 μM Spermine-NONOate+100 mM L-NAME (T4) or 1 mM db-cAMP + 0.1 mM IBMX (T5) or 100μM H-89 (T6) or 100 μM Spermine-NONOate+100 μM H-89 (T7) in combination to induce acrosome reaction. The extent of AR was assessed by dual-staining of spermatozoa with trypan blue/Giemsa stain. AR-associated tyrosine-phosphorylated proteins were detected by SDS-PAGE followed by immunoblotting using monoclonal anti-phosphotyrosine antibody. Significant (P<0.05) number of spermatozoa were acrosome reacted in Spermine-NONOate (T2) treated cells but it was significantly (P<0.05) lower than LPC (T1) induced AR. Addition of Spermine-NONOate + L-NAME (T4) resulted in non significant (P>0.05) decrease in acrosome reaction. On addition of H-89 + Spermine-NONOate (T7) to sperm culture medium, resulted in significant (P<0.05) decrease in the percent acrosome reaction. Conversely, addition of db-cAMP+IBMX (T5, cAMP analogue) resulted in the significantly (P<0.05) higher number of acrosome reacted spermatozoa. Pattern of sperm protein tyrosine phosphorylation was also different in NO induced acrosome reaction compared to that of LPC. The present study concluded that nitric oxide is involved in acrosome reaction of buffalo spermatozoa by causing the tyrosine phosphorylation of proteins mainly p17 and p20 and through activation of cAMP/PKA pathway.     

Secretion patterns and effect of prostate-derived granules on the sperm acrosome reaction of rabbit buck

There is increasing evidence that the particulate fraction of seminal plasma plays an important role in reproduction of several mammalian species. However, the origin and role of these granules in the physiology of rabbit spermatozoa is partially unknown. The aim of this study was to investigate the implication of prostate gland in the production and secretion of granules into the rabbit semen and the role of prostate-derived granules in the sperm acrosome reaction. Light and electron microscopy of the prostate gland showed that the anterior and middle tracts of the prostate (namely the proprostate and prostate, respectively) are chiefly implicated in the secretion of granules of different size: smaller granules (SG; 0.5 μm) and large granules (LG; 4 μm). Two major patterns of secretion were identified, based on electron microscope views: storage granules (large granules) seem to empty inner smaller granules directly into the duct by exocytosis, or the storage vesicle itself is released in toto into the ducts (diacytosis). In vitro experiments using granules from vasectomized rabbits, to exclude testicular origin of granules, showed that granules reduce the acrosome reaction of Percoll-selected spermatozoa, independently of the size. Interestingly, spermatozoa incubated with heat-treated granules showed a higher sperm acrosome reaction rate, suggesting a potential role of granule-derived proteins in this process. Inhibition of the acrosome reaction is a crucial event in rabbit reproduction; ejaculated spermatozoa have to wait for a long time (8-16 h) for egg availability in the female tract after mating. Taking together, our results demonstrate that prostate granules secreted either by exocytosis or diacytosis can preserve spermatozoa fertilizing ability, by preventing sperm acrosome reaction. The type of granule-derived proteins or other macromolecules implicated in this process should be further investigated.     

Effect of heparin on in vitro capacitation of boar sperm

Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 microM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 microg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.     

F-actin in acrosome-reacted boar spermatozoa.

Biochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction. F-actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe: incubation with a fluorescein-phalloidin complex and an anti-fluorescein antibody, followed by labeling with protein A-gold complex. Gold particles, indicating the presence of F-actin, were localized on the sperm surface of the acrosome-reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma membrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP-40 extraction, the SDS-PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spermatozoa is polymeric.     

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

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

Gamete membrane fusion in hamster spermatozoa with reacted equatorial segment

Mammalian spermatozoa must undergo many changes to be able to fertilize the oocyte. One of these changes, the acrosome reaction, has been established as a requisite for gamete membrane fusion to occur; it consists of the fusion and vesiculation of the sperm plasma membrane with the outer acrosomal membrane of the principal segment of the acrosome. Reaction of the equatorial segment has occasionally been observed. The objective of the present work was to determine whether the presence of the sperm plasma membrane over the equatorial segment is necessary for gamete membrane fusion to occur. Golden hamster spermatozoa were capacitated in vitro in TAPL 10K, and the maximum possible percentage of acrosome reaction was determined at 82.79% + 1.69% SD (P = 0.27; r = 0.21). Ultrastructural studies showed that 93.6% of the reacted spermatozoa in this population had their principal and equatorial segments reacted. The fertilizing ability of these spermatozoa was assayed using zona-free hamster oocytes. The percentage of fertilized ova obtained was 98.8% (308/312). Ultrastructural studies snowed the presence of spermatozoa with reacted equatorial segment inside the cytoplasm of immature oocytes. The evidence presented in this work demonstrates that the plasma membrane of spermatozoa with reacted equatorial segment retains its ability to fuse with the oocyte.     

Role of intra-acrosomal antigenic molecules acrin 1 (MN7) and acrin 2 (MC41) in penetration of the zona pellucida in fertilization in mice

In this study the role of two intra-acrosomal molecules, acrin 1 (MN7) and acrin 2 (MC41), during in vitro fertilization (IVF) was examined. The pertinent monoclonal antibodies mMN7 and mMC41 specifically recognize a 90 kDa protein (acrin 1) localized to the entire acrosome and a 200 kDa protein (acrin 2) localized to the cortex region of the anterior acrosome, respectively. Experiments were designed to assess the effects of mMN7 and mMC41 on fertilization in mice using TYH medium containing mMN7 or mMC41 at 0.0, 0.025, 0.05 and 0.1 mg ml-1. Under these conditions, capacitated spermatozoa inseminated the cumulus-invested oocytes. Acrosome-reacted spermatozoa inseminated the zona pellucida-free oocytes. The antibodies had no effect on sperm motility and primary binding to the zona pellucida, but significantly inhibited the rate of fertilization of zona pellucida-intact oocytes in a dose-dependent manner. A significantly small number of spermatozoa remained attached to the zona pellucida at 5 h after insemination in the presence of mMC41. mMC41 and mMN7 antibodies did not affect the fertilization rate of zona pellucida-free oocytes. Confocal laser scanning microscopy with indirect immunofluorescence traced the effect of the monoclonal antibodies on the zona pellucida-induced acrosome reaction, and revealed that mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the zona pellucida after the acrosome reaction but before penetration of the zona pellucida. Thus, the intra-acrosomal antigenic molecules acrin 1 and acrin 2 are essential for distinct events before sperm penetration of the zona pellucida in mice.     

Effects of angiotensin II on the acrosome reaction in equine spermatozoa

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