Thapsigargin stimulates acrosomal exocytosis in hamster spermatozoa

Thapsigargin (TG), a plant-derived sesquiterpene lactone, inhibits several isoforms of both the sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPases. Thus, intracellular Ca²⁺ stores found in the endoplasmic reticulum can be released by this compound. The mammalian sperm acrosome reaction (AR) depends on influx of extracellular Ca²⁺. However, few reports have presented evidence for the involvement of putative Ca²⁺ stores and intracellular Ca²⁺ mobilization in the AR. Thus, we designed experiments to evaluate the effect of TG on the hamster sperm AR. Thapsigargin stimulated—in a dose-dependent manner—the AR of spermatozoa previously capacitated for at least 3 hr, not affecting sperm motility. A maximal stimulatory effect was apparent 3 min after addition of TG to spermatozoa previously capacitated for 4 hr and was dependent on external Ca²⁺ since ethyleneglycol-bis-(b-amino-ethyl ether) N,N′-tetra-acetic acid added 1 min before TG completely inhibited AR stimulation. The Ca²⁺ channel blockers diltiazem and nifedipine also abolished the TG-stimulatory effect when added to capacitated spermatozoa 10 min before the inhibitor. In addition, the trypsin inhibitors p-nitrophenyl-p′-guanidine-benzoate hydrochloride and benzamidine added to the sperm suspensions 10 min before TG inhibited by 70–80% the TG-induced AR. These results indicate that putative Ca²⁺ stores release may be involved in stimulation of extracellular Ca²⁺ influx required for the occurrence of the AR. In addition, a sperm trypsin-like protease may be part of the mechanism by which TG induces the hamster sperm AR. Mol. Reprod. Dev. 51:84–91, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of an acrosomal matrix protein in hamster and bovine spermatids and spermatozoa.

Experiments have been carried out characterizing an Mr 22,000 protein present in the acrosomes of hamster and bull spermatozoa. The Mr 22,000 protein is resistant to solubilization in detergent solutions containing high or low salt and has a pI of -5.2. With various lectins, the protein from hamster sperm was shown to be sparingly glycosylated with N-acetylglucosamine, mannose, and galactose while that from the bull demonstrated a slight reactivity for galactose. Using a specific monoclonal antibody (MAB 4/18), the Mr 22,000 polypeptide has been localized exclusively to the acrosomes of mature testicular and epididymal hamster and bovine sperm. Acrosomal components of differentiating bovine and hamster spermatids in tissue sections did not react with the monoclonal antibody, although the protein was present in immunoblots of round spermatids. In bovine sperm, MAB 4/18-staining at the ultrastructural level with immunogold-labeled second antibody was present as a reticulum throughout the acrosomal cap and as punctate aggregates in the equatorial segment. In hamster sperm, MAB 4/18-reactivity was present along the periphery of the acrosome in conjunction with matrix components (M1 and M2), as well as along the inner acrosomal membrane. These observations indicate that the acrosomes of bovine and hamster sperm possess an immunologically related Mr 22,000 protein and suggest that differences in MAB 4/18-staining of spermatids and spermatozoa is a result of epitope modification and/or a change in accessibility of the epitope to the antibody probe during the course of spermiogenesis. Based on its localization and solubility properties, we suggest that the Mr 22,000 protein, in conjunction with other polypeptides, forms a structural framework to maintain acrosomal shape and/or compartmentalize acrosomal contents.     

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.     

Golden hamster perivitelline spermatozoa do not show proacrosin/acrosin at the inner acrosomal membrane.

It has been suggested that acrosin may function in penetration of the zona pellucida and of the highly structured extracellular matrix of the perivitelline space. In this study we investigated whether golden hamster perivitelline spermatozoa contain proacrosin/acrosin, as evidenced by the silver enhanced immunogold technique using the monoclonal antibody antiacrosin C2E5. None of the 197 spermatozoa recovered from the perivitelline space showed proacrosin/acrosin associated with the acrosomal region, suggesting that acrosin would not play a role in the penetration of the perivitelline extracellular matrix.     

Lipids of plasma membrane and outer acrosomal membrane from bovine spermatozoa

Plasma membrane (PM), primarily from the anterior sperm head, and outer acrosomal membrane (OAM), were isolated from ejaculated bovine spermatozoa, and the major lipid classes were characterized. Whole sperm (WS) lipids were analyzed for comparison. PM was removed by nitrogen cavitation and purified by sucrose density-gradient centrifugation. The OAM was removed by centrifugation through hyperosmotic sucrose and recovered by sucrose density-gradient centrifugation. The PM contained primarily spherical vesicles from the region overlying the OAM and was enriched 9- and 13-fold in 5'-nucleotidase and alkaline phosphatase activity, respectively, compared to the original cavitate. The OAM was recovered as caplike structures with associated ground substance. Protein, phospholipid, and cholesterol (PR, PL, and CH as micrograms/5 x 10(9) sperm) were 300, 467, and 93 for PM and 276, 111, and 25 for OAM, respectively. Corresponding values for WS (mg/5 x 10(9) sperm) were 31.4, 6.63, and 0.72. The PR/PL (w/w) and CH/PL (mol/mol) ratios were 0.66 and 0.38 for PM; 2.48 and 0.26 for OAM; and 4.39 and 0.22 for WS. Cholesterol was the only free sterol detected by gas/liquid chromatography in WS, PM, and OAM, with traces of CH sulfate present in all three preparations. Glycolipid tentatively identified as sulfogalactolipid was detected by thin-layer chromatography (TLC) in PM but not OAM. Phospholipid composition of WS and membranes was determined by TLC. Cardiolipin (3% of total PL) was present in WS only. Choline, ethanolamine, and inositol phosphoglycerides (CP, EP, PI, PIP, PIPP); sphingomyelin (SP); phosphatidylserine (PS); and lysophosphatidylcholine (LPC) were present in WS, PM, and OAM. Approximately 50% of total PL was CP in all preparations; SP was 13% of PL in PM and 17% in OAM (p less than 0.05); EP was 7% of PL in PM and 10% in OAM (p less than 0.05). The differences in composition between PM and OAM is discussed with respect to capacitation and ability of sperm to undergo the acrosome reaction.     

Molitity and acrosomal changes in ionophore-treated bovine spermatozoa and their relationship with in vitro penetration of zone-free hamster oocytes

Frozen-thawed spermatozoa from Friesian bulls held at stud in Ireland were used to assess the effect of ionophore on motility, acrosome reaction and heterologous in vitro fertilization. Bovine spermatozoa penetrated zone-free hamster oocytes following treatment with calcium ionophore in the absence of bovine serum albumin (BSA) and in the presence of 10 mM caffeine. Sperm velocity was stimulated in concentrations of caffeine <2.5 mM following dilution with medium containing BSA. Sperm attachment to the plasmalemma showed no association with penetration rates of zona-free hamster oocytes. Penetrated oocytes in regimens with >0.1 mM ionophore did not progress through Meiosis II. Increasing concentrations of ionophore induced the acrosome reaction more rapidly, although this was associated with reduced motility. Hyperactive motility was observed in calcium ionophore-treated spermatozoa which were capable of penetrating zona-free hamster oocytes. Sperm velocity remained unchanged. whereas the track:vector ratio, a measurement of curvilinear movement, was reduced. This work may have important implications for the assessment of bovine fertility and cytogenetic analysis of bovine sperm.     

Assessment of acrosomal integrity of vervet monkey spermatozoa after cryopreservation

Cryopreservation of Vervet monkey semen caused a highly significant reduction in the percentage of normal acrosomes and a highly significant increase in the percentage of mildly damaged, severely damaged, and lost acrosomes. This was demonstrated by staining ten post-thaw semen samples, which enabled a visualization of the acrosomal damage. The incidence of post-thaw intact acrosomes in this cryopreservation method was found to be similar to that reported for man and cynomolgus monkey semen.     

Regulation of acrosomal matrix dispersion in digitonin-permeabilized guinea pig spermatozoa

Digitonin-permeabilized guinea pig spermatozoa undergo acrosomal matrix dispersion in response to 2.0 mM CaCl2. In this report, the effects of pH and metal ions on matrix dispersion in permeabilized spermatozoa are examined. Calcium-induced dispersion of the acrosomal matrix was dependent on the calcium concentration; the response was not observed at concentrations of CaCl2 less than 50 microM. Magnesium could not substitute for calcium and, in fact, had a retarding effect on the calcium-induced response. Matrix dispersion was also found to be pH-dependent. The induction of matrix dispersion was inhibited at pH 5.6 and pH 9.5 relative to the responses observed at pH 6.3 and pH 7.8. Nigericin induced acrosomal matrix dispersion in the absence of added calcium, indicating a possible role of Na+/H+ exchange across the outer acrosomal membrane in initiating the matrix modification. Sodium was required for the action of nigericin; the ionophore was ineffective in medium in which choline chloride or sucrose was substituted for NaCl. In contrast, the calcium-induced dispersion of the acrosomal matrix occurred in the absence of sodium. Furthermore, low concentrations of calcium inhibited an adenosine triphosphatase activity associated with isolated acrosomal apical segments. These data are consistent with the hypothesis that calcium induces alkalinization of the acrosome, leading to matrix dispersion. However, permeabilized spermatozoa incubated at either pH 9.5 or in the presence of 50 mM NH4Cl at pH 7.5 failed to undergo spontaneous matrix dispersion, suggesting that elevated intraacrosomal pH alone was not sufficient to initiate the reaction. The proposed alternative hypothesis is that calcium initiates matrix dispersion by a mechanism in which elevated intraacrosomal pH may be a secondary response.     

Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at ?196°C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.     

Cholesterol-loaded cyclodextrin inhibits premature acrosomal reactions in liquid-stored rabbit spermatozoa

The effect of pretreatment of rabbit sperm cells with different concentrations of cholesterol-loaded cyclodextrin (CLC) on the occurrence of premature acrosome reactions during 72 h of liquid storage was investigated in three successive experiments. The aim of the first experiment was to establish a liquid storage model to facilitate premature acrosome reactions in rabbit sperm cells and, therefore, examined the relative effects of different dilution rates (1:5, 1:25 or 1:50) and storage temperatures (4°C or 35°C) on the occurrence of premature acrosome reactions. Increasing both dilution rate (from 1:5 to 1:25; P<0.05) and storage temperature (from 4°C to 35°C; P<0.0001) significantly enhanced the percentage of sperm cells that underwent premature acrosome reactions during storage. Therefore, a constant dilution rate of 1:25 and storage temperature of 35°C was employed for the rest of the study. The second experiment examined the effect of different CLC concentrations (0, 0.5, 1.0 and 3.0mg per 120×10(6) spermatozoa) on the occurrence of premature acrosome reactions in sperm cells. CLC supplementation of the extender inhibited (P<0.001) premature acrosome reactions in sperm cells in a dose-dependent manner during 72 h of storage. In the third experiment, the ability of CLC-pretreated sperm cells to undergo acrosome reactions induced by lysophosphatidylcholine (LPC) was evaluated following 72 h of storage. A considerable proportion of sperm cells pretreated with CLC (between 68.7 and 91.8%) underwent the acrosome reaction in response to LPC following 72 h of liquid storage. However, the ability of the sperm cells to undergo the acrosome reaction varied with regards to the dose levels of CLC pretreatment (P<0.001). In conclusion, CLC supplementation prevents premature acrosome reactions in liquid-stored rabbit spermatozoa.     

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.     

First attempt to characterize 3H-fucose acrosomal label in spermatozoa

Summary Acrosomes of rabbit spermatozoa were labelled by tritiated fucose introduced into the cells during spermatogenesis. The labelling was analysed simultaneously by autoradiography and biochemically. In compact intact acrosomes the labelling was confined strictly to the acrosomal region of the sperm head. In swollen and detached acrosomes the autoradiographic grains were associated mostly with the acrosomal cap. Only in some cells a small proportion of radioactivity was detected to be associated with denuded sperm heads. In acrosomal extracts a considerable share of radioactivity coincided with gel filtration fractions showing esterase activity (BAEE-Nalpha-benzoyl-l-arginine ethyl ester splitting), akin to that exhibited by acrosin.