Ionophore A23187-induced Acrosome Reaction Research Articles

Characterization of acrosin and acrosin binding protein as novel CRISP2 interacting proteins in boar spermatozoa.

Previously, we reported that cysteine-rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine-rich secretory protein 2protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning. This study aimed to identify cysteine-rich secretory protein 2 interacting partners.These binding partner interactions were investigated under different conditions, namely, non-capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187-induced acrosome reaction. The incubated pig sperm samples were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands were excised and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC-MS for protein identification. The most prominent cystein-rich secretory protein 2 interacting proteins that appeared in both independent LC-MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with cystein-rich secretory protein 2in pig sperm. Blue native gel electrophoresis and native immunoblots revealed that cystein-rich secretory protein 2was present within a ∼150kDa protein complex under all three conditions. Interrogation of cystein-rich secretory-protein 2-immunoreactive bands from blue native gels as well as cystein-rich secretory protein 2immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein-rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co-immunoprecipitation and immunoblotting indicated that cystein-rich secretory protein 2 interacted with pro-acrosin (∼53kDa) and Aacrosin binding proteinunder all three conditions and additionally to acrosin (∼35kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein-rich secretory protein 2was assessed via in situ proximity ligation assays. The colocalization signal of cystein-rich secretory protein 2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of cystein-rich secretory protein 2and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post-acrosomal sheath region upon capacitation. These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.

The fate of porcine sperm CRISP2 from the perinuclear theca before and after in vitro fertilization†.

In a previous study, we reported that porcine sperm cysteine-rich secretory protein 2 (CRISP2) is localized in the post-acrosomal sheath-perinuclear theca (PT) as reduction-sensitive oligomers. In the current study, the decondensation and removal of CRISP2 was investigated during in vitro sperm capacitation, after both the induction of the acrosome reaction and in vitro fertilization. Confocal immunofluorescent imaging revealed that additional CRISP2 fluorescence appeared on the apical ridge and on the equatorial segment (EqS) of the sperm head following capacitation, likely due to cholesterol removal. After an ionophore A23187-induced acrosome reaction, CRISP2 immunofluorescence disappeared from the apical ridge and the EqS area partly not only owing to the removal of the acrosomal shroud vesicles, but to its presence in a subdomain of EqS. The fate of sperm head CRISP2 was further examined post-fertilization. In vitro matured porcine oocytes were co-incubated with boar sperm cells for 6-8h and the zygotes were processed for CRISP2 immunofluorescent staining. Notably, decondensation of CRISP2, and thus of the sperm PT, occurred while the sperm nucleus was still fully condensed. CRISP2 was no longer detectable in fertilized oocytes in which sperm nuclear decondensation and paternal pronucleus formation were apparent. This rapid dispersal of CRISP2 in the PT is likely regulated by redox reactions for which its cysteine-rich domain is sensitive. Reduction of disulfide bridges within CRISP2 oligomers may be instrumental for PT dispersal and elimination.

The Melatonin Attenuates Alloxan Induced Post-Diabetic Testicular Damage and Oxidative Effects in Rats

The aim of this study was to examine the protective effect of melatonin (MLT) on the alloxan-induced post-diabetic testicular damage in rats. Forty-eight Sprague Dawley male rats were divided into four groups (n=12). Group C received 0.9% saline for 45 days, as control. Group DM (Diabetes Mellitus) was injected by 120 mg/kg single dose alloxan intraperitoneally (IP). Group MLT was treated by 10 mg/ kg/d MLT IP for 45 d. Group DM+MLT was treated by 10 mg/kg/d MLT IP for 45 days following alloxan injection. Six animals on days 15 and the remaining 6 on days 45 were sacrificed in each group after the initiation of the study. Testis tissues and blood samples were collected to investigate blood glucose, oxidant and antioxidant statues, sperm parameters, and histopathological and 8-hydroxy-2′-deoxyguanosine (8-OHdG)-immunopositive cell examinations. MLT treatment of diabetic rats prevented the increases in malondialdehyde (MDA) levels and 8-OHdG concentrations, and the decreases in the GSH and SOD levels observed in DM groups. Alloxan administration depressed the sperm concentration, the number of progressively motile spermatozoa, premature acrosome reaction (AR) and calcium ionophore A23187-induced AR rates, without any change in dead and/or abnormal sperm rates in DM group. Histopathological and immunohistochemical observations also revealed that MLT-treated diabetic rats had an enhanced histological architecture and DNA damage in testis. These findings suggest that MLT treatment prevents the testicular damage by declining the oxidative stress, but it did not recover the depressed sperm parameters in diabetic rats.

Desalted and lyophilized bovine seminal plasma delays induction of the acrosome reaction in frozen-thawed bovine spermatozoa in response to calcium ionophore

Cryopreservation is partially damaging and induces capacitation-like changes in spermatozoa. Seminal plasma (SP) contains a variety of biochemical components, such as protein and lipids, which are specific for the regulation of sperm cell function including those effective for decapacitation of spermatozoa. Therefore, this study tested the hypothesis that desalted and lyophilized SP could prevent premature capacitation (cryocapacitation) of Japanese Black bull spermatozoa. Seminal plasma was desalted by using Sephadex G-25 desalting column and lyophilized before added to semen extender at final concentrations 0, 2.5, 12.5, and 25 mg/mL. Frozen-thawed sperm progressive motility, acrosomal integrity, abnormal morphology, and the calcium ionophore A23187–induced acrosome reaction were assessed. Protein and lipid compositions in SP were analyzed by SDS-PAGE and thin-layer chromatography, respectively. The results revealed that progressive motility, intact acrosome, and abnormal morphology were not substantially modified by addition of SP. Stimulation of spermatozoa with calcium ionophore A23187 resulted in a time-dependent induction of the acrosome reaction, which was delayed by the desalted and lyophilized SP. There was no difference in the protein profile of SP before and after gel filtration. In total, 19 protein bands with molecular masses ranging from 5.2 to 185.8 kDa were detected and those of 185.8, 80, 34, 20.8, 18.8, 17.5, and 10 kDa were considered as novel proteins. Neutral lipids and phospholipids before and after gel filtration were the same, and the detected neutral lipid spots were monoacylglycerol, cholesterol, 1,2- and 1,3-disaturated diacylglycerol, 1,2- and 1,3-saturated, unsaturated diacylglycerol, whereas the detected phospholipid spots were sphingomyelin, phosphatidylcholine, phosphatidylserine, and three species of phosphatidylinositol, phosphatidylethanolamine, cerebroside, and polyglycerol phosphatide. The results suggest that premature capacitation during freeze-thaw processes could be reduced by adding desalted and lyophilized SP.

AB219. The use of anti-VDAC2 antibody for the combined assessment of human sperm acrosome integrity and ionophore A23187-induced acrosome reaction

Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca transmembrane transport.

The Use of Anti-VDAC2 Antibody for the Combined Assessment of Human Sperm Acrosome Integrity and Ionophore A23187-Induced Acrosome Reaction

Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca2+ increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca2+ transmembrane transport.

The effect of anti-eppin antibodies on ionophore A23187-induced calcium influx and acrosome reaction of human spermatozoa

Before a spermatozoon can fertilize an oocyte it must undergo a cascade of biochemical and physiological changes that facilitate its binding and penetration into the oocyte. Epididymal protease inhibitor (eppin) has been found to play a critical role in male fertility through an immunological approach. In this study, we used an anti-eppin antibody to clarify the effect of eppin on human sperm functions during fertilization. Immunofluorescence studies were performed on ejaculated human spermatozoa in uncapacitated, capacitated and ionophore-treated states. Human spermatozoa were incubated in the presence or absence of anti-eppin antibody under capacitating conditions and with A23187. The effects of the antibody were evaluated on sperm motility, protein phosphotyrosine content and free intracellular calcium. Immunofluorescence results demonstrated that eppin is located on the acrosome and tail. After the acrosome reaction eppin is found on the equatorial segment and tail. We found that blocking eppin with antibodies significantly inhibited the human sperm acrosome reaction induced by A23187 in a dose-dependent manner. Finally, fluo-3 analysis demonstrated that the A23187-induced elevation of sperm intracellular calcium concentration was markedly reduced after incubation with anti-eppin antibody. However, the tyrosine phosphorylation of sperm proteins did not change. These results demonstrate that eppin can modulate intracellular calcium concentrations and subsequently affect the calcium ionophore A23187-induced acrosome reaction.

Synthesis, Processing, and Subcellular Localization of Mouse ADAM3 during Spermatogenesis and Epididymal Sperm Transport

To elucidate synthesis, processing, and subcellular localization of mouse ADAM3 (cyritestin) during spermatogenesis and epididymal sperm transport, we carried out immunoblotting and immunohistochemical analysis of testicular germ cells, and epididymal and vas deferens sperm, using affinity-purified anti-ADAM3 antibody. ADAM3 was initially synthesized as a 110-kDa precursor in round spermatids, and the precursor was then processed into a 42-kDa mature protein during the sperm transport into and/or once in the epididymis. The mature ADAM3 was localized on the anterior part of capacitated sperm heads and was rapidly removed from the head region during the calcium ionophore A23187-induced acrosome reaction. These results demonstrate that the mature form of ADAM3 is involved in the binding of sperm to the egg zona pellucida, not in the membrane fusion between sperm and egg.

An anti-actin monoclonal antibody inhibits the zona pellucida-induced acrosome reaction and hyperactivated motility of human sperm.

We report an inhibitory effect of an anti-actin monoclonal antibody (mAb) on the human zona pellucida (ZP)-induced acrosome reaction (AR). Motile sperm were incubated with native human ZP for 2 h in medium containing either the anti-actin mAb, an irrelevant control mAb or cytochalasins B or D (40 micromol/l). Sperm bound to the ZP were recovered and the AR was determined by fluorescein-labelled Pisum Sativum agglutinin. Anti-mouse immunoglobulin G (mIgG) Dynabeads, immunofluorescence and immunogold were used to detect the location of the anti-actin mAb in sperm. The anti-actin mAb significantly inhibited the ZP-induced AR (equivalent to cytochalasins), the ionophore A23187-induced AR and hyperactivation of sperm in medium. After incubation with anti-actin mAb, anti-mIgG beads bound to the head of >50% of sperm recovered after binding to the ZP and 10% of sperm remaining in the medium. The proportion of sperm that bound anti-mIgG beads after recovery from binding to the ZP in the presence of the anti-actin mAb was significantly correlated with the ZP-induced AR in the absence of the antibody. Immunofluorescence and immunogold demonstrated entry of the anti-actin mAb into sperm. This study suggests that the sperm plasma membrane becomes permeable to the anti-actin mAb during capacitation and initiation of the AR.

Effects of Genistein Isoflavone (4',5',7-Trihydroxyisoflavone) and Dexamethasone on Functional Characteristics of Spermatozoa.

Caudal epididymal spermatozoa were used to study the influence of genistein isoflavone and dexamethasone (dxm) on the functional characteristics of spermatozoa. The effects of genistein alone and in combination with dxm on sperm motility, sperm morphology, spontaneous acrosome reaction (AcR), and ionophore A23187-induced AcR were investigated. The FITC-PSA/Hoechst 33258 staining procedure was used to assess sperm cell viability and AcR status and thus to differentiate between true AcR and acrosome degeneration. The overall results indicated that (1) lower doses of genistein alone, or in combination with dxm, did not significantly influence sperm motility or sperm morphology; (2) ionophore A23187 induced AcR in rat spermatozoa; (3) there appeared to be no direct correlation between sperm motility and AcR, (4) higher doses of genistein, alone or in combination with dxm, significantly interfered with percentage sperm motility and caused significant detachment of sperm heads but did not cause morphological defects; and (5) higher doses of genistein caused significant decrease in sperm acrosome reactivity with long duration of exposure. In view of the fact that sperm capacitation and AcR are physiological prerequisites for successful fertilization of oocytes, the findings suggest that chronic exposure of spermatozoa to high doses of genistein could be associated with infertility problems through suppression/inhibition of AcR and sperm motility. Dexamethasone did not appear to influence the effect of genistein on the functionality of postspermatogenic spermatozoa.

Difference of acrosomal serine protease system between mouse and other rodent sperm

A fraction of acrosomal proteins dispersed during calcium ionophore A23187-induced acrosome reaction was prepared from cauda epididymal sperm of wild-type and acrosin-deficient mice, rat, and hamster. The acrosome-reacted sperm were further extracted by Nonidet P-40 to obtain the detergent-soluble protein fraction. Activities of serine proteases in the two protein fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of gelatin. A mixture of 42- and 41-kDa gelatin-hydrolyzing proteases was found in both fractions of the wild-type mouse sperm, whereas the acrosin-deficient mouse sperm contained the active 42-kDa protease and apparently lacked the activity of the 41-kDa protease. However, exogenous bovine pancreatic trypsin compensated for the absence of acrosin in the protein fractions of the mutant mouse sperm; the gelatin-hydrolyzing activity of the 41-kDa protease appeared when the sperm proteins of the mutant mice were treated with pancreatic trypsin. Two-dimensional polyacrylamide gel electrophoresis revealed that the 42- and 41-kDa proteases were distinguished from acrosin by the isoelectric point and immunoreactivity with affinity-purified antibody against an oligopeptide corresponding to the N-terminal amino acid sequence of mouse proacrosin. Moreover, the gelatin-hydrolyzing proteins corresponding to these two proteases were not detected in rat and hamster sperm, in spite of the treatment of the sperm extracts with pancreatic trypsin, and the total amount of gelatin-hydrolyzing activities in mouse was much smaller than those in rat and hamster. These results may reflect the difference of the serine protease system for the sperm penetration through the egg zona pellucida between mouse and other rodent animals, possibly explaining why the acrosin-deficient mouse sperm are capable of penetrating the zona pellucida. Dev. Genet. 25:115–122, 1999. © 1999 Wiley-Liss, Inc.

Difference of acrosomal serine protease system between mouse and other rodent sperm

A fraction of acrosomal proteins dispersed during calcium ionophore A23187-induced acrosome reaction was prepared from cauda epididymal sperm of wild-type and acrosin-deficient mice, rat, and hamster. The acrosome-reacted sperm were further extracted by Nonidet P-40 to obtain the detergent-soluble protein fraction. Activities of serine proteases in the two protein fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of gelatin. A mixture of 42- and 41-kDa gelatin-hydrolyzing proteases was found in both fractions of the wild-type mouse sperm, whereas the acrosin-deficient mouse sperm contained the active 42-kDa protease and apparently lacked the activity of the 41-kDa protease. However, exogenous bovine pancreatic trypsin compensated for the absence of acrosin in the protein fractions of the mutant mouse sperm; the gelatin-hydrolyzing activity of the 41-kDa protease appeared when the sperm proteins of the mutant mice were treated with pancreatic trypsin. Two-dimensional polyacrylamide gel electrophoresis revealed that the 42- and 41-kDa proteases were distinguished from acrosin by the isoelectric point and immunoreactivity with affinity-purified antibody against an oligopeptide corresponding to the N-terminal amino acid sequence of mouse proacrosin. Moreover, the gelatin-hydrolyzing proteins corresponding to these two proteases were not detected in rat and hamster sperm, in spite of the treatment of the sperm extracts with pancreatic trypsin, and the total amount of gelatin-hydrolyzing activities in mouse was much smaller than those in rat and hamster. These results may reflect the difference of the serine protease system for the sperm penetration through the egg zona pellucida between mouse and other rodent animals, possibly explaining why the acrosin-deficient mouse sperm are capable of penetrating the zona pellucida.

Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen.

The aim of this study was to determine the relationship between calcium ionophore A23187-induced acrosome reaction (AR) and sperm fertilizing ability. Semen samples remaining after preparation for standard IVF were studied in 109 patients who had sperm concentrations > or =20 x 10(6)/ml. Ionophore-induced AR was performed on motile spermatozoa selected by centrifugation on a Percoll gradient. Semen analysis was performed using standard methods. Patients with higher (>50%, n = 76) fertilization rates had significantly higher ionophore-induced AR than patients with lower (<50%, n = 33) fertilization rates (49 +/- 14 versus 38 +/- 21%, P < 0.05). When the data from all patients were analysed by logistic regression, only the percentage sperm motility in insemination medium and ionophore-induced AR were significantly related to fertilization rates. Similar results were also obtained when the data from a subgroup of patients with poor (<15% normal) sperm morphology were analysed. However, when patients with normal sperm morphology > or =15% were analysed separately, only sperm count and the percentage of spermatozoa with progressive motility in semen were significantly related to fertilization rates. In conclusion, ionophore-induced AR was significantly related to fertilization rates in vitro mainly in patients with teratozoospermic semen. Tests for ionophore-induced AR may provide additional information about sperm fertilizing ability but may not indicate specific defects of the physiological AR.

Inhibition of domestic cat spermatozoa acrosome reaction and zona pellucida penetration by tyrosine kinase inhibitors.

Spermatozoa from teratospermic domestic cats (> 60% morphologically abnormal spermatozoa per ejaculate) consistently exhibit lower levels of oocyte penetration in vitro than their normospermic (< 40% abnormal spermatozoa per ejaculate) counterparts. This could be caused by structural abnormalities or intracellular defects resulting in disruption of normal cellular functions. Spermatozoa from teratospermic cats also are compromised in the ability to capacitate and undergo the acrosome reaction (AR) in vitro. Further, we recently identified two tyrosine phosphorylated proteins (95- and 160-kDa) localized over the acrosome region in domestic cat spermatozoa. Phosphorylation of these proteins is reduced in teratospermic compared with normospermic ejaculates. To begin to understand the relationship between tyrosine phosphorylation and sperm function, we examined the effects of two protein tyrosine kinase inhibitors (tyrphostin RG-50864 and genistein) on (1) sperm motility; (2) protein tyrosine phosphorylation; (3) the ionophore A23187-induced AR; (4) the spontaneous and zona pellucida (ZP)-induced AR, and (5) the ability of spermatozoa from normospermic cats to penetrate conspecific ZP-intact oocytes. Over a wide range of concentrations, neither inhibitor affected sperm percentage motility during incubation (P > 0.05). Preincubation with either inhibitor reduced tyrosine phosphorylation of both (95- and 160-kDa) sperm proteins. Although both inhibitors blocked the ZP-induced AR, neither influenced the spontaneous AR nor the A23187-induced AR, suggesting that tyrosine phosphorylation may be involved in physiologic AR. No differences (P > 0.05) were observed in the ability of control or inhibitor-treated spermatozoa to bind to or penetrate the outer ZP layer. However, percentages of oocytes with treated spermatozoa in the inner ZP (tyrphostin, 8.7%; genistein, 20.4%) and perivitelline space (tyrphostin, 0%; genistein, 2.3%) were less (P < 0.001) than untreated controls (inner ZP, 62.7%; perivitelline space, 10.2%). These results (1) demonstrate that ZP-induced acrosomal exocytosis in domestic cat spermatozoa is regulated via a tyrosine kinase-dependent pathway and (2) suggest that defects in these signaling pathways may represent one of the causes for compromised sperm function in teratospermic males.

Relationship between the zona pellucida (ZP) and ionophore A23187-induced acrosome reaction and the ability of sperm to penetrate the ZP in men with normal sperm-ZP binding

To determine whether the proportions of sperm undergoing the acrosome reaction (AR) on the zona pellucida (ZP) or on exposure to the calcium ionophore A23187 correlate with sperm-ZP penetration in men with normal sperm-ZP binding. Sperm samples from 14 fertile, 50 normozoospermic, and 21 teratozoospermic men with normal sperm-ZP binding were used to study ZP and ionophore-induced AR and sperm-ZP penetration. University research unit associated with a clinical infertility and IVF program. The proportion of sperm undergoing the AR induced by ZP and A23187, the number of sperm bound to and penetrating into the ZP, the proportion of ZP penetrated by sperm, and semen characteristics were analyzed. There were highly significant correlations between the percentage AR of sperm bound to the ZP and both the proportion of ZP penetrated and average number of sperm penetrating per ZP (Spearman r = 0.697 and 0.703). In contrast, A23187-induced AR did not correlate with either ZP-induced AR or sperm-ZP penetration. Although sperm concentration in semen correlated with both ZP-induced AR and sperm-ZP penetration by Spearman test, it was not significant in multivariate regression analyses. Ionophore A23187-induced AR does not correlate with either ZP-induced AR or sperm-ZP penetration. The ZP-induced AR is correlated highly with sperm-ZP penetration. Testing ZP-induced AR may be useful for assessing infertile men with normal sperm-ZP binding. In particular, it will identify failure of ZP penetration because of specific defects of the AR on the ZP.

In vitro fertilisation of Chinese hamster oocytes by spermatozoa that have undergone ionophore A23187-induced acrosome reaction, and their subsequent development into blastocysts.

To enhance potential use of the Chinese hamster, Cricetulus griseus, in developmental and cytogenetic studies of mammalian gametes and embryos, techniques for in vitro fertilisation and embryo culture were developed in the species. Spermatozoa were recovered from the vasa deferentia of mature males, and incubated in modified TYH medium for 1 h at 37 degrees C under 5% CO2 in air. They were then treated with ionophore A23187 (20 microM) for 10 min to induce the acrosome reaction. Following ionophore treatment, superovulated oocytes were collected from hormonally stimulated females and incubated with the acrosome-reacted spermatozoa for 2 h at 37 degrees C under 5% CO2 in air. In this study, 245 oocytes were used for insemination, and 198 (80.8%) were found to be penetrated by sperm; among them, 194 ova (98.0%) were determined to be monospermic. The monospermic ova were then cultured in TYH supplemented with 1 mM hypotaurine under the same gas phase. Within 30 h of fertilisation, 182 ova (93.8%) cleaved to the 2-cell stage, and subsequently 163 ova (84.0%) developed beyond the 2-cell stage. Thus, obstinate developmental arrest at the 2-cell stage ('2-cell block') was not observed in this species. Ultimately, 65.5% of monospermic ova reached morula to blastocyst stages.

A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona pellucida: lack of relationship with ionophore A23187-induced acrosome reaction.

Acrosome reactions induced by the calcium ionophore A23187 and zona pellucida (ZP) were studied. Sperm samples were obtained from fertile men or men with normal semen analysis and normal sperm-ZP binding. Oocytes were obtained, with the consent of the patients, after the failure of fertilization in vitro. Motile spermatozoa selected by a swim-up technique were incubated with 10 microM A23187 for 1 h, four oocytes for 2 h or solubilized ZP (4 ZP/microliters) for 2 h. Spermatozoa bound to the ZP were dislodged and collected in a small volume of phosphate-buffered saline by aspirating the oocytes with a glass pipette with an inner diameter (120 microns) slightly smaller than the diameter of the oocyte. The acrosome status of the spermatozoa was determined using fluorescein-labelled Pisum sativum agglutinin. The proportion of spermatozoa undergoing the acrosome reaction on the ZP at 2 h varied over a wide range (5-99%), but the agreement between results for the same semen sample exposed to different groups of oocytes was good: the standard deviations of the differences being 9%. Pre-incubation of spermatozoa for 2 h did not increase the ZP-induced acrosome reaction. Re-incubation of ZP with the same sperm suspension for 2 h after removing ZP-bound spermatozoa from the first 2 h incubation produced a significantly lower ZP-induced acrosome reaction in the second incubation (22 +/- 16%) than in the first incubation (30 +/- 14%; P < 0.001, n = 20). There was no significant difference in the ZP-induced acrosome reaction with oocytes with ZP which had or had not been penetrated by spermatozoa during the in-vitro fertilization insemination. Pre-incubation of spermatozoa with solubilized ZP blocked sperm-ZP binding. However, the acrosome reaction induced by solubilized ZP (4 ZP/microliters) was significantly lower than the acrosome reaction induced by intact ZP (10 +/- 5 and 30 +/- 13% respectively, n = 11, P < 0.001), but there was a high correlation (Spearman r = 0.822, P < 0.01) between the results. On the other hand, although the average of the acrosome reaction was similar for A23187 (42%) and for ZP (43%), there was no significant correlation between the results for the two stimuli (n = 60). In conclusion, a useful method for assessing the ZP-induced acrosome reaction has been developed using oocytes which failed to fertilize in vitro. The lack of a relationship between the result of the chemical (A23187) and physiological (ZP) stimuli for the acrosome reaction in the same subjects questions the biological basis of using A23187 for tests of sperm function. Solubilized human ZP in a concentration that blocks sperm-ZP binding is a less efficient inducer of the acrosome reaction than is intact ZP. It is possible that the three-dimensional structure of the ZP is important for induction of the acrosome reaction or that spermatozoa which bind to the ZP are more likely to acrosome react. Assessment of the physiological acrosome reaction for diagnosis of sperm defects which interfere with the fertilization process should be concentrated on the spermatozoa which are capable of binding to the ZP.

Acrosomal autoantigens of guinea pig sperm. I. The purification of an aspermatogenic protein, AP2.

A soluble aspermatogenic autoantigen (AP2) capable of inducing experimental allergic orchitis (EAO) in the guinea pig has been isolated from the soluble acrosomal contents (SAC) released from guinea pig cauda epididymal sperm during the in vitro Ca+2 ionophore A23187-induced acrosome reaction. AP2 purification was achieved by heat inactivation of SAC enzymatic activity, followed by chaotropic solubilization and ultracentrifugation, gel filtration on Sephadex G-50, preparative isoelectric focusing, and SDS-polyacrylamide gel electrophoresis. Approximately 80 micrograms of AP2 was obtained from 1 to 2 x 10(10) cauda epididymal sperm. AP2 has a m.w. of 9500 +/- 1500 as determined by unreduced SDS-PAGE and an isoelectric point of 5.52 +/- 0.11. Amino acid analysis of AP2 indicates that it is a protein. AP2 has no detectable hexose or hexosamine as determined by gas liquid chromatographic analysis and is therefore probably not a glycoprotein. Five micrograms of AP2 are capable of inducing severe EAO in 100% of guinea pigs tested, whereas 0.5 to 2.0 micrograms induces mild lesions consisting primarily of aspermatogenesis in 66% of guinea pigs tested.