Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum

Outer dense fibers (ODF) are specific subcellular components of the sperm flagellum. The functions of ODF have not yet been clearly elucidated. We have investigated the protein composition of purified ODF from bovine spermatozoa and found that one of the most abundant proteins is a 30-32-kDa polypeptide. This protein was analyzed by sequencing peptides derived following limited proteolysis. Peptide sequences were found to match VDAC2 and VDAC3. VDACs (voltage-dependent, anion-selective channels) or eukaryotic porins are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes. In mammals, three VDAC isoforms (VDAC1, -2, -3) have been identified by cDNA cloning and sequencing. Antibodies against synthetic peptides specific for the three mammal VDAC isoforms were generated in rabbits. Their specificity was demonstrated by immunoblotting using recombinant VDAC1, -2, and -3. In protein extracts of bovine spermatozoa, VDAC1, -2, and -3 were detected by specific antibodies, while only VDAC2 and -3 were found as solubilized proteins derived from purified bovine ODFs. Immunofluorescence microscopy of spermatozoa revealed that anti-VDAC2 and anti-VDAC3 antibodies clearly bound to the sperm flagellum, in particular to the ODF. Transmission electron immunomicroscopy supported the finding that VDAC2 protein is abundant in the ODF. Since the ODF does not have any known membranous structure, it is tempting to speculate that VDAC2 and VDAC3 might have an alternative structural organization and different functions in ODF than in mitochondria.     

Inhibition of tyrosine phosphorylation of sperm flagellar proteins,outer dense fiber protein‐2 and tektin‐2, is associated with impaired motility during capacitation of hamster spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor, tyrphostin‐A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin‐A47‐treated spermatozoa exhibited circular motility, which was associated with a distinct hypo‐tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000–60,000. In this study, we provide evidence on the localization of capacitation‐associated tyrosine‐phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo‐tyrosine phosphorylated major proteins of tyrphostin‐A47‐treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein‐2 and the 51 kDa protein as tektin‐2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo‐tyrosine‐phosphorylation status of outer dense fiber protein‐2 and tektin‐2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR‐tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa. Mol. Reprod. Dev. 77: 182–193, 2010. © 2009 Wiley‐Liss, Inc.     

FSCB phosphorylation in mouse spermatozoa capacitation

It is generally accepted that spermatozoa capacitation is associated with protein kinase A-mediated tyrosine phosphorylation. In our previous study, we identified the fibrous sheath CABYR binding protein (FSCB), which was phosphorylated by PKA. However, the phosphorylation status of FSCB protein during spermatozoa capacitation should be further investigated. To this aim, in this study, we found that phosphorylation of this 270-kDa protein occurred as early as 1 min after mouse spermatozoa capacitation, which increased over time and remained stable after 60 min. Immunoprecipitation assays demonstrated that the tyrosine and Ser/Thr phosphorylation of FSCB occurred during spermatozoa capacitation. The extent of phosphorylation and was closely associated with the PKA activity and spermatozoa motility characteristics. FSCB phosphorylation could be induced by PKA agonist DB-cAMP, but was blocked by PKA antagonist H-89.Therefore, FSCB contributes to spermatozoa capacitation in a tyrosine-phosphorylated format, which may help in further elucidating the molecular mechanism of spermatozoa capacitation.     

Pentoxifylline induced signalling events during capacitation of hamster spermatozoa: significance of protein tyrosine phosphorylation.

To fertilize the oocyte, mammalian spermatozoa must undergo capacitation and acrosome reaction. These events are believed to be associated with various biochemical changes primarily mediated by cAMP, Ca2+ and protein kinases. But the precise signaling mechanisms governing sperm function are not clear. To study this, we used pentoxifylline (PF), a sperm motility stimulant and a cAMP-phosphodiesterase inhibitor, during capacitation and acrosome reaction of hamster spermatozoa. PF induced an early onset of sperm capacitation and its action involved modulation of sperm cell signaling molecules viz, cAMP, [Ca2+]i and protein kinases. The PF-induced capacitation was associated with an early and increased total protein phosphorylation coupled with changes in the levels of reactive oxygen species. Protein kinase (PK)-A inhibitor (H-89) completely inhibited phosphorylation of a 29 kDa protein while PK-C inhibitor (staurosporine) did not inhibit phosphorylation. Interestingly, PF induced protein tyrosine phosphorylation of a set of proteins (Mr 45-80 K) and a greater proportion of PF-treated spermatozoa exhibited protein tyrosine phosphorylation, compared to untreated controls (82 + 9% vs 34 +/- 10%; p < 0.001); tyrosine-phosphorylated proteins were localized specifically to the mid-piece of the sperm. The profile of protein tyrosine phosphorylation was inhibitable by higher concentrations (> 0.5 mM) of tyrosine kinase inhibitor, tyrphostin A47. However, at lower (0.1-0.25 mM) concentrations, the compound interestingly induced early sperm capacitation and protein tyrosine phosphorylation, like PF. These results show that protein tyrosine phosphorylation in the mid-piece segment (mitochondrial sheath) appears to be an early and essential event during PF-induced capacitation and a regulated level of tyrosine phosphorylation of sperm proteins is critical for capacitation of hamster spermatozoa.     

Effect of reactive oxygen species on capacitation and associated protein tyrosine phosphorylation in buffalo (Bubalus bubalis) spermatozoa

In the present study, the effect of two particular reactive oxygen species (ROS), superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) on buffalo (Bubalus bubalis) sperm capacitation and associated protein tyrosine phosphorylation was studied. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50 x 10(6)/mL and incubated at 38.5 degrees C for 6h with or without heparin (10(g/mL; a positive control), or xanthine (X; 0.5mM)-xanthine oxidase (XO; 0.05 U/mL)-catalase (C; 2100 U/mL) system that generates O(2)(-) or NADPH (5mM) that stimulates the endogenous O(2)(-) production or H(2)O(2) (50 microM). The specific effect of O(2)(-), H(2)O(2) and NADPH on buffalo sperm capacitation and protein tyrosine phosphorylation was assessed by the addition of superoxide dismutase (SOD), catalase and diphenylene iodonium (DPI), respectively, to the incubation medium. Each of X+XO+C system, NADPH and H(2)O(2) induced a significantly higher percentage (P<0.05) of capacitation in buffalo spermatozoa compared to control. However, DPI inhibited this NADPH-induced capacitation and protein tyrosine phosphorylation and suggested for existence of an oxidase in buffalo spermatozoa. Using immunoblotting technique, at least seven tyrosine-phosphorylated proteins (20, 32, 38, 45, 49, 78 and 95 kDa) were detected in capacitated buffalo spermatozoa. Out of these, the tyrosine phosphorylation of p95 was induced extensively by both O(2)(-) as well as exogenous source of H(2)O(2) and using specific activators and inhibitors of signaling pathways, it was found this induction was regulated through a cAMP-dependent PKA pathway. Further, immunofluorescent localization study revealed that these ROS-induced tyrosine-phosphorylated proteins are mostly distributed in the midpiece and principal piece regions of the flagellum of capacitated spermatozoa and suggested for increased molecular activity in flagellum during capacitation. Thus, the study revealed that both O(2)(-) and H(2)O(2) promote capacitation and associated protein tyrosine phosphorylation in buffalo spermatozoa and unlike human and bovine, a different subset of sperm proteins were tyrosine-phosphorylated during heparin- and ROS-induced capacitation and regulation of these ROS-induced processes were mediated through a cAMP/PKA signaling pathway.     

CABYR, a novel calcium-binding tyrosine phosphorylation-regulated fibrous sheath protein involved in capacitation

To reach fertilization competence, sperm undergo an incompletely understood series of morphological and molecular maturational processes, termed capacitation, involving, among other processes, protein tyrosine phosphorylation and increased intracellular calcium. Hyperactivated motility and an ability to undergo the acrosome reaction serve as physiological end points to assess successful capacitation. We report here that acidic (pI 4.0) 86-kDa isoforms of a novel, polymorphic, testis-specific protein, designated calcium-binding tyrosine phosphorylation-regulated protein (CABYR), were tyrosine phosphorylated during in vitro capacitation and bound (45)Ca on 2D gels. Acidic 86-kDa calcium-binding forms of CABYR increased during in vitro capacitation, and calcium binding to these acidic forms was abolished by dephosphorylation with alkaline phosphatase. Six variants of CABYR containing two coding regions (CR-A and CR-B) were cloned from human testis cDNA libraries, including five variants with alternative splice deletions. A motif homologous to the RII dimerization domain of PK-A was present in the N-terminus of CR-A in four CABYR variants. A single putative EF handlike motif was noted in CR-A at aas 197-209, while seven potential tyrosine phosphorylation-like sites were noted in CR-A and four in CR-B. Pro-X-X-Pro (PXXP) modules were identified in the N- and C-termini of CR-A and CR-B. CABYR localizes to the principal piece of the human sperm flagellum in association with the fibrous sheath and is the first demonstration of a sperm protein that gains calcium-binding capacity when phosphorylated during capacitation.     

Phosphorylation and consequent stimulation of the tyrosine kinase c-Abl by PKA in mouse spermatozoa; its implications during capacitation

Upon ejaculation, spermatozoa undergo a series of post-translational modifications in a process known as capacitation in order to prepare for fertilization. In the absence of capacitation, fertilization cannot occur. Spermatozoa are unusual in that one of the hallmarks of capacitation is a global up-regulation in phosphotyrosine expression, which is known to be mediated upstream by PKA. Little is known about the signaling events downstream of PKA apart from the involvement of SRC, as a key mediator of PKA-induced tyrosine phosphorylation in the sperm tail. Here we describe the presence of c-Abl in mouse spermatozoa. In vitro analysis confirmed that PKA can up-regulate c-Abl kinase activity. In vivo, this tyrosine kinase was found to associate, and become threonine phosphorylated by PKA in the sperm flagellum. By treating spermatozoa with hemolysin we could demonstrate that a significant proportion of the tyrosine phosphorylation associated with capacitation could be suppressed by the c-Abl inhibitor, Gleevac. This is the first report of c-Abl being up-regulated by PKA for any cell type. We present a model, whereby these kinases may operate together with SRC to ensure optimal levels of tyrosine phosphorylation in the sperm flagellum during the attainment of a capacitated state.     

Failure of the expression of phospholipid hydroperoxide glutathione peroxidase in the spermatozoa of human infertile males

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) was intensely expressed in mitochondria in the midpiece of human spermatozoa by immunostaining with anti-PHGPx monoclonal antibodies. The PHGPx not only reduced phospholipid hydroperoxide but also scavenged hydrogen peroxide in human spermatozoa. We found a dramatic decrease in the level of expression of PHGPx in the spermatozoa of some infertile males by immunoblotting with anti-PHGPx monoclonal antibodies. These individuals accounted for about 10% of the group of 73 infertile males that we examined. All seven patients with PHGPx-defective spermatozoa were classified as suffering from oligoasthenozoospermia, a defect in which both the number and the motility of spermatozoa are significantly below normal. Males with PHGPx-defective spermatozoa accounted for 26% of the 27 infertile males with oligoasthenozoospermia. No defects in expression of PHGPx in spermatozoa were observed in 31 fertile volunteers. After a 3-h incubation, the relative number of motile spermatozoa with low-level expression of PHGPx was significantly lower than that of spermatozoa with normal expression of PHGPx. The PHGPx-defective spermatozoa failed to incorporate rhodamine 123, revealing a loss of mitochondrial membrane potential. Ultrastructual analysis of mitochondria by electron microscopy demonstrated that the morphology of mitochondria in PHGPx-defective spermatozoa was abnormal. The results suggest that failure of the expression of mitochondrial PHGPx in spermatozoa might be one of the causes of oligoasthenozoospermia in infertile men.     

Effect of two intracellular calcium modulators on sperm motility and heparin-induced capacitation in cryopreserved bovine spermatozoa

Spermatozoa require a preparatory process called capacitation to fertilize mature oocytes. Two events related to capacitation of mammalian spermatozoa are an increase in intracellular Ca(2+) and protein tyrosine phosphorylation. The sites that regulate intracellular Ca(2+) concentration are plasma membrane and mitochondria. There are different systems for mitochondrial Ca(2+) influx and efflux. Our aim was to study the involvement of mitochondrial Ca(2+) cycle during heparin-induced capacitation in cryopreserved bovine spermatozoa. Samples were incubated at 38°C for 45 min, in TALP medium, in the presence of: (a) heparin (H), a well known capacitation inducer; (b) H+CGP 37157, a specific inhibitor of mitochondrial Ca(2+) efflux; (c) H+RU 360, a specific inhibitor of Ca(2+) influx to the mitochondria and (d) H+CGP 37157+RU 360. In every treatment, capacitation (by CTC), progressive motility (by optical microscopy), viability (by the eosin/nigrosin technique) and protein tyrosine phosphorylation (by Western Immuno-blotting), were evaluated. The addition of CGP 37157 (20 μM) decreased progressive motility (p<0.05), without affecting capacitation or protein tyrosine phosphorylation, indicating the importance of calcium efflux for maintaining progressive motility. RU 360 (5 μM) significantly reduced capacitation without affecting progressive motility, sperm viability or protein tyrosine phosphorylation, showing that inhibition of the mitochondrial calcium uptake, negatively affect the capacitation process. The addition of both inhibitors showed the effect of RU 360. According with these results, there would exist a differential participation of the income and outcome mitochondrial calcium carriers, in the capacitation process. In conclusion, this research demonstrates the importance of normal mitochondrial calcium cycle in the achievement of sperm capacitation and the maintenance of progressive motility in cryopreserved bovine spermatozoa.     

Phospholipid hydroperoxide glutathione peroxidase: expression pattern during testicular development in mouse and evolutionary conservation in spermatozoa

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility.     

Characterization of the major proteins of bovine seminal fluid by two-dimensional polyacrylamide gel electrophoresis

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5–7.8 and at molecular weights (Mr) between 6 and 100 kDa. Many isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a Mr of 16.5 kDa and in the range of pl of 4.7–5.0; BSP-A2 at 16 kDa and at a pl of 4.9–5.2; BSP-A3 at 15 kDa and at a pl of 4.8–5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9–4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8–5.0 and decreased its Mr to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their Mr nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content. © 1994 Wiley-Liss, Inc.     

Glycodelin-S in human seminal plasma reduces cholesterol efflux and inhibits capacitation of spermatozoa

Tight control of sperm capacitation is important for successful fertilization. Glycodelin-S is one of the most abundant glycoproteins in the human seminal plasma. However, its function is unclear. We investigated the role of glycodelin-S on capacitation of human spermatozoa. Binding kinetics experiments demonstrated the presence of two saturable and reversible binding sites of glycodelin-S on human spermatozoa. Differently glycosylated other isoforms of glycodelin, glycodelin-A and -F, did not compete with glycodelin-S for these binding sites, suggesting that the glycodelin-S binding sites are different from those of the other isoforms. Indirect immunofluorescent staining revealed specific binding of glycodelin-S around the sperm head. This immunoreactivity was greatly reduced in spermatozoa that had migrated through the cervical mucus surrogates. Glycodelin-S at physiological concentrations significantly reduced the bovine serum albumin and cyclodextrin-induced cholesterol efflux and down-regulated the adenylyl cyclase/protein kinase A/tyrosine kinase signaling pathway, resulting in suppression of capacitation. Deglycosylation abolished glycodelin-S binding and the effect of glycodelin-S on bovine serum albumin-induced capacitation. This indicates that the carbohydrate moiety of glycodelin-S is critical for the function of the molecule. It is concluded that glycodelin-S in seminal plasma maintains the uncapacitated state of human spermatozoa.     

Microfilaments appear in boar spermatozoa during capacitation in vitro

Boar spermatozoa were incubated in a capacitation medium and examined for the presence of filamentous actin by using the fluorescent probe NBD-phallacidin. F-actin was not observed in uncapacitated sperm, but developed in most regions of the cell during the capacitation period. Fluorescent staining was most intense in the flagellum. When fresh seminal plasma was added to capacitated sperm and the sperm was further incubated, F-actin was no longer observed. In view of previous experiments which indicated that plasma membrane proteins (PMPs), including a major integral PMP, move out of the sperm head into the flagellum during capacitation and that this movement is inhibited by the microfilament poison cytochalasin D (Peterson, Saxena, Saxena, and Russell: Biol. Reprod., in press, '86), we suggest that actin-PMP interactions play a major role in capacitating boar spermatozoa.     

酪氨酸磷酸化蛋白在体外获能豚鼠精子上的分布与表达

为研究豚鼠精子获能过程中蛋白酪氨酸磷酸化的变化规律,将豚鼠精子悬浮于改良的TALP获能培养基中,在5% CO2 孵箱37 ℃培养,以精子与金霉素(CTC) 荧光结合类型为指标评价精子获能状态,用免疫荧光技术和Western blot方法检测酪氨酸磷酸化的蛋白在精子上的分布以及酪氨酸磷酸化水平的变化。结果显示,随着获能的进行,发生蛋白酪氨酸磷酸化的精子占总精子的百分比增加,由未获能前的36％增至获能7h时的92％。酪氨酸磷酸化的蛋白分布变广,由精子头部扩增至精子头部、鞭毛主段和中段。另外,有80,45,40kDa的三种蛋白发生酪氨酸磷酸化,其中40kDa的蛋白酪氨酸磷酸化水平自精子体外培养后呈递增趋势,45kDa的蛋白酪氨酸磷酸化自培养3h后发现并呈递增趋势,而80kDa的蛋白酪氨酸磷酸化水平在精子培养3h时最高,后呈递减趋势。     

Cyclic AMP-dependent tyrosine phosphorylation in tammar wallaby (Macropus eugenii) spermatozoa

Despite considerable advances in our understanding of the molecular mechanisms regulating eutherian sperm function, there is a paucity of such knowledge for the Metatheria. In eutherian spermatozoa, the attainment of functional competence is associated with a redox-regulated, cAMP-mediated tyrosine phosphorylation cascade, activated during capacitation. In this report we investigate whether tammar wallaby (Macropus eugenii) spermatozoa possess a similar signal transduction pathway. Western blot analysis of phosphotyrosine expression in caudal and ejaculated populations of tammar spermatozoa revealed that elevation of intracellular cAMP levels, but not exposure to oxidants or NADPH, induced a dramatic increase in the overall level of tyrosine phosphorylation. Washed, ejaculated spermatozoa exhibited more pronounced increases in tyrosine phosphorylation than unwashed sperm populations. Localisation of tyrosine phosphorylation by immunocytochemistry showed that phosphotyrosine residues were principally located along the tammar sperm flagellum, and occasionally at a small region of the sperm head, adjacent to the acrosome. Associated with the tyrosine phosphorylation of tammar spermatozoa, was a change in sperm head conformation to a T-shaped orientation, further implying the importance of these pathways to normal tammar sperm function. Redox activity, as detected by lucigenin-dependent chemiluminescence, was stimulated by NADPH in caudal sperm preparations but not ejaculated spermatozoa. However, neither sperm population responded to treatment with NADPH with changes in intracellular cAMP or tyrosine phosphorylation. In conclusion, tammar spermatozoa possess the same cAMP-mediated, tyrosine phosphorylation-dependent signal transduction cascade that has been associated with capacitation in eutherian spermatozoa. However in Metatherian spermatozoa we could find no evidence that this pathway was redox regulated.     

Role of translation by mitochondrial‐type ribosomes during sperm capacitation: An analysis based on a proteomic approach

Mammalian spermatozoa contain a complex population of mRNAs, some of which have been demonstrated to be translated de novo by mitochondrial‐type ribosomes using D‐chloramphenicol (CP), a specific inhibitor of mitochondrial translation. However, little is known about the functions of these mRNAs in mature sperm. In the present study, differential proteomic approaches were applied to study sperm protein profiles translated by mitochondrial‐type ribosomes using the inhibitor CP and 44 proteins were identified with lower expression in CP‐treated sperm in comparison to capacitated sperm (ratio ≥ 1.5, p<0.05). Results of Western blot and real‐time PCR suggest that four proteins were translated by mitochondrial‐type ribosomes. Bioinformatics analysis indicated that 26 of 44 proteins were involved in some critical processes correlated to sperm–egg interaction event. In addition, Mups, whose functions in reproduction have never been studied, were chosen for further study. Our results showed that Mups proteins were localized to the acrosome and flagellum of precapacitated sperm, and were also expressed in the equatorial segment of capacitated sperm. The depletion of Mups using neutralizing antibodies significantly inhibited capacitation in a dose‐dependent manner, subsequently inhibited acrosome reaction and sperm–egg fusion. In summary, mitochondrial translation during capacitation can store proteins beneficial for sperm–egg interaction.     

Alteration of sperm thiol-disulfide status and capacitation in the guinea pig

Epididymal spermatozoa of the guinea pig were incubated under conditions known to promote a rapid synchronous capacitation in a large proportion of the spermatozoa (Ca²⁺-free medium with lysophosphatidylcholine, LC) or in Ca ²⁺-free medium without LC. To study the effects of altered thiol-disulfide status and content, incubations were conducted with reagents that maintain and increase thiol groups (DTT, GSH), maintain and increase disulfide groups (diamide, GSSG), or which irreversibly block thiol groups by alkylation (NEM). The permeable DTT inhibited LC-induced capacitation and at high concentrations diminished the percentage of acrosome reactions in capacitated spermatozoa. The permeable diamide exhibited a stimulatory effect upon capacitation. The largely impermeable GSH and GSSG exhibited effects similar to their respective permeable counterparts but their effects were moderate and required extremely high concentrations. The DTT inhibition of LC-induced capacitation was reversible by washing and a further 1 hr incubation. In this final incubation after removal of DTT by washing, LC was absent too so its stimulatory effect must have been accomplished prior to washing and in the presence of DTT. NEM-alkylation of the existing thiol population did not affect LC-induced capacitation but alkylation of the increased thiol population after prior DTT treatment was inhibitory of capacitation. These results suggest that the maintenance and/or formation of disulfide groups on enzymes or structural proteins may be a component of the capacitation process. In contrast, the formation and maintenance by alkylation of increased thiol groups but not the maintenance of existing thiol groups, is inhibitory of capacitation. The relevance of these findings to a role for a thiol-sensitive proteinase in capacitation is discussed.     

Solubilization and purification of alpha-mannosidase, a marker enzyme of vacuolar membranes in Saccharomyces cerevisiae

Yeast alpha-mannosidase, a marker enzyme of vacuolar membranes, was solubilized and purified from commercial bakers' yeast. The alpha-mannosidase was solubilized efficiently with 10 mM Na2CO3. A high pH (greater than 8.5) and a sufficient amount of a detergent such as 0.2% (w/v) Triton X-100 were required to keep the enzyme in a soluble state. This suggested that the enzyme is either a peripheral membrane protein or an ecto-type integral membrane protein. After 4,300-fold purification by conventional chromatography, the alpha-mannosidase gave a single band on nondenaturing polyacrylamide gel electrophoresis, but could be fractionated into active isoforms, which consisted of 107-, 73-, and 31-kDa polypeptides, with a Mono Q anion exchange fast protein liquid chromatography system. Apparent molecular weight of the native enzyme was determined as 560,000. It suggested that the composition of isoforms will be described as (107 kDa)n (73 kDa)6-n (31 kDa)6-n, where n is 0-6. The 107- and 73-kDa polypeptides were purified further under denaturing conditions. One-dimensional peptide map analysis and immunological analysis of these polypeptides indicated that they are closely related proteins. Immunoblotting of crude cell lysates revealed that the 107-kDa polypeptide appeared first, and then the 73-kDa polypeptide appeared along growth phase. It suggested that proteolytic conversion of the 107-kDa polypeptide occurs to form the 73- and 31-kDa polypeptides and leads to formation of isoforms of the enzyme.