Protein serine and threonine phosphorylation,hyperactivation and acrosome reaction in in vitro capacitated hamster spermatozoa

Monoclonal antibodies against phosphoserine and phosphothreonine were used in the present study to investigate the changes in serine and threonine phosphorylation respectectively during capacitation of hamster spermatozoa. Immunoblot analysis of hamster spermatozoa capacitated in TALP, a medium that supports capacitation, showed that a set of four proteins of molecular weight 56, 63, 66, and 100 kDa was phosphorylated both at the serine and threonine residues. In addition, five other proteins of molecular weight 32, 39, 45, 53, and 61 kDa were phosphorylated specifically at the threonine residues. Of these nine proteins, the 100 kDa protein showed a time dependent or capacitation-dependent decrease in intensity which coincided with the percentage acrosome-reacted spermatozoa. In contrast, the 49 and 63 kDa threonine phosphorylated proteins showed increased phosphorylation coinciding with capacitation. H8 (a serine and threonine kinase inhibitor) had a transient effect on the phosphorylation of these two phosphothreonine proteins but inhibited acrosome reaction substantially all through the treatment period. Okadaic acid (OA) (a serine and threonine protein phosphatase inhibitor) inhibited hyperactivation but had no effect on acrosome reaction. In fact, OA stimulated acrosome reaction. Finally the immunofluorescence studies indicated localization of the serine phosphorylated proteins in tail as well as in head of the capacitated hamster spermatozoa whereas the threonine phosphorylated proteins were localized mostly in the tail of the spermatozoa. The findings of the present study suggest that serine/threonine phosphorylation and the enzymes responsible for regulating the level of phosphorylation play an important role in capacitation and capacitation-associated events namely hyperactivation and acrosome reaction. However, further studies are needed in order to establish the exact role of these proteins in capacitation of spermatozoa.     

Hamster sperm capacitation: role of pyruvate dehydrogenase A and dihydrolipoamide dehydrogenase

Recently, we demonstrated that pyruvate dehydrogenase A2 (PDHA2) is tyrosine phosphorylated in capacitated hamster spermatozoa. In this report, using bromopyruvate (BP), an inhibitor of PDHA, we demonstrated that hamster sperm hyperactivation was blocked regardless of whether PDHA was inhibited prior to or after the onset of hyperactivation, but the acrosome reaction was blocked only if PDHA was inhibited prior to the onset of the acrosome reaction. Further, inhibition of PDHA activity did not inhibit capacitation-associated protein tyrosine phosphorylation observed in hamster spermatozoa. It is demonstrated that the essentiality of PDHA for sperm capacitation is probably dependent on its ability to generate effectors of capacitation such as reactive oxygen species (ROS) and cAMP, which are significantly decreased in the presence of BP. MICA (5-methoxyindole-2-carboxylic acid, a specific inhibitor of dihydrolipoamide dehydrogenase [DLD]), another component of the pyruvate dehydrogenase complex (PDHc), also significantly inhibited ROS generation and cAMP levels thus implying that these enzymes of the PDHc are required for ROS and cAMP generation. Furthermore, dibutryl cyclic adenosine monophosphate could significantly reverse the inhibition of hyperactivation observed in the presence of BP and inhibition of acrosome reaction observed in the presence of BP or MICA. The calcium ionophore, A23187, could also significantly reverse the inhibitory effect of BP and MICA on sperm acrosome reaction. These results establish that PDHA is required for hamster sperm hyperactivation and acrosome reaction, and DLD is required for hamster acrosome reaction. This study also provides evidence that ROS, cAMP, and calcium are involved downstream to PDHA.     

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.     

Inhibition of in vitro capacitation of hamster spermatozoa by nitric oxide synthase inhibitors.

In an attempt to understand the role of nitric oxide(NO) in sperm capacitation, in the present study, hamster spermatozoa were used to evaluate the effects of NO on motility, viability, hyperactivation, capacitation and protein tyrosine and serine phosphorylation using specific inhibitors of nitric oxide synthase (NOS); namely L-NAME (N-nito-L-aginine methyl ester) and 7-Ni (7-nitroindazole). The results indicated that L-NAME inhibits sperm motility, hyperactivation and acrosome reaction where as 7-Ni inhibits only hyperactivation and acrosome reaction thus implying that NOS inhibitors exhibit subtle differences with respect to their effects on sperm functions. This study also provides evidence that NOS inhibitors inhibit sperm capacitation by their ability to modulate protein tyrosine phosphorylation. However, the inhibitors had no effect on the protein serine phosphorylation of hamster spermatozoa during capacitation. Thus, these results indicate that NO is required     

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.     

Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP‐dependent hyperactivation in the spermatozoa of Japanese Black bulls

The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen‐stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell‐permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine‐phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen‐stored spermatozoa were classified as having a high‐grade acrosome condition before incubation, sperm tyrosine‐phosphorylated proteins, including the 33‐kDa tyrosine‐phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41‐ and 33‐kDa sperm tyrosine‐phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine‐phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen‐stored sperm were classified as having a low‐grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine‐phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine‐phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP‐dependent hyperactivation for the spermatozoa of individual Japanese Black bulls. Mol. Reprod. Dev. 77:910–921, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Activity of pyruvate dehydrogenase A (PDHA) in hamster spermatozoa correlates positively with hyperactivation and is associated with sperm capacitation

Unravelling the molecular basis of capacitation is crucial to our understanding the basis of acquisition of fertilization competence by spermatozoa. In two recent studies, we have demonstrated that dihydrolipoamide dehydrogenase, which is a post-pyruvate metabolic enzyme and one of the components of pyruvate dehydrogenase complex, undergoes capacitation-dependent tyrosine phosphorylation, and that the activity of the enzyme correlates with capacitation events in the hamster spermatozoa. However, it is not clear as to whether other components of the pyruvate dehydrogenase complex are also crucial for sperm capacitation. In this report, we have identified pyruvate dehydrogenase A2 (PDHA2), a constituent of pyruvate dehydrogenase A (PDHA), which is a component of pyruvate dehydrogenase complex that exhibits tyrosine phosphorylation during hamster spermatozoal capacitation. This is the first report showing that hamster sperm PDHA2 is a testis-specific phosphotyrosine that is associated with the fibrous sheath of hamster spermatozoa. The localization of PDHA2 in spermatozoa was investigated using antibodies to PDHA, which is the active tetrameric protein that consists of a homodimer of PDHA2 and PDHB. Both immunofluorescence and confocal studies indicated a unique non-canonical, extramitochondrial localization for PDHA in the principal piece of hamster spermatozoa. It was also observed that PDHA colocalized with AKAP4 in the fibrous sheath of the spermatozoon. The enzymatic activity of PDHA was positively correlated with hyperactivation but not with the acrosome reaction. Given the localization of PDHA and the evidence that its activity correlates positively with hyperactivation and that its PDHA2 subunit exhibits capacitation-associated protein tyrosine phosphorylation, it appears that PDHA2 is associated with the process of capacitation.     

Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro.

The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.     

Novelty of the pyruvate metabolic enzyme dihydrolipoamide dehydrogenase in spermatozoa: correlation of its localization, tyrosine phosphorylation, and activity during sperm capacitation

Spermatozoa are cells distinctly different from other somatic cells of the body, capacitation being one of the unique phenomena manifested by this gamete. We have shown earlier that dihydrolipoamide dehydrogenase, a post-pyruvate metabolic enzyme, undergoes capacitation-dependent tyrosine phosphorylation, and the functioning of the enzyme is required for hyperactivation (enhanced motility) and acrosome reaction of hamster spermatozoa (Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70, 887-899). In this report we have investigated the localization of this mitochondrial enzyme in spermatozoa revealing non-canonical extra-mitochondrial localization of the enzyme in mammalian spermatozoa. In hamster spermatozoa, dihydrolipoamide dehydrogenase along with its host complex, the pyruvate dehydrogenase complex, are localized in the acrosome and in the principal piece of the sperm flagella. The localization of dihydrolipoamide dehydrogenase, however, appears to be in the mitochondria in the spermatocytes, but in spermatids it appears to show a juxtanuclear localization (like Golgi). The capacitation-dependent time course of tyrosine phosphorylation of dihydrolipoamide dehydrogenase appears to be different in the principal piece of the flagella and the acrosome in hamster spermatozoa. Activity assays of this bi-directional enzyme suggest a strong correlation between the tyrosine phosphorylation and the bi-directional enzyme activity. This is the first report of a direct correlation of the localization, tyrosine phosphorylation, and activity of the important metabolic enzyme, dihydrolipoamide dehydrogenase, implicating dual involvement and regulation of the enzyme during sperm capacitation.     

Capacitation-associated protein tyrosine phosphorylation starts early in buffalo (Bubalus bubalis) spermatozoa as compared to cattle

A comparative study was conducted on protein tyrosine phosphorylation events of capacitating sperm of two ruminant species, cattle and buffalo. Ejaculated cattle and buffalo bull spermatozoa were suspended separately in sp-TALP medium at 50 × 10⁶ mL⁻¹ and incubated at 38.5 °C with 5% CO₂ in air in the absence or presence of heparin for a period of 6 h. The extent of sperm capacitation after various periods of incubations was assessed by lysophosphatidyl choline-induced acrosome reaction followed by a triple-staining technique and capacitation-associated tyrosine-phosphorylated proteins were detected by immunoblotting technique using a monoclonal antiphosphotyrosine antibody. In the same media, over a time-period, a significant increase in capacitation percentage was observed even in control group of buffalo spermatozoa as compared to a non-significant increase in that of cattle sperm. In both cattle and buffalo spermatozoa, at 6 h, four proteins of molecular weight 49, 45, 32, and 20 kDa (designated as p49, p45, p32, and p20) were tyrosine phosphorylated. However, in buffalo, two additional proteins of 38 and 30 kDa were also tyrosine phosphorylated. In a time-course study, p20 appeared as early as at 0 h in capacitated buffalo spermatozoa as compared to 4 h in cattle. Further, in heparin-treated buffalo spermatozoa, with a time-dependent increase in tyrosine phosphorylation of some proteins, there was time-dependent dephosphorylation of some other proteins that was never seen in heparin-treated cattle sperm. Thus, the present findings revealed that though buffalo sperm takes more time than cattle for capacitation but its associated protein tyrosine phosphorylation event starts very early as compared to cattle.     

Glutathione-S-transferase: Role in buffalo (Bubalus bubalis) sperm capacitation and cryopreservation

In this study, glutathione-S-transferase Mu3 (GST) has been reported to play an important role in sperm capacitation, acrosome reaction, and fertilization. The freshly ejaculated buffalo spermatozoa were in vitro capacitated using heparin (10 μg/mL) or cryopreserved in egg yolk citrate extender. Glutathione-S-transferase was identified and characterized in terms of their isozymic forms, tyrosine phosphorylation, and immunolocalization patterns in cryopreserved buffalo spermatozoa in comparison with freshly ejaculated and in vitro capacitated spermatozoa. Two-dimensional gel electrophoresis, immunoblot, immunocytochemistry, and enzyme activity analyses were done to characterize GST in this study. Five and eight isozymic forms of GST were detected in cryopreserved and capacitated spermatozoa, respectively. Differential tyrosine phosphorylation of these enzymes was observed in cryopreserved and capacitated spermatozoa. The tyrosine phosphorylation of this enzyme involved cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent pathways during in vitro capacitation of the spermatozoa. Differential immunolocalization patterns of GST were observed in freshly ejaculated, capacitated, and cryopreserved spermatozoa. Glutathione-S-transferase Mu3 enzyme activity was found to be significantly (P < 0.05) different in freshly ejaculated, capacitated, and cryopreserved spermatozoa. Activity of GST was significantly (P < 0.05) increased with the progression of capacitation. The cryopreserved spermatozoa showed significantly (P < 0.05) greater enzyme activity compared with fresh spermatozoa and was equal to 2-hour capacitated spermatozoa. The cryopreserved spermatozoa showed significant (P < 0.05) loss of GST enzyme protein. Tyrosine phosphorylated GST showed significantly (P < 0.05) greater activity compared with their dephosphorylated forms. The information generated in this study can be used to understand the molecular mechanism of the effects of GST on capacitation. Regulation of GST during sperm cryopreservation could be a good target to improve fertility of cryopreserved spermatozoa for their use in assisted reproductive technologies.     

Protein tyrosine phosphorylation of a heparin-binding sperm membrane mitogen (HBSM) is associated with capacitation and acrosome reaction

Protein tyrosine phosphorylation in spermatozoa is associated with epididymal maturation and though to be central for attainment of a capacitated state and expression of hyperactivated motility. Heparin, the most highly sulfated glycosaminoglycans, was also the most potent at stimulating the acrosomal reaction in bovine epididymal spermatozoa. Studies using radiolabeled inorganic phosphate showed 11-fold increase (32)Pi incorporation in heparin-binding sperm membrane protein (HBSM) during spermatozoal capacitation, and the phosphorylation occurs at the tyrosine residue. Epididymal spermatozoa were induced to undergo capacitation and acrosome reaction by 70% when the cells were incubated in BWW medium supplemented with heparin. The spermatozoa pre-treated with anti-HBSM antibody showed 46% reduction in the hyperactivated motility and lowers the acrosome reaction. This was confirms by measuring the hydrolysis of benzoyl-l-arginine ethyl ether (BAEE) by the acrosomal enzyme; acrosin. The preliminary finding suggests that HBSM may play an important role in the sperm capacitation and acrosome reaction.     

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.     

Phosphorylation of Shc proteins in human sperm in response to capacitation and progesterone treatment

Several authors have demonstrated the involvement of tyrosine kinases during sperm capacitation and acrosome reaction. Shc proteins (p46^Shc, p52^Shc, and p66^Shc) are cytoplasmic substrates of activated tyrosine kinases and are widely expressed in mammalian somatic tissues. Experiments were designed to demonstrate the presence of Shc in spermatozoa and to study its involvement in the signal transduction events leading to acrosome reaction. Anti-Shc antibodies strongly reacted with the acrosomal region of methanol-fixed human sperm. Only one Shc isoform (p52^Shc) was detected on Western blot. To study the degree of phosphorylation of Shc during capacitation and acrosome reaction, sperm samples were divided into two groups: noncapacitated and capacitated/progesterone treated. Lysates from both groups were immunoprecipitated with anti-phosphotyrosine antibodies and the precipitated (ie, phosphorylated) proteins were tested with anti-Shc antibodies. The intensity of p52^Shc was clearly increased in capacitated/progesterone-stimulated cells. Mol. Reprod. Dev. 50:113–120, 1998. © 1998 Wiley-Liss, Inc.     

Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm.

Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place. Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2, 2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 microM, and with an EC50 of 100 microM. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium. In contrast, addition of 500 microM dibutyryl cAMP (dbcAMP), alone or together with 100 microM 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium. These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium. When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX. The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation.     

The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation

It is generally accepted that incubation with heparin is required to induce capacitation of ejaculated bovine spermatozoa in vitro. The capacitation process implicates many biochemical events, and is correlated with modified sperm motility and the phosphorylation of specific proteins on tyrosine residues. To better understand the molecular basis of heparin-induced capacitation, bovine spermatozoa were incorporated with a radioactive substrate of protein kinases [gamma32P]-ATP, to observe protein phosphorylation dynamics over time. Sperm motion parameters including the percentage of motile spermatozoa, amplitude of lateral head displacement (ALH) and flagellar beat cross frequency (BCF) were assessed to determine whether the protein phosphorylation patterns induced by heparin also promote changes in motility. Capacitation was confirmed using the chlortetracycline fluorescence assay and the appearance of 'pattern B' stained spermatozoa. Evaluation of the different motility parameters during capacitation reveal that heparin has a marked negative effect, over time, on the percentage of motile spermatozoa, consistent with hyperactivation. Indeed, the presence of heparin greatly increases the BCF of bull spermatozoa and induces a significant increase in the ALH compared to spermatozoa incubated without heparin. We detected several sperm proteins that are phosphorylated over time. A 45 kDa protein is the most intensely phosphorylated of the sperm proteins. However, it is visible regardless of the presence of heparin. Interestingly, a second phosphorylated protein of approximately 50 kDa undergoes more intense phosphorylation with heparin than without. In summary, the present study demonstrated that heparin induces physiological changes in several sperm motility parameters including ALH, BCF and the percentage of motile spermatozoa. Heparin also increases the intensity of phosphorylation of a 50 kDa sperm protein. These results suggest that capacitation of bovine spermatozoa and capacitation-associated motility changes may be regulated by a mechanism that includes protein phosphorylation, and that a presently unknown protein kinase is involved.     

Identification of the major tyrosine phosphorylated protein of capacitated hamster spermatozoa as a homologue of mammalian sperm a kinase anchoring protein.

The molecular basis of mammalian sperm capacitation is unique in that, it is associated with a protein kinase A (PKA) dependent upregulation of protein tyrosine phosphorylation. Therefore, PKA activity during capacitation would be crucial for the downstream events of protein tyrosine phosphorylation, and mechanisms may exist to ensure that PKA phosphorylates its specific substrate. This could be achieved by bringing PKA close to its substrate, a function normally carried out by an A-kinase anchoring protein (AKAP). We showed previously that cauda epididymidal spermatozoa of hamster undergo a capacitation-dependent increase in protein tyrosine phosphorylation. In the present study, evidence is provided that two major tyrosine phosphorylated proteins of molecular weight 97 and 83 kDa are the hamster homologues of mouse pro-AKAP82 and AKAP82, and have been designated as hamster pro-AKAP83 and AKAP83 respectively. Hamster AKAP83 resembled the mouse AKAP82 with respect to its molecular weight, pI (pH 5-5.5) and cDNA and amino acid sequences. Sequence analysis indicated that the primary structure of pro-AKAP83 was highly conserved and exhibited 91% identity with mouse and rat AKAP82. Further, the functional domains, namely the region involved in binding the regulatory subunit of PKA and the proteolytic cleavage site between pro-AKAP83 and AKAP83, were identical with that observed in rat and mouse pro-AKAP82 and AKAP82. Immunoblot analysis using polyclonal hamster anti-AKAP83 antibodies indicated that AKAP83 was present both in caput and cauda epididymidal spermatozoa. The antibody also identified the pro-AKAP82 and AKAP82 in mouse caput and cauda epididymidal spermatozoa. Immunofluorescence studies indicated that AKAP83 in hamster spermatozoa was localized along the length of principal piece of the tail. It was also demonstrated that hamster pro-AKAP83/AKAP83 gene expression was testis specific and was not expressed in other organs in either sex. This is the first report implicating AKAP in capacitation in rodents.