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.     

Signals of seminal vesicle autoantigen suppresses bovine serum albumin-induced capacitation in mouse sperm

Capacitation is the prerequisite process for sperm to gain the ability for successful fertilization. Unregulated capacitation will cause sperm to undergo a spontaneous acrosome reaction and then fail to fertilize an egg. Seminal plasma is thought to have the ability to suppress sperm capacitation. However, the mechanisms by which seminal proteins suppress capacitation have not been well understood. Recently, we demonstrated that a major seminal vesicle secretory protein, seminal vesicle autoantigen (SVA), is able to suppress bovine serum albumin (BSA)-induced mouse sperm capacitation. To further identify the mechanism of SVA action, we determine the molecular events associated with SVA suppression of BSA's activity. In this communication, we demonstrate that SVA suppresses the BSA-induced increase of intracellular calcium concentration ([Ca2+]i), intracellular pH (pH(i)), the cAMP level, PKA activity, protein tyrosine phosphorylation, and capacitation in mouse sperm. Besides, we also found that the suppression ability of SVA against BSA-induced protein tyrosine phosphorylation and capacitation could be reversed by dbcAMP (a cAMP agonist).     

A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis

Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating medium) yielded high rates of protein tyrosine phosphorylation. Either HCO3(-) or BSA was required to support these changes, with the combination of both providing the most intense results. When a membrane-permeable form of cAMP and a phosphodiesterase inhibitor (IBMX) were added to MW in the absence of HCO3(-) and BSA, the tyrosine phosphorylation results obtained in our capacitating conditions could not be replicated, suggesting either effects apart from cAMP were responsible for tyrosine phosphorylation, or that stallion sperm might respond differently to these reagents as compared to sperm from other mammals. Sperm incubation in capacitating conditions was also associated with high percentages (P相似文献   

Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation.

We previously demonstrated that mouse sperm capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca2+, and NaHCO(3), all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO-(4) to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO-(4) and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO-(3) is necessary for the capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO-(3) or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO-(3) effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm.     

Remodeling of the actin cytoskeleton during mammalian sperm capacitation and acrosome reaction

The sperm acrosome reaction and penetration of the egg follow zona pellucida binding only if the sperm has previously undergone the poorly understood maturation process known as capacitation. We demonstrate here that in vitro capacitation of bull, ram, mouse, and human sperm was accompanied by a time-dependent increase in actin polymerization. Induction of the acrosome reaction in capacitated cells initiated fast F-actin breakdown. Incubation of sperm in media lacking BSA or methyl-beta-cyclodextrin, Ca(2+), or NaHCO(3), components that are all required for capacitation, prevented actin polymerization as well as capacitation, as assessed by the ability of the cells to undergo the acrosome reaction. Inhibition of F-actin formation by cytochalasin D blocked sperm capacitation and reduced the in vitro fertilization rate of metaphase II-arrested mouse eggs. It has been suggested that protein tyrosine phosphorylation may represent an important regulatory pathway that is associated with sperm capacitation. We show here that factors known to stimulate sperm protein tyrosine phosphorylation (i.e., NaHCO(3), cAMP, epidermal growth factor, H(2)O(2), and sodium vanadate) were able to enhance actin polymerization, whereas inhibition of tyrosine kinases prevented F-actin formation. These data suggest that actin polymerization may represent an important regulatory pathway in with sperm capacitation, whereas F-actin breakdown occurs before the acrosome reaction.     

Identification of capacitation associated tyrosine phosphoproteins in buffalo (Bubalus bubalis) and cattle spermatozoa

At ejaculation mammalian sperm lack fertilizing ability as they are released in a functionally immature form. The capacity to fertilize eggs is only acquired after they have been educated in the female reproductive tract and this phenomenon is termed as capacitation. Sperm capacitation includes a cascade of biochemical modifications, including cholesterol efflux, Ca(2+) influx and cAMP/PKA-dependent/independent protein tyrosine phosphorylation which is specifically considered as the biochemical marker for capacitation. The identification of tyrosine phosphoproteins shall be useful in delineating their physiological role in different events associated with sperm capacitation. The present study was conducted to identify the tyrosine phosphoproteins in the capacitated buffalo and cattle spermatozoa using 2D immunoblotting and mass spectrometry. Among several proteins identified in the buffalo capacitated sperm, serine/threonine-protein phosphatase PP1-gamma catalytic subunit, MGC157332 protein, alpha-enolase, 3-oxoacid CoA transferase 2 and actin-like protein 7A were identified as new tyrosine phosphorylation substrates in mammalian spermatozoa. Cattle sperm also contain proteins such as serine/threonine-protein phosphatase PP1-alpha catalytic subunit and membrane metallo-endopeptidase-like 1 which have not been reported as tyrosine phosphorylated in any other species. Though the presence of serine/threonine-protein phosphatase PP1-alpha catalytic subunit was demonstrated for the first time in mammalian sperm, further studies are required for its existence and possible role in different sperm functions.     

Implication of cAMP during porcine sperm capacitation and protein tyrosine phosphorylation

Second messengers are involved in sperm fertilizing potential, as both motility and the acrosome reaction are influenced by cAMP. Moreover, the activity of cyclic nucleotides is implicated in the appearance of tyrosine phosphorylated sperm proteins, which is associated with capacitation in the mammalian spermatozoa. Nevertheless, the involvement of the cAMP/protein kinase A (PK-A) pathway during pig sperm capacitation may be different from that observed in other mammals. The objective of the present study was to clarify the cAMP/PK-A pathway during the capacitation of porcine spermatozoa and to evaluate this impact on the p32 sperm tyrosine phosphoprotein appearance. The presence of p32 was assessed after incubating fresh pig sperm with IBMX/db-cAMP, H-89, a PK-A inhibitor or bistyrphostin, a tyrosine kinase inhibitor, in capacitating (CM) or non-capacitating conditions (NCM) by immunoblotting SDS-extracted and separated sperm proteins using an anti-phosphotyrosine antibody. When pig spermatozoa were incubated in CM supplemented with H-89 (50 microM) or bistyrphostin (1.2 microM), capacitation decreased significantly (P < 0.001). The p32 sperm tyrosine phosphoprotein, previously shown to be associated with capacitation of porcine sperm though not necessarily an end point of this phenomenon, was not modulated by IBMX/db-cAMP (100 microM/1 mM), H-89 (50 microM) nor bistyrphostin (1.2 microM). Our results indicate, therefore, that pig sperm are regulated somewhat differently than as described for other mammals, because although the cAMP/PK-A and tyrosine kinase pathways are involved in capacitation, they do not influence the appearance of p32.     

Tyrosine phosphoproteome of hamster spermatozoa: Role of glycerol‐3‐phosphate dehydrogenase 2 in sperm capacitation

Capacitation confers on the spermatozoa the competence to fertilize the oocyte. At the molecular level, a cyclic adenosine monophosphate (cAMP) dependent protein tyrosine phosphorylation pathway operates in capacitated spermatozoa, thus resulting in tyrosine phosphorylation of specific proteins. Identification of these tyrosine‐phosphorylated proteins and their function with respect to hyperactivation and acrosome reaction, would unravel the molecular basis of capacitation. With this in view, 21 phosphotyrosine proteins have been identified in capacitated hamster spermatozoa out of which 11 did not identify with any known sperm protein. So, in the present study attempts have been made to ascertain the role of one of these eleven proteins namely glycerol‐3‐phosphate dehydrogenase 2 (GPD2) in hamster sperm capacitation. GPD2 is phosphorylated only in capacitated hamster spermatozoa and is noncanonically localized in the acrosome and principal piece in human, mouse, rat, and hamster spermatozoa, though in somatic cells it is localized in the mitochondria. This noncanonical localization may imply a role of GPD2 in acrosome reaction and hyperactivation. Further, enzymatic activity of GPD2 during capacitation correlates positively with hyperactivation and acrosome reaction thus demonstrating that GPD2 may be required for sperm capacitation.     

Effects of methyl-beta-cyclodextrin-mediated cholesterol depletion in porcine sperm compared to somatic cells

In this study, the use of methyl-beta-cyclodextrin (MBCD) to support capacitation of sperm cells was studied. Sperm were incubated with MBCD or alternatively capacitated in an in vitro fertilization medium. The effects of these incubations on phospholipid scrambling (using merocyanin), cholesterol depletion, GM-1 localization (using cholera-toxin B (CTX)), and membrane deterioration were assessed. For comparison, this was also tested in MBCD-treated MDCK cells. In MDCK cells, upto 71% of cholesterol was depleted, which coincided with a more diffuse CTX staining without any obvious effects on cell viability. In sperm, a similar depletion of 53% cholesterol was found after a 10 mM MBCD treatment. However, no merocyanin response was observed in viable sperm after MBCD treatments (indicating a lack of membrane changes associated with sperm capacitation). In contrast to MDCK, cells >1 mM MBCD caused plasma membrane disintegration and rendered sperm immotile. At higher concentrations also acrosome disruption was noted. CTX staining was absent at < 0.1 mM MBCD incubations but appeared at higher MBCD levels and was found to be specific for deteriorated cells that showed morphological signs of acrosome disruption. No significant plasma membrane deterioration, acrosome disruption, and sperm immotility nor CTX staining and only a modest (< 15%) cholesterol depletion were observed in conventionally capacitated sperm, where 40% of the intact sperm showed merocyanin staining. Taken together, the results indicate that membranes of sperm are more sensitive to MBCD-mediated cholesterol depletion than MDCK cells and that the use of MBCD to support sperm capacitation cannot be recommended due to its spermicidal effects.     

Mouse Uterine 24p3 Protein as a Suppressor of Sperm Acrosome Reaction

Under in vitro conditions, incubation with 0.3% bovine serum albumin (BSA) and 1.8 mM CaCl₂ induces mouse sperm capacitation and increases the consequential acrosome-reaction. The effect of mouse uterine 24p3 protein on such stimulated sperm has been investigated to understand the biological function of the 24p3 protein. Variations in the intracellular pH (pHi), calcium concentration, cAMP levels and tyrosine phosphorylation in cytosol were determined and on in vitro mouse fertilization was evaluated. The presence of 24p3 protein reduced the response of sperm to BSA and calcium by suppressing the elevation of intracellular pH, calcium uptake, cAMP accumulation and protein tyrosine phosphorylation of BSA/calcium-stimulated sperm and showed inhibitory effect on mouse in vitro fertilization. The results indicated the inhibition of the BSA-stimulated sperm acrosome reaction by 24p3 protein then suppressed sperm fertilization. We suggested that the 24p3 protein acts as an in vitro inhibitor of the acrosome reaction in BSA stimulated sperm and this might be an anti-fertilization factor in vitro.     

Semen-coagulating protein, SVS2, in mouse seminal plasma controls sperm fertility

Mammalian seminal plasma is known to contain a decapacitation factor(s) that prevents capacitation and thus, the fertility of sperm. This phenomenon has been observed in experiments conducted in vitro that assessed the inhibition of epididymal sperm fertility by seminal plasma or by the purified decapacitation factor. However, the phenomenon of decapacitation has not yet been characterized in vivo. In the present study, we demonstrate that seminal vesicle protein secretion 2 (SVS2), which is a 40-kDa basic protein and a major component of the copulatory plug, enters the uterus and interacts with ejaculated sperm heads after copulation. The SVS2-binding region of sperm changed from the postacrosomal region to the equatorial segment, while the sperm migrated through the uterus and finally disappeared in the oviduct. Furthermore, SVS2 reduced the fertility of epididymal sperm. The sperm treated with SVS2 decreased the percentage of fertilized oocytes from 60% to 10%. The capacitation state was assessed by protein tyrosine phosphorylation and the comprehensiveness of the acrosome reaction. SVS2 functioned to maintain sperm in the uncapacitated state and to reverse capacitated sperm to the uncapacitated state. We found that the fertility of ejaculated sperm is associated with SVS2 distribution in the female reproductive tract. These results indicate that SVS2 functions as a decapacitation factor for mouse sperm.     

Nitric oxide interacts with the cAMP pathway to modulate capacitation of human spermatozoa

This study aimed to demonstrate nitric oxide production by human spermatozoa and to characterize the interaction between nitric oxide and cAMP-related pathway in the control of human sperm capacitation and protein tyrosine phosphorylation. Spermatozoa were incubated in Tyrode's medium with or without bovine serum albumin (BSA), and nitric oxide was measured with the spin trap sodium N-methyl-D-glucamine dithiocarbamate. Under noncapacitating conditions, spermatozoa produced low levels of nitric oxide. However, under capacitating conditions, prominent nitric oxide adduct signals were obtained and a time-dependent increase of nitric oxide production was observed. When spermatozoa were incubated in Tyrode+BSA medium with nitric oxide-releasing compounds, intracellular cAMP concentrations increased to levels higher than those of spermatozoa incubated in Tyrode+BSA alone. In contrast, incubation with nitric oxide synthase inhibitors (N(G)-nitro-L-arginine methyl ester or N(G)-monomethyl L-arginine) decreased intracellular sperm cAMP concentrations. The inhibitory effect observed with N(G)-nitro-L-arginine methyl ester on capacitation and tyrosine phosphorylation of two sperm proteins (105, 81 kDa) was overcome by the presence of cAMP analogs or of a phosphodiesterase inhibitor. These results indicate that nitric oxide is produced by capacitating human spermatozoa and that it may act as a cellular messenger by modulating the cAMP pathway involved in capacitation and protein tyrosine phosphorylation.     

(S)-α-Chlorohydrin Inhibits Protein Tyrosine Phosphorylation through Blocking Cyclic AMP - Protein Kinase A Pathway in Spermatozoa

α-Chlorohydrin is a common contaminant in food. Its (S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 μM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5'-triphosphate (ATP) levels, 3'-5'-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH.     

Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP-dependent pathway

Changes of protein tyrosine phosphorylation in ejaculated boar sperm incubated in vitro were examined with the use of antiphosphotyrosine antibodies and immunoblotting. The intracellular levels of cAMP were modulated by treatment with various combinations of caffeine, 3-isobutyl-1-methylxanthine (IBMX), and dibutyryl cyclic AMP (dbcAMP), and acrosome reactions (ARs) were induced via treatment with divalent cation ionophore A23187. Proteins of M_r 34, 38, 40, and 44 (p34 . . . p44) were strongly phosphorylated on tyrosine residues in freshly prepared sperm samples and at the same level during all subsequent treatments. Incubation of sperm in vitro for various periods of time induced an increase of tyrosine phosphorylation of p20, p93, and p175. The tyrosine phosphorylation of p93, p175, and several other sperm proteins was up-regulated in a concentration-dependent manner following treatment of the sperm with dbcAMP, caffeine, or IBMX alone, or with combinations of caffeine and IBMX, respectively, with dbcAMP; the tyrosine phosphorylation of p20 was not correlated with treatment of sperm with cAMP-elevating reagents. The percentage of sperm cells undergoing spontaneous ARs was not affected by the manipulation of cAMP levels and was not correlated with protein tyrosine phosphorylation. In contrast, the addition of calcium to the incubation media decreased protein tyrosine phosphorylation and elevated percentage of spontaneous ARs. The induction of ARs with A23187 caused a significant decrease of tyrosine phosphorylation of p93, p175, and p220/230, indicating that dephosphorylation on protein tyrosine residues might be associated with calcium influx during physiological ARs as well. Proteins p93 and p175 were effectively solubilized in greater than 9M urea/1% triton and in SDS sample buffer, but to only a small extent in triton, while p20 was virtually completely extractable with triton. In conjunction with the previously reported isolation of active tyrosine kinase sp42 from triton extracts of noncapacitated boar sperm cells (Berruti and Porzio, 1992: Biochim Biophys Acta 1118:149–154), our results suggest that a cAMP-dependent event is required for tyrosine phosphorylation of triton-insoluble proteins such as p93 and p175. On the other hand, the tyrosine phosphorylation of p20 (and potentially other triton-soluble substrates) might not strictly require such cAMP up-regulation. We discuss the differences in the regulation of cAMP-dependent tyrosine phosphorylation in mouse, human, and boar sperm, and suggest that sensitivity to calcium and distinct basal levels of cyclic nucleotide PDE might correspond to species-specific reproduction strategies in mammals. Mol. Reprod. Dev. 51:304–314, 1998. © 1998 Wiley-Liss, Inc.     

Kinases, phosphatases and proteases during sperm capacitation

Fertilization is the process by which male and female haploid gametes (sperm and egg) unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential steps, including sperm migration through the female genital tract, sperm penetration through the cumulus mass, sperm adhesion and binding to the zona pellucida, acrosome exocytosis, sperm penetration through the zona and fusion of the sperm and egg plasma membranes. However, freshly ejaculated sperm are not capable of fertilizing an oocyte. They must first undergo a series of biochemical and physiological changes, collectively known as capacitation, before acquiring fertilizing capabilities. Several molecules are required for successful capacitation and in vitro fertilization; these include bicarbonate, serum albumin (normally bovine serum albumin, BSA) and Ca(2+). Bicarbonate activates the sperm protein soluble adenylyl cyclase (SACY), which results in increased levels of cAMP and cAMP-dependent protein kinase (PKA) activation. The response to bicarbonate is fast and cAMP levels increase within 60?s followed by an increase in PKA activity. Several studies with an anti-phospho-PKA substrate antibody have demonstrated a rapid increase in protein phosphorylation in human, mouse and boar sperm. The target proteins of PKA are not known and the precise role of BSA during capacitation is unclear. Most of the studies provide support for the idea that BSA acts by removing cholesterol from the sperm. The loss of cholesterol has been suggested to affect the bilayer of the sperm plasma membrane making it more fusogenic. The relationship between cholesterol loss and the activation of the cAMP/PKA pathway is also unclear. During early stages of capacitation, Ca(2+) might be involved in the stimulation of SACY, although definitive proof is lacking. Protein tyrosine phosphorylation is another landmark of capacitation but occurs during the late stages of capacitation on a different time-scale from cAMP/PKA activation. Additionally, the tyrosine kinases present in sperm are not well characterized. Although protein phosphorylation depends upon the balanced action of protein kinases and protein phosphatase, we have even less information regarding the role of protein phosphatases during sperm capacitation. Over the last few years, several reports have pointed out that the ubiquitin-proteasome system might play a role during sperm capacitation, acrosome reaction and/or sperm-egg fusion. In the present review, we summarize the information regarding the role of protein kinases, phosphatases and the proteasome during sperm capacitation. Where appropriate, we give examples of the way that these molecules interact and regulate each other's activities.     

Na+/K+ATPase regulates sperm capacitation through a mechanism involving kinases and redistribution of its testis‐specific isoform

Incubation of bovine sperm with ouabain, an endogenous cardiac glycoside that inhibits both the ubiquitous (ATP1A1) and testis‐specific α4 (ATP1A4) isoforms of Na⁺/K⁺ATPase, induces tyrosine phosphorylation and capacitation. The objectives of this study were to investigate: (1) fertilizing ability of bovine sperm capacitated by incubating with ouabain; (2) involvement of ATP1A4 in this process; and (3) signaling mechanisms involved in the regulation of sperm capacitation induced by inhibition of Na⁺/K⁺ATPase activity. Fresh sperm capacitated by incubating with ouabain (inhibits both ATP1A1 and ATP1A4) or with anti‐ATP1A4 immunoserum fertilized bovine oocytes in vitro. Capacitation was associated with relocalization of ATP1A4 from the entire sperm head to the post‐acrosomal region. To investigate signaling mechanisms involved in oubain‐induced regulation of sperm capacitation, sperm preparations were pre‐incubated with inhibitors of specific signaling molecules, followed by incubation with ouabain. The phosphotyrosine content of sperm preparations was determined by immunoblotting, and capacitation status of these sperm preparations were evaluated through an acrosome reaction assay. We inferred that Na⁺/K⁺ATPase was involved in the regulation of tyrosine phosphorylation in sperm proteins through receptor tyrosine kinase, nonreceptor type protein kinase, and protein kinases A and C. In conclusion, inhibition of Na⁺/K⁺ATPase induced tyrosine phosphorylation and capacitation through multiple signal transduction pathways, imparting fertilizing ability in bovine sperm. To our knowledge, this is the first report documenting both the involvement of ATP1A4 in the regulation of bovine sperm capacitation and that fresh bovine sperm capacitated by the inhibition of Na⁺/K⁺ATPase can fertilize oocytes in vitro. Mol. Reprod. Dev. 77: 136–148, 2010. © 2009 Wiley‐Liss, Inc.     

Regulation of tyrosine kinase activity during capacitation in goat sperm

Protein tyrosine phosphorylation is a key event accompanying sperm capacitation. Although this signaling cascade generates an array of tyrosine-phosphorylated polypeptides, their molecular characterization is still limited. It is necessary to differentiate the localization of the tyrosine-phosphorylated proteins in spermatozoa to understand the link between the different phosphorylated proteins and the corresponding regulated sperm function. cAMP plays a pivotal role in the regulation of tyrosine phosphorylation. The intracellular cAMP levels were raised in goat spermatozoa by the addition of the phosphodiesterase inhibitor, IBMX in conjugation with caffeine. Tyrosine phosphorylation was significantly up-regulated following treatment with these two reagents. Treatment of caudal spermatozoa with IBMX and caffeine, time dependent up-regulated phosphorylation of the protein of molecular weights 50 and 200 kDa was observed. Increased phosphorylation was observed with a combination of IBMX and caffeine treatment. Tyrosine phosphorylation in caput spermatozoa was not affected significantly under these conditions. The expression level of tyrosine kinase in sperm was examined with specific inhibitors and with anti-phosphotyrosine antibody. The indirect immunofluorescence staining was carried out on ethanol permeabilized sperm using anti-phosphotyrosine antibody. Western blot analysis was done using two separate PKA antibodies: anti-PKA catalytic and anti-PKA RIα. Almost no difference was found in the intracellular presence of the PKA RIα and RIIα subunits in caput and caudal epididymal spermatozoa. However, the catalytic subunit seemed to be present in higher amount in caudal spermatozoa. The results show that caprine sperm displays an enhancement of phosphorylation in the tyrosine residues of specific proteins under in vitro capacitation conditions.     

Baboon serum is superior to human or bovine serum albumin for baboon sperm capacitation and zona binding

Background  Baboon in vitro fertilization requires capacitated sperm in appropriate media. In this study, we compared the effect of baboon serum (Bas), human serum albumin (HSA) and bovine serum albumin (BSA) on baboon sperm capacitation.
Methods  Five males (n = 5) were electroejaculated and 43 oocytes retrieved from super-ovulated female baboons (n = 10). Each sperm sample was assessed for initial motility and concentration before and after swim-up. For swim-up, each sperm sample was incubated separately in Biggers–Whitten–Whittingham media containing either BaS, HSA, BSA or without protein supplementation (control). After swim-up, each sperm aliquot was incubated with two to three oocytes. The number of sperm bound to the zona was evaluated after overnight incubation.
Results  Sperm motility and zona binding was significantly higher after capacitation in media supplemented with BaS than in HSA or BSA or in media without protein supplementation ( P  < 0.05).
Conclusion  Baboon serum is superior to HSA or BSA for baboon sperm capacitation and zona binding.     

Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm

The effect of various capacitation inducers, i.e. heparin, superoxide anion, bicarbonate, adenosine, and caffeine, and their role in intracellular mechanisms involved in capacitation, were studied in cryopreserved bovine sperm. Capacitation was determined by epifluorescence chlortetracycline, protein tyrosine phosphorylation, and the ability of capacitated sperm to undergo an acrosome reaction and fertilize in vitro matured oocytes. Participation of membrane adenylate cyclase and protein kinases (protein kinase A, protein kinase C, and protein tyrosine kinase) was evaluated indirectly (with specific inhibitors). Involvement of reactive oxygen species (ROS) was determined with scavengers of superoxide anion, hydrogen peroxide, or nitric oxide. Percentages of capacitated (27-29%) and acrosome-reacted sperm (23-26%) did not differ (P > 0.05) among various capacitation inducers. Significantly higher rates of IVF were obtained with heparin (43%) or bicarbonate plus caffeine (45%), when compared with control samples (17%). Adding the membrane adenylate cyclase inhibitor diminished capacitation rates with heparin (8%) or adenosine (10%). There was differential protein kinase participation in response to inducers; protein kinase inhibitors diminished cleavage rates in heparin-capacitated sperm relative to controls. There were differences between and within the studied inducers in protein tyrosine phosphorylation patterns. We inferred that capacitation in cryopreserved bovine sperm was promoted through diverse pathways. Mechanisms triggered by heparin, or caffeine plus bicarbonate-induced capacitation, involved activation of intracellular pathways to optimize fertilizing capability of cryopreserved bovine sperm.     

Sperm capacitation in vitro in the eld's deer

Sperm capacitation was examined in the endangered Eld's deer (Cervus eldi thamin). Sperm motility and viability (percentage of sperm cells with intact membranes) were assessed in vitro over time after attempting to induce capacitation in TALP alone and TALP supplemented with calcium (10 mM CaCl2), dibutyryl cAMP (1 mM dbcAMP), or fetal calf serum (20% FCS). Sperm aliquots were evaluated at 0, 3, 6, 9, and 12 h for motility, viability, and ability to acrosome react after exposure to calcium ionophore (A23187, CI; 10 microM) or lysophosphatidylcholine (LC; 100 microg/mL). Fresh sperm aliquots in TALP + 10 mM CaCl2 exposed to CI had fewer (P < 0.05) intact acrosomes than the TALP control (TALP alone) or dbcAMP and FCS treatments after 9 h. Mean (+/- SEM) percentage of intact acrosomes of spermatozoa incubated in medium with increased CaCl2 declined (P < 0.05) from 80.2 +/- 2.6% (0 h) to 49.7 +/- 7.3% after prolonged incubation (9 h). The proportion of capacitated fresh spermatozoa was not influenced by LC treatment. Capacitation was not induced (P > 0.05) by any of the presumptive sperm capacitators after freeze-thawing. Likewise, neither CI nor LC induced the acrosome reaction (AR) in these spermatozoa, suggesting that the freeze-thawing process may have caused membrane damage. Results revealed that the supplementation of medium with CaCl2 evokes capacitation in some spermatozoa. However, Eld's deer spermatozoa appear remarkably resistant to conventional stimulators of capacitation and the AR.