The SLO3 sperm-specific potassium channel plays a vital role in male fertility

Here we show a unique example of male infertility conferred by a gene knockout of the sperm-specific, pH-dependent SLO3 potassium channel. In striking contrast to wild-type sperm which undergo membrane hyperpolarization during capacitation, we found that SLO3 mutant sperm undergo membrane depolarization. Several defects in SLO3 mutant sperm are evident under capacitating conditions, including impaired motility, a bent “hairpin” shape, and failure to undergo the acrosome reaction (AR). The failure of AR is rescued by valinomycin which hyperpolarizes mutant sperm. Thus SLO3 is the principal potassium channel responsible for capacitation-induced hyperpolarization, and membrane hyperpolarization is crucial to the AR.     

Involvement of a Na+/HCO-3 cotransporter in mouse sperm capacitation

Mammalian sperm are incapable of fertilizing eggs immediately after ejaculation; they acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes sperm undergo in the female reproductive tract that render sperm able to fertilize constitute the phenomenon of "sperm capacitation." We have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins and that these events are regulated by an HCO(3)(-)/cAMP-dependent pathway involving protein kinase A. Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. Here we present evidence that, in addition to its role in the regulation of adenylyl cyclase, HCO(3)(-) has a role in the regulation of plasma membrane potential in mouse sperm. Addition of HCO(3)(-) but not Cl(-) induces a hyperpolarizing current in mouse sperm plasma membranes. This HCO(3)(-)-dependent hyperpolarization was not observed when Na(+) was replaced by the non-permeant cation choline(+). Replacement of Na(+) by choline(+) also inhibited the capacitation-associated increase in protein tyrosine phosphorylation as well as the zona pellucida-induced acrosome reaction. The lack of an increase in protein tyrosine phosphorylation was overcome by the presence of cAMP agonists in the incubation medium. The lack of a hyperpolarizing HCO(3)(-) current and the inhibition of the capacitation-dependent increase in protein tyrosine phosphorylation in the absence of Na(+) suggest that a Na(+)/HCO(3)(-) cotransporter is present in mouse sperm and is coupled to events regulating capacitation.     

Capacitation induces tyrosine phosphorylation of proteins in the boar sperm plasma membrane.

Capacitation (activation) of mammalian spermatozoa is accompanied by protein phosphorylation, elevation of the intracellular calcium concentration and an increased plasma membrane fluidity. The subcellular localization of tyrosine phosphorylation during capacitation have not yet been elucidated. The aim of this study was to investigate whether boar sperm capacitation induces tyrosine phosphorylation of plasma membrane proteins. Capacitation induced tyrosine phosphorylation of 3 proteins (27, 37, and 40 kDa), which coincided with an increase in the plasma membrane fluidity. The importance of the induced tyrosine phosphorylation in sperm binding to the zona pellucida and the induction of the acrosome reaction is discussed.     

cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.     

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.     

Inhibition of capacitation-associated tyrosine phosphorylation signaling in rat sperm by epididymal protein Crisp-1

Ejaculated sperm are unable to fertilize an egg until they undergo capacitation. Capacitation results in the acquisition of hyperactivated motility, changes in the properties of the plasma membrane, including changes in proteins and glycoproteins, and acquisition of the ability to undergo the acrosome reaction. In all mammalian species examined, capacitation requires removal of cholesterol from the plasma membrane and the presence of extracellular Ca2+ and HCO3-. We designed experiments to elucidate the conditions required for in vitro capacitation of rat spermatozoa and the effects of Crisp-1, an epididymal secretory protein, on capacitation. Protein tyrosine phosphorylation, a hallmark of capacitation in sperm of other species, occurs during 5 h of in vitro incubation, and this phosphorylation is dependent upon HCO3-, Ca2+, and the removal of cholesterol from the membrane. Crisp-1, which is added to the sperm surface in the epididymis in vivo, is lost during capacitation, and addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner, thus inhibiting capacitation and ultimately the acrosome reaction. Inhibition of capacitation by Crisp-1 occurs upstream of the production of cAMP by the sperm.     

Ion Permeabilities in Mouse Sperm Reveal an External Trigger for SLO3-Dependent Hyperpolarization

Unlike most cells of the body which function in an ionic environment controlled within narrow limits, spermatozoa must function in a less controlled external environment. In order to better understand how sperm control their membrane potential in different ionic conditions, we measured mouse sperm membrane potentials under a variety of conditions and at different external K⁺ concentrations, both before and after capacitation. Experiments were undertaken using both wild-type, and mutant mouse sperm from the knock-out strain of the sperm-specific, pH-sensitive, SLO3 K⁺ channel. Membrane voltage data were fit to the Goldman-Hodgkin-Katz equation. Our study revealed a significant membrane permeability to both K⁺ and Cl⁻ before capacitation, as well as Na⁺. The permeability to both K⁺ and Cl⁻ has the effect of preventing large changes in membrane potential when the extracellular concentration of either ion is changed. Such a mechanism may protect against undesired shifts in membrane potential in changing ionic environments. We found that a significant portion of resting membrane potassium permeability in wild-type sperm was contributed by SLO3 K⁺ channels. We also found that further activation of SLO3 channels was the essential mechanism producing membrane hyperpolarization under two separate conditions, 1) elevation of external pH prior to capacitation and 2) capacitating conditions. Both conditions produced a significant membrane hyperpolarization in wild-type which was absent in SLO3 mutant sperm. Hyperpolarization in both conditions may result from activation of SLO3 channels by raising intracellular pH; however, demonstrating that SLO3-dependent hyperpolarization is achieved by an alkaline environment alone shows that SLO3 channel activation might occur independently of other events associated with capacitation. For example sperm may undergo stages of membrane hyperpolarization when reaching alkaline regions of the female genital tract. Significantly, other events associated with sperm capacitation, occur in SLO3 mutant sperm and thus proceed independently of hyperpolarization.     

Capacitation is associated with tyrosine phosphorylation and tyrosine kinase-like activity of pig sperm proteins

Capacitation represents the final maturational steps that render mammalian sperm competent to fertilize, either in vivo or in vitro. Capacitation is defined as a series of events that enables sperm to bind the oocyte and undergo the acrosome reaction in response to the zona pellucida. Although the molecular mechanisms involved are not fully understood, sperm protein phosphorylation is associated with capacitation. The hypothesis of this study is that protein tyrosine phosphorylation and kinase activity mediate capacitation of porcine sperm. Fresh sperm were incubated in noncapacitating or capacitating media for various times. Proteins were extracted with SDS, subjected to SDS-PAGE, and immunoblotted with an antiphosphotyrosine antibody. An M(r) 32 000 tyrosine-phosphorylated protein (designated as p32) appeared only when the sperm were incubated in capacitating medium and concomitant with capacitation as assessed by the ionophore-induced acrosome reaction. The p32 was soluble in Triton X-100. Fractionation of sperm proteins with Triton X-114 demonstrated that after capacitation, this tyrosine phosphoprotein is located in both the cytosol and the membrane. Enzyme renaturation of sperm proteins was conducted in gels with or without either poly glu:tyr (a tyrosine kinase substrate) or kemptide (a protein kinase A substrate). An M(r) 32 000 enzyme with kinase behavior was observed in all gels but was preferentially phosphorylated on tyrosine, as assessed by phosphorimagery and by thin layer chromotography to identify the phosphoamino acids. Indirect immunolocalization showed that the phosphotyrosine residues redistribute to the acrosome during capacitation, which is an appropriate location for a protein involved in the acquisition of fertility.     

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.     

Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa.

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to produce fertilization of the oocyte in vivo and in vitro. Although this process results from a poorly understood series of morphological and molecular events, protein tyrosine phosphorylation has been associated with sperm capacitation in several mammalian species, but it still remains to be demonstrated in ram spermatozoa. Studies of capacitation in ram spermatozoa are of great interest, since several reports have suggested that the reduced fertility of cryopreserved spermatozoa is due to their premature capacitation. In this work, we report for the first time, to our knowledge, that tyrosine phosphorylation of ram sperm membrane proteins is related to the capacitation state of these cells. Capacitation induced tyrosine phosphorylation of some plasma membrane proteins of ram spermatozoa freed from seminal plasma by a dextran/swim-up procedure. It has also been proved that cold-shock induces protein tyrosine phosphorylation as well as a decrease in plasma membrane integrity. Addition of seminal plasma proteins prior to cold-shock not only improved sperm survival but also promoted a decrease in protein tyrosine phosphorylation.     

Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction

Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pH_i) and Ca²⁺ ([Ca²⁺]_i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K⁺ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K⁺ or by addition of solubilized zona pellucida (sZP). Moreover, K⁺ and sZP were also able to increase [Ca²⁺]_i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K⁺ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca²⁺]_i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction.     

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.     

Seminal CD38 Enhances Human Sperm Capacitation through Its Interaction with CD31

Human sperm have to undergo a maturational process called capacitation in the female reproductive tract. Capacitation confers upon the sperm an ability to gain hypermotility and undergo acrosome reaction. Previous studies have suggested that seminal plasma proteins induce the capacitation of sperm in the female reproductive tract for the successful fertilization of the oocyte. However, the function of seminal plasma proteins in capacitation remains largely unclear. To the end, we found that soluble CD38 (sCD38) in seminal plasma increases the capacitation of sperm via specific interactions between sCD38 and the CD31 on the sperm. Upon the association of sCD38 with CD31, tyrosine kinase Src phosphorylates CD31, a process blocked by Src inhibitors. Shc, SHP-2, Grb2, and SOS, as well as Src kinase were found to associate with the phosphorylated CD31. The sCD38-induced phosphorylation of CD31 initiates a cascade reaction through the phosphorylation of Erk1/2, which results in the acrosome reaction, and sperm hypermotility. These processes were prevented by Src, Ras and MEK inhibitors. Taken together, these data indicate that the sCD38 present in seminal plasma plays a critical role in the capacitation of sperm.     

Suppression effect of seminal vesicle autoantigen on platelet-activating factor-induced mouse sperm capacitation

Mammalian sperm gain the ability to fertilize an egg successfully by the capacitation process. An unregulated capacitation process causes sperm to undergo a spontaneous acrosome reaction (AR) and resulting in loss of their fertilization activity. Thus, functional sperm activation is tightly regulated by a capacitation and suppression (decapacitation) mechanism. Factors, such as platelet-activating factor (PAF) present in both sperm and the female genital tract, are able to stimulate sperm capacitation. Seminal plasma is thought to have the ability to suppress sperm capacitation; however, the regulatory mechanisms of seminal plasma protein on sperm capacitation are not well understood. Recently, we demonstrated that seminal vesicle autoantigen (SVA), a major seminal vesicle secretory protein, is able to suppress mouse sperm capacitation. To further study the suppression spectra of SVA on sperm capacitation, we investigated the effect of SVA on PAF-induced mouse sperm capacitation-related signals. Here, we demonstrate that SVA decreases the [Ca(2+)](i) to suppress the PAF's effects on [Ca(2+)](i), the cAMP level, protein tyrosine phosphorylation, and capacitation. The inhibition of PAF-induced protein tyrosine phosphorylation and capacitation by SVA can be reversed by cAMP agonists. Characterization of the interactions of SVA with PAF by TLC overlay and tryptophan fluorescence spectrum analyses indicates that SVA is capable of binding PAF with an apparent dissociation constant K(d) > 50 microM. Together with these results, we demonstrate that SVA deceases [Ca(2+)](i) and cross-talks with PAF-induced intracellular signals to regulate mouse sperm capacitation.     

Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.     

Signaling pathways in sperm capacitation and acrosome reaction.

The binding to the egg's zona pellucida stimulates the spermatozoon to undergo acrosome reaction, a process which enables the sperm to penetrate the egg. Prior to this binding, the spermatozoa underago in the female reproductive tract a series of biochemical transformations, collectively called capacitation. The first event in capacitation is cholesterol efflux leading to the elevation of intracellular calcium and bicarbonate to activate adenylyl cyclase (AC) to produce cyclic-AMP, which activates protein kinase A (PKA) to indirectly phosphorylate certain proteins on tyrosine. During capacitation, there is also an increase in protein tyrosine phosphorylation dependent actin polymerization and in the membrane-bound phospholipase C (PLC). Sperm binding to zona-pellucida causes further activation of cAMP/PKA and protein kinase C (PKC), respectively. PKC opens a calcium channel in the plasma membrane. PKA together with inositol-trisphosphate activate calcium channels in the outer acrosomal membrane, which leads to an increase in cytosolic calcium. The depletion of calcium in the acrosome will activate a store-operated calcium entry mechanism in the plasma membrane, leading to a higher increase in cytosolic calcium, resulting in F-actin dispersion which enable the outer acrosomal and the plasma membrane to come into contact and fuse completing the acrosomal reaction.     

Iberiotoxin and clofilium regulate hyperactivation,acrosome reaction,and ion homeostasis synergistically during human sperm capacitation

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K⁺ currents in human sperm (I_KSper) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca²⁺, K⁺, Cl⁻, and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K⁺]_i, [Cl⁻]_i, and pH_i, but a decrease in [Ca²⁺]_i. Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K⁺]_i, [Cl⁻]_i, and pH_i, and the decrease in [Ca²⁺]_i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.