Chloride Is essential for capacitation and for the capacitation-associated increase in tyrosine phosphorylation

After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na(+), HCO(3)(-), Ca(2+), and a cholesterol acceptor; however, little is known about the function of Cl(-) during this important process. To determine whether Cl(-), in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl(-) was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl(-) did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl(-)-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl(-) acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl(-) transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na(+)/K(+)/Cl(-) cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl(-) movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm.     

Bicarbonate is required for migration of sperm epididymal protein DE (CRISP-1) to the equatorial segment and expression of rat sperm fusion ability

Numerous studies have demonstrated that sperm capacitation is a bicarbonate-dependent process. In the rat, capacitation has not been studied as much as in other species, mainly because of the difficulties in carrying out functional assays with this animal model. In the present study, we have examined the influence of bicarbonate in the overall rat sperm capacitation process by analyzing involvement of the anion in 1) protein tyrosine phosphorylation, 2) migration of epididymal protein DE (also known as CRISP-1) from the dorsal region to the equatorial segment of the sperm head that occurs during capacitation, and 3) ability of sperm to fuse with the egg. Incubation of sperm under capacitating conditions produced a time-dependent increase in protein tyrosine phosphorylation. This phosphorylation did not occur in the absence of HCO3- and rapidly increased by either exposure of sperm to HCO3- or replacement of the anion by a cAMP analog (dibutyryl-cAMP) and a phosphodiesterase inhibitor (pentoxifylline). The absence of HCO3- also produced a significant decrease in the percentage of cells showing migration of DE to the equatorial segment. This parameter was completely restored by addition of the anion, but dibutyryl-cAMP and pentoxifylline were not sufficient to overcome the decrease in DE migration. Sperm capacitated in the absence of HCO3- were unable to penetrate zona-free eggs independent of the presence of the anion during gamete coincubation. Exposure of these sperm to bicarbonate, or replacement of the anion by dibutyryl-cAMP and pentoxifylline, only partially restored the sperm fusion ability. Altogether, these results indicate that, in addition to its influence on protein tyrosine phosphorylation, bicarbonate is required to support other rat sperm capacitation- associated events, such as migration of DE to the equatorial segment, and expression of the ability of sperm to fuse with the egg.     

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.     

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.     

Src Kinase Is the Connecting Player between Protein Kinase A (PKA) Activation and Hyperpolarization through SLO3 Potassium Channel Regulation in Mouse Sperm

Plasma membrane hyperpolarization is crucial for mammalian sperm to acquire acrosomal responsiveness during capacitation. Among the signaling events leading to mammalian sperm capacitation, the immediate activation of protein kinase A plays a pivotal role, promoting the subsequent stimulation of protein tyrosine phosphorylation that associates with fertilizing capacity. We have shown previously that mice deficient in the tyrosine kinase cSrc are infertile and exhibit improper cauda epididymis development. It is therefore not clear whether lack of sperm functionality is due to problems in epididymal maturation or to the absence of cSrc in sperm. To further address this problem, we investigated the kinetics of cSrc activation using anti-Tyr(P)-416-cSrc antibodies that only recognize active cSrc. Our results provide evidence that cSrc is activated downstream of PKA and that inhibition of its activity blocks the capacitation-induced hyperpolarization of the sperm plasma membrane without blocking the increase in tyrosine phosphorylation that accompanies capacitation. In addition, we show that cSrc inhibition also blocks the agonist-induced acrosome reaction and that this inhibition is overcome by pharmacological hyperpolarization. Considering that capacitation-induced hyperpolarization is mediated by SLO3, we evaluated the action of cSrc inhibitors on the heterologously expressed SLO3 channel. Our results indicate that, similar to SLO1 K⁺ channels, cSrc blockers significantly decreased SLO3-mediated currents. Together, these results are consistent with findings showing that hyperpolarization of the sperm plasma membrane is necessary and sufficient to prepare the sperm for the acrosome reaction and suggest that changes in sperm membrane potential are mediated by cSrc activation.     

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.     

Na+/K+ATPase as a signaling molecule during bovine sperm capacitation

A heteromeric integral membrane protein, Na+/K+ATPase is composed of two polypeptides, alpha and beta, and is active in many cell types, including testis and spermatozoa. It is a well-known ion transporter, but binding of ouabain, a specific inhibitor of Na+/K+ATPase, to Na+/K+ATPase in somatic cells initiates responses that are similar to signaling events associated with bovine sperm capacitation. The objectives of the present study were to demonstrate the presence of Na+/K+ATPase in bovine sperm and to investigate its role in the regulation of bovine sperm capacitation. The presence of Na+/K+ATPase in sperm from mature Holstein bulls was demonstrated by immunoblotting and immunocytochemistry using a monoclonal antibody developed in mouse against the beta 1 polypeptide of Na+/K+ATPase. Binding of ouabain to Na+/K+ATPase inhibited motility (decreased progressive motility, average path velocity, and curvilinear velocity) and induced tyrosine phosphorylation and capacitation but did not increase intracellular calcium levels in spermatozoa. Furthermore, binding of ouabain to Na+/K+ATPase induced depolarization of sperm plasma membrane. Therefore, binding of ouabain to Na+/K+ATPase induced sperm capacitation through depolarization of sperm plasma membrane and signaling via the tyrosine phosphorylation pathway without an appreciable increase in intracellular calcium. To our knowledge, this is the first report concerning the signaling role of Na+/K+ATPase in mammalian sperm capacitation.     

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   

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.     

Participation of the Cl-/HCO(3)- exchangers SLC26A3 and SLC26A6, the Cl- channel CFTR, and the regulatory factor SLC9A3R1 in mouse sperm capacitation

Sperm capacitation is required for fertilization and involves several ion permeability changes. Although Cl(-) and HCO(3)(-) are essential for capacitation, the molecular entities responsible for their transport are not fully known. During mouse sperm capacitation, the intracellular concentration of Cl(-) ([Cl(-)](i)) increases and membrane potential (Em) hyperpolarizes. As in noncapacitated sperm, the Cl(-) equilibrium potential appears to be close to the cell resting Em, opening of Cl(-) channels could not support the [Cl(-)](i) increase observed during capacitation. Alternatively, the [Cl(-)](i) increase might be mediated by anion exchangers. Among them, SLC26A3 and SLC26A6 are good candidates, since, in several cell types, they increase [Cl(-)](i) and interact with cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel present in mouse and human sperm. This interaction is known to be mediated and probably regulated by the Na(+)/H(+) regulatory factor-1 (official symbol, SLC9A3R1). Our RT-PCR, immunocytochemistry, Western blot, and immunoprecipitation data indicate that SLC26A3, SLC26A6, and SLC9A3R1 are expressed in mouse sperm, localize to the midpiece, and interact between each other and with CFTR. Moreover, we present evidence indicating that CFTR and SLC26A3 are involved in the [Cl(-)](i) increase induced by db-cAMP in noncapacitated sperm. Furthermore, we found that inhibitors of SLC26A3 (Tenidap and 5099) interfere with the Em changes that accompany capacitation. Together, these findings indicate that a CFTR/SLC26A3 functional interaction is important for mouse sperm capacitation.     

Sperm from beta1,4-galactosyltransferase I-null mice exhibit precocious capacitation

Mammalian sperm must undergo a physiological maturation, termed capacitation, before they are able to fertilize eggs. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. In this paper, we describe the capacitation phenotype of sperm lacking the long isoform of beta1,4-galactosyltransferase I (GalT I), a sperm surface protein that functions as a receptor for the zona pellucida glycoprotein, ZP3, and as an inducer of the acrosome reaction following ZP3-dependent aggregation. As expected, wild-type sperm must undergo capacitation in order to bind the zona pellucida and undergo a Ca(2+) ionophore-induced acrosome reaction. By contrast, GalT I-null sperm behave as though they are precociously capacitated, in that they demonstrate maximal binding to the zona pellucida and greatly increased sensitivity to ionophore-induced acrosome reactions without undergoing capacitation in vitro. The loss of GalT I from sperm results in an inability to bind epididymal glycoconjugates that normally maintain sperm in an 'uncapacitated' state; removing these decapacitating factors from wild-type sperm phenocopies the capacitation behavior of GalT I-null sperm. Interestingly, capacitation of GalT I-null sperm is independent of the presence of albumin, Ca(2+) and HCO(3)(-); three co-factors normally required by wild-type sperm to achieve capacitation. This implies that intracellular targets of albumin, Ca(2+) and/or HCO(3)(-) may be constitutively active in GalT I-null sperm. Consistent with this, GalT I-null sperm have increased levels of cAMP that correlate closely with both the accelerated kinetics and co-factor-independence of GalT I-null sperm capacitation. By contrast, the kinetics of protein tyrosine phosphorylation and sperm motility are unaltered in mutant sperm relative to wild-type. These data suggest that GalT I may function as a negative regulator of capacitation in the sperm head by suppressing intracellular signaling pathways that promote this process.     

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).     

Inwardly rectifying K(+) channels in spermatogenic cells: functional expression and implication in sperm capacitation

To fertilize, mammalian sperm must complete a maturational process called capacitation. It is thought that the membrane potential of sperm hyperpolarizes during capacitation, possibly due to the opening of K(+) channels, but electrophysiological evidence is lacking. In this report, using patch-clamp recordings obtained from isolated mouse spermatogenic cells we document the presence of a novel K(+)-selective inwardly rectifying current. Macroscopic current activated at membrane potentials below the equilibrium potential for K(+) and its magnitude was dependent on the external K(+) concentration. The channels selected K(+) over other monovalent cations. Current was virtually absent when external K(+) was replaced with Na(+) or N-methyl-D-glucamine. Addition of Cs(+) or Ba(2+) (IC(50) of approximately 15 microM) to the external solution effectively blocked K(+) current. Dialyzing the cells with a Mg(2+)-free solution did not affect channel activity. Cytosolic acidification reversibly inhibited the current. We verified that the resting membrane potential of mouse sperm changed from -52 +/- 6 to -66 +/- 9 mV during capacitation in vitro. Notably, application of 0.3-1 mM Ba(2+) during capacitation prevented this hyperpolarization and decreased the subsequent exocytotic response to zona pellucida. A mechanism is proposed whereby opening of inwardly rectifying K(+) channels may produce hyperpolarization under physiological conditions and contribute to the cellular changes that give rise to the capacitated state in mature sperm.     

Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter

Regulation of intra- and extracellular ion activities (e.g. H(+), Cl(-), Na(+)) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na(+)/HCO(3)(-) cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO(3)(-) transporters are known: a neutral Na(+)/ HCO(3)(-) cotransporter, a K(+)/ HCO(3)(-) cotransporter, and a Na(+)-dependent Cl(-)-HCO(3)(-) exchanger. We report the cloning and characterization of a Na(+)-coupled Cl(-)-HCO(3)(-) exchanger and a physiologically unique bicarbonate transporter superfamily member. This Drosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughout Drosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl(-), Na(+), H(+), and HCO(3)(-) but does not require HCO(3)(-). Transport is blocked by the stilbene 4,4'-diisothiocyanodihydrostilbene- 2, 2'-disulfonates and may not be strictly electroneutral. Our functional data suggest this Na(+) driven anion exchanger (NDAE1) is responsible for the Na(+)-dependent Cl(-)-HCO(3)(-) exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva.     

Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.     

Cl- is required for HCO3- entry necessary for sperm capacitation in guinea pig: involvement of a Cl-/HCO3- exchanger (SLC26A3) and CFTR

Our previous study demonstrated the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in transporting bicarbonate that is necessary for sperm capacitation; however, whether its involvement is direct or indirect remains unclear. The present study investigated the possibility of a Cl-/HCO3- exchanger (solute carrier family 26, number 3 [SLC26A3]) operating with CFTR during guinea pig sperm capacitation. Incubating sperm in media with various concentrations of Cl- resulted in varied percentages of capacitated sperm in a concentration-dependent manner. Depletion of Cl-, even in the presence of HCO3-, abolished sperm capacitation and vice versa, indicating the involvement of both anions in the process. Capacitation-associated HCO3--dependent events, including increased intracellular pH, cAMP production, and protein tyrosine phosphorylation, also depend on Cl- concentrations. Similar Cl- dependence and inhibitor sensitivity were observed for sperm-hyperactivated motility and for sperm-egg fusion. The expression and localization of CFTR and SLC26A3 were demonstrated using immunostaining and Western blot analysis. Taken together, our results indicate that Cl- is required for the entry of HCO3- that is necessary for sperm capacitation, implicating the involvement of SLC26A3 in transporting HCO3-, with CFTR providing the recycling pathway for Cl-.     

Muscarinic activation of Na+-dependent ion transporters and modulation by bicarbonate in rat submandibular gland acinus

To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, we measured the membrane voltage (V(m)) and intracellular concentrations of Ca(2+), Na(+) ([Na(+)](c)), Cl(-), and H(+) (pH(i)) in rat submandibular gland acini (RSMGA). After a transient depolarization induced by a short application of acetylcholine (ACh; 5 muM, 20 s), RSMGA showed strong delayed hyperpolarization (V(h,ACh); -95 +/- 1.8 mV) that was abolished by ouabain. In the HCO(3)(-)-free condition, the V(h,ACh) was also blocked by bumetanide, a blocker of Na(+)-K(+)-2Cl(-) cotransporter (NKCC). In the presence of HCO(3)(-) (24 meq, bubbled with 5% CO(2)), however, the V(h,ACh) was not blocked by bumetanide, but it was suppressed by ethylisopropylamiloride (EIPA), a Na(+)/H(+) exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [Na(+)](c) was totally blocked by bumetanide in the absence of HCO(3)(-), but only by one-half in the presence of HCO(3)(-). ACh induced a prominent acidification of pH(i) in the presence of HCO(3)(-), and the acidification was further increased by EIPA treatment. Without HCO(3)(-), an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pH(i) during the application of NH(4)(+) (20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO(3)(-). In summary, the ACh-induced anion secretion in RSMGA is followed by the activation of NKCC and NHE, resulting an increase in [Na(+)](c). The intracellular Na(+)-induced activation of electrogenic Na(+)/K(+)-ATPase causes V(h,ACh). The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO(3)(-).     

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.