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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-.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel, mutations of which cause cystic fibrosis, a disease characterized by defective Cl − and HCO 3 − transport. Although >95% of all CF male patients are infertile because of congenital bilateral absence of the vas deferens (CBAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense debates. Here we report that CFTR is detected in both human and mouse sperm. CFTR inhibitor or antibody significantly reduces the sperm capacitation, and the associated HCO 3 − -dependent events, including increases in intracellular pH, cAMP production and membrane hyperpolarization. The fertilizing capacity of the sperm obtained from heterozygous CFTR mutant mice is also significantly lower compared with that of the wild-type. These results suggest that CFTR in sperm may be involved in the transport of HCO 3 − important for sperm capacitation and that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CBAVD.

We investigated whether GABA activates phospholipase A2 (PLA2) during acrosomal exocytosis, and if the MEK-ERK1/2 pathway modulates PLA2 activation initiated by GABA, progesterone or zona pellucida (ZP). In guinea pig spermatozoa prelabelled with [14C]arachidonic acid or [14C]choline chloride, GABA stimulated a decrease in phosphatidylcholine (PC), and release of arachidonic acid and lysoPC, during exocytosis. These lipid changes are indicative of PLA2 activation and appear essential for exocytosis since inclusion of aristolochic acid (a PLA2 inhibitor) abrogated them, along with exocytosis. GABA activation of PLA2 seems to be mediated, at least in part, by diacylglycerol (DAG) and protein kinase C since inclusion of the DAG kinase inhibitor R59022 enhanced PLA2 activity and exocytosis stimulated by GABA, whereas exposure to staurosporine decreased both. GABA-, progesterone- and ZP-induced release of arachidonic acid and exocytosis were prevented by U0126 and PD98059 (MEK inhibitors). Taken together, our results suggest that PLA2 plays a fundamental role in agonist-stimulated exocytosis and that MEK-ERK1/2 are involved in PLA2 regulation during this process.

Phospholipase A2 (PLA2) is activated in spermatozoa in response to progesterone and Ca 21 ionophores, but to our knowledge, no study has yet reported zona pellucida (ZP)-induced activation of PLA2. We investigated whether PLA2 is involved in ZP-stimulated acrosomal exocytosis, if Ca 21 is required for activation of PLA2, and signal transduction pathways modulating PLA2 using guinea pig sperm as a model. Spermatozoa were capacitated and labeled in low-Ca 21 medium with [ 14 C]choline chloride or [ 14 C]arachidonic acid and were then exposed to millimolar Ca 21 and various reagents and stimulated with ZP. Precapacitated spermatozoa exposed to millimolar Ca 21 and stimulated with ZP experienced increases in arachidonic acid (AA) and lysophosphatidylcholine (lysoPC) levels and a parallel decrease in phosphatidylcholine level; these changes are indicative of PLA2 activation. Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid, a PLA2 inhibitor, before treatment with ZP. Stimulation with ZP in medium without added Ca 21 or in medium with millimolar Ca 21 and EGTA or La

To investigate whether GABA/progesterone (P(4)) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa(2+) for 5.5 h and then labeled with [(32)P]pi for 1 h. Samples were washed through a three-step gradient Percoll, adjusted to 5x10(7) cells/mL and exposed to 2 mmol/L Ca(2+), 5 micromol/L GABA, 10 micromol/L P(4) and other agents. Lipids were separated by t.l.c. and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The results showed that (i) when spermatozoa were treated with GABA,(32)P-label diminished rapidly in phosphatidylinositol 4, 5-bisphosphate (PIP(2)), phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic acid (PA). The loss of label from PPI was almost completed by 10 min. The time-course of the AR was much slower than PPI when spermatozoa reached a maximal response by 15 min; (ii) the pattern of PPI hydrolysis and stimulation of AR was similar for the three agonists tested; their potency followed the order A23187progesteroneor =GABA; (iii) GABA-induced PIP(2) hydrolysis and rise in PA and the AR were prevented by inclusion of 10 mmol/L neomycin; (iv) the loss of PIP(2) labeling and the increase in PA labeling abolished when spermatozoa were exposed to EGTA or Ca(2+) channel blocker. These results indicate that GABA or P(4)-induced PPI breakdown is an important and essential event in the series of changes to membrane fusion during the AR of guinea pig spermatozoa and this effect is mediated via calcium by activation of phosphatidylinositol-specific phospholipase C.

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-.