After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. It is well established that capacitation requires Na⁺, HCO₃^-, Ca²⁺ and a cholesterol acceptor; however, little is known about the function of Cl^- during this important process. In this study, it is shown that Cl^- is essential for capacitation and that Cl^- act upstream of the cAMP/PKA signaling pathway. To investigate the Cl^- transporters involved, 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⁺, Clcotransporters (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.

Other key issue in sperm capacitation that remains unsolved despite many years of research is the possibility that transmembrane adenylyl cyclases (tmACs) have a role in sperm physiology. If tmACs were involved in the increase of cAMP in sperm, a role of G proteins in these responses would be plausible. Uncertainties exist about the activation of sperm adenylyl cyclase(s) by heterotrimeric G proteins. In this regard, there is some debate as to whether Gα _s is present in spermatozoa. In this work, Gα_s was detected on membranes purified from mouse sperm by [³² P] ADP-ribosylation with cholera toxin followed by immunoprecipitation with a specific antibody. In addition, immunofluorescence studies identified a specific signal in the sperm acrosomal region. Moreover, forskolin was able to induce acrosome reaction after incubating sperm in capacitating conditions and a higher accumulation of cAMP was observed after incubating sperm with forskolin. In this context, the clarification of the role of the ACs and G proteins will constitute a pivotal step for further investigation on sperm capacitation. Forskolin induces acrosome reaction only on capacitated sperm. These data suggest that a tmAC might be involved in the acrosome reaction and that capacitation is needed to couple this signal to other pathways related to the exocytic process.