Calcineurin-mediated Dephosphorylation of Synaptotagmin VI Is Necessary for Acrosomal Exocytosis

Abstract

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. In general, exocytosis is initiated by a cytosolic calcium increase. In this report we show that calcium affects several factors during human sperm acrosomal exocytosis. By using an antibody that specifically recognizes synaptotagmin VI phosphorylated at the polybasic region of the C2B domain, we showed that a calciumdependent dephosphorylation of this protein occurred at early stages of the acrosomal exocytosis in streptolysin O-permeabilized sperm. We identified the phosphatase as calcineurin and showed that the activity of this enzyme is absolutely required during the early steps of the secretory process. When added to sperm, an inhibitor-insensitive, catalytically active domain of calcineurin was able to rescue the effect of the specific calcineurin inhibitor cyclosporin A. This same domain dephosphorylated recombinant synaptotagmin VI C2B domain, validating this protein as a new substrate for calcineurin. When sperm were treated with catalytically active calcineurin before stimulation, exocytosis was inhibited, an effect that was rescued by the phosphomimetic synaptotagmin VI C2B-T418E,T419E mutant domain. These observations indicate that synaptotagmin must be dephosphorylated at a specific window of time and suggest that phosphorylated synaptotagmin has an active role at early stages of the acrosomal exocytosis.

K+ and Cl− Channels and Transporters in Sperm Function

“…Plasma membrane–outer acrosome hybrid vesicles are liberated as a result of the multiple fenestrations that occur in this irreversible reaction, which leads to acrosomal content release of hydrolytic enzymes. Not surprisingly, the fusion machinery conserved in many neuroendocrinal secretory cells and regulated by Ca 2+ is present in sperm and involved in the AR (Bello, Zanetti, Mayorga, & Michaut, 2012; Castillo Bennett, Roggero, Mancifesta, & Mayorga, 2010). At present, where the AR takes place in the female tract and the identity of the molecules inducing this exocytotic reaction is controversial (Jin et al, 2011; Visconti & Florman, 2010; Yanagimachi, 1998).…”

Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive

“…In the male reproductive system, animal experiments have revealed that CsA and FK506 cause deleterious effects on spermatogenesis and epididymal sperm maturation (3,4). Further, in vitro treatment of spermatozoa with these drugs impairs sperm motility and the acrosome reaction (5,6). These data suggest important roles of calcineurin in male fertility; however, the existence of several isoforms expressed in the testis hampered the clarification of their functions and pharmacological processes.…”

Participation of protein kinases and phosphatases in the progesterone‐induced acrosome reaction and calcium influx in human spermatozoa

“…There is virtually no information regarding the involvement of Ser/Thr phosphatases on the effects of P on spermatozoa. There is one report about the effect of PP2B inhibitors on the calcium‐triggered AR (Castillo Bennett et al ., ). Nothing has been reported in relation to calcium influx.…”