Calpain‐1 and Cholesterol Regulate the Dynamics of the Lipid Membrane Domains Essential for Suitable Capacitation and Acrosomal Reactions

Calpain-1 and -2 are calcium-dependent cysteine proteases associated with sperm processes, such as capacitation, the acrosomal reaction, and motility, making them important for the acquisition of fertilizing capacity among spermatozoa. Moreover, their inhibition significantly reduces in vitro fertilization. Guinea pig spermatozoa express only calpain-1, which has been implicated in spectrin cytoskeletal remodeling and NOX2 and NOX4 activation during capacitation. However, little is known about the mechanisms by which calpains participate in capacitation and the acrosomal reaction. The interaction of spectrin with phospholipids maintains the asymmetry of the plasma membrane. Since spectrin is a significant target of calpain, calpain may be involved in the dynamics of phospholipids and other membrane lipids during the capacitation process. Therefore, this work aims to elucidate the role of calpain in capacitation and acrosomal reactions, as well as its relationship with the dynamics of different membrane lipids related to capacitation and the acrosomal reaction. The results show that calpain-1 inhibition by calpeptin significantly reduced the number of spermatozoa that underwent capacitation and acrosomal reactions. Inhibition of calpain-1 also blocked protein phosphorylation at Tyr, as well as calcium influx and actin polymerization, which required for successful capacitation. Calpain inhibition also prevented phosphatidylserine translocation and the dynamics of caveolin-1, both processes associated with capacitation and the acrosome reaction. Sperm capacitation in the presence of cholesterol prevented the dynamics of phosphatidylserine, GM1 and caveolin-1. However, calpain-1 inhibition did not prevent cholesterol or GM1 ganglioside dynamics. The results of this investigation provide strong evidence for the mechanisms by which calpain-1 regulates capacitation and the acrosome reaction in guinea pig spermatozoa, suggesting that calpain-1 is a key player in optimal capacitation and the acrosome reaction.