Cdc42 is essential for CatSper function in the principal piece of mouse sperm flagellum

ROMAROWSKI A; DALOTTO-MORENO T; DE LA VEGA-BELTRAN JL; SCHIAVI-EHRENHAUS LJ; VISCONTI PE; BUFFONE MG; LUQUE GM; XU X; ORTA G; JABLOÑSKI M; KRAPF D; DARSZON A; GERVASI MG; STIVAL C; BALESTRINI PA; GILIO N; KRAPF D

Congreso; XX Jornadas Anuales de la Sociedad Argentina de Biología (SAB). XVII Jornadas de la Sociedad Uruguaya de Biociencias (SUB) - Segundas Jornadas Rioplatenses de Biología; 2018

Sperm acquire the ability to fertilize in the female genital tract in a process called capacitation, where bicarbonate and calcium (Ca2+) stimulation of the soluble adenylyl cyclase leads to the activation of the cAMP/PKA pathway. During capacitation sperm undergo a change in the motility pattern called hyperactivation. Ca2+ is the primary second messenger that triggers this motility and it depends on CatSper channels. It has been described that CatSper1 proteins form a unique pattern of four linear ??stripes?? along the principal piece of the flagellum and colocalizes with Ca2+ signaling molecules. By using super-resolution microscopy, the localization of the small GTPase Cdc42 in the mouse sperm flagellum was determined. Cdc42 forms four columns along the principal piece resembling the localization of CatSper. PKA-dependent phosphorylation was completely abrogated in the presence of specific Cdc42 inhibitors. This inhibition was bypassed when a membrane permeable analog of cAMP was added. The rise in intracellular Ca2+ that occurs during capacitation as a result of CatSper activation, was suppressed with Cdc42 pharmacological inhibition. In the presence of Cdc42 inhibitors, sperm underwent an increase in tyrosine phosphorylation upon incubation in nominal zero Ca2+ medium, as CatSper KO sperm. Furthermore, Cdc42 inhibition significantly impaired the depolarization caused by EGTA addition, an indirect CatSper activity assay. Both results suggest that Cdc42 modulates CatSper activity in mouse sperm. When sperm were capacitated in the presence of Cdc42 inhibitors, a strong decrease in percentage of hyperactivation, as well as in the percentage of fertilized eggs was observed. All together, these results indicate that Cdc42 is participating in a molecular complex with CatSper channels modulating the levels of intracellular Ca2+ and ultimately, the development of hyperactivation and fertilizing ability.