Cdc42 is localized in a highly organized signaling distribution complex that is critical for mouse sperm hyperactivation.

BUFFONE MG; KRAPF D; PUGA MOLINA LC; XU X; LUQUE GM; VISCONTI PE; KRAPF D; STIVAL C; DARSZON A; GERVASI MG; GILIO N; DALOTTO-MORENO T; ROMAROWSKI A

Holderness

Congreso; Gordon Research Conference and Seminar: Fertilization and Activation of Development.; 2017

Sperm acquire the ability to fertilize in the female genital tract in a process called capacitation. From a molecular point of view, 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 (HA) which is critical to fertilization. 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?? running down the principal piece of the flagellum. CatSper Ca2+ domains orchestrate the timing and extent of complex phosphorylation cascades because it colocalizes with Ca2+ signaling molecules. Our central hypothesis is that Cdc42 is an essential component of the highly organized signaling complex that controls intracellular Ca2+ through CatSper channels during mammalian sperm capacitation. This spatial distribution is critical for the development of HA. Using super-resolution microscopy, we observed that Cdc42 localized in the sperm tail and form a pattern of four linear ??stripes?? running down the principal piece, which resembles the localization of CatSper. By using specific inhibitors of Cdc42, we detected that PKA-dependent phosphorylation were completely abrogated. This inhibition was bypassed by using membrane permeable analogs of cAMP. The rise in intracellular Ca2+ that occurs during capacitation as a result of CatSper activation, was suppressed with Cdc42 inhibition. When sperm where incubated in the presence of Cdc42 inhibitors, we observed a strong decrease in percentage of HA, as well as in the percentage of fertilized eggs. In order to determine whether Cdc42 is modulating CatSper activity we used different approaches. In the presence of Cdc42 inhibitors, sperm underwent an increase in tyrosine phosphorylation upon incubation in nominal zero Ca2+ media, such as CatSper KO sperm. Furthermore, the membrane potential of epididymal sperm was measured with a fluorescent cyanine dye using a protocol that unveils the activity of CatSper by suddenly removing external Ca2+ after adding 3.5 mM EGTA. Cdc42 inhibitor significantly suppressed the depolarization caused by EGTA addition, suggesting that Cdc42 inhibits CatSper channels in mouse sperm. To corroborate these results, we recorded CatSper currents from cauda epididymal sperm using a voltage-ramp protocol and Cs+ as the main conducting ion in the absence of divalent cations. Inhibition of Cdc42 abrogated >40% of the CatSper currents at negative and positive potentials. All together, these results suggest that Cdc42 is participating in a molecular complex with CatSper channels modulating the levels of intracellular Ca2+ and ultimately, the development of HA.