The early molecular events leading to COFILIN phosphorylation during mouse sperm capacitation are essential for acrosomal exocytosis

ROMAROWSKI A.; SCHIAVI-EHRENHAUS LJ; JABLOÑSKI M; KRAPF D; LUQUE GM; BUFFONE MG.

Congreso; 47th American Society of Andrology (ASA) Annual Meeting; 2022

Actin cytoskeleton reorganization during sperm capacitation is essential for the occurrence of acrosomal exocytosis (AR). We previously showed that actin remodeling in the head of mouse sperm is regulated by RHOA/C and RAC1, two members of the Rho-family of small GTPases. Their activation promotes LIM-kinase 1 (LIMK1) phosphorylation and activation. LIMKs are activated through Rho-associated activated kinases (ROCKs) and p21-activated kinases (PAKs). We described that ROCK1, a downstream effector of RHOA/C, contributes to LIMK1 phosphorylation. RAC1-dependent LIMK1 activation is controlled by PAKs, but their expression and function in mouse sperm remains unclear. COFILIN is the only known substrate of LIMKs. Phosphorylation of COFILIN in Ser3, inhibits its actin severing activity. COFILIN displays a transient increase in Ser3 phosphorylation during the first 10 min of capacitation followed by a rapid return to the basal level. These results strongly suggest that COFILIN phosphorylation is controlled by other enzymes such as Ser/Thr phosphatases. In somatic cells, Slingshot family of protein phosphatases (SSHs) dephosphorylate and reactivate Ser3-pCOFILIN. We hypothesize that COFILIN is subject to a dual regulation by LIMKs and SSHs to control its actin-depolymerizing activity. In the present work, we investigated the participation of PAK4 and SSH1 phosphatase in the signaling pathway leading to actin remodeling during mouse sperm capacitation.•Western Blot. •Immunofluorescence. •Fluorescence staining of actin filaments.•AR assessment. We demonstrate that within the first minutes of exposure upon capacitating conditions, the activity of RHOA/C and RAC1 is essential for LIMK1 and COFILIN phosphorylation. However, we observed that the signaling pathway involving RAC1 and PAK4 is the main player in controlling actin polymerization in the sperm head necessary for the AR. Moreover, we show that the transient phosphorylation of COFILIN is influenced by SSH1. Its activity is regulated by two pathways. On one hand, RHOA/C and RAC1 activity promotes SSH1 phosphorylation (inactivation). On the other hand, the activating dephosphorylation is driven by Okadaic acid (OA) sensitive-phosphatases. This regulatory mechanism is independent of PP2B and emerges as a new finely-tuned modulation that is, so far, exclusively observed in mouse sperm. However, persistent phosphorylation of COFILIN by SSH1 inhibition or OA did not altered actin polymerization and the AR.Altogether, our work highlights the role of small GTPases in modulating actin dynamics required for AR.