It is well accepted that PKA is a key enzyme

It is well accepted that PKA is a key enzyme activated at the beginning of the capacitation process [69]. As mentioned above phosphorylation/inactivation of cofilin in sperm is a PKA dependent process [58]. We could not see any change in cofilin phosphorylation level by adding the PKA activator 8Br-cAMP to the pyk2 [58]. In previous work activation of PKA by adding 8Br-cAMP to human sperm demonstrated activation of Src to phosphorylate/inactivate gelsolin [41]. Also, 8Br-cAMP enhances F-actin formation which is inhibited by inhibition of Src [41] or by inhibition of PKA [70]. We also showed that inhibition of PKA caused almost complete inhibition of human sperm hyperactivated motility [71], and inhibition of F-actin formation inhibits mouse and human sperm motility [20]. Thus, 8Br-cAMP stimulates F-actin formation due to the enhanced phosphorylation/inactivation of gelsolin [41], but it does not influence cofilin phosphorylation at the beginning of the capacitation process probably because the intracellular concentration of cAMP is already high at the beginning of the capacitation process [72]. Elevation of intracellular Ca2+ significantly reduced p-cofilin which was not reversed by inhibiting PLC [58], indicating that this dephosphorylation is PLC-independent. In contrast, gelsolin dephosphorylation/activation depends on PLC activity [41]. It has been shown elsewhere that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent dephosphorylation activated by SSH1L [73]. CaMKII negatively regulates SSH1L activity by phosphorylation/activation of LIMK1, which regulates the subcellular localization of SSH1L [74]. Inhibition of sperm CaMKII during incubation under capacitation conditions prevents F-actin formation resulting in an increase in spontaneous acrosomal exocytosis [19]. We suggest that inhibition of CaMKII would allow SSH1L activation and p-cofilin dephosphorylation/activation which will prevent F-actin formation resulting in an increase in spontaneous acrosomal exocytosis. It has been shown elsewhere that PI3K mediates the activation of SSH1L [75]. We showed elsewhere that towards the end of sperm capacitation CaMKII undergoes dephosphorylation/inactivation whereas PI3K is activated [47]. Thus, it is possible that inactivation of CaMKII and activation of PI3K towards the end of the capacitation together with the increase in [Ca2+]i activates SSH1L which dephosphorylates/activates cofilin to disperses F-actin allowing the acrosomal exocytosis to take place. We suggest a model for the effects of gelsolin and cofilin on actin during capacitation (Fig. 1). The two proteins affect F-actin levels at different times in the capacitation process: inhibition of cofilin maintains relatively high levels of F-actin at the early stages of capacitation, whereas gelsolin inhibition enables high F-actin in the late steps of the capacitation process. The fact that cofilin phosphorylation/inactivation is independent of PIP2, whereas gelsolin phosphorylation/inactivation does, further support the suggested time course for these phosphorylation. The levels of PIP2 are relatively low at the beginning of the capacitation process [18], therefore it is not expected that PIP2 will regulate cofilin activity. In addition, PIP2 levels are enhanced in the sperm head during capacitation, leading to phosphorylation/inactivation of gelsolin in the head allowing F-actin increase in the sperm head [18]. Although both gelsolin and cofilin translocate from the tail to the head during capacitation, gelsolin phosphorylation/inactivation, but not cofilin phosphorylation is regulated by PIP2 in the head; however, it is not clear how cofilin phosphorylation is regulated. The fact that translocation of cofilin from the tail to the head depends upon its phosphorylation suggests that cofilin is phosphorylated in the tail. The phosphorylation dependency of cofilin translocation is supported by other studies. It was shown in NIH3T3 cells that subcellular localization of cofilin depends on the phosphorylation state of Ser-3, in which non-phosphorylated cofilin accumulates within the nuclei [76]. However, in HL-60 cells, cofilin translocate to the plasma membrane concomitantly to activation-induced cofilin dephosphorylation [77]. Tyrosine phosphorylation of gelsolin is mediated by PKA/Src activities and serine-phosphorylation of cofilin is also mediated by PKA which probably phosphorylates/activates LIMK [56], which phosphorylates/inactivates cofilin [50].