Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. In previous studies, we demonstrated that the increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP2). Here, we showed for the first time the involvement of PIP2/gelsolin in human sperm motility before and during capacitation. Activation of gelsolin by causing its release from PIP2 inhibited sperm motility, which could be restored by adding PIP2 to the cells. Reduction of PIP2 synthesis inhibited actin polymerization and motility, and increasing PIP2 synthesis enhanced these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP2 and F-actin. During capacitation there was an increase in PIP2 and F-actin levels in the sperm head and a decrease in the tail. In sperm with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends on its binding to PIP2. Activation of phospholipase C by Ca(2+)-ionophore or by activating the epidermal-growth-factor-receptor inhibits tyrosine phosphorylation of gelsolin. In conclusion, the data indicate that the increase of PIP2 and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result the decrease of gelsolin in the tail allows keeping high level of F-actin in the tail, which is essential for the development of HA motility.

The spermatozoon is capable of fertilizing an oocyte only after undergoing several biochemical changes in the female reproductive tract, referred to as capacitation. The capacitated spermatozoon interacts with the egg zona pellucida and undergoes the acrosome reaction, which enables its penetration into the egg and fertilization. Actin dynamics play a major role throughout all these processes. Actin polymerization occurs during capacitation, whereas prior to the acrosome reaction, F-actin must undergo depolymerization. In the present study, we describe the presence of the actin-severing protein, cofilin, in human sperm. We examined the function and regulation of cofilin during human sperm capacitation and compared it to gelsolin, an actin-severing protein that was previously investigated by our group. In contrast to gelsolin, we found that cofilin is mainly phosphorylated/inhibited at the beginning of capacitation, and dephosphorylation occurs towards the end of the process. In addition, unlike gelsolin, cofilin phosphorylation is not affected by changing the cellular levels of PIP2. Despite the different regulation of the two proteins, the role of cofilin appears similar to that of gelsolin, and its activation leads to actin depolymerization, inhibition of sperm motility and induction of the acrosome reaction. Moreover, like gelsolin, cofilin translocates from the tail to the head during capacitation. In summary, gelsolin and cofilin play a similar role in F-actin depolymerization prior to the acrosome reaction but their pattern of phosphorylation/inactivation during the capacitation process is different. Thus, for the sperm to achieve high levels of F-actin along the capacitation process, both proteins must be inactivated at different times and, in order to depolymerize F-actin, both must be activated prior to the acrosome reaction.