The sperm acrosome reaction and penetration of the egg follow zona pellucida binding only if the sperm has previously undergone the poorly understood maturation process known as capacitation. We demonstrate here that in vitro capacitation of bull, ram, mouse, and human sperm was accompanied by a time-dependent increase in actin polymerization. Induction of the acrosome reaction in capacitated cells initiated fast F-actin breakdown. Incubation of sperm in media lacking BSA or methyl-beta-cyclodextrin, Ca(2+), or NaHCO(3), components that are all required for capacitation, prevented actin polymerization as well as capacitation, as assessed by the ability of the cells to undergo the acrosome reaction. Inhibition of F-actin formation by cytochalasin D blocked sperm capacitation and reduced the in vitro fertilization rate of metaphase II-arrested mouse eggs. It has been suggested that protein tyrosine phosphorylation may represent an important regulatory pathway that is associated with sperm capacitation. We show here that factors known to stimulate sperm protein tyrosine phosphorylation (i.e., NaHCO(3), cAMP, epidermal growth factor, H(2)O(2), and sodium vanadate) were able to enhance actin polymerization, whereas inhibition of tyrosine kinases prevented F-actin formation. These data suggest that actin polymerization may represent an important regulatory pathway in with sperm capacitation, whereas F-actin breakdown occurs before the acrosome reaction.

In order to fertilize, the mammalian spermatozoa should reside in the female reproductive tract for several hours, during which they undergo a series of biochemical modifications collectively called capacitation. Only capacitated sperm can undergo the acrosome reaction after binding to the egg zona pellucida, a process which enables sperm to penetrate into the egg and fertilize it. Polymerization of globular (G)-actin to filamentous (F)-actin occurs during capacitation, depending on protein kinase A activation, protein tyrosine phosphorylation, and phospholipase D activation. F-actin formation is important for the translocation of phospholipase C from the cytosol to the sperm plasma membrane during capacitation. Prior to the occurrence of the acrosome reaction, the F-actin should undergo depolymerization, a necessary process which enables the outer acrosomal membrane and the overlying plasma membrane to come into close proximity and fuse. The binding of the capacitated sperm to the zona pellucida induces a fast increase in sperm intracellular calcium, activation of actin severing proteins which break down the actin fibers, and allows the acrosome reaction to take place.

The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H(2)O(2)) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H(2)O(2) in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 microM H(2)O(2). At much higher concentrations of H(2)O(2) (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H(2)O(2) after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H(2)O(2). These two agents, but not heparin, induced Ca(2+)-dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H(2)O(2) (50 microM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H(2)O(2) are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H(2)O(2) activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.

A convenient assay for interferons based on reduction of cytopathic effect was developed. The number of manipulations and the lengths of the various incubation steps were reduced to a minimum. The assay is simple to perform and can be completed within 16 h. Moreover, it can be used with various types of cells and a variety of viruses.

Mammalian spermatozoa should reside in the female reproductive tract for a certain time before gaining the ability to fertilize. During this time, the spermatozoa undergo a series of biochemical processes collectively called capacitation. We recently demonstrated that actin polymerization is a necessary step in the cascade leading to capacitation. We demonstrate here for the first time a role for phospholipase D (PLD) in the induction of actin polymerization and capacitation in spermatozoa. The involvement of PLD is supported by specific inhibition of F-actin formation during sperm capacitation by PLD inhibitors and the stimulation of fast F-actin formation by exogenous PLD or phosphatidic acid (PA). Moreover, PLD activity is enhanced during capacitation before actin polymerization. Protein kinase A (PKA), known to be active in sperm capacitation, and protein kinase C (PKC), involved in the acrosome reaction, can both activate PLD and actin polymerization. We suggest that PKA- and PKC-dependent signal transduction pathways can potentially lead to PLD activation; however, under physiological conditions, actin polymerization depends primarily on PKA activity. Activation of PKA during capacitation causes inactivation of phospholipase C, and as a result, PKC activation is prevented. It appears that PKA activation promotes sperm capacitation whereas early activation of PKC during capacitation would jeopardize this process.