Sperm capacitation is characterized by a series of significant biochemical and biophysical modifications. Unlike the case with most other mammalian species, ram spermatozoa are difficult to capacitate in vitro. We have already suggested that unusually high levels of intracellular phosphodiesterases would account for cAMP levels that are too low to initiate tyrosine phosphorylation of flagellar proteins that are indicative of capacitation. In this study, we have 1) investigated the presence of the epidermal growth factor receptor (EGFR) and ERK1/2, a specific subset of the mammalian mitogen-activated protein kinase (MAPK) family, in ram spermatozoa and their involvement in capacitation; 2) searched for possible cross talk between the EGF effect and PKA pathway; and 3) explored a possible relationship between the EGF effect and the MAPK family that may underlie modulation of ram sperm capacitation. Indirect immunofluorescence evidenced the presence of EGFR and ERK in fresh ram spermatozoa. Western blot analysis confirmed both that EGFR is in the active form and that phosphorylation of Tyr845 increased after incubation with EGF. The proportion of CTC capacitated-sperm pattern and protein tyrosine phosphorylation significantly increased in the presence of EGF as well as the phosphorylation state (activation) of ERK. The specific inhibition of EGFR, PKA, or MEK reduced capacitation and protein tyrosine phosphorylation induced by EGF. We propose a working model for the molecular mechanism of the signaling cascade involved in ram sperm capacitation. These findings should improve our understanding of the biochemical mechanisms involved in the acquisition of mammalian sperm functional competence and, ultimately, fertility.
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Vol. 87 • No. 4