Caffeine induces ram sperm hyperactivation independent of cAMP-dependent protein kinase

We have previously shown that a cocktail-containing phosphodiesterase inhibitors (theophylline and caffeine), a phosphatase inhibitor (okadaic acid) and dibutyryl-cAMP promoted specific protein tyrosine phosphorylation in ram spermatozoa during incubation in capacitating conditions. Here, we show, for the first time, that this cocktail induced a progressive time-dependent increase in the capacitated-sperm subpopulation. The addition of either the analogue of adenosine, 2-chloro-2'-deoxyadenosine (Cl-Ado) or caffeine provided a significant increase in the proportion of capacitated spermatozoa and total tyrosine phosphorylation. Computer-assisted semen analysis was used to identify hyperactivated spermatozoa by setting maximum threshold for linearity (< or =45%) and minimum for amplitude of lateral head displacement (> or =3.5 microm). Our results showed that ram spermatozoa can be capacitated in vitro without displaying hyperactivated movement. Among the above-mentioned compounds, only caffeine was able to induce hyperactivation that achieved the maximal response at 8 min of incubation, with a significant increase in hyperactivated spermatozoa of 44.4 +/- 5.6% related to control samples. Flow cytometry analyses showed that caffeine induced a significant increase in the content of calcium in viable spermatozoa during the time-course of incubation in capacitating conditions. BAPTA-AM, a cell-permeable calcium chelator, did not suppress the caffeine-dependent hyperactivation. Quantitative analysis revealed that the addition of caffeine or Cl-Ado accounted for an increase in intracellular cAMP level. However, this increase in cAMP does not seem to be responsible for the caffeine-induced hyperactivation because the cAMP-elevating agents (cocktail) did not promote hyperactivation either, although they greatly induced capacitation and protein tyrosine phosphorylation. The inhibition of PKA with H89 reduced both capacitation and protein tyrosine phosphorylation although hyperactivation increased. These results suggest that calcium from internal stores would be enough to initiate the hyperactivated movement, and that protein tyrosine phosphorylation implicated in ram sperm hyperactivation would be regulated by calcium rather than by PKA-dependent cAMP.