REGULATION OF PROTEIN-TYROSINE PHOSPHORYLATION IN BOAR SPERM THROUGH A CAMP-DEPENDENT PATHWAY

Changes of protein tyrosine phosphorylation in ejaculated boar sperm i ncubated in vitro were examined with the use of antiphosphotyrosine an tibodies and immunoblotting. The intracellular levels of cAMP were mod ulated by treatment with various combinations of caffeine, 3-isobutyl- 1-methylxanthine (IBMX), and dibutyryl cyclic AMP (dbcAMP), and acroso me reactions (ARs) were induced via treatment with divalent cation ion ophore A23187. Proteins of M-r 34, 38, 40, and 44 (p34... p44) were st rongly phosphorylated on tyrosine residues in freshly prepared sperm s amples and at the same level during all subsequent treatments. Incubat ion of sperm in vitro for various periods of time induced an increase of tyrosine phosphorylation of p20, p93, and p175. The tyrosine phosph orylation of p93, p175, and several other sperm proteins was up-regula ted in a concentration-dependent manner following treatment of the spe rm with dbcAMP, caffeine, or IBMX alone, or with combinations of caffe ine and IBMX, respectively, with dbcAMP; the tyrosine phosphorylation of p20 was not correlated with treatment of sperm with cAMP-elevating reagents. The percentage of sperm cells undergoing spontaneous ARs was not affected by the manipulation of cAMP levels and was not correlate d with protein tyrosine phosphorylation. In contrast, the addition of calcium to the incubation media decreased protein tyrosine phosphoryla tion and elevated percentage of spontaneous ARs. The induction of ARs with A23187 caused a significant decrease of tyrosine phosphorylation of p93, p175, and p220/230, indicating that dephosphorylation on prote in tyrosine residues might be associated with calcium influx during ph ysiological ARs as well. Proteins p93 and p175 were effectively solubi lized in greater than 9M urea/1% triton and in SDS sample buffer, but to only a small extent in triton, while p20 was virtually completely e xtractable with triton. In conjunction with the previously reported is olation of active tyrosine kinase sp42 from triton extracts of noncapa citated boar sperm cells (Berruti and Porzio, 1992: Biochim Biophys Ac ta 1118:149-154), our results suggest that a cAMP-dependent event is r equired for tyrosine phosphorylation of triton-insoluble proteins such as p93 and p175. On the other hand, the tyrosine phosphorylation of p 20 (and potentially other triton-soluble substrates) might not strictl y require such cAMP up-regulation. We discuss the differences in the r egulation of cAMP-dependent tyrosine phosphorylation in mouse, human, and boar sperm, and suggest that sensitivity to calcium and distinct b asal levels of cyclic nucleotide PDE might correspond to species-speci fic reproduction strategies in mammals. (C) 1998 Wiley-Liss, Inc.