CAPACITATION OF MOUSE SPERMATOZOA .2. PROTEIN-TYROSINE PHOSPHORYLATION AND CAPACITATION ARE REGULATED BY A CAMP-DEPENDENT PATHWAY

In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129 -1137) we demonstrated that the tyrosine phosphorylation of a subset o f mouse sperm proteins of M(r) 40,000-120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosi ne phosphorylation is regulated in sperm during this process is the su bject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the cap acitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin re quirement for protein tyrosine phosphorylation can be completely overc ome by the addition of biologically active, but not inactive, cAMP ana logues. Addition of the active cAMP analogues to sperm incubated in me dia devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inab ility of these media to support capacitation, as assessed by the abili ty of the cells to acquire the pattern B chlortetracycline fluorescenc e, to undergo the zona pellucida-induced acrosome reaction and, in som e cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-depend ent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and RP-cAMPS) block the capacitation-dependent increases in pro tein tyrosine phosphorylation in sperm incubated in media supporting c apacitation. Capacitation, as assessed by the aforementioned endpoints , also appears to be inhibited by H-89 in a concentration-dependent ma nner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the ca pacitated state in mouse sperm. They also demonstrate that both protei n tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PK A in sperm is, to our knowledge, the first demonstration of such an in terrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.