ROLE OF CAMP IN THE REACTIVATION OF DEMEMBRANATED RAM SPERMATOZOA

Ejaculated ram sperm were demembranated with Triton X-100, separated f rom the detergent-soluble matrix, and reactivated [San Agustin and Wit man (1993): Cell Motil. Cytoskeleton 24:264-273]. The percent motility of models prepared from freshly washed sperm was comparable to that o f the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motilit y of freshly washed sperm. Cyclic AMP was similar to 100 times more ef fective than cGMP. The requirement for cAMP could be bypassed by addit ion of porcine heart cAMP-dependent protein kinase (PKA) catalytic sub unit to the reactivation medium, demonstrating that cAMP was acting vi a PKA. The cAMP stimulation of reactivation was not affected by inclus ion of the PKA inhibitor PKI(5-24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5-24) prior to r eactivation. The cytosol-free models retained >90% of the sperm PKA ac tivity; therefore, the PKA appears to be anchored to internal sperm st ructures. This PKA could not be extracted by cAMP or Triton X-100 alon e, but only by cAMP and Triton X-100 in combination. We conclude that cAMP-dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptid e arg-lys-arg-ala-arg-lys-glu, which has been reported to be a selecti ve inhibitor of cGMP-dependent protein kinase. (C) 1994 Wiley-Liss, In c.