Protein kinase A (PKA) activity and Src family kinases (SFK) inhibitors effect in stallion sperm under capacitating conditions

VIVANI L; GRADIL C; MUTTO A; VISCONTI P

BIOLOGY OF REPRODUCTION

SOC STUDY REPRODUCTION

Lugar: Madison; Año: 2012

0006-3363

After ejaculation, mammalian spermatozoa must undergo a series of complex and poorly understood cellular events known as ?capacitation? in order to be able to fertilize an oocyte. Among these biochemical changes, an increase in tyrosine phosphorylation of a subset of sperm proteins has been correlated with the sperm capacity to fertilize an egg and found to be regulated by a protein kinase A (PKA) dependent pathway. Since PKA is a serine/threonine (ser/thr) kinase, it is not clear how it mediates protein tyrosine phosphorylation during sperm capacitation. Recently, the activity of Src family of kinases (SFK) has been shown to regulate phosphorylation pathways associated with mouse sperm capacitation. However, little is known on the regulation of stallion sperm physiology. In order to better understand the molecular mechanisms involved in stallion sperm capacitation, the objectives of our study were: (1) To analyze PKA activation in stallion sperm under capacitating conditions and; (2) to evaluate the effect of inhibitors of the Src family kinases on stallion sperm phosphorylation events associated with capacitation. Our results are consistent with an increase on the levels of phosphorylation of PKA substrates in stallion sperm upon capacitation. We have confirmed the presence of c-Src, the prototypic member of SFK, in stallion sperm extracts. Moreover, the addition of SFK inhibitors SU6656 and SKI606 (bosutinib) to the capacitating media inhibited both protein tyrosine phosphorylation and sperm motility in a concentration dependent manner. However, contrary to what was observed for mouse sperm, in stallion sperm, SFK inhibitors did not affect phosphorylation of PKA substrates suggesting a role of SFK downstream of PKA activity in horse sperm capacitation.