Identification of capacitation-associated phosphoproteins in porcine sperm electroporated with ATP-gamma-(32)P.

Abstract

Our objectives were to incorporate ATP-gamma-(32)P into boar sperm to radiolabel endogenous phosphoproteins and compare phosphorylation patterns from sperm incubated in capacitating (CM) and non-capacitating conditions (NCM). Sperm were electroporated (1000 V/cm, 125 microF/cm, 65 Omega/cm, 0.3 msec) with ATP-gamma-(32)P which moderately decreased sperm viability (P < 0.01), but did not affect motility (P = 0.34) or the appearance of spontaneous acrosome reactions (P = 0.49). Sperm incubated in CM for 3 hr underwent capacitation, determined by the ability to undergo ionophore-induced acrosome reactions (P </= 0.05). Furthermore, more sperm in CM than in NCM exhibited chlortetracycline (CTC) pattern B (capacitated) fluorescence (P </= 0.01). SDS-PAGE, autoradiography and phosphoimagery of extracted, (32)P-labeled sperm proteins revealed a subset of phosphoproteins (Mr 28,000-60,000) from cells incubated in CM, whereas only two phosphorylated proteins were evident from sperm in NCM (44 and 57 kDa). The appearance of phosphoproteins increased concomitant with capacitation (P </= 0.05). In NCM, the 44 kDa protein was unaffected by time (P > 0.05) and the 57 kDa phosphoprotein increased after capacitation (P </= 0.05). Computer-assisted analysis revealed that the percentage of motile sperm in either medium decreased with time, and CM only transiently maintained motility over NCM (P >/= 0.02). ATP-gamma-(32)P can, therefore, be incorporated into porcine sperm to radiolabel endogenous phosphoproteins, and the different profiles from sperm incubated in NCM versus CM suggest that capacitation is mediated by signaling events involving protein phosphorylation.