Our objectives were to incorporate ATP‐γ‐³²P 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 μF/cm, 65 Ω/cm, 0.3 msec) with ATP‐γ‐³²P 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, ³²P‐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‐γ‐³²P 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. Mol. Reprod. Dev. 54:292–302, 1999.

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