2-APB-potentiated channels amplify CatSper-induced Ca2+ signals in human sperm

Ca2+ (i) signalling is pivotal to sperm function. Progesterone, the best-characterized agonist of human sperm Ca2+ (i) signalling, stimulates a biphasic [Ca2+](i) rise, comprising a transient and subsequent sustained phase. In accordance with recent reports that progesterone directly activates CatSper, the [Ca2+](i) transient was detectable in the anterior flagellum (where CatSper is expressed) 1-2 s before responses in the head and neck. Pretreatment with 5 mu M 2-APB (2-aminoethoxydiphenyl borate), which enhances activity of store-operated channel proteins (Orai) by facilitating interaction with their activator [STIM (stromal interaction molecule)] 'amplified' progesterone-induced [Ca2+](i) transients at the sperm neck/midpiece without modifying kinetics. The flagellar [Ca2+](i) response was unchanged. 2-APB (5 mu M) also enhanced the sustained response in the midpiece, possibly reflecting mitochondrial Ca2+ accumulation downstream of the potentiated [Ca2+](i) transient. Pre-treatment with 50-100 mu M 2-APB failed to potentiate the transient and suppressed sustained [Ca2+]i elevation. When applied during the [Ca2+]i plateau, 50-100 mu M 2-APB caused a transient fall in [Ca2+](i), which then recovered despite the continued presence of 2-APB. Loperamide (a chemically different store-operated channel agonist) enhanced the progesterone-induced [Ca2+](i) signal and potentiated progesterone-induced hyperactivated motility. Neither 2-APB nor loperamide raised pH(i) (which would activate CatSper) and both compounds inhibited CatSper currents. STIM and Orai were detected and localized primarily to the neck/midpiece and acrosome where Ca2+ stores are present and the effects of 2-APB are focussed, but store-operated currents could not be detected in human sperm. We propose that 2-APB-sensitive channels amplify [Ca2+](i) elevation induced by progesterone (and other CatSper agonists), amplifying, propagating and providing spatio-temporal complexity in [Ca2+](i) signals of human sperm.