Membrane hyperpolarization activates trout sperm without an increase in intracellular pH

Sperm from trout, like other sperm, are immotile in the seminal tract and initiate motility upon dilution into an appropriate fertilizing environment. Trout sperm motility is inhibited by high extracellular [K+] and can be activated by dilution of extracellular [K+]. Activation of trout sperm by the dilution of extracellular [K+] suggests regulation by membrane potential. Using the membrane potential-sensitive fluorescent dye 3,3'-dipropylthiocarbocyanine iodide (diS-C3-(5)) we directly measured the K+ contribution to the membrane potential. Manipulating the membrane potential with Cs+ and the ionophore valinomycin can override K+ regulation. We show that trout sperm can also be activated in the presence of inhibitory [K+] by the addition of divalent cations. Activation by divalent cations is explained by the cations' ability to mask membrane surface potential and thus alter the potential sensed by membrane voltage sensors. Using the surface potential-sensitive dye, 1-anilino-8-naphthosulfonate (ANS), we directly measure the divalent cations' ability to mask surface potential. We propose a model where membrane hyperpolarization is the trigger that initiates the cascade of events leading to trout sperm activation. An increase in intracellular pH has been suggested to be a conserved step in the activation of sperm motility. We show that increasing intracellular pH by procedures that activate sea urchin and mammalian sperm does not activate trout sperm. In contrast, there is a decrease in intracellular pH upon activation of trout sperm motility. Artificially decreasing intracellular pH is not sufficient for activation of motility in trout sperm in an inhibitory [K+]. Thus, unlike some other sperm, changes in intracellular pH do not regulate trout sperm motility.