Serotonin–Induced Sperm Hyper-Motility In Pacific Oyster (Crassostrea Gigas) Associates With K+ Efflux and Membrane Hyperpolarization

Abstract

Serotonin (5-HT) induces sperm hyper-motility in bivalves. This process has been suggested to be associated with K⁺ efflux due to higher concentrations of K⁺ ions in testicular fluid compared to that of seawater. This hypothesis was supported by inhibition of 5-HT–induced sperm hyper-motility in artificial seawater (ASW) containing high extracellular K⁺ ions or in the presence of a voltage-dependent K⁺ channel blocker (4-AP). Here, we studied changes of sperm membrane potential to elucidate 5-HT–induced sperm hyper-motility signaling in Pacific oyster (Crassostrea gigas). Sperm motility was partially initiated (48.34 ± 7.80%) in ASW, and decreased at 5 min post-activation (p < 0.05). In the presence of 10^–5 M 5-HT, sperm motility was recorded 81.63 ± 3.55%, which remained unchanged within 60 min post-activation. After sperm activation in ASW with or without 5-HT, fluorescence intensity of membrane potential-sensitive fluorescent (DiSC₃(5)) was decreased to lower than that of the resting stage, indicating membrane hyperpolarization. Induction of membrane hyperpolarization, using valinomycin or in K⁺-free ASW (KF-ASW) could not trigger sperm hyper-motility, suggesting that hyperpolarization itself did not induce sperm hyper-motility. Next, we showed that membrane hyperpolarization was due to K⁺ efflux. The fluorescence intensity of DiSC₃(5) was increased in ASW or KF-ASW containing 4-AP, suggesting membrane depolarization due to inhibition of K⁺ efflux. The valinomycin–induced membrane hyperpolarization was changed to depolarization by subsequent additions of KCl, suggesting that changes in the electrochemical gradient of K⁺ ions resulted in the retention of intracellular K⁺ ions. Observed membrane depolarization in the presence of 4-AP or high K⁺ ions was associated with inhibition of 5-HT–induced sperm hyper-motility. Taken together, this study shows that 5-HT–induced sperm hyper-motility was associated with membrane hyperpolarization due to K⁺ efflux.