Mechanisms regulating sperm motility activation are generally known in oviparous fishes, but are poorly understood in viviparous species. The mechanism of osmotic-shock induced signaling for oviparous fishes is not suitable for viviparous fishes which activate sperm motility within an isotonic environment. In addition, the presence of sperm bundles in viviparous fishes further complicates study of sperm activation mechanisms. The goal of this study was to establish methodologies to detect intracellular Ca²⁺ signals from sperm cells within bundles, and to investigate the signaling mechanism of sperm activation of viviparous fish using Redtail Splitfin (Xenotoca eiseni) as a model. Motility was assessed by classification of bundle dissociation and computer-assisted sperm analysis, and intracellular Ca²⁺ was assessed using the fluorescent probe Fura-2 AM. Bundle dissociation and sperm motility increased with extracellular Ca²⁺ and pH levels. Intracellular Ca²⁺ signals were detected from sperm within bundles, and increased significantly with extracellular Ca²⁺ and pH levels. Major channel blockers known to inhibit Ca²⁺ influx (NiCl₂, ruthenium red, GdCl₃, SKF-96365, nimodipine, verapamil, methoxyverapamil, mibefradil, NNC 55–0396, ω-Conotoxin MVIIC, bepridil, and 2-APB) failed to inhibit Ca²⁺ influx, except for CdCl₂, which partially inhibited the influx. We propose a novel mechanism for motility regulation of fish sperm: an alkaline environment in the female reproductive tract opens Ca²⁺ channels in the sperm plasma membrane without osmotic shock, and the Ca²⁺ influx functions as a second messenger to activate motor proteins controlling flagella movement.

Summary Sentence

Extracellular alkalinization can induce Ca²⁺ influx, which activates sperm within bundles of a viviparous fish.