ALKALINIZATION MEDIATED CA2+ INFLUX ACTIVATES SPERM MOTILITY FROM LIVE-BEARING FISH

Signal regulations for sperm activation of egg-laying fishes have been widely studied, however, such mechanisms for live-bearing fishes are poorly understood, despite the importance of artificial insemination of live-bearing fishes to activities such as development of germplasm repositories via sperm cryopreservation for biomedical research models and imperiled species, and induced spawning of aquarium species. Live-bearing species often produce sperm bundles which are another challenge for mechanistic studies. We studied intracellular calcium (Ca²⁺) signaling during sperm activation of live-bearing fish using Redtail Splitfin (Xenotoca eiseni) as a research model, and established methodologies for studying cell signaling with sperm bundles. Real-time Ca²⁺ imaging analysis with Fura 2/AM was used to investigate intracellular Ca²⁺ signals during sperm activation. We found that the percentage of activated bundles and motility increased with extracellular Ca²⁺ concentration ([Ca²⁺]_e) and pH levels ([pH]_e). Intracellular calcium concentrations ([Ca²⁺]_i) of sperm within bundles significantly (P < 0.0001) increased with [Ca²⁺]_e (Fig. 1), and such increases stimulated by 10 - 50 mM [Ca²⁺]_e were 633 - 1940% higher than 0.5 - 2 mM [Ca²⁺]_e. This [Ca²⁺]_e-dependent increase of [Ca²⁺]_i was confirmed in free sperm cells from dispersed bundles. When [Ca²⁺]_e remained at 2 mM, [Ca²⁺]_i increased significantly (P < 0.0001) with [pH]_e, and the [Ca²⁺]_i stimulated by 8.5 - 9.5 > pH 8.0 > pH 6.5 - 7.5). Furthermore, we tested the effect on Ca²⁺ signaling of most recognized test agents: non-selective Ca²⁺ channel blockers (CdCl₂, NiCl₂, and ruthenium red), a mechano-sensitive channel blocker (GdCl₃), a store-operated calcium channel blocker (SKF-96365), L-type voltage-dependent Ca²⁺ channel (VDCC) blockers (nimodipine, verapamil, and methoxyverapamil), and T-type VDCC blockers (mibefradil and NNC 55-0396). The CdCl₂ (200 µM) inhibited (P = 0.0304) Ca²⁺ signals for 25%, but all other blockers failed to inhibit intracellular Ca²⁺ signals. These findings strongly suggest that sperm activation and bundle dissociation of live-bearing fish X. eiseni are due a Ca²⁺ influx that can be mediated by increased extracellular alkalinization. However, the influx does not appear to be mediated through any commonly known Ca²⁺ channels. This raises the possibility of a novel mechanism at work in X. eiseni. Findings from this study can provide a foundation for standardization of the artificial insemination, and further investigation of molecular activation mechanisms using sperm from live-bearing fishes.