Regulation of sperm motility in Eastern oyster (Crassostrea virginica) spawning naturally in seawater with low salinity

Abstract

Oyster aquaculture is expanding worldwide, where many farms rely on seed produced by artificial spawning. As sperm motility and velocity are key determinants for fertilization success, understanding the regulation of sperm motility and identifying optimal environmental conditions can increase fertility and seed production. In the present study, we investigated the physiological mechanisms regulating sperm motility in Eastern oyster, Crassostrea virginica. Sperm motility was activated in ambient seawater with salinity 4-32 PSU with highest motility and velocity observed at 12-24 PSU. In artificial seawater (ASW) with salinity of 20 PSU, sperm motility was activated at pH 6.5-10.5 with the highest motility and velocity recorded at pH 7.5-10.0. Sperm motility was inhibited or totally suppressed in Na+, K+, Ca2+, and Mg2+-free ASW at 20 PSU. Applications of K+ (500 μM glybenclamide and 10-50 mM 4-aminopyridine), Ca2+ (1-50 μM mibefradil and 10-200 μM verapamil), or Na+ (0.2-2.0 mM amiloride) channel blockers into ASW at 20 PSU inhibited or suppressed sperm motility and velocity. Chelating extracellular Ca2+ ions by 3.0 and 3.5 mM EGTA resulted in a significant reduction and full suppression of sperm motility by 4 to 6 min post-activation. These results suggest that extracellular K+, Ca2+, and Na+ ions are involved in regulation of ionic-dependent sperm motility in Eastern oyster. A comparison with other bivalve species typically spawning at higher salinities or in full-strength seawater shows that ionic regulation of sperm motility is physiologically conserved in bivalves. Elucidating sperm regulation in C. virginica has implications to develop artificial reproduction, sperm short-term storage, or cryopreservation protocols, and to better predict how changes in the ocean will impact oyster spawning dynamics.

Fig 1

Effect of salinity on sperm motility (%, A) in Eastern oyster, Crassostrea virginica. Sperm motility was activated in high salinity seawater diluted to salinities of 4 to 32 PSU. Average motility at each salinity (B) and time post-activation (C) is displayed. D shows a second-order polynomial regression for the effects of salinity on sperm motility at 15 min post-activation. Sperm head trajectories at each salinity are shown (E). Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 5). Treatments with different superscripts significantly differ (p < 0.05). A motility of 0% was indicated by asterisk.

Fig 2

Effect of salinity on sperm velocity (μm/s, A) in Eastern oyster, Crassostrea virginica. Sperm motility was activated in high salinity seawater diluted to salinities of 4 to 32 PSU. Average velocity at each salinity (B) and time post-activation (C) is displayed. D shows a second-order polynomial regression for the effects of salinity on sperm velocity at 15 min post-activation. Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 5). Treatments with different superscripts significantly differ (p < 0.05). A velocity of 0 μm/s was indicated by asterisk.

Fig 3

Effect of pH on sperm motility (%, A) in Eastern oyster, Crassostrea virginica. Sperm motility was activated in artificial seawater buffered with 20 mM MES, HEPES or Tris, pH 6.5–10.5. Average motility at each pH (B) and time post-activation (C) is displayed. D shows a second-order polynomial regression for the effects of pH on sperm motility at 15 min post-activation. Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 4). Treatments with different superscripts significantly differ (p < 0.05). A motility of 0% was indicated by asterisk.

Fig 4

Effect of pH on sperm velocity (μm/s, A) in Eastern oyster, Crassostrea virginica. Sperm motility was assessed in artificial seawater buffered with 20 mM MES, HEPES or Tris, pH 6.5–10.5. Average motility at each pH (B) and time post-activation (C) is displayed. D shows a second-order polynomial regression for the effects of pH on sperm velocity at 15 min post-activation. Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 4). Treatments with different superscripts significantly differ (p < 0.05). A velocity of 0 μm/s was indicated by asterisk.

Fig 5

Effect of potassium (K⁺) ions on sperm motility (%, A-C) and velocity (μm/s, D-F) in Eastern oyster, Crassostrea virginica. Sperm was activated in K⁺-free artificial seawater (ASW) and ASW containing a voltage-gated (4-aminopyridine, 4-AP) or an ATP-sensitive (glybenclamide, G) K⁺ channel blocker. Average motility in each treatment (B) and time post-activation (C) is displayed. Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 5). Treatments with different superscripts significantly differ (p < 0.05).

Fig 6

Effect of calcium (Ca²⁺) ions on sperm motility (%, A-D) and velocity (μm/s, E-H) in Eastern oyster, Crassostrea virginica. Sperm was activated in artificial seawater (ASW), Ca²⁺-free ASW and ASW containing Ca²⁺ channel blockers: mibefradil (M), nifedipine (N), or verapamil (V). Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 4). Treatments with different superscripts significantly differ (p < 0.05). Motility of 0% and velocity of 0 μm/s were indicated by asterisk.

Fig 7

Effect of sodium (Na⁺) ions on sperm motility (%, A-C) and velocity (μm/s, D-F) in Eastern oyster, Crassostrea virginica. Sperm was activated in artificial seawater (ASW), Na⁺-free ASW and Na⁺-free ASW or ASW containing Na⁺ channel blockers: amiloride (A). Motility and velocity with each treatment (B, E) and time post-activation (C, F) is displayed. Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 5). Treatments with different superscripts significantly differ (p < 0.05).

Fig 8

Effect of magnesium (Mg²⁺) ions on sperm motility (%, A-C) and velocity (μm/s, D-F) in Eastern oyster, Crassostrea virginica. Sperm was activated in artificial seawater (ASW) or Mg²⁺-free ASW. Motility and velocity with each treatment (B, E) and time post-activation (C, F) is displayed. Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 5). Treatments with different superscripts significantly differ (p < 0.05).