pH controls spermatozoa motility in the Pacific oyster (Crassostrea gigas)

Abstract

Investigating the roles of chemical factors stimulating and inhibiting sperm motility is required to understand the mechanisms of spermatozoa movement. In this study, we described the composition of the seminal fluid (osmotic pressure, pH, and ions) and investigated the roles of these factors and salinity in initiating spermatozoa movement in the Pacific oyster, Crassostrea gigas The acidic pH of the gonad (5.82±0.22) maintained sperm in the quiescent stage and initiation of flagellar movement was triggered by a sudden increase of spermatozoa external pH (pHe) when released in seawater (SW). At pH 6.4, percentage of motile spermatozoa was three times higher when they were activated in SW containing 30 mM NH₄Cl, which alkalinizes internal pH (pHi) of spermatozoa, compared to NH₄Cl-free SW, revealing the role of pHi in triggering sperm movement. Percentage of motile spermatozoa activated in Na⁺-free artificial seawater (ASW) was highly reduced compared to ASW, suggesting that change of pHi triggering sperm motility was mediated by a Na⁺/H⁺ exchanger. Motility and swimming speed were highest in salinities between 33.8 and 42.7‰ (within a range of 0 to 50 ‰), and pH values above 7.5 (within a range of 4.5 to 9.5).

Keywords: Ions; Motility; Salinity; Seminal fluid; Spermatozoa; pH.

Fig. 1.

Effects of salinity on percentage of motile spermatozoa and VAP after 1 and 5 min after activation. Motile spermatozoa (A, P=0.000), VAP (B, P=0.003); VAP, Velocity of the Average Path. VAP in salinities of 3.9 ‰, 8.9 ‰ (1 and 5 min after activation), and 14 ‰ (1 min after activation) was not measured because of low number of motile spermatozoa. VAP was not significantly different among salinities for spermatozoa activated for 1 min. Different letters refer to significantly different results (mean±s.e.m.; lower case letters, 1 min after sperm activation; upper case letters, 5 min after sperm activation; n=5 oysters). Data were compared using one-way ANOVA and Fisher a posteriori test.

Fig. 2.

Effects of pH on percentage of motile spermatozoa and VAP after 1 and 5 min after activation. Motile spermatozoa (A, P=0.000), VAP (B, P=0.000); VAP, Velocity of the Average Path. Different letters refer to significantly different results (mean±s.e.m.; lower case letters, 1 min after sperm activation; upper case letters, 5 min after sperm activation; n=5 oysters). Data were compared using one-way ANOVA and Fisher a posteriori test.

Fig. 3.

Effect of NH₄Cl (0 or 30 mM) on percentage of motile spermatozoa and VAP after 1 and 5 min after activation. Motile spermatozoa (A, P=0.000), VAP (B); VAP, Velocity of the Average Path. VAP was not significantly different for both NH₄Cl concentrations (B). Different letters refer to significantly different results (mean±s.e.m.; lower case letters, 1 min after sperm activation; upper case letters, 5 min after sperm activation; n=5 oysters). Data were compared using one-way ANOVA and Fisher a posteriori test.

Fig. 4.

Effects of ion deprivation on percentage of motile spermatozoa and VAP after 1 and 5 min after activation. Motile spermatozoa (A, P=0.006 at 1 min and P=0.000 at 5 min after activation), VAP (B); VAP, Velocity of the Average Path. VAP in Na⁺-free ASW was not measured because of low number of motile spermatozoa. No movement of spermatozoa was observed in ion-free ASW. Sperm VAP was not significantly affected by a lack of Ca²⁺, K⁺ or Mg²⁺ in ASW. ASW, artificial seawater; different letters refer to significantly different results (mean±s.e.m.; lower case letters, 1 min after sperm activation; upper case letters, 5 min after sperm activation; n=5 oysters). Data were compared using one-way ANOVA and Fisher a posteriori test.