Influence of different cellular concentrations of boar sperm suspensions on the induction of capacitation and acrosome reaction

Abstract

We aimed to analyze the influence of different cellular concentrations of boar sperm suspensions on the induction of capacitation and acrosome reaction. When spermatozoa were incubated at 100 or 200 mill/ml, significant increases in protein tyrosine phosphorylation in the p32 protein were observed, compared to those at 50 mill/ml. In addition, sperm concentration-dependent increases were observed in plasma membrane lipid disorganization (50 mill/ml vs. 200 mill/ml), induction of the acrosome reaction (50 mill/ml vs. 100 mill/ml and 200 mill/ml), and sperm viability (50 mill/ml vs. 100 mill/ml and 200 mill/ml). Our data indicate that an increase in sperm concentration stimulates the induction of capacitation and acrosome reaction in boars.

Keywords: Acrosome reaction; Boar spermatozoa; Capacitation.

Fig. 1.

Effect of sperm concentration during incubation on protein tyrosine phosphorylation (A) and phosphorylation of PKA substrates (B). Spermatozoa were incubated at concentrations of 50, 100 or 200 mill/ml in TCM for 4 h at 38.5°C. Subsequently, spermatozoa were lysed and western blotting was performed using an anti-phosphotyrosine antibody (A; upper panel) or an anti-phospho-PKA substrate antibody (B; upper panel). The immunoblots are representative of 8 independent experiments (n = 8); α-tubulin levels are shown as loading control. The bars represent the mean ± SEM of the optical densities of p32 (A; lower panel) and phospho-PKA substrates (B; lower panel) in arbitrary units. Bars marked with * differ from the 50 mill/ml concentration significantly (P < 0.05).

Fig. 2.

Effect of sperm concentration during incubation on plasma membrane lipid organization. Spermatozoa were incubated at 50, 100 or 200 mill/ml in TCM for 4 h at 38.5°C. Following this, plasma membrane lipid organization was assessed by flow cytometry using Merocyanine 540 (M540). Panel (A) shows the percentage of viable spermatozoa with high M540 fluorescence (mean ± SEM; n = 6). Bars marked with * differ from the 50 mill/ml concentration significantly (P < 0.05). Panel (B) shows representative plots acquired at 50 and 200 mill/ml.

Fig. 3.

Effect of sperm concentration during incubation on acrosome reaction and viability. Spermatozoa were incubated at concentrations of 50, 100 or 200 mill/ml in TCM for 4 h at 38.5°C. Following this, acrosome reaction and viability were simultaneously assessed by flow cytometry using PNA-FITC and PI. Panel (A) shows the percentage of live acrosome-reacted spermatozoa, and panel (B) shows the percentage of viable spermatozoa (mean ± SEM; n = 8). Bars marked with * differ from the 50 mill/ml concentration significantly (P < 0.05). Panel (C) shows representative plots acquired at 50 and 200 mill/ml.