Image_1_Changes in Protein O-GlcNAcylation During Mouse Epididymal Sperm Maturation.JPEG

<p>After leaving the testis, sperm undergo two sequential maturational processes before acquiring fertilizing capacity: sperm maturation in the male epididymis, and sperm capacitation in the female reproductive tract. During their transit through the epididymis, sperm experience several maturational changes; the acquisition of motility is one of them. The molecular basis of the regulation of this process is still not fully understood. Sperm are both transcriptionally and translationally silent, therefore post-translational modifications are essential to regulate their function. The post-translational modification by the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) can act as a counterpart of phosphorylation in different cellular processes. Therefore, our work was aimed to characterize the O-GlcNAcylation system in the male reproductive tract and the occurrence of this phenomenon during sperm maturation. Our results indicate that O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAcylation, is present in the testis, epididymis and immature caput sperm. Its presence is significantly reduced in mature cauda sperm. Consistently, caput sperm display high levels of O-GlcNAcylation when compared to mature cauda sperm, where it is mostly absent. Our results indicate that the modulation of O-GlcNAcylation takes place during sperm maturation and suggest a role for this post-translational modification in this process.</p

Membrane potential measurements of wild-type and SLO3 mutant sperm in capacitated and non-capacitated conditions.

<p>Curves shown are GHK fits to (A) wild-type and (B) SLO3 mutant sperm, prior to and one hour after being subjected to capacitating conditions. The measured membrane potential values seen for capacitated wild-type sperm are likely to represent the average values for a mixed population of sperm (see text). Note that in (B) there is no predicted decrease in P_Na in SLO3 mutant sperm subjected to capacitating conditions. The curves correspond to mean n = 11 experiments ± S.E.M. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060578#pone.0060578.s005" target="_blank">table S1</a> for membrane potential values.</p

[Na⁺]_i is decreased in SLO3 mutant sperm and in sperm treated with the SLO3 inhibitor clofilium.

<p>Cauda epididymal sperm from wild type or SLO3 mutant mice were recovered and loaded with CoroNaRed in media lacking BSA and HCO₃⁻ which does not support capacitation (Non Cap). After thirty minutes incubation, the sperm were washed once and resuspended in the same media or in media containing BSA and HCO₃⁻ (Cap) in the absence or in the presence of clofilium (50 µM) (for wild-type sperm). After 1 hour incubation, PI was added and the sperm population analyzed by flow cytometry. A) SLO3 wild-type sperm: PI vs CoroNa Red two-dimensional dot plots of sperm incubated in non capacitating conditions (Non Cap), in media that support capacitation (Cap) or in media that support capacitation in the presence of clofilium (Cap+Clofilium 50 µM). The left merged panel combined data from Non Cap and Cap dot plots, the right merged panel combined the Cap and the Cap+clofilium dot plots. B) SLO3 mutant sperm: PI vs CoroNa Red two-dimensional dot plots of sperm incubated in non capacitating conditions (Non Cap) or in media that support capacitation (Cap). Live sperm populations in each case were then analyzed for their individual [Na⁺]_i CoroNa Red fluorescence histograms (Non Cap and Cap); the merged panel combined both data. C) Summary for plots showed in A and B. The bars represent the mean n = 3 experiments ± S.E.M. NS indicates No statistical significance (P≥0.05), **indicates statistical significance (P≤0.01).</p

SLO3 mutant sperm do not hyperpolarize even at 180 min in capacitating conditions.

<p>Membrane potential measurements (A) and representative plots of florescence emission traces (B) at different capacitation times for wild-type and SLO3 mutant sperm. The bars represent the mean of n = 12 experiments. *indicates (P≤0.05); **indicates (P≤0.01). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060578#pone.0060578.s006" target="_blank">table S2</a> for membrane potential values.</p

Plots of membrane voltages recorded at different [K⁺]_e under non-capacitated conditions.

<p>Curves shown are GHK fits to wild-type (A) and SLO3 mutant sperm (B). The green line in both plots is a GHK best fit that omits P_Cl. Thus, the only way to obtain an accurate fit of the curves with the GHK equation is by including P_Cl. The relative values of P_K:/P_Cl in non-capacitated sperm are similar to the ones seen in other cell types (see text). All permeabilities given are relative to P_K in SLO3 mutant sperm at pH 7.4, which is assigned a value of 1.00. The curves correspond to mean n = 11 experiments. (C) The predicted permeabilities and p values for each permeability in the GHK fit are shown. All measured membrane potential values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060578#pone.0060578.s005" target="_blank">table S1</a>.</p

Amiloride or the reduction of external Na⁺ blocks most P_Na, leaving the membrane dominated by P_K.

<p>Amiloride treatment (A) and low external Na⁺ (B) leave the sperm membrane dominated by P_K in both wild-type and in SLO3 mutant sperm. After the addition of amiloride the P_K to P_Na ratio is somewhat larger in wild-type than in the SLO3 mutant, reflecting the activity of SLO3 K⁺ channels in the membrane (see text). Although the SLO3 channel is absent in SLO3 mutant sperm, the dominance of P_K over all other ion permeabilities in SLO3 mutant sperm is additional evidence for the presence of a K⁺ leak conductance in SLO3 mutant sperm plasma membrane. GHK fits did not require inclusion of P_Cl (see text). Since we cannot accurately predict the internal sodium concentration when external Na⁺ is reduced to 1 mM, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060578#pone-0060578-g006" target="_blank">figure 6B</a> is fitted with least squares linear regression to compare the resulting slopes with that of a theoretical line illustrating pure potassium selectivity (red). Permeability values predicted by the GHK equation for A are given in (C). The curves correspond to mean n = 4 experiments ± S.E.M. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060578#pone.0060578.s010" target="_blank">table S6</a> for membrane potential values.</p