The SLO3 sperm-specific potassium channel plays a vital role in male fertility

Abstract

Here we show a unique example of male infertility conferred by a gene knockout of the sperm-specific, pH-dependent SLO3 potassium channel. In striking contrast to wild-type sperm which undergo membrane hyperpolarization during capacitation, we found that SLO3 mutant sperm undergo membrane depolarization. Several defects in SLO3 mutant sperm are evident under capacitating conditions, including impaired motility, a bent "hairpin" shape, and failure to undergo the acrosome reaction (AR). The failure of AR is rescued by valinomycin which hyperpolarizes mutant sperm. Thus SLO3 is the principal potassium channel responsible for capacitation-induced hyperpolarization, and membrane hyperpolarization is crucial to the AR.

Fig 1. SLO3 channel is absent in mutant mice as illustrated by western blot analysis

Equal amounts of membrane protein from testis of wild type (wt)(+/+), heterozygous mutant (+/−) and homozygous mutant (−/−) mice were stained with anti-SLO3 antibody (methods). The SLO3 protein was undetectable in homozygous mutants (−/−) and present in approximately half the wt amount in heterozygous (+/−) mice.

Fig 2. SLO3 mutant sperm lack capacitation induced hyperpolarization and, in contrast to wt, depolarize after capacitation

Membrane potentials before and after in vitro capacitation are illustrated for wt and SLO3 homozygous mutant (−/−) sperm (error bars show SEM). Sperm membrane potential was measured using the fluorescence dye DiSC3 as described in methods. Uncapacitated wt sperm have a membrane potential value of −47 ± 3.3 mV and hyperpolarize to −62.2 ± 2.9 mV (n =4) after capacitation. Uncapacitated SLO3 mutant sperm have a membrane potential of −48 mV ± 3.9 mV and depolarize during capacitation to −38.8 ± 2.8 mV (n = 6). Membrane potential differences between wt and mutant sperm are significant for all pairwise comparisons at the 0.05 % level (paired t-test).

Fig 3. Mutant SLO3 sperm lack pH sensitive K⁺ currents

Representative whole cell voltage clamp recordings and current/voltage relationships from testicular sperm are shown for wt (A) and SLO3 mutant sperm (B). Whole cell currents were evoked by 10 mV voltage steps from −80 to +60 mV at a holding potential of −50 mV. Currents are shown in control conditions and after bath application of 40 mM NH₄Cl. In control experiments with wt sperm we noted that the relative increase in outward current amplitude after alkalization with NH₄Cl was somewhat smaller than that reported in a previous paper (Navarro et al, 2007). However this may be due to the different stages of sperm that were analyzed; in our study testicular sperm were used, while in the previous study, more mature epididymal sperm were used. The bar graphs in (C) show a comparison of outward current amplitudes evoked at +60 mV for wt (n=4) and mutant SLO3 sperm (n=4), before (control) and after (NH₄Cl) exposure to 40 mM NH₄Cl.

Fig 4. Mutant SLO3 sperm display less progressive motility

Percentage of progressive motility is shown for wt and mutant SLO3 ^−/− sperm. Sperm showing progressive motility were counted as a percentage of total sperm using computer assisted sperm analysis (CASA). While 31.3 ± 0.7 % of wt sperm showed progressive motility, only 9 ± 2.1 % of mutant sperm showed progressive motility for both wt (n=3) and mutant (n=3) animals; approximately 1000 sperm were counted for each animal). Differences were significant (p < 0.05).

Fig 5. Acrosome reaction (AR) induced by A23187 is impaired in SLO3 mutant sperm, & rescued by valinomycin

The percentage of spontaneous AR is similar in wild-type (WT) and SLO3 mutant sperm after exposure to capacitating conditions. However, the addition of A23187 greatly increases AR in WT (p≪.001) but has no significant effect on SLO3 mutant sperm (p=0.09). The addition of valinomycin, however, to A23187 greatly increases the AR reaction in SLO3 mutant sperm (p<0.02). 200 sperm for each condition were counted as acrosome reacted (no staining in the acrosomal region) or as acrosome intact (dark blue staining over the acrosomal region). [Numbers of animals for each condition: WT spontaneous=13; WT A23187=9; WT valinomycin=3; WT A23187+valinomycin=3; SLO3 mutant (SLO3): SLO3 spontaneous=11; SLO3 A23187=8; SLO3 valinomycin=3; SLO3 valinomycin=A23187=3]

Fig 6. Sperm from SLO3 mutants are not capable of fertilization under in vitro conditions

Results of in vitro fertilization (IVF) experiments performed with wt(n = 2 animals) and SLO3 mutant sperm from 2 animals a. % of two stage cell embryos observed after IVF using wt or SLO3 mutant sperm. Capacitated wt and mutant SLO3 mutant sperm were incubated with superovulated oocytes with ZP intact from C57BL/6 wt mice (145 and 134 eggs respectively), and with the ZP removed (94 and 88 eggs respectively). The percentage (respectively for wt and SLO3 mutant sperm) of two-stage embryos was: 79% (114/145) and 1.5% (2/134) respectively in intact-ZP eggs, and 65% (61/94) and 2% (2/88) in ZP-free eggs. b. representative micrographs of intact-ZP eggs after 24 hs of fertilization with wt or SLO3 mutant sperm. c. representative micrographs of eggs with ZP removed after 24 hrs of fertilization with wt or mutant SLO3 sperm.