KCNQ1 Potassium Channel Expressed in Human Sperm Is Involved in Sperm Motility, Acrosome Reaction, Protein Tyrosine Phosphorylation, and Ion Homeostasis During Capacitation

Figures

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  Figure 1. Expression and localization of KCNQ1 and KCNE1 in human sperm. (A) Human sperm were lysed after capacitation for 3h. Sperm proteins were separated and analyzed using 10% SDS-PAGE and Western blotting, using antibodies for KCNQ1 (Abcam) and KCNE1 (Abcam). The figure is representative of 3 separate experiments. The full uncropped immunoblots was provided in the supplementary data (Supplementary Figure 1). (B) Indirect immunofluorescence of KCNQ1 (Santa Cruz) and KCNE1 (Invitrogen) in human sperm before and after capacitation. KCNQ1 (red), KCNE1 (green), and co-localization of KCNQ1 and KCNE1 (merge). The negative control cells were incubated with normal IgG as the primary antibodies. The nuclei of spermatozoa were stained blue with DAPI. The figure is representative of 3 separate experiments.

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  Figure 2. Chromanol 293B effect on human sperm AR and viability. (A) The human sperm AR was evaluated using PSA-FITC staining. Sperm were treated with different reagents (DMSO or 100μM chromanol 293B) for 3h during capacitation, and the sperm were then treated with 15μM progesterone for 15min to induce AR. To detect spontaneous AR before capacitation, sperm were stained with PSA-FITC immediately after discontinuous Percoll gradient centrifugation and washing. AR indicates acrosome-reacted sperm, while acrosome integrity (AI) indicates uncapacitated sperm. (B) After PSA-FITC staining, sperm were examined by fluorescence microscopy. The AR ratio is calculated by dividing the number of AR sperm with the total number of sperm. For 100 μM chromanol 293B group compared to vehicle control group, *p<0.05. Propidium iodide (PI) is used to stain dead sperm. Percentage of not viable cells (NVC%) was calculated. Values represent the means ± SEM of at least 6 experiments.

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  Figure 3. Effect of chromanol 293B on protein tyrosine phosphorylation during human sperm capacitation. (A) Western blotting results showing the effect of chromanol 293B on protein tyrosine phosphorylation during human sperm capacitation. Sperm before capacitation incubation were used as the control group. Other sperm were treated with the vehicle control (DMSO), 20μM chromanol 293B, and 100μM chromanol 293B under capacitated conditions for 30min and 3h. Thereafter, sperm were lysed and then the proteins were resolved using 10% SDS-PAGE. Protein tyrosine phosphorylation was detected using a primary anti-phosphotyrosine antibody by Western blotting. Subsequently, the blot was stripped and probed with an anti-β-tubulin antibody as a loading control. The figure is representative of 6 separate experiments. The full uncropped immunoblots was provided in the supplementary data (Supplementary Figure 2). (B) The ratio of protein tyrosine phosphorylation levels to that of the loading control, β-tubulin. Values represent the means ± SEM of 6 experiments. For 100μM chromanol 293B group compared to vehicle control group, *p<0.05. (C) Indirect immunofluorescence of protein tyrosine phosphorylation in…

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  Figure 4. Effect of chromanol 293B on ion homeostasis during human sperm capacitation. (A) Intracellular K+ concentration ([K+]i) was detected using the K+ fluorescence probe PBFI-AM. The fluorescence intensity was directly proportional to [K+]i. (B) Human sperm membrane potential (Vm) was analyzed using the fluorescence probe DisC3(5). The fluorescence intensity was directly proportional to Vm. (C) Intracellular Ca2+ concentration ([Ca2+]i) was detected using the Ca2+ fluorescence probe Fluo3-AM. The fluorescence intensity was directly proportional to [Ca2+]i. (D) Intracellular Cl− concentration ([Cl−]i) was detected using the Cl− fluorescence probe MQAE. The fluorescence intensity was inversely proportional to [Cl−]i. (E) Intracellular pH (pHi) was analyzed using the pH fluorescence probe BCECF-AM. The fluorescence intensity was directly proportional to pHi. All the data were acquired at 3min intervals for 30min during capacitating incubation. After capacitation for 3h, the fluorescence signal was acquired again. Asterisks indicate the significant differences between samples and vehicle control at the same time point (*p<0.05). The exact P value were provided in the supplementary data…

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  Figure 5. Schematic representation of how KCNQ1 potassium channel regulates human sperm function. When human sperm are treated with chromanol 293B, the K+ outflow currents mediated by KCNQ1 are inhibited, which causes a [K+]i increase and the membrane depolarization. Vm changes regulate other voltage-gated channels and affect the electrical driving force for ions. Thus, [Cl−]i increases, while the pHi and [Ca2+]i decreases. It is generally known that during capacitation, Ca2+ and HCO3− activate soluble adenylate cyclase (sAC) which converts ATP into cAMP. Then PKA can be activated. PKA subsequently activates target proteins and protein tyrosine phosphorylation levels increase. KCNQ1 in human sperm participates in ion homeostasis regulation and affects capacitation.