Pharmacological inhibition of KSper impairs flagellar pH homeostasis of human spermatozoa.

Background: Sperm-specific potassium channel (KSper) comprised of pore-forming subunit SLO3 and auxiliary subunit LRRC52 is of importance for sperm fertility. The deficiency of KSper in both mice and humans resulted in severe impairments of sperm functions including sperm hyperactivity and acrosome reaction. Previous reports suggested that mouse KSper modulated sperm function possibly by affecting sperm intracellular pH (pH_i). However, the precise signaling mechanism of human KSper (hKSper) on the regulation of sperm functions was largely unclear.

Objective: To explore the regulatory role of hKSper on sperm flagellar pH_i.

Materials and methods: More than 50 sperm donors were recruited during a period of 1 year. As reported in our previous work, we quantitatively measured flagellar pH_i by employing a single-cell pH fluorescent recording on human spermatozoa loaded with pH indicator pHrodo Red. Three different hKSper antagonists including clofilium, quinidine, and a polyclonal antibody of LRRC52 (LID1) were utilized to evaluate the effect of hKSper inhibition on sperm flagellar pH_i.

Results: Given the predominant role of hKSper on the regulation of membrane potential (Em), we first detected a considerable depolarization (about 25-30 mV) of Em evoked by clofilium and quinidine. Subsequently, it was shown that flagellar pH_i values of human spermatozoa were significantly decreased by the treatment of clofilium (50 µM, from 7.13 ± 0.11 to 6.43 ± 0.12), quinidine (500 µM, from 7.00 ± 0.11 to 6.64 ± 0.08) and LID1 (20 µg/mL, from 6.98 ± 0.16 to 6.67 ± 0.22). Moreover, we found that when human spermatozoa were pre-incubated with a high K⁺ solution (135 mM), both the depolarization of Em and the acidification of flagellar pH_i evoked by clofilium and quinidine were abolished. In addition, we found that extracellular substitution of N-methyl-D-glucamine for Na⁺ abolished pH_i acidification induced by hKSper inhibition.

Discussion and conclusion: Our results demonstrate that hKSper inhibition evokes flagellar pH_i acidification of human spermatozoa, suggesting that flagellar pH_i maintenance is an important signaling mechanism of hKSper on the regulation of sperm functions.