Bicuculline

Sperm were capacitated in PFM for 4 h, and GABA solution was added to each suspension at the final concentrations of 0 (PBS), 0.1, 1, 10, and 100 μM, and the samples (10⁷ cells/ml) were incubated for 30 min at 38.5°C under 5% CO₂ in the air. Thereafter, each sample was smeared onto glass slides and air‐dried. After 60 min of blocking using Blocking One, the acrosome reaction was assessed by staining with FITC‐PNA (1:500) and Hoechst 33342 (1:2000) in a light‐shielded humidity chamber. Thereafter, the slides were washed with PBS and covered with mounting medium and glass coverslips. Bicuculline (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan), a GABA_A receptor antagonist, was dissolved in DMSO and added to sperm suspensions in the presence of PBS or GABA (1 μM). All the groups contained 0.1% DMSO. Sperm acrosomal disappearance rates were evaluated by calculating the number of PNA‐negative sperm among total sperm. We regarded sperm that lost more than 80% of their acrosomes to be acrosome reaction sperm. The acrosome‐reacted sperm were counted under a fluorescence microscope (BZ‐X710; Keyence). Duplicate counting of at least 100 sperm cells was performed. The percentage of sperm with no fluorescence over the acrosomal region was calculated as the number of PNA‐negative sperm cells divided by the total counted sperm.