Ab230417

To detect the role of ABHD2 in sperm capacitation, spermatozoa from fertile (control) participants were incubated in a 5% CO₂ incubator at 37°C for 3.5 hours to enable sperm capacitation. Then, the spermatozoa were incubated with 3 μM progesterone for 2 hours with or without pretreatment with 100 μmol/L ABHD2 antibody (ab230417, Abcam, Cambridge, UK) or the PKA inhibitor H89 (MCE, Shanghai, China) for 15 minutes. Then, the effects of progesterone on cAMP, PKA, and acrosome reaction in spermatozoa were studied.

The spermatozoa samples were precipitated and lysed using cooled radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor to lyse the cells and then centrifuged at 10,000 × g and 4°C for 12 minutes to collect the protein. The protein was quantified by the bicinchoninic acid (BCA) method (Beyotime Biotechnology, Shanghai, China), and the loading buffer was added. After mixing, the cells were soaked in 100°C water for 5 minutes and cooled on ice. After sodium dodecyl sulfate-PAGE, the protein was transferred onto a polyvinylidene difluoride membrane, blocked in 5% nonfat milk for 1 hour, incubated with ABHD2 antibody (1:1000, ab230417, Abcam) overnight at 4°C, washed in PBS 3 times (10 minutes each), and incubated with horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG secondary antibody for 1 hour at room temperature. Then, enhanced chemiluminescence (ECL) with a substrate chromogenic solution (Thermo Fisher Scientific) was used to detect the signal by using the Bio-Rad Gel Doc XR+ (BioRad Laboratories, Hercules, CA, USA), and intensity was quantified using ImageJ (National Institutes for Health, Bethesda, MD, USA).