Physiological basis of contraceptive function of a GSK3α-selective inhibitor, manifested during epididymal sperm maturation

Glycogen synthase kinase 3 alpha (GSK3α) has emerged as a prospective target to develop male contraceptives. Global or testis-specific loss of GSK3α, but not GSK3β, results in male infertility. Previous studies demonstrated that the GSK3α-selective inhibitor BRD0705 induces reversible infertility in heterozygous mice. Here, we show that BRD0705 induces similar infertility in wild-type male mice. Fertility outcomes remain largely unaltered following superovulation in female mice mated with BRD0705-treated males. Further, no evidence of foetal resorption was observed in these females. Sperm count in treated mice were not altered; however, morphological defects appeared during their passage through the epididymis. The GSK3α inhibitor affected pathways related to the enzyme's catalytic activity in epididymal sperm. Phosphoproteomic analysis of sperm from inhibitor-treated mice showed that phosphorylation of over 50 proteins involved in motility, metabolism, and fertility, among others, were significantly altered. Among the highly hypophosphorylated proteins, four are likely substrates of GSK3 (viz., AKAP4, NT5C1B, SPATA18, and TRA2B). Analysis of the seminiferous tubules of BRD0705-treated mice showed normal spermatogenesis, suggesting that the drug did not affect developing spermatogenic cells. However, in vitro exposure of post-meiotic germ cells demonstrated their susceptibility to this inhibitor. BRD0705, therefore, shows only a non-genomic effect at the post-translational level. These findings support GSK3α inhibition via BRD0705 as a non-hormonal, on-demand and reversible male contraceptive strategy.