GSK3α-mediated regulation of the RNA demethylase fat mass and obesity-associated protein (FTO) during murine...

Idiopathic male infertility is a rising global concern characterised by ejaculatory defects, absence or low sperm count with abnormal morphology, and poor sperm motility. Deciphering the aetiology of male infertility requires a fundamental understanding of multiple spermatogenic events, including sperm maturation. Glycogen synthase kinase 3 paralog-α (GSK3α) plays a critical role in sperm maturation, specifically during epididymal motility, capacitation and hyperactivation. Methylation modulates mRNA transport, stability, turnover, and translational efficiency to meet cellular requirements. The mRNA demethylase, fat mass and obesity-associated protein (FTO), is a target for phosphorylation by GSK3α, suggesting the potential role of this enzyme in male fertility. This study focuses on the high-affinity spatiotemporal interaction between FTO and GSK3α to delineate the post-transcriptional modifications in the murine testis. Expression of Gsk3a and Fto increases temporally starting with day 18-20 postpartum testis, coinciding with meiosis I and gradually peaks by day 25-34 with the formation of spermatids and completion of spermatogenesis. Co-immunoprecipitation of GSK3α and GSK3β with FTO using respective antibodies and super-resolution microscopy shows a preferential interaction of GSK3α with FTO. Moreover, Gsk3a knockout mice showed significantly low m6A levels in testis, presumably due to enhanced FTO activity. In silico protein-protein docking and molecular dynamics analysis demonstrated an energetically favourable, consensus phosphorylation motif-dependent high-affinity interaction between FTO and GSK3α, further validating our observation. A specific missense mutation (Cys326 > Ser) permitted an additional GSK3-phosphorylation site in FTO, leading to teratozoospermia in a patient. Collectively, this study affirms GSK3α as a spatiotemporal regulator of FTO function inside the mammalian testis.