METTL3‐Mediated m6A Modification Promotes the Stability of PLCζ mRNA in Sperm

Male infertility is a global health concern, with sperm dysfunction being a major contributing factor. Phospholipase C zeta (PLCζ), a key sperm-specific factor essential for oocyte activation, is closely linked to fertilization failure. This study investigated the role of METTL3-mediated N6-methyladenosine (m6A) modification in regulating PLCζ expression and sperm function. The mRNA and protein expression levels of PLCζ, METTL3, METTL14, and WTAP in sperm were measured using qRT-PCR and western blotting, respectively. The morphology of the sperm was evaluated by Giemsa staining. The zinc ion content of the semen was measured using the 5-Br-PAPS colorimetric method, and the citrate concentration and the total m6A level were measured using commercial kits. The m6A modification level of PLCζ mRNA in mouse sperm and the binding interaction between METTL3 and PLCζ mRNA were analyzed by RNA immunoprecipitation (RIP). Finally, the stability of PLCζ mRNA was examined using an actinomycin d-mediated transcription inhibition assay. The results showed that PLCζ expression was downregulated in the semen of patients with low fertilization rates (LFR), and its level correlated positively with reduced zinc/citrate content. These patients also exhibited a marked decrease in global m6A modification levels. Inhibition of METTL3 downregulated PLCζ expression in mouse sperm and was accompanied by impaired prostate secretory function. Mechanistically, METTL3 inhibition reduced the stability of PLCζ mRNA by decreasing its m6A modification. In conclusion, METTL3 enhances the stability of PLCζ mRNA through m6A methylation, thereby maintaining its normal expression level in sperm and ultimately supporting fertilization capacity and secretory homeostasis of the prostate.