METTL14 facilitates the process of sexual reversal via m6A RNA methylation in Pelodiscus sinensis

The Chinese soft-shelled turtle (Pelodiscus sinensis, P. sinensis) demonstrates noteworthy sexual dimorphism, where the males grow more rapidly and significantly larger than females under equivalent conditions. Estradiol (E2) administration can catalyze transformation from male to pseudo-female (PF), during which m6A RNA methylation undergoes considerable alterations. Nevertheless, the function of m6A methylation, specifically, the methyltransferase 14, N6-adenosine-methyltransferase non-catalytic subunit gene (METTL14) during this sex reversal process remains unclear. Within this study, we characterized the METTL14 gene, which was predominantly expressed within the ovary and demonstrated notable expression in PF individuals. Interference of METTL14 results in altered expression of methylation-related genes, yielding elevated RSPO1 expression and diminished AMH expression. Administration of E2 and METTL14-RNAi elicits 7994 differentially expressed genes (DEGs) during sexual differentiation, and KEGG enrichment analysis highlighted that METTL14 profoundly affects embryonic development through pathways including steroid hormone biosynthesis, ovarian steroidogenesis, tryptophan metabolism, and Glycolysis/Gluconeogenesis. Gene set enrichment analysis (GSEA) indicated that METTL14-RNAi triggers reduced expression of steroid hormone biosynthesis and ovarian steroidogenesis pathways while increasing the PPAR signaling pathway. In conclusion, METTL14-RNAi results in significant up-regulation of RSPO1 and down-regulation of AMH, inducing substantial alterations in pathways associated with hormone and metabolism. These findings propose that METTL14 may play a facilitating role during E2-induced sex reversal in P. sinensis, offering a novel avenue for further exploration into all-male breeding.