Mechanism Of GnRH Regulating Adenohypophysis Gonadotropin Synthesis And Secretion Through FTO-Mediated M~6A Modification

The hypothalamic-pituitary-gonadal axis precisely controls reproductive activity in mammals.The pulsatile release of GnRH from the hypothalamus regulates reproductive activities such as gonadotropin development and gametogenesis by regulating the synthesis and secretion of gonadotropins from the pituitary gland.Reproductive hormones and their analogues have been widely used in the regulation of artificial reproduction in animals,but due to the limited knowledge of the mechanism of GnRH regulation of gonadotropin synthesis and secretion,the application effect in practical production is still unstable,and the relevant theoretical and technical"bottlenecks"need to be broken.The molecular mechanism by which GnRH regulates gonadotropin synthesis and secretion is very complex.The transcriptional regulation and post-transcriptional modification of Fshb and Lhb genes are the key steps to achieve their functions.The systematic analysis of the epigenetic mechanism of RNA methylation modification,in regulating gonadotropin synthesis and secretion can help to reveal the molecular mechanism of reproductive endocrinology and regulate the reproductive process of animals more precisely.In recent years,m~6A modification has been shown to play a key role in reproductive activities such as oocyte maturation,spermatogenesis,early embryonic development,and steroid hormone synthesis.However,the molecular mechanisms involved in the regulation of gonadotropin synthesis and secretion by m~6A modifications remain unclear.To elucidate the potential function of m~6A modifications in the regulation of gonadotropin synthesis and secretion by GnRH,transcriptomic and m~6A modification analyses were performed in rat adenohypophysis before and after GnRH treatment.Based on the differential gene expression profile and differential m~6A modification profile,the m~6A demethylase FTO and m~6A target gene Foxp2 were screened,which may respond to GnRH signal and participate in the regulation of gonadotropin synthesis and secretion;The primary rat adenohypophysis cells and LβT2 cell line were used as experimental materials.The study successively clarified the regulatory roles of FOXP2and FTO on gonadotropin synthesis and secretion,verified the interactions between FTO and Foxp2 m RNA,and elucidated the molecular mechanism by which FTO mediates m~6A modification to regulate FOXP2 expression and thus promote gonadotropin synthesis and secretion,through RIP,Me RIP-q PCR,RT-q PCR,ELISA and other experiments.The specific results are as follows:1.The differential gene expression profile of rat pituitary before and after GnRH treatment was successfully constructed.709 differentially expressed genes were identified,of which 438 were up-regulated and 271 were down-regulated,and the differentially expressed genes were mainly enriched in c AMP signaling response,steroid hormone synthesis,neuroactive ligand-receptor interaction and other physiological activities and signaling pathways.2.The differential m~6A modification profile of rat adenohypophysis before and after GnRH treatment was successfully constructed.6413 differential m~6A peaks were identified,of which 3764 were up-regulated and 2649 were down-regulated.A total of51.5%of the differential m~6A peaks were enriched in the 3’UTR region and mainly showed the"GGACU"motif.3.Two hundred and fifty differentially expressed genes after GnRH treatment were mined and identified to have both m~6A modification differences.Among them,the expression of demethylase FTO was significantly up-regulated;the expression of transcription factor FOXP2 was significantly up-regulated and the level of m~6A modification in its m RNA 3’UTR region was significantly down-regulated.4.GnRH significantly decreased the m~6A modification level of total RNA and promoted the expression of FTO and FOXP2 in gonadotropin cells.5.The up-regulated expression of FTO enhanced Foxp2 m RNA stability and promoted FOXP2 expression by reducing m~6A modification on Foxp2 m RNA and blocking the binding of YTHDF3 to m~6A modification on Foxp2 m RNA.6.The up-regulated expression of FOXP2 promoted gonadotropin synthesis and secretion by activating the c AMP/PKA signaling pathway.In summary,the m~6A modification in the pituitary was used as the entry point to investigate the regulation of gonadotropin synthesis and secretion by FOXP2,and the interactions between FTO,YTHDF3 and Foxp2 m RNA were systematically analyzed and annotated in this study.We have analyzed the molecular mechanism by which FTO attenuates the degradation of Foxp2 m RNA by YTHDF3 through mediating m~6A modification on Foxp2 m RNA,thereby regulating Fshb gene and Lhb gene expression through the c AMP/PKA signaling pathway.And revealed the potential mechanism of GnRH regulating the gonadotropin synthesis and secretion through FTO-mediated m~6A modification.These studies systematically interpreted the regulatory mechanism of gonadotropin synthesis and secretion in the pituitary at the epigenetic level and provided a theoretical basis for the application of reproductive hormones in the regulation of artificial reproduction in animals.