The Mechanism Of RNA M6A Modification Regulating Sertoli Cell Proliferation And Functional Differentiation In Adolescent Rat Testis

Objective: The process of mammalian spermatogenesis involves a complex maturation process of the germinal epithelium in the testis,including proliferation of germinal tubule cells before puberty,precise regulation of the differentiation of various cells within the testis by the hypothalamic-pituitary-gonadal axis at puberty,and the orderly maintenance of the microenvironment of spermatogenesis in the adult testis.Sertoli cells are important cells in the germinal epithelium,which proliferate rapidly under the influence of hormones such as FSH before puberty and stop proliferating in adulthood,gradually specializing into mature Sertoli cells that maintain the development of germ cells and regulate the blood-testis barrier.Recent studies have shown that N6-methyladenosine(m6A)modification-related enzymes,such as METTL3/14/WTAP,are involved in mammalian spermatogenesis and play an important role.m6 A modification-related enzymes dynamically modify and recognize m6 A,thereby affecting downstream RNA stability,intracellular localization,nuclear output,degradation,and protein translation.This paper aims to discuss the functional and physiological significance of m6A-modified transcripts in the proliferation and maturation transition of Sertoli cells in the germinal epithelium,caused by differential expression of m6 A modification-related enzymes between immature and mature Sertoli cells,and to clarify the dominant role of key m6 A differentially modified genes in the transition of Sertoli cells from a proliferative state to a mature state.FSH,as a key factor in the proliferation of Sertoli cells,triggers changes in signaling pathways during the transition from proliferation to differentiation of Sertoli cells,and its specific mechanism of action is still unknown.Considering the difference in serum levels of FSH between juvenile and adolescent rats,we decided to further investigate the role of RNA m6 A modification in FSH-promoted Sertoli cell proliferation and development before puberty in rats,and to clarify the key regulatory factors involved in the transition of Sertoli cells from the proliferative state to the mature state.Methods: 1、Extract immature Sertoli cells from 10-day-old SD rats and mature Sertoli cells from 20-day-old SD rats,compare their transcription levels of m6 A modificationrelated enzymes by qPCR,and compare the protein levels of m6 A modification-related enzymes by Western blotting.2、Purify and culture immature Sertoli cells from 10-dayold SD rats and mature Sertoli cells from 20-day-old SD rats,extract total RNA,perform m6A-RIP and conventional transcriptome sequencing,compare the transcriptome differences and m6 A modification differences between P10 and P20 Sertoli cells.3、Statistical analysis of differential m6 A modification patterns in P10 and P20 rat Sertoli cells.4、Correlation analysis of Me RIP-Seq and RNA-Seq data for differential m6A-modified transcripts in P10 and P20 rat Sertoli cells.5、Use the joint GEO database to perform intersection and union analysis of genes related to FSH involvement in Sertoli cell proliferation and development.6、Treat Sertoli cells with FSH to detect the expression levels of methylation-related enzymes in Sertoli cells.7、Use the CCK-8 method to screen the reasonable time and concentration of the Mettl3 inhibitor STM2457 to treat Sertoli cells,and detect the m6 A level after inhibitor treatment by ELISA.8、Use the EdU method to detect the proliferation ability of P10 Sertoli cells after STM2457 treatment,and use PI staining to detect their cell cycle.9、Treat P20 Sertoli cells with the Mettl3 inhibitor STM2457,detect the levels of BTBrelated proteins by Western blotting,and detect the distribution of BTB proteins by immunofluorescence.10、Use the GEO data in combination with m6A-RIP to screen key factors that promote Sertoli cell proliferation by FSH,and use SELECT-qPCR to identify the critical m6 A modification site of Wee1.11、Treat P10 Sertoli cells with FSH and the Mettl3 inhibitor STM2457,detect the transcription level of Wee1 by qPCR,and detect the abundance of critical m6 A modification sites of Wee1 by SELECT-qPCR,and detect the expression level of Wee1 protein by Western blotting.12、Treat P10 Sertoli cells with FSH and the Mettl3 inhibitor STM2457 in combination,and detect the expression level of Wee1 protein by Western blotting.13、After co-treatment of Sertoli cells with FSH and ACTD,The stability of Wee1 transcripts was examined by qPCR.14、Western blotting analysis was performed to measure the expression levels of downstream effector proteins of Wee1,including p-Cdc2-Y15,and other G2/M phase regulatory proteins in P10 Sertoli cells treated with FSH.Conclusion: 1、There are a large number of m6A-modified differentially expressed genes in Sertoli cells of P10 and P20 SD rats,which are closely related to the physiological state of Sertoli cells.2、FSH affects the m6 A modification level of a specific site in Wee1 through Mettl3.The m6 A modification of this site determines the stability and protein level of Wee1 transcripts,thereby regulating the downstream cell cycle G2/M transition and affecting the proliferation process of Sertoli cells.