Study On The Transcriptional Activity Of FOXO3 Inhibiting Transcription Factor SOHLH1 On Oocyte-specific Gene Bmp15/Gdf9

Objective:The activation of follicles does not depend on gonadotropins,but on the signal exchange between oocytes and granulosa cells.Our research group has found that the transcription factor SOHLH1 can affect the follicular development by up-regulating c-Kit,affecting the communication between oocytes and granulosa cells.However,it is still unclear which transcription factors are regulated by other paracrine factors BMP15 and GDF9 in follicles.In this study,we explored whether FOXO3 and SOHLH1 have a regulatory effect on Bmp15 and Gdf9,and explored the reasons why BMP15 and GDF9 proteins could not be expressed in the primordial follicular phase.Methods:In this study,6-8w C57BL/6J mouse ovary was used as the research object.Ch IP assay was used to find the binding sites of SOHLH1 and FOXO3 on the promoter sequences of Bmp15 and Gdf9 genes.By constructing Bmp15,Gdf9 promoter expression vector and FOXO3 eukaryotic expression The vector,the dual luciferase reporter gene was used to study the transcriptional regulatory activities of the FOXO3 and SOHLH1 proteins on the promoter sequences of Bmp15 and Gdf9 genes.Results: 1.The pc DNA3.0-FOXO3 expression plasmid,p GL3.0-Bmp15 fluorescent plasmid and p GL3.0-Gdf9 fluorescent plasmid were successfully constructed.2.FOXO3 overexpression plasmid was transfected into HEK293 T cells for 24 h and 48 h,and FOXO3 protein was expressed in the nucleus and cytoplasm of HEK293 T cells.3.The SOHLH1 protein was expressed in the nucleus and cytoplasm of HEK293 T cells after transfection of SOHLH1 into HEK293 T cells for 24 h and 48 h.4.SOHLH1 has a positive regulatory effect on the full-length region of Bmp15 and Gdf9 promoters.5.FOXO3 inhibited the positive regulation of SOHLH1 on the full-length region of Bmp15 and Gdf9 promoters,and the inhibitory effect was stronger with the increase of FOXO3 content.6.SOHLH1 protein can bind to the E-box site of Bmp15 promoter(-170 bp,80bp),(-450 bp,-220bp),(-745 bp,-560 bp)and(-1410 bp,-1200 bp);SOHLH1 binds to the E-box site of the Gdf9 promoter(-525 bp,-340 bp),(-960 bp,-755bp)and(-1080 bp,-790bp),and SOHLH1 cannot bind to the Gdf9 promoter (-200 bp,0bp)Interval binding;8.FOXO3 can bind to Bmp15 promoter(120bp,183bp),(-84 bp,-75bp),(-442 bp,-395bp),(-546 bp,-470bp)and(-674 bp,-665bp)9.FOXO3 binds to the Gdf9 promoter(-278 bp,-271bp),(-1004 bp,-997bp).Conclusion:1.SOHLH1 can activate Bmp15 and Gdf9 promoter regions through transcription,affect the signal exchange between granulosa cells and oocytes,and regulate follicular development.2.In the primordial follicle stage,FOXO3 can affect the transcriptional activation of Bmp15 and Gdf9 by SOHLH1 through competitive binding of transcriptional regulatory sites.