| Objective:Ovary is one of the most critical organs in the production system of female mammals.As the basic structure and functional unit in ovarian development,follicle is consist of an oocyte and surrounding granular cells.Follicle development starts from primordial germ cell(PGC)during embryonic period.PGC forms primordial follicle at birth,undergoes primary follicle,secondary follicle,antral follicle,and finally differentiates to mature and ovulate.The proper number of initial follicles and the rate of follicle atresia ensure the normal process of early follicle development and remain the female reproductive age at an appropriate length.The number of primordial follicles no longer increases after formation,which is determining the initial number of follicles in female mammals;Abnormal rate of follicle atresia can be caused by a large number of cell death.Insufficient initial follicle number or accelerated rate of early follicular atresia can exhaust follicle and premature ovarian failure,leading to a shortening of female reproductive age.Sohlh1,one of the candidate genes for premature ovarian failure,plays an important role in the early stages of follicle development.As a reproductive transcription factor,it has the capabilities to specifically bind the E-Box site(CANNTG)of promoter and proceed transcriptional regulation.In female mammals,Sohlh1 only expresses in embryonic ovarian oogonium and oocytes of primordial follicles and primary follicle.The results showed that the atresia of Sohlh1 did not affect the number of initial follicles,but it could accelerate the rate of early follicular follicles atresia until the knockout mice were of premature ovarian failure.We hypothesized that Sohlh1 may influence the development of early follicles by avoiding early follicle accelerated atresia,but the mechanism of early follicle accelerated atresia that due to the absence of Sohlh1 is still not clear.The apoptosis of cells in follicle is the primary cause leading to follicular atresia.BCL2L10,GDF9 and BMP15 play an important roles in follicular growth and atresia.At the same time,we found that there were multiple Sohlh1 binding sites(E-Box)in Bcl2l10,Gdf9 and Bmp15 promoter regions,which may be directly regulated by Sohlh1.Bcl2l10,as an anti-apoptotic factor,is one of BCL2 protein family members,and its expression overlaps with the original and primary follicular phase as the same as Sohlh1.Bcl2l10 is capable of expressing on the mitochondrial membrane of oocyte and binding to the apoptotic factor Bax to maintain the permeability of mitochondrial membrane,and eventually plays an anti-apoptotic role in the mitochondrial apoptosis pathway of oocyte.Gdf9 and Bmp15 are both growth factors of the TGF? family,and their expressions overlap in early follicular phase with Sohlh1's in oocyte.Gdf9 and Bmp15 are paracrined to the intercellular space.They are essential factors in the early follicular by forming a heterodimer,binding to granular cell surface receptors,and activating SMAD pathway in granulosa cells to regulate granulosa cell growth and differentiation.According to the previous studies,we assume that Sohlh1 is likely to regulate atresia of primordial follicles and primary follicles by affecting Bcl2l10,Gdf9 and Bmp15,and finally impacts the number of follicles and the reproductive age of female mice.This study aims to discover the morphology and molecular mechanisms of the Sohlh1(-/-)mice early follicle atresia and sift candidate genes leading to follicular atresia: Bcl2l10,Gdf9,and Bmp15.It will provide a theoretical basis for further clarifying Sohlh1 gene function and clinical diagnosis and treatment of premature ovarian failure and infertility.Methods: In this study,the littermate PD7.5 Sohlh1 knockout mice and the wild type C57BL/6J mice were studied to analyze the differences of early follicle development,apoptosis,and ultrastructure.In order to screen the candidate genes causing the abnormal development of early follicles,gene chip analysis was performed on the ovary of PD7.5 Sohlh1 knockout mice and the wild type C57BL/6J mice;Luciferase reporter assays and chromatin immunoprecipitation experiment were conducted to determine whether candidate genes,Bcl2l10,Bmp15,and Gdf9,were transcriptionally regulated by Sohlh1.Results: 1.Morphological observation revealed that PD7.5 WT mice had primordial,primary,and secondary follicular structures,and the number of early follicles was high.Compared with WT littermates,PD7.5 Sohlh1(-/-)mice had primordial follicle atresia rapidly.No normal primary follicles were observed,while abnormal primary follicles with none oocytes and cubic type granulosa cells were surrounding occasionally.Severe congestion presented at medulla of ovary and Ovarian hilum.2.The TUNEL test found that there was a significant difference in the apoptosis of ovarian cells between WT mice and PD7.5 Sohlh1(-/-)mice.3.Apoptosis and necrosis in the atresic ovary could be observed in knockout mice via transmission electron microscope.Apoptosis occured in atresic follicles precedes in granulosa cells.However,follicular development was stable in PD7.5 WT mice.4.Gene expression profile chip technology for ovary indicated that knockout mice,compared with WT,was lack of Sohlh1 that would lead to difference expression in 828 genes.After cluster analysis was implemented,the chip results showed that differentially expressed genes were primarily concentrated in the categories associated with follicular development,including reproductive related,apoptosis and inflammation related,cell cycle and meiotic related,and metabolism related.5.q PCR results have proved a portion of follicle atresia associated genes differentially expression and they were consistent with the trend of gene chip results,including reproductive related transcription factors(Figla,Nobox,Pou5f1),members of the family of TGF-beta(Gdf9,Bmp15),other reproductive related gene(Nlrp5,Nlrp14,Oosp1,Fshr),cell cycle(Cdk4,Ccnd2),and apoptosis related gene(Bax,Bad,Bcl2l10).In knockout mice,the expression was significantly down-regulated,while the expression of other apoptotic genes(Bcl2,Caspase3)was up-regulated.6.Several Sohlh1 binding sites were recognized in the Gdf9,Bmp15,Bcl2l10 gene promoter regions.Dual luciferase report assays were conducted to confirm that Sohlh1 was capable of activating Gdf9,Bmp15,Bcl2l10 promoter regions.7.The chromatin immunoprecipitation Ch IP experiment indicated that Sohlh1 was able to bind multiple sites containing E-Box sites at upstream of Gdf9,Bmp15 and Bcl2l10 promoter regions.Conclusion: 1.Apoptosis and necrosis of cells in the ovary is an vital cause of accelerated follicular atresia in Sohlh1 knockout mice,and the follicle atresia begins with apoptosis in the oocyte.2.In PD7.5 mouse ovaries,atresia of Sohlh1 results in828 genes expression differences.The candidate genes associated with follicular atresia were sifted and classified,including reproductive related,apoptosis related,inflammatory response related,cell cycle and meiosis related,and metabolism related.Abnormal expression of these genes may be an important cause of accelerated follicular atresia in Sohlh1 knockout mice.3.Sohlh1 is able to specifically bind to multiple sites in the promoter region of early follicles development related gene,Bmp15,Gdf9,and Bcl2l10,and has transcriptional activation. |
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