The Role Of TOX3 In Ovarian Follicle Development

Follicle is the basic functional unit of female reproduction,consisting of germ cells and somatic cells.Follucular development was a process that the functional unit develops until ovulation.TOX3 was one of the most important susceptibility genes identified in the gemome-wide association study(GWAS)in polycytic ovary syndrome(PCOS)by our study group.However,the functional role of TOX3 during follicular development has yet to be known.In this study,we explored the role of TOX3 in follicular development using TOX3 knockout mice,TOX3-oocyte considitional knockout mice,TOX3-granulosa cell conditional knockout mice and human granulosa cell lines.Chapter ? TOX3 did not affect oocyte maturationObjective:TOX3,a member of the TOX high mobility group-box protein group family,contains an HMG-box structural domain and is predicted to be able to affect downstream gene transcription by influencing transcription factors binding to DNA.The functional role of TOX3 during follicle development is scarsly reported.In this chapter,we first investigated the expression pattern of TOX3 in the ovary of mice and explored the specific role of TOX3 during follicle development with the help of TOX3 knockout mice model and TOX3-oocyte conditional knockout mouse model.Methods:The expression pattern of TOX3 in wild type mouse ovaries was first analyzed.TOX3 KO-first mice were constructed using gene targeting technology,which can be used to observe TOX3-Null phenotype.Next,the flox locus knock-in mice of TOX3(TOX3fl/fl mice)was obtained by crossing TOX3 KO-first mice with Flp mice.In this chapter,to obtain oocyte-specific knockout TOX3 mice,TOX3fl/fl mice were crossed with Gdf9-Cre and Zp3-Cre instrumental mice to generate oocyte-specific knockout mice from primordial follicles and primary follicles,respectively.The effect of TOX3-specific knockout on reproductive phenotype was explored with the help of hematoxylin-eosin staining,immunohistochemical staining and fertility testing.Results:Real-time quantitative PCR and western blot showed that TOX3 was ubiquitously expressed in various tissues of mice,mainly in brain,lung,ovary and testis.Immunohistochemical staining of mouse ovaries showed that TOX3 was highly expressed in mouse ovarian epithelium,early follicular oocytes and granulosa cells,with granulosa cells predominating.Heterozygous mice showed no significant abnormalities in follicular development or fertility.We proceeded to succesfully construct two strains of TOX3 oocyte-specific knockout mice,Tox3fl/fl;Gdf9-Cre mice,which knock out TOX3 from the primordial follicular stage in oocytes,and Tox3fl/fl;Zp3-Cre mice,which knock out TOX3 from the primary follicular stage in oocytes.Normal developing follicles at all levels were visible by H&E staining of mouse ovarian sections,and there were no significant abnormalities in ovarian appearance and follicular morphology.Conclusion:TOX3 was lethal by systemic knockout,and normal follicle development was noticed at birth.TOX3 is indispensable for oogenesis.Chapter ? TOX3 regulates primordial follicle activation by regulating inhibin synthesisObjective:The normal development of ovarian follicles ensures the functioning of the normal female reproductive cycle,a process that cannot be achieved without the synergistic interactions between germ cells and somatic cells.The granulosa cells are the most important members of the follicular somatic cells,providing essential nutrients,growth factors and steroid hormones to the oocytes during follicular development.Abnormal granulosa cell function affects follicular development directly,thus causing reproductive dysfunction.In this chapter,we use TOX3 granulosa cell-specific knockout mice to investigate the mechanism of action of TOX3 in regulating follicle development.Methods:In this study,we obtained conditional knockout mice that knocked out TOX3 in granulosa cells from the embryonic stage by crossing TOX3fl/fl mice with Foxl2-Cre instrumental mice.The mice were observed for ovarian appearance and weighed;ovarian morphology of mice at different days of age was observed using hematoxylin-eosin staining and serially sectioned for follicle counting;the oocyte-corona-cumulus complex was removed from the oviductal jugular after 44 h of PMSG+hCG injection for 16 h.The number of ovulated oocytes was counted after hyaluronidase digestion;female mice were tested for fertility.Granulosa cells were extracted from mice and the cell cycle was analyzed by flow cytometry.The apoptosis level of granulosa cells was analyzed by TUNEL staining.Immunofluorescence staining was used to investigate whether TOX3 affects primordial follicle activation.Transcriptome sequencing was performed using 5-day-old mouse ovaries and combined with chromatin immunoprecipitation sequencing to analyze the downstream genes regulated by TOX3.Gene set enrichment analysis(GSEA),GO and KEGG were used to analyze the enrichment pathway of differential genes to explore the specific mechanisms affecting follicle development after TOX3-specific knockout.Results:a.After succesfully constructing an early mouse granulosa cell conditional knockout TOX3 mouse model,the knockdown efficiency was verified at DNA,mRNA and protein levels.b.3-day-old TOX3fl/fl;Foxl2-Cre mice ovaries did not exhibit significant differences.At 5 days of age,there was an increase in the number of primordial follicles and a decrease in the number of primary follicles in TOX3fl/fl;Foxl2-Cre mice.At 14 and 21 days of age,the number of secondary and antral follicles were significantly lower in TOX3fl/fl;Foxl2-Cre mice.c.TOX3fl/fl;Foxl2-Cre mice ovulated significantly less than TOX3fl/fl mice.d.TOX3fl/fl;Foxl2-Cre mice showed a significantly subfertility phenotype,as evidenced by fewer births and fewer litters per litter.e.Cell cycle assays suggested that the granulosa cells of TOX3fl/fl;Foxl2-Cre mice showed a G2/M phase block.Real-time quantitative PCR and western blot assays showed a significant increase in the cell cycle-related protein p21.f.TUNEL staining showed an increase in the proportion of granulosa cells with apoptosis after TOX3 knockout and a decrease in the proportion of granulosa cells with apoptosis when TOX3 was overexpressed.g.Immunofluorescence assays showed that TOX3fl/fl;Foxl2-Cre mice showed significantly lower nucleation rate of FOXO3a,suggesting that primordial follicle activation was inhibited after TOX3 knockout in granulosa cells.h.Transcriptome sequencing using 5-day-old TOX3fl/fl mice and TOX3fl/fl;Foxl2-Cre mice ovaries revealed several early granulosa cell differentiation-related genes expressed differently.i.The chromatin immunoprecipitation sequencing(ChIP-seq)experiments combined with transcriptome sequencing in the granulosa cells showed that TOX3 could directly bind to the INHA gene promoter and regulate its expression to affect follicle development.Conclusion:TOX3 deletion in early granulosa cells of mice affects primordial follicle activation and causing subfertility of females.Further studies revealed TOX3 regulates early follicle development by directly enhancing transcription of INHA.Chapter ? TOX3 affects estradiol synthesis by enhancing expression of CYP19A1Objective:In the ovary,granulosa cells are essential to endocrine functions and play a crucial role in follicle development.In the previous chapter,we found that conditional knockdown of TOX3 in granulosa cells resulted in a reduced proportion of primordial follicle activation and impaired follicular development.In this chapter,we continue to investigate the mechanism played by TOX3 during steroidogenesis in granulosa cellsMethods:In this study,mouse serum steroid hormone levels were measured by radioimmunoassay.Primary mouse granulosa cells were counted and ceiled,and steroid hormone levels were measured in the cell culture medium.Then estrogen levels were corrected by protein concentration in the corresponding well plates.To inquire thoroughly on the mechanism of TOX3 regulating estradiol production,human granulosa cell line with the ability to synthesize estradiol were used.Lentivirus transfection was used to establish granulosa cell lines with stable overexpression or knockout of TOX3.Cell viability was detected by CCK-8 assay.TUNEL staining and flow cytometry were used to detect apoptosis rate.Electrochemiluminescence was used to detect estrogen levels in granulosa cell culture system.Transcriptome sequencing was used to investigate the effect of TOX3 on the function of estradiol synthesis in granulosa cells.The expression of estrogen synthesis-related genes was detected by real-time quantitative PCR and western blot.The transcriptional regulatory function of TOX3 in granulosa cells was detected by ChIP-qPCR.Results:a.Serum levels of estradiol and testosterone in TOX3fl/fl;Foxl2-Cre mice did not differ from those in the control group,and progesterone levels were significantly lower.There was no difference in estrogen synthesis levels in the supernatant of mouse granulosa cells cultured in vitro.b.A TOX3 overexpression stable cell line was successfully constructed,and the granulosa cells overexpressing TOX3 showed a significant higher cell viability than control group.c.TUNEL staining and apoptosis assay suggested that apoptosis was reduced after TOX3 overexpressed and was increased after TOX3 knocked out.d.Estradiol synthesis was increased when TOX3 was overexpressed and decreased when TOX3 was knocked out.e.Transcriptome sequencing of TOX3 overexpression stable cell lines showed significant changes in 2508 genes.GSEA analysis enriched to steroid hormone synthesis,cell cycle and DNA replication pathways.The significant alterations of several genes related to estrogen synthesis in the steroid hormone synthesis pathway contains CYP19A1,BMP6 and FSHR.These differentially expressed genes were verified by real-time quantitative PCR and western blot.f.ChIP-qPCR experiments showed that TOX3 binds to the promoter region of CYP19A1,suggesting that TOX3 affects estrogen synthesis by regulating the expression of CYP19A1 directly.Conclusion:This study found that TOX3 regulates estrogen synthesis in granulosa cells by directly regulating the transcription of CYP19A1.