Studies On The Effects And Mechanisms Of Oocyte MTOR Signaling In The Control Of Oogenesis And Follicular Development

The quality of oocytes determines the reproductive potential of a woman and the health of the offspring.Therefore,the production of an egg fully competent to support fertilization and the development of a healthy individual is key to the success of reproduction and the ensurance of woman's reproductive health.Egg production and follicular development are well-integrated processes,which proceed in an orderly way under the control of both the autonomous genetic program intrinsic to the oocytes(generally referred to as intrinsic factors)and the extrinsic factors(e.g.,different hormones and growth factors,metabolites and nutrients,as well as stress)surrounding the oocyte in the follicular microenvironment.However,how the oocyte integrates these intrinsic and extrinsic factors to promote its own development,as well as those of the granulosa cells,remains largely unclear.The mechanistic target of rapamycin(MTOR)pathway is an evolutionarily conserved energy sensing pathway within the cell.Given the well known functions of MTOR in irritating the cues from both the intra-and extra-cellular nutrient,stress,hormones and growth factors,we propose that this pathway fulfills a similar role in oocytes,which is crucial for promoting the development of both the oocyte and granulosa cells.We therefore explored the effects and mechanisms of oocyte MTOR signaling in the control of oocyte and follicle development and maturation by conditional knockout of Mtor in oocytes.Prior to this endeavor,we first investigated the localization and activation status of MTOR pathway in the ovarian follicles at different stages of development.Immunohistochemical analysis revealed that the proteins of MTOR pathway are localized and activated in specific patterns with the ovaries.MTOR and the active p-MTOR are expressed in the oocytes of follicles at all developmental stages,with higher expression observed in oocytes and granulosa cells of secondary and onward follicles.The active form of p-RPS6 and p-4EBP1,two major downstream effectors of MTOR,are only expressed at higher levels in oocytes of secondary and onward follicles and large antral follicles,respectively.p-RPS6 and p-4EBP1 start to express in some granulosa cells of secondary follicles,and are enriched in cumulus cells and preantral granulosa cells of large antral follicles.These specific patterns of expression and activation of MTOR-pathway proteins in the ovaries hence indicate that this pathway may participate in the control of different events during oogenesis and follicular development.Gdf9-CRE mediated oocyte-specific deletion of Mtor causes female infertility.Development of the first wave of follicles in these cKO mice are defective with restricted growth of the secondary follicles,and reduced levels of expression of BMP 15 and GDF9,which results in almost anovulation.Deterioration of the follicular defect was found in the cKO ovaries as the mice turning older,and the cKO females display typical syndromes of primary ovarian insufficiency(POI).Oocytes of the early stage growing follicles express excessive levels of ?H2AX and degenerate;and associated with the striking shrinkage in the size of the ovaries,the levels of estradiol and anti-Mullarian hormone in circulation are decreased,while levels of luteinizing hormone are increased.At the age of six months,there are essentially no normal follicles present in the ovaries,and many unusual-looking follicles resembling in appearance to the testicular seminiferous tubules were prevalent in the cKO ovarian cortex.Transcriptome sequencing revealed that the expression of genes characteristic to granulosa cells are reduced,while some genes specific to testicular Sertoli or Leydig cells are expressed in these cKO ovaries.Granulosa cells in these cKO follicles turns into Sertoli-like cells in appearance and express CLAUDIN5,a component of testicular tight junctions.These data thus indicate that MTOR signaling in oocytes of primordial follicles is indispensable for granulosa cell fate determination and identity maintenanceZp3-CRE mediated oocyte-specific deletion of Mtor also causes female nearly infertile,however,it does not affect folliculogenesis.These cKO mice can ovulate,but most of the ovulated oocytes fail to complete the first meiosis normally,and are nearly unable to be fertilized and to support the preimplantation development.Proteomic and transcriptomic analyses revealed that the expression a cohort of genes associated with MTOR signaling-related metabolic pathways,cytoskeleton,spindle and cell cycle are downregulated in the cKO oocytes.These changes,especially the downregulation of expression of PRC1,a regulator of cytokinesis,are probably the key causes for the meiotic defects.Therefore,MTOR signaling in the oocyte of follicles at primary and onward stages plays crucial roles in the control of meiosis and development competence.Taken together,this thesis reveals the crucial roles of oocyte MTOR signaling in follicular development.The effects of this pathway on oocyte and granulosa cell development are follicle stage-specific.During the initial stages of follicle development,oocyte MTOR signaling maintains granulosa cell identity and promotes follicle growth by preventing the accumulation of excessive levels of DNA damages and stimulating the expression key oocyte-derived paracrine factors.While at later stages of follicular development,particularly during the process of oocyte meiotic maturation,it controls oocyte quality by regulating the expression of genes essential for completion of meiosis and acquisition of developmental competence in oocytes.These observations hence open new avenues for dissecting the causes for POI-one of the leading reproductive diseases in women,and for metabolic disorder-oriented decline in egg quality that is prevalent in patients with polycystic ovarian syndrome,obesity or diabetes.