Characterization of human oocyte development and the role of mitochondrial activity in in vitro germ cell differentiation

Abstract

The mammalian ovary performs two primary functions: the production of germ cells and the release of steroid hormones. The ovarian cortex contains many follicles, each composed of a single oocyte surrounded by somatic support cells; these somatic support cells consist of granulosa and theca cells, which become steroidogenic as the follicle grows. These cell types together form the basis for human oocyte support and maturation, as well as participate in the hypothalamic-pituitary-gonadal (HPG) axis to maintain hormone production and signaling. During embryonic development, ovarian follicles are formed when migrating mitotic primordial germ cells (PGCs) reach the gonad, associate with a layer of pre-granulosa cells, initiate meiosis, and subsequently remain in meiotic arrest until sexual maturity. Upon follicle activation, the pre-granulosa cells proliferate and begin hormone generation, the follicle grows in size, and the oocyte is ovulated for possible fertilization. Because follicular assembly and cell specification is established during embryonic development, the signals governing these transitions are not fully elucidated and only recently tools have become available to study these complex cellular differentiation stages and interactions. Through the use of a quantitative proteomics analysis of the human ovary during the first and second trimesters of development, I established a baseline protein expression profile during the establishment of the ovary when PGCs are newly differentiating into oogonia through the stages of follicle formation and into adulthood. This screen allowed me to validate markers of germ cells not previously identified in human cells such as MAEL and TEX11 and show that they could be used in stem cell models of in vitro germ cell differentiation. Additionally, I provide evidence for the in vitro differentiation of granulosa cells from human pluripotent stem cells. This global protein expression profile also enabled me to further probe signaling cascades that have not previously been investigated in human folliculogenesis, including the finding for support of canonical WNT signaling in the oocyte at the time of follicle assembly; it also allowed me to investigate the transition of metabolism and metabolic control and identify that the fetal ovary is highly reliant on mitochondrial activity during PGC differentiation and follicle formation. To highlight the role of mitochondria in germ cell differentiation, I further utilized newly validated techniques for germ cell differentiation from pluripotent stem cells; by carefully tracking mitochondrial activity during the initial stages of PGC differentiation I found that the mitochondrial activity of the precursor stem cells to PGCs influences differentiation, and that stem cells that are more slowly proliferating have less active mitochondria yet preferentially differentiate into PGCs in vitro. The compilation of a well-defined source of stem cell derived oocytes and in vitro derived granulosa cells will allow the observation of human follicle formation in vitro and enable detailed mechanistic studies related to proper follicle maturation and relevant pathologies.