Regulation of Ovarian Granulosa Cell Proliferation and Differentiation by the Notch Signaling PathwayPublic Deposited
The follicle is the functional unit of the mammalian ovary and serves as a tightly controlled microenvironment where granulosa cells nurture the growth and maturation of the oocyte. Early events in follicular development are autonomous of the hypothalamic-pituitary-gonadal axis and are instead controlled by local factors that are used in bidirectional communication between oocytes and somatic cells. Following puberty, gonadotropins will eventually become the principle regulators of follicular growth, maturation, and ovulation. It is becoming increasingly appreciated that gonadotropin actions are achieved through interactions with local intraovarian signals. This thesis explores the functions of one such local intraovarian regulator, the Notch signaling pathway, at multiple distinct stages of ovarian follicular development. The Notch pathway is a highly conserved cell-contact dependent juxtacrine signaling system and is an important regulator of primordial follicle formation in the mouse ovary. Using a reporter mouse line for identification of Notch active cells, continued Notch activity was observed in granulosa cells of follicles belonging to both the gondatropin independent and dependent stages of development. Based on this observation, I hypothesized that the Notch signaling pathway regulates distinct follicular functions at multiple stages of development to promote the overall health of the follicle. Active Notch signaling in granulosa cells was associated with greater proliferative capacity at the prepubertal age of postnatal day (PND) 21. The mitogenic function of Notch signaling was further confirmed using conditional knockout lines of the ligand Jag1 from germ cells (J1KO) or the receptor Notch2 from granulosa cells (N2KO) where suppressed proliferation in the ovary was observed at PND19. Significantly extending the understanding of Notch signaling during the gonadotropin-independent stage of follicular growth, a potential mechanism for Notch signaling propagation across multiple layers of granulosa cells in growing follicles was described. Activation of Notch signaling in granulosa cells led to increased expression of Jag1 ligand, which can transactivate Notch receptors on neighboring granulosa cells, consistent with a lateral induction mechanism. Notch activity and expression of Notch ligands and receptors in the ovary were shown to be positively regulated following LH-receptor activation by exogenous human chorionic gonadotropin (hCG) in prepubertal mice during the periovulatory period. The localization of JAG1, the most abundantly expressed Notch ligand in the mouse ovary, underwent a striking shift from oocytes to somatic cells following hormone stimulation. The function of JAG1 in ovarian steroidogenic somatic cells was investigated using a long-term primary granulosa cell culture system. Disruption of Notch signaling using siRNA knockdown of Jag1 resulted in a suppression of granulosa cell differentiation into the steroidogenic phenotype and a retention of proliferative capacity. The loss of JAG1 led to a maintenance of MAPK/ERK and PI3K/AKT pathway activation, which are associated with proliferation in granulosa cells. These results indicate that the Notch signaling pathway can influence the broader signaling network in granulosa cells in complex way. During the highly dynamic periovulatory period, active Notch signaling favors the promotion of the mature steroidogenic preovulatory phenotype, at the expense of continued proliferation. Collectively, this thesis work describes a multi-faceted role of Notch signaling as a regulator of granulosa cell proliferation and differentiation throughout the gonadotropin independent and dependent stages of follicular development.