Notch Signaling Regulates Differentiation and Steroidogenesis in Female Mouse Ovarian Granulosa Cells

The Notch pathway is a highly conserved juxtacrine signaling mechanism that is important for many cellular processes during development, including differentiation and proliferation. Although Notch is important during ovarian follicle formation and early development, its functions during the gonadotropin-dependent stages of follicle development are largely unexplored. We observed positive regulation of Notch activity and expression of Notch ligands and receptors following activation of the luteinizing hormone-receptor in prepubertal mouse ovary. JAG1, the most abundantly expressed Notch ligand in mouse ovary, revealed a striking shift in localization from oocytes to somatic cells following hormone stimulation. Using primary cultures of granulosa cells, we investigated the functions of Jag1 using small interfering RNA knockdown. The loss of JAG1 led to suppression of granulosa cell differentiation as marked by reduced expression of enzymes and factors involved in steroid biosynthesis, and in steroid secretion. Jag1 knockdown also resulted in enhanced cell proliferation. These phenotypes were replicated, although less robustly, following knockdown of the obligate canonical Notch transcription factor RBPJ. Intracellular signaling analysis revealed increased activation of the mitogenic phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways following Notch knockdown, with a mitogen-activated protein kinase kinase inhibitor blocking the enhanced proliferation observed in Jag1 knockdown granulosa cells. Activation of YB-1, a known regulator of granulosa cell differentiation genes, was suppressed by Jag1 knockdown. Overall, this study reveals a role of Notch signaling in promoting the differentiation of preovulatory granulosa cells, adding to the diverse functions of Notch in the mammalian ovary. (Endocrinology 159: 184-198, 2018) A s key structural and functional units of the female gonad, ovarian follicles are responsible for the growth and ovulation of high-quality oocytes that ensure fertility, as well as for the production of steroid and peptide hormones important for reproductive physiology. The formation, growth, and function of these follicles are controlled by networks of intraovarian and endocrine signals. Local factors, acting through paracrine and juxtacrine mechanisms, are important for follicle formation and early development to the secondary follicle stage. These include, but are not limited to, estrogen (1-3), progesterone (4),