(358) Somatic Cell Fate Specification and Separation in the Fetal Ovary

Yu-Ying Chen¹; Karina Rodriguez¹; Adriana Alexander¹; Saniya Rattan¹; Chang Liu¹; Xin Xu²; Brian Papas³; Humphrey H-C Yao¹

1.     Reproductive & Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, NC, 27709, USA

2.     Epigenetics & Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, NC, 27709, USA

3.     Integrative Bioinformatics Support Group, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, NC, 27709, USA


Abstract Text: Proper differentiation of somatic cell types in the fetal ovary lays the foundation for future ovarian function in adulthood. Defects in the formation and differentiation of these cells can lead to conditions including polycystic ovarian syndrome (PCOS), primary ovarian insufficiency (POI), and infertility in women. Understanding how each cell type is established is crucial in developing methods to intervene in ovarian diseases caused by cellular dysfunction. The ovary comprises two major somatic cell lineages: granulosa cells, which secrete hormones and provide nutrients to germ cells, and interstitial cells, which produce hormones and maintain ovarian structural integrity. While it is known that granulosa cells and interstitial cells originate from a common somatic progenitor in the fetal ovary, a critically unanswered question is how the two somatic cell lineages are specified apart from each other during ovarian development. To address this question, we profiled the transcriptomes of mouse ovarian cells at the onset of ovary formation and identified two major somatic populations: one positive for the nuclear receptor Nr2f2 and the other negative for Nr2f2. To determine the fate of the Nr2f2-positive progenitor cells, we performed lineage tracing by permanently marking these cells at the beginning of ovary formation and tracking their developmental trajectory. We found that Nr2f2-positive cells from the fetal ovary give rise to the interstitial population in the adult ovary. Next, to elucidate the signaling pathway differentiating Nr2f2-positive and Nr2f2-negative populations, we compared the two populations using single-cell RNA sequencing. We observed an inverse enrichment of Notch signaling components: the Nr2f2-positive population expressed Notch receptors and downstream transcription co-activators, whereas the Nr2f2 -negative population exhibited high level of the Notch antagonist Numb. These observations led us to hypothesize that active Notch signaling in Nr2f2-positive cells at the onset of ovary formation specifies interstitial lineage fate, whereas inactive Notch signaling in Nr2f2-negative cells promotes granulosa cell fate. To test this hypothesis, we first performed lineage tracing to permanently label Notch-active cells in the fetal ovary and follow their developmental trajectory. We discovered that Notch-active cells in the fetal ovary, similar to Nr2f2-positive cells, eventually differentiate into interstitial cells in the adult ovary, supporting that Nr2f2-positive and Notch-active cells belong to the same interstitial progenitors. Furthermore, to explore whether Notch signaling directs interstitial fate determination, we ectopically activated Notch pathway in the Nr2f2-negative population, in which Notch is normally inactive. Ectopic activation of Notch pathway resulted in a significant increase in interstitial cells and a decrease in granulosa cells and germ cells, disrupted ovarian morphology, and upregulation of pathways associated with epithelial-to-mesenchymal transition. These findings underscore the importance of balanced Notch signaling in determining cell type composition during ovarian development, and support a model that active Notch signaling in Nr2f2-positive cells specifies the interstitial cell lineage, separating them from the Nr2f2-negative granulosa cell lineage.