Single cell analysis of ovarian immune cells during homeostasis and hormonal flux reveals dynamic changes in NK and B cell populations in the periovulatory phase

The mammalian ovary is the dynamic end-organ of the hypothalamic pituitary ovarian axis. In this coordinated system, ovarian cells undergo continuous cycles of apoptosis, proliferation, and differentiation. These changes parallel fluctuations in ovarian hormones such as estradiol (E2); however, the ovarian immune microenvironment during high-E2 and low-E2 states is not fully understood. We induced a high-E2 state in the mouse ovary by stimulation via gonadotropin treatment. Single-cell RNA sequencing and flow cytometry analysis of ovarian leukocytes revealed abundant mature NK cells, B1 and B2 cells, CD8+ T cells, CD4+ T cells, mature CD4-CD8-T cells, T regulatory cells, and distinct myeloid subsets, including Trem2+ and Apoe+ macrophages. In vivo labeling of circulating cells determined that the vast majority of ovarian leukocytes were tissue-resident. Following gonadotropin treatment, the frequency of NK cells increased two-fold, while B1 cell frequency was reduced by half. Consistently, flow cytometric analysis revealed an increase in mature CD11b+ NK cells following gonadotropin treatment. Gonadotropin treatment also increased cell-cell signaling by myeloid cells at the expense of NK cells. Our findings reveal a diverse resident immune landscape in the ovary that responds robustly to hormonal changes. These findings have implications for a role for immune cells in ovarian physiology and functional dysregulation.