Single-cell analysis of ovarian immune cells reveals dynamic changes in NK and B-cell populations after ovarian stimulation.

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; however, the ovarian immune microenvironment during high- and low-estradiol states remains incompletely defined. We induced a high-estradiol state in the mouse ovary by gonadotropin stimulation. Single-cell RNA sequencing and flow cytometry 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 indicated that the vast majority of ovarian leukocytes were tissue resident. Following gonadotropin treatment, the frequency of NK cells doubled, whereas that of B1 cells was reduced by half. Consistently, flow cytometry demonstrated an increase in mature CD11b+ NK cells after gonadotropin treatment. Cell-cell communication analysis further showed that gonadotropin treatment increased signaling by myeloid cells at the expense of NK cells. Collectively, these findings reveal a diverse, resident immune landscape in the ovary that responds robustly to hormonal changes, with implications for immune regulation in ovarian physiology and in pathological states.