Single cell transcriptomic analysis of the ovarian immune microenvironment reveals hormone-dependent changes

Abstract

Abstract The ovary is a dynamic organ, where, driven by the estrous cycle, cells undergo continuous waves of apoptosis, proliferation, and differentiation. These changes parallel fluctuations in ovarian hormones such as estradiol (E2), however, the immune microenvironment during high-E2 and low-E2 states has not been described. We induced a high-E2 state by superovulation with i.p. injection of gonadotropins (GN) into 10 week old C57BL/6 mice. Flow cytometry and single-cell RNA sequencing revealed abundant NK cells, B cells, CD8 +T cells, CD4 +T cells, CD3 +TCRβ +CD4 −CD8 −“double negative” T cells, and several distinct myeloid subsets. The frequency of NK cells in the CD45+ compartment was 14% in GN-treated mice and 7% in controls, suggesting increased proliferation or recruitment of NK cells in response to E2. Conversely, mature B cells represented 8% of CD45 +cells in GN-treated mice and 18% in controls. To determine whether CD45 +cells were recruited from circulation or ovarian resident cells, we injected fluorescently-labelled α-CD45 i.v. into live mice and performed flow cytometry on ovaries, spleen, and blood. The majority (88–96%) of ovarian CD45 +cells were unlabeled, suggesting that they were ovary-resident. To assess population dynamics during the naturally occurring low-E2 state of reproductive aging, we performed flow cytometry on ovaries of 40 week old mice. Interestingly, we identified significant increases in the number of macrophages and double-negative T cells in aged ovaries. Our findings reveal a diverse resident immune landscape in the ovary that responds robustly to hormonal changes. These findings may have implications for immune-mediated causes of infertility such as primary ovarian insufficiency and endometriosis. Support by a grant from the NIH 3P30GM118228-05S3