(341) cGAS-STING Pathway Induction in Ovarian Follicles Elicits an Inflammatory Response via Type I Interferons and Compromises Development

Prianka H. Hashim¹, Aubrey K. Converse¹, Fatimah Aljubran¹, Jennifer L. Gerton², Francesca E. Duncan

1. Department of Obstetrics & Gynecology, Northwestern University, Chicago, IL, USA

2. Stowers Institute for Medical Research, Kansas City, MO, USA

Abstract Text:

The ovarian follicle, composed of an oocyte and surrounding somatic cells, is a non-renewable unit requiring tight developmental control to eliminate poor-quality oocytes and prioritize fertilization of high-quality oocytes. The cGAS-STING pathway is a known mediator of the innate immune response in many tissues activated by the presence of cytosolic double-stranded DNA (dsDNA) and resulting in the transcription of Type I interferons and pro-inflammatory cytokines. To determine if the cGAS-STING pathway is present in ovarian follicles, localization of pathway machinery was assessed at both the RNA and protein level. cGAS is expressed in oocytes and STING in granulosa and theca cells, with cGAS expression significantly increasing during the primary to secondary follicle transition. Utilizing cGAS knockout animals, follicle counts were performed with no significant differences observed in follicle numbers or class distributions compared to Wild-Type animals at 6 weeks and 12 months of age. Additionally, cGAS knockout animals were fertile similar to controls indicating that the cGAS-STING pathway is not constantly active or upregulated with age. This leaves questions of why the machinery of the cGAS-STING pathway is present in ovarian follicles. We hypothesize that the cGAS-STING pathway, a cytosolic DNA-sensing mechanism known to trigger inflammation, may act as a surveillance mechanism in follicles to detect double-stranded DNA generated in response to exogenous stimuli and promote removal of compromised follicles.To assess the function and responsiveness of the cGAS-STING pathway, early secondary follicles were isolated from mice and treated with Q-VD-OPH & ABT-737 or a vehicle control for up to 10 days to induce pathway activation. Cell viability staining after short-term treatment demonstrated that treatment was not toxic to follicles. This treatment activated the cGAS-STING pathway, confirmed by increased phospho-IRF3, -TBK1, and -STING expression relative to controls. To determine whether follicles produced a cGAS-dependent inflammatory signature after treatment, follicles from cGAS Wild-Type or Knockout mice treated with Q-VD-OPH & ABT-737 or a vehicle control were submitted for RNASeq 24 hours after treatment. Results indicate that wild-type treated follicles produce an interferon-dependent inflammatory response that is not replicated in treated cGAS KO follicles. An ex vivo follicle growth assay was utilized to investigate the impact of pathway activation on follicle and oocyte outcomes. Short- and long-term treatment of cultured wild-type follicles have significantly decreased follicle survival, growth, and increased atretic oocytes compared to controls. GV oocytes collected from treated follicles on the terminal day of culture were significantly smaller than GV oocytes from vehicle-treated follicles. Follicles cultured from knockout animals suggest a decreased responsiveness to pharmacological activation with increased survival relative to controls, indicating specificity of these compounds to the cGAS-STING pathway. We have demonstrated that the cGAS-STING pathway is functional and responsive in early secondary follicles in response to pharmacologic induction. Early secondary follicles remain responsive with both short- and long-term compound exposure. We speculate that the cGAS-STING pathway functions as an active surveillance mechanism to remove poor quality oocytes from the growing pool. Future studies will investigate what genes and pathways contribute to decreased follicle survival and growth upon cGAS activation after Q-VD-OPH and ABT-737 treatment, how responsiveness of the cGAS-STING pathway changes with reproductive age and how knockout animals may use alternative mechanisms to respond to exogenous stimuli.

Funding: This work was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (R01HD105752).