Ovulatory Dysfunction and Compromised Granulosa Cells: Where does Adar fit?

Abstract

Mural granulosa cells (mGCs) undergo waves of proliferation throughout the reproductive lifespan, and facilitate a timely gene expression response to the surge of luteinizing hormone in order to achieve ovulation and corpus luteum formation. High-throughput RNA sequencing has highlighted the amplitude of non-encoded RNA polymorphisms, particularly adenosine-to-inosine (A-to-I) transitions, in numerous tissues, but never in the somatic cells of the ovary. Such edits are catalyzed by the family of editing enzymes, adenosine deaminases acting on RNA (ADAR). Editing dependent and independent functions of adenosine deaminases acting on dsRNA affect codon usage, splice sites, transcript stability, and miRNA biogenesis. Physiological consequences of disrupting ADAR in murine cardiomyocytes and liver tissue include apoptosis and fibrosis. Utilizing AdarFL/FL /AromataseCre/+ and wild-type control littermate female mice, the current thesis examines the role of Adar in mGCs. RNAScope was performed to confirm Adar depletion in mGCs. Fertility was assessed in a 7-month breeding trial, and morphology and state of fibrosis of the ovaries were evaluated using H&E and Picrosirius Red staining. Vaginal lavages were performed to confirm mating and visualize vaginal epithelium patterning. Ovulation was assessed via intraperitoneal injection of PMSG and hCG. Morphology of AdarFL/FL /AromataseCre/+ ovaries at 16, 18, and 20 hours post-hCG administration was evaluated with H&E staining. Together, these data have shown that Adar in mGCs is critical for female fertility in mice. Mice lacking Adar exhibited extreme infertility with very few pups born despite recorded breeding attempts. Increased fibrosis was observed in ovaries of breeding trial mice lacking Adar compared to controls while H&E revealed intact follicles of varying sizes and the presence of luteal tissue. Estrous cycle pattern coordination was disrupted, and ovulation was delayed following exogenous gonadotropin administration. Oocytes were found trapped in follicles of a range of luteinization states in AdarFL/FL /AromataseCre/+ ovaries. A lack of Adar in mGCs disrupts coordination of ovulation and luteinization, underscoring the fundamental role of Adar in granulosa cell physiology. Further studies to distinguish editing-dependent and editing-independent roles of ADAR in mGCs are warranted. Future investigations regarding the increased fibrosis observed in AdarFL/FL /AromataseCre/+ breeding trial ovaries will determine the earliest age of significant fibrosis accumulation in AdarFL/FL /AromataseCre/+ ovaries. Long term, studies may develop to evaluate the role of ADAR in balancing inflammation and immunity in mGCs to promote female fertility.