Circular RNA Role in Ovarian Follicular Development and Fertility

Project Summary Ovarian follicles undergo a series of dynamic changes following the ovulatory surge of luteinizing hormone including cumulus expansion, oocyte maturation, ovulation and luteinization. Post-transcriptional gene regulatory events are critical for mediating LH follicular responses and among all non-ribosomal RNA isoforms, circular RNA (circRNA) are the most abundantly expressed form of RNA, yet remain the least studied. Functionally, circRNA can act as miRNA sponges, protein sponges/decoys, regulators of transcription and translation. The role of circRNA in context to ovarian follicular development is relatively unknown, although their presence is known and our recent RNA sequencing studies of ovarian granulosa cells before and after the LH surge indicates the significant upregulation of circRNA, some of which have probable roles in preovulatory follicular development. Uniquely, KHDRBS1 (KH RNA Binding Domain Containing, Signal Transduction Associated 1) RNA element binding sites were identified in a large proportion of these LH-induced circRNA granulosa cell transcripts. KHDRBS1 is a recognized RNA binding protein with circRNA biogenic activity in other tissues and our preliminary results indicate high expression in ovarian granulosa cells. Moreover, global Khdrbs1 knockout female mice were reported to be severely sub-fertile. In this proposal, we will test the hypothesis that KHDRBS1 is a key regulator of LH induced circRNA biogenesis and of periovulatory events associated with ovulation. To investigate this hypothesis the following aims are proposed 1) Examine effect granulosa cell-specific KHDRBS1 loss has on LH-regulated circRNA biogenesis and fertility and 2) Examine LH-regulated circRNA molecular mechanism(s) of action and their functional impact on periovulatory follicular events. Aim 1 will use a granulosa cell-specific Khdrbs1 knockout mice (Khdrbs1fl/fl :Aromatase cre) female mice to address the role of this protein in circRNA biogenesis and its impact on fertility. In Aim 2, a complete functional analysis of several LH-regulated circRNA will be completed, to elucidate their mechanisms of action and whether these unique RNA molecules have roles in LH mediated events such as cumulus expansion, oocyte maturation, ovulation and luteinization. Studies in other systems indicate that circRNA are important modulators of cell differentiation and abnormal expression of circRNA has been associated with female reproductive diseases (i.e., primary ovarian insufficiency and polycystic ovarian syndrome).