Investigating the physiological significance of follicular extracellular vesicle miRNAs: From gonadotropin control of biogenesis to application in oocyte vitrification - Project Summary As the average age of first-time mothers in the United States increases, the prevalence of assisted reproductive technologies (ARTs) including oocyte cryopreservation, continues to rise. Cryopreservation has been demonstrated to reduce expression of genes associated with cell cycle progression, and use of cryopreserved ova is correlated with lower live birth rates compared with fresh eggs. Recently, extracellular vesicles (EVs, lipid-bound vesicles secreted by cells which contain regulatory molecules) have shown promise in supporting the recovery or function of damaged cells. In reproductive EVs, miRNAs have been of particular interest owing to their potential ability to regulate key signaling pathways associated with developmental competence. Supplementation of EVs from ovarian follicular fluid (ffEV) have been shown to improve in vitro blastocyst production and, in our laboratory, enhance the domestic cat cumulus-oocyte complex’s (COC) ability to resume meiosis following vitrification. In this proposal, we describe a series of studies aimed at elucidating the physiological role(s) of miRNAs in ffEVs and exploring their therapeutic potential using the domestic cat as a model for human ARTs. As exogenous gonadotropin stimulation protocols are known to modify follicular gene expression and function, including composition of ffEVs, we will apply microfluidic technology to mimic gonadotropin exposure patterns on granulosa cells in vitro. Specific Aim 1 improves our knowledge of the in vitro generation of EVs by comparing the molecular (miRNA, mRNA, protein) composition of EVs produced under ‘natural estrus’ versus ‘ovarian stimulation’ conditions in vitro against in vivo derived ffEVs, and their efficacy in modulating COC gene expression, developmental competence, and embryo quality. Beyond improving our understanding of gonadotropin control of EV biogenesis, this approach aims to improve our ability to consistently produce high quality EVs in vitro, which is vital to their future application to ARTs. Specific Aim 2 will elucidate the functional relevance of miRNAs enriched in ffEVs using a two-pronged approach: selectively inhibiting three highly expressed endogenous miRNA in ffEVs (via miRNA inhibitors), and loading three under-expressed exogenous miRNA (via miRNA mimics). The proposal targets heat shock 70 kDA protein expression to modulate the cell stress-response and developmental competence. We will evaluate the bioavailability and intracellular localization of miRNA modified-ffEVs and (in single and multiple miRNA combinations) their ability to alter COC gene and protein expression and subsequent influence on vitrified oocyte cryo-recuperation and embryonic development. Cumulatively, these studies will generate new insight into miRNA-mediated intrafollicular communication and the downstream effects of follicular fluid EVs on oocytes, develop a new system for biomimetic reproductive EV production in vitro, and assess the utility of ffEVs for future therapeutic application to ARTs, including oocyte vitrification. ,
