# Investigating the physiological significance of follicular extracellular vesicle miRNAs: From gonadotropin control of biogenesis to application in oocyte vitrification

> **NIH NIH R01** · SMITHSONIAN INSTITUTION · 2024 · $255,458

## Abstract

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 utili...

## Key facts

- **NIH application ID:** 10801855
- **Project number:** 1R01HD110572-01A1
- **Recipient organization:** SMITHSONIAN INSTITUTION
- **Principal Investigator:** Jennifer Nagashima
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $255,458
- **Award type:** 1
- **Project period:** 2024-09-17 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10801855

## Citation

> US National Institutes of Health, RePORTER application 10801855, Investigating the physiological significance of follicular extracellular vesicle miRNAs: From gonadotropin control of biogenesis to application in oocyte vitrification (1R01HD110572-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10801855. Licensed CC0.

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