(316) The Microenvironment Matters: Follicular Fluid and Microfluidic Chip-Derived Granulosa Cell Extracellular Vesicles Influence Oocyte Maturation and Embryo Development in the Domestic Cat

The Microenvironment Matters: Follicular Fluid and Microfluidic Chip-Derived Granulosa Cell Extracellular Vesicles Influence Oocyte Maturation and Embryo Development in the Domestic Cat

Kathryn M. Storey¹; Ahmed Gad²; Nucharin Songsasen¹; Dawit Tesfaye²; and Jennifer B. Nagashima¹

1. Center for Species Survival, Smithsonian’s National Zoo and Conservation Biology Institute, Front Royal, VA, USA

2. College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA

Abstract Text:

Recently, extracellular vesicles (EVs) sourced from ovarian follicular fluids have been shown to play critical roles in follicular communication and oocyte development, including influencing post-thaw oocyte meiotic resumption and in vitro embryo production. To harness the potential role of EVs in assisted reproduction applications, a greater understanding of the hormonal microenvironment impact on EV biogenesis and subsequent function, plus the ability to consistently produce high-quality EVs in vitro, are needed.

Toward these goals, we utilized a microfluidic chip culture system to generate EVs from granulosa cells (gcEVs) under gonadotropin stimulation conditions mimetic of natural estrus (NE-gcEVs) compared with gcEVs produced without hormone addition (control gcEVs), and those collected from fresh follicular fluid (ffEVs). Briefly, granulosa cells were isolated by mechanical scraping from >1.5mm diameter (large antral stage) cat (Felis catus) ovarian follicles. P2 cells were seeded into the microfluidic chips and then incubated for 5 days under 2 ul/min flow with regulated, pulsatile release of 3-5 IU/L FSH and 2.5-20 IU/L LH, representative of natural estrus, including mimicry of the LH surge. Cell viability after 5 days was >90% under flow conditions and spent medium was collected for EV isolation. EV yield was 4.74e+9 particles/follicle for ffEVs and averaged 23,200 particles/cultured cell for gcEVs. Median diameters were 192.1±136.9nm (ffEVs), 133.6±75.2nm (gcEVs, cultured 48 hours), and 237.7±156.1nm (gcEVs, cultured 108 hours). Transmission electron microscopy images confirmed lipid-bound membranes on fresh ffEVs and generated gcEVs.

To test the hypothesis that oocytes exposed to NE-gcEVs or ffEVs (ie EVs from gonadotropin-primed cells) have improved embryo development rates compared with control gcEVs or no EV supplementation, immature cumulus-oocyte complexes were assigned to five treatment groups in four replicates (N=23 cats, n = 9-12 oocytes/treatment/replicate): Minimum Essential Medium Eagle (MEM) [base], 60 μg/mL EVs from follicular fluid (ffEVs), control granulosa cells (control gcEVs), or hormonally stimulated granulosa cells (NE-gcEVs) in MEM, and Quinns Advantage Protein Plus Blastocyst Medium [Quinn, positive control]. Following IVM, oocytes were subjected to in vitro fertilization (IVF) using domestic cat epididymal sperm and subsequently incubated in MEM-based medium with/without EVs depending on treatment group for embryo culture for an additional 3 days to evaluate development. Embryos were produced in all treatments, with cleavage rate per oocyte determined at 48 hours post IVF, averaging (Mean ±SD) 72±7% for base, 27±9% for ffEV, 40±17% for control gcEV, 46±31% for NE-gcEV, and 36±22% for Quinn. Embryos reached the morula stage in each group, with an average of 41±16% for base, 20±11% for ffEV, 19±11% for control gcEV, 28±18% for NE-gcEV, and 21±18% for Quinn. No statistically significant differences (P>0.05) were observed in cleavage or morula rates with the current sample size, though gcEVs yielded higher cleavage rates compared to ffEVs.

Cumulatively, these data indicate protein-free conditions, such as in the base treatment group, may have a slightly positive impact on the very early stage of cat embryo development. Previous work by our laboratory demonstrated larger impacts of EV supplementation, which contains protein cargo, on later-stage embryonic development. Thus, future research will evaluate the impacts of EVs in more extended embryo culture, as well as understand how EV molecular cargo changes with varying gonadotropin stimulation conditions. Understanding these EV-mediated cell-cell communication and cargo delivery mechanisms have the potential to improve oocyte quality in assisted reproductive technologies for both human and endangered species fertility preservation efforts.