(279) Extracellular vesicles of follicular fluid preserve follicular integrity and maintain estradiol production in ovarian tissue cultured in vitro

L.V. S. Ñaupas¹, K. Cañón-Beltrán², Y.N. Cajas³, R. Mazzarella⁴, A. Cordova-Gomez ⁵, R. H. Céliz Ygnacio⁶,  E.M. González⁷, A. Gutierrez-Adán ⁴, D.Rizos⁴, A. P. R. Rodrigues¹

1. Laboratory of Manipulation of Oocytes and Ovarian Pre-Antral Follicles, Faculty of Veterinary Medicine, State University of Ceará, Fortaleza, CE, Brazil.

2. Department of Biochemistry and Molecular Biology, Veterinary Faculty, Complutense University of Madrid (UCM), Madrid, Spain.

3. Department Agrarian Production, Technical University of Madrid, Madrid, Spain.

4. Department of Animal Reproduction, National Institute for Agriculture and Food Research and Technology (INIA), Madrid, Spain.

5. Peruvian University Cayetano Heredia, Faculty of Sciences and Engineering, Lima, Peru.

6. National Institute of Innovation Agraria – INIA, Lima, Perú.

7. Department of Anatomy and Embryology, Veterinary Faculty, UCM, Madrid, Spain.

Abstract Text:

Cryopreservation followed by in vitro culture of ovarian tissue is a promising strategy to preserve the fertility of cancer patients and to conserve genetic resources from endangered or high-value animals. Although in vitro culture (IVC) is an alternative to transplantation - which carries the risk of reintroducing cancer cells in humans and is costly in animals- IVC protocols still yield a low percentage of follicles reaching advanced stages. The use of extracellular vesicles from follicular fluid (EVs-FF), which facilitate intercellular communication, is emerging as a promising approach to improve these protocols, with demonstrated potential in oocyte maturation and embryonic development. In this study, we tested the hypothesis that supplementing the in vitro culture medium with ovarian tissue from prepuberal sheep with ovine and bovine EVs-FF improves follicular survival and development. To test this hypothesis, EVs were previously isolated and characterized by the Nanosight technique and transmission electron microscopy. For in vitro culture, ovaries from five sheep were fragmented and allocated into four groups: fresh (CTR, n=10); cultured in vitro in alpha minimum essential medium (α-MEM) without EVs-FF (IVc-ø, n=10); with 1 x 10^6/mL of ovine EVs-FF (IVc-O, n=10); and with 1 x 10⁶/mL of bovine EVs-FF (IVc-B, n=10) for 12 days. Fragments were analyzed by classical histology (follicular morphology and development) and qPCR-RT for receptor for follicle stimulating hormone (FSH-R) and Bone morphogenetic protein 15 (BMP15), genes related to folliculogenesis. Furthermore, estradiol (E₂) levels in the culture medium were measured on days 2, 4, 6, 8, 9, 10, and 12 by enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using SPSS® software, and the Kruskal-Wallis H test was applied for comparisons, considering significant differences when P < 0.05. The results showed that the percentage of normal follicles in IVc-O remained similar (P > 0.05) to CTR and higher (P < 0.05) than IVc-ø. There was a significant increase (P < 0.05) of developing follicles in all cultured groups. IVc-O presented higher levels of E₂ (P < 0.05) compared to IVc-ø and IVc-B, whose levels decreased after day 4. The FSH-R gene had higher expression (P < 0.05) in IVc-O, and BMP15 showed higher expression (P < 0.05) in CTR, IVc-O, and IVc-B compared to IVc-ø. In conclusion, supplementation with EVs-FF in the culture of prepubertal sheep ovarian tissue has positive effects on morphological preservation, E₂ maintenance, and the expression of genes related to folliculogenesis, with effects that appear to be species-specific.