Volume: 56 Issue: 2
Year: 2025, Page: 261-267, Doi: https://doi.org/10.51966/jvas.2025.56.2.261-267
Received: Nov. 25, 2024 Accepted: Feb. 13, 2025 Published: June 30, 2025
Cumulus cells, derived from granulosa cells, play a vital role in supporting the maturation and development of oocytes. Exosomes are nanoparticles encapsulating bioactive molecules such as proteins, nucleic acids, enzymes, and metabolites, which are known to modulate cellular signaling pathways. The exosomes present in follicular fluid enhance the maturation of cumulus-oocyte complexes (COCs). In the current study, a total of 292 culture grade COCs collected from slaughterhouse ovaries were subjected to in vitro maturation (IVM) using two protocols: Group I (control), with 146 COCs matured at 38.5ºC for 24 h (physiological IVM) and Group II (EXO) with 146 COCs matured under identical conditions but supplemented with 1μL of exosomes per 100 μL of maturation medium. Both groups were incubated in a controlled environment of 5 per cent CO2 and 95 per cent relative humidity. Following maturation, cumulus expansion rates (Grade A 62.10 ± 3.99 vs 50.00 ± 2.24 %) and IVM rates (91.33 ± 1.69 vs 75.93 ± 2.85 %) were noted to be significantly higher (p≤ 0.01) in the exosome supplemented group (Group II) compared to the control (Group I). The relative expression of hyaluronan synthase 2 (HAS2), a key enzyme in cumulus expansion, depicted a significant upregulation of 2-fold (fold change FC= 2.10; p ≤ 0.05) correlating with enhanced morphological cumulus expansion. Additionally, the expression of caveolin 1 (CAV1), a gap junctional protein, exhibited a 1.4-fold increase (FC= 1.41; p > 0.05), though not statistically significant. These findings suggest a beneficial role of exosome supplementation during IVM in promoting cumulus cell functionality and developmental competence of bovine oocytes. The study underscores the potential of exosomes as a valuable supplement for improving the success rate of bovine in vitro embryo production.
Keywords: Cumulus cell, cumulus oocyte complex, exosomes, cumulus expansion, hyaluronan synthase 2, caveolin 1, in vitro maturation, bovine, real-time PCR
Aaron, J.W., Hsueh, T.E.Y., Phillip, A.B.C., Jones, S. and Thomas, H.J. 1984. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocrine Rev. 5: 76-127.
Allworth, A.E. and Albertini, D.F. 1993. Meiotic maturation in cultured bovine oocytes is accompanied by remodeling of the cumulus cell cytoskeleton. Dev. Biol. 158: 101-112.
Arya, T., Abhilash, R.S., Jayakumar, C., Amritha, A., Naicy, T., Harshan, H.M. and Revathy, M.M. 2023. Developmental competence of bovine oocytes in maturation media supplemented with follicular fluid exosomes. J. Vet. Anim. Sci. 54: 465-471.
Baena, V. and Terasaki, M. 2019. Three-dimensional organization of transzonal projections and other cytoplasmic extensions in the mouse ovarian follicle. Sci Rep. 9: 1-13.
Chen, C., Zhang, Z., Gu, X., Sheng, X., Xiao, L. and Wang, X. 2023. Exosomes: new regulators of reproductive development. Matr. Today Bio. 19: 1-13.
Defamie, N. and Mesnil, M. 2012. The modulation of gap junctional intercellular communication by lipid rafts. Biochem. Biophys. Acta. 1818: 1866-1869.
Fukui Y. 1990. Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro. Mol. Reprod. Dev. 26: 40–46.
Furnus, C.C., de Matos, D.G. and Moses D.F. 1998. Cumulus expansion during in vitro maturation of bovine oocytes: relationship with intracellular glutathione level and its role on subsequent embryo development. Mol. Reprod. Dev. 51: 76–83.
Gabrys, J., Gurgul, A., Szmatoła, T., Mitka, K.B., Andronowska, A., Karnas, E., Kucharski, M., Puchałka, J.; Kochan, W.J. and Poniewierska, B.M. 2024. Follicular fluid-derived extracellular vesicles influence on in vitro maturation of equine oocyte: impact on cumulus cell viability, expansion and transcriptome. Int. J. Mol. Sci. 25: 1-23.
Gao, J., Li, A., Hu, J., Feng, L., Liu, L.and Shen, Z. 2023. Recent developments in isolating methods for exosomes. Front. Bioeng. Biotechnol. 10: 1-17.
Gutnisky, C., Dalvit, G.C., Pintos, L.N., Thompson, J.G., Beconi, M.T. and Cetica, P.D. 2007. Influence of hyaluronic acid synthesis and cumulus mucification on bovine oocyte in vitro maturation, fertilisation and embryo development. Reprod. Fert. Dev. 19: 488-497.
Hung, W.T., Hong, X., Christenson, L.K., and McGinnis, L.K. 2015. Extracellular vesicles from bovine follicular fluid support cumulus expansion. Biol. Reprod. 93: 1-9.
Javadi, M., Rad, J.S., Pashaiasl, M., Farashah, M.S.G. and Roshangar, L. 2022. The effects of plasma derived extracellular vesicles on cumulus expansion and oocyte maturation in mice. Reprod. biol. 22: 1-8.
Kiss, A.L. and Botos, E. 2009. Endocytosis via caveolae: alternative pathway with distinct cellular compartments to avoid lysosomal degradation? J. Cell. Mol. Med. 13: 1228–1237.
Kobayashi, K., Yamashita, S. and Hoshi, H. 1992. Influence of epidermal growth factor and transforming growth factor α on in vitro maturation of cumulus cell enclosed bovine oocytes in a defined medium. J. Reprod. Fert. 100: 439- 446.
Leibfried-Rutledge, M.L., Critser, E.S., Parrish, J.J. and First, N.L. 1989. In vitro maturation and fertilization of bovine oocytes.Theriogenology. 31: 61–74.
Loos, D.F., Van Vliet, C.P., Maurik, V. and Kruip, A.M.T. 1989. Morphology of immature bovine oocytes. Gamete Res. 24: 197-204.
Matsuno, Y., Onuma, A., Fujioka, Y.A., Yasuhara, K., Fujii, W., Naito, K. and Sugiura, K. 2017. Effects of exosome like vesicles on cumulus expansion in pigs in vitro. J. Reprod. Dev.63: 51-58.
McKenize, K.A. and Cohen, B.D. 2009. Investigation of human follicle stimulating hormone residency in membrane microdomains. J. Fed. Am. Soc. Exp. Biol. 23: 1-7.
Nevoral, J., Orsak, M., Klein, P., Petr, J., Dvorakova, M., Weingartova, I., Vyskocilova, A., Zamostna, K., Krejcova, T. and Jílek, F. 2014. Cumulus cell expansion, its role in oocyte biology and perspectives of measurement: a review. Sci. Agric. Bohemica. 45: 212–225.
Parkes, W.S., Amargant, F., Zhou, L.T., Villanueva, C.E., Duncan, F.E. and Pritchard, M.T. 2021. Hyaluronan and collagen are prominent extracellular matrix components in bovine and porcine ovaries. Genes. 12: 1-23.
Revathy, M.M., Unnikrishnan, M.P., Jayakumar, C., Abhilash, R.S., Ramnath, V. and Sreeranjini, A.R. 2023. Assessment of cumulus cell expansion rate and first polar body extrusion rate in bovine oocytes supplemented with bovine follicular fluid exosomes. Int. J. Vet. 8: 149-152.
Richard, S. and Baltz, J.M. 2014. Prophase I arrest of mouse oocytes mediated by natriuretic peptide precursor c requires GJA1 (connexin-43) and GJA4 (connexin-37) gap junctions in the antral follicle and cumulus oocyte complex. Biol. Reprod. 90: 1-10.
Rispoli, L.A., Payton, R.R.,Gondro, C., Saxton, A.M., Nagle, K.A., Jenkins, B.W.,Schrick, F.N. and Edwards, J.L. 2013. Heat stress effects on the cumulus cells surrounding the bovine oocyte during maturation: altered matrix metallopeptidase 9 and progesterone production. Reproduction. 146: 193–207.
Rodrigues, T.A., Tuna, K.M., Alli, A.A., Tribulo, P., Hansen, P.J., Koh, J. and Paula-Lopes, F.F. 2019. Follicular fluid exosomes act on the bovine oocyte to improve oocyte competence to support development and survival to heat shock. Reprod. Fert. Dev. 31: 888-897.
Sasseville, M., Gagnon, M.C., Guillemette, C., Sullivan, R., Gilchrist, R.B. and Richard, F.J. 2009. Regulation of gap junctions in porcine cumulus oocyte complexes: contributions of granulosa cell contact, gonadotropins, and lipid rafts. J. Mol. Endocrinology. 23: 700-710.
Schoenfelder, M. and Einspanier, R. 2003. Expression of hyaluronan synthases and corresponding hyaluronan receptors is differentially regulated during oocyte maturation in cattle. Biol. Reprod. 69: 269–277.
Shimada, M., Hernandez-Gonzalez, I., Gonzalez-Robayna, I. and Richards, J.S. 2006. Paracrine and autocrine regulation of epidermal growth factor-like factors in cumulus oocyte complexes and granulosa cells: key roles for prostaglandin synthase 2 and progesterone receptor. Mol. Endocrinology. 20: 1352-1365.
Sidrat, T., Khan, A., Joo, M.D., Wei, Y., Lee, K.L., Xu, L. and Kong, I.K.2020. Bovine oviduct epithelial cell derived culture media and exosomes improve mitochondrial health by restoring metabolic flux during pre implantation development. Int. J. Mol. Sci. 2: 1-20.
Snedecor, G.W. and Cochran, W.G. 1994. Statistical Methods. (8th Ed.). The Iowa State University press, Iowa, 564p.
Turathum. B., Gao, E.M. and Chian, R.C. 2021. The function of cumulus cells in oocyte growth and maturation and in subsequent ovulation and fertilization. Cells. 10: 2292-2298.
Vincencio, J.M., Yellon, D.M., Sivaraman, V., Das, D., Boi-Doku, C., Arjun, S., Zheng, Y., Riquelme, J.A., Kearney, J., Sharma, V., Multhoff, G., Hall A.R. and Davidson, S.M. 2015. Plasma exosomes protect the myocardium from ischemia reperfusion injury. J. Am. Coll. Cardiology. 65: 1525-1536.
Wang, X., Zhang, Z., Qi, Y., Zhang, Z., Zhang, Y., Meng, K., Yuan, J. and Quan, F. 2024.Study of the uptake mechanism of two small extracellular vesicle subtypes by granulosa cells. Anim. Reprod. Sci. 270: 1-13.
Wei, Y., Idrees, M., Sidrat, T., Joo, M., Xu, L., Ko, J. and Kong, I. 2022. BOEC–Exo addition promotes in vitro maturation of bovine oocyte and enhances the developmental competence of early embryos. Animals. 12: 1-15.
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Parvathi, P., Abhilash, R.S., Jayakumar, C., Harshan, H.M., Magnus, K.P., Radhika, G., Kurian, E., Prathima, P., and Saifudeen, S.M. 2024.Molecular investigations on exosome-enriched in vitro maturation of bovine cumulus cells: Insights from cumulus cell dynamics. J. Vet. Anim. Sci. 56 (2):261-267