Features of the free radical status of the follicular fluid of mares in the process of folliculogenesis
Abstract
Background. The maturation of germ cells is a complex and multilevel process. To increase the effectiveness of assisted reproductive technologies in horses, a detailed study of the mechanisms of regulation of folliculogenesis is necessary.
Purpose. A lifetime study of the state of free radical status and the level of transcription factors regulating the antioxidant defense system in the follicular fluid (FF) of mares of the late transition period and the season of sexual cyclicity.
Materials and methods. The study was performed on mares, crossbreeds of heavy-duty, riding and Vyatka breeds, 6-12 years old in the spring transition period and the period of normal sexual cyclicity. 4 follicle groups were formed: large (≥31mm) follicles in the spring transition period, without signs of luteinization (group 1) and with signs of luteinization (group 2); large estrous follicles (>35 mm) in the ovulatory period without hormonal treatment (group 3) and with hormonal treatment (group 4). Photometric methods were used to determine the concentration of lipid peroxidation products – TBK-reactive products and protein oxidation products – carbonyl derivatives of proteins; the activity of antioxidant enzymes – superoxide dismutase and glutathione peroxidase and the level of endogenous antioxidant glutathione. The relative amounts of Nrf2, HIF-1a, VEGF and NFkB proteins were analyzed using the wester blot method.
Results. In the follicles of group 3, the concentration of TBK-reactive products and carbonyl derivatives of proteins increased significantly and exceeded the values of groups 1, 2 and 4. The content of non-protein SH groups reached a maximum level in the SH-group 4, exceeding the indicators of groups 1 and 3. The activity of superoxide dismutase and glutathione peroxidase significantly increased in group 4 relative to group 1. The level of Nrf2 increased in the SH-group 4, HIF1a – 2 and 3 groups, NFkB and VEGFA – in group 2.
Conclusion. The growth of the follicle and its preparation for ovulation in cycling mares in the summer is accompanied by the development of oxidative stress, which leads to the activation of the redox-sensitive factor Nrf2, followed by an increase in the activity of antioxidant enzymes superoxide dismutase and glutathione peroxidase and the level of endogenous antioxidant glutathione. For large follicles with signs of luteinization in the spring transition period, an increase in the level of VEGF and NFkB is characteristic in the FF, which may reflect the development of local hypoxia and inflammation and cause the development of the follicle along the path of luteinization without ovulation.
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References
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Fu, X., Shi, L., Liu, P., Jiao, Y., Guo, S., Chen, Q., Zheng, Q., Chen, X., & Wang, Y. (2023). Expression and clinical significance of HIF 1α in follicular fluid and granulosa cells in infertile PCOS patients. Reproductive Sciences, 30, 2263–2274. https://doi.org/10.1007/s43032-022-01135-2. EDN: https://elibrary.ru/ELENTM
Galli, C., Duchi, R., Colleoni, S., Lagutina, I., & Lazzari, G. (2014). Ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice. Theriogenology, 81, 138–151.
Gloire, G., & Piette, J. (2009). Redox regulation of nuclear post translational modifications during NF kappaB activation. Antioxidants & Redox Signaling, 11, 2209–2222.
Gomard, T., Michaud, H. A., Tempé, D., Thiolon, K., & Pelegrin, M., Piechaczyk, M. (2010). An NF kappaB dependent role for JunB in the induction of proinflammatory cytokines in LPS activated bone marrow derived dendritic cells. PLOS ONE, 8, e9585.
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Hyde, K. A., Aguiar, F. L. N., Alves, B. G., Alves, K. A., Gastal, G. D. A., Gastal, M. O., & Gastal, E. L. (2022). Preantral follicle population and distribution in the horse ovary. Reproduction and Fertility, 3, 90–102. https://doi.org/10.1530/raf-21-0100. EDN: https://elibrary.ru/XKLDII
Kang, K. A., & Hyun, J. W. (2017). Oxidative stress, Nrf2, and epigenetic modification contribute to anticancer drug resistance. Toxicological Research, 33, 1–5.
Luciano, A. M., Goudet, G., Perazzoli, F., Lahuec, C., & Gérard, N. (2006). Glutathione content and glutathione peroxidase expression in in vivo and in vitro matured equine oocytes. Molecular Reproduction and Development, 73, 658–666.
Mihara, M., Uchiyama, M., & Fukazawa, K. (1980). Thiobarbituric acid value on fresh homogenate of rat as a parameter of lipid peroxidation in aging, CCL intoxication and vitamin E deficiency. Biochemical Medicine, 23, 302–311.
Mohammed, A. A., Al Suwaiegh, S., & Al Shaheen, T. (2019). Effects of follicular fluid components on oocyte maturation and embryo development in vivo and in vitro. Advances in Animal and Veterinary Sciences, 7, 346–355.
Murakami, K., Kotani, Y., Nakai, H., & Matsumura, N. (2020). Endometriosis associated ovarian cancer: the origin and targeted therapy. Cancers (Basel), 12(6), 1676.
Park, J. Y., Chung, T. W., Jeong, Y. J., Kwak, C. H., Ha, S. H., Kwon, K. M., Abekura, F., Cho, S. H., Lee, Y. C., Ha, K. T., Magae, J., Chang, Y. C., & Kim, C. H. (2017). Ascofuranone inhibits lipopolysaccharide induced inflammatory response via NF kappaB and AP 1, p ERK, TNF α, IL 6 and IL 1β in RAW 264.7 macrophages. PLOS ONE, 12, e0171322.
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Tang, Z., Xu, R., Zhang, Z., Shi, C., Zhang, Y., Yang, H., Lin, Q., Liu, Y., Lin, F., Geng, B., & Wang, Z. (2021). HIF 1α protects granulosa cells from hypoxia induced apoptosis during follicular development by inducing autophagy. Frontiers in Cell and Developmental Biology, 9, 1–12. https://doi.org/10.3389/fcell.2021.631016. EDN: https://elibrary.ru/QXOZWG
Tatemoto, H., Muto, N., Sunagawa, I., Shinjo, A., & Nakada, T. (2004). Protection of porcine oocytes against cell damage caused by oxidative stress during in vitro maturation: role of superoxide dismutase activity in porcine follicular fluid. Biology of Reproduction, 71, 1150–1157.
Tucker, K. E., Cleaver, B. D., & Sharp, D. C. (1993). Does resumption of follicular estradiol synthesis during vernal transition in mares involve a shift in steroidogenic pathways? Journal of Biological Reproduction, 1, 519.
Watson, E. D., Thomassen, R., Steele, M., Heald, M., Leask, R., Groome, N. P., & Riley, S. C. (2002). Concentrations of inhibin, progesterone and oestradiol in fluid from dominant and subordinate follicles from mares during spring transition and the breeding season. Journal of Animal Reproduction Science, 74, 55–67.
Weber, D., Davies, M. J., & Grune, T. (2015). Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: focus on sample preparation and derivatization conditions. Redox Biology, 5, 367–380. https://doi.org/10.1016/j.redox.2015.06.005. EDN: https://elibrary.ru/UOUOQP
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