Eugenol influences the expression of messenger RNAs for superoxide dismutase and glutathione peroxidase 1 in bovine secondary follicles cultured in vitro

Abstract

Summary This study aimed to investigate the effects of eugenol on growth, viability, antrum formation and mRNA expression of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1) and peroxiredoxin 6 (PRDX6) in bovine secondary follicles cultured in vitro. To this end, bovine ovaries were collected from a local slaughterhouse and in the laboratory the follicles were isolated from the ovarian cortex. The follicles were then cultured in TCM-199+ alone or supplemented with different concentrations of eugenol (0.5, 5.0 and 50.0 μM). Follicular diameters and antrum formation were evaluated on days 0, 6, 12 and 18. Viability analysis was performed using calcein and ethidium homodimer. Real-time PCR was used to quantify mRNA levels for SOD, CAT, GPX1 and PRDX6 in cultured follicles. Follicular diameters and mRNA levels in follicles cultured in vitro were compared using analysis of variance and Kruskal–Wallis tests, while follicular survival and antrum formation were compared using the chi-squared test (P < 0.05). The results showed that secondary follicles cultured with eugenol maintained similar morphology and viability to follicles cultured in the control group. A progressive increase in follicular diameter was observed between days 0 and 12 for all treatments, except for follicles cultured with 50 µM eugenol. Eugenol (5.0 and 50.0 μM) increased mRNA levels for GPX1 in cultured follicles, but 0.5 μM eugenol reduced mRNA levels for SOD. The addition of eugenol did not influence mRNA expression for CAT and PRDX6. In conclusion, eugenol supplementation reduces mRNA levels for SOD and increases mRNA levels of GPX1 in bovine secondary follicles cultured in vitro.

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue

“…The antioxidant capacity of eugenol was confirmed by its ability to decrease the H 2 O 2 production. Eugenol not only decreases the excess of ROS ( 24 ) but also enhances the activity of enzymatic antioxidants ( 25 ). Ighodaro and Akinloye ( 31 ) showed that eugenol mitigates the effects of H 2 O 2 via the regulation of glutathione peroxidase levels.…”

“…Furthermore, eugenol is chemically stable during culture, with direct and indirect effects against ROS ( 24 ). Based on a previous study, eugenol supports the viability of bovine large secondary follicles in vitro ( 25 ). However, to the best of our knowledge, the effect of eugenol on in-vitro early folliculogenesis, i.e., survival and activation of the primordial follicle and initial growth of these follicles, is still unknown.…”

Effects of N-acetylcysteine on Growth, Viability, and Ultrastructure of In Vitro Cultured Bovine Secondary Follicles

“…For follicular isolation, the method of Vasconcelos et al [ 8 ] was followed. Fragments (1–2 mm) of the ovarian cortex were removed with the aid of a sterile scalpel blade and placed in TCM-199 medium supplemented with HEPES (0.05 mM/mL) and penicillin (100 IU)/streptomycin (0.1 mg/mL).…”

“…It is important to highlight that in-vitro-cultured follicles are not in their natural environment and need proper conditions to survive and to develop. In cows, various reports have demonstrated that secondary follicles cultured for a period of 18 days grow up to early antral follicles, but an increase in the atresia rate is commonly reported at this stage [ 5 , 7 , 8 ].…”

“…These authors showed that the oocytes had swollen Golgi apparatus, a large number of lipid droplets, and mitochondria with vacuolization and degeneration of the cristae and matrix. To minimize the adverse effects of oxidative stress, various studies have reported the importance of adding antioxidant substances to in vitro culture media [ 5 , 8 , 9 ]. N-acetylcysteine (NAC) is a substance that can be used to optimize in vitro conditions during follicle growth [ 14 ].…”