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

Abstract

This study evaluated the effects of different concentrations (10, 20, or 40 μM) of eugenol (EUG 10, EUG 20, or EUG 40), ascorbic acid (50 μg/mL; AA) or anethole (300 μg/mL; ANE 300) on the in-vitro survival and development of goat preantral follicles and oxidative stress in the cultured ovarian tissue. Ovarian fragments from five goats were cultured for 1 or 7 days in Alpha Minimum Essential Medium (α-MEM+) supplemented or not with AA, ANE 300, EUG 10, EUG 20 or EUG 40. On day 7 of culture, when compared to MEM, the addition of EUG 40 had increased the rate of follicular development, as observed by a decrease in the proportion of primordial follicles alongside with an increase in the rate of normally developing follicles. Furthermore, EUG 40 significantly increased both follicular and oocyte diameters. Subsequently, ovarian fragments from three goats were cultured for 1 or 7 days in α-MEM+ supplemented or not with AA, ANE 300 or EUG 40. All tested antioxidants, except ANE 300, were able to significantly decrease the levels of reactive oxygen species in the ovarian tissue, but EUG 40 could most efficiently neutralize free radicals. All ovarian tissues cultured in the presence of antioxidants, especially EUG 40, presented a significant decrease in H3K4me3 labeling, indicating a silencing of genes that play a role in the inhibition of follicular activation and apoptosis induction. When compared to cultured control tissues, both EUG 40 and ANE 300 significantly increased the intensity of calreticulin labeling in growing follicles. The mRNA relative expression of ERP29 and KDM3A was significantly increased when the culture medium was supplemented with EUG 40, indicating a response to ER stress experienced during culture. In conclusion, EUG 40 improved in-vitro follicle survival, activation and development and decreased ROS production, ER stress and histone lysine methylation in goat ovarian tissue.

Trimethylation profile of histones H3 lysine 4 and 9 in late preantral and early antral caprine follicles grown in vivo versus in vitro in the presence of anethole

“…Unlike H3K9me3, which is involved in gene silencing, increased H3K4me3 is associated with gene expression activation (Ren et al, 2022;Yu et al, 2018), which suggests that in the present study, the anethole treatment promoted a shift from a repressive to a more active chromatin state. Previous observations by our group revealed that anethole increased H3K4me3 fluorescence intensity in early PA follicles included in ovarian tissue grown in vitro compared with follicles grown in vivo (Silva et al, 2022). Similar results were obtained by using ascorbic acid, which altered H3K4me3 fluorescence intensity without altering H3K9me3 fluorescence intensity during in vitro maturation of porcine COC (Yu et al, 2018).…”

“…However, an increase of H3K4me3 in the regulatory regions of STAR, CYP17, and CYP19A1 in GC (Bahrami & Maleki, 2019;Sha, Jiang, et al, 2020), and genes related to survival and growth in oocytes and suppression of oxidative stress (Ren et al, 2022;Yu et al, 2018) et al, 2015), might also be involved. Previous work from our laboratory has suggested that the antioxidant effects of anethole on PA and EA follicles in culture may involve KDM genes and H3K4me3 (Sá et al, 2017;Silva et al, 2022). In addition, the increase in H3K4me3 herein observed might have been a result of an increase in the expression of the CXXC finger 1 protein (CFP1), a DNA-binding subunit of the histone methyltransferase SETD1.…”

“…During IVFC, suboptimal conditions in the culture environment may alter the methylation pattern (Silva et al, 2022). Thus, assessing the methylation profile during in vivo folliculogenesis (Seneda et al, 2008;Sha, Jiang, et al, 2020) may be a useful marker for the optimization of IVFC conditions (Silva et al, 2021(Silva et al, , 2022. In this study, the methylation profiles of T A B L E 2 Percentages of morphologically intact, extruded, and degenerated follicles and antrum formation, mean (± SEM) follicular diameters and daily growth rate in vitro of caprine follicles.…”

“…PerkinElmer) using the wavelengths 530 nm (excitation) and 595 nm (emission). The ROS concentration was expressed in micromoles of H 2 O 2 by interpolation with a curve of H 2 O 2 (Silva et al, 2022).…”

“…Methylation of the lysine residues K4 and K9 of the histone H3 in GC is involved in the regulation of folliculogenesis through the modulation of transcription and other chromatin functions (Baumann et al, 2015;Seneda et al, 2008). During IVFC, suboptimal conditions in the culture environment may alter the methylation pattern (Silva et al, 2022). Thus, assessing the methylation profile during in vivo folliculogenesis (Seneda et al, 2008;Sha, Jiang, et al, 2020) may be a useful marker for the optimization of IVFC conditions (Silva et al, 2021(Silva et al, , 2022.…”