Effects of dexamethasone on growth, viability and ultrastructure of bovine secondary follicles cultured in vitro

Abstract

Summary This study aimed to evaluate the effects of dexamethasone on development, viability, antrum formation and ultrastructural integrity of bovine secondary follicles cultured in vitro for 18 days. Bovine ovaries were obtained from slaughterhouses and secondary follicles of ~150–200 µm diameter were isolated and cultured in the laboratory in TCM-199+ alone or supplemented with different concentrations of dexamethasone (1, 10, 100 and 1000 ng/ml). Follicle viability was evaluated after the culture period, using calcein-AM (viable) and ethidium homodimer (nonviable). Follicle diameters and antrum formation were evaluated at days 0, 6, 12 and 18. Before or after in vitro culture, follicles were fixed for histological and ultrastructural analysis. Follicle diameters were evaluated using analysis of variance and Kruskal–Wallis test, while chi-squared test was used to evaluate the percentage of viable follicles and antrum formation (P < 0.05). Follicles cultured for 6 days with all treatments increased their diameters significantly, but there was no significant difference between treatments at the end of the culture period. In vitro cultured follicles showed antral cavity formation at the end of the culture period, but no influence of dexamethasone was seen. Ultrastructural analysis showed that follicles cultured with dexamethasone (1, 10, 100 and 1000 ng/ml) had well preserved granulosa cells. However, oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone showed signs of degeneration. It can be concluded that follicles cultured in vitro in the presence of dexamethasone demonstrated continuous in vitro growth, but oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone had poor ultrastructure.

Glucocorticoids, Stress and Delta-9 Tetrahydrocannabinol (THC) during Early Embryonic Development

“…These results also demonstrate the significance of the TNF-α system in bovine follicles, suggesting that apoptotic pathways present in cows and mice are similar [86]. Barroso et al (2020) continued to evaluate the effects of DEX on bovine folliculogenesis by assessing its influence on growth, viability, antrum formation, morphology and ultrastructure of secondary follicles cultured in vitro. DEX (1-1000 ng/mL) exposure over an 18-day culture period did not alter the growth, antrum formation or survival of bovine secondary follicles in vitro [87].…”

“…Barroso et al (2020) continued to evaluate the effects of DEX on bovine folliculogenesis by assessing its influence on growth, viability, antrum formation, morphology and ultrastructure of secondary follicles cultured in vitro. DEX (1-1000 ng/mL) exposure over an 18-day culture period did not alter the growth, antrum formation or survival of bovine secondary follicles in vitro [87]. In agreement with the findings of Silva et al (2017) for primordial follicles enclosed in ovaries, the ultrastructure of follicular granulosa cells of secondary follicles cultured in the presence of DEX (1, 10, 100 and 1000 ng/mL) were well maintained [87].…”

“…On the other hand, adversities were not present at a DEX concentration of 1 ng/mL, where the integrity of oocytes, follicular cells and their connections were well maintained. Barroso et al (2020) delved further than Silva et al (2017) by evaluating the effects of glucocorticoids on the oocytes from secondary follicles, while also demonstrating that lower concentrations are non-harmful and potentially beneficial to preserving quality [86,87].…”

“…Mitigating apoptosis is also relevant in oocytes during maturation or cryopreservation, given that it will impact their competence to successfully produce embryos [ 18 , 52 , 60 , 85 ]. Both endogenously and exogenously produced glucocorticoids have been shown to alter apoptotic events and cell viability in mice, rats, and bovine [ 18 , 52 , 60 , 80 , 86 , 87 ]. Glucocorticoid-induced apoptosis has been shown to act through various mechanisms, including the upregulation of Fas receptor Fas ligand (Fas-Fas L) [ 60 ] and tumor necrosis factor (TNF)-α [ 18 , 86 ] in oocytes, GCs, CCs, and embryonic cells of rodents and bovine [ 18 , 52 , 60 , 85 ].…”

“…DEX (1–1000 ng/mL) exposure over an 18-day culture period did not alter the growth, antrum formation or survival of bovine secondary follicles in vitro [ 87 ]. In agreement with the findings of Silva et al (2017) for primordial follicles enclosed in ovaries, the ultrastructure of follicular granulosa cells of secondary follicles cultured in the presence of DEX (1, 10, 100 and 1000 ng/mL) were well maintained [ 87 ]. However, oocytes contained within secondary follicles exposed to DEX concentrations of 10, 100 and 1000 ng/mL had ultrastructural abnormalities such as reduced microvilli and connections between oocyte and GCs, likely as a result of apoptosis.…”