Epigallocatechin-3-gallate maintains follicular survival similar to fresh control after in vitro culture of ovine ovarian tissue

Epigallocatechin-3-gallate (EGCG) is natural polyphenol with antioxidant potential (JIANG et al., International Journal of Clinical and Experimental Medicine, 9, 2.479-2.485, 2016). Studies showed that this substance decreases oxidative stress (ORTIZ-LOPEZ et al., Neuroscience, 322, 208-220, 2016) and has lower toxicity (FIORINI et al., Liver Transplantation, 11, 298-308, 2005) in different types of cell. However, it is not known whether EGCG could influence on the in vitro culture of ovine ovarian follicles. The aim of this study was to verify the effect of epigallocatechin-3-gallate on the morphology and activation of ovine preantral follicles cultured in ovarian tissue. Ovine ovaries (n = 10) were fragmented (approximately 3x3x1 mm), being one fragment fixed for histology (fresh control) and the remaining fragments were individually cultured in 1 mL of medium for 7 days at 39o C with 5% CO2. The basic culture medium (control medium: α-MEM+ ) consisted of α-Minimum Essential Medium (α-MEM) supplemented with 10 ng/mL insulin, 5.5 µg/mL transferrin, 5.0 ng/mL selenium, 2 mM glutamine, 2 mM hypoxanthine, 1.25 mg/mL de BSA, 100 µg/mL penicillin and 100 µg/mL streptomycin. To evaluate the effects of EGCG, ovarian fragments were cultured in α-MEM+ alone or added by different concentrations of EGCG (0.01, 0.1, 1, 10 or 100 µg/mL – diluted in ultrapure water). After culture, the fragments were destined to histological analysis and classified as morphologically normal when the follicles did not show cytoplasmic shrinkage or nuclear pyknosis or organized granulosa cells and/or theca cells; atretic follicles showed one of those parameters. Follicular activation (transition from primordial to developing follicles) was also evaluated. Chemicals used in the culture and histology were purchased from Sigma Chemical Co. (St. Louis, USA) and Dinâmica (Sao Paulo, Brazil), respectively. The percentages of morphologically normal, primordial and growing follicles were submitted to ANOVA and Tukey test (P 0.05) to that observed in fresh control. However, 100 µg/mL EGCG reduced (P < 0.05) the percentage of morphologically normal follicles when compared to α- MEM+ and other EGCG concentrations. In all treatments (α-MEM+ and different concentrations of EGCG), it was observed a reduction (P < 0.05) in the percentage of primordial follicles concomitant with an increase (P < 0.05) in the percentage of developing follicles, compared to fresh control. In conclusion, 1 µg/mL EGCG maintain follicular survival similar to fresh control. In addition, all EGCG concentrations, except 100 µg/mL, showed follicular survival similar to α-MEM+ after in vitro culture of ovine preantral follicles.