Resveratrol mantains survival and promotes cell proliferation in ovine preantral follicles cultured in situ

Some studies have demonstrated that resveratrol, a fitoalexin produced by several plants and present in grapes, reduces follicular atresia (KONG et al., J Endocrinol Invest., v. 34, p. 374-81, 2011) and reactive oxygen species (KWAK, et al., Theriogenology, v. 78, p. 86-101, 2012) after rat ovarian follicle culture. However, there are no reports about the effect of resveratrol on the in vitro culture of ovine ovarian preantral follicles. Thus, the aim of this study is to evaluate the effect of resveratrol on the morphology, activation and cell proliferation of ovine preantral follicles cultured in situ. After collection of ovine ovaries (n=10) in the slaughterhouse, ovarian cortex was fragmented and one fragment was immediately fixed and destined to histology (fresh control). The remaining fragments were cultured in vitro for 7 days in α-Minimal Essential Medium (α-MEM – GIBCO, Invitrogen, St Louis, EUA) supplemented with insulin, transferrin, selenium, hypoxanthine, glutamine, ascorbic acid and BSA (α- MEM+ - control medium) or in α-MEM+ added by different concentrations of resveratrol (2; 10 or 30 µM). After culture, preantral follicles were morphologically analysed and classified as normal or atretic according to the absence or presence of cytoplasmic shrinkage, nuclear pycnosis and/or disorganization of granulosa cells, as well as classified as primordial or growing follicles (intermediate, primary and secondary follicles). Immunohistochemical analysis was also perfomed for detection of Proliferation Cell Nuclear Antigen (PCNA; 1:500; Santa Cruz Biotechnology; Santa Cruz, CA, EUA). The PCNA positive cells (brown) were counted in the sections and expressed as percentage. The percentage of normal, primordial and growing follicles were compared by ANOVA and Tukey’s test (P<0.05). After 7 days of culture, there was a significant reduction in the percentage of morphologically normal follicles compared to the fresh control. The concentration of 2 µM resveratrol showed percentages of normal follicles similar to the control medium and significantly higher than other resveratrol concentrations. In addition, there was a significant reduction in the percentage of primordial follicles and an increase in the percentage of growing follicles (follicular activation) in all treatments compared to the fresh control. Moreover, it was possible to observe a significant increase in the percentage of PCNA positive cells in follicles cultured in 2 µM resveratrol (50.7%) in comparison to fresh control (34.0%) and α-MEM+ (4.3%). In conclusion, resveratrol at 2µM maintains survival, promotes activation and cell proliferation of ovine preantral follicles cultured in situ.