Utilização de substâncias antioxidantes (tocoferol e ternatina) no cultivo in vitro de folículos pré-antrais caprinos

Abstract

The aim of this study was to evaluate the effects of tocopherol and ternatin on morphology and activation of goat preantral follicles cultured in vitro and realize a review including the origin of oxidative stress and reactive oxygen species, as well as the mechanisms that protects the organism against free radicals, implications of them in mammalian ovary and embryo and the role of nitric oxide in this process. In the first work, the ovarian cortex was divided into small pieces and one fragment was immediately fixed (control). The remaining fragments were in vitro cultured for 1 or 5 days at 39oC and 5% CO2, in Minimum Essential Medium (MEM) supplemented and added or not by 5, 10 or 15 mM of a-tocopherol or ternatin. Control and cultured ovarian fragments were fixed in 10% formaline or in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer for histological and ultrastructural analysis, respectively. Follicles were classified as primordial or developing, as well as normal or degenerated. When compared with control, in vitro culture led to a decrease in the percentages of morphologically normal preantral follicles in all treatments with a-tocopherol or ternatin (P < 0.05) after 5-days culture. Furthermore, when compared with control, culture of ovarian cortex for 5 days increased the percentages of follicular activation in all treatments (P < 0.05). In comparison with MEM alone, addition of α- tocopherol or ternatin in the culture medium did not affect follicular activation (P > 0.05), except at day 1 of culture, when concentrations of 5 and 15 mM were used respectively and showed a significantly increase of follicular activation, in comparison with others treatments. Ultrastructural analysis showed that follicles cultured for 5 days in medium containing antioxidants were degenerated. This degeneration did not occur in follicles treated with MEM alone or control, which maintained their ultrastructural integrity. In conclusion, this study demonstrated that α-tocopherol and ternatin can promote follicular activation, however the addition of antioxidants in the tested concentrations reduced the follicular viability after in vitro culture. The second work verified that reactive oxygen (ROS) or nitrogen (RNS) species or free radicals are products of normal organic metabolism and their accumulation can generate the oxidative stress. Then, this stress causes cellular damage, leading to death. This process is involved with cellular degeneration and/or apoptosis during embryos development and in different organs, such as the mammalian ovary. In murine ovary, ROS are produced during luteal regression and they are involved in the process of follicular activation and spontaneous meiosis resumption in oocytes. Although these are physiological processes, accumulation of free radicals in the mammalian ovary may unchain pathologies. In the embryo, free radicals are normally produced due to normal metabolism. However, in in vitro environment with high oxygen concentrations, embryo development can be damaged. Nitric oxide is considered a free radical and takes part in pathologic and physiologic processes, such as inflammation, ovulation, embryo implantation and uterine contraction. To protect the biological systems from cellular damage caused by free radicals, the organism uses enzymatic mechanism, which involves, for instance, catalase, susperoxide-dismutase and glutation peroxidase and others that include antioxidants substances, such as vitamin C, vitamin E, b-carotene etc.