Avaliação do efeito da punicalagina no cultivo in vitro de tecido ovariano de bovinos

Abstract

The biotechnique of in vitro culture (IVC) of pre-antral ovarian follicles has stood out for its ability to provide growth and development of pre-antral follicles. One of the main limitations of IVC of ovarian follicles is the excessive formation of reactive oxygen species (ROS) which cause oxidative stress (OS). In order to counteract the effects of OES on IVC, the addition of antioxidant substances has attracted attention. Therefore, this study evaluated the effects of Punicalagin on activation, growth, follicular survival, stromal cell density, collagen content in bovine ovarian tissue, as well as mRNA levels for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), peroxiredoxin 6 (PRDX6), nuclear factor erythroid 2 (NRF2) and biochemical analysis to quantify the antioxidant activity of the enzymes CAT, SOD, GPX and the thiol level using the Bradford method. For this purpose, fragments of ovarian cortex (3x3x1 mm) were fixed in 4% paraformaldehyde (uncultured control) or cultured in vitro in 500 µL of control medium (α-MEM⁺) alone or supplemented with 1, 10 or 100 µM Punicalagin at 38.5°C with 5% CO² for 6 days, the partial medium (60%) was changed every 2 days of culture. Statistical analysis was carried out using GraphPad Prisma (9.0) software, and the percentage of primordial and developing follicles, as well as normal or degenerated follicles, was analyzed using the Chi-square test. Analysis of the results revealed that the 10 or 100 µM concentrations of Punicalagin showed higher rates of normal follicles compared to the control group. In addition, there was an increase in the number of growing follicles when compared to the uncultured control group. With regard to the density of stromal cells, the presence of 100 µM Punicalagin in the culture medium kept the density of these cells similar to those observed in uncultured tissues. The ECM showed a decrease in collagen fibers in the culture medium supplemented with 1 or 100 µM Punicalagin compared to the non-cultured control group, while the concentration of 10 µM maintained the collagen fibers in the cultured tissues. Tissues cultured in the presence of 10 µM Punicalagin also showed high levels of mRNA for NRF2, GPX1 and PRDX6 compared to tissues treated with 1 or 100 µM Punicalagin. As for the expression of protein levels, the fragments cultured with Punicalagin 10 µM showed higher thiol levels, as well as greater activity of the enzymes SOD, CAT and GPX compared to the control group. In summary, supplementation with Punicalagin atspecific concentrations had an effect on follicular morphology and development, stromal cell density, ECM, mRNA expression and antioxidant proteins in bovine ovarian tissue cultured in vitro for six days.