Analysis of functional activity of granulosa cells of porcine ovarian follicles

Abstract

Functioning of the granulosa cells of animal ovarian follicles (OF) determines the growth and development of the OF and the oocyte formed in it, which determines their use in modeling the composition of media for oocyte maturation in vitro in cell reproductive technologies. The purpose of the study is to determine the peculiarities of functional activity of granulosa cells in follicles of different diameters (< 3, 3–5, 5–8 mm) using complex testing of granulosa cell viability parameters (apoptosis level, production of reactive oxygen species, functional activity of mitochondria and lipidome) and hormonal status of follicle fluid (estradiol, testosterone, anti-Müllerian hormone level) in order to optimize the composition of the culture media for in vitro maturation of pig oocytes. The experiments found that the proportion of apoptotic cells in the OF increased as the diameter of the OF increased (by 11% in OFs 5–8 mm in diameter, by 4% in OFs < 3 mm in diameter). There was an increase in the number of high reactive oxygen species cells (by 10% in 5–8 mm diameter OF compared with < 3 mm diameter OF) and high mitochondrial activity (by 12% in 5–8 mm diameter OF compared with < 3 mm diameter OF). Correlations were found between the group of cells with high generation of reactive oxygen species and the level of apoptosis (0.500, p < 0.05), as well as with high levels of mitochondrial activity (0.500, p < 0.05). Estradiol concentration in follicular fluid increased as the OF grew (1.50 ng/ml in OF < 3 mm diameter and 2.09 ng/ml in OF 3–5 mm diameter, p < 0.01), being almost unchanged in OF 5–8 mm diameter (2.11 ng/ml), as was the testosterone concentration. Correlations were found between the proportion of the cells with high NileRed/LD FI (a marker of lipid droplet functional activity) and the levels of estradiol (0.998, p < 0.05), testosterone (0.500, p < 0.05), and anti-Müllerian hormone (–0.500, p < 0.05) in the dynamics of folliculogenesis, as well as between estradiol concentration and the proportion of cells with high mitochondrial activity (0.746, p < 0.05). Correlations were detected between the concentrations of estradiol and anti-Müllerian hormone, testosterone and anti-Müllerian hormone (–1.000, p < 0.05), between the level of apoptotic cells and the concentration of anti-Müllerian hormone in follicular fluid (–1.000, p < 0.05). The results of the monitoring of the viability and functional activity of porcine OF granulosa cells supplement the available data on the peculiarities of their functioning in the dynamics of folliculogenesis and can be used in modeling the systems of in vitro maturation of donor oocytes of Sus Scrofa Domesticus.