Effects of thyroid hormones on the functional state of bovine granulosa cells in vitro

Abstract

Any dysfunction of the thyroid gland causes abnormal changes in the functioning of the reproductive system, primarily the ovaries. Therefore, the question of the possible direct effect of thyroid hormones on the bovine ovarian function by modulating the functional state or functional activity of granulosa cells seems relevantPurpose: to study in vitro the effect of thyroxine and triiodothyronine on the proliferative and steroidogenic activity, as well as apoptotic changes of bovine granulosa cells.Materials and methods. Granulosa cells were isolated from follicles with a diameter of 1-5 mm and precultured for two days in a medium containing 10 % serum. The cells were then placed in serum-free medium containing thyroxine (25-200 ng/mL) or triiodothyronine (0.5-4.0 ng/mL) and incubated for another 48 h. After culturing, the content of estradiol-17β and progesterone in the media was determined by ELISA. The proliferative activity and apoptotic changes in the cells were assessed by immunocytochemical assay, based on the expression level of proliferating cell nuclear antigen PCNA and pro-apoptotic protein Bax, respectively.Results. It was found that the proportion of cells with a positive reaction to PCNA increased 1.1 times (P<0.01) compared with that in the control at a triiodothyronine concentration of 1 ng/ml and did not change with its further increase to 4 ng/ml. In addition, the introduction of triiodothyronine at a concentration of 1 ng/ml into the medium led to a decrease in the relative number of Bax-positive cells from 25.6 ± 0.3% to 23.3 ± 0.6 % (P<0.01). A further increase in this concentration to 4 ng/ml enhanced the observed anti-apoptotic effect 1.1 times (P<0.05). The pattern of the effect of thyroxine on the proliferative activity and apoptotic changes of granulosa cells in culture was similar to that for triiodothyronine. Concurrently, the growth-stimulating and anti-apoptotic effects of thyroxine were achieved at a concentration of 50-200 ng/ml. At the same time, both thyroid hormones did not affect the secretion of estradiol-17β or progesterone by the cells.Conclusions. Thus, thyroxine and triiodothyronine can stimulate in vitro the proliferation of bovine granulosa cells, as well as inhibit the expression of the proapoptotic Bax protein in these cells, which is not associated with the regulation of the production of ovarian steroid hormones. Overall, these data suggest that thyroid hormones at physiological concentrations are able to exert a regulatory effect on the growth and atresia of bovine small antral follicles and, therefore, directly modulate the ovarian activity.