Modulating effect of growth hormone on the functional state of cultured cells from hen preovulatory follicles
Somatotropic hormone (STH) is an important positive modulator of ovarian function in mammals. Local production of STH and the expression of the corresponding specific receptors were also detected in hen ovarian follicles, which indicates the participation of this hormone in the endocrine/paracrine control of folliculogenesis in birds. Nevertheless, the role of STH in the regulation of growth of avian follicles at the final stage of maturation is still not clear.Objective: To study in vitro the effect of STH on the proliferative activity and apoptotic changes of granulosa and theca cells from preovulatory follicles of domestic hens.Materials and methods. Young laying hens aged 34-35 weeks with a long clutch were used in the experiments. Granulosa and theca cells were isolated from the largest yellow follicle in the hierarchy (F1). The cells were cultured in a medium containing 10% fetal bovine serum until a monolayer was formed, and then for 24 h in the medium without serum in the absence (control) or in the presence of STH at various concentrations (1-100 ng/ml). 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. The proportion of PCNA-positive granulosa cells increased 1.3-1.8 times (P<0.01-0.05) as compared to control with increasing the content of STH in the medium to 10-100 ng/ml. Furthermore, within this concentration range, the studied hormone reduced 1.2-1.6 times (P<0.05) the relative number of granulosa cells with the positive reaction to Bax. The sensitivity of theca cells to the growth-stimulating effect of STH was lower than that of granulosa cells. Such the effect of STH led to an increase in the proportion of PCNA-positive thecal cells by 1.2-1.3 times (P<0.05) and was detected only at concentrations of 25 and 100 ng/ml. Meanwhile, STH (25-100 ng/ml) increased 1.3 times (P<0.05) the level of Bax expression in theca cells.Conclusions. The results of the present study indicate the stimulating effect of STH in vitro on the proliferative activity of granulosa and theca cells from the most mature hen preovulatory follicle. In addition, STH is able to reduce the expression of the pro-apoptotic protein Bax in granulosa cells and increase this expression in thecal cells. Thus, the data obtained indicate the possible participation of STH in the regulation of growth and development of follicles at the final stage of maturation during the period of maximum egg-laying intensity in laying hens.