Effects Of Sex Steroids On Proliferation Of The Hypothalamic Neurons In Embryonic Chickens

Hypothalamus represents one of the most important endocrine gland to control the complicated physiological functions. In the hypothalamuic-pituitary-gonadal axis (HPG), the hypothalamus secrets gonadotropin-releasing hormone (GnRH) to stimulate secretion of pituitary gonadotropins including follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which subsequently promote the gonads to secrete sex steroid hormones. Gonadal hormones such as 17β-estradiol (E₂) and testosterone (T) impose direct or indirect effects on hypothalamus via feedback action. In this study, the hypothalamus of chicken embryos was adopted to evaluate the effects of androgen and estrogen on cell proliferation and the mechanism was explored.In this study we established a glia-neuron cell primary coculture model in which neurons could grow upon or among glial cells, keep proliferation and survival for more than two weeks. After culture in DMEM supplemented with 2.5% striated fetal calf serum for three days, the medium was replaced with serum-free medium supplemented with 10μg/mL insulin,5μg/mL transferrin and 3×10^-8 M selenite. In this culture system, neuronal cells underwent morphological changes. With the days went on, neurons sent out an extensive network of processes and developed large phase bright cell bodies. Neurons were immunostained positively for the neuron-specific enolase (NSE), while glias were positive for the glial fibrillary acid protein (GFAP).We adopted this culture model to evaluate the effects of testosterone (T) and 17β-estradiol (E₂) on proliferation of hypothalamic neurons and compared the sexual differences in the proliferating effects induced by sex steroid hormones. The number of neurons was counted and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). PCNA-LI (labeling index, percentage of PCNA-positive neurons to the total neurons) was determined for neurons. Estrogen receptor immunoreactive (ER-IR) neuron numbers was counted after the ER immunocytochemistry. Results showed that hypothalamic neuron number was increased by E₂ (10^-8-10^-6M) treatment in both the male and female. The PCNA-LI of neurons was significantly greater in E₂-treated groups (10^-8-10^-6M) than in the control group (P<0.05). Another, this stimulating effect was inhibited by tamoxifen (10^-8-10^-6M). The similar results displayed on male chicken hypothalamic neurons that were treated with T (10^-7-10^-6M). However, in female only high concentration of T (10^-6M) manifested significant effect on neuron proliferation. ER-IR neuron numbers was higher in E₂ or T-treated groups than in the control group. These results indicate that gonadal sex hormones could promote proliferation of embryonic hypothalamic neurons and also stimulate the expression of ER on neurons.To reveal the mechanism of T-induced proliferating effect on neurons, hypothalamic neurons were exposed to T (10^-6M) alone or in combination with flutamide, tamoxifen or letrozole. Results showed that flutamide (100-1000ng/mL), tamoxifen (10^-8-10^-6M) and letrozole (10^-8-10^-6M) decreased the proliferation of neurons that was stimulated by T. Similar changes in PCNA and BrdU incorporation were demonstrated in which flutamide, tamoxifen or letrozole decreased T-stimulated increase in PCNA-LI and BrdU-LI. The above results suggested that T stimulated proliferation of cultured hypothalamic neurons through both androgenic and estrogenic actions in embryonic chickens.In conclusion, the above studies indicated that:â‘ the hypothalamic neuron culture model was sensitive to sex steroid hormones and could be applied to evaluated effects of exogenous hormones or drugs on hypothalamic neurons.â‘¡E₂ could promote embryonic hypothalamic neuron proliferation through estrogenic actions and also stimulate the expression of ER on neurons.â‘¢T stimulated neuron proliferation through both androgenic and estrogenic actions, and the effect was more sensitive in male chicken embryo.