Effects Of Endocrine Disrupting Chemicals On Gonadal Development Of Embryonic Chickens

During the past 50 years, a variety of synthetic chemicals and their by-products have been released to the environment as a consequence of efforts to increase agricultural productivity or modern manufacturing, which caused new problems for environment. Some of these chemicals are similar to endogenous hormone in structure and could interrupt the synthesis, excretion, availability, action and biotransformation of endogenous hormones. These chemicals were named Endocrine disrupting chemicals (EDCs). In recent years, much attention has been focused on the potential for a wide range of xenobiotic chemicals to interact with and disrupt the endocrine systems of animal and human populations. Datas showed that EDCs could interrupt the action of E2, thyroxine, catechol and testosterone. In clinic it manifested reproductive abnormal, birth defect, metabolic disorder and some cancer. The present study chose chicken embryo as experiment animal and evaluated the effects of hormones, polychlorinated biphenyls (PCBs) and diethylstilbestrol (DBS) on embryonic chicken gonad development and gamete differentiation in chicken embryos by in vivo and in vitro methods.In vitro studies: A serum-free chicken ovarian germ-somatic cell coculture model was established in vitro. The cellular suspension obtained from the left ovary contains germ cells (mainly are oogonia and primary oocytes) and somatic cells (granulosa, interstitial and epithelial cells etc.). The diameter of germ cells was greater than somatic cells. After culture for 24h, somatic cells have attached at the bottom of the culture plate, growing as shapes of shuttle and almost spread around the whole bottom at 48h of culture. Germ cells as round or oval in shape were found in the free surface of the aggregate of somatic cells and their diameters were between 15-25 um. Germ cells were immunostained positively for the c-kit at the cell surface. In the germ-somatic cells coculture model, germ cells showed high activity of proliferation by immunostaining with PCNA after culture for 48h and could survive for at least 10 days in serum-free medium.At first, whether this system is sensitive to hormones was tested. Cells were challenged with FSH (0.25-1.0 ID/ml) or E2 (10"8~10"5M) alone and in combination. The number of germ cells was counted and the proliferating cells were immunolocalized by a specific antibody againstproliferating cell nuclear antigen (PCNA). PCNA-LI (labeling index, percentage of PCNA-positive germ cells to the total germ cells) was determined for germ cells. Results showed that both FSH (0.25-1.0 lU/ml) and E2 (10~7~10~5 M) alone induced marked increment in germ cell number (PO.05). Compared with the control group, germ cells in the FSH-treated group and Entreated group were more distinct in figure and had higher cuboidal form. The PCNA-LI of germ cell was significantly greater in FSH-treated groups (0.25-1.0 ILJ/ml) and E2-treated groups (10^-10 5M), compared with vehicle-treated group (PO.05). Furthermore, FSH manifested a synergistic effect with E2 in stimulating germ cell proliferation. These results indicated that this model was sensitive to hormone and could be applied to evaluated effects of exogenous hormone, drug and toxicant on germs cell and somatic cells.Effects of PCBs on embryonic chicken ovarian development in vitro were evaluated by the germ-somatic cell coculture system. At the same time of culture, cells were challenged with a mixture of PCBs (Aroclor 1254, 0.1-10 ug/ml). Results showed that lower PCBs at 0.1-1 u.g/ml manifested mainly estrogenic effect to increase germ cell proliferation, while at 10 ug/ml PCBs imposed severe toxicity on germ cells and somatic cells. PCBs induced condensed nuclear chromosome in germ cells and caused cell exfoliation and breakdown within initial hours of treatment. Many cell pieces were released into the medium. Results of 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release assay after treatment for 6h showed significant reduction in remaining a...