| Objective1. To study effects of the ovarian reserve function of female mice induced by brominated diphenyl ethers-209;2. This study was designed to set up the method of separating cultured granulosa cells from the ovary of nonfertile mice.To study granulosa cells'biological properties, provides an ideal cell sources for follow-up experiment.3. Using BDE-209 in vitro treatment of granulosa cells.To observe on the proliferation and development of granulosa cells.Methods1. Female mice were randomly divided into two groups. The control group:the mice were filled the stomach with peanut oil without BDE-209.The experimental group:mice were filled the stomach with peanut oil contained 0.003 g/ml BDE-209.After 30 days,they were sacrificed at diestrus and made for vaginal smears to watch growth,development in mice organ coefficient, estrous cycle, serum hormone levels, ovarian follicular development and histopathology results.2. Pregnant mare serumgonadotropin (PMSG) favored follicles to grow in the sexual matu-ration of female mice. We obtained ovarian granulosa cells by combined mechanical trypsin,di-gestion and low-speed centrifugation.Cell purity identified by flow cytometric analysis. Through the observation of cell morphology, MTT method assays cells value-added, depicts the growth curve and observes the biological characteristics of ovaty granulosa cells in vitro.3. Make 0μg/mL BDE-209 as the control group A, and 10,20,40μg/mL BDE-209 respectively as the experimental group B, C, D.Granulosa cells in each groups were treated 24 hours, and used CCK-8 for cell survival and inhibition, atomic force microscope for cell surface structure.Results1. The weight of mice were increased during the stomach perfusion,but the trend of experimental mice was slowly during the period of filling stomach.With time, the body weight of the experimental group are lighter than that of the control group(P<0.05,P<0.01).The weight of the control group uterus declined, compared with the other group(P<0.05), but the weight and organ coefficient of ovaries were different (P>0.05). 2. Hormone estradiol (estradiol, E2) levels in the experimental group in serum decreased compared with that of the control group(P<0.05), FSH(follicle stimulating hormone, FSH) was low between two groups(P<0.05); luteinizing hormone (luteinizing hormone, LH) and FSH/ LH make no different (P> 0.05). Estrogen cycle of experimental mice disorders, M and D repeated appearance.There are three mice did not appear in estrogen period.during 14 days.3. BDE-209 decreased primordial and primary/secondary follicle populations,but increased atretic population. The number of granulosa cells reduced in the experimental group, diffuse and loose structure was not clear, granular cell and membrane cell gap widened signifycantly, atretic follicles increased. Primary follicles and secondary follicles decreased.4. The method of mechanical separation, trypsin digestion and low-speed centrifugation could be used to get a large number of stable, purified mouse granulosa cells.The number of 106 cells get from 6 mice with 85% purity on average. Granulosa cells in vitro and biological characteristics:most of the cultured granulosa cells were adherent and round 24 hours after inoculation, the exponentialphase of growth was after 72 h, fusiform-shaped after inoculation.5. BDE-209 can inhibit the mouse ovarian granulosa cell survival. BDE-209 at a concentration of 10μg/mL was without significantly different (P> 0.05), at middle and high concentrations (20μg/mL,40μg/mL)the cell proliferation was apparently divers higher than that of the control group(P<0.01).6. Mouse granulosa cells morphology was observed by Atomic forcemicroscopy.The form of mouse granulosa cells structure was normal, the nuclear was complete,the cell height was similar.With BDE-209 exposure concentration increased, the cell nucleus was destroyed,nuclear broken, nuclear cracked more, collapse, and there was no normal cell with normal form.Conclusion1. The ovarian reserve function in mice reduced and follicle development were hindered after thirty days of stomach irrigation female mice with BDE-209.2. More than 85% of highly purified granulosa cells could be obtained by mechanical method combined with trypsin digestion and brachytely centrifugation.3. BDE-209 inhibited the growth of mouse ovarian granulosa cells and had a dose-effect relationship.4. BDE-209 may do injury mouse ovarian granulosa cells. 5. Combination with inside and outside experimental, BDE-209 has adverse effect for mice ovarian reserve function... |
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