The Effects Of Dexamethasone And Carbon Nanotubes On Progesterone Secretion Of Ovarian Granulosa Cells And Preliminary Study On Its Molecular Mechanism

Ovarian granulosa cells(GCs)are a layer of cells located in the peripheral follicle surrounding the oocyte.They are the main functional cells of the ovary.The proliferation,differentiation,apoptosis and function of GCs can play crucial roles in the processes of follicle development,ovulation,luteinization and steroid hormone secretion.It is a convenient and reliable cell model to explore the ovary endocrine function and regulation mechanism by separating and cultivating GCs.This is widely used in the study of reproductive endocrinology and toxicology.Recently the effects of endocrine disruptors on reproductive health have become a hot topic in the research field.The glucocorticoids drugs are widely used in clinic,and the nanoparticles have entered our daily life.They are both potential endocrine disruptors that can cause reproductive toxicity,leading to female reproductive system disease or tumorigenesis.Consequently,they are of important significance in screening endocrine disruptors,evaluating the effects on female reproductive health and studying the mechanism.In this paper,we use in vitro cultured rat granulosa cells as experimental materials to discover the progesterone secretion influence of dexamethasone,and multi-walled carbon nanotubes by Radioimmunoassay,Western Blot,and activity analysis methods,and the mechanism was also discussed.Part 1 The preliminary research of the progesterone secretion influence and mechanism induced by dexamethasone in ovary granulosa cellsObjectives:To discuss the progesterone secretion influence and mechanism induced by dexamethasone in ovary GCs;to study the influence of reproductive endocrinology and follicle development by dexamethasone,providing references for the evaluation of the dexamethasone influence on ovarian function.Methods:Expose the ovary GCs to different doses of dexamethasone,discuss the progesterone secretion influence,involved protein expression,redox state by Radioimmunoassay,Western Blot,flow cytometry,and activity analysis detection methods;expose the female rats to dexamethasone at the dose of 200 mg/kg/rat/day for 7 days by intraperitoneal injection;detect the serum of endocrine hormones by RIA and Elisa.Prepare ovarian slice to observe follicular changes.Results:The treatment of preovulatory GCs with dexamethasone in vitro stimulated progesterone production.Further experiments demonstrated that dexamethasone treatment could change the level of intracellular StAR protein expression in preovulatory GCs,and P450scc was not changed obviously.Dexamethasone decreased the levels of ROS but increased the GSH levels in parallel to the charges in progesterone production.Dexamethasone treatment induced caspase-3 activation,although no obvious apoptosis was observed.Inhibition of caspase-3 activity significantly decreased progesterone production stimulated by dexamethasone.While a significant decrease in serum levels of progesterone and AMH was observed in female rats following dexamethasone treatment.Dexamethasone sped up the ovary follicle development.Conclusions:Dexamethasone can improve the redox state of GCs,induce caspase-3 activation,facilitate StAR protein expression and stimulate progesterone production.The decrease of AMH in serum and changes in ovarian morphology demonstrate that dexamethasone may affect ovarian endocrine and reserve function.Part 2 The preliminary research of the progesterone secretion influence and mechanism induced by carbon nanotubes in ovary granulosa cellsObjectives:To discuss the progesterone secretion influence and mechanism induced by both of the short and long multi-well carbon nanotubes in ovary GCs;to provide references to the reproductive toxicity of carbon nanotubes.Methods Expose the ovary GCs to different doses of both of the short and long multi-well carbon nanotubes,discuss the progesterone secretion influence,involved protein expression,redox state by Radioimmunoassay,Western Blot,and activity analysis detection methods;Results:The treatment of preovulatory ovary GCs with carbon nanotubes in vitro inhibited progesterone production.Further experiments demonstrated that carbon nanotubes increased the levels of ROS and down-regulated the level of intracellular StAR protein expression in preovulatory ovary GCs,and P450scc was not changed obviously.After withdrawing the carbon nanotubes exposition,the StAR protein expression could change to normal levels.Conclusios:Both of the short and long multi-well carbon nanotubes can inhibit progesterone production of ovary granulosa cells in vitro,while increasing the levels of ROS and down-regulating the StAR proteins expression;withdraw the carbon nanotubes exposition,the StAR proteins expression can change to normal level.