Effects Of CART And Insulin On The Production Of CAMP In Sheep Ovarian Granulosa Cells FSH Signal Transduction

Objective:This experiment use the method of gradient liquid added amount of CART and insulin on granulosa cells in vitro culture so as to explore the effect of different concentrations of CART and insulin on granulosa cell proliferation, E2secretion and intracellular cAMP production.In order to determine the role of CART and insulin on granulosa cells via cAMP signal transduction pathway, this experiment study the mechanism of action of CART and insulin on granulosa cells.Method:Firstly, adding different concentrations of liquid culture of insulin (0,1,10ng/mL) to determine the optimum concentration of insulin on granulosa cell proliferation and the secretion of E2. Then use the insulin concentration to study the effects of CART on granulosa cell proliferation and the secretion of E2by adding different concentrations of CART (0,0.01,0.1,1ng/mL). To determine the best concentration of insulin which influence the production of cAMP in the granulosa cells by using different concentrations of insulin cultured granulosa cells. Finally use the insulin concentration to study the effects of the production of cAMP in the granulosa cells by adding different concentrations of CART. This experiment measure the concentration of E2and cAMP by using double antibody sandwich method (Elisa test).Results:First, the maximum number of granulosa cells and the maximum concentration of E2in the culture medium when the concentration of insulin in the culture medium is10ng/mL. The number cells of each hole less than50000without adding insulin.Second, the number of granulosa cells and the content of E2in liquid culture were obviously reduced when CART was added into the medium. The number of granulosa cells were little changed and the content of E2were decreased about1times when the concentration in the culture solution of CART improved from0.01ng/mL to1ng/mL.Third, the content of cAMP in granulosa cells was significantly increased when the culture of insulin added solution. The contents of cAMP in the intracellular were little changed when the concentration of insulin was increased from1ng/to10ng/mL. Fourth, the content of cAMP in granulosa cells decreased with the increase of the concentration of CART when the concentration of insulin is10ng/mL in the culture.Conclusion:First, the number of cell increased significantly when added the insulin into the cultured of ranulosa cells in vitro culture. But the number of cells will reduce a little without addition of insulin and the number of cells will little change when improve the concentration of insulin. This shows that the growth and proliferation of granulosa cells and insulin existence the dose-dependent effect. Between the secretion of estrogen granule cells and the production of cAMP exist the same phenomenon. This same phenomenon illustrates that the effect of insulin on granulosa cells may not through the cAMP signal transduction pathway and the influence of insulin for the secretion of estrogen granulosa cells is no direct relationship. Second, CART can inhibit the proliferation of granulosa cells. The increase of CART concentration did not make corresponding reduction in the number of cells. This shows that CART does not directly inhibit granulosa cell proliferation but there is a complex mechanism of action between them. CART can also inhibit the secretion of estrogen granulosa cells and the production of cAMP. The content of cAMP in cells were decreased with the increase of the concentration of CART in culture solution, but the decrease of estrogen is not a linear relationship with the concentration of CART. This shows that the effect of CART on granulosa cells probably through cAMP signal transduction pathway, but the effect of CART on the secretion of estrogen granulosa cells does not pass cAMP signal transduction pathway.