Expression Of Cart Protein In Pig Ovarian Follicles And Effects On Folliclar Granulosa Cells In Vitro Culture

Research purpose:To study whether Cocain-and Amphetamine-Regulated Transcript (CART) has expression and Immunohistochemical localization of intrafollicular CART protein, and to optimize the culture system of pig follicular granulosa cells in vitro.Design and methods:1.To study CART expression in pig ovary follicle and orientate its location by immunohistochemistry.2.To optimize the serum-free in vitro culture system of pig ovarian granulosa cells by adjusting the concentration of FSH and insulin, through counting cells and measuring estragen concentration with Elisa method.3.To investigate CART effects on the development of pig granulosa cells by adding different concentrations of CART (0,0.01,0.1,1μM) in the above optimized in vitro culture system.Results and analyses:(1) To orientate CART in pig ovarian follicle by immunohistochemistry, the results showed that CART was expressed on the granulosa cells and theca interna cells, there was more expressed quantity in small follicles than that in large follicles.(2) In the serum-free culture system, the experimental group with no adding FSH and insulin had the least granulosa cells and lowest secretion of estrogen which proved that FSH and insulin could promote the development and estragen secretion of pig granulosa cells and had a dose-dependent effects. When the concentrations in culture system of FSH and insulin were25ng/mL and100ng/mL, respectively, the number of granulose cell was biggest. However, When added5ng/mL FSH and10ng/mL insulin in the cell culture system,estrogen concentration was the highest(P<0.05), which was the optimum adding concentrations for cell culture in vitro for pig granulosa cells.(3) Compared with the experimental groups with adding CART, the experimental group with no adding CART had the largest number of granulosa cells and secretion of estrogen (P<0.01).Compared with the experimental groups with adding CART, the experimental group with no adding CART had the largest number of granulosa cells and secretion of estrogen (P<0.01).The number of the granulosa cells and estrogen secretion decreased in different levels with adding different CART concentrations in culture system, this proved CART had inhibiting effects on pig ovarian follicular granulosa cell proliferation and estrogen secretion. When the additive concentration of CART was0.1uM, estrogen had the lowest secretion concentration compared with other experimental groups (P<0.01), when the additive concentration of CART was1μM, estrogen secretion increased comparing with the experimental group of0.1μM, the difference was very significant (P<0.01).