Effects Of Icariin And Epimedium Polysaccharide On Follicular Development And Antioxidation

In the mammalian ovary,cell death and survival-promoting factors regulate follicular development and atresia,which include intraovarian regulators(intracellular proteins,cytokines and gonadal steroids)and hormones(gonadotropins).Hundreds of primordial follicles are exist on the ovary in mammals;however,only a limited amount of follicles will develop to the preovulatory stage and ovulate.The most of primordial follicles will be removed by a degenerative mechanism called "atresia".Activation by death ligands or eliminated of critical survival-promoting growth factors is the main cause leading to apoptosis,and well known stress inducers also could cause apoptosis,like oxidative stress,radiation,toxicants and drugs.Recent studies indicate that the apoptosis of ovarian granulose cells may play an important role in follicular atresia.Epimedium(EP),the largest herbaceous genus of Berberidaceae,used for promoting animal empathema impotence,spermatorrhea,infertility,amenorrhea menopause symptoms.Icariin(ICA)and Epimedium Polysaccharide(EPS)were the most important contents of Epimedium.Epimedium could stimulate the granulose cells of rat secrets estradiol.Icariin can promote follicles development and advanceds estrus in mice,increase the antioxidant enzyme SOD activity levels in serum of rat.Epimedium Polysaccharide also can improve the reproductive index of mice,and used as an antioxidant.However study about the effect of ICA and EPS on the development of oocytes and antioxidiant in follicular granulose cells in mice has been rarely reported.The aim of this experiment was to research the effect of ICA and EPS on development of oocytes,and estimate the anti-apoptosis and antioxidant effect of ICA and EPS on follicular granulose cells in mice,in order to find the effect of ICA and EPS promote the development of ovarian follicles,and provide reference to new experiment to the apply of EP and its effective ingredients and oocytes in vitro culture.The results are showed as follows:1.Effect of ICA and EPS on the development and maturation of murine oocyte in vitroHealthy 4-week mice were dosed with ICA(0.1,0.3,0.5,0.7 mg),EPS(2,4,6,8 mg)and saline(control)via continuous orally administrated for 7 and 14 d.Mice from each group were killed and oocytes from ovarian antral follicles were separated and conducted for maturation in vitro,recorded and caculated the rates of pbI extrusion after 12 h,then maturation oocytes of every group for fertilization in vitro,observed fertilization rate at 6 h.The results showed that the pbl extrusion rates of ICA 0.5 mg groups were significantly increased compared with saline control(75%to 63.3%;P<0.05),and also higher than the pbI extrusion rates of ICA 0.3 mg treatment groups which highest in 14 d(68.1%;P<0.05);only the pbI extrusion rates of EPS(8 mg)was significance higher(80%;P<0.05)after 7 d treatment.Continuous orally administrated for 7 d the fertilization rates of ICA 0.3 mg groups were significantly increased(69.4%to 41.1%;P<0.05),after 14d,the fertilization rates of ICA and EPS groups were not significantly increased(P>0.05);2.Effect of ICA and EPS on the development of female murineAfter 7 d orally administrated with different dosages of ICA(5 and 7 mg)and EPS(6 and 8 mg),normal saltwater(control).Weight gain,body and organ weights of mice were measured at 8 days.the weight gain and body weight of all treatment groups have a slight decline compared with control,but there was no significant difference,liver,kidney,lung,spleen and heart index were not impacted by the drug treatments(P>0.05).All drug treatment groups advanced the oestrum.3.Effect of ICA and EPS on the apoptosis of murine follicular cellsMice treated with ICA(5 and 7 mg)and EPS(6 and 8 mg),control treated with saltwarter for 7-day,follicular cells were isolated and tested the apoptosis of follicular cells by FCM.ICA(5 and 7 mg)and EPS(6 mg)groups were significance lowered the levels of apoptosis in follicular cells compared to control groups,the early apoptosis rate of follicular cells of all the treatment groups were significance decreased(P<0.05).Quantitative PCR analysis of the relative expression of Bcl-2 and Bax mRNA in follicular cells,ovaries and uterus,after treatment with ICA 0.7 mg group for 7 days,the ratio of Bcl-2 levels to Bax levels was significantly increased(P<0.05),the ratio of Bcl-2 levels to Bax levels of EPS 6 mg has not abvious change compared to control group,while EPS 8 mg group was significance lower(P<0.05).The gene expression rate of Bcl-2 levels to Bax levels in ovary and uterus of treatment groups were not significance different compared to control group(P>0.05).4.Effect of ICA and EPS on ROS and O2-of murine follicular cellsMice treated with ICA(5 and 7 mg)and EPS(6 and 8 mg),control group treated with saltwater for 7-day,follicular cells were isolated,tested the ROS via DCFH-DA by FCM and measurement the O2-by Cellular Superoxide Anion Colorimetric Quantitative Determination Kit.ROS and O2-levels increased significantly in follicular cells of EPS(8 mg)(P<0.05),the ROS level of EPS(8 mg)group was higher significantly(P<0.05),but ROS and O2-levels of ICA(5 and 7 mg)groups did not show significance different compared to the control group(P>0.05).5.Effect of ICA and EPS on the antioxidant of murine follicular cellsMice treated with ICA(5 and 7 mg)and EPS(6 and 8 mg),control group treated with saltwater for 7-day,follicular cells were isolated and tested the activities of three antioxidant enzymes—SOD,GSH-PX and CAT-were determined using commercial kits.the activities of the antioxidant enzymes(GSH-Px and CAT)increased in follicular cells of mice administered with ICA(5 and 7 mg)and EPS(6 and 8 mg)groups,especially EPS 8 mg group significantly increased(P<0.05),the activities of SOD in all drug treatment groups had not big difference compared with control(P>0.05).Quantitative PCR analysis showed that mRNA levels of SOD1,SOD2 and GPX1 in follicular cells,ovaries and uterus,ICA 7 mg group significantly increased the mRNA levels of SOD land SOD2 in follicular cells and GPX1 in uterus(P<0.05),EPS 8 mg group significantly lower the mRNA levels of SOD2 and GPX1 in follicular cells,but significantly higer in uterus(P<0.05).