The Study Of The Role Of Uncoupling Protein 2 In Female Reproduction

It is well known that age-related change in oocyte be association with oxidants damaging.The study show that hyeroxidant can induce embryo degenerative process .These data suggest that ROS be involve in the regulation of reproductive process,but it mechanism has not been clarified.Uncoupling protein2 (UCP2) belong to the family of transporters present in the inner membrane of mitochondria. Ucp2 is widely expressed in mammalian tissues, and the presence of the corresponding protein was demonstrated in macrophages, spleen, gut, lung, and white adipose tissue mitochondria as well as in pancreatic islets and thymocytes. The phenotypic analysis of Ucp2(-/-) mice indicates that this gene does not play a role in either thermogenesis or the regulation of body fat content but is strongly implicated in the regulation of ROS production, as was proposed previously for proteins able to uncouple respiration. In addition, very recent studies support a role of UCP2 in immunity and inflammatory responsiveness.A recent publication has presented evidence for Ucp2 expression in the reproductive tract of female mice. In addition, it has already led to other publications in which a role for UCP2 has been proposed in mechanisms of antioxidant injury. Accordingly, we decided to reinvestigate the putative presence of UCP2 in the reproductive tract of female and also to investigate the role of UCP2 in the reproductive tract of female.Part I:the expression of UCP2 in endometrium and decidual and its relationto infertility Objective:To investigate the expression and localization of uncoupling protein 2(UCP2) in human endometrium during menstrual cycle and deciduas in early pregnancy and evaluate its significance in the implantation of blastocyst.Methods:1 Samples:21 samples of normal endometrium were obtained from fertile women in Department of Gynaecology ,NanFang hospital,9 cases in proliferative phases, 12 cases in secretary phases.22 samples in secretary phases were from infertile women, 13 patients with tubal factor (7 patients with hydrosalpinges),4 patients with endometriosis,5 patients with anovulation.7 samples of decidual were from early pregnancy women undergoing induced abortion.2 Methods: the expression of UCP2 mRNA in human endometrium was detected by in-situ hybridation technique.The amount of UCP2 protein in human endometrium during menstrual cycle was anaylized by western blot. Results:1 UCP2 mRNA and protein were detected in the human peri-implantation endometrium. UCP2 mRNA was located in the cytoplasm of glandular epithelial cells and trophocyte on the surface of chorinic villi.2 UCP2 mRNA and protein level had periodical change during the menstrual cycle and pregnancy(protein level, 19.1 + 3.2,36.4±3.9,47.9 ±4. 2). The expression of UCP2 mRNA was mainly shown in secretory phase endometrium and ducidua in early pregnancy.3 The result of western blot showed that tha level of UCP2 protein increased significantly in the secretary endometrium with hydrosalpinges compared to normal fertile women or other infertile women.Part II:the expression of UCP2 in ovary and its relation to age Objective:To study the expression and localization of UCP2 in human ovaries tissue and evaluate its role in the follicular development and its relationship to female age. Methods: 1 33 samples of ovaries were obtained from patients in NanFang hospitalundergoing ovariotomy, 11 cases =^37years,22 cases > 37years. The samples were taken into liquid nitrogen for in-situ hybridation and western blot analysis.2 Methods: the expression of UCP2 mRNA in human ovaries was detected by in-situ hybridation technique.The amount of UCP2 protein in human ovaries during menstrual cycle was anaylized by western blot.Results:1 In human ovaries,the UCP2 mRNA specially located in granulose cells in follicule and cells in vascular wall and remain consistent through the whole menstrual cycle.2 The expression of UCP2 protein in human ovaries is correlated with female age.The level of UCP2 protein from advanced-age women (>37 years) was lower than that of younger group (^ 37 years),(24.2± 1.9 vs 34.1 ±3. 1, P < 0. 05) .Part III:the expression of UCP2 in human granulose cell and its relation tothe development of early embryo Objective:To study the expression of UCP2 in human luteinizing granulose cells from patients undergoing in-vitro fertilization-embryo transfer (IVF-ET),to investigate the relationship between the expression of UCP2 and reactive oxygen species (ROS) level or transmitochondrial electrochemical gradient in cell and evaluate its relation to oocyte development.Methods:53 samples of human GCs were recovered from follicular aspirates obtained during transvaginal ultrasound-guided oocyte retrieval for in vitro fertilization embryo transfer.The expression of UCP2,the level of reactive oxygen species and the mitochondrial membrane potential were measured by flow cytometer.Results:the expression of UCP2 in luteinizing granulose cells correlated inversely to ROS level and mitochondrial membrane potential in granulose cells.(r = -0.578,P <0.01 and r =-0.787, P <0.01).The patient whose UCP2 was under the mean levelmight have high level ROS in granulose cells and its oocytes development potential might .be impaired.Part IV:the induction of UCP2 in granulose and the effect on antioxidantcapability Objective:1 To investigate the induction effect of hydrogen peroxide on the expression of UCP2 in in-vitro cultured granulose cell and the change of antioxidant capability in cell after inhibition of the expression of UCP2 in granulose cell.2 It has been known that oxidantal injury related to the age-related change of oocyte.That the mechanism of antioxidant had been elucidated would make for the manoeuvre to human reproductive.MethodsAdd hydrogen peroxide at different concentration (0,0.1mmol/L,0.25 mmol/L,0.5 mmol/L) to in vitro cultured granulose cells, Western blot were used to analyze the expression of UCP2 in cells.After inhibition of UCP2 with adenosine triphosphate( ATP) at 1 mmol/L,the apoptosis rate was analyzed by flow cytometer.Results1 Hydrogen peroxide obviously induced the expression of UCP2 in in-vitro cultured granulose cell. The level of UCP2 protein was highest when hydrogen peroxide concentration is at 0.25mmol/L.2 To inhibit the expression of UCP2 with ATP under the condition of hydrogen peroxide (0.25mmol/L), the apoptosis rate significantly increased than control group(4. 81 + 0. 50 vs 1.24+1.02).Conclusions1 UCP2 express in the reproductive tract of female where UCP2-ROS regulation system may be the main mechanism of antioxidant injury.UCP2 is a member of mitochondrial transport proteins that promote proton leakage across the inner mitochondrial menbrane,uncoupling oxidative phosphorylation from adenosine triphosphate production. UCP2 negatively regulated reactive oxygen species generation. UCP2 might exert protective effects by diminishing ROS production through activation of mitochondrial UCP2.2 UCP2 was synthesized and secreted in human peri-implantation endometrium, UCP2 would play a regulative role in the induction of human endometriial decidual and have an important function in the implantation of the blastocyst.3 The expression of UCP2 in granulose cells in follicule and its relation to female age suggest that UCP2 might involve in the follicle development regulation.4 there is a UCP2-ROS system in granulose cell. Oxidants could activated UCP2 and promote feedback down-regulation of mitochondrial ROS production.The exceptional UCP2-ROS system will have a adverse effect on follicular development.