Establishment Of Mouse Model Of Ovarian Oxidative Stress And The Screening Of Ovarian Protective Antioxidants

With the quickening of the pace of life, the social competition is increasingly fierce. No matter work or learning is full of pressure, and excessive stress and anxiety lead to increasing psychological pressure for modern career women. The incidence of reproductive diseases is increasing, and the human reproductive diseases caused by ovarian oxidative stress are growing. Ovarian oxidative stress not only affects the generation of the egg, follicle development, oocyte maturation and follicle atresia but also reduces the quality of oocyte and embryo in early embryo development, which make the fertilization rate, embryo quality and pregnancy rate significantly reduced. Nevertheless, the relationship between oxidative stress and such disorders is not clear and cannot be adequately investigated in human for self-evident ethical reasons. So it is very important to establish the model of ovarian oxidative stress, which has important practical significance for revealing the ovarian oxidative stress mechanism and the development of new anti oxidative stress drugs. In this study, oxidant3-NPA is used to establish an ovarian oxidative stress model and assessed this model. Using this ovarian oxidative stress model induced by3-NPA, comparing the protective effect of different antioxidants on ovarian and screening four kinds of ovarian protective antioxidants. This has important significance for revealing the mechanism of ovarian oxidative stress and researching the action mechanism of anti-ovarian oxidative stress drugs. A mouse model of ovarian oxidative stress can be established by intraperitoneal injections of3-NPA.1. Establishment of a mouse model of ovarian oxidative stressThe aim of this study was to establish an animal model that could mimic the characteristics of ovarian degeneration induced by oxidative stress in vivo and investigate the effect of ovary oxidative stress on the follicle and oocyte development. Kunming female mice were dosed with3-NPA (6.25,12.5and25mg/kg) and saline (control) via intraperitoneal injection for7days. After dosing, ovaries were collected for histological evaluation of follicle numbers, granulosa cell apoptosis and atresia, and for measurement of weight, ROS levels, mRNA expression levels (Bcl-2, Bax, SOD2, CAT and GPX), and antioxidant enzymes (T-SOD, CAT and GPX). All data were also measured in other organs. The results indicated that at the7-day time point,3-NPA (12.5mg/kg) increased significantly the percentage of atretic large follicles, decreased the weight of ovaries, and reduced the number of large follicles compared with controls (P<0.05). Moreover, the number of ovulated oocytes and early embryonic development capacity were decreased significantly compared to controls (P<0.05). In addition,3-NPA (12.5mg/kg) also decreased significantly, the ratio of Bcl-2to Bax, increased the mRNA expression of (SOD2, CAT and GPX) and ROS levels in granulosa cell (P<0.05).Collectively, these data indicate that3-NPA induce granulosa cell apoptosis, large follicle atresia, and increase ROS levels in ovary. Therefore, it is suggested that we have established an in vivo model of ovarian oxidative stress that is useful for studying the mechanism of ovarian oxidative damage induced by free radicals and for the screening of novel antioxidants.2. The screening of ovarian oxidative stress protective antioxidantsThis experiment adopts the ovarian oxidative stress model by3-NPA and compares the antioxidant protective effects of different antioxidants on ovarian oxidative stress. In this thesis, antioxidant activities of ten antioxidants such as Vitamin C, Vitamin E Carotene, Glutathione, Gallic acid, Curcumin, Proanthocyanidin, Resveratrol, L-cysteine and Coenzyme Q were measured. In the study of antioxidant activity in vitro, ROS level and activity of the antioxidant enzyme such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities in ovarian tissue were used as the measurement index of the antioxidant function. The results showed that the ten antioxidants exhibited different antioxidant abilities, L-cysteine,Gallic acid, Curcumin, Proanthocyanidins, Resveratrol and Carotene can significantly reduce ROS levels in ovarian tissue (P<0.05) and Curcumin, Resveratrol, Procyanidin and carotene can significantly increase the weight of the ovary (P<0.05) compare with model group. Eight antioxidants such as GSH, Vitamin E, L-cysteine, Gallic acid, Vitamin C, Coenzyme Q, Resveratrol and Carotene can make compensatory elevated glutathione peroxidase (GSH-Px) activity in ovarian tissue significantly decreased (P<0.05). GSH, Vitamin E, L-cysteine,curcumin, Vitamin C, coenzyme Q, resveratrol and carotene eight kinds of antioxidants can make compensatory elevated T-SOD in ovarian tissue significantly lowered (P<0.05). GSH, Vitamin E, procyanidin, coenzyme Q and carotene five kinds of antioxidants are able to make compensatory elevated CAT activity in ovarian tissue reduced significantly in ovarian tissue (P<0.05). The results of the experiment showed that ovarian oxidative stress can lead to ovarian tissue oxidative damage, promote the development of ovary related disease. Antioxidant supplements can dramatically reduce ovarian oxidative damage and improve ovarian tissue antioxidant function. The study demonstrated that the mouse model of ovarian oxidative stress was successfully established, and the granulosa cell apoptosis, large follicle atresia, and increased ROS levels in ovary could be observed significantly in this model. By comparing the protective effect of different antioxidants on ovarian oxidative damage, and finding that phenol antioxidants have strong antioxidant activity and have a very good protection against ovarian oxidative damage.3. Study on comparison of different antioxidants antioxidant activity in vitroTo explore the protective effect of different antioxidants PC、VC、NAC against the damage on mouse granulosa cells induced by3-NPA. There were five groups in this experiment, namely negative control group,3-NPA group, PC (50μg、100μg、200μg/mL)、 VC (25μg、50μg、100μg/mL)、NAC (25μg、50μg、100μg/mL). The model of the damage on mouse granulosa cells induced by3-NPA was made by culture in vitro. Morphological changes in granulosa cells were observed. The survival rate was detected by MTT method. The levels of SOD and MDA were measured by chemical colorimetry methods. The apoptosis rate of granulosa cells was detected by TUNEL method. Results:The oxidative damages in granulosa cells induced by3-NPA in PC、VC、NAC groups were relieved as observed under microscope. Compared with3-NPA, the survival rate in PC、VC、NAC groups increased from53.7%to85.3%,63.8%and60.5%respectively (P<0.05). The apoptosis rate in PC、VC、NAC groups decreased from38.2%to13.4%、23.6%and25.8%respectively(P<0.05).The SOD activity decreased significantly in PC、VC、NAC groups, the level of MDA in PC and NAC groups decreased significantly(P<0.05). The levels of mRNA anti-stress genes and apoptosis-related genes decreased significantly in PC、VC、 NAC groups(P<0.05).Conclusion:PC and VC has significantly protective effects against oxidative damage induced by3-NPA in mouse granulosa cells.The mechanism of protection may be related to anti-oxidation and apoptosis reduction.3. Ovarian oxidative stress on reproductive performanceThe aim of this study was to explore the influence of the ovarian oxidative stress on the embryonic development in female mouse.Female mice were dosed with3-NPA (12.5 mg/kg) and saline (control) via intraperitoneal injection for7days.After super-ovulation treatment, the females were paired with a male (one to one) overnight and were examined for the presence of a vaginal plug in the following morning. The day on which a vaginal plug was observed as considered as Day0.5of gestation (0.5d), the pregnant mice were killed on day7.5、12.5and18respectively.The embryos were isolated and collected. The number and weight of embryos were measured; the dead embryos were counted. Results: compared with control group, the3-NPA treatment group decreased the growth of pregnant mice body weight and the number of embryo, resulted in the increased of the rate of the dead embryo. Conclusion:Ovarian oxidative stress can reduce the fertility and reproductive capability.