| According to the world health organization predicts,infertility is becoming the third largest disease after cancer and cardiovascular disease in the 21st century.The disease was caused by the global warming,environmental pollution,people’s accelerated pace of life,epidemics and other factors.The causes of female infertility was complex and diverse,involving environmental and genetic factors.Recently,with the rapid development of the gene knockout or transgenic technology,it provide a powerful means for revealing pathological mechanism in female infertility,which may provide a new diagnostic and therapeutic targets for infertility treatment.NRAGE(neurotrophin receptor-interacting MAGE homolog)is a member of MAGE(Melanoma antigen-encoding gene,MAGE)family,it is located on X chromosome.Previous studies found that NRAGE plays an important roles in cell apoptosis,cell cycle,and the development of cell growth and many other processes.However,only a few related studies about its roles in female reproductive system.but previous studies show that the circadian clock plays essential roles for normal folliculogengsis in female mice.And we also known that the NRAGE belongs to the circadian clock,this results suggests that NRAGE is involved in the regulation of the female reproductive system.Furthermore,the most important is that during the breeding of the mice,we found that under the same environmentally controlled had normal reproductive ability,but the knockout of NRAGE mice was infertility and heterozygous female mice reproductive capacity is extremely low.Based on these researches and findings,we mainly used the gene knockout heterozygous female mice(homozygous females mice was extremely rare to get)to study the influence of NRAGE knockout on the reproductive system.The main experimental contents and results are as follows:1.Clear the expression of NRAGE in gonadal axis(hypothalamus,pituitary and ovary)in wild type female.Through the RT-PCR and Q-PCR experiments,we found that NRAGE was high expression in hypothalamus,pituitary and ovary,which is far higher than the expression in the cerebral cortex(It has been reported that NRAGE is highly expressed in the cerebral cortex).Meanwhile,the gene expression in the oviduct and uterus were abundant,which was significantly higher than other organizations(such as the heart,liver and kidney and other tissues).Western blot analysis further to support the qPCR results,NRAGE was detected immunohistochemically in ovary,with predominant expression in the luteal cells,the most intensive staining was observed in the oocytes,thecal cells and interstitial cells,and granulosa cells with some weak staining.2 Reproductive competition and fertility analysis,detection of mice vaginal opening time,estrous cycle and serum hormone E2,FSH,LH.To further clear the influence of NRAGE knockout on female reproduction,we used five pairs of wild-type and heterozygous female mice to finish the reproductive competition and fertility experiments.The results showed that forty percent of heterozygous mice was completely sterile,and the body weight of sterile mice was significantly lighter than the control group.In addition,the first production of reproductive age of fertile mice had extended trend when compared with wide-type mice,the average litter size and survival rates exhibited a decreased trend in the fertile heterozygous mice.Meanwhile,the litters production of heterozygous mice was significantly lower than the control group(NRAGE+/-:2.0±1.87vs NRAGE+/+:4.75±0.96,p<0.05).Considering low fecundity of NRAGE knockout female mice,we examined the heterozygous mice’s vaginal opening time,estrous cycle and the level of the serum hormones.The results showed significantly delayed vaginal opening time(NRAGE+/-:40.86±8.25 天 vs.NRAGE+/+:33.74 ± 4.84 天,p<0.05),shorter estrus cycle(NRAGE+/-:11.90 ± 14.36%vs.NRAGE+/+:38.10±6.73%),and longer interval(NRAGE+/-:25.40±12.7%vs.NRAGE+/+:20.95±7.22%,p>0.05)in heterozygous mice,and irregular estrous cycles,while the serum hormone E2,FSH and LH showed no significance difference when compared with wild type mice.3.Morphological observation of ovary and uterus,and follicle countBecause of normal levels of hormone in NRAGE knockout mice,we analyzed the morphology of ovaries and uterus of heterozygous mice.The results indicated that the morphology of ovaries and uterus of heterozygous mice with 4-week-old showed no difference compared with wide type mice(n=2).But we found that 6-month-old heterozygous mice showed different phenotypes,of which fifty percent totally sterile and these sterile female mice exhibited small size,light weight,and ovaries and uterus dysplasia.Fertile heterozygous females mice showed no difference in the body weight,ovaries and uterus,but the number of antral follicles were significantly reduced(NRAGE+/-:0.28±0.20 vs.NRAGE+/+:0.80 ± 0.26,p<0.01).4.SuperovulationTo further explore the reasons for low fertility in mice whether caused by the ovulation disorder,4-week-old mice(n=5)were used for superovulation test.The results showed that the heterozygous females low responsed to exogenous hormone,and ovulation was significantly reduced(NRAGE+/-:16.4±5.77 vs.NRAGE+/+:28.6 ± 9.24,p<0.05).Through the HE staining,we found that ovaries of heterozygous mice were small,antral follicles were significantly reduced(NRAGE+/-:0.28 ± 0.05 vs.NRAGE+/+:0.62±0.19,p<0.05),and a decreased uterine response that(including endometrial thickness and uterine weight)after superovalation treatment.5.TUNEL AssayBecause of the decrease number of antral follicles in heterozygous mice,the present study through TUNEL assay to detect the decrease number of antral follicles whether caused by NRAGE knockout causing apoptosis capacity improved,it was found that follicles apoptosis ability show no significantly diference both at the age of the 4-week-old or 6-month-old in heterozygous mice when compared with wild type mice.The main conclusions:1.NRAGE gene was highly expressed in female gonads axis.2.NRAGE gene knockout heterozygous mice exhibited a reduced fertility phenotype,of which forty percent was completely sterile,the first production of reproductive age of fertile mice had extended trend compared with wid type mice,and its production of litters were reduced,in addition,heterozygous mice had prolonged and irregular estrous cycles,and delayed vaginal opening time,the number of antral follicles decrease significantly,and its litters were significantly lower than control group.3.NRAGE gene knockout mice had poor response to gonadotropin,its performance was reduced numbers of ovulation and thin endometria. |
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