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Methoxychlor View to Implantation in Mice Exposed to HOXA-10 Gene Expression

Author: DiQingZhi
Tutor: ChenBiLiang；MaXiangDong
School: Fourth Military Medical University
Course: Obstetrics and Gynaecology
Keywords: MXC HOXA-10 Reproductive toxicity Embryo dination Immunofluorescence Real-time fluorescent PCR western blotting
CLC: R114
Type: Master's thesis
Year: 2009
Downloads: 47
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Abstract

Since environmental pollution has threatened human health seriously, to study the blight of persistent organic pollutants on human health and multiply has become a global focus of common concern. Harmful environmental factors can influence on human body chronically and synthetically, interfere in any process of reproductive development and do harm to reproductive health. It is proved that occupational or environmental exposure of a variety of chemical substances may affect human reproductive function. It can change human bearing ability evidently, induce libido lowering or losing, infertility, offspring deformities and tumors. MXC is a kind of chemical substance with the largest amount and toxicity which people put in environment initiatively. Recently studies show that long-term exposure to MXC can lead to decline of bearing ability.HOXA-10 gene belongs to abdominal Group B HOX genes. It is involved in the development of female reproductive system, form of normal endometrial shape. And it plays very important roles in endometrial proliferation and differentiation, development of endometrial receptivity, embryo orientation and attachment, pinopod formation, endometrial decidualization, and enhancement of vessel permeability.Purposes:(1) Research on the impact of MXC toxicosis to mice embryo nidation.(2) Research on the impact of MXC to HOXA-10mRNA expression and protein expression in embryo nidation .(3) Discuss the possible mechanisms of MXC influencing on embryo nidation to further explore the methods of treatment.Methods:(1) Take intraperitoneal injection of MXC to make 40 sexually mature female Kunming mice toxic; according to the proportion of 2:1, make female and male mice copulating; kill the mice in the fifth day of pregnancy, and observe the situation of embryo nidation.(2) To compare the uterus morphosis of the mice of each group by taking HE staining method.(3) FQ-PCR was used to detect the concentration of HOXA-10 mRNA in the uterine tissue in 4 groups of mice.(4) Immunofluorescence and western blotting methods were used to detect the concentration of HOXA-10 protein expression in the uterine tissue in 4 groups of mice.Results:(1) The uterine morphosis and the number of the embryo nidation of MXC toxicosis mice have changed. The contrast group has a large number of embryo nidation, and it distributes evenly, while toxicosis group distributes unevenly and the number of embryo nidation decreases more or less. The medium and high dosage group has distinct difference from contrast group (p<0.05). The low dosage group has less number of embryo nidation than the contrast group, but this number has no statistical significance. And the low dosage group also has distinct difference from the medium and high dosage group(p<0.05).(2) It is obvious after HE staining that there is no obvious abnormity in uterus deciduas of the embryo nidation and the flesh thickness between the toxicosis group and the contrast group.(3) Immunofluorescence shows that uterine glandular organ and decidua fluoresce green, and present positive reaction, while the smooth muscle doesn’t present obvious positive reaction. Although all the groups have been colored, the contrast group’s fluorescence is much brighter than the others. The toxicosis group’s fluorescence weakened with the bigger toxicosis dosage, but there is no statistical significance.(4) FQ-PCR shows obviously the differences of HOXA-10 mRNA in the uterine tissue between every dosage group. Both the medium and the high dosage group significantly lower the expression level of HOXA-10 mRNA(p<0.05).Compare with the contrast group, the low dosage group has a lower expression level of HOXA-10 mRNA, but this has no statistical significance.(5) The result of western blotting shows that the protein expression quantities and HOXA-10 mRNA in every dosage group are all the same.Discussions:(1) After MXC toxicosis, the number of mice embryo nidation reduces, and especially in the medium and high dosage group it reduces obviously, which has statistical significance. According to animal experiment, it can be concluded that MXC’s low Estrcgen like effect changes the uterine internal environment, disturbs the reception and window phase of normal uterus, thus disturbs embryo nidation, and lower the rate of embryo nidation. (2) HE results show that although MXC affects the embryo nidation, it doesn’t affect the uterine tissue’s normal anatomic structure in the nidation spot. Due to the results, the MXC toxicosis mainly changes the uterine internal environment in the nidation period, but has little effect on uterine tissue after the nidation.(3) The result of immunofluorescence and FQ-PCR show that: HOXA-10 gene is expressed in pregnant uterine glandular organs and decidua tissues. With the increasing dosage of MXC toxicosis, the number of embryo nidation and the expression amount of HOXA-10mRNA decreases. The mechanism of MXC’s impact on embryo nidation is to lower the expression amount of HOXA-10mRNA, and to further affect the uterine internal environment in the nidation period, which makes the open and duration time of the uterine reception and window phase non-synchronously with the embryonic development, thus the rate of nidation reduces.(4) The result of western blotting shows that MXC toxicosis can reduce the expression of HOXA-10 protein, but each impact doesn’t all the same. Both the protein expression quantities and HOXA-10 mRNA are the same, we believe this kind of protein is influenced by the embryo dination of HOXA-10 gene.

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