| At the present time, the problem of environmental chemicals that exhibit estrogenic activities is of great concern. Numerous studies indicate that estrogens and environmental chemicals with estrogenic activity cause endocrine-disrupting effects, including altered reproductive functions[1,2], in various animals.The organochlorine pesticide methoxychlor(MXC), which is a DDT substitute, is a proestrogen that is activated in vivo into an active estrogenic metabolite[3]. It has been used extensively to control pests in household as well as agricultural settings. MXC is less acutely toxic than DDT, is rapidly eliminated from the body, and is less likely to be stored in body fat[4]. However,research has shown that MXC has deleterious effects on fertility of animals. These effects include the ability to block implantation[5,6,7]. But the action mechanism of MXC in inhibiting implantation has not been made clear.The 9kD cytosolic calcium-binding protein (Calbindin-D9k) belongs to a family of intracellular proteins, Uterine Calbindin-D9k has been postulated to control myometrial activity related to intracellular calcium levels[8]. In addition, evidence has been presented for an absolute requirement for Calbindin-D9k during the early phase of embryo implantation[9].Many researchs have demonstrated that in vivo exposure to environmental estrogens, such as MXC causes Calbindin-D9k expression significantly increase in a dose-dependent manner in immature rat uterus, so Calbindin-D9k can be used as a biomarker for detection of environmental estrogenic chemicals such as MXC; however, few studies have been pursued regarding MXC's effects on Calbindin-D9k expression during implantation of embryo.The aim of this study was to use the MXC poisoned mouse as a experimental animal model search for unrecognized molecules mechanism of MXC in inhibiting implantation. We constructed MXC poisoned mouse models during early pregnancy and observed the expression of Calbindin-D9k mRNA and protein of pregnant mouse uteri. In addition, the effects of MXC onαsubunit of Estrogen receptor (ERα) and Progesterone receptor (PR) expression of mouse uterine endometriums were also further studied.Our exprement were divided into two parts.Part 1. Methoxychlor poisoned pregnant mouse models were constructed,the numbers of embryo implantation were observed and effects of MXC on ERαand PR expression of mouse uteri were measured.Eighty successfully mated female mice were randomly divided into four groups (n=20 per group). Three MXC exposed groups of twenty animals were treated orally with MXC at three different doses:100, 200 and 400 mg/kg body weight(BW). One group of mice were given orally with sesame oil alone as a vehicle control. MXC were administered in a sesame oil vehicle, once per day during days 1-3 of pregnancy (vaginal plug=Day 0). Pregnant mice were euthanatized on day 5 of pregnancy and their uteri were rapidly excised. The numbers of embryo implantation were examined and the pregnancy rate was counted. The expression of ERαand PR mRNA were measured by RT-PCR , the expression of ERαand PR protein were analyzed by immunohistochemical staining and image analysis method.Results:①A fter treatment with MXC for 3 d, the pregnancy rate in mid and high doses of MXC(200,400 mg/kg BW) groups was significantly lower than that in the control group(P<0.05), whereas, no significant alterations were observed in low dose of MXC(100 mg/kg BW) group compared to the control group.②The average number of embryo implantation per animal was decreased significantly in mid and high doses of MXC(200,400 mg/kg BW) group compared to the control group after 3 days of treatment with MXC (P<0.01). Treatment with a low dose of MXC(100 mg/kg BW) appeared to decrease the average number of embryo implantation, but the decrease was not significant.③With the increase of the administered MXC dose,the ERαmRNA and protein expression levels of pregnant mouse uteri were also increased correspondingly, there were significant differences in mid and high doses of MXC groups compared to the control group(P<0.05).④treatment with MXC significantly decreased PR mRNA expression at mid and high doses(200,400 mg/kg BW) (P<0.05), treatment with low(100 mg/kg BW) dose of MXC did not have significant effects in the PR mRNA expression. The expression of PR protein showed a similar change to that of PR mRNA.Part 2. Effects of methoxychlor on the Calbindin-D9k gene expression in the mouse uterus during implantation period.We established the MXC contaminated models for early pregnant mouse as the same as the first experiment. The expression of Calbindin-D9k mRNA was measured by RT-PCR, Western blot analysis and immunohistochemical staining were used to examine the expression of Calbindin-D9k protein.Results:After the pregnant mice were treated with MXC for 3 d, the Calbindin-D9k mRNA levels of their uteri were significantly decreaseced at 200 and 400 mg/kg BW doses compared to the control group (P<0.05), treatment with 100 mg/kg BW MXC appeared to decrease Calbindin-D9k mRNA, but the decrease was not significant(P>0.05). On day 5 of pregnancy, Calbindin-D9k protein was mainly located in the luminal and glandular epithelial cells. In parallel with its mRNA levels, the protein levels of Calbindin-D9k were significantly decreased by the treatments with MXC at 200 and 400 mg/kg BW(P<0.05), but no significant alteration was observed in the protein level at 100 mg/kg BW MXC treatmentConclusion: Taken the exprement results together, we can draw the conclusions as follows:①MXC can decrease the pregnancy rate and the number of embryo implantation in the mouse;②MXC can induce the expression of ERαin mouse uterine endometrium; MXC can decrease the expression of progesterone receptor in mouse uterus;③MXC can reduce the expression of Calbindin-D9k gene in the mouse uterus during implantation period;④MXC down-regulateing Calbindin-D9k expression was mediated by PR; the decrease caused by MXC in Calbindin-D9k level may be a potential mechanism of the antiimplantation effect of MXC in the mouse.
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