| Objective:The objective of this study is to investigate the effect of cadmium (Cd) on female gonads , which are the effect of cadmium (Cd) on the estrus cycle and the function of sexual hormones secreting of both pituitary and ovary in female animals , and to explore relationship between the potential estrogenicity of cadmium and it's toxicities of female gonads in order to provide the scientific support for the study on toxicities of female gonads endocrine disrupt of cadmium.Methods:(1)Thirty Sprague-Damley (SD) female rats were weighted and randomly assigned to three groups based on body weight and were administrated subchronically with subcutaneous injection (daily dose is respectively 0, 0. 5,1. 0 mg/Kg (count Cd2+) BWT, 5 days a week, for 6 weeks ) . 20 days later, estrous cycle of each animal was observed with daily vaginal smears untilthe end of the test (for 10 days).(2)The changes of serum luteinizing hormone (LH), follicle stimulating hormone(FSH), progesterone (P) and estradiol(E2) levels were observed with the radio immunoassays in female rats administrated subacutely with intraperitoneal injection for 7 days (daily dose is respectively 0,0.25,1.0 mg/Kg(count Cd2+) BWT) or subchronicaly with subcutaneous injection(daily dose is respectively 0,0.25,1.0 mg/Kg BWT) for 14 days. Another 30 SD female rats administrated subchronically with subcutaneous injection (daily dose is respectively 0,0.5, 1.0 mg/Kg BWT, 5 days a week, for 6 weeks ) have been observed for the determination of the serum FSH and LH levels before and after the injection of the gonadotrpin-release hormones (GnRH).(3)Sprague-Damley rats were ovariectomized (a range of 10~16 days prior to receptor preparation ).After rats were sacrificed ,uteri were excised ,trimmed of excess fat and mesentery, weighted and immediately placed in ice-cold TEMD buffer(10 mmol / L Tris-HCl, 1.5 mmol / L EDTA, 20 mmol / L Na2MoO4, 5mmol / L MgCU 6mmol/L P -Mercaptoethanol, PH 7. 4). Unless otherwise stated, all procedures are performed at 4℃. The uterine tissue was homogenized in TEMD buffer(1:10(W/V)). The resulting homogenate was transferred to pre-cooled(4℃) high-centrifuge tubes and centrifuged at 800 X g for 10 min to obtain cytosolic pellet. The cytosolic fraction is removed and then centrifuged at 15, 000xg for 30 min. After centrifugation, theER-rich supernatant was decanted into 10 ml conical tubes and was stored at -74 until used in competition assays.(4)Radioligand binding assay(RBA) (estrogen receptor competitive-binding assay .Supernatant in ovariectomized SD rats was used as the source of ER ) in vitro was used. The effects of cadmium on estrogen binding were performed using a sing-dose ligand-binding assay. Extract from uterus in ovariectomized SD rats were treated with various concentrations of cadmium(0, 10-3, 10-5 or 10-7M)for various pre-incubation time(0, 30, 60, 90 min) using orthogonal experimental design with orthogonal layout of 1,6(45) (the experimental was repeated 5 times) . The ability of the ER to binding hormone was then assayed by incubating the extract with M [3H]estradiol in the presence or absence of a 200-fold molar excess of diethylatilbestrol for 18h at 4 ℃ . Following the incubation period, 200 ul of a cold dextran-coated charcoal (DCC, 1% activated charcoal, 0. 1% Dextran T-70 in TE buffer) was added to each tube to separate the bound ligand from the free ligand. These tubes were incubated in an icewater bath for 15 min and vortexed one time at 5-min. Tubes were subsequently centrifuged (3500rpm) at 4℃ for 15 min. The .resulting supernatant was decanted into vials contain 11 ml scintillation cocktail. Radioactivity was measured on a Packard Tri-Carb 2300TR Liquid Scintillation Analyzer (Packard instrument Company). The specific binding was determined as the difference between total and non-specific binding.(5)To determine whether cadmium altered the binding affinity of estradiol to the receptor, a multiple-dose ligand-binding assay was performed. The cytosol ER extracted fro... |
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