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The Effects of Lead Acetate on the Expression of Androgen Binding Protein, Transferrin and Inhibin mRNA in Rat Sertoli Cells

Author: ZhaoShiGuang
Tutor: ChenXiaoYu
School: Zhengzhou University
Course: Health Toxicology
Keywords: Lead acetate Sertoli cells Androgen binding protein Inhibin Transferrin
CLC: R114
Type: Master's thesis
Year: 2008
Downloads: 48
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Abstract


In recent years, environmental pollution has become a very prominent issue, heavy metals is one of the important class of contaminants. Lead is a heavy metal earliest human started using one of the elements, because of its wide range of applications, but also caused environmental pollution. Multiple systems can lead to the body and organ damage and affect reproductive function in animals and humans. At home and abroad in the past about the lead male reproductive toxicity studies have focused on the testicles, because testosterone is the main male gonads, and is the most xenobiotics target organ of male reproductive toxicity. Testicular Sertoli cells is a male animal is unique within the seminiferous tubules of somatic cells in the male reproductive system plays an important role. Sertoli cells in vitro model as a rapid, economical, less interference, the experimental results and the reliability of the results targeted and reproducible characteristics, are widely used exogenous toxins in reproductive toxicology studies. Supporting cells called germ cells of the \cell differentiation and maturation, has a fine coordinating role to ensure the orderly conduct of normal spermatogenesis. Sertoli cells secrete androgen binding protein (androgen binding protein, ABP), transferrin (transferrin, Tf) and inhibin (inhibin, INH) and so on. ABP androgen capable of transporting and storage, so that the seminiferous tubules to maintain high levels of androgens, spermatogenesis and sperm cells have the ability to combine and metabolic ABP; Tf living sperm cells can provide physiological activities and biochemical processes needed to iron , and directly on cell growth stimulation; INH to inhibit FSH (follicle stimulating hormone, FSH) secretion, not only in the regulation of pituitary - testicular axis balance plays an essential role, and germ cells in the testis differentiation during development plays a direct role. ABP, Tf and INH are widely used as a sign of support for cell function. Lead can cause abnormal secretion of FSH, impact support concentration of intracellular second messengers, and can act directly on Sertoli cells to support affect intracellular second messenger formation. These changes may affect gene expression within supporting cells, resulting in secretion of Sertoli cell dysfunction, resulting in male reproductive toxicity. This study was isolated and cultured in vitro rat Sertoli cells and exposed to different concentrations of lead acetate, from the mRNA level of the test substance in supporting cells ABP, INH and Tf impact. In order to investigate the Sertoli cells caused by lead acetate in the male reproductive dysfunction role for the further study of the reproductive toxicity of lead in providing a theoretical basis. Method: 1. Establish rat Sertoli cells in primary culture model. 18 to 21-day-old SD rat testis followed by trypsin and collagenase digestion, separation and purification of supporting cells. At 35 ℃, 5% CO 2 were cultured under conditions and support by Feulgen staining cells were identified. Using the MTT assay described support cell growth curve. DMEM/F-12 medium was formulated with solvent working solution of lead acetate, treated with different doses on Sertoli cells exposed to 24h, lead acetate to determine the maximum non-cytotoxic dose. (2) lead acetate on ABP, INH and Tf gene transcription. Lead acetate concentration 0 mol / L, 10 -8 mol / L, 10 -7 mol / L, 10 -6 mol / L and 10 -5 mol / L on isolated primary cultured rat Sertoli cells exposed to 24h, the total extraction of intracellular RNA, reverse transcription polymerase chain reaction (RT-PCR), in order to GAPDH as an internal reference, testing support intracellular ABP, INH and TfmRNA levels. 3 Statistical analysis. Using SPSS 12.0 statistical software ANOVA (analysis ofvariance, ANOVA), Bonfferoni pairwise comparisons, Levene homogeneity of variance test was used for statistical analysis (significance level α = 0.05). Results: In this study, Sertoli cells were isolated and cultured in vitro grow well, and the supporting cells relatively high purity, can be used for in vitro study. Exposed to different concentrations of lead acetate in vitro cultured Sertoli cells 4d 24h, found that the highest concentrations of lead acetate was 10 -5 mol / L when no cytotoxicity. 2.RT-PCR Results: ① to 10 -8 mol / L ~ 10 -5 mol / L lead acetate exposed Sertoli cells 24h, ABP mRNA expression levels relative higher, but the difference was not statistically significant (P> 0.05). ② to 10 -8 mol / L ~ 10 -5 mol / L lead acetate exposed Sertoli cells 24h, TfmRNA in 10 -8 mol / L, 10 -7 mol / L and 10 -6 mol / L dose groups relative expression were higher, 10 -5 mol / L dose groups relative expression level lower than the control group, but each dose group relative expression of genes compared with the control group, the difference was not statistically significant (P> 0.05). ③ The 10 -8 mol / L ~ 10 -5 mol / L lead acetate acts on Sertoli cells after 24h, INH mRNA in 10 -8 mol / L and 10 -7 mol / L dose groups relative expression level lower than the control group, the difference was statistically significant (P <0.05). 10 -6 mol / L dose groups relative expression level than the control group, 10 -5 mol / L dose groups relative expression level was higher, but the difference not statistically significant (P> 0.05). Conclusions: 1. Acetate of lead on primary cultured rat Sertoli cells in the 10 -8 mol / L ~ 10 -5 mol / L dose range was not observed cytotoxicity . (2) lead acetate in 10 -8 mol / L ~ 10 -5 mol / L dose range on Sertoli cells androgen binding protein and transferrin had no effect on the transcriptional expression lead acetate at this dose does not affect androgen binding protein and transferrin secretion. 3 lead acetate in 10 -8 mol / L and 10 -7 mol / L dose can be reduced Sertoli cell inhibin transcription. Lead acetate in 10 -6 mol / L and 10 -5 mol / L dose for transcriptional expression of inhibin no effect. Lead acetate can change inhibin secretion of inhibin in the reproductive system effects in rats.

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