The Effects Of Lead Acetate On The Expression Of Androgen Binding Protein, Transferrin And Inhibin Mrna In Rat Sertoli Cells

In recent years, environmental pollution has become a prominent issue. Heavy metal is one of the most important pollutants. Lead is one kind of the heavy metal that began to use at the early period in the human history. It is applied widely, and it pollutes the environment. Lead has adverse effects on several systems and organs in body. It also can affect the reproductive function of animal and human.The previous studies about male reproductive toxicity of lead mainly focused on testis. Testis is the most important male gonad and the main target organ of most of xenobiotics. The Sertoli cell is the only body cell in the mammalian testicle's seminiferous tubule. Sertoli cells play a very important role in male reproductive system. Primary culture of Sertoli cells in vitro is characterized by its rapid speed, less cost, less interference, and its results are more target, reliability and reproducibility. It is applied in the study of the reproductive toxicology of xenobiotics.Sertoli cells are called "nursing cell" of spermatogenic cells. Sertoli cells have multiple functions such as providing the cytoarchitecture support and energy and nourishment for developing germ cells. Sertoli cells can also synthesize and secrete many different gene products, which have important biologic functions. Sertoli cells' gene products include androgen binding protein (ABP), transferrin (Tf) and inhibin (INH ) and so on. ABP can especially combine with testosterone to maintain a high concentration of androgen in the testicle's seminiferous tubule, which facilitates the development and maturation of spermatogenic cells. Tf is a major secretory product of Sertoli cells. Tf is postulated to transport Fe³⁺ to the developing germ cells sequestered by the blood-testis barrier and promote spermatogenic cells's growth and maturation. INH can act on pituitary gonadotropes to suppress FSH secretion. Furthermore, INH appears to have local effects within the testis to suppress spermatogonial numbers. ABP, INH and Tf are so far regarded as the best endocrine marker of Sertoli cells to evaluate spermatogenesis in the testes. Lead can disrupt the secretion of FSH, and this will change the concentration of second messenger in Sertoli cells. Lead can act on Sertoli cells directly and change the concentration of second messenger, too. These changes may disrupt the expression of gene products in Sertoli cells. The abnormity of Sertoli cell's function may be the mechanism of lead's toxic effect on male reproductive system.Therefore, the goal of the present study was to determine the effects of different concentration of lead acetate on cell proliferation and expressions of ABP, INH and Tf in primary cultured Sertoli cells isolated from immature Sprague- Dawley rats to explore the molecular mechanisms of reproductive malfunction of male mammal caused by lead acetate.Methods:1. A method was set up for obtaining a large number of viable Sertoli cells from Sprague- Dawley rats of 18～20 days of age. Minced whole testes from SD rats were sequentially treated with 0.25% pancreatin for 15～20 min and 0.1% collagenase for 20～25 min, then washed Sertoli cells sequently, were cultured in DMEM/F-12 media with 10% fetal bovine serum incubated at 35℃in a humidified incubator in an atmosphere of 5% CO₂. On the 4th day of culture, contaminating spermatogenic cells were lysed with a hypotonic solution of 20mM Tris- HCL for 5 min. Feulgen staining had been used to identify the purification and viability of Sertoli cells. Subsequently we established the growth curve of Sertoli cells in vitro and ascertained the maximum dose of non- cytotoxicity of lead acetate on Sertoli cells via MTT method. Sertoli cells were treated with different concentrations of lead acetate for 24h. The concentration of lead acetate in DMEM/F-12 media were 0 mol/L, 10^-9mol/L, 10^-8 mol/L, 10^-7 mol/L, 10^-6 mol/L and 10^-5 mol/L.2. The Sertoli cells cultured for 4 days in 100 mL culture bottles were treated with different concentrations of lead acetate and then transferred into the incubator with 5% CO₂ humidified air to continue to incubate for 24h at 35℃. According to the results of Sertoli cells toxicity of lead acetate by MTT method, Sertoli cells exposed to 10^-8 mol/L, 10^-7 mol/L, 10^-6 mol/L and 10^-5 mol/L were used for the total RNA extraction. 0mol/L was taken as control. The total RNA extraction was used in the subsequent reverse transcription. ABP, INH, Tf and GAPDH were amplified by PCR kit. GAPDH was used as control of the quality of the RNAs. The levels of ABP, INH and Tf expressions were measured by densitometric analysis and standardized by comparison to the GAPDH control using a digital imaging and analysis system.3. Statistical analysis results were presented as mean±SD. Statistical comparisons referred to Bonfferoni test for multiple comparisons when significant difference were detected by one- way analysis of variance (ANOVA). A difference at P<0.05 was considered statistically significant.Results:1. Sertoli cells isolated from rat testis for primary culture grew well in vitro. The viability of Sertoli cells was nonsignificantly reduced at the different concentrations of lead acetate treatment for 24h. The lead acetate didn't have cytotoxicity at the concentration of 10^-5mol/L.2. The levels of the expression of ABP mRNA at different concentrations were all higher than that of the control group, but the increase were not statiscally significant (P>0.05). The expression of Tf mRNA increased at the dose of 10^-8 mol/L, 10^-7 mol/L and 10^-6 mol/L while the expression decreased at the dose of 10^-5 mol/L. But these changes compared with control group were not statiscally significant (P>0.05). The expression of INH mRNA decreased at the dose of 10^-8 mol/L and 10^-7 mol/L, and these changes were statiscally significant (P<0.05). But the changes at the dose of 10^-6 mol/L and 10^-5 mol/L were not statiscally significant (P>0.05).Conclusions:1. The lead acetate don't have cytotoxicity at the concentration from 10^-8 mol/L to 10^-5mol/L.2. The lead acetate has no impact on the expression of ABP mRNA and Tf mRNA at the concentration from 10^-8 mol/L to 10^-5mol/L.3. The lead acetate can decrease the expression of INH mRNA at the concentrations of 10^-8 mol/L and 10^-7 mol/L and has no impact on the expression of INH mRNA at the concentrations of 10^-6 mol/L and 10^-5mol/L.