The Effect Of HgCl₂ On The Expression Of Androgen Binding Protein And Inhibin In Rat Sertoli Cells

In nearly half of century, the study showed that human fertility has dropped significantly. The incidence of infertility patients especially with male infertility is increasing year by year. It has become a global medical and sociological issues that impact human development and health. The environmental pollution has been the main reason leading to the problem. Heavy metals is one of the more important environmental pollutants can lead to reproductive disorders. Mercury and its compounds are widely used in chemical industry, military production, medicine and agricultural production, these environmental pollution and hazards have been received wide attention. The provious studies show that testis is an important target organ of exogenous chemical substances, and epidemiological study on the crowd also showed that mercury can through the blood-testis barrier, and accumulate in the testes. Accordingly, it can affecte the quantity and quality of sperm and the spermatogenic process, in which can induce male infertility. All These damages may be arise by affecting the function, structure, and the number of Sertoli cells firstly. Important function of Sertoli cells and Sertoli cell culture model that make it widely used in reproductive toxicity of exogenous compounds in the study.ObjectiveIn this study, primary cultured rat Sertoli cells as a model will use to explore the expression of androgen binding protein (ABP) and inhibin(INH) on the effect of mercuric chloride(HgCl2) in the levels of molecular and protein, in order to provide a theoretical basis to further study of reproductive toxicity of inorganic mercury mechanism.Methods1. Cell Culture:18 to 20-day-old male clean grade Sprague Dawley rats were isolated by trypsin-collagenase digestion method and cultured at 37℃in humidified incubator in an atmosphere of 5% CO2. The purification of Sertoli cells be used by Tris-HCl hypotonic liquid after 48 hours.2. The cell viability was analysed by the method of MTT:The purified Sertoli cell were plated in 96-well plates at (2-4)×105/ml, respectively added culture medium containing different concentrations of HgCl2(0,10-10,10-9,10-8,10-7,10-6,10-5, 10-4 mol/L). Each concentration for six parallel way, the OD value was measured by Microplate Reader at 492 nm wavelength.3. The level of mRNA expression of ABP and INH was determined by reall time-quantitative RT-PCR:when the Sertoli cell cells covered around 80 percent of the culture flask, added HgCl2 working fluid containing different concentrations (0,10-8,10-7,10-6,10-5 mol/L). after 24h, extracted RNA and determined the level of mRNA by reall time-quantitative RT-PCR.4. The protein expression of ABP and INH in Sertoli cells was determined by ELISA:Taken the supernatant with different concentrations of HgCl2 working fluid (0,10-8,10-7,10-6,10-5 mol/L) to be threted after 24h and measured the OD value of each hole using the microplate reader at 450nm wavelength.Results1. The results of cell culture:The Sertoli cells survival rate above 95%. After 24 hour the cells began to grow and 48 hours to spread, which purification more than 90% be treatmented by Tris-HCl.2. The results of cell viability:The OD value showed that exposure of Sertoli cells to different concentrations of HgCl2 can inhibit cells growth in the dose of 10-4 mol/L, it had significant statistically differences compared with the control group(P<0.05).3. The mRNA expression of ABP and INH:the level of ABP had statistically differences compared with the control in the dose of 10-7 and 10-6 mol/L(P<0.05). The level of INH had statistically differences compared with the control in the dose of 10-7 mol/L,10-6 mol/L and10-5 mol/L(P<0.05).4. The protein expression of ABP and INH:The results showed that the protein of ABP had statistically differences compared with the control groups in the dose of 10-6 mol/L(P<0.05). The protein of INH had statistically differences compared with the control groups in the dose of 10-7 mol/L,10-6 mol/L and 10-5 mol/L(P<0.05).Conclusion The study suggested that mercury chloride can influencbe the secretory function of Sertoli cells. It can promote the expression of ABP mRNA and protein, but can inhibit the expression of INH mRNA and protein.