| In recent years,the capacity of the human reproductive in global scope decreased significantly,the incidence of infertility patients especially with male infertility is growing year by year.It has become a global medical and sociological issue that impact human development and health.This phenomenon mainly attributed to the social competition,the employment pressure and the deterioration of the environment and so on.The growing environmental pollution is the main reason leading to the phenomenon.A wide range of environmental pollutants can lead to reproductive disorders,Heavy metals is one of the environmental pollutants.Mercury is non-essential for humanbody,it is liquid at room temperature.Mercury and its compounds are widespread in nature,its usage is extremely broad.In recent years, scholars at home and abroad study methylmercury much more than inorganic mercury, and the study of inorganic mercury is concentration in nerve toxicity and kidney toxicity,but the study of reproductive toxicity is less.Epidemiological study on the crowd showed that the accumulation of mercury found in the testes,it also affected the quantity and quality of sperm and the spermatogenic process,and it can through the blood-testis barrier,which can induce male infertility.Acute toxicity tests also showed that the level of mercury in the mice testicular tissue has increased significantly and appeared a clear and pathological change before the symptoms of nervous toxicity,it showed that the threshold dose of mercury injuded testicular physiological function lower than the threshold dose caused nerve toxicity,so mercury is more sensitive to the testicular.In addition,mercury can also cause the changes of animal endocrine secretion.Testosterone levels in serum of the rabbits exposed to mercuric chloride were significantly lower than that before the exposure, and the testosterone levels in serum of male workers exposed to mercury also declined, accompanied with sexual dysfunction.Testosterone levels of Leydig cells exposed mercury acetate were significantly decreased and it had a dose-response relationship. But its mechanism is not reported.This study will use MA-10 mouse Leydig tumor cells(mLTC-1) as a model,and observation the effect of HgCl2 on the steroidgenesis in mouse Leydig tumor cells and its mechanism,it will provide a theoretical basis for the mechanisms of reproductive and developmental toxicity in the furture.Methods1.The cell viability of mLTC-1 affected by HgCl2:The mLTC-1 cells of logarithmic phase were plated in 96-well plates,added culture medium containing different concentrations of HgCl2(0,10-10,10-9,10-8,10-7,10-6,10-5 and 10-4 mol/L) respectively.Each concentration for six parallel ways,the OD value was measured by MTT.2.The secretion of progesterone of mLTC-1 cells:The mLTC-1 cells of logarithmic phase were plated in 96-well plates,each concentration for six parallel way,did the following step,respectively:(1)added diffenrent concentrations of HCG(0.0 IU/ml,0.1 IU/ml,1.0 IU/ml,10.0 IU/ml,100.0 IU/ml),after 4h,progester -one of supernatant was measured by the RIA;(2) Exposed on the mixture of 10.0 IU/ml HCG and 10-7 mol/L HgCl2,10.0 IU/ml HCG as the control,after 1h,2h,3h, 6h,12h,24h,progesterone of supernatant was measured by the RIA;(3) added FBS -free mudium containing the different concentration of HgCl2(0,10-8,10-7,10-6and 10-5mol/L) and 10IU/ml HCG,after 2h,progesterone of supernatant was measured by the RIA.3.The level of mRNA expression of StAR and P450scc in mLTC-1 cells was determined by semi-quantitative RT-PCR:FBS-free medium containing different concentrations HgCl2(0,10-8,10-7,10-6and 10-5mol/L) were added to mLTC-1 cells, after 2h,extracted RNA,the level of mRNA expression of StAR and P450scc in mLTC-1 cells was determined by semi-quantitative RT-PCR. Results1.The results of MTT assay showed that exposure of mLTC-1 cells to different concentrations of mercury chloride can promote cells growth in the dose of 10-5 mol/L and inhibit cells growth in the dose of 10-4 mol/L,they had significant statistically differences compared with the control group.Also it had no remarked effect on the viability of mLTC-1 in the other dose.2.The RIA results showed that:(1) given HCG stimulation,the secretion function of mLTC-1 cells incesased with the concentration of HCG,but when the concentration of HCG reached 10.0 IU/ml,the secretion function of mLTC-1 cells enter the platform,so10.0 IU/ml was the best concentration;(2) the progesterone secretion of mLTC-1 exposed on HCG and the mixture of HCG and mercury chloride reached its peak after 6h,followed by the downward trend,it is close to zero after 24h. The secretion of mLTC-1 cells in mixed stimulation group decreased than that of the HCG control group at all time points,and it had statistically differences compared with the control at 2h and 12h(P＜0.05),but there was no statistically difference at other time points(P＞0.05);(3) after stimulationg,the progesterone secretion of mLTC-1 in all dose group is lower than that of the control groups,just the 10-5 mol/L and 10-8 mol/L dose groups had statistically differences compared with the control(P＜0.05).3.The level of mRNA express of StAR and P450scc:Semi-quantitative RT-PCR showed that the level of mRNA express of StAR and p450scc in the mLTC-1 cells had statistically differences compared with the control in the dose of 10-5～10-7 mol/L mercury chloride and had no statistically differences compared with the control in the dose of 10-8 mol/L mercury chloride.ConclusionsThis study demonstrated that it has the dose-effect relationgship and the time-effect relationship of HgCl2 on the steroidgenesis in mLTC-1,and it may reduce secretion of progesterone by inhibiting the expression of StAR and P450scc. |
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