|
A wide range of environmental contaminants can lead to reproductive disorders, heavy metal is one of the environmental contamints. Mercury can damage nervous system, blood system, digestive system, reproductive system and so on. Mercury accumulation in the testis affect sperm quality, quantity and sperm production, and induce male infertility.ObiectiveThis study will use adult rat Leydig cells as a model, by observing the effect of mercuric chloride (HgCl2) on the steroidgenesis in Leydig cells to further explore the mechanism of inorganic mercury toxicty on male reproductive system.Methods1. Isolation, purification and identification of Leydig cells.2. Selection of Leydig cells adherent time:Leydig cells were cultured 2 h,3 h,6 h,12 h,24 h,24 h later, collect the non-adherent cells and culture medium to a new culture bottle, add serum-containing DMEM/F12 culture medium to cultivate. The original cells cultured in serum-free culture medium. Observe cell number and morphology, respectively, and determine the best adherent time.3. The effect of cell viability of Leydig cells exposed to HgCl2:Leydig cells were added culture medium with different concentrations of HgCl2 respectively. The OD values were measured by MTT in the Microplate Reader.4. The secretion of progesterone of Leydig cells:Leydig cells were inoculated, in logarithmic growth phase did the following steps, respectively.①Added culture medium with different concentrations of human chorionic gonadotropin (0.0IU/ml, 0.1IU/ml, 1.0IU/ml,10.0IU/ml and 100.0IU/ml). The progesterone of supernatant was measured by radioimmunoassay (RIA) four hours later.②Exposed to the mixture of 10-6mol/L HgCl2 and 10.0 IU/ml HCG,10.0 IU/ml HCG as control group, after 1h,2h,3h,6h,12h,24h, progesterone of supernatant was measured by RIA, respectively.③Added serum-free medium containing different concentration of HgCl2 (0 mol/L,10-8mol/L,10-7mol/L,10-6mol/L and 10-5mol/L), the progesterone of supernatant was measured by RIA three hours later.5. The determination of the level of mRNA expression of growth phase of primary Leydig cells, added serum-free medium containing different concentration of HgCl2 (0 mol/L,10-8 mol/L,10-7 mol/L,10-6 mol/L and 10-5 mol/L), afer three hours, extracted RNA, with GAPDH as an internal reference, the level of mRNA expression of StAR, P450scc and 3β-HSD in Leydig cells was determined by real-time quantitative PCR.Results1. The results of identification showed that the viability of Leydig cells was more than 90%, the purity of secondary purified Leydig cells was more than 97%. The purity and viability all reach the test requirements.2. The determination of Leydig cells adherent time:The results showed that the numbers of cells of 6 h,12 h,24 h groups are agree in amount, the morphology of 6 h group is better than others, so we determined 6 h as the best adherent time.3. The results of MTT assay showed that HgCl2 can inhibit cells growth at the dose of 10-4 mol/L (P<0.05). The other groups had no significant statiscally differences compared with the control group, so it had no remarked effect on the viability of Leydig cells in these doses.4. The RIA results:①After given stimulating of HCG, the secretion function of Leydig cells increased with the concentration of HCG, but when the dose of HCG exceed 10 IU/ml, the secretion of Leydig cells tends to be worse. So 10 IU/ml was the best concentration.②The progesterone secretion of of Leydig cells reached its peak after 6 h, the secretion of Leydig cells in mixed stimulation group lower than that of the HCG control group at all time points, and only at 3 h and 12 h time points the differences between mixed stimulation group and HCG control group were statistically significant (P<0.05). Progesterone can be transformed into other steroid hormones under the catalysis of cytochrome P450 17a-hydroxylase during testosterone synthesis, so we select 3 h as exposure time.③With the HCG stimulated, the secretion of progesterone of all dose groups decreased, in which 10-7 mol/L and 10-6 mol/L dose groups had statistically differences compared with the control group (P<0.05).5. The results of real-time quantitative PCR:The mRNA relative expression levels of StAR, P450scc and 3(3-HSD in Leydig cells exposed to HgCl2 at the dose of 10-8 mol/L had no statistically significant (P>0.05) compared with the control group, while the mRNA relative expression levels of StAR, P450scc and 3β-HSD in cells exposed to HgCl2 at the dose of 10-5mol/L～10-7 mol/L had statistically significant (P <0.05) compared with the control group.ConclusionsIn this experimental condition, it did not affect the viability of primary Leydig cells at the dose of 10-8mol/L～10-5 mol/L HgCl2. It had inhibited cells growth above the dose of 10-4 mol/L HgCl2. At the dose of 10-5mol/L～10-7 mol/L HgCl2, it can inhibit the mRNA expression of StAR, P450scc and 3β-HSD.
|