| Objective:The present study was designed to culture ovarian granulosa cells of 26-day-old mices in vitro;observe the changes of granulosa cells morphology and their hormone secretion function after the cells esposed to cadmium at different time and different concentrations; investigate Sf-1 gene expression and its DNA methylation on the mouse ovary granulosa cells hormone synthesis inhibited by cadmium.and to provide new ideas for further study of the effects of Cd on ovarian granulosa cells function and the mechanisms. Methods:The 26-day-old female ICR mices’ ovarian granulosa cells were extracted under the sterile condition in vitro,when the cells were adherent stably and their shapes were stable. exposured the harvested ovaries granulosa cells to Cd(Cd Cl2) at different concentrations of 0,10.20,40 μM, and different exposure time of 2 h、4 h、6 h、8 h. then completed the following detections when the exposure ended. 1、Observed the morphology change of granulosa cells under an inverted microscope. 2、Detected estrogen(E2) and progesterone(P4) in the medium respectively by enzyme linked immunosorbent assay(ELISA). 3、Detected Sf-1 m RNA level by real time PCR after exposure. 4、Detected Sf-1 protein level by western blot after exposure. 5、As soon as the 6h exposure finished,extracted genomic DNA from the cultured ovarien granulosa cells to analyze Sf-1 gene DNA methylation by Sequenom Mass ARRAY platform, which was composed of matrix-assisted laser desorption ionization time-of-flight(MA LDI-TOF) mass spectrometry and combined with RNA base-specific cleavage. Results:1.Effect of Cd on ovarie granulosa cells morphology: The morphology of the ovary granulosa cells changed,with the increase of cadmium exposure time and exposure doses.Compared with control group, the morphology of ovaries granulosa cells changed, the number of cells reduced,Their synapses were away, shapes became round, refractivity became strong,we could see some died cells in high-dose group.2.(1)Effect of Cd on E2 expression in ovarie granulosa cells:①The test results under the same exposure time,different exposure doses were as follows. The E2 levels were reduced with exposure dose increased(trend test p<0.05).In 2 h and 4 h experimental groups, the E2 levels among 20 μM and 40 μM group, compared with control group, were significantly reduced(p<0.05). In 6 h and 8 h experimental groups, the E2 level among 10 μM,40 μM,20 μM groups,compared with control group, were significantly reduced(p<0.05).②The test results under the same exposure doses,different exposure time were as follows.The E2 levels were increased with exposure time increased(trend test p<0.05). In 10 μM experimental group, the E2 level among 4 h group,compared with 2 h group, were significantly increased(p<0.05); In control,20 μM,40 μM experimental groups,the E2 level among 4h,8h groups,compared with 2h group,were significantly increased(p<0.05).(2)Effect of Cd on P4 expression in ovarie granulosa cells:①The test results under the same exposure time,different exposure doses were as follows.The test results are as follows.The P4 level among 10 μM and 20 μM groups,compared with control group, were significantly increased(p<0.05) in 2 h experimental group; In 4 h experimental group, the P4 levels were reduced with exposure dose increased(trend test p<0.05),the P4 level among 20 μM and 40 μM groups,compared with control group, were significantly increased(p<0.05); The differences of the P4 level among all dose groups had no statistical significance(p>0.05) in 6 h and 8 h experimental groups. ②The test results under the same exposure doses,different exposure time were as follows.In control、10 μM、40μΜ experimental group, the P4 levels were increased with exposure dose increased(trend test p<0.05).Under the exposure dose of 0 μM(control), the P4 level among 8h group,compared with 2 h group, was significantly increased(p<0.05); Under the exposure dose of 10 μM, the P4 levels among 4 h、6 h、8 h groups,compared with 2 h group,were significantly reduced(p<0.05); Under the exposure dose of 20 μM,the P4 levels among 4 h group,compared with 2 h group, was significantly reduced(p<0.05) in 20 μM experimental group.3. Effect of Cd on Sf-1 m RNA expression in ovarie granulosa cells:(1)The test results under the same exposure time,different exposure doses were as follows.The differences of the Sf-1 m RNA level among all dose groups compared with control group, had no statistical significance(p>0.05),after exposured 2 h; After exposured 4 h、6 h、8 h respectively, the Sf-1 m RNA levels among all dose groups, compared with control group,were significantly reduced(p<0.05);and under these exposure time, the Sf-1 m RNA levels were reduced with the exposure dose increased(trend test p<0.01).(2)The test results under the same exposure doses,different exposure time were as follows.Trend test showed: under the exposure doses of 10 μM、20 μM、40 μM, the Sf-1 m RNA levels were reduced with the exposure time increased(p<0.05). After the 10 μM group treated 8h, the Sf-1 m RNA level compared with the level after treated 2 h, their difference was statistically significant(p<0.05); After the 20 μM group treated 6 h, the Sf-1 m RNA levels compared with the level after treated 2 h, their difference was statistically significant(p<0.05); After the 40μM group treated 6 h、8 h, the Sf-1 m RNA levels compared with the level after treated 2 h, their difference was statistically significant(p<0.05).4. Effect of Cd on SF-1 protein expression in ovarie granulosa cells:(1)The test results under the same exposure time,different exposure doses were as follows.After exposured 2 h,the differences of the SF-1 protein level among all dose groups,compared with control group,had no statistical significance(p>0.05);After treated 4 h, the SF-1 protein levels among 20 μM and 40 μM groups, compared with control group, were significantly reduced(p<0.05); After treated 4 h、6 h、8 h respectively,the SF-1 protein levels among all treated groups,compared with control group, were significantly reduced(p<0.05); Trend test showed: under these exposure time(4 h、6 h、8 h), the SF-1 protein levels were reduced with the exposure dose increased(p<0.01).(2)The test results under the same exposure doses,different exposure time were as follows. The SF-1 protein levels were reduced with the exposure time increased(trend test p<0.01),under the exposure doses of 10 μM,20 μM,40μM. After the 10 μM group treated 6 h、8 h respectively, the SF-1 protein levels,compared with the level after treated 2 h, their difference was statistically significant(P<0.05); After the 20 μM and 40 μM groups treated 4 h、6 h、8 h respectively,the SF-1 protein levels,compared with the level after treated 2 h, their difference was statistically significant(p<0.05).5. Effect of Cd on SF-1 DNA methylation pattern:We actually detected nine CG sites by using Sequenom DNA methylation detection platform, however, since the methylation rates of the 7,8,9 CG sites were 0 in some dose groups.finally,we choosed 1-6 CG sites for analysis, Data analysis showed that the total methylation rates of SF-1 gene in all groups were low, and compared with the control group, the DNA methylation rates of SF-1 gene promoter region CG sites in DAC treatment group and the Cd Cl2 exposure groups had no significant changing, the difference was not statistically significant(p>0.05). Conclusions: 1、Under the experimental conditions, Cadmium exposed mice ovarie granulosa cells can change their morphology and reduce the E2 and P4 secretion in some dose range. 2、After ovarian granulosa cells exposed to cadmium(10-40 μM)in vitro, the expression level of Sf-1 gene m RNA and SF-1 protein were inhibited, this may be an important mechanism in Cadmium-induced sex hoymones secretion disorder in ovarian granulosa cells. 3、After ovarian granulosa cells exposed to cadmium, the rate of Sf-1 DNA methyla- tion were low, the way that Cadmium regulates the expression of Sf-1 may not be by changing the Sf-1 gene promoter DNA methylation patterns... |
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