Effect Of Heavy Metals On The Apoptosis Of Granulosa Cells And Apoptosis-related Genes In Mice

Cd and Pb are normal environment pollutants. Pb-associated changes in the nervous system, the hematopoiesis, the kidney, the liver, cardiovascular system, and the reproductive system have also been described. This study was done to correlate Cd and Pb to the apoptosis of granulose cells. The occurrence of apoptosis is related to apoptosis-related genes which can be divided into two types:the former induces apoptosis,for example, Bax、Bad、Bim、Bik、p53、Bcl-Xs, etc.The latter inhibits it such as Bcl-2、Bcl-X、Mcl-1and so on. Bcl-2and Bax play an important role in apoptosis. This research is to detect the expression level of the two genes during apoptosis, then we explore the effect of heavy metals on apoptosis from molecular level.Twenty five-day-old mice were divided into five groups:intraperitoneal (i.p.) injection of either lead acetate0.5mg/kg、1mg/kg、2mg/kg、4mg/kg for five days and cadmium chloride0.5mg/kg、1mg/kg、2mg/kg、4mg/kg for48hours or or an equal volume of ultrapure water. After that, each group was injected PMSG. The mice were killed after48hours. The ovaries were extracted out and with punctured with18syringe needle to collect granulose cells and its apoptotic rate was detected by flow cytometry. The results showed that the proportion of apoptotic cells in2mg/kg was the highest. There is a very significant difference between it and the control group as well as0.5mg/kg(P<0.01). Significance also exists in it and the other two groups (P<0.05).There is no significance between the other groups (p>0.05).There is no significance between the group of the mice which were given intraperitoneal injection of different concentration of cadmium chloride (p>0.05).Accroding to Bcl-2and Bax gene sequences of mice in Genebank, the specific primers for PCR were designed crossing the exons. Taking the ovary out, extracting the total RNA, cDNA was amplified using RT-PCR. We detected the level of expression of Bcl-2and Bax in the ovary with semi-quantitative analysis. The results showed that within2mg/kg,the values of Bcl-2and Bcl-2/Bax decreased gradually with the increasing concentration of Pb. Every experiment group had a very significant difference with the control group(P<0.01).When the concentration of Pb was4mg/kg,the expression of Bcl-2and Bcl-2/Bax was a little higher,but it’s still lower than the control group.The level of Bax in0.5mg/kg is the highest.It had a very significant difference with the control group and the other experiment groups(P<0.01). There was a significant difference between the experiment group2mg/kg and4mg/kg(P<0.05).The other groups had no significant differences (P>0.05).Compared with Bcl-2, the level of Bax is higher.