| Objective: Research cadmium exposure to newborn rat ovary on oocytes apoptosis and its possible molecular mechanism,to further explore the role of SCF in this process and its signaling pathways.Method: Four day old female SD rats,ovaries cultured for four days in vitro,37?,5%CO2.1.Cadmium dose were 0?M,0.5?M,5?M,10?M,50?M,after cadmium exposure,we investigated apoptosis in oocytes by transmission electron microscope and terminal deoxynucleotidyl tranferase-mediated d UTP nick end labeling(TUNEL).We used real time PCR and Western blot to detect gene Bax,Bcl-2,Bcl-xl,Scf,c-kit m RNA and protein expression level in ovarian tissue.2.The intervention experiment of exogenous SCF :Test 1: control,100ng/m L SCF Test 2: control,Cd10?M,100ng/m L SCF+ 10?M Cd;Test 3: control,10?M Cd,100ng/m L SCF+ 10?M Cd,100ng/m L SCF+10?M Cd +2?g/m L ACK-2;Test 4: control,100ng/m L SCF + 10?M Cd,100ng/m L SCF+10?M Cd+10?M LY294002.After cultured,we investigated apoptosis in oocytes by transmission electron microscope and terminal deoxynucleotidyl tranferase-mediated d UTP nick end labeling(TUNEL).We used real time PCR and Western blot to detect Bax,Bcl-2,Bcl-xl m RNA and protein expression level in ovarian tissue.Results: 1.Effect of newborn rat ovary exposed cadmium on oocytes apoptosis and related gene expression.(1)Transmission electron microscopy showed that the high dose Cd(5-10?M)expoure groups observed oocyte nucleus shrive,heterochromatin increased,apoptotic bodies and steatosis,mitochondria vacuoles,swelling.50?MCd exposure group showed a loose organizational structure,cell and organelle swelling,dissolution,intact cells were not visible.(2)By using TUNEL assay,execpt 50?M Cd exposure group,oocytes apoptosis rate of other groups were significantly higher than that of control group(P<0.05).(3)Compared with control group,execpt 0.50?M Cd exposure group,Bax m RNA and protein expression in other groups were significantly up-regulated(P<0.05).(4)Compared with control group,Bcl-2 m RNA expression in all of the Cd expoure groups were significantly decreased(P<0.05),while the protein expression were significantly decreased(p<0.01).(5)Compared with control group,Bcl-xl m RNA and protein expression in all of the Cd expoure groups were significantly decreased(P<0.05).2.Effect of newborn rat ovary exposed cadmium on Scf and c-kit m RNA and protein expression.(1)Compared with control group,Scf m RNA and protein expression in 0.5?M Cd exposure group were significantly increased(p<0.01),while other Cd expoure groups were significantly decreased(P<0.01).(2)Compared with control group,c-kit m RNA and protein expression in Cd(5-50?M)exposure groups were significantly decreased(p<0.01),while c-kit protein expression in 0.5?M Cd exposure group was significantly increased(p<0.01).3.Effect of different intervention groups on newborn rat ovary oocyte apoptosis and related gene expression.(1)Effect of exogenous SCF on newborn rat ovary oocyte apoptosis and related gene expression.1)Transmission electron microscopy revealed that SCF group oocytes nuclei obviously,nucleus chromatin distributed evently,mitochondria and endoplasmic are rich in the cytoplasm.2)By using TUNEL assay,the oocyte apoptosis rate in SCF group was significantly decreased than that of control group.3)Compared with control group,Bax m RNA expression in SCF group was significantly decreased(p<0.01),while Bcl-2,Bcl-xl m RNA expression in SCF group was significantly increased(p<0.01).4)Compared with control group,BAX protein expression in SCF group was significantly decreased(p<0.01),while BCL-2 protein expression in SCF group was significantly increased(p<0.01).(2)Effect of exposure SCF and Cd on newborn rat ovary oocyte apoptosis and related gene expression.1)Transmission electron microscopy revealed that SCF+Cd group oocytes nuclei obviously,nucleus chromatin distributed evently,mitochondria and endoplasmic are rich in the cytoplasm.2)By using TUNEL assay,the oocyte apoptosis rate in SCF+Cd group was significantly decreased than that of Cd group.3)Compared with Cd group,Bax m RNA expression in SCF+Cd group was significantly decreased(p<0.01),while Bcl-2,Bcl-xl m RNA expression in SCF+Cd group was significantly increased(p<0.01).4)Compared with Cd group,BAX protein expression in SCF+Cd group was significantly decreased(p<0.01),while BCL-2 protein expression in SCF+Cd group was significantly increased(p<0.01).Compared with control group,BCL-XL protein expression in SCF+Cd group was significantly increased(p<0.01).(3)Effect of exposure SCF?Cd and ACK-2 on newborn rat ovary oocyte apoptosis and related gene expression.1)Transmission electron microscopy revealed that oocytes vacuoles changes,apoptotic bodies,steatosis and mitochondria reducing were visible in SCF+Cd +ACK-2 group.2)By using TUNEL assay,the oocyte apoptosis rate in SCF+Cd+ACK-2 group was significantly increased than that of SCF+Cd group(P<0.01).3)Compared with SCF+Cd group,Bax m RNA expression in SCF+Cd+ACK-2 group was significantly increased(p<0.01),while Bcl-2,Bcl-xl m RNA expression in SCF+Cd +ACK-2 group was significantly decreased(p<0.01).4)Compared with SCF+Cd group,BAX protein expression in SCF+Cd+ACK-2 group was significantly increased(p<0.01),while BCL-2 protein expression in SCF+Cd+ACK-2 group was significantly decreased(p<0.05).4.Effect of exposure SCF?Cd and LY294002 on newborn rat ovary oocyte apoptosis and related gene expression. 1)Transmission electron microscopy revealed that oocytes structure changes,muclear membrane shrivel,apoptotic bodies,mitochondria reducing were visible in SCF+Cd + LY294002 group.2)By using TUNEL assay,the oocyte apoptosis rate in SCF+Cd+ LY294002 group was significantly increased than that of SCF+Cd group(P<0.01).3)Compared with SCF+Cd group,Bax m RNA expression in SCF+Cd+ LY294002 group was significantly increased(p<0.01),while Bcl-xl m RNA expression in SCF+Cd + LY294002 group was significantly decreased(p<0.01).4)Compared with SCF+Cd group,BAX protein expression in SCF+Cd+ LY294002 group was significantly increased(p<0.01),while p-AKt protein expression in SCF+Cd+LY294002 group was significantly decreased(p<0.05).Conclusion: 1?Cadmium exposed newborn rat ovary could induce apoptosis of oocytes in ovary,up-regulated the expression of Bax and down-regulated the expression of Bcl-2,Bcl-xl may be one of the possible molecular mechanism.2?Cadmium exposed newborn rat ovary can inhibit the Scf and c-kit expression.3?Exogenous SCF could suppress that cadmium exposure to newborn rat ovaries induced oocytes apoptosis through down-regulated the expression of Bax and up-regulated the expression of Bcl-2,Bcl-xl.4?SCF/c-kit-PI3K-AKt pathway may be involved in newborn rats ovary exposed to cadmium induced oocytes apoptosis. |
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