The Effects Of SCF/C-KIT On The Newborn Rat Ovary Primordial Follicular Development Inhibited By Cadmium

Objective:Cadmium(Cd)is a common pollutant,which has reproductive toxicology on the ovary.The present study was designed to examine whether or not SCF/C-KIT expression and their gene DNA methylation is involved in Cd-induced primordial follicles development disorder.Methods:Sprague-Dawley rats’ ovaries were harvested on postnatal day4(PND4),and then cultured in the medium consisted of 1:1 combination of DMEM/F12 and α-minimal essential medium(α-MEM),at 37°C,5%CO2 for 4 days.1.Effects of Cd on the ovary: the harvested ovaries were exposured to Cd(Cd Cl2)at different concentration of 0,0.5,5,10,50μM.After 4 days incubation,ovaries were fixed and embedded in paraffin.Serial sections were stained with hematoxylin and eosin for histology and morphological evaluation.Follicles at different stages were counted and oocyte diameter,corresponding to those follicles,was measured with microscope.The percentage of PCNA-positive granulose cell was investigated by immunohistochemistry;Estrogen(E2)in the medium were detected respectively by chemiluminescent immunoassays(CLIA)while progesterone(P4)by enzyme linked immunosorbent assay(ELISA).2.Effect of Cd on SCF/C-KIT expression in ovarie: ovaries were exposured to Cd at the dosage of 0,0.5,5,10,50μM.Total RNA and protein were extracted from ovaries to detected scf/c-kit m RNA and protein level by real time PCR and western blot.3.The intervention experiment of exogenous SCF: ovaries were cultured in the following models:(1)control,100ng/m L SCF groups;(2)control,10μM Cd,10μM Cd+100ng/m L SCF groups;(3)control,10μM Cd,10μM Cd+100ng/m L SCF+2μg/m L ACK-2groups.After cultured,count the number of follicles;measured the diameter of oocyte;counted pcna-positive cells;detected the level of E2 and P4.All of them used the same methods as described above.4.Effect of Cd on DNMTs expression in ovary: ovaries were cultured in the groups:control,0.5μM Cd,10μM Cd,50μM Cd,5μM 5-aza-Cd R,10μM Cd+ 5μM 5-aza-Cd R.After cultured,dnmts m RNA were determined by RT-PCR and DNMTs protein were detected by western blot.5.Effect of Cd on SCF/C-KIT DNA methylation pattern: ovaries were cultured in the following groups: 0,0.5μM Cd,10μM Cd,50μM Cd,5μM 5-aza-Cd R,10μM Cd+ 5μM 5-aza-Cd R.Genomic DNA were extracted from the cultured ovaries to analyze scf/c-kit gene DNA methylation by Sequenom Mass ARRAY platform,which was composed of matrix-assisted laser desorption ionization time-of-flight(MALDI-TOF)mass spectrometry and combined with RNA base-specific cleavage.Results:1.Effects of Cd on the ovary:(1)Composition ratio of follicle: compared with control group,the ratio of primordial follicles of ovaries in 10μM group was increased(P<0.05);(2)Diameter of oocyte: compared with control group,the oocyte diameter of primordial and developing follicles in 0.5μM group were significantly increased(P<0.01)but decreased in 10μM group(P<0.05).Compared with control group,the oocyte diameter of developing follicle in 5μM group increased(P<0.05),while primordial follicles’ had no significant change(P>0.05).(3)Rate of PCNA-positive cell: compared with control group,the rate of PCNApositive granulose cell increased in 0.5μM group(P<0.05)and reduced significantly in other groups(P<0.01);(4)The level of E2 and P4: compared with control group,the level of E2 in 10μM and50μM group is lower(P<0.05,P<0.01),but the P4 level was no difference in different groups(P>0.05).2.Effect of Cd on SCF/C-KIT expression in ovarie:(1)scf/c-kit m RNA : compared with control group,both scf and c-kit m RNA were significantly increased in 0.5μM group(P<0.01,P<0.01)and decreased significantly in other groups(P<0.01,P<0.01);(2)scf/c-kit protein : compared with control group,both scf and c-kit protein were significantly increased in 0.5μM group(P<0.01,P<0.01)while decreased significantly in other groups(P<0.01,P<0.01);3.The intervention experiment of exogenous SCF:(1)Effect of SCF on ovary: compared with control group,in SCF group,the composition ratio of the developing follicle was increased(P<0.01);both oocytes in primordial and developing follicle were significantly increased(P<0.01,P<0.01);the expression of PCNA in granulose cell were increased(P<0.05);the level of E2 was higher(P<0.01).But P4 in groups were not different(P>0.05).(2)Effect of SCF and Cd on ovary: there were no difference in composition ratio of follicle,oocyte diameter,rate of pcna-positive and level of E2 and P4 between control group and Cd+SCF group.Compared with 10μM group,in Cd+SCF group,the composition ratio of the developing follicle was significantly increased(P<0.01);the oocyte diameter in at different stages were significantly larger(P<0.01,P<0.01);the rate of pcna-positive granulose cell were increased(P<0.05);the level of E2 was higher(P<0.01),but P4 in groups were not different(P>0.05).(3)Effect of SCF,Cd,ACK-2 on ovary: compared with 10μM group,the composition ratio of primodial follicle in Cd+SCF+ACK-2 group was increased,but there was no significant difference(P>0.05);the oocyte diameter in primordial follicle was significantly smaller(P<0.05),while not significantly change in developing ones(P>0.05);the rate of pcna-positive granulose cell were decreased(P<0.01).The level of E2 and P4was were not different between 10μM Cd group and Cd+SCF+ACK-2 group.4.Effect of Cd on DNMTs expression in ovary:(1)Effect of Cd on dnmts m RNA:compared with control group,both dnmt1,dnmt3α,dnmt3βm RNA were significantly increased in 0.5μM and 10μM group(P<0.01,P<0.01),but decreased in 50μM group(P<0.05);(2)Effect of Cd on DNMTs protein:compared with control group,DNMT1 protein increased in 10μM group,but decreased in other Cd-exprosed groups.DNMT3αprotein increased in 10μM group and decreased in 50μM group.The level of dnmt3βprotein in different group were no significant differece(P>0.05).5.Effect of Cd on SCF/C-KIT DNA methylation pattern in ovarie:(1)scf gene DNA methylation: the rate of scf DNA methylation were low in different groups,but compared with control group,there are no significant difference(P>0.05,P>0.05);(2)c-kit gene DNA methylation: the rate of scf DNA methylation were low in different groups,but compared with control group,there are no significant difference(P>0.05,P>0.05).Conclusions:1.Neonatal exposure to 5-50μM cadmium can induce development disorder of primordial follicle and reduce the E2 secretion.2.Neonatal exposure to 5-50μM cadmium can inhibit the scf and c-kit expression,but have no effect on scf and c-kit DNA methylation pattern.3.The Inhibition of scf and c-kit expression may be involved in Cadmium-induced ovarian developmental disorder.4.Neonatal exposure to 5-50μM cadmium can make DNMTs expression disorder.There are not enough evidence to deny that the change of DNA methylation patter is associated with cadmium bad effects on follicle development and hormone secretion.