B(a)P暴露干扰细胞周期调控蛋白影响孕早期小鼠卵巢黄体功能

该研究旨在探讨苯并(a)芘Benzo(a)pyrene,B(a)P]对孕早期小鼠卵巢黄体功能的影响及机制。体内模型:将昆明小鼠每晚按雌雄3:1的比例合笼,次晨查得阴栓记为孕第1天(d1);将其随机分为对照组和B(a)P处理组,每日早晨称重后以0.1 mL/10 g动物体质量灌胃给予0.2 mg/(kg·d)的B(a)P,对照组灌胃等体积的玉米油,收取d4、d7小鼠卵巢组织。体外模型:培养小鼠卵巢颗粒细胞KK-1,将其分为对照组(0.1%DMSO)、HCG组(1.0 IU/mL HCG)、HCG+BPDE(1.0 IU/mL HCG和0.5μmol/L BPDE)联合处理组,处理细胞24 h后进行后续检测。ELISA检测小鼠血清雌激素(E2)、孕激素(P4)水平;qRT-PCR检测体内外卵巢雌、孕激素合成限速酶3β-HSD、17β-HSD和P450SCC的mRNA水平;免疫组化检测卵巢组织切片中Ki67、PCNA的表达,CCK-8检测KK-1细胞增殖情况;Western blot、免疫组化和免疫荧光检测周期相关蛋白CyclinA1、CDK2、CDK4、CyclinB1以及GAS1的表达情况。透射电镜和Mitotracker探针观察线粒体形态。与对照组相比,B(a)P暴露导致孕早期小鼠血清中E2、P4水平明显降低;同时,卵巢雌、孕激素合成限速酶3β-HSD、17β-HSD和P450SCC mRNA水平下调;CCK-8结果显示,BPDE暴露导致细胞活力下降;体内B(a)P暴露导致卵巢黄体中Ki67、PCNA表达下调;Western blot、免疫组化和免疫荧光结果显示,B(a)P或BPDE暴露下调细胞周期相关因子CyclinA1、CDK2、CDK4、CyclinB1及GAS1水平;电镜和免疫荧光结果显示,BPDE暴露导致线粒体形态异常。B(a)P及其代谢物BPDE干扰细胞周期调控,影响线粒体功能,进而导致孕早期小鼠卵巢黄体功能异常。

Exposure to Benzo(a)pyrene in Early Pregnant Mice Impairs Ovarian Corpus Luteum Function by Interfering with Cell Cycle Regulation Proteins

This study was aimed to explore the effects and mechanism of B(a)PBenzo(a)Ppyrene]on the function of ovarian corpus luteum during the early pregnancy of mice.For in vivo model,the female and male Kunming mice were mated to produce pregnancy at a ratio of 3:1 every night.At the next morning,the female mice with vaginal copulation plugs were recorded as"pregnant day 1(d1)".The pregnant mice were randomly divided into control group and B(a)P-treated group.The B(a)P-treated group received 0.2 mg/(kg·d)B(a)P daily by oral gavage at 0.1 mL/10 g of body weight from d1,and the control group received corn oil.The pregnant mice were sacrificed by cervical dislocation on d4 and d7,and the ovaries were collected immediately.In vitro model,the cultured mouse ovarian granule KK-1 cells were divided into three groups and treated with vehicle alone(control group,0.1%DMSO),1.0 IU/mL HCG(HCG group)and 1.0 IU/mL HCG plus 0.5μmol/L BPDE(HCG+BPDE group)simultaneously for 24 h.ELISA was used to detect the levels of serum estrogen and progesterone.The mRNA levels of 3β-HSD,17β-HSD and P450SCC were determined by qRT-PCR.Immunohistochemistry was used to detect Ki67 and PCNA expression in ovarian tissue sections,and CCK-8 was used to detect the proliferation of KK-1 cells.Western blot,immunohistochemistry and immunofluorescence were used to measure the expression levels of CyclinA1,CDK2,CDK4,CyclinB1 and GAS1.The morphology of mitochondria was observed by transmission electron microscope and Mitotracker probe.B(a)P decreased the levels of serum estrogen and progesterone compared with the control group.The mRNA levels of 3β-HSD,17β-HSD and P450SCC were down-regulated by B(a)P.The cell activity was weakened after BPDE exposure in vitro.B(a)P exposure resulted in the down-regulation of Ki67 and PCNA expression.B(a)P or BPDE exposure decreased the expression of CyclinA1,CDK2,CDK4,CyclinB1 and GAS1.Electron microscopy and immunofluorescence results revealed that BPDE exposure could lead to abnormal mitochondria morphology.B(a)P exposure might disturb the cell cycle and affect mitochondrial function,which leaded to luteal dysfunction in early pregnancy mice.