| Objective: In recent years,with the development of aluminum industry in Guangxi,the influence of aluminum exposure on reproductive health has attracted people’s attention,and oocytes are not only the basis of female fertility,but also the key factors affecting the development of later embryos.Therefore,this study explored the effect of aluminum exposure on mouse oocyte maturation through in vitro culture,in order to understand the theoretical basis of its toxic effects,and hope to provide reference for the prevention and treatment of female infertility.Methods:(1)Germinal vesicle(GV)oocytes were collected and cultured in 0 m M,0.1 m M,1 m M,and 5 m M aluminum-containing medium for 12 h until Metaphase II(MII).The development of mouse oocytes was determined according to the extrusion rate of the first polar body,and the aluminum exposure level of the subsequent experimental group was screened according to the development of mouse oocytes.(2)GV stage oocytes were cultured for 8 h until Metaphase I(MI).The spindle distribution,chromosome arrangement and microfilament expression of mouse oocytes were detected by fluorescence staining.The mitochondrial distribution,mitochondrial membrane potential and intracellular calcium ion levels were detected by fluorescence staining.The distribution of Golgi apparatus,endoplasmic reticulum and lysosome were detected by fluorescence staining.The effects of aluminum exposure on the subcellular structure and function of mouse oocytes were discussed above.(3)Mouse oocytes were cultured from GV stage for 8 hours to MI stage,The effects of aluminum exposure on oxidative stress,DNA damage and apoptosis of mouse oocytes were investigated by immunofluorescence assay and western blotting assay respectively.(4)Mouse oocytes at GV stage were cultured for 8 h to MI stage,and the expression levels of Sod,Cat,Gpx and Bax/bcl-2 were detected to further verify the toxic effect of aluminum exposure on oocyte development.Results:(1)Compared with the control group,there was no significant difference in the first polar body extrusion rate between 0.1 m M and 1 m M experimental group(Control: 89.37%±4.93%,0.1 m M: 82.69%±3.93%,1 m M: 77.30%±4.91%),while the PB1 extrusion rate in the 5 m M group was significantly decreased(5 m M: 39.18%±4.25%),5 m M concentration was selected as the exposure level for subsequent experiments.(2)Compared with the control group,the acetylation level of tubulin increased significantly(Control: 41.99±2.00,5 m M: 70.24±2.25,P<0.001).The spindle in the control group was mostly barreled or fusiform,and in the experimental group appeared abnormal microtubule assembly and bipolar disappearance.Spindle abnormal rate of the experimental group was significantly higher than the control group(Control: 23.97%±2.21%,5 m M: 57.36%±2.66%,P<0.001).The proportion of irregular chromosome arrangement increased in experimental group(Control: 27.83%±1.48%,5 m M: 51.87%±2.64%,P<0.01).Cortical microfilaments expression increased in experimental group(Control: 1.00,5 m M: 1.23±0.05,P<0.05)and there was no significant difference in the cytoplasmic microfilaments(Control: 1.00,5 m M: 1.09±0.02,P>0.05).(3)The endoplasmic reticulum of oocytes in the control group was mostly perinuclear distribution,the abnormal distribution rate was 23.70%±1.89%,while the endoplasmic reticulum of oocytes in the experimental group was clustered or dispersed distribution,the abnormal distribution rate was 55.80%±1.04%(P<0.001).The abnormal distribution rate of Golgi apparatus in the experimental group was higher than that in the control group(Control: 30.76%±2.26%,5 m M: 65.50%±2.29%,P<0.01).The number of lysosomes in experimental group was decreased,and the fluorescence signal was lower than that in control group(Control: 1.00,5 m M: 0.34±0.03,P<0.01).Mitochondria in the control group were perinuclear distribution,abnormal distribution rate was 24.63%±1.77%.Most mitochondria in the experimental group were clustered or homogeneous distribution,abnormal distribution rate was 60.00%±1.65%(P<0.001).Mitochondrial membrane potential decreased in experimental group(Control: 1.00,5 m M: 0.77±0.01,P<0.01).The oocyte calcium level in the experimental group was significantly higher than that in the control group(Control: 1.00,5 m M: 1.61±0.14,P<0.05).(4)ROS level in the experimental group was significantly higher than that in the control group(Control: 1.00,5 m M: 2.12±0.10,P<0.01),and Sod1,Cat and Gpx1 genes were significantly up-regulated in the experimental group(Sod1: Control: 1.00 vs 5 m M: 1.47±0.11,P<0.05;Cat: Control: 1.00 vs 5 m M: 1.32±0.03,P<0.01;Gpx1: Control: 1.00 vs 5 m M: 1.36±0.05,P<0.05).Compared with the control group,the oocyte γ-H2 AX fluorescence signal in the experimental increased,and oocytes had significant DNA damage(Control: 1.00,5 m M: 1.39±0.02,P<0.01).The early apoptosis rate of oocytes increased in the experimental group(Control: 14.97%±1.65%,5 m M: 52.00%±1.64%,P<0.001).The level of BAX and Bax/bcl-2 increased in the experimental group(BAX: Control: 1.00 vs 5 m M: 1.16±0.02,P<0.05;Bax/bcl-2: Control: 1.00 vs 5 m M: 1.36 ± 0.03,P<0.01).Conclusion:(1)Certain concentration of aluminum exposure could affect the development of mouse oocytes.The abnormal distribution of cortical microfilaments and spindle indicated that aluminum exposure could hinder the normal meiosis process of oocytes by disrupting the cytoskeleton.(2)Organelle dysfunction indicated that aluminum had effect on proteins synthesis,transport and digestion(3)Mitochondrial dysfunction and imbalance calcium homeostasis indicated that aluminum exposure caused limited energy supply.In addition,DNA damage and early apoptosis caused by oxidative stress were also the main manifestations of Al toxicity on oocytes. |
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