| Objective: Nickel is a kind of heavy metal widely distributed in natural environment and used in modern industry and life.Nickel divalent is the most widely present form and has been reported to have toxic effects on various tissues.The purpose of this study was to investigate the toxic effects of heavy metal nickel exposure on female reproductive system and its internal mechanism.Methods: Female ICR mice aged 4 weeks were randomly divided into control,low-dose,medium-dose and high-dose groups,with 35 mice in each group: 0,5,10,20 mg/kg/d of nickel sulfate were respectively exposed in vivo for 3 weeks.H.E.staining was used to detect the changes of various ovarian follicles.Immunohistochemistry was used to detect the apoptosis of follicles and the expression of inflammatory and fibrosis related factors.Sirius red staining was used to detect the content of total collagen in ovarian tissue.The expression of related proteins in ovary was detected by WB technique.MII oocytes were obtained by superovulation method.Oocyte indexes were detected by immunofluorescence staining.Morphological changes of oocyte mitochondria were detected by transmission electron microscopy.Results: Compared with the control group,the body weight of mice in high-dose group was significantly decreased from 3 d to 21 d(p < 0.05),and the ovarian weight and organ coefficient of mice in each dose group were significantly decreased after exposure(p < 0.05).The number of follicles before ovulation in the high-dose group was lower than that in the control group,but there was no significant difference.The number of atresia follicles in medium and high dose groups was significantly increased(P < 0.01),accompanied by increased apoptosis of granulosa cells.Compared with the control group,the expression levels of pro-inflammatory factors TNF-α,Nf-κB and IL-6 in ovary were significantly increased(P < 0.05),while the expression levels of anti-inflammatory factors IL-4 and IL-10 were significantly decreased(P < 0.05).Sirius red staining showed that the total amount of collagen in ovary in medium-dose and high-dose groups was significantly increased compared with that in control group(P < 0.05),and the expressions of fibronectin,α-SMA,MMP2 and TGF-β1 were significantly increased(P < 0.05).The number of GV stage oocytes obtained from ovary was significantly decreased(P < 0.01),and the number of MⅡ stage oocytes obtained from oviduct superovulation was also significantly decreased compared with the control group(P < 0.05).In oocytes,the content of reactive oxygen species was significantly increased(P < 0.05),the degree of histone methylation was significantly decreased(P < 0.05),the degree of DNA damage was significantly increased(P < 0.05),and the proportion of early apoptosis was significantly increased(P < 0.05).Transmission electron microscopy showed that the exposure of nickel sulfate destroyed the normal morphology of mitochondria,caused swelling of external lumen and crest disappearance.Mitochondrial related probe detection showed that the proportion of abnormal mitochondrial distribution in high dose group was significantly increased(P < 0.01),and mitochondrial membrane potential was significantly decreased in medium and high dose groups(P < 0.05).Immunofluorescence staining of histone methylation showed that H3K4me3 and H3K9me3 decreased significantly in high-dose group(P < 0.05).Conclusion: Exposure to nickel sulfate induces ovarian fibrosis and inflammation through TGF-β1 and Nf-κB pathways,disrupts ovarian homeostasis,interferes with follicular development and decreases oocyte development.The damage of mitochondrial structure and function in oocytes leads to the increase of intracellular reactive oxygen species,DNA damage and early apoptosis,which results in the decrease of oocyte quality.Changes in histone methylation levels suggest that exposure may interfere with genome reprogramming,resulting in abnormal gametes. |
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