The Molecular Mechanism Of Fluoride-Induced Reproductive Disorders Through Damaged Mitochondrial Respiratory Chain In Female Mice

The female reproduction is one of the most basic forms of animal life activities.It not only guarantees the reproduction of animal races,but also is closely related to the healthy development of animal husbandry.A large number of studies have confirmed that fluoride can act on different stages of human and animal reproductive process and show strong reproductive toxicity.In this study,the experimental animal models of fluoride-exposed female mice were established by adding different doses of sodium fluoride?calculated by fluoride ion:0 mg/L,25 mg/L,50 mg/L and 100 mg/L?to mice drinking water.The molecular mechanism of fluoride-induced reproductive disorders through damaged mitochondrial respiratory chain in female mice was researched by transmission electron microscopy,real-time PCR,immunohistochemistry,Western-blot and parallel reaction monitoring?PRM?.The specific results are as follows:1.Fluoride exposure inhibited follicular development and significantly reduced litter size in miceThe development of follicles in mice ovary was observed and analyzed,and the result showed that the number of mature follicles and growing follicles in the ovary of fluoride exposed mice was significantly lower than the control group,while the number of atresia follicles was significantly higher compared with the control group.This indicates that long-term excessive fluoride exposure seriously affects the development of follicles,which can further trigger female reproductive disorders.In addition,the litter size of mice were analyzed and found that fluoride exposure significantly reduced the litter size in mice.These results indicate that long-term excessive fluoride exposure has strong female reproductive toxicity.2.Fluoride exposure damaged the morphological structure of ovarian tissue in miceHE staining results showed that the number of layers of granulosa cells decreased,granulosa cells exfoliated,nuclei condensed and showed different degrees of concentration granules,suggesting that the histomorphological structure of ovary was damaged by fluoride.The mitochondria in ovarian granulosa cells showed cristae rupture,dissolution and even mitochondrial vacuolation by transmission electron microscopy.This suggests that fluoride exposure seriously damages the ultrastructure of mitochondria in granulosa cells and has toxic effects on mitochondria.3.Fluoride exposure damages mitochondrial respiratory chain and decreases ATP content in ovarian tissue of mice by induced oxidative stressReal-time PCR showed that the expression levels of NDUFV2,SDHA and CYC1in ovarian tissues increased significantly?P<0.01?,while the expression levels of COX IV decreased significantly?P<0.05 and P<0.01?.The results of immunohistochemistry and Western-blot at protein level were consistent with those at mRNA level.This suggests that fluoride exposure can interfere with the expression of mitochondrial respiratory chain complex subunits and has the potential to damage mitochondrial respiratory chain.The content of ROS in the ovaries of fluoride-exposed mice increased significantly?P<0.01?,while the activities of SOD and GSH-Px decreased significantly?P<0.01?,suggesting that fluoride exposure induced oxidative stress in the ovaries of mice.The content of ATP in ovaries of fluoride-exposed mice was significantly lower than the control group?P<0.05 and P<0.01?,which also confirmed that mitochondrial respiratory chain was damaged by fluoride,oxidative stress is one of the main causes of mitochondrial respiratory chain damage.4.Fluoride exposure inhibits proliferation of ovarian granulosa cells in miceTo investigate the effect of mitochondrial respiratory chain damage on the proliferation of granulosa cells,BrdU was injected into the mice to detect the proliferation of granulosa cells.The results showed that fluoride exposure significantly inhibited the proliferation of granulosa cells.Real-time PCR,immunohistochemistry and Western-blot showed that the expression of major genes including JNK,STAT5,CDK2 and CDK4 in JNK/STAT pathway,which regulates cell proliferation in ovarian tissue of fluoride-exposed mice,was significantly different at the level of gene expression and protein expression,which further indicated that the proliferation of ovarian granulosa cells was inhibited by fluoride.TUNEL staining showed that fluoride exposure could seriously damage the DNA of ovarian granulosa cells,and single cell gel electrophoresis assay also confirmed that granulosa cells DNA was damaged.These results suggest that fluoride exposure inhibits the proliferation of ovarian granulosa cells and induces DNA damage of granulosa cells by damaging the mitochondrial respiratory chain,which is one of the main causes of follicular dysplasia.5.Analysis of mitochondria-associated proteins in fluoride exposure mice ovary of PRMPRM was used to detect and analyze ovarian mitochondrial related proteins.The results showed that fluoride exposure could induce differential expression of 283ovarian proteins.We chose mitochondrial-related proteins for analysis and found that10 of them were increased in protein expression,such as Fabp3 and Atp2b4,which were involved in energy metabolism pathway,MGARP and VAP1 which were involved in follicular development and cell proliferation,while the three proteins Ptges3,Prdx4 and Cyp11a1 which were decreased in expression were involved in early embryonic development,oxidative stress and sex hormone synthesis,respectively.The disorder of these proteins can affect the biological process of ovarian mitochondria and follicular development.6.Fluoride exposure interferes with MMP-9/TIMP-1 system affecting embryo implantationMMP-9 and TIMP-1 were detected by real-time PCR and Western blot in mouse embryo implantation interface.The results showed that MMP-9 in the fluoride exposure group on the 3^ir,4^th and 5^th day of embryo implantation.The mRNA expression level of TIMP-1 gene in embryo implantation interface tissue was significantly increased?P<0.01?,and the expression levels of protein were also significantly increased?P<0.01?.This suggests that fluoride exposure can overexpress MMP-9 and TIMP-1 genes,and the decrease of litter size in mice is related to the overexpression of MMP-9/TIMP-1 system induced by fluoride.In summary,fluoride exposure induces oxidative stress which damage to the mitochondrial respiratory chain and inhibits the proliferation of ovarian granulosa cells,affects follicular development and interferes with embryo implantation,resulting in decreased litter size in mice,causing females reproductive disorders.In this study,the mitochondrial respiratory chain was used as an entry point to elucidate the mechanism of action of mitochondrial respiratory chain in fluoride injury,revealing the molecular mechanism of reproductive failure in female mice induced by fluoride,and providing a theoretical basis for studying the reproductive toxicity of fluoride.