Effects Of Fluorosis On Infiltration Of Different Immune Cells In Mice Testis

[Objective]This study was aimed to provide more evidence for the study of reproductive toxicity mechanism of fluorine and understand the testicular inflammation caused by fluorosis by comparing the infiltration status of different immune cells in mice testes between fluorosis model and experimental autoimmune orchitis(EAO)model.[Method]12 healthy male mice of each strain(ICR,B ALB/c and c57 strains)were divided randomly into control group and EAO group,6 mice in each group.0.2 mL emulsified homogenates were subcutaneously injected in each mouse for three times at an interval of two weeks.Mice that were injected with an equal volume of an emulsion of phosphate buffered saline(PBS)with complete Freund's adjuvant(CFA)alone served as the controls.The mice in EAO group were subcutaneously injected with syngeneic testicular germ cells emulsified with the CFA.At 50 days after the first immunization,epididymal cauda and vas deferens of mice were collected for evaluation of the semen quality.And the sections of testis from mice were prepared.For histopathological observation,the sections were stained with hematoxylin-eosin(HE)kit.BALB/c strain mice were easy to establish EAO model after evaluating the establishment of EAO model.Then 30 BALB/c mice were divided randomly into 5 groups,6 mice in each group.The control group and EAO group were drank distilled water.Low fluoride group,middle fluoride group and high fluoride group were drank distilled water containing 25 mg/L,50 mg/L and 100 mg/L sodium fluoride(NaF),respectively.EAO was induced in EAO group by using the same method as above.At 150 days after the first immunization,we collect femur for the detection of F-concentration in bone with fluoride ion electrode method,sera for identifying the testicular antigens that specifcally reacted with sera from each group and the anti-testicular autoantibody-reacting sites.The testes of mice were prepare for making sections to observe testicular lesion by HE staining and the infiltration of inflammatory cells by immunofluorescence technique and examining the expression of cytokines by enzyme linked immunosorbent assay(ELISA).[Results]The results of EAO model in different strains mice showed that compared to the control group,the Sperm count in EAO group of BALB/C mice was significantly lower(P<0.05),but the Sperm count in the EAO group of ICR and C57 mice have no significant change.However,the sperm motility and viability in the EAO group of three strains mice observably deCTeased(P<0.05).EAO group of BALB/C mice showed severe EAO,but ICR and C57 mice did not develop severe EAO.The results of infiltration of testicular immune cells exposed to fluoride indicated that:1.Detection results of F-concentration in mice bone showed that low dose NaF group significantly increased compared with the control group(P<0.05);middle dose NaF group and high dose NaF group significantly increased compared with the control group(P<0.01).2.The results the growth performance test demonstrated that compared with the control group,the body weight gain of mice in high dose NaF group markedly reduced(P<0.05);The testicular index of EAO group decreased observely(P<0.05).3.Estimation of semen quality revealed that compared with the control group,sperm counts in high dose NaF group and EAO group notably decreased(P<0.05,P<0.01);Sperm motility was significantly decreased in middle dose NaF group,high dose NaF group and EAO group(P<0.05,P<0.01).The sperm viability of middle dose NaF group,high dose NaF group and EAO group was significantly reduced(P<0.01).4.HE staining of testicular tissue sections of showed that compared with the control group,there were significant spermatogenic disturbance in middle,high dose NaF group and EAO group(P<0.05,P<0.01).5.The results of IL-6,TNF-?,IFN-? and IL-1? showed that compared with the control group,the content of IL-6,TNF-? and IFN-? in high dose NaF group and EAO group significantly increased(P<0.05,P<0.01).However,there were no significant difference in IL-1? expression in all groups.6.Identifying the testicular antigens that specifcally reacted with sera from each group and the anti-testicular autoantibody-reacting sites indicated that the sera of three NaF-treated groups discovered anti-germ cell autoantibody which were similar to some anti-testicular autoantibody in EAO group;Spermatid and sperms in seminiferous tubules were stained with sera of middle,high dose NaF group and EAO group.7.Immunofluorescence staining demonstrated that compared with the control group,the expression of CD68 increased significantly in middle,high dose NaF group(P<0.05,P<0.01).There was no significant difference in CD56 expression of every group.The expression of CD1lc significantly increased in middle,high dose NaF group and EAO group(P<0.05,P<0.01).The expression of CD3 was markedly increased in three NaF-treated groups and EAO group(P<0.01).We could observe infiltration of macrophages,DC and T cells in three NaF-treated groups,only DC and T cells in EAO group.We did not discover infiltration of NK in all groups.[Conclusion]1.BALB/c strain mice are more likely to induce EAO than ICR and c57 mice.2.It can be seen from that results of infiltration of testicular immune cells by fluorne:Fluornde-induced testicular inflammation is similar to EAO.