Effects Of Fluoride On Acrosome Formation During Spermatogenesis In Rats Testes

[Objective] Numerous studies at home and abroad have proved that fluoride can reduce sperm quality and affect spermatogenesis,but whether fluoride can induce acrosome formation and structural damage is rarely reported.In this study,the effects of fluoride on acrosome formation in spermatogenesis were studied by detecting the acrosome ultrastructure and the key regulatory genes and proteins expression changes during acrosome formation in rats after fluorosis,providing theoretical basis for exploring the mechanism of fluorine-induced male reproductive toxicity.[Method] In this study,40 healthy SD male rats(160-180 g)were randomly divided into four groups after 1 week of adaptive feeding,namely control group(0 mg/L Na F),low fluoride group(25 mg/L Na F),medium fluoride group(50 mg/L Na F)and high fluoride group(100 mg/L Na F),10 rats per group.After 8 weeks,8 rats in each group were randomly selected and sacrificed by anesthesia.Tissues such as testis,epididymis,kidney,liver,spleen and femur were collected and weighed,fixed or frozen for reserve.q RT-PCR was used to detect m RNA expression levels of the genes required for acrosome formation marker genes and nuclear lamina structure in rat testis,and Western blot,immunohistochemistry and immunofluorescence techniques were used to detect acrosome formation and nuclear lamina structure related proteins in testes tissue and the ultrastructure of acrosome was observed by transmission electron microscopy.[Results] 1.Compared with the control group,the visceral coefficients of testis,epididymis,kidneys,liver and spleen of the all treatment group rats treated with sodium fluoride were not significantly changed.The results of electrode method for determination of fluoride ion content in femur of rats showed that the fluoride ion content in femur of rats in all fluoride treatment group was significantly increased compared with the control group.2.The results of q RT-PCR showed that the m RNA expression level of Zpbp1 in each fluoride treatment group were very significantly decreased compared to the control group;compared to the control group,the m RNA expression of Spaca1 in all treatment groups was significantly decreased;and the m RNA expression level of Dpy19l2 was significantly decreased in the middle and high fluoride groups.However,the expression levels of Gba2,Pick1,Gopc and Hrb were not significantly altered.3.Western Blot,immunofluorescence and immunohistochemistry were used to further detect the differentially expressed genes.The results showed that the protein expression levels of ZPBP1 and SPACA1 in each treatment group were very significantly decreased compared to the control group,and the protein expression level of DPY19L2 was significantly decreased.4.The results of transmission electron microscopy showed that normal structure of sperm cells could be observed in the control group,the acrosome was attached to the front of the nucleus and the structure of the nucleus lamina was continuous.Compared to the control group,each sodium fluoride treatment group showed the acrosome membrane fracture,and discontinuous and partially missing of the nuclear lamina.5.The expression levels of nuclear lamina structure related genes Lmna/c,Lmnb1,Lmnb2,Man1,Lap2,Baf,Emerin,Lbr and Hp1 were detected by Western Blot and q RT-PCR.The results showed that the m RNA expression of Lmnb2 in each sodium fluoride treatment group was significantly decreased compared to the control group,while the expression levels of Lmna/c,Lmnb1,Man1,Lap2,Baf,Emerin,Lbr and Hp1 were not significantly altered;and the protein expression of LMNB2 in the medium and high fluorine groups was significantly decreased compared to the control group.[Conclusion] Fluoride can affect the acrosome formation during spermatogenesis in rats,down-regulate the transcription and expression of key proteins ZPBP1,SPACA1 and Dpy19L2 during acrosome formation,alter the ultrastructure of acrosome and nuclear lamina,resulting in destruction of acrosome membrane and discontinuity and partial deletion of the nuclear lamina,and reduce the m RNA and protein expression of LMNB2.Our results reveal the mechanism of fluoride interference with sperm formation and male reproductive toxicity.