Effects Of Fluoride On The Sperm Acrosome Response In Rat Epididymis

[Objective] Previous studies have shown that high concentrations of fluoride can interfere with sperm fertilization,however,the underlying mechanism of fluoride on sperm fertilization is still unclear.Hence,we aimed to investigate the effects of fluoride on rat acrosome reaction in rats by in vitro fertilization experiments and expression of key proteins in sperm acrosome reaction,in order to further explore the mechanism of fluorine to male reproductive toxicity and implement scientific target medicine to provides the theory basis for the design.[Method] 40 healthy adult male SD rats,weighting about 160g-180 g,were purchased from the Experimental Animal Center of Shanxi Medical University(Taiyuan,China),along with the standard diet.After acclimatization for one week,the animals were randomly divided into 4 groups including the control group(drinking the distilled water)and three Na F-treated groups(drinking the distilled water containing 25,50,and 100 mg/L Na F,respectively).The drinking water intake was recorded to calculate the fluoride intake daily.One week before sampling,four healthy SD female rats aged 8 weeks were purchased and injected with PMSG and h CG successively for superovulation treatment.After 8 weeks,the semen of 3male rats were randomly selected from each group and combined with the eggs.7 male rats in each group were sacrificed randomly,epididymis,testis and thighbone were collected.Real-time PCR,Western-blot and immunofluorescence were used to evaluate the expression levels of proteins related to acrosome reaction and epididymosomes.[Results] 1.During the 8 weeks of SD male rats,the water consumption of each group did not change significantly.Compared with the control group,the fluoride intake of drinking water increased in a dose-dependent manner.Meanwhile,the fluoride content of bone significantly increased in a dose-dependent manner.2.Morphological observation of the rat cauda epididymidis shows that the epithelial cells of the rat epididymis were closely arranged in control group and 25 mg/L Na F treatment group;the epithelial cells in the epididymal lumen were not closely arranged and the cell nucleus was missing in 50 and 100 mg/ L Na F treatment groups.3.In vitro fertilization experiments,the cumulus cell layer of the egg was almost completely decomposed in the control group and 25 mg/L Na F treated group,and only a small amount of unseparatedgranulosa cells adhered to the surface of the egg.However,the cumulus cell layer of the egg was not completely decomposed in 50 and 100 mg/L Na F treated groups.The large number of unseparated granulosa cells still adhered to the surface of the egg,and the sperm stayed outside the radiation crown.4.The results showed that the m RNA expression level of Prss21 was significantly lower in the 50 and100 mg/L Na F treatment groups than in the control group.Meanwhile,the m RNA expression levels of Acr in each Na F treatment groups were significantly reduced.Nevertheless,Na F treatment did not significant alter the m RNA expression levels of Mif,Atp2b4,Dcxr in the epididymis.5.Compared with the control group,ACR protein expression levels in the epididymis of rats in the 50 and 100 mg/L Na F treatment groups were significantly decreased,and PRSS21 protein expression levels in each Na F treatment group were decreased.And there was no significant difference in the protein expression levels of ACR in the testes between each treatment group.6.Compared with the control group,the m RNA expression level and protein expression level of SPAM1 were significantly decreased in the 50 and 100 Na F mg/L treatment groups.Immunofluorescence analysis showed that the fluorescence intensity of SPAM1 in the epididymis of the 50 and 100 mg/L Na F treatment groups was significantly reduced.Meanwhlie,the fluorescence intensity of SPAM1 of sperm in the 50 and 100 mg/L Na F treatment groups was significantly reduced.7.Compared with the control group,the m RNA and protein expression level of CD9 were significantly decreased in 50 and 100 mg/L treatment groups.Meanwhlie,the fluorescence intensity of CD9 was significantly reduced in 50 and 100 mg/L Na F treatment groups.The m RNA and protein expression level of CD81 were significantly decreased in 100 mg/L treatment groups.The fluorescence intensity of CD81 was significantly reduced in 50 and 100 mg/L Na F treatment groups.While the expression level of Mfge8 and Hsc70 m RNA in each group showed no significant changes.[Conclusion] The results showed that fluoride can interfere with the sperm acrosome reaction in rats,reducing the ability of sperm to break down the cumulus cell layer,resulting in a large number of rat sperm stay on the surface of the cumulus cell layer,which in turn affects the sperm fertilization process.Fluoride can down-regulate the key proteins SPAM1,ACR,PRSS21,CD9 and CD81 in rat epididymis,which is may interference with sperm acrosome reaction and even fertilization ability.