The Study Of Sperm Mitochondrial DNA Damage In Mice With Fluoride Exposure

[Objective] The paper studied the effect of fluoride on mtDNA and nDNA of mouse sperm, speculated the mechanism, laid a theoretical foundation for further proof of the reproductive toxicity of fluoride.[Method] One hundred 3 week old male ICR mice were randomly divided into 4 groups,25 rats each group, respectively control group, low fluorine group, the fluoride group and high fluorine group. Challenge viral dosage of 0 mg/L and 25 mg/L,50 mg/L and 100 mg/L NaF. Dynamic observed and recorded drinking water intake and growth condition of mice during the fluoride exposure. The samples were collected after 60 days, analyzed the sperm routine. We used transmission electron microscopy (TEM) to observe the ultrastructure of sperm, flow cytometry (FCM) to detect mitochondrial membrane potential and nDNA integrity. Simultaneously extracted total DNA of sperm, utilized real time PCR technique measured the copy number of sperm mtDNA. We adopted Long-PCR technique to detect mice sperm mtDNA integrity, PCR amplification for mice sperm mitochondrial MTCYB and MTATP6 and product sequencing for screening gene mutation.[Result] 1. The sperm density, motility and viability of 60 days fluoride exposure mice showed a downward trend, compared with the control group. The sperm density of medium fluoride group and high fluorine group appeared significant differences (P< 0.05), viability and motility were extremely significantly (P< 0.01).2. Used flow cytometry detected the mitochondrial membrane potential of mice sperm and the integrality of nDNA. Results showed that, compared with the control group, the mitochondrial membrane potential of high fluorine group mice sperm decreased significantly (P< 0.05), while the nDNA integrity also decreased significantly (P< 0.05).3. We detected the mitochondrial gene rRNA 16S and nuclear gene GAPDH of mice sperm by QRT-PCR, and then calculated the relative copy number of mtDNA. The results showed that the copy number of mtDNA in fluoride exposure mice appeared an upward trend compared with the control group, among them the high fluorine group was significantly increased(p<0.05).4. Long-PCR technique was used to detect the integrity of mice sperm mtDNA, while we didn't found mtDNA fragment deletion in any of the samples.5. Observed the ultrastructure of mice spermatozoa under transmission electron microscope. The results showed that the nuclear chromatin condensation of sperm head in high fluoride group mice, mitochondria vacuoles or disordered arrangement of the midway of tail transverse. The chance of damage and deletion of the main and the tail section peripheral dense fibrous and 9+2 structure increased compared with control group. There was no significant change in low fluoride group and medium fluoride group.6. The gene mutation site had not found in MTCYB and MTATP6 gene PCR product sequencing results.[Conclusion] Fluoride could reduce the quality of sperm in mice, induced the decrease of sperm nDNA integrity and the increased the copy number of mtDNA. However, we had not found the loss of mtDNA integrity and gene mutation. The reason and mechanism still needs further studied.