Study On Apoptosis Effect Induced By Fluoride In Male Mouse Leydig Cells And Its Mechanism

Objective:Fluorine(F)is one of the nonmetal trace elements which are indispensable to organisms. Having strong electronegative, fluorine widely exists in the environment by fluorine. Appropriate fluoride is beneficial to prevent carins, to maintain nerve transmission, as well as to support calcium and phosphate of organisms. And yet, excessive intake of fluorine produces potent toxicity to some systematical function of organisms. In previous researches, the damage of skeletal tissue by fluorine was research emphasis. But with fluoride's extensive application in industry, agriculture, and daily life, recent researches pay more attention to the nonskeletal toxicity of fluorine. So, studies on male reproductive toxicity induced by fluoride were attached more importance recently.The study had in vivo experiment and in vitro experiment. In vivo experiment was done to study the influence of fluoride on the testis apoptosis in mouse. In vitro experiment was done to observe the influence of different cryopreserved and resuscitative conditions on the biological characteristics of leydig cells, and optimize the culture techniques. To explore the effect of different concentrations of fluoride on proliferation and apoptosis of Leydig cell myoblasts in vitro. To investigate relationship between proliferation and apoptosis and the expression of related genes in the testis.Methods:Experimental Kunming mouse were exposed to sodium fluoride(NaF) added to drinking water, Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was employed to detect apoptosis. To obtain a simple and effective method and eligible condition to isolate and culture mouse Leydig cells with two enzymes were used in present experiments, after subculturing, freezing and thawing, Leydig cells were observed by inverted microscopy for morphology and growth behavior. Leydig cell cultured in vitro were collected by trypsinization to form monoplast suspension, and then centrifuged to float again. The cellular numbers were counted and the cell suspension density adjusted, and the cells were inoculated into 96-shadow mask according to 1×106 per milliliter. All cells were cultured in the normal way. While cell fusion ratio arrived 80% and cells did not differentiate. Cells were divided into 4 groups which were DMEM-F12 medium respectively containing0,5,10,20mg/ L. After each group was treated for 0,24,48,72,96,120 hour, MTT was used to detect cell proliferation in each group. AnnexinV-PI double staining with flow cytometry(FCM) was applied to detect Leydig cell apoptosis in each group after treatment for 48h. The expression of caspase-3, caspase-9, bcl-2 and bax protein and apoptosis cell were detected by immunohistochemical method.Results:(1) TUNEL-positive cells could be detected in fluoride-treated rat testis. The apoptotic rates of fluoride—treated testis cells were higher than that of control significantly.(2) The results showed that the viability and purity of leydig cells were up to more than 92 % digested with Trypsin andⅣcollagenage, the better cultural condition was 37℃,5%CO2, under inverted microscope, the cultivated cells exhibited as polygon or fusiform. Higher recovery rate were also happened when gradual cooling down and DMSO were employed.(3) After the cells were treated for 24 hours and 48 hours, fluoride (20 mg/L) significantly inhibit proliferation of Leydig cells, but 5 mg/L and10mg/L have a less effect on Leydig cells. After the cells were treated for 72 hours, fluoride (10 mg/L) significantly inhibit proliferation of Leydig cells. After the cells were treated for 96h and 120 h, fluoride(5 mg/L,10 mg/L,20 mg/L) significantly inhibit Leydig cells proliferation.(4) When the cells were treated for 48 hours by fluoride, Leydig cells of every group were induced apoptosis. The Leydig cells late apoptosis rate of 10 mg/L was highest, while the Leydig cells early apoptosis rate of 20 mg/L was significantly highest.(5) Immunocytochemistry showed that:positive rate of Caspase-3, Caspase-9, bax increased with the increasing of the concentration of caesalpinia sappan aqueous extract, while the gray scale lowered. Positive rate of bcl-2 decreased with the increasing of the concentration of caesalpinia sappan aqueous extract, while the gray scale increased.Conclusions:There is tendency of apoptosis in testis of fluorosis in mouse. It is most similitude with change spot of pathology. There may exist apoptosis mechanism in testis lesion of fluorosis. These results will provide a basis for isolation and in vitro culture of mouse testis leydig cells. Fluoride can efficiently inhibit the proliferation of Leydig cell, and promote the apoptosis of Leydig cell. The results suggest that proliferation and apoptosis were both involved in the development of the testis from the fluorosis. Caspase-3,Caspase-9,p53 was correlated with the modulation of proliferation respectively; bcl-2 and bax play an important role in apoptosis regulation.