| The findings from animal experiments and epidemiological studies approve that fluorine not only can damage the structure of the testis and the epididymis directly, but also can disturb the function of hypothalamic-pituitary-testicular axis(HPTA) and the microenvironment in the testis, then results in reproductive injury indirectly.In the testis, there are only two cell types in the seminiferous epithelium, sertoli cells and spermatogenic cells. As the one and only somatic cells in the seminiferous epithelium, sertoli cells are very useful to keep balance of microenvironment in the testis. Firstly, they form the blood-testis barrier by tight junction between adjacent sertoli cells, which can prevent external chemicals from impacting on spermatogenic cells. Secondly, sertoli cells can synthesize and secrete a variety of active substances, of which we pay much attention to are androgen binding protein(ABP) and inibin B(INH B). ABP can bind and transport androgen to maintain high androgen level in the testis and make it available for spermatogenesis. As to INH B, not only keep balance of the HPTA by restraining the secretion of follicle stimulating hormone (FSH)specialy, it can also promote the differentiation and development of spermatogenic directly. As the important locatioan and function of sertoli cells, they become the target cells of many testicular toxicants.Objective:The present study was performed in primary culture of rat sertoli cells, after exposuring to sodium fluoride(NaF), the expressions of ABP and INH B mRNA from sertoli cells were measured using RT-PCR technology, the amount of ABP and INH B in medium was tested by ELISA. The object is to provide some clues for the mechanism of the male endocrine disorder resulted by sodium fluoride, in order to offer experimental evidence and related information to the prevention of fluorosis.Methods:1 Sertoli cells from testicular tissue of 18～20-day-old Sprague-Dawley rats were sequentially treated with 0.25% pancreatin and 0.1% collagenase, then the seprated sertoli cells were incubated in a humidified atmosphere of 5% CO2 at 37℃. MTT assay was used to evaluate the viability of the cultured cells and determine the sodium fluoride concentrations of exposure,2.5μg/ml,5.0μg/ml, 10.0μg/ml and 20.0μg/ml, serum-free medium as the control group.2 Cultured sertoli cells exposured to NaF at the concentrations determined by MTT assay for 24h was used for the total RNA extraction, then examined the levels of ABP and INH B mRNA by RT-PCR technology. The level of GAPDH mRNA was used as control normalization. The levels of ABP and INH B expressions were measured by densitometric analysis and standardized by comparison to the GAPDH control using a digital imaging and analysis system.3 After the cultured sertoli cells exposured to NaF at the concentrations as described above, ELISA methed was employed to test the levels of ABP and INH B in culture medium.4 Statistical analysis referred to Bonfferoni test for multiple comparisons when significant difference were detected by one-way analysis of variance (ANOVA). A difference at P<0.05 was considered statistically significant.Results:1 Sertoli cells isolated from rat testis for primary culture grew well in vitro, the livability exceeded 95%, as well as the purity. According to the growth curve of Sertoli cells, they had the strongest viability and metabolized exuberantly from the third to the seventh day.2 Compared to the control group, the level of expression of ABP mRNA in 2.5μg/ml was significantly increased(P<0.05). The 5.0μg/ml group was increased, the others were lower than the control group, but there were not statiscally significant (P>0.05).The levels of expression of INH B mRNA in the 2.5μg/ml and 5μg/ml groups were significantly higher than that of the control group, and the 10μg/ml and 20μg/ml groups were not found to be significantly different(P>0.05), though they were decreased.3 The levels of ABP in culture medium in the treated groups were not found to be significantly lower than that of the control group(P>0.05). Compared to the control group, the contents of INH B in culture medium in the 2.5μg/ml and 5.0μg/ml groups were increased, the 10μg/ml and 20μg/ml groups were decreased, but the changes were not statiscally significant (P>0.05).Conclusions:Fluoride in the lower concentrations can effect the expression of ABP and INH B and there is no effect on the levels of ABP and INH B in culture medium at the concentrations in present study. |
PDF Full Text Request