Effects Of Fluoride And Sulfur Dioxide Exposure On The Blood-testis Barrier Of Male Mice

[Objective]Fluoride and sulfur dioxide, two well-known environmental contaminants, have been implicated to have adverse effects on male reproductive health in humans and animals. Blood-testis barrier (BTB), as a physical barrier of the testis to restrict the diffusion of various endogenous and exogenous toxic chemicals in mammal, dysfunction of which has been considered as one of the important pathways involved in chemicals-induced reproductive toxicity. However, no sufficient data have been found on fluoride and sulfur dioxide inducing disruption of the BTB. Therefore, the objective of this study is to explore if the impairments of male reproduction induced by fluoride and sulfur dioxide are caused by disturbing BTB, and then provide the basic evidence for further consummating rationale of fluoride and sulfur dioxide reproductive toxicity.[Methods]To simulate the actual environmental pollution,48 healthy Kunming male mice were randomly divided into four groups of twelve mice each and respectively exposed to 100mg NaF/L in the drinking water, SO2 in ambient air (10ppm SO2,3hr/day), or both NaF and SO2 together for eight consecutive weeks. During exposuring periods, the situation changes of activity, food and water intake, weight in mice were regularly observed and recorded. After 56 days exposure, testes were collected for the tests of qRT-PCR, Western-blot, TEM and HE staining. Cauda epididymis was also collected to investigate changes in sperm count and sperm morphology of each group. The effects of fluoride and sulfur dioxide on the blood-testis barrier were later evaluated.[Results] 1. Compared to the control group, the body weight gain is apparently decreased in all treatment groups, and there has significantly difference between the NaF+SO2 group and the control group, while there is no statistical difference in relative weight of testis and epididymis among the four groups.2. Significant decreases in sperm count were observed in mice of all treatment groups. Moreover, there were significant changes in sperm malformation between the treatment groups and the control group.3. Testis HE staining showed that, mice of the control group showed normal seminiferous tubules, Sertoli cells and various spermatogenic cells. However, decreased cell layers and vacuoles in Sertoli cells were observed with fluoride and/or sulfur dioxide treatment.4. The ultrastructural changes of the BTB in testes of male mice after fluoride and sulfur dioxide treatment were studied by TEM. The image of the control group showed a classic basal ectoplasmic specialization structure, actin filament bundles sandwiched between opposing plasma membranes and the endoplasmic reticulum, and a tight junction in the BTB. However, compared with the controls, disassembly of the TJ, disorganization of the ER, the absence of actin filament bundles and the widened intercellular space between the two adjacent Sertoli cell plasma membranes were observed in the groups treated with fluoride and/or sulfur dioxide.5. qRT-PCR results of mice testes mRNA expression of F11R, CAR, Occludin, ZO-1, N-cadherin, a-catenin,β-catenin, Connexin-43 and Desmoglein-2 showed that:mice exposure to NaF reduced the mRNA expression of Occludin, ZO-1, N-cadherin, a-catenin and Connexin-43, while mice of the SO2 and NaF+SO2 treatment group showed no statistical difference, but mRNA expression of Desmoglein-2 in all treatment groups was decreased significantly. There did not have any significant difference in the mRNA expression of the other genes such as F11R, CAR and P-catenin.6. The protein expression levels of Occludin, ZO-1, N-cadherin, a-catenin, Connexin-43 and Desmoglein-2 in testes of mice were evaluated using western-blotting technology. NaF and SO2 decreased basal ectoplasmic specialization proteins (N-cadherin and a-catenin), gap junction protein (Connexin-43) and desmosome protein (Desmoglein-2), whereas the tight junction proteins (Occludin and ZO-1) did not change, and that the most dramatic declines of the changed proteins were detected in the NaF group.[Conclusion]Fluoride and sulfur dioxide interfere with the spermatogenesis, disturb the structure of BTB, and decrease the expression of BTB related proteins (N-cadherin, a-catenin, Connexin-43, Desmoglein-2). This suggest that fluoride and sulfur dioxide may impaire male reproduction by disturbing the integrity and function of BTB, and that fluoride toxicity on male reproduction should be deliver more attention.