Effects And Molecular Mechanisms Of PFOS On Blood-testis Barrier Structure And Functions

Perfluorooctane sulfonate (PFOS) is known as a new environmentally persistentorganic pollutant (POPs) with strong accumulation, which induces organism healthhazards and has drawn increasing attention in recent years. Perfluorooctane sulfonateexhibits toxicity on liver, nerve, immune and reproductive systems. In recently, manyin vivo and in vitro studies focus on PFOS-induced reproductive toxicity via theendocrine disruption. However, there is no data to date available about the directtoxicity of PFOS on the testis and blood-testis barrier (BTB). Blood testis barrier iscommon structure in the mammalian testis and plays key role during spermatogenesis,which is important target for xenobiotics. To explore the effects and mechanisms ofPFOS on BTB is helpful to fully understand PFOS-induced male reproductivedisorder.In this study, we investigated the effects and mechanisms of PFOS on BTB invivo and in vitro. The changes of structure and functions of BTB were evaluated. Thelevels of expression and location of junction proteins were also estimated. Theimportant cell and molecular targets of PFOS in testis were also assessed. Importantly,the key mechanism of PFOS-induced disrupt of BTB were fully explored. Thepresent study will help reveal the toxicological and physiological importance ofPFOS-induced male reproductive disruption and establish the protection strategy forhumans. Part I: Effects of PFOS on reproductive system in male ICRmouseObjective: To reveal the direct toxic effects of PFOS on testis and assess the celltarget of PFOS-induced disruption of testis. Methods: The animal reproductivedisruption model was established via administration of PFOS by oral to adult maleICR mice. The body weight and testicular and epididymal organ coefficients of micewere analyzed. The levels of serum reproductive hormones were detected byradioimmunoassay. The changes of testicular morphology and target cells of PFOSwere evaluated by light and electron microscopy. The sperm parameters wereanalyzed by computer-assisted sperm analysis (CASA). Results:1. General toxicity:There were no significant changes of bodyweights and testicular and epididymalorgan coefficients between the control and PFOS-treated groups.2. Serumreproductive hormones: Compared with the control group,2.5mg/kg/d and up doseof PFOS significantly decreased the serum levels of testosterone (T)(p<0.05for2.5mg/kg/d group, p<0.01for25and50mg/kg/d groups). The changes of estradiol (E2),follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were relativelyslight.3. Testicular morphology: Under light microscopy,2.5mg/kg/d and up dose ofPFOS significantly induced vacuolization of Sertoli cells. The immature germ cellswere observed in the lumen of seminiferous tubule in the50mg/kg/d group. Underelectron microscopy,2.5mg/kg/d and up dose of PFOS significantly inducedvacuolization of Sertoli cells and the lipid droplet were significantly increased in thecytoplasm.4. Sperm parameter: The sperm counts were significantly decreased in thePFOS-treated groups with the dose ranged from2.5to50mg/kg/d and withdose-dependent manner (p<0.05for2.5mg/kg/d group, p<0.01for25and50mg/kg/d groups). More over, the motility of sperm were decreased in50mg/kg/dgroups (p<0.05) and the rapid cell percentage were also decreased PFOS (p<0.05for25mg/kg/d group, p<0.01for50mg/kg/d group). Part II: Effects of PFOS on BTB structure and functionsObjective: To reveal the direct toxic effects of PFOS on BTB structure andfunctions. Methods: The changes of BTB morphology were analyzed by electronmicroscopy. The changes of BTB functions were assessed by biotin tracer. Thechanges of expression and location of BTB junction proteins were evaluated byimmunoblotting and immunohistochemisty. Results:1. Ultrastructure of BTB:Compared with the control group, the significant changes including absence of actinfilament bundles, disassembly of TJ and vacuoles were observed among thePFOS-treated groups with dose ranged form2.5to50mg/kg/d.2. BTB functions:Compared with control group, the biotin tracer passed though the BTB and enteredinto the adluminal compartment of seminiferous tubule among the PFOS-treatedgroups with dose range from2.5to50mg/kg.3. Serum and testicular PFOS levels:Compared with control group, PFOS were detected in the serum and testes among thePFOS-treated groups. The positive correlation between serum and testes PFOS levelwas also observed and its coefficient correlations was0.9676(Pearson, P<0.0001).4.The changes of expression and location of junction proteins: The significant decreasein ZO-1(from2.5to50mg/kg/d groups, p<0.01), Claudin-11and Occludin (p<0.05for2.5mg/kg/d group, p<0.01for25and50mg/kg/d groups), Connexin-43(2.5and50mg/kg/d groups, p<0.05) and p-Connexin-43(from2.5to50mg/kg/d groups, p<0.01) expression in testes were observed. More over, the locations of these proteins inBTB were also decreased among the PFOS-treated groups with dose ranged from2.5to50mg/kg/d.5. Activation of MAPK: The significant increases in p-Erk andp-p-38expression in mice testes were observed (p-Erk, p<0.01for25and50mg/kg/d groups; p-p-38, p<0.05for2.5mg/kg/d group, p<0.01for25and50mg/kg/d groups). Part III: Molecular mechanisms of Sertoli cells as targets inPFOS-induced disruption of BTB structure and functionObjective: To reveal the role of Sertoli cell in PFOS-induced disruption of BTBstructure and function. To assess the relationship between the activation of MAPKsignal pathway and the toxic effects of PFOS on BTB. Methods: The target cells forPFOS were estimated by cytotoxicity assays. The expressions of PFOS transporters intarget cells were analyzed by RT-PCR. The expressions and locations of BTB relatedjunction proteins were evaluated by immunoblotting and immunofluorescence. Thekey signal pathway of MAPK related to PFOS-induced disruption of BTB wasanalyzed by using specific MAPK signal pathway inhibitors. Results:1. Target cellsfor PFOS: Compared with other testicular cells, the Sertoli cell line (TM4) was moresensitive to PFOS toxicity.2. The expressions of PFOS transporters: PFOStransporters OAT1, OATP1and OATP3were expressed in TM4cells.3. Theexpressions and locations of BTB related junction proteins: Compared with controlgroup, PFOS significantly decreased the expression of Claudin-11in TM4cells.(50,100and150μM group, p<0.01). Similarly, ZO-1, Occludin, Connexin-43andp-Connexin-43were decreased by PFOS with concentrations ranged from100to150μM (p<0.01for ZO-1, Occludin and p-Connexin-43; p<0.05for Connexin-43). Asincrease of PFOS concentrations, the locations of Claudin-11and Connexin-43werealso decreased.4. The relationship between the activation of MAPK signal pathwayand PFOS-induced disruption of BTB: Compared with control group, the p-38MAPK signal pathway inhibitors significantly recovered PFOS-induced the decreaseof expressions and locations of BTB related junction proteins.5. PFOS significantlydecreased tight junction and the expressions of junction proteins including ZO-1,Claudin-11, Occludin, Connexin-43and p-Connexin-43in primary Sertoli cells (20and30μM group, p<0.05or p<0.01). The p-38MAPK signal pathway inhibitorssignificantly recovered PFOS-induced the decrease of tight junction and expressionsof BTB related junction proteins in primary Sertoli cells. 1. PFOS induces reproductive disorder on male ICR mouse and Sertoli cell is animportant target for PFOS.2. PFOS not only disrupted the structure and function of BTB but also decreased theexpression and locations of BTB related junction proteins, which implied that thejunction proteins might be the important molecular targets for PFOS.3. PFOS targeted Sertoli cell and activated p-38MAPK signal pathway via decreasethe expressions and locations of BTB related proteins, which may be a possiblemolecular mechanism for PFOS-induced disruption of structure and function ofBTB.