Estrogen Receptors Are Involved In PFOS-induced Reproductive Toxicity In Male Mice

Perfluorooctane sulfonate (PFOS), which belongs to the perfluorinated compounds (PFCs) family, has been widespreadly used for different industrial and commercial purposes in the past decades and is ubiquitous in the environment. PFOS has been reported to have toxic effects on various organs in several fish and mammalian models, especially the adverse effects on reproductive system including reducing fertility and interfering with serum testosterone levels. Epidemiological studies have also indicated that the declined semen quality may be related to PFOS exposure. Although such researches confirmed the toxicity of PFOS on reproductive system, the specific reproductive toxicity especially the toxicity targets and the underlying mechanisms have not been elucidated. In addition, several lines of evidence have indicated that estrogen receptors (ERs) are involved in the regulatory mechanisms of spermatogenesis, and our previous studies showed that PFOS exposure could interfere with the expression of ERs in mice testis, then are ERs involved in the regulation of PFOS-induced male reproductive damage?Thus, in this project, male mice and spermatocyte-derived cell line will be used to investigate the effects of PFOS on reproductive system such as spermatogenesis, fuction and structure of testis in male mice. The involvement of ERs in this process and the exact mechanism involved will also be studied. Results derived from this project may provide a basis for further investigation to reproductive toxic effects of PFOS, it may also provide a theoretical basisi for the government to make strategy of PFOS contamination controlling.Part Ⅰ:The reproductive toxicity of PFOS in male mice ObjectiveTo explore the reproductive toxic effects of PFOS on adult male mice. Methods36 male C57 mice were divided randomly into three experimental groups. Mice were weighed by the electronic balance and then orally received 0.5 or 10 mg/kg/d PFOS every day. Control mice were administered equal volume of corn oil containing 0.1% DMSO by oral gavage. The mice were weighed and then killed after treatment for 35 days. Computer-aided sperm analysis (CASA) was used to analyze the sperm counts; Radioimmunoassay was used to detect the serum reproductive hormone levels; Testicular morphology was observed under the light microscope; TdT-mediated dUTP Nick-End Labeling (TUNEL) was used to assess apoptosis in the testis; Immunohistochemistry and western blot were used to evaluate the expression and location of proteins involved in cell proliferation and apoptosis together with ERa and ERβ.ResultsExposure to 10mg/kg/d PFOS resulted in a significant decrease in body weight, testis weight, testis organ coefficient, sperm counts and serum testosterone level. The change of estradiol was relatively slight. Under microscopical investigation, 10mg/kg/d PFOS decreased the number of germ cells and caused vacuolization of some Sertoli cells. TUNEL assay showed that PFOS could induce testicular germ cell apoptosis. Besides, 10mg/kg/d PFOS exposure increased the expression levels of Bax、 cleaved caspase-3、cleaved caspase-9 and ERβ while decreased that of PCNA and ERa in testis.0.5mg/kg/d PFOS exposure only elevated the expression levels of ERβ.ConclusionPFOS can induce reproductive disorder including decreasing sperm counts in male mice. Increased germ cell apoptosis and altered expression levels of ERa, ERβ, proteins involved in cell proliferation and apoptosis might be important reasons for the decreased sperm counts.Part Ⅱ:Involvement of estrogen receptors in impaired spermatogenesis induced by PFOSObjectiveTo explore the role of ERs in PFOS-induced apoptosis on mouse spermatocyte GC-2 cell line.MethodsThe effects of PFOS on GC-2 cell viability, apoptosis and cell cycle were assessed by MTT assay and flow cytometry; To explore the mechanism of PFOS-induced GC-2 cell apoptosis, effects of PFOS on the expression levels of ERa% ERβ、p-ERKl/2 and proteins involved in cell proliferation and apoptosis in GC-2 cells were examined by western blot. Furthermore, ICI 182780 (ERs antagonist), PPT (ERa specific agonist) and DPN (ERb specific agonist) were used to confirm the involvement of ERs in PFOS-induced apoptosis; To investigate whether PFOS acts through ERs classical genomic pathway, effects of PFOS on ERs transcriptional activity were tested by reporter gene assay. To analyze how PFOS interfering with ERs expression, levels of miRNAs targeting ERs were examined by RT-PCR.ResultsPFOS inhibited cell proliferation, caused the arrest of cells in G0/G1 phase and induced apoptosis in GC-2 cells. Compared with control group, PFOS treatment increased the protein expression of ERβ, Bax, cleaved caspase-3 and cleaved caspase-9 while decreased that of ERa, pERKl/2, Bcl-2, PCNA and cyclin D1. The addition of selective ERa agonist PPT partly reversed PFOS-induced alteration in cell proliferation, apoptosis and expression of proteins involved while selective ERβ agonist DPN accelerated it. Reporter gene assay showed that PFOS had no significant effect on ERs transcriptional activity. In addition, PFOS induced an increase in the expression level of miR-145 and miR-206 while a decrease in miR-92.ConclusionPFOS inhibites cell proliferation and induces apoptosis which might be associated with the activation of ERs genomic pathway. This involves decreased p-ERK1/2 level, followed by interfered expression of downstream Bax、Bcl-2、 caspase-9 and caspase-3 which are involved in the intrinsic apoptotic pathway together with the downregulation of cell cycle regulator cyclin D1. In addition, the treatment of PFOS changes the expression levels of miR-145、miR-206 and miR-92, which might partly explain the altered expression of ERs.Part Ⅲ:The effects of PFOS on spermatogenesis in ERβKO miceObjectiveTo explore the effect of ERβ deficiency on PFOS-induced spermatogenesis impairment in ERβKO male mice.Methods11 male ERβKO mice were randomly divided into two groups with control group 5 and PFOS group 6; 12 male WT mice were randomly divided into two groups with 6 each group. Mice in PFOS group were administered a single dose of PFOS (8mg/kg/d) by oral gavage. Mice in control group received equal volume of corn oil containing 0.1% DMSO. Both control and PFOS-treated mice were weighed and killed at 35d after exposure. CAS A was used to examine the sperm counts; Radioimmunoassay was used to assess the serum testosterone and estradiol levels; Testicular morphology was observed by the light microscope; TUNEL was used to evaluate apoptosis in the testis; Immunohistochemistry and western blot were used to determine the expression and location of proteins involved in cell proliferation and apoptosis.ResultsNo significant differences in sperm counts, testicular morphology, germ cell apoptosis, serum testosterone and estradiol levels were observed between WT and ERβKO mice in control groups. After PFOS exposure, sperm counts, testosterone level and PCNA expression decreased while germ cell apoptosis, expression levels of Bax and cleaved caspase-3 increased in both WT and ERβKO mice. Meanwhile, PFOS treatment didn’t affect body weight, testis weight and estradiol levels in both kinds of mice. Further comparison of the above data between WT and ERβKO mice exposed to PFOS showed that the germ cell apoptosis index (AI) in WT mice was significantly higher than in ERβKO mice. Except this, no other significant difference was observed between WT and ERβKO mice in PFOS-treated groups.ConclusionLoss of ERβ protein expression has no influence on sperm counts, testicular morphology, serum testosterone and estradiol levels in male mice. Compared with WT mice, germ cell apoptosis induced by PFOS in ERβKO mice significantly decreased, which implies the possible protective effect of ERβ deficiency on PFOS-induced germ cell apoptosis.