Reproductive Toxicology 51 (2015) 114–124
Contents lists available at ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
2,2 ,4,4 -Tetrabromodiphenyl ether disrupts spermatogenesis, impairs mitochondrial function and induces apoptosis of early leptotene spermatocytes in rats Shaoping Huang a,b , Yiqiang Cui a , Xuejiang Guo a , Lei Wang a,∗ , Suying Li a , Ying Lu a , Ye Bi a , Xiaoyan Huang a , Min Lin a , Yankai Xia a , Shoulin Wang a , Xinru Wang a , Zuomin Zhou a,∗ , Jiahao Sha a a b
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Jiangsu, China Department of Human Anatomy and Neuroscience, Medical School, Southeast University, Nanjing, Jiangsu, China
a r t i c l e
i n f o
Article history: Received 15 September 2014 Received in revised form 14 December 2014 Accepted 10 January 2015 Available online 2 February 2015 Keywords: Apoptosis Mitochondria PBDEs Spermatogenesis Testis
a b s t r a c t Our objective was to explore molecular markers and mechanism of BDE47 on spermatogenesis in mammals. Adult male SD rats were gavaged daily with corn oil containing 0, 0.001, 0.03, 1 or 20 mg BDE47/kg bw for eight weeks. Testes morphology was analyzed using electron microscopy, TUNEL, immunohistochemistry and morphometry. Differential proteome profile and western blotting were applied to determine molecular markers and protein expression. GC1-spg cells (mouse spermatogonial cells) were used to verify mechanism of BDE47. Data showed BDE47 reduced tubular epithelial thickness, impaired mitochondrial function and induced apoptosis in early leptotene spermatocytes. Proteomic study identified 70 differential spots corresponding to 64 proteins. 20 proteins related to apoptosis, 15 located in mitochondria. Exposure of GC1-spg cells showed BDE47 induced apoptosis, impaired mitochondria and decreased Bcl-2 in cells. Data indicate that BDE47 disrupts spermatogenesis, impairs mitochondrial function and induces apoptosis of early leptotene spermatocytes in rats probably via mitochondrial pathway. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Spermatogenesis consists of three phases: self-renewal of spermatogonial stem cells and differentiation, meiotic division of spermatocytes, and spermiogenesis. Spermatogenic cells are highly sensitive to physical and chemical environmental factors. In the past decades, studies had reported that some environmental factors are associated with deleterious effects on spermatogenesis [1]. For example, bisphenol A exposure at an environmentally relevant dose induces meiotic abnormalities in adult male rats [2]; bis-(2-ethylhexyl) phthalate (DEHP) impaired spermatogenesis in zebrafish by inducing a mitotic arrest of germ cells [3]; fenvalerate, a widely used pyrethroid insecticide, induces germ cell apoptosis in
∗ Corresponding authors at: State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, Jiangsu Province, China. Tel.: +86 25 86862905; fax: +86 25 86862908. E-mail addresses: [email protected] (L. Wang), [email protected] (Z. Zhou). http://dx.doi.org/10.1016/j.reprotox.2015.01.009 0890-6238/© 2015 Elsevier Inc. All rights reserved.
mouse testis through the Fas/FasL signaling pathway [4]. Furthermore, studies had reported that DEHP, BPA and fenvalerate were negative correlated to human sperm quality [5–7]. For several decades, polybrominated diphenyl ethers (PBDEs) were mass-produced for widespread application as flame retardants, and added to a wide variety of products such as computer housing, textiles, furniture, and electrical appliances [8]. As a result, the levels of PBDEs and their residues in the environment and human tissues have increased in recent decades. Of all PBDEs, 2,2 ,4,4 -tetrabromodiphenyl ether (BDE47) was one of the most prevalent congeners in animal and human tissues [9,10]. Studies have demonstrated that a low concentration of BDE47 (∼15 g/L) can result in developmental toxicity to turbot embryos [10]. Exposure of perinatal rats to low levels of BDE47 as found in humans resulted in neurodevelopmental defects [11]. Schreiber et al. [12] reported that PBDEs inhibited the differentiation of human fetal neural progenitor cells into neurons. In addition, BDE47 was also toxic to human hematopoietic stem cells via mitochondrial damage and apoptosis [13]. BDE47 was found in frog testes and in human semen [14,15]. Our previous study demonstrated that BDE47 dose-dependently reduced the
S. Huang et al. / Reproductive Toxicology 51 (2015) 114–124
spermatogenesis, induced apoptosis of spermatocytes and reactive oxygen species (ROS) in seminiferous tubules. Also, BDE47 induced apoptosis related proteins FAS/FASL, p-p53 and caspase 3 in rat testis [16]. But what kind of the molecular markers that are related to impaired spermatogenesis effected by BDE47 are still unknown. Furthermore, as to the spermatocytes, include different stages such as leptotene, zygotene, pachytene, diplotene and diakinesis, which stage of the spermatocytes was sensitive to BDE47 is still not clear. So, objective of this study is to explore the sensitive stage of the spermatogenesis that is effected by BDE47 and to explore the molecular markers relating to the impaired spermatogenesis after BDE47 exposure. This study will provide an insight to understand the molecular mechanism of BDE47 on the germ cells during the spermatogenesis. 2. Materials and methods 2.1. Animals and treatment Fifty adult male SD rats (200 ± 15 g) were obtained from the Laboratory Animal Center of Nanjing Medical University (Nanjing, China). Animals were maintained in a 12/12-h light/dark cycle at 20–22 ◦ C and 50–70% humidity. Two rats per cage were housed in a plastic Macrolon cage with a bedding of wood shavings, and food and water ad libitum. The rats were divided into five groups of 10 rats and gavaged daily with corn oil (Sigma, St. Louis, USA) containing 0, 0.001, 0.03, 1 or 20 mg BDE47/kg bw (Chem Service, Inc., West Chester, USA) for 8 weeks. The study was approved by the Animal Ethical Committee, Nanjing Medical University, China (study number: 20090598). Testes were collected and fixed for histological examination, TUNEL analysis and electron microscopy, or frozen at −80 ◦ C.
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After incubation of the homogenate at 4 ◦ C for 1 h, insoluble matter was removed by centrifugation at 40,000 × g at 4 ◦ C for 1 h. Protein concentration was determined by Bradford method using BSA as a standard [20]. 2.6. Two-dimensional electrophoresis (2-DE) Immobilized pH gradient (IPG) strips (length, 24 cm; pH 3–10, nonlinear; Amersham Bioscience, Uppsala, Sweden) were loaded with 180 g protein. After isoelectric focusing, the IPG strips were equilibrated, electrophoresed in an Ettan DALT Twelve Electrophoresis System (GE Healthcare, San Francisco, USA), and visualized by silver staining. In this experiment, 12 gels were generated from every four individual rat testes of the control, 0.001 and 0.03 mg BDE47/kg bw groups. The stained gels were scanned, and the protein spots were detected, quantified, compared and statistically analyzed by ImageMasterTM 2D Platinum Software (Version 5.0, Amersham Bioscience, Swiss Institute of Bioinformatics, Geneva, Switzerland). Expression level of each protein spot was determined on the basis of the relative volume (vol%) of each spot in the gel, the values obtained from four individual experiments of every group were pooled to calculate the mean and standard derivations (SD) of each differential expressed protein spot. P values corresponding to each differential spot between groups were assessed using independent t-tests, and considered statistically significant if
Contents lists available at ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
2,2 ,4,4 -Tetrabromodiphenyl ether disrupts spermatogenesis, impairs mitochondrial function and induces apoptosis of early leptotene spermatocytes in rats Shaoping Huang a,b , Yiqiang Cui a , Xuejiang Guo a , Lei Wang a,∗ , Suying Li a , Ying Lu a , Ye Bi a , Xiaoyan Huang a , Min Lin a , Yankai Xia a , Shoulin Wang a , Xinru Wang a , Zuomin Zhou a,∗ , Jiahao Sha a a b
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Jiangsu, China Department of Human Anatomy and Neuroscience, Medical School, Southeast University, Nanjing, Jiangsu, China
a r t i c l e
i n f o
Article history: Received 15 September 2014 Received in revised form 14 December 2014 Accepted 10 January 2015 Available online 2 February 2015 Keywords: Apoptosis Mitochondria PBDEs Spermatogenesis Testis
a b s t r a c t Our objective was to explore molecular markers and mechanism of BDE47 on spermatogenesis in mammals. Adult male SD rats were gavaged daily with corn oil containing 0, 0.001, 0.03, 1 or 20 mg BDE47/kg bw for eight weeks. Testes morphology was analyzed using electron microscopy, TUNEL, immunohistochemistry and morphometry. Differential proteome profile and western blotting were applied to determine molecular markers and protein expression. GC1-spg cells (mouse spermatogonial cells) were used to verify mechanism of BDE47. Data showed BDE47 reduced tubular epithelial thickness, impaired mitochondrial function and induced apoptosis in early leptotene spermatocytes. Proteomic study identified 70 differential spots corresponding to 64 proteins. 20 proteins related to apoptosis, 15 located in mitochondria. Exposure of GC1-spg cells showed BDE47 induced apoptosis, impaired mitochondria and decreased Bcl-2 in cells. Data indicate that BDE47 disrupts spermatogenesis, impairs mitochondrial function and induces apoptosis of early leptotene spermatocytes in rats probably via mitochondrial pathway. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Spermatogenesis consists of three phases: self-renewal of spermatogonial stem cells and differentiation, meiotic division of spermatocytes, and spermiogenesis. Spermatogenic cells are highly sensitive to physical and chemical environmental factors. In the past decades, studies had reported that some environmental factors are associated with deleterious effects on spermatogenesis [1]. For example, bisphenol A exposure at an environmentally relevant dose induces meiotic abnormalities in adult male rats [2]; bis-(2-ethylhexyl) phthalate (DEHP) impaired spermatogenesis in zebrafish by inducing a mitotic arrest of germ cells [3]; fenvalerate, a widely used pyrethroid insecticide, induces germ cell apoptosis in
∗ Corresponding authors at: State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, Jiangsu Province, China. Tel.: +86 25 86862905; fax: +86 25 86862908. E-mail addresses: [email protected] (L. Wang), [email protected] (Z. Zhou). http://dx.doi.org/10.1016/j.reprotox.2015.01.009 0890-6238/© 2015 Elsevier Inc. All rights reserved.
mouse testis through the Fas/FasL signaling pathway [4]. Furthermore, studies had reported that DEHP, BPA and fenvalerate were negative correlated to human sperm quality [5–7]. For several decades, polybrominated diphenyl ethers (PBDEs) were mass-produced for widespread application as flame retardants, and added to a wide variety of products such as computer housing, textiles, furniture, and electrical appliances [8]. As a result, the levels of PBDEs and their residues in the environment and human tissues have increased in recent decades. Of all PBDEs, 2,2 ,4,4 -tetrabromodiphenyl ether (BDE47) was one of the most prevalent congeners in animal and human tissues [9,10]. Studies have demonstrated that a low concentration of BDE47 (∼15 g/L) can result in developmental toxicity to turbot embryos [10]. Exposure of perinatal rats to low levels of BDE47 as found in humans resulted in neurodevelopmental defects [11]. Schreiber et al. [12] reported that PBDEs inhibited the differentiation of human fetal neural progenitor cells into neurons. In addition, BDE47 was also toxic to human hematopoietic stem cells via mitochondrial damage and apoptosis [13]. BDE47 was found in frog testes and in human semen [14,15]. Our previous study demonstrated that BDE47 dose-dependently reduced the
S. Huang et al. / Reproductive Toxicology 51 (2015) 114–124
spermatogenesis, induced apoptosis of spermatocytes and reactive oxygen species (ROS) in seminiferous tubules. Also, BDE47 induced apoptosis related proteins FAS/FASL, p-p53 and caspase 3 in rat testis [16]. But what kind of the molecular markers that are related to impaired spermatogenesis effected by BDE47 are still unknown. Furthermore, as to the spermatocytes, include different stages such as leptotene, zygotene, pachytene, diplotene and diakinesis, which stage of the spermatocytes was sensitive to BDE47 is still not clear. So, objective of this study is to explore the sensitive stage of the spermatogenesis that is effected by BDE47 and to explore the molecular markers relating to the impaired spermatogenesis after BDE47 exposure. This study will provide an insight to understand the molecular mechanism of BDE47 on the germ cells during the spermatogenesis. 2. Materials and methods 2.1. Animals and treatment Fifty adult male SD rats (200 ± 15 g) were obtained from the Laboratory Animal Center of Nanjing Medical University (Nanjing, China). Animals were maintained in a 12/12-h light/dark cycle at 20–22 ◦ C and 50–70% humidity. Two rats per cage were housed in a plastic Macrolon cage with a bedding of wood shavings, and food and water ad libitum. The rats were divided into five groups of 10 rats and gavaged daily with corn oil (Sigma, St. Louis, USA) containing 0, 0.001, 0.03, 1 or 20 mg BDE47/kg bw (Chem Service, Inc., West Chester, USA) for 8 weeks. The study was approved by the Animal Ethical Committee, Nanjing Medical University, China (study number: 20090598). Testes were collected and fixed for histological examination, TUNEL analysis and electron microscopy, or frozen at −80 ◦ C.
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After incubation of the homogenate at 4 ◦ C for 1 h, insoluble matter was removed by centrifugation at 40,000 × g at 4 ◦ C for 1 h. Protein concentration was determined by Bradford method using BSA as a standard [20]. 2.6. Two-dimensional electrophoresis (2-DE) Immobilized pH gradient (IPG) strips (length, 24 cm; pH 3–10, nonlinear; Amersham Bioscience, Uppsala, Sweden) were loaded with 180 g protein. After isoelectric focusing, the IPG strips were equilibrated, electrophoresed in an Ettan DALT Twelve Electrophoresis System (GE Healthcare, San Francisco, USA), and visualized by silver staining. In this experiment, 12 gels were generated from every four individual rat testes of the control, 0.001 and 0.03 mg BDE47/kg bw groups. The stained gels were scanned, and the protein spots were detected, quantified, compared and statistically analyzed by ImageMasterTM 2D Platinum Software (Version 5.0, Amersham Bioscience, Swiss Institute of Bioinformatics, Geneva, Switzerland). Expression level of each protein spot was determined on the basis of the relative volume (vol%) of each spot in the gel, the values obtained from four individual experiments of every group were pooled to calculate the mean and standard derivations (SD) of each differential expressed protein spot. P values corresponding to each differential spot between groups were assessed using independent t-tests, and considered statistically significant if
