Androgen Receptor-Mediated Apoptosis in Bovine Testicular
Induced Pluripotent Stem Cells in Response to Phthalate Esters
Running title: Effect of phthalates on iPSCs derived from testis cells
Shin-Wei Wang1-5*, Deng-Chyang Wu2-5*, Ying-Chu Lin4,6, Sheng-Wen Wang2-5, Chun-Chieh Wu3,5, Chee-Yin Chai3,5, Jau-Nan Lee3,5, Eing-Mei Tsai1,3-5, Cheng-Lung Lin1-5, Rei-Cheng Yang3,5, Ying-Chin Ko3,5,7, Hsin-Su Yu3,5, Yoshinobu Murayama8, Hiroyuki Miyoshi9, Yukio Nakamura9, Shinichi Hashimoto10, Kouji Matsushima10, Kohsuke Kato11, Kyosuke
Nagata11, Richard Eckner12, Shigeo Saito1,3,4,13** and Kazunari K. Yokoyama1,3,4**
1Graduate Institute of Medicine, 2Cancer Center, 3College of Medicine, 6School of Dentistry, 4Kaohsiung
Medical University and 5Hospital, Kaohsiung, Taiwan; 7Graduate Institute of Clinical Medical Science,
College of Medicine, China Medical University, Taichung, Taiwan; 8College of Engineering, Nihon
University, Koriyama, Fukushima, Japan; 9RIKEN BioResource Center, Tsukuba, Ibaraki, Japan; 10Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo,
Tokyo, Japan; 11Graduate School of Comprehensive Human Sciences and Institute of Basic Medical
Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan; 12Dept of Biochemistry & Molecular Biology,
UMDNJ-New Jersey Medical School, Newark, NJ, USA; 13Saito Laboratory of Cell Technology, Yaita,
Tochigi, Japan.
*These authors contributed equally to this work.
**Correspondence should be addressed to Kazunari K. Yokoyama ([email protected]), and Shigeo Saito
(saict1@ maple.ocn.ne.jp), Graduate Institute of Medicine, Kaohsiung Medical University (Tel: 886-7-312-1101, ext.2729; Fax: 886-7-313-3849).
Funding
This research was supported by a grant from National Science Council in Taiwan (NSC; NSC-100-2320-B-037-020; NSC-101-2320-B-037-047-My3; NSC-101-2314-B-037-004-My2), National Health Research Institutes (NHRI; NHRI-Ex101-10109BI) and the Kaohsiung Medical University Research Foundation (KMU-ER006, KMU-EM-93-3) to KKY.
Acknowledgements
We thank Drs. Minamihashi, A. and Yamamoto, Y. for their kind supply of the sample of bovine testes and Ms. Chen, W. and Ku, C. for their technical support.
Conflict of interest statement, No declared.
ABBREVIATIONS
BBP Butylbenzyl phthalate
DBP Di-n-butyl phthalate
DEHP Di (2-ethylhexyl) phthalate EDC Endocrine disrupting chemicals
ESCs Embryonic stem cells
iPSCs Induced pluripotent stem cells MEF Mouse embryonic fibroblast
qRT PCR Quantitative real time polymerase chain reaction
Abstract
phthalate derivatives has been linked to testicular impairment and male subfertility. However, the effects of phthalate esters on induced pluripotent stem cells (iPSCs) are
unclear.
OBJECTIVES: iPSCs were generated from bovine testicular cells, and the effects of
phthalate esters were measured.
METHODS: iPSCs were generated by electroporation of Pou-domain transcription factor
Oct4. Supplementation with leukemia inhibitory factor and bone morphogenetic protein 4 maintained and stabilized the expression of stemness genes and pluripotency. Androgen receptor (AR)- mediated apoptosis and AR signaling after exposure to phthalates esters including di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP), and di (2-ethylhexyl) phthalate (DEHP) were assessed in IPSCs.
RESULTS: Bovine iPSCs displayed the normal karyotype, expressed stemness genes, and
differentiated into cell types of all three germ layers in teratomas. DEHP repressed the expression of AR and the ability of iPSCs to commit to apoptosis; this was mediated by increased expression of p21Cip1. DBP and BBP had no effect. Loss of Wnt/β-catenin signaling, especially activities of the Frizzed receptor and Disheveled, was responsible for
the effect of DEHP on AR-mediated apoptosis.
CONCLUSIONS: iPSCs from testicular cells are useful for screening the toxicity of
environmental disruptors in terms of maintenance of stemness and pluripotency.
KEY WORDS: Bovine iPSCs, Testiscular cells, endocrine disruptor, Wnt, Androgen
The identification of the genotoxic or mutagenic risk of endocrine-disrupting chemicals (EDCs) is critical for developing therapeutic agents for many human and domestic animal diseases (Colborn et al. 1993). Thus, the characterization of the genetic traits that arise during exposure to EDCs during development is a matter of concern. The new regenerative approach using embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) is promising for developing stem-cell-based therapy and for developing novel therapeutic agents against human and animal diseases. The genetic traits and alterations in pluripotency and stemness resulting from developmental exposure to EDCs and iPSCs, especially environmental hormones, such as phthalate derivatives, during development have not been characterized fully.
Phthalates are synthetic compounds used widely as plasticizers, solvents, and additives in many consumer products. Several previous studies have reported that main cellular target of phthalates in the male reproductive organs is Serotoli and Leydig cells of the testis (Jobling et al. 1995; Lyche et al. 2009). The long-branched di-(2-ethylhexyl) phthalate (DEHP) and its metabolites have been shown to exert estrogen receptor α (ERα)-agonistic and ERβ-antagonistic activities. By contrast, di-n-butyl phthalate (DBP) and butyl benzyl phthalate (BBP) have ERα- agonistic activities and androgen receptor (AR)-antagonistic activities. DEHP and its metabolites can cause oxidative DNA damage, to the testes by inducing apoptosis in testicular cells (Casals-Cases and Desvergne, 2011). BBP induced necrosis in human granulosa cells, mediated through its effects on the aryl hydorcarbon receptor, have been reported recently (Chen et al. 2012). However, the effects of EDCs on
apoptosis and necrosis of ESCs and the induced pluripotent stem cells are unknown.
modified livestock and hold great promise for cell or organ therapies and drug screening, and as human disease models. Although many attempts have been made to establish ESCs in large domestic species, teratoma formation in all three germ layers has not yet been
confirmed except in the goat (Behboodi et al., 2011).
Pluripotent cells have been established from other embryonic and adult tissues using cell culture systems (Saito et al. 2013). Embryonic germ cells (Matsui et al., 1992) have been isolated from the primordial germ cells of midgestation embryos, and multipotent germline stem cells have been generated from explanted neonatal and adult mouse testicular cells, but at very low efficiency (Kanatsu-Shinohara et al., 2004; Guan et al., 2006). iPSCs have been generated by the addition of several combinations of transcription factors (OCT4,
MYC, KLF4, and SOX2)(Takahashi and Yamanaka, 2006).
In this paper, we report and characterize the stemness and pluripotency of bovine iPSCs generated by electroporation of OCT4. To understand the effects of environmental hormones of phthalate derivatives on testicular iPSCs, we have investigated AR-mediated apoptosis of iPSCs. Moreover, we examined the global impact of phthalates on molecular signaling cascade of AR-mediated apoptosis and identify the unveiled molecular target of phthalates. Thus, we suggest that iPSCs are useful for screening for toxicity of environmental hormones that might affect the early development and pluripotency of stem cells in domestic animals and that this screening may provide a good model to study the effects of EDCs on human
development.
Materials and Methods
Chemical Co. (Milwaukee, WI, USA). Caspase 3 assay kit and bezyloxycarbonyl-Val-Ala-Asp-Ch2F (Z-VAD) were from Promega Inc. (Madison, WI, USA). Trypan blue stain solution (0.5%) was from Nacalai Tesque (Tokyo, Japan). Biotin-conjugated 16-2´-deoxyuridine-5´-triphosphate, proteinase K, and the blocking reagent were from Roche Diagnostics (Mannheim, Germany). The pCMV-Flagh-OCT3/4 (RDB6598) was obtained from the RIKEN DNA Bank (Tsukuba, Ibaraki, Japan) and the pEGFP plasmid was generated as described elsewhere (Saito et al. 2003).
The plamids IRESneo-AR, WT-ARE-luciferase, mutARE-luciferase, and pGK-CAS-FZD7 were kindly gifted by Drs. Ben H. Park (The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA), Patrice J. Morin (National Institute on Aging, NIH, Baltimore, USA), and Karl Willert (University of California, San Diego, CA, USA), respectively. Constructs of small interfering RNA (siRNA) against p21Cip1 and AR were
obtained from Invitrogen (Paisley, UK).
Culture of bovine testicular cells. The testicular tissues from a bull calf were cut into 1-3
mm3 pieces, were then isolated by enzymatic digestion with 0.25% trypsin-EDTA (Gibco, Grand Island, NY, USA) for 10 min, and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10 ng/mL human inhibitor factor (LIF) (Sigma, St. Louis, MO, USA) and supplemented with 10% fetal bovine serum (FBS), and antimycotics-antibiotics (AM-AB; Gibco). After 2-3 passages, compact colonies were picked and split into other
dishes at a 1: 3 ratio in the same medium.
Generation of iPSCs. The dissociated testicular cells (5 x 105) were used for transfection with the OCT4 gene as described elsewhere (Saito et al., 2003) with 10 direct-current
electrical pulses of 20 V intensity at an interval of 50 ms. Cells in 2 mm cuvettes containing 200 μL of DMEM and 10 μg of plasmid DNA were treated in an electroporator (CUY 21 Vitro-EX, BEX Co., Ltd. Tokyo, Japan). The cells were then cultured and selected with G418 (100 μg/mL). Two days after selection, the cells were replated onto mitomycin-C-treated mouse embryonic fibroblasts (MEFs) cells using the standard medium supplemented with bone morphogenetic protein 4 (BMP4) (5 ng/mL; Sigma). The transfected cells were grown in the same medium until iPSCs were detected on day 17. The iPSC colonies were then picked up manually and replated onto a new feeder layer (first passage). The bovine iPSCs were then subcultured with trypsin-EDTA treatment, and the medium was replaced every 2 days. The bovine iPSCs (2 x 105) were incubated for 24 h in the presence of the phthalate esters DBP, BBP, or DEHP (Sigma-Aldrich) at the indicated doses and then
harvested.
Stemness assay and karyotyping. Alkaline phosphatase activity and immunostaining were
examined as described previously (Saito et al. 2005). The antibodies used were directed against: Oct4 (sx-5279; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Nanog (AF1997; R&D Systems, Minneapolis, MN, USA), Sox2 (AB5603; Millipore, Billerica, MA, USA), SSEA-1 (MAB4301; Millipore), SSEA-4 (MAB4304; Millipore), and Fluorescently-labeled secondary antibodies A11034 and A11029 were from Invitrogen (Carlsbad, CA, USA). Nuclei were detected with 0.5 μg/mL 4', 6-diamidino-2-phenylindole (DAPI, D3571, Invitrogen) for 1 h. Metaphase mitotic chromosomes were prepared using a conventional air-drying technique. GTG (G-banding) staining was performed as described elsewhere (Ford et al. 1980).
Cell viability, apoptosis, and necrosis. The number of viable cells was determined by
staining with thiazole orange and propidium iodine (Cell Viability Kit; Becton Dickinson and Company, BD Bioscience, San Jose, CA, USA) following the manufacturer’s protocol. To differentiate apoptosis from cell necrosis, cells were identified using flow cytometric analysis of cells stained with fluorescein isothiocyanate (FITC)-labeled annexin V to identify apoptotic cells and propidium iodine to label the permeable cells (FITC Annexin V Apoptosis Detection Kit II; BD BioScience). The percentages of apoptotic and necrotic cells were determined using a Bioluminescent Cell Viability Kit II (ADP/ATP). A terminal deoxynucleotidyl transferase dUTP nick end labeling assay (Apoptosis Detection System; Promega Co., Madison, WI, USA) and caspase-3/7 assay were conducted as described
elsewhere (Tang et al. 2002).
RNA extraction and quantitative polymerase chain reaction (PCR). RNA was extracted
from cells in the presence of the indicated dose of DEHP, DBP, or BBP and dimethyl sulfoxide (DMSO) as described elsewhere (Nakade et al., 2006). RNA was purified using an RNeasy Mini kit (2074104; Qiagen, Hilden, Germany), and reverse transcription (RT) was performed using Superscript III reverse transcriptase (18080-093; Invitrogen) and primers (see Supplemental Material, Table S1). PCR was performed using GoTaq® Green Master Mix (M7122; Promega). To avoid contamination by feeder cells, we chose primer pairs that do not amplify mouse transcripts. Real-time quantitative RT-PCR (qRT-PCR) was performed in a PRISM™ 7700 system as described elsewhere (Nakade et al., 2006; Amersham Biosystems, Foster City, CA, USA). We designed the primers using the
public-domain Primer 3 program of GENETYX-Mac Ver. 14 software (Hitachi Software, Tokyo, Japan). The respective pairs of primers are listed in Supplemental Material, Table S1.
Transfection and luciferase assay. IRESneo-AR, and pGK-CAS-FZD7 and their control
vectors (IREneo and pGK-CAS), and AR and its mutant-reporter DNAs (WT ARE, mutARE) were transfected into bovine iPSCs at 400 ng of total DNA per well of a 24-well plate (5 × 104 cells/well) using 2 µL of LipofectamineTM-2000 reagent (Invitrogen, Paisley, UK) and cultured in the presence of the indicated amount of phthalate ester, and Luciferase activity was measured using an assay kit system (Dual-Glo; Promega Co.) as described elsewhere (Jin et al. 2002; Nakade et al. 2006). The reporter plasmids comprised either three copies of a wild-type consensus binding site for AR or three copies of mutated binding sites of AR conjugated to a firefly luciferase reporter (Garay et al. 2012). Twenty-four hours after phthalate treatment, luciferase activity was measured using a commercial luciferase assay system (Dual-Glo; Promega). AR activity is expressed as the ratio of the luciferase activity in cells transfected with ARE-luciferase divided by the luciferase activity of its mutant reporter plasmid.
In vitro differentiation analysis. Bovine iPSCs were harvested using trypsin and large
clumps of cells (around 100 cells) isolated after centrifugation were plated in six-well dishes in differentiation medium. To induce ectodermal (neuronal) differentiation, the cells were cultured in medium (DMEM, 10 ng/mL basic finroblast growth factor (bFGF), 10 ng/mL epidermal growth factor, 10 ng/mL platelet-derived growth factor, and 1% AM-AB) for 7 days, and then cultured in growth medium (DMEM, 10% FBS, and 1% AM-AB) for 7-14
days. To induce mesodermal (cardiomyocyte) differentiation, cell colonies were placed in suspension culture in the differentiation medium (DMEM, 10% FBS, 100 μM ascorbic acid, and 1% AM-AB) for 10 days. The cell clumps placed in gelatin-coated dishes in the same medium, and the adherent cardiomyocytes were observed 7 days after replating. To induce endodermal differentiation, the cells were differentiated in the medium (DMEM, 100 ng/mL activin-A and 1% AM-AB) for 7 days, and then transferred to growth medium (DMEM, 10% FBS and 1% AM-AB), and allowed to differentiate for 7 days. The antibodies used were: mouse astrocyte-specific anti-glial fibrillary acidic protein (GFAP) antibody (Sigma), mouse neuron-specific anti-ß-tubulin Ш (Tuj1) antibody (Sigma), mouse cardiomyocyte-specific anti-human Nkx 2.5 antibody (CosmoBio, Tokyo, Japan) and mouse endoderm-specific anti-human α-fetoprotein protein (CosmoBio). FITC-conjugated rabbit secondary antibody against mouse IgG (Sigma) was used for immunostaining.
Teratoma formation assay. Bovine iPSCs (106) in DMEM plus 10% FBS were injected
under the kidney capsule of SCID mice using a 27G needle. Six to eight weeks after injection, the tumors were surgically dissected, fixed with 4% formaldehyde, and embedded in paraffin, and 4 μm sections were cut and stained with hematoxylin and eosin. The antibodies used were; rabbit anti-human muscle-specific actin (M0635; Dako, Glostrup, Denmark), rabbit anti-human S-100 (N1573; Dako), rabbit anti-human epithelial membrane antigene (EMA)(M0613; Dako), and rabbit anti-human cytokeratin (M3515; Dako). Periodic acid Schiff (PAS) staining was performed according to the manufacturer’s specification (NovaUltra Special Staining Kits; Woodstock, MD, USA).
Statistical analysis. Differences between the treated and control cells were analyzed by
Student’s t-test. P < 0.05 was considered significant.
Results
Stemness of iPSCs from bovine testicular cells. After three passages (15-21 days) of the
bovine testicular cells without a feeder cell layer, we observed compact, elliptically shaped colonies (Figure 1A). These colonies expressed some pluripotency marker genes, such as KLF4, MYC, STAT3, DNMT1, SUZ12, and MEF2A but did not express other stemness genes like OCT4, SOX2, and NANOG (Figure 1B). To generate bovine iPSCs, we used electroporation under the optimal condition of 10 electrical pulses of 20 V at 50 ms intervals. Seventeen days after electroporation, small packed domed colonies were detected on the mitotic-inactivated MEF cells. These colonies comprised small, rapidly dividing cells with a high nuclear/cytoplasmic ratio and large nucleoli (Takahashi and Yamanaka, 2006). The estimated reprogramming efficiency of our one-factor method was 0.3 %, which is 20-fold higher than that of the one-factor approach to reprogramming murine neural stem cells (Kim et al., 2009). When we continued to culture for more than 4 weeks, the cells displayed strong alkaline phosphatase activity (Figure 1A). Immunofluorescence staining confirmed that the iPSCs induced by OCT4 (1F-iPSCs) expressed stemness makers such as Oct4, Nanog, Sox2, SSEA-1, and SSEA-4 (Figure 1B). These markers were more intense in the dense patches of cells. Examination of chromosomal abnormalities in iPSCs revealed normal distributions of the 60 chromosomes, including the XY sex chromosomes at passage 15 by the G-banding (Figure 1C). At the transcript level of stemness genes, the 1F-iPSCs expressed ESC markers, including OCT4, SOX2, MYC, KLF4, MEF2a, SUZ12, STAT3, and
DNMT1 (Figure 1B).
Pluripotency. To confirm the developmental potential of the bovine 1F iPSCs in vitro, the
cell clumps were stimulated to differentiate into the three germ layers, after which GFAP-positive astrocytes and Tuj1-GFAP-positive neurons, α-fetoprotein-GFAP-positive endodermal cells, and Nkx 2.5-specific cardiomyocyte precursor cells were detected in most of the differentiated cell colonies (Figure 2A). To assess the pluripotency of the bovine 1F iPSCs in vivo, we transferred the cells into immunodeficient SCID mice. Bovine iPSCs generated benign cystic teratomas containing mature tissues in all three germ layers (Figures 2B). Immunohistochemical staining for the neural marker S-100 and muscle actin, and PAS to identify the ectodermal, mesodermal, and endodermal lineages, respectively, confirmed the differentiation of all three germ layers.
Effects of phthalate esters. We next examined the stability of stemness genes, the
differentiation potency and apoptosis in bovine testicular cells and its iPSCs from the same origin by exposing the cells to the EDCs DBP, BBP and DEHP. At low concetrations (10-7 M to 10-6 M), these three phthalates esters did not affect the pluripotency or the expression of stemness genes including OCT4, SOX2, and NANOG in the bovine iPSCs established here (data not shown). However, the phthalates induced significant cytotoxicity in iPSCs compared with the original testicular cells even at low concentration 10–6 M to 10–8 M. Phthalates induced significantly greater necrosis activity in testicular cells compared with iPSCs (4-7-fold ; see Supplemental Material, Figure S1A, B). Phthalate esters induced greater apoptosis activity in iPSCs than in testicular cells (3-6-fold; Figure 3A). DEHP had a
greater effect than DBP or BBP on apoptosis in iPSCs (19-24% vs 15-17%). We explored further this stringer effect of DEHP on apoptosis in iPSCs. Exposure to DEHP caused greater apoptotic activity, as determined by the number of annexin V-positive cells, and caspase 3 activity than did exposure to BDP or BBP (4.1-8.2-fold; Figure 3B and C).
Regulation of AR, p21, and apoptosis. We next compared the effects of phthalate
derivatives on expression of AR because the iPSCs were derived from testicular cells. Previous studies have reported that the AR plays a role in regulating apoptosis in prostate cancer (Balk and Knudson, 2008; Lin et al. 2006). AR-mediated apoptosis is inhibited by increased expression of p21Cip1 (cycling-dependent kinase inhibitor 1) and by phosphorylation of the AR induced by Akt or motigen-activated protein kinase (Garay et al. 2012; Lin et al. 2001). We designed the bovine-specific primers to detect the mRNA of bovine AR to avoid the noise caused by contamination by the feeder of MEFs. BBP and DBP increased the expression of AR 2.9- 3.5-fold, but DEHP decreased AR expression significantly to 38% of the control of value with DMSO (Figure 4A, and see also Supplemental Material, Figure S2A). Expression of the apoptosis-related gene Bax gene was also increased 4-fold, but expression of the antiapoptotic gene Bcl-2 was reduced to 30% of the control value in iPSCs incubated with DEHP. The expression p21Cip1 increased by 12-fold in iPSCs treated with DEHP, but increased only moderately in iPSCs treated with DBP or BBP (3.5- and 4.5-fold, respectively). No significant increase in AKT1 or AKT2
expression was observed (Figure 4A).
the Wnt/β-catenin pathway (Wang et al. 2004). We next examined the expressions of the Wnt pathway by exposing iPSCs to each phthalates derivative (Figure 4, see also Supplemental Material, Figure S2A). BBP decreased the expression of WNT5a significantly (11-fold) and WNT3a moderately (2.4-fold). By contrast, DBP increased the expressions of every WNT including WNT3a (8-fold), WNT1 (4.5-fold), WNT5a (4.4-fold), WNT5b (2.7-fold), and WNT7a (4.2-fold). DEHP increased the expression of only WNT3a (4.7-fold). FZD1 and FDZ7 were induced significantly by DBP (7-fold and 2.2-fold, respectively), and BBP increased the expression of FZD1, 3, and 7 (2.5~2.7-fold). By contrast, DEHP inhibited the expression of FZD7 significantly (Figure 4B). BBP increased the expression of Disheveled (DVL) 1, 2, and 3 by 4.0-4.7-fold. DBP increased the expression of DVL1 and 2 by 6.3-7.5-fold). However, DEHP did not alter the expression of any Dvls. Both DBP and BBP increased the Wnt/β-catenin canonical signaling by inactivation of GSK3β (25-30%) and by activating CTNNB1 (4.3-5.7-fold) and TCF3 (2.5-fold). By contrast, DEHP inactivated FZD and then inhibited the Wnt/β-catenin signal (Figure 4C).
To confirm the link between the different effects on the expression of the AR and apoptosis, we introduced a knockdown vector against AR and exposed the cells to BBP or DBP. The siRNA against AR reduced the expression of AR (Figure 5A and B) and inhibited apoptosis, but the scrambled siRNA had no effect (Figure 5C). We also tested the effects of the siRNA against p21Cip1 on DEHP-induced apoptosis (Figures 5D-F). The siRNA against p21Cip1, but not the scrambled siRNA, attenuated apoptotic activity completely (Figure 5F), Moreover, under the condition of reduced expression of AR caused by exposure to DEHP, forced expression of AR and FZD7 rescued the expression of AR (3-4.5-fold)(Figure 6A and B) and increased AR-luciferase activity (3.5-5.5-fold)(Figure 6B). The control vectors for
AR or FDZ7 had no effect (Figure 6A and B). These results suggest that apoptosis was induced even in the presence of DEHP (Figure 6C).
Discussion
These studies have several important implications, First, the introduction of OCT4 alone was sufficient to reprogram bovine testicular cells to generate iPSCs in the presence of LIF and BMP4, thus replacing the need for expression of SOX2, KLF4, and MYC. Second, EDCs such as DEHP, DBP, and BBP increased necrosis of bovine testicular cells and triggered greater apoptosis in bovine testicular iPSCs, Third, DHEP induced significant apoptosis by downregulating the AR through upregulation of p21Cip1, but BBP and DBP did not have this effect. Fourth, DEHP inactivated the expression of the Frizzled receptor, but BBP and DBP did not. Our study clearly that the expression of the reprogramming factor OCT4 is sufficient to induce pluripotency in bovine testis cells. ESCs are particularly sensitive to the dosage of changes of OCT4; for example, a 50% increase or decrease in the level of Oct4 causes their differentiation into cells expressing markers of the endoderm and mesoderm or trophectoderm, respectively (Niwa et al. 2000). Therefore, Oct4 is a critical factor in nuclear reprogramming and cellular self-renewal. To our knowledge, the generation of bovine iPSCs through the transfection of only OCT4 has not been reported other than in a recent study in which four reprogramming factors were used (Han et al. 2011). It is widely accepted that Oct4 is essential for the identity of the pluripotent stem cells in mammalian embryos (Niwa et al., 2000; Jaenisch and Young 2008; Wering et al. 2008). Contradictory data were also reported to show that OCT4 is not essential for the acquisition and maintenance of pluripotency in the generation of pig iPSCs (Montserrat et al. 2012) or for the self-renewal
of mouse somatic stem cells (Jengner et al. 2007). Therefore, the requirement for OCT4 might be species specific or cell-type specific, depending on the origin of the stem cells. Here, it is evident that OCT4 alone was sufficient to induce pluripotency in bovine testis cells.
The expression of the pluripotency markers, including OCT4, NANOG, SOX2, STAT3, MYC, KLF4, TERT, and DNMT3A, was maintained in the bovine iPSCs. When FGF2 was used instead of both LIF and BMP4, the bovine iPSCs differentiated toward neural progenitor cells (data not shown). This indicates that bovine iPSCs differ from human iPSCs, whose characteristics include a dependence on FGF2/activin signaling and a flat morphology, which reduce the tolerance to single-cell dissociation by trypsin. The morphology of these iPSCs resembled that of mouse ESCs/iPSCs rather than human ESCs/iPSCs. Mouse ESCs and iPSCs express SSEA-1 but not SSEA-4, whereas human ESCs and iPSCs express SSEA-4 but not SSEA-1 (Hanna et al., 2010). Pig iPSCs are also positive for SSEA-4 but not SSEA-1, and exhibit a similar morphology to that of human ESCs/iPSCs (Montserrat et al. 2011; Wu et al., 2009). Interestingly, bovine iPSCs express both SSEA-1 and SSEA-4, and SSEA-1 expression is observed in both equine and bovine embryonic stem-like cells, as we were described (Saito et al. 2002, 2003, 2008). In addition to SSEA-1, we found a strong signal for SSEA-4, which has not been reported previously in bovine ES-like cells (Saito et al. 2003). Therefore, our iPSCs are more similar to naïve iPSCs than to iPSCs derived from fibroblasts (Han et al. 2011). We found that bovine testis cells could be reprogrammed more easily than fibroblasts.
We used these bovine iPSCs to examine the effects of EDCs such as the phthalate derivatives DEHP, BBP, and DBP on the ability of bovine testicular iPSCs to maintain
pluripotency in vivo and in vitro. Low dose of DEHP, DBP, and BBP delayed cell proliferation of bovine iPSCs to 10-15 % of the level in cells not exposed to phthalates (data not shown) and induced necrosis and apoptosis. Phthalate derivatives such as BBP, DBP, and DEHP increased necrosis activity in bovine testicular cells but induced apoptosis in bovine iPSCs. DEHP had a greater effect on apoptosis in iPSCs compared with BBP and DBP. Therefore, we focused on the signaling cascades of AR-mediated apoptosis because the iPSCs were derived from testicular cells. Exposure to DEHP decreased the expression of AR to 30% in iPSCs, but BBP and DBP increased expression of AR (2.8-3.4-fold)(Figure 4A). DEHP increased apoptosis of bovine iPSCs by 3-5-fold (Figure 3). This difference in
the expression of AR is critical to AR-mediated apoptosis.
We also found that AR-mediated apoptosis was regulated by p21Cip1. This signaling was also mediated by inactivation of the Wnt receptor Frizzled after exposure to DEHP but not to BBP or DBP, indicating that AR-mediated apoptosis involves activation of Wnt/β-catenin signaling. Therefore, we speculate that phthalates downregulate the expression of AR in bovine iPSCs, which can lead to cell toxicity including apoptosis, via; (i) activation of p21Cip1, and (ii) inactivation of the Wnt receptor Frizzled. To clarify the signal cascade involved in AR-mediated apoptosis, we introduced an siRNA against AR or p21Cip1 and forced the expression of FZD7 which rescued AR-mediated apoptosis in response to DEHP, but not in response to BBP or DBP. Our results show that DEHP and BBP or DBP exert different effects on the signaling process involved in AR-mediated apoptosis. We cannot rule out other factors that can affect the regulation of AR such as TGFβ-Smad 3 and IL6-Stat3, whose levels were also increased by these phthalate esters (see Supplemental
Conclusions
We here generated iPSCs from bovine testicular cells using electroporation of OCT4. The established iPSCs were exposed to phthalate esters including DBP, BBP and DEHP, and AR-mediated apoptosis and its signaling were measured. DEHP repressed the expression of AR in iPSCs, which committed them to apoptosis through increased expression of p21Cip1, but DBP and BBP had no effect. DHEP inactivated the Frizzled receptor, which are crucial to AR-mediated apoptosis. Testicular iPSCs are useful for screening of EDCs and for
investigating the maintenance of stemness and pluripotency.
REFERENCES
Behboodi E, Bondarareva A, Begin I, Rao K, Pierson JT, Wylie C, et al. 2011. Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage
embryos. Mol Repod Dev 78: 201-211.
Casals-Casas D, Desvergne B. 2011. Endocrine disruptors: from endocrine to metabolic disruptor. Ann. Rev Physol 73: 135-162.
Chen HS, Chiang PH, Wang YC, Kao MC, Shieh TH, Tsai CF, et al. 2012. Benzyl butyl phthalate induces necrosis by AhR mediation of CYP1B1 expression in human granulosa cells. Reprod Toxicol 33:67-75.
Colborn T, vom Saal FS, Soto AM. 1993. Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ Health Perspect 101: 378-384.
Ford CE, Pollock DL, Gustavsson I. 1980. Proceedings of the first international conference for the standardization of banded karyotypes of domestic animals. Hereditas 92:
Garay JP, Karakas B, Abukhdeir AM, Cosgrove DP, Gustin JP, Higgins MJ, et al. 2012. The growth response to androgen receptor signaling in ERα-negative human breast cells is dependent on p21 and mediated by MAPK activation. Breast Cancer Res 14: R27. Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH, et al. (2006). Pluripotency of
spermatogonial stem cells from adult mouse testis. Nature 440: 1199-1203.
Han X, Han J, Ding F, Cao S, Lim SS, Dai Y, et al (2011). Generation of induced pluripotent stem cells from bovine embryonic fibroblast cells. Cell Res 21: 1509-1512. Hanna JH, Saha K, Jaenisch R. (2010). Pluripotency and cellular reprogramming: facts,
hypothesis, unresolved issues. Cell 143: 508-525.
Jaenisch R, Young R. 2008. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132: 567-582.
Jin C, Li H, Murata T, Sun K, Horikoshi M, Chiu R, et al. 2001. JDP2. A repressor of AP-1, recruits a histone deacetylase 3 complex to inhibit the retinoic acid-inducible differentiation of F9 cells. Mol Cell Biol 22: 4815-4826.
Jengner CJ, Camargo FD, Hochedlinger K, Welstead GG, Zaidi S, Gokhale S, et al. 2007. Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell 1: 403-415.
Jobling S, Reynolds T, White R, Parker MG, Sumpter JP. 1995. V ariety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ Health Perspect 103: 582-587.
Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, et al. 2004. Generation of pluripotent stem cells from neonatal mouse testis. Cell 119: 1001-1012. Kim JB, Sebastiano V, Wu G, Arauzo-Bravo MJ, Sasse P, Gentile L, et al. 2009. Oct4
induced pluripotency in adult neural stem cells. Cell 136: 411-419.
Lin HK, Yeh S, Kang HY, Chang C. 2001. Akt suppresses androgen-induced apoptosis by
98:7200-Lin Y, Kokontis J, Tang F, Godfrey B, Liao S, Lin A, et al. 2006. Androgen and its receptor promote Bax-mediated apoptosis. Mol Cell Biol 26: 1908-1916.
Lyche JL, Gutleb AC, Bergman A, Eriksen GS, Murk AJ, Ropstad E, et al. 2009.
Reproductive and developmental toxicity of phthalates. J Toxicol Environ Health B Crit Rev 12: 225-249.
Matsui Y, Zsebo K, Hogan BL. 1992. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70: 841-847.
Martino-Andrade AJ, Chahoud I. 2010. Reproductive toxicity of phthalate esters. Mol Nutr Food Res 54: 148-157.
Montserrat N, Bahima EG, Batlle L, Häfner S, Miguel A, Rodrigues C, et al. 2011. Generation of pig iPS cells: a model for cell therapy. J Cardiovasc Transl Res 14: 121-130.
Montserrat G, de Onate L, Garreta E, Gonzales F, Adamo A, Euizabal C, et al. 2011. Generation of feeder free pig induced pluripotent stem, cells without Pou5f1. Cell Transplantation 21: 815-825.
Nakade K, Pan J, Yoshiki A, Ugai H, Kimura M, Liu B, et al. 2006. JDP2 suppresses adipocyte differentiation by regulating histone acetylatyion. Cell Death and Differ 14: 1398-1405.
Niwa H, Miyazaki J, Smith AG. 2000. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24: 472-476. Saito S, Ugai H, Sawai K, Yamamoto Y, Minamihashi A, Kurosawa K, et al. 2002. Isolation
of embryonic stem-like cells from equine blastocysts and their differentiation in vitro. FEBS Lett 531: 389-396.
Saito S, Sawai K, Ugai H, Moriyasu S, Minamihashi A, Yamamoto Y, et al. 2003. Generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells. Biochem Biophys Res Commun 309: 104-113.
maintenance and induction of the differentiation in vitro of equine embryonic stem cells. In K. Turksen (ed), Embryonic Stem Cell Protocols (pp. 59-79). Totowa, New Jersey; Humana Press.
Saito S, Sawai K, Murayama Y, Fukuda K, Yokoyama K. 2008. Nuclear transfer to study the nuclear reprogramming of human stem cells. Methods Mol Biol 438: 1651-1669. Saito S, Lin Y-C, Murayama Y, Hashimoto K, Yokoyama K. 2013. Human amnion-derived
cells as a reliable source of stem cells. Curr Mol Med In press.
Schrantz N, da Silva Correia J, Fowler B, Ge Q, Sun Z, Bokoch GM. 2004. Mechanism of
p21-activated kinase 6-mediated inhibition of androgen receptor signaling. J Biol Chem 279: 1922-1931.
Seandel M, James D, Shmelkov SV, Falciatori I, Kim J, Chavara S, et al. 2007. Generation of functional multipotent adult stem cells from GPR 125+ germline progenitors. Nature
449: 346-350.
Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663-676.
Tang F, Tang G, Xiang J, Dai Q, Rosner MR, Lin A. 2002. The absence of
mediated inhibition of c-Jun N-terminal kinase activation contributes to tumor necrosis
factor alpha-induced apoptosis. Mol Cell Biol 22: 8571-8579.
Wang G, Wang J, Sadar MD. 2008. Crosstalk between the androgen receptor and catenin in castrate-resistant prostate cancer. Cancer Res 68: 9918-2997.
Wernig M., Zhao JP, Pruszak J, Hedlund E, Fu D, Soldner F, et al. 2008. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci USA 105: 5856-5861. Wu Z, Chen J, Ren J, Bao L, Liao J, Cui C, et al. 2009. Generation of pig induced
Figure legends
Figure 1. Generation of iPSCs from bovine testicular cells. (A) Typical morphology of bovine iPSC colonies generated with OCT4 on day 25 after electroporation (Magnification ×100; upper left panel). Alkaline phosphatase staining of bovine iPSCs (lower left panel), and immunocytochemical analysis of pluripotency and surface markers (Oct4, Nanog, Sox2, SSEA-1, and SSEA-4 indicated in green) in bovine iPSCs. Nuclei were stained with DAPI (indicated in blue). (Magnification × 200). (B) Bovine iPSC gene expression. RT-PCR analysis of transcripts of “stemness” genes (OCT4, SOX2, MYC, KLF4, STAT3, SUZ12, DNMT1, and MEF2A) in bovine testis cells and iPSCs. (C) G-banding karyotype analysis of the bovine iPSC cell line. Bovine iPSCs showed the normal distribution of 60 chromosomes,
including the XY sex chromosomes, at passage 15.
Figure 2. Pluripotency of bovine iPSCs. (A) In vitro differentiation of and marker
expression by bovine iPSC-derived ectodermal, mesodermal, and endodermal precursor cells. Immunostaining with antibody directed against the astrocyte-specific antigen GFAP (ectodermal differentiation), neuron-specific antigen Tuj1 (ectodermal differentiation), cardiomyocyte-specific antigen NKX 2.5 (mesodermal differentiation), or α-fetoprotein (endodermal differentiation). (B) Teratoma formation 6-8 weeks after the transplantation of bovine iPSCs into SCID mice. The teratomas were sectioned and stained with hematoxylin and eosin. Immunohistochemical staining was performed with antibodies specific for S-100 (nerve bundles), muscle-specific-actin (mesenchymal cells and myofibroblasts), or PAS (secretory cells). (Magnification x400). In panel a, the red and yellow arrows indicate blood vessels and nerve bundles, respectively. In panel b, the red arrows indicate glands. Staining for S-100 indicates nerve bundles (panel c) and staining for muscle-specific actin indicates mesenchymal cells and myofibroblasts (panel d). PAS staining indicates secretory cells
Figure 3. Apoptosis induced by phthalate derivatives in bovine iPSCs. (A) Apoptotic
activity was measured by propidium iodide staining and (B) FITC-labeled annexin V staining followed by flow cytometry to identify the apoptotic cells, as described in Materials and Methods. (C) Caspase-3 activity was measured in testicular cells and iPSCs. DEHP, BBP or DBP was added at a dose of 10-6 to 10-8 M and staurosporin were added to testicular cells or iPSCs at a dose of 10-7 M for 48 h, and their apoptotic activity was measured. The
data are presented as means ± SD. *P <0.05, **P <0.01; ***P <0.001. Student’s t-test.
Figure 4. Relative expression of genes of AR-mediated apoptosis and Wnt signaling.
Real-time PCR was performed using the bovine specific primers listed in Supplemental Material, Table S2. (A) Relative expression of AR, p21Cip1, AKT1, AKT2, Bax, and Bcl-2 in iPSCs. (B) Relative expression of GSK3β, CTNNB1, and APC in iPSCs. (C) Relative expression of the Wnt ligands Frizzled and Disheveled in iPSCs. Lane 1, control DMSO (0.001%); lane 2, 10-6 M BBP; lane 3, 10-6 M DBP; lane 4, 10-6 M DHEP. The values are expressed as mean ±
SEM. n ≥ 3, *P <0.05, **P <0.01; ***P <0.001.
Figure 5. Effects of the knockdown expression vector of AR siRNA or p21Cip1 siRNA on gene expression and apoptosis. Bovine iPSCs were treated with AR siRNA (A) or p21Cip1 siRNA (C), or their scrambled nonspecific siRNAs, and DMSO for 12 h during exposure to each phthalate revivative. The expression levels were measured after another 12 h incubation in triplicate by qRT-PCR and were corrected for GAPDH RNA level. (B, D) Apoptotic cells were quantified by staining with annexin V as described in Materials and Methods. The value measured with DMSO was defined as 1. Lane 1, control DMSO (0.001%); lane 2, 10-6 M BBP; lane 3, 10-6 M DBP; lane 4, 10-6 M DHEP. The values are expressed as mean ± SEM. n ≥3; *P <0.05, **P <0.01; ***P <0.001.
Figure 6. Effects of forced expression of AR and Frizzled FZD7 on gene expression and
apoptosis in iPSCs. (A) Four hundred nanograms of IRESneo-AR or pGK-CAS-FZD7 and each control vector were introduced into bovine iPSCs, the cells were cultured for 24 h, then 10-6 M DEHP was added and the cells were cultured for another 24 h. The relative gene expression of AR was quantitated by qRT-PCR as described in Materials and Methods. (B) Effect of DNA concentration of IRESneo-AR and pPGK-CAS-FZD7 on AR promoter-luciferase activity (100-400 ng) as described in Materials and Methods. (C) Effect of AR and FZD7 expression on apoptosis. Apoptotic cells were quantified as described in Materials and Methods. The value measured with DMSO was defined as 1. The value are expressed as mean ± SEM, n ≥3, *P <0.05, **P <0.01; ***P <0.001. (D) Schematic model of
