4-Bromodiphenyl ether delays pubertal Leydig cell development in rats

Accepted Manuscript 4-Bromodiphenyl ether delays pubertal Leydig cell development in rats Xianwu Chen, Yaoyao Dong, Erpo Tian, Lubin Xie, Guimin Wang, Xiaoheng Li, Xiuxiu Chen, Yong Chen, Yao Lv, Chaobo Ni, Yinghui Fang, Ying Zhong, Ren-Shan Ge PII:

S0045-6535(18)31476-0

DOI:

10.1016/j.chemosphere.2018.08.008

Reference:

CHEM 21926

To appear in:

ECSN

Received Date: 23 June 2018 Revised Date:

31 July 2018

Accepted Date: 2 August 2018

Please cite this article as: Chen, X., Dong, Y., Tian, E., Xie, L., Wang, G., Li, X., Chen, X., Chen, Y., Lv, Y., Ni, C., Fang, Y., Zhong, Y., Ge, R.-S., 4-Bromodiphenyl ether delays pubertal Leydig cell development in rats, Chemosphere (2018), doi: 10.1016/j.chemosphere.2018.08.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT 1

4-Bromodiphenyl ether delays pubertal Leydig cell development in rats

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Xianwu Chen

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Xiaoheng Li c, Xiuxiu Chen a, Yong Chen c, Yao Lv b, Chaobo Ni c, Yinghui Fang b,

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Ying Zhong d,**, Ren-shan Ge a,c,*

, Yaoyao Dong

1, b

, Erpo Tian d, Lubin Xie a, Guimin Wang c,

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1, a

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a

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of Anesthesiology, the Second Affiliated Hospital and Yuying Children’s Hospital,

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Wenzhou Medical University, Wenzhou, Zhejiang 325027, China

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d

Jinjiang Maternity and Child Health Hospital, Chengdu, Sichuan 610000, China

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These authors contributed equally to this work.

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Department of Obstetrics and Gynecology, b Department of Pharmacy, c Department

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Running title: BDE-3 blocks Leydig cell differentiation.

*Correspondence: Ren-Shan Ge, M.D., Department of Anesthesiology, The Second

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Affiliated Hospital and the Yuying Children's Hospital of Wenzhou Medical

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University, Wenzhou, Zhejiang 325027, China, Tel: 001-86-1390-577-7099,

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[email protected] (RS Ge)

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**Co-corresponding author. Ying Zhong, Jinjiang Maternity and Child Health

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Hospital Chengdu, Sichuan 610000, China, Email: [email protected]

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Abstract Polybrominated diphenyl ethers are a class of brominated flame retardants that

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are potential endocrine disruptors. 4-Bromodiphenyl ether (BDE-3) is the most

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abundant photodegradation product of higher polybrominated diphenyl ethers.

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However, whether BDE-3 affects Leydig cell development during puberty is still

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unknown. The objective of this study was to explore effects of BDE-3 on the pubertal

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development of rat Leydig cells. Male Sprague Dawley rats (35 days of age) were

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gavaged daily with BDE-3 (0, 50, 100, and 200 mg/kg body weight/day) for 21 days.

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BDE-3 decreased serum testosterone levels (1.099 ± 0.412 ng/ml at a dose of 200

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mg/kg BDE-3 when compared to the control level (2.402 ± 0.184 ng/ml, mean ± S.E.).

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BDE-3 decreased Leydig cell size and cytoplasmic size at a dose of 200 mg/kg,

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decreased Lhcgr, Star, Dhh, and Sox9 mRNA levels at ≥ 100 mg/kg and Scarb1,

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Cyp11a1, Hsd17b3, and Fshr at 200 mg/kg. BED-3 also decreased the

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phosphorylation of AKT1, AKT2, ERK1/2, and AMPK at 100 or 200 mg/kg. BDE-3

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in vitro induced ROS generation, inhibited androgen production, down-regulated

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Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Srd5a1, and Akr1c14 expression in immature

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Leydig cells after 24-h treatment. In conclusion, the current study indicates that

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BDE-3 disrupts Leydig cell development via suppressing AKT, ERK1/2, and AMPK

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phosphorylation and inducing ROS generation.

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Key words: 4-Bromodiphenyl ether; Leydig cell; Sertoli cell; Leydig cell

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development; testosterone.

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Introduction Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame

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retardants used in many fields, including electronics, furniture, and building materials.

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PBDEs are officially added to a list of persistent organic pollutants by the United

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Nations Environment Programs in 2009 (Wang et al., 2015; Wei et al., 2018). In the

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environment, as the photo-degradation product of higher brominated PBDEs,

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4-bromodiphenyl ether (BDE-3) is the most fundamental mono-brominated diphenyl

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ether (Raff and Hites, 2007; Wei et al., 2018).

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Some early studies showed that PBDEs possess the endocrine disrupting activity

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and

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carcinogenicity (Yogui and Sericano, 2009), thyroid toxicity (Gascon et al., 2011;

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Vuong et al., 2018), and liver toxicity (Linares et al., 2015). Besides, several studies

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demonstrated that BDE-3 could cause the reproductive toxicity, by reducing sperm

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mobility and suppressing spermatogenesis (Akutsu et al., 2008; Abdelouahab et al.,

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2011; You et al., 2017). The spermatogenesis requires high level of testicular

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testosterone, which is produced by Leydig cells in males. However, whether BDE-3

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affects Leydig cell development and steroidogenesis is largely unclear.

et

al.,

2016),

teratogenicity,

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have developmental

In the current study, we evaluated the effects of BDE-3 on rat Leydig cell

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development during the pubertal period. In rats, the postnatal development of Leydig

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cells can be conceptually divided into four phases: stem, progenitor, immature, and

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adult Leydig cells (Ye et al., 2017). Stem Leydig cells exist in the interstitium of the

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testis during the whole lifespan to maintain the homeostasis of Leydig cell number. 3

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lineage via sequential proliferation and differentiation. In the Leydig cell lineage,

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progenitor Leydig cells are first observable until postnatal day 14 and they become

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abundant on postnatal day 21. They transform into immature Leydig cells at postnatal

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day 28, and finally finish the maturation into adult Leydig cells 56 days postpartum

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(Ye et al., 2017). When compared to the final phase (adult Leydig cells), immature

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Leydig cells express all the necessary steroidogenic proteins to synthesize testosterone,

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such as luteinizing hormone (LH) receptor (LHCGR)(Shan and Hardy, 1992),

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extracellular cholesterol-transporting protein (scavenger receptor class B member 1,

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SCARB1) and intracellular cholesterol-transporting protein (steroidogenic acute

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regulatory protein, STAR), cytochrome P450 cholesterol side-chain cleavage enzyme

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(CYP11A1),

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17α-hydroxylase/17,20-lyase (CYP17A1), and 17β-hydroxysteroid dehydrogenase 3

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(HSD17B3)(Payne et al., 1997; Ye et al., 2011; Li et al., 2016). Immature Leydig cells

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also possess high levels of testosterone-metabolizing enzymes, steroid 5α-reductase 1

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(SRD5A1)(Ge and Hardy, 1998a), which turns testosterone into dihydrotestosterone,

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and

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dihydrotestosterone into 5α-androstanediol (DIOL) (Ge and Hardy, 1998b). Therefore,

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rat immature Leydig cells mainly secrete DIOL (Ge and Hardy, 1998b). When

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immature Leydig cells differentiate into adult Leydig cells, SRD5A1 is silenced and

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these mature Leydig cells primarily produce testosterone (Ge and Hardy, 1998b).

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Interestingly, when progenitor Leydig cells develop into immature Leydig cells,

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dehydrogenase

1

(HSD3B1),

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3β-hydroxysteroid

3α-hydroxysteroid

dehydrogenase

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(AKR1C14),

which

converts

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enzyme, begins expressed and trends higher when Leydig cells mature (Phillips et al.,

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1989b). Therefore, this enzyme is a good biomarker for Leydig cells in immature

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phase and beyond. Leydig cell development not only relies on its intrinsic factors but

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also on Sertoli cell-secreted growth factors, such as dessert hedgehog (DHH) (Ye et

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al., 2017), which is regulated by follicle-stimulating hormone (FSH) after it binds to

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FSH receptor (FSHR) on the surface of Sertoli cells (Makela et al., 2011).

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In the present study, we exposed male pubertal rats to BDE-3 for 21 days and

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treated immature Leydig cells with BDE-3 to investigate its effects on Leydig cell

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development and dissect the underlying mechanisms.

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1. Materials and methods

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2.1 Chemicals and animals

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BDE-3 was purchased from Alfa Aesar (Shanghai, China). Resin (Permount,

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SP15-100) was purchased from ThermoFisher Scientific (Waltham, UK). SYBR

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Green qPCR Kit and BCA Protein Assay Kit were purchased from Takara (Otsu,

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Japan). Trizol Kit was purchased from Invitrogen (Carlsbad, CA). Immulite2000 Total

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Testosterone Kit was purchased from Sinopharm Group Medical Supply Chain

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Services Co (Hangzhou, Zhejiang, China). Radio immunoprecipitation Assay buffer

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was obtained from Bocai Biotechnology (Shanghai, China). [3H] Androstanediol was

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purchased from Dupont-New England Nuclear (Boston, MA). Unlabeled DIOL and

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testosterone were obtained from Steraloids (Newport, RI). All other reagents were

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ACCEPTED MANUSCRIPT obtained from Sigma-Aldrich (St. Louis, MO). Male Sprague Dawley rats (28 days of

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age) were purchased from Shanghai Animal Center (Shanghai, China). The animal

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study was approved by the Institutional Animal Care and Use Committee of Wenzhou

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Medical University and was performed in accordance with the Guide for the Care and

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Use of Laboratory Animals. The list of antibodies is provided in Supplementary Table

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S1.

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2.2 Animal administration

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Twenty-four 28-day-old male Sprague Dawley rats were raised in a 12-h

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dark/light cycle with temperature of 23 ± 2 oC and relative humidity of 45–55%.

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Water and food were provided ad libitum. Animals were adjusted to the new

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environment for 7 days before they were randomly divided into 4 groups: control (0

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mg/kg/day, n = 6), low dose (50 mg/kg/day, n = 6), middle dose (100 mg/kg/day, n = 6)

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and high dose (200 mg/kg/day, n = 6). BDE-3 was dissolved in corn oil and was daily

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gavaged 0, 50, 100, or 200 mg/kg to rats starting on postnatal day 35 for 21 days,

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respectively. Body weight of each rat was recorded every 2 days. Rats were

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euthanized on postnatal day 56 by CO2. Trunk blood was collected and put in a gel

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glass tube and the tube was centrifuged at 1500 × g for 10 min to collect serum

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sample. All serum samples were labeled and stored at -20 oC until hormone

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(testosterone, LH, and FSH) analysis. Furthermore, each pair of testes was separated

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and weighted. One testis of each animal was frozen in the liquid nitrogen and stored at

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-80 oC for subsequent gene and protein expression analysis. The contralateral testis

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ACCEPTED MANUSCRIPT was collected and fixed in Bouin’s solution for subsequent immunohistochemical

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analysis. All studies were approved by the Wenzhou Medical University’s Animal

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Care and Use Committee.

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1.3 ELISA for serum LH and FSH levels

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Serum LH and FSH levels were detected with ELISA kits according to the

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manufacturer’s instruction (Chemicon, CA, USA) as previously described (Wu et al.,

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2017b). In brief, aliquots of sample and assay diluent were added to the pre-coated

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plate. The plate was incubated at room temperature for 2 h. Peroxidase-conjugated

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IgG anti-LH or anti-FSH solution was added into each well and incubated at room

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temperature for 2 h. Finally, substrate buffer was added into each well, and incubated

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in the dark place at room temperature for 30 min. The enzyme reaction was stopped

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by a stop solution. The measurement of LH or FSH level was obtained by a

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microplate reader at 550 nm with correction wavelength at 450 nm.

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2.4 Immunohistochemical staining of the testis

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One testis from each rat was used for immunohistochemical staining as

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previously described (Wu et al., 2017a). Six testes per group were selected and testis

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samples were prepared and embedded in paraffin in a tissue array as previously

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described (Wu et al., 2017a). Tissue-array was dehydrated in ethanol and xylene and

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then embedded in paraffin. Transverse section (6 µm thick) was attached on an

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adhesive glass slide. Immunohistochemical staining process began with an antigen

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retrieval with a heating water bath containing 10 mM (pH 6.0) of citrate buffer for 10

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min, followed by the elimination of endogenous peroxidase with 3% H2O2 in

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methanol for 10 min. Sections were incubated with CYP11A1 (a biomarker of all

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Leydig cells) or HSD11B1 (a biomarker for immature and mature Leydig cells) or

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SOX9

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Diaminobenzidine was selected to illustrate the antibody-antigen complexes,

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positively labeling Leydig cells by brown color in the cell cytosol (CYP11A1 or

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HSD11B1) or labeling Sertoli cells by brown color in the cell nucleus (SOX9). Mayer

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hematoxylin was added to counterstain the sections. The sections were then

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dehydrated in graded concentrations of alcohol and cover-slipped with resin.

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Non-immune rabbit IgG was used as the negative control. The Leydig cell number

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after CYP11A1 and HSD11B1 or the Sertoli cell number after SOX9 staining was

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counted. The density of CYP11A1 or HSD11B1 in the individual Leydig cell or

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SOX9 in the individual Sertoli cell was measured.

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2.5 Counting Leydig and Sertoli cell number in the testis

biomarker

for

Sertoli

cells)

polyclonal

antibody

overnight.

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To count CYP11A1- or HSD11B1-positive Leydig cells or SOX9-positive Sertoli

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cells, a fractionator technique was used as previously described (Mendis-Handagama

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et al., 1989). Each testis was cut in 8 discs, of which two discs were randomly

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selected. Each disc was cut in 4 pieces, of which one piece was randomly selected.

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These pieces of testis were embedded in paraffin in a tissue-array. Paraffin block was

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sectioned in 6 µm thick section. Ten sections were randomly sampled from each testis

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per rat. Sections were used for immunohistochemical staining of CYP11A1 or

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HSD11B1 or SOX9. Total microscopic fields per section were counted. The total

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ACCEPTED MANUSCRIPT number of Leydig/Sertoli cells was calculated by multiplying the number of

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Leydig/Sertoli cells counted in a known fraction of the testis by the inverse of the

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sampling probability.

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2.6 Computer-assisted image analysis of cell size and nuclear size

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Leydig cells were identified by staining HSD11B1 as above. The Leydig cell size,

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nuclear size, and cytoplasmic size were calculated as previously described (Liu et al.,

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2016). Six randomly selected fields in each of three non-adjacent sections per testis

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were captured using a BX53 Olympus microscope (Tokyo, Japan). Cell size and

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nuclear size were estimated using the image analysis software with the main area

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measurement mode (Image-Pro Plus; Media Cybernetics, Silver Spring, MD). More

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than 50 Leydig cells were evaluated in each testis. The cell size and nuclear size were

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calculated as µm3 and cytoplasmic size was calculated by cell size minus nuclear size.

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2.7 Semi-quantitative measurement of CYP11A1 and HSD11B1 and SOX9

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CYP11A1 and HSD11B1 are the proteins of Leydig cells, and SOX9 is the

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protein of Sertoli cells. Immunohistochemical stainings of CYP11A1, HSD11B1, and

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SOX9 were performed as above. Target protein density and adjacent area background

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density were measured using the image analysis software with main density as the

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measurement mode as previously described (Liu et al., 2016). The net density was

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calculated by subtracting the background density value from the density value of the

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target protein. More than 50 Leydig cells were evaluated in each testis and the protein

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density of each sample was averaged.

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2.8 Immature Leydig cell isolation

Male Sprague Dawley rats (28 days of age) were purchased and shipped to the

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laboratory and adjusted for one week. Then, rats were euthanized by CO2. Testes were

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removed and immature Leydig cells were purified as described previously (Ge and

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Hardy, 1998b). In brief, testis was perfused with collagenase via the testicular artery,

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digested with collagenase and DNase for 15 min. The digested cells were filtered

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through 100 µm nylon mesh, and the cell pellets were separated under Percoll gradient.

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The cell fraction with the density of 1.070-1.088 g/ml was collected and washed. Purity

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of immature Leydig cell fraction was measured by a histochemical staining for

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HSD3B1 activity, using 0.4 mM etiocholanolone as the steroid substrate and 0.2 mM

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NAD+ as cofactor (Payne et al., 1980). More than 95% immature Leydig cells were

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intensely stained. Three different preparations were performed.

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2.9 Immature Leydig cell culture

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After isolation, the purified immature Leydig cells were seeded into 24-well

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culture plated with cell density of 0.5 × 106 cells/well. Immature Leydig cells were

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cultured in 0.5 ml DMEM: F12 medium (phenol-free) in the presence of 0.05-50 µM

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BDE-3 (BDE-3 was dissolved in ethanol and ethanol was the control) for 24 h. Media

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were collected for DIOL measurement. Cells were harvested for isolation of total

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RNAs.

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2.10 Serum testosterone assay

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Testosterone Kit according to manufacturer’s instruction (Siemens, Germany) as

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previously described (Guo et al., 2017). The minimal detection limit of testosterone

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was 0.2 ng/ml. The intraassay and interassay coefficients of variation for testosterone

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were within 10%.

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2.11 Medium DIOL assay

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DIOL concentration in the medium was measured with a tritium-based

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radioimmunoassay (RIA) as previously described (Ge and Hardy, 1998b), using the

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commercial RIA kit (Hangzhou Pterosaur Biotech, Hangzhou, China). Intraassay and

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interassay coefficients of variation for DIOL were within 10%.

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2.12 Measurement of intracellular ROS levels

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ROS production was measured with the fluorescence dye 2’,7’-dichlorofluorescin

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diacetate (DCFH-DA) assay kit (Shanghai Qcbio Science & Technologies Co.,

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Shanghai, China) according to the manufacturer's instruction. Briefly, isolated

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immature Leydig cells were plated into the 6-well plate at the density of 5×105 cells

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per well and stabilized for 24 h. Then, cells were incubated with 0, 5, or 50 µM

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BDE-3 for 24 h. Thereafter, cells were collected by digestion with trypsin, after which

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cells were suspended and incubated with 200 µL DCFH-DA for 20 min at 37 °C in

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dark. Phosphate buffered saline (PBS) was used for washing and fluorescence

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intensity was determined by a DTX800 Multimode Detector (Beckman Coulter,

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Fullerton, CA) with excitation of 485 nm and emission of 535 nm to determine ROS

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levels.

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2.13 Annexin V and PI assay for apoptosis Immature Leydig cells were seeded into a 6-well plate in a density of 5 × 105

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cells per well. Cells were incubated with 0, 5, or 50 µM BDE-3 for 24 h. An Annexin

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V-FITC/PI Apoptosis Detection Kit (Nanjing KeyGEN Biotech, Nanjing, China) was

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used to evaluate both infant and terminal cellular apoptosis under the manufacturer’s

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instruction. Leydig cells were collected by digestion with trypsin, washed with cold

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PBS, and resuspended in 200 µL annexin V-binding buffer. Cells were further stained

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with FITC-labeled annexin V and PI. Fluorescence of cells was detected by the Flow

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Cytometer as above.

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2.14 RNA isolation and real-time PCR (RT-qPCR)

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Total RNAs were purified from the testes of rats or immature Leydig cells treated

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with BDE-3 using the Trizol Kit according to the manufacturer’s instruction, and the

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concentration of RNAs was measured by reading OD value at 260 nm. The first

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strand (cDNA) was reverse-transcribed and used as the template for subsequent qPCR

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measurement as previously described (Li et al., 2014). The levels of Leydig cell

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mRNAs (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1,

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Srd5a1, Akr1c14, and Nr5a1) and Sertoli cell mRNAs (Dhh, Sox9, and Fshr) were

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measured using a SYBR Green qPCR Kit. The mRNA name and primer sequences

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were listed in Supplementary Table S2. The qPCR reaction mixture (25 µl) consists of

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10 µl SYBR Green mix, 1 µΜ forward and reverse primers, 400 ng cDNAs, and

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ACCEPTED MANUSCRIPT RNase-free water. The reaction program was set as follows: 95 oC for 5 min, followed

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by 40 cycles of 95 oC for 10 s, and 60 oC for 30 s. The Ct value was calculated and the

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level of a mRNA was calculated against a standard curve as previously described (Li

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et al., 2014). All mRNA levels were adjusted to Rps16 (the internal control).

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2.15 Western blotting

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Western blotting was performed as previously described (Wu et al., 2017a). In

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brief, testis was homogenized in ice and boiled in equal volume of a sample loading

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buffer. Protein sample (50 µg protein each sample) was electrophoresed on 10%

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polyacrylamide gel containing sodium dodecyl sulfate. After electrophoresis, proteins

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were transferred onto a nitrocellulose membrane, and the membrane was incubated

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with 5% nonfat milk for 1 h to block the nonspecific binding. Then, the membrane

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was incubated with primary antibodies against the following antigens: LHCGR,

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SACRB1, STAR, CYP11A1, HSD3B1, HSD11B1, HSD17B3, FSHR, SOX9, DHH

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and ACTB. The membrane was washed and incubated with a 1:5000 dilution of goat

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anti-rabbit antiserum that was conjugated to horseradish peroxidase. Immunoreactive

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bands were visualized by an ECL kit (Amersham, Arlington Heights, IL). The

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intensity of proteins was quantified using Image J software. The Leydig and Sertoli

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cell proteins were adjusted to ACTB, a house-keeping protein.

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2.16 Statistical analysis

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All data are presented as mean and standard errors (S.E.). Statistical significance

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was analyzed using one-way ANOVA followed by ad hoc Turkey’s multiple 13

ACCEPTED MANUSCRIPT comparisons between groups. Statistical analysis was performed using GraphPad

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Prism (version 6, GraphPad Software Inc., San Diego, CA). A P < 0.05 was

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considered statistically significant.

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2. Results

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3.1 General toxicological parameters of BDE-3

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To analyze the general toxicological effects of BDE-3, body, testis, and

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epididymal weights were recorded at the end of BDE-3 treatment (Table 1). BDE-3

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did not affect rat body, testis, and epididymal weights when compared to the control

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(Table 1). There were no change in mortalities and no abnormal activities observed in

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rats of any group.

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3.2 BDE-3 lowers serum testosterone levels in vivo

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We exposed rats to BDE-3 (0, 50, 100, and 200 mg/kg) via gavage from postnatal

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day 35 to 56 (Fig.1A). When compared to control, BDE-3 dose-dependently

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decreased serum testosterone levels with a statistic significance being recorded in 200

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mg/kg BED-3 group (Fig.1B). BDE-3 did not affect serum LH and FSH levels at any

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doses although there was a slight increase of FSH levels at 200 mg/kg (Fig.1C-D).

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This suggests that BDE-3 delays Leydig cell development.

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3.3 BDE-3 does not affect Leydig cell number in vivo

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We identified Leydig cells using CYP11A1 (a general biomarker of all Leydig

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cells in this lineage (Ye et al., 2017) and HSD11B1 (a specific biomarker of Leydig 14

ACCEPTED MANUSCRIPT cells at mature stages) (Phillips et al., 1989a). Leydig cells were shown by the brown

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cytosolic staining. We enumerated CYP11A1- (Supplementary Fig.S1A-C) and

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HSD11B1-positive (Supplementary Fig.S1D-F) Leydig cells in the interstitium of the

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testis. We found that BDE-3 did not alter CYP11A1- or HSD11B1-positive Leydig

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cell number, suggesting that BDE-3 does not affect Leydig cell proliferation. We

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identified Sertoli cells using SOX9, a biomarker of the Sertoli cell, and found that

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BDE-3 had no effect on SOX9-positive Sertoli cell number (Supplementary

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Fig.S1G-I), indicating that BDE-3 does not cause Sertoli cell death.

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3.4 BDE-3 decreases Leydig cell size and its cytoplasmic size

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When the Leydig cell matures, its cell and cytoplasmic size increase (Shan et al.,

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1993). We measured the cell, nuclear, and cytoplasmic sizes of the Leydig cell and

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found that BDE-3 decreased Leydig cell size and its cytoplasmic size without

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affecting its nuclear size at a dose of 200 mg/kg (Fig.2). This indicates that BDE-3

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delays Leydig cell maturation.

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3.5 BDE-3 decreases Leydig and Sertoli cell mRNA levels in vivo

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We measured Leydig cell mRNA (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1,

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Cyp17a1, Hsd17b3, Hsd11b1, Insl3, and Nr5a1) and Sertoli cell mRNA (Fshr, Dhh,

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and Sox9) levels in the testis after BDE-3 treatment. BDE-3 significantly lowered

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Lhcgr, Star, Sox9, and Dhh levels at ≥ 100 mg/kg and Scarb1, Cyp11a1, Hsd3b1,

316

Hsd17b3, and Fshr levels at 200 mg/kg. However, it did not affect Cyp17a1, Hsd11b1,

317

Insl3, and Nr5a1 levels (data not shown)(Fig.3). These results suggest that BDE-3 15

ACCEPTED MANUSCRIPT 318

affects both Leydig and Sertoli cell gene expression.

319

3.6 BDE-3 decreases Leydig and Sertoli cell protein levels in vivo We measured the levels of Leydig cell proteins (LHCGR, SCARB1, STAR,

321

CYP11A1, HSD3B1, and HSD17B3) and Sertoli cell proteins (FSHR, DHH, and

322

SOX9) in the testis after BDE-3 treatment. These protein levels paralleled with their

323

respective mRNA levels (Fig.4).

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We further measured CYP11A1 and HSD11B1 protein levels in the individual

325

Leydig cell as well as SOX9 levels in the individual Sertoli cell using the

326

semi-quantitative immunohistochemical staining in a tissue-array (Fig.5). BDE-3

327

decreased CYP11A1 density at 200 mg/kg while it did not affect the HSD11B1

328

density. BDE-3 also decreased SOX9 density at ≥ 100 mg/kg. The data further support

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the Western blotting result.

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3.7 Effects of BDE-3 on kinase phosphorylation

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Many studies have demonstrated that AKT and ERK1/2-AMPK pathways

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participate in the development of Leydig cells (Manna et al., 2006; Manna et al., 2007;

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Shiraishi and Ascoli, 2007; Ahn et al., 2012; Tartarin et al., 2012; Liu et al., 2017;

334

Taibi et al., 2017). In the current study, we investigated the downstream signals of

335

BDE-3. BDE-3 significantly decreased the ratios of pAKT2/AKT2 and pERK/ERK at

336

≥ 100 mg/kg as well as pAKT1/AKT1 and pAMPK/AMPK at 200 mg/kg. It

337

decreased their phosphorylated protein levels without affecting the levels of their total

338

proteins (Fig.6). These results suggest that ERK1/2-AMPK, AKT1 and AKT2

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pathways are involved in the BDE-3-mediated suppression of Leydig cell

340

development.

341

3.8 BDE-3 inhibits androgen production and increases ROS in vitro In order to investigate whether BDE-3 directly affects Leydig cells, we cultured

343

immature Leydig cells with different concentrations of BDE-3. Immature Leydig cells

344

primarily secreted DIOL (Ge and Hardy, 1998b). As shown in Fig.7A, BDE-3

345

significantly inhibited DIOL production at 5 and 50 µΜ. We measured the apoptotic

346

rate after BDE-3 treatment and found that BDE-3 did not increase the apoptotic rate at

347

the concentrations tested (Fig.7B-C). Since ROS levels are negatively associated with

348

androgen production in Leydig cells (Zhou et al., 2013), we measured ROS levels

349

after BDE-3 treatment. BDE-3 significantly increased ROS levels at 5 and 50 µM

350

when compared to the control (Fig.7D-E). These data suggest that BDE-3 stimulates

351

ROS generation, thus inhibiting androgen production.

352

3.9 BDE-3 decreases Leydig cell steroidogenic enzyme mRNA levels in vitro

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Because the secretion of DIOL in immature Leydig cells relies on the LHCGR

354

receptor signaling (Lhcgr), cholesterol transportation (Scarb1 and Star), androgen

355

biosynthesis (Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3), and androgen metabolism

356

(Srd5a1 and Akr1c14), we measured the effect of BDE-3 on the respective levels of

357

these mRNAs (Fig.8). We found that BDE-3 significantly lowered Hsd3b1 level at

358

0.05 µM, Akr1c14 level at 0.5 µΜ, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, and

359

Hsd17b3 levels at ≥ 50 µM, suggesting that BDE-3 down-regulated the expression of

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androgen biosynthetic and metabolizing enzyme genes at much lower concentration.

361 362

DISCUSSION Previous study has shown that PBDEs disrupted the synthesis of sex hormones as

364

the reproductive toxicants (Abdelouahab et al., 2011). The results of the present study

365

for the first time demonstrated that BDE-3 significantly delayed pubertal Leydig cell

366

development in rats. The following facts support this notion: 1) Exposure to BDE-3

367

from postnatal day 35 for 21 days lowered serum testosterone levels; 2) BDE-3

368

decreased Leydig cell size and cytoplasmic size in vivo; 3) BDE-3 down-regulated

369

Leydig cell specific gene and protein expression both in vivo and in vitro; 4) BDE-3

370

suppressed DIOL formation in immature Leydig cells in vitro. BDE-3 might exert its

371

action via indirect Sertoli cells and/or via direct induction of Leydig cell oxidative

372

stress. We demonstrated herein that the blockade of phosphorylation of AKT1 and

373

AKT2 as well as ERK1/2-AMPK might be associated with BDE-3 mediated

374

suppression of Leydig cell development.

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The current study showed that BDE-3 caused the inhibtion of the androgen

376

production when it was exposed to rats during the puberty. BDE-3 decreased

377

androgen production by several mechanisms: by down-regulating the expression of

378

key Leydig cell genes and proteins such as LHCGR, SCARB1, STAR, HSD3B1, and

379

HSD17B3 (Fig.3 and Fig.4), and by affecting the maturity of the Leydig cell indicated

380

by reduction of Leydig cell size and cytoplasmic size (Fig.2) without changing the

381

Leydig cell number (Supplementary Fig.S1). These effects may not be mediated by

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the pituitary-secreted LH and FSH, since serum LH and FSH levels were not changed

383

(Fig.1). A direct effect of BDE-3 on the development of testis might be involved. The development of Leydig cells depends on both the extrinsic and intrinsic

385

factors. One of extrinsic factors is SOX9 in the Sertoli cell (Ye et al., 2017). Sertoli

386

cell maturation is also controlled by the transcription factor, SOX9 (Rahmoun et al.,

387

2017). Indeed, the null mutation of SOX9 causes sexual reversal in both humans and

388

mice (Koopman, 1999; Barrionuevo et al., 2006). SOX9 controls many Sertoli cell

389

gene expression, including Dhh (Rahmoun et al., 2017). DHH is a critical factor to

390

regulate Leydig cell development. Indeed, Dhh-null mice exhibit deficiency of adult

391

Leydig cells and significant reduction of teststosterone levels (Clark et al., 2000).

392

Interestingly, BDE-3 treatment significantly lowered the levels of both SOX9 and

393

DHH (Fig.4), and low DHH levels could delay the Leydig cell development.

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BDE-3 may also directly act on Leydig cells. Indeed, treatment of BDE-3 on

395

purified immature Leydig cells significantly increased ROS production (Fig.7),

396

down-regulated Leydig cell steroidogensis-related gene expression (Fig.8), and

397

supprssed androgen secretion (Fig.7). Several studies showed that overproduction of

398

ROS can damage testis function (Tseng et al., 2006), induce germ cell apoptosis

399

(Zhang et al., 2013) and inhibit androgen production by Leydig cells (Weissman et al.,

400

2005). Mitochondrion is an important regulatory subcellular component that regulates

401

the steroid biosynthesis and it is sensitive to oxidative stress (Allen et al., 2004; Hales

402

et al., 2005; Li et al., 2017). Oxidative stress can perturb the mitochondrion and

403

inhibit STAR expression (Diemer et al., 2003). In the current study, we found that

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ACCEPTED MANUSCRIPT BDE-3 significantly induced ROS production in immature Leydig cells (Fig.7).

405

Indeed, BDE-3 significantly down-regulated Star expression at 50 µΜ (Fig.8). A

406

similar down-regulation of Star expression was also observed after in vivo 100 and

407

200 mg/kg BDE-3 treatment (Fig.3). However, the concentrations of BDE-3 tested

408

herein were not enough to induce Leydig cell apoptosis but significantly decrease

409

androgen production (Fig.7).

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The development of Leydig cells is mainly regulated by LH via cAMP-PKA

411

dependent signaling pathway. LH stimulates steroidognesis after binding to LHCGR

412

to activiate PKA and regulate the transcription of STAR, CYP11A1 or other

413

steroidogenic enzymes (Miller and Bose, 2011). However, several studies also

414

showed that ERK1/2 and AKT signaling pathways participated in Leydig cell

415

development (Manna et al., 2006; Renlund et al., 2006; Manna et al., 2007; Shiraishi

416

and Ascoli, 2007). Some growth factors such as epidermal growth factor and

417

insulin-like growth factor 1 and LH can regulate ERK1/2 or AKT. Epidermal growth

418

factor binds to its receptor, causing the activation of ERK1/2 in Leydig cells

419

(Hirakawa and Ascoli, 2003; Shiraishi and Ascoli, 2007, 2008) and epidermal growth

420

factor receptor is the partial mediator of the LHCGR-provoked activation of ERK1/2

421

cascade in immature Leydig cells (Hirakawa and Ascoli, 2003; Shiraishi and Ascoli,

422

2007, 2008). Moreover, activation of PKA can phosphorylate ERK1/2 (pERK1/2),

423

which in turn activates steroid hormone biosynthesis via activating STAR, a key

424

protein for cholesterol transportation into the inner membrane of the mitochondrion

425

(Poderoso et al., 2008; Wen et al., 2018). The decreased phosphorylation of ERK1/2

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could be due to the downregulation of LHCGR, thus leading to lower level of STAR. AKT is also a key regulator of Leydig cell development. Three isoforms of AKT

428

in mammals were identifed, AKT1-AKT3. AKT1 is the major isoform in many

429

mammalian tissues and regulates organ development and survival; AKT2 is highly

430

expressed in insulin-responsive tissues and mainly regulates glucose metabolism;

431

AKT3 is expressed primarily in the brain (Hay, 2011). AKT1-null mice exhibit

432

morphological abnormalities in the testis (Chen et al., 2001), while there is no

433

significant decrease in the testis weight in AKT2/AKT3-null mice (Dummler et al.,

434

2006). These data indicate that AKT1 plays a critical role in testicular function. AKT

435

is mainly regulated by insulin-like growth factor 1 (Tai et al., 2009) and kit ligand

436

(Rothschild et al., 2003). Null mutaion of insulin-like growth factor 1 in mice caused

437

the down-regulation of several Leydig cell biomarkers such as Star, Cyp11a1, Hsd3b1,

438

and Cyp17a1, induced Leydig cell hypoplasia, and lowered testosterone synthesis

439

(Baker et al., 1996; Hu et al., 2010). Blockade of kit ligand signaling also led to the

440

reduced testosterone production in Leydig cells (Rothschild et al., 2003). AKT

441

phosphorylation relies on the activation of phosphatidylinositol 3-kinase (Tai et al.,

442

2009; Zhou et al., 2016). Phosphatidylinositol 3-kinase is activated by insulin-like

443

growth factor 1 and kit ligand (Rothschild et al., 2003). Therefore, the reduced

444

phosphorylation of AKT1 and AKT2 could lead to the suppression of AKT signaling,

445

thus resulting in the delay of Leydig cell development. Although the direct interaction

446

between the phosphrylation of AKT and Star expression in Leydig cells has not been

447

studied, an increase of pAKT1 and Star levels in pig ovary after female pigs exposed

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ACCEPTED MANUSCRIPT 448

to heat stress indicates that the phsophrylation of AKT positively regulates Star

449

expression in steroidogenic tissues (Abass et al., 2009). Besides, AMP-activated protein kinase (AMPK) plays an important role in

451

cellular energy homeostasis (Tartarin et al., 2012), especially in cholesterol

452

homeostasis (Kahn et al., 2005; Hardie et al., 2006; Kola et al., 2006). Several studies

453

have shown that AMPK is related to steroidogenic enzyme promoter activities and

454

gene expression by reducing cyclic AMP (Ahn et al., 2012), such as STAR, HSD3B1,

455

CYP17A1 (Tartarin et al., 2012). Although we have not performed a complete

456

characterization of the pathways by which the BDE-3 reduces ERK1/2, or AKT, or

457

AMPK phosphorylation, the data presented herin showed that ERK1/2 and AKT, and

458

AMPK pathways are associated with Leydig cell development (possible mechanisms

459

of BDE-3 in the suppression of Leydig cell development and steroidogenesis is

460

proposed in Fig.9).

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In conclusion, pubertal male rats exposed to BDE-3 developed the delay of

462

puberty with the decreased testosterone production. The effects of BDE-3 on Leydig

463

cell development may be mediated by the indirect Sertoli cell mediate mechanism and

464

by direct suppression of the phosphorylation of ERK1/2, AKT1, AKT2, and AMPK

465

and induction of ROS generation.

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Conflict of interest

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The authors declared that no competing interests exist.

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ACCEPTED MANUSCRIPT 470

Acknowledgements Supported by NSFC (81730042 to R.S.G., 81601264 to X.H.L.), Health &

472

Family Planning Commission of Zhejiang Province (2017KY483 to X.H.L., 11-CX29

473

to R.S.G.), Zhejiang Provincial NSF (LY15H160065 to G.W), and Wenzhou Bureau of

474

Science and Technology (ZS2017009 to R.S.G.).

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Taibi, N., Dupont, J., Bouguermouh, Z., Froment, P., Ramé, C., Anane, A., Amirat, Z.,

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Postnatal exposure of the male mouse to 2,2',3,3',4,4',5,5',6,6'-decabrominated diphenyl ether:

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Figure Legend

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Fig.1. Effects of BDE-3 on serum hormone levels

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Panel A, BDE-3 regimen; Panel B-D, serum testosterone (T), LH, and FSH levels,

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respectively. LC# =Leydig cell (LC) number; SC# =Sertoli cell number. Mean ± S.E.,

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n = 6. * P < 0.05 indicates significant difference when compared to the control (0

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mg/kg).

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Fig.2. Leydig cell, cytoplasmic, and nuclear sizes in rat testis sections after the

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treatment of BDE-3 (0-200 mg/kg)

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Mean ± S.E., n = 6. *P < 0.05, **P < 0.01 indicate significant differences when

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compared to the control (0 mg/kg BDE-3), respectively.

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Fig.3. BDE-3 down-regulates both Leydig and Sertoli cell gene expression

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Leydig cell genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, and Hsd17b3; Sertoli cell

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genes: Fshr, Dhh, and Sox9. Mean ± S.E., n = 6. *P < 0.05, **P < 0.01 indicate

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significant differences when compared to the control (0 mg/kg), respectively.

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Fig.4. Protein levels in the testis in rats with or without BDE-3 treatment

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(A) Western blot gel image (upper pane: Leydig cell proteins; lower panel: Sertoli cell

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proteins); (B) Quantification of protein levels. Mean ± S.E., n = 6. *P < 0.05, **P <

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0.01 indicate significant differences when compared to the control (0 mg/kg), 32

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respectively.

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Fig.5. Semi-quantitative measurement of CYP11A1, HSD11B1, and SOX9

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density in the rat testis sections after BDE-3 treatment

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Panels A and B: CYP11A1 staining; Panels D and E: HSD11B1 staining; Panels G

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and H: SOX9 staining; Panels A, D, and G: control (0 mg/kg); Panels B, E, and H:

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200 mg/kg BDE-3; Panels C, F and I: quantification of CYP11A1, HSD11B1, and

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SOX9 densities, respectively. Black arrowhead points to the CYP11A1-positive

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Leydig cell; black arrow points to the HSD11B1-positive Leydig cells; and * points to

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the SOX9-positive Sertoli cell. Mean ± S.E., n = 6. *P < 0.05, **P < 0.01, ***P <

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0.001 indicate significant differences when compared to the control (0 mg/kg),

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Fig.6. Protein levels of kinases and phosphorylated kinases in rat testis after the

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BDE-3 treatment

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(A) Western blotting gel image; (B) Quantification of protein levels. Mean ± S.E., n =

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6. *P < 0.05, **P < 0.01 indicate significant differences when compared to the control

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(0 mg/kg), respectively.

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Fig.7. Androgen secretion and ROS production and apoptotic rate of immature

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Leydig cells after BDE-3 treatment

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Panel A, DIOL level; Panel B, quantitation of apoptosis; Panel C, apoptosis analysis; 33

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Panel D, quantitation of ROS levels; Panel E, count of ROS. Mean ± S.E., n = 4. *P <

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0.05, *** P < 0.001 indicate significant differences when compared to the control (0

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μM), respectively.

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Fig.8. BDE-3 down-regulates Leydig cell gene expression in immature Leydig

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cells in vitro

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Immature Leydig cell genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1,

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Hsd17b3, Srd5a1, and Akr1c14. Mean ± S.E., n = 4. *P < 0.05, **P < 0.01, ***P <

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0.001 indicate significant differences when compared to the control (0 μ M),

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Fig.9. Illustration of the working mechanisms for BDE-3 in the suppression of

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Leydig cell development and sterodogenesis.

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Cholesterol is required to be transported into Leydig cells by SCARB1, and further by

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STAR into the mitochondrion, where CYP11A1 catalyzes the first reaction to generate

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pregnenolone. Pregnenolone is further converted into testsoterone by a series of

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enzymes (HSD3B1, CYP17A1, and HSD17B3) in the smooth endoplasmic reticulum.

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BDE-3 inhibits the phosphorylaton of AKT1, AKT2, ERK1/2, and AMPK amd

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generates ROS, leading to the dysfunction of Leydig cells.

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Supplementary Fig.S1. Immunohistochemical staining of CYP11A1, HSD11B1,

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and SOX9 in rat testis sections and enumeration of Leydig and Sertoli cell 34

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CYP11A1: A, B, C; HSD11B1: D, E, F; SOX9: G, H, I. (A), (D), and (G): control (0

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mg/kg BDE-3); (B), (E,) and (H): 200 mg/kg BDE-3. Representative images were

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used. Black arrowhead points to the CYP11A1-positive Leydig cell (LC); black arrow

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points to the HSD11B1-positive Leydig cell; and * points to the SOX9-positive

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Sertoli cell (SC). Mean ± S.E., n = 6. No significant difference between two groups

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was observed. Scale bars = 50 µm.

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Parameters 0

5

100

200

Before BDE-3

150.7±24.94

152.0±9.30

146.6±17.20

133.1±22.89

After BDE-3 (PND56)

240.1±24.31

211.1±24.34

234.8±11.42

212.4.0±26.49

2.78±0.52

2.76±0.17

3.13±0.24

2.90±0.22

Testis weight (g) After BDE-3 (PND56) Epididymis weight (g) After BDE-3 (PND56)

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Body weight (g)

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0.66±0.14 0.58±0.11 0.60±0.07 0.53±0.11 PND = postnatal day. Mean ± S.E., n = 6. No significant difference between any group.

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ACCEPTED MANUSCRIPT ► 4-Bromodiphenyl ether delays pubertal Leydig cell development. ► 4-Bromodiphenyl ether down-regulates Leydig cell gene expression. ► 4-Bromodiphenyl ether blocks Leydig cell some kinase phosphorylation.

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► 4-Bromodiphenyl ether induces Leydig cell ROS generation.