Danio rerio

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Ecotoxicology and Human Environmental Health

Dydrogesterone causes male bias and accelerates sperm maturation in zebrafish (Danio rerio) Wen-Jun Shi, Yu-Xia Jiang, Guo-Yong Huang, Jian-Liang Zhao, JinNa Zhang, You-Sheng Liu, Lingtian Xie, and Guang-Guo Ying Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02556 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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Dydrogesterone causes male bias and accelerates sperm maturation in zebrafish (Danio rerio)

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Wen-Jun Shi†,§, Yu-Xia Jiang†,§, Guo-Yong Huang†, Jian-Liang Zhao†, Jin-Na Zhang†,§, You-Sheng

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Liu†, Ling-Tian Xie†, Guang-Guo Ying†, *

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†

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Chemistry, South China Normal University, Guangzhou 510006, China

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§

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Pollution and Control, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences,

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The Environmental Research Institute, MOE Key Laboratory of Environmental Theoretical

State Key Laboratory of Organic Geochemistry, CAS Research Centre of PRD Environmental

Guangzhou 510640, China

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*Corresponding author (GG Ying)

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Tel.: +86 20 39310796; Fax: +86 20 85290200.

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E-mail address: [email protected]; [email protected]

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ABSTRACT: Synthetic progestins are widely used in human and veterinary medicine. They can

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enter aquatic environments mainly via wastewater discharge and agricultural runoff, thus affecting

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fish populations in receiving waters. Here we investigated the chronic effects of dydrogesterone

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(DDG) on zebrafish from 21 days post fertilization (dpf) to 140 dpf at 3.39, 33.1 and 329 ng L-1.

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The results showed that the male ratio increased with exposure concentration, and after 120 d of

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exposure to 329 ng L-1 98% of the fish were males. The DDG exposure during sex differentiation

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significantly increased the transcription of dmrt1 (1.83 fold) and apoptosis related genes, but

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suppressed the transcription of cyp19a1a (3.16 fold). Histological analysis showed that the exposure

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to DDG at 329 ng L-1caused 61.5% of mature spermatocytes in males, while the exposure to DDG

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at 33.1 ng L-1 resulted in 14.7% of atretic follicles in females. Microarray analysis identified

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spermatogenesis related gene ontology (endothelial barrier and immune response) in the testes at all

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concentrations. Genes from phagosome, lysosome and sphingolipid metabolism pathways were

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enriched and could be responsible for sperm maturation. The findings from this study demonstrate

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that DDG in the aquatic environment can cause male bias and accelerate sperm maturation in

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zebrafish, resulting in potential high ecological risks to fish populations.

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INTRODUCTION Dydrogesterone (DDG) is one of the commonly used progestins in contraception and therapy

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of menstrual disorders and endometriosis in human and animals. 1 The consumption of DDG was

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approximately 34 kg in Switzerland in 2010 and 209 kg in UK in 2006. 2,3 In France, the

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consumption of DDG reached 744.70 kg in 2004. 4 Though the consumption of DDG is not clear in

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China, DDG has been detected in the effluents of wastewater treatment plants (WWTPs) and

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receiving waters. 5 The measured concentrations of DDG were up to 35.1 ng L-1 in WWTP effluents,

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and 9.6 ng L-1 in river water in China. 5 Furthermore, some extreme values (up to 2790 ng L-1) for

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DDG have been reported in the flush water of swine farms. 5 The lowest observed effect

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concentration (LOEC) for DDG on gene expressions in zebrafish embryos has been reported to be

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as low as 4.8 ng L-1, 4 which is below the reported levels in surface water (9.6 ng L-1) and in

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effluents of WWTP (35 ng L-1). 5 This clearly suggests that DDG may pose a risk for fish at

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environmentally realistic concentrations. DDG is relatively easy to be degraded in the exposure

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medium. 4 In addition, the half-life of DDG is estimated to be approximately 5 - 7 hr in humans. 6

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In general, DDG is purposed to resemble progesterone mainly in its progestogenic activity, but

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less in the androgenic, anti-androgenic and glucocorticoid activities. 4 An in vivo study showed that

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DDG increased the frequency of mature spermatocytes in zebrafish, implying DDG might have

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potential androgenic activity. 4 An in vitro study using the GripTiteTM 293 cell line derived from

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human embryonic kidney cell (HEK 293) demonstrated a negligible androgenic activity of DDG by

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GeneBLAzer® Nuclear Receptor Cell-based assays. 1 Previous studies showed some androgenic

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progestins such as norgestrel (NGT), and levonorgestrel (LNG) can cause skewed sex ratio in

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zebrafish. 7,8 LNG and NGT caused male bias in zebrafish by affecting transcription of cytochrome

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P450, family 19, subfamily A, polypeptide 1a (cyp19a1a) and double sex/mab-3 related 3


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transcription factor 1 (dmrt1). 7,8 Genes such as dmrt1, forkhead transcription factor gene L2 (foxl2)

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and cyp19a1a have been demonstrated to be associated with the process of sex differentiation in

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zebrafish. 9 In addition, the oocyte apoptosis is the mechanism of gonadal differentiation in

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zebrafish. 10 For instance, the synthetic estrogen 17α-ethinylestradiol (EE2) disturbs the gonadal

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differentiation via affecting apoptosis pathway. 11 However, the effects of DDG on the sex

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differentiation and sex determination in fish have yet to be determined.

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Previous studies showed that progestins can affect the gonad development in fish. 4,7-8,12 These

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studies have addressed the acute and chronic effects of progestins on the frequency of gonadal

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maturity stages. 8,12 However, the mechanism of histological alterations in gonads after exposure to

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progestins is still not clear. Compared with the acute exposure, chronic exposure at environmentally

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realistic concentrations from fish larval stages to sexual maturity will provide better understanding

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of the effects of progestins like DDG in fish.

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The aim of this study was to investigate the chronic effects of DDG on gonad and brain in

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zebrafish. To achieve our goal, the transcriptional expression of genes related to gonad

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differentiation and apoptosis was examined in the zebrafish larvae at 35 dpf. In addition, microarray

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analysis was conducted and histology of the gonads was evaluated in the sexually mature zebrafish

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(140 dpf). The study can help us better understand the mechanisms for the effects of DDG on sex

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differentiation and gonadal development.

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MATERIALS AND METHODS Chemical. Dydrogesterone (DDG, CAS number: 152–62–5; purity: 98%) was obtained from

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US Pharmacopeia (Rockville, MD). The stock solution of DDG was prepared in dimethyl sulfoxide

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(DMSO), and then stored at -20 °C in the dark. 4



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Test Organisms and Exposure Experiment. Zebrafish were maintained in the Aquatic

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Toxicology Laboratory of South China Normal University. Zebrafish embryos were obtained from

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spawning adults placed in groups of 6 males and 3 females. Normally developed embryos were

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selected for the exposure. The embryos were collected and transferred into petri dishes with 50 mL

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charcoal-filtered dechlorinated tap water, and kept at 26 ± 1 °C with a photoperiod of 14 h: 10 h

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(light: dark). The zebrafish larvae were fed homogenate of brine shrimp twice daily from 5 dpf until

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18 dpf. Larvae were fed with newly hatched brine shrimp after 18 dpf (Shandong, China).

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The experimental set-up consisted of one solvent control (SC, 0.001% (v/v) of DMSO) and

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three DDG groups: 5 (L treatment), 50 (M treatment), and 500 ng L−1 (H treatment) with the same

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DMSO concentration. Each treatment had four replicates. At 21 dpf, 25 larvae per replicate were

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randomly selected and exposed to DDG in 5 L glass aquaria with 3 L of exposure media. The

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exposure media were renewed daily. Dead and malformed fish in all aquaria were removed during

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the exposure. At 35 dpf, 9 larvae from each replicate (n = 4) were randomly taken and anesthetized

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with 0.01% tricaine methane sulfonate (MS-222, Sigma–Aldrich) and were pooled as a composite

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sample and transferred to RNAlater for later qPCR analysis. At 60 dpf, the larvae from each

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aquarium were transferred to the 10 L glass tanks each containing 6 L of exposure media to

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continue exposure until 140 dpf. At 140 dpf, all fish were anesthetized with 0.01% MS-222. The

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body length (cm) and weight (g) were measured. The plasma samples from three individual fish of

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the same sex were pooled as a composite sample using glass capillary and pooled as a composite

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sample. The blood samples were transferred into heparin sodium-rinsed centrifuge tubes. After

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centrifugation (7,000 g for 5 min at 4 °C), the plasma samples were stored at -80 °C for hormone

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analysis.

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All remaining fish were dissected. The sampling time continued for approximately four hours. 5


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Two male and one female fish per replicate were randomly taken and dissected out for the gonads.

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The gonads were fixed in Bouin’s solution for 24 h prior to histological analysis. For RNA

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extraction, the gonads and brain of three female and four male fish per replicate were pooled and

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preserved in RNAlater at −80 °C. All experiments were conducted with the approval of the Ethics

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Committee of South China Normal University for the Care and Use of Laboratory Animals.

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Chemical Analysis. The exposure concentration of DDG in each aquarium was determined

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according to our previous method. 13 The concentrations of DDG at two different sampling dates

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(i.e., 60 dpf for juvenile stage; 140 dpf for adult stage) were measured. The limits of detection and

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quantification for DDG were 0.05 ng L-1 and 0.18 ng L-1, respectively. The recovery was 101% ±

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3%. For detailed information about chemical analysis, please refer to the Supporting Information

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(SI Text S1) and our previous work. 13

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Microarray Analysis. Microarray analysis was performed to investigate the global

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transcription profile following the exposure to DDG. Single-color zebrafish oligo microarray in a 4

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× 44 K slide format (Agilent, design ID 026437) was used in the microarray analysis as described

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previously. 14 The testis and brain samples stored at RNAlater were sent to Capital-Bio Inc. (Beijing,

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China) for microarray analysis. In total, 16 microarrays were analyzed for testis or brain (SC, L, M,

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and H treatments, 4 biological replicates per treatment). Capital-Bio cRNA Amplification and

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Labeling Kit (CapitalBio, Beijing, China) was used to label the samples with a fluorescent dye (Cy5

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and Cy3-dCTP) to produce yields of labeled cDNA. Hybridization was performed at 42 °C for

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overnight in an Agilent hybridization oven with a rotation speed of 20 rpm. The arrays were washed

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and scanned by Agilent G2565CA Microarray Scanner (Agilent, Santa Clara, USA). 15

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Quantitative Real-time PCR assay. Details on total RNA extraction, reverse-transcription of

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RNA into cDNA and qPCR reaction were described in a previous study. 16 Synthesis of cDNA was 6



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carried out from 500 ng of total RNA using ReverTra Ace® qPCR RT Master Mix with gDNA

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Remover (Toyobo, Japan) in a total volume of 50 µL. The qPCR analysis was conducted on the

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Applied Biosystems ViiATM 7 Dx (ABI) using the THUNDERBIRD SYBR® qPCR Mix (Toyobo,

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Japan). The intron/exon boundary-spanning primers were preferred to minimize DNA

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contamination. Primer-BLAST was employed to check the specificity for the primers (Table S1). In

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order to ensure specificity of the primers, a melting curve analysis (60 - 95 °C) was completed on

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each sample at the end of qPCR reactions. The ribosomal protein L (RpL13α) and elongation factor

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1 alpha (Ef1−α) showed high gene expression stabilities after chronic exposure to DDG (SI, Figure

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S1). The average Ct value (threshold cycle) of the reference genes was employed to normalize the

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expression of mRNA for the target genes. Relative mRNA expression was determined by the

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delta-delta CT method. 17

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Sex Hormone Analysis. The determination of sex hormone concentration was performed as

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previously described. 18 The detailed information was provided in the Supporting Information (SI

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Text S1). The concentrations of estradiol (E2) and 11-ketotestosterone (11-KT) were measured

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using ELISA kits (Cayman Chemical Company, Ann Arbor, MI, USA).

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Histological Examination. Histological preparation was performed as described in previous

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studies. 14,19 Briefly, the fixed gonad samples were dehydrated through a graded series of ethanol

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solutions, and the dehydrated samples were embedded in paraffin. Three longitudinal sections (4

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µm) from different regions of the ovary and testis were cut through the gonadal region. The sections

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were stained by hematoxylin-eosin, and examined under a Nikon Eclipse 50 i light microscope

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(Nikon, Japan). Testis and ovary staging and histopathological alterations were evaluated according

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to OECD guidelines and previous studies. 14,19,20

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Data Statistics and Analysis. The microarray data had been submitted to the Gene Expression 7


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Omnibus (GEO) database in accordance with the minimum information about a microarray

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experiment (MIAME) standards with an accession No.GSE116641. GeneSpring software V13

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(Agilent) was used to analyze the microarray data. Due to the relatively low number of genes from

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the L and M treatments selected based on the false discovery rate (FDR) analysis (< 0.05, see also

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the statistical analysis section in Text S2 in SI), differentially expressed genes (DEGs) were

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selected according to the threshold values (≥ 2) as compared with solvent control and the p value

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calculated following t-test. 15,21-24 Database for Annotation, Visualization, and Integrated Discovery

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(DAVID) was used instead of Kyoto Encyclopedia of Genes and Genomes (KEGG) as DAVID can

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combine the functionally descriptive data with intuitive graphical displays, which can facilitate the

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interpretation of the DEGs and the determination of significantly gene ontology (GO) categories

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and biological pathways. 25 The significantly altered biological pathways and GO categories were

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identified based on the p values (≤ 0.05).

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Hierarchical clustering (HAC) maps were constructed using the cluster 3.0 Software. The

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chi-square test was used to examine the differences in the sex ratio between the solvent control and

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other treatments. One way analysis of variance (ANOVA) was used to analyze the data followed by

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Tukey multiple comparison tests. Prior to ANOVA analysis, Kolmogorov–Simirnov and Levene’s

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tests were used to test the assumptions of normality and homogeneity of variances, respectively.

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The statistical analyses were conducted in SPSS (version 13.0). Data were considered significantly

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different at p < 0.05.

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RESULTS and DISCUSSION

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Measured DDG Exposure Concentrations. The concentrations of DDG were measured at the

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initiation of exposure (T0) and prior to water renewal (T24) during the juvenile and adult stages (See 8



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Chemical Analysis section). The measured concentrations of DDG were close to the nominal values

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at T0, but remained approximately 25% - 30% of their nominal concentrations at T24 (SI Table S2).

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The average measured concentrations of DDG for 5, 50, and 500 ng L-1 treatments were 3.79, 37.9

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and 374 ng L-1 during juvenile stage, and 2.97, 28.3, and 285 ng L-1 during adult stage, respectively.

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For simplicity, the average of the measured concentrations of DDG (i.e., 3.39, 33.1, 329 ng L-1 for

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the L, M and H treatments, respectively) during juvenile and adult stages was used for the

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presentation of the data throughout the study. In the present study, the decrease trend and magnitude

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of DDG concentrations during the exposure for juvenile and adult fish were similar to those

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observed in other studies on the effects of progestins in zebrafish. 4,12

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Sex Differentiation and Transcriptional Alterations. Results on the sex ratio of

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DDG-exposed populations are presented in Figure. 1A. DDG exposure severely disturbed sex

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differentiation in the H treatment with approximately 98% males. Previous studies have

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demonstrated that NGT and LNG caused 100% males due to their strong androgenic activity. 7,26 In

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the present study, DDG affected the sex ratio of the zebrafish populations, leading to a shift towards

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males. To the best of our knowledge, it is the first time to show that the exposure to DDG resulted

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in male-biased zebrafish populations.

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To further understand the possible mechanisms underlying the effects of DDG on sex

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differentiation, we examined the transcription of genes related to sex differentiation and apoptosis

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pathway at 35 dpf. The results showed that DDG suppressed the transcription of cyp19a1a

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(3.16-fold decrease) but stimulated the expression of dmrt1 mRNA (1.83-fold increase) in the H

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treatment (Figure. 1B). It is generally believed that dmrt1 is predominantly expressed in testis

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during sex differentiation in fish. 9 The over expression of dmrt1 mRNA would probably lead to

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differentiation of juvenile ovary into testis, which is consistent with the observed male-biased 9


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zebrafish populations in the present study. Moreover, dmrt1 promotes the early formation of testes

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by suppressing the ovarian developmental pathway via the repression of aromatase transcription and

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estrogen production in the gonads, 9 which was also evidenced by the low transcription of cyp19a1a

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in the DDG-exposed zebrafish. Previous studies also demonstrated that the male-biased zebrafish

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populations caused by exposure to NGT and LNG had low expression of cyp19a1a mRNA. 16,26,27

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Therefore, the up-regulation of dmrt1 and down-regulation of cyp19a1a at least partially explained

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the male bias in zebrafish populations chronically exposed to DGG.

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The suppression of cyp19a1a is most likely to decrease the production of aromatase and

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estrogen. The depletion of aromatase and estrogen could induce oocyte apoptosis during sex

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differentiation, therefore resulting in male bias. 10 Caspases (including casp1b, casp9, and casp3a)

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are a family of enzymes playing major roles in apoptosis. 28,29 In the present study, DDG

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significantly increased the transcription of casp1b, casp3a and casp9 in the H treatment. It was

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possible that the up-regulation of casp1b, casp9, and casp3a was closely related to sex

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determination in zebrafish (Figure 1B). Therefore, it is implied that over expression of dmrt1

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mRNA suppressed the transcription of cyp19a1a which activated the apoptosis pathway, thereby

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resulting in enhanced oocyte apoptosis and accelerating the differentiation of the juvenile ovary into

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testis in zebrafish. 10

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Blood Plasma Hormone Levels. Chronic exposure to DDG had no significant effect on the

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plasma concentrations of 17β-estradiol (E2) in females and 11-ketotestosterone (11-KT) in males

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(SI Figure S5), which were consistent with a previous study. 4 However, several studies have

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showed that progestins (LNG, norethindrone (NET) and megestrol acetate (MTA)) decreased the

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plasma hormone levels in fish, which might be due to the differences in disrupting potency of the

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chemicals and fish species 4,8,18. 10



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Global Gene Expression Analysis. Based on the sex ratio results, the brain and testis in male

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fish were selected for microarray analysis. Volcano plots showed that more DEGs were identified in

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the H treatment in both the testis and brain (Figure 2A, B). The number of up-regulated DEGs by

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DDG showed a concentration dependent pattern in the testis. In contrast, the number of

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up-regulated DEGs by DDG in the brain decreased with increasing DDG concentrations (Figure 2A,

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B, SI Figure S6).

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The Venn diagrams depicted the overlap of DEGs and Gene Ontology (GO) at all

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concentrations (Figure 2C, D). In the testis, there were 6 overlapped DEGs mainly involved in

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immune responses (mhc1uea and mhc1ufa), endothelial barrier and regulation of cell migration

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(tnfrsf19) (SI Table S3). Similarly, in the brain, there were 17 overlapped DEGs, which were

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mainly involved in the rhythmic processes (clocka, antl2, antl2b, cry5, cyr1ab, per1b, nr1d2b,

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si:ch211-132b12.7, nfil3-5, nfil3-6 and mapk8b) and in responses to chemical stimuli (cyp2ad6,

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nr1d4b and ddb2) (SI Table S3). Overlapping DEGs were identical to those overlapping GOs both

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in the brain and testis of male fish.

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Confirmation by qPCR. The DEGs predicted by microarray analysis were validated by qPCR

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in both the testis and the brain of male fish (Table 1; SI Table S11). In the testis, the qPCR results

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of p2rx4b, il1rapl1a and tnfrsf19 were consistent with microarray results, while the fold change of

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mhc1uea, mhc1ufa and trim66 from the qPCR data was higher than that of the microarray data. The

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transcriptional alterations of asah1b, casp1b, casp3a, and casp9 were also consistent with the

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microarray results (Table 1). In the brain, the qPCR results showed the transcriptional levels of

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clocka, nr1d4b, arntl2, cyp2ad6, cry1ab, cry5, ddb2, nr1d2b, gabrr1 and zgc:112266 were

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significantly enhanced, which was consistent with the microarray results. The fold change of nfil3-5

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and si:ch211-132b12.7 by qPCR was slightly higher than that by microarray analysis, while the 11


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fold change of nfil3-6, si:ch211-168n16.1 and nfil3-6 by qPCR analysis was relatively lower than

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that by microarray analysis (Table 1).

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We further evaluated the transcription of the overlapping DEGs in the ovary and brain of

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female fish. In the ovary, the suppression of mhc1uea and mhc1ufa was observed in the L treatment,

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while up-regulation of mhc1uea, mhc1ufa, asah1b and casp3a was observed in the H treatment

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(Figure 3 A). In the brain, the transcriptional alteration of the overlapping DEGs in females was

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very similar to that of the males (Figure 3 B). DDG exposure increased transcription of clocka and

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arntl2, but decreased the transcription of per1b, cry5, nr1d2b and cyp2ad6. Heatmap analysis

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revealed a significant distinction between brain and gonad (Figure 3 C). The target genes from the

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brain were clustered on the top, except for tnfrsf19. The genes in females in the M treatment were

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clustered near the genes of male in the H treatment, indicating that DDG was androgenic to

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zebrafish and therefore indirectly corroborated the observed male-biased zebrafish populations.

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Histological Alterations and Functional Genomics Analysis in the Gonads. The

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histological alterations in gonads are presented in Figure 4. The typical histological sections of

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gonads of zebrafish in the SC and the H treatment in the testes, and in the SC and M treatment in

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the ovaries treatment are shown in Figure 4 A-D. Other DDG treatment groups are presented in

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Figure S3 and S4. Compared with the control gonads, DDG exposure significantly increased the

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percentage of mature spermatids in the testes in the H treatment, and atretic follicles (AF) in the L

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and M treatments in the ovaries (Figure 4 E, F).

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In the present study, GOs related to immune response, endothelial barrier and regulation of cell

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migration were enriched at all concentrations. A previous study showed 21 days of exposure to

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progesterone (P4) and drospirenone (DRSP) mainly affected GOs related to the ion channel in the

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ovary of adult zebrafish, 12 which was different from our results. Endothelial barrier, regulation of 12



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cell migration, and phagosome are essential for maintaining the spermatogenesis in the testis. 30,31

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Phagosome plays important roles in tissue remodeling by engulfing the regressing tissues (e.g., the

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residual body) during spermatogenesis. 32,33 Defective elimination of residual bodies in mammals is

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associated with impairment of sperm quantity. 34 Moreover, endothelial barrier is established at the

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end of meiotic phase, which controls the movement of spermatocytes from the basal to the

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adluminal compartment via disassembles and reassembles. 31 Therefore, the enriched GOs from the

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above mentioned three processes in the testes from DDG-exposed zebrafish implied that DDG

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might influence spermatogenesis.

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In the testis, interestingly, exposure to DDG increased the percentage of mature spermatids in

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the H treatment. Similar results were observed in zebrafish exposed to 1263 ng L-1 DDG for 21

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days.4 In the present study, lysosome, phagosome and sphingolipid metabolism pathways were

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mostly affected in the H treatment by KEGG analysis (Table 2). Since the mechanisms of the

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effects of progestins on spermatogenesis in fish are largely unknown, it is assumed that these three

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pathways are responsible for the alterations of spermatogenesis caused by exposure to DDG based

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on the following evidence. First, these three pathways are involved in regulation of spermatogenesis,

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especially for the acrosome biogenesis. 30,35 The acrosome is regarded as a modified lysosome or a

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novel lysosome-related organelle (LRO). 35 In the present study, microarray analysis showed DDG

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exposure up-regulated the transcription of neu1, naga and asah1b in the H treatment. Neu1 and

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naga encode glycosidase which is located on the surface of acrosome and participates in the

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regulation of spermatogenesis, epididymis maturation and acrosome reaction. 36,37 Meanwhile, the

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activity of glycosidase encoded by naga in the seminal plasma is considered as an indicator of

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sperm cell membrane integrity during semen maturation in the reproductive tract of the common

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carp (Cyprinus carpio). 37 Therefore, the over expression of neu1 and naga mRNA might accelerate 13


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the formation of acrosome, resulting in more mature spermatids, as evidenced from our histological

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observations. Secondly, during acrosome biogenesis, the lysosome fuses with an autophagosome to

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form LRO. 37 Previous studies showed that interference with autophagosome-lysosome fusion, a

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key step in acrosome biogenesis, could affect acrosome biogenesis and sperm quality in zebrafish.

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35,38

In the present study, asah1b and cers2a, two genes involved in the regulation of autophagy,

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39,40

were identified in the sphingolipid metabolism pathway. In addition, stx17, a gene encoding the

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protein required for autophagosome-lysosome fusion, 41 was enriched in Golgi transport GOs

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functioning in regulating the acrosome biogenesis. 35 Its expression was significantly enhanced in

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the H treatment. Therefore, the elevated expression of stx17 suggested that DDG promoted the

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autophagosome-lysosome fusion, and accelerated the formation of acrosome biogenesis. Finally,

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once spermatids are differentiated into spermatozoa, the bulk cytoplasmic contents are segregated

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into the residual body, which is phagocytosed via the phagosome pathway. 32 In the present study,

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DDG exposure increased transcription of target genes (e.g., mhc1ufa and fc50e01) involved in the

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phagosome pathway by microarray analysis. The activation of the phagosome pathway implied a

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more efficient removal of the residual bodies and a more accelerated production of spermatozoa, as

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evidenced from our histological observations. Taking together, it is reasonable to conclude that

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DDG increased the level of sperm maturation via sphingolipid metabolism and lysosome pathways,

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and enhanced the efficient removal of residual bodies via the phagosome pathway.

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In the ovary, DDG increased the frequency of AF. The ovarian follicle atresia is an apoptotic

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process leading to reabsorption of maturing oocytes. 42 The elevated transcription of casp3a in the

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ovary in all treatments implied that DDG was likely to cause apoptosis, which was supported by the

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histological data. Moreover, it is known that mhc1uea and mhc1ufa are expressed on the surface of

307

the regression tissues (e.g., the ovarian follicle) and then induce the T cells and macrophages to 14



Page 16 of 29

308

clear and engulf the regression tissues. 32,33,43 The over expression of mhc1uea and mhc1ufa mRNA

309

implied more removal of regression tissues, indicating more AF occurred, which was supported by

310

the increased frequency of AF. On the contrary, the lower expression of mhc1uea and mhc1ufa in

311

the L treatment implied the less removal of residues, which increases the space stress and induces

312

the apoptosis as well. 43,44 Therefore, the increased frequency of AF was also noticed in the L

313

treatment.

314

Functional Genomics Analysis in Brain. The majority of the overlapping DEGs were

315

involved in circadian rhythm network. Up-regulation of clocka and arntl2 and down-regulation of

316

per1b, cry1ab and nr1d2b were fitted well to the circadian rhythm network in both female and male

317

brains. 45 This was consistent with those from previous studies. 4,12,46 Interestingly,

318

si:ch211-132b12.7 was ascribed to be a negative regulator of circadian rhythm by microarray

319

analysis. The suppressed transcription of si:ch211-132b12.7 was most likely to stimulate the

320

expression of clocka, arntl2 and arntl2b, which was observed in the present study. It is worth noting

321

that the transcriptional expressions of genes in the circadian rhythm were altered even at –3.39 ng

322

L-1, suggesting that circadian rhythm pathways are vulnerable to the DDG exposure, and these

323

genes are sensitive to the exposure to DDG and other progestins in general as well. 4,12,46

324

Circadian rhythm network is vital to mediate crucial cellular and physiological processes, such

325

as cell cycle, metabolism, hormone secretion, and reproduction. 47 For instance, endogenous

326

circadian clocks in hypothalamic−pituitary axis participate in regulating the release of leuthine

327

hormone (LH) from pituitary gonadotropin cells in rodents. 48 In the present study, it seemed that

328

circadian rhythm might affect other physiological processes in zebrafish. The microarray analysis

329

showed two genes (nr1d4a and nr1d4b) involved in both the rhythmic process and in response to

330

steroid hormone were identified after chronic exposure to DDG. 45 Therefore, exposure to DDG 15


Page 17 of 29


331

might affect the metabolism of steroid hormones via the alterations in the transcriptional genes in

332

the circadian rhythm network in the brain. It is known that steroid hormones play an important role

333

in sex differentiation and gonad development. However, whether the circadian rhythm network

334

affects the reproductive system of teleost requires further investigation. Previous studies suggested

335

disorder of the circadian rhythm affected fish reproduction in zebrafish and Japanese medaka,

336

probably via the HPG-Liver axis. 46,49 In the present study, the HPG axis related pathways were not

337

significantly enriched, and hormone levels in blood plasma were not changed (SI Figure S5).

338

Furthermore, the transcriptional changes of the genes of the circadian rhythm in the brain were not

339

well coordinated with the histological changes in the gonads. Slight transcriptional alteration of

340

genes related to circadian rhythm network was observed at the period of sex differentiation (35 dpf)

341

(SI Figure S10). Future research on the correlation between the circadian rhythm network and fish

342

reproduction is still warranted.

343

Intriguingly, the transcription of cyp2ad6 was stimulated at all concentrations in the brain. Up

344

to now, the catalytic or biological functions of cyp2ad6 have yet to be determined in fish. 50 In the

345

present study, the cyp2ad6 was enriched in the arachidonic acid metabolic process by GO analysis.

346

It is showed that cyp2ad6 could have similar catalytic functions to cyp2j2 which is important in the

347

oxidation of arachidonic acid in human, 51 and this was manifested by our GO analysis. The

348

transcriptional alteration of cyp2ad6 implied that DDG might influence arachidonic acid

349

metabolism in zebrafish.

350

Our results showed that exposure to DDG induced multiple transcriptional responses, caused

351

male bias and affected the gonadal development in fish. Progestins such as DDG, NGT and LNG

352

cause male bias and have similar effects on the transcription of dmrt1 and cyp19a1a during sex

353

differentiation in zebrafish but with different potentials. These progestins may simultaneously exist 16



354

in contaminated aquatic environments. The effects (for example, on the endocrine system) of the

355

mixture of progestins in natural fish populations deserve further research.

Page 18 of 29

356 357

ASSOCIATED CONTENT

358

Supporting Information. The detail materials and methods (Text S1), statistical analyses

359

(Text S2), primer sequences (Table S1), measured concentrations (Table S2), overlapping GOs and

360

DEGs (Table S3), the period of exposure, endpoints and number of replicates (Table S4),,

361

overlapping GOs (Table S5-S10), confirmation of the DEGs (Table S11), top 10 pathways at three

362

concentrations (Table S12-17), list of DEGs (p < 0.05; FC ≥2) at three concentrations (Table

363

S18-23), stability analysis of RpL13a and EF1-α (Figure S1), condition factors and gonadosomatic

364

index at 140 dpf (Figure S2), typical histological sections of gonad (Figure S3, 4), plasma

365

concentrations (Figure S5), number of DEGs in testis and brain (Figure S6), transcriptional

366

alteration of target genes in brain and gonad (Figure S7-8), schematic representations of the

367

spermatid mature (Figure S9), transcriptional alteration of genes in circadian rhythm network at 35

368

dpf (Figure S10).

369 370

ACKNOWLEDGEMENTS

371

The authors would like to acknowledge the financial support from the National Natural Science

372

Foundation of China (U1401235 and 41273119) and National Water Pollution Control Program

373

(2014ZX07206-005), as well as Guangdong Provincial Key Research Program (2015B020235012)

374

and Guangdong Natural Science Foundation (2015A030313738).

375 376

Notes 17


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377


The authors declare no competing financial interest.

378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418

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509

Table 1. Confirmation of the differentially expressed genes in brain and testis of male

510

zebrafish by qPCR. Fold change a Organ

Gene

3.39 ng L-1 vs SC b

33.1 ng L-1 vs SC

329 ng L-1 vs SC

qPCR

Micro array

qPCR

Micro array

qPCR

Micro array

Testis

mhc1uea mhc1ufa p2rx4b il1rapl1a tnfrsf19 asah1b casp1b casp3a casp9

-10.71*** -9.75*** -2.20*** -1.24* 1.51** 0.50 -0.66 -0.38 0.30

-7.07* -7.76* -1.96* -1.13** 1.89* 0.40 -0.48 -0.56 0.14

-9.00*** -8.50*** -1.22* -1.20* 1.47** 0.21 -0.73 0.49 -0.36

-6.48* -7.06* -1.51** -1.05** 1.05* 0.63 -0.19 0.19 -0.35

1.82* 1.60* -0.73 -0.67 1.72** 1.10* -0.62 0.65 0.53

1.50** 1.16* -1.10* -1.28*** 1.11* 1.04** -0.22 0.25 0.07

Brain

clocka nr1d4b arntl2 cry5 cyp2ad6 per1b si:ch211-13 2b12.7 zgc:112266 ddb2

1.71*** c 3.56*** 2.16*** -1.56* -1.76** -2.07***

1.42** 3.88** 1.78*** -1.50** -1.82** -1.73**

1.90*** 2.92*** 1.76** -1.87*** -0.85 -2.11***

1.39* 3.23** 1.91* -1.62** -1.17*** -2.39*

2.23*** 4.85*** 2.31*** -1.59* -1.25* -2.13***

1.74** 4.53*** 2.07*** -1.65*** -1.76*** -2.06***

-2.23**

-1.58**

-1.92**

-1.39*

-2.70***

-1.86**

-1.98** -1.38*

-1.84* -1.40*

-1.42* -1.85***

-2.00* -1.54***

-2.31** -1.18*

-2.74** -1.37***

511

a

512

2−∆∆CT method and ratio of array spot intensity, respectively;

513

b

SC: solvent control;

514

c

Data are shown as mean of four replicates;

515

* p < 0.05, ** p < 0.01, and *** p < 0.001.

Fold changes (log2) of qPCR and microarray compared to solvent controls was determined by

516

21


Page 23 of 29


517

Table 2. The top 10 affected signaling pathways in testis of zebrafish from the H treatment as

518

compared to the solvent control. Signaling Pathway

FDR a

p value

DEGs b

0.62989

actb2, hbl4, sec61al2, fc50e01, mhc1uea, mhc1uxa2.c tubb1.d

0.62989

neu1, cers2a, asah1b. smpd3.

0.62989

neu1, naga, ctsc, fc50e01, asah1b, cd164.

0.62989

aldh7a1.

0.06196

0.70939

adrb3a. ltc4s, cyp2p9, ggt1l2.2.

Calcium signaling pathway

0.07089

0.70939

mylk3, tnnc1b. p2rx4b, cacna1i, adrb2b, pdgfra, mylk4b.

Biosynthesis of amino acids

0.07273

0.70939

cth, gapdh, aldh7a1. gpt2l.

Cell adhesion molecules (CAMs)

0.07821

0.70939

mhc1uea, lrrc4.1, mpz, mhc1uxa2. cntn1a.

Neuroactive ligand-receptor interaction

0.08800

0.70939

adrb3a, c5ar1. p2rx4b, gria3a, vipr1b, nmur1, chrm4a, adrb2b.

Nicotinate and nicotinamide metabolism

0.10005

0.70939

enpp1, nt5c2b.

Phagosome

0.00649

Sphingolipid metabolism

0.02354

Lysosome

0.03485

Lysine biosynthesis

0.03669

Arachidonic acid metabolism

**

*

* *

519

* p < 0.05 and ** p < 0.01;

520

a

FDR: False discovery rate;

521

b

DEGs: Differentially expressed genes;

522

c

Red color: up-regulation;

523

d

Green color: down-regulation.

524 525

22



Page 24 of 29

526

Figure Captions

527

Figure 1. Sex differentiation and transcriptional alterations. (A) The sex ratio of zebrafish after

528

exposure to dydrogesterone (DDG) from 21 dpf to 140 dpf. The sex of adult fish was determined by

529

the morphology of its gonad. N represents the total number of fish from four replicate aquariums in

530

each treatment. Abbreviations: SC: solvent control; L: 3.39 ng L-1; M: 33.1 ng L-1; H: 329 ng L-1. (B)

531

Transcriptional alterations of dmrt1, cyp19a1a, foxl2, casp1b, casp3a, casp9, esr and ar in juvenile

532

zebrafish after 14 days (from 21 to 35 dpf) of exposure to DDG compared to solvent control. Data

533

are shown as mean ± SD of four replicates. * p < 0.05, ** p < 0.01, and *** p < 0.001. L: 3.39 ng

534

L-1; M: 33.1 ng L-1; H: 329 ng L-1.

535 536

Figure 2. Multivariate analysis of the transcriptomics data. (A-B) Volcano plots depicting fold

537

change (log2, x-axis) and statistical significance (–log10 p value, y-axis) in the testis (A) and brain

538

(B) of male zebrafish. The fold change cut-off is +/− 1 and −log10 p value cut-off is 1. The upper

539

corners of the plot represent the genes that show both statistical significance and large fold changes.

540

Red dots: significant up-regulation; Green dots: significant down-regulation; Black dots: no

541

significant alteration. (C) Venn diagram of the overlapping DEGs (C1) and GOs (C2) at all

542

concentrations in the testis compared to solvent control. (D) Venn diagram of the overlapping

543

DEGs (D1) and GOs (D2) at all concentrations in the brain of male zebrafish compared to solvent

544

control. One hundred percent (100%) stands for the sum of all the numbers in the pie charts.

545

Abbreviations: Key: SC: solvent control; L: 3.39 ng L-1; M: 33.1 ng L-1; H: 329 ng L-1.

546 547

Figure 3. The qPCR analysis of DEGs in females. (A, B) Transcriptional alteration of target

548

genes in the ovary (A) and brain of females (B). Data are shown as mean ± SD of four replicates. * 23


Page 25 of 29


549

p < 0.05, ** p < 0.01, and *** p < 0.001. Abbreviations: Key: SC: solvent control; L: 3.39 ng L-1;

550

M: 33.1 ng L-1; H: 329 ng L-1. (C) Hierarchical clustering analysis of qPCR data in the brain and

551

gonads of females (F-) and males (M-). L: 3.39 ng L-1; M: 33.1 ng L-1; H: 329 ng L-1. Due to

552

insufficient number of females in the H treatment (male-biased populations), no data on qPCR are

553

available for females.

554 555

Figure 4. Histological alterations in the gonads at 140 dpf. (A, B) Typical histological sections

556

of testis of adult male zebrafish in SC (A) and H treatment (329 ng L-1 of DDG) (B). Examples of

557

spermatozoa maturation stages include Sg (spermatogonia), Sc (spermatocytes), St (spermatids) and

558

Sz (spermatozoa). (C, D) Typical histological sections of ovary of adult female zebrafish in SC (C)

559

and M treatment (33.1 ng L-1) (D). Examples of oocyte developmental stages include PO

560

(perinucleolar oocyte), CO (cortical alveolar oocyte), EV (early vitellogenic oocyte), LV (Mid-late

561

and Late- vitellogenic oocyte), POF (postovulatory follicles) and AF (atretic follicles). (E)

562

Percentage of mature (MA) and immature (IMA) spermatocytes in the testis of zebrafish at 140 dpf.

563

MA includes St and Sz, and IMA includes Sg and Sc. (F) Percentage of oocytes at different

564

developmental stages in the ovary of zebrafish at 140 dpf. Bar from top to bottom refers to AF, POF,

565

LV, EV, CO and PO. Data are shown as mean values of four replicates. * p < 0.05, ** p < 0.01, and

566

*** p < 0.001. Abbreviations: SC: solvent control; L: 3.39 ng L-1; M: 33.1 ng L-1; H: 329 ng L-1.

567

24



Figure 1

%

A 1.0

Male Female

0.5

N=49 N=54 N=51 *** 0.0 N=53 SC H M L 570 571

B4 Fold Change

568 569

Page 26 of 29

2

SC L M ** H

**

** **

0 -2 ***

-4 a 1 2 b a 9 r r 1 rt xl 1 3 sp es a 19a dm fo casp casp ca p cy

25


Page 27 of 29

572 573


Figure 2

574 575

26



576 577

Page 28 of 29

Figure 3

578 579

27


Page 29 of 29

580 581


Figure 4

582 583

28


Danio rerio

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