Genetic evidence for estrogenicity of bisphenol A in zebrafish gonadal differentiation and its signalling mechanism

Journal Pre-proof Genetic Evidence for Estrogenicity of Bisphenol A in Zebrafish Gonadal Differentiation and Its Signalling mechanism Weiyi Song, Huijie Lu, Kun Wu, Zhiwei Zhang, Esther Shuk-Wa Lau, Wei Ge

PII:

S0304-3894(19)31840-0

DOI:

https://doi.org/10.1016/j.jhazmat.2019.121886

Reference:

HAZMAT 121886

To appear in:

Journal of Hazardous Materials

Received Date:

20 September 2019

Revised Date:

9 December 2019

Accepted Date:

10 December 2019

Please cite this article as: Song W, Lu H, Wu K, Zhang Z, Shuk-Wa Lau E, Ge W, Genetic Evidence for Estrogenicity of Bisphenol A in Zebrafish Gonadal Differentiation and Its Signalling mechanism, Journal of Hazardous Materials (2019), doi: https://doi.org/10.1016/j.jhazmat.2019.121886

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Genetic Evidence for Estrogenicity of Bisphenol A in Zebrafish Gonadal Differentiation and Its Signalling Mechanism

Running title: Estrogenic activities of BPA and its action mechanisms in zebrafish

Weiyi Song†, Huijie Lu†, Kun Wu†, Zhiwei Zhang†, Esther Shuk-Wa Lau‡ and Wei Ge*† [email protected]/[email protected] Centre of Reproduction, Development and Aging (CRDA), Faculty of Health

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Sciences, University of Macau, Taipa, Macau 999078, China ‡

School of Life Sciences, The Chinese University of Hong Kong, Shatin, New

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Territories, Hong Kong 999077, China.

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Corresponding author:: Wei Ge, Faculty of Health Sciences, University of Macau,

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Graphical abstract

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Taipa, Macau, China; Tel: +853-8822-4996; ORCID identifier: 0000-0002-4296-15

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Highlights 

BPA can mimic the effect of estrogens on ovarian differentiation in zebrafish.

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Esr2a plays a critical role in mediating the feminizing effect of BPA.

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The LE zebrafish line provides a model for evaluating estrogenic effects of EDCs. The nER mutants provide valuable tools for evaluating the actions of e-EDCs.

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Abstract

Bisphenol A (BPA) can induce endocrine disorders in humans and animals. In this

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study, we used several zebrafish mutants deficient in estrogen production and signalling, which could be valuable for evaluating estrogenic activities and

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mechanisms of EDCs. With low endogenous estrogens, the all-male aromatase mutant (cyp19a1a-/-) is expected to be more responsive to estrogenic exposure, and mutants of nuclear estrogen receptors (nERs; esr1-/-, esr2a-/- and esr2b-/-) alone or in

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combination would allow us to evaluate the action mechanisms of estrogenic EDCs. Exposure to BPA could rescue the all-male phenotype of the cyp19a1a-/- mutant,

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delayed gonadal development in both sexes, resulting in infertility or subfertility, and caused follicle atresia in females and impairment of spermatogenesis in males. To understand the mechanisms of these effects, we tested BPA in cyp19a1a and nER

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mutants of different combinations. The feminizing effect of BPA on sexual differentiation was dependent on nERs, in particular esr2a. As for males, nERs were also involved in BPA-induced impairment of spermatogenesis. Taken together, with genome editing technology our study provides the most comprehensive genetic evidence for estrogenic activities of BPA in zebrafish and its action mechanisms. This study also establishes a powerful platform for studying other EDCs with estrogenic activity. 2

Key words: Genome editing, aromatase, estrogen receptors, bisphenol A, gonadal

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differentiation, zebrafish

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1. Introduction

Bisphenol A (BPA) is one of the most-produced chemicals in the world [1, 2],

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with annual production reaching more than 7 million tons by 2020, and release exceeding one million pounds per year [3]. As an estrogenic endocrine-disrupting

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chemical (e-EDC) [4], the activities of BPA in living organisms and its safety to humans have been major issues in endocrinology with great concerns in public health. Recent studies show that numerous endocrine disorders and reproductive

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malfunctions in humans and animals are associated with BPA. BPA exposure in different animals can induce various adverse effects on both reproductive and non-

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reproductive functions, such as changes in steroid levels, gonadal size, gametogenesis and fertility [5-9] as well as aberrations in morphology, metabolism, immunity and behavior [10-12]. Treatment of rodents with BPA during critical periods of gonadal

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development caused developmental retardation of gonads, testicular degeneration, infertility, accelerated puberty onset, and premature birth [10, 13-17]. In humans, there is a positive association between urinary BPA concentration and implantation failure in women [18] and lower semen quality in men [19]. Various in vitro cellular and biochemical assays have been developed to study potential action mechanisms of BPA, including nER-ligand (nERs, nuclear estrogen receptors) binding assays, nER-expressing cell lines and nER-responsive reporter 3

gene assays [20-22]. These assays have provided critical information on receptor specificity and potency of BPA [20]. However, data from such in vitro studies are often difficult to be extrapolated to the intact endocrine system and complex signalling pathways in vivo. Small model fish species (zebrafish, medaka and fathead minnow) are popular and valuable in vivo models for studying EDCs. Various transgenic fish lines have been developed for assessing estrogenic activities of BPA and other e-EDCs, and most of them are based on GFP expression driven by estrogen-responsive promoters or endogenous promoters of ER-regulated genes [23-29]. These transgenic fish are

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useful tools for quick and sensitive screening of chemicals for estrogenic activities. However, this approach does not provide much information on whether and how

EDCs disrupt developmental and physiological processes in vivo such as gonadal differentiation and maintenance.

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To evaluate the effects of e-EDCs on reproduction, several key endpoints have been established and validated for assessing reproductive disruption such as

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vitellogenin (VTG) expression, sex ratio, secondary sexual characteristics and intersex [30, 31]. Among these, sex ratio is specifically related to gonadal sex differentiation,

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and female-biased sex ratio is often regarded as a direct indicator of feminization of fish exposed to e-EDCs [32, 33].

Despite numerous studies on BPA interference with reproduction in vivo using

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different animal models, controversy remains on its feminizing effects on sexual differentiation due to inconsistent results from different studies. For instance, femalebiased sex ratios (79% and 92%) were found in zebrafish fed with a diet containing

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BPA at 1000 and 2000 mg⋅kg-1 [34]. However, treatment of Japanese medaka for 40 days with BPA in water at 6-600 μg/L did not cause any significant alteration in sex

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ratio [35]. A study on Xenopus larvae reported that exposure to BPA at 27 μg/L resulted in a female-biased sex ratio [36]; however, no significant deviation from the expected 1:1 sex ratio was observed in the same species after exposure to BPA in the range of 0.83-497 μg/L [37]. Different factors may have contributed to the inconsistent in vivo results, such as species, developmental stage, age, experimental design (e.g., exposure dosage and duration), and even funding source [38]. One hypothetical cause could be that most in vivo studies so far have used wild type (WT) 4

females, which produce high levels of endogenous estrogens. Depending on the stage of animals used, these may interfere with and mask the effects of e-EDCs tested because most e-EDCs have weaker estrogenic activities compared with natural estrogens [4, 39]. We have recently generated a series of zebrafish mutants for evaluating feminizing effects of EDCs and understanding their signalling mechanisms in vivo. For example, the fish line without ovarian aromatase (cyp19a1a-/-) [40] likely has increased responsiveness to estrogenic exposure in terms of ovarian differentiation due to low background level of endogenous estrogens. We named this mutant as low-

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estrogen line (LE line). In addition, the lines without nERs (single, double and triple knockouts for the three nERs) [41] are useful tools for understanding how each

receptor is involved in mediating estrogenic actions of e-EDCs in vivo. The quadruple knockout of ovarian aromatase and nERs (cyp191a1-/-;esr1-/-;esr2a-/-;esr2b-/-), called

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non-responsive line (NR line), could be valuable for distinguishing nER-dependent and -independent activities of e-EDCs.

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Using these genetic tools, we undertook this project to systematically evaluate the feminizing effect of BPA on zebrafish gonadal differentiation and the underlying

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signalling mechanism. To our knowledge, this represents the first systematic genetic study in vivo at such large scale on an e-EDC and its action mechanism. Our data not only confirmed the estrogenic activity of BPA and its impact on gonadal

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differentiation, but also provided critical clues to the receptors involved in the process. This approach will also provide a powerful platform for dissecting nER-

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dependent and -independent activities of BPA and other e-EDCs in the future.

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2. Materials and methods 2.1 Chemicals 2,2-Bis(4-hydroxyphenyl) propane (BPA, CAS:80-05-7, 99+%), 17β-estradiol

(E2, CAS: 50-28-2, 99+%) and 3-aminobenzoic acid ethyl ester (MS-222) were purchased from Sigma (St. Louis, MO). The ELISA kit for zebrafish VTG was from Biosense (Bergeb, Norway) and E2 from GE Healthcare (Piscataway, NJ), respectively. Chemicals were dissolved in 100% ethanol to obtain stock solutions of 5

BPA (20 mM) and E2 (500 μM) and stored at -20oC. The final concentrations of ethanol in the treatment water were less than 0.05% v/v. 2.2 Fish maintenance The fish lines were raised in the ZebTEC multilinking rack system (Tecniplast, Buguggiate, Italy) under constant conditions (temperature 28°C, pH 7.3, conductivity 400 µS/cm, and photoperiod 14L:10D). The fish were fed twice a day by the Tritone automatic feeder system (Tecniplast) and supplemented with live brine shrimp (Artemia) twice daily. All experimental procedures were approved by the Animal Ethics Committee of University of Macau (Approval no. AEC-13-002) and the

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animals handled according to the Animal Protection Act enacted by the Legislative Council of Macao Special Administrative Region under Article 71 (1) of the Basic Law. 2.3 Fish lines

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The mutant of ovarian aromatase (cyp19a1a-/-) with vasa-EGFP transgene

[Tg(vas:EGFP)cyp19a1a-/-] and different nER mutant lines (esr1-/-, esr2a-/- and esr2b) were created in our previous studies (cyp19a1aumo5, esr1umo10, esr2a umo11,

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esr2bumo13; see Table S1 for allele sequences) [40, 41]. The transgenic fish line

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Tg(vas:EGFP) expresses EGFP in the germ cells under germ cell-specific vasa promoter [42]. From these fish lines, we first produced an offspring with four genes at heterozygous state (cyp19a1a+/-;esr1+/-;esr2a+/-;esr2b+/-). By self-crossing of the

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heterozygote, we generated a variety of fishes with different mutant combinations, including double mutants with two nERs present (cyp19a1a-/-;esr1-/-, cyp19a1a/-

;esr2a-/- and cyp19a1a-/-;esr2b-/-), triple mutants with one nER (cyp19a1a-/-;esr1;esr2a-/-, cyp19a1a-/-;esr1-/-;esr2b-/-, and cyp19a1a-/-;esr2a-/-;esr2b-/-), and quadruple

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/-

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mutants without any nERS (cyp19a1a-/-;esr1-/-;esr2a-/-;esr2b-/-). 2.4 Experimental design and sample collection To evaluate the impacts of BPA on sex differentiation, gonadal development and

maturation, we first tested it in low-estrogen LE line without ovarian aromatase (cyp19a1a-/-). In cyp19a1a-deficient fish, the serum E2 concentration was significantly lower than that in the WT fish [43]. Newly fertilized eggs from the breeding between Tg(vas:EGFP)cyp19a1a+/- and Tg(vas:EGFP)cyp19a1a-/- were 6

collected and maintained in an environmental chamber until 20 dpf (days postfertilization). The treatments were performed in BPA-free tanks with 10 L water. In the first experiment, about 400 juvenile fish at 20 dpf were randomly selected and distributed to six treatment groups (~60-70 fish/dose). The fish were exposed to a range of environmentally relevant concentrations of BPA (0.01, 0.1, 1 and 10 μM) from 20 to 150 dpf, the period that covers gonadal differentiation and maturation (Fig. S1). As control, a group of fish (n≈60) was treated with E2 (0.5 nM) (positive control) and a group (n≈60) was exposed to ethanol only (negative control). During the exposure period, the water was renewed daily with new BPA and E2 to maintain

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relatively constant concentrations. The dosage used for BPA (0.01 to 10 μM) covered the range between the detectable levels in rivers (< 0.01 μM) and the levels in the

effluents from wastewater treatment plants and landfill sites (up to 75 μM), which are the major source of BPA contamination in aquatic environments [44, 45]. The fish

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were sampled (15-20 fish per group) at 65, 100 and 150 dpf during the exposure

period for histological analysis. After termination of the treatment at 150 dpf, the

the females induced by E2 and BPA.

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remaining fish were maintained to 210 dpf in clean water to assess the reversibility of

In the second and third exposure tests, we conducted the experiments in four

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groups (BPA 0, 1 and 10 μM; E2 0.5 nM) (40 fish/group) and sampled all the fish at 65 dpf for sex ratio calculation.

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At each sampling time, the fish were euthanized with an overdose of MS-222 (200 mg/L) followed by photographic recording of each fish and the fluorescent signal of GFP in the gonads as well as morphometric measurement of body size

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(weight and standard length). Each fish was genotyped by high-resolution melting analysis (HRMA) on genomic DNA extracted from the tail fin [46].

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To illustrate whether the effects of BPA on gonadal differentiation and

maturation involve nERs and the roles of each nER in mediating BPA actions, we then performed BPA treatment (10 μM) from 20 to 60 dpf on cyp19a1a-/- mutant with different combinations of nER mutations. The fish were sampled for histological analysis for sex ratio and gonadal development, and cyp19a1a-/- mutant alone was used as the control. The number of fish in each group is shown in Table S3. 2.5 High resolution melt analysis (HRMA) 7

Genotyping was performed on genomic DNA by using HRMA according to the protocol we described previously [47]. Briefly, the fin cut from the tail of each fish was incubated in 40 μL NaOH (50 mM) at 95°C for about 15 min. The solution was cooled down at 4°C for 5 min followed by addition of 4 μL Tris-HCl (1 M, pH 8.0). The sample was centrifuged at maximum speed for 1 min, and the supernatant was used for HRMA. Primers flanking the target site were used to amplify the genomic region by real-time qPCR on the CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, CA). The detailed information on target sites was reported previously [40, 41]. The conditions for amplification/duplex formation were as follows: denaturation

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at 95°C for 3 minutes; 40 cycles of reaction (denaturation at 95°C for 15 sec; annealing at 62°C for 15 sec; extension at 72°C for 20 sec); denaturation at 95°C for

15 sec; melt curve 70°C-95°C, with a 0.2°C increment each step. HRMA analysis was carried out using the Precision Melt Analysis software (Bio-Rad). Different genotypes

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(+/- and -/-) could be identified by distinctive melt curves (Fig. S7). 2.6 Blood E2 and VTG determination

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For determining E2 and VTG levels, the blood was sampled from each fish (~1015 µL) either directly from the heart or non-lethally from the tail according to a

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reported protocol [48]. To analyse E2 levels in the serum, the blood samples were incubated at 4°C for half an hour followed by centrifugation at 5000 rpm for 10 min. The supernatant was transferred to a new microtube and diluted at 1:50 with ELISA

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buffer. The levels of E2 were then determined by ELISA following manufacturer’s protocol (GE Healthcare). Eight fish (six month) were sampled for each genotype for analysis.

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To determine VTG levels in the plasma, the blood was sampled into a heparin sodium-rinsed tube. After centrifugation at 3000 rpm for 30 min, the supernatant was

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collected, and the protein concentration quantified using the 2-D Quant Kit (GE healthcare). Three fish were sampled for control and BPA treatment group (10 μM) respectively. The level of VTG in the plasma was measured using a zebrafish VTG ELISA Kit (Biosense). The supernatants were added into a 96-well plate after proper dilution according to manufacturer’s instructions. The absorbance of the reaction solution was read on the Infinite M200 PRO microplate reader (TECAN, Männedorf,

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Switzerland) at 492 nm. The concentrations of VTG in samples were expressed as VTG/total protein (ng/μg). 2.7 Fertility test To investigate the effects of BPA on male fertility after 100 days of exposure, six homozygous mutant males (cyp19a1a-/-; all males) were randomly selected from each group (BPA 1 and 10 μM, E2 0.5 nM and negative control) to breed with untreated WT (+/+) females, respectively. Similarly, six induced cyp19a1a-/- females (BPA 10 μM and E2 0.5 nM groups) were also selected to breed with untreated WT males. The spawning rate was determined by the ratio of spawned fish number/six. The The test

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fertilization rate was assessed by the ratio of fertilized eggs/spawned eggs.

was repeated ten times every other day in 20 days (120-140 dpf), and each time we sampled six fish randomly from each treatment group to mate with different WT

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males and females (n=10 tests).

Similar treatment (BPA 10 μM) scheme was also conducted on the quadruple

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mutant male fish (cyp19a1a-/-;esr-/-1;esr2a-/-;esr2b-/-) at 100 dpf to investigate if the adverse effect of BPA on male fertility involved nER-mediated pathways.

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2.8 RNA extraction and real-time qPCR

Total RNA was isolated from different tissues with TRIzol (Invitrogen, Carlsbad,

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CA) according to the manufacturer’s protocol and our previous reports [49, 50], and the concentration was determined on the NanoDrop 2000 (Thermo Scientific, Waltham, MA). Reverse transcription (RT) reaction was performed on total RNA of

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each sample in a final volume of 10 μL reaction solution containing 100 U M-MLV reverse transcriptase, 1×reverse transcription buffer, 0.5 μg oligodeoxythymidine,

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0.5 mM each deoxynucleotide triphosphate and 0.1 mM dithiothreitol (Invitrogen) at 37°C for 2 h.

Semiquantitative RT-PCR was used to detect the expression of all three nERs in

tissues, including gonad, muscle and tail of WT fish at 30 dpf. The assay was carried out on CFX96 real-time system (Bio-Rad, Hercules, CA) in a volume of 20 μL containing 9 μL of cDNA template, 0.2 μM of each primer and 10 μL of 2 x SuperMix (Bio-Rad). The reactions were performed for 25 cycles under the following 9

conditions: 95°C for 20 s, 60°C for 20 s, and 72°C for 30 s. The expression of housekeeping gene ef1a was included as control [51]. The specific primers used in RT-PCR and qPCR were listed in Table S2. The expression levels of esr1, esr2a, esr2b, and ef1α in the testis of cyp19a1a-/adult fish without one form of nER were determined by real-time qRCR on CFX96 real-time system. The reaction was performed as described above for 38 cycles of 95°C for 20 s, 60°C for 20 s, 72°C for 30s, and 84°C for 8s for signal detection. A melting curve analysis was performed at the end of the reaction to demonstrate the specificity of the reaction. Results were normalized to the housekeeping gene ef1α

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[51] by the 2-△△Ct method [52]. Each sample was analysed in triplicate. 2.9 Histological analysis and sex ratio

In the pilot experiment, 15~20 fish were sampled at each time point (65, 100 and 150 dpf) for histological analysis. In the following repeating experiments, 40 fish

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were sampled for sex ratio in each group at 65 dpf. The whole fish or dissected

gonads were immediately fixed in Bouin’s solution for 48 h followed by dehydration

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and embedding in paraffin on the EG1150H tissue embedding system (Leica, Wetzlar, Germany). The samples were sectioned at 5-μm thickness along the sagittal plane of

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the gonad using Leica RM2235 microtome. The sections were then mounted on slides, deparaffinized, rehydrated and stained with hematoxylin and eosin (H&E). The stained sections were dehydrated and mounted with Canada balsam on Leica ST5020

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multistainer. All stained sections were examined on the ECLIPSE Ni-U microscope (Nikon, Tokyo, Japan) and micrographs recorded by the Digit Sight DS-Fi2 digital

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camera (Nikon).

The sex of each sample was determined by histology at 60-65 dpf for sex ratio calculation. The effect of long-term BPA exposure on testis development and

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spermatogenesis was evaluated by the amount of sperm production (area of mature sperm/area of testis). Briefly, the amount of mature sperm was quantified with ImagePro Plus 6.0 software on three sections of each individual, and five individuals were sampled for each treatment group. The effect of long-term BPA exposure on ovarian development was evaluated by follicle composition in terms of areas occupied by different stages of follicles. The average areas occupied by primary growth (PG, stage I), pre-vitellogenic (PV, stage II) and vitellogenic (EV-FG, stage III) follicles were 10

calculated based on quantification on three separate sections per fish, and five individuals were examined and analyzed at each time point. 2.10 Statistical analysis Data were analyzed by Student’s t test or one-way ANOVA followed by the Tukey multiple comparison test using Prism 6 (GraphPad Software, San Diego, CA). All values were expressed as means ± SEM based on multiple independent tests and/or biological repeats (n≥3). The significance level was set at P<0.05.

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3. Results 3.1 Effect of BPA on growth and development

To ensure that the treatment scheme we adopted did not generate significant

toxicological effects on development and growth, we first examined if BPA had any

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impact on body growth of both aromatase mutant (cyp19a1a-/-) and heterozygous

control (cyp19a1a+/-). As shown in Figure S2, no significant difference in standard

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body length and body weight was observed at 65 dpf between the control and BPA treatment group at any concentrations tested (F=1.463, P= 0.1675 for Body length;

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F=1.761, P=0.079 for Body weight). 3.2 Effect of BPA on sex ratio

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When examined at 65 dpf, the cyp19a1a-/- mutant showed all-male phenotype, in agreement with our recent report [40].

To confirm low level of estrogens in

cyp19a1a-/- mutant, we measured the serum E2 levels in adult fish. As shown in

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Figure 1A, no significant difference was observed between WT females and cyp19a1a+/- females.

The E2 levels were significantly lower in males as compared

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to those in females and there was no difference between WT males and cyp19a1a+/male either. For cyp19a1a-/- males, their E2 levels were even lower than those in control males (WT and +/-) (Fig. 1A). Treatment of the heterozygous control fish (cyp19a1a+/-) with BPA increased

female ratios at different concentrations (0.01-10 µM) to 66-90% at 65 dpf as compared to 50% in the control group (0 µM). Interestingly, treatment of the mutant (cyp19a1a-/-) with BPA showed no effect at low concentrations (0.01-1 µM) at 65 dpf, 11

but a phenomenal effect at 10 µM with 88% females as compared to 0% in the control. As the positive control, treatment with E2 (0.5 nM) also increased female ratio to 73% (Fig. 1B). We further repeated the experiment twice focusing on 1 and 10 µM BPA and statistical analysis of three independent experiments further confirmed the results described in Figure 1B (Fig. 1C). 3.3 Effect of BPA on sexual differentiation Our previous studies showed that zebrafish sexual differentiation started around 25 dpf and completed by 35 dpf for females and 45 dpf for males [50, 53]. To provide evidence for BPA interference with sexual differentiation, we performed histological

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analysis at 65 dpf.

As shown in Figure 2A, all individuals of cyp19a1a-/- mutant in the control group (0 μM BPA) were males, which exhibited normal spermatogenesis in testis without any notable abnormalities. As positive control, treatment with E2 at 0.5 nM during

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sexual differentiation rescued the all-male phenotype with more than half of the

treated individuals developing ovaries with perinucleolar (PN) oocytes at PG stage.

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Interestingly, exposure to 10 μM BPA also induced ovarian formation in cyp19a1a-/mutant, similar to the effect of E2. However, no effect on sex ratio was observed with

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1 μM BPA although it obviously retarded spermatogenesis. In agreement with histological observations, the intensity of GFP signal in the gonads (ovary) of most E2 and BPA (10 µM)-treated fish [Tg(vas:EGFP)cyp19a1a-/-] was much stronger than

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that in the control with testis (Fig. 2A). 3.4 Effect of BPA on puberty onset

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In zebrafish, the appearance of cortical alveoli in PV follicles (stage II) is considered a marker of female puberty onset, which normally occurs around 45 dpf when the standard length and body weight reach 1.8 cm and 100 mg respectively [50].

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As described above, both BPA (10 μM) and E2 (0.5 nM) could rescue the all-male phenotype of cyp19a1a-/- mutant by inducing ovarian formation; however, they both suppressed subsequent ovarian growth and follicle activation (PG-PV transition; 65 dpf), therefore delaying or blocking puberty onset in both aromatase mutant fish (cyp19a1a-/-) and their sibling control (cyp19a1a+/-). In addition, the ovaries of BPA (10 μM) and E2 (0.5 nM)-treated fish contained large numbers of dark basophilic oocytes, an indication of cell degeneration. BPA also seemed effective at 1 µM in 12

postponing puberty onset in cyp19a1a+/- control females as shown by the delayed development of PV follicles (Fig. 2B and C). 3.5 Effect of BPA on post-pubertal folliculogenesis In teleosts, endogenous estrogens not only play an important role in ovarian differentiation, but also are essential for ovarian maturation. To examine if BPA interferes with ovarian maturation in cyp19a1a-/- females after rescuing the all-male phenotype of the mutant, we further investigated the developmental status of gonads at 100 dpf in cyp19a1a-/- fish.

Females appeared in the group treated with 10 μM

BPA as described above; however, all follicles in the ovary were arrested at the PG

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stage at 100 dpf (Fig. 3A). There was no significant progression in follicle

development compared with that at 65 dpf (Fig. 2B). In contrast, the follicles in the

ovaries of E2-treated fish could develop to or beyond the previtellogenic (PV) stage, a marker for puberty onset (Fig. 3A). The feminization of fish was further illustrated by

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the enhanced GFP signals in the gonads [Tg(vas:EGFP) cyp19a1a-/-] that developed into ovaries in these two groups.

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We also examined the effects of BPA and E2 on the heterozygote (cyp19a1a+/-) as a control. A full range of follicles from PG to FG (full-grown) stage was observed

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in the ovaries of control (0 µM BPA) and low concentration (1 µM BPA) groups whereas the follicles from 10 µM BPA group were predominantly at PG stage with only a few entering early PV stage (Fig. 3B and C).

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3.6 Effects of BPA on post-pubertal spermatogenesis In addition to ovarian differentiation and folliculogenesis, BPA also significantly

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affected spermatogenesis in both control (cyp19a1a+/-) and mutant (cyp19a1a-/-) fish. When observed at 65 dpf, BPA at low concentration (1 µM) significantly reduced the

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size of testis and delayed spermatogenesis. Most of the spermatogenic cells were cystic spermatogonia with only a few cysts entering the stage of meiotic spermatocytes and no mature spermatozoa in both cyp19a1a+/- and cyp19a1a-/- fish (10 µM BPA). In contrast, the testes in the control group showed normal spermatogenesis with all essential stages including proliferation (spermatogonia), meiosis (spermatocytes) and spermiogenesis (mature spermatozoa) (Fig. 4A and B). The effect of 1 µM BPA diminished with age (100 and 150 dpf) (Fig. 4C). BPA had more prominent and long-lasting effect on spermatogenesis at 10 µM. The 13

spermatogenesis seemed to be blocked at early stage with all spermtogenic cells being cystic spermatogonia and there was little sign of meiotic spermatocytes. As positive control, treatment with E2 also resulted in dysgenesis of testis with few mature sperm at all times examined (Fig. 4A-C). 3.7 Effects of BPA on reproductive performance To examine if long-term BPA exposure has adverse effects on reproductive performance, we determined fecundity of BPA-induced mutant females by mating with WT males or WT females mated with BPA-treated mutant males. The mutant females induced by BPA (10 µM) or E2 (0.5 nM) were infertile, and

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so were males treated with 10 µM BPA. By comparison, the males treated with 1 µM BPA and 0.5 nM E2 were both fertile; however, their fertilities were significantly

reduced compared with the control (Fig. 4D). The eggs fertilized by the sperm from

BPA (1 µM) and E2 (0.5 nM)-treated males showed normal developmental potential

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with the same rates as that of the control (Fig. 4E).

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3.8 Effects of BPA on sex maintenance

Endogenous estrogens are essential for maintaining female status in adults. To explore whether the estrogenic activity of BPA could also maintain feminization in

exposure to BPA.

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adults, we examined gonadal development and status at 150 dpf after long-term

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In cyp19a1a-/- mutant fish, all individuals in control and 1 μM BPA group were males with well-developed testes containing large number of mature spermatozoa in the lumen (Fig. 5A). However, long-term exposure to 10 μM BPA led to no or fewer

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mature spermatozoa compared to the control group (azoospermia or oligozoospermia) in both genotypes, and E2 treatment resulted in even more serious disruption of

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spermatogenesis (Fig. 5B and C). As for females, the ovaries from 10 μM BPA group were full of follicles at late PV stage without accumulation of yolk in cyp19a1a-/mutant; however, the follicles exhibited abnormal morphology with obvious signs of atresia or disintegration. The atretic follicles were often characterized with proliferation of the somatic cells and breakdown of the zona radiata (Fig. 5B). As control, the ovaries from E2-exposed mutant females were also characterized by increased atretic follicles, mostly at PV stage (Fig. 5C). 14

In cyp19a1a+/- fish, treatments with E2 and 10 µM BPA also caused significant dysgenesis of testis with few mature spermatozoa as observed in cyp19a1a-/- mutant males. Different from the ovaries in BPA-induced cyp19a1a-/- mutant females, the ovaries of cyp19a1a+/- fish treated with 0.5 nM E2 and 10 µM BPA contained both PV and vitellogenic follicles with yolk accumulation although many follicles were undergoing atresia (Fig. S3). Despite the lack of vitellogenic follicles in 10 µM BPAtreated mutant fish (cyp19a1a-/-), the expression of vitellogenin (VTG) molecules was significantly increased by BPA treatment, as shown by increased plasma VTG levels (Fig. S4A).

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3.9 Effects of BPA on secondary sexual characteristics Normally, male zebrafish shows a slim body shape with brownish coloration and an invisible urogenital papilla, while female zebrafish has a round body shape with silverish coloration and a protruding urogenital papilla.

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In addition to inducing VTG expression (Fig. S4A), treatment of cyp19a1a-/mutant fish with BPA (10 µM) or E2 (0.5 nM) induced typical secondary sexual

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characteristics of females in most fish treated, including brownish coloration, round body shape and visible urogenital papilla (84% for BPA and 78% for E2) (Fig. 5D

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and Fig. S4B). In contrast, all individuals in control and 1 µM BPA groups were males with typical male secondary sexual characteristics.

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3.10 Reversibility of BPA effects on gonads

As described above, both BPA (10 µM) and E2 (0.5 nM) showed strong feminizing effects on gonadal differentiation during exposure period (20-150 dpf). To

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test the reversibility of such effects, we examined the treated cyp19a1a-/- mutant fish at 210 dpf, which was 60 days after termination of the treatments. The results showed

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that the feminizing effects of both BPA and E2 appeared reversible in the absence of treatments.

As shown in Figure 5E and F, the follicles in the ovaries of induced

females were all undergoing atresia, and testicular tissues showing active spermatogenesis with mature spermatozoa started to appear in the gonads, mostly at the edge of the ovaries. 3.11 Signalling mechanism of BPA in inducing ovarian differentiation

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To determine whether and how nERs are involved in BPA-mediated feminization of gonadal differentiation, we first examined the expression of three nERs (esr1, esr2a and esr2b) at 30 dpf in the gonads of WT fish undergoing sex differentiation. All three nERs were expressed in the gonads (Fig. S5). As reference, their expression could also be detected in the muscle, but not in the tail. To examine whether the loss of single nER would affect the expression of the other two forms, we then analyzed the expression levels of nERs in the testis of each double mutant combination (cyp19a1a-/- with single nER mutation). We did not observe significant difference in expression level of the other two nER subtypes after knocking out one

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nER with cyp19a1a-/- (Fig. S6). We then performed a series of experiments on BPA actions by using aromatase

mutant (cyp19a1a-/-) in combination with single (esr1-/-, esr2a-/- and esr2b-/-), double

(esr1-/-;esr2a-/-, esr1-/-;esr2b-/- and esr2a-/-;esr2b-/-) and triple (esr1-/-;esr2a-/-;esr2b-/-)

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nER mutants. The cyp19a1a-/- mutant in the presence of one or two nERs would

provide valuable information about the receptor types involved in BPA signalling.

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Consistent with the results described earlier, treatment of cyp19a1a-/- alone with BPA rescued the all-male phenotype with 68% fish being females. The ovaries of

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these females were full of follicles at PG stage (Fig. 6A). However, when the same treatment was performed on the quadruple mutant, which lacks endogenous estrogens and nERs, no female individuals (0%) were found (Fig. 6A and D). Histological

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analysis at 60 dpf showed that most individuals (85%) in quadruple mutant group had typical testis structure and the rest had perinucleolar oocytes scattered among testicular tissues (ovotestis). These oocytes disappeared completely in all samples

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examined at 80 dpf (Fig. 6A).

To characterize the nERs involved in mediating BPA actions, we performed

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further experiments by using three different triple knockouts without cyp19a1a and two nERs, but with one nER present. We found no females in cyp19a1a-/-;esr1/-

;esr2a-/- and cyp19a1a-/-;esr2a-/-;esr2b-/- mutants; in contrast, nearly half of

cyp19a1a-/-;esr1-/-;esr2b-/- mutant fish were females, comparable to the control of cyp19a1a-/- mutant alone (Fig. 6B and D). These results strongly suggested a critical role for esr2a in mediating the feminizing effect of BPA during gonadal differentiation. 16

To further test this hypothesis, we performed the third set of experiments on all three double knockouts with mutations of cyp19a1a and one nER. Consistent with the results from the triple knockouts, the absence of Esr2a (cyp19a1a-/-;esr2a-/-) resulted in the lowest ratio of females compared to Esr1 and Esr2b, whose absence did not change the sex ratios compared to the control of single cyp19a1a-/- mutant (Fig. 6C and D). 3.12 Signalling mechanism of BPA in disrupting spermatogenesis Interestingly, the absence of nERs also rescued the adverse effect of BPA on male fertility. In contrast to BPA (10 µM)-treated cyp19a1a-/- mutant males that were

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infertile (Fig. 4D) with few mature spermatozoa in the testicular tubular lumen (Fig. 5B), the BPA-treated quadruple mutant males (cyp19a1a-/-;esr1-/-;esr2a-/-;esr2b-/-) could spawn normally at 100 dpf with WT females with fecundity comparable to

those mated with WT control males (Fig. 6E). This was also supported by histological

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examination at 80 dpf, which showed normal spermatogenesis in the testes of the

quadruple mutant males with abundant mature spermatozoa in the tubular lumen (Fig.

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6A; fish 5 and 6).

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4. Discussion

Zebrafish offers obvious biological advantages over mammalian models for

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studying e-EDCs due to its labile sex determination and differentiation, high fecundity, rapid development, and easy amenability to drug treatment [54]. Numerous studies have been performed on BPA impact on zebrafish at different time points of

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life cycle [55-60]. Exposure of zebrafish embryos to different concentrations of BPA induced a significant skew in sex ratio towards females [5, 61]. Plasma levels of VTG

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increased significantly in both male and female zebrafish after exposure to BPA [6264]. Since sex ratio and VTG induction are key endpoints to evaluate e-EDC-induced feminization in fish [31, 65, 66], these studies confirmed estrogenic actions of BPA in fish species.

Despite increasing importance of in vivo studies on e-EDCs, there are some drawbacks in the application of animal models such as zebrafish. First, due to the existence of aromatase enzyme that produces high levels of endogenous estrogens in 17

females, the feminizing effect of estrogenic exposure could be easily masked, especially to e-EDCs which often have weak estrogenic activities as compared to natural estrogens. This could be one of the major reasons for discrepancies among different studies about feminizing effects of EDCs. Second, most e-EDCs have both nER-dependent and -independent activities [67]; however, there is a lack of appropriate methods to distinguish these two activities in vivo. Third, despite some studies on receptor specificity of e-EDCs in vitro [20, 21, 68], it is difficult to perform such studies in vivo under physiological conditions. To overcome these shortcomings, we undertook this study by using a low-estrogen mutant zebrafish deficient of ovarian

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aromatase (cyp19a1a-/-) for evaluating feminizing effects of BPA on gonadal differentiation and development [40], and the zebrafish lacking one or more nER(s) for dissecting receptor mechanisms underlying BPA action.

As shown by our data, the control zebrafish (heterozygote cyp19a1a+/-) had a

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female ratio of about 50%. Treatment of the heterozygote with E2 (0.5 nM) and BPA (0.01-10 μM) led to feminization to different degrees (16-40% increase in female

ratio). This agrees with the data of our previous study and others [5, 61]. However, the

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response could be easily dampened by the high background female ratio (50%) due to endogenous estrogens. In contrast, the low-estrogen mutant (cyp19a1a-/-) showed all-

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male phenotype with 0% females, providing a clean and consistent baseline for demonstrating estrogenic activities of any chemicals. As expected, the female ratios in the cyp19a1a-/- mutant exposed to E2 (0.5 nM) and BPA (10 μM) reached 78 and 88%

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respectively as compared to 0% in the control, much cleaner and higher than the responses of 16%-40% detected in the control fish with aromatase (cyp19a1a+/-).

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This result provides unequivocal evidence for estrogenic activity of BPA in inducing gonadal differentiation in the zebrafish model with its potency at 10 µM equivalent to

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or stronger than that of E2 at 0.5 nM. Interestingly, we did not find any females at lower BPA concentrations (0.01-1

nM). Therefore, the ovarian aromatase mutant (cyp19a1a-/-) provides a clean background for testing feminizing effects of e-EDCs with increased responsiveness, but it does not seem to offer higher sensitivity.

Our hypothetical explanation is that

without endogenous estrogens, the expression levels of nERs or other estrogen signalling molecules may be low, resulting in a lower sensitivity of the animal to estrogenic exposure. We will test this hypothesis in future studies by priming the fish 18

with low levels of estrogens prior to the treatments at 20 dpf, followed by examining nER expression levels and responsiveness to low BPA concentrations.

It has been

well documented that estrogens can up-regulate nERs in various cell and animal systems [69, 70]. After inducing ovarian formation, BPA suppressed its growth and blocked follicle activation, resulting in delayed or failed puberty onset. The females induced by both E2 and BPA in the cyp19a1a-/- mutant were therefore infertile. The impairments of follicle development, including follicle cell proliferation and zona radiata breakdown, agree well with previous reports [6, 61, 71]. The retardation of follicle development in

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BPA-induced mutant females (cyp19a1a-/-) was probably due to the weak estrogenic activities or toxicity of BPA. These results suggest that the estrogenic activity of BPA at 10 μM can drive gonadal differentiation towards ovary but is not potent enough to maintain its further development.

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As for males, a concentration-dependent suppression of testicular development and spermatogenesis was observed after long-term exposure to BPA. BPA

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significantly inhibited spermatogenesis even at lower concentrations. Our results support the previous reports in both zebrafish [31] and other species such as fathead

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minnows [72] and mice [15]. However, the mechanisms underlying the impairment of spermatogenesis by BPA are unclear.

Our data provided strong evidence for the

involvement of nERs in mediating BPA action.

The lack of nERs in the quadruple

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mutant could rescue the phenotypes induced by BPA in cyp19a1a-/- including spermatogenesis and male fertility.

Numerous in vitro studies have shown that BPA exerts many of its effects by

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binding to nERs to change the expression of estrogen-responsive genes, although its estrogenic potency is lower than that of E2 by several orders [73-75]. Besides the

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classical nER signalling pathways, some estrogenic effects of BPA may be mediated via nER-independent mechanisms. For example, G-protein-coupled receptor (GPER30) and orphan nuclear estrogen-related receptor γ (ERRγ) have been shown to bind BPA with high specificity [76-79]. In addition, BPA can also act by interfering with other nuclear hormone receptors, such as androgen, thyroid hormone and glucocorticoid receptors [80-82] as well as pregnane X receptor (PXR) [83]. these studies, it remains largely unknown how these receptors are involved in 19

Despite

mediating BPA actions in vivo in any physiological processes.

Pharmacological

approaches using aromatase inhibitors and estrogen receptor antagonists have been attempted with certain success [23, 55, 84-86]. For example, BPA-induced estrogenic responses in an estrogen-responsive transgenic zebrafish Tg(ERE:Gal4ff)(UAS:GFP) were inhibited by nER inhibitor ICI 182780, indicating an estrogen receptordependent mechanism [23]. However, all pharmacological drugs used were initially developed in mammalian species and they have not been well validated in fish models. To address the question of how BPA signals in vivo and the roles of nERs, we

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generated unique zebrafish lines deficient of cyp19a1a and various nERs (esr1, esr2a and esr2b) alone or in combination. Using these genetic tools, we demonstrated clearly that BPA exposure induced ovarian differentiation and female sexual

characteristics in the absence of ovarian aromatase and therefore low level of

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endogenous estrogens. Interestingly, the feminizing effects of BPA were abolished when using the quadruple knockouts of aromatase and all three nERs (cyp19a1a/-

;esr1-/-;esr2a-/-;esr2b-/-), strongly implicating nERs in mediating BPA-induced As discussed above, the lack of nERs also ameliorated the

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ovarian formation.

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impairment of BPA on spermatogenesis.

To further characterize the specific role of each nER in the action of BPA, we tested BPA induction of ovarian differentiation in cyp19a1a mutant plus single, Interestingly, all our results pointed to esr2a as the

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double or triple nER mutations.

indispensable form of nER for the feminizing effect of BPA on sexual differentiation. Whenever esr2a gene was absent (cyp19a1a-/-;esr2a-/-, cyp19a1a-/-;esr1-/-;esr2a-/-,

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cyp19a1a-/-;esr2a-/-;esr2b-/- and cyp19a1a-/-;esr1-/-;esr2a-/-;esr2b-/-), the feminizing effect of BPA on gonadal differentiation was blocked or suppressed, and the effect

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was obvious as long as esr2a was present (cyp19a1a-/-, cyp19a1a-/-;esr1-/-, cyp19a1a/-

;esr2b-/-, cyp19a1a-/-;esr1-/-;esr2b-/-). The function of Esr2a might be compensated

weakly by the other two forms (Esr1 and Esr2b). Our finding is consistent with our previous observation in zebrafish that esr2a

showed the highest level of expression among the three during folliculogenesis and was the only form expressed in both the oocyte and follicle cells. By comparison, esr1 and esr2b were expressed only in the somatic follicle cells [87]. 20

In vitro studies

in mammals also indicated that BPA had a higher affinity for ERβ than ERα in target cells [39, 73, 88, 89].

However, in reporter cell lines stably expressing zebrafish

nER subtypes, BPA exhibited significant difference in activating the three nERs in comparison to human nERs. BPA displayed higher potency for stimulating Esr1 compared to Esr2a and 2b [59, 90-92]. These data suggest that in vitro studies on cultured cells may not be easily extrapolatable to physiological processes in vivo such as gonadal differentiation.

An in vivo study in the mouse showed that in utero

exposure to BPA during mid-gestation disrupted oocyte meiosis in the fetal ovaries, which mimicked the effect of ERβ null mutant. Interestingly, treatment of the ERβ null mouse with BPA produced no additional aberrant effects, suggesting that BPA

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exerts its effect on the early oocyte by interfering with the actions of ERβ [9]. Our

study in zebrafish now provides new genetic evidence for dominant roles of esr2a in mediating the feminizing effects of BPA on gonadal differentiation.

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In conclusion, the low-estrogen mutant zebrafish provides an excellent model for evaluating estrogenic effects of EDCs, especially in the reproductive system. Using this mutant line, we provided unequivocal in vivo evidence for estrogenic effect of

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BPA on sexual differentiation. In addition, long-term exposure to BPA also had adverse effects on other aspects of reproduction, including delayed or failed puberty

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onset, retarded sexual maturation, impaired gametogenesis (folliculogenesis and spermatogenesis) and failed spawning. On the other hand, the zebrafish nER mutants provide valuable tools for understanding how each nER is involved in mediating the

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actions of e-EDCs. Using these mutants (single, double and triple receptor mutants), we demonstrated an indispensable role for esr2a in mediating the feminizing action of

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BPA in gonadal differentiation. The aromatase and nER mutants together provide a

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novel and powerful genetic platform for studying e-EDCs in the zebrafish model.

Author Contribution Statement Weiyi Song : perform research Huijie Lu: mutant generation Kun Wu: analyzed data 21

Zhiwei Zhang: edited the paper Esther Shuk-Wa Lau: software Wei Ge: design experiment

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Conflict of interest: The authors declare no conflict of interest.

Acknowledgments

This study was supported by grants from the University of Macau (MYRG2015-

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00227-FHS, MYRG2016-00072-FHS, MYRG2017-00157-FHS and CPG2014-

00014-FHS) and The Macau Fund for Development of Science and Technology

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(FDCT114/2013/A3, FDCT089/2014/A2 and FDCT173/2017/A3) to W.G. We thank the Histology Core of the Faculty of Health Sciences for technical

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support, and Ms. Crystal Hang Ieng Leong and Ms. Fong Heitong for fish

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maintenance.

22

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Figure legends

Fig. 1. Serum estradiol level in cyp19a1a-/- mutant and its sex ratio response to BPA exposure. (A) Adult serum estradiol levels of different genders and genotypes of cyp19a1a.

The values are expressed as mean ± SEM (n=8) and analysed by one-way

ANOVA followed by the Tukey HSD for multiple comparisons. Different letters indicate statistical significance (p< 0.05). (B) Female ratios of homozygous (cyp19a1a-/-) and heterozygous (cyp19a1a+/-) zebrafish in response to BPA and E2 30

treatments from 20 to 65 dpf (around 60~70 fish per group). (C) Statistical analysis on sex ratio response to BPA and E2 exposure. The values are expressed as mean ± SEM from three independent experiments (n=3) and analyzed by one-way ANOVA

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followed by the Tukey HSD for multiple comparisons. * P<0.05; *** P<0.001.

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Fig. 2. Effect of BPA on sexual differentiation and female puberty onset. (A) GFP expression of representative fish in the gonads of the transgenic line Tg(vas:EGFP).

Gonadal histology of cyp19a1a-/- mutant fish treated with BPA (1 and 10 μM) and E2 (0.5 nM) from 20 to 65 dpf. Fish 1-4 are representative individuals of BPA and E2 groups respectively. (B) Gonadal histology of heterozygote cyp19a1a+/- fish treated with BPA and E2 at same timepoint. (C) Follicle composition in the ovary of cyp19a1a+/- fish treated with BPA and E2 groups respectively. Three separate sections were quantified and averaged for each fish, and five fish were used for statistical analysis for each group (mean ± SEM; n = 5). PG (stage I), primary growth follicle; PV (stage II), pre-vitellogenic follicle; EV (stage III), early vitellogenic; sg,

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spermatogonia; sc, spermatocytes; sz, spermatozoa; Asterisks indicate degenerative

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Fig. 3. Expression of GFP in the gonads of the transgenic line Tg(vas:EGFP). Gonadal histology of cyp19a1a-/- mutant (A) and heterozygous control (cyp19a1a+/-) (B) treated with BPA (1 and 10 μM) and E2 (0.5 nM) at post-pubertal stage (100 dpf).

The fish shown (1-4 for each genotype) were representative

individuals of different groups. The photos at the bottom are magnified images of the boxed areas. (C) Follicle composition in the ovary of cyp19a1a+/- fish treated with 33

BPA and E2. Three separate sections were quantified and averaged for each fish, and the averages of five fish were used for statistical analysis for the control and treated fish (mean±SEM; n=5). PG (stage I), primary growth follicle; PV (stage II), previtellogenic follicle; EV-FG (stage III), early vitellogenic; FG, full-grown. sg,

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spermatogonia; sc, spermatocytes; sz, spermatozoa.

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Fig. 4. Effect of BPA on spermatogenesis in males, sperm production and spawning rate. (A-B) Gonadal histology of homozygous cyp19a1a-/- and heterozygous cyp19a1a+/- fish treated with BPA (1 and 10 μM) and E2 (0.5 nM) from 20 to 65 dpf. (C) Sperm production (area of mature sperm/area of testis) of treated homozygous mutant males were also calculated at 65, 100 and 150 dpf. The average of the three sections was calculated to represent the individual. Five fish in each group were used for statistical analysis (mean±SEM; n = 5). One-way ANOVA, followed by the Tukey HSD for multiple comparisons, different letters in each dataset indicate

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statistical significance (p< 0.05). (D) Fertility test of cyp19a1a-/- mutant fish treated

with BPA and E2. For males, six treated fish (cyp19a1a-/-) were randomly selected from each treatment group to mate with untreated WT females. Similarly, six

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induced females (cyp19a1a-/-) were randomly sampled from each group to mate with WT males. The spawning rate is defined as the ratio of successful spawning in each The test was repeated ten times at 2-day interval (from

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test (spawned fish/six fish).

120 to 140 dpf), each time with six new fish sampled from each group (n=10).

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were analyzed by One-way ANOVA and the Tukey HSD for multiple comparisons. Different letters in each dataset indicate statistical significance (p< 0.05). (E)

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Fertilization rate of cyp19a1a-/- mutant fish treated with BPA and E2, n=10. One-way

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ANOVA (p＞0.05, no significance). sz, spermatozoa. WT, wild type.

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Fig. 5. Effects of long-term exposure to BPA and E2 on gonads, secondary sexual characteristics in cyp19a1a-/- mutant, and their reversibility. (A-C) Gonadal histology

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of cyp19a1a-/- mutant fish treated with BPA and E2 at 150 dpf. (D) Urogenital papilla (1-4) and body colour (5-8) of cyp19a1a-/- mutant fish in each treatment group at 150 dpf. Morphology and gonadal histology of induced female individuals (cyp19a1a-/-) at 210 dpf, two months after cessation of drug administration (150 to 210 dpf). One representative individual is shown for BPA (10 μM) (E) and E2 (0.5 nM) (F) group, respectively. sz, spermatozoa. PV, previtellogenic follicle. 37

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Fig. 6. Characterization of estrogen receptors involved in feminizing effect of BPA on gonadal differentiation. Gonadal histology of cyp19a1a-/- mutant and quadruple (A), triple (B) and double (C) knockouts with different combinations of nER mutations. The fish were treated with BPA (10 μM) for 40 days from 20 to 60 dpf (80 dpf was also included for the quadruple knockout). Representative individuals are shown for different mutant combinations. (D) Sex ratios of cyp19a1a-/- mutant with different 38

nER combinations after exposure to BPA (10 μM). Data were pooled from three separate experiments. In total, 788 fish were genotyped for different mutation combinations.

“N” represents the fish number in the corresponding genotype. (E)

Spawn rate of quadruple mutant males (NR; cyp19a1a-/-; esr1-/-; esr2a-/-; esr2b-/-) treated with BPA (10 μM) at 100 dpf. To see whether the absence of all estrogen receptors can delete the effect of BPA on spawning. Independent-samples t-test (p＞

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0.05, n=10). ns, no significance.

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