Unexpected Observations: Exposure to Aromatase Inhibitor

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Ecotoxicology and Human Environmental Health

Unexpected Observations: Exposure to Aromatase Inhibitor Prochloraz did not Alter Vitellogenin Content in Zebrafish Ova, but Inhibited Growth of Larval Offspring Yao Dang, Qian Sun, Robert J. Letcher, and Chunsheng Liu Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00486 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 28, 2018

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Environmental Science & Technology Letters

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Unexpected Observations: Exposure to Aromatase Inhibitor Prochloraz

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did not Alter Vitellogenin Content in Zebrafish Ova, but Inhibited

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Growth of Larval Offspring

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Yao Dang¶, Qian Sun¶, Robert J. Letcher‡, Chunsheng Liu¶,†,*

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¶Hubei

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Huazhong Agricultural University, Wuhan 430070, China

Provincial Engineering Laboratory for Pond Aquaculture, College of Fisheries,

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†Collaborative

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Hunan Province, Changde 415000, China

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‡Ecotoxicology

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National Wildlife Research Centre, Carleton University, Ottawa, Ontario K1A 0H3,

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Canada

Innovation Centre for Efficient and Health Production of Fisheries in

and Wildlife Health Division, Environment and Climate Change Canada,

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*Author for correspondence:

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Tel: 86 27 87282113

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Fax: 86 27 87282114.

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Email: [email protected] (C. Liu)

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ACS Paragon Plus Environment


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ABSTRACT: Sparse information exists for evaluation of effects on ova derived from

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estrogen inhibition in fish. In this study, two exposure experiments were performed.

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Firstly, adult zebrafish were exposed to aromatase inhibitor prochloraz (PCZ) (0, 3, 30 or

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300 μg/L) for 21 days, and developmental toxicity of larval offspring was evaluated. PCZ

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markedly decreased 17β-estradiol (E2) and vitellogenin (vtg) concentrations in plasma of

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females, and suppressed spawned egg number. PCZ exposure caused a bioaccumulation

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in ova and resulted in a significant decreased in the body length of larval offspring in 300

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μg/L PCZ, but unexpectedly it did not alter vtg content in ova. Secondly, adult zebrafish

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were exposed to 0 or 300 μg/L PCZ for 21 days, and responses of transcriptome and

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proteome in ova were characterized. Although 21 day treatment with PCZ resulted in

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bioaccumulation of PCZ in ova, it was not responsible for the observed inhibition of body

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length of larval offspring as evidenced by PCZ microinjection. Furthermore, exposure of

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adult zebrafish to PCZ significantly down-regulated mRNA expression of the retinol

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binding protein 4 (rbp4) and changed protein concentrations related to mitochondrial

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energy metabolism in ova, and those changes might be responsible for the inhibition

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growth of larval offspring.

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■INTRODUCTION

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The widespread presence of endocrine-disrupting chemicals (EDCs) in various

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(aquatic) environmental media has been a critical contributor to the concerns of the

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public about the possible long-term effects on fish, including population declines.1 In the

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past few decades, a number of environmental EDCs have been reported to interference

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with sex hormones in exposed aquatic biota, and therefore much attention has been paid

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to these environmental pollutants given that effects could be manifested in the functional

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alteration of the reproductive system and the subsequent aggravation of fish population

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declines.1-3

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Estrogens are pivotal to the function and regulation of sexual differentiation,

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gonadal maturation and reproduction.4 In fish, estrogens stimulate the liver to produce the

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yolk protein precursor vitellogenin (vtg), which is an essential ingredient for

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vitellogenesis in oocytes4 and providing the necessary nutrients for embryogenesis in

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offspring.5 Previous studies have confirmed that inhibition of estrogen synthesis in fish

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could induce a cascading series of events, including the reduction of plasma vtg,

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fecundity, and impairment of oocyte development.6 For example, exposure of fathead

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minnows (Pimephales promelas) to the non-steroidal aromatase inhibitor fadrozole

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caused a dose-dependent decrease of 17β-estradiol (E2) and vtg concentrations in female

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plasma, retarded ovary maturation as shown by a histological assessment of ovaries, and

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reduced the cumulative number of eggs spawned.7 Similarly, exposure to another 3



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aromatase inhibitor, the imidazole fungicide prochloraz (PCZ), significantly decreased

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concentrations of E2 and vtg in plasma and reduced fecundity in fathead minnow and

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Japanese medaka (Oryzias latipes), and led to a compensatory up-regulation of

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hypothalamic–pituitary–gonadal axis genes.8, 9 However, little information is available to

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evaluate the effects of estrogen inhibition on ova (unfertilized eggs) in fish.

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In this study, we hypothesized that inhibition of estrogen synthesis in female fish

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would decrease vtg content in released ova and further affect larval development of

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offspring. To test the hypothesis above, adult zebrafish (Danio rerio) were exposed to

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PCZ, an aromatase inhibitor in zebrafish,10-13 at different concentrations for 21 days, and

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the reproductive toxicity and effect on developmental endpoints of larval offspring were

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evaluated. Here, PCZ was used as a model chemical since results of previous studies

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demonstrated that exposure to PCZ in fish significantly decreased E2 concentrations in

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plasma and fecundity.8,

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concentration of PCZ and its metabolite in ova were examined to explore possible toxic

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mechanisms.

9, 11, 12, 14

Furthermore, transcriptome, proteome and the

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■MATERIALS AND METHODS

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Zebrafish Maintenance and the 21-Day Fish Reproduction Assay. The

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information related to chemicals and reagents were provided in the Supporting

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Information. Sexually mature sixteen week-old zebrafish (AB strain) were raised in

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flow-through aquariums using previously reported protocol.12 The exposure experiments 4


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consisted of two phases (Figure S1). Firstly, a fish short term reproduction assay was

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performed.15 Sexually mature zebrafish were exposed to 0, 3, 30 or 300 μg/L PCZ for 21

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days, and fecundity was evaluated. During the final three days of the exposure, ova were

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collected for the quantification of PCZ and the metabolite 2,4,6-trichlorophenol

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(2,4,6-TCP). Zebrafish were paired to spawn in clean water (without PCZ) and embryos

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were collected and cultured to evaluate developmental toxicity of offspring. After

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exposure, females and males (these fish have never been used for mating spawn and ova

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collection) were euthanized with 0.03% MS-222, and body mass and gonads were

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weighed to determine the gonadal-somatic index (GSI). Blood was collected from the

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caudal vein in each fish for the measurement of sex steroid hormones and vtg. The related

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details were provided in the Supporting Information.

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Secondly, based on the results of Phase 1, in Phase 2 the zebrafish were cultured as

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above and sexually mature zebrafish were exposed to 0 or 300 μg/L PCZ for 21 days with

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3 replicate aquariums in each concentration (15 males and 15 females per aquarium).

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During the final three days of exposure, fish from the exposure groups were mated with

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unexposed fish from the control group in breeding tanks containing clean water (without

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PCZ) to determine the source of abnormal phenotype in offspring. Mating groups

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consisted of unexposed males and unexposed females (control group); exposed males and

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unexposed females (exposed male group, EM group); exposed females and unexposed

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males (exposed female group, EF group). Resultant embryos were collected, cultivated,

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and endpoint parameters were assessed. The ova were immediately collected as described 5



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above during the final three days of the exposure, and then frozen in liquid nitrogen and

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stored at -80C for subsequent measurement of vtg contents, and analyses of the

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transcriptome and proteome.

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Measurements of Sex Steroid Hormones in Plasma. To obtain plasma, the whole

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blood of zebrafish were centrifuged (5,000 × g，5 min, at 4℃) and supernatants were

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collected. Plasma from 3-6 fish of the same sex was pooled together to form a replicate

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sample (10 μL) (n = 3 per group) for the measurement of sex steroid hormones (E2 and

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T). The contents of E2 and T in plasma were determined by use of Cayman EIA kits

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following previously reported methods.16 The details were provided in the Supporting

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Information.

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Vtg Content Measurement in Plasma and Released Ova. Zebrafish plasma was

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obtained from each group as above. A 10 μL plasma sample pooled from 3-6 females was

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diluted using dilution buffer provided in the vtg ELISA kit and was considered as one

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replicate sample. In order to validate our hypothesis, the ova from control and 300 μg/L

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PCZ groups were collected for the measurement of vtg contents. The ova were

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homogenized on ice in 0.5 mL of saline (0.9% sodium chloride) and then ova

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homogenates were centrifuged at 12, 000 g for 15 min at 4 °C. The ova supernatants were

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diluted using dilution buffer. The diluted plasma and ova supernatant were used to

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measure the vtg content according to the manufacturer’s instructions with the vtg ELISA

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kit. The detection limit was 0.12 ng vtg/mL dilution buffer.

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LC/MS Quantification of PCZ and 2,4,6-TCP in Released Ova and PCZ 6


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Microinjection Validation. In this study, quantification of PCZ and its metabolite

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2,4,6-TCP in ova was performed. Ova from 3-6 females from the same aquarium were

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pooled as a biological replicate (about 50 mg per replicate) and 3 replicates per

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concentration. The method limits of quantification of PCZ and 2,4,6-TCP were 0.05 µg/g

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and 0.5 µg/g, respectively, and the instrument limits of detection of PCZ and 2,4,6-TCP

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were 0.5 μg/L and 5 μg/L, respectively. The mean recoveries of PCZ and 2,4,6-TCP for

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zebrafish ova were 90 to 95 % and 75 to 85 %, respectively. The details were provided in

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the Supporting Information.

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In order to determine whether accumulated PCZ in ova due to parental exposure of

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PCZ was responsible for the observed inhibition of growth in offspring, the content of

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PCZ in each ovum from the 300 µg/L exposure group was calculated (1.6 ng PCZ/ovum).

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After that, zebrafish embryos which were produced by unexposed adult zebrafish were

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used for PCZ microinjection experiment, where the microinjection dose of PCZ was

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theoretically equal to the calculated dose of PCZ (ng PCZ/ovum) in each ovum above.

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The details were provided in the Supporting Information.

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Transcriptomics and Proteomics. In this study, the ova transcriptomics and

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proteomics sequencing were performed to identify responses of mRNA and protein

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in .released ova following PCZ exposure of the female. Ova from three females in each

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aquarium were collected and pooled as a biological replicate (n=3 replicates) for RNA

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sequencing by use of methods described previously.17, 18 The overall protocol included

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RNA isolation, cDNA library construction, sequencing and bioinformatics analysis. This 7



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information is found in the Supporting Information. For proteomics analysis, ova

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collected from three-six females in same aquarium were pooled as one sample (about 150

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mg), and each group contained three biological replicates. Quantitative proteomic

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analysis was conducted in accordance with the previously reported methodology.19, 20 The

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details were provided in the Supporting Information.

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Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Validation.

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Expression of retinol binding protein 4 (rbp4) that is involved in larval development and

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its expression was significantly changed after PCZ exposure was validated by use of

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qRT-PCR. qRT-PCR were carried out with Minimum Information for Publication of

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Quantitative Real-Time PCR Experiment (MIQE) Guidelines.21, and the details were

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provided in the Supporting Information. Relative expression of target gene compared

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with the control was calculated by 2−ΔΔCt method.22

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Statistical Analysis. The Kolmogorov−Smirnov test and Levene’s test were

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performed to check normal distribution and homogeneity of variances of data,

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respectively. Significant differences (p < 0.05) between the control group and exposure

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groups were determined by use of one-way analysis of variance (ANOVA) followed by

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the Tukey test. To analyze the data including vtg content of ova, embryo microinjection,

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transcriptome and proteome, a one-tailed independent-sample t test was used to

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determine significance between the control group and the PCZ treatment group.

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Differentially expressed mRNAs and proteins in transcriptomics and proteomics were

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selected based on padj < 0.05 and p < 0.05, respectively. All data were expressed as 8


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mean ± SEM.

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■RESULTS AND DISCUSSION

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Reproductive Endocrine Disruption Effects of Zebrafish Exposed to PCZ. PCZ is an

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inhibitor of cytochrome P450 c17α-hydroxylase/17,20-lyase (CYP17) and aromatase

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(CYP19) activity in vertebrates, which are key steroidogenic enzymes involved in the

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synthesis of T and the catalyzed conversion of T to E2, respectively.14 After exposure for

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21 days, the highest dose of PCZ (300 µg/L) significantly reduced plasma concentrations

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of E2 (734.83 ± 82.44 pg/ml, p = 0.04), GSI (9.65 ± 0.85, p = 0.001) and average

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production of eggs (19.92 ± 1.46, p = 0.006) in female zebrafish compared with the

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plasma concentrations of E2 (1174.50 ± 66.55 pg/ml), GSI (16.23 ± 1.27) and the

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average production of eggs (64.74 ± 5.19) in the control group. In males, PCZ did not

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change GSI, or the plasma concentrations of E2 or T (Figure 1A-D). Results of previous

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studies have confirmed that short-term exposure of adult fish (e.g., fathead minnow,

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Japanese medaka and zebrafish) to PCZ (300 μg/L) prominently depressed plasma

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concentrations of E2 in females, and decreased GSI and production of eggs.8, 9, 11, 12, 14

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PCZ was an effective inhibitor of E2 production in fish,23 and female fish were more

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sensitive to anti-estrogenic effects of the PCZ than male fish,12, 14 which are consistent

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with our present results. E2 acts directly on oogonial proliferation,24 and promotes

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formation of cortical alveolar oocytes and induces hepatic vtg production in female fish,25

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and exerts pivotal roles in the regulation of ovary development and reproduction. 9



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Because vtg acts as the precursor for egg yolk synthesized in liver in response to

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endogenous estrogen, its production is closely correlated to plasma E2 concentrations.26

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Results of this study affirmed this relationship, as plasma vtg concentrations were

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significantly decreased (4.77 ± 0.44 ng/mL, p = 0.026) in females from 300 μg/L

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exposure group compared with the control group (13.23 ± 1.32 ng/mL) (Figure 1E).

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Ovarian follicle enlargement needs uptake of vtg from the plasma by the oocyte for

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production of yolk, facilitating oocyte maturation.25 Plasma concentrations of vtg in

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female fish could therefore indirectly reflect egg production.27, 28 It was hypothesized that

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the lower egg production and reduced GSI in the present study were the result of

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inhibited ovarian E2 synthesis, and reduced liver vtg synthesis.

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PCZ Effects on Larval Offspring. To the best of our knowledge, we report for the

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first time that for adults of a fish species exposed to PCZ, there was growth inhibition of

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larval offspring. No significant differences in survival rate, hatching rate and

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malformation incidence of the larval offspring were found following exposure of the

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parent zebrafish to different concentrations of PCZ (0, 3, 30 and 300 μg/L) for 21 days

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(Figure S2). However, compared with control group (3.91 ± 0.01 mm), prominent

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inhibition of the body length of larval offspring was observed (3.42 ± 0.02 mm, p =

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0.001) with exposure to 300 µg/L PCZ (Figure 1F). Because the body length of larvae

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was evidenced as one of main limitation factors at the first feeding of fish,29 inhibition of

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the body length of larval offspring might affect larval viability during the first stages of

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exogenous feeding and further cause a potential threat to fish population. Furthermore, a 10


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significant decrease of body length in offspring was observed (3.55 ± 0.02 mm. p =

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0.001) in EF group while body length (3.85 ± 0.01 mm, p > 0.05) of offspring in EM

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group was not changed (Figure 2A). These results suggested that body length inhibition

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of larval offspring observed was attributed to the PCZ-exposed females. PCZ exposure

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inhibited the E2 synthesis and subsequently decreased vtg content in female plasma.

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Because vtg contains amino acids, lipids, metal ions, phosphates and carbohydrates, and

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thus provides the necessary nutrients for embryonic development of fish offspring.5 We

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hypothesized that vtg content in released ova would be decreased due to exposure of

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female adult fish to PCZ, resulting growth inhibition of larval offspring. To test our

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hypothesis, vtg content was determined in ova derived from female fish exposed to 300

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μg/L PCZ. The results were unexpected. There was no significant difference (t test, p >

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0.05) in vtg content of ova between the control and 300 μg/L PCZ groups (Figure 2B),

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indicating that PCZ did not alter vtg concentration per ova, but instead the number of ova

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produced (fecundity, Figure 1A) was reduced. It was hypothesized that vtg was important

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for the development and maturation of ovum, and thus the decrease in vtg concentration

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in plasma might lead to reduction of egg production due to the ability of zebrafish to

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maintain vtg content of ova at a threshold level. Our results may indicate a correlation

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between maternal vtg concentrations with PCZ exposure and a subsequent reduction in

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fecundity.

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Concentration-Dependent Accumulation of PCZ in Zebrafish Ova and

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Microinjected Embryos. We detected a dose-dependent accumulation of PCZ in the 11



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zebrafish ova (Table S4). On the basis of results of a previous study,30 2,4,6-TCP is

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generally considered as a primary metabolite of PCZ, and a phenolic compound (e.g.,

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2,4,6-TCP) that could further undergo rapid phase I and phase II metabolism to generate

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more water-soluble glucuronide and sulfate conjugates.31 In this study, concentrations of

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2,4,6-TCP in zebrafish ova were less than the method limits of quantification, which

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could possibly be due to (1) the low transformation rate of 2,4,6-TCP in the zebrafish

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tissues, (2) negligible bioconcentration of 2,4,6-TCP in the zebrafish ovary and/or (3)

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2,4,6-TCP being easily metabolized. Embryo microinjection with PCZ was performed for

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validating whether accumulated PCZ in ova could inhibit larval growth. No significant

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differences in body length of larvae (t-test, p > 0.05) were observed between the control

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group and the microinjected group (Figure S3). These results demonstrated that observed

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growth inhibition of larval offspring could not be attributed to accumulated PCZ in ova.

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Transcriptome and Proteome in Ova: To further explore potential molecular

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mechanisms leading to growth inhibition of larval offspring, we examined the expression

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profiles of mRNA and protein in released ova derived from PCZ-exposed females. The

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transcriptomics results showed that the expression of 24 genes was significantly altered

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(17 up-regulated and 7 down-regulated, padj 0.05). Therefore, we

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predominantly focused on the function of differentially expressed genes associated with

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larva development. Retinol binding protein 4 (rbp4) is a transporter of retinol in 12


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vertebrate plasma and was shown to be expressed in the yolk syncytial layer during early

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embryogenesis.32 Knockdown of rbp4 in the yolk syncytial layer of zebrafish caused

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significantly shortened yolk extension.32 Yolk extension is closely related to the process

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of body axis formation33 and thus shortened yolk extension could result in decrease the

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body length. Therefore, the PCZ-induced down-regulation of rbp4 in the ova might be

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responsible for the subsequent decrease of body length in larval offspring. The proteomic

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results showed that the abundances of 73 proteins were significantly changed (71

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up-regulated and 2 down-regulated, p < 0.05) in 300 μg/L PCZ group. The functions of

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these proteins were mainly associated with citrate cycle, pyruvate metabolism,

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glycerolipid metabolism, amino acid metabolism, protein processing in endoplasmic

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reticulum, ribosome biogenesis and assembly (Table S5). These results suggested that

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PCZ exposure might have the potential to disrupt mitochondrial energy metabolism, three

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major nutrients for metabolism, protein biosynthesis and/or other biological processes in

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ova by altering the abundances of proteins involved in these biological processes.

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Therefore, PCZ exposure might disrupt mitochondrial energy metabolism process in fish

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offspring and further lead to inhibition growth of larval offspring. It should be noted that

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parental exposure to PCZ significantly decreased mRNA content of rbp4 in ova, but no

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significant change in the protein content of rbp4 was observed. In this study,

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transcriptomics and proteomics sequencing were performed using 0-hpf ova, and the

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mRNAs and proteins in 0-hpf ova might be from parental transfer. At least, to the best of

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our knowledge, the mRNAs and proteins in yolk of embryos should be from parental 13



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transfer. Therefore, we hypothesized that the mRNA and protein levels of some gene in

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0-h ova might be not correlated since translation process from mRNA to protein for the

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gene, at least in yolk of embryos, might not occur.

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Overall and to our knowledge, the present study for the first time clarified that

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exposure of adult zebrafish to the aromatase inhibitor PCZ can inhibit body length of

280

larval offspring, but unexpectedly it did not alter the vtg content in ova. We further found

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accumulated PCZ in ova was not responsible for the inhibition of larval offspring as

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evidenced by PCZ microinjection, and the decreased body length of larval offspring

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might be attributed to down-regulated expression of rbp4 and dysfunction of

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mitochondrial energy metabolism in ova.

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Supporting Information Available

287

Chemicals and reagents, zebrafish maintenance and the 21-day fish reproduction assay,

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Measurement of sex steroid hormones in plasma, LC/MS quantification of PCZ and

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2,4,6-TCP in ova, PCZ microinjection validation, RNA Sequencing, quantitative

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real-time polymerase chain reaction (qRT-PCR) validation, quantitative proteomic

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analysis, the schematic chart of PCZ exposure experiment (Figure S1), developmental

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endpoints (survival rate, hatching rate and malformation incidence) of larvae offspring at

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96-hpf (Figure S2), PCZ microinjection validation (Figure S3), concentration of PCZ and

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its metabolite 2,4,6-TCP in ova (Table S4), list of differentially expressed proteins in ova

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(Table S5). This information is available free of charge on the ACS Publications website. 14


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Acknowledgement

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This work was supported by National Key R&D Program of China (2017YFF0211203)

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and National Natural Science Foundation of China (21622702).

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Figure 1. Phase 1: Effects of the aromatase inhibitor prochloraz (0, 3, 30, and 300μg/L treatment groups) in a 21-day reproduction assay in zebrafish (A) average production of eggs per female during the 14-day of pre-exposure and the 21-day of exposure (n = 3); (B) female and male GSI (n = 12); (C) plasma E2 concentration (n = 3); (D) plasma T concentration (n = 3);(E) plasma vtg concentration in female (n = 3);(F) 96-hpf body length of larval offspring derived from adult zebrafish exposed to various concentrations of PCZ for 21 days (n = 60). Data are presented as the mean ± SEM. Different small cap letters indicate significant

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differences among groups (p < 0.05)

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Figure 2. Phase 2: (A) 96-hpf body length of larval offspring derived from control group (n = 30); exposed female fish (300 µg/L PCZ) mated with unexposed male fish (EF group) (n = 30) or exposed male fish (300 µg/L PCZ) mated with unexposed female fish (EM group) (n = 30); (B) Vtg contents in ova between control and 300 μg/L groups (n = 3); (C)Heat map of differentially expressed genes in ova in response to 300 μg/L PCZ exposure. Red and green boxes colors depict relative up- and down-expression in PCZ exposure group relative to the control. The fold change was calculated based on the Log2 transformation. (D) mRNA levels of rbp4 (RNA-sequence and qRT-PCR) in ova derived from 300 μg/L PCZ exposure group (n = 3). All data shown are mean ± SEM. Different small cap letters indicate significant differences among groups (p < 0.05). 20


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Unexpected Observations: Exposure to Aromatase Inhibitor

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