Effects of Exposure to 17α-Ethynylestradiol during Sexual Differentiation on the Transcriptome of...

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Article

Effects of exposure to 17#-ethynylestradiol during sexual differentiationon the transcriptome of the African clawed frog (Xenopus laevis)

Amber R. Tompsett, Steve Wiseman, Eric Higley, John P Giesy, and Marcus HeckerEnviron. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es400436y • Publication Date (Web): 03 Apr 2013

Downloaded from http://pubs.acs.org on April 8, 2013

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Hypothalamus

Pituitary

Thyroid Liver

Gonads

Steroid synthesis Steroid signaling

TH signaling TH metabolism

Retinoic acid signaling

Steroid metabolism

Testicular development Spermatogenesis

Figure 1

Cholesterol synthesis

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y = 1.0826x - 1.1316 R² = 0.7849

0

0.5

1

1.5

2

2.5

3

3.5

1.5 2 2.5 3 3.5 4

qPCR Fold-change

(Absolute value)

RNA-Seq Fold-change (Absolute value)

Figure 2

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Effects of exposure to 17α-ethynylestradiol during sexual differentiation on the

transcriptome of the African clawed frog (Xenopus laevis)

Amber R. Tompsetta,*

, Steve Wisemana, Eric Higley

a, John P. Giesy

a,b,c, and Markus Hecker

a,d

a Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada

b Dept. of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK

c Dept. of Biology and Chemistry, City University of Hong Kong, Hong Kong, SAR, PR China

d School of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK

*Corresponding author. E-mail: [email protected]; Postal address: Toxicology Centre,

University of Saskatchewan, 44 Campus Drive, Saskatoon, SK, S7N 5B3; Telephone: 1-306-

966-4680; Fax: 1-306-966-4796

Abstract

Exposure to estrogens during the period of sexual differentiation is known to adversely

affect the development of testes in African clawed frogs (Xenopus laevis), but little is known

about the molecular changes that coincide with the development of altered phenotypes.

Therefore, the transcriptome-level effects of exposure to 17α-ethynylestradiol (EE2) during

sexual differentiation of X. laevis were evaluated by use of Illumina sequencing coupled with

RNA-Seq expression analysis. Overall, a number of processes were affected by 17α-

ethynylestradiol, including steroid biosynthesis, thyroid hormone signaling and metabolism,

testicular development, and spermatogenesis. Some of the altered pathways, such as thyroid

hormone signaling and testicular development, could be linked with biological effects on

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metamorphosis and gonadal phenotypes, respectively, that were observed in frogs that were

exposed to 17α-ethynylestradiol throughout metamorphosis and the early post-metamorphic

period. Thus, early changes at the transcriptome-level may have been predictive of pathologies

that did not manifest until later in development. To validate the quantitative capacity of RNA-

Seq, a subset of transcripts identified to have altered abundances in individuals exposed to 17α-

ethynylestradiol was also evaluated by use of quantitative polymerase chain reaction (qPCR).

While small sample sizes (n=3) limited the ability to draw conclusions pertaining to differences

in qPCR-derived abundances of transcripts between control and exposed tadpoles, there was a

significant relationship (r2=0.78) between fold-changes for RNA-Seq and qPCR.

Introduction

Some amphibians, including the African clawed frog (Xenopus laevis), display

phenotypic plasticity during larval development and can be feminized or demasculinized by

exposure to estrogenic chemicals immediately prior to and during the period of sexual

determination and differentiation.1,2,3,4,5

If alterations in sexual development result in changes in

morphology or physiology, these alterations can affect sex ratios, fertility, or fecundity, which

can potentially affect community and population-level fitness.6,7

Estrogenic substances, such as

17β-estradiol (E2) and 17α-ethynylestradiol (EE2), enter aquatic environments through various

sources, including discharge of liquid effluents from wastewater treatment facilities8 and runoff

containing animal manure.9 Thus, there is the potential for exposure of larval amphibians to

these compounds during the sensitive period of sexual determination and differentiation.

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In general, exposure of genetic male X. laevis to potent estrogens during the sensitive

period results in abnormal development of testes, mixed sex phenotypes, and reversal of

phenotypic sex, depending upon exposure concentrations and the specific estrogen

utilized.1,3,10,11

While phenotypic effects of exposure to estrogens have been relatively well-

characterized, there has been little effort to determine the molecular events that orchestrate

estrogen-impacted sexual differentiation. Under normal circumstances, development of genetic

females is at least partially controlled by the female-specific sex-linked gene DM-W. However,

since genetic males lack the DM-W gene, alterations in phenotypic sex caused by estrogens are

not mediated by DM-W.12

At the tissue level, abnormal sexual development is due to to

inhibition of migration of primordial germ cells from the cortex to the medulla of the primordial

gonad,7 but no efforts have been made to determine molecular changes that may be associated

with the inhibited migration.

Recent advances in next generation sequencing technologies, including Illumina’s

polymerase-based sequence by synthesis technique, have made feasible sequencing of entire

transcriptomes. When results of sequencing are coupled with software to construct and evaluate

transcriptomes, it is possible to create “digital microarrays” that are more specific and have

greater coverage than traditional microarrays.13

This open format approach is useful for

discovering novel biological responses without prior knowledge of these responses and could

ultimately be used to form definitive linkages between molecular responses and effects on

individuals or populations.13,14,15

In the context of estrogen-induced effects on sexual

development, sequencing and transcriptome analyses would be useful to help determine

molecular changes associated with impacts on sexual differentiation.

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The current study was designed to elucidate the molecular mechanisms associated with

alterations in sexual differentiation and development of genetic male X. laevis exposed to the

model estrogen EE2. To do so, samples of mRNA from exposed and unexposed male tadpoles

that were undergoing sexual differentiation were sequenced with an Illumina sequencer and then

evaluated by use of RNA-Seq expression analysis to compare transcriptomes of exposed and

unexposed tadpoles. Results of the RNA-Seq analysis were then validated for a subset of

regulated transcripts by use of quantitative polymerase chain reaction (qPCR). To determine the

possible biological relevance of changes at the transcriptome level, data were also evaluated in

context with other biological data that was collected from a group of X. laevis tadpoles that were

grown through metamorphosis and early post-metamorphic development (89 d) in water

containing the same concentration of EE2.11

Experimental

Prior to commencement of experiments, approval for the use of X. laevis and all

experimental procedures was obtained from the University Committee on Animal Care and

Supply Animal Research Ethics Board at the University of Saskatchewan (Animal Use Protocol

#20090066). Sexually-mature, adult X. laevis were bred and eggs were collected as previously

described.11

Concentrations of EE2 in exposure water were validated during the experiment by

use of high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS)

following methods described elsewhere.11,16

Feeding, water renewals, and basic water quality

measurements were performed as has been previously described.11

Water quality parameters

over the course of the experiment were all within an acceptable range for culture of X. laevis.

Average values were temperature of 22.6 ± 0.1 °C, conductivity of 1.70 ± 0.02 mS/cm, 7.3 ± 0.1

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mg dissolved oxygen/L, pH of 7.8 ± 0.1 standard units, and 0.02 ± 0.01 mg ammonia/L. Nitrite

was never detected above the method detection limit of 0.02 mg/L. Nitrate was never detected

above the method detection limit of 0.25 mg/L.

For the current study, two treatments, a solvent control (0.0025% ethanol) and 0.84 µg

EE2/L dissolved in the ethanol carrier, were utilized, and each was replicated in triplicate tanks.

Each tank was initially stocked with 25 fertilized eggs. Tadpoles were sampled after 31 d of

exposure, when the average tadpole had reached the period of sexual differentiation

(Nieukwoop-Faber (NF) Stage 53). At that time, 2-4 tadpoles were removed from each tank and

euthanized in an overdose of MS-222. Differing numbers of tadpoles were taken from each tank

due to slight, but not significant, differences at the tank level in percent hatch and average

developmental stage of tadpoles. Only individuals that could be definitely identified as NF stage

53 were sampled. The remaining tadpoles (about 15 per tank) were allowed to complete

metamorphosis and were subjected to analyses that have been reported previously.11

After euthanization, the body and tail of each tadpole were separated for subsequent

analyses. The tail portion of the tadpole was preserved at -20 °C until genomic DNA was

extracted for use in a multiplex PCR assay that determines genetic sex in X. laevis individuals.12

This assay was performed as previously described.11

The body portion of the tadpole was flash

frozen in liquid nitrogen and stored at -80 °C until RNA was extracted to be utilized for analyses

of transcripts. RNA was extracted, purified, and stored as has been previously described.17

Analyses of abundances of transcripts were only performed on tadpoles with a male

genotype. For both RNA-Seq and qPCR analyses of abundances of transcripts, 3 individuals

from the solvent control and 3 individuals from the 0.84 µg EE2/L treatment were utilized.

Sample size was limited to 3 individuals per treatment because there were only 3 genetic male

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tadpoles sampled from the solvent control. For determining the RNA-Seq, samples were pooled

by treatment, and for qPCR, samples were evaluated individually. A pooled sample was utilized

for the RNA-Seq as a screen for effects at the treatment level, and qPCR was used to validate the

results of the RNA-Seq.

Samples used for RNA-Seq were sequenced on an Illumina Hi-Seq 2000 Sequencer

(Illumina, San Diego, CA, USA). Each sample consisted of 5 µg of RNA from 3 individuals that

was pooled into a 15 µg RNA sample. One sample consisted of pooled RNA from 3 individuals

from the solvent control treatment, and the other RNA sample consisted of pooled RNA from 3

individuals from the 0.84 µg EE2/L treatment. The integrity of RNA was evaluated with a 2100

Bioanalyzer (Agilent, Clara, CA, USA), then RNA was prepared and sequenced by the National

Research Council of Canada Plant Biotechnology Institute (Saskatoon, SK, Canada). Briefly,

cDNA libraries were prepared using an mRNA-Seq Sample Prep Kit (Illumina) according to the

manufacturer’s protocol. Libraries were then sequenced as 2x100bp paired-end reads on a Hi-

Seq 2000 Sequencer (Illumina). Each pooled sample was sequenced in an individual lane to

maximize the number of reads per sample.

The raw sequence data were uploaded to the National Center for Biotechnology’s

Sequence Read Archive and are freely available at: http://www.ncbi.nlm.nih.gov/Traces/sra/.

For processing, individual files containing sequence information from the Illumina sequencing

run were imported into CLC Genomics Workbench V 5.1 software (CLC Bio, Aarhus, Denmark)

for each RNA sample sequenced, and subsequent analyses, unless otherwise noted, were

performed using the CLC Genomics Workbench. Sequences were trimmed of adapter sequences

and ambiguous nucleotides and filtered for quality using a modified-Mott trimming algorithm.

Reads with lengths of less than 30 nucleotides were discarded. For each sample of RNA,

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sequences that met quality criteria were then mapped to a reference transcriptome by use of

RNA-Seq with individual gene expression values normalized as reads per kilobase of exon

model per million mapped reads (RPKM).18

The reference transcriptome consisted of all 11,654

full-length X. laevis cDNAs currently available in the National Centre for Biotechnology

Information sequence database (http://www.ncbi.nlm.nih.gov). Once the RNA-Seq had been

performed for each sample, an expression analysis was performed to compare expression of the

two RNA samples to one another. Reference transcripts with fewer than 10 mapped gene reads

in at least one sample of RNA were eliminated from the expression analysis. Fold-changes were

calculated based upon RPKM and a significant change in the abundance of a transcript was

defined as a fold-change greater than or equal to ±2-fold. The ±2-fold significance level was

chosen because it is a conservative value for determining both statistical and biological

differences in abundances of transcripts according to previous studies.19,20,21

Separate lists of transcripts that were up-regulated or down-regulated at least ±2-fold

were compiled into FASTA files. These transcript files were imported into Blast2GO

software22,23

and the identity and gene ontology (GO) of each transcript was determined. The

pathways affected by exposure to EE2 were determined by analysis of both the GO terms and

Kyoto Encyclopedia of Genes and Genomes (KEGG) enzyme mapping using Blast2GO.

Primers for qPCR were designed for genes of interest from the RNA-Seq analysis (Table

S.1) using published X. laevis mRNA sequences (National Center for Biotechnology

Information, http://www.ncbi.nlm.nih.gov) as has been previously described,17

and forward and

reverse DNA oligos were purchased (Invitrogen, Burlington, ON, Canada). Primers were

validated and qPCR was performed according to previously published methods17

with minor

modifications. Briefly, for each primer set, efficiency was calculated based upon a standard

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curve consisting of cDNA dilutions (1:2, 1:4, 1:8, 1:16, 1:32, 1:64) of a pool of cDNA from 5

random X. laevis tadpoles, and qPCR experiments were performed for primer sets with

coefficients of determination of at least 0.99 and efficiencies of 1.9-2.1, where efficiency=10^(-

1/slope of standard curve). To perform qPCR, cDNA was synthesized from 1 µg of each tadpole RNA

sample, diluted 1:5, and used as template in a Quantitect SYBR Green PCR Kit (Quiagen)

according to the manufacturer’s protocol.

Abundances of transcripts were quantified according to the Mean Normalized Expression

(MNE) method24

using ribosomal protein L8 (rpl8) as a reference gene. Treatment means for

qPCR data are expressed as mean fold-change compared to solvent control tadpoles. No

statistical analyses of differences in abundances of transcripts between the solvent control and

EE2 treated tadpoles were performed for qPCR due to limited sample size (n=3

tadpoles/treatment), which limited power to determine statistical differences.

In the current study, both RNA-Seq and qPCR expressions were reported as fold-change

relative to control. However, methods for calculation and normalization for the two techniques

were not the same. RNA-Seq expression was calculated on the basis of RPKM, which reflects

the molar concentration of transcripts in the sequenced sample.18

Fold-changes derived from

qPCR were calculated by use of the Mean Normalized Expression method24

after being corrected

for primer efficiency. These values are indicative of expression of the target gene normalized to

that of an internal control gene. To evaluate the quantitative capacity of RNA-Seq, the degree of

agreement between fold-change values calculated by RNA-Seq and qPCR was determined by

use of a least squares linear regression.

Results and Discussion

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To our knowledge, this is the first report of molecular pathways in X. laevis affected by

exposure to EE2 during sexual differentiation, as determined by use of transcriptomic

techniques. Specifically, transcriptomes of genetic male tadpoles exposed to EE2 during sexual

determination and differentiation were evaluated 31d post-fertilization at NF stage 53, which is

after sexual determination has taken place (NF stages 50-52) and during the period of sexual

differentiation (NF stages 53-59).25

While analyses of phenotypic sex could not be performed in

the current study, the phenotypic effects of exposure to the concentration of EE2 utilized were

determined in a group of animals that was grown through metamorphosis and early post-

metamorphic development (89 d).11

Although results of previous studies indicated that exposure

to ~1 µg E2/L had the potential to cause up to 50% reversal of phenotypic sex of genetic

males,10,26

exposure to 0.84 µg EE2/L in the 89 d study resulted in the reversal of phenotypic sex

of 7% of genetic male individuals.11

Thus, it is unlikely that individual tadpoles utilized in the

current study were undergoing complete reversal of phenotypic sex. However, the histological

analyses performed on the 89 d experiment group did indicate that 83% of these animals would

exhibit some degree of atypical sexual development of the gonads, including 11% of individuals

developing as mixed sex and 72% of individuals developing as abnormal males.11

As such,

molecular changes identified by the RNA-Seq in the current study are probably not indicative of

changes associated with reversal of phenotypic sex so much as with abnormal sexual

development of genetic males.

A total of 72,835,794 and 91,814,234 sequence reads were obtained for RNA isolated

from tadpoles exposed to the solvent control or 0.84 µg EE2/L, respectively. When these reads

were mapped to the reference transcriptome of 11,654 X. laevis cDNAs, 67% and 72% of reads

mapped to the transcriptome for tadpoles exposed to the solvent control and to 0.84 µg EE2/L,

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respectively. Reads that mapped to the reference transcriptome were subjected to an analysis of

expression by use of RNA-Seq to determine which transcripts from the reference transcriptome

were of 2-fold greater or lesser abundance. Within this limit, the transcripts of 126 genes were

of greater abundance and the transcripts of 399 genes were of lesser abundance in tadpoles

exposed to 0.84 µg EE2/L, compared to those exposed to the solvent control (Table S.2). These

figures correspond to 1.1% and 3.4% of the total number of transcripts in the reference

transcriptome that were up- and down-regulated, respectively. The up- and down-regulated

transcripts were further subjected to GO and KEGG pathway analyses.

The RNA-Seq analysis, coupled with GO and KEGG analyses, indicated that a number of

biological processes were up-regulated in individuals exposed to 0.84 µg EE2/L. For those

transcripts that were of greater abundances, a number of processes and pathways that are either

known to be affected by exposure to potent estrogens or known to be involved in sexual

development were indicated. The up-regulated pathways and processes included steroid and

xenobiotic signaling and metabolism, development of gonads, and biosynthesis of steroids

(Figure 1). Transcripts representative of these pathways encode nuclear receptors, enzymes, and

transcription factors (Table 1), and these pathways are discussed below.

Table 1: Selected transcripts altered by at least 2-fold in genetic male Xenopus laevis exposed to

0.84 µg 17α-ethynylestradiol/L.

Transcript

ID

Gene Identity

Accession

Number

Functiona

RNA-Seq

fold-

changeb

qPCR

fold-

changeb

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3β-hsd

3β-hydroxysteroid

dehydrogenase

BC106482.1 SB

+2.4 NM

11β-hsd

11β-hydroxysteroid

dehydrogenase

NM_001092593.1 SB +2.4 +1.3

ahr1a

Aryl hydrocarbon receptor

1α

NM_001171912.1 SXSM -2.3 -1.5

celf1

CUGBP, Elav-like family

member 1

BC072134.1 TDS -3.3 NM

cyp2k4-l Cytochrome P450 2K4-like BC060406.1 SXSM -2.4 -1.2

ftzf1

Fushi tarazu transcription

factor-1

BC169770.1 GD +2.7 +1.6

gstp1

Glutathione-S-transferase pi-

1

NM_001088783.1 SXSM -2.2 -1.6

rara Retinoic acid receptor α BC169958.1 RAS -2.2 -1.8

sult6b1 Sulfotransferase 6B1 BC129612.1 THSM -3.7 -3.0

trh

Thyrotropin-releasing

hormone

M34699.1 THSM -2.9 -2.0

a SB=steroid biosynthesis; SXSM=steroid and xenobiotic signaling and metabolism;

GD=gonadal development; RAS=retinoic acid signaling; TDS=testicular development and

spermatogenesis; THSM=thyroid hormone signaling and metabolism

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b Fold-change values for RNA-Seq are normalized using the number of reads per kilobase of

exon model per million mapped reads (RPKM). Fold-change values for qPCR are normalized to

the expression of a reference transcript. NM indicates that a transcript was not evaluated by use

of qPCR. All fold-changes are expressed as change in expression relative to controls.

Abundances of transcripts of two genes involved in steroid and xenobiotic signaling and

metabolism, cyp2a6 and nr1h4-like, were greater in genetic male tadpoles exposed to EE2

compared to the controls. The protein product of cyp2a6 is involved in metabolism of nicotine,

coumarin, and some pharmaceuticals, and has been shown to be inducible in human cell lines

exposed to E2.27

The nr1h4-like transcript, which is also called for2 in X. laevis, codes for an

orphan nuclear receptor related to the farnesoid X receptor (FXR) in mammals and is thought to

be involved in metabolism of endogenous and exogenous compounds and development of the

liver.28

Thus, greater expression of cyp2a6 and nr1h4-like transcripts in exposed tadpoles was

perhaps induced by exposure to EE2.

Two transcripts involved in biosynthesis of steroids, 3β-hsd and 11β-hsd, had greater

abundances in genetic male tadpoles exposed to 0.84 µg EE2/L. The 3β-hsd transcript codes for

an enzyme that is important for regulating the balance of synthesis of different steroid hormones,

since this enzyme can catalyze multiple reactions during steroidogenesis. One reaction catalyzed

by the 3β-hsd enzyme produces androstenedione, the precursor to estrogens and androgens, and

modeling has shown that alterations in activity of 3β-hsd are likely to affect production of

androstenedione.29

The 11β-hsd transcript codes for an enzyme that is involved in androgen

synthesis in fish, and the expression of this gene is greater after exposure to the anti-androgen

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flutamide, even though exposure phenotypically feminized the fish.30

This result, coupled with

phenotypic data collected from X. laevis that were exposed to EE2 for 89 d, which indicated that

exposure to EE2 demasculinized or feminized genetic male X. laevis,11

indicates that greater

transcription of genes involved in androgen biosynthesis does not necessarily indicate

masculinization, and paradoxically might be associated with feminization. In fact, qPCR

performed on livers of frogs from the 89 d experiment indicated that the abundance of transcripts

of the androgen receptor were greater in frogs exposed to EE2, even after 89 d.31

Exposure to EE2 affected abundances of transcripts involved in the development of

gonads in genetic males. The abundance of transcripts of ftzf1 was greater in genetic male

tadpoles exposed to EE2. The ftzf1 transcript codes for a transcription factor involved in cellular

differentiation and development of germ cells in male and female amphibians. It has been shown

to be expressed in immature oocytes in X. laevis that were transfected with the Rana rugosa ftzf1

gene32

and in developing germ cells of male and female R. rugosa.33

Thus, this gene seems to be

involved in development of gonads in males and females of some amphibians, and altered

expression of this gene might contribute to abnormal development of gonads in amphibians.

However, additional targeted studies are required to elucidate whether up-regulation of this gene

would have any impacts on sexual differentiation of either the testis or ovary.

For those transcripts that were down-regulated in comparison to controls, a number of

pathways and processes that have been shown to be affected by exposure to estrogens or that are

involved in sexual development were identified as altered. Specifically, pathways and processes

related to steroid and xenobiotic signaling and metabolism, development of testes, retinoic acid

signaling, thyroid hormone signaling and metabolism, biosynthesis of cholesterol, and

biosynthesis of steroids were affected (Figure 1). Transcripts representative of these pathways

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and processes included nuclear receptors, enzymes, and transcription factors (Table 1). While

some of the individual genes that were impacted could be mechanistically significant, the down-

regulated processes are perhaps more interesting when anchored to the phenotypic and biological

data gathered from X. laevis individuals that were exposed to EE2 throughout larval and early

post-metamorphic development (89 d).11

In the 89 d experiment, exposure to EE2 adversely impacted development of testes in

83% of individuals exposed to 0.84 µg EE2/L.11

In the RNA-Seq expression analysis, three

transcripts involved in development of the testis and spermatogenesis, celf1, dmrt1β, and gtsf1,

were down-regulated by at least 2-fold in genetic males exposed to EE2. Because complete

reversal of sex of genetic males was relatively rare (7%) at this concentration of EE2, it is

possible that the down-regulation of these genes was at least partially responsible for the

development of the majority (76%) of genetic males with abnormal and mixed sex phenotypes.11

Development could also have been affected by lesser abundances of transcripts of 5α-reductase

(5ar). The 5ar transcript codes for an enzyme that catalyzes conversion of testosterone to

dihydrotestosterone, so reductions in the abundance of transcripts of 5ar might lead to lesser

circulating concentrations of dihydrotestosterone. Lesser abundances of transcripts of 5ar have

previously been linked to mixed sex conditions in Silurana (Xenopus) tropicalis.34

Based upon the abundances of transcripts determined in the RNA-Seq analysis, the

processes of thyroid hormone signaling and metabolism and retinoic acid signaling might have

been down-regulated in genetic male X. laevis exposed to EE2. These two pathways are known

to interact via the formation of thyroid hormone receptor-retinoic acid receptor (THR-RAR)

hetero complexes35

that can induce expression of genes through a common response element.36

Normally, X. laevis complete resorption of the tail and metamorphose at NF stage 66, about 6-8

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weeks after hatch. Exposure to 0.84 µg EE2/L for 89 d significantly delayed metamorphosis of

tadpoles into juvenile frogs by 17 d relative to control individuals.11

Thyroid hormones are

responsible and necessary for orchestrating metamorphosis,37,38

and exposures to inhibitors of the

synthesis of thyroid hormone delay metamorphosis.39

Because a number of genes involved in

thyroid hormone signaling and metabolism were already down-regulated at NF stage 53, it is

possible that the delays to metamorphosis observed in the 89 d exposure were due, at least in

part, to a general down-regulation of the thyroid hormone axis that was detectable weeks before

metamorphosis was to occur. In addition, inhibition of synthesis of thyroid hormones impairs

differentiation of testes and leads to female-biased sex ratios.40

While the sex ratio of animals

from the grow-out experiment was not biased toward females, significant effects on

differentiation of testes were observed.11

Two biological processes down-regulated in genetic male tadpoles exposed to EE2 that

could not be anchored to the phenotypic data collected during the 89 d study were synthesis of

cholesterol and steroid and xenobiotic signaling and metabolism. In the 89 d study, endpoints

that are associated with these two pathways were not monitored, so it is not possible to draw

conclusions about the possible downstream phenotypic effects of a down-regulation of these

processes. Synthesis of cholesterol has been shown to be induced after exposure to potent

estrogens,41,42,43

but most studies have quantified concentrations of cholesterol, not abundances

of transcripts of genes related to cholesterol biosynthesis. In addition to the changes in synthesis

of cholesterol, a number of transcripts that are known to be involved in Phase I and II

biotransformation reactions (cyp2c20, cyp2k4-l, gstp1) were down-regulated by exposure to EE2.

CYP2 enzymes play a role in metabolism of endogenous hormones, including E2.44

In addition,

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inhibitions of members of the CYP2K family of enzymes have been observed in Atlantic salmon

(Salmo salar) hepatocytes exposed to nonylphenol, a weak estrogen.45

A subset of transcripts that were of significantly greater or lesser abundances was

evaluated by use of qPCR to validate the results of the RNA-Seq experiment. Overall, for the

subset of transcripts whose abundances were measured by use of qPCR (11β-hsd, acat2, ahr1a,

cyp2k4, cyp26a1, fdps, ftzf1,gstp1, rara, sult6b1, and trh), the trends in direction of fold-change

were in agreement with the direction of change from the RNA-Seq expression analysis. In

addition, there was a significant linear relationship between the fold-changes from the RNA-Seq

and the fold-changes from qPCR (Figure 2; Least-squares linear regression, p<0.001; r2=0.78),

even though calculation of the fold-changes for the two methods is fundamentally different.

Previous research has also indicated that both qPCR and microarray fold-change expression

values correlate well (r2

values ranging from 70-95%) with those calculated from next generation

sequencing experiments.46,47,48

In addition, a comparison of qPCR to Illumina digital gene

expression values based upon tag annotation showed that around 70% of transcripts had similar

directions of change in abundances of transcripts.49

Overall, exposure to 0.84 µg EE2/L during sexual determination and differentiation

affected the transcriptome of genetic male X. laevis at 31 d in a manner that could be linked with

phenotypic data that were collected from a parallel group of X. laevis that were exposed to the

same concentration EE2 until after the completion of metamorphosis (89 d). RNA-Seq identified

pathways, processes, and individual genes that might have been associated with altered

phenotypes and delays to metamorphosis that were observed in frogs after 89 d. This technique

of analysis of the transcriptome was also helpful in determining de novo other pathways and

processes that were affected by exposure to EE2 and could be useful for developing targeted

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molecular endpoints for evaluation in future experiments. Since the concentration of EE2

utilized was within 2-fold of maximal estrogen equivalent concentrations that have been

previously measured in surface waters in the United States50,51

, the results of the RNA-Seq may

also be useful for developing predictive tools for assessing possible effects of worst-case

scenario exposure of amphibians to estrogens in the environment.

Acknowledgements

ART was partially supported by scholarships from the Toxicology Graduate Program and the

Graduate Students’ Association at the University of Saskatchewan and by teaching fellowships

from the College of Graduate Studies at the University of Saskatchewan, the Northern

Ecosystems Toxicology Initiative (NETI), and AREVA Resources. JPG was supported by the

Canada Research Chair program, an at large Chair Professorship at the Department of Biology

and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong,

and The Einstein Professor Program of the Chinese Academy of Sciences. MH was supported

by the Canada Research Chair program. The authors would like to thank S. Pryce for laboratory

assistance.

Supporting Information Available

Additional information regarding the primers utilized for qPCR and the identities and ontologies

of regulated genes from the RNA-Seq experiment is included as Supporting Information to this

manuscript. This information is available free of charge via the Internet at http://pubs.acs.org/.

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Figure 1: Schematic representation of processes and pathways in genetic male Xenopus laevis

tadpoles that were affected by exposure to 17α-ethynylestradiol during sexual differentiation.

Simplified schematics of the hypothalamic-pituitary-gonad-liver axis and the hypothalamic-

pituitary-thyroid axis are shown in white boxes connected with black arrows. Processes and

pathways that were affected by exposure to EE2 are shown in colored boxes connected by white

arrows to the biological compartments along the axes. Blue boxes indicate pathways that were

generally down-regulated. Red boxes indicate pathways that were differentially regulated (i.e.

some transcripts up-regulated, some transcripts down-regulated).

Figure 2: Comparison of fold-change values from RNA-Seq expression analysis and qPCR. The

relationship between the fold-change values from RNA-Seq and qPCR was linear and significant

(Least-squares linear regression, p<0.001; r2=0.78).

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