Efficient removal of 17α-ethinylestradiol (EE2) from water ...
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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254x94mm (300 x 300 DPI)
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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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