Original Paper

HORMONE Horm Res 2006;65:200–209 Received: October 31, 2005

RESEARCH DOI: 10.1159/000092408
Accepted: January 31, 2006
Published online: March 28, 2006

Changes in Gene Expression during

Wolffian Duct Development
Sabine E. Hannema a Cristin G. Print b D. Stephen Charnock-Jones c Nick Coleman d
Ieuan A. Hughes a
Departments of a Paediatrics, Addenbrooke’s Hospital, b Pathology, and c Obstetrics and Gynaecology, The Rosie Hospital,
University of Cambridge, and d MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, UK

Key Words matrix synthesis were reflected in regulation of other

Testosterone ! Wolffian duct ! Androgen receptor !
Microarray
Abstract
Background: Wolffian ducts (WDs) are the embryonic
precursors of the male reproductive tract. Their develop-
ment is induced by testosterone, which interacts with the
androgen receptor (AR). The molecular pathways under-
transcripts. Several genes were previously suggested to
be regulated by androgens or contained functional or
putative androgen/glucocorticoid response elements,
indicating they may be direct targets of androgen signal-
ling. Conclusion: Our results suggest novel cohorts of
signals that may contribute to androgen-dependent WD
development and provide hypotheses that can be tested
by future studies.
Copyright © 2006 S. Karger AG, Basel

lying androgen-dependent WD development are largely

unknown. We aimed to identify AR target genes impor-
tant in this process. Methods: RNA was isolated from rat
WDs at E17.5 and E20.5. Affymetrix GeneChip expres-
sion arrays were used to identify transcripts up- or down-
regulated more than 2-fold. Regulation of seven tran-
scripts was confirmed using quantitative PCR. Results:
Transcripts from 76 known genes were regulated, includ-
ing modulators of insulin-like growth factor and trans-
forming growth factor-! signalling. By controlling these
modulators, androgens may indirectly affect growth fac-
tor signalling pathways important in epithelial–mesen-
chymal interactions and organ development. Caveolin-1,
also upregulated, may play a role in modifying as well
as mediating AR signalling. Differentiation of WD epithe-
lium and smooth muscle, innervation and extracellular
Introduction
Wolffian ducts (WDs) are the embryonic structures
that form the epididymis, vas deferens and seminal ves-
icle. In the male foetus, the WDs are stabilised and stim-
ulated to grow by testosterone, whereas in the female foe-
tus, they regress, in the absence of androgens [1]. To exert
its effects, testosterone binds to the androgen receptor
(AR). AR activity is essential to WD development; severe
mutations that completely disrupt AR activity are associ-
ated with the absence of epididymides/vasa deferentia,
whereas milder mutations that allow residual AR activ-
ity are associated with WD development [2]. However, it
is largely unknown how the AR stimulates growth and
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0301–0163/06/0654–0200$23.50/0 Juliana Children’s Hospital
Fax +41 61 306 12 34 Sportlaan 600
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differentiation of the WD. Since AR acts as a ligand-de-
pendent transcription factor, it is likely that induction of
expression of specific target genes plays an important
role.
Epithelial–mesenchymal interactions are essential in
embryonic development of many organs, and are also
thought to be important in WD development. Because
AR appears in the mesenchyme before it is expressed in
the epithelium [3], androgens are thought to initially in-
duce their effects on the epithelium via signals from the
mesenchyme. This idea is supported by studies showing
that, in tissue recombinants, seminal vesicle mesenchyme
can induce proliferation and differentiation of AR nega-
tive epithelium from androgen insensitive tfm mice [4].
The AR may control epithelial–mesenchymal signalling,
either directly by regulating expression of genes encoding
signalling molecules, or indirectly by regulating genes that
modulate signalling.
In order to identify genes that are important in andro-
gen-induced WD development, we compared gene ex-
pression in the WD at E17.5 and E20.5. These time points
were chosen because testosterone levels in the foetal rat
show a peak between day E17 and E20 [5], and the larg-
est morphological changes, which are expected to be pre-
ceded by changes in gene expression, occur between E19
and E20. Using Affymetrix GeneChip arrays and quan-
titative PCR, we identified 76 transcripts from known
genes that were up- or downregulated more than 2-fold
and may play a role in androgen signalling.
Methods
Animals
Time-mated Wistar rats (63–70 days old) were purchased from
Charles River (Margate, UK). The morning after mating is referred
to as day E0.5. At E17.5 and E20.5, pregnant females were killed
using CO2 and cervical dislocation, and pups were removed. Pups
used for histology were immersed in ice-cold neutral buffered
formalin (Sigma-Aldrich, Ltd., Poole, UK). Pups used for RNA
isolation were placed in icecold PBS. Following decapitation, the
testes and WDs were dissected en bloc from male pups and placed
in PBS on ice. WDs and efferent ducts were isolated and stored in
RNAlater (Ambion, Huntingdon, UK) at 4 ° C. This work was car-
ried out in an animal facility designated under the Animals (Sci-
entific Procedures) Act 1986 with the approval and under the su-
pervision of the Named Animal Care and Welfare Officer for the
facility.
RNA Isolation
WDs and efferent ducts from one litter were transferred from
RNAlater into TRIzol Reagent (Invitrogen Life Technologies,
Paisley, UK) containing 112.5 "g/ml GlycoBlue (Ambion). Ho-
Gene Expression in the Wolffian Duct
mogenisation was achieved by passing the WDs through a 25
gauge needle until they had disintegrated and total RNA was pre-
pared following the instructions provided with TRIzol Reagent.
Following RNA cleanup through RNeasy mini spin columns (Qia-
gen, Ltd., Crawley, UK) and precipitation, RNA integrity was
assessed on an Agilent 2100 Bioanalyser (Agilent Technologies,
Stockport, UK).
Affymetrix Oligonucleotide Gene Arrays
Two independent samples of RNA from day E17.5 and two
from day E20.5 (10 "g each, from WDs from approximately 25
pups) were used as template to synthesise double stranded cDNA
using the SuperScript Choice System (Invitrogen Life Technolo-
gies) according to the manufacturer’s instructions, but the T7-
(dT)24 primer (Sigma-Aldrich, Ltd.) was used for first strand syn-
thesis. Biotin-labelled cRNA was synthesised using the ENZO Bio-
Array High Yield RNA transcript labelling kit (Cambridge
Bioscience, Cambridge, UK) and purified using RNeasy mini spin
columns (Qiagen, Ltd.). Fragmentation and hybridisation to
GeneChip Rat Expression Array 230A (Affymetrix, Santa Clara,
Calif., USA) were carried out by the MRC geneservice (Hinxton,
UK). This array contains 15,866 probe sets, of which 10,467 are
ESTs. Two arrays were used for each time point. Images were as-
sessed for quality and then numerified using the Affymetrix micro-
array software MAS 5.0.
Data Analysis
Raw transcript abundance data (‘average differences’ for probe
sets) were globally scaled to a target intensity of 100 and log base
10 transformed. The data were then normalised in an intensity-de-
pendent manner using the Loess function of the R statistical soft-
ware system (rweb.stat.umn.edu/R/doc/html). To do this, an index
array was chosen, which was, based on Euclidian distance, the most
similar to the other three arrays. The overall distributions of log
base 10 transcript abundance values of the other three arrays were
adjusted to match that of the index array, as previously described
[6]. Only a very small degree of normalisation of the array data was
required. A second analysis was performed using log scale robust
multi-array analysis, an alternative method that discards mismatch
probe information [7], rather than average differences between per-
fect match and mismatch probes. This alternative method pro-
duced almost identical results to the Affymetrix MAS 5.0 software.
Therefore, only data produced by the MAS 5.0 software will be de-
scribed in this paper. To identify the transcripts that were most
likely to have been regulated, the ratio between the average abun-
dance of each transcript on the two E20.5 arrays and the average
abundance on the two E17.5 arrays was calculated. Transcripts with
ratios of 12.0 (upregulated) or !0.5 (downregulated) were further
analysed. For these transcripts, ratios between the abundance on
each E20.5 array and that on each E17.5 array were determined
(four ratios) and abundance values and ‘p values’ (which indicate
accuracy of the reported transcript abundance signal for each chip,
not to be confused with comparisons between chips) calculated for
each probe set by MAS 5.0, were inspected. Upregulated transcripts
for which all four ratios were 11.5, the average abundance on the
two E20.5 arrays was 130 and both p values on day E20.5 were
!0.05, were included in the results, as well as downregulated tran-
scripts for which all four ratios were !0.67, the average abundance
on the two E17.5 arrays was 130 and both p values on day E17.5
were !0.05. Data from probe sets known to hybridise with more
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Horm Res 2006;65:200–209 201
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than one sequence from the same or from different gene families
were not included in the results.
Quantitative PCR
Two samples of RNA from day E17.5 and two from day E20.5
(1 "g each) were used as a template to synthesise cDNA using Su-
perscript II reverse transcriptase (Invitrogen Life Technologies)
and 2.5 "M random hexamers, according to the manufacturer’s
instructions. The ABI PRISM 7700 Sequence Detection System
(TaqMan; Applied Biosystems, Warrington, UK) was used to per-
form real-time PCR for a total of seven transcripts, chosen because
of potential functional interest, according to the manufacturer’s
protocol. Primers and probes were proprietary oligonucleotides ob-
tained from Applied Biosystems ‘Assays-on-Demand’; probes con-
tained a 5" 6-FAM reporter and a 3" non-fluorescent quencher and
minor groove binder. All reactions were carried out in triplicate.
Standard curves were prepared for the endogenous control, 18S
ribosomal RNA, and for each target gene. For each experimental
sample, the amount of target and endogenous control was deter-
mined relative to these curves. The amount of target was divided
by the amount of endogenous control, and these normalised values
were used to calculate fold changes in gene expression.
Identification of Putative GREs
To identify putative GREs in the promoter regions of seven
genes (Igfbp2, Igfbp6, caveolin, lumican, lipocalin 2, transgelin and
serine protease 11), 3,000 bp of sequence upstream from the tran-
scription initiation site of these genes was obtained from Ensembl
(www.ensembl.org). AliBaba2 software was used to predict the
presence of transcription factor binding sites in this sequence [8].
Histology
After fixation for approximately 24 h in neutral buffered forma-
lin, rat foetuses were embedded in paraffin. Sections (5 "m) were
cut and stained with haematoxylin and eosin.
Results
Between E17.5 and E20.5 the WD undergoes consid-
erable growth and starts to coil (fig. 1). Both epithelium
and mesenchyme undergo growth, approximately to the
same extent, as demonstrated by figure 1, although this
was not quantified. The changes that occur during this
time course are expected to be regulated and accompa-
nied by changes in gene expression. On each of the ar-
rays 62–66% of all transcripts was expressed. The ma-
jority of these were expressed at similar levels at E17.5
and E20.5. For 266 probe sets, the ratio between the av-
erage abundance on the two arrays from one time point
and the average on the arrays from the other time point
was more than 2. Using the criteria described in the
Methods section, we narrowed down this number fur-
ther to 132 transcripts that showed consistent expres-
sion and regulation. Seventy-six of these were transcripts
202 Horm Res 2006;65:200–209
from known genes (tables 1, 2). On the day when expres-
sion of a gene was highest, variation between the two
replicate arrays of the same time point was 9% on aver-
age. Some of the transcripts were represented by mul-
tiple probe sets on the array and in these cases results
from the different probe sets correlated well (table 1; for
example glutamine synthetase 1 and thymus cell antigen
1#). However, one of the upregulated transcripts was
represented by a second probe set, which did not show
upregulation, and this transcript was therefore excluded
from the results.
Changes in expression of seven genes were investigat-
ed using quantitative PCR. These were chosen because
of potential functional interest and included both genes
that were highly regulated (transgelin, 5.1! up) and genes
that were not as highly regulated (caveolin 1, 2.5! up),
as well as genes of high and low abundance (such as re-
spectively Igf-binding protein 2 and lipocalin 2). 18S Ri-
bosomal RNA was used as an endogenous control. Quan-
titative PCR showed fold changes very similar to those
found using microarrays (fig. 2), although the microarrays
indicated slightly lower fold changes than the quantita-
tive PCR for lumican and lipocalin 2. This was not due
to the normalisation procedure used for the array data;
fold changes calculated using the data before normalisa-
tion were very similar (5.2 and 4.9, respectively). The fold
changes referred to in the discussion is those based on the
array data.
Tissue Growth and Remodelling
Several of the identified genes encode proteins that
form part of the cytoskeleton or extracellular matrix (ta-
ble 1). Nearly all of these are upregulated, probably be-
cause these proteins are required for growth of cells and
interstitial tissue. Some of the cytoskeletal genes are epi-
thelial, such as cytokeratin 8 (CK8) (2.1! up). Others are
mesenchymal, including smooth muscle $-actin (3.5!
up), smooth muscle %-actin (2.6! up), transgelin (also
known as SM22$) (5.1! up) and calponin-1 (2.6! up).
Their upregulation may be a sign of smooth muscle dif-
ferentiation. Upregulation of several transcripts, such as
tropoelastin (3.3! up), lysyl oxidase (2.6! up) and fibu-
lin-5 (2.8! up) suggests synthesis of elastic fibres, which
are abundant in the wall of the adult vas deferens. F-spon-
din (3.0! up), growth associated protein 43 (2.2! up)
and dihydropyrimidinase-like 3 (2.2! up) may play a
role in innervation of the WD.
Other genes may be important in the (growth factor)
signalling required to coordinate growth and differentia-
tion of the WD. Several of the genes listed in table 1 have
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 Fig. 1. Changes in the WD between day E17.5 and E20.5. a, b Macroscopic changes. WDs were dissected from
rat embryos on day E17.5 (a), and E20.5 (b). Photos show growth and coiling of the WD. Scale bars: 1 mm.
c, d Microscopic changes. Whole embryos were removed from the uterus and immersed in cold formalin on day
E17.5 (c) and E20.5 (d), and embedded in paraffin. Haematoxylin and eosin stained sections show growth of the
distal part of the WD, destined to become the vas deferens. Scale bars: 100 "m.

previously been implicated in control of the Igf and Tgf!
pathways. This is discussed in more detail below. Genes
involved in fat and steroid metabolism (table 1) may in-
fluence signalling by steroid hormones. In addition, they
may be important in metabolic control.
Role of the Androgen Receptor
Several of the identified genes were previously sug-
gested to be regulated by androgens or harbour a func-
tional GRE in their promoter region (table 1 footnotes 5,
7 and table 2 footnote 3). To investigate whether the sev-
en genes confirmed to be regulated by quantitative PCR
may be direct targets of AR signalling, we used AliBaba2
software [8] to screen for putative GREs in putative pro-
moter regions. We studied 3,000 bp of sequence upstream
of the transcription initiation site of the seven transcripts.
Several putative GREs were detected in all seven genes,
but most of these did not closely resemble the consensus
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Table 1. Changes in transcript abundance in the WD between E17.5 and E20.5

Gene Transcripta Fold changeb E17.5c E20.5c Gene Transcripta Fold changeb E17.5c E20.5c

Cytoskeleton Other
Transgelin NM_031549 5.1! up 1.2 5.8 Lipocalin 2f NM_130741 5.0! up 0.7 3.2
$-Actinin 2 associated LIM protein AF002281 4.1! up 2.4 9.9 Flavin containing monooxygenase 1d NM_012792 3.3! up 1.9 6.1
Smooth muscle $-actin BI282702 3.5! up 3.6 12.2 Coagulation factor XIIIa NM_021698 3.1! up 0.5 1.5
Calponin 1 NM_031747 2.6! up 0.5 1.2 BM388525 3.3! up 0.5 1.7
Smooth muscle %2-actin NM_012893 2.6! up 0.3 0.7 Xanthine dehydrogenase NM_017154 3.0! up 0.4 1.1
Myosin heavy polypeptide 9 NM_013194 2.3! up 1.0 2.2 S100 protein !-polypeptide NM_013191 2.9! up 0.6 1.8
Nexilin AA799423 2.1! up 0.8 1.7 Branched chain aminotransferase 1 AI102790 2.7! up 0.9 2.4
Basic keratin complex 2 gene 8 BF281337 2.1! up 2.7 5.4 Annexin 1 NM_012904 2.5! up 4.7 11.8
Tissue type transglutaminasedd BI275994 2.5! up 2.3 5.8
Growth factor related/signalling Annexin 2 NM_019905 2.4! up 9.6 22.7
CD44 antigen BI302830 4.6! up 0.4 1.7 Glutathione-S-transferase "1d M28241 2.4! up 3.8 8.8
Igf binding protein 6 NM_013104 3.2! up 1.3 4.1 GPI-anchored ceruloplasmin AF202115 2.3! up 0.7 1.6
Igf binding protein 2d NM_013122 2.7! up 7.2 19.4 Glutamine synthetase 1f BI275294 2.2/2.3/ 2.6/ 6.1/
Serine protease 11 NM_031721 2.7! up 1.6 4.2 2.3! upe 5.8/ 13.5/
Caveolin 1d NM_031556 2.5! up 1.7 4.2 6.2 13.5
Calpain 6 NM_031808 2.4! up 7.6 17.9 Growth associated protein 43d NM_017195 2.2! up 0.5 1.1
Ectonucleoside triphosphate Beta-1,3-glucuronyltransferase 1 NM_054003 2.1! up 0.6 1.3
diphosphohydrolase 2 NM_172030 2.4! up 1.2 2.7 AA925375 2.5! up 0.6 1.5
Connective tissue growth factor NM_022266 2.4! up 0.6 1.3 Myeloid-associated differentiation
e
Calcitonin receptor-like L27487 2.1/2.7! up 1.0/ 2.2/ marker NM_183332 2.1! up 3.9 8.2
1.2 3.3 Cyclin-dependent kinase inhibitor 2c NM_131902 2.1! up 1.9 4.0
Dihydropyrimidinase-like 3 BI294841 2.1! up 6.3 12.9 Cysteine dioxygenase 1 NM_052809 2.1! up 6.1 12.6
AF389425 2.2! up 0.6 1.2 Annexin 4 NM_024155 2.1! up 0.5 0.9
WAP four-disulphide core domain 1 BI279661 2.1! up 0.6 1.3 Ribonuclease, RNase A family 4 ?g 2.1! up 1.1 2.3
Plasticity-related gene 2 NM_181634 2.2! down 2.3 1.0 Glutathione-S-transferase "3d NM_031154 2.1! up 2.5 5.0
Opioid receptor kappa 1 L22536 5.1! down 1.2 0.2 Putative eps protein BE110691 2.0! up 2.1 4.3
Transcription factors Heat shock 20-kDa protein D29960 2.0! up 1.1 2.2
Interferon regulatory factor 1 NM_012591 2.0! up 1.0 1.9 Cysteine and glycine-rich protein 1 NM_017148 2.0! up 6.4 12.9
Cartilage homeoprotein 1 NM_012921 2.8! down 1.8 0.6 Lysozymef NM_012771 2.0! up 2.4 4.8
Act. leukocyte cell adhesion
Fat and steroid metabolism molecule BE118251 2.0! down 12.2 6.0
Gonadotropin-regulated long chain Thymus cell antigen 1# AI145313 2.0/2.3! 5.0/ 2.5/
acyl-CoA synthetase NM_134389 3.9! up 0.8 3.1 downe 5.5 2.3
Insulin induced gene 1 NM_022392 2.6! up 1.6 4.0 Complement component factor h NM_130409 2.5! down 1.4 0.6
d BE120766 2.6! down 1.4 0.5
Low density lipoprotein receptor BI294974 2.6! up 1.2 3.1 Integrin $8

Extracellular matrix
a
Lumican NM_031050 5.1! up 3.0 15.4 Transcript ID for GenBank.
b
Tropoelastinf J04035 3.3! up 1.9 6.5 The ratio between average transcript abundance at E20.5 and at E17.5.
c
Average abundance of transcripts on day E17.5 and E20.5. For the purpose of
Dermatopontin BI278545 2.7! up 1.8 4.8 this table, abundance values from each array were scaled to a median of 1.
BI285485 3.1! up 1.5 4.5 d
Genes previously suggested to be regulated by androgens [14, 37–43].
F-spondin AA801238 3.0! up 0.9 2.6 e
Transcripts represented by multiple probe sets on the array.
f
Fibulin 5 NM_019153 2.8! up 1.4 3.8 Genes thought to harbour a functional GRE in the promoter region [9,
Lysyl oxidased BI304009 2.6! up 3.4 8.6 44–47].
g
Cartilage link protein 1f NM_019189 2.0! down 1.3 0.6 The accession number of this transcript was not available in the annotation
file provided by Affymetrix.
TGF! induced BG379319 2.1! down 31.1 14.9

GRE; some only consisted of a half site. An example of a
putative GRE that was similar to the consensus GRE is
5"-GAAACAtttTGTTTT-3" at –2,258 from the transcrip-
tion initiation site of serine protease 11. Two overlapping
functional GREs (5"AGAACAGGGTGTCCTGTTCT-
3") have previously been described in the promoter region
of the mouse lipocalin 2 gene at –520 and –515 [9]. A
similar but not identical sequence (5"-GGAACAGGGT-
GTCCCATTCT-3") was identified in the putative pro-
moter of the rat lipocalin 2 gene at –534. Two putative
GREs were identified further upstream, 5"-TGCACAgag-
TGTTGG-3" at –1,821, and 5"-TTCACTtccTGTTCT-3"
at –1,506, but these sequences differed from the consen-
sus GRE at four base pairs.
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Table 2. Genes expressed in the WD at
E17.5 or E20.5 only Gene Transcript IDa Present at Abundanceb

Hemoglobin &1 BE108901 E17.5 2.1

AMH type 2 receptor NM_030998 E17.5 1.3
Neurofilament 3 medium NM_017029 E17.5 0.6
11!-Hydroxysteroid dehydrogenase 1c NM_017080 E20.5 0.9
Copper-containing amine oxidase 3 AI070137 E20.5 1.0
Adenylyl cyclase-associated protein 2 AW919109 E20.5 0.9/1.4d
Mast cell protease 2 AA957923 E20.5 0.9
Preproenkephalin related sequence NM_017139 E20.5 6.8
S100 calcium binding protein A9 NM_053587 E20.5 0.9

a
Transcript ID for GenBank.
b
Average abundance of transcripts on day E17.5 or E20.5. For the purpose of this table
abundance values from each array were scaled to a median of 1.
c
Genes previously suggested to be regulated by androgens [48].
d
Transcript represented by multiple probe sets on the array.

proteins, transport proteins, RNA-binding proteins and

11 chaperones [10]. In addition several proteins underwent
E17.5
10 E20.5
posttranslational modification, both in vivo and in mouse
foetal vas deferens cells exposed to androgens [10, 11].
9
However, regulation of lower abundance proteins may
8
not have been detected in this study, because of the lim-
Relative abundance

7
ited sensitivity of the 2D gels used for the proteomic ana-
6
lysis. A study of the effects of linuron, an herbicide and
5 weak anti-androgen, on WD development, identified sev-
4 eral more potential AR targets, using microarrays and
3 real-time RT-PCR [12]. Prenatal exposure to linuron re-
2 sults in several abnormalities of the WD, including de-
1 creased coiling and absence of parts of the epididymis.
0 Genes up- or downregulated in the malformed WDs in-
A RT A RT A RT A RT A RT A RT A RT
clude genes involved in Egf, Igf1, Bmp, Fgf and Notch
Tg Ibp6 Ibp2 Caveolin Lumican Lip2 Sp11
signalling [12]. However, for some of these genes the mi-
croarrays and real-time PCR gave conflicting results,
which makes it difficult to interpret the results from this
Fig. 2. Quantitative PCR confirms the regulation of a subset of
transcripts. Shown are abundance values of seven transcripts at
study. Furthermore, it is not clear whether the anti-an-
E17.5 (black symbols) and E20.5 (white symbols) as determined by drogenic effect of linuron is responsible for the altered
microarrays (A) and real-time RT-PCR (RT). All values were nor- gene expression, or whether linuron directly affects other
malised to the average value on day E17.5. Two biological replicates signalling pathways.
were used for these experiments (circles and triangles); 18S ribo- This study was set up to further investigate which
somal RNA was used as an endogenous control.
genes may be important in androgen-induced WD devel-
opment. The use of Affymetrix GeneChip Expression Ar-
rays allowed us to compare expression of a broad range
Discussion of genes, including genes with low expression levels. How-
ever, with large numbers of genes investigated the num-
Several studies have previously investigated targets of ber of false positives may also increase. Ideally, a large
AR signalling in the WD. Proteomic analysis of the rat number of biological and technical replicates are used for
WD identified 13 proteins whose expression levels change statistical analysis to maximise the chance of identifying
between E17 and E21, including enzymes, cytoskeletal truly regulated genes. However, this was not feasible since
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such a study would require many animals. The use of two
biological replicates did not allow statistical analysis, but
we did use stringent criteria to define ‘regulated genes’.
These criteria included consistent regulation; as discussed
above, variation between the biological replicates was
only 9% on average for the regulated genes. Technical
replicates were not performed, but regulation of approxi-
mately 10% of genes was confirmed using a different
method, quantitative RT-PCR. Although only a small
percentage of transcripts was quantified using RT-PCR,
the fact that there was 100% agreement suggests that the
microarrays reliably detected fold changes in gene expres-
sion.
It is important to note that both epithelial and mesen-
chymal transcripts were upregulated, consistent with
growth of both these tissues, which is also apparent on
microscopical examination (fig. 1c, d). It is, therefore, un-
likely that the changes in transcript abundance identified
in this study are simply the result of a greatly changed
balance between epithelial and mesenchymal transcripts.
However, the exact ratio of epithelial to mesenchymal
cells at each time point was not measured and might have
changed slightly, contributing to some of the changes in
gene expression we identified.
The study was designed to study the physiology of an-
drogen-induced WD development. A disadvantage of
this design is that it cannot distinguish which genes are
regulated by androgens. The administration of androgens
or anti-androgens to the pregnant dams might have been
useful in identifying androgen-regulated genes, but this
might also have had other undesired side effects on the
pregnancy and embryonic development. We chose not to
use an in vitro model, since a previous study showed that
development of the WD in vitro is slower than in vivo,
and not accompanied by all the changes in protein regu-
lation that take place in vivo [10]. Thus we cannot con-
clude which genes are direct AR targets, but the fact that
several genes have previously been suggested to be regu-
lated by androgens and some have GREs in their pro-
moter suggests that these may be potential AR target
genes. Further studies are necessary to confirm whether
the AR binds to these GREs and can induce gene tran-
scription.
Another limitation of the current study is the fact that
it does not provide functional data. To discover the func-
tion of the identified genes it would be useful to first study
in more detail the time course and site of their expression.
RNA silencing or knockout studies may provide more
definite clues about the role of these genes in WD devel-
opment. Until such studies are performed hypotheses can
206 Horm Res 2006;65:200–209
be formulated using the current knowledge about the
function of the identified genes.
Signalling in the WD
Caveolin-1 (2.5! up), for example, may be involved
in AR signalling in the WD. Upon ligand binding, the AR
can interact with caveolin-1, and caveolin-1 potentiates
ligand-dependent AR activation [13]. Caveolin-1 might
play a role in phosphorylation or in nuclear translocation
of the AR, and may also bring AR in the proximity of ty-
rosine kinase Src [13]. Interestingly, caveolin-1 not inter-
acts only with the AR, it is also upregulated by testoster-
one in several prostate cancer cell lines, in part through
transcriptional regulation. It mediates testosterone-in-
duced cell survival of these cells after serum starvation
[14]. These studies support a possible role for caveolin-1
in androgen-induced survival and growth of the WD.
Other genes may control WD development through
regulation of the Igf signalling pathway. The importance
of this pathway is illustrated by the finding that Igf1 null
mice show severe reproductive abnormalities [15]. They
have a disproportionately small corpus and cauda epi-
didymis, vas deferens and seminal vesicles, and the cau-
da epididymis lacks the numerous ductal convolutions
that are characteristic of this region [15]. Studies in pre-
pubertal mice have shown Igf1 transcripts in myofibro-
blastic cells surrounding the epididymis, whereas type 1
Igf receptor transcripts were detected in the epithelium
of the duct [15], suggesting that Igf1 may be involved in
epithelial–mesenchymal interactions in the WD. Both
Igfbp2 (2.7! up) and Igfbp6 (3.2! up) can influence (Igf-
induced) cell proliferation. The actions of Igfbps, in turn,
are modulated by proteases, such as serine protease 11
(2.7! up), which is thought to cleave Igfbps [16]. Yet an-
other level of control is exerted by activators and inhibi-
tors of proteases. WAP four-disulphide core domain 1
(Wfdc1) (2.1! up), also known as ps20, is a serine prote-
ase inhibitor which exhibits growth- and differentiation-
regulatory functions, perhaps by altering growth factor
bioavailability [17, 18]. Wfdc1 was originally identified
as a potential mediator of epithelial–mesenchymal inter-
actions in the urogenital sinus [17]. It is expressed in sev-
eral adult rat tissues, including smooth muscle of the vas
deferens and seminal vesicles [18].
Another family of proteins that may be important in
WD development is the Tgf! super gene family. Tgf!2
knockout mice have urogenital defects; all five males ex-
amined by Sanford et al. [19] had ectopic testes and one
had unilateral testicular hypoplasia with lack of an epi-
didymis and vas deferens dysgenesis. Tgf! receptor type 3
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null mice, which show decreased sensitivity to Tgf!2,
are poorly fertile when they survive until after birth, al-
though it is unclear what the direct cause of reduced fer-
tility is [20]. Several of the regulated genes play a role in
controlling Tgf! signalling. Dermatopontin (2.7! and
3.1! up) for example, enhances Tgf!-induced promoter
activation; it may act by anchoring Tgf! to the extracel-
lular matrix, thereby preserving it and ensuring a con-
tinuous supply to cells [21]. Lumican (5.1! up) is a pro-
teoglycan widely expressed in connective tissue matrices.
Fibroblasts from lumican-deficient mouse embryos do
not show growth suppression in response to Tgf! to the
same extent as wild-type cells do, suggesting lumican may
also play a role in Tgf! signalling [22]. Serine protease 11
(2.7! up) not only can cleave Igfbps, as discussed above,
but also binds several members of the Tgf! family includ-
ing Tgf!1, Tgf!2 and Bmp4 [23]. It can inhibit signalling
by Tgf! proteins, although the mechanism of this inhibi-
tion is unclear; it does not seem to involve degradation
of the Tgf! proteins [23]. Connective tissue growth factor
(2.4! up) is an immediate early gene, highly induced by
Tgf!, and may act as an effector molecule [24]. It is
thought to stimulate cell proliferation and survival, extra-
cellular matrix synthesis and angiogenesis, although it has
also been implicated in apoptosis (reviewed in [24]). Fur-
thermore, it augments the activity of growth factors such
as Fgfs [25], also implicated in WD development [26].
Interferon regulatory factor 1 (2.0! up) is another factor
mediating growth regulation by Tgf!1 [27].
Depending on the cell type, Tgf! receptors type 1 and/
or 2 can interact with an integral plasma membrane pro-
tein CD44 (4.6! up), which is the principal vertebrate
receptor for hyaluronan, an extracellular matrix proteo-
glycan. CD44 is important in prostate development [28].
In experiments using organ cultures, anti-CD44 antibody
inhibits testosterone-induced ductal branching morpho-
genesis. The role that CD44 plays in ductal branching
morphogenesis is not clear, but depends on interaction
with hyaluronan [28]. Interestingly, both CD44 and hy-
aluronan have been found in mammalian adult epidid-
ymis and vas deferens [29, 30]. Furthermore, CD44 is
required for limb outgrowth [31]. It is expressed in the
apical ectodermal ridge, where it presents Fgfs to limb
mesenchyme [31]. It is possible that CD44 fulfils a sim-
ilar role in the WD. Fgfs are expressed in the WD and
may be important in control of growth, since overex-
pression of Fgfs leads to hyperplasia and other abnor-
malities of the epididymis, vas deferens and seminal
vesicles [32].
Gene Expression in the Wolffian Duct
Taken together, the Igf and Tgf! signalling pathways
seem to play an important role in WD development. Our
results show changes in factors that modulate these path-
ways; this may be more important than changes in the
levels of growth factors themselves. A similar hypothesis
has been postulated for androgen-dependent develop-
ment of the prostate [26].
Tissue Differentiation
Other genes may be important in differentiation of
mesenchyme to smooth muscle. Many of the identified
cytoskeletal genes encode known components of muscle
in general or of WD smooth muscle specifically. Disrup-
tion of the calponin gene (2.6! up) results in altered me-
chanical properties of smooth muscle from adult mouse
vas deferens [33]. Caveolin-1 (2.5! up) has also been
found in smooth muscle of the urogenital tract and ca-
veolin-1 knockout mice display urogenital changes, in-
cluding fluid accumulation in the seminal vesicles, which
may be the results of primary smooth muscle cell dysfunc-
tion [34].
Cysteine-rich LIM-only protein 1 (CRP1) (2.0! up)
may play a role in the induction of these smooth muscle
marker genes. CRP1 potentiates transcriptional activity
of serum response factor in complex with GATA6 by sev-
eral orders of magnitude on promoters of smooth muscle
$-actin, smooth muscle %-actin, calponin-1 and transgelin
[35]. Furthermore, cotransfection of serum response fac-
tor, GATA6 and CRP1 in murine pluripotent mesenchy-
mal cells results in induction of smooth muscle $-actin,
smooth muscle %-actin, transgelin and calponin mRNA
and protein [35]. Interestingly, AR itself can also induce
transcription of myogenic genes through interaction with
serum response factor [36].
In conclusion, we have identified a pattern of co-ordi-
nated transcriptome regulation which is likely to accom-
pany and underlie WD differentiation. Our results sug-
gest several novel cohorts of signals that may contribute
to WD development. Further studies are required to con-
firm the identified changes at the protein level, to deter-
mine the time course over which they occur in more de-
tail, and to investigate in which cells they take place. The
results generate hypotheses that can be tested using in
vitro or gene knock-out studies. Such studies will need to
distinguish which genes are direct and indirect targets of
androgen action and what is their functional role. Identi-
fication of androgen responsive genes expressed in the
developing male reproductive tract may also improve our
understanding of the causes of hypospadias and other
genital anomalies of unknown cause.
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Horm Res 2006;65:200–209 207
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 Acknowledgements
We are grateful to Doreen Christmas, Nicola Johnson, Pam
Stacey and Cris Burton for their help.
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