Dicer1 Ablation in the Mouse Epididymis Causes
Dedifferentiation of the Epithelium and Imbalance in Sex
Ida Bjo ¨rkgren1,2, Lauri Saastamoinen1, Anton Krutskikh3, Ilpo Huhtaniemi3, Matti Poutanen1,4,
Petra Sipila ¨1,4*
1Department of Physiology, Institute of Biomedicine, University of Turku, Turku, Finland, 2Turku Graduate School of Biomedical Sciences, Turku, Finland, 3Institute of
Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, London, United Kingdom, 4Turku Center for Disease Modeling, (TCDM),
University of Turku, Turku, Finland
Background: The postnatal development of the epididymis is a complex process that results in a highly differentiated
epithelium, divided into several segments. Recent studies indicate a role for RNA interference (RNAi) in the development of
the epididymis, however, the actual requirement for RNAi has remained elusive. Here, we present the first evidence of a
direct need for RNAi in the differentiation of the epididymal epithelium.
Methodology/Principal Findings: By utilizing the Cre-LoxP system we have generated a conditional knock-out of Dicer1 in
the two most proximal segments of the mouse epididymis. Recombination of Dicer1, catalyzed by Defb41iCre/wt, took place
before puberty, starting from 12 days postpartum. Shortly thereafter, downregulation of the expression of two genes
specific for the most proximal epididymis (lipocalin 8 and cystatin 8) was observed. Following this, segment development
continued until week 5 at which age the epithelium started to regress back to an undifferentiated state. The
dedifferentiated epithelium also showed an increase in estrogen receptor 1 expression while the expression of androgen
receptor and its target genes; glutathione peroxidase 5, lipocalin 5 and cysteine-rich secretory protein 1 was
downregulated, indicating imbalanced sex steroid signaling.
Conclusions/Significance: At the time of the final epididymal development, Dicer1 acts as a regulator of signaling pathways
essential for maintaining epithelial cell differentiation.
Citation: Bjo ¨rkgren I, Saastamoinen L, Krutskikh A, Huhtaniemi I, Poutanen M, et al. (2012) Dicer1 Ablation in the Mouse Epididymis Causes Dedifferentiation of
the Epithelium and Imbalance in Sex Steroid Signaling. PLoS ONE 7(6): e38457. doi:10.1371/journal.pone.0038457
Editor: Joel R. Drevet, Clermont-Ferrand Univ., France
Received January 20, 2012; Accepted May 5, 2012; Published June 6, 2012
Copyright: ? 2012 Bjo ¨rkgren et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by research grants from The Academy of Finland (project number 138561, http://www.aka.fi), and Sigrid Juse ´lius Foundation
(http://www.sigridjuselius.fi/foundation). IB is supported by the Turku Doctoral Programme of Biomedical Sciences (http://www.tubs.utu.fi). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
After development in the testis, the spermatozoa travel through
the epididymis where they mature, gaining motility and the ability
to fertilize the oocyte. Despite consisting of a single long duct, the
epididymis is a complex organ, highly convoluted and divided into
several anatomical and functional segments, i.e. initial segment
(IS), caput (CAP), corpus (COR) and cauda (CAU). Each segment
synthesizes and secretes a specific set of proteins, thus creating the
unique luminal environment needed for the sperm maturation
process [1–3]. The most proximal segments (IS and CAP) have
been proven essential for sperm maturation as the disruption of
their development or function often leads to male infertility [4–7].
The epididymis develops from the mesonephric tubules and the
proximal Wolffian duct (WD) . During the embryonic stage,
and before epithelial differentiation, mesenchymal androgen
receptor (AR), along with inhibin beta A (Inhba), facilitates the
elongation and convolution/coiling of the tubule [9–11]. Devel-
opment of the epididymis continues after birth with differentiation
of the epithelial cells into principal, basal and narrow/clear cells
[2,2,12,12,13]. At the onset of spermatogenesis, the epididymal
epithelium develops segment-specific gene expression . From
studies with genetically modified mice, it has become evident that
leucine-rich repeat-containing G protein-coupled receptor 4
(LGR4, also known as GPR48) is needed for the postnatal
epididymal coiling and the differentiation of IS [15,16]. In
addition, the proto-oncogene Ros1 (ROS1, also known as c-ros)
is necessary for the formation of IS . Recent studies with
epididymal AR knock-out mice have revealed that androgen
signaling is required for the formation of IS and differentiation of
principal and basal cells [6,7,10]. However, there are still many
unresolved issues regarding the regulatory pathways responsible
for differentiation of the epididymal segments and their specific
Many aspects of development are regulated by RNA interfer-
ence (RNAi). Small non-coding RNAs bind to complimentary
mRNA sequences and cause translational silencing and mRNA
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cleavage . One class of small non-coding RNAs is the
microRNAs (miRNAs) which are initially produced as longer
precursors that need to be processed by the RNaseIII enzyme
Dicer1 to become fully functional . The ,22 nt-long mature
miRNAs then control protein expression and modulate diverse
cellular events such as differentiation, proliferation, apoptosis and
cell metabolism [19,20]. Consequently, Dicer1 deficient mice die
already on embryonic day 7.5 owing to complete loss of
pluripotent stem cells . To study the effect of RNAi in the
development and function of specific organs, Dicer1 conditional
knock-out (Dicer1 cKO) mice can be used. The need for Dicer1 in
the differentiation of several cell types, including sensory epithelial
cells, T cells and pancreatic b-cells, has been shown in Dicer1 cKO
mice by crossing Dicer1fl/flmice with those expressing Cre in the
cells of interest [12,22–24]. Furthermore, a requirement for Dicer1
in the patterning of the liver and colon was also confirmed with
Dicer1 cKO mice. Hepatocyte specific Dicer1 ablation compro-
mises region-specific protein expression while Dicer1 depletion in
the developing colon leads to a disorganized epithelium [25,26].
Previous studies on human and rat epididymides have shown
that miRNAs are differentially expressed at juvenile and adult
stages [27,28], indicating a role for miRNAs in the postnatal
development of the epididymis. To study the function of RNAi in
the developing epididymis we generated epididymis-specific Dicer1
cKO mice by crossing Dicer1fl/flmice with Defb41iCremice. This
resulted in the elimination of Dicer1 expression from the pre-
pubertal epididymis. Our results demonstrate the importance of
Dicer1 in sex steroid signaling and in maintenance of the
differentiated state of the epididymal epithelium.
Generation of Dicer1fl/fl; Defb41iCre/wtMice
Quantitative RT-PCR of the mouse epididymis showed a
continuous expression of Dicer1 from birth into adulthood
(Figure 1C). As the full knock-out of Dicer1 is embryonically lethal
, we generated a Dicer1 cKO mouse line by crossing Dicer1fl/fl
mice  with a mouse line expressing iCre under the Defensin
beta 41 (Defb41) promoter. The Dicer1flallele consists of two loxP
sites flanking exon 24, which contains a major part of the second
RNaseIII domain (Figure 1A). The heterozygous Defb41iCremouse
line did not show any phenotypic defects or fertility problems and
expressed iCre in the epithelium of the most proximal part of the
epididymis, IS and CAP. Recombination of Dicer1 was observed in
12 day-old Dicer1fl/fl; Defb41iCre/wtmouse IS and CAP by genomic
PCR (Figure 1B) and qRT-PCR studies revealed a significant
reduction in Dicer1 expression levels at the age of 2 months
Morphology of the Epididymis and Fertility of Dicer1fl/fl;
Macroscopic evaluation of 2 month-old Dicer1fl/fl; Defb41iCre/wt
mice epididymides revealed an underdeveloped IS and, in
addition, the mice frequently presented with enlarged efferent
ducts (Figure 2A, B). The IS of control mice can be clearly
visualized owing to the endogenous b-galactosidase activity in the
segment. The much smaller IS of Dicer1fl/fl; Defb41iCre/wtmice
could not be distinguished from CAP with X-gal staining
(Figure 2A). Furthermore, the intense vasculature typical of WT
IS was missing from the Dicer1 cKO IS. Histological evaluation
showed a division of the epididymis into different segments
(Figure 2B) but the epithelial cell layer of both IS and CAP was
disorganized (Figure 3H). Dicer1fl/flmice have a similar phenotype
to WT mice epididymides and were used as controls throughout
the study. Dicer1 cKO epididymides were significantly smaller than
those of control mice (30.461.5 mg, control: 35.460.7 mg,
P#0.01). No significant difference in the weight of 6 month-old
Dicer1 cKO and control mice epididymides was observed.
However, the epithelial cell layer of the 6 month-old Dicer1fl/fl;
Defb41iCre/wtmouse was further disturbed, with neoplastic changes
in the efferent ducts causing their progressive obstruction (Figure
S1). Even though sperm were detected in the CAU, 2- to 3-month-
old Dicer1fl/fl; Defb41iCre/wtmale mice failed to produce offspring
when mated with WT females (Table 1). The number of sperm
was reduced in Dicer1fl/fl; Defb41iCre/wtmouse epididymides as
histological staining showed some tubular cross sections with no
sperm. At 6 months of age the number of tubular cross sections
without sperm was further increased due to the obstruction of the
Dicer1fl/fl; Defb41iCre/wtmouse efferent ducts. The testis of the 6
month-old Dicer1fl/fl; Defb41iCre/wtmouse also displayed disruption
of the seminiferous epithelium owing to fluid back-pressure (data
not shown). Further morphological analyses revealed that the
number of sperm with angulated tails was not significantly
increased in 2 month-old Dicer1fl/fl; Defb41iCre/wtmouse CAU
(22.963.4% of all sperm, control: 16.361.2%).
Effects of Dicer1 cKO on Epididymal Cell Differentiation
At the age of one month, the IS of both control and Dicer1fl/fl;
Defb41iCre/wtmice was distinguishable from the CAP by its high,
columnar-shaped epithelial cells (Figure 3C, D). However, the
height of the epithelium of Dicer1 cKO IS was reduced below that
of control mouse IS. In the 45 day-old mouse, the epithelial cells of
the Dicer1 cKO IS had regressed to an undifferentiated state with a
disorganized epithelial cell pattern (Figure 3E, F), resembling those
of the epididymis of 14 day-old mice (Figure 3A, B). Furthermore,
the height of the epithelial cells of IS was significantly reduced
(average cell height in control IS: 45 mm, in Dicer1 cKO IS:
28 mm, P#0.0001) in 2 month-old Dicer1fl/fl; Defb41iCre/wtmice.
However, the phenotype of the Dicer1 cKO IS varied between
individuals from a severely disorganized epithelial cell layer to a
thin epithelial cell layer resembling that of COR (Figure S2) in
To study further the effect of Dicer1 ablation on epithelial cell
differentiation, the presence of different epithelial cell types of the
epididymal epithelium was analyzed by immunohistochemistry.
Phalloidin conjugated to TRITC was used to stain the F-actin of
principal cells, and antibodies against vacuolar H+-ATPase (V-
ATPase) and Keratin 5 (Krt5) were used to stain clear/narrow
cells and basal cells, respectively. The results indicated that all cell
types were present in the Dicer1 cKO epididymis (Figure 4). The
epithelial cell types were found in similar numbers and locations in
the Dicer1 cKO epithelium as in control animals. However, a-actin
staining revealed an increase in muscle cell layer thickness
(Figure 4G, H). The Dicer1fl/fl; Defb41iCre/wtmice displayed three
layers of muscle cells surrounding the epididymal duct, whereas
control mice typically had one.
Genes specifically expressed in the mature proximal epididymis,
IS and proximal CAP; lipocalin 8 (Lcn8), brain expressed
myelocytomatosis oncogene (Bmyc) and cystatin 8 (Cst8), showed
a marked downregulation in their expression in IS and CAP of 2
month-old Dicer1fl/fl; Defb41iCre/wtmice. The only exceptions were
Ros1 and G protein-coupled receptor 64 (Gpr64, also known as
HE6) that showed no significant difference in expression levels
compared with the control (Figure 4I). The expression of Cst8,
Lcn8 and Ros1 is initiated at postnatal days 20, 21 and 16,
respectively [5,30,31]. The qRT-PCR results from different time
points during epididymal development showed significantly
reduced expression of both Cst8 and Lcn8 in Dicer1fl/fl; Defb41iCre/wt
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mice, from 21 days postpartum onward (Figure S3A, B). However,
the expression levels of Ros1 did not differ significantly from that of
control mice (Figure S3C).
Cell Proliferation and Apoptosis
Immunohistochemical studies showed a marked increase in cell
proliferation throughout the proximal epididymis of 2 month-old
Dicer1fl/fl; Defb41iCre/wtmice (Figure 5B). The number of Ki-67
positive cells was on average 2 times higher in Dicer1 cKO IS
(15.263.0 cells/mm, control: 6.460.6 cells/mm, P#0.05) and
almost 6 times higher in Dicer1 cKO CAP (11.161.7 cells/mm,
control: 1.960.5 cells/mm, P#0.001) compared with those of
control mice epididymides (Figure 5C). Although the epithelium
was highly proliferative, it did not lead to a marked increase in the
size of the Dicer1fl/fl; Defb41iCre/wtmouse epididymides. On the
contrary, at the age of 2 months the Dicer1 cKO epididymis was
significantly smaller than that of the control mouse. Furthermore,
the number of apoptotic cells was increased in Dicer1 cKO IS and
CAP (Dicer1 cKO IS: 0.8960.39 cells/mm, control IS: 0.0760.07
cells/mm; Dicer1 cKO CAP: 1.1260.34 cells/mm, control CAP:
0.0760.07 cells/mm, P#0.05) (Figure 5D–F). The difference seen
in IS was not statistically significant owing to high variation
between the individual mice.
Expression of Sex Steroid and Fibroblast Growth Factor
Receptors in the Dicer1 cKO IS and CAP
To study whether the observed epididymal phenotype is caused
by changes in sex steroid signaling, the expression of Ar, estrogen
receptor 1 (Esr1, also known as ERa) and estrogen receptor 2 (Esr2,
also known as ERb) was analysed by qRT-PCR and immunohis-
tochemistry in 2 month-old Dicer1 cKO and control epididymides.
The qRT-PCR results showed a significantly weaker expression of
Ar in Dicer1 cKO IS and CAP (Figure 6E), and downregulation of
AR target gene expression, cysteine-rich secretory protein 1
(Crisp1), glutathione peroxidase 5 (Gpx5) and lipocalin 5 (Lcn5), was
also observed (Figure 6F). Esr2 showed a similar reduction in
mRNA expression levels as Ar, while the expression of Esr1 did not
significantly differ between Dicer1 cKO and control IS and CAP
(Figure 6E). However, immunohistological staining displayed an
altered ESR1 expression pattern in Dicer1 cKO IS. ESR1 was
foremost expressed in the narrow cells of control mouse IS,
whereas the Dicer1 cKO IS showed equal expression of the
receptor in almost all epithelial cells (Figure 6A, B). Furthermore,
immunohistochemical analyses clearly showed weaker AR staining
in a number of the Dicer1 cKO epididymides analysed (Figure 6D).
Nevertheless, the AR findings were variable and some Dicer1 cKO
epididymides had an AR staining equal to that of the controls.
Furthermore, the relative expression values of fibroblast growth
factor receptors 1–4 (Fgfr1–4) were not statistically different when
comparing qRT-PCR results from Dicer1fl/fl; Defb41iCre/wtmice
with control mouse IS and CAP (data not shown).
At birth, the epididymis consists of a long convoluted duct
lacking both cell type and segment specific markers. In the mouse
Figure 1. The epididymis specific Dicer1 knock-out. (A) Schematic diagram of the Dicer1 locus with loxP sites flanking exon 24 (e24). Arrows
indicate the location of genotyping primers used for analyzing the deletion of e24. (B) Genomic PCR of 12 day-old mice showing the intact locus
(Dicer1fl) and the recombinant locus with e24 deleted (Dicer1flD). Exon 24 is only deleted in the Dicer1fl/fl; Defb41iCre/wtmouse initial segment (IS) and
caput (CAP) while the iCre locus is detected in all segments of the epididymis. (C) Expression of Dicer1 mRNA in the whole epididymis of 1–42 day-old
wild-type mice. (D) Dicer1 mRNA expression levels in the efferent ducts (ED), the different segments of the epididymis and testis (TE) of 2 month-old
control and Dicer1 conditional knock-out (cKO) mice. Expression levels are presented relative to Ppia (testis) and L19 (epididymis) expression. COR,
corpus; CAU, cauda. Statistical significance was calculated from the expression levels of 3 control and 4 Dicer1fl/fl; Defb41iCre/wtmouse samples using
the unpaired t-test. Statistical significance of changes is indicated as follows: **, P#0.01. Schematic picture modified from Harfe et al., 2005 .
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epididymis, differentiation of the epithelial cell types takes place
between day 14 and day 21  after which segment-specific gene
expression can be observed . Nonetheless, very little is known
about the regulatory pathways responsible for the epididymal
segmentation and the differentiation of epithelial cell types. In this
study, we demonstrate that post-transcriptional regulation via
RNAi signaling is an important regulator in the development of
the proximal epididymal segments.
In our Dicer1fl/fl; Defb41iCre/wtmouse model the ablation of Dicer1
begins 12 days pp but, at the time of the final cell type
differentiation during puberty, the epididymal epithelium of
Dicer1fl/fl; Defb41iCre/wtmice still resembles that of control mice.
Studies revealed a significant reduction in the expression of IS
specific genes at 21 days pp, and about 2 weeks later, the
epithelium began to regress. At 45 days of age, the Dicer1 KO IS
morphologically resembled that of an undifferentiated pre-
pubertal epididymis. Despite the morphological changes, Dicer1
ablation did not affect cell type differentiation as all major
epithelial cell types were detected in the epididymides of 2 month-
old Dicer1fl/fl; Defb41iCre/wtmice. However, the function of the
principal cells was compromised as they displayed a significant
reduction in segment specific gene expression.
When studying the different epithelial cell types, we detected a
thicker layer of smooth muscle cells surrounding the duct of the
Dicer1fl/fl; Defb41iCre/wtmouse epididymis. As Dicer1 ablation is not
likely to occur in the stromal tissue, the observed increase in
smooth muscle cell number may indicate altered epithelial-
mesenchymal signaling. It has previously been shown that cross
talk between different cell types is essential for normal epididymal
functions, and for example the communication between clear cells
and other epithelial cell types is required to maintain luminal pH
. Interestingly, there are species-specific differences in
thickness and distribution of the epididymal smooth muscle cell
layer [34–36]. Further investigations regarding smooth muscle cell
differentiation could thus benefit from comparative studies of
Previous studies have shown that ROS1 is one of the master
regulators of IS development, as the lack of both ROS1 and its
negative regulator, protein tyrosine phosphatase SHP-1, causes a
defect in IS differentiation [5,37]. This affects also the regulation
of sperm volume as the majority of sperm from the Ros1 KO CAU
present with angulated tails . However, Dicer1 cKO IS still has
,70% of the Ros1 expression of the control IS and the
dedifferentiated IS does not cause a significant increase in sperm
tail angulation. Furthermore, males with one intact Ros1 allele
have normal epididymal development . These data indicate
that the epididymal phenotype, observed in the present study, is
independent of ROS1 signaling. Other proteins that have a
regulative role in IS are the Fibroblast growth factors. They
control gene expression by binding to FGFRs in the epithelium of
IS [39,40]. Studies in other male reproductive organs also indicate
a role for FGFRs in regulating stromal-epithelial induction of cell
proliferation and tissue homeostasis . To assess the effect of
FGF signaling on the observed epididymal phenotype, we studied
the expression of Fgfrs in the epididymis. However, no changes
were observed and it is therefore unlikely that FGF signaling
contributes to the dedifferentiation of the epithelium or to the
increased smooth muscle cell layer thickness of the Dicer1fl/fl;
Sex steroids are important regulators of epididymal develop-
ment and function. In the epididymis, epithelial expression of Ar
starts around E14.5–15.5 [10,42] and is required for IS
development  and for the differentiation of principal and basal
cells in the proximal epididymis [6,10]. A further role for androgen
signaling is observed after orchidectomy, where androgen
depletion leads to extensive apoptosis throughout the epididymal
Figure 2. Morphology of 2 month-old mice proximal epididy-
mides. (A) X-gal staining detecting endogenous b-galactosidase
activity in a control mouse initial segment (IS) and proximal corpus
(COR). Dicer1fl/fl; Defb41iCre/wtmice display no staining of initial segment
and have enlarged efferent ducts (ED). (B) HE staining of the efferent
ducts and the proximal epididymis of control and Dicer1fl/fl; Defb41iCre/wt
mice. Scale bars 1.5 mm.
Table 1. Fertility of Dicer1fl/fl; Defb41iCre/wtmale mice.
No. of MalesTotal No. of Copulatory PlugsTotal No. of Litters Average No. of Pups per Litter
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epithelium of both prepubertal and postpubertal animals . In
male reproductive organs, estrogens are produced in the testis,
spermatozoa and epididymis [44,45]. Esr1 is highly expressed
throughout the efferent ducts, where it regulates fluid reabsorption
of non-ciliated epithelial cells [46,47]. Less is known about the
function of ESR1 in the epididymis, although high expression of
Esr1 is detected in the narrow cells of IS and throughout the
epithelium of CAP . Recent studies have indicated a role for
ESR1 in luminal pH maintenance [48,49] as well as in smooth
muscle contractility . Esr2 is transcribed in all cells of the
epithelium, however, its role in the epididymis is still unknown
since the full knock-out of Esr2 does not display any difference in
morphology or function of the epididymis compared with that of
WT mice .
Figure 3. Differentiation of the epididymal epithelium. Hematoxylin and eosin staining of control and Dicer1fl/fl; Defb41iCre/wtmouse
epididymides (A, B) The undifferentiated epithelium of the proximal epididymis of a 14 day-old control and a Dicer1fl/fl; Defb41iCre/wtmouse. (C, D) 33
day-old control and Dicer1fl/fl; Defb41iCre/wtmouse showing initiated differentiation of the initial segment (IS). (E) The fully developed IS of a 45 day-old
control mouse. (F) The epithelium of a Dicer1fl/fl; Defb41iCre/wtmouse IS resembling that of the 14 day-old mouse. (G, H) The epididymis of an adult, 2
month-old, control and Dicer1fl/fl; Defb41iCre/wtmouse. CAP, caput. Scale bars 100 mm.
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The Dicer1fl/fl; Defb41iCre/wtmouse model presents with a
significant downregulation in Ar, known AR target genes and
Esr2 mRNA expression in the proximal epididymis. However, on
the basis of the current study, we cannot distinguish whether the
down-regulation of the target genes is due to a direct lack of AR
signaling or an effect of the observed epithelial cell dedifferenti-
ation. ESR1, on the other hand, displayed an altered expression
pattern in the Dicer1 KO IS, with expression not only in narrow
cells, as in IS of control mice, but throughout the epithelium. As
ESR1 is known to promote cell proliferation , high ESR1
levels in the Dicer1 cKO IS could explain the observed increase in
cell proliferation in that epididymal segment.
Studies on excessive estrogen signaling in males suggest that an
imbalance in the testosterone-estrogen ratio could be of more
importance than the actual increase in estrogen signaling .
Administration of a single high dose of diethylstilbestrol (DES), a
potent synthetic estrogen, to neonatal rats, caused a reduction in
the epithelial height of the epididymis. However, the increased
estrogen levels gave rise to an additional downregulation of AR in
the entire epididymis and thus an imbalance in sex steroid
signaling. When the estrogen-testosterone ratio was adjusted by
co-administration of testosterone, no phenotypic abnormalities
were observed . The reduction in Ar expression observed in
mice could therefore not only have
significant consequences for epididymal development but also be
directly caused by the increased ESR1 levels. However, a recent
study on a proximal epididymis-specific AR cKO, also showed
ESR1 expression in all cell types of the proximal epididymal
epithelium . This would indicate a role for AR in the repression
of Esr1 expression. AR cKO epididymides also showed similar
features to those of the Dicer1fl/fl; Defb41iCre/wtmice with an altered
epididymal stroma showing a disrupted smooth muscle cell layer
or a thickened mesenchyme [7,55]. In light of these studies, AR
and ESR1 expression in the epididymis seem to be tightly linked
and although our current results show an imbalance in sex steroid
receptor ratio we cannot with certainty say if the imbalance was
initiated by a lowered AR expression or an increase in ESR1
Adams et al. were the first to show that Esr1 is a direct target of
miR-206 . Since then, around 14 evolutionarily conserved
miRNAs have been found to target directly the 39-UTR of
mammalian Esr1 . There is also evidence of a miRNA-induced
downregulation of Esr1 after testosterone treatment in the female
mouse liver . In light of these previous studies the upregulation
of ESR1 in Dicer1 KO IS might be directly ascribable to the
ablation of mature miRNAs. Interestingly, previous studies have
shown induction of miRNA expression by AR in vivo .
Androgen Responsive Elements (AREs) are also found in the
promoter region of several miRNAs [60,61]. Although AR would
not induce expression of all epididymal miRNAs, the observed
phenotype of Dicer1fl/fl; Defb41iCre/wtmice could be partially AR
dependent. To understand better the role of the RNAi pathway
during epididymal development, miRNA expression data from
different developmental time points is needed. These results could
be further compared to already available array data from the
human and rat epididymis [27,28] to demonstrate evolutionarily
conserved RNAi regulation.
In conclusion, the present study shows the importance of the
RNAi pathway in the postnatal development of the proximal
epididymis. Ablation of Dicer1 in the epididymal epithelium causes
a regression in cell differentiation. The epithelium maintains cell
type identity but lacks a segment-specific gene expression pattern.
Furthermore, the epididymal phenotype of Dicer1fl/fl; Defb41iCre/wt
mice resembles that of mice with an imbalanced ratio of sex steroid
receptors, AR cKO mice and mice with excess estrogen signaling.
Figure 4. Immunohistochemical staining of the different epithelial cell types. Staining of initial segment (A, B) and initial segment and
caput (C–H) of two-month-old control and Dicer1fl/fl; Defb41iCre/wt mouse epididymides. (A, B) Staining of principal cell F-actin by phalloidin-TRITC.
(C, D) Expression of vacuolar H+-ATPase (V-ATPase) in narrow and clear cells (marked with arrows). (E, F) Expression of keratin 5 (Krt5) in basal cells. (G,
H) Smooth muscle cells. Dicer1fl/fl; Defb41iCre/wt mice display a thicker smooth muscle cell layer than that of control mice. Inserts are 36
enlargements of the stained cells. (I) qRT-PCR for proximal epididymis-specific gene expression. Expression of lipocalin 8 (Lcn8), brain expressed
myelocytomatosis oncogene (Bmyc), cystatin 8 (Cst8), Ros1 proto-oncogene (Ros1) and G protein-coupled receptor 64 (Gpr64) in the initial segment
and caput of 2 month-old control and Dicer1 conditional knock-out (cKO) mouse epididymides. Values relative to L19 expression. Statistical
significance was calculated from the expression levels of 3 control and 3 Dicer1fl/fl;Defb41iCre/wt mouse (Ros1:8 control and 9 Dicer1fl/fl;Defb41iCre/
wt mouse) samples using the unpaired t-test. Statistical significance of changes is indicated as follows: ns, not significant; *, P#0.05; **, P#0.01. Scale
bars 100 mm.
Figure 5. Cell proliferation and apoptosis. Ki67 immunostaining;
(A) Control; (B) Dicer1fl/fl; Defb41iCre/wtmouse. TUNEL labeling; (D)
Control; (E) Dicer1fl/fl; Defb41iCre/wtmouse. Arrows mark apoptotic cells.
(C, F) Comparison of number of proliferating cells and apoptotic cells in
the initial segment (IS) and caput (CAP) of control (white bars) and
Dicer1fl/fl; Defb41iCre/wtmice (grey bars). The number of proliferating and
apoptotic cells were calculated from ten tubular cross sections of 5
control and 5 (Ki67 stained) and 8 (TUNEL labeled) Dicer1fl/fl; Defb41iCre/
wtmice epididymides and the total cell number was divided by the
circumference of the tubular cross sections (mm). Statistical significance
of changes, calculated using the unpaired t-test, is indicated as follows:
ns, not significant; *, P#0.05; ***, P#0.001. Scale bars 100 mm.
Dicer1 in the Epididymal Development
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This suggests that Dicer1-dependent pathways are important
regulators of balanced estrogen-androgen action in the mouse
Materials and Methods
All mice were handled in accordance with the institutional
animal care policies of the University of Turku (Turku, Finland),
and every effort was made to minimize suffering of the animals.
Mice were specific pathogen-free, fed with complete pelleted chow
and tap water ad libitum in a room with controlled light (12 hours
light, 12 hours darkness) and temperature (2161uC). Animal
experiments were approved by the Finnish Animal Ethics
Committee (license number: 20072-Sipila ¨
Poutanen), and the institutional policies on animal experimenta-
tion fully met the requirements as defined in the NIH Guide on
To inactivate Dicer1 conditionally from the epithelial cells of the
proximal epididymis, Dicer1fl/flmice  were crossed with a
heterozygous Defb41iCreknock-in (KI) mouse line. The KI was
Figure 6. Expression of Sex steroid receptors. (A) Immunostaining of two-month-old control mouse initial segment (IS) shows expression of
Estrogen receptor 1 (ESR1) only in the narrow cells (indicated by arrows) while (B) the Dicer1fl/fl; Defb41iCre/wtmouse has ESR1 expression in most cells
of the epithelium. (C, D) Staining for Androgen receptor. (D) Androgen receptor expression varied between Dicer1fl/fl; Defb41iCre/wtmice epididymides.
Some samples had similar expression levels as the control while others showed areas of more weakly stained tissue. CAP, caput. Scale bars 100 mm.
(E) Expression of androgen receptor (Ar) and estrogen receptor 1 and 2 (Esr1 and Esr2) as well as (F) AR target gene: glutathione peroxidase 5 (Gpx5),
lipocalin 5 (Lcn5), cysteine-rich secretory protein 1 (Crisp1) mRNA in IS and CAP of 2 month-old control and Dicer1 conditional knock-out (cKO) mice
epididymides. Expression values relative to L19 expression. Statistical significance was calculated from the expression levels of 3 control and 4 Dicer1fl/
fl; Defb41iCre/wtmouse samples using the unpaired t-test. Statistical significance of changes is indicated as follows: ns, not significant; *, P#0.05; ***,
Dicer1 in the Epididymal Development
PLoS ONE | www.plosone.org8 June 2012 | Volume 7 | Issue 6 | e38457
generated by inserting the iCre cDNA allelle into the translation
initiation site of the Defb41 gene. A detailed characterization of the
Defb41iCreKI mouse line will be described elsewhere. Defb41iCre/wt
TTGTCTTACCAGGTTTCCTCCT, Tm 56uC, for the wild-
type (WT) allele and iCre Fw: TCTCCAACCTGCTGACTGTG
and iCre Re: AGGGACACAGCATTGGAGTC, Tm 59uC, for
the iCre allele. Dicerfl/flmice were genotyped as previously
described . For detection of the floxed allele, IS together with
CAP, COR together with CAU and testes from 12 day-old mice
were collected. After DNA extraction, the recombined allele was
detected by genomic PCR as previously described . Dicer1fl/fl;
Defb41iCre/wtmice and the controls, Dicer1fl/flmice, were obtained
from the same litters. The genetic background of these mice was
mixed C57Bl/6N and SV129.
Sperm Morphology and Fertility of Dicerfl/fl; Defb41iCre/wt
To study male fertility, 2–3 month-old male Dicer1fl/fl;
Defb41iCre/wtmice and control mice were mated with FVB/N
female mice. The female mice were superovulated by intraperi-
toneal injections of 5 IU pregnant mare serum gonadotropin
(PMSG, NHPP, Dr. Parlow) and 5 IU human chorionic
gonadotrophin (hCG, Pregnyl, Schering-Plough) 26 and 2 hours
before mating, respectively. Females were checked for copulatory
plugs the following morning. The mated females were followed for
3–4 weeks to determine the number of litters and offspring
produced by each male. For sperm analyses, CAU of two-month-
old control and Dicer1fl/fl; Defb41iCre/wtmice were dissected and
incubated in 300 ml of HTF medium (William A. Cook Australia
Pty. Ltd, Brisbane, Australia) at 37uC, 5% CO2 for 30 min, to
allow the sperm to swim out. The sperm were spread on
microscope slides and stained using the Papanicolaou technique
(Haematoxylin, OG-6 and EA-50). The morphology of 100
sperm/sample from 4 control and 5 Dicer1fl/fl; Defb41iCre/wtmice
samples was analyzed.
Histology, Immunohistochemistry and b-galactosidase
Epididymides from 19 day - 6 month-old control and Dicer1fl/fl;
Defb41iCre/wtmice were fixed overnight in 4% paraformaldehyde
(PFA), embedded in paraffin and prepared for histological analyses
by standard procedures. Haematoxylin and eosin (HE) staining
was performed by standard procedures. Epithelial cell height was
measured by using the Leica IM500 imaging software. To detect
the different epithelial cell types of the epididymis, the slides were
incubated with the following antibodies overnight at 4uC: rabbit
monoclonal anti-Keratin 5 1:100 (RM-2106, Thermo Scientific),
rabbit polyclonal anti-V-ATPase 1:100 (a gift from S. Breton,
Program in Membrane Biology, MGH Simches Research Center,
Boston, MA) and mouse monoclonal anti-a-actin 1:500 (sc-32251,
Santa Cruz Biotechnology). All the primary antibodies were
diluted in PBS supplemented with 1% bovine serum albumin. The
antibody-antigen complexes were visualized by incubation for
30 min at room temperature with 1:200 Alexa Fluor 488- and
594-conjugated goat anti-mouse and goat anti-rabbit antibodies
(Invitrogen). For detection of F-actin, the epididymides of 2
month-old mice were fixed for 15 min in 4% paraformaldehyde
and rapidly frozen. The frozen sections (8 mm) were immuno-
stained with Phalloidin-TRITC 1:400 (P1951, Sigma-Aldrich). All
slides used for the above mentioned stainings were counterstained
with 49,6-diamino-2-phenylindole dihydrochloride (DAPI, Sigma)
and mounted in Mowiol 4–88 (Sigma). For sex steroid receptor
detection the following antibodies were used: rabbit polyclonal
anti-AR (N-20) 1:1000 (sc-816, Santa Cruz Biotechnology), mouse
monoclonal anti-estrogen receptor a 1:200 (M7040, Dako) and
mouse monoclonal anti-estrogen receptor b1 1:100 (M7292,
Dako). The antibody-antigen complexes were visualized by using
anti-rabbit and anti-mouse HRP labeled polymer (EnVision,
Dako) combined with DAB+ chromogen system (Dako).
For b-galactosidase staining two-month-old mice epididymides
were fixed in 0.2% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA
in PBS for 30 minutes. The tissues were washed overnight and
stained for 2 hours at 37uC in 2 mM MgCl2, 0.01% NaDeox-
ycholate, 0.02% Tergitol-type NP-40, 5 mM K4Fe(CN)6, 5 mM
K3Fe(CN)6, 1 mg/ml X-gal in PBS.
RNA Isolation and qRT-PCR
For analyzes of gene expression, 1 day – 42 day-old WT mice
(mixed background, C57Bl/6N and SV129) and 21 day - 2
month-old control and Dicer1fl/fl; Defb41iCre/wtmice were used. The
epididymides, efferent ducts and testes were dissected out and
weighed. The epididymis of control and Dicer1fl/fl; Defb41iCre/wt
mice was cut into three segments; IS and CAP, COR and CAU.
All tissues were frozen in liquid nitrogen and stored at 280uC.
Total RNA was isolated from the tissues using Tri Reagent
according to the manufacturer’s instructions (Molecular Research
Center, Inc.). For RT-PCR, 1 mg of total RNA was treated with
deoxyribonuclease I (DNaseI, Amplification Grade, Invitrogen)
and reverse-transcribed by using the DyNAmo cDNA synthesis kit
(Thermo Scientific). The cDNA was diluted 1:50–1:100 for
quantitative PCR. Quantitative PCR was performed using the
DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific). All
samples were run in triplicate reactions and standards in duplicate
reactions. L19 and Ppia were used as endogenous controls to
equalize for the amounts of RNA in the epididymis and testis,
respectively. Primer sequences and qRT-PCR conditions for
analyzing the expression of Dicer1, Ros1, Bmyc, Lcn8, Cst8, Gpr64,
Ar, Esr1, Esr2, Gpx5, Lcn5, Crisp1 and Fgfr 1–4 are described in
Cell Proliferation and Apoptosis
To detect cell proliferation, epididymis sections were stained
with rat monoclonal anti-Ki67 1:500 (M7249, Dako). The
antibody-antigen complexes were visualized by using 1:200 rabbit
polyclonal anti-rat (Dako) combined with anti-rabbit HRP-labeled
polymer and DAB+ chromogen system. Terminal Uridine
Deoxynucleotidyl Nick End Labeling (TUNEL) was used to detect
apoptotic cells. Labeling was performed by using 0.8 U/ml TdT
(Terminal transferase, recombinant, Roche) and Biotin-16-dUTP
(Roche), 1 h at 37uC. The reaction was visualized with Biotin
coupled with ExtrAvidine-Peroxidase, 1:200 (Sigma-Aldrich)
combined with DAB+ chromogen system. Proliferating and
apoptotic cells were counted from ten tubular cross sections of 5
control and 5–8 Dicer1fl/fl; Defb41iCre/wtmouse IS and CAP,
respectively. The number of proliferative and apoptotic cells was
then divided by the circumference (mm) of the tubular cross
For statistical analyses of organ weights, epithelial cell heights,
animal fertility, sperm morphology, cell proliferation/apoptosis
and qRT-PCR, the GraphPad Prism 5 software (GraphPad
Software, Inc., La Jolla, CA) was used. Unpaired t-test was used to
determine statistical significances, assigning P#0.05 as the limit of
Dicer1 in the Epididymal Development
PLoS ONE | www.plosone.org9 June 2012 | Volume 7 | Issue 6 | e38457
ducts. Hematoxylin and eosin staining of efferent ducts of six-
month-old (A) control and (B) Dicer1fl/fl; Defb41iCre/wtmice. Arrow
marks neoplastic changes. Scale bar 100 mm.
Changes in the epithelium of the efferent
segment. Haematoxylin and eosin staining of (A) initial segment
(IS) and (D) corpus (COR) of a 2 month-old control mouse and (B,
C) IS of two individual 2 month-old Dicer1fl/fl; Defb41iCre/wt
mice. Scale bar 100 mm.
Varied phenotype of the Dicer1 cKO initial
epididymal development. Expression of proximal epididymis
specific genes in the initial segment and caput of 21 days – two-
month-old control and Dicer1 conditional knock-out (cKO) mice.
Expression of (A) cystatin 8 (Cst8), (B) lipocalin 8 (Lcn8) and (C)
Ros1 proto-oncogene (Ros1) relative to L19 expression. Statistical
significance was calculated from the expression levels of 3 control
Segment-specific gene expression during
and 3 Dicer1fl/fl;Defb41iCre/wt mouse samples from each time
point, using the unpaired t-test. Statistical significance of changes
is indicated as follows: *, P#0.05; **, P#0.01; ***, P#0.001.
Dicerfl/flmice were a generous gift from BD Harfe, Department of
Molecular Genetics and Microbiology, University of Florida, Gainesville.
Defb41iCreKI mice were generated in collaboration with Turku Center for
Disease Modeling, TCDM, Turku, Finland, (www.tcdm.fi), and we thank
the TCDM personnel for skillful assistance in various stages of this study.
We thank T. Leinonen for technical assistance with the histology
specimens, and Leena Strauss for help in histological analyses.
Conceived and designed the experiments: IB LS AK MP PS. Performed
the experiments: IB LS AK. Analyzed the data: IB LS AK IH MP PS.
Wrote the paper: IB IH MP PS.
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