Cell Tissue Res (2012) 349:717–731
DOI 10.1007/s00441-012-1381-0
REVIEW
Region-specific gene expression in the epididymis
Clémence Belleannée & Véronique Thimon &
Robert Sullivan
Received: 29 November 2011 / Accepted: 10 February 2012 / Published online: 18 March 2012
# Springer-Verlag 2012
Abstract The epididymis is responsible for post-testicular
sperm maturation, which consists in the acquisition of for-
ward motility and fertilizing ability. This organ is composed
of three main anatomical regions — the caput, corpus and
cauda epididymidis — which possess distinct gene expres-
sion profiles, ensuring different epididymal functions essen-
tial to the different steps of sperm maturation. Since many
genes display spatially restricted expression in the epididy-
mis, this organ constitutes a model of choice to study the
mechanisms that govern region-specific gene expression.
Factors such as steroid hormones, lumicrine factors and
temperature affect the pattern of gene expression in the
epididymis. Recently, the contribution of small RNAs in
epididymal gene regulation has been investigated and con-
stitutes a promising avenue for clinical application with
regard to male fertility.
Keywords Epididymis . Sperm maturation . Gene
expression . Lumicrine factors . Small RNAs
Introduction
Immature testicular spermatozoa need to transit throughout
the epididymis to acquire their motility and fertilizing
C. Belleannée (*) : R. Sullivan (*)
Département d’Obstétrique-Gynécologie, Faculté de Médecine,
Centre de Recherche du Centre Hospitalier de l’Université Laval
(CHUQ), Université Laval,
2705 Boulevard Laurier, T1-49,
Quebec City, QC G1V 4G2, Canada
e-mail: clemence.belleannee@crchuq.ulaval.ca
e-mail: robert.sullivan@crchul.ulaval.ca
V. Thimon
Département de Biologie, Université de la Martinique,
B.P. 7209 97271, Schoelcher, Cedex
ability. During the maturation of male germ cells in this
long convoluted tubule, the sperm plasma membrane is
subjected to sequential biochemical modifications as a result
of subtle interactions with components of the extracellular
milieu (Olson and Hinton 1985; Cornwall et al. 1990;
Veeramachaneni et al. 1990; Vreeburg et al. 1991;
Dacheux et al. 2003, 2009; Turner 2008; Cornwall 2009;
Belleannée et al. 2011b). The composition of this milieu
varies along the different segments of the epididymis and
relies on an exquisite timing and fine regulation of epidid-
ymal gene expression that is one of the most prominent
features of this organ (Krull et al. 1993; Pera et al. 1994;
Gebhardt et al. 1999; Belleannée et al. 2011a; Guyonnet et
al. 2011). The epididymis is divided into three anatomical
regions: the caput, the corpus and the cauda (Fig. 1). In
rodents, an additional segment called initial segment (IS) is
located between the efferent ducts (ED) and the caput epi-
didymidis. Each of these segments possesses a distinct pat-
tern of gene expression related to physiological functions
important for the different steps of sperm maturation
(Johnston et al. 2005, 2007; Dubé et al. 2007; Thimon et
al. 2007; Turner et al. 2007; Guyonnet et al. 2009). One of
the key questions that have been investigated for years by
pioneers is: what are the factors responsible for the regula-
tion and segmentation of epididymal gene expression ? The
role of testicular factors found in the efferent ductal fluid —
the so-called lumicrine factors, androgens and estrogens
have been extensively studied and their contribution in the
regulatory process of the epididymis is indubitable (Robaire
and Viger 1995; Hinton et al. 1998; Hess et al. 2011).
Whereas androgens and lumicrine factors mostly influence
the expression of the IS and caput-specific genes, these
factors are less efficient in the more distal corpus and
cauda regions of the epididymis (Lan et al. 1998; Sipila
et al. 2006). Therefore, contribution of other factors (i.e.,
temperature, pressure, small RNAs) may be necessary to
718 Cell Tissue Res (2012) 349:717–731

Fig. 1 Schematic
representation of the human
male reproductive tract and
excurrent ducts. The human
epididymis (in greek, epi for
“on” and didymoi for “testis”)
is composed of three main
anatomical regions: the caput,
the corpus and the cauda. This
single coiled tubule connects
the efferent ducts from the rear
of each testicle to the vas
deferens. Its unraveled length is
approximately 6 m

explain the whole regulation system found in the epidid-
ymis. Recent work on human epididymis has highlighted
the possible role of small RNAs, hence bringing unex-
plored aspects of gene regulation in this organ: RNA
stability and post-transcriptional regulation (Zhang et al.
2010, 2011). This review aims to take stock of the
contribution of the different factors mentioned above
and establish their respective roles in the regulation
of gene expression in the different segments of the
epididymis.
Morphology of the epididymis
The epididymis is an important organ of the male excurrent
duct system, since its main functions are: (1) to receive and
concentrate immature spermatozoa from the testis, (2) to
transport and maturate them thanks to subtle interactions
with secreted luminal products and (3) to store functional
sperm prior to release in the vas deferens at the time of
ejaculation. This organ is a single convoluted tubule that
connects the ED to the vas deferens. Its unraveled length has
Cell Tissue Res (2012) 349:717–731
been estimated as 6 m in humans and, depending on species,
it takes between 1 and 2 weeks for a spermatozoon to travel
across the entire length of the organ. The acquisition of
sperm-fertilizing ability occurs at a location in the epididy-
midis that is species-dependent (Dacheux and Paquignon
1980). At first glance, the epididymis displays an elementary
structure: a single coiled tubule. However, physiological,
biochemical and molecular studies performed on this
organ over decades have unveiled an extraordinary com-
plexity both at the structural and functional levels. The
epididymis can be divided into four main regions based
on their anatomical properties: the IS (only present in
rodents), the caput, the corpus and the cauda (Fig. 1).
Each of these regions possesses distinct gene expression
profiles as established by several microarray-based stud-
ies (Johnston et al. 2005, 2007; Zhang et al. 2006;
Dubé et al. 2007; Jelinsky et al. 2007; Thimon et al.
2007; Guyonnet et al. 2009). This compartmentalized
gene expression triggers segment-specific protein secretions
into the luminal fluid and, therefore, generates sperm micro-
environments optimized for each step of sperm maturation:
whereas luminal secretions from caput and corpus epididymi-
des are beneficial for the acquisition of sperm motility and
fertilizing ability (Turner 1995), cauda epididymal secretions
are important for the proper storage of spermatozoa for several
days in conditions that preserve their fertility (Jones and
Murdoch 1996). The composition of the intra luminal milieu
is controlled by the surrounding pseudostratified epithelium,
which is composed of six cell types possessing distinct phys-
iological functions: narrow and apical cells found in the prox-
imal region of the epididymis and principal cells, clear cells,
basal cells and halo cells found all along the organ (Joseph et
al. 2011). Only the latest category is described in this review
(Fig. 2). For additional information regarding narrow and
apical cells, please refer to Joseph et al. 2011.
Principal cells
Principal cells are found all along the epididymis and
represent about 80% of the epithelial cells. The main func-
tion of principal cells is to synthesize and release proteins,
via either merocine or apocrine secretion. This latest type of
secretion involves membranous vesicles called epididymo-
somes, which play a role in the transfer of proteins such as a
zona pellucida binding protein dicarbonyl/l-xylulose reduc-
tase (P26H/P34H/DCXR), macrophage migration inhibitory
factor (MIF), epididymal secretory protein E5 (HE5/CD52),
A disintegrin and metalloprotease 7 (ADAM7), ubiquitin,
type 5 glutathione peroxidase (GPX5), enzymes of the pol-
yol pathway and hyaluronidase PH-20 (SPAM1/PH-20) on
the sperm surface (Sullivan et al. 2007; Caballero et al.
2010). Whereas some of these proteins are involved in the
acquisition of sperm-fertilizing ability (Montfort et al.
719
2002), others may contribute to the acquisition of sperm
forward motility (Frenette et al. 2004, 2006), to the protec-
tion of male gametes against reactive oxygen species
(Vernet et al. 2004), or to the elimination of defective
spermatozoa (Sutovsky et al. 2001). Furthermore, principal
cells exhibit major changes along the epididymis with re-
gard to both their structure and functions (Hermo and
Robaire 2002; Joseph et al. 2011). Indeed, these cells exhibit
a tall columnar shape in the proximal regions that is replaced
by a squared appearance in the distal regions and which
coincides with important modifications of both their secre-
tory and endocytic apparatus. In addition, principal cells
specifically express several channels and transporters, such
as cystic fibrosis transmembrane conductance regulator
(CFTR) (Ruz et al. 2004), aquaporin 9 (AQP9) (Pastor-
Soler et al. 2001), pannexins (Turmel et al. 2011), chloride
channel protein 3 (ClC3) (Isnard-Bagnis et al. 2003) and
membrane-associated carbonic anhydrases CAIV and CAII
(Kaunisto et al. 1995), which mediate the transport of
elements that are important to epididymal physiology (i.e.,
ATP, chloride, water and bicarbonate).
Clear cells
Clear cells are mitochondria-rich cells that are found in an
increasing proportion from the proximal to the distal region of
the epididymis. These cells express the vacuolar proton pump
ATPase (V-ATPase), whose activity contributes to luminal
acidification in order to keep spermatozoa in a quiescent state
prior to ejaculation (Breton et al. 1996). In a steady-state
condition, the V-ATPase is distributed between sub-apical
vesicles and the apical plasma membrane (Shum et al.
2009). However, in response to stimuli provided by the lumi-
nal environment (high pH, low bicarbonate concentration,
angiotensin II, purinergic agonists), the V-ATPase is recruited
into the plasma membrane, which elongates to form extended
microvilli (Pastor-Soler et al. 2003; Beaulieu et al. 2005;
Shum et al. 2008; Belleannée et al. 2010). Functional studies
have shown that the increase in the length of microvilli is
correlated with the increase of proton secretion into the lumen
(Shum et al. 2008). Furthermore, clear cells are thought to be
endocytic cells that remove proteins from the epididymal fluid
(Vierula et al. 1995), in addition to cytoplasmic droplets that
detach from spermatozoa during epididymal sperm matura-
tion (Hermo et al. 1988).
Basal cells
Basal cells are pyramidal-shaped cells located at the base of
the epithelium. It has been recently established that these cells
are capable of projecting narrow cytoplasmic extensions that
cross the tight junctions at the apical pole of the epithelium in
order to be in direct contact with the luminal fluid and to
720
Fig. 2 Schematic organization of the different cell types found in a
representative cross-section of the epididymis. Principal cells are the
main cell type found in the epididymal epithelium and are involved in
protein secretion and apocrine secretion of epididymosomes into the
intraluminal fluid via the formation of apical blebs. Clear cells are
large endocytic cells whose microvilli extend toward the lumen to
regulate the intraluminal pH. Basal cells are located against the
“sense” its composition (Shum et al. 2008). Indeed, the
detection of luminal angiotensin II by the angiotensin II
type 2 receptor (AGTR2) expressed in basal cells triggers a V-
ATPase-dependent proton secretion by clear cells, as a result
of a cross-talk between basal cells and adjacent clear cells. In
addition, basal cells express different markers such as the Yf
subunit of the Glutathione-S-transferase (GST)(Veri et al.
1993) and the CuZn-superoxide dismutase (SOD)
(Nonogaki et al. 1992), suggesting a role for these cells in
the protection against reactive oxygen species.
Halo cells
Halo cells are found all along the epididymis but are more
abundant in the proximal regions (Nashan et al. 1989;
Cell Tissue Res (2012) 349:717–731
basement membrane and “sense” the composition of the lumen via
narrow body extensions. Halo cells are immune cells located across the
epididymis. Dendritic cells form a dense network at the base of the
epithelium and project long dendrites between epithelial cells of the
epithelium. A thick layer of smooth muscle cells surrounds this cell
structure
Flickinger et al. 1997; Seiler et al. 1999). They represent a
mixture of different immune cells such as helper T lympho-
cytes, cytotoxic T lymphocytes and macrophages and their
number increases during ageing (Serre and Robaire 1999).
The existence of these immunocompetent cells is unques-
tionable but their exact characteristics and their immuno-
logical functions remain to be clarified.
Epithelial cell cross-talk
Whereas these different cell types exert independent and
specific functions within the epididymal epithelium, they
also exhibit interrelated roles, since they are able to com-
municate with each other to create the proper luminal envi-
ronment for sperm maturation and storage (Shum et al.
Cell Tissue Res (2012) 349:717–731
2011). For instance, principal cells communicate with clear
cells via the ATP/adenosine purinergic pathways to regulate
the recruitment of V-ATPase to the apical pole of clear cells
(Belleannée et al. 2010), basal cells communicate with clear
cells via angiotensin II and nitric oxide production to control
the V-ATPase-dependent proton secretion (Shum et al.
2008) and basal cells communicate with principal cells after
stimulation with lysylbradykinin to induce anion secre-
tion by principal cells (Leung et al. 2004; Cheung et al.
2005). The existence of such cross-talk between epidid-
ymal epithelial cells underscores the necessity to con-
duct integrated studies to better mimic what happens in
this organ in vivo.
Blood–epididymal barrier
The different epididymal cell types of the epithelium form a
monolayer surrounding the lumen and are linked by exten-
sive tight junctions at the apical pole, thus creating a blood–
epididymal barrier (Cyr et al. 2007). The existence of major
differences in the concentration of organic and inorganic
compounds between the luminal fluid and blood reflects
the effectiveness of the blood–epididymal barrier to selec-
tively exclude or concentrate ions and molecules (Hinton
and Howards 1981; Cyr et al. 1995). In addition, this blood–
epididymal barrier participates in the confinement of auto-
antigenic spermatozoa in an immunoprivileged environ-
ment. However, the existence of “gaps” that allow basal
cell extensions to reach the lumen (Shum et al. 2008),
suggests that this barrier is not totally sealed and requires
additional support to protect billions of spermatozoa from
the “host” immune system. Recently, an impressive network
of dendritic cells closely attendant to the epididymal epithe-
lium has been discovered all along the epididymis (Da Silva
et al. 2011). These cells, named eDCs for “epididymal
dendritic cells”, surround the base of the epithelium and
protrude long and narrow dendrites between epithelial cells
toward the lumen of the proximal epididymal regions. In
addition, CD11c+and CD11b+eDCs isotypes are potent
antigen-presenting cells in vitro, since they are capable of
inducing T-cell proliferation in the presence of ovalbumin
(Da Silva et al. 2011). Whereas the potential contribution of
eDCs to the immune tolerance of autoantigenic spermatozoa
in the epididymis needs to be investigated, these cells are
most likely at the interplay between the reproductive and
immune systems.
The epididymis, an exquisite model for studying
the regulation of gene expression
The epididymis is a valuable model system to decipher the
mechanisms that govern region-specific gene expression,
721
since many epididymal genes are subjected to a spatially
restricted expression along this organ (Rodriguez et al.
2001). Indeed, despite its apparent simple structure, the
epididymis is composed of several lobules delimited by
connective-tissue septa that exhibit distinct gene-expression
signatures (Turner et al. 2003). The gene-expression pattern of
different segments of the epididymidis has been investigated
using the microarray technology in human (Zhang et al. 2006;
Dubé et al. 2007; Thimon et al. 2007), mouse (Johnston et al.
2005, 2007), rat (Jelinsky et al. 2007) and boar (Guyonnet et
al. 2009). These data determine that the high degree of gene-
expression segmentation in the epididymis is a common fea-
ture shared between these species. In addition, the expression
of 12% and 13% of epididymal genes has been assessed to be
highly segmented (fold change above 4) in human and mouse
epididymides respectively (Johnston et al. 2005; Zhang et al.
2006). Despite the heterogeneity of both the segmentation and
the methods of analysis used in these different studies, some
common characteristics can be determined. Among the most
described in the literature, the expression of several gene
families such as beta-defensins, aquaporins and lipocalins is
strongly affected by tight region-specific regulation along the
epididymis.
Beta-defensins
Beta-defensins are small antimicrobial peptides that are
conserved among species and mainly expressed in the testis
and the epididymis (Patil et al. 2005; Oh et al. 2006). So far,
39 human, 52 mouse and 43 rat beta-defensin genes have
been identified (Patil et al. 2005). Whereas it is well-
established that beta-defensins have antimicrobial and host
defense activities, there are increasing evidences showing
their contribution to sperm maturation, motility, capacitation
and male fertility (Rao et al. 2003; Zanich et al. 2003; Zhou
et al. 2004; Yudin et al. 2005; Hall et al. 2007; Tollner et al.
2011). The expression of beta-defensin genes exhibits a
pronounced segmentation in the different species studied
(Johnston et al. 2005; Jelinsky et al. 2007; Guyonnet et al.
2009). In humans, most defensin genes are more expressed
in the corpus region (Fig. 3). This is the case for Def106b
whose rat homologous, Def15, has been shown to be involved
in the regulation of sperm motility in addition to its antimi-
crobial activity (Zhao et al. 2011). In addition, whereas Defb1
is highly expressed in the caput epididymidis, Defb125 and
Defb126 are much more representative of the cauda epididy-
midis (Fig. 3). Interestingly, amongst beta-defensins the most
studied, DEFB126, is localized on the sperm surface and has
been reported to be involved in sperm penetration through the
cervical mucus and binding to the zona pellucida (Liu et al.
2001; Rao et al. 2003; Tollner et al. 2008). Furthermore,
mutations on the human Defb126 gene have been shown to
be associated with impaired sperm functions and subfertility
722
Fig. 3 Regionalized expression of beta-defensin genes in the human
epididymis according to the transcriptomic analysis performed by
Thimon et al. 2007. The mean signal intensity of 20 defensin genes
is shown along the different segments of epididymides provided by
three donors
(Tollner et al. 2011). The high level of gene expression and
elevated degree of segmentation of beta-defensins in the epi-
didymis suggest region-specific roles of these peptides. Given
their dual functions related to both host defense mechanisms
and to sperm maturational events, beta-defensins might be
moonlighting proteins that exert different functions depending
on the region where they are expressed.
Lipocalins
Lipocalins constitute a family of proteins involved in the
transport of small hydrophobic ligands such as retinoids,
steroids and fatty acids. Among the ten members of the
lipocalin family expressed in the epididymis (Lcn 2, Lcn
5, Lcn 6, Lcn 8, Lcn9, Lcn 10, Lcn 11, Lcn 12, Lcn13 and
prostaglandin D2 synthase (Ptgds)), Lcn5, Lcn 9, Lcn 10,
Lcn 12 and Lcn 13 are specifically expressed in this organ
(Suzuki et al. 2007). In addition, the expression of lipocalin
genes is highly regionalized in the epididymis: whereas in
rodents most of the lipocalins are expressed in the IS (Lcn 2,
Lcn 8, Lcn 9 and Lcn 10) and the caput epididymidis (Lcn
2, Lcn 5 and Lcn 10), Ptgds is mostly expressed in the
corpus and cauda epididymidis. In human, we observed a
different pattern of lipocalin expression according to micro-
array analyses (Fig. 4). Several lipocalins (Lcn 6, Lcn 8, Lcn
10, Lcn 12) are significantly more expressed in the corpus
Cell Tissue Res (2012) 349:717–731
Fig. 4 Regionalized expression of nine lipocalin genes in the human
epididymis according to the transcriptomic analysis performed by
Thimon et al. 2007. The mean signal intensity is shown along the
different segments of epididymides provided by three donors
epididymidis, whereas Lcn 2 is much more expressed in the
caput epididymidis. Ptgds is by far the more highly
expressed lipocalin in the human epididymis.
Aquaporins
Aquaporins play an essential role in water transport across
the plasma membrane. In the epididymis, water transport
contributes to sperm concentration all along the epididymis.
This function is critical for the male reproductive tract, since
the impairment of solute and fluid transport in ED modifies
the properties of the luminal environment and affects fertil-
ity (Hess 2000; Zhou et al. 2001). Several members of the
aquaporin family (Aqp1, 3, 5, 7, 9 and 11) have been well-
described in the male reproductive tract and their expression
at the protein level is highly segmented in the different
regions of the epididymis (Da Silva et al. 2006; Hermo
and Smith 2011). In human, whereas Aqp9 mRNA is much
more abundant in the caput and corpus epididymidis, Aqp1
expression is significantly enhanced in the distal region of
the epididymis (Fig. 5) (Thimon et al. 2007). The high
expression of AQP9 in the proximal epididymidis is consis-
tent with the important water resorption that occurs in this
region.
The expression in a region-specific manner of several
genes in the epididymis is a singular feature found in this
Cell Tissue Res (2012) 349:717–731
Fig. 5 Regionalized expression of four aquaporin genes in the human
epididymis according to the transcriptomic analysis performed by
Thimon et al. 2007. The mean signal intensity is shown along the
different segments of epididymides provided by three donors
organ. Since principal cells exhibit important morphological
and functional changes with regard to their secretory/endo-
cytic apparatus along the epididymis, it is likely that seg-
mented expression of some epididymal genes might initiate
and coincide with these cell transformations. This is sup-
ported by the fact that the expression of members of junc-
tional complexes (i.e., cadherins, catenins, claudins and
occludins) involved in the blood epididymal barrier forma-
tion and structural cell shaping is highly segmented along
the epididymis (Cyr et al. 1995). The expression profile of
some genes revealed by transcriptomic analyses [i.e.,
Rnase10, glutathione peroxidase 5 (Gpx-5), Clusterin]
matches the level of protein found in the epididymal fluid
from the corresponding regions (Guyonnet et al. 2011).
However, the highly segmented gene expression profile
found in humans contrasts with the proteome analysis of
human epididymal fluid, which exhibits only a few differ-
ences of protein composition from one region to another
(Dacheux et al. 2006; Thimon et al. 2007). This discrepancy
may be attributed to the resolution window allowed by
classical 2D-electrophoresis, which makes difficult the
detection of highly hydrophobic proteins such as aquapor-
ins, poorly expressed, or small proteins or polypeptides such
as beta-defensins. Whereas these limitations do not exist in
transcriptomic analyses, gene-expression profiles cannot
723
reflect regulations occurring at the post-transcriptional
level. Therefore, transcriptomic and proteomic analyses
are complementary methods, which help the understand-
ing of the molecular mechanisms essential to epididymal
physiology.
Regulation by steroid hormones
Both orchidectomy, which consists of the removal in the
testicle and efferent duct ligation provoke dramatic changes
on the morphology and functions of the epididymis (Brooks
1976; Fawcett and Hoffer 1979; Fan and Robaire 1998; Ezer
and Robaire 2003; Turner et al. 2007). Therefore, the phys-
iology of the epididymis highly relies on testicular and
circulating factors that the epididymis is unable to synthe-
size itself. Testosterone produced by Leydig cells is one of
the best-known and studied factors affecting epididymal
functions such as metabolism, ion transport, synthesis and
secretion of epididymal proteins and sperm maturation,
transport and storage (Bilinska et al. 2006). This circulating
hormone is also present in the intraluminal milieu of the ED
and of the epididymis at a very high concentration. After
leaving the testis through the ED, testosterone is incorpo-
rated into principal cells of the epididymis by endocytosis of
the complex testosterone–androgen binding protein (ABP)
(Danzo et al. 1977). Testosterone can be quickly metabo-
lized: whereas the enzymes 5-alpha reductase converts tes-
tosterone into the most biologically active androgen
dihydrotestosterone (DHT), the aromatase P450 converts
testosterone into the estrogenic compound estradiol (E2).
Both androgens and estrogens have been shown to regulate
epididymal gene expression.
Androgens
Among the most important hormonal factors studied, andro-
gens regulate epididymal gene expression via androgen-
dependent signaling pathways involving MEK, ERK1/2
and CREB in epididymal cell lines (Hamzeh and Robaire
2011), or via the formation of an androgen–Androgen re-
ceptor (AR) complex that regulates transcription of andro-
gen response elements (AREs) containing-genes (He et al.
1999; Janne et al. 2000; Lamb et al. 2001; Heinlein and
Chang 2002), this latest mechanism being by far the most
described. The AR is encoded by a gene located on the X
chromosome and is a ligand-inducible transcription factor
composed of a N-terminal regulatory domain, a DNA-
binding domain and a ligand-binding domain (Li and Al-
Azzawi 2009). Several androgenic ligands can bind the AR,
whose selectivity is regulated by coactivators or corepres-
sors (Malovannaya et al. 2011). The DNA-binding domain
724
of the complex androgenic ligand–AR specifically binds to
AREs located in the promoter sequences of target genes.
Both experimental and in silico approaches have been
developed to identify target genes affected by androgens in
the epididymis. The first category includes removal of the
source of testosterone by bilateral orchidectomy (remove
both circulating and intraluminal androgens) and subsequent
androgen replacement (Brooks 1987; Danzo 1995; Fan and
Robaire 1998; Yeung et al. 1999; Cheuk et al. 2000; Ezer
and Robaire 2003), treatment with AR antagonists (Dhar et
al. 1982; Kaur et al. 1992; Rulli et al. 1997) and treatment
with steroid 5-a-reductase inhibitors to determine solely the
role of DHT (Henderson et al. 2004). Amongst androgen-
dependent genes, the expression of cystein-rich secretory
protein 1 (Crisp1) (Kohane et al. 1983), Gpx-5 (Rigaudiere
et al. 1992), glutathione peroxidase 3 (Gpx-3) (Schwaab et
al. 1998), carbonic anhydrase (Kaunisto et al. 1999), angio-
tensinogen (Leung et al. 2000) and transforming growth
factor (Tgf) (Desai and Kondaiah 2000) is modified in the
epididymis after ochidectomy. This list has been reinforced
by the use of high-throughput microarray studies following
androgen withdrawal that have been carried out on rat (Ezer
and Robaire 2003; Hamzeh and Robaire 2010) and mouse
epididymides (Chauvin and Griswold 2004; Sipila et al.
2006; Snyder et al. 2009). In rat, the expression of 6–7%
of the probe sets expressed on the epididymis is signif-
icantly induced or reduced after bilateral orchidectomy and
this effect is reversed for more than two-thirds of these genes
after DHT treatment (Hamzeh and Robaire 2010), showing
their androgen-dependent expression. These genes encode for
growth factors (Egf, Igf1, Igfbp3), factors involved in cell
proliferation (Bmp4, Edn1), proteins implicated in gap junc-
tions (Gja1, Gjb3, Gja4) and interleukin receptors engaged in
immune response (Il13ra2, Il1rl1) (Hamzeh and Robaire
2010). Results from microarray analyses indicate that the
highest number of androgen-regulated genes is found in the
caput epididymidis (Chauvin and Griswold 2004; Sipila et al.
2006; Hamzeh and Robaire 2010), a region where the selec-
tive disruption of AR expression in two transgenic mouse
models triggers epididymal obstruction and male infertility
(Krutskikh et al. 2011; O’Hara et al. 2011). Since the AR is
expressed along the entire length of the epididymis, the
regionalized responsiveness of the organ to androgens might
depend on the regional expression of AR coregulators (Sipila
et al. 2011) as well as on the androgen gradient concentration
found in the epididymis (Robaire and Viger 1995).
Estrogens
Estrogens are present at a very high concentration in the
testis and are generated from the aromatization of testoster-
one by the cytochrome P450 aromatase. Aromatase activity
has been detected both in Leydig cells (Hess et al. 2001) and
Cell Tissue Res (2012) 349:717–731
in epididymal spermatozoa (Hess et al. 1995; Joseph et al.
2011). Estrogens mediate their biological effects after their
interaction with ESR1 or ESR2 nuclear receptors and sub-
sequent recognition of estrogen response elements (EREs)
located in the promoters of target genes (Joseph et al. 2011).
The loss of fertility in Esr1−/− male mice underscores the
importance of the response elicited by estrogens and medi-
ated by ESR1 in reproductive function (Eddy et al. 1996).
Whereas less genes are regulated by estrogens compared to
androgens in the mouse epididymis (Hamzeh and Robaire
2010), estrogen regulates the expression of genes involved
in solute and water transport (Hess et al. 1997, 2001;
Bilinska et al. 2006; Snyder et al. 2009), a function that is
essential for normal reproductive performance (Hess et al.
1997). Among these genes, Aqp9 contains both AREs and
EREs and its expression is regulated by both estrogen and
androgen (Pastor-Soler et al. 2010; Joseph et al. 2011). This
suggests that the balance of estrogen and androgen may be
important to ensure proper epididymal functions. In addi-
tion, the physiological role of estrogens can be precluded
following their sulfonation by the enzyme estrogen sulfo-
transferase (EST). Interestingly, this enzyme is present in
both the epithelium and the intraluminal fluid of the epidid-
ymis and might control the pool of bioactive luminal estro-
gens in this organ (Frenette et al. 2009).
Regulation by lumicrine factors
Preventing testicular factors from entering the epididymis
triggers epididymal gene expression changes (Hinton et al.
1998; Lan et al. 1998; Hermo et al. 2000), a decrease in
protein synthesis and secretion (Holland et al. 1992) and
epithelial cells apoptosis specifically in the IS (Nicander et
al. 1983; Abe and Takano 1989; Fan and Robaire 1998).
This latest phenomenon cannot be reversed by androgen
replacement (Fawcett and Hoffer 1979). Therefore, it is
suggested that factors originating from Sertoli and/or germ
cells — the so-called lumicrine factors — reach the epidid-
ymis via the rete testis and ED and regulate epithelial cell
functions in the IS. The influence of lumicrine factors on the
epididymis has been investigated following efferent duct
ligation (EDL), a technique that does not affect blood sup-
ply. Several genes such as proenkephalin (Garrett et al.
1990), retinoic acid binding protein (Zwain et al. 1992),
cystatin related epididymal specific (Cornwall et al. 1992),
gamma-glutamyl transpeptidase (Palladino and Hinton
1994), A-raf (Winer and Wolgemuth 1995), glutathione
peroxidase (Rigaudiere et al. 1992; Vernet et al. 1997), 5-
alpha -reductase (Viger and Robaire 1996), polyomavirus
enhancer activator 3 (Lan et al. 1997) and ADAM7
(Cornwall and Hsia 1997) have been shown to depend on
testicular factors. Whereas the identity of lumicrine factors
Cell Tissue Res (2012) 349:717–731
responsible for such a regulation is for the most part un-
known, there is some evidence showing the involvement of
members of the fibroblast growth factor (FGF) family and
spermatic factors in the regulation of epididymal physiology
(Cotton et al. 2008; Reyes-Moreno et al. 2008).
Growth factors
Most of the IS-specific genes are regulated by lumicrine
factors, such as growth factors that are continuously pro-
duced in the testis (Lan et al. 1998). For instance, treatment
with fibroblast growth factor 2 (FGF2) but not epidermal
growth factor (EGF) is capable of restoring the activity of
the enzyme γ-glutamyl transpeptide (GGT) that is affected
by EDL in rats (Lan et al. 1998). This observation suggests
that cellular pathways might be regulated by luminal FGF2
via FGF receptors (Fgfrs) in the IS. Among the FGF recep-
tors type 1, 2, 3 and 4 that are found at the protein level in
the rat IS, the transcript of the isoform Fgfr1 IIIc is present
in principal cells of the epididymal epithelium, making this
receptor a good candidate for the regulation of FGFR sig-
naling pathways in this region (Kirby et al. 2003). In
human, Fgfr1 is expressed all along the epididymis, whereas
the agonist Fgf2 is significantly more expressed in the cauda
epididymidis (unpublished data). Overall, it has been pro-
posed that the activation of the FGFR pathway by testicular
FGF maintains the expression of pro-survival pathways and
protective genes in the IS.
Spermatic factors
In addition to factors found in the luminal fluid, spermato-
zoa themselves have been shown to affect epididymal epi-
thelial cell functions (Garrett et al. 1990; Reyes-Moreno et
al. 2008). For instance, the expression of proenkephalin in
the rat epididymis depends on the presence or absence of
spermatozoa controlled by the alkylating agent busulphan.
This was the first evidence showing that spermatozoa, or a
spermatozoa-associated factor, could regulate epididymal
gene expression (Garrett et al. 1990). In addition, incubation
of ejaculated spermatozoa washed free of seminal plasma
affects the protein neosynthesis/secretion and cell prolifera-
tion of primary cultures from the caput, corpus and cauda
epididymidis in a temperature-dependent manner (Reyes-
Moreno et al. 2008). Whereas the identity of spermatic
factors involved in this process remains unknown, several
spermatic proteins have been shown to indirectly regulate
epididymal functions. For instance, spermatozoa exhibit an
aromatase activity that may generate estrogen in the epidid-
ymal fluid (Hess et al. 1995), which in turn controls the
expression of several epididymal genes. Moreover, the ger-
minal angiotensin I-converting enzyme (gACE) is present
on the surface of testicular spermatozoa, and released into
725
the epididymal fluid in the proximal epididymidis (Metayer
et al. 2002). This enzyme belongs to the renin–angiotensin
system and converts angiotensin I into the active vasopres-
sor angiotensin II, while it inactivates the vasodilator bra-
dykinin. Both angiotensin II and bradykinin are present in
the epididymal fluid and regulate epididymal functions such
as luminal acidification and water transport (Shum et al.
2008; Belleannée et al. 2009). Overall, these investigations
underscore the role of spermatic factors on epididymal gene
expression and functions, which in turn have an impact on
sperm maturation.
Regulation by thermodynamic factors
In addition to the luminal factors described above, temper-
ature and pressure are two parameters that regulate epidid-
ymal gene expression. In most mammals, testes and
epididymides are located in the scrotum, where the temper-
ature is slightly lower than that of the rest of the body
(usually one or two degrees Celsius below the body temper-
ature). This cooler temperature is of major importance, since
it influences epididymal physiology. For instance, tempera-
ture regulates sperm storage and survival in the cauda epi-
didymidis and epididymal epithelial cell secretion (Foldesy
and Bedford 1982; Esponda and Bedford 1986; Bedford
1991; Bedford and Yanagimachi 1991; Regalado et al.
1993; Reyes-Moreno et al. 2008). Whereas this latest
temperature-dependent mechanism is not mediated by an-
drogen, both factors can act cooperatively (Regalado et al.
1993; Kirchhoff et al. 2000). Moreover, specific gene ex-
pression in the epididymis is controlled by temperature
(Esponda and Bedford 1986; Regalado et al. 1993; Pera et
al. 1996). For instance, bcl-2 and bax mRNAs have been
shown to be overexpressed following cryptepididymis (Jara
et al. 2002), a surgical method used to assess the effect of a
rise of temperature on epididymal functions and sperm
maturation (Bedford 1978). These two genes belong to the
Bcl-2 family and it has been suggested that they may
participate in the temperature-dependent apoptosis observed
in the cauda epididymidis (Jara et al. 2002).
In addition to temperature, intraluminal flow rate and
pressure are factors that could affect epididymal physiology
as well. Indeed, several parameters such as intratubular
pressure (Johnson and Howards 1976) and fluid movements
in the epididymis (Turner et al. 1990) are affected following
vasectomy and could therefore be responsible for the epi-
didymal gene-expression changes observed after such a
surgery in humans (Thimon et al. 2008; Sullivan et al.
2011). Using a high-throughout microarray approach, it
has been established that whereas gene-expression pattern
along the human epididymis is highly regionalized in nor-
mal as well as in epididymal tissues after vasectomy, several
726
genes show a level of expression highly affected by vasec-
tomy (Thimon et al. 2008). For example, the expression of
CRISP1, which is associated with the sperm surface and
implicated in sperm–egg fusion and sperm decapacitation
(Roberts et al. 2003; Ellerman et al. 2006) is significantly
modified after vasectomy (Thimon et al. 2008; Legare et al.
2010). Other genes encoding DCXR, Niemann–Pick dis-
ease, type C2 (NPC2), DEFB126, ADAM7 and epididymal
sperm-binding protein 1 (ELSPBP1) exhibit an altered expres-
sion in a region-dependent manner following vasectomy
(Thimon et al. 2008; Sullivan et al. 2011). Whether this is
directly owed to a change of luminal flow or change of
pressure following the surgical procedure is not known and
would need to be further investigated.
Regulation by small RNAs
The importance of small noncoding RNAs as regulators of
transcription, RNA stability and translation arouses a grow-
ing interest in a broad range of biological systems (Plasterk
2006). Among endogenous small RNAs, small interfering
RNAs (siRNAs), microRNAs (miRNAs) and piwi-
associated RNAs (piRNAs) have been characterized in a
number of eukaryotes (Valencia-Sanchez et al. 2006).
miRNAs are small, ∼21-nucleotide (nt) RNAs that operate
through post-transcriptional silencing of partially comple-
mentary mRNAs by targeted destruction and/or translational
repression. miRNAs are produced from the cleavage of
doublestranded hairpined RNA precursors by the ribonucle-
ase III Dicer enzymes (Bartel 2009). miRNAs are involved
in numerous pathways and biological functions and have
been shown to be important players for the acquisition and
maintenance of male fertility. For instance, deletion of Dicer
in mice Sertoli cells or male germ cells leads to infertility
due to impairment of spermatogenesis, progressive testicu-
lar degeneration and meiotic and spermiogenic defects
(Papaioannou and Nef 2009; Papaioannou et al. 2009,
2010; Romero et al. 2011). Interestingly, more than 200
miRNAs have been identified in human epididymis
(Zhang et al. 2010). The negative correlation found between
the decreasing number of miRNAs and the increasing num-
ber of mRNAs expressed in the epididymis during the life-
span (from new-born to aged human epididymis) suggests
that epididymal miRNAs might regulate gene expression in
an androgen-dependent manner in this organ (Zhang et al.
2010). In addition, based on a mammalian microRNA ex-
pression atlas performed on 26 distinct organ systems and
cell types, some miRNAs such as mir-205 and miRNAs
belonging to the miR-888 and miR-371 clusters are signif-
icantly enriched in human reproductive systems (Landgraf
et al. 2007). Based on in silico studies, the six members of
the miR-888 cluster are predicted to target several genes
Cell Tissue Res (2012) 349:717–731
controlling sperm maturation and male fertility such as
sperm-associated antigen 6 (Spag6), sperm-associated anti-
gen 1 (Spag1), sperm associated antigen 8 (Spag8), round
spermatid basic protein 1 (Rsbn1) and transmembrane epi-
didymal protein 1 (Teddm1) (Li et al. 2010). Despite the fact
that these findings have to be experimentally validated, this
suggests a significant role for miRNAs in the regulation of
epididymal functions.
Conclusion
One of the unique features of the epididymis is its segmen-
tation, which occurs before the appearance of spermatozoa
in the epididymal lumen during postnatal development (Sun
and Flickinger 1979). This event coincides with changes of
cell morphology, function and gene expression profiles
along the duct. Indeed, a plethora of epididymal genes are
expressed in specific regions of the epididymis. Whereas the
predominant factor that regulates epididymal function and
gene expression is androgen, there is increasing evi-
dence showing that estrogens, lumicrine factors, sperma-
tozoa and small RNAs play important roles in the
regulation and maintenance of the epididymal segmen-
tation that is essential to ensure the different steps of
sperm maturation.
Acknowledgements The authors would like to acknowledge the
contribution of France Couture from the Graphic core facility of the
CRCHUQ for the figure designs. The work from our laboratory
reported in this review was supported by CIHR funding held by Dr
Robert Sullivan.
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