† Materials and Methods
Epididymal cell structure and function
Regulation of epididymal cell function
Epididymal luminal environment
New insights into epididymal biology
Gail A. Cornwall1
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
1Correspondence address. E-mail: email@example.com
table of contents
background: The epididymis performs an important role in the maturation of spermatozoa including their acquisition of progressive
motility and fertilizing ability. However, the molecular mechanisms that govern these maturational events are still poorly defined. This review
focuses on recent progress in our understanding of epididymal function including its development, role of the luminal microenvironment in
sperm maturation, regulation and novel mechanisms the epididymis utilizes to carry out some of its functions.
methods: A systematic search of Pubmed was carried out using the search term ‘epididymis’. Articles that were published in the English
language until the end of August 2008 and that focused on the specific topics described above were included. Additional papers cited in the
primary reference were also included.
results: While the majority of these findings were the result of studies in animal models, recent studies in the human epididymis are also
presented including gene profiling studies to examine regionalized expression in normal epididymides as well as in those from vasectomized
conclusions: Significant progress has been made in our understanding of epididymal function providing new insights that ultimately
could improve human health. The data also indicate that the human epididymis plays an important role in sperm maturation but has
unique properties compared with animal models.
Key words: epididymis / sperm maturation / human / rodent
Spermatozoa leaving the testis and entering the long convoluted
tubule known as the epididymis are non-functional gametes. It is
only during transit through the epididymis that spermatozoa
undergo maturation and acquire progressive motility and the ability
to fertilize ova. Because spermatozoa are, for the most part, syntheti-
cally inactive, maturation involves the interaction of spermatozoa with
proteins that are synthesized and secreted in a region-dependent
manner from the epididymal epithelium. Despite considerable effort,
the molecular and biochemical events that are integral for epididymal
sperm maturation are unknown.
The importance of understanding epididymal function and sperm
maturation is emphasized by the fact that up to 40% of infertile men
exhibit idiopathic infertility that may reflect sperm maturational dis-
orders. Unfortunately, owing to the lack of alternative therapies,
these patients and their partners require assisted reproductive tech-
niques (ART) such as intracytoplasmic sperm injection (ICSI), which
utilizes spermatozoa independent of maturational status, to achieve
a pregnancy. Although effective, because natural selection processes
& The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Human Reproduction Update, Vol.15, No.2 pp. 213–227, 2009
Advanced Access publication on January 8, 2009doi:10.1093/humupd/dmn055
that prevent suboptimal spermatozoa from fertilizing ova are
bypassed, there can be increased risks of genetic abnormalities
being transmitted to the offspring (Cox et al., 2002; Merlob et al.,
2005; Georgiou et al., 2006; Fedder et al., 2007; Sanchez-Albisua
et al., 2007). If the mechanisms of sperm maturation were established,
it is possible that sperm could be matured in vitro providing an alterna-
tive therapy to current reproductive technologies.
The significance of the lack of understanding regarding the func-
tional role of the epididymis in sperm maturation is also under-
scored by the lack of contraceptives for men. Although much
work has been put into developing hormonal methods that inter-
fere with sperm production in the testis, these approaches have
been hampered by cumbersome regimes, extended times before
efficacy is achieved, and possible side effects of the administered
hormones. Interest has shifted to include identifying epididymal mol-
ecules that could serve as targets for non- steroidal-based male
contraceptives with the idea that sperm production would occur
normally but the spermatozoa would be non-functional. Clearly, if
we are to improve human health by developing new and better
ways to improve as well as prevent fertility, further research into
the epididymis is needed.
The purpose of this review is to provide a general background of
the epididymis followed by a brief overview of recent progress in
the field including advances in our understanding of the epididymal epi-
thelium and its regulation, composition and function of the luminal
fluid, as well as changes occurring in spermatozoa during epididymal
transit. Because of the limited availability of epididymal tissue from
healthy men of reproductive age, the lack of appropriate in vitro
models, and the constraint to manipulate the human epididymis
experimentally, the majority of these studies have been carried out
in rodent models. However, as discussed below, genomic and proteo-
mic analyses of the human epididymis have revealed valuable new
information which lends support to the view that, although there
may be species differences with regard to where in the epididymis
spermatozoa acquire their functions, the human epididymis does
serve a role in the functional maturation of spermatozoa.
Materials and Methods
A systematic search of Pubmed was carried out using the search term ‘epi-
didymis’. Articles that were published in the English language until the end
of August 2008 and that focused on the specific topics described above
were included. Additional papers cited in the primary reference were
also taken into account. This review incorporates background material
presented in a previous review (Robaire et al., 2006) but includes recent
advancements as well as topics not covered or presented with a different
emphasis such as the human epididymis and components of the epididy-
mal luminal environment.
On the basis of histological and ultrastructural differences, the epididy-
mis can be grossly divided into three regions including the caput
(head), corpus (body) and cauda (tail) epididymidis. The most proxi-
mal epididymal region, in some species such as the mouse, is also
known as the initial segment (Fig. 1). Each epididymal region carries
out distinctive functions with the caput and corpus carrying out
early and late sperm maturational events, respectively, while the
cauda region primarily serves as a storage site for functionally
The epididymis is derived from the Wolffian duct and at birth con-
sists mainly of mesenchymal tissue. The epididymis undergoes con-
siderable remodeling including duct elongation and convolution so
that by puberty the epididymis has acquired its fully differentiated
state consisting of a highly tortuous tubule lined by epithelial cells
(Rodriguez et al., 2002). The development of a fully differentiated epi-
thelium is dependent not only on androgens but also requires the
influence of luminal factors from the testis (Rodriguez et al., 2002).
Considering that the adult epididymis exhibits region-specific charac-
teristics, it is not surprising that homeobox genes, such as Hox
genes that control segmental patterning during development, are
expressed during epididymal development and participate in the
appearance of segment-specific differences (Rao and Wilkinson,
2002). Although studies have established circulating androgens and
luminal factors as playing a necessary role in the development of the
epididymis, less is known of other factors that mediate the series of
morphogenic events that result in the formation of the adult epididy-
mis (Lei et al., 2003; Zhang et al., 2004).
Figure 1 Mouse and human epididymides.Mouse: 1, initial segment,
proximal caput; 2, midcaput; 3, distal caput; 4, corpus; 5, cauda.
Adapted (regional divisions by the author) from Trevor Cooper,
University of Muenster (Yeung and Cooper, 2002), with permission of
author and publisher, Springer. Human: ED, efferent ducts; 1, anterior
caput; 2, posterior caput; 3, anterior corpus; 4, midcorpus; 5, posterior
corpus; 6, anterior cauda; 7, posterior cauda; DD, ductus deferens.
Reprinted by permission of the author (Dacheux et al., 2006), UMR
INRA-CNRS and publisher, Elsevier Limited. Scale bar, 1 cm.
Several studies have implicated epithelial-mesenchymal interactions as
integral for epididymal morphogenesis. Indeed, early studies showed
when proximal regions of Wolffian duct epithelium were cultured
on seminal vesicle mesenchyme, the epithelium differentiated into
seminal vesicle epithelium (Higgins et al., 1989). Bone morphogenetic
proteins (BMP), members of the transforming growth factor b (TGFb)
superfamily, and their receptors may be involved in this interaction
since disruption of the Bmp4, Bmp7 and Bm8a and Bmp8b genes
results in epididymal degeneration that is region-specific (Zhao
et al., 1998, 2001; Chen et al., 1999; Hu et al., 2004). C-ros,
(ROS1), a member of the tyrosine kinase receptor family, may also
play an essential role in epididymal development, since mice lacking
the rosIgene lack theinitial
(Sonnenberg-Riethmacher et al., 1996). Because during kidney devel-
opment, ROS1 is thought to regulate the extracellular matrix, a
storage site for growth factors (Liu et al., 1996), a similar mechanism
of action may also occur in the epididymis. Mice with mutations in the
SH2 domain protein tyrosine phosphatase (SHP-1) gene [‘motheaten’
(me), ‘viable motheaten’ (mev)] exhibit an aberrant proximal epididy-
mal region similar to that in the c-ros knockout (Keilhack et al., 2001).
Since SHP-1 and c-ROS are coexpressed in the epididymis and interact
in vitro, SHP-1 was proposed to function as a regulator of c-ros signal-
ing (Keilhack et al., 2001).
LGR4, a leucine-rich repeat domain containing G protein-coupled
receptor (GPCR) 4 also appears critical for epididymal development
since in the LGR4 (Lgr4) knockout mouse the epididymal tubule,
especially in the caput, fails to elongate and convolute and the resulting
duct is surrounded by a thick condensation of mesenchymal cells
(Mendive et al., 2006). The abnormal arrangement of the epithelium
and mesenchyme in the LGR4 knockout suggested that altered inter-
actions between these two compartments may cause the phenotype
(Mendive et al., 2006). The LGR4 hypomorphic mutant mouse
(Lgr4Gt) also exhibits altered post-natal development of the epididymis
that lacks the initial segment. Examination of the epididymal ultrastruc-
ture demonstrated disruption of the extracellular matrix with an
increase in laminin (Hoshii et al., 2007). Thus Lgr4, as postulated for
c-ros, may regulate epididymal morphogenesis via maintenance of
the extracellular matrix.
Most recently, inhibin beta A, a mesenchyme-derived paracrine
factor, was shown to control the coiling of the epithelium in the
anterior Wolffian duct, the Anlage of the adult epididymis, thus pro-
viding evidence in vivo that interactions between the epithelial and
mesenchyme compartments are essential for proper development
of the epididymis (Tomaszewski et al., 2007). These studies also
demonstrated that the regulation of epididymal coiling was not directly
controlled by androgens since the Inhba knockout embryos exhibited
normal androgenic parameters.
segment andare sterile
Epididymal cell structure and function
The adult epididymis consists of a pseudostratified epithelium of
several cell types including principal, basal, clear, narrow, apical and
halo cells (Fig. 2). The primary cell type throughout the tubule is the
principal cell which constitutes ?80% of the epithelium and is, by
far, the most studied since it is responsible for the bulk of the proteins
that are secreted into the lumen. Less is known regarding the function
of the remaining cell types; however, narrow, apical and clear cells
contain the vacuolar Hþ-ATPase and secrete protons into the
lumen and thus participate in its acidification (Pietrement et al.,
2006; Kujala et al., 2007), while clear cells are also endocytic cells
and may be responsible for clearance of proteins from the epididymal
lumen. Basal cells do not access the luminal compartment and are in
close association with the overlying principal cells, as indicated by the
presence of basal cell cytoplasmic extensions between principal cells,
and thus may regulate its functions (Veri et al., 1993; Seiler et al.,
1999). Halo cells appear to be the primary immune cell in the epidi-
dymis, while apical cells may also endocytose luminal components.
It is likely that the individual cell types within the epithelium may
perform separate as well as integrated functions within the epididymis.
In support of this view, recent studies demonstrated that basal cells
regulate principal cell electrolyte transport by releasing paracrine
factors, specifically by the release of prostaglandin PGE2 (Cheung
et al., 2005). Thus, cell–cell interactions within the epithelium can
directly affect the luminal environment and ultimately sperm matu-
ration. The principal cells also form tight junctions with one another
and as such form the blood–epididymis barrier. This barrier creates
an immunoprotective site within the epididymal lumen that is necess-
ary for sperm maturation. Several androgen-dependent transmem-
brane proteins including occludin and claudins contribute to the
formation of these tight junctions (Cyr et al., 2007). Gap junctions
formed by a family of integral proteins known as connexins, are also
present between adjacent principal cells both at their apical and
lateral surfaces. These structures, which consist of aligned intercellular
pores, allow the transport of molecules ,1 kDa.
Within the principal cells, gene expression and subsequently protein
synthesis and secretion are tightly regulated and region-specific such
that neighboring cells may express very different subsets of genes
and gene products. This region-dependent expression contributes to
the distinctive luminal protein profile within each epididymal region
which is thought to be integral for sperm maturation. While previously
it was thought that varying patterns of gene expression along the
tubule was loosely associated with different epididymal regions,
Turner et al. (2003 demonstrated that the presence of connective
tissue septa further subdivides the caput, corpus and cauda epididymi-
dis into discrete intra-regional segments and that region-specific gene
expression may in fact be highly ordered and compartmentalized
within these precise segments. By using size exclusion dyes and radio-
labeled molecules, these authors further demonstrated that the con-
nective tissue septa may also act as barriers restricting the
movement of molecules from the interstitial space of one segment
to the next. This would allow segment-specific paracrine signaling to
occur between stromal and epithelial cells that could regulate the
tightly controlled segment-specific expression of genes. Supporting
this view, microperifusion studies have demonstrated that the effects
of perifused growth factors including epidermal growth factor (EGF),
fibroblast growth factor (FGF2) and vascular endothelial growth
factor (VEGFA) on epithelial cell mitogen-activated protein kinase
(MAPK) signaling was restricted to the perifused region only and not
neighboring epididymal segments, presumably reflecting a functional
barrier created by the connective tissue septa. However, when
growth factors were simultaneously perifused with collagenase, that
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Submitted on December 17, 2007; resubmitted on September 30, 2008; accepted
on October 20, 2008
New insights into epididymal biology and function