Human Reproduction Update, Vol.10, No.4 pp. 349–369, 2004
Advance Access publication June 10, 2004.
doi:10.1093/humupd/dmh026
Ectoplasmic specialization, a testis-specific cell – cell
actin-based adherens junction type: is this a potential
target for male contraceptive development?
Nikki P.Y.Lee and C.Yan Cheng1
Population Council, 1230 York Avenue, New York, NY 10021, USA
1
To whom correspondence should be addressed. E-mail: [email protected]
The seminiferous tubule of the mammalian testis is largely composed of Sertoli and germ cells, which coordinate
with Leydig cells in the interstitium and perform two major physiological functions, namely spermatogenesis and
steroidogenesis respectively. Each tubule is morphologically divided into (i) the seminiferous epithelium composing
Sertoli and germ cells, and (ii) the basement membrane (a modified form of extracellular matrix); underneath this
lies the collagen fibril network, the myoid cell layer, and the lymphatic vessel, which collectively constitute the
tunica propia. In the seminiferous epithelium, of rodent testes each type A1 spermatogonium (diploid, 2n) differentiates into 256 elongated spermatids (haploid, 1n) during spermatogenesis. Additionally, developing germ cells must
migrate progressively from the basal to the luminal edge of the adluminal compartment so that fully developed
spermatids can be released into the lumen at spermiation. Without this timely event of cell movement, spermatogenesis cannot reach completion and infertility will result. Yet developing round elongating/elongated spermatids must
remain attached to the epithelium via a specialized Sertoli– germ cell actin-based adherens junction (AJ) type
known as ectoplasmic specialization (ES), which is crucial not only for cell attachment but also for spermatid movement and orientation in the epithelium. However, the biochemical composition and molecular architecture of the
protein complexes that constitute the ES have only recently been studied. Furthermore, the signalling pathways that
regulate ES dynamics are virtually unknown. This review highlights recent advances in these two areas of research.
It is expected that, if adequately expanded, these studies should yield new insights into the development of novel
contraceptives targeted to perturb ES function in the testis. The potential to specifically target the ES may also
mean that contraceptive action could be achieved without perturbing the hypothalamic–pituitary –testicular axis.
Key words: 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide/ectoplasmic specialization/male contraception/Sertoli –germ cell AJ
dynamics/testis
Introduction
The testis is an organ in which millions of germ cells are produced daily. For instance, it is known that ,150 £ 106 sperm
are produced per day in a typical man (Johnson et al., 1970).
The functional unit of the mammalian testis that produces germ
cells is the seminiferous tubule where spermatogenesis occurs.
Throughout spermatogenesis, a single type A1 spermatogonium
(2n, diploid) divides and differentiates into 256 elongated spermatids (1n, haploid) in the seminiferous epithelium (Russell
et al., 1990). Spermatogenesis, however, is regulated largely by
FSH released from the pituitary gland and testosterone produced
by Leydig cells in the interstitium via steroidogenesis (for a
review, see de Kretser and Kerr, 1988). Figure 1 (left panel) is
a schematic drawing of the seminiferous epithelium in the rat
testis that illustrates the relative relationship of germ cells at
different stages of their development with Sertoli cells and
the different junctional types between Sertoli and germ cells in
the epithelium. The right panel of Figure 1 is a schematic drawing that illustrates the three known protein complexes and their
associated peripheral proteins found at the apical ectoplasmic
specialization (apical ES) in the seminiferous epithelium of the
rat testis, which is a testis-specific cell –cell actin-based adherens
junction (AJ) type (see below for a detailed description and definition of apical and basal ES). The electron micrographs shown
in Figures 2A–C are the magnified view of the seminiferous epithelium using cross-sections of an adult rat testis, which also
illustrate the intimate relationships between Sertoli and germ
cells (Figure 2B) and the ultrastructural feature of the apical ES
(Figure 2C) (see also Figure 2D and Figure 1). The schematic
drawing and micrographs shown in Figures 1 and 2 are typical
of the testis in adult rats, mimicking many of the same features
found in other mammals including mice and men.
Human Reproduction Update Vol. 10 No. 4 q European Society of Human Reproduction and Embryology 2004; all rights reserved
349
N.P.Y.Lee and C.Y.Cheng
Figure 1. A schematic drawing that illustrates the relative location of different junction types, namely occluding tight junctions (TJ), anchoring junctions and
communicating gap junctions in the seminiferous epithelium of the rat testis (left panel) and the molecular architecture of the three known structural protein
complexes at the site of apical ectoplasmic specialization (ES) between Sertoli cells and elongating/elongated spermatids (right panel). The cadherin/catenin
complex, the nectin/afadin complex, and the a6b1-integrin/laminin g3 complex are the three adherens junction (AJ) protein complexes found at the site of apical ES (for reviews, see Cheng and Mruk, 2002; Takai and Nakanishi, 2003). These three complexes in turn structurally associate with other adaptors and signalling proteins, constituting the multi-protein complexes. As such, ES also functions as a signalling platform in addition to its structural role in adhering germ
cells onto Sertoli cells in the seminiferous epithelium.
Q
Figure 2. Cross-section of a seminiferous tubule from an adult rat testis. The functional unit that carries out spermatogeneis in the testis is the seminiferous
tubule, which is composed of Sertoli and germ cells that constitute the seminiferous epithelium (A). Underneath the Sertoli cell is the tunica propria, which is
composed of both an acellular zone, namely the basement membrane (asterisk), collagen fibril network (arrowhead), and a cellular zone, namely the peritubular
myoid cell layer and the lymphatic vessel (A). (B) Micrograph showing the cross-section of a seminiferous tubule at lower magnification illustrating the intimate physical relationship between Sertoli cells (SC) and germ cells, such as spermatogonia, pachytene spermatocytes (PS), round spermatids (RS) and elongating/elongated spermatids (EGS), at different stages of their development in the seminiferous epithelium before the release of fully developed spermatids
(sperm) into the tubule lumen that occur during spermiation at late stage VIII of the epithelial cycle. Ectoplasmic specialization (ES) is an actin-based AJ
located at the basal compartment between Sertoli cells at the site of blood–testis barrier (BTB) (basal ES, see also Figure 1, left panel) and at the apical compartment between Sertoli cells and spermatids (apical ES). Apical ES can be found between Sertoli cell and an adjacent elongating spermatid (EGS), which is
circled in B. A magnified view of the apical ectoplasmic specialization (ES) between an elongating spermatid (EGS) and a Sertoli cell encircled in B is shown
in C. (C) Electron micrograph showing an elongated spermatid (N, nucleus) associated with a Sertoli cell having the typical apical ES structural features. Apical
ES is typified by the presence of packed actin filaments (asterisk in C) sandwiched between the cisternae of the endoplasmic reticulum (ER) and the closely
apposed plasma membranes of Sertoli and germ cells (arrowheads in C). It is known that cadherins, integrins and nectins are transmembrane proteins found at
the site of apical ES in the testis as shown in a schematic drawing in D. In the Sertoli cell side of the ES, there is a layer of actin bundles underneath the Sertoli
cell membrane, which is followed by ER. A microtubule is also found near the site of ES, likely having a role in spermatid translocation across the seminiferous
epithelium. LD ¼ lipid droplet; M ¼ mitochondrion; V ¼ vacuole. Bars ¼ 2.5 mm (A), 10 mm (B), and 1 mm (C). Figure 2b was adapted from Lee et al
(2004) reproduced by permission of the American Society of Andrology.
350
Ectoplasmic specialization dynamics in the testis
351
N.P.Y.Lee and C.Y.Cheng
The seminiferous epithelium can be subdivided into (i)
the basal compartment and (ii) the adluminal compartment based
on the relative location of the blood-testis barrier (BTB)
(see Figure 1, left panel), which is also known as the seminiferous epithelium barrier (Figures 1 and 2A, B) (de Kretser and
Kerr, 1988; Russell et al., 1990; Byers et al., 1993). The BTB is
constituted largely by tight junctions between Sertoli cells (also
known as the Sertoli cell barrier), however, both basal ES, desmosome-like junctions, and gap junctions are also found at the
BTB site in the rat and mouse testis (de Kretser and Kerr, 1988;
Russell et al., 1990; Byers et al., 1993). Immediately underneath
the basal compartment lies the tunica propria (Figure 1, left
panel, Figure 2A, B), which is composed of both an acellular
zone (i.e. basement membrane and collagen fibril network) and a
cellular zone (i.e. myoid cells, lymphatic endothelium). Within
the seminiferous epithelium, specialized testis-specific cell –cell
actin-based AJ, such as the ectoplasmic specializations (ES)
(Figures 1, 2C and D) and tubulobulbar complexes, and cell –
cell intermediate filament-based desmosome-like junctions
(Figure 1) (note: all three are anchoring junction types, see
Table I), are responsible for anchoring developing germ cells
onto the epithelium at different stages of the epithelial cycle.
Cell junctions in the testis are classified primarily based on their
relative locations and the attachment sites used for constituting
the cytoskeleton (for a review, see Cheng and Mruk, 2002) (see
Table I).
In the rat testis, spermatogenesis per se involves extensive
junction restructuring when immature germ cells, such as preleptotene and leptotene spermatocytes, initially residing at the basal
compartment, need to migrate to the adluminal compartment
traversing the BTB for further development at late stage VIII
and early stage IX of the epithelial cycle (Russell, 1977c) (see
Figures 1 and 2). Furthermore, spermatocytes must continue
their journey migrating to the adluminal edge of the lumen
while differentiating into round and elongated spermatids. As
such, the testis per se is a novel in vivo model to study the
biology and regulation of junction dynamics. Obviously, such
studies, if adequately expanded, will also shed new light on
male contraceptive development. For instance, if the key regulatory molecules that are important for the junction restructuring
event are identified and characterized, they can become the
prime targets for male contraceptive development. In this
review, the biology and regulation of ES are discussed. We will
not discuss desmosome-like junctions since less is known
regarding this junction type, for example its molecular composition, despite earlier morphological studies (Russell, 1977a). A
review on the tubulobulbar complex (TBC, a testis-specific
actin-based adherens junction type) is also not provided here
since it has recently been discussed and eminently reviewed
(Russell, 1993). But in this context, it is of interest to note that
TBC is another testis-specific AJ type in the seminiferous epithelium found at the site of BTB between Sertoli cells (basal
TBC) as well as surrounding the heads of elongating/elongated
spermatids (apical TBC) (Russell, 1993) (see also Figure 1, left
panel). Apical TBC appears as a pair of tubular structures surrounded by actin with a balloon-like terminal bulbar structure
restricted to the concave side of the elongating/elongated
spermatid head, which is not visible until several days prior to
spermiation at stage VII and is crucial to prevent premature
release of elongating/elongated spermatids from the epithelium
(for reviews, see Russell, 1993; Vogl et al., 1993). However, the
precise structure of basal TBC at the BTB site is not well understood, and the precise biochemical composition of both basal
and apical TBC is not known except for a recent report illustrating that cofilin, an actin-severing protein, is associated with the
apical TBC in the rat seminiferous epithelium (Guttman et al.,
2004). As such, it is clear that much research is needed to dissect this testis-specific AJ type biochemically. Yet ES is the
most studied testis-specific AJ type in the seminiferous epithelium. Interestingly, an in-depth review on the protein complexes that constitute ES and their regulation is currently
missing. As such, it is the goal of this review to fill in this gap.
Ectoplasmic specialization in the seminiferous epithelium:
basic structure and functions
ES is an actin-based testis-specific AJ type between Sertoli cells
at the basal compartment at the site of BTB as well as between
Sertoli and germ cells at the adluminal compartment of the seminiferous epithelium in the rat testis (Figure 1, left panel) (for
reviews, see Russell, 1980; Vogl et al., 2000; Toyama et al.,
2003). Since most of the earlier morphological studies of the ES
were performed in rats (Russell, 1977b; Russell et al., 1990)
including several recent biochemical studies (Lee et al., 2003,
2004; Lui et al., 2003b; Siu et al., 2003; Siu and Cheng, 2004),
the structures of ES discussed herein are primarily based on the
Table I. Classification of anchoring junctions in the testis
Anchoring junction type
Attachment site
Associated cell types/cellular structures
AJ: cell–cell
ES
TBC
Focal adhesion: cell –matrix
Actin filament
Sertoli cells and Sertoli cells (basal ES, basal TBC)
Sertoli cells and spermatids (apical ES and apical TBC)
Actin filament
Sertoli cells and extracellular matrix
Germ cells and extracellular matrix
Sertoli cells and Sertoli cells
Sertoli cells and non-spermatid germ cells
Sertoli cells and extracellular matrix
Germ cells and extracellular matrix
Desmosome-like junction: cell–cell
Intermediate filament
Hemidesmosome: cell-matrix
Intermediate filament
This Table was prepared based on several earlier reviews (Russell, 1980; Byers et al., 1993; Cheng and Mruk, 2002).
AJ ¼ adherens junction; ES ¼ ectoplasmic specialization; TBC ¼ tubulobulbar complex.
352
Ectoplasmic specialization dynamics in the testis
rat model. In the rat testis, the basal ES is situated near the BTB
site between adjacent Sertoli cells throughout the epithelial cycle
(for reviews, see Russell, 1980; Vogl et al., 2000; Toyama et al.,
2003). Likewise, the apical ES is also present throughout the
seminiferous epithelial cycle; it first appears between Sertoli
cells and round spermatids in late stage VII and early stage VIII
in the rat testis (Figure 1) (de Franca et al., 1993; for reviews,
see Russell, 1980; Vogl et al., 2000; Toyama et al., 2003). It
persists in the seminiferous epithelium and associates with spermatids up to step 19 in the mouse before the appearance of the
tubulobulbar complex (for reviews, see Russell, 1980; Vogl
et al., 2000; Toyama et al., 2003).
Structurally, apical ES consists of a narrow layer of hexagonally packed, parallel actin bundles sandwiched between Sertoli
cell plasma membrane and a cistern of endoplasmic reticulum
(ER) on the Sertoli cell side of the plasma membrane between
an apposing Sertoli cell and an elongating/elongated spermatid
(Figure 2C, D, see also Figure 1) (for reviews, see Russell,
1980; Vogl et al., 2000; Toyama et al., 2003). A typical apical
ES found between a Sertoli cell and the head of an elongating
spermatid is also shown in an electron micrograph in Figure 2C.
Figure 2D is a schematic drawing that illustrates the ultrastructural feature of the apical ES in the seminiferous epithelium of
an adult rat testis. It is noted that the basic ultrastructures of the
basal and apical ES are virtually identical; however, ribosomes
are found in the cytoplasmic side of ER in the basal, but not apical, ES, suggesting their constituent proteins could be different
(for a review, see Vogl et al., 2000). Also, the intercellular
space between two apposing Sertoli cells having the basal ES is
sealed by tight junctions at the BTB site (Figure 1, left panel)
(for reviews, see Russell, 1993; Vogl et al., 2000; Toyama et al.,
2003). Furthermore, at the apical ES, actin bundles and ER are
only found on the Sertoli cell side of the plasma membrane;
tight junctions are not present, and little is known about the integral membrane junctional molecules in the apposing elongating/elongated spermatids (for reviews, see Russell, 1980, 1993;
Vogl et al., 2000; Toyama et al., 2003). However, recent studies
have shown that nectin-2, nectin-3, N-cadherin, laminin g3,
RhoB GTPase, Rab8B, zyxin and others are found at the apical
ES site and are associated with elongating/elongated spermatids
(Lee et al., 2003, 2004; Lui et al., 2003c; Siu et al., 2003; Lau
and Mruk, 2003; Siu and Cheng, 2004). The primary function of
ES is to facilitate germ cell movement, with an additional
anchoring function to retain germ cells, in particular spermatids,
in the epithelium until spermiation (for reviews, see Russell,
1980; Vogl et al., 2000; Toyama et al., 2003). In addition, it
likely plays a role in the BTB regulation (for a review, see Vogl
et al., 2000). For instance, the intimate relationship between
basal ES and tight junction (TJ) was demonstrated by freeze –
fracture technique, suggesting that they are present side-by-side
in the BTB (Parreira et al., 2002) (Figures 1, 2). Although ES
was first identified in the testis almost three decades ago (for
reviews, see Russell, 1980; Vogl et al., 2000; Toyama et al.,
2003), the precise mechanism(s) that regulates ES dynamics has
yet to be elucidated. Recent studies have shed new light on the
molecular architecture of the constituent protein complexes at
the apical ES site. Also, the underlying mechanisms that regulate
this AJ type in the testis have recently been identified and partially characterized.
Components of ES
Multi-protein complexes
To date, three multi-protein complexes, namely the cadherin/
catenin, the nectin/afadin/ponsin, and the integrin/laminin complexes are found in the ES (for reviews, see Cheng and Mruk,
2002; Takai and Nakanishi, 2003). For the cadherin/catenin and
the nectin/afadin complexes, both are detected at the apical
and basal ES sites, whereas the integrin/laminin complex is
restricted mostly to the apical ES site. Table II summarizes
some of the biochemical features and functions of different
classes of proteins at the ES site and their interacting partners,
some of which are discussed in this section. It must be noted
that many of the proteins found at the ES site are common to AJ
in other epithelia, and a deletion of these proteins can be fatal,
as shown in results of gene knockout experiments in mice
summarized in Table III.
The cadherin/catenin complex
The cadherin/catenin complex is one of the best studied AJ
structural complexes in the testis (for a review, see Cheng and
Mruk, 2002) (Table II).
Cadherin. Neural (N) and epithelial (E)-cadherins are classic
cadherins, belonging to the cadherin superfamily (for a review,
see Yagi and Takeichi, 2000). Classic cadherins, including
N- and E-cadherins, interact with both p120ctn and b-/g-catenin
(see Figure 1) (for a review, see Perez-Moreno et al., 2003).
Both N- and E-cadherins are found in the seminiferous epithelium of the testis in rats and/or mouse and are products of
Sertoli and germ cells; and germ cells express more E-cadherin
than Sertoli cells (Wu et al., 1993; Byers et al., 1994; Chapin
et al., 2001; Johnson and Boekelheide, 2002b; Lee et al., 2003).
In short, these data suggest that cadherins residing on Sertoli and
germ cells can indeed confer cell adhesion function. This concept was rather provocative when it first emerged (Byers et al.,
1994; Lee et al., 2003), since ultrastructural observations on the
ES had suggested that Sertoli cells contributed most, if not all,
of the adhesive devices for the ES (Figure 2C). Another recent
report indeed supports this concept. For instance, nectin-3 was
shown to reside exclusively in elongating/elongated spermatids
that can form heterotypic interactions with nectin-2 residing in
Sertoli cells at the site of apical ES (Ozaki-Kuroda et al., 2002).
While N-cadherin was found at the site of apical and basal ES
using fluorescent microscopy (Wine and Chapin, 1999; Johnson
and Boekelheide, 2002b; Lee et al., 2003), these classic cadherins apparently were utilizing intermediate filament as the underlying cytoskeleton for attachment (Mulholland et al., 2001;
Johnson and Boekelheide, 2002b). Yet we have recently completed a detailed biochemical analysis using co-immunoprecipitation techniques and lysates of testes and seminiferous tubules
(note: these tubules were freed of Leydig and myoid cell contamination) to investigate this further. It was shown that virtually
all N-cadherin in the Leydig and myoid cell-free seminiferous
tubule lysates were using actin as the attachment site (Lee et al.,
2003) similar to those found in other epithelia (for reviews, see
Gumbiner, 2000; Yagi and Takeichi, 2000; Perez-Moreno et al.,
2003). Yet , 5% of E-cadherin in seminiferous tubule lysates
was indeed associated with vimentin, an intermediate filament
353
N.P.Y.Lee and C.Y.Cheng
Table II. Proteins found at the ectoplasmic specialization site: their functions, properties, and interacting partners
Proteins
Mr (kDa) Binding partnersa
Transmembrane proteins
E-Cadherin
120
N-Cadherin
127 –135
Functions/properties
Selected references
170
70 –80
83 –85
actin, Rab8B, vimentin, zyxin
actin, axin, a-catenin, b-catenin, p120ctn,
cortactin, Fer kinase, ILK, a4-integrin,
b1-integrin, Src, vimentin, WASP, zyxin
N-cadherin, b1-integrin, vinculin
actin, b1-integrin, paxillin
actin, N-cadherin, b-catenin, cortactin,
p-FAK-Tyr397, a4-integrin, a6-integrin,
ILK, paxillin, vinculin laminin g3
BI-integrin, mmp-2
actin, nectin-3
actin, afadin, espin, nectin-2
Adhesion; signalling Chapin et al., 2001
Adhesion; signalling Salanova et al., 1995; Chapin et al., 2001
Adhesion; signalling Palombi et al., 1992; Salanova et al., 1995;
Chapin et al., 2001; Mulholland et al., 2001;
Siu et al., 2003
Adhesion
Koch et al., 1999; Sin and Cheng 2004
Adhesion
Bouchard et al., 2000; Ozaki-Kuroda et al., 2002
Adhesion
Ozaki-Kuroda et al., 2002; Mueller et al., 2003
100
actin, axin, WASP, zyxin
Adaptor
Afadin
a-Catenin
b-Catenin
205
100 –104
92
g-Catenin
82
nectin-3
N-cadherin, b-catenin
actin, axin, N-cadherin, a-catenin, p120ctn,
cortactin, b1-integrin, ILK, WASP, zyxin
Fer kinase, Rab8B
p120ctn
65 –120
axin, N-cadherin, b-catenin, Fer kinase,
paxillin, plectin, WASP, vinculin, zyxin
Cortactin
80 –85
Fimbrin
Paxillin
68
68 –70
Vinculin
130
actin, N-cadherin, b-catenin, ERK2,
b1-integrin, ILK, paxillin, Src
actin
actin, p120ctn, cortactin, FAK, a6-integrin,
b1-integrin, tubulin, vinculin
actin, p120ctn, ERK 2, espin, ILK, a4-integrin,
b1-integrin, pFAK-Tyr397, paxillin
ZO-1
220 –225
n.k.
Adaptor
60
actin, axin, N-cadherin, cortactin, ERK2,
Fer kinase, tubulin, WASP, zyxin
n.k.
n.k.
cortactin, c-Src, vinculin
n.k.
paxillin
Tyr kinase
a4-Integrin
a6-Integrin
b1-Integrin
Laminin g3
Nectin-2
Nectin-3
Adaptors
a-Actinin
Signalling proteins
c-Src
80 –165
118 –145
118
Csk
ERK1
ERK2
pERK1/2
FAK
50
44
42
44/42
120
pFAK-Tyr397
Fer kinase
120
94
Fyn
ILK
59
59
b1-integrin, c-Src, vinculin
actin, N-cadherin, g-catenin, p120ctn,
Rab 8B, c-Src, vimentin
actin
N-cadherin, b-catenin, cortactin,
b1-integrin, vinculin
n.k.
PKA
40 –55
Motor proteins
Cytoplasmic dynein 1– 2 MDa actin
Myosin VIIa
250
actin, Keap1
Proteins or molecules with special functions
Espin
110
actin, vinculin
Adhesion
Lau and Mruk, 2003; Lee et al., 2003, 2004
Adhesion; signalling Wine and Chapin, 1999; Chapin et al., 2001;
Chen et al., 2003; Lee et al., 2003, 2004
Russell and Goh, 1988; Vogl, 1989;
Lee et al., 2004
Adaptor
Ozaki-Kuroda et al., 2002
Adaptor
Byers et al., 1994; Lee et al., 2004
Signalling; adaptor
Byers et al., 1994; Wine and Chapin, 1999; Chapin
et al., 2001; Lee et al., 2003; Lee et al., 2004
Signalling; adaptor
Cowin et al., 1986; Byers et al., 1994;
Chen et al., 2003; Lau and Mruk, 2003
Signalling; structural Wine and Chapin, 1999; Chapin et al., 2001;
Johnson and Boekelheide, 2002a;
Chen et al., 2003; Lee et al., 2004
Actin-binding
Chapin et al., 2001
Adaptor
Adaptor, signalling
Adaptor
Tyr kinase
MAP kinase
MAP kinase
MAP kinase
Tyr kinase
Tyr kinase
Tyr kinase
Tyr kinase
Ser/Thr kinase
Ser/Thr kinase
Motor proteins
Motor proteins
Actin-linker
Gelsolin
91
actin
Actin-severing
Keap1
PI(4,5)P2
68
0.971
myosin VIIa
actin
Nrf2 regulator
Gelsolin inhibitor
354
Grove and Vogl, 1989
Wine and Chapin, 1999; Chapin et al., 2001;
Mulholland et al., 2001
Grove and Vogl, 1989; Wine and Chapin, 1999;
Mulholland et al., 2001; Chapin et al., 2001;
Beardsley and O’Donnell, 2003; Siu et al., 2003
Stevenson et al., 1986; Balda and
Anderson, 1993
Nishio et al., 1995; Wang et al., 2000; Chapin
et al., 2001; Chen et al., 2003; Lee et al., 2004
Wine and Chapin, 1999
Chapin et al., 2001
Chapin et al., 2001
Chapin et al., 2001
Wine and Chapin, 1999; Mulholland et al., 2001;
Siu et al., 2003
Siu et al., 2003
Chen et al., 2003
Maekawa et al., 2002
Chapin et al., 2001; Mulholland et al., 2001;
Beardsley and O’Donnell, 2003
Lonnerberg et al., 1992
Hall et al., 1992; Miller et al., 1999;
Guttman et al., 2000
Velichkova et al., 2002
Bartles et al., 1996; Chen et al., 1999;
O’Donnell et al., 2000
Rousseaux-Prevost et al., 1997;
Guttman et al., 2002
Velichkova et al., 2002
Guttman et al., 2002
Ectoplasmic specialization dynamics in the testis
Table II Continued
Proteins
PLCg
Scinderin
Testin I, II
mmp 2
GTPases
Cdc42
Rab8B
Rac1
RalA
Ras
RhoB
Cytoskeletal proteins
Actin
Mr (kDa) Binding partnersa
Functions/properties
Selected references
148
80
37, 40
actin
n.k.
n.k.
Hydrolyse PI(4,5)P2
Actin-severing
Signalling
64
Laminin
Protease
Guttman et al., 2002
Pelletier et al., 1999
Zong et al., 1994; Grima et al., 1997,
Grima et al., 1998
Sin and Cheng, 2004
22
24
n.k.
actin, E-cadherin, g-catenin, Fer kinase,
a-tubulin, b-tubulin, vimentin
n.k.
n.k.
n.k.
n.k.
GTPase
GTPase
Chapin et al., 2001
Chen et al., 2003; Lau and Mruk, 2003
GTPase
GTPase
GTPase
GTPase
Chapin et al., 2001
Chapin et al., 2001
Chapin et al., 2001
Lui et al., 2003b
axin, E-cadherin, N-cadherin, b-catenin,
cortactin, espin, Fer kinase, fimbrin,
Fyn, gelsolin, eNOS, iNOS, nectin-2, nectin-3,
paxillin, PI(4,5)P2, PLCg, Rab8B, Src,
vinculin, WASP, zyxin
axin, eNOS, iNOS, paxillin, Rab8B, Src,
WASP, zyxin
Structural
Russell, 1977b; Chapin et al., 2001;
Ozaki-Kuroda et al., 2002; Guttman et al., 2002;
Maekawa et al., 2002; Chen et al., 2003; Lee and
Cheng, 2003
Motility; structural;
Germ cell viability;
Spermatid adhesion
Structural proteins;
signalling
Russell, 1977b; Vogl, 1988; Chapin et al., 2001;
Lee and Cheng, 2003; Fleming et al., 2003a,b;
Lee et al., 2004
Amlani and Vogl, 1988; Chen et al., 2003;
Lau and Mruk, 2003; Lee et al., 2004
21
24
21
24
42
Tubulin
55
Vimentin
55
axin, E-cadherin, N-cadherin, Fer kinase,
eNOS, iNOS, Rab8B, WASP, zyxin
a
Binding partners were assessed by either co-localization studies using immunofluorescent microscopy, or by biochemical studies using co-immunoprecipitation
techniques with and without chemical cross-linker(s). Binding partners identified by biochemical means are underlined. Although we intended to provide an
extensive list on all the known ectoplasmic specialization-associated proteins, some important information may be missing from this Table, and readers should
refer to other reviews listed in this paper for additional information.
n.k. ¼ not known; ERK ¼ extracellular signal-regulated kinase; ILK ¼ integrin-linked Kinase; Nos ¼ nitric oxide synthase; WASP ¼ Wiskott–Aldrich
syndrome protein; MMP ¼ matrix metalloprotase-2.
structural protein (Lee et al., 2004). Furthermore, when lysates
of testes (with Leydig and myoid cells in addition to Sertoli and
germ cells) were used for co-immunoprecipitation, it was shown
that as much as 50% of the E- and N-cadherins were indeed
associated with vimentin in the testis (Lee et al., 2004). Collectively, these analyses reveal that classic cadherins at the ES are
using actin predominantly as attachment sites in the seminiferous
epithelium with only , 5% of E-cadherin using vimentin as its
attachment site, whereas cadherins found in the interstitial cells
and at the basal portion of the tubules are using intermediate
filament as attachment sites (Lee et al., 2003).
Catenins. a-Catenin is a 100–104 kDa actin-binding and
a-actinin-binding protein (Knudsen et al., 1995) (for a review,
see Rudiger, 1998). The 92 kDa b-catenin and 82 kDa g-catenin,
also called plakoglobin, can interact with cadherins at the highly
conversed intracellular cytoplasmic domain called catenin binding domain (CDB) (Pierceall et al., 1995; Takayama et al.,
1996). But their interactions with actin are mediated via a-catenin (for a review, see Zhurinsky et al., 2000). b- and g-catenins
share similar structural features, yet b-catenin is predominantly
localized at the site of AJ whereas g-catenin is found in both AJ
and desmosome-like junctions (for a review, see Zhurinsky et al.,
2000) (Figure 1). b- or g-catenin in turn interacts with a-catenin,
linking cadherins to the underlying actin filament via a-catenin
and/or a-actinin (for a review, see Zhurinsky et al., 2000). All
three catenins are found in the testis (Cowin et al., 1986; Byers
et al., 1994; Janssens et al., 2001; Lee et al., 2003). b-Catenin
co-localizes with N-cadherin at the site of ES in the basal compartment of the epithelium (Lee et al., 2003); it also co-localizes
with classic cadherin at non-ES sites, possibly at the desmosome-like junctions (Mulholland et al., 2001). a-Catenin was
localized to the basal ES and myoid cells (Byers et al., 1994;
Janssens et al., 2001). g-Catenin was found at the site of basal
ES (Cowin et al., 1986; Byers et al., 1994). The three catenins
have been shown to form a complex with E- or N-cadherin in
the testis (Lau and Mruk, 2003; Lee et al., 2003, 2004). A transmembrane phosphatase called PTPm was shown to associate
with the catenin/cadherin complexes and might take part in the
signalling event (Brady-Kalnay et al., 1995), yet this possibility
remains to be investigated in the testis.
p120 ctn. More than four isoforms of p120 ctn with Mr ,65 –
120 kDa are found in different tissues, including the testis, due
to alternative splicing (Mo and Reynolds, 1996; Johnson and
Boekelheide, 2002a). p120ctn binds to cadherin at a site different
from the b-/g-catenin binding site (Figure 1) (for a review, see
Anastasiadis and Reynolds, 2000). It is still not known whether
p120ctn facilitates or inhibits cell adhesion function (for a
review, see Anastasiadis and Reynolds, 2000). p120ctn is localized to apical and basal ES, to the same site of N-cadherin and
b-catenin, and is also found in desmosome-like junctions, colocalizing with plectin (Wine and Chapin, 1999; Golenhofen and
Drenckhahn, 2000; Johnson and Boekelheide, 2002a).
355
N.P.Y.Lee and C.Y.Cheng
Table III. Studies on effects of deletion of ectoplasmic specialization (ES) proteins that are also found in other epithelia by gene knockout experimentsa
Proteins
Transmembrane proteins
E-Cadherin2/2
N-Cadherin2/2
a4-Integrin2/2
a6-Integrin2/2
b1-Integrin2/2
Nectin-22/2
Adaptors
Afadin2/2
a-Catenin2/2
b-Catenin2/2
g-Catenin2/2
Signalling proteins
c-Src2/2
Csk2/2
FAK2/2
Fer kinase2/2
Fyn2/2
Motor proteins
Cytoplasmic dynein2/2
Proteins or molecules with special functions
Gelsolin2/2
PLCg2/2
Cytoskeletal proteins
Vimentin2/2
Viability
Fertility
Selected references
embryonic lethality (E4.5)
embryonic lethality (E10)
embryonic lethality (E12.5)
neonatal lethality
embryonic lethality (E5.5)
viable
n.k.
n.k.
n.k.
n.k.
n.k.
infertile
Larue et al., 1994
Radice et al., 1997
Yang et al., 1995
Georges-Labouesse et al., 1996
Stephens et al., 1995
Bouchard et al., 2000
embryonic lethality (E10)
embryonic lethality
embryonic lethality (E9.5)
embryonic lethality (E12)
n.k.
n.k.
n.k.
n.k.
Ikeda et al., 1999
Torres et al., 1997
Haegel et al., 1995
Ruiz et al., 1996
postnatal lethality
embryonic lethality (E10)
embryonic lethality (E8.5)
viable
viable
n.k.
n.k.
n.k.
fertile
fertile
Soriano et al., 1991
Imamoto and Soriano, 1993
Ilic et al., 1995
Craig et al., 2001
Maekawa et al., 2002
embryonic lethality (E8.5)
n.k.
Harada et al., 1998
viable
embryonic lethality (E9)
fertile
n.k.
Witke et al., 1995
Ji et al., 1997
viable
fertile
Colucci-Guyon et al., 1994
a
This Table compiles some results using gene knockout experiments to investigate the role of proteins found in other epithelia, which are also present in the ES
in the rat testis. This list is not intended to be exhaustive, but it illustrates the physiological significance of many ES-associated proteins.
E ¼ age of embryo post coitus; n.k. ¼ not known because mice died before birth.
The nectin/afadin/ponsin complex
Nectin/afadin/ponsin is a recently identified putative ES structural unit (for reviews, see Cheng and Mruk, 2002; Takai and
Nakanishi, 2003) (Table II). Nectin is an AJ-integral membrane
protein that interacts with afadin, which in turn binds to the
underlying actin filament (Figure 1). Ponsin is an afadin-binding
protein also found in the testis (Mandai et al., 1999; Lebre et al.,
2001) (Figure 1).
Nectin. Four nectins, namely nectin-1, -2, -3 and -4, are
known to date (for a review, see Takai and Nakanishi, 2003). It
is structurally associated with afadin at its cytoplasmic domain
(for a review, see Takai and Nakanishi, 2003). Recent studies
have shown that nectin-2 and nectin-3 found in spermatids can
mediate adhesive interactions with nectin-2 found in Sertoli cells
(Bouchard et al., 2000; Ozaki-Kuroda et al., 2002; Mueller et al.,
2003). Nectin-2, -3 and -4, but not nectin-1, are found in the
testis (for a review, see Takai and Nakanishi, 2003). Nectin-2
and -3 were shown to co-localize with actin and espin to ES
(Ozaki-Kuroda et al., 2002; Mueller et al., 2003). Nectin-22/2
mice are infertile with defective Sertoli –spermatid junctions,
resulting in sperm malformation (Bouchard et al., 2000; OzakiKuroda et al., 2002; Mueller et al., 2003). Interestingly, nectin-3
immunostaining was still detected in apical ES in nectin-22/2
mice but with disturbed and diminished distribution (Ozaki-Kuroda et al., 2002; Mueller et al., 2003).
Afadin. Afadin has two splice variants, namely l-afadin
(afadin) and s-afadin (a rat homologue of human AF-6 protein,
which lacks the actin filament-binding domain at its C-termi-
356
nus), with an apparent Mr of 205 and 190 kDa respectively
(Mandai et al., 1997). Afadin is expressed by both Sertoli and
germ cells and is localized to the site of apical and basal ES
(Ozaki-Kuroda et al., 2002). Interestingly, afadin level was
found to be diminished at the site of Sertoli-spermatid junctions, but not the Sertoli–Sertoli cell junctions in nectin-22/2
mice (Ozaki-Kuroda et al., 2002), suggesting that nectin-2 is
required for the proper localization of afadin at the apical, but
not basal, ES. This also implicates the presence of another yetto-be identified nectin(s) or nectin-associated protein(s) at the
site of basal ES.
Ponsin. Ponsin/SH3P12/CAP belongs to an adaptor family
consisting of at least two other members: vinexin and Argbinding protein 2 (ArgBP2) (for a review, see Kioka et al.,
2002). It has at least 13 splice variants, and the Mr 93 kDa variant
is the major afadin binding partner at the AJ site (Mandai et al.,
1999). Ponsin is an afadin-binding protein (Mandai et al., 1999).
It remains to be determined if ponsin is present at the site of ES
in the seminiferous epithelium but studies by northern blots have
positively identified ponsin in the testis (Mandai et al., 1999).
The integrin/laminin complex
The integrin/laminin complex was initially identified as a junction complex at the site of cell –substratum focal adhesion (for a
review, see Hynes, 2002). Yet the integrin/laminin complex is
one of the first AJ structural protein complexes found at the site
of ES in the testis (Koch et al., 1999; Palombi et al., 1992;
Salanova et al., 1995; Siu and Cheng, 2004) (Table II).
Ectoplasmic specialization dynamics in the testis
Integrin. Integrin is a transmembrane protein receptor, composed of a and b subunits. To date, eight b and 18 a subunits
have been found in mammalian epithelia, and most integrin
receptors are restricted to the focal adhesion site between cell
and extracellular matrix (for a review, see Hynes, 2002). Interestingly, a6b1- and a4b1-integrins are two putative integral
membrane proteins at the site of apical ES, co-localzing with
actin, on the Sertoli cell side (Palombi et al., 1992; Salanova
et al., 1995, 1998; Chapin et al., 2001; Mulholland et al., 2001).
The interacting partners of b1-, a4- and a6-integrins in the testis
can be found in Table II. The findings that Sertoli –germ cells
are using components of the focal adhesion complex (a cell –
matrix actin-based anchoring junction type in other epithelia, for
a review, see Cheng and Mruk, 2002) to constitute apical ES (a
testis-specific cell –cell actin-based AJ type) are significant since
integrins at the focal adhesion site at the level of cell – matrix are
crucial to permit rapid cell-matrix restructuring, such as between
fibroblasts/macrophages and extracellular matrix, to facilitate
cell movement. Since the event of spermiation also requires
extensive junction restructuring, it is not surprising that apical
ES is utilizing one of the most efficient protein complexes at
focal adhesion sites in other epithelia, namely integrin/laminin,
to regulate ES dynamics.
Laminin. Laminin is a heterotrimer composed of three different chains, one each of the a, b and g chain, to constitute a
functional laminin protein, which is the putative binding partner
for an integrin receptor in many epithelia (for a review, see
Colognato and Yurchenco, 2000). As such, laminin is anticipated
to be the binding partner for a6b1- and a4b1-integrin at the apical ES site. However, laminin is restricted to the basement membrane in most epithelia. Recently, a non-basement membrane
form, laminin g3 (Mr 170 kDa), was found in the testis, which
was found at the site consistent with its localization at the apical
ES (Iivanainen et al., 1999; Koch et al., 1999). Subsequent
studies using fluorescent microscopy with dual fluorescent
probes against b1-integrin and laminin g3 have localized both
proteins to the same site at the apical ES in the seminiferous
epithelium of the adult rat testis (Siu and Cheng, 2004).
More important, these data were also verified by co-immunoprecipitation technique using seminiferous tubules without Leydig
and myoid cell contamination, since an anti-b1-integrin can pull
out laminin g3 from the tubule lysates, whereas an anti-laminin
g3 can also pull out b1-integrin but not N-cadherin and nectin-3
(Siu and Cheng, 2004). Collectively, these data clearly illustrate
that b1-integrin can form a bona fide complex with laminin g3
(Siu and Cheng, 2004). Yet the other two chains, namely a and
b, that are required to assemble a functional laminin receptor for
a6b1 integrin in the seminiferous epithelium, remain to be
identified. In this context, it is of interest to note that besides
the a6b1-/a4b1-integrin/laminin a(?)b(?)g3, which is found at
the apical ES site, recent studies have shown that other signalling molecules that are usually restricted to the focal adhesion
site in other epithelia are also present in the apical ES. This
will be discussed in ‘Signalling proteins’ below.
Adaptors
Adaptors are proteins that recruit other signalling and structural
proteins to the site of junctions, such as ES. They are also
crucial to tether integral membrane proteins, such as cadherins,
integrins and nectins, to the underlying cytoskeleton network,
such as the actin-based microfilament. Yet few reports are found
in the literature that have investigated the role of adaptors in ES
dynamics. The known adaptors at the site of ES include a-,
b- and g-catenins, afadin, a-actinin, cortactin, fimbrin, paxillin,
vinculin, zonula occludens-1 (ZO-1), and several others (Table
II) (for reviews, see Russell and Goh, 1988; Vogl et al., 2000;
Cheng and Mruk, 2002).
Signalling proteins
Signalling proteins in the ES are mostly protein kinases, phosphorylating the downstream target proteins at either Tyr, Ser or
Thr residues. The putative interacting partners of several signalling molecules found in the seminiferous epithelium of the testis
are listed in Table II.
Cellular homologue of a viral protein in Rous Sarcoma virus (RSV)
(c-Src) and Fyn
c-Src and Fyn belong to the Src family and have apparent Mr
ranging between 52 and 62 kDa (for a review, see Thomas and
Brugge, 1997). c-Src (Nishio et al., 1995; Wine and Chapin,
1999; Wang et al., 2000) and Fyn (Maekawa et al., 2002) have
been localized to the ES in the seminiferous epithelium.
C-terminal Src kinase (Csk)
Csk is a 50 kDa protein that phosphorylates c-Src at the
C-terminal Tyr residue (for a review, see Bjorge et al., 2000).
Csk is localized to ES (Wine and Chapin, 1999), almost at the
same site of Src, demonstrating a kinase-substrate relationship of
these two kinases in regulating ES dynamics.
Focal adhesion kinase (FAK)
FAK is a 120 kDa focal adhesion-associated non-receptor PTK
and the only other member of this family known to date is cell
adhesion kinase b (CAKb) (for a review, see Parsons, 2003).
FAK has been localized to ES in studies using immunohistochemistry (Wine and Chapin, 1999; Siu et al., 2003). Interestingly, pFAK-Tyr397 is intensely localized to apical ES in stage
VII through early stage VIII but not during spermiation in late
stage VIII of the epithelial cycle, whereas most of the FAK is
restricted to the basal compartment (Siu et al., 2003). It is noted
that pFAK-Tyr397 co-localizes with vinculin to the same site in
ES (Siu et al., 2003). pFAK is also an important PTK that mediates the b1-integrin-induced signalling function, regulating ES
dynamics in the rat testis (Siu et al., 2003).
Fes-related protein (Fer) kinase
Fer kinase is a 94 kDa cytoplasmic protein tyrosine kinase
(PTK) (for a review, see Greer, 2002). The truncated form of
Fer kinase is called FerT (51 kDa), which is restricted to pachytene spermatocytes (Fischman et al., 1990; Keshet et al., 1990).
Fer kinase and FerT are non-receptor PTK in the testis (Fischman et al., 1990; Keshet et al., 1990; Chen et al., 2003).
Fer kinase is a stage-specific protein, being at its highest levels
in stages I –XIII (Chen et al., 2003); it largely associates with
spermatocytes and elongating, but not elongated, spermatids in
stages IX– XIV, and is restricted to round spermatids in stages
357
N.P.Y.Lee and C.Y.Cheng
I–VIII (Chen et al., 2003). Fer kinase is also associated with
ES, apparently both the basal and apical ES site, as demonstrated
by immunohistochemistry and immunoprecipitation technique
(Chen et al., 2003), in contrast to FerT, which is restricted to
pachytene spermatocytes (Keshet et al., 1990; Hazan et al.,
1993). Interestingly, Fer kinase2/2 mice are viable and fertile
(Craig et al., 2001), seemingly suggesting that while Fer kinase
is a crucial PTK in spermatogenesis, its function can be superseded by other kinases in the testis. As such, these negative
findings by gene knockout experiments do not negate the
significance of Fer kinase, they indeed reinforce the significance
of PTK in AJ dynamics.
of research that deserves much attention in future studies in
order to delineate the mechanism(s) that regulates ES dynamics
in the testis. Table III also summarizes results of recent findings
using gene knockout mice, illustrating the crucial function of
many proteins found in the ES as well as in AJ sites in other
epithelia.
Motor proteins
ILK is a 59 kDa serine/threonine kinase that binds to the cytoplasmic tail of b1-integrin (Hannigan et al., 1996). ILK has
been localized to apical ES, virtually to the same site of b1integrin and vinculin (Chapin et al., 2001; Mulholland et al.,
2001; Beardsley and O’Donnell, 2003). In the testis, ILK and
b1-integrin form an interacting complex (Mulholland et al.,
2001). Recent studies have shown that this is an important signalling molecule that mediates the integrin downstream signalling function, affecting ES dynamics in the testis (Mulholland
et al., 2001).
Three types of motor proteins, namely dynein, kinesin and
myosin, are found in locomotive cells, such as fibroblasts and
macrophages, facilitating cellular movement. In the testis, with
the exception of kinesin (a microtubule-based motor protein),
both dynein and myosin are found in the apical ES and these
proteins apparently function as transporters to assist spermatid
translocation across the seminiferous epithelium during spermatogenesis (Redenbach and Vogl, 1991; Redenbach et al., 1992;
Beach and Vogl, 1999). In essence, motor proteins work in concert with other partner proteins, such as Kelch-like ECH Associating Protein 1 (Keap1), that they use microtubule found in
Sertoli cells at the apical ES site as a track to facilitate the
movement of elongating/elongated spermatids across the epithelium (Figure 3) (Table II) (Redenbach and Vogl, 1991; Beach
and Vogl, 1999; Guttman et al., 2000; Velichkova et al., 2002).
Protein kinase A (PKA)
Cytoplasmic dynein
PKA has four regulatory (R) subunits (RIa, RIb, RIIa and RIIb)
and three catalytic (C) subunits (a, b, and g), which have similar
Mr ranging between 40 and 55 kDa (for reviews, see Chin et al.,
2002; Robinson-White and Stratakis, 2002). PKA is found in
the testis and different subunits have distinct distribution in the
seminiferous epithelium (Pariset et al., 1989; Lonnerberg et al.,
1992). For instance, the expression of RIb is highest in stages
VIII–XI, whereas RIIa and RIIb are highest in stages VII –VIII
(Lonnerberg et al., 1992). It is noteworthy that the relative
abundance of different subunits changes during germ cell maturation, suggesting their differential role in spermatogenesis. For
instance, RII subunits are predominantly expressed in late-stage
germ cells, such as elongating spermatids (Pariset et al., 1989;
Landmark et al., 1993). While the function of PKA in ES
dynamics remains to be studied, it is a known crucial regulator
of Sertoli cell TJ dynamics (Li et al., 2001). Likewise, PKG
was recently shown to be a crucial regulator of Sertoli cell TJ
dynamics (Lee and Cheng, 2003).
Cytoplasmic dynein is a microtubule-associated motor protein
complex with Mr , 1000 –2000 k Da (for review, see King,
2000). It is known to generate the minus-end-directed movement
along the microtubules (for reviews, see King, 2000; Kamal and
Goldstein, 2002). Cytoplasmic dynein associates with the cytoplasmic face of ER at the site of ES, co-localizing with actin
(Collins and Vallee, 1989; Hall et al., 1992; Maeda et al., 1998;
Miller et al., 1999; Guttman et al., 2000). It was postulated that
cytoplasmic dynein was used as a vehicle and the microtubule as
a track to assist spermatid movement across the seminiferous
epithelium (Redenbach and Vogl, 1991; Redenbach et al., 1992;
Beach and Vogl, 1999; Guttman et al., 2000) (Figure 3). However, the positive-end-directed microtubule-based motor protein,
likely a kinesin family member, remains to be found at the ES
site in the testis.
Integrin-linked kinase (ILK)
Remarks
While several reports are found in the literature confirming the
presence of signalling molecules at the site of ES, mostly by
immunohistochemistry as reviewed herein, how these proteins
regulate ES integrity and restructuring during spermatid movement is largely unknown. Much of the information in this area
of research relies on studies of the same proteins found in other
epithelia many of which are confined to the cell-matrix focal
adhesion sites, such as FAK and ILK. Nonetheless, these recent
findings have opened new opportunities to study ES dynamics.
For instance, inhibitors can now be used to assess whether a disruption of these kinases can perturb spermatid movement or
spermiation. If it does, these inhibitors can become the basis of
developing novel male contraceptives. Obviously, this is an area
358
Myosin VIIa
Myosin VIIa, which hydrolyses ATP and can bind actin, is a
250 kDa motor protein in a large family with at least 15 different
classes (for a review, see Sellers, 2000). Two forms of myosin
VII, namely VIIa and VIIb, which lacks the coiled-coil domains,
are known to date (Chen et al., 2001). Myosin VIIa is a motor
protein in the testis (Hasson et al., 1995). It co-localizes with
Keap1, a negative regulator of transcription factor NF-E2Related Factor 2 (Nrf2) (Itoh et al., 1999), at the site of ES
(Velichkova et al., 2002).
Proteins with specific functions
Espin
Espin is an ES-associated protein of Mr ,110 kDa (Bartles et al.,
1996). It has three actin-binding sites that link actin filaments to
each other and to cell membranes at the site of ES (Bartles et al.,
Ectoplasmic specialization dynamics in the testis
Figure 3. A schematic diagram depicting the regulatory mechanism utilized by ectoplasmic specialization (ES) in the up-and-down movement of developing
spermatids during spermatogenesis. Germ cell movement involves the minus end-directed microtubule-based motor protein, such as cytoplasmic dynein
(dynein), and a yet-to-be-identified positive end-directed microtubule-based motor protein, which is likely a kinesin family member. On the other hand, an
actin-based motor protein, such as myosin VIIa, may also have a role in germ cell movement since myosin VIIa and Keap1 are crucial protein partners to affect
cell movement. The intrinsic polarity of the microtubule (indicated by the plus and minus signs), which functions as a track, makes it ideal for the directed
movement of spermatids across the seminiferous epithelium ER, Endoplasmic reticulum. This figure was prepared based on the following reports: Redenbach
and Vogl (1991); Redenbach et al. (1992); Beach and Vogl (1999); Miller et al. (1999); Guttman et al. (2000); Velichkova et al. (2002).
1996; Chen et al., 1999). It is co-localized with actin and vinculin to the site of basal and apical ES (Bartles et al., 1996; Chen
et al., 1999; O’Donnell et al., 2000). It is noted that actin bundles start to appear at the time of espin accumulation (Chen
et al., 1999) illustrating that it is a crucial actin bundling
protein.
co-existing in ES. Indeed, treatment of ES-enriched fractions
either with exogenous PLCg or with a synthetic peptide corresponding to the PI(4,5)P2 binding region of gelsolin can lead to
actin filament disassembly, releasing gelsolin (Guttman et al.,
2002).
Phosphatidylinositol 4,5-bisphosphate
Gelsolin and scinderin
Gelsolin and scinderin, an apparent Mr of 91 and 80 kDa respectively, are actin-severing proteins belonging to the same family
with at least six other members, such as advillin (for a review,
see Kwiatkowski, 1999). Scinderin, also called adseverin, is
the closest homologue of gelsolin (for review, see Kwiatkowski,
1999). Gelsolin co-localizes with actin to the same site in ES
(Rousseaux-Prevost et al., 1997; Guttman et al., 2002). In
addition, scinderin (Pelletier et al., 1999), and two other gelsolin
regulators, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and
phosphoinositide-specific phospholipase C (PLC)-g, are also
found in the same site (Guttman et al., 2002), suggesting
that gelsolin and its regulators are physically and intimately
PI(4,5)P2 belongs to a group of phospholipids with at least seven
known derivatives (for a review, see Yin and Janmey, 2003).
PI(4,5)P2 co-localizes with actin, gelsolin, and PLCg to the
same site in mechanically isolated spermatids (Guttman et al.,
2002). PI (4, 5) P2 is a crucial regulator of junction dynamics
via its effects on intracellular calcium level and PKC.
PLC
PLC is member of a large protein family consisting of at least
11 members in mammals, such as b1 to b4, g1 and g2, d1 to d4
and 11. The molecular weights of PLC range from 85 to
150 kDa and PLCg has an apparent Mr of 148 kDa (for review,
see Rebecchi and Pentyala, 2000). It hydrolyses PI(4,5)P2 to two
359
N.P.Y.Lee and C.Y.Cheng
second messengers, namely inositol 1,4,5-trisphosphate (IP3),
which releases calcium from the intracellular store, and diacylglycerol (DAG), a PKC activator (for review, see Rebecchi and
Pentyala, 2000). Other studies have shown that PKC is a crucial
regulator of setor; cell tight junction dynamics (Li et al 2001).
Remarks
As reviewed in sections ‘Motor proteins’ and ‘Proteins with
specific functions’, it is obvious that ES is equipped with the
necessary motor and regulatory proteins to induce spermatid
movement in the epithelium. Yet it remains unclear how these
proteins can interact together to facilitate spermatid movement
(see ‘The role of ES in germ cell movement across the seminiferous epithelium’ below).
Cytoskeletal proteins
Cytoskeletal proteins, such as actin, tubulin and vimentin, are
constituents of cytoskeletons (Table II). They are crucial not
only to confer cell shape and cell rigidity, but also to facilitate
cell movement by changes in polymerization/depolymerization
and via the intricate interactions with different proteins, such as
adaptors and signalling molecules, at the ES site. Recent studies
using adenovirus to induce overexpression of g-tubulin in Sertoli
cells in vivo have shown that in addition to its structural role as
a cytoskeleton, it is crucial for the attachment of the heads of
elongating/elongated spermatids onto the epithelium at the site
of apical ES (Fleming et al., 2003a,b). Additionally, overexpression of Sertoli cells with g-tubulin apparently induces germ cell
apoptosis, suggesting that g-tubulin may be crucial to cell viability in the epithelium (Fleming et al., 2003b). It is not certain
based on these data whether g-tubulin overexpression recruits
signalling molecules harmful to cell viability to the ES sites
where spermatids attach onto the epithelium, or whether
g-tubulin per se can induce apoptosis via a yet-to-be defined
mechanim(s), such as by disrupting the intracellular trafficking
system. Nonetheless, this latest finding has illustrated that cytoskeletal proteins may have potential functional roles in the seminiferous epithelium other than their structural function. Figure 1
shows the three cytoskeletal elements in the testis and their
interacting partners (see also Table II).
The role of ES in germ cell movement across the
seminiferous epithelium
The events of junction restructuring that take place in the seminiferous epithelium during spermatogenesis have made the testis
as one of the most dynamic organs in the mammalian body. As
such, the testis is equipped with some of the most flexible cell –
cell junctions to facilitate germ cell movement in the seminiferous epithelium (for reviews, see Russell, 1980; Cheng and
Mruk, 2002) (Table I). Furthermore, desmosome-like junctions
that are found between Sertoli cells and spermatogonia/spermatocytes are not detected between Sertoli cells and spermatids
beyond step 9 (Russell, 1977a). Instead, they are equipped with
ES (Russell, 1977b). In short, this seemingly suggests that the
mechanism(s) that regulates the movement of preleptotene and
leptotene spermatocytes across the blood-testis barrier that
occurs at late stage VIII through early stage IX of the epithelial
360
cycle in the rat (Russell, 1977c) is regulated differently from
those involved for spermatid movement at the ES site. Indeed,
recent studies have shown that ES is crucial for the spermatid
movement across the epithelium. Such up-and-down movement
of spermatids across the epithelium during the epithelial cycle is
likely regulated by the intricate interactions of several motor
proteins that work in concert with the underlying cytoskeletons
at the ES (Miller et al., 1999; Guttman et al., 2000; Velichkova
et al., 2002). A brief review is presented herein.
As described above and shown in Figure 2C, D, apical ES is
composed of thin layers of hexagonally packed actin filaments
and endoplasmic reticulum (ER) on the Sertoli cell side lying
against the heads of elongating/elongated spermatids in the epithelium (Figure 3) (for reviews, see Russell, 1980; Vogl et al.,
2000). At the cytoplasmic face of the ER, microtubules are also
arranged parallel to the long axis of the Sertoli cell with the plus
end pointing to the basal epithelium and the minus end directed
to the apical side (Redenbach and Vogl, 1991) (Figure 3). It was
postulated that the intrinsic polarity of microtubules at the cytoplasmic side of the ER provides a guide for the directed movement of spermatids across the epithelium from the basal to the
adluminal compartment and vice versa during the epithelial
cycle (Vogl, 1988; Redenbach and Vogl, 1991; Fleming et al.,
2003a,b; Kann et al., 2003) (Figure 3). Yet microtubules alone
are not sufficient for spermatid translocation unless motor proteins that act as vehicles provide the needed driving force, moving spermatids along the microtubules that act as a track. Indeed,
cytoplasmic dynein, a minus end-directed motor protein (for a
review, see Kamal and Goldstein, 2002), was recently identified
in ES (Hall et al., 1992; Miller et al., 1999; Guttman et al.,
2000). In addition, cytoplasmic dynein links to ER, which is part
of the structural component of ES (Guttman et al., 2000). As
such, forces generated by cytoplasmic dynein can assist spermatid translocation. At this point, the precise mechanism by which
motor proteins generate the needed driving force is not known.
Yet based on studies in migrating fibroblasts or macrophages,
changes in the rigidity and fluidity of a migrating cell can be the
result of polymerization/depolymerization of the actin network,
coupling with the intricate interactions between focal adhesion
complexes, GTPases, and ATPases. Thus it is tempting to postulate that the mechanical forces known to be generated by fibroblasts for cell movement are likely utilized by Sertoli cells to
uplift spermatids, yet this possibility must be vigorously tested
experimentally. No plus end-directed motor protein has been
identified to date in ES, even though it is likely to be a kinesin
family protein (for a review, see Kamal and Goldstein, 2002).
Myosin VIIa, another actin filament-dependent motor protein, is
also found in ES (Velichkova et al., 2002) (Figure 3). As yet,
the functional role of myosin VIIa in spermatid movement is not
known. While the event of spermatid movement across the seminiferous epithelium remains obscure, it is obvious that different
motor proteins, which interact with the underlying cytoskeletons,
play a crucial role. The significance of microtubule-based cell
vertical mobility in the seminiferous epithelium utilizing the ES
has recently been demonstrated in two recent studies in which
rats were transfected with adenovirus carrying g-tubulin inducing overexpression of g-tubulin in Sertoli cells in vivo (Fleming
et al., 2003a,b). For instance, overexpression of g-tubulin in Sertoli cells in vivo led to retention of spermatids and residual
Ectoplasmic specialization dynamics in the testis
bodies in the epithelium by disrupting the events of spermatid
release and residual body elimination (Fleming et al., 2003b), as
well as inducing germ cell apoptosis (Fleming et al., 2003b),
possibly as a result of intracellular trafficking disruption. Needless to say, many open questions remain unanswered. For
instance, unlike fibroblasts or macrophages, Sertoli and germ
cells are not actively migrating cells per se; since the movement
of developing germ cells must co-ordinate with their developmental status, what signal(s) is involved that dictates the timely
movement of germ cells across the epithelium in a given stage
of the epithelial cycle? Also, what is the mechanism that regulates the opening and closing of junctions to facilitate cell movement? Do germ cells elicit any signals to Sertoli cells requesting
the needed uplifting driving force? To answer these questions,
the molecular architecture of the motor proteins regulating the
structural complex and the pertinent signalling and structural
proteins at the site of ES must be delineated. Figure 3 illustrates
a current concept of germ cell movement at the site of ES.
Regulation of ES dynamics
The precise mechanism(s) that regulates ES dynamics is not
known. Yet recent studies have shown that ES can be regulated
by several parameters via different molecules and pathways.
(Figure 4).
Phosphorylation and dephosphorylation
The importance of phosphorylation in the regulation of ES was
implicated by localization studies using antibodies specific to
phosphor-Tyr, -Ser or -Thr residues, which yielded a pattern of
localization, resembling the location of ES in the seminiferous
epithelium (Wine and Chapin, 1999; Chapin et al., 2001;
Mulholland et al., 2001; Maekawa et al., 2002). These studies
have shown that many of the ES constituent proteins are phosphorylated. In addition, several PTK, such as c-Src, Csk and
FAK, and serine/threonine kinase, such as ILK, are also localized
to the ES (Wine and Chapin, 1999; Mulholland et al., 2001; Siu
et al., 2003). Equally important, the putative substrates of protein
kinases, such as paxillin and p120ctn, are also found at the same
site (Wine and Chapin, 1999). Collectively, these findings implicate the role of phosphorylation and dephosphorylation in the
regulation of ES dynamics. For instance, the non-phosphorylated
form of FAK was found to be restricted to the basal ES, whereas
most of the pFAK-Tyr397 was localized at the apical ES (Siu
et al., 2003), suggesting that there is a rapid intracellular trafficking system in the seminiferous epithelium to facilitate such intracellular protein compartmentalization. Future research must
include the identification of downstream pathway(s) following
phosphorylation of specific target proteins by kinases.
Calcium ions
The importance of calcium ions in maintaining cadherinmediated adhesion function is well documented (for a review,
see Steinberg and McNutt, 1999). Furthermore, calcium ions can
activate gelsolin activity, changing it from an inactive state to an
active state, inducing its inherent actin filament-severing ability
(for a review, see Kwiatkowski, 1999). Also, hydrolysis of
PI(4,5)P2 by PLCg releases calcium ions from ER at the site
of ES, subsequently stimulating gelsolin activity (Guttman et al.,
2002).
GTPases
RhoB, Rac1, Cdc42 and Rab8B are found at the site of ES in
the testis (Chapin et al., 2001; Lau and Mruk, 2003; Lui et al.,
2003b). In light of their roles in intracellular trafficking in other
epithelia, GTPases are likely to be important molecules in ES
dynamics; however, much work is needed in this area to define
the significance of GTPases in the ES. Indeed, recent studies
have shown that ES dynamics are regulated, at least in part, by
the integrin/RhoB GTPases/ROCK/LIM kinase/cofilin signalling
pathway since the use of an inhibitor specific to ROCK can
indeed perturb the cell adhesive function between elongating/
elongated spermatids and Sertoli cells in the seminiferous epithelium (Lui et al., 2003b). Furthermore, recent studies from
different laboratories have shown that many effectors and regulatory molecules pertinent to GTPase functioning are found in
the testis, which are associated specficially with either Sertoli or
germ cells (for a review, see Lui et al., 2003a). This is certainly
an area of research that deserves much attention.
Actin-severing proteins
An efficient mechanism to induce rapid cell – cell dissociation is
by disrupting the underlying cytoskeletons in the Sertoli cell
side at the site of ES since actin is being used (Pelletier et al.,
1999). As the main attachment site for ES-associated structural
protein complexes, such as the cadherin/catenin, nectin/afadin
and integrin/laminin (see above) molecules that regulate ES
dynamics. Gelsolin (Guttman et al., 2002) and scinderin are
putative actin-severing proteins found at the site of ES; collectively, these results suggest the importance of actin-severing proteins and their regulators in ES dynamics.
AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide]:
a potential male contraceptive
Whereas multi-protein complexes, such as cadherin/catenin,
nectin/afadin and integrin/laminin, that confer cell adhesion
function, are found at the site of ES in the seminiferous epithelium (for reviews, see Cheng and Mruk, 2002; Takai and
Nakanishi, 2003), it is not yet known how these proteins affect
ES dynamics. Obviously, one of the possible mechanisms is
mediated by changes in the interactions between these protein
complexes and the underlying actin-based cytoskeletal network
regulated by some of the known (or yet-to-be-defined) signal
transduction pathways. This in turn will determine the cell
adhesion function of these protein complexes, regulating ES
dynamics. If this information is known, ES can become a prime
target for male contraceptive development. For instance, one can
perturb cell adhesion function at this site by compromising a key
signalling molecule, inducing premature loss of developing elongating/elongated spermatids from the epithelium without affecting the hypothalamic –pituitary –testicular axis. To address this
open question, an AJ restructuring in vivo model was recently
developed in which AF-2364 was used to identify the downstream ES-regulating pathways (Figure 4).
361
N.P.Y.Lee and C.Y.Cheng
AF-2364 is a novel chemical entity having potent aspermatogenic effects in male rats, apparently exerting its effects primarily on the anchoring junctions, namely the ES the TBC, and
desmosome-like junctions, by inducing germ cell loss from the
seminiferous epithelium (Cheng et al., 2001; Grima et al.,
2001). This conclusion was reached based on a recently completed study to assess the kinetics of germ cell loss following
AF-2364 treatment. For instance, it was shown that in adult rats
treated with a single dose of AF-2364 (50 mg/kg body weight by
gavage), elongated/elongating spermatids were depleting from
the seminiferous epithelium with a half-time of , 6.5 h (i.e. half
of the tubules scored had elongating/elongated spermatids in the
tubule lumen when compared to normal testes) versus 3 day and
6.5 day for round spermatids and spermatocytes respectively,
when the loss of these germ cells from the epithelium was
tracked by morphometric analysis (Chen et al., 2003). These
results thus support the notion that the apical ES (and perhaps
the apicial TBC) between elongating/elongated spermatids and
Sertoli cells apparently is the most susceptible AJ structure to
the AF-2364 treatment, to be followed by the desmosome-like
junctions at the interface between round spermatids/spermatocytes and Sertoli cells (Chen et al., 2003). Indeed, this postulate
was supported by a recent electron microscopy study which has
demonstrated a disruption of the Sertoli-germ cell anchoring
junctions in the seminiferous epithelium of the rat testis within
4 h after AF-2364 treatment (50 mg/kg body weight, by gavage)
(Lee et al., 2004). Most importantly, cell adhesion function in
other organs, such as kidney, liver and brain, apparently was not
affected by AF-2364 since histological analysis of these organs
at the time when tubules were virtually devoid of germ cells had
shown that the epithelia in these organs remained relatively
intact (Grima et al., 2001). Obviously, one can argue it is
equally plausible that the AF-2364-induced germ cell loss from
the epithelium is mediated via other mechanism(s), such as germ
cell death or simply a direct toxic effect, with secondary effects
on cell junctions, such as the ES, the apical TBC or the desmosome-like junctions. Yet recent findings seem to argue against
such possibilities. First, when Sertoli cells were cultured alone
in vitro or co-cultured with germ cells in chemically defined
media, such as Ham’s F12/Dulbecco’s modified Eagle’s medium, addition of AF-2364 between 1 and 500 ng/ml could induce
the production of b1-integrin and vinculin (but AF-2364 had no
effects on germ cell vinculin level when added to germ cells cultured alone and had no effects on germ cell viability) dose
dependently (Siu et al., 2003), mimicking the response observed
in vivo when rats were treated with AF-2364 by gavage (Siu
et al., 2003). Yet the DNA content and the cell viability (monitored by Trypan Blue staining and Erythrosine Red dye exclusion test to assess Sertoli and germ cell viability respectively) of
these cultures at the end of the experiment remained unaltered
with and without AF-2364 treatment (Siu et al., 2003),
suggesting that AF-2364 is non-toxic to either Sertoli or germ
cells at doses that could induce b1-integrin production. Likewise, AF-2364 can induce RhoB GTPase protein production in
Sertoli-germ cell co-cultures without affecting cell viability,
similar to its effects in vivo (Lui et al., 2003b). This argument is
further strengthened by the observation that the blood–testis
barrier in rats treated with AF-2364 was not damaged when
assessed by fluorescent microscopy at the time when virtually all
362
the elongating/elongated/round spermatids and spermatocytes
were depleted from the seminiferous epithelium (Mruk and
Cheng, 2004). Second, electron microscopy studies have shown
that germ cells indeed began to dislodge from Sertoli cells in the
seminiferous epithelium within 4 h after receiving a single dose
of AF-2364 (50 mg/kg body weight by gavage) in adult rats by
developing tiny intercellular spaces at the interface between
round spermatids and Sertoli cells in the seminiferous epithelium, which are not found in the normal testis (Lee et al.,
2004), further implicating the site of action of AF-2364 as being
at the cell –cell interface. Furthermore, these rats were recovered
fully within 250 days after the AF-2364 treatment, where the
seminiferous epithelium was indistinguishable from normal rat
testes (Cheng et al., 2001; Grima et al., 2001), suggesting that
the loss of germ cells from the epithelium is not likely secondary
to a toxic effect on cells in the seminiferous epithelium. Third,
following treatments of adult rats with AF-2364 by gavage using
different regimens, there is a lag of ,20 –30 days before infertility can be detected, suggesting that this compound has no
apparent effects on the sperm reserve in the epididymis (Cheng
et al., 2001; Grima et al., 2001). If AF-2364 is indeed toxic to
germ cells, such as epididymal sperm found in the epididymis,
its infertility effects would have been detected much sooner than
20 days. Furthermore, more recent biochemical studies have
shown that AF-2364 may affect the production of adaptor proteins at the ES site, such as zyxin and axin, failing to recruit the
needed proteins to the ES site to maintain its integrity, thereby
causing germ cell loss from the epithelium (Lee et al., 2004).
Fourth, serum microchemistry analysis to monitor hepatotoxicity
[liver function tests: levels of serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT),
and alkaline phosphatase] and nephrotoxicity [kidney function
tests: serum levels of blood urea nitrogen (BUN), creatinine,
albumin, g-globulins, phosphorus, calcium and potassium] has
shown that rats treated with AF-2364 at doses effective to induce
reversible male infertility in rats displayed no alteration in both
liver and kidney functions, as well as no significant changes in
serum testosterone, FSH, and LH levels (Cheng et al., 2001;
Grima et al., 2001). This latter observation thus suggests that the
hypothalamic –pituitary –testicular hormonal axis is not affected
by AF-2364-induced male infertility. However, it remains to be
determined whether AF-2364 treatment can alter the testicular
androgen levels, such as in the seminiferous tubule fluid and rete
testis fluid, or other yet-to-be-defined testicular function.
Although it is unlikely that AF-2364 can alter the intratesticular
androgen microenvironment, since it had no apparent effects on
the serum testosterone, FSH and LH levels (Cheng et al., 2001;
Grima et al., 2001), if it did, this might indicate that AF-2364
may exert its effects by disrupting the microenvironment in the
epithelium. This can in turn affect cell attachment in the seminiferous epithelium. Lastly, pathology analysis of tissues, including liver, kidney, prostate, brain and others have shown no signs
of damage to epithelia in these organs except for the seminiferous epithelium in the testis (Grima et al., 2001). Furthermore,
recent in vitro studies have shown that AF-2364 only affected
germ cell adhesion function onto Sertoli cell epithelium without
affecting Sertoli cell tight junction-barrier permeability (Mruk
and Cheng, 2004). Taken collectively, these results appear to
suggest that the multi-protein complexes at the site of ES may
Ectoplasmic specialization dynamics in the testis
act as a receptor complex for AF-2364 and since other epithelia
are lacking these ES structures, cell adhesive function in these
epithelia is not affected by AF-2364 treatment. It is obvious that
this postulate should be vigorously tested in future studies by
targeted depletion of genes that encode ES structural protein
complexes, such as with the use of RNA silencing technique or
antisense oligos, to assess whether their transient depletion can
render the loss of responses to AF-2364. Perhaps it is equally
plausible that AF-2364 exerts its effects by affecting other testicular factors (e.g. androgens) that are needed for the maintenance of germ cell attachment onto the seminiferous epithelium.
This is why an assessment of the intratesticular androgen level
in AF-2364-treated rats as described above is worthy of research.
Nonetheless, in light of these findings, this in vivo model was
used to investigate the underlying signalling molecules that regulate AJ structuring in the testis.
The integrin/pFAK/PI 3-kinase/p130 Cas/c-Jun N-terminal kinase
(JNK) pathway
Interestingly, the protein levels of b1- and b2-integrin were
induced during AF-2364-mediated AJ disruption in the testis
following treatment of rats with AF-2364 (50 mg/kg body
weight) by gavage (Lui et al., 2003b; Siu et al., 2003),
suggesting an activation of integrin. This result also suggests
that integrin is one of the molecules upstream by which
AF-2364 exerts its effects in the seminiferous epithelium. It is
obvious that future studies should include an assessment on the
phosphorylation status of b1-integrin (and possibly the precise
location of activation via phosphorylation) in the seminiferous
epithelium of the rat testis during AF-2364-induced AJ
disruption. Nonetheless, this induction of integrin was also
accompanied by an induction of the downstream signal transducers including pFAK-Tyr397, phosphatidylinositol 3-kinase (PI
Figure 4. A current molecular model for the regulation of adherens junction (AJ) dynamics at the site of ectoplasmic specialization (ES) in the testis. In brief,
the AJ dynamics at the ES site can be regulated by kinases, phosphatases and GTPases (for a review, see Cheng and Mruk, 2002). In addition, an optimal calcium ion [Ca2 þ ] level is required for the normal functioning of cadherins (for a review, see Steinberg and McNutt, 1999). Besides, gelsolin, an actin-severing
protein, can regulate ES dynamics by cleaving the actin filament at the site of ES, causing ES opening (Guttman et al., 2002). It is known that the integrity of
the integrin and the cadherin-mediated protein complexes and the functionality of their underlying actin network at the site of ES are regulated by at least three
signalling pathways: (I) integrin b1/pFAK/PI 3-kinase via MAP kinase or p130 Cas (Siu et al., 2003), (II) integrin b1 or b2/RhoB/ROCK/LIMK/cofilin (Lui
et al., 2003b), and (III) the Rab8B GTPase-mediated signalling pathway (Lau and Mruk, 2003).
363
N.P.Y.Lee and C.Y.Cheng
3-kinase) and protein encoded by Crkas gene also called Crk-associated protein (p130 Cas) (Siu et al., 2003). As such, these
results seemingly suggest that AF-2364 induces integrin, which in
turn activates pFAK and its downstream effector proteins, which
can in turn affect the actin-based cytoskeletal network (Siu et al.,
2003) via the JNK signalling pathway (Siu and Cheng, 2004;
unpublished observations). Collectively, these results illustrate
that the integrin/pFAK/PI 3-kinase/p130 Cas/JNK mitogen-activated protein (MAP) kinase is likely one of the AJ regulatory
pathways operating at the site of ES (Figure 4).
The integrin/RhoB/ROCK/LIMK/cofilin pathway
The levels of b1- and b2-integrins, RhoB, ROCK, LIMK and
cofilin were also induced during AF-2364-mediated AJ disruption in the testis (Lui et al., 2003b), suggesting that this is yet
another putative signalling pathway that regulates ES dynamics.
This is because an activation of cofilin, a calcium-independent
actin-severing (or -depolymerizing) protein, can change the
intracellular F-actin levels thereby inducing loss of cell adhesive
function (Lui et al., 2003b). Perhaps the most important of all,
when Y-27632, a ROCK-specific inhibitor, is administered
in vivo prior to AF-2364 treatment by blocking ROCK, it can
effectively delay the AF-2364-induced elongated/elongating
spermatid loss from the seminiferous epithelium (Lui et al.,
2003b). The study using Y-27632 clearly confirms that the testis
is using the RhoB/ROCK/LIMK pathway to regulate ES
dynamics (Figure 4). Furthermore, AF-2364 had no apparent
effects on the RhoB protein levels in liver, brain and kidney in
contrast to the testis, which was significantly induced by AF2364 treatment (Lui et al., 2003b). This result thus explains that
the lack of response of cell adhesion molecules to AF-2364 outside the seminiferous epithelium is due to the lack of the protein
complexes unique to the apical ES or the tubulobulbar complex
found between elongating/elongated spermatids and Sertoli cells
in the seminiferous epithelium of the rat testis. In this context, it
is of interest to note that a recent study has shown that cofilin is
specifically localized to the tubulobulbar complex site (a testisspecific AJ type) between elongating/elongated spermatids and
Sertoli cells in the seminiferous epithelium of the rat testis
(Guttman et al., 2004). In short, these data illustrate that
AF-2364 exerts its effects at the AJ sites between elongating/
elongated spermatids and Sertoli cells via the integrin/RhoB/
ROCK/LIMK/cofilin pathway.
Remarks
Using the AF-2364-induced germ cell loss from the epithelium,
it was shown that ES dynamics are regulated by two putative
signalling pathways as reviewed herein. Yet these results must
now be validated using another in vivo model of AJ disruption.
It is obvious that the androgen depletion-induced loss of spermatids from the epithelium in the rat (for a review, see McLachlan
et al., 2002) can be a useful model to reassess these recent findings. Once these results are confirmed, some of the signalling
molecules in these pathways can also become the prime targets
of male contraceptive development by compromising cell
adhesion function in the seminiferous epithelium.
364
Can ES be a target for male contraceptive development?
As briefly reviewed and discussed above, the ES in the seminiferous epithelium apparently is the primary target of AF-2364
when it is administered orally. This compound apparently exerts
its effects by first activating integrin (Lui et al., 2003b; Siu et al.,
2003), cadherins, catenins (Chen et al., 2003; Lee et al., 2003)
and testin (Cheng et al., 2001) (and possibly other yet-to-beidentified proteins) in the seminiferous epithelium at the site of
the apical ES and/or the tubulobulbar complex, which in turn
can activate the downstream Rho B/ROCK/LIM kinase/cofilin
(Lui et al., 2003b) or the pFAK/PI 3-kinase/p130Cas/JNK signalling pathway (Siu et al., 2003; Siu and Cheng, 2004). In this
context, it is of interest to note that unless AF-2364 can freely
diffuse into the seminiferous epithelium across the BTB or it
enters the seminiferous epithelium by non-receptor-mediated
endocytosis, its entry into the epithelium has to be receptormediated (note: AF-2364 does not apparently exert its effect as a
toxicant based on the recently published reports summarized in
previous Section). While the precise molecular architecture of
the multi-protein complex that can act as a possible receptor-like
protein complex for AF-2364 is not known, testin is possibly the
ES-protein that provides the necessary linkage (or cross-talk?)
between the integrin/laminin and cadherin/catenin protein complexes at the ES site. This postulate was reached because of
some of the unusual features of testin in the seminiferous epithelium. First, testin is localized to the heads of elongating/elongated spermatids at the site of apical ES in the seminiferous
epithelium (Zong et al., 1992; Grima et al., 1995), and is confined to the Sertoli cell side when visualized by immunogold
electron microscopy technique (Grima et al., 1997). Also, the
expression of testin is the highest at early stage VIII just prior to
spermiation (Zong et al., 1994). Second, testin can be induced
by several orders of magnitude in the testis when the Sertoligerm cell adhesion function (but not the Sertoli –Sertoli tight
junction function) is disrutped (Cheng et al., 1989; Grima et al.,
1997, 1998; Grima and Cheng, 2000), suggesting that it is an
important signalling molecule at the Sertoli-germ cell AJ site.
Furthermore, an induction of testin is noted during AF-2364induced germ cell loss from the epithelium, yet this induction
occurs at the time before any physical damage to the epithelium
is detected (Cheng et al., 2001; Chen et al., 2003; Lee et al.,
2003). Third, and perhaps the most important of all, recent
studies by immunoprecipitation have shown that testin is physically associated with E-cadherin and b1-integrin, the corresponding integral membrane proteins of the cadherin/catenin and
the a6b1-integrin/laminin g3 protein complexes (unpublished
observations). Obviously, this hypothesis must be vigorously
tested in future studies such as with the use of cross-linkers, fluorescent microscopy, electron microscopy, RNA silencing technology, and pertinent biochemical and molecular analyses. In
this context, it is of interest to note that, besides testin, other
yet-to-be-identified proteins may also provide the linkage
between the multi-protein complexes at the ES site, such as ponsin. For instance, ponsin is known to interact with both afadin
and vinculin, and vinculin is part of the cadherin–catenin protein complex in many epithelia (Weiss et al., 1998; Mandai
et al., 1999). Indeed, it has been proposed that ponsin and perhaps vinculin provide the functional cross-talk and physical link-
Ectoplasmic specialization dynamics in the testis
age between the cadherin/catenin and the nectin/afadin protein
complexes in cell epithelium at the AJ site (Mandai et al.,
1999). Nonetheless, based on this information, we reason that
AF-2364 fails to activate integrin and/or cadherin found in other
organs (Cheng et al., 2001; Grima et al., 2001; Lui et al.,
2003b; Siu et al., 2003), such as the brain, kidney and liver, and
that this is likely due to the lack of testin in these organs (note:
integrins and many other components of the ES proteins, such as
vinculin and ponsin but not testin, are found in these other
organs). Once the upstream protein complex(es) is activated by
AF-2364, the signalling events can go downstream via either
one of the two pathways as described above. The fact that the
use of Y-27632, a ROCK inhibitor, can effectively delay the
AF-2364-induced germ cell loss from the epithelium (Lui et al.,
2003b) has clearly illustrated the significance of this integrin/RhoB/ROCK/LIM kinase/cofilin signalling pathway in regulating ES dynamics (Figure 4). A more recent finding has suggested
that AF-2364 can also activate Rab8B GTPase (Lau and Mruk,
2003), which is known to be crucial to intracellular trafficking
pertinent to AJ regulation. Ironically, the signalling molecules in
this pathway (Lui et al., 2003b) and those found in the integrin/pFAK/PI 3-kinase/p130cas/JNK pathway (Siu et al., 2003; Siu
and Cheng, 2004) (Figure 4), and possibly the downstream effector proteins of Rab8B GTPase (Lau and Mruk, 2003) can now be
tackled to perturb cell adhesion function at the site of ES,
thereby inducing premature detachment of elongating/elongated
spermatids from the epithelium. Needless to say, it is crucial that
these three signalling pathways be vigorously investigated using
other in vivo models of AJ disruption. In the context, it is of
interest to note that a photoaffinity biotinylated analogue of lonidamine [1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid],
which is also an analogue of AF-2364, was recently shown to
bind to the stress fibres of Sertoli cells (Wulser et al., 2003), consistent with its association with the ES and/or tubulobulbar complex structure. In short, these findings have shown that AF-2364
is a candidate compound to perturb cell adhesion function at the
ES and/or the tubulobulbar complex, illustrating the feasibility
that ES can be a prime target for male contraceptive development (Cheng et al., 2001; Grima et al., 2001).
Apparently, the use of estrogen and estrogen/androgen
implants to induce loss of spermatid binding to the epithelium
in vivo (for reviews, see Cheng and Mruk, 2002; McLachlan
et al., 2002) is an obvious choice to validate the three signalling
pathways that regulate ES dynamics. For instance, androgen
depletion in rats using estrogen/androgen implants can induce
sloughing of spermatids from step 8 and beyond, suggesting that
the function of ES is maintained by androgens (McLachlan et al.,
1994; O’Donnell et al., 2000) (for a review, see McLachlan
et al., 2002). However, it must be noted that in these earlier
studies, the ES structures between Sertoli cells and spermatids
apparently remained normal, at least at the microscopic level
(O’Donnell et al., 2000), suggesting that while ES structures are
preserved, their cell adhesion functionality is lost. Yet it is of
interest to note that sloughing of spermatids does not occur in
humans receiving similar treatments, suggesting that there may
be species differences in the regulation of spermatid adhesion in
the epithelium (for a review, see McLachlan et al., 2002). Interestingly, a more recent study using testosterone pretreatment for
1 week, followed by estrogen/testosterone implants plus FSH
antibody administration for another week, can induce retainment
instead of dislodging of elongated spermatids in the epithelium
(Beardsley and O’Donnell, 2003). This apparently is mediated
by perturbing the final disengagement of spermatids from Sertoli
cells at stage VIII involving b1-integrin but not integrin-linked
kinase (ILK) without affecting the ES structures at the light and
electron microscopic levels (Beardsley and O’Donnell, 2003),
thereby inducing spermatid retainment in the epithelium. In
short, much work is needed to define the role of hormones, such
as androgens and FSH, on the integrity, maintenance and functioning of ES in the epithelium, and how the interactions of
different proteins at the ES site confer cell adhesion function.
Conclusion and future perspectives
ES is a specialized testis-specific AJ type. Cadherins, integrins,
laminins and nectins are the integral membrane proteins at the
ES site (Table II). These proteins in turn interact with other
adaptors, signalling proteins and cytoskeletal proteins, conferring
structural and adhesive function, and regulate germ cell movement (Table II). They also act as a platform for signal transduction. As such, a highly coordinated yet complex biochemical
system is maintained at the site of ES to fine-tune the complicated process of spermatid movement in the seminiferous epithelium via the timely association and dissociation of the ES
integral membrane proteins, adaptors, signalling molecules and
cytoskeletal proteins. This mechanism is also important to spermiation during which rapid junction restructuring occurs to
release sperm into the tubule lumen. Yet most of the studies conducted thus far are limited to the constituent proteins of the
ES-associated protein complexes, so less is known about the
regulatory mechanism(s) utilized by the ES to regulate these
events. Even less is known regarding the structural composition
of the tubulobulbar complex and its regulation. It is obvious that
future studies should include better designs of experiments in
which the coordinated changes of multiple target proteins at the
site of ES are assessed instead of limiting to a single or a single
set of molecules. This can possibly be achieved using Sertoligerm cell co-cultures in vitro as a model to study ES dynamics
in initial and preliminary experiments. Once positive results are
identified, these can be further expanded to the in vivo level for
validation. For instance, it is now known that functional ES
(Cameron and Muffly, 1991; Lui et al., 2004; Mruk and Cheng,
2004) and desmosome-like junctions (Cameron and Muffly,
1991; Enders and Millette, 1988; Lui et al., 2004; Mruk and
Cheng, 2004) are formed in these cocultures in vitro. Indeed, we
have used this in vitro system in conjunction with the AF-2364
in vivo model to demonstrate that Sertoli-germ cell AJ dynamics
are regulated by the intricate interactions of proteases and protease inhibitors (Mruk et al., 1997, 2003; Siu and Cheng, 2004).
For instance, recent in vivo studies (Siu and Cheng, 2004) have
validated earlier in vitro results (Mruk et al., 1997, 2003) regarding the significance of proteases and protease inhibitors in
Sertoli-germ cell AJ dynamics. For instance, a blockage of the
metalloprotease activity by specific inhibitors can indeed inhibit
the AF-2364-induced germ cell loss from the seminiferous
epithelium in adult rats (Siu and Cheng, 2004). Furthermore, a
more recent study using this in vitro system has demonstrated
the significance of interactions between IQGAP1, Cdc42, and
365
N.P.Y.Lee and C.Y.Cheng
the cadherin/catenin complex in the regulation of Sertoli-germ
cell AJ dynamics (Lui et al., 2004). These results can now be
applied to the in vivo level. Using such an approach, we can
limit the unnecessary use and suffering of live animals. Needless
to say, many open questions remain unanswered. For instance:
How do the three known protein complexes in the ES shown in
Figure 1 cross-talk to each other, co-ordinating the events pertinent to germ cell movement (Figures 3 and 4)? What are the
protein complexes that constitute the basal ES and their molecular architecture versus the apical ES? What are the differences in
the biochemical/molecular composition between the ES and the
tubulobulbar complex? If AF-2364 exerts its effects primarily at
the apical ES without affecting cell adhesion function in other
epithelia, what is the nature and/or architecture of the integrin/
testin/cadherin protein complex that may possibly act as a
receptor-like protein complex for AF-2364? While Figure 1
summarizes the most recent molecular architecture of the
ES-integral membrane protein complexes and their peripheral
signalling and adaptor proteins (see also Table II), this information will undoubtedly be updated rapidly. Furthermore, are
there any cross-talks between the known mechanical (i.e. involving motor proteins, see Figure 3) and signalling pathways
(i.e. involving signal transducers, see Figure 4) that regulate ES
dynamics? This review has discussed some of the recent findngs
in the field, some of which can become the prime subject areas
for future research. Also, many molecules reviewed herein can
be the target of active male contraceptive research. For instance,
a specific blockade of the key signalling molecules in the RhoB
or Rab8B pathway is likely to compromise ES adhesion function
in the testis.
Acknowledgements
We are indebted to the thoughtful and stimulating comments and
discussion of Dr Dolores Mruk during the preparation of this
review. While every effort was made to cite important contributions
of our colleagues and other investigators in the field, many important articles and work were not cited and discussed because of page
limit, and we ask for their understanding. This work was supported
in part by grants from the National Institutes of Health (NICHD,
U01 HD45908 to C.Y.C.; U54 HD29990, Project 3 to C.Y.C.), the
CONRAD Program (CICCR CIG-96-05-B and CIG-01-72 to
C.Y.C.), and the Noopolis Foundation.
References
Amlani S and Vogl AW (1988) Changes in the distribution of microtubules
and intermediate filaments in mammalian Sertoli cells during spermatogenesis. Anat Rec 220,143–160.
Anastasiadis PZ and Reynolds AB (2000) The p120 catenin family: complex
roles in adhesion, signaling and cancer. J Cell Sci 113,1319–1334.
Balda MS and Anderson J (1993) Two classes of tight junctions are revealed
by ZO-1 isoforms. Am J Physiol 264,C918–C924.
Bartles JR, Wierda A and Zheng L (1996) Identification and characterization
of espin, an actin-binding protein localized to the F-actin-rich junctional
plaques of Sertoli cell ectoplasmic specializations. J Cell Sci 109,
1229–1239.
Beach SF and Vogl AW (1999) Spermatid translocation in the rat seminiferous epithelium: coupling membrane trafficking machinery to a junction
plaque. Biol Reprod 60,1036– 1046.
Beardsley A and O’Donnell L (2003) Characterization of normal spermiation
and spermiation failure induced by hormone suppression in adult rats.
Biol Reprod 68,1299–1307.
Bjorge JD, Jakymiw A and Fujita DJ (2000) Selected glimpses into the activation and function of Src kinase. Oncogene 19,5620–5635.
366
Bouchard MJ, Dong Y, McDermott BM, Lam DH, Brown KR, Shelanski M,
Bellve AR and Racaniello VR (2000) Defects in nuclear and cytoskeletal morphology and mitochondrial localization in spermatozoa of
mice lacking nectin-2, a component of cell–cell adherens junctions. Mol
Cell Biol 20,2865– 2873.
Brady-Kalnay SM, Rimm DL and Tonks NK (1995) Receptor protein tyrosine phosphatase PTPm associates with cadherins and catenins in vivo.
J Cell Biol 130,977–986.
Byers SW, Jegou B, MacCalman C and Blaschuk OW (1993) Sertoli cell
adhesion molecules and the collective organization of the testis. In
Russell LD and Griswold MD (eds) The Sertoli Cell. Cache River Press,
Clearwater, FL, pp 461–476.
Byers SW, Sujarit S, Jegou B, Butz S, Hoschutzky H, Herrenknecht K,
MacCalman C and Blaschuk OW (1994) Cadherins and cadherin-associated molecules in the developing and maturing rat testis. Endocrinology
134,630–639.
Cameron DF and Muffly KE (1991) Hormonal regulation of spermatid
binding. J Cell Sci 100,623– 633.
Chapin RE, Wine RN, Harris MW, Borchers CH and Haseman JK (2001)
Structure and control of a cell –cell adhesion complex associated with
spermiation in rat seminiferous epithelium. J Androl 22,1030–1052.
Chen B, Li A, Wang D, Wang M, Zheng L and Bartles JR (1999) Espin contains an additional actin-binding site in its N terminus and is a major
actin-bundling protein of the Sertoli cell –spermatid ectoplasmic specialization junctional plaque. Mol Biol Cell 10,4327–4339.
Chen YM, Lee NPY, Mruk D, Lee WM and Cheng CY (2003) Fer kinase/
FerT and adherens junction dynamics in the testis: an in vitro and in vivo
study. Biol Reprod 69,656– 672.
Chen Z, Hasson T, Zhang D, Schwender BJ, Derfler BH, Mooseker MS and
Corey DP (2001) Myosin-VIIb, a novel unconventional myosin, is a
constituent of microvilli in transporting epithelia. Genomics 72,
285–296.
Cheng CY and Mruk DD (2002) Cell junction dynamics in the testis: Sertoligerm cell interactions and male contraceptive development. Physiol Rev
82,825–874.
Cheng CY, Grima J, Stahler MS, Lockshin RA and Bardin CW (1989) Testins are two structurally related Sertoli cell secretory proteins whose
secretion is tightly coupled to the presence of germ cells. J Biol Chem
264,21386–21393.
Cheng CY, Silvestrini B, Grima J, Mo MY, Zhu LJ, Johansson E, Saso L,
Leone MG, Palmery M and Mruk D (2001) Two new male contraceptives exert their effects by depleting germ cells prematurely from the
testis. Biol Reprod 65,449–461.
Chin KV, Yang WL, Ravatn R, Kita T, Reitman E, Vettori D, Cvijic ME,
Shin M and Iacono L (2002) Reinventing the wheel of cyclic AMP.
Novel mechanisms of cAMP signaling. Ann NY Acad Sci 968,49–64.
Collins CA and Vallee RB (1989) Preparation of microtubules from rat liver
and testis: cytoplasmic dynein is a major microtubule associated protein.
Cell Motil Cytoskeleton 14,491– 500.
Colognato H and Yurchenco PD (2000) Form and function: the laminin
family of heterotrimers. Dev Dyn 218,213– 234.
Colucci-Guyon E, Portier MM, Dunia I, Paulin D, Pournin S and Babinet C
(1994) Mice lacking vimentin develop and reproduce without an obvious
phenotype. Cell 79,679–694.
Cowin P, Kapprell HP, Franke WW, Tamkun J and Hynes RO (1986) Plakoglobin: a protein common to different kinds of intercellular adhering
junctions. Cell 46,1063–1073.
Craig AWB, Zirngibl R, Williams K, Cole LA and Greer PA (2001) Mice
devoid of fer protein-tyrosine kinase activity are viable and fertile but
display reduced cortactin phosphorylation. Mol Cell Biol 21,603– 613.
de Franca LR, Ghosh S, Ye SJ and Russell LD (1993) Surface and surfaceto-volume relationships of the Sertoli cell during the cycle of the seminiferous epithelium in the rat. Biol Reprod 49,1215–1228.
de Kretser DM and Kerr JB (1988) The cytology of the testis. In Knobil E
and Neill J (eds) The Physiology of Reproduction. Raven Press, New
York, pp 837 –932.
Enders GC and Millette CF (1988) Pachytene spermatocyte and round spermatid binding to Sertoli cells in vitro. J Cell Sci 90,105–114.
Fischman K, Edman JC, Shackleford GM, Turner JA, Rutter WJ and Nir U
(1990) A murine fer testis-specific transcript (ferT) encodes a truncated
Fer protein. Mol Cell Biol 10,146–153.
Fleming SL, Shank PR and Boekelheide K (2003a) g-Tubulin overexpression
in Sertoli cells in vivo I. Localization to sites of spermatid head attachment and alterations in Sertoli cell microtubule distribution. Biol Reprod
69,310–321.
Ectoplasmic specialization dynamics in the testis
Fleming SL, Shank PR and Boekelheide K (2003b) g-Tubulin overexpression
in Sertoli cells in vivo II. Retention of spermatids, residual bodies, and
germ cell apoptosis. Biol Reprod 69,322–330.
Georges-Labouesse E, Messaddeq N, Yehia G, Cadalbert L, Dierich A and
Le Meur M (1996) Absence of integrin a6 leads to epidermolysis bullosa and neonatal death in mice. Nat Genet 13,370– 373.
Golenhofen N and Drenckhahn D (2000) The catenin, p120ctn, is a common
membrane-associated protein in various epithelial and non-epithelial
cells and tissues. Histochem Cell Biol 114,147–155.
Greer P (2002) Closing in on the biological functions of fps/fes and fer. Nat
Rev Mol Cell Biol 3,278–289.
Grima J and Cheng CY (2000) Testin induction: the role of cyclic 30 , 50 adenosine monophosphate/protein kinase A signaling in the regulation
of basal and lonidamine-induced testin expression by rat Sertoli cells.
Biol Reprod 63,1648–1660.
Grima J, Zhu LJ, Zong SD, Catterall JF, Bardin CW and Cheng CY (1995)
Rat testin is a newly identified component of the junctional complexes
in various tissues whose mRNA is predominatly expressed in the testis
and overy. Biol Reprod 52,340–355.
Grima J, Zhu LJ and Cheng CY (1997) Testin is tightly associated with testicular cell membrane upon its secretion by Sertoli cells whose steadystate mRNA level in the testis correlates with the turnover and integriy
of inter-testicular cell junctions. J Biol Chem 272,6499–6509.
Grima J, Wong CSC, Zhu LJ, Zong SD and Cheng CY (1998) Testin
secreted by Sertoli cells is associated with the cell surface and its
expression correlates with the disruption of Sertoli–germ cell junctions
but not the inter-Sertoli tight junction. J Biol Chem 273,21040–21053.
Grima J, Silvestrini B and Cheng CY (2001) Reversible inhibition of spermatogenesis in rats using a new male contraceptive, 1-(2,4-dichlorobenzyl)indazole-3-carbohydrazide. Biol Reprod 64,1500–1508.
Grove BD and Vogl AW (1989) Sertoli cell ectoplasmic specializations: a
type of actin-associated adhesion junction? J Cell Sci 93,309–323.
Gumbiner BM (2000) Regulation of cadherin adhesive activity. J Cell Biol
148,399–404.
Guttman J, Kimel GH and Vogl AW (2000) Dynein and plus-end microtubule-dependent motors are associated with specialized Sertoli cell junction plaques (ectoplasmic specializations). J Cell Sci 113,2167–2176.
Guttman J, Janmey P and Vogl AW (2002) Gelsolin-evidence for a role in
turnover of junction-related actin filaments in Sertoli cells. J Cell Sci
115,499–505.
Guttman J, Obinata T, Shima J, Griswold M and Vogl AW (2004) Nonmuscle cofilin is a component of tubulobulbar complexes in the testis.
Biol Reprod 70,805–812.
Haegel H, Larue L, Ohsugi M, Fedorov L, Herrenknecht K and Kemler R
(1995) Lack of b-catenin affects mouse development at gastrulation.
Development 121,3529–3537.
Hall ES, Eveleth J, Jiang C, Redenbach DM and Boekelheide K (1992) Distribution of the microtubule-dependent motors cytoplasmic dynein and
kinesin in rat testis. Biol Reprod 46,817–828.
Hannigan GE, Leung-Hagesteijn C, Fitz-Gibbon L, Coppolino MG, Radeva
G, Filmus J, Bell JC and Dedhar S (1996) Regulation of cell adhesion
and anchorage-dependent growth by a new b1-integrin-linked protein
kinase. Nature 379,91–96.
Harada A, Takei Y, Kanai Y, Tanaka Y, Nonaka S and Hirokawa N (1998)
Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic
dynein. J Cell Biol 141,51–59.
Hasson T, Heintzelman MB, Santos-Sacchi J, Corey DP and Mooseker MS
(1995) Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type 1B. Proc Natl Acad Sci USA
92,9815–9819.
Hazan B, Bern O, Carmel M, Lejbkowicz F, Goldstein RS and Nir U (1993)
ferT encodes a meiosis-specific nuclear tyrosine kinase. Cell Growth
Differ 4,443–449.
Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell
110,673–687.
Iivanainen A, Morita T and Tryggvason K (1999) Molecular cloning and
tissue-specific expression of a novel murine laminin g3 chain. J Biol
Chem 274,14107– 14111.
Ikeda W, Nakanishi H, Miyoshi J, Mandai K, Ishizaki H, Tanaka M, Togawa
A, Takahashi K, Nishioka H, Yoshida H et al. (1999) Afadin: a key
molecule essential for structural organization of cell–cell junctions of
polarized epithelia during embryogenesis. J Cell Biol 146,1117–1131.
Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S,
Fujimoto J, Okada M and Yamamoto T (1995) Reduced cell motility
and enhanced focal adhesion contact formation in cells from FAKdeficient mice. Nature 377,539–544.
Imamoto A and Soriano P (1993) Disruption of the csk gene, encoding a
negative regulator of Src family tyrosine kinases, leads to neural tube
defects and embryonic lethality in mice. Cell 73,1117– 1124.
Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD and
Yamamoto M (1999) Keap1 represses nuclear activation of antioxidant
responsive elements by Nrf2 through binding to the amino-terminal
Neh2 domain. Genes Dev 13,76–86.
Janssens B, Goossens S, Staes K, Gilbert B, van Hengel J, Colpaert C,
Bruyneel E, Mareel M and van Roy F (2001) aT-Catenin: a novel
tissue-specific b-catenin-binding protein mediating strong cell –cell
adhesion. J Cell Sci 114,3177–3188.
Ji Q, Winnier GE, Niswender KD, Horstman D, Wisdom R, Magnuson MA
and Carpenter G (1997) Essential role of the tyrosine kinase substrate
phospholipase C-g1 in mammalian growth and development. Proc Natl
Acad Sci USA 94,2999–3003.
Johnson AD, Gomes WR and Vandemark NL (1970) The Testis Volume 1:
Development, Anatomy, and Physiology. Academic Press, New York.
Johnson KJ and Boekelheide K (2002a) Dynamic testicular adhesion junctions are immunologically unique. I. Localization of p120 catenin in rat
testis. Biol Reprod 66,983–991.
Johnson KJ and Boekelheide K (2002b) Dynamic testicular adhesion junctions are immunologically unique. II. Localization of classic cadherins
in rat testis. Biol Reprod 66,92–1000.
Kamal A and Goldstein LSB (2002) Principles of cargo attachment to
cytoplasmic motor proteins. Curr Opin Cell Biol 14,63–68.
Kann ML, Soues S, Levilliers N and Fouquet JP (2003) Glutamylated tubulin: diversity of expression and distribution of isoforms. Cell Motil
Cytoskeleton 55,14–25.
Keshet E, Itin ., Fischman K and Nir U (1990) The testis-specific transcript
(ferT) of the tyrosine kinase FER is expressed during spermatogenesis
in a stage-specific manner. Mol Cell Biol 10,5021–5025.
King SM (2000) The dynein microtubule motor. Biochim Biophys Acta
1496,60–75.
Kioka N, Ueda K and Amachi T (2002) Vinexin, CAP/ponsin, ArgBP2: a
novel adaptor protein family regulating cytoskeletal organization and
signal transduction. Cell Struct Funct 27,1–7.
Knudsen KA, Soler AP, Johnson KR and Wheelock MJ (1995) Interaction of
a-actinin with the cadherin/catenin cell–cell adhesion complex via
a-catenin. J Cell Biol 130,67–77.
Koch M, Olson PF, Albus A, Jin W, Hunter DD, Brunken WJ, Burgeson RE
and Champliaud MF (1999) Characterization and expression of the laminin g3 chain: a novel, non-basement membrane-associated, laminin
chain. J Cell Biol 145,605–617.
Kwiatkowski DJ (1999) Functions of gelsolin: motility, signaling, apoptosis,
cancer. Curr Opin Cell Biol 11,103–108.
Landmark BF, Oyen O, Skalhegg BS, Fauske B, Jahnsen T and Hansson V
(1993) Cellular location and age-dependent changes of the regulatory
subunits of cAMP-dependent protein kinase in rat testis. J Reprod Fertil
99,323–334.
Larue L, Ohsugi M, Hirchenhain J and Kemler R (1994) E-cadherin null
mutant embryos fail to form a trophectoderm epithelium. Proc Natl
Acad Sci USA 91,8263–8267.
Lau ASN and Mruk DD (2003) Rab8B GTPase and junction dynamics in the
testis. Endocrinology 144,1549–1563.
Lebre A, Jamot L, Takahashi J, Spassky N, Leprince C, Ravise N, Zander C,
Fujigasaki H, Kussel-Andermann P, Duyckaerts C, Camonis JH and
Brice A (2001) Ataxin-7 interacts with a Cbl-associated protein that it
recruits into neuronal intranuclear inclusions. Hum Mol Genet 10,
1201–1213.
Lee NPY and Cheng CY (2003) Regulation of Sertoli cell tight junction in
the rat testis via the nitric oxide synthase/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway: an in vitro study. Endocrinology 144,3114–3129.
Lee NPY, Mruk DD, Lee WM and Cheng CY (2003) Is the cadherin/catenin
complex a functional unit of cell –cell actin-based adherens junctions in
the rat testis? Biol Reprod 68,489– 508.
Lee NPY, Mruk DD, Conway AM and Cheng CY (2004) Zyxin, axin, and
WASP are adaptors that link the cadherin/catenin protein complex to
cytoskeleton at adherens junctions in the seminiferous epithelium of the
rat testis. J Androl 25,200–215.
Li JCH, Mruk DD and Cheng CY (2001) The inter-Sertoli tight junction permeability barrier is regulated by the interplay of protein phosphatases
and kinases: an in vitro study. J Androl 22,847–856.
367
N.P.Y.Lee and C.Y.Cheng
Lonnerberg P, Parvinen M, Jahnsen T, Hansson V and Persson H (1992)
Stage- and cell-specific expression of cyclic adenosine 30 , 50 -monophosphate-dependent protein kinases in rat seminiferous epithelium. Biol
Reprod 46,1057–1068.
Lui WY, Lee WM and Cheng CY (2003a) Rho GTPases and spermatogenesis. Biochim Biophys Acta 1593,121–129.
Lui WY, Lee WM and Cheng CY (2003b) Sertoli-germ cell adherens junction dynamics in the testis are regulated by RhoB GTPase via the
ROCK/LIMK signaling pathway. Biol Reprod 68,2189–2206.
Lui WY, Mruk DD, Lee WM and Cheng CY (2003c) Adherero junction
dynamics in the testis and spermatogenesis. J Andol 24,1–14.
Lui WY, Mruk DD and Cheng CY (2004) Interactions among IQGAP1,
Cdc42 and the cadherin/catenin protein complex regulates Sertoli-germ
cell adherens junction dynamics in the testis. J Cell Physiol (in press).
Maeda S, Nam SY, Fujisawa M, Nakamuta N, Ogawa K, Kurohmaru M and
Hayashi Y (1998) Localization of cytoplasmic dynein light-intermediate
chain mRNA in the rat testis using in situ hybridization. Cell Struct
Funct 23,9–15.
Maekawa M, Toyama Y, Yasuda M, Yagi T and Yuasa S (2002) Fyn tyrosine kinase in Sertoli cells is involved in mouse spermatogenesis. Biol
Reprod 66,211–221.
Mandai K, Nakanishi H, Satoh A, Obaishi H, Wada M, Nishioka H, Itoh M,
Mizoguchi A, Aoki T, Fujimoto T, Matsuda Y, Tsukita S and Takai Y
(1997) Afadin: a novel actin filament-binding protein with one PDZ
domain localized at cadherin-based cell-to-cell adherens junction. J Cell
Biol 139,517– 528.
Mandai K, Nakanishi H, Satoh A, Takahashi K, Satoh K, Nishioka H,
Mizoguchi A and Takai Y (1999) Ponsin/SH3P12: an l-afadin- and
vinculin-binding protein localized at cell–cell and cell-matrix adherens
junctions. J Cell Biol 144,1001–1017.
McLachlan RI, Wreford NG, Meachem SJ, de Kretser DM and Robertson
DM (1994) Effects of testosterone on spermatogenic cell populations in
the adult rat. Biol Reprod 51,945– 955.
McLachlan RI, O’Donnell L, Meachem SJ, Stanton PG, de Kretser, Pratis K
and Robertson DM (2002) Hormonal regulation of spermatogenesis in
primates and man: insights for development of the male hormonal contraceptive. J Androl 23,149–162.
Miller MG, Mulholland D and Vogl AW (1999) Rat testis motor proteins
associated with spermatid translocation (dynein) and spermatid flagella
(kinesin-II). Biol Reprod 60,1047–1056.
Mo YY and Reynolds AB (1996) Identification of murine p120cas isoforms
and heterogeneous expression of p120cas isoforms in human tumor cell
lines. Cancer Res 56,2633–2640.
Mruk DD and Cheng CY (2004) Sertoli–Sertoli and Sertoli-germ cell interactions and their significance in germ cell movement in the seminiferous
epithelium during spermatogenesis. Endocr Rev, (in press).
Mruk DD, Zhu LJ, Silvestrini B, Lee WM and Cheng CY (1997) Interactions
of proteases and protease inhibitors in Sertoli-germ cell cocultures
preceding the formation of specialized Sertoli-germ cell junctions
in vitro. J Androl 18,612–622.
Mruk DD, Siu MKY, Conway AM, Lee NPY, Lau ASN and Cheng CY
(2003) Role of tissue inhibitor of metalloproteases-1 in junction
dynamics in the testis. J Androl 24,510–523.
Mueller S, Rosenquist TA, Takai Y, Bronson RA and Wimmer E (2003)
Loss of nectin-2 at Sertoli-spermatid junctions leads to male infertility
and correlates with severe spermatozoan head and midpiece malformation, impaired binding to the zona pellucida, and oocyte penetration.
Biol Reprod, 69,1330–1340.
Mulholland D, Dedhar S and Vogl AW (2001) Rat seminiferous epithelium
contains a unique junction (ectoplasmic specialization) with signaling
properties both of cell/cell and cell/matrix junctions. Biol Reprod 64,
396 –407.
Nishio H, Tokuda M, Itano T, Matsui H, Takeuchi Y and Hatase O (1995)
pp60c-src expression in rat spermatogenesis. Biochem Biophys Res
Commun 206,502–510.
O’Donnell L, Stanton PG, Bartles JR and Robertson DM (2000) Sertoli cell
ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat. Biol Reprod 63,99– 108.
Ozaki-Kuroda K, Nakanishi H, Ohta H, Tanaka H, Kurihara H, Mueller S,
Irie K, Ikeda W, Sakai T, Wimmer E, Nishimune Y and Takai Y (2002)
Nectin couples cell–cell adhesion and the actin scaffold at heterotypic
testicular junctions. Curr Biol 12,1145– 1150.
Palombi F, Salanova M, Tarone G, Farini D and Stefanini M (1992) Distribution of b1 integrin subunit in rat seminiferous epithelium. Biol
Reprod 47,1173–1182.
368
Pariset C, Feinberg J, Dacheux JL, Oyen O, Jahnsen T and Weinmanm S
(1989) Differential expression and subcellular localization for subunits
of cAMP-dependent protein kinase during ram spermatogenesis. J Cell
Biol 109,1195–1205.
Parreira GG, Melo RCN and Russell L (2002) Relationship of Sertoli–Sertoli
tight junctions to ectoplasmic specialization in conventional and en face
views. Biol Reprod 67,1232–1241.
Parsons JT (2003) Focal adhesion kinase: the first ten years. J Cell Sci 116,
1409– 1416.
Pelletier RM, Trifaro JM, Carbajal ME, Okawara Y and Vitale ML (1999)
Calcium-dependent actin filament-severing protein scinderin levels and
localization in bovine testis, epididymis, and spermatozoa. Biol Reprod
60,1128–1136.
Perez-Moreno M, Jamora C and Fuchs E (2003) Sticky business: orchestrating cellular signals at adherens junctions. Cell 112,535–548.
Pierceall WE, Woodard AS, Morrow JS, Rimm D and Fearon ER (1995)
Frequent alterations in E-cadherin and a- and b-catenin expression in
human breast cancer cell lines. Oncogene 11,1319–1326.
Radice GL, Rayburn H, Matsunami H, Knudsen KA, Takeichi M and
Hynes RO (1997) Developmental defects in mouse embryos lacking
N-cadherin. Dev Biol 181,64– 78.
Rebecchi MJ and Pentyala SN (2000) Structure, function, and control of
phosphoinositide-specific phospholipase C. Physiol Rev 80,1291– 1335.
Redenbach DM and Vogl AW (1991) Microtubule polarity in Sertoli cells: a
model for microtubule-based spermatid transport. Eur J Cell Biol
54,277–290.
Redenbach DM, Boekelheide K and Vogl AW (1992) Binding between
mammalian spermatid-ectoplasmic specialization complexes and microtubules. Eur J Cell Biol 59,433–448.
Robinson-White A and Stratakis CA (2002) Protein kinase A signaling.
Cross-talk with other pathways in endocrine cells. Ann NY Acad Sci
968,256–270.
Rousseaux-Prevost R, Delobel B, Hermand E, Rigot JM, Danjou P,
Mazeman E and Rousseaux J (1997) Distribution of gelsolin in human
testis. Mol Reprod Dev 48,63–70.
Rudiger M (1998) Vinculin and a-catenin: shared and unique functions in
adherens junctions. Bioessays 20,733–740.
Ruiz P, Brinkmann V, Ledermann B, Behrend M, Grund C, Thalhammer C,
Vogel F, Birchmeier C, Gunthert U, Franke WW and Birchmeier W
(1996) Targeted mutation of plakoglobin in mice reveals essential functions of desmosomes in the embryonic heart. J Cell Biol 135,215–225.
Russell L (1977a) Desmosome-like junctions between Sertoli and germ cells
in the rat testis. Am J Anat 148,301–312.
Russell L (1977b) Observations on rat Sertoli ectoplasmic (‘junctional’)
specializations in their association with germ cells of the rat testis. Tissue Cell 9,475–498.
Russell L (1977c) Movement of spermatocytes from the basal to the adluminal compartment of the rat testis. Am J Anat 148,313–328.
Russell L (1993) Morphological and functional evidence for Sertoli-germ
cell relationships. In Russell LD and Griswold MD (eds) The Sertoli
Cell. Cache River Press, Clearwater, FL, pp 365 –390.
Russell L, Ettlin RA, Sinha Hikim AP and Clegg EJ (1990) Histological and
Histopathological Evaluation of the Testis. Cache River Press, Clearwater, FL, pp 1–52.
Russell LD (1980) Sertoli-germ cell interrelations: a review. Gamete Res
3,179– 202.
Russell LD and Goh JC (1988) Localization of actinin in the rat testis: preliminary observations. In Parvinen M, Huhtaniemi I, and Pelliniemi LJ
(eds) Development and Function of the Reproductive Organs. VIIth
Ares-Serono Symposia Series.
Salanova M, Ricci G, Boitani C, Stefanini M, de Grossi S and Palombi F
(1998) Junctional contacts between Sertoli cells in normal and aspermatogenic rat seminiferous epithelium contain a6b1 integrins, and their
formation is controlled by follicle-stimulating hormone. Biol Reprod
58,371–378.
Salanova M, Stefanini M, de Curtis I and Palombi F (1995) Integrin receptor
a6b1 is localized at specific sites of cell-to-cell contact in rat seminiferous epithelium. Biol Reprod 52,79–87.
Sellers JR (2000) Myosins: a diverse superfamily. Biochim Biophys Acta
1496,3–22.
Siu MKY and Cheng CY (2004) Interactions of proteases, protease inhibitors, and the b1 integrin/laminin g3 protein complex in the regulation of
ectoplasmic specialization dynamics in the rat testis. Biol Reprod,
70,945–964.
Ectoplasmic specialization dynamics in the testis
Siu MKY, Mruk DD, Lee WM and Cheng CY (2003) Adhering junction
dynamics in the testis are regulated by an interplay of b1-integrin
and focal adhesion complex-associated proteins. Endocrinology 144,
2141–2163.
Soriano P, Montgomery C, Geske R and Bradley A (1991) Targeted disruption of the c-Src proto-oncogene leads to osteopetrosis in mice. Cell
64,693–702.
Steinberg MS and McNutt PM (1999) Cadherins and their connections:
adhesion junctions have broader functions. Curr Opin Cell Biol
11,554–560.
Stephens LE, Sutherland AE, Klimanskaya IV, Andrieux A, Meneses J,
Pedersen RA and Damsky C (1995) Deletion of b1 integrins in mice
results in inner cell mass failure and peri-implantation lethality. Genes
Dev 9,1883–1895.
Stevenson BR, Siliciano JD, Mooseker MS and Goodenough DA (1986)
Identification of ZO-1: a high molecular weight polypeptide associated
with the tight junction (zonula occludens) in a variety of epithelia.
J Cell Biol 103,755–766.
Takai Y and Nakanishi H (2003) Nectin and afadin: novel organizers of
intercellular junctions. J Cell Sci 116,17–27.
Takayama T, Shiozaki H, Shibamoto S, Oka H, Kimura Y, Tamura S, Inoue
M, Monden T, Ito F and Monden M (1996) b-Catenin expression in
human cancers. Am J Pathol 148,39–46.
Thomas SM and Brugge JS (1997) Cellular functions regulated by Src family
kinases. Annu Rev Cell Dev Biol 13,513–609.
Torres M, Stoykova A, Huber O, Chowdhury K, Bonaldo P, Mansouri A,
Butz S, Kemler R and Gruss P (1997) An a-E-catenin gene trap
mutation defines its function in preimplantation development. Proc Natl
Acad Sci USA 94,901–906.
Toyama T, Maekawa M and Yuasa S (2003) Ectoplasmic specializations in
the Sertoli cell: new vistas based on genetic defects and testicular toxicology. Anat Sci Int 8,1–16.
Velichkova M, Guttman J, Warren C, Eng L, Kline K, Vogl AW and Hasson
T (2002) A human homologue of Drosophila kelch associates with myosin-VIIa in specialized adhesion junctions. Cell Motil Cytoskeleton
51,147–164.
Vogl AW (1988) Changes in the distribution of microtubules in rat Sertoli
cells during spermatogenesis. Anat Rec 222,34–41.
Vogl AW (1989) Distribution and function of organized concentrations of
actin filaments in mammalian spermatogenic cells and Sertoli cells. Int
Rev Cytol 119,1–56.
Vogl AW, Pfeiffer DC, Redenbach DM and Grove BD (1993) Sertoli cell
cytoskeleton. In Russell LD and Griswold MD (eds) The Sertoli Cell.
Cache River Press, Clearwater, FL, pp 39 –86.
Vogl AW, Pfeiffer DC, Mulholland D, Kimel G and Guttman J (2000)
Unique and multifunctional adhesion junctions in the testis: ectoplasmic
specializations. Arch Histol Cytol 63,1–15.
Wang W, Wine RN and Chapin RE (2000) Rat testicular Src: normal distribution and involvement in ethylene glycol monomethyl ether-induced
apoptosis. Toxicol Appl Pharmacol 163,125–134.
Weiss EE, Kroemker M, Rudiger AH, Jockusch BM and Rudiger M (1998)
Vinculin is part of the cadherin–catenin junctional complex: complex
formation between a-catenin and vinculin. J Cell Biol 141,755–764.
Wine RN and Chapin RE (1999) Adhesion and signaling proteins
spatiotemporally associated with spermiation in the rat. J Androl
20,198–213.
Witke W, Sharpe AH, Hartwig JH, Azuma T, Stossel TP and Kwiatkowski
DJ (1995) Hemostatic, inflammatory, and fibroblast responses are
blunted in mice lacking gelsolin. Cell 81,41–51.
Wu JC, Gregory CW and DePhilip RM (1993) Expression of E-cadherin in
immature rat and mouse testis and in rat Sertoli cell cultures. Biol
Reprod 49,1353–1361.
Wulser MJ, Abu-Yousif A, Ender GC, Heckert LL, Datta A, Georg GI and
Tash JS (2003) Localization of binding of the indazole-3-carboxylic
acid male contraceptive, lonidamine, using a biotinylated UV-crosslinking analogue in rat tissues and isolated cells. Biol Reprod (Suppl) Abstr
638.
Yagi T and Takeichi M (2000) Cadherin superfamily genes: functions,
genomic organization, and neurologic diversity. Genes Dev
14,1169–1180.
Yang JT, Rayburn H and Hynes RO (1995) Cell adhesion events mediated
by a4 integrins are essential in placental and cardiac development.
Development 121,549–560.
Yin HL and Janmey P (2003) Phosphoinositide regulation of the actin cytoskeleton. Annu Rev Physiol 65,761–789.
Zhurinsky J, Shtutman M and Ben-Ze’ev A (2000) Plakoglobin and
b-catenin: protein interactions, regulation and biological roles. J Cell Sci
113,3127–3139.
Zong SD, Bardin CW, Phillips D and Cheng CY (1992) Testins are localized
to the junctional complexes of rat Sertoli and epididymal cells. Biol
Reprod 47,568–572.
Zong SD, Zhu LJ, Grima J, Aravindan GR, Bardin CW and Cheng CY
(1994) Cyclic and postnatal developmental changes of testin in the rat
seminiferous epithelium—an immunohistochemical study. Biol Reprod
51,843–851.
Submitted on November 13, 2003; resubmitted on February 2, 2004;
accepted on April 7, 2004
369