review REVIEW

Spermatogenesis 1:2, 105-115; April/May/June 2011; © 2011 Landes Bioscience

Regulation of blood-testis barrier dynamics

by desmosome, gap junction, hemidesmosome
and polarity proteins
An unexpected turn of events
C. Yan Cheng,1,* Elissa W.P. Wong,1 Pearl P.Y. Lie,1 Michelle W.M. Li,1 Dolores D. Mruk,1 Helen H.N. Yan,1 Ka-Wai Mok,1
Jayakanthan Mannu,2 Premendu P. Mathur,2 Wing-yee Lui,3 Will M. Lee,3 Michele Bonanomi4 and Bruno Silvestrini4
1
The Mary M. Wohlford Laboratory for Male Contraceptive Research; Center for Biomedical Research; The Population Council; New York, NY USA; 2Center for Bioinformatics;
School of Life Sciences; Pondicherry University; Pondicherry, India; 3School of Biological Sciences; The University of Hong Kong; Hong Kong, China;
4
S.B.M. Srl; Via Domenico Tardini; Rome, Italy

Key words: testis, spermatogenesis, seminiferous epithelial cycle, blood-testis barrier, Sertoli cell,
estrogens, bisphenol A, environmental toxicants, adjudin

The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such
as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between
endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal
ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous
tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that
meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the
apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades,
the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the
BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome
and gap junction that serve as a signaling platform to coordinate the “opening” and “closing” of the TJ-permeability
barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by
polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and
c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or
protein degradation). These events not only serve to destabilize the existing “old” BTB above preleptotene spermatocytes
in transit in “clones” at the BTB, but also contribute to the assembly of “new” BTB below the transiting spermatocytes.
Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring
events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible
route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB
also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the
transit of spermatocytes, can be the “gateway” to making the BTB so vulnerable to toxicants and/or chemicals, causing
male reproductive dysfunction.

Introduction
In the mammalian testis, the blood-testis barrier (BTB) forms
during puberty in humans (at ~12–14 years of age) and a func-
tional BTB is established by ~17–21 day postpartum in rats,1,2
coinciding with the differentiation of Type B spermatogo-
nia to prepare spermatocytes for meiosis I and meiosis II.3
This timing also coincides with terminal differentiation of
*Correspondence to: C. Yan Cheng; Email: Y-Cheng@popcbr.rockefeller.edu
Submitted: 03/14/10; Revised: 04/04/11; Accepted: 04/05/11
DOI: 10.4161/spmg.1.2.15745
Sertoli cells which cease to proliferate by ~15–17 days of age post-
partum in rats.4 Thus the Sertoli cell population remains rela-
tively constant in adult mammals with ~30 million Sertoli cells
per testis in adult rats at 150 and 240 days of age post-partum,5
and each Sertoli cell is known to support ~30–50 developing
germ cells in the seminiferous epithelium.6,7 However, recent
studies have shown that Sertoli cells are capable of dividing
even in adult animals including rats8 and mice9 under experi-
mental and even under physiological conditions in humans.9,10
The BTB anatomically divides the seminiferous epithelium into
the basal and apical (or adluminal) compartments (see Fig. 1).
This ultrastructure is created by specialized junctions between
adjacent Sertoli cells near the basement membrane, and it is

www.landesbioscience.com Spermatogenesis 105

 Figure 1. A schematic illustration of the blood-testis barrier (BTB) in the seminiferous epithelium of adult mammalian testis. This is a schematic
representation of the seminiferous epithelium in the adult rat testis of a stage VII tubule. Anatomically, the BTB divides the seminiferous epithelium
(composed of only Sertoli and germ cells, lying on top of the basement membrane, which is a modified form of extracellular matrix63,64) into the basal
and apical (adluminal) compartments. The BTB is constituted by coexisting TJ, basal ES, desmosome and gap junction. The most obvious feature of
the BTB is the tightly packed actin filament bundles which are lying parallel to the plasma membrane and are sandwiched in between cisternae of
endoplasmic reticulum and the opposing plasma membranes of the two adjacent Sertoli cells. This tripartite structure is known as the basal ES. This
feature is also found at the Sertoli cell-spermatid (step 8–19 spermatids in rats) interface known as the apical ES in the apical compartment, which is
the only anchoring device once it appears during spermiogenesis. At the BTB, TJ and gap junction coexist with basal ES, whereas desmosome is an
intermediate filament-based anchoring junction type at the BTB, illustrating that the actin- and the intermediate filament-based cytoskeletons are
working in concert to regulate BTB dynamics during spermatogenesis. The unusually organized network of actin filament associated with the plasma
membrane at the BTB and apical ES are maintained via the intricate interactions of: (1) several actin regulatory proteins (e.g., Eps8, an actin bundling
and capping protein) and Arp2/3 protein complex (an actin nucleation protein causing the branching of actin filaments, thereby disrupting the actin
filament bundles at the ES, replacing them with branched actin network) and (2) the properly organized cell adhesion protein complexes at the TJ
(e.g., occludin-ZO-1) and basal ES (e.g., N-cadherin-β-catenin), which are maintained by the signaling action of the protein complexes at the desmo-
some (e.g., desmoglein-2/desmocollin-2/c-Src) and the gap junction (e.g., connexin43-plakophilin-2-c-Src) as well as the polarity proteins (e.g., PAR3,
PAR6, 14-3-3, Cdc42).

crucial to confer cell polarity, to limit paracellular transport of
biomolecules, ions, electrolytes and water from the basal to the
apical compartment, and to maintain an immunological barrier
to sequester post-meiotic germ cell development from the sys-
temic circulation.1,11 Thus, spermiogenesis and spermiation take
place in a specialized microenvironment at the apical compart-
ment of the seminiferous epithelium composed of only Sertoli
cells and developing germ cells.1,12,13 An earlier study has shown
that when the BTB assembly was delayed by ~4 wk via treatment
of neonatal rats with a toxicant diethylstilbestrol, spermatogo-
nial differentiation and the onset of meiosis would be delayed,
and their resumption correlate with the establishment of an
intact and functional BTB.2 A more recent study has shown that
spermatogenesis failed to reinitiate in rats treated with adjudin

106 Spermatogenesis Volume 1 Issue 2

[1-(2,4-dichorobenzyl)-1H-indazole-3-carbohydrazide] at high
doses (e.g., 250 mg/kg b.w.) that irreversibly disrupted the BTB
integrity, even though the population of spermatogonial stem
cells/spermatogonia did not reduce significantly.14 Interestingly,
rats treated with adjudin at a lower dose, such as at 50 mg/kg b.w.
which also perturbed the BTB integrity transiently and reversibly,
managed to re-initiate spermatogonial differentiation and sper-
matogenesis when the BTB functionality was restored.14 These
findings thus illustrate the significance of the BTB in maintain-
ing spermatogenesis. Unlike other blood-tissue barriers, such as
the blood-brain barrier and the blood-ocular barrier which are
constituted by TJ-permeability barrier of the endothelial cells in
the microvessels in the brain and the eye, respectively,11,15,16 the
BTB is constituted by coexisting tight junction (TJ, also known
as zonula occludens), basal ectoplasmic specialization [basal ES,
a testis-specific adherens junction (AJ, also known as zonula
adherens) type], desmosome (also known as macula adherens)
and gap junction1 (see Fig. 1). More importantly, in addition to
TJ protein complexes (e.g., occludin-ZO-1, JAM-A-ZO-1, clau-
din-ZO-1), the BTB is composed of adhesion protein complexes
found in other junction types, including adherens junction (AJ,
e.g., N-cadherin/β-catenin; nectin-afadin), desmosome (e.g.,
desmoglein-2-desmocollin-2) and gap junction (e.g., connexin
43-plakophilin-2) (see Fig. 1).1,17,18 Yet the roles of desmosome
and gap junction at the BTB remain unknown for decades since
their first discoveries in the 1970s.19-21 Nevertheless, recent stud-
ies have shown that they are crucial to provide the necessary
cross-talk between different junction types at the BTB. Thus, the
“opening/disruption” and “closing/reassembly” of different junc-
tion types can be coordinated temporally and spatially, thereby
facilitating the transit of preleptotene spermatocytes across the
BTB at stage VIII of the seminiferous epithelial cycle while
maintaining the immunological barrier.
What are the Roles of the Desmosome
and Gap Junction at the BTB?
Introduction. A desmosome (also known as macula adherens) is
an intermediate filament-based cell-cell anchoring junction type
in which the integral membrane proteins (known as the desmo-
somal cadherins: desmogleins and desmocollins) are anchored
to intermediate filaments (composed of vimentin in adult tes-
tes) via the cytolinker desmoplakin.22,23 Desmosomal cadherin-
desmoplakin interactions are further reinforced by the armadillo
proteins plakoglobin and plakophilin, forming a distinctive
electron dense ultrastructure under electron microscopy at the
Sertoli-Sertoli cell interface at the BTB.17,24-26 In the testis, besides
the BTB, desmosomes can also be found at the Sertoli cell-sper-
matid (limited to step 1–7 spermatids) or Sertoli-spermatocyte/
spermatogonium interface.19,27 A gap junction, on the other hand,
is a cell-cell actin-based communicating junction type.21,28-30 A
functional gap junction communication channel is composed of
two opposing connexons (the functional units of gap junction
with each connexon known as a hemichannel) between adjacent
Sertoli cells at the BTB [or between Sertoli cells and spermatids
(step 1–7 spermatids only), spermatocytes or spermatogonia].21,28
www.landesbioscience.com Spermatogenesis
Each connexon is composed of a hexamer of gap junction pro-
teins known as connexins (Cx). At least 20 connexins are found
in humans and rodents, such as Cx43, Cx26 and Cx33. The gap
junction channels were shown to permit the diffusion of solutes
of <1–1.5 kDa in molecular mass.31-33 More recent studies have
shown that substances of large molecular sizes, such as siRNA
duplexes, polypeptides and cyclic nucleotides can also pass
through the gap junction channels with selective permeability.34
Thus, while desmosomes contribute to the cell adhesion func-
tion at the BTB, gap junction communication channels provide
the means for signaling to coordinate cellular events at the BTB.
However, recent findings illustrate that both desmosome and gap
junction provide an unusual platform for cell signaling at the
BTB, which are discussed in the following sections.
Role of desmosomes. Recent studies have unequivocally
demonstrated that desmosomes are performing some unique and
significant roles at the BTB. First, it was shown that while the
knockdown of desmoglein-2 or desmocollin-2 alone in the Sertoli
cell epithelium by RNAi using specific siRNA duplexes failed to
perturb the Sertoli cell TJ-permeability barrier, the knockdown
of either desmoglein-2 alone or desmoglein-2 and desmocollin-2
combined caused mis-localization of ZO-1, moving from the
Sertoli-Sertoli cell surface to cell cytosol.35 More importantly, a
knockdown of both desmoglein-2 and desmocollin-2 by RNAi
was found to perturb the Sertoli cell TJ-permeability function
in addition to a mis-localization of both c-Src (a nonreceptor
protein tyrosine kinase) and coxsackievirus and adenovirus
receptor (CAR, an integral membrane protein and a cell adhe-
sion molecule at the BTB,35a,37,58 which likely interacts with the
N-cadherin-based adhesion protein complex at the basal ES
via β-catenin as its binding partner37), moving from the cell-
cell interface to cell cytosol.35 Second, the mis-localization of
CAR in the Sertoli cell epithelium induced by the knockdown
of desmoglein-2 and desmocollin-2 was shown to be the result
of an increase in endocytosis of CAR,35 thereby destabilizing
the cell adhesion function at the BTB. Third, studies by co-
immunoprecipitation have shown that while desmoglein-2 did
not structurally interact with occludin, ZO-1 or Cx43, it was
tightly associated c-Src; 35 and other studies have shown that
c-Src interacted with Cx43,36 CAR,37 occludin, N-cadherin
and ZO-1.38 Thus, the desmoglein-2/desmocollin-2/c-Src des-
mosomal protein complex at the BTB may be an important
regulatory complex with c-Src being served as the dominant
regulator to confer proper phosphorylation status of proteins at
the BTB. The knockdown of desmoglein-2/desmocollin-2 by
RNAi in the Sertoli cell epithelium led to improper localization
of c-Src which moved away from the cell-cell interface to cyto-
sol,35 thereby “messing up” the homeostasis of c-Src signaling
function, leading to improper phosphorylation status of crucial
proteins at the Sertoli-Sertoli cell interface (e.g., CAR, ZO-1)
and affecting their kinetics of endocytosis. All these contribute
to a destabilization of the adhesion function at the site, which in
turn, affect the TJ-permeability barrier. Collectively, these find-
ings illustrate that besides conferring adhesion function at the
BTB, desmosome serves as a signaling platform at the BTB via
its interaction with c-Src.
107
 Role of gap junctions. It is known that gap junctions that lie
behind the junctional complexes (composed of TJ, AJ and desmo-
somes) in multiple epithelia provide the necessary cross-talk and
signaling function for the coordination of cellular events to main-
tain the homeostasis of an epithelium.30,33,39 However, the func-
tion of gap junctions at the BTB remains largely unknown until
recently because they are found to be intermingled with TJ, basal
ES and desmosome instead of being separate entities as in other
epithelia (see Fig. 1). It was found that in Sertoli cell epithelium
with an established functional TJ-barrier and having ultrastruc-
tures that mimicked the Sertoli cell BTB in vivo, a knockdown
of Cx43 or its adaptor protein plakophilin-2 (also an adaptor for
desmosome, illustrating a tight structural association between
actin-based gap junction and intermediate filament-based desmo-
some at the BTB in the mammalian testis) using specific siRNA
duplexes failed to perturbed the Sertoli cell TJ-barrier function.36
However, a simultaneous knockdown of Cx43 and plakophilin-2
by RNAi led to a disruption of the Sertoli cell TJ-permeability
barrier, possibly caused by the mis-localization of occludin and
ZO-1, with these proteins moving from the Sertoli-Sertoli cell
interface to cell cytosol, thereby destabilizing cell adhesion at
the TJ and leading to barrier disruption.36 More important, even
though the knockdown of Cx43 alone did not perturb the Sertoli
cell TJ-barrier function,36 subsequent studies using the [Ca 2+]-
switch model and the bisphenol A (BPA) model showed that Cx43
knockdown hindered these cells from “re-assembling” a disrupted
TJ-barrier.40 These findings thus illustrate that while Cx43 may
not be critical for the assembly of a “new” TJ-barrier, it is essential
for the maintenance of an established TJ-barrier, , such as at the
BTB in adult mammalian testes, via its effects on TJ reassembly
during spermatogenesis when the BTB is transiently disrupted
during the epithelial cycle (e.g., at stage VIII to facilitate the
transit of preleptotene spermatocytes), as manifested by a loss of
intracellular [Ca 2+] or following the exposure of Sertoli cell epi-
thelium to an environmental toxicant BPA.40 Similar to a number
of BTB protein complexes (e.g., desmoglein-2-based complex),35
the Cx43-based gap junction protein complex also contains c-Src
as an integrated component,36 illustrating that c-Src is likely to
modify the phosphorylation status of connexins at the gap junc-
tion, thereby regulating the kinetics of their endocytic vesicle-
mediated protein trafficking events.
What are the Roles of Polarity Proteins
and Hemidesmosome on BTB Dynamics?
Polarity proteins. Earlier studies in Drosophila melanogaster and
Caenorhabditis elegans have shown that cellular asymmetry and
apico-basal polarity are established and maintained by polarity
proteins.41-44 There are three modules of polarity protein com-
plexes known as (1) the PAR (partitioning defective) protein
complex [e.g., PAR3/PAR6/aPKC (atypical protein kinase C,
a nonreceptor Ser/Thr protein kinase)], (2) the Crumbs pro-
tein complex [e.g., Crumbs-3/PALS1 (protein associated with
(LIN-7)-1)/PATJ (PALS1-associated tight-junction protein)] and
(3) the Scribble complex [e.g., Scribble/LGL1/2 (Lethal giant lar-
vae 1/2)/DLG1 (Discs large 1)]. Apico-basal polarity is conferred
108 Spermatogenesis
by the differential localization of polarity complexes: the PAR-
and the Crumbs-based complexes are localized predominantly to
TJ and the Scribble-based protein complex displays basolateral
localization. Since each of these protein complexes recruit their
own binding partners (including adaptors, kinases, phospha-
tases and scaffolding proteins), the mutual exclusion between
the Scribble-based complex and the TJ localized polarity protein
complexes thus confers apico-basal polarity and asymmetrical
distribution of proteins and organelles within an epithelial cell.
These polarity proteins have at least one homolog found in mam-
mals.41,44 Indeed, recent studies have shown that many of these
proteins are expressed by Sertoli and/or germ cells,45 regulating
cell polarity in the seminiferous epithelium. For instance, Sertoli
cell nuclei are restricted to the basal compartment near the base-
ment membrane, and the heads of developing spermatids are all
pointing toward the basement membrane, illustrating cell polar-
ity in the seminiferous epithelium.44
Besides conferring Sertoli cell and spermatid polarity in the
testis, polarity proteins (e.g., PAR3, PAR6) were recently shown
to be involved in cell adhesion at the Sertoli cell-elongating
spermatid interface (known as apical ES) and Sertoli-Sertoli
cell interface at the BTB.45 For instance, a knockdown of either
PAR3 or PAR6 led to a redistribution of JAM-A and α-catenin at
the Sertoli-Sertoli cell interface, moving from cell surface to cell
cytosol, thereby destabilizing cell adhesion at the TJ-barrier.45
These findings also implicate polarity proteins may be involved
in endocytic vesicle-mediated protein trafficking events, such
as endocytosis, transcytosis, recycling and endosome-mediated
protein degradation. In fact, subsequent studies have shown that
polarity proteins (e.g., 14-3-3 also known as PAR5 and Cdc42
also called cell division control protein 42 and a member of the
Rho GTPases are integrated components of the PAR-based polar-
ity protein complex) are involved in protein endocytosis at the
Sertoli cell BTB, since a knockdown of 14-3-3 by RNAi led to a
significant increase in the kinetics of endocytosis of JAM-A and
N-cadherin.46 Additionally, overexpression of a Cdc42 dominant
negative mutant in Sertoli cells abolished the TGFβ3-induced
acceleration in occludin and CAR endocytosis in the Sertoli cell
epithelium, thereby blocking the TGFβ3-induced disruption of
the Sertoli cell TJ-barrier.47 These findings thus illustrate that
polarity proteins are crucial for the endocytic vesicle-mediated
protein trafficking events at the Sertoli BTB. Taking these data
collectively, it is likely that the predominant localization of polar-
ity proteins at the BTB (e.g., PAR6 14-3-3) and their stage-
specific expression during the seminiferous epithelial cycle are
crucial for the desmosome- and gap junction-mediated effects on
protein distribution, which require changes in protein endocyto-
sis, transcytosis and/or recycling. The net result of these changes
thus regulates cell adhesion at the apical ES, and basal ES at the
BTB.
Hemidesmosome. Hemidesmosome is an intermediate fil-
ament-based cell-matrix anchoring junction type found at the
Sertoli cell-basement membrane interface in the mammalian
testis.1 Its composition and function in the testis remain largely
unknown, except that β1-integrin and α2-laminin chain appear
to be the putative components of the hemidesmosome.48 Most
Volume 1 Issue 2
 Figure 2. A molecular modeling study to assess the docked complex of
estradiol-17β with the Cx43-based gap junction communication chan-
nel. (A) Topographic view of the Cx43-gap junction protein complex
with estradiol-17β. (B) Side view of the Cx43-gap junction protein
complex with estradiol-17β. (C) Ball and stick model for the interaction
residues and secondary structure representation for protein between
estradiol-17β and Cx43. The SOSUI WWW server65 was used to predict
transmembrane helices for connexin 43 (Cx43)-based gap junction (GJ)
communicating channel. The characteristic feature of the Cx43-GJ chan-
nel is the spanning of transmembrane helices in the cell membrane that
creates the “pore” which allows passage of small molecules (see “A” and
“B”). The crystal structure of human Cx26 at 3.5 Å resolution in humans
is known,66 and this information was used for the molecular modeling of
Cx43. Initially, the amino acid sequence of Cx43 [UniProtKB ID: P08050]
was retrieved from UniProtKB database (www.uniprot.org).67 The crystal
structure of Cx26 was retrieved from Protein Data Bank (PDB ID: 2ZW3).68
The MODELLER 9v7 program69 was used for sequence alignment and
model building.70 The sequence and structural alignment of Cx43 to
human Cx26 was carried out using ‘align2d’ program in MODELLER 9v7.
This alignment study illustrated 45% sequence identity in the trans-
membrane region between the two proteins, which suggested that the
most important pore region is conserved between Cx26 and Cx43. The
produced alignment was then used to generate hemichannel struc-
ture with ‘model-multichain’ program in MODELLER 9v7. The quality of
the structure was assessed by submitting the modeled structure into
PROCHECK.71 The loop building and energy minimization were carried
out with Swiss-PdbViewer. The energy computations were done by GRO-
MOS96 implemented in Swiss-PdbViewer. The modeled three dimen-
sional structure of Cx43 was prepared using Schrödinger Suite 2009 Pro-
tein Preparation Wizard. All the hydrogens were added to the backbone
of the structure and bond orders was assigned appropriately. Exhaustive
sampling method was used for the optimization of the hydrogen bond-
ing network, in which the orientation of hydroxyl groups, amide groups
of Asn and Gln, and imidazole ring of His residues were optimized. The
RMSD (root mean square deviation) of atom displacement for terminat-
ing the minimization was set to be 0.30 Å. The minimization was carried
out with OPLS2005 force field. The ligand structure of estradiol-17β (CID:
5757), bisphenol A (CID: 6623) (Fig. 3) and adjudin (CID: 9819086) (Fig.
4) were retrieved from NCBI-PubChem database. LigPrep (Version 2.3,
Schrödinger, LLC, New York, NY 2009) was used to generate 3D structure
with correct chiralities for each ligand used in this study. Grid files repre-
sent the volume of the receptor that can be searched for ligand docking.
Grid box was set on the centriod of all the residues in the Cx43 and no
constraints were selected. The Standard Precision (SP) mode of Glide
(Version 5.5, Schrödinger, LLC, New York, NY 2009) was used for the flex-
ible ligand docking. The best binding conformation was selected based
on the one having the lowest docking energy from the generated dock-
ing solutions. Molecular docking of estradiol-17β into Cx43 generated 32
solutions, in which the best binding conformation having the docking
score of -5.40 kcal/mol. The size of binding site surface area of Cx43 was
864.562 Å, and the size of ligand surface was 271.946 Å. These docking
studies revealed that side chain oxygen atom of Glu42 (E42, Chain A)
makes a hydrogen bond formation with estradiol-17β in the bond length
of 1.790 Å. In addition, Gly2 (G2, Chain B), Asp3 (D3, Chain B), Trp4 (W4,
Chain A and B), Ser5 (S5, Chain A), Ile31 (I31, Chain A), Ile34 (I34, Chain
A), Leu35 (L35, Chain A), Leu37 (L37, Chain A), Gly38 (G38, Chain A) and
Val41 (V41, Chain A) were involved in non-bonded interactions such as
van der Waals forces. It is important to note that estradiol-17β binds with
amino acid residues of N-terminal helix (NTH) and Trans membrane helix
1 (TMH1) of chain A and B alone and it leaves empty space in the pore
region. Thus, it is possible that additional units of estradiol-17β may also
bind with remaining chains to alter the Cx43 gating mechanism.

www.landesbioscience.com Spermatogenesis 109


importantly, a knockdown of β1-integrin in Sertoli cell epithe-
lium (without any residual elongating/elongated spermatids and
thus no apical ES was present) not only perturbed the hemides-
mosome function, but also led to a disruption of the Sertoli cell
TJ-barrier function. This effect resulted from the induction of
occludin endocytosis and its redistribution from cell surface to
cell cytosol, thereby disrupting cell adhesion function at the
BTB.48 It remains to be determined the involvement of polarity
proteins in this event. These findings thus demonstrate that a
disruption of β1-integrin function at the hemidesmosome would
110 Spermatogenesis
Figure 3. A molecular modeling study to assess the docked complex of
bisphenol A (BPA) with Cx43-based gap junction communication chan-
nel. (A) Topographic view of the Cx43-gap junction protein complex
with BPA. (B) Side view of the Cx43-gap junction protein complex with
BPA. (C) Ball and stick model for the interaction residues and secondary
structure representation for protein between BPA and Cx43. Molecular
docking of bisphenol A into Cx43 shows formation of strong interac-
tions by making two hydrogen bonds in the complex. The side chain
oxygen atom of Asp3 (D3, Chain A) and backbone nitrogen atoms of
Gly2 (G2, Chain A) were involved in hydrogen bond formation at the
bond distance of 1.587 Å and 1.748 Å, respectively. In addition to these
residues, Gly2 (G2, Chain B and F), Asp3 (D3, Chain B–F), Trp4 (W4,
Chain A and F), Ser5 (S5, Chain F), Ala6 (A6, Chain B, E and F) and Leu7
(L7, Chain B and C) were involved in non-bonded interactions, such as
van der Waals forces. Asp3 (D3) and Trp4 (W4) are the major residues
involved in the interaction in this docked complex. Glide docking score
of the complex was -4.07 kcal/mol. The binding site surface and ligand
surface area of the docked complex were 1494.841 Å and 253.687 Å,
respectively. It is noted that the residues of N-terminal helix (NTH) in
Cx43 alone was involved in the interactions with BPA. The compound
BPA appears to occupy largest binding site surface area in the Cx43
and can block the channel based on the molecular modeling, which is
consistent with recent findings using a functional GJ communication
assay.40
affect TJ-barrier function, illustrating a physiological link exists
between junctions at the BTB and hemidesmosome.
Does the Gap Junction that Provide the Necessary
Cross-Talk between Different Junction Types
at the BTB also Make the BTB More Vulnerable
to Environmental Toxicants?
As briefly discussed above regarding the role of desmosome and
gap junction at the BTB, these two junction types at the BTB
likely provide the signaling function and cross-talk between
different junction types, maintaining the immunological bar-
rier during the epithelial cycle. Furthermore, molecular mod-
eling studies have shown that toxicants (e.g., BPA), estrogens
(e.g., estradiol-17β) and male contraceptives (e.g., adjudin) that
can modulate Sertoli cell BTB function can interact with the
gap junction communicating channel with putative docking
domains (see Figs. 2–4). These interactions can possibly lead
to the blockage of Cx43-based gap junction-based communica-
tion, as well as the penetration of the BTB by utilizing the gap
junction communication junction as a “gateway” and gaining
entry into the apical compartment (Figs. 2–4). Indeed, BPA
was recently shown to perturb gap junction communication
based on a functional dye-transfer assay.40 Based on these find-
ings and those discussed above,1,18,49 we herein propose a hypo-
thetical model (see Fig. 5) summarizing the molecular basis
of BTB restructuring during spermatogenesis. In brief, during
stage VIII of the epithelial cycle, “new” TJ-fibrils are being
established behind the preleptotene spermatocytes in transit at
the BTB via testosterone-induced de novo protein synthesis,50-53
as well as testosterone- or cytokine-mediated transcytosis and
recycling of integral membrane protein complexes from the
“old” BTB site above the transiting spermatocytes.54-56 Once
the “new” BTB is established, the “old” BTB site undergoes
Volume 1 Issue 2
 Figure 4. A molecular modeling study to assess the docked complex of
adjudin with Cx43-based gap junction communication channel.
(A) Topographic view of the Cx43-gap junction protein complex with
adjudin. (B) Side view of the Cx43-gap junction protein complex with
adjudin. (C) Ball and stick model for the interaction residues and sec-
ondary structure representation for protein between adjudin and Cx43.
Docking of adjudin into Cx43 shows strong interactions in the pore lin-
ing residues of N-terminal helix (NTH). The amino acid residues such as
Gly2 (G2, Chain B) and Asp3 (D3, Chain A and B) were involved in multi-
ple hydrogen bond formation with adjudin (C). The backbone nitrogen
atom of Gly2 (G2, Chain B) and side chain nitrogen atom of Asp3 (D3,
Chain B) were shown to have hydrogen bond interaction with adjudin
in the bond distances of 2.305 Å and 1.940 Å, respectively. Whereas the
side chain oxygen of Asp3 (D3, Chain A) and backbone nitrogen atom
of Asp3 (D3, Chain A) were also involved in hydrogen bond formation
in the bond distance of 1.703 Å and 2.227 Å, respectively. In addition
to hydrogen bonds, Gly2 (G2, Chain A, C, D and F), Asp3 (D3, Chain C),
Trp3 (W3, Chain A and B), Ser5 (S5, Chain A) and Ala6 (A6, Chain A) were
involved in non-bonded interaction to further stabilize the interaction.
The docking score of this binding complex was -4.47 kcal/mol. The size
of surface area for binding site and ligand were 978.172 Å and 290.038
Å, respectively. This docking result has shown that adjudin binds with
pore lining N-terminal helix residues of chain (A–D) and (F) except chain
(E). Hence, it has the possibility that adjudin can block the gating of
this channel protein. From these docking studies, we have observed
that large size of both binding site surface in BPA and ligand surface in
adjudin makes very strong binding into N-terminal helix of Cx43. On the
other hand, docking of estradiol-17β reveals very little docking energy
when compared to docking of other two compounds, which indicates
estradiol-17β can make very stable binding into Cx43-based GJ com-
munication channel. It can also be noted that both NTH and TMH1
amino residues are involved in the binding pocket for estradiol-17β
interactions. Our docking results disclose that each of the compounds
is forming at least one hydrogen bond with the binding site residues in
the Cx43-based communication channel. As a whole we concluded that
all three compounds can regulate the function of Cx43 by binding with
pore lining residues of this protein.

complete degeneration induced by cytokines (e.g., TNFα,

TGFβ3, IL-1α).54-57 Furthermore, TJ, basal ES, desmosome and
gap junction proteins expressed by germ cells (e.g., preleptotene
spermatocytes) during their transit at the BTB can also form
inter-locking interactions with the corresponding proteins in
Sertoli cells, even “loosely” associated as recently proposed.58
This thus avoids the formation of a “disrupted” or an “open”
configuration at the BTB to elicit an unwanted immunologi-
cal response in the host immune system. In short, these events,
as discussed in above sections and recently published,35,36,40 are
regulated and coordinated by gap junctions and desmosomes
at the BTB, so that different junction types can be “disrupted”
and “re-established” orderly and perhaps in sequence, illustrat-
ing the physiological significance of gap junctions and desmo-
somes at the BTB.
Recent studies using a bisphenol A (BPA) model (note: BPA
is an estrogenic environmental toxicant that interacts with estro- this BPA-induced Sertoli cell TJ-barrier disruption is mediated
gen receptor in mammalian cells59-61) have shown that the BPA- by a blockade of the gap junction function due to the physical
induced disruption of the Sertoli cell TJ-barrier, both in vitro interaction of BPA with the Cx43-based gap junction channel.
and in vivo in immature rats,62 is likely mediated by a disrup- Using molecular modeling approach, it was found that similar
tion of the gap junction communication channel function as to estradiol-17β (Fig. 2), BPA (Fig. 3) was capable of interacting
demonstrated by a functional dye-transfer assay.40 These find- with Cx43-based gap junction channel via a putative “docking”
ings also provide the first functional proof regarding the role of domain in the Cx43-based connexon. These findings thus pro-
gap junctions at the BTB. They also prompted us to examine if vide a molecular basis that BPA-induced blockage of gap junction

www.landesbioscience.com Spermatogenesis 111

 Figure 5. For figure legend, see page 113.

channel-mediated dye transfer40 is likely to be the result of BPA
“docking” in the functional channel, disrupting the necessary
cross-talk maintained by the Cx43-based gap junctions at the
BTB. The molecular docking findings (Figs. 2 and 3) also illus-
trate that BPA can possibly traverse the BTB to enter the apical
compartment via the gap junction channels.
A recent study has shown that rats treated with adjudin at
50 mg/kg b.w. (by gavage) displayed a transient BTB disruption
(wherein BTB was disrupted by ~6–12 wk, but it was fully recov-
ered by 20 wk post adjudin treatment), but when adjudin was
used at a higher dose at 250 mg/kg b.w. (by gavage), BTB was
disrupted by 2 wk but did not recover up to 30 wk at the end of

112 Spermatogenesis Volume 1 Issue 2

 Figure 5 (See opposite page). A schematic illustration of a molecular/biochemical model on the regulation of BTB restructuring during the seminifer-
ous epithelial cycle of spermatogenesis in adult mammalian testes. This model was prepared based on recent findings in studies in the rat testis (see
text). On the left part, an intact and functional BTB is shown in which the BTB is constituted by actin-based TJ-, basal ES- and gap junction-protein
complexes, as well as the intermediate filament-based desmosome. The adhesion function of these protein complexes are maintained by the actin
filament bundles sandwiched in between cisternae of endoplasmic reticulum and the apposing plasma membranes of two adjacent Sertoli cells
provided by the basal ES, and the integrity of the actin filament bundles is maintained by their intriguing interactions with Eps8 and Arp2/3 protein
complex. Intermediate filaments at the desmosome also contribute to the integrity of the BTB, and it is obvious that there are cross-talks between
intermediate filaments and actin-based cytoskeleton to maintain the BTB function. Both protein kinases and polarity proteins are also involved in
maintaining the proper phosphorylation status and the cellular distribution of the adhesion protein complexes at the BTB. During stage VIII of the
epithelial cycle when BTB undergoes restructuring to accommodate the transit of preleptotene spermatocytes (which are interconnected by intercel-
lular bridges as “clones”72-75), “new” TJ-fibrils that create a “new” BTB are being assembled behind the transiting spermatocytes via de novo synthesis
of BTB proteins under the influence of androgen and estrogen. Furthermore, proteins at the “old” BTB site also undergo endocytosis facilitated by
polarity proteins (e.g., 14-3-3, PAR6). These protein trafficking events are perhaps triggered by c-Src- and/or FAK-mediated phosphorylation under the
influence of cytokines (TGFβ3, TNFα, IL-1α) and steroids (e.g., testosterone, estradiol-17β), so that they can be transcytosed and recycled to the “new”
BTB site, while some unwanted endocytosed proteins are targeted to degradation via the endosome- or ubiquitin-mediated pathways. Through this
mechanism, the integrity of the immunological barrier can be maintained because there is no “actual” “opening” or “degeneration” of the BTB at any
time during the transit of preleptotene spermatocytes at the site. Furthermore, TJ, basal ES, desmosome and gap junction integral membrane proteins
are also expressed on the cell surface of spermatocytes, so that they can form interlocking interactions with the corresponding proteins on the Sertoli
cell plasma membrane to maintain “loosely” associated protein complexes during their transit at the BTB.

the experimental period suggesting a permanent BTB damage.14
Interestingly, spermatogenesis failed to “re-initiate” in the high-
dose treated group while spermatogenesis re-initiates gradually in
the low-dose adjudin treated group. Thus, we sought to examine
if adjudin could also interact with the Cx43-based gap junction
channel via a “docking” domain. As shown in Figure 4, adju-
din was found to interact with the gap junction channel. These
observations are significant since gap junctions at the BTB,
unlike other blood-tissue barriers, are not sequestered from the
junctional complexes behind the TJ and the AJ and desmosome
plaques. Instead, they are intermingled with other junction types
and are immediately accessible to toxicants (e.g., BPA) or chemi-
cals (e.g., adjudin) when the host, including rodents and humans,
is exposed to harmful substances that exert their effects via the
gap junction. This thus renders the BTB (and the testis) more
vulnerable to environmental toxicants than the TJ-permeability
barrier of the microvessels in the interstitium. This apparently
new concept also opens a possible new window of research to
“manage” toxicant-induced male reproductive dysfunction by
protecting or sequestering gap junctions and desmosomes from
these toxicants.
Concluding Remarks and Future Perspectives
Herein, we review some unexpected findings based on recent
reports regarding the role of gap junctions and desmosomes that
coexist with TJ and basal ES at the BTB, illustrating these junc-
tions provide the necessary cross-talks and serve as the signal-
ing platform to maintain the homeostasis of different junction
types in the seminiferous epithelium during the cyclic restruc-
turing of the BTB throughout the seminiferous epithelial cycle
3.
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Studies in the authors’ laboratories were supported by grants from
the National Institutes of Health (R01 HD056034 to C.Y.C.;
R01 HD056034-02-S1 to C.Y.C.; U54 HD029990, Project 5
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