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- Original Article -
Disruption of ectoplasmic specializations between Sertoli cells
and maturing spermatids by anti-nectin-2 and anti-nectin-3
antibodies
Yoshiro Toyama, Fumie Suzuki-Toyota, Mamiko Maekawa, Chizuru Ito, Kiyotaka Toshimori
Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba 260-8670,
Japan
Abstract
Aim: To understand the biological functions of the ectoplasmic specializations between Sertoli cells and maturing
spermatids in seminiferous epithelia. Methods:
In order to disrupt the function of the ectoplasmic specializations,
nectin-2, which is expressed at the specialization, was neutralized with anti-nectin-2 antibody micro-injected into the
lumen of the mouse seminiferous tubule. Anti-nectin-3 antibody was also micro-injected into the lumen in order to
neutralize nectin-3, which is expressed at the specialization.
Results: The actin filaments at the specialization disappeared,
and exfoliation of maturing spermatids was observed by electron
microscopy. Conclusion: Nectin-2 was neutralized
by anti-nectin-2 antibody and nectin-3 was neutralized by anti-nectin-3 antibody, respectively. Inactivated nectin-2
and nectin-3 disrupted the nectin-afadin-actin system, and finally the actin filaments disappeared. As a result, the
specialization lost the holding function and detachment of spermatids was observed. One of the functions of the
specialization seems to be to hold maturing spermatids until
spermiation. (Asian J Androl 2008 Jul; 10: 577_584)
Keywords: ectoplasmic specialization; Sertoli cell; spermatogenic cell; testis; actin; nectin; mice
Correspondence to: Dr Yoshiro Toyama, Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba
University, Chiba 260-8670, Japan.
Tel: +81-43-226-2020 Fax: +81-43-226-2021
E-mail: ytoyama@faculty.chiba-u.jp
Received 2007-06-29 Accepted 2007-09-17
DOI: 10.1111/j.1745-7262.2008.00357.x
1 Introduction
Two types of ectoplasmic specializations are observed
in the seminiferous epithelium. One exists between
adjoining Sertoli cells, and the other exists between Sertoli
cells and maturing spermatids. Both types of
specializations in the seminiferous epithelium were formerly called
junctional specializations. The widely used term
"ectoplasmic specializations" was proposed by Russell [1].
The ectoplasmic specialization between adjoining Sertoli
cells is equipped with more than 100 tight junctional
strands and forms one of the tightest junctions. The
junctional strands function as the blood-testis barrier,
establishing a specialized environment essential for germ
cell development. The strands also function as an
immunological barrier, but the function is incomplete [2].
The function of the specialization between the Sertoli
cell and the maturing spermatids is not known. Many
hypotheses have been proposed: shaping of the sperm
head; holding maturing spermatids until spermiation;
releasing spermatozoa at the time of spermiation;
alignment of the maturing spermatids in the seminiferous
epithelium; and providing a scaffold for the microtubules.
As unique structures, both types of specializations
accompany layers of actin filaments [3] and the
subsurface cisternae of the smooth endoplasmic reticulum.
Knowledge of the molecules expressing in both
speciali-zations is accumulating [4, 5].
Nectins, Ca2+-independent immunoglobulin (Ig)-like
cell-cell adhesion molecules, play roles in cell adhesion,
migration, and polarization [6]. They constitute a family
of four members, nectin-1, nectin-2, nectin-3 and nectin-4,
and are associated with actin filaments through afadin.
Nectin-1, nectin-2 and nectin-3 are ubiquitously expressed
in a variety of cells, including fibroblasts, epithelial cells,
and neurons [7]. Nectin-2 and nectin-3 are also expressed
in cells that lack cadherins, such as B cells and monocytes.
Human nectin-4 is expressed mainly in the placenta.
Nectin-1 and nectin-2 were originally isolated as
poliovirus receptor-related proteins and named PRR-1 and
PRR-2, respectively. They were later renamed HveC
and HveB, respectively.
Nectins have an extracellular domain containing three
Ig-like loops, a transmembrane domain and a
cytoplasmic tail domain. Furthermore, the tail domain has a
conserved motif of four amino acid residues
(Glu/Ala-X-Tyr-Val) at the carboxyl terminus and this motif binds
the PDZ domain of afadin. Actin filaments bind the
F-actin-binding domain in afadin [7], forming the
nectin-afadin-F-actin system.
Sertoli cells express nectin-2 [8_10] and spermatids
express nectin-3 [9, 11]. In Sertoli cells, two nectin-2
molecules form a homo-cis-dimer, whereas, in the
spermatids, two nectin-3 molecules form a
homo-cis-dimer [6]. At the Sertoli cell-maturing spermatid
ectoplasmic specialization, the nectin-2 dimer in the Sertoli
cell and the nectin-3 dimer in the spermatid form a
hetero-trans-dimer that is much stronger in binding than the
homo-trans-dimer [6].
In the present study, nectin-2 in the Sertoli cells was
inactivated by anti-nectin-2 antibody. Similarly, nectin-3
in the spermatids was inactivated by anti-nectin-3 antibody.
In both experiments, the Sertoli-maturing spermatid
ectoplasmic specialization was disrupted and maturing
spermatids were exfoliated from the seminiferous epithelium.
The Sertoli-Sertoli ectoplasmic specializations, however,
were not affected.
2 Materials and methods
Adult ICR mice were used in this study. Animal
handling was approved by the Animal Research Committee of
Chiba University (Chiba, Japan).
2.1 Immunohistochemistry
Animals were anesthetized with pentobarbital and fixed
with Bouin's fluid by perfusion through the left ventricle.
The testes were removed, immersed in the same fixative,
processed for paraffin embedding and cut at a thickness
of 5 µm. Sections were autoclaved at 120ºC for 5 min to
activate the antigen. Non-specific binding of the antibody
was blocked in 10% fetal bovine serum in
phosphate-buffered saline (PBS) for 30 min at room temperature. The
sections were then incubated with goat anti-nectin-2
antibody (1:100 dilution; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) at 4ºC overnight. The antibody was raised
against a peptide mapping near the N-terminus in the
extracellular domain. After washing with PBS, samples were
incubated with biotinylated rabbit antigoat IgG (1:500
dilution; Dako Japan, Kyoto, Japan) for 1 h, followed by
incubation with streptavidin_biotin peroxidase complex
solution (DAKO LSAB2 system; Dako, Japan) for 30 min.
Immunohistochemical reactions were visualized using
3,3'-diaminobenzidine (DAB) and
H2O2. The sections were
then counterstained with hematoxylin. Sections for the
negative control were processed in the same manner, but
the anti-nectin-2 antibody treatment was omitted.
2.2 Confocal laser scanning microscopy
Animals were perfused through the left ventricle with
4% paraformaldehyde in PBS. The testes were removed,
immersed in the same fixative for an additional 4 h, rinsed
with PBS, embedded in optimal cutting temperature
compound and cut at a thickness of 20 µm on a cryostat. The
sections were incubated with a blocking solution for 1 h
at room temperature to block the non-specific binding of
the antibody, then incubated with rat anti-nectin-3
antibody (1:500 dilution; Abcam, Cambridge, UK) at 4ºC
overnight. The antibody was raised against the
recombinant fragment corresponding to the mouse nectin-3
extracellular domain. After washing with PBS, the slides were
incubated with Alexa Fluor 488 goat antirat IgG
(0.5 µg/mL; Invitrogen, Carlsbad, CA, USA) and
propidium iodide (1 µg/mL; Sigma, St. Louis, CA, USA) in the
blocking solution for 1 h at room temperature. After washing
with PBS, the samples were mounted with PermaFluor
(Immunon, Pittsburgh, PA, USA) and observed using a
confocal laser scanning microscope (LSM 410; Zeiss,
Jena, Germany). Sections processed in the absence of
the anti-nectin-3 antibody were used for the negative
control.
2.3 Micro-injection experiments
Anti-nectin-2 antibody, anti-nectin-3 antibody and
anti-afadin antibody were used in the micro-injection
experiments. Ten microliters of the antibody solution,
890 µL of medium CZB [12], and 100 µL of 1% Trypan
blue was mixed thoroughly and filtered with a Millipore
filter (0.22 µm; Millipore, Bedford, MA, USA). The
solution contained 0.01% sodium azide.
The mixture was micro-injected into the lumen of the
seminiferous tubule according to the method introduced
by Brinster and Zimmermann [13] and Koh et
al. [14]. Briefly, the scrotum and tunica vaginalis were cut with
surgical scissors under deep anesthesia. The tunica
albuginea was exposed and a small cut (approximately
0.1 mm in length) was made in the tunica. A glass
pipette (inner diameter, 70 µm) filled with the antibody
mixture was inserted into the lumen of the seminiferous
tubule through the cut in the tunica. Approximately 3 µL
of the mixture was injected into the lumen. The
topography of the seminiferous tubules showing blue in color,
because of the Trypan blue in the mixture, was recorded,
and the tunica vaginalis and the scrotum were sutured.
One seminiferous tubule in the right testis was selected
for the micro-injection. The left testis remained intact.
Antibodies against nectin-2, nectin-3, and afadin were
micro-injected.
After 1, 2, 5 and 12 h, the animals were anesthetized
and perfusion fixed with glutaraldehyde. The testes were
processed for electron microscopy. Semithin sections
stained with toluidine blue were observed with a light
microscope, and ultrathin sections stained with uranyl
acetate and lead citrate were observed with an electron
microscope (JEM 1200EX II; JEOL, Tokyo, Japan). As
the seminiferous tubule often makes hairpin bends in the
course, it is common to observe several cross-sections
of a seminiferous tubule in a single histological section.
As negative control experiments, the same amounts
of the mixtures without the antibody were micro-injected
into the seminiferous tubules. Briefly, 10 µL of the
antibody solution without antibody, 890 µL of medium CZB,
and 100 µL of 1% Trypan blue was mixed thoroughly
and filtered with a Millipore filter (0.22 µm). The
solution contained 0.01% sodium azide.
3 Results
3.1 Immunohistochemistry and confocal laser
microscopy
Nectin-2 was expressed around the heads of
maturing spermatids, as well as the basal part of the
seminiferous epithelium (Figure 1A). The localization around the
heads corresponded to the ectoplasmic specialization
between the Sertoli cells and maturing spermatids; the
localization at the basal part corresponded to the
ectoplasmic specialization between adjoining Sertoli cells. In
the negative control sections, the signal for nectin-2 was
not detected (Figure 1B).
Nectin-3 was expressed around the heads of
maturing spermatids (Figure 2A), corresponding to the
ectoplasmic specialization between the Sertoli cells and
maturing spermatids. Nectin-3 was not expressed at the
ectoplasmic specialization between the adjoining Sertoli
cells (Figure 2A). The negative control sections showed
no reactivities (Figure 2B).
3.2 Micro-injection experiments
The seminiferous tubules micro-injected with the
solution without antibody (negative control experiments)
showed minor ultrastructural changes, such as the
presence of enlarged vacuoles in the Sertoli cells and in the
spermatocytes. The intercellular space tended to be
enlarged (Figure 3). The ectoplasmic specialization
between the Sertoli cells and the maturing spermatids in
the control experiments was intact together with the
actin layers and subsurface cistern of the endoplasmic
reticulum (Figure 4). The ectoplasmic specialization
between adjoining Sertoli cells was also intact.
Five hours after micro-injection of anti-nectin-2
antibody, the histological sections of the seminiferous
tubules showed similar features to the control testis,
except for the exfoliation of maturing spermatids (Figure
5). Electron microscopy at this stage showed that part
of the actin layer in the ectoplasmic specialization
between the Sertoli cells and maturing spermatids was
disrupted. By 12 h, the actin layer had completely
disappeared (Figures 6, 7), and maturing spermatids were
observed in the lumen of the seminiferous tubules
because of detachment. The ectoplasmic specialization
covering the acrosomal cap of a step 8 spermatid was
also affected by the anti-nectin-2 antibody (Figure 6).
Micro-injection of anti-nectin-3 antibody showed
similar structural changes. By 5 h after the injection,
maturing spermatids were exfoliated (Figure 8), and by
12 h after injection the actin layer at the ectoplasmic
specialization between Sertoli cells and spermatids had
completely disappeared (Figures 9, 10).
After micro-injection of anti-afadin antibody, no
drastic changes in the ultrastructure were observed. The
ultrastructure was similar to that in the negative control
animals.
The Sertoli-Sertoli ectoplasmic specialization was not
affected by either anti-nectin-2, anti-nectin-3, or
anti-afadin antibodies. The specialization showed an intact
ultrastructure with actin filaments and the subsurface
cistern (Figure 11).
4 Discussion
The antibody solutions used in the present study were
micro-injected into the lumen of seminiferous tubules.
The solution filled the lumen, penetrated into the
intercellular space in the adluminal compartment of the
seminiferous epithelium and stopped penetrating at the
adluminal edge of the ectoplasmic specialization between
Sertoli cells. The solution also penetrated into the
intercellular space at the ectoplasmic specialization between
Sertoli cells and maturing spermatids. This was already
shown by Toyama et al. [15], who micro-injected
cytochrome c into the lumen of the seminiferous tubules of
mouse and rat and electron microscopically traced the
intercellular space after a 3,3'-diaminobenzidine reaction.
Nectins have carboxyl termini in the cytoplasmic
domain and N-termini in the extracellular domain. As the
anti-nectin-2 antibody used in the present study was
raised against the amino acid residues near the N-terminus,
the epitope was exposed in the intercellular space at the
Sertoli cell-maturing spermatid ectoplasmic
specialization and the antibody bound the epitope of nectin-2. As
a result, nectin-2 generated a conformational change and
appeared to disrupt the actin filaments in the Sertoli cells
through the nectin-afadin-actin system.
As disruption of the actin filaments at the
ectoplasmic specialization between Sertoli cells and maturing
spermatids was not observed in the negative control
experiments, the disruption was a result of the injection
of the anti-nectin-2 antibody. Both the antibody mixture
and the control mixture contained 0.01% sodium azide,
therefore the disruption was not an adverse effect of
sodium azide.
Micro-injected anti-nectin-3 antibody, as well as
anti-nectin-2 antibody, penetrated into the intercellular space
in the adluminal compartment. The anti-nectin-3
antibody used in the present study was also raised against
the recombinant fragment, corresponding to the mouse
nectin-3 extracellular domain. The seminiferous tubules
micro-injected with anti-nectin-3 antibody showed
similar adverse effects, including disruption of the actin
filaments in Sertoli cells at the Sertoli-maturing spermatid
ectoplasmic specialization and exfoliation of maturing
spermatids from the seminiferous epithelium. The
presence of afadin and F-actin in the spermatid at the
specialization has not yet been ascertained. The results of
the present study suggest that nectin-3 in the spermatids
affected the actin filaments in the Sertoli cells through
nectin-2 and afadin. A possible but highly speculative
explanation to these adverse effects is as follows.
Anti-nectin-3 antibody bound the epitope of nectin-3, which is
exposed in the intercellular space at the specialization,
and changed nectin-3, which in turn affected nectin-2,
which was forming the hetero-trans-dimer with
nectin-3. Nectin-2, affected by the inactivated nectin-3 in the
spermatids, seemed to disrupt the actin filaments in the
Sertoli cells through the nectin-afadin-actin system, as
mentioned above. Nectin-3 and nectin-2 finally disrupted
the actin filaments at the ectoplasmic specialization
between Sertoli cells and maturing spermatids. This is
supported by the fact that nectin-2 at the ectoplasmic
specialization between Sertoli cells and maturing spermatids
completely disappeared in nectin-3 knockout mice [11].
The close relationship between nectin-2 and nectin-3 was
also reported in nectin-2 and nectin-3 knockout mice
[8_11]. Male infertility, because of malformed spermatozoa,
was the prominent feature. The heads of spermatozoa
from both nectin-2 and nectin-3 knockout mice were
heterogeneous in shape and had malformed acrosomes
and large vacuoles. The tails were coiled up around the
heads and the mitochondrial sheaths were disorganized.
The mitochondria were detached from the outer dense
fibers in the middle piece and came together in the heads,
forming the so-called "stratified mitochondrial sheaths".
Similar abnormalities were observed in spermatozoa from
Golgi-associated PDZ- and coiled-coil motif-containing
protein-deficient mice [16]. The state of the
spermatozoa from these mice is known as a globozoospermia,
lacking acrosomes and posterior rings in their heads.
The seminiferous tubules micro-injected with
anti-afadin antibody showed no remarkable changes, as
expected. As afadin has neither a transmembrane
domain nor an extracellular domain, it is reasonable that the
antibody, micro-injected into the intercellular space at
the Sertoli-maturing spermatid specialization, did not bind
any epitope in afadin.
Vacuoles in the Sertoli cells and enlarged
intercellular spaces were observed in all experimental systems
(Figures 5_10), including the negative controls (Figures
3, 4). As the solution for all experimental systems
contained 0.01% sodium azide, it might cause the enlarged
vacuoles and intercellular spaces.
The present study suggests that the ectoplasmic
specialization between Sertoli cells and maturing spermatids
holds the spermatids until spermiation, as was put
forward by Brokelmann [17], Nicander [18], and Fawcett
[19]. Similar results, deletion of the ectoplasmic
specialization between Sertoli cells and maturing spermatids,
were reported in mouse testes treated with estrogen or
estrogen-like chemicals, such as bisphenol A [15, 20].
The researchers also concluded that the ectoplasmic
specialization shapes the nucleus of the mature spermatids,
as the chemicals induced malformation of the heads of
mature spermatids.
Injected antibodies directly bind and inactivate the
proteins that are expressed at the ectoplasmic
specialization between Sertoli cells and maturing spermatids,
therefore this experimental system, a micro-injection system,
as well as the generation of knockout mice, are useful
tools for understanding the function of the specialization.
However, one disadvantage of this experimental system
is that antibody concentration in the lumen cannot be
controlled.
The ectoplasmic specialization between adjoining
Sertoli cells equipped with tight junctions was not affected
by the antibodies used (anti-nectin-2, anti-nectin-3, and
anti-afadin antibodies). Because the antibody did not bind
the antigen, the conformational change in the antigen
(nectin-2, nectin-3, and afadin) did not occur. As a result,
the ultrastructure and function of the specialization
remained intact. Explanations for this are as follows. As
anti-nectin-2 antibody micro-injected into the lumen of
the seminiferous tubule penetrated into the intercellular
spaces in the adluminal compartment of the
seminiferous epithelium and was stopped at the uppermost strand
of the tight junction, just like cytochrome c
micro-injected into the lumen [15], the antibody did not reach the
epitope. The anti-nectin-3 antibody was not localized at
the specialization, as shown in Figure 2A. Because afadin
has neither a transmembrane domain nor an extracellular
domain, the antibody did not bind the epitope.
The function of the blood-testis barrier was also intact.
This is supported by the fact that normal
spermatogenesis, except for detachment of maturing spermatids, was
observed. Without the blood-testis barrier,
spermatogenesis is arrested at meiosis. Although spermatozoa
were malformed and infertile, nectin-2 and nectin-3
knockout mice showed spermatogenesis [8_11],
suggesting that the function of the blood_testis barrier in these
mice was normal.
Acknowledgment
This study was supported by a Grant-in-Aid for
Scientific Research from the Japan Society for Promotion of
Science to Y.T. (17590151). The authors thank Dr Eric
Spicer, ESTI, USA for careful reading of the manuscript.
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