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The Population Council, Center for Biomedical Research, New York, New York 10021
Address all correspondence and requests for reprints to: C. Yan Cheng, Ph.D., The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: yan{at}popcbr.rockefeller.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin, cathepsin L, N-cadherin,
ß-catenin, zonula occludens 1 (ZO-1), sertolin,
prostaglandin D2 synthetase (PGD-S), and
myotubularin (5, 6, 7, 8, 9, 10, 11, 12, 13). However, a model to study the events
of junction disassembly is lacking. If such a model is available, it
will be useful to study the biology of junction restructuring during
spermatogenesis in multiple ways. First, this model can be used to
investigate the events leading to spermiation. Second, it can be used
to identify target genes that are involved in the breakdown of the
inter-Sertoli TJs to permit the passage of preleptotene spermatocytes.
Third, it can become a potential in vitro assay system to
screen contraceptives targeted to prevent or induce junction
disassembly. It is known for almost 4 decades that cadmium can induce disruption of the BTB in the testis, which can be prevented by zinc (14). Although the precise mechanism is not clearly defined, some biochemical alterations were detected in the testis after cadmium exposure, such as reduced activity of testis-specific anhydrase and glutathione disulfide reductase, and increased activity of glutathione peroxidase (15). Earlier studies have shown that Leydig cells may be the target of cadmium toxicity (16). More recent in vitro studies, however, suggest that the Sertoli cell is the most vulnerable target of CdCl2 and is more sensitive than Leydig cells to cadmium-induced damage (17). In vivo injection of CdCl2 (30 µmol/kg BW) to rats induced an increase in BTB permeability, indicating a disruption of inter-Sertoli TJs (18, 19). Studies by confocal microscopy illustrated that CdCl2 disrupted TJ-associated microfilaments in rat Sertoli cells, which, in turn, damaged the BTB (20). CdCl2 also induced apoptosis in testicular tissue after ip injection (21). This event was characterized by an activation of endonuclease as a result of the loss of intracellular calcium (22). The CdCl2-induced damage of the inter-Sertoli TJ permeability barrier was proposed as a model for toxicological investigations of the BTB in vitro (23). In addition, disruption of paracellular barrier by CdCl2 was detected in intestinal epithelial cell lines, Caco-2 and IEC-18 (24). Despite these earlier studies, it is not known whether CdCl2-induced disruption of inter-Sertoli TJ can become a model to study the events of junction disassembly, because it must be able to fulfill the following criteria. First, at the doses used to disrupt the inter-Sertoli TJs, CdCl2 must be noncytotoxic to the treated cells. Second, the effect must be reversible, so that the disrupted inter-Sertoli TJs are capable of resealing. Third, the transient induction of target genes when inter-Sertoli TJs are reassembled after the removal of CdCl2 should be similar to the changes detected when inter-Sertoli TJs are assembled shortly after their isolation from the testes. If a suitable in vitro model of junction disassembly is found, not only will it be useful to study this aspect of junction restructuring during spermatogenesis, but it will enhance our ability to screen compounds that can disrupt this cellular event, leading to the discovery of new agents to disrupt spermatogenesis and regulate male fertility.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of Sertoli cell cultures
Primary Sertoli cells were prepared from 20-day-old Sprague
Dawley rats by sequential enzymatic treatment as previously described
(25, 26). Cells were resuspended in serum-free Ham’s F-12
nutrient mixture and F12/DMEM (1:1, vol/vol; Life Technologies, Inc., Grand Island, NY) supplemented with 15 mM
HEPES, 1.2 g/L sodium bicarbonate, 10 µg/ml bovine insulin, 5 µg/ml
human transferrin, 2.5 ng/ml epidermal growth factor, 20 mg/liter
gentamicin, and 10 µg/ml bacitracin and cultured at either low or
high cell density at 35 C in a humidified atmosphere with 5%
CO2/95% air. For low cell density cultures,
Sertoli cells were plated at 5 x 104
cells/cm2 in either 100-mm petri dishes with 9-ml
F12/DMEM or Matrigel (Collaborative Biochemical Products, Bedford,
MA)-coated (diluted 1:7 with F12/DMEM, vol/vol) bicameral units to
allow the formation of monolayer cultures without the assembly of
inter-Sertoli TJs when monitored by transepithelial electrical
resistance (TER) across the Sertoli cell epithelia on Matrigel-coated
bicameral units and other criteria as previously described
(27). Under this culture condition, it was found that
adherens (AJ) and gap junctions (GJ) were formed (9). For
high cell density cultures, Sertoli cells were plated on
Matrigel-coated 24-well dishes or bicameral units at a density of
1.2 x 106 cells/cm2
as previously described (27) to allow the formation of TJ,
AJ, and GJ, mimicking the in vivo morphology of Sertoli
cells. In selected culture experiments, 24 h after Sertoli cells
were isolated and plated (day 1) the culture medium was supplemented
with FSH (100 ng/ml) and T (T; 1 x
10-9 or 2 x
10-7
M). About 48 h after plating (on day 2 of
culture), cultures were hypotonically treated with 20
mM Tris-HCl, pH 7.4, for 2.5 min to lyse
contaminating germ cells (28) to obtain Sertoli cell
cultures with greater than 95% purity.
Effects of CdCl2 on the assembly and
maintenance of inter-Sertoli TJs in vitro
To examine the effects of CdCl2 on the
assembly and maintenance of inter-Sertoli TJ permeability barrier
in vitro, primary Sertoli cells were isolated as described
above and cultured at 1.2 x 106
cells/cm2 on Matrigel (1:7)-coated bicameral
units (Millipore Corp., Bedford, MA) as previously
described (26). TJ assembly was monitored by TER
measurement across the Sertoli cell epithelia using a Millicell
Electrical Resistance System as previously described (12, 27). Briefly, a short (
2 sec) pulse of current (20 µA)
was passed through the Sertoli cell epithelia between two silver-silver
chloride electrodes, and resistance was calculated from the change in
voltage across the Sertoli cell epithelia. The resistance was
multiplied by the effective growth area of the filter (0.6
cm2) to yield the areal resistance
(ohms · cm2). The net value of TER was computed by
subtracting the background, which was measured on the Matrigel-coated
cell-free chambers, from values of Sertoli cell-plated chambers. To
minimize temperature-induced fluctuation when recording TER, cultures
were stabilized at room temperature for 20 min before TER measurement.
CdCl2 at 0.1–10 µM was
added to both the basal and apical compartment of the bicameral unit on
days 1 and 4 and incubated for 8 h to assess the effect of
CdCl2 on the assembly and maintenance of the
inter-Sertoli TJ, respectively. It is noted that in preliminary
experiments we had examined whether there was any difference in TER
across the Sertoli cell epithelia if CdCl2 at 5
µM was added to the apical, basal, or both
chambers of the bicameral units on day 1 of culture (see Fig. 1A
). It was found that the inter-Sertoli
TJ permeability barrier responded similarly to the
CdCl2 treatment regardless of where this compound
was added. As such, CdCl2 was added to both
chambers in all experiments. The inter-Sertoli TJs were assembled by
day 4, as manifested by a stable TER across the cell epithelia.
Controls were Sertoli cells cultured in F12/DMEM alone or in the
presence of FSH and two different concentrations of T. After
CdCl2 exposure, all experimental and control
cultures were washed with fresh F12/DMEM three times, and cultures were
incubated for an additional 4–7 days. TER readings were recorded daily
before media were replaced. Each time point had triplicate cultures,
and each experiment was repeated at least twice using different batches
of cells.
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Effects of T on the cellular gene expression by Sertoli cells
Sertoli cells were prepared as described above and cultured at
1.2 x 106 cells/cm2
on Matrigel-coated dishes in F12/DMEM supplemented with insulin,
epidermal growth factor, transferrin, and bacitracin. On day 3, Sertoli
cells were exposed to T at either 1 x
10-9 or 2 x
10-7 M for
2–24 h in the absence of ovine FSH. Cultures were terminated at
specific time points by RNA STAT-60 for RNA extraction.
Semiquantitative RT-PCR was then performed to monitor the effects of T
on steady state messenger RNA (mRNA) levels of occludin, uPA, and
E-cadherin.
Semiquantitative RT-PCR to assess changes of target gene expression
during CdCl2-induced junction disassembly
and reassembly
When the inter-Sertoli TJ permeability barrier was disrupted by
CdCl2, as manifested by a decline in TER across
the Sertoli cell epithelia, cell samples at specified time points were
terminated for total RNA extraction. RT-PCR was performed to monitor
changes in the expression of target genes, such as E-cadherin (an AJ
protein), occludin (a TJ-integral membrane protein), and urokinase
plasminogen activator (uPA, a serine protease). Semiquantitative RT-PCR
was performed essentially as previously described (7, 8, 9, 10, 27). Approximately 3 µg total RNA were reverse transcribed
into complementary DNAs with 1 µg
oligo(deoxythymidine)15 using a Moloney murine
leukemia virus RT kit (Promega Corp., Madison, WI) in a
final reaction volume of 25 µl. In preliminary experiments, PCR
products were examined over a range of 20–28 amplification cycles to
ensure linearity using different concentrations of RT products and
primers. PCR was performed by combining 3 µl of the RT product with
0.3 µg of each sense and antisense primer of the selected target
gene, and with rat ribosomal S16 primer pairs (Table 1), 5 µl 10 x PCR buffer, 3 µl
MgCl2 (25 mM), 8 µl deoxy (d)-NTPs
(200 µM each of dATP, dGTP, dCTP, and dTTP), 2.5 U
Taq DNA polymerase, and sterile double distilled water to a
final reaction volume of 50 µl. The cycling parameters for PCR were
as follows: denaturation at 94 C for 1 min, annealing at 58-62 C for 2
min, and extension at 72 C for 3 min, for a total of 22–24 cycles,
which was followed by a 15-min extension at 72 C in a
Perkin-Elmer Corp. thermal cycler (Norwalk, CT). To
enhance the detection limit and to yield data for quantitative
analysis, PCR was performed in the presence of
-32P-labeled primer. Briefly, the sense
primers of E-cadherin, occludin, uPA, and S16 were labeled at the
5'-end with [-32P]dATP (SA, 6000 Ci/mmol;
Amersham Pharmacia Biotech, Arlington Heights, IL) using
T4 polynucleotide kinase (Promega Corp., Madison, WI). Approximately, 3 x
106 cpm were used per PCR reaction. To ensure the
linearity of both target gene and S16 during their amplification by
PCR, 10-µl aliquots of PCR products at 20, 22, 24, 26, and 28 cycles
were withdrawn and resolved onto 5% T (total acrylamide)
concentration (gm/100 ml = acrylamide + methylene bisacrylamide
polyacrylamide gels using 0.5 x TBE (44.5
mM Tris-borate and 1
mM EDTA, pH 8.0) as running buffer. PCR products
were visualized by ethidium bromide staining, and autoradiography was
performed using Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY). Autoradiograms were densitometrically
scanned at 600 nm using an Ultro-Scan XL enhanced laser densitometer
(Pharmacia Amersham Pharmacia Biotech) to yield
semiquantitative data for statistical analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Similar results were
obtained when the assembly of TJs was assessed by other techniques,
such as restricted diffusion of [3H]inulin or
[125I]BSA from the apical to the basal chamber
of the bicameral unit, and polarized secretion of Sertoli cell
proteins, such as transferrin, androgen-binding protein, and testin
(26, 30). As such, TER measurement was selected to assess
the effects of CdCl2 on the assembly and
maintenance of inter-Sertoli TJs, because this method is relatively
simple to perform, and the data obtained are semiquantitative. It is
also noted that this assay was widely used by cell biologists in the
field since its introduction (31, 32). After Sertoli cells
were exposed to CdCl2 at either 5 or 10
µM for 8 h on day 1, there was a delay in
the assembly of inter-Sertoli TJs when CdCl2 was
present in the medium at 5 µM compared with
control cultures (Fig. 1A). Also, the inter-Sertoli TJ was less
"tight" in the presence of 5 µM
CdCl2. For instance, on day 3, cultures treated
with 5 µM CdCl2 had a TER
of 15 vs. 35 ohm · cm2 in controls
(Fig. 1A). In contrast, CdCl2 at 10
µM virtually abolished inter-Sertoli TJ
assembly (Fig. 1A). As Sertoli cells isolated from 20-day-old rats used
in the present study were nondividing when assessed by
[3H]thymidine incorporation studies (Mruk, D.,
and C. Y. Cheng, unpublished observation), the number of Sertoli
cells should be consistent throughout the culture period if they stay
viable. Thus, the measurement of DNA content in these cultures is a
reliable index to assess cell viability. Apparently, the observed
effects of CdCl2 (5–10
µM) on the inter-Sertoli TJ permeability
barrier was not the result of cell cytotoxicity, because the DNA
content of CdCl2-treated cultures was not
significantly different from that of controls (Fig. 1B).
Effects of CdCl2 on the maintenance of
inter-Sertoli TJs in vitro in the absence and presence of FSH and T
When Sertoli cells were cultured in serum-free F12/DMEM
without FSH or T, inter-Sertoli TJs were assembled by day 4 as
manifested by a stable TER across the Sertoli cell epithelia. To study
the effects of CdCl2 on the maintenance of
inter-Sertoli TJs, Sertoli cells were exposed to different
concentrations of CdCl2 ranging from 0.1–5
µM for 8 h on day 4 when inter-Sertoli TJs were
assembled, and CdCl2 was then removed thereafter
by rinsing Sertoli cells with fresh F12/DMEM. It was noted that the TER
across the cell epithelia in CdCl2-treated
cultures declined dose dependently, indicating the inter-Sertoli TJs
were being disrupted (Fig. 2A). As shown
by the colorimetric DNA assay, the CdCl2-induced
TJ disruption was unlikely to be due to cell toxicity, as DNA contents
of treated cultures on day 8 were not significantly different
from those of controls (Fig. 2B). Interestingly, when Sertoli cells
were cultured under the same condition but in the presence of both FSH
(100 ng/ml) and T (1 x
10-9 M), the
CdCl2-induced disruption of the inter-Sertoli TJ
permeability barrier could be resealed when CdCl2
was present at 0.1–1 µM, a dosage capable of disrupting
inter-Sertoli TJs (Figs. 3A
vs. 2A). When T was present at 2 x
10-7
M, it could prevent CdCl2
(at 0.1–1 µM) from inducing disruption of
inter-Sertoli TJs unless it was present at a concentration of 5
µM (Fig. 3C). Based on these observations, it
is logical to conclude that a high concentration of T, such as 2
x 10-7
M, could counteract the
CdCl2-induced damage to the inter-Sertoli TJ
permeability barrier in vitro, but the inclusion of T at
1 x 10-9
M in CdCl2-treated Sertoli
cells, this can possibly become an in vitro model to study
junction disassembly. The DNA contents of treated cultures were not
significantly different from those of controls, except that treated
with 5 µM in the presence of T at 2 x
10-7
M (Fig. 3, B and D). When the results in
Fig. 3D were compared with those shown in Figs. 1B and 2B, it seems
that the presence of T coupled with a high concentration of
CdCl2, such as 5 µM, can
induce cell cytotoxicity.
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, A and B)
and 2 x 10-7
M, respectively (Fig. 4, C and D), and cultured
for an additional 24 h in the absence of FSH. It was found that T
alone at 1 x 10-9
and 2 x 10-7
M elicited a mild, but statistically significant,
increase in occludin steady state mRNA level at 10–24 and 8–24 h,
respectively, compared with cultures at time zero. It was noted that
such a T-induced occludin expression was not detected in the control
experiment without T (Fig. 4, E and F), illustrating that the increase
in occludin expression shown in Fig. 4 was not the result of an
endogenous induction in these Sertoli cells. Although the assembly of
inter-Sertoli TJs was known to induce occludin expression, its steady
state mRNA level remains elevated between days 2–4.5 (33, 40), further suggesting that the increase shown in Fig. 4 was a
result of T stimulation rather than an endogenous change. Moreover,
when these same samples were analyzed for E-cadherin and u-PA
expression by semiquantitative RT-PCR, T had no apparent effect on
their cellular expression (data not shown).
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Effects of CdCl2 on the expression of
E-cadherin. E-Cadherin is a Ca2+-dependent
cell adhesion molecule known to participate in the assembly of AJs
(34); however, CdCl2-induced
disruption did not alter the expression of E-cadherin while the
inter-Sertoli TJs were being disrupted and reassembled (Fig. 5, A and B, and Fig. 3A).
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, A and B, and Fig. 3A). These results
are in contrast to the cellular expression of E-cadherin, whose steady
state mRNA level remained constant when inter-Sertoli TJs were
disassembled and reassembled (Fig. 6 vs. Fig. 5).
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, A and B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin (6, 12);
and cell adhesion molecules, such as N-cadherin and ß-catenin
(6). However, these studies are corroborative in nature,
proof that their involvement in junction assembly is not established.
Still, these results illustrate that germ cell migration is a dynamic
cellular event, requiring the participation of an array of molecules. A
more recent study in which transforming growth factor-ß is shown to
abolish the timely expression of ZO-1 and occludin when inter-Sertoli
TJs are being assembled and that it can perturb the inter-Sertoli TJ
permeability barrier (40) provides the first proof that
these earlier descriptive studies to delineate the events of junction
reassembly and their regulation are physiologically important.
Nevertheless, an in vitro model to study the events of
junction disassembly is still lacking. Administration of CdCl2 to rats in vivo by ip was shown to induce vascular permeability; this, in turn, led to edema, hemorrhage, and eventually necrosis (41, 42, 43, 44). Other in vivo studies also demonstrated that CdCl2 caused irreversible disruption of TJ-associated microfilaments in rat Sertoli cells (21). In addition to the damage in the testis, CdCl2 disrupted vascular endothelial integrity in different organs (for review, see Ref. 45). Likewise, CdCl2 disrupted the endothelial TJ permeability barrier in vitro, such as human umbilical vascular endothelial cells (for review, see Ref. 46), and the Sertoli cell permeability barrier in vitro (23).
In this report, the addition of CdCl2 to Sertoli
cells inhibited not only the assembly but also the maintenance of
inter-Sertoli TJs dose dependently. This
CdCl2-induced disruption was irreversible in
serum-free medium; however, its effect became reversible in FSH- and
T-supplemented medium. More important, the disruptive effect of
CdCl2 could be counteracted using a
pharmacological dose of T at 2 x
10-7 M. These
results led us to speculate that T may play a role in regulating TJ
assembly through an as yet to be defined signal transduction pathway.
However, we have not investigated the potential protective effects of
FSH alone on CdCl2-induced inter-Sertoli TJ
permeability barrier disruption. In this study, T stimulated Sertoli
cell occludin expression in vitro, and the effect was more
profound at 2 x 10-7
than at 1 x
10-9
M, indicating that T may exert its action by
inducing TJ integral proteins, which subsequently become the building
blocks of TJs. Although 2 x
10-7
M (200 nM) T, used in this
study, is about 100-fold higher than the level of T in the systemic
circulation (2 nM), the levels of T in rete
testis fluid and seminiferous tubular fluid range between 50 and 200
nM (47), illustrating that the
microenvironment that surrounds developing germ cells is indeed
equipped with such a high level of T. On the other hand, occludin is
known to be induced, and its protein is accumulated at the site of
inter-Sertoli TJs at 15–18 days of age when the BTB is being assembled
in vivo (48, 49). It is therefore possible that
the timely opening and closing of TJs during spermatogenesis that
facilitate the passage of preleptotene spermatocytes through the BTB
are regulated at least in part by testicular hormones. For instance, T
may exert its effects in regulating the BTB by modulating de
novo synthesis of TJ integral proteins, such as occludin. This
speculation is further strengthened by a recent observation that the
expression of claudin-11 in vitro is regulated by FSH and
tumor necrosis factor- (50), suggesting a critical role
of testicular hormone and signaling molecules in remodeling the BTB
during spermatogenesis. An earlier report also demonstrated that
treatment of rats with androgens delayed the onset of infertility after
their exposure to CdCl2 (44).
Apparently, the effects of CdCl2 on the
inter-Sertoli TJ permeability barrier are unlikely to be due to cell
toxicity, because the DNA content of the
CdCl2-treated cultures was not significantly
different from that of control cultures. Moreover, the
CdCl2-induced disruption of the inter-Sertoli TJ
barrier could be resealed after CdCl2 was
removed, illustrating that cells were still viable after the
CdCl2 treatment. However, we consistently
observed that when Sertoli cells were cultured in the presence of T at
2 x 10-7
M and exposed to 5 µM
CdCl2, these cultures had a DNA content
significantly lower than that of Sertoli cell cultures exposed to 0,
0.1, or 1 µM CdCl2 at the
end of the experiment (day 8), illustrating that cell viability was
reduced by CdCl2 treatment in the presence of a
high concentration of T. These results seemingly suggest that although
the presence of 2 x
10-7
M T could protect the inter-Sertoli TJ
permeability barrier from the disruptive effects of
CdCl2, its presence apparently also made the
Sertoli cell more vulnerable to CdCl2 treatment,
in particular when CdCl2 was present at a
concentration as high as 5 µM, possibly by
altering some as yet to be defined cellular properties.
The CdCl2-induced inter-Sertoli TJ disassembly also associates with changes in cellular expression of E-cadherin, occludin, and uPA. It has been reported that the assembly of TJs in vitro requires the participation of adhesion molecules, such as E-cadherin. For instance, addition of anti-E-cadherin antibody to MDCK cells impaired resealing of the Ca2+-depletion-induced TJ permeability barrier disruption (31). Studies by Northern blots, immunoblots, and immunohistochemistry have shown that expression of E-cadherin is the highest in the rat testis at 14 days of age just before the establishment of BTB (51), illustrating its important role in inter-Sertoli TJ assembly. Apart from its involvement in the assembly of BTB, E-cadherin has also been shown to mediate an early adhesion event between epithelial cells that is required for the assembly of desmosomes and adherens junctions (52). Taking these data collectively, it is clear that E-cadherin plays a crucial role in the assembly of AJs, GJs, and TJs. The CdCl2-induced inter-Sertoli TJ disruption may be mediated by its initial disruption in AJs even though a decline in E-cadherin expression was not detected, as a disruption of AJs may not require any changes in the synthesis of their building blocks. This speculation is reached based on several recent studies suggesting that E-cadherin is the target of CdCl2. First, CdCl2 causes redistribution of E-cadherin in Caco-2 cells when examined by immunohistochemistry (for review, see Ref. 46). Second, studies by immunohistochemistry using MDCK and LLC-PK1 cells cultured in vitro have shown that E-cadherin becomes diffusely localized in the cytoplasm rather than at the cell-cell border in Cd2+-treated cells, reflecting the fact that E-cadherin is translocated from the site of AJs to cytoplasm instead of a disruption in its expression or synthesis upon CdCl2 treatment (for review, see Ref. 46). Of course, the biochemical mechanism by which this molecular translocation is initiated and regulated remains to be determined. The fact that the Cd2+-mediated E-cadherin redistribution is similar to that induced by Ca2+ depletion strongly suggests that Cd2+ may compete with Ca2+ to the same binding sites in E-cadherin, which causes its subsequent dissociation.
It has been reported that integral TJ proteins, such as occludin and claudins, play an important role in forming paracellular seal (for review, see Ref. 35). Besides claudins, occludin is another TJ integral protein found in the TJ fibrils (53). It is concentrated in TJs of mouse/rat, but not human/guinea pig, Sertoli cells (48). An immunolocalization study in the mouse revealed that occludin is localized at the basal region of seminiferous tubules by postnatal day 14 when BTB is being assembled (49). In vitro studies from our laboratory have shown that occludin is a biochemical marker to monitor inter-Sertoli TJ assembly (33, 40). Moreover, addition of a synthetic peptide corresponding to the second external loop of occludin can perturb the inter-Sertoli TJ assembly in vitro and spermatogenesis in vivo (33). Recent studies using glycerol-treated rat testes have shown that the glycerol-induced disruption of inter-Sertoli TJs is associated with a dissolution of actin filaments and a loss of occludin staining at the TJs, which, in turn, leads to an increase in BTB permeability (54). These results further illustrate that occludin is a crucial component of the paracellular seal in TJs. Based on these studies, it is logical to speculate that a disruption of the inter-Sertoli TJ leading to an increase in paracellular permeability would be associated with a decline in occludin expression. Indeed, CdCl2-induced inter-Sertoli TJ permeability barrier disruption is accompanied by reduced occludin expression. When these CdCl2-disrupted TJs were resealing, a steady increase in occludin expression was detected, indicating that de novo synthesis of occludin is required to rebuild the disrupted TJs.
On the other hand, proteases have been implicated in tissue restructuring during testicular development and maturation. This speculation is further strengthened by the fact that there are changes in protease activities in the epithelium at different stages of the spermatogenic cycle. For instance, the expression of uPA, a Sertoli cell product, is highest in Sertoli cells at stages VII–VIII (38). A recent study has demonstrated a significant, but transient, induction of uPA expression within 1 h after coculture of Sertoli cells with germ cells at the time germ cells adhered to Sertoli cells (6). It was suggested that proteases might be used to disrupt specific sites on Sertoli cell surface upon which germ cells can adhere to allow the subsequent AJ formation (5, 6). When Sertoli cells were cultured at a high cell density, a transient, but significant, increase in uPA expression was also detected while inter-Sertoli TJs were being assembled and when TJ assembly was completed (12). This pattern of changes in expression of selected cellular genes is consistent with the present report (12). It is possible that the increased uPA expression during CdCl2-induced inter-Sertoli TJ disruption is an indication of its involvement in junction complex dissolution.
In summary, these results illustrate that CdCl2-induced inter-Sertoli TJ disruption can be used as a model to study cell junction disassembly because this model fulfills the following criteria: 1) CdCl2 is noncytotoxic to Sertoli cells at doses that can disrupt the inter-Sertoli TJs; 2) its effect on TJ permeability barrier is reversible and dose dependent; 3) an actual disruption of the TJ has indeed taken place, as the time it took for the junction to reassemble mimics that of the untreated cells, but is different from the Ca2+-induced TJ "leakiness," which could be restored within 90 min; and 4) this process of junction reassembly mimics the reassembly of inter-Sertoli TJs and AJs as previously reported (7, 9, 12) when the cellular expressions of several target genes are examined.
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Acknowledgments |
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Footnotes |
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Received October 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin, and
testins: biosynthesis and differential regulation by germ cells. Mol
Cell Endocrinol 89:127–140[CrossRef][Medline]
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 D. D. Mruk, A. S. N. Lau, O. Sarkar, and W. Xia Rab4A GTPase Catenin Interactions Are Involved in Cell Junction Dynamics in the Testis J Androl, September 1, 2007; 28(5): 742 - 754. [Abstract] [Full Text] [PDF]
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 T. J Kaitu'u-Lino, P. Sluka, C. F H Foo, and P. G Stanton Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro Reproduction, June 1, 2007; 133(6): 1169 - 1179. [Abstract] [Full Text] [PDF]
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 W. Xia, D. D. Mruk, and C. Y. Cheng C-type natriuretic peptide regulates blood-testis barrier dynamics in adult rat testes PNAS, March 6, 2007; 104(10): 3841 - 3846. [Abstract] [Full Text] [PDF]
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 W. Xia, D. D. Mruk, W. M. Lee, and C. Y. Cheng Differential Interactions between Transforming Growth Factor-beta3/TbetaR1, TAB1, and CD2AP Disrupt Blood-Testis Barrier and Sertoli-Germ Cell Adhesion J. Biol. Chem., June 16, 2006; 281(24): 16799 - 16813. [Abstract] [Full Text] [PDF]
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G. A. Tarulli, P. G. Stanton, A. Lerchl, and S. J. Meachem Adult Sertoli Cells Are Not Terminally Differentiated in the Djungarian Hamster: Effect of FSH on Proliferation and Junction Protein Organization Biol Reprod, May 1, 2006; 74(5): 798 - 806. [Abstract] [Full Text] [PDF]
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A. Florin, M. Maire, A. Bozec, A. Hellani, S. Chater, R. Bars, F. Chuzel, and M. Benahmed Androgens and Postmeiotic Germ Cells Regulate Claudin-11 Expression in Rat Sertoli Cells Endocrinology, March 1, 2005; 146(3): 1532 - 1540. [Abstract] [Full Text] [PDF]
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 D. D. Mruk and C. Y. Cheng Sertoli-Sertoli and Sertoli-Germ Cell Interactions and Their Significance in Germ Cell Movement in the Seminiferous Epithelium during Spermatogenesis Endocr. Rev., October 1, 2004; 25(5): 747 - 806. [Abstract] [Full Text] [PDF]
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N. P.Y. Lee and C. Y. Cheng Adaptors, Junction Dynamics, and Spermatogenesis Biol Reprod, August 1, 2004; 71(2): 392 - 404. [Abstract] [Full Text] [PDF]
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M. K.Y. Siu and C. Y. Cheng Interactions of Proteases, Protease Inhibitors, and the {beta}1 Integrin/Laminin {gamma}3 Protein Complex in the Regulation of Ectoplasmic Specialization Dynamics in the Rat Testis Biol Reprod, April 1, 2004; 70(4): 945 - 964. [Abstract] [Full Text] [PDF]
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 C.-h. Wong, D. D. Mruk, W.-y. Lui, and C. Y. Cheng Regulation of blood-testis barrier dynamics: an in vivo study J. Cell Sci., February 15, 2004; 117(5): 783 - 798. [Abstract] [Full Text] [PDF]
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D. D. Mruk, M. K.Y. Siu, A. M. Conway, N. P.Y. Lee, A. S.N. Lau, and C. Y. Cheng Role of Tissue Inhibitor of Metalloproteases-1 in Junction Dynamics in the Testis J Androl, July 1, 2003; 24(4): 510 - 523. [Abstract] [Full Text] [PDF]
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N. P. Y. Lee and C. Yan Cheng Regulation of Sertoli Cell Tight Junction Dynamics in the Rat Testis via the Nitric Oxide Synthase/Soluble Guanylate Cyclase/3',5'-Cyclic Guanosine Monophosphate/Protein Kinase G Signaling Pathway: an in Vitro Study Endocrinology, July 1, 2003; 144(7): 3114 - 3129. [Abstract] [Full Text] [PDF]
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W.-y. Lui, W. M. Lee, and C. Y. Cheng Sertoli-Germ Cell Adherens Junction Dynamics in the Testis Are Regulated by RhoB GTPase via the ROCK/LIMK Signaling Pathway Biol Reprod, June 1, 2003; 68(6): 2189 - 2206. [Abstract] [Full Text] [PDF]
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W.-y. Lui, W. M. Lee, and C. Y. Cheng Transforming Growth Factor {beta}3 Regulates the Dynamics of Sertoli Cell Tight Junctions Via the p38 Mitogen-Activated Protein Kinase Pathway Biol Reprod, May 1, 2003; 68(5): 1597 - 1612. [Abstract] [Full Text] [PDF]
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W.-Y. Lui, D. Mruk, W. M Lee, and C. Y. Cheng Sertoli Cell Tight Junction Dynamics: Their Regulation During Spermatogenesis Biol Reprod, April 1, 2003; 68(4): 1087 - 1097. [Abstract] [Full Text] [PDF]
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N. P.Y. Lee, D. Mruk, W. M. Lee, and C. Y. Cheng Is the Cadherin/Catenin Complex a Functional Unit of Cell-Cell Actin-Based Adherens Junctions in the Rat Testis? Biol Reprod, February 1, 2003; 68(2): 489 - 508. [Abstract] [Full Text] [PDF]
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M. K. Y. Siu, W. M. Lee, and C. Y. Cheng The Interplay of Collagen IV, Tumor Necrosis Factor-{alpha}, Gelatinase B (Matrix Metalloprotease-9), and Tissue Inhibitor of Metalloproteases-1 in the Basal Lamina Regulates Sertoli Cell-Tight Junction Dynamics in the Rat Testis Endocrinology, January 1, 2003; 144(1): 371 - 387. [Abstract] [Full Text] [PDF]
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 C. Y. Cheng and D. D. Mruk Cell Junction Dynamics in the Testis: Sertoli-Germ Cell Interactions and Male Contraceptive Development Physiol Rev, October 1, 2002; 82(4): 825 - 874. [Abstract] [Full Text] [PDF]
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N. P.Y. Chung, D. Mruk, M.-y. Mo, W. M. Lee, and C. Y. Cheng A 22-Amino Acid Synthetic Peptide Corresponding to the Second Extracellular Loop of Rat Occludin Perturbs the Blood-Testis Barrier and Disrupts Spermatogenesis Reversibly In Vivo Biol Reprod, November 1, 2001; 65(5): 1340 - 1351. [Abstract] [Full Text] [PDF]
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