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First published online 9 April 2008
doi: 10.1242/dev.018572
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1 Program in Genes and Development, The University of Texas Graduate School of
Biomedical Sciences at Houston, Houston, TX 77030, USA.
2 Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer
Center, Houston, TX 77030, USA.
3 Department of Cancer Genetics, The University of Texas M.D. Anderson Cancer
Center, Houston, TX 77030, USA.
4 UMR 6175 Physiologie de la Reproduction Institut National de la Recherche
Agronomique, Centre National de la Recherche Scientifique, Université
de Tours, Haras Nationaux Nouzilly, France.
5 Department of Pharmacology, Graduate School of Medicine, Kyoto University,
Kyoto, 606-8501, Japan.
Author for correspondence (e-mail:
rrb{at}mdanderson.org)
Accepted 10 March 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: β-Catenin, Wt1, Testis, Sertoli cell, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Gubbay et al., 1990;
Koopman et al., 1991;
Lovell-Badge and Robertson,
1990; Vidal et al.,
2001). Sry is expressed only during a short window of
time in the XY gonad, from E10.5 to E12.5
(Hacker et al., 1995;
Koopman et al., 1990).
However, Sox9 expression in the XY gonad starts from E11.5 and is
maintained throughout embryogenesis (Kent
et al., 1996; Kobayashi et
al., 2005; Morais da Silva et
al., 1996). After the testis is determined, hormones secreted by
Sertoli cells and other differentiated cell types in the XY gonad drive sex
differentiation and make XY embryos morphologically very different from XX
embryos. Sertoli cells secrete anti-Müllerian hormone (AMH), also called
Müllerian-inhibiting substance (MIS) to induce the regression of
Müllerian ducts, the precursor of the female reproductive tract
(Behringer et al., 1994).
Testosterone, produced by Leydig cells, promotes Wolffian duct differentiation
into male reproductive tract organs and virilizes the external genitalia.
Insulin-like 3, also produced by Leydig cells, together with AMH and
testosterone, mediate transabdominal testicular descent into the scrotum
(Kobayashi and Behringer,
2003; Nef and Parada,
1999; Nef and Parada,
2000; Zimmermann et al.,
1999).
β-Catenin is an intracellular protein that plays important roles in
both intercellular adhesion and the canonical Wnt signaling pathway
(Morin, 1999;
Peifer and Polakis, 2000). It
has been demonstrated to be involved in multiple developmental processes and
in tumorigenesis. In intercellular adhesion, β-catenin is a component of
the cadherin/catenin complexes, which mediate Ca2+-dependent
homophilic interactions (Aberle et al.,
1996). In the canonical Wnt signaling pathway, β-catenin is a
central player involved in the transduction of extracellular signals to the
nucleus. In the absence of Wnts, cytoplasmic β-catenin is phosphorylated
by glycogen synthase kinase 3β (GSK3β) and casein kinase I (CKI)
bound to the adenomatosis polyposis coli (APC) complex and is targeted for
degradation through the ubiquitination pathway
(Aberle et al., 1997;
Morin, 1999;
Peifer and Polakis, 2000).
Activation of the Wnt pathway inhibits GSK3β, resulting in the
stabilization and cytoplasmic accumulation of β-catenin. The stabilized
β-catenin translocates into the nucleus and forms a heterodimeric complex
with the Tcf/Lef family of DNA-binding proteins to regulate the transcription
of downstream target genes, such as Myc and cyclin D1
(He et al., 1998;
Tetsu and McCormick, 1999).
β-Catenin was previously reported to be expressed in the plasma membrane
and cytoplasm of germ cells during testis development. Suppression of
Wnt/β-catenin signaling is necessary for the normal development of
primordial germ cells because stabilization of β-catenin in germ cells
causes delayed cell cycle progression and results in germ cell deficiency
(Kimura et al., 2006). The
expression of β-catenin and its potential function in other cell types
during testis development has not been studied.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Gao et al., 2006;
Harada et al., 1999;
Hayashi and McMahon,
2002).
Mouse crosses to generate Sox9flox/-; AMH-Cretg/+ mice
Male studs were heterozygous for the Sox9 flox allele
(Chaboissier et al., 2004) and
also hemizygous for a Sertoli cell-specific Cre transgene, AMH-Cre.
Female mice were homozygous for the Sox9 flox allele and also
hemizygous for an oocyte-specific Cre transgene, Zp3-Cre
(de Vries et al., 2000).
Expression of the Zp3-Cre transgene in oocytes of Sox9
flox/flox females leads to recombination of the Sox9 flox alleles
into deletion alleles. When crossed to male studs, mice with the genotype of
Sox9flox/-; AMH-Cretg/+, Sox9+/-;
AMH-Cretg/+, Sox9flox/-; +/+, and
Sox9+/-; +/+ were generated, regardless of their
Zp3-Cre genotype.
Primary Sertoli cell isolation
A modified method was used to isolate primary Sertoli cells from the testes
of 3-week-old animals (van der Wee et al.,
2001). Testes were decapsulated under the dissection microscope.
The seminiferous tubules were pooled and washed with phosphate-buffered saline
(PBS) three times. The tubules then were incubated with 2 mg/ml collagenase I
(Sigma) and 0.5 mg/ml DNase I (sigma) in DMEM for 30 minutes at 37°C on a
shaker. The tubules were then washed twice with DMEM and further digested with
2 mg/ml collagenase I, 0.5 mg/ml DNase I and 1 mg/ml hyaluronidase type III
(Sigma) for 20-30 minutes at 37°C on a shaker. The tubules were allowed to
settle and were washed twice with DMEM. The tubules were further digested with
2 mg/ml collagenase I, 0.5 mg/ml DNase I, 2 mg/ml hyaluronidase, 1 mg/ml
trypsin for 40-60 minutes at 37°C on a shaker. This final digestion step
resulted in a cell suspension containing primarily Sertoli cells and type A
spermatogonia. The dispersed cells were then washed twice with DMEM and placed
into culture dishes in DMEM containing 10% fetal calf serum and incubated at
37°C and 5% CO2. Spermatogonia were unable to attach to the
dish and were removed after the medium change on the next day. 4OH-Tamoxifen
(Sigma, H7904) was dissolved in ethanol to generate a 1 mM stock solution and
further diluted to appropriate concentrations prior to use. Recombination was
initiated by adding 4OH-TM to cultured Sertoli cells at a final concentration
of 1 µm. After 3 days culture, total RNA and protein were extracted as
described below.
Western blot analysis
Sertoli cells were washed with ice-cold PBS and lysed at 4°C by shaking
robustly in radioimmunoprecipitation assay buffer [RIPA: PBS containing 1%
NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/l EDTA (pH 8.0), 1 mmol/l
sodium orthovanadate (pH 10.0), 2 mmol/l phenylmethylsulfonyl fluoride and
protease inhibitor cocktail tablet]. Lysates were centrifuged at 16,000
g for 10 minutes, and the supernatants were subjected to
protein content determination using a detergent compatible protein assay kit
(Bio-Rad, Hercules, CA) according to the manufacturer's instructions. An equal
volume of 2x SDS sample loading buffer containing 100 mmol/l Tris-HCl
(pH 6.8), 10% glycerol, 4% SDS, 0.05% Bromophenol Blue and 5%
β-mercaptoethanol was added to cell lysates before heating at 95°C
for 5 minutes. Aliquots of 20 µg total cell protein were loaded onto 10%
SDS-polyacrylamide gels. Proteins were separated by electrophoresis at
constant voltage (80-110 V) and electrotransferred to nitrocellulose
membranes. Membranes were blocked overnight at 4°C in PBST containing 5%
nonfat dried milk and incubated at room temperature for 1 hour with two
anti-β-catenin antibodies (a rabbit anti-β-catenin antibody 1:1000,
Sigma C 2206, recognizes amino acids 768-781 of human or mouse β-catenin;
a mouse monoclonal anti-active β-catenin antibody 1:1000, Upstate
#05-665, recognizes the active form of β-catenin dephosphorylated on
Ser37 or Thr41) and a mouse anti-β-actin monoclonal antibody (1:10,000,
Sigma, A1978). Membranes were washed with PBST buffer three times and
incubated at room temperature for 1 hour with an IRDye 800CW conjugated goat
anti-rabbit IgG (LI-COR, 926-32211) and an Alexa Fluor 680 goat anti-mouse IgG
(Molecular Probes, A21058) at dilutions of 1:2500. After washing, the blots
were visualized by scanning using Li-Cor Odyssey Imager (Li-Cor, Lincoln,
NE).
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Tissue collection and histological analysis
Testes were dissected from at least four mutant males at various
time-points. Tissues from three to four control littermates were also
collected. Tissues were fixed in 4% paraformaldehyde overnight, embedded in
paraffin wax and sectioned at 4 µm. After de-waxing, the sections were
stained with Hematoxylin and Eosin for histological analyses.
Immunohistochemistry/fluorescence
Immunohistochemical analysis was carried out using the Vectastain ABC
(avidin-biotin-peroxidase) kit (Vector Laboratories) as recommended by the
manufacturer. Endogenous peroxidase activity was destroyed using 3% hydrogen
peroxide for 20 minutes. Antigen recovery was performed by boiling samples in
0.01 M sodium citrate buffer (pH 6.0) for 20 minutes. Sections were incubated
with 5% bovine serum albumin in PBS for 30 minutes at room temperature and
then incubated for 1 hour at room temperature with either anti-WT1 antibody
(Santa Cruz, sc-192), anti-SOX9 antibody (Chemicon, AB5535), anti-3β-HSD
(3β-hydroxysteroid dehydrogenase) antibody (Santa Cruz, sc-30821),
anti-AMH antibody (Santa Cruz, sc-6886) or anti-GCNA1 (germ cell nuclear
antigen 1) antibody (provided by Dr George Enders) at dilutions of 1:100,
1:200, 1:200, 1:100 and 1:50, respectively. After three washes with PBS, the
sections were incubated with biotinylated secondary antibody (Santa Cruz) at a
dilution of 1:250 for 45 minutes at room temperature. After incubation with
avidin-biotin-peroxidase complex for 45 minutes, the sections were washed with
PBS. The color was developed with 3,3'-diaminobenzidine (DAB) or
3-amino-9-ethylcarbazole (AEC) substrate. Samples were counterstained with
Harris Hematoxylin. For β-catenin immunofluorescence, rabbit
anti-β-catenin antibody (Sigma, C 2206) was used at a dilution of 1:2000
for 1 hour and washed three times in PBS. An Alexa Fluor 488 nm goat
anti-rabbit (Molecular Probes, A11008) was used at a dilution of 1:800 for 1
hour and washed twice with PBS. Sections were mounted with Vectashield
mounting medium with DAPI (Vector Laboratories, H1200).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
we observed β-catenin localized to the plasma membrane and cytoplasm of
germ cells at all embryonic stages examined
(Fig. 1). We also observed
expression of β-catenin in Sertoli cells, starting from E15.5
(Fig. 1G,I,K). β-Catenin
was localized mainly on Sertoli cell membranes, but not in the nucleus, as
shown by a double staining of β-catenin and Sertoli cytoplasmic protein
AMH (Fig. 1H,J,L).
β-Catenin deletion in Sertoli cells does not affect testis development
To determine the essential role of β-catenin in Sertoli cells of the
developing testis, we specifically deleted β-catenin in Sertoli cells by
crossing mice with a floxed β-catenin conditional null allele
(Brault et al., 2001) to
AMH-Cre mice that express Cre only in Sertoli cells after E13.5
(Lecureuil et al., 2002). In
Catnbfx/fx; AMH-Cre male mice at E16.5, no β-catenin
expression was detected in Sertoli cells but expression of β-catenin in
germ cells were unaffected, suggesting that β-catenin was specifically
deleted in Sertoli cells (Fig.
2A). Surprisingly, we observed no gross abnormalities of the
testes and reproductive tracts of newborn and adult mutant males. In
Catnbfx/fx; AMH-Cre males at P0, the testicular cords were
normally formed. Well-organized Sertoli cells and germ cells surrounded by a
layer of peritubular myoid cells could be found in each cord. In the testes of
the adult mutant mice, the seminiferous tubules contained all of the different
stages of germ cells (spermatogonia, spermatocytes, spermatids and
spermatozoa), suggesting spermatogenesis occurred normally. Sertoli and Leydig
cell identity were confirmed by immunostaining of the Sertoli cell marker SOX9
and the Leydig cell marker 3βHSD (Fig.
2B).
|
). In
Catnblox(e3)/+; AMH-Cretg/+ testes, we detected
the accumulation of β-catenin protein in Sertoli cells, starting from
E13.5. The accumulation of β-catenin initiated in a few Sertoli cells at
E13.5 and was found in almost all Sertoli cells in the testicular cords by
E15.5. The stabilized β-catenin was localized in both the cytoplasm and
nucleus of Sertoli cells (Fig.
3). Catnblox(e3)/+; AMH-Cretg/+ mice were viable. No gross abnormalities of the external genitalia were observed in seven-week-old Catnblox(e3)/+; AMH-Cretg/+ males, but testis size was much smaller in all of the mutants than that of control littermates. Fifty percent of the Catnblox(e3)/+; AMH-Cretg/+ males (10/20) were found to have a fully or partially retained uterus in addition to a vas deferens, epididymis, and seminal vesicles, suggesting that the normal regression of the Müllerian ducts caused by AMH was affected (Fig. 4A). Histological analyses revealed severe abnormalities in the testes of Catnblox(e3)/+; AMH-Cretg/+ mice. Mutant testes completely lacked normal tubular structures and consisted mainly of masses of eosinophilic cells, which were identified as Leydig cells by staining with anti-3β-HSD antibody. Only a few degenerated tubules were found in the Catnblox(e3)/+; AMH-Cretg/+ testes. However, spermatogenesis was completely absent in these tubules and only rare clumps of Sertoli cells remained (Fig. 4B).
To determine when defects in the testes first occurred, Catnblox(e3)/+; AMH-Cretg/+ testes from animals of various developmental stages were examined, from E12.5 to birth. No obvious differences in the structure of the testes were observed before E14.5 (Fig. 5). From E15.5 to P0, control testes exhibited progressive testicular cord maturation and expansion of interstitial spaces, which contribute to the growth of testis size. During the same time period in Catnblox(e3)/+; AMH-Cretg/+ testes, we observed no obvious growth in testis size and increasingly disrupted tubular structure. In control testes, GCNA-1 positive germ cells were localized in the center of testicular cords, surrounded by well-organized Sertoli cells. 3βHSD-positive Leydig cells were sparsely dispersed in the interstitial spaces. However, in mutant testes, Sertoli cells were disorganized and germ cells were scattered outside of the cords. Masses of Leydig cells were found in the interstitial spaces of the mutant testes. At P0, mutant testes consisted largely of clusters of eosinophilic Leydig cells and a few aberrant cord-like structures. Very few germ cells remained in the cord remnants (Fig. 6).
Stabilization of β-catenin causes loss of SOX9 and AMH expression but does not affect WT1 expression in Sertoli cells
SOX9 is a transcription factor that is expressed in Sertoli cells and
essential for testis formation (Chaboissier
et al., 2004). Amh is a downstream target of SOX9 and
responsible for the regression of the Müllerian ducts in males
(Behringer et al., 1994;
De Santa Barbara et al., 1998).
We examined how their expression was affected by stabilization of
β-catenin. SOX9 protein was detected in Sertoli cells of both control and
mutant testes at E12.5. We observed decreased levels of SOX9 in mutant testes
at E13.5 and almost no SOX9 was detected in Catnblox(e3)/+;
AMH-Cretg/+ testes at E14.5. We observed similar changes of
AMH expression in Sertoli cells of the mutant mice. Previous study showed that
Wt1 deletion in Sertoli cells causes the loss of both SOX9 and AMH
expression (Gao et al., 2006).
To determine whether the loss of SOX9 and AMH expression in our
Catnblox(e3)/+; AMH-Cretg/+ mice is a secondary
effect due to changes in Wt1 expression, we performed
immunohistochemical analysis of WT1 and found that the expression of WT1 in
Sertoli cells was not affected by β-catenin stabilization
(Fig. 7).
Stabilization of β-catenin in Sertoli cells alters testicular gene expression
To elucidate putative mechanisms associated with the development of the
phenotypes in Catnblox(e3)/+; AMH-Cretg/+
mutants, we performed quantitative RT-PCR analysis in the testes of
Catnblox(e3)/+; +/+ and Catnblox(e3)/+;
AMH-Cretg/+ mice at E14.5. Compared with
Catnblox(e3)/+; +/+ control testes, we found that
transcripts of several important markers of Sertoli cells, such as Gata4,
Sf1, Dhh and Fgf9, were not significantly changed in the
Catnblox(e3)/+; AMH-Cretg/+ testes. However,
the transcripts of Dax1 (Nr0b1 - Mouse Genome Informatics),
the X-linked orphan nuclear hormone receptor the precise dose of which is
required for proper testis development, were upregulated by 70%.
Interestingly, the expression of Wnt4, an ovarian somatic marker, was
upregulated by 100%. We also compared the expression levels of several
molecules that are involved in Sertoli cell-Sertoli cell junction formation
and found that the connexin 43 (Cx43) transcripts were reduced by 50%
in the mutant testes while the expression levels of occludin and claudin 11
were not significantly changed. Consistent with the immunohistochemical
analysis data, our real-time PCR analysis showed that Wt1 expression
levels were not changed by the stabilization of β-catenin
(Table 1).
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). Sertoli cells lose the expression of SOX9 and AMH in both
Wt1 and β-catenin stabilized mutant mice. To determine whether
the Wt1 conditional knockout phenotype is related to misregulated
β-catenin signaling, we performed β-catenin immunostaining on testes
of Wt1fx/-; AMH-Cretg/+ mice. Although
expression of β-catenin was only found mainly on Sertoli cell membrane in
control testes, we observed nuclear localization of β-catenin in Sertoli
cells of Wt1 conditional knockout testes
(Fig. 8A).
To determine the quantitative changes of β-catenin expression after
Wt1 deletion in Sertoli cells, we isolated primary Sertoli cells from
the testes of 3-week-old mice and used an inducible in vitro system to measure
expression level changes of β-catenin upon Wt1 deletion. The
CAGGCre-ER transgenic mouse line has been shown to be a good
inducible Cre line in which Cre-mediated recombination is tamoxifen-inducible
and dose dependent (Hayashi and McMahon,
2002). We crossed Wt1+/-;
CAGGCre-ERtg/+ males with Wt1fx/fx females
to obtain Wt1fx/-; CAGGCre-ERtg/+ male mice and
isolated primary Sertoli cells from their testes. We were able to obtain over
90% purity as assessed by immunostaining for WT1
(Fig. 8B). To determine the
efficiency of tamoxifen-induced recombination in the isolated Sertoli cells,
we performed quantitative RT-PCR and found Wt1 mRNA levels were
reduced by 
6-fold after 3 days treatment with 1 µm 4OH-tamoxifen
(Fig. 8C). To determine the
expression level change of β-catenin protein upon Wt1 deletion,
we performed western analysis and found that the level of total β-catenin
was upregulated more than twofold in Wt1fx/-;
CAGGCre-ERtg/+ Sertoli cells after 4OH-tamoxifen treatment. To
exclude the possibility that the increase in β-catenin levels may be due
to the effect of 4OH-tamoxifen treatment, we also included
Wt1fx/fx; +/+ Sertoli cells as a control and found that
the level of β-catenin was not affected by the drug treatment
(Fig. 8D). To determine whether
the increase of total β-catenin protein was due to an increase in the
transcription of β-catenin, we performed quantitative RT-PCR and found
that β-catenin mRNA levels were not changed upon Wt1 deletion in
Sertoli cells (data not shown). To determine whether the increase of total
β-catenin protein in Sertoli cells was due to a decrease in the
degradation of β-catenin, we performed western analysis using an antibody
specific for the active form of β-catenin and found that the level of
active β-catenin was upregulated by about twofold in
Wt1fx/-; CAGGCre-ERtg/+ Sertoli cells after
4OH-tamoxifen treatment (Fig.
8D). Given that the level of β-catenin is controlled both by
protein synthesis and degradation, we reasoned that the increase of
β-catenin protein might be due to reduced degradation of β-catenin
upon Wt1 deletion.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Morin, 1999;
Peifer and Polakis, 2000).
Expression of β-catenin was observed in Sertoli cells starting from
E15.5. The β-catenin protein was found mainly on the Sertoli cell
membrane, but was undetectable in the nucleus. Despite the expression of
β-catenin in Sertoli cells, our data suggest that β-catenin is not
essential for the maintenance of Sertoli cell function and testicular
structure. Conditional knockout of β-catenin in Sertoli cells using
AMH-Cre mice caused no obvious abnormalities. Mutant mice developed
normal testicular structure and had normal spermatogenesis. However,
stabilization of β-catenin in Sertoli cells caused severe testicular cord
disruption and germ cell depletion, suggesting that β-catenin signaling
must be tightly controlled in Sertoli cells for proper Sertoli cell function
and the maintenance of testis structure. Although testes of
Catnblox(e3)/+; AMH-Cretg/+mice were
indistinguishable morphologically from control testes at E13.5 before the
expression of the AMH-Cre transgene, a dramatic and progressive
disruption of testicular cord structure was observed beginning at E15.5
following β-catenin stabilization. With the disruption of testicular
cords, germ cells leaked into the interstitial spaces and initiated meiosis
(data not shown). In the end, the germ cells all died because of the loss of
their niche. Adult mutant testes were much smaller than control testes and
consisted of Leydig cells, fibroblast-like cells and a few aberrant tubule
remnants. Leydig cells developed normally in mutant testes, except that we
observed a high proportion of Leydig cells, most probably owing to the
collapse of testicular cords and the depletion of germ cells. Leydig cells in
mutant testes did not proliferate excessively because we did not observe a
significant increase in the number of Leydig cells positive for the mitotic
cell marker H3P from E13.5 to E18.5 (data not shown).
|
; Vidal et al.,
2001). Amh is a direct downstream target gene of
Sox9 (De Santa Barbara et al.,
1998). AMH protein is an important secretory protein of Sertoli
cells, which is necessary to induce the regression of the Müllerian ducts
in male fetuses (Behringer et al.,
1994). Stabilization of β-catenin in Sertoli cells caused the
loss of both SOX9 and AMH, two important Sertoli cell markers. Wnt4
is an ovarian somatic marker that acts as a partial anti-testis gene by
repressing aspects of male development in the female gonad
(Heikkilä et al., 2002;
Vanio et al., 1999).
Interestingly, the stabilization of β-catenin caused an upregulation of
Wnt4 transcript levels.
A previous study showed that Wt1 is required to maintain SOX9 and
AMH expression in Sertoli cells. Sertoli cell-specific deletion of
Wt1 leads the loss of the expression of both SOX9 and AMH
(Gao et al., 2006). Here, we
found that stabilization of β-catenin also caused the loss of SOX9 and
AMH expression in Sertoli cells. However, the expression of WT1 was not
altered. This indicates that the expression of WT1 by itself is not sufficient
to maintain SOX9/AMH expression in Sertoli cells. This could be explained as:
(1) the expression of SOX9/AMH is regulated positively by WT1 and negatively
by β-catenin, independently; or (2) WT1 regulates SOX9/AMH expression
through the inhibition of β-catenin. Given the data in which we showed
up-regulation of β-catenin in Wt1 knockout Sertoli cells, the
latter scenario seems more likely.
Stabilization of β-catenin affects inter-Sertoli cell contacts
We detected no increased apoptosis or decreased cell proliferation in
Sertoli cells of Catnblox(e3)/+; AMH-Cretg/+
mice, suggesting that testicular cord disruption is not a result of programmed
cell death or reduced proliferation of Sertoli cells
(Fig. 9). As we observed a
progressive disruption of well-formed tubules upon the stabilization of
β-catenin in mutant testes, we speculate that β-catenin
stabilization might cause the dysregulation of genes that are important for
Sertoli cell-cell contacts or Sertoli cell-germ cell contacts. A major role of
Sertoli cells is to provide a nurturing environment for germ cells. As
testicular cords start to form, Sertoli cells are evenly distributed and form
a single layer surrounding the germ cells. Mature Sertoli cells form three
types of intercellular junctions, cadherin-based adherens junctions,
occludin-based tight junctions and connexin-based gap junctions. All of these
junctions are involved in forming the blood-testis barrier, which physically
divides the seminiferous tubules into basal and apical compartments
(Mruk and Cheng, 2004).
Claudin 11 and occludin are integral components of tight junctions between
Sertoli cells and both are expressed in Sertoli cells early in fetal
development (Cyr et al., 1999;
Hellani et al., 2000). Claudin
11 knockout male mice and occludin knockout male mice are sterile
(Gow et al., 1999;
Saitou et al., 2000). In our
studies, claudin 11 and occludin mRNA levels were not significantly reduced in
mutant testis. Connexin 43 (CX43) is a predominant testicular gap junction
protein, which is involved in the formation of gap junctions between adjacent
Sertoli cells and between Sertoli and germ cells in the seminiferous
epithelium (Batias et al.,
1999; Decrouy et al.,
2004; Perez-Armendariz et al.,
2001). CX43 is essential for the control of Sertoli cell
proliferation and maturation. Sertoli cell-specific deletion of Cx43
causes spermatogenesis defects and the formation of Sertoli cell clumps inside
seminiferous tubules (Brehm et al.,
2007; Sridharan et al.,
2007). We observed a significant downregulation of Cx43
mRNA expression in Catnblox(e3)/+; AMH-Cretg/+
testes prior to histological abnormalities. Testicular cords in the mutant
mice were subsequently disrupted. Those few remaining degenerated cords were
poorly organized and mainly appeared as clumps of Sertoli cells, which could
partially be a result of reduced Cx43 expression. However,
dysregulation of other cell contact molecules must also be involved as Sertoli
cell specific deletion of Cx43 does not cause embryonic testis
defects. These changes could be a direct result of the stabilization of
β-catenin, but also could be secondary to the misregulation of Sox9,
Wnt4 and Dax1.
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|
). In our study, we combined a Wt1
conditional null allele with a tamoxifen-inducible Cre allele and investigated
the expression level changes of β-catenin upon Wt1 deletion in
isolated primary Sertoli cells. We found that the level of total and active
β-catenin was dramatically upregulated in Wt1fx/-;
CAGGCre-ERtg/+ Sertoli cells upon 4OH-tamoxifen treatment. One
caveat of our in vitro study is that Sertoli cells at 3 weeks of age might be
different from embryonic Sertoli cells. But, importantly, we found that
Wt1 deletion also caused upregulation of β-catenin in Sertoli
cells in vivo. β-Catenin immunostaining on the testes of
Wt1fx/-; AMH-Cretg/+ mice showed nuclear
localization of β-catenin in the Sertoli cells of Wt1
conditional knockout testes, whereas the expression of β-catenin was only
found on the Sertoli cell membrane in control testes. The negative regulation
of β-catenin by Wt1 in Sertoli cells is further supported by the
data that stabilization of β-catenin in Sertoli cells has similar
phenotypes to Wt1 conditional knockout mutants. The negative
regulation of β-catenin by Wt1 is probably achieved by enhancing
the degradation of β-catenin protein as we did not observe any
significant changes in the levels of β-catenin mRNA upon Wt1
deletion.
Signals regulate later stages of testis development
It has been shown that Sox9/Fgf9 and Wnt4 signaling play
antagonistic roles during sex determination
(Kim et al., 2006). In
undifferentiated XY gonads, Sry expression initiates the male pathway
by upregulating Sox9. Sox9 upregulates Fgf9, which initiates
a Sox9/Fgf9 positive-feedback loop that acts as an antagonist of
Wnt4 and accelerates commitment to the male pathway. In
undifferentiated XX gonads, the Sox9/Fgf9 feedback loop is not
established and Wnt4 commits gonadal development to the female
pathway. After sex is determined, the signals required for the maintenance of
testis development are relatively unknown. A previous study found that
Wt1 is required for the maintenance of Sox9 expression in
Sertoli cells during later stages of testis development
(Gao et al., 2006). In this
study, we found stabilization of β-catenin also caused the inhibition of
Sox9 expression. Loss of Sox9 expression appears in both the
Wt1 deletion and β-catenin stabilized mutants. Surprisingly, we
found that Sox9 is not essential for the testis development after
E14.5. We used AMH-Cre combined with Zp3-Cre to create
Sox9 flox/-Sertoli cell knockout mice and we did not detect any
abnormalities in mutant testes at E18.5
(Fig. 10). Mutant Sertoli
cells expressed AMH and β-catenin normally suggesting that Sox9
is not essential for the maintenance of Sertoli cell identity during later
stages of testis development. This could be due to other Sox family
members, such as Sox8, that might act redundantly or compensate for
Sox9 during later stages of testis development
(Chaboissier et al., 2004).
In this study, we identified that β-catenin signaling as an important
regulator for the later stages of testis development. Upregulation of
β-catenin signaling causes not only the loss of the male marker SOX9/AMH
expression, but also overexpression of the female marker Wnt4, which
thus alters Sertoli cell identity and causes the disruption of testicular
cords. Wt1, which is expressed in Sertoli cells throughout testis
development, functions as a negative regulator of the β-catenin signaling
pathway to commit XY gonads to the male development pathway. As we used
AMH-Cre to study the roles of Wt1 and β-catenin in
Sertoli cells after sex determination, it is not clear whether our finding is
also applicable for earlier stages of testis development (i.e. testis
determination). A previous study found that R-spondin1 mutation
caused female to male sex reversal in humans and it was proposed that
R-spondin1 might function through the stabilization of β-catenin
signaling during female sex determination
(Parma et al., 2006). Similar
experiments using earlier expressed Cre lines (to delete β-catenin in
bipotential XX gonads or stabilize β-catenin in bipotential XY gonads)
will be required to determine whether β-catenin signaling is also
involved in sex determination.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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