1 Department of Medicine, Division of Endocrinology, Metabolism, and Molecular
Medicine. Northwestern University, Feinberg School of Medicine, Chicago, IL
60611, USA
2 Department of Pathology. Northwestern University, Feinberg School of Medicine,
Chicago, IL 60611, USA
3 Department of Developmental Biology, National Institute for Basic Biology,
Okazaki, Aichi 444-8585, Japan
* Author for correspondence (e-mail: ljameson{at}northwestern.edu)
Accepted 19 November 2002
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SUMMARY |
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Key words: Dax1, Sex differentiation, Gonadal development, Mouse
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INTRODUCTION |
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Mutations of DAX1 in humans cause the X-linked clinical syndrome, adrenal
hypoplasia congenita (AHC) (Muscatelli et
al., 1994). This syndrome is characterized by impaired development
of the adult zone of the adrenal cortex, leading to adrenal insufficiency and
by hypogonadotropic hypogonadism, caused by impaired production of
hypothalamic GnRH and pituitary gonadotrope production of luteinizing hormone
(LH) and follicle-stimulating hormone (FSH) (reviewed by
Achermann et al., 2001
). More
recently, individuals with this syndrome have been shown to have gonadal
dysgenesis that is independent of the gonadotropin deficiency (Ozisik et al.,
2002). Treatment with exogenous gonadotropins stimulates testosterone
production but does not appear to normalize spermatogenesis in the few
individuals who have been carefully studied
(Reutens et al., 1999
).
Targeted mutagenesis of the genes encoding orphan nuclear receptors has
proven useful for clarifying their functions, especially as there are no known
ligands that can be used as agonists or antagonists. Disruption of
Sf1, for example, causes adrenal and gonadal agenesis and leads to
impaired gonadotropin production, indicating that it plays a crucial role in
the development of these glands, as well as regulating the expression of a
variety of target genes involved in steroidogenesis and reproduction
(Ingraham et al., 1994;
Parker et al., 2002
). Targeted
mutagenesis of Dax1 (also known as Ahch; Nr0b1
Mouse Genome Informatics) has also been performed, using a Cre-loxP
strategy to circumvent the X-linked infertility in males
(Yu et al., 1998
). The
phenotype of these mice is similar to that of individuals with the AHC
syndrome, although the adrenal and pituitary abnormalities are less pronounced
in mice (Yu et al., 1998
;
Babu et al., 2002
). The testes
of the Dax1-deficient mice were initially shown to be small with
degeneration of the seminiferous epithelium and loss of germ cells
(Yu et al., 1998
). Subsequent
studies revealed multiple abnormalities including clusters of poorly
differentiated Sertoli cells within the lumen of the seminiferous tubules and
efferent ducts, hyperplasia of epithelial cells within the rete testis and
efferent ducts and Leydig cell hyperplasia
(Jeffs et al., 2001b
). The
lumen of the rete testis, normally a patent passage formed by the union of
seminiferous tubules and the epididymus, is obstructed by these cells, leading
to dilation and pressure atrophy of seminiferous tubules. In the area around
the rete testis, the basal lamina that encircles the seminiferous tubules is
intermittently disrupted. Clusters of undifferentiated PTM cells surround the
seminiferous tubules and Leydig cells are ectopically located within the
tubules (Jeffs et al., 2001b
).
These features led us to hypothesize that Dax1 deficiency causes a
defect during early gonadal development.
Development of the gonad is a complex and highly orchestrated process
(Tilmann and Capel, 2002;
Yao et al., 2002a
). The
indifferent gonad arises from the ventral surface of the urogenital ridge
(Kaufman, 1995
). At 10.5 dpc,
the primordial germ cells (PGCs) migrate from the allantois into the gonad of
both sexes (Hogan, 1994
). A
wave of Sry expression is initiated at 10.5 dpc in the male gonad and begins
the process of sexual dimorphism (Hacker et
al., 1995
). Soon after Sry expression, the pre-Sertoli cells at
the coelomic surface proliferate and invade the male gonad where they cluster
near the germ cells (Schmahl et al.,
2000
). Peritubular myoid (PTM) cells surround and enclose the
Sertoli and germ cells, creating discrete testis cords. It is hypothesized
that PTM cells are among the cells that originate from the mesonephros
(Martineau et al., 1997
;
Tilmann and Capel, 1999
). When
the PTM and Sertoli cells make contact, they secrete a matrix that forms the
basal lamina, separating the testis cords from the interstitial compartment
(Tung et al., 1984
). Delay or
disruption of testis cord formation could result in gonadal dysgenesis or
infertility. In this report, we examined early gonadal development in
Dax1-deficient mice to assess whether alterations might account for
the testicular dysgenesis seen in adult mice and in humans with DAX1
mutations. We identify a marked deficiency of PTM cells, associated with
incomplete formation of testis cords, providing the likely cause of the
progressive pathogenesis and infertility seen in animals of reproductive
age.
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MATERIALS AND METHODS |
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Histology, immunohistochemistry, and in situ hybridization
For immunohistochemistry, embryos were fixed in 10% phosphate-buffered
formalin, embedded in paraffin, and 3 µm sagittal sections were cut using a
Jung microtome (Leica, Heerbrugg, Switzerland). Tissue sections were viewed
with a Zeiss Axioskop (Thornwood, NY). Standard histological techniques were
used for Hematoxylin and Eosin staining. Sections were deparaffinized in
xylenes and descending ethanols, followed by antigen retrieval in sodium
citrate buffer. Sections were blocked in normal serum (5%) for 45 minutes and
incubated with primary antibody for 4 hours at room temperature or 4°C
overnight. Rabbit anti-laminin (Sigma, 1:200), goat anti-GATA-4 (Santa Cruz,
1:200), Dax1 (1:3,000) and Sf1 (K. Morohashi, 1:500) were used as primary
antibodies, with specific secondary antibodies at 1:200 dilution (Jackson
Immunoresearch). After washing with PBS+0.1% Triton X, sections were incubated
in secondary antibody for 2 hours at room temperature, washed again and
mounted with Vectashield mounting medium (Vector Laboratories, Burlingame,
CA). In situ hybridization was performed using a standard protocol
(Wilkinson, 1998). Probes for
Amh, Scc, Dhh and Sox9 were graciously provided by B. Capel (Duke University),
Andy McMahon (Harvard University) and Peter Koopman (University of
Queensland). In all cases, littermates were used as wild-type controls.
Cell death and proliferation
To determine if cells were dividing, 0.1 mg/g body weight of
5-Bromo-2'-deoxyuridine (BrdU 10 mg/ml, Sigma-Aldrich), a thymidine
analog incorporated during S phase was injected i.p. into pregnant mothers.
Two hours post-injection, embryos were removed, fixed in 10% formalin and
embedded. After sectioning, slides were deparaffinized and treated with sodium
borate (pH 8.5) to quench endogenous fluorescence. To increase penetration of
the antibody, slides were treated with pepsin (10 mg/ml, Sigma-Aldrich) in
0.01 N HCl at 37°C for 1 hour, followed by incubation with DNase (100
U/ml) at 37°C for 1 hour to further increase nuclear penetration. Rat
anti-BrdU:FITC antibody (Serotec, Kidlington, Oxford, UK) was applied to
sections for 2 hours at room temperature. When bright-field microscopy was
employed, sections were then incubated with biotinylated rabbit-anti-rat
secondary antibody, followed by RTU horseradish peroxidase streptavidin and
finally development with DAB peroxidase substrate (all from Vector
Laboratories Burlingame, CA), Slides were then washed and covers were applied.
When colocalization with BrdU was used, slides were treated by antigen
retrieval instead of pepsin/DNase and the anti-BrdU antibody was applied at
the time of incubation with the secondary antibody. Cell count values were
made on eight wild-type and 10 Dax1KO gonads.
Cell death was determined by labeling the fragmented DNA generated by endonucleases during apoptosis. Instructions provided in the Fluorescein-FragEL Cell Death Detection Kit (Oncogene, La Jolla, CA) were followed. Briefly, sections were deparaffinized through xylenes and ethanols, and permeabilized with proteinase K for 20 minutes. After equilibration with reaction buffer, sections were treated with the TdT labeling reaction for 1.5 hours at 37°C, washed and mounted. Sections were viewed with a Zeiss Axioskop.
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RESULTS |
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Histological examination of wild-type and Dax1-deficient gonads was performed during the time that spans testis cord formation and early gonadal differentiation (11.5-17.5 dpc; Fig. 2). The normal gonad is histologically indifferent at 11.5 dpc, and there are no apparent differences between wild-type male, female or Dax1-deficient gonad at 11.5 dpc (Fig. 2A,B). Thus, Dax1 is not required for gonadal development from the urogenital ridge. At 12.5 dpc, sexual dimorphism is first apparent histologically. On the ventral surface of the male gonad, the coelomic vessel is present and filled with red-blood cells (arrowheads, Fig. 2C). This large arterial vessel extends capillaries dorsally into the substance of gonad within the peritubular space. Individual testis cords are identifiable and PTM cells circumscribe their borders (asterisks, arrow; Fig. 2C). As observed in the whole-mount analyses, Dax1-deficient testes have a patent coelomic vessel, yet few capillaries are present the intertubular space (Fig. 2D). Within the Dax1-deficient testis, Sertoli and germ cells are heterogeneously clustered without discernable separation between the cords (Fig. 2D). Further inspection suggests that the testis cords lack organization because of a marked reduction in PTM cells in the Dax1KO testis.
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At 13.5 dpc, the testis cords are larger and more clearly defined in the wild-type gonad (asterisk, Fig. 2E). PTM cells become differentiated and flatten, forming a concentric layer of cells that encircles the testicular cord (arrows, Fig. 2E). Differentiated Sertoli cells are present at the periphery of the tubule and their apical cytoplasm is polarized opposite to the PTM cells (Fig. 2E). In the Dax1-deficient testis, germ cells and Sertoli cells are seen within the dorsal surface of the gonad (Fig. 2F). These cells cluster together at 13.5 dpc, but few PTM cells are observed (arrow, Fig. 2F). Although the phenotype encompasses a spectrum of cellular disorganization, very few discrete testis cords are discernable from peritubular tissue in the Dax1-deficient testis.
By 14.5 dpc, the normal male testis is more sphere-shaped (Fig. 2G). The testis cords become further differentiated as the triangular-shaped Sertoli cells line the outer-edge of the cord in clear opposition to the PTM cells (arrows, Fig. 2G). By contrast, testis cords remain rudimentary in Dax1-deficient testis (Fig. 2H). Occasionally, scattered PTM cells are present in Dax1-deficient gonads at 14.5 dpc (arrow, Fig. 2H). This occurs predominately at the ventral surface, leaving the dorsal half of the gonad more disorganized (Fig. 2H). Most cells remain round and undifferentiated in Dax1-deficient gonads. At 17.5 dpc, testis cords are round, uniform in size and resemble early seminiferous tubules (asterisk, Fig. 2I). In the Dax1-deficient testis, individual tubules are larger in size and pleomorphic in shape, without clearly defined borders (Fig. 2J).
The basal lamina is discontinous or absent in Dax1 deficient
testis
Beginning at 12.5 dpc, PTM and Sertoli cells cooperate to deposit a layer
of basal lamina that defines the edges of individual testis cords
(Tung et al., 1984). Because
PTM cells are reduced in the Dax1-deficient testis, we assessed the
formation of the basal lamina. In wild-type males at 12.5 dpc, laminin is
present in the peritubular space throughout the gonad as the nascent cords are
first identifiable (Fig. 3A).
In the Dax1-deficient gonads, laminin expression is disorganized and
patchy as the peritubular space is less identifiable
(Fig. 3B). By 13.5 dpc, the
edge of individual testis cords is clearly identifiable by a sharp, continuous
border of laminin (Fig. 3C). In
Dax1-deficient gonads, laminin is expressed heterogeneously
throughout the gonad (Fig. 3D).
Although rudimentary cords are periodically identifiable, only fragments of
the circumferential border are positive for laminin
(Fig. 3D). By 14.5 dpc, testis
cord-like structures are more easily identifiable in the
Dax1-deficient testis. However, these cords are smaller, irregular in
shape, and there are frequent breaks in laminin localization
(Fig. 3F). In some cases, a
testis cord is observed to encircle an island of laminin (inset of
Fig. 3F).
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Sertoli cells are present but disorganized in Dax1-deficient
testis
In the developing male gonad, Sertoli cells initiate formation of the
testis cords (Schmahl et al.,
2000). In the wild-type gonad, Sertoli cells are restricted to the
testis cord, which create a scalloped pattern on the mesonephric side of the
gonad when labeled by in situ hybridization for the Sertoli-specific genes
Dhh, Amh or Sox9 (Fig.
4A,C,E,G). In the Dax1-deficient gonad, expression of
these markers is present, but staining is not discretely localized to the
testis cords (Figs. 4B,D,F,H).
All somatic cells in the male gonad express Gata4, but Sertoli cells express
much higher levels than cells within the peritubular space
(Viger et al., 1998
). Sections
were counterstained for laminin to clearly identify the testis cords (Gata4
positive, Laminin negative). In both wild-type and Dax1KO testis,
Gata4-positive Sertoli cells are identifiable, despite differences in
organization within the gonad (Fig.
4I,J).
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Fetal Leydig cell development is arrested in Dax1-deficient
males
The production of androgens by Leydig cells is critical for male sexual
differentiation (Habert et al.,
2001). Although the origin of these cells is unknown, immature
Leydig cells are postulated to originate from within the gonad
(Habert et al., 2001
). One of
the earliest distinguishing characteristics of immature Leydig cells is
expression of side-chain cleavage (Scc), a steroidogenic enzyme required for
androgen synthesis (Yao et al.,
2002b
; Habert et al.,
2001
). In the wild-type gonad, Scc-positive cells are arranged
exclusively in columns between the testis cords
(Fig. 5A,C). By in situ
hybridization, these Scc-positive columns extend completely from the coelomic
to mesonephric surface of the gonad (Fig.
5C). In the Dax1-deficient gonads, Scc-positive cells on
the coelomic surface are located in the interstitium of structures that
resemble testis cords (Fig.
5B). Overall, the number of Scc-positive cells on the coelomic
surface appears similar to the wild-type male gonad. However, when viewed from
the lateral aspect (Fig. 5D),
very few Scc-positive cells can be identified. Sertoli cells weakly express
Sf1, but its expression is strikingly upregulated in Leydig cells, probably
because of the actions of Dhh (Yao et al.,
2002b
). These Sf1-positive Leydig cells are evident as columns
that extend from the coelomic to the mesonephric surface in the normal testis
in the same pattern as observed for Scc expression (arrows,
Fig. 5E). In the
Dax1-deficient testis, these cells are restricted to the coelomic
surface and are almost completely excluded from the more dorsal part of the
gonad (above the broken line in Fig.
5F).
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Abnormal peritubular myoid cell development due to Dax1
deficiency
Testis cords require PTM cells for complete differentiation
(Clark et al., 2000;
Pierucci-Alves et al., 2001
).
This key myoepithelial cell-type is present exclusively in males.
Histologically, few PTM cells are present in the Dax1-deficient
gonad, suggesting a defect in PTM development
(Fig. 2). DNA fragmentation was
analyzed in both normal and Dax1-deficient gonads from 11.5 to 13.5 dpc to
determine if PTM cells undergo accelerated apoptosis in the
Dax1-deficient gonad. However, there was no significant difference in
cell death between Dax1-deficient and wild-type mice
(Fig. 6A-C). Rates of
proliferation were analyzed using BrdU, a thymidine analog that is
incorporated during the S phase of mitosis. BrdU was injected into pregnant
females to identify cells actively dividing cells. There was no difference in
proliferation at 11.5 dpc, a time when Sertoli cells proliferate at the
coelomic epithelium (data not shown). By 13.5 dpc in normal mice, each testis
cord was surrounded by dividing PTM cells that were identified by location and
cellular morphology, as specific markers are not currently available (arrows,
Fig. 6D). Only a one-third as
many dividing PTM cells were identified in the Dax1-deficient gonad
compared with wild type (Fig.
6E,F). There were no differences in number of proliferating germ
cells between wild-type and knockout testis.
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DISCUSSION |
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The development of testis cords is a central feature of gonadal
differentiation in the male, as the testis cord is the antecedent structure of
the seminiferous tubule (Kaufman,
1995). Moreover, testis cords are one of the earliest structural
features that discriminate testis versus ovary development from the
bipotential gonad. In the Dax1-deficient mouse, gonadal development
is apparently normal until the point of testis cord formation, suggesting that
Dax1 plays a role in gonadal differentiation rather than gonadal patterning
(e.g., Sf1) or sex determination (e.g., Sry). The fact that DAX1
mutations in humans result in hypogonadism and infertility rather than sex
reversal or pseudohermaphroditism is consistent with DAX1 action as a mediator
of sexual differentiation.
Defective testis cord formation is most likely to reflect abnormal function
of either Sertoli cells or PTM cells, which comprise the major somatic cell
types of this structure. Germ cells are less likely involved as Dax1 is not
expressed in germ cells, and testis cords and seminiferous tubules form
normally in the absence of germ cells
(Orr-Urtreger et al., 1990).
There is no deficiency or overabundance of Sertoli cells at any stage of
development in the Dax1-deficient testis. At 11.5 dpc, Sertoli cell
proliferation is normal, as assessed by BrdU, and expression of the
Sertoli-specific genes Sox9, Dhh and Amh is relatively
unaffected in the Dax1-deficient testis. The critical difference
between wild-type and Dax1-deficient testis is that Sertoli and germ cells are
not arranged in testis cords, which is probably secondary to a defect in
testis cord organization. Thus, although some as yet unrecognized defect may
exist in Dax1-deficient Sertoli cells, there is no indication of a
cell-autonomous abnormality in Sertoli cells. This view is supported by the
fact that transgenic expression of DAX1 in Sertoli cells fails to alter testis
pathology, although fertility is improved
(Jeffs et al., 2001a
).
The origin of fetal Leydig cells is debatable. Electron microscopic studies
suggest that Leydig cells develop from a population of cells that migrate into
the gonad from the mesonephros
(Merchant-Larios and Moreno-Mendoza,
1998). The secreted morphogen Dhh is required for Leydig cell
development, as Dhh knockout mice are deficient in fetal Leydig cells
(Clark et al., 2000
). The
transmembrane receptor Ptch transduces the Dhh signal and is expressed by
Leydig cells (Yao et al.,
2002b
). Using gonad-mesonephros co-culture experiments, it appears
that Ptch(+) cells originate within the gonad by 11.5 dpc
(Yao et al., 2002b
),
suggesting that Leydig cells may originate from within the gonad. Deficiency
of Dax1 restricts Leydig cells to coelomic edge of the gonad. These findings
suggest that at least one population of Leydig cells may arise from the
coelomic epithelium and that Dax1 may contribute to the
development/differentiation of this cell type. Humans with DAX1
mutation are still able to produce testosterone when stimulated by exogenous
chorionic gonadotropin, indicating that Leydig cells are present and
functional in the adult. Thus, similar to Sertoli cells, Leydig cells are
present and appear to function normally, but are aberrantly located in the
Dax1-deficient testis.
Differentiation of testis cords also requires PTM cells
(Clark et al., 2000). These
myoepithelial cells surround the testis cord, separating it from the
peritubular space and contributing to the production of the basal lamina.
Examination of the adult testis from the Dax1-deficient mouse
revealed disruption of the basal lamina and ectopic localization of Leydig
cells within the seminiferous tubules
(Jeffs et al., 2001b
). There
was also a deficiency of differentiated PTM cells, a feature that is also
present during early testis development. Although there are no specific
markers for developing PTM cells, the paucity of PTM appears to result from
decreased proliferation rather than increased apoptosis. There are at least
three possible reasons for decreased numbers of PTM cells: (1) Dax1 may play a
role in PTM cell replication; (2) Dax1 may be required for progenitors of PTM
cells to migrate into the gonad, where they later replicate; or (3) Dax1 may
be required for the production of a mitogen that induces PTM migration and/or
replication. Pending further studies needed to address these possibilities, we
propose the following model (Fig.
7). Dax1 is expressed in Sertoli cells and other somatic cells
immediately beneath the coelomic epithelium. As Sertoli cells appear
relatively normal, it is more likely that other somatic cells, including a
potential common progenitor for PTM, and Leydig cells are affected by Dax1
deficiency. A common lineage of these two cell types is likely as they are
both deficient in the Dhh knockout mouse and disorganized or absent in the
Dax1-deficient testis (Clark et
al., 2000
; Jeffs et al.,
2001b
). Thus, Dax1 may be required for the development or normal
responsiveness of these progenitor cells. It is striking that Dax1 is not
expressed in dividing cells (Fig.
7), suggesting that it may act to differentiate cells rather than
initiate cell division. Thus, once PTM cells are generated, Dax1 might induce
their differentiation.
|
Dax1 was initially proposed as an ovarian determining gene, an idea not
supported by deletion of both Dax1 alleles in XX mice
(Yu et al., 1998). However,
most models of sex determination still portray Dax1 as an `anti-testis' gene
(Goodfellow and Camerino,
2001
). This view is based on many lines of evidence. First,
duplication of the DAX1 locus in humans is associated with dysgenetic
testes and male to female sex-reversal
(Bardoni et al., 1994
). Second,
transgenic overexpression of Dax1 prevents Sry-mediated sex-reversal of XX
mice (Swain et al., 1998
). Our
data indicates that Dax1 is necessary for normal testis differentiation. It is
difficult to reconcile these findings easily with the effects of Dax1
overexpression or duplication. It is possible that the transgenic expression
models do not exactly replicate the spatial or temporal pattern of Dax1
expression, thereby altering the fate of cells in a manner that precludes
normal testis development. Or, Dax1 overexpression may secondarily alter other
developmental pathways that interfere with gonadal development. Alternatively,
Dax1 deficiency may eliminate a key progenitor cell or differentiation step
without revealing Dax1 functions in other cell types or at later developmental
stages. In light of our results, the view that Dax1 is an antitestis gene
oversimplifies its varied functions. It is apparent, however, that gonadal
development is highly sensitive to the dose and pattern of Dax1 expression,
underscoring the importance of future studies that further define its
mechanism of action.
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ACKNOWLEDGMENTS |
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