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Division of Endocrinology, Metabolism, and Molecular Medicine (B.J., M.I., F.A.M., J.L.J.), Northwestern University Medical School, Chicago, Illinois 60611; Departments of Pathology, Molecular and Cellular Biology, and Molecular and Human Genetics (M.M.M.), Baylor College of Medicine, Houston, Texas 77030; and Southern Illinois University School of Medicine (L.D.R.*), Department of Physiology, Carbondale, Illinois 62901
Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, 303 East Chicago Avenue, Tarry Building 15-709, Chicago, Illinois 60611-3008. E-mail: ljameson{at}northwestern.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutations in the DAX1 gene cause the X-linked cytomegalic form of AHC, a disorder characterized by sites of endocrine dysfunction that reflect the normal tissue-specific expression of DAX1: the adrenal cortex, ventral medial hypothalamus, anterior pituitary gonadotropes, and gonads (5, 6). Affected male infants typically present with primary adrenal failure owing to the failure of the mature adult zone of the adrenal cortex to develop (7). The failure of pubertal development in AHC patients kept alive by adrenal steroid replacement therapy led to the recognition that hypogonadotropic hypogonadism (HHG) is also an integral feature of the syndrome (8). The HHG is caused by combined defects in the production of hypothalamic GnRH and pituitary gonadotropins (2, 3, 8, 9).
Infertility in patients with AHC has been attributed to gonadotropin deficiency. Although administration of exogenous human CG (hCG) has been shown to induce a normal T response in most AHC patients, spermatogenesis has not been induced (10, 11), and only rare spermatogonia have been described in testicular biopsies (10). These findings suggest DAX-1 may have a direct role in testicular development and function outside the hypothalamo-pituitary axis, a hypothesis first supported by the characterization of Dax1-deficient mice. Mutant male mice are both infertile and hypogonadal despite normal serum levels of T, gonadotropins, and adrenal steroids (12).
Histologic examination of the testes of 12-wk-old Dax1-deficient male mice reveals a progressive degeneration of the germinal epithelium, manifesting as a loss of spermatogenesis and dilation of the seminiferous tubules (12). A subsequent analysis of sperm production and function in mutant mice identified low epididymal sperm counts and decreased sperm motility (13). In addition, fewer sperm from Dax1-deficient mice were able to undergo an immediate acrosome reaction, and fertilized fewer eggs in vitro than wild-type sperm (13). Interestingly, in experimental models of infertility, ligation of the efferent ductules also results in dilated seminiferous tubules (14, 15, 16), reduced sperm motility, and an impaired acrosome reaction (17) similar to that observed in Dax1-deficient male mice. These observations suggest the possibility that infertility in Dax1 mutant males may result from a blockage of the testicular duct system. To test this hypothesis, we examined the male reproductive tract in Dax1-deficient mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Light and electron microscopy
To fix the testicular duct system for light and electron
microscopy, 5-, 12-, 52-, and 104-wk-old Dax1-deficient and
wild-type male mice were anesthetized with pentobarbital and perfused
through the heart (18) with 0.05 M
sodium cacodylate buffer containing 5% gluteraldehyde. After fixation,
the testes, efferent ducts, and epididymis were excised and cut into 1
mm3 tissue blocks. These tissue blocks were
postfixed in osmium:ferrocyanide (19), dehydrated,
infiltrated, and embedded in Araldite 502 epoxy resin (Electron
Microscopy Sciences, Ft. Washington, PA). For light microscopy of these
tissues, 1-µm sections were mounted onto glass slides and stained
with 1% toluidine blue. For electron microscopy, thin sections showing
silver-gold interference colors were prepared using an ultra-microtome
with a diamond knife (Delaware Diamond Knives, Wilmington, DE) and
examined with an H500 transmission electron microscope (75 kV,
Hitachi, Tokyo, Japan).
Immunohistochemistry
For immunohistochemical analysis, 12-wk-old mice were killed by
cervical dislocation. Testes were quickly dissected and fixed in 4%
paraformaldehyde overnight at 4 C. Excess fixative was removed with a
70% ethanol/1% ammonium hydroxide solution, dehydrated, and embedded
in paraffin for 3 µm sectioning.
Immediately before immunohistochemistry, testicular sections were microwaved at high power for 10 min in 0.01 M sodium citrate buffer (pH 6.0) and left to cool to room temperature. After washing in a T-PBS bath (0.1% Tween-20 in sodium PBS) for 5 min, sections were immersed in 3% hydrogen peroxide in absolute methanol for 20 min to quench endogenous peroxidase activity. Nonspecific background was reduced with the aid of CAS Block (Zymed Laboratories, Inc., San Francisco, CA). For immunodetection of Leydig cell-specific steroidogenic acute regulatory protein (StAR) expression, tissue was incubated with a rabbit antihuman StAR antibody (residues 63–285; 1:100 dilution in T-PBS, 1 h, room temperature) kindly provided by Dr. J. F. Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA), followed by the application of an antirabbit biotinylated secondary antibody (Zymed Laboratories, Inc.). To detect Sertoli cell-specific expression of GATA-1, sections were incubated with a rat antimouse GATA-1 (N6) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:100 dilution in CAS Block, 4 C overnight) followed by an antirat biotinylated secondary antibody (Santa Cruz Biotechnology, Inc.). Final visualization of both StAR and GATA-1 expression was achieved with the serial addition of streptavidin-peroxidase and DAB substrate-chromogen. For negative controls, the primary antibody was omitted.
Lipid histochemistry
To detect lipid accumulation in the rete testis, 12-wk-old mice
were killed by cervical dislocation, their testes rapidly excised,
immersed in Tissue-Tek embedding matrix (Sakura Finetek USA, Inc.,
Torrance, CA) and frozen on dry ice. Frozen tissue specimens were then
placed in a Microtome Cyrostat HM 505 E (MICROM Laborgeräte GmBH,
Walldorf, Germany) and 50 µm cyrosections taken. These sections were
immediately stained in a saturated solution of oil red O
(Sigma, St Louis, MO) for 10 min, followed by two washes
in water and mounting (20).
Tumor histopathology
A subset of aged Dax1-deficient male mice (>1 yr
old) exhibited testicular tumors. The affected testis was quickly
removed and fixed in Bouin’s solution (25% formaldehyde, 5% glacial
acetic acid in saturated picric acid) overnight at 4 C. Excess fixative
was removed with a 70% ethanol/1% ammonium hydroxide solution and the
testis dehydrated and embedded in paraffin for 3 µm sectioning.
Sections were subjected to either periodic acid Schiff staining or
immunochemical characterization courtesy of the Special Histology
Laboratory, Northwestern Memorial Hospital (Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In contrast, dilated
and degenerating seminiferous tubules were evident in mutant mice of
all ages. (Fig. 1B). The dilation of tubules appeared transient, with
considerable variability in the proportions of normal, dilated, and
degenerating seminiferous tubules between different mutant males of
different ages. Vacuolation of the seminiferous epithelium and lipid
accumulation were common in affected tubules, especially near the rete
and in all tubules in older animals, indicative of a breakdown in
Sertoli-germ cell junctions (21).
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, C and D). Electron microscopic analysis demonstrates that the Leydig
cells in the interstitial tissue near the rete are hyperplastic, with
large mitochondria containing numerous tubular cristae (Fig. 2). In contrast, the Leydig cells in the
seminiferous tubules are smaller and contain smaller mitochondria.
Neither type of Leydig cell exhibits the cytoplasmic swirls of saccular
endoplasmic reticulum that are characteristic of normal adult-type
murine Leydig cells (22), but both stained positive for
the Leydig cell-specific StAR protein (Fig. 1D).
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and 3). The peritubular myoid cells are
relatively undifferentiated, there is no basement membrane between the
myoid cells and Sertoli cells, and both cell types send microvillous
processes into the intracellular space separating them.
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). In Dax1-deficient male
mice of all ages, however, the rete is blocked by proliferation of a
unique population of cells (Fig. 4B). Their distinct tripartite
nucleoli and the immunohistochemical detection of the transcription
factor GATA-1 (Fig. 4C) characterize these cells as Sertoli cells,
despite having a slightly irregular shape owing to their loss of
contact with a basement membrane. This was confirmed by subsequent
electron microscopic analysis that identified vacuolated mitochondria
characteristic of murine Sertoli cells (Fig. 5) (23).
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, inset), and accordingly this tissue is
stained heavily by oil Red O compared with the rest of the testis (Fig. 4D). Macrophages phagocytosing germ cells were observed in the
seminiferous tubules and rete testis. Many of the macrophages observed
in the interstitial tissue contained crystalloid structures resembling
those that have been previously described as containing cholesterol
(24) (Fig. 6). Cholesterol
crystals are also observed in several Leydig and Sertoli cells (Fig. 3), suggesting a steroidogenic defect.
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). In Dax1-deficient male
mice, however, the epithelium of the efferent duct is frequently
overgrown and consists of several layers, blocking the passage of sperm
(Fig. 7B). The epithelia also contain numerous basally positioned lipid
deposits not observed in the wild-type. These phenomena are most
pronounced in the efferent duct closest to the rete testis (proximal)
and lessen in severity through the middle and distal ductules. The
epididymides appear normal in the mutant mice.
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, C
and D). These Sertoli cells possess the characteristic infolded nucleus
and vacuolated mitochondria found in mice and appear to originate from
the basal lamina, displacing the epithelial cells (Figs. 7D and 8).
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). The vast
majority of the tumors were bilateral and characterized visually under
the light microscope as sex cord-stromal tumors (Fig. 9). These tumors
were either derived from cells of the Sertoli/granulosa cell lineage
(Fig. 9, A–C) or Leydig cells (interstitial cells; Fig. 9, D and E).
The former tumors were reminiscent of juvenile granulosa cell tumors
that are seen in young infants and girls (25). These
granulosa cell tumors in the Dax1-deficient males are
similar to the testicular tumors observed in inhibin-
knockout mice
that arise as early as 4 wk of age (26). In both cases,
the tumors cause tubular degeneration and a block of spermatogenesis
(Fig. 9B) and are mitotically active (Fig. 9C). The Leydig cell
neoplasia (Fig. 9D) and Leydig cell hyperplasia (Fig. 9E) is similar to
the findings in Müllerian-inhibiting substance (MIS) knockout
(27) or MIS/inhibin- double knockout mice
(28). Most Dax1-deficient male mice
exhibited Leydig cell hyperplasia (Fig. 9E), and three mice with Leydig
cell neoplasia (Fig. 9D) were observed. These diagnoses were confirmed
immunohistochemically (Table 1)
(25). Neither tumor stained positively for alkaline
phosphatase, confirming that these tumors were sex cord-stromal and not
germ cell in origin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A major feature of the current study was the novel observation of
normally compartmentalized testicular cell types in abnormal locations.
Even at 5 wk of age, Sertoli cells had accumulated in the rete testis
and efferent ducts, whereas Leydig cells were present in seminiferous
tubules near the rete testis. These phenomena might be explained by a
defect in the embryological development of the excurrent duct system.
Dax1 transcripts are detected in the gonadal ridge and
mesonephros from E11.5 until E12.5, after which there is a rapid
decline in Dax1 expression in the testis (29).
Dax1 expression, however, is preserved in a group of cells
at the junction between the mesonephros and the gonad, the mesonephric
tubules (6), which eventually forms the rete testis and
efferent ducts (30). Given the temporal and spatial
expression of Dax1 in the male embryo, it appears
possible that Dax1 has a significant role in the migration
and differentiation of the various testicular cell types from the
mesonephros and also plays a role in the development of the
excurrent duct system. We also observed dividing Leydig cells inside
seminiferous tubules directly adjacent to disrupted peritubular tissue
and within the peritubular myoid cell layer (Fig. 3). Indeed, the
intratubular Leydig cells in Dax1-deficient male mice
resemble fetal Leydig cells, and the myoid cells in the peritubular
tissue are relatively undifferentiated. Our findings corroborate the
connection between peritubular myoid and Leydig cell lineages observed
in the Desert hedgehog-deficient mouse. In male mice lacking the
Desert hedgehog gene, the peritubular basal lamina is absent
and intermingling of Leydig and Sertoli cells is observed
(31). Further studies are required to evaluate the
embryonic contribution to the testicular defects observed in the
Dax1-deficient male mice.
Although we observe proliferation of Sertoli cells in the rete testis and efferent duct, the mitogen(s) that are responsible for this stimulation are unknown. E2 has been demonstrated to induce Sertoli cell proliferation (32) by increasing the secretion of TGF-ß, which acts synergistically with FSH to promote DNA synthesis (33). We previously examined the consequences of Dax1 deficiency on Leydig cell steroidogenesis in vivo (34). The transcript, protein, and enzymatic activity of aromatase (Cyp19), the enzyme responsible for the conversion of T to E2 in the male, was significantly increased in Dax1-deficient Leydig cells and was accompanied by a 40-fold increase in intratesticular E2 (34). These findings are consistent with the repression of steroidogenic factor-1-mediated transactivation of the Cyp19 promoter by DAX-1 in transient transfection studies in vitro (34). Based on the observations made in the present study, it is reasonable to postulate that the abnormally high intratesticular levels of E2 found in adult Dax1-deficient male mice may promote Sertoli cell proliferation and blockage of the rete testis and efferent ducts, indirectly accounting for many of the testicular abnormalities seen in the mutant mice. This idea is supported by the finding that the antiestrogenic compound tamoxifen partially reverses the testicular pathology observed in Dax1-deficient male mice; tamoxifen restored fertility and reduced Leydig cell hyperplasia (34). However, the fact that the abnormal pathology remains in the rete testis of tamoxifen-treated Dax1-deficient male mice supports the possibility that an embryonic defect, in addition to the overexpression of aromatase, is involved in the testicular pathology found in the mutant mice.
The granulosa cell tumors identified in aged Dax1-deficient
males are similar to those observed in inhibin- knockout mice
(26). In turn, the Leydig cell tumors are similar to the
findings in MIS knockout (27) or MIS/inhibin- double
knockout mice (28). However, while the latter gene
targeting strategies have indicated that inhibins and MIS synergize to
influence sex cord-stromal tumor development, we have not found any
alterations in the expression of TGF-ß family members in
Dax1-deficient testes (data not shown). Rather, the raised
levels of intratesticular E2 in Dax1-deficient male mice may
be primarily responsible for the testicular tumorigenesis. Estrogens
are well known to stimulate the development of Leydig cell tumors in
the mouse, and the exogenous E, diethylstilbestrol, has been
extensively studied as a model for the induction of Leydig cell tumors
in this species (35, 36). Furthermore, the transgenic
overexpression of aromatase in mice led to the development of
testicular Leydig cell tumors (37). Sertoli and granulosa
cells are derived from the same precursor cells (29) and
E2 causes proliferation of both cell types (32). It
follows that, although granulosa cell tumors in the testis are unusual
(25), the appearance of proliferating neoplastic granulosa
cells in Dax1-deficient mice supports the idea that the
proliferation of the Sertoli/granulosa cell lineage is defective in
this model.
In addition to the possible role of raised intratesticular E2 as a
contributing factor for the pathology observed in the rete testis and
efferent ducts, Zhang et al. (38) identified
that DAX-1 interacts with ER and ERß, inhibiting their activation.
These interactions could play a significant role in the development and
function of the testis, especially given developmental studies
suggesting that Dax-1 and ERs are coexpressed in reproductive tissues
during embryogenesis and into adulthood (5, 39). Indeed,
studies of the targeted inactivation of the gene encoding ER suggest
that estrogens, and more specifically functional ER, are essential
for normal male fertility (40, 41). Similar to the
Dax1-deficient male mouse, the so-called ERKO mouse is
infertile. Their testes atrophy by 150 d of age (42),
and sperm concentrations are significantly reduced in the epididymis
(40). The rete testis and efferent ducts are also abnormal
in ERKO mice (41). However, this defect is not caused
by a proliferation of cells as in the Dax1 knockout mouse
but rather by a decrease in fluid absorption. Our data raises the
possibility that overactivity of ERs caused by the loss of Dax-1
function might also result in impaired development and dysfunction of
the rete testis and efferent ductules. Although we have not observed
any differences of ER or ERß expression in the testis of
Dax1-deficient male mice (data not shown), ER activity in
this model warrants further research.
Other than the up-regulation of aromatase in Leydig cells purified from
Dax1-deficient male mice, no alteration was observed in the
expression of the steroidogenic enzymes required for the biosynthesis
of T (34). In the current study, however, significant
lipid (particularly cholesterol) deposits, indicative of impaired
steroidogenesis, was observed in all cell types located in the vicinity
of the rete testis. For example, lipid accumulation is particularly
pronounced in Leydig cells immediately adjacent to the Sertoli cells in
the rete (Fig. 4B, inset). This may result from the
secretion of various paracrine factors from the Sertoli cells, which en
masse, inhibit nearby Leydig cell steroidogenic function. The area near
the rete testis also contains an unusually high number of
hemosiderin-positive macrophages containing cholesterol crystals.
Macrophages are known to perform several nonimmune functions including
the production of 25-hydroxycholesterol that stimulates T production by
neighboring Leydig cells (43, 44). Cholesterol appears to
be collected in macrophages located near the rete testis, rather than
dispersed to Leydig cells, suggesting that T production is impaired.
The physiological consequences of this area of steroidogenic deficiency
near the rete are apparently negligible given that serum levels of T
are normal in mutant male mice. Although technically difficult,
investigation of the cell-to-cell interactions near the rete testis may
reveal further significant cellular consequences of Dax1
disruption.
To date, the histology of the rete testis has not been examined in patients with AHC, and intratesticular levels of E have not been assessed. Although the observations in the rete testis and efferent ducts of the Dax1-deficient male mice suggest a significant embryonic component to the observed pathology, there may be an opportunity to restore fertility to some AHC patients by correcting the steroidogenic defects. As described previously, the ER antagonist tamoxifen partially reversed the testicular pathology observed in Dax1-deficient male mice (34). Tamoxifen has been shown to improve total sperm count, motility, and functional sperm fraction in some men with idiopathic oligozoospermia (45, 46). Thus, while the etiology of infertility in males with AHC is multifactorial, a potential therapeutic approach might involve correction of aberrant steroidogenesis, as well as gonadotropin deficiency.
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Acknowledgments |
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Footnotes |
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1 The authors would like to dedicate this research to the memory of
Professor Lonnie D. Russell, following his untimely death on 11 July
2001. Lonnie Russell was Professor in the Department of Physiology,
Southern Illinois University School of Medicine. A well-renowned expert
in the field of testicular morphology and spermatogenesis, Professor
Russell authored numerous texts related to the testis and microscopy,
as well as the local history and beauty of Southern Illinois, where he
made his home. Lonnie welcomed collaboration in the pursuit of
scientific inquiry and was always in possession of a smile and sense of
humor. His warmth and hospitality will be sorely missed and fondly
remembered. 
Abbreviations: AHC, Adrenal hypoplasia congenita; HHG, hypogonadotropic hypogonadism; hCG, human CG; MIS, Müllerian-inhibiting substance; StAR, steroidogenic acute regulatory protein.
Received March 20, 2001.
Accepted for publication June 27, 2001.
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References |
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-inhibin is a tumour-suppressor gene with gonadal specificity
in mice. Nature 26:313–319
and ß during mouse embryogenesis. Mech Dev 81:163–167[CrossRef][Medline]
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