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Reproductive Endocrinology |
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235; and the Department of Internal Medicine, University of Bonn, Bonn, Germany
Address all correspondence and requests for reprints to: Michael J. McPhaul, M.D., Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8857.
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Abstract |
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To assist in the evaluation of patients with possible AR defects, we previously employed the use of a recombinant adenovirus to deliver an androgen-responsive gene into fibroblast cultures to assay AR function in normal subjects and patients with complete forms of androgen resistance. Although these studies demonstrated measurable differences between these two groups of subjects, we did not assay samples from patients with partial defects of androgen action. In the current study, we have modified this method to examine AR function in three groups of patients with known or suspected defects of AR function: patients with Reifenstein syndrome, patients with spinobulbar muscular atrophy, and patients with severe forms of isolated hypospadias. When assayed using this method, the AR function of patients with Reifenstein syndrome was intermediate between that of normal control subjects and that of patients with complete testicular feminization. Using the parameters established by the aforementioned experiments, we found that defective AR function can be detected in fibroblasts established from patients with spinobulbar muscular atrophy and in some patients with severe forms of isolated hypospadias, including two with a normal AR gene sequence. These results suggest that this method may have some utility in screening samples to detect defects of AR function, particularly when viewed in the context of other AR assays results.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone
(DHT). Defects in the development of the normal male phenotype can be
caused by alterations in the synthesis or metabolism of androgens or to
defects in the androgen receptor (AR) gene (1). In a substantial
proportion of patients with abnormalities of virilization (such as
severe isolated hypospadias), no such defects have been identified
(2). The diagnosis of patients with defects of AR function in the larger population of individuals with abnormalities of male development presents a considerable challenge and requires the use of data from the family history, endocrine testing, and measurement of androgen binding in cultures of genital skin fibroblasts. Even with such information, a proportion of mutations in the DNA-binding domain of the AR receptor would not be detected.
The diagnosis of patients with abnormalities of the AR would be facilitated by the availability of a test that assessed the function of the AR expressed in fibroblasts cultured from individual patients. As androgen-responsive genes have not been characterized in fibroblasts, we have used a recombinant adenovirus to deliver a model androgen-responsive reporter gene [mouse mammary tumor virus (MMTV)-luciferase] into genital skin fibroblast cultures. Our previous studies demonstrated the utility of this assay in discriminating subjects with normal AR function from those with the complete defects of AR function (complete testicular feminization) (3). In the current study, we have applied this assay to measure AR function in genital skin fibroblast cultures from three groups of patients with known or potential defects of AR function: men with the Reifenstein phenotype, spinobulbar muscular atrophy (SBMA), and isolated severe hypospadias.
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Materials and Methods |
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The fibroblast cultures assayed in the current investigation were from four distinct groups. Patients with Reifenstein syndrome displayed endocrine and clinical profiles and a family history consistent with X-linkage. The genetic defects in 7 of the 11 individual strains studied have been identified (4, 5) (our unpublished observations). Patients with SBMA have a progressive degeneration of the spinal and bulbar motor neurons and clinical evidence of androgen resistance. These clinical features are not present at birth, but are first evident in adulthood and have been traced to an expansion of the triplet repeat (CAG) encoding the glutamine homopolymeric segment within the amino-terminus of the AR (6). The samples from patients with severe isolated hypospadias (perineal or penoscrotal) were established from individuals that were without endocrine features or family history suggestive of an AR defect (7). Normal control strains were established from samples of foreskin or genital skin fibroblast biopsies from patients with unrelated disorders, as outlined below.
Androgen-responsive reporter gene delivery via a recombinant adenovirus
The recombinant adenovirus contains the firefly luciferase gene under the control of the MMTV-long terminal repeat (8). This long term repeat contains sequences that confer responsiveness to the AR and other related members of the steroid receptor family (including the glucocorticoid receptor).
Fibroblast strains were plated in six-well plates (3.5-cm
diameter wells) and cultured in MEM containing 10% FCS until
confluency. Experiments were initiated by infection of the confluent
monolayers (
2 x 105 cells/well) with approximately
107 plaque-forming units of the recombinant adenovirus in
MEM for 1 h at 37 C. After infection, the medium was replaced with
fresh MEM containing 10% FCS, and the incubations were continued for
24 h. After this 24-h incubation, the medium was removed and
replaced with MEM containing 5% charcoal-stripped serum alone or the
hormone to be assayed. Incubations were conducted with 2 nmol/L
mibolerone (Mb), 2 nmol/L DHT, or 4 nmol/L T. The hormone stimulation
was continued for 72 h, at which time the cells were harvested and
assayed to determine luciferase activity. As noted below, in some
incubations the 5-reductase inhibitor finasteride was included at a
final concentration of 0.1 µmol/L.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have characterized the AR expressed in fibroblast strains
established from a number of patients with Reifenstein syndrome and
have identified amino acid substitutions in seven patients (4, 5)
(unpublished observations). The mutations are localized to the AR
hormone-binding and DNA-binding domains and have variable effects on
ligand binding by the receptor (see Table 1
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. Initial experiments measured the activities of the reporter
gene that are observed after stimulation with a saturating
concentration of Mb, as previously reported (3). The results obtained
for this group of patients appeared to define two subject categories.
In one group (represented by strains 691, 217, 1103, 1104, and 497), AR
function is reduced and falls between that observed for normal subjects
(>10-fold induction) and that in patients with complete testicular
feminization (1.1-fold induction) (3). Samples from the second group of
patients (strains 787, 38, and 593) showed a strikingly different
pattern, as the level of reporter gene induction after stimulation with
Mb alone was well within the range defined for fibroblasts from normal
subjects with normal AR function.
In prior studies of mutant ARs using transfection assays (8), we noted
that AR function could be restored for some mutant ARs by the use of
potent, nonmetabolizable androgen and suggested that the use of such
ligands to stimulate the AR in functional assays might obscure defects
of the AR caused by instability of the AR-hormone complex. To test this
possibility, assays were performed using the physiologic ligands DHT
and T to stimulate receptor function. As depicted in Table 1, the
strains that demonstrated abnormalities of AR function using Mb (1061,
691, 497, and 217) also demonstrated similar abnormalities using T or
DHT as ligand. The levels of AR function observed in strains 38, 787,
and 593 remained in the normal range and were not markedly reduced in
cultures stimulated with DHT or T compared to those in cultures
stimulated with Mb.
The amino acid substitution mutations identified in strains 787, 38,
and 593 caused a rapid dissociation of ligand from the receptor. In
addition, our previous studies demonstrated that in transfection assays
the mutant AR predicted for one patient (patient 38) was more
responsive to DHT than to T (9). This finding prompted us to repeat
these experiments including finasteride to block the conversion of T to
DHT in the genital skin fibroblast cultures. As shown in Table 1, this
modification of our assay procedure had two effects. First, consistent
with previous results, the inclusion of finasteride dramatically
reduced the activity of the AR predicted for patient 38; finasteride
had a similar effect on the activity of the mutant AR expressed in the
787 and 593 fibroblast strains. The second effect was a subtle decrease
in activation of the reporter gene in normal fibroblast strains. In
contrast to the marked changes noted in the 38, 787, and 593 cell lines
stimulated with T in the presence of finasteride (>70% reduction),
the effect on the activity of the AR in normal fibroblasts decreased by
an average of 50% (range, 24–67%). An important point is that the
level of AR activity measured in the normal fibroblast samples remained
in the normal range (>10-fold stimulation), even when measured after
stimulation with T in the presence of finasteride. All of the
Reifenstein samples showed low levels of AR function when assayed with
T in the presence of finasteride.
AR function is reduced in fibroblast strains established from patients with SBMA
Expansions of the glutamine repeat segment in the amino-terminus of the AR are associated with degeneration of anterior motor neurons and clinical signs of androgen resistance, including gynecomastia (Kennedy’s disease) (5). Transfection assays of complementary DNAs encoding ARs containing such expanded glutamine repeats have demonstrated that these mutant ARs possess a reduced capacity to activate androgen-responsive genes (9, 10, 11), and these deficits are even more profound when the levels of immunoreactive AR are assessed (11).
To assess whether the expected reduction of AR function could be
detected in fibroblast strains established from patients with this
disorder, we infected the monolayers with the adenovirus carrying the
MMTV-luciferase reporter gene. As before, AR function was assessed
after following stimulation with saturating doses of Mb. As shown in
Table 2, in all instances the level of AR function was
reduced compared to that in normal control fibroblasts. In most cases,
the level of AR function was comparable to that in patients with
Reifenstein syndrome, although in some (e.g. strain
814), the level of AR function was markedly reduced and approached the
level observed in samples from patients with complete androgen
resistance.
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Hypospadias is one of the most common of human congenital defects (12, 13). Because this abnormality is a prominent component of some partial androgen resistance phenotypes, it has been suggested that a proportion of patients with severe forms of isolated hypospadias (i.e. not clearly part of a familial condition) might be caused by mutations of the AR.
To address this question, we investigated the level of AR function in
eight patients with severe forms of isolated hypospadias (6). As shown
in Table 3, AR function was normal after stimulation
with Mb in two strains, was reduced in two others, and was virtually
absent in one. As indicated in Table 3, the strain with near-absent AR
function (strain 931) was later discovered to harbor a mutation in the
DNA-binding domain of the receptor (G566V). Our studies indicate that
strains 938 and 939 do not have amino acid substitutions in the
AR-coding region (2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using assays of Mb-stimulated AR function, the Reifenstein patients comprise a heterogeneous group. In the first group, exemplified by patient 691, the level of AR function is intermediate between that measured in fibroblasts of subjects and that in fibroblasts of patients with complete testicular feminization. By contrast, using this same assay, fibroblasts from three other patients with Reifenstein syndrome (787, 38, and 593) demonstrate levels of AR function that are well within the normal range.
This result suggested either that the level of AR function measured in patients with Reifenstein syndrome had no direct relationship to the observed phenotype or that the assay method used in these experiments was somehow obscuring this relationship. Our previous studies in transfected cells (9) and the nature of the AR defect in the 787 and 38 fibroblast strains (accelerated dissociation rate) suggested that the latter might be the case.
For this reason, the assays of AR function were repeated for normal
control subjects and selected Reifenstein patients using single
additions of saturating doses of Mb, DHT, or T in both the presence and
absence of the 5-reductase inhibitor, finasteride. As shown in Table 1, the inclusion of finasteride in the incubations had a discernible
effect on the level of induction of the reporter gene in normal
subjects. Although this effect was somewhat variable, average decreases
of 50% (range, 24–67%) were observed for assays performed with T in
the presence of finasteride compared to parallel assays performed in
the absence of finasteride. By contrast, when similar assays were
performed on the mutant ARs expressed in fibroblast strains 787, 38,
and 593, decreases of over 70% were observed in the samples stimulated
with T in the presence of finasteride compared to those in which no
finasteride was included. Importantly, in these latter assays, the
levels of AR function were well below the range established as
representing normal AR function. These observations suggest that
defective AR function can be detected in fibroblast strains from most
patients with the Reifenstein phenotype, although modifications of our
previously described assay were necessary to detect defects in patients
that harbor hormone-binding domain mutations that cause isolated
abnormalities of ligand dissociation from the mutant AR.
The decreases observed in AR function in fibroblasts from normal
controls measured after stimulation with T in the presence of
finasteride are probably caused by inhibition of the formation and
action of DHT in the fibroblasts. Although not tested rigorously in
these experiments, the variability in the magnitude of this change
seems to correlate with the varying levels of 5-reductase measured
in the different strains. Of interest, the decreases observed in the
effects of finasteride on Mb- and DHT-stimulated AR function for
strains 787 and 593 are inconsistent with the lack of effects seen in
the normal strains. This suggests that finasteride may have a more
direct effect on the function of these mutant receptors, one that is
not evident in the assays of normal fibroblast strains.
The results of the assays of the SBMA and hypospadias patients are interesting for different reasons. Using the adenovirus reporter gene assay, the levels of AR function in the SBMA samples are substantially reduced. In patients 711 and 814, AR function is reduced to a level approaching that observed in patients with complete forms of androgen resistance. Although decreases in AR function have been found after transfection of complementary DNAs encoding mutant ARs with such expanded repeats (10, 11, 12), the depressed levels of AR function seen here are much greater that those reported to date and suggest that such transfection assays may underestimate the degree to which AR function is impaired in vivo. Of note, the normal levels of AR measured in these samples by binding and immunoblot assays do not support a role for reductions in the levels of AR alone in the pathogenesis of androgen resistance associated with SBMA (15).
The assays of AR function in patients with isolated hypospadias represent our first attempts to employ this assay to identify fibroblasts with abnormalities of AR function in a population with clinical features suggestive of defects in androgen action, but in whom supporting endocrine data or a suggestive family history are not available. The level of Mb-stimulated AR function appears normal in four strains and abnormal in three strains. Although an AR gene mutation has been identified in one strain that exhibits reduced AR function (strain 931; G566V), the AR gene sequence is normal in the other two. These findings suggest that the observed deficit of AR function present in the remaining two strains (938 and 939) may be caused by an abnormality not localized to the AR gene itself.
In summary, we provide evidence for the utility of an adenovirus-based reporter gene assay in the identification of patients with partial forms of androgen resistance. In addition to identifying abnormalities of AR function in a range of patients with Reifenstein syndrome caused by amino acid substitutions in the DNA- and hormone-binding domains of the AR, this assay is able to detect the abnormalities of AR function present in patients with SBMA. Finally, our data suggest that abnormalities of AR function exist in a small proportion of patients with phenotypic abnormalities suggesting androgen resistance, such as severe hypospadias, and a normal AR gene sequence. Such individuals would be candidates to harbor defects in a gene(s) other than the AR that act to impair normal AR function.
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Footnotes |
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Received September 6, 1996.
Revised February 13, 1997.
Accepted February 20, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase 2 deficiency,
testicular feminization, and related disorders. In: Scriver CR, Beaudet
Al, Sly WS, Valle D, eds. The metabolic and molecular basis of
inherited disease, 7th ed. New York: McGraw-Hill; 2967–2998.
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