Regulation of the androgen receptor by androgen in normal and androgen-resistant genital skin fibroblasts

J. steroid Eiochem.Vol. 18. No. 4. pp. 383-390.1983 Printed in Great Britain. All rights reserved

0022-473l/83/040383-08$03.00/O Copyright 0 1983Pergamon Press Ltd

REGULATION OF THE ANDROGEN RECEPTOR BY ANDROGEN IN NORMAL AND ANDROGEN-RESISTANT GENITAL SKIN FIBROBLASTS MORRIS KAUFMAN*, LEONARDPINSKY*. RICKI HOLLANDER* and JOHN D. BAILEY? *Cell Genetics Laboratory, Lady Davis Institute for Medical Research, Sir Mortimer B. DavisJewish General Hospital, Montreal H3T lE2; the Centre for Human Genetics, Department of Biology, McGill University, Montreal H3A 1Bl and TThe Hospital for Sick Children, Department of Pediatrics, University of Toronto, Toronto MSG 1X8

SUMMARY Normal genital skin fibroblast (GSF) monolayers incubated with serum-free medium containing 3 nM [3H]-5a-dihydrotestosterone (DHT) at 37°C for 20 h have about 35% more specific DHT-binding than replicates incubated in serum-free medium with C3H]-DHT for only 1h to saturate basal specific androgen-receptor activity. If, after 19 h, spent medium is replaced by fresh medium with 3 nM C3H]-DHT for 1 h, specific DHT binding is 85% more than basal. The acquisition of increased binding is temoerature deuendent (37 > 27°C) and cvcloheximide (2 uM) suouressible. The increased bindinn activiiy is considered to represent an gugmentation of andiogen ‘recdptor concentration because it has the same equilibrium dissociation constant (K, _ 0.5 nM), rate constant of dissociation (k- 1 - 6 x lo-” min- I) and ligand specificity as basal androgen-receptor activity, and because basal DHT-binding activity is stable in cells preincubated in androgen-free or serum-free medium alone for up to 72 h before assay. Prolonged incubation with methyltrienolone (R1881), a nonmetabolizable synthetic androgen, causes a greater, more persistent increment of androgen receptor activity than does equimolar DHT. The fibroblasts from two subjects with receptor-positive, partial androgen resistance lose their basal receptor activity during prolonged incubation with DHT, but augment it normally with R1881. This suggests that defective DHT metabolism is somehow involved in the pathogenesis of their androgen resistance.

INTRODUCTION Serially subcultured genital skin fibroblasts have been very useful in identifying constitutional defects of the androgen-receptor system that result in various states of androgen resistance (insensitivity) [l, 23. Three types of defects have been recognized: (i) receptornegative (no or barely measurable receptor activity); (ii) receptor-deficient (easily measurable but less than normal); and (iii) receptor-positive (15-50 fmol DHT$ bound specifically/mg protein). Since the concentration of GSF receptor activity is not a good predictor of clinical phenotype, various qualitative receptor defects have been sought. Two have been found: (i) receptor thermolability as expressed by decreased

$ The following trivial names and abbreviations have been used: dihydrotestosterone (DHT), 17/?-hydroxy5c+androstan-3-one; methyltrienolone (R1881), 17/?-hydroxy- 17a-methylestra-4,9,11-trien-3-one; dexamethasone, 9a-fluoro- 16a-methyl- 1la,17,21- trihydroxy- 1,4-pregnadiene-3,2-dione; androstanediol, 5a-androstane-3a(3/?), l’la-dial; Sa-reductase, NADPH:A4-3-ketosteroid-Sa-oxidoreductase. Correspondence to: Dr Leonard Pinsky. Lady Davis Institute for Medical Research, Sir Mortimer B. DavisJewish General Hospital, 3755 Cote Saint Catherine Road, Montreal, Quebec H3T lE2 Canada.

androgen-receptor activity in intact fibroblast monolayers assayed at 42 versus 37°C [3,4]; and (ii) excessive dissociability of DHT-receptor complexes both within intact fibroblasts [4] and after extraction from the cells [S]. Defects in translocation of the complexes from cytoplasm to nucleus, in the intranuclear distribution of the complexes, or at any of the theoretical post-receptor steps in the sequence that effects androgen responsiveness have not yet been found. In this paper, we delineate further a recently recognized [6] regulatory behavior of the androgen-response system in normal GSF-augmentation of the androgenreceptor activity in response to prolonged incubation with androgen-and describe additional aspects of the misbehavior of this regulatory property in the GSF of two subjects with receptor-positive partial androgen resistance.

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DESCRIPTIONS OF ANDROGEN-RESISTANTSUBJECTS Patient 99900 was born with ambiguous genitalia after a normal pregnancy. He has one healthy sister and no family members are similarly affected. The parents are not consanguineous. At 11 months he was referred to the Hospital for Sick Children, Toronto, for investigation of severe penoscrotal hypospadias.

The penis was bound by chordee to the median raphe of a bitid scrotum, The width of the glans was 1.2 cm. After release of the chordee. the stretched penile length was 3 cm. The testes, I ml in volume, were descended. Retrograde urethrography revealed a normal male urethra and a slightly enlarged prostatic utricle. The karyotype was 46.XY. At 3 years his plasma testosterone and androstenedione levels were < 10 and 29 ngjdl, respectively; three days after a single i.m. injection of hCG (1500 USP units/M’) the testosterone rose normally to 9S ngjdl. while the androstenedione remained unchanged. Patient 14679 is the only child of nonconsanguineous parents. No family members are similarly affected. The pregnancy was uncomplicated except for a urinary tract infection in the seventh month. At 14 days he was referred to the University of Tennessee Health Sciences Center, Memphis, for investigation of ambiguous genitalia. The penile phallus. bound by ventral chordee, measured 3.0 x 1.3cm and was encircled by pigmented, well-rugat~d labioscrotal folds. The urethra opened at the phallic mid-shaft. The testes were descended, the right completely. Cenitography revealed a “sac-like structure” that communicated with the right vas deferens. Laparotomy showed that the “structure” was a dilated prostatic utricle and revealed that the left vas deferens extended to the left testis. The karyotype is 46,XY. ACTH (Cortrosyn: 0.25 mg/4 h, i.v.) induced normal elevations in the plasma levels of cortisol, progesterone, 17-hydroxyprogesterone. dehydroepiandrostcrone (DHEA) and DHEA sulfate. After hCG (3000 USP units. M’,~ day. i.m.. for 4 days) the plasma testosterone. 5a-dihydrotestosterone and ~tndrost~nediolle levels rose normally from 153. 50, and 35 to 480. 108. and 88 ng,‘dl. respectiveI>,.

MATERIALS 4ND METHODS

The fibroblast s&rains were derived from pieces of foreskin, scrotum and labium majus skin donated by normal volunteers or androgen-resistant subjects with informed consent according to protocols approved by the Ethics Committee of the Hospital.

Before initiating the studies reported below. the mutant strains were ascertained to be normal in the following respects by methods previously described in detail: apparent equilibrium dissociation constant of the basal DHT-receptor activity determined by a fiveor six-point Scatchard analysis with varying concentrations of C3H]-DHT [73: rate constant of dissociation of DHT--receptor complexes as measured in intact cells [4] and in partially purified preparations of the complexes [5]: and 5ol-reductase activity using testosterone as the substrate [S].

Genital skin fibroblast monolaycrs (rcprescnting strains that had undergone 10 30 mean population doublings) are grown to confluence in 5cm petri dishes with Eagle’s MEM (Earle’s salts) supplemented by 2 mM glutamine, I mM pyruvatc. 1x non-essential amino acids and equal parts of fetal and newborn calf serum (lo”,, vIv). The activit! is determined. essentially as described 13.91. by subtracting “nonspecific” binding, measured in duplicate with 3 nM [ 1.2,4.5,6.7-“HJ&dihydrotestosterone (I 30 Kmmol; New England Nuclear Corporation) in the presence of excess (0.6 /tM) radioinert DHT. from “total” binding, measured in triplicate. that occurs with 3 nM [“HI-DHT alone, The androgen is dissolved in serum-free (s-f) medium that is additionally buffered to pH 7.4 with I5 mM HEPES (N-7-hydroxyethyl piperaz~ne-.~-~-ethane-sulplionic acid; Sigma Chemical Corp.). The specific activity is saturated within 3@45 min of incubation [IO, 131.and it is not affected by preincubation of the cells in androgen-free medium for up to 72 h before assay [9, i I. I?]. Not more than 4OY, of the initial 3 nM C3H]-DHT is metabolized during a I h incubation [3. I I. 131. and the equilibrium dissociation constant of the activity is about 0.5 nM [9. 12, 13]. The individual results on replicates in the “total” and “‘nonspecific” groups seldom differ from their respective means by more than IO”,,, and nonspecific binding is seldom more than lo”,, of total binding. The one-point assay yields a measure of specific binding activity within lo”,, of the maximum binding capacity (B,,,,) that is extrapolated from the least squares regression-fitted line joining the points of a Scatchard plot.

At the outset of this study we wished to assess the effect on specific androgen-binding activity of prolonged preincubation with a known initial concentration of androgen. For this purpose we used serumfree medium as the vehicle. and all the experiments in which exposure to androgen occurred for 20 h or less were conducted this way. Thus. at various times before assay, confluent monoiayers are washed free of growth medium. divided into three groups. and fed: Medium i (s-f medium with 15 mM HEPES): Medium II (Medium I plus 3 nM [“HI-androgen); or Medium III (Medium II plus O.h/tM radioinert androgen). To initiate the assay for basal androgenbinding activity, five monolayers that had been fed Medium 1 are washed. divided into two sets and fed either Medium II (“total” binding in triplicate) or Medium 111 (“nonspecific” binding in duplicate) for I h. Likewise monolayers that had been fed Medium II or If1 are washed and refed their respective media for 1 h to measure total and nonspecific binding after androgen preincubation for various times. In other experiments the effect of prolonged incubati~?n in s-f

385

Androgen regulation of androgen receptor medium with androgen on androgen-binding activity was assessed without replacement of the spent medium by a fresh supply of Medium II or III. In order to avoid the possibility that very prolonged preincubation in s-f medium might interfere with the ability of the cells to respond to simultaneous preincubation with androgen, most of the experiments in which exposure to androgen exceeded 24 h were performed with growth medium (10% serum) as the vehicle. In fact, the choice of vehicle had no effect on the results. To determine the effect of a protein synthesis inhibitor on the response to prolonged incubation with androgen, replicate cultures were preincubated and assayed with 2pM cycloheximide (Sigma Chemical Company), a concentration known to inhibit synthesis of the receptor activity. The effect of temperature on the response was assessed in rephcate cultures preincubated and assayed at 27 and 37°C. Prolonged incubation with DHT or R1881

To determine the equilibrium dissociation constant and the relative affinity for competitor ligands of the basal and augmented androgen-binding activities, replicate monolayers were preincubated for 1 or 19 h with 3 nM radioinert DHT or R1881. A fresh supply of serum-free medium with 3 nM androgen was provided at the 19th h in order to ensure saturation of the augmented DHT-binding sites. For uniformity the same step was included after 19 h pre-incubation with R1881, even though R1881 is not catabolized in this interval (see Results below). Finally a 2 h exchange assay was performed as described [6] with varying concentrations of C3H]-DHT or [ 17cc-methyl-3H]Rl881 (87 Ci/mmol) plus or minus excess radioinert ligand, or with a fixed concentration of either plus 20-fold concentration

of various competitors.

The same chromatographi~ system Cl41 used to separate testosterone from its See-reduced metabolites in the Scr-reductase assay also served to assess the rate of C3H]-DHT or C3H]-Rl881 consumption from the

medium of monolayers. [‘“Cl-DHT was used routinely to monitor recovery and, in several experiments, re~ystallization to constant isotopic ratio revealed that the DHT spot was >900,:, pure. Protein was measured by the method of Lowry et ~I.[151 and radioactivity by liquid scintillation in an Intertechnique (SL-30) spectrometer at an efficiency of 40%. Student’s paired-t-test was used to compare the differences between means. RESULTS The left panel of Fig. 1 contains the results in aggregate of 46 experiments done on 18 control GSF

strains, of which 14 originated from foreskin and four from labium majus skin. The 1 h result [27 + 1.4 (SEM) fmol/mg protein ; designated 10051/l represents the plateau achieved by saturation of basal specific DHT-receptor activity within 3@45 min of initiating an incubation with 2-3 nM C3H]-DHT at 37°C [ 133. In replicate monolayers incubated for 5 or 20 h without a medium change, specific DHT-binding is augmented by 35 2 5 (P -C 0.001) and 34 & 10% (P < O.OOl),respectively. The latter set of results reflects the fact that there is considerable interexperimental variation from the prototypic outcome in which there is an intermediate increment of binding activity in the 5 h monolayers compared to their 20 h replicates. Monolayers offered 3 nM C3H]-DHT for 1 h at the 19th h (either in fresh serum-free medium or in a small aliquot of alcohol) have 85 & 8% more than basal specific DHT-binding (see “19 I I”, Fig. l), while those given serum-free medium alone, or alcohol alone, do not differ from their 20 h replicates. This suggests that metabolic consumption of DHT leads to an underestimate of the extent to which specific DHT-binding is augmented. To characterize the process of augmentation two types of experiments were performed. Figure 2 reveals that incubation at 27 versus 37°C minimizes augmentation but not basal DHT binding: in six assays (on

Fig. 1. The effect of prolonged incubation with 3nM [‘HI-DHT expressed as o/oof basal activity after incubation for 1h at 37°C. containing 3 nM C3H]-DHT was provided for 1h at the 19th h. The column equals number of observations. The bars

on specific DHT-binding activity 19 + 1 means that fresh medium number in parentheses above each equal + SEM.

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2. The effect of temperature on the response to prolonged incubation with 3 nM [3H]-DHT. 4 f I, 11 + 1, and 19 + 1 indicate that fresh medium containing 3 nM C3H]-DHT was provided for 1h at the 4th, 1Ith and 19th h. respectively.

five strains). mean basal DHT-binding at 37 and 27°C were 15.5 and 22.3 fmol/mg protein, respectively (P > 0.05). We also found that 2 PM cycloheximide elirnill~~ted ~~u~mentation but did not affect basal DHT-binding (results not shown). To characterize the quality of the augmcntcd DHTbinding activity, equilibrium binding kinetics, dissodation kinetics and ligand specificity were assessed. The basal and augmented binding activities have identical K, values ( -0.3 nM). and the li_ 1 of the augmented activity in a strain that increased from 30 to 68fmol,/mg protein was 7 x 10e3 min-’ (basal: The order of hgand 6 5 0.3 x lO,“j min’ )[4.5]. specificity, as determined by an exchange assay in studies with 20-fold concentrations of competitor, is the same for the basal and augmented activities: DHT > 17~~-estr~~diol = androstan~diol > progestrronc = dcx~lrnetl~~isone. In contrast to controls, the GSF of mutant subjects 999~ and 14679 do not augment their specific basal DHT-binding activities (22 fr 2.2 and 32 k 2.5 fmoi/ mg protein, respectively) even when they are offered fresh medium containing 3 nM [3H]-DHT at the 19th h (Fig. I). Indeed. they lose DHT-binding activity rapidly so that by 20 h their remaining activities

are only 23 i: 7 (P < 0.001) and 12 i: 4u/;, (P < 0.005) of basal, respectively (Fig. I). The ability of prolonged incubation with a nonmetabolizable androgen. R 1881. to provoke augmentation of androgen-binding activity was characterized in order to compare it with DHT. Figure 3 shows that [3H]-R1881 can induce a doubling of [3H]-R1881binding activity without a change in KD values. The basal and augmented R 1881-binding activities also have the same ligand specificity (Table 1) and li _ , values (11 + 0.9 x toe3 mm’ at UC: ft = 15). Figures 4 and 5 demonstr~~te that prolonged incubation with R18Xl is able to augment androgen”binding activity, not only to a greater extent and in more persistent fashion than DHT in control strains, but also in the strains from the two androgen-resistant subjects (99900 and 14679).

Table I. Per cent inhibition of basal and augmented specific R 188l-binding activities* by a X-fold concentration of various inbibitots (compared to RI88 1 = 1007~~) inhibitor DHT Androstaned~ol Progesterone 17/J-estradiol

Basal

Augmented

88 55 27 6

86 58 20 0

* Basal and augmented activities measured with 3nM C3H]-R1881 by the exchange assay were 36 and 77 fmolimg protein. respectively.

387

Androgen regulation of androgen receptor

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Fig. 4. The effect of prolonged incubation with 3 nM [3H]-R1881 on specific Rl881-binding in control strains expressed as y0 of basal activity after incubation for 1 h at 37°C. The number in parentheses above each column equals number of observations. The bars equal + SEM. The results were equal in s-f or growth medium.

90

0

HOURS

Fig. 6. The effect of prolonged incubation with [jH]-DHT or [3H]-R1881 on specific androgen binding in replicate monolayers of a labium majus skin strain (top) and a foreskin strain (bottom). The number above each column is the % remaining of the indicated initial concentration of androgen in the medium. The value of 114 (upper right panel) is the computed estimate of C3H]-R I88 1 recovery in the 72 h incubation medium, using [‘%Z]-DHT as the internal marker. 4 + 1 and 16 + 1 mean that fresh medium containing the initial concentration of ligand was provided for 1 h at the 4th and 16th h, respectively.

To compare their augmentation ability directly, concurrent studies with DHT and R188 1 were performed. The top panels of Fig. 6 show that in the first 24 h each androgen provoked a similar extent and time-course of augmented androgen-binding activity in a labium majus skin strain, despite the fact that only 12% of the initial 2.7nM DHT in the medium remained at the 24th h. Thereafter, the GSF incubated with DHT lost binding activity, while those incubated with R1881 increased still more. It is noteworthy, moreover, that in the 48 h period (2472 h), when residual DHT concentration declined from 12 to 5%, only 50% of the DHT-binding activity was lost. The bottom panels of Fig. 6 depict the ineffectiveness of DHT, relative to R1881, in augmenting androgenreceptor activity of a control foreskin strain (DFF). It is important to notice, moreover, that continuous incubation with DHT did not augment binding ac-

tivity in this experiment despite the fact that, starting with 3.2 nM, the concentrations of DHT remaining in the medium after 1, 5, and 17 h were very similar to those remaining after 1, 5 and 24 h in the experiment (upper left panel, Fig. 6) wherein binding activity nearly tripled. A similar result, indicating that augmentation is not

99900

20282

dl_l 1

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Fig. 5. The effect of prolonged representative

24

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incubation with 3 nM [3H]-R1881 on specific Rl881 control strain (20282) and the two mutant strains.

binding

in a

librium

binding

specificity observations

Fig. 7. The effect of prolonged incubation with C3H]-DHT on specific DHT binding in a control foreskin strain (7777) and a mutant strain. The number above each column is the “” remaining of the indicated initial concentration of [3H]-DHT in the medium.

a simple function of DHT catabolism as measured in the medium, is presented in the left panel of Fig. 7. Notice that at the 20th h the control foreskin strain (7777) augmented its binding activity by 33”,, despite the fact that. starting with 1.5 nM DHT, it consumed DHT from its medium at a rate that left it with only 0.24 nM DHT. Of equal relevance is the observation that 52 h after this strain had attained its peak binding activity (29 fmol/mg protein at 20 h), it still retained 38”,, (11 fmoljmg protein) despite the fact that the residual concentration of DHT in its medium during this period declined from 0.24 to 0.06 nM. In contrast, the mutant strain (99900. right panel, Fig. 7) lost 80”” of its basal binding activity in only 19 h, during which time the residual DHT concentration in its medium declined from 1.5 to 0.18 nM.

DISCUSSION

In a preliminary paper [6] we reported that preincubation of GSF monolayers with 3 nM C3H]-DHT in serum-free medium for 19 h before initiating a standard 1 h assay of specific DHT-binding yielded twice as much activity compared to replicates maintained in serum-free medium alone. Since basal specific DHTbinding activity is stable in cells preincubated in androgen-free medium for at least 72 h before assay C9.11. 121, DHT was not thought to act simply by preventing the loss of binding activity from cells maintained for 19 h in serum-free medium alone. Furthermore. the augmented DHT-binding activity had the same k- , as basal DHT-binding activity, and an equal extent of augmentation could be measured by a 2 h exchange assay with 3 nM C3H]-DHT following preincubation in serum-free medium containing 10 nM radioinert DHT. For the latter two reasons, we interpreted the augmented binding activity to represent an increased concentration of specific DHTreceptor activity. In this paper we report that the augmented DHT-binding activity also has the same equi-

kinetics basal

support

tKL,)

and

DHT-binding the

previous

order

of

activity. interpretation.

ligand These In

shown that preincubation with R I88 1 can augment specific R 188 I -binding activity. and that the basal and augmented activities have the same K,. km, and order of ligand specificity. It thus appears that prolonged incubation with DHT. a natural metabolizable androgen. or R 188I. a synthetic nonmetabohzable androgen. can provoke GSF to increase their concentration of androgen receptor molecules. The mechanism, presumably common, by which these two androgens increase specific androgen-receptor activity, remains to be defined. However, some clues can be gleaned from the data presented above. First, R1881 augments androgen-receptor activity in control strains to a greater degree, and in a more persistent manner. than does equimolar DHT. This implies that metabolic disposition of DHT limits augmentation. By metabolic disposition we refer to consumption of DHT (and generation of its derivatives) as measured in the medium on the one hand. and to the intracellular fdte of DHT (and its derivatives) on the other. A 20 h incubation starting with 3 nM [“HIDHT can result in sufficient metabolic consumption of the ligand so that its resultant concentration is insufficient to saturate the augmented receptor activity. In this circumstance, provision of additional ligand at the 19th h would be expected to increase the measurable level of augmentation. Indeed, we observed the expected to an equal extent whether C3H]DHT was added to the spent medium, or whether the latter was replaced by fresh serum-free medium containing [3H]-DHT. By contrast. one cannot correlate the various degrees of augmentation seen in different experiments on control strains with the degrees to which DHT is consumed from their respective media. nor with any other property of the strains. Furthermore. control strains lose their augmented DHTbinding activity much more slowly than the mutant strains lose their basal DHT-binding activity. and this disparity also cannot be correlated with any aspect of DHT disposition as measured in the medium. We have previously reported defective augmentation of DHT-receptor activity in two other subjects with receptor-positive androgen resistance [6]. both of whom form DHT-receptor complexes that do not “activate” normally as judged from the fact that they dissociate more rapidly than normal within GSF 141. and in an abnormal (biphasic) fashion when they are extracted from the cells [S]. This suggests that an unknown primary receptor defect may be responsible for whatever abnormal intracellular metabolism of DHT is yet to be discovered in the GSF of the two mutant subjects discussed here. In any event. we interpret augmentation to be adaptive. As such. defective augmentation may be pathogenetic since physiologic fluctuation in the level of free DHT during pre- and post-natal life may penalize mutant cells that cannot addition,

HOURS AT 37oc

as the

we have now

Androgen regulation of androgen receptor

augment their DHT-receptor activities during times of relative DHT abundance. A second aspect of the phenomenon of androgendependent augmentation of androgen-receptor activity that needs elucidation is the extent to which decreased degradation, increased neosynthesis, and recruitment from a receptor intermediate, alone or in combination, is responsible for the increased concentration of receptor activity. The t+ of the uniiganded androgen receptor in GSF grown in regular medium is about 12 h [16], and we have found equal degrees of augmentation whether prolonged incubation with androgen occurs in serum-free or growth medium. Thus, if occupancy of androgen-binding sites protected receptor molecules against degradation, and their de noro synthesis continued at the steady-state rate, one could expect preincubation with androgen to increase receptor activity by no more than 50”;; in the first 12 h. But. in many experiments, DHT induces such an increase in only 5 h, and RlgXl does so by lOO?din the same period. Therefore, it is unlikely that decreased degradation (stabilization) is the sole origin of the augmented androgen-receptor activity. The ability of cycloheximide (2 /lM) to nullify androgeninduced augmentation of androgen-receptor activity is compatible with the notion that increased neosynthesis of the androgen receptor contributes to the increment. However, the effect of cycloheximide might also be explained by interruption of basal receptor synthesis and inhibition of basal protein degradation [17] without elimination of androgen-dependent stabilization of the receptor. Recruitment of typical receptors from a pool of proreceptor molecules, to explain augmentation at a rate that is not attributable to receptor stabilization alone, needs direct support. It is noteworthy, however that the comparatively lowaffinity (Type 11) estrogen-binding “sites” in rat uterus, which are 4-fold more numerous, have been postulated to serve as a reservoir for classical (Type I) estrogen-receptor activity in that organ [18]. Finally, it is necessary to compare our data with relevant ones reported by others. Griffin[3] found that DHT-receptor activity in normal GSF monolayers, and in those from subjects with four different types of receptor-deficient androgen resistance, increased by about 50% on incubation with C3H]-DHT for 16 h at 26°C rather than for 1 h at 37°C despite the fact that only two of the four receptor-deficient types had a form of androgen receptor that was thermolabile at 42°C in the 1 h assay. In the light of our results, it would appear that none of his patient fibroblast strains was augmentation-defective. Mowszowicz et u&19] concluded that the androgen receptor in human genital skin is not regulated by androgens on the grounds that receptor activity is equal in men and women, and does not fluctuate with age in males. It must be recalled that our measurements were performed on serially subcultured. dermis-derived genital skin fibroblasts, while theirs were performed on preparations of cytosol derived from the heterogeneous

389

cell types present in full-thickness biopsies of genital skin from which only the subcutaneous fat was removed. Acknowledgements-We are grateful for faithful assistance to Hoda Karam. Carmen Gil Estaban, Leslie Long-Simpson and Datevik Sumbulian. This work was supported by the Medical Research Council of Canada Group Grant in Medical Genetics.

REFERENCES 1. Griffin J. E. and Wilson J. D.: The syndromes of androgen resistance. N. Engl. J. Med. 302 (1980)

198-209. 2. Brown T. R. and Migeon C. J.: Cultured human skin

fibroblasts: a model for the study of androgen action. Mol. Cell. Biochem. 36 (1981) 3-22. 3. Griffin J. E.: Testicular feminjzation associated with a thermolabile androgen receptor in cultured human fibroblasts. J. clin. Invesr. 64 (1979) 1624.-t631. 4. Pinsky L.. Kaufman M. and Summitt R. L.: Congenital androgen insensitivity due to a qualitatively abnormal androgen receptor. Am. J. Med. Genet. 10 (1981) 91-99. 5. Kaufman M. and Pinsky L.: Defective activation of androgen-receptor complexes: a marker of androgen insensitivity. Molrc. cell. Endocr. 25 (1982) 151-162. 6. Kaufman M., Pinsky L. and Feder-Hollander R.: Defective upregulation of the androgen receptor in human androgen insensitivity. Nutztre 293 (1981) 735-737. 7. Kaufman M., Straisfeld C. and Pinsky L.: Male pseudohermaphroditism presumably due to target organ unresponsiveness to androgens. J. clin. Inoust. 58 (1976) 34.5-350. 8. Leshin M., Griffin J. E. and Wilson J. D.: Hereditary male pseudohermaphroditism associated with an unstable form of Sr-reductase. J. c/in. Incest. 62 (1978) 685-691. 9. Keenan B. S.. Meyer W. J. III, Hadjian A. J. and Migeon C. J.: Androgen receptor in human skin fibroblasts: characterization of a specific I ‘?/&hydroxy-5aandrostan-3-one-protein complex in cell sonicates and nuclei. Steroids 25 (1975) 535-552. 10. Collier M. E.. Griffin J. E. and Wilson J. D.: Intranuclear binding of [3H]-dihydrotestosterone by cultured human fibroblasts. Endocrinolou . 1 103 119781 1499-1505. 11. Kaufman M., Straisfeld C. and Pinsky L.: Expression of androgen-responsive properties in human skin fibroblasts of genital and nongenital origin. Somnr. C&i Genet. 3 (I 977) 1J-25.

12. Kaufman M.. Pinsky L., Baird P. A. and McGillivray B. C.: Complete androgen insensitivity with a normal amount of Sa-dihydrotestosterone-binding activity in labium majus skin fibroblasts. Am. d. Med. Genet. 4 (1979) 401-411. 13. Brown T. R., Rothwell S. W. and Migeon C. J.: Comparison of methyltrienolone and dihydrotestosterone binding and metabolism in human genital skin fibroblasts.>. steroid ~~oc~em. 14 (1981) iO13-1022. 14. Pinsky L.. Kaufman M., Straisfefd C., Zilahi B. and Hail C. St-G.: .5a-Reductase activity of genital and nongenital skin fibroblasts from patients with 5a-reductase deficiency, androgen insensitivity, or unknown forms of male pseudohermaphroditism. Am. J. Med. Genef. 1 (1978) 407-416.

15. Lowry 0. H., Rosebrough N. J.. Farr A. L. and Randall R. H.: Protein determination with the Folin phenol reagent. J. hiol. CIzem 193 (1951) 265.-275.

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