Male Pseudohermaphroditism Presumably Due to Target

Organ Unresponsiveness to Androgens

DEFICIENT 5a-DIHYDROTESTOSTERONEBINDING

IN CULTUREDSKIN FIBROBLASTS

MoRRIS KAUFMAN,CIPIA STRAISFELD, and LEONARDPINSKY

From the Cell Genetics Laboratory, Lady Davis Institute for Medical Research,Jewish General Hospital, Montreal, Quebec H3T IE2, and the Department of

Pediatrics, Faculty of Medicine, McGill University,Montreal, Quebec H3C3G1

A B S T R A C T Maximum specific 5a-dihydrotestoster-one (DHT) binding activity (B.ax) has been measuredin intact confluent monolayers representing fibroblaststrains derived from nongenital and genital (labiummajus) skin of normal individuals and of 11 patientsfulfilling the clinicogenetic criteria of complete testicularfeminization (TF). Nine labium majus strains fromadult females had a mean Bmas value three times greaterthan that of seven nongenital strains from adult females(33 vs. 11 fmol/mg cell protein). The Bmax results for13 adult nongenital strains varied from 5.6 to 23.3 fmol/mg protein; the values for males and females had verysimilar means and ranges. The variation could not becorrelated with the chronologic age of adult skin ex-plant donors or with the in vitro age (mean populationdoubling level) of the cultures assayed. The Bmaxactivities of three nongenital strains from normal in-fants (two male, one female) did not exceed 5 fmol/mgprotein. Seven of eight nongenital TF strains had Bmaxvalues below 2 fmol/mg protein; the value for theeighth coincided with the lower limit of normal adults.The lower limit of DHT binding in normal labiummajus strains was 15 fmol/mg protein. Three of fivelabial strains from patients with TF had Bm.x valuesclose to zero; the other two fell between 10 and 15fmol/mg protein. It is apparent that labial skin fibro-blast strains from clinically homogeneous patients with

Abstracts of portions of this work have appeared in Am.J. Hum. Genet. 27: 53A (1975) and Clin. Res. 23: 624A(1975).

Received for publication 9 December 1975 and in revisedform 26 March 1976.

TF have highly variable degrees of DHT bindingdeficiency, and that they permit a more reliable diag-nosis of severe and intermediate degrees of DHT bind-ing deficiency than do strains of nongenital skin fibro-blasts.

INTRODUCTIONTarget cell unresponsiveness to androgen has beenidentified as the cellular fault in three types of humanmale sexual maldevelopment caused by mendelian genemutation. The prototype disorder is X-linked (1) com-plete testicular feminization (TF) ' in which thereis neither normal male differentiation of the Wolffianducts, urogenital sinus or genital tubercle, folds andswellings prenatally, nor genital or somatic virilizationat puberty. These patients are resistant to testosteroneand its immediate derivative, 5a-dihydrotestosterone(DHT, 5a-androstan-17P-ol-3-one) (2). Familial in-complete male pseudohermaphroditism (FIMP), type 1is like TF in pattern of genetic transmission, the uni-versality of target organs affected, and presumably inconcordant resistance to testosterone and DHT, but itsdefect is incomplete in severity and variable in expres-sion (3). All patients with TF and most with FIMP,type 1 have female breast development because of ele-vated plasma estradiol levels secondary to defective

'Abbreviations used in this paper: Bma., maximum bind-ing capacity; DHT, 5a-androstan-17fi-ol-3-one; FIMP,familial incomplete male pseudohermaphroditism; Kd, ap-parent dissociation constant; MP, male pseudohermaphro-ditism; 5a-reductase, A'-3-ketosteroid-5a-oxidoreductase;TF, testicular feminization.

The Journal of Clinical Investigation Volume 58 August 1976 345550345

TABLE I

Clinical Features of Patients Referred with the Diagnosis of Testicular Feminization

Spontaneouspubertal Family history

feminizationUnambiguous with sparse/ Delayed

female absent sexual Fully affected menarcheexternal hair, without and (or)genitalia hypokalemia/ Maternal decreased

Patients Age Cell strains at birth hypertension Siblings relatives sexual hair

yr

1 25 JJX* + + + ? ?2 21 JAX + + + ? ?3 16 JRX + + + ? ?4 3/12 TMN, TML + ? + (X2) ?5 22 CVL + + + - + (X3)6 17 TRA, TRL + + (adopted)7 17 SFL + + + (X8) +8 18 RDL + + + (X3) ? +9 29 TOD + + - -

10 10 HCD + ? - - +11 19 GHA + + + ? ?

* The final letter of the cell strain codes denotes the anatomic source of the skin explant from which the strain was developed:A abdominal (suprapubic); D, deltoid area of the arm; L, labium majus; N, nongenital (source unknown); X, axilla.

feedback regulation of luteinizing hormone secretion atthe pituitary-hypothalamic level (3). FIMP, type 2resembles the most severe expression of the type 1 dis-order from the external genital viewpoint, but differsfrom the other two in having an autosomal recessiveetiology, normal Wolffian duct differentiation, no gyne-comastia, and prominent somatic and genital virilizationat puberty (4, 5). The phenotype of this disorder is ex-plainable by reduced conversion of testosterone to DHT,due to deficient A'-3-ketosteroid-5a-oxidoreductase (5a-reductase) activity, in DHT-dependent targets such asthe urogeni,tal sinus and external genital primordiabefore birth, and the prostate and facial hair follicles atpuberty (5).

Fibroblasts cultured from normal human skin metabo-lise testosterone to DHT as well as other 5a-reducedproducts (6, 7) and they synthesize a specific receptorprotein that binds DHT in the cy.toplasm and trans-locates it into the nucleus (8). Skin fibroblasts culturedfrom genital skin (prepuce, labium majus, and scrotum)perform both functions to a greater extent than dothose from various nongenital skin sites (7-10). Thepreservation of such differentiative properties in seriallysubcultured skin fibroblasts predicts that these cells willexpress the mutations responsible for the three knowntypes of androgen-unresponsive male sexual maldevelop-ment. The prediction has been fulfilled. Thus, preputialskin fibroblasts of patients with type 2 FIMP have de-ficient 5a-reductase activity (7), and skin fibroblasts(both genital and nongenital) from patients with TF

lack or have very low levels of the specific receptorprotein (s) responsible for binding DHT (10, 11).

The purpose of this study was to measure DHTbinding activity in genital and nongenital skin fibro-blast cell strains derived from a group of 11 patientsreferred with the diagnosis of TF. It is important tonote that a majority of the cell strains studied representpatients whose testes were removed either before theadvent of accurate radioreceptor or radioimmunoassaysfor the sex hormones and gonadotropins, or, before ex-perience with these assays for accura.te differentialdiagnosis of male pseudohermaphroditism (MP) waswidely disseminated. Furthermore, none of the cellstrains belongs to a patient whose anabolic unrespon-siveness to androgen has been assessed. Nevertheless, allof the patients whose strains were chosen for studywere considered extremely likely to have complete TF,according to exclusive clinicogenetic criteria definedbelow. In addition, five of the patients (or an affectedrelative) had normal adult male levels of plasma tes-tosterone.

METHODSOrigin of cell strains

Testicular feminization. Essential clinical data on the11 patients are summarized in Table I. Additional featurescommon to the group were normal testis differentiation,absence of female internal genitalia, a blind (usually short)vagina, and absence of prepubertal adrenocortical failure.Spontaneous pubertal feminization (that is essentially nor-mal except for primary amenorrhea and faulty develop-

346 M. Kaufman, C. Straisfeld, and L. Pinsky

TOTAL

0-~~~~~

0 NONSPECIFI

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/ __o_°

x

iU,

az

0a

:IC

1 2 3 4 5 6 7

FREE[3H]DHT (nM)

5 10 15 20 25 30 35 40

SOUND[3H]DHT(fmol per mg cell protein)

FIGURE 1 (A) A representative curve of [3H] DHT binding in NEL, a normal adult female

labium majus skin fibroblast strain. (B) Specific binding is plotted according to Scatchard

(27). The intercept at the abscissa defines Bmas; the reciprocal of the slope defines the

apparent Kd.

ment of sexual hair, and that is not accompanied by hypoka-kalemic hypertension) in an XY individual born with un-

ambiguously female external genitalia and absent or vestigialWolffian duct derivatives, despite normal testis differentia-tion, was sufficient to discount all known genetic disordersof male sexual maldevelopment other than classical com-

plete TF. This discriminator was applicable to 8 of the 11patients; 2 of the other 3 (patients 4 and 10) were pre-

pubertal at the time of referral but had fully or partially(12) affected adult maternal relatives. The remainingpatient (patient 6, Table I) had a nonmosaic XYY karyo-type in multiple tissues. It is likely that this was causallyunrelated to her TF phenotype because this aneuploidy isrelatively common (1.5 per 1,000) among newborn maleswithout sexual anomalies (13), and her pituitary-hypo-thalamic resistance to androgen was documented (14). Thefull process of excluding other causes of MP with unam-

biguously female external genitalia at birth follows; normaltestis differentiation excluded covert sex chromosome aneu-

ploidy and pure XY gonadal dysgenesis (15). Congenitallipoid adrenal hyperplasia (16) and A'-isomerase-3#-hy-droxysteroid dehydrogenase deficiency (17) were discountedbecause, although each can result in males with unambigu-ously female external genitalia at birth, almost all suchpatients have life-threatening adrenocortical failure beforepuberty. 17, 20-Cholesterol desmolase deficiency (18) hasbeen described in only one family, and the three affectedmales had ambiguous external genitalia. The lack ofvirilization or hypokalemic hypertension, respectively, amongour postpubertal patients indicated that none had 17-keto-reductase (19) or 17a-hydroxylase (20-22) deficiency. 5a-reductase deficiency was excluded because of normal Wolf-fian duct differentiation, external genital ambiguity at birth,and significant genital and somatic virilization at puberty.

Conttrols. Most of the control strains were developed inour laboratory from small skin samples obtained with in-formed consent. These explants were obtained from volun-teers by full-thickness punch biopsy (4 mm) of skin in thedeltoid or upper medial regions of the arm, or from skinincisions made at the time of surgery. Four strains were

obtained from the stock of control strains in "The HumanGenetic Mutant Cell Respository" maintained at the Insti-

tute for Medical Research, Camden, N. J. (Dr. A. Greene).The age of nongenital skin donors varied from infancy to60 yr. The labial skin donors were all postpubertal. Noneof the controls was known to have a disorder of sex steroidhormone metabolism or responsiveness.

Cell cultureThe materials and methods used for developing cell strainsfrom small samples of skin and maintaining them in serialsubculture have been described previously (23, 24). Our cul-tures were screened for mycoplasma by a sensitive test(25) capable of revealing such contamination when stan-dard microbiological culture methods fail to do so. Occa-sional cultures were found to be contaminated. This didnot affect the results obtained on control or patient cellstrains because uncontaminated sister strains yielded thesame results as their contaminated partners, and purpose-ful contamination induced no change from results obtainedon uncontaminated replicates. To avoid the possible influenceof population density of a culture on its specific DHT bind-ing activity, all monolayers were grown to confluence be-fore their DHT binding was determined. The cell strainsused in this study had population doubling times of 24-36 h.The population doubling time of a strain is one measure ofits in vitro age (26). There was no obvious relationshipbetween the population doubling time or the calculated invitro age of a strain and its DHT binding activity, an

observation made by others (8).

DHTbinding assay

The principle of the assay is to use a set of replicateconfluent monolayers for measuring "total" [3H] DHT bind-ing in the absence of excess (200 X) cold DHT, and "non-specific" [3H]DHT binding in the presence of excess coldDHT. The latter is low-affinity, high-capacity binding at-tributable to various macromolecules in the cell. The differ-ence between "total" and "nonspecific" ['H] DHT repre-sents "specific" DHT binding at that concentration. Themaximum binding capacity (Bmax) of the specific DHTbinding activity in a strain and the apparent affinity withwhich it binds DHT (Kd, dissociation constant) were deter-

Deficient Skin Fibroblast DHT Binding in Male Pseudohermaphroditism

A

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LABIALFIGURE 2 The distribution of mean specific dihydrotes-tosterone binding maxima in fibroblast strains derived fromnongenital and labial skin of controls and males withcomplete TF. Notice that the results for nongenital andlabial skin strains are plotted on axes which differ by afactor of 2. TRL and TRA, TML and TMNare pairs oflabial and nongenital strains, respectively, derived fromsingle patients. All of the patient strains and many of thecontrol strains were assayed three or more times. Eachassay was performed at least in quadruplicate. Replicatesseldom deviated from the mean by more than 10 percent.The use of probe sonication to effect total cell disruptionin place of gentle cell disruption by Dounce homogenizationhas enabled us to detect small amounts of DHT binding(30) in some control nongenital strains that had previouslyappeared to lack it (10). Nevertheless, some TF strainshad so little specific DHTbinding that negative mean valuesresulted when nonspecific was subtracted from total binding.

mined by exposing a series of replicate monolayers to vary-ing concentrations of ['H]DHT, each in the presence andabsence of a 200-fold excess of cold DHT, and subjectingthe data to Scatchard analysis (27) (Fig. 1). Specific DHTbinding activity is high-affinity (Kd -1 nM), low-capacity(5-70 fmol/mg cell protein) binding attributable to one ormore specific cytoplasmic proteins synthesized by the cells(8).

The assay procedure used was that described by Keenanet al. (8) with four modifications: (a) the serum-freemedium used for incubating the monolayers with DHT wasadditionally buffered (to pH 7.4) with 15 mMHepes; (b)bovine gammaglobulin was excluded from the solution usedto wash the monolayers before scraping them from thedishes with rubber policemen; this allowed us to determinecellular protein content accurately and to express DHTbind-ing activity per milligram cell protein; (c) the cells wereruptured by direct exposure for 10 s to the 1-cm probe of asonicator (Measuring and Scientific Equipment Co., Lon-don, England) set to an amplitude of 2 tm; and (d) dex-tran-coated charcoal adsorption of "unbound" (free) DHTrather than molecular sieving was used to isolate it from

"bound" DHT. Pellets of dextran-coated charcoal wereprepared by centrifuging (2,000 g, 20 min, 4C) 1 ml of asuspension of 0.5% charcoal and 0.05% dextran T-70(Pharmacia Fine Chemicals, Inc., Piscataway, N. J.) in0.02 M Tris-HCI (pH 7.5, 4°C), 0.4 M KCI, 1.5 mMEDTA, and 2 mMmercaptoethanol. The cell extract super-nates (0.5 ml) were added to the pellets and the mixturewas resuspended by vigorous vortexing for 3 s. After stor-age at 4°C for 10 min, the suspensions were centrifugedas above, and portions of the supernate, containing bound['H] DHT (80 Ci/mmol), were removed for protein de-termination (28) and scintillation counting (29).

RESULTSThe mean specific dihydrotestosterone Bmax for the in-dividual skin fibroblast cell strains of various classes areshown in Fig. 2.

Controls. The values for 16 control nongenital strains(column 1) varied ninefold and had a mean of 11.2fmol/mg cell protein. This large range of normal varia-tion was not obviously related to either the precise ana-tomical origin of the nongenital skin sample from whicha strain was developed, or the in vitro age (mean popu-lation doubling level) of a strain at the time of itsassay. Nor could the variation be related to the sex or

simply correlated with the chronologic age of a skindonor. Thus, while it is notable that the three strainswith the lowest values were derived from infants ofboth sexes at approximately 1 yr of age, the fourthand fifth lowest values represent strains derived from a

60-yr-old man and a 51-yr-old woman, respectively.Seven adult female strains ranged from 6 to 21 (x =

11.7) fmol/mg protein, while six adult male strainsranged from 6 to 23 (x = 14.2).

The Bmax values for nine adult labial strains (column3, Fig 2) varied from 17 to 53 and had a mean of 33.2fmol/mg cell protein. As above, neither donor age norin vitro age could account for the variation observedamong the strains. The difference between the labial andnonlabial strains of adult females had a P < 0.001 (31).

Testicular feminization. Data on nongenital strainsfrom eight patients referred with the diagnosis of TFare shown in column 2 of Fig. 2. Of the seven strainswhose Bmax values were below normal, three (JJX,JAX, JRX) were from adult siblings with typical TF.The nongenital TF strain (HCD) whose Bmax valuewas in the normal range was derived from deltoidskin of a 10-yr-old girl whose maternal grandmothernever developed axillary hair.

Results of labial strains from five patients referredwith the diagnosis of TF are given in column 4 ofFig. 2. Three of the strains had values clearly belowthe range of controls and two were intermediate. It isnoteworthy that two (TRL and TML) of the threestrains well below the controls could be paired withnongenital strains (TRA and TMN) developed simul-taneously from the same patients. In both cases the

348 M. Kaufman, C. Straisfeld, and L. Pinsky

* CONTROL* TESTICULAR FEMINIZATION

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* S

0@

* * HCD *EDL

I

JJX.' -MN TML;f CVLJAXE-JRX

values for the nongenital strains fell below the lowerlimit of the controls. Unfortunately, we do not havenongenital strains from the two patients whose labialstrain results (SFL and RDL) were close to the lowerlimit of the controls.

DISCUSSION

As a group serially subcultured adult labial skin fibro-blast strains have significantly more specific DHT bind-ing than strains derived from nongenital skin of bothsexes varying in age from 1 to 60 yr. Six of nine labialstrains had Bnax values exceeding 25 fmol/mg cell pro-tein, whereas none of the nongenital strains did, in-cluding seven from adult females. Keenan et al. (8)have previously alluded to the tendency of foreskinstrains to have more DHT binding than nongenitalstrains.

The difference between genital and nongenital strainsin respect to DHT binding recalls the fact that genitalstrains metabolise testosterone more rapidly than thosefrom nongenital skin (7, 9). The variability amongstrains within *each class in respect to DHT binding issurpassed by their variability in rates of testosteronemetabolism (7) and amounts of 5a-reductase activity(32). The origin of this variability is not known, butone clue is derived from studies indicating that single-cell clones isolated from one strain can differ in theirpatterns of testosterone metabolism (33) and in theiramounts of DHTbinding (1).

The lower limit of DHT binding in our control non-genital strains is so close to the lowest detectable levelthat the diagnosis of even a severe DHT binding de-ficiency would carry a significant risk of error. In con-trast, the spread between the lower limit of our controllabial strains and the lowest detectable level is largeenough to permit not only a confident diagnosis ofcomplete DHT binding deficiency but also the strongsuspicion of intermediate degrees of such a deficiency.A decisive precedent for this situation is found in theuse of cultured skin fibroblasts to diagnose 5a-reductasedeficiency in patients with type 2 FIMP (4). Here,the enzyme activity in nongenital fibroblasts from thepatients falls within the range of normal nongenitalfibroblasts so that the phenotype can be recognizedonly in genital (foreskin) fibroblasts (7).

It is clear from the present study that two of thelabial strains derived from patients with the phenotypeof complete TF have a lesser degree of DHT bindingdeficiency than the three others (column 4, Fig. 2). Theexistence of such heterogeneity has been affirmed byAmrhein et al. (34), who have recently discovered afamily with complete TF whose cultured skin fibroblastshave quantitatively normal DHT binding. Of the eightnongenital TF strains that we have studied, the one

(HCD, column 2, Fig. 2) with normal DHT bindingmay represent a patient of the latter type.

If those patients who have the typical phenotype ofTF despite quantitatively normal fibroblast DHT bind-ing can be shown to have cytoplasmic DHT receptorsthat are qualitatively normal, then a defect in target cellresponsiveness to DHT centripetal to the step of cyto-plasmic binding is possible. It is important to realize,however, that defective nuclear localization or chromatinbinding may be secondary to selective dysfunction ofcytoplasmic receptors that retain a normal ability tobind DHT (35).

Measurement of specific DHT binding in culturedgenital skin fibroblasts should be an early step in thediagnosis and management of individuals or familieswith MP. The result can indicate the priorities thatshould be attached to standard tests for evaluating thetestis-pituitary-hypothalamic axis, may influence genderassignment when anatomic considerations permit achoice, and will inevitably accelerate the accumulationof knowledge on the heterogeneity of those forms ofmale sexual maldevelopment that are due to deficits ordefects in the molecular apparatus that underlies normalandrogen responsiveness.

ACKNOWLEDGMENTSFor referring patients or providing skin biopsies and cellcultures, we are grateful to the following: P. Baird, De-partment of Medical Genetics, University of British Co-lumbia, Vancouver; J. Hamerton, Children's Hospital ofWinnipeg, Manitoba; R. Volpe, Wellesley Hospital, To-ronto; E. de Koos, Montreal General Hospital; and B.Lambert, L'Hopital Ste-Justine, G. H. Arronet, Royal Vic-toria Hospital, and M. Clamen and S. Jacobson, JewishGeneral Hospital, all of Montreal.

We thank Lyon Schwartzben and Cyril Light for faithfultechnical assistance.

This work was supported by a grant (MT-2830) fromthe Medical Research Council of Canada.

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Deficient Skin Fibroblast DHT Binding in Male Pseudohermaphroditism 349

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350 M. Kaufman, C. Straisfeld, and L. Pinsky