CLINICAL, MORPHOLOGICALAND BIOCHEMICAL STUDIES OFA VIRILIZING TUMORIN THE TESTIS *

By KENNETHSAVARD,t RALPH I. DORFMAN,BILLY BAGGETT,t LINDA L.FIELDING, LEWIS L. ENGEL,§ HARRYT. McPHERSON,LEONARDM.

LISTER,II DAVID S. JOHNSONA EDWIN C. HAMBLENANDFRANKL. ENGEL

(IProm the Worcester Foundation for Experimental Biology, Shrewsbury, Mass.; The JohnCollins Warren Laboratories, Collis P. Huntington Memorial Hospital of Harvard Uni-

versity at Massachusetts General Hospital, and the Department of Biological Chem-istry, Harvard Medical School, Boston, Mass.; and the Departments of Medicine,

Obstetrics and Gynecology, and Pathology, Division of Endocrinology,Duke University Medical Center, Durham, N. C.)

(Submitted for publication May 18, 1959; accepted November 12, 1959)

Although there is considerable indirect evidenceindicating that virilizing testicular tumors exerttheir effects by producing androgenic steroid hor-mones, few attempts have been made to determinethe exact nature of the compounds produced. Onetype of tumor which exerts particularly markedvirilizing effects when found in prepubertal boysis that classified as an interstitial cell tumor of thetestes (1-21). It has been well established that

* This work was supported by Contracts no. DA-49-007-MD-184, Army Medical Research and DevelopmentBoard, Office of the Surgeon General, Department of theArmy; no. AT(30-1)-918, United States Atomic EnergyCommission and a grant from the Charles A. King Trust.This is 'Publication no. 990 of the Cancer Commissionof Harvard University and was supported by Grants no.CG-1393-C7 and C-2833-Cl, United States Public HealthService; P-95 (T) American Cancer Society Inc.; and agrant from the Jane Coffin Childs Memorial Fund forMedical Research. Portions of the study on this casehave been reported in abstract form in: Fed. Proc. 1956,15, 346; J clin. Endocr. 1956, 16, 970 and 1629; Abstractsof the Twentieth International Physiological Congress,1956, Brussels; Expos. ann. Biochim. med. 1957, 12, 219;Southern Society for Clinical Research, January 25, 1958,New Orleans; Hormone Production in Endocrine Tu-mors, Ciba Found. Coll. Endocr. 1958, 12, 62.

t Investigator, Howard Hughes Medical Institute;present address: Departments of Biochemistry and Medi-cine, University of Miami School of Medicine, Miami,Fla.

t: Present address: Department of Pharmacology, Uni-versity of North Carolina School of Medicine, ChapelHill, N. C.

§ Permanent Faculty Fellow of the American CancerSociety.

If Present address: University of Maryland School ofMedicine, Baltimore, Md.

fI Present address: Snodgrass Laboratory of Pathol-ogy and Bacteriology, St. Louis, Mo.

such tumors cause elevated levels of urinary 17-ketosteroids. The morphology of these tumorsleads one to predict that they are producing tes-tosterone, since normal interstitial cells seem tohave this function (22, 23). A few studies of thequalitative nature of the urinary steroids in pa-tients with this type of tumor seem to confirm sucha conclusion (13, 24). Accordingly, when theopportunity arose to study a patient bearing atesticular -tumor thought to be of this type, itseemed worthwhile to attempt to establish theexact nature of the steroids produced. The studypresented here was therefore undertaken.

This paper presents a description of the patient,histological observations on the tumor, the re-sults of urinary steroid assays and the results ofexperiments in which slices of the tumor were in-cubated with C14-labeled steroid precursors.

EXPERIMENTALAND RESULTS

I. Clinical data

D. W. (Duke no. D-99663), a 5-year-and-7-month oldwhite male, was first seen at Duke Hospital on November1, 1954, because of precocious growth and development,increasing libido, and personality changes. His birth andearly development had been normal, but at 3 years of ageprogressive statural growth spurt and enlargement ofthe penis were noted. At 4 years of age, genital hairappeared, followed shortly by axillary, body and facialhair, mild facial acne and progressive deepening of thevoice. During one year a gradual, painless enlargementof the right testis had been noted, accompanied by in-creasingly frequent penile erections and masturbation,apparently without ejaculation. The child became ag-gressive, somewhat withdrawn and uninterested in previ-ous playmates and activities.

Physical examination revealed normal vital signs witha blood pressure of 112/60 mmHg. His height was 135

534

VIRILIZING TESTIS TUMOR

TABLE II

Urinary levels of individual 17-ketosteroids

PostoperativePreoperative 2 days

mg/24 hrs mg/24 hrsAndrosterone 2.4 0Etiocholanolone 2.0 0.1Androstane-3, 17-dione and 0.1 0.1

etiocholane-3,17-dioneDehydroepiandrosterone 1.4 01 1,B-Hydroxyetiocholanolone 0.2 0

[and A5(M1)-etiocholenolone]11-Ketoetiocholanolone 0.2 01 13-Hydroxyandrosterone 1.6 0

[and z90(")-androstenolone]11 -Ketoandrosterone 0.2 0.1

FIG. 1. THE PATIENT PRIOR TO OPERATION; AGE, FIVE

YEARS AND SEVEN MONTHS.

cm and his weight 33.6 kg. Anthropometric measure-ments corresponded to those of an average boy approxi-mately 11 years of age. The muscular development wasrather Herculean (Figure 1). The dental age was esti-mated to be 6.5 years. There was slight facial acne,moderate axillary hair, and abundant heavily pigmentedand coarse pubic hair. Hirsutism was noted over the ex-tremities, back and upper lip. The penis, in a flaccidstate, measured 11.5 cm in circumference and 12.5 cm inlength. The right testis was enlarged (4.5 X 3.7 X 3.2cm). It was uniformly abnormally firm, slightly irregu-lar in contour and freely movable within the well rugated

TABLE I

Urinary steroid and gonadotropin excretion

Postoperative

Preoperative 2 days 1.5 years

mg/24 hrs mg/24 hrs mg/24 hrs17-Ketosteroids (25)* 13.6 1.4 1.6Neutral acylable lipids (26)* 138 50Fluorogenic phenols (27)* 78 80 13Pregnanetriolt 4.1 0.4Corticosteroids (28)1 1.4 1.1 1.1Gonadotropins§ <1 7

* Estimations carried out on extracts of urine boiledwith 15 volumes per cent hydrochloric acid for ten minutes.

t Carried out in the laboratory of Dr. Judson J. Van Wyk.t Estimations carried out on extracts of urine incubated

with mammalian 69-glucuronidase (Ketodase, Warner-Chilcott Laboratories) at pH 5.1.

§ Rat uterine units per 24 hours.

and pigmented scrotum. The cord structures on that sidewere normal to palpation, and there was no local ordistant lymphadenopathy. The left testis was normal insize, shape and consistency (2 X 0.7 X 0.7 cm). Theprostate was enlarged and comparable in size to thatof an adolescent. No other physical abnormalities werenoted.

On psychological consultation the child was thought tobe advanced both intellectually and in his social adjust-ments, corresponding approximately to the age of 8 years.The intelligence quotient was 135 and the social quotient146.

The hemogram and urinalysis were normal, and theserological test for syphilis was negative. Blood chemi-cal analyses (nonprotein nitrogen, total proteins and al-bumin/globulin ratio, fasting blood sugar, serum cho-lesterol and electrolytes) were normal. The basal me-tabolic rate was + 3 per cent. X-rays of the chest andskull and intravenous pyelograms revealed no abnormali-

FIG. 2. THE SECTIONED SURFACEOF THE TUMOR, TESTISAND CORD STRUCTURESARE ILLUSTRATED. The identifica-tion tag is 2 cm in length.

535

SAVARDET AL.

'/ the cell boundaries were very clearly defined. Occasion-ally, syncytial masses of cytoplasm were formed with 2

~ i ~ ~ to 5 nuclei. The nuclei were round, vesicular and showed;:.~~~~~~.: ~~~~ no wrinkling of the nuclear membrane. There was a

fine granular chromatin network with 1 to 3 prominentnucleoli. Occasional large hyperchromatic nuclei werepresent, but generally there was little variation and mi-

*-~~~~~-.i~~~toses were rare. With Helly's fixation the cytoplasmwas brightly eosinophilic, whereas with 10 per cent

.-formalin fixation the cytoplasm appeared light pink withprominent unstained vacuoles.

-~~~~~~~.~~~~~The most striking cytological feature was the presenceof a variety of cytoplasmic inclusions. With hematoxylin

_ .~~~~~~~4 ~~~~~~and eosin and Helly's fixation most of the cells showedvery finely divided, granular, brown inclusions clustered

.i -m~t about the nucleus (Figure 4). This material was in-distinguishable from the "lipochromei pigment present in

~~~. -~~~~normal interstitial cells; however, it was not sudanophilic,A By.~.ys nor was there any sudanophilic material in any of the

cells. This material reacted positively with the Schiffreagent and was particularly well demonstrated by phos-rphotungstic acid hematoxylin stains. A second type of

,t!Y~as^^Docytoplasmic inclusion was present in most of the cells.<9;_-**;¢These structures varied considerably in size measuring9:..;2**-bi , 8 < from 1.7 to 5.1 , in diameter. They were usually clus-

FIG. 3. THE MARGIN OF THE TUMORIS SHOWNAT ITS L:-JUNCTION WITH THE SURROUNDINGNORMALTESTICULAR jiTUBULES. Note the fibrous septa dividing the tumor intolobules. (Hematoxylin and eosin, X205.)

ties. The bone age was greater than 10 but less than14 years. Urinary steroid and gonadotropin analysesare reported below (Tables I and II).

On November 16, 1954, a right radical orchiectomyand biopsy of the left testis were performed.

Pathological anatomy. Gross: The right testis meas-ured 2.5 X 3.0 X 3.5 cm and weighed 25 g. On sectiona grossly well encapsulated, homogeneous soft tumor,lobulated by delicate fibrous septa, was observed (Fig-ure 2). The sectioned surface of the tumor measured

V 23 x 32 mm, bulged and was dark brown with a distinctgreen cast. The surrounding testicular tissue wasMcompressed.

Microscopic examination. There was an incomplete,thin connective tissue capsule. Where the capsule wasincomplete, scattered small clumps of tumor cells ex- itended for a short distance into the surrounding tissue.The tumor was made up of solid sheets of cells brokenup by occasional thin, vascular, fibrous tissue septa (Fig-ure 3). A delicate reticulum supporting frameworkfurther separated the tumor into clumps of 2 to 12 cells.The tumor cells were irregular, polyhedral in shape, 14 FIG. 4. THIS MICROPHOTOGRAPHILLUSTRATES THEto 35 A in diameter (average, 22 AL), and occasionally LIPOCHROMEGRANULES CLUSTERED ABOUT THE NUCLEUShad indistinct cell boundaries, although in most areas OF VIRTUALLY EVERYCELL. (Periodic acid-Schiff, X1,021.)

536

I

537VIRILIZING TESTIS TUMOR

tered about the periphery of the cytoplasm and occasion- j

ally filled the entire cell. They were highly refractileand were surrounded by a clear halo. Some of thesegranules had an internal structure in which the outer

wall was stained dark blue with phosphotungstic acidhematoxylin. Just internal to this was a narrow, clear,unstained area. A solid mass of dark blue materialmade up the remainder of the inclusion. Others were

solid and homogeneous. These were not well seen in tformalin-fixed tissue by any technique used but werereadily apparent in Helly-fixed tissue, particularly withMasson trichrome and phosphotungstic acid hematoxylinstains as well as with hematoxylin and eosin (Figure 5).A third type of inclusion was present, consisting of

very rare rod-shaped structures which were indistinguish-able from crystalloids of Reinke. Their most strikingtinctorial reaction was their oxyphilia rendering themred with the Masson trichrome technique. Some of thesewere seen crossing a cell boundary and extending intoan adjacent cell; others lay completely within the cyto- .

plasm of a single cell (Figure 6).The seminiferous tubules and interstitial cells of the

left testis showed development corresponding to the pa-tient's chronological age. There was no evidence of X

$6 V~~~*

FIG. 6. THE CELL IN THE CENTER OF THE FIELD CON

TAINS TWOELONGATEDCRYSTALLOIDS OF REINKE. (Mas-

A!iHeyDo; t ^ --R ffi i son trichrome, X1,021.)

4 t s.4: ;Xs}J! 9 iS Elf 2 >'9 4 spermatogenesis. The pathological diagnosis was inter-*i stitial cell tumor of the right testis.

.'f Follow-up. Since discharge from the hospital therehas been no regression in any of the somatic featurespreviously depicted. In March and June, 1955, no. es-

X ..................................sential differences were noted. While the left testis was

normal in size and consistency, the strong sexual drive

persisted. The latter was considerably reduced by May,1956, and the child's social adjustment was better. His

height was 149 cm and his weight 39.5 kg, gains of 14

icm and 5.9 kg, respectively, since operation. At this time*d̂:;A d it was noted that the remaining left testis had grown re-

markably in size in the preceding year, now measuring

Wok* .̂**.A d d X 5~5.5 X 3 cm. The testicular size, consistency and con-

*;9-Rx-<_̂htour, and the prostate gland, were now comparable to

s1t Aft k^. 4 *that of a normal late-adolescent male. He was last seen

ings similar to those noted six months earlier except fora further gain of 6 cm in height. The bone age was esti-mated as 12 years, an advance of almost two years since

FIG. 5. LARGE CYTOPLASMIC GRANULESCAN BE SEEN operation. Daily urinary gonadotropin excretion was 7

DISTRIBUTED ABOUTTHE PERIPHERY OF THE TUMORCELLS. rat uterine units, within the range for a normal adult.

Nuclear detail is also well shown. (Phosphotungstic The postoperative urinary steroid and gonadotropin lev-nif 1vAPmntnvv1x 021-) els are recorded in Tables I and II.aClU nCTINILVILY1111, ^JL)V"JL.,/

SAVARDET AL.

II. Urinary steroids

Estimations of steroid concentrations were car-ried out on 24-hour urine specimens collected be-fore and at various periods after the removal ofthe tumor. The values obtained are shown inTable I. These values as well as those of gonado-tropins are in agreement with values previouslyobserved in this syndrome. The excretion of cor-ticosteroids measured by the Silber and Portermethod (28) was in the normal range for a 5year old boy and was unchanged after removalof the tumor. Paper chromatograms of the corti-costeroid fraction in the toluene/methanol: water(75: 25) system (29) showed the presence oftetrahydrocortisone and tetrahydrocortisol as thepreponderant corticosteroids before and after theoperation.' No unusual reducing compoundswere detected.

The daily excretion of 17-ketosteroids, neutralacylable lipids (hydroxysteroids) and pregnane-triol was elevated before removal of the tumor. Afurther sample of urine was boiled with 15 vol-umes per cent of hydrochloric acid, and the etherextracts were chromatographed in the Bush lig-roin/methanol: water (96: 4), and ligroin: tolu-ene (67: 33)/methanol-water (70: 30) systems.The chromatograms were sprayed with Zimmer-mann reagent and the amount of each compoundestimated by comparison of size and intensity ofthe spot with the sizes and intensities resultingfrom known amounts of the compound chromato-

1 The chemical names for the steroid compounds usedin this paper are as follows: 3a-hydroxyandrostan-17-one(androsterone); 3a,11j3-dihydroxyandrostan-17-one (1158-hydroxyandrosterone) ; 11 6-hydroxy-4-androstene-3,17-dione (ll1,-hydroxyandrostenedione); 4-androstene-3,11,-17-trione (adrenosterone); 4-androstene-3,17-dione (an-drostenedione); 3a-hydroxyetiocholane-17-one (etiocho-lanolone) ; 17,3-hydroxy-4-androsten-3-one (testosterone);11p,17,f-dihydroxy-4-androsten-3-one (118-hydroxytestos-terone); 17,j-hydroxy-4-androstene-3,11-dione (11-keto-testosterone); 3,8-hydroxy-5-androsten-17-one (dehydro-epiandrosterone) ; 3a,17a,21-trihydroxypregnane-11,20-di-one (tetrahydrocortisone) ; 3a,11,8,17a,21-tetrahydroxy-pregnan-20-one (tetrahydrocortisol); pregnane-3a,17a,-20a-triol (pregnanetriol); 4-pregnene-3,20-dione (proges-terone); 17a-hydroxy-4-pregnene-3,20-dione (17a-hy-droxyprogesterone) ; 11,8 -hydroxy-4-pregnene-3,20-dione(llfi-hydroxyprogesterone) ; 6f-hydroxy-4-pregnene-3,20-dione (6,6-hydroxyprogesterone); 6j3-acetoxy-4-pregnene-3,20-dione (6,-acetoxyprogesterone); 3p8-hydroxy-5-preg-nen-20-one (5-pregnenolone).

graphed at the same time. This method has beenfound to be accurate within 15 per cent (30).The daily excretion of individual 17-ketosteroidsestimated by this method is presented in Table II,in which the artifacts produced by the hydrolysiswith acid are included with their known pre-cursors.

Elevated excretion of both C,9O2 and C,903compounds, androsterone and 11,8-hydroxyandro-sterone in particular, is to be noted. The excre-tion of all steroids measured after removal of thetumor was normal. The level of fluorogenicphenols gave no indication of elevated estrogenexcretion and was unchanged by removal of thetumor; the reduction of this level 1.5 years afterthe tumor was removed is unexplained.

III. In vitro studies

The incubation of slices of this tumor was un-dertaken as a means of direct biochemical assess-ment of its steroid production. This techniqueutilizes small amounts of tissue and C14-labeledsteroid precursors such as acetate-i-C14, and isone which is of proven value in this type of study(31). In order to facilitate extraction of the traceamounts of radioactive steroids from the incuba-tion medium and tissue, it is necessary to addknown amounts of crystalline nonradioactive ster-oids. In the case of each substance, the totalsteroid (radioactive and carrier) may then be iso-lated and purified and its radioactivity per milli-gram (specific activity) determined after suitablepurification. By calculation back to the originalweight of carrier, the amount of radioactivity as-sociated with each carrier steroid may be ascer-tained. This in turn reflects the relative amountof each steroid formed during the experiment.Other methods for the microdetermination of theabsolute amounts of endogenous steroid hormonespresent in endocrine glands have recently been de-veloped (32-36); these however require exactingmanipulations for certainty in the identificationand quantitative measurement of the minuteamounts of the compounds found. The incuba-tion of specific C'4-labeled steroids with tissuesand the isolation of radioactive products, alsoby means of carrier dilution, provide an assess-ment of the tissue's capacity to effect certain spe-cific transformations.

538

VIRILIZING TESTIS TUMOR

The chemical methods employed consisted firstin making a total lipid extract which was separatedinto nonpolar (fatty constituents, and so forth)and polar lipids. The latter fraction containingthe carrier steroids was separated into acidic, phe-nolic and neutral polar lipids, and on occasion,into ketonic and nonketonic fractions. Theseprocedures, described below in greater detail, af-forded extracts containing the steroids relativelyfree from extraneous material. These extractswere then subjected to countercurrent distribu-tions (ccd) and paper chromatographic separa-tions in a variety of systems. Each steroid so iso-lated was free from steroidal and other contami-nants, and was judged chemically pure by meltingpoint and other criteria. The achievement ofradiochemical purity required further procedures,which are described below.

METHODS1. General

Countercurrent distributions. All 4- and 8-transferdistributions were carried out in separatory funnels.Lower layers were transferred, and equal volumes of thetwo phases were used. The 24-transfer distributionswere carried out in a 25-tube, stainless steel Craig ap-

paratus and those of greater numbers of transfers were

carried out in the glass apparatus (H. 0. Post ScientificInstrument Co.).

Paper chromatography. Chromatograms of the Zaffa-roni type (37) and modifications (38, 39) were run

at room temperature on Whatman no. 1 filter paper. Inall systems employed, propylene glycol was used as thestationary phase. The following solvents were used as

moving phases: ligroin (38), toluene (37), toluene:cyclohexane (50: 50), and toluene: methyl cyclohexane(50: 50) (39). Chromatograms of the type describedby Bush (29) were run at 37° C on Whatman no. 2 filterpaper.

Compounds were located on the developed chromato-grams by visualization with ultraviolet light (A4-3-keto-steroids), and by spraying with alkaline blue tetrazolium(reducing steroids) (40), alkaline m-dinitrobenzene(all ketosteroids) (38), or antimony trichloride reagent(A5-3,8-hydroxysteroids) (41). After locating the ster-oids on the preparative chromatograms, the zones con-

taining compounds or mixtures of compounds were cutout, cut into small pieces, and extracted repeatedly withhot methanol, then chloroform, or a mixture of equalparts of methanol and chloroform. The combined ex-

tracts were filtered to remove paper fibers and evapo-

rated to dryness under reduced pressure. The extractsof paper previously saturated with propylene glycol were

dissolved in chloroform and the chloroform washed sev-

eral times with 1/10 volumes of water to remove theglycol.

When required, zones of radioactivity on the chromato-grams were located by means of contact radioautography.This consisted in placing the chromatogram (or a rep-resentative portion) in contact with Kodak MedicalX-ray film (no screen) for the required exposure time(42), and developing the film in the usual manner.

Measurement of radioactivity. All samples were platedat infinite thinness in metal dishes and counted in a win-dowless gas-flow counter. A minimum of 2,000 countswas accumulated on each. All counts were correctedfor background.

Ultraviolet absorption. All ultraviolet absorption spec-tra and values of absorbance were measured in 95 percent ethanol.

Formation of derivatives. Acetylations were carriedout at room temperature with acetic anhydride and an-hydrous pyridine (1: 1); excess reagents were evapora-ted under a stream of air. Oxidations were carried outwith chromium trioxide in glacial acetic acid at roomtemperature for two hours (43). Formation of oximeswas carried out in the conventional way.

If. IncubationThe tumor was received within a few minutes of ex-

cision and chilled in cold physiological saline. It wassectioned and a representative portion used for the bio-chemical studies; the remainder was taken for pathology.Slices were prepared with a Stadie-Riggs slicer and dis-tributed among four incubation flasks. These flaskscontained as their final contents 0.93 g of tumor tissue,20 ml of fresh human serum, 0.02 mmole of potassiumfumarate, 10 mg of glucose, 500 units of pregnant mareserum gonadotropin (Equinex-Ayerst), 500 units ofpregnancy urine gonadotropin (APL-Ayerst) and 5units of porcine pituitary gonadotropin (FSH-Armour).The radioactive substances (the first three shown to bechromatographically homogeneous), added one to eachflask, were: A) testosterone-4-C1' (0.92 mg, 3.82 X 10'cpm); B) progesterone-4-C1' (2 mg, 350,000 cpm); C)cholesterol-4-C1' (2.2 mg, 7.5 X 10' cpm); and D) so-dium acetate-i-C14 (1 mc); the radioacetate was addeddissolved in water, and the steroids in 0.2 ml of propyleneglycol. Incubation was carried out with shaking forthree hours at 370 C with 95 per cent 02 and 5 per centCO, as gas phase. The flask contents were frozen untilanalyzed.

III. Addition of carrier steroids, extraction and prelimi-nary purificationOn removal of the incubation mixtures for storage,

chromatographically pure steroids in varying amounts(5 to 20 mg) were added to each of the incubation flasks(Table III).

Each incubation mixture (tissue, medium and addedcarrier) was extracted individually in a Waring blendorwith 25 volumes of chloroform: methanol (2: 1) mixture(44). After removal of tissue debris by filtration, 1/5volume of water was added and the mixture shaken; theupper aqueous layer was removed and extracted twicewith 1/2 volume of chloroform. The combined lowver

539

SAVARDET AL.

TABLE III

Carrier steroids added to incubation mixtures

Experiment-C'4substrateC. Cholesterol*

Steroids A. Testosterone B. Progesterone I II D. Acetate

mg mg mg mg mgEstrone 5Estradiol-17,7 5Estriol 5Testosterone 10 10 20 104-Androstenedione 10 10 20 10110-Hydroxyandrostenedione 10 lot 20 101 1f-Hydroxytestosterone 10 20Adrenosterone 10 5tDehydroepiandrosterone 20 105-Androstene-3,6,17j-diol 20Progesterone 10 20 101 7a-Hydroxyprogesterone 10 20 5t1 1f3-Hydroxyprogesterone 10t 5t1 1-Ketoprogesterone 10t5-Pregnenolone 20 103f3,17a-Dihydroxy-5-pregnen-20-one 206,B-Hydroxyprogesterone 5t

* The extract from this incubation was divided into two equal parts; estrogens were added before the division andthe two resulting phenolic fractions were combined later in the fractionation; other carriers were added after divisionof the extract.

t Added at a later stage in the fractionation procedure; for details see text.

layers were evaporated to dryness and the residue sub-jected to an 8-transfer ccd between ligroin (bp 65 to750 C) and 90 per cent aqueous methanol. The individualtubes from the distribution of each incubation were evapo-rated to dryness, weighed, assayed for radioactivity andfor the carrier ketosteroids (by absorption at 240 mas orby the antimony trichloride reagent). The tubes con-taining the carrier steroids and associated radioactivepolar material from each incubation were combined anddissolved in toluene. Each pooled sample was separatedinto the respective phenolic, acidic and neutral fractionsby shaking the toluene solution with 1 N sodium hydrox-ide (27). The aqueous alkali solution was adjusted topH 8.5 and shaken with ether to remove the phenols.The neutral material remained in the toluene solution.The neutral polar fractions obtained in the incubationsof testosterone (A), progesterone (B) and cholesterol(C) were subjected to a separation with Girard reagentT into ketonic and nonketonic fractions. The extractfrom the acetate-i-C1' experiment (D) was not sub-jected to this separation.

IV. Separation and purification of carrier steroids

Since somewhat different methods were used for thepurification of the individual carrier steroids in each ex-periment, they will be discussed separately. The stepsinvolved in establishing the radiochemical purity (45) ofeach apparently labeled compound will be presented foreach compound. Where derivatives of the carriers wereprepared (acetates, oximes, and so forth) the values ofspecific activity, unless otherwise noted, are not cor-rected for changes in molecular weight, and are givenas determined.

V. Incubation experimentsEXPERIMENTA. Incubation of testosterone-4-C14:

The ketonic fraction from this incubation was chromato-graphed in ligroin/propylene glycol for 96 hours. Eachsteroid carrier zone was located and recognized by itsrelative position (37), eluted and rechromatrographed.17a-Hydroxyprogesterone, testosterone and adrenosteronewere not completely separated by this chromatography.Separation was carried out by acetylation of the mixtureand the complete removal of testosterone acetate bychromatography in ligroin/propylene glycol; the tworemaining steroids were separated by chromatographyon paper in toluene: methyl cyclohexane (50: 50)/propyl-ene glycol.

l1,i-Hydroxytestosterone: This compound did not mi-grate from the starting line as the free steroid in ligroin/propylene glycol in 100 hours. It was chromatographedin toluene/propylene glycol for 12 hours to yield a con-taminated product, 3.7 mg in weight and specific activity7,370 cpm per mg. One and two-tenths mg of this wasdiluted with 30 mg of 1 1,8-hydroxytestosterone carrier,acetylated, and chromatographed in ligroin/propyleneglycol (24 hours), to provide a diluted 11,8-hydroxy-testosterone acetate (mp 151 to 1520 C, from acetone:hexane) with a specific activity of 114 cpm per mg (or129 corrected to the free compound). Oxidation withchromic acid and chromatography in ligroin/propyleneglycol gave 11-ketotestosterone acetate (mp 164 to 165° C,acetone: hexane) with a specific activity of 112 cpm permg (127 as the free compound). Saponification withpotassium carbonate in ethanol (46) gave 11-ketotestos-terone (mp 184 to 1850 C); specific activity 131 cpm per

540

VIRILIZING TESTIS TUMOR

mg (130 as ll,-hydroxytestosterone). The specific ac-

tivity of the initially isolated 1l,8-hydroxytestosteronewas calculated as 3,300 cpm per mg; this value is usedin the calculation of those data for this compound inTable IV.

11,8-Hydroxyandrostenedione: As initially isolated andcrystallized (mp 1940 C), this compound had a specificactivity of 6,500 cpm per mg. Two samples of 0.5 mg

each were diluted with 50 and 70 mg of carrier and puri-fied as follows. Sample 1 was chromatographed (ligroin/propylene glycol, 116 hours) and recrystallized fromacetone: hexane (59 cpm per mg); oxidized to adreno-sterone, chromatographed, and recrystallized from metha-nol (mp 220 to 2250 C; 60 cpm per mg); recrystallizedfrom acetone: hexane (mp 219 to 2200 C; 59 cpm per

mg); converted to the dioxine and recrystallized frommethanol (mp 209 to 2100 C; 56 cpm per mg, correctedto the molecular weight of adrenosterone). Sample 2was chromatographed and crystallized (48 cpm per mg);oxidation followed by chromatography and crystalliza-tion gave adrenosterone (mp 2180 C; 46 cpm per mg).The initially isolated steroid was radiochemically pure

at 6,500 cpm per mg.

Adrenosterone: As isolated and recrystallized (mp2120 C) the specific activity was 278 cpm per mg and as

may be seen from the following, was slightly contaminated.The dioxime (mp 222° C; 258 cpm per mg) was chro-matographed (toluene/propylene glycol, 34 hours) andrecrystallized from methanol (mp 218 to 2200 C; 190cpm per mg, calculated as adrenosterone, 209 cpm permg); the material in the mother liquor had the same

specific activity. Adrenosterone was considered pure at209 cpm per mg.

Androstenedione: This compound was recrystallizedfrom ethyl acetate to give crystals (mp 172 to 173° C;19,700 cpm per mg) and a crystalline residue from themother liquors (17,400 cpm per mg). This product wasnot further manipulated and was considered radiochemi-cally pure.

Testosterone: The original substrate, diluted as noted,was recovered (mp 151 to 152.5° C; 250,000 cpm per mg).

Radioautography of the initial chromatograms havingrevealed no zones other than those associated with theabove steroids, it is presumed that no other importanttransformations of testosterone occurred in this study.Total radioactivity associated with each carrier steroidat the above specific activities is given in Table IV,column A, corrected to the amount present at the additionof the original carrier.

EXPERIMENTB. Incubation of progesterone-4-C04:The chromatography of the neutral ketonic fraction fromthis incubation was somewhat encumbered by the presenceof a strongly fluorescing material whose presence seri-ously distorted the initial chromatograms in ligroin/propylene glycol. In order to eliminate this materialrepeated chromatography was necessary; quantitativemeasurement of several carrier steroids showed that but1.0 to 1.2 mg remained. This was assumed to be truefor all others and at this point additional amounts ofeach carrier were added (10 mg) to the recombined frac-tions, and all were rechromatographed in ligroin/propyl-ene glycol, and separated as described above; 1ljl-hy-

TABLE IV

Results of incubation studies*

Experiment-CULsubstrate

A. Testosterone B. Progesterone D. Acetate

Added carriers 3.82 X 106 cpm 3.5 X 105 cpm 1.3 X 109 cpm

cPm % cpmt % cPm ScEstrone NoneEstradiol-1 7j1 NoneEstriol NoneTestosterone 2.500 X 106 66.0 0.11 X 105 3.1 0.30 X 10' 0.0034-Androstenedione 0.197 X 106 5.0 0.30 X 105 8.6 6.50 X 10' 0.065

1j3-Hydroxyandrostenedione 0.065 X 106 1.7 0.13 X 10' 3.7t 3.47 X 10' 0.0351 10-Hydroxytestosterone 0.033 X 106 0.8Adrenosterone 0.002 X 106 0.05 0.06 X 106 0.0006TDehydroepiandrosterone 0.04 X 106 0.00045-Androstene-3i, 1 7fl-diolProgesterone 2.03 X 10' 58.0 None17a-Hydroxyprogesterone 1.14 X 10' 32.6 0.63 X 105 0.00611 1-Hydroxyprogesterone None None1 1-Ketoprogesterone None5-Pregnenolone None3P, 1 7a-Dihydroxy-5-pregnen-20-one60-Hydroxyprogesterone None

Total 74 106 0.11

* Amount of radioactivity and per cent of added substrate isolated by carrier-dilution.t These values are based upon a further dilution of the original carriers by a ratio of 1 to 10; this dilution is explained

in the text.$ This value is not comparable to others, as carrier was added late in the fractionation scheme.

541

SAVARDET AL.

droxyandrostenedione, 11,l-hydroxy- and 11-ketopro-gesterone were added at this point for the first time.

11,8-Hydroxyandrostenedione: This compound was

recognized as a transformation product of progesterone-

C4 late in the study and was seen first as a radioactivezone of slow mobility [slower than 11f8-hydroxyproges-terone and 17a-hydroxyprogesterone (38)]. To the ma-

terial eluted from this radioactive area, 5 mg of carrier11j8-hydroxyandrostenedione was added and the wholerechromatographed in ligroin/propylene glycol; the re-

sulting zone was eluted and the steroid recrystallizedfrom acetone: hexane to constant specific activity (mp195.5 to 196.50 C; 127 cpm per mg); 2.57 mg of this ma-

terial was diluted with 4.38 mg of additional carrier andoxidized, chromatographed, and crystallized from metha-nol to give adrenosterone (mp 213 to 214° C; 47 cpmn per

mg). The dioxime was formed and had a specific ac-

tivity of 43 cpm per mg. The 11,-hydroxyandrostene-dione obtained after the initial addition of carrier was

considered radiochemically pure at approximately 127cpm per mg. Comparison of specific activity with thoseof other isolated compounds is not valid because thecarrier in this case was not added at the same stage as

in the others. The total amount of this compoundformed, shown as counts per minute in Table IV, mustbe considered minimal.

11,-Hydroxyprogesterone: As isolated following thesecond addition of carriers, this compound (14 mg) hada specific activity of 44 cpm per mg but was contami-nated with traces of 17a-hydroxyprogesterone. Re-crystallization from ethyl acetate gave 10.4 mg (mp 182.5to 183.50 C; 8 cpm per mg). Oxidation with chromicacid followed by chromatography and crystallization frommethanol gave 11-ketoprogesterone (mp 170.5 to 172.50C; 5 cpm per mg). Formation of the oxime and re-

crystallization (ethyl acetate) gave crystals (mp 226 to2290 C) ounting 2 cpm per mg. 11,-Hydroxyproges-terone was therefore considered to be devoid of sig-nificant radioactivity.

17a-Hydroxyprogesterone: Nine and five-tenths mg ofcrystalline steroid was isolated counting 1,050 cpm per

mg. Of this, 1.85 mg was diluted with 18 mg of addi-tional carrier and the oxime formed and recrystallizedfrom methanol (mp 244 to 2460 C). The oxime had a

specific activity of 96 cpm per mg; the mother liquorsyielded a residue which had essentially the same specificactivity. 17a-Hydroxyprogesterone was considered pure

at 1,050 cpm per mg specific activity.Testosterone: Separated by chromatography from the

above as the acetate (mp 139.5 to 140.50 C), this com-

pound counted 109 cpm per mg, and was slightly con-

taminated. Saponified to testosterone (mp 149 to 1500 C),the compound counted 104 cpm per mg. Converted totestosterone oxime (mp 212 to 214.50 C), the compoundcounted 95 cpm per mg. Testosterone was thereforeconsidered radiochemically pure at 104 cpm per mg.

11-Ketoprogesterone: This compound as isolated andcrystallized (methanol, mp 171 to 1720 C) had a specificactivity of 6 cpm per mg. Formation of the oxime andcrystallization eliminated the radioactivity.

Androstenedione: This compound was freed of most ofits contaminating progesterone-4-C1' by the second chro-matography in ligroin/propylene glycol. Recrystalliza-tion from ethyl acetate gave apparently contaminatedcrystals (mp 170.5 to 1710 C) with a specific activity of319 cpm per mg; the semicrystalline residue from themother liquors counted 260 cpm per mg. The crystals,7.1 mg, were treated to form the oxime (mp 208 to 2100C; 202 cpm per mg), which was further purified bychromatography (toluene/propylene glycol, 25 hours) andcrystallized (mp 212 to 213.50 C). It counted 260 cpmper mg with a similar specific activity in the motherliquor residue. Despite the variations in specific ac-tivities, no evidence of radioimpurities was apparent.The androstenedione was considered pure at a specificactivity of 275 cpm per mg.

Progesterone: The diluted substrate was recovered andrecrystallized (acetone, mp 120 to 120.5° C). Its spe-cific activity was 1,840 cpm per mg.

Radioautographs of the initial chromatograms revealedno zones of radioactivity other than those associatedwith the above compounds. The areas between pro-gesterone and 4-androstenedione notably were free fromradioactivity; the epimers 20a- and 20,6-hydroxy-4-preg-nen-3-ones, normal derivatives of progesterone in theovary (34), have mobilities intermediate between thoseof the former compounds.

The data in Table IV for this Experiment B are cal-culated from the above specific activities and from theassumption that at the point of the second addition ofcarriers, 1.0 mg of each of the original steroid carrierswas present.

EXPERIMENTC. Incubation of cholesterol-4-C14:The mixture from this incubation was extracted in theusual way. The chloroform: methanol extract obtained atthe outset was halved and to the separate portions, I andII, were added the steroid carriers listed in Table III.Each portion was treated as described above (SectionIII. In vitro studies) ; the phenolic fractions from por-tions I and II were combined.

EXPERIMENTC-I. The total ketonic material fromportion I containing the added a,,a-unsaturated ketosteroidcarriers, weighed 122 mg and contained 80,000 cpm. Itwas separated on paper chromatograms in ligroin/propylene glycol as described in Experiments A and B.

11,8-Hydroxytestosterone: This compound as isolated,was rechromatographed in toluene/propylene glycol,acetylated, and the 1 1p-hydroxytestosterone acetatechromatographed in ligroin/propylene glycol. The puri-fied steroid was recrystallized several times (acetone:hexane) and finally melted at 127 to 1280 C, and had aspecific activity of 20 cpm per mg. The mother liquorresidue counted 78 cpm per mg. The small amounts ofsubstance precluded further manipulations and, therefore,definite radiochemical purity was not achieved.

11,8-Hydroxyandrostenedione: This compound, follow-ing rechromatography in ligroin/propylene glycol (96hours), was recrystallized from acetone: hexane to con-stant melting point (195 to 1960 C) and a specific ac-tivity of 71 cpm per mg in the final crystals (8.0 mg).

542

VIRILIZING TESTIS TUMOR

Recrystallization from ethyl acetate: hexane gave crystals(5.6 mg, 69 cpm per mg) and an oily mother liquorresidue (1.5 mg) counting 156 cpm per mg, suggestingthat radiochemical purity had not been achieved.

17a-Hydroxyprogesterone: Following the separation oftestosterone from this compound by the acetylation pro-

cedure, the 17a-hydroxyprogesterone was rechromato-graphed several times in toluene: methyl cyclohexane(50: 90)/propylene glycol and recrystallized from metha-nol to constant specific activity (50 cpm per mg) ofboth the final crystals and mother liquor residue.

Testosterone: Isolated as the acetate, this compoundcounted 25 cpm per mg. The mother liquor was notstudied.

Androstenedione: Isolated and purified by rechroma-tography, androstenedione failed to crystallize. The oilyproduct had a specific activity of 110 cpm per mg; thisvalue is considered maximal.

Progesterone: This compound, purified as in Experi-ment A, was crystallized from acetone. The progesteroneas crystals (mp 119 to 1200 C) counted 193 cpm per mg;

the progesterone as found in the last mother liquorcounted 163 cpm per mg. The radioactivity in this car-

rier, as in all others, was considered too low to warrantfurther attention, in contrast to the activities in Experi-ments A, B and D.

EXPERIMENTC-II. 5-Androstenediol: The nonke-tonic fraction of portion II, containing the carrier 5-andro-stenediol and its associated radioactivity, weighed 29 mg

and was chromatographed in toluene: methyl cyclohexane(50: 50)/propylene glycol for 48 hours. The androstene-diol was located (antimony trichloride reagent), elutedand the chromatography repeated. Crystallization of theeluted steroid (acetone: hexane) yielded 7.3 mg of5-androstenediol, mp to 175 to 1770 C, specific activity90 cpm per mg. After further addition of carrier (14.3mg) to the above, the substance was acetylated and crys-

tallized to give the diacetate, mp 157 to 1580 C, specificactivity 14 cpm per mg. Recrystallization from acetonegave crystals and a residue in the mother liquors count-ing, respectively, 13 and 17 cpm per mg.

3,8,17a-Dihydroxy-5-pregnen-20-one: The ketonic frac-tion weighing 45.8 mg and containing 99,700 cpm, was

dissolved in hot methanol and allowed to crystallize.Two crops of steroid were obtained, which were com-

bined and recrystallized to constant melting point (226 to2360 C); the last crystals had a specific activity of 90cpm per mg. Acetylation and crystallization of thelatter from acetone gave 5.7 mg of 3,8,17a-dihydroxy-5-pregnen-20-one acetate, mp 221 to 2250 C, specific ac-

tivity 93 cpm per mg and a mother liquor residue, 2.4mg and 370 cpm per mg. Dilution of the crystals (4.0mg) with carrier steroid acetate (4.0 mg) and recrystal-lization from acetone gave crystals (4.7 mg) with a

specific activity of 52 cpm per mg, which did not changeon recrystallization from methanol. The two motherliquor residues from these steps (1.3 mg each), how-ever, counted 108 and 72 cpm per mg, respectively. Thus,despite apparent constant specific activity of the crystals,the mother liquors gave evidence of radiochemical con-

tamination. Further purification was not possible be-cause of the lack of material.

Dehydroepiandrosterone and 5-pregnenolone: The to-tal ketonic fraction, less the crystalline 3,8,17a-dihydroxy-5-pregnen-20-one, containing 28,000 epm, was chromato-graphed on paper in methyl cyclohexane/propylene glycolfor 41 hours. Two zones were visualized by the anti-mony trichloride reagent, eluted, and each was rechroma-tographed in the same solvent system.

The slower zone which gave a positive Zimmermannreaction was crystallized from hexane to give dehydro-epiandrosterone (190 cpm per mg) which was dilutedwith an equal weight of carrier, acetylated and crystal-lized from hexane to give crystals counting 91 cpm permg. This material was recrystallized and the crystalsobtained (mp 168 to 1690 C, corrected) counted 68 cpmper mg; the mother liquor residue counted 110 cpm permg. Recrystallization of 2.0 mg of the crystals gave0.8 mg of specific activity 74 cpm per mg and a newmother liquor residue (0.7 mg) counting 140 cpm permg. Radiochemical purity could not, therefore, bedemonstrated.

The faster zone was eluted, and after addition of car-rier steroid (10.9 mg), was recrystallized to constantmelting point (186 to 1880 C) and a final specific activityof 208 cpm per mg; the mother liquor residues were ateach step of higher specific activity. Acetylation andcrystallization from methanol gave crystals of pregneno-lone acetate (mp 148 to 1490 C), whose specific activitywas 125 cpm per mg; crystallized once more, the acetatecounted 70 cpm per mg. The two last mother liquorresidues counted 290 and 750 cpm per mg, respectively.The product was considered contaminated.

In contrast to Experiments A, B and D, little radio-activity was associated with the above steroid carriersat the point of their isolation from the chromatograms.This low radioactivity precluded the further addition ofcarrier-diluent and prevented use of recrystallization pro-cedures for demonstrating and achieving radiochemicalpurity. The uncharacterized radioactivity present in theextracts was not associated with any chromatographicallydiscrete compounds; it tended to smear throughout thepaper chromatograms of both Experiments C-I and C-II.Whether this material is a product of a biochemical trans-formation cannot be inferred from this study; it may beentirely due to artifact formation caused by the manipula-tory procedures. The values obtained in this study havenot been included in the tabulation of the results fromthe other three studies (Table IV).

EXPERIMENTD. Incubation of acetate-1-C14: Thepolar lipids [tubes 5 to 8 from the distribution between lig-roin and methanol: water (90: 10) ] were combined (2.62 X10o cpm) and washed free of acetate-i-C1' by means of a4-transfer ccd between water and chloroform, using thestripping procedure described by Weisiger (47). Theupper phase of tube 0 was saturated aqueous sodium ace-tate (pH 7.0) instead of water. The resulting lipid frac-tion contained 2.68 X 10' cpm; apparently no appreciableamount of labeled acetate was present. This fractionwas subjected to a phenolic separation (27), giving a

543

SAVARDET AL.

neutral fraction containing 2.20 X 106 cpm and a phenolicfraction (86,000 cpm) which was subjected to a ccd be-tween toluene and 1 N sodium hydroxide, using fivefunnels and complete stripping. There was a peak inradioactivity which moved with the alkali and was ex-tractable with ether at pH 8.5. The material in thispeak, having the properties of phenols, contained 15,000cpm. This purified phenolic fraction was subjected to a99-transfer ccd in methanol: water (70: 30) /chloroform:carbon tetrachloride (40: 60). The distribution wasanalyzed for radioactivity and for the estrogens by thefluorimetric method. Estrone, estradiol-17,8, and es-triol were identified, but no peak in radioactivity was as-sociated with any of the three estrogens. It was there-fore concluded that none of the three was labeled.

Five mg each of 17a-hydroxyprogesterone, 11p-hy-droxyprogesterone, 6p8-hydroxyprogesterone and adreno-sterone were added as additional carriers to the neutralfraction. This was subjected to a 150-transfer ccd incyclohexane: ethyl acetate (50: 50)/ethanol: water (50:50). The peaks of radioactivity (K2 = 0.32 and K =1.02) contained all of the neutral carrier steroids exceptprogesterone and 5-pregnenolone which were found to-gether in an area containing no peak of radioactivity(K = 2.56). It was, therefore, concluded that theselatter two compounds were not appreciably labeled.

The contents of the tubes in each of the two peaks ofradioactivity containing the steroid carriers were com-bined, resulting in a pool of polar (K = 0.32) material(Pool A) and one of less polar (K = 1.02) material(Pool B). Pool A contained all of the 118-hydroxy-androstenedione and adrenosterone. Pool B containedall of the androstenedione, dehydroepiandrosterone, and17a-hydroxyprogesterone; each pool contained part ofthe 6f3-hydroxyprogesterone, 11,8-hydroxyprogesterone andtestosterone. Pool A was acetylated and chromatographedin the cyclohexane: toluene (50: 50) /propylene glycolsystem. Four zones were located on the chromatogramand eluted. These contained 113-hydroxyandrostenedione,1 1p-hydroxyprogesterone, adrenosterone, and a mixtureof testosterone acetate and 6,8-acetoxyprogesterone. Thismixture of acetates was saponified with 1 N potassium hy-droxide in methanol overnight at room temperature. Thesaponified material was chromatographed in the ligroin:toluene (67: 33)/methanol: water (70: 30) system.Three zones were located and eluted: testosterone, 6f8-hy-droxyprogesterone, and a less polar compound havingthe RF of allopregnane-3,6,20-trione, the maj or productof saponification of 6fl-acetoxyprogesterone with excessalkali (48).

Pool B was subjected to a digitonin separation (49).The ",f-fraction" was chromatographed in the ligroin:toluene (67: 33)/methanol: water (70: 30) system toyield dehydroepiandrosterone. The "a-fraction" wasacetylated and chromatographed in the ligroin/propyleneglycol system. Three zones were located on the chro-matogram and eluted; they were androstenedione, a mix-ture of llf-hydroxyprogesterone and 17a-hydroxypro-

2 K = distribution coefficient.

gesterone; and a mixture of the acetates of testosteroneand 6,8-hydroxyprogesterone. The mixture of ll,-hy-droxyprogesterone and 17a-hydroxyprogesterone was re-solved in the ligroin: toluene (67:33)/methanol: water(70: 30) system. The mixture of acetates of testosteroneand 6,8-hydroxyprogesterone was saponified and chro-matographed in the same way as the corresponding frac-tion from Pool A. Again testosterone, 6,8-hydroxypro-gesterone, and allopregnane-3,6,20-trione were recovered.

At this point, all of the carriers from the two peaks ofradioactivity in the 150-transfer ccd had been recoveredchromatographically pure. In the cases in which thecompound was recovered in both pools, the two portionswere combined. Each carrier isolated was assayed forweight and radioactivity; additional unlabeled carrierwas then added to each, and tests of radiochemical puritywere applied. The details of these procedures are givenfor each compound.

l1,8-Hydroxyandrostenedione: Analysis by ultravioletabsorption at 240 mu showed a recovery of 3.11 mg con-taining 109,000 cpm. An additional 50 mg of carrier wasadded and this was crystallized three times from aqueousethanol yielding crystals having specific activities of1,890, 1,850, and 1,880 cpm per mg (calculated, 2,050cpm per mg) and a final mother liquor residue, specific ac-tivity 1,660 cpm per mg. Crystallization from benzeneyielded crystals having a specific activity of 1,850 cpmper mg, and a mother liquor residue of 1,920 cpm permg. The crystals from benezene were subjected to a 99-transfer ccd in methanol: water (70: 30) /chloroform:carbon tetrachloride (20: 80). The single peak in ra-dioactivity coincided with the peak in material havingultraviolet (240 m/u) absorption (K = 0.73), and therewas no evidence of lack of homogeneity (45). The meanspecific activity in the region of the peak was 2,120 ±218 (standard deviation) cpm per mg. 11,8-Hydroxy-androstenedione was considered radiochemically pure atthis point.

Adrenosterone: The adrenosterone isolated was meas-ured by ultraviolet absorption (238 m/u), and contained2.66 mg and 32,000 cpm. An additional 10 mg of un-labeled adrenosterone was added and a 99-transfer ccdwas carried out using methanol: water (70: 30) /chloro-form: carbon tetrachloride (10: 90). Analysis by ul-traviolet absorption showed a single peak (K = 0.62),but radioactivity extended from tube 0 through the peakin adrenosterone, thereby demonstrating lack of purity.The contents of the tubes containing the adrenosteronewere pooled and crystallized four times from aqueousethanol, yielding crystals having specific activities ateach step of 395, 300, 279, and 289 cpm per mg and aresidue in the last mother liquor, specific activity of308 cpm per mg. Crystallization from ethyl acetate:hexane gave crystals, specific activity 275 cpm per mgand a mother liquor residue of specific activity 264 cpmper mg. The final crystals and residue in the motherliquor were combined and subjected to a 49-transfer ccdin cyclohexane: ethyl acetate (60: 40)/ethanol: water(50: 50). The single peak in radioactivity coincidedwith the single peak in material absorbing at 238 mg

544

VIRILIZING TESTIS TUMOR

(K = 0.71). The material was radiochemically pure asjudged by the specific activity in the region of the peakwhich was constant at 277 ± 24 cpm per mg. Theadrenosterone was considered radiochemically pure.

li,8-Hydroxyprogesterone: The ll(-hydroxyprogeste-rone isolated was analyzed by ultraviolet absorption at242 mA, and consisted of 3.08 mg and 24,000 cpm; 10 mgof carrier was added. A 99-transfer ccd in methanol:water (70: 30)/chloroform: carbon tetrachloride (20: 80)showed a clear dissociation of radioactivity (K = 0.30)from the 11,8-hydroxyprogesterone as measured by ul-traviolet absorption (K = 0.41). Hence, the ll1(-hy-droxyprogesterone was not labeled.

6,8-Hydroxyprogesterone: The portion of the 6,6-hy-droxyprogesterone carrier recovered unchanged was re-chromatographed in ligroin: toluene (67: 33)/methanol:water (70: 30). The compound as isolated from thischromatogram was still not pure, as shown by an atypicalultraviolet absorption spectrum. The absorbance at 237mA indicated a maximum content of 0.13 mg; it con-tained 950 cpm. To this was added 8.6 mg of unlabeledcarrier. A 99-transfer ccd in methanol: water (80: 20)/chloroform: carbon tetrachloride (10: 90) showed a cleardissociation of the radioactivity (K = 1.57) from the car-rier (K = 1.41). The transformation product of 6,8-hy-droxyprogesterone, allopregnane-3,6,20-trione, as recov-ered from the chromatograms, contained only 3,700 cpm.This low level of radioactivity together with the lack ofactivity in the small amount of 6(8-hydroxyprogesteronerecovered made further work on this fraction seemunwarranted.

Androstenedione: The pool of this compound recoveredfrom the incubation mixture contained 2.04 mg as meas-ured by ultraviolet absorption at 240 mAt, and 133,000 cpm.To this was added 60 mg of unlabeled androstenedione.Two crystallizations from aqueous ethanol furnishedcrystals having specific activities of 1,980 and 1,900cpm per mg (calculated 2,140 cpm per mg) and a finalmother liquor, specific activity of 1,960 cpm per mg.Crystallization from acetone: cyclohexane yielded crystalsof specific activity 1,880 cpm per mg and a motherliquor residue of 1,960 cpm per mg. These latter crys-tals were subjected to a 99-transfer ccd in methanol:water (80: 20)/carbon tetrachloride. The single peakin radioactivity coincided with the peak in ultravioletabsorption (K = 0.72); the mean specific activity in theregion of the peak was 2,130 ± 194 cpm per mg. Theandrostenedione was therefore considered radiochemi-cally pure at this point.

17(3-Hydroxyprogesterone: The pool of 17a-hydroxy-progesterone contained 1.61 mg (239 mA) and 22,100cpm; 10 mg of 17a-hydroxyprogesterone carrier wasadded and the whole subjected to a 99-transfer ccd inmethanol: water (70: 30)/chloroform: carbon tetrachlo-ride (10: 90). The peak in radioactivity coincided withthe peak in the 17a-hydroxyprogesterone as measured byultraviolet absorption (K = 0.49). The mean specificactivity around the peak was 1,900 ± 167 cpm per mg.The material from this peak was crystallized twice from

aqueous ethanol to give crystals having specific activitiesof 1,670 and 1,740 cpm per mg (calculated 1,900 cpm permg). Crystallization from benzene: cyclohexane gavecrystals, 1,740 cpm per mg and a mother liquor resi-due, 1,750 cpm per mg. The 17a-hydroxyprogesteronewas considered radiochemically pure at this point.

Dehydroepiandrosterone: The recovered dehydroepi-androsterone was analyzed by the Zimmermann reac-tion (25) and contained 2.11 mg and 3,800 cpm; 10 mg ofunlabeled dehydroepiandrosterone was added and thewhole was subjected to a 99-transfer ccd in methanol:water (80: 20)/carbon tetrachloride. A single peak(Zimmermann) of carrier was found (K = 1.25); therewere two peaks in radioactivity-one (K = 8.00) con-tained 745 cpm; the larger peak corresponded to thepeak in dehydroepiandrosterone (K = 1.25), but con-tained other labeled material since it failed to fit thetheoretical distribution curve. This peak containedabout 2,100 cpm. The contents of the tubes containingthe dehydroepiandrosterone were combined and crystal-lized five times from aqueous ethanol giving crystalscontaining 87, 102, 86, 84, and 90 cpm per mg and a finalmother liquor residue having 110 cpm per mg. Crystal-lization from acetone: hexane yielded crystals containing85 cpm per mg and a mother liquor residue of 75 cpm permg. The last crystals were subjected to a 24-transferccd in ethyl acetate: cyclohexane (30: 70)/ethanol: water(55: 45). The peak in radioactivity coincided with thepeak in dehydroepiandrosterone (Zimmermann reaction,K = 1.04). Constant specific activity, 80 ± 12 cpm permg in the region of the peak, indicated radiochemicalpurity. The dehydroepiandrosterone was then consid-ered radiochemically pure.

Testosterone: The testosterone as isolated from theincubation mixture was impure as indicated by its ultra-violet absorption spectrum. It was, therefore, rechro-matographed in ligroin: toluene (67: 33)/methanol: wa-ter (70: 30). It then had a satisfactory spectrum indi-cating a recovery of 1.12 mg and contained 4,800 cpm;10 mg of carrier testosterone was added and the wholesubjected to a 99-transfer ccd in methanol: water (80:20)/chloroform: carbon tetrachloride (10: 90). A singlepeak in material absorbing at 240 mu coincided with thesingle peak in radioactivity (K = 1.19) ; however, theexperimental points for radioactivity did not agree wellwith the theoretical curve, thus indicating the presenceof a radioactive impurity. The peak contained 9.73 mgand 3,380 cpm. The contents of the tubes containingthe testosterone were crystallized five times from aqueousethanol. The specific activities of the crystals were 306,312, 255, 196, and 206 cpm per mg, and that of the resi-due in the final mother liquor, 276 cpm per mg. Crystal-lization from acetone: hexane yielded crystals contain-ing 223 cpm per mg and a residue in the mother liquorof 210 cpm per mg. In view of these results and therelatively high level of activity associated with theclosely related androstenedione, the testosterone was con-sidered labeled.

545

SAVARDET AL.

DISCUSSION

Methods for radiochemical purity

In the demonstration of radiochemical purityof the isolated steroid carriers, it has been our

experience that radioactive contamination is rela-tively unlikely when the radioactive starting ma-

terial is a steroid closely related to the products;thus, in Experiments A and B, achievement of ra-

diochemical purity presented no serious difficul-ties. This was not true when the radioactivestarting material was a substance far removed(in the biosynthetic sense) from the products un-

der study, as in Experiments C and D in whichcholesterol-C'4 and acetate-C'4 were incubated.In one, only a small percentage of the radioactivitywas ultimately associated with the steroid car-

riers, while in the other (Experiment C), demon-stration of radiochemical purity was not achieved;the purification problem in these latter cases ap-

pears to have been compounded by the large va-

riety and unknown nature of the radioactive im-purities, particularly in the cholesterol-C14 study.

We have found that after separation and iso-lation of the carrier steroids in chemically pure

state, several purification measures may be usedto demonstrate radiochemical purity, the maincriterion being constancy of specific activity of thesteroid at each step through a series of physicaland chemical manipulations. The greater thenumber of such manipulations without significantchange in specific activity, the greater the likeli-hood that the compound is radiochemically pure.

Conversely, decreases in specific activity throughthese manipulations signify contamination of thecarrier by some trace impurity.

Among the various manipulations currentlyemployed for purification, countercurrent distri-butions have proven particularly useful, sincenonhomogeneity can be readily detected when theradioactive compound and the steroid carrierhave only slightly different partition coefficientsin the system employed (45). Formation of suit-able derivatives is also of value, since two com-

pounds (steroid carrier and radioactive com-

pound) which have very similar physical proper-

ties may form derivatives having less similarproperties. In this regard, the steroid carrierderivative selected should be one which is basedupon the most characteristic chemical feature

of the carrier and which effects the maximum al-teration in its physical properties. Crystalliza-tion (of steroid carriers and derivatives), how-ever, has been the manipulation most widely usedas the criterion for radiochemical purity, since atrace contaminant is likely to be completely solu-ble in the solvent used to crystallize the largeramount of carrier. Unfortunately, with steroids,this ideal situation is often complicated by adsorp-tion of impurities on crystals and even mixedcrystal formation (this phenomenon may explainthe tenacity of the contaminants encountered inExperiment C). Purification to constant specificactivity in a particular solvent may be consideredto have been achieved when specific activity re-mains constant through several recrystallizations.When only small quantities of carrier are avail-able, however, the above is not always possible;in such cases we have found it necessary to carryout specific activity measurements on both crys-tals and mother liquors from each crystallizationin order to determine if constant specific activityhas been achieved in that particular solvent.

These criteria served to provide the values givenin Table IV for Experiments A, B and D; thesame criteria applied to the steroids in the cho-lesterol-C'4 study (Experiment C) indicated thatthe associated radioactivity was largely due to un-known impurities. To be noted is the value ofthe isolation of several carriers devoid of radio-activity; such negative results lend considerableconfidence to the positive values obtained in thesame study.

Biochemical considerations

The elevated level of the urinary 17-ketosteroidsseen in the patient preoperatively is consistentwith the classical clinical features of interstitialcell tumors of the testis in childhood (1-21).Analysis of the individual 17-ketosteroids revealedandrosterone and etiocholanolone (Table II) to bepresent in the largest amounts. Metabolic stud-ies in humans (50) show that the principal pre-cursors of these two urinary C1,02 steroids areandrostenedione, testosterone and dehydroepian-drosterone, and that the two metabolites are usu-ally excreted in approximately equal amounts, asthey are in our patient (Table II). The elevatedlevels of the 11-oxygenated 17-ketosteroids in the

546

VIRILIZING TESTIS TUMOR

urine are most impressive since the occurrence ofan oxygen substituent at carbon-11 of a steroidof mammalian origin has long been consideredindicative of that steroid's adrenal cortical origin.The high concentration of 11-keto- and 11,8-hy-droxyandrosterone (1.8 mg per 24 hours) indi-cates that at least one of the likely precursors(511) i,8-hydroxyandrostenedione, adrenoster-one or 11,8-hydroxytestosterone-was formed bythe tumor; these three latter compounds havebeen isolated from adrenal tissue either as con-stituents or as metabolic products (52-55). Theurinary 17-ketosteroid pattern thus suggests thatthe principal C,90, androgen elaborated was tes-tosterone or androstenedione (or perhaps dehy-droepiandrosterone), and that one of the moreweakly androgenic C,90, compounds-11,8-hy-droxyandrostenedione, 1 1,8-hydroxytestosterone,or adrenosterone (56, 57)-was formed in les-ser amounts. The preoperative levels of urinarypregnanetriol suggest also the likelihood of theformation by the tumor of 17a-hydroxyprogester-one (50).

The results of the acetate incubation study,given in Table IV, indicate that androstenedioneand testosterone are indeed the major steroidselaborated by the tissue under the conditions ofincubation and, no doubt, account for the virilizingproperties of this tumor. The accumulation ofC14 in both these compounds amounts to 0.07 percent of the added acetate. 1i-,8-Hydroxyandro-stenedione and its companion adrenosterone ( 11/-hydroxytestosterone was not sought) accumulated0.04 per cent. Thus, in the acetate experiment,the ratio of C,9O, to C,,O3 steroids is in excellentagreement with the quantitative relationship ofthese two classes of steroids found in the urine.

In the study with acetate-C14, little or no radio-activity was associated with progesterone while asignificant amount accumulated in 17a-hydroxy-progesterone. Perfused stallion testes have beenshown to incorporate acetate-C14 into both pro-gesterone and 17a-hydroxyprogesterone (58),and a human embryonal testicular tumor, undertissue-slice conditions, elaborated progesterone(31) in addition to androstenedione and testoster-one. Our incubation studies suggest that whilethese two C21-compounds appear to act in thistumor as intermediates (see below) in the biosyn-thetic pathway to the androgens (Experiment B),

both may well have been secreted by this tumorin vivo, their production being reflected by theelevated urinary titers of pregnanetriol and neu-tral acylable lipids (Table I), although the lattermay be made up of C,9-diol compounds. Therelatively minor amount of dehydroepiandroster-one obtained in the incubation with acetate-C14(Table IV), suggests that this substance wastransformed rapidly by the tissue and thereforeaccumulated little or no radioactivity in the in vivostudy.

The incubation of the tumor tissue with cho-lesterol-C14 was undertaken with the notion thatan efficient utilization and transformation of la-beled cholesterol into steroid hormone productswould occur. This premise has proved to be false.This does not necessarily mean that cholesterolis not an intermediate in the synthesis of steroidhormones in this tumor. Although the tissue wascapable of active steroid synthesis as seen in theother experiments, no significant amount of cho-lesterol-C'4 was transformed into recognizableC,9 or C,, steroid products. Dilution of the radio-active cholesterol by the tissue and medium choles-terol, or its failure to penetrate cell membranescould explain the negative results. However, thecholesterol-C14 did undergo some chemical trans-formations to more polar material, as indicated bythe large amounts of radioactivity found in thepolar fractions accompanying the steroid carriers.This unidentified material, which may have beena biochemical transformation product or an arti-fact, accompanied the carrier steroids throughseveral purifications., including the formation ofderivatives.

The results of the incubation of testosterone-C14(Experiment A) are clear-cut. The major trans-formation product of testosterone was androstene-dione, known to be a normal secretory productof human (59) and dog testicular tissue (60).The presence in this tumor of the enzyme, 11,8-hydroxylase (61), was demonstrated by the iso-lation of radioactive 11,8-hydroxyandrostenedioneand 11,8-hydroxytestosterone (54). The signifi-cance of this reaction in testicular tumor tissue isdiscussed below. The formation of radioactiveadrenosterone in minor amounts points to thepresence of the widely occurring 11-hydroxyde-hydrogenase (62) in this tumor tissue or possiblyin the plasma used in the incubating medium. It

547

SAVARDET AL.

should be noted that no radioactive productsother than the four described were found in thisexperiment and that the 11p,-hydroxylase activityof the tissue is demonstrated by the fact that 7.6per cent of the added testosterone was convertedto 11-oxygenated steroids.

The isolation of radioactive 17a-hydroxyproges-terone, testosterone and androstenedione from theprogesterone-C'4 incubation experiment (TableIV, Experiment B) serves to indicate the pathwayfor the formation of androgens by this tumor tis-stue. Slaunwhite and Samuels (63) described thetransformation of progesterone into androstenedi-one and testosterone by rat testicular tissue andpostulated that 17a-hydroxyprogesterone is an in-termediate. These transformations have been con-firmed by the present study. by Solomon, VandeWiele and Lieberman (64) in bovine ovarian tis-sue, and by Lynn and Brown (65) and one of theauthors in normal rat testis tissue, which has alsobeen shown to transform 1 7a-hydroxyprogester-one-C14 into radioactive androstenedione and tes-tosterone (66). The presence of 11,8-hydroxy-androstenedione among the transformation prod-ucts of progesterone is consistent with the resultsof the testosterone incubation. The absence of11,8-hydroxyprogesterone and 11-ketoprogester-one from the products derived from progesteroneis interesting in view of the isolation of 11-oxy-genated C,9 compounds in this and other incuba-tions. This may suggest that the tumor 11,8-hy-droxylase exhibits a substrate preference (61).A final point of interest is the apparent failure ofthis tumor tissue to convert progesterone to eitherof the two substances, 20a- or 20,8-hydroxy-4-pregnen-3-one. They have been shown to be pro-duced from progesterone by human (66) and bo-vine (67) ovarian tissue in vitro, and by the evis-cerated rat in vivo (68), and normally accompanyprogesterone in the blood of pregnant women andin human follicles, corpora lutea and possibly inplacental blood (35).

Clinical considerations

There are 22 previously reported cases of so-called interstitial cell tumors of the testis occurringin childhood and producing isosexual precocity(1-21). In the present case the demonstrationin the tumor of certain biochemical activities

(1 1,8-hydroxylase activity, formation of 1 1/3-hy-droxyandrostenedione and related compounds),which more closely resemble those of adrenal cor-tical than of testicular tissue, requires reappraisalof the status of the interstitial cell tumor of thetestis.

As with the previous 22 cases, our patient dis-played clinical abnormalities attributable to theoverproduction of androgens: precocious staturaland isosexual growth, advanced skeletal matura-tion, excessive muscular development, facial acneand hirsutism. Precocity began between the agesof three and six years in almost all reported cases,including the present one, and the tumor was uni-lateral and benign in all but one case. Correlatedwith this, in our case and nine previous cases sostudied, urinary 17-ketosteroids were elevated forthe age, ranging from 2.9 to 64 mg per 24 hours.Gynecomastia was observed in three previous casesbut not in the present one, where urinary estrogenwas in the normal range and none was formed bythe tumor tissue in vitro (Experiment D). Aclue to the gynecomastia in the three cases maybe in the recent demonstration of the conversionof androgen to estrogen by human endocrine (69)and nonendocrine tissues (70) and the observationof gynecomastia following androgen administra-tion (71).

The origins of these tumors are not clear.First, there are no precise morphologic criteriafor differentiating adrenal cortical cells from in-terstitial cells of Leydig (11, 72), and secondly,adrenal cortical rests do occur in the testes. Thisis not surprising since the adrenal cortex arisesembryologically in the dorsal mesenteric root inclose proximity to the urogenital ridge containingthe gonads (73). In this way it is conceivablethat these tumors can resemble adrenal cortical tis-sue both morphologically and biochemically. Onthe other hand, massive replacement of testiculartissue by aberrant adrenal cortical cells in a patientwith congenital virilizing adrenal cortical hyper-plasia has been reported by Wilkins and co-work-ers (74). Subsequently, this has been confirmed,and in 1954, Hedinger reported a similar patientin whomcortisone administration induced markedregression of testicular size (75). Staubitz andco-workers in 1953 reported a patient with bi-lateral "interstitial cell tumors" with precocity be-ginning at three years of age, but in whomurinary

548

VIRILIZING TESTIS TUMOR

17-ketosteroids were 73.9 mg per 24 hours beforebilateral orchiectomy and still elevated at 69.6mg per 24 hours three months postoperatively(76), suggesting that this was an instance ofadrenogenitalism with bilateral adrenal corticalrests in the testes; for this reason it has not beenincluded in our list of previously reported casesof interstitial cell tumors of the testis. Becauseof insufficient data for diagnosis, another case withbilateral testicular tumors reported by Garveyand Daniel has been excluded from our series(77). We have accepted the case reported byRezek and Hardin (19) with bilateral testiculartumors, primarily on the basis of a decrease in17-ketosteroids from 91 (60 per cent "f3-ketoster-oids") to 2.1 (no "/3-ketosteroids") mg per 24hours 16 months postoperatively. However, thepreoperative level of 3,8-hydroxy-17-ketosteroidsin the urine is suggestive evidence for tumor for-mation from adrenal-cortical cell rests. Sand-blom's patient has been included perhaps incor-rectly in our series, since at four years of age andone year postoperatively the urinary 17-keto-steroids varied between 10 and 20 mgper 24 hoursas compared to 64 mg per 24 hours before sur-gery (8). There thus appears, probably forwant of completeness in their work-up, an ob-scure picture of the "testicular" origins of thesetumors; in some instances they possess the dis-tinct character of adrenal cortical rests, while inthose included in our series, they show no suchmorphological properties.

There is one histological finding that has longbeen considered specific for Leydig cells, namelythe presence of crystalloids of Reinke (78).These also occur in the ovarian hilus cells buthave never been described in adrenal cortical tis-sue. Recently Sternberg, Segaloff and Gaskillhave reaffirmed that these crystalloids are indeed"specific identification tags. distinguishing hilusand Leydig cells from morphologically similarcells such as lutein and adrenal cortical cells" (79).Although absent in each of the previously reportedcases of prepubertal interstitial cell tumors, crys-talloids of Reinke were clearly demonstrated inthe tumor of our patient. Thus, on the one handthis tumor had this morphologically identifyingstigma of testicular origin, while on the otherhand, its biochemical activity suggested certainadrenal cortical properties. Here the enigma

must rest, but we are forced to re-open the ques-tion as to tissue specificity of both the crystalloidsof Reinke and the capacity to elaborate 1 1f-hy-droxysteroids. Perhaps it should be re-empha-sized that while 11,8-hydroxylase activity hasnever been previously observed in normal testicu-lar tissue, no distinct search has been made untilvery recently; it was not found in normal stalliontestis tissue (58) and could not be demonstratedin an arrhenoblastoma (66). The apparent ab-sence of 11,8-hydroxylase from normal testicularand ovarian tissues may however reflect a failureof analytical techniques to detect extremely lowconcentrations of enzymatic activity. The processof tumor formation could bring about an increasein concentration of an enzyme normally consideredabsent from the tissue, and seemingly could causethe tumor to "acquire" an enzyme not usually as-sociated with the parent tissue. The recent re-port of the enzyme 21-hydroxylase (likewise gen-erally associated with adrenal cortical tissue) inan induced tumor of testicular interstitial cell ori-gin in the mouse (80) suggests such a possibility.

Our patient's postoperative course during twoyears of observation has been of considerable in-terest. His excessive libido gradually subsidedand social adjustment improved, but there was noregression of the physical manifestations of pre-cocity; indeed, he continued to mature. Growthcontinued at approximately the same rate as pre-operatively, but the bone age advanced only one totwo years. The remaining left testis, small pre-operatively, increased to late-adolescent size withnormal consistency. Urinary gonadotropins,which were absent preoperatively, reached thenormal adult range of 7 rat uterine units. Theurinary 17-ketosteroid levels fell precipitouslyafter operation to approximately 1.0 mg per 24hours, but during the last year of observation havevaried between 3.0 and 7.4 mg per 24 hours. Theabove data suggest that postoperatively the pa-tient entered true puberty with its attendant go-nadotropin production, growth spurt and matura-tion of the remaining testis. This simulates thesexual maturation which has been observed dur-ing cortisone therapy of patients with the adreno-gentital syndrome in whom somatic maturation,reflected by a bone age of 11 to 12 years, had takenplace prior to treatment (81). It would appearthat, irrespective of chronological age, some re-

549

550 SAVARI

lationship exists between the level of somaticand skeletal development and the initiation of thehypothalamic-pituitary discharge which leads toelaboration of gonadotropin. The normal adoles-cent values of urinary gonadotropins, 17-ketoster-oids and pregnanetriol postoperatively in ourpatient are evidence against either developmentof a new tumor in the remaining testis or ofadrenal hyperplasia as a basis for the subsequentcontinued somatic and sexual development.

Inadequate data are available on the postopera-tive course of the 22 previously reported cases.In general, the literature reports little or no re-gression of the precocious physical characteristics.In two patients with preoperative heights of 152cm there was no further growth postoperatively,probably reflecting closure of the epiphyses priorto surgical excision of the tumors. However,in five patients whose preoperative height was lessthan 127 cm and whose bone age was 12 years orless, a deceleration of growth rate and genitaldevelopment was specifically noted. One otherpatient, with a preoperative height of 120.7 cm

[CH3 COO] H

CH3

co

-OH

HO H

D ET AL.

and a bone age of 10 years, grew 20 cm duringthe first 17 months after operation, and the re-maining testis appeared to be enlarging. Whilethe latter patient's course is suggestively similarto that of our own case, no testicular biopsy norgonadotropin assays were reported. In three pa-tients with gynecomastia, breast enlargement dis-appeared in two and persisted in one.

This protracted study has served to demon-strate that the virilizing properties of this tumormay be ascribed to two principal androgenic ster-oids, testosterone and 4-androstenedione, whoseformation in the tumor is consistent with themechanism of androgen biosynthesis recentlypostulated for normal testicular tissue (63) (Fig-ure 7). The demonstration of the presence of theenzyme 11,8-hydroxylase points to certain hithertounsuspected endocrine and embryological con-siderations of this tumor. The implications ofthese observations as to the nature of the hor-mone-secreting cells of this tissue must await theextension of these studies to similar virilizing tu-mors of the testis.

K)al Ac,Pr,Te 3t Te

FIG. 7. PROBABLE PATHWAYOF STEROID BIOSYNTHESIS IN THE TESTICULAR TUMOR. Broken arrows indicatepresumed transformations; Te = produced from testosterone-4-C1' in Experiment A; Pr = produced from pro-gesterone-4-C14 in Experiment B; Ac = produced from acetate-1-C14 in Experiment D. I = cholesterol; II =progesterone; III = 17ca-hydroxyprogesterone; IV = dehydroepiandrosterone; V = 4-androstene-3,17-dione; VI =

testosterone; VII = 11,8-hydroxy-4-androstene-3,17-dione; VIII = lljS,17,8-dihydroxy-4-androstene-3-one (11,8-hy-droxytestosterone) IX = adrenosteroiie.

VIRILIZING TESTIS TUMOR

SUMMARY

An interstitial cell tumor in the testis was re-

moved from a highly virilized boy. The preopera-

tive levels of urinary 11,8-hydroxyandrosterone,11,8-hydroxyetiocholanolone, 11-ketoandrosterone,androsterone, etiocholanolone and pregnanetriolwere abnormally high and fell promptly followingsurgery. After incubation of tumor slices withacetate-1 -C14, the following radioactive steroidswere found by carrier dilution: testosterone,4-androstene-3,17-dione, (in small amount) de-hydroepiandrosterone, 17a-hydroxyprogesterone,adrenosterone and 1 1,8-hydroxyandrostenedione;estrone, estradiol-17P, estriol, progesterone and1 1,/- and 6,8-hydroxyprogesterone were isolateddevoid of radioactivity. Incubation with proges-

terone-4-C14 yielded radioactive 17a-hydroxypro-gesterone, androstenedione, testosterone and 11,8-hydroxyandrostenedione. Incubation with testos-terone-4-C14 yielded radioactive androstenedione,1 1,8-hydroxytestosterone, 1 1fB-hydroxyandrostene-dione and adrenosterone. Cholesterol-4-C14 failedto give rise to significant amounts of radioactivesteroid products.

A critique of methods for the study of radio-chemical purity of steroids is presented.

The morphological, biochemical and clinical as-

pects of this twenty-third reported case of viriliz-ing interstitial cell tumor of the testis are discussed.

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