CLINICAL, MORPHOLOGICAL AND BIOCHEMICAL STUDIES OF A VIRILIZING TUMOR IN THE TESTIS

Kenneth Savard, … , Edwin C. Hamblen, Frank L. Engel

J Clin Invest. 1960;39(3):534-553. https://doi.org/10.1172/JCI104067.

Research Article

Find the latest version: https://jci.me/104067/pdf CLINICAL, MORPHOLOGICAL AND BIOCHEMICAL STUDIES OF A VIRILIZING TUMOR IN THE TESTIS * By KENNETH SAVARD,t RALPH I. DORFMAN, BILLY BAGGETT,t LINDA L. FIELDING, LEWIS L. ENGEL,§ HARRY T. McPHERSON, LEONARD M. LISTER,II DAVID S. JOHNSONA EDWIN C. HAMBLEN AND FRANK L. ENGEL (IProm the Worcester Foundation for Experimental Biology, Shrewsbury, Mass.; The John Collins 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 evidence such tumors cause elevated levels of urinary 17- indicating that virilizing testicular tumors exert ketosteroids. The morphology of these tumors their effects by producing androgenic steroid hor- leads one to predict that they are producing tes- mones, few attempts have been made to determine tosterone, since normal interstitial cells seem to the exact nature of the compounds produced. One have this function (22, 23). A few studies of the type of tumor which exerts particularly marked qualitative nature of the urinary steroids in pa- virilizing effects when found in prepubertal boys tients with this type of tumor seem to confirm such is that classified as an interstitial cell tumor of the a conclusion (13, 24). Accordingly, when the testes (1-21). It has been well established that opportunity arose to study a patient bearing a testicular -tumor thought to be of this type, it * This work was supported by Contracts no. DA-49- 007-MD-184, Army Medical Research and Development seemed worthwhile to attempt to establish the Board, Office of the Surgeon General, Department of the exact nature of the steroids produced. The study Army; no. AT(30-1)-918, United States Atomic Energy presented here was therefore undertaken. Commission and a grant from the Charles A. King Trust. This paper presents a description of the patient, This is 'Publication no. 990 of the Cancer Commission of Harvard University and was supported by Grants no. histological observations on the tumor, the re- CG-1393-C7 and C-2833-Cl, United States Public Health sults of urinary steroid assays and the results of Service; P-95 (T) American Cancer Society Inc.; and a experiments in which slices of the tumor were in- grant from the Jane Coffin Childs Memorial Fund for cubated with C14-labeled steroid precursors. Medical Research. Portions of the study on this case have been reported in abstract form in: Fed. Proc. 1956, 15, 346; J clin. Endocr. 1956, 16, 970 and 1629; Abstracts EXPERIMENTAL AND RESULTS of the Twentieth International Physiological Congress, I. Clinical data 1956, Brussels; Expos. ann. Biochim. med. 1957, 12, 219; D. W. no. Southern Society for Clinical Research, January 25, 1958, (Duke D-99663), a 5-year-and-7-month old New white male, was first seen at Duke Hospital on November Orleans; Hormone Production in Endocrine Tu- 1, 1954, of mors, Ciba Found. Coll. Endocr. 1958, 12, 62. because precocious growth and development, increasing libido, and personality changes. His birth and Investigator, Howard Hughes Medical t Institute; early development had been normal, but at 3 years of age present address: Departments of Biochemistry and Medi- progressive statural growth spurt and enlargement of cine, University of Miami School of Medicine, Miami, the penis Fla. were noted. At 4 years of age, genital hair appeared, followed shortly by axillary, body and facial t: Present address: Department of Pharmacology, Uni- hair, mild facial acne and progressive deepening of the versity of North Carolina School of Medicine, Chapel voice. During one year a gradual, painless enlargement Hill, N. C. of the right testis had been noted, accompanied by in- § Permanent Faculty Fellow of the American Cancer creasingly frequent penile erections and masturbation, Society. apparently without ejaculation. The child became ag- If Present address: University of Maryland School of gressive, somewhat withdrawn and uninterested in previ- Medicine, Baltimore, Md. ous playmates and activities. fI Present address: Snodgrass Laboratory of Pathol- Physical examination revealed normal vital signs with ogy and Bacteriology, St. Louis, Mo. a blood pressure of 112/60 mm Hg. His height was 135 534 VIRILIZING TESTIS TUMOR 535

TABLE II Urinary levels of individual 17-ketosteroids

Postoperative Preoperative 2 days mg/24 hrs mg/24 hrs Androsterone 2.4 0 Etiocholanolone 2.0 0.1 Androstane-3, 17-dione and 0.1 0.1 etiocholane-3,17-dione Dehydroepiandrosterone 1.4 0 1 1,B-Hydroxyetiocholanolone 0.2 0 [and A5(M1)-etiocholenolone] 11-Ketoetiocholanolone 0.2 0 1 13-Hydroxyandrosterone 1.6 0 [and z90(")-androstenolone] 11 -Ketoandrosterone 0.2 0.1

and pigmented scrotum. The cord structures on that side were normal to palpation, and there was no local or distant lymphadenopathy. The left testis was normal in size, shape and consistency (2 X 0.7 X 0.7 cm). The prostate was enlarged and comparable in size to that FIG. 1. THE PATIENT PRIOR TO OPERATION; AGE, FIVE of an adolescent. No other physical abnormalities were YEARS AND SEVEN MONTHS. noted. On psychological consultation the child was thought to cm and his weight 33.6 kg. Anthropometric measure- be advanced both intellectually and in his social adjust- ments corresponded to those of an average boy approxi- ments, corresponding approximately to the age of 8 years. mately 11 years of age. The muscular development was The intelligence quotient was 135 and the social quotient rather Herculean (Figure 1). The dental age was esti- 146. mated to be 6.5 years. There was slight facial acne, The hemogram and urinalysis were normal, and the moderate axillary hair, and abundant heavily pigmented serological test for syphilis was negative. Blood chemi- and coarse pubic hair. Hirsutism was noted over the ex- cal analyses (nonprotein nitrogen, total proteins and al- tremities, back and upper lip. The penis, in a flaccid bumin/globulin ratio, fasting blood sugar, serum cho- state, measured 11.5 cm in circumference and 12.5 cm in lesterol and electrolytes) were normal. The basal me- length. The right testis was enlarged (4.5 X 3.7 X 3.2 tabolic rate was + 3 per cent. X-rays of the chest and cm). It was uniformly abnormally firm, slightly irregu- skull and intravenous pyelograms revealed no abnormali- 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 hrs 17-Ketosteroids (25)* 13.6 1.4 1.6 Neutral acylable lipids (26)* 138 50 Fluorogenic phenols (27)* 78 80 13 Pregnanetriolt 4.1 0.4 Corticosteroids (28)1 1.4 1.1 1.1 Gonadotropins§ <1 7 * Estimations carried out on extracts of urine boiled with 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 FIG. 2. THE SECTIONED SURFACE OF THE TUMOR, TESTIS with mammalian 69-glucuronidase (Ketodase, Warner- Chilcott Laboratories) at pH 5.1. AND CORD STRUCTURES ARE ILLUSTRATED. The identifica- § Rat uterine units per 24 hours. tion tag is 2 cm in length. 536 SAVARD ET 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 prominent nucleoli. Occasional large hyperchromatic nuclei were present, but generally there was little variation and mi- ~~~toses*-~~~~~-.i were rare. With Helly's fixation the cytoplasm was brightly eosinophilic, whereas with 10 per cent .-formalin fixation the cytoplasm appeared light pink with prominent unstained vacuoles. ~~~~~The-~~~~~~~. most striking cytological feature was the presence of a variety of cytoplasmic inclusions. With hematoxylin _ .~~~~~~~4 ~~~~~~and eosin and Helly's fixation most of the cells showed very 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 Schiff reagent and was particularly well demonstrated by phos- photungsticr 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 9*i , 8 < , measuring :..;2*-b from 1.7 to 5.1 in diameter. They were usually clus-

FIG. 3. THE MARGIN OF THE TUMOR IS SHOWN AT ITS L:- JUNCTION WITH THE SURROUNDING NORMAL TESTICULAR ji TUBULES. Note the fibrous septa dividing the tumor into lobules. (Hematoxylin and eosin, X205.)

ties. The bone age was greater than 10 but less than 14 years. Urinary steroid and gonadotropin analyses are reported below (Tables I and II). On November 16, 1954, a right radical orchiectomy and 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 section a 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 distinct green cast. The surrounding testicular tissue wasM compressed. Microscopic examination. There was an incomplete, thin connective tissue capsule. Where the capsule was incomplete, scattered small clumps of tumor cells ex- i tended for a short distance into the surrounding tissue. The tumor was made up of solid sheets of cells broken up by occasional thin, vascular, fibrous tissue septa (Fig- ure 3). A delicate reticulum supporting framework further separated the tumor into clumps of 2 to 12 cells. The tumor cells were irregular, polyhedral in shape, 14 FIG. 4. THIS MICROPHOTOGRAPH ILLUSTRATES THE to 35 A in diameter (average, 22 AL), and occasionally LIPOCHROME GRANULES CLUSTERED ABOUT THE NUCLEUS had indistinct cell boundaries, although in most areas OF VIRTUALLY EVERY CELL. (Periodic acid-Schiff, X1,021.)I VIRILIZING TESTIS TUMOR 537

tered about the periphery of the cytoplasm and occasion- j ally filled the entire cell. They were highly refractile and were surrounded by a clear halo. Some of these granules had an internal structure in which the outer wall was stained dark blue with phosphotungstic acid hematoxylin. Just internal to this was a narrow, clear, unstained area. A solid mass of dark blue material made up the remainder of the inclusion. Others were solid and homogeneous. These were not well seen int formalin-fixed tissue by any technique used but were readily apparent in Helly-fixed tissue, particularly with Masson trichrome and phosphotungstic acid hematoxylin stains 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 striking tinctorial reaction was their oxyphilia rendering them red with the Masson trichrome technique. Some of these were seen crossing a cell boundary and extending into an 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 TWO ELONGATED CRYSTALLOIDS OF REINKE. (Mas- son trichrome, A!iHeyDo; t ^ --R ffi i X1,021.)

diagnosis was inter- 4 4:t s. ;Xs}J! 9 iS Elf 2 >'9 4 spermatogenesis. The pathological *i stitial cell tumor of the right testis. there .'f Follow-up. Since discharge from the hospital has been no regression in any of the somatic features previously depicted. In March and June, 1955, no. es- differences were noted. While the left testis was X ...... sentialnormal 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 testis re- ^d:;* A d it was noted that the remaining left had grown markably in size in the preceding year, now measuring *^.**.A d d X 5~5.5 X 3 cm. The testicular size, consistency and con- Wok to *;9-Rx-<_^htour, and the prostate gland, were now comparable that of a normal late-adolescent male. He was last seen s1t Aft k^. 4 * ings similar to those noted six months earlier except for a further gain of 6 cm in height. The bone age was esti- mated as 12 years, an advance of almost two years since 7 FIG. 5. LARGE CYTOPLASMIC GRANULES CAN BE SEEN operation. Daily urinary gonadotropin excretion was DISTRIBUTED ABOUT THE PERIPHERY OF THE TUMOR CELLS. rat uterine units, within the range for a normal adult. Nuclear detail is also well shown. (Phosphotungstic The postoperative urinary steroid and gonadotropin lev- els are recorded in Tables I and II. aClUnif 1vAPmntnvv1nCTINILVILY1111, x^JL)V"JL.,/021-) 538 SAVARD ET AL. II. Urinary steroids graphed at the same time. This method has been Estimations of steroid found to be accurate within 15 per cent (30). concentrations were car- The ried out on 24-hour urine specimens collected be- daily excretion of individual 17-ketosteroids fore and at various periods after the removal of estimated by this method is presented in Table II, in the tumor. The values obtained are shown in which the artifacts produced by the hydrolysis with are Table I. These values as well as those of gonado- acid included with their known pre- tropins are in agreement with values previously cursors. observed in this syndrome. The excretion of cor- Elevated excretion of both C,9O2 and C,903 ticosteroids measured by the Silber and Porter compounds, androsterone and 11,8-hydroxyandro- sterone in is to be method (28) was in the normal range for a 5 particular, noted. The excre- tion year old boy and was unchanged after removal of all steroids measured after removal of the of the tumor. Paper chromatograms of the corti- tumor was normal. The level of fluorogenic no costeroid fraction in the toluene/methanol: water phenols gave indication of elevated estrogen excretion (75: 25) system (29) showed the presence of and was unchanged by removal of the tumor; the reduction of this tetrahydrocortisone and tetrahydrocortisol as the level 1.5 years after the tumor preponderant corticosteroids before and after the was removed is unexplained. operation.' No unusual reducing compounds III. In vitro studies were detected. The daily excretion of 17-ketosteroids, neutral The incubation of slices of this tumor was un- acylable lipids (hydroxysteroids) and pregnane- dertaken as a means of direct biochemical assess- triol was elevated before removal of the tumor. A ment of its steroid production. This technique further sample of urine was boiled with 15 vol- utilizes small amounts of tissue and C14-labeled umes per cent of hydrochloric acid, and the ether steroid precursors such as acetate-i-C14, and is extracts were chromatographed in the Bush lig- one which is of proven value in this type of study roin/methanol: water (96: 4), and ligroin: tolu- (31). In order to facilitate extraction of the trace ene (67: 33)/methanol-water (70: 30) systems. amounts of radioactive steroids from the incuba- The chromatograms were sprayed with Zimmer- tion medium and tissue, it is necessary to add mann reagent and the amount of each compound known amounts of crystalline nonradioactive ster- estimated by comparison of size and intensity of oids. In the case of each substance, the total the spot with the sizes and intensities resulting steroid (radioactive and carrier) may then be iso- from known amounts of the compound chromato- lated and purified and its radioactivity per milli- gram (specific activity) determined after suitable 1 The chemical names for the steroid compounds used purification. By calculation back to the original in this paper are as follows: 3a-hydroxyandrostan-17-one (androsterone); 3a,11j3-dihydroxyandrostan-17-one (1158- weight of carrier, the amount of radioactivity as- hydroxyandrosterone) ; 11 6-hydroxy-4-androstene-3,17- sociated with each carrier steroid may be ascer- dione (ll1,-hydroxyandrostenedione); 4-androstene-3,11,- tained. This in turn reflects the relative amount 17-trione (adrenosterone); 4-androstene-3,17-dione (an- of each steroid formed during the experiment. drostenedione); 3a-hydroxyetiocholane-17-one (etiocho- Other methods for the microdetermination of the lanolone) ; 17,3-hydroxy-4-androsten-3-one (testosterone); 11p,17,f-dihydroxy-4-androsten-3-one (118-hydroxytestos- absolute amounts of endogenous steroid hormones terone); 17,j-hydroxy-4-androstene-3,11-dione (11-keto- present in endocrine glands have recently been de- testosterone); 3,8-hydroxy-5-androsten-17-one (dehydro- veloped (32-36); these however require exacting epiandrosterone) ; 3a,17a,21-trihydroxypregnane-11,20-di- manipulations for certainty in the identification one (tetrahydrocortisone) ; 3a,11,8,17a,21-tetrahydroxy- pregnan-20-one (tetrahydrocortisol); pregnane-3a,17a,- and quantitative measurement of the minute 20a-triol (pregnanetriol); 4-pregnene-3,20-dione (proges- amounts of the compounds found. The incuba- terone); 17a-hydroxy-4-pregnene-3,20-dione (17a-hy- tion of specific C'4-labeled steroids with tissues droxyprogesterone) ; 11,8 -hydroxy-4-pregnene-3,20-dione and the isolation of radioactive products, also (llfi-hydroxyprogesterone) ; 6f-hydroxy-4-pregnene-3,20- by means of carrier dilution, provide an assess- dione (6,6-hydroxyprogesterone); 6j3-acetoxy-4-pregnene- 3,20-dione (6,-acetoxyprogesterone); 3p8-hydroxy-5-preg- ment of the tissue's capacity to effect certain spe- nen-20-one (5-pregnenolone). cific transformations. VIRILIZING TESTIS TUMOR 539

The chemical methods employed consisted first When required, zones of radioactivity on the chromato- in making a total lipid extract which was separated grams were located by means of contact radioautography. into nonpolar (fatty constituents, and so forth) This consisted in placing the chromatogram (or a rep- resentative portion) in contact with Kodak Medical and polar lipids. The latter fraction containing X-ray film (no screen) for the required exposure time the carrier steroids was separated into acidic, phe- (42), and developing the film in the usual manner. nolic and neutral polar lipids, and on occasion, Measurement of radioactivity. All samples were plated into ketonic and nonketonic fractions. These at infinite thinness in metal dishes and counted in a win- procedures, described below in greater detail, af- dowless gas-flow counter. A minimum of 2,000 counts was accumulated on each. All counts were corrected forded extracts containing the steroids relatively for background. free from extraneous material. These extracts Ultraviolet absorption. All ultraviolet absorption spec- were then subjected to countercurrent distribu- tra and values of absorbance were measured in 95 per tions (ccd) and paper chromatographic separa- cent ethanol. tions in a variety of systems. Each steroid so iso- Formation of derivatives. Acetylations were carried out at room temperature with acetic anhydride and an- lated was free from steroidal and other contami- hydrous pyridine (1: 1); excess reagents were evapora- nants, and was judged chemically pure by melting ted under a stream of air. Oxidations were carried out point and other criteria. The achievement of with chromium trioxide in glacial acetic acid at room radiochemical purity required further procedures, temperature for two hours (43). Formation of oximes which are described below. was carried out in the conventional way. If. Incubation METHODS The tumor was received within a few minutes of ex- 1. General cision and chilled in cold physiological saline. It was Countercurrent distributions. All 4- and 8-transfer sectioned and a representative portion used for the bio- distributions were carried out in separatory funnels. chemical studies; the remainder was taken for pathology. Lower layers were transferred, and equal volumes of the Slices were prepared with a Stadie-Riggs slicer and dis- two phases were used. The 24-transfer distributions tributed among four incubation flasks. These flasks were carried out in a 25-tube, stainless steel Craig ap- contained as their final contents 0.93 g of tumor tissue, paratus and those of greater numbers of transfers were 20 ml of fresh human serum, 0.02 mmole of potassium carried out in the glass apparatus (H. 0. Post Scientific fumarate, 10 mg of glucose, 500 units of pregnant mare Instrument Co.). serum gonadotropin (Equinex-Ayerst), 500 units of Paper chromatography. Chromatograms of the Zaffa- pregnancy urine gonadotropin (APL-Ayerst) and 5 roni type (37) and modifications (38, 39) were run units of porcine pituitary gonadotropin (FSH-Armour). at room temperature on Whatman no. 1 filter paper. In The radioactive substances (the first three shown to be all systems employed, propylene glycol was used as the chromatographically homogeneous), added one to each stationary phase. The following solvents were used as flask, were: A) testosterone-4-C1' (0.92 mg, 3.82 X 10' moving phases: ligroin (38), toluene (37), toluene: cpm); B) progesterone-4-C1' (2 mg, 350,000 cpm); C) cyclohexane (50: 50), and toluene: methyl cyclohexane cholesterol-4-C1' (2.2 mg, 7.5 X 10' cpm); and D) so- (50: 50) (39). Chromatograms of the type described dium acetate-i-C14 (1 mc); the radioacetate was added by Bush (29) were run at 37° C on Whatman no. 2 filter dissolved in water, and the steroids in 0.2 ml of propylene paper. glycol. Incubation was carried out with shaking for Compounds were located on the developed chromato- three hours at 370 C with 95 per cent 02 and 5 per cent grams by visualization with ultraviolet light (A4-3-keto- CO, as gas phase. The flask contents were frozen until steroids), and by spraying with alkaline blue tetrazolium analyzed. (reducing steroids) (40), alkaline m-dinitrobenzene (all ketosteroids) (38), or antimony trichloride reagent III. Addition of carrier steroids, extraction and prelimi- (A5-3,8-hydroxysteroids) (41). After locating the ster- nary purification oids on the preparative chromatograms, the zones con- On removal of the incubation mixtures for storage, taining compounds or mixtures of compounds were cut chromatographically pure steroids in varying amounts out, cut into small pieces, and extracted repeatedly with (5 to 20 mg) were added to each of the incubation flasks hot methanol, then chloroform, or a mixture of equal (Table III). parts of methanol and chloroform. The combined ex- Each incubation mixture (tissue, medium and added tracts were filtered to remove paper fibers and evapo- carrier) was extracted individually in a Waring blendor rated to dryness under reduced pressure. The extracts with 25 volumes of chloroform: methanol (2: 1) mixture of paper previously saturated with propylene glycol were (44). After removal of tissue debris by filtration, 1/5 dissolved in chloroform and the chloroform washed sev- volume of water was added and the mixture shaken; the eral times with 1/10 volumes of water to remove the upper aqueous layer was removed and extracted twice glycol. with 1/2 volume of chloroform. The combined lowver 540 SAVARD ET AL.

TABLE III Carrier steroids added to incubation mixtures Experiment-C'4substrate C. Cholesterol* Steroids A. Testosterone B. Progesterone I II D. Acetate

mg mg mg mg mg Estrone 5 Estradiol-17,7 5 Estriol 5 Testosterone 10 10 20 10 4-Androstenedione 10 10 20 10 110-Hydroxyandrostenedione 10 lot 20 10 1 1f-Hydroxytestosterone 10 20 Adrenosterone 10 5t Dehydroepiandrosterone 20 10 5-Androstene-3,6,17j-diol 20 Progesterone 10 20 10 1 7a-Hydroxyprogesterone 10 20 5t 1 1f3-Hydroxyprogesterone 10t 5t 1 1-Ketoprogesterone 10t 5-Pregnenolone 20 10 3f3,17a-Dihydroxy-5-pregnen-20-one 20 6,B-Hydroxyprogesterone 5t * The extract from this incubation was divided into two equal parts; estrogens were added before the division and the two resulting phenolic fractions were combined later in the fractionation; other carriers were added after division of 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- V. Incubation experiments jected to an 8-transfer ccd between ligroin (bp 65 to EXPERIMENT A. Incubation of testosterone-4-C14: 750 C) and 90 per cent aqueous methanol. The individual The ketonic fraction from this incubation was chromato- tubes from the distribution of each incubation were evapo- graphed in ligroin/propylene glycol for 96 hours. Each rated to dryness, weighed, assayed for radioactivity and steroid carrier zone was located and recognized by its for the carrier ketosteroids (by absorption at 240 mas or relative position (37), eluted and rechromatrographed. by the antimony trichloride reagent). The tubes con- 17a-Hydroxyprogesterone, testosterone and adrenosterone taining the carrier steroids and associated radioactive were not completely separated by this chromatography. polar material from each incubation were combined and Separation was carried out by acetylation of the mixture dissolved in toluene. Each pooled sample was separated and the complete removal of testosterone acetate by into the respective phenolic, acidic and neutral fractions chromatography in ligroin/propylene glycol; the two by shaking the toluene solution with 1 N sodium hydrox- remaining steroids were separated by chromatography ide (27). The aqueous alkali solution was adjusted to on paper in toluene: methyl cyclohexane (50: 50)/propyl- pH 8.5 and shaken with ether to remove the phenols. ene glycol. The neutral material remained in the toluene solution. l1,i-Hydroxytestosterone: This compound did not mi- The neutral polar fractions obtained in the incubations grate from the starting line as the free steroid in ligroin/ of testosterone (A), progesterone (B) and cholesterol propylene glycol in 100 hours. It was chromatographed (C) were subjected to a separation with Girard reagent in toluene/propylene glycol for 12 hours to yield a con- T into ketonic and nonketonic fractions. The extract taminated product, 3.7 mg in weight and specific activity from the acetate-i-C1' experiment (D) was not sub- 7,370 cpm per mg. One and two-tenths mg of this was jected to this separation. diluted with 30 mg of 1 1,8-hydroxytestosterone carrier, IV. Separation and purification of carrier steroids acetylated, and chromatographed in ligroin/propylene Since somewhat different methods were used for the glycol (24 hours), to provide a diluted 11,8-hydroxy- purification of the individual carrier steroids in each ex- testosterone acetate (mp 151 to 1520 C, from acetone: periment, they will be discussed separately. The steps hexane) with a specific activity of 114 cpm per mg (or involved in establishing the radiochemical purity (45) of 129 corrected to the free compound). Oxidation with each apparently labeled compound will be presented for chromic acid and chromatography in ligroin/propylene each compound. Where derivatives of the carriers were glycol gave 11-ketotestosterone acetate (mp 164 to 165° C, prepared (acetates, oximes, and so forth) the values of acetone: hexane) with a specific activity of 112 cpm per specific activity, unless otherwise noted, are not cor- mg (127 as the free compound). Saponification with rected for changes in molecular weight, and are given potassium carbonate in ethanol (46) gave 11-ketotestos- as determined. terone (mp 184 to 1850 C); specific activity 131 cpm per VIRILIZING TESTIS TUMOR 541

mg (130 as ll,-hydroxytestosterone). The specific ac- specific activity. Adrenosterone was considered pure at tivity of the initially isolated 1l,8-hydroxytestosterone 209 cpm per mg. was calculated as 3,300 cpm per mg; this value is used Androstenedione: This compound was recrystallized in the calculation of those data for this compound in from ethyl acetate to give crystals (mp 172 to 173° C; Table IV. 19,700 cpm per mg) and a crystalline residue from the 11,8-Hydroxyandrostenedione: As initially isolated and mother liquors (17,400 cpm per mg). This product was crystallized (mp 1940 C), this compound had a specific not further manipulated and was considered radiochemi- activity of 6,500 cpm per mg. Two samples of 0.5 mg cally pure. each were diluted with 50 and 70 mg of carrier and puri- Testosterone: The original substrate, diluted as noted, fied as follows. Sample 1 was chromatographed (ligroin/ was recovered (mp 151 to 152.5° C; 250,000 cpm per mg). propylene glycol, 116 hours) and recrystallized from Radioautography of the initial chromatograms having acetone: hexane (59 cpm per mg); oxidized to adreno- revealed no zones other than those associated with the sterone, chromatographed, and recrystallized from metha- above steroids, it is presumed that no other important nol (mp 220 to 2250 C; 60 cpm per mg); recrystallized transformations of testosterone occurred in this study. from acetone: hexane (mp 219 to 2200 C; 59 cpm per Total radioactivity associated with each carrier steroid mg); converted to the dioxine and recrystallized from at the above specific activities is given in Table IV, methanol (mp 209 to 2100 C; 56 cpm per mg, corrected column A, corrected to the amount present at the addition to the molecular weight of adrenosterone). Sample 2 of the original carrier. was chromatographed and crystallized (48 cpm per mg); EXPERIMENT B. Incubation of progesterone-4-C04: oxidation followed by chromatography and crystalliza- The chromatography of the neutral ketonic fraction from tion gave adrenosterone (mp 2180 C; 46 cpm per mg). this incubation was somewhat encumbered by the presence The initially isolated steroid was radiochemically pure of a strongly fluorescing material whose presence seri- at 6,500 cpm per mg. ously distorted the initial chromatograms in ligroin/ Adrenosterone: As isolated and recrystallized (mp propylene glycol. In order to eliminate this material 2120 C) the specific activity was 278 cpm per mg and as repeated chromatography was necessary; quantitative may be seen from the following, was slightly contaminated. measurement of several carrier steroids showed that but The dioxime (mp 222° C; 258 cpm per mg) was chro- 1.0 to 1.2 mg remained. This was assumed to be true matographed (toluene/propylene glycol, 34 hours) and for all others and at this point additional amounts of recrystallized from methanol (mp 218 to 2200 C; 190 each carrier were added (10 mg) to the recombined frac- cpm per mg, calculated as adrenosterone, 209 cpm per tions, and all were rechromatographed in ligroin/propyl- mg); the material in the mother liquor had the same 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 Sc Estrone None Estradiol-1 7j1 None Estriol None Testosterone 2.500 X 106 66.0 0.11 X 105 3.1 0.30 X 10' 0.003 4-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' 0.035 1 10-Hydroxytestosterone 0.033 X 106 0.8 Adrenosterone 0.002 X 106 0.05 0.06 X 106 0.0006T Dehydroepiandrosterone 0.04 X 106 0.0004 5-Androstene-3i, 1 7fl-diol Progesterone 2.03 X 10' 58.0 None 17a-Hydroxyprogesterone 1.14 X 10' 32.6 0.63 X 105 0.0061 1 1-Hydroxyprogesterone None None 1 1-Ketoprogesterone None 5-Pregnenolone None 3P, 1 7a-Dihydroxy-5-pregnen-20-one 60-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. 542 SAVARD ET AL. droxyandrostenedione, 11,l-hydroxy- and 11-ketopro- Androstenedione: This compound was freed of most of gesterone were added at this point for the first time. its contaminating progesterone-4-C1' by the second chro- 11,8-Hydroxyandrostenedione: This compound was matography in ligroin/propylene glycol. Recrystalliza- recognized as a transformation product of progesterone- tion from ethyl acetate gave apparently contaminated C4 late in the study and was seen first as a radioactive crystals (mp 170.5 to 1710 C) with a specific activity of zone of slow mobility [slower than 11f8-hydroxyproges- 319 cpm per mg; the semicrystalline residue from the terone and 17a-hydroxyprogesterone (38)]. To the ma- mother liquors counted 260 cpm per mg. The crystals, terial eluted from this radioactive area, 5 mg of carrier 7.1 mg, were treated to form the oxime (mp 208 to 2100 11j8-hydroxyandrostenedione was added and the whole C; 202 cpm per mg), which was further purified by rechromatographed in ligroin/propylene glycol; the re- chromatography (toluene/propylene glycol, 25 hours) and sulting zone was eluted and the steroid recrystallized crystallized (mp 212 to 213.50 C). It counted 260 cpm from acetone: hexane to constant specific activity (mp per mg with a similar specific activity in the mother 195.5 to 196.50 C; 127 cpm per mg); 2.57 mg of this ma- liquor residue. Despite the variations in specific ac- terial was diluted with 4.38 mg of additional carrier and tivities, no evidence of radioimpurities was apparent. oxidized, chromatographed, and crystallized from metha- The androstenedione was considered pure at a specific nol to give adrenosterone (mp 213 to 214° C; 47 cpmn per activity of 275 cpm per mg. mg). The dioxime was formed and had a specific ac- Progesterone: The diluted substrate was recovered and tivity of 43 cpm per mg. The 11,-hydroxyandrostene- recrystallized (acetone, mp 120 to 120.5° C). Its spe- dione obtained after the initial addition of carrier was cific activity was 1,840 cpm per mg. considered radiochemically pure at approximately 127 Radioautographs of the initial chromatograms revealed cpm per mg. Comparison of specific activity with those no zones of radioactivity other than those associated of other isolated compounds is not valid because the with the above compounds. The areas between pro- carrier in this case was not added at the same stage as gesterone and 4-androstenedione notably were free from in the others. The total amount of this compound radioactivity; the epimers 20a- and 20,6-hydroxy-4-preg- formed, shown as counts per minute in Table IV, must nen-3-ones, normal derivatives of progesterone in the be considered minimal. ovary (34), have mobilities intermediate between those 11,-Hydroxyprogesterone: As isolated following the of the former compounds. second addition of carriers, this compound (14 mg) had The data in Table IV for this Experiment B are cal- a specific activity of 44 cpm per mg but was contami- culated from the above specific activities and from the nated with traces of 17a-hydroxyprogesterone. Re- assumption that at the point of the second addition of crystallization from ethyl acetate gave 10.4 mg (mp 182.5 carriers, 1.0 mg of each of the original steroid carriers to 183.50 C; 8 cpm per mg). Oxidation with chromic was present. acid followed by chromatography and crystallization from EXPERIMENT C. Incubation of cholesterol-4-C14: methanol gave 11-ketoprogesterone (mp 170.5 to 172.50 The mixture from this incubation was extracted in the C; 5 cpm per mg). Formation of the oxime and re- usual way. The chloroform: methanol extract obtained at crystallization (ethyl acetate) gave crystals (mp 226 to the outset was halved and to the separate portions, I and 2290 C) ounting 2 cpm per mg. 11,-Hydroxyproges- II, were added the steroid carriers listed in Table III. terone was therefore considered to be devoid of sig- Each portion was treated as described above (Section nificant radioactivity. III. In vitro studies) ; the phenolic fractions from por- 17a-Hydroxyprogesterone: Nine and five-tenths mg of tions I and II were combined. crystalline steroid was isolated counting 1,050 cpm per EXPERIMENT C-I. The total ketonic material from mg. Of this, 1.85 mg was diluted with 18 mg of addi- portion I containing the added a,,a-unsaturated ketosteroid tional carrier and the oxime formed and recrystallized carriers, weighed 122 mg and contained 80,000 cpm. It from methanol (mp 244 to 2460 C). The oxime had a was separated on paper chromatograms in ligroin/ specific activity of 96 cpm per mg; the mother liquors propylene glycol as described in Experiments A and B. yielded a residue which had essentially the same specific 11,8-Hydroxytestosterone: This compound as isolated, activity. 17a-Hydroxyprogesterone was considered pure was rechromatographed in toluene/propylene glycol, at 1,050 cpm per mg specific activity. acetylated, and the 1 1p-hydroxytestosterone acetate Testosterone: Separated by chromatography from the chromatographed in ligroin/propylene glycol. The puri- above as the acetate (mp 139.5 to 140.50 C), this com- fied steroid was recrystallized several times (acetone: pound counted 109 cpm per mg, and was slightly con- hexane) and finally melted at 127 to 1280 C, and had a taminated. Saponified to testosterone (mp 149 to 1500 C), specific activity of 20 cpm per mg. The mother liquor the compound counted 104 cpm per mg. Converted to residue counted 78 cpm per mg. The small amounts of testosterone oxime (mp 212 to 214.50 C), the compound substance precluded further manipulations and, therefore, counted 95 cpm per mg. Testosterone was therefore definite radiochemical purity was not achieved. considered radiochemically pure at 104 cpm per mg. 11,8-Hydroxyandrostenedione: This compound, follow- 11-Ketoprogesterone: This compound as isolated and ing rechromatography in ligroin/propylene glycol (96 crystallized (methanol, mp 171 to 1720 C) had a specific hours), was recrystallized from acetone: hexane to con- activity of 6 cpm per mg. Formation of the oxime and stant melting point (195 to 1960 C) and a specific ac- crystallization eliminated the radioactivity. tivity of 71 cpm per mg in the final crystals (8.0 mg). VIRILIZING TESTIS TUMOR 543

Recrystallization from ethyl acetate: hexane gave crystals tamination. Further purification was not possible be- (5.6 mg, 69 cpm per mg) and an oily mother liquor cause of the lack of material. residue (1.5 mg) counting 156 cpm per mg, suggesting Dehydroepiandrosterone and 5-pregnenolone: The to- that radiochemical purity had not been achieved. tal ketonic fraction, less the crystalline 3,8,17a-dihydroxy- 17a-Hydroxyprogesterone: Following the separation of 5-pregnen-20-one, containing 28,000 epm, was chromato- testosterone from this compound by the acetylation pro- graphed on paper in methyl cyclohexane/propylene glycol cedure, the 17a-hydroxyprogesterone was rechromato- for 41 hours. Two zones were visualized by the anti- graphed several times in toluene: methyl cyclohexane mony trichloride reagent, eluted, and each was rechroma- (50: 90)/propylene glycol and recrystallized from metha- tographed in the same solvent system. nol to constant specific activity (50 cpm per mg) of The slower zone which gave a positive Zimmermann both the final crystals and mother liquor residue. reaction was crystallized from hexane to give dehydro- Testosterone: Isolated as the acetate, this compound epiandrosterone (190 cpm per mg) which was diluted counted 25 cpm per mg. The mother liquor was not with an equal weight of carrier, acetylated and crystal- studied. lized from hexane to give crystals counting 91 cpm per Androstenedione: Isolated and purified by rechroma- mg. This material was recrystallized and the crystals tography, androstenedione failed to crystallize. The oily obtained (mp 168 to 1690 C, corrected) counted 68 cpm product had a specific activity of 110 cpm per mg; this per mg; the mother liquor residue counted 110 cpm per value is considered maximal. mg. Recrystallization of 2.0 mg of the crystals gave Progesterone: This compound, purified as in Experi- 0.8 mg of specific activity 74 cpm per mg and a new ment A, was crystallized from acetone. The progesterone mother liquor residue (0.7 mg) counting 140 cpm per as crystals (mp 119 to 1200 C) counted 193 cpm per mg; mg. Radiochemical purity could not, therefore, be the progesterone as found in the last mother liquor demonstrated. counted 163 cpm per mg. The radioactivity in this car- The faster zone was eluted, and after addition of car- rier, as in all others, was considered too low to warrant rier steroid (10.9 mg), was recrystallized to constant further attention, in contrast to the activities in Experi- melting point (186 to 1880 C) and a final specific activity ments A, B and D. of 208 cpm per mg; the mother liquor residues were at EXPERIMENT C-II. 5-Androstenediol: The nonke- each step of higher specific activity. Acetylation and tonic fraction of portion II, containing the carrier 5-andro- crystallization from methanol gave crystals of pregneno- stenediol and its associated radioactivity, weighed 29 mg lone acetate (mp 148 to 1490 C), whose specific activity and was chromatographed in toluene: methyl cyclohexane was 125 cpm per mg; crystallized once more, the acetate (50: 50)/propylene glycol for 48 hours. The androstene- counted 70 cpm per mg. The two last mother liquor diol was located (antimony trichloride reagent), eluted residues counted 290 and 750 cpm per mg, respectively. and the chromatography repeated. Crystallization of the The product was considered contaminated. eluted steroid (acetone: hexane) yielded 7.3 mg of In contrast to Experiments A, B and D, little radio- 5-androstenediol, mp to 175 to 1770 C, specific activity activity was associated with the above steroid carriers 90 cpm per mg. After further addition of carrier (14.3 at the point of their isolation from the chromatograms. mg) to the above, the substance was acetylated and crys- This low radioactivity precluded the further addition of tallized to give the diacetate, mp 157 to 1580 C, specific carrier-diluent and prevented use of recrystallization pro- activity 14 cpm per mg. Recrystallization from acetone cedures for demonstrating and achieving radiochemical gave crystals and a residue in the mother liquors count- purity. The uncharacterized radioactivity present in the ing, respectively, 13 and 17 cpm per mg. extracts was not associated with any chromatographically 3,8,17a-Dihydroxy-5-pregnen-20-one: The ketonic frac- discrete compounds; it tended to smear throughout the tion weighing 45.8 mg and containing 99,700 cpm, was paper chromatograms of both Experiments C-I and C-II. dissolved in hot methanol and allowed to crystallize. Whether this material is a product of a biochemical trans- Two crops of steroid were obtained, which were com- formation cannot be inferred from this study; it may be bined and recrystallized to constant melting point (226 to entirely due to artifact formation caused by the manipula- 2360 C); the last crystals had a specific activity of 90 tory procedures. The values obtained in this study have cpm per mg. Acetylation and crystallization of the not been included in the tabulation of the results from latter from acetone gave 5.7 mg of 3,8,17a-dihydroxy-5- the other three studies (Table IV). pregnen-20-one acetate, mp 221 to 2250 C, specific ac- EXPERIMENT D. Incubation of acetate-1-C14: The tivity 93 cpm per mg and a mother liquor residue, 2.4 polar lipids [tubes 5 to 8 from the distribution between lig- mg and 370 cpm per mg. Dilution of the crystals (4.0 roin and methanol: water (90: 10) ] were combined (2.62 X mg) with carrier steroid acetate (4.0 mg) and recrystal- 10o cpm) and washed free of acetate-i-C1' by means of a lization from acetone gave crystals (4.7 mg) with a 4-transfer ccd between water and chloroform, using the specific activity of 52 cpm per mg, which did not change stripping procedure described by Weisiger (47). The on recrystallization from methanol. The two mother upper phase of tube 0 was saturated aqueous sodium ace- liquor residues from these steps (1.3 mg each), how- tate (pH 7.0) instead of water. The resulting lipid frac- ever, counted 108 and 72 cpm per mg, respectively. Thus, tion contained 2.68 X 10' cpm; apparently no appreciable despite apparent constant specific activity of the crystals, amount of labeled acetate was present. This fraction the mother liquors gave evidence of radiochemical con- was subjected to a phenolic separation (27), giving a 544 SAVARD ET AL. neutral fraction containing 2.20 X 106 cpm and a phenolic gesterone; and a mixture of the acetates of testosterone fraction (86,000 cpm) which was subjected to a ccd be- and 6,8-hydroxyprogesterone. The mixture of ll,-hy- tween toluene and 1 N sodium hydroxide, using five droxyprogesterone and 17a-hydroxyprogesterone was re- funnels and complete stripping. There was a peak in solved in the ligroin: toluene (67:33)/methanol: water radioactivity which moved with the alkali and was ex- (70: 30) system. The mixture of acetates of testosterone tractable with ether at pH 8.5. The material in this and 6,8-hydroxyprogesterone was saponified and chro- peak, having the properties of phenols, contained 15,000 matographed in the same way as the corresponding frac- cpm. This purified phenolic fraction was subjected to a tion from Pool A. Again testosterone, 6,8-hydroxypro- 99-transfer ccd in methanol: water (70: 30) /chloroform: gesterone, and allopregnane-3,6,20-trione were recovered. carbon tetrachloride (40: 60). The distribution was At this point, all of the carriers from the two peaks of analyzed for radioactivity and for the estrogens by the radioactivity in the 150-transfer ccd had been recovered fluorimetric method. Estrone, estradiol-17,8, and es- chromatographically pure. In the cases in which the triol were identified, but no peak in radioactivity was as- compound was recovered in both pools, the two portions sociated with any of the three estrogens. It was there- were combined. Each carrier isolated was assayed for fore concluded that none of the three was labeled. weight and radioactivity; additional unlabeled carrier Five mg each of 17a-hydroxyprogesterone, 11p-hy- was then added to each, and tests of radiochemical purity droxyprogesterone, 6p8-hydroxyprogesterone and adreno- were applied. The details of these procedures are given sterone were added as additional carriers to the neutral for each compound. fraction. This was subjected to a 150-transfer ccd in l1,8-Hydroxyandrostenedione: Analysis by ultraviolet cyclohexane: ethyl acetate (50: 50)/ethanol: water (50: absorption at 240 mu showed a recovery of 3.11 mg con- 50). The peaks of radioactivity (K2 = 0.32 and K = taining 109,000 cpm. An additional 50 mg of carrier was 1.02) contained all of the neutral carrier steroids except added and this was crystallized three times from aqueous progesterone and 5-pregnenolone which were found to- ethanol yielding crystals having specific activities of gether in an area containing no peak of radioactivity 1,890, 1,850, and 1,880 cpm per mg (calculated, 2,050 (K = 2.56). It was, therefore, concluded that these cpm per mg) and a final mother liquor residue, specific ac- latter two compounds were not appreciably labeled. tivity 1,660 cpm per mg. Crystallization from benzene The contents of the tubes in each of the two peaks of yielded crystals having a specific activity of 1,850 cpm radioactivity containing the steroid carriers were com- per mg, and a mother liquor residue of 1,920 cpm per bined, resulting in a pool of polar (K = 0.32) material mg. The crystals from benezene were subjected to a 99- (Pool A) and one of less polar (K = 1.02) material transfer ccd in methanol: water (70: 30) /chloroform: (Pool B). Pool A contained all of the 118-hydroxy- carbon tetrachloride (20: 80). The single peak in ra- androstenedione and adrenosterone. Pool B contained dioactivity coincided with the peak in material having all of the androstenedione, dehydroepiandrosterone, and ultraviolet (240 m/u) absorption (K = 0.73), and there 17a-hydroxyprogesterone; each pool contained part of was no evidence of lack of homogeneity (45). The mean the 6f3-hydroxyprogesterone, 11,8-hydroxyprogesterone and specific activity in the region of the peak was 2,120 ± testosterone. Pool A was acetylated and chromatographed 218 (standard deviation) cpm per mg. 11,8-Hydroxy- in the cyclohexane: toluene (50: 50) /propylene glycol androstenedione was considered radiochemically pure at system. Four zones were located on the chromatogram this point. and eluted. These contained 113-hydroxyandrostenedione, Adrenosterone: The adrenosterone isolated was meas- 1 1p-hydroxyprogesterone, adrenosterone, and a mixture ured by ultraviolet absorption (238 m/u), and contained of testosterone acetate and 6,8-acetoxyprogesterone. This 2.66 mg and 32,000 cpm. An additional 10 mg of un- mixture of acetates was saponified with 1 N potassium hy- labeled adrenosterone was added and a 99-transfer ccd droxide in methanol overnight at room temperature. The was carried out using methanol: water (70: 30) /chloro- saponified material was chromatographed in the ligroin: form: carbon tetrachloride (10: 90). Analysis by ul- toluene (67: 33)/methanol: water (70: 30) system. traviolet absorption showed a single peak (K = 0.62), Three zones were located and eluted: testosterone, 6f8-hy- but radioactivity extended from tube 0 through the peak droxyprogesterone, and a less polar compound having in adrenosterone, thereby demonstrating lack of purity. the RF of allopregnane-3,6,20-trione, the maj or product The contents of the tubes containing the adrenosterone of saponification of 6fl-acetoxyprogesterone with excess were pooled and crystallized four times from aqueous alkali (48). ethanol, yielding crystals having specific activities at Pool B was subjected to a digitonin separation (49). each step of 395, 300, 279, and 289 cpm per mg and a The ",f-fraction" was chromatographed in the ligroin: residue in the last mother liquor, specific activity of toluene (67: 33)/methanol: water (70: 30) system to 308 cpm per mg. Crystallization from ethyl acetate: yield dehydroepiandrosterone. The "a-fraction" was hexane gave crystals, specific activity 275 cpm per mg acetylated and chromatographed in the ligroin/propylene and a mother liquor residue of specific activity 264 cpm glycol system. Three zones were located on the chro- per mg. The final crystals and residue in the mother matogram and eluted; they were androstenedione, a mix- liquor were combined and subjected to a 49-transfer ccd ture of llf-hydroxyprogesterone and 17a-hydroxypro- in cyclohexane: ethyl acetate (60: 40)/ethanol: water (50: 50). The single peak in radioactivity coincided 2 K = distribution coefficient. with the single peak in material absorbing at 238 mg VIRILIZING TESTIS TUMOR 545

(K = 0.71). The material was radiochemically pure as aqueous ethanol to give crystals having specific activities judged by the specific activity in the region of the peak of 1,670 and 1,740 cpm per mg (calculated 1,900 cpm per which was constant at 277 ± 24 cpm per mg. The mg). Crystallization from benzene: cyclohexane gave adrenosterone was considered radiochemically pure. crystals, 1,740 cpm per mg and a mother liquor resi- li,8-Hydroxyprogesterone: The ll(-hydroxyprogeste- due, 1,750 cpm per mg. The 17a-hydroxyprogesterone rone isolated was analyzed by ultraviolet absorption at was considered radiochemically pure at this point. 242 mA, and consisted of 3.08 mg and 24,000 cpm; 10 mg Dehydroepiandrosterone: The recovered dehydroepi- of carrier was added. A 99-transfer ccd in methanol: androsterone was analyzed by the Zimmermann reac- water (70: 30)/chloroform: carbon tetrachloride (20: 80) tion (25) and contained 2.11 mg and 3,800 cpm; 10 mg of showed a clear dissociation of radioactivity (K = 0.30) unlabeled dehydroepiandrosterone was added and the from the 11,8-hydroxyprogesterone as measured by ul- whole was subjected to a 99-transfer ccd in methanol: traviolet absorption (K = 0.41). Hence, the ll1(-hy- water (80: 20)/carbon tetrachloride. A single peak droxyprogesterone was not labeled. (Zimmermann) of carrier was found (K = 1.25); there 6,8-Hydroxyprogesterone: The portion of the 6,6-hy- were two peaks in radioactivity-one (K = 8.00) con- droxyprogesterone carrier recovered unchanged was re- tained 745 cpm; the larger peak corresponded to the chromatographed in ligroin: toluene (67: 33)/methanol: peak in dehydroepiandrosterone (K = 1.25), but con- water (70: 30). The compound as isolated from this tained other labeled material since it failed to fit the chromatogram was still not pure, as shown by an atypical theoretical distribution curve. This peak contained ultraviolet absorption spectrum. The absorbance at 237 about 2,100 cpm. The contents of the tubes containing mA indicated a maximum content of 0.13 mg; it con- the dehydroepiandrosterone were combined and crystal- tained 950 cpm. To this was added 8.6 mg of unlabeled lized five times from aqueous ethanol giving crystals carrier. A 99-transfer ccd in methanol: water (80: 20)/ containing 87, 102, 86, 84, and 90 cpm per mg and a final chloroform: carbon tetrachloride (10: 90) showed a clear mother liquor residue having 110 cpm per mg. Crystal- dissociation of the radioactivity (K = 1.57) from the car- lization from acetone: hexane yielded crystals containing rier (K = 1.41). The transformation product of 6,8-hy- 85 cpm per mg and a mother liquor residue of 75 cpm per droxyprogesterone, allopregnane-3,6,20-trione, as recov- mg. The last crystals were subjected to a 24-transfer ered from the chromatograms, contained only 3,700 cpm. ccd in ethyl acetate: cyclohexane (30: 70)/ethanol: water This low level of radioactivity together with the lack of (55: 45). The peak in radioactivity coincided with the activity in the small amount of 6(8-hydroxyprogesterone peak in dehydroepiandrosterone (Zimmermann reaction, recovered made further work on this fraction seem K = 1.04). Constant specific activity, 80 ± 12 cpm per unwarranted. mg in the region of the peak, indicated radiochemical Androstenedione: The pool of this compound recovered purity. The dehydroepiandrosterone was then consid- from the incubation mixture contained 2.04 mg as meas- ered radiochemically pure. ured by ultraviolet absorption at 240 mAt, and 133,000 cpm. Testosterone: The testosterone as isolated from the To this was added 60 mg of unlabeled androstenedione. incubation mixture was impure as indicated by its ultra- Two crystallizations from aqueous ethanol furnished violet absorption spectrum. It was, therefore, rechro- crystals having specific activities of 1,980 and 1,900 matographed in ligroin: toluene (67: 33)/methanol: wa- cpm per mg (calculated 2,140 cpm per mg) and a final ter (70: 30). It then had a satisfactory spectrum indi- mother liquor, specific activity of 1,960 cpm per mg. cating a recovery of 1.12 mg and contained 4,800 cpm; Crystallization from acetone: cyclohexane yielded crystals 10 mg of carrier testosterone was added and the whole of specific activity 1,880 cpm per mg and a mother subjected to a 99-transfer ccd in methanol: water (80: liquor residue of 1,960 cpm per mg. These latter crys- 20)/chloroform: carbon tetrachloride (10: 90). A single tals were subjected to a 99-transfer ccd in methanol: peak in material absorbing at 240 mu coincided with the water (80: 20)/carbon tetrachloride. The single peak single peak in radioactivity (K = 1.19) ; however, the in radioactivity coincided with the peak in ultraviolet experimental points for radioactivity did not agree well absorption (K = 0.72); the mean specific activity in the with the theoretical curve, thus indicating the presence region of the peak was 2,130 ± 194 cpm per mg. The of a radioactive impurity. The peak contained 9.73 mg androstenedione was therefore considered radiochemi- and 3,380 cpm. The contents of the tubes containing cally pure at this point. the testosterone were crystallized five times from aqueous 17(3-Hydroxyprogesterone: The pool of 17a-hydroxy- ethanol. The specific activities of the crystals were 306, progesterone contained 1.61 mg (239 mA) and 22,100 312, 255, 196, and 206 cpm per mg, and that of the resi- cpm; 10 mg of 17a-hydroxyprogesterone carrier was due in the final mother liquor, 276 cpm per mg. Crystal- added and the whole to a 99-transfer ccd in subjected lization from acetone: hexane yielded crystals contain- methanol: water (70: 30)/chloroform: carbon tetrachlo- ride (10: 90). The peak in radioactivity coincided with ing 223 cpm per mg and a residue in the mother liquor the peak in the 17a-hydroxyprogesterone as measured by of 210 cpm per mg. In view of these results and the ultraviolet absorption (K = 0.49). The mean specific relatively high level of activity associated with the activity around the peak was 1,900 ± 167 cpm per mg. closely related androstenedione, the testosterone was con- The material from this peak was crystallized twice from sidered labeled. 546 SAVARD ET AL.

DISCUSSION of the carrier and which effects the maximum al- Methods for radiochemical purity teration in its physical properties. Crystalliza- tion (of steroid carriers and derivatives), how- In the demonstration of radiochemical purity ever, has been the manipulation most widely used of the isolated steroid carriers, it has been our as the criterion for radiochemical purity, since a experience that radioactive contamination is rela- trace contaminant is likely to be completely solu- tively unlikely when the radioactive starting ma- ble in the solvent used to crystallize the larger terial is a steroid closely related to the products; amount of carrier. Unfortunately, with steroids, thus, in Experiments A and B, achievement of ra- this ideal situation is often complicated by adsorp- diochemical purity presented no serious difficul- tion of impurities on crystals and even mixed ties. This was not true when the radioactive crystal formation (this phenomenon may explain starting material was a substance far removed the tenacity of the contaminants encountered in (in the biosynthetic sense) from the products un- Experiment C). Purification to constant specific der study, as in Experiments C and D in which activity in a particular solvent may be considered cholesterol-C'4 and acetate-C'4 were incubated. to have been achieved when specific activity re- In one, only a small percentage of the radioactivity mains constant through several recrystallizations. was ultimately associated with the steroid car- When only small quantities of carrier are avail- riers, while in the other (Experiment C), demon- able, however, the above is not always possible; stration of radiochemical purity was not achieved; in such cases we have found it necessary to carry the purification problem in these latter cases ap- out specific activity measurements on both crys- pears to have been compounded by the large va- tals and mother liquors from each crystallization riety and unknown nature of the radioactive im- in order to determine if constant specific activity purities, particularly in the cholesterol-C14 study. has been achieved in that particular solvent. We have found that after separation and iso- These criteria served to provide the values given lation of the carrier steroids in chemically pure in Table IV for Experiments A, B and D; the state, several purification measures may be used same criteria applied to the steroids in the cho- to demonstrate radiochemical purity, the main lesterol-C'4 study (Experiment C) indicated that criterion being constancy of specific activity of the the associated radioactivity was largely due to un- steroid at each step through a series of physical known impurities. To be noted is the value of and chemical manipulations. The greater the the isolation of several carriers devoid of radio- number of such manipulations without significant activity; such negative results lend considerable change in specific activity, the greater the likeli- confidence to the positive values obtained in the hood that the compound is radiochemically pure. same study. Conversely, decreases in specific activity through these manipulations signify contamination of the Biochemical considerations carrier some trace by impurity. The elevated level of the urinary 17-ketosteroids Among the various manipulations currently seen in the patient preoperatively is consistent employed for purification, countercurrent distri- with the classical clinical features of interstitial butions have proven particularly useful, since cell tumors of the testis in childhood (1-21). nonhomogeneity can be readily detected when the radioactive compound and the steroid carrier Analysis of the individual 17-ketosteroids revealed have only slightly different partition coefficients androsterone and etiocholanolone (Table II) to be in the system employed (45). Formation of suit- present in the largest amounts. Metabolic stud- able derivatives is also of value, since two com- ies in humans (50) show that the principal pre- pounds (steroid carrier and radioactive com- cursors of these two urinary C1,02 steroids are pound) which have very similar physical proper- androstenedione, testosterone and dehydroepian- ties may form derivatives having less similar drosterone, and that the two metabolites are usu- properties. In this regard, the steroid carrier ally excreted in approximately equal amounts, as derivative selected should be one which is based they are in our patient (Table II). The elevated upon the most characteristic chemical feature levels of the 11-oxygenated 17-ketosteroids in the VIRILIZING TESTIS TUMOR 547 urine are most impressive since the occurrence of both may well have been secreted by this tumor an oxygen substituent at carbon-11 of a steroid in vivo, their production being reflected by the of mammalian origin has long been considered elevated urinary titers of pregnanetriol and neu- indicative of that steroid's adrenal cortical origin. tral acylable lipids (Table I), although the latter The high concentration of 11-keto- and 11,8-hy- may be made up of C,9-diol compounds. The droxyandrosterone (1.8 mg per 24 hours) indi- relatively minor amount of dehydroepiandroster- cates that at least one of the likely precursors one obtained in the incubation with acetate-C14 (511)i,8-hydroxyandrostenedione, adrenoster- (Table IV), suggests that this substance was one or 11,8-hydroxytestosterone-was formed by transformed rapidly by the tissue and therefore the tumor; these three latter compounds have accumulated little or no radioactivity in the in vivo been isolated from adrenal tissue either as con- study. stituents or as metabolic products (52-55). The The incubation of the tumor tissue with cho- urinary 17-ketosteroid pattern thus suggests that lesterol-C14 was undertaken with the notion that the principal C,90, androgen elaborated was tes- an efficient utilization and transformation of la- tosterone or androstenedione (or perhaps dehy- beled cholesterol into steroid hormone products droepiandrosterone), and that one of the more would occur. This premise has proved to be false. weakly androgenic C,90, compounds-11,8-hy- This does not necessarily mean that cholesterol droxyandrostenedione, 1 1,8-hydroxytestosterone, is not an intermediate in the synthesis of steroid or adrenosterone (56, 57)-was formed in les- hormones in this tumor. Although the tissue was ser amounts. The preoperative levels of urinary capable of active steroid synthesis as seen in the pregnanetriol suggest also the likelihood of the other experiments, no significant amount of cho- formation by the tumor of 17a-hydroxyprogester- lesterol-C'4 was transformed into recognizable one (50). C,9 or C,, steroid products. Dilution of the radio- The results of the acetate incubation study, active cholesterol by the tissue and medium choles- given in Table IV, indicate that androstenedione terol, or its failure to penetrate cell membranes and testosterone are indeed the major steroids could explain the negative results. However, the elaborated by the tissue under the conditions of cholesterol-C14 did undergo some chemical trans- incubation and, no doubt, account for the virilizing formations to more polar material, as indicated by properties of this tumor. The accumulation of the large amounts of radioactivity found in the C14 in both these compounds amounts to 0.07 per polar fractions accompanying the steroid carriers. cent of the added acetate. 1i-,8-Hydroxyandro- This unidentified material, which may have been stenedione and its companion adrenosterone ( 11/- a biochemical transformation product or an arti- hydroxytestosterone was not sought) accumulated fact, accompanied the carrier steroids through 0.04 per cent. Thus, in the acetate experiment, several purifications., including the formation of the ratio of C,9O, to C,,O3 steroids is in excellent derivatives. agreement with the quantitative relationship of The results of the incubation of testosterone-C14 these two classes of steroids found in the urine. (Experiment A) are clear-cut. The major trans- In the study with acetate-C14, little or no radio- formation product of testosterone was androstene- activity was associated with progesterone while a dione, known to be a normal secretory product significant amount accumulated in 17a-hydroxy- of human (59) and dog testicular tissue (60). progesterone. Perfused stallion testes have been The presence in this tumor of the enzyme, 11,8- shown to incorporate acetate-C14 into both pro- hydroxylase (61), was demonstrated by the iso- gesterone and 17a-hydroxyprogesterone (58), lation of radioactive 11,8-hydroxyandrostenedione and a human embryonal testicular tumor, under and 11,8-hydroxytestosterone (54). The signifi- tissue-slice conditions, elaborated progesterone cance of this reaction in testicular tumor tissue is (31) in addition to androstenedione and testoster- discussed below. The formation of radioactive one. Our incubation studies suggest that while adrenosterone in minor amounts points to the these two C21-compounds appear to act in this presence of the widely occurring 11-hydroxyde- tumor as intermediates (see below) in the biosyn- hydrogenase (62) in this tumor tissue or possibly thetic pathway to the androgens (Experiment B), in the plasma used in the incubating medium. It 548 SAVARD ET AL. should be noted that no radioactive products (1 1,8-hydroxylase activity, formation of 1 1/3-hy- other than the four described were found in this droxyandrostenedione and related compounds), experiment and that the 11p,-hydroxylase activity which more closely resemble those of adrenal cor- of the tissue is demonstrated by the fact that 7.6 tical than of testicular tissue, requires reappraisal per cent of the added testosterone was converted of the status of the interstitial cell tumor of the to 11-oxygenated steroids. testis. The isolation of radioactive 17a-hydroxyproges- As with the previous 22 cases, our patient dis- terone, testosterone and androstenedione from the played clinical abnormalities attributable to the progesterone-C'4 incubation experiment (Table overproduction of androgens: precocious statural IV, Experiment B) serves to indicate the pathway and isosexual growth, advanced skeletal matura- for the formation of androgens by this tumor tis- tion, excessive muscular development, facial acne stue. Slaunwhite and Samuels (63) described the and hirsutism. Precocity began between the ages transformation of progesterone into androstenedi- of three and six years in almost all reported cases, one and testosterone by rat testicular tissue and including the present one, and the tumor was uni- postulated that 17a-hydroxyprogesterone is an in- lateral and benign in all but one case. Correlated termediate. These transformations have been con- with this, in our case and nine previous cases so firmed by the present study. by Solomon, Vande studied, urinary 17-ketosteroids were elevated for Wiele and Lieberman (64) in bovine ovarian tis- the age, ranging from 2.9 to 64 mg per 24 hours. sue, and by Lynn and Brown (65) and one of the Gynecomastia was observed in three previous cases authors in normal rat testis tissue, which has also but not in the present one, where urinary estrogen been shown to transform 1 7a-hydroxyprogester- was in the normal range and none was formed by one-C14 into radioactive androstenedione and tes- the tumor tissue in vitro (Experiment D). A tosterone (66). The presence of 11,8-hydroxy- clue to the gynecomastia in the three cases may androstenedione among the transformation prod- be in the recent demonstration of the conversion ucts of progesterone is consistent with the results of androgen to estrogen by human endocrine (69) of the testosterone incubation. The absence of and nonendocrine tissues (70) and the observation 11,8-hydroxyprogesterone and 11-ketoprogester- of gynecomastia following androgen administra- one from the products derived from progesterone tion (71). is interesting in view of the isolation of 11-oxy- The origins of these tumors are not clear. genated C,9 compounds in this and other incuba- First, there are no precise morphologic criteria tions. This may suggest that the tumor 11,8-hy- for differentiating adrenal cortical cells from in- droxylase exhibits a substrate preference (61). terstitial cells of Leydig (11, 72), and secondly, A final point of interest is the apparent failure of adrenal cortical rests do occur in the testes. This this tumor tissue to convert progesterone to either is not surprising since the adrenal cortex arises of the two substances, 20a- or 20,8-hydroxy-4- embryologically in the dorsal mesenteric root in pregnen-3-one. They have been shown to be pro- close proximity to the urogenital ridge containing duced from progesterone by human (66) and bo- the gonads (73). In this way it is conceivable vine (67) ovarian tissue in vitro, and by the evis- that these tumors can resemble adrenal cortical tis- cerated rat in vivo (68), and normally accompany sue both morphologically and biochemically. On progesterone in the blood of pregnant women and the other hand, massive replacement of testicular in human follicles, corpora lutea and possibly in tissue by aberrant adrenal cortical cells in a patient placental blood (35). with congenital virilizing adrenal cortical hyper- plasia has been reported by Wilkins and co-work- Clinical considerations ers (74). Subsequently, this has been confirmed, and in 1954, Hedinger reported a similar patient There are 22 previously reported cases of so- in whom cortisone administration induced marked called interstitial cell tumors of the testis occurring regression of testicular size (75). Staubitz and in childhood and producing isosexual precocity co-workers in 1953 reported a patient with bi- (1-21). In the present case the demonstration lateral "interstitial cell tumors" with precocity be- in the tumor of certain biochemical activities ginning at three years of age, but in whom urinary VIRILIZING TESTIS TUMOR 549 17-ketosteroids were 73.9 mg per 24 hours before must rest, but we are forced to re-open the ques- bilateral orchiectomy and still elevated at 69.6 tion as to tissue specificity of both the crystalloids mg per 24 hours three months postoperatively of Reinke and the capacity to elaborate 1 1f-hy- (76), suggesting that this was an instance of droxysteroids. Perhaps it should be re-empha- adrenogenitalism with bilateral adrenal cortical sized that while 11,8-hydroxylase activity has rests in the testes; for this reason it has not been never been previously observed in normal testicu- included in our list of previously reported cases lar tissue, no distinct search has been made until of interstitial cell tumors of the testis. Because very recently; it was not found in normal stallion of insufficient data for diagnosis, another case with testis tissue (58) and could not be demonstrated bilateral testicular tumors reported by Garvey in an arrhenoblastoma (66). The apparent ab- and Daniel has been excluded from our series sence of 11,8-hydroxylase from normal testicular (77). We have accepted the case reported by and ovarian tissues may however reflect a failure Rezek and Hardin (19) with bilateral testicular of analytical techniques to detect extremely low tumors, primarily on the basis of a decrease in concentrations of enzymatic activity. The process 17-ketosteroids from 91 (60 per cent "f3-ketoster- of tumor formation could bring about an increase oids") to 2.1 (no "/3-ketosteroids") mg per 24 in concentration of an enzyme normally considered hours 16 months postoperatively. However, the absent from the tissue, and seemingly could cause preoperative level of 3,8-hydroxy-17-ketosteroids the tumor to "acquire" an enzyme not usually as- in the urine is suggestive evidence for tumor for- sociated with the parent tissue. The recent re- mation from adrenal-cortical cell rests. Sand- port of the enzyme 21-hydroxylase (likewise gen- blom's patient has been included perhaps incor- erally associated with adrenal cortical tissue) in rectly in our series, since at four years of age and an induced tumor of testicular interstitial cell ori- one year postoperatively the urinary 17-keto- gin in the mouse (80) suggests such a possibility. steroids varied between 10 and 20 mg per 24 hours Our patient's postoperative course during two as compared to 64 mg per 24 hours before sur- years of observation has been of considerable in- gery (8). There thus appears, probably for terest. His excessive libido gradually subsided want of completeness in their work-up, an ob- and social adjustment improved, but there was no scure picture of the "testicular" origins of these regression of the physical manifestations of pre- tumors; in some instances they possess the dis- cocity; indeed, he continued to mature. Growth tinct character of adrenal cortical rests, while in continued at approximately the same rate as pre- those included in our series, they show no such operatively, but the bone age advanced only one to morphological properties. two years. The remaining left testis, small pre- There is one histological finding that has long operatively, increased to late-adolescent size with been considered specific for Leydig cells, namely normal consistency. Urinary gonadotropins, the presence of crystalloids of Reinke (78). which were absent preoperatively, reached the These also occur in the ovarian hilus cells but normal adult range of 7 rat uterine units. The have never been described in adrenal cortical tis- urinary 17-ketosteroid levels fell precipitously sue. Recently Sternberg, Segaloff and Gaskill after operation to approximately 1.0 mg per 24 have reaffirmed that these crystalloids are indeed hours, but during the last year of observation have "specific identification tags. distinguishing hilus varied between 3.0 and 7.4 mg per 24 hours. The and Leydig cells from morphologically similar above data suggest that postoperatively the pa- cells such as lutein and adrenal cortical cells" (79). tient entered true puberty with its attendant go- Although absent in each of the previously reported nadotropin production, growth spurt and matura- cases of prepubertal interstitial cell tumors, crys- tion of the remaining testis. This simulates the talloids of Reinke were clearly demonstrated in sexual maturation which has been observed dur- the tumor of our patient. Thus, on the one hand ing cortisone therapy of patients with the adreno- this tumor had this morphologically identifying gentital syndrome in whom somatic maturation, stigma of testicular origin, while on the other reflected by a bone age of 11 to 12 years, had taken hand, its biochemical activity suggested certain place prior to treatment (81). It would appear adrenal cortical properties. Here the enigma that, irrespective of chronological age, some re- 550 SAVARID ET AL. lationship exists between the level of somatic and a bone age of 10 years, grew 20 cm during and skeletal development and the initiation of the the first 17 months after operation, and the re- hypothalamic-pituitary discharge which leads to maining testis appeared to be enlarging. While elaboration of gonadotropin. The normal adoles- the latter patient's course is suggestively similar cent values of urinary gonadotropins, 17-ketoster- to that of our own case, no testicular biopsy nor oids and pregnanetriol postoperatively in our gonadotropin assays were reported. In three pa- patient are evidence against either development tients with gynecomastia, breast enlargement dis- of a new tumor in the remaining testis or of appeared in two and persisted in one. adrenal hyperplasia as a basis for the subsequent This protracted study has served to demon- continued somatic and sexual development. strate that the virilizing properties of this tumor Inadequate data are available on the postopera- may be ascribed to two principal androgenic ster- tive course of the 22 previously reported cases. oids, testosterone and 4-androstenedione, whose In general, the literature reports little or no re- formation in the tumor is consistent with the gression of the precocious physical characteristics. mechanism of androgen biosynthesis recently In two patients with preoperative heights of 152 postulated for normal testicular tissue (63) (Fig- cm there was no further growth postoperatively, ure 7). The demonstration of the presence of the probably reflecting closure of the epiphyses prior enzyme 11,8-hydroxylase points to certain hitherto to surgical excision of the tumors. However, unsuspected endocrine and embryological con- in five patients whose preoperative height was less siderations of this tumor. The implications of than 127 cm and whose bone age was 12 years or these observations as to the nature of the hor- less, a deceleration of growth rate and genital mone-secreting cells of this tissue must await the development was specifically noted. One other extension of these studies to similar virilizing tu- patient, with a preoperative height of 120.7 cm mors of the testis.

[CH3 COO] H co

CH3-OH

HO H

K) al Ac,Pr,Te 3t Te FIG. 7. PROBABLE PATHWAY OF STEROID BIOSYNTHESIS IN THE TESTICULAR TUMOR. Broken arrows indicate presumed 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 551

SUM MARY 7. Urban, N. Riickbildungserscheinungen bei Pubertas praecox. Mschr. Kinderheilk. 1942, 90, 11. An interstitial cell tumor in the testis was re- 8. Sandblom, P. Precocious sexual development pro- moved from a highly virilized boy. The preopera- duced by an interstitial cell tumor of the testis. tive levels of urinary 11,8-hydroxyandrosterone, Acta endocr. (Kbh.) 1948, 1, 107. 11-ketoandrosterone, 9. Fraser, K. B. Interstitial cell tumours of the testis. 11,8-hydroxyetiocholanolone, The male sex hormone. Aust. N. Z. J. Surg. 1949, androsterone, etiocholanolone and pregnanetriol 19, 48. were abnormally high and fell promptly following 10. Melicow, M. M., Robinson, I. N., Ivers, W., and surgery. After incubation of tumor slices with Rainsford, L. K. Interstitial cell tumors of the acetate-1 -C14, the following radioactive steroids testis. J. Urol. 1949, 62, 672. 11. James, D. C., and Shupe, R. D. Interstitial cell tumor were found by carrier dilution: testosterone, of the testicle with report of a case. J. Urol. 1950, 4-androstene-3,17-dione, (in small amount) de- 63, 718. hydroepiandrosterone, 17a-hydroxyprogesterone, 12. Newns, G. H. Precocious sexual development due adrenosterone and 1 1,8-hydroxyandrostenedione; to an interstitial-cell tumour of the testis. Brit estrone, estradiol-17P, estriol, progesterone and J. Surg. 1952, 39, 379. 13. Cook, C. D., Gross, R. E., Landing, B. H., and 1 and were isolated 1,/- 6,8-hydroxyprogesterone Zygmuntowicz, A. S. Interstitial cell tumor of devoid of radioactivity. Incubation with proges- the testis. Study of a 5-year-old boy with pseudo- terone-4-C14 yielded radioactive 17a-hydroxypro- precocious puberty. J. clin. Endocr. 1952, 12, 725. gesterone, androstenedione, testosterone and 11,8- 14. Anagnostou, J., and Frangopoulos, G. J. Pubertas hydroxyandrostenedione. Incubation with testos- praecox caused by interstitial-cell tumour of tes- terone-4-C14 yielded radioactive androstenedione, ticle. Brit. med. J. 1953, 1, 861. 15. Demetriadis, T., et al. Arch. med Sci. (Athens) 1 1,8-hydroxytestosterone, 1 1fB-hydroxyandrostene- 1948, quoted by Aragnostou, J., and Frangopoulos, dione and adrenosterone. Cholesterol-4-C14 failed B. J. (14). to give rise to significant amounts of radioactive 16. Thamdrup, E. Macrogenitosomia caused by inter- steroid products. stitial cell tumor of the testis. A case in a 7Y2- A critique of methods for the study of radio- year-old boy. Acta Paediat. 1953, 42, 369. 17. Hertz, R., Cohen, M. I., Lewis, L. G., and Firminger, chemical purity of steroids is presented. H. I. Sexual precocity in a 5-year-old boy with The morphological, biochemical and clinical as- interstitial-cell tumor of the testis. J. clin. Endocr. pects of this twenty-third reported case of viriliz- 1953, 13, 1248. ing interstitial cell tumor of the testis are discussed. 18. Pomer, F. A., Stiles, R. E., and Graham, J. H. In- terstitial-cell tumors of the testis in children. New Engl. J. Med. 1954, 250, 233. REFERENCES 19. Rezak, P., and Hardin, H. C., Jr. Bilateral inter- 1. Sacchi, E. Di un caso di gigantismo infantile (Pedo- stitial cell tumor of the testicle: Report of one macrosomia) con tumore del testicolo. Riv. sper. case observed fourteen years. J. Urol. 1955, 74, Freniat. 1895, 21, 149. 628. 2. Rowlands, R. P., and Nicholson, G. W. Growth of 20. Jolly, H. Sexual Precocity. Springfield, Charles C the left testicle with precocious sexual and bodily Thomas, 1955, p. 62. development (macro-genito-somia). Guy's Hosp. 21. Jungck, E. C., Thrash, A. M., Ohlmacher, A. P., Rep. 1929, 79, 401. Knight, A. M., Jr., and Dryenforth, L. Y. Sexual 3. Stewart, C. A., Bell, E. T., and Roehlke, A. B. An precocity due to interstitial-cell tumor of the testis: interstitial-cell tumor of the testis with hyper- Report of two cases. J. clin. Endocr. 1957, 17, " genitalism in a child of five years. Amer. J. 291. Cancer 1936, 26, 144. 22. Hooker, C. W. The postnatal history and function 4. Somerford, A. E. A case of interstitial cell tumor of the interstitial cells of the testis of the bull. of the testis in a boy of eleven years. Brit. J. Urol. Amer. J. Anat. 1944, 74, 1. 1941, 13, 13. 23. Pollock, W. F. Histochemical studies on the inter- 5. Huffman, L. F. Interstitial cell tumor of the testicle: stitial cells of the testis. Anat. Rec. 1942, 84, 23. Report of a case. J. Urol. 1941, 45, 692. 24. Venning, E. H., Hoffman, M. M., and Browne, 6. Werner, A. A., Spector, H. I., Vitt, A. E., Ross, J. S. L. Isolation of androsterone sulfate. J. biol. W. L., and Anderson, W. A. D. Pubertas precox Chem. 1942, 146, 369. in a six-year-old boy produced by a tumor of the 25. Nathanson, I. T., and Wilson, H. Factors affecting testis, probably of interstitial cell origin. J. clin. colorimetric urinary 17-ketosteroid determinations. Endocr. 1942, 2, 527. Endocrinology 1943, 33, 189. 552 SAVARD ET AL.

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