Pure gonadal dysgenesis

Pure gonadal dysgenesis

Pure Gonadal Dysgenesis Studies of In Vitro Androgen Metabolism LESLIE I. ROSE, M.D. RICHARD H. UNDERWOOD, Ph.D. GORDON H. WILLIAMS, M.D.’ GERALDIN...

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Pure Gonadal Dysgenesis Studies of In Vitro Androgen Metabolism

LESLIE I. ROSE, M.D. RICHARD H. UNDERWOOD,

Ph.D.

GORDON H. WILLIAMS, M.D.’ GERALDINE S. PINKUS. M.D. Boston, Massachusetts

From the Departmentof Medicine,EndocrineMetabolicUnit, and Departmentof Pathology, Peter Bent BrighamHospital,HarvardMedical School, Boston,Massachusetts.Requestsfor reprintsshouldbe addressedto Dr. Leslie 1. Rose, Peter Bent BrighamHospital,721 HuntingtonAvenue,Boston, Massachusetts 02115. Manuscript accepted December 10, 1973. ‘Investigator of the Howard Hughes Medical Institute.

In the syndrome of pure gonadal dysgenests, clttoromegaty is frequently present even when there are no other manifestations of androgen excess. A few cases have been reported with marked virilism, and the source of the androgen excess has been shown to be the streak gonads. Although the streak gonads contain several cell types, the luteln-Leydig cells have been theorized to be a source of testosterone synthesis. We report a case of pure gonadal dysgenesis with clitoromegaly in which pure nests of the lutetn-Leydig cells were obtained from a gonadoblastoma and in which the conversion of 14C-androstenedtone to 14C-testosterone by these cells was compared with that In an area of the gonad that contained no lutein-Leydig cells (germinoma). The data suggest that the lutein-Leydtg cells contain 17-hydroxysterotd oxidoreductase and thus are capable of converting androstenedione to testosterone. The lutein-Leydig cells demonstrated a tenfold greater conversion of ‘%-androstenedione- to 14C-test&terone than the area that contained no lutein-Leydig cells. Thus, the lutein-Leydig cells are a potential source of gonadal testosterone in this pattent. Pure gonadal dysgenesis is a rare disorder of unknown etiology in which differentiation of the primitive gonad into either a testicle or an ovary does not seem to occur. Patients with this syndrome present as phenotypic females with primary amenorrhea, tall stature, eunuchoid proportions, minimal breast development, scanty axillary and pubic hair, infantile uteri, and male, female or mosaic chromosomal patterns [ 11. Clitoromegaly may be observed. This syndrome is often confused with Turner’s syndrome [2]. The diagnosis of Turner’s syndrome, however, should probably be reserved for phenotypic females with vestigal streak gonads, underdeveloped external genitalia and a combination of somatic abnormalities including shortness of stature, webbed neck, cubitus valgus and shield-like chest. The patient we describe and other patients with pure gonadal dysgenesis are tall and eunuchoid, and do not have webbed necks. Malignant tumors may develop in the gonads of patients with this syndrome. The tumors are usually gonadoblastomas or germinomas [3-51. In virilized patients with gonadoblastomas, the stromal Leydig or lutein-type cells have been theorized to be the source of androgens. We report a case of pure gonadal dysgenesis with clitoromegaly in which tissue from a gonadoblastoma that contained

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only nests of Leydig or lutein-type ed with 14C-androstenedione and testosterone was measured. For from an adjacent germinoma that dig or lutein-type cells was similarly

cells was incubatconversion to 14Ccomparison, tissue contained no Leystudied.

CASE REPORT

Figure 1. Photograph of patient with pure gonadal dysgenesis (XV).

Figure 2. Left streak gonad and fallopMn tube. The germinoma forms an ovoid mass at the medial aspect of the gonad (indicated by arrow). The gonadoblastoma comprises most of the remaining tissue. 958

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The patient (Figure i), a 23 year old black phenotypic woman, was admitted to the Peter Bent Brigham Hospital for evaluation of primary amenorrhea. She was raised as a female and her growth and development were normal to age 12 years. At that time, her growth rate increased so that she was taller than her peers, but breast enlargement was only minimal and axillary and pubic hair was scanty. At age 16 years, she consulted a physician because of amenorrhea and was given two intramuscular injections of an unknown medication, following which she had scant menstrual flow over a 2 day period. Since that time, she has had no menses. Her family history is significant in that she is taller than both her parents and her siblings. She has three maternal half-sisters whose breast development was normal and who have regular menses. Two of her half-sisters have delivered normal healthy children. Physical examination disclosed a eunuchoid phenotypic female with a height of 71 inches, span of 76 inches and distance from floor to symphysis pubis of 40 inches. Her blood pressure was 120/60 mm Hg, pulse rate 66/min, respirations 16/min and weight 76.4 kg. Examination of the integument showed scant pubic and axillary hair, no striae, buffalo hump, bruises or increased sweating. There was no webbing of the neck and no cubitus valgus deformity. Minimal breast tissue was palpable. The remainder of the examination was unremarkable with the exception of the pelvic examination which disclosed clitoromegaly with normal appearing labia and a vaginal canal 12 cm in length. A uterus was palpable through the vaginal canal. On exploratory laparotomy, a uterus with’fallopian tubes was found with streak gonads present in the broad ligaments. The gonads were removed and examined. Streak gonads were present bilaterally. The left gonad contained a germinoma (dysgerminoma; seminoma) which had arisen in the gonadoblastoma. The right gonad showed hilus cell hyperplasia. Grossly, the left gonad measured 4 by 1 by 1 cm in over-all dimension and contained a 1.0 cm nodule at its medial aspect (Figure 2). The serosal surface was unremarkable. On cut section, the nodule measured 1.4 by 1.0 by 1.0 cm and consisted of fleshy, grayish tan tissue,’ which did not extend through the capsule but was poorly demarcated from adjacent gonadal tissue. The gonadal tissue was grayish white to pale tan, with focal yellow areas, and showed diffuse calcific stippling. Microscopically, the periphery of the gonad was comprised of ovarian-type stroma devoid of ova or follicular derivatives. No testicular tissue was identified. The nodule was a germinoma which was composed of nests of germ cells with scattered foci of calcification, separated by connective tissue septums infiltrated with lymphocytes and occasional plasma cells (Figure 3). The adjacent gonadal tissue was interpreted as a gonadoblastoma. It contained aggregates of large, Volume 57

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rounded cells of the Leydig or lutein-type with abundant eosinophilic cytoplasm, a few nests of immature cells of the Sertoli or granulosa type associated with foci of hyalinization and calcification, and in the stroma, multiple “mulberry-like” foci of calcification (Figure 4). No of crystalloids Reinke were identified. Germ cells were not identified in association with the immature Sertoli or granulosa cells; therefore, the diagnosis of gonadoblastoma could not be considered unequivocal. The right gonad measured 4.5 by 0.7 by 0.4 cm and on cut section consisted of grayish tan, slightly firm tissue with scattered small foci of calcification. Microscopically, ovarian-type stroma was present, but no ova, follicular derivatives or testicular tissue was evident. There were scattered calcium deposits and nodular aggregates of hilus cells, some of which contained yellowish brown pigment and were present in relation to smooth muscle bundles, vessels and nonmedullated nerve fibers. No crystalloids of Reinke were seen. A small number of irregular cleft-like spaces, lined by low cuboidal epithelium, and several tubular structures, which had smooth muscle walls and were lined by ciliated and nonciliated epithelium, were also present, consistent with rete tubules and tubules of mesonephric origin, respectively. Fallopian tubes were present bilaterally. Each measured 5.5 cm in length and 0.4 cm in diameter. Both tubes were well developed histologically. METHODS In Vivo Studies. Twenty-four urine samples were collected daily from 7 AM to 7 AM for determination of 17-ketosteroids, 17-hydroxycorticosteroids and total gonadotrophins. The adequacy of urine collections was determined by the creatinine value. Urine 17-hydroxycorticosteroids were measured as Porter-Silber chromogens by the method of Reddy [6], and urinary 17-ketosteroids were measured employing the Zimmerman reaction as described by Peterson and Pierce [ 71. In Vitro Steroid Incubations. All radioactive steroids were purchased from New England Nuclear Corporation, 575 Albany Street, Boston, Mass. 4-‘4C-androstenedione, specific activity 50 mCi/mmol, and 1,2-3H-androstenedione, specific activity 52 Cilmmol, were purified before use by paper chromatography in the system Skellysolve:benzene: methanol:water 71214~1v/v and found to be 98 to 99 per cent pure and were used without further purification. Testosterone, melting point 154’ to 156O, and androstenedione, melting point 172’-173’ (Steraloids, Pawling. N.Y.) were used without further purification. LiquifluoP (New England Nuclear Corporation, Pilot Chemical Division, No. NEF-903) was used for prep’lring liquid scintillation counting solution as described. Samples of germinoma and gonadoblastoma tissue were taken and minced. A sample of gonadoblastoma tissue was boiled for 10 minutes in 1 ml distilled water and used as a control. Then all three sample tissues (control, germinoma and gonadoblastoma) were incubated for 1 hour at 37’C in a 95 per cent oxygen15 per cent carbon dioxide, atmosphere in 2 ml of a pH 7.4 Krebs-Ringer bicarbonate buffer containing 2 mg of dextrose and 0.048 &i “C-androstenedione. After 1 hour, 8 ml methanol was December

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Germinoma in left streak gonad. The tumor is Figure 3. comprised of primitive germ ceils with intervening stroma infiltrated by lymphocytes and plasma cells. Hematoxylin and eosin stain; original magnification X 320. reduced by 50 per cent

added to each sample to stop the steroidogenesis. The samples were diluted to 10 ml and a 1 per cent (0.1 ml) aliquot was removed. 3H-testosterone, 15,000 dpm, was added to the remaining 99 per cent (9.9 ml) of every sample. The doubly labeled testosterone was then extracted and purified to constant 3H:14C ratio by paper chromatography in several different solvent systems. The complete procedure of extraction, purification and chromatography has been described by Rose et al. elsewhere [a]. 3H and 14C were assayed simultaneously in a Nuclear Chicago Unilux liquid II scintillator counter. Efficiencies the in tritium channel were 31 per cent for 3H and 9 per cent ?or 14C. In the 14C channel the efficiency was 52 per cent for j4C and the discriminator settings were adjusted to give no contribution of 3H into this channel. The contributions from 3H and 14C were calculated according to the method of Okita et al. [9].

Figure 4. Gonadobiastoma in left streak gonad. Nest of with immature Sertoli and/or granulosa cells (arrow) focal hyalinization. Surrounding stroma contains clusters of iutein and/or teydig type cells and foci of calcification. Hematoxylin and eosin stain; original magnification X 200, reduced by 50 per cent. 1974

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Testosterone Formation by Gonadal Tissue

TABLE I A.

ET AL.

tn Vitro Conversion of 4-1%Androstenedione

(X-A)

to 4-X-Testosterone

(X-T)

‘4C-A (dpm incubated)

(dpm after system 3)

3H-T (indicator % recovered)

Corrected for “H recovered

% Conversion %‘-A to WT 0.19 1.91

14C.T Incubated Gonadal Tissue

(w)

by Gonadal Tissue

Germinoma Gonadoblastoma

18.7 25.4

104,000 104,000

26.6 238.8

15.6 13.4

170.6 1,770.8

Boiled gonadoblastoma (control)

42.1

104,000

0

14.6

...

B. ‘Ratio of 3H:X

0

During Paper Chromatographic Purification of Testosterone Chromatography System

Gonadal Tissue Germinoma Gonadoblastoma Boiled gonadoblastoma

(control)

1

2

3

34.7 8.7 73

100.3 9.9 503

102.4 9.9 1,840

RESULTS

COMMENTS

Urinary 17-hydroxycorticosteroid excretion was 5.3 mg/24 hours and 17-ketosteroid excretion was 12.4 mg/24 hours. Urinary gonadotrophins were positive at 100 muw on three separate occasions. White blood cell chromosomes showed a 46/XY pattern on two different occasions. Of the incubated 14C-androstenedione 9 1.9 per cent was recovered unchanged from the germinoma as contrasted to only 85.6 per cent recovered from the gonadoblastoma. The control incubation showed a 94.9 per cent recovery of “C-androstenedione. The results of the in vitro studies are summarized in Table I. The germinoma showed a 0.19 per cent conversion to 14C-testosterone, whereas the gonadoblastoma showed a 1.91 per cent conversion. Purification of the 14C-testosterone was achieved by paper chromatography in three different solvent systems and formation of the acetate derivative prior to the last chromatography. The small amount of ‘“Ctestosterone available at the final stage of the purification procedure was insufficient for purposes of crystallizing to constant specific activity with authentic testosterone. However, specificity of the final : measurement of testosterone (as testosterone acetate after chromatography in system 3) was indicated by the constant 3H*‘4C . ratio over the last two chromatographies in between which the acetate derivative was prepared (Table I).Statistical radioactivity counting errors were determined according to the method of Horton and Tait [lo] _ Every sample vial was counted. for a minimum total of 2,000 counts. Thus, the maximum error on the measurement of 14C-androstenedione with a background of 10 cpm was 2.5 per cent. The counting error on the tritium assay was 1.O per cent, with a background of i2 cpm in the 3H-testosterone channel.

Judd et al. [ 1 l] have shown a threefold gradient of testosterone concentration between gonadal and peripheral vein blood in a 24 year old patient with pro-

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gressive hirsutism and pure gonadal dysgenesis (XX). They theorized that the luteinized stromal cells and hilus cells found in the streak gonads might be the source of the gonadal testosterone [ 111. The present study permits further evaluation of the cell type ‘involved in the. production of androgens in patients with this syndrome. We present data on a patient with pure gonadal dysgenesis (XV) and clitoromegaly in whom streak primitive gonads were found. The left gonad contained a germinoma (seminoma; dysgerminoma) which had arisen in a gonadoblastoma. Tissue was taken from the germinoma which contained no lutein or Leydig-like cells and from an area of the gonadoblastoma which contained many eosinophilic lutein or Leydig-like cells and incubated separately with 14Candrostenedione. The tissue, gonadoblastoma, containing lutein or Leydig-like cells showed a tenfold greater conversion to 14C-testosterone than did the germinoma, thus confirming the Leydig or lutein-type cells in this primitive gonad as a potential gonadal source of the testosterone in this patient. The hyperplastic hilus cells present in the right streak gonad possibly may have contributed to testosterone production in this patient. However, hilus cell hyperplasia is not consistently associated with virilization [ 12- 151. Studies by Bardin et al. [ 161 in a virilized case of XY pure gonadal dysgenesis with Leydig cell hyperplasia showed that the Leydig cells produced a large amount of testosterone from labeled acetate. This is similar to our findings, with the further clarification that although the lutein-Leydig cells in our case did

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exhibit more 17dehydrogenase activity than the nonlutein-Leydig cell tissue, both tissues showed conversion of androstenedione into testosterone. Bartlett et al. [ 171 incubated gonadal tumor tissue (germinoma arising in a gonadoblastoma) from a patient with XY pure gonadal dysgenesis and were unable to show any estrogen synthesis from the precursors progesterone, 17-alpha-hydroxyprogesterone, pregnenolone, 17-alpha-hydroxypregnenolone or 19hydroxyandrost-4-ene-3,17-dione; but they were able to show conversion of both pregnenolone and progesterone to testosterone by the tumor. Griffiths et al. [ 181, however, were able to demonstrate synthesis of both 17-beta-estradiol and testosterone from progesterone in the dysgenetic gonadal tumor (XV) they studied. Therefore, it should be noted that not all dysgenetic gonadal tumors exhibit the same steroidal metabolic pathways. Thus, the reports cited and our present study confirm that the dysgenic gonad is a potential site of tes-

DYSGENESIS-ROSE

ET AL.

tosterone production. The lutein-Leydig-like cells contain 17-hydroxysteroid oxidoreductase and are potential gonadal sources for the androgen excess leading to clitoromegaly or virilism. Our patient also demonstrated tumor formation in one of the streak gonads. In the study of Teter and Boczkowski [3], 8 of 25 patients with pure gonadal dysgenesis were found to have tumors. (One of these patients was XO/XY and may represent mixed gonadal dysgenesis rather than pure gonadal dysgenesis.) All eight of these patients as well as ours were chromatin-negative. This emphasizes the necessity for complete surgical removal of both streak gonads in patients with XY pure gonadal dysgenesis. ACKNOWLEDGMENT We wish to thank Robert Scully, M.D. for reviewing the microscopic slides of the tissues and Mary T. Dunning for excellent technical assistance. This work was supported by Schering Pharmaceuticals.

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6. 7.

8.

9.

10.

11.

Sohval AR: The syndrome of pure gonadal dysgenesis. Am J Med 38: 615, 1965. Turner HH: A syndrome of infantilism, congenital webbed neck and cubitus valgus. Endocrinology 23: 566, 1938. Teter J, Boczkowski K: Occurrence of tumors in dysgenetic gonads. Cancer 20: 1301, 1967. Scully RE: Gonadoblastoma. Cancer 25: 1340. 1970. Schellhas HF. Trujillo JM, Rutledge FN, Cork A: Germ cell tumors associated with XY gonadal dysgenesis. Am J Obstet Gynecol 109: 1197, 1971. Reddy WJ, Jenkins D, Thorn GW: Estimation of 17-OHCS in the urine. Metabolism 1: 5, 1952. Peterson RE, Pierce CE: Determination of urinary neutral 17-ketosteroids, Lipis and the Steroid Hormones in Clinical Medicine (Sunderman FW. Sunderman FW Jr, eds), Philadelphia, J. B. Lippincott Co., 1960, p 158. Rose LI, Underwood RH, Parker DE, Williams GH: A4-3-ketosteroid 5a reductase in the adult human testis. J Clin Endocrinol Metab 36: 995, 1973. Okita GT, Kabara JJ, Richardson F, Leroy GB: Assaying compounds containing 3H and 14C. Nucleonics 15: 111, 1957. Horton R, Tait JF: In vivo conversion of dehydroisoandrosterone and testosterone in man. J Clin Endocrinol Metab 27: 79, 1967. Judd HL, Scully RE, Atkins L, Neer RM, Kliman B: Pure go-

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nadal dysgenesis with progressive hirsutism. N Engl J Med 282: 881, 1970. Greenbfatt RB, Carmona N, Higdon L: Gonadal dysgenesis with androgenic manifestations in the tall eunuchoid female. J Clin Endocrinol Metab 16: 235. 1956. Gordon GS, Overstreet EW, Trout HF. Winch G: A syndrome of gonadal dysgenesis. J Clin Endocrinol Metab 15: 1, 1955. Dougherty CM, Thompson JD: Adrenal cortical and Leydig cell nodules in dysgenetic gonads. Am J Obstet Gynecol 80: 317, 1960. Guimet P, Mathieu J, Tatin J: Etude histologique de I’ovaire dans un cas de Syndrome de Turner. Ann Endocrinol (Paris) 15: 499, 1954. Bardin CW, Rosen S, LeMaire WJ, Tjio JH, Gallup J, Marshall J, Savard K: In vivo and in vitro studies of androgen metabolism in a patient wtth pure gonadal dysgenesis and Leydig cell hyperplasia. J Clin Endocrinol Metab 29: 1429, 1969. Bartlett DJ. Grant JK, Pugh MA, Aherne W: A familial feminizing syndrome. J Obstet Gynecol Br Commonw 75: 199, 1968. Griffiths K, Grant JK, Browning MCK, Whyte WG. Sharp JL: Steroid synthesis in vitro by tumor tissue from a dysgenetic gonad. J Endocrinol 34: 155, 1966.

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