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A histochemical study of steroid 3β-ol dehydrogenase activity in some steroid-producing tumors
A histochemical study of steroid 3fi·OI dehydrogenase activity in some steroid-producing tumors B. GOLDBERG, PH.D. G. E. SEEGAR JONES, M.D. J.D. WOODRUFF, M.D. Baltimore, Maryland
T H E s E results represent part of a histochemical study undertaken to elucidate steroid biogenesis in the human tissues, ovary, adrenal, placenta, and testis. The steroid 3 [3-ol dehydrogenase, described by Samuels1 and demonstrated histochemically by Wattenberg/ was chosen as it is apparently an essential enzyme in the degradation of cholesterol for the synthesis of progesterone, androgens, or estrogens (Fig. 1). With the use of dehydroepiandrosterone as a substrate it was possible, by gas liquid chromatography, to correlate the histochemical reaction with the generation of £1 4 -androstenedione in the substrate, thus confinning the validity of the histochemical reaction. 3 To test the over-all enzymatic activity of the tissues studied, parallel assays of the DPN (diphosphopyridine nucleotide) diaphorase, and lactic dehydrogenase activity were made, and routine fat stains were performed. The histochemical studies indicated that the steroid 3 [3-ol dehydrogenase activity
was high in those cells presumably concerned with progesterone production but seldom readily detectable in loci of normal tissues presumably concerned with androgen or estrogen production. 3 As these findings are somewhat different from others reported4 and at variance with the findings in rodents/· 5 • 6 further investigation of human tissues seemed necessary. It has been possible to study 3 estrogenproducing tumors arising from 3 different organ sites (ovary, adrenal, and testis) as well as one ovarian tumor producing androgens and one ovarian tumor producing progesterone. The histochemical findings are reported and the results discussed. Methods The surgical tissues examined in this study were frozen as soon as feasible after excision (a few minutes to within an hour) as previously described. 7 For steroid 3 [3-ol dehydrogenase, sections v;ere cut at 24 fL
and incubated from 1 to 15 hours in the incubation mixture described by Wattenberg2 or in a simplified medium which appeared equally satisfactory (0.15 M phosphate buffer, pH 7.4, DHA, and Nitro BT). The methods of N achlas and co-workers8 • 9
From the Department of Gynecology and Obstetrics and the Department of Pathology, The fohns Hopkins University School of Medicine and Hospital. Supported by a research Grant A-3806-(Cl) from the United States Public Health Service.
v;ere used for most other dehydrogenases
studied. Reduced coenzyme I (DPNH) as substrate was used at a concentration of 0. 7 1003
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The three estrogen-producing tumors, an ovarian thecoma, a testicular interstitial cell tumor associated with gynecomastia, and a feminizing adrenal tumor all failed to show any activity for a steroid 3 {J-ol dehydrogenase although they are otherwise enzymically active and show positive fat stains (Figs. 2, 3, and 4). The case reports and the pathologic aspects of the tumors are discussed in detail below. Case 1. The patient, H. W., was a 28-year-old Negro woman whose major complaint was amenorrhea of 10 months' duration. Her menses had begun at the age of 15 years and were regu· Jar, every 28 days, and of 8 to 10 days' duration with heavy flow and cramps. She had been married 12 years. She had had 5 pregnancies and 5
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Fig. 1. Scheme depicting sex hormone biogenesis.
living children, the oldest 11 years and the youngest 22 months. Her menses had returned after a nonnal delivery and remained regular until the sudden cessation II months prior to initial hospitalization. Aside from the fact that she had gained 36 pounds, she had had no symptoms indicating a pregnancy. The physical examination showed a well-developed, somewhat obese Negro woman with blood pressure of 130/90 mm. Hg, no hirsutism or acne, no breast masses or secretion, and no abdominal masses, ascites, or tenderness. Tht~ pelvic examination was significant in that the vaginal mucosa showed a normal estrogenic reaction. The fundus was in l degree retroposition and, although difficult to outline, seemed to he enlarged. There was an 8 to 10 em. semisolid cystic mass in the right adnexal area and neither ovary was palpable. A total urinary estrogen level by the Brown method1 " was 18 pg per 24 hours; 6 p.g estrone, 3 p.g estradiol, and 9 /J-g estriol. The maturation index was 0 j73 j27 and the endometrium was proliferative in type (Fig. 5). The pathology examination showed a right ovarian tumor of 105 grams, 7 em. in diameter. On cut surface, no normal ovarian tissue was
Volume 86 Numbo" 8
identified. The tissue was reddish in color and small cysts, 3.5 mm. in diameter, were scattered throughout. Microscopically the tumor showed the char· acteristic water-silk appearance associated with some granulosa cell tumors (Fig. 2, A). Fine fat droplets were demonstrated throughout the tumor. No steroid 3 fi-ol dehydrogenase was de-
Steroid 3fi-ol dehydrogenase activity 1005
teetable although the lactic dehydrogenase activity was marked (Fig. 2, B, C, D). Case 2. The patient, M. G., was a 3-year-old Puerto Rican child. Five months prior to admission, a vaginal discharge had been noted and shortly thereafter bilateral areolar and nipple enlargement. The pediatrician ordered an x-ray bone survey, and a bone age of 6 to 7 years was
A
B
Fig. 2. Case 1. A, Ovarian granulosa cell tumor; associated estrogen production. Hematoxylin and eosin stain showing water-silk pattern. B, Stained with hematoxylin and oil red 0 photographed with blue filter to show fat droplets. Minimal to moderate amounts of lipid present.
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Fig. 2. C, 15!-' section incubated with sodium lactate and DPN. pH 74. :i 7' C" lor '·, hour. Tumor cells show pronounced lactic d~hydrogenase activity. ( X208.) D, '2.5!-' sPrtion incubated overnight in presence of DHA and DPN. There is uo evidence of steroid 'Vl-ol dehydrogenase activity in sections similar to those shown in A and B above. (·· 208.)
determined. The rectal examination was negative for tumor. A vaginal smear was thought to shovv a questionable estrogen effect and the uri~ nary 17-ketosteroid determinations were 21 and 28 mg. per 24 hours. The intravenous pyelogram was normaL A menstrual period occurred 2 months after onset of vaginal discharge. In spite of cortisone suppression therapy, repeat 17-ketosteroid determinations were 32 and 33 mg. per 24 hours. Total estrogens were 33 J..tg per 24 hours (estrone 10, estradiol 4, and estriol 19), and 35 J..tg per 24 hours, with similar distributions. The diagnosis of an adrenal tumor was made and she was referred to Johns Hopkins Hospital for surgery. The physical examination showed a child who was large for 3 years, alert, and precociously developed. There was beginning axillary hair with some pubic hair. Both breasts were well developed with erect nipples and deep areolar pigmentation. There was no hirsutism; no abdominal or pelvic masses were palpable by rectal examination. A retroperitoneal carbon dioxide
insufflation study revealed a discrete mass in the right suprarenal area. At operation a tumor was removed Vlith the right adrenal gland. The pathology examination showed a soft yellowish brown tumor of the right adrenal measuring 4. 7 by 1.8 em. On cut surface the adrenal tumor seemed to be completely encapsulated and normal adrenal gland was identified outside of the capsule. Microscopically the tumor cells were fairly uniform in size with relatively pyknotic nuclei of varying size and abundant eosinophilic cytoplasm. Although some anaplasia and pleomorphism were present, the diagnosis of a benign adrenal adenoma was made because of lack of capsule or vessel invasion (Fig. 3, A). A section through the normal adrenal and adjoining fragment of the adrenal tumor is shown after incubation in DHA for steroid dehydrogenase activity (Fig. 3, C). The normal tissue showed considerable activity while the tumor was quite inactive. In contrast, incubation with another DPN -linked substrate, lactic acid,
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Steroid 3,8-ol dehydrogenase activity 1007
B
Fig. 3. Case 2. Adrenal tumor inducing precocious puberty (all sections similarly oriented). A, Stained with hematoxylin and orange G and photographed with blue filter. Cortex of normal adrenal at bottom of photo, adrenal capsule in center, and tumor at upper third. (x40.) Fat is most prominent in the normal adrenal but is also present in large amounts in the tumor. B, Secti.on similar to 3A, 25.u, incubated for 45 minutes with sodium lactate and DPN at pH 7.4. The tumor cells show more lactic dehydrogenase activity than the normal adrenal. (x40.) C, Section, 25~t, incubated 3 hours with DHA as indicated in 2. D. The steroid 3/3-ol dehvdrogenase activity is confined essentially to. the nom1al adrenal cortex shown in lower part of figure, oriented with glomerular zone up; the tumor, which, as in 3A and B, is in upper part of photograph, is inert. ( x40.)
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A Fig. 4. Case 3. Feminizing testicular interstittal eel! turnor. A, Hernatoxylin and eosin stain showing well-differentiated cells, fairly abundant eosinophilic cytoplasm, and eccentric nuclei. B, 151" section from another portion of this tumor incubated for 8 minutes with 0.7 mg. per milliliter DPNH in 0.15 M phosphate buffer at pH 7A. Tumor cells have a very active DPN diaphorase. (x208). C, 25Jl section from same block as shown in 4, B incubated with DHA for 2 hours as in 2, C. No discernibl•· steroid 3fl·ol dehydrogenase activity. i:<'W8.)
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Volume 86 Numbe. 8
resulted in clear-cut acttvity in both nonnal tissue and tumor cells (Fig. 3, B). Cas-e 3. This patient, H. T., a 44-year-old man noticed a tumor of the left testis about 1 year prior to operation but was unable to persuade 2 different urologists that it existed. He stated that it had not increased in size. Six weeks prior to his admission, swelling of the left breast occurred. A urinary chorionic gonadotrophin assay was negative. The physical examination was remarkable only in that the left breast was flypertrophied, typical of gynecomastia. There was a 3 mm. mass in the left testis just beneath the globus major. Laboratory studies are missing as the preoperative specimens for estrogens were lost. Only the tumor was removed, and 6 weeks after operation, gynecomastia had regres:;ed. The tumor was encapsulated and about 8 mm. in diameter. The microscopic examination showed large pleomorphic cells with abundant eosinophilic cytoplasm. The diagnosis was made of an interstitial cell tumor of the testis (Fig. 4, A). The histochemical studies showed no detectable steroid 3 {3-ol dehydrogenase activity in the tumor cells, when DHA was the substrate (Fig. 4, C). The lactic dehydrogenase activity \vas appar·~nt at 15 minutes and the DPN diaphorase was even more active (Fig. 4, B).
Fig. 5·, Case 1. Hematoxylin and eosin stain of endometrium showing actively prolif~:rating tissue from patient whose ovarian tu· mor study is shown in Fig. 2. (x208.)
Steroid 3{3-ol dehydrogenase activity 1009
An arrhenoblastoma showed only minimal steroid 3{3-ol dehydrogenase activity in the Leydig-like cells. Case 4. The patient, J. C., a 15-year-old girl, was excreting 18 to 22 mg. of 17-ketosteroids daily, and was markedly virilized; Reinke crystals were present in the tumor tissue, all strong presumptive evidences that testosterone was being produced. The findings in this patient are being reported in detail elsewhere, therefore, no further clinical data will be included. The left ovary measured 10.5 by 5 by 2.5 em. It was pearly white and typical of an "oyster ovary" described in the Stein syndrome. On cut surface the cortex was replaced by numerous cysts and no definite tumor nodule could be made out. Microscopically the tumor occupied the hilus of the ovary and was composed of two types of tissue; an adenomatous area and islands of pale staining cells with prominent, rather small nuclei. Many Reinke crystals were present in the tumor tissue. A diagnosis was made of arrhenoblastoma (adenoma testiculare) in ovaries compatible with the diagnosis of the Stein syndrome. The steroid 3 {3-ol dehydrogenase was minimal, although fat was demonstrated in the Leydig-like cells and DPN diaphorase showed ac· tivity throughout (Fig. 6, A, B, C, and D).
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A Fig. 6. Case +. Masculinizing arrh<:>noblastoma found in ovary of 15-year-uld patient. A. Sr·rtiun stained with hematoxylin .md oil red () and photographed with yellow filter, showing fat deposition in Leydig-like cells in the ovarian tuwor. ( ss :~cti\·e. (
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A luteinized granulosa cell tumor, which was associated with evidence of marked progesterone secretion, as judged by the decidual reaction in the endometrium (Fig. 7), gave a 1narked reaction for the steroid 3{3-o! dehydrogenase in those areas of the tumor which were luteinized. U nluteinizec! cell areas wen' inactive.
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Case 5. The patient, C. (:. was :1 :i-vt·ar-uid child wlws•· n>~nplaint wa' \·agirul disch~rg•', breast dt~velopnwnt, and abdominal m·dling ol 6 weeks' durarion. On physical examination no axillary hair was present hut the breasts wen~ hnwrtrophicd 'vith erect nipples and sorue areolar pigmentation. There was no nipple discharge. The external genitals showed no pubic hair, wme hypf'rtrophy and pigmentation of the labia, and a mucoid vaginal discharge. The abdomm was distended; th<'t'l' was a fluid wave aud a midline lower abdominal mass which was smooTh, tirm, and freely movabl1•. The rectal examination was confirmatory, hut it was impossihl" to determine whether the mass arose from th(' uterus or ovary. The maturation index wa~ o 1 9~)/ l. Total estrogt·ns w•·n• :!.(J/ ftg per 2'~ hours; estrone O.l:i, est r:Hli< d 0. 7-'i, and t~striol 1.19 ftg per !.4 hours. At operation a large left ovarian tumor was found with 1,:iOO mi. of bloody ascites. No cvidenn· of Illetastasis was seen. The left ovctrian tumor weighed 290 grams and measun·d 12 hy I 0 by 8 em. It sPemed cornpleff'ly encapsulated, of soft consistency, and was yellow in color Pxcept for hemorrhagic art> as. On cut ,,,,.t ion there were several small cystic areas :·) trltll. in dianlf'ter filled \vilh blood , lot; the remaining solid portion of th<, llltlltJl \\'as divided intu lobules by dPnse comwdiH· tissur· s•·pt<'- In sfllllt' areas tlw tumor \las :1 bright golden yt'llow, in others light tan, and still other ar••as appt>ared lwnwrrhagic. The tumor rdls Wt'l'l' morphologically predominantly granulosa et'lls but there wc·n· manv thecal elements presf'nt. Both types oi ct>lls showt•d extmsive luteinization (Fig. 8, A. and B). 'T'ht~ endornetrillnl
Fig. 6. B
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Steroid 3,.8-ol dehydrogenase activity 1011
Fig. 5. C
Fig. 6. D
decidual reaction with focal areas of hemorrhage (Fig. 7). With DHA as a substrate the tumor in this patient showed large areas of inactive cells interspersed with areas of intense activity comparable to that found in a corpus luteum (Fig. 8, D). Thrcughout there were strands of ceils with less intense activity. All areas of the tumor showing activity were composed of luteinized cells. The assay for LDH showed a more general reac1 ion but the areas of luteinization were most
intensely reactive. The assay with DPNH showed essentially the same pattern as with lactic acid (Fig. 8, C, left).
Fig. 7. Case 5. Hematoxylin and eosin-stained section of endometrial stroma showing marked decidual reaction. (x208.) The associated ovarian tumor is shown in Fig. 8.
Comment
Data have been accumulated which show that the biogenesis of sex horm~es, progesterone, testosterone, and estradiol, may proceed by cholesterol degradation to pregnenolone and from this compound to
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A Fig. 8. Case 5. A, Hematoxylin and l'osin stain ,f >.!ranulosa '.-II tl!JJJ<>J of ovary, with marked luteinization, in 5·-yr•ar-old p.tli<·ni showing Hurk<'r el!"mcnts l:wltm and to the right. S!"rtion on th<> leh. 1'iP.. inct~hat•·d with DPNJ I f.,, i.'i minutes: S!"ction on the right, '2'iJ.L, incubatf'd with DHA for 5", hour.' The DPN diaphorase activity is more widespread 1. ·, D. High-JXIW<'< photomicrograph of section "" right in R. l.. Tht·J ,. is mMk<'d \fl., ,J 1
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decreased activity. The diffprence itl .-.cti,·ity i' ,~omparahk 10 thd.t lw tween th(' lutein Cf•lls of a normal q,rrllt' illtt•ttll• .. ,,,.) th•· tlw, ·' iutt·rn:; i
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progesterone. testosterone, and finally r~st ta ~ clio!, in this sequence 11 ' 1 ~, i\ suggestion ha:-; hePn made that dehydroepiandrosterom· SO, (DHA-50 4 ) may be the preferential precursor for testosterone and estradiol. also by way of L; 1-androstenc-3, 17 -diom·. For either of these pathways, a steroid ·; P-ol dehydrogenase appears involved. Histochemical data show that steroid :l,B-ol dehydrogenase is readily demonstrable in those tissues which arc considered to be producing relatively large amounts of progesterone, e.g., the corpus luteun1, placenta, and adrenal."· J. "·" Jn human tissues which are presumably producing androgens and estrogens, this enzyme activity is either absent or low." The data reported in the present paper
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'teroid-produt·iru~: !t!!ltl)r5~ !H) :1/)-u! t!(·!!)dtfl· gPnase activity '"1s fn11nd ill Litntor., fro111 patients who t'xhihitnl climcal ,., irkm t' nt •·xcessin' estrogen prnd twnon ,\ n ,u rlwno-
blastoma as~ociated with lltarkl'd din!< a~ \·irilization had minim:1l aniYit\ ln n•n t rast, a. lllteini;rt>rl ~Tanulnsa n·ll ltlltinr "! the ovary, associated witlt marked dccidttdi reaction in tht· t'ndometmllll. had maxitnal hut Ill thf' lutc·inii't·d :rr·t'a' nnh This activitY \\-as comparable tu that st't'll in the most acti\·e tissue' which we h;n•· •·ncounten·d. n>rpora ltttt'a. The failure to demonstrate ihi~ dctivit\ might be the result of an enzyuw deficiencY a reduced supplv of sorrH' ratf'-limitin~ en. factor. nr !hf' fH'csenCt' nf •·xr·t·"i\·t· .nnnttllt'
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Steroid 3(1-ol dehydrogenase activity 1013
Volume :!16 Number 8
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of inhibitor. It might signify a different biogenetic pathway in the human gonads for estrogen and testosterone synthesis from that for, or by way of, progesterone. Howard13 has suggested such an alternate pathway for estrogens which might not necessitate a 3[:?-ol dehydrogenase activity. If DHA-S0 4 , which Burstein and Dorfman 14 have indicated might arise directly from cholesterol without the preliminary t:- 0 pregnen-3[:?-ol, 20-one formation, was the substrate, C-19 and ring A oxidation might occur without steroid 3[:?-ol dehydrogenase
REFERENCES
1. Samuels, L. T., Helmreich, M. L., Lasater, M. B., and Reich, H.: Science 113: 490, 1951. 2. Wattenberg, L. W.: J. Histochem. 6: 225, 1958. 3. Goldberg, B., Jones, G. E. S., and Turner, D. A.: Steroid 3/3-ol dehydrogenase activity in human endocrine tissues. (In press.) 4. Deane, H. W., Lobel, B. L., and Romney, S.: AM. J. OusT. & GYNEC. 83: 281, 1962. 5. Levy, H., Deane, H. W., and Rubin, B. L.: Endocrinology 65: 932, 1959. 6. Niemi, M., and Ikonen, M.: Endocrinology 70: 167, 1962. 7. Fitz-Williams, W. G., Jones, G. E. S., and Goldberg, B.: Stain Techno!. 35: 195, 1960.
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1
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activity and the end result wotdd lw t·~trorw which could then be converted to estradiol by reduction at the 17- position 1Fig. !I. Admittedly, the evidence for this pathwa::is meager at best, but it pn'sents a possibility for estrogen synthesis from cholesterol that is not dependent on steroid :1(:/-ol dehydrogenasP activity. As such. it appt'at' to be in better accord with the histochelllical data. It is understood that all of the entymir systems necessary to produce a 1l of t ht· theoretical reactions depicted have heen demonstrated in all steroid-producing tissues. Howewr, the fluctuations in these the enzvmPs m· ----- cofartors ----------- which -------- ac! iv;c~ fl' them must surely determine which way th<' equilibrium will be shifted. The technique of histochemistry is a valuable t
~--------
8. Nachlas, M. M., Walker, D. G., and Seligman, A. M.: J. Biophys. & Biochem. Cytol. 4: 467, 1958. 9. Nachlas, M. M., Walker, D. G., and Seligman. A. M.: .J. Biophys. & Biochem. Cytol. 4: 29, 1958. 10. Brown, J. B.: Biochem. J. (W: 185, 1955. 11. Mason, N. R., and Samuels, L. T.: Endocrinology 68: 899, 1961. 12. Smith, 0. W., and Ryan, K. J.: Endo~rin ology 69: 970, 1961. 13. Howard, E.: Fed. Proc. 21: 2, 1962. 14. Burstein, S., and Dorfman, R. I.: Acta endocrinol. 40: 188, 1962. 601 North Broadway Baltimore, Maryland
T H E s E results represent part of a histochemical study undertaken to elucidate steroid biogenesis in the human tissues, ovary, adrenal, placenta, and testis. The steroid 3 [3-ol dehydrogenase, described by Samuels1 and demonstrated histochemically by Wattenberg/ was chosen as it is apparently an essential enzyme in the degradation of cholesterol for the synthesis of progesterone, androgens, or estrogens (Fig. 1). With the use of dehydroepiandrosterone as a substrate it was possible, by gas liquid chromatography, to correlate the histochemical reaction with the generation of £1 4 -androstenedione in the substrate, thus confinning the validity of the histochemical reaction. 3 To test the over-all enzymatic activity of the tissues studied, parallel assays of the DPN (diphosphopyridine nucleotide) diaphorase, and lactic dehydrogenase activity were made, and routine fat stains were performed. The histochemical studies indicated that the steroid 3 [3-ol dehydrogenase activity
was high in those cells presumably concerned with progesterone production but seldom readily detectable in loci of normal tissues presumably concerned with androgen or estrogen production. 3 As these findings are somewhat different from others reported4 and at variance with the findings in rodents/· 5 • 6 further investigation of human tissues seemed necessary. It has been possible to study 3 estrogenproducing tumors arising from 3 different organ sites (ovary, adrenal, and testis) as well as one ovarian tumor producing androgens and one ovarian tumor producing progesterone. The histochemical findings are reported and the results discussed. Methods The surgical tissues examined in this study were frozen as soon as feasible after excision (a few minutes to within an hour) as previously described. 7 For steroid 3 [3-ol dehydrogenase, sections v;ere cut at 24 fL
and incubated from 1 to 15 hours in the incubation mixture described by Wattenberg2 or in a simplified medium which appeared equally satisfactory (0.15 M phosphate buffer, pH 7.4, DHA, and Nitro BT). The methods of N achlas and co-workers8 • 9
From the Department of Gynecology and Obstetrics and the Department of Pathology, The fohns Hopkins University School of Medicine and Hospital. Supported by a research Grant A-3806-(Cl) from the United States Public Health Service.
v;ere used for most other dehydrogenases
studied. Reduced coenzyme I (DPNH) as substrate was used at a concentration of 0. 7 1003
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mg. per milliliter in 0.15 M phosphate buffer (Na2HP04 and KH 2 P04 , pH 7.1). The length of incubation time (8 minutes to 18 hours) and section thickness ( 10 to 25 p.) were varied, depending on the intensity of reaction obtained. Results
The three estrogen-producing tumors, an ovarian thecoma, a testicular interstitial cell tumor associated with gynecomastia, and a feminizing adrenal tumor all failed to show any activity for a steroid 3 {J-ol dehydrogenase although they are otherwise enzymically active and show positive fat stains (Figs. 2, 3, and 4). The case reports and the pathologic aspects of the tumors are discussed in detail below. Case 1. The patient, H. W., was a 28-year-old Negro woman whose major complaint was amenorrhea of 10 months' duration. Her menses had begun at the age of 15 years and were regu· Jar, every 28 days, and of 8 to 10 days' duration with heavy flow and cramps. She had been married 12 years. She had had 5 pregnancies and 5
€$TRADIOL
Fig. 1. Scheme depicting sex hormone biogenesis.
living children, the oldest 11 years and the youngest 22 months. Her menses had returned after a nonnal delivery and remained regular until the sudden cessation II months prior to initial hospitalization. Aside from the fact that she had gained 36 pounds, she had had no symptoms indicating a pregnancy. The physical examination showed a well-developed, somewhat obese Negro woman with blood pressure of 130/90 mm. Hg, no hirsutism or acne, no breast masses or secretion, and no abdominal masses, ascites, or tenderness. Tht~ pelvic examination was significant in that the vaginal mucosa showed a normal estrogenic reaction. The fundus was in l degree retroposition and, although difficult to outline, seemed to he enlarged. There was an 8 to 10 em. semisolid cystic mass in the right adnexal area and neither ovary was palpable. A total urinary estrogen level by the Brown method1 " was 18 pg per 24 hours; 6 p.g estrone, 3 p.g estradiol, and 9 /J-g estriol. The maturation index was 0 j73 j27 and the endometrium was proliferative in type (Fig. 5). The pathology examination showed a right ovarian tumor of 105 grams, 7 em. in diameter. On cut surface, no normal ovarian tissue was
Volume 86 Numbo" 8
identified. The tissue was reddish in color and small cysts, 3.5 mm. in diameter, were scattered throughout. Microscopically the tumor showed the char· acteristic water-silk appearance associated with some granulosa cell tumors (Fig. 2, A). Fine fat droplets were demonstrated throughout the tumor. No steroid 3 fi-ol dehydrogenase was de-
Steroid 3fi-ol dehydrogenase activity 1005
teetable although the lactic dehydrogenase activity was marked (Fig. 2, B, C, D). Case 2. The patient, M. G., was a 3-year-old Puerto Rican child. Five months prior to admission, a vaginal discharge had been noted and shortly thereafter bilateral areolar and nipple enlargement. The pediatrician ordered an x-ray bone survey, and a bone age of 6 to 7 years was
A
B
Fig. 2. Case 1. A, Ovarian granulosa cell tumor; associated estrogen production. Hematoxylin and eosin stain showing water-silk pattern. B, Stained with hematoxylin and oil red 0 photographed with blue filter to show fat droplets. Minimal to moderate amounts of lipid present.
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Fig. 2. C, 15!-' section incubated with sodium lactate and DPN. pH 74. :i 7' C" lor '·, hour. Tumor cells show pronounced lactic d~hydrogenase activity. ( X208.) D, '2.5!-' sPrtion incubated overnight in presence of DHA and DPN. There is uo evidence of steroid 'Vl-ol dehydrogenase activity in sections similar to those shown in A and B above. (·· 208.)
determined. The rectal examination was negative for tumor. A vaginal smear was thought to shovv a questionable estrogen effect and the uri~ nary 17-ketosteroid determinations were 21 and 28 mg. per 24 hours. The intravenous pyelogram was normaL A menstrual period occurred 2 months after onset of vaginal discharge. In spite of cortisone suppression therapy, repeat 17-ketosteroid determinations were 32 and 33 mg. per 24 hours. Total estrogens were 33 J..tg per 24 hours (estrone 10, estradiol 4, and estriol 19), and 35 J..tg per 24 hours, with similar distributions. The diagnosis of an adrenal tumor was made and she was referred to Johns Hopkins Hospital for surgery. The physical examination showed a child who was large for 3 years, alert, and precociously developed. There was beginning axillary hair with some pubic hair. Both breasts were well developed with erect nipples and deep areolar pigmentation. There was no hirsutism; no abdominal or pelvic masses were palpable by rectal examination. A retroperitoneal carbon dioxide
insufflation study revealed a discrete mass in the right suprarenal area. At operation a tumor was removed Vlith the right adrenal gland. The pathology examination showed a soft yellowish brown tumor of the right adrenal measuring 4. 7 by 1.8 em. On cut surface the adrenal tumor seemed to be completely encapsulated and normal adrenal gland was identified outside of the capsule. Microscopically the tumor cells were fairly uniform in size with relatively pyknotic nuclei of varying size and abundant eosinophilic cytoplasm. Although some anaplasia and pleomorphism were present, the diagnosis of a benign adrenal adenoma was made because of lack of capsule or vessel invasion (Fig. 3, A). A section through the normal adrenal and adjoining fragment of the adrenal tumor is shown after incubation in DHA for steroid dehydrogenase activity (Fig. 3, C). The normal tissue showed considerable activity while the tumor was quite inactive. In contrast, incubation with another DPN -linked substrate, lactic acid,
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Steroid 3,8-ol dehydrogenase activity 1007
B
Fig. 3. Case 2. Adrenal tumor inducing precocious puberty (all sections similarly oriented). A, Stained with hematoxylin and orange G and photographed with blue filter. Cortex of normal adrenal at bottom of photo, adrenal capsule in center, and tumor at upper third. (x40.) Fat is most prominent in the normal adrenal but is also present in large amounts in the tumor. B, Secti.on similar to 3A, 25.u, incubated for 45 minutes with sodium lactate and DPN at pH 7.4. The tumor cells show more lactic dehydrogenase activity than the normal adrenal. (x40.) C, Section, 25~t, incubated 3 hours with DHA as indicated in 2. D. The steroid 3/3-ol dehvdrogenase activity is confined essentially to. the nom1al adrenal cortex shown in lower part of figure, oriented with glomerular zone up; the tumor, which, as in 3A and B, is in upper part of photograph, is inert. ( x40.)
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A Fig. 4. Case 3. Feminizing testicular interstittal eel! turnor. A, Hernatoxylin and eosin stain showing well-differentiated cells, fairly abundant eosinophilic cytoplasm, and eccentric nuclei. B, 151" section from another portion of this tumor incubated for 8 minutes with 0.7 mg. per milliliter DPNH in 0.15 M phosphate buffer at pH 7A. Tumor cells have a very active DPN diaphorase. (x208). C, 25Jl section from same block as shown in 4, B incubated with DHA for 2 hours as in 2, C. No discernibl•· steroid 3fl·ol dehydrogenase activity. i:<'W8.)
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resulted in clear-cut acttvity in both nonnal tissue and tumor cells (Fig. 3, B). Cas-e 3. This patient, H. T., a 44-year-old man noticed a tumor of the left testis about 1 year prior to operation but was unable to persuade 2 different urologists that it existed. He stated that it had not increased in size. Six weeks prior to his admission, swelling of the left breast occurred. A urinary chorionic gonadotrophin assay was negative. The physical examination was remarkable only in that the left breast was flypertrophied, typical of gynecomastia. There was a 3 mm. mass in the left testis just beneath the globus major. Laboratory studies are missing as the preoperative specimens for estrogens were lost. Only the tumor was removed, and 6 weeks after operation, gynecomastia had regres:;ed. The tumor was encapsulated and about 8 mm. in diameter. The microscopic examination showed large pleomorphic cells with abundant eosinophilic cytoplasm. The diagnosis was made of an interstitial cell tumor of the testis (Fig. 4, A). The histochemical studies showed no detectable steroid 3 {3-ol dehydrogenase activity in the tumor cells, when DHA was the substrate (Fig. 4, C). The lactic dehydrogenase activity \vas appar·~nt at 15 minutes and the DPN diaphorase was even more active (Fig. 4, B).
Fig. 5·, Case 1. Hematoxylin and eosin stain of endometrium showing actively prolif~:rating tissue from patient whose ovarian tu· mor study is shown in Fig. 2. (x208.)
Steroid 3{3-ol dehydrogenase activity 1009
An arrhenoblastoma showed only minimal steroid 3{3-ol dehydrogenase activity in the Leydig-like cells. Case 4. The patient, J. C., a 15-year-old girl, was excreting 18 to 22 mg. of 17-ketosteroids daily, and was markedly virilized; Reinke crystals were present in the tumor tissue, all strong presumptive evidences that testosterone was being produced. The findings in this patient are being reported in detail elsewhere, therefore, no further clinical data will be included. The left ovary measured 10.5 by 5 by 2.5 em. It was pearly white and typical of an "oyster ovary" described in the Stein syndrome. On cut surface the cortex was replaced by numerous cysts and no definite tumor nodule could be made out. Microscopically the tumor occupied the hilus of the ovary and was composed of two types of tissue; an adenomatous area and islands of pale staining cells with prominent, rather small nuclei. Many Reinke crystals were present in the tumor tissue. A diagnosis was made of arrhenoblastoma (adenoma testiculare) in ovaries compatible with the diagnosis of the Stein syndrome. The steroid 3 {3-ol dehydrogenase was minimal, although fat was demonstrated in the Leydig-like cells and DPN diaphorase showed ac· tivity throughout (Fig. 6, A, B, C, and D).
1010
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A Fig. 6. Case +. Masculinizing arrh<:>noblastoma found in ovary of 15-year-uld patient. A. Sr·rtiun stained with hematoxylin .md oil red () and photographed with yellow filter, showing fat deposition in Leydig-like cells in the ovarian tuwor. ( ss :~cti\·e. (
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A luteinized granulosa cell tumor, which was associated with evidence of marked progesterone secretion, as judged by the decidual reaction in the endometrium (Fig. 7), gave a 1narked reaction for the steroid 3{3-o! dehydrogenase in those areas of the tumor which were luteinized. U nluteinizec! cell areas wen' inactive.
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Case 5. The patient, C. (:. was :1 :i-vt·ar-uid child wlws•· n>~nplaint wa' \·agirul disch~rg•', breast dt~velopnwnt, and abdominal m·dling ol 6 weeks' durarion. On physical examination no axillary hair was present hut the breasts wen~ hnwrtrophicd 'vith erect nipples and sorue areolar pigmentation. There was no nipple discharge. The external genitals showed no pubic hair, wme hypf'rtrophy and pigmentation of the labia, and a mucoid vaginal discharge. The abdomm was distended; th<'t'l' was a fluid wave aud a midline lower abdominal mass which was smooTh, tirm, and freely movabl1•. The rectal examination was confirmatory, hut it was impossihl" to determine whether the mass arose from th(' uterus or ovary. The maturation index wa~ o 1 9~)/ l. Total estrogt·ns w•·n• :!.(J/ ftg per 2'~ hours; estrone O.l:i, est r:Hli< d 0. 7-'i, and t~striol 1.19 ftg per !.4 hours. At operation a large left ovarian tumor was found with 1,:iOO mi. of bloody ascites. No cvidenn· of Illetastasis was seen. The left ovctrian tumor weighed 290 grams and measun·d 12 hy I 0 by 8 em. It sPemed cornpleff'ly encapsulated, of soft consistency, and was yellow in color Pxcept for hemorrhagic art> as. On cut ,,,,.t ion there were several small cystic areas :·) trltll. in dianlf'ter filled \vilh blood , lot; the remaining solid portion of th<, llltlltJl \\'as divided intu lobules by dPnse comwdiH· tissur· s•·pt<'- In sfllllt' areas tlw tumor \las :1 bright golden yt'llow, in others light tan, and still other ar••as appt>ared lwnwrrhagic. The tumor rdls Wt'l'l' morphologically predominantly granulosa et'lls but there wc·n· manv thecal elements presf'nt. Both types oi ct>lls showt•d extmsive luteinization (Fig. 8, A. and B). 'T'ht~ endornetrillnl
Fig. 6. B
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Steroid 3,.8-ol dehydrogenase activity 1011
Fig. 5. C
Fig. 6. D
decidual reaction with focal areas of hemorrhage (Fig. 7). With DHA as a substrate the tumor in this patient showed large areas of inactive cells interspersed with areas of intense activity comparable to that found in a corpus luteum (Fig. 8, D). Thrcughout there were strands of ceils with less intense activity. All areas of the tumor showing activity were composed of luteinized cells. The assay for LDH showed a more general reac1 ion but the areas of luteinization were most
intensely reactive. The assay with DPNH showed essentially the same pattern as with lactic acid (Fig. 8, C, left).
Fig. 7. Case 5. Hematoxylin and eosin-stained section of endometrial stroma showing marked decidual reaction. (x208.) The associated ovarian tumor is shown in Fig. 8.
Comment
Data have been accumulated which show that the biogenesis of sex horm~es, progesterone, testosterone, and estradiol, may proceed by cholesterol degradation to pregnenolone and from this compound to
1012 Goldberg, Seegar Jones, and Woodruff \m
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A Fig. 8. Case 5. A, Hematoxylin and l'osin stain ,f >.!ranulosa '.-II tl!JJJ<>J of ovary, with marked luteinization, in 5·-yr•ar-old p.tli<·ni showing Hurk<'r el!"mcnts l:wltm and to the right. S!"rtion on th<> leh. 1'iP.. inct~hat•·d with DPNJ I f.,, i.'i minutes: S!"ction on the right, '2'iJ.L, incubatf'd with DHA for 5", hour.' The DPN diaphorase activity is more widespread 1. ·, D. High-JXIW<'< photomicrograph of section "" right in R. l.. Tht·J ,. is mMk<'d \fl., ,J 1
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decreased activity. The diffprence itl .-.cti,·ity i' ,~omparahk 10 thd.t lw tween th(' lutein Cf•lls of a normal q,rrllt' illtt•ttll• .. ,,,.) th•· tlw, ·' iutt·rn:; i
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progesterone. testosterone, and finally r~st ta ~ clio!, in this sequence 11 ' 1 ~, i\ suggestion ha:-; hePn made that dehydroepiandrosterom· SO, (DHA-50 4 ) may be the preferential precursor for testosterone and estradiol. also by way of L; 1-androstenc-3, 17 -diom·. For either of these pathways, a steroid ·; P-ol dehydrogenase appears involved. Histochemical data show that steroid :l,B-ol dehydrogenase is readily demonstrable in those tissues which arc considered to be producing relatively large amounts of progesterone, e.g., the corpus luteun1, placenta, and adrenal."· J. "·" Jn human tissues which are presumably producing androgens and estrogens, this enzyme activity is either absent or low." The data reported in the present paper
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'teroid-produt·iru~: !t!!ltl)r5~ !H) :1/)-u! t!(·!!)dtfl· gPnase activity '"1s fn11nd ill Litntor., fro111 patients who t'xhihitnl climcal ,., irkm t' nt •·xcessin' estrogen prnd twnon ,\ n ,u rlwno-
blastoma as~ociated with lltarkl'd din!< a~ \·irilization had minim:1l aniYit\ ln n•n t rast, a. lllteini;rt>rl ~Tanulnsa n·ll ltlltinr "! the ovary, associated witlt marked dccidttdi reaction in tht· t'ndometmllll. had maxitnal hut Ill thf' lutc·inii't·d :rr·t'a' nnh This activitY \\-as comparable tu that st't'll in the most acti\·e tissue' which we h;n•· •·ncounten·d. n>rpora ltttt'a. The failure to demonstrate ihi~ dctivit\ might be the result of an enzyuw deficiencY a reduced supplv of sorrH' ratf'-limitin~ en. factor. nr !hf' fH'csenCt' nf •·xr·t·"i\·t· .nnnttllt'
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Steroid 3(1-ol dehydrogenase activity 1013
Volume :!16 Number 8
Fig. 8. B
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1014 Goldberg, Seegar Jones, and Woodruff \Jn,
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of inhibitor. It might signify a different biogenetic pathway in the human gonads for estrogen and testosterone synthesis from that for, or by way of, progesterone. Howard13 has suggested such an alternate pathway for estrogens which might not necessitate a 3[:?-ol dehydrogenase activity. If DHA-S0 4 , which Burstein and Dorfman 14 have indicated might arise directly from cholesterol without the preliminary t:- 0 pregnen-3[:?-ol, 20-one formation, was the substrate, C-19 and ring A oxidation might occur without steroid 3[:?-ol dehydrogenase
REFERENCES
1. Samuels, L. T., Helmreich, M. L., Lasater, M. B., and Reich, H.: Science 113: 490, 1951. 2. Wattenberg, L. W.: J. Histochem. 6: 225, 1958. 3. Goldberg, B., Jones, G. E. S., and Turner, D. A.: Steroid 3/3-ol dehydrogenase activity in human endocrine tissues. (In press.) 4. Deane, H. W., Lobel, B. L., and Romney, S.: AM. J. OusT. & GYNEC. 83: 281, 1962. 5. Levy, H., Deane, H. W., and Rubin, B. L.: Endocrinology 65: 932, 1959. 6. Niemi, M., and Ikonen, M.: Endocrinology 70: 167, 1962. 7. Fitz-Williams, W. G., Jones, G. E. S., and Goldberg, B.: Stain Techno!. 35: 195, 1960.
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activity and the end result wotdd lw t·~trorw which could then be converted to estradiol by reduction at the 17- position 1Fig. !I. Admittedly, the evidence for this pathwa::is meager at best, but it pn'sents a possibility for estrogen synthesis from cholesterol that is not dependent on steroid :1(:/-ol dehydrogenasP activity. As such. it appt'at' to be in better accord with the histochelllical data. It is understood that all of the entymir systems necessary to produce a 1l of t ht· theoretical reactions depicted have heen demonstrated in all steroid-producing tissues. Howewr, the fluctuations in these the enzvmPs m· ----- cofartors ----------- which -------- ac! iv;c~ fl' them must surely determine which way th<' equilibrium will be shifted. The technique of histochemistry is a valuable t
~--------
8. Nachlas, M. M., Walker, D. G., and Seligman, A. M.: J. Biophys. & Biochem. Cytol. 4: 467, 1958. 9. Nachlas, M. M., Walker, D. G., and Seligman. A. M.: .J. Biophys. & Biochem. Cytol. 4: 29, 1958. 10. Brown, J. B.: Biochem. J. (W: 185, 1955. 11. Mason, N. R., and Samuels, L. T.: Endocrinology 68: 899, 1961. 12. Smith, 0. W., and Ryan, K. J.: Endo~rin ology 69: 970, 1961. 13. Howard, E.: Fed. Proc. 21: 2, 1962. 14. Burstein, S., and Dorfman, R. I.: Acta endocrinol. 40: 188, 1962. 601 North Broadway Baltimore, Maryland