Histochemical studies of steroid 3β-ol dehydrogenases of the human ovaries

Histochemical

studies of steroid .3~-ol

dehydrogenases ICHIRO

TAKI,

of the human ovaries

M.D.*

HIROSHI

IIJIMA,

MASAYO

UETSUKI,

M.D.* M.D.*

NOBUAKI

HAMANAKA,

M.D.*

MASAAKI

MORISHITA,

D.D.S.‘”

MASAHIKO Osaka,

MORI,

D.D.S.“”

Japan

The activity of steroid 3P-01 dehydrogenase and, glucose-6-phosphate in normal and pathologic human ouaries was studied using seven kinds of steroids as the substrate. In thetaand granulosa-lutein cells of the corpus luteum, no correlation was found between the distributions of glucose-6-phosphate and steroid 3p-01 dehydrogenases. The activity of both enzymes showed similar distribution and intensity in normal follicle and cystic follicle of the polycystic ovary. An intense steroid dehydrogenase activity for androstane-3P-01, I7-one and 5-androstene-3P-01, 17-one in a luteiniting cyst associated with a chorionic tumor was suggestive of an abnormal steroid metabolism in the ovary.

transferred to a cryostat at -20’ C. and sectioned 12 to 18 p thick with a sliding microtome. The sections were placed on clear slides, dried at room temperature, and incubated in the following mixture: Steroid (10 mM. in acetone), 0.5 ml.; nicotinamide adenine dinucleotide, 7.0 mg. ; phosphate buffer (pH 8.0)) 6.5 ml.; and Nitro BT (5 mg. per 3 ml. in distilled water), 3.0 ml. The following seven kinds of steroid employed as the substrates were used :

R E c E N T progress in histochemical methods, particularly in the detection of steroid dehydrogenase activity, seems to afford an important approach to study the endocrine function of the human ovary.3p B-1o, 13sI6 It has been suggested that constant and prominent glucose-6-phosphate dehydrogenase activity in luteal cells indicates an interaction with steroid biosynthesis.l?, I5 Therefore, it is worthwhile to investigate correlative distribution patterns of both enzymes in ovaries which are either normal or abnormal. Material Surgically centa, and immediately

1. 5-Androstene-S/3-01, 17/j-01 ( 2”). 2. Androstane-3P-ol, 17p-01 (3”). 3. 5-Androstene-3P-ol, 17-one (dehydroepiandrosterone) 3, (4*). 4. Androstane-3P-01, 17-one (epiandrosterone)

and methods removed human ovaries, plachorionic tumors were frozen at -70’ C. in dry ice. They were

(‘3”). 5. 5-Pregnen-3/3-01, 20-one (pregnenolone) (Si). 6. 5-Androstene-S/3-01, 17,!3-01 (20”). 7. 5-Androstene-3,@-ol, 17-one (2 1”).

From the Department of Obstetrics and Gynecology, Osaka University Medical School and the Department of Oral Surgery, Osaka University Dental School. This investigation was supported in part by Research Grant No. DRG-725 from the Damon Runyon Memorial Fund.

*The number corresponds to that appearing of Goldberg, Jones, and Woodruff.9 107

in the paper

108

Taki

May I, 1967 J. Obst. & Gym.

et al. Am.

The incubation was performed at 37’ C. for the desired time. Following incubation, the sections were fixed in neutral 10 per cent formalin for 10 minutes and mounted in glycerin. Formazan deposition produced in the sections by steroid dehydrogenase activity appeared generally prominent when the substrates 3, 4, and 7 were used as compared with the others. The other sections obtained from the same materials were incubated for the histochemical demonstration of glucose-6-phosphate dehydrogenase activity, the procedures of which had been reported previously.15 In total, 17 growing follicles, 14 Graafian follicles, 11 corpora lutea, 9 corpora albicantia, 13 luteinized cysts, and 32 cystic follicles of polycystic ovaries and ovarian tumors were examined. They were collected from 27 cases under normal and pathologic conditions. Besides, 9 cases of chorionic tumors and placentas obtained by artificial termination of pregnancy ( 1.5 and 3 months) and at term were studied. Results The results

were

phate dehydrogenase and 3p-01 dehydrogenases. Glucose-6-phosphate dehydrogenase activity of immature follicles was much more conspicuous in theta interna than in other tissue components. 3/3-01 dehydrogenase and isomerase showed very low activity in the theta interna and negligible in granulosa cells. Primary follicles showed almost negative reaction of 3/3-01 dehydrogenases. Graafian follicles. Fourteen Graafian follicles were examined. They had more intense staining reactions for glucose-6-phosphate dehydrogenase and 3p-01 dehydrogenases than younger growing follicles, but stainability of 3p-01 dehydrogenase was still low as compared with the corpus luteum. In almost all cases, granulosa and theta interna cells of Graafian follicles indicated only a trace or no formazan deposition for 3/3-01 dehydrogenases in substrates 1, 2, 3, 5, and 6, and they showed low or only trace stainability for substrates 4 and 7. Theta interna cells were characterized by the presence of the highest activity of glucose-6-phosphate dehydrogenase, though granulosa cells were slightly positive for this enzyme.

Atretic follicles or cysts of polycystic ovary. Thirty-two cystic follicles were ob-

summarized

in Table I. Primary and growing follicles. Seventeen growing follicles and many primary follicles were observed. They were rather low in staining reaction for both glucose-6-phos-

Table I. Comparison in ovarian

of distribution

Substrate

Theta

G-6-PD 1 2 3 4 5 6 7 Propyl alcohol? Isopropyl alcohol+ Negative;

tDetails

and activities * for steroid

3/3-01 dehydrogenase

structures Graafian

*-,

served. The finding of enzyme histochemistry for steroid dehydrogenases and glucose6-phosphate dehydrogenase was nearly the same as observed in the Graafian follicle.

will

cell

follicle

Polycystic

Glanulosa cell

Theta

2 --+I -

+3 -

- 4 --+ -k --+ +1 + -+1 * +1

-+ k

+1

+3 -

-

+1 +l +1 +l

? -

+I

+

+ 1, trace; be published

cell

ovary

Corpus

Granulosa cell

- 4 --?: -* -k +l -+1 + +l

+-

+1

+-

Theta

---+I - - +1 f - +1 t+1

+4

+1

weak

activity;

elsewhere.

+2,

moderate

activity:

+3, marked

2 - +1 ++2

t +2 - 3

+2

+l-

most

intense

activity.

+l2 -+1 +

3

+ - --tl +l3 2

-

cyst Lute-irked cell

+1-2

+

+1 + +4

5 -cl +4,

cell +4

+3-4

+l

activity;

Theta

4

+l +3

+2 2 -

Luteinized

Granulosa cell +2 -

f - +l +l +l3 +13 2 - +1 +l +23

+ +l,

cell

+1

luteum

+1 -

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Steroid 3/3-01 dehydrogenases

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Fig. 1. Components of follicle cyst in polycystic ovary. (x100.) a, Glucose-6-phosphate dehydrogenase activity was characteristically high in the theta interna and positive in the granulosa cell. The localization of the enzyme is similar to that of normal follicle. b, Androstane-3&o& 17&01 (2). A slight formazan deposition appeared in the theta interna. c, Androstane-3/3-o& 17-one (epiandrosterone, 4). Moderate formazan deposition was found in the theta interna cell and slight deposition in the granulosa cell. d, 5-Pregnen-3P-01, 20-one (pregnenolone, 5). Formazan deposition is very slight in the theta intema celll. e, 5-Androstene3/3-o& 17-one (7). Formaaan deposition is slight to moderate in the theta interna cell and slight in the granulosa cell. f, Secondary alcohol dehydrogenase with propyl alcohol as a substrate. A positive reaction was found in the theta and granulosa cells.

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1, 1967

Am. J. Obst. & Gynec.

Fig. 2. Corpus luteum on the fifteenth day of menstruation. (x100.) a, Glucose-6-phosphate dehydrogenase. The theta-lutein cell was characterized by the presence of the most intense activity. The granulosa-lutein cell showed a moderate reaction. b, 5-Androstene-SP-ol, 17p-01 (1). Note a slight formazan deposition in the lutein cell tissue. c, 5-Androstene-36-01, 17-one (dehydroepiandrosterone, 3). Note a high stainability in the granulosa-lutein, while a low in the theta-lutein. d, Androstane-J/3-01, 17-one (epiandrosterone, 4). A similar distribution pattern as in e was found. e, 5-Androstene-3P-01, 17-one (21). The highest formazan deposition was seen in the granulosa-lutein and a slight one in the theta-lutein was found. f, Secondary alcohol dehydrogenase with propyl alcohol. Note moderate activities in both the lutein tissues.

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Fig. 3. Corpus luteum obtained from a patient with chorioadenoma. (x100.) a, Glucose-6phosphate dehydrogenase activity was high in lutein structure, but indistinguishable in both the thetaand granulosa-lutein cells. b, 5-Androstene-3/3-o& 17-one (dehydroepiandrosterone, 3). Note a slight to moderate formazan deposition in lutein tissue. c, Androstane-J/3-01, 17one (epiandrosterone, 4). d, 5-Androstene-3/3-o& 17-one (7). In both substrates, 4 and 7, no difference existed in stainability between thetaand granulosa-lutein cells. Scattered cells which showed the highest stainability among slightly stained lutein tissue were characteristic.

1

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In reactions with substrates 1, 2, 3, 5, and 6, almost all cysts of polycystic ovary presented negative or trace stainability in theta interna and granulosa cells, but only one case showed a slightly positive reaction in In substrates 4 and 7, both components. theta interna cells revealed a slightly positive staining and granulosa cells showed trace or negative staining, except for one case which showed slight to moderate staining. Secondary alcohol dehydrogenase activity in propyl alcohol was low in theta interna and granulosa cells. The highest glucose-6-phosphate dehydrogenase activity was constantly present in theta interna cells as it was in normal Graafian follicles (Fig. 1, a through f) . Corpora lutea and albicantia. Eleven mature and two immature corpora lutea and nine corpora albicantia were examined. The distribution of steroid dehydrogenases in corpora lutea was even in both theta-, and granulosa-lutein cells without difference in stain-

May I, 1967 Am. J. Obst. & Gynec.

ability between them. Glucose-6-phosphate dehydrogenase in corpora lutea was characteristically prominent in theta-lutein cells. Steroid dehydrogenase reactions in lutein tissue were low in substrate 1, 2, 5, and 6; and moderate to high in substrates 3, 4, and 7. In the latter group of substrates enzymatic stainability in granulosa-lutein cells was higher than that in theta-lutein cells, e.g., low to moderate in theta-lutein cells with formazan crystal formation, and moderate to high in granulosa-lutein cells without crystal formation. In the one case of pregnancy, corpus luteum at 2 months gestation showed a more intense reaction to the 3 steroid p-01 dehydrogenase than the menstrual corpora lutea. In corpus luteum obtained from a patient with a chorionepithelioma, steroid dehydrogenase of lutein cells showed a low reaction in all substrates used, with exception of highly positive reactions in scattered cells for substrate 6. This case indicated the strongest glucose-6-phosphate dehydrogenase

Fig. 4. Luteinized cyst obtained from a patient with choriocarcinoma. (x100.) a, Glucose-6phosphate dehydrogenase activity in the theca interna was the highest and it was moderate in the granulosa cell. b, Androstane-3P-01, 1 f-one (epiandrosterone, 4). c, 5-Androstene-3/3-o& l7-one (7). Formazan was more intensely deposited in the lutein cell than in the granulosa cell, in the latter appearing in fine granules or crystals.

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activity in lutein cells, the origin of which was not identified (Fig. 3, a through d) . Glucosed-phosphate dehydrogenase activity was slightly to moderately reactive in granulosa-lutein cells and most intensely reactive in theta-lutein cells in the normal menstrual cycle or in pregnancy (Fig. 2, a through f) . Corpora albicantia showed a decreasing order of steroid and glucose-6-phosphate dehydrogenase stainability. They revealed only slightly positive reactions in substrates 3, 4, and 7, and negative ones in 1, 2, 5, and 6. The hyalinized area in the central portion was absent for the dehydrogenase activity. Glucose-6-phosphate dehydrogenase reaction was noticeable only in degenerative lutein cells. Luteinized follicle and trophoblastic tumors. Thirteen luteinized follicles obtained from patients with associated chorionic tumors showed similar staining properties for steroid dehydrogenase and glucose-6-phosphate dehydrogenase to those observed in the corpus luteum during the menstrual cycle. The enzymatic stainability for substrates 1, 2, 3, 5, and 6 were low or trace in theta interna and luteinizing granulosa cells. Formazan deposition in luteinized granulosa cells formed large crystals. In substrate 6, enzymatic stainabilities were moderate to high in theta interna cells and low to moderate in granulosa cells. Glucose-6-phosphate dehydrogenase activity was markedly deposited in theta interna and moderately present in granulosa cells as similarly observed in menstrual corpus luteum (Fig. 4, a through c). Ovarian adenocarcinoma. One case of ovarian adenocarcinoma showed positive glucose-6-phosphate dehydrogenase activity in neoplastic epithelium containing the highest number of active dotted cells. All steroid dehydrogenase activity in the present experiment was absent in neoplastic epithelium, irrespective of the presence of scattered tumor cells with the highest glucose-6-phosphate dehydrogenase activity. Normal placenta and trophoblastic tumors. Normal placental villi showed a high glucose-6-phosphate dehydrogenase activity,

Fig. 5. Villi in hydatidiform mole obtained from a 27-year-old patient. (x100.) a, Glucose-6-phosphate dehydrogenase. b, Androstane-3P-01, 17-one (epiandrosterone, 4) . C, 5-Androstene-3/3-o& 17one (7). Neoplastic villi showed stronger activities of dehydrogenases and isomerases than in normal placental villi.

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Taki et al.

a moderate steroid dehydrogenase activity for substrate 4, low activities for substrates 1, 2, 3, and 7, and a trace for 5 and 6. Neoplastic villi of hydatidiform moles, showed a similar enzymatic pattern to that of normal placental villi, and they were of higher activity for glucosed-phosphate dehydrogenase than normal villi. Neoplastic trophoblasts of chorionepithelioma revealed an intense glucose-6-phosphate dehydrogenase and varying activities with steroid dehydrogenases (Fig. 5, a through c) . Comment The present experiments confirmed that glucose-6-phosphate dehydrogenase in thecalutein cells retained a constantly high activity, while the granulosa-lutein cells presented a lower reaction. In contrast, steroid 3/3-01 dehydrogenase activity in theta-lutein cells was always low and that in the granulosa-lutein cells was positive in varying intensities. There was no definite correlation in activity level and localization between glucose-6-phosphate and steroid 3 p-01 dehydrogenases in corpus luteum under apparently normal conditions. It was also recognized that both types of lutein cells were definitely involved in steroid biosynthesis, showing constantly positive steroid 3/?-01 dehydrogenase activity. A strongly positive glucose-6-phosphate dehydrogenase activity in theta-lutein cells signified the production of NADPH in the pentose cycle. Recently, histochemical procedures for detection of 3a, 3,8, S/3, llcu, 11,6, 12a, 16cy, 16/3, 1 ICY, 17/3, 2Oq 20/3, 21, and 24 hydroxysteroid dehydrogenases have been described using Nitro BT with or without coenzymes (1, 2, 3, 4, 5, 14, 16). The description was almost always of the enzyme distribution in the ovary under normal conditions or during the menstrual cycle, but Goldberg, Jones, and Woodruff9 have detected 3p-01 dehydrogenase in steroid-producing tumors and in a

May 1, 1967 Am. J. Obst. & Gynec.

luteinized cyst caused by chorionic tumors. In the present results, steroid 3p-01 dehydrogenase activities for various substrate steroids exhibited a regular distribution in theta interna and granulosa of physiologic follicles as well as in those of cystic follicle found in polycystic ovary. Formazan deposition in granulosa-lutein cells and luteinized cells of luteinized cysts was characterized by the development of fine crystallization. The latter was not detected in ordinary granulosa cells and theta interna cells. The crystallization was most conspicuous in the luteinized cysts of chorionic tumors, as if to suggest a different property of the cellular elements. The significance of histochemical distribution of steroid 3/S01 dehydrogenase with seven kinds of substrate steroids might be in the appearance of the enzyme in individual cells of steroid-producing components and not in the stainability in each tissue section, particularly in the abnormal condition such as chorionic tumors which are supposed to imply systemic hormone imbalance. Corpus luteum obtained from a 41-year-old patient with invasive mole revealed scattered cells in the granulosa-lutein structure which showed strong stainability in substrate steroids, androstane-3/3-o& 1 ‘/-one and 5-androstene-3/3-ol, 17-one. It was assumed that the presence of those scattered cells was suggestive of an unusual steroid biosynthesis due to the neoplastic condition, Distribution of the strong glucose-6-phosphate dehydrogenase activity in the villi of normal placenta seemed related to the nucleic acid synthesis and steroid metabolism. Trophoblastic cells in chorionic tumors also showed strong glucose-6-phosphate dehydrogenase and positive steroid 3/?-01 dehydrogenase activity. This might suggest that no fundamental divergence in steroid production existed between neoplastic and nonneoplastic trophoblasts.

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Baillie, A. H., Calman, K. C., Ferguson, M. M., and Hart, D. M.: Histochemie 5: 384, 1965. 4. Baillie, A. H., Calman, K. C., Ferguson, M. M.. and Hart. D. M.: T. Endocrinol. 34: 1, 1966. ’ Balogh, K.: J. Histochem. Cytochem. 12: 670, 1964. Deane, H. W., Lobe& B. L., and Romney, S. L.: AM. J. OBST. & GYNEC. 83: 281, 1962. Ferguson, M. M.: J. Endocrinol. 32: 365, 1965. Goldberg, B., Jones, G. E. S., Turner, D. A., Sarlos, I. J., and Horton, E. H.: AM. J. OBST. & GYNEC. 86: 349, 1963. 9. Goldberg, B., Jones, G. E. S., and Woodruff, J. D.: AM. J. OBST. & GYNEC. 86: 1003, 1963. 10 Goldberg, B., Jones, G. E. S., and Borkowf, ”

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3-Chome