Biosynthetic studies of human ovarian arrhenoblastomatous tissue in vitro I. Sulfatase and sulfokinase activity

237

BIOSYNTHETIC STUDIES OF I-IUMAN OVARIAN ARRBENOBLASTOMATOUSTISSUE IN VITRO I. SULFATASE AND SULFOKINASE ACTIVITY (1) Eugene C. Sandberg, R. Clifton Jenkins and Harry M. Trifon Department of Gynecology and Obstetrics Stanford University School of Medicine Palo Alto, California

Received June 2, 1966.

ABSTRACT Virilizing ovarian arrhenoblastomatoustissue was studied for the presence of alcoholic and phenolic steroid sulfatase and sulfokinase activity by independently incubating this tissue with the following tritiated substrates: dehydroisoandrosteronesulfate, estrone sulfate, dehydroisoandrosteroneand estrone. The influence of human menopausal urinary gonadotropin, human chorionic gonadotropin and adrenocorticotropin on sulfatase and sulfokinase activity by this tissue in vitro was examined. Considerable hydrolysis of both sulfurylated substrates (65-95s) was noted. Only minor sulfurylationof dehydroisoandrosterone(1%) was detected and there was no demonstrable sulfurylationof estrone. Except for an apparent reduction in the degree of esterification of dehydroisoandrosterone in the presence of adrenocorticotropin,the addition of tropic hormones did not significantly influence the yields from these reactions. During the past six years considerable evidence has been accumulated suggesting that the sulfate esters of numerous steroidal compounds may have physiologic importance in the human well beyond the mere expedition of hormone inactivationand excretion occasioned by their formation. Demonstrations of the endocrine secretion of dehydroisoandrosterone sulfate (2,3) and of peripheral interconversionof this compound with its unconjugated derivative in vivo (3,4)

are

pertinent

in this regard. Of confirmatory importance are the in vivo and in vitro demonstrations of metabolism of steroidal sulfates without loss of the sulfate moiety (5,6,7,8,9)

and demonstrations of utilization

of dehydroisoandrosteronesulfate in the formation of urinary estrogen in normal (10,ll) and abnormal (12) human pregnancy. While the usefulness and the importance of the findings have

STEROIDS

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not yet been demonstrated, numerous non-hepatic human tissues have recently been shown to have sulfatase and/or sulfokinase activity relative to steroidal compounds in vitro. These include the adrenal., testis, carcinomatous breast tissue, normal and abnormal placenta, fetal membranes, renal cortex, endometrium,myometriumand normal and abnormal ovarian tissue. Incubations of the normal human ovary have shown that this organ has the ability to sulfurylate dehydroisoandrosterone (13),

androstenediol(l3),estrone (14) and estradiol (15).

Sulfatase activity in vitro has been demonstrated in normal human ovarian tissue for dehydroisoandrosteronesulfate (16,17)

and estrone

sulfate (X7), and in polycystic (18) and arrhenoblastomatous(16) ovarian tissue for dehydroisoandrosteronesulfate. The recent availability of a virilizing ovarian arrhenoblastoma has afforded the opport~ity to confirm the observation above (16) and to extend the study to the synthesis as well as the cleavage of steroidal sulfates by this tumor tissue. The results of our ineubations demonstrate that this tumor has the capacity to hydrolyze both dehydroisoandrosteronesulfate and estrone sulfate and to sulfurylate de~y~o~soan~osterone

but not estrone.

The only significant effect from the addition of tropic hormones to the incubations was a reduction in the degree of sulfurylationof dehydroisoandrosteronein the presence of adenocorticotropin. MATERIALS AND METHODS Abbreviations and trivial names. Dehydroisoandrosterone= 36hydroxy-androst-5-en-17-one,dehydroisoandrosteronesulfate = 36 sulfoxy-androst-5-en-17-one,estrone = 3-hydroxy-estra-1,3,5(10)-trien17-one, estrone sulfate = 3-sulfoxy-estra-1,3,5(10)-trien-17-one, estradiol = estra-1,3,5(X0)-triene-3,L7@ -dial, androstenediol= androst-5-en-34, 17<-dial, Tris = 2-amino-2 (hydroxy-methyl)-1,3propanediol, HMG = human menopausal urinary gonadotropin (Cutter Laboratories), HCG = human chorionic gonadotropin {Squibb and Sons), ACTH = adrenocorticotropin(Abbott Laboratories),ATP = adenosine triphosphate. Clinical station. The patient was a 16 year old, unmarried female complaining of amenorrhea, acne and mild hirsutism of two years duration. Menstruation had commenced at the age of eleven and normal monthly menses occurred for the next three years. The amenorrhea which followed was associated with the gradual development of acne and mild hirsutism of the chin, cheeks, periareolar areas and abdomen. There had been no temporal hair loss or decrease in the size of the

Aug.

1966

STEROIDS

239

breasts. Preoperative examination additionally revealed a low pitched voice, well developed skeletal musculature and mild clitoral enlargement. Pelvic examination failed to reveal the 5 cm diameter, left ovarian mass which was subsequentlydetected by pelvic pneumography. The vaginal smear revealed minimal estrogen effect and the small quantity of cervical mucus present failed to form a fern pattern on drying. Urinary 17-ketosteroidand 17-hydroxycorticosteroidassays were in the mid-normal range. Operative exploration of the pelvis confirmed the pneumographic findings and a left salpingo-oophorectomyand elective appendectomy were performed. Histologic sections of this solid tumor showed the typical cord-like arrangement of irmnaturecells seen in a moderately differentiated arrhenoblastoma. Swollen, pale,lutein-like ("Leydig") cells were scattered lightly through the tissue. The patient noted vaginal bleeding of several days duration commencing one month following surgery and regular menses have continued at monthly intervals since. A substantialreduction in facial hirsutism has been noted since surgery and the patient has also lost a great deal of her heavy musculature and has, both physically and socially, assumed more feminine traits. All of the tumor except that utilized for microscopic examination was immediately frozen and prior to incubation was thawed and sliced using a Stadie-Riggs hand microtame. Radioactivity quantitation. Radioactivity was measured using a liquid scintillation spectrometer (Packard Instrument Company, Model 3214) adjusted to simultaneouslyprovip a 39% counting efficiency for C. All samples were counted in 3H and a 65% counting efficiency for five milliliters of a standard fluorescence solution (4 g. diphenyloxazole and 0.1 g. 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene per liter of toluene) and were counted for a sufficient period of time to obtain a statistical accuracy of f 3 percent. For purposes of steroid solubilization, one milliliter of absolute ethanol was added to each sample containing sulfurylated compounds. Internal standards were employed to correct for quenching. Absolute quantities of 3H and 14C existing in the same sample were calculated by the discriminator ratio method using the formulae of Okita et al (19). Chromatographicmethods and steroid localization. Celite column chromatographywas performed as described by Kelly et al (20). Descending paper chromatographywas performed at room temperature using Whatman #l paper prdviously washed in redistilled methanol. Non-radioactive, authentic, standard compounds were always run on parallel strips to verify the chromatographicbehavior of the solvent system. Chromatographic systems utilized in this study are listed in Table 1. Localization of radioactivity in column eluates was performed by aliquot counting. Localization of radioactivity on paper chromatograms was accomplished using a Vanguard Model 880 chromatogrsm scanner. Non-radioactivedehydroisoandrosteroneand estrone were localized by the color reaction of Zimmermann (21) while their sulfate esters were localized by that of Crepy and Judas (22). Preparation and purification of substrates and standards. 7&3H-dehydroisoandrosterone(SA 1.7 c/mmole, New England Nuclear Corpora-

240

STEROIDS

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Table 1.

Chromatographic systems employed.

system

support

I

Celite

Iso-octane: t-butanol: methanol: water (20:8:8:4)

II

Celite

n-heptane: methylcellosolve (5:l)

III

Celite

n-hexane: ethyl acetate: methanol: water (19:1:9:1)

IV

Celite

n-hexane: ethyl acetate: methanol: water (17:3:7:3)

V

Celite

Ligroin B: benzene: methanol: water (6:2:5:1)

VI

Celite

Iso-octane: t-butanol: 1 M bones hydroxide (2:5:5)

VII

Paper

8 hours

Cyclohexane: benzene (1:l) (paper impregnated with propylene glycol: methanol, 1:l)

VIII

Paper

2.5

Ligroin B: benzene: methanol: water (33:17:40:10)

IX

Paper

6 hours

Toluene: n-butanol: 10% ammonium hydroxide (3:1:2)

X

Paper

33 hours

Isopropyl ether: ligroin B: t-butanol: 10% ammonium hydroxide (5:2:3:10)

Duration

hours

Solvents

tion) and 6-7-%I-estrone (SA 40.6 c/mmole, New England Nuclear Corporation) were chromatographitiali& purified using systems I, II and III and I, IV and V respectively. 4- C-dehydroiatandrosterone(SA 40.7 mc/mtnole, New England Nuclear Corporation) and 16- C-estrone (SA Zlmc/mmole, Volk Chemical Company) were chromatographicallypurified using systems I and VIII, and V and VIII respectively. Only a single peak of radioactivity was noted in each instance. Non-radioactive deh~roisoandrosterone (Calbiochem)was recrystallized from acetone-petroleumether. The sulfate ester of each of the above materials, radioactive and non-radioactive,was synthesized in the manner of Levitz (23). Pyridine sulfate was prepared by reacting dry pyridine with concentrated sulfuric acid (3:l molar ratio) in chloroform. The resulting precipitate was washed with chloroform and rapidly transferred to a dessicator for storage. Pyridine sulfate (250 mgm) was crushed in dry pyridine (2.0 ml) and acetic anhydride (0.2 ml) and the mixture was stirred for thirty (25-lOO,iz), minutes. Radioactive dehy~o~so~drosterone or estrone solubilized in a minimum of dry pyridine (less than 1 ml), was then added to the mixture and this was stirred constantly overnight. Sodium

Aug.1966

STEROIDS

bicarbonate (20 ml of a lO$ solution) was added and the aqueous material was extracted with two volumes of diethyl ether. The aqueous phase was adjusted to pH 11 with 2N NaOB.and extracted twice with two volumes Of n-BuOH. The solvent was evaporated under vacuum. M-3H-dehydroisoandrosterone sulfate and 16-14C-estrone sulfate were chromatographically purified using systems VI and IX and 4-14C-dehydroisoandrosteronesulfate and 6-7-3%estrone sulfate were chromatographicallypurified using systems VI and X. con-r~ioact~ve sulfate esters of de~~oiso~~osterone and estrone were synthesized in the identical fashion. For each gram of unconjugated compound, approximatelytwo grams of pyridine sulfate, one ml of acetic anhydride and a total volume of eight ml of dry pyridine were used. Dehydroisoandrosteronesulfate was recrystallized twice from methanol-ether while estrone sulfate was first chromatographically purified using system VI and then recrystallized twice from this same combination of solvents. The purity of each radioactive material was examined by combining an aliquot of each with the appropriate cold carrier and reerystallizing twice. In every instance, the specific activities of crystals and mother liquor were within four percent of the mean value of all determinations for a given steroid. Radioactive materials were stored in absolute ethanol at 4'C. Nonradioactivematerials were stored at room temperature with the exception of the moderately unstable estrone sulfate which was stored in a dessicator in the dark at -2OOC. Prior to use in incubations,each radioactive material employed as substrate was diluted with cold carrier to provide a final tissue: substrate ratio of 50,000 (w/w)* Sulfurylated substrates were partitioned between ether and water immediatelybefore use to remove any products resulting from spontaneous hydrolysis during storage. The ether phase was never noted to contain more than 5% of the original radioactivity even after storing the radioactive material for several months. Incubations for determination of sulfatase activity. 5.3 x lo5 dpm (4~) of jH-dehydroisoandrosteronesulfate was incubated with 200 m&n of sliced tissue in 2.0 ml of Tris buffer at pH 7.4 with and without the addition of BMG (100 Cutter units), HCG (1000 T.V.) and ACTH (8 U.&P. units). Boiled tissue and tissue-less incubations, without tropic hormone additions, were used as controls. An identical set of incubationswas performed in which 3H-estrone sulfate (5.3 x 105 dpm) (4,ug) was employed as substrate. Incubation of these twelve different mixtures was continued for two hours in air at 37OC. At the conclusion of incubation, four volumes of absolute ethanol were added to each vessel, the contents were separately homogenized using Potter-Elvenhjemhomogenizers and the homogenates were stored at 4OC overnight. 1.7 x 104 dpm of 14C-dehydroisoandrosterone was then added to incubations in which 3H-dehydroiso drosterone sulfate had been the substrate and a similar amount of %C-estrone was pded to incubations containing 3H-estrone sulfate as substrate. The 1 C-labeled standard of the anticipated primary product of sulfatase activity was added in each instance forpurposes of product identificationand correction of extraction and chromatographiclosses

242

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Incubations for determination of sulfokinase activity. 4.1 x lo6 dpm (4%) of 3H-dehydroisoandrosteronewas incubated with 200 mgm of sliced tissue in 2.0 ml of 0.1 M phosphate buffer (pH 7.4) containing MgC12 (lmole), K2SO4 (lpole) and ATP (2ymoles). Separate identical incubations were performed following the addition of BMG, HCG, and ACTH in the quantities previously mentioned. Boiled tissue and tissueless incubations,without tropic hormone additions, were again used as controls. An ide tical set of incubationswas performed in which 3Hestrone (4.1 x 108 dpm, 4pg) was employed as substrate. As in the previous experiment, these twelve different mixtures were incubated for two hours in air at 37OC. Subsequently, addition of absolute ethanol, homogenization and overnight refrigeration were performed as in the previous experiment. 1.6 x lo4 dpm of 14C-dehydroisoandrosteronesulfate was thereafter added to incubations in which 3H-de ydroisoandrosteronehad been the substrate and a similar amount of lbC-estrone sulfate was added to incubations which had contained 3H-estrone as substrate. The addition of a 14~ labeled standard of the anticipated primary product of sulfokinase activity was for the same purpose as noted in the previous experiment. Analysis. The contents of each incubation were centrifuged and the precipitates were washed twice with 8% ethanol and recentrifuged. The supernatantsrelated to each incubation were combined and evaporated to the aqueous phase under vacuum. Each was consecutively extracted with two volumes of ether and four volumes of n-butanol to separate unconjugated from conjugated compounds. The ether extracts from the incubations for sulfatase activity were sequentially and separately chromatographedusing systems VII and VIII and radioactivity with the mobility of the primary anticipated product in each instance (dehydroisoandrosteroneor estrone) was eluted. The butanol extracts from the incubations for sulfokinase activity were sequentially and separately chromatographedusing systems IX and X and radioactivity with the mobility of the anticipated primary product (dehydroisoandrosterone sulfate or estrone sulfate) was eluted. Radioactivity coinciding with the mobility of the anticipated primary product from each incubation was then mixed with approximately 20 mgm of the appropriate, purified cold carri r. individually recrystallized to a constant 3H:1fC raEF:h m~~Sfrd~,w~~pressed as the percentage of substrate radioactivity converted to primary product radioactivity, were then calculated and corrected for losses. RESULTS Sulfatase activity. Table 2 reveals the 3H:14C ratios found following the addition of standard 14C-dehydroisoandrosteroneto 3Hdehydroisoandrosteroneformed from 3H-dehydroisoandrosteronesulfate by the sulfatase activity of this sxrhenoblastomatoustissue. Similar ratios for incubations containing gonadotropic and adrenocorticotropic hormones are included. Table 3 shows the ratios found in estrone com-

Aug. 1966

STEROIDS

243

Table 2. 3,. . 14 C ratios noted in dehydroisoandrosteroneformed from various incubations of arrhenoblastomatoustissue with 3H-d hydroisoandrosterone sulfate followed by the addition of standard lEC-dehydroisoandrosterone (before chromatography)and non-radioactivedehydroisoandrosterone(before recrystallization). Tissue alone

Tissue plus HMG

Tissue plus HCG

Tissue plus ACTH

lSt chromatogram

23.2

20.2

20.2

22.4

2nd chromatogrsm

24.5

21.9

21.3

25.0

st 1 recrystallization

24.5

22.6

20.9

24.5

2nd recrystallization

24.1

21.9

20.7

24.2

3,. . l4 C ratios noted in estrone formed from various incubations Table 3. of srrhenoblastomatoustissue with 3H-estrone sulfate followed by the addition of standard l4C-estrone (before chromatography)and non-radioactive estrone (before recrystallization). Tissue alone

Tissue plus HMG

Tissue plus HCG

Tissue plus ACTH

st 1 chromatogram

27.8

27.9

27.7

28.8

2nd chromatogrsm

28.1

28.9

27*9

28.7

1st recrystallization

28.3

28.8

28.5

29.0

2nd recrystallization

28.1

29-3

27.9

29.4

posed of standard 14C-estrone plus 3H-estrone formed from 3H-estrone sulfate under the same conditions. Both tables include the ratios i found in chromatographiceluates as well as those noted following reverse isotope dilution and recrystallization. The ratio in the eluate from the second chromatogrsm and the ratios in the two groups of crystals did not vary from the value of their mean by more than 2% for any of the incubations.

244

8:2

STEROIDS

Table 4. Sulfatase activity. Percentages of sulfuryleted substrate radioactivity hydrolyzed by ~rhe~oblastomatous tissue in vitro, corrected for extract&e and chromatographielosses. (No hydrolysis noted in boiled tissue or tissue-less control incubations.)

Substrate

Tissue alone

Tissue plus HMG

Tissue plus HCG

Tissue plus ACTH

76.5

69.3

65.5

76.5

92.0

95.9

91.1

96.2

sulfate 3H-estrone sulfate

The corrected yield of primary product formed from sulfatase activity on the sulfurylated substrates in each of the incubations is tabulated in Table 4.

76% of 3H-dehydroisoandrosteronesulfate was con-

verted to 3H-dehydroisoandrosteroneby this virilizing tissue both with and without the presence of ACTH.

Slightly lower percentage conversions

were apparent in incubations containing HMG and HCG but these differences are probably without significance. The only radioactivity seen on chromatography of the ether extracts other than that having the mobility of anticipated primary product was noted from all non-control incubationswherein 3H-dehydroisoandrosterone sulfate was the substrate. A very small amount of radioactivity which was more polar than 3H-dehydroisoandrosteronebecame evident on the second chromatogram. Further chromatographyrevealed that its mobility was identical with that of androstenediol but further efforts at identificationwere not undertaken. 92% of 3H-estrone sulfate was converted to 3H-estrone and no significant difference in yield was noted among incubations containing tropic hormones. Boiled tissue and tissue-less control incubations revealed no conversion of either substrate. Sulfokinase activity. Table 5 reveals the 3H:14C ratios found 14 following the addition of standard C-dehydroisoandrosteronesulfate to the 3X-dehydroisoandrosteronesulfate formed from 3H-dehydroisoandrosterone by this tumor tissue. The ratios found in both chromatographic eluates and crystals are given and similar ratios for incubations containing gonadotropic and adenocorticotropichormones are

Aug. 1966

STEROIDS

245

Table 5. %:14 C ratios noted in dehydroisoandrosteronesulf te formed from various incubations of arrhenoblastomatousti sue with 3H-dehydroisoandrosteronefollowed by addition of standard l&C-dehydroisoandrosterone sulfate (before chromatography)and non-radioactivedehydroisoandrosterone sulfate (before recrystallization). Tissue alone

Tissue plus HMG

Tissue plus HCG

Tissue plus ACTH

2.60

2.34

1.94

1.00

2nd c~omatogr~

2.58

2.15

1.92

0.91

st recrystallization 1

2.64

2.29

2.07

0.96

nd recrystallization 2

2.70

2.36

2.13

1.01

P

chrcmatogrsm

Table 6. Sulfokinase activity. Percentages of substrate radioactivity sulfurylatedby arrhenoblastomatoustissue in vitro, corrected for extractive and chromatographiclosses. (No sulfurylationnoted in boiled tissue and tissue-less controlincubations,)

Substrate

Tissue alone

3H-dehydroisoandrosterone 1.1

Tissue plus H&E 1.0

Tissue plus HCG

Tissue plus ACTH

o-9

0.4

3H-estrone

included. The ratios for a given incubation did not vary more than 6.2% from the mean value for that incubation. Corrected yields of the primary product formed from sulfokinase activity sre detailed in Table 6.

Only 1% of the 3H-dehydroisoandro-

sterone was sulfurylatedby this virilizing tissue and no significant difference in yield was noted in those incubations containing HMG or HCG. Less than one-half this degree of conversion appeared to occur in the incubation containing ACTH.

In view of the low yields obtained

in these incubations and the lack of opportunity to corroborate this finding, we are uncertain of its significance. No s~~ylation

of 3H-estrone was noted in any incubation with

or without tropic hormone additives and boiled tissue and tissue-less control incubations revealed no sulfurylationof either substrate.

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No peaks of radioactivity other than those associated with the anticipated primary product were noted on chromatography of the butanol extracts. DISCUSSION Warren and French (16) have previously reported the hydrolysis of dehydroisoandrosteronesulfate by human arrhenoblastomatoustissue in vitro. The results of the incubations described here confirm their finding and additionally demonstrate that this virilizing ovarian tissue has the capacity to hydrolyze estrone sulfate, revealing the existence in this tissue of two sulfatases for steroidal compounds. This is a logical extension of our previous observation (17) that norma1 human ovarian tissue is capable of converting both of these sulfurylated substrates to their unesterified forms. The results of these incubations additionally indicate that gonadotropic and adrenocorticotropichormones do not significantly influence the activity of either sulfatase under the conditions and in the concentrationsemployed. This is consistent with our observation that similar concentrations of HCG do not effect sulfatase activity in -in vitro incubations of homogenized normal human placental, ovarian or endometrial tissue relative to either dehydroisoandrosteronesulfate or estrone sulfate. Additionally, Fulkkinen and Hakkarainen (24) were unable to detect any alteration in steroid sulfatase activity in the liver of rats treated with gonadotropic hormones. Sulfokinase activity relative to dehydroisoandrosteronewas also detected in this virilizing tissue. While the percentage of substrate conversion was small, it was adequate for detection of product and, moreover, was comparable to that noted by Wallace and Silberman (13) in demonstrating the presence of this same enzyme in normal ovarian tissue. Gonadotropins, as used in these incubations, had no influence on the activity of this enzyme and while the data obtained suggests that adrenocorticotropinhas an inhibitory effect on sulfokinase activity for dehydroisoandrosteronekinetic studies would be required to validate this finding. Sulfurylation of estrone by this tissue could not be demonstrated. This in vitro capacity of ~rhenoblastomatous tissue to synthe-

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STEROIDS

size and cleave dehydroisoandrosteronesulfate has also been demonstrated for normal human adrenal and testicular tissue (18,25,26) while normal human ovarian tissue has been shown to be capable of synthesizing and cleaving both dehydroisoandrosteronesulfate and estrone sulfate. The significance of such capabilities in in vivo metabolism is as yet unknown. It seems unlikely that the ability of these tissues to both synthesize and cleave steroidal sulfates is restricted to the substrates studied. It is not unreasonable to anticipate that these activities exist relative to a large number of steroidal sulfates in these organs and others as well.

If such should be the case, biologic

regulation of these reversible reactions might well serve as one in a series of controls governing the production and end organ concentration of physiologically active hormone. ACKNOWLEDGEMENTS This project was supported by United States Public Health Service Grant No. AM-08220 from the National Institutes of Health. Human menopausal gonadotropin (Pergonal)was kindly supplied by Cutter Laboratories, Berkeley, California, U.S.A. REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Previously published in abstract form in the PROGRAM OF THE 47TH MEETING OF THE ENDOCRINE SOCIETY, JUNE 17-19, New York 1965, P. 125. Baulieu, E., C. R. ACAD. SCI. (Paris) 251, 1421 (1960). Vande Wiele, R.L., MacDonald, P.C., Gur'Fpde,E. and Lieberman, S., RECENT PROGR. HORMONE RES. 2, 275 (1963). Sandberg, E., Gurpide, E., and Lieberman, S., BIOCHEMISTRY3, 1256 (1964). Calvin, H.I., Vande Wiele, R.L. and Lieberman, S., BIOCHEMISTRY 2, 648 (1963). Calvin, H.I. and Lie,berman,S., BIOCHEMISTRY~, 259 (1964). Baulieu, E.E., Corpechot, C., and Emiliozzi, R., STEROIDS Z!,429 (1963). Lebeau, M.C., Alberga, A. and Baulieu, E.E., BIOCHEM BIOPHYS. RES. COMM. 17, 570 (1964). Roberts, K.D., Bandi, L., Calvin, H.I., Drucker, W.D. and Lieberman, S., BIOCHEMISTRY3, 1983 (1964). Baulieu, E.E. and Dray, F., J. CLIN. ENDOCR. 23, 1298 (1963). Warren, J.C. and Timberlake, C.E., OBSTET. AND GYNEC. 23, 689 (1964). Siiteri, P.K. and MacDonald, P.C., STEROIDS 2, 713 (1963). Wallace, E. and Silberman, N., J. BIOL. CHEM. 3, 2809 (1964).

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14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

STEROIDS

Holcenberg, J. and Rosen, S.W., PROG. 46TH RETINA BORIC Sec., JUNE 18-20, San Francisco 1964, Abst. 157, p. 101. Lindberg, M.C., McClenaghan, C. and Hermann, W.L., CLIN. RES. 13, 132 (1965) (Abst.). Warren, J.C. and French, A.P., J. CLIN. ENDOCR. 25, 278 (1965). Sandberg, E.C. and Jenkins, R,C., BIOCHIM. BIOPHYS. ACTA 113, lgo (1966). Burstein, S. and Dorfman, R.I., J. BIOL. CHEM. 238, 1656 (1963). Okita, C&T., Kabara, J.J., Richardson, F. and LeRoy, G-V., NUCLEONICS 2, 111 (1957). Kelly, W.G., Bandi, L., Shoolery, J.N. and Lieberman, S., BIOC~ISTR~ I, 172 (1962). Z&, 47 (1936). Zimnermann, W., HOPPE-~~~~S Z. PHYSIOL Cm. Cre'py,0. and Judas, O., REV. FRANC. ETUDES CLIN. BIOL. 2, 284 (1960). Levitz, M., STROPS 1, 117 (1963). Pulkkinen, M.O. and Hakkarainen, H., ACTA ENDOCR. (Kbh.) 48, 313 (1965). Adams, J.B., BICCHIM. BIOPHYS, ACTA 7l, 243 (1963). Dixon, R., Vincent, V. and Kase, N., ST~O~S 6, 757 (1965).