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Biosynthetic studies of human ovarian arrhenoblastomatous tissue in vitro II. Formation of androgens from dehydroisoandrosterone sulfate
249
BIOSYNTKETIC STUDIES OF BUMAN OVARIAN ARRHENOBLASTOMATOUSTISSUE IN VITRO II. FORMATION OF ANDROGENS FROM DEHYDROISOANDROSTERONESULFATE (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 Virilizi ovarian arrhenoblastomatoustissue s simultaneously incubated with % -dehydroisoandrosteronesulfate and 3%C-progesterone with and without the addition of human menopausal urinary gonadotropin, human chorionic gonadotropin and adrenocorticotropin. A small and indeterminant yield of testosterone was obtained from dehydroisoandrosterone sulfate while androstenedionewas obtained from both substrates in a yield of 0.1 to 0.7%. An ancillary incubation using polycystic ovarian tissue was also performed. Similar results were obtained with the exception that no testosterone was recovered. The influence of gonadotropins and adrenocorticotropinon the conversions of these substrates to androstenedione was inconclusive. No sulfurylatedproducts were noted. The ovarian srrhenoblastomain the human has long been recognized as a tumor capable of causing the various physiological and morphological changes associated with androgenization. Reversal of the changes, to a variable degree, has followed tumor extirpation. That the tumor itself is the source of the elevated plasma levels of androgens found in the tumor's host (2,3) is surmised from the fact that active androgens have been extracted from this tissue (2,4),that in vitro studies have demonstrated the tissues' ability to formulate androstenedionesnd testosterone from both pregnenolone and progesterone
(5,6) and that significantlymore testosterone has been demonstrated in the tumor's venous than arterial blood (7). While the formulation of androstenedioneand testosterone has generally been considered to occur via progesterone in both normal and androgenizing ovarian tissue, recent evidence would suggest that these steroids may be formulated to some extent from A 5-3601 compounds via
250
STEROIDS
dehydroisoandrosterone(6,8).
8:2
Recognizing the normal existence of a
large body pool of dehydroisoandrosteronesulfate (g), a moderately great plasma concentration of this same material (10) as well as previous demonstrations of considerable steroid alcohol sulfatase activity in arrhenoblastomatoustissue (11,12), it is not unreasonable to consider that this sulfurylated steroid might serve as a physiological substrate for active androgen production by virilizing human ovarian tissue. This consideration is supported by the recent demonstration by Aakvaag et al. (13) of the conversion of dehydroisoandrosterone sulfate to androstenedione and testosterone by the normal canine ovary and testis perfused in vivo. The potential of the hypothesis was examined by incubating androgenizing arrhenoblastomatoustissue with radiolabelled dehydroisoandrosterone sulfate. The tissue was simultaneously incubated with radiolabelled progesterone for comparative purposes. The influence of gonadotropic and adrenocorticotropichormones on this in vitro system was also studied. An ancillary incubation was performed using polycystic ovarian tissue. Physiologically active androgen was formed from both substrates by both tissues. The influence of tropic hormones on these reactions was inconclusive. MATERIALS AND METHODS Abbreviations and trivial names. Pregnenolone = 34 -hydroxypregn-5-en-20-one,progesterone = pregn-4-en-3, 20-dione, 170(-hydroxyprogesterone = 17*-hydroxy-pregn-4-en-3, 20-dione, androstenedione= a&cost-4-en-3, 17-dione, testosterone = 176 -hydroxy-androst-k-en-3one, testosterone acetate = 176 -acetoxy-androst-4-en-3-one,dehydroisoandrosterone= 38 -hydroxy-androst-5-en-17-one, dehydroisoandrosterone sulfate = 38-sulfoxy-androst-5-en-17-one, cholesterol = cholest-5-en-3@-01, Tris = 2-amino-2(hydroxy-methyl)-1,3-propanediol, RMG = human menopausal urinary gonadotropin (Cutter Laboratories), HCG = human chorionic gonadotropin (Squibb and Sons), ACTH = adrenocorticotropin (Abbott Laboratories), NAD = nicotinamide-adeninedinucleotide, JXADP= nicotinamide-adeninedinucleotide phosphate. Clinical summations. The ovarian arrhenoblastomatoustissue used in this experiment was obtained from a sixteen year old female with amenorrhea, anovulation, hirsutism, acne and masculine musculature. Her clinical status and course are described in the accompanying paper. The tumor was frozen immediately following extirpation and was thawed immediately prior to incubation. Polycystic ovarian tissue used in the ancillary experiment was obtained from a fifteen year old female with a history of primary
Aug.
1966
STEROIDS
251
amenorrhea, obesity since the age of three and rapidly developing hirsutism of the face, abdomen and extremities for the preceding eighteen months. Pubic and axillary hair had appeared at the age of eight and breast development had also begun at that time. She was muscular and obese and, although her breasts were of normal size, she exhibited hirsutism, a male escutcheon and an enlarged clitoris. Pelvic exsmination revealed a normal vagina and uterine cervix while pelvic pneumography demonstrated a small uterus of normal contour and ovaries bilaterally enlarged to four by seven centimeters. Urinary 17-ketosteroidand 17-hydroxycorticosteroidassay values were in the normal range and suppressed normally following dexamethasone administration. The sella turcica, visual fields and plasma protein bound iodine were normal. Vaginal smears revealed a total lack of cornified cells. Bilateral ovarian wedge resection was performed and the excised tissue, both grossly and histologically,was compatible with the ovaries of the Stein-Leventhal syndrome. The adrenal glands were palpably normal. In the four month interval following surgery the patient experienced only a single, three day episode of vaginal bleeding of minimal volume. There has been no substantialphysical change since operation. The precise diagnosis remains uncertain and it is additionally uncertain whether the excess ovarian tissue encountered was the main source of the steroidal agents responsible for her clinical androgenization. Radioactivity quantitation and chromatography. The methods utilized for radioactivity quantitation and chromatographicsteroidal separation and localization have been previously described (12). Radioactive samples were counted for a sufficient period of time to obtain a statistical accuracy of i 3 percent. Non-radioactivecompounds with a A4-3 ketone configurationwere localized on paper by viewing under ultraviolet light. Solvent systems utilized in this study are listed in Table 1. Preparation and purification of radioactive substrates. 743H-dehydroisoandrosteronesulfate used in these incubationswas part of that synthesized for use in the experiment described in the accompanying paper. This material was partitioned between ether and water immediately prior to use in order to remove any unconjugated compound appearing as a result of spontaneous hydrolysis during storage. 4-14C-progesterone(SA 45.8 mc/mtnole,New England Nuclear Corporation) was chromatographicallypurified using systems I and II. Only one peak of radioactivity was noted. This had the mobility of standard progesterone in each instance. No further efforts to verify the purity of this compound were undertaken. Formation of derivatives. Acetylation was performed at room temperature by the overnight reaction of the steroid (20 mg or less) with acetic anhydride (0.6 ml) in the presence of dry pyridine '(1.2ml). The product was extracted by partitioning the reaction solvent against a small volume of methylene chloride. The latter was washed with small volumes of lN HCl, 5% NaHCO3 and water and evaporated under vacuum. Hydrolysis was accomplished by dissolving the steroid ester in 1 ml of absolute ethanol, combining this with 10 ml of 15% (v/v) aque-
STEROIDS
252
Table 1.
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Chronatographic systems employed.
system
support
I
Celite
Iso-octane: methylcellosolve (5:Q
II
Celite
Ligroin C: 8%
III
Paper
30 hours
Iso-propyl ether: ligroin B: t-butanol: 10% smmoniurn hydroxide (5:2:3:10)
IV
Paper
4$- hours
Toluene: n-butanol: 10% ammonium hydroxide (3:1:2)
V
Paper
3-4 hours
Ligroin C: 8%
VI
Paper
2 hours
Cyclohexane: benzene (1:l) (paper impregnated with propylene glycol: methanol, (1:l)
VII
Paper
9 hours
n-heptane: d~ethyls~foxide (1:l) (paper impregnated with dimethylsulfoxide:methanol, 1:l)
VIII
Celite
n-heptane: methy~cellosol~e (lo:31
Ix
Celite
&o-octane: t-butanol: methanol: water (25:10:10:5)
Duration
Solvents
methanol (1:l)
methanol (1:l)
ous HCl and incubating overnight at 37OC. The product was isolated by extracting the aqueous reaction mixture with three volumes of ether. This was washed with water until neutral and evaporated under vacuum. Reduction was achieved by reacting the steroid (in 2 ml absolute ethanol) with an equal weight of NaE%l+(in 0.85 ml water) overnight at room temperature. Several drops of 6N acetic acid were then slowly added and, following the addition of a few milliliters of water, the reaction product was extracted with ether. The ether was washed with water and evaporated under vacuum. Oxidation was carried out by dissolving the steroid in 90% acetic acid (2 to 4 ml) and reacting this with a 1.5% solution of Cr03 (6 to 8 ml), also in 9% acetic acid, at room temperature for two hours. One and one-half volumes of water were added and the product was extracted with ether. This was washed with 5% NaHC03 and water and evaporated to dryness under vacuum. Incubation of arrhenoblastomatousovarian tissue. A 5% homogenate of this tissue was prepared in 0.25 M Tris buffer. 65 mg of tissue was used in each incubation, all incubations be'ng perfor ed in duplicate. Each incubation vessel contained 3.97 x 106 dpm of lk.progesterone dissolved in six drops of absolute ethanol. An appropriate sulfate was quantity of pure, non-radioactive deh~oiso~~ostexone added so that equimolar quantities (7.5 nylmoles) of the substrates
Aug. 1966
STEROIDS
253
could be simultaneouslyincubated. NAB (O.lpole), NADP (O.lpole), glucose-6-phosphate(l.O)rmole),glucose-6-phosphatedehydrogenase (0.1 Kornberg unit), KC1 (l.OFole), and MgC12 (0.5 pole) were added to each vessel and the volume was brought to 2.3 ml with O.25M Tris buffer. Incubation was carried out in an atmosphere of 95% 02 and 5% CO2 at 37OC for two hours. Identical, separate incubations were simultaneouslyperformed following the addition of HMG (100 Cutter units), HCG (1000 I.U.) and ACTH (8 USP U.). Both boiled tissue and tissue-less control incubationswere utilized. Incubation of polycystic ovarian tissue. Precisely the same conditions were employed for these incubations as for those with arrhenoblastomatoustissue with the exception that 280 mg of polycystic ovarian tissue in a 20$ homogenate was used in each. Quantities of substrates, co-factors and tropic hormone additives were the same and boiled tissue and tissue-less control incubationswere similarly utilized. Analysis. Enzyme activity was halted by the addition of four volumes of absolute ethanol. Following refrigeration overnight, duplicate incubations were combined giving a single sample for each of the incubation conditions. Each sample was thereafter treated identically in the following fashion. The precipitate was separated by centrifugationand washed twice with 8% ethanol. The supernatants were combined and evaporated to the aqueous phase which was then partitioned against an equal volume of diethyl ether to separate unconjugated from conjugated compounds. The conjugated compounds were extracted from the aqueous phase with four volumes of n-butanol. The ether phase was partitioned against a l/3 volume of lN NaOH. The ether phase, containing unconjugated neutral steroids, was washed with water and these washings were added to the NaOH phase. The NaOH phase, containing any existing unconjugated phenolic steroids, was brought to pH 8.5 with 6N HCl and the phenolic compounds were extracted into an equal volume of benzene. The benzene was washed with water until neutral. The ether, butanol and benzene extracts were evaporated under vacuum and an aliquot of each residue was counted. RESULTS
Arrhenoblastomatousovarian tissue. Table 2 demonstrates the percentages of the initial 3H and 14C substrate radioactivity recovered in each of the solvent extracts for each of the incubation conditions. 52-57s of the ether insoluble 3H-dehydroisoandrosteronesulfate was converted to ether soluble, unconjugated, neutral steroids in all incubations except the controls in which insignificantconversions were noted. This confirms our previous demonstration of considerable steroid alcohol sulfatase activity in this tissue and the insignificant influence of gonadotropic and adrenocorticotropichormones on the activity of this enzyme under the in vitro conditions utilized (12).
STEROIDS
254
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Table 2. Percentages of initial substrate radioactivity re overed in various extracts from incubateons of ~rhenoblastoma with % -dehydroisoandrosteronesulfate and l'C!-progesterone. Tissue Tissue alone plus HMG
Tissue @us HCG
Tissue plus ACTH
No Boiled tissue tissue
3H l4 C
56.9
56.2
55.5
52.3
0.5
95.7
96.8
97.6
96.4
97.6
96.5
3,
40.6
41.6
45.8
43.6
96.0
100.7
EiutanoL14c
2.3
2.0
1.1
1.1
1e.8
0.5
3H
05.
0.7
0.4
0.3
0.1
0.1
o. 6
0.8
0.4
0.4
0.4
0.5
Ether
Benzene 14c
0.6
95-97% of the l4C radioactivity was recovered as ether soluble material from all reaction conditions as anticipated. The further interrogation of this fraction will be described later. Butanol extracts contained essentially the remainder of the incubated radioactivity. Nearly total recovery of the tritium-labeled, sulfurylated substrate was obtained from control incubations. Each butanol extract was sequentially chromatographedusing systems III and IV. All l4C radioactivity in these fractions moved with the solvent front and was felt to represent unconjugated steroidal material which was retained in the aqueous phase during ether extraction and subsequently partitioned into butanol. This presumably occurred due to the presence of some unknown tissue component inasmuch as considerably 14 less C contaminationwas noted in the butanol extract of the tissueless control incubation. The only other peak of radioactivity was composed solely of tritiated material and had the chromatographicmobility of dehydroisoandrosteronesulfate in both chromatographic systems. There was no evidence of conversion of this sulfurylated substrate to a similarly sulfurylated product. Benzene extracts were considered to contain negligible quantities of radioactivity in all instances and were not examined further. The ether extract from each of the incubationswas chromato-
255
STEROIDS
Aug. 1966
Table 3. Percentages of initial substrate radioactivityrecovered following c~~ato~aphy (system V) of ether extracts from incubatio s of arrhenoblastomawith 3H-dehydroisoandrosteronesulfate and lcC progesterone.
Tissue alone
Tissue plus BMG
Tissue plus HCG
Tissue plus ACTH
3H
1.2
1.3
0.9
1.1
Peak A 24,
o.*
0.3
0.2
0.2
48.6
47.0
46.5
Peak B 14,
2 8 .
2.8
4.8
2.4
3H
0.1
0.1
0.1
0.1
7.1
3.4
7*2
3~
graphed using system V.
43.7
Boiled No tissue tissue
0.1
0.1
1.7
0.2
0.1
-
Peaks of radioactivity coinciding with the
mobility of androstenedione(peak A), dehydroisoandrosterone(peak B) and testosterone-l'i'o(-hydroxy-progesterone (peak C) were localized and tiluted. Table 3 demonstrates the percentages of the initial %I and
14
C substrate radioactivityrecovered from these peaks. Radioactive material with the mobility of androstenedionein
each incubation (peak A) was re~~~ato~aphed
using system VI. Radio-
activity with the mobility of androstenedionein this system was eluted and mixed with a precise amount of authentic cold carrier. Each of these mixtures was recrystallized from acetone: petroleum ether to a constant
3,. 14C ratio as noted in Table 4. Specific activities relative to +I and l4C were determined for the crystals from the fourth recrystallization in each instance. Yields of androstenedione,ignoring losses incurred during extraction and purification,were calculated by isotope dilution and are expressed in Table 4 as a percentage of the initial substrate radioactivity. Approximately 0.6% of 3H-dehydroisoandrosteronesulfate was converted to androstenedione,this yield being uninfluenced by the presence of gonadotropic or adrenocorticotropichormone. 0.1% of 14Cprogesterone was converted to androstenedionein three of the four incubations. Only half this degree of conversion was noted in the pre-
256
8:2
STEROIDS
Table 4. 3,.14 C ratios and percentage yields of an rostenedione recovered from incubations4 of arrhenoblastomawith 9H-dehydroisoandrosterone sulfate and C-progesterone (following reverse isotope dilution). Tissue Tissue alone plus HMG
Tissue plus HCG
Tissue plus ACTH
3HS14C . ratios: 1st recrystallization
28.5
28.3
43.1
28.5
2nd recrystallization
33.9 34.3
62.9 69.3
30.5
3rd recrystallization
32.5 33.7
4th recrystallization
33.5
34.3
70.1
31.2
31.2
Percentage yields from:
3H-dehydrosioandrosterone sulfate 0.69 14C-progesterone 0.11
0.64
0.52
0.56
0.10
0.04
0.10
sence of HCG, this being the basis for the accentuated 3H:14C ratio noted in androstenedione in that incubation. The tritiated radioactivity in peak B (Table 3) was accepted as being composed principally of dehydroisoandrosterone,the product of steroid alcohol sulfatase activity on the sulfurylated substrate. The yield of material with this mobility was not substantiallyaltered by the presence of the tropic hormones employed. 14C-labelled material in this peak was considered to represent either a contaminant or a metabolic derivative of progesterone. Neither of these possibilities was verified as the peak was not examined further. Peak C was re-chromatographedusing system VII which separated non-radioactive standards of testosterone and 17u<-hydroxy-progesterone run simultaneously. Only two peaks of radioactivity were noted. That having the mobility of 17&-hydroxy-progesterone contained an amount of l4C equivalent to approximately 6% of the
14
C-labelled substrate in
all non-control incubations except that containing HCG, wherein only half that amount was noted. This material contained no tritium. Approximately 13 mgm of authentic testosterone carrier was added to the peak of radioactivity coinciding with the mobility of testosterone from
Aug. 1966
257
STEROIDS
each incubation and each mixture was recrystallized three times from acetone:petroleumether. The quantity of 14C!in these groups of crystals was too small to permit statistical accuracy in counting and, additionally, it was impossible to achieve a constant specific activity relative to tritiwn, presumably because of incomplete purification. The remaining material was insufficientto allow continued handling of the product of each incubation separately and, consequently, testosterone crystals and mother liquors from the various non-control incubationswere combined. The mixture was acetylated, chromatographed using system VIII and recovered testosterone acetate was recrystallized twice from ether:petroleumether. The testosterone ester was hydrolyzed and recrystallized from acetone:ether:petroleumether. The resulting material was oxidized, chromatographedusing system IX and recrystallized twice from acetone:petroleumether. These crystals had the chromatographic mobility and melting point of standard androstenedione. The specific activities of all crystals were within 4.146of their mean value (Table 5).
Table 5. Specific activities relative to 3H (dpm/mM) of derivatives of combined testosterone obtained from incub tions of arrhenoblastoma with 3H-dehydroisoandrosteronesulfate and l-&-progesterone(following reverse isotope dilution).
Following acetylation: st 1 recrystallization nd 2 recrystallization Following hydrolysis: st 1 recrystallization Following oxidation: st 1 recrystallization nd 2 recrystallization
19160 18830
18460
17760 17760
STEROIDS
258
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Table 6. Percentages of initial.substrate radioactivity recovered in various extracts from incubations of polycystic ovarian tissue with 3H-dehydroisosndrosteronesulfate and 14C-progesterone. Tissue Tissue alone plus RMG
Boiled NO tissue tissue
Tissue plus HCG
Tissue plus ACTH
6.7
7.6
6.0
0.5
3H
7.2
14c
85.9
80.9
85.0
77.5
82.8
83.7
3H
91.5
88.8
89.7
91.3
97*0
87.2
14C
5.0
5.0
3.9
5.6
4.2
0.4
3H
183
1.3
1.7
0.3
0.1
0.5
Ether
Butanol
Benzene 14C
1.0
Polycystic ovarian tissue.
Table 6 demonstrates the per-
centages of initial 3H and 14C substrate radioactivity recovered in each of the solvent extracts of each of the incubation conditions in the ancillary experiment employing polycystic ovarian tissue. Although only 6-7s hydrolysis of the sul_furylatedsubstrate occurred with this tissue, gonadotropic and adrenocorticotropichormones were again noted to have insignificant influence on the activity of the enzyme under the conditions employed. Chromatography of the butanol extract again revealed that all tritiated material had the chromatographicmobility of dehydroiso14 androsterone sulfate and that C-labelled radioactivity in this extract c~omatographed as ~conj~ated
material. Neither the butanol.
nor the benzene extracts were examined further. The ether extracts were chromatographedas in the experiment with arrhenoblastomatoustissue. Peaks of radioactivity chromatographically identical to those in the previous experiment were obtained and these were similarly designated as peaks A, B and C.
The
percentages of substrate radioactivity recovered in each are shown in Table 7. Radioactive material with the mobility of androstenedione (peak A) was handled as previously described. Authentic cold
Aug.
STEROIDS
1966
259
Table 7. Percentages of initial substrate radioactivity recovered following chromatography (system V of ether extracts from incubations of po ycystic ovarian tissue with 4H-dehydroisoandrosteronesulfate and 1tC-progesterone.
Tissue Tissue alone plus HMG
Peak A
Peak B
Peak c
Tissue plus HCG
Tissue plus ACTH
3,
0.4
0.7
0.7
0.6
14,
4.3
6.6
2.4
5.4
3H l4 C
. 49
53 .
6.1
5.2
2.1
1.1
1.4
1.3
3H
_
_
14c
40.0
47.0
49.8
47.4
Boiled No tissue tissue --
0.1
0.3
0.5
0.6
3H.14 Table 8. . C ratios and percentage yields of androstenedione recovered from incubations of po ystic ovarian tissue with 3H-dehydroisoandrosteronesulfate andl% C-progesterone (following reverse isotope dilution). Tissue plus HMG
Tissue plus HCG
0.472
0.426
1.298
0.450
0.475
0.434
1.294
0.461
0.21
0.39
0.25
2.25
1.40
2.57
Tissue alone
Tissue plus ACTH
3H:14C ratios: st 1 recrystallization nd 2 recrystallization
Percentage yields from: 3H-dehydroisoandrosterone sulfate 0.16 14 C-progesterone 1.59
carrier was added and the mixture was recrystallized to a constant 3H.14 . C ratio from acetone:petroleumether (Table 8). Yields were calculated in the same manner as for arrhenoblastomatoustissue. A smaller percentage of dehydroisoandrosteronesulfate and a
STEROIDS
260
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larger percentage of progesterone were converted to androstenedione by this tissue than by arrhenoblastomatoustissue. The 3H.14 . C ratio in androstenedioneformed in the incubation containing HCG was again higher than that noted in other incubations. This was apparently due, however, to a greater production of that compound from dehydroisoandrosteronesulfate rather than being due to a decreased production from progesterone as was noted with arrhenoblastomatous tissue. Slightly greater yields of androstenedione appear to have been obtained from progesterone in the presence of HMG and ACTH. To further verify the radiochemical purity of this product, precise weights of crystals from the second recrystallizationfor 3 14, each incubation were mixed and theoretical values for the H: ratio and specific activities relative to 3H and 14C were calculated for the mixture. The material was then reduced with NaBH4,chromatographed using system IX and recrystallized twice from methanol:acetone: petroleum ether. Acetylation was then performed and the product was chromatographedusing system VIII and recrystallized twice from methanol:benzene. The chromatographicmobility and melting point of the crystalline material were consistent with those for 38,
17@ -
diacetoxy-androst-&en. Theoretical and actual values for the 3H: 14 C ratios and specific activities of each group of crystals were constant as noted in Table 9. Radioactive material with the chromatographicmobility of dehydroisoandrosterone(peak B) was not examined further. Peaks of radioactivity having the mobility of testosterone-1W -hydroxy-progesterone(peak C) were combined and authentic testosterone carrier was added. The mixture was acetylated and chromatographed using system VIII. Insignificantradioactivity accompanied the eluted testosterone acetate and it was apparent that testosterone had not been formulated from either substrate. Radioactive material with the 14 mobility of 17cl(-hydroxy-progesterone contained C in an amount equi14 C-labelled substrate used in the four valent to 3C$ of the total incubations. No tritium was present in this material and no other peaks of radioactivity were detected.
STEROIDS
Aug.1966
261
Table 9. Results obtained after combining androstenedionefrom all incubations of polycystic ovarian tissue, following reverse isotope dilution and recrystallizationsnoted in Table 8.
Calculated values in mixture of crystals
0.608
328
540
320
531
325
538
Following reduction: lSt recrystallization
0.599
2nd recrystallization
0.603
Following acetylation: recrystallization
0.606
2nd recrystallization
0.604
P
DISCUSSION Despite the fact that four times more polycystic ovarian tissue than arrhenoblastomatoustissue was used per incubation vessel, the steroid alcohol sulfatase activity in arrhenoblastomatoustissue was significantlymore impressive. Similar but inconsistent comparisons have been previously noted (11). Gonadotropic and adrenocorticotropic hormonal additions in the concentrationsand under the conditions utilized, did not appear to modify the degree of hydrolysis of dehydroisoandrosteronesulfate by either of these tissues. We have similarly failed, in other experiments, to detect any influence of HCG on the hydrolysis of dehydroisoandrosteronesulfate or estrone sulfate by homogenates of normal human ovarian, placental or endometrial tissue. Androstenedionewas formed from both dehydroisoandrosterone sulfate and progesterone by both arrhenoblastomatousand polycystic ovarian tissues. Arrhenoblastomatoustissue formed testosterone from dehydroisoandrosteronesulfate but polycystic ovarian tissue did not. In all instances, conversion was small but adequate for demonstration and confirmation. In contrast to the results obtained from previous incubations of these forms of ovarian tissue (5,14), neither formed testosterone from progesterone under these conditions.
STEROIDS
262
8:2
Arrhenoblastomatoustissue formed androstenedionemore efficiently from dehydroisoandrosteronesulfate than from progesterone and effected a greater percentage conversion of dehydroisoandrosterone sul_fateto androstenedione than did polycystic ovarian tissue. Polycystic ovarian tissue, on the other hand, formed androstenedionemore efficiently from progesterone than from dehydroisoandrosteronesulfate and effected a greater
percentage conversion of progesterone to andro-
stenedione than did ~rhenobl~s~omatous tissue. At face value this might suggest
a difference in preferentiality for androgen precursors
by these histologically different but physiologically similar ovarian tissues but such a conclusion cannot be safely drawn from a single, & vitro observation. The presence in the incubation of HCG did not influence the conversion of dehydroisoandrosteronesulfate to androstenedioneby sxrhenoblastomatoustissue but was associated with an increased formation of an~ostenedione from this substrate by polycystic ovarian tissue. Moreover, it would appear that the presence of HCG resulted in a reduced yield of androstenedionefrom progesterone by arrhenoblastomatous tissue while HMG and ACTH were associated with an increased conversion of progesterone to androstenedioneby polycystic ovarian tissue. These variations lack confirmatory support and in this in vitro situation in which corrections for losses were incomplete, recovery of products was small and apparent tropic hormone influence was modest, the variations noted are felt to be equivocal and inconclusive. Ability of these tissues to metabolize a steroidal.sulfate without hydrolysis was not adequately tested by the substrate employed and the apparent lack of such metabolism in these incubations is not presented as evidence against such a capability. Whether or not dehydroisoandrosteronesulfate served as a precursor for any of the androgens formulated by these ovarian tissues in vivo is, of course, impossible to determine or reconstruct from these studies. The results, however, demonstrate that these abnormal human ovarian tissues have an innate capacity to utilize a sulfurylated substrate in androgen formation, a capacity which, as previously mention-
Aug. 1966
STEROIDS
263
ed, has been detected in normal canine ovarian and testicular tissue in vivo (13) and most recently in normal human testicular tissue (15) and human fetal adrenal tissue (16) in vitro. The presence of such an enzyme system in each of the three major androgen producing endocrine glands would suggest the probability that the system is put to some physiologic use. The observation of a lower concentrationof dehydroisoandrosteronesulfate in venous than arterial blood of both normal and polycystic ovaries (7) would appear to support this rationale. However, even though it has been adequately demonstrated in in vivo experiments that dehydroisosndrosteronesulfate serves as an important precursor of urinary estrogens in human pregnancy (17,188) evidence of in vivo utilization of dehydroisoandrosteronesulfate in the production of androgenic or estrogenic hormones in the non-pregnant state has not yet been obtained.
ACKNOWLEDGEMENTS This project was supported by United States Public Health Service Research Grant No. AM-08220 from the National Institutes of Human menopausal gonadotropin (Pergonal)was kindly supplied by Cutter Laboratories, Berkeley, California, U.S.A. REFERENCES 1.
2.
Presented in part at the Sixth Pan-American Congress of Endocrinology, Mexico City, October 10-15, 1965 and published in abstract form in Excerpta Medica, InternationalCongress Series, No. 99, Excerpta Medica Foundation, Amsterdam, p.E-167. Simmer, H., and Hillemanns, H.G., ARCH. FUR GYNAK Q6, 541 (u62).
3. 4. 5. 6. 7. 8. 9.
Dignam, W-J., Pion, R.J., Lamb, E.J. and Simmer, H.H., ACTA ENDOCR. (Kbh) 3, 254 (1964). Anliker, R., Rohr, 0. and Ruzicka, L., ANN. CHEM. LIEBIG'S 603, 109 (1957). Savard, K., Gut, M., Dorfman, R.I., Gabrilove, J.L. and Soffer, L.J., J. CLIN. ENDOCR. 21, 165 (1961). Kase, N. and Conrad, S.H., AM. J. OBST. AND GYNEC. 2, 1251 (1964). Simmer, H.H., In discussion of Mahesh, V.B. and Greenblatt, R.B., RECENT PROGR. HORMONE RES. 20, 341 (1.964). Mahesh, V.B. and Greenblatt, R.B., RECENT PRCGR. HORMONE RES. 20, 341 (1964). Sandberg, E., Gurpide, E. and Lieberman, S., BIOCHEMISTRY3, 1256 (1964).
264
10. 11.
12. 13. 14. 15. 16.
17. 18.
STEROIDS
8:2
Baulieu, E.E., Mauvais-Jarvis, P. and Corpe/chot,C., J. CLIN. ENDOCR, 23, 374 (1963). Warren, J.C. and French, A.P., J. CLIN. ENDOCR. 25, 278 (1965). Sandberg, E.C. and Jenkins, R.C., Accompanying article. Aakvaag, A., Hagen, A.A. and Eik-Nes, K.B., BIOCHIM. BIOPHYS. ACTA 86, 622 (1964). Axelrod, L.R. and Goldzeiher, Jew., J. CLIN. ENDOCR. 22, 431 (1962). Dixon, R., Vincent, V. and Kase, N., STEROIDS 6, 757 (1965). Villee, C.A., EXCERPTA MEDICA, INTERNATIONAL CONGRESS SERIES NO. 99, VIth Pan American Congress of Endocrinology,Mexico City, October 10-15, 1965, Abstract No. 406, p. E-183. Baulieu, E.E. and Dray, F., J. CLIN. ENDCCR. 3, 1298 (1963). Siiteri, P.K. and MacDonald, P.C., STEROIDS 2, 713 (1963).
BIOSYNTKETIC STUDIES OF BUMAN OVARIAN ARRHENOBLASTOMATOUSTISSUE IN VITRO II. FORMATION OF ANDROGENS FROM DEHYDROISOANDROSTERONESULFATE (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 Virilizi ovarian arrhenoblastomatoustissue s simultaneously incubated with % -dehydroisoandrosteronesulfate and 3%C-progesterone with and without the addition of human menopausal urinary gonadotropin, human chorionic gonadotropin and adrenocorticotropin. A small and indeterminant yield of testosterone was obtained from dehydroisoandrosterone sulfate while androstenedionewas obtained from both substrates in a yield of 0.1 to 0.7%. An ancillary incubation using polycystic ovarian tissue was also performed. Similar results were obtained with the exception that no testosterone was recovered. The influence of gonadotropins and adrenocorticotropinon the conversions of these substrates to androstenedione was inconclusive. No sulfurylatedproducts were noted. The ovarian srrhenoblastomain the human has long been recognized as a tumor capable of causing the various physiological and morphological changes associated with androgenization. Reversal of the changes, to a variable degree, has followed tumor extirpation. That the tumor itself is the source of the elevated plasma levels of androgens found in the tumor's host (2,3) is surmised from the fact that active androgens have been extracted from this tissue (2,4),that in vitro studies have demonstrated the tissues' ability to formulate androstenedionesnd testosterone from both pregnenolone and progesterone
(5,6) and that significantlymore testosterone has been demonstrated in the tumor's venous than arterial blood (7). While the formulation of androstenedioneand testosterone has generally been considered to occur via progesterone in both normal and androgenizing ovarian tissue, recent evidence would suggest that these steroids may be formulated to some extent from A 5-3601 compounds via
250
STEROIDS
dehydroisoandrosterone(6,8).
8:2
Recognizing the normal existence of a
large body pool of dehydroisoandrosteronesulfate (g), a moderately great plasma concentration of this same material (10) as well as previous demonstrations of considerable steroid alcohol sulfatase activity in arrhenoblastomatoustissue (11,12), it is not unreasonable to consider that this sulfurylated steroid might serve as a physiological substrate for active androgen production by virilizing human ovarian tissue. This consideration is supported by the recent demonstration by Aakvaag et al. (13) of the conversion of dehydroisoandrosterone sulfate to androstenedione and testosterone by the normal canine ovary and testis perfused in vivo. The potential of the hypothesis was examined by incubating androgenizing arrhenoblastomatoustissue with radiolabelled dehydroisoandrosterone sulfate. The tissue was simultaneously incubated with radiolabelled progesterone for comparative purposes. The influence of gonadotropic and adrenocorticotropichormones on this in vitro system was also studied. An ancillary incubation was performed using polycystic ovarian tissue. Physiologically active androgen was formed from both substrates by both tissues. The influence of tropic hormones on these reactions was inconclusive. MATERIALS AND METHODS Abbreviations and trivial names. Pregnenolone = 34 -hydroxypregn-5-en-20-one,progesterone = pregn-4-en-3, 20-dione, 170(-hydroxyprogesterone = 17*-hydroxy-pregn-4-en-3, 20-dione, androstenedione= a&cost-4-en-3, 17-dione, testosterone = 176 -hydroxy-androst-k-en-3one, testosterone acetate = 176 -acetoxy-androst-4-en-3-one,dehydroisoandrosterone= 38 -hydroxy-androst-5-en-17-one, dehydroisoandrosterone sulfate = 38-sulfoxy-androst-5-en-17-one, cholesterol = cholest-5-en-3@-01, Tris = 2-amino-2(hydroxy-methyl)-1,3-propanediol, RMG = human menopausal urinary gonadotropin (Cutter Laboratories), HCG = human chorionic gonadotropin (Squibb and Sons), ACTH = adrenocorticotropin (Abbott Laboratories), NAD = nicotinamide-adeninedinucleotide, JXADP= nicotinamide-adeninedinucleotide phosphate. Clinical summations. The ovarian arrhenoblastomatoustissue used in this experiment was obtained from a sixteen year old female with amenorrhea, anovulation, hirsutism, acne and masculine musculature. Her clinical status and course are described in the accompanying paper. The tumor was frozen immediately following extirpation and was thawed immediately prior to incubation. Polycystic ovarian tissue used in the ancillary experiment was obtained from a fifteen year old female with a history of primary
Aug.
1966
STEROIDS
251
amenorrhea, obesity since the age of three and rapidly developing hirsutism of the face, abdomen and extremities for the preceding eighteen months. Pubic and axillary hair had appeared at the age of eight and breast development had also begun at that time. She was muscular and obese and, although her breasts were of normal size, she exhibited hirsutism, a male escutcheon and an enlarged clitoris. Pelvic exsmination revealed a normal vagina and uterine cervix while pelvic pneumography demonstrated a small uterus of normal contour and ovaries bilaterally enlarged to four by seven centimeters. Urinary 17-ketosteroidand 17-hydroxycorticosteroidassay values were in the normal range and suppressed normally following dexamethasone administration. The sella turcica, visual fields and plasma protein bound iodine were normal. Vaginal smears revealed a total lack of cornified cells. Bilateral ovarian wedge resection was performed and the excised tissue, both grossly and histologically,was compatible with the ovaries of the Stein-Leventhal syndrome. The adrenal glands were palpably normal. In the four month interval following surgery the patient experienced only a single, three day episode of vaginal bleeding of minimal volume. There has been no substantialphysical change since operation. The precise diagnosis remains uncertain and it is additionally uncertain whether the excess ovarian tissue encountered was the main source of the steroidal agents responsible for her clinical androgenization. Radioactivity quantitation and chromatography. The methods utilized for radioactivity quantitation and chromatographicsteroidal separation and localization have been previously described (12). Radioactive samples were counted for a sufficient period of time to obtain a statistical accuracy of i 3 percent. Non-radioactivecompounds with a A4-3 ketone configurationwere localized on paper by viewing under ultraviolet light. Solvent systems utilized in this study are listed in Table 1. Preparation and purification of radioactive substrates. 743H-dehydroisoandrosteronesulfate used in these incubationswas part of that synthesized for use in the experiment described in the accompanying paper. This material was partitioned between ether and water immediately prior to use in order to remove any unconjugated compound appearing as a result of spontaneous hydrolysis during storage. 4-14C-progesterone(SA 45.8 mc/mtnole,New England Nuclear Corporation) was chromatographicallypurified using systems I and II. Only one peak of radioactivity was noted. This had the mobility of standard progesterone in each instance. No further efforts to verify the purity of this compound were undertaken. Formation of derivatives. Acetylation was performed at room temperature by the overnight reaction of the steroid (20 mg or less) with acetic anhydride (0.6 ml) in the presence of dry pyridine '(1.2ml). The product was extracted by partitioning the reaction solvent against a small volume of methylene chloride. The latter was washed with small volumes of lN HCl, 5% NaHCO3 and water and evaporated under vacuum. Hydrolysis was accomplished by dissolving the steroid ester in 1 ml of absolute ethanol, combining this with 10 ml of 15% (v/v) aque-
STEROIDS
252
Table 1.
8:2
Chronatographic systems employed.
system
support
I
Celite
Iso-octane: methylcellosolve (5:Q
II
Celite
Ligroin C: 8%
III
Paper
30 hours
Iso-propyl ether: ligroin B: t-butanol: 10% smmoniurn hydroxide (5:2:3:10)
IV
Paper
4$- hours
Toluene: n-butanol: 10% ammonium hydroxide (3:1:2)
V
Paper
3-4 hours
Ligroin C: 8%
VI
Paper
2 hours
Cyclohexane: benzene (1:l) (paper impregnated with propylene glycol: methanol, (1:l)
VII
Paper
9 hours
n-heptane: d~ethyls~foxide (1:l) (paper impregnated with dimethylsulfoxide:methanol, 1:l)
VIII
Celite
n-heptane: methy~cellosol~e (lo:31
Ix
Celite
&o-octane: t-butanol: methanol: water (25:10:10:5)
Duration
Solvents
methanol (1:l)
methanol (1:l)
ous HCl and incubating overnight at 37OC. The product was isolated by extracting the aqueous reaction mixture with three volumes of ether. This was washed with water until neutral and evaporated under vacuum. Reduction was achieved by reacting the steroid (in 2 ml absolute ethanol) with an equal weight of NaE%l+(in 0.85 ml water) overnight at room temperature. Several drops of 6N acetic acid were then slowly added and, following the addition of a few milliliters of water, the reaction product was extracted with ether. The ether was washed with water and evaporated under vacuum. Oxidation was carried out by dissolving the steroid in 90% acetic acid (2 to 4 ml) and reacting this with a 1.5% solution of Cr03 (6 to 8 ml), also in 9% acetic acid, at room temperature for two hours. One and one-half volumes of water were added and the product was extracted with ether. This was washed with 5% NaHC03 and water and evaporated to dryness under vacuum. Incubation of arrhenoblastomatousovarian tissue. A 5% homogenate of this tissue was prepared in 0.25 M Tris buffer. 65 mg of tissue was used in each incubation, all incubations be'ng perfor ed in duplicate. Each incubation vessel contained 3.97 x 106 dpm of lk.progesterone dissolved in six drops of absolute ethanol. An appropriate sulfate was quantity of pure, non-radioactive deh~oiso~~ostexone added so that equimolar quantities (7.5 nylmoles) of the substrates
Aug. 1966
STEROIDS
253
could be simultaneouslyincubated. NAB (O.lpole), NADP (O.lpole), glucose-6-phosphate(l.O)rmole),glucose-6-phosphatedehydrogenase (0.1 Kornberg unit), KC1 (l.OFole), and MgC12 (0.5 pole) were added to each vessel and the volume was brought to 2.3 ml with O.25M Tris buffer. Incubation was carried out in an atmosphere of 95% 02 and 5% CO2 at 37OC for two hours. Identical, separate incubations were simultaneouslyperformed following the addition of HMG (100 Cutter units), HCG (1000 I.U.) and ACTH (8 USP U.). Both boiled tissue and tissue-less control incubationswere utilized. Incubation of polycystic ovarian tissue. Precisely the same conditions were employed for these incubations as for those with arrhenoblastomatoustissue with the exception that 280 mg of polycystic ovarian tissue in a 20$ homogenate was used in each. Quantities of substrates, co-factors and tropic hormone additives were the same and boiled tissue and tissue-less control incubationswere similarly utilized. Analysis. Enzyme activity was halted by the addition of four volumes of absolute ethanol. Following refrigeration overnight, duplicate incubations were combined giving a single sample for each of the incubation conditions. Each sample was thereafter treated identically in the following fashion. The precipitate was separated by centrifugationand washed twice with 8% ethanol. The supernatants were combined and evaporated to the aqueous phase which was then partitioned against an equal volume of diethyl ether to separate unconjugated from conjugated compounds. The conjugated compounds were extracted from the aqueous phase with four volumes of n-butanol. The ether phase was partitioned against a l/3 volume of lN NaOH. The ether phase, containing unconjugated neutral steroids, was washed with water and these washings were added to the NaOH phase. The NaOH phase, containing any existing unconjugated phenolic steroids, was brought to pH 8.5 with 6N HCl and the phenolic compounds were extracted into an equal volume of benzene. The benzene was washed with water until neutral. The ether, butanol and benzene extracts were evaporated under vacuum and an aliquot of each residue was counted. RESULTS
Arrhenoblastomatousovarian tissue. Table 2 demonstrates the percentages of the initial 3H and 14C substrate radioactivity recovered in each of the solvent extracts for each of the incubation conditions. 52-57s of the ether insoluble 3H-dehydroisoandrosteronesulfate was converted to ether soluble, unconjugated, neutral steroids in all incubations except the controls in which insignificantconversions were noted. This confirms our previous demonstration of considerable steroid alcohol sulfatase activity in this tissue and the insignificant influence of gonadotropic and adrenocorticotropichormones on the activity of this enzyme under the in vitro conditions utilized (12).
STEROIDS
254
8:2
Table 2. Percentages of initial substrate radioactivity re overed in various extracts from incubateons of ~rhenoblastoma with % -dehydroisoandrosteronesulfate and l'C!-progesterone. Tissue Tissue alone plus HMG
Tissue @us HCG
Tissue plus ACTH
No Boiled tissue tissue
3H l4 C
56.9
56.2
55.5
52.3
0.5
95.7
96.8
97.6
96.4
97.6
96.5
3,
40.6
41.6
45.8
43.6
96.0
100.7
EiutanoL14c
2.3
2.0
1.1
1.1
1e.8
0.5
3H
05.
0.7
0.4
0.3
0.1
0.1
o. 6
0.8
0.4
0.4
0.4
0.5
Ether
Benzene 14c
0.6
95-97% of the l4C radioactivity was recovered as ether soluble material from all reaction conditions as anticipated. The further interrogation of this fraction will be described later. Butanol extracts contained essentially the remainder of the incubated radioactivity. Nearly total recovery of the tritium-labeled, sulfurylated substrate was obtained from control incubations. Each butanol extract was sequentially chromatographedusing systems III and IV. All l4C radioactivity in these fractions moved with the solvent front and was felt to represent unconjugated steroidal material which was retained in the aqueous phase during ether extraction and subsequently partitioned into butanol. This presumably occurred due to the presence of some unknown tissue component inasmuch as considerably 14 less C contaminationwas noted in the butanol extract of the tissueless control incubation. The only other peak of radioactivity was composed solely of tritiated material and had the chromatographicmobility of dehydroisoandrosteronesulfate in both chromatographic systems. There was no evidence of conversion of this sulfurylated substrate to a similarly sulfurylated product. Benzene extracts were considered to contain negligible quantities of radioactivity in all instances and were not examined further. The ether extract from each of the incubationswas chromato-
255
STEROIDS
Aug. 1966
Table 3. Percentages of initial substrate radioactivityrecovered following c~~ato~aphy (system V) of ether extracts from incubatio s of arrhenoblastomawith 3H-dehydroisoandrosteronesulfate and lcC progesterone.
Tissue alone
Tissue plus BMG
Tissue plus HCG
Tissue plus ACTH
3H
1.2
1.3
0.9
1.1
Peak A 24,
o.*
0.3
0.2
0.2
48.6
47.0
46.5
Peak B 14,
2 8 .
2.8
4.8
2.4
3H
0.1
0.1
0.1
0.1
7.1
3.4
7*2
3~
graphed using system V.
43.7
Boiled No tissue tissue
0.1
0.1
1.7
0.2
0.1
-
Peaks of radioactivity coinciding with the
mobility of androstenedione(peak A), dehydroisoandrosterone(peak B) and testosterone-l'i'o(-hydroxy-progesterone (peak C) were localized and tiluted. Table 3 demonstrates the percentages of the initial %I and
14
C substrate radioactivityrecovered from these peaks. Radioactive material with the mobility of androstenedionein
each incubation (peak A) was re~~~ato~aphed
using system VI. Radio-
activity with the mobility of androstenedionein this system was eluted and mixed with a precise amount of authentic cold carrier. Each of these mixtures was recrystallized from acetone: petroleum ether to a constant
3,. 14C ratio as noted in Table 4. Specific activities relative to +I and l4C were determined for the crystals from the fourth recrystallization in each instance. Yields of androstenedione,ignoring losses incurred during extraction and purification,were calculated by isotope dilution and are expressed in Table 4 as a percentage of the initial substrate radioactivity. Approximately 0.6% of 3H-dehydroisoandrosteronesulfate was converted to androstenedione,this yield being uninfluenced by the presence of gonadotropic or adrenocorticotropichormone. 0.1% of 14Cprogesterone was converted to androstenedionein three of the four incubations. Only half this degree of conversion was noted in the pre-
256
8:2
STEROIDS
Table 4. 3,.14 C ratios and percentage yields of an rostenedione recovered from incubations4 of arrhenoblastomawith 9H-dehydroisoandrosterone sulfate and C-progesterone (following reverse isotope dilution). Tissue Tissue alone plus HMG
Tissue plus HCG
Tissue plus ACTH
3HS14C . ratios: 1st recrystallization
28.5
28.3
43.1
28.5
2nd recrystallization
33.9 34.3
62.9 69.3
30.5
3rd recrystallization
32.5 33.7
4th recrystallization
33.5
34.3
70.1
31.2
31.2
Percentage yields from:
3H-dehydrosioandrosterone sulfate 0.69 14C-progesterone 0.11
0.64
0.52
0.56
0.10
0.04
0.10
sence of HCG, this being the basis for the accentuated 3H:14C ratio noted in androstenedione in that incubation. The tritiated radioactivity in peak B (Table 3) was accepted as being composed principally of dehydroisoandrosterone,the product of steroid alcohol sulfatase activity on the sulfurylated substrate. The yield of material with this mobility was not substantiallyaltered by the presence of the tropic hormones employed. 14C-labelled material in this peak was considered to represent either a contaminant or a metabolic derivative of progesterone. Neither of these possibilities was verified as the peak was not examined further. Peak C was re-chromatographedusing system VII which separated non-radioactive standards of testosterone and 17u<-hydroxy-progesterone run simultaneously. Only two peaks of radioactivity were noted. That having the mobility of 17&-hydroxy-progesterone contained an amount of l4C equivalent to approximately 6% of the
14
C-labelled substrate in
all non-control incubations except that containing HCG, wherein only half that amount was noted. This material contained no tritium. Approximately 13 mgm of authentic testosterone carrier was added to the peak of radioactivity coinciding with the mobility of testosterone from
Aug. 1966
257
STEROIDS
each incubation and each mixture was recrystallized three times from acetone:petroleumether. The quantity of 14C!in these groups of crystals was too small to permit statistical accuracy in counting and, additionally, it was impossible to achieve a constant specific activity relative to tritiwn, presumably because of incomplete purification. The remaining material was insufficientto allow continued handling of the product of each incubation separately and, consequently, testosterone crystals and mother liquors from the various non-control incubationswere combined. The mixture was acetylated, chromatographed using system VIII and recovered testosterone acetate was recrystallized twice from ether:petroleumether. The testosterone ester was hydrolyzed and recrystallized from acetone:ether:petroleumether. The resulting material was oxidized, chromatographedusing system IX and recrystallized twice from acetone:petroleumether. These crystals had the chromatographic mobility and melting point of standard androstenedione. The specific activities of all crystals were within 4.146of their mean value (Table 5).
Table 5. Specific activities relative to 3H (dpm/mM) of derivatives of combined testosterone obtained from incub tions of arrhenoblastoma with 3H-dehydroisoandrosteronesulfate and l-&-progesterone(following reverse isotope dilution).
Following acetylation: st 1 recrystallization nd 2 recrystallization Following hydrolysis: st 1 recrystallization Following oxidation: st 1 recrystallization nd 2 recrystallization
19160 18830
18460
17760 17760
STEROIDS
258
8:2
Table 6. Percentages of initial.substrate radioactivity recovered in various extracts from incubations of polycystic ovarian tissue with 3H-dehydroisosndrosteronesulfate and 14C-progesterone. Tissue Tissue alone plus RMG
Boiled NO tissue tissue
Tissue plus HCG
Tissue plus ACTH
6.7
7.6
6.0
0.5
3H
7.2
14c
85.9
80.9
85.0
77.5
82.8
83.7
3H
91.5
88.8
89.7
91.3
97*0
87.2
14C
5.0
5.0
3.9
5.6
4.2
0.4
3H
183
1.3
1.7
0.3
0.1
0.5
Ether
Butanol
Benzene 14C
1.0
Polycystic ovarian tissue.
Table 6 demonstrates the per-
centages of initial 3H and 14C substrate radioactivity recovered in each of the solvent extracts of each of the incubation conditions in the ancillary experiment employing polycystic ovarian tissue. Although only 6-7s hydrolysis of the sul_furylatedsubstrate occurred with this tissue, gonadotropic and adrenocorticotropichormones were again noted to have insignificant influence on the activity of the enzyme under the conditions employed. Chromatography of the butanol extract again revealed that all tritiated material had the chromatographicmobility of dehydroiso14 androsterone sulfate and that C-labelled radioactivity in this extract c~omatographed as ~conj~ated
material. Neither the butanol.
nor the benzene extracts were examined further. The ether extracts were chromatographedas in the experiment with arrhenoblastomatoustissue. Peaks of radioactivity chromatographically identical to those in the previous experiment were obtained and these were similarly designated as peaks A, B and C.
The
percentages of substrate radioactivity recovered in each are shown in Table 7. Radioactive material with the mobility of androstenedione (peak A) was handled as previously described. Authentic cold
Aug.
STEROIDS
1966
259
Table 7. Percentages of initial substrate radioactivity recovered following chromatography (system V of ether extracts from incubations of po ycystic ovarian tissue with 4H-dehydroisoandrosteronesulfate and 1tC-progesterone.
Tissue Tissue alone plus HMG
Peak A
Peak B
Peak c
Tissue plus HCG
Tissue plus ACTH
3,
0.4
0.7
0.7
0.6
14,
4.3
6.6
2.4
5.4
3H l4 C
. 49
53 .
6.1
5.2
2.1
1.1
1.4
1.3
3H
_
_
14c
40.0
47.0
49.8
47.4
Boiled No tissue tissue --
0.1
0.3
0.5
0.6
3H.14 Table 8. . C ratios and percentage yields of androstenedione recovered from incubations of po ystic ovarian tissue with 3H-dehydroisoandrosteronesulfate andl% C-progesterone (following reverse isotope dilution). Tissue plus HMG
Tissue plus HCG
0.472
0.426
1.298
0.450
0.475
0.434
1.294
0.461
0.21
0.39
0.25
2.25
1.40
2.57
Tissue alone
Tissue plus ACTH
3H:14C ratios: st 1 recrystallization nd 2 recrystallization
Percentage yields from: 3H-dehydroisoandrosterone sulfate 0.16 14 C-progesterone 1.59
carrier was added and the mixture was recrystallized to a constant 3H.14 . C ratio from acetone:petroleumether (Table 8). Yields were calculated in the same manner as for arrhenoblastomatoustissue. A smaller percentage of dehydroisoandrosteronesulfate and a
STEROIDS
260
8:2
larger percentage of progesterone were converted to androstenedione by this tissue than by arrhenoblastomatoustissue. The 3H.14 . C ratio in androstenedioneformed in the incubation containing HCG was again higher than that noted in other incubations. This was apparently due, however, to a greater production of that compound from dehydroisoandrosteronesulfate rather than being due to a decreased production from progesterone as was noted with arrhenoblastomatous tissue. Slightly greater yields of androstenedione appear to have been obtained from progesterone in the presence of HMG and ACTH. To further verify the radiochemical purity of this product, precise weights of crystals from the second recrystallizationfor 3 14, each incubation were mixed and theoretical values for the H: ratio and specific activities relative to 3H and 14C were calculated for the mixture. The material was then reduced with NaBH4,chromatographed using system IX and recrystallized twice from methanol:acetone: petroleum ether. Acetylation was then performed and the product was chromatographedusing system VIII and recrystallized twice from methanol:benzene. The chromatographicmobility and melting point of the crystalline material were consistent with those for 38,
17@ -
diacetoxy-androst-&en. Theoretical and actual values for the 3H: 14 C ratios and specific activities of each group of crystals were constant as noted in Table 9. Radioactive material with the chromatographicmobility of dehydroisoandrosterone(peak B) was not examined further. Peaks of radioactivity having the mobility of testosterone-1W -hydroxy-progesterone(peak C) were combined and authentic testosterone carrier was added. The mixture was acetylated and chromatographed using system VIII. Insignificantradioactivity accompanied the eluted testosterone acetate and it was apparent that testosterone had not been formulated from either substrate. Radioactive material with the 14 mobility of 17cl(-hydroxy-progesterone contained C in an amount equi14 C-labelled substrate used in the four valent to 3C$ of the total incubations. No tritium was present in this material and no other peaks of radioactivity were detected.
STEROIDS
Aug.1966
261
Table 9. Results obtained after combining androstenedionefrom all incubations of polycystic ovarian tissue, following reverse isotope dilution and recrystallizationsnoted in Table 8.
Calculated values in mixture of crystals
0.608
328
540
320
531
325
538
Following reduction: lSt recrystallization
0.599
2nd recrystallization
0.603
Following acetylation: recrystallization
0.606
2nd recrystallization
0.604
P
DISCUSSION Despite the fact that four times more polycystic ovarian tissue than arrhenoblastomatoustissue was used per incubation vessel, the steroid alcohol sulfatase activity in arrhenoblastomatoustissue was significantlymore impressive. Similar but inconsistent comparisons have been previously noted (11). Gonadotropic and adrenocorticotropic hormonal additions in the concentrationsand under the conditions utilized, did not appear to modify the degree of hydrolysis of dehydroisoandrosteronesulfate by either of these tissues. We have similarly failed, in other experiments, to detect any influence of HCG on the hydrolysis of dehydroisoandrosteronesulfate or estrone sulfate by homogenates of normal human ovarian, placental or endometrial tissue. Androstenedionewas formed from both dehydroisoandrosterone sulfate and progesterone by both arrhenoblastomatousand polycystic ovarian tissues. Arrhenoblastomatoustissue formed testosterone from dehydroisoandrosteronesulfate but polycystic ovarian tissue did not. In all instances, conversion was small but adequate for demonstration and confirmation. In contrast to the results obtained from previous incubations of these forms of ovarian tissue (5,14), neither formed testosterone from progesterone under these conditions.
STEROIDS
262
8:2
Arrhenoblastomatoustissue formed androstenedionemore efficiently from dehydroisoandrosteronesulfate than from progesterone and effected a greater percentage conversion of dehydroisoandrosterone sul_fateto androstenedione than did polycystic ovarian tissue. Polycystic ovarian tissue, on the other hand, formed androstenedionemore efficiently from progesterone than from dehydroisoandrosteronesulfate and effected a greater
percentage conversion of progesterone to andro-
stenedione than did ~rhenobl~s~omatous tissue. At face value this might suggest
a difference in preferentiality for androgen precursors
by these histologically different but physiologically similar ovarian tissues but such a conclusion cannot be safely drawn from a single, & vitro observation. The presence in the incubation of HCG did not influence the conversion of dehydroisoandrosteronesulfate to androstenedioneby sxrhenoblastomatoustissue but was associated with an increased formation of an~ostenedione from this substrate by polycystic ovarian tissue. Moreover, it would appear that the presence of HCG resulted in a reduced yield of androstenedionefrom progesterone by arrhenoblastomatous tissue while HMG and ACTH were associated with an increased conversion of progesterone to androstenedioneby polycystic ovarian tissue. These variations lack confirmatory support and in this in vitro situation in which corrections for losses were incomplete, recovery of products was small and apparent tropic hormone influence was modest, the variations noted are felt to be equivocal and inconclusive. Ability of these tissues to metabolize a steroidal.sulfate without hydrolysis was not adequately tested by the substrate employed and the apparent lack of such metabolism in these incubations is not presented as evidence against such a capability. Whether or not dehydroisoandrosteronesulfate served as a precursor for any of the androgens formulated by these ovarian tissues in vivo is, of course, impossible to determine or reconstruct from these studies. The results, however, demonstrate that these abnormal human ovarian tissues have an innate capacity to utilize a sulfurylated substrate in androgen formation, a capacity which, as previously mention-
Aug. 1966
STEROIDS
263
ed, has been detected in normal canine ovarian and testicular tissue in vivo (13) and most recently in normal human testicular tissue (15) and human fetal adrenal tissue (16) in vitro. The presence of such an enzyme system in each of the three major androgen producing endocrine glands would suggest the probability that the system is put to some physiologic use. The observation of a lower concentrationof dehydroisoandrosteronesulfate in venous than arterial blood of both normal and polycystic ovaries (7) would appear to support this rationale. However, even though it has been adequately demonstrated in in vivo experiments that dehydroisosndrosteronesulfate serves as an important precursor of urinary estrogens in human pregnancy (17,188) evidence of in vivo utilization of dehydroisoandrosteronesulfate in the production of androgenic or estrogenic hormones in the non-pregnant state has not yet been obtained.
ACKNOWLEDGEMENTS This project was supported by United States Public Health Service Research Grant No. AM-08220 from the National Institutes of Human menopausal gonadotropin (Pergonal)was kindly supplied by Cutter Laboratories, Berkeley, California, U.S.A. REFERENCES 1.
2.
Presented in part at the Sixth Pan-American Congress of Endocrinology, Mexico City, October 10-15, 1965 and published in abstract form in Excerpta Medica, InternationalCongress Series, No. 99, Excerpta Medica Foundation, Amsterdam, p.E-167. Simmer, H., and Hillemanns, H.G., ARCH. FUR GYNAK Q6, 541 (u62).
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