- Email: [email protected]
Steroid biosynthesis in vitro by testes of rainbow trout, Salmo gairdneri
GEKER4L
AND
Steroid
COMPARATIVE
Biosynthesis
in Vitro RUOICHI
National
8, 305-313 (1967)
ENDOCRINOLOGY
Science Museum,
by Testes of Rainbow ARAI
AND
BUN-ICHI
Ueno, Tokyo; and National Chiba-shi, Japan Received August
Trout,
Saltno
g~ir~~
TAMAOKI Institute
of Radiological
Sciencesj
17, 1966
After incubation of progesterone-4-Y! with the testicular homogenate of rainbow trout (Salmo gairdneri), radioactive 17a-hydroxyprogesterone, 17a,20/3-dihydroxypregn+en-3-one, androst-4-ene-3,17-dione and testosterone were obtained as metabolites of progesterone. Testosterone, llfi-hydroxytestosterone and ll-ketotestosterone were confirmed as metabolites of androstil-ene-3,17-dione-4-W by the fish testicular homogenate, but neither llP-hydroxyandrost-4-ene-3,17-dione nor adrenosterone was detected as its metabolite. The incubation products were respectively identified by their mobilities on thin layer chromatograms, chemical characteristics in comparison with the corresponding authentic preparations, and finally on the basis of constant specific activities throughout repeated crystallizations with respective reference preparations. These studies demonstrated the presence of 17~ hydroxylase, 20,&hydroxysteroid dehydrogenase, 17a-hydroxyprogesterone C&L lyase, 17P-hydroxysteroid dehydrogenase, lip-hydroxylase and 11/3-hydroxysteroid dehydrogenase in rainbow trout testes. The biosynthetic studies suggest a pathway of testosterone formation from progesterone in the testicular tissue of this fish similar to that which is reported for mammalian testes. However, rainbow trout testes furthermore forms 11-ketotestosterone from androst-4-ene-3,17-dione probably through testosterone and lip-hydroxytestosterone, which sequence of bioconversion was not observed for normal mammalian testes.
It was reported that several steroids occuring in the testes of fish were identified as being similar to those found in mammalian endocrine glands. Progesterone,2 androstenedione, testosterone and/or es-
tradiol-17,p have been isolated from elasmobranches (Chieffi and Lupo, 1961; Simpson et al., 1963). Existence of l?crhydroxylase, 21-hydroxylase, 20/3-hydroxysteroid dehydrogenase and the pregneneC,,-C,, lyase was demonskated in the testis and semen of the dogfish (Simpson et al., 1964a,b). As to teleosts, the same steroids that! have been obtained from elasmobraach testes have been identified in teleostean t.estes (Chief%, 1962; Grajcer and Idler, 1963) and in t’he hermaphroditic gonads of Xerranus scriba (Lupo and Chief!?, 1965). Arai et al. (1964) reported bioconversion of progesterone to 17a-hydroxyprogesterone and androstenedione in the testes of Japanese date, Triboiadon F,akonensis. These findings are certainly in agreement with the concept that umversal
’ This investigation was supported in part by Research Grant CA-07083 from National Institutes of Health, U. S. Public Health Service. ‘The following are the systematic names for ihe steroids mentioned in the text: progesterone, pregn-4-ene-3,20-dione; 17a-hydroxyprogesterone, 17a-hydroxypregn-4-ene-3,20-dione ; Compound S, 17a,21-dihydroxypregn-4-ene-3,2O-dione; androstenedione, androst-4-ene-3,17-dione; testosterone, 17P-hydroxyandrost-4-en-3-one; llp-hydroxytestosterone, 11/3,17/3-dihpdroxyandrost-4-en-3-one; llketotrstosterone, 17P-hydroxyandrost-4-ene-3,11dione ; IGa-hydroxytestosterone, 16a,l7/3-dihydroxyandrost-4-en-3-one ; adrenosterone, androst4-ene-3,11,17-trione and ll,&hydroxyandrostenedione. ll,B-hydroxyandrost-4-ene-3,17-dione. 305
306
ARAI
AND
metabolic pathways exist for androgen synthesis up to testosterone. From the standpoint of comparative endocrinology, the present paper deals with steroidogenesis in testes of rainbow trout in breeding season, using radioactive substrates progesterone-4J4C and androstenedione-4-W. MATERIALS RADIOACTIVE
AND
SUBSTRATES
METHODS AND
CARRIER
STEROIDS
Progesterone-4-14C (83 pc/mg) and androstenedione-4-l’C (71 pc/mg) were purchased from New England Nuclear Corp. (Boston, Mass.). llP-Hydroxytestosterone and its l&isomer were a gift from Dr. L. L. Engel of Harvard Medical School (Boston, Mass.) and other steroids were commercially obtained. Radiochemical purities of the substrate steroids were confirmed by thin-layer chromatography shortly before use. TISSUE
PREPARATION
AND
INCUBATUSN
The adult male of the rainbow trout, Salmo gairdneri in breeding season, 2 years of age, was caught at Oizumi Trout Hatchery, the Tokyo University of Fisheries, on November 5, 1964. From the rainbow trout, 1020 gm in body weight, 45 gm of testes was obtained and kept in cold Krebs-Ringer phosphate bicarbonate solution (pH 7.0). The testes were entirely free from the contamination of adrena. tissue, as interrenal tissue of the fish in the kidney was located differently from the testes. Ten grams of the tissue was homogenized in 3O ml of Krebs-Ringer phosphate bicarbonate solution with a loose-fitting Teflon homogenizer between 0” and 5°C. Ten milliliters of the testicular homogenate was mixed with 10 ml of Krebs-Ringer solution containing NADPH as cofactor and then transfered into the incubation flasks, in which the radioactive steroid precursor (progesterone-4-“C, 9.2 x 10“ cpm or androstenedione-4-‘4C, 10.2 X lOI cpm) had been added with two drops of propylene glycol. Final volume of the incubation mixture was 10 ml per flask with 12 pM of NADPH. Incubation was carried out with shaking for 30 min in air at 16”C, temperature of water in which the fish had been kept. EXTRACTION
AND
SEPARATION
OF
THE
TAMAOKI
The residual aqueous layer containing the protein precipitates was extracted twice more with the total volume of 40 ml of methylene dichloride. The extracts were combined and dried over anhydrous sodium sulfate, filtered through glass wool, and concentrated with stream of nitrogen gas at 40°C. An aliauot of the extract. to which were added nonradioactive carrier steroids, was analyzed by thin-layer chromatography in which a coating of fluorescent silica gel GF (E. Merck, Darmstadt, Germany) was used, with n-heptaneethyl ether (9:2, v/v) system being used to remove less polar lipoidal contaminants. Thereafter the same chromatogram was again developed with System A, benzene-acetone (8:2, v/v) system, at 15”-20°C. Zones of carrier A4-3-ketosteroids were detected under an ultraviolet lamp (wavelength, 253 mp) and also by color reagents. Radioactive zones were detected by autoradio’graphic method of exposing X-ray film to the coated phase of the thin-layer plate for several days and also by windowless flow-gas counter with a scanning device (Vanguard Autoscannel 880, Technical Measurement Corp., North Haven, Conn.) Detected radioactive areas were scraped off from the plate and eluted from absorbent with a mixture of chloroform and ethanol (1: 1, v/v), after packing scraped silica gel powder as a small column. Each radioactivity thus separated from the zones was measured by a liquid scintillation spectrometer. Radioactive products were subjected to acetylation with acetic anhydride and pyridine (1: I, v/v) and oxidation with 0.5% CrOa in 90% acetic acid for 10 min. They were then chromatographed on thin layer in System A and System B, or benzene-chloroform-ethyl ethermethanol (2:2:1:1, by vol.) system, and their behaviors on a thin-layer chromatogram were compared with those of the standard preparations identically treated with the above stated procedures. Finally, their identities were confirmed bv reoeated crvstallizations with various solvent system after addition of corresponding authentic preparation to them. Criterion on the constancy of specific activity of crystals was based on a coefficient of variation of less than 5%, at least in the last three consecutive crystallizations, considering also the specific activity of solid in mother liquor. I
-
PRODUCTS
Immediately after incubation, 20 ml of methylene dichloride was added to each flask, and the flasks were shaken vigorously to arrest further enzymatic reactions. The mixture was centrifuged at ZOOO x g for 20 min. The methylene dichloride layer was separated and reserved as an extract.
QUANTITATION
OF RADIOACTIVITIES
OF THE
PRODUCTS
Suitable aliquots of radioactive compound solutions were transfered into the counting glass vials (Wheaton Glass, Co., Millville, N. J.) and, after evaporation of the solvents, 11 ml of scintillation grade toluene solution containing 0.4%
ANDROGEN
SYNTHESIS
of 2,5-diphenyloxazole or PPO and 001% of 1,4bis-2(5phenyloxazolyl)-benzene or POPOP was added to each vial. Radioactivities were then measured with a liquid scintillation spectrometer (System 725, Nuclear Chicago, Des Plaines, Ill.) for a sufficient period of time to reduce the counting error to below 5%. Average efficiency of counting radioca,rbon was about 70%. CHEMICAL
DERIVilTION
IN
FISH
307
TESTES
17a+hydroxyprogesterone, and Compound S were added to the extract. The mixture was chromatographed on thin layer of silica gel in System A at, 15”-20°C. The radioactive metabolites were isolatecl from the chromatogram and identified after extensive purification, as summarized in Table 1. Idenmtification of Androste?ledio?~,e-?j(I:
OF THE STEXOIDS
Samples were acetylated in the following manner: the radioactive steroid was dissolved in pyridine, to which an equal volume of acetic anhydride was added, and the mixture was left overnight at room t,emperature. Then, the reagents were removed by a stream of nitrogen gas. For oxidation, samples were dissolved in 1 ml of 0.5% Cr03 in 66% acetic acid and kept for 10 min at room temperature. Three ml of distilled water were added to stop further reaction, and then extraction was carried out three times with 5 ml of methylene dichloride each time. The methylene dichloride extract was washed twice wit’h 2 ml of water, dried with anhydrous sodium sulfate, and then evaporated to dryness. Another type of oxidation was carried out with chromium trioxide-pyridine complex (Poos et al., 1953) for 1 hour at room temperature.
The eluate of the radioactive fraction (Zone II) corresponding to carrier androstenedione on the initial thin-layer ehromatogram was concentrated, and an aliquot was reohromatographecl in System A. Repeated chromatography yielded a single radioactive peak which showed the: mobility identical with authentic androstenedione. The radioackive substance thus obtained could not be acetylat’ed wit’h pyridine and acetic anhydride. After treatment with CrO, in acetic acid, the radioactive substance referable to androst.enedione remained uncha,nged. Carrier androstenedione (10.5 mg) was added to the purified radioactive substance and the mixture was crystallized from several solvent mixture,, and as shown in Table 2: RESULTS speoific activities of the crystals were Inciibation Study of Progesterone-&Y2 as found constant through crystaliizathon with methanol-water, et.hanol-water, acetonethe Substrate water and dioxane-water, respectively. Of the 1.7 gm of homogenized tissue incubated with progesterone-4-l%, as Identification of 17cr-Nydroxypropreviously desc’ribed, 99.0% of the initial gesterone-‘“6 radioactivity or 9.1 X lo4 cpm was recovered by the extraction from the incuThe radioactive area (Zone III) corbation mixture. About 50 pg each of pro- responding to standard 17ma-hydroxyprogestrone, andro&enedione, testosterone, gestrone an t,he init.ial thin-layer ehroTABLE DISTRIBGTION
1
OF RADIOACTIVITY DURING PRELIMIKARY SEPARATION. AND RADIOACTIVE METABOLITES 0~ PROGESTERONE-4J*C FINALLY IDE~~TIFIED Metabolite
Zone
I
0.58
zone Zone Zone Zone
II III IV V
0.50 0.36 0.29 0.18
2.2 2.3 5.2 7.6 72.1
x x x X X
10” 10” 103 lo3 103
Progesterone Androstenedione 17ol-Hydroxyprogesterone Testosterone 17~,20fl-Dihydroxypregn-4-en-3-one
a Calculated from the chromatogram developed by benzene-acetone (8 : 3) system. h Calculated from radioactivity of the fraction to total radioactivity recovered after incubation cpm).
Hieidh (%)
24 2.5 5.7 8.4 79.2 (9,1 X IW
308
ARAI
IDENTIFICATION
Zone
II III Ivb Vc
Added
OF ZONES
carrier
steroid
Androstenedione 17ol-Hydroxyprogesterone Testosterone acetate 17a-Hydroxyprogesterone
II, III,
Id
AND
TAMAOKI
TABLE 2 IV AND V MENTIONED Specific
activity
2nd-
3rd-
(cpm/mg) 4tha
153 152 155 153 268 262 270 257 212 207 199 200 144 146 148 144
IN TABLE Calculated specific activity
MeaIl *SE
153 264 205 146
k + + k
1 BY RECRYSTALLIZATION
(oPdw
1 3 3 1
150 288 200 153
Identified
as
Androstenedione 17cr-Hydroxyprogesterone Testosterone 17ar,20p-Dihydroxypregn4-en-a-one
Q Crystallization with different solvent mixture (see text). b The radioactive substance obtained from Zone IV was acetylated before crystallization. c The radioactive substance obtained from Zone V was oxidized after this substance was chromatographically identified as 17&,2Op-dihydroxypregn-l-en-3-one, but not as 17cu,20~dihydroxy-pregn-4-en-3-one and others.
matogram was eluted. This radioactive crystallized four times with authentic tessubstance was not acetylated with a mix- tosterone acetate, with solvent mixtures ture of pyridine and acetic anhydride. such as methanol-water, ethanol-water, Oxidation of the substance with CrOs in acetone-water, and dioxane-water, and acetic acid yielded a radioactive product constancy of specific activities of the which had a mobility identioal with obtained crystals was confirmed (Table 2). authentic androstenedione on thin-layer chromatogram developed by System A. Identification of 17cr,~O,~-Dihydroxypregn17a-Hydroxyprogesterone (27.2 mg) was The radioactive area (Zone V) which added as carrier and the mixture was crystallized with methylene dichloride-nwas a little less polar than Compound S on the initial thin-layer chromatogram heptane, acetone-water, methanol-water and ethyl acetate-n-heptane, where con- was oxidized with chromium trioxidestant specific activity was observed (Table pyridine complex, and yielded the radioactive products corresponding to 17a-hy2). droxyprogesterone and androstenedione in Identification of Testosterone-W? 32.7% and 22.5%, respectively. On the The radioactive area (Zone IV) cor- other hand, it was found by acetylation responding to carrier testosterone on the experiment that the radioactive substance initial thin-layer chromatogram was ex- seemed to retain an acetylable hydroxyl substance, tracted, and an aliquot of the extract was group. To this radioactive rechromatographed in System A. In every 17@,2O/?-dihydroxypregn-4-en-3-one and its repeated chromatography, a single radio- 20a-epimer were added, and the mixture active peak which had a mobility identical was chromatographed in System A and with authentic testosterone was observed. System B in which systems 17n,20a-dihyAnother aliquot was acetylated with droxypregn-4-en-3-one was found in more pyridine and acetic anhydride mixture and polar than its 20j?-epimer and separable chromatographed in System A, where a each other. The radioactivity was located single radioactive product corresponding to at the same position as 17a,20@-dihydroxystandard testosterone acetate was detected. pregn-4-en-S-one on thin-layer chromatoOxidation of the initial radioactive sub- gram developed by System A. The product with 17a-hydroxyprogesterone stance with CrOB in acetic acid provided identical a compound identical to androstenedione which was obtained by oxidation of the on thin-layer chromatogram. The acety- radioactive substance was pooled, mixed lated radioactive substance was finally with standard 17a-hydroxyprogesterone and
ANDROGEN
SYNTHESIS
IN
TABLE DISTRIBUTION
I 11 III IV
TESTES
SEPARATION, AND FINALLY IDE~VT~FIED
Radioactivity
1.9 79.3 1.5 4.6
x x x X
RADIOACTIVE P ieldb (%:
Rietabolite
(CPrn)
0.50 0.29 0.11 0.06
309
3
OF RADIOACTIVITY DURING PRELIMIKARY METABOLITES OF ANDROSTENEDIONE-~-~~C RP
Zone Zone Zone Zone
FISH
Androstenedione Testosterone 11-Ketotestosterone I@-Mydroxytestosterone
103 103 103 lo3
2.0 85.8 1.7 55
a Calculated from the chromatogram developed by benzene-acetone (8 : 2) system. b Calculated from radioactivity of the fract,ion to total radioactivity recovered (9.24 X lo2 cpm).
after
incubation
crystallized four times. Specific activities of the crystals were constant through crystallization with methylene dichloride-nheptane, met,hanol-water, acetone-water and dioxane-water (Table 2) .
Idendification of ll-Iietotestostero~e-14C Two radioactive areas or Zones III and IV were detected around more polar posit’ion than Compound S on thin-layer chromatogram developed by System A. About Incubation Study of Androstenedione-.J- 50 kg each of ll-ketotestosterone, Il/3-hydroxyt’estosterone and I&-hydroxytestosterWI? as the Substrate one were added to the elute of the radioThe homogenized testicular tissue (1.7 active area (Zone III) which was less polar gm) was incubated with androstenedionethan the other (Zone IV)) and chromato4J4C. TotaI radioactivity extracted from graphed in System B. A single radioactive incubation mixture was 9.24 X lOa cpm or peak corresponding to the carrier I:-keto90.4% of the radioactivity initially added. testosterone was obtained, When this radioTo the extract were added about 50 pg active substance was acetylated and chroeach of the nonradioactive carrier stermatographed in System A, it provided a. oids, progesterone, 17a-hydroxyprogestersingle peak showing mobility identical with one, Compound S, androstenedione and ll-ketotestosterone acetate. Oxidation of testosterone. After removing lipid, the this radioactive substance with CrO, in steroidal extract was chromatographed on acetic acid yielded a single peak coincided thin layer developed by System A. The distribution of radioactivity in relation to with authentic adrenosterone on thin-layer carrier steroids were summarized as shown chromatogram deveioped by System A. Repeated crystallizations of this radioin Table 3. Purification and identification active substance with authentic il-ketoof the product as testosterone was identical to the previous case where progesterone testosterone showed a constant. specific was used as substrate. activit.y within experimental error, 2s TABLE IDENTIFICATION
OF
ZOXES
111
AND
IV
Specific activity Zone
Added carrier steroid
III IV
11-Ketotestosterone Adrenosterone
I St= Znd=
61 136
60 142
4
MENTIONED
3rda
61 137
IN TABLE
(cpm/mg) 4th=
3 BY ]~ECRYSTALLIZATI~N
___-
C$$t;d nhn ZkSE!
59 60 5 1 141 139 + 1
activity icPm/w)
61 141
Identified
2s
ll-Ketotestosterone 1 I@-Hydroxytestosterone
a Recrystallized from different solvent system (see text). b The radioactive substance obtained from Zone IV was oxidized after this substance was chromatographically identified as Pip-hydroxyt,estosterone, but not as ll~hydroxytestosterone~ ll&hydroxyandrostenedione and adrenosterone.
310
ARAI
AND
shown in Table 4. The solvents used for repeated crystallization were methylene dichloride-n-heptane, chloroform-n-heptane, acet.one-n-heptane, and dioxane-n-heptane.
TAMAOKI
Shikita and Tamaoki, 1965a; Noumura et al., 1966), guinea pig (Lynn and Brown, 1958), and fish (Arai et al., 1964; Simpson et al., 1964). Transformation of 17c~hydroxyprogesterone into androstenedione Identification of ll@-Hydroxytestosand testosterone (Schoen, 1964) and bioterone-W synthesis from androstenedione to testosThe other radioactive area (Zone IV) on terone (Dominguez, 1961; Sharma et al., the initial thin-layer chromatogram was 1965) have been also reported. eluted and rechromatographed in System A In the present study, 17a-hydroxyproand System B. In each case, a single radiogesterone, 17,c,2Ofi-dihydroxypregn-4-en-3active peak was observed. This radioactive one, androstenedione and testosterone were substance was mixed with carrier steroids, obtained as metabolites of progesterone-4such as 11-keto-, lip-hydroxyand 16y~- YJ, and testosterone, I@-hydroxytestoshydroxy-testosterone, and then chromatoterone and 11-ketotestosterone were isographed in System B. A single radioactive lated as metabolites of androstenedione-4l”C. These findings suggest that the pathpeak occured in a position identical with llg-hydroxytestosterone, but not with the way, which involves the following sequence: + others. Furthermore, the radioactive sub- progesterone + 178a-hydroxyprogesterone androstenedione + testosterone, exists in stance was chromatographed with authentic the testes of the rainbow trout as those of llfl-hydroxytestosterone and its l&epimer normal mammals. However, isolation of in System B, in which ll,p-hydroxytestos11/3-hydroxytestosterone and ll-ketotestosterone was distinguishable from its llaterone seems to indicate a different pathepimer. The radioactive substance ran with the mobility identical with authentia 11/3- way of steroid conversion in the fish testes to those of mammalian testes. Hereafter, hydroxytestosterone. Acetylation of this the following three steroids are further dissubstance gave a single peak corresponding in comparison to 11/Ghydroxytestosterone 17&acetate on cussed in detail, particularly pathway of thin-layer chromatogram developed by with the main biosynthetic androgen established in testes of mammals. System A. This radioactive substance was 1’7a,[email protected]. This oxidized with CrO, in acetic acid solution and then chromatographed to yield a steroid has been isolated from the testes of young bulls (Neher and Wettstein, 1960) single peak which ran at the same rate and the blood plasma. of Pacific salmon with authentic adrenosterone. The radioac(Idler et al., 1960a). The reduction of 20tive product obtained by oxidation was ketone of 17a-hydroxyprogesterone to mixed with adrenosterone, and pooled, was recrystallized. Specific activities of the 178a,20fl-dihydroxypregn-4-en-3-one ported in testicular tissue of guinea pig crystals throughout t.he crystallization with (Lynn and Brown, 1958), rat (Dominguez methylene dichloride-n-heptane, acetone-net al., 1961); Sohoen, 1964), and dogfish heptane, benzene-n-heptane and dioxane-nheptane remained constant, as shown in (Simpson et al., 1964). It was reported that 17a-hydroxylation was followed by the reTable 4. duction of 20-ketone in the synthesis of DISCUSSION 17a,2&-dihydroxy compound and that Slaunwhite and Samuels (1956) described 17~~,20#a- or 17a,20,/?-dihydroxy compound the transformation of progesterone into was not subjected to the side-chain cleavandrostenedione and testosterone by rat age in the testicular tissue of rat (Shikita testicular tissue and postulated that 17a- and Tamaoki, 1965b). In the present hydroxyprogesterone was an intermediate. study, 17a-hydroxyprogesterone and 17cu, These transformations have been confirmed 2O+dihydroxypregn-4-en-3-one were isolated and identified. This suggests that in testes of men (Axelrod, 1965), rat (Lynn and Brown, 1956; Shikita et al., 1964; 20&reduction of 17a-hydroxy steroid oc-
ANDROGEN
SYNTHESIS
curred in the fish testes and that 17~ hydroxyprogesterone was mostly consumed into catabolic pathway to t.he dihydroxy compound and t,he rest was utilized for test.osterone formation under the incubation condition adopted. alp-Hydroxytestosterone. lip-Hydroxytestosterone was formed from testosterone or androstenedione in testicular t,umor of men (Savard et al., 1960; Dominguez, 1961), adrenal tumor of mice (Bloch et al., 1960) and normal human (Chang et al., 1963), and bovine (Axelrod and Arroyave, 1953) adrenals. From these results, it was postulated that 1lb-hydroxylase activity was limited to adrenal cSortex, though tesGcular tumors produced llp-hydroxytestosterone or 11/S-hydroxyandrostenedione (Engel et al., 1966). Therefore, the finding of Il,!?-hydroxylase activity in the normal testes of fish is of particular interest because no lip-hydroxylase activity could be demonstrated in normal testes of any species of animals previously, even though 21-hydroxylase in normal testes was demonstrated (Dominguez et al., 1960; Simpson et al., 1964). Dominguez (1961) suggested that two
IX
FISH
TESTES
311
kinds of II@-hydroxylase exist because of different substyat’e specificities to Cls and C,, steroids. In the present study, conversion of androstenedione-14C to different II.oxygenated steroids was demonstrated, but the formation of Il-oxysteroids from progesterone-W2 used as sub&rate could not be found. These results are in agreement with those of previous investigaticns (Dominguez, 1961; Chang et al., 1963) i and suggest that lip-hydroxylase in fish testes prefer C,, st,eroid as the substrate and furthermore that the immediate metabohte of lip-hydroxytestosterone seems LO be testosterone, as no Il,&hydroxyandrostenedione was detected in the present, experiment. I~-ki'etotestost~rone. This steroid was firstly reported from the blood plasma of Pacific salmon (Idler et al.! 1960) and found to be biosynthesized in uivo in Pacific salmon by Idler and Truscott (1963), who suggested a pathway of the androgen biosynthesis, which invoives the following sequence: 17ar-hydroxyprogesterone -+ androstenedione --)I lI@hydroxyandrostenedione -+ adrenosterone -+ Il-ketotestosterone. In their report, testost,erone 0
OH
c 0
0 IV
1
P, A
0
IX
VI
A
1. Biosymhetic pathway for steroid hormones in the testes of fish. Steroids in brackets were not detected in fish testes. Pr = produced fromm progesterone-4-“C in experiment ; A = produced from androstenedione-4-Y in experiment ; I = progesterone ; II = 17a-hydroxyprogesterone ; III = androstenedione; IV = testosterone; V = ll/%hydroxytestosterone; VT = ll-ketotestosterone ; VII == 17a,20P-dihydroxypregn-4-en-3-one; VIII = 11,bhydroxyandrostenedione ; IX = adrenosterone. FIG.
312
ARAI
AND
did not appear to be a better precursor for production of ll-ketotestosterone than did 17a-hydroxyprogesterone. In the present study, however, significant amount of testosterone, 11,8-hydroxytestosterone and 11-ketotestosterone were obtained from androstenedione-W, but neither l&L?-hydroxyandrostenedione nor adrenosterone was detected. These results suggest that llketotestosterone was synthesized more likely from testosterone through llghydroxytestosterone in the testes of rainbow trout as shown in Fig. 1 by solid lines rather than via other pathways mentioned by the broken lines (Idler et al., 1963). Recently, it was demonstrated that llketotestosterone is at least ten times more active than testosterone in producing male secondary sexual characters to female medaka, Oryxias latipes (Arai, 1967). ACKNOWLEDGMENTS The authors wish to thank Drs. Mikio Shikita, Katsumi Wakabayashi and Nobuo Egami, National Institute of Radiological Sciences, for their kind cooperation and encouragement during this investigation. The authors are also indebted to Dr. Denzaburo Inaba of the Tokyo University of Fisheries for his kind offer of experimental materials and facilities and to Dr. L. L. Engel of Harvard Medical School, Boston, Mass. for his kind supply of ll,&hydroxytestosterone and its 1 la-epimer. ADDENDUM After the manuscript was submitted, the following two papers related to the subject were published. KOERNER, D. R. (1966). lip-Hydroxysteroid dehydrogenase of lung and testis. Endoc&ology 79, 935-938; GOTTFRIED, H., AND LUSIS, 0. (1966). Steroids of invertebrates: the in vitro production of 11-ketotestosterone and other steroids by the eggs of the slug, Ation ater rujus (Linn.). Nature 218, 1488-1489. REFERENCES ARAI, R. (1967). Androgenic effects of ll-ketotestosterone on some sexual characteristics in the teleost, Oryzias latipes. Annot. Zool. Japon 40, l-5. ARAI, R., SHIKITA, M., AND TAMAOKI, B. (1964). 1n vitro bioconversion of progesterone-4-Cl4 to 17a-hydroxyprogesterone and androst-4-ene-3,17dione in testicular tissue of Tribolodon hakonen.& Gunther. Gen. Comp. Endocrinol. 4, 68-73.
TAMAOKI
L. R. (1965). Metabolic patterns of biosynthesis in young and aged human Biochim. Biophys. Acta 97, 551-556. AXELROQ L. R., AND ARROYAVE, G. (1953). Biosynthesis and characterization of llp-hydroxytestosterone. J. Amer. Chem. Sot. 75, 572% 5731. BLOCH, E., COHEN, A. I., AND FORTH, J. (1960). Steroid production in vitro by normal and adrenal tumor-bearing male mice. J. N&Z. Cancer Inst. 24, S-107. CIEANG, E., MITTELMAN, A., AND DAO, T. L. (1963). Metabolism of 4-CY-testosterone in normal human adrenal homogenate. J. Biol. Chem. 238, 9113-917. CHIEFFI, G. (1962). Integration of reproductive functions II, Endocrine aspects of reproduction in elasmoblanch fishes. Gen. Comp. Endocrinol. Suppl. 1, 2?5-285. CHJYEFFI, G., AND LUPO, C. (1961). Identification of estradiol-17P, testosterone and its precursors from Scylliorhinus stellaris testes. Nature 190, 16~9-170. DOMINGUEZ, 0. V. (1961). Biosynthesis of steroids by testicular tumors complicating congenital adrenocortical hyperplasia. J. C&n. Endocrinol. Metnb. 21, 663-674. DOMINGUEZ, 0. V., ACEVEDO,H. F., HUSEBY, R. A., AND SAMIJEL~S, L. T. (1960). Steroid 21-hydroxylase in normal testes and malignant interstitial cell tumors. J. Biol. Chem. 235, 2608-2612. ENGEL, L. L., LANMAN, G., S~CJLLY, R. E., AND VILLEE, D. B. (1966). Studies on an interstitial cell tumor of the testis: Formation of cortisd“C from acetate-l-%. J. C&n. Endocrinol. Metab. 286, 381-392. GRAJCER, D., AND’ IDLER, D. R. (1963). Conjugated testosterone in the blood and testes of spawned Fraser River sockeye salmon (Oncorhynchus nerka). Can. J. Biochem. Physiol. 41, 2330. IDLER, D. R., FAGERLUND, U. H. M., AND RONALD, A. P. (19’69a). Isolation of pregn-4-ene-17ar,20/3diol-a-one from the plasma of Pacific salmon (Oncorhynchus nerka). Biochem. Biophys. Res. Commun. 2, 133-137. IDLER, D. R., SCHMIDT, P. J., AND RONALD, A. P. (1960b). Isolation and identification of ll-ketotestosterone in salmon plasma. Can. J. Biothem. Physiol. ,38, 1053-1057. IDLER, D. R., AND TRUSCOTT, B. (1963). In viva metabolism of steroid hormones by sockeye salmon (A) Impaired hormone clearance in mature and spawned Pacific salmon (0. nerka) (B) Precursors of 11-ketotestosterone. Can. J. Biochem. Physiol. 41, 87b887. LUPO, C., AND CHIEFFI, G. (1965). Identifica-
AXELROD, steroid testes.
ANDROGEN
SYNTHESIS
tion of steroid hormones in the gonadal extract of the synchronous hermaphrodite teleost, Serranus scriba. Gen. Comp. Endocrinol. 5, 6~9% 699. LYXN,
W. S., AND BROWN, R. (1956). Mechanism of in vitro steroid oxidation. Biochim. Biophys. Acta 21, 4OS405. LYNX, W. S., AXD BROXVN, R. H. (1958). The conversion of progesterone to androgens by testes. J. Biol. Chem. 232, 1015-1030. NEHER, R., AKD WETTSTEIN, A. (1960). Steroide und andere Inhaltsstoffe aus Stierhoden. Helv. Chim.
Acta
43,
162%1639.
NOUMURA, T., WEISZ, J., AND In vitro conversion of
LLOYD,
c.
W.
(1966).
7-3H-progesterone to androgens by the rat testis during the second half of fet,ai life. Endocrinology 78, 245-253. Poos, G. I., ARTH, G. E., BEYLER, R. E., .4iiD SARETT, L. H. (1953). Approaches to the total synthesis of adrenal steroids. V. 4b-methyi-7-ethylenedioxy-l,2,3,4,4aLu,413,5,6,7, 8,10,10a,&dodecahydrophenanthrene-4/3-ol1 -one and related tricyciic derivatives. J. Amer. Chem. SKARMA,
Sot.
75, 422429. C., DORFMAN,
R. I., AKD SCYUTHREN: A. L. (1965). Steroid biosynthesis in vitro by feminizing testes. Endocrinology 76, 966-978. SAVARD, K., DORFMAN, R. I., BAGETT, B., FIELDING, L. L., ENGEL, L. L., MCPHERSON, H. T., LISTER, L. M., JOHNSON, D. S., HAMBLEN, E. C., D.
IN
FISH
3x3
TESTES
F. L. (1960). Clinical, Morphological and biochemical studies of a virilizing tumor in the testis. J. C&n. kvest. 39, 534-553. SCHOEN, E. J. (1964). Effects o’f local irra,diatio:r on testicular androgen biosynthesis. En&c+ nology 7#5, 5665. AND ENGEL,
SHIKITA,
M.,
KAKIZAKI,
H.,
AND
TAMAOXI,
B.
(1964). The pathway of formation of testosterone from 3P-hydroxypregn-5-en-%-one by rat testicular microsomes. Steroids 4, 5’21-531. SHIKITA, M., AND TAMAOKI, B. (1965a). Testosterone formation by subcellular particles ot’ rat testes. Endocrinology 76, 563-569. SHIKITA, M., ASD TAMAOKI, B. (M%b). 20~~ Hydroxysteroid dehydrogenase of testes. Riochemistry 4, 1189-l-1195. SIMPSON, T. H., WRIGHT, R. S., AND GOTTFFZIED,I%. (1963). Steroid in the semen of dogfish (Squub~s acanthius). J. Endocrinol. 26, 4891498. SIMPSON, T. H., WRIGHT, 33. S., AND RKX-F~EW, J. (1964a). Steroid biosynthesis in the semen of dogfish (SqualzLs acanthios). J. Endockok 31, 1l-20. SIMPSON, T. H., WRIGHT, R. S., AND HCNT, S. V. (1964’n). Steroid biosynthesis in the testis of dogfish (Squalus acanthias). J. Endocrinol. 31, 29-38.
W. R., JR., AND SAMUELS, 1,. T. (1956). Progesterone as a precursor of testicular androgens. J. Biol. Chem. 2220, 34358.
SLAUNWHITE,
AND
Steroid
COMPARATIVE
Biosynthesis
in Vitro RUOICHI
National
8, 305-313 (1967)
ENDOCRINOLOGY
Science Museum,
by Testes of Rainbow ARAI
AND
BUN-ICHI
Ueno, Tokyo; and National Chiba-shi, Japan Received August
Trout,
Saltno
g~ir~~
TAMAOKI Institute
of Radiological
Sciencesj
17, 1966
After incubation of progesterone-4-Y! with the testicular homogenate of rainbow trout (Salmo gairdneri), radioactive 17a-hydroxyprogesterone, 17a,20/3-dihydroxypregn+en-3-one, androst-4-ene-3,17-dione and testosterone were obtained as metabolites of progesterone. Testosterone, llfi-hydroxytestosterone and ll-ketotestosterone were confirmed as metabolites of androstil-ene-3,17-dione-4-W by the fish testicular homogenate, but neither llP-hydroxyandrost-4-ene-3,17-dione nor adrenosterone was detected as its metabolite. The incubation products were respectively identified by their mobilities on thin layer chromatograms, chemical characteristics in comparison with the corresponding authentic preparations, and finally on the basis of constant specific activities throughout repeated crystallizations with respective reference preparations. These studies demonstrated the presence of 17~ hydroxylase, 20,&hydroxysteroid dehydrogenase, 17a-hydroxyprogesterone C&L lyase, 17P-hydroxysteroid dehydrogenase, lip-hydroxylase and 11/3-hydroxysteroid dehydrogenase in rainbow trout testes. The biosynthetic studies suggest a pathway of testosterone formation from progesterone in the testicular tissue of this fish similar to that which is reported for mammalian testes. However, rainbow trout testes furthermore forms 11-ketotestosterone from androst-4-ene-3,17-dione probably through testosterone and lip-hydroxytestosterone, which sequence of bioconversion was not observed for normal mammalian testes.
It was reported that several steroids occuring in the testes of fish were identified as being similar to those found in mammalian endocrine glands. Progesterone,2 androstenedione, testosterone and/or es-
tradiol-17,p have been isolated from elasmobranches (Chieffi and Lupo, 1961; Simpson et al., 1963). Existence of l?crhydroxylase, 21-hydroxylase, 20/3-hydroxysteroid dehydrogenase and the pregneneC,,-C,, lyase was demonskated in the testis and semen of the dogfish (Simpson et al., 1964a,b). As to teleosts, the same steroids that! have been obtained from elasmobraach testes have been identified in teleostean t.estes (Chief%, 1962; Grajcer and Idler, 1963) and in t’he hermaphroditic gonads of Xerranus scriba (Lupo and Chief!?, 1965). Arai et al. (1964) reported bioconversion of progesterone to 17a-hydroxyprogesterone and androstenedione in the testes of Japanese date, Triboiadon F,akonensis. These findings are certainly in agreement with the concept that umversal
’ This investigation was supported in part by Research Grant CA-07083 from National Institutes of Health, U. S. Public Health Service. ‘The following are the systematic names for ihe steroids mentioned in the text: progesterone, pregn-4-ene-3,20-dione; 17a-hydroxyprogesterone, 17a-hydroxypregn-4-ene-3,20-dione ; Compound S, 17a,21-dihydroxypregn-4-ene-3,2O-dione; androstenedione, androst-4-ene-3,17-dione; testosterone, 17P-hydroxyandrost-4-en-3-one; llp-hydroxytestosterone, 11/3,17/3-dihpdroxyandrost-4-en-3-one; llketotrstosterone, 17P-hydroxyandrost-4-ene-3,11dione ; IGa-hydroxytestosterone, 16a,l7/3-dihydroxyandrost-4-en-3-one ; adrenosterone, androst4-ene-3,11,17-trione and ll,&hydroxyandrostenedione. ll,B-hydroxyandrost-4-ene-3,17-dione. 305
306
ARAI
AND
metabolic pathways exist for androgen synthesis up to testosterone. From the standpoint of comparative endocrinology, the present paper deals with steroidogenesis in testes of rainbow trout in breeding season, using radioactive substrates progesterone-4J4C and androstenedione-4-W. MATERIALS RADIOACTIVE
AND
SUBSTRATES
METHODS AND
CARRIER
STEROIDS
Progesterone-4-14C (83 pc/mg) and androstenedione-4-l’C (71 pc/mg) were purchased from New England Nuclear Corp. (Boston, Mass.). llP-Hydroxytestosterone and its l&isomer were a gift from Dr. L. L. Engel of Harvard Medical School (Boston, Mass.) and other steroids were commercially obtained. Radiochemical purities of the substrate steroids were confirmed by thin-layer chromatography shortly before use. TISSUE
PREPARATION
AND
INCUBATUSN
The adult male of the rainbow trout, Salmo gairdneri in breeding season, 2 years of age, was caught at Oizumi Trout Hatchery, the Tokyo University of Fisheries, on November 5, 1964. From the rainbow trout, 1020 gm in body weight, 45 gm of testes was obtained and kept in cold Krebs-Ringer phosphate bicarbonate solution (pH 7.0). The testes were entirely free from the contamination of adrena. tissue, as interrenal tissue of the fish in the kidney was located differently from the testes. Ten grams of the tissue was homogenized in 3O ml of Krebs-Ringer phosphate bicarbonate solution with a loose-fitting Teflon homogenizer between 0” and 5°C. Ten milliliters of the testicular homogenate was mixed with 10 ml of Krebs-Ringer solution containing NADPH as cofactor and then transfered into the incubation flasks, in which the radioactive steroid precursor (progesterone-4-“C, 9.2 x 10“ cpm or androstenedione-4-‘4C, 10.2 X lOI cpm) had been added with two drops of propylene glycol. Final volume of the incubation mixture was 10 ml per flask with 12 pM of NADPH. Incubation was carried out with shaking for 30 min in air at 16”C, temperature of water in which the fish had been kept. EXTRACTION
AND
SEPARATION
OF
THE
TAMAOKI
The residual aqueous layer containing the protein precipitates was extracted twice more with the total volume of 40 ml of methylene dichloride. The extracts were combined and dried over anhydrous sodium sulfate, filtered through glass wool, and concentrated with stream of nitrogen gas at 40°C. An aliauot of the extract. to which were added nonradioactive carrier steroids, was analyzed by thin-layer chromatography in which a coating of fluorescent silica gel GF (E. Merck, Darmstadt, Germany) was used, with n-heptaneethyl ether (9:2, v/v) system being used to remove less polar lipoidal contaminants. Thereafter the same chromatogram was again developed with System A, benzene-acetone (8:2, v/v) system, at 15”-20°C. Zones of carrier A4-3-ketosteroids were detected under an ultraviolet lamp (wavelength, 253 mp) and also by color reagents. Radioactive zones were detected by autoradio’graphic method of exposing X-ray film to the coated phase of the thin-layer plate for several days and also by windowless flow-gas counter with a scanning device (Vanguard Autoscannel 880, Technical Measurement Corp., North Haven, Conn.) Detected radioactive areas were scraped off from the plate and eluted from absorbent with a mixture of chloroform and ethanol (1: 1, v/v), after packing scraped silica gel powder as a small column. Each radioactivity thus separated from the zones was measured by a liquid scintillation spectrometer. Radioactive products were subjected to acetylation with acetic anhydride and pyridine (1: I, v/v) and oxidation with 0.5% CrOa in 90% acetic acid for 10 min. They were then chromatographed on thin layer in System A and System B, or benzene-chloroform-ethyl ethermethanol (2:2:1:1, by vol.) system, and their behaviors on a thin-layer chromatogram were compared with those of the standard preparations identically treated with the above stated procedures. Finally, their identities were confirmed bv reoeated crvstallizations with various solvent system after addition of corresponding authentic preparation to them. Criterion on the constancy of specific activity of crystals was based on a coefficient of variation of less than 5%, at least in the last three consecutive crystallizations, considering also the specific activity of solid in mother liquor. I
-
PRODUCTS
Immediately after incubation, 20 ml of methylene dichloride was added to each flask, and the flasks were shaken vigorously to arrest further enzymatic reactions. The mixture was centrifuged at ZOOO x g for 20 min. The methylene dichloride layer was separated and reserved as an extract.
QUANTITATION
OF RADIOACTIVITIES
OF THE
PRODUCTS
Suitable aliquots of radioactive compound solutions were transfered into the counting glass vials (Wheaton Glass, Co., Millville, N. J.) and, after evaporation of the solvents, 11 ml of scintillation grade toluene solution containing 0.4%
ANDROGEN
SYNTHESIS
of 2,5-diphenyloxazole or PPO and 001% of 1,4bis-2(5phenyloxazolyl)-benzene or POPOP was added to each vial. Radioactivities were then measured with a liquid scintillation spectrometer (System 725, Nuclear Chicago, Des Plaines, Ill.) for a sufficient period of time to reduce the counting error to below 5%. Average efficiency of counting radioca,rbon was about 70%. CHEMICAL
DERIVilTION
IN
FISH
307
TESTES
17a+hydroxyprogesterone, and Compound S were added to the extract. The mixture was chromatographed on thin layer of silica gel in System A at, 15”-20°C. The radioactive metabolites were isolatecl from the chromatogram and identified after extensive purification, as summarized in Table 1. Idenmtification of Androste?ledio?~,e-?j(I:
OF THE STEXOIDS
Samples were acetylated in the following manner: the radioactive steroid was dissolved in pyridine, to which an equal volume of acetic anhydride was added, and the mixture was left overnight at room t,emperature. Then, the reagents were removed by a stream of nitrogen gas. For oxidation, samples were dissolved in 1 ml of 0.5% Cr03 in 66% acetic acid and kept for 10 min at room temperature. Three ml of distilled water were added to stop further reaction, and then extraction was carried out three times with 5 ml of methylene dichloride each time. The methylene dichloride extract was washed twice wit’h 2 ml of water, dried with anhydrous sodium sulfate, and then evaporated to dryness. Another type of oxidation was carried out with chromium trioxide-pyridine complex (Poos et al., 1953) for 1 hour at room temperature.
The eluate of the radioactive fraction (Zone II) corresponding to carrier androstenedione on the initial thin-layer ehromatogram was concentrated, and an aliquot was reohromatographecl in System A. Repeated chromatography yielded a single radioactive peak which showed the: mobility identical with authentic androstenedione. The radioackive substance thus obtained could not be acetylat’ed wit’h pyridine and acetic anhydride. After treatment with CrO, in acetic acid, the radioactive substance referable to androst.enedione remained uncha,nged. Carrier androstenedione (10.5 mg) was added to the purified radioactive substance and the mixture was crystallized from several solvent mixture,, and as shown in Table 2: RESULTS speoific activities of the crystals were Inciibation Study of Progesterone-&Y2 as found constant through crystaliizathon with methanol-water, et.hanol-water, acetonethe Substrate water and dioxane-water, respectively. Of the 1.7 gm of homogenized tissue incubated with progesterone-4-l%, as Identification of 17cr-Nydroxypropreviously desc’ribed, 99.0% of the initial gesterone-‘“6 radioactivity or 9.1 X lo4 cpm was recovered by the extraction from the incuThe radioactive area (Zone III) corbation mixture. About 50 pg each of pro- responding to standard 17ma-hydroxyprogestrone, andro&enedione, testosterone, gestrone an t,he init.ial thin-layer ehroTABLE DISTRIBGTION
1
OF RADIOACTIVITY DURING PRELIMIKARY SEPARATION. AND RADIOACTIVE METABOLITES 0~ PROGESTERONE-4J*C FINALLY IDE~~TIFIED Metabolite
Zone
I
0.58
zone Zone Zone Zone
II III IV V
0.50 0.36 0.29 0.18
2.2 2.3 5.2 7.6 72.1
x x x X X
10” 10” 103 lo3 103
Progesterone Androstenedione 17ol-Hydroxyprogesterone Testosterone 17~,20fl-Dihydroxypregn-4-en-3-one
a Calculated from the chromatogram developed by benzene-acetone (8 : 3) system. h Calculated from radioactivity of the fraction to total radioactivity recovered after incubation cpm).
Hieidh (%)
24 2.5 5.7 8.4 79.2 (9,1 X IW
308
ARAI
IDENTIFICATION
Zone
II III Ivb Vc
Added
OF ZONES
carrier
steroid
Androstenedione 17ol-Hydroxyprogesterone Testosterone acetate 17a-Hydroxyprogesterone
II, III,
Id
AND
TAMAOKI
TABLE 2 IV AND V MENTIONED Specific
activity
2nd-
3rd-
(cpm/mg) 4tha
153 152 155 153 268 262 270 257 212 207 199 200 144 146 148 144
IN TABLE Calculated specific activity
MeaIl *SE
153 264 205 146
k + + k
1 BY RECRYSTALLIZATION
(oPdw
1 3 3 1
150 288 200 153
Identified
as
Androstenedione 17cr-Hydroxyprogesterone Testosterone 17ar,20p-Dihydroxypregn4-en-a-one
Q Crystallization with different solvent mixture (see text). b The radioactive substance obtained from Zone IV was acetylated before crystallization. c The radioactive substance obtained from Zone V was oxidized after this substance was chromatographically identified as 17&,2Op-dihydroxypregn-l-en-3-one, but not as 17cu,20~dihydroxy-pregn-4-en-3-one and others.
matogram was eluted. This radioactive crystallized four times with authentic tessubstance was not acetylated with a mix- tosterone acetate, with solvent mixtures ture of pyridine and acetic anhydride. such as methanol-water, ethanol-water, Oxidation of the substance with CrOs in acetone-water, and dioxane-water, and acetic acid yielded a radioactive product constancy of specific activities of the which had a mobility identioal with obtained crystals was confirmed (Table 2). authentic androstenedione on thin-layer chromatogram developed by System A. Identification of 17cr,~O,~-Dihydroxypregn17a-Hydroxyprogesterone (27.2 mg) was The radioactive area (Zone V) which added as carrier and the mixture was crystallized with methylene dichloride-nwas a little less polar than Compound S on the initial thin-layer chromatogram heptane, acetone-water, methanol-water and ethyl acetate-n-heptane, where con- was oxidized with chromium trioxidestant specific activity was observed (Table pyridine complex, and yielded the radioactive products corresponding to 17a-hy2). droxyprogesterone and androstenedione in Identification of Testosterone-W? 32.7% and 22.5%, respectively. On the The radioactive area (Zone IV) cor- other hand, it was found by acetylation responding to carrier testosterone on the experiment that the radioactive substance initial thin-layer chromatogram was ex- seemed to retain an acetylable hydroxyl substance, tracted, and an aliquot of the extract was group. To this radioactive rechromatographed in System A. In every 17@,2O/?-dihydroxypregn-4-en-3-one and its repeated chromatography, a single radio- 20a-epimer were added, and the mixture active peak which had a mobility identical was chromatographed in System A and with authentic testosterone was observed. System B in which systems 17n,20a-dihyAnother aliquot was acetylated with droxypregn-4-en-3-one was found in more pyridine and acetic anhydride mixture and polar than its 20j?-epimer and separable chromatographed in System A, where a each other. The radioactivity was located single radioactive product corresponding to at the same position as 17a,20@-dihydroxystandard testosterone acetate was detected. pregn-4-en-S-one on thin-layer chromatoOxidation of the initial radioactive sub- gram developed by System A. The product with 17a-hydroxyprogesterone stance with CrOB in acetic acid provided identical a compound identical to androstenedione which was obtained by oxidation of the on thin-layer chromatogram. The acety- radioactive substance was pooled, mixed lated radioactive substance was finally with standard 17a-hydroxyprogesterone and
ANDROGEN
SYNTHESIS
IN
TABLE DISTRIBUTION
I 11 III IV
TESTES
SEPARATION, AND FINALLY IDE~VT~FIED
Radioactivity
1.9 79.3 1.5 4.6
x x x X
RADIOACTIVE P ieldb (%:
Rietabolite
(CPrn)
0.50 0.29 0.11 0.06
309
3
OF RADIOACTIVITY DURING PRELIMIKARY METABOLITES OF ANDROSTENEDIONE-~-~~C RP
Zone Zone Zone Zone
FISH
Androstenedione Testosterone 11-Ketotestosterone I@-Mydroxytestosterone
103 103 103 lo3
2.0 85.8 1.7 55
a Calculated from the chromatogram developed by benzene-acetone (8 : 2) system. b Calculated from radioactivity of the fract,ion to total radioactivity recovered (9.24 X lo2 cpm).
after
incubation
crystallized four times. Specific activities of the crystals were constant through crystallization with methylene dichloride-nheptane, met,hanol-water, acetone-water and dioxane-water (Table 2) .
Idendification of ll-Iietotestostero~e-14C Two radioactive areas or Zones III and IV were detected around more polar posit’ion than Compound S on thin-layer chromatogram developed by System A. About Incubation Study of Androstenedione-.J- 50 kg each of ll-ketotestosterone, Il/3-hydroxyt’estosterone and I&-hydroxytestosterWI? as the Substrate one were added to the elute of the radioThe homogenized testicular tissue (1.7 active area (Zone III) which was less polar gm) was incubated with androstenedionethan the other (Zone IV)) and chromato4J4C. TotaI radioactivity extracted from graphed in System B. A single radioactive incubation mixture was 9.24 X lOa cpm or peak corresponding to the carrier I:-keto90.4% of the radioactivity initially added. testosterone was obtained, When this radioTo the extract were added about 50 pg active substance was acetylated and chroeach of the nonradioactive carrier stermatographed in System A, it provided a. oids, progesterone, 17a-hydroxyprogestersingle peak showing mobility identical with one, Compound S, androstenedione and ll-ketotestosterone acetate. Oxidation of testosterone. After removing lipid, the this radioactive substance with CrO, in steroidal extract was chromatographed on acetic acid yielded a single peak coincided thin layer developed by System A. The distribution of radioactivity in relation to with authentic adrenosterone on thin-layer carrier steroids were summarized as shown chromatogram deveioped by System A. Repeated crystallizations of this radioin Table 3. Purification and identification active substance with authentic il-ketoof the product as testosterone was identical to the previous case where progesterone testosterone showed a constant. specific was used as substrate. activit.y within experimental error, 2s TABLE IDENTIFICATION
OF
ZOXES
111
AND
IV
Specific activity Zone
Added carrier steroid
III IV
11-Ketotestosterone Adrenosterone
I St= Znd=
61 136
60 142
4
MENTIONED
3rda
61 137
IN TABLE
(cpm/mg) 4th=
3 BY ]~ECRYSTALLIZATI~N
___-
C$$t;d nhn ZkSE!
59 60 5 1 141 139 + 1
activity icPm/w)
61 141
Identified
2s
ll-Ketotestosterone 1 I@-Hydroxytestosterone
a Recrystallized from different solvent system (see text). b The radioactive substance obtained from Zone IV was oxidized after this substance was chromatographically identified as Pip-hydroxyt,estosterone, but not as ll~hydroxytestosterone~ ll&hydroxyandrostenedione and adrenosterone.
310
ARAI
AND
shown in Table 4. The solvents used for repeated crystallization were methylene dichloride-n-heptane, chloroform-n-heptane, acet.one-n-heptane, and dioxane-n-heptane.
TAMAOKI
Shikita and Tamaoki, 1965a; Noumura et al., 1966), guinea pig (Lynn and Brown, 1958), and fish (Arai et al., 1964; Simpson et al., 1964). Transformation of 17c~hydroxyprogesterone into androstenedione Identification of ll@-Hydroxytestosand testosterone (Schoen, 1964) and bioterone-W synthesis from androstenedione to testosThe other radioactive area (Zone IV) on terone (Dominguez, 1961; Sharma et al., the initial thin-layer chromatogram was 1965) have been also reported. eluted and rechromatographed in System A In the present study, 17a-hydroxyproand System B. In each case, a single radiogesterone, 17,c,2Ofi-dihydroxypregn-4-en-3active peak was observed. This radioactive one, androstenedione and testosterone were substance was mixed with carrier steroids, obtained as metabolites of progesterone-4such as 11-keto-, lip-hydroxyand 16y~- YJ, and testosterone, I@-hydroxytestoshydroxy-testosterone, and then chromatoterone and 11-ketotestosterone were isographed in System B. A single radioactive lated as metabolites of androstenedione-4l”C. These findings suggest that the pathpeak occured in a position identical with llg-hydroxytestosterone, but not with the way, which involves the following sequence: + others. Furthermore, the radioactive sub- progesterone + 178a-hydroxyprogesterone androstenedione + testosterone, exists in stance was chromatographed with authentic the testes of the rainbow trout as those of llfl-hydroxytestosterone and its l&epimer normal mammals. However, isolation of in System B, in which ll,p-hydroxytestos11/3-hydroxytestosterone and ll-ketotestosterone was distinguishable from its llaterone seems to indicate a different pathepimer. The radioactive substance ran with the mobility identical with authentia 11/3- way of steroid conversion in the fish testes to those of mammalian testes. Hereafter, hydroxytestosterone. Acetylation of this the following three steroids are further dissubstance gave a single peak corresponding in comparison to 11/Ghydroxytestosterone 17&acetate on cussed in detail, particularly pathway of thin-layer chromatogram developed by with the main biosynthetic androgen established in testes of mammals. System A. This radioactive substance was 1’7a,[email protected]. This oxidized with CrO, in acetic acid solution and then chromatographed to yield a steroid has been isolated from the testes of young bulls (Neher and Wettstein, 1960) single peak which ran at the same rate and the blood plasma. of Pacific salmon with authentic adrenosterone. The radioac(Idler et al., 1960a). The reduction of 20tive product obtained by oxidation was ketone of 17a-hydroxyprogesterone to mixed with adrenosterone, and pooled, was recrystallized. Specific activities of the 178a,20fl-dihydroxypregn-4-en-3-one ported in testicular tissue of guinea pig crystals throughout t.he crystallization with (Lynn and Brown, 1958), rat (Dominguez methylene dichloride-n-heptane, acetone-net al., 1961); Sohoen, 1964), and dogfish heptane, benzene-n-heptane and dioxane-nheptane remained constant, as shown in (Simpson et al., 1964). It was reported that 17a-hydroxylation was followed by the reTable 4. duction of 20-ketone in the synthesis of DISCUSSION 17a,2&-dihydroxy compound and that Slaunwhite and Samuels (1956) described 17~~,20#a- or 17a,20,/?-dihydroxy compound the transformation of progesterone into was not subjected to the side-chain cleavandrostenedione and testosterone by rat age in the testicular tissue of rat (Shikita testicular tissue and postulated that 17a- and Tamaoki, 1965b). In the present hydroxyprogesterone was an intermediate. study, 17a-hydroxyprogesterone and 17cu, These transformations have been confirmed 2O+dihydroxypregn-4-en-3-one were isolated and identified. This suggests that in testes of men (Axelrod, 1965), rat (Lynn and Brown, 1956; Shikita et al., 1964; 20&reduction of 17a-hydroxy steroid oc-
ANDROGEN
SYNTHESIS
curred in the fish testes and that 17~ hydroxyprogesterone was mostly consumed into catabolic pathway to t.he dihydroxy compound and t,he rest was utilized for test.osterone formation under the incubation condition adopted. alp-Hydroxytestosterone. lip-Hydroxytestosterone was formed from testosterone or androstenedione in testicular t,umor of men (Savard et al., 1960; Dominguez, 1961), adrenal tumor of mice (Bloch et al., 1960) and normal human (Chang et al., 1963), and bovine (Axelrod and Arroyave, 1953) adrenals. From these results, it was postulated that 1lb-hydroxylase activity was limited to adrenal cSortex, though tesGcular tumors produced llp-hydroxytestosterone or 11/S-hydroxyandrostenedione (Engel et al., 1966). Therefore, the finding of Il,!?-hydroxylase activity in the normal testes of fish is of particular interest because no lip-hydroxylase activity could be demonstrated in normal testes of any species of animals previously, even though 21-hydroxylase in normal testes was demonstrated (Dominguez et al., 1960; Simpson et al., 1964). Dominguez (1961) suggested that two
IX
FISH
TESTES
311
kinds of II@-hydroxylase exist because of different substyat’e specificities to Cls and C,, steroids. In the present study, conversion of androstenedione-14C to different II.oxygenated steroids was demonstrated, but the formation of Il-oxysteroids from progesterone-W2 used as sub&rate could not be found. These results are in agreement with those of previous investigaticns (Dominguez, 1961; Chang et al., 1963) i and suggest that lip-hydroxylase in fish testes prefer C,, st,eroid as the substrate and furthermore that the immediate metabohte of lip-hydroxytestosterone seems LO be testosterone, as no Il,&hydroxyandrostenedione was detected in the present, experiment. I~-ki'etotestost~rone. This steroid was firstly reported from the blood plasma of Pacific salmon (Idler et al.! 1960) and found to be biosynthesized in uivo in Pacific salmon by Idler and Truscott (1963), who suggested a pathway of the androgen biosynthesis, which invoives the following sequence: 17ar-hydroxyprogesterone -+ androstenedione --)I lI@hydroxyandrostenedione -+ adrenosterone -+ Il-ketotestosterone. In their report, testost,erone 0
OH
c 0
0 IV
1
P, A
0
IX
VI
A
1. Biosymhetic pathway for steroid hormones in the testes of fish. Steroids in brackets were not detected in fish testes. Pr = produced fromm progesterone-4-“C in experiment ; A = produced from androstenedione-4-Y in experiment ; I = progesterone ; II = 17a-hydroxyprogesterone ; III = androstenedione; IV = testosterone; V = ll/%hydroxytestosterone; VT = ll-ketotestosterone ; VII == 17a,20P-dihydroxypregn-4-en-3-one; VIII = 11,bhydroxyandrostenedione ; IX = adrenosterone. FIG.
312
ARAI
AND
did not appear to be a better precursor for production of ll-ketotestosterone than did 17a-hydroxyprogesterone. In the present study, however, significant amount of testosterone, 11,8-hydroxytestosterone and 11-ketotestosterone were obtained from androstenedione-W, but neither l&L?-hydroxyandrostenedione nor adrenosterone was detected. These results suggest that llketotestosterone was synthesized more likely from testosterone through llghydroxytestosterone in the testes of rainbow trout as shown in Fig. 1 by solid lines rather than via other pathways mentioned by the broken lines (Idler et al., 1963). Recently, it was demonstrated that llketotestosterone is at least ten times more active than testosterone in producing male secondary sexual characters to female medaka, Oryxias latipes (Arai, 1967). ACKNOWLEDGMENTS The authors wish to thank Drs. Mikio Shikita, Katsumi Wakabayashi and Nobuo Egami, National Institute of Radiological Sciences, for their kind cooperation and encouragement during this investigation. The authors are also indebted to Dr. Denzaburo Inaba of the Tokyo University of Fisheries for his kind offer of experimental materials and facilities and to Dr. L. L. Engel of Harvard Medical School, Boston, Mass. for his kind supply of ll,&hydroxytestosterone and its 1 la-epimer. ADDENDUM After the manuscript was submitted, the following two papers related to the subject were published. KOERNER, D. R. (1966). lip-Hydroxysteroid dehydrogenase of lung and testis. Endoc&ology 79, 935-938; GOTTFRIED, H., AND LUSIS, 0. (1966). Steroids of invertebrates: the in vitro production of 11-ketotestosterone and other steroids by the eggs of the slug, Ation ater rujus (Linn.). Nature 218, 1488-1489. REFERENCES ARAI, R. (1967). Androgenic effects of ll-ketotestosterone on some sexual characteristics in the teleost, Oryzias latipes. Annot. Zool. Japon 40, l-5. ARAI, R., SHIKITA, M., AND TAMAOKI, B. (1964). 1n vitro bioconversion of progesterone-4-Cl4 to 17a-hydroxyprogesterone and androst-4-ene-3,17dione in testicular tissue of Tribolodon hakonen.& Gunther. Gen. Comp. Endocrinol. 4, 68-73.
TAMAOKI
L. R. (1965). Metabolic patterns of biosynthesis in young and aged human Biochim. Biophys. Acta 97, 551-556. AXELROQ L. R., AND ARROYAVE, G. (1953). Biosynthesis and characterization of llp-hydroxytestosterone. J. Amer. Chem. Sot. 75, 572% 5731. BLOCH, E., COHEN, A. I., AND FORTH, J. (1960). Steroid production in vitro by normal and adrenal tumor-bearing male mice. J. N&Z. Cancer Inst. 24, S-107. CIEANG, E., MITTELMAN, A., AND DAO, T. L. (1963). Metabolism of 4-CY-testosterone in normal human adrenal homogenate. J. Biol. Chem. 238, 9113-917. CHIEFFI, G. (1962). Integration of reproductive functions II, Endocrine aspects of reproduction in elasmoblanch fishes. Gen. Comp. Endocrinol. Suppl. 1, 2?5-285. CHJYEFFI, G., AND LUPO, C. (1961). Identification of estradiol-17P, testosterone and its precursors from Scylliorhinus stellaris testes. Nature 190, 16~9-170. DOMINGUEZ, 0. V. (1961). Biosynthesis of steroids by testicular tumors complicating congenital adrenocortical hyperplasia. J. C&n. Endocrinol. Metnb. 21, 663-674. DOMINGUEZ, 0. V., ACEVEDO,H. F., HUSEBY, R. A., AND SAMIJEL~S, L. T. (1960). Steroid 21-hydroxylase in normal testes and malignant interstitial cell tumors. J. Biol. Chem. 235, 2608-2612. ENGEL, L. L., LANMAN, G., S~CJLLY, R. E., AND VILLEE, D. B. (1966). Studies on an interstitial cell tumor of the testis: Formation of cortisd“C from acetate-l-%. J. C&n. Endocrinol. Metab. 286, 381-392. GRAJCER, D., AND’ IDLER, D. R. (1963). Conjugated testosterone in the blood and testes of spawned Fraser River sockeye salmon (Oncorhynchus nerka). Can. J. Biochem. Physiol. 41, 2330. IDLER, D. R., FAGERLUND, U. H. M., AND RONALD, A. P. (19’69a). Isolation of pregn-4-ene-17ar,20/3diol-a-one from the plasma of Pacific salmon (Oncorhynchus nerka). Biochem. Biophys. Res. Commun. 2, 133-137. IDLER, D. R., SCHMIDT, P. J., AND RONALD, A. P. (1960b). Isolation and identification of ll-ketotestosterone in salmon plasma. Can. J. Biothem. Physiol. ,38, 1053-1057. IDLER, D. R., AND TRUSCOTT, B. (1963). In viva metabolism of steroid hormones by sockeye salmon (A) Impaired hormone clearance in mature and spawned Pacific salmon (0. nerka) (B) Precursors of 11-ketotestosterone. Can. J. Biochem. Physiol. 41, 87b887. LUPO, C., AND CHIEFFI, G. (1965). Identifica-
AXELROD, steroid testes.
ANDROGEN
SYNTHESIS
tion of steroid hormones in the gonadal extract of the synchronous hermaphrodite teleost, Serranus scriba. Gen. Comp. Endocrinol. 5, 6~9% 699. LYXN,
W. S., AND BROWN, R. (1956). Mechanism of in vitro steroid oxidation. Biochim. Biophys. Acta 21, 4OS405. LYNX, W. S., AXD BROXVN, R. H. (1958). The conversion of progesterone to androgens by testes. J. Biol. Chem. 232, 1015-1030. NEHER, R., AKD WETTSTEIN, A. (1960). Steroide und andere Inhaltsstoffe aus Stierhoden. Helv. Chim.
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NOUMURA, T., WEISZ, J., AND In vitro conversion of
LLOYD,
c.
W.
(1966).
7-3H-progesterone to androgens by the rat testis during the second half of fet,ai life. Endocrinology 78, 245-253. Poos, G. I., ARTH, G. E., BEYLER, R. E., .4iiD SARETT, L. H. (1953). Approaches to the total synthesis of adrenal steroids. V. 4b-methyi-7-ethylenedioxy-l,2,3,4,4aLu,413,5,6,7, 8,10,10a,&dodecahydrophenanthrene-4/3-ol1 -one and related tricyciic derivatives. J. Amer. Chem. SKARMA,
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R. I., AKD SCYUTHREN: A. L. (1965). Steroid biosynthesis in vitro by feminizing testes. Endocrinology 76, 966-978. SAVARD, K., DORFMAN, R. I., BAGETT, B., FIELDING, L. L., ENGEL, L. L., MCPHERSON, H. T., LISTER, L. M., JOHNSON, D. S., HAMBLEN, E. C., D.
IN
FISH
3x3
TESTES
F. L. (1960). Clinical, Morphological and biochemical studies of a virilizing tumor in the testis. J. C&n. kvest. 39, 534-553. SCHOEN, E. J. (1964). Effects o’f local irra,diatio:r on testicular androgen biosynthesis. En&c+ nology 7#5, 5665. AND ENGEL,
SHIKITA,
M.,
KAKIZAKI,
H.,
AND
TAMAOXI,
B.
(1964). The pathway of formation of testosterone from 3P-hydroxypregn-5-en-%-one by rat testicular microsomes. Steroids 4, 5’21-531. SHIKITA, M., AND TAMAOKI, B. (1965a). Testosterone formation by subcellular particles ot’ rat testes. Endocrinology 76, 563-569. SHIKITA, M., ASD TAMAOKI, B. (M%b). 20~~ Hydroxysteroid dehydrogenase of testes. Riochemistry 4, 1189-l-1195. SIMPSON, T. H., WRIGHT, R. S., AND GOTTFFZIED,I%. (1963). Steroid in the semen of dogfish (Squub~s acanthius). J. Endocrinol. 26, 4891498. SIMPSON, T. H., WRIGHT, 33. S., AND RKX-F~EW, J. (1964a). Steroid biosynthesis in the semen of dogfish (SqualzLs acanthios). J. Endockok 31, 1l-20. SIMPSON, T. H., WRIGHT, R. S., AND HCNT, S. V. (1964’n). Steroid biosynthesis in the testis of dogfish (Squalus acanthias). J. Endocrinol. 31, 29-38.
W. R., JR., AND SAMUELS, 1,. T. (1956). Progesterone as a precursor of testicular androgens. J. Biol. Chem. 2220, 34358.
SLAUNWHITE,