Estradiol-17,3, Estrone, and Progesterone in the Ovaries of Dogfish (Squalus Suckle Yi)* by Herbert H

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ESTRADIOL-17,3, ESTRONE, AND PROGESTERONE IN THE OVARIES OF DOGFISH (SQUALUS SUCKLE YI)* BY HERBERT H. WOTIZ,t CHARLES R. BOTTICELLI, FREDERICK L. HISAW, JR., AND A. G. OLSEN§ BOSTON UNIVERSITY SCHOOL OF MEDICINE, HARVARD UNIVERSITY, AND UNIVERSITY OF WASHINGTON Communicated by Frederick L. Hisaw, February 23, 1960 Although ovarian extracts having estrogenic activity have been reported for representatives of all classes of vertebrates and certain invertebrates, little is known of the chemical nature of the active agents in any instance with the exception of mammals. Evidence based on countercurrent partitioning, chromatography, and bioassay methods has been presented for the presence of estradiol-17,3 in ovarian eggs of the dogfish (Squalus suckleyi)1 and similar procedures have been employed for identifying estradiol-17gl, estrone, and estriol obtained from the ovaries of laying hens2 and the lungfish (Protopterus annectens).3 The present paper presents a more detailed study of the steroid hormones of the ovary of the dogfish (S. suckleyi). Procedures and Results.-One third of the total acetone extracts (2100 ml) from 7.9 Kg of whole ovaries of nonpregnant dogfish (S. suckleyi) was evaporated to dryness by a gentle stream of warm air passing over the solution which was contained in a large evaporating dish. The residue was then taken up in ethyl ether and the resulting solution kept at 50C for a week. At this time a heavy precipitate was found and the supernatant ether layer was carefully decanted. The ether solution was evaporated to an oily residue in vacuo. The oil was dissolved in a sufficient volume of 70 per cent ethanol and extracted three times with ligroin (b.p. 30-60'C). The ethanolic layer of the above fractionation was diluted with water to contain 35 per cent of alcohol. After standing in the cold room (40C) overnight, three layers were formed. The lowest of these, the alcoholic layer, was removed and washed three times with ligroin. The washings were combined with the two upper layers, and backwashed with 35 per cent ethanol. A sample from the pooled alcoholic extracts was bioassayed and gave positive results for estrogenic activity. The 35 per cent alcoholic extract was evaporated to dryness and the residue partitioned with 29 transfers between 70 per cent ethanol and 50 per cent chloroform-50 per cent carbon tetrachloride. Five fractions were made by combining the contents of tubes 0-2, 3-9, 10-19, 20-25, and 26-29. Each fraction was taken to dryness in vacuo and the residue dissolved in 10 ml of methanol. A 1.0 ml portion of each fraction was used in the preparation of serial dilutions for bioassay by the Astwood4 method. In the final tests, the results for which are shown in Table 1, each animal received extract equivalent to 1.8 grams of fresh ovarian tissue in 0.1 ml of sesame oil. Estrogenic activity was found only in the fractions from tubes 3-9 and 10-19. Estimates based on the activity in tubes 10-19, later identified as estradiol-17,3, indicated that the whole extract (7.9 Kg) contained at least 948 ,.ug of estradiol or 120 ,ug per kilogram of fresh ovarian tissue. The material from tubes 10-19 was evaporated to dryness and the gummy residue dissolved in 30 ml of benzene, This solution was applied to an alumina 580 Downloaded by guest on October 6, 2021 VOL. 46, 1960 ZOOLOGY: WOTIZ ET AL. 581

TABLE 1 BIO-ASSAY OF FRACTIONS OBTAINED BY PARTITIONING 35 PER CENT ALCOHOLIC EXTRACT BETWEEN 70 PER CENT ETHANOL AND A 50-50 PER CENT MIXTURE OF CHLOROFORM AND CARBON TETRACHLORIDE Average Uterine Wet Per Cent Increase Fraction Weight over Control Control 21.8 (25)* 0-2 21.2 (4) 0 3-9 28.9 (4) 32.6 10-19 40.8 (4) 87.1 20-25 20.9 (4) 0 26-29 21.9 (4) 0 * Number of 22-day old rats, weighing 55-60 grams, used in each assay group. column (Woelm alumina activity grade I). The column was eluted with ligroin (200 ml), benzene (100 ml), ethyl acetate (250 ml) and chloroform (200 ml). Each fraction was bioassayed and estrogenic activity was found only in the chloroform eluate. After evaporation of the solvent approximately 2 mg of a yellow, oily substance remained. The oily residue from the chloroform eluate was again dissolved in a minimal quantity of benzene and applied to another alumina column. This column was z WAVELENGTH IN MICRONS

a- s0- Is- 4 40

oJ 3700 3000 2000 So71600 0001200 KM am 66 WAVE NUMBERS IN CM FIG. 1.-Infra-red spectrograph of authentic extradiol-17jB dinaphthoate and experimental estradiol-17,3 dinaphthoate (------). eluted with benzene containing increasing amounts of methanol. The sixth 100 ml fraction (5 per cent methanol) on evaporation yielded a small residue of white crystals in oil. The semicrystalline material was re-precipitated from aqueous methanol yielding 0.88 mg of an amorphous substance melting from 148-1550C. This was reacted with 1-naphthoyl-chloride as described by MacCorquodale, Thayer, and Doisy.5 The derivative was recrystallized from ethanol, yielding 1.03 mg of white crystals melting from 192-1970C. A mixed melting point with authentic estradiol dinaphthoate (mp 1970C) showed no depression. The re- mainder of the material was dissolved in carbon disulfide and an infrared spectrum was recorded on a Perkin-Elmer model 21 double beam spectrophotometer. This was compared with a spectrum of approximately 1 mg of authentic estradiol dinaphthoate and the two spectra were found to be identical (Fig. 1). The dark brown residue obtained on combining the contents of tubes 3-9 from the first countercurrent distribution was dissolved in 50 ml of 1 N sodium hydroxide and washed three times with 25 ml portions of ether. The alkaline residue was partially neutralized to pH 7.5 and again extracted with three 50 ml portions of ether. This last extract, now straw-yellow in color, was taken to dryness in vacuo. Downloaded by guest on October 6, 2021 582 ZOOLOGY: WOTIZ ET AL. PROC. N. A. S.

Since it was uncertain whether the estrogenic activity remaining in this portion of the extract was due to tailing of the estradiol peak in the countercurrent partition or to the presence of estrone, this residue was chromatographed on paper in a toluene-propylene glycol system.6 After development of the chromatogram for 16 hours the areas corresponding to estradiol and estrone, as determined by com- parison with a simultaneously developed control strip, were eluted and a portion thereof was bioassayed. The substance eluted near the starting line and corresponding to estradiol pro- duced 56 per cent average increase in uterine wet weight when a 1/50 aliquot was divided equally and given to two rats. This portion representing estradiol tailing in the first partition was not further investigated. The material 36-39 cm from the origin and corresponding in travel rate to authentic estrone was eluted from the strip and a l/50 portion caused a 66 per cent average increase in uterine wet weight when tested on two rats. This material was further purified by formation of the Girard T complex, washing the water soluble phase with ether followed by acid hydrolysis and reextraction of the phenolic material

WAVELENGTH IN MICRONS 15 3 4 56 7 a 9 10 II 12 3 14 0 (.n X '~~~~~a.= b~~e-9-.A X ;. (

60 6 4 I I 40 3t00-.o~~ ~200 ~ ~ lo I 60 10 20 lo 0 60o40 WAVE~~~~NUMBERINCM- 20. 20- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~20 0. 0 3700 300o00w0 W60 1400 1200 1000 600 650 WAVE NUMBERS IN CM.-' FIG. 2.-Infra-red spectrograph of authentic estrone benzoate ( ) and experimental estrone benzoate (------). with ether. The amorphous white residue was then reacted with benzoyl chloride in pyridine at 0°C. After leaving the mixture overnight it was poured over cracked ice and extracted with ether. The ether layer was then washed successively with dilute hydrochloric acid, 10 per cent sodium bicarbonate solution and water. Following these washings the solution was taken to dryness in vacuo. The dried residue was applied in 2 ml of benzene to a 10 X 50 mm alumina column (Woelm, neutral, alumina activity grade II). The column was eluted with 75 ml of ligroin followed by 200 ml of benzene. The eluates were collected in 25 ml portions and the ninth eluate after evaporation left a white semicrystalline residue. This material was dissolved in a few drops of carbon disulfide and an infrared absorption spectrum was recorded. This spectrum was identical with one obtained from authentic estrone benzoate (Fig. 2). The ligroin fractions were combined and subjected to procedures for the isola- tion of progestational substances. They were evaporated to an oily black residue and then applied to an alumina column (15 X 250 mm) in benzene. The column was eluted with petroleum ether, benzene, chloroform, and alcohol. A total of eleven 100 ml fractions were collected and individually taken to dryness in vacuo and an Downloaded by guest on October 6, 2021 VOL. 46, 1960 ZOOLOGY: WOTIZ ET AL. 583

aliquot (1/20) of each was tested for progestational activity by the method of Hooker and Forbes.7 A positive reaction was given by fractions five, seven, eight, nine, and eleven. These fractions of the crude material were combined in two lots, four through seven in one (A) and eight through eleven in the other (B) and each applied to an alumina column (100 g) in benzene. A control column of 100 g of alumina was developed in like fashion after adsorption of 1.0 mg progesterone and 400 Ag each of 20a and 20,3-hydroxy-pregn4-en-3-one. Identification of the control substances after chromatography was carried out by infrared spectroscopy. The portions of both experimental chromatograms that showed progestational activity and correspond to authentic progesterone were combined and dissolved in 0.5 ml of benzene. This solution was applied to a 2 cm paper strip and the chromatogram was developed in a ligroin, propylene glycol system for 33/4 hours. A control strip containing progesterone was run simultaneously. On staining the dried control strip with Zimmermann's reagent the color appeared 13-16 cm from the origin. The chromatogram containing the extract was cut and the area from 13-16 cm was eluted with several portions of methanol. The material obtained from the eluate, when bioassayed by the Hooker-Forbes method, gave positive reactions thus further indicating the presence of progesterone. Conclusions.-The results obtained in this study show conclusively that estradiol-17,3 and estrone are present in the ovaries of the dogfish, Squalus suckleyi. They also support previous evidence' for the presence of estradiol-17j# in the ova of this species. Estriol was not identified and it seems that in this respect the dogfish may differ from the lungfish, Protopterus annectens.3 That progesterone was also isolated is strongly indicated but the evidence is not as conclusive as that for the estrogens. Although the chromatographic data and bioassays were in full agreement the amount of material was not sufficient for a more thorough identification. There also were indications of other progestational substances, particularly 20a-hydroxy-pregn4-en-3-one. The presence of the same steroid hormones in the ovaries of an elasmobranch fish and mammals suggests that they may be found in all vertebrates and at the same time raises questions regarding function and possible significance in the evolu- tionary development of the ovary. * Aided by grants from the National Science Foundation and the United States Public Health Service. t Senior Research Fellow, United States Public Health Service. 1 Present address: Department of Zoology, Oregon State College, Corvallis, Oregon. § Present address: Department of Zoology, Brandeis University, Waltham, Massachusetts. ' Wotiz, H. H., C. Botticelli, F. L. Hisaw, Jr., and I. Ringler, J. Biol. Chem., 231, 589-592 (1958). 2 Layne, D. S., R. H. Common, W. A. Maw, and R. M. Fraps, Nature, 181,351-352 (1958). 3Dean, F. D., and I. C. Jones, J. Endocrin., 18, 366-371 (1959). 4Astwood, E. B., Endocrinology, 23, 25 (1938). 5 MacCorquodale, D. W., S. A. Thayer, and E. A. Doisy, J. Biol. Chem., 115, 435 (1936). 6 Jellinck, P. H., Nature, 171, 750 (1953). 7 Hooker, C. W., and T. R. Forbes, Endocrinology, 41, 158 (1947). Downloaded by guest on October 6, 2021