The CHEMICAL NATURE of the OVARIAN and the GONADO-
TROPIC HORMONES H. L. FEVOLD University of Wisconsin, Madison, Wisconsin
In the last decade the sex hormones have commanded much attention both with regard to their physiological action and to their chemical nature. The importance of these substances i n clinical use for corrective treatmenl and i n the diagnosis of pregnancy and pathological cases, makes the chemical extraction and puri$cation of these active principles of the highest importance, to say nothing of the purely scienti$c interest which i s attached to such work. Consequently many interesting and valuable data have accumulated from variow sources and have greatly increased our knowledge of the general chemical behazior of these hormones. Further detailed studies of the chemical nature of these substances are being carried on at the present time, especially a two or three of the hormones which have now been isolated i n crystalline form.
blood and urine of pregnancy, the urine of castrate women, and in the urine of certain pathological cases such as hydatidiform mole, genital carcinoma, and chorio-epithelioma. Whether the gonadotropic substances obtained from these various sources are the same is doubtful, as will be brought out later. THJ3 CESTROGENIC HORMONES
The follicular hormone has been extracted from a number of sources and has a wide distribution. It is present, as stated above, in greatest amounts in the ovaries, in placentae, in the placental fluids, and in blood and urine of pregnancy. Small amounts are found in normal female and male urine and substances of the same nature are also found in the plant world [(I) to (lo)]. Various methods of extraction have been + + + + + + used but in general the principle is the same, namely, an extraction with some fat solvent such as ethyl ether, HE sex hormones which will be discussed in this chloroform, benzene, butyl, and in some cases ethyl paper are those which are generally known and are alcohol. The product has then been purified by various secreted by the ovarv, the anterior lobe of the methods. In 1929 Doisy and Butenandt, working indehypophysis and the placenta. They are present in the pendently, isolated the hormone in crystalline form from blood particularly during pregnancy and at this time urine of pregnancy (11), (12). This crystalline material some of them are excreted in large amounts in the urine. Doisy called "theelin." Subsequently Laqueur (13) Pregnancy urine has been an excellent source of a num- and co-workers accomplished the same crystallization. ber of these substances. Since that time, due to their work, our knowledge of Of the estrogenic substances, we have the follicu- the chemical nature of this hormone has become much lar hormone as found principally in the follicles of more detailed and exact. The crystalline hormone, theelin, is readily soluble the ovary, the placenta, its associated fluids, and blood and urine of pregnancy. Two modifications of this in ethyl alcohol, chloroform, and benzene, less soluble hormone have been isolated from pregnancy urine by in ethyl ether and ethyl acetate and very difficultly several investigators, namely, a ketonic form and a soluble in petroleum ether, so much so that it may be triatomic alcoholic form. In our discussion we shall precipitated in crystalline form from various solvents use the nomenclature adopted by Doisy, namely by the addition of petroleum ether. In water, theelin "theelin" for the ketonic form and "theelol" for the is slightly soluble, 100 cc. of saturated aqueous solualcoholic form. From the placenta and urine of preg- tion containing 1.5 mg. of the substance. It is acidic nancy Collip has isolated another estrogenic substance in nature due either to a phenolic OH or to the enolizawhich he has called "emminin" and which is apparently tion of the ketonic group in the molecule. It is stable very similar to theelol. There are two corpus luteum to acids and alkalies and to beat. I t is sensitive to hormones known, namely, "relaxin" which is obtained atmospheric oxygen, especially in ethyl alcohol solufor chemical work chiefly from the corpora lutea of tion. Theelin has been found to have the empirical forthe sow but is also present in the blood of various animals during pregnancy. The second corpus luteum mula C,sH2d02. The melting point is given as 250°C. hormone, "corporin" or "progestin," is also extracted It is optically active and is dextro rotatory ([a]?f156 chiefly from sow's corpora lutea. The anterior lobe in chloroform). The crystalline material gives a mono or anterior lobe-like gonadotropic substances are found acetate and benzoate, an oxime and a mono methyl in the anterior lobe itself, in human placenta, in the ether. I t is concluded that the substance contains
T
three double bonds which are saturated by catalytic hydrogenation, only one of which takes up iodine, however. The substance contains one hydroxyl group and one keto group which shows tautomerism to an en01 form (Id), (15). Marrian isolated a second estrogenic substance from pregnancy urine and found its formula to be CmHzaOa (16). This substance contained three hydroxyl groups. One of these hydroxyls was found to be acidic, due presumably to a phenolic grouping since the phenol reactions were positive. It was different physiologically from theelin in that its biological activity was much less. Later Doisy also isolated this second substance and called i t "theelol" (17), (18). He separated i t from theelin due to differential solubility in alkali and ether, theelol being more soluble in the former. Butenandt also obtained this product and found that the activity of theelol, when brought to constant melting point (276'C.) by repeated aystallizations was only 20,000 rat units per gram while that of theelin was 8,000,000. The formulas of these two differ by one molecule of water and Butenandt was able, by heating with KHSO, in a vacuum, to change theelol (C18H1403)to theelin (Cl8HBO1) with a corresponding increase in biological activity, namely from 20,000 rat units per gram to 8,000,000 units per gram (19). This is the best proof a t the present time that the crystalline theelin is actually the pure follicular hormone. It also demonstrates that theelin and theelol are two closely related substances existing in different forms. Butenandt suggests that theelol may be a precursory stage of theelin or possibly a modification to a more convenient form for excretion (19). The yield of theelol from urine is a t least five times greater than theelin, but Doisy considers that this may be an artifact brought about during purification (18). Butenandt, on the other hand, believes that the two exist in that proportion which is not changed by the process of purification. At the present time neither theelin nor theelol has been obtained from any other source than pregnancy urine, although Collip has isolated from placenta a crystalline substance, "emminin," which is somewhat similar to theelol. He also obtained the same substance from the urine of pregnancy. It is nearly identical with theelol as far as melting points and carbon and hydrogen content are concerned. However, the physiological activity does not seem to agree entirely with that of theelol. Collip gives the activity of emminin as 60,000 rat units per gram. Whether emminin is the same substance as theelol or is still another modification of theelin remains for further work to establish (20). HORMONES OF THE CORPORA LUTEA
Two corpus luteurn hormones have been studied and described; they are relaxiu (21) and corporin (22) or progestin (23), (24). Relaxin, which produces relaxation of the pelvic ligaments of certain animals does not seem to be present in all mammals. Thus
it occurs in the blood and placenta of pregnant rabbits, sows, dogs, cats, guinea pigs, and mares, but it hasnot been found in the blood of cows and women. The corpora lutea of sows have proved to be the best source of this substance. Corporin has also been studied chiefly as obtained from the corpora lutea of the sow. Its distribution in blood, urine, placenta, and other tissues has not been studied chiefly because of the presence of large amounts of follicular hormone which interferes with the biological assay of this substance. Because of the chemical similarity of the follicular hormone, and carporin, no satisfactory method of separating these two substances quantitatively has been developed. Relaxin, as is true with most of the sex hormones, has been studied chiefly in extract form and the purest preparations are still undoubtedly not pure. Consequently very little is known as to its exact chemical nature. It has, however, been purified to the point where the nitrogen content of the product is constant and the biological activity of the product also seems to be the same in different preparations. Relaxin is extracted from corpora lutea of the sow or from other tissues by means of a d d alcohol. The extract is subsequently purified by the elimination of proteins, phospholipius, and other fatty materials by various methods, with the result that a water-soluble extract containing the active material in concentrated form is obtained. The hormone may then be precipitated from solution with picric acid and subsequently puriiied with the result that a product is obtained which contains from 25 to 30 guinea-pig units per milligram. It apparently has a peptide-like structure. The purified product contains 11.5% nitrogen, has both basic and acidic properties and has a definite iswlectric point of 5.4-5.5. Proteolytic enzymes break down the material to a point where i t is no longer active. It is unstable to heat, to oxidation, to alkalies, and to formaldehyde. It is soluble in glacial acetic acid, only slightly soluble in distilled water, but soluble in acidified or alkaline aqueous solution. It is insoluble in organic solvents such as ether, acetone, alcohol, and ethyl acetate (25). Corporin is quite different in its general chemical characteristics from relaxin. It is quite similar to theelin in its solubilities and general behavior with certain important exceptions. It is soluble in alcohol, ethyl ether, acetone, and similar solvents. It is also quite readily soluble in aqueous ethyl alcohol above 33% alcohol and in petroleum ether. Butenandt states that theelin is very di&ultly soluble in this medium. Corporiu is more soluble in petroleum ether than in 50% aqueous methyl alcohol but more soluble in 90% aqueous methyl alcohol than in petroleum ether. Another definite difference in the chemical behavior of theelin and corporin is found in their respective reactions to alkali, theelin being quite stable while corporin is very labile. Both are destroyed by atmospheric oxygen but both are stable a t fairly high temperatures (24), (26).
cipitation with alcohol or acetone, thereby concentrating it greatly, still further purification is complicated by the lability of the active principle to acids and alkali and to relatively low temperature (60-80°C.). It is readily soluble in water and purifications have been effected by precipitation by saturation of the aqueous solution with ammonium sulfate (35), by absorption on charcoal or diatomaceous earth [(34), p. 1461, by precipitation with uranyl acetate (36). or by precipitation with tannic acid (35). Fischer removed much inactive material from the aqueous solution by precipitation with barium acetate and barium hydroxide and obtained a preparation which contained 130-140 mouse units per milligram (37). Katzman and Doisy obtained a preparation which assayed 3800 mouse units per milligram. They absorbed the active material on finely divided benzoic acid and further purified the,product by fractional precipitation with acetone (38). Others have investigated the placenta as a source of gonad-stimulating substances. Zondek (39) did not consider the placenta a good source but Wiesner and Marshall (40) obtained sulfosalicylic acid extracts GONADOTROPIC HORMONES which were active while Collip, el al. (41), extracted There are several sources of anterior lobe or anterior placentae with acetone and obtained a final alcohollobe-like hormones whicb have a gonad-stimulating insoluble product which was very active and which effect. Substances of this type are present in the was similar physiologically to the substance obtained anterior lobe, the placenta, blood of pregnant ani- from pregnancy urine.. Collip's method did not yield mals, and human female urine. The active principles active extracts when applied to pituitary material. from these various sources have been studied by many In this laboratory the same method has been applied investigators who have purified the active substances to placenta as has been worked out for pituitary powder with good success. The biological results obto various degrees. Zondek and Aschheim were perhaps the first to make tained with the use of placental extracts are different use of pregnancy urine as a source of gonad-stimu- from those obtained with anterior lobe in that it is lating substance. They developed a method of cou- not possible to increase the size of the ovaries much centrating the active material by precipitation from over that of normal ovaries of mature animals by the the urine with alcohol. The precipitate was further use of the former, whereas enormous ovaries can be purified by removing toxic substances by washing with produced by pituitary preparations. In this respect ethyl ether. It was then taken up in water and cen- placental extracts are similar to prolan B. Gonad-stimulating material has also been extracted trifuged free of insoluble material and the resulting solution was used for injection. I t was found that from the anterior lobe itself by the use of various the substance from gravid urine brought the animal solvents. Thus Evans and Long (42) succeeded in to sexual maturity with growth of follicles in the ovary, producing changes in the reproductive tract by the together with development of corpora lutea. It was injection of alkaline extract of anterior lobe substance. also found that when the urines from women who This consisted of a powerful stimulus to lutein cell had been castrated for some time, and the urines from formation. Similar (alkaline) extracts have been used women with genital carcinoma or women past the by various investigators; Brouba and S i o n n e t (43) menopause, were used as a source, the physiological observed similar effects; Leonard found that alkaline response was different. In tbese cases the injected extracts produce ovnlation in rabbits (44) while Weisanimals came to sexual maturity but the ovaries con- ner and Marshall (40) state that the estrogenic propertained only follicular growth. They called the active ties can be enhanced by removing the proteins by prepreparation from gravid urine prolan B, while that cipitation with sulfosalicylic acid. Recently Bugwhich gave only follicular growth was called prolan A. bee, et al. (45), used alkaline solutions to extract the There were no chemical differences in the two prepa- gonad-stimulating substance from sheep pituitaries. rations as far as Zondek and Aschheim have reported, Fevold, et al. (46), obtained good results by the use of but the physiological action was quite different [(29)to aqueous pyridine as an extractive. This extract produced follicular growth together with luteinization. (34) 1. . .. Since their work several investigators have attempted Evans and Simpson (47) reported tbat acid extracts to purify the active principle from pregnancy urine. were effective in producing sexual maturity in young While it can easily be removed from the urine by pre- animals. Bellerby (48) described a method based
Corporin has recently been obtained in crystalline form but a detailed chemical study of the crystalline product has not yet been made due to the lack of sufficient material (27). Fevold, et al. (26), obtained a third fraction from corpus luteurn tissue of the sow which produced vaginal mucification in various animals. This preparation was ether-insoluble, alcohol- and water-soluble. The active substance was definitely destroyed by alkali. I t has later been shown that mncifxation can be produced by very small amounts of the follicular hormone theelin (28). Although the extract had been tested for estrogenic action such small amounts were present that it was only by testing a large amount of the preparation tbat it was shown to be present. The fact tbat the activity was destroyed by alkalies does not comespond to the commonly recognized stability of the follicnlar hormone to such reagents. However, it seems probable that the production of the mucification reaction was due to the small amonnts of follicular hormone in this fraction since the same reaction can be produced with the crystalline hormone, theelin.
on extraction with 0.1% acetic acid. Hewitt (49) used the same extractive with good results. WallenLawrence and Van Dyke (50) extract the pituitary glands by means of acetate b d e r a t pH of 4.5 with very good yield of active material. Hill and Parkes (51) induced ovulation in the hypophysectomized rabbit by the administration of acid extracts of the anterior lobe of the pituitary gland. It is thus apparent that the gonad-stimulating substances can be extracted from the anterior lobe with alkaline or acidified solutions with almost equal success. Any difference in the physiological response of these extracts has not been entirely clear, although Evans (47) states that acidified extractives are better for follicular stimulating substance while alkaline extracts are better for luteinizing hormone, which he believed a t this time to be the same as the growth hormone. The question has always come up as to whether the physiological changes which are produced in the ovary by pituitary extracts are due to one substance or two. Is the production of follicles in the ovary caused by the same principle which causes the luteinization of the follicles? Evans and Simpson (47) state that the stimulus for follicular growth may not he the same as that causing luteinization. Zondek (29), (30), (31), (32) offers indirect evidence for the presence of two substances by his preparations from urine, prolans A an& B, the former producing only follicular growth in the ovary while the latter also produces luteinization. Clans (52) reported the separation of the two substances from pituitary material. Fevold, et al. (46), reported the partial fractionation of their aqueous pyridine extract into a follicular stimulating fraction which in small doses produce mainly follicular growth and a fraction which was inactive on immature rats but which produced great luteinization when added to the follicular stimulating fraction. The follicular stimulating fraction has been further purified and i t has been possible by the use of this fraction to produce large ovaries in rats and rabbits which contain only follicular development while the unfractionated pyridine extract produces large luteinized ovaries. On the other hand Bellerby (48) does not believe i t is necessary to postulate two substances. WallenLawrence and Van Dyke (50) do not believe that the theory of two substances has s&icient support. They believe that a small dose causes follicular growth while larger doses cause luteinization and that there is wide variation in the response of various animals. Evans (53) in a recent paper does not find evidence for two substances. Evidence seems to be accumulating which tends to show that the gonad-stimulating substances from various sources may not be the same. The evidence consists of the fact that the physiological response to preparations from various sources is different. For instance, it is possible by the use of the active material from pituitary glands to grow enormous ovaries in immature rats. On the other hand, the amount of
growth which can be produced by placental extracts and by preparations from pregnancy urine seems to be limited. Thus Collip (54) was not able to produce ovaries in immature rats larger than normal adult ovaries by the use of placental extracts. Similarly Evans (53) found that the response of rats was proportional to dosage when pituitary material was injected while this was not the case when urinary preparations were used. In the latter case the weight of the ovaries approached a maximum of about 60 mg. and increased dosage did not increase the size of the ovaries beyond this limit. Hypophysectomized animals have been found to be very unreactive to urinary preparations but still reactive to preparations from the pituitary gland itself (51), (55), (56). WallenLawrence and Van Dyke (50) find that pregnancy urine preparations were as active in males as females, while this is not true of anterior lobe substance. These investigators also find a difference in heat stability of the two preparations, pituitary preparations being more stable than those from urine. Evans (53) has put forth the theory, supported by experimental data, that the material which is obtained from urine does not act on the ovary hut is an activator for the anterior lobe causing it to secrete the substance which affects the ovaries. This would explain the inactivity of urinary preparations on hypophysectomized animals. It seems probable that the active principle found in pregnancy urine is the same as that found in the placenta and is apparently secreted by placental tissue. This is supported by the fact that the physiological response is similar with preparations from either source. Also great amounts of the active substance are found in the urine of patients with pathological types of placental tissue such as hydatidiform mole and chorioepithelioma which would also lend weight to the placental origin of this material. The anterior lobe then secretes another substance or substances which are the true anterior lobe hormones. Prolan A would seem to he of pituitary origin since no other known source is present in the body a t the time of excretion. If this is true i t would seem that the anterior lobe must secrete a second luteinizing substance since anterior lobe preparations ordinarily produce both follicular and lutein development in the ovary. Friedman (57) has shown that ovulation can be induced in the rabbit by a single intravenous injection of urine of pregnant women. Bellerhy (58),Hill and Parkes (59), and Jares (60) obtained the same results by injections of anterior lobe extracts. This reaction has become the basis of a test for pregnancy which is in quite general clinical use. It is generally agreed that i t depends on the presence of the gonadstimulating substances which are present in the urine of the patients a t that period. In attempting to determine experimentally what hormone is responsible for the ovulation reaction Leonard (44) showed that it could he produced in rabbits by preparations from pregnancy urine, by prolan A, by anterior lobe extract, and by a growth hormone preparation from the an-
terior lobe which had no or very little gonad stimulating effect. As we have seen, these preparations give different physiological responses when tested by their effect on the ovaries of immature animals and probably the active principles do not come from the same source. However, they give the same result, qualitatively a t least, when tested by the ovulation reaction. The possibility, therefore, arises that ovulation may be produced by a separate hormone which is present in all these preparations. The identification of the substance which causes ovulation, either as a separate hormone or identical with some other known substance, still remains to be accomplished. Leonard's findings are suggestive, but more positive proof is needed. While these substances give different physiological reactions, no definite differences in chemical characteristics have been reported. The active material from any source is insoluble in organic solvents and is precipitated from aqueous solution by such solvents as alcohol or acetone. The active principles do not dialyze and are unstable to strong acids or alkalies. Fischer and Ertel (37) give the chemical reactions of their active preparations from urine as follows: positive biuret, M i o n , Pauly, and Molisch reactions, but the Adamkiewicz-Hopkins reaction is negative. The active substance is precipitated by saturated ammonium sulfate, uranyl acetate, and by phosphotungstic, phosphomolybdic and tannic acid, especially in acidified solution. Picric acid, ammonium molybdate, copper acetate, and silver nitrate cause no precipitation. Their preparation shows strong reducing properties and they conclude that a carbohydrate is present which is probably a hexose combined with a protein component. Reiss, et al. (61) state that the substances are-destroyed by trypsin but not by polypeptidases which would indicate a substance of high molecular weight. LITERATURE CITED
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(9)
(50) .WALLRN-LAUXENCE, Z., AND VAN DYKE, H. B., "The gonad-sbmulatmg substances of the anterior lobe of the pituitary body and of pregnancy urine," J. Pharrn. &Ex$. Ther., 53, 93 (1931). (51) HILL, M., AN? PARKES,A. S., "Induction of ovulation in the hypophysedomlzed r a b b ~ tby administration of anterior lobe extracts," J. Physiol., 71, 36 (1931). (52) CLAWS,P. E., "Separation of anterior lobe substances and study of their individual effects," Phys. Zool., 4,36 (1931). M. E.. "Rela(53) EVANS.H. M., MEYER,K., ANDSIMPSON. tion of prolan t o the anterior hypophyseal hormones." Am. J. Physiol.. 100,141 (1932);, (54) COLLIP. J. B.. Placental hormones." Proc. Calif. Aced. Mcd., 1,38 (1930). (55) REICHERT,F. L., PENCHBRZ,R. I.. SIMPSON.M. E., MEYER, K., AND EVANS.H. M., "Relative ineffectiveness of proIan in hypophysectomized animals," Am. J. Physiol., 100, 157 (1932). (56) HI+,M., AND PARKES, A. S., "The action of the ovulation-produnng hormone on the hypophysectomized rabbit," J. Physiol., 71,36 (1931).
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~ ~ ~ ~ Y ~ i , of, ovulation $ in the C. W., "The relation of the anterior lobe t o (58) BELLERBY. o v u ~ a t i o n ,J. ~ physi,,l,, 67, xxii (1929), (59) HEL, M,, AND A, S,, ovulation induced in the hypophysectomized rabbit by anterior lobe extracts,H ;bid., 69, axiii (1930). (60) Jams, J., "Studies on ovulation induced by extracts of the hypophysis and by urine of pregnant women," Anat. Rec., 45, 264 (1930). , F., "Ueber (61) Rerss, M., S c a a a a ~ s nA.,ANDHAW~OWITZ, die InaMiv~enmgdes aus Schwangerenharu gewonnen Hypophysenvorderlappenhormons durch proteolytische Enzyme," En&krinologie, 8, 22 (1931).
