I. The Estrogens

FERTILITY AND STERILITY VOLUME

9

NOVEMBER-DECEMBER

1958

NUMBER

6

Pacemaker Action of Ovarian Hormones in Reproductive Processes I. The Estrogens

Joseph Thomas Velardo

Velardo et al. 48 reported that the metabolites of the ovarian sex steroid hormones interfere with mating, implantation, the course of pregnancy, and litter size in adult albino rats of known reproductive vigor. Recently, Hisaw/7 and Velardo and Sturgis49 • 51- 52 and their collaborators extended and confirmed the original findings of Szego and Roberts 89-4° that uterine growth induced by estradiol-17,8 can be suppressed by a number of glucocorticoids, mineralocorticoids, and structurally modified and halogenated adrenal corticosteroids. More recently, Hisaw, 21 Velardo42 and Sturgis 50 characterized the uterine growth-promoting action of estradiol-17,8 and its five metabolites: estrone, 16a-hydroxyestrone, 16,8-hydroxyestrone, estriol, and 16-epiestriol. These

PREVIOUSLY,

From the Depiutment of Anatomy, Yale University School of Medicine, New Haven, Conn. Presented at the Fourteenth Annual Meeting of the American Society for the Study of Sterility at Beverly Hills, Calif., April 18 to 20, 1958. These studies were carried out during the tenure of a Lederle Medical Faculty Award. Aided in part by a research grant from the United States Public Health Service (RG-4577C), National Institutes of Health. 479

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Fertility & Sterility

same investigators ascertained that 16-epiestriol shares in common with estriol the property of suppressing uterine growth induced by estradiol17fi.21· 42 · 50 These several, independent observations indicated that ( 1) the estrogens act in concert; ( 2) the observed metabolic alterations of the reproductive tract represent the physiologic expression of the sum total of all present biologic hormones and/ or their metabolites acting in concert on the receptor organs; and ( 3) the Ll1· 3 • 5-estratrienes (estradiol-17ft and its five metabolites) act directly on the uterus. Furthermore, it was reported that estriol and 16-epiestriol exert their suppressing effects independently of the ovaries, adrenal glands, and the pituitary gland. 42 Therefore, it appeared more than likely that a pacemaker action among the ovarian hormones might well regulate the physiologic status of the female reproductive tract. The present report is primarily concerned with an elucidation of the hormonal factors involved in uterine growth. In this aim, a thorough review of the quantitative relationships of ovarian sex steroid hormones was undertaken with a view toward piecing together the metabolic career and physiologic actions of the estrogens and progesterones. Furthermore, the thought that a pacemaker action might well occur among the ovarian sex steroids seemed worthy of exploration. Therefore, this paper is concerned with a progress report on the experiments performed and the results obtained therefrom in respect to the hormonal factors operative in the regulation of uterine growth under highly standardized and rigidly controlled experiments. EXPERIMENTAL PROCEDURES Adult albino rats, 100 days of age, purchased from the Charles River Breeding Laboratories, weighing approximately 200 Gm. were used in all experiments involving ovariectomized rats. In experiments utilizing ovariectomized and ovariectomized-adrenalectomized-hypophysectomized rats, it was found more suitable to use rats 105 days of age and weighing 235 Gm. These animals survived the triple surgery much better than the younger rats. All animals were started on experiment 7 days after ovariectomy. The hypophysectomies were performed 1 week prior to the ovariectomies, and the adrenalectomies were started from 3 to 5 minutes after the removal of the ovaries. The ovariectomized rats were housed six to a 9X9X18" wire-mesh cage, and fed the standard Purina "Lab" chow and tap water ad libitum. The adrenalectomized-ovariectomized rats were similarly maintained, except

Vol. 9, No. 6, 1958

481

PACEMAKER ACTION OF OVARIAN HORMONES: I

for the substitution of 1 per cent NaCl drinking solution in place of the tap water. The ovariectomized-adrenalectomized-hypophysectomized rats received the Purina "Lab" chow, were maintained in similar cages, and received two drinking solutions: 1 per cent NaCl and 1 per cent glucose . The hormones were dissolved in sesame oil; each dosage of hormone was contained in either 0.1 or 0.2 cc. of vehicle, and injected subcutaneously daily for three days. When two or more hormones were injected, they were administered at separate sites. Seventy-two hours after the initial injection or injections, necropsies were performed, and the uteri were removed and weighed to the nearest 0.2 mg. on a Roller-Smith torsion balance. Parallel quantitative data were obtained in each experiment on wet and dry weights of the uteru.s, percentage water, total nitrogen, and solids within the uterus. Although only uterine weights are used for the purpose of this paper, their presentation is in essential agreement with the biochemical ESTRADIOL -17 p data. The curves depicted for dry uterine weights follow those of the total nitrogen data quite precisely. RESULTS Potentiality of the Six Naturally Occurring Estrogens for Promotion of Uterine Growth

The research of Marrian and coworkers,28-32 Watson,54- 55 Bauld,3-4 and Brown11-15 are responsible for the advancement and clarification of our knowledge of the relationships that exist among the estrogens. Figure 1 represents a summary of Marrian's research which has led us to a more comprehensive view of the biochemical career of estradiol-17,8 and the A1 • 3· 5 -estratrienes. This exhibit indicates that estrone may undergo hydroxylation at the sixteenth position (a and ,8), and that the two hydroxyestrones may then be reduced at C-17 to form estriol and 16-epiestriol respectively (Fig. 1). It

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Fig. 2. Curves depicting the uterine growth-promoting effects of different dosages of estradiol-17(3 in ovariectomized albino rats. Rats 100 days of age and weighing approximately 200 Gm. were ovariectomized. Beginning 7 days later the dosage of estradiol-17(3 indicated was given once daily for 3 days and the animals killed 72 hours after the first injection. Each point on these and subsequent curves represents the arithmetical average of uterine wet and dry weight for at least 7 to 21 animals. Fig. 3. Uterine growth-promoting effects of different dosages of estrone. These data when juxtaposed with those of estradiol-17(3 indicate that estradiol-17(3 is very much more potent than estrone in the promotion of uterine growth in the ovariectomized rat under rigidly controlled and highly standardized experimental procedures.

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Fig. 4. Action of 16a-hydroxyestrone on uterine growth. It can readily be noted that the hydroxylated form of estrone is much less active than estrone in promoting uterine growth. Fig. 5. Uterine growth-promoting effects of 16/3hydroxyestrone. Comparatively these data reveal that this estrogen is slightly less active than 16a-hydroxyestrone. This is especially obvious when one compares the dry uterine weights. (See Fig. 4.)

5

484

VELARDO

Fertility & Sterility

is of interest to point out that our knowledge of the naturally occurring estrogens up to 195421 was indeed fragmentary, and the picture today is yet incomplete, but three new naturally occurring estrogens have been isolated and characterized biologically since 1954. The recent isolation, identification, and availability of the 16-hydroxyestrones and 16-epiestriol have aided considerably in establishing a more realistic point of view of the estrogens and their regulation of uterine growth. Previously it was reported that estradiol-17,8 > estrone > estriol in the promotion of uterine growth. 21 Before dissecting the modus operandi of the role of the estrogens in uterine growth, it may prove helpful to examine the uterine growth-promoting action of the six naturally occurring estrogens under highly standardized and rigidly controlled conditions. Such information may well serve as a base line for any and all comparisons of the combined actions of the estrogens, a phase of this research which will be discussed subsequently. Therefore, the potentiality of each estrogen for the promotion of uterine growth was determined by establishing a dose-response curve (Figs. 2-7). These data reveal that maximal uterine growth for the 72-hour period was obtained with a daily subcutaneous injection of 1.0 J.Lg. estradiol-17,8 (Fig. 2),* 5.0 J.Lg. estrone (Fig. 3), 50.0 J.Lg. 16a-hydroxyestrone (Fig. 4), 50.0 J.Lg. 16,8-hydroxyestrone (Fig. 5), t 20.0 J.Lg. estriol (Fig. 6), and 20 J.Lg. 16-epiestriol (Fig. 7).f Additional studies on the biochemistry of the uteri from these experiments indicate that the increase in uterine luminal fluid, nitrogen, and solid content of the uteri parallels quite closely the curves depicted for estradiol-17,8 and its five A1 • 3 • 5-estratriene metabolites. Thus, in summary, these data demonstrate that estradiol-17,8 >estrone > 16a-hydroxyestrone > 16,8-hydroxyestrone > estriol > 16-epiestriol in the promotion of uterine growth. Interactions Among the Estrogens in the Promotion of Uterine Growth

Hisaw, Velardo, and Goolsby, 21 and Velardo and Sturgis50 established the fact that both estriol and 16-epiestriol share in common the property of suppressing the uterine growth-promoting effects of estradiol-17,8 and *The estradiol-17,8, estrone, and estriol used in this study were kindly furnished by Dr. Preston L. Perlman, Department of Biochemistry, The Schering Corporation, Bloomfield, N. J. f 16a- and 16,8-hydroxyestrones were generously furnished by Dr. Thomas F. Gallagher. t 16-epiestriol was made available through the cooperation of Dr. Max N. Huffman.

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Fig. 6. These curves depict the slight uterine growth-promoting action of estriol. Fig. 7. These curves indicate that 16-epiestriol is the weakest of the six naturally occurring estrogens in the uterine growth-promoting reaction.

486

VELARDO

m

Fertility & Sterility

RESTRICTIVE ACTION OF ESTRIOL ON ESTRADIOL-17B·AND ESTRONE IN UTERINE GROWTH

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Fig. 8. Restrictive action of estriol on estradiol-17B and estrone in uterine growth. Note that the restrictive action of estriol is quite marked on the wet weights as well as on the dry weights of the uterine tissue. (Confirmation of Hisaw, Velardo, and Goolsby. 21 )

estrone. The present experiments were undertaken in an effort to ascertain ( 1) if the same obtains in another strain of rat, and ( 2) to extend these experiments to include some biochemical determinations on the uteri. Results from these experiments indicate that estriol is quite active in suppressing the uterine growth-promoting action of estrone, and estradiol17{3, and combinations of estrone and estradiol-17{3 (Fig. 8). Biochemical data likewise reveal that the nitrogen, solid, and water content parallels the dry weight data in a most precise manner. Interactions of Estrogens in Ovariectomized-AdrenalectomizedHypophysectomized Rats It may prove helpful to review a recently completed group of experiments which have been designed to shed some light on the modus operandi of the suppressive effects of the estriols. Current interest in this laboratory is being focused on the mechanism of

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Fig. 9. Exhibit showing that the action of 16-epiestriol on estradiol-1713 in uterine growth is not mediated by way of the adrenal glands nor the adenohypophysis. ( ~ struck through= ovariectomized; ADX struck through= adrenalectomized; AP struck through = hypophysectomized.) Fig. 10. Dry weight data of corresponding uteri represented in Fig. 9.

488

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Ferti I ity & Sterility

action of the suppressive effects of the estriols on the more active estrogens. Numerous approaches to this problem have been initiated by several investigators. These include studies on ( 1 ) the structural specificity of steroids in the growth and inhibition of the uterus, 22-25 · 34· 39 -40 · 45 ( 2) the hormone-enzyme relationships,6-9· 35-36· 41 · 46 · 53 and ( 3) the endocrine organs involved in the growth and suppression of the uterus. 42 The following is a progress report of the current research in this laboratory on the mechanism or mechanisms involved in the restriction of uterine growth. It should be stated at the outset that it was previously postulated that estriol might in some way stimulate the release of adrenocorticotropic hormone ( ACTH) and consequently adrenocortical steroids. 21 This line of reasoning seemed consistent with the fact that the antiuterotropic activity might well be due to the inhibitory effects of the adrenal corticosteroids.34· 39· 40· 42-45 · 47 • 48 • 51 · 52 Therefore, it seemed that some critical information could be obtained if this assumption were tested. These experiments reveal that: 1. Estradiol-17,8 is an activ~ uterine-growth promoting agent in: (a) Ovariectomized rats. (b) Ovariectomized-adrenalectomized rats. (c) Ovariectomized-adrenalectomized-hypophysectomized rats. 2. Estriol and 16-epiestriol both inhibit uterine growth induced by estradiol-17,8 in: (a) Ovariectomized rats-from the 0.1 /Lg. estradiol-17,B controls of 243 mg. down to 156 mg. (dry uterine weights 40 mg. down to 27 mg.). (b) Ovariectomized and adrenalectomized-from 237 mg. down to 142 mg. (dry uterine weights 38 mg. down to 26 mg.). (c) Ovariectomized-adrenalectomized-hypophysectomized- 228 mg. down to 135 mg. (dry uterine weights 36 mg. down to 25 mg.). These data are summarized in Figs. 9 and 10. Since the results obtained with estriol parallel those of 16-epiestriol quite precisely, for conciseness the graphs pertaining to the results obtained with estriol are not presented. Relationship of the Estrogens During the Menstrual Cycle The central theme of this paper is directly concerned with an elucidation of the available knowledge which points to the fact that a pacemaker action among the ovarian sex steroid hormones regulates uterine growth. The

-

Vol. 9, No. 6, 1958

PACEMAKER ACTION OF OVARIAN HORMONES: I

489

latter experiments may be interpreted as the necessary base-line controls for the objective background of this study. If the premise is true that the estrogens act in concert and that a pacemaker action exists among the estrogens, then it appears that such evidence must be borne out in ( 1) the

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-DAY OF MENSTRUAL CYCLE Fig. 11. Urinary values of estradiol-17B, estrone, and estriol during the menstrual cycle. Note that all values are given in micrograms (!Lg.) per 24-hour urine. (After J. B. Brown, Con£. Soc. Endocrinology and Royal Society of Medicine, London, 1954.) Fig. 12. Urinary values of estradiol-17B estrone, and estriol during the last four weeks of pregnancy in the human female. Note that all values are given in milligrams (mg.) per 24-hour urine. (After J. B. Brown. 14 )

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VELARDO

Fertility & Sterility

metabolic career of the estrogens and ( 2) the circulating ratios of the estrogens and/ or their metabolites at a specific time in a physiologic system. Therefore, it appears necessary to investigate and examine the best quantitative data available on the circulating estrogens. Most steroid biochemists are in essential agreement that the methods for the determination and quantitation of the estrogens announced by Brown, Bauld, and Marrian at the British Empire Cancer Campaign (held in London, June, 1956) are the best available today. Consequently, particular emphasis will be placed upon the quantitative data on the urinary estrogens as determined by the Edinburgh school. Brown's study on the urinary estrogens during the normal menstrual cycle reveals that approximately 10 per cent of excreted urinary estrogen is estradiol-17fi, 30 per cent estrone, and 60 per cent estriolP The daily averages of these three estrogens are presented in Fig. 11. It should be noted that these data are given in terms of micrograms ( p.g. ) of estrogens per 24hour urine. Special emphasis should be given to the daily ratios of the estrogens, and not the over-all amounts of each of these estrogens. Of equal interest are the studies by Brown14 on the determination of the estrogens during pregnancy. These studies indicate that estriol is the predominant urinary estrogen, whereas estrone and estradiol-17fi are present in slightly more than trace amounts. That this is true is borne out by the graphic presentation of the urinary estrogens during the last four weeks before delivery (Fig. 12) . These data are given in milligrams (mg. ) estrogens per 24-hour urine. It is unfortunate that comparable quantitative data for the circulating estrogen in blood are not yet available, but current research in the United Kingdom and the United States offers promise of the perfection of this much needed technic in the foreseeable future. Therefore, for the present we must use and interpret urinary measurements with caution. This is particularly true if one recalls the fact that we can only account for 16 per cent of exogenously administered estradiol-17fi or estrone, and only 57 per cent of injected estriol. Data on the Plasma Progesterones During the Menstrual Cycle

In an effort to have a more thorough understanding of the ovarian sex steroid hormones during the menstrual cycle and pregnancy, a consideration of progesterone appears entirely essential. Furthermore, it is well-known that there are numerous interactions between estrogen and progesteroneP The most reliable quantitative data on daily fluctuation of progestogens

Vol. 9, No. 6, 1958

TABLE 1.

PACEMAKER ACTION OF OVARIAN HORMONES: I

491

Systemic Plasma levels of Progesterones During the Human Menstrual Cycle 19 and Pregnancy 11

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are those obtained by Forbes 18 • 19 and Zander. 57 Since progesterone has not been detected in the urine, :and since it is quite misleading to rely upon the values of urinary C-21 related metabolites of progesterone (there are at least ten such urinary metabolites27 ), it is impossible to relate the urinary values of the catabolites of progesterone to the urinary values of the estrogens. Moreover, the metabolites of the estrogens are all biologically active as estrogenic substances, whereas those of progesterone are inactive progestationally. Therefore, it is entirely justifiable to rely upon the biological assay of the progestogens (progesterone; A\ 3-pregnen-20o:-ol; and A\ 3-pregnen-20,8-ol, all progestationally active in this bio-assay) for the purpose of this study. Forbes studied the systemic plasma progesterone levels at frequent intervals for approximately six weeks from each of four young women. Waking temperatures were recorded daily throughout the same periods. His results are summarized in Table 1. Effects of Reconstituted Ratios of Urinary Estrogens and Plasma Progesterones "Typical" of the Normal Menstrual Cycle on Uterine Growth in Rats

The response of the uterus of the ovariectomized rat to reconstituted ratios of the urinary estrogens during Days 1-13 of the "typical" 28-day menstrual cycle of the human indicates that the extent of uterine growth is dependent upon the changing ratios of these estrogens (Fig. 13). * During *The best available evidence indicates that 0.1 p.g. estradiol-17,8 is a physiologic level of estrogen for the duplication of the uterine-growth response in the ovariectomized rat. Therefore, all ratios were scaled to the dosages consistent with the uterine-growth response of the rat. For example, if the ratios of estradiol-17,8, estrone, and estriol were 1:1:37, then 0.1 p.g. estradiol 17,8, 0.1 p.g. estrone, and 3.7 p.g. estriol were administered. Each estrogen was contained in 0.1 cc. sesame oil, and administered at separate sites. The dosage of the progesterones was calculated in terms of progesterone per blood volume of the human female. Since 0.25 to 4.5 mg. progesterone daily for 3 days elicits physiologic progestational responses in the rat, the exact amount of plasma progesterones (in progesterone equivalents) was administered. Progesterone was likewise given in 0.1 cc. sesame oil vehicle, and was injected at a separate site, thus preventing unnecessary accumulation of hormone vehicle.

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14

Vol. 9, No. 6, 1958

PACEMAKER ACTION OF OVARIAN HORMONES: I

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Days 15-27, the ratios of the urinary estrogens alone and in combination with plasma progesterones were reconstituted and studied for their action on the uterus. Interestingly enough, there was no significant difference in the results produced between the two types of treatments, both producing much less growth than that induced by the urinary ratios of the proliferative phase. In summary, examination and reconstitution of urinary ratios of these estrogens and progesterones from young women during the ( 1 ) proliferative, (2) progravid, and (3) menstrual phases revealed that 1>2>3 in the promotion of uterine growth. Thus, it appears that the circulating estrogens set the pace for the growth of the uterus (Fig. 13). More specifically, these results suggest that estriol is quite suspect as the pacemaker of uterine growth in that it has the ability to modulate the action of the two more active estrogens, estradiol-17,8 and estrone. Witness the data on the restrictive action of the estriols (Fig. 8) and the quantitative data on the urinary estrogens per 24-hour urine (Fig. 11). Effects of Reconstituted Ratios of Urinary Estrogens "Typical" of Late Pregnancy on Uterine Growth in Rats

In an earlier study it was assumed that estriol modulated the action of the more active estrogens in the human female during pregnancy. 21 Therefore, it appeared of added interest to reconstitute the ratios of the estrogens during late pregnancy, and ascertain their effects in the ovariectomized rat. Results from these experiments add further credence to the thought that estriol restricts in a large measure the growth of the uterus (Fig. 14). During pregnancy, estriol is the predominant estrogen, whereas estrone and estradiol-17,8 are quite minimal. The uterine-growth response to the reconstituted estrogens "typicaf' of late pregnancy seems quite reminiscent of that usually produced with estriol. As a matter of fact, the uterine response to these ratios is the least obtained throughout all of these studies. Comparatively, these data when juxtaposed with those of the "typical" menstrual cycle reveal that the uterine growth induced by the reconstituted estrogens from the proliferative phase > progravid phase > menstrual phase > late pregnant state. Experiments now in progress are concerned with ascertaining the uterine growth-promoting effects of the reconstituted ratios of the urinary estrogens and plasma progesterones typical of the whole course of pregnancy in the human female.

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DISCUSSION It is evident from these experiments that the uterine-growth promoting action of the estrogens is a more complicated process than hitherto envisaged. Previously, it had been thought that an endocrine receptor organ was influenced by one or two hormones alone. From the present study it is quite apparent that the uterus is not governed by one or maybe two hormones, but more correctly it now appears that the several metabolic alterations of the reproductive tract are influenced by the sum total of all the biologic hormones and/ or their metabolites acting in concert. Giving added credence to this concept is the recently acquired evidence which showed that estradiol-17,8 and its five known metabolites not only possess different estrogenic activities but also markedly different physiologic properties as well. That this is true is strikingly demonstrated in· the actions of these estrogens on the lactic dehydrogenase-diphosphopyridine nucleotide oxidase ( LDHDPNH-oxidase) system of the uterus. Recently, Bever, Velardo, and Hisaw 6 showed that estradiol-17,8 ( 0.1 to 1.0 f-tg. daily for three days) and estrone ( 0.5 to 2.0 f-tg.) induced mild to moderate activation of the LDH component of this enzyme system of the uterus of the rat, whereas estriol ( 0.5 to 2.0 f-tg.) induced strong activation of LDH. Conversely, estradiol-17,8 ( 0.1 to 1.0 f-tg.) and estrone ( 0.5 to 2.0 f-tg.) effected strong activation of the DPNH-oxidase component, but estriol ( 0.5 to 2.0 f-tg. ) had only a mild to moderate action on the flavoprotein portion of this enzyme. The recent studies by Villee and Hagerman53 and Talalay and Williams-Ashman41 will undoubtedly sharpen our concept of these enzyme-6. 1 •3 •5-estratriene relationships. These insights into the biochemical pathway of estrogen-effects all the way down into the microsomal level of the cell are indeed quite revealing. There are a number of other facts which support the contention that each of the estrogens may well have a specific action or a greater action th~m its closely related active estratrienes: ( 1) estradiol-17,8 and estrone are very active uterine growth promoters; ( 2) estriol is the most active in the imbibition of uterine luminal fluid; ( 3) estriol and 16-epiestriol share in common the property of suppressing the effects of estradiol-17,8 and estrone in uterine growth; and ( 4) the combined actions of the estrogens are neither augmentative nor synergistic. It may prove of interest to pursue the action of each of these estrogens alone and together in ratios consistent with those found in the urine (and blood in the future) on adenohypophyseal secretions of

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the gonadotropins. Such a study may well prove helpful to lay bare certain of the gynecologic dysfunctions which now escape corrective therapy. The scope of the present report strongly suggests that ( 1 ) a pacemaker action exists among the estrogens, and ( 2) the estriols figure quite importantly in .the pacemaker action. It should be emphasized that the metabolic alterations of the reproductive tract are not exclusively governed by the estrogens. This study represents only the beginning of an attempt to dissect with clarity the factors responsible for uterine growth. Furthermore, it seems more than convincing that the endocrine receptor organs are influenced by the sum total of all the biological hormones and/or their metabolites acting in concert at a specific time and in a physiologic system free of pathologic variants. Moreover, midtwentieth century medicine appreciates the fact that the whole metabolic career of the gonadal hormones may be influenced by way of the hypothalamus-pituitary-thyroid-adrenal-gonad axis. 26 • 43 This is quite demonstrably brought out in the following endocrine complexities: 1. In both frank myxedema and hyperthyroidism there is consistently observed a marked dysfunction of the female reproductive system which is manifested by either amenorrhea or menorrhagia. 20 • 51 2. In the treatment of the Stein-Leventhal syndrome, it was found that ovarian wedge resection did not invariably reverse the course of the disease, and that in some cases adrenal hyperfunction may be the basic cause involved in the several clinical entities associated with the Stein-Leventhal syndrome.1. 16, 3s, 56 3. A relationship exists between estrogen and cortisone in inoperable mammary carcinoma. 37 4. In anovulatory cycles, hypothalamic amenorrhea, and uterine hypoplasia and hyperplasia, a whole array of endocrine factors have been held suspect. All of these conditions point to the fact that there are numerous interrelationships among the hormones of the thyroid, adrenals, and ovaries. The year 1956 saw three long awaited breakthroughs in our paucity of information. First, Baggett et aP have amassed a great deal of information which shows that testosterone is the precursor of estradiol-17,8. Secondly, Bradlow et al. 10 have performed a group of experiments which reveal that triiodothyronine, a thyroid hormone, influences the metabolism of testosterone. Thirdly, Sturgis' laboratory51 -52 showed that purified adrenocorticotropin

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and purified adrenal hormones suppressed the effects of estradiol-17,8 in a group of experiments in ovariectomized and adrenalectomized rats. Thus, in conclusion, these experiments point up the facts that ( 1) the several metabolic alterations of the female reproductive tracts are influenced by the combined actions of all the hormones and/ or their metabolites acting in concert, and ( 2) the methodical, clinical dissection of gynecologic disturbances perforce must take into consideration a careful analysis of the functional activity of the entire endocrine environment.

SUMMARY 1. A detailed evaluation of the naturally occurring estrogens (estradiol17,8 and its five .!11 •3 •5 -estratrienes) was undertaken in an effort to explore the possible relationships of the estrogens in the promotion of uterine growth. 2. The potentiality of each of the naturally occurring estrogens was determined by establishing a dose-response curve. These data reveal that estradiol-17,8 > estrone > 16a-hydroxyestrone > 16,8-hydroxyestrone > estriol > 16-epiestriol in the promotion of uterine growth. 3. These data clearly indicate that several interactions exist among the estrogens. Both estriol and 16-epiestriol suppress the uterine growth promoting actions of estradiol-17,8 and estrone. That the suppressive effects of the estriols are seemingly directly on estradiol-17,8 is borne out by the fact that the estriols suppress the more active estrogens in ovariectomized, adrenalectomized, and hypophysectomized rats. 4. When estradiol-17,8, estrone, and estriol are administered in different ratios, and in concert, these results show that the extent of uterine growth is definitely influenced by the ratios of these <11 •3 •5-estratrienes. Extensive examination and reconstitution of urinary ratios of these estrogens from young women during the (a) proliferative, (b) progravid, and (c) menstrual phases, and (d) in late pregnancy revealed that a > b > c > d in the promotion of uterine growth. The high titers of estriol and 16-epiestriol found in the urine of pregnant women may explain the suppressive effects on uterine growth normally induced by the more active estrogens. The addition of the ratios of plasma progesterones to the ratios of the estrogens did not appreciably modify the response of the uterus. 5. These data support the contention that a pacemaker action exists

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among the estrogens, and that the estriols are quite suspect as the key estrogens responsible for the pacemaker action. 6. Finally, it seems that the hormonal action observed is not the result of one hormone alone. Specifically, additional evidence from these experiments gives added credence to the thought that the metabolic alterations of the uterus are due to all of the hormones and certain of their metabolites acting in concert. ·,.-:.••'

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

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Hyperadrenocorticism and the Stein-Leventhal syndrome. ]. Clin. Endocrinol. & Metab. 14:766, 1954. BAGGETT, B., et al. The conversion of testosterone 3-C14 to C14 estradiol-17,8 by human ovarian tissue. ]. Biol. Chem. 221:931, 1956. BAULD, W. S. Method for determination of oestriol, oestrone and oestradiol-17,B in human urine by partition chromatography and colorimetric estimation. Biochem. ]. 68:488, 1956. BAULD, W. S. Some errors in colorimetric estimation of oestriol, oestrone and oestradiol by Kober reaction. Biochem. ]. 56:426, 1954. BENSON, R. C., KoLB, F. 0., and TRAUT, H. F. Hirsutism, defeminization, and virilization: The endocrine basis for treatment. Obst. & Gynec. 5:307, 1955. BEVER, A. T., VELARDO, J. T., and HisAw, F. L. Action of estrogens on lactic dehydrogenase-DPNH oxidase system of rat uterus. Endocrinol. 58:512, 1956. BEVER, A. T., VELARDO, J. T., and HisAw, F. L. The effect of desoxycorticosterone acetate, testosterone and cortisone acetate on the lactic dehydrogenase-DPNH oxidase system of ovariectomized rat uteri. Anat. Rec. 117:554, 1953. BEVER, A. T., VELARDO, J. T., and HisAw, F. L. Effect of estrogens on the lactic dehydrogenase-DPNH oxidase system of ovariectomized rats. Fed. Proc. 12:15, 1953. BEVER, A. T., VELARDO, J. T., and HISAW, F. L. The effect of progesterone on the lactic dehydrogenase-DPNH oxidase system of ovariectomized rat uteri. ]. Clin. Endocrinol. & Metab.18:835, 1953. BRADLOW, H. L., et al. Interaction of hormonal effects: Influence of triiodothyronine on androgen metabolism. Science 124:1206, 1956. BROWN, J. B. Some observations on Kober colour and fluorescence reactions of natural estrogens. ]. Endocrinol. 8:196, 1952. BROWN, J. B. "A New Method for the Determination of Oestrogens in Urine and Its Application to a Study of the Oestrogen Excretion in the Menstrual Cycle." In The Technique and Significance of Oestrogen Determination. Memoirs of the Society for Endocrinology, No. 3, pp. 1-10, 1955. BROWN, J. B. Chemical method for determination of oestriol, oestrone and oestradiol in human urine. Biochem. ]. 60:185, 1955. BROWN, J. B. Urinary excretion of oestrogens during pregnancy, lactation, and the re-establishment of menstruation. Lancet 1(XX):104, 1956. BROWN, J. B. The relationship between urinary oestrogens and oestrogens produced in the body. ]. Endocrinol. 16:202-212, 1957. BuxToN, C. L., and VAN DE WIELE, R. Wedge resection for polycystic ovaries. New England]. Med. 251:293, 1954.

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17. CouRRIER, R. "Interactions between Estrogens and Progesterone." In Vitamins and Hormones. New York, Acad. Press, 1951, Vol. 8, p. 179. 18. FoRBES, T. R. Septemic levels of plasma progesterone during pregnancy in women and monkeys. Endocrinology 49:218, 1951. 19. FoRBES, T. R. Septemic plasma progesterone levels during the human menstrual cycle. Am.]. Obst. & Gynec. 69:180, 1950. 20. GoLDSMITH, R. E., et al. The menstrual pattern in thyroid disease. /. Clin. Endocrinol. & Metab. 12:846, 1952. 21. HISAW, F. L., VELARDO, J. T., and GooLSBY, C. M. Interactions of estrogens on uterine growth. /. Clin. Endocrinol. & Metab. 14:1134, 1954. 22. HuGGINS, C., and JENSEN, E. V. Significance of the hydroxyl groups of steroids in promoting growth. /. Exper. Med. 100:241, 1954. 23. HuGGINS, C., and JENSEN, E. V. The depression of estrone-induced uterine growth by phenolic estrogens with oxygenated functions at position 6 or 16: The impeded estrogens. ]. Exper. Med. 102:335, 1955. 24. HuGGINS, C., and JENSEN, E. V. The depression of growth of the uterus, adrenals. and ovaries by fluorinated steroids in the pregnane series. /. Exper. Med. 102: 347, 1955. 25. HuGGINS, C., JENSEN, E. V., and CLEVELAND, ANNE S. Chemical structure of steroids in relation to promotion of growth of the vagina and uterus of the hypophysectomized rat. ]. Exper. Med. 100:255, 1954. 26. KuPPERMAN, H. S., et al. The diagnostic and therapeutic use of intravenous estrogen in anovulation. Fertil. and Steril. 9:26, 1958. 27. LmBERMAN, S., and TEICH, S. Recent trends in the biochemistry of the steroid hormones. Pharm. Rev. 5:285, 1953. 28. MARRIAN, G. F. Technique and Significance of Oestrogen Determinations. Memoirs of the Society for Endocrinology. Cambridge, England, Cambridge Univ. Press, 1955, No. 3, p. 49. 29. MARRIAN, G. F. The urinary estrogens and their quantitative determination. Cancer 10:704, 1957. 30. MARRIAN, G. F., and BAULD, W. S. Isolation of fourth Kober-chromagen from urine of pregnant women. Proc. Biochem. Soc. in Biochem. ]. 58:36, 1954. 31. MARRIAN, G. F., and BAULD, W. S. Isolation of 16 epi oestriol from urine of pregnant women. Biochem.]. 59:1.S6, 1955. 32. MARRIAN, G. F., et al. 16-Hydroxyoestrone in urine of pregnant women. Biochem./. 66:60, 1957. 33. MARRIAN, G. F., WATSON, E. J. D., and PANATTONI, M. Isolation of ketonic dihydroxy-Kober-chromagen from urine of pregnant women. Biochem. ]. 65: 12,. 1957. 34. RoBERTS, S., and SZEGO, C. M. Steroid interaction in the metabolism of the reproductive target organs. Physiol. Rev. 33:593, 1953. 35. RosA, C. G., and VELARDO, J. T. Localization of the lactic dehydrogenase-DPNH oxidase system in the uterus of the rat during the estrous cycle. Anat. Rec. 121: 358, 1954. 36. RosA, C. G., and VELARDO, J. T. The influence of estradiol-17B upon the lactic dehydrogenase-DPNH oxidase system (LDH) in the vagina of the castrated rat. Anat. Rec. 120:771, 1954. 37. SMITH, 0. W., and EMERSON, K., JR. Urinary estrogens and related compounds in postmenopausal women with mammary cancer: Effect of cortisone treatment. Proc. Soc. Exper. Biol. & Med. 85:264, 1954.

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38. STEIN, I. F., and CoHEN, M. R. Surgical treatment of bilateral polycystic ovaries. Am.]. Obst. & Gynec. 88:465, 1939. 39. SZEGO, C. M. Pituitary-adrenal cortical antagonism to estrogenic stimulation of the uterus of the ovariectomized rat: Observations on structural specificity of crystalline steroids. Endocrinology 50:429, 1952. 40. SZEGO, C. M., and RoBERTS, S. Steroid action and interaction in uterine metabolism. Rec. Progr. Hormones Res. 8:419, 1953. 41. TALALAY, P., and WILLIAMs-AsHMAN, H. G. Activation of hydrogen transfer between pyridine nucleotides by steroid hormones. Proc. Nat. Acad. Sc. 44:15, 1958. 42. VELARDO, J. T. Suppressive effects of estriol and 16 epi-estriol on estradiol-178 in ovariectomized, adrenalectomized and hypophysectomized rats. Anat. Rec. 180: 384,1958. 43. VELARDO, J. T. "The Anatomy and Endocrine Physiology of the Female Reproductive Tract." In The Endocrinology of Reproduction. New York, Oxford, 1958, Chap. VII, pp. 101-212. 44. VELARDO, J. T. Steroidal aspects of pregnane inhibition of progesterone in decidual development. Am.]. Physiol. 188:317, 1957. 45. VELARDO, J. T. Inhibition of estradiol-17,8 induced uterine growth by .6.1, 9a fluoro-17 hydrocorticosterone. Am.]. Physiol. 186:468, 1956. 46. VELARDO, J. T. Inhibitory action of progesterone and aminopterin on estradiolinduced alkaline phosphatase activity in the reproductive tract of the ovariectomized rat. Anat. Rec. 120:770, 1954. 47. VELARDO, J. T., HISAW, F. L., and BEVER, A. T. Inhibitory action of desoxycorticosterone acetate, cortisone acetate and testosterone on uterine growth induced by estradiol-17B. Endocrinology 59:165, 1956. 48. VELARDO, J. T., et al. Effect of various steroids on gestation and litter size in rats. Fertil. & Steril. 7:301, 1956. 49. VELARDO, J. T., and STURGIS, S. H. Modification of estradiol-17B induced uterine growth by metacortandralone and metacortandracin. Am. ]. Physiol. 188:259, 1955. 50. VELARDO, J. T., and STURGIS, S. H. Interaction of 16 epi-estriol and estradiol-17B on uterine growth. Proc. Soc. Exper. Biol. & Med. 90:609, 1955. .51. VELARDO, J. T., and STURGIS, S. H. Suppression of uterine growth by purified hydrocortisone acetate, 9a-fluorohydrocortisone acetate and corticotropin. ]. Clin. Endocrinol. & Metab. 16:496, 1956. 52. VELARDO, J. T., and STURGIS, S. H. Antagonism of glucocorticoids and ACTH on estradiol-17B induced uterine growth. Surgical Forum 6:457, 1956. 53. VILLEE, C. A., and HAGERMAN, D. D. Compounds with antiestrogenic activity in vitro. Endocrinology 60:552, 1957. 54. WATSON, E. J. D., and MARRIAN, G. F. Detection of 16-oxoestradiol-17,8 in urine of pregnant women. Proc. Biochem. Soc. in Biochem. ]. 61 :xxiv, 1955. 55. WATSON, E. J. D., and MARRIAN, G. F. Observations on occurrence of 16 epioestriol in urine. Biochem.]. 68:64, 1956. 56. WILKINS, L., et al. Suppression of androgen excretion by cortisone in case of congenital adrenal hyperplasia: Preliminary report. Bull. Johns Hopkins Hasp. 86: 249, 1950. 56. ZANDER, J. Progesteron in menschlichem Blut und Gewebe: I. Progesteron im peripheren venosen blut der Frau. Klin. Wchnschr. 88:691, 1955.