In recent years, many potent progestins have been made available for clinical use. In this paper, research on estrogenic and anti-estrogenic activities of progestins is presented.
Estrogenic activities of test compounds were measured by the uterotrophic effect in immature mice⁵⁹⁾ and by vaginal cornification in spayed rats.⁶⁰⁾ From experimental data obtained, synthetic progestins were classified in two groups, namely, estrogenic and nonestrogenic progestins. 17α-ethynyl-19-nortestosterone (ENT), 17α-methyl-19-nortestosterone (MNT), 17α-ethynyl-5 (10) -estrenolone (EEO), 17α-ethynyl-4-estrene-3β, 17β-diol-diacetate (EEDDA) and 17α-ethynyl-estrenol (EEL) which showed marked uterotrophic effects were classified in the former, and progesterone, 6-dehydro-retroprogesterone (Δ⁶RP), 6α-methyl-17α-acetoxyprogesterone (MAP), 6-dehydro-6-methyl-17α-acetoxyprogesterone (Δ⁶MAP), 6-dehydro-6-chloro-17α-acetoxyprogesterone (Δ⁶CAP) and 17α-allyl-estrenol (AlEL) which showed no such effect, in the latter. By vaginal smear test, estrogenic effects were also observed with ENT and MNT. But the cornified cell-appearance induced by ENT was slightly different from that induced by MNT. To clarify these differences, 17α-ethynyl-estradiol (EED) and 17α-methyl-estradiol (MED) were also examined. Similarity of the effects on the cornified cell-appearance was observed between ENT and EED, and between MNT and MED. Since these estrogenic progestins were proved capable of converting into 17α-substituted estradiol in vivo, the resulted compound might be expected to play some role on the estrogenic effect. However, 17α-ethynyl-5α-androstane-17β-ol-3-one, which theoretically did not convert to EED, showed cornification in the Allen-Doisy test. This suggests that estrogenic effects of progestins are due not only to converted estrogen but also to the other metabolites and original compouuds.
Besides estrogenic effects, all tested progestins have inhibitory activities upon estrogen-induced uterine growth in immature mice. Mouse uterus, fully grown by the administration of 0.3μg of estrone, decreased its weight with simultaneous administration of synthetic progestins. The decrease was parallel with the dose of administered progestins.However, when estrogenic progestins were used for the inhibitory agents, maximal effect was obtained with the dose of 30-300μg and a larger dose had no more inhibitory effect. The maximally depressed uterine weight obtained with the combined administration was almost identical with maximally grown uterine weight by a single administration of these estrogenic progestins. (Fig. 8) The depressed uterine weight with large doses of progestin was not affected by the additional 1-3μg of estrone (Table 7). These antagonizing effects were similarly observed in the hexesterol treated mice (Table 6).
Antagonizing effects of estrogenic progestins in the dose of 10-100μg against estrogeninduced vaginal cornification in spayed rat, were not recognized. However, 100μg of nonestrogenic progestins, progesterone and Δ⁶CAP, showed antagonizing effects (Table 11 & 12).
While androgens were also reported to increase the uterine weight of immature mice, uterotrophic activities of estrogenic progestins were compared with that of estrogens and androgens. Histrogically, most remarkable changes were observed in endometrial epithelium. Estrone as well as EED induced high columnar, estrogenic progestins columnar, non-estrogenic progestins and testosterone cuboidal, and estrone in combination with progesterone columnar epithelium (Fig. 5 & 6, Photo. 1-7). Radio-autography of dissected uterus revealed that the ³H-thymidine uptake in the uterus was increased markedly by estrogens,

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Effects of clomiphene citrate on the pituitary ovarian axis were investigated under various conditions using intact, ovariectomized and androgen-sterilized female rats. The compound was administered subcutaneously as a sesame oil suspension. In immature or ovariectomized rat, clomiphene was estrogenic (uterotrophic) at the dosages over 10μg/rat/day. In mature and androgenized rats, however, the compound was anti-estrogenic (anti-uterotrophic) at the same dosage range as that producing estrogenicity. Clomiphene inhibited ovarian hypertrophy of the parabiotic female mouse united with a castrated male partner at the dosages of 10μg/mouse/day or more. Spontaneous ovulation of cycling female rats was completely inhibited in 2 rats out of 5 by 300/μg/rat given 2 days before the expected ovulation. In hypophysectomized immature rats, clomiphene facilitated superovulation induced by PMS followed by HCG at 1 or 10μg/rat/day but inhibited at 100μg/rat/day or more. In androgen-sterilized rats, the pituitary LH content was elevated at the dosages of over 100μg/rat/day. Clomiphene increased plasma LH level in ovariectomized mature rats at the dosages of 1 or 10μg/rat/day without corresponding reduction of pituitary LH content. These results indicate the dual effect of clomiphene citrate on the rat pituitary ovarian axis; namely, at lower dosages (less than 10μg/rat/day) the compound facilitates the production and release of pituitary LH and increases the ovarian sensitivity to gonadotropin, but at higher dosages (more than 100μg/rat/day) it inhibits gonadotropin secretion and ovulation. It is also suggested that the manifestation of estrogenicity or anti-estrogenicity of the compound depends on the presence or absence of functional ovaries.

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In recent years, the progress of the studies on the progestins has been remarkable and many kinds of progestins were put on the market. Their roles in gynecology and obstetrics have become important on the development of progestins, and numerous reports have appeared concerning their effects on mammalian gestation period. But only few reports have been made concerning the mammalian placenta.
In our department, studies on the progestins have been done but many questions still remain fundamentally and clinically.
The following studies were carried out in order to observe the morphological changes of the rat placenta and pregnancy maintenance ability in ovariectomized rat after administration of several progestins. For many years, the placentas in many kinds of animals were observed anatomically and physiologically by many investigators.
In these experiments, rat placentas were chosen. In rats, abortion occurred in almost all cases by ovariectomy. The histochemical and histological changes of succinic dehydrogenase were investigated during abortion. The pregnant rats were divided into 2 groups, according to the day of pregnancy. They were sacrificed and autopsied on the 1st, 2nd and 3rd day, respectively, after ovariectomy.
The results of this study are as follows :
All fetuses died within 48 to 52 hours after ovariectomy on the 13th, 14th and 15th day of pregnancy. Progestin could prolong the duration of pregnancy, to some extent. Its activity was remarked in the late pregnancy, but in early pregnancy it was not so remarkable. This activity of pregnane derivatives was stronger than that of estrane derivatives.
It was difficult to observe the morphological changes of rat placenta. The authors observed the blood vessel system in labyrinth chiefly. Twenty four hours after ovariectomy, the morphological changes of the placenta were very slight only in blood vessels of the labyrinth. After 48 hours, its changes became remarkable. Individual cells of labyrinth and spongy zone were strongly atrophic and there were bleeding areas in the labyrinth.
The atrophic changes at the onset of abortion appeared in all layers of the labyrinth. For the above-mentioned findings, it could be concluded that the labyrinth was the most important layer for the metabalism between the fetus and mother.
The effects of 17α-ethynyl-19-nor testosterone (EtNT) and 17α-hydroxy progesterone caproate (17-OHPC) were tested. Atrophic changes of placenta after the administration of 17α-OHPC were weaker than that of EtNT. The activity changes of succinic dehydrogenase were weak 24 or 48 hours after ovariectomy.

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The effects of treatment with desiccated thyroid, glucocorticoid and chloroquine on patients with chronic thyroiditis were observed. The clinical course of the disease was studied with special attention to changes of goiter and thyroid antibody in the serum. The titer of circulating antibody was determined by tanned red cell agglutination technic using thyroglobulin as antigen (TRC) and complement fixation test using thyroid microsome as antigen (CF).
With desiccated thyroid, the goiter was less reduced and antibody titer was not decreased. With glucocorticoid, the goiter was reduced and the complement fixation antibody decreased significantly, but these were restored to their original state when this drug was discontinued. With chloroquine, the complement fixation antibody decreased to some extent, goiter however was not reduced. Thyroglobulin antibody was not changed by either therapy.
For the purpose of investigating the immunological effect of these drugs, the changes in the complement fixation ability of aggregated r-globulin, which was shown to be a cytotoxic effect, under the influence of these drugs were studied in vitro. When chloroquine was administered (32-128mg/100ml), its complement fixation ability was decreased remarkably.
As the treatment of chronic thyroiditis, use of glucocorticoid was effective, but was not practical because of the rebound phenomena after discontinuance of the drug. Therefore, chloroquine was used at the same time when glucocorticoid was decreased. By this combined use of glucocorticoid and chloroquine, rebound phenomena of CF titer and goiter were suppressed when glucocorticoid was discontinued. Accordingly, the administration of chloroquine to chronic thyroiditis was effective clinically and immunochemically.
As mentioned above, in the treatment of chronic thyroiditis, TRC antibody titer remai.ned constant, but CF titer changed in accordance with changes of the goiter. When a patient with hyperthyroidism was treated with ¹³¹I, TRC titer did not change, While CF titer elevated temporarily as the goiter became smaller. In the experimental thyroiditis which was produced by prolonged sensitization with thyroidextract in rat, TRC titer rose in all cases, but CF titer tended to elevate in rat which showed marked histological findings.
These results suggested that CF antibody titer indicates the presence of active inflamatory change in thyroid tissue, while TRC antibody titer reflects sensitized state in antibody producing tissue.

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One of the significant clinical problems frequently caused by the administration of glucocorticoid is “Steroid Diabetes.” To elucidate this adverse reaction, the histological and histochemical changes of islets of Langerhans of the rats were examined by treating them with glucocorticoid.
All the male rats employed for the experiment weighed between 200 and 300gr. and were of pure strain of “Sprague-Dawley.” After 24 hours of fasting, they were sacrificed for the examination for a period of 24 hours after glucocorticoid injection.
The blood glucose concentration was determined. The reactivity of islets for the glucocorticoid treatment was represented morphologically by the determination of the amount of beta granula, the concentration of protein bound S-S and SH groups and stainable zinc of beta cells. It was further taken into consideration for the evaluation of hyperglycemia of the rats.
In order to evaluate the grade of hyperplasia and hypertrophy of the islets, not only the number and the determination of the size of the islets in the aggregate in 100 square mm of pancreas were taken into account, but also radioautography was performed with ³H-thymidine.
In the radioautography the grade was shown with the radioactive index.
1) In the group of rats into which were injected 10mg. of cortisone acetate once a day continually for long time, the blood glucose concentration indicated a wavy curve, which marked two maxima in the term 5 to 15 days and 35 days after the experiment.
At the early stage of this series, the blood sugar level ranged from 130 to 150mg/dl. The amount of beta granula, the concentration of protein bound S-S and SH groups and zinc of beta cells decreased, while the number and the size of islets increased remarkably.
At the latter stage, the blood sugar level rose. The granula and the concentration of S-S and SH groups and of zinc increased more than those under the normal state.
In this stage, the correlation coefficient between the concentration of S-S and SH groups and the blood sugar level was much larger than the coefficient between the size of islets in the aggregate and the blood sugar level. It is suggested that the hyperglycemia at the latter stage of this series is influenced by the restraint of the release of insulin from the islets, and as one of the factors for the cause of this restraint is regarded to be the increase of stainable zinc of B cells.
2) In the group of rats, which were treated with gradually increased doses, no particular changes in islets were observed except the increase of the number and the size.The blood sugar level revealed a range of between 80 and 90mg/dl.
3) The radioactive index of islets of rats, which were treated with glucocorticoid, suggests that the enlargement of the islets was caused by the mitosis of B cells.

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Biological activities of 17α-ethyny1-4-estrene-3β, 17β-diol diacetate (EEDDA) were investigated in comparison with those of 17α-ethynyl-19-nortestosterone (ENT), 17α-methy1-19-nortestosterone (MNT), 17α-ethynyl-5 (10) -estrene-17β-ol-3-one (EEO) and 17α-ethynyl-4-estrene-17β-ol (EEL).
Subcutaneous administration of EEDDA produced progestational activity in the McPhail assay and the endometrial carbonic anhydrase test. But the dose-response curves of EEDDA in these assays were characteristic. The maximal response was obtained in a total dose of 500-1000μg, and as the dose was increased above 1000μg, the responses of endometrium were reversed. No such reversal was obtained following treatment with progesterone and other estrogenic progestins at the dose-range in this experience.
Administration of EEDDA produced a marked increase in the weights of uteri in immature mice. Since this effect was similar to those of the above-mentioned steroids, EEDDA should belong in the category of estrogenic progestin. In Allen-Doisy test, estrual changes in the vaginal smears were observed in the dose of 0.1mg.
The slope of dose-response-curve obtained with EEDDA, as well as with other estrogenic progestins, in uterotrophic activity was shallow and was of the impeded type. These results might be due to their inherent antiestrogenic activities.
Slight androgenic-myotrophic activity was observed after EEDDA administration in castrated male rats.
Anti-inflammatory and thymolytic activities were not manifested by EEDDA treatment in adrenalectomized immature male rats.
Long term administration of EEDDA resulted marked inhibition in rat hypothalamo-hypophyseo-gonadal axis, but no suppression in the adrenal weight.
Studies on the metabolism of EEDDA in rat revealed that this was partly converted to 17α-ethynyl-4-estrene-3β, 17β-diol in the small intestin and to ENT in the liver. It was suggested that this conversion was responsible for the resemblance of biological activities between EEDDA and ENT.

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Pregnant Mare's Serum Gonadotrophin (PMS) and Human Chorionic Gonadotrophin (HCG) were used in 110 anovulatory women who were suffering from various types of ovarian insufficiency. As a result, in 65 out of 110 patients (59.1%) ovulation was induced. Clinically, it was clear that larger amounts of PMS were needed in the second course than in the first course. This evidence is presumably due to the inhibition of PMS activity by anti-PMS appeared in the blood.
It has been discussed that PMS might act as an antigen in the human body because it is a heteroproteohormone. The follwoing experiments were performed immunologically : 5 c.c. of blood were obtained from sixteen patients selected randomly out of the patients treated with gonadotrophin. The sera were incubated at 56°C for 30 min. Anti-PMS titers were determined by using the haemagglutination reaction before, through, and after the treatment. The anti-PMS titers could not be detected before the treatment in all cases, and with some individual differences, at least 1 : 128 of the titers were observed in all cases and continued for about 20 weeks. The blood obtained from eleven patients treated repeatedly 8-30 weeks after the first therapy was tested with the same method. Generally, titers increased rapidly, maintained a high level and continued for a longer period than the term after the first course.
In order to investigate whether or not the anti-PMS which was detected by haemagglutination reaction could neutralize the biological activity of PMS, the following experiments were performed biologically : 1 c.c. of serum obtained from the patient, whose serum indicated the titer of 1 : 1,024, inactivated the biological activity of 2 IU of PMS completely in the uterine weight of CF#1 immature mouse. This same serum was diluted by the two fold method until 1 : 8, and was tested with the same method. As a result, it was recognized that 1 c.c. of and serum up to 1 : 64 in titer could not neutralize the biological activity of PMS 2 IU. The sera obtained from six patients who had different anti-PMS titers from 1 : 1,024 showed similar results. From these results, it can be said that anti-PMS of titer 1 : 128 neutralizes 2 IU of PMS; that is, approximately 5,000 IU of PMS injected into the human body (50 kg body weight) are inactiviated by anti-PMS of 1 : 128, and do not stimularte the ovary.
Since these results apparently indicated that anti-PMS formation in serum is a big problem in the gonadotrophic therapy, it is extremely important to devise the method of suppression of anti-PMS. The following two methods were tested : an amount of 0.5 mg of corticosteroid (dexamethasone) was administered daily for 20 days orally (total 10 mg), and 100 mg of cyclophosphamide were injected intravenously every 5 days for 5 days (total 500 mg) together with gonadotrophic therapy. As a result, anti-PMS formation was apparently suppressed, and disappeared in a short period. Although, on the application of cyclophosphamide in this field further studies are necessary, the concomitant use of corticosteroid is unquestionably favorable to induce the ovulation.
Lastly, it should be emphasized that gonadotrophic therapy with PMS in anovulatory women should be carefully carried out with corticosteroid under a rational schedule aimed at the conception.

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