H. L. Fevold

Production of a Premenstrual Endometrium in Castrated Monkeys by Ovarian Hormones.

The several theories explaining menstrual phenomena in primates for the most part agree that it depends upon hormonal function of the ovaries. Some authors emphasize follicular hormone, some corpus luteum, while others believe that both are concerned. Van Herwerden 1 found that in Cercocebus cynomolgus menstruation may occur without ovulation and Corner 2 and Allen 3 have established the same fact for Macacus rhesus. Allen also discovered that rather scanty menstruation in castrate and sexually immature monkeys usually followed after a certain degree of uterine growth had been induced by the injection of follicular hormone. These authors agree, however, that the uterine endometrium under these conditions is not typical of the normal premenstrual endometrium found only when a corpus luteum is present. Novak 4 suggested that the physiology of menstruation could perhaps be solved if follicular and corpus luteum extracts of known potency were administered to experimental animals in the same sequence that they normally occur in the menstrual cycle. Allen 3 has shown that physiologically active preparations of the follicular hormone do not promote typical premenstrual development of the uterine endometrium. We have made corpus luteum extracts which, in proper combination with follicular hormone, produce many physiological reactions ascribed to the normal corpus luteum (Hisaw, 5 Weichert, 6 Hisaw el al 7 ). This paper reports the experimental use of these corpus luteum preparations on the production of premenstrual development of the uterine endometrium of castrate Macacus rhesus monkeys. Five sexually mature female monkeys were castrated. They were first brought into full oestrum by the injection of follicular hormone (kindly furnished by E. R. Squibb & Sons) and then given a series of injections of corpus luteum extracts.

Effects of Hypophyseal Extracts on Sexually Immature Monkeys.

Information concerning the effects of anterior pituitary materials on the gonads of primates is very limited. E. Allen 1 and Hartman 2 have demonstrated the gonad stimulating effect of hypophyseal implants using dogs, monkeys, and pigs as donors, and Courrier, et al., 3 have shown that similar effects can be obtained with alkaline hypophyseal extracts. We wish to report the results of injecting aqueous pyridine extracts of anterior pituitary material in immature female Macacus rhesus monkeys. 21 Aqueous pyridine extracts as prepared by Fevold, et al. 4 were used. The preparations were injected subcutaneously in 0.5 cc. doses twice daily for periods of 14 to 16 days and each cc. of extract was equivalent to 1 gm. of dried pituitary powder. As small a dose as 8 gm. equivalent of pituitary was found to be sufficient in one animal. The first noticeable change observed was the appearance, in 2 to 3 days of injection period, of a purplish coloration in the skin of the peri-anal region and a reddening of the nipples of the mammae. The maximum coloration and peri-anal swelling was reached in about 8-12 days, the vaginal smear at this time showing predominantly cornified cells. When the injections are continued further the coloration decreases somewhat but the oedemmatous condition in the inguinal region and the posterior part of the thigh becomes progressively greater. No blood could be detected in the vagina although uterine bleeding (follicular menstruation) followed the cessation of injections. The effects of the treatment on the internal sexual organs were very marked. The ovaries increased in weight as much as 1800%, the increase being due to development of a number of large follicles. Corpora lutea were entirely absent except in one doubtful case where there seemed to be slight luteinization.

The Corpus Luteum Hormone. II. Methods of Extraction

1 N PREVIOUS papers (Weichert, 1928; Hisaw, Meyer, and Weichert, 1928; Hisaw, 1927 and 1929) several physiological properties of extracts prepared from the corpora lutea of the sow were described, and a few of the chemical characteristics were also briefly given (Hisaw, 1929). This paper reports in some detail the chemical procedure used in the preparation of active extracts as well as certain chemical properties of the substance, in so far as they have been determined.

Effects of Progesterone on the Female Genital Tract after Castration Atrophy.

The effects of oestrin-free progestin on the atrophic endometria of 2 castrated monkeys were described by one of us. 1 These animals had been castrated for 37 days and were given 4 RbU of oestrin-free corpus luteum extract daily for 10 days. The endometria of both monkeys showed unquestionable premenstrual development. Although these preparations did not produce eornifkation of the vaginas of castrated rats it could not be stated with certainty that they were entirely free of oestrin and that the observed effects were due only to the action of progestin. We wish here to present results of similar experiments using synthetic progesterone.† A young adult monkey that had been castrated for 242 days was given 4 mg. of progesterone daily for 18 days, a vaginal biopsy was taken on the eighth day, a thread was placed through the uterus on the eleventh day and the animal was killed on the nineteenth. Nucleated epithelial cells appeared in the vaginal smear and were present in considerable numbers by the eighth day, after which they gradually decreased and were almost absent at the conclusion of the experiment. The face showed coloration by the seventh day and the pale sexual skin developed and maintained a deep red color. The uterus, at laparotomy on the eleventh day, measured 11 by 9 mm. at its greatest diameters and at autopsy 17 by 13.5 mm. This treatment produced a fully developed premenstrual endometrium and epithelial proliferations 1 characteristic of an implantation site. The cervical glands showed active secretion though the epithelial cells in most places were not as tall as those seen following injections of oestrin. The vaginal mucosa, though thin, showed some stimulation both at the time of biopsy on the eighth day and at autopsy.

Pituitary Hormone Antagonism.

It is known that certain hypophyseal extracts will inhibit the increase in the size of the ovaries of immature rats, which occurs when gonad stimulating extracts are administered. In a previous note (Leonard 1 ), it was stated that the growth hormone of the pituitary was not the inhibiting substance and it was suggested that a sex stimulating fraction or something associated with it, was the antagonistic agent. In further attempts to identify the inhibiting substance with known extracts of the pituitary, several gonad stimulating fractions and a thyroid stimulating fraction were used. The results obtained are reported here. Antuitrin S† was injected subcutaneously into immature female rats of similar age and weight, in doses which produced ovaries weighing approximately 30 mg., in 5 days. Similar rats were treated for the same period with the same amount of Antuitrin S plus intraperitoneal injections of the several pituitary extracts to be tested for their inhibiting power. These were as follows: follicle-stimulating hormone (F.S.H.) and luteinizing hormone (L.H.) prepared from sheep pituitary (Fevold, et al. 2 ), luteinizing hormone from horse pituitary, unfractionated pregnant mare serum, follicle stimulating urine (F.S.U.) prepared by alcoholic precipitation, and thyroid stimulator from the Schering Corporation.‡ These preparations were all tested and found to be active. It is quite expedient that the fraction to be tested for inhibition be given intraperitoneally rather than subcutaneously or otherwise the presence of the inhibiting substance may not be detected (Leonard 2 ). The results of the experiments are summarized in Table I. It is readily seen that the luteinizing fraction prepared from sheep pituitary was the only one to give a complete and definite inhibition of ovarian growth. The follicle stimulating substances from pituitary or urinary sources failed; the pregnant mare serum, the thyroid stimulator and the luteinizing hormone from horse pituitary also failed.