Frederick L. Hisaw

Experimental relaxation of the pubic ligament of the guinea pig.

The extraordinary separation of the pubic bones of the guinea pig in late pregnancy has been recorded by several investigators. Todd 1 has made the most recent contribution to this subject and has thoroughly discussed previous research as well as described the gradual changes that take place at the symphysis during normal pregnancy and the subsequent return to the post-parturient condition typical of multiparous females. The writer has been interested in this phenomena for the last four years, chiefly from the standpoint of its physiological explanation and possible correlation with a similar condition which has been studied in the pocket gopher. 2 In the pocket gopher the pubic bones are resorbed at the symphysis before pregnancy occurs, and the reaction is governed by the ovary, while in the guinea pig relaxation of the pubis occurs during pregnancy and little or no bone is resorbed, but the connective tissue at the symphysis is greatly increased, allowing a marked separation of the bones. It has been possible to produce changes in the pubic ligament of virgin guinea pigs by experimental procedure and these are apparently identical with those occurring normally during pregnancy. If 2 cc. of blood serum of pregnant rabbits or guinea pigs are injected subcutaneously into virgin guinea pigs during early post oestrum a noticeable relaxation of the pubic ligament can be discerned within six to eight hours by movements at the pubis. These movements gradually become more pronounced during the next eighteen hours and the ligament may not return to its normal condition for two or three days. The blood of males and nonpregnant females does not bring about this reaction, and the blood of parturient females loses its effectiveness very noticeably within the first eight hours, negative results being usually obtained from blood drawn 24 hours after the young are born.

The physiology of menstruation in Macacus rhesus monkeys

Abstract Over forty monkeys were used in this series of experiments to determine the physiologic factors responsible for the development of the primate endometrium. The experiments may be divided into two groups: (1) those dealing with the action of estrin and corporin on the endometrium after castration, and (2) those in which endometrial modifications were induced through the action of anterior pituitary preparations on the ovaries. It is possible by both of these methods to produce endometrial conditions typical of the different phases of the menstrual cycle. The estrin effects are characteristic of the first half or follicular part of the cycle and corporin the last half or lutein part. The histologic and cytologic changes produced by estrin and corporin are so different that their individual actions can be easily distinguished. Postoperative menses can be inhibited by estrin following oophorectomy during the follicular half of the cycle but becomes progressively more difficult with the development of the lutein condition. Attempts to inhibit bleeding of well-developed premenstrual endometria by the injection of estrin were not successful. When estrin is administered for an extended period following castration atrophy of the endometrium there is first marked mitotic activity which gradually subsides and may be almost absent by the end of thirty days. Such endometriums will bleed if estrin injections are discontinued or the dosage materially lowered and may even do so during treatment when injections are continued for a long time. Corporin will inhibit postcastration bleeding of either a follicular or a lutein endometrium and will also inhibit bleeding which invariably follows discontinuance of a prolonged estrin treatment. The dosage required for inhibition of bleeding seems higher than that which produces characteristic corporin effects in the endometrium. When corporin is administered for an extended period, following an estrin treatment, the endometrium develops the modifications found in the lutein half of the normal cycle, and the glands finally become secretorially exhausted and remain in this condition as long as treatment is continued. Few or no mitoses are found and this is especially true of well-developed premenstrual conditions. Corporin can also produce its characteristic effects, without previous estrin stimulation, in endometriums which have undergone castration atrophy. When corporin and estrin are injected simultaneously ½ Rb.U. of corporin is at least partially effective in the presence of 100 R.U. of estrin. Epithelial proliferations from the surface mucosa and distal portions of the glands were found in several instances following traumatization, as a result of biopsy, during the presecretory corporin phase of experimental cycles. These proliferations were not observed to follow biopsies of estrin endometriums or biopsies after prolonged corporin treatment. The significance of these epithelial growths is not definitely known, though they are very similar and appear morphologically identical with those seen at the implantation site of a fertilized ovum.

Estradiol-17β and Progesterone in Ovaries of Starfish (Pisaster ochraceous).

Summary Ovaries of starfish (Pisaster ochraceous) were examined for sex steroids. A low level of estrogenic activity, approximately 0.1 μg or less/kg of ovarian tissue, was determined by the Astwood 6 hour assay method. The biologically active material was identified as estradiol 17β by countercurrent distribution, paper chromatography and bioassay. Presence of progesterone was tentatively identified by methods of column chromatography and Hooker-Forbes assay.

Pseudopregnancies from Electrical Stimulation of the Cervix in the Diestrum.

The artificial induction of a pseudopregnant state in the rat by means of electrical or mechanical stimulation of the cervix uteri is a common laboratory procedure. It has long been supposed that these stimuli to be effective must be applied at or near the time when the female is in a sexually receptive mood (i. e., late proestrus or estrus). We wish to report some observations on the application of an electrical stimulus to the cervix of adult female rats during the diestrous phase of the cycle. The cervix was exposed with a speculum and the electrodes, spaced at 2 mm, were applied at any place on the body of the cervix. The stimulus, a faradic current taken from an induction coil, was moderately intense and always produced convulsive contractions in the hind quarters The stimulus was applied continuously over a period of 5 to 10 sec. In a group of 26 virgin females known to be having regular 4 to 5 day cycles 13 were stimulated on the 1st day of diestrous, and 13 on the second day. The number of animals becoming immediately pseudopregnant, those showing one additional estrus before becoming pseudopregnant and those showing no disturbance of the estrous cycle are listed in Table I. The total number of pseudopregnancies obtained in the above groups in which the stimulus was applied during the interval was 20 or 76%. Thirty-six females in late proestrus or estrus were stimulated in the same manner: 31 or 86% became pseudopregnant immediately and the remaining 5 continued to show cycles of normal length. The above figures are all on the basis of a single stimulation for each animal.

Effect of Oestrin on Gonad Stimulating Power of the Hypophysis.

Smith and Engle 1 showed that the anterior lobe of the hypophysis of the guinea pig in oestrum is less potent in its gonad stimulating power than the hypophysis of animals in the dioestrum. Burch and Cunningham 2 reported that injection of a commercial placental extract, containing considerable amounts of oestrin, into adult, castrate, female rats tends to increase the gonad stimulating power of the pituitaries of such animals, as compared with non-injected castrate controls of approximately the same weight. The period of injection in their experiment was 6 days and the dosage employed was from Thirty-four immature female rats varying in age from 30 to 40 days were injected with 2 R.U., 0.1 cc., per day of an oil soluble oestrous hormone prepared from the amniotic liquor of the cow.† Twenty-eight litter-mate sisters of these experimental animals were injected with 0.1 cc. of Mazola oil and used as controls. The period of injection varied from 30 to 70 days, after which the animals were sacrificed, ovaries weighed and pituitaries implanted into female rats varying in age from 20 to 30 days. Two pituitaries were implanted simultaneously into each recipient. The criterion of gonad stimulating power of the hypophyseal implants was the time necessary for the opening of the vaginas of the recipients. This method is not satisfactory unless the vaginas of the recipients being compared open 4 or more days apart. The ovaries of the oestrin injected donors weighed 40% less than the ovaries of the control litter-mate donors. The pituitaries of the oestrin injected rats opened the vaginas of the recipients from 9 to 25 days after implantation, whereas the vaginas of control recipients which received pituitaries from the control donors opened 4 to 5 days after implantation.

ESTRADIOL-17-BETA, ESTRONE, AND PROGESTERONE IN THE OVARIES OF LAMPREY (PETROMYZON MARINUS).

Summary Ovaries of cyclostomes (Petromyzon marinus) were examined for sex steroids. The ripe ovaries contain a minimum of 16 μg estradiol-17β/kilo of fresh ovarian tissue determined by the Astwood 6 hour assay. The biologically active material was identified as estradiol-17β and estrone by countercurrent distribution, paper chromatography and bioassay. Presence of progesterone was tentatively identified by methods of column chromatography and Hooker-Forbes assay.

Inhibition of Ovulation and Associated Histological Changes.

The idea that the corpus luteum is responsible for the inhibition of ovulation seems to have been first elaborated by Beard 1 and by Prenant. 2 The experimental proof of the inhibitory action of the corpus luteum has been attacked in many different ways. The two most general methods have been based on anatomical and physiological modifications of the reproductive tract, correlated with the presence or absence of a functional corpus luteum, and, changes produced by the injection of extracts of lutein tissue. Corner and Hurni 3 reported negative results in an attempt to inhibit ovulation by injecting corpus luteum extracts in normal white rats. Loeb 4 failed to obtain consistent positive results in the guinea pig, while Papanicalaou 5 recently reported inhibition of ovulation in the same animal. Pearl and Surface 6 were able to stop hens from laying by injections of water extracts of a dried commercial preparation. Kennedy 7 described positive results on the rabbit for extracts made from a desiccated commercial preparation which were injected for a period of time before mating, resulting in the inhibition of ovulation. Parkes and Bellerby 8 reported inhibition of ovulation in mice by injections of an emulsion of an ether extract of the corpus luteum of the cow. They noted that not all of their extracts were potent, and that those that were soon deteriorated. Many different kinds of extracts have been employed by various investigators but the results obtained through their use are so contradictory that they cannot be discussed in this brief report. An acid alcohol extract of the fresh corpus luteum of the sow has been prepared in this laboratory and refined to the extent that very little protein remains. This preparation is capable of sensitizing the uterus of spayed rats and guinea pigs so that placentomata may be produced (Weichert). 9 It will relax the pubic ligaments of virgin guinea pigs in oestrum (Hisaw)10; and will inhibit ovulation and oestrum in the normal female albino rat. Inhibition of oestrum was best effected by subcutaneous injections of an amount equivalent to 3 gm. of fresh tissue given every 4 or 5 hours from the beginning of oestrum until the experiment was ended. Rats injected in this manner did not come into oestrum for 6 to 12 days, the length of time for which ovulation was inhibited depending upon the amount of the extract injected. Attempts are now being made to inhibit ovulation for a greater period and to determine at what stage of the dioestrous interval injections may be most effective.

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.

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.