D. L. Thomson

Effect of Oestrin on Ovaries and Adrenals

The enlargement of the ovary observed during gestation in the rat is solely due to the corpora lutea attaining a much larger size than those of the normal cycle or those produced in non-pregnant animals by administration of gonadotropic substances; yet there has been little investigation of the factors regulating the size of the individual corpus luteum. We have shown 1 that the mere distension of the uterine cavity with paraffin leads to prolonged vaginal dioestrus and inhibits the involution of the corpora lutea of pregnancy even though the embryos and their placentae be removed; these corpora, however, showed histological signs of fatty degeneration and were unable to maintain the mammary glands in a fully developed condition. The hypophysis is not essential for the maintenance of the corpora lutea of gestation. 2 , 3 The administration of gonadotropic hormones produces larger corpora in the pregnant animal than in the non-pregnant. 4 Since the gonadotropic hormones lead not only to formation of new follicles and corpora lutea but also to involution of corpora already present, we have repeated and modified these experiments. Six rats received 3 injections of 50 units of A.P.L. (anterior-pituitary-like hormone of pregnancy urine) on 3 successive days at about mid-pregnancy. The animals were sacrificed 7 days later; their ovaries were found to contain very large numbers of corpora lutea, all of which were as large as normal corpora of gestation. There is, therefore, some factor in the pregnant animal which transforms experimentally-produced corpora lutea into corpora lutea of gestation, and maintains them throughout the whole period of pregnancy. There is evidence that oestrin is produced in large quantities in the pregnant rodent. 5

Effect of hypophysectomy upon pregnancy and lactation.

The considerable enlargement and structural changes of the pituitary during pregnancy and lactation seem to indicate that the internal secretion of this organ plays a very important rôle during this period. Therefore repeated attempts have been made to determine whether pregnancy can be maintained after hypophysectomy, but the results of such experiments are contradictory. Whereas Aschner 1 found that abortion takes place in hypophysec-tomized pregnant dogs, Allan and Wiles 2 observed that pregnancy and parturition are not interfered with in the hypophysectomized cat, although lactation is impossible. Pencharz and Long, 3 working with rats, stated that pregnancy is not interrupted by removal of the hypophysis, but the process of parturition becomes impossible and the foetuses die in utero after a somewhat prolonged gestation period. Repeating these experiments on rats we were able to confirm the statement that pregnancy is usually prolonged (up to 26 days). If the pituitary is removed between the tenth and fourteenth day of gestation, death and resorption of the foetuses may occur; but when the pregnancy proceeded normally until term, in 22 out of 24 cases the mechanism of parturition was not interfered with, and the litters were born alive; in the 2 exceptional cases hemorrhage occurred at term and the foetuses died in utero. We further established that the milk secretion always sets in normally at birth, but stops after a few hours, so that the hypophysectomized mother is unable to nurse her young. As has been pointed out previously, 4 milk secretion will also stop immediately if the pituitary is removed in various stages of lactation. These experiments indicate that the endocrine functions of the pituitary are not indispensable during the second part of pregnancy and parturition in the rat.

Effect of Hypophysectomy upon Pregnancy and Lactation in Mice

In the rat, hypophysectomy during lactation leads promptly to cessation of milk secretion; 1 hypophysectomy during the second half of pregnancy does not usually prevent the birth of living young nor the development of the mammary glands, in fact milk secretion begins normally at parturition but ceases within 26 hours. 2 Caesarian section shortly before term regularly induces lactation, but fails to do so if the pituitary is removed. 3 Mammary development in virgin rats under the influence of ovarian hormone secretion stimulated by the anterior-pituitary-like hormone leads to milk secretion when the luteinized ovaries are removed, but this reaction also does not occur in the absence of the pituitary. 4 These findings are in accord with the view of Riddle, 5 Turner, 6 and others that a specific hormone of the anterior pituitary is responsible for the actual initiation of lactation, and that a sudden decrease in the amount of ovarian hormones in the circulation is a stimulus for the production of this hormone (Nelson and Smelser 7 ), but they also indicate that the contents of the pregnant uterus may take the place of the anterior pituitary in this reaction. We have recently succeeded in hypophysectomizing albino mice by a parapharyngeal operation, almost identical with the modified Smith procedure used by us in rats and described elsewhere. 8 In the mouse the pituitary is situated somewhat more caudally, relative to the spheno-occipital synchondrosis; the bone is therefore drilled exactly over this synchondrosis. We have experienced no trouble from hemorrhage, no mice have been lost during the operation, and the post-operative mortality has been somewhat lower than in rats. Six mice, 3 months of age, were hypophysectomized on the second or fourth day of lactation. All recovered from the operation and attempted to rear their litters, but milk secretion failed and within 24 hours the stomachs of the young were empty.

Preparation of a Purified and Highly Potent Extract of Growth Hormone of Anterior Pituitary Lobe

Since the demonstration by Long and Evans 1 of the growth hormone of the anterior pituitary, various workers have confirmed the existence in alkaline extracts of the anterior lobes of animals of a growth-promoting substance. Van Dyke and Wallen-Lawrence 2 have suggested the use of totally hypophysectomized animals as a test object for determining the potency of extracts. The latter also made definite progress in the preparation of potent extracts which were less crude than those previously used. We have been successful in preparing highly potent extracts of the anterior lobe of oxen. These extracts have been tested for growth-promoting effects upon totally hypophysectomized rats. Growth has started almost immediately following the institution of therapy and has continued at a fairly uniform rate (see chart). It was first observed that an alkaline extract (anterior lobes extracted with 10 volumes of 1% NH4OH) which had been partially deproteinized, by the addition of acetic acid to pH 6.5 and filtering, had a slight growth-promoting effect in hypophysectomized rats. Such extracts were then saturated with ammonium sulphate in the presence of 1% NH4OH, and the resultant precipitate collected, extracted with dilute ammonia and dialyzed. After dialysis the solution was concentrated at low temperature and pressure. During the concentration process a semi-crystalline substance separated out at a pH of 7.5 to 8. This was removed and an alkaline extract of it was found to be highly potent in stimulating growth in hypophysectomized rats. From these leads the following method has been evolved. The anterior lobe tissue is treated with several volumes of dilute alkali: 0.5 to 1% NaOH or 1% NH4OH.

Effect of Prolonged Administration of the Anterior Pituitary-Like Hormone on Pituitary and Thyroid

Changes in the anterior lobe of the pituitary after administration of the anterior pituitary-like hormone (A.P.L.) of pregnancy urine or placenta have been observed by numerous investigators, 1 , 2 , 3 but their results are contradictory, both as regards the histological nature of the changes thus produced and the effectiveness or ineffectiveness of A.P.L. in the male. Zondek 3 found that when prolan is given to female rats over a long period the ovaries, which have increased in size in the beginning, will become smaller again. These observations have been confirmed in this Department, and it has also been found that the same retrogression in size takes place in the sex organs of the male if A.P.L is given over a very long period. 4 We thought that this decrease in sensitivity to A.P.L. which sets in gradually in chronic experiments might be at least partly responsible for the discrepancies in the results of those investigators who studied the effect of A.P.L. on the pituitary. Our experiments seem to confirm this view. We found in several series of experiments in which injections were given from the 27th to the 67th day of life, that the size of the pituitary runs parallel to the size of the ovaries in the A.P.L. treated female rat. In 16 rats observed at the time when the enlargement of the ovaries was most conspicuous (0.547-0.616 gm. as compared with 0.032 in the untreated control females of the same age) the pituitary was also at its maximum size (0.0115-0.012 gm. as compared with 0.004-0.0045 gm. in the control animals). This increase in weight is due solely to the enlargement of the anterior lobe.

Anterior Pituitary and Lactation

By injection of the anterior-pituitary-like hormone (A.P.L.) of pregnancy urine Evans and Simpson 1 were successful in producing marked development of the mammary glands in virgin rats, but were unable to produce milk secretion; Bradbury 2 obtained similar results in mice. We have confirmed these results by histological examination, which shows that no secretion takes place in the glands, although the alveoli are as numerous as in late pregnancy. We further observed that the development of the mammary gland in these animals runs parallel to the increase in the weight of their ovaries. We found, however, that removal of the intensely luteinized ovaries of these rats will lead to abundant milk secretion in their mammary glands within 36 hours (13 experiments, all positive). We further observed that if the pituitary was removed simultaneously with the ovaries, milk secretion did not set in (4 experiments). These experiments seem to indicate that removal of the luteinized ovaries will lead to milk secretion in the fully developed mammary gland of the A.P.L. treated rat, but only in the presence of the pituitary.

Further observations on the effect of hypophysectomy on lactation.

The removal of the pituitary from a lactating rat leads promptly to a cessation of milk secretion. 1 Immature female rats treated with the anterior-pituitary-like hormone long enough to induce full development of their mammary glands secrete milk when the luteinized ovaries are removed, but not if the pituitary is removed at the same time. 2 These observations seem to support the view of Riddle 3 and others who believe that lactation is controlled by a pituitary hormone, prolactin. Less easily reconciled with this view was the observation 4 that pregnant rats hypophysectomized 10 days before term showed milk secretion for a few hours after parturition. It seemed possible that in these last experiments pituitary function was partly replaced by some stimulus from the pregnant uterus. We found in 10 experiments that if the uterus was emptied by Caesarian section late in pregnancy, lactation set in within 36 hours; similar observations have been made in the guinea pig 5 and the goat. 6 In 6 other experiments, however, in which the pituitary was removed when the uterus was emptied, no milk secretion was observed. This indicates that milk secretion is not possible in the rat if both the fetuses and the pituitary are absent.

Further Studies on Production of Thecal Luteinization by Means of A. P. L

We have reported that if A.P.L (anterior-pituitary-like hormone) is given to very young rats (6-8 days old) no follicle maturation or corpus luteum formation occurs, but the thecal cells increase in size and store large amounts of fat, so that their appearance becomes very similar to that of real corpus luteum cells. Normal reactions to A.P.L—that is, follicle maturation and corpus luteum formation—is regularly found in prepubertal rats if the injections are started around the 21st day of life. We found, however, that rats treated daily with this hormone, beginning at the age of 6 days, react with thecal luteinization only, even when they reach the age of 21 days or more. 1 In the present experimental series, we wished to see (1) if the formation of thecal lutein cells precedes the appearance of the squamous vaginal reaction and, (2) how long these rats continue to react to A.P.L. with exclusively thecal luteinization and continuous oestrus. For this purpose, 43 female rats were injected with increasing amounts of A.P.L. (6-25 units daily), beginning at the age of 6 days. We examined the vaginal smears every day and killed an animal every few days in order to study the histological changes produced in the ovary. These experiments showed that at the time when the smears first became oestrous—on the seventh to the thirteenth day of treatment—the formation of thecal corpora lutea was already very conspicuous. All the animals killed during the next 4 to 5 weeks showed a similar reaction, inasmuch as in all of them only thecal cells turned into lutein cells. As the treatment progressed, severe follicular atresia set in, so that most of the primary follicles disappeared and histological preparations in these stages showed almost nothing but small islands of luteinized thecal cells.

Further Studies on Loss of Sensitivity to Anterior Pituitary-Like Hormone of Pregnancy Urine

It is well known that both the anterior pituitary sex hormone and the anterior pituitary-like hormone of pregnancy urine (A.P.L.) lose their ovary-stimulating effect after a certain time, if given daily over a long period. Hisaw 1 has recently been able to show that this loss of sensitivity may be obtained in rabbits even by the administration of subthreshold doses of anterior pituitary sex hormone, and we 2 ,3 found that the loss of sensitivity is limited to the gonadotropic preparation with which the animals have been treated previously. Animals which became insensitive to A.P.L. remained responsive to pituitary implants and vice versa. In an attempt to determine whether this loss of sensitivity is due to the formation of specific hormone inhibiting substances as postulated by Collip 4 or simply to an insensitivity of the ovarian tissue itself, we performed the following experiments. Five female rats, 21 days of age at the beginning of the experiment, have been injected daily with 100 R.U. of A.P.L. subcutaneously for a period of 8 months. Then they and 7 untreated females of the same size received 100 R.U. of A.P.L. intravenously. All animals were killed 1 hour after the injection. The blood of each animal was collected separately and injected in 3 doses of approximately 1.5 cc. each on 3 consecutive days into an immature rat 21 days of age, and thus tested for its A.P.L. content. The blood of all 7 previously untreated rats led to corpus luteum formation in the immature females, while the blood of the A.P.L.-insensitive animals was ineffective with one exception. The odds against obtaining such a result by chance greatly exceed 100 to 1. From this experiment we conclude that A.P.L. disappears more rapidly from the blood of the A.P.L-insensitive rat than from the blood of the normal animal.

Replacement of Gonadotropic Action of Pituitary in the Hypophysectomized Rat

We have pointed out that the administration of anterior-pituitary-like hormone (A.P.L.) to the hypophysectomized rat cannot prevent the degenerative changes in the ovary which invariably occur after the removal of the pituitary. 1 We also found that it is impossible to induce maturation of follicles and corpus luteum formation in the hypophysectomized rat with A.P.L. injections. In the absence of the hypophysis, A.P.L. leads only to the luteinization of thecal cells but it does not act upon the ovary as it would under normal conditions. The present experiments show us that it is possible to obtain both follicle maturation and corpus luteum formation in the hypophysectomized rat if certain pituitary extracts are administered simultaneously with A.P.L. In the first series, 6 rats were hypophysectomized when 22 days old, and from that time they received 0.5 cc. of a 0.5% aqueous ammonia extract (1 cc. = 1/10 gm. of anterior lobe tissue), and 25-50 units of A.P.L. daily for 9 days. They all showed squamous vaginal smears on the fourth to the sixth day, and the histological appearance of their ovaries was the same as that of normal immature females receiving A.P.L. Numerous corpora lutea and mature follicles had been formed in these immature rats in the absence of living pituitary tissue. In the second series, 7 postpuberal female rats, weighing 72-129 gm., were hypophysectomized and treated with 100 units of A.P.L and 1/4 cc. of the same ammoniacal pituitary extract daily for 14 to 24 days. At autopsy they showed numerous corpora lutea, some of them quite recent (Fig. 1). Their ovaries were enlarged, weighing between 43 and 156 mg. Similar results were also obtained with the combination of A.P.L. and aqueous acetic acid extract of the pituitary.