J. B. Collip

SOME EFFECTS OF PROLONGED ADMINISTRATION OF ŒSTRIN IN RATS

This article records the results of experiments with the prolonged (over 132 days) administration of estrone in rats. It is known that the administration of large doses of estrone to female rats may lead to hypertrophy of the anterior lobe within a few days. 6 castrate female rats (3-4 months old) received daily subcutaneous injections of estrone for 331 days. The animals were sacrificed after a further 30 days during which time all were either untreated or injected with solutions of crystalline progesterone. At postmortem the horns of the uteri were found to be nearly solid pearly white and hard. Squamous metaplasia of the epithelium was present in 5 cases. The pituitaries were enlarged in all cases and large cavernous adenomata of the anterior lobe were present in 3 rats. All showed multiple mammary milk cysts; breast changes included an adenofibroma. It is unlikely that heredity factors played an important role in producing the above changes. The incidence of spontaneous tumors of any kind is very low in this rat colony.

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.

Effect of Large Doses of Progesterone in the Female Rat

Summary Daily administration of 4 mg. of synthetic progesterone to iiormally cyclic iemale rats causes immediate cessation of cycles, without vaginal mucification. There was ovarian atrophy, hypertrophy of the pituitary, and slight atrophy of the thymus. It has no effect n-hatsoever upon the condition of the mammary gland. Since cestrin is also without any direct effect upon the mammary gland (as shown in hypophysectomized animals) the effect of ovarian hormones on the mamma must be largely dependent upon the presence of pituitary secretions.

Effect of Testosterone on the Mammary Gland

The fact that derivatives of the male hormone may exert an action on the female sex organs is known from the work of Butenandt and Hanisch, 1 who showed that androstenedione which is inactive in castrate females produces vaginal oestrus in the presence of an ovary, and from the work of Parkes, 2 who found that the same is true in the case of androstanediol. The action of these compounds might possibly be explained by a transformation of these substances into oestrin by the ovary. This would seem all the more likely since Collip, Browne and Thomson 3 , 4 , 5 have shown that the ether-insoluble fraction of oestrin extracted from pregnancy urine (Emmenin) is relatively inactive in the spayed female, while it is much more active in the presence of an ovary. Several compounds of the androsterone-testosterone series, however, do also cause vaginal cornification in ovariectomized female rats, as shown recently by Deanesly and Parkes 6 and Korenchevsky and coworkers, 7 and it is still to be seen whether this is due to a transformation of these compounds in the female organism into female hormones by tissues other than the ovary, or whether male hormone may stimulate the genital organs of the female directly. It seems of interest in this connection that we were able recently to obtain milk secretion, that is the development of a female characteristic even in the male, both normal and castrate, by the administration of testosterone, the most active compound of the male hormone series, first isolated by Laqueur, et al. from testes, The same hormone also causes milk secretion in normal and ovariectomized females. In all our experiments we used crystalline synthetic testosterone-benzoate.∗

Effect of Combined Administration of Oestrone and Progesterone in Adult Ovariectomized Rats

Previous experiments 1 , 2 , 3 , 4 have shown that the mucification of the vagina which is usually observed in pregnant rats may be elicited during lactation if large doses of oestrin are administered. Since administration of similar doses of oestrin in ovariectomized rats would lead to cornification of the vaginal epithelium, the mucification reaction was ascribed to the simultaneous action of oestrin and corpus luteum hormone. Similarly the epithelial lining of the uterus shows only a moderate degree of progestational proliferation during lactation or pseudo-pregnancy in the rat, while in the second half of pregnancy the proliferation of the endometrium and the growth of the uterus in toto is much more marked and of a different nature. Various reasons which are discussed in more detail in the papers quoted above led us to the conclusion that the endometrial changes during lactation, pseudo-pregnancy, and early pregnancy, are mainly due to the hormone of the corpus luteum, and we called this reaction “the first stage of progestational proliferation.” The “second stage of progestational proliferation” is that observed during late pregnancy, which we believed to be caused by the simultaneous action of corpus luteum hormone and oestrin. We concluded from indirect evidence that oestrin is produced in large quantities during late pregnancy in the rat but not during lactation or early pregnancy. This would explain why vaginal mucification and the second stage of progestational proliferation appears in late pregnancy only and not during early pregnancy or lactation.

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 the Testis on the Mammary Gland

Summary Development and secretion of mammary parenchyma is normally demonstrable in male rats beginning at puberty. Castration prevents the proliferation of the mammary gland in the immature male, just as it does in the female. In adult males, in which the mammary gland is already developed, castration is followed by involution of the gland. Testosterone in doses sufficient to produce mammary gland development does not cause vaginal cornification either in the normal or the ovariectomized immature female rat.∗

Anti-Gonadotropic Substances

Summary From these experiments we conclude that the chronic administration of gonadotropic extracts from the pituitary or from pregnancy urine leads to the formation of substances inhibitory to their action, and that a passive resistance to both these hormones may be produced by the administration of serum obtained from animals chronically injected with these gonadotropic substances.