Prolactin Preparations Research Articles

Lipemia-producing activity of pituitary gland: separation of lipemia-producing component from other pituitary hormones.

SummaryA method for fractionation of hog pituitary glands by saline extraction, fractional precipitation with acetone at pH 4.3, and ion-exchange chromatography, is described. The final fraction has high lipemia-producing activity in the rabbit and is free of recognized pituitary hormones. Lipemia-producing activity of this fraction is enhanced by simultaneous injection of ACTH. Production of lipemia in the rabbit by injection of ACTH together with commercial preparations of TSH, prolactin or FSH is also described. In the combination ACTH + commercial TSH, evidence is presented that ACTH acts in synergism with some “contaminating” substance in commercial TSH preparation, rather than with TSH itself.

The response of rabbit mammary glands to locally administered prolactin.

SUMMARY The response of rabbit mammary glands to intraductally-injected prolactin preparations has been investigated with a view to developing an assay procedure for prolactin, based on milk formation in mammals. Milk was clearly detectable in the glands by the 3rd day after injection of prolactin, and there was evidence of an increase in response with increased dose, but the variability so far encountered has been high. Intraductal injection of pituitary extracts rich in activities other than that of prolactin did not lead to milk formation.

DIABETOGENIC ACTION OF PROLACTIN

IT HAS been difficult to establish whether the effects on carbohydrate metabolism of different preparations of prolactin are due to the hori mone itself or to other pituitary hormones which may be present in small amounts in these preparations. Thus, the clearly marked anti-insulin effect of impure pituitary preparations (Young, 1936; Marks, 1936) is reduced so as to be infinitesimal in purified preparations (Young, 1948, b, c). Riddle and his associates, in several papers published between 1937 and 1947, reported that prolactin had hyperglycemic and diabetogenic activity; later they (Riddle et al., 1947) found that prolactin preparations which were free of adrenocorticotropin (ACTH) did not modify the blood sugar of the fasting pigeon, and the slight increase in blood sugar observed in animals which were fed seemed to be due to a greater ingestion of food. Hepatic glycogen, however, was found to be increased by prolactin in normal pigeons, and even more so in hypophysectomized animals.

Localization of radioactivity after administration of labeled prolactin preparations to the female rat.

PREVIOUS reports have indicated that anterior pituitary hormone preparations can be labeled with radioactive isotopes (Sonenberg, Keston and Money, 1950 and 1951). In these earlier studies, hormone preparations with adrenocorticotropic activity were labeled with traces of radioactive iodine, I131. Preparations labeled in this manner were then used to investigate the localization of radioactivity after being administered to rats. The technique of trace labeling proteins has been extended to other preparations containing pituitary hormones. This report presents the results of studies with prolactin preparations labeled with radioactive iodine.

Lack of effect of lactogenic hormone on mammary adenocarcinoma in mice.

Many clinical observations have indicated that the growth of mammary cancer in women is accelerated by pregnancy and lactation (1,2). The effect of lactation alone is not known, although Geschickter (3) believes that it does not influence the growth rate of in filtrating cancer of the breast. Pregnancy stimulation is usually ascribed to the high estrogen titre, a situation which does not apply during lactation(3). In mice, estrogens exert a carcinogenic effect on the mammary gland (4), but the estrogen preponderance of pregnancy apparently does not accelerate the tumor growth(5). The influence of lactation, with its high lactogenic titre, has not been established, although it has been shown that a crude prolactin preparation did not modify the growth rate of these tumors (6). With the recent availability of homogeneous pituitary lactogenic hormone, it was thought worth while to test this substance for its possible effect upon the growth of mammary adenocarcinoma in mice. Procedure. Virgin female mice of the C3H strain, 5 to 6 months old, were used. To obviate any ovarian influence half the group was oophorectomized. After a 7 week recovery period, uniform fragments of a transplantable C3H strain mammary adenocarcinoma were implanted by trocar in the dorsal subcutaneous tissues. Eight days later subcutaneous injections were begun. Half of each group, castrated and intact, received two 0.1 cc injections daily, each containing 1 mg of purified lactogenic hormone assaying 30 I.U. per mg(7). The control groups received equal volumes of normal saline. Periodic recordings were made of body weights and tumor diameters. Daily vaginal smears of the hormone-treated mice showed anestrous periods of 12 to 15 days occurred regularly throughout the experiment, thus demonstrating the continued potency of the hormone.

PROLACTIN ASSAY BY A COMPARISON OF THE TWO CROP-SACS OF THE SAME PIGEON AFTER LOCALINJECTION11,

RECENTLY, data were presented (Hall, 1944) suggesting that consistent assay results, within the limits of error desired, were not obtained when prolactin preparations were compared with the international standard using the systemic crop-sac weight method and groups of 10 to 12 pigeons. Bates and Riddle (1941) had a similar experience with this assay method in comparing their preparation No.657 with the Standard. This completely objective method was the best available, and improvements in the manner of comparing prolactin preparations by it have been suggested (Hall, 1944). In the hope of 4 obtaining more accuracy in prolactin assay, the ‘micro’ methods originating with Lyons and Page (1935), have also been investigated. The present work adds nothing to the original ideas on which the method was based. The inherent possibilities in this unique method have, perhaps, been more fully explored.

ABILITY OF ADRENAL CORTICAL HORMONES, PROLACTIN AND THYROXIN TO SUSTAIN WEIGHT OF BODY AND VISCERA OF HYPOPHYSECTOMIZED PIGEONS

AEARLIER study by Schooley, Riddle and Bates (1941) showed that during the 10 days following the removal of the anterior pituitary gland of pigeons there is a marked loss in body weight (20 per cent) and a notably greater loss in the weight of the intestine (43 per cent) and pancreas (54 per cent). These weight losses, except that in the pancreatic tissue, were prevented and even ovepcompensated by moderate daily dosage with highly purified prolactin. Maintenance of the pancreatic weight seemed to require thyrotropin in addition to prolactin. Most of the preparations of prolactin used in that study had no detectable effect in maintaining the weight of the thyroids, gonads or adrenals; three of the preparations used,apparently as effective as others, had been heated to 6o°C. for 5 hours to in activate any ‘growth’ hormone.

Preparation of Pituitary Adrenotropic Hormone.

This is a preliminary description of a relatively simple procedure devised for the isolation of purified and highly active preparations of adrenotropic hormone from whole hog pituitary glands. The extraction and fractionation procedures consist of the following steps: (1) Extraction and isolation of a fraction designated as crude prolactin by the procedure originally developed by Lyons1 and adopted by White, Bonsnes and Long2 as the first step for the subsequent preparation of crystalline prolactin. This “crude prolactin” contains adrenotropic hormone activity. (2) Solution of the crude prolactin preparation in 2% concentration at pH 9.0 and removal of precipitates which form by careful adjustment of pH to 8.0, 6.6 and 5.4. (3) Supernatant obtained in Step 2, after removal of pH 5.4 insoluble material (prolactin), is freed from traces of prolactin by the addition of 7 ml of saturated (NH4)2SO4 to each 100 ml of solution. The precipitate is separated and discarded. (4) Acetone is added to the supernatant t...

Excessive Uterine Bleeding: Antigonad Effect of Prolactin

In june, 1937, the writer began clinical investigation of the effects of prolactin when administered during phases of excessive uterine bleeding. Thirty-five cases were reported in 1938 (1). During the clinical investigation of the effects of the lactogenic hormone on phases of excessive uterine bleeding, several preparations of prolactin have been used.1 Information has been received from Lyons (2) that none of these has been pure lactogenic hormone. The prolactin used in the current series however, is sufficiently pure and potent to be used effectively for controlling excessive uterine bleeding, and the following report seems to justify its wider usage for this purpose. In every case in which there was no pathological condition such as fibromyomata, intrauterine papillomata, or persistent thick-walled cysts, the lactogenic hormone therapy was satisfactory. Cessation of excessive bleeding in cases of fibromyomata has been obtained in variable percentages in each series of patients.

SEROLOGICAL PROTECTION AGAINST THE DIABETOGENIC SUBSTANCE OF THE ANTERIOR PITUITARY GLAND

A number of investigators have observed that, when dogs are treated with diabetogenic anterior pituitary extract, the symptoms of diabetes may make only a transitory appearance despite the daily administration of very large amounts of extract. Young [1937] found that the diabetic condition could be induced to reappear when the amount of extract injected daily was suitably increased, and did not believe that the refractory state resulted from the appearance in the blood of antihormones. However, Dohan & Lukens [1939] found that when a dog had been treated with daily injections of crude anterior pituitary extract for over 9 months, its serum possessed anti-diabetogenic activity. Administration of such serum to completely depancreatized dogs induced a marked decrease in the intensity of glycosuria. Young [1938], on the other hand, using preparations of prolactin with marked glycotropic (anti-insulin) activity, was able to produce sera with marked anti-prolactin action but with no detectable

CHANGES IN THE FAT PERCENTAGE AND FAT YIELD OF DAIRY COWS WITH INJECTIONS OF AN ANTERIOR PITUITARY PREPARATION1

SEVERAL INVESTIGATORS have shown that certain extracts from the anterior pituitary have marked effects on the fat metabolism of various laboratory animals 1–5). The present report concerns the effect of anterior pituitary extracts on the fat production of dairy cattle. The anterior pituitary extract used in this study was the alkaline extract described by Best and Campbell (3) and was prepared in this laboratory according to the method outlined in that report. No quantitative assay was made on the preparation. Tests were made, however, to demonstrate the liver-fat activity of the extracted material. The preparation was injected for 5 day-periods in 500 mg. amounts. For purposes of comparison a prolactin preparation prepared in this laboratory by the method of Bates and Riddle (6) was given in 500 mg. amounts to the same 4 cows during another 5-day period.

Mammary Growth in Hypophysectomized Male Mice Receiving Estrogen and Prolactin.

SummaryThe mammary rudiments grew in hypophysectomized male mice receiving either of 2 purified preparations of prolactin and estrogen. The injection of these preparations of prolactin alone was not followed by mammary growth.

ANNUAL VARIATION IN THE RESPONSE OF CROP-SACS AND VISCERA OF PIGEONS TO PROLACTIN

SINCE OUR FIRST PUBLICATION (I) ON THE ASSAY of prolactin by means of the weight of the crop-sacs of doves and pigeons we have studied various factors (2) which modify the extent to which a specific quantity of prolactin increases the weight of the cropsacs. The present study was undertaken to determine the re producibility throughout the year of the crop-sac weights obtained at any one time or season. A constant amount of the same preparation of prolactin was used in all tests, and groups of 10 birds were tested at intervals of 4 to 6 weeks during two years. During the first year of study large and unanticipated variations in crop-sac weights were observed at certain periods, and it was therefore decided to continue the study throughout a second year in order to verify the findings of the first year.

A Study of Hormonal Factors Which Influence the Production of Insulin

1. Insulinotropic effects were obtained with a protein fraction of the anterior pituitary, with estradiol and with stilbestrol. 2. Varying diabetogenic effects were obtained with highly purified prolactin preparations, with progesterone, with testosterone and with a fraction, probably also protein, from the anterior pituitary.

LACTATION

Summary1. In most species oestrogens mainly stimulate mammary duct growth. In the guinea‐pig and goat they promote complete alveolar development. The mouse is the only animal in which treatment with progesterone has been found to promote mammary duct growth. In other species simultaneous treatment with oestrogens and progesterone is necessary for complete alveolar development. It is difficult to say whether or not oestrogens per se can promote alveolar development, because the adrenal cortex produces progesterone.2. Androgens cause duct and alveolar growth in some species.3. Some now believe that, under the influence of oestrogen, the anterior pituitary produces a “mammogenic” hormone which promotes mammary duct growth.4. The presence of the anterior pituitary is essential for the initiation and maintenance of lactation. The anterior lobe is believed to secrete a specific lactogenic hormone (prolactin), but other anterior lobe hormones probably participate in the control of normal lactation.5. Oestrogens may inhibit lactation during pregnancy, but it is probable that the pregnant uterus is also concerned in the inhibition.6. The adrenal‐cortical hormone is necessary for lactation, since adrenal‐ectomized animals do not lactate, and, further, prolactin will not initiate or maintain lactation in hypophysectomized animals unless corticosterone or adrenotrophic hormone is also given.7. The thyroid gland is essential for normal lactation: thyroxine stimulates nilk secretion in the cow.8. Posterior pituitary extracts cause emptying of the bovine udder but do not affect the formation of milk.9. Oestrogen treatment enriches milk in fatty and non‐fatty solids, and so does thyroxine treatment. Repeated injections of certain prolactin extracts increase the fat content of Cow's milk; certain other prolactin preparations increase the lactose content.10. No true secretory nerves innervate the mammary gland. Lactation is disturbed in the cat if the sympathetic chains are extirpated.11. The extraction of milk from the bovine udder depends on the reflex erection of udder tissues caused by stimulation of the teat, which also appears to cause the release of a lactogenic hormone or hormones from the hypophysis.12. The mammary gland synthesizes lactose from blood glucose and also, probably, lactic acid. It is also possible that blood amino acids are de‐aminated by the mammary gland and used for lactose synthesis.13. The precursor of milk fat is the neutral fat of the blood. The mammary gland probably oxidizes long‐chained fatty acids to the short‐chained acids of milk fat.14. It is improbable that milk protein arises from blood amino acids. Plasma proteins are probably utilized for the synthesis of caseinogen, the phosphorus coming from the blood inorganic phosphate.15. Various enzymes have been detected in active mammary tissue, which is a particularly rich source of the alkaline phosphomonoesterase. Changes in the secretory function of the mammary gland are usually accompanied by striking changes in the phosphomonoesterase content of milk.

FURTHER EXPERIMENTS ON THE CONTINUED TREATMENT OF LACTATING COWS WITH ANTERIOR PITUITARY EXTRACTS

Earlier experiments have shown that when repeated injections of a prolactin preparation are given to cows in declining lactation, a substantial stimulation of milk production occurs, though later this stimulus declines and finally vanishes, despite continued treatment [Folley & Young, 1939]. Further experiments have now been carried out to determine if the initial response would disappear with continued treatment with other pituitary preparations, and if the development of such resistance to the action of the extract was associated with the appearance, in the blood of the treated cows, of anti-prolactin [Young, 1938 c; Bischoff & Lyons, 1939] activity. Folley & Young [1938] found that the stimulating influence on milk production of cows in declining lactation of a single injection of various anterior pituitary fractions was more closely correlated with their glycotropic (anti-insulin) activities than with their prolactin1 content. It therefore seemed desirable to investigate the influence on milk production

IMMUNOLOGIC INVESTIGATION OF HYPOPHYSEAL MAMMOTROPIC PREPARATIONS

THE ONLY EXPERIMENTS dealing with antisubstances to the lactogenic or mammotropic hormone that we are aware of are those of Kabak and Stulova (I) who induced a refractory state to prolactin in chronically injected pigeons, and Young (2) and Strangeways (3) who produced antisera against preparations of prolactin that still contained blood proteins. In our investigation we were interested in determining a), whether our mammotropic preparations were antigenic, b), whether they were species' specific antigens, c), whether the antibodies formed against them were cap' able of inhibiting their physiological activity, and d), whether their antihor'monal properties were species'specific.The beef and sheep mammotropic preparations used were made according to methods previously published (4). They stimulated pigeon crop growth in a total dose of 0.1 mg. when given subcutaneously for 4 days (macro'test), and caused milk secretion in post'estrous virgin guinea pigs in a total dose of 1.0 mg.

The production of antisera to preparations of prolactin containing the glycotropic (anti-insulin) factor of the anterior pituitary gland

The production of antisera to preparations of prolactin containing the glycotropic (anti-insulin) factor of the anterior pituitary gland

METHODS FOR THE ASSAY OF PROLACTIN

Prolactin is a hormone produced in the anterior lobe of the pituitary, probably by the eosinophilic cells. It was early found to stimulate lactation in the mammary gland and proliferation of the mucosa of the crop-sacs of doves and pigeons. Purification studies have shown it to be protein-like in chemical and physical properties. It is found in the fraction which is insoluble in water and quite soluble in 60–70 p.c. ethanol or acetone at pH 4.5–5.5, but is precipitated by ethanol of 85–90 p.c. concentration. Six additional physiological responses to our most potent preparations of prolactin have been obtained and studied in our laboratory; these are discussed by Riddle in a later paper in this volume. Some of these other responses may be due to some contaminating hormone, and since none of them seems promising for quantitative assay, we will confine our descriptions to mammary gland and crop-sac methods.

On the Protein Nature of Prolactin and of Follicle-Stimulating Hormones

SummaryProlactin is a protein-like substance which at pH 8.0 is almost completely destroyed by trypsin in 2 hours at 37°C. In confirmation of previous work though using the bird testis instead of the rodent ovary to test potency, it is found that the follicle-stimulating hormone obtained from the anterior pituitaries of cattle is very rapidly destroyed by trypsin under the above-named conditions.The methods used in the separation and purification of prolactin suggest that this hormone is a protein, but since it has not yet been obtained in a pure state the possibility of its adsorption on inert protein is not thus excluded. It is therefore desirable to learn whether prolactin is affected by tryptic digestion.That prolactin is rapidly destroyed by trypsin is shown by the data of Table I. Almost complete destruction in 2 hours by a purified trypsin is obtained. The prolactin preparation used in this test was relatively pure and highly potent. Both the control and digest were, at the beginning of the test, a...