MM)! I WWII ‘ l 145 390 THS YRS E‘ffifi? BF CEETMN ANTERWR WWARY MPMMQHS 5H MM SEWING?! mm m MERE OF M. BF 5-. MfiflflEGAN STATE BULLEGE ‘NILLZAM LOUIS MEDLEMAN 1941 THESIS LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 ICIProj/AocGPros/ClRC/Dateomjndd THE EFFECT OF CERTAIN ANTERIOR PITUITARX - PREPARATIONS ON MILK SECRETION by William Louie fieuleman A.THESIS Submitted to the Graduate School at Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER or sermon Department of Physiology and.Pharmacology 1941 T HES‘S ACKNOWLEDGEMENTS The writer wishes to express his deepest appreciation to Dr. J. F. Sykes for guidance in the work reported in this paper, and for his constructive criticism in the pre- paration or this manuscript. He also wishes to acknowledge the courtesy of Dr. 0. F. Huffman, of the Dairy Department, for placing at his disposal the experimental animals used in this experiment“ To Mr. G. G. Lightfoot and.Mr. G. W. Duncan 0! the Chemistry Experimental Station, the writer wishes to express appreciation for their cooperation and assistance in carrying out the various chemical analyses, and to the members or the Department of Physiology and Pharmacology, he wishes to express his indebtedness for their assistance and advice given in the course or this work. Itch ‘ ( l n in“ d‘ I 5,30 TABLE OF CONTENTS INTRODUCTION REVIEW OF THE LITERATURE The Fat-Metabolism Hormone EXPERIMENTAL PROCEDURE EXPERIMENTAL RESULTS Results of Assay of the Hormone Preparations Effect of Hormone Preparations on Lactation Results of Blood and Urine Analysis DISCUSSION OF RESULTS CONCLUSION REFERENCES Page 11 14 14 24 853 INTRODUCTION The physiological activities of the mammary gland, as governed by the hormones, may be divided into two phases. The first phase is concerned with the development of the mammary gland from puberty through pregnancy. It has been proved that this phase is primarily controlled by an ovarian-pituitary re- lationship. The second phase of mammary activity is concerned mainly with the secretory processes which occur following parturition. That this part of the cycle is not dependent upon the ovaries is shown by the fact that castration does not terminate lacta- tion. In fact, lactation sometimes follows oophorectomy. From the studies upon the anterior pituitary, it has been found that there are certain hormones which are responsible for initiating and continuing lactation. The presence of a lactogenic hormone, prolactin, has been well established, and therefore no attempt was made to show that this hormone does initiate lactation. This investigation was undertaken in an attempt to deter- mine the effect of prolactin and the fat-metabolism hormone, whether given separately or combined, upon total milk yield and fat production. IREVIEW OF THE LITERATURE The phenomenon of lactation has been known from antiq- uity, but it has only been in recent years that the causes have been investigated. Prior to 1928, lactation was generally accepted as a catabolic process, ensuing after parturition, and.was without an active stimulus. However, since the his- toric work of Grater and Stricker (1929), more and more evidence has accumulated showing that the anterior hypophysis and its secretions play a very important part in lactation. Grater and Stricker found that in mature, normal dairy cows, crude pituitary extracts were capable of initiating lactation. With the knowledge that anterior pituitary extracts would stimulate lactation, many workers sought methods of extracting the lac- togenic fraction of the pituitary. Riddle, Bates and Dykshorn (1932) prepared the first prolactin by iso-electric precipi- tation. From five insoluble fractions prepared, the pH 5 fraction was found to be most potent in the lactogenic prin- ciple. The other insoluble fractions contained varying amounts of the gonadotropic fraction with little or no lactogenic po- tency. In 1935, the same workers improved the original method of preparation so that the follicle-stimulating and thyreo- tropic fractions were eliminated. The amount of inert protein and other contaminants was reduced and the lactogenic potency was considerably increased by these procedures. On the basis of biological tests, it was shown that the lactogenic hormone was a distinct entity which stimulated milk secretion. This preparation was likewise shown to stimulate the growth and secretion of the crop gland of pigeons, and these latter effects 3 are used as a basis for assaying the potency of various lacto- genic preparatione., Evidence concerning the specificity of this hormone was also presented by Lyons and Catchpole (1933) and.Lyons, et a1., (1933, 1937) who called the hormone mammo- tropin. Gardner and Turner (1933) have likewise prepared a lactogenic hormone, galactin, and fully described the mammary changes following its administration. At the present time it is recognized that mammotropin, galactin and prolactin have similar effects. Riddle, Bates and Dykshorn (1932) first prepared pro- lactin by aqueous extraction at pH 2. By repeated iso- electric precipitation in an acid medium, it was believed that the growth hormone would be eliminated. Later (1933), an aqueous alkaline extraction at pH 9 was adopted because it was more complete. Gardner and Turner (1933) followed a similar extraction procedure. Lyons and Catchpole (1933) used 66% acetone extractions. Lyons (1937) has prepared prolactin, free from the adrenocorticotropic hormone, by an aqueous- acetone precipitation. Bates and Riddle (1935) published a method whereby a 60% to 70% ethanol extraction at pH 9 gave still more satisfactory yields. A 90% ethanol precipitation from this extract yielded a precipitate which contained 80% to 90% of the prolactin. The contaminants, mainly thyreotro- pic and gonadotropic hormones, were present in smaller propor- tions; subsequent ice-electric precipitations at pH 3-4 re- moved all detectable traces of the thyreotropic and gonado- tropic fractions. The pH 3-4 insoluble fraction was further concentrated to a unitage of 5-10 units per mg. by repeated precipitations at pH 6 from 70% alkaline ethanol. Extracts containing prolactin have also been made by Young (1938) after a.modification of the Bates and Riddle procedure (1935). Young's procedure is mainly a cold saline extraction at pH 8 and.precipitation at pH 5.5. By using these various extracts, the effect of the anterior pituitary on lactation, as first observed by Grater and Stricker, has been further investiga- ted and substantiated; not only for dairy cows, but also for many other species as well (Corner, 1930; Allan and Wiles, 1932; Asdell, 1932; Catchpole et a1., 1933; Evans, 1933; Gardner and Turner, 1933; Graham, 1934; De Fremary, 1936; Asimov and Krouze, 1937; Folley and Young, 1938; Bergman and Turner, 1940). The effect of the anterior pituitary on lactation has likewise been determined by removal of the gland and by re- placement therapy. In all species, if ablation is effected during the second half of pregnancy, there is only a very brief period in which.1actation occurs following birth or abortion (Selye, et a1., 1933; McPhail, 1935, 1935a; Pencharz and Lyons, 1934; Genes and Turner, 1936). If animals are hy- pophysectomized during lactation, secretion generally stops within a very short time (2 days to one week) (McPhail, 1935; Hill, et a1., 1935; Gomez and Turner, 1937). Administration of crude pituitary extracts to pregnant hypophysectomized an- imals will initiate lactation and maintain it following par- turition (Gomez and Turner, 1936; Lyons, et a1., 1933; Nelson 5 and Gaunt, 1936). However, the partially purified pituitary extract (prolactin, galactin) by itself is incapable of either continuing lactation or initiating milk secretion in bypophys- ectomised animals (Gomez and Turner, 1936, 1936a; Nelson and Gaunt, 1936, 1937; Reece, 1939). In all cases, prolactin is only capable of initiating secretion when given in conjunction with other hormones which may affect lactation in an indirect manner and which are lacking in the hypOphysectomized animal. Most of the experimental data presented have been obtain- ed from studies involving small animals. The usual laboratory animal, however, does not allow any very exact quantitative estimation of milk. Allan and Wiles (1932) and Evans (1933) reported increased secretion as a result of injections of anterior pituitary extracts in goats. Evans, however, noted that virgin goats will not respond to the lactogenic principle. This has been found, by the Russian and English workers, to apply to heifers. In fact, in all species so far examined, prolactin will only stimulate secretion in the mature gland (Asdell, 1931; Catchpole, et a1., 1933; Lyons, et a1., 1933; Nelson and Pfiffner, 1931). Recently the effects of anterior pituitary preparations have been studied on established lactap tion in cattle. The Russian workers headed by Asimov and his associates (1937) produced increased lactation with single and repeated injections of alkaline extracts of the anterior pitu- itary. Their results indicated that the injections were more effective given during early lactation, or more particularily, at the peak of lactation. They also showed that crude extracts were more effective than purified prolactin. 6 Folley and.¥oung likewise studied the effects of anterior pituitary extracts in cattle. It was found that single injec- tions of crude pituitary extracts, as well as partially puri- fied.prolactin preparations, temporarily increased.milk pro- duction (Folley and Ibung, 1937, 1938, 1939). Young's preps, ration (1938) of prolactin, modified from the Bates and Riddle procedure (1935), produced increased secretion upon single in- jections in cattle. He showed that this preparation contained, in addition to prolactin, significant amounts of thyreotropic, ldiabetogenic and glycotropic hormones. When these materials were separated from prolactin, it was found that the purified prolactin was much.1ess active, as judged by secretion, than when it was combined with these other fractions, although an intense crop gland response was obtained. On the other hand, injections of extracts containing thyreotropic, diabetogenic and glycotropic activity showed little crop gland response but stimulated milk secretion to some extent.‘ In view of these results, it seems probable that prolactin may not be'a specif- ic lactogenic hormone and that the lactogenic properties of anterior pituitary preparations may be due to a number of an- terior lobe hormones. These important facts were noted by both the Russian and English workers. They likewise have shown that although milk production is stimulated by the in- jections, the degree of stimulation can only be carried to a certain maximum, as continued treatment will not cause a greater increase. In fact, a decrease in production sets in, which can not be offset despite continued injections. This latter effect may be due to the formation of antihormones which.have been shown to develop during continued injections (Young, 1938). The type of milk secreted, in so far as was determined, indicated that the milk produced under this stimuv lation was essentially normal. Only minor variations in fat and other constituents were noted. At present, there are two general methods of assaying prolactin: the crop gland method and the lactation, or mammary gland method. The latter method of assay (lactation) is rather difficult to carry out and is seldom used, as the usual labora- tory test animals do not allow a quantitative estimation of the milk. Further, the amount of milk secreted depends not only on the prolactin dosage, but also upon the amount and maintenance of mammary tissue. The crop-sac stimulation method may be further subdivided into, (a) the weight method (Riddle, Bates and Dykshorn, 1933), (b) minimum stimulation method (McShan and Turner, 1936) and (0) local stimulation method of Lyons and Page (1935). The weight method of bioassay consists of giving four daily intra~ muscular injections to pigeons 2 to 3 months old. Ninety-eight hours after the first injection the glands are excised, all fat removed and the glands weighed. Because of the large num- ber of factors which must be controlled, the weight method is unsatisfactory for use in a small laboratory where it is not possible to keep large pigeon colonies. Other variables, too numerous to discuss adequately at this point, also affect the results (Bates and Riddle, 1936, 1939; Bates, et a1., 1939). S The minimum stimulation method of McShan and Turner (1936) is more generally used because of its simplicity. Approximately 20 common pigeons, weighing from 260 to 340 grams, are used for one assay. Prolactin, at different dosage levels, is injected once daily for 4 days into the breast muscle. On the fifth day, the crop glands are examined for proliferation. A.pigeon unit is defined as the total amount of hormone in- jected during a period of 4 days which will cause a minimum but definite proliferation of the crop glands of 50% I 11% of 20 common pigeons weighing 300 I 40 grams (McShan and Turner, 1936). The local stimulation method of Lyons and Page (1935) is a very valuable test because of the small amounts of hormone used. The results can be obtained in 48 hours. One injection is made over the crop gland on one side. The inject- ed fluid must cause a blister or raised area on the skin. The examination is made 48 hours later, and only the area directly under the site of injection is stimulated. The Fat-Metabolism Hormone In 1930, Burn and Ling showed that subcutaneous injec- tions of anterior lobe preparations caused an increaseraf the urinary acetone bodies in fasting rats. In the rat, dog, and man, it has also been noted that accompanying the urinary ac- etone increase, the blood acetone bodies also increased (Anselmino and Hoffmann, 1931; Mirsky, 1936; Best and Campbell, 1936). Furthermore, it was noted by Best and Campbell (1936, 1938) that this fat-metabolism hormone also possessed a ”liver fat'' activity. The liver fat activity caused an intense fatty 9 infiltration of the liver in normal, fasting, white rats. The evidence concerning the fate of the blood lipids following injections of anterior pituitary preparations has not been ascertained too definitely as some workers report an increase, while others note a decrease in blood lipids (Evans, 1933; Strauber, 1937; Houchin and Turner, 1939). On the basis of fractionation, Campbell and Keenan (1940) reported that the liver fat activity of anterior pituitary preparations is not necessarily associated with ketogenic activity, liver fat ac- tivity being stable to heat at pH 3.5 while ketogenic activity is stable at pH 8. These preparations are relatively free from other known anterior pituitary hormones. The preparation of the fat-metabolism fraction as made by Best and Campbell (1936) is a modified procedure of the Burn and Ling (1930) and Anselmino and Hoffmann (1931) methods. Very briefly, this procedure is as follows: the acetone—dried pituitary powder is extracted with 20 volumes of N/2O NaOH and precipitated.at pH 5.2. After two such treatments, the fil- trates are combined and the fraction precipitated by adding 2 volumes of absolute alcohol. The precipitate is dissolved in faintly alkaline aqueous solution and brought to neutrality before injection. . Three methods of assay have been proposed for fat- metabolism preparations. Shipley and Long (1938) used male rats weighing from 120 to 150 grams. The rats were fasted for 48 hours previous to the test. At that time, the degree of ketc- nemia was determined. The extract was then given intraperitoneally 10 and a second.blood sample taken 4 or 5 hours later. A posi- tive test was indicated when there was an increase of 4 mg.% in the blood acetone body content. Campbell (1938), in assay- ing the potency of the anterior pituitary extracts which in- crease liver fat, used mice. The mice were fasted before the injection, and 7 hours after the injection, the livers were excised and analyzed for liver fat. The method of Houchin and Turner (1939) assays for liver fat activity as well as fat- metabolism activity. Healthy female guineaspigs, weighing from 160 to 210 grams, are given a single intraperitoneal injection. Six hours later the pigs are sacrificed. The livers are re- moved, freed of blood, adipose and connective tissue and an- alyzed for liver fat. Blood samples are analyzed for plasmar fat. 11 EXPERIMENTAL PROCEDURE The lactogenic hormone, prolactin, and the fat-metabolism hormone were prepared by the methods of Bates and Riddle (1935) and Best and Campbell (1936) respectively. As both preparations have been outlined rather briefly in the preced- ing paragraphs and as the detailed procedures may be found in the references cited, no method of preparation will be given here. The preparation outlined by Best and Campbell for use on rats has been used in this work. The hormone preparations were examined for the lacto- genic and fat-metabolism activity, as well as to determine the presence of other principles. The prolactin was assayed for lactogenic activity according to the method of McShan and Turner (1936). Assays to determine the glyccgropic and diabe- togenic activity of the prolactin preparations were run by the method of Young (1938). Prolactin was also assayed for fat- metabolism activity according to the method of Houchin and Turner (1939), and by determining the liver-fat activity. The plasma-fat, liver-fat and lactogenic activity of the fat- metabolism hormone were also determined. Plasmapfat activity was demonstrated by the method of Houchin and Turner (1939). Liver-fat activity was determined by the procedure of Campbell (1938), but, in this case guinea—pigs were used instead of mice. Lactogenic potency was determined by the methodcof McShan and Turner (1936). The hormones were prepared for injection by solution in weak alkali. The solution was adjusted to pH 7.0-7.5. All 12 pH adjustments were made by a Beckman pH Meter. Solutions were made up as needed and then only a few hours before the injection period during which time they were stored at 6° C. The cows were injected subcutaneously just previous to milking. Care was taken so that all injections were given at approxi- mately the same time of day over the injection period. In a few instances the injections raised a welt at the site of in- jection, but there were no other detectable reactions. The injection period in all instances was five days, during which time one injection per day was given. Four cows in declining lactation were selected for the injections of these hormones. The effect of each hormone was first determined in separate injection periods, and followb ing this the preparations were combined and their effects in this form ascertained. All cows received a ration of alfalfa hay and corn silage, and were fed liberally at a level in ex- cess of the Morrison standards. The animals were milked twice daily. Records on these animals were obtained for periods up to 10 days preceding the first injections and for varying periods following treatment. Daily milk records were obtained and the fat per cent determined on 3 day composite samples. From this data the daily amount of 4% fat-corrected milk was calculated to facilitate comparisons. Blood and urine samples were obtained on the day previous to the initial injection, just before feeding time each day during the injection period and for several days following 15 the injections. Blood sugars were determined by the Somogyi modification (1926) of the Shaffer and Hartman method (1921), plasma-fat by the method of Allen (1930, 1933) and blood ke- tones by the method of Van Alyke (1917). Urinary ketones only were determined on the urine samples. 14 EXPERIMENTAL RESULTS 1. Results of Assay of the Hormone Preparations The assay of prolactin showed this preparation to be a very strong lactogenic stimulant having a potency of 2 units per mg. Glycotropic and diabetogenic assays were negative for this preparation. The prolactin showed no fat-metabolism pro- perties as judged by the liver-fat or plasma-fat activities on guineappigs. The fat-metabolism preparation proved to possess a potent liver-fat activity, while plasma-fat activity was present to a slight degree. This preparation did show very mild lactogenic properties as determined by the crop-sac stim- ulation method. The results of the assays are tabulated in Table I. 2. Effect of Hormone Preparations on Lactation When prolactin was given alone at a level of 500 mg. (1000 units) per day, 3 of the 4 cows showed a rise in milk production. The fourth cow did not show the expected rise. The increase in production was not great in any case. A maximum increase of 2.5 pounds was observed in #D5 while #A23 and #285 showed increases of 2.0 and 2.5 pounds, res- pectively. The increase in production was observed, in all cases where such increases occurred,oon the second day of in- jection, but did not reach the maximum until about the third day of injection. In 2 cows (#A23 and #285) the effect of the hormone did not disappear even when the injections.were stopped. Number A23 returned to approximately the pre- injeotion level of production in about 30 days, and #285 was 15 #4 Ah“ fl. 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