145 904 EFFECTS 0F HYDROCORTlSONE ACEYATE, OXYTOCEN AND PROLACTEN ON LACTAT‘EONAL PERFORMANCE OF RATS AFTER PARTURITWN Thesis hr #219 Daemo- m" M. S. MICHPGAN STATE UNWERSETY P'gmachandra Keshavrac Taiwaéker 3959' EFFECTS OF HDROCORTISONE ACETATE, OXITOCIN AND PROLACTIN 0N LACTATIONAL PERFORMANCE OF RATS.AFTER.PARTURITION BY PURNACHANDRA KESHAVRAO TALWALKER AN ABfl'BACT Submitted to the College of Science and Arts, Michigan State University of Agriculture and Applied Science, in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology & Pharmacology 1959 1. ABSTRACT In experiment ‘I, the effects of administmtion'cf hydrocorti-sone acetate;-~prolactin and ontocin, singular-1y and in~combination-,~ on lactational performance of rats were studied. Ninety mature female rats of the‘Carworth strain were bred and on the 4th day‘postpartum-the litters were reduced to six young each. The rats were: divided. into nine groups, . each group containing ten rate. ,All in- . Jections were given subcutaneously, in 0.1 ml saline, once daily, except oxytocin which was given twice daily. The injections were given from the 4th davr to 17th day postpartum On the 18th day the dams were killed and the pituitary, adrenals and ovaries were removed, cleaned and weighed. Each group received the following treatment: a. Controls, saline 0.85%. b. maroccrtisone acetate 0.25 mg. c. marocortisone acetate 0.5 mg. d. I-Udrocortisone acetate 1.0 mg. e. Oxytocin l‘ LU. ' f. Prolactin 1, mg. g. Hydrocortisone acetate 0.25 mg and prolactin 1 mg. and oxytocin l LU. 11 PURNACHANDRA KESHAVBAO TALWALKER ABSTRACT 2. h. Hydrocortisone acetate 0.5 mg. and prolactin 1 mg. and oxytocin l I.U. i. Hydrocortisone acetate 1 mg. and prolactin 1 mg. and oxytocin l I.U. When injected individually into lactating rats, hydrocortisone acetate, oxytocin and prolactin, only hydrocortisone acetate at the 0.5 mg. dose level increased milk secretion significantly, whereas oxytocin and prolactin had no effect. When all hormones were injected in combination, increases in lactation were no greater than with hydrocortiscne acetate alone. (Hydroccrtisone acetate was effective in increasing milk secretion probably because during lactation the adrenal cortex secretes less than Optimal amounts of glucocorticoids and hence small amounts of additional glucocorticoid have beneficial effects in increasing milk secretion. Oxytocin and prolactin were not effective in increasing milk secretion. During lactation these two hormones are secreted in optimum amounts and hence additional amounts have no effect.) 111 PURNACHANDRA KESHAVRAO TALKALKER ABSTRACT 5. 4. Dams receiving saline, oxytocin and prolactin alone each gained in average body weight by approximately 20-25 gms., while those receiving 0.25 or 0.5 mg. hydrocortisone acetate gained in average body weight by only 7-10 gms. Dams injected with 1.0 mg. hydro- cortisone acetate lost about 8 gms. in average body weight during the injection period. No significant difference was observed in the average weight of the pituitaries between the different groups. However, the average weights of the adrenals and ovaries were significantly lower than controls in the groups which received 0.5 and 1.0 mg. hydro- cortisone acetate. 5. (in experiment II, the effect of prolactin on lactational performance of cephorectomieed rats was studied. Twenty mature female rats of the Garworth strain were bred. 0n the 2nd daypostpartum both.the ovaries were removed while the rats were under light ether anesthesiaJ They were divided into two groups of ten rats each.f Litters were reduced to six young each on the 6th day postpartum. Group I iv PURNACHANDRA KESHAVRAO TALWALKER ABSTRACT received 0.85% saline and group II received.prolactin) (1 mg. daily) during the 4-17 day-period of lactation. 0n the 18th day, the dams were killed and the adrenals, ovaries and pituitaries were removed, cleaned and weight. Dams and litters were weighed daily. 6. (No significant difference was observed in average litter weight gain during the 4-18 day-period between the oOphorectomized and intact groups. This shows! that sufficient prolactin is secreted after parturition to luteinize the ovaries (lutsotrOpic action) and to maintain optimal lactation. This also shows that the functional ovaries of the rat after parturition do not inhibit milk production.) EFFECTS or Hrnaocon'nsons ACETATE, omocm AND Pnomcrm 0N LACTATIONAL PERFOMNCE or RATS AFTER PARTURITION BY mmmcwmm xssmvnm TALWALKER A THESIS psubmitted to the College of Science and Arts, Michigan State University of Agriculture and Applied Science,in partial fulfillment of the requirements for the degree of MABTER.0F SCIENCE Department of Physiology a Pharmacology 1959 ACKNO WLEDGEMEN T8 The author feels highly grateful to Dr. J. Meites, Professor, Department of Physiology and Pharmacology, for his kind help and guidance throughout the whole course of this study. His inspiration and encouragement in finishing this project are greatly appreciated. The writer also wishes to express his appreciation to'Dr. B. V. Alfredson, Head of the Department of Physiology and Pharmacology, for providing an Opportunity to carry out postgraduate studies at this University. Special thanks are due to Mr. C. S. Nicoll for assisting during the course of the experiments. Many thanks are also due to Dr. E. P..Reineke, Dr. L. F. Woltsrink, Dr. R. F. Johnson, Dr. C. P. Cairy, Dr. J. E. Nellor and Mr. G. C. Gerritsen, all of the Department of Physiology and Pharmacology, and Dr. C. A. Hoppert, Department of Chemistry, whose knowledge and experience we of real value to the author. The author also wishes to thank Mr. Merlyn Swab for the help rendered in care of experimental animals. I am also indirectly indebted to the following for gifts of hormones supplied to Dr. J. Meites: prolactin, (20 I.U./mg.), Endocrinology Study Section, NIH; oxytocin, Dr. D. McGinty, Parke Davis and 00., Detroit, Michigan; vii hydrocortisone acetate, Dr. L. Michaud, Merck-Sharpe and Dohme, Hailey, N:_J., This work was supported in part by a NIH‘grant to Dr.- JrMeites‘ and by Michigan Agricultural Experiment Station project 105. viii II III IV TABLE OF CONTENTS INTRODUCTION. . . . . . . . . . . . REVIEW OF LITERATURE. . . . . . . . l. Endocrine control of postpartum lactation in hypOphysectomized animals . . .'. . . . . . . . . 2. Neural control of anterior and posterior pituitary secretions during lactation. .-. .'. . . . 5. Endocrine control of postpartum lactation in adrenalectomized animals . . . . . . . . . . . . 4. GalactOpoietic studies in laboratory and farm animals . . MATERIALS AND METHODS ‘. . . . . . . EXPERIMENTAL RESUDTS 1. Effects of hydrocortisone acetate, oxytocin and prolactin on lactation inratseeeeeeeeeeee 2. Effects of prolactin on lactation in cephcrectomized rats . . . . DISCUSSION. . . . . . . . . . . . . SUMMARY AND CONCLUSIONS . . . . . . BIBLIOGRAPHI............ ix Page 13 16 25 31 45 47 Table l. 2. 3. . 4. 5. 6. Figure l. 2. LIST OF TABLES Title Page Effects of hydrocortisone acetate,' . prolactin and oxytocin on body, adrenal, ovarian and pituitary weight of lace Eating rats 0 e e e e e e e e e e e e e e e 32 Effects of hydrocortisone acetate, prolactin and oxytocin in combination on body, adrenal, ovarian and pituitary weight or lactating rats. 0 e e e e e e e e 35 Effects of hydrocortisone acetate, prolactin and oxytocin on body weights or litters. e e e e e e e e e e e e e e e e 34 Effects of hydrocortisone-acetate, prolactin and oxytocin in combination on body weights of litters. . . . . . . . . 55 Effect of prolactin on body weights of litters of cephorectomized rats . . . . . . 56 Effect of prolactin on body, adrenal, ovarian and pituitary weight of lac- tating rats 0 e e 0.0 e e e e e e e e e e e 37 LIST OF FIGURES Title Page Graph showing the effects of hydro- cortisone acetate on litter weight gains in the rat. . . . . . . . . . . . . . 58 Graph showing the effects of hydro- cortisone acetate, prolactin and oxytocin in combination on litter weight galnSln the rate 0 e e e e e e e e e 39 INTRODUCTION It is well known that completion of mammary growth alone does not initiate lactation; thus, factors other than those necessary for mammary growth must be present to initiate and later, to maintain lactation. Although this subject is still not free from controversy, there is sufficient evidence to show that several endocrine factors are essential for the maintenance of established lactation. Dolley and.Young (1940) and Bergman and.Turner (1940) independently suggested the term “galactOpoiesis' to designate the stimulation of established lactation, and thus they drew a distinction between the initiation of lactation (lactogenesis) and its maintenance, once established. ‘ It is very well known that lactation in most mammals rapidly reaches a peak and subsequently declines over a /, long period of time. Various studies have been carried out in order to determine the factors responsible for the decline in lactation. These studies include: 1) Administration of hormones during the decline to stimulate milk production in farm and laboratory animals. 2) Removal of an endocrine gland or glands and replacement therapy to maintain normal lactation. 3) Maintenance of mammary structure and functional integrity by hormonal administration after removal of milking stimulus. l 4) Biochemical 'studies ,. 5) .Nutritional studies. The removal of the hypOphysis in any stage of lacta- tion in laboratory animals has been shown to cause a rapid and abrupt cessation of lactation (Gomez and Turner, 1956a; Hill gt 51., 1955). However, it was shown that lactation could be maintained in hypOphysecto- mixed animals by administration of crude hypOphyseal extracts (Gomez and Turner,l956b; Hbussay, 1935). ‘These earlier studies showed the importance of the role of the pituitary in maintaining lactation. waever, it is not still precisely known how many hormones of the pituitary are involved in normal maintenance of lactation. Variations in kind and amount of hormonal requirements have been found to vary in different species. The importance of the adrenal cortical hormones for the maintenance of lactation is very well established. Adrenalectamy during lactation results in cessation of lactation, but replacement with.adrenal cortical hormones can maintain lactation.partially or completely. waever, the precise role of the various adrenal cortical hormones in maintaining lactation is not known. It is very well established that the maintenance of the lactating“mammary gland in a state of functional integrity is dependent upon_the”continued application of suckling or milking stimulus (Selye, 1959; Bruce; 1958;-Nicolland Meites, 1959). There is evidence that the suckling w’ stimulus during lactaticn~causes the release'of oxytccin (Petersen gt al.,-1942; Cross-and Harris, 1950, 1951), prolactin (Reece and.Turncr, 1957; Meites and Turner, . 1948) and ACTH (Tabachnick and'Trentin, 1951; Gregoire, 1946) from the pituitary. It has been reported that injections of prolactin (Williams, 1945; Johnson, 195?) or oxytocin (Johnson, 1957; Benson and Dolley, 1956) to lactating mother rats after removal of their litters inhibit involutionary changes in the mammary glands. It // has been shown that cortisone administration can signifi- cantly retard mammary involution in rats after litter removal, and can maintain milk secretion at a higher level during the declining phase (Johnson and.Meites, 1958). Meites and Nicoll (1959) have shown that injections of hydrocortisone acetate, oxytocin and.prolactin, in combination, can retard mammary involution as long as 70" 75 days after litter removal on the 4th.day postpartum. (These studies suggested that secretion of oxytocin, prolactin and ACTH or dorticoids may be limiting factors in the lactational performance of rats after parturition. Therefore it was of interest to know whether these three*/ hormones administered singularly or in combination could increase normal milk production in the rat. In the rat, prolactin has been shown to have luteo~ trepic activity. Lyons (1958b) suggested that prolactin injected into rats bearing corpora leutea of lactation, might be utilized by the corpora lutea-tc promote the // secretion of progesterone, thus stimulating-mammary growth and antagonizing lactation. Therefore it was of interest to determine the effects of ovariectomy and injections of prolactin into ovariectomized rats on milk yields. I REVIEW OF LITERKTURE Reviewing the literature is a very informative and interesting experience. One gains a feeling of gratitude towards those pioneers in the field of the physiology of milk secretion who were so instrumental in laying the ground work for a better understanding of such.a complex process as milk secretion on the basis of keen observation, curiosity and.painstaking effort. From this background comes much of the knowledge we presently have of this subject. The literature on lactation is voluminous. This ‘well indicates the importance of this subject, not only from a commercial point of view but also from the point of view of acquiring basic understanding of the process of milk secretion. In recent years great advances have been made, and much is known about the general and some specific aspects. However, many of the fundamental processes still remain to be elucidated. l. Endocrine Control of Postpartum Lactation lig_§ipgphysectomiged Animals. The earliest reports on the maintenance of lactation after hypOphysectomy are those of Gomez (1939, 1940) who found that rats hypOphysectomized during lactation could rear their litters, if given anterior pituitary extract, adrenal_cortical extract, glucose and posterior pituitary extract. He used posterior pituitary extract at a time when the role of posterior pituitary in lactation was not fully understood. Recently, experiments have been carried out in hypo- physectomized rats to determine which.hormones of the pituitary are necessary for maintaining lactation adequate to support average-sized litters. It is now commonly accepted that hypOphysectomized test animals should be used in lactational studies in order to eliminate possible disturbing factors due to the action of endogenous pituitary hormones. Nelson 33 5;. (1945) showed that lactation could not be induced in the hypophysectomized guinea pig with.laoto- genie hormone and deoxycorticosterone. They also reported that they could induce and.partially maintain lactation in hypOphyseotomized guinea pigswith.laotogenic hormone and l7-hydroxy-ll-dehydrocortioosterone. Gowie (1957) showed that slight maintenance of milk secretion, in rats hypOphysectomized on the 4th.day of lactation, could be obtained by administration of crude / anteriorvpituitary extracts or prolactin alone (25 I.U. twice daily). Slightly higher milk yield was obtained with .0. a larger dose of prolactin (so-LU. twice daily). Neither GH nor ACTH when-administered alone had any replacement value, although a slight replacement‘was obtained whengthey were administered together. In'no case was complete main- tenance of lactation achieved, although yields of almost ml: the normal were obtained with prolactin and ACTH, or prolactin'and GH. Bintarningsih 33 9A. (1957) were able to obtain considerable maintenance ofmilk secretion in the hypo- physeotomized rat with prolactin and cortisol acetate or. / prednisclcne acetate. Lyons gt _a_l_. (1968) lupOphysectomized rats on the 12th day of pregnancy. In these animals en. mammary gland develOped in response to placental and ovarian hormones. Normally such animals after parturition secrete a small amount of milk for a day before regressing. They found that they could maintain lactation by injecting prolactin and prednisclcne acetate, and assumed that the endogenous secretion of cxytocin continued because the milk ejection reflex was found to be regenerated within ten days after hypOphysectcmy. They showed that OH or thyroxine administrations had no effect in enhancing lactation; the milk yield, however, of the rats injected with prolactin and either ACTH or cortisol was only about 50% of normal. The hypopnvsectcmized mother rats showed gains in body weight when GH and thyroxine were given. But those rate ‘which received prolactin plus cortisone lost considerable body weight. Thus in hypOphysectomised rats prolactin, ACTH and possibly GH appear to be components of a pituitary / complex concerned in the maintenance of milk secretion. 2. Neural Control of.Anterior and Posterior Pituitary Becretignc during Lactation. Much.of the milk present in the mammary gland just before suckling or milking is in the alveoli and ducts from which it can be removed by the-squeezing action of a contractile effector tissue, the myoepithelium, which.torms a network surrounding each alveclus.' This is a reflex response to the stimulation of-teat or other stimuli, often psychic, to which the animal has been conditioned. .Thus the active but unconscious participation of the nursing mother is essential if the milk is to be made available to the young. Once it was believed that this milk-ejection reflex consisted of a purely neural arc (Harris, 1958). Gains (1915) reported that pups’suckling an anes- thetized bitch usually obtained no milk. Thus anesthesia inhibited the milk-ejection reflex. However this inhibition . was overcome by the injection of posterior pituitary extract. Turner and Slaughter (1930) suggested that milkvejecticn reflex might act through the posterior pituitary gland. Ely and Petersen (1941) showed that milk-ejection could occur in response to the normal milking stimulus in one half of the'bovine udder in which.two nerves, believed to carry all the efferent fibers to the gland, had been cut. 10 Petersen “and 'Ludwick' (1942) showed that blood collected from cows after stimulation produced ejection of milk when perfused through the isolated cow udder. These studies indicated that this reflex consists of'a neurohormonal are. This involves the'rel-ease-of a hormone, probably oxytocin from the posterior pituitary which causes contraction, of the myoepithelium surrounding the alveoli and thus squeezing out the milk (Harris, 1958). There is now-considerable evidence that the suckling stimulus during lactation also causes releasepof prolactin (Reece and Turner, 1957; Meites and Turner, l9“) and _ ACTH (Tobachnick and Trentin, 1951; Gregoire, 1946) from the anterior pituitary, which are essential for the Iaintenance of normal lactation. Thus the suckling stimu- lus appears to play a central role in the two main phases of lactation -- milk secretion and milk ejection. '. Harris, (1955) suggested that there is considerable evidence in favor of the view that anterior pituitary secretions are stinulat ed by the liberation from the hypothalamus of ' some Immoral substance (s) into the primary plexus of the hypophyual portal system. It is believed that this substance is then carried in the portal circulation to the anterior lobe. Various suggestions have been made regarding the nature of the humoral substance (s). ll Benson and Folley"(l956, 1957) suggested that oxytocin ‘/ might be a humeral link for the release of prolactin or the whole galactopoietic complex from-the anteriorvpituitary. The basis for such.apeculation was mainly the ability of oxytccin to retard mammary gland involution in the rat and galactOpoietic effect Observed with oxytocin in ruminants . by many other workers (Adams and.a11en, 1952; Sprain gt 51., 1954;.Dcnker gt 31.,1954) and in the rat by Johnson (1958). However, oxytocin injections have_not been shown to alter pituitary prolactin content in rats (Meites and Turner, 1948; Johnson and Heites,,1957), guinea pigs or rabbits (neitcc and Turner, 1948). Grosvenor and Turner (1958) /, reported that injection of oxytocin in anesthetized I lactating rats failed to alter pituitary prolactin content from the control prenursing level. Donovan and Vendor Verff ten Bosch (1957) reported that milk secretion could be maintained in rabbits in which the hypOphyseal portal system was destroyed and which received only oxytocin. These studies are in contrast to the views held by Benson and Folley that oxytocin is the humoral link in anterior pituitary secretions. ' Heites (1959) and Meites, Nicoll and Talwalker (in . press) have recently shown that acetylcholine, epinephrine \, and serctinin can initiate milk secretion in virgin female 12 rats primed with estrogen.* These three substances also retarded mammary involution after litterremoval on the 4th day postpartum.- This suggests that probably one or more of these substances may be involved in the hmmoral link between the hypothalamus and anterior pituitary, resulting in the release of prolactin and perhaps other hormones favorable to lactation. 13 5. Endocrine Control of Postpartum Lactation in. Adrenalectgmized Animals. Carr (1951) reported that removal of adrenals resulted in complete cessation of lactation‘in rats. These results / were confirmed‘by Swingle andeiffner (1952), Brownell g§_§l. (1955), Gaunt (1955), and Britton and Kilns (1956). Meites _e_t_ 9;. (1942) showed that adrenalectomy did not prevent initiation of milk secretion at parturition, but lactation was only of short duration, in rats. In these earlier studies both.partial and complete cessation of milk secretion after adrenalectomy had been reported. 0n the other hand it has been shown by many workers that lactation can be maintained in adrenal- ectomized.rats by administrating adrenal cortical extracts J (Nelson and Gaunt, 1957; aohnltx, 1957; Gomez and.Turner, 19:57). The characterization and ‘synthesis of different adrenal cortical hormones stimulated further studies in order to ascertain the role of the adrenals in lactation. Gaunt (1941) reported that deoxycorticosterone was incapable of maintaining lactation in adrenaleotomized rats. However, Nelson and Gaunt (1957) observed that deoxyccrticosterone had a slightly beneficial effect upon established lactation in_the rats. They concluded that 14 alterations in the electrolyte and water balance had an adverse effect upon-lactation. ' Gomez and.Turner (1957), while studying the effect of adrenotrOpic principle on lactation, suggested that a carbohydrate metabolism disturbance appears to be more important than water and electrolyte balance in maintaining lactation.- Gaunt.gt‘§l.-(1942) found that in adrenalecto-_/ mixed-lactating rats complete restoration of lactation could be obtained by the administration of 17-hydroxy-ll- dehydro corticosterone (cortisone). They concluded that while restoration of normal electrolyte metabolism was helpful for maintenance of normal lactation, the limiting factor for maximum lactation appeared to be glucocorticcids . which are concerned with carbohydrate metabolism. Volley and Cowie (1944) reported that they could obtain better maintenance of lactation in adrenalectomised rats with deoxycorticosterone compared with.ll-oxygenated ~/ steroids. However they could not obtain complete mainten- ance with.deoxycorticosterone alone (Cowie and.Folley, 1947). chie (1952) reported complete maintenance of lactation in adrenalectomized rats by administering cortisone and deoxyccrticosterone together. I chie and Tindal (1957) showed that in lactating goats there was a rapid inhibition of milk secretion after l5 adrenalectomy. They could obtain partial to complete maintenance of lactation by implanting tablets of cortisone or cortisone acetate and-deoxycorticosterone acetate. Deoxycorticosterone acetate appeared to be the more critical component of the combination. 16 4. Galactopoietic Studiesgin Laboratory and Farm.Animals. Following the demonstration of lactogenic pr0perties of‘anteriorvpituitary extracts by Stricker and Grater‘ (1928) in ovariectomized, pseudOpregnant rabbits, galacto- poietic studies of anterior pituitary extracts were carried out in laboratory and farm animals.- The earliest report is that of Asimov and.Krauze (1957) who showed that injections of ox~anterior-pituitary into cows produced a marked temporary increase in milk 'production. They found that the treatment was most effective during the first 4 months of lactation during ‘which time the yield was increased slightly above the natural peak value. Asdell Efiuél' (1956) reported that in goats the anterior pituitary extracts were effective during the declining phase but not at the peak of lactation. Similar results have been obtained in cows. Folley and Young (1958, 1959, 1940) showed that they could obtain higher milk.yeild during the declining phase by administering anterior pituitary extracts on milk secretion in cows was only temporary even when injections were continued over a longer period of time. They also reported that they 17 could not delay_the onset.of decline of lactation by administering crude saline extracts. Folley and‘Young (1945) obtained asigmoid dose response curve for single injections of crude ox-anterior-pituitary extracts in cows. ' Riddle gt 31. (1955)were the first to extract the lactogenic hormone following its discovery by Stricker and Gruter (1928). This hormonal principle caused. enlargement and secretion of the pigeon crap gland. After this discovery various prolactin preparations were prepared and studies were made to determine its galacto- poietic effect. ‘ Folley and Young (1958, 1959, 1940) showed that single injections of partially purified prolactin preparations in _ cows were ineffective in causing increases in milk production), during the declining phase of lactation. This was in con- trast with the marked galactOpoietic effect shown with single injection of anterior pituitary extracts. However they could obtain higher milk yield with repeated injections of high doses of prolactin preparations, but in some cows this initial stimulating effect soon disappeared though the treatment was continued. They also found that their _ prolactin preparation showed no correlation between prolactin content and galactOpoietic effect. 18‘ It became apparent that prolactin was not the only ‘ pituitary hormone involved in the maintenance of lactation, and‘the galactOpoietic activity of crude anterior pituitary extract was not due to only a single hormone. Folley and 'IounS (1941) suggested the concept of a pituitary galacto- poietic hormone-complex.- Recent advances in identification, purification and knowledge of chemical and biochemical preperties of anterior pituitary hormones, has made it possible to study their galactopbietic activity, singulariy and in combination, in order to elucidate the pituitary galactOpoietic hormone-complex. Roy (1947) studied the relative galact0poietic activities of anterior pituitary extracts, prolactin, thyroxine, ACTH, whole anterior pituitary extracts plus thyroxine and ACTH plus prolactin in cows. He found that all hormonal preparations were active in stimulating milk production in cows during the declining phase of lactation. . However, the responses to the combined treatment were found to be greater than any single treatment. Cotes 33 al. (1949b) showed that single injections of 40 mg. purified prolactin had no effect on the milk yield of lactating cows. However, these cows were responsive to unfractioned ox pituitary extracts. Balmain and Polley (1952) studying the galactOpoietic 19 effects of various anterior'gpituitary-extracts, found that diabetogenic anteriozrjpituitary fractions showed, marked galactOpoietic- activity in the-cow. Cotes e: al.‘(1949a) showedthatpurified growth hormone (GH) from ox anterior pituitary had diabetogenic effects in the intact'cat. Cotes _e_t. a1. (1949b) reported that single injection of purified GB (50 mg.) resulted in marked temporary-increase in milk yield during thegdeclining phase of lactation in cows. , This galactopoieticeff'ect of GH- in cows has been confirmed. by other workers (Donker and-Petersen, 1951, 1952) in . singleinjection tests,_~and alsoby Chung gt a}... (1955), wronn and Sykes *(1963) and Brumby and Hancock (1955) who observed substantial increases in milk yield in response to series of daily injections. Jordan and Shaffhausen (1954) hawercported marked increases in milk production with GH in lactating ewes: ' . Shaw (1955) injected cows in various stag“ of lactation with 50 to 100 mg GH daily for periods varying from a few days to seven weeks. He could elicit and maintain a 25-50 per cent increase in milk production. during the injection period. The fat percentage in milk was found to be increased by 120 during a 14 day injection period. It was also fou’nd that administration of GH prior to or following parturition resulted in lasting effects in milk production after 6!! was' discontinued. Shaw suggested that the action of OH on lactation may be that of increasing the availability of milk precursors in the blood, increasing the efficiency of milk secretion or producing an increase in growth ofmammary tissue.“ ' Meites and Reineke (1955) studiedthe effect of GH on lactation in goats. They obtained about a 12-16per cent increase in milk production by injecting 50 mg. of GE daily for 5 days. These goats were receiving Protamone, a thyroid-active material, before 61! treatment was started. ' Keites and Reineke (1955) showed that the marked decrease in milk production following removal of Protamone could be corrected partially by GK. They interpreted these results as suggesting that the galactoPoietic actions of thyroid- active substances may be mediated through an increased secretion of GR by the pituitary. However, additional studies by these workers (unpublished) indicated that the effects of GE and Protamone were exerted independently. Hutton (195'?) studied the effect of GH on the yield and composition of cow's milk. The results obtained showed a highly significant linear relationship between the log weight of a single injection of GH and increase in the milk yield obtained in cows during the decline of lactation. When he decreased the doses of (GH, he obtained a significant increase in fat yield relative-to the yield of non-fatty solids. Administration of GH also appeared to increase the efficiency of' conversion of food to' milk. Hutton suggested that the galactopoietic effect of GH appearedto be due to itsability to increase the functional efficiency of the alveola‘ cells-.- In contrast to thepositive findings with GH in cows reported by many workersythere is a report by Flux, (1955) who found decreasesin milk yield after single injections of purified growth hormone in the cow (40 mg.) and goat (20 and 50 mg). The results in goats are also in contrast to that reported, by Meites (1955) who obtained a 12-15% increase with GH. The virtually negative results obtained by Hutton may be due to lower doses of GH used (20 and 50 as)- Meites in his experiment used 50 mg. daily for five days. Meites (1957) reported that injections of one mg. GH in rats for 12 days starting on the 5th day postpartum had ‘/ .no galactOpoietic effect. The mothers receiving GH injections benefited by a significant increase in body weight. Grosvenor and Turner (1959a) claimed about a 40% increase in milk production in rats by daily injection of one mg- of GH during the 7-l5th days postpartum. However their method of measuring milk yield is cpen to criticism. They considered the~amount of milk obtained (stomach.content) by a litter of'6 during 50 minutes nursing on the 14th day postpartum as an index of lactation, and yet found no_ increase in growth by these litters. ‘They also reported that the milk yield was‘greater when oxytoxin was injected in combination with GH. ‘ ' It thus appears that GH may be a limiting factor in_ the decline of lactation in the cow (Cotes gtflal., 1949b; Holley, 1955; Shaw, 1955; banker and Petersen, 1951-1952; Chung 23 51., 1955; Wrenn and Sykes, 1955; and Brumby and Hancock, 1955), cheap (Jordan, 1959), and goat (Meites, 1955). However it does not appear to be a limiting factor./ in the decline of lactation in the rat. ‘ Roy (1947) reported that injections of ACTH] or ACTH and prolactin caused significant increases in milk pro- duction in cows. However Cotes gt al. (1949b) found that single injections of approximately 100 or 200 I.U. of ACTH in cows caused temporary decreases in milk production rather than an increase. Similar results were reported by Flux gt al. (1954). Shaw (1955) reported that administrae tions of purified.ACTH.(100-500 I.U.), hydrocortisone alcohol, ' cortisone acetate or hydrocortisone acetate (5 gms.) caused pronounced decreases in milk production in cows. He also found that the decline in milk production due to ACTH administration could be corrected by simultaneous adminis- tration of GH in cows. Meites (1955) reported that-administration of cortisone, 100 mgz/day for 5 days did not cause any reduction in milk production in goats. Johnson and Meites (1958) showed that daily injection of 0.5 mg. cortisone acetate in rats // resulted in-a significant rise in milk production, as' judged by growth;rate of litters during an 18 day postpartum period. Apparently pure corticosteroids vary in their effectiveness in replacing the adrenals in lactation. ACTH might favor or antagonize lactation, depending upon the , quantity and quality of steroidal output by the adrenals. Although.thyroideactive substances were not used in, the present study, they are briefly considered here since they have been shown to increase milk production in‘ ruminants. The earlier experiments by Graham (1954a) showed that either thyroxine injection (1954b) or feeding of dried thyroid gland (1954a) increased the milk and fat yeild of dairy cows. These results were confirmed with goats and cows by manyother workers (Jack and Bechdel, ‘1935; Holley and White, 1956; Hennan 9;; 5;” 1958). On the other hand complete thyroidectomy or thiourea feeding have beenshown to reduce milk secretion in cows (Blaxter £3 51., 1949). Experiments with iodinated casein, first reported by Reineke (1942); Reineke and.Turner (1942) and confirmed.by others indicated that properly prepared 24 iodinated casein has galactoPoietic effects in ruminants. However great variability in milk production has been obtained by feeding iodinated casein or injecting thyroxine. These variations are due-to a number of factors such as the dose employed, physiological status of the cow, length of treatment, nutritional status, etc. (Blaxter, at 51., 1949). Chen 2E El. (1955) reported that the thyroid was unnecessary for mammary develOpment and milk secretion in , hypophysectomized, cephorectcmized,_adrenalectonized and.. / thyroidectomized rats.~ Lyons gt al.(l958) and Cowie (1957) found that thyroxine had no effect in enhancing milk secretion alone or in combination with prolactin and (ACTH or cortisone in hypOphysectomized rats. Meites (unpublished) observed that thyroxine had no effect in retarding mammary involution and maintaining ‘ .milk secretion after litter'removal on the 4th.day post- partum in rats. On the other hand Desclin (1949) and Grosvenor and.Turner (1959b) reported increases in milk secretion in rats following daily administrations of l-thyroxine. The amount of milk obtained by a litter of 6 young during 50 minutes suckling on the l4th.day postpartum was used as the index of response by Grosvenor and Turner. This method of accessing the milk yield is cpen to criticism, since the young did not show any increase in growth rate despite an apparent increase in milk intake. II METERIALS AND METHODS It is difficult to accurately measure lactational responses in small laboratory animals when compared to larger animals such as the cow and goat, in which, by use of milking machines, the milk can be almost quantitatively 'withdrawn. Since it is difficult to measure milk output, it is necessary to employ survival of litters and litter growth.curves as measures. -In these experiments litter growth rates have been employed. This is not an absolute but only relative index of lactational response, since the daily weight loss due to excreta and insensible perspirar tion are not measured. Cowie (1946) used a lOgistic equation and.plotted a mean growth curve for litters of lactating dams from birth to 16 days of age and obtained a sigmoid curve. 'The points from the 6th to 11th days of lactation, in this curve fall on a straight line. He found that during this 5 day period the average increment in litter weight was approximately constant as well as at a maximum for each rat. The weight gain of litters during this period, Cowie termed the “litter growth index" and he used it as a quantitative measure for lactational responses in rate. This is not believed to be a true reflection of milk yield.but is 25 26 estimated.by Cowie to be roughly 50-60 per cent of the true output. Johnson (1957) showed that cortisone administration. ‘/ during the initial phase of lactation (0-5th day) did not have any significant effect on litter growth. However significant increases over that of the controls were. obtained when cortisone was administered during later periods of lactations, 6thr10th.days.and llthrl8th.days. Therefore in this experiment it was decided to carry out _ hormonal treatments during the 4thrl7th days of lactation. In Experiment I, 90 mature female albino rats of the . Carworth strain were bred. Once pregnancy was established, the dams were placed in individual cages. These cages .‘were designed to make the dam's ration inaccessible to the young throughout the experimental period. This insured that the young received food only from their mothers. The food and water was available to dams ag_1ibitum. The .animals were kept in an air-conditioned room with.a temperature of 74110 F throughout the experiment. Constant artificial lighting was provided in the room from 7:50 A.M. until 9:50 P.M. The rats weighed between 220-275 gms. at the beginning of the experiment. From the day of parturition to the 18th.day of lactation the rats and litters were weighed daily at approximately the same_hour of each day. On the 4th day after parturition the litters were reduced to 6 young each. This gives a better chance for survival of the young and standardizes the number per group. The litters were.randomly divided into 9 groups of uniform weight. All injections were administered sub- cutaneously, daily,the 4th day postpartum to the 17th“ day postpartum. 0n the 18thday the dams were killed. The ovaries and adrenals were removed, dissected free_from connective tissue and their weights were recorded. Similarly the pituitary was removed and weighed on a Roller-Smith balance. ‘ The following injection schedule was carried out once daily except for oxytocin which was injected.twioe daily: (1) Controls, saline (0.85%)., (2) Hydrocortisone acetate, 0.25 mg. (5) Hydrocortisone acetate, 0.5 mg. (4) Hydrocortisone acetate, 1.0 mg. (5) Prolactin (20 I.U.), 1 mg. (6) Oxytocin (20 I.1?./mg.), 1 I.U. (7) Hydrooortisone acetate, 0.25 mg.; prolactin, 1 mg. and oxytocin, l I.U. (8) Hydrocortisone acetate, 0.5 mg.; prolactin, 1 mg. and oxytocin, l I.U. 28 (9) Hydrocortisone acetate, 1 mg.; prolactin, 1 mg. and oxytocin, 1 I.U. All injections were given in a 0.1 cc. solution. InExperiment II, 20 mature female albino rats of_the Carworth strain were bred. 0n the 2nd day after parturi- tion both.the ovaries were removed under light ether anesthesia. The rats were divided into two groups. Group I received saline daily while Group II received 1 mg. prolactin (20 I.U./ mg.) daily during the 4thrl7th days postpartum. Dams and litters of six young each.were weighed daily. 0n the 18th day postpartum the dams were killed and the adrenals, ovaries and.pituitary were removed and weighed on a Roller-Smith.balance. III EXPERIMENTAL RESULTS 1. Effegts of Hydrocortisone Acetate,_0xytocin andTProlactin on Lactatigna; Performance in Rats The results of this experiment are summarized in Tables 1-4. Dams receiving saline, oxytocin or prolactin gained in average body weight by about 20-25 gms. during . the 18 days period.» Dams receiving 0.25 or 0.5 mg. hydro- cortisone acetate gained in average body weight by only 7-10 gms. However the dams which.received 1.0 mg. hydro- cortisone acetate lost in average body weight by about 8 gms. during the 18 days period. No significant differences were found in the average weights of pituitaries in the groups. However the average weights of the adrenals and ovaries are significantly lower than the controls in the groups which received 0.5 and 1.0 mg. hydrocortisone respectively. There was no significant difference in average litter growth rate, total and daily, during the 6-10 day period (Cowie's litter growth index) between the controls and the groups receiving oxytocin, prolactin, or hydrocortisone acetate at a dose of 0.25 mg. These.groups also did not show any significant difference in their average litter growth rate during the ll-lBth days or 4-18th days from the controls. 29 The group which received 0.5 mg. hydrocortisone acetate daily, showed significant increases over the controls in average litter growth rate, total and daily, during the first 6-10 days period (Cowie's litter growth index), 11-18 days period and 4-18 days period. This group-gained approximately 18 per cent more in average body weight during 4-18 day period than the controls. The group which received 1 mg. hydrocortisone acetate showed a significant decrease in average litter weight gain during the 4-18 days period. However there wasgno ._.__,_,...o—‘ eignificant difference during the first 6-10 days between the groups receiving 0.5 and 1.0 mg. of hydrocortisthMMH acetate, respectively. ‘ There were no significantly greater increases in average litter growth rate when the rats were given 1 I.U. oxytocin twice daily and 1 mg. prolactin in addition to 0.25, 0.5 or 1.0 mg. hydrocortisone acetate. Thus oxytocin and prolactin do not appear to have any ‘ synergistic effects with.hydrocortisone acetate in pro- i noting average litter growth rate. The magnitude of the response with 0.5 mg. hydrocortisone acetate was the same when given alone or in combination with.l mg. prolactin and l I.U. oxytocin. Figure 1 shows the average daily weight gains of the 31 young of the control group and the groups receiving 0.25, 0.5 and 1.0 mg. hydrocortisone acetate, respectively. Figure 2 shows the average daily weight gains of the _ young of the control group and the groups receiving 0.25, 0.5 and 1.0 mg. hydrocortisone acetate, respectively, in addition to 1 mg. prolactin and l I.U. oxytocin twice daily. 2. Effects of Prolactip_on Lagtational Performance in Qgpgprectomigedfifiats The results of this experiment are summarized in Tables 5 and 6. Dams in both.groups gained an average of , 20 gms. in body weight during the lB-day postpartum.period. No significant difference in the average weight of the _// pituitary were observed between groups. Also no signifi- cant differences in average littir*growth.rate, total and daily, during the first 6-10 days (Cowie's litter growth index), ll-lSth days and 4-18thdays postpartum were observed between the two groups. mm some who we aoaac pamuomum as macaw no; mama mo popes: n mm.aa No.0“ NN.HH o.meu H.0HH Aoav memos co.om mm.o ma.ca m.mcm w.amm «.me cons» .a.H H cacooaxo mHHmo .ME H :HpomHonm memos .me o.H odomenoooammm A: mm.HH no.0“ H.HH m.oen m.mn mm.mm om.m m.nm N.mmm m.mom a.oa Aoev memos ooeao .o.H a cacocaso mHHmc .me H oHpomHonm mHHmp .ma m.o coopraooonpam Km oo.HH mm.ow am.aw N.OHH a.mw Aoav aaaco Na.cm no.3 mH.aN N.mwm N.mom m.oa ocean .D,H H namesake AHHmc .ms H meomHoam .aeace .ms mm.o ooomeaoooauam AN Hd.HH mm.oH 3N.Hw o.mfi www.mfi oa.mm mw.: mo.am m.oam m.Hmm m.ae .Aoav mecca condom. \ mHoapooo fiH .zm .mnm 00H\.mm .Rm .msm 00H\.ms .zm .mem 00H\.Ma .mam .meu AvaHm pm uncapmoaa pom aspnmoumom aduamapmofi soaammpmom adpammpmom moHpHaounmm pm oaHm. .oz macaw once we so monocco case we so .oe case we cc .ca once we no .oz son .ca poncho mo .93 .>4 mumvaouHm .nd Hammond .b4 .9: sea .>4 .nd spam udecposH mo auanoz mampHdaHm use :mHam>o .Hooeaod .auom do coHpmerSOO :H oHoouaHo use oHpomHonm .oumaoo< onoernoooneam mo muoommm m mummy amospmoap mo ooHacm Hopes ++ Nong opxoam HcpuHH + on some on» me Hones dampcmpm so HoppHH Hog warm m .mdoam Hem whoppHH Mo Hence: a Hw.oH no.0“ mm.oH Hm.mu am.oH mm.mH oH.0H H.mmH Hm.m m.ma mm.m H.oo AOHV aHHco .mc H meowHoam Aw Ha.ou mm.me oa.on Ho.au ma.on NH.oH am.OH w.mmH N .m m.aa mo.m m.mo HOHVHHHco coHan .D.H H mHoopaxo Am ma.on H.0HH mm.on om.mu Ho.ou NH.mH V om.OH o.amH Hm.OH m.mw oc.m a.mo AOHV aHHS .ma H . esomeooooaram A: Hw.on am.mu mo.om mm.mn mo.on Hm.nH ; mm.mH m.:wH Nc.HH o.mm Ha.OH m.mm HOHV aHHcs .os m.o coomenooonwmm Am Hm.ou aa.wn aa.on mo.mn am.on am.on Ha.HH a.mmH aH.HH 3.0m o.OH 0.0m AOHV HHHco .ms mm.o coomeHooonomm Am no.0“ mm.mu om.ou Hm.mn; Hm.on ..NH.¢H no.0H H.amH oc.c a.ma mm.m m.oa .AOHV aHHco ooHHcm mHonpooo AH .95% nimsm, .maw limam \flflflw ‘JmEM uncapmona pom aHch Hcooa a.HHciH Hosea +HHHco Hence .oz cacao ++mmcc nomHua name ouleHH mace HHOHIw comm museum HeupHH ommno>4 whoppHH Mo mpthoz zoom no eHOOpzxo can :HpomHon .opmpood coomeHoooawzm mo mpoomwm m anm< Honooen .>n .oa Eco .se "i' comm wnHpmpomnncoNHaonooHonmoo mo concHoa HocoHcon coo .Hccooo4 .aoom co oHoccHoom Ho cocoon w aHm' controls 0.25 mg. 0.50 mg. 2.00 mg. iii 1 l J 1 1 1 8 10 12 14 16 DAYS POSTPARTUM Effect of Hydrocortisone acetate on Litter Weight Gains in the Rat 38 FIGURE 2 A controls B 0.25 lge I'D ‘. Prol. 1.0 use . m 1 love C 0.50 lg. H3 0 Prcl. 1.0 mg. + ox 1 I.U. ,C o 1.00 mg. so + Prol. 1.0 mg. + oz 1 I.U. f" 40 P L l l I i )- )- 6 8 10 12 14 16 18 DAYS POSTRARTUM Effect of Hydorcortisone acetate, Prolactin and Oxytocin on Litter Weight Gains in the Rat 39 IV DISCUSSION It has been very well established that the adrenals are essential for the maintenance of lactation in rats (Nelson and.Gaunt, 1957; Cowie and Folley, 1947) and in goats (Cowie and Tindal, 1957). Lyons (1958) observed that in hypOphysectomized rats prolactin and.ACTH or a glucocorticoid are the minimum hormones necessary to maintain lactation. It has also been reported that the suckling stimulus causes release oanCTH from the pituitary, which would subsequently induce release of adrenal cortical steroids (Tabachnick and Trentin, 1951; Gregoire, 1946). From these studies it appears that ACTH or a corticoid could be a limiting factor in the decline of lactation. .However, the galactOpoietic studies with ACTH or corticoids in ruminants have been reported to have either inhibitory effect (Cotes 23.2l‘: 1949b; Flux! t 31., 1954; Shaw, 1955) or no effect (Meites, 1955), the exception being the report by Roy (1947) who obtained stimulation of milk secretion in the cow. The results of the present study indicate that hydrocortisone acetate can increase the milk secretion during the 5-10 day period (Cowieus litter growth.index)/ and during the remaining period of lactation in rats. These findings confirm the results obtained by 40 41 Johnson and.Heites (1958)“with cortisone-acetate. The daily dose of 0.5 mg. hydrocortisone acetate appears to be Optimal. With a lower dose (0.25 mg. daily) of hydro-g cortisone acetate there was slight stimulation but this was not significant.‘ waever at‘a higher dose level (1 mg. daily) the rat mothers lost considerable body weight and.there was a slight decrease in milktsecretion as judged bylitter growth rate.- Johnson (1958) showed b/ that when cortisone wasadministered in combination with GH and prolactin to lactating rate, they did not lose ' body weight, but the lactational response was of the same magnitude as with cortisone alone. Also Lyons (1958) reported that the hypOphysectomized lactating rats given // prolactin and prednisclcne acetate lost considerably more in body weight than rats also given GH. However, the litter growth rates in both cases were essentially similar. These results present one additional support to the earlier findings that the adrenal cortical hormones are ' essential for the maintenance of milk secretion in rats. Hydrocortisone acetate was effective in increasing milk / secretion during lactation, probably because the adrenal cortex of the rat secretes less than optimal amounts of glucocorticcids. It is prObable that ruminants, in contrast, already secrete sufficient amounts of adrenal 42 cortical hormones for lactation, and hence additional amounts are harmful, producing catabolic rather than anabolic effects. It is known that physiological doses of adrenal glucocorticcids are essential for normal “/ metabolism of carbohydrate, protein and.fat, whereas large amounts are detrimental to these processes. It has been reported that injections of prolactin to lactating mother rats after removal of their litters A inhibit involutionary changes in the mammary gland '/ (Williams, 1945; Johnson, 1957). It has also been reported that in hypOphysectomized rats prolactin together with.an adrenal corticoid is essential for the maintenance of milk secretion (Cowie, 1957; ‘>< Bintarningsih gt gl., 1957; Lyons 31 al., 1958). The results of the present study indicate that daily in- Jections of 1 mg. of prolactin (20 I.U./mg.) had no effect on the lactational performance in rats. These results corroborate the negative findings in ruminants, but are in disagreement with the positive findings reported by Johnson (1958) in the rat. The principal difference between this experiment and that of Johnson (1958) is that a different prolactin preparation was employed and the diets were not the same. In the rat prolactin has been shown to have luteotrOpic activity. Moore and.Nolbandov (1955) have also reported that prolactin is luteotrOpic in sheep. 'Lyons (1958b) suggested that prolactin, injected in rats bearing corpora lutea during lactation, might be utilized by the corpora lutea. This would promote secretion of progesterone, and thus stimulate mammary growth and antagonize lactation. However, prolactin, at the dose level used, did not show any stimulating effect on milk secretion in either cephorectomized or intact lactating rats. Therefore it appears that during lactation the suckling stimulus releases prolactin in sufficient amounts to luteinize ovaries and maintain lactation. The presence of lutsinized ovaries in the lactating rat were shown not to interfere in any way with.milk.production even when the rats were injected with.prolactin, which.presumably stimulated greater luteal secretion by the ovaries. The suckling stimulus during lactation has been shown to cause the release of oxytocin from the neurohypOphysis (Petersen and Ludwick, 1942; Cross and Harris, 1950, 1951). Oxytocin acts on the myoepithelial cells surrounding the "u alveoli, causing contraction, and thus squeezes out the milk from the lumen of the mammary alveoli. This milk ejection effect of oxytocin has been very well established. There are various reports that regular injections of oxytocin have galactOpoietic effects in lactating cows (Adams and Allen, 1952; Sprain 33 5%., 1954; Donker 23 gl., 1954). Johnson (1958) reported that oxytocin had galact0poietic ‘< effects in rats during lactation. It has been also shown that oxytocin inhibits mammary involution after litter removal on the 4th day of parturition in rats (Benson and Folley, 1957; Meites and Nicoll, 1959). In the present study injections of oxytocin 1 I.U. twice daily in lactating rats appear to have no galactOpoietic effect. This is in contrast to the findings of Johnson (1958). ‘K The same doses of oxytocin were used in both experiments, and the same strain of rats were employed. HOwever, the diets were not the same in the two experiments and this might have accounted for the difference in results. 1. 2. V SUMMARY AND CONCLUSIONS The effects of administering 0.25, 0.5 or 1.0 mg. of hydrocortisone acetate, l-mg. (20 I.U./mg.) prolactin or 1 I.U. oxytocin, singularly or all 5 in combina- tion were studied on the lactational performance of postpartum rats. In a second experiment the effects of prolactin on lactational performance of postpartum rats ovariectomized on the 2nd.postpartum day were observed. The growth rate of litters of 6 young each during the 4th918th.days postpartum was used as an index of milk production. When hydrocortisone acetate, oxytocin or prolactin were injected individually in lactating rats, only hydrocortisone acetate at the 0.5 mg. dose level increased milk secretion significantly. When all three hormones were injected in combination, the increases in lactation were no greater than with hydrocortisone acetate alone. Hydrccortisone acetate was effective in increasing milk secretion, perhaps because the adrenal cortex of the rat secretes less than Optimal amounts of hormones during lactation. Oxytocin and prolactin were not effective in increasing 45 3. milk secretion. These two hormones are probably secreted in sufficient amounts during lactation in the rat, and hence additional amounts of oxytocin and prolactin might have no effect. Prolactin at the dose level used did not induce a significant increase in milk secretion over that of saline injected controls in ovariectomized lactating rats. Also no significant difference in milk secretion was observed between the ovariectomized and intact controls. These results suggest that the suckling stimulus during lactation causes release of sufficient prolactin from anterior pituitary in sufficient amounts to luteinize the ovaries and maintain milk secretion. 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