THE MAINTENANCE OF MILK SYNTHESIS DURING EXTENDED LACTATioN Thesis for the Degree of Ph. D. MICHiGAN STATE UNWERSITY WILLIAM W. THATCHER 1968 a“... LIBRARY Michigan Sate University ‘ This is to certify that the thesis entitled THE MAINTENANCE OF MILK SYNTHESIS DURING EXTENDED LACTATION presented by William W. Thatcher has been accepted towards fulfillment of the requirements for Ph . D 0 degree in~ Dairy [hm December 12, 1968 0-169 ABSTRACT THE MAINTENANCE OF MILK SYNTHESIS DURING EXTENDED LACTATION by William w. Thatcher The primary objective of these studies was to determine some of the physiological and endocrine factors that may limit lactational performance in rats. Intensive nursing, which was maintained by replac- ing all l6—day-old litters every N days with lZ-day-old foster litters and supplying at least one pup per mammary gland, failed to prevent declines in mammary DNA, RNA and litter weight gains (LWG) between days 20 and 36 of lac- tation. Intense nursing maintained pituitary prolactin during extended lactation but pituitary ACTH decreased 68%. Adrenal corticosterone content and concentration decreased significantly from day 16 to 32 of lactation. Peripheral plasma concentrations of corticosterone in lactating rats were higher than the resting levels measured in virgin rats. However, corticosterone con— centrations in plasma did not change from day 16 to 32 of lactation. Measurements of corticoid binding globulin activity in pooled plasma samples indicated William W. Thatcher that binding activity of rats killed at day 32 of lactation was greater than the activity found in rats killed at day 16 and 24. Adrenal corticosterone content of rats at day 16 of lactation was significantly correlated with mammary gland DNA (r=O.59, P<0.01), RNA (r=O.60, P<0.01) and litter weight gain (r=O.4U, P<0.05). The decrease in adrenal cor— ticosterone content from day 16 to 32 of lactation was cor- related significantly with the decrease in mammary gland DNA (r=0.35, P<0.01) and RNA (r=O.M5, P<0.0l) content. Oxytocin injections during advanced lactation caused increases in DNA and RNA content of the mammary gland but did not significantly increase litter weight gain or synthetic activity per cell (RNA/DNA). Hydrocortisone acetate, prolactin and growth hor- mone were given singularly and in all possible combinations to determine if these treatments would maintain mammary nucleic acid content and lactational performance during extended lactation. Hydrocortisone acetate maintained mammary DNA and RNA content and reduced the normal decline in litter weight gain. Prolactin given with hydrocortisone acetate reduced the DNA response, but stimulated synthetic activity of the mammary cells (RNA/DNA). Prolactin and growth hormone did not exert a galactopoietic influence on milk yield. The chronic secretion of rat prolactin from five isotransplanted pituitaries under the kidney capsule of lactating rats did not increase litter weight gain William w. Thatcher although the mammary gland DNA and RNA content and RNA/ DNA ratio were significantly increased. Prolactin is probably not rate limiting to milk synthesis in rats be- cause pituitary prolactin content did not decrease during extended lactation and neither ovine prolactin injections nor rat prolactin from isotransplanted pituitaries in- creased milk yield. A daily dose of 50 ug of 9—fluoroprednisolone acetate (Predef) from day 16 to 32 of lactation maintained mammary gland DNA and RNA content and increased milk yield. A delay in the onset of injections until day 24, or injection of 50 ug of Predef daily from day 16 to 23 and 100 pg of Predef daily from 2A to 32 days of lac— tation maintained LNG and increased mammary gland nucleic acid content. Results of in £1359 incubations indicated that Predef injections for 16 days increased the ability of rat livers to metabolize cortisol into polar metabo— lites while the formation of non—polar metabolites was inhibited. Adrenocortical secretions may be rate limiting to milk synthesis during advanced lactation in rats because pituitary ACTH and adrenal corticosterone content de— crease during this time, corticosteroid binding globulin activity of plasma increases (although total circulating levels of corticosterone did not change) whereas the de— clines in mammary gland nucleic acid content and litter weight gain can be prevented with exogenous hydrocortisone acetate or Predef. THE MAINTENANCE OF MILK SYNTHESIS DURING EXTENDED LACTATION By William w; Thatcher A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1968 Dedicated to My Parents Muriel and William Thatcher ii BIOGRAPHICAL SKETCH William Watters Thatcher was born on January 12, 19u2, in Baltimore, Maryland. He attended public schools in Baltimore County and graduated from Kenwood High School in June, 1959. In September, 1959, he enrolled at the University of Maryland, majoring in Animal Hus- bandry, and received a Bachelor of Science degree in June, 1963. In June, 1963, he enrolled in the graduate school of the University of Maryland. He received the Master of Science degree in September, 1965, majoring in Dairy Science. In September, 1965, he was awarded a graduate re— search assistantship by the Department of Dairy, Michigan State University. He was granted a NIH Predoctoral Fellowship in March, 1968 and completed the requirements for the Ph.D. degree on December 12, 1968. iii ACKNOWLEDGMENTS The author expresses his deepest appreciation to Dr. H. A. Tucker, for his guidance, his inspiration and his help during the course of the research and the preparation of this manuscript. The author is indebted to Dr. H. D. Hafs for his frequent assistance and encouragement during the graduate program. Appreciation is extended to Dr. G. D. Riegle for aid and advice on the adrenal gland aspects of this research and to Dr. J. L. Gill for his counsel on experi- mental design and analysis of these experiments. The criticisms of Dr. R. J. Brunner and the above listed committee members on this manuscript are appreciated. The author wishes to thank Dr. L. J. Boyd, Dr. R. M. Cook and Dr. R. S. Emery for their cooperative assistance. The author is indebted to his student colleagues, A. J. Hackett, J. A. KOprowski, M. J. Paape, D. E. Pritchard, Y. N. Sinha, L. V. Swanson and R. P. Wettemann for their interest, assistance and stimulation. The gifts of hormones from the Endocrinology Study Section of the National Institutes of Health and the Upjohn Company are much appreciated. iv The author is grateful for the financial support provided by Michigan State University through a Graduate Research Assistantship and by the National Institutes of Health for a Predoctoral Fellowship. The patience, understanding and encouragement of the author's wife, Jeanne throughout my entire graduate program is deeply appreciated. TABLE DECICATION . . . BIOGRAPHICAL SKETCH ACKNOWLEDGMENTS . . LIST OF TABLES. . . LIST OF FIGURES . . LIST OF APPENDICES . INTRODUCTION . . . REVIEW OF LITERATURE. Terminology. . Measurements of In Hypophysectomized and Adrenal- OF CONTENTS Lactational Performance Factors Affecting Lactational Performance. ectomized Animals. In Intact Animals. Milk Ejection Reflex. Release and Synthesis of Hormones . Intensity of Nursing Stimuli and the Extension of Lactation MATERIALS AND METHODS Experimental Animals and Design . 0 Experiment I.--Lactationa1 Performance of Intensely Nursed Rats During Ex- tended Lactation Experiment II. --Lactational Performance of Intensely Nursed Rats Injected with Oxytocin During Extended Lactation . . . . vi Page ii iii iv ix xii xiii UTUOM FHO~QU1 l9 l9 19 20 Page Experiment III.--Lactational Performance of Intensely Nursed Rats Injected with Hydrocortisone Acetate (H), Prolactin (P), and Growth Hormone (G) During Extended Lactation . . . 22 Experiment IV.--Lactational Performance of Intensely Nursed Rats Bearing Isotransplanted Pituitaries During Extended Lactation. . . . . . . 22 Experiment V.—-Lactational Performance of Intensely Nursed Rats Injected with 9—Fluoroprednisolone Acetate (Predef) During Extended Lactation . 23 Experiment VI.-—Measurement of Adrenal Function and Corticoid Binding Globulin (CBG) Activity of Intensely Nursed Rats During Extended Lac- tation. I O O O O O O O o O 214 Biochemical Parameters Measured in Mammary Glands . . . . . . . . . 25 Bioassay of Pituitary Hormones. . . . . 3O Prolactin. . . . . . . . . . . 3O ACTH Bioassay . . . . . . . . . 32 I2_Vitro Metabolism of Cortisol by the Liver O O O O I O O O O O O O 3Ll Preparation of Liver Microsomes . . . 3A Ig'Vitro Incubation Procedure . . . . 35 Fluorometric Assay of Plasma and Adrenal Corticosterone . . . . . . . . . 38 Plasma Corticosterone Procedure . . . 38 Adrenal Corticosterone Procedure . . . A2 Measurement of Corticosteroid—Binding Globulin (CBG) Activity . . . . . . U3 RESULTS AND DISCUSSION . . . . . . . . . #9 Experiment I.--Lactational Performance of Intensely Nursed Rats During Extended LaCtation. o o s o o o s o o 0 L19 vii Page Experiment II.—-Lactationa1 Performance of Intensely Nursed Rats Injected with Oxytocin During Extended Lactation . . 56 Experiment III.-—Lactational Performance of Intensely Nursed Rats Injected with Hydrocortisone Acetate (H), Prolactin (P) and Growth Hormone (G). . . . . 6O Experiment IV.--Lactational Performance of Intensely Nursed Rats Bearing Iso- transplanted Anterior Pituitaries During Extended Lactation . . . . 79 Experiment V. --Lactational Performance of Intensely Nursed Rats Injected with 9-Fluoroprednisolone Acetate (Predef) . 83 Experiment VI.--Measurement of Adrenal Function and Corticoid Binding Globulin (CBG) Activity of Intensely Nursed Rats During Extended Lactation . lOO SUMMARY AND CONCLUSIONS. . . . . . . . . 109 BIBLIOGRAPHY o o o s o o s o o o o o 113 APPENDICES . . . . . . . . . . . . . I25 viii Table LIST OF TABLES Nucleic Acid Content of Mammary Glands and Litter Weight Gain of Intensely Suckled Rats During Extended Lactation . . . Nucleic and Lactic Acid Content of Mammary Glands and Litter Weight Gain of In- tensely Suckled Rats During Extended Lactation . . . . . . . . . Prolactin and ACTH Content of Pituitaries of Intensely Suckled Rats During Extended Lactation . . . . . . . . . . Nucleic Acid Content of Mammary Glands and Litter Weight Gain of Intensely Suckled Rats Injected with Oxytocin Between Day 16 and 32 . . . . . . . . . . Organ Weights of Intensely Suckled Rats Injected with Oxytocin Between Day 16 and 3 2 o o o o s o o s o o 0 Nucleic Acid Content of Mammary Glands and Litter Weight Gain of Intensely Suckled Rats Injected with Hydrocortisone (H), Prolactin (P) and Growth Hormone (G) Between Day 16 and 32 . . . . . . . Organ Weights of Intensely Suckled Rats Injected with Hydrocortisone (H), Pro- lactin (P) and Growth Hormone (G) Be- tween Day 16 and 32. . . . . . Nucleic Acid Content of Mammary Glands and Litter Weight Gain of Intensely Suckled Rats Bearing Isotransplanted Pituitaries Between Day 3 and 32 . . . . . . Organ Weights of Intensely Nursed Rats Bearing Isotransplanted Pituitaries Be— tween Day 3 and 32 . . . . . . . . ix Page 50 52 53 58 59 61 67 80 82 Table 10. ll. 12. l3. 1“. 15. 16. 17. 18. Page Nucleic Acid Content of Mammary Glands and Litter Weight Gain of Intensely Nursed Rats Injected with 9-Fluoroprednisolone Acetate (Predef) Between Day 16 and 32. . 85 Organ Weights of Intensely Nursed Rats In- jected with 9-F1uor0prednisolone Acetate (Predef) Between Day 16 and 32 . . . . 88 Nucleic Acid Content of Mammary Glands and Litter Weight Gain of Intensely Nursed Rats Injected with 9-F1uor0prednisolone Acetate (Predef) Between Day 16 and 32. . 91 Organ Weights of Intensely Nursed Rats In— jected with 9-F1uor0prednisolone Acetate (Predef) Between Day 16 and 32 . . . . 92 Amount of Cortisol Metabolites Produced from Liver Preparations of Intensely Nursed Rats Injected with Saline and 9- Fluoroprednisolone Acetate (Predef) Between Day 16 and 32 . . . . . . . 94 Amount of Cortisol Metabolites Produced Per mg of Protein from Liver Preparations of Intensely Nursed Rats Injected with Saline and 9-F1uoroprednisolone Acetate (Predef) Between Day 16 and 32 . . . . 96 Amount of Cortisol and Cortisol-Water- Soluble Metabolites Produced from Liver Preparations of Intensely Nursed Rats Injected with Saline and 9-Fluoro- prednisolone Acetate (Predef) Between Day 16 and 32 . . . . . . . . . . 98 Liver Weights and Protein Measurements of Liver Microsomes and 105,000xg Superna— tant Fluid of Intensely Nursed Rats In- jected with Saline and 9-F1uoropredni- solone Acetate (Predef) Between Day 16 and 32 . . . . . . . . . . . . 99 Nucleic Acid Content of Mammary Glands and Litter Weight Gain of Intensely Suckled Rats During Extended Lactation . . . . lOl Table Page 19. Adrenal and Plasma Corticosterone Levels in Intensely Suckled Rats During Ex- tended Lactation . . . . . . . . 103 20. Correlation Coefficients of Mammary Gland Nucleic Acid Content and Litter Weight Gains with Adrenal Corticosterone Con- tent of Intensely Suckled Rats During Extended Lactation . . . . . . . 106 xi LIST OF FIGURES Figure 1. Separation of Protein—bound and Non- protein-bound Corticosterone n-14c During Gel Filtration . . . 2. Litter Weight Gain of Intensely Suckled Rats Injected with Hydrocortisone (H), Prolactin (P) and Growth Hormone (G) Between Day 16 and 2N . . . . 3. Litter Weight Gain of Intensely Suckled Rats Injected with Hydrocortisone (H), Prolactin (P) and Growth Hormone (G) Between Day 2“ and 32 . . . . . . . A. DNA Content of Mammary Glands of Intensely Suckled Rats Injected with Hydrocortisone (H), Prolactin (P) and Growth Hormone (G) Between Day 16 and 32. . . . . 5. Interaction of Hydrocortisone (H), Pro- 1actin (P) and Growth Hormone (G) with DNA Content of Mammary Glands of In- tensely Suckled Rats Injected Between Day 16 and 32. . . . . . . . 6. RNA Content of Mammary Glands of Intensely Suckled Rats Injected with Hydrocorti- sone (H), Prolactin (P) and Growth Hor- mone (G) Between Day 16 and 32 . . . . 7. Interaction of Hydrocortisone (H), Pro- lactin (P) and Growth Hormone (G) with RNA/DNA Ratio of Mammary Glands of In- tensely Suckled Rats Injected Between Day 16 and 32. . . . . . . . . xii Page 47 62 6A 68 71 73 76 LIST OF APPENDICES Appendix Page I. Composition of Rat Feed . . . . . . 126 II. Orcinol Reagent Used in the RNA Pro- cedure . . . . . . . . . . 129 III. Incubation Media for ACTH Assay . . . 131 IV. 0.1M Phosphate-glycerol Buffer pH7.N Used in 12_Vitro Metabolism Studies . 13“ V. 0.1M Phosphate Buffer pH7.U Used in In'Vitro Metabolism Studies. . . . 136 VI. Scintillation Fluid #1 . . . . . . 138 VII. Scintillation Fluid #2 . . . . . . 1A0 VIII. Fluorescent Reagent for Corticosterone Determination . . . . . . . . 1A2 IX. Phosphate Buffers Used in the CBC Procedure. . . . . . . . . . 14A xiii INTRODUCTION Milk secretion is initiated immediately after parturition in the bovine at a relatively high level. Milk yield normally increases during the initial 4 to 6 weeks of lactation and then decreases progressively until the end of lactation. Any means of minimizing this normal decline in milk production would represent a sizeable contribution of animal protein for human consumption as well as a marked gain in operational efficiency to dairy farmers. The primary objective of these studies was to determine some of the physiological and endocrine factors that may limit lactational performance. The rats were the experimental animals. Changes in milk yield and biochemical composition of the mammary gland were mea- sured in response to both nursing intensity and exogenous administration of hormones believed to be intimately associated with milk production. Based on the results of these experiments, a subsequent study was conducted to determine if adrenal function became limiting as lactation advanced. REVIEW OF LITERATURE Terminology Milk secretion involves both intracellular synthesis of milk in alveolar epithelium of the mammary gland and subsequent passage of milk from the cytoplasm into the alveolar lumen. The term lactogenic describes those factors responsible for the initiation of milk secretion whereas galactopoietic indicates the ability of factors to enhance an already established milk secretion (Cowie, 1961). GalactOpoiesis is used in a general sense to infer the maintenance of milk secretion. The act of suckling influences milk secretion through the synthesis and re- lease of certain anterior pituitary hormones that are in turn either directly or indirectly involved in the stimu- lation of mammary alveoli to produce milk. Milk removal from the mammary gland involves both active and passive withdrawal of milk. The suckling pro- cess initiates active milk expulsion, the so-called milk ejection reflex. During this reflex suckling elicits sensory afferent nerve impulses which travel from the teats to the central nervous system to cause a release of oxytocin from the posterior pituitary. Oxytocin is D) subsequently transported in the blood to the mammary gland. At the mammary gland oxytocin stimulates con- traction of the myoepthelial cells which surround the alveoli and small milk ducts and thus forces the milk into the large milk ducts, gland cisterns and sinuses. The external physical force of suckling causes the passive withdrawal of milk from the large milk ducts, cisterns and sinuses of the mammary gland to the exterior of the body. Measurements of Lactational Performance It is more difficult to measure accurately lac- tational response in small laboratory animals than in larger animals such as the cow and goat. In the latter species milk can be weighed gravimetrically over a long period of time whereas in laboratory animals milk pro- duction is usually measured indirectly as some function of litter weight gain. Cowie and Folley (19A6) used a logistic equation. They plotted the mean growth curve for litters of lactating rats from the time of birth to 16 days of age and obtained a sigmoid curve. From day 6 to day 11 of lactation, daily litter weight gain was maximum. The gain during this five—day period was termed "litter growth index" and has been used as a quantative measure for lactational responses in rats. The two extremes of the sigmoid curve were defined as the initial and declining phases of the lactation curve. However, it should be pointed out that a litter weight gain is not an absolute but a relative index of lac- tational response. In litter growth curves, the daily milk yield is not directly calculable, since the daily weight loss due to excreta and perspiration are not measured. In addition there is some question as to whether each pup is able to convert milk to body weight at the same rate. Grosvenor and Turner (1959a) used as an index of lactational performance the amount of milk obtained (stomach content) in a litter of six during 30 minutes nursing. Each litter was isolated from its mother for 10 hours. During this interval any milk present in stomachs of the young was digested and the mammary glands of the mother rat became turgid with milk. At the end of isolation, mother and young were reunited and the litter permitted to nurse 30 minutes. After the nursing period, the litter was weighed, killed by decapitation and sto- mach contents weighed. The weight of milk obtained was then expressed as a percent of litter body weight. Tucker and Reece (1963a) determined DNA and RNA content of the mammary gland throughout lactation and observed marked changes in these parameters that paral- leled changes in functional activity. Later Tucker (1966) correlated litter weight gain with total DNA, RNA and RNA/DNA, and observed the highest correlation coefficient between litter weight gain and total RNA (r=O.93). He suggested, that at a given time in a lactation, total RNA could be used as an index of lac- tational performance. Factors Affecting Lactational Performance In Hypophysectomized and Adrenalectomized Animals To evaluate the hormones required to maintain lac- tation, one test system would be experimental animals devoid of endogenous hormones that might participate in the lactational process. However, attempts to maintain full lactation in these types of animals is rather diffi- cult because the process of lactation involves the inter— action of several hormones. HypOphysectomy caused a marked depression of lac- tation in rats, goats, and sheep (Cowie, 1957), (Lyons et al., 1958), (Cowie and Tindal, 1960), (Cowie et al., 196“), (Denamur and Martinet, 1961). Bintarningsih et al. (1957) and Cowie and Tindal (1961) were able to obtain partial maintenance of milk secretion in hypophysectomized rats with injections of prolactin and either cortisol acetate or prednisolone acetate. In goats the combi- nation of ovine prolactin, bovine growth hormone, tri— iodo—L-thyroxine, zinc protamine insulin, and tablet implants of dexamethazone maintained milk secretion at the pre-Operative level when therapy was started immedi- ately after hypophysectomy (Cowie et al., 196A). Early investigations established that rats adrenalectomized early in lactation were unable to lac- tate sufficiently to maintain their young (Gaunt et al., 19u2) (Cowie and Folley, 1947). The predominant secre— tions of the rat adrenal cortex are aldosterone and corticosterone (Peron, 1960). Supplementation of adrenal— ectomized lactating rats with either aldosterone (Cowie and Tindal, 1955) or aldosterone and cortiocosterone (Anderson and Turner, 1963) maintained lactational per- formance at about 70-80% of normal. In a classical study Lyons (1958) developed in hypophysectomized, ovariectomized, and adrenalectomized rats a mammary lobular—alveolar system with a combination of estrogen, progesterone, adrenal corticoids, prolactin and growth hormone. Milk secretion based upon histo- logical appearance of the mammary gland was subsequently initiated by the injection of adrenal corticoids and pro- lactin. These studies in animals devoid of the sources of hormones thought to be involved in lactation, showed that it was possible to mimic the mammary growth re- sponse of pregnancy and to duplicate lactogenesis associ- ated with parturition. In Intact Animals Maintenance of milk secretion is a response to the coordinated actions of several pituitary hormones. Grosvenor and Turner (1959c) suggested that an increase in milk production associated with exogenous adminis— tration of a hormone(s) indicates a sub—optimal secretion of that hormone(s). On the other hand such responses to hormones may represent pharmacological responses and be unrelated to the secretion rate of the hormone. Johnson and Meites (1958) reported that daily in— jections of cortisone acetate in rats produced a signifi- cant rise in lactational performance during the first 18 days post-partum. In an experiment in which injections of hydrocortisone acetate, oxytocin and prolactin were given singularly and in all combinations, Talwalker _t _l. (1960) found that only hydrocortisone acetate increased milk synthesis significantly. Apparently suckling released optimal levels of oxytocin because administration of this hormone in combination with the corticoid did not increase litter weight gains. Exogenous prolactin was not galac- topoietic in the intact rat. This effect of prolactin in the rat was confirmed by Macdonald and Reece (1961). Several workers (Cotes et a1., 1949) (Flux et a1., 1954) (Shaw 33 al., 1955) (Brush, M. G., 1960) (Campbell §£.§l-: 196“) reported that adrenalcorticotropic hormone (ACTH) or glucocorticoids caused temporary decreases rather than increases of milk production in cows. How— ever, Roy (1947) reported that injection of ACTH increased milk production. Folley and Young (1940) showed that a single in— jection of partially purified prolactin in cows did not increase milk production during the declining phase of lactation. Similarly Cotes et a1. (1949) demonstrated that single injections of 40 mg of purified prolactin had no effect on the milk yield of lactating cows. Meites (1957) and Macdonald and Reece (1961) re- ported that injections of growth hormone in rats had no galactOpoietic effect although the mothers increased in body weight. Many workers (Cotes 32 31., 1949) (Donker and Petersen, 1951) (Donker and Petersen, 1952) (Chung 33 31., 1953) (Wrenn and Sykes, 1953) (Shaw, 1955) (Hutton, 1957) demonstrated that growth hormone is galactopoietic when administered to cows. Shaw (1955) suggested that exogenous growth hormone may increase the availability of milk precursors in the blood, increase the efficiency of milk secretion, and increase the amount of mammary tissue in the cow. Using milk yield (stomach contents) during a 30— minute nursing period as an estimate of lactational per- formance, a series of experiments from the laboratory of C. W. Turner over the last decade indicated that growth hormone, thyroxine, prolactin, parathyroid hormone, corticosterone and aldosterone increased milk secretion in the rat (Grosvenor and Turner, l959abc) (Djojosoebagio and Turner, l964ab) (Kumaresan and Turner, 1965) (Hahn and Turner, 1966) (Kumaresan 33 31., 1966). However, several of these reported stimulatory responses due to hormones such as growth hormone (Grosvenor and Turner, 19593) and prolactin (Kumaresan 3E 31., 1966) are in direct conflict with the results of others (Meites, 1957) (Talwalker 3: 31., 1960) (Macdonald and Reece, 1961) who used litter weight gain as an estimate of lactational performance. The stomach contents procedure evaluates lactational performance for a half-hour period of time, whereas litter weight gain estimates performance over a much longer period of time. The apparent discrepancy between results from the Turner group and results from others appears to be due to the method of measuring lac— tation. If growth hormone causes a 40% increase in milk yield as suggested by Grosvenor and Turner (l959a), then the stimulation should be reflected in a sizeable increase in mammary nucleic acid content (a quantative index of functional activity of the target tissue). Such an experi- ment should resolve the question whether growth hormone exerts a galactOpoietic effect on the rat mammary gland. Milk—ejection Reflex Ott and Scott (1910) were first to demonstrate the milk ejection reflex after an injection of unfractionated lO posterior pituitary extracts. Gaines (1915) described in detail the similarity of response to natural milk ejection and the ability of oxytocin to cause milk let- down when anesthesia blocked normal milk ejection in the bitch. Ely and Peterson (1941) suggested that a neurohormonal reflex mechanism existed whereby afferent stimuli from the teats during suckling or milking caused the release of neurohypophyseal oxytocin. Using the contractile response of the estrogenized uterus to esti- mate reflex liberation of oxytocin, Fuchs and Wagner (1963) observed that suckling of one or two rabbit pups released only 1-2 mu of oxytocin. However, suckling of an entire litter of rabbits (four pups) released 50—100 mu of oxytocin. The milk ejection reflex is partially dependent on afferent impulses originating from the sensory receptors of the mammary gland. The degree of dependency on these impulses to maintain milk production (synthesis and re- moval of milk) varies among species. Complete sensory denervation of the mammary gland had no detectable effect upon lactation in sheep (as cited by Grosvenor, 1964a) and goats (Denamur and Martinet, 1959ab, 1960) even when no supplemental oxytocin was given. In fact, Linzell (1960) transplanted entire goat mammary glands to the sides or necks of animals, which completely destroyed the peripheral nerve supply to the gland, and he noted 11 that milk secretion was 70 to 90 percent of that expected from glands 13_situ. In contrast severance of the spinal cord resulted in almost total cessation of milk synthesis in the cat (Beyer 33 31., 1962) and rabbit (Mena and Beyer, 1963). However, lactation in spinal cord—tran- sected rabbits is partially maintained if oxytocin is injected periodically, and further improvement occurs if ACTH or prolactin is administered with oxytocin (Mena and Beyer, 1963). Grosvenor (l964a) reported that oxytocin and prolactin injections in the spinal cord— sectioned rat partially maintained lactation. Release and Synthesis of Hormones Since prolactin and glucocorticoids are required to maintain milk synthesis in the spinal sectioned and in the hypophysectomized rat, it was of interest to determine how lactation and the suckling stimulus affects release and synthesis of hormones associated with lactation. Pituitary prolactin concentrations during lactation exceeded those observed during the estrous cycle, pseudo- pregnancy, or pregnancy in rats (Reece and Turner, 1936) (Grosvenor and Turner, 1958) (Minaguchi and Meites, 1967). Hurst and Turner (1942) reported a marked rise in pro- lactin content of the pituitary glands of post-partum rats. It reached a peak on day 3 of lactation and de— clined precipitously thereafter. Grosvenor and Turner l2 (1958a) measured prolactin concentrations in pituitaries from rats which had been deprived of their litter for 10 hours. Prolactin concentration increased from day 2 to day 6 of lactation but it declined toward the end of lactation. These results agreed with the original ob- servations of Reece 32,313 (1939). Relative to chronic nursing stimulation, Meites and Turner (1942ab) reported that parturient rats and rabbits which were not suckled for the first week or 10 days post—partum contained less pituitary prolactin than suckled controls. Recently, Tucker 33 31. (1967b) ob- served that chronically nursed rats contained signifi— cantly more prolactin than non—nursed controls. In addition increasing chronic nursing intensity from two to six pups during lactation increased pituitary prolactin concentration in lactating rats (Tucker 33 31., 1967a). Reece and Turner (1937a) provided the first direct evidence that the suckling stimulus caused prolactin re— lease. They reported a decrease in pituitary prolactin concentration in rats when mother and pups were reunited for a 3-hour period following a l2-hour interval of non- suckling. Ligation of the galactophores did not prevent the decrease in prolactin due to suckling, which sug- gested that the suckling stimulus rather than removal of milk was the factor responsible for prolactin release (Reece and Turner, 1937b). Later, Grosvenor and Turner 13 (1957ab, 1958b), Grosvenor (1965), Grosvenor 33 31. (1965) and Moon and Turner (1959) showed that 30 minutes of suckling, following 10 hours isolation, were sufficient to induce a significant decrease in pituitary prolactin if applied during the first two—thirds of lactation, but failed to do so on day 21 of lactation. Grosvenor and Turner (1957ab) determined that one—half of the pre- suckling level of prolactin was restored within 2.5 hours post-suckling. Complete restoration was not accomplished by 9.5 hours post—suckling. However, a more comprehen- sive inventigation indicated that complete restoration was achieved within 8 hours after suckling (Convey, 1968). Grosvenor 33 31. (1967) reported that 2 minutes of suckling was as effective as 30 minutes in evoking the discharge of prolactin. Neither 2 nor 30 minutes of suckling by six pups induced a fall in pituitary prolactin if the preceding period of non—suckling was of 16 hours duration. As previously described suckling can cause a release of prolactin from the anterior pituitary and of oxytocin from the posterior pituitary. It is not surprising that Petersen (1942) postulated a causal link between the two processes. He suggested that oxytocin from the posterior pituitary may be responsible for the rapid decrease in pituitary prolactin observed after suckling. Grosvenor and Turner (1958b) demonstrated that oxytocin injections 30 or 60 times physiological levels did not alter pro- lactin content in pituitaries of lactating rats. Likewise, l4 oxytocin did not affect the production of prolactin from anterior pituitaries cultured 13 31333 (Nicoll and Meites, 1962). Benson and Folley (l956ab, 1957) reported that oxytocin retarded the involution of the secretory epithelium which occurs after removal of the young. They suggested that oxytocin stimulated secretion of prolactin from the anterior pituitary. These obser- vations on maintenance of mammary structure were subse- quently confirmed by others including Meites (1958, 1959), Ota and Yokoyama (1958), McCann 33 31. (1959) and Meites and Nicoll (1959). The results of Ota 33 31. (1962, 1965) indicated that administration of oxytocin preserved only the structural integrity of the mammary tissue. On the other hand, prolactin injections not only prevented structural involution but also maintained some functional activity of the tissue. Since the above experiments were performed in intact rats, the possibility existed that oxytocin may have exerted some effect via the anterior pituitary. When oxytocin was injected with prolactin and ACTH into hypopys— ectomized lactating rats, milk secretion (based on histo- logical ratings of the mammary tissue) was observed in 92% of the rats (Meites and HOpkins, 1961). In comparison, milk secretion was observed in only 21% of the rats that received only prolactin and ACTH. These results were 15 interpreted to mean that the favorable effects of oxytocin in retarding mammary involution were exerted directly on the mammary gland. Using involution of the thymus as an index of in- creased ACTH secretion, Gregorie (1947) showed that suckling maintained ACTH secretion in ovarietomized rats. The results reported by Tucker 33 31. (1967a) indicated a trend for pituitary ACTH to decrease with advancing stages of lactation. Various measurements of adrenal corticol function such as adrenal weight, cholesterol and ascorbic acid content, and peripheral plasma levels of corticoids have been used to estimate changes in ACTH secretion of lactating rats. Reports on rats are contra- dictory as to whether adrenal weights increase or decrease during lactation. Bearn 33 31. (1960) indicated that dur- ing lactation there was a significant decrease in adrenal weight when compared with virgin controls at estrus, but there was no significant change when compared with con— trols at other stages of the estrous cycle. In contrast, Poulton and Reece (1957) reported that adrenal activity increased during lactation, based on the evidence that adrenal cholesterol concentrations were maintained at a low level throughout lactation. Anderson and Turner (1962) measured adrenal ascorbic acid levels during pregnancy and lactation in rats. Lower adrenal abcorbate levels found during early lactation were interpreted to 16 represent a high level of adrenocortical function. After day 6 of lactation adrenal function decreased to the level of virgin controls. However, there is some question as to the validity of using adrenal ascorbate measurements as an estimate of increased corticotropin release (Hedner and Rerup, 1962). To my knowledge, direct measurements of adrenal content of corticoids in the lactating rat during advanced lactation are not available. Chronic suckling stimulation (12 pups/litter) dur- ing lactation caused a progressive increase in plasma corticosterone from parturition to day 12 of lactation. And these elevated levels were maintained to day 21 of lactation (Gala and Westphal, 1965). Concurrently, there was a decrease in corticoid binding globulin (CBG) activity of blood plasma from parturition to day 3 of lactation, and this low level was maintained until day 21. Further- more, rats receiving a lower nursing intensity (4 pups/ litter) had smaller plasma levels of corticosterone and greater CBG activity compared with more intensely nursed rats. The ACTH activity of anterior pituitaries of goats and ewes declined significantly from pre—milking levels to a minimum value 30 minutes after milking (Denamur 33 31., 1965). Within 90 minutes post—milking, pre- milking levels were regained. In contrast, pituitary ACTH did not change after an 8-hour non-nursing period 17 followed by a 1-hour suckling period in rats (Taleisnik and Orias, 1966). A significant depletion of growth hormone and thyroid stimulating hormone from the anterior pituitary, following an overnight non-nursing period with subse- quent acute nursing stimulation, occurred in lactating rats (Grosvenor, l964b, Grosvenor 33 31., 1968). Further- more, varying the chronic nursing intensity from 0 to 2, 6, or 12 pups progressively decreased pituitary growth hormone content whereas mammary development and metabolic activity increased (Tucker and Thatcher, 1968). These results suggested that growth hormone and thyroid stimu- lating hormone secretion rates are probably enhanced during lactation. Intensi3y of Nursing Stimuli and the Extension of Lactation Milk secretion is essentially a continuous process whereas the act of suckling or milking is intermittent. It is therefore possible that the nursing stimulus triggers the release of additional amounts of the galactopoietic hormones to maintain mammary function for several hours. Maintenance of an intense nursing stimulation, which may increase the secretion of galactOpoietic hormones, has been used in several studies to extend lactation. 18 According to Selye and McKeown (1934) regular replacement of young with foster litters prolonged lactation. With the use of foster litters, Nicoll and Meites (1959) partially maintained lactation to 70 days in rats. A steady decline in milk yield (litter weight gain) was evident in these animals. In marked contrast, Bruce (1958, 1961) observed in three rats almost constant lactational performance for periods up to one year when a frequent litter replacement regime was employed. Tucker and Reece (1963b) using litter replacements every 21 days maintained structural integrity but not func- tional integrity of the mammary gland. However, Moon (1962) noted that by day 28 of lactation, after replace— ment of original litters with foster litters on day 14 of lactation, there was a significant reduction in cell numbers (DNA) as well as in milk production. MATERIALS AND METHODS Experimental Animals and Design Experimental animals were primiparous rats of the Sprague-Dawley strain. They were maintained at 2431C and subjected to illumination between 5 AM and 7 PM. All rats received a 21.2% protein diet (Appendix I) and water 33 libitum. Experiment l.--Lactationa1 Performance of Intensely Nursed Rats During Ex- tended Lactation The purpose of this experiment was to determine if intensive nursing would maintain mammary cell numbers and secretory activity during an extended lactation. A second objective was to relate changes in mammary cell number and synthetic activity with prolactin and ACTH content in the pituitary. On the third day of lactation thoracic teats of 48 rats were ligated, litter size adjusted to six pups, and mother rats weighed and assigned to one of four groups to be killed either on day 16, 20, 28, or 36 of lactation. Both l6-day-old original litters and foster litters were replaced every 4 days with l2-day-old foster litters. Cumulative litter weight gains were recorded between 19 20 days 13 and 16 of age for all litters. Final body weights of lactating rats were recorded at time of killing, and the nucleic acid content determined on six abdominal- inguinal mammary glands. Twelve anterior pituitaries were collected from each of two additional groups of rats killed on either day 20 or 36 of lactation. Prolactin potency of the 12 pooled pituitaries of each group was estimated by the method of Reece and Turner (1937a). The ACTH potency of the two groups of pituitaries was determined by the method of Saffran and Schally (1965). Litter weight gain and nucleic acid content of mammary tissue were also deter- mined in these two groups. In addition, lactic acid con— tent of mammary glands was determined (Barker and Summer- son, 1941) as another index of mammary function (Cowie and Folley, 1961). Results were analyzed by analysis of variance. Experiment II.--Lactationa1 Performance of Intensely Nursed Rats Igjected with Oxytocin During Extended Lactation The purpose of experiment II was to determine if oxytocin injections would increase lactational performance. On the third day of lactation thoracic teats of 24 rats were ligated, litter size adjusted to six pups and mother rats weighed and assigned to one of two treatment groups. Oxytocin (Syntocinon Sandoz, Pharmaceuticals) 21 injections were initiated at day 16 of lactation and con- tinued to day 32 of lactation. One unit of oxytocin was given three times daily at 8:00 AM, 4:00 PM and 12:00 PM. Controls received 0.1 ml of 0.85% sodium chloride. At day 16 of lactation original litters were replaced with 8—day—old foster litters and subsequent 16-day-old litters, at day 24 of lactation, were again replaced with 8-day-old foster litters. This litter replacement regimen was intended to maintain an intense suckling stimulus throughout the treatment periods. Litter weights were recorded daily during lactation. Cumulative litter weight gains were calculated from day 16 to 24 and from day 24 to 32 of lactation. All rats were killed on day 32 of lactation by decapitation and the six abdominal-inguinal glands removed, weighed, trimmed and stored in 0.25 M sucrose at -200 until analyzed for nucleic acid content. Body weights, an— terior and posterior pituitary, adrenal, ovary, and uterus weights were recorded at autopsy. Unless stated otherwise, these experimental details (teat ligations, litter size adjustments, litter replace- ment regimen, litter weight gains and routine autopsy procedures) were applicable in all subsequent experiments. 22 Experiment III.--Lactationa1 Eerformance of Intensely Nursed Rats Injected with Hydrocortisone Acetate (H), Prolactin (P), and Growth Hormone (G) During Ex- tended Lactation The purpose of Experiment III was to determine if the exogenous administration of hydrocortisone-2l—acetate (Sigma Chemical Co.), NIH-P—SB ovine prolactin, and NIH-GH-SB ovine growth hormone singularly or in all possible combinations would maintain milk synthesis during extended lactation. Sixty-three rats were divided among eight treat— ments (H, P, G, H and G, H and P, P and G, H and P and G, saline) in a 2X2X2 factorial experiment. All hormone injections were initiated at day 16 of lactation and continued daily to day 32 of lactation (autOpsy). In- jections were given daily at 8:00 AM and 5:00 PM. The total daily doses injected were 0.5 mg of H, 2 mg of P and 1 mg of G. Controls received 0.85% sodium chloride. Due to a limited amount of available prolactin there were unequal numbers of rats in the treatment groups. Conse— quently, a least squares statistical analysis of the data was performed. Experiment IV.——Lactational Performance of Intensely Nursed Rats Bearing Iso- transplanted Pituitaries During Extended Lactation Experiment IV was conducted to verify that prolactin was not limiting to lactational performance. 23 On the third day of lactation, five anterior pituitaries from mature female rats were transplanted under the left kidney capsule of each of 14 lactating recipient rats. Kidneys of sham-operated controls (11) were manipulated surgically in a manner similar to that of rats receiving pituitary transplants. Both groups of rats were killed on day 32 of lactation. Experiment V.--Lactationa1 Performance of Intensely Nursed Rats Injected with 9—Fluoroprednisolone Ace- tate (Predef) During Ex- tended Lactation In an initial experiment, injections of Predef (Upjohn Co.) were given twice daily from day 16 to 32 of lactation at doses of either 10 pg, 50 pg or 100 pg per day. The numbers of rats in the 10 pg, 50 pg, 100 pg and saline injected control groups were 9, 10, 8 and 10, respectively. A second experiment was conducted in which the doses of Predef were varied during the injection period. In one group of rats, Predef was injected at a daily dose of 50 pg from day 16 to 23, and at 100 pg from day 24 to 32 of lactation. A second group of rats received saline injections (0.85%) between day 16 and 23 and 50 pg of Predef daily between day 24 and 32 of lac- tation. A third group (controls) received saline in— jections (0.85%) throughout the 16-32 day interval. At 24 the time of autOpsy the livers of rats from the first and third groups were weighed and prepared for subse- quent 13 vitro incubations. 13 vitro incubations were conducted to evaluate the ability of the liver of Predef— and saline-treated rats to metabolize cortisol. The procedural details for liver preparations and 13 vitro metabolism studies will be outlined in a subse— quent section (page 34). Experiment VI.--Measurement of Adrenal Function and Corticoid Binding3Globulin (CBG) Activity of Intensely Nursed Rats Dur- Inngxtended Lactation To determine if adrenal function and CBG activity change during lactation, 20 rats per group were killed on day 16, 24 and 32 of lactation. An additional group of 20 virgin rats was killed in metestrus to determine if the experimental procedures resulted in resting plasma levels of corticosterone comparable to that reported in the literature. Rats were maintained at 24C:lC and grouped (4 per cage) for at least 2 weeks under conditions of controlled lighting (fluorescent illumination from 7:30 AM to 8:30 PM) in an isolated room. After the 2- week conditioning period, female rats were co—habited with male rats. When diagnosed pregnant by palpation rats were housed in individual cages. Routine rat room maintenance and experimental manipulations were carried out between the hours of aagdfl‘ 25 1:00 and 4:00 PM. Vaginal smears were taken from the virgin rats daily. Rats were killed by decapitation within 10 seconds from the time the cage was first handled. In order to reduce plasma corticosterone vari- ations due to circadian rhythm, rats were decapitated between 8:00 and 8:30 AM. Trunk blood was collected in heparinized 50 ml beakers, transferred to 15 ml centrifuge tubes and centrifuged at 12,350xg for 10 minutes at 50. Plasma was frozen at -200 until analyzed for corticosterone. Adrenal glands were trimmed of accessory fat and connective tissue, and frozen in 2 ml of homogenization fluid (0.85% NaCL in 20% ethanol) for subsequent corticosterone analyses. Corticosterone concentrations in individual rat plasma samples were measured by the method of Silber 33 31. (1958). Corticosterone determinations of adrenal glands from each rat were determined by the procedure of Moncloa 33 31. (1959). Gel filtration was used to mea- sure CBG activity as described by Doe 33 31. (1964). Estimates of CBG activity were made on plasma samples pooled within treatments. Biochemical Parameters Measured in Mammary Glands The six abdominal-inguinal mammary glands were removed, dissected free of extraparenchymal fat and connective tissue and weighed. The glands were covered 26 with ice—cold 0.25 M sucrose and stored at —20C for subsequent analyses. At the time of analysis the samples were thawed at 4C. The mammary glands were homogenized for 2 minutes in distilled water at 4C to produce final concentrations of 50 mg of mammary tissue per ml for subsequent analysis. The analytical procedure for nucleic acids was based on modifications of the Schmidt and Thannhauser (1945) procedure described by Tucker (1964): 1. Two ml of the 100 mg mammary gland homogenate was pipetted into a 16 ml polypropylene centrifuge tube. Ten ml of 95 per cent ethyl alcohol was added, the tube capped and shaken at room temperature for 12 hours. 2. The tube was then centrifuged at 35,600xg for 15 minutes at 50 and the supernatant fluid discarded. 3. Two m1 of anhydrous ether was added and mixed thoroughly with the tissue residue and centri- fuged as above. Supernatant material was dis- carded and the tissue residue dryed in a fume hood to form a firm tissue pellet. 4. Ten ml of methanol: chloroform (2:1) was added to the sample pellet, shaken for 24 hours, centrifuged and the supernatant fluid discarded. 10. 27 Ten ml of anhydrous ether was added, shaken at room temperature for 12 to 24 hours, centri- fuged and the supernatant fluid discarded. Five ml of ice-cold 10% trichloracetic acid (TCA) was added to the residues of the mammary gland, mixed, centrifuged and the supernatant ,pfigg fluid discarded. This step was repeated. : y Five ml of ice-cold 75% ethanol saturated 4 with sodium acetate was added, mixed, centri- fuged and the supernatant fluid discarded. i 3 Two m1 of 1N potassium hydroxide was pipetted into each sample, the tubes capped with rubber stoppers and placed in a 370 oven for 15 hours. The tubes were cooled in ice water, 0.3 ml of ice-cold 6N hydrochloric acid and 5 ml of ice- cold 10% perchloric acid (PCA) added, mixed, centrifuged and the supernatant fluid (RNA fraction) of the mammary homogenate saved. Five ml of ice-cold 5% PCA was added, mixed, centrifuged and the supernatant fluid saved and pooled with the previous RNA fraction. This step was repeated. The combined supernatant fluids (RNA fraction) of the mammary homogenates were brought up to 20 ml with 5% PCA and mixed. One ml of the RNA fraction was then pipetted into a clean 16 ml test tube containing 2 m1 of 5% PCA ll. l2. 13. 28 and 3 ml of freshly prepared orcinol reagent (Appendix II) was added. The tube was then capped and boiled for 30 minutes. Color development was measured in a Beckman DB spectrophotometer (Beckman Instrument Co.) after 15 minutes at 670 mp. The RNA content s n _ IL. .bfi‘q ~. of the sample was calculated from a standard curve derived from pure yeast RNA (Worthington “Lu-’4 ”.3...“ Biochem. Corp.). Five ml of ice-cold 5% PCA was added to the j precipitate of the mammary gland sample from ' step nine, mixed, incubated at 70C for 15 minutes, cooled to SC, centrifuged and the supernatant fluid (DNA fraction) poured into 25 ml graduated test tube. Five ml of ice—cold 5% PCA was added to the precipitate, mixed, centrifuged and the super- natant fluid poured into the 25 ml graduated test tube. This step was repeated. The supernatant fluid (DNA) from the mammary gland homogenate was brought up to 25 ml with 5% PCA in a graduated test tube. The Optical density was read in a Beckman DB spectrophoto- meter (Beckman Instrument Co.) at 268 mp, and the DNA content of the sample was calculated from a standard curve derived from pure highly polymerized DNA (Worthington Biochemical Corp.). 29 The Barker and Summerson (1941) chemical assay was used to measure the lactic acid content of the mammary gland. The following procedure was adopted: 1. Mammary gland homogenate containing 200 mg of tissue (4 ml) was pipetted into a 25 ml test tube containing 2 m1 of 20 per cent ZnSOu ° 7H20 and 8.32 ml of 0.3N Ba(OH)2. The contents of the tube were mixed-and allowed to stand for 30 minutes, re-mixed and centrifuged at 85xg for 15 minutes at room temperature. 2. Eight m1 of supernatant fluid were pipetted into a tube containing 1 ml of H20 and 1 m1 of 20 per cent CuSOu. One gram of powdered Ca(OH)2 was added to the tube. 3. The test tube was sealed with parafilm, mixed and allowed to stand for 30 minutes at room temperature with intermittent shaking. After centrifugation 1 ml of supernatant fluid was pipetted into a 25 ml test tube. Six ml of ice-cold H280“ was added slowly from a buret to the tube which was held in an ice bath. 4. The test tube was boiled 5 minutes and then immediately cooled in ice water. A 0.05 ml volume of 4% CuSOu . 5H 0 and 0.10 ml of 1.5% 2 p—hydroxydiphenyl in 0.5% NaOH was added to 30 the tube and mixed immediately. The contents were then heated at 30C for 30 minutes with shaking at 15—minute intervals. After the 30-minute incubation, the contents were boiled in a water bath for 90 seconds and cooled to room temperature. real Optical density was read in a DB spectrophotometer E V (Beckman Instrument Co.) at 565 mp. A standard curve , was obtained by taking different concentrations of lactic i acid (Sigma Chemical Co.) standard (in a 4 ml volume) 1 , “j‘ through the same steps as the unknown sample starting at step one. Bioassay of Pituitagy Hormones The 12 rat anterior pituitaries, from each of two groups of rats which were killed on either day 20 or 36 of lactation and stored at —20C, were thawed and homo- genized in a Potter-Elvehjem homogenizer in 10 m1 of 0.85% NaCl in pyrogen-free distilled water. The volume of the homogenate was adjusted to a final concentration of 4 mg of rat pituitary per ml. Homogenates were centri- fuged for 15 minutes at 600xg at 5C and supernatant fluids used in bioassays for prolactin and ACTH. Prolactin Prolactin potency was assayed in White King pigeons by the method of Reece and Turner (1937a). Pigeons of both sexes were obtained from Cascade Squab Farm, Grand 31 Rapids, Michigan, at 5-8 weeks of age and housed in a room artificially illuminated between 6 AM and 8 PM. They were fed mixed grain and water 33 libitum. The breast feathers were plucked on the day following arrival and pigeons were used for bioassay 2 days later. Low doses of pituitary homogenate (0.1 mg/day) and standard NIH-P-S6 ovine prolactin (0.25 pg/day) were injected into right and left crop sac areas, respectively, of 8 birds. High doses of pituitary homogenates (0.4 mg/ day) and standard prolactin l pg/day) were injected similarly into 8 additional birds. Total doses injected over a 4-day period for the low and high doses of pitui» tary homogenate were 0.4 mg and 1.6 mg, respectively. Comparable 4-day total doses for standard prolactin were 1 pg and 4 pg. The test materials were injected intradermally in a 0.1 ml volume with a 1.0 ml tuberculin syringe and a 27 gauge needle. Twenty-four hours after the last in- jection pigeons were decapitated, crop sacs removed and the area of the response rated visually in terms of Reece-Turner (R-T) units. A crop sac response of 2.1 cm in diameter constituted one R—T unit. The scale ranged from 0.0 to 4.0 R—T units, increasing in multiples of 0.25. The prolactin potency of the unknown samples was computed from a parallel line assay comparison with 1...... -14.,— IF-mm.-. 1.49,..- . . .. 7'” 32 standard prolactin (Bliss, 1952). Calculation of the potencies and the criteria for the validity of an assay, such as the test for nonparallelism, lambda (A), standard error and 95% confidence interval were performed based on the unknowns and standards assayed at one time. ACTH Bioassay ] “ The method of Saffran and Schally (1965) was used to assay ACTH potencies of the two groups of pituitaries. Eight male adult rats were required per assay. Each rat was anesthetized with sodium pentabarbital at a dose A 41 of 5 mg per 100 g of body weight. Adrenals were removed from each rat and carefully freed of adhering fat with fine scissors. Each pair of adrenals was weighed and the glands placed into a petri dish containing some in- cubation medium (Appendix III). Each adrenal pair was cut into quarters with scissors; thus each donor male rat supplied eight quarters. One quarter from each of eight rats was placed on one of eight sectors of a filter paper circle previously moistened with incubation medium. Thus, each sector contained a quarter of an adrenal from each of eight rats--a procedure which minimized rat to rat variation in subsequent incubations. The eight adrenal quarters in each sector were weighed together and placed in 25-ml Erlenmeyer flasks containing 1.5 m1 of incubation medium. Each flask was flushed with a mixture of 95% 02-5% C02 and shaken in 33 a water bath at 380 for 1 hour. At the end of the pre- incubation period flasks were removed from the bath and the medium aspirated. An additional 0.5 ml of incu— bation medium was added to each flask, mixed with the adrenal quarters and the medium discarded. An addi- tional 1.4 ml quantity of incubation medium was added to each flask. Two flasks were given the low dose (3 mp/0.l ml) of standard (ACTH, Mann Research Laboratory, Inc.) and two additional flasks the high dose (12 mp/0.l ml) of standard ACTH. The remaining two pairs of flasks re- ceived the low and high doses of pituitary homogenates. The low and high doses of pituitary homogenate for the rats lactating 20 days were 0.13 mg/0.1 ml and 0.4 mg/ 0.1 ml, respectively. The corresponding doses for the day 36 of lactation treatment group was 0.4 mg/0.l ml flh-aux _4 .....__.. ._ -Am‘...‘ i i if “‘3“ and 1.6 mg/0.l ml, respectively. Flasks were gassed with 95% 02-5% C0 and replaced in the water bath for a 2-hour 2 incubation at 380. After this incubation the flasks were removed and a 1 ml aliquot of medium transferred from each flask to a corresponding 2 ml volumetric tube con- taining purified redistilled methylene chloride. Tubes were then stoppered, shaken and centrifuged at 480xg to separate the aqueous and methylene chloride phases. An aliquot of the lower methylene chloride phase was removed and Optical density was measured in a Beckman 34 DB spectrOphotometer (Beckman Instrument Co.) at 240 and 255 mp against a blank which contained methylene chloride that had been extracted with 1 ml of incubation medium. The response at each dose was estimated by: optical density2u0 - optical denSlty255 x 10 1 mg adrenal ACTH potency and assay validity tests were calculated by the method of Bliss (1952). [Weir—HT?¢_. “ c.-_s ’V‘AJ q ”a N1; .4‘ In Vitro Metabolism of Cortisol 3y the Liver To determine if livers from Predef-treated rats metabolized cortisol to a greater degree than livers from saline-injected controls, the following 13_31333_proce- dures were developed with the cooperation of Dr. Robert Cook (MSU Dairy Department). Preparation of Liver Microsomes At the time of necropsy, each liver was weighed and a 0.5 g section frozen at -2OC for subsequent liver pro- tein measurements. The remaining part of the liver was homogenized, for two 3—second intervals with a Waring Blender, in 4 volumes of 0.1 M phosphate buffer contain- ing 20% glycerol (phosphate-glycerol buffer), pH 7.4 (Appendix IV). From the time of necropsy to the time of homogenization, livers were stored in ice-cold phosphate-glycerol buffer. The homogenate was poured into 40 m1 polypropylene tubes and centrifuged at 9000xg for 20 minutes at 50. Tubes were removed and the supernatant fluid decanted and saved. The 9000xg supernatant fluid was then centri- fuged in 10 ml tubes at 105,000xg for 60 minutes at 5C. The subsequent supernatant fluid was decanted and frozen at -2OC in plastic capped tubes. Microsome pellets if; "* 7y?“ “1 {5 (105,000xg pellet) were rinsed once with the phosphate- glycerol buffer and the pellets transferred to a glass grinding vessel containing a volume of phosphate-glycerol im 2“ i buffer equal to 0.5 ml times the number of microsome pellets. The pellets were homogenized with a Teflon power driven pestle by two movements up and down the. vessel. The microsome homogenate was then frozen in a screw cap plastic tube at —2OC. On the day of assay microsome preparations within treatments were pooled by pipetting 2 m1 aliquots from each microsome preparation. The 105,000xg supernatant preparations within each treatment were also pooled. In Vitro Incubation Procedure Pooled liver microsomes and 105,000xg supernatant fluids from the Predef- and saline—treated rats were each incubated with 1 pM (1.0912 pCi/mg), 2 pM (0.548 pCi/mg) and 5 pM (0.219 pCi/mg) of cortisol (Nuclear- Chicago Corp. and Sigma Chemical Co.). Isocitric dehydrogenase, isocitric acid and NADP were used as a NADPH generating system during the incubation. 36 Prior to the assay 30 mls of 0.1 M phosphate buffer, pH 7.4 without glycerol (Appendix V), was gassed for 5 minutes with 95% 02—5% C0 The incu— 2. bations were carried out in 50 m1 glass stoppered round bottom tubes. The treatment flask contained: mls of 0.1 M phosphate buffer ml of Mg012 (15 pM) m1 of NADP (1.00 pM) ml of isocitric acid (10 pM) ml of isocitric dehydrogenase (3 units) 2 or 5 pM of cortisol-cortisol-l4C ml of pooled microsomes ml of pooled 105,000xg supernatant I—‘l—‘l—‘OOOON o as. o o o o o NFJHFJUI OO O ml Total U1 The reaction blank flasks contained: 1 ml of Mg012 (15 pM) 1 ml of NADP (1.00 pM) .1 ml of isocitric acid (10 pM) 2 ml of isocitric deh drogenase (3 units) 0 5 r 5 pM of cortisol-1 C-cortisol ml of 0.1 M phosphate buffer 5.0 ml Total The incubations were initiated by the addition of pooled microsomes and pooled 105,000xg supernatant fluid to the reaction flask. Both the reaction and blank flasks were gassed immediately for 30 seconds with 95% 02-5% C02 and then stoppered. A11 flasks were in- cubated for 1 hour at 370. Reactions were terminated by the addition of 1 m1 of l N H01. The incubation media and contents were directly shaken with 30 ml of methylene chloride for 30 minutes, and centrifuged at l651xg for 37 15 minutes to separate the aqueous and methylene chloride phases. The aqueous phase was aspirated into a 10 ml graduated test tube and the volume recorded. The radio- activity in 0.2 ml of the aqueous phase was counted in a Model 6725 Nuclear Chicago liquid scintillation spectro— meter using the scintillation fluid listed in Appendix VI. Twenty—four ml of the methylene chloride extract was pipetted into a 50 ml conical centrifuge tube and taken to dryness under nitrogen in a sand bath at 45C in a fume hood. The dried residue was redissolved in 1 ml of ethanol. Fifty pl of the ethanol extract was spotted on Eastman silica gel thin layer chromatography plates (Eastman Chromatogram sheet 6060) and chromato- graphed in chloroform: methanol: water (90:10:l) for 45 minutes. The cortisol substrate spot was visualized with ultraviolet light and marked on the plate. Excluding the cortisol substrate spot, 1 cm strips were cut from the origin to the solvent front. Each strip, including the substrate spot, was placed directly in scintillation vials and radioactivity was quantified in 10 ml of the scintil— lation fluid described in Appendix VII. The distribution of DPM from the origin to the sol- vent front was a quantative measurement of the amount of cortisol metabolized as well as the amount of polar or non-polar metabolites formed. Since the total amount of cortisol and its specific activity were known, direct 38 calculations were made of the amount of cortisol meta— bolites produced per hour. The amount of aqueous cortisol and its metabolites were also calculated by isotope dil- ution. The amounts of liver, microsomal, and 105,000xg supernatant proteins were measured by the method of Lowry (1951) and used to calculate the specific activity of the enzyme system. Fluorometric Assay of Plasma and Adrenal Corticosterone A modification of the method of Silber 33 31. (1958) was used to measure corticosterone in rat plasma. The procedure of Moncloa 33 31. (1959) was followed to measure corticosterone in rat adrenals. A Turner model 111 fluoro- meter, equipped with filters providing narrow band excit- ation light at 470 mp and emission at 530 mp, was used in all corticosterone measurements. These procedures possess- ed the degree of sensitivity that was required to measure corticosterone concentrations in plasma of individual rats. Plasma Corticosterone Procedure Corticosterone-4-luC (9148 DPM) (Nuclear Chicago Corp.) was added to and mixed with a 0.5 m1 plasma sample. The sample was brought to a volume of 1 ml with double distilled water. Each sample was analyzed in duplicate in a heavy walled 15 ml ground glass stoppered centrifuge 39 tube. Six m1 of redistilled methylene chloride was pipetted into the tube and the contents extracted by a gentle rotation for 3 minutes and then centrifuged at 1651xg for 3 minutes. The upper steroid hormone-free aqueous layer was aspirated and discarded. Partial purification of the steroid extract was accomplished by a vigorous extraction of the methylene chloride for 30 seconds with 0.75 ml of cold 0.1 N NaOH. The samples were centrifuged at l65lxg for 3 minutes and the 0.1 N NaOH layer was discarded. Five ml of the original methylene chloride extract was transferred to a clean 15 ml glass stoppered centrifuge tube from which 1 ml was pipetted into a scintillation vial. Four m1 of fluorescing reagent (Appendix VIII) was added to the 4 ml methylene chloride extract. Cortico- sterone was extracted into the fluorescing reagent by shaking the tubes vigorously for 1 minute. The mixture was then centrifuged at l651xg for 2 minutes after which the methylene chloride layer was discarded. The reagent— hormone mixture was allowed to develop for 15 minutes from the end of the extraction before fluorometric determi- nations were recorded. A reagent blank, consisting of 1 ml of distilled water, was carried through the entire assay procedure for each assay. This served as a cor- rection factor for background fluorescence. Plasma corticosterone concentrations were calculated from a 40 linear regression equation based on the fluorescence of 4 corticosterone standards (0.02 pg, 0.04 pg, 0.08 pg and 0.16 pg dissolved in 20 pl ethanol) which were analyzed with every assay. Extraction efficiency was determined from the radioactivity in the 1 ml of methy- lene chloride after being dried under nitrogen and counted in the liquid scintillation spectrometer. To verify the relative accuracy of the fluorometric procedure for corticosterone quantitation in rat plasma, the fluorometric assay and the colorimetric blue tetra- zolium reaction of Elliot 33 31. (1954) were used to determine the corticosterone concentration of a pooled plasma sample. For the blue tetrazolium reaction 50 ml of plasma was mixed with 25,000 DPM of corticosterone-4-luC and extracted with 200 ml of methylene chloride for 6 minutes by gentle rotation in a separatory funnel. The methylene chloride extract was washed vigorously with 50 m1 of cold 0.1 N NaOH and reduced to dryness in a vacuum evaporator. Using methylene chloride the residue was spotted on a silica gel thin-layer chromatography plate. The tube was rinsed with ethanol and these washings were also spotted on the plate. After two dimensional chroma- tography in hexanezethyl acetate (5:2) and chloroform: methanolzwater (188:12zl) the corticosterone spot was identified by autoradiography over night using Kodax Medical X—ray film (Eastman Kodax Co.). The area on the 41 silica gel plate containing the lLAC-corticosterone was scraped into a 12 ml centrifuge tube. Then the cortico- sterone was eluted from the silica gel with a double washing of chloroformzmethanol (2:1). The eluate was evaporated under nitrogen at 450, and the residue re- suspended in 2.0 m1 of ethanol, and corticosterone was rwn; measured by the blue tetrazolium reaction. Extraction é efficiency was determined by measuring the radioactivity 5 present in an aliquot of the 2 m1 sample used to quantify E the mass. Mass of corticosterone was corrected for losses i i of radioactivity. Triplicate corticosterone analyses of 0.5 ml aliquots of the same plasma pool were determined by the fluoro- metric procedure outlined previously. The level of corticosterone per 100 ml of plasma, estimated by the fluorimetric method, was 21.55:0.ll pg (mean and standard error) and 23.66 pg using the blue tetrazolium method. Since the fluorimetric value was 91% of the blue tetra- zolium value (considered 100%), I considered these values close enough to justify the use of the fluorimetric method. The degree of precision of the fluorimetric assay was determined by repeated analyses of a pooled plasma sample from stressed rats. Six determinations were made on the plasma over a period of 2 weeks. The resultant estimate was 84.52:0.83 pg corticosterone/100 ml of plasma (mean and standard error of the mean). 42 Adrenal Corticosterone Procedure Adrenal glands were thawed and the two glands trans— ferred to a glass grinding vessel for homogenization in 4 ml of 0.85% saline in 20% ethanol (homogenization fluid). The homogenate was transferred to a 20 ml test tube; and diluted to 10 ml with the homogenization fluid. Dupli— cate aliquots of the homogenate (0.5 ml and 1.0 ml) were assayed. The samples were pipetted into 15 ml glass ”c (9148 stoppered centrifuge tubes. Corticosterone-4-l DPM) (Nuclear Chicago Corp.) was added to each sample and mixed thoroughly. Each sample was adjusted to 1 ml volume with the homogenization fluid. Five ml of petroleum ether was added to each sample and the contents extracted vigorously for 1 minute and then centrifuged at l651xg for 3 minutes. The upper petroleum ether layer was aspirated and discarded. Six ml of redistilled methylene chloride was added to the remain— ing aqueous phase and the contents extracted gently for 3 minutes by a rotary motion and then centrifuged for 3 minutes. The upper steroid hormone—free aqueous layer was then aspirated and discarded. The methylene chloride extract was extracted vigorously for 30 seconds with 0.75 ml of cold 0.1 N NaOH, centrifuged and the NaOH layer discarded. One ml of the methylene chloride was saved for measurements of radioactivity. Four ml of the methylene chloride extract was extracted with 4 ml of 1 WW .- C“!l1”"“‘ . , 4. _ l . .. , 43 fluorescing reagent (Appendix VIII) for 1 minute. After centrifugation for 3 minutes and aspiration of the methylene chloride fraction, the fluoresence of the reagent-hormone mixture was measured in the fluorometer 15 minutes after the extraction. A reagent blank of 1 ml of homogenizing fluid was carried through the en- tire procedure for each assay as a correction for back- ground fluorescence. Adrenal corticosterone contents and concentrations were calculated from a linear regression equation based on the fluoresence of 4 corticosterone standards (0.02 pg, 0.04 pg, 0.08 pg and 0.16 pg) which were included in every assay. Extraction efficiency was determined from the radioactivity in the 1 m1 of methylene chloride that was dried under nitrogen and counted in the scintillation counter. Measurement of Corticosteroid—Binding Globulin (CBG) Activity The procedure of Doe 33 31. (1964), used for the measurement of CBG activity in humans, was adopted for use in the rat plasma of the present study. This method has been used in many species for studies of CBG activity (Seal and Doe, 1965). In the present study inadequate plasma was available to measure CBG activity in individual rats. Consequently, plasma samples were combined within treatments by adding 0.5 ml of plasma from each rat to a common pool. The activity of CBG was determined in 44 duplicate for each plasma pool for rats killed at either day 16, 24 or 32 of lactation. Prior to use, 35 g of Sephadex (Pharmacia Co.) G-50 medium was mixed with 400 ml of 0.2 M phosphate buffer pH 7.4 (Appendix IX). The suspension of Sephadex was equilibrated at 4C for 16 hours at which time the fines were decanted. Three columns (2x40 cm) were packed at 40 to a height of 28 cm. Two m1 of each plasma sample was mixed with: 40 p1 of corticosterone-4-lu0 solution (0.05 pc with a mass of 0.32 pg) and 40 pl of a unlabelled solution containing 3.24 pg of corticosterone. Each sample was equilibrated by gentle shaking at 37C for 1 hour and then equilibrated at 4C for 1 hour. After the equilibration periods, 1 ml of plasma from each sample was placed on separate Sephadex columns being careful not to allow the columns to run com- pletely dry. To wash any residual plasma into the Sepha- dex two 1 ml aliquots of buffer were allowed to run into the column. Then the flow of elution buffer (0.1 M phosphate buffer pH 7.4 Appendix IX) was initiated. The 1 ml of equilibrated plasma layered on the column con- tained a total mass of 1.92:0.03 pg corticosterone. This total mass of corticosterone (measured fluori— metrically) is composed of an endogenous source as well as the mass from the added radioactive and from the added non—radioactive corticosterone. Approximately 3 m1 fractions were collected by means of an automatic fraction 45 collector. When elution was complete, the Sephadex was resuspended in a flask with phosphate buffer and used to pack the columns the following day for duplicate analyses. All elution fractions were monitored for protein by absorbancy at 280 mp with a Beckman DB spectrophoto— meter (Beckman Instrument Co.). Elution of the protein and protein-bound radioactivity was accomplished in four fractions each with an absorbancy greater than 0.10. The fractions in this group were pooled and the volume adjusted to 25 ml with buffer. A 5 ml aliquot of the pooled eluates was extracted with 5 ml of methylene chloride for 15 seconds by vigorous mixing on a Vortex shaker. After a 3—minute centrifugation the aqueous upper phase was aspirated and discarded. An additional 5 ml of methylene chloride was added to the tube and the contents mixed vigorously for 15 seconds on a Vortex mixer. Following centrifugation for 3 minutes, 8 ml of the methylene chloride extract was transferred to a scintillation vial and the contents evaporated under a flow of air at 45C. The radioactivity was counted in a liquid scintillation spectrometer. The total amount of radioactivity present in each of the original equilibrated plasma samples was deter- mined by dilution of 0.1 ml with 5 ml of phosphate buffer. This diluted and equilibrated plasma was ex- tracted and counted for radioactivity as described above. 46 The amount of protein—bound corticosterone per ml of plasma was obtained by multiplying the fraction of the added counts which were protein—bound times the cortico- sterone content of the sample (added plus endogenous). This value may be converted to pg bound per 100 ml by multiplying by 100. The calculation used is as follows: protein bound CPM total CPM Total corticosterone pg/ml x X100 = i protein-bound corticosterone pg/100 ml A plot of separation of the protein—bound and non— protein-bound corticosterone-4-luC is shown in Figure l. 3“ . The use of gel filtration for assay of CBG content in plasma depends upon the separation of the protein bound fraction from the unbound fraction of corticosterone by molecular exclusion based upon the wide difference in molecular size of the two fractions. The radioactivity was eluted in two peaks. The first peak contained the plasma proteins as measured by ultraviolet absorbancy at 280 mp and the protein bound hormone. The second peak of radioactivity represents unbound corticosterone-4-luC whose elution from the column was retarded by virtue of its small molecular weight. The low amount of radio- activity between the two peaks demonstrates that there was very little dissociation of the corticosterone-4-luC from the CBG during elution through the column. 47 .COHmepHHm How wcfihso 0:HI: ocopopmoofipaoo oczonlcfiopopglcoc pew oesonlcfiopopd mo COHpmprom .H ohsmfim 48 mum—232 ZO_._.U0.05) between day 16 and 20 nor between day 28 and 36. Total mammary DNA did not change significantly (P>0.05) between day 16 (36.2 mg) and 20 (37.2 mg). How— ever, mammary DNA declined linearly (P<0.01) between day 20 and 36 (27.0 mg) of lactation. Mammary RNA did not change significantly (P=0.10) between day 16 (206.7 mg) and 20 (226.5), but a linear decline (P<0.01) of 54% was observed between day 20 and 36. Mammary RNA/DNA ratios were 5.7, 6.1, 4.8, and 3.8 for groups killed on day 16, 20, 28, and 36, respectively. Thus, changes in ratio paralleled very closely total RNA changes. Since body weight of mother rats increased during lactation (P<0.01), correcting for body weight merely magnifies the differ- ence among groups for nucleic acid changes. 49 50 .meeeeafi Ham eoe owe no ma one ma mzmo coozpon oopeooop ohms mcflmw pzwwoz poupflfi o>fipmasesoo .cmoE mo mm new cmon .meeeeaa eeemoe eaouseeuma sea: edemaaee eHo meme ea meeeeaqm m.HHm.m e.mum.om w.aum.mm H.mum.mm exmv deem sewed: seeped m.on.m m.oum.: H.oua.e H.0He.m azm\0.05) between mammary gland weights; there- fore, lactic acid content per gram of mammary tissue was not significantly different between the two groups (P>0.05). Pituitaries of rats killed at day 20 and 36 of lactation contained 0.018 and 0.015 IU of prolactin/mg of pituitary, respectively, and 22.5 and 7.3 milliunits of ACTH/mg of pituitary, respectively (Table 3). 52 TABLE 2. Nucleic and lactic acid content of mammary glands and litter weight gain of intensely a suckled rats during extended lactation. Days of Lactationb 20 36 Number of rats l2 12 Body wt. (g) 242 :3 265 :5 Total DNA (mg) 28.6:1.2 23.6 31.4 Total RNA (mg) l72.7i9.8 81.3 :7.4 RNA/DNA 6.02:0.21 3.39:0.22 Total Lactic Acid (mg) 5.9 :0.3 5.0 30.4 Litter weight gain0 (g) 29.27:2.54 -4.06:2.51 aLitters 16 days old replaced with l2-day-old foster litters. bMean and SE of mean. CCumulative litter weight gains were recorded be- tween day 13 and 16 of age for all litters. 53 .mhoppfia powwow oaonsmoumfi eeaz ooomaaoe oao memo ea meoeeaqm mo.o w.oauo.m o.H m.e em.o omo.u:oo. woo.o mfio.o m.m om Hm.o w.awue.m :.m m.mm 02.0 emo.-eoo. Hao.o mao.o o.m om a amm Ho mm soeoooa a emm Ho mm socoeoa Amev .oz eoaooeoea mhmpflspfio mm mama “we\moaeoaaaaev mega Ams\aHv eaoomaoea .mea maLSU mpmp poaxosm mmaomcopcfl mo .QOHpmpomH popcopxo moflpmpfiSpHo mo pcopcoo meo< pew empomaopm .m mqm<8 54 The losses of RNA during extended lactation were much greater in magnitude than losses in DNA, and may suggest that factors controlling protein synthesis limited milk production more than factors influencing cell numbers. Nonetheless the significant decline in DNA indicates the mammary gland has undergone a partial cellular involution although a strong sucking stimulus was maintained. The combination of reduced cell numbers and less synthetic activity of the remaining cells at least partially accounted for the marked decline in LWG. In agreement with the present study, Moon (1962) reported a decrease in mammary DNA during advanced lactation. In contrast, Tucker and Reece (1963b) observed that DNA con- tent did not decrease significantly from 21 to 61 days of lactation, but that RNA content decreased significantly and this latter effect probably contributed to the decrease in milk production. The nursing intensity of six pups/six glands used in my experiment has been shown to be suffi— cient to stimulate mammary growth (DNA) to a maximal value on day 16 of lactation (Tucker, 1966). However, at day 36 mammary cell numbers declined to a level which was less (P<0.01) than day 16 of lactation. The lower nursing in- tensity used by Tucker and Reece (1963b) does not produce as much mammary growth as that obtained in the present study (Tucker, 1966). Perhaps one reason why Tucker and Reece (1963b) did not observe decreased levels of 55 mammary DNA may be that it is easier to maintain a basal level of cells than to maintain the abnormally large number of cells produced by intense nursing. Other workers, (Nicoll and Meites, 1959), (Grosve- nor, 1961), (Tucker and Reece, 1963b), and (Moon, 1962) using nursing stimuli of less intensity than in the pre- sent study, observed a decline in milk production of the rat with advancing lactation. However, Bruce (1961) reported, that lactation was prolonged by foster litters in three rats for 9—12 months, and the litters continued to gain at about the same rate throughout the experiment. The report of Bruce (1961) is in disagreement with the present experiment in which milk production virtually ceased at day 36 of lactation. The nursing intensity in my experiment was of a greater magnitude (six pups/six glands and litters exchanged every four days). Conse- quently, the great demands of the litters for milk in the current study may have been large enough to deplete the body of certain factors needed for milk production. Lactic acid production increases in mammary glands when milk is not removed, but no increases occur when milk is removed (Cowie and Folley, 1961). The similar lactic acid content of mammary glands from rats killed at 20 and 36 days of lactation suggest that frequent litter exchange permitted adequate milk removal. These results further suggested that increased anaerobic 56 oxidation was not a limiting factor to maximal milk synthesis during extended lactation. The intense nursing stimulus maintained pituitary prolactin content at least until day 36 of lactation. However, intense nursing failed to prevent a decline of 68% in pituitary ACTH between day 20 and 36 of lactation. This decline in ACTH during extended lactation agrees with the observations of Tucker 33 _1. (1967a) in which pituitary ACTH content declined between day l and 16 of lactation. Furthermore, in the present experiment, de- 1%. clines in ACTH paralleled declines observed in mammary nucleic acid content and LWG during extended lactation. If reduced pituitary ACTH content reflects an actual de- cline in synthesis of the hormone, ACTH may be one of the hormonal factors that limits milk synthesis during extended lactation. A decrease in ACTH synthesis and release would cause a decrease in adrenocortical function with advancing lactation. Experiment II.--Lactationa1 Performance of Intense1y Nursed Rats Injected with Oxytocin During Extended Lactation As shown in Experiment I intensive nursing stimu- lation failed to maintain maximal milk production during extended lactation in the rat. Therefore, an experiment was conducted to determine if inadequate milk ejection limited milk production during extended lactation. 57 There were no significant differences (P>0.05) between oxytocin- and saline—injected rats in LWG from 16 to 24 or from 24 to 32 days of lactation (Table 4). However, there was a marked decline (P<0.01) in LWG from the first 8-day period (day 16 to 24) to the second 8-day period (day 24 to 32) for both treatments (47% for oxytocin— and 72% for saline—treated rats). The oxytocin-treated group contained 37% more DNA and 44% more RNA than the saline—treated group (P<0.01), but ratios of RNA/DNA did not differ (P>0.05) between treatments. Oxytocin—treated rats had a greater (P<0.05) gain in body weight from day 3 to 32 of lactation than con- trols. There was no significant difference (P>0.05) be- tween treatments with regard to weights of anterior and posterior pituitaries, adrenals or uteri (Table 5). Although the oxytocin injections retarded mammary cell losses (DNA) and declines in metabolic activity (RNA), synthetic activity per cell (RNA/DNA) remained unchanged and these conditions were not sufficient to prevent a 47% decline in litter weight gain. The mainte— nance of mammary DNA is in agreement with the results of Ota 33 31. (1965) who reported that the administration of oxytocin preserved the structural integrity of the mammary tissue in weaned rats. However, they were unable to retard decreases in RNA content and RNA/DNA ratio with oxytocin. This contradiction to my results may be due to 58 TABLE 4. Nucleic acid content of mammary glands and litter weight gain of intenselya suckled rats injected with oxytocin between day 16 and 32. Oxytocinb . b 3 IU/day Saline Number of rats 12 ll Litter weight gainC 16-24 days (g) 74.8:6.6 86.7:2.7 Litter weight gain0 ‘ 24—32 days (g) 39.913.8 24.0:8.9 Total DNA (mg) 33.2:l.2 24.3:l.6 Total RNA (mg) l42.5:10.l 99.0:9.4 RNA/DNA 4.3:0.2 4.0+0.3 aLitters 16 days old replaced with 8-day-old foster litters. bMean and SE of mean. CCumulative litter weight gains were recorded be— tween day 8 and 16 of age for all litters. 59 TABLE 5. Organ weights of intenselya suckled rats injected with oxytocin between day 16 and 32. b Oxytocin b 3 IU/day Saline Number of rats 12 ll Anterior pituitary (mg) 9.2: 0.6 9.4: 0.5 Posterior pituitary (mg) 2.9: 0.2 2.43 0.2 Adrenal (mg) 51.0: 0.1 50.7: 1.8 Uterus (mg) 276 :16 293 :17 Body weight gain (g) 3—32 days 19.2: 3.4 8.2: 3.5 aLitters 16 days old replaced with 8—day old foster litters. bMeans and SE of mean. 60 differences between weaned and lactating rats. Kumaresan and Turner (1966) reported that oxytocin therapy increased LWG 13% between day 7 to 20 of lactation. They postulated that inadequate removal of milk from the gland partially limited lactation. However, my results indicate that oxytocin injections do not increase LWG in advanced lac- tation (16-32 days). The results of the present study and the observation in Experiment I, that lactic acid con- tent did not change during extended lactation, suggest that the milk—ejection reflex is adequate during advanced lactation. Experiment III.--Lactationa1 Performance of Intensely Nursed Rats Injected with Hydrocortisone Acetate (HLL Prolactin (P) and Growth Hormone (GT Since neither intense suckling (Experiment I) nor exogenous oxytocin (Experiment II) influenced the decline in milk synthesis during advanced lactation the present experiment was designed to test if adrenal glucocorticoids, prolactin or growth hormone would prevent the declines in milk synthesis. As shown in Table 6 and Figures 2 and 3 rats re- ceiving any combination of H (H, HG, HP, HGP) had a larger (P<0.01) 16-24 day LWG reSponse (95 g) than those rats not receiving H (S, G, P, GP) (80 g). The mean 24— to 32-day LWG response for the corticoid- and non corticoid- injected rats was 68.9 g and 38.7 g, respectively. As .meooeHH Ham pom omm mo ma pom m mmmo somepon poopooos opoz mcflmw pgwfioz popufia o>HpmHSESQU .cmoe mo mm new comic .meoppfia nopmom oHouzmplm spas ooomadoh oao when ma whopufiqm .dsopw poo mama mo hooEdZO 61 m.+e.a a.mHo.mm w.oHa.aH --- -1- Aoav ammwmmm m.HH.e H.mwm.em a.oHe.m 3.:Hw.oa :.ewm.ow Ame m .o :.Hm.: o.mHe.em o.HHH.mH H.eHm.mz m.aHm.om Ame a m.H:.a o.mwm.om e.oH:.HH m.eHw.em a.eum.ee Aoav o :.Hm.e H.mum.mw e.oum.HH w.ewe.me :.ewo.am Ame a .o .m :.H:.o H.3Ho.we m.oum.mH e.mum.ae m.ewo.om Ame d .m N.HN.m a.mna.we m.ona.ma m.mnm.ee m.mna.moa “HHS o .e N.Hm.: m.mHm.He o.oHo.mH m.mHm.me o.eHm.em AOHV m m.Ho.m m.mHe.Hm m.oum.m m.:um.mm m.mue.ae oAOHV oeaamm azo\I u I _ 20. o _ on. o I I I I _ _ go _ _ QC \.\ -m 1 \Q\\\\ 10_ l \ 1 __ 1 \\ ob I? 4. 1N. 1 IM_ 1 IV_ .l In.“- I 73 .mm ocm ma zoo coozpmo va onEpog gpzopw ocm Amv napomHOLQ .Amv mcomfiphooohohn spa: ompoomcfi mums oonoSm mammcopcfl mo mocmaw mngEmE wo pcmucoo I u I on OI _I OI _ 0 AT .4 O Q2Q\ VSE 78 with G. If paired adrenal weights are corrected for body size (mg/100 g body weight) differences in adrenal weights were eliminated. No significant differences (P>0.05) among hormone treatments were detected for weights of anterior and posterior pituitaries, ovaries or uteri. The results of this experiment indicate that in— jections of glucocorticoids partially retard declines in LWG in the intact, lactating rat during advanced stages of lactation. These results are in agreement with those of Johnson and Meites (1958) and Talwalker 33 a1. (1960) [mum- in uni-2. &! who demonstrated a galactopoietic effect of corticoids in early lactation. Growth hormone and prolactin were not galactOpoietic during prolonged lactation in the rat. This is in agreement with other experiments conducted dur- ing early lactation (Meites, 1957) (Macdonald and Reece, 1961). Hydrocortisone acetate maintained mammary DNA con- tent during extended lactation. The interaction between H and P was such that P reduced the DNA response, but P stimulated the synthetic activity of the mammary cells when given with H. In fact, these results suggest that exogenous prolactin when given with a corticoid may re- duce mitotic activity in the mammary gland. Associated with the corticoid stimulation of milk synthesis (LWG) was a reduction in body weight gain during the treatment period. Corticoid injections were able to 79 reduce the body growth response associated with growth hormone injections. The metabolic effects of adrenal corticoids such as protein catabolism and translocation, mobilization of fatty acids from adipose tissue, and increased blood levels of glucose may be associated with the maintenance of milk precursors in the blood for milk synthesis. EXperiment IV.--Lactationa1 Performance of Intensely Nursed Rats Bearing Isotransplanted Anterior Pituitaries During Extended Lactation Since the prolactin in Experiment III was of ovine orgin and administered only twice a day, it was of interest to chronically administer rat prolactin to rats through the use of anterior pituitary isotransplants. Vaginal smears of rats containing five anterior pituitary transplants or sham—Operated controls indicated that both groups were in an anestrus state throughout lac- tation. The 16— to 2A— and 2H- to 32-day LWG responses for the rats with pituitary transplants were 79.A and 40.“ g, respectively (Table 8). The comparable responses for the sham—Operated rats were 83.3 and 2A.S g, re- spectively. There were no significant differences (P>0.05) in the 16— to 24- or the 2A- to 32-LWG responses between the two groups of rats. 80 TABLE 8. Nucleic acid content of mammary glands and litter weight gain of intenselya suckled rats bearing isotransplanted pituitaries between day 3 and 32. Pituitary Sham Transplant Operation Number of rats 14 ll Litter weight gainc 16-2u days (g) 79.u: 5.9 83.3: u.3 Litter weight gainc 2U-32 days (g) 40.“: 5.3 2U.5: 6.8 Total DNA (mg) 29.2: 1.1 2H.Oi 1.7 Total RNA (mg) 138.2:10.0 98.2:10.7 RNA/DNA n.7i 0.2 u.o: 0.2 aLitters 16 days old replaced with 8-day—old foster litters. bMean and SE of mean. CCumulative litter weight gains were recorded be- tween days 8 and 16 of age for all litters. . -’ 1'M3‘. 7‘77““ hunt-4*: ~| ' p / ~4' h —. I T 81 Anterior pituitary transplants significantly in- creased (P<0.05) mammary gland DNA content 22%, RNA con— tent N1% and RNA/DNA ratios 18% over non-transplanted control rats. The larger percent increase of total RNA than DNA and the increased synthetic activity per cell indicated that mammary glands of rats bearing anterior pituitary transplants had a greater potential to secrete milk than the controls. But as shown in Table 8 it did not result in increased LWG. Anterior pituitary and ovarian weights of rats with five anterior pituitary transplants were significantly less (P<0.05) than those of sham-operated controls (Table 9). There were no differences in body weight gains, adrenal or uterine weights. There is substantial evidence that anterior pitui- taries transplanted to the kidney capsule synthesize and release copious amounts of prolactin (Meites and Nicoll, 1966). In my experiment the decrease in pituitary weight and the increase in mammary DNA and RNA of rats having five transplanted pituitaries indicated that the trans- planted pituitaries were functional during lactation. These data are in agreement with Sinha and Tucker (1968) who observed that prolactin secreted from five anterior transplants in virgin rats depressed the in_§i£u_pitui- tary weight and increased mammary DNA and RNA content. Malven and Sawyer (1966) observed that exogenous prolactin hastened corpora lutea regression in hypOphysectomized 82 TABLE 9. Organ weights of intenselya nursed rats bearing isotransplanted pituitaries between day 3 and 32. Pituitary Sham b Transplant Operation Number of rats 1“ 11 Body wt. gain 18-32 days (g) 6.2: 3.0 —1.0: 2.6 Anterior Pituitary (mg) 8.5: 0.3 9.6: 0.3 Posterior Pituitary (mg) 2.7: 0.2 2.8: 0.2 Adrenal (mg) 51.8: 1.7 5A.O: 2.6 Ovary (mg) 6U.9: 3.1 78.3: 5.2 Uterus (mg) 266 :17 316 :20 aLitters 16 days old replaced with 8—day-old foster litters. bMean and SE of mean. 83 rats. A decrease in ovarian weight was attributable to induced luteolysis. A possible explanation for the de- crease in ovarian weights in my experiment is that pro- lactin secreted from the pituitary transplants caused luteolysis of the post-partum corpora lutea. Conse- quently, this regression may account for the decrease in ovarian weights. In the present eXperiment, constant secretion of rat prolactin from five anterior pituitary transplants failed to elicit a galactOpoietic effect in the lactating rat. This verifies the results of Experiment III in which injections of ovine prolactin were not beneficial to milk yield although mammary nucleic acid content was elevated. Similarly, Cowie 32 a1. (1960) also reported that pituitary grafts under the kidney capsule had no effect on milk yield during early lactation. On the basis of Experiment III and IV, I concluded that the stimulatory effect of prolactin on cell numbers and synthetic activity of the mammary gland is simply not sufficient to maintain persistent milk production during advanced lactation. Experiment V.—-Lactational Performance of Intensely Nursed Rats Injected with 9-Fluoroprednisolone Acetate (Predef) Although rats receiving hydrocortisone acetate were more persistent in their lactational ability than non-corticoid—treated animals (Experiment III), signifi- cant declines in lactational ability still occurred as 8U lactation advanced. The present experiments were de- signed to determine if the synthetic glucocorticoid, 9-fluor0prednisolone acetate (Predef) would minimize this decline. The 16- to 2U-day cumulative LWG responses of 105.8 and 115.0 g for rats given daily doses of 50 and 100 ug of Predef, respectively, were larger (P<0.01) than the 87.7 and 71.4 g LWG responses to a lO-ug dose of Predef or of saline, respectively (Table 10). The 10 ug dose caused a greater (P<0.05) LWG response than the saline injections. During the second 8-day period (2A to 32 days) the stimulatory effect of Predef on lac- tational performance is even more readily apparent. The 50 and 100 ug doses of Predef caused an approximate l2U% increase (P<0.01) in LWG above values for saline-injected controls. In addition, the 10 pg dose caused a signifi- cant increase (P<0.05) in LWG over that of the saline control rats. However, in Spite of the Predef there was a decline in LWG from the first to the second 8-day period of lactation. Rats receiving injections of 50 and 100 ug of Predef had more (P<0.01) mammary tissue than either the 10 ug— or saline—treated rats. The 50 pg dose of Predef main- tained mammary DNA at a level higher (39.9 mg, P<0.01) than that found in either the lO-ug (26 mg) or saline- control (2U.1 mg) rats. However, the 100—ug dose 85 .mpmppfia HHO coo omm mo ma ocm w mhmo cmmzpmo coopoooh mpoz mcflmw pgwfloz Loppfia m>prHSESQO .cmoE mo mm ocw com: o .mpmppfia powwow oaoummolm npfiz ooowadop oHo mmmo ma whoppflqm s.O HO.O m.O “H.m m.O HO.O m.oHO.m aZO\0.05), but RNA content was stimulated (P;0.08). Consequently, the synthetic activity of the remaining mammary gland 87 cells (RNA/DNA) was increased. In comparison, the 50 pg dose of Predef stimulated cell numbers (DNA) and total synthetic activity (RNA) of the mammary gland. However, the synthetic activity per cell (RNA/DNA) was reduced. These results suggest that the synthetic activity of the mammary cells is stimulated preferentially at the lower dose level, whereas the 50 pg dose maintained the structural integrity in addition to the synthetic activity of the mammary gland. The 100 pg dose of Predef appeared to overstimulate the mammary gland as reflected by a loss of cell numbers (DNA) and a higher synthetic activity of the remaining cells in comparison with the 50 pg treat- ment group. Increasing the dose of Predef, from zero before day 16 to 100 pg between day 16 and 32 of lactation, caused a linear decline (P<0.01) in body weight gain from day 18 to 32 of lactation (Table 11). As was demonstrated in Experiment III, glucocorticoids have the ability to di- vert nutrients from body growth to the mammary gland for the synthesis of milk. Adrenal weights from rats injected with 100 pg of Predef were smaller (P<0.05) than the adrenal weights of the 0-, 10- and SO-pg treatment rats. The 50—pg and lOO-pg Predef treatment rats had smaller (P<0.05) posterior pituitary weights than the 10 pg or saline control rats. Perhaps Predef injections at these doses r”... 88 .Qmmg MO mm GEM Gmmzo, .mpmppHH powwow oHoummolw Sufi: omomaaop oHo mzmo ma whoppfiqm Hm“ 5mm NH“ mam m.mHH Omm O.HHH Omm Hmsv magma: m.O HH.OO H.m HO.HH O.m Hm.me m.O HO.mO Hmev Hpaso m.m Hm.HO O.H HO.OO m.: Hm.Om m.m Hm.OO Hmsv HOOOOOO m.O HO.N H.O HO.N m.O HO.m O.O HO.m Hmsv OHOOHOOHO HOHHOOOOO O.O HH.O O.O HO.O O.O HH.OH O.O Hm.O stv HHOOHOOHO LOHHOOOO O.m HO.mmu O.m HO.mH- O.O HO.H- O.m “H.O Hmv OOOO mmIOH cflmw .ps zoom O OH O OH mums co pmpssz 00H om OH ocflamm ozmo\mmoopm w: .mm Ocm OH OOO smmzpmp AOOOOHOV mumpmom ocoaomflcompooHOSHMIm £pH3 ompoomcfi hump comps: mmaomzopcfl mo wpcwfioz compo .HH mqm0.05) among the treatments. Of the Predef doses administered, the 50 pg dose appeared to be optimum for maximal lactational perfor- mance since LWG was near maximum, mammary cell numbers were maintained, adrenal weights were not depressed, and body weight losses were not excessive during the treatment period. Nonetheless, Predef injections only partially retarded declines in milk synthesis. As pre— viously shown, the LWG responses from 2A to 32 days of lactation were less (P<0.01) than the LWG from 16 to 2A days regardless of the dose of Predef injected. It be- came of interest to determine if Predef injections gradu- ally altered corticoid requirements of the mammary gland with time. Perhaps, the initial corticoid injections in the present experiment induced enzymes that metabolized the injected hormone to a form that was less active and/or more readily excreted by the body. The following experi- ment was designed to determine if the observed declines in litter weight gain could be minimized by increasing the dose of Predef injected during advancing lactation to compensate for any increased losses. In addition, a preliminary in vitro study was conducted to measure any differences in cortisol metabolism by the liver which may have been induced by Predef injections. 90 The first group of rats were injected with saline from day 16 to 32. A second group of rats received saline injections from 16 to 23 days of lactation and 50 pg of Predef daily from 2A to 32 days of lactation. The third group of rats received 50 pg of Predef daily between day 16 and 23 and 100 pg between day 2“ and 32. The 2u— to 32-day LWG of the second and third groups in— creased 95% above that of saline controls (Table 12). Injections of 50 pg of Predef daily from day 2“ to 32 of lactation resulted in as much LWG during this period as if the 50 pg were injected from day 16 to 23 and then the dose was doubled between day 2A and 32 (group three). Consequently, either-a delay in the onset of injections to day 2“ of lactation or an increase in the dose during the treatment period, eliminated the decline in LWG as lactation advanced. The rats receiving Predef had more mammary gland DNA (P<0.05), more RNA (P<0.01) and greater RNA/DNA ratios than saline-injected controls. Rats in group three, which received the highest dose of Predef for the longest time period, had adrenal weights which were significantly less (P<0.01) than either groups one or two (Table 13). The blood level of glucocorticoids regulates the secretion of ACTH which in turn controls the functional status of the adrenal cortex. The depressed adrenal weights of rats in group three probably reflected suppressed ACTH 91 now omm mo 0H .mmeOHH HHm ocm w mmmo zoozpon oooHooop who: msfimm psmfimz hoppfia o>HpmHsesoo .cmoE mo mm osm comic .mpoppfla Hopmom CHOIzmolw Sufi: ooomamog oHo mmmo ma whoppflqm m.O HO.m m.oHO.O H.OHH.m azoxazm m.MHHO.OOH O.OH~.OON O.OHA.OHH Hmsv <2m Hmpoe m.H Hm.Hm O.HHO.mm m.HHm.Om stv azo HOOOB m.s HO.mO H.mHO.mm m.mHO.mH va OOOO mmuzm OcHOm pgmHmz pmpOHH O.O Hm.mO O.mHO.OO O.OHH.HO Ocmmw MmmmmmwaprH OH mH OH mums co pmossz OOH Om Om O O O cmempm m: Ommuqm mmumH ammusm mmuOH ammuzm mmuOH :OHpmpomq co mama .mm Ocm OH OOO cmozpmo Amooopmv mamboom mQOHochooHQOHOSHMIm Spas ompoowcfi mums moompsc zaomcopcfi no chm unwflmz HoppHH ocm nonmaw mmeEmE mo usoucoo oHom oHoHosz .mH mqmo O.H HO.mO O.H HO.OO O.H HO.OO stv HmcmpOa H.o Hm.m m.o H:.m m.o Hm.m Amev zpmpHSHHm LOHpopmom m.O HO.O 0.0 Hm.OH m.O H0.0H Hmev HHOOHOOHO OOHLOOOO m.O HO.OH- O.m Hm.OH- O.H HH.O OOOO mmumH :Omm .pz zoom OH MH OH mums Oo meEOz OOH Om OO O O O OOOOLO m: Ommuzm mmuOH nmmnzm mmnOH Ommuzm mmuOH OOHpmpomq co mama .mm Ocm OH OOO cmmzpmn Homompmv Opmpmom ocoaowflcooaooposHmnm ssz oopooncfi mums oompsc whaomcopcfi mo mpngmz compo .mH mqm0.05) among rats in the three groups. The livers of rats from groups one and three were prepared for in_vi§§g incubations as described in materials and methods. The results of a preliminary experiment indi- cated that an incubation with 1 ml each of microsome and 105,000xg supernatant preparations increased the amount of cortisol metabolized by luu% in comparison with an incubation with only microsomes. Consequently, all subse- quent incubations were performed with 1 m1 of the micro— some fraction plus 1 ml of the 105,000xg supernatant fraction. The liver preparations of saline- and Predef—treated rats at the various levels of cortisol substrate produced inconsistent results with respect to the total mass of cortisol metabolites produced per hour (Table 1A). For example, liver preparations of Predef-treated rats did not consistently produce greater or lesser amounts of cortisol metabolites than livers of saline-treated rats at the 1, 2 and 5 pm substrate levels. However, the percent non- polar and polar metabolites produced within substrate levels between treatments did show a consistent trend. For example, a larger percentage of polar metabolites for the Predef-treated rats was observed at the l, 2 and 9H .moUHHoompoE Hmpou mo pcoopomm HONOO OOH HOOHO Hm OOH s: O OOOOOO AOOOO OOm HOOHO Om mOm s; m OOOOOO HONOO OHH HOO O OH OOH s; H OOOOOO HOHOO OO HOOOO OOH OHN an O OOHHOO HHOOO OOH HOOOO mO HON a: m OOHHOO OHOOOO OOH OAOOOO NO mOH a: H OOHHOO Loon pom poo: poo poo: poo ooosoopa ooodoopa ooodoOHQ oooom Cowumpooa mopHHoompoE mopHHoompoE mopHHoompoE opmgpmosm mo mmmo mmIoH HOmepoo Homflppoo Homflupoo Homfippoo Eopm psoEpmoppopm Hoaoq mo m1 Hmfioalcoc mo m: mo w: Hmpoe .mm ocm OH moo sooZHoo Hmooopmv oompooo ocoHochooHQoposHmnm oco mcHHmw csz oopoomcH mpmp oommss OHomcoch mo mcoHumpmomgo Ho>HH Eosm ooosoomo mopHHooouoE HomeHoo mo pcsoeo .OH mqmOH Scum cfiouomo mo me Log ooosoopm moufiaonmpoe Homfiupoo mo pcdoeo .mH mqmqb 97 The total amount of cortisol and its metabolites found in the aqueous extraction phase of the livers was larger for the Predef- than for the saline—treated rats (Table 16). In addition the specific activity for the Predef enzyme system was greater than the saline system at all substrate levels. Since enzyme assays were per- formed on pooled liver preparations within treatments, there is no estimate of the variation within treatments. Total liver weights, liver protein content and pro- tein per gram of liver did not differ (P>0.05) between Predef-treated and saline control rats (Table 17). Liver weights per 100 grams of body weight were significantly larger (P<0.025) in Predef-injected rats than in saline— injected controls. The pooled microsomal protein content of saline-treated rats was 1AA% higher than the microsomal protein content of Predef-injected rats. The first series of Predef experiments indicated that relative to saline-injected controls, daily injections of either 10, 50 or 100 pg of Predef for 16 days exerted a galactopoietic effect on milk synthesis. However, significant declines (P<0.01) in LWG were observed as lactation advanced. If the injections of Predef were delayed to day 24 of lactation or if the dose of Predef was increased during the treatment period, the declines in LWG during advanced lactation were eliminated. These results in vivo suggested that the adrenal corticoid 98 O.H O.mm E: m mmoopm m.H m.OH E: m mooopm m.o O.oa E: H moooum w.o w.om E: m ocHHmm m.o H.MH E: m mcHHmm m.o m.w E: H ocfiaom momma msoosom CH Campopo ommog msoosom CH Hzon\ oooom COpruoma mo wE\Lzoc\ooosoopQ ooozoopa mopfiaonmpoe mummmeSO mo mzmo mmIOH mopflaoompoe ocm HOOHpHoo mo m1 ocm Homfippoo mo w: HOOHuhoo Eopm pcoEpmopuopm .mm ocm OH moo coozuoo Amooopmv ouMpoom oQOHochoopooposHmnm ocm ocfifimm cpfiz oopooncfi mpOH oomgsc maomsopcfi mo mCOOpopmaopa po>fia SOLO ooozoogd movaHoompoE oHQBHoOILmOOZIHomeHoo ocm Homflppoo mo pcsoeo .OH mqm0.05) between day 16 (32.2 mg) and 2H (31.0 mg) but declined (P<0.01) to 25.5 mg by day 32 of lactation. Mammary RNA content declined linearly (P<0.01) from day 16 to 32 of lactation. Mammary RNA/DNA ratios were 5.3, A.“ and 3.6, respectively. The changes in ratio paralleled very closely total RNA changes. These results, which confirm the results of Experiment I, indicated that lactational performance declined during the advancing stages of lac- tation. The mean resting level of plasma corticosterone for a group of virgin rats killed at metestrus was 10.65 pg per 100 ml of plasma (Table 19). This plasma level of corticosterone was in close agreement with the resting corticosterone levels reported by Guillemin (1958) for female rats. These results substantiated that the manage— ment procedures used in this experiment allowed one to measure resting levels of corticosterone in rat plasma. The total corticosterone content per two adrenals for the groups killed at 16, 2M and 32 days of lactation was 2.01, 1.66 and 1.35 pg, respectively, a significant linear decline (P<0.01). The adrenal corticosterone con— tent at day 32 of lactation was not significantly differ- ent (P>0.05) than the level found in the virgin rats (1.23 pg). When expressed as pg per 100 mg of adrenal 103 .cmoe mo mm osm com: o .mpoppfla powwow oHOIOmoIO opfiz ooomHQmH oHo mOoo OH mpoppfiqm mO Om MO III mammaa mo He OOH\OcOOO mochopmoofippoo m: O.HH O.Hm O.OH m.mm O.OH m.mm H.HH O.Om .Oz OOOO O0 O OOH\Os HOcmtOO OO.HH O.OO OO.HH O.OO OO.HH O.OO OO.OH O.OO HOeO HmemOOO OO.HHOO.OH OO.HHNO.OH OO.OHOO.OH OO.OHOO.OH OEOOHO me He OOH \ocohopmooflpgoo w; OH.OHOO.N mm.OHOO.O OO.OHOO.O OH.OHOm.m HOOOOOO O0 we OOH \ocohopmoofipgoo m; HH.OHOO.H OH.OHOO.H Om.OHHO.m OH.OHOO.H mHmempOO m \mcopopwoofippoo w; ON ON ON ON OOOH Oo HOOEOZ mm Om OH o coapouoma mo whom mpwp cfiwpfl> p .coapmpoma ooocopxo wcfimso mpOp ooHXoSO mmaomcopcfi CH mao>oa ocopopmoofippoo OEOOHQ ocm Homopo< .mH mqm0.05) for adrenal weights to decrease with advancing stages of lactation. Peripheral plasma concentrations of corticosterone for rats killed at day 16, 2A and 32 of lactation (18.76, 18.32 and 15.97 Hg per 100 ml of plasma, respectively) were not significantly different (P>0.05) from each other. Measure- ments of CBG activity, at each stage of lactation, were made on pooled plasma samples. The quantities of corticosterone bound to CBG per 100 ml of rat plasma were 63, 58 and 75 pg at day 16, 2M and 32 of lactation, respectively (Table 19). The results of Holzbauer (1957) indicated a positive correlation between the quantities of cortical hormones present in the adrenal and its secretory activity at any given time. Thus, a high adrenal corticosterone concen- tration was associated with high secretory activity, a low concentration with low secretory activity. Comparisons of secretion rates and the stores of corticosterone in rat adrenals provided direct evidence for this relationship (Holzbauer, 1957). The correlation between cortico- sterone stores and secretaion rate in the rat adrenal provides a method in which information can be obtained on the activity of the adrenal cortex immediately before extirpation of the gland. With this relationship in mind, the results of the present study suggest that the secretion 105 rate of corticosterone decreased from day 16 to day 2“ and was at its lowest level at day 32 of lactation. In fact, the adrenal corticosterone content at day 32 was comparable to that found in the virgin non—lactating state. However, there were no significant (P>0.05) changes in peripheral plasma levels of corticosterone that reflected this decrease in adrenal secretion rate with advancing stages of lactation. Perhaps the target tissues of the body utilize the increased amount of corticoid hormone that is secreted into the plasma pool at the peak of lactation (day 16). If the target tissues of the body promptly utilized any additional amount of corticosterone available at different stages of lactation, then the peripheral plasma levels may remain at a con- stant level. The basal corticoid level during lactation appears to be at a level of equilibrium higher than that measured in virgin rats. The mammary gland is a tissue that has an apparent corticoid requirement to maintain lactation in the rat (Experiments III and V). Consequently, the mammary tissue may be utilizing the additional corti- coid secreted at the peak of lactation. If such a relation- ship does exist then the secretion rate or the cortico- sterone content of adrenal gland may be correlated with some end organ response in the mammary gland. The within-stage, among—rat correlation coefficients between total adrenal corticosterone content and mammary gland RNA, DNA and LWG are listed in Table 20. The largest 106 TABLE 20. Correlation coefficients of mammary gland nucleic acid content and litter weight gains with adrenal corticosterone content of in- tenselya suckled rats during extended lac— tation. Correlation Coefficients Stage of lactation RNA DNA LWG l6 0.60b 0.59b 0.4uc 2H 0.26 0.11 -0.02 32 0.43 0.29 0.35 Pooled within stage of lac- d d tation 0.A5 0.35 0.27 aLitters 16 days old replaced with 8—day—old foster litters. bCorrelation coefficients significantly different from zero (P<0.01). 0Correlation coefficients significantly different from zero (P<0.05). dPooled correlation coefficients within stages of lactation significantly different from zero (P<0.01). 107 correlation coefficients were between adrenal cortico- sterone content and mammary RNA (0.60), DNA (0.59) and LWG (0.UU) at day 16 of lactation. At day 16 adrenal corticosterone content was the greatest and the mammary gland was stimulated maximally. Decreased adrenal secretion rates at day 2“ and 32 of lactation may cause a general decrease in the responsiveness of the mammary tissue. Such a decrease may account for the absence of a significant correlation coefficient between adrenal corticosterone content and the mammary gland traits mea- sured at these two stages of lactation. The pooled cor- relation coefficients of adrenal corticosterone content with mammary gland RNA and DNA within stages of lac- tation were significant (P<0.01). I conclude that the declines in adrenal corticosterone content may be related to changes in mammary gland function. An additional factor for consideration is the possible interaction of peripheral plasma corticosterone and CBG activity. The mass of corticosterone bound per 100 ml of plasma appeared to be higher at day 32 of lac- tation (75 ug%) than at day 16 (63 pg%) or 24 (58 pg%). Gala and Westphal (1965) reported that CBG activity is depressed during lactation but at four days post-weaning it rises to a level comparable to that found in virgins. The higher CBG activity at day 32 of lactation (75 Ug% compared with 63 pg% and 58 pg%) may reflect an increase 108 in binding activity during advanced lactation that ap- proaches the activity found in the virgin rat. Based on the binding affinity for cortisol, the level of CBG activity reported for the virgin rat is 105 pg% (Seal and Doe, 1965). Since the binding affinity for corticosterone in the rat is comparable to the affinity for cortisol (Seal and Doe, 1965), the 75 pg of corti- costerone bound per 100 m1 of plasma at day 32 of lac- tation in the present experiment may be a level that approaches the CBG activity of the virgin rat. If the CBG activity is greater at day 32 of lactation, then the amount of free plasma corticosterone available to the mammary gland may be less, regardless of the constant peripheral blood levels between 16, 2A and 32 days. SUMMARY AND CONCLUSIONS Intensive nursing, which was maintained by replac- ing all l6-day-old litters every A days with 12-day-old foster litters and supplying at least one pup per mammary gland, failed to prevent declines in mammary DNA, RNA and litter weight gains (LWG) between days 20 and 36 of lactation. Intense nursing maintained pituitary pro— lactin during extended lactation but ACTH decreased 68%. Adrenal corticosterone content and concentration decreased significantly from day 16 to 32 of lactation, indicating that ACTH stimulation of the adrenal gland decreases in rats during extended lactation. Peripheral plasma concentrations of corticosterone in lactating rats were higher than the resting levels measured in the virgin state. However, corticosterone concentrations in the plasma did not change from day 16 to 32 of lactation. Measurements of corticoid binding globulin activity in pooled plasma samples indicated that the binding activity of rats killed at day 32 of lactation was greater than the activity found in rats killed at day 16 and 2A. Adrenal corticosterone content of rats at day 16 of lac— tation was significantly correlated with mammary gland 109 110 DNA (r=0.59, P<0.01), RNA (r=0.60, P<0.01) and litter weight gain (r=0.AA, P<0.05). The decrease in adrenal corticosterone content from day 16 to 32 of lactation was correlated significantly with the decrease in mammary gland DNA (r=0.35, P<0.01) and RNA (r=0.A5, P<0.01) content. Oxytocin injections during advanced lactation caused marginal increases in the DNA and RNA content of the mammary gland but did not significantly increase litter weight gain or the synthetic activity per cell (RNA/DNA). Consequently, milk removal appeared to be adequate for maintenance of milk synthesis during ad- vanced lactation. Hydrocortisone acetate, prolactin and growth hor— mone were given singularly and in all possible combi- nations to determine if these treatments would maintain mammary nucleic acid content and lactational perfor- mance during extended lactation. Hydrocortisone acetate maintained mammary DNA and RNA content and reduced the normal decline in litter weight gain. Prolactin given with hydrocortisone acetate reduced the DNA response, but stimulated the synthetic activity of the mammary cells (RNA/DNA). Prolactin and growth hormone did not exert a galactopoietic influence on milk yield. Chronic secretion of rat prolactin from five iso- transplanted pituitaries under the kidney capsule of 111 lactating rats did not increase litter weight gain al— though mammary gland DNA and RNA content and RNA/DNA ratio were significantly increased. Prolactin is probably not rate limiting to milk synthesis in the rat because pituitary prolactin content did not de- crease during extended lactation and neither ovine pro- lactin injections nor rat prolactin from isotransplanted pituitaries increased milk yield. A daily dose of 50 pg of 9—fluoroprednisolone acetate (Predef) from day 16 to 32 of lactation main- tained mammary gland DNA and RNA content and increased milk yield. A delay in the onset of injections until day 2“, or the injection of 50 pg of Predef daily from day 16 to 23 and 100 pg of Predef daily from 2A to 32 days of lactation maintained LWG and increased the mammary gland nucleic acid content. Results of in vitro incubations indicated that Predef injections for 16 days increased the ability of rat livers to metabolize cortisol into polar metabolites while the formation of non-polar metabolites was inhibited. I conclude that adrenocortical secretions may be rate limiting to milk synthesis during advanced lactation in the rat because pituitary ACTH and adrenal cortico- sterone content decrease during this time, corticosteroid binding globulin activity of the plasma increased (al- though total circulating levels of corticosterone did 112 not change), and the declines in mammary gland nucleic acid content and litter weight gain can be prevented with hydrocortisone acetate or Predef supplementation. BIBLIOGRAPHY BIBLIOGRAPHY Anderson, R. R. and Turner, C. W. 1962. Adrenal ascorbic acid levels during pregnancy and lactation in rats and mice. Endocrinology, 70:796. Anderson, R. R. and Turner, C. W. 1963. Aldosterone and corticosterone in maintenance of lactation in adrenalectomized rats. Proc. Soc. Exptl. Biol. and Med., 112:997. Barker, S. B. and Summerson, W. H. 19Ul. The colori- metric determination of lactic acid in biological material. J. Biol. Chem., 138:535. Bearn, J. G., Gould, R. 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Ca Pantothenate 2.5 g Riboflavin 1.5 g Nicotinic acid (Niacin) 15.0 g Vitamin B-12 (0.1% mannitol trituration) 3.0 g DL alpha tocopherol acetate, 250 IU vitamin E/g 8.8 g Menadione (Vitamin K) 1.0 g DL methionine 227.0 g Limestone 16.0 lb Dicalcium phosphate 17.5 lb Iodized salt 5.0 lb Manganous sulphate (MnSou . H O) 32.5% manganous 168.9 g 128 Ferrous sulfate (Fe Sou . 7H20) 20.9% iron 215.2 g Calcium carbonate (CaC03) 40.4% calcium 83.8 g Zinc carbonate, basic (ZnCO3) 56.0% zinc 40.2 g Cupric sulfate (CuSOu . 5H20) 25.45% COpper 12.9 g Cobalt chloride (CoCl2 6H20) 24.77% cobalt 4.7 g Potassium iodine (KI) 76.45% iodine 2.2 g This ration gave the following analysis: protein 21.2% fat 3.9% crude fiber 2.5% productive net energy 902 C/lb APPENDIX II ORCINOL REAGENT USED IN THE RNA PROCEDURE l. 130 A stock solution of 1.6% ferric chloride (FeCl2 . 6H20) was prepared by adding 1.6 g of ferric chloride to 1 liter of concentrated hydrochloric acid. A 1% solution of orcinol in 1.6% ferric chloride solution was prepared 1 hour before use. “1 1" f i . g APPENDIX III INCUBATION MEDIA FOR ACTH ASSAY Stock gpock Stock Stock Stock Stock Solution Solution Solution Solution Solution Solution 132 A 5.75% KCL in distilled water B 8.08% CaCl .2H 0 in distilled 2 2 water C 10.55% KH P0“ in distilled water 2 D 19.10% MgSOu.7H2O in distilled water E 200 ml of .9% NaCl containing 0.520 g of glucose F 100 ml of 1.3% NaHCO3 (solution gassed for 30 minutes with 95% C02+5%02) Preparation of incubation media 1. "Krebs Ringer Salt" solution is prepared by adding tog 4 ml 3 ml 1 m1 1 ml 36 ml The total ether: of solution A of solution B of solution C of solution D of distilled water volume of 45 m1 is designated as a "Krebs Ringer Salt Solution." 2. The incubation media (Krebs—Ringer-Bicarbonate with Glucose) is prepared as follows: 133 42 m1 of solution F 18 ml of "Krebs Ringer Salt" solution 200 ml of solution E The total contents of 260 ml is gassed with 95% 02 and 5% CO2 for 10 minutes prior to use. APPENDIX IV 0.1M PHOSPHATE-GLYCEROL BUFFER pH7.4 USED IN 1E.VITRO METABOLISM STUDIES 135 Solution A: 0.2M sodium phosphate, monobasic Solution B: 0.2M sodium phosphate, dibasic, heptahydrated 0.1M phosphate buffer with 20% glycerol pH7.4: 19 ml solution A 81 ml solution B 40 ml glycerol 60 ml distilled water ,_H.. 4 . -.-r“_. a. Lug-KM: ." ‘I. APPENDIX V 0.1M PHOSPHATE BUFFER pH7.4 USED IN 15‘VITRO METABOLISM STUDIES 137 Solution A: 0.2M sodium phosphate, monobasic Solution B: 0.2M sodium phosphate, dibasic heptahydrated 0.1M phosphate buffer, pH7.4: 19 ml solution A 81 ml solution B 100 ml distilled water . ! 1'»).er . ‘I‘ } APPENDIX VI SCINTILLATION FLUID #1 139 4 g of PPO or 2, 5—Diphenyloxazole--scinti1- lation grade (Nuclear Chicago) and 50 mg of POPOP or 1, 4-bis[2-(5—Phenyl- oxayolyl)] benzenescintillation grade (Nuclear Chicago) in 1 liter of toluene Radioactivity measured in the following system: 0.2 ml of aqueous phase 5.0 ml of ethyl alcohol 5.0 m1 of scintillation fluid r...” ._... APPENDIX VII SCINTILLATION FLUID #2 141 1. 7.5 g PPO 75 mg POPOP 120 g Napthalene 500 ml Xylene 500 ml Dioxane I ~ ”An: mu”? nl' uni—unaw—O i. {-4 5““ - APPENDIX VIII 1 . FLUORESCENT REAGENT FOR CORTICOSTERONE DETERMINATION 143 Sulfuric Acid: Ethanol (3:1) mixture This mixture was cooled in a flask under tap H20 and allowed to stand at least 12 hours before use. Sulfuric Acid: 96.4%, 1.84 sp gr J. T. Baker Chemical Co. Phillipsburg, N. J. Ethanol — 200 proof — "Gold Shield Alcohol" Commercial Solvents Corp., Terre Haute, Indiana -ux—q..__ APPENDIX IX PHOSPHATE BUFFERS USED IN THE CBG PROCEDURE 145 Solution A: 0.4M monobasic sodium phosphate Solution B: 0.4M dibasic sodium phosphate 0.2M phosphate buffer, pH7.4 19 ml of solution A 81 ml of solution B 100 m1 of distilled water 0.1M phosphate buffer, pH7.4 OO.- 19 m1 of solution A 81 m1 of solution B 3 300 ml of distilled water