INFLUENCE OF D!FFERENT PHYSIOLOGICAL PARAMETERS 0N PROMOTIN BiNDlNG ACTIVETY IN PROLACTIN TARGET TISSUES Dissertation. far the Degree of Ph. D. MECHlfiA‘N S‘E'A'E'E Ufié‘JERSE‘fi’ MARIE CfixTE-EEREHE GE'LATO 3.975 This is to certify that the thesis entitled Influence of Different Physiological Parameters on Prolactin Binding Activity in Prolactin Target Tissues presented by Marie Catherine Gelato has been accepted towards fulfillment of the requirements for Ph .D. degree in Physiology Date November 26, 1974 0-703. ll . 6 Iv ? HUM} & SflNS . ‘BCUK ENDERYINB.| ' \ Llapmv smacks munch" MICHIBAN ‘ . A_.~.;. ._ -__._ .id ‘9’". ‘_ - c '1 y.. I‘Nflj’f‘mfll. 3“» ..-. puma» ‘ ‘Mhnvx'iY‘ z‘l ' A (WW ‘0‘ Mu' . ‘ x" Q. 11mm; of ‘ ' “ ”than and Tril'r‘c.. I ”Ned 9‘1. :11. w. M. “L”! W” mam-awe ;.-u \‘L... I. met with in H3!“ ‘ : . ' aim" _ ‘ ‘ 3%,“ Show ”mi “‘2 1'.~i‘::_ -‘ —s'.;:.: low ' '--*. 'Er- - 9‘“ in the ‘35:.“ van, ‘ Kw" j'ln Nadia: sctlaiiy «u:- mnsmx‘r ”a mum. 23.9? _ {tissue ‘r-Jm lands». and ecu“. exti‘mq w-I‘e‘n' ‘ activity for prolacun in immune rat wane: -. -.§S¢ binding of adult term 9 rat Mat 3 and n» ’ fluted. fin the day of vaginal warning the Man‘- J?" g *9 m curios m (wearable to binding «this; i.“ “w ,7 in ntun Mic rats In Molt cycling binding Octlvity in the overt» norm : ABSTRACT 0\ INFLUENCE OF DIFFERENT PHYSIOLOGICAL w PARAMETERS 0N PROLACTIN BINDING ACTIVITY IN PROLACTIN TARGET TISSUES By Marie Catherine Gelato l. Binding of 125I-radiolabelled prolactin was demonstrated for liver, ovarian and mammary gland microsomal membrane prepara- tions. Non-labelled prolactin readily displaced the labelled pro- lactin in all three membrane preparations whereas LH, TSH or GH did not cross react with the labelled prolactin in these prepara- tions. These data show that the binding of 125I-radiolabelled pro- lactin is specific in the tissues measured. 2. Prolactin binding activity was measured in microsomal membranes of ovarian tissue from immature and adult cycling female rats. The binding activity for prolactin in immature rat ovaries was less than l/2 the binding of adult female rat ovaries and in- creased as the rats matured. 0n the day of vaginal opening the bind- ing for prolactin in the ovaries was comparable to binding activity observed during estrus in mature female rats. In adult cycling female rats, prolactin binding activity in the ovaries fluctuated W' Marie Catherine Gelato and was highest on the days of diestrus during the estrous cycle. It is concluded that the increase in prolactin binding activity in the ovaries of immature rats is associated with the appearance of corpora lutea and that prolactin has a role in ovarian function during the estrous cycle of the rat. 3. Prolactin binding activity was measured in ovarian and mammary gland microsomal membrane preparations during pregnancy and lactation. The binding activity of prolactin in the ovaries was significantly increased on days 3 and 6 of gestation and on days 4 and lo of lactation. Prolactin binding activity in the mammary tissue remained unchanged throughout pregnancy and increased by 2-fold on days 4 and lo of lactation. These data suggest that the binding activity of prolactin is correlated with physiological requirements for the hormone during pregnancy and lactation. 4. Prolactin binding activity was measured in liver, kidney, and adrenal microsomal membrane preparations of 15, 23, 28, 33, 38, 43 and 75 day-old female rats. The binding activity in the liver increased and reached a peak at 43 days of age whereas the pro- lactin binding activity in the kidney and adrenal tissue steadily decreased until 43 days of age in these rats. Estradiol benzoate injected at l‘pg for 5 days in immature female rats significantly increased prolactin binding activity by 4-5 fold in the liver tissue. It is concluded that estrogen secretion near the time of puberty may stimulate prolactin binding activity in the liver, and that the functions of prolactin may be more important in the kidney and adrenals of the immature rat than in the adult rat. Marie Catherine Gelato 5. Prolactin binding activity was measured in microsomal membranes of liver tissue from intact, ovariectomized, ovariectomized- thyroidectomized, and ovariectomized-thyroidectomized rats injected with thyroxine (T4) or estradiol benzoate (EB). Thyroidectomy and ovariectomy each reduced prolactin binding activity in liver tissue significantly. The combination of ovariectomy and thyroidectomy decreased prolactin binding activity more than thyroidectomy or ovariectomy alone. Doses of 2.5‘ug or lO,pg T4/100 9 3w daily re- turned prolactin binding activity in the thyroidectomized rats to intact control values, and in the ovariectomized-thyroidectomized rats to the ovariectomized values. A dose of 2 ug EB/rat increased prolactin binding activity above that of intact controls. Scatchard analysis showed that ovariectomy and thyroidectomy decreased the number of prolactin binding sites in the liver as compared to those in intact controls or in ovariectomized-thyroid- ectomized rats treated with EB and T4. It is concluded that the thyroid and ovaries are important regulators of prolactin binding activity in the liver of the rat. 6. Prolactin binding activity was measured in liver and mammary gland microsomal membranes of ovariectomized rats treated with estradiol benzoate (E8) (5 and 20.n9) or estradiol benzoate (5.ug) and progesterone (4 mg) for 10 days. Estradiol benzoate or the combination of EB and progesterone significantly increased prolactin binding activity in the liver tissue approximately 4-fold as compared to controls. The prolactin binding activity in the mam- mary tissue was significantly decreased by EB and the combination i Marie Catherine Gelato of EB and progesterone. One week after the treatment was terminated the effects of EB and EB and progesterone were still present in the liver and mammary tissue when assayed in a second group of ovariectomized rats. It is concluded that EB (5 or 204ug) or a combination of EB and progesterone are able to stimulate prolactin binding activity in the liver and depress binding of prolactin in the mammary tissue. 7. The effects of adrenalectomy and hydrocortisone acetate treatment on prolactin binding activity in liver microsomal membranes was measured in ovariectomized rats. Adrenalectomy for either 6 or 24 days slightly lowered prolactin binding activity in the liver. Hydrocortisone acetate treatment, 1 mg daily for 10 days, produced a slight depression in prolactin binding activity; however, 100 pg hydrocortisone acetate/loo 9 BW daily to adrenalectomized rats significantly decreased the binding of prolactin in the liver. These results suggest that adrenalectomy had a tendency to lower prolactin binding activity in the liver of female rats, whereas treatment with hydrocortisone acetate produced a more marked reduction in prolactin binding activity. INFLUENCE OF DIFFERENT PHYSIOLOGICAL PARAMETERS ON PROLACTIN BINDING ACTIVITY IN PROLACTIN TARGET TISSUES By Marie Catherine Gelato A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology l975 ACKNOWLEDGEMENTS I would like to take this opportunity to express my gratitude and deep appreciation to the several people who have helped make my graduate career a truly meaningful experience. To my advisor, Dr. Joseph Meites, who provided not only the materials for the work in this thesis to be done but an atmosphere that helped me grow as a person and a student, I am very grateful. An expression of thanks to my Guidance Committee, Dr. v.0. Collings, Dr. H. Hafs, Dr. T. Jenkins, Dr. R. Bernard and Dr. T. Brody, for their help in the preparation of this manuscript and for their willingness to listen and give assistance whenever it was needed. A special thanks to Dr. C. Martin Norbom of Hunter College whose encouragement throughout my graduate career has never ceased. My gratitude is expressed to Steve Marshall who was instru- mental in establishing the radioreceptor assay utilized in the studies presented in this thesis and to Gary Kledzik, Michael Boudreau, John Bruni, Greg Mueller and Dr. G. Riegle for their assistance in per- forming some of the studies in this presentation. I would like to thank Dr. Gordon Campbell for his assistance and patience in explain- ing and performing the Scatchard analysis. Appreciation is also ex- pressed to Mrs. Claire Twohy for her technical assistance and her “sparking" personality which livened many dull days and to my coworkers for helping me see the bright side of most situations. I am especially grateful to Mrs. Pam Rashid for her pleasant attitude and willingness to help prepare this manuscript. My thanks to Mrs. Amylou Davis for her help in filling out forms and meeting deadlines. I would also like to thank Dr. J. Meites, Dr. R. Bernard and the Department of Physiology for providing me with funds during my graduate education. Finally, to my parents, Mr. and Mrs. I. Gelato, and my brother, Ronnie, a sincere thanks for their constant support and understanding throughout my education. iii TABLE OF CONTENTS LIST OF TABLES ......................... LIST OF FIGURES ......................... INTRODUCTION .......................... LITERATURE REVIEW ........................ I. General Hypothalamic Control of Anterior Pituitary Function .................... A. Anatomy of the Hypothalamus .............. B. Anatomy of the Hypothalamo-Pituitary Connections ...................... C. Hypothalamic Hypophysiotropic Hormones ........ II. Hypothalamic Control of Prolactin Secretion ........ A. Hypothalamic Prolactin Release-Inhibiting Factor (PIF) ..................... B. Hypothalamic Prolactin-Releasing Factor (PRF) ..................... C. Role of Catecholamines, Serotonin and Acetylcholine ................... III. Functions of Prolactin .................. A. Mammary Gland .................... B. Pigeon Crop Sac .................... C. Ovaries ....................... D. Male Reproductive System ............... E. Metabolic Function ............... . . . F. Salt and Water Balance ................ G. Adrenal Function ................... IV. Prolactin Interactions with Other Hormones ........ A. Effects of Estrogen, Testosterone and Progesterone on Prolactin Secretion .......... B. Thyroid Hormones ................... iv Page Vll'al \lU'l-hww Page C. Glucocorticoids ................... 46 D. Growth Hormone .................... 46 E. Gonadotropins .................... 48 V. Protein Hormone Receptors ................ 49 A. ACTH and Angiotensin ................. 49 B. Growth Hormone and Other Hormones .......... Sl C. Insulin ....................... 53 O. Gonadotropins, LH and FSH .............. 55 E. Prolactin ...................... 58 MATERIALS AND METHODS ..................... 64 I. Animals ......................... 64 11. Surgical Procedures ................... 65 A. Thyro-parathyroidectomy ............... 65 B. Ovariectomy ..................... 66 C. Adrenalectomy .................... 66 III. Preparation of Hormones ................. 67 A. Hormones for Injection ................ 67 B. Hormones for Cross-reactivity Studies ........ 68 IV. Radioreceptor Assay for Prolactin ............ 68 A. Preparation of Tissue Samples ............ 68 B. Iodination of Ovine Prolactin ............ 68 C. Assay Procedure ................... 70 V. Scatchard Analysis .................... 71 VI. Statistical Analysis ................... 76 EXPERIMENTAL .......................... 77 I. Demonstration of Specific Binding of Prolactin to Liver, Ovarian and Mammary Gland Membrane Preparations. . 77 A. Objectives ...................... 77 B. Procedures ...................... 77 C. Results ....................... 79 D Conclusions ..................... 79 II. III. VI. ‘Prolactin Binding Activity in Ovarian Tissue During Prepuberal Development and the Estrous Cycle in the Rat. . A. Objectives ...................... B. Procedures ...................... C. Results ........................ D. Conclusions ...................... Prolactin Binding Activity in Ovaries and Mammary Tissue During Pregnancy and Lactation in the Rat ......... A. Objectives ...................... B. Procedures ...................... C. Results ........................ D. Conclusions ...................... Normal Development and Effects of Estrogen on Prolactin Binding Activity in Liver, Adrenals and Kidneys of Immature Female Rats ................... A. Normal Development and Effects of Estrogen on Prolactin Binding Activity in Liver of Immature Female Rats ...................... l. Objectives .................... 2. Procedures .................... 3. Results ...................... B. Ontogeny of Prolactin Binding Activity in the Adrenal Glands and Kidneys .............. l. Objectives .................... 2. Procedures .................... 3. Results ...................... C. Conclusions ...................... Effects of the Thyroid and Ovaries on Prolactin Binding Activity in Rat Liver ............... A. Objectives ...................... B. Procedures ...................... C. Results ........................ D. Conclusions ...................... Effects of Ovarian Hormones on Prolactin Binding Activity in Liver and Mammary Tissues . . . ....... A. Effects of Estrogen or Estrogen and Progesterone on Prolactin Binding Activity in Liver ........ vi Page 97 97 97 97 98 99 99 100 104 105 107 107 108 109 118 i_‘£-‘ A 1. Objectives .................... 2. Procedures .................... 3. Results ...................... 8. Effects of Estrogen or Estrogen and Progesterone on Prolactin Binding Activity in Mammary Tissue ..... l. Objectives .................... 2. Procedures .................... 3. Results ...................... C. Conclusions ...................... VII. Effects of Adrenals on Prolactin Binding Activity in Liver of Female Rats ............. A. Effects of Adrenalectomy and Hydrocortisone Treatment on Prolactin Binding Activity in Liver . . . 1' Objectives .................... 2. Procedures .................... 3. Results ...................... B. Effects of Adrenalectomy and Hydrocortisone Acetate Replacement Therapy on Prolactin Binding Activity in the Liver ............. 1. Objectives .................... 2. Procedures .................... 3. Results ...................... C. Conclusions ...................... GENERAL DISCUSSION ....................... REFERENCES ........................... APPENDIX ............................ vii Page l18 ll9 120 120 120 l23 124 124 128 128 128 129 130 130 130 131 132 132 135 144 179 “-7-, Vw Table II. III. IV. VI. VII. VIII. IX. XI. XII. LIST OF TABLES Page Prolactin Binding Activity in Ovarian Membranes During Prepuberal Development in the Rat ......... 88 Prolactin Binding Activity in Ovarian Membranes During the Estrous Cycle in the Rat ........... 89 Prolactin Binding Activity in Ovarian Membranes During the Estrous Cycle in the Rat ........... 90 Prolactin Binding Activity in Ovarian Membranes During Pregnancy and Lactation in the Rat ......... 93 Prolactin Binding Activity in Mammary Gland Membranes During Pregnancy and Lactation in the Rat ........................ 95 Prolactin Binding Activity in Liver Homogenates of Untreated and Estradiol Benzoate Treated Female Rats ........................ lOl Prolactin Binding Activity in Adrenal and Kidney Homogenates of Growing Female Rats ............ l02 Prolactin Binding Activity in Liver Homogenates of Thyroidectomized (Tx) and Thyroxine (T4) Treated Female Rats .................... l12 Prolactin Binding Activity in Liver Homogenates of Ovariectomized (va)-Thyroidectomized (Tx) Rats Given or not Given Thyroxine (T4) .......... ll3 Prolactin Binding Activity in Liver Homogenates of Ovariectomized (0vx)-Thyroidectomized (Tx) Rats Given Thyroxine (T4) and/or Estradiol Benzoate (EB) ................. ll4 Scatchard Analysis .................... l15 Prolactin Binding Activity in Liver Homogenates of Ovariectomized (va) Rats Treated with Estrogen (E8) or Estrogen and Progesterone (Prog) and Killed 24 hours After Last Injection ............... lZl viii Table XIII. XIV. XV. XVI. XVII. Page Prolactin Binding Activity in Liver Homogenates of Ovariectomized (va) Rats Treated with Estrogen (EB) or Estrogen and Progesterone (Prog) and Killed 7 Days After Last Injection ............ 122 Prolactin Binding Activity in Mammary Tissue Homogenates of Ovariectomized (0vx) Rats Treated with Estrogen (EB) or Estrogen and Progesterone (Prog) and Killed 24 Hours After Last Injection ................... 125 Prolactin Binding Activity in Mammary Tissue Homogenates of Ovariectomized (0vx) Rats Treated with Estro en (EB) or Estrogen and Progesterone (Prog , and Killed 7 Days After Last Injection ................... 126 Prolactin Binding Activity in Liver Homogenates of Ovariectomized (va) Rats Adrenalectomized (Adx) or Hydrocortisone Acetate Treated Rats ....... 133 Prolactin Binding Activity in Liver Homogenates of Ovariectomized (va)-Adrenalectomized (Adx) Rats Given or Not Given Hydrocortisone Acetate (HC). . . . 134 Figure LIST OF FIGURES Page Prolactin-Receptor Interaction .............. 63 Competitive Displacement of 1251 Radiolabelled Ovine Prolactin From Mammary Gland Membranes of Female Rats ................. 8O Competitive Displacement of 1251 Radiolabelled Ovine Prolactin from Ovarian Membranes of Rats ...... 81 Competitive Displacement of 1251 Radiolabelled Ovine Prolactin from Liver Membranes of Female Rats ...................... 82 Competitive Displacement of 1251 Radiolabelled Ovine Prolactin from Mammary Gland, Ovarian and Liver Membranes ............... 84 Normal Development and Effects of Estrogen on Prolactin Binding Activity in Liver Tissue of Immature Female Rats ................. 103 Ontogeny of Prolactin Binding Sites in Kidney and Adrenal Tissues of Immature Female Rats ....... 106 Competitive Displacement of 1251 Radiolabelled Ovine Prolactin from Liver Membranes of Intact, Ovariectomized-Thyroidectomized, and Ovariectomized-Thyroidectomized Rats Injected with Estrogen and Thyroxine ............... 116 Scatchard Plot of Data Obtained from Liver Membranes of Intact Control, Ovariectomized- Thyroidectomized and Ovariectomized- Thyroidectomized Rats Injected with Estrogen and Thyroxine .................. 117 INTRODUCTION Part of the definition of the term hormone states that hormones are substances carried by the circulating blood to another part of the body where they evoke systemic functions by acting on specific tissues and organs. In order for this hormone system to be specific there would have to be "recognition" by the target tissue for the particular hormone. Substantial evidence now exists that the initial interaction of polypeptide hormones with their target cells is with a hormone specific receptor site on the target cell. The receptor molecule has been localized on the plasma membrane of the cell. Recently membrane receptors have been isolated for prolactin, lutein- izing hormone, follicle stimulating hormone, insulin and others. The study of hormone receptors has provided another tool for further characterization of hormone action. In the last year the development of a specific radioreceptor assay for prolactin (Shiu gt_gl., 1973) has made it possible to measure the binding activity of prolactin in target tissues, and with the use of Scatchard analysis to estimate the number of receptors per mg of tissue and determine the binding affinity of prolactin for these receptors. The major emphasis of this thesis is on the physiological characterization of prolactin receptor binding activity in several target organs, mainly the liver and ovaries. " presented follow three lines of Investigation: "- of specific binding for prolactin in liver. ovarian ‘ 7 gland tissues and demonstration of no cross reactivity :‘fiudluiones (2) the development of prolactin receptor bind- _:§£%f5“land possible correlation with prolactin levels in the fifjjhfgififihrement of prolactin receptor binding activity in ?80es during different physiological conditions, such as J Lu LITERATURE REVIEW I. General Hypothalamic Control of Anterior Pituitary Function A. Anatomy of the Hypothalamus The physiological relationship between the hypothalamus and the anterior pituitary is made clearer by an understanding of their anatomy. The role of the hypothalamus in pituitary function is depicted in the colored illustrations of the nervous system by Netter (1968). The hypothalamus is described as the most ventral portion of the diencephalon. It is bordered anteriorly by the lamina terminalis, posteriorly by the interpeduncular fossa, dorsally by the hypo- thalamic sulcus in the third ventricle and ventrally by the tuber cinereum. There are three distinct hypothalamic regions. In a cephalocaudal direction they are: anterior or supraoptic area, middle or tuberal area and a caudal or mammillary area. The supra- optic area contains two sharply defined hypothalamic nuclei, the paraventricular nucleus and supraoptic Nucleus. In the tuberal area the hypothalamus reaches its widest extent and the medial portion forms the central gray substance of the ventricular wall. At the lower end the periventricular arcuate complex form the base of the third ventricle. Also located in the tuberal region are the 7_.—-—a—v——-a dorsomedial, ventromedial, and lateral hypothalamic nuclei. The mammillary portion consists of the mammillary bodies and the dorsally located cells of the posterior hypothalamic nucleus. B. Anatomy of the Hypothalamo-Pituitary Connections The pituitary gland is attached to the brain by the infundi- bulum or tuber cinereum, an extension of the third ventricle which is prolonged downward as the pituitary stalk. Part of the tuber cinereum and the uppermost part of the neurohypophysis is called the median eminence. It is in this region, the infundibulum and median eminence that the hypophysial portal blood vessels originate. There are no direct neural connections between the hypothalamus and anterior pituitary; rather, the connection is by way of the hypophysial portal blood vessels. Popa and Fielding (1930) first described the pituitary portal circulation and erroneously indicated that the flow of blood was from the anterior pituitary to the hypothalamus. Subsequent studies suggested that the flow was from the hypothalamus to the anterior pituitary (Hislocki and King, 1936; Houssay gt 11., 1935). Green and Harris (1949) directly observed the pituitary portal circulation in living rats under the microscope.‘ They indicated that the portal vessels originated in the median eminence and infundibular stem, and stated that the blood flowed caudally toward the anterior pituitary. These observations were confirmed in the dog by Torok (1954) and in the mouse by Worthington (1955). More detail on the anatomy of the pituitary portal system was given by Daniel (1966). ”,0.- u | ~ ’eIa 4 pl Ia. h p I" (W) C. Hypothalamic Hypophysiotropic Hormones Several books and reviews have been written on hypothalamic control of anterior pituitary function and the hypothalamic hypo- physiotropic hormones (Meites, 1970a; Szentagothai gt_gl., 1972; Burgus and Guillemin, 1970; McCann, 1971; Reichlin and Mitnick, 1973; Vale §t_gl,, 1973; Schally §t_gl,, 1973). Only some of the most important and classical references on this topic will be re- ported. The hypothalamus regulates secretion of the anterior pitui- tary hormones by producing specific hypophysiotropic hormones. Taubenhaus and Soskin (1941) suggested that the rat hypothalamus secretes an acetylcholine-like substance into the portal vessels to elicit pituitary LH release. Later, Markee §t_gl,, (1948) and McDermott gt gl. (1951) proposed that epinephrine, acetylcholine, and histamine might be involved in the release of gonadotropins, ACTH and other pituitary hormones. The direct influence of the hypothalamus on anterior pituitary function was first demonstrated by Harris and Jacobson (1952). They observed the return of estrous cycles in hypophysectomized rats when the anterior pituitary was removed and transplanted under the median eminence. However, when the anterior pituitary was transplanted under the temporal lobe of the brain in hypophysectomized rats, normal estrous cycles were not resumed. Harris (1955) proposed the "chemotransmitter hypothesis", according to which neurohormones released from the hypothalamus were responsible for regulating pituitary function. These neurohormones of which Harris (1955) wrote were later revealed to be a special class of polypeptides. Saffran and Schally (1955) and Guillemin and Rosenberg (1955) were among the first to report that crude acidic hypothalamic extracts caused release of ACTH jg_yjt§g and jg_yjyg. Saffran and Schally (1955) called the active substance "corticotropin releasing factor“ (CRF). McCann gt 31. (1960) demonstrated the presence of LRF (luteinizing hormone releasing factor) in acid extracts of rat hypothalami. Talwalker, Ratner and Meites (1963) and Pasteels (1961-63) provided evidence for PIF (prolactin inhibiting factor) activity. GHRF activity was first demonstrated by Deuben and Meites (1963). Evidence for the existence of other hypophysiotropic hormones followed, such as TRH (Shibusawa gt_gl., 1959; Schreiber, 1963; Guillemin gt_gl,, 1962), FSH-RF (Igarashi and McCann, 1964; Mittler and Meites, 1962), MIF and MRF (Kastin and Schally, 1966; Taleisnik and Orias, 1964), GIF (Krulich gt_gl., 1968) and PRF in birds (Kragt and Meites, 1965; Meites and Nicoll, 1966). Very recently the structures for several of the hypophysio- tropic hormones have been elucidated. Boler gt 91, (1969) and Burgus gt_g1, (1969) first reported the structure of thyrotropic releasing factor (TRH or TRF), and subsequently the amino acid sequence of LRF (Matsuo _§._l., 1971), MIF (melanocyte stimulating hormone inhibiting factor) (Nair gt 11., 1971) and GIF (somatostatin) (Brazeau gt gl., 1973) were reported. II. Hypothalamic Control of Prolactin Secretion A. Hypothalamic Prolactin Release-Inhibiting Factor (PIF) The regulation of prolactin secretion is unique in that it is the only anterior pituitary hormone that is chronically inhibited by the mammalian hypothalamus under most conditions. Any disturbance in the connection of the hypothalamus with the anterior pituitary can result in increased release of prolactin. Everett (1954; 1956) demonstrated that transplantation of the pituitary underneath the kidney capsule resulted in sustained release of prolactin, whereas release of all other anterior pituitary hormones was sharply re- duced. This work was later confirmed by Nikitovitch-Hiner and Everett (1958) and Chen g; gl. (1970). Various other experimental techniques have been employed to demonstrate hypothalamic inhibition of prolactin secretion, such as placement of lesions in the median eminence or "hypophysiotropic area" of the hypothalamus (Chen gt_gl., 1970; Nelsch 33 gl., 1971), culture or incubation of the anterior pituitary jg_yjtgg_(Meites gt_gl., 1961) and administration of appropriate drugs (Meites, 1962; Meites gt 51,, 1963). Inhibition of pituitary prolactin release by the mammalian hypothalamus is exerted via the action of a PIF. Culture of anterior pituitary tissue in vitro has demonstrated that the anterior pituitary can synthesize and release prolactin autonomously when removed from the hypothalamus and other body influences (Meites, 1959a). Addition of crude acid extracts of rat hypothalamic tissue resulted in de- creased release of prolactin (Talwalker gt_gl,, 1963; Pasteels ££.£l-: IOU nu l‘. I er ..'I I‘- '01 'Cq T -v ,_—— -~—' “ ~anon __._‘W 1963). Kragt and Meites (1967) demonstrated a negative dose-response relationship between the quantity of hypothalami c extract added and the amount of prolactin released i_nvi_tr3. This work was later con- firmed by Chen (1969) with the use of a specific radioimnunoassay for rat prolactin. There have been several i_n m demonstrations of PIF activity. Grosvenor gal. (1964) injected hypothalamic extracts into post— Partum lactating rats and reported that they inhibited prolactin re— lease following suckling. Kurashima gt fl (1966) reported prevention 01’ pituitary prolactin depletion in response to cervical stimulation during estrus by hypothalamic extracts. Injection of crude extract 0f rat hypothalamus reduced serum prolactin in cycling and lactat- ing rats (Amenomori and Meites, 1970) and decreased serum prolactin in normal and orchidectomized male rats (Watson g_t_ _1., 1971). PIF activity in the hypothalamus can be altered by several "Bans including use of central acting drugs, hormones, and physio— 1091 cal stimuli. Perphenazine (Danon _e_ta_l_., 1963), reserpine (Ratner e_t_fl., 1965), haloperidol (Dickerman fig” 1972a), Na pento- bar‘bital (Huttke fig” 1971a), epinephrine and acetylcholine (Mittler and Meites, 1967), estrogen (Ratner and Meites, 1964), progesterone, testosterone and cortisol (Sar and Meites, 1968), a norethynodrel- (menstranol combination (enovid) (Minaguchi and Meites, 1967a) and the suckling stimulus (Ratner and Meites, 1964; Minaguchi and Meites, 1 967b) were found to decrease hypothalamic PIF activity in rats. EE‘scbcornine (Huttke t 1., 1971b), L-Dopa and monoamine oxidase inhibitors (Pargyline, iproniazid, and Lilly compound-15641)(Lu and 4.0-. \ Meites, 1971) and prolactin itself (Chen e_t__1., 1967; Clemens and Meites, 1968; Voogt and Meites, 1971) were shown to increase PIF activity in the hypothalamus. Work by Lu and Meites (1972) demon- strated that L-DOpa, the immediate precursor of dopamine, increased PIF activity in the hypothalamus and elicited the presence of PIF activity in the systemic blood of rats. These results together with numerous other studies confirmed the view that drugs which increased hypothalamic catecholamines also stimulated synthesis and release of PIF, whereas drugs that reduced catecholamines in the brain depressed hypothalamic PIF activity (Meites fig” 1972). 3- Hypothalamic Prolactin-Releasing Factors (PRF) Unlike the mamalian hypothalamus which inhibits prolactin secretion, the avian hypothalamus appears to exert a stimulatory in- fluence on prolactin secretion and apparently contains a prolactin-re- leasing factor (PRF). Kragt and Meites (1965) demonstrated that an extract of pigeon hypothalamus stimulated prolactin release by the pigeon 9115“" tary 13M. Hypothalamic extracts from the chicken, quail, trlcalmed blackbird, duck and turkey (Meites, 1967; Nicoll, 1965; Go""da'i and Tixier-Vidal, 1966; Chen $11,, 1968) also induced re- lease of prolactin when incubated with pituitaries from these species. The eX'lstence of a PRF in the manmalian hypothalamus is probable but less Clear. Meites fill, (1960a) reported initiation of lactation by inJection of crude hypothalamic extracts into estrogen-primed rats. Injecliions of crude cerebral extracts also initiated lactation in s . Ome rats. ThlS work was later confirmed by Mishkinsky gt fl. (1968) . 1 r0 '- 1. O. '0 'l :4 Ice p. H \1 'O. ( o ivy-V 10 Nicoll fig. (1970) observed both prolactin inhibiting and prolactin releasing activity in rat hypothalamic extract when incubated with rat pituitary. PIF activity was reported to be present predominantly in the dorsolateral part of the preoptic area, and PRF activity mainly in the median eminence and a narrow basal portion of the preoptic area (Krulich fig” 1971). Valverde and Chieffo (1971) reported only PRF activity in an extract of porcine hypothalamus. However, others have failed to show PRF activity in comparable systems (Meites gt a_l_.. 1972). Synthetic pyro-glutamyl-histidyl-proline amine (TRH or TRF) has been observed by several laboratories to induce prolactin release. TRH has been shown to increase release of prolactin in the cow (Convey fl a_l., 1972; Kelly fig” 1973a). rat (Tashjian _tgi., 1971), human (Hwang fig” 1971; Bowers gig” 1971; Jacobs e_t._a_l_., 1971) and monkey (Josimovich fig. , 1974). Recently our laboratory re- Por‘ted that in the rat TRH was able to induce prolactin release i_nm (Muener, Chen and Meites, 1973) and gum (Dibbet Egg” 1972). This work raises the possibility that TRH and PRF in manuals are the same, However, under most physiological conditions, TSH and prolactin are not released together (Meites, 1973), i.e. hot and cold tempera- t“"‘es (Mueller e_tflu 1974), during suckling, administration of L-Dopa (Chen gig” in press), etc. It is possible that TRH is similar to PRF structurally. C- Role of Catecholamines, Serotonin and Acetylcholine In the last few years much attention has been given to the role 0 f neurotransmitters on prolactin and gonadotropic secretion. The 11 substances which serve as neurotransmitters in the brain are acetyl- choline and three monoamines, i.e. dopamine, norepinephrine and sero- tonin. A more thorough review of the localization of these transmitters in the brain is presented by Cooper Q fl., (1970), Anton-Tay and Hartman (1971), and Fuxe and Hokfelt (1969). With the development of histochemical fluorescence techniques for identifying biogenic amines in the brain, it has been demonstrated that norepinephrine and serotonin are highly concentrated in the hypothalamus and midbrain (Vogt, 1954; Brodie t al., 1959). Anden g g. (1964) showed that the median eminence is rich in dopaminergic nerve terminals and Bjorklund gig. (1970) demonstrated noradrenergic terminals in the median eminence. Chemical assays showed relatively large amounts of norepinephrine and dopamine in the median eminence (Rinne and Sonninen, 1968). More recently, Piezzi gtgl. (1970) have observed that the bovine median eminence also contains high concentrations of serotonin. Since the techniques for detection of cholinergic neurons are not as sensitive 0" as specific as histochemical fluorescence, much less is known about the distribution of these fibers in the brain. However, Shute and LEWIS (1969) have demonstrated cholinergic fibers in the lateral Preoptic and mammillary regions of the hypothalamus. These amines may be released into the hypothalamO-pituitary pc"‘11a1 system from neurons whose cell bodies lie in the medial hypo- tha'lamus and whose nerve endings are terminated in the median eminence and the infundibulum near the primary capillary loops of the portal system (Fuxe and Hokfelt, 1969). Halasz (1969) has shown that the medial basal area of the hypothalamus, the so-called hypophysiotropic area. is essential for tonic release of anterior pituitary hormones. . ow"..—.—‘e 12 Coppola fig. (1966) and others postulated that a sympathetic tonus, originating in the hypothalamus, normally acted to stimulate the release of FSH and LH while restraining the secretion of prolactin. In the absence of this tonus, FSH and LH secretion were suppressed while prolactin release was stimulated. Evidence for this postulate came from pharmacological studies in which certain drugs which inter- ferred with or enhanced catecholamine activity were used, and gonado- tropin secretion was monitored. Drugs such as reserpine, a catechola— mine depletor, and chlorpromazine, a potent adrenergic blocker, were shown to stimulate deciduoma formation (Barraclough and Sawyer, 1959), block ovulation (Coppola g g. , 1966), induce pseudopregnancy (COppola gglu 1965), and induce lactation in rabbits (Meites, 1957; Kanematsu a fl., 1963). These reports suggest an increase in Pro] actin secretion and a decrease in gonadotropin secretion. Early work from our laboratory indicated that injections of epinephrine and norepinephrine induced lactation in estrogen-primed 1"alts and rabbits (Meites, 1959a; 1962; Meites gtfl” 1963). The Observations that such drugs as reserpine and chlorpromazine were able to stimulate pseudopregnancy and lactation (Meites, 1959a) sug- gested that they induced prolactin release. Ratner g; g. (1965) fl"‘tl'ler demonstrated that reserpine decreased hypothalamic production of P1 F, providing an explanation of how reserpine evoked increased pr‘°"a<:tin secretion. Mizuno gig fl. (1964) further demonstrated that injections of iproniazid, a monoamine oxidase inhibitor and the"QT’Ore depressor of catecholamine metabolism, inhibited post- Dartmn lactation in rats, suggesting decreased secretion of prolactin. T his Provided the first evidence that brain catecholamines are 13 inhibitory to anterior pituitary secretion of prolactin. Further work by Lu £91. (1970) showed that reserpine, chlorpromazine, °(-nethyl-meta-tyrosine and °(-methyl-para-tyrosine, which are all catecholamine depressants, produced increases in serum prolactin levels indicating that they decreased hypothalamic PIF activity. On the other hand drugs as L-dopa (the inmediate precursor of dopamine) or monoamine oxidase inhibitors (pargyline, iproniazid, or Lilly compound-15641), each known to enhance hypothalamic catecholamine activity, significantly decreased serum prolactin values (Lu and Meites, 1971). Lu and Meites (1972) later showed that L-Dopa in- creased PIF activity in the hypothalamus. Another neuroleptic drug, haloperidol , reduced brain catecholamines and also markedly Elevated serum prolactin by decreasing PIF in the hypothalamus (Di ckerman Q fl., 1974). These reports led to the concept that (ll/pothalamic catecholamines, including dopamine and norepinephrine, am: as neurotransmitters to increase the release of PIF, which in turn enters the pituitary portal vessels to inhibit pituitary pro- lactin release (Meites gtflq 1972). There is evidence to suggest that the catecholamines have a di r‘l-‘-'<:t effect on the anterior pituitary to alter prolactin release. Gala and Reece (1965) observed that some doses of epinephrine in- creased prolactin release by rat pituitary. However, Jacobs g a_1_. “968), MacLeod (1969), and Birge g g. (1970) reported that cate- ch01amines including dopamine, norepinephrine and epinephrine i"""ibi ted prolactin release by rat pituitary tissue i_n vitro and conc‘lmed that catecholamines may represent the undefined hypothalamic P . IF. Koch gt fl. (1970) demonstrated a biphasic effect of catecholamines 14 on prolactin release. High doses of catecholamines produced inhibi- tion, intermediate doses no effect and low doses caused stimula- tion of prolactin release. Recent reports suggest that catechola- mines, particularly dopamine, may be a PIF. Shaar ggl, (1973) demonstrated that dopamine when incubated with pituitary halves inhibited prolactin release as compared to controls without dopa- This work has been confirmed by Samli and MacLeod (1974), Subsequently, Shaar mine. 0.ieda gta_1_. (1974) and Dibbet gtfl. (1974). and Clemens (1974) have shown that hypothalamic extracts subjected to catecholamine absorption on alumina gel lost their ability to inhibit prolactin release. Their results indicated that the pro- lactin inhibiting activity of hypothalamic extracts can be totally accounted for by the endogenous catecholamines normally present In the hypothalamus and further indicate that a catecholamine may be a PIF. These same workers presented evidence that dopamine is Present in the portal blood system (Shaar, personal conmunication). However, Takahara gt _a1. (1974) have evidence that a non-catechola- mine PIF is present in the hypothalamus. Indoleamines and catecholamines appear to work in opposition 0" control of pituitary prolactin release (Meites gtg1., 1972). "he" serotonin is given centrally, it increases prolactin (Kamberi fi LL, 1970; 1971). Systemic injection of 5-hydroxytryptophan, a p"'e<:ursor of serotonin, also increases serum prolactin (Lu and M11185, 1973). It is interesting to note that indoleamines exert an inhibitory effect on pituitary secretion of gonadotropins (Kamberi fig” 1970; 1971; Fraschini, 1970), in opposition to the effefits of the catecholamines (Kamberi g 31., 1970; Schneider and "ccann, 1970). o ‘-.‘\_~_.__ 15 Early investigations with cholinergic drugs indicated that atropine, a cholinergic blocking agent, could inhibit ovulation in the rat (Everett gt_gl,, 1949) and in the rabbit (Sawyer gt_g1,, Other drugs which act like atropine also blocked ovulation 1949). Kamberi and Bacleon (1973) reported that injections (Sawyer, 1963). of stropine into the third ventricle inhibited the proestrous surge of gonadotropins and prolactin. Cholinergic drugs have been shown to influence lactation and pseudopregnancy. Atropine in high doses given systemically induced lactation (Meites _‘g_1., 1960b) and pseudopregnancy (Gitsch and Everett, 1958), whereas low doses inhibited lactation (Jacobson a flu 1950) and pseudopregnancy (Grosvenor and Turner, 1958). Pilocar— Pine, a cholinomimetic agent, or physostigmine, an acetylcholine eSterase inhibitor, induced lactation in rats (Meites fig” 1960). However lactation is not a specific indicator of prolactin secre- tion. Recent work from our laboratory (Grandison gt __l_., 1974) has Shown that injections of acetylcholine into the lateral ventricle of female rats significantly decreased serum prolactin. Systemic Injections of either pilocarpine or physostigmine also decreased serum prolactin levels in female and male rats. The mechanisms by which acetylcholine inhibits prolactin release is not clear at p"VFASent. Previous work has shown that acetylcholine has no direct effect on pituitary prolactin release (Talwalker a fl” 1963). There is 1‘-he possibility that the effects of acetylcholine are mediated th”‘0th the hypothalamus and affect the PIF/PRF system. III. Functions of Prolactin A. Manmary Gland Eighty-two different actions have been reported for prolactin. Historically the best-known category of actions consists of effects related to reproduction (Nicoll and Bern, 1972). The most widely known of these in manlnals is stimulation of mamnary gland develop- ment and lactation, and in avian species stimulation of pigeon crop milk production. Classical experiments by Lyons and co-workers (1958) used (Upophysectomized, ovariectomized, and adrenalectomized rats as a model, and showed that injections of estrogen and GH produced ductal growth whereas injections of estrogen, progesterone, GH and pro- lactin elicited lobulo-alveolar growth. These studies were con- firmed by the £11m experiments of Elias (1957) and Rivera (1964). Undeveloped mamnary glands from mice and guinea pigs were CUItured for 5-7 days with combinations of insulin, estrogen, pro- gesterone, GH and prolactin, and these showed lobulo-alveolar growth. Pro] actin and GH can induce manmary lobulo-alveolar growth equi- va‘lent to that seen in pregnancy in the absence of ovarian hormones. ThUs transplantation of a pituitary mammtropic tumor that secreted hi 9"! amounts of prolactin, GH and ACTH into adreno-orchidectomized rats of the inbred Fischer strain resulted in manmary lobulo- a1"eolar growth equivalent to that seen during pregnancy “:1 ifton and Furth, 1960). Related experiments by Talwalker and "eitES (1961) showed that prolactin and GH injections into rats 17 which were adrenalectomi zed and ovari ectomi zed or adrenalectomi zed, ovariectomized and hypophysectomized were able to stimulate lobulo- alveolar growth. Turner (1939), Lyons fig. (1958) and Meites and Hopkins (1961) demonstrated that ovarian hormones have no ability to stimulate manmary gland development in the absence of anterior pitui- tary hormones. It has been suggested by Meites and Nicoll (1966) that the gonadal steroids stimulate secretion of anterior pituitary hormones (prolactin and GH) and synergize with the pituitary hormones to sensitize the mamnary tissue to these hormones. After development of mamary glands, the minimal requirements for initiating or maintaining lactation appear to be prolactin and ACTH or adrenal cortical hormones (Meites, 1966). Combinations of Prolactin and adrenal glucocorticoids were observed to initiate lactation in hypophysectomized rats (Folley, 1956), and in hypo- Physectomized, adreno-gonadectomized rats (Lyons g g“ 1958). Adrenal glucocorticoid hormones alone initiated lactation in the Pregnant rat (Talwalker and Meites, 1961) and cow (Tucker and Meites, 1965), and either prolactin or hydrocortisone acetate induced lacta- tion in the pregnant rabbit (Meites, 1966). There is an increase in Secretion of prolactin and adrenal cortical hormones at the end of pregnancy, permitting the onset of lactation. The milking stimulus together with milk removal from the manmary gland serves to maintain postpartum lactation (Meites ggg1., 1972). A look at the cellular actions of prolactin provides some clues as to how it stimulates manmary gland development and milk produc- ti on. More recently Turkington (19723) has reviewed the molecular biology of prolactin. He reported the ability of prolactin to 18 stimulate RNA synthesis leading to the production of specific milk proteins. Ovine prolactin can induce synthesis of casein, o(-1act- albumin and ,6 -lactoglobulin in mamary epithelial cells formed in culture (Turkington, 1968). The induction of milk protein synthesis is prevented by actinomycin D and this suggests that prolactin may require new DNA-directed RNA synthesis for milk protein synthesis. RNA and DNA content increase between day 18 or pregnancy and day 3 of lactation and the combination of insulin, prolactin and hydro- cortisone stimulate RNA synthesis in both prepartum and postpartum tissue (Mohrenweiser and Emery, 1973). When lactating rats are hypophysectomized the manmary gland undergoes several changes. Gland weight decreases and levels and synthesis of RNA and DNA are retarded (Baldwin and Martin, 1968). DNA levels and rates of sYnthesis were maintained by prolactin which also partially main- Rates of casein and cytoplasmic protein syn- For main- tained gland weight. thesis were maintained at normal levels by prolactin. teflance of optimal conditions of the mamary gland, both prolactin and cortisol seemed necessary. In mamary gland explants addition 01: prolactin to the insulin-hydrocortisone medium allows daughter c9115 to complete their ultrastructural differentiation; this includes appearance of secretory protein granules (Mills and Topper, 1970; Gr‘een and Topper, 1970). There is ample evidence that prolactin is a true metabolic hormone with respect to its action on the mamary 91am. It was of interest therefore to measure prolactin receptor b1 "ding activity of the mamary gland of the rat throughout preg- nancy and lactation as part of this thesis. (-3 LI B. Pigeon Crop Sac In a review by Riddle (1963) the pigeon crop sac was described. Only two limited lateral areas of the total crop are involved in "milk" production. Histological examination showed that only the mucosa hypertrophies and forms crop-milk. The deeper lying cells persistently divide and contribute to a thickening of the crop wall. These cells form globules of fat, begin to disintegrate, and the desquamated dead cells form the whitish masses called crop milk. Riddle and Braucher (1931) demonstrated that the crop-sac response is controlled by an anterior pituitary hormone. Riddle, Bates and Dykshorn (1932; 1933) isolated and named the hormone ”prolactin" and showed that it evoked a crop-sac response as well as stimulated milk secretion in mamnals. Nicoll and Sherry (1967) demonstrated that the prolactin-induced stimulation of crop-sac mucosa is mediated by RNA and protein synthesis. Riddle and coworkers described a systema- tic method for quantitative assay of prolactin based on weight in- Cv‘ease of the crop sac after 4 daily intramuscular injections of pro- Another assay developed by Lyons and Page (1935) involved Until the advent of a lactin. Intracutaneous injection over the crop sac. "adioinlnunoassay for prolactin (Niswender 3531., 1969), the pigeon Cr‘Op sac response was the standard assay procedure for prolactin and is still used as a bioassay for prolactin. As reviewed by Bern and Ni coll (1968) one of the main uses of the pigeon crop sac assay has lBeen to distinguish between prolactins from various vertebrates 8“ch as teleosts and poikilothermic tetrapod’s and manlnals. 20 C. Ovaries Prolactin is the major luteotropin in the rat. In hypo- physectomized rats with fresh corpora lutea, prolactin administration maintained functional corpora lutea and deciduoma formation (Astwood, 1941; Evans gt_g1,, 1946; Ahmad gt_g1,, 1969). Increased levels of prolactin can maintain functional corpora lutea for prolonged periods of time. Everett (1956) showed that removal of the anterior pituitary in rats from its connection with the diencephalon and placing it under the kidney capsule caused a prolonged secretion of prolactin and maintenance of luteotropic activity. Subsequent studies by Nikitovitch-winer and Everett (1958) in which anterior pituitary grafts were placed under the kidney and in the anterior chamber of the eye, also demonstrated a prolonged luteotropic acti- vity in rats. Chlorpromazine, reserpine (Barraclough and Sawyer, 1959) and perphenazine (Ben-David, 1968), are all stimulators of prolactin secretion (Lu gt_g1., 1970; Danon _t__1,, 1963), and all prolong luteotropic activity. Injections of ovine lactogen for 5-7 days from the day of estrus induced pseudopregnancy in normal or hysterectomized rats (Anderson, 1968). Saito gt 31. (1970) showed that prolactin administration to hypophysectomized rats, soon after ovulation had been induced by PMS and HCG, also induced pseudo- pregnancy. The above studies indicate that prolactin is able to stimulate Progesterone secretion from rat corpora lutea. That prolactin was able to maintain progesterone secretion was shown by MacDonald and GPeep (1968). The mechanism of action of prolactin on corpora lutea 21 involves ovarian enzymes. Hashimota and Hiest (1969) explained the luteotrophic action of prolactin by its antagonistic action to luteal 20-0(-hydroxysteroid dehydrogenase and in part by its mobilization of a progesterone precursor. The enzyme, 204’(-hydroxysteroid dehydrogenase (206(HD) converts progesterone to a ZOcn-dihydro derivative which has no progesterone activity. A 3-4 fold increase in 20°(HD activity in the ovary is induced by treatment of rats with various ergot alkaloids, and this increase is prevented by exogenous prolactin (Lamprecht gglg1., 1969). Prolactin administration to rats bearing three day old corpora lutea increases progesterone secretion, decreases the synthesis of 200(HD, and influences the rate of cholesterol turnover in lutein tissue (Armstrong gt_g1,, 1969). In a subsequent study by Armstrong gglg1. (1970) 30 day old female rats were treated with PMS and HCG to induce ovulation and then they were cervically stimulated to induce pseudopregnancy. Hypophy- sectomy of these rats decreased progesterone secretion and increased the level of ZOCA-hydroxypregn4-en-3-one, an inactive progesterone derivative. Prolactin treatment begun a few hours after hypophy- sectomy considerably increased progesterone and decreased the levels of the inactive progesterone derivative, and increased free and esterified cholesterol levels. The activity of two other enzymes involved in progesterone metabolism, Set-reductase and 3l3-hydroxyster- oid dehydrogenase, is decreased after prolactin and administration (Zmigrod gt_g1., 1972). In addition to influencing progesterone metabolism, prolactin also causes cholesterol accumulation in both interstitial and luteal compartments of the ovary in intact and hypophysectomized innature rats (Zarrow and Clark, 1969). Behrman gt_g1., 22 (1970) and Behrman and Greep (1972) demonstrated that prolactin was able to induce enzymes controlling luteal cholesterol ester turnover, sterol acyl transferase and sterol esterase. In addition to being the main luteotropin in the rat, there is evidence that prolactin also may influence luteal function in the mouse, hamster, ferret, rabbit, cow, pig, and sheep. In 1935, Dresel reported that injections of prolactin into cycling mice produced persistent leucocytic vaginal smears and a suspension of estrus. Prolactin is part of the luteotropic complex in the hamster (Greenwald, 1967a). Pseudopregnancy was induced in cyclic hamsters with a combination of prolactin and FSH (Grady and Greenwald, 1968). In the ferret, isolation of the hypophysis by pituitary stalk sec- tion does not interfere with luteal function (Donovan, 1963). Stalk sectioned animals in whom ovulation is induced by sterile mating show typical pseudopregnant changes, including the ovaries which contain large secretory corpora lutea. Donovan (1967) re- ported that the factor secreted by the isolated hypophysis to main- tain luteal function was prolactin. Prolactin was the only hormone to support luteal function in the hypophysectomized ferret. Unlike the rat, mouse and ferret, the role of prolactin in larger animals is not clear. It may act synergistically with other hormones to main- tain luteal function. In the hypophysectomized rabbit, prolactin is not able to maintain corpora luteal function (Kilpatrick gt_g1,. 1964). Hilliard and Sawyer (1966) showed that although prolactin was not able to acutely stimulate steroidogenesis in the rabbit ovary, it could enhance the responsiveness of the preovulatory ovary to LH and thus increase basal progesterone secretion. They concluded 23 that prolactin affects steroidogenesis in the rabbit ovary by maintaining availability of steroid precursors. A later study by Hilliard gt_gl, (1968) showed that LH accelerates the synthesis and release of 206K -hydroxypregn—4-en-3-one (20e<-0H) from rabbit ovarian interstitial tissue, concomitant with a loss of interstitial cholesterol. Hhen ovarian cholesterol is depleted by LH, less steroid is released, and chronic prolactin treatment promotes chol- esterol storage, restores the basal output of 20°<-0H and enhances sensitivity of the ovary to LH in the intact rabbit. In the hypo— physectomized rabbit it is necessary for prolactin and estrogen or LH to be administered in order to elevate cholesterol stores and induce progestin release. Bartosik t al. (1967) and Romanoff gt_g1. (1966), using perfused bovine ovaries in vitro, demonstrated that prolactin infusion caused an increase in progesterone secretion rate and an increase in acetate-1-14C incorporation into progesterone. Prolactin infusion into ovaries jg_gitg was seen to increase the secretion rate of progesterone in sheep (Domanski and Dobrowolski, 1966; Domanski gt_g1,, 1967). Hixon and Clegg (1969) reported that 50 mg of prolactin was able to significantly increase ovarian progesterone secretion in the hypophysectomized ewe. Cook gt 31. (1969) showed that prolactin was able to enhance progesterone secre- tion in the pig but had no effect on luteal function in the ewe. The most dramatic effect of prolactin on corpora lutea func- tion is seen during pregnancy. Cutuly (1941) showed that rats hypophysectomized 1 to 5 days after mating and treated with lacto- genic hormone, were able to implant and maintain pregnancy for periods ranging from 6 days to term. Cutuly (1941) suggested that 24 lactogenic hormone was capable of stimulating corpora lutea function. Meites and Shelesnyak (1957) administered large doses of prolactin and observed a lengthening of pregnancy by extending functional activity of corpora lutea. Hypophysectomy at days 1, 4, or 8 of pregnancy in the hamster resulted in regression of corpus luteum (Greenwald, 1967b). Treatment with prolactin and FSH increased vascularity of the corpus luteum and maintained pregnancy in these hamsters. Prolactin and FSH seem to be the luteotropic complex in the mouse as well as the hamster (Choudary and Greenwald, 1969). Pregnant mice hypophysectomized on day 6 and injected with 500 ug prolactin and 200 ug FSH maintained pregnancy. In dwarf mice pro- lactin seems to be the sole hormone necessary to maintain pregnancy and support corpora luteal function (A. Bartke, l966a;A. Bartke, 197k: A. Bartke, 1973). Robson gt_g1. (1971) reported that in mice, prolactin is the major hormone to support pregnancy during the pre- implantation period and after implantation LH seems to be necessary. In rabbits hypophysectomized during the second week of gestation, severe follicular involution and degeneration of corpora lutea became apparent within seven days (Spies gt_g1., 1968). Prolactin in combination with either FSH and more effectively with estrogen restored ovarian luteal and interstitial tissue comparable to that of intact pregnant rabbits. In the rat, prolactin is the major if not sole stimulus for luteal function until days 7 to 8 of pregnancy, when placental prolactin and pituitary LH become essential members of the luteo- tropic complex. From day 12 of pregnancy until parturition, LH is no longer required and placental prolactin alone may be sufficient 25 by itself to maintain the corpus luteum (Neill and Smith, 1974). The following reports seem to substantiate the above statement. Clemens _t_g1. (1969a) showed that prolactin was necessary to main- tain pregnancy in the rat from days 1 to 6. The pregnant rats were given hypothalamic implants of prolactin and this appeared to induce luteal regression and cessation of progesterone secretion. Rats hypophysectomized on day 5 or 6 of pregnancy and given prolactin, were only able to maintain pregnancy until day 10 (Greenwald and Johnson, 1968; Yang gt_gl,, 1973). If prolactin and ICSH or FSH or estrogen were given, rats maintained pregnancy until term show- ing that prolactin by itself is essential during the first half of pregnancy. Morishige and Rothchild (1973; 1974) have shown that blocking prolactin secretion with ergocornine caused regression of corpora lutea until days 7-8 of pregnancy; after this period inhibition of pituitary prolactin secretion did not induce luteolysis. LH anti- serum did not induce luteolysis until days 7-8, when it became highly effective. They also observed that in addition to hypophyseal LH, placental prolactin is necessary for maintenance of corpora lutea at days 7-8. In the absence of hypophyseal prolactin but in the presence of hypophyseal LH, removal of the uterus and thus placental prolactin at days 7-8, caused regression of corpora lutea. The corpus lutea of the rat are maintained at day 12 of pregnancy or later without hypophyseal support (Astwood and Greep, 1938). Moreover, Madhwa Raj and Moudgal (1970) showed that LH antiserum became ineffective in inducing abortion at day 12. The maintenance of corpora lutea and progesterone secretion during the last half 26 of pregnancy is probably accounted for by secretion of a placental prolactin (Neill and Smith, 1974). Recent work using a radioreceptor assay has reported 2 peaks of placental prolactin during pregnancy, one at days 10-14 and another at days 17-21 (Kelly gt_g1., 1973). It also appears that prolactin is necessary to maintain corpora luteal function during lactation, since inhibition of lactation and corpora luteal function was induced by a hypothalamic implant of prolactin in postpartum rats (Clemens gt_gl,, 1969b). Prolactin has the ability to be luteolytic as well as luteo- tropic in rats (Malven and Sawyer, 1966). The luteolytic effect of prolactin in the rat seems to be exerted on corpora lutea which have lost their capacity to secrete progesterone (Lam and Rothchild, 1973). In hypophysectomized rats, administration of prolactin either 2 or 5 days after hypophysectomy enhances luteal regression (Mac- Donald and Greep, 1969; Saito e_t fl” 1970), whereas if prolactin administration is started soon after hypophysectomy it maintains the corpora lutea (Malven, 1969; Saito gt g1., 1970). The function of prolactin in the normal cycling rat may be to induce luteolysis. Huttke and Meites (1971) demonstrated that the rise of prolactin during the estrous cycle of the rat serves to induce luteolysis of the older crop of corpora lutea during each cycle. This work was later confirmed by Gelato gt_g1. (1972) and by Grandison and Meites (1972) in mice. Since one of the most important functions of pro- lactin is to regulate corpora luteal function it was thought of interest to study the ontogeny of prolactin receptor binding activity in ovaries from pubescence through adulthood, and during pregnancy and lactation. 27 0. Male Reproductive System The role of prolactin in the male has not been clearly defined. In a recent report by Negro-Vilar gt_g1. (1973), changes in serum prolactin as well as the gonadotropins were monitored during sexual development of the male rat. The serum hormone levels were correlated with organ growth rates and they found that the initial increase in testicular growth was preceded by a sharp rise in FSH and prolactin at 25 days of age. In immature hypophysectomized rats, pituitary transplants induced a significant increase in testicular growth (Negro-Vilar and Seed, 1972). They assumed prolactin was responsible for the testicular growth by activation of steroidogenic pathways leading to androgen biosynthesis. Hafiez gt_g1. (1971) and Musto gt_g1. (1972) showed that prolactin administration can increase 3-B-hydroxysteroid dehydrogenase and l7-B-hydroxysteroid dehydro- genase activity in the testis of dwarf mice. Injections of prolactin to hypophysectomized rats raised testosterone to detectable levels in half of the rats tested (Hafiez gt 11., 1972). In the same study LH increased the levels of testosterone in all rats, and when a combination of prolactin and LH were given there was a greater increase in testosterone than when LH alone was given. Prolactin has also been shown to promote accumulation of cholesterol esters in the mouse testis (Bartke, 1971a). Prolactin may influence testicular function as there is evidence reported that it stimulates testosterone production and synergizes with LH. Bartke (1971b) reported that LH produces more androgen in hypophysectomized rats when prolactin is present. Several reports have shown some effects of prolactin on I.‘ 28 spenmatogenesis in rats and mice (A. Bartke, 1965; A. Bartke, 1966a; A. Bartke and C.H. Lloyd, 1970a; A. Bartke, 1971b). However, these effects may be secondary to the ability of prolactin to stimulate androgen synthesis. In addition to influencing testicular function, prolactin can influence the male accessory organs. Prolactin stimulated growth of seminal vesicles in tissue culture when added to medium (Bengmark and Hesselsjo, 1964). In castrate male mice prolactin caused an increase in seminal vesicle weight (Bartke and Lloyd, 1970b), and when testosterone was given in combination with prolactin the weight of these organs was increased further (Bartke, 1967). Castrate guinea pigs given testosterone and prolactin had seminal vesicles twice the size of the castrates receiving only testosterone (Antliff gt g1., 1960). Prolactin also synergizes with testosterone to increase the size of the prostate. Gonadectomized-hypophysectomized rats treated with either LH or STH alone produced no response in prostate weight but testosterone caused a doubling in ventral and anterior prostate weight (Chase gt_g1., 1957). In these same animals prolactin plus testosterone produced a significant increase over the effect of testosterone alone. A similar report was presented by Grayhack (1963) who also showed that prolactin and testosterone synergize to increase prostate weight. In the absence of testosterone, prolactin can synergize with other hormones to stimulate prostatic growth. In immature castrate rats prolactin and ACTH synergize to increase ventral prostate weight (Tullner, 1963). If these immature rats are hypophysectomized as well as castrated, prolactin, ACTH and thyroxine can act to restore the weight of the ventral prostate. Fri 29 Prolactin has also been shown to increase citric acid content of the lateral prostate in hypophysectomized-castrate rats in combina- tion with testosterone (Grayhack, 1963; Grayhack and Lebowitz, 1967). More recently Moger and Geschwind (1972) reported that prolactin synergized with testosterone to increase fructose and citric acid content of the dorsolateral prostate, seminal vesicles and coagulat- ing glands. They also showed that prolactin was able to increase zinc uptake in the absence of testosterone. In prostatic tissue homogenates of fourteen-week-old rats, prolactin stimulated adenyl cyclase activity whereas testosterone had no effect (Golder ggig1., 1972). This may be a possible mechanism of action for prolactin. It seems apparent that prolactin may indeed be an important "co- factor" in the male reproductive system. E. Metabolic Function Prolactin has been reported to be a somatic as well as a metabolic hormone (Riddle, 1963; Bern and Nicoll, 1968; Nicoll and Bern, 1972). Licht and Jones (1967) showed that prolactin increased food consumption and increased lean body weight in adult male lizards. These same effects of prolactin were seen in juvenile lizards along with a decrease in hepatic lipid content (Licht and Hoyer, 1968). Licht (1967) and Tassava (1969) demonstrated that injections of pro- lactin or pituitary grafts were able to stimulate tail regeneration in the lizard. A more recent report by Callard and Chan (1972) indi- cated a synergistic effect of prolactin and corticosterone to restore liver weight and glycogen content in the hypophysectomized lizard. 30 A somatic role of prolactin in tadpoles has been well esta- blished. Berman gt_gl. (1964) and later Enemar gt_g1. (1968) re— ported that mammalian prolactin caused increased body weight and increased tail length in tadpoles. It seems that prolactin is antagonistic to thyroxine in tadpoles and favors the retention of larval structures (Bern and Nicoll, 1968). Bern gt_g1. (1967) countered the tail-resorbing influence of thyroxine added to aquarium water by injections of prolactin. Later Brown and Frye (1969a) showed that prolactin inhibited metamorphosis and promoted further growth in thyroidectomized tadpoles. In subsequent work, Brown and Frye (1969b) showed that prolactin was ineffective in stimulating growth in post-metamorphic frogs. Vellano gt_g1. (1970) were able to show that in castrate or castrate-thyroidectomized newts, prolactin administration increased tail height. They con- cluded that height of tail in the newt is prolactin dependent. As well as stimulating growth in amphibians, prolactin has also been reported to increase carbohydrate content of the liver (Brown and Brown, 1971) and induce arginase activity in larval liver (Guarda- bassi gt g1., 1970). Early work by Bates gt_gl. (1937) and Schooley gt_g1. (1941) demonstrated that prolactin had the ability to maintain body weight in hypophysectomized pigeons and in normal young pigeons, and was most effective in stimulating appetite and increasing intestinal, pancreatic, and liver tissue weight. Prolactin also synergized with other hormones to promote organ and body growth. Miller and Riddle (1943) reported that prolactin, desoxycorticosterone and thyroxine in combination had a greater effect than any of these hormones given 31 alone. One unit of prolactin daily reduced the amount of weight lost to less than half in untreated hypophysectomized pigeons, whereas when prolactin, desoxycorticosterone and thyroxine were given the hypophysectomized pigeons regained all their lost weight. In a later study Bates £3.21- (1962) reported that adequate doses of either prolactin or CH alone or together increased organ weight and body weight in hypophysectomized pigeons. An even greater increase was observed when thyroxine and prednisone were given together with GH and prolactin. They noted synergisms in the order of 50 fold when all 4 hormones were given. Prolactin not only increased liver size but stimulated liver function in the pigeon. Goodridge and Ball (1967) treated pigeons with prolactin and found an increased rate of hepatic fatty acid synthesis and increased rate in conversion of glucose to fatty acids. They also observed an increased utilization of glucose by tissues of prolactin-treated birds, and an elevated level of enzymes involved in lipogenesis. Prolactin is somatotrophic in other birds as well as the pigeon. In the Spotted Munia prolactin enhances body growth and influences spleen and liver weight (Chandala and Thapliyal, 1968; Chandola and Thapliyal, 1973). Increases in body fat were the result of prolactin administration to the white-throated Sparrow (Meier and Martin, 1971). In mammals there is strong evidence indicating that prolactin is a metabolic hormone capable of stimulating growth! Bates gt_91, (1935) were able to stimulate growth in dwarf mice with a crude prolactin preparation. In a subsequent study Bates and co-workers (1942) demonstrated that pituitary extracts containing prolactin e. 32 caused a 30% increase in body weight of dwarf mice. Knobil (1959) observed striking stimulation of growth in young normal rats which was comparable to that seen in response to growth hormone. In male rats hypophysectomized for 7 days, prolactin caused an increase in body weight and cartilage width as compared to saline injected hypophysectomized rats (Cargill Thompson and Crean, 1963). As re- ported in the pigeons, prolactin also synergizes with other hormones in the rat (Bates gt g1., 1964; Milkovic gt_g1., 1964). Prolactin alone caused an increase in nose to tip of tail length in normal rats, and when prolactin, GH and ACTH were given in combination a rapid increase in body weight of nearly 4 g per day was seen. Rats with transplantable mammotropic tumors, which secrete large amounts of GH, prolactin and ACTH, developed diabetes as well as enlarged livers (Bates gt_g1., 1966; Wilson, 1969). Prolactin appears to affect the metabolism of carbohydrate and fat in both liver and adipose tissue. Hinegrad gt_g1. (1959) removed epididymal fat pads from male rats and incubated with ovine prolactin. Prolactin in this preparation increased the production of C02 from glucose and increased the incorporation of glucose carbon into long chain fatty acids. It's interesting to note that prolac- tin and GH worked differently. GH also increased glucose oxidation to C02 but this increase was not accompanied by an increase in fatty acid synthesis from glucose. They felt that the data indicated prolactin stimulated phosphogluconate oxidative pathway in glucose utilization. Similar results were reported on the effect of prolactin on adipose tissue by Moore and Ball (1962) and Beck gt Q1. (1964). They saw the same effect of prolactin and also noted the difference 33 between GH and prolactin, that is GH did not increase the incorpora- tion of glucose carbons into long chain fatty acids in adipose tissue as did prolactin. Nejad gt_gl, (1962) studied the conversion of glucose carbon to fatty acids and C02 in liver slices of hypophy- sectomized rats. In hypophysectomized rats the conversion of glu- cose to fatty acids and 002 was impaired. Administration of 300 ug GH for 14 days was not able to repair this defect but when 100 ug prolactin with 3 ug l-thyroxine (which was ineffective alone) were given, the lipogenesis was repaired. It is of interest to point out here that prolactin affects the metabolism of the mammary gland in the same way it does adipose tissue and liver, as cited above (Heitzman, 1968; Wang gt_g1,, 1971; Strong gt_g1., 1971). In mice prolactin has been reported to induce liver glyco- genolysis (Elghamry gt g1,, 1966), increase xanthine oxidase acti- vity in liver and mammary gland (Chandra and Cole, 1961) and stimu- late hepatic RNA synthesis as well as increase body weight by 16% (Chen gt_g1,, 1972). Rats bearing mammotropic tumors which secrete large amounts of prolactin, GH and ACTH, showed increased incorpora- tion of acetate into long chain fatty acids, increased protein con- tent and increased acetyl-CoA carboxylase activity which is the rate limiting enzyme in fatty acid biosynthesis (MacLeod gt g1., 1968). Placental lactogen stimulated the incorporation of glycine-Z-C14 into liver protein of rats (Burt gt_g1,, 1969). The authors discussed the point that pregnant animals have higher incorporation of glycine-l-C14 than non-pregnant animals, and liver in pregnancy has higher nucleic acid content which may be related to the action of human placental lactogen. Turkington (1972b) also observed large increases in rates of RNA formation in liver during pregnancy and lactation. 34 Houssay and Penhas (1956) demonstrated a diabetogenic effect of prolactin in dogs that were hypophysectomized-adrenalectomized and had 15-18% of their pancreas removed. In a later study, DeBodo and Altszuler (1958) reported that prolactin seemed to improve the insulin hypersensitivity of hypophysectomized dogs. Continued administration of prolactin in hypophysectomized dogs caused eleva- tion of post-absorptive blood sugar levels toward normal and decreased the responsiveness to insulin. These authors point out that pro- lactin differs from GH in that it does not produce severe hypo- glycemia after first treatment. More recent work by Rothgeb gt 11. (1971) showed again that prolactin administration in the dog in- creased the concentration of glucose in the post-absorptive state but produced no change in insulin. Prolactin also increased the rate of glucose production and uptake in the post-absorptive state. Again mention is made of the difference between prolactin and GH. In the GH treated dog insulin was very high and this was not so with prolactin treatment. However, prolactin like GH in the dog increased glucose turnover, liver glycogen and plasma free fatty acids (Hinkler gt 21,, 1971). This effect of prolactin is not due to GH contamina- tion since the amount of GH believed to be in the prolactin prepara- tion was injected without any effect. In the cow prolactin admini- stration caused an increase in non-esterified fatty acids (Hilliams gt_g1,, 1966), and feeding and arginine infusion in cows resulted in a significant increase in plasma prolactin (McAtee and Trenkle, 1971). Human studies also point to the possibility of prolactin being a metabolic hormone. In 1958, Bergenstal and Lipsett observed that administration of ovine prolactin to human subjects who had been 35 hypophysectomized caused a retention of nitrogen and an increase in urinary amino acid nitrogen excretion, indicating these patients were in a positive nitrogen balance. In humans both placental lactogen and pituitary prolactin seem to induce carbohydrate intolerance and are thought to be physiological antagonists of insulin (Beck and Daughaday, 1967; McGarry gt_g1,, 1968). An intravenous injection of insulin into men and women caused a significant increase in plasma prolactin and rather low levels of glucose (10 mg/100 ml) also stimulated prolactin release (Wilson gt_g1,, 1972). The meta- bolic effects of human prolactin were assessed in a recent study in women. Berle (1973) reported that administration of human prolactin resulted in an increase in plasma B-hydroxybutyrate, a product of fatty acid degradation, and free fatty acids and a decrease in pyruvate and lactate indicating an effect on glucose metabolism and utilization. There seem to be some common threads in the action of pro- lactin in these different species. Prolactin is able to stimulate growth in amphibians, birds, rats and mice, and can stimulate liver protein synthesis in rats and mice and influence carbohydrate and lipid metabolism in pigeons, rats, dogs, cows and man. It is quite possible that in avian and mammalian species there exists a "Metabolic Complex" of hormones consisting of GH, prolactin, adrenal corticoids and thyroid hormones. This is suggestive in the work of Bates gt_g1, (1974). Part of the data to be presented in this thesis deals with studies measuring prolactin binding to liver membrane preparations. 36 F. Salt and Hater Balance Osmoregulation in lower vertebrates and mammals has been reported to involve prolactin. Burden (1956) showed that killfish were unable to survive in fresh water without the pituitary. Pick- ford and Phillip (1959) demonstrated that the pituitary factor able to overcome the effects of hypophysectomy in euryhaline fish was prolactin. According to Ball (1969) euryhaline teleosts are unable to survive in fresh water after hypophysectomy for more than a limited period, but they can live for much longer in sea water or in a fish Ringer solution. Prolactin seems to be specific since injections of ADH, oxytocin, GH, TSH or ACTH are without effect in hypophysectomized teleosts (Schreibman and Kallman, 1966). Blood osmotic pressure and sodium concentration fall after hypophysectomy in teleost. When the hypophysectomized fish are given injections of prolactin, they are able to maintain sodium levels near normal (Ball and Ensor, 1965; Ball and Ensor, 1967; Dharmamba, 1970). Prolactin has been reported to have the same sodium retaining effect in intact fish (Utida gt g1., 1971). Intact seawater adapted fish were given prolactin injections and showed significantly high plasma sodium concentrations. In some cases the sodium levels rose so high it be- came toxic to the fish. In the teleost pituitary there is a specific region which is a source of prolactin-like hormone (Bern and Nicoll, 1968). The erythrosinophilic or eta cells are organized into a distinct rostral lobe. Ectopic transplants of rostral lobe in hypophysectomized fish permit survival in fresh water (Ball, 1965). The rostral lobe 37 comprises 8% of the total gland and increases to 42% in specimens held for long periods in fresh water (Blanc-Livini and Abraham, 1970). Prolactin in the rostral lobe also increases in fish held in freshwater for extended periods of time. This evidence indicates that prolactin is an important hormone in teleosts for sodium regulation. It's interesting to note that Hirano gt_g1, (1973) reported an increase in labelled thymidine incorporation into DNA of the urinary bladder of the flounder after prolactin administra- tion. The increase in sodium absorption after prolactin treatment fellowed closely the time course of thymidine incorporation. It's possible therefore, that prolactin action (sodium retention) in teleosts may be mediated through RNA and protein synthesis. In the domestic duck prolactin has been postulated to aid in adaptation from freshwater to an estuarine environment. Prolactin infused intravenously in domestic duck caused an increase in nasal fluid output (Peaker gt_gl,, 1970). Therefore it may stimulate the inactive salt gland to start secreting at a high rate. Further work by Ensor and Phillips (1970) showed that the pituitary pro- lactin levels in the domestic duck increased after 2-3 days of salt loading. These workers concluded that prolactin may have a direct effect on salt gland of these birds and cause it to excrete sodium. These reports indicate a species difference in prolactin's osmo- regulatory action. The overall effect of prolactin in mammals seems to be sodium retention and promotion of antidiuresis, which is similar to its action in the teleosts. Rats given a single injection of either bovine or ovine lactogenic hormone show a decrease in the rate of 38 urinary excretion of sodium, which was interpreted to be due to a direct effect of these hormones on the renal tubules (Lockett and Nail, 1965). Lockett (1965; 1967) reported a direct effect of lactogenic hormones on the renal tubules in a perfused kidney preparation in cats. The perfusion of the cat kidneys with lacto- genic hormone resulted in an increased renal blood flow and glomer- ular filtration rate followed by a retention of sodium. It's inter- esting to note that in Lockett's preparation, GH was also anti- diuretic and caused the retention of sodium but did not alter renal blood flow or glomerular filtration rate. Prolactin in rats has been reported also to influence clearance of insulin and para-amino hippuric acid (Matthews, 1963). Ensor gt g1., (1972) demonstrated that dehydration in non-lactating rats was correlated with a 25% de- crease in pituitary prolactin. In lactating female rats this fall was increased to 50%. Further experiments injecting ovine prolactin into intact female rats showed prolactin to be antidiuretic. Relkin and Adachi (1973) and Relkin (1973) reported that rats maintained on a low sodium diet had increased plasma and pituitary prolactin, whereas no change was seen in GH or TSH. They proposed that pro— lactin may enhance the aldosterone secretory rate in sodium deprived rats since aldosterone secretion was increased in these rats. Pallmore gt 91, (1970) reported that the pituitary is necessary to maintain aldosterone secretion in rats. The important hormone could possibly be prolactin. Salt loading (400 meg NaCl) in the ewe appears to negate the sodium retaining action of aldosterone. If these animals were injected with sheep pituitary prolactin the sodium retaining action of aldosterone was restored in spite of the high 39 salt (Burstyn gt_g1,, 1972). Further work by the same group of investigators (Horrobin gt_gl,, 1973) showed that ewes given 80 meg of NaCl, aldosterone promotes sodium retention. If cortisol is given along with the aldosterone it antagonizes the action of aldo- sterone and the animals lose sodium. Prolactin is able to override the effects of cortisol and aldosterone, again causing sodium re- tention. Therefore it's possible that prolactin may sensitize the renal tubules to the action of aldosterone. Rats treated with 2- bromo-°(-ergokryptine had an increased sodium excretion as well as an increased fluid volume (Richardson, 1973). The actions of 2- bromoairergokryptine on the kidney were attributed to the ability of the drug to decrease prolactin secretion. In the human, Horrobin gt 31, (1971) reported that a single injection of prolactin produced a significant decrease in water ex- cretion, sodium excretion, and increased plasma sodium levels. They suggested that prolactin may act on the proximal tubule. More recent studies on humans (Buckman and Peake, 19733.8uckman and Peake, 1973b) showed that plasma prolactin was decreased after administration of hypotonic fluid and increased after administration of hypertonic fluid. The reports in humans seem somewhat conflicting. It is hard to fit the prolactin responses to hypo- and hyper-tonic fluids into the proposed action of prolactin on the kidney tubules to enhance sodium retention. Relkin (1974) reported this same phenomenon in rats. He saw a large increase in prolactin when he infused a 3% NaCl solution into rats and a decrease in prolactin with infusion of hypotonic NaCl solution. It's quite possible that the mechanisms of prolactin action as an osmoregulator are many sided, that is, 4O prolactin may respond to both a change in sodium concentration and a change in osmolality. In light of the present work, it was felt of interest to determine whether or not prolactin binds specifically to rat kidney homogenates. G. Adrenal Function Tullner (1963) and Bates gt_g1, (1964) observed that prolactin was able to augment the adrenal weight response to ACTH. This appeared to indicate that prolactin may influence adrenal function. A clinical report by Ingvarsson (1969) Showed that prolactin may cause sensitization of an ACTH refractory adrenal cortex. In a female rheumatoid arthritic patient, dependent on steroids, prolactin ad- ministration lessened the dosage of steroids required for treatment. After prolactin administration, a low excretion of 17-ketogenic steroids was found and a considerable increase in excretion of 17- ketogenic steroids in response to administration of ACTH after prolactin treatment. Nitorsch and Kitay (1972) demonstrated that prolactin decreased adrenal Set-reductase activity in hypophysecto- mized rats. Adrenal sit-reductase converts corticosterone to reduced metabolites. The data suggest that prolactin plays an alternate physiological role in regulating hormone secretion by preventing 'the intra-adrenal conversion of corticosterone to reduced metabo- lites. More recently Lis gt_g1, (1973) reported that prolactin was able to stimulate corticosterone synthesis. Using isolated rat adrenal cells jg_vitro they observed that prolactin administered jg_vivo partially restored the corticosterone biosynthesis in 41 adrenals from hypophysectomized rats. Further the combined treatment to hypophysectomized rats of ACTH and prolactin gave higher maximal response than treatment with ACTH alone. In addition to affecting adrenal corticoids, Piva gt_g1, (1973) showed that prolactin also influences adrenal progesterone. Dexamethasone treated castrated female rats given an intravenous injection of ovine prolactin ex- hibited a marked increase in adrenal progesterone as measured by progesterone plasma levels. Prolactin was almost three times as effective as either human LH or ovine GH. With these reports on the ability of prolactin to influence adrenal steroid production, it was thought of interest to measure specific binding of prolactin to adrenal membrane preparations. IV. Prolactin Interactions with Other Hormones The endocrine system is a highly integrated affair, and ex- cesses or deficiencies in one gland may alter the rate of production of hormones by others (Turner and Bagnara, 1971). Almost every physiologic adjustment within the endocrine system is effected by a balance between hormones acting together or in sequence. For example, complete and normal functioning of mammary glands requires estrogens, progesterone, insulin, prolactin, oxytocin, adrenal steroids, thyroid hormones and possibly others. The estrous and menstrual cycles require a multitude of hormones acting in concert to produce the observed changes (Schwartz and McCormack, 1972). Hormones in the body fluids never act alone, and one may be modified (potentiated, limited or secretion pattern altered) by others that 42 are present. There is evidence that progesterone, emanating from the placenta, prevents premature expulsion of the fetus by block- ing the response of the uterine musculature to other hormones (Turner and Bagnara, l97l). Synergisms are an important phenomenon in endocrinology. Some hormones increase the effectiveness of others that are present with them in low concentrations. Bates gt_al, (l964) and Milkovic gt 31, (l964) reported that for normal growth in rats several hormones are needed, such as growth hormone, prolactin, ACTH, and thyroxine. They also observed that the combination of hormones in physiological doses was much more effective in stimulating growth than any of the hormones administered alone in large quantities. A. Effects of Estrogen, Testosterone and Progesterone on Prolactin Secretion Estradiol can directly stimulate rat pituitary prolactin re- lease when added to pituitary culture jg_yjt§9 (Meites, l966). Injections of estrogen jg_yjyg_depressed hypothalamic PIF activity in the rat (Ratner and Meites, l964). This work indicated that estrogen acts to promote prolactin release both via the hypothalamus and by a direct action on the anterior pituitary. There are several reports which show that estrogen administration jg_yi!g_in rats increases serum and pituitary prolactin levels. Implants of minute quantities of estrogen in the median eminence of rats with DMBA- induced mammary tumors caused an increase in pituitary and serum [Jrolactin levels as compared to rats implanted with cholesterol “ 43 (Nagasawa gt al., 1969). In ovariectomized rats estrogen also stimulated prolactin secretion and it appears that small doses of estrogen are more effective than large doses (Chen and Meites, l970). The ability of estrogen to stimulate prolactin secretion in ovariectomized rats was confirmed by Blake gt_gl, (l972) and Kalra £3 91, (l973). Kalra §t_gl, (l973) also observed that estrogen administration was able to stimulate the release of prolactin when injected on the morning of estrus into intact female rats and en- hance prolactin release in castrate males. Data to be presented in this thesis will demonstrate that estrogen can also influence pro- lactin receptor binding activity in liver and mammary tissue. In culture of rat pituitaries progesterone failed to stimulate prolactin release (Meites, 1966) whereas Meites (1959) reported that jg_yjyg_administration of large doses of progesterone increased pituitary prolactin content and elicited mammary growth and secretion in rats. More recent work on the progesterone effect on prolactin release indicated that in ovariectomized rats it can partially counteract the stimulatory action on prolactin release (Chen and Meites, 1970). Blake gt_gl, (1972) observed that progesterone did not alter prolactin secretion in castrate female rats. However, Kalra et al. (1973) reported that 5, lO, and 25 mg of progesterone administered to ovariectomized rats stimulated prolactin release and a lower dose of l.5 mg progesterone had no effect on prolactin. This may account for the discrepancy between the work of Kalra gt_gl, (l973) and Blake §t_gl, (I972). Certainly there is no doubt that prolactin enhances progesterone synthesis in several species as has been reviewed elsewhere in this literature survey (see Section 11.8. Ovaries). 44 Testosterone appears to have no direct action on the pituitary to enhance prolactin secretion (Meites, 1966), whereas jg_yiyg_ administration of testosterone has been observed to increase pro- lactin content slightly in the pituitary and stimulate mammary growth and secretion in rats (Meites, l959). Kalra gt El: (l973) reported that testosterone pr0pionate in doses of 0.5 mg to 2 mg per rat significantly increased serum prolactin levels in castrate male and female rats. Testosterone pr0pionate was more effective in male rats at 0.5 mg and l mg doses. As reviewed (see II. Functions of Prolactin C. Males) in this thesis, prolactin is able to stimu- late testosterone synthesis in male rats and synergize with testos- terone to stimulate growth of male accessory sex organs. B. Thyroid Hormones Early work suggested that thyroidectomy (McQueen-Williams, 1935) or thiouracil feeding (Meites and Turner, 1947) diminished pituitary prolactin content in rats. Administration of thyroid hormones jn_yjyg_on the other hand has been reported to stimulate pituitary prolactin secretion and milk production (Meites, l966). Lu gt_al, (l972) recently confirmed that thyroidectomy decreased serum and pituitary prolactin levels. The in vitro data on thyroid hormones and prolactin release support the jn_vjyg_results. Incor- poration of small amounts of thyroxine and triiodothyronine into a culture system significantly increased prolactin release over that of pituitary tissue not cultured with those hormones (Nicoll and Meites, l963). Chen and Meites (1969) reported that jg_vivo 45 injection of thyroxine did not alter hypothalamic PIF activity in the rat suggesting that thyroxine acts directly on the pituitary to stimulate prolactin release. Subsequent work by Dibbet gt_gl, (1973) showed that pituitaries from male thyroidectomized rats released significantly less prolactin into medium 199 than intact controls, whereas thyroxine treated male rats released slightly more prolactin into the medium than controls. In addition they again showed that triiodothyronine directly stimulates prolactin release when added to incubation medium containing pituitary halves. In addition to influencing prolactin release by thyroid hor- mones, there is evidence to show that prolactin and thyroxine may act synergistically on some organ systems. Bates g__gl, (1962) and Miller and Riddle (1943) demonstrated that prolactin and thyroxine among other hormones are able to act synergistically in the pigeon to stimulate body and visceral growth. In the rat prolactin and thyroxine in combination restored lipogenesis in the liver after hypophysectomy (Nejad gt gl,, 1962). Addition of thyroxine to cul- ture medium of rat mammary glands containing suboptimal amounts of prolactin resulted in improved development of mammary glands (Singh and Bern, 1969). This same work was confirmed jg_y1yg using rabbits by Nilsson gt_gl, (l970). They observed that development of mammary gland was incomplete in thyroidectomized rabbits injected with prolactin. In light of the present work on prolactin-thyroid interaction it was thought of interest to determine the effects of hypo- and hyperthyroidism on prolactin receptor binding activity in female rats. ..I- \ 4"..- «n. luau w: I- . 'I-a: ' II I] n '1'- ‘ i 46 C. Glucocorticoids In the rat, Meites (1966) reported that cortisol administered jg_!j!g_was able to increase prolactin content in the pituitary and stimulate mammary growth and secretion. Morishige and Leathem (1973) observed that in protein deficient pregnant and non-pregnant rats, adrenalectomy decreased serum prolactin. Exogenous corticosterone treatment restored serum prolactin levels to normal. These authors suggested that corticosterone may restore hypophysial prolactin secretion to normal, although Nicoll and Meites (1964) were not able to demonstrate a direct effect of either cortisol or corticosterone on pituitary prolactin release. At parturition, in rats, prolactin and corticoids synergize to initiate lactation and promote milk secretion both i_nylm (Meites, 1966) and jn_yitrg_(Turkington, 1972). In avian species corticosterone and prolactin act synergistically to stimulate gonadal growth (Meier gt 21,, 1971) and fat storage (Meier and Martin, 1971). Evidence will be reported in this thesis to show that adrenal corti- coids influence prolactin receptor binding activity in target tissues. D. Growth Hormone There are no data available as of yet that show that growth hormone can alter the secretion of prolactin. However, there are nuany conditions during which both hormones are released or depressed 0'“ act synergistically to produce specific effects. f“, lm 47 A classical example of synergism between the two hormones is in stimulating development of mammary glands. In ovariectomized- hypophysectomized rats given injections of GH and prolactin, con- siderable lobulo—alveolar growth was observed (Talwalker and Meites, 1961). For a more detailed discussion of this topic the reader is referred to Lyons §__al, (1958) and Meites (1966). Growth hormone and prolactin also synergize with other hormones such as ACTH and thyroxine to stimulate body growth (Bates e__gl,, l964; Milkovic et al., 1964) and lipogenesis from glucose in the liver (Nejad £3 11.. 1962) of rats as well as avian species (Bates et al., l962). There are many similarities between the two hormones. They are both produced by the acidophils of the anterior pituitary, and there are several mammotropic pituitary tumors which secrete large amounts of both prolactin and growth hormone (MacLeod gt_al,, l968; Meites, 1972). A review by McGarry gt_al, (1968) described the similarities in function of the two hormones. Both prolactin and growth hormone are able to stimulate free fatty acid synthesis, in- crease urinary calcium, induce carbohydrate intolerance, increase glucose utilization, cause nitrogen retention, stimulate body growth and were renotropic in rats or humans. Prolactin and growth hormone not only resemble each other in some of their functional aspects, but also respond similarly to certain treatments. In female rats both growth hormone (Dickerman ital” 1972b) and prolactin (Voogt e_t_a_l_., 1970) increase at vaginal opening. During the estrous cycle of rats prolactin peaks late in the afternoon of proestrus (Huttke and Meites, 1970) and growth hormone peaks on the day of estrus (Dickerman e_t 1L, 1972b). I ". V Eda-j“ V. I P’ f" it: ‘4 ‘I 3f?” ., ,u: ‘I' (‘3 ,. a...“ :35. r 1", 48 Surgical procedures such as ovariectomy decrease serum prolactin (Chen and Meites, 1970) and decrease plasma growth hormone (Dicker- man, 1971) in rats, whereas estrogen treatment increases both hormones (Chen and Meites, 1970; Dickerman, l97l). Thyroidectomy in rats also decreases prolactin (Lu _t_gl,, 1972) and growth 1., 1972b). hormone (Dickerman gt The mechanisms underlying the similarities between these two hormones have yet to be unraveled. There is evidence that hormones such as human growth hormone, ovine prolactin, and human chorionic somatomammotropin, which are active as growth promoting and lacto- genic hormones, have similarities in their amino acid sequences (Li, 1972). E. Gonadotropins In many physiological states there seems to be a divergence between prolactin and gonadotrophin secretion by the pituitary, including the puberal state (Kragt and Maskin, 1972; Voogt gt_gl,, 1970), suckling (Amenomori gt_gl,, 1970; Diebel and Bogdanove, 1970), and after castration or estrogen administration (Chen and Meites, 1970; Kalra gt_gl,, 1973). When prolactin secretion is low, gonado- trophin secretion is high, and vice versa. Clemens gt 91, (1969b) reported that implantation of prolactin into the median eminence of postpartum lactating rats permitted cycling to resume in otherwise diestrous rats. Earlier work by the same investigators (Clemens and Meites, 1968) showed that implantation of prolactin but not of cocoa butter in the median eminence of rats in early pregnancy 49 resulted in termination of pregnancy and resumption of estrous cycles. The above results indicate that prolactin may influence secretion of the gonadotropins by the pituitary. Subsequent work by Voogt gt_gl, (1969) demonstrated that prolactin implanted into the median eminence of immature female rats stimulated pituitary FSH release. In pseudopregnant rats a prolactin implant increased serum LH and FSH and caused termination of pseudopregnancy (Voogt and Meites, 1971). This antagonism between prolactin and gonado- trophin secretion was also reported by Ben-David gt_al, (1971a). They showed that when the pituitary is secreting high amounts of gonadotrophin, prolactin secretion is suppressed. V. Protein Hormone Receptors A. ACTH and Angiotensin For a hormone to activate a target tissue, it must first bind to some constituent of the cell. This first step in polypeptide hormone action had been studied indirectly for many years by measur- ing this effect of the hormone. Early work showed reversible bind— ing of tritiated vasopressin to bladder tissue, localization of 13II-insulin in sarcolemma and soluble fractions of muscle cells, and that the first step in TSH action on thyroid and of insulin on rat diaphragm was rapid, firm reversible binding to a superficial site, presumably receptors (Lefkowitz gt 31,, l97l). This conclusion for TSH was based on the demonstration that thyroid slices incubated With TSH at 1°C then washed and incubated at 37°C without hormone, 50 showed persistent effects attributable to TSH stimulation. It was not until 1969 that two groups of researchers using 125I-ACTH and 125I-angiotensin, respectively, that methods applicable for direct study of the interaction of peptide hormones and their specific receptors on target cells were demonstrated. Lefkowitz §t_gl, (1969; l970a)reported specific binding of IZSI-ACTH to adrenal tissue extracts. This binding was proportional to the amount of extract added, was not altered by insulin, and could be correlated to ACTH's ability to stimulate adenyl cyclase activity. Subsequent work by this group (Lefkowitz gt_g1,, l970b)led to the development of a radioreceptor assay for ACTH utilizing the hormone- receptor complex. This assay was reported to have an absolute sensitivity of 1 pg of ACTH. The further characterization of ACTH receptors revealed that the membrane fraction of the adrenal extract displayed the greatest ability to bind 125I-ACTH; only labeled ACTH bound to the fraction, FSH, HPL, and insulin did not; the use of Scatchard analysis showed two sets of receptors for ACTH, high affinity, low capacity and low affinity, high capacity; proteolytic enzymes, phospholipase, and sulfhydryl reagents all decreased ACTH binding whereas RNAase and DNAase were without an effect which suggested these receptors may be partially protein and/or lipid (Lefkowitz gt al., 197l). At about the same time Goodfriend and Lin (1969) demonstrated specific binding of labeled angiotensin to tissue fragments of rat uterus, rabbit aorta, bovine adrenal cortex, and a cell free parti- culate preparation from bovine adrenal cortex. Similar physical- chemical characterization work was done on angiotensin receptors as 51 had been reported for ACTH. Lin and Goodfriend (1970) reported that the binding of angiotensin was not altered by oxytocin, serotonin, acetylcholine, vasopressin or bradykinin; binding of angiotensin to uterus was maximum in 20 minutes at 10°C; binding was temperature and pH dependent; and heating and freeze-thawing decreased the specific binding of angiotensin to uterine tissue, although freeze- thawing had less of an effect on adrenal particles. Further work by Goodfriend and Lin (1970) showed binding for angiotensin II and angiotensin I and this binding did not correlate with ATPase or adenyl cyclase activity unlike the binding of ACTH. These reports on the binding of ACTH and angiotensin were the beginnings of an approach to hormone research that has been ex- tended by dozens of laboratories to many other peptide hormones. The general approach to the study of hormone-receptor interaction has been physical-chemical characterization and somewhat less of an approach has been physiological characterization. B. Growth Hormone and Other Hormones Analogous to the system described by Lefkowitz gt_al, (1970) for a radioreceptor assay for ACTH, Lesinak gt_g1, (1973) used cultured human lymphocytes to establish a radioreceptor assay for human growth hormone (HGH). They demonstrated that ‘25I-HGH binds specifically to cultured human lymphocytes since other species of growth hormone, as well as bovine TSH and pork insulin did not alter binding. The authors proposed this system as a quantitative bio- logical assay. More recently Tsushima gt_gl, (1974) reported use 52 of rabbit liver membrane preparations for a radioreceptor assay to measure GH. The use of this type of assay system has revealed that plasma as well as pituitary immuno-reactive HGH comprises at least two discrete components, "big" HGH and "little" HGH (Gorden gt_gl,, 1973). Apparently big HGH component has much less activity in the radioreceptor assay than in radioimmunoassay, whereas little HGH component has similar activity in both assays. It is possible that this tool can have important clinical applications. Human growth hormone also has been reported to bind specifi- cally to rat and rabbit liver microsomal membrane preparations (Posner gt al., 1974) and to membrane fractions of 7,12-dimethyl- benzanthracene (DMBA) induced rat mammary tumors (Kelly gt $1,, 1974a). The ontogeny of growth hormone receptor binding activity in the liver of female rats revealed that specific binding increased as the animals reached sexual maturation, and during late pregnancy the binding for GH was 222% of control levels (Kelly et_al,, 1974b). Binding and receptor interaction have been reported for several other peptide hormones, such as glucagon binding to rat liver plasma membranes (Robdell gt_gl,, 1971), ADH binding to porcine renal membranes (Campbell gt_gl,, 1972), calcitonin binding to purified renal plasma membranes of rat (Marx gt g1,, 1972), parathyroid hormone interaction with rat renal plasma membranes (Malbon and Zull, 1974), oxytocin receptors in uterus of rat and sow (Soloff and Swartz, 1974), TRH binding to plasma membranes of bovine anterior pituitary (Borden and Labrie, 1973), and binding of somatomedin to skeletal, liver and placental membranes (Hintz gt_g1,, 1974). All hormone- receptor binding reactions were shown to be specific and in the case 53 of TRH, ADH, glucagon and parathyroid hormone binding was corre- lated with stimulation of adenyl cyclase activity. C. Insulin Insulin-receptor interaction has been described for liver cell membranes, fat cell membranes and human lymphocytes. House and Heideman (1970) demonstrated binding of 125I-insulin to membranes from hepatic parenchymal cells. Maximum binding was observed to occur in less than 2.5 minutes at 0°C. Cuatrecasas gt Q1, (1971) further described the binding of insulin to liver cell membranes as a saturable process which is a time dependent reaction following second order kinetics. Radiolabeled insulin was only displaced from the membranes by unlabeled insulin. Hormones such as glucagon, GH, albumin or biologically inactive, reduced or oxidized chains of insulin had no ability to alter the binding of 125I-insulin. A more quantitative study on the interaction of insulin and its receptor in liver plasma membranes revealed two classes of receptors, a high affinity, low capacity site and a low affinity, high capacity site (Kahn gt al., 1974). At 30° the binding of insulin to liver membranes was a rapid reaction and by 15 minutes binding had reached almost 90% of its maximum value. The amount of binding was increased at 4°, although it took longer to reach a steady state. This indi- cates that the binding reaction is temperature dependent. Insulin binding to fat cell membranes and human lymphocytes is very similar to binding in liver cell membranes. There also seems to be two types of receptor sites in fat cells and lymphocytes that 54 is high affinity, low capacity and low affinity, high capacity (Gavin gt gl,, l973; Hammond gt_gl,, 1972). Binding is also tempera- ture dependent and is increased at lower temperatures in fat cells and lymphocytes. Insulin receptors in liver, fat cells and lymphocytes respond similarly to treatment with various enzymes. Trypsin destroys bind- ing of insulin to its receptors in its target tissues, whereas treatment of these receptors with phospholipase A and C enhances insulin binding (Cuatrecasas gt al., 1971; Cuatrecasas, 1971; Krug gt_a1,, 1972). Apparently perturbation of phospholipids of liver and fat cell membranes by digestion with phospholipase C or A re- sults in the appearance of new binding sites for insulin (Krug gt al., 1972). \ There are two other substances, wheat germ agglutinin and con- canavalin A, both plant lecithins, which interact with insulin re- ceptors. The wheat germ agglutinin enhances the specific binding of insulin to fat cells and liver membranes, whereas higher con- centrations inhibit binding of insulin (Cuatrecasas and Tell, 1973). The enhancement is not due to unmasking of receptors and the inhibition occurs because the wheat germ agglutinin in high con- centrations binds to a site on the insulin macromolecule (Cuatrecasas, 1973). Concanavalin A only displaces insulin from its receptor on fat cells and liver membranes. Both plant proteins have insulin- like activity (Cuatrecasas and Tell, 1973). Some physiological characterization of insulin-receptor interaction has been done. Kahn gt_gl, (1973) reported that obese- hyperglycemic mice bind less insulin to liver membranes than their 55 thin litter mates. They showed that this was due to a decrease in the number of receptors and correlated well with the insulin re- sistance these obese mice exhibit. There is no change in insulin binding in the liver during development in the rat but receptor binding does increase significantly during pregnancy (Kelly gt_gl,, 1972). These same workers (Kelly gt_al,, 1974b) also demonstrated insulin binding to kidney membranes as well as DMBA induced mammary tumors (Kelly gt_gl,, 1974a). whether binding in the kidney and these DMBA tumors is related to function has yet to be determined. 0. Gonadotropins, LH and FSH Hormone-receptor interactions have been studied in the testis for human luteinizing hormone (HLH), human chorionic gonadotropin (HCG) and human follicle stimulating hormone (HFSH) and in the ovary for HLH and HCG. The receptor preparations used for the testis vary from crude homogenates to purified plasma membrane prepara- tions (Catt and Dufau, 1973). In these receptor preparations, interstitial cells, intact cells and homogenized cell fractions, HLH and HCG, show the same amount of binding (Cat and Dufau, 1973). Human FSH on the other hand binds more appreciably to the semini- ferous tubule homogenate (Bhalla and Reichert, 1974). Means (1973) reported that HFSH bound membrane fraction of tubular testicular cells, indicating that the tubular binding sites for HFSH are located on the surface of the cell. Autoradiographic studies have shown this to be true for the binding sites of HLH and HCG on the interstitial cells. In the case of all three hormones, HLH, HCG and 56 HFSH, the binding reaction to their respective receptors is tempera- ture dependent with higher initial association at 37°C, the optimum pH is in the range of 7.5, and is not significantly altered by calcium (Catt and Dufau, l973; Means, l973; Bhalla and Reichert, 1974). Altering the structure of HCG by removing the sialic acid or galactase residues and removing sialic acid from FSH does not affect the binding of these hormones to their receptors (Catt and Dufau, l973; Means, 1973). However, Dufau gt_al, (1974) have reported that the disulfide bonds form an important component of the receptor for HCG in the testis and appear to be essential fbr hormone-receptor interaction. Solubilization of the gonadotropin receptor in the testis showed similar binding as the homogenate of receptors but the affinity of the receptors for 125I-HCG was reduced by 50% (Dufau and Catt, 1973). The binding of HCG and HFSH has been correlated with stimulation of adenyl-cyclase system. The synthesis and release of cyclic AMP into incubation medium of rat testis was detectable after addition of HCG, this same response was seen for HCG and testosterone synthesis (Catt and Dufau, 1973). An early response to FSH was observed to be accumulation of cAMP and an increase in protein kinase activity (Means, 1973). The stimulation of adenyl cyclase and protein kinase activity seems to be a common result in the interaction of protein hormones and their receptors. The binding of HLH and HFSH in the testis appears to be age dependent. HFSH binds more to testis homogenates of immature rats than adult rats (Means and Vaitukaitis, 1972) whereas HLH has 57 higher specific binding in testis homogenates of 130 day old rats than either 5 day or 22 day old rats (Sharpe gt_al,, 1973). The binding of HLH correlates well with increase in Leydig cells seen at around 50 days in the rat. The receptors for HLH and HCG in the corpus luteum of the human, bovine and rat respond similarly as the gonadotropin receptors in the testis. The binding reaction for the gonadotropins in the corpus luteum is temperature dependent with equilibrium being reached within 30 minutes at 37°C, the optimum pH is 7.5, and calcium or magnesium don't appreciably alter the binding (Lee and Ryan, 1972; Cole gt 21,, 1973; Haour and Saxena, 1974). In the rat corpus luteum, trypsin,tu(-chymotrypsin, and phospholipase C and D in- hibited binding of HCG and HLH (Lee and Ryan, 1972), whereas in the bovine corpus luteum only pepsin and phospholipase A were able to inhibit the binding of HCG. The use of these enzymes indicates that the receptors in the corpus luteum are lipoproteins and possibly covered by glycoproteins. The binding of radiolabeled HCG and HLH were considered to be specific since both unlabeled HCG and HLH administered 19_gjyg_ and jg_yjtgg_inhibited the binding of HLH and HCG by the corpus luteum (Lee and Ryan, 1972; Braendle gt_al,, 1972). The subunits of LH (ovine and human) and HCG are considerably less potent than the native hormones in inhibiting the binding of 125I—HLH by corpus luteum (Lee and Ryan, 1973). This is also true for gonadotropin (LH, HCG, and FSH) receptors in the testis (Catt and Dufau, l973; Means, 1973). 58 Initial radioautography studies revealed that binding of 125I-HLH and HCG was predominantly localized to the surface corpus luteum (Midgley, 1973). Subsequent work with electron microscopic radioautographs showed that the majority of the silver grains (from 125I-HCG) in luteal and thecal cells was located at the plasma mem- branes of these cells (Han gt_gl,, 1974). These studies were con- firmed with localization of LH (HCG) binding sites by fractionation of subcellular organelles (Rajaniemi gt_al,, 1974a). This analysis demonstrated that specific radioactivity (IZSI-HCG) was predominantly with fractions representing mostly plasma membrane particles. Luteal and testis binding sites have been applied to the devel- opment of radioligand-receptor assays for HCG, LH and FSH (Saxena £3 21,, 1974; Catt _t._l., 1971; Reichert and Bhalla, 1974). These receptor assays are not as sensitive as radioimmunoassays for LH but they provide a powerful tool for measuring biological activity of these hormones (Catt and Dufau, 1973). Reichert and Bhalla (1974) used a rat testis tubule receptor assay to compare the properties of FSH from several species. A more practical applica- tion of this type of receptor assay used plasma membranes of bovine corpora lutea of early pregnancy to measure HCG and in this way, detect pregnancy within 6 to 8 days after conception (Saxena gt 91,, 1974). E. Prolactin Radioautographic studies have shown that an intravenous in- jection of 1251- or I311-prolactin becomes distributed throughout the body (Birkinshaw and Falconer, 1972; Rajaniemi gt_gl,, 1974b). In 59 both males and females uptake was shown by liver and kidney. Female rats and mice showed weak labeling of prolactin in the ovaries and mammary tissue and male rats displayed some uptake by the testis, prostate and seminal vesicles (Rajaniemi gt 21,, 1974b). Midgley (1973) using autoradiographic analysis demonstrated specific binding of 125I-ovine prolactin to the corpora lutea of rats. He also re- ported that the functional state of the corpora affected the bind- ing of prolactin, that is, new corpora bound more prolactin than old corpora. These radioautographic studies demonstrated that prolactin bound to the surface of the cell in the tissues, and indi- cated that the receptors for prolactin on its target tissues are located on the membrane. With these radioautographic reports providing a basis, the direct studyof prolactin and its receptor interaction was started. Turkington and Frantz (1972) using tissue homogenates reported specific binding for prolactin in mammary glands, liver, kidney, and seminal vesicles. The 125I-ovine prolactin binding by these tissues was not altered by either LH or TSH. Subsequent work by Turkington t al. (1973) and Friesen gt a1, (1973) demonstrated the specific binding of radiolabeled prolactin to purified plasma mem- branes of adrenals and ovaries in addition to liver, kidney and lactating mammary glands. The pigeon crop sac also binds prolactin and this binding induces proliferation (Mishkinsky §t_gl,, 1972). The binding of 125I-labeled prolactin to mammary gland particles was complete by 20 minutes at either 4°C or 37°C, although at 37°C more prolactin was bound (Frantz _t_gl,, 1974). Scatchard analysis of prolactin binding to mammary gland particles revealed a Kd of 60 9.1x10'9 mol/l and the number of binding sites were estimated to be 15.5xio-‘3 mol/mg protein (Frantz _t__i_., 1974). These same workers reported that treatment of mammary gland particles with trypsin decreased the specific binding of prolactin whereas neuro- aminidase, RNAase and DNAase had no effect. This suggests that pep- tide bonds are required for prolactin-binding activity. Heating of the binding particles at 70°C for 10 minutes completely removed hormone-displaceable binding activity further suggesting its macromolecular nature (Frantz gt__l,, 1974). Solubilization of prolactin receptors from mammary gland increased the affinity of the receptor five-fold over the particulate receptor (Shiu gt 91,, 1974). An application of prolactin binding sites in mammary gland has been the development of a radioreceptor assay for prolactin (Shiu st 21:: 1973). The assay is able to measure prolactin in several species as well as distinguish between prolactin preparations of varying potencies. Probably the most important clinical application of this system is the determination of prolactin receptor binding activity in mammary carcinomas. Kelly et_al, (1974a)demonstrated that DMBA induced rat mammary tumors specifically bound 125I-ovine prolactin and this binding was correlated to the growth response of these tumors to prolactin, that is, prolactin dependent tumors displayed the greatest amount of binding. Turkington (1974), using three different types of experimental mammary carcinomas, showed the same phenomenon. The DMBA-induced carcinomas which are pro- lactin dependent displayed greatest binding; the R3230AC carcinoma, which is responsive to prolactin for milk protein synthesis but not growth, showed some binding; and the C3HBA carcinoma, a relatively 61 autonomous tumor, had no detectable binding. Prolactin reisptors have also been demonstrated in an estrogen-receptor deficient rat mammary carcinoma and these receptors are similar to those found in lactating rat mammary tissue (Costlow gt_gl,, 1974). These results provide evidence for prolactin receptors in mammary carcinomas and suggest that the degree of prolactin dependence of such carcinomas may be characterized by the relative number of prolactin receptors present. This is a powerful tool for the clinician which may provide him with an easy test of responsiveness of human breast cancers to hormones and evaluation of specific treatments. The topic of this thesis is the study of prolactin receptors in mammary glands, ovaries and liver tissue with the major emphasis on physiological characterization. Recently one such report has~ appeared on prolactin receptors in the liver (Kelly gt_gl,, 1974b). They found that receptors increase with age, during pregnancy and lactation and females have more receptors for prolactin than males. There are certain properties that all protein hormone receptors have in common, as the foregoing discussion has revealed. These properties are: 1) high affinity for binding a specific hormone; 2) a requirement for a high degree of structural specificity in the hormone which is bound; 3) the hormone binds rapidly and the popula- tion of receptors is easily saturable with hormone, but binding is reversible; 4) a restricted distribution among various cell types of the animal, being present in highest concentration in the hormonal target organ; 5) the receptor is macromolecular in nature, with protein and lipid moieties; and 6) formation of the hormone-receptor complex serves to activate hormone-dependent processes in the target 62 cell (Turkington gt al., 1973). Hormone-receptor interaction can be summarized by the following schematic representation using pro- lactin binding to mammary alveolar cells as a model: A23 31% ml 5356755 :oEuEBE .8333. .. 5328:. ._ 925.; 3.33.... 5.: 3205.: 3:88;. .afluduudnuddnu. w24808w! (Im(..a mauauaz A]. 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Amy H_ metamoeo ram.o.u o.op Adv H mseomo_o m.o.H O.“ Amy maepmm sm.o.u m.m +Aev metamooea dmao-H do me_eeem oemmwoem N e_oau eo sea pom mgu cw «Foam maocumu mg» mcweso mmcoLnEmz cowoaso ea xoeseoo< meeoeem eeooeeoea .HHH e_eae 91 III. Prolactin Binding Activity in Ovaries and Mammary Tissue During Pregnancy and Lactation in the Rat A. Objectives Prolactin is an essential hormone during pregnancy (Neill and Smith, 1974) and lactation (Meites, 1966). It is necessary for maintenance of the corpora lutea during the first six days of ges- tation (Clemens gt_al,, 1969a), and together with the adrenal gluco- corticoids initiates and maintains lactation (Meites, 1966). Since prolactin is important for both the ovaries and mammary tissue, it was of interest to measure prolactin binding activity in these tissues during pregnancy and lactation. 8. Procedures 1. Animals Sprague-Dawley female rats weighing 225-250 grams were housed 4 to a cage together with 1 male rat. The day sperm were found in the vaginal lavage was considered day 1 of pregnancy. Control females were followed for 2 consecutive estrous cycles. Rats were killed on the day of estrus and on 1, 3, 6, 12, 16 and 20 days of pregnancy. Two additional groups of pregnant rats were maintained for the full gestation period, and their litter size was adjusted to six pups on day l of parturition and suckled for either 4 or 10 92 days. At the end of the experiment both the ovaries and mammary glands were removed and stored frozen until assayed. 2. Tissue preparation and assay procedures Ovaries from 4 animals were pooled and homogenized in 0.2 M sucrose solution and microsomal membranes were prepared. The mammary glands from individual rats also were homogenized and micro- somal membranes were collected. Both ovaries and mammary tissue were diluted to 200 )Jg/lOO m and specific binding of 1251-radio- labelled prolactin was measured. 3. Statistical Analysis An analysis of variance was followed by Duncan's Multiple Range Test for a comparison among the means. C. Results From day l of pregnancy (Table IV, group 2) there was an in- crease in the binding of prolactin to the ovaries, reaching a peak on days 3 and 6 of pregnancy (Table IV, groups 3 and 4). By day 12 of pregnancy (Table IV, group 5) the prolactin binding in the ovaries has significantly decreased reaching its lowest level on day 20 of pregnancy (Table IV, group 7). As the animals suckled, the binding of prolactin again significantly increased (Table IV, groups 8 93 mzeumm .m> P as $23 .2, 8:38.. a as see 8.3%.. mzeumm .m> coppopuog a. man mom use mp .~_ >.8 .m> m use m 93: $2.3m .m> m 25 m 93.5 tom 86%“. a come some toe mcowgocmEsmumu mo .oz + Keane 3V op a tioufie A3 a .m momma 3v ON g edmmo 3v 8— 8 momma :V N. 8 Lemma :V e a 15H_d :V m a esN.o.H m.e Aav P .N e.o.H o.e +Amv aseomm .F mcwwmwmiwmmwuwmm a xummwwwwuowoummma pom on» cw cowyopuob new zucmcomea mcweao mmcmensmz eaeeaso ed zoeseoo< meeoeem eeooa_oee .>H e_oae 94 and 9) although the values did not reach the levels of binding seen in the first half of gestation. The prolactin binding activity in the mammary tissue (Table V) during pregnancy was relatively low and showed little change during the entire period. On days 4 and 10 of lactation (Table V, groups 8 and 9) prolactin binding activity increased by about 100%. 0. Conclusions These results show that prolactin binding activity in the ovaries is the highest during the first half of gestation in the rat, and that prolactin binding is significantly increased in the mam- mary tissue during lactation. Pituitary prolactin has been demonstrated to be essential for maintaining luteal function in the rat during the first 6 days of gestation (Clemens gt_al,, 1969a; Morishige and Rothchild, 1974) and also during lactation (Clemens gt_al,, 1969b). The binding of prolactin in the ovaries follows this same pattern, i.e., the binding is highest during the first half of gestation and lactation. Shiu gt_al, (1973) reported that rat placental lactogen increases during the second half of gestation and Neill and Smith (1974) proposed that placental lactogen supports progesterone secre- tion during the second half of gestation. It is possible that placental lactogen binds to the ovaries during the second half of gestation and therefore pituitary prolactin binding is decreased. 95 3:03 .358 :o 3 35358 no Eda? amdmmm op a emo.o.u e.~ a .m mo.o.H o.” om .N ToH o; 2 .o P.o.H m.o Np .m _eumd e 3 Fame; m a _dmo; _ & _.o.u o._ ascend ._ ammo-Hm~— mo Ansoem\muos we .ocv mcwucwm uwmpumam n cowuouuog use aucocmmsa mo mama you we» cw copumuuog new xucocmmgm mcwsso mmcogasmz eea.¢ xeaEEaz e? »o_>_eo< me_oeem e_ooepoea .> apnea 96 The manmary tissue, unlike the ovaries, shows very little binding of prolactin during pregnancy but the binding signifi- cantly increased at lactation. Placental lactogen is similar to pituitary prolactin in that it maintains corpora lutea and stimu- lates growth and milk secretion by rat mammary gland (Neill and Smith, 1974). Shiu _e_t _l. (1973) demonstrated that human placental So the lactogen also binds to lactating rabbit mammary glands. low pituitary prolactin binding in the mammary gland during the later half of pregnancy when the glands are very well developed could be due to the saturation of these sites by placental lactogen. The binding for prolactin in the lactating manlnary glands was low even though significantly increased over the levels observed during pregnancy. One explanation for this is that as the rats suckle, the levels of endogenous prolactin are highly elevated and the bi nding sites for prolactin become partially saturated, result- ing in low binding activity. The effects of high endogenous levels 0" Prolactin on binding in different target organs has yet to be deteI"Inined. 97 IV. Normal Deve10pment and Effects of Estrogen on Prolactin Binding Activity in Liver, Adrenals and Kidneys of Inmature Female Rats A. Normal Development and Effects of Estrogen on Prolactin Binding Activity in Liver of Immature Female Rats 1. Objectives Prolactin binding activity in the liver had been reported to increase with age and reach a peak shortly after puberty in the female rat (Kelly 32191., 1974b). Our laboratory had preliminary indications that estrogen could stimulate prolactin binding acti- vi ty in the liver. Since binding was low in the inmature animal, it was thought of interest to determine whether inmature rats were I"esvonsive to estrogen stimulation in terms of changes in prolactin binding activity in the liver. 2 - Procedures a - Animals Immature female Sprague-Dawley rats 10, 18, 23, 28, 33, 38 and 70 days of age were treated with either 1 ug estradiol benzoate :ln 0‘ 1 cc corn oil or with the vehicle alone. The rats were in- Jected for 5 days at the end of which time they were killed and the ‘ 1 r. . '1 ivers were removed and stored frozen. 98» b. Tissue preparation and assay procedure Liver microsomal membrane preparations were made and diluted to 300 pg protein per 100 pl diluent, as described previously. Specific binding of 125I-radiolabeled prolactin was determined for each sample. c. Statistical Analysis The data were analyzed using an analysis of variance for unequal sample size, followed by Duncan's Multiple Range test for comparison among means. The data for the controls were also subjected to an unweighted least squares fit for an exponential function, whereas the data for the treated animals were analyzed by an unweighted linear regression (Sokal and Rohlf, 1969). 3 . Results These data demonstrate that prolactin binding activity in- creases with age (Figure 6) and this developmental pattern follows an exponential growth curve (correlation coefficient 0.98). The adult level emerges about the time of sexual maturation, since at the time of vaginal opening (38-40 days, Figure 6) the sharpest increase was seen and thereafter the curve reached a plateau. The estrogen treated rats (Figure 6) showed a marked increase 1" binding as compared to the control rats of corresponding age 99 (Table VI). However, those rats treated at 10 days of age and killed at 15 days of age did not respond to estrogen treatment, as can be seen in Table VI. Their binding was at approximately the same level as the untreated controls. It is interesting to note that the prolactin binding activity of the estrogen treated rats displayed a linear pattern (correlation coefficient of 0.97) of development, indicating that estrogen modified the pattern observed iri the untreated rats. [3. Ontogeny of Prolactin Binding Activity in the Adrenal Glands and Kidneys 1. Objectives He had observed that the adult pattern of prolactin binding activity in the liver emerged at the time of puberty in the female rat- Since both the adrenal glands and the kidneys had been shown to specifically bind 125I-radiolabeled prolactin, the question arose as to whether their development of prolactin binding activity would fol low the same pattern as in the liver. The present study was undertaken to answer this question. 100 2. Procedures a. Animals The adrenal glands and kidneys were removed from the 23, 28, 33, 38, 43 and 75 day old control rats of the previous study (see A. 2. Procedures a. Animals). b. Tissue preparation and assay procedure Adrenal glands from 4 animals were pooled and a membrane frac- tiori was prepared. A total of 4 kidneys were pooled for each mem- brarue preparation. This was done in order to have enough protein to assay. The adrenal membranes were diluted to 100 pg protein/ 100 .ul and the kidney membranes to 1000 pg protein/100 pl. Specific binciing of 125I-prolactin was measured and each sample was assayed in (quadruplicate (Materials and Methods, section IV). c. Statistical analysis The data were analyzed by using an unweighted least squares fit for an exponential function (Sokal and Rohlf, 1969), and the dl'fference between means was tested by analysis of variance followed by Duncan's Multiple Range test. 101 N;Hogp mauog_ AKVK mamedm eomfim are? cauea_ eouam Afiva Néumdp eouee Afivz 9_H92 Ndumd AEVE o;Hmd_ momfim sza fiomtm Fammm Ame: umpomepimm mpoeucoo Aaaocm\mpos mo .ocv sxee-ammp to aeeee_m e_t_ooam N axon e_ om< muse «Pusan emanate mpoonzmm Fowcosumm ecu emanates: to monogamoeo: cm>w4 cw xuw>Puo< mcmucpm cwuompoga .H> m—aoh 102 .coms sea gouge unaccoum acacvssmumu so; new: mmPanm mo cmaszzr Aepdmnd Acmdum; 3 “Seamus Acmdnpa 2 Atudmmg A3 Fame 3 Asmdums Azmameg 8 Aemduea A255“ __ 8 ismduaa LS ~H ~_ 8 mamcvwx mpocmsu< Sade-um- eo meeeeem oee_ooem a axon ea am< mama uposmm mcwzoge mo mwuocmmoso: accepx new paeoeea e. su_>eoo< me_oe_m eeooa_oea .~H> opnmh 103 ED CONTROL uslo-PRL PERCENT SPECIFIC BINDING O 1523333384350 15 DAYS OF AG E Figure 6. Normal development and effects of estrogen on prolactin binding activity in liver tissue of immature female rats. 104 3. Results Prolactin binding activity (Figure 7) in the adrenal glands and kidneys decreased exponentially (correlation coefficients - 0.98 and -0.97, respectively) with age. This pattern was the reverse of what was seen in the liver (Figure 6). The decline continued until puberty and then prolactin plateaued to adult levels. The binding in the adult rat adrenal gland was approximately 1/6 the binding seen in the immature animal at 23 days of age (Table VI), and this binding at 23 days was significantly different (P 0.01) from the binding observed at either 43 or 75 days of age. The kidneys of 28 day old rats (Table VII) displayed binding of radiolabeled prolactin about 7-fold greater than in the adult female rat 75 days old (P 0.01 for 28 days vs. 75 days). The binding of prolactin in the adrenal glands of immature rats was much greater than observed in either the liver (Table VI) or kidneys (Table VII) of immature female rats. It should be pointed out that adrenal binding is expressed as per 100.u9 Protein whereas liver binding is expressed as per 300.u9 Protein and kidney binding as per 1000.09 protein. Thus the adrenal gland binds by far more radiolabeled prolactin per ug of protein than either liver or kidney tissue. 105 C. Conclusions These data show that prolactin binding activity in the liver increases with age and reaches a plateau at puberty, whereas the binding in the adrenal glands and kidneys decreases with age and also plateaus at puberty. The binding activity in the liver correlates well with the pattern of prolactin secretion in immature rats since the highest levels of serum prolactin were seen at puberty and thereafter (Voogt et_al,, 1970). It is difficult to interpret the high binding in the immature adrenals and kidneys as compared to the low binding in the adult. One can speculate that the function of prolactin may be more important in these tissues during prepuberal development than in the adult. In the liver, estrogen was able to increase prolactin binding activity in the immature animal, although the animals treated at 10 days of age did not respond to the estrogen treatment. Ten days of age could possibly be a period when the mechanism(s) involved in prolactin binding activity in the liver may be refractory to the effects of estrogen. The mechanism(s) of estrogen stimulation are not yet known. Estrogen increases prolactin levels in 21 day old female rats (Voogt gt 11., 1970) as well as in adult female rats (Chen gt 21,, 1970), and it also influences liver size (Leathem, 1961). It is possible that at puberty estrogen stimulates not only prolactin secretion but also prolactin receptor binding activity in the liver. 106 20 22 I. 14 A0516 NAL IO 2 KIDNEY /\ : ”510901. PERCENT SPECIFIC BINDING 0 IS 2323331050 15 DAYS Of AGE Figure 7. Ontogeny of prolactin binding activity in kidney and adrenal tissues of immature female rats. 107 V- Effects of the Thyroid and Ovaries on Prolactin Binding Activity i n Rat Liver A - Objectives Recent reports by several laboratories (Turkington and Frantz, 1972 ; Friesen gt _a_l_., 1973) and our own laboratory have demonstrated that prolactin binds specifically to liver tissue, indicating the presence of prolactin receptors in this tissue. The nature and functions of prolactin receptors in the liver, as well as the mechan- isms regulating their induction and maintenance have not yet been determined. Among the endocrine glands that may influence liver functions are the thyroid and ovaries. Thyroid hormones are known to be Protein anabolic (Friesen and Lipner, 1971), and can stimulate GH SQCPetion (Dickerman egg” 1972b). Liver size and protein content are reduced after thyroidectomy (Turner and Bagnara, l97l). Estrogen also has been reported to influence liver size and function (Leathem, 196‘ ) 9 and can increase prolactin (Meites £311., 1972) and GH secretion (Dickerman 3311,, 1972b). It was of interest therefore, to determine the effects of thyroidectomy, ovariectomy, thyroxine and etitrogen on prolactin receptor binding activity in the liver of female rats. 108 B . Procedures 1 . Animals Mature, virgin female Sprague-Dawley rats weighing 200-225 grams each were used. Three separate experiments were performed. In experiment 1, intact controls and thyroidectomized rats were injected sc daily wi th 0.2 ml of 0.85% NaCl for 30 days. Two other groups of thyroid- ectomized rats were injected sc with 2.5 or 10 pg L-thyroxine (T4) per 1 00 911 body weight for 30 days. In the second experiment, ovariectomized and ovariectomized-thyroidectomized rats were in- jeCted sc with 2.5 or 10 pg T4 per 100 gm body weight. Treatment was begun 31 days after surgery and continued for 15 days. In the thi rd experiment, the animals were injected sc daily as follows: (1 ) intact female rats, 0.85% NaCl; (2) ovariectomized-thyroidecto- "fi zed rats, 0.85% NaCl; (3) ovariectomized-thyroidectomized rats, 2‘ 5 ”9 T4 per 100 gm body weight (4) ovariectomized-thyroidectomized rats . 2 pg estradiol benzoate (E8) in corn oil and (5) ovariectomized- thyroidectomized rats, T4 and E8. 2 - Tissue preparation and assay procedure for prolactin binding activity. The liver tissue from each rat was homogenized in 0.3 M s "cmse and microsomal membranes were collected according to the methods previously described (see Materials and Methods. IV- 109 Radioreceptor Assay). Each liver membrane sample used for the assay contained 300 pg of protein as determined by Lowry protein method (1955). The specific binding of 125I-labeled ovine prolactin to these membranes was determined by the methods outlined in section IV, Radi oreceptor Assay, Materials and Methods. Each sample was assayed in quadruplicate. 3- Statistical analysis of data The data were first analyzed by an analysis of variance for unequal sample size (Sokal and Rohlf, 1969), followed by Duncan's Mu‘l ti ple range test for comparison of means among all groups (Duncan, 1955). 4- Scatchard Analysis This analysis was carried out according to the procedures out] Tried in section V. Scatchard Analysis (Materials and Methods). C ~ Results In experiment 1 (Table VIII), thyroidectomy (group 2) reduce“ pro] actin binding activity to less than a third of that present in intact contra], (group 1). Doses of 2.5 and 10 pg T4/100 on body Weight (groups 3 and 4) restored prolactin binding activity to the 110 intact control level. The effects of the two doses of 14 on specific pro] actin binding activity in the liver were not statistically different from each other. Rats both ovariectomized and thyroidectomized (group 2, Tab‘l e IX) showed significantly less prolactin binding activity than rats only ovariectomized (group 1). Treatment with either dose of T4 (groups 3 and 4) restored liver prolactin binding activity to level 5 present in the ovariectomized rats (group 1). In the third experiment (Table X) thyroidectomy and ovariectomy (group 2) again significantly decreased prolactin binding activity in the liver as compared to intact controls (group 1). Replacement therapy with either 2.5 pg T4/lOO gm body weight (group 3) or 2 pg EB (group 4) partially restored prolactin binding activity. However, both the T4 (group 3) and the EB (group 4) treated rats had signi- 1:icahtly less binding than the intact control rats (group 1). When ovar-i ectomized-thyroidectomized rats were treated with both T4 and EB (group 5), prolactin binding activity was increased above that of the intact controls. The prolactin binding activity in intact contJ‘Ols in this experiment was about twice that seen in intact controls in experiment 1 (Table VIII). The iodinated preparations Of o‘I‘ine prolactin used for the assays in experiments 1, 2, and 3 Were not the same, and as mentioned previously (see Materials and Methods, section IV. Radioreceptor Assay), the iodinated ovine pm] a(Itin used in experiment 3 was repurified. This latter is be- 1 . 1e\Ied to be mainly responsible for the higher specific binding 3 . ee" 1n the third experiment. 111 Data were obtained for the competitive inhibition of labeled hormone by different amounts of unlabeled prolactin with receptor preparations from intact control and experimental animals (Fig. 8). A Scatchard analysis is shown in Figure 9 on the data from intact control animals and reveals the presence of a low concentration (2- ‘l 'I 3x10'14M/3OO pg protein) of binding sites for prolactin of very high affinity (Ka = 5.03x1012M'1). The combination of ovariectomy and thyroidectomy resulted in approximately a 9-fold reduction in the number of these binding sites. Replacement with T4 and E8 in the ovariectomized-thyroidectomized animals restored the number of binding sites to levels slightly higher than found in controls (3- 65x10‘14M/3oo pg protein). The affinity constants of the sites in the control and experimental groups were not substantially di fferent. These data from Scatchard analysis are sunmarized in TabIe XI. D - Conclusions These results indicate that either thyroidectomy or ovariectomy deer‘ease prolactin binding activity in the liver. Thyroidectomy Was "lore effective in this respect than ovariectomy. Injections of T4 r‘eturned prolactin binding activity in the thyroidectomized rats to the level of the intact controls, and in the ovariectomized- thyro‘idectomized rats to the level of ovariectomized rats. Estrogen re ' plaCement together with T increased prolactin binding activ1ty 4 ab 0V9 that of intact control levels. Scatchard analysis revealed 3:95 .550 :o 3 “.2358 me 86 Va... 112 a.o.fl_m.m “so 3m 5m oop\ae aa.op + xe .a as H 3 E g .5 2:3 as. 3 + a; .m em.o.H 8., Amy xe .N «to H ea 5 oozed“. 335 .P page- H to qsogm\mumg to .o: acaccwm umeumam a use acmEuomsP mama dynamo caboose Ache mcwxocxgh can Axpv coNPEouumewogxgp to mooaeomoeoz eo>ed es seeseoo< meeoeem Ame .~H~> opoae 113 8:96 smfio So on 389:8 mo 86 .2 a N5 H as 2: 3m 8 8:: 2:: + on + 55 . a to H 3 a: 3m 5m 8:: a: 3 + on + as .m to“: E x7125 .N N.O.H m.m Ame weapon + x>o ._ 4mg mcm>oimmpm mo asosm\muoc mo .0: meeeeem oeeeooam a + oeoeoaoee a. Achy m:_xosxgh cm>pw aoz so cm>wu mama Axhv chPanumuwossghiAx>ov vm~Psouumwgo>o mo mouocmmoso: Lo>m4 cw xu_>_pu< acwucpm 4mm .xH e_aoh 114 328.3 335 S cocoa—=8 mo 85:? a: H N8 2: 8 3N 8a S 33 + on + 8 .m .8; H 8.8 a: 8:8 8: N + 5 + .25 .e «no H 98 a: o: 8 8 8:3 8:3. + on + 55 .m and H 3 A8 on + 55 .N N; H mi 2: 8:58 835 ._ omooimm H to aaosm\muos to .o: mcwucwm ow» owqm a use acosuomsh Ammo mueo~=mm _owuocumu so\ucm Ache mcwxos85p cm>mo mama Axhv vm~wsouumupogxghifix>ov vowesouomwgo>o mo mmumcmmosoz sm>wb cw anp>wpu< mc—ucpm cpuuoposa .x epoch 115 Neo_xap.m aF-OPXmme.m _em.o mm .ee Npo_x_c.m mp-o_xoce.N Feo.o xso .xe N_opxmo.m ep-o_xm__.N eme.o float e_ocoeoo eoaoeu Ammfioev Amcv :wuuoPoea :wpuoposa n_-mo_ozv opoaee_m e_aeee_m agate ox Ezswxmz Esewxoz acmEpmmep memNPae< esaeooaom .Hx e_eae Figure 8. 116 need-n "i 0 man.- M O O O c . Binding of 125I-ovine prolactin to liver membranes of ovariectomized- thyroidectomized, intact controls and ovariectomized-thyriodectomized rats injected with T4 and EB as a function of unlabelled o-prolactin (noted on abscissa). The Sgdinate represents the binding of 1 I-o-Erolactin as a percent of the total 1 5I-o-prolactin in the incubation. Each tube contained 300 ug protein and 70,000 cpm I25l- o-prolactin. Non-specific binding was 2% of the total radioactivity added. Figure 9. 117 M/m- Scatchard plot of data for competitive displacement of 251-ovine prolactin with unlabelled o prolactin from liver membranes of ovariectomized-thyroidec- tomized, intact controls, and ovariec- tomized-thyroidectomized rats injected with T4 and E8. The ordinate represents ratio of nanograms bound/free hormone, and abscissa the nanograms of o-prolactin bound to liver membranes. The affinity constant (Ka) is the negative slope of the line. The intercept on the abscissa is the total amount of o-prolactin bound to the receptors. 118 that the differences in prolactin binding activity were due to an alteration in the number of "receptor" binding sites in the liver. Since thyroidectomy can decrease liver proteins generally (Turner and Bagnara, 1971), this decrease also may have included receptor proteins for prolactin. The mechanism(s) by which the ovaries influence prolactin receptors in the liver is unknown, although estrogens can increase pituitary prolactin release (Meites gt al., 1972) and can alter liver function (Leathem, 1961). VI. Effects of Ovarian Hormones on Prolactin Binding Activity in Liver and Mammary Tissues A. Effects of Estrogen or Estrogen and Progesterone on Prolactin Binding Activity in Liver 1. Objectives Estrogen and progesterone have been reported to stimulate prolactin secretion (Chen and Meites, 1970; Kalra §t__1,, 1973). Since estrogen greatly increases prolactin secretion, it was thought of interest to determine if estrogen and progesterone could also stimulate prolactin binding activity in the liver. 119 2. Procedures a. Animals Approximately 45 Sprague-Dawley female rats weighing 200-225 grams were used in these experiments. The rats were ovariectomized and treatment was begun one week after surgery. The rats were assigned to the following groups: (1) controls, corn oil, 0.2 ml, (2) estradiol benzoate (EB), 5,pg/0.2 ml, (3) EB, 20.pg/0.2 m1, (4) EB, 5.u9/O.l m1 and progesterone, 4 mg/0.l ml. The treatments were given sc daily for 10 days. At the end of the treatment period, half of the rats were killed 24 hours after the last injection and the other half were killed 7 days after the last injection. b. Tissue preparation and assay procedure Liver microsomal membrane preparations were made and all tissue was aliquoted at 300 pg protein/100 pl. The specific binding of 125I-radiolabeled prolactin was measured for each rat. The samples were assayed in triplicate. c. Statistical analysis The data were analyzed by analysis of variance followed by Duncan's Multiple Range Test for comparisons among the means. 120 3. Results Table XII shows that estrogen in both dose levels (groups 2 and 3) as well as the combination of EB and Progesterone (group 4) significantly increased prolactin binding activity as compared to the ovariectomized controls (group 1). The effects of the two doses of EB and the combination of EB and Progesterone on the specific prolactin binding activity in the liver were not statistically different from one another. In the second half of this experiment (Table XIII), in which the rats were killed 7 days after the last treatment, the specific prolactin binding in the liver of ovariectomized rats treated with E8 (groups 2 and 3) or E8 + prog (group 4) was still significantly higher than the controls. Although the binding was slightly less than that seen 24 hrs after the last injection (Table XII), the stimulatory effects of estrogen were still present. 8. Effects of Estrogen or Estrogen and Progesterone on Prolactin Binding Activity in Mammary Tissue 1. Objectives Estrogen and progesterone stimulate the development of mammary glands (Meites, 1966) as well as influence prolactin secretion (Chen gt al., 1970; Kalra et al., 1973) in the rat. It was therefore 121 :o 5.8 + x>c 3 882.8 mo 86 Van « ~.H mm mmv as e .moga + .3 .mm + 56 .e a N H 8 E a: 8 .8 + 56 .m ae._.H pm Ame 8:.m .Nu + x>o .N m.o.H m.~ Ame _wo :eou + x>o .F emaciam to Agnosm\mbos to .ocv meeeeem oeemwoam N ‘ seasooooe eeeeoonem one; eooe< ”Lao; aN eo__e¥ eea .Amoeav ecosmummaosa use camospmm so Ammo cmmocumm new: educate mama Ax>ov umNPEouuuaeo>o to mmuocmmoeox cose; e, Hoeseoo< meeoeem e_ooapoca .HHx epoch 122 78 58 + 56 3 889.8 mm 86 V9. a NHmN +l «.N am +1 N m.p so 8 a 8.: + min .3 + x>o E 9;. .8 + 86 E 2.8 .3 + 56 “me —_c coco + x>o omaoiumNP to actuate e_eeooam N Aazosm\mpoe to .ocv pcmsuomeh cowpumnca ammo sebum mama A um—pwx new “mosey .mcosoummmoea use cmmogumm so Ammo :maosumm gar: taboos» mama Ax>ov vaNEouompsm>o we mmumcmmoeo: posed e. seeseoo< meeeeem e_ooa_oea .HHHx e_ae» 123 of interest to observe the effects of estrogen and progesterone on prolactin binding activity in the mammary tissue. 2. Procedures a. Animals The mammary tissue was removed from the same ovariectomized rats as in the previous study (VI. A. 1. Procedures a. Animals) treated with varying doses of estrogen or the combination of estrogen and progesterone. b. Tissue preparation and assay procedure Microsomal membrane preparations were made from mammary tissue and diluted to 200 pg protein/100 p1. Specific binding of 1251- radiolabeled prolactin was measured and each sample was assayed in quadruplicate. c. Statistical analysis The data were analyzed using analysis of variance followed by Duncan's Multiple Range Test for comparisons among the means. 124 3. Results Treatment of ovariectomized rats with either 5 or 20 pg EB (Table XIV, groups 2 and 3) or a combination of E8 + Prog (group 4) significantly reduced prolactin binding activity by approximately 1/2 in mammary tissue. The effects of the two doses of EB and the combination of E8 and Prog on specific prolactin binding activity in the mammary tissue were not statistically different from one another. Table XV shows that when the other half of the rats were killed one week after the last injection, EB (groups 2 and 3) and the come bination of EB and Prog (group 4) also significantly reduced pro- lactin binding activity in the manmary tissue. However, prolactin binding in the controls (group 1, Table XV) was higher than in the controls (group 1) in Table XIV, when killed 24 hours after the last injection. It is possible that the longer period of ovariectomy (Table XII) increased the prolactin binding activity in this mammary tissue. C. Conclusions These data demonstrate that estrogen (5 or 20 MO) or a combination of estrogen and progesterone are able to stimulate prolactin binding activity in the liver and decrease binding of prolactin in the mammary tissue. As mentioned before, the mechanism(s) 125 2o 58 + 56 on 888.8 3 8.9:... a8éHma fiv84.8& + :.m .8” + xso .e .88 H 3 E a: 8 .8 + 55 .m ap.o.H.N.o Ame ma.m .mu + xso .N eoH€_ svzofi8+x3 ; umaoium to quosm\muog mo .ocv aeeeeem oeemwoam N ceaseaoeh =o_ooone~ one; sooc< mesa: aN oo...g ego .Amosav ocogmummmosa can comosumm so Ammo cmaocumu gap: embeds» mung Ax>cv coupeouuuwso>o so monogamoso: damask NuaEEe: es No.>_eo< ae.oe.m e.eoa_oca .>_x epoch 126 pro :gou + x>o op vmsaasoo ma Fo.odwaa 1.8 H as 3 a... e .88 + am .mm+x>o .e a~.o.H_N.o Ame 8: ON .mm + xso .m aNo.o.H_N.o Ame 8:.m .mm + xso .N m.o.H_N.m Ame pee ecoo + xse ._ uzaoim to Aaaosm\muog to .aev aeeeeem e_mmwoam N peasoaoce cowpumncm ammo soum< man: A voP_px new .Amosav mcogmummmosa can :mmosumu so Ammo cmmogumu gov: educate mama Ax>ov uoNFEouumpgo>o to mmuocmmoso: 88: raga: 5 3:58 8:8; £822.. .: ozfi 127 involved in the effect of estrogen on prolactin binding activity remains to be defined. Evidence from our previous work indicates that estrogen increases the number of binding sites in the liver (see Experimental section V. 3. Results). Estrogen has also been shown to alter liver size and function (Leathem, 1961), as well as to stimulate prolactin release (Chen and Meites, 1970). The effects of estrogen and estrogen and progesterone on mammary tissue seem to fall in line with early work reported by Meites and Sgouris (1953). They suggested that although large doses of estrogen increase pituitary prolactin secretion, they render the mammary glands less sensitive to the lactational action of prolactin. Large doses of estrogen, or combinations of estrogen and progesterone, stimulated mammary growth but prevented prolactin from initiating lactation in castrated rabbits. Hhen estrogen administration was terminated, prolactin initiated copious lactation. Further work by Meites (l970b) demonstrated that large doses of estrogen were able to inhibit growth of DMBA-induced rat mammary tumors even though serum prolactin levels were high. Since these tumors are responsive to prolactin, the author concluded that estrogen may possibly inhibit the action of prolactin on the mammary tumor. The evidence we have reported here suggests that large doses of estrogen may inhibit the action of prolactin on the mammary gland by decreasing prolactin binding activity. Another possibility is that the high serum pro- lactin produced by estrogen treatment may result in greater satura- tion of prolactin binding sites in the mammary gland, and hence less measurable prolactin binding. 128 In a recent article, Kelly gt al. (1974a) reported prolactin binding activity in DMBA-induced rat mammary tumors. The data showed that prolactin dependent tumors had the highest prolactin binding, and a negative relationship was observed with prolactin binding activity in the liver, i.e., the liver prolactin binding was lowest in those animals bearing mammary tumors with highest prolactin bind- ing. He also have observed a reciprocal relationship between liver and mammary tissue binding in response to estrogen and progesterone treatment, i.e., when the binding in the liver was high, that in the mammary tissue was low. VII. Effects of Adrenals on Prolactin Binding Activity in Liver of Female Rats A. Effects of Adrenalectomy and Hydrocortisone Treatment on Prolactin Binding Activity in Liver 1. Objectives The adrenal glucocorticoids influence protein synthesis and carbohydrate metabolism of the liver (Litwack and Singer, 1972) as well as the general body metabolism of rats (Frieden and Lipner, 1971). There is also evidence that glucocorticoids may reduce prolactin secretion in rats whereas adrenalectomy may increase prolactin secretion (Ben David et_ 1., 1971b; also Mueller, unpub- lished). Since we had observed such a profound effect of thyroid 129 and ovarian hormones on prolactin binding activity in the liver, it was of interest to investigate the effects of another class of metabolic hormones, this is, the glucocorticoids, on prolactin binding activity in the liver. 2. Procedures a. Animals Virgin, female Sprague-Dawley rats weighing 200-225 gms were used. The animals were ovariectomized or ovariectomized-adrenalec- tomized for 14 days at which time treatment was begun. The ovariec- tomized rats were treated sc with either 0.85% saline or 1 mg hydro- cortisone acetate and the ovariectomized-adrenalectomized animals were injected sc with 0.85% NaCl. The treatment was continued for 10 days. The ovariectomized-adrenalectomized animals were main- tained on 0.9% saline drinking water for the duration of the experi- ment. b. Tissue preparation and assay procedure Liver tissue was homogenized in 0.3 M sucrose and microsomal membranes were prepared and diluted to 300 pg protein/100 pl, as described previously. Specific binding of 125I-radiolabeled prolactin was measured. Each sample was assayed in quadruplicate. 130 c. Statistical analysis The data were analyzed using analysis of variance for unequal sample size (Sokal and Rohlf, 1969). 3. Results Adrenalectomy (Table XVI, group 2) and hydrocortisone acetate treatment (group 3) each lowered prolactin binding activity in the liver. Analysis of variance showed marginal significance (P£.0.05) since the effect of either treatment was not very pronounced. 8. Effects of Adrenalectomy and Hydrocortisone Acetate Replacement Therapy on Prolactin Binding Activity in the Liver 1. Objectives The previous experiment had shown a tendency for adrenalectomy and a large dose of hydrocortisone acetate to decrease prolactin binding activity. The present study was designed to investigate whether a replacement dose of hydrocortisone would restore prolactin binding to control level. 131 2. Procedures a. Animals Sprague-Dawley virgin female rats weighing approximately 300 gms were used. The rats were ovariectomized for 40 days and then adrenalectomized on day 41. Rats ovariectomized or ovariectomized-adrenalectomized received a sc injection of 0.85% saline and another group of ovariectomized-adrenalectomized rats was given 100,pg hydrocortisone/100 gm body weight sc daily. Treat- ment was begun on the same day the adrenalectomies were performed and continued for 6 days. b. Tissue preparation and assay procedure Liver microsomal membrane preparations were made and specific binding of 125I-radiolabeled prolactin was measured as described previously. Each sample was assayed in triplicate. c. Statistical analysis The data were analyzed using an analysis of variance for unequal sample size followed by Duncan's Multiple Range Test for comparison among the means. 132 3. Results In this second experiment, adrenalectomy (Table XVII, group 2) again lowered prolactin binding activity, although this was not significant as compared to the controls. Hydrocortisone treatment (group 3), however, produced a significant decrease in prolactin binding activity. C. Conclusions These results show that adrenalectomy had a tendency to lower prolactin binding activity in liver of female rats, whereas treatment with hydrocortisone acetate produced a more marked reduction in prolactin binding activity than was observed by adrenalectomy alone. Generally, the glucocorticoids are considered to be protein cata- bolic hormones and therefore decrease protein synthesis (Frieden and Lipner, 1971), although they stimulate gluconeogenesis in the liver (Litwack and Singer, 1972). This may be one explanation for the effects of a glucocorticoid hormone on prolactin binding activity, that is, it possibly decreases the receptor proteins for prolactin. 133 o.o.H m.¢ Ame abandon mcomwueouogvx; + x>o .m N.o.H N.a Ace xe<-x>o .N c.o.H e.c Ame xso .p page- H to asoem\muos to .o: meeee_m omwwooam N oea oeoeoaoce mung educate mpopmu< acomwugouosu»: so Axuov kumsouompgo>o mo mmuocomoso: cose; e. sopseoo< newoeem e_ooo_oca .H>x e_oae 134 295:8 2.23. + 56 B oflogou mo 85 Va and H on 8: 8 8 8:8 8:8. + x2 + :5 .m eoHQm :Hé<+§o.N m.o.H o.o Hoe oeHHoa + xso .H ammo- H to ooogm\muog Ho .o: mcHoch ommwooom a one pcoeuoosh Huzv ououoo< ocomHugouosoxz co>Ho «oz so =o>Ho muom onov omanouooHso>o co moooeoooeoz cosHo e. NeH>Hoo< oeHoeHN eHooaHoea .HHHx e_eaH GENERAL DISCUSSION The data presented in this thesis demonstrate that 1251- radiolabeled prolactin binds specifically to ovarian, mammary and liver microsomal membranes, and that prolactin binding activity changes under different physiological conditions in all tissues studied. This suggests that binding sites for prolactin vary with the physiological requirements for the hormone, such as an increase in prolactin binding activity during lactation in the mammary gland. The ontogeny of prolactin binding activity does not follow the same pattern in all tissues that specifically bind prolactin. In liver and ovarian tissues the binding for prolactin increased as the animal continued to develop, and reached adult levels at or about the time of vaginal opening in the rat. 0n the other hand, kidney and adrenal tissues showed significantly higher binding for prolactin in the immature rat, followed by a steady decrease in binding activity as the rat continued to mature. The levels of prolactin binding activity in the kidney and adrenals also reach adult levels at about the time of vaginal opening. Kelly gt_gl, (1974b) reported similar findings for prolactin binding activity in the liver of rats, but no reports have as yet appeared on prolactin binding activity in the ovaries, kidneys or adrenal glands of immature rats. 135 136 The increase in binding for prolactin in the ovaries of developing rats correlates well with the appearance of corpora lutea in the ovaries at vaginal opening or at first ovulation. Accord- ing to Schwartz gt_a1, (1974), until the first ovulation there are only follicles present in the developing ovaries. As the rat approaches sexual maturation, there is follicular maturation and ovu- lation. These authors also suggested that there is an increase in the number of receptor sites for the gonadotropins in the ovaries as the rat matures, resulting in increased sensitivity of the ovaries to these hormones. The binding data presented on prolactin in the ovaries tends to support this statement. Vaginal opening appears to be an important time for prolactin binding activity. This is also the period when prolactin serum levels rise and begin to show cyclic fluctuations (Voogt gt_al,, 1970). Estrogen levels in the serum increase preceding vaginal opening as well (Ramirez, 1973). In immature rats, estrogen stimu- lated prolactin binding activity in the liver, and preliminary obser- vations indicated that estrogen decreased prolactin binding activity in the kidney and adrenals (Marshall, Gelato and Meites, unpublished data). It is possible therefore that the pattern of prolactin binding activity in these tissues of the immature rat is related to the increase in estrogen at about the time of vaginal opening. However, the relatively high amount of prolactin binding activity in the adrenals and kidneys of 20-30 day old female rats suggests that prolactin has some functions on these organs at this time. Prolactin has been shown to influence both kidney and adrenal function in 137 the adult rat. It has been reported that a decrease in prolactin caused an increase in sodium and potassium excretion by the kidney (Richardson, 1973) and Lis gt_al, (1973) observed that prolactin was able to partially restore corticosterone biosynthesis in the adrenals of hypophysectomized rats. The immature animal may be more sensi- tive to these actions of prolactin. The most pronounced effect of prolactin as a luteotropic agent is seen during early pregnancy or pseudopregnancy. Data presented in this thesis show that prolactin binding activity is the highest during the first six days of gestation when pituitary prolactin is known to be essential for the support of progesterone secretion from the corpus luteum (Clemens gt_gl,, 1969a). During the second half of gestation binding of prolactin in the ovaries goes down and reaches a low level just before parturition. At this time there is another prolactin-like hormone to be considered. Placental lactogen in the rat shows two peaks during gestation, one at days 10-12 and another at days 17-21 (Shiu gt 31,, 1973). Neill and Smith (1974) reported that a placental prolactin is probably responsible for support of the corpus luteum during the second half of gestation. It is possible that the high levels of placental lacto- gen during the second half of gestation as measured by Shiu gt__l, (l973) compete with pituitary prolactin for binding sites on the corpus luteum and therefore binding for pituitary prolactin is decreased. Competition between radiolabeled ovine prolactin and human placental lactogen has been demonstrated by Shiu gt 91, (1973) for binding sites on lactating mammary gland membranes. The 138 ovarian preparation for binding studies used in the present study were membrane fractions of whole ovaries, and the corpora lutea were not separated. Therefore the binding activity of prolactin may have been different if only the corpora lutea had been measured during pregnancy. Prolactin is the major luteotropic hormone in the rat, but it has also been shown to be luteolytic (Malven and Sawyer, 1966). These authors reported that prolactin was luteolytic or luteotropic depending on the time prolactin was administered after hypophysectomy. Prolactin given two or more days after hypophysectomy had a luteo- lytic action whereas when given soon after hypophysectomy it was luteotropic. Although these authors demonstrated a luteolytic action for prolactin, it's physiological significance was not known. In lactating rats two generations of corpora lutea are present, one re- sulting from pregnancy and another from parturition-induced ovulation (Discussion in Malven and Sawyer, 1966). Normally the corpora lutea of pregnancy regress after parturition. If lactation is pre- vented, the corpora lutea of pregnancy regress more slowly. Pre- sumably the suckling induced prolactin surges cause the regression of the non-functional corpora lutea of pregnancy. Recent work indi- cated that the role of prolactin during the estrous cycle in the rat may be luteolysis. Huttke and Meites (1971) demonstrated that blockade of the proestrous surge of prolactin by ergocornine resulted in an accumulation of old corpora lutea in the ovaries of rats. This work has been confirmed in the rat (Gelato gt_al,, 1972) and mouse 139 t al., 1972). The binding for prolactin during the (Grandison cycle is relatively high and suggests a function for prolactin during this time. An interesting question is why is prolactin luteotropic at one time and luteolytic at another? The answer apparently lies in the ovary and how it responds to prolactin. Prolactin inhibits enzymes such as 20¢(—hydroxysteroid dehydrogenase (Hashimoto and Hiest, 1969), Sat-reductase and 3/3-hydroxysteroid dehydrogenase (Zmigrod gt_al,, 1972) which metabolize progesterone to an inactive dihydro compound. In this way prolactin is a luteotropic agent. The mechanism(s) for the action of prolactin as a luteolytic agent are not clearly defined. Malven and Sawyer (1966) proposed that there is a decreased responsiveness of the corpora lutea to pro- lactin with time. Hashimoto and Hiest (1969) reported that the ratio of prolactin to LH may dictate either support or luteolysis of corpora lutea. In subsequent work, Malven (1969) suggested that the luteolytic action of prolactin may involve some undefined con- trol mechanism over stromal cell development, since the distinctive histological characteristic of a luteolytic response to prolactin is the relative increase in the stromal cell population. The ovarian binding studies reported here were done on whole ovaries. Corpora lutea were not separated from other ovarian tissue. The data show that prolactin binds to ovaries of immature animals which have no corpora lutea. Midgley (1973) demonstrated that prolactin binds to interstitial ovarian tissue as well as luteal tissue. Therefore it is possible that prolactin may be influencing other structures 140 of the ovary other than the corpora lutea. Binding studies separat- ing corpora lutea from other ovarian tissues may provide some interest- ing clues on the actions of prolactin on the ovaries during the cycle. The actions of prolactin on the mammary gland have been well defined. Prolactin becomes attached to its receptor located on the mammary cell membrane (Turkington, 1972a; Shiu §t_al,, 1973) and initiates a series of cellular events involving DNA and RNA directed synthesis of protein kinase, leading to mammary growth and the pro- duction of milk proteins. The cellular events following the action of prolactin on the corpora lutea have not been studied as exten- sively as the mammary gland, but it is known that prolactin stimu- lates progesterone secretion and this effect is mediated by depression of enzymes involved in the metabolism of progesterone (Hashimoto and Hiest, 1969; Zmigrod £3 31,, 1972). Data presented in this thesis indicate that as the physiological requirement by the ovaries and the mammary gland for the action of prolactin are increased, the binding activity of prolactin is increased. Clemens gt al, (1969a) reported that pituitary prolactin is essential during the first six days of gestation to support progesterone secretion from the cor- pus luteum. The binding activity for pituitary prolactin throughout pregnancy was found to be highest on days 3 and 6 of gestation. At parturition prolactin serum levels increase and lactation begins. Prolactin is necessary for milk secretion in the rat, and as the need for prolactin is increased, the number of receptor sites for this hormone in the mammary gland are also increased. Frantz 141 £5431, (1974) showed that lactating mouse mammary glands bind more prolactin than non-lactating glands. Recent work by Kelly gt_al, (1974a) demonstrated that DMBA induced rat mammary tumors which are dependent on prolactin for growth have more binding sites for prolactin than DMBA tumors which are not dependent on prolactin. Similar work by Turkington (1974) also showed that tumors in rats and mice which were most responsive to prolactin for growth had the greatest number of receptors for prolactin. Another tissue that shows increased prolactin binding acti- vity as its functions increase is the liver. Turkington (1972b) observed an increase in liver function during gestation as evidenced by stimulation of RNA and protein synthesis. Kelly gt_al, (1974b) found that prolactin binding activity in the liver of pregnant rats was 3-fold higher than in non-pregnant rats. This elevation in prolactin binding activity could possibly reflect stimulation of liver function by prolactin. There are indications that prolactin stimulates protein synthesis in the liver (Burt gt_al,, 1969; Chen gt al., 1972). These studies, together with the data presented in this thesis, provide evidence that the number bf binding sites for prolactin in a tissue are indicative of physiological functions for this hormone on these tissues. Some hormonal treatments can alter prolactin binding activity in tissues. Thyroidectomy significantly reduced prolactin binding activity in the liver and replacement therapy with thyroxine re- turned binding to intact levels. Hydrocortisone acetate treatment significantly decreased prolactin binding activity in the liver. 142 Unpublished observations (Marshall, Gelato and Meites) in our labora- tory indicate that thyroxine and hydrocortisone acetate produce similar effects on prolactin binding activity in the kidney, adrenals and liver. Thyroidectomy decreases the amount of protein in the liver (Turner and Bagnara, 1971) and thus may reduce the binding sites for prolactin since they are considered to be protein in nature (Frantz gt al., 1974). This is supported by Scatchard analysis which showed that the actual number of binding sites for prolactin in the livers of thyroidectomized rats decreased as compared to intact controls, and there was little change in the affinity constant of these sites. Thyroxine is an anabolic hormone whereas hydrocortisone acetate is a catabolic hormone (Frieden and Lipner, 1971). The effects of thyroxine and hydrocortisone acetate on prolactin binding activity in the liver, kidneys and adrenal glands could be related to their function as regulators of protein synthesis. Thus, in order for a certain level of prolactin binding activity to be maintained in these tissues, both thyroid and adrenal hormones may be necessary, and an imbalance in these hormones could result in a change in the numbers of binding sites. Estrogen has been considered to be one of the most important regulators of prolactin secretion (Meites gt al., 1972). A recent report by Leung and Sasaki (l973) implicated prolactin as a possible regulator of estrogen receptors in mammary tissue. Data presented in this thesis show that estrogen may be important in regulating prolactin binding activity in the liver and mammary gland. In the 143 liver, prolactin binding activity was increased 4-5 fold after administration of estrogen to adult ovariectomized and immature female rats. Estrogen has been shown to influence liver function and is implicated in liver protein synthesis (Leathem, 1961). Thus estrogen may increase prolactin binding activity in the liver as a result of increasing general protein synthesis. On the other hand, mammary tissue as well as kidney and adrenal tissues did not show stimulation but inhibition of prolactin binding activity after administration of estrogen. The reason for the decrease in prolactin binding activity in the mammary gland after estrogen administration is not clear, but may be due to the relatively high doses of estrogen employed. High doses of estrogen have been shown to inhibit the action of prolactin on the mammary gland (Meites gt_al,, 1972), reducing lactation and mammary tumor growth. Lower doses of estro- gen may increase prolactin binding sites in the mammary gland al- though this remains to be demonstrated. In DMBA induced rat mammary tumors, low levels of estrogen stimulate growth of the tumors and high doses inhibit growth (Meites, l970b). Further work needs to be done in order to elucidate the actions of different doses of estrogen on prolactin binding activity. The relatively high amounts of prolactin binding activity in tissues such as the liver of adult and immature rats and the adrenals and kidneys of immature rats indicates that these tissues are target organs for prolactin. Prolactin has been shown to be a somatic as well as metabolic hormone in mammals, and is generally considered to be a "growth hormone” in avian species (Nicoll and 144 Bern, 1972). There is a substantial amount of data linking prolactin to metabolic function such as stimulation of hepatic RNA synthesis (Chen e_“al., 1972), stimulation of glucose utilization (Berle, 1973), stimulation of growth in rats (Knobil, 1959) and many other functions reviewed elsewhere in this thesis. Another function that has been attributed to prolactin is that of an osmoregulator, and this has been recognized as an important role of prolactin in teleosts. Work by Lockett (1965; 1967), Horrobin gt al, (1971), Buckman and Peake (l973a; l973b), and Relkin (1973) indicate an osmo- regulatory role for prolactin in mammals as well. At least 82 sepa- rate functions have been ascribed to prolactin (Nicoll and Bern, 1972) and the advent of receptor physiology may be the key to separat- ing "function" from "fiction". Research on prolactin receptors is a new area and much work needs to be done in order to determine their physiological signifi- cance and their role in target tissues. Receptors are believed to be the intermediates between the presence of a hormone at a target tissue site and initiation of cellular events in the tissue. The studies presented in this thesis suggest a relation between prolactin binding activity and physiological functions in several tissues. REFERENCES REFERENCES Ahmad, N., H.R. Lyons and 5. Ellis, 1969. Luteotrophic activity of rat hypophysial mammatrophin. Endocrinology 85:378-380. Amenomori, Y. and J. Meites, 1970. Effect of a hypothalamic extract on serum prolactin levels during the estrous cycle and lacta- tion. Proc. Soc. Exptl. Biol. Med. 134:492-495. Anden, N.E., A. Carlsson, A. Dahlstrom, K. Fuxe, N.A. Hillarp and K. Larsson, 1964. Demonstration and mapping out of nigro- neo-striatal dopamine neurons. Life Sci. 3:523-530. Anderson, R.R., 1968. Lactogenic hormone requirement for pseudo- pregnancy in normal and hysterectomized rats. Proc. Soc. Exp. Biol. Med. 127:723-725. Antliff, H.R., M.R.N. Prassad and R.K. Meyer, 1960. Action of prolactin on seminal vesicles of guinea pig. Proc. Soc. Exp. Biol. Med. 103:77-80. Anton-Tay, F. and R.J. Hurtman, 1971. Brain monoamines and endocrine function. In; Frontiers in Neuroendocrinology, 1971. Edited by L. Martini and H.F. Ganong. PP. 45-66. Oxford University Press, New York. Armstrong, D.T., L.S. Miller and K.A. Knudsen, 1969. Regulation of lipid metabolism and progesterone production in rat corpora lutea and ovarian interstitial elements by prolactin and luteinizing hormone. Endocrinology 85:393-401. Armstrong, D.T., K.A. Knudsen and L.S. Miller, 1970. Effects of prolactin upon cholesterol metabolism and progesterone bio- synthesis in corpora lutea of rats hypophysectomized during pseudopregnancy. Endocrinology_86:634-641. Astwood, 8.8., 1941. The regulation of corpus luteum function by hypophysial luteotrophin. Endocrinology_28:309-320. Astwood, E.8. and R.O. Greep, 1938. A corpus luteum-stimulating substance in the rat placenta. Proc. Soc. Exp. Biol. Med. 38:713-716. 145 146 Baldwin, R.L. and R.J. Martin, 1968. Effects of hypophysectomy and several hormone replacement therapies upon patterns of nucleic acid and protein synthesis and enzyme levels in lactating rat mammary glands. J. Dairy Sci. 51:748-753. Ball, J.N., 1965. Effects of autotransplantation of different regions of the pituitary gland on freshwater survival in the teleost Poecilia latipinna. J. Endocrinol. 33:v-vi. Ball, J.N., 1969. Prolactin (fish prolactin or paralactin) and growth hormone. In; Fish Physiology. Edited by H.S. Hoar and D.J. Randall. PP. 207-240. Academic Press, New York. Ball, J.N. and D.M. Ensor, 1965. Effect of prolactin on plasma sodium in the teleost, Poecilia Latipinna. J. Endocrinol. 32:269-270. Ball, J.N. and D.M. Ensor, 1967. Specific action of prolactin on plasma sodium in the hypophysectomized Poecilin Latipinna (Teleostei). Gen. Comp. Endocrinol. 8:432-440. Barden, N. and F. Labrie, 1973. Receptor for thyrotropin- releasing hormone in plasma membranes of bovine anterior pituitary gland. J. Biol. Chem. 248:7601-7606. Barraclough, C.A. and C.H. Sawyer, 1957. Blockade of the release of pituitary ovulating hormone in the rat by chlorpromazine and reserpine: possible mechanisms of action. Endocrinology_ 61:341-351. Bartke, A., 1965. Influence of luteotrophin on fertility of dwarf mice. J. Reprod. Fertil. 10:93-103. Bartke, A., l966a. Reproduction of female dwarf mice treated with prolactin. J. Reprod. Fertil. 11:203-206. Bartke, A., l966b. Influence of prolactin on male fertility in dwarf mice. J. Endocrinol. 35:419-420. Bartke, A., 1967. Influence of pituitary homografts on the weight of seminal vesicles in castrated mice. J. Endocrinol. 38:195-196. Bartke, A., 1971a. Effects of prolactin and luteinizing hormone on the cholesterol stores in the mouse testis. J. Endocrinol. 49:317-324. Bartke, A., 1971b. Effects of prolactin on spermatogenesis in hypophysectomized rats. J. Endocrinol. 49:311-316. 147 Bartke, A., 1971c. The maintenance of gestation and the initiation of lactation in the mouse in the absence of pituitary pro- lactin. J. Reprod. Fertil. 27:121-124. Bartke, A., 1973. Differential requirement for prolactin during pregnancy in the mouse. Biol. Reprod. 9:379-383. Bartke, A. and C.H. Lloyd, 1970a. Influence of prolactin and pituitary isografts on spermatogenesis in dwarf mice and hypophysectomized rats. J. Endocrinol. 46:321-329. Bartke, A. and C.H. Lloyd, l970b. The influence of pituitary homo- grafts on the weight of the accessory reproductive organs in castrated male mice and rats and on mating behavior in male mice. J. Endocrinol. 46:313-320. Bartosik, 0., E.B. Romanoff, D.J. Hatson and E. Scricco, 1967. Luteotropic effects of prolactin in the bovine ovary. Endocrinology.81:186-194. Bates, R.R., T. Laanes and 0. Riddle, 1935. Evidence from dwarf mice against the individuality of growth hormone. Proc. Soc. Exp. Biol. Med. 33:446-450. Bates, R.H., 0. Riddle, E.L. Lahr and J.P. Schooley, 1937. Aspects of splanchnomegaly associated with action of prolactin. Am. J. Physiol. 119:603-609. Bates, R.R., T. Laanes, E.C. MacOowell and O. Riddle, 1942. Growth in silver dwarf mice with and without injections of anterior pituitary extracts. Endocrinology_3l:53-58. Bates, R.H., R.A. Miller, and M.M. Garrison, 1962. Evidence in the hypophysectomized pigeon of a synergism among prolactin, growth hormone, thyroxine and prednisone upon weight of the body, digestive tract, kidney and fat stores. Endocrinology_7l: 345-360. Bates, R.R., S. Milkovic and M.M. Garrison, 1964. Effects of prolactin, growth hormone and ACTH and in combination, upon organ weights and adrenal function in normal rats. Endocrinology_74:714-723. Bates, R.H., R.O. Scow and P.E. Lacy, 1966. Induction of permanent diabetes in rats by pituitary hormones from a transplantable mammotropic tumor. Concomitant changes in organ weights and the effects of adrenalectomy. Endocrinology 78:826-836. Bates, R.H. and M.M. Garrison, 1974. Hormonal interactions among GH, ACTH, cortisol and dexamethasone upon size of kidney, liver, and adrenal. Proc. Soc. Exp. Biol. Med. 146:725-731. 148 Beck, 0.0., A. Gonda, M.A. Hamid, R.0. Morgen, D. Rubinstein and E.E. McGarry, 1964. Some metabolic changes induced by pri- mate growth hormone and purified ovine prolactin. Metabolism 13: Suppl. 1108-1134. Beck, P. and H.H. Daughaday, 1967. Human placental lactogen: studies of its' acute metabolic effects and disposition in normal man. J. Clin. Invest. 46:103-110. Behrman, H.R., G.P. Orczyk, G.J. MacDonald and R.O. Greep, 1970. Prolactin induction of enzymes controlling luteal cholesterol ester turnover. Endocrinology 87:1251-1256. Behrman, H.R., and R.0. Greep, 1972. Hormonal dependence of cholesterol ester hydrolase in the corpus luteum and adrenal. Horm. Metab. Res. 4:206-209. Ben-David, M., 1968. The role of the ovaries in perphenazine- induced lactation. J. Endocrinol. 41:377-385. Ben David, M., A. Danon and F.G. Sulman, 1971a. Evidence of antagonism between prolactin and gonadotrophin secretion: effect of methallibure on perphenazine-induced prolactin secretion in ovariectomized rats. J. Endocrinol. 51:719-725. Ben-David, M., A. Danon, I. Benveniste, F. Heller and F.G. Sulman, 1971b. Results of RIA of rat pituitary and serum prolactin after adrenalectomy and perphenazine treatment in rats. J. Endocrinol. 50:599-606. Bengmark, S. and R. Hesselsjo, 1964. Endocrine dependence of rat seminal vesicle tissue in tissue culture. Urol. Int. 17: 84-92. Berle, P., 1973. Comparative studies on the metabolic effects of some parameters of carbohydrate and lipid metabolism after intravenous administration of human placental lactogen, human prolactin and growth hormone. Acta Endocrinol. (Kbh). Suppl. 173:104. Berman, R., H.A. Bern., C.S. Nicoll and R.C. Strohman, 1965. Growth- promoting effects of mammalian prolactin and growth hormone in tadpoles of Rana Catesbeiana. J. Exptl. 2001. 156:353-360. Bern, H.A., C.S. Nicoll and R.C. Strohman, 1967. Prolactin and tadpole growth. Proc. Soc. Exp. Biol. Med. 126:518-520. Bern, H.A. and C.S. Nicoll, 1968. The comparative endocrinology of prolactin. Recent Progr. Horm. Res. 24:681-720. Bhalla, V.K. and L.E. Reichert, Jr., 1974. Properties of follicle stimulating hormone-receptor interactions. J. Biol. Chem. 249:43-51. 149 Birge, C.A., L.S. Jacobs, C.T. Hammer and H.H. Daughaday, 1970. Catecholamine inhibition of prolactin secretion by isolated rat adenohypophyses. Endocrinol. 86:120-130. Birkinshaw, M. and I.R. Falconer, 1972. The localization of pro- lactin labelled with radioactive iodine in rabbit mammary tissue. J. Endocrinol. 55:323-334. Bjorklund, A., B. Falck, F. Hromek, C. mean and K.A. West, 1970. Identification and terminal distribution of the tubero- hypophyseal monoamine fibre systems in the rat by means of stereotaxic and microspectrofluorometric techniques. Brain Res. 17:1-24. Blake, C., R. Norman and C.H. Sawyer, 1972. Effects of estrogen and/or progesterone on serum and pituitary gonadotropin levels in ovariectomized rats. Proc. Soc. Exp. Biol. Med. 141:1100-1103. Blanc-Livini, N. and M. Abraham, 1970. The influence of environ- mental salinity on the prolactin and gonadotropin secreting regions in the pituitary of Mugie (Teleostii). Gen. Comp. Endocrinol. 14:184-197. Boler, J., F. Enzmann, K. Folkers, E.Y. Bowers and A.V. Schally, 1969. The identity of chemical and hormonal properties of the thyrotropin releasing hormone and pyroglutamyl-histidyl proline amide. Biochem. Biophys. Res. Comm. 37:705-710. Bowers, C.Y., H.G. Friesen, P. Hwang, H.J. Guyda and K. Folkers, 1971. Prolactin and thyrotropin release in man by synthetic pyroglutamyl-histidyl-prolinamide. Biochem. Biophys. Res. Comm. 45:1033-1041. Braendle, M., M. Breckwaldt, D. Graeaglin‘and'HJC.H. Heise, 1973. Distribution and binding of I -human chorionic gonadotropin (HCG) in different organs of pseudopregnant female rats. Fertil. Steril. 24:126-130. Brazeau, P., H. Vale, R. Burgus, N. Ling, M. Butcher, J. Rivier and R. Guillemin, 1973. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:77-79. Brodie, 8.8., S. Specton and P.A. Shore, 1959. Interaction of drugs with norepinephrine in the brain. Pharmacol. Rev. 11:548-864. Brown, P.S. and B.E. Frye, 1969a. ’Effects of prolactin and growth hormone and metamorphasis of tadpoles of the frog, Rana pipiens. Gen. Comp. Endocrinol. 13:126-138. Brown, P.S. and B.E. Frye, 1969b. Effects of hypophysectomy, pro- lactin and growth hormone on growth of post-metamorphic frogs. Gen. Comp. Endocrinol. 13:139-145. 150 Brown, P.S. and S.C. Brown, 1971. Growth and metabolic effects of prolactin and growth hormone in the red-spotted newt, noto- phthalmus viridescens. J. Exptl. 2001. 178:29-34. Buckman, M.T. and G.T. Peake, 1973a. Osmolar control of prolactin secretion in man. Program of Fifty-Fifth Meeting of The Endocrine Society, Abst. 2, A-49. Buckman, M.T. and G.T. Peake, l973b. Osmolar control of prolactin secretion in man. Science 181:755-757. Burden, C.E., 1956. The failure of hypophysectomized Fundulus heteroclitus to survive in fresh water. Biol. Bull. 110: 8-28. Burgus, R., T.F. Dunn, D. Desideri and R. Guillemin, 1969. Structure moleculaire du facteur hypothalamique hypophysiotrope TRF d'origine ovine: evidence par spectrometrie de masse de la sequence PCA-His-Pro-NHz. Compt. Rend. Acad. Sci. [D](Paris) 269:1870—1873. Burgus, R. and R. Guillemin, 1970. Hypothalamic Releasing Factors. Ann. Rev. Biochem. 39:499-526. Burstyn, P.G., D.F. Horrobin and M.S. Manku, 1972. Saluretic action of aldosterone in the presence of increased salt intake and restoration of normal action by prolactin or by oxytocin. :1; Endocrinol. 55:369-376. Burt, R., P.S. Pegram and M.M. Leake, 19?3. Effect of placental lactogenic hormone on glycine-l-C incorporation into liver protein of the rat. Am. J. Obst. Gynecolog. 103:44-47. Callard, I.P. and D.K.0. Chan, 1972. Hormonal effects on liver glycogen and blood sugar levels in the Iguanid lizard Dipsosaurus dorsalis. Gen. Comp. Endocrinol. 18:552-556. Campbell, B.J., G. Woodward and V. Borberg, 1972. Calcium-mediated interactions between the anti-diuretic hormone and renal plasma membranes. J. Biol. Chem. 247:6167-6175. Cargill Thompson, H.E.C. and G.P. Crean, 1963. Studies on the effect of hormone administration on body weight and on tibial epi- physial cartilage width in intact, hypophysectomized and adrenalectomized rats. J. Endocrinol. 25:473-482. Catt, K.J., M.L. Dufau and T. Tsuruhara, 1971. Studies on a radio- ligand-receptor assay system for luteinizing hormone and chrionic gonadotropin. J. Clin. Endocrinol. Met. 32:860-863. 151 Catt, K.J. and M.L. Dufau, 1973. Interactions of LH and hCG with testicular gonadotropin receptors. In; Receptors for Reproductive Hormones, edited by B.W. O'Malley and A.R. Means, PP. 379-418. Plenum Press, New York. Chandola, A. and J.P. Thapliyal, 1968. Further studies on the regulation of the body weight of Spotted Munia, Lonchura punctulata. Gen. Comp. Endocrinol. 11:272-277. Chandola, A. and J.P. Thapliyal, 1973. Effect of growth hormone and prolactin on the body weight and thyroid activity of Spotted Munia. Ann. Endocrinol. (Paris) 33:583-591. Chandra, P. and R.D. Cole, 1961. The effect of prolactin on some purine metabolizing activities. Endocrinol. 69:319-323. Chase, M.D., I.I. Geschwind and H.A. Bern, 1957. Synergistic role of prolactin in response of male rat sex accessories to androgen. Proc. Soc. Exp. Biol. Med. 94:680-683. Chen, C.L., 1969. Effect of hypothalamic extract, pituitary hormones and ovarian hormones on pituitary prolactin secretion. Ph.D. Dissertation, Michigan State University. Chen, C.L., H. Minaguchi and J. Meites, 1967. Effects of trans- planted pituitary tumors on host pituitary prolactin secretion. Proc. Soc. Exp. Biol. Med. 126:317-320. Chen, C.L., E.J. Bixler, A.I. Weber and J. Meites, 1968. Hypothalamic stimulation of prolactin release, from the pituitary of turkey hens and poults. Gen. Comp. Endocrinol. 11:489-494. Chen, C.L. and J. Meites, 1969. Effects of thyroxine and thiouracil on hypothalamic PIF and pituitary prolactin levels. Proc. Soc. Exp, Biol. Med. 131:576-578. Chen, C.L. and J. Meites, 1970. Effects of estrogen and progesterone on serum and pituitary prolactin levels in ovariectomized rats. Endocrinol. 86:503-505. Chen, C.L., Y. Amenomori, K.H. Lu, J.L. Voogt and J. Meites, 1970. Serum prolactin levels in rats with pituitary transplants or hypothalamic lesions. Neuroendocrinol. 6:220-227. Chen, H.J. and J. Meites, 1974. Effects of biogenic amines and TRH on release of prolactin and TSH in the rat. Endocrinol. in press. Chen, H.W., D.M. Hamer, H. Heiniger and H. Meier, 1972. Stimula- tion of hepatic RNA synthesis in dwarf mice by ovine prolactin. Biochem. Biophys. Acta 287:90-97. 152 Choudary, J.B. and 6.5. Greenwald, 1969. Luteotropic complex of the mouse. Anat. Rec. 163:373-384. Clemens, J.A. and J. Meites, 1968. Inhibition by hypothalamic prolactin implants of prolactin secretion, mammary growth and luteal function. Endocrinol. 82:878-881. Clemens, J.A., M. Sar and J. Meites, 1969a. Termination of preg- nancy in rats by a prolactin implant in median eminence. Proc. Soc. Exp. Biol. Med. 130:628-630. Clemens, J.A., M. Sar and J. Meites, 1969b. Inhibition of lactation and luteal function in postpartum rats by hypothalamic im- plantation of prolactin. Endocrinol. 84:868-872. Clifton, K.H. and J. Furth, 1960. Ducto-alveolar growth in mammary glands of adreno-gonadectomized rats bearing mammotropic pituitary tumors. Endocrinol. 66:893-897. Cole, F.E., J.C. Wied, G.T. Schneider, J.B. Halland, W.L. Geary and B.F. Rice, 1973. The gonadotropin receptor of the human corpus luteum. Am. J. Obstet. Gynecol. 117:87-95. Convey, E.M., H.A. Tucker, V.G. Smith and J. Zolman, 1972. Prolactin, thyroxine and corticoid after TRH. J. Anim. Sci. 35:258. Cook, 8., C.C. Kaltenbach, G.D. Niswender, H.W. Norton and A.V. Nalvandov, 1969. Short-term ovarian responses to some pitui- tary hormones infused jn_vivo in pigs and sheep. J. Anim. Sci, 29:711-718. Cooper, J.R., F.E. Bloom and R.H. Roth, 1970. The_Biochemical. Basis of Negropharmagology, Oxford University Press, London. Coppola, J.A., R.G. Leonardi, W. Lippmann, J.W. Perrine and I. Ringler, 1965. Induction of pseudopregnancy in rats by de- pletors of endogenous catecholamines. Endocrinol. 77:485-490. Coppola, J.A., R.G. Leonardi and W. Lippmann, 1966. Ovulatory failure in rats after treatment with brain norepinephrine depletors. Endocrinol. 78:225-228. Costlow, M.E., R.A. Buschow and W.L. McGuire, 1974. Prolactin receptors in an estrogen receptor-deficient mammary carcinoma. Science 184:85-86. Cuatrecasas, P., 1971. Unmasking of insulin receptors in fat cells and fat cell membranes. J. Biol. Chem. 246:6532-6542. Cuatrecasas, P., 1973. Interaction of concanavalin A and wheat germ agglutinin with the insulin receptor of fat cells and liver. J. Biol. Chem. 248:3528—3534. 153 Cuatrecasas, P., B. Desbuiquois and F. Krug, 1971. Insulin-receptor interactions in liver cell membranes. Biochem. Biophys. Res. Comm. 44:333-339. Cuatrecasas, P. and G.P.E. Tell, 1973. Insulin-like activity of concanavalin A and wheat germ agglutinin-direct interactions with insulin receptors. Proc. Nat. Acad. Sci. 70:485-489. Cutuly, E., 1941. Implantation following mating in hypophysecto- mized rats injected with lactogenic hormone. Proc. Soc. Exp. Biol. Med. 48:315-318. Daniel, P. M. 1966. The anatomy of the hypothalamus and pituitary gland. In: Neuroendocrinology, edited by L. Martini and W. F. Ganong, I: —15- 80. Academic Press, New York. Danon, A., S. Dikstein and F.G. Sulman, 1963. Stimulation of pro- lactin by perphenazine in pituitary-hypothalamus organ culture. Proc. Soc. Exp. Biol. Med. 114:366-368. DeBodo, R.C. and N. Altszuler, 1958. Insulin hypersensitivity and physiological insulin antagonists. Physiol. Rev. 38:389-445. Deuben, R. R. and J. Meites, 1963. In vitro stimulation of growth hormone release from anterior _pituitary by extract of rat hypothalamus. Fed. Proc. 22:571. Dharmamba, M., 1970. Studies of the effects of hypophysectomy and prolactin on plasma osmolarity and plasma sodium in Tilapia Massambica. Gen. Comp. Endocrinol. 14:256-269. Dibbet, J.A., J. F. Bruni, G. P. Mueller, H. J. Chen and J. Meites, 1973. In vivo and In Vitro stimulation of prolactin (PRL) secretion by synthetic_ TRH in rats. The Fifty-Fifth Meeting of Endocrine Society, abst. 182, A-139. Dibbet, J.A., M.J. Boudreau, J.F. Bruni and J. Meites, 1974. Possi- ble role of dopamine in modifying prolactin response to TRH. Xha Fifty-Fifth Meeting of the Endocrine Society, abst. 262, - 86. Dickerman, E., 1971. Radioimmunoassay for rat growth hormone; fur- ther studies on the control of growth hormone secretion in the rat. Ph.O. Dissertation, Michigan State University. Dickerman, S., J. Clark, E. Dickerman and J. Meites, 1972a. Effects of haloperidol on serum and pituitary prolactin and on hypo- thalamic PIF in rats. Neuroendocrinology,9:332-340. Dickerman, E., S. Dickerman and J. Meites, 1972b. Influence of age, sex and estrous cycle on pituitary and plasma GH levels in rats. Ln: Growth and Growth Hormone, edited by A. Pecile and E. E. Muller, PP. 252- 260. Excerpta Medica, Amsterdam. 154 Dickerman, S., G. Kledzik, M. Gelato, H.J. Chen and J. Meites, 1974. Effects of haloperidol on serum and pituitary prolactin, LH and FSH, and hypothalamic PIF and LRF. Neuroendocrinology 15: 10-20. Diebel, N.D. and E.M. Bogdanove, 1970. Post-partum changes in LH and FSH secretion in the rat. 52nd Meeting of Endocrine Society, p. 56. Domanski, E. and H. Dobrowolski, 1966. Perfusion of an organ jn_situ as a method in endocrinological investigations. Excerpta Medica, Inter. Congress. Hormonal Steroids, Abst. 471, 259. Domanski, E., L. Skrzeczkowski, E. Stupnicka, R. Fitko and H. Dobrowolski, 1967. Effect of gonadotrophins on the secretion of progesterone and oestrogens by the sheep ovary perfused jg_situ. J. Reprod. Fert. 14:365-372. Donovan, B.T., 1963. The effect of pituitary stalk section on luteal function in the ferret. J. Endocrinol. 27:201-211. Donovan, B.T., 1967. The control of corpus luteum function in the ferret. Archives D'Anatomie Microscopigue Suppl. 3-4, 56: 281-291. Dresel, I., 1935. The effect of prolactin on the estrous cycle of non-parous mice. Science 82:173. Dufau, M.L. and K.J. Catt, 1973. Extraction of soluble gonadotrophin receptors from rat testis. Nature (New Biol.) 242:246-248. Dufau, M.L., D. Ryan and K.J. Catt, 1974. Disulphide groups of gonadotropin receptors are essential for specific binding of human chorionic gonadotropin. Biochim. Biophys. Acta 343: 417-422. Duncan, 0.8., 1955. Multiple range and multiple F tests. 819- metrics 11:1-42. Elghamry, M.I., A. Said and S.A. Elmongy, 1966. The effect of lactogenic hormone on liver glycogen and blood glucose in ovariectomized mice. Naturwissenchaften 53:530. Elias, J.J., 1957. Cultivation of adult mouse mammary glands in hormone-enriched synthetic medium. Science 126:842-844. Enemai, A., B. Essvik and R. Klang, 1968. Growth promoting effects of ovine somatotropin and prolactin in tadpoles of Rana temporaria. Gen. Comp. Endocrinol. 11:328-331. 155 Ensor, D.M. and J.G. Phillips, 1970. The effect of salt loading on the pituitary prolactin levels of the domestic duck and juvenile herring or lesser black-backed gulls. J. Endocrinol. 48:167-172. Ensor, D.M., M.R. Edmondson and J.G. Phillips, 1972. Prolactin and dehydration in rats. J. Endocrinol. 53:Lix-Lx. Evans, H.M., M.E. Simpson, w.R. Lyons and K. Tarpeinen, 1941. Anterior pituitary hormones which favor the production of traumatic uterine placentomata. Endocrinol. 28:933-945. Everett, J.N., 1954. Luteotrophic function of autografts of the rat hypophysis. Endocrinol. 54:685-690. Everett, J.N., 1956. Functional corpora lutea maintained for months by autografts of the rat hypophysis. Endocrinol. 58:786-796. Everett, J.H., C.H. Sawyer and J.E. Markee, 1949. A neurogenic timing factor in the control of the ovulatory discharge of luteinizing hormone in the cyclic rat. Endocrinol. 44: 234-250. Folley, S.J., 1956. The Physiology ond Biochemistry of Loctotjon. Thomas, Springfield, Illinois. Frantz, W.L., J.H. MacIndoe and R.H. Turkington, 1974. Prolactin receptors: characteristics of the particulate fraction bind- ing activity. J. Endocrinol. 60:485-497. Fraschini, F., 1970. Role of indolamines in the control of the secretion of pituitary gonadotropins. In: Neurochemical Aspects of Hypothalamic Function. L. Martini and J. Meites (eds.). Academic Press, N.Y. 141-159. Frieden, E. and H. Lipner, 1971. Biochemical Enooccjnology of the Vertebrates. Prentice Hall, Inc., New Jersey. Friesen, H.G., G. Talis, R. Shiu and P. Hwang, 1973. Studies on human prolactin: chemistry, radioreceptor assay and clinical significance. In; Human Prolactin, edited by J.L. Pasteels and C. Robyn. PP. 11-23. Excerpta Medica, Amsterdam. Fuxe, K. and T. Hokfelt, 1969. Catecholamines in the hypothalamus and the pituitary gland. 1n; Frontiers in Neuroendocrinology, 1969, edited by L. Martini and H.F. Ganong. PP. 47-96. Oxford University Press, New York. Gala, R.R. and R.P. Reece, 1965. Influence of neurohormones on anterior pituitary lactogen production jn_vitro. Proc. Soc. Exp. Biol. Med. 120:220-222. 156 Gavin, J.R. III, P. Gorden, J. Roth, J.A. Archer and D.N. Buell, 1973. Characteristics of the human lymphocyte insulin re- ceptor. J. Biol. Chem. 248:2202-2207. Gelato, M.C., K.H. Lu and J. Meites, 1972. Inhibition of luteolysis by iproniazid during the estrous cycle in rats. Program 5th Annual Meeting, The Society for the Study of Reproduction, East Lansing, Mich., p. 80. Gitsch, E. and J.N. Everett, 1958. Influence of the anticholinergic drug, Pathilon, on the reproductive cycle of the female rat. Endocrinol. 62:400-409. Golder, M.P., A.R. Boyns, M.E. Harper, and K. Griffiths, 1972. An effect of prolactin on prostatic adenylate cyclase activity. Biochem. J. 128:725-727. Goodfriend, T. and S.Y. Lin, 1969. Angiotensin receptors. Clin. Res, 17:243. Goodfriend, T.L. and S.Y. Lin, 1970. Rece tors for angiotensin I and II. Circ. Res. 26-27 (Suppl. I :163-174. Goodridge, A.G. and E.G. Ball, 1967. The effect of prolactin on lipogenesis in the pigeon: In_vivo studies. Biochem. 6: 1676-1682. Gorden, P., M.A. Lesniak, C.M. Hendricks and J. Roth, 1973. "Big" growth hormone components from human plasma: decreased reactivity demonstrated by radioreceptor assay. Science 182: 829-831. Gourdji, D. and A. Tixier-Vidal, 1966. Mise en evidence d'un control hypothalamique stimulant de la prolactine hypophysaire chez le canard. Compt. Rend. Acad. Sci. 263:162-165. Grady, K.L. and G.S. Greenwald, 1968. Gonadotropic induction of pseudopregnancy in the cyclic hamster. Endocrinol. 83:1173-1180. Grandison, L. and J. Meites, 1972. Luteolytic action of prolactin during estrous cycle of the mouse. Proc. Soc. Exp. Biol. Med. 140:323-325. Grandison, L., M. Gelato and J. Meites, 1974. -Inhibition of pro- lactin secretion by cholinergic drugs. Proc. Soc. Exp, Biol. Med, 145:1236-1239. Grayhack, J.T., 1963. Pituitary factors influencing growth of the prostate. Nat. Cancer Inst. Monogr. 12:189-199. 157 Grayhack, J.T. and J.M. Lebrowitz, 1967. Effect of prolactin on citric acid of lateral lobe of prostate of Sprague-Dawley rat. Invest. Urol. 5:87-94. Green, M.R. and Y.J. Topper, 1970. Some effects of prolactin, in- sulin and hydrocortisone on RNA synthesis by mouse mammary gland, jn_vitro. Biochim. Biophys. Acta 204:441-448. Greenwald, G.S., 1967a. Further observations on the luteotropic complex of the hamster. Archives D'Anatomie Microscopique 56:Suppl. 3-4, p. 281-291. Greenwald, G.S., 1967b. Luteotropic complex of the hamster. Endocrinol. 80:118-130. Greenwald, G.S. and D.C. Johnson, 1968. Gonadotropic requirements for the maintenance of pregnancy in the hypophysectomized rat. Endocrinol. 83:1052-1064. Grosvenor, C.E. and C.H. Turner, 1958. Effects of oxytocin and blocking agents upon pituitary lactogen discharge in lactat- ing rats. Proc. Soc. Exp. Biol. Med. 97:463-465. Grosvenor, C.E., S.M. McCann and M.D. Nallar, 1964. Inhibition of suckling-induced release of prolactin in rats injected with acid extract of bovine hypothalamus. Program 46th Meeting Endocrine Society, San Francisco, p. 96. Guardabassi, A., M. Olivero, E. Campantico, M.T. Renaudo, C. Giunta and R. Bruno, 1970. On the early appearance of arginase activity in the liver of Bufo bufo Larvae after prolactin treatment. Gen. Comp. Endocrinol. 14:148-151. Guillemin, R., E. Yamazaki, M. Jutisz and E. Sakiz, 1962. Presence dans un extrait de tissue hypothalamiques d'une substance stimulant 1a secretion de 1'hormone hypophysaire thyrotrope. Compt. Rend. 25:1018-1020. Hafiez, A.A., J.E. Philpatt and A. Bartke, 1971. The role of pro- lactin in the regulation of testicular function: the effect of prolactin and luteinizing hormone on 3B-hydroxysteroid dehydrogenase activity in the testis of mice and rats. J, Endocrinol. 50:619-623. Hafiez, A.A., C.H. Lloyd and A. Bartke, 1972. The role of prolactin in the regulation of testis function: the effects of prolactin and luteinizing hormone on the plasma levels of testosterone and androstenedione in hypophysectomized rats. J. Endocrinol. 52:327-332. 158 Halasz, B., 1969. The endocrine effects of isolation of the hypo- thalamus from the rest of the brain. In; Frontiers In Neuroendocrinology 1969, edited by M.E. Ganong and L. Martini, 307-343. Oxford University Press, London. Hammond, J.M., L. Jarett, I.K. Maiz and H.H. Daughaday, 1972. Heterogeneity of insulin receptors on fat cell membranes. Biochem. Biophys. Res. Comm. 49:1122-1128. Han, S.S., H.J. Rajaniemi, M.I. Cho, A.N. Hirshfield and A.R. Midgley, Jr., 1974. Gonadotropin receptors in rat ovarian tissue. 11. Subcellular localization of LH binding sites by election microscopic radioautography. Endocrinol. 95:589-598. Haour, F. and 8.8. Saxena, 1974. Characterization and solubilization of gonadotropin receptor of bovine corpus luteum. J. Biol. Chem. 249:2195-2205. Harris, G.H., 1955. Neural Control of the Pituitary Gland. Arnold, London. Harris, B.W. and D. Jacobsohn, 1952. Functional grafts of the an- terior pituitary gland. Proc. Roy. Soc. (London) Ser. B. 139: 263-279. Hashimota, I. and H.G. Niest, 1969. Luteotrophic and luteolytic mechanisms in rat corpora lutea. Endocrinol. 84:886-892. Heitzman, R.J., 1968. The hormonal induction of glucose 6-phosphate metabolizing enzymes in the mammary gland of the rabbit. J. Endocrinol. 41:xvi-xvii. Hilliard, J. and C.H. Sawyer, 1966. Effect of prolactin on steroid- ogenesis and cholesterol storage in rabbit ovary. Excerpta Medica, Inter. Congress Hormonal Steroids, abst. 335, 195. Hilliard, J., H.G. Spies, L. Lucas and C.H. Sawyer, 1968. Effect of prolactin release and cholesterol storage by rabbit ovarian interstitium. Endocrinol. 82:122-131. Hintz, R.L., L.E. Underwood, D.R. Clemmons, S.J. Voina, R.H. Marshall and J.J. Van Nyk, 1974. Separate receptors for insulin and somatomedin in skeletal and non-skeletal tissue. Fifty-sixth Meeting, Endocrine Society, Abst. 34:A-72. Hirano, T., S. Hayashi and S. Utida, 1973. Stimulatory effect of prolactin on incorporation of [3H] thymidine into the urinary bladder of the flounder (Kareius Bicoloratus). J. Endocrinol. 56:591-597. Hixon, J.E. and M.T. Clegg, 1969. Influence of the pituitary on ovarian progesterone output in the ewe: effects of hypophysec- tomy and gonadotropic hormones. Endocrinol. 84:828-834. 159 Horrobin, D.F., I.J. Lloyd, A. Lipton, P.G. Burstyn, N. Durkin and K.L. Muiruri, 1971. Actions of prolactin on human renal func- tion. Lancet 2:352-354. Horrobin, D.F., M.S. Manku and P.G. Burstyn, 1973. Saluretic action of aldosterone in the presence of excess cortisol: restoration of salt-retaining action by prolactin. J. Endocrinol. 56: 343-344. House, P.D.R. and M.J.W. Weideman, 1970. Characterization of an [125IJ-insu1in binding plasma membrane fraction from rat liver. Biochem. Biophys. Res. Comm. 41:541-548. Houssay, B.A., A. Biasotti and R. Sammartino, 1935. Modifications fonctionelles de 1'hypophyse apres les lesions infundibulo- tuberiemes ches 1e crapaud. Compt. Rend. Soc. Biol. 120: 725-726. Houssay, B.A. and J.C. Penhas, 1956. Diabetogenic action of pitui- tary hormones on adrenalectomized hypophysectomized dogs. Endocrinol. 59:637-641. Hwang, P., H.J. Guyda and H.G. Friesen, 1971. Human prolactin (HPr): purification and clinical studies. Clin. Res. 19:772. Igarashi, M. and S.M. McCann, 1964. A hypothalamic follicle stimu- lating hormone-releasing factor. Endocrinol. 74:446-452. Ingavarsson, C.G., 1969. The action of prolactin on the adreno- cortical function. Acta Rheum. Scand. 15:18-20. Jacobs, L.S., C.A. Birge, C. Hammer and W.H. Daughaday, 1968. The effect of epinephrine on synthesis and release of rat pitui- tary growth hormone and prolactin jn_vitro. Clin. Res. 16: 441. Jacobs, L.S., P.J. Snyder, J.F. Wilber, R.D. Utiger and W.H. Daughaday, 1971. Increased serum prolactin after administra- tion of synthetic thyrotropin releasing hormone (TRH) in man. J. Clin. Endocr. 33:996-998. Jacobson, A., H.A. Salhanick and M.X. Zarrow, 1950. Induction of pseudopregnancy and its inhibition by various drugs. Am. J. Physiol. 161:522-527. Josimovich, J.B., G. Weiss, and D.L. Hutchinson, 1974. Sources and disposition of pituitary prolactin in maternal circulation, amniotic fluid, fetus and placenta in the pregnant Rhesus monkey. Endocrinol. 94:1364-1371. Kahn, C.R., D.M. Neville and J. Rath, 1973. Insulin-receptor inter- action in the obese-hyperglycemic mouse. J. Biol. Chem. 248:244-250. 160 Kahn, C.R., P. Freychet and J. Roth, 1974. Quantitative aspects of the insulin-receptor interaction in liver plasma membranes. J. Biol. Chem. 249:2249-2257. Kalra, P.S., C.P. Fawcett, L. Krulich, and S.M. McCann, 1973. The effects of gonadal steroids on plasma gonadotropins and pro- lactin in the rat. Endocrinol. 92:1256-1268. Kamberi, I.A., R.S. Mical and J.C. Porter, 1970. Effect of anterior pituitary perfusion and intraventricular injection of cate- cholamines and indoleamines on LH release. Endocrinol. 87: 1-12. Kamberi, I.A., R.S. Mical and J.C. Porter, 1971. Effects of mela- tonin and serotonin on the release of FSH and prolactin. Endocrinol. 88:1288-1293. Kamberi, I.A. and F.S. Bacleon, 1973. Role of cholinergic synapses in neutral circuits controlling the gonadotropin secretion. Fifty-Fifth Annual Meeting, The Endocrine Society. Chicago, 111. June 20-23. Abst. 175, p. A136. Kanematsu, S., J. Hilliard and C.H. Sawyer, 1963. Effect of reser- pine and chlorpromazine on pituitary prolactin content and its hypothalamic site of action in rabbits. Acta Endocrinol. 44:467-474. Kastin, A.J. and A.V. Schally, 1966. MSH activity in pituitaries of rats treated with hypothalamic extracts. Gen. Comp. Endocrinol. 7:452-456. Kelly, P.A., K.N. Bedirian, R.D. Baker, and H.G. Friesen, 1973a. Effect of synthetic TRH on serum prolactin, TSH and milk pro- duction in the cow. Endocrinol. 92:1289-1293. Kelly, P.A., R.P.C. Shiu, M.C. Robertson and H.G. Friesen, 1973b. Studies of rat chorionic mammotropin by radioreceptor assay. Fed. Proc. 32: abst. 213. Kelly, P.A., C. Bradley, R.P.C. Shiu, J. Meites and H.G. Friesen, 1974a. Prolactin binding to rat mammary tumor tissue. Proc. Soc. Exp. Biol. Med. 146:816-824. Kelly, P.A., B.I. Posner, T. Tsushima and H.G. Friesen, 1974b. Studies of insulin, growth hormone and prolactin binding: ontogenesis, effect of sex and pregnancy. Endocrinol. 95: 532-639. Kilpatrick, R., D.T. Armstrong and R.D. Greep, 1964. Maintenance of the corpus luteum by gonadotrophins in the hypophysectomized rabbit. Endocrinol. 74:453-461. 161 Knobil, E., 1959. Discussion of paper by Knobil, E. and R.D. Greep: The physiology of growth hormone with particular reference to its action in the rhesus monkey and the “spies specificity" problem. Rec. Progr. Horm. Res. 15:106. Koch, Y., K.H. Lu and J. Meites, 1970. Biphasic effects of cate- cholamines on pituitary prolactin release jn_vitro. Endo- crinol. 87:673-675. Kragt, C.L. and J. Meites, 1965. Stimulation of pigeon pituitary prolactin release by pigeon hypothalamic extract jn_vitro. Endocrinol. 76:1169-1176. Kragt, C.L. and J. Meites, 1967. Dose-response relationships between hypothalamic PIF and prolactin release by rat pituitary tissue in vitro. Endocrinol. 80:1170-1173. Kragt, C.L. and J.F. Maskin, 1972. Puberty-physiological mechanisms of control. J. Anim. Sci. 34:Suppl. I, 1-15. Krug, U., F. Krug and P. Cuatrecasas, 1972. Emergence of insulin receptors on human lymphocytes during jn_vitro transformation. Proc. Nat. Acad. Sci. 69:2604-2608. Krulich, L., A.P.S. Dhariwal and S.M. McCann, 1968. Stimulatory and inhibitory effects of purified hypothalamic extracts of growth hormone-release from rat pituitary 1n_vitro. Endocrinol. 83: 783-790. Krulich, L., M. Quijada and P. Illnu, 1971. Localization of pro- lactin-inhibiting factor, p-releasing factor (PRF), growth hormone-RF (GRF) and GIF activities in the hypothalamus of the rat. Program 53rd Meeting, Endocrine Society, San Fran- cisco, Calif. P. 83. Kuroshima, A., A. Arimura, C.Y. Bowers and A.V. Schally, 1966. Inhibition by pig hypothalamic extracts of depletion of pitui- tary prolactin in rats following cervical stimulation. Endocrinol. 78:216-217. Lam, P. and I. Rothchild, 1973. Absence of the luteolytic effect of prolactin in the pregnant rat after hypophysectomy and hysterectomy on day 12. J. Endocrinol. 56:609-610. Lamprecht, S.A., H.R. Lindner and J.F. Strauss III, 1969. Induction of ZOK-hydroxysteroid dehydrogenase in rat corpora lutea by pharmacological blockade of pituitary prolactin secretion. Biochim. Biophys. Acta. 187:133-143. Leathem, J.H., 1961. Nutritional effects on endocrine secretions. In: Sex and Internal Secretions, Vol. I, edited by W.C. Young. PP. 666-704. The Williams and Wilkins Co., Baltimore, Maryland. 162 Lee, C.Y. and R.J. Ryan, 1972. Luteinizing hormone receptors: specific binding of human luteinizing hormone to homogenates of luteinized rat ovaries. Proc. Nat. Acad. Sci. 69:3520-3523. Lee, C.Y. and R.J. Ryan, 1973. Luteinizing hormone receptors in luteinized rat ovaries. In: Receptors for Reproductive Hormones, edited by B.W. DTMalley and A.R. Means. PP. 419- 430. Plenum Press, New York. Lefkowitz, R.J., I. Patson, and J. Roth, 1969. In; The role of adenylcyclase and cyclic 3;5'-AMP in biological systems, edited by I.W. Rall, M. Rodbell and P. Condliffe. PP. 88-95. NIH Fogarty International Center Proceedings No. 4. Bethesda, Maryland. Lefkowitz, R.J., J. Roth, W. Pricer and I. Pastan, 1970a. A TH receptors in the adrenal: specific binding of ACTH-12 I and its relation to adenyl cyclase. Proc. Natl. Acad. Sci. 65: 745-752. Lefkowitz, R.J., J. Roth and I. Pastan, l970b. Radioreceptor assay of adrenocorticotropic hormone: new approach to assay of polypeptide hormones in plasma. Science 170:633-635. Lefkowitz, R.J., J. Roth and I. Pastan, 1971. ACTH-receptor inter- action in the adrenal: a model for the initial step in the action of hormones that stimulate adenyl cyclase. Ann. N.Y. Acad. Sci. 185:195-209. Lesniak, M.A., J. Roth, P. Gorden and J.R. Gavin, III, 1973. Human growth hormone radioreceptor assay using cultured human lymphocytes. Nature (New Biol.) 241:20-22. Leung, 8.5. and C.H. Sasaki, 1973. Prolactin and progesterone effect on specific estradiol binding in uterine and mammary tissues jn_vitro. Biochem. Biophys. Res. Comm. 55:1180-1187. Licht, P., 1967. Interaction of prolactin and gonadotropins on appetite, growth and tail regeneration in the lizard, Anolis carolinensis. Gen. Comp. Endocrinol. 9:49-63. Licht, P. and R.E. Jones, 1967. Effects of exogenous prolactin on reproduction and growth in adult males of the Lizard Anolis carolinesis. Gen. Comp. Endocrinol. 8:228-244. Licht, P. and H. Hoyer, 1968. Somatotropic effects of exogenous prolactin and growth hormone in juvenile lizards. Gen. Comp. Endocrinol. 11:338-346. Lin, S.Y. and T.L. Goodfriend, 1970. Angiotensin receptors. Am. J. Physiol. 218:1319-1328. 163 Litwack, G. and S. Singer, 1972. Subcellular actions of glucocorti- coids. In: Biochemical Actions of Hormones, Vol. II, edited by G. Litwack, pp. 114-165. Academic Press, New York. Lis, M., C. Gilardeau, and M. Chretien, 1973. Effect of prolactin on corticosterone production by rat adrenals. Clin. Res. 21:1027. Lockett, M.F., 1965. A comparison of the direct renal actions of pituitary growth and lactogenic hormones. J. Physiol. 181: 192-199. Lockett, M.F., 1967. Hormonal effects on isolated perfused cat kidneys. Med. J. Aust. 2:298-300. Lockett, M.E. and 8. Nail, 1965. A comparative study of the renal actions of growth hormone and lactogenic hormones in the rat. J. Physiol. 180:147-156. Lowry, 0.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265. Lu, K.H., Y. Amenomori, C.L. Chen and J. Meites, 1970. Effects of central acting drugs on serum and pituitary prolactin levels in rats. Endocrinol. 87:667-672. Lu, K.H. and J. Meites, 1971. Inhibition by L-DDPA and monoamine oxidase inhibitors of pituitary prolactin release; stimulation by methyldopa and d-amphetamine. Proc. Soc. Exp. Biol. Med. 137:480-483. Lu, K. H. and J. Meites, 1972. Effects of L- -Dopa on serum prolactin and PIF in intact and hypophysectomized, pituitary-grafted rats. Endocrinol. 91: 868-872. Lu, K. H, C. J. Shaar, K. H. Kortright and J. Meites, 1972. Effects of synthetic TRH on Ln thro and Ln vao prolactin release in the rat. Endocrinol. 91:1540-1545. Lu, K.H. and J. Meites, 1973. Effects of serotonin precursors and melatonin on serum prolactin release in rats. Endocrinol. 93: 152-155. Lyons, H.R. and E. Page, 1935. Detection of mammotropin in the urine of lactating women. Proc. Soc. Exp. Biol. Med. 32:1049-1050. Lyons, W.R., C.H. Li and R.E. Johnson, 1958. The hormonal control of mammary growth and lactation. Recent Progr. Horm. Res. 14: 219-254. MacDonald, G.J. and R.D. Greep, 1968. Maintenance of progesterone secretion from rat corpora lutea. Perspect. Biol. Med. 11: 490-497. 164 MacDonald, G.J. and R.0. Greep, 1969. Prolactin-induced morphological luteal regression unaffected by LH. Proc. Soc. Exp. Biol. Med. 131:905-907. MacLeod, R.M., 1969. Influence of norepinephrine and catecholamine- depleting agents on the synthesis and release of prolactin and growth hormone. Endocrinol. 85:916-923. MacLeod, R.M., M.B. Bass, S.C. Huang and M.C. Smith, 1968. Inter- mediary metabolism in the liver and adipose tissue of rats with hormone-secreting pituitary tumors. Endocrinol. 82: 253-265. Madhwa Raj, H.G. and N.R. Moudgal, 1970. Hormonal control of gesta- tion in the intact rat. Endocrinol. 86:874-889. Malbon, C.C. and J.F. Zull, 1974. Interactions of parathyroid hormone and plasma membranes from rat kidney. Biochim. Biophys. Res. Comm. 56:952-958. Malven, P.V., 1969. Luteotrophic and luteolytic responses to pro- lactin in hypophysectomized rats. Endocrinol. 84:1224-1229. Malven, P.V. and C.H. Sawyer, 1966. A luteolytic action of prolactin in hypophysectomized rats. Endocrinol. 79:268-274. Markee, J.E., C.H. Sawyer and W.H. Hollinshead, 1948. Adrenergic control of the release of luteinizing hormone from the hypo- physis of the rabbit. Recent Progr. Hormone Res. 2:117-131. Marx, S.J., S. Fedak and 6.0. Aurback, 1972. Preparation and characterization of a hormone-responsive renal plasma mem- brane fraction. J. Biol. Chem. 247:6913-6918. Matsuo, H., Y. Baba, R.M.G. Nair, A. Arimura and A.V. Schally, 1971. Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem. Biophys. Res. Comm. 43:1334—1339. Matthews, B.F., 1963. Effects of hormones, placental extracts and hypophysectomy on insulin and para-amino-hippurate clearance in the anaesthetized rat. J. Physiol. 165:1-9. McAtee, J. W. and A. Trenkle, 1971. Effects of feeding, fasting, glucose or arginine on plasma prolactin levels in the bovine. Endocrinol. 89:730-734. McCann, S, 1971. Mechanism of action of hypothalamic-hypophyseal stimulating and inhibiting hormones. Ln: Frontiers In Neuro- endocrinology 1971. Edited by L. Mart1ni and W. F. Ganong. PP. 209-236. Oxford University Press, London. 165 McCann, S.M., S. Taleisnik and H.M. Friedman, l960. LH releasing activity in hypothalamic extracts. Proc. Soc. Exp. Biol. Med. l04z432-434. McDermott, W.V., E.S. Fry, J.R. Brobeck and C.N.H. Long, 1951. Mechanism of control of adrenocorticotrophic hormone. Yale J. Biol. Med. 23:52-65. McGarry, E.E., D. Rubinstein and J.C. Beck, 1968. Growth hormones and prolactins: biochemical, immunological and physiological similarities and differences. Ann. N.Y. Acad. Sci. 148:559-571. McQueen-Williams, M., 1935. Decreased mammotropin in pituitaries of thyroidectomized (Maternalized) male rats. Proc. Soc. Exp. Biol. Med. 33:406-407. Means, A.R., 1973. Specific interaction of 3H-FSH with rat testis binding sites. In; Receptors for Reproductive Hormones, edited by B.W. O'Malley and A.R. Means. PP. 43l-448. Plenum Press, New York. Means, A.R. and J. Voitukaitis, 1972. Peptide hormone "receptors": specific binding of H3-FSH to testis. Endocrinol. 90:39-46. Meier, A.H. and 0.0. Martin, l97l. Temporal synergism of corti- costerone and prolactin controlling fat storage in the white throated sparrow, zonotrichia albicollis. Gen. Comp. Endo- crinol. 17:311-318. Meier, A., D. Martin and R. MacGregor, 1971. Temporal synergism of corticosterone and prolactin controlling gonadal growth in sparrows. Science 173:1240-1242. Meites, J., l957. Induction of lactation in rabbits with reserpine. Proc. Soc. Exp, Biol. Med. 96:728-730. Meites, J., 1959a. Mammary growth and lactation. In; Reproduction in Domestic Animals, edited by H.H. Cole and P.T. Cupps, Vol. I. PP. 539-593. Academic Press, New York. Meites, J., 1959b. Induction and maintenance of mammary growth and lactation in rats with acetylcholine or epinephrine. Proc. Soc. Expt. Biol. Med. 100:750-754. Meites, J., 1962. Pharmacological control of prolactin secretion and lactation. In; Pharmacological Control of Release of Hormones Including Antidiabetic Drugs, edited by R. Guillemin. PP. 151-181. Pergamon Press, London. Meites, J., 1966. Control of mammary growth and lactation. In: Neuroendocrinology, Vol. I, edited by L. Martini and W.F. Ganong. Academic Press, New York. PP. 669-707. 166 Meites, J., l967. Control of prolactin secretion. Archives D'Anatomie Microscopigue et de Morphologie Experimentale 56:5l6-529. Meites, J., l970a. Direct studies of the secretion of the hypo- thalamic hypophysiotropic hormones (HHH). In; Hypophysio- tropic Hormones of the Hypothalamus: Assay and Chemistry, edited by J. Meites. PP. 261-278. The Williams and Wilkins Co., Baltimore, Meites, J., l970b. The relation of estrogen and prolactin to mammary tumorigenesis in the rat. In; Estrogen Target Tissues and Neoplasia, edited by T.L. Dao. PP. 275-286. University of Chicago Press, Chicago. Meites, J., 1973. Control of prolactin secretion in animals. In; Human Prolactin, edited by J.L. Pasteels and C. Robyn. PP. l05-ll8. Excerpta Medica, Amsterdam. Meites, J. and C.W. Turner, l947. Effect of thiouracil and estrogen on lactogenic hormone and weight of pituitaries of rats. Proc. Soc. Exp. Biol. Med. 64:488-492. Meites, J. and J. Sgouris, 1953. Can the ovarian hormones inhibit the mammary responses to prolactin? Endocrinol. 53:]7-21. Meites, J. and M.C. Shelesnyak, 1957. Effects of prolactin on dura- tion of pregnancy, viability of young and lactation in rats. Proc. Soc. Exp. Biol. Med. 94:746-749. Meites, J., P.K. Talwalker and C.S. Nicoll, l960a. Initiation of lactation in rats with hypothalamic or cerebral tissue. Proc. Soc. Exp. Biol. Med. 103:298-300. Meites, J., C.S. Nicoll, P.K. Talwalker and T.F. Hopkins, l960b. Induction and maintenance of mammary growth and lactation by neurohormones, drugs, non-specific stresses and hypo- thalamic tissue. Acta Endocrinol. Suppl. 51:1l37. Meites, J. and T.F. Hopkins, l96l. Mechanisms of oxytocin action in retarding mammary involutions: study of hypophysectomized rats. J. Endocrinol. 22:207-213. Meites, J., R.H. Kahn and C.S. Nicoll, l96l. Prolactin production by rat pituitary in vitro. Proc. Soc. Exp. Biol. Med. 108: 440-443. Meites, J., C.S. Nicoll and P.K. Talwalker, 1963. The cnetral nervous system and the secretion and release of prolactin. In; Advances in Neuroendocrinology, edited by A.V. Nalbandov. PP. 238-277. University of Illinois Press, Urbana. 167 Meites, J. and C.S. Nicoll, 1966. Adenohypophysis: Prolactin. Ann. Rev. Physiol. 28:57-88. Meites, J., K.H. Lu, W. Wuttke, C.W. Welsch, H. Nagasawa and S.K. Quadri, 1972. Recent studies on functions and control of prolactin secretion in rats. Recent Progr. Hormone Res. 28: 471-516. Midgley, A.R. Jr., 1973. Autoradiographic analysis of gonadotropin binding to rat ovarian tissue sections. In; Receptors for Reproductive Hormones, edited by B.W. O'Malley and A.R. Means. PP. 365-378. Plenum Press, New York. Milkovic, S., M. Garrison and R. Bates, 1964. Study of the hormonal control of body and organ size in rats with mammotropic tumors. Endocrinol. 75:670-679. Miller, R.A. and Riddle, 0., 1943. Ability of adrenal cortical hormones, prolactin and thyroxin to sustain weight of body and viscera of hypophysectomized pigeons. Endocrinol. 32: 463-474. Mills, E.S. and Y.J. Topper, 1970. Some ultrastructural effects of insulin, hydrocortisone and prolactin on mammary gland explants. J. Cell. Biol. 44:310-328. Minaguchi, H. and J. Meites, l967a. Effects of suckling on hypo- thalamic LH-releasing factor and prolactin-inhibiting factor, and on pituitary LH and prolactin. Endocrinol. 80:603-607. Minaguchi, H. and J. Meites, 1967b. Effects of a norethynodrel- mestranol combination (Enovid) on hypothalamic and pituitary hormones in rats. Endocrinology 81:826-834. Mishkinsky, J., K. Khazan and F.G. Sulman, 1968. Prolactin re- leasing activity of the hypothalamus of post-partum rats. Endocrinol. 82:611-613. Mishkinsky, J.S., Y. Givant, F.G. Sulman, A. Eshkol and B. Lunenfeld, 1972. Uptake of 125I-labelled prolactin by rat mammary gland and pigeon crop mucosa. J. Endocrinol. 52:387-396. Mittler, J.C. and J. Meites, 1964. “In vitro stimulation of pitui- tary follicle-stimulating hormone release by hypothalamic extract. Proc. Soc. Exp. Biol. Med. 117:309-313. Mittler, J.C. and J. Meites, 1967. Effects of epinephrine and acetylcholine on hypothalamic content of prolactin-inhibiting factor. Proc. Soc. Exp. Biol. Med. 124:310-311. 168 Mizuno, H., P.K. Talwalker and J. Meites, 1964. Inhibition of mammary secretion in rats by iproniazid. Proc. Soc. Exp,_Biol. Meg, 115:604-607. Moger, W.H. and 1.1. Geschwind, 1972. The action of prolactin on the sex accessory glands of the male rat. Proc. Soc. Exp. Biol. Med. 141:1017-1021. Mohrenweiser, H.W. and R.S. Emery, l973. Hormonal control of pre- cursor pools, ribonucleic acid synthesis and cellular mor- phology in mammary tissue pieces during lactogenesis. J, Dairy Sci. 56:436-445. Moore, R.0. and E.C. Ball, 1962. Studies on the metabolism of adipose tissue: some jn_vitro effects of a prolactin prepara- tion alone and in combination with insulin or adrenalin. Endocrinol. 71:57-67. Morishige, W.K. and J.H. Leathem, 1973. Effect of adrenalectomy on corticosterone maintenance of pregnancy in dietary protein deprivation: influence on hypophysial and serum prolactin. Fertil. Steril. 24:527-533. Morishige, W.K., G.J. Pepe and I. Rothchild, 1973. Serum luteinizing hormone, prolactin and progesterone levels during pregnancy in the rat. Endocrinol. 93:1527-1530. Morishige, W.K. and I. Rothchild, 1974. Temporal aspects of the regulation of corpus luteum function by luteinizing hormone, prolactin and placental luteotrophin during the first half of pregnancy in the rat. Endocrinol. 95:260-274. Mueller, G.P., H.J. Chen and J. Meites, l973. Ig_vivo stimulation of prolactin release in the rat by synthetic TRH. Proc. Soc. Exp. Biol. Med. 144:613-615. Mueller, G.P., H.T. Chen, J.A. Dibbet, H.J. Chen and J. Meites, 1974. Effects of warm and cold temperature on release of TSH, GH and prolactin in rats. Proc. Soc. Exp. Biol. Med., in press. Musto, N., A.A. Hafiez and A. Bartke, 1972. Prolactin increases 17 B-hydroxy-steroid dehydrogenase activity in the testis. Endocrinol. 91:1106-1112. Nagasawa, H., C.L. Chen and J. Meites, 1969. Effects of estrogen implant in median eminence on serum and pituitary prolactin levels in the rat. Proc. Soc. Exp. Biol. Med. 132:859-861. Nair, R.M.G., A.J. Kastin, and A.V. Schally, 1971. Isolation and structure of hypothalamic MSH Release-Inhibiting Hormone. Biochem. Biophys. Res. Comm. 43:1376-1381. 169 Negro-Vilar, A. and W.A. Saad, 1972. Influence of prolactin-secreting pituitary homografts on male accessory organs. IV International Congress of Endocrinology, Washington D.C., June 1972. P. 73, #184. Negro-Vilar, A., L. Krulich and S.M. McCann, 1973. Changes in serum prolactin and gonadotropins during sexual development of the male rat. Endocrinol. 93:660-664. Neill, J.D. and M.S. Smith, 1974. Pituitary-ovarian interrelation- ships in the rat. In: Current Topics in Experimental Endo- crinology, edited by V.H.T. James and L. Martini, pp. 73-106. Academic Press, New York. Nejad, M.S., I.L. Charkoff and R. Hill, 1962. Hormonal repair of defective lipogenesis from glucose in the liver of the hypo- physectomized rat. Endocrinol. 71:107-112. Netter, F.H., 1968. The Hypothalamus, suppl. to Vol. I. Nervous System,_The Ciba Collection of Medical Illustrations. Ciba Pharmacentical Products, Inc., Summit, M.J. Nicoll, C.S., 1965. Neural regulation of adenohypophyseal pro- lactin secretion in tetrapods: indication from in vitro studies. J. Exp. Zool. 158:203—210. Nicoll, C.S. and J. Meites, 1963. Prolactin secretion jg_vitro: effects of thyroid hormones and insulin. Endocrinol. 72: 544-551. Nicoll, C.S. and J. Meites, 1964. Prolactin secretion in vitro: effects of gonadal and adrenal cortical steroids. Proc. Soc. Exp. Biol. Med. 117:579-583. Nicoll, C.S., R.P. Fiorindo, C.T. McKennee and J.A. Parsons, 1970. Assay of hypothalamic factor which regulate prolactin secre- tion. In; Hypophysiotropic Hormones of the Hypothalamus: Assay and Chemistry, edited by J. Meites. PP. 115-144. The Williams and Wilkins Co., Baltimore. Nicoll, C.S. and H.A. Bern, 1972. On the actions of prolactin among the vertebrates: is there a common denominator? In; Lactogenic Hormones, edited by C.E.W. Wolstenholme and J. Knight, pp. 299-324. Churchill Livingstone, London. Nikitovitch-Winer, M.B. and J.W. Everett, 1958. Functional restitution of pituitary grafts re-transplanted from kidney to median eminence. Endocrinology 63:916-930. Nilsson, A., T. Nilsson and A. Norgren, 1970. Studies on mammary respiratory metabolism and mammary development after thyroid- ectomy in the rabbit. Acta Physiol. Scand. 80:4A. 170 Niswender, G.D., C.L. Chen, A.R. Midgley, Jr., J. Meites and S. Ellis, 1969. Radioimmunoassay for rat prolactin. Proc. Soc. Exp. Biol. Med. 130:793-797. Ojeda, S.R., F.G. Haims and S.M. McCann, 1974. Sites and mechanism of action of dopamine in controlling prolactin release. Egg, Proc. abst. 193. Pallmore, W.P., R. Anderson and P.J. Muliow, 1970. Role of the pituitary in controlling aldosterone production in sodium- depleted rats. Endocrinol. 86:728-734. Pasteels, J.L., l96l. Sécrétion de prolactin par l'hyposphyse en culture de tissues. Compt. Rend. Soc. Biol. 253:2140-2142. Pasteels, J, 1963. Administration d' extracts hypothalamiques a 1' hypophyse de Rat in vitro, dans le but d' en contraler la secretion de prolactine. Compt. Rend. 254: 2664- 2666. Peaker, M., J.G. Phillips and A. Wright, 1970. The effect of pro- lactin on the secretory activity of the nasal salt-gland of the domestic duck. J. Endocrinol. 47:123-127. Piezzi, R.S., F. Larin, and R.J. Wurtman, 1970. Serotonin, 5- hydroxy-indoleacetic acid (5-HIAA) and monoamine oxidase in the bovine median eminence and pituitary gland. Endocrinol. 86:1460-1462. Pickford, B.E. and J.G. Phillips, 1959. Prolactin, a factor in promoting survival of hypophysectomized killfish in fresh water. Science 139:454-455. Piva, P., P. Gagliano, M. Motta and L. Martini, 1973. Adrenal progesterone: factors controlling its secretion. Endocrinol. 93:1178-1184. Popa, G.T. and U. Fielding, 1930. A portal circulation from the pituitary to the hypothalamus. J. Anat. (London) 65:88-91. Posner, B.I., P.A. Kelly, R.P.C. Shiu and H.G. Friesen, 1974. Studies of insulin, growth hormone and prolactin binding: tissue distribution, species variation and characterization. Endocrinol. 95:521-531. Ramirez, V.D., l973. Endocrinology of puberty. In: Handbook of Physiology. Endocrinology, Vol. II, edited by R. 0. Greep and E.B. Astwood. PP. 1-28. American Physiological Society, Washington, D.C. Ranjaniemi, H.J., A.N. Hirshfield and A.R. Midgley, Jr., 1974a. Gonadotropin receptors in rat ovarian tissue. I. Localiza- tion of LH binding sites by fractionation of subcellular organelles. Endocrinol. 95:579-588. 171 Rajaniemi, H., A.0ksanen and T. Vanha-Perttula, 1974b. Distribution of 1251-pro1actin in mice and rats. Studies with whole body micro-autoradiography. Horm. Res. 5:6-20. Rathgeb, I., B. Winkler, R. Steele and N.Altszuler, 1971. Effect of ovine prolactin administration on glucose metabolism and plasma insulin levels in the dog. Endocrinol. 88:718-722. Ratner, A. and J. Meites, 1964. Depletion of prolactin-inhibiting activity of rat hypothalamus by estradiol or suckling stimu- lus. Endocrinol. 75:377-382. Ratner, A., P.K. Talwalker and J. Meites, 1965. Effect of reserpine on prolactin-inhibiting activity of rat hypothalamus. Endocrinol. 77:315-319. Reichert, L.E. Jr. and V.K. Bhalla, 1974. A comparison of the properties of FSH from several species as determined by a rat testis tubule receptor assay. Gen. Comp. Endocrinol. 23: 111-117. Reichlin, S. and M. Mitnick, 1973. Biosynthesis of hypothalamic hypophysiotropic factors. In; Frontiers In Neuroendocrino- logy 1973, edited by W.F. Ganong and L. Martini. PP. 61-88. Oxford University Press, London. Relkin, R., 1973. Effect of sodium deprivation and pinealectomy on pituitary and plasma prolactin in the rat. J. Endocrinol. 59:383-384. Relkin, R., 1974. Effects of alterations in serum osmolality on pituitary and plasma prolactin levels in the rat. Neuro- endocrinology 14:61-64. Relkin, R. and M. Adachi, 1973. Effects of sodium deprivation on pituitary and plasma prolactin, growth hormone and thyrotropin levels in the rat. Neuroendocrinology_11:240-247. Richardson, B.P., 1973. Evidence for a physiological role of pro- lactin in osmoregulation in the rat after its inhibition by 2-bromo-0L-ergokryptine. Br. J. Pharmacol. 47:623-624. Riddle, 0., 1963. Prolactin in vertebrate function and organization. J. Nat. Cancer Inst. 31:1039-1110. Riddle, 0. and P.F. Braucher, 1931. Studies on physiology of re- production in birds; control of special secretion of the crop-gland in pigeons by an anterior pituitary hormone. Am. J. Physiol. 97:617-625. Riddle, 0., R.W. Bates and S.W. Dykshorn, 1932. A new hormone of the anterior pituitary. Proc. Soc. Exp. Biol. Med. 29:1211-1212. 172 Riddle, 0., R.W. Bates and S.W. Dykshorn, 1933. The preparation, identification and assay of prolactin - a hormone of the anterior pituitary. Am. J. Physiol. 105:191-216. Rinne, U.K. and V. Sonninen, 1968. The occurrence of dopamine and norepinephrine in the tubero-hypophyseal system. Experientia 24:177-178. Rivera, E.M., 1964. Interchangeability of adrenocrotical hormones in initiating mammary secretion j§_vitro. Proc. Soc. Exp. Biol. Med. 116:568-572. Robdell, M., H.M.J. Krans, S.L. Pohl and L. Birnbaumer, 1971. The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. J. Biol. Chem. 246:1861-1871. Robson, J.M., F.M. Sullivan and C. Wilson, 1971. The maintenance of pregnancy during the pre-implantation period in mice treated with phenelzine derivatives. J. Endocrinol. 49:635-648. Rodbard, D., 1973. Mathematics of hormone-receptor interaction. I. Basic principles. In; Receptors for Reproductive Hormones, edited by B.W. O'Malley and A.R. Means. PP. 289- 326. Plenum Press, New York. Romanoff, E.B., M. Inaba, 0. Watson, E. Scricco and G. Pincus, 1966. Biosynthesis of steroids in bovine ovaries perfused i_n vitro. Excerpta Medica, Inter. Congress Hormonal Steroids, abst. 475, 260-261. Saffran, M. and A.V. Schally, 1955. Release of corticotrophin by anterior pituitary tissue jg_vitro. Can. J. Biochem. Physiol. 33:408-415. Saito, M., A. Arimura, S. Sawano and A.V. Schally, 1970. Luteo- trophic and luteolytic effects of prolactin in hypophy- sectomized rats. Endokrinologie 56:129-139. Samli, M.H. and R.M. MacLeod, 1974. Interaction of thyrotropin releasing hormone and dopamine on the secretion of prolactin and 4C-glucose oxidation by rat anterior pituitary incu- bated jg_vitro. Fed. Proc., abst. 195. Sar, M. and J. Meites, 1968. Effects of progesterone, testosterone, and cortisol on hypothalamic prolactin-inhibiting factor and pituitary prolactin content. Proc. Soc. Exp, Biol. Med. 127: 426-429. Sawyer, C.H., 1963. Discussion of paper by Munson, P.L.: Pharma- cology of neuroendocrine blocking agents. In: Advances in Neuroendocrinology, edited by A.V. Nalbandov. PP. 444-459. University of Illinois Press, Urbana. 173 Sawyer, C.H., J.E. Markee and B.F. Townsen, 1949. Cholinergic and adrenergic components in the neurohumoral control of the release of LH in the rabbit. Endocrinol. 44:18-37. Saxena, B.B., S.H. Hasan, F. Haour and M. Schmidt-Gollwitzer, 1974. Radioreceptor assay of human chorionic gonadotropin: detec- tion of early pregnancy. Science 184:793-795. Schally, A.V., A. Arimura and A.J. Kastin, 1973. Hypothalamic Regulatory Hormones. Science 179:341-350. Schneider, H.P.G. and S.M. McCann, 1970. Mono- and indoleamines and control of LH secretion. Endocrinol. 86:1127-1133. Schooley, J.P., 0. Riddle and R.W. Bates, 1941. Replacement therapy in hypophysectomized juvenile pigeons. Am. J. Anat. 69:123- 158. Schreiber, V., 1963. Hypothalamo-hypophyseal system. Czech. Acad. Sci. Prague, 187-276. Schreibman, M.P. and K.D. Kallman, 1966. Endocrine control of freshwater tolerance in teleosts. Gen. Comp. Endocrinol. 6: 144-155. Schwartz, M.D. and C.E. McCormack, 1972. Reproduction: gonadal function and its regulation. Ann. Rev. Physiol. 34:425-472. Schwartz, N.B., C.H. Anderson, L.G. Nequin and C.A. Ely, 1974. Follicular Maturation. In; The Control of the Onset of Puberty, edited by M.M. Grumbach, G.D. Grave and F.E. Mayer. PP. 367-385. John Wiley and Sons, New York. Shaar, C.J., E.B. Smalstig and J.A. Clemens, 1973. The effect of catecholamines, apo-morphine, and monoamine oxidase on rat anterior pituitary prolactin release 19_vitro. Fall Meetings of American Society for Pharmacology and Experimental Therapeutics, abst. 562, p. 256. Shaar, C.J. and J.L. Clemens, 1974. Effect of aluminum oxide cate- cholamine adsorption and monoamine oxidase on the inhibition of rat anterior pituitary prolactin release by hypothalamic extracts jg_vitro. Fed. Proc. abst. 192. Sharpe, R. M, M. Hartog, M. G. Ellwood and P. S. Brown, 1973. Age- dependent differences in the binding of [1311] LH by rat testis homogenates. J. Reprod. Fertil. 35: 529- 532. Sherry, W.E. and C.S. Nicoll, 1967. RNA and protein synthesis in the response of pigeon crop-sac to prolactin. Proc. Soc. Exp. Biol. Med. 126:824-829. 174 Shibusawa, K., T. Yamamoto, K. Mishi, C. Ave, S. Tomie and K. Shirota, 1959. TRF concentrations in various tissues follow- ing anterior hypothalamic lesions. Endocrinol. Japan 6: 149-152. Shiu, R.P.C., P.A. Kelly and H.G. Friesen, 1973. Radioreceptor Assay for Prolactin and Other Lactogenic Hormones. Science 180:968-970. Shiu, R.P.C. and H.G. Friesen, 1974. Solubilization and purification of a prolactin receptor from rabbit mammary gland. The Fifty-Sixth Meeting of Endocrine Society, abst. 168, A-l39. Shute, C.C.D., 1969. Distribution of choline esterase and choliner- gic pathways. In; The Hypothalamus. edited by L. Martini, M. Motta, and F. Fraschini. Academic Press, N.Y. PP. 167- 79. Singh, D.V. and H.A. Bern, 1969. Interaction between prolactin and thyroxine in mouse mammary gland lobulo-alveolar. J. Endo- crinol. 45:579-583. Sokal, R.R. and F.J. Rohlf, 1969. Biometry. W.H. Freeman and Co., San Francisco. Soloff, M.S. and T.L. Swartz, 1974. Characterization of a proposed oxytocin receptor in the uterus of the rat and sow. J. Biol. Chem. 249:1376-1381. Spies, H.G., J. Hilliard and C.H. Sawyer, 1968. Maintenance of corpora lutea and pregnancy in hypophysectomized rabbits. Endocrinol. 83:354-367. Strong, C., R. Oils and I.A. Forsyth, 1971. The effects of prolactin on fatty acid synthesis by rabbit mammary gland 1n_vitro. J. Endocrinol. 51:xxxii-xxxiii. Szentagothai, J., Bela Flerko, Bela Mess and Bela Halasz, 1972. Hypothalamic Control of the Anterior Pituitary. Akademiai Kiado, Budapest. Takahara, J., A. Arimura and A.V. Schally, 1974. Suppression of prolactin release by a purified porcine PIF preparation and catecholamines infused into a rat hypophysial portal vessel. Endocrinol. 96:462-465. Taleisnik, S. and R. Orias, 1965. A MSH releasing factor in hypo- thalamic extracts. Amer. J. Physiol. 208:293-296. Talwalker, P.K. and J. Meites, 1961. Mammary lobulo-alveolar growth induced by anterior pituitary hormones in adreno-ovariectomized and adreno-ovariectomized-hypophysectomized rats. Proc. Soc. Exp. Biol. Med. 107:880-883. 175 Talwalker, P.K., A. Ratner and J. Meites, 1963. In vitro inhibition of pituitary prolactin synthesis and release by hypothalamic extract. Am. J. Physiol. 205:213-218. Tashjian, A.H., Jr., M.J. Barowsky and D.K. Jensen, 1971. Thyro- tropin releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture. Biochem. Biophys. Res. Comm. 43:516-523. Tassava, R.A., 1969. Survival and limb regeneration of hypophy- sectomized newts with pituitary xenografts from larval axolotls, Ambystoma Mexicanum. J. Exp. 2001. 171:451-457. Taubenhaus, M. and S. Soskin, 1941. Release of luteinizing hormone from anterior hypophysis by an acetylcholine-like substance from the hypothalamic region. Endocrinol. 29:958-964. Torok, B., 1954. Lebendbeobachtung des hypophysenk-reislaufes an hunden. Acta Morphol. Acad. Sci. Hung, 4:83-89. Tsushima, T., H.G. Friesen, T.W. Chang and M.S. Raben, 1974. Studies by radioreceptor assay (RRA) of a factor with growth hormone-like activity in incubation media of spargana of spirometra mansonoides. Fifty-Sixth Meeting, Endocrine Society, abst. 28:A-69. Tullner, W.H., 1963. Hormonal factors in the adrenal-dependent growth of the rat ventral prostate. Nat. Cancer Inst. Monogn, 12:211-224. Turker, A. and J. Meites, 1965. Induction of lactation in pregnant heifers with 9-fluoro-prednisolone acetate. J. Dairy Sci. 48: 403. Turkington, R.W., 1968. Induction of milk protein synthesis by placental lactogen and prolactin jn_vitro. Endocrinol. 82: 575-583. Turkington, R.W., 1972a. Molecular biological aspects of prolactin. In; Lactogenic Hormones, edited by B.E.W. Wolstenholme and J. Knight. PP. 111-136. Churchill Livingstone, London. Turkington, R.W., 1972b. Human prolactin. An ancient molecule provides new insights for clinical medicine. J. Medicine 53: 389-394. , Turkington, R.W., 1974. Prolactin receptors in mammary carcinoma cells. Cancer Res. 34:758-763. 176 Turkington, R.W. and W.L. Frantz, 1972. The biochemical action of prolactin. In: Prolactin and Carcinogenesis, edited by A.R. Boyns and K. Griffiths. PP. 39-53. Alpha Omega Alpha Publishing, Wales. Turkington, R.W., W.L. Frantz and G.C. Majumder. l973. Effector- receptor relations in the action of prolactin. In; Human Prolactin. edited by J.L. Pasteels and C. Robyn. PP. 24- 35. Excerpta Medica, Amsterdam. Turner, C.W., 1939. The mammary glands. In: Sex and Internal Secretions. 2nd edition E. Allen (532). Williams and Wilkins Co., Baltimore. P. 740. Turner, C.D. and J.T. Bagnara, 1971. General Endocrinology. W.B. Saunders Co., Philadelphia. Utida, S., S. Hatai, T. Hirano and F.I. Kamemato, 1971. Effect of prolactin on survival and plasma sodium levels in hypophy- sectomized medaka Oryzias latipes. Gen. Comp. Endocrinol. 16: 566-573. Vale, W., G. Grant and R. Guillemin, 1973. Chemistry of the hypo- thalamic releasing factors - studies on structure-function relationships. In; Frontiers in Neuroendocrinology 1973, edited by W.F. Ganong and L. Martini. PP. 375-414. Oxford University Press, London. Valverde, C. and V. Chieffo, l97l. Prolactin releasing factors in porcine hypothalamic extracts. Program 53rd Meeting, Endo- crine Society, San Francisco, Calif. PP. 84. Vellano, C., V. Mazzi and M. Sacerdote, 1970. Tail height, a prolactin-dependent ambisexual character in the newt (Tri- turus cristatus carnifex Laur). Gen. Comp. Endocrinol. 14: 535-541. Vogt, M., 1954. The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. J. Physiol. (London) 123:451-481. Voogt, J.L., J.A. Clemens and J. Meites, 1969. Stimulation of pituitary FSH release in immature female rats by prolactin implant in median eminence. Neuroendocrinology_4:157-163. Voogt, J.L., C.L. Chen and J. Meites, 1970. Serum and pituitary prolactin levels before, during, and after puberty in female rats. Am. J. Physiol. 218:396-399. 177 Voogt, J.L. and J. Meites, 1971. Effects of an implant of prolactin in median eminence of pseudopregnant rats on serum and pitui- tary LH, FSH and prolactin. Endocrinol. 88:286-292. Wang, D.V., R.C. Hallowes, J. Bealing, C. Strong and R. Dils, 1971. Effect of prolactin and growth hormone on fatty acid syn- thesis in mouse mammary gland explants in organ culture. 9, Endocrinol. 51:xxx-xxxi. Watson, J.T., L. Krulich, and S.M. McCann, 1971. Effect of crude rat hypothalamic extract on serum gonadotropin and prolactin levels in normal and orchidectomized male rats. Endocrinol. 89:1412-1418. Welsch, C.W., M.D. Squiers, E. Cassell, C.L. Chen and J. Meites, 1971. Median eminence, lesions and serum prolactin influence of ovariectomy and ergocornine. Am. J. Physiol. 221: 1714-1717. Williams, W.F., A.G. Weisshaar and G.E. Lauterbach, 1966. Lacto- genic hormone effects on plasma non-esterified fatty acids and blood glucose concentrations. J. Dairy,Sci. 49:106-107. Wilson, J.T., 1969. Pituitary mammotropic tumor in rats: evidence for a dosage effect on organ weight and liver drug metabolism. J. Nat. Cancer Inst. 43:1067-1072. Wilson, R.G., I. Percy-Robb, V.K. Singhal, A.P.M. Forrest, E.M. Cole, A.R. Boyns and K. Griffiths, 1972. Response of plasma prolactin and growth hormone to insulin hypoglycaemia. Lancet 2:1283-1285. Winegrad, A.I., W.N. Shaw, F.D. W. Lukens and W.C. Stadie, 1959. Effect of prolactin jn_vitro on fatty acid synthesis in rat adipose tissue. J. Biol.cChem, 234:3111-3114. Winkler, B., I. Rathgeb, R. Steele and N. Altszuler, 1971. Effect of ovine prolactin administration on free fatty acid meta- bolism in the normal dog. Endocrinology 88:1349-1352. Wislocki, 6.8. and E.L. Smith, 1936. The permeability of the hypophysis and hypothalamus to vital dyes, with a study of the hypophyseal vascular system. Am. J. Anat. 58:421-427. Witorsch, R.J. and J.I. Kitay, 1972. Pituitary hormones affecting adrenal 5*“f-reductase activity: ACTH, growth hormone and prolactin. Endocrinol. 91:764-769. Worthington, W.C., Jr., 1955. Some observations on the hypophyseal portal system in the living mouse. Bull. Johns Hopkins Hosp. 97:343-357. 178 Wuttke, W. and J. Meites, 1970. Effects of ether and pentobarbital on serum prolactin and LH levels in proestrous rats. Proc. Soc. Exp. Biol. Med. 135:648-652. Wuttke, W. and J. Meites, 1971. Luteolytic role of prolactin during the estrous cycle of the rat. Proc. Soc. Exp. Biol. Mgg, 137:988-991. Wuttke, W., M. Gelato and J. Meites, 1971a. Mechanisms of pento- barbital actions on prolactin release. Endocrinol. 89: 1191-1194. Wuttke, W., E. Cassell and J. Meites, 1971b. Effects of ergocornine on serum prolactin and LH, and on hypothalamic content of PIF and LRF. Endocrinol. 88:737-741. Yang, W.H., M.R. Sarran and C.H. Li, 1973. The effect of ICSH-B and its combination with prolactin on the maintenance of pregnancy in the rat. Acta Endocrinol. 72:173-181. Zarrow, M.X. and J.H. Clark, 1969. Gonadotropin regulation of ovarian cholesterol levels in the rat. Endocrinol. 84: 340-346. Zmigrod, A., H.R. Lindner and S.A. Lamprecht, 1972. Reductive pathways of progesterone metabolism in the rat ovary. Acta Endocrinol. 69:141-152. APPENDIX CURRICULUM VITAE AND LIST OF PUBLICATIONS NAME: DATE OF BIRTH: PLACE OF BIRTH: PRESENT ADDRESS: FUTURE ADDRESS: EDUCATION: Degree Year B.A. 1965-1969 M.S. 1969-1971 Ph.D. 1972-1975 HONORS: CURRICULUM VITAE GELATO, Marie C. Ju1y 7, 1947 New York City, New York Department of Physiology Michigan State University East Lansing, Michigan 48824 Max Planck Institute for Biophysical Chemistry Department of Neurobiology 34 Gottingen-Nikolausberg West Germany Institution Hunter College Michigan State Univ. Michigan State Univ. Major Field of Study Biol. Sciences Physiology Neuroendocrinology (l) Elected associate member of Sigma Xi, Spring, 1973. (2) Elected full member of Sigma Xi, Spring, 1974. POSITIONS HELD: (a) Post-doctoral Fellow, Max Planck Institute, January, 1975. (b) Teaching Assistant in Physiology, Michigan State University, 1970-present. (c) Instructor, Department of Physiology, Michigan State Univer- sity, Summer term, 1972. (d) Research Assistant in PhysioloQY. Michigan State University, 1970. 179 180 TALKS PRESENTED AT SCIENTIFIC MEETINGS: Meetings Year Topic 5th Annual Meeting of 1972 Inhibition of Luteolysis Society for the Study by Iproniazid during the of Reproduction Estrous Cycle in Rats RESEARCH PUBLICATIONS 1. W. Wuttke, M. Gelato, and J. Meites. Mechanisms of Pentobarbital Actions on Prolactin Release. Endocrinology 89:1191, 1971. W. Wuttke, M. Gelato, and J. Meites. Effects of Na-Pentobarbital on Hypothalamic PIF, LRF, and FSH, RF and on Serum Prolactin, LH and FSH. Brain-Endocrine Interaction. Median Eminence: Structure angzgunction. Int. Symp. Munich 1971, p. 267-79 (Karger, Basel, M. Gelato, S.K. Quadri, and J. Meites. Inhibition of Prolactin Release by a Thalidomide-Related Compound (CG 603). Proc. Soc. Exp. Biol. Med. 140:167, 1972. S. Dickerman, G. Kledzik, M. Gelato, J. Chen and J. Meites. Effects of Haloperidol on Serum and Pituitary Prolactin, LH and FSH, on Hypothalamic PIF and LRF, and on Ovulation in Rats. Neuro- endocrinology 15:10-20 L. Grandison, M. Gelato and J. Meites. Inhibition of Prolactin Secretion by Cholinergic Drugs. Proc. Soc. Exp. Biol. Med. 145: 1236-1239 M. Gelato, S. Marshall, M. Boudreau, J. Bruni, G.A. Campbell and J. Meites. Effects of Thyroid and Ovaries on Prolactin Binding Activity in Rat Liver. Endocrinology (in press). M. Gelato, S. Marshall, M. Boudreau and J. Meites. Estrogen Stimulation of Prolactin Binding Activity in the Liver of Immature and Mature Female Rats. (in preparation). M. Gelato, G. Kledzik, 5. Marshall, G.D. Riegle and J. Meites. Prolactin Binding Activity in the Ovaries during the Estrous Cycle, Pregnancy and Lactation. (in preparation). "7111111111111lefiljlfllfifllll’lfllflljlllm