; .‘w'Cu' “353“: '- . ASU tin .. ‘ ; ,é' LIBRARY Michigan State University llllllllllllllllllllllllllllllllIlllllllIlllllllllllllllllll 3 1293 10490 0893 This is to certify that the thesis entitled Hormone Control of Prolactin Receptor Activity in Male Rat Accessory Sex Organs, Carcinogen-Induced Mammary Tumors and Pigeon Crop-Sacs presented by Gary Steven Kledzik has been accepted towards fulfillment of the requirements for Ph.D. Physiology degree in Date November 11 , 1976 0-7639 ‘9' IINSINO BY ”0A6 & SONS' 800K OMEN INC. L's-aw muons spams—rout Imam! ABSTRACT HORMONE CONTROL OF PROLACTIN RECEPTOR ACTIVITY IN MALE RAT ACCESSORY SEX ORGANS, CARCINOGEN-INDUCED MAMMARY TUMORS AND PIGEON CROP-SACS BY Gary Steven Kledzik 1. Specific binding of (1251) iodoprolactin was demonstrated in particulate membrane fractions of rat ventral prostates, carcinogen-induced mammary tumors, and pigeon crop—sacs. The specific binding was time and temperature dependent. Unlabeled prolactin readily dis— placed the binding of (1251) iodoprolactin, whereas LH, FSH, TSH or GH showed no such competition. By contrast, membranes obtained from rat testis or seminal vesicles did not appreciably bind labeled prolactin. 2. Injections of testosterone propionate into intact 30 and 60 day old rats had little effect on specific prolactin binding in testes or seminal vesicles, but significantly increased prolactin binding per 200 ug of membrane protein derived from ventral prostates. There was no significant difference between the binding values of the two age groups. Gary Steven Kledzik 3. Castration reduced the specific binding of (1251) iodoprolactin in ventral prostates and injections of testosterone propionate into castrated rats increased prostatic prolactin binding. Scatchard analysis revealed that the concentration of high affinity prolactin binding sites in ventral prostates was decreased by castration and returned toward normal by testosterone injections. The dissociation constants were not significantly altered in the castrated group. 4. £g_gi££g binding of (1251) iodoprolactin was inhibited in prostatic tissue removed from intact rats 2 hours after a subcutaneous injection of unlabeled prolactin, but not in ventral prostates from rats killed 26 or 74 hours after injection. Prolactin injected together with testosterone propionate into castrated rats produced no greater increase in specific prolactin binding than testosterone alone. 5. Administration of ergocornine to intact or castrated rats did not influence the binding of labeled prolactin, and injections of 2.0 ug estradiol benzoate into castrated rats followed by 3 injections of ergocornine within 26 hours also did not significantly influence prolactin binding in the ventral prostate. However, injections of 25 ug estradiol benzoate into castrates followed by ergocornine decreased prostatic prolactin binding at a 5% level of significance. Gary Steven Kledzik 6. £2.33E59 binding of (1251) iodoprolactin was significantly reduced in membrane preparation of ventral prostates obtained 5, 15 or 30 minutes after an intra- venous injection of unlabeled prolactin. Binding of labeled prolactin to ventral prostates removed 1 hour to 8 days after an injection of unlabeled prolactin was not signifi— cantly different from control values. A single subcutaneous injection of testosterone propionate increased the binding of labeled prolactin to ventral prostates removed 3, 4 or 5 days later. The binding of labeled prolactin removed 12.5 hours to 2 days and from 6 days to 20 days after testosterone injection did not differ significantly from control values. 7. Specific prolactin binding in ventral prostates of 10 and 20 month old rats was markedly reduced as com- pared to 2% month-old rats. Serum prolactin levels were significantly greater in 20 month old rats, and serum testosterone levels significantly less in 10 and 20 month- old rats, as were found in 2% month-old rats. 8. Crop sacs from juvenile pigeons contains approximately twice as much prolactin binding activity as crOp sacs from mature pigeons. Proliferation of the crOp sac in response to prolactin is associated with an increase in binding of prolactin. Parent and prolactin injected pigeons, each with proliferated crop sac epithelium, Gary Steven Kledzik exhibited 4-5 times as much specific prolactin binding as nonproliferated crops from juvenile or mature birds. 9. A significant negative correlation was noted between administered doses of estrogen and the subsequent binding of (125 I) iodoprolactin to rat mammary tumor mem- branes. Injections of 10 or 25 ug estradiol benzoate daily for 10 days effectively inhibited mammary tumor growth and significantly reduced specific prolactin binding to mammary tumor cell membranes. HORMONE CONTROL OF PROLACTIN RECEPTOR ACTIVITY IN MALE RAT ACCESSORY SEX ORGANS, CARCINOGEN-INDUCED MAMMARY TUMORS AND PIGEON CROP-SACS BY Gary Steven Kledzik A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1976 DEDICATION Dedicated to the memory of my brother Rollie Pompili and good friend Suzy Pangborn and to my family. ii ACKNOWLEDGMENTS I wish to sincerely thank Dr. Joseph Meites for his guidance and support throughout my graduate studies. It _was indeed a honor and a pleasure for me to have been his student. I also wish to thank Drs. W. D. Collings, E. Convey, G. D. Riegle, and the members of my guidance committee: Drs. C. Welsch, H. Hafs, W. Frantz, K. Moore, R. Pittman and R. Bernard for their advice and encourage- ment. Appreciation is expressed to all my present and former colleagues eSpecially to Carol Bradley, Steve Marshall, Drs. K. Quadri, S. Dickerman, and M. Gelato. iii LIST OF TABL LIST OF FIGU INTRODUCTION REVIEW OF LI I. II. III. TABLE OF CONTENTS ES 0 O O O O O O O O 0 O O O O O O O 0 RES 0 O O O O O O O O O O O O O O O O TERATURE O O O O O O O O O O O O O O 0 Functional Neuroanatomy of Hypothalamic- Hypophyseal System . . . . . . . . A. General Anatomy of the Hypothalamus . . . . . . . . . B. Anatomy of the Pituitary Gland . . C. Hypothalamic Hypophyseal Vascular Connection . . . . . . Control of Prolactin Secretion . . . . Hypothalamic Inhibition . . . . . . Hypothalamic Stimulation . . . . . Role of Neurotransmitters . . . Prolactin Short Feedback Loop . . Exteroceptive Stimuli . . . . . . . Effects of Estrogen, Testosterone and Ergot Derivatives on Prolactin Secretion . . . . . . WMUOCD> Functions of Prolactin . . . . . . . . Mammary Gland . . . . . . . . . . Mammary Tumors . . . . . . . . . . Ovaries . . . . . . . . . . . . . Crop Sac . . . . . . . . . . . . . Male Accessory Sex Organs . . . . Osmoregulation . . . . . . . . . . Adrenals and Liver . . . . . . . . O’HMUOUIII’ iv Page viii ix 10 12 18 19 21 23 23 26 28 30 31 34 35 IV. MATERIALS AN III. IV. EXPERIMENTAL I. II. Page Peptide Hormone Binding to Receptors . . . 37 A. Hormone Labeling . . . . . . . . . . . 37 B. Receptor Preparations . . . . . . . . 38 C. Incubation of Hormone and Receptor . . . . . . . . . . . . . . 39 D. Affinity Constants and Binding Capacity . . . . . . . . . . . . . . 40 E. Prolactin Binding . . . . . . . . . . 42 F. Receptors for Other Polypep- tide Hormones . . . . . . . . . . . 49 D METHODS . . . . . . . . . . . . . . . . 52 Animals 0 O O O O O O O O O O O O O O O O 52 Prolactin Radioreceptor Assay . . . . . . 53 A. Preparation of Prolactin-Binding Subcellular Figctions . . . . . . . 53 B. Preparation of 5I-Labeled PrOlactin O O O O O O O O O O O O O 53 C. Assay Procedure . . . . . . . . . . . 54 Hormones and Drugs . . . . . . . . . . . . 55 Statistical Analysis . . . . . . . . . . . 56 O O O O I O O O O O I O O O O O O O O O O 57 Prolactin Binding Activity in Ventral Prostates, Seminal Vesicles and Testes of 40 and 70 Day Old Male Rats: Effects of Testosterone . . . . . 57 A. Objectives . . . . . . . . . . . . 57 B. Materials and Methods . . . . . . . . 57 C. Results . . . . . . . . . . . . . . . 58 D. Conclusions . . . . . . . . . . . . . 62 Effects of Castration, Testosterone, Estradiol and Prolactin on Specific Prolactin Binding Activity in Ventral Prostates of Male Rats . . . . . 64 A. Objectives . . . . . . . . . . . . . 64 B. Materials and Methods . . . . . . . . 65 C. Results . . . . . . . . . . . . . . . 67 D. Conclusions . . . . . . . . . . . . . 73 III. IV. VI. VII. Effects of Ergocornine Injections on Specific Prolactin Binding Activity Rats in Ventral Prostates of Male Objectives . . . . . . . Materials and Methods . Results . . . . . . . . Conclusions . . . . . . UOCD> Time Course Effects of Single Injec- tions of Testosterone or Prolactin on Prolactin Binding Activity in Ventral Prostates of Intact Rats Objectives . . . . . . . Materials and Methods Results . . . . . . . . . Conclusions . . . . . . cow» Serum Prolactin, Testosterone and Prolactin Receptors in Ventral Prostates of Aging Male Rats . Objectives . . . . . Materials and Methods . Results . . . . . . . . . Conclusions . . . . . . coma, Prolactin Binding Activity in the Crop Sacs of Juvenile, Mature, Parent and Prolactin Injected Pigeons O O O O O O O O I A. Objectives . . . . . B. Materials and Methods . C. Results . . . . . . . . D. Conclusions . . . . . . Effects of High Doses of Estrogen on Prolactin Binding Activity and Growth Growth of Carcinogen Induced Mammary Cancers in Rats . Objectives . . . . . . Materials and Methods . Results . . . . . . . . . Conclusions . . . . . . COCOS, vi Page 74 74 75 76 78 79 79 79 80 83 84 84 85 86 86 88 88 89 90 95 96 96 96 98 102 GENERAL DISCUSSION . . . . . . . . . REFERENCES . . . APPENDIX A. Colorimetric Protein Assay B. Radioiodination of Prolactin CURRICULUM VITAE vii Page 106 113 149 151 153 LIST OF TABLES Table Page 1. Effects of prolactin injections on prolactin specific binding activity in ventral prostates of intact rats . . . . . . . . . . . . . . . . 68 2. Effects of testosterone propionate (TP) and/or prolactin (PRL) or estradiol benzoate (EB) on specific PRL binding activity in ventral prostates of castrated (Cast) rats . . . . . . . . . . . . . . . . . . . . . 69 3. Effects of prolactin (PRL), testo- sterone propionate (TP), ergo- cornine (ERG) and estradiol benzoate (EB) on specific pro- 1actin binding activity in ventral prostates of castrated (Cast) rats . . . . . . . . . . . . . . . . . 71 4. Effects of ergocornine injections on serum prolactin and specific pro- lactin binding activity in ventral prostates . . . . . . . . . . . . . . . . . . 77 5. Serum prolactin, testosterone and specific prolactin binding activity in ventral prostates of aging male rats . . . . . . . . . . . . . . 87 6. Specific prolactin binding activity in 600 ug of crop sac microsomal protein . . . . . . . . . . . . . . . . . . . 94 viii LIST OF FIGURES Figure Page 1. Time course of the binding of (1251) iodoprolactin to prostatic membrane preparations . . . . . . . . . . . . . . . . . 59 2. Effect of membrane protein concen- Eggtion on the specific binding of I-labeled prolactin . . . . . . . . . . . . 6O 3. Competitive displacement of (1251) iodoprolactin binding to prostatic membranes by various concentrations of unlabeled hormones . . . . . . . . . . . . 61 4. The effects of testosterone propionate (TP) injections on Specific prolactin binding activity in testes, seminal vesicles and ventral prostates of 40 and 70 day old rats . . . . . . . . . . . . 63 5. Time course effects of a single intra- venous injection of unlabeled pro- lactin on in vitro binding of labeled prolact1n . . . . . . . . . . . . . . 81 6. Time course effects of a single sub- cutaneous injection of testosterone propionate on in vitro binding of labeled prolactin . . . . . . . . . . . . . . 82 7. Time course of the binding of (125I) iodoprolactin to pigeon crop sac membrane preparations . . . . . . . . . . . . 91 8. Effect of membrane protein concen- tration on the specific binding of (1 51) iodoprolactin . . . . . . . . . . . . . 92 ix Figure Page 9. Competitive displacement of specific (1251) iodoprolactin to 600 ug of particulate membrane protein from proliferated crop sacs by various concentrations of unlabeled hormones . . . . . . . . . . . . . . 93 10. Effects of estrogen treatment on tumor growth 0 I O O O O O I O O O O O O I C O O O O 99 11. Time course of specific binding of (1251) iodoprolactin to tumor membranes at 37C, 24C and 4C 0 O O O I O O O O I O O O O O 100 12. Competition of (125I) iodoprolactin and unlabeled hormones for binding to tumor membranes obtained from a pooled source . . . . . . . . . . . . . . . . . . . . 101 13. Scatchard analysis derived from a com- petitive inhibition curve . . . . . . . . . . 103 14. Effects of graded doses 3 EB on the Specific binding of (1 I) iodopro- lactin to mammary tumor membranes . . . . . . 104 INTRODUCTION A current view of the molecular basis of hormone action is that receptors concentrated in target cells rapidly bind a hormone and, as a consequence activates the hormone dependent cellular events (Litwack, 1972, 1975; O'Malley and Means, 1973). Although numerous reports describe macromolecules that appear to be intimately involved in selective uptake and retention of hormones in presumed target tissues, their relationship to biochem- ical mechanisms by which hormones influence cellular function is far from clear (Williams-Ashman and Reddi, 1971; Cuatrecasas et al., 1975). Thus, it should be realized that the term "receptor" is used for convenience and inherently implies a lack of knowledge of the precise structure and function of these substances. However, "receptor" in the context used currently in hormonal studies, does not apply to a variety of unrelated sub- stances that bind hormones with low Specificity and avidity nor does it apply to a host of enzymes catalyzing hormone metabolism. Most contemporary workers use the term "receptor" to describe those molecules in target cells that serve to recognize a specific hormone. Recognition is demonstrated by high affinity binding of the hormone to a receptor of limited capacity (Roth et al., 1975; Cuatrecasas et al., 1975). Receptors for steroid hormones have been localized in cytoplasmic and nuclear fractions of hormone responsive cells and have been well characterized (Gorski et al., 1968; Jensen and DeSombre, 1973; O'Malley and Means, 1973; McGuire et al., 1975). The present discussion will focus on receptors for protein hormones and, in particular those for prolactin. The experimental approach to the study of hormone-receptor interactions has been to measure the binding of a radioactively labeled hormone to intact or fractionated target cells. The fundamental principle is that the binding of radiolabeled hormone to specific receptor sites can be competitively inhibited only through native hormone or closely related analogs. REVIEW OF LITERATURE I. Functional Neuroanatomy of Hypothalamio—Hypophyseal System A. General Anatomy of the Hypothalamus The hypothalamus, though somewhat vaguely defined, comprises the most ventral portion of the diencephalon where it forms the floor and lower walls of the third ventricle. On the floor of the brain, the hypothalamus is bound cranially by the optic chiasam, laterally by the optic tracts and caudally by the mammillary bodies. Extending from the basal surface is a clear visible protuberance, the tuber cinereum prolonged as the pituitary stalk. An inconspicuous groove, the hypothalamic sulcus, separates the hypothalamus from the dorsally located thalamus (Jenkins, 1972; Haymaker et al., 1969; Martini et al., 1970). In a craniocaudal direction, individual nuclei of the hypothalamus have been artificially divided into three groups. It is noteworthy that many hypothalamic nuclei are not as clearly defined as they appear to be in diagrams. Often they are diffuse and merge with surrounding tissue or with one another (Schreiber, 1963). The anterior 3 hypothalamus, a region identical with what some authors term the supraoptic region (Netter, 1968), contains the paraventricular, supraoptic and suprachiasmic nuclei. In the middle hypothalamus or tuberal area the periventricular and arcuate nuclei envelope the base of the third ventricle. Also found in the middle region are the lateral, dorsol- medial and ventromedial nuclei. The posterior hypothalamus contains three nuclei surrounding the prominent mammillary bodies; the pre mammillary, the supramammillary and the tuberomammillary nuclei. The neural pathways entering or leaving the hypothalamus are numerous. In addition to well defined tracts, they undoubtedly include many isolated fibers con— necting hypothalamic nuclei with each other and with other parts of the central nervous system. Ascending fibers from the brain stem and descending fibers from the forebrain comprise the major afferent pathways of the hypothalamus. The forebrain projections to the hypothalamus include those from olfactory and hippocampal areas. The hippocampus (and septum) probably channel sensory and neocortical input to the hypothalamus (Martini et al., 1970). Major efferent hypothalamic connections include an ascending bundle to basal forebrain areas and a descending tract to autonomic motoneurons. Most pertinent to the present discussion are efferent fibers which deliver oxytocin and antidiuretic hormone (ADH) to the posterior pituitary and efferent fibers that terminate near the pituitary stalk. This latter group of fibers are presumed to contain hypothalamic hormones (releasing factors) that influence the synthesis and release of anterior pituitary hormones (Martini et al., 1970; Martini and Ganong, 1966; Meites, 1970a). B. Anatomy of the Pituitary Gland The pituitary or hypophysis is a composite gland of neural and epithelial components situated underneath the hypothalamus. In some species the pituitary lobes are encased in the sella turcica, a concavity of the sphenoid bone (Atwell, 1926). The posterior pituitary, i.e., the neurohypOphysis, derived from neural tissue, consists of the pars neurosa (posterior lobe) and its upperward exten- sion, the infundibular stem. The glial cells of the neuro- hypophysis arencalled pituicytes (Bucy, 1932). These were once thought to be the secretory cells of the posterior pituitary. However, most workers now believe that hormones of the posterior pituitary are products of hypothalamic neurosecretory cells. The neurosecretory material moves along axons of the hypothalamo-hypophyseal tract into the pars neurosa where it is stored and released as needed (Turner and Bagnara, 1971). The anterior portion (adenohypOphysis) makes up the epithelial component of the pituitary. The pars distalis (anterior lobe) forms the greater part of the adenohypophysis and is situated in the sella turcica (if formed). The anterior pituitary also includes the pars tuberalis forming the ventral side of the pituitary stalk and the pars intermedia. The pars intermedia is regarded as a separate (middle) lobe of the pituitary in some sub- mammalian species. Histologically, the epithelial cells of the anterior pituitary can be divided into three main groups according to their staining properties: acidophils, basophils and chromophobes. The numerical relationship of the cell types varies considerably in different functional states, e.g., sex differences, lactation, castration. Prolactin and growth hormone are thought to originate in acid-stained cells; gonadotrophins and thyrothropin in basophils and ACTH in chromophobes (Ganong, 1969). The pars tuberalis of the adenohypophysis and the infundibular stem of the neurohypophysis make up the pituitary stalk. The region corresponding to the upper- most portion of the stalk is often referred to as the median eminence (Tilney, 1936). C. Hypothalamic Hypophyseal Vascular Connection In contrast to the direct innervation of the neurohypophysis there is no neural pathway between the hypothalamus and the anterior pituitary. An alternate pathway by which the nervous system might influence the pituitary gland was provided by the discovery of hypophyseal portal veins (Popa and Fielding, 1930). Other studies later established that the direction of blood flow is from the median eminence to sinusoids of the pars distalis (Houssay et al., 1935; Green and Harris, 1947). The sig- nificance of this hypothalamic-hypophyseal vascular link was unappreciated until Harris and others (Harris and Jacobson, 1952; Nikitovitch-Winer and Everett, 1958) demonstrated that the anterior pituitary loses most of its histological characteristics and secretory capacity when the portal vessels are interrupted. When anterior pitui- tary tissue was grafted to the hypothalamus, particularly to regions near the median eminence, it was noted that the histological appearance and functional activity of the transplants were partially maintained (Halasz et al., 1962; Knigge, 1962). In 1955 Harris proposed the "chemotrans- mitter hypothesis" suggesting that blood-borne substances introduced into capillaries of the hypophyseal portal system are responsible for neural regulation of anterior pituitary function. In recent years, evidence has accumulated support- ing the existence of hypothalamic hypophysiotropic hor- mones. Although not totally supported, it is presumed that there are chemically distinct hypothalamic hormones for each of the known adenohypophyseal hormones. Three such substances, thyrotropin—releasing hormone (TRH), luteinizing hormone-releasing hormone (LHRH) and somatostatin (SIF) have been isolated from hypothalamic tissue and shown to have marked influence on anterior pituitary function. However, all these hormones have been reported to influence the release of more than one pitui- tary hormone and to exert other neural effects (on behav- ior). Efforts are being made to determine the physiological significance of these observations. II. Control of Prolactin Secretion A. Hypothalamic Inhibition There is substantial evidence that prolactin syn- thesis and release are chronically inhibited by the mam- malian hypothalamus. The anatomical connections between the hypothalamus and the anterior pituitary are critical in this consideration. Thus, excised pituitaries trans- planted to the kidney capsule (Everett, 1954; Chen et al., 1970) or incubated in gi££g_(Meites et al., 1961) produce a hypersecretion of prolactin. Moreover, isolation of the pituitary by stalk section (Dempsey et al., 1940), lesions in the median eminence that destroy the hypophysiotropic area (Chen et al., 1970; Welsch et al., 1971), or adminis- tration of certain CNS depressing drugs (Meites, 1962; Meites et al., 1963) enhance prolactin release. Studies showing that extracts of hypothalamic tissue inhibited pituitary prolactin synthesis and release provided presumptive evidence for the presence of a prolactin-inhibitory-factor (PIF) in the hypothalamus. Crude hypothalamic extracts from a variety of species (Shally et al., 1967) added to pituitary incubations (Meites et al., 1961; Talwalker et al., 1963) or cultures (Pasteels, 1961) suppressed the autonomous secretion of prolactin. Kragt and Meites (1967) were able to demon- strate a negative dose-response between the amount of hypothalamic extract added to an incubation and the subse- quent release of prolactin into the medium. Recently, Nicoll (1971) reported that hypothalamic PIF activity can act to prevent Ca++ influx into prolactin secreting cells and thus inhibit the spontaneous release of secretory granules in vitrg. Several other workers have demonstrated hypothalamic PIF activity in zitgg. Grosvenor et a1. (1964) reported that injected bovine hypothalamic extracts prevented a suckling—induced release of prolactin in rats and Kurashima et a1. (1966) reported that porcine hypo- thalamic extracts inhibited prolactin release in rats following cervical stimulation. Rat hypothalamic extracts reduced serum prolactin in cycling and lactating rats (Amenomori et al., 1970) and decreased serum prolactin in intact male rats (Watson et al., 1971). Kamberi et al. (1971a) infused hypothalamic extracts into a hypophyseal portal blood vessel and noted a dose related inhibition of prolactin secretion. Hypothalamic PIF content is readily altered by various drugs, hormones and other stimuli. Perphenazine 10 (Danon et al., 1963), reserpine (Ratner et al., 1965), haloperidol (Dickerman et al., 1972, 1974), sodium pento— barbital (Wuttke et al., 1971), epinephrine and acety- choline (Mittler and Meites, 1967), estrogen (Ratner and Meites, 1964), progesterone, testosterone and contisol (Sar and Meites, 1968), a norethynodrel—mentranol combina- tion (Enovid) (Minaguchi and Meites, 1967) and the suckling stimulus (Ratner and Meites, 1964; Minaguchi and Meites, 1967) were reported to decrease hypothalamic PIF activity and hence raise serum prolactin levels in rats. Agents reported to increase hypothalamic PIF activity include prolactin itself (Chen et al., 1967; Clemens and Meites, 1968; Voogt and Meites, 1971), ergocornine (Wuttke et al., 1971), L-dopa and a variety of monoamine oxidase inhibitors (Lu and Meites, 1971). Furthermore L-dopa was reported to raise PIF activity in systemic blood of hypophysectomized and intact rats (Lu and Meites, 1972) and Kamberi et al. (1971b) reported that a single injection of dopamine into the third ventricle of rats increased PIF activity in hypophyseal portal blood. B. Hypothalamic Stimulation Unlike mammals, the predominant influence of the avian hypothalamus is stimulatory to prolactin release and apparently contains a prolactin-releasing factor (PRF). Whereas mammalian pituitaries autografted or cultured in vitro produced a marked secretion of prolactin, transplanted ll chicken pituitaries (Ma and Nalbandov, 1963) or cultured pigeon pituitaries (Nicoll and Meites, 1962a) show no increase in prolactin release. Subsequently Kragt and Meites (1965) demonstrated that extracts of pigeon hypo- thalamic stimulated prolactin release by pigeon pituitaries in vitro. Likewise, crude hypothalamic extracts from chicken, quail (Meites, 1967), tricolored blackbird (Nicoll, 1965), duck (Gourdji and Tixter-Vidal, 1966), and turkey (Chen et al., 1968) induced in vitro prolactin release from their respective pituitaries. Meites et a1. (1960) first reported that crude rat hypothalamic extracts could induce lactation in estrogen— primed female rats. This was later confirmed by Mishkinsky et a1. (1968). However, these studies could not definitely conclude that the extracts contained a PRF since many other agents, including cerebral cortical extracts, initi- ated lactation in these rats. The in vitro studies of Nicoll et a1. (1970) provided evidence that rat hypothalamic extracts contained both PIF and PRF activities. Crude hypothalamic extracts added to rat pituitary incubations initially inhibited prolactin release and then later increased prolactin release. Using a somewhat different incubation system, Meites (1970) observed only inhibition of prolactin release over a similar time period. Krulich et a1. (1971) sectioned rat hypothalamic and reported PRF activity predominantly in the median eminence and PIF 12 activity localized in the dorsolateral preoptic area. However, this report is suspected since numerous other studies indicate that median eminence lesions enhance prolactin release suggesting that this area contains PIF activity (Meites et al., 1972) and that lesions in the preoptic area have no significant effect on prolactin secretion (Everett and Quinn, 1966; Kordon, 1966). Tashjian et a1. (1971) first reported that syn- thetic pyro—glutamyl-histidyl-proline—amine (TRH) induced release of prolactin in vitro from clonal strains of rat pituitary tumor cells. Ensuing studies demonstrated that TRH markedly stimulates prolactin and thyrotropin secretion in humans (Hwang et al., 1971; Bowers et al., 1971; Jacobs et al., 1971), cows (Convey et al., 1972; Kelley et al., 1973), monkeys (Josimovich et al., 1974) and rats (Mueller et al., 1973; Dibbet et al., 1972). These results suggest that TRH might be responsible for hypothalamic PRF activity. However, under many physiological conditions, thyrotropin and prolactin are not released together (Meites, 1973) suggesting that prolactin releasing activity of the hypo- thalamus cannot be solely accounted for by the existence of TRH. Indeed, Valverde (1972) reported PRF activity in porcine hypothalamic distinct from TRH. C. Role of Neurotransmitters There is considerable evidence that synaptic mediators have an important role in control of anterior l3 pituitary function (COppola, 1968; Wurtman, 1970). Sub- stances generally thought to serve as neurotransmitters in the mammalian central nervous system include catecholamines (dopamine and norepinephrine), serotonin and acetylcholine (Cooper et al., 1974). Norepinephrine and serotonin are highly concentrated in the hypothalamus (Vogt, 1954; Brodie et al., 1959) and the median eminence is especially rich in dopaminergic nerve terminals (Anden et al., 1964; Fuxe and Hokfelt, 1969; Carlsson et al., 1962). Assay techniques for acetylcholine are much less sensitive and as a result, little is known about the central distribution of chol- inergic neurons (Cooper et al., 1974). However, Shute (1969) demonstrated the presence of acetylcholine in the hypothalamus. A role for catecholamines in regulating prolactin secretion was suggested by Kanematsu et a1. (1963), who reported that reserpine, a recognized depleter of stored monoamines (Pletscher et al., 1955; Holzbauer and Vogt, 1956), induced lactation and depressed pituitary prolactin concentration in ovariectomized rabbits. Similarly, Mizuno et a1. (1964) observed that iproniazid, an inhibitor of catecholamine metabolism (Jarvik, 1970) inhibited post- partum lactation in rats. Since lactation is not a specific response to prolactin alone, these early studies were of uncertain significance (Meites et al., 1963). However, subsequent work indicated that drugs that inhibit 14 catecholamine synthesis and release, including reserpine, chlorpromazine, alpha-methyl-para-tyrosine, alpha-methyl- metatyrosine, methyldopa and d-amphetamine, all signifi- cantly increased serum prolactin levels. Administration of catecholamine precursors and monoamine oxidase inhibitors resulted in a reduction of serum prolactin (Lu et al., 1970; Lu and Meites, 1971). These observations support the hypothesis that catecholamines act as neurotransmitters to increase release of hypothalamic PIF, which in turn depresses prolactin release (Meites et al., 1972). An alternate hypothesis proposes that hypothalamic catecholamines may be released into hypophyseal portal blood vessels, transferred to the anterior pituitary and exert a direct effect on prolactin secretion (Fuxe and Nilsson, 1967; Anton-Tay et al., 1971; MacLeod, 1976). Since Fuxe (1964) and Hokfelt (1967) first identified dopaminergic nerve endings in the median eminence, there has been growing evidence that dopamine may be a physio- logical inhibitor of prolactin secretion. The work of MacLeod (1969), confirmed by Birge et a1. (1970), Koch et a1. (1970) and Shaar et al. (1974), demonstrated that dopamine directly inhibited in 21359 secretion of prolactin. Although other catecholamines directly inhibited prolactin release in gitrg, dopamine was most potent in this respect and was effective at concentrations known to exist in the hypothalamus (Shaar and Clemens, 1974b). Kamberi et a1. 15 (1971) reported that catecholamines, principally dopamine, suppressed serum prolactin when injected into the third ventricle of rats but were ineffective when infused into hypophyseal portal blood. However, Takahara (1974) later suggested that the inability of infused catecholamines to alter prolactin release was related to their oxidative destruction. When dissolved in a glucose solution and infused into portal blood, dopamine markedly decreased serum prolactin. Systemically administered L-dopa, the immediate precursor of dopamine, reduced serum prolactin from pituitaries in situ (Lu and Meites, 1972) or when autografted to the kidney capsule (Lu and Meites, 1972; Donoso et al., 1974) and in rats with median eminence lesions (Donoso et al., 1973). Presumably, L-dOpa is metabolically converted to dopamine which acts directly on the pituitary to inhibit prolactin release. Dopamine receptor blockers stimulate prolactin secretion in vivo (Meites and Clemens, 1972) and inhibit the action of dopamine in vitro (MacLeod, 1974), and dopamine agonists inhibit prolactin release in give and in vitro (Mueller et al., 1976; Smalstig et al., 1974). Recently Takalara et a1. (1974) have reported that porcine hypothalamic extracts contain sufficient amounts of catecholamines to account for all the PIF activity. Similarly, Shaar and Clemens (1974a) showed that rat hypothalamic extracts sub- jected to monoamine oxidation or catecholamine adsorption 16 by aluminum oxide (alumina) had no PIF activity in vitrg. However, these findings do not unequivocally mean that PIF is a catecholamine since other hypothalamic preparations showing PIF activity are devoid of catecholamines (Schally et al., 1973; Greibrokk et al., 1974). Also, there is some question as to whether treatment of hypothalamic extracts with alumina or monoamine oxidase may not have removed polypeptides (PIF, PRF, TRH, etc.) as well as catecholamines. Serotonin and its metabolite melatonin act to increase prolactin release in contrast to catecholamines. Kamberi et a1. (1971) observed a significant rise in serum prolactin after central administration of serotonin or melatonin, and Lu and Meites (1973) noted that systemic injections of serotonin precursors that pass the blood- brain barrier (tryptophan and 5-hydroxtryptophan) increase serum prolactin. Serotonin antagonists have been reported to depress the suckling-induced release of prolactin in rats (Kordon et al., 1973) and reduce serum prolactin in estrogen—primed female rats (Chen and Meites, 1974). These results suggest a role for serotonin in hypothalamic con- trol of prolactin release. Gala et a1. (1970) implanted atropine, a cholinergic blocker, into the hypothalamus of rats and induced deciduomata formation suggesting that acetylcholine may be involved in control of prolactin secretion. Later Libertun and McCann (1973) and McLean and Nikitovitch-Winer (1975) 17 reported that atrOpine.inhibited prolactin release. These results were challenged by Grandison et a1. (1974) who showed that acetylcholine injected into cerebral ventricles or cholinergic drugs (pilocarpine and physostigmine) administered systemically, decreased serum prolactin. These authors dismiss the reports by the previous investi- gators as toxic and nonspecific excitatory effects of the large doses of atropine used. Earlier work has shown that acetylcholine has no direct effect on pituitary prolactin release (Talwalker et al., 1963). But recently, Grandison and Meites (1975) have suggested that the prolactin inhibit- ing action of the cholinergic system might be mediated by catecholamines. Thus prior administration of drugs that depress catecholamine activity prevented pilocarpine inhibition of prolactin release. Several amino acids, including gamma-aminobutyric acid (GABA) and glycine have been proposed as possible central neurotransmitter agents (Krnjevic, 1974; Defeudis, 1975). Endogenous GABA is concentrated in the diencephalaic regions of the rat brain (Cooper et al., 1974) and when exogenously administered into lateral ventricles of rats increases serum prolactin levels (Mioduozewski, 1976). Ondo and Pass (1976) have reported similar effects of GABA and additionally noted that glycine also effectively ele- vated serum prolactin. The distribution of glycine in the Central nervous tissue has not yet been established (Cooper 18 et al., 1974). It is noteworthy that a current abstract from Schally's laboratory (1976) has indicated that GABA may be responsible for hypothalamic PIF activity. Thus the reported effects of GABA on prolactin secretion are contradictory and the physiological Significance of these observations are not yet established. D. Prolactin Short Feedback Loop Perhaps all pituitary hormones act to depress their own secretion by a "short feedback loop" (Motta et al., 1969). Sgouris and Meites (1953) first hypothesized that prolactin may act to inhibit its own secretion. Subse- quent studies have supported this hypothesis albeit the mechanism of the feedback and its physiological significance remain to be determined. Systemically injected prolactin (Sinha and Tucker, 1968), transplanted pituitaries (Welsch et al., 1968) or prolactin secreting tumors (MacLeod, 1966, 1968; Chen et al., 1967), all significantly reduced the prolactin content of in situ rat pituitaries. More- over, small implants of prolactin into the hypothalamus of rats decreased pituitary prolactin (Clemens and Meites, 1968) and reduced serum prolactin levels (Voogt and Meites, 1971). Similar implants inhibited lactation and caused mammary gland regression (Clemens et al., 1969a), prevented deciduomata formation and interrupted pseudopregnancy (Chen et al., 1968) and pregnancy (Clemens et al., 1969b). Rats bearing pituitary tumors or implants of prolactin in 19 the median eminence have been reported to increase hypo- thalamic PIF activity (Meites, 1970), and prolactin injections have been Shown to activate tuberoinfundibular dopaminergic neurons in rats (Fuxe and Hokfelt, 1969). Nicoll (1971) has reported that prolactin added to pituitary incubates in zitrg has no effect on prolactin release. These observations suggest that prolactin acts by way of the hypothalamus to inhibit its own secretion. However, a possible direct action of prolactin on the pituitary cannot be dismissed. Indeed Spies and Clegg (1971) have reported that pregnancy inhibited by pituitary or hypo- thalamic implants of prolactin in rats can be maintained by exogenous prolactin suggesting the pituitary as a possible site for prolactin feedback in autoregulation. E. Exteroceptive Stimuli Many exteroceptive stimuli can influence prolactin release. A variety of direct and indirect studies have suggested that the nursing stimulus produce a rapid release of pituitary prolactin. Early studies noted that suckling limited to a few nipples maintained milk secretion in all the mammary glands (Selye, 1934; Selye et al., 1934). A role for prolactin in the initiation and maintenance of lactation is now well established (Meites, 1961, 1966; Cowie, 1969). Within minutes after suckling, prolactin release increases and is maintained at high levels for 2-3 hours in rats (Amenomori et al., 1970). Frequent nursing 20 is necessary to sustain prolactin secretion during lacta— tion (Folley, 1952; Turner, 1939). Although nursing serves as a major stimulus for prolactin release during lactation, other exteroceptive stimuli are also important (Neill, 1974). The mere presence of pups during the last half of lactation is sufficient stimuli to release prolactin (Grosvenor and Mena, 1971). Olfactory stimuli is the likely substitute for suckling since prolactin was not released when the mother could see and hear the pups but not smell them. Physical stresses of many types have been reported to affect lactation and prolactin release (Nicoll et al., 1960). Restraint stress, ether anesthesia, continuous lighting or extreme heat are among those nonspecific stresses that elevate serum prolactin levels (Euker et al., 1975; Mueller et al., 1974; Wuttke et al., 1971; Kledzik et al., unpublished). Recently Mueller et a1. (1976) observed that restraint stress raises hypothalamic serotonin and serum prolactin levels suggesting a causal relationship. Stresses such as cold (Mueller et al., 1974) and starvation (Campbell et al., 1975) lower serum prolactin. Riegle et al. (unpublished) has noted that restraint chronically applied, may also lower prolactin levels. 21 F. Effect of Estrogen, Testosterone and Ergot Derivatives on Prolactin Secretion It has long been recognized that estrogens can markedly influence the secretion of prolactin. There is evidence that estrogens act directly upon the pituitary and/or indirectly through the hypothalamus to promote prolactin synthesis and release. Nicoll and Meites (1962b) first reported that estrogens directly stimulate prolactin release when added to rat pituitaries incubated in gitgg. Other studies (Nicoll and Meites, 1964; Lu et al., 1971) subsequently confirmed these results. Estrogens stereo- taxically implanted into the pituitary promote pseudo- pregnancy, mammary growth and lactation and mammary cancer growth, suggesting a direct stimulation of pituitary pro- lactin release (Ramirez and McCann, 1964; Kanematsu and Sawyer, 1963; Nagasawa et al., 1969). Moreover, estrogens have been reported to directly stimulate prolactin secretion from pituitaries autographed to the kidney capsule (Chen et al., 1970). The observation that estrogen injections reduce hypothalamic PIF activity in rats provides evidence for an indirect action of estrogen on pituitary prolactin release. Although estrogen implanted in the median eminence promote prolactin release (Ramirez and McCann, 1964; Nagasawa et al., 1969) the possibility that it is trans- ported by portal blood to affect the pituitary directly cannot be excluded. 22 Ovariectomy decreases pituitary prolactin production albeit estrogen reverses this effect (Catt and Moffat, 1967; MacLeod et al., 1969). The proestrous surge of prolactin in rats seems to be dependent on estrogen since it is blocked by prior administration of antiestrogen compounds or estrogen antiserum (Neill et al., 1971). Other evidence suggests that estrogens sensitize the prolactin release mechanisms to exteroceptive stimuli. For example, the stress induced prolactin release (Neill, 1970) or the ease at which cervical stimulation induces pseudopregnancy (Everett, 1966) are greater during the estrogen-dominated phase of the estrus cycle. Whereas estrogen can directly stimulate prolactin release from incubated pituitaries, Nicoll and Meites (1964) reported that testosterone had no effect on prolactin release in 31339. However, when injected into ovariectomized rats, testosterone reduced hypothalamic PIF content and increased pituitary prolactin concentration (Sar and Meites, 1968). Kalra et al. (1973) reported that testosterone proprionate significantly increased serum prolactin in castrated male and female rats. Perhaps the most useful agents in suppressing prolactin release are the ergot alkaloids. A number of ergot derivatives (dopamine agonists) have been shown to inhibit prolactin secretion, as evidenced by inhibition of lactation and reduced serum prolactin (Shaar and 23 Clemens, 1972). Ergocornine increases hypothalamic PIF activity (Wuttke et al., 1971), but also can act directly on the pituitary to inhibit prolactin release (Lu et al., 1971). Rat pituitaries incubated in vitro with ergocornine show an increase in prolactin stores but a significant decrease in prolactin release (Lu et al., 1971). Addi- tionally ergocornine has been reported to counteract estrogen stimulation of prolactin secretion in 31339 and in vivo. III. Functions of Prolactin A. Mammary Gland The role of prolactin in mammary gland development and secretory activities has been extensively studied. Many workers (Turner, 1939; Lyons et al., 1958; Meites and Hopkins, 1961) have noted that mammary glands of hypophys- ectomized animals Show little or no growth response to ovarian hormones. The definitive studies of Lyons et a1. (1958) and Nandi (1958, 1959, 1961) in rodents provided much of the information on the hormonal requirements of the mammary gland. Lyons and colleagues (1958) reported that in triply-operated rats (hypophysectomized, ovari— ectomized and adrenalectomized) normal mammary development could be induced by injections of growth hormone combined with adrenal steroids and estrogen, but progesterone and prolactin were necessary for lobulo-alveolar development. 24 The same hormones have been reported to produce mammary gland growth in hyposectomized mice (Nadi, l959)--although in one strain of mice, growth hormone was found to be interchangeable with prolactin (Nadi, 1961). Subsequently Talwalker and Meites (1961) were able to induce moderate lubulo-alveolar growth in the absence of ovarian or adrenal steroids by multiple daily injections of growth hormone and prolactin. However, these observations do not negate a role for steroid hormones in mammogenesis but suggest that steroids may in some way sensitize the mammary tissue to the action of pituitary hormones. Nagasawa and Yanai (1971) have recently provided support for this idea. They reported that minute implants of estrogen over the mammary glands of ovariectomized rats resulted in localized lobulo- alveolar development when the levels of circulating pro- lactin were raised. However, if estrogen implants were too concentrated, mammary growth was, in many cases, retarded (Nagasawa and Yanai, 1972). Mammary gland tissue cultured in_vitrg_containing hormone supplements have generally confirmed the 12.2132 studies on hormonal requirements for mammary gland growth (Rivera, 1964). Stricker and Grueter (1928) demonstrated that anterior pituitary extracts injected into ovariectomized pseudopregnant rabbits initiated lactation. Ensuing studies with purified prolactin revealed some differences in lacto- genic responses obtained with prolactin and with pituitary 25 extracts. Even though prolactin or adrenocorticoid hormoes alone can induce lactation in pregnant rabbits, a combina- tion of these two hormones increases the intensity of lactation (Friesen, 1966; Meites et al., 1963). Prolactin and adrenal corticoids appear to be minimal requirements for lactation in guinea pigs and rats (Cowie and Lyons, 1959; Meites, 1966), while growth hormone and cortisol are as effective as prolactin and cortisol in C3H mice (Nadi, 1958). Hypophysectomy during lactation results in a com- plete cessation of milk production (Folley and Malpress, 1948). Hormonal replacement in hypophysectomized lactating rats has only been partially successful in restoration of milk secretion. Large doses of prolactin produce milk yields of 25% normal while prolactin administered with ACTH or corticoids results in 50% restoration (Cowie, 1957). Complete milk secretion was restored after hypophysectomy in rabbits injected with either prolactin or growth hormone (Cowie, 1969) and in hypophysectomized goats with pro- lactin, growth hormone, triiodothyrosmine, insulin and corticosteroid injections (Cowie, 1964). Prolactin has little or no stimulatory effect on existing lactation in cows (Smith et al., 1974) but does increase milk production in goats (Meites, 1961) and rabbits (Cowie, 1969). Small weight gains have been 26 reported in rats injected with prolactin, suggestive of a galactopoietic effect (Meites, 1961). B. Mammary Tumors The two most important hormones in mammary tumori- genesis in mice and rats are believed to be prolactin and estrogen (Meites, 1972). Although carcinogen-induced rat mammary tumors can develop in the presence of normal serum prolactin levels (Meites, 1972), hypothalamic lesions (Clemens et al., 1968; Welsch et al., 1969), pituitary grafts (Welsch et al., 1968), or central acting drugs (Welsch and Meites, 1970; Quadri et al., 1973) that enhance prolactin secretion promote tumor growth. Drugs which inhibit prolactin release retard mammary tumor growth (Cassell et al., 1971; Quadri et al., 1973). However, if high levels of prolactin or ovarian hormones are present before the administration of a carcinogen the induction of the mammary tumor is inhibited in rats (Clemens et al., 1968; Welsch et al., 1969; Kledzik et al., 1974). Growth of mammary tumors in rats can be maintained, at least temporarily, by prolactin alone even in the absence of the ovaries and adrenals (Pearson et al., 1969), but estrogen has no growth promoting action on mammary tumors in the absence of the pituitary (Sterental et al., 1963). Whereas low doses of estrogen are stimulatory to mammary tumor development and growth in the intact rat (Huggins et al., 1962), large doses of estrogen have an inhibitory 27 effect despite their ability to increase blood prolactin levels (Meites, 1972; Huggins et al., 1962). Recent studies have suggested that high doses of estrogen may interfere directly with the stimulatory action of prolactin on mammary tumor tissue (Meites et al., 1971). Welsch and Rivera (1972) reported that prolactin stimulated DNA syn- thesis in rat mammary tumor organ cultures but high doses of estrogen inhibited DNA synthesis and also suppressed -induced DNA synthesis. Moreover, a Significant negative correlation has been reported between administered doses of estrogen and the subsequent binding of prolactin to mammary tumor cell membranes (Kledzik et al., 1976). Frequently spontaneous mammary tumors appear in old female rats (Meites et al., 1972) and unlike carcinogen- induced mammary adenocarcinomas, they usually occur as single benign fibroadenomas. Apparently prolactin is also a major factor in growth and development of these mammary tumors. Thus bilateral median eminence lesions (Welsch et al., 1970) or multiple pituitary homografts (Welsch et al., 1970), significantly enhanced serum prolactin levels and spontaneous mammary tumor incidence in rats. Moreover, Quadri and Meites (1971) reported marked regression of spontaneous mammary tumors in old female rats treated with ergot drugs. Similar effects of hypothalamic lesions have been reported in mice (Bruni and Montemurro, 1971). In addition, Meites et a1. (1972) have occasionally noted 28 spontaneous mammary tumors in rats bearing transplanted pituitary tumor that secrete large amounts of prolactin and growth hormone. C. Ovaries It is generally agreed that prolactin is necessary for progesterone secretion from the corpora lutea in the rat, mouse, hamster and ferret (Hilliard, 1973). In other species, a luteotropic role for prolactin has not been established. Recent studies have indicated that maintenance of progesterone secretion involves a luteotropic complex rather than a single hormone. In rats and ferrets, this complex apparently consists of prolactin and LH whereas in mice and hamster, prolactin and FSH are the minimally required luteotropins (Choudary and Greenwald, 1969; Greenwald, 1969). In the absence of mating or surrogate cervical stimulation, the corpora lutea of the rat secrete only small amounts of progesterone during the estrous cycle and then regress. However, cervical stimulation prolongs the luteal phase and causes the corpora lutea to become fully functional. If the cervical stimulation does not lead to pregnancy, a "pseudopregnant" period of progesterone secretion lasts for 12-14 days (Hashimoto et al., 1968). Hypophysectomy during pseudopregnancy causes regression of functional corpora lutea but daily injections of pro- 1actin will prevent this (Astwood, 1941). Moreover, during 29 pseudOpregnancy, mammary gland growth and development is suggestive of sustained prolactin release (Freyer and Evans, 1923; Schutze and Turner, 1933). Confirming these earlier indirect studies, Freeman and Neill (1972) reported daily biphasic surges of prolactin release throughout pseudopregnancy. These surges of prolactin appear to maintain the morphological integrity of the corpora lutea and regulate the precursor pools of progesterone (Everett, 1954). Prolactin has been reported to increase the corpora luteal levels of sterol acyl-transferase and sterol esterase that function in the metabolism of cholesterol (Behrman et al., 1970). LH acts to convert the cholesterol to progesterone. Additionally prolactin may act to inhibit enzymes that catabolize progesterone (Weist and Kidwell, 1969; Zmigrod et al., 1972). A similar pattern of pituitary prolactin release occurs during the first half of pregnancy in rats but after about day 12 prolactin secretion declines and is maintained at low levels until the end of gestation. Just prior to parturition there is a significant increase in prolactin secretion presumably as a result of increased estrogen secretion (Yoshinaga et al., 1969). This increase in prolactin release is believed to participate in the initi- ation of lactation occurring at parturition (Meites, 1961). During the second half of pregnancy in the rat the luteo- tropic function is assumed by a placental factor (Astwood 30 and Greep, 1938). This is evidenced by the failure of the corpora lutea to regress following hypophysectomy after day 12 of pregnancy (Pencharz and Long, 1933). Paradoxically prolactin can also act to destroy the corpora lutea, at least in mice and rats. Malven (1969) reported that prolactin administered to hypophys- ectomized rats can have luteotropic or luteolytic actions, depending on the time of injection. When administered within 56 hours after hypophysectomy, prolactin had the luteotropic effect of progesterone stimulation. Yet when injections are delayed, prolactin had a luteolytic effect. Wuttke and Meites (1971) noted that ergot drugs that inhibit endogenous prolactin release do not interfere with normal estrous cycles or ovulation. However, the old corpora of the previous cycles do not degenerate. These observations suggested that the rise in prolactin seen during the estrous cycle serves to induce luteolysis of the older crop of corpora lutea. A similar role for pro- lactin has been reported in mice (Grandison and Meites, 1972). D. CrOp Sac Members of the avian family Columbidae (doves and pigeons) have a large thoracic food storage organ, the crop sac (Nicoll, 1974). Near the end of the incubation period, the crop sac of the adult bird rapidly proliferates and thickens. The hypertropied mucosa cells gather fat 31 globules and are eventually Sloughed into the crop sac lumen as "crop milk." Crop milk is used by the parent birds to feed their hatchlings. Riddle and Braucher (1931) reported that crop sac proliferation is sensitive to anterior pituitary control. Subsequently, Riddle et a1. (1932, 1933) identified and isolated prolactin as the pituitary hormone responsible for crop sac proliferation. Although the crop sac can respond to prolactin in the absence of gonadal, adrenal or thyroid hormones (Riddle and Dykshorn, 1932; Schooley et al., 1937) the crop response to prolactin in hypophysectomized pigeons is augmented by thyroxin, growth hormone or adreno- corticoid injections (Bates et al., 1962). Nicoll and Sherry (1967) reported that prolactin stimulates RNA and protein synthesis in pigeon crop sacs. Over the past four decades the crop sac response to injected prolactin has become the classical bioassay for this hormone (Riddle et al., 1931, 1933; Lyons, 1937; Nicoll, 1967). In the methods most generally used, pro- lactin has been injected intradermally or systemically once daily and the crop sac removed on the 5th day and measured for growth response. E. Male Accessory Sex Organs Several reports have suggested a role for prolactin in controlling prostatic function. Prostatic atrophy was more marked after hypophysectomy than after castration 32 (Huggins and Russell, 1946; Lostroh and Li, 1957), and the prostatic growth response to exogenous androgen was smaller after hypophysectomy than after orchiodectomy (Grayhack et al., 1955; Vander Laan, 1953). Simultaneous treatment with prolactin enhanced the response of the prostate to testo- sterone in hypophysectomized rats (Grayhack et al., 1955; Grayhack, 1963) and prolactin and testosterone synergized in maintaining rat prostatic tissue in organ culture (Lasnitzki, 1972). Moger and Geschwind (1972) noted that prolactin alone was able to increase 652m uptake by the prostates of castrated male rats. Asano (1965) reported that prostatectomy increased prolactin secretion and pituitary prolactin content in rats, and more recently (Asano et al., 1971) observed that injections of antiserum to prolactin decreased prostatic weights in rabbits. However, these interesting findings remain unconfirmed. Similar activity of prolactin on the seminal vesicles has been described. Pasqualini (1953) reported a significant increase in seminal vesicle secretion of castrated rats after testosterone followed by prolactin, and Chase et a1. (1957) observed growth of seminal vesicles of castrated rats with injections of prolactin alone or in combination with testosterone. Bengmark and Hesselsjo (1963, 1964) reported that prolactin stimulates the pro- liferation of rat seminal vesicle cells in tissue culture. However, Okamoto et a1. (1960) reported that prolactin 33 increased prostatic weight but not seminal vesicle weights of hypophysectomized or hypophysectomized-castrated rats. There is also ample evidence that prolactin influ- ences testicular growth and function. In immature hypo- physectomized rats, pituitary transplants increased testic- ular growth (Negro-Vilar and Saad, 1972). Bartke (1965, 1966a, 1966b) studied strains of infertile mice genetically deficient in growth hormone and prolactin. Injections of prolactin into these mice increased their spermatoza yield and rendered them fertile (Bartke and Lloyd, 1970). Similar effects on spermatogenesis were noted in hypophysectomized genetically normal mice (Bartke, 1971) and a synergism between LH and prolactin was observed. Hafez et al. (1971, 1972a, 1972b) reported that prolactin raised enzymatic activity in the testes of dwarf mice and that prolactin injections combined with LH into hypophysectomized rats raised testosterone secretion in 2119 and testosterone synthesis in gitrg. It is also noteworthy that Boynes et a1. (1972) injected an ergot drug that lowered endo- genous prolactin while increasing LH release and observed a lowered plasma testosterone level in male rats. Recently McCann et a1. (1974) noted that an initial elevation in serum prolactin at day 25 in male rats is associated with the beginning growth of accessory sex organs and that a second rise in prolactin after day 50 was associated with further accessory organ growth. 34 F. Osmoregulation Many studies have indicated that prolactin has important osmoregulatory functions. In seagulls and ducks, prolactin stimulates secretion by nasal or orbital glands concerned with elimination of excess salt (Nicoll, 1974). In certain fish and amphibians, prolactin influences the permeability of gills and the functional activities of kidney and bladder to promote sodium retention (Nicoll and Bern, 1972; Nicoll, 1974). Recent studies have suggested that prolactin may also facilitate sodium reabsorption by the mammalian kidney. Thus Lockett et a1. (1965) reported that prolactin injections reduced urinary water and sodium excretion in rats and cats. Relkin and Adachi (1973) reported increased plasma prolactin levels in rats maintained on a low sodium diet. Marshall et a1. (1975) reported that whereas uni- lateral nephrectomy and water deprivation elevated circu— lating prolactin levels Significantly, salt loading had no effect. Ensor et a1. (1972) noted that prolactin injections stimulated drinking and furthered water retention in dehy- drated rats. Moreover, Ensor et a1. (1972) observed that lactating rats were more resistant to dehydration than nonlactating rats. Humans have also been shown to decrease water and sodium loss and to increase plasma sodium levels in response to prolactin injections (Horrobin et al., 1971). However, hypertonic solutions have been reported 35 to increase blood prolactin levels and hypotonic solutions were reported to have the opposite effect in rats (Relkin, 1974) and man (Buckman and Peake, 1973a, b). These fore- going observations suggested that prolactin may have a more complex osmoregulatory role in mammals than just promotion of sodium retention by the kidney. Burstyn et a1. (1972) reported prolactin and aldo- sterone synergism in sheep. Very high salt intake abolished the sodium retaining effect of aldosterone but prolactin injections restored aldosterone activity. Similarly, Horrobin et a1. (1973) reported that prolactin injections could restore the water retaining ability of ADH previously blocked by cortisol treatment. G. Adrenals and Liver Although prolactin is not generally regarded as a regulator of adrenal and liver function, some evidence has accumulated suggesting a trophic influence of prolactin on these organs. The reported presence of high affinity prolactin binding sites in liver (Posner et al., 1974) and adrenals (Marshall at al., 1975) supports this View. Many hormones have been reported to inhibit adrenal Sa-reductase activity (Witorsch and Kitay, 1972). Adrenal Sa-reductase converts corticosterone to reduced metabolites. Accordingly, hormones which inhibit 5d-reductase activity promote an increase in corticosterone output. Witorsch et a1. (1972) reported that estrogen lowers adrenal 36 5a-reductase activity in ovariectomized rats only in the presence of the pituitary. Of several pituitary hormones observed to lower reductase activity in hypophysectomized rats, only prolactin was inhibitory in castrated females (Witorsch and Kitay, 1972). This suggests that prolactin is involved in ovarian control of adrenal reductase activity. More recently, Lis et a1. (1973) reported that prolactin or ACTH injected into hypophysectomized rats partially restored the capacity of adrenal cells to syn- thesize corticosterone in gitrg and combinations of pro- lactin and ACTH were more effective than either hormone alone. Furthermore, Piva et a1. (1973) demonstrated that prolactin enhances adrenal progesterone secretion in dexamethasone treated castrated female rats and Relkin et al. (1973a, b) reported that prolactin enhances aldo- sterone secretion in response to sodium deprivation. Many hepatic functions have been attributed to prolactin in a variety of Species. These include effects on carbohydrate, lipid and protein metabolism (Riddle, 1963; Bern and Nicoll, 1968). In general, prolactin has been reported to stimulate protein synthesis in mice (Chen et al., 1972) and rats livers (Turkington, 1972); to reduce hepatic lipid content in lizards (Licht and Hoyer, 1968), pigeons (Goodridge and Ball, 1967) and dogs (Winkler et al., 1971); to promote hepatic glycogen 37 synthesis in lizards (Callard and Chan, 1972) and mice (Elghamry et al., 1966). IV. Peptide Hormone Binding to Receptors A. Hormone Labeling In order to detect a small number of receptor sites in a cell preparation, a hormone must be labeled to high specific activity without destroying its biological activity. Most receptor studies involving protein hor- 1251 has been the mones have used radioiodine labeling. isotope of choice for a number of practical reasons (Yalow et al., 1968). It is available virtually in a carrier-free state, has a long useful half-life, and is efficiently detected. 3H-labeled peptide hormones have also been used successfully in receptor studies. However, the theoretical advantage of a substituted atom rather than an added iodine is cutweighed by a much lower specific activity. Similarly, 14C and 35 S do not provide labeled hormones of high specific activity even where it is pos- sible to substitute many atoms (Roth, 1973). Until recently, radioiodination of hormones were carried out using high concentrations of strong oxidizing agents such as chloramine-T (Hunter et al., 1962) or iodine monochloride (Kahn, 1965). Although these methods can produce radioiodinated hormones of high specific activity, the strong oxidizing agents may drastically alter 38 protein structure and biological activity. Nevertheless, these methods have been used by many investigators for studies of receptor-hormone interactions. More recently, gentler techniques of radioiodination, employing enzymes such as lactoperoxidase have been developed (Marchalonis, 1969; Thorell et al., 1971). These enzymatic methods can yield labeled hormones with high specific activity while preserving the biological activities. B. Receptor Preparations A variety of cell preparations have been the source of "receptors" for protein hormone binding studies. Whole tissue homogenates (Danzo et al., 1972; Leidenbergee et al., 1972) as well as intact cells isolated from the circulation (Lin et al., 1970; Galvin et al., 1972) grown in culture (Lesniak et al., 1973; Krug et al., 1972; Roth et al., 1972) or dispersed by mechanical (Dufau et al., 1971; Catt et al., 1971) or enzymatic treatment (Cuatrecasas, 1971; Kahn et al., 1973) have been used successfully. Cell preparations previously exposed to enzymes such as trypsin or phospholipase show a significant loss in receptor activity suggesting that receptors are proteins associated with membrane lipids (Posner, 1975). Receptors obtained from subcellular sources have been especially concentrated in membrane fractions (Freychet et al., 1971; Cuatrecasas, 1972). For some studies, receptors have been solubilized and freed from the membranes by detergents. However, many 39 detergents are difficult to remove from proteins and occasionally cause irreversible denaturation or micelle artifacts capable of entrapping labeled hormone (Blecher et al., 1974; Galvin et al., 1972). Solubilized receptors have been partially purified by column chromatography and estimates of their molecular weights range from 40,000 for the epinephrine receptor to several million for the ACTH receptor (Lefkowitz et al., 1969, 1972). It has been shown that several hormones bind to saturable sites in their target tissues. However, virtually all radio-labeled hormones can bind nonspecifically to many biological and inert materials as well as to specific receptor sites (Cuatrecasas et al., 1975). Specificity is demonstrated when the binding of a labeled hormone is com— petitively inhibited only by the native hormone or closely related analogs. The degree of inhibition is predictable by the relative biological potencies of the unlabeled com- petitor. This approach is similar to hapten inhibition used to Show the specificity of antigen-antibody reactions. C. Incubation of Hormone and Receptor The binding of a labeled hormone to its specific receptor site is dependent on concentration, temperature and time (Cautrecasas et al., 1975; Posner, 1975; Roth, 1973). Increasing the concentration of either labeled hormone or receptors increases the rate and extent of association. Maximal binding occurs sooner at higher 40 temperatures, but can be achieved at lower temperatures provided the incubation period is prolonged. A narrow pH range of 7.0-7.4 is generally optimal for binding. Dis- sociation of labeled hormone from receptor sites can be demonstrated by dilution, addition of excess unlabeled hormone or a change in pH and accelerated at increased temperature. Although hormone—receptor interactions should ideally be studied in physiologic conditions (37°C, pH 7.4, etc.), practical considerations often dictate other- wise. Incubation temperatures below 37° reduce hormone and/or receptor degradation and slow dissociation of hor- mone from receptor. Thus at lower temperatures the separation of bound and free radioactivity can be accom- plished with little dissociation. D. Affinity Constants and Binding Capacity The affinity of a hormone for its receptor as well as the number of receptor sites in a membrane preparation have been estimated from competitive dose-response curves by a variety of mathematical methods (Feldman, 1972; Weber, 1965; Weder et al., 1974). These methods are based on the following assumptions: (1) labeled and unlabeled hormone react identically; (2) all binding sites are equivalent; (3) there is no cooperativity effect between binding sites; (4) the binding follows the law of mass action. Of the various available methods, one of the most commonly used 41 is that described by Scatchard (1949). At equilibrium, a Scatchard analysis of hormone-receptor interaction can be HR H —HR 0 concentration of hormone; R0, the initial concentration of described as: = Ka (RC-HR) where H0 is the initial receptor; HR is the concentration of hormone-receptor complexes and Ka is the affinity constant. A graphical representation plots the ratio of bound/free hormone as a function of that bound. For a single class of binding sites this yields a straight line. The binding parameters can be easily determined from the Scatchard plot. The affinity constant, Ka’ equals the negative value of the slope and the number of binding sites corresponds to the intercept on the abscissa. When there is a second class of binding sites, a curved relationship is seen. This is usually the case in hormone binding experiments since radio-labeled hormones bind to specific receptor sites and also to nonspecific Sites having lower affinities and higher capacities. However, when the nonspecific binding is subtracted at each point, a linear plot of the specific binding can be resolved (Chamness et al., 1975). Nonspe- cific binding can be determined as the binding of labeled hormone in the presence of excess unlabeled hormone used to saturate high affinity receptor sites. E. Prolactin Binding The tissue distribution of radiolabeled prolactin was first reported by Cox (1951). Twenty minutes after an 42 intravenous injection into C H mice, the highest concen- 3 trations of radioactivity were found in the liver and kidney and appreciable amounts in mammary glands, adrenals and ovaries. A later autoradiographic study (Birkinshaw et al., 1972) noted similar results in rabbits with the highest uptake ratios of tissue plasma iodoprolactin in kidneys and lactating mammary glands. In the lactating mammary tissue the radioactivity was found to be localized on or near the alveolar secretory cell membrane proximal to the vascular supply. At about the same time, Mishkinsky et a1. (1972) observed high concentrations of radioactivity in mammary glands of lactating rats after the injection of 125I-labeled prolactin or NalZSI. In both cases, the uptake was suppressed by KI-I2 indicating that the radio- activity did not necessarily represent labeled-prolactin. 125 However, injections of either I-labeled prolactin or 125 125 125 I-labeled HCG but neither I-labeled albumin nor Na I into pigeon crop sacs resulted in a significant retention of radioactivity. Although both iodoprolactin and iodo— HCG were bound to the crop sac, mucosa proliferation was elicited by prolactin and inhibited by HCG. This was interpreted as evidence for the binding of prolactin and HCG to the same receptor sites. Rajaniemi et a1. (1974) reported the localization of injected iodoprolactin in various tissues of mice and rats. In the kidney, radioactivity was confined to the 43 proximal tubular cells whereas in the liver the labeling was diffuse over parenchymal and Kupffer cells. In the testis, labeling was localized around Leydig cells with no indication of tubular distribution. The ventral prostate epithelium was strongly labeled but the labeling was weak in epithelium of preputial gland and seminal vesicles. The radioactivity concentrated in the ovary was localized in the corpora lutea and the mammary epithelium showed some labeling during pregnancy and lactation. Midgley (1973) applied radioiodinated prolactin topically to rat ovarian sections and noted that the localization of radioactivity was affected by the functional state of the corpora lutea, that is, newly formed corpora bound more radioactivity than old corpora. In this section, each of the foregoing studies have been limited to the identification of prolactin receptor activity by its ability to bind radioactivity pre- sumably representing iodoprolactin. Recently, other investigators have focused attention on the following properties of prolactin receptors predicted from theoretical considerations: (1) they have strict structural specificity for prolactin; (2) they exhibit high affinity binding in accordance with the physiological concentrations of pro- lactin; (3) they exist in limited numbers in target cells and are easily saturated. Turkington et a1. (1972, 1973) and Frantz (1974) identified specific prolactin binding 44 of high affinity and low capacity in plasma membranes of mouse or rat mammary gland, liver, kidney, midbrain, ovary, adrenal and seminal vesicle cells. The prolactin binding to mammary cell membranes was further characterized and found to be sensitive to trypsin or heating at 70°C indi- cating a receptor of protein nature. Furthermore, the prolactin concentration at which specific binding was observed was similar to that which induces casein synthesis. This provides supportive evidence for a physiological role of prolactin binding. The fact that the greatest specific binding was detectable in plasma membranes is consistent with an earlier study showing that sepharose-coupled pro- lactin retains its biological activity, indicating a mem- brane site of action (Turkington, 1970). Shiu and Friesen (1974a, 1976) have successfully blocked the biological action of prolactin with an antiserum to purified prolactin receptors from rabbit mammary glands. Apparently, the antibodies block the receptor sites and render them inacces- sible to prolactin. Other studies have identified and characterized the prolactin binding in various tissues and species (Posner et al., 1974; Shiu et al., 1974b). Several studies have hinted that prolactin receptor levels may be regulated by endocrine factors. This regu- lation may serve to determine the degree of tissue sensi— tivity to prolactin and provide a partial explanation for hormonal synergism and antagonism. Whether a hormone 45 induces or represses prolactin binding sites seems to depend upon the tissue involved and the existing hormonal milieu. Kelly et al. (1974) observed significant develOp- mental changes in prolactin binding sites in rabbit and rat liver membranes. Prolactin binding activity was found to be similar in male and female rabbit livers and developed gradually before the onset of puberty. In contrast, liver membranes obtained from female rats Showed a marked increase in Specific prolactin binding during a time associated with the start of puberty and further increased during pregnancy. Liver membranes from male rats at all ages studied showed much lower binding activity than corresponding female livers. This suggests a role for ovarian hormones in pro- lactin receptor development. Indeed, Gelato and colleagues (1975) subsequently reported that ovariectomy reduces and estrogen increases prolactin binding to liver membranes of female rats. Other workers (Posner et al., 1974b) induced prolactin binding sites in male livers by daily injections of estrogens or prevented this induction by prior adminis- tration of anti—estrogen compounds (Kelly et al., 1975). A noteworthy correlation has been reported between the plasma estrogen levels and the number of ovarian prolactin binding sites during the rat estrous cycle (Saito et al., 1975). Binding was lowest during metestrus, increased during diestrus and reached a maximum at proestrus. 46 Unlike the ontogeny of prolactin receptors in rat livers, binding sites in the kidney and adrenals of rats decreased as puberty approached (Gelato, 1975). Supportive data showing that estrogen has an inhibitory effect on prolactin binding in kidneys and adrenals was provided by Marshall et a1. (1976). Similarly, estrogen tended to depress prolactin binding in ventral prostates (Kledzik et al., 1976; Aragona et al., 1975) and mammary tissue (Gelato, 1975) of rats. Differential effects of testo- sterone on the binding of prolactin in male rats have also been observed. Testosterone reduces receptor activity in kidney and adrenals (Marshall et al., 1976), but increases it in ventral prostates (Kledzik et al., 1976; Aragona et al., 1975). Evidence suggesting a role for prolactin receptors in salt and water regulation has been reported (Marshall et al., 1975). A concept that multiple hormone interactions are involved in control of prolactin receptors is supported by a number of recent experiments. Ovariectomy (OVX) and/or thyroidectomy (THX) significantly reduced hepatic prolactin binding activity. The combined surgery decreased binding more than either alone. Injections of thyroxin restored binding in THX rats to intact control levels and in OVX-THX rats to OVX values (Gelato et al., 1975). Hypophysectomy results in a loss of prolactin receptor activity in female livers and prevented an estrogen-induced increase in male 47 livers (Posner et al., 1974). A kidney pituitary implant, capable of maintaining circulating prolactin levels in hypophysectomized rats, partially prevented the decrease in prolactin receptor levels in female rats and induced the receptors in males consequent to hypophysectomy (Posner et al., 1975). Multiple injections of estrogen, progester- one, hydrocortisone, triiodotyronine (T3) or estrogen plus T3 had no effect on hepatic prolactin binding in the absence of the pituitary, but a single injection of pro- lactin to hypophysectomized rat produced a marked increase within 18 hours. Repeated injections of prolactin alone or combined with estrogen and T3 had no greater effect than a single prolactin injection (Costlow et al., 1975). Administration of prolactin to intact rats did not further increase the normal prolactin binding activity measured in livers (Costlow et al., 1975). These results suggest that prolactin regulates its own receptor activity in rat livers and that part of the inductive effect of estrogen is through an ability to stimulate prolactin. The binding of prolactin to membrane fractions of pigeon crop sacs has been Shown to correlate well with the growth response of the crop sac to prolactin (Kledzik et al., 1975b). ‘A similar relationship between the growth of carcinogen-induced rat mammary cancers to prolactin and subsequent prolactin binding has been reported (Kelly et al., 1974). Other studies (Turkington, 1974; Costlow et 48 al., 1974, 1975) have noted the existence of high affinity prolactin receptor sites in mammary tumors responsive to prolactin and little, if any, receptor activity in prolactin- independent tumors. Growth of rat mammary tumors in response to endocrine ablation has been better correlated to a combination of estrogen and prolactin receptor levels than to either receptor concentration alone (DeSombre et al., 1976). This supports earlier studies (Meites, 1972; Bradley et al., 1976) suggesting that rat mammary tumors may be dependent upon one or both of these hormones for growth. The high affinity binding sites in mammary cell membranes have been utilized to develop a specific radioligand-receptor assay for prolactin and other lacto- genic hormones (Turkington, 1971; Shiu et al., 1973). This assay offers important advantages that complement the conventional prolactin bioassays. Technically, radioligand assays are more sensitive and precise than bioassays and more conveniently applied to the measurement of large numbers of samples. Moreover, receptor assays allow the biological activity at target cells to be evaluated without the effect of metabolism in yizg (Catt et al., 1972). Shiu et al. (1973) used such a prolactin receptor assay to determine the serum concentrations of prolactin in many species and the potency of various prolactin preparations. 49 Additionally he was able to identify and measure a placental lactogen secreted during pregnancy in rats. F. Receptors for Other Polypeptide Hormones In the past few years many reports have appeared describing membrane receptors for other polypeptide hor- mones in various tissues. Like those involving prolactin receptors, most of these studies have determined whether the specificity, affinity and number of binding sites are compatible with the known biological activities of the hormones. This section will not attempt a comprehensive review of all such work but rather illustrate a general survey of hormone-receptor interactions studied to date. Lefkowitz and colleagues (1969) first described the specific binding of radiolabeled ACTH to adrenal tissue extracts. Especially important was the demonstration that ACTH binding was quantitatively related to biological potency. At about the same time, Goodfriend and Lin (1969) identified angiotensin receptors in adrenal, aorta and uterine tissue. Subsequently, these authors (Goodfriend and Lin, 1970) noted that analogs inhibited the binding of labeled angiotensin proportional to their ability to stimulate smooth muscle contraction. Ensuing studies demonstrated the specific binding of labeled calcitonin (Marx et al., 1972), ADH (Campbell et al., 1972) and parathyroid hormone (Malbon and Zull, 1974) to renal 50 plasma membranes, glucagon to liver and fat cells (Rodbell et al., 1971; Robinson et al., 1971), and oxytocin to uterine fractions (Soloff and Swartz, 1974). Hintz et a1. (1974) reported the binding of somatomedin to skeletal, liver and placenta membranes while others noted TRH (Bardon and Labrie, 1973) and LHRH receptors (Spona, 1972) in pituitary cells. Often the hormone-receptor inter- actions were correlated with adenyl cyclase activity sug- gestive of cellular stimulation. Insulin receptors have been well characterized in hepatic (Freychet et al., 1972; Roth et al., 1975), placental (Posner, 1974) and adipose tissue (Cuatrecasas, 1971, 1972). Kahn et a1. (1973) noted that livers from hyperglycemic obese mice bound less insulin than livers from thin litter mates. Although the cause of this reduced insulin binding is not yet clear, it does provide some physiological support for these studies. More recently Posner et a1. (1974) demonstrated insulin binding activity in normal mammary tissue and Kelly et a1. (1974) demonstrate insulin binding in carcinogen induced mammary tumors. Tissues reported to contain growth hormone receptors include liver (Posner et al., 1974), lymphocytes (Lesniak et al., 1973) and mammary tumors (Kelly et al., 1974). A growth hormone radio-receptor aSsay using liver membranes has revealed the existence of so-called "big" and "little" growth hormones. Apparently they are indistinguishable in 51 a radioimmunoassay system but the "big" growth hormone has much less binding activity to a receptor preparation. The thyroid has been reported to have binding sites for thyro- tropin (Winand and Kohn, 1972) and the gonads contain receptors for follicle stimulating hormone (Means and Vaitukaitus, 1972), luteinizing hormone (Catt et al., 1971) and chorionic gonadotropin (Dufau and Catt, 1973). Many of the studies cited above undoubtedly reflect true hormone-receptor interactions; there is good corre- lation between biological and physiochemical data. However, unexpected hormone binding activity was found in tissues not generally regarded as targets. These studies have created interesting possibilities for unknown endocrine functions likely to be elucidated by further work. MATERIALS AND METHODS I. Animals Rats used for all studies, except Experiment V (Serum Prolactin, Testosterone and Prolactin Receptors in Ventral Prostates in Aging Male Rats) were of the Sprague-Dawley strain purchased from Spartan Research Animals Inc. (Haslett, Michigan). Male rats of the Long- Evans strain obtained from the Blue Spruce Farms (Altamont, New York) were used in Experiment V. All rats were housed in a light (14 hours/day) and temperature (25C 1 1C) con- trolled environment, and fed a diet of Purina Rat Chow (Ralston Purina Co., St. Louis, Missouri) and tap water ad libitum. All castrations, intravenous injections and mammary tumor measurements were performed under ether anesthesia. Pigeons used in Experiment VI (Prolactin Binding Activity in Crop Sacs of Juvenile, Mature, Parent and Prolactin Injected Pigeons) were of the White Carneau strain obtained from Meadowbrook Farms (Fenton, Michigan). Pigeons that received daily injections were housed in a temperature (25C i 1C) and light (14 hours/day) controlled 52 53 room and maintained on Rydes Pigeon Feed (Rydes Inc., Mason, Michigan) and tap water ad libitum. II. Prolactin Radioreceptor Assay A. Preparation of Prolactin-Binding Subcellular Fractions All tissues were excised from decapitated animals, weighed and immediately frozen on dry ice. Subsequently the tissues were thawed and homogenized at 4°C in .3 M sucrose using a Brinkman Polytron-PTlO or ground glass homogenizer. The homogenates were centrifuged at 14,500 x g for 20 minutes in a Sorvall RC-ZB centrifuge and the resultant supernatant centrifuged at 105,000 x g for 90 minutes in a Sorvall OTD-2 ultracentrifuge to obtain a membrane rich pellet (Gray and Whittaker, 1960; Shiu et al., 1973). Each pellet was resuspended in tris buffer (25 mM tris, 10 mM CaClz, pH 7.6) and its protein content colorimetrically determined by the method of Lowry et a1. (1951). A detailed description of this protein assay is presented in Appendix A. B. Preparation of 125I-labeled Prolactin Ovine prolactin (NIH S-lO, 25.6 IU/ug) was radio- iodinated at room temperature by a lactoperoxidase-H202 method similar to that reported by Thorell and Johansson (1971). A description of the reagents used and the iodination procedures is presented in Appendix B. The 54 reaction mixture was fractionated on a column of Sephadex G 50 (0.9 cm x 20 cm) eluted with .025 M tris-HCl contain— ing 10 mM CaCl at pH 7.8. Fractions of approximately 0.5 2 ml were diluted with 1% bovine serum albumin (BSA)-tris buffer to give approximately 70,000 cpm per 100 ul in a Nuclear Chicago automatic gamma counter with a 3 inch scintillation detector. Each fraction was tested for bind- ing activity to stock membrane preparations of rat liver and kidney. For the purpose of choosing the best radio- active fraction for binding studies, it was previously determined that liver and kidney membranes would yield essentially the same results as any tissue showing specific binding. The amount of radioactivity that could be dis- placed by excess unlabeled prolactin (l ug/tube) was con- sidered to represent specific binding. The fractions demonstrating the highest specific binding were repurified on a Sephadex G-100 column (0.9 cm x 50 cm) and again tested for binding activity. Only the repurified fractions with the highest specific binding were used in the subse- quent experiments. C. Assay Procedure Subcellular fractions were assayed in quadruplicate in 12 x 75 mm disposable culture tubes. Each tube con- tained 0.1 ml (1251) iodoprolactin diluted in 1% BSA-tris buffer and a subcellular preparation (usually the 105,000 x g particulate membrane fraction) containing a 55 predetermined protein concentration in 0.4 m1 of tris buffer. Parallel incubations were performed containing the same reactants together with excess unlabeled ovine prolactin (l ug/tube). Again the final incubation volume was 0.5 ml. The incubations were terminated by the addition of 3 m1 tris buffer and the bound and free (lZSI) iodoprolactin was separated by centrifugation at 800 x g for 30 minutes. The resultant pellets were counted in an automatic gamma counter for 60 seconds each. Specific prolactin binding was the difference between cpm bound in the absence of excess unlabeled prolactin and that bound in its presence. III. Hormones and Drugs The following hormones and drugs were used: ovine prolactin (NIH-S-lO, 25.6 IU/mg); ovine GH (NIH-S-ll, 0.56 IU/mg); ovine LH (NIH-S-lS, 0.99 NIH—LH-S-l units/mg); ovine FSH (NIH-S-7, 1.15 NIH-FSH-S-l units/mg); ovine TSH (NIH-S-6, 2.47 USP units/mg); estradiol benzonate (EB) and testosterone propionate (TP) (Nutritional Biochemicals Corporation, Cleveland, Ohio) and ergocornine methane- sulfonate (ERG) (Sandoz Pharmaceuticals, Hanover, New Jersey). The 7,12-dimethylbenz (a)-anthracene (DMBA) was kindly provided by Dr. Paul Schurr, The Upjohn Co., Kalamazoo, Michigan. Purified rat prolactin (H-lO-lO-B) obtained from Dr. S. Ellis (NASA, Ames Research Center, Moffett Field, 56 California) was iodinated and used for the determination of serum prolactin by the radioimmunoassay method of Niswender et a1. (1969). Serum prolactin values are expressed in terms of NIAMDD-rat-prolactin-RP-l. 3H-l,2 testosterone (New England Nuclear, Cambridge, Mass.) was the labeled ligand used for the testosterone immunoassay established by Smith and Hafs (1973) with values expressed in terms of Sigma Chemical Company (St. Louis, Missouri) testosterone. IV. Statistical Analysis Unless otherwise stated, all data were statistically evaluated by analysis of variance and individual means compared by the Student—Newman-Keuls test at a 1% level of significance. EXPERIMENTAL I. Prolactin Binding Activity in Ventral Prostates, Seminal Vesicles and Testes of 40 and 70 Day Old Male Rats: Effects of Testosterone A. Objectives Many reports have suggested a role for prolactin alone or as a synergist with testosterone in maintaining the weight and functional integrity of male accessory sex organs (see Review of Literature, III. Functions of Prolactin, E. Male Accessory Sex Organs). Since the bind- ing of prolactin to high affinity membrane sites is believed to initiate prolactin dependent cellular events, it was of interest to determine whether Specific prolactin binding sites existed in ventral prostates, seminal vesicles and testes of immature and mature male rats and, if so, to investigate the effects of testosterone on pro- lactin binding activity. B. Materials and Methods Intact male rats 30 or 60 days of age were injected SC with either 1 mg testosterone prOpionate (TP) in 0.1 m1 corn oil or with vehicle alone (controls) for 10 days. 57 58 On the 11th day all rats were killed and their testes, seminal vesicles and ventral prostates removed, dissected free of fat and other adherent tissue and frozen on dry ice. Just prior to homogenization, the capsule surrounding each testis was removed and discarded. Subcellular fractions of decapsulated testes, seminal vesicles and ventral prostates were prepared and the specific binding of labeled prolactin was determined as previously described (see Materials and Methods, I. Prolactin Binding Assay). Using a pooled source of prostatic membranes the time course of binding at 4C, the effect of membrane protein concentration and the binding specificity for prolactin were determined. C. Results Figure 1 illustrates the time course of specific binding of labeled prolactin to prostatic membranes. Since a high level of specific binding at 4C was observed at 48 hours, this time was selected for subsequent incubations. 1251) iodo- Figure 2 shows that the specific binding of ( prolactin increased linearly with the amount of membrane protein added up to 400 ug protein per reaction tube. In order to insure an adequate number of assay replications for each tissue sample, 200 ug of membrane protein was chosen for the routine incubations. Figure 3 shows that levels of TSH, LH or FSH as high as 1000 ng each were 125 unable to displace I labeled prolactin from the prostatic 59 BINDONG or ‘2‘I-Pni [cpm no“! :wsteev9?§?i§¥ Fig. l.——Time course of the binding of (1251) iodoprolactin to prostatic meTBEane preparations. Approximately 72,000 cpm of ( I) iodoprolactin were incubated with 200 ug membrane protein at 4C. Specific binding (SB) was the difference between cpm bound in the absence of excess unlabeled prolactin (TB) and that bound in its presence (NSB). 60 :6" 2 X E 10* a .3 -| a a; m- 2‘. .8 6- 2; O .2 4' :5 8. U) 2- ' I I 260 400 600 800 ug PROTEIN Fig. 2.—-Effect of membrane prggein concentration on the specific binding of 1 I-labeisd prolactin. Approximately 62,000 cpm of ( 5I) iodoprolactin were incubated with prostatic membranes at 4C for 48 hours. Determination of specific binding was as described in Figure 1. 61 mm u'” “mm Iowa 8 u t a 4 I an to 100 noon www.mw Fig. 3.--Competitive displacement of (1251) iodoprolactin binding to prostatic membranes by various concen- trations of unlabeled hormones. The ordinate reflects the amount of labeled-prolactin specific- ally bound expressed as the percent of that bound in the absence of competing hormone. The abscissa represents the log of the amount of unlabeled hor- mone present in each reaction tube. All membranes were incubated at 4C for 48 hours with approxi- mately 72,000 cpm (1251) iodoprolactin. 62 membrane preparation. By contrast, unlabeled prolactin at concentrations greater than 0.4 ng per reaction tube readily displaced the labeled prolactin. The competition seen with levels of GH greater than 500 ng is believed to be due to slight prolactin contamination in this NIH preparation. The effects of TP injections on prolactin binding activity in testes, seminal vesicles and ventral prostates is shown in Figure 4. In each tissue the highest binding per 200 ug of protein was observed in the 105,000 x g membrane fraction. Injection of 1 mg TP for 10 days into intact 30 and 60 day old rats had little effect on prolactin binding activity in testes or seminal vesicles but signifi- cantly increased prolactin binding per 200 ug of protein derived from ventral prostates. There was no significant difference between the binding values of the two age groups. It is noteworthy that relative binding differences between control and TP treated groups were seen in equal amounts of protein from the 14,500 x g pellets although fewer counts were bound. D. Conclusions This study shows that particulate membrane frac- tions of rat ventral prostates have a greater prolactin binding ability than rat testes or seminal vesicles. No significant binding difference was noted in preparations obtained from 40 or 70 day old rats. Testosterone 63 10" § 12" Specific Binding labeled-Pd (Pinon!) 7 fit— ‘lPCTP CTPC C Lima-9"“ fears a” "ME-v” “'73" 788‘"! SIIINAL VII‘I’RAL VISICLI PROSTAYI Fig. 4.-—The effects of testosterone propionate (TP) injections on specific prolactin binding activity in testes, seminal vesicles and ventral prostates of 40 and 70 day old rats. Each bar represents the mean binding activity in tissues obtained from 10 animals. Two ventral prostates from rats within the same treatment group were pooled before homogenization and subsequently treated as a single sample. Testes and seminal vesicles were assayed individually. Each sample was assayed in quadruplicate. The standard error of the mean is indicated by the ventricle line in the middle of the bar. Membranes were incubated with approxi- mately 62,000 cpm of (1251) iodoprolactin for 48 hours at 4C. 64 increased specific prolactin binding in ventral prostates but had no significant effect on binding in testes or seminal vesicles. The specific binding of prolactin at 4C was shown to be time dependent and to increase linearly with the amount of membrane protein. The binding specificity for prolactin was demonstrated by displacement with unlabeled prolactin and inability of TSH, LH, FSH or GH to readily compete with labeled prolactin. II. Effects of Castration, Testosterone, Estradiol and Prolactin on Specific Prolactin Binding Activity—in Ventral Prostates of Male Rats A. Objectives The previous study indicated that testosterone increases specific prolactin binding to ventral prostates of rats. In order to further verify any testosterone influence on prolactin binding activity in ventral prostates it was necessary to demonstrate an effect of testosterone deficiency (castration). Since it was proposed that pro- lactin synergizes with testosterone in controlling pro- static function, the effect of prolactin alone or combined with testosterone on prolactin binding activity was ascer- tained. Other workers (Posner et al., 1974; Gelato et al., 1975) have reported that estrogen increases specific ‘ prolactin binding in male and female rat livers, therefore it was also of interest to determine the effects of 65 estrogen on prolactin binding activity in ventral pros- tates. B. Materials and Methods In the first experiment, intact male rats 30 days of age were injected SC with either 1 mg unlabeled prolactin dissolved in 0.85% NaCl (saline) adjusted to pH 8.2 with 0.1 N NaOH, or with saline at pH 8.2 (controls) for 5 days. The control rats were killed 2 hours after the last injection. The rats injected with prolactin were killed 2, 26 or 74 hours after their last injection. All ventral prostates were excised and frozen. In the second experiment mature male rats, 200-225 g were castrated and treatment was begun on the following day. The castrated rats were injected SC for 10 days as follows: (a) castrate controls, 0.1 m1 corn oil; (b) 1 mg prolactin/0.1 ml saline at pH 8.2; (c) 0.5 mg testosterone propionate (TP)/0.1 ml corn oil; (d) 1 mg TP/0.1 m1 corn oil; (e) 1 mg TP/0.l ml corn oil and 1 mg prolactin/0.1 ml. An intact control group received injections of 0.1 m1 corn oil. All rats were killed 26 hours after the last injection. In the third experiment, 5 groups of rats were treated as in experiment 2, except that 1 mg prolactin was given together with 0.5 mg TP, and treatment with 1 mg TP alone was deleted. Also, the rats were injected for 5 instead of 10 days. ’These rats were killed 26 hours after 66 the last injection and their ventral prostates were removed and frozen. Four additional groups in this experiment were treated as follows: (a) intact controls, 0.1 ml corn oil; (b) castrated controls, 0.1 ml corn oil; (c) castrated and 2 ug estradiol benzoate (EB)/O.l ml corn oil; (d) castrated and 25 ug EB/O.l ml corn oil. Each of these 4 groups also received 3 injections of 1 mg ergocornine/kg BW over a 26 hour period following their treatments in order to reduce endogenous blood levels of prolactin. Ergocornine (a dopamine agonist) has previously been shown to counteract an estrogen stimulation of prolactin release (Lu et al., 1971). These rats were killed 4 hours after the last ergocornine injection, and blood was collected from the decapitated trunk. The ventral prostates were excised and frozen. The blood was allowed to clot at 4C and the serum separated and stored at —20C until radioimmunoassayed for prolactin by the method of Niswender et a1. (1969). Specific prolactin binding activity was determined in each of the foregoing experiments before the beginning of succeeding experiment. Data was evaluated by analysis of variance and individual means compared by the Student- Newmans-Keuls test. Scatchard functions were derived from competitive displacement of specifically bound (125I) iodoprolactin by graded doses of unlabeled prolactin. The slopes (generated by linear regression) of the bound/free prolactin ratio as 67 a function of free prolactin yielded the negative reciprocal of the apparent dissociation constant (-l/Kd). The x- intercept produced the total labeled prolactin binding capacity. C. Results Table 1 shows that injection of 1 mg unlabeled prolactin for 5 days into intact 30 day old rats resulted 125I labeled prolactin bound in a significant decrease in when the rats were killed 2 hours after the last injection. Prolactin binding in prostates from intact rats killed 26 or 74 hours after the last prolactin injection did not differ significantly from the controls. There were no differences in the weight of the ventral prostates among the 4 groups of experiment 2. Castration for 10 days (Table 2) resulted in a significant decrease in prolactin binding per 200 ug particulate membrane protein. Injections of 1 mg prolactin for 10 days into castrates had no significant effect on prostatic binding of labeled prolactin. However, daily injections of 0.5 or 1.0 mg TP into orchidectomized rats significantly raised prolactin binding. The prostatic prolactin binding activity in castrated rats injected daily with the combination of 1 mg TP and 1 mg prolactin was not significantly different from that seen with 1 mg TP alone. Scatchard analysis of competitive displacement of labeled prolactin by graded doses of unlabeled prolactin 68 HHm NHV mo Emu oom.~m pamumsflxoummm auHB omumnsosfl mum3 mmcanEmz .ao.o v m an mamme Hmsuo Eoum ucwummuao mawcmoHMHcmflmo .Uv um muson we now cHuomHoumoooH n .mumofiamsupmsv CH owhmmmm comm “memEMm omaoom mo uwnEdzm 2mm H some mm ommmmumxm mosam> HH< ONO H OOmm OHm H OOmm oOOO H OOOH ovm H mmh¢ O.~ H m.om Ame OH HOOV as H .cHHomHoHa O.H H O.OO Ame OH HONV as H .cHHomHon m.~ H O.OO Hmc OH Amy me H .cHHomHon m.~ H O.OH mime OH Ame mHoHHOoo samuoum on com o (m cos: n E o ACOHuomncfl umma mumm mo .02 Hmumm mnsomv usmEummuB 3m m OOHEs unmwmz mumumoum amuucw> mufl>fluom mcflpcfln .mumu UOMUGH mo mmumumoum Hmuucm> ca oamwommm sfluomaoum so mGOHuommca :Huomaoum mo muommmmlu.a manna 69 .Ho.o v m H6 Hmnuo comm Eoum ucmHmMMHU waucmoHMHsmHm mum muawuomummsm ucmHmMMHo £HH3 mcmw2m.m.© .me H Gmmzo .mumowamsuomsv CH Um>Mmmm comm “mmHQEMm pmHoom mo HmQEszn .oH Hm mHsoz OH How Ammooofl HHmmHv mo Emu ooo.mw mHmumEonummm £HH3 pmumnsocfl mum3 mmcmunfimzm mnmm H Omom mH.O H “.mmH OH + my musm.mew mOHm H HNOH m0.0H H ~.OOH OH me me H .Hmmo cvhv H mmmm mp.m H «.mOH OH me me m.O .Hmmo HOHm H OMOH HVHBO H 0.0H HOV OH Hmm as H .Hmmo HOO H «mm Hvm.O H 0.0 HOV OH mHouucoo .Hmmo m.ommm H mmhm o.m H n.vn namv oH mHOHHcoo .HomucH aHmHOHa m: OOm 3m m OOmes mHmm Ho .02 mama OH Hon cocoon Emu oucmflmz mumumoum Hmuucm> HcmEummHB m.mpmu Hummov omumuummo mo mmumumoum Hmnpcm> CH wuflbfluom mCHocHQ Hmm OHMHommm so Hmmv mumoNsmn Howpmuumm Ho Hammv :HHUMHOHQ H0\psm Away mumsoflmoum msouwumoummu mo muowMMM|a.m magma 7O 15) revealed the presence of a low concentration (8.8 x 10- moles/200 ug protein) of binding sites with high apparent ll affinity (Kd = 7.1 x 10- M) in the intact control mem— brane preparations. The concentration of high affinity binding sites was greatly reduced by castration (1.2 x 10-15 moles/200 ug protein), and treatment for 5 days with 0.5 mg testosterone propionate returned the concentration of bind- 15 moles/200 ug ing sites towards normal values (5.7 x 10— protein). The dissociation constants were not significantly altered in the castrated groups. Table 3 again shows that castration reduced pro- lactin binding, and that injections of 0.5 mg TP for 5 days into castrates returned prolactin binding to intact values. Prolactin injections alone clearly had no significant effect on the binding of labeled prolactin and again failed to augment the action of TP. Administration of ergocornine to intact or castrate controls did not influence the bind- ing of labeled prolactin, and injections of 2.0 ug EB for 5 days into castrate rats followed by 3 injections of ergocornine in 26 hours also did not significantly influ- ence prolactin binding in the ventral prostate. However, injections of 25 ug EB into castrates followed by ergo- cornine decreased prostatic prolactin binding at a 5% level of significance. Neither dose of EB followed by ergocornine had a significant effect on the ventral prostate weight of castrated rats. The circulating levels of 71 wmm mem H Nov m.~ H H.mH co.~ H n.mH Hmv w + mm ms N Hummuv HmH - Ovm O.O - n.HH O.O - O.mH HMO O omm m + + U + + mHOHucoo Hummuv u . n . . I . 0mm HOOH + ommv m H + m HH OH H + m OO O + mHoHucoo mochH - . - . Hmm as H m.oovm + vmmo mv n + m moa m + m9 m5 m.o Hummuv mva H mmv Um.o H o.mH Hmv o Hum 08 H Hummov movm H Hmvn m~.oH H m.NHH m m9 m8 m.o Hummuv movm H mvm m.m H m.¢~ o>.o H m.~H mHmv m mHouusoo Hummov n.0mov H ovum m.~ H m.nH oo.v H m.mn m mHouucoo uomucH sHmHOHQ ms bow HE\mc 3m ooa\me mumn mhmw m Dodson Emu .Hmm Edumm .HcmHmz mumumoum mo .02 How uswsummue .mumu Hummov cmumnummo mo mmumumoum Hmuucm> :H >HH>HHUM mcHocHn :HuowHoum UHMHowmm so Ammo mumoNcmn HoHomHHmm can memv mchHooome .Hmav mHMGOHQOHQ wcoumumoumwu .Aqmmv cHHomHoum mo muommmmuu.m mHnms 72 .UoHHmm HcoEHmmuH amp m map mcHonHOH poHHmm H503 mm m Hw>o meHH m wmuomwsw was mcHsHooomHm 3m mx\me mac: .mo.o v m an mHouucoo Eoum ucmumwwflw >HucmoHMHcmHm ma cmmE 0:90 .Ho.o v m Hm Hwnuo comm Eoum Hcmummmflo waucmoHMHcmHm mum mumwuomnmmsm usmeMMHU SHH3 mamm2m.w.c.o .Uv Hm muson mv How :HHUMHOHQOUOH IAHmNHV mo Emu 00H.hn mHmHmEonummm £HH3 pmumnsocH mHm3 mmcmunamzn .mHmoHHmsuomsU cH pmmmmmm comm “mmHmEmm anoom mo HwnEszm .Emm H com: mm ommmmnmxm mmsHm> Had m.mnh H va m.H H m.m~ UH.m H «.ma Amy m 0mm + mm on mm Hummov :Hmuoum on com HE\@s 3m ooa\mE mHmH mhmc m canon Emu .Hmm Edumm .ucmflm3 mumumoum mo .02 How usmEHmmHB a .wmscflusooll.m manna 73 prolactin were consistently low (< 25 ng/ml) in the groups given ergocornine as well as in the intact and castrate controls not given ergocornine. D. Conclusions This study demonstrates that castration decreases and testosterone increases prolactin binding to a particu- late membrane fraction of rat ventral prostate. Scatchard analysis revealed that the concentration of high affinity prolactin binding sites was decreased by castration and returned toward normal by testosterone treatment. The first experiment clearly demonstrates that injections of unlabeled prolactin can inhibit £2.2iEEE binding of labeled prolactin. However, since the binding of labeled prolactin was at the control level 26 and 74 hours after the last prolactin injection, it appears that prolactin does not markedly alter the detection of pros- tatic prolactin binding activity after 26 hours. This does not preclude the possibility that prolactin can influence prolactin binding in other tissues or in prolactin deficient rats. Indeed recent studies (Posner et al., 1975; Costlow et al., 1975) have suggested that prolactin can increase prolactin binding to hepatic membranes of hypophysectomized rats. Experiments 2 and 3 show that prolactin in com- bination with TP did not increase prolactin binding or prostatic weight over that produced by TP alone. It is 74 possible that the doses of TP used were too large and masked any stimulatory action by prolactin. However, it seems unlikely that 0.5 mg alone (experiment 3) stimulated maximal prolactin binding since 1.0 mg TP produced even greater prolactin binding activity (experiment 2). The results of experiment 3 suggest that large doses of estrogen injected into castrates can decrease prostatic prolactin binding. Since other studies (Posner et al., 1974; Gelato et al., 1975) have suggested that estrogen increases prolactin binding in rat livers, it appears that the mechanisms regulating prolactin binding sites can be different in different tissues. It subse- quently has been shown that estrogen in female rats and testosterone in male rats decrease specific prolactin binding activity in kidneys and adrenal glands while castration increased binding in kidney of both sexes and in adrenals of female rats (Marshall at al., 1976). III. Effects of Ergocornine Injections on Specific Prolactin Binding Activity in Ventral Prostates of Male Rats A. Objectives The preceding study suggested that high levels of circulating prolactin can conceal the detection of pro- lactin binding sites. Presumably the binding of endogenous prolactin renders the receptor sites inaccessible for in vitro binding of labeled prolactin. The previous work also 75 suggested that 26 hours after the last injection of pro- lactin, the available prolactin binding sites have returned to control values. However, unlike injected prolactin that wanes with a half-life of approximately 5 minutes (Koch et al., 1971), other agents, notably estrogen, will stimulate the release of endogenous prolactin many hours after its last injection. Ergocornine has been reported to inhibit prolactin release and to counteract estrogen stimulation of prolactin secretion (Wuttke et al., 1971; Lu and Meites, 1971). Although it was shown in the previous study that injections of ergocornine given over 26 hours did not significantly alter prolactin binding activity, it was not known whether these injections altered the binding capacity or affinity for prolactin. An increase in one of these parameters might compensate for a reduction in the other. As a result a change in prolactin binding activity might not be observed. The present study was undertaken to see the effects of once daily injections of ergocornine on prolactin binding activity and to determine whether multiple injections of ergocornine given over 26 hours influenced the binding capacity or affinity for prolactin in ventral prostates. B. Materials and Methods Intact male rats, 200—225 g, were injected once daily with either 1 mg or 2 mg ergocornine/kg BW or with the vehicle (0.87% NaCl containing 3% ethanol) alone 76 (controls) for 2 or 20 days. All rats were killed approxi- mately 2 hours after the last injection and blood collected from the decapitated trunks. The ventral prostates were removed and frozen on dry ice. Serum was separated from the blood and later radioimmunoassayed for circulating prolactin (Niswender et al., 1969). Particulate membrane fractions of the ventral prostates were assayed for specific prolactin binding activity. In a second experiment, 3 injections of 1 mg ergocornine/kg BW or vehicle alone (controls) were adminis- tered to intact male rats, 200-225 g, at 8 hour intervals. All rats were killed 4 hours after the last injection. Blood was collected and later assayed for prolactin. Ventral prostates were removed and subsequently assayed for prolactin binding activity. Membranes from ergocornine treated or control rats were separately pooled and used for Scatchard analysis. Binding capacity and affinity for prolactin were derived from the competitive displacement 12 of ( 5I) iodoprolactin binding by unlabeled prolactin. C. lResults Table 4, experiment 1 shows that once daily injections of 1 mg or 2 mg ergocornine/kg BW for 2 or 20 days had no significant effect on the specific binding of labeled prolactin to prostatic membranes. Moreover, 3 injections of 1 mg ergocornine/kg BW given within 24 hours had no significant effect on specific prolactin binding 77 .Ho.o v m an mHouucoo Scum HGMHMMMHo haucmoHMHsmHm mum memo: Q .o« Hm muse: OH How cHHomH IOHQOUOH HHmmHv Emu ooo.om aameEonummm £HH3 UOHMQDUGH mum3 mmsmunEmzw .zmm H sum: mm pommmumxm mmfiam> Ham vm¢ H mvm¢ mmH H mwmw mm H wmvv 0mm H Hhm¢ mom H «Haw mmn H ommm mam H mhmv ~.H H.O OH Ame HO mchuooomHm m.o H «.mH ca maouucoo +l n Ham mx\wmoov musom vm How musom m mum>m omuomnsH Hm HcmEHHmmxm H.OH 0H Ame NV mcHCHooomHm m o O 'H m.o H m.m 0H Ame av mcHGHooomHm ABm mx\mm0Uv mama m How haHmo mono pmuommsH am.o H h.@ ca Ame NV mcwsuooomum Qw.o H m.m OH Ame av mcHsnooomum 0.0 H O.mH OH mHoHHcoo ABm ox\mmoov mama om How maHmn 00:0 wmuomnsH "H HamEHHmmxm cfimuoum ms com mpcson emu HHE\vcv mumm CHHOMHOHm Esuwm mo .02 .mmumumoum Hmuucm> CH muH>HHom mswoan sHuomaoum UHMHowmw can CHHUMHOHQ Edumm so mcoHHowncH mcHsuooomHm mo muommmmll.v magma 78 (experiment 2) and no apparent effect on the binding capacity or affinity for prolactin. The dissociation con- 125 stants of the ( I) iodoprolactin binding to membrane preparations of control and ergocornine treated rats were 11 M and 2.6 x 10’11 2.0 x 10- M and the prolactin binding capacities per 200 ug protein were 3.1 and 3.9 fmoles respectively. In all cases serum prolactin levels were significantly lower in ergocornine treated rats than in control animals at the time of killing. D. Conclusions This study demonstrates that a daily reduction in circulating prolactin induced by single injection of ergo- cornine does not significantly influence the detection of prolactin binding sites. However, this does not preclude the possibility that a more extreme or prolonged reduction in circulating prolactin has an effect on prolactin binding activity. Deprivation of prolactin may contribute to the marked reduction in prolactin binding sites observed in hypophysectomized rats (Posner et al., 1974). Most importantly this study shows that injections of ergocornine decrease serum prolactin levels for 24 hours but do not alter the membrane affinity for prolactin or the number of prolactin binding sites. This supports the use of ergocornine in those studies where high levels of circulating prolactin could mask a treatment effect. For example, it was previously shown that large doses of 79 estrogen reduced prolactin binding activity in ventral prostates of castrated rats. If ergocornine had not been used to offset estrogen stimulation of prolactin release, the reduced binding of labeled prolactin may have been interpreted as representing competition by endogenous prolactin and not the effect of estrogen. IV. Time Course Effects of Single Injections of Testosterone or Prolactin on Prolactin Binding Activity in Ventral Prostates of'Intact Rats A. Objectives The foregoing studies indicated that testosterone stimulated and high levels of circulating prolactin apparently masked prolactin binding sites in ventral pros- tates. The present study was undertaken to investigate the time course of these effects. B. Materials and Methods Intact male rats, 225-250 grams, were given a single tail vein injection of 1 mg ovine-prolactin dis- solved in 0.85% NaCl at pH 8.2 (saline) or with saline alone (controls). Control rats were killed by decapitation either 5 minutes or 8 days later. Rats injected with prolactin were killed at various time intervals from 5 minutes to 8 days. All ventral prostates were removed, 80 frozen and subsequently assayed for binding of 1251- labeled prolactin. In a second study, intact male rats were injected SQ with 1 mg testosterone propionate (TP) dissolved in corn oil or corn oil alone (controls). Control rats were killed either 5 minutes or 20 days later. Rats given TP were killed at various times from 12.5 hours to 20 days after injection. Ventral prostates were excized, frozen and later assayed for prolactin binding activity. C. Results 52:) Figure 5 shows that the in zitgg binding of (12 iodoprolactin was significantly reduced in membrane prepa- rations obtained 5, 15 or 30 minutes after an intravenous injection of 1 mg unlabeled_prolactin. (The binding of labeled prolactin to ventral prostates removed 1 hour to 8 days after an injection of unlabeled prolactin was not significantly different from control values. Figure 6 shows that a single, subcutaneous injection of 1 mg TP significantly increased the binding of labeled prolactin to ventral prostates removed 3, 4 or 5 days later. The binding of labeled prolactin to ventral prostates removed from 12.5 hours to 2 days and from 6 days to 20 days after TP injection did not differ significantly from control values. Fig. 81 ‘7 f I ? I E Y‘ 0‘ l srecmc amomc no Matteo-Puma...” u 1 " 5" Is” 30" 1h 2h 4h ah 12h corn. Id 24 4d ad 33” nmsArnanNucnou t 5.--Time course effects of a single intravenous injection of unlabeled prolactin on in vitro binding of labeled prolactin. Specific binding is expressed as a percgntage of the approximately 68,000 total cpm (1 51) iodoprolactin used in each incubation. A * indicates a significant difference as compared to controls (p < 0.01), while the vertical line at each dose represents the SEM. The numbers atop each bar indicate the number of individual prostates, each assayed in quadruplicate. Fig. 82 PRL (Dunn!) o 3‘ u I I l I I l N I U (II A n 1 l SPECIFIC BINDING of LABELS!)- l .n I 5' 125k 1d 2d 3d 4d 54 6d 8d 0d 12d 154 20d 20‘ CWT. CONT. TIME AFIEI INJECYION 6.--Time course effects of a single subcutaneous injection of testosterone propionate on in vitro binding of labeled prolactin. Specific binding is expressed as a percsntage of the approximately 68,000 total cpm (1 5I) iodoprolactin used in each incubation. A * indicates a significant differ- ence as compared to controls (p < 0.01) while the vertical line at each dose represents the SEM. The numbers atop each bar indicate the number of individual prostates, each assayed in quadrupli- cate. 83 D. Conclusions These results demonstrate the time course effects of single injections of prolactin on testosterone on pro- lactin binding activity in ventral prostates. In agree- ment with an earlier study (Kledzik et al., 1976), high levels of circulating prolactin reduced the subsequent binding of labeled prolactin. The present study suggests that unlabeled prolactin is bound to prostatic membranes in ziyg within minutes and that the available prolactin binding sites are at control values within an hour. An explanation for time discrepancies between this and an earlier study (Kledzik et al., 1976) in which injected prolactin decreased the binding of labeled prolactin to prostates removed 2 hours after injection, is the way in which unlabeled prolactin was administered. In the current study, unlabeled prolactin was injected intravenously whereas earlier prolactin was administered by a slower subcutaneous route. Shiu and Friesen (1974) have reported that 50% of labeled prolactin is dissociated from mammary gland binding sites in yitrg at 37C within 5 hours. If injected prolactin had increased its own binding sites and then masked any immediate detection of them, it seems likely that an increase in the binding of labeled prolactin would have been observed at one of the many intervals tested. In general, I believe these results lend further support to the view that prolactin injections do not 84 markedly influence prolactin binding activity in ventral prostates beyond that of masking the detection of the binding sites. The time course of testosterone stimulation of prolactin binding is consistent with reported effects of testosterone in ventral prostates. Fujii and Viller (1967, 1968) have noted that a single SC injection of TP produces maximal increases in protein synthesis and prostatic weight 3 to 4 days after injection. The present results show maximal stimulation of prolactin binding activity in prostates 3 to 5 days after TP injection. Since prolactin binding sites have been reported to be largely protein in nature (Posner, 1975), it is encouraging to find that testosterone stimulation of prolactin binding activity coincides temporally with stimulation of protein synthesis. V. Serum Prolactin, Testosterone and Prolactin Receptors in Ventral Prostates of Aging Male Rats A. Objectives Senescence has been regarded by many investigators (Roth and Adelman, 1975) as altered cellular responsiveness to biochemical stimuli. The binding of a hormone to spe- cific receptor sites is thought to initiate the cellular response to that hormone. Recent studies (Kelly et al., 1974; Gelato, 1975) demonstrated early developmental changes in prolactin binding sites, however, prolactin 85 binding activity in old aged rats has not yet been deter- mined. The purpose of the present study was to investigate the effects of aging upon the prolactin binding activity in ventral prostates and to correlate the binding with serum prolactin and testosterone levels. B. Materials and Methods Long-Evans male rats, 2%, 10 or 20 month old were killed by decapitation. Blood was collected from the trunk and the ventral prostates excised and frozen. The blood was allowed to clot at 4C and the serum separated and stored frozen until assayed (in collaboration with H. H. Huang and S. Marshall) for prolactin by the method of Niswender et a1. (1969). Serum testosterone was deter- mined (in collaboration with J. F. Bruni) by the method of Smith and Hafs (1973). Specific prolactin binding activity was determined in membrane fractions of each ventral prostate. In a later experiment, 3 injections of 1 mg ergocornine/kg BW were given to 2% and 20 month old rats over a 24 hour period before killing. A 2% month old con- trol group received vehicle (0.87% saline-3% ethanol) injections only. All rats were killed 4 hours after the last injection. Blood collected from the decapitated trunks was later assayed for serum prolactin levels. Ventral prostates were removed, and subsequently assayed for prolactin binding activity in membrane fractions. 86 C. Results Table 5 (experiment 1) shows that a Specific prolactin binding in ventral prostates of 10 and 20 month old rats was reduced to approximately 50% and 3% of that seen in 2% month old rats. There was no difference in serum pro- lactin between 28 and 10 month old groups but by 20 months, the circulating prolactin had risen 3-fold. Serum testo- sterone was significantly less in 10 month old rats as compared to 28 month old animals, and an even greater reduction of serum testosterone was found in 20 month old rats. Table 5 (experiment 2) shows again that prolactin binding activity was markedly reduced in ventral prostates of 20 month old rats as compared to 2% month old rats. Serum prolactin levels were similar in these groups that received ergocornine injections. As can be seen in the 2% month old groups, ergocornine alone had no significant effect on prolactin binding activity. D. Conclusions These results demonstrate that aging is associated with significant decreases in binding of labeled prolactin to ventral prostates, increases in serum prolactin and reductions in serum testosterone. It seems unlikely that the decrease in specific prolactin binding was a result of increased endogenous prolactin occupying the binding sites. A significant reduction in binding of labeled 87 .msHHHHx mnemmn ooHHma Hsos Hm m Hm>o pwHmeHcHfiom mum3 3m mx\msHsHooomHm me H mo mGOHHommsH doused .Ho.o v m an Hmzuo comm Eoum HGmHmHMHv >HusmoHMHsmHm mum mHmHHomHOQSm HsmHOMMHv 5HH3 mcmmzo.n .Uw Hm mason mv How CHHUMHOHQOUOHIHHmNHV mo Hm HcmEHmexmv Emu ooo.mm cam AH HsmEHHmmxwv Emu ooo.om mHmHmEonummm nHHz mHMOHHmsucmsv :H omumnsosH onm3 mOCMHQEOZM .zmm H :00: mm ommmwumxm mmsHm> HH¢ nHvH H Hom nHm.o H mm.o ¢.m H o.mm m cmuomnsH 0mm .sucoe om hmv H mHmm mm.v H Nb.¢H ~.m H m.mm h powuomnsH wmm .nucofi m.~ mmm H mHmm mv.m H mh.m m.m H m.vm h mHouusoo .nusoe m.~ m HawEHHmmxm Uvm H omH am. H ¢~.N no.mv H m.mMH m canoe om nmmm H mHvH nNm. H mh.m m.mH H m.mm m nusoe OH «mm H mmmm mm. H ¢.m >.HH H N.H¢ m canoe m.m H HcmEHHmmxm ammuoum as com HHE\msV HHE\msv mumm mod musson Emu msoumumoumme Esnmm :HuomHoum Ednmm mo .02 .mumn mHmE qumm mo mmumumoum Hmuucm> sH >HH>HHom musan cHHUMHOHm OHMHommm can msoumumoummu .GHHUMHOHQ Esumm .m anme 88 prolactin was noted in ventral prostates from 10 month old rats as compared to prostates from 2% month old animals although no difference in serum prolactin was found. More- over, an age related decrease in prolactin binding was seen in rats injected with ergocornine used to minimize endo- genous prolactin competition. Since previous experiments established that castration decreases and testosterone increases prolactin binding in ventral prostates, the reduction in serum testosterone observed in 10 and 20 month old rats probably was a major factor in the reduction of prolactin binding activity observed in these old age groups. VI. Prolactin Binding Activity in the Crop Sacs of Juvenile, Mature, Parent and Prolactin Injected Pigeons A. Objectives Prolactin is the endogenous stimulus for the for- mation of "crop milk" near the end of the pigeon's incu- bation cycle, and prolactin injections have been shown to cause rapid proliferation of the epithelial lining of the crOp sac (Beans and Meyer, 1931; Riddle and Braucher, 1931). The crop sac response to systemic or locally injected prolactin has become the traditional bioassay for this hormone (Riddle et al., 1933; Lyons, 1937; Nicoll, 1967). Since specific prolactin binding sites have been reported to be present in a variety of prolactin responsive tissues (Turkington and Frantz, 1972; Posner et al., 1974), it was 89 of interest to determine the prolactin binding activity of pigeon crop sacs. In this study crop sacs from juvenile, mature, parent pigeons with crop milk, and pigeons injected with prolactin, were removed and assayed for specific binding of radioiodinated prolactin. B. Materials and Methods All birds were of the White Carneau Strain. No sex determination was made, as it has been shown that sex does not influence the crop sac response to prolactin (Riddle and Braucher, 1931). The juvenile pigeons used in this investigation were 4 weeks old, the mature pigeons were 6 months old and 8-12 month old parent pigeons were used 7-10 days after hatching of their young. Six juvenile, 5 parent and 6 mature pigeons were killed on the day of arrival by cervical dislocation. The cr0ps were excised, washed in cold tap water, frozen on dry ice and stored at -20C for less than 7 days. The remaining 8 mature birds were injected subcutaneously in the loose skin of the lower abdomen once daily for 4 days with 250 mg ovine prolactin dissolved in 0.85% NaCl adjusted to pH 8.3 with 0.1 N NaOH. On the fifth day, the 8 pigeons given prolactin injections were killed, and their crop sacs removed and frozen. All frozen crop sacs were later thawed, minced in .3 M sucrose with a Waring minicup blender and particulate membrane preparations prepared as 90 previously described (see Materials and Methods, I. Prolactin Binding Assay). The time course of binding at 4C, the effect of membrane protein concentration and the binding specificity for prolactin were determined in pooled membrane fractions obtained from proliferated crop sacs. C. Results Figure 7 shows that the highest level of specific prolactin binding at 4C was at 48 hours and Figure 8 shows that the specific binding of labeled prolactin increased linearly with the amount of membrane protein. Figure 9 shows the results of competitive displacement of labeled prolactin by various concentrations of unlabeled hormones. Only unlabeled prolactin readily displaced the binding of labeled prolactin to 600 ug of membrane protein. Similar effects of incubation time, membrane protein concentration and binding specificity were previously described for prolactin binding activity in ventral prostates. Table 6 shows the specific cpm bound per 600 ug membrane protein in crop sacs of juvenile, mature, parent and mature pigeons injected with prolactin. CrOp sacs from juvenile pigeons contained approximately twice as much binding activity as an equivalent amount of protein from mature pigeons. Neither group showed visible proliferation of the cr0p sac epithelium. Injections of prolactin for 4 days into mature pigeons produced a 5-6 fold increase in 91 _I (D l ><:::° I25 BINDING of l-PHOLACTIN(cpm no“) "8 7‘ 1‘ Fig. 7.--Time course of the binding of (1251) iodoprolactin to pigeon crop sac membrgne preparations. Approxi- mately 79,000 cpm of ( 2 I) iodoprolactin were incubated with 600 ug membrane protein at 4C. Specific binding (SB) is the difference between cpm bound in the absence of excess unlabeled prolactin (TB) and that bound in its presence (NSB) . 92 ”a 5 £ 01 fi 3's- Po . 3 3H . g g z- s a 260 ' as r a» ' so. NWTEIN Fig. 8.--Effect of membrane prpggin concentration on the specific binding of ( I) iodggrolactin. Approximately 79,000 cpm of ( I) iodoprolactin were incubated with crop sac membranes at 4C for 48 hours. Specific binding was determined as described in Figure 7. Fig. 9.--Competitive displacement of specific ( 93 i? mum POM.“ 3 I as I a 4 a an an Ice W mumps»... 125I) iodoprolactin to 600 ug of particulate membrane protein from proliferated crop sacs by various concentrations of unlabeled hormones. The ordinate represents the amount of radioactivity specifically bound to the membranes as a % of control in which no unlabeled hormone was present. The abscissa represents the log of the amount of unlabeled hormone present in each reaction tube. All membranes were incubated at 4C Sgr 48 hours with approximately 79,000 cpm of (1 I) iodopro- lactin. 94 Table 6.--Specific prolactin binding activity in 600 ug of crop sac microsomal protein. b . No. of cpm bounda’ Type Of Pigeon Pigeons 600 ug protein Juvenile 6 2204 i 466* Mature 6 1153 i 316 Parent 5 5072 i 529** Prolactin-Injected 8 6739 t 467** aMean 1 SEM bMembranes were igggbated at 4C for 48 hours with approximately 79,000 cpm I-o-prolactin. *P < 0.05 as compared to mature pigeons. **P < 0.01 as compared to all other groups. 95 specific cpm bound and the crop sacs appeared to be as well proliferated as in the parent pigeons. Proliferated crops from parent pigeons had prolactin binding activity considerably higher than in crops from unstimulated juvenile or mature birds, although less than in crop sacs of the prolactin injected pigeons. D. Conclusions These results show that the pigeon crop sac con- tains specific binding sites for prolactin, and that pro- liferation of the crop sac in response to prolactin is associated with an increase in prolactin binding activity. Crop sacs from mature pigeons have been reported to be more responsive to prolactin than crop sacs of young birds (Riddle et al., 1933). However, the present study demonstrated that unstimulated crOp sacs from adult pigeons had less binding activity than in the crops of juvenile birds. Difference in crop response between adult and juvenile birds to prolactin may have little to do with changes in membrane binding, but may reflect an immaturity of the intracellular mechanisms governing crop epithelial proliferation in the juvenile pigeon. 96 VII. Effects of High Doses of Estrogen on Prolactin Binding Activity and Growth of Carcinogen Induced Mammary Cancers in Rats A. Objectives Prolactin and/or low doses of estrogen are stimu- latory to mammary tumor growth in intact rats (Huggins et al., 1962; Meites, 1972). Large doses of estrogen increase serum prolactin but have an inhibitory effect on mammary tumor growth (Meites, 1972). Recent studies have sug- gested that large doses of estrogen may directly interfere with the stimulatory action of prolactin on mammary tumor tissue (Meites et al., 1971; Welsch and Rivera, 1972). Prolactin binding sites have been shown to be present in carcinogen-induced rat mammary tumors and a direct relation- ship has been reported between the growth response of these tumors to prolactin and their prolactin binding activity (Kelly et al., 1974). Since the action of prolactin on target tissues is thought to begin with the specific bind- ing to membrane receptor sites, it was of interest to determine the effects of large doses of estrogen on pro- lactin binding activity in mammary tumor tissue. B. Materials and Methods Mammary tumors were induced in 55-60 day old virgin female rats by a single intravenous injection of a lipid emulsion containing 5 mg of 7,12-dimethylhenz(a)anthracene (DMBA). Approximately 2% months later, when each rat had 97 developed at least one mammary tumor 1 cm in diameter or larger, the rats were randomly divided into groups and given daily SC injections for 10 days as follows: 1, 0.1 ml corn oil (controls); 2, 2.0 ug estradiol benzoate (EB) in 0.1 ml corn oil; 3, 25.0 ug EB in 0.1 ml corn oil. Immediately prior to, and at 5 day intervals during the treatment period, each mammary tumor was measured with calipers to the nearest mm for length, width, and depth. The sum of these measurements was determined for each tumor. Differ- ences between diameter sums before and after the treatment period were recorded as the growth index. Each rat also received 3 injections of 100 ug ergocornine/100 g BW during a 24 hour period after the 10 days of treatment in order to reduce circulating levels of prolactin and thereby minimize competition for prolactin binding sitesby the endogenous prolactin. All rats were killed approximately 4 hours after the last ergocornine injection and blood was collected from the decapitated trunk. Each palpable mammary tumor was excised, frozen on dry ice and stored at -20C until assayed 2 weeks later for prolactin binding activity. The blood was allowed to clot at 4C and the serum was separated and stored at -20C until radioimmunoassayed for circulating prolactin. The entire experiment was subsequently repeated with the following treatments: Group 1, 0.1 ml corn oil (controls); Group 2, 0.2 ug EB in 0.1 ml corn oil; Group 3, 98 10 ug BB in 0.1 ml corn oil; Group 4, 25 ug BB in 0.1 ml corn oil. C. Results Analysis of covariance disclosed that the data obtained from the two experiments could be combined for further statistical treatment, and hence the results are presented as from a single experiment. Figure 10 shows that mammary tumors in control rats increased .46 i .09 cm in their growth index during the 10 day treatment period. Daily injections of 0.2 ug EB resulted in a gain in tumor growth index of .63 t .70 cm, and rats given 2.0 ug EB daily showed an increase of .39 i .10 cm. By contrast, rats injected with 10.0 ug EB or 25.0 ug EB daily showed a significant decrease in tumor growth index of .31 i .13 cm and .39 i .11 cm, respectively, during the treatment period. 251) The time course of specific binding of (1 iodoprolactin to tumor membranes at 37C, 24C and 4C is shown in Figure 11. Because the highest level of specific binding observed was between 45 and 69 hours at 4C as well as at 14 hours at 24C, a convenient time of 48 hours at 4C was selected for subsequent incubations. Figure 12 shows that of the various polypeptide hormones tested, only unlabeled prolactin readily displaced the binding of 12 ( 5I) iodoprolactin to tumor membranes. A Scatchard plot of a competitive displacement response curve revealed the 99 E .8 I o .6 I x .§ 1 — .4 ' .= ‘5 2 .2 6 ”=82 II=3O "=36 a 0 g n=3o n=44 3. ~’-‘ I» + a . . 5.04 I . E a :! 26 (10 (L2 210 10 25 Dose of Estradiol Benzoate lug] Fig. 10.--Effects of estrogen treatment on tumor growth. The growth index is the difference between the sums of tumor length, width and depth before and after treatment. Analysis of variance was used to determine variations among groups, and the least-significant-difference test was used for all comparisons between treated and control groups; a * indicates a significant difference vs. controls at p < 0.01. The vertical lines at each dose represent the SEM. 100 B 3 /‘\\ g a. '5 E .3 I " ‘~ 7’/ 0 ‘\ g 11‘. a A A -- .e 2 6.5 14 21 4s 69 118 Incubation time I hours 1 Fig. ll.--Time course of specific binding of (1251) iodoprolactin to tumor membranes at 37C, 24C and 4C. The membrane preparations used for incubations were derived from a pooled source of 38 untreated DMBA-induced mammary tumors. Each reaction tube, incubated in quadruplicate, con- tained 300 ug mgmbrane protein and approximately 100,000 cpm (12 I) iodoprolactin. Parallel incubations were performed in the presence of excess (1 ug/tube) unlabeled oPRL. Specific binding, expressed as a % of total radioactivity used in each incubation, is the difference between cpm bound in the absence of excess unlabeled PRL and that bound in its presence. Since all values were replicates of a common membrane source the SEM at each point was negligible (< 0.1%). 101 0 3 l-oPRI. (cpm x 109) 125 u BINDING OF L 1 I l J L 1 1 1 l 1 ‘l 2 5 10 40 100 500 2000 UNLABELED HOMES Monograms) Fig. 12.--Competition of (1251) iodoprolactin and unlabeled hormones for binding to tumor membranes obtained from a pooled source. LH, FSH, TSH and GH were tested only at 1,000 ng per reaction tube. Incubations were carried out in quadruplicate at 4C for 48 hours. 102 presence of prolactin binding sites in tumor membranes with a dissociation constant (Kd) of 4.0 x 10.9 moles and a binding capacity of 130 femtomols per 300 ug protein (Figure 13). The effects of the administered doses of EB on specific prolactin binding activity in the DMBA-induced mammary tumors is shown in Figure 14. A linear regression analysis indicated a significant negative correlation (P < 0.01) between estrogen dose and the specific binding 1251) iodoprolactin to tumor membranes. However, of ( further statistical analysis showed that only the two largest doses of EB significantly reduced specific pro- lactin binding activity as compared to controls (P < 0.01). The circulating levels of prolactin in the ergocornine treated rats at the time of killing were determined to be consistently low (< 20 ng/ml) in all groups. D. Conclusions Specific binding sites for prolactin were detected in membrane preparations obtained from DMBA-induced mam- mary tumors in this study, in agreement with a previous report (Kelly et al., 1974). The binding of (1251) iodoprolactin was time and temperature dependent and sensitive to competitive displacement by as little as 0.5 ng unlabeled prolactin. A significant negative correlation was noted between administered doses of estrogen and the subsequent binding of prolactin to tumor cell membranes. 103 o 50 IOO f Moles Bound Fig. 13.--Scatchard analysis derived from a competitive inhibition curve. The ordinate represents the ratio of bound/free oPRL and the abscissa the number of femtomoles oPRL bound to tumor mem- branes. The abscissa intercept represents the total oPRL binding capacity for 300 ug of membrane protein. 104 [X] L. Specific Binding of “SI-oPRL 1 III-82 “=30 "=36 "'30 “844 0.0 0.2 2.0 10.0 25.0 Dose of Estradiol Bcnzoato lug] Fig. 14.--Effects of gigged doses of EB on the specific binding of ( I) iodoprolactin to mammary tumor membranes. Binding ranged from low (< 2%) to high (> 10%) in each group with no single tumor showing more than 18% specific binding. Vari- ations among groups and mean comparisons were statistically analyzed as described in Figure 10. A * indicates a significant difference vs. controls at p < 0.01 while the vertical lines at each dose represent the SEM. 105 Injections of 10 or 25 ug estradiol benzoate daily for 10 days effectively inhibited mammary tumor growth and signifi- cantly reduced specific prolactin binding to mammary tumor membranes. Thus, despite elevated circulating prolactin levels which normally result from high estrogen adminis- tration, the growth promoting action of prolactin on mammary tumors appears to be diminished. GENERAL DISCUSSION These data indicate that particulate membrane fractions of rat ventral prostates, pigeon crop sacs and DMBA-induced mammary tumors in rats contain specific bind- 125I) iodoprolactin. In contrast, membranes ing sites for ( obtained from rat testis or seminal vesicles did not appreciably bind labeled prolactin. Several reports have suggested that prolactin accentuates the effects of androgens in stimulating growth and function of prostates (Grayhack et al., 1955; Grayhack, 1963; Chase et al., 1957) and there is evidence that prolactin increases prostatic binding of testosterone. Thus, Lawrence and Landau (1965) observed that hypophysec- tomy decreased in givg uptake of radioactive testosterone by rat prostates, and Farnsworth (1972) reported that prolactin increased binding of testosterone by prostatic slices in yitrg. The data presented in this thesis demon— strates that castration decreases and testosterone increases the concentration of high affinity prolactin binding sites in ventral prostates. The influence of testosterone on the binding activity of prolactin, and of prolactin on 106 107 that of testosterone lends support to the view that they exert synergist actions on prostates. Although other studies have indicated that pro- 1actin increases prolactin binding activity in livers of hypophysectomized rats (Posner et al., 1975; Costlow et al., 1975), the present data suggests no such effect on ventral prostates of intact or castrated rats. Moreover, pro- lactin in combination with testosterone did not increase prolactin binding over that produced by testosterone alone. However, injections of unlabeled prolactin were able to significantly decrease the detectable prolactin binding sites within minutes following an intravenous injection. Presumably as a result of the higher levels of circulating prolactin, more prolactin binding sites were occupied and thus unavailable for the binding of labeled prolactin. Ergocornine was used to counteract any stimu- lation of prolactin release that would conceal an effect of treatment on prolactin binding activity. The pioneering studies of Huggins et a1. (1940, 1941) established the efficacy of estrogen therapy in androgen-dependent prostatic cancer. Although it has been shown that estrogen depresses blood testosterone levels (Alder et al., 1968), estrogen also is a potent stimulator of pituitary prolactin release (Chen and Meites, 1970; Nagasawa et al., 1969). Since prolactin has been reported to increase the affinity of the prostate for testosterone 108 (Farnsworth, 1972), it seems possible that prostatic tissue exposed to high circulating prolactin can more effectively concentrate the lower levels of androgen. The present data demonstrates that prolactin binding in the ventral prostate is reduced after a decrease in testosterone, i.e., after castration. Hence, if prolactin binding reflects biological activity, the ability of the prostate to concentrate testosterone may decrease with estrogen therapy even in the presence of high circulating prolactin. In addition, large doses of estrogen injected into castrated rats tend to further decrease prolactin binding. A role for prolactin in prostatic cancer has not been clearly established, but it is possible that the effectiveness of estrogen therapy on prostatic cancer is mediated in part by influencing prostatic prolactin bind- ing. Unlike ventral prostates, prolactin significantly stimulates prolactin binding activity in membrane fractions of pigeon crop sacs. The increase in prolactin binding activity in response to prolactin is associated with pro- liferation of the crop mucosa normally occurs during the second half of the incubation period. Near the end of incubation, and continuing approximately 2 weeks after hatching, secretion of "crop milk" is produced by desquama- tion of fat-laden epithelial cells (Beams and Meyer, 1931; Riddle and Braucher, 1931). Proliferation of the crop sac 109 mucosa and formation of "crop milk" in parent pigeons occurs in response to release of endogenous prolactin (Beams and Meyer, 1931; Riddle and Braucher, 1931). Using a cholchicine method, Lahr and Riddle (1938) reported increased mitotic rates in the pigeon crop sac throughout the 18 days of incubation. Injections of prolactin to virgin pigeons produced a crop response not unlike that in parent pigeons, and resulted in an increased mitotic rate. Whether the increase in mitosis is a direct consequence of prolactin binding has not yet been established. However, it appears to be significant that the increased prolactin binding activity is associated with proliferation of crop mucosa. In recent years, the existence of high affinity estrogen receptor proteins in mammary cancers has been demonstrated (Jensen et al., 1972) and the presence of such receptors appears to be of value in determining estrogen dependency of such cancers. Although Braunsberg et a1. (1973) has indicated that tumor regression induced by high estrogen therapy cannot be correlated with estradiol uptake and retention in human mammary tumor tissue, other workers (McGuire et al., 1975) have suggested a good correlation with tumor estrogen receptor values. Because the other important hormone in rat mammary tumor deve10p- ment and growth is prolactin, it was assumed for many years that the effectiveness of high estrogen treatment was 110 mediated by an inhibition of pituitary prolactin release. However, such an assumption was shown to be incorrect when Chen et a1. (1970) demonstrated that large as well as small doses of estrogen elevated serum prolactin levels in rats. Subsequently, Meites (1972) suggested that high estrogen doses may inhibit mammary tumor growth by interfering with the peripheral action of prolactin on the tumor tissue. The present results clearly indicate that large doses of estrogen significantly reduce prolactin binding to tumor membranes. Previous work in our laboratory also suggested that high doses of estrogen in vitro can decrease prolactin binding to slices of DMBA-induced mammary tumor (Bradley et al., 1975). Thus, despite elevated circulating pro- lactin levels which normally result from high estrogen administration, the growth-promoting action of prolactin on mammary tumors appears to be diminished. Prolactin binding activity in DMBA-induced mammary tumors previously has been shown to be correlated with the growth response to administered prolactin (Kelly et al., 1974). In general, the tumors with the greatest amount of prolactin receptor activity exhibited the greatest growth response to administered prolactin, and vice versa. If prolactin receptors are reduced in the mammary tumors by administration of high doses of estrogen, then the circu- lating prolactin would be expected to be less effective in promoting growth of the tumors, as actually observed in the 111 present work. This could then result in less tumor DNA synthesis from labeled thymidine, as reported by Welsch et a1. (1972). However, we also have observed that adminis- tration of relatively large doses of prolactin can overcome the inhibition by high doses of estrogen on DMBA-induced mammary tumor growth (Meites et al., 1971). The mechanism(s) involved is not readily apparent at present. It is possible that large doses of administered prolactin increase prolactin receptors or favorably alter estrogen receptor activity in the mammary tumor tissue. Prolactin has been reported to increase estrogen receptors in DMBA-induced mammary tumors in rats (Vignon and Rochefort, 1974). Throughout these studies I was careful to describe the specific binding of prolactin by membrane fractions as receptor or binding activity rather than receptors per se. This was to stress the uncertainty of any cellular events subsequent to binding and to emphasize that tissue binding of prolactin does not necessarily mean that prolactin receptors are present in every cell. Although it is gen- erally assumed that there is a direct relationship between the amount of receptor binding activity and the magnitude of a target tissue response to a hormone, it is possible that target cells have spare or redundant receptors. Moreover binding activity may not reflect absolute biological acti- vity, esPecially in wasting tissue (e.g., regressing mammary tumors), since the relation of membrane fractions to viable cells is known. It also is possible that prolactin 112 responsive tissues contain a mixed population of cells with respect to prolactin receptors and that the effects of testosterone, estrogen and prolactin.on prolactin binding activity were to alter the relative number or com— position of receptor containing cells. For example, a change in intracellular membranes relative to plasma membranes can alter the receptor content expressed on the basis of membrane protein. Thus binding differences in response to hormones may reflect changes in cells without prolactin receptors and/or cells with prolactin receptors. Likewise, binding comparisons of different tissues may reflect the tissue heterogeneity of cell type or composition and not the cellular sensitivities to prolactin. It seems likely that unequivocal results could be obtained by utilizing pure cell preparations and comparing the number of sites per specific binding cell. These possibilities need to be explored. REFERENCES REFERENCES Alder, A.; H. Burger; J. Davis; A. Dulmanis; B. Hudson; G. Sarfath; and W. Straffon. 1968. Carcinoma of prostate: response of plasma luteinizing hormone and testosterone to oestrogen therapy. Br. Med. J. 1:28-30. Amenomori, Y.; C. L. Chen; and J. Meites. 1970. Serum prolactin levels in rats during dufferent repro- ductive states. Endocrinology 86:506-510. Amenomori, Y., and J. Meites. 1970. Effect of a hypo- thalamic extract on serum prolactin levels during the estrous cycle and lactation. Proc. Soc. Exptl. Biol. Med. 134:492-495. Anden, N. E.; A. Carlsson; A. Kahlstrom; K. Fuxe; N. A. Hillarp; and K. Larsson. 1964. Demonstration and mapping out of nigro-neostriatal dopamine neurons. Life Sci. 3:523-530. Anton-Tay, F., and R. J. Wurtman. 1971. Brain monoamines and endocrine functions. In: Frontiers in Neuro- endocrinology, edited by L. Martini and W. F. Ganong, pp. 45-66. Oxford University Press, New York. Aragona, C., and H. G. Friesen. 1975. Specific prolactin binding sites in the prostate and testis of rats. Endocrinology,97:677-684. Asano, M. 1965. Basic experimental studies on the pituitary prolactin-prostate inter-relationships. J. Urol. 93:87-93. Asano, M.; S. Kanzaki; E. Sekiguchi; and T. Tasaka. 1971. Inhibition of prostatic growth in rabbits with antiovine prolactin serum. J. Urol. 106:248-252. Astwood, E. B. 1941. The regulation of corpus luteum function by hypOphysial luteotrophin. Endocrin- ology 28:309-320. 113 114 Astwood, E. B., and R. O. Greep. 1938. A corpus luteum- stimulating substance in the rat placenta. Proc. Soc. Exp. Biol. Med. 38:713-716. Atwell, W. J. 1926. The development of the hypophysis cerebri in man, with special reference to the pars tuberalis. Am. J. Anat. 37:159-193. 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. Bartke, A. 1965. Influence of luteotrophin on fertility of dwarf mice. J. Reprod. Fertil. 10:93-103. Bartke, A. 1966a. Reproduction of female dwarf mice treated with prolactin. J. Reprod. Fertil. 11: 203-206. Bartke, A. 1966b. Influence of prolactin on male fer- tility in dwarf mice. J. Endocrinol. 35:419-420. Bartke, A. 1971. Effects of prolactin and luteinizing hormone on the cholesterol stores in the mouse testis. J. Endocrinol. 49:317-324. Bartke, A., and C. W. Lloyd. 1970a. Influence of pro- 1actin and pituitary isografts on spermatogenesis in dwarf mice and hypophysectiomized rats. J. Endocrinol. 46:321-329. _ Bartke, A., and C. W. Lloyd. 1970b. The influence of pituitary homografts on the weight of the acces- sory reproductive organs in castrated male mice and rats and on mating behavior in male mice. J. Endocrinol. 46:313-320. Bates, R. W.; 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 71: 345-360. Beams, H. W., and R. K. Meyer. 1931. The formation of pigeon "milk." Physiol. 2001. 4:486-500. 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. 115 Bengmark, S., and R. Hesselsjo. 1963. The combined effect of prolactin and androsterone on the growth of rat seminal vesicle ig vitro. Urol. Intern. 16:387- 390. Bergmark, S., and R. HesselsjB. 1964. Endocrine dependence of rat seminal vesicle tissue in tissue culture. Urol. Intern. 17:84-92. Bern, H. A., and C. S. Nicoll. 1968. The comparative endocrinology of prolactin. Recent Progr. Horm. Res. 24:681-720. - Birge, C. A.; L. 8. Jacobs; C. T. Hammer; and W. H. Daughaday. 1970. Catecholamine inhibition of prolactin secretion by isolated rat adenohypophyses. Endocrinology 86:120-130. Birkinshaw, M., and I. R. Falconer. 1972. The localization of prolactin labeled with radioactive iodine in rabbit mammary tissue. J. Endocrinol. 55:323-334. Blecher, M.; N. A. Giorgio; and C. B. Johnson. 1974. Hormone receptors. IV. Solubilization and purifi- cation of a liver plasma membrane glucagon-binding protein. In: Advances in Enzyme Regulation, Vol. 12, edited-By G. Weber, pp. 289-309. Academic Press, New York. 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. Boyns, A. R.; E. N. Cole; M. P. Golder; V. Danutra; M. E. Harper; B. Brownsey; T. Cowley; G. E. Jones; and K. Griffiths. 1972. Prolactin studies with the prostate. £2: Prolactin and Carcinogenesis, Proceedings of the 4th Tenovous Workshop, edited by A. R. Boyns and K. Gri-fiths, pp. 207-216. Alpha Omega Alpha, Cardiff, Wales. Bradley, C. J.; G. A. Campbell; S. Marshall; J. Meites; and W. Collings. 1975. Rapid alterations in prolactin binding activity of tissue slices in response to estrogen. Fed. Proc. 34:343. Bradley, C. J.; G. S. Kledzik; and J. Meites. 1976. Prolactin and estrogen dependency of rat mammary cancers at early and late stages of development. Cancer Res. 36:319-324. 116 Braunsberg, H.; V. H. T. James; W. T. Irvine; W. C. Jamieson; and R. A. Sellwood. 1973. Prognostic significance of oestrogen uptake by human breast cancer tissue. Lancet 1:163-165. Brodie, B. B.; S. Pecton; and P. A. Shore. 1959. Inter- action of drugs with norepinephrine in the brain. Pharmacol. Rev. 11:548-864. Bruni, J. E., and D. G. Montemurro. 1971. Effects of hypothalamic lesions on the genesis of spontaneous mammary gland tumors in the mouse. Cancer Res. 31:854-863. 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. J. Endocrinol. 55:369-376. Buckman, M. T., and G. T. Peake. 1973a. OSmolar control of prolactin secretion in man. Program of Fifty- Fifty Meeting of the Endocrine Society, Abst. 2, A-490 Buckman, M. T., and G. T. Peake. 1973b. Osmolar control of prolactin secretion in man. Science 181:755- 757. Bucy, P. C. 1932. The hypophysis cerebri. I3: Cytology and Cellular Pathology of the Nervous System, edited by W. Penfield, pp. 707-738. Harper, New York. 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. Borbert. 1972. Calcium-mediated interactions between the anti- diuretic hormone and renal plasma membranes. g. Biol. Chem. 247:6167-6175. Campbell, G. A.; M. Kurcz; S. Marshall; and J. Meites. 1975. Anterior pituitary (AP) function and response to synthetic LRH-TRH during acute and chronic starvation. In: Symposium of the Inter- national Society of Psychoneuro-endocrinology, pp. 243-251. Publishing House of the Hungarian Academy of Sciences, Budapest. 117 Carlsson, A.; A. Dahlstrom; K. Fuxe; and N. A. Hillays. 1962. Cellular localization of brain monoamines. Acta Physiol. Scand. 56:Suppl. 196, 1-28. Cassell, E. E.; J. Meites; and C. W. Welsch. 1971. Effects of ergocornine and ergocryptine on growth of 7, 12-dimethy1-benzanthracene-induced mammary tumors in rats. Cancer Res. 31:1051-1053. Catt, K. J.; M. L. Dufau; and T. Tsuruhara. 1971. Studies on a radio-ligand-receptor assay system for luteinizing hormone and chorionic gonadotropin. J. Clin. Endocrinol. Met. 32:860-863. Catt, K. J.; M. L. Dufau; and T. Tsuruhara. 1972. Radioligand-receptor assay of luteinizing hormone and chorionic gonadotropin. J. Clin. Endocrinol. 34:123-132. Catt, K., and B. Moffat. 1967. Isolation of internally labeled rat prolactin by preparative disc electro- phoresis. Endocrinology 80:324-328. Chamness, G. C., and W. L. McGuire. 1975. Scatchard plots: common errors in correction and interpretation. Steroids 26:530-542. 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.; Y. Amenomori; K. H. Lu; J. L. Voogt; and J. Meites. 1970. Serum prolactin levels in rats with pituitary transplants or hypothalamic lesions. Neuroendocrinology 6:220-227. 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.; H. Minaguchi; and J. Meites. 1967. Effects of transplanted pituitary tumors on host pituitary prolactin secretion. Proc. Soc. Exp. Biol. Med. 126:317-320. Chen, C. L., and J. Meites. 1970. Effects of estrogen and progesterone on serum and pituitary prolactin levels in ovariectomized rats. Endocrinology 86: 503-505. 118 Chen, H. J., and J. Meites. 1974. Effects of biogenic amines and TRH on release of prolactin and TSH in the rat. Endocrinology 96:10-14. Chen, H. W.; D. H. Jamer; H. Heiniger; and H. Meier. 1972. Stimulation of hepatic RNA synthesis in dwarf mice by ovine prolactin. Biochem. Biophys. Acta 287:90-97. Choudard, J. B., and G. S. Greenwald. 1969. Luteotropic complex of the mouse. Anat. Record 163:373-388.. 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. Inhibition of lactation and luteal function in postpartum rats by hypothalamic implantation of prolactin. Endocrinology,84:868-872. Clemens, J. A.; M. Sar; and J. Meites. 1969b. Termination of pregnancy in rats by a prolactin implant in median eminence. Proc. Soc. Exp. Biol. Med. 130: 628-630. » Clemens, J. A.; C. W. Welsch; and J. Meites. 1968. Effects of hypothalamic lesions on incidence and growth of mammary tumors in carcinogen-treated rats. Proc. Soc. Exp. Biol. Med. 127:969-972. Convey, E. M.; H. A. Tucker; V. G. Smith; and J. Zolman. 1972. Prolactin, thyrozine and corticoid after TRH. J. Anim. Sci. 35:258. Cooper, J. R.; F. E. Bloom; and R. H. Roth. 1974. The Biochemical Basis of Neuropharmacology. Oxford University Press, LondOn. Coppola, J. A. 1968. The apparent involvement of the sympathetic nervous system in the gonadotrophin secretion of female rats. J. Reprod. Fertil. Suppl. 4, 35. Costlow, M. E.; R. A. Buschow; and W. L. McGuire. 1974. Prolactin receptors in an estrogen receptor- deficient mammary carcinoma. Science 184:85-86. Costlow, M. E.; R. A. Buschow; and W. L. McGuire. 1975. Prolactin stimulation of prolactin receptors in rat livers. Life Sci. 17:1457-1465. 119 Costlow, M. E.; R. A. Buschow; N. J. Richert; and W. L. McGuire. 1975. Prolactin and estrogen binding in transplantable hormone-dependent and autonomous rat mammary carcinoma. Cancer Res. 35:970-974. Cowie, A. T. 1957. The maintenance of lactation in the rat after hypophysectomy. J. Endocrinol. 16: 135-147. Cowie, A. T. 1964. Complete restoration of lactation in the goat after hypophysectomy. J. Endocrinol. 28:267-279. Cowie, A. T. 1969. Variations in the yield and composi- tion of the milk during lactation in the rabbit and the galactopoietic effect of prolactin. J. Endocrinol. 44:437-450. - Cowie, A. T., and W. R. Lyons. 1959. Mammogenesis and lactogenesis in hypophysectomized, ovariectomized, adrenalectomized rats. J. Endocrinol. 19:29-32. Cox, P. L. 1951. The preparation and distribution of iodoprolactin labeled with I 131. Anat. Rec. 109:285. Cuatrecasas, P. 1971. Insulin-receptor interactions in adipose tissue cells: direct measurement and properties. Proc. Natl. Acad. Sci. USA 68:1264- 1268. I Cuatrecasas, P. 1972. Properties of the insulin receptor isolated from liver and fat cell membranes. g. Biol. Chem. 247:1980-1991. Cuatrecasas, P.; M. D. Hollenberg; K. J. Chang; and V. Benn Bennet. 1975. Hormone receptor complexes and their modulation of membrane function. In: Recent Progr. in Hormone Research 31, edited by R. O. Greep, pp. 37-93. Academic Press, New York. Danon, A.; S. Dikstein; and F. G. Sulman. 1963. Stimu- lation of prolactin by perphenazine in pituitary- hypothalamic organ culture. Proc. Soc. Exp. Biol. Mgd. 114:366-368. Danzo, B. J.; A. R. Midgley; and L. J. Kleinsmith. 1972. Human chorionic gonadotropin binding to ovarian tissue in vitro. Proc. Soc. Exp. Biol. Med. 139: 88-92. 120 Defeudis, F. V. 1975. Amino acids as central neurotrans- mitters. Ann. Rev. Pharmacol. 15:105-130. Dempsey, E. W., and U. U. Uotila. 1940. The effect of pituitary stalk section upon reproductive phenomena in the female rat. Endocrinology 27:575-579. DeSombre, E. R.; G. S. Kledzik; S. Marshall; and J. Meites. 1976. Estrogen and prolactin receptor concen- trations in rat mammary tumors and response to endocrine ablation. Cancer Res. 36:354-358. 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-l39. Dickerman, S.; J. Clark; E. Dickerman; and J. Meites. 1972. Effects of haloperidol on serum and pitui- tary prolactin and on hypothalamic PIF in rats. Neuroendocrinology 9:332-340. Dickerman, S.; G. S. Kledzik; M. Gelato; J. J. Chen; and J. Meites. 1974. Effects of haloperiodol on serum and pituitary prolactin, LH and FSH, and hypothal- amic PIF and LRF. Neuroendocrinology 15:10-20. Donoso, A. O.; A. M. Banzan; and J. C. Barcaglioni. 1974. Further evidence on a direct action of L-dopa on prolactin release. Neuroendocrinology 15:236-239. Donoso, A. O.; W. Bishop; and S. M. McCann. 1973. The effects of drugs which modify catecholamine syn- thesis on serum prolactin in rats with median eminence lesions. Proc. Soc. Exp. Biol. Med. 143: 360-363. . 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.; K. L. Catt; and T. Tsuruhara. 1971. Retention of ig vitro biological activities by desialylated human luteinizing hormone and chorionic gonadotropin. Biochem. Biophys. Res. Comm. 44: 1022-1027. Eighamry, 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. 121 Ensor, D. M.; M. R. Edmondson; and J. G. Phillips. 1972. Prolactin and dehydration in rats. J. Endocrinol. Euker, J. S.; J. Meites; and G. D. Riegle. 1975. Effects of acute stress on serum LH and prolactin in intact, castrate and dexamethasone-treated male rats. Endocrinology 96:85-92. Everett, J. W. 1954. Luteotrophic function of autografts of the rat hypophysis. Endocrinology 54:685-690. Everett, J. W. 1966. The control of the secretion of prolactin. In: The Pituitary Gland, Vol. 2, edited by G. W. Harris and B. T. Donovan, pp. 166- 178. University of California Press, Berkeley. Everett, J. W., and D. L. Quinn. 1966. Differential hypothalamic mechanisms inciting ovulation and pseudopregnancy in the rat. Endocrinology 78: 141-150. Farnsworth, W. E. 1972. Prolactin and the prostate. lg: Prolactin and Carcinogenesis, Proceedings of the 4th Tenovous Workshop, edited by A. R. Boyns and K. Griffiths, pp. 217-225. Alpha Omega Alpha, Cardiff Wales. Feldman, H. A. 1972. Mathematical theory of complex ligand-binding systems of equilibrium: some methods for parameter fitting. Anal. Biochem. 48:317-388. Frantz, W. L.; J. H. Macindoe; and R. W. Turkington. 1974. Prolactin receptors: characteristics of the particu- late fraction binding activity. J. Endocrinol. 60:485-497. Freeman, M. E., and J. D. Neill. 1972. The pattern of prolactin secretion during pseudopregnancy in the rat: a daily nocturnal surge. Endocrinology 90: 1292-1294. Freychet, P.; R. Kahn; and J. Roth. 1972. Insulin inter- -actions with liver plasma membranes: independence of binding of the hormone and its degradation. J. Biol. Chem. 247:3953-3958. Freychet, P.; J. Roth; and D. M. Neville. 1971. Inigéin receptors in the liver: specific binding of I- insulin to the plasma membrane and its relation to insulin bioactivity. Proc. Natl. Sci. USA 68:1833-1837. ‘ 122 Freyer, M. E., and H. M. Evans. 1923. Participation of the mammary gland in the changes of pseudopregnancy in the rat. Anat. Record (abst.) 25:108. Friesen, H. G. 1966. Lactation induced by human placental lactogen and cortisone acetate in rabbits. Endocrinology,79:212-215. Folley, S. J. 1952. Lactation. £2: Marshall's Physiology of Reproduction, edited by A. S. Parkes, pp. 525- 554. Longmans and Green, London. Folley, S. J., and F. H. Malpress. 1948. Hormonal control of mammary growth. 32‘ The Hormones, Vol. 1, edited by G. Pincus and K. V. Thimann, pp. 695-739. Academic Press, New York. Fujii, T., and C. A. Villee. 1967. Stimulatory effects of RNA on the growth of the seminal vesicle of immature rats. Proc. Natl. Acad. Sci. USA 57: 1468-1473. Fujii, T., and C. A. Villee. 1968. Effect of testosterone on ribonucleic acid metabolism in the prostate, seminal vesicle, liver and thymus of immature rats. Endocrinology 82:463-467. Fuxe, K., and T. Hokfelt. 1969. Catecholamines in the hypothalamus and the pituitary gland. £2: Fron- tiers in Neuroendocrinology, 1969, edited by L. Martini and W. F. Ganong, pp. 47-96. Oxford University Press, New York. Fuxe, K., and O. Nilsson. 1967. Activity changes in the tuber-infundibular dopamine neurons of the rat during various states of the reproductive cycle. Life Sci. 6:2057-2061. Fuxe, K. 1964. Cellular localization of monoamines in the median eminence and the infundibular stem of some animals. A. Zellforsch 61:710-715. Gala, R. R.; P. B. Markarian; and J. O'Neill. 1970. The influence of neural blocking agents implanted into the hypothalamus of the rat on induced decidumata formation. Life Sci. 9:1055-1059. Ganong, W. F. 1971. Review of Medical Physiology. Lange Medical Publications, Los Altos. 123 Gavin, J. R.; J. Roth; and P. Jen. 1972. Insulin recep- tors in human circulating cells and fibroblasts. Proc. Natl. Acad. Sci. USA 69:747-756. Gelato, M. C. 1975. Influence of different physiological parameters on prolactin binding activity in pro- lactin target tissues. Ph.D. dissertation, Michigan State University. Gelato, M.; S. Marshall; M. Broudreau; J. Bruni; G. A. Campbell; and J. Meites. 1975. Effects of thyroid and ovaries on prolactin binding activity in rat liver. Endocrinology 96:1292-1296. Goodridge, A. G., and E. G. Ball. 1967. The effect of prolactin lipogenesis in the pigeon: ig_vivo studies. Biochem. 6:1676-1682. Gorski, J.; D. O. Toft; G. Shyamala; G. Smith; and A. Notides. 1968. Hormone receptors: studies of the interaction of estrogen with the uterus. Recent Progr. Hormone Res. 24:45-80. Gourdju, D., and A. Tixier-Vidal. 1966. Mise en evidence d'un control hypothalamique stimulant de la pro- lactine hypophysaire chezle canard. Compt. Rend. Acad. Sci. 263:162-165. Goodfriend, T., and S. Y. Lin. 1969. Angiotensin receptors. Clin. Res. 17:243. Goodfriend, T. L., and S. Y. Lin. 1970. Receptors for angiotensin I and II. 'Circ. Res. 26-27 (Suppl. I): 163-174. Grandison, L.; M. Gelato; and J. Meites. 1974. Inhibition of prolactin secretion by cholinergic drugs. Proc. Soc. Exp. Biol. Med. 145:1236-1239. 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., and J. Meites. 1976. Evidence for adrenergic mediation of cholinergic inhibition of prolactin release. Endocrinology 99:775-779. Gray, E. G., and V. P. Whittaker. 1960. The isolation of synaptic vesicles from the central nervous system. J. Physiol. 153:35-37. 124 Grayhack, J. T. 1963. Pituitary factors influencing growth of the prostate. Nat. Cancer Inst. Monogr. 12:189-199. Grayhack, J. T.; P. L. Bunce; J. W. Kearns; and W. W. Scott. 1955. Influence of the pituitary on pro- static response to androgen in the rat. Bull. Johns HOpkinS Hosp. 96:154-164. Green, J. D., and G. W. Harris. 1947. The neurovascular link between the neurohypophysis and adenohypophysis. J. Endocrinol. 5:136-146. Greenwald, G. S. 1969. Evidence for a luteotropic complex in the hamster and other species. 22‘ Progress in Endocrinology, edited by Carlis Gual, pp. 921- 926. Excerpta Medica, Amsterdam. Greibrokk, T.; B. L. Currie; K. Nils-Gunnar Johansson; J. J. Hausen; and K. Folkers. 1974. Purification of a prolactin inhibiting hormone and the revealing of hormone D-GHIH which inhibits the release of growth hormone. Biochem. and Biophys. Res. Comm. 59: 704-709. Grosvenor, C. E.; S. M. McCann; and M. D. Nallar. 1964. Inhibition of suckling-induced release of pro- lactin in rats injected with acid extract of bovine hypothalamus. Program 46th Meeting Endocrine Society, San Francisco, p. 96. Grosvenor, C. E., and F. Mena. 1971. Effect of suckling upon the secretion and release of prolactin from the pituitary gland of the lactating rat. J. Animal Sci. 32, Suppl. 1:115-136. Hafiez, A. A.; A. Bartke; and C. W. Lloyd. 1972. The role of prolactin in the regulation testis function: the synergistic effects of PRL and luteinizing hormone on the incorporation of (1-14C) acetate into testosterone and cholesterol by testes from hypophysectomized rats in vitro. J. Endocrinol. 53:223-230. Hafiez, A. A.; C. W. 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. _ 125 Hafiez, A. A.; J. E. Philpatt; and A. Bartke. 1971. The role of prolactin in the regulation of testicular function: the effect of prolactin and luteinizing hormone on 38-hydroxysteroid dehydrogenase activity in the testis of mice and rats. J. Endocrinol. 50: 619-623. Halasz, B., and J. Szentagothai. 1962. The trophic dependence of the anterior pituitary on the hypothalamus. £2: Hypothalamic Control of the Anterior Pituitary, edited by J. Szentagothai et al., pp. 266-276. Publ. House Hung. Acad. Sci., Budapest. Harris, G. W., and D. Jacobsohn. 1952. Functional grafts of the anterior pituitary gland. Proc. Roy. Soc. (London) Ser. B. 139:263-279. Hashimoto, I.; D. M. Henricks; L. L. Anderson; and R. M. Melampy. 1968. Progesterone and pregn-4-en-200- ol-3one in ovarian venons blood during various reproductive states in the rat. Endocrinology 82:333-341. Haymaker, W.; E. Anderson; and W. J. H. Nauta (eds.). 1969. The Hypothalamus. Charles C. Thomas Pub- lishing, Springfield. Hilliard, J. 1973. Corpus luteum function in guinea pigs, hamsters, rats, mice and rabbits. Biol. Reprod. 8:203-221. Hintz, R. L.; L. E. Underwood; D. R. Clemmons; S. J. Voina; R. N. Marshall; and J. J. Van wyk. 1974. Separate receptors for insulin and somatomedin in skeletal and nonskeletal tissue. Fifty-sixth Meeting, Endocrine Society, Abst. 34:A-72. H6kfelt, T. 1967. The possible ultrastructural identifi- cation of tuberoinfundibular dopamine-containing nerve endings in the median eminence of the rat. Brain Res. 5:121-123. Holzbauer, M., and M. Vogt. 1956. Depression by reserpine of the noradrenalin concentration in the hypo- thalamus of the cat. J. Neurochem. 1:8-11. Horrobin, D. F.; I. J. Lloyd; A. Lipton; P. G. Burstyn; N. Durkin; and K. L. Muiruri. 1971. Actions of prolactin on human renal function. Lancet 2:352- 354. 126 Horrobin, D. F.; M. S. Manku; and P. G. Burstyn. 1973. Salurectic action of aldosterone in the presence of excess contisol: restoration of salt-retaining action by prolactin. J. Endocrinol. 56:343-344. Houssay, B. A.; A. Biasotti; and R. Sammartino. 1935. Modifications fonctionelles de 1'hyp0physe apres 1es lesions infundibulatuberiemes ches 1e crapaud. Compt. Rend. Soc. Biol. 120:725-726. Huggins, C., and P. J. Clark. 1940. The effect of cas- tration and of estrogen injection on the normal and on the hyperplastic prostate glands of dogs. J. Exp. Med. 72:747-761. ‘ Huggins, C., and C. V. Hodges. 1941. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastic carcinoma of the prostate. Cancer Res. 1:293-297. Huggins, C.; R. C. Moon; and S. Moril. 1962. Extinction of experimental mammary cancer. I. Estradiol-17B and progesterone. Proc. Natl. Acad. Sci. 48:379- 384. Huggins, L. B., and P. S. Russell. 1946. Quantitative effects of hypophysectomy on testis and prostate of dogs. Endocrinology 39:1-7. Hunter, W. M., and F. C. Greenwood. 1962. Preparation of iodine-131 labeled human growth hormone of high specific activity. Nature 194:495-497. Hwang, P.,; H. J. Guyda; and H. G. Friesen. 1971. Human prolactin (HPr): purification and clinical studies. Clin. Res. 19:772. Jacobs, L.; P. J. Snyder; J. F. Wilber; R. D. Utiger; and W. H. Daughaday. 1971. Increased serum prolactin after administration of synthetic thyrotropin releasing hormone (TRH) in man. J. Clin. Endocr. 33:996-998. Jarvik, M. E. 1970. Drugs used in the treatment of psychiatric disorders. In: The Pharmacological Basis of Therapeutics, edited by L. S. Goodman and A. Gilman, pp. 151-203. The MacMillan Co., London. Jenkins, T. W. 1972. Functional Mammalian Neuroanatomy. Lea and Febriger, Philadelphia. 127 Jensen, E. V.; G. E. Block; S. Smith; K. Kyser; and E. R. DeSombre. 1972. Estrogen receptors and hormone dependence. In: Estrogen Target Tissues and Neo- plasia, edited—by T. L. Dao, pp. 23-58. The University of Chicago Press, Chicago. Jensen, E. V., and E. R. DeSombre. 1973. Estrogen- receptor interaction. Science 182:126-134. 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. Endocrinology,94:1364-1371. Kahn, C. R.; D. M. Neville; and J. Roth. 1973. Insulin- receptor interaction in the obese-hyperglycemic mouse. Biochem. Biophys. Res. Comm. 48:135-142. Kalra, P. 8.; C. P. Fawcett; L. Krulich; and S. M. McCann. 1973. The effects of gonadal steroids on plasma gonadotropins and prolactin in the rat. Endocrinology»92:1256-1268. Kanematsu, S.; J. Hilliard; and C. H. Sawyer. 1963. Effect of reserpine and chlorpromazine on pituitary prolactin content and its hypothalamic site of action in rabbits. Acta Endocrinol. 44:467-474. Kanematsu, S., and C. H. Sawyer. 1963. Effects of intra- hypothalamic and intrahypophysial estrogen implants on pituitary prolactin and lactation in the rabbit. Endocrinology 72:243-252. Kamberi, I. A.; R. S. Mical; and J. C. Porter. 1971a. Pituitary portal vessel infusion of hypothalamic extracts and release of LH, FSH, and prolactin. Endocrinology 88:1294-1299. Kamberi, I. A.; R. S. Mical; and J. C. Porter. 1971b. Effect of anterior pituitary perfusion and intra- ventricular injection of catecholamines on prolactin release. Endocrinology 88:1012-1020. Kamberi, I. A.; R. S. Mical; and J. C. Porter. 1971c. Effects of melatonin and serotonin on the release of FSH and prolactin. Endocrinology 88:1288-1293. Kelly, P. A.; K. N. Bedirian; R. D. Baker; and H. G. Friesen. 1973. Effect of synthetic TRH on serum prolactin, TSH and milk production in the cow. Endocrinology,92:1289-1293. 128 Kelly, P. A.; C. Bradley; R. P. C. Shiu; J. Meites; and H. G. Friesen. 1974. Prolactin binding to rat mammary tumor tissue. Proc. Soc. Exp. Biol. Med. 146:816-824. Kelly, P. A.; B. I. Posner; and H. G. Friesen. 1974. Effects of hypophysectomy, ovariectomy and cyclo- heximide on binding sites for lactogenic hormones. Clin. Res. 22:732-736. Kelly, P. A.; B. I. Posner; T. Tsushima; and H. G. Friesen. 1974. Studies of insulin, growth hormone and pro- lactin binding: ontogenesis, effect of sex and pregnancy. Endocrinology 95:532-539. Kledzik, G. S.; G. J. Bradley; 8. Marshall; G. A. Campbell; and J. Meites. 1976. Effects of high doses of estrogen on prolactin binding activity and growth of carcinogen-induced mammary cancers in rats. Cancer Res. 36:3265-3269. Kledzik, G. S.; C. J. Bradley; and J. Meites. 1974. Reduction of carcinogen-induced mammary cancer incidence in rats by early treatment with hormones or drugs. Cancer Res. 34:2953-2956. Kledzik, G. S.,; S. Marshall; G. A. Campbell; M. Gelato; and J. Meites. 1976. Effects of castration, testosterone, estradiol and prolactin on specific prolactin binding activity in ventral prostate of male rats. Endocrinology 98:373-379. Kledzik, G. S.; S. Marshall; M. Gelato; G. Campbell; and J. Meites. 1975. Prolactin binding activity in the crop sacs of juvenile, mature, parent and prolactin-injected pigeons. Endocrine Res. Comm. 2:345-355. Knigge, K. M. 1962. Gonadotropic activity of neonatal pituitary glands implanted in the rat brain. Am. J. Physiol. 202:387-391. Koch, Y.; Y. F. Chow; and J. Meites. 1971. Metabolic clearance and secretion rats of prolactin in the rat. Endocrinology 89:1303-1308. Koch, Y.; K. H. Lu; and J. Meites. 1970. Biphasic effects of catecholamines on pituitary prolactin release i3 vitro. Endocrinology 87:673-675. 129 Kordon, C. 1966. Recherches sur le controle hypothamamique des fonctions gonadotropes femelles du rat. Ph.D. dissertation, University of Paris. Kordon, C.; C. A. Blake; J. Terkel; and C. H. Sawyer. 1973. Participation of serotonin-containing nervous in the suckling-induced rise in plasma prolactin levels in lactating rats. Neuroendo- crinology 13:213-223. Kragt, C. L., and J. Meites. 1965. Stimulation of pigeon pituitary prolactin release by pigeon hypothalamic extract in vitro. Endocrinology 76:1169-1176. Kragt, C. L., and J. Meites. 1967. Dose-response rela- tionships between hypothalamic PIF and prolactin release by rat pituitary tissue i2 vitro. Endocrinology 80:1170-1173. Krnjevic, K. 1974. Chemical nature of synaptic trans- mission in vertebrates. Physiol. Rev. 54:418-540. Krug, D.; F. Krug; and P. Cuatrecasas. 1972. Emergence of insulin receptors on human lymphocytes during in vitro transformation. Proc. Natl. Acad. Sci. USA’69:2604-2610. Krulich, L.; M. Quijada; and P. Illnu. 1971. Localization of prolactin-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 Francisco, Cali- fornia, p. 83. Kuroshima, A.; A. Arimura; C. Y. Bowers; and A. V. Schally. 1966. Inhibition by pig hypothalamic extracts of depletion of pituitary prolactin in rats following cervical stimulation. Endocrinology 78:216-217. Lahr, E. L., and O. Riddle. 1938. Proliferation of crop- sac epithelium in incubating and in prolactin- injected pigeons studied with the cholchicine method. Amer. J. Physiol. 123:614-619. Lasnitzki, I. 1972. The effect of prolactin on rat prostate glands in organ culture. £2: Prolactin and Carcinogenesis, Proceedings of the 4th Tenovous WorkshOp, edited by A. R. Boyns and K. Griffiths. Alpha Omega Alpha, Cardiff, Wales. 130 Lawrence, A. M., and R. L. Landau. 1965. Impaired ventral prostate affinity for testosterone in hypophys- ectomized rats. Endocrinology 77:1119-1125. Lefkowitz, R. J.; I. Pastan; and J. Roth. 1965. Hormones, receptors, and adenyl cyclase activity in mammalian cells. In: The Role of Adenyl Cyclase and Cyclic 3'5'-AMP_in Biological Systems, edited by T. W. Rodbell and P. Condliffe, pp. 88-95 (discussion). NIH, Bethesda. Lefkowitz, R. J.; E. Haber; and D. O'Hara. 1972. Identi- fication of the cardiac beta-adrenergic receptor protein: solubilization and purification by affinity chromatography. Proc. Natl. Acad. Sci. USA 69: 2828-2830. Leidenberger, F., and L. E. Reichert. 1972. Studies on the uptake of human chorionic gonadotropin and the homogenates and interstitial tissue. Endocrinology 91:135-143. 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. Libertun, C., and S. M. McCann. 1973. Blockade of the release of gonadotropins and prolactin by sub- cutaneous or intraventicular injection of atropine in male or female rats. Endocrinology 92:1714- 1724. 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. Lis, M.; C. Gilardeau; and M. Chretien. 1973. Effect of prolactin on corticosterone production by rat adrenals. Clin. Res. 21:1027. Litwack, G. (ed.). 1972. Biochemical Actions of Hormones, Vol. II. Academic Press, New York. Litwack, G. (ed.). 1975. Biochemical Actions of Hormones, Vol. III. Academic Press, New York. 131 Lockett, M. F., and B. Nail. 1965. A comparative study of the renal actions of growth hormone and lactogenic hormones in the rat. J. Physiol. 180:147-156. Lostroh, A. J., and C. H. Li. 1957. Stimulation of the Lowry, Lu, K. Lu, K. Lu, K. Lu, K. Lu, K. Lyons, Lyons, Ma, R. sex accessories of hypophysectomized male rats by non-gonadotropic hormones of the pituitary gland. Acta Endocrinol. 25:1-16. O. H.; N. J. Rosebrough; A. L. Farr; and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265. H.; Y. Amenomori; C. L. Chen; and J. Meites. 1970. Effects of central acting drugs on serum and pituitary prolactin levels in rats. Endocrinology 87:667-672. H.; Y. Koch; and J. Meites. 1971. Direct inhibition by ergocornine of pituitary prolactin release. Endocrinology 89:229-233. H., and J. Meites. 1971. Inhibition by L-dopa and monoamine oxidase inhibitors of pituitary prolactin release; stimulation by methyldopa and d- amphetamine. Proc. Soc. Exp. Biol. Med. 137:480- 483. H., and J. Meites. 1972. Effects of L-dopa on serum prolactin and PIF in intact and hypophys- ectomized, pituitary grafted rats. Endocrinology 91:868-872. H., and J. Meites. 1973. Effects of serotonin precursors and melatonin on serum prolactin release in rats. Endocrinology 93:152-155. W. R. 1937. The preparation and assay of mammo- trophin. Cold Spring Harbor Symp. Quant. Biol. 5:198-209. W. R. 1958. Hormonal synergism in mammary growth. Proc. R. Soc. Bl49:303-310. C._S., and A. V. Nalbandov. 1963. Physiology of the pituitary gland as affected by transplantation or stalk section (discussion). I2: Advances in Neuroendocrinology, edited by A. V. Nalbandov, pp. 306-312. University of Illinois Press, Urbana, Ill. 132 Marshall, 8.; M. Gelato; and J. Meites. 1975. Serum prolactin levels and prolactin binding activity in adrenals and kidneys of male rats after dehy- dration, salt loading and unilateral nephrectomy. Proc. Soc. Exp. Biol. Med. 149:185-188. Marshall, 8.; G. S. Kledzik; M. Gelato; G. A. Campbell; and J. Meites. 1976. Effects of estrogen and testosterone on specific prolactin binding in the kidneys and adrenals of rats. Steroids 27:187-195. MacLeod, R. M. 1968. Influence of norepinephrine and catecholamine-depleting agents on the synthesis and release of prolactin and growth hormone. Endocrinology 85:916-923. MacLeod, R. M. 1974. Regulation of pituitary function by catecholamines. In: Mammary Cancer and Neuro- endocrine Therapy, edited by B. A. Stoll, pp. 139- 159. Butterworth and Co., New York. MacLeod, R. M. 1976. Regulation of prolactin secretion. In: Frontiers of Neuroendocrinology, Vol. 4, edited by L. Martini and W. F. Ganong, pp. 169-194. Raven Press, New York. MacLeod, R. M.; A. Abad; and L. L. Eidson. 1969. In vivo effect of sex hormones on the in vitro synthESIS of prolactin and growth hormonE—in normal and pituitary tumor-bearing rats. Endocrinology 84: 1475-1483. MacLeod, R. M.; G. W. DeWitt; and M. C. Smith. 1968. Suppression of pituitary gland hormone content by pituitary tumor hormones. Endocrinology 82:889-894. Malbon, C. C., and J. F. Zull. 1974. Interactions of parathyroid hormone and plasma membranes from rat kidney. Biochem. Biophys. Res. Comm. 56:952-958. Malven, P. V. 1969. Luteotrophic and luteolytic responses to prolactin in hypophysectomized rats. Endocrinology 84:1224-1229. Marchalonis, J. J. 1969. An enzymatic method for the trace iodination of immunoglobulines and other proteins. Biochem. J. 113:299-305. Martini, L., and W. F. Ganong (eds.). 1966. Neuroendo- crinology. Academic Press, New York. 133 Martini, L.; M. Motta; and F. Fraschini (eds.). 1970. The Hypothalamus. Academic Press, New York. Marx, S. J.; S. Fedak; and G. D. Aurback. 1972. Prepara- tion and characterization of a hormone-responsive renal plasma membrane fraction. J. Biol. Chem. 247:6913-6918. McCann, S. M.; S. Ojeda; and A. Negro-Vilar. 1974. Sex steroid, pituitary and hypothalamic hormones during puberty in experimental animals. In: Control of the Onset of Puberty, edited by M. M. Grumbach, G. G. Gilman, and F. E. Mayer, pp. 1-19. John Wiley & Sons, New York. McGuire, W. L.; P. P. Carbone; and E. P. Vollner (eds.). 1975. Estrogen Receptors and Human Breast Cancer. Raven Press, New York. McLean, B. K., and M. B. Nikitovitch-Winer. 1975. Cholinergic control of the nocturnal prolactin surge in the pseudopregnant rat. Endocrinology 97:763-770. Means, A. R., and J. Voitukaitis. 1972. geptide hormone "receptors": specific binding of H -FSH to testis. Endocrinology 90:39-46. Meites, J. 1961. Hormonal induction of lactation and galactopoiesis, Chapter 8. In: Milk: The Mammary Gland and Its Secretion, Volume I, edited by S. K. Kon and A. T. Cowie. Academic Press, New York. Meites, J. 1962. Pharmacological control of prolactin secretion and lactation. In: Pharmacological Con- trol 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, edited by L. Martini and W. F. Ganong, pp. 669-707. Academic Press, New York. Meites, J. 1967. Control of prolactin secretion. Archives D'Anatomie Microscopique et de Morphologie Experimentale 56:516-5291 Meites, Meites, Meites, Meites, Meites, Meites, Meites, Meites, Meites, Meites, 134 J. 1970. Direct studies of the secretion of the hypothalamic hypophysiotropic hormones (HHH). In: Hypophysiotropic Hormones of the Hypothalamus: Assay and Chemistry, edited by J. Meites, pp. 261- 278. The Williams and Wilkins Co., Baltimore. J. 1972. Relation of prolactin and estrogen to mammary tumorigenesis in the rat. J. Natl. Cancer Inst. 48:1217-1224. J. 1973. Control of prolactin secretion in animals. In: Human Prolactin, edited by J. L. Pasteels afid C. Robyn, pp. 105-118. Excerpta Medica, Amsterdam. J.; E. Cassell; and J. Clark. 1971. Estrogen inhibition of mammary tumor growth in rats; counter- action by prolactin. Proc. Soc. Exp. Biol. Med. 137:1225-1227. J., and J. A. Clemens. 1972. Hypothalamic control of prolactin secretion. Vitamins and Hormones 30: 165-221. J., and T. F. Hopkins. 1961. Mechanisms of oxytocin action in retarding mammary involutions: study of hypophysectomized rats. J. Endocrinol. 22:207-213. J.; T. F. Hopkins; and P. K. Talwalker. 1963. Induction of lactation in pregnant rabbits with prolactin cortisol acetate or both. Endocrinology 73:261-264. J.; R. H. Kahn; and C. S. Nicoll. 1961. Prolactin production by rat pituitary in vitro. Proc. Soc. Exp. Biol. Med. 108:440-443. 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. l2: Recent Progr. Hormone Res. 28, edited by R. O. Greep, pp. 471-516. Academic Press, New York. J.; C. S. Nicoll; and P. K. Talwalker. 1963. The central nervous system and the secretion and release of prolactin. £23 Advances in Neuroendo- crinology, edited by A. V. Nalbandov, pp. 238-277. University of Illinois Press, Urbana. 135 Meites, J.; P. K. Talwalker; and C. S. Nicoll. 1960a. Initiation of lactation in rats with hypothalamic or cerebral tissue. Proc. Soc. Exp. Biol. Med. 103:298-300. 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. Minaguchi, H., and J. Meites. 1967. Effects of a nore- thynodrel-mestranol combination (Enovid) on hypothalamic and pituitary hormones in rats. Endocrinology»81:826-834. Mioduozewski, R.; L. Grandison; and J. Meites. 1976. Stimulation of prolactin release in rats by GABA. Proc. Soc. Exp. Biol. Med. 151:44-46. Mishkinsky, J. S.; Y. Givant; F. G. Sulmigé A. Eshkol; and B. Lunenfeld. 1972. Uptake of I-labeled pro- lactin by rat mammary gland and pigeon crop mucosa. J. Endocrinol. 52:387-396. Mishkinsky, J.; K. Khazan; and F. G. Sulman. 1968. Pro- lactin releasing activity of the hypothalamus of post-partum rats. Endocrinology 82:611-613. Mittler, J. C., and J. Meites. 1967. Effects of epinephrine and acethylcholine on hypothalamic content of prolactin-inhibiting factor. Proc. Soc. Exp. Biol. Med. 124:310-311. Mizuno, H.; P. K. Talwalker; and J. Meites. 1964. Inhibition of mammary secretion in rats by iproniazid. Proc. Soc. Exp: Biol. Med. 115:604- 607. Moger, W. H., and I. I. Greshwind. 1972. The action of prolactin on the sex accessory glands of the male rat. Proc. Soc. Exp. Biol. Med. 141:1017-1021. Motta, M.; F. Fraschini; and L. Martini. 1969. Short feedback mechanisms in the control of anterior pituitary function. In: Frontiers in Neuroendo- crinology, edited by L. Martini and W. F. Ganong, pp. 211-253. Oxford University Press, New York. 136 Mueller, G. P. 1976. Relation of biogenic amines, tem- perature and stress to the release of anterior pituitary hormones. Ph.D. dissertation, Michigan State University. Mueller, G. P.; H. T. Chen; J. A. Dibbet; H. J. Chen; and J. Meites. 1974. Effects of warm and cold tem- perature on release of TSH, GH and prolactin in rats. Proc. Soc. Exp. Biol. Med. 147:698-700. Mueller, G. P.; H. J. Chen; and J. Meites. 1973. In vivo stimulation of prolactin release in the rat—By synthetic TRH. Proc. Soc. Exp. Biol. Med. 144: 613-615. Mueller, G. P.; C. P. Twohy; H. T. Chen; J. P. Advis; and J. Meites. 1976. Effects of L-tryptophan and restraint stress on hypothalamic and brain serotonin turnover, and pituitary TSH and prolactin release in rats. Life Sci. 18:715-724. Nandi, S. 1958. Endocrine control of mammary-gland development and function in the C3H/HeCRgl mouse. J. Natl. Cancer Inst. 21:1039-1043. Nandi, S. 1959. Hormonal control of mammogenesis and lactogenesis in the C3H/HeCRgl mouse. Univ. Calif. Publs. Zool. 65:1-7. Nandi, S., and H. A. Bern. 1961. The hormones responsible for lactogenesis in BALB/cCRgl mice. Gen. Comp. Endocr. 1:195-201. Nagasawa, H.; C. L. Chen; and J. Meites. 1969. Effects of estrogen implant in median eminance on serum and pituitary prolactin levels in the rat. Proc. Soc. Exp, Biol. Med. 132:859-861. Nagasawa, H., and R. Yanai. 1971. Increased mammary gland response to pituitary mammotropic hormones by estrogen in rats. Endocr. Jap. 18:53-57. Nagasawa, H., and R. Yanai. 1972. Inhibitory effect of estrogen on mammary growth and its counteraction by pituitary isografts in mice. Endocr. Jap. 19: 107-110. Negro-Villar, 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. 137 Neill, J. D. 1970. Effect of stress on serum prolactin and luteinizing hormone levels during the estrous cycle of the rat. Endocrinology 87:1192-1197. Neill, J. D. 1974. Prolactin: its secretion and control. In: Handbook of Physiology, Section 7: Endocrinology, edited by R. O. Greep and E. B. Astwood, pp. 469- 488. American Physiological Society, Washington, D.C. Neill, J. D.; M. E. Freeman; and S. A. Tillson. 1971. Control of the proestrus surge of prolactin and luteinizing hormone secretion by estrogen in the rat. Endocrinology 85:1448-1453. Netter, F. H. 1968. The Hypothalamus, Suppl. to Vol. I. Nervous System, The Ciba Collection of Medical Illustrations. Ciba Pharmacentical Products, Inc., Summit, New Jersey. Nicoll, C. S. 1965. Neural regulation of adenohypophyseal prolactin secretion in tetrapods: indications from In vitro studies. J. Exptl. 2001. 158:203-210. Nicoll, C. S. 1967. Bioassay of prolactin. Analysis of the pigeon crop-sac response to local prolactin injection by an objective and quantitative method. Endocrinology 80:641-647. Nicoll, C. S. 1971. Aspects of neural control of prolactin secretion. In: Frontiers in Neuroendocrinology, edited by L. Martini and W. F. Ganong, pp. 291-330. Oxford University Press, New York. Nicoll, C. S. 1974. Physiological actions of prolactin. In: Handbook of Physiology, Section 7: Endocrinology, edited by R. O. Greep and E. B. Astwood, pp. 253- 292. American Physiological Society, Washington, D.C. 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 G. E. W. Walstenholme and J. Knight, pp. 299-324. Churchill Livingstone, London. Nicoll, C. S.; R. P. Fiorinda; C. T. McKennee; and J. A. Parsons. 1970. Assay of hypothalamic factor which regulate prolactin secretion. In: Hypophysiotropic Hormones of the Hypothalamus: Assay and Chemistry, edited by J. Meites, pp. 115-144. The Williams and Wilkins Co, Baltimore. 138 Nicoll, C. S., and J. Meites. 1962a. Prolactin secretion In vitro: comparative aspects. Nature 195:606-607. Nicoll, C. S., and J. Meites. 1962b. Estrogen stimulation of prolactin production by rat adenohypophysis In vitro. Endocrinology 70:272-277. 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.; P. K. Talwalker; and J. Meites. 1960. Initiation of lactation in rats by nonspecific stresses. Amer. J. Physiol. 198:1103-1106. 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. Niswender, G. D.; C. L. Chen; A. R. Midgley; J. Meites; and S. Ellis. 1969. Radioimmunoassay for rat prolactin. Proc. Soc. Exp. Biol. Med. 130:793-797. Okamoto, H. R.; M. R. N. Prasad; and R. K. Meyer. 1960. Action of prolactin on sex accessories of hypo- physectomized rats. Proc. Soc. Exp. Biol. Med. 103:77-80. O'Malley, B. W., and A. R. Means (eds.). 1973. Advances in Experimental Medicine and Biology: Receptors for Reproductive Hormones. Plenum Press, New York. Ondo, J. G., and K. A. Pass. 1976. The effects of neurally active amino acids on prolactin secretion. Endocrinology 98:1248-1252. Pasqualini, R. Q. 1953. La funcion la gonadotrofina c (luteotrofina, prolactina) en el macho. Prensa Med. Arg. 40:2658—2660. Pasteels, J. L. 1961. Secretion de prolactin par l'hyposphyse en culture de tissues. Compt. Rend. Soc. Biol. 253:2140-2142. Pearson, O. H.; P. Llerena; L. Llerena; A. Molina; and T. Butler. 1969. Prolactin-dependent rat mammary cancer: a model for man? Trans. Assoc. Amer. Physicians 82:225-238. 139 Pencharz, R. I., and J. A. Long. 1933. Hypophysectomy in the pregnant rat. Am. J. Anat. 53:117-135. Piva, F.; P. Gagliano; M. Motta; and L. Martini. 1973. Adrenal progesterone: factors controlling its secretion. Endocrinol. 93:1178-1184. Pletscher, A.; P. A. Shore; and B. B. Brodie. 1955. Serotonin release as a possible mechanism for reserpine action. Science 122:374-375. 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. 1974. Insulin receptor in human and animal placental tissue. Diabetes 23:209-217. Posner, B. I. 1975. Peptide hormone receptors: character- istics and applications. Can. J. Physiol. and Pharmacol. 53:689-703. Posner, B. I.; P. A. Kelly; and H. G. Friesen. 1975. Prolactin receptors in rat liver: possible induction by prolactin. Science 188:57-59. Posner, B. I.; P. A. Kelly; and H. G. Friesen. 1976. Induction of a lactogenic receptor in rat liver: influence of estrogen and the pituitary. Proc. Natl. Acad. Sci. USA 71:2407-2410. 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. Endocrinology 95:521-531. Quadri, S. K.; J. L. Clark; and J. Meites. 1973. Effects of LSD, pargyline and haloperidol and mammary tumor growth in rats. Proc. Soc. Eyp. Biol. Med. 142:22-26. Quadri, S. K.; G. S. Kledzik; and J. Meites. 1973. Effects of 1-dopa and methyldopa on growth of mammary cancers in rats. Proc. Soc. Exp. Biol. Mpg. 142:759-761. Quadri, S. K., and J. Meites. 1971. Regression of spon- taneous mammary tumors in rats by ergot drugs. Proc. Soc. Exp. Biol. Med. 138:999-1001. 140 Rajaniemi, H.; A. Oksanené and T. Vanha-Perttula. 1974b. Distribution of 5I-prolactin in mice and rats. Studies with whole body micro-autoradiography. Horm. Res. 5:6-20. Ramirez, V. D., and S. M. McCann. 1964. Induction of prolactin secretion by implants of estrogen into the hypothalamo-hypophysial region of female rats. Endocrinology,75:206-214. Ratner, A., and J. Meites. 1964. Depletion of prolactin- inhibiting activity of rat hypothalamus by estradiol or suckling stimulus. Endocrinology 75:377-382. Ratner, A.; P. K. Talwalker; and J. Meites. 1965. Effect of reserpine on prolactin-inhibiting activity of rat hypothalamus. Endocrinology 77:315-319. Relkin, R. 1974. Effects of alterations in serum osmolality on pituitary and plasma prolactin levels in the rat. Neuroendocrinology 14:61-64. Relkin, R., and M. Adachi. 1973a. Effects of sodium deprivation on pituitary and plasma prolactin, growth hormone and thyrotropin levels in the rat. Neuroendocrinology 11:240-247. Relkin, R., and M. Adachi. 1973b. Prolactin secretion and sodium deprivation. Federation Proc. (Abst.) 32:1031. Riddle, O. 1963. Prolactin in vertebrate function and organization. J. Nat. Cancer Inst. 31:1039-1110. Riddle, O.; R. W. Bates; and S. W. Dykshorn. 1932. A new hormone of the anterior pituitary. Proc. Soc. Exp. Biol. Med. 29:1211-1212. Riddle, O., and P. F. Braucher. 1931. Studies on physi- ology of reproduction 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., and S. W. Dykshorn. 1932. Secretion of crop milk in the castrated male pigeon. Proc. Soc. Exp. Biol. Med. 29:1211. Riddle, O.; 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. 141 Rivers, E. M. 1964. Interchangeability of adrenocrotical hormones in initiating mammary secretion in 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. Robinson, C. A.; B. R. Boshell; and W. J. Reddy. 1971. The binding of glucagon and insulin to plasma membranes. Diabetes 20:340-347. Roth, G. S., and R. C. Adelman. 1975. Age related changes in hormone binding by target cells and tissues; possible role in altered adaptive responsiveness. Exp. Geront. 10:1-11. Roth, J. 1973. Peptide hormone binding to receptors: a review of direct studies In vitro. Metabolism 22:1059-1073. Roth, J.; R. Kahn; M. A. Lesniak; P. Gordon; et a1. 1975. Receptors for insulin, MSILA-s, growth hormone: applications to disease states in man. In: Recent Progress in Hormone Research, 31, edited—by R. O. Greep, pp. 95-139. Academic Press, New York. Roth, J., and M. Lesniak. 1972. In: Insulin Action, Proceedings of Symposium, Toronto, 1971, pp. 186- 187, 202-203. Academic Press, New York. Saito, T., and B. B. Saxena. 1975. Specific receptors for prolactin in the ovary. Acta Endocrinologica 80:126-137. 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. Scatchard, G. 1949. The attraction of proteins for small molecules and salts. Ann. N.Y. Acad. Sci. 51: Schally, A. V.; A. Arimura; and A. J. Kastin. 1973. Hypothalamic regulatory hormones. Science 179: 341-350. 142 Schally, A. V.; T. W. Redding; G. L. Linthicum; and A. Dupont. 1976. Inhibition of prolactin release in vitro and In vivo by natural hypothalamic and _— synthetic gamma-aminobutyric acid. 58th Annual Meeting of the Endocrine Society, Abst., 321. San Francisco, California. Schooley, J. P.; O. Riddle; and R. W. Bates. 1937. Effective stimulation of crop-sacs by prolactin in hypophysectomized and in adrenalectomized pigeons. Proc. Soc. Exp. Biol. Med. 36:408. Schreiber, V. 1963. Hypothalamo-hypophyseal system. Czech. Acad. Sci., Prague, 187-276. Schultze, A. B., and C. W. Turner. 1933. Experimental initiation of milk secretion in the albino rat. J. Dairy Sci. 16:129-133. Selye, H. 1934. On the nervous control of suckling. Am. J. Physiol. 107:535-538. Selye, H.; J. B. Collip; and D. L. Thompson. 1934. Nervous and hormonal factors in lactation. Endocrinology 18:237-248. Shaar, C. J., and J. Clemens. 1972. Inhibition of lacta- tion and prolactin secretion in rats by ergot alkaloids. Endocrinology 90:285-289. Shaar, C. J., and J. L. Clemens. 1974a. Effect of alumi- num oxide catecholamine adsorption and monoamine oxidase on the inhibition of rat anterior pituitary prolactin release by hypothalamic extracts in vitro. Fed. Proc. Abst. 192. _— Shaar, C. J., and J. A. Clemens. 1974b. The role of catecholamines in the release of anterior pituitary prolactin In vitro. Endocrinology 95:1202-1212. Shally, A. V.; A. J. Kastin; W. Locke; and C. Y. Bowers. 1967. In: Hormones in the Blood, edited by C. H. Gray and H. L. Bacharach, pp. 492-526. Academic Press, New York. 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. 143 Shiu, R. P. C., and H. G. Friesen. 1974a. Solubilization and purification of a prolactin receptor from rabbit mammary gland. The Fifty-Sixth Meeting of Endocrine Society, Abst. 168, A-139. Shiu, R. P. C., and H. C. Friesen. 1974b. Properties of a prolactin receptor from the rabbit mammary gland. Biochem. J. 140:301-311. Shiu, R. P. C., and H. G. Friesen. 1976. Blockade of prolactin action by an antiserum to its receptors. Science 192:259-261. Shiu, R. P. C.; P. A. Kelly; and H. G. Friesen. 1973. Radioreceptor assay for prolactin and other lactogenic hormones. Science 180:968-970. Shute, C. C. D. 1969. Distribution of choline esterase and cholinergic pathways. In: The Hypothalamus, edited by L. Martini, M. Motta, and F. Fraschini, pp. 167-179. Academic Press, New York. Sguris, J. T., and J. Meites. 1953. Differential inacti- vation of prolactin by mammary tissue from pregnant and parturient rats. Am. J. Physiol. 175:319-321. Sinha, Y. N., and H. A. Tucker. 1968. Pituitary prolactin content and mammary development after chronic administration of prolactin. Proc. Soc. Exp, Biol. Men. 128:84-88. Smalstig, E. B.; B. D. Sawyer; and J. A. Clemens. 1974. Inhibition of rat prolactin release by apomorphine In vivo and In vitro. Endocrinology 95:123-129. Smith, 0. W., and H. D. Hafs. 1973. Competitive protein binding and radioimmunoassay for testosterone in bulls and rabbits; blood serum testosterone after injection of LH or prolactin in rabbits. Proc. Soc. Exp. Biol. Med. 142:804-410. Smith, V. G.; T. W. Beck; E. M. Convey; and H. A. Tucker. 1974. Bovine serum prolactin, growth hormone, cortisol and milk yield after ergocrytine. Neuroendocrinology 15:172-181. 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. 144 Spies, H. G., and M. T. Clegg. 1971. Pituitary as a possible site of prolactin feedback in autoregu- lation. Neuroendocrinology 8:205-212. Spona, J. 1973. LH-RH stimulated gonadotropin release mediated by two distinct pituitary receptors. FEBS Letters 35:59-62. Sterental, A.; J. M. Dominguez; C. Weissman; and 0. Pearson. 1963. Pituitary role in the estrogen dependency of experimental mammary cancer. Cancer Egg. 23:481-484. Stricker, P., and R. Grueter. 1928. Action du lobe anterieur de l'hypophyse sur la montee' laiteuse. Compt. Rend. Soc. Biol. 99:1978-1980. 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 hypophyseal portal vessel. Proc. Soc. Exp. Biol. Med. 146:831-835. Talwalker, P. K., and J. Meites. 1961. Mammary lobula- alveolar growth induced by anterior pituitary hormones in adreno-ovariectomized and adreno- ovariectomized-hypophysectomized rats. Proc. Soc. Exp. Biol. Med. 107:880-883. 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.; N. J. Barowsky; and D. K. Jensen. 1971. ThyrotrOpin releasing hormones: direct evidence for stimulation of prolactin production by pituitary cells in culture. Biochem. Biophys. Res. Comm. 43:516-523. Thorell, J. I., and B. G. Johansson. 19125 Enzymatic iodination of polypeptides with I to high specific activity. Biochem. Biophys. Acta 251: 363-369. Tilney, F. 1936. The development and constituents of the human hypophysis. Bull. Neurol. Inst. N.Y. 5: 387-436. 145 Turkington, R. W. 1970. Stimulation of RNA synthesis in isolated mammary cells by insulin and prolactin bound to sepharose. Biochem. Biophys. Res. Comm. 41:1362-1367. Turkington, R. W. 1971. Measurement of prolactin activity in human plasma by new biological and radioreceptor assays. J. Clin. Invest. 50:94. Turkington, R. W. 1972. Molecular biological aspects of prolactin. In: Lactogenic Hormones, edited by G. E. W. Wolstenholme and J. Knight, pp. 111-136. Churchill Livingstone, London. Turkington, R. W. 1974. Prolactin receptors in mammary carcinoma cells. Cancer Res. 34:758-763. Turkington, R. W., and W. L. Frantz. 1972. The biochemical action of prolactin. In: Prolactin and Carcino- genesis, 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. 1973. Effector-receptor relations in the action of pro- lactin. 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, edited by E. AITen, C. H. Danforth and E. A. Doisy, pp. 740-766. Williams and Wilkins, Baltimore. Turner, C. D., and J. T. Bagnara. 1971. General Endo- crinology. W. B. Saunders Co., Philadelphia. Valverde, R. C.; V. Chieffo; and S. Reichlin. 1972. Prolactin releasing factor in porcine and rat hypothalamic tissue. Endocrinology 91:982-993. Vanderlaan, W. P. 1953. Direct effect of the anterior pituitary on the ventral prostate in rats. I. Clin. Invest. 32:609. Vignon, F., and H. Rochefort. 1974. Regulation des "recepteurs" des oestroge'nes dans les tumeurs mammaires: effet de la prolactine in vivo. Comptes Rendu 278:103-106. '__ 146 Vogt, M. 1954. The concentration of sympathin in differ- ent parts of the central nervous system under normal conditions and after the administration of drugs. J. Physiol. (London) 123:451-481. Voogt, J. L., and J. Meites. 1971. Effects of an implant of prolactin in median eminence of pseudopregnant rats on serum and pituitary LH, FSH and prolactin. Endocrinology 88:286-292. Watson, J. T.; L. Krulich; and S. M. McCann. 1971. Effect of crude rat hypothalamic extract on serum gonado- tropin and prolactin levels in normal and orchidectomized male rats. Endocrinology 89:1412- 1418. Weber, G. 1965. In: Molecular Biophysils, edited by B. Pullman and—M. Weissbluth, pp. 369-396. Academic Press, New York. Weder, H. G.; J. Schildknecht; R. A. Lutz; and P. Kesselring. 1974. Determination of binding parameters from Scatchard plots, theoretical and practical considerations. Eur. J. Biochem. 42: 475-481. Weist, W. G., and W. R. Kidwell. 1969. The regulation of progesterone secretion by ovarian dehydrogenases. In: The Gonads, edited by K. W. McKeans, pp. 295- 326. Appleton, New York. Welsch, C. W.; J. A. Clemens; and J. Meites. 1968. Effects of multiple pituitary homo-grafts or progesterone on 7, lZ-dimethylbenz(a)anthracene-induced mammary tumors in rats. J. Natl. Cancer Inst. 41:465-471. Welsch, C. W.; J. A. Clemens; and J. Meites. 1969. Effects of hypothalamic and amygdaloid lesions on develop- ment and growth of carcinogen-induced mammary tumors in the female rat. Cancer Res. 29:1541- 1549. Welsch, C. W.; T. W. Jenkins; and J. Meites. 1970. Increased incidence of mammary tumors in the female rat grafted with multiple pituitaries. Cancer Res. 30:1024-1029. Welsch, C. W., and J. Meites. 1970. Effects of reserpine on development of 7, l2-dimethylbenzanthracene- induced mammary tumors in female rats. Experientia 26:1133-1134. 147 Welsch, C. W.; H. Nagasawa; and J. Meites. 1970. Increased incidence of spontaneous mammary tumors in female rats with induced hypothalamic lesions. Cancer Res. 30:2310-2313. Welsch, C. W.; A. Negro-Villar; and J. Meites. 1968. Effects of pituitary homografts on host pituitary prolactin and hypothalamic PIF levels. Neuroendo- crinology 3:238-245. Welsch, C. W., and E. M. Rivera. 1972. Differential effects of estrogen and prolactin on DNA synthesis in organ cultures of DMBA-induced rat mammary carcinoma. Proc. Soc. Ex IiBiol. Med. 139:623-629 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-Ashman, H. G., and A. H. Reddi. 1971. Actions of vertebrate sex hormones. Ann. Rev. of Physiol. 33:31-82. Winand, R. J., and L. o. Kohn. 1972. The binding of (3H) thyrotropin and an 3H-labeled exophthalmogenic factor by plasma membranes of retro-orbital tissue. Proc. Natl. Acad. Sci. USA 69:1711-1718. Winkler, B.; I. Rathgeb; R. Steele; and N. Altszuler. 1971. Effect of ovine prolactin administration on free fatty acid metabolism in the normal dog. Endocrinology 88:1349-1352. Witorsch, R. J., and J. I. Kitay. 1972. Pituitary hor- mones affecting adrenal Sa-reductase activity: ACTH, growth hormone and prolactin. Endocrinol. 91:764-769. Wurtman, R. J. 1970. The role of brain and pineal indoles in neuroendocrine mechanism. In: The Hypothalamus, edited by L. Martini, M. Motta and F. Fraschins, pp. 153-165. Academic Press, New York. Wuttke, W.; E. Cassell; and J. Meites. 1971. Effects of ergocornine on serum prolactin and LH, and on hypothalamic content of PIF and LRF. Endocrinol. 88:737-741. Wuttke, W.; M. Gelato; and J. Meites. 1971. Mechanisms of pentobarbital actions on prolactin release. Endocrinology 89:1191-1194. 148 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. Med. 137:988-991. Yalow, R. S., and S. A. Berson. 1970. General principles of radioimmunoassay. In: Radioisotopes in Medicine: In Vitro Studies, edited by R. L. Hayes, F. A. Goswitz and B. E. P. Murphy, pp. 7-42. 0.8. Atomic Energy Commission, Springfield. Yoshinaga, K.; R. A. Hawkins; and J. F. Stocker. 1969. Estrogen secretion by the rat ovary in vivo during the estrous cycle and pregnancy. Endacrinology 85:103-112. Zmigrod, A.; H. R. Lindner; and S. A. Lamprecht. 1972. Reductive pathways of progesterone metabolism in the rat ovary. Acta Endocrinol. 69:141-152. APPENDICES APPENDIX A COLORIMETRIC PROTEIN ASSAY APPENDIX A COLORIMETRIC PROTEIN ASSAY This is the colorimetric assay of Lowry et a1. (1951) in which a phosphomolybdute-phoSphotungstate complex is reduced by a copper-protein complex producing a blue color. All reagents are prepared from stock solutions immediately prior to use. Reagents: 1. Assay: 1. Alkaline copper tartarate; mix 2% CuSO4 5 H20 (2 m1), 4% tartarate (2 ml) and 3% Na2CO3 in 0.1 NaOH (96 m1). Phenol reagent (Folin and Ciocalteu) diluted with equal volume of distilled H20. Stock protein standard (bovine serum albumin) 2 mg/ml tris buffer (25 mM tris, 10 mM CaClz, pH 7.6). Each protein standard or unknown sample was assayed in triplicate. 20-300 ug of protein standard or the unknown solution is diluted in tris buffer so as to have a total volume of 0.5 ml. Blank tubes contain tris buffer alone. 149 150 Five ml of alkaline copper tartarate was added, mixed and allowed to stand at room temperature for 10 minutes. 0.5 ml of diluted phenol is added to each tube, immediately mixed and allowed to react for 20 minutes at room temperature. The optical density of each sample was measured at 750 mu in a Beckman DB-G spectrophotometer initially zeroed with the blank solution. APPENDIX B RADIOIODINATION OF PROLACTIN APPENDIX B RADIOIODINATION OF PROLACTIN Reagents: 1. Ovine-prolactin (NIH-S-lO; 25.6 IU/mg) 2. Carrier free Na1251 (Amersham/Searle, Chicago, IL). 3. Lactoperoxidase (grade A, Calbiochem, La Jolla, CA). 4. 30% hydrogen peroxide (Mallinckrodt Chemicals, St. Louis, MO). 5. Sephadex G-50, G-100 (Pharmacia Fine Chemicals, Piscataway, NJ) expanded in tris-HCl buffer con- taining 10 mm CaCl2 at pH 7.8. Procedure: 1. Add 1.0 mCi Na125 I to serum vial containing 5 ug o-prolactin in 20 ul distilled water. 2. Add 30 ng lactOperoxidase in 10 ul of distilled water and 10 ul of 30% hydrogen peroxide diluted 1:30,000. Shake gently for 50 seconds. 3. Add 200 pl of 16% sucrose solution. Withdraw entire reaction mixture and carefully layer on a sephadex G-50 column (.9 x 20 cm) coated with 1% egg albumin in tris buffer. 151 152 Collect .5 ml aliquots in disposable culture tubes (12 x 75 mm) containing .5 ml 1% bovine serum albumin (BSA)-tris buffer. Count each tube in a Nuclear Chicago well scin- tillation counter (DS 303V) for 15 seconds to obtain iodination profile. Dilute the peak fractions in 1% BSA-tris buffer so that 100 pl gives approximately 60-70,000 cpm in a Nuclear Chicago automatic gamma counter (Model 1085, with 3 inch crystal). Test each fraction for its ability to bind spe- cifically to stock membranes of rat kidney and liver. Repurify the best binding fraction on a Sephadex G-100 column (.9 x 50 cm). Again collect .5 m1 aliquots, dilute to 60-70,000 cpm and test each peak fraction for specific binding. Use only the fractions with the highest specific binding to assay receptor activity. CURRICULUM VITAE NAME: DATE OF BIRTH: PLACE OF BIRTH: MARITAL STATUS: PRESENT ADDRESS: FUTURE ADDRESS: EDUCATION: Degree Ignn 3.8. 1971 Ph.D. 1976 CURRICULUM VITAE Gary Steven Kledzik September 23, 1949 Toledo, Ohio Single Department of Physiology Michigan State University East Lansing, MI 48824 (517) 353-6358 Medical Research Council Group in Molecular Endocrinology Centre Hospitalier De L'Universite Laval Quebec GIV 4 G2, Canada (418) 656-8253 Institution Major Field of Study Michigan State U. Biology Michigan State U. Physiology HONORS AND SOCIETY MEMBERSHIPS: a) Elected Member Sigma Xi, 1973. b) Recipient of the Graduate Office Scholarship Award, Dept. of Physiology, Michigan State University, 1975. c) Sigma xi Graduate Student Award for Meritorious Research in Physiology, 1976. 153 POSITIONS HELD: 154 a) Laboratory Technician in Neuroendocrinology, Department of Physiology, Michigan State University, East Lansing, MI, January, 1971 to December, 1971. b) Research Assistant in NeuroendoCrinology, Department of Physiology, M.S.U., East Lansing, MI, December, 1971 to October, 1976. c) Instructor of Anatomy and Physiology, Lansing Community College, Lansing, MI, September, 1974 to May, 1976. TALKS PRESENTED AT SCIENTIFIC MEETINGS: Meeting Date 57th Annual FASEB Meeting 1973 58th Annual FASEB Meeting 1974 57th Annual Meeting of 1975 The Endocrine Society 1976 Meeting of The 1976 American Association of Cancer Research PUBLICATIONS PAPERS: Quadri, S. K.; G. S. Kledzik; J. Meites. Topic Inhibition of Prolactin Release in Rats by Pyrogallol Reinitiation of Estrous Cycles in Light-Induced Constant Estrous Rats by Drugs Effects of Castration, Testosterone, Estradiol and Prolactin on Specific Binding Activity in Ventral Prostates of Male Rats Effects of High Doses of Estrogen on Prolactin Binding Activity and Growth of Carcinogen- Induced Mammary Cancers in Rats 1973. Effect of L-Copa and Methyldopa on Growth of Mammary Cancers in Rats. Proc. Soc. Exp. Biol. Med. 142:759-761. 155 Papers--Continued Quadri, S. K.; G. S. Kledzik; J. Meites. 1973. Reiniti- ation of Estrous Cycles in Old Constant-Estrous Rats by Central-Acting Drugs. Neuroendocrinology 11:248-255. Quadri, S. K.; G. S. Kledzik; J. Meites. 1974. Enhanced Regression of DMBA-Induced Mammary Cancers in Rats by Combination of Ergocornine with Ovariectomy or High Doses of Estrogen. Cancer Res. 34:499-501. Quadri, S. K.; G. S. Kledzik; J. Meites. 1974. Counter- action by Prolactin of Androgen-Induced Inhibition of Mammary Tumor Growth in Rats. J. Natl. Cancer Inst. 52:875-878. - Dickerman, S.; G. Kledzik; M. Gelato; H. H. Chen; J. Meites. 1974. Effects of Haloperidol on Serum and Pitui- tary Prolactin, LH and FSH, and Hypothalamic PIF and LRF. Neuroendocrinology 15:10-20. Kledzik, G. S.; J. Meites. 1974. Reinitiation of Estrous Cycles in Light-Induced Constant Estrous Female Rats by Drugs. Proc. Soc. Exp. Biol. M39. 146: 989-992. Kledzik, G. S.; C. J. Bradley; J. Meites. 1974. Reduction of Carcinogen-Induced Mammary Cancer Incidence in Rats by Early Treatment with Hormones or Drugs. Cancer Res. 34:2953-2956. Kledzik, G.; S. Marshall; M. Gelato; G. Campbell; J. Meites. 1975. Prolactin Binding Activity in the Crop Sacs of Juvenile, Mature, Parent and Prolactin-Injected Pigeons. Endocr. Res. Communications 2(485): 345-355. Bradley, C. J.; G. S. Kledzik; J. Meites. 1976. Prolactin and Estrogen Dependency of Rat Mammary Cancers at Early and Late Stages of Development. Cancer Res. 36:319-324. DeSombre, E. R.; G. Kledzik; S. Marshall; J. Meites. 1976. Estrogen and Prolactin Receptor Concentrations in Rat Mammary Tumors and Response to Endocrine Ablation. Cancer Res. 36:354-358. Kledzik, G. S.; S. Marshall; G. A. Campbell; M. Gelato; J. Meites. 1976. Effects of Castration, Testo- sterone, Estradiol and Prolactin on Specific Prolactin Binding Activity in Ventral Prostate of Male Rats. Endocrinology 98:373-379. 156 Papers--Continued Marshall, 8.; G. S. Kledzik; M. Gelato; G. A. Campbell; J. Meites. 1976. Effects of Estrogen and Testo- sterone on Specific Prolactin Binding in the Kidneys and Adrenals of Rats. Steroids 27:187-195. Kledzik, G.; C. Bradley; 8. Marshall; G. A. Campbell; J. Meites. 1976. Effects of High Doses of Estrogen on Prolactin Binding Activity and Growth of Carcinogen-Induced Mammary Cancers in Rats. Cancer Res. 36:3265-3268. Marshall, 8.; J. F. Bruni; G. A. Campbell; G. S. Kledzik; J. Meites. Micro-Radioimmunoassay of Rat Prolactin, Luteinizing Hormone and Follicle Stimulating Hormone. Endocrinology (submitted). Bradley, C. J.; G. S. Kledzik; G. A. Campbell; S. Marshall; J. Meites. Changes in Specific Prolactin Binding During Growth of Mammary Cancers in Rats. Proc. Son. Exp. Biol. Med. (Submitted). ABSTRACTS: Quadri, S. K.; G. S. Kledzik; J. Meites. 1973. Enhanced Regression of Mammary Cancers in Rats by Combina- tion of High Estrogen or Ovariectomy and Ergo- cornine. Proceedings oI_Ing.AACR. ABST 71, p. 18. Kledzik, G. S., and J. Meites. 1974. Reinitiation of Estrous Cycles in Light-Induced Constant Estrous Rats by Drugs. Fed. Proc. ABST 33. Kledzik, G. S.; S. Marshall; M. Gelato; J. Meites. 1975. Effects of Castration, Testosterone, Estradiol and Prolactin on Specific Prolactin Binding Activity in Ventral Prostate of Male Rats. 57th Annual Meeting 2: the Endocrine Society ABST 56, New York. Kledzik, G. S.; C. Bradley; S. Marshall; G. A. Campbell; J. Meites. 1976. Effects of High Doses of Estrogen on Prolactin (PRL) Binding Activity and Growth of Carcinogen-Induced Mammary Cancers in Rats. American Assoc. oI Cancer Research, Toronto. nICHIcaN STATE UNIV. LIBRARIES IIIIIMINIIIIIIIIlIIIIIIIIIIIIIIIIIIIIHIIIIIIIIINHI 31293104900893