swmss on PROLACTIN SECRETION gimme Thai: for H1. Degree of Ph. D. MICHIGAN STATE UNIVERSITY Charles SamueI Nicoll 1962 This is to certify that the thesis entitled Studies on Prolactin Secretion I_n. Vitro presented by Charles S. Nicoll has been accepted towards fulfillment of the requirements for __P_h_£'__degree in Ph 3101083' Date June 18, 1962 0-169 LIBRARY Michigan State University STUDIES ON PROLACTIN SECRETION g5 VITRO BY Charles Samuel Nicoll ' AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Pharmacology 1962 ABSTRACT STUDIES ON PROLACTIN SECRETION _I_1\1 VITRO By Charles Samuel Nicoll Prolactin secretion ig_vitro by explants of anterior pituitary (AP) glands from rats and other species was investigated. The AP explants were cultured by modified watch glass techniques in medium 199 for 3 to 6 days. The cultures were incubated in air or 95% O2 — 5% CO2 atmos- phere at 35°C. The prolactin activities in the culture medium samples were determined by the local "micro" pigeon crop method. The results of these studies were as follows: 1. Explants from the APs of rats actively secreted pro- lactin ig,vitro in synthetic 199 medium. Prolactin activity was detectable in the medium of cultures which were incubated for periods up to 21 days. 'The AP glands from lactating rats produced about twice as much prolactin in vitro aS- the glands from non-lactating mature females. The AP fragments which were incubated in air atmosphere synthesized about as much prolactin during each day of culture as they contained at the Charles Samuel Nicoll beginning of culture. The AP fragments incubated in the 95% O2 - 5% CO2 atmosphere produced from 3 to 11 times their initial prolactin content during each day Of ig_vitro life and explant survival was much better than in cultures in air atmosphere. These results demonstrate that AP glands can actively secrete prolactin in a system which is virtually free of hypothalamic or other i§_vivo influences. This indicates that prolactin secretion is an autonomous characteristic of the AP. Explants from the APs of female guinea pigs, rabbits and mice and of male mice and Cynomalogus monkeys ac- tively secreted prolactin in vitro. The glands of male and female pigeons released very little prolactin into the culture medium. These results demonstrate that the APs of male and female mammals can secrete prolactin' autonomously and indicate that there may be a basic physiological difference in prolactin secretion by mam- malian and avian pituitaries. Medium incubated without any tissue or with several non-pituitary tissues did not have prolactin activity and the culture medium did not potentiate the pigeon Charles Samuel Nicoll crop response to prolactin. Cutting the AP glands of rats into 6 explants resulted in about a 20% reduction in wet weight indicating a loss of 1/5 of the tissue. Incubation in 199 medium for 3 days caused a further loss in wet weight of 8%“ At the end of 3 days of incubation, the explants con- tained about 84.5% of their initial prolactin content. Incubation of rat AP fragments with hypothalamic or cerebral explants, or with homogenates of rat hypo- thalamic or cerebral tissue added to the medium, greatly reduced the prolactin activity in the medium samples. An acid extract of rat hypothalamic tissue signifi- cantly reduced prolactin secretion i§_vitro whereas a similar extract of cerebral tissue had no effect. The results with the extracts indicate that hypothalamic tissue may contain an acid extractable material which specifically inhibits prolactin secretion. Neither oxytocin nor pitressin influenced prolactin secretion when added to the medium at concentrations of 0.1 U and 1.0 U per ml respectively. Charles Samuel Nicoll Addition of estradiol to the 199 medium at concentra- tions of 0.05 and 0.5 ugm per ml increased prolactin secretion ig_vitro. No stimulation occurred with 2 ugm estradiol per ml. Thyroxine and triiodothyronine stim- ulated prolactin production in_vitro at concentrations of 0.1 ugm per ml. Cortisol, at a concentration of 10 ugm per ml depressed prolactin secretion i§_vitro and 20 ugm/ml of corticosterone had no effect on prolactin production. Neither testosterone nor progesterone influenced prolactin secretion i2_vitro at a concentra- tion of 2 ugm/ml. Concentrations of the latter steroids of 5 and 10 ugm per ml were toxic to the AP explants as was corticosterone at concentrations of 30 and 50 ugm -per ml. These results indicate that estradiol, thyroid hormones and cortisol can influence prolactin secretion by a direct action on the AP cells. The lack of an effect of progesterone and testosterone ig_vitro indi- cates that the effects of these steroids on prolactin secretion in vivo may be indirect. Insulin did not affect prolactin secretion ;Q_yitgg indicating that insulin is not essential for prolactin synthesis. 9. Charles Samuel Nicoll A dose-response relationship was established for NIH prolactin in White King squabs for prolactin doses from 2 to 60 ugm. With 10 pigeons per dose a regression equation was obtained in which y = 0.13 + 1.64 x, where y represents the pigeon crop response to x ugm of pro— lactin. The index of precision of the assay method was 0.34 and the variance ratio was highly significant (F = 190.4) thus demonstrating the high degree of acceptability of the assay method. STUDIES ON PROLACTIN SECRETION g VITRO BY Charles Samuel Nicoll A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Pharmacology 1962 Dedicated to my Mother and my Father ACKNOWLEDGEMENTS The author wishes to express his deep gratitude to Dr. Joseph Meites, Professor in the Department of Physiology and Pharmacology, for his inspiration, guidance, advice and assistance throughout the course of this and other studies and during the preparation of this manuscript. He also wishes to express his appreciation to Dr. B. V. Alfredson, head of the Department of Physiology and Pharmacology, for providing facilities and laboratory space for conducting these experiments. The author also wishes to thank Dr. E. P. Reineke, Dr. W. D. Collings, Dr. L. F. Wolterink and Dr. J. E. Nellor for advice and information pertaining to these and other studies. Special gratitude is due to Mrs. Carol Blackwell for invaluable technical assistance. The writer is also indebted to Dr. P. J. Clark, Profes- sor of the Department of Zoology and to Dr. J. H. Stapleton, Department of Statistics for statistical advice. The advice of Drs. M. A. Richardson of the Brucilla Laboratory, Michi- gan State University, and R. H. Kahn, Department of Anatomy, University of Michigan, Ann Arbor, Michigan, on culture pro- cedures is gratefully acknowledged. The author is also iii indebted to Dr. W. N. Mack of the Department of Microbiology and Public Health, Michigan State University for the monkey pituitary glands and to Mr. M. Swab for attending to the experimental animals. Mr. K. Irish must also be thanked for his services in constructing apparatus. The author also wishes to express his gratitude to the Department of Physiology and Pharmacology of Michigan State University for a research assistantship which was granted from September 1959 to September 1960 and to the National Institutes of Health for the predoctoral research fellowship which was granted from September 1960 until completion of the work. The author is also grateful for N.I.H. grants which provided funds for this research. iv TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . 4 I. Evidence for Neural Influence on Adenohypo- physial Function . . . . . . . . . . . . . . 4 II. Evidence for Hypothalamic Regulation of the Pars Distalis . . . . . . . . . . . . . . . . 6 III. Evidence for Hypothalamic Regulation of Prolactin Secretion . . . . . . . . . . . . . 12 A. The Suckling Stimulus 12 B. Effects of Drugs, Hormones and Non- specific Agents on Prolactin Secretion 14 C. Hypothalamic Lesions 16 D. Pituitary Stalk Section 20 E. Pituitary Transplantation 22 1. Luteotrophic Activity of Pituitary Transplants 23 2. Pituitary Transplants and Mammary Growth and Secretion 25 IV. Hormone Production by Pituitary in_Vitro . . 27 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 33 I. Animals . . . . . . . . . . . . . . . . . . . 33 II. Culture Methods . . . . . . . . . . . . . . . 33 III. The Culture Medium . . . . . . . . . . . . . 41 IV. The Culture Procedure . . . . . . . . . . . . 43 V. The Assay Procedure . . . . . . . 47 VI. The Matched Pair Culture and Assay Procedure. 49 VII. Hormone Preparation . . . . . . . . . . . . . 50 VIII. Statistical Procedures . . . . . . . . . . . 53 EXPERIMENTAL . . . . . . . . . . . . . . . . . I. II. III. IV. VI. VII. VIII. IX. XI. XII. COMPARISON OF IN_VITRO PROLACTIN PRODUCTION BY PITUITARY EXPLANTS FROM MATURE FEMALE AND POSTPARTUM LACTATING RATS . . . . . . . . PROLACTIN SECRETION IN;VITRO BY LONG TERM CULTURES OF RAT PITUITARY TISSUE . . . EFFECTS OF 95% 02 - 5% coz ATMOSPHERE 0N EXPLANT SURVIVAL AND PROLACTIN SECRETION I—N VITRO O O O C O I O O O O O O O O O O ASSAYS TO DETERMINE OF 199 MEDIUM INCUBATED WITH AND WITHOUT TISSUES HAD PROLACTIN ACTIVITY AND THE EFFECTS OF 199 MEDIUM WITH INSULIN ON THE PIGEON CROP RESPONSE TO P ROLACT I N O O O O O O I O O O O C O O O O DETERMINATION OF THE QUANTITY OF GLANDULAR TISSUE LOST IN THE PROCEDURE OF DISSECTING THE ANTERIOR PITUITARIES OF RATS INTO 6 EXPLANTS O O O O O O O O O O O O O O O 0 EFFECTS OF IN_VITRO INCUBATION ON THE WEIGHT AND PROLACTIN CONTENT OF ANTERIOR PITUITARY EXPLANTS . O O O C O O O O O O O I O O O PROLACTIN SECRETION IN_VITRO BY PITUITARY GLANDS FROM DIFFERENT SPECIES . . . . . . EFFECTS OF HYPOTHALAMUS AND CEREBRUM ON PROLACTIN SECRETION IN_VITRO . . . . . . EFFECTS OF NEUROHYPOPHYSIAL HORMONES EFFECTS OF ESTROGEN . . . . . . . . . . . EFFECTS OF PROGESTERONE AND TESTOSTERONE EFFECTS OF HYDROCORTISONE AND CORTICOSTERONE. vi Page 54 54 60 65 72 77 79 85 96 ll3 127 152 165 Page XIII. EFFECTS OF THYROXINE AND TRIIODOTHYRONINE . . 179 XIV. EFFECTS OF INSULIN . . . . . . . . . . . . . 194 XV. THE BIOASSAY OF PROLACTIN . . . . . . . . . . 202 GENERAL DISCUSSION . . . . . . . . . . . . . . . . . 218 SUMMARY . . . . . . . . . . . . . . . . . . . . . . 240 REFERENCES . . . . . . . . . . . . . . . . . . . . . 245 vii 10. 11. 12. LIST OF TABLES Page Components of 100 M1 of "199" Culture Medium . . 42 Comparison of ig_Vitro Prolactin Production by Pituitaries of Mature Female and Postpartum Lactating Rats . . . . . . . . . . . . . . . . . 60 ;n_Vitro Prolactin Production by Rat Pituitary Explants Over Two Successive Seven-Day Periods . 64 Comparison of Prolactin Production by Pitui- taries from Mature Female Rats Cultured in Air and 95% O2 - 5% C02 Atmosphere . . . . . . . . . 68 Assays To Determine if 199 Medium Incubated with and without Tissues Had Prolactin Activity and the Effects of 199 Medium on the Pigeon Crop Response to Prolactin . . . . . . . . . . . . . 76 The Wet Weight of Uncultured and Incubated Rat Anterior Pituitary Tissue . . . . . . . . . . . 83 Effect of in_Vitro Incubation on the Prolactin Content of Rat Anterior Pituitary Explants . . . 84 Comparison of Pituitary Prolactin Content and iQ_Vitro Prolactin Secreting Capacity of An- terior Pituitaries from Different Species . . . 89 Prolactin Content of Media from 6-Day Cultures of Monkey and Rabbit Anterior Pituitary Tissue . 90 Effects of Hypothalamus and Cerebrum on Prolactin Secretion in_Vitro . . . . . . . . . . 109 Effect of 0.1 U per m1 of Oxytocin on Prolactin Secretion in_Vitro . . . . . . . . . . . . . . . 121 Summary of the in_Vitro Effects of 0.1 U per m1 of Oxytocin . . . . . . . . . . . . . . . . . . 122 viii Table Page 13. Effect of 1.0 U per m1 of Pitressin on Pro- lactin Secretion ig_Vitro . . . . . . . . . . . 123 14. Summary of the in.Vitro Effects of 1.0 U per m1 of Pitressin . . . . . . . . . . . . . . . . . . 124 15. Effects of 0.5 ugm per m1 of Estradiol in 199 Medium on Prolactin Secretion in_Vitro . . . . . 140 16. Effect of 0.05 ugm per ml of Estradiol on Prolactin Secretion i§_Vitro . . . . . . . . . . 141 17. Summary of the in_Vitro Effects of 0.05 ugm per ml of Estradiol . . . . . . . . . . . . . . . . 142 18. Effect of 0.5 ugm per m1 of Estradiol on Prolactin Secretion in_Vitro . . . . . . . . . . 143 19. Summary of the in.Vitro Effects of 0.5 ugm per m1 of Estradiol . . . . . . . . . . . . . . . . 144 20. Effect of 2.0 ugm per m1 of Estradiol on Prolactin Secretion ig_Vitro . . . . . . . . . . 145 21. Summary of the in_Vitro Effects of 2.0 ugm per m1 of Estradiol . . . . . . . . . . . . . . . . 146 22. Effect of 2.0 ugm per m1 of Progesterone on Prolactin Secretion ig_Vitro . . . . . . . . . . 156 23. Summary of the in.Vitro Effects of 2.0 ugm per m1 of Progesterone . . . . . . . . . . . . . . . 157 24. Effect of 2.0 ugm per m1 of Testosterone on Prolactin Secretion in_Vitro . . . . . . . . . . 158 25. Summary of the iQ_Vitro Effects of 2.0 ugm per m1 of Testosterone . . . . . . . . . . . . . . . 159 26. Effect of 10 ugm per m1 of Cortisol on Prolactin Secretion ig_Vitro . . . . . . . . . . . . . . . 172 ix Table 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. Page Summary of the ig.Vitro Effects of 10 ugm per ml of Hydrocortisone . . . . . . . . . . . . . . 173 Effect of 20 ugm per m1 of Corticosterone on Prolactin Secretion in_Vitro . . . . . . . . . . 174 Summary of the in.Vitro Effects of 20 ugm per m1 of Corticosterone . . . . . . . . . . . . . . 175 Effect of 0.1 ugm per m1 of Thyroxine on Pro- lactin Secretion in_Vitro . . . . . . . . . . . 186 Summary of the in_Vitro Effects of 0.1 ugm per m1 of Thyroxine . . . . . . . . . . . . . . . . 187 Effect of 0.1 ugm per m1 of Triiodothyronine on Prolactin Secretion ig_Vitro . . . . . . . . 188 Summary of the in_Vitro Effects of 0.1 ugm per m1 of Triiodothyronine . . . . . . . . . . . . . 189 Effect of 2.0 U per m1 of Insulin in 199 Medium on Prolactin Secretion in_Vitro . . . . . . . . 199 Effect of 2.0 U per m1 of Insulin in 199 Medium on Prolactin Secretion in_Vitro Continued . . . 200 Summary of the in_Vitro Effects of 2.0 U per m1 of Insulin . . . . . . . . . . . . . . . . . . . 201’ Dose-Reseponse Data for NIH Prolactin in White King Pigeons in Terms of Reece-Turner Units . . 210 Work Form for the Analysis of Variance of a Dose-Response Curve with Two or More Responses at Each Dose . . . . . . . . . . . . . . . . . . 212 Analysis of Variance of Dose-Response Curve for , NIH Prolactin in 60 White King Pigeons . . . . . 213 Analysis of Variance of the Responses of Six Groups of 10 Pigeons Which Were Injected with 10 ugm NIH Prolactin . . . . . . . . . . . . . . 214 Table 41. 42. Page Pigeon Crop Responses to Prolactin Standards Administered at Different Intervals During the Course of the Investigations . . . . . . . . . . 215 Analysis of Variance of the Assay Results from 6 Groups of Cultures . . . . . . . . . . . . . . 222 xi 10. 11. LIST OF FIGURES The glass and plastic Petri dish preparations. The plexiglass chamber used for gassing the cultures with the 95%02 — 5% CO2 atmosphere The plexiglass hood . . . . . . . . . . . . . . A schematic representation of the paired culture and assay procedure . . . . . . . . . . . . . . Photomicrograph of an explant of rat anterior pituitary tissue cultured in air atmosphere for 6 days. X 130 . . . . . . . . . . . . . . . . . Photomicrograph of an explant of rat anterior pituitary tissue cultured in 95% 0 - 5% CO atmosphere for 6 days. X 130 . .2. . . . g . . Photomicrograph of fresh, uncultured rat anterior pituitary tissue. X 130 . . . . . . . Photomicrograph of fresh, uncultured rat anterior pituitary tissue. X 450 . . . . . . . Photomicrograph of an explant of rat anterior pituitary tissue cultured in 95% O2 - 5% CO2 atmosphere for 6 days. X 450 . . . . . . . . . Photomicrograph of an explant of anterior pituitary tissue from a male Cynomalogus monkey cultured for 6 days in 95% 0 - 5% CO atmos- phere. X 450 . . . . . . .2. . . . I . . . . . Photomicrograph of an explant of anterior . pituitary tissue from a mature female guinea pig cultured for 6 days in 95% 02 - 5% C02 atmos— phere. X 450 . . . . . . . . . . . . . . . . . xii Page 36 4o 46 51 69 70 71 91 92 93 94 Figure Page 12. Photomicrograph of an explant of pigeon anterior pituitary tissue cultured for 6 days in 95% 02 - 5% CO2 atmosphere. X 450 . . . . . . . . . . . 95 13. Dose-response curve of NIH prolactin in White King pigeons showing the upper and lower 99% confidence limits . . . . . . . . . . . . . . . 211 14. A scatter diagram showing the relationship between explant weight and total IU of prolactin in the medium samples . . . . . . . . . . . . . 223 xiii INT RODUCTI ON Considerable research in the last three decades has been devoted to evaluating the endocrine function of the anterior pituitary gland. It is becoming increasingly apparent that the function of the pars anterior is regulated to a considerable extent by the central nervous system (CNS). The hypothalamus is evidently the portion of the CNS which is most intimately associated with governing adendhypophysial secretion. The modus operandi of hypothalamic control of the anterior pituitary gland has been shown to be of a neuro- -humora1 nature rather than by direct innervation of the adenohypophysial cells. Neurosecretory cells in the hypo- thalamus liberate chemical mediators which are conveyed by / the hypothalamic-hypophysial portal blood to the pars anterior where they influence the secretion of the endo- crine products of the gland. It is now generally accepted that the secretion of adrenocorticotropin (ACTH), thyrotropin (TSH), follicle stimulating hormone (FSH), luteinizing hormone (LH) and somatotropin (STH) is contingent upon humoral stimulatory agents of hypothalamic origin. The secretion of prolactin by the pars anterior is exceptional in this respect since it seems to be independent of hypothalamic stimulation. Neural regulation of prolactin secretion appears to be accomplished by an hypothalamic engendered neurohumor which inhibits prolactin secretion. Procedures which prevent the hypothalamic neurohumors from gaining access to the adeno— hypophysis, such as pituitary transplantation, severance of the hypothalamic-hypophysial stalk or destruction of regions of the hypothalamus by electrolytic lesions, result in a pronounced reduction in the secretion of all of the hormones of the pars anterior except prolactin. Indeed, prolactin secretion is apparently favored by such measures. It has not been established, however, whether pro- lactin secretion is in fact an autonomous characteristic of the anterior pituitary or whether secretion of the hor- mone depends upon some extrahypothalamic stimulation which can manifest itself when hypothalamic inhibition is re- moved. It also remains undetermined whether the hormones which alter anterior pituitary function, such as estrOgens and thyroid hormones, accomplish this by an action on the adenohypophysis, on the hypothalamus or on both locations. It was of interest, therefore, to determine whether anter- ior pituitary fragments could secrete prolactin in an organ culture system where ig_yiy9_inf1uences could be virtually eliminated. Since a number of hormones are known to in- fluence prolactin secretion i vivo, the effects of these hormones on prolactin production in_vitro were also examined to determine if they influenced prolactin secretion by a direct action on the adenohypophysis. REVIEW OF THE LITERATURE The outlook of early endocrinologists was considerably influenced by the remarkable advances in the field of neuro- physiology which occurred in the late nineteenth and early twentieth centuries. To establish the endocrine function of an organ, endocrinologists were compelled to eliminate all possible neural influences from their experimental situations. They became accustomed, therefore, to regard- ing the functions of the nervous and endocrine systems as being quite independent of one another. The paramount pos- sibility of interrelation and interaction between these two co-ordinative systems consequently eluded the attention of physiologists generally. Many of the early researchers who investigated the functional activity of adenohypophysial transplants in hypophysectomized animals erroneously concluded that nor- mally functioning transplants were obtained (see Harris, 1955). This supported the concept, which was generally accepted for a time, that the pituitary performed its several functions without significant influence by the other organ systems of the body. A number of observations, however, indicated that the pars anterior is influenced by the central nervous system. I. Evidence for Neural Influence on Adenohypophysial Function Marshall (1942) was one of the first to emphasize the relationships between the external environment and repro- ductive processes. He drew attention to the fact that a variety of environmental factors, such as food, light and temperature, are of importance in conditioning sexual periodicity and suggested that they operated through the central nervous system on the anterior pituitary. The influence of exteroceptive stimuli on thyroid and adrenal function was also recognized, and the mechanism by which certain psychic states influenced the activity of the tar- get organs of the adenohypophysial trophic hormones re- mained unexplained. These observations, together with other phenomena, such as reflex induction of ovulation in rabbits consequent to the stimulus of coitus, and numerous endo- crinopathies associated with hypothalamic disease such as Frohlichs adiposo-genital syndrome, prompted the investi- gation of the possibility that the activity of the pars distalis is influenced by the CNS. II. Evidence for Hypothalamic Regulation of the Pars Distalis Since the adenohypophysis originates as an evagination of the Rathke's pouch, which migrates from the roof of the mouth to become attached to the floor of the third ventri- cle, it seemed probable that the hypothalamus is the part of the CNS most directly concerned with the regulation of the adenohypophysis (Harris, 1955). Control of the function of the adenohypophysis by direct innervation of the cells was discounted due to the paucity of nerve fibers in the pars anterior (Rasmussen, 1938). On the other hand, there exists a network of fine blood vessels in the hypothalamic area at the base of the brain whidh coalesces into larger vessels leading down the pituitary stalk from the hypo- thalamus to the pituitary gland, where they again ramify to supply the blood sinuses in the adenohypophysis. This portal circulation, first described by Popa and Fielding (1930), is ideally suited to convey materials of hypothala- mic origin directly to the anterior pituitary. The possi- bility was therefore investigated that the neurosecretory’ cells of the hypothalamus release humoral agents which are conveyed by the portal blood vessels to the anterior pituitary where they activate the secretion of hormones from the gland. This possibility was first proposed by Harris (1937) and Brooks (1938). The investigation of hypothalamic regulation of the adenohypophysis has employed a variety of procedures includ— ing pituitary transplantation, sectioning of the pituitary stalk, electrical stimulation of the CNS and hypothalamus, 'induction of hypothalamic lesions and administration of drugs. Electrical stimulation of the hypothalamus can induce in- creased secretion of adrenocorticotropin (ACTH), thyrotro- pin (TSH) and gonadotropins (FSH—LH) (Harris, 1955). Stim- ulation of the pituitary gland directly is ineffective in this respect. The electrical stimuli are believed to activate the release of humoral agents from the neuro- secretory cells of the hypothalamus into the portal circu- lation. The neurOhumors are then conveyed to the pars distalis where they stimulate the secretion of the tropic hormones. The procedure of inducing relatively discrete electro- lytic lesions in the hypothalamus has disclosed that ACTH (deGroot and Harris, 1950), TSH (Greer, 1952) and gonado- tropin (Dey, 1943) secretion can be impaired. Massive lesions depress growth hormone (STH) secretion (Reichlin, 1961). The lesions studies indicate that localized centers in the hypothalamus may be concerned with the regulation of ACTH, TSH and FSH-LH secretion. Injury to these centers either destroys the neurosecretory cells specifically con- cerned with regulating the secretion of each trophic hor- mone, or impairs the neural areas which govern the activity of the neurosecretory elements. Transplantation of the adenohypophysis to an ectopic site in hypophysectomized animals or severence of the pituitary stalk, with measures to prevent re-establishment of the vascular connections between the hypothalamus and the pituitary (e.g., by insertion of a wax paper barrier), are procedures which divorce the pituitary gland from hypothalamic neurohumoral regulation. These measures have been found to cause a profound reduction in ACTH, TSH, FSH-LH and STH secretion (Harris, 1955). Harris (1950) demonstrated that severence of the pituitary stalk of female rats resulted in a cessation of estrous cycles. Many of the rats, however, showed a re— sumption of cyclic activity after a few days. When serial sections of the hypothalmo-hypophysial region of these rats were examined histologically after perfusion oftjn vascular system with India ink, it was found that the portal cir- culation had become re—established in the rats which showed a restoration of estrous cycles. The animals which did not return to estrous activity had more or less permanently disrupted portal connections. When pituitary grafts are placed beneath the median eminence of the hypothalamus of hypophysectomized rabbits (Jacobsohn, 1954) or rats (Harris and Jacobsohn, 1952), thus enabling the portal blood supply to become re-established, regular estrous cycles return. The animals became pregnant when mated and delivered normal young. Similar grafts in other sites did not support reproductive function. Nikitovitch-Winer and Everett (1958) showed that when pituitary transplants were re-transplanted from the kidney capsule to the median eminence of rats, estrous cycles were resumed and thyroid and adrenal function returned to normal. When the grafts were re-transplanted under the temporal lobe of the brain or were permitted to remain on the kidney there were no indications of a return of FSH-LH, ACTH or TSH secretion. It is therefore apparent that when the adenohypophysis is separated from its hypo- thalamic connnections, and then placed in a position which 10 enables regeneration of the hypothalamo-hypophysial portal supply, normal functional activity of the gland returns. These observations provide some of the most cogent evidence that hypothalamic regulation of the adenohypophysis is accomplished by a humoral mechanism. Secretion of the adenohypophysial hormone, prolactin, is rather unusual in certain respects. Prolactin does not appear to participate in a feedback mechanism with its target organs, a feature which it Shares with STH. In addition, prolactin secretion is apparently not depressed by the procedures which disrupt hypothalamic regulation of the pars distalis, such as transplantation, pituitary stalk section or hypothalamic lesions. In contrast, the produc- tion of all the other anterior pituitary hormones is greatly impaired by such disruptive measures. Indeed, prolactin secretion is apparently favored by measures which remove hypothalamic control from the adenohypophysis. The hypo- thalamic neurohumoral mechanism controlling prolactin secretion is therefore considered to be of an inhibitory nature, whereas all of the other adenohypophysial hormones apparently require neurohumoral stimulators. Hypothalamic regulation of prolactin secretion is considered in more detail in the following sections. 11 III. Evidence for Hypothalamic Regulation of Prolactin Secretion A. The Suckling Stimulus The first experimental indications that an extero- ceptive stimulus influences prolactin secretion emerged from the observations of Selye (1934). He found that continued suckling of rats with ligated galactophores maintained the secretory activity and structural integrity of their mammary glands. Secretory activity in the mam- mary glands of unsuckled rats ceased rapidly and involu- tion occurred shortly thereafter. Retardation of mammary involution in rats with excised nipples when other intact glands were nursed gave further evidence that suckling influenced prolactin secretion. This led to the suggestion that prolactin was released in response to suckling (Selye et al., 1934). Further evidence of the role of suckling in prolactin secretion was gained from studies on the ef- rects of suckling on non-lactating cyclic female rats (Selye and McKeown, 1934). Induction of pseudopregnancy, mammary growth and initiation of lactation was observed' in these virgin females. These observations on suckling and the maintenance of mammary integrity have been amply 12 confirmed by a number of studies (see Cross, 1961). The influence of exteroceptive stimuli on prolactin secretion is further illustrated by the observations that irritation of the nipples with turpentine retards mammary involution in rats and mice (Hooker and Williams, 1940; Mixner and Turner, 1941) and electrical stimulation of the nipples of estrogen primed rats initiates lactation (Maqsood and Meites, 1961). More cogent evidence that suckling stimulates prolactin secretion was provided by studies on the effects of suck- ling on the pituitary prolactin content of lactating rats. After a period of nonsuckling, a rapid reduction of pitui- tary prolactin content from the prenursing level was observed in response to a brief period of suckling (Reece and Turner, 1937; Grosvenor and Turner, 1957). Regular suckling increases the pituitary prolactin content and maintains it at a higher level than in glands of nonsuckled postpartum rabbits (Meites and Turner, 1948). The observa- tion that suckling causes degranulation of pituitary acido- phils is further evidence that this stimulus influences pro- lactin secretion (Desclin, 1947) since the acidophils are generally associated with prolactin secretion (see Purves, 1961). 13 The suckling stimulus apparently influences the secretion of other anterior pituitary hormones. Gregoire (1947) and Tabachnick and Trentin (1951) presented evi— dence that nursing stimulates ACTH discharge. Ovariectomy does not result in the appearance of castration cells in the pituitaries of lactating rats when they are suckled (Desclin, 1947), suggesting that nursing inhibits gonado- tropin secretion. Resumption of postpartum estrous cycles is hastened when litter size, and consequently the inten— sity of the nursing stimulus, is reduced in mice (Parks, 1926) and rats (Rothchild, 1960a). Clap (1937) observed that the interval from parturition to first estrous was prolonged in cows with increased frequency of milking or sudkling. Evidence has been presented for a reciprocal relation between the secretion of FSH—LH and prolactin (see Meites et al., 1962), and it has been suggested that suckling promotes prolactin secretion primarily by depres- sing gonadotropin secretion (Donovan, 1960). In addition to the well-established fact that suckling stimulates oxytocin release from the neurohypophysis (Petersen, 1942; Cross and Harris, 1952), there is evidence that nipple stimulation promotes antidiuretic hormone (ADH) secretion 14 in a number of species (see Cross, 1961), as judged by (inhibition of urine flow during application of the milking stimulus. It is thus apparent that the exteroceptive stimulus of suckling can considerably alter anterior and ( posterior pituitary function and these effects are pre— sumably mediated via the CNS. B. Effects of Drugs, Hormones and Nonspecific Agents on Prolactin Secretion Many drugs, hormones and nonspecific factors have been tested in our laboratory to determine whether they can in— duce prolactin secretion, as indicated by (a) initiation of mammary secretion in estrogen-primed rats and rabbits and pseudopregnant rabbits and (b) maintenance of secretory - activity and mammary integrity in postpartum rats after litter removal. The tranquilizing drugs reserpine, chlor- promazine and meprobamate were found to be effective in promoting prolactin secretion in rats and rabbits (see Meites et al., 1962). These drugs have occasionally been observed to promote breast growth and lactation in women (Sulman and Winnik, 1956; Winnik and Tennenbaum, 1955) and are reported to induce pseudopregnancy in rats (Barra— clough and Sawyer, 1959). Kanematzu et al. (1961) recently 15 reported that reserpine produced a depletion of pituitary prolactin content three days after a single injection into rabbits, whereas Meites (1958a) found an increase seven days after injection. This suggests that reserpine in- creases both the synthesis and release of prolactin, but the latter effect diminishes by the seventh day. The action of reserpine may depend on its demonstrated ability to inhibit hypothalamic function (Gaunt et al., 1954; Bein, 1956), thereby removing the inhibition on prolactin secre- tion. The other tranquilizers may operate in a similar manner. Reserpine and chlorpromazine can inhibit FSH-LH release (Gaunt et al., 1954; Barraclough and Sawyer, 1959) and stimulate prolactin and ACTH discharge (Harwood and Mason, 1957). ‘ The neurohormones epinephrine, nor—adrenaline, acetyl- choline and serotonin were effective in stimulating pro- lactin secretion (Meites, 1959a; Meites et al., 1959b, 1960b). Anti-adrenergic and cholinergic drugs were also effective in this respect as were numerous other agents such as morphine sulfate, hypothalamic extracts and Guille- \ min's corticortropin releasing factork(CRF) (Meites, Nicoll and Talwalker; 1962). CRF is a polypeptide of hypothalamic l6 origin which Guillemin and colleagues have isolated and which they believe to be the principal neurohumoral agent responsible for ACTH secretion (Guillemin, 1962). The mechanism by which most of these agents elicit prolactin secretion is unknown. The materials may have acted directly on the pituitary gland, on the hypothalamus or elsewhere in the CNS. Most of these drugs and other agents have been reported to cause ACTH discharge (Ganong and Forsham, 1960) and induce pseudopregnancy in rats (Swingle et al., 1951b). Since nonspecific stresses can induce prolactin secretion, as judged by initiation of lactation (Nicoll et al., 1960) and pseudopregnancy (Swingle et al., 1951b), it is quite likely that most of the factors which induce prolactin and ACTH secretion were operating as stressful agents. C. Hypothalamic Lesions The studies on the effects of hypothalamic lesions on prolactin secretion have disclosed that lesions do not depress production of this hormone. Indeed when prolactin secretion was affected by hypothalamic lesions, augmented production of the hormone was evidenced. Electrolytic lesions in the supraoptico-hypophysial tract (Cross and (m. 17 Harris, 1952) or in the hypothalamo-pituitary area (Donovan and van der Werff ten Bosch, 1957) of lactating rabbits results in failure of milk ejection without interference with milk synthesis. Injection of posterior pituitary extracts or oxytocin enabled the young to obtain normal milk yields as judged by litter growth rate. Lesions in the dorsal and posterior regions of the hypothalamus did not interfere with either milk synthesis or removal (Cross and Harris, 1952). The effects of hypothalamic lesions on prolactin secretion, as judged by lactational performance in post- partum rats, have been studied by several investigators. Yokoyama and Ota (1959a, 1959b, 1960) observed that the litters of lactating rats bearing hypothalamic lesions died within seven days after the operation despite vigorous suckling, and no milk was found in the stomachs of the young. Nevertheless, milk could be expressed from the mother's nipples even 10 days after lesioning, indicating a selective impairment of the regulation of oxytocin secretion without disturbing the production of milk. In a preliminary study McCann, Mack and Gale (1959) asserted that hypothalamic lesions in the supraoptico-hypophysial 18 tract inhibited the release of both prolactin and oxytocin. Since injections of oxytocin partially restored lactation in these rats, they suggested, in concurrence with the view of Benson and Folley (1956, 1957a), that oxytocin is the neurohumor which stimulates the release of lactogenic hor— mone from the pars distalis. The mammary glands of these rats were apparently not examined, however, to determine whether milk secretion had in fact been suppressed. In a subsequent report Gale et a1. (1961) re-examined the effects of median eminence lesions in lactating rats. They observed that prolactin injections did not restore lacta- tion, whereas cortisol was partially effective. They also observed a significant decrease in adrenal weight and con- cluded that the lesions interfered with ACTH rather than with prolactin secretion. McCann and Friedman (1960) reported that lesions induced in the hypothalami of rats during proestrus or estrus re- sulted in pseudopregnancy, indicating increased prolactin or luteotropin (LTH) secretion. They concluded that the role of oxytocin in LTH discharge is a doubtful one since the secretion rate of LTH is augmented in the presence of lesions which abolish oxytocin secretion. Degranulation of l9 pituitary acidophils, subsequent to the placement of hypo-V, thalamic lesions in rats has been observed by Soulairac and Desclaux (1947). This can be considered as indirect evidence for increased prolactin release since the acido- phils are associated with prolactin secretion (Purves, 1961). Lactation has been induced in rabbits and cats by in- duction of hypothalamic lesions thus indicating augmented prolactin secretion. Haun and Sawyer (1960) placed lesions in several regions of the hypothalamus of overiectomized rabbits whose mammary glands had been developed by estro— gen treatment. Lactation occurred only in animals with lesions in the medial basal tuberal region, suggesting that this area may inhibit release of prolactin in rabbits. Since this same area apparently controls the release of LH (Haun and Sawyer, 1960), they suggested, in agreement with the View of Everett (1954, 1956), that the neurohumoral factor(s) which traverses the hypophysial portal system to induce discharge of LH, also inhibits prolactin release. In post—y partum cats whose mammary glands had regressed for a period of up to 2 months after litter removal, Grosz and Rothballer (1961) sectioned the tuber cinereum transversely just behind 20 the optic chiasma and observed the onset of milk secretion within 3—12 days. Since stimulation of this area induces estrus in the cat (Sawyer, 1960), these authors suggested that this region exerts a reciprocal control on the release of FSH and prolactin. D. Pituitary Stalk Section A number of investigators have studied hypothalamic regulation of the hypophysis by transecting the pituitary stalk, which connects the pituitary gland to the hypothala- mus. This procedure disrupts the pathway of communication between the diencephalon and the pars distalis without disturbing the anatomical location of the gland. Studies on the effects of pituitary stalk section on prolactin secretion have been inconclusive and sometimes contradic- tory, and the degree of regeneration of the hypophysial portal vessels was usually not determined. Herold (1939) and Desclin (1940) reported that pituitary stalk section in rats during late pregnancy or early lactation resulted in failure of milk secretion as judged by growth of the suckling young. Dempsey and Uotila (1940) observed no inhibition of milk secretion in stalk sectioned rats. Jacobsohn and Westman (1945) found that mammary involution 21 after stalk section in lactating rats was slower than after weaning or hypophysectomy. Secretory activity was found to persist in the mammary alveoli even though most of the parenchyma had regressed. Retardation of mammary involu- tion and maintenance of secretory activity are indicative of augmented, or at least, persistent prolactin secretion (Meites, 1959b). Evidence that pituitary stalk section does not inter- fere with prolactin secretion in rabbits is provided by the studies of Jacobsohn (1949). Transaction of the stalk of pregnant and lactating rabbits disclosed that mammary secretory activity was maintained and involution was re- tarded for up to 33 days after surgery. Despite vigorous suckling of the young, most died within 6-8 days of the operation. The completeness of stalk severence was deter- mined by examining serial sections of the midbrain, pituitary and adjacent areas. When hypophysial stalk sec— tion was incomplete, the mammary glands were similar to those of unoperated rabbits and the young nursed normally. In a similar study, Donovan and van der Werff ten Bosch (1957) observed no reduction in milk volume provided oxy- tocin was administered to ensure milk ejection. A decrease 22 in litter growth rate was noted, however, which was attrib- uted to a change in milk composition. Augmented prolactin secretion consequent to pituitary stalk section, as evi- denced by induction of lactation, has been observed in women with breast cancer (Eckles et al., 1958). It may be concluded from the above studies that removal of hypothalamic regulation of the pituitary gland, by severence of the pituitary stalk, does not depress prolac- tin secretion and may even foster the production of this hormone. Lactation failure consequent to pituitary stalk section appears to be primarily due to oxytocin deficiency rather than to inadequacy of prolactin. E. Pituitary Transplantation The clearest evidence that an organ is an endocrine gland is that the organ will maintain its normal function if transplanted to a distant site in the body. Many of the early studies with pituitary transplants in hypophysec- tomized animals claimed that functionally active glands were obtained. This supported the concept that the pars distalis functioned without major influence from the other organ systems of the body. Several reports, however, noted that hypophysectomized animals bearing pituitary 23 transplants showed reduced adrenal cortical, gonadal and thyroidal activity (Harris, 1955; Everett, 1962). When more thorough studies were performed, in which the com- pleteness of hypophysectomy was carefully checked at autopsy, it was found that the pituitary gland differed markedly from other endocrine glands with respect to transplantation. Adenohypophysial transplants in com- pletely hypophysectomized animals show a marked reduction in TSH, ACTH, STH and gonadotropin secretion (Harris, 1955; Everett, 1962). The earlier claims of functionally active transplants in hypophysectomized animals are therefore probably in error due to incompleteness of hypophysectomy. l. Luteotropic activity Qf_pituitary transplants. Prolactin is the only pituitary hormone which is known to exert a luteotropic action in rats and mice (Astwood, 1941; Evans et al., 1941). Induction of pseudopregnancy is there- fore an excellent indicator of prolactin secretion by the adenohypophysis. Desclin (1949) first studied the effects of pituitary grafts on the luteal bodies of rats. He reported that administration of estrogen to hypophysecto- mized rats with single pituitary grafts under the kidney capsule evoked hypertrophy of the corpora lutea and 24 mucification of the vagina comparable to that seen in pregnancy. Everett (1954, 1956) confirmed Desclin's observation and further demonstrated that prolactin secre- tion was independent of estrogen treatment. He observed that the transplants could maintain continuous secretory function of the corpora lutea for up to 90 days in hypo- physectomized rats, and suggested that prolactin secretion was actually favored by such isolation. In a subsequent study Desclin (1950) noted that estrogen induced more intensive luteinization of the ovaries of hypophysectomized rats bearing pituitary grafts, and the grafts showed a greater degree of degranulation of the acidophils and disappearance of the basophils. He concluded that estro- gen increased prolactin secretion by the pituitary grafts. The luteotrophic action of pituitary transplants in rats has been thoroughly confirmed (Alloiteau, 1958; Quilligan and Rothchild, 1960; Sanders and Rennels, 1957). Pituitary transplants in intact mice (Muhlbock and Boot, 1959) and rats (Quilligan and Rothchild, 1960) change the normal sequence of estrous cycles into a series of pseudopregnancies. Nikitovitch—Winer (1960) recently ob— served that massive destruction of the hypothalamus did not 25 interfere with LTH secretion by rat pituitary autografts. This indicates that materials of hypothalamic origin, which may be conveyed to the transplant by the systemic circulation, are not responsible for prolactin secretion by the grafts. Montemurro and Gardner (1961) recently noted that an increase in LTH secretion does not occur when the hypothalamus is transplanted together with the pituitary to the kidney capsule of intact mice. Most of the mice maintained normal estrous cycles for up to 54 days following transplantation. This study provides further evidence that the hypothalamus inhibits prolactin secretion. 2. Pituitary transplants and mammary growth and secretion. Desclin (1956a, 1956b) observed that single pituitary transplants to the kidney capsule induced lobulo- alveolar growth of the mammary glands of hypophysectomized rats; however mammary secretion was not observed. Meites and Hopkins (1960) compared the effects of single pituitary grafts on mammary secretion and lobulo-alveolar development in estrogen primed, intact and hypophysectomized rats. The grafts induced a much higher incidence of mammary secretion and better lobulo-alveolar development in the intact as 26 compared with the hypophysectomized animals. The low inci- dence of mammary secretion in the hypophysectomized rats was attributed to a deficiency of ACTH and this suspicion was confirmed by subsequent studies (Meites, Nicoll and Talwalker, 1962). The effects of pituitary transplants on lactation in postpartum rats have been studied by several workers. Benson et a1. (1958, 1959) implanted intact lactating rats with 3-6 pituitaties (derived from the mother's own 7-day— old pups) under the kidney capsule. The rats were mated and on the fourth day of their subsequent postpartum lac- tation, they were hypophysectomized and injected with oxytocin to allow the young to obtain milk. Some secretion was obtained, and this was slightly enhanced by STH injected and even more so by ACTH. In a similar experiment Cowie et a1. (1960) transplanted pituitaries from 7—8-day—old rats to the anterior eye chamber or under the kidney cap- sule of lactating rats. The rats were mated and then hypo- physectomized after parturition. Lactation could be partially maintained in these rats if the mothers were injected with oxytocin. Substantial increases in milk production occurred when ACTH was injected, while STH had 27 a lesser effect. Rothchild (1960a) also reported partial maintenance of lactation and litter growth rate when hypophysectomized rats with single pituitary transplants were injected with oxytocin 2-6 times daily. Milk pro— duction, as estimated by litter growth rate, was not returned to normal in any of these experiments. In a related study, Meites and Hopkins (1960) reported that single pituitary transplants to the kidney capsule retarded mammary involution in intact postpartum rats after litter removal. Since mammary growth, initiation and maintenance of lactation, and retardation of mammary involution are all dependent upon prolactin, these studies provide fur- ther evidence that pituitary transplants secrete prolactin and that secretion of this hormone appears to be an auton- omous characteristic of the mammalian pituitary gland. IV. Hormone Production by Pituitary in_Vitro Although there have been numerous culture studies of pituitary tissue, only a few investigators have attempted to study adenohypophysial hormone secretion and its regu- lation by tissue culture techniques. Anderson and Haymaker (1935) cultured anterior and posterior lobe tissue from 28 young rats and found that only melanophore stimulating hormone (MSH) showed evidence of increased activity in the culture medium. Definite hormonal restorative effects were noted upon the thyroids, ovaries and adrenals of hypophysectomized rats when material from a large number of cultures was assayed. The effects, however, were no greater than those produced by an extract of fresh, non- cultured pituitaries. Cutting and Lewis (1938) cultured pituitaries of rats for periods of 50-80 days and assayed the nutrient fluid for trophic hormone activity at various intervals of culture. Evidence of TSH, ACTH and gonado- tropin activity was obtained only within the first 10 days of culture and was accounted for by release of the gland's initial content. Anterior pituitary explants, when cul- tured adjacent to osteogenic cells, were observed to promote growth of the cells suggesting STH activity (Gaillard, 1937). Substituting liver or salivary gland for adenohypophysial explants was ineffective in this respect. More recently, Guillemin and Rosenberg (1955) and Guillemin (1956) reported that explants of dog and rat pituitary tissue failed to produce detectable quantities of ACTH after the 4th day in culture. Addition of hypothalamic 29 explants or homogenates to the cultures re-established detectable ACTH activity in the medium. Substituting cerebral cortex, liver or spleen for hypothalamic tissue did not stimulate ACTH secretion. Although pitressin pre- parations had ACTH—hypophysiotrophic activity in this in_ vitro system, purified vasopressin was ineffective. These observations provide further evidence for the existence of a specific material of hypothalamic origin which is neces- sary to stimulate and sustain ACTH secretion. Schaberg and DeGroot (1958) cultured adrenals from 5 day old rats in contact with anterior pituitary fragments. The cultured adrenals exhibited morphological changes and produced cor- ticosteroids in a manner characteristic of ACTH stimulation. Evidence of ACTH activity by this system.was demonstrated even after 12 days in culture. The discrepancies between the latter work and that of Guillemin and Rosenberg (1955) and Guillemin (1956) may be due to differences in the sensi- tivities of the systems employed. Florsheim et a1. (1957) attempted to demonstrate the existence ofenihypothalamic stimulator of thyrotropin secre- tion by organ cultures of mouse adenohypophysis. TSH secre- tion was not detected in the medium after the first week of 30 culture and addition of hypothalamic fragments did not re- instate discernable TSH production. The hypothalamic frag- / v ments did, however, stimulate corticotropin secretion as was observed by Guillemin and Rosenberg (1955) and Guille— min (1956). Suspension cultures of human pituitary cells have been studied for hormonal activity. Samples of the suspensions were found to contain detectable quantities of STH, ACTH and gonadotropins (Thompson et al., 1959). Kobayashi et a1. (1961) have reported that explant and monoloyer cultures of rat pituitary produce little FSH in_ vitro. Addition of hypothalamic extracts to the cultures increased FSH levels in the cells and culture medium. Extracts of the cerebral cortex were ineffective in this respect. This study provides convincing evidence for the existence of a material of hypothalamic origin which is necessary to maintain FSH secretion. In all of the above studies on ip_vitro secretion of adenohypophysial hormones, hormonal activity, when detected, was found only for the first few days of culture. With the possible exception of MSH (Anderson and Haymaker, 1935), significant in_vitro hormonal synthesis was not demonstrated in any of the studies. This is in accord with the abundant 31 evidence that secretion of most of the anterior pituitary hormones requires hypothalamic mediated stimulation. Pro? lactin is a notable exception in this respect since removal of hypothalamic regulation from the pituitary apparently favors prolactin secretion. The unusual in_yiyg behavior of prolactin might be expected to be manifested ig_vitro where the pars distalis can be maintained in an environment free of hypothalamic or other in_yiyg_influence. Studies were therefore undertaken to determine if anterior pituitary fragments, from rats and other species, would secrete detectable quantities of prolactin into a synthetic medium when the explants were maintained by organ culture tech- niques. The effects of other hormones on prolactin secre- tion ig_vitro were also studied since the endocrine products of several glands apparently influence prolactin secretion in yiyg. The objectives of these studies, in more detail, were as follows: 1. To determine if explants of anterior pituitary (AP) tissue from rats and other species could actively secrete prolactin in_vitro and to compare the prolactin secreting capacities of explants from the APs of lactating and non- lactating rats. 32 To investigate the influence of explants, homogenates and acid extracts of hypothalamic and cerebral tissue on prolactin secretion in_vitro. To determine the effects of oxytocin and vasopressin on prolactin secretion ig_vitro since the neurohypo- physial hormones are considered by many investigators to be the neurohumoral regulators of AP function. To study the effects of the endocrine products of the gonads, thyroid and adrenal cortex on prolactin secre- tion in_vitro. To determine if insulin affects prolactin secretion by rat AP explants. To evaluate the local, "micro" method of prolactin bio- assay in the pigeon crop to determine the acceptability and precision of the assay procedure. MATERIALS AND METHODS I. Animals Most of the 550 rats used in these experiments were of the Carworth CFN strain. In some of the preliminary studies, multiparous females weighing 270—300 grams were used. Three to four-month—old females weighing 180-220 grams were employed for most of the remaining experiments. Description of other animals used for the culture studies appears with each experiment where necessary. The animals were housed in an air conditioned room under uniform temperature (76 ilo F) and lighting conditions. Mature or 6-10 week old White Carneau pigeons (Palmetto Pigeon Plant, Sumpter, N.C.) were used as assay animals in the initial studies. The remaining assays were performed in 4-8 week old White King squabs (Cascade Squab Farm, Grand Rapids, Mich.). A total of 963 pigeons were used for these studies. II. Culture Methods The watch glass procedure of Fell and Robison (1929), as modified by Chen (1954), was used in the initial studies. This preparation consists of a 3 inch watch glass supported 33 34 by moist cotton or paper pulp inside a 4 inch Petri dish. The culture medium is placed in the watch glass and a raft of siliconized lens paper or cellulose acetate is placed on the medium. The raft supports the tissue explants at the medium-atmosphere interface. This enables the tissues to obtain nutrients from the culture medium and maintains them in a moist condition while providing optimal conditions for respiratory exchange. Although the Petri dish is closed in this system, an airtight seal is not obtained; therefore, the atmosphere inside the dish can exchange readily with the external gases. In the preliminary studies rafts of cellulose acetate or urethane foam sponge were used. These did not prove to be satisfactory since the cellulose acetate rafts frequently sank in the medium and the urethane foam rafts often floated too high on the medium. Explant survival consequently suffered. A modification of the method of Trowell (1959) was therefore adopted. This consisted of using rafts of #46 grid stainless steel mesh (United Surgical Supply Co.) in- stead of lens paper or cellulose acetate. The rafts were~ made by cutting 1 cm x 2.6 cm rectangles from the sheets of stainless steel mesh. When in contact with the sides of 35 the watch glasses, the ends of the rectangles supported the raft at the surface of the medium. The explants were placed on sterile, washed lens paper rectangles (1 cm x 2 cm) and the lens paper was then placed upon the rafts. The ends of the lens paper draped over the sides of the rafts into the medium where they could act as wicks in the event of evaporative loss of the culture fluid. This system proved to be highly satisfactory from the standpoint of explant survival and hormone production; however, the size of the Petri dishes limited to 24 the number of cultures which could be simultaneously incubated in the culture chamber. In addition to this objection, the process of cleaning and sterilizing the watch glasses and Petri dishes was rather laborious, and the problem of residual contamin- ation from the watch glasses could not be eliminated with absolute certainty. Further modifications were necessary to eliminate these problems. Instead of using the watch glass — Petri dish prepara- tions to hold the medium, rafts and tissues, sterile, disposable plastic Petri dishes alone were used. These plastic Petri dishes (Falcon Plastics, Inc.) have a diameter of 3.5 cm and a height of 1 cm. The stainless steel mesh 36 Figure 1. The glass and plastic Petri dish prepara- tions. Upper left: a 10 cm x 1.5 cm sterile plas- tic Petri dish used for cutting AP glands into 6 explants. Upper right: the standard watch glass- Petri dish preparation for explant culture. Lower left: an open 3.5 cm x 1 cm sterile plastic Petri dish with a stainless steel mesh platform in front. Lower right: a 3.5 cm x 1 cm sterile plastic Petri dish containing 3 m1 of medium and the stainless steel mesh platform. 37 rectangles were modified for use in the plastic dishes by bending each end at right angles to form a platform 1 cm wide, 1.8 cm long and 4 mm high. When these platforms were placed inside the plastic Petri dishes with 3 m1 of medium they supported the explants, which were again placed on the lens paper rectangles, at the surface of the medium. This system proved to be highly satisfactory in all respects. Explant survival and hormone production was excellent; as many as 120 cultures could be incubated at one time and the dishes were discarded after use. The platforms were not discarded. The glass and plastic Petri dish preparations are shown in Figure 1 along with one of the 10 cm x 1.5 cm plastic Petri dishes which were used for cutting the pitui- tary glands. A few of the initial cultures were incubated in air atmosphere; however, explant survival and hormone production left much to be desired. The remainder of the cultures were therefore performed in an atmosphere of 95% 02 - 5% CO2 in a plastic chamber. The chamber was constructed from a plastic mouse cage (10 x 10 x 8.5 inches) with an open top. The cage was inverted and placed on a sheet of 1/4 inch thick glass plate which was covered with a thin sheet of 38 polyethylene plastic. This provided a fairly good gas tight seal. The chamber was equipped with two gas inlets and two ' outlets. The O2 - CO2 gas mixture was passed through the chamber at a rate of about 200 cc per minute. Before enter— ing the chamber the gas mixture was humidified by passing through a sintered glass filter which was immersed in distilled water in a glass cylinder. The cylinder was equipped with ground glass joints. The sintered glass fil- ter caused the gas to form innumerable small bubbles which greatly enhanced humidification of the gas mixture. The filter also trapped any particulate material present in the gas. The culture dishes were placed on square platforms of 1/4 inch thick plywood (9 1/4 x 9 1/4 inches) which were equipped with 3/4 inch legs on each corner. The plywood platforms were covered with Saran Wrap (Dow Chemical Corp.) and 6 were stacked one upon another in the culture chamber. Each of these platforms can accommodate 4 of the glass or 20 of the plastic Petri dishes. A plexiglass chamber was later constructed for gassing the cultures. This chamber measured 12.5 inches across the front, 9.5 inches in height and was 12 inches deep. The chamber was equipped with a 39 door which was held in place with screw clamps. The door had rubber weatherstripping insulation around the edges. The plexiglass used for this chamber was 1/4 inch thick. The shelves inside the chamber were constructed from 1/8 inch plexiglass and were perforated with numerous 1/4 inch diameter holes to facilitate thorough mixing of the gas. The Shelves were supported inside the chamber by strips of 1/4 x 1/4 inch plexiglass which were attached to the inside walls. They could therefore be removed and replaced readily by sliding them along the strip supports. Each of the shelves in this new chamber could accommodate up to 30 of the small sterile plastic Petri dishes. The chamber was equipped with 2 gas inlets and 2 outlets. One of the out- lets was attached to a water manometer and when the chamber was in operation the gas inflow and outflow was adjusted to give an inter—chamber pressure of 3-5 mm Hg above atmos- pheric pressure. Difficulties have been experienced with the door and insulation of the chamber; therefore further modification is warranted. This chamber is shown in Figure 2. All cultures were incubated at 35 I 1°C. Standard recommendations were followed in washing and sterilizing all glassware and instruments (Merchant et al., 4O Figure 2. The plexiglass chamber used for gassing the cultures with the 95% 02 — 5% C02 gas mixture. The cylinder containing water and the sintered glass filter, which was used for humidifying the gas mixture, is shown at the left. The water manometer is shown on the right of the chamber. 41 1960; Paul, 1960). The water used for the final rinses of the washed glassware and instruments was purified by distil- lation, passing through a deionizing resin column, then de- organified by percolation through another resin column. For preparation of the culture medium this water was purified further by a single distillation from glass. The stainless steel platforms were cleaned by soaking in 1/2 concentrated hydrochloric acid for 15 minutes, then washed in running tap water for 30 minutes. The rafts were then rinsed 4 times in the purified water, immersed in absolute ethanol for 30 minutes, immersed in diethyl ether for a further 30 minutes and finally allowed to dry in air. The platforms were placed in a Petri dish and sterilized. All materials were sterilized by autoclaving except those which would be destroyed by the high temperatures, such as the insulin and antibiotic stocks. Such labile materials were sterilized by passing them through a Milli- pore bacteriological filter with a pore size of 0.45u (Millipore Filter Corp.). III. The Culture Medium Synthetic Medium "199" was used exclusively for these studies. This medium contains all of the known essential 42 metabolites for cellular nutrition and maintenance ig_yitrg_ but usually does not support cellular proliferation (Mer- chant et al., 1960). The "199" was obtained from Difco or Microbiological Associates, Inc. in bottles containing 100 ml of sterile, lO-fold concentrated stock solution. The ingredients used for the preparation of 100 ml of culture medium, with a pH of 7.4, for use in O - C0 atmosphere, 2 2 are shown in Table 1. Table l PREPARATION OF 100 ML OF "199" CULTURE MEDIUM Component Volume 199 10x stock . . . . . . . . . . . . . . . . . 10 ml Antibiotic stock (5.0 mg streptomycin and 2,500 U penicillin per ml) . . . . . . . . . . 2 ml Zn-free insulin stock (200 U/ml) . . . . . . . . 1 ml 5.6% NaHCO3 . . . . . . . . . . . . . . . . . . 6 ml Purified water . . . . . . . . . . . . . . . . . 81 ml The final medium therefore had a concentration of 2U Zn-free insulin, 0.1 mg streptomycin sulfate and 50 U peni- cillin per m1. Insulin was used in the medium for most of the culture studies; however, it was found to be unnecessary 43 for prolactin secretion in_vitro and was therefore omitted from the medium in the last few experiments. The NaHCO3, Zn-free insulin and antibiotic stocks were sterilized by millipore filtration and stored in sterile, plastic 25 ml bottles until used. The antibiotic stock was frozen at 20°C and the insulin and bicarbonate solution were stored in a refrigerator at 4°C. The Zn-free insulin stock was prepared by dissolving the insulin in a minimum of 0.002 N HCl and diluting to volume with purified water. Steroid hormones were added to the medium by first preparing con- centrated stock solutions of the steroids in absolute ethanol. The ethanol stock was then added to give the desired concentration of the hormone with an alcohol con- centration of 0.5% in the medium. Control medium for these studies contained an equivalent concentration of ethanol. The streptomycin sulfate and penicillin G potassium used in the culture medium were obtained from Nutritional Bio- chemicals Corporation. IV. The Culture Procedure The animals were decapitated and their heads were dropped for a few seconds into a beaker containing WD% ethanol. This moistens the hair of the heads and reduces 44 the chances of contamination of the cultures with air- borne rat hair. Usually 4 rat heads were handled together. The skull was exposed by cutting the skin with scissors and the top of the skull was removed with bone cutting scissors. The scissors were pre—soaked in 70% ethanol before use. The pituitary gland was then exposed by reflecting the brain with a pair of sterile forceps. A second pair of sterile forceps was used to separate and discard the posterior lobe of the pituitary and then the anterior lobe was removed. The gland was then placed in a sterile plastic 10 cm x 1.5 cm Petri dish containing a few drops of "199" medium. Each anterior lobe was halved longitudinally and each half cut into 3 explants of about 2 mm diameter. Scalpels with #11 disposable blades (Crescent Mfg. Co.) were used for the dissection. The plastic Petri dishes were found to be much more satisfactory for dissecting the pituitary glands than glass Petri dishes containing moist filter paper. When the latter method was used, fragments of the paper often adhered to the explants and handling of the tissue was con— sequently encumbered. The explants were then placed on the 45 lens paper rectangles and transfered to a raft inside a culture dish into which the medium had previously been added. The culture dishes containing the explants were then transfered to the gassing chamber inside the incuba- tor. The entire procedure, from removal of the glands to placing the tissues in the culture dishes, was performed inside a plexiglass hood (Figure 3). Sterility inside the hood was maintained by a 15 watt ultraviolet germicidal lamp whidh was usually left on when the hood was not in use. Immediately before use the inside of the hood was washed with 70% ethanol. The medium from the few initial cultures which were incubated in air atmosphere was usually changed daily and only 1 ml of medium was placed in each dish. The duration of these cultures was usually 6 or 7 days. Medium from the cultures in 02 - CO2 atmosphere was changed every 3 days and 3 m1 of medium was used in each dish. Many of the cultures in 02 - C02 were incubated for 6 days; however, it was found that a 3 day incubation period was entirely satis- factory for most experiments. The medium from each culture dish was collected separately and stored in a freezer until used for assay. Medium from the cultures in air atmosphere 46 Figure 3. The plexiglass hood wherein the AP glands were removed from the animals heads and prepared for incubation. The ultraviolet lamp is shown on the inside rear wall of the hood. Two flourescent lamps are shown on the top. 47 was 1yophilized and redissolved in water to a final volume suitable for administration of the entire quantity to 3 or 5 pigeons. A 2 m1 aliquot of the medium from most of the cultures in the O2 - CO2 atmosphere was assayed in 5 birds and the total activity of the medium sample was calculated. The medium was pooled for assay in only a few of the cul- tures in air atmosphere. The medium samples from all of the remaining cultures were assayed separately without pooling. These studies involved a total of 542 cultures; however, the medium was not assayed from all of these. Upon termination the explants from most of the cultures were weighed on a Mettler balance and fixed in Bouin's fluid or 10% neutral buffered formalin. The tissues were then embedded in paraffin, sectioned at 6u and stained with hematoxylin and eosin. Attempts were made to differentially stain the explants by using several different procedures; however none of these gave satisfactory results. V. The Assay Procedure The culture medium was assayed for prolactin activity) by the sensitive pigeon crop method of Lyons (1937) as modified by Reece and Turner (1937). The samples were injected daily in 0.1 ml volumes for 4 days in the same 48 intradermal site. Two samples were assayed in the same pigeon by injecting one sample over the left crop half and the other over the right side. The pigeon crop responds to prolactin by a proliferation of the mucosal epithelial cells. When prolactin is injected intradermally the mucosa thickens under the site of injection. The area and thickness of the response is a linear function of the logarithm of the dose of prolactin (see Experiment No. XV). The degree of the response is rated by a subjective method in units from 0.25 to 4.00 at intervals of 0.25. This subjective rating sys- tem takes both the area and the thickness of the response into consideration. A dose-response standard curve was not established for the initial Studies in which White Carneau pigeons were used. Prolactin standards were injected, however, which gave responses within the range observed with the responses from the medium samples. A rough estimate of the potency of the medium samples in International Units was therefore calculable. This procedure does not give results which are quantitatively exact; however, the relative differences are of value for comparative purposes. A dose-response standard curve was established with 49 N.I.H. prolactin for the White King squabs and the values for the remainder of the assays were determined from this standard. At frequent intervals the birds were injected with prolactin standards to check for variations in sensi- tivity. As shown in Experiment XV, the sensitivity of the pigeons did not vary appreciably from that observed when the standard dose-response relationship was established. The same standard curve can therefore be used with reason- able certainty for conversion from Reece-Turner to Inter- national Units in all of the cases where this was performed. Periodically the prolactin content of fresh rat anterior pituitary tissue was determined. Pituitary glands from the same type of rats as used in the culture studies were homogenized with a ground glass homogenizer in physiological saline or medium "199" and injected into assay pigeons. VI. The Matched Pair Culture and Assay Procedure When the effect of a particular agent on in_vitro pro— lactin secretion was investigated, a paired culture and assay procedure was used. This consisted of placing the explants from one-half of each anterior pituitary gland in a culture dish containing control medium and the explants v‘, 50 from the remaining halves in a dish containing the experi- mental medium. The experimental medium contained the material which was being tested. The control and experi- mental media were otherwise identical in composition. The control medium was assayed by injection over the right crop half of the assay pigeons and the experimental medium was injected over the left crop half. This permitted the influence of the agent on prolactin production by an equi— valent amount of pituitary tissue from the same rats, cultured under the same conditions, to be ascertained in the same assay animals. A schematic representation of the paired culture and assay procedure is shown in Figure 4. When there was a difference in the degree of explant sur- vival between the control and the experimental cultures, as judged by the histological appearance of the tissues, the media samples were not assayed. VII. Hormone Preparations The sources, forms, trade names and potencies of the hormones used in these studies are shown as follows: 51 QOHU commflm anaemz Hmucmsflumoxm puss smfio musuaso Sufism: Houucoo and: Swan musuaso 58 N. .60 e. Ewan ousuaso mo 3ma> mnam / Ki Q 0 Ai mucmamxm iv Q Q Q Q mam “mm \ nonopmoonm momma can musuaso Umuflmm mflu mo coflumucmmmumwn owumfioaom .¢ musmflm Hormone “Prolactin Thyroxine Triiodothyronine VPitressin Oxytocin Insulin (Estradiol Testosterone 'Progesterone Cortisol Corticosterone 52 Source, Trade Name, Form and Potency Endocrine Study Section, National Insti— tutes of Health. Powder form. 15 IU/mg. Ovine. Smith, Kline and French Labs., Philadel- phia, Pa. Elythrin: Sodium ~L-thyroxine pentahydrate powder. Smith, Kline and French Labs. Powder form. Parke, Davis and Co., Detroit, Mich. Powder form. 55 pressor and 4 oxytocic units per mg. Parke, Davis and Co. Pitocin. Aqueous solution in sterile ampuole. 10 IU/ml. Eli Lilly and Co., Indianapolis, Ind. Zn-free powder. 20 U/mg. American Steroids Co., Puerto Rico. Non- esterified crystalline powder. American Steroids Co. Non-esterified crystalline powder. Nutritional Biochemical Corp. Non- esterified crystalline powder. Merck and Co., Rahway, N.J. Non-esterified crystalline powder. Hydrocortisone. The Upjohn Co., Kalamazoo, Mich. Non—q esterified crystalline powder. 53 VIII. Statistical Procedures The students "t" test and the ”t" test for matched observations were used for analysis of the data from these studies (Snedecor, 1956). The two-sided evaluation was used to determine the level of significance in all cases. Standard procedures for the analysis of bioassay data (Bliss, 1952) were used for the dose-response relationship established with standard N.I.H. prolactin preparations in the assay pigeons. The means and standard errors of the means are shown in the usual manner (X I 8;) and the standard error of the mean was calculated by the formula: SX g-V n(n - l) EXPERIMENTAL EXPERIMENT I, COMPARISON OF IN_VITRO PROLACTIN PRODUCTION BY PITUITARY EXPLANTS FROM MATURE FEMALE AND POSTPARTUM LACTATING RATS. In order to determine if anterior pituitary tissue from rats would secrete prolactin in_vitro, the glands from mature female non-lactating and postpartum lactating rats were cultured by the watch glass technique. Six cultures were incubated in air atmosphere with the explants on cel- lulose acetate rafts. Explants from the pituitaries (APs) of 3 mature female rats (180-200 gm body weight) were placed in each of 4 culture dishes. An additional culture dish contained explants from 3 APs and another from 4 glands from 15 day postpartum lactating rats. The medium in each dish was changed daily and collected separately over the 7 day culture period. Medium from the first day of culture was discarded to remove possible contamination from cel- lular debri. The medium from the 4 cultures of mature female rat APs was pooled to give 2 samples each containing medium from 2 culture dishes. All of the culture fluid samples were lyophilized and redissolved in purified water to give a final volume suitable for administration to 3 or 54 55 5 pigeons. The medium from the cultures of APs from mature female rats was assayed by injection over the left crop sac v” of the pigeons and the culture fluid from the APs of lac- tating rats was injected over the right crop half. Each pooled sample of medium from the mature female rat APs was thus matched with a sample from the lactating rat APs. In conjunction with the above assays, medium samples from similar cultures of rat AP tissue, obtained from Dr. R. H. Kahn*, were also tested for prolactin activity by the same assay procedure. Kahn used rats of the Sprague-Dawley (S.D.) strain and cultured the APs in Trowell T—8 medium by a watch glass procedure which utilizes stainless steel rafts (Merchant et al., 1960). He incubated the pituitaries for 3 days in an atmosphere of 95% O2 - 5% C02. The number of APs per culture dish in Kahn's experiment is shown in Table 2 with the other assay results. Mature White Carneau pigeons were used for these assays. Results and Discussion The data presented in Table 2 show that prolactin ac-‘ tivity was detected in the medium of all of the cultures. *Department of Anatomy, University of Michigan, Ann Arbor, Michigan. 56 Furthermore, the assay results disclose that the medium samples from lactating rat pituitaries produced substan- tially more prolactin than the glands from the mature female animals. Prolactin production by the APs from lactating rats, on a per pituitary per day basis, was 100% greater than that of the mature females. This difference was Sig- nificant (P < 0.05) and cannot be attributed entirely to a difference in the quantity of tissue per pituitary. An- terior pituitaries from similar mature female rats weighed about 8.7 mg and pituitary glands from similar lactating Carworth rats have previously been found to weigh about 11 mg (Johnson and Meites, 1958), or approximately 26% greater than glands from mature female rats. It is therefore apparent that about 74% of the excess prolactin production by the APs from the lactating rats, must be accounted for by factors other than a difference in the quantity of pituitary tissue. Everett and Baker (1945) have reported that the pitui- tary glands of lactating rats show about a 60% increase in acidophil count above the prelactating level around the 16th day postpartum. Assuming that the same relative differences in acidophil count existed in the pituitary glands of the V- 57 non-lactating and lactating rats used in this culture ex- periment, these data indicate that most of the difference in the in_vitro prolactin secreting capacities of the pituitaries is due to a difference in the number of pro- lactin producing cells. The pituitary acidophils are believed to be the cells which produce prolactin (see Purves, 1961). The 60% increase in acidophils can account for all but 14% of the difference which cannot be attrib- uted to a difference in the quantity of tissue. This 14% difference may be due to a difference in the prolactin synthesizing capacity of the individual pituitary acido- phil cells and suggests that the acidophils from the pituitary glands from lactating rats secrete more prolactin per cell than those of the non-lactating animals. Meites and Turner (1948) reported that the pituitary glands of lactating rate contain 140% more prolactin at the 16th dayxpostpartum than the glands from non-lactating females. The 100% difference in prolactin secretion be— tween non—lactating and lactating rat pituitaries observed in this ig_vitro study supports the belief that the pituitary prolactin content can be indicative of ig_yiyg_prolactin secretion rate. 58 Histological examination of the explants which were cultured for 7 days disclosed that the tissue fragments had large areas of central necrosis. All of the explants pos- sessed a thin outer rim of viable cells (Figure 5) and there was no obvious difference in the degree of cellular survival between the explants from the mature female or the lactating rats. The prolactin levels found in the medium from Kahn's cultures, which were incubated in Trowell's medium in an atmosphere of 95% 0 - 5% CO 2 2, are not appreciably greater than the levels reported here with 199 medium in air atmos— phere. When we incubated rat AP explants in 199 medium in 02 — C02 atmosphere, the explants produced about 8 times more prolactin than similar cultures in air atmosphere (see Experiment III). Since the APs in Kahn's cultures produced only a fraction of the amount of prolactin found in our cultures under similar conditions, it appears that Kahn's culture conditions were less satisfactory for prolactin secretion than our methods. Since explant survival was equally good in both instances, and it seems unlikely that the difference in the strain of rats could account for the difference in prolactin production, the difference may be 59 due to the composition of the culture media. Trowell's medium contains the antibiotic, chloramphenicol. Strep- tomycin and penicillin were used in the cultures with 199 medium. Since chloramphenicol has been shown to exert a pronounced depressant action on protem synthesis (Rendi, 1959), it is possible that this antibiotic is responsible for the lower levels of prolactin synthesis in Kahn's cultures. 60 moo.o H em.o m¢.o u wocmHTMMAU omoum>¢ meHMDHsuHm umH msaumuoma mo ommuoem vm.o u mmfiumufisufim umn mamamm enzyme mo wmmnm>¢ pmaoom mmnmflc musuaso m Scum Esacmzs oa.o+ m¢.o om.H om.m mcaumuuma m m .o.m mm.o oo.H oo.m maaumuomauaoz m m .o.m a o¢.o+ ms.o o~.m om.m mcaumuomu m a .o.m mm.o oo.H oo.¢ assumuomaucoz m a .o.m m mH.o+ oa.o ~¢.~ mm.s mcaumuumu m m zeo mm.o mm.a ma.a mcaumuomaucoz m m Izmo m S~.o+ om.o ma.m me.m mcflumuoma m a zmo mo.o mm.o mm.m mcaumnomaucoz m o szmo a oucmnom mmm\m¢ m4 I an mom mom Hmuoa >mmm¢ mmflumu w. you nasuam umm mo .02 ham mo wuss Snaomz mouam mo :Amuum mnsuaso CH mHHCD GHUUMHOHQ HOCHDBIOOOOM MO .02 .OZ mfi_mM.mO ZOmHm¢mSOU N magma 61 EXPERIMENT ll- PROLACTIN SECRETION IN 11139 BY LONG TERM CULTURES OF RAT PITUITARY TISSUE. Three cultures were incubated for 14 days and another 3 for 21 days in air atmosphere to determine if explants of rat anterior pituitary tissue could secrete prolactin for prolonged periods in_yit£g, All of the cultures Contained explants from 3 mature female rat APs except one of the 14- day dishes which contained tissue from 3 lactating rat APs. One of the l4-day cultures contained estradiol in the medium at a concentration of 0.5 per ml. The medium was changed daily in the l4-day cultures. Each dish contained 1 ml of culture fluid. The medium used in the 21 day culture was buffered with NaZHPO4 and was changed every 3 days. Three ml of nutrient fluid was placed in each dish in these cul- tures. The explants were supported by cellulose acetate rafts in all of these cultures. The medium from days 2—7 of the l4-day cultures was collected separately from each dish for assay. Medium from days 7-14 was similarly collected. The medium collected. during the last 3-day culture interval (days 19—21) of the 3dweek culture was pooled for assay. All medium samples were 1yophilized, redissolved in water and each was assayed in 3 mature White Carneau pigeons. 62 Results and Discussion The results of the l4-day culture are presented in Table 3. The pituitary explants obviously secreted con- siderably less prolactin during the second 7 day culture period than they produced during the first week of culture. The average prolactin production per pituitary per day during the first 7 days was 0.29 Reece-Turner units (RTU) for the 3 cultures. During the second week the cultures produced an average of 0.15 RTU per AP per day. This amounts to about a 50% reduction in prolactin production and is probably the result of death of the cells in the explant. Survival of the cells at the end of the l4-day culture was not as good as at the end of 7 days. The pooled medium sample from the last 3 days of the 21—day culture gave a total response of 4.4 RTU. The APs there- fore produced 0.63 RTU/gland or 0.21 RTU per AP per day. Histological examination of the explants from the 21-day culture revealed that very few viable cells remained and that tissue maintenance was even poorer than that observed at the end of 14 days. This was somewhat surpris- ing since the prolactin activity of the 21-day cultures is intermediate between the values obtained at the end of 7 63 and 14 days of culture. With only one sample, however, no valid conclusions can be drawn from this observation regard- ing the relative quantities of prolactin produced. This experiment demonstrates that rat AP tissue can secrete pro- lactin for prolonged periods in_vitro without participation of hypothalamic or other ig_yiyg_inf1uences. Recently Pasteels (1961a) reported the results of his study on prolactin i2_vitro during a three week culture period. He cultured the pituitary from one male rat in natural, growth promoting medium and observed that the cel- lular elements proliferated vigorously. The prolactin level in the medium was found to increase linearly with time. This observation of increasing prolactin activity in the medium with the duration of culture is probably due to the continued increase in the number of cells which produce the hormone rather than to a change in the prolactin secret- ing capacity per cell. No data were presented, however, which would permit the increase in prolactin secretion with the duration of culture to be compared with the increase in the cell population. 64 mcaumu na.o SH.H om.m a om.o om.a ma.v o mma :omu m Hoao Imuumo oamEmm mm.o ma.a mm.e a om.o SH.N om.o o + mma masons m mHmEom 60.0 No.0 mm.H S N~.o mm.H mn.m o mmH manna: m . Thou musu mmo\m< ma smO\m< m< Hmuoe Iago Hmuoa Iago mom mom mo mom Hmm mo ssaoms 2A mSmo asflomz ca mama umm was mean: cfiuomaoum muss: cauomaoum ssaomz mmme mo .02 mMmm m>HmmMUUDm 039 mm>0 mBZ.mM m GHQMB 65 EXPERIMENT III. EFFECTS OF 95% 02 - 5% C02 ATMOSPHERE ON EXPLANT SURVIVAL AND PROLACTIN SECRETION IN_VITRO. Since explant survival in air atmosphere was not en— tirely satisfactory (Figure 5), most of the remaining cul- tures were incubated in an atmosphere of 95% 02 — 5% C02. This procedure greatly improved explant survival and pro- lactin production by the pituitary explants. Assay results from 7 cultures in air atmosphere and 5 in 02 - CO2 are shown in Table 4. Each of the cultures contained explants from the pituitaries of 3 mature female rats on cellulose acetate rafts. The medium from 4 of the air atmosphere cultures was pooled to give 2 samples, each containing medium from 2 cultures. All of the medium samples were concentrated by lyophilization and redissolving in water for injection. Each sample was injected into 3 birds. All of the cultures were incubated for 6 days. The medium from the air atmosphere cultures was changed daily and 1 m1 of medium was used per dish. Medium from the cultures in 02 - CO2 was changed after 3 days and each dish contained 3 ml. Medium from the first day of culture was not discarded in any of these cultures. The medium from 2 of the O2 - CO2 cultures was collected separately over 66 the first and second three day culture periods and the medium from the second 3 days of culture (days 4-6) was assayed. In all other cases all of the medium from the 6 days of culture was assayed. The anterior pituitary glands from 10 mature female rats were assayed for the determination of AP prolactin content by homogenizing in physiological saline and in- jecting into 5 assay birds. Mature White Carneau pigeons were used for these assays. Results and Discussion The assay results shown in Table 4 demonstrate that the pituitaries incubated in 02 - CO2 atmosphere secrete considerably more prolactin into the culture medium than the explants cultured in air atmosphere. The air cultures a produced an average of 0.13 RTU per AP per day, whereas the 02 - C02 cultures secreted 1.05 RTU/AP/day. This difference was highly significant (P < 0.001). Explant survival in the 02 — C02 atmosphere was excellent (Figure 6) and the explants compared favorably with fresh, uncultured pituitary tissue in histological appearance (Figure 7). The beneficial effects of gassing on explant survival are thus reflected in the 8-fold greater prolactin production by the 67 pituitary tissue. The assay results on the adenohypophyses of mature female rats disclosed that the glands contained an average of 1.25 I 0.21 RTU per anterior lobe. When compared to the daily prolactin production of the glands in 02 - CO2 atmos— phere, which averaged 1.05 RTU/gland/day, it is evident that the pituitary explants in yit£g_synthesized about as much prolactin during each day of culture as they contained when introduced into the culture system. A fair degree of net hormone synthesis by the glands in 02 — CO2 atmosphere was therefore realized. The procedure of dissecting the pituitary glands was found to result in about a 20% loss in glandular tissue as judged by difference in pre— and post-dissection weight (see Experiment V). This indicates that ig_yitrg_prolactin production, on a per pituitary basis, should actually be 1/5 higher than reported since only about 4/5 of each gland was introduced into the culture dishes. When this differ- ence is taken into consideration, it is evident that pro- lactin production each day, on a per pituitary basis, wOuld actually be equivalent to the initial content of the tissue. This represents a substantial degree of hormone synthesis. Hoo.o v m 68 mm.oa u moo I NC .m> Ham u Umaoom mmusuasu moo.o H mo.H u .>¢ 00.0 H ma.o n .>¢ m Eoum anomzs mN.H mm.m mm.HH m m m m mm.o mm.a mh.h m so m no.a Hm.m mo.m m m m v mo.o mm.o mm.m m am e mo.H mm.o oo.mH m o m m ma.o SH.H om.m m m m mm.o mn.m mm.hH m o m m wa.o mm.o om.m m m m om.o m¢.m mm.oH m o m H «N.o m¢.H mm.¢ m m H hon w \m< mm Hmuoe \Mm m< Hmuoe Hm mom mmmm< cons» mm< .02 new mom >mm®4 mam. .02 m Mom IHDU mo Thou Mom MO was» ssaomz mouflm mama .oz Iago Suave: mouam .oz Iago as means cauomaoum ca moans :auumaoum mmmmmmozem Noo I No mmmmmmozfim MH¢ ZH mmmDBADU Nflfl m mmmmmmOZBd NOU Rm I NC fimm QZ¢ MH< ZH QHMDBQDU mfifim ma<2mm mmDBmz 20mm mMHmaom mmmum>< m z N N o m sumssms passes oo I o o o m mumEEmE mam MOSHSO NOO I No b N N O m QOHU COOWHAH 00 l O O O m @502 HH< 0 mmcommmm mcowmflm mammae onmammoEuN coflquDUCH Hmuoe mo .02 . mo whoa Mme ZO ZDHQHZ mmH m0 mEUmmmm mmB Q24 NBH>HBU¢ ZHBUfiAOMm ZHBUfiAOMQ OE mmZOmmmm momU ZOMUHA Qfim mmDmmHB BDOEBHB 02¢ EBH3_QWB¢mDOZH ZDHQMZ mma mH HZHSmMBmQ OB mwfimmfi m OHQMB 77 EXPERIMENT V, DETERMINATION OF THE QUANTITY OF GLANDULAR TISSUE LOST IN THE PROCEDURE OF DISSECTING THE ANTERIOR PITUITARIES OF RATS INTO 6 EXPLANTS. In order to determine the quantity of AP tissue that is destroyed by cutting the APs of rats into 6 explants, the glands of 11 mature female rats were subjected to the dis— section procedure. Each whole anterior lobe was weighed on a Roller-Smith balance before being placed in a plaStic Petri dish which contained a few drops of 199 medium. Each gland was cut into 6 pieces and the fragments were blotted on Whatman No. 1 filter paper, which was moistened with 199 medium, before being reweighed. Results and Discussion The average weight of the 11 APs before dissection was found to be 9.52 I 0.43 mg. After dissection the average weight of the 6 explants of each gland was 7.61 i 0.47 mg. This represents an average weight loss of 1.90 I 0.17 mg which is a highly significant reduction in tissue wet weight (t = 11.2; P < 0.001). The average percent loss in tissue weight was 20.47 i 2.04 mg (t = 10.01; P < 0.001) which was also highly significant. The loss of 1/5 of the AP wet weight is probably due to crushing some of the cells 78 by the scalpel blade as the glands are cut into 6 explants. The blade may also tear some of the cells from the organ and rupture others causing the lost tissue to be left in the Petri dish. Manipulation of the tissue may also result in a loss of water which would contribute to the weight difference. 79 EXPERIMENT VI, EFFECTS OF IN_VITRO INCUBATION ON THE WEIGHT AND PROLACTIN CONTENT OF ANTERIOR PITUITARY EXPLANTS. An experiment was performed with the APs of 16 mature female rats to ascertain the effects of culturing on explant wet weight and prolactin content. Two APs were paired to provide tissue for determination of fresh gland prolactin content and for culturing. Each gland of each AP pair was halved and one-half was cut into 3 explants. The 6 explants from the 2 AP halves of each AP pair were cultured for 3 days in the plastic Petri dish preparations in 02 - CO2 atmosphere. The remaining 2 AP halves of each AP pair were weighed on a Mettler balance and prepared for aSsay. At the end of the culture period the 6 explants from each dish were weighed on the Mettler balance and prepared for assay. The explants from 2 of the culture dishes were combined in one case and the uncultured AP halves from the same pitui- taries were also combined. In all other cases the explants from each culture dish and the fresh AP halves were treated separately. The fresh and cultured AP tissue was prepared for assay by homogenizing in 199 medium at a concentration of 3 mg of AP per 0.4 ml of medium. The homogenates of the explants 80 from each AP pair were injected over the left crop half of 3 White King squabs and the homogenates of the 2 uncultured AP halves of the same AP pair were injected over the right crop half. The fresh tissue and explant content assays were, therefore, paired. Each bird received 3 mg of AP tissue by this assay procedure. The results of this study are shown in Tables 6 and 7. The standard dose-response plot (Experiment XV) was used to determine the potencies of these assays in IU. Results and Discussion The two pituitary halves of the 6 pituitary pairs had an average weight of 8.17 mg. After dissection and incu- bation the explants were found to have an average weight of 5.84 mg per cultured pituitary. Each AP therefore lost an average of 2.33 mg which represents an average weight reduction of 28.5 percent. This loss in weight is highly significant (P < 0.001). The previous study demonstrated that the dissection procedure caused a tissue loss which amounted to 20.47 percent. It is evident, therefore that) incubation per se in 199 medium results in a wet weight loss of 8.03%. This slight difference is significant (t = 2.13; P < 0.05). The change in wet weight is probably 81 due to death of some of the explant cells with a consequent loss of the intracellular contents into the medium. Loss of red blood cells from the explants into the medium and a change in the degree of hydration of the explants may also have contributed to the weight reduction. The assay results disclosed that the uncultured AP samples contained an average of 1.24 IU of prolactin where- as the explants contained 1.02 IU on the basis of 100 mg of tissue wet weight. This represents a reduction in prolactin content of 15.53% which is significant at the 1% level (t = 3.33). When the loss in weight of the explants due to incubation in 199 medium is considered, the reduction in prolactin content is increased by 7.47% and amounts of 16.69%. Meites, Nicoll and Talwalker (1962) reported that AP transplants in the kidney capsule of intact female rats contain only about 1.5% as much prolactin as the ig_§itu_APs of the engrafted hosts. Since the AP transplants were secreting substantial quantities of prolactin, as judged by mammary growth and lactation, it is apparent that the trans- plant cells were releasing prolactin at such a high rate that the intracellular prolactin pool could only be main- tained at a low level by the synthetic capacity of the cells. The results of the prolactin content of the explants are of 82 interest since one would expect the explants to behave in a manner similar to the transplanted AP with respect to pro- lactin release. The prolactin which escaped from the trans- plant cells must diffuse for a distance of only a few microns before being carried away by the circulation. Dif- fusion problems are therefore relatively unimportant in the transplant. In the cultured explants, however, the prolac- tin must diffuse through an extracellular distance which may amount to as much as 1 mm, after being released from the explant cells, before escape into the medium is possible. Since diffusion is not facilitated by circulation in the explants, the resistance to prolactin diffusion would pre- sumably be rather large and consequently the synthetic capacity of the cells would be able to establish a rela- tively large intracellular, or perhaps more correctly, intra-explant prolactin pool. The permeability properties of the explant cells to prolactin, therefore, may not be appreciably different from those of the transplant cells. Part of the reduction in prolactin content of the AP ex- plants and transplants is undoubtedly due to death of some of the cells; however it is not feasible to ascertain to what extent cellular death contributes to the lowered prolactin levels. 83 am.o + em.m u m mm.o H SH.m u m ads I w 13 u w Em A m OK 13 m a In H m mg: Tom m 4 ad a m mg 6.3 m 4 m6 H m To 4.2 m 4 Em A m We 4.3 m 4 m4“. H m SK NEH N a We N a 1m m.mm v m \uQMMOB mmm MMMMMWM me mfi me unmNOB mmm mm>amm ucmamxm mo .02 Im< mo .02 \unmflm3 mfl Hmuoa mo .02 m< mo .02 MDmmHB m< Eua>auo¢ cfluomaoum DH Hmuoe *DH cHuomHOHm .: I III Ammmmmmozfid NOD Rm I NC fimm ZH mmMDBADU M.mm QZfl BZMBZOU ZHBU¢ m.m v>.m H m.nH n.mm om.H H m.h m uomnuxw pfio< o.om «0.0 H o.m m.m¢ m¢.m H ¢.m m mumammoaom m.mm mm.H H o.aa m.m¢ mm.a H m.m m mmusuasonou 05Hm> msam> Houucou Eouflmnwo Houucou msEmamnuomhm Eoum Eoum cofluooomm coauosomm mmusuasu X x mo .02 mnsuaso m<\DBm .Eoflowz CH muw>fiuo< qHHUMHOHm mmwnm>¢ omeH> mm onemmomm zHeoHeo< szoaqomm Mama wusuaso .wmmm< Hmm mcowmfim m>fim .nmflo mom m< N\H Eonm mucmamxm nuHS mmusuasu >MQ mouse OMBH>.WW ZOHBMMUmm ZHBUdAOMm ZO ZHUOBNXO m0 AS mmm D H.O m0 Bommmm HH mHQMB 122 mm.mH mo.o om.m vm.ma + om.m + m¢.ma mm.>a Dem Hmuoe mm.mH .m.z I no.0 + OH.H . oa.oo mm.ao m4 me OOH 006 :Huumaonm DH mm.mH mo.o ok.m mm.ma + sm.o + am.m nm.~ m< umm :Huomaoum DH m©H.OH .m.z m¢.m sa.sa + v.0 + mn.m mm.m ma unmwmz unmamxm mocwumm mocm. savoumxo Houucoo v m u Imam x. unmanao "mo mmmum>< mmmum>¢ mmmnm>< wusuaso zHooewxo mo a: mmm a H.o mo meommmm omeH>.mm was mo wmHBU< ZHBOHm .Amflo mom m< «\H nuflz mwnsuaso ham mouse ma mHQMB Sufism: com .02 Hflmm musuaso omeH> mm onemmomm zHeo¢ mmmum>¢ mmmnm>¢ manuaso szmmmeHm so a: mum D O.H mo meommmm omeH> mm mme mo wmmzzom va magma 125 Conclusions The failure of oxytocin to influence prolactin secretion ig_vitro is in agreement with the observations that oxytocin administration does not alter prolactin secretion i vivo, as judged by the effects of oxytocin on the AP prolactin content (Meites and Turner, 1942, 1948; Grosvenor and Turner, 1958a). The results of this ig_vitro study also corroborate the other evidence, cited previously, which indicates that oxytocin has no effect on prolactin secretion. It is therefore concluded that the hypothesis of Benson and Folley (1956, 1957a, 1957b) that oxytocin is responsible for stimulating prolactin re- lease, is untenable. Guillemin (1956) reported that addition of a vasopressin preparation to the medium of cultures of rat or dog AP tissue resulted in stimulation of ACTH release. When synthetic vasopressin was added to these cultures, no ACTH-hypophysio- tropic effect was obtained. The ACTH stimulatory action of the vasopressin preparation was therefore attributed to con- tamination with CRF. Since no effect on prolactin secretion was realized with pitressin in this ig_vitro study, it is evident that ADH does not influence prolactin secretion. The results also suggest that the pitressin did not contain 126 detectable quantities of the hypothetical hypothalamic pro- lactin inhibiting factor (PIF) as a contaminant although, as shown in Experiment VIII, hypothalamic acid extracts may contain the PIF. 127 EXPERIMENT X, EFFECTS OF ESTROGEN. In vivo experiments have established that estrogens increase prolactin secretion in a variety of species as judged by increased pituitary prolactin content, stimula- tion of mammary growth and the initiation of lactation (Reece and Turner, 1937; Meites and Turner, 1948). Estro- gen also induces pseudopregnancy in rats (Wolf, 1935; Merckel and Nelson, 1940) indicating enhanced LTH or pro- lactin secretion. Although moderate doses of estrogen induce a pronounced rise in pituitary prolactin content, higher doses are less effective in this respect (Meites and Turner, 1948). Large doses of estrogens also depress milk secretion in lactating animals (Meites, 1959b). These latter observa— tions have been interpreted by Folley and Malpress (1948) as indicative of a differential effect of estrogen on pro- lactin secretion. They suggested that low doses of estrogen stimulate prolactin secretion whereas higher levels exert a depressant effect. Since it has not been established whether estrogen augments prolactin secretion by an action at the pituitary level, or indirectly through an effect on the hypothalamus, it was ofinterest to determine if estrogen 128 could influence prolactin secretion in_vitro by a direct action on the pituitary cells. The effects of different levels of estrogen were also tested to determine if a dif- ferential effect could be demonstrated. Methods Rats of the Carworth (CFN strain) or hybrids of Car- worth and Michigan State University Department of Chemistry rats were used for the first study. All of the rats were mature nulliparous females weighing 180-200 gms. Six APs were used to provide explants for the 2 culture dishes of each culture pair. Three of the 6 explants from each of the 6 AP halves were transferred to a raft containing control medium. The remaining 3 explants from each of the 6 APs were placed on a similar raft and cultured with medium con- taining estradiol. Thus each culture dish of each culture pair contained 18 explants equivalent to 3 rat APs obtained from 6 rats. Media from both dishes of each culture pair was assayed in the same pigeons by the paired assay proce- dure described previously. This permitted the effects of estrogen on prolactin production from an equivalent amount of pituitary tissue from the same rats, cultured under the same conditions, to be ascertained in the same assay animals. 129 Estradiol was added to the medium from a stock solution of the steroid in absolute ethanol. The final concentra- tions in the medium were 0.5 ugm estradiol per ml in 0.5% ethanol. The control medium contained an equivalent con- centration of ethanol. Rafts of urethane foam sponge were used in culture pairs 1, 2 and 3. Lens paper supported by urethane foam sponge was used for rafts in pairs 6 and 7 and in pairs 8, 9 and 10 stainless steel rafts were employed. All cultures were incubated for 6 days in 02 - CO2 atmos- phere. The medium which was collected after the second 3— day culture interval (days 4-6) of culture pairs 1-5 was used for assay. All of the medium from the 6 days of incu- bation from culture pairs 6-8 was used for assay. A 2 ml aliquot of medium from each culture dish was assayed in 5 mature White Carneau pigeons and the total activity of the medium sample calculated. The explants from this study were not weighed at the end of culture. For the determination of the effects of different con- centrations of estradiol on prolactin secretion in_yi££g, the plastic Petri dish - stainless steel raft preparations were used and the APs were obtained from 180-200 gm CFN female rats. Estradiol was added to the 199 medium in the 130 same manner as before at concentrations of 0.05, 0.5 and 2 ugm per ml. Eight culture pairs were used at each dose level. Essentially the same paired culture and assay pro- cedure was used for this study as was used in the first study. Each culture dish at the low dose level contained one explant (1/6 of an AP) and the dishes with the inter— mediate concentration each contained 2 explants (1/3 of an AP). For the high dose level one-half of an AP was placed in each culture dish (3 explants). A different quantity of tissue was used in the culture dishes at each dose level in an attempt to ascertain the effects of the quantity of tissue cultured on prolactin secretion in_yi££2_by comparison of the prolactin levels of the control medium samples. This was unsuccessful, however, as will be shown subsequently. The cultures in this experiment were incubated for 3 days in 02 - CO2 atmosphere and 3 ml of medium was used in each dish. Insulin was not added to the 199 medium for this second study with estradiol although it was present in the medium used in the first study. The medium was assayed for prolactin activity by injection of 1.2 ml of the nutrient fluid from each dish into 3 White King squabs. The prolac- tin levels in the medium samples were converted to IU by use 131 of the standard dose-response plot (Experiment XV). To determine if estrogen influenced the pigeon crop response to prolactin, 10 mature White Carneau pigeons were injected over the right crop sac with a total dose of 20 ugm pro- ()6) lactin per bird in 0.5% ethanol solution. The left crop halves of the same pigeons were injected with the same pro- lactin dose in 0.5% ethanol containing 0.5 ugm estradiol per ml. Upon termination of the cultures in this second estradiol study, the explants were weighed before being fixed for histological examination. Results and Discussion The data from the first experiment, which are shown in Table 15, demonstrate that the estradiol medium samples from each culture pair contained higher prolactin activity then the control medium samples in all 8 cases. Prolactin production by the APs in estradiol medium averaged 0.89 RTU per AP per day of culture and the average of the control cultures was 0.74 RTU per AP per day. This difference of 0.15 RTU represents a 31.94 percent increment in prolactin activity and is significant at the 2% level. Histological examination of the explants disclosed that tissue survival was excellent and no differences were apparent in the degree 132 of survival of explants from cultures in estradiol medium when compared with their appropriate control explants. The standard 20 ugm dose of prolactin gave an average response of 2.38 t 0.22 RTU per bird while the same standard with 0.5 ugm/ml of estradiol produced an average response of 2.2 i 0.25 RTU per bird. This difference was not significant; therefore, estradiol did not influence the pigeon crop response to prolactin at the dose level used. The data from the experiment with the low level of estradiol in the second study are shown in Table 16 and summarized in Table 17. It will be seen that estradiol had no apparent effect on the weight of the AP explants. The explants cultured in the control and estradiol cultures had an average weight of 0.66 and 0.63 mg respectively. The average difference in explant weight of 0.04 I 0.06 mg was not significant. The prolactin activity of the estradiol medium samples was found to be significantly greater than that of the control samples on the basis of IU/100 mg or total RTU. Estradiol at a concentration of 0.05 ugm per m1 increased prolactin secretion by an average of 36.90% on the basis of IU per 100 mg of explant weight (P < 0.05) and by 28.40%»on the basis of total RTU (P < 0.05). The 133 difference in prolactin activities in the estrogen and con- trol medium samples was not significant on the basis of IU/AP. The results from the experiment with estradiol at a concentration of 0.5 ugm per ml are shown in Table 18 and summarized in Table 19. In this study, as in the previous experiment, the estradiol did not effect the weight of the pituitary explants. The average difference in explant weight of 0.01 I 0.09 mg is not significant. The prolactin content of the estradiol medium samples was not significantly different from the control samples on the basis of IU per cultured AP. On the basis of IU per 100 mg of explant weight, the medium from the estradiol cultures averaged 58.10%ihigher in prolactin content than the control cultures (P < 0.05). When expressed as total RTU, the estradiol medium samples averaged 29.20% higher in prolactin activity than the control samples (P < 0.05). Table 20 shows the data obtained from the experiment with estradiol at a concentration of 2 ugm per ml and the results are summarized in Table 21. At this dose level estradiol did not affect the weight of the explants. The average difference in explant weight between the control 134 and estradiol cultures was only 0.06 I 0.05 mg and was not significant. Although the prolactin activity of the estra- diol medium samples was higher than the levels found in the control medium samples by an average of 18.54% and 15.18% on the basis of IU/AP and IU per 100 mg explant weight respectively, these differences were not significant. On the basis of total RTU, however, the prolactin levels in the estrogen medium samples were significantly higher than the control samples by 7.44% (P'< 0.05). Conclusions drawn from the statistical analysis have been based primarily on the criterion of IU per 100 mg of explant weight, since this is probably the most definitive of the parameters for assessing the effectiveness of the experimental treatment. It is therefore concluded that estrogen significantly increased the prolactin levels in the medium samples at the 0.05 and 0.5 ugm per m1 dose levels but was without effect at the 2 ugm/ml concentration. Explant survival was excellent in all cases in this second study with estradiol at different concentrations and no differences were apparent in the histological appearance of explants from the control and estradiol cultures. It is evident, therefore, that the higher prolactin levels found 135 in the estradiol medium samples from the experiments with the 0.05 and 0.5 ugm/ml concentrations, cannot be attributed to differences in explant survival. Since estradiol did not influence the pigeon crop response to prolactin at the 0.5 ugm dose level, and no differences in explant survival were detectable, it seems evident that estradiol increased the prolactin activity of the medium samples by an action on the AP explants. Presumably the estradiol acted directly on the pituitary acidophils to effect an increase in pro- lactin synthesis. Support for this interpretation is pro- vided by the observations that estrogens increase the 5“ respiratory metabolism of rat (Victor and Anderson, 1937) ,1; and human (Gaul and Villee, 1959) AP tissue ig_yit£g, The stimulatory action of the 0.05 ugm/ml level of estradiol on prolactin secretion in_vitro in the second study was about the same as that found with 0.5 ugm per ml in the first trial. Although the increase in prolactin synthesis induced by 0.5 ugm/ml of estradiol in the second study was greater than that produced by the same level in the first trial or by the 0.05 ugm per ml concentration in the second experiment, these increases did not differ from one another. Since the high concentration of estradiol (2 136 ugm/ml) did not stimulate prolactin secretion ip_vitro, these data indicate that the lower dose levels of estradiol are more effective in promoting prolactin secretion than higher levels. The ineffectiveness of the higher level of estrogen, in augmenting prolactin synthesis, is in agreement with the in vivo observation that high doses of estrogen are less effective in increasing the pituitary prolactin content in guinea pigs, rats and rabbits (Meites and Turner, 1948). The average prolactin levels in the medium samples of the control cultures in the second study, with the 0.05, 0.5 and 2.0 ugm/ml concentrations of estradiol, were found to be 39.7 t 5.3, 13.6 t 2.16 and 17.2 i 2.5 IU per 100 mg of ’explant weight respectively. The prolactin levels of the control medium samples from cultures with the intermediate and high concentrations of estradiol were significantly lower than at the lower level (P < 0.001 and P < 0.01 respectively). The difference between the control averages in prolactin synthesis of the cultures from the studies with the inter- mediate and high levels of estradiol are not significant. The average weight of the explants from the control cultures from the experiments with the low, intermediate and high concen- trations of estradiol were 0.66, 1.66 and 2.18 mg respectively. 137 It is felt, however, that the marked differences in pro- lactin production observed in these studies cannot be attributed to the difference in the quantity of cultured tissue. The differences in prolactin synthesis by the controls in these culture experiments is probably due to some dif- & ference in the culture conditions. The culture with the 0.5 and 2.0 ugm/ml levels of estradiol were incubated one week after the cultures with 0.05 ugm/ml. If an undetected reduction in either incubation temperature or gas flow rate had occurred, even though slight in degree and of relatively short duration, during incubation of these cultures, a pro- nounced reduction in hormone synthesis would probably occur without influencing explant morphology. Another possible explanation for the lower levels of prolactin synthesis in the control cultures from the studies with 0.5 and 2.0 ugm/ m1 of estradiol would be the presence in the medium of a minute amount of a material which was toxic or otherwise inhibitory to protein synthesis. It is conceivable that such a toxic material could have gained entry into the medium and, although insufficient in amount to cause cellu- lar death, could be adequate to suppress prolactin synthesis. 138 The results of Kahn's culture studies (Experiment I) illus- trate this point. Kahn used antibiotic chloramphenicol in his cultures. Although his explants were incubated in 02 - CO2 atmosphere and they were well maintained, the AP fragments synthesized only about as much prolactin as the cultures in air atmosphere in the author's studies. Chlor- amphenicol exerts a pronounced depressant effect on protein synthesis (Rendi, 1959). It is recognized that other possibilities exist which could account for these differences in prolactin synthesis; however, it is the opinion of the author that the sugges- tions advanced are the most probable. Despite this marked between-experiment difference in prolactin production the within—experiment comparisons which have been made between the control and estradiol cultures are valid since the conditions of culture, the composition of the medium (apart from the presence of estradiol) and the assay conditions were identical for each experiment. An assay of AP tissue from 190-200 gm CFN female rats, which was performed at about the same time as the assays on the medium samples from the study with different doses of estradiol, disclosed that the tissue had a prolactin content 139 of 1.04 I 0.17 IU per 100 mg wet weight. Comparison of this value with the average prolactin levels in the control culture medium samples from the low, intermediate and high concentrations of estradiol (39.7, 13.8 and 17.2 IU per 100 mg of explant weight respectively) reveals that in all three studies the pituitary explants synthesized substantial quantities of prolactin. The AP explants in the control cultures evidently synthesized, during each day of the 3 day culture, about 13, 4 and 5 times as much prolactin as the explants contained at the beginning of culture. The prolactin synthesizing capacity of the AP explants in the control cultures from the study with the 0.05 ugm/ml level of estradiol admirably illustrates the autonomous nature of prolactin secretion by the adenohypophysis. 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L IL - n . — A02\DH mHv ZHBU1 ma.o + x 00H ¢®.H 212 0000mm0unu> 0mmmoo n x 0:0Hum>u0mno m0 .0: H0000 ma0>0a 0000 m0 .0: H x m + N N I a H0HH0 0uHmomEOU IAMII I U H 0008 How N NV 00Hu00HHOU 0\NA>NV I N» N H I 0 H0009 m Amy AN I H I Hmuouv H0Uchs0m x I a 0:008 0000 N uzonm mGOHHMH>0Q m 0QHH 09090 00008 Nm\< 0 ANV Nm I 0 INéNHVN N I x 0000 m0 H0uumom c leeemu ix eonc I mix eoch 0000 N I . m mem >w x voHH I Nxx mova E0000Hm m 0umsom m0umsom m0 85m m0 GOHuMHH0> 0002 m00Hm0Q m0 00HDOm mmOQ mo¢m 94 mmmZmemm HMOZ m0 039 mBHS m>MDU mmzommmmlmmOQ < m0 MUZ m0 mHm>Q¢Z¢ HEB mom Zmom MMOZ mm mamdfi 213 Hm.0 00.00 mm 00000 000momaoo ma.mNN MH.mNN H 0008 000 0000000000 Ho.H 06.00 mm H0009 Hm.o Nm.0H 0m 00005 0000 00000 000000H>00 .0.2 00.0 NN.o 00.0 v 0000 00000 00008 0000 00 0000000 00. 6m.000 00.00 mm.Hv H 0 .0000 00 00000 0%mwmww V 0 000000 0000000 Eoww00m 00000000> V m 0002 00 500 0000000 00 000000 mzomem OZHM mBHEB om ZH ZHBUMDU mmzommmmlmmoa m0 mUZ¢HM<> m0 mHmMA 0002 00 800 0000000 mo 000000 zH000qomm 0H0 20: OH 00H: nmeomsz 0003 monz mzomem 0H mo mmDomw 0H0 0o mmmzommmm 000 0o 0020H0<> 0o 0Hqumz< ow mHm4B 215 60.0 0 05.0 m 6 mcHHmm H6\m\m 6 50.0 H m~.H m 0H asHume 00H H6\0H\NH 6 mm.0 H H6.H 0H m maHHmm H6\m\0H 0 0H.O H M6.H 0H 0H 000600 00H H6\0~\0 m 6H.o H 00.H m m wcHHmm H6\0H\0 m mm.o H mm.H 0H 0H mcHHmm H6\em\e H 0%”; ”Mummy 2.0% 2.30m 33 ”WNW“ mZOHafiwHBmm>ZH WEB m0 mmMDOU mmB GZHMDQ mH<>MMBZH BzmmmmmHQ Ed QmmmBmHZHznfl QO¢QZ m0 mHmWA€Z¢ Nfi mam¢B 223 .mmamEmm 850608 030 :0 :0uomaonm mo DH 0000» 6:0 u£m0m3 unmamxw cmm3umn m0£mc0000000 mgu mc03onm Emnm00p Hmuumum ¢ .00 musm0m oz .00on: 0.20.088 0 0 0 m 0 0 I n d d H O n1 m I N I N I N m I. m n 00.0 n 0 N 60.0 .. x 60.0 n 0 1 0 224 medium sample to the amount of tissue present in the culture dish (i.e. explant weight) provides a valid index for anal- yzing the data. The regression curve and equation for the relation between explant weight and total IU of prolactin in the medium samples are also shown in Figure 14. The regression equation, y = 0.46 x - 0.46, where y equals the total IU of prolactin and x equals the explant weight, indicates that with one mg of explant tissue, no prolactin would be detect- able in the medium. Although this conclusion may be correct for these selected data, it cannot be extrapolated to all of the data from these studies. In Experiment X, substan- tial prolactin activity was found in medium samples from cultures which contained as little as 0.5 mg of AP tissue. The average total prolactin levels in these 52 medium samples was 0.78 IU. Since each medium sample had a volume of about 2.8 ml at the end of the 3 day culture period, the average prolactin concentration in the medium samples was about 0.28 IU/ml. In Experiment VI, it was shown the rat AP explants contain 1.02 IU of prolactin per 100 mg of tissue wet weight. If we assume that rat AP tissue has a density of about 1, it is evident that the prolactin 225 concentration in the explants was approximately 10.2 IU per ml of AP tissue. It is therefore obvious from these esti- mates that there is no indication that the explants were secreting prolactin against a concentration gradient. The prolactin concentration differential of 10.2 IU/ml in the explant and 0.28 IU/ml in the medium indicates that the prolactin molecules could probably diffuse quite readily from the explants into the medium. Since no data are available for comparison of the intracellular and extracellular prolactin concentrations within the explant, the intra-explant resistance to pro- lactin diffusion can not be determined. Diffusion of pro- lactin away from the AP cells is not facilitated by cir- culation in the explants, therefore a considerable extra- cellular concentration of prolactin might be present within the AP fragments. This might influence the rate of prolactin secretion by the explant cells by a mass action effect; however we cannot determine to what extent this phenomenon might have influenced prolactin secretion in this in vitro system. Li and codworkers (see Li, 1961) have isolated a pro- lactin preparation from ovine AP glands which has a molecular 226 weight of about 24,000 and a maximum biological activity of 35 IU per mg. If it is assumed that this prolactin prepar- ation is the "pure" hormone, and that the prolactin secreted by the rat AP explants has the same molecular weight and biological activity as the "pure" ovine preparation, then we can perform some rather interesting, though highly specu- lative, calculations. The average amount of prolactin produced by the AP fragments in the 8 control cultures from the experiment with estradiol at a concentration of 0.05 ugm per ml, was 39.7 IU per 100 mg of explant weight. This is the highest degree of prolactin synthesis encountered in the 3 day culture studies. If the maximum biological activity of the rat prolactin is taken as 35 IU/mg, then each 100 mg of rat AP explant tissue synthesized about 1.14 mg of prolactin. Each gram of rat AP explant tissue therefore synthesized approximately 11.4 mg of the protein hormone. With a molecular weight of 24,000, each mole of prolactin would weigh 24 x 106 mg. The value of 11.4 mg of prolactin is therefore equivalent to 4.75 x 10..7 moles of the hor— mone. With Avogadro's No. of 6.02 x 1023 molecules/mole, it is evident that one gram of rat AP explant tissue 227 synthesized approximately 2.86 x 1017 molecules of the hormone in 3 days. Thompson (personal communications) reported that one gram of human AP cells, derived from a suspension culture, contained about 3 x 108 cells. If we further assume that rat AP parenchymal cells weigh about the same as these human cells, one gram of rat AP explant tissue would also contain 3 x 108 cells. This estimate ignores the contri- bution of non-parenchymal elements (e.g. red blood cells) to the weight of the rat AP tissue; however it is not known to what extent the non-parenchymal elements contributed to Thompson's estimate of the number of cells per gram of human AP. For these highly conjectural estimates therefore, the value of 3 x 108 cells per gram of AP tissue can prob— ably be used with as much validity as any other figure. If it is assumed that approximately one—third of the rat AP cells were the prolactin producing acidophils, and that the acidophils account for 1/3 of the weight of the tissue,then one gram of rat AP explant tissue would have contained 1 x 108 prolactin synthesizing cells. It was previously estimated that one gram of rat AP explant tissue synthesized approximately 11.4 mg of 228 prolactin, and this was equivalent to 2.86 x 1017 molecules of the hormone. Using the value of l x 108 acidophils per gram of AP explant, it can be estimated that each acidophil cell synthesized 11.4 x 10-3 mg of prolactin in 3 days and this is equivalent to 2.86 x 109 molecules of the hormone. Each acidophil cell, therefore, produced an average of 1.58 x 10—9 mg of prolactin or 3.96 x 107 molecules of the hormone per hour during the 3 days of ig.yit£g_incubation. Setlow and Pollard (1962) have estimated that a bacillus of Eschericha Coli synthesizes 3.6 x 106 protein molecules per hour during the process of replication. Corner (1947) estimated that each cell in the corpora lutea of pseudo- pregnant rabbits produces 5.4 x 1010 molecules of proges- terone per hour. It is therefore evident that the estimated prolactin synthetic rate of 39.6 million molecules per acidophil cell per hour, in the ig_yi§£9_system, is not at all unreasonable. When the molecular weight and biological activity of rat prolactin are known, and the number of functional acidophils in the in_yit£9_system can be deter- mined, a more precise estimate of the prolactin synthesizing capacity of the acidophil cells of the AP explants will be possible. 229 Reece and Turner (1937) and Meites and Turner (1942) have reported that the AP glands of lactating rats contain more prolactin than the glands of non-lactating mature fe- male rats. They also observed that the APs of mature females contained higher prolactin levels than the glands of immature rats. The observation from Experiment I, that AP explants from lactating rats secrete more prolactin in_vitro than the glands of non-lactating females, supports the conclusions of the ig_yiyg_studies that the prolactin content of the pars anterior can be a useful indicator of the amount of hormone secreted by the gland. Meites, Kahn and Nicoll (1961) have shown that APs from mature female rats secrete more prolac- tin in.vitro than the glands of immature rats. This con- stitutes additional evidence that the AP prolactin content can reflect the secretion rate of the hormone. Further support for this concept was obtained from the comparative study (Experiment VII) where it was shown that the prolactin content of the APs of mice, rats and guinea pigs was reflec- ted in their relative capacities to secrete the hormone ig_ vi__’2r<_>- It must be emphasized that the pituitary prolactin con- J tent per se merely reflects the intracellular hormone pool 230 which is the result of the differential between the rates of synthesis and release of the hormone. It is evident, however, that the AP prolactin content can be a useful, although not invariable, indicator of the secretion rate of the hormone. Suppression of in_vitro prolactin secretion by an acid extract of hypothalamic tissue (Experiment VIII), while a similar extract of cerebral tissue was without effect, pro- vides rather convincing evidence for the existence of a material of hypothalamic origin which inhibits prolactin secretion. It cannot be ascertained as yet, however, whether the prolactin inhibiting factor (PIF) is also responsible for stimulating LH secretion as suggested by several investigators (Everett, 1954, 1956; Haun and Sawyer, 1960; McCann, 1962a). Secretion of gonadotropins and prolactin (LTH) appears to be reciprocally related in many circumstances. During pseudopregnancy and lactation, when prolactin is presumably secreted actively, there is usually little or no indication of gonadotropin secretion, as evidenced.by quiescence of the ovarian follicular apparatus. Injection of numerous agents, such as estrogen, tranquilizing drugs (Barraclough 231 and Sawyer, 1959) and morphine (Barraclough and Sawyer, 1955), suppresses FSH-LH secretion while stimulating pro- lactin release. Conversely, there are no physiological manifestations of prolactin secretion during normal estrous cycles of rats and mice. Several observations, however, indicate that secretion of gonadotropins and prolactin may not necessarily be mutually exclusive events under all conditions. Electrical stimulation of the uterine cervix of imma- ture rats promotes FSH secretion, as evidenced by hastened sexual maturation (Swingle et al., 1951c), and induces ovulation and pseudopregnancy in mature rats (Haterius, 1932), indicating that LH and prolactin secretion are in- creased. Cervical stimulation also initiates lactation in estroqen primed rats (Meites et al., 1959). Ovulation and pseudopregnancy are readily induced in rabbits by stimula- tion of the genitals indicating that LH and LTH secretion are increased. Epinephrine and acetylcholine are reported to induce LH release (Teubenhaus and Soskin, 1942; Sawyer et al., 1949) but can also induce mammary secretion in rats and rabbits (Meites, 1959; Meites et al., 1960), and pseudopregnancy in rats (Swingle et al., 1951b). Rats, 232 mice and guinea pigs ovulate within 1 or 2 days after par- turition, indicating FSH and LH release (Asdell, 1946). At the same time these animals show a marked increase in prolactin secretion (Meites, 1959). Rabbits also enter a state of estrus Shortly after parturition and readily ovulate in response to coitus. Although these observations indicate that the secre- tion of prolactin and gonadotropins may occur simultan- eously, other explanations are feasible for the apparent lack of reciprocal secretion of these hormones under these conditions. Induction of ovulation and pseudopregnancy could be the result of a concomitant increase of LTH and LH secretion for a brief period, followed by a diminished secretion of LH. It is also possible that LH secretion is first stimulated, and when ovulation occurs, LTH secretion commences concomitant with a reduction in LH secretion. The occurrence of ovulation shortly after parturition, when prolactin is apparently secreted actively, may be accom- plished by a brief period of reduced LTH secretion during which sufficient LH is réleased to induce follicular rupture. Complete elucidation of this complex phenomena, however, will require considerable additional research. 233 The site of action of the endocrine products of the gonads, thyroid and adrenal cortex, in altering AP function, may be on the hypothalamus or the adenohypophysis or on both components of the hypothalamo-hypophysial apparatus. Thyroid hormones can apparently depress TSH secretion by an action on the AP (see D'Angelo, 1962). Other evidence indicates that estrogens depress gonadotropin secretion (Rose and Nelson, 1957) and cortisol inhibits ACTH secre- tion (Rose and Nelson, 1956) by acting directly on the AP. The stimulatory action of estradiol and thyroid hormones, and the depressant effect of cortisol, on prolactin secre- tion observed in these ig_yi££2_studies, provides further evidence that estrogenic, thyroidal and adrenal cortical hormones can alter adenohypophysial function by an action on the AP cells. Endroczi et a1. (1961) reported that injection of cortisone acetate in agar into the tuberal region of the hypothalamus of cats and rats depresses adrenal steroid / secretion. Injections into the reticular formation and the top of the mammilary body had a slight inhibitory effect. Control injections into other parts of the hypo- thalamus were ineffective. These observations indicate 234 that the depressant action of corticoids on ACTH secretion may be mediated in part by an effect on the hypothalamus. L Other experimental observations indicate that estrogens depress gonadotropin secretion (Flerko and Szentagothai, 1957; Flerko, 1962; Lisk, 1960; Davidson and Sawyer, 1961a) and thyroid hormones inhibit TSH release (Yamada, 1959; see also D'Angelo, 1962) by actions on the hypothalamus. It is possible, therefore, that the stimulatory action of thyroid hormones (Weichert and Boyd, 1933-34; Grosvenor, 1961; Moon, / 1962) and estrogens (Reece and Turner, 1936; Meites and Turner, 1948) on prolactin secretion ig_yiyg_may be the result of an action of these hormones on the AP and the hypothalamus. The recent observation of Kanematsu and Sawyer (1962), that intrahypothalamic implants of estrogen increase the AP pro- lactin content of rabbits, indicates that estrogens can alter prolactin synthesis via the diencephalon. The relative importance of the hypothalamic and hypophysial actions of thyroidal and estrogenic hormones, in augmenting prolactin secretion in_yiy9, however, cannot be fully assessed at 1 present. The depressant action of cortisol on prolactin secretion in vitro does not conform with the conclusions of Johnson 235 and Meites (1955) that glucocorticoids increase prolactin secretion 1 vivo. In the in vivo situation, corticoids could conceivably stimulate prolactin secretion via the hypothalamus or by some other means. It seems unlikely, however, that corticoids could increase prolactin secretion by a direct action on the AP. The lack of an effect of corticosterone on prolactin secretion, even with a concen- tration of 20 ugm per m1, indicates that the depressant effect of cortisol is not due to nonspecific steroid toxicity. Failure of progesterone and testosterone to alter pro- lactin secretion i§_vitro, while these steroids can appar— ently increase prolactin secretion in_yiyg (Reece and Mixner, 1939; Reece and Bivins, 1942; Meites and Turner, 1948) indicates that the in_yiyg_effects of these steroids on prolactin secretion may be via the hypothalamus. Testosterone pellets in the hypothalami of dOgs depress gonadotropin secretion (Davidson and Sawyer, 1961b) and injection of progesterone into the hypothalami of chickens (Ralph and Fraps, 1961) induces ovulation. It is apparent, therefore, that these gonadal steroids can influence AP function by actions on the hypothalamus. Hypothalamic 236 mediated stimulation of prolactin secretion by progesterone and testosterone is therefore quite conceivable. It would be of interest to determine if intra-hypothalamic implants // of testosterone and progesterone could induce pseudopreg— nancy or initiate lactation in suitably prepared animals. The ig_vitro procedures used in these studies on pro- lactin secretion have several advantages and disadvantages. The organ culture method permits the function of the AP to be studied in a highly simplified system where a great degree of experimental control is possible. The effects of numerous extraneous, and otherwise uncontrolable factors can be virtually eliminated from the experiments. Incuba- tion of AP fragments in chemically defined medium, however, cannot be considered as physiological. It is possible that the effects of the various hormones on prolactin secretion observed in these in yitro studies would be somewhat modified in the presence of physiological levels of the other hormones. For example, although pro- gesterone and testosterone had no demonstrable effect on prolactin secretion in_vitro when they were incorporated into the medium individually, if these steroids were tested in a system containing quantities of the other hormones 237 (estrogen, thyroxine, etc.) they may have influenced pro- lactin production. An ig.yiygiillustration of such endo- crine interaction is provided by a recent study of McCann (1962b). He observed that progesterone alone, even in very high doses, does not depress the high plasma LH levels in ovariectomized rats. When progesterone was administered along with a dose of estrogen, a greater depression of LH secretion was achieved than was obtained with either steroid alone. Thorough evaluation of the effects of hormones, individually at different concentrations, and in various combinations with other hormones, on prolactin secretion in_ vitro presents a task of herculean proportions for future endeavors. It will becflfinterest to determine if interactions can be obtained from combinations of the hormones which were demonstrated to alter prolactin production in these ig_vitro studies. Would the stimulatory action of estrogen and thy- roxine on prolactin secretion be synergistic when combina- tions of these two hormones are tested? Can the depresSant effects of cortisol be overcome with estrogens and thyroid hormones? The observation that progesterone inhibits the estrogen—induced rise in AP prolactin content in vivo 238 (Meites and Turner, 1948) will be worthy of further inves- tigation in this i§_yi§£g system. Possibly progesterone, though ineffective by itself, would block the estrogen- induced increase in prolactin secretion. This ig_vitgg system provides a convenient method for studying numerous other aspects of AP physiology. Cul- tures of adenohypophysial fragments could be used with much facility for further studies on the hypothalamic neurohumoral agents which apparently regulate AP function. Studies on the physico-chemical characteristics of the hormones which are actually secreted by the AP cells may be possible using culture techniques. Previously, such investigations relied