STUDIES OF THE NEUROENDOCRINE CONTROL OF PROLACTIN SECRETION IN RATS a i'". Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY MADHABANANDA SAR 1968 C3 ' 3 P a y r ’ 7 l k “ kl . | '. 1 ‘ ' ’ a I of I ‘4‘: ”1.4.4. L 4' .rl :4 h This is to certify that the thesis entitled Studies of the Neuroendocrine Control of Prolactin Secretion in Rats presented bg Madhabananda Sar has been accepted towards fulfillment of the requirements for Ph D degree in Bhysiology ajor professor Date February 23) 1968 0-169 3’ BIN‘DING BY ‘5'!" "DAB 3 SONS' BOOK BINDERY INC. LIBRARY BINDE RS SIIIIEPOI‘I. mama” ABSTRACT STUDIES OF THE NEUROENDOCRINE CONTROL OF PROLACTIN SECRETION IN RATS by Madhabananda Sar 1. Changes in pituitary prolactin release and hypothalamic prolactin inhibiting factor (PIF) content during the estrous cycle of rats, Anterior pituitaries removed from proestrous and estrous rats contained significantly more prolactin than anterior pituitaries from diestrous rats; the former released significantly more prolactin when incubated i2 vitro than the latter, and proestrous and estrous rats had significantly less PIF in the hypothalamus than diestrous rats. It was concluded that ovarian estrogen secreted during proestrus and estrus increases prolactin release by depressing hypothalamic PIF production and by directly stimulating the pituitary. 2. Effects of progesterone, testosterone and cortisol on hypothalamic PIF content, pituitary prolactin concentration and mammary development in ovariectomized rats. Each of the The 3 steroids were each injected for 10 or 20 days. 3 steriods significantly decreased hypothalamic PIF content, increased pituitary prolactin concentration and induced mammary growth. Pre- viously it had been reported that none of these steroids directly stimulated pituitary prolactin secretion in vitro. The present obser- vations indicate therefore, that the stimulatory effect of the 3 hormones on pituitary prolactin secretion is mediated through the hypothalamus. Madhabananda Sar 3. Effects of long-term implantation (65 or 130 days) of diethylstilbestrol (DES) on pituitary prolactin secretion, hypothalamic PIF content and mammary development of intact female rats. Pituitary prolactin content was increased significantly and this was associated with a decrease in hypothalamic PIF content in rats implanted for either 65 or 130 days. Pituitary size was increased to a much greater extent in rats treated with DES for 130 as compared to rats treated for 65 days. DES implantation induced extensive mammary gland growth associated with development of hyperplastic nodules resembling those of preneoplastic nodules in the mammary glands of cancer susceptible mice. The present results suggest that the mechanism of "mammotrOpic" pituitary tumor deve10pment by estrogen in rats involves a depression of PIF production by the hypothalamus and possibly a direct action on the pituitary as well. The resultant increase in prolactin secretion is believed to be important in devel- Opment of mammary tumors. 4. Effects of suckling on hypothalamic PIF content and pituitary release of prolactin, GH and TSH in postpartum lactating rats. The results indicate that a single suckling period of 3 hours after a period of 10-12 hours non-suckling on the 4th or 10th day postpartum results in a significant decrease in hypothalamic PIF content, a reduction in pituitary prolactin (33-70%) and GH(51-54%) change in TSH concentration. Half an hour concentration but no of suckling after a period of 10 hours non-suckling on postpartum day 10 neither reduced hypothalamic PIF content nor decreased pituitary prolactin concentration. This suggests that-% hour of suckling is not sufficient to suppress hypothalamic inhibition of prolactin release in Madhabananda Sar these rats and hence did not result in any significant fall in pituitary prolactin content. The stimulation by suckling of GH release suggests that this may be partially responsible for the increase in food intake during lactation. 5. Effects of thyroidectomy on pituitary prolactin and hypothalamic PIF levels. When female rats were thyroidectomized for 30 days, there was a significant decrease in pituitary prolactin concentration but no change in hypothalamic PIF content. Since thyroid hormones had previously been shown to directly stimulate pituitary prolactin release but to have no effect on hypothalamic PIF content, these results suggest that it is the absence of thyroid hormones after thyroidectomy, affecting the pituitary directly, which is reSponsible for the fall in pituitary prolactin content. 6. Effect of thyroidectomy and thyroxine on the grOWth of a transplantable mammotropic tumor (MtTW ) and a hormone dependent- mammary tumor (MTWIS) in female Wistar rats. Thyroidectomy 2 weeks before transplantation of pituitary and hormone dependent mammary tumors completely inhibited their growth. Following transplantation of tumor tissue into the host rats, admin- istration of thyroxine (10 ug/lOO g b.w.) for 45 days to the tumor bearing rats had no significant effect on pituitary tumor growth but slightly depressed growth of mammary tumors. The slight inhibition of mammary tumor growth may be due to a direct inhibitory effect of thyroxine on the mammary tissue. These results Suggest that thyroid hormones are necessary for growth of these tumors upon transplantation. Madhabananda Sar 7. Effects of prolactin in immature and mature male rats on tesces and accessories. Prolactin injections (.5 mg) for 21 days significantly increased the weight of the testis, ventral prostate and seminal vesicles but decreased the weight of the adrenals in young rats treated from age 3 to 23 days. No significant effects on testes and accessory organs were observed when 3 mg of prolactin was administered daily for 10 days into adult male rats approximately 3-3% months old. These results suggest that prolactin has a gonadotropic effect in young but not in mature male rats. 8. Hypothalamic FSH-RF content in the female rat before and after puberty. The FSH releasing activity of the hypothalamus of immature rats remained fairly constant before the establishment of puberty. There appeared to be a drop in hypothalamic FSH-RF content on the day of vaginal opening (38 days) and thereafter the hypothalamic FSH-RF content rose to prepubertal levels. This suggests that FSH-RF may have a role in regulation of FSH secretion by the anterior pituitary at the time of onset of puberty. STUDIES OF THE NEUROENDOCRINE CONTROL OF PROLACTIN SECRETION IN RATS by Madhabananda Sar A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1968 m&-~q. .. »_' G a 0014 1 ‘yr:o~¢5 DEDICATED TO MY FATHER AND MY BROTHERS ii ACKNOWLEDGEMENTS The author wishes to express his sincere and deep gratitude to Dr. Joseph Meites, Professor of Physiology for his guidance, advice and assistance throughout the course of this study and during the preparation of this thesis. He also expresses appreciation to Dr. W.D. Collings, Dr. E.P. Reineke, Dr. M.L. Calhoun, Dr. R.L. Anderson and Dr. P.O. Fromm for serving as members of the doctoral guidance committee, and for reading the manuscript. The author is indebted to fellow graduate students, other faculty members and staff of the Department of Physiology for their helpful cooperation during the course of this study. Special thanks are due Dr. W.L. Frantz for providing a Special Research Assistantship from December 1963 to September 1964. The author also wishes to express his gratitude to the Department of Physiology, Michigan State University for providing a graduate teaching assistantship from Fall 1964 to Winter 1968. Thanks are also due to the National Institute of Health, which provided funds and made available purified anterior pituitary hormones (Prolactin, GH, TSH & FSH) for the research carried out by the author. Sincere thanks are due to "Mohini" the author's wife, who supplied encouragement and much tolerance during the entire period of graduate study, without which he would not have successfully completed this work. iii II. III. IV. II. III. TABLE OF CONTENTS INTRODUCTION ......................................... REVIEW OF LITERATURE ................................. Neural Regulation of Adenohypophysial Function ....... Hypothalamic Regulation of Prolactin Secretion ....... A. Hypothalamic Lesions ............................. B. Transplantation ....................... . .......... C. Prolactin Secretion in yitgg .. ................... D. Prolactin Inhibiting Factor (PIF) ................ Influence of Suckling Stimulus on Prolactin Secretion. Effects of Steroids on Prolactin Secretion ........... A. Estrogen ........................ . ................ B. Testosterone ................ ....... .............. C. Progesterone ..................................... D. Cortisol (Glucocorticoids) ..... . ................. MATERIALS AND METHODS ..... ........................... Animals .... .......................................... Incubation Technique .... .............. . ...... . ....... A. Preparation of Acid Extract of Hypothalamus ...... B. Incubation .............. . ....... . ................ Bioassays ............................................ A. Prolactin ................. .......... ............. B. Growth Hormone (CH) .............................. C. Thyroid Stimulating Hormone (TSH) ................ iv 10 ll 12 16 16 17 18 20 23 23 23 23 24 25 25 25 26 TABLE OF CONTENTS - continued IV. Histological Preparations ............................ 26 v. Statistical Analysis ................................. 27 EXPERIMENTAL ......................................... 28 1. Changes in Pituitary Prolactin Release and Hypothalamic PIF Content During the Estrous Cycle of Rats 00...... OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 28 II. Effects of Progesterone, Testosterone and Cortisol on Hypothalamic PIF Content, Pituitary Prolactin Concen- tration and Mammary Development in Ovariectomized Rats ..................... . ............................ 36 III. Effects of Long-term Implantation (65 or 130 days) of Diethylstilbestrol (DES) on Pituitary Prolactin Secretion, Hypothalamic PIF Content and Mammary Development of Intact Female Rats .................... 48 IV. Effects of Suckling on Hypothalamic PIF Content and Pituitary Release of Prolactin, GH and TSH in Post- partum Lactating Rats ................................. 64 V. Effects of Thyroidectomy on Pituitary Prolactin and Hypothalamic PIF Levels .............................. 76 VI. Effects of Thyroidectomy and Thyroxine on the Growth of Transplantable Mammotropic Tumor (MtTW ) and a Hormone Dependent Mammary Tumor (Mtw ) in Female . 15 Wistar Rats ................ . ......................... 81 VII. Effects of Prolactin in Immature and Mature Male Rats on Testes and Accessories .. ..................... 88 VIII. Hypothalamic FSH—RF Content in the Female Rat Before and After Puberty .................................... 94 GENERAL DISCUSSION ......... .......................... 100 REFERENCES ........................................... 102 APPENDIX ............................................. 120 Method used for transforming Reece-Turner Units (RTU) into Intemational Units (IU). I OCOOOOOOOOOOOOOOI. 00000 120 Table l. Prolactin content of rat anterior pituitaries during the estrous cycle ............. . ............. . ............ 2- Effect of estrous cycle on pituitary prolactin release in vitro ......... . ............. .... ...... . ......... . ..... 3, PIF content of hypothalamus during the estrous cycle ..... 4. Prolactin concentration of anterior pituitaries (AP) from control, progesterone (P), testosterone propionate (TP) and cortisol acetate (CA) treated ovariectomized rats 5. Effects of progesterone, cortisol and testosterone on mammary lobulo-alveolar growth in ovariectomized rats 6. Hypothalamic PIF content of control, progesterone (P), testosterone propionate (TP) and cortisol acetate (CA) treated ovariectomized rats ............................. 7. Effects of diethylstilbestrol (DES) on body and organ weights in female Wistar rats ........................... 8. Effects of diethylstilbestrol implantation on mammary lobulo-alveolar growth in female Wistar rats ..... ....... 9. Assays of pituitary prolactin content in normal and DES implanted rats ............... . ..... . ............... ..... 10. Hypothalamic PIF content in normal and DES implanted rats 11. AP prolactin concentration after a single period of suckling in lactating rats ........... ................... 12. PIF content of hypothalamus during a single period of Suckling in lactating rats ... ..................... ..... 13. Pituitary CH content after a single period of suckling or non_SUCk1i—ng ‘00... OOOOOOOOOO OOOOOOOOO¢OOOOOOOOQ ...... 14. CH concentration of AP after a single period of suckling or non-suckling in lactating rats ....... .. ........... ... 15. Effect of a single period of suckling on pituitary TSH LIST OF TABLES concentration in lactating rats ..... ...... . ..... . ....... vi Page 30 31 33 39 4O 46 51 54 58 59 67 68 70 71 72 l6. l7. l8. 19. 20. 21. 22. LIST OF TABLES - continued Prolactin content of anterior pituitaries from control and thyroidectomized female Sprague-Dawley rats ......... PIF content of hypothalami from control and thyroidectomized female Sprague-Dawley rats .... ......... Effect of thyroidectomy and thyroxine on tumor growth and organ weights in female Wistar rats ........ . ............ Effects of prolactin on organ weights in neonatal male Sprague-Daney rats 0... 000000 00.000.00.0'000000000.0.0.00 Effects of prolactin on organ weights in mature male rats OOOOOOOOOOOOOOOOO D ..... 30.00. OOOOOOOOOOOOOOOOOO .00 FSH-RF content in the hypothalamus of immature female rats .0000 ..... O ...... 00.00.00.000. 0000000 .0. OOOOOOOOOOOO FSH-RF content in the hypothalamus of female rats before and after puberty .................. ...... ..... . ..... .... vii 77 78 85 9O 91 97 98 LIST OF FIGURES Figure Page 1. Ix) 10. 11. 12. 13. Photomicrograph of whole mount preparation of right inguinal mammary gland from control ovariectomized rat injected with corn oil ..... ............. ..... ...... ...... 41 Photomicrograph of a section of mammary gland from control ovariectomized rat injected with oil only ..... .......... 41 Photomicrograph of whole mount preparation of right inguinal mammary gland from ovariectomized rat injected with 10 mg progescerone daily for 21 days ..... .......... 42 Photomicrograph of a section of mammary gland from ovariectomized rat injected with 10 mg progesterone daily for 21 days ........... .......... ...... ..... ....... 42 Photomicrograph of whole mount preparation of right inguinal mammary gland from ovariectomized rat injected with 2 mg testosterone propionate daily for 10 days ..... 43 Photomicrograph of whole mount preparation of right inguinal mammary gland from ovariectomized rat injected with 2 mg cortisol acetate daily for 10 days ..... ....... 43 Photomicrograph of a section of mammary gland from ovariectomized rat injected with 2 mg testosterone propionate daily for 10 days ................. . .......... 44 Photomicrograph of a section of a mammary gland from ovariectomized rats treated with 2 mg cortisol acetate daily for 10 days ........................... ...... ...... 44 Photomicrograph of ovary from an intact rat after implantation of DES for 65 days .................. ....... 52 Photomicrograph of ovary from an intact rat after implantation of DES for 130 days ........... ......... .... 52 Photomicrograph of ovary from intact control rat (after 65 days) ....... . ...... . ................ . ................ 53 Photomicrograph of ovary from intact control rat (after 130 dayS) a900.000.00.0000000000000 oooooooooooooooooo ..-... 53 Photomicrograph of hyperplastic alveolar nodule from a mammary gland of a rat after 65 days of DES implantation 55 viii 14. 16. 17. 18. LIST OF FIGURES - continued Photomicrograph of hyperplastic alveolar nodule from a mammary gland of a rat after 130 days of DES implantation Photomicrograph of a whole mount preparation of right inguinal mammary gland from control rat (after 65 days) Photomicrograph of whole mount preparation of right inguinal mammary gland from control rat (after 130 days) in intact GrOWth of transplanted pituitary tumor MtTW fgyroxine control rats and intact rats injected with Effects of thyroxine on growth of mammary tumor in intact rats bearing an Mt'l‘w15 tumor ...... ...... ...... ix 55 57 57 84 84 Introduction Hypothalamic control of anterior pituitary function is well established. The hypothalamic mechanism is necessary to stimulate the secretion of all anterior pituitary hormones with the exception of prolactin which is chronically inhibited. A prolactin inhibiting factor (PIF) in the hypothalamus has been demonstrated. This factor has been shown to be reduced under several physiological conditions, wnich result in increased prolactin release, i.e. suckling, adminis- tration of estrogen, reserpine, etc- Demonstration of such changes helps in understanding the physiological role of PIF. It was of interest, therefore, to correlate alterations in prolactin secretion with changes in hypothalamic PIF content under different physiological states which had not previously been studied. An attempt was made to determine whether changes in hypotha- lamic PIF and pituitary prolactin levels could be detected during different stages of the estrous cycle. Since progesterone, testos- terone and cortisol had been shown to have no effect on pituitary prolactin release in 31339, it was of interest to see if their stim- ulatory effect on pituitary prolactin secretion in 3132 was mediated through the hypothalamus. Studies were also carried out to determine the effects of long-term implantation of diethylstilbestrol on pituitary growth, pituitary prolactin levels, hypothalamic PIF content and growth of mammary gland in intact female rats. The influence of a single suckling period during post partum lactation was determined on hypothalamic PIF content, and on pituitary 1 prolactin, CH and TSH content. In addition to above, the following problems were investigated: (1) the effects of thyroidectomy on hypothalamic PIF content and on pituitary prolactin content in female rats (2) the effects of thyroidectomy and thyroxine on growth of mammotropic (MtTWlS) and mammary (MtW) tumors in female Wistar rats (3) the effects of prolactin on the testes and accessory organs in neonatal and adult male rats. Finally, changes in FSH-RF activity in the hypothalamus of female rats before and after puberty were determined to evaluate the possible role played by this neurohormone in initiation of puberty. Review of Literature General Introduction It is now generally recognized that the central nervous system plays an essential role in the regulation of synthesis and release of anterior pituitary hormones- Marshall(l936, 1942) was probably the first to emphasize the part played by the nervous system in regulating anterior pituitary function, based on his observation that environ- mental stimuli could alter gonadotropin secretion. The concept of a neurovascular pathway between the hypothalamus and the anterior lobe of the pituitary was deveIOped from the work of Green and Harris (1947), Harris (1955) and others. The hypothalamic control of anterior pituitary function, unlike that of posterior pituitary function, is not mediated by a direct nervous connections. The link between the brain and the anterior pituitary is provided by the portal vessels. This portal circulation, first recognized by Popa and Fielding (1930), carries neurohormones from the median eminence region to the anterior hypOphysis. Green (1948) reported that the direction of blood flow in the portal vessels is downward from the median eminence to the anterior pituitary. Daniel and Prichard (1956) described "short and long" portal vessels in several Species, which supply blood to Specific regions of the pars distalis of the pituitary. The short portal vessels drain blood from the neural lobe proper into the anterior lobe whereas the long ones originate in the median eminence and stalk. The findings of Adams et a1. (1964, 1966) supports the theory that the neurohumors secreted by the hypothalamic nuclei are carried through blood in the portal vessels to one specific type of secretory cells of the pituitary gland, since particular groups of cells with similar functions are grouped to— gether in Specific vascularities of the hypophysis. I. Neural regulation gf adenohypophysial function. The secretion of all adenohypophysial hormones is thought to be influenced by hypothalamic neurohumors. The various methods employed to study hypothalamic regulation include electrical stimulation and lesioning of the CNS and hypothalamus, pituitary stalk section, pitu- itary transplantation to a site far removed from hypothalamus, hypo- thalamic lesions and in yitgg culture and incubation systems. Some investigators have also studied the effects of drugs and hormones on CNS and hypothalamus. Hypothalamic stimulation was found to result in increased discharge of luteinizing hormone (Harris, 1937). Markee et al. (1946) induced ovulation in 3 out of 4 rabbits by bipolar stimulation of the hypothalamus. Stimulation of the tuber cinereum resulted in an ovulatory response in the rabbit whereas direct pituitary stimulation did not, Suggesting that the pituitary gland lacks a secretomotor nerve supply (Harris, 1948). Subsequent studies on hypothalamic stimulation indicated that secretion of ACTH (De Groot and Harris, 1950; Goldfien and Ganong, 1962) and TSH (Harris and Wood, 1958) are also increased. Harris (1950) demonstrated that sectioning of the pituitary stalk of female rats results in a cessation of estrous cycle. He further presented evidence, using injections of India ink, that hypophysial portal vessels are able to regenerate after section of the pituitary stalk and begin to do so as soon as one day after operation. This regeneration can be correlated with the resumption of cyclic activity and can be prevented by interposition of an impermeable barrier between the pituitary and hypothalamus. This is direct evidence that gonadotropin secretion by the anterior pituitary of the rat is dependent on the hypothalamus (Harris, 1950). The earlier work on pituitary transplants was reviewed by Harris (1955). There is now considerable evidence that tranSplan- tation of the hypophysis to a distant site removed from the sella turcica shows very little functional activity. The gonads of the animals possessing hypophysial tissue without contact with the brain are atrophic. Everett (1956) and Desclin (1956) reported on the maintenance of the corpus luteum by the tranSplanted pituitary, indicating a considerable amount of luteotropin secretion from a pituitary tranSplant. Nikitovitch—Winer and Everett (1958a) ob- served maximum impairment of gonadotropin function and less pro- nounced impairment of ACTH and TSH release in animals bearing an autotransplant of the pituitary gland. On the other hand when pituitary grafts were retransplanted from the kidney to the median eminence, restitution of normal function appeared. Hertz (1959) rePorted that somatotropin production and release are not completely dependent upon an intact hypothalamic-pituitary system. Grafting of 4 pituitaries under the kidney capsule of the weanling, hypophy- sectomized rat, resulted in maintenance of subnormal body growth at about two-thirds of the normal rate. Meites and Kragt (1964) also observed body weight gains in young hypOphysectomized rats with a single subcutaneous pituitary tranSplant. It is now believed that limited growth occurs in animals bearing pituitary grafts removed from brain control. These observations further support the theory that neural control over the pituitary gland is mediated by a neurovascular link. Hypothalamic control of pituitary function has been studied by placement of lesions at different regions of the hypothalamus. Hypothalamic lesions were found to interfere with the secretion of all anterior pituitary hormones with the exception of prolactin secretion which is augmented (McCann and Friedman, 1960). Greer (1952) and Bogdanove and Halmi (1953) observed that certain hypothalamic lesions may prevent the hypertrophy of the thyroid that follows the administration of goitrogenic drugs. . The neurohumoral mediators regulating the secretion of ante- rior pituitary hormones are now referred to as "releasing factors". These neurohumors have all been partially purified from crude extracts of hypothalamus and appear to be polypeptides or amines in nature. Either crude acid extracts of hypothalamic tissue, including median eminence, or purified extracts, have been shown to stimulate release of anterior pituitary hormones in XEEEE (Saffron et a1. 1955; Schreiber et al., 1961; Deuben and Meites, 1964; Mittler and Meites, 1964; Piacsek and Meites, 1966) or in 2139 (Harris, 1960; Campbell et al., 1961, 1964; David et al., 1965; Vernikos-Danellis, 1965a; Meites and Fiel, 1965). LRF (Nallar and McCann, 1965) and FSH—RF (Negro—Vilar and Meites, 1967) have been demonstrated in the plasma of hypophysectomized rats. Recently, Fink et al. (1966) collected blood from the cut ends of the pituitary stalk of normal proestrous rats and hypophysectomized female rats and assayed for LRF activity. They found significantly greater LRF activity in hypophysial portal lihxxithanin the systemic blood of the same animals, as previously prOposed by Harris (1955). The identifications of each of these neurohumors in hypophysial portal blood together with their chemical characterization and purification will provide strong evidence for neuroendocrine control of pituitary function. 11. Hypothalamic regulation 9f prolactin secretion. Considerable evidence is now available that the primary influ- ence of the central nervous system (CNS) over prolactin secretion is inhibitory. This has been demonstrated by placement of hypothalamic lesions, pituitary stalk section, pituitary tranSplantation, culture or incubation of anterior pituitary tissue and treatment with a variety of CNS-depressent drugs. A "prolactin inhibiting factor" (PIF) has been demonstrated in hypothalamic extracts. A. Hypothalamic lesions. Prolactin secretion can be induced by certain hypothalamic lesions in the rat. Cross and Harris (1952) reported that a lesion in the supraoptico hypophysial tract of the median eminence caused a marked retardation of milk removal without disturbing the secretory activity of the mammary glands in rabbits. Similar results were also reported by Donovan and Van der Werff ten Bosch (1957) in lactating rabbits. In subsequent studies, Yokoyama and Ota (1959) demonstrated that lactating rats have the ability to secrete prolactin in the absence of suckling, but all pups died when allowed to suckle those mother rats bearing hypothalamic lesion due to lack of oxytocin and ACTH release. Median eminence lesions also resulted in a retardation of mammary involution following litter removal from lactating dams, indicating persistent prolactin secretion (McCann and Friedman, 1960). Gale (1963) and Gale and Larsson (1963) reported that median eminence lesion in goats resulted in a marked decline in milk production. Administration of a combination of ACTH, STH, triiodothyronine and insulin restored milk yields to pre-lesion levels; prolactin had no effect. These investigators concluded that median eminence lesions caused a decrease in release of all anterior pituitary hormones except prolactin. Recently hypothalamic lesions in the median eminence, hypo- physial stalk or paraventricular nuclei of male rats were shown to produce lobulo-alveolar development and secretion in the mammary gland (De Voe et al., 1966). Stalk section has also been shown to initiate milk secretion in cats (Grosz and Rothballer, 1961) and to maintain corpus luteum function in the ferret (Donovan, 1963). On the other hand, Beyer et al. (1962) found that lactation failed after placing lesions in the caudal hypothalamus of the cat. They con- sidered that afferent pathways for prolactin secretion might have been interrupted. In subsequent studies, Beyer and Mena (1965) demonstrated that this was in fact caused by failure of oxytocin secretion. B. Transplantation. Evidence that prolactin secretion can continue in the absence of a hypothalamic influence have been provided by tranSplantation of the adenohypophysis to a favorable site at a distance from the hypothalamus, and subsequent occurrence of a luteotrOphic action and pseudopregnancy. Desclin (1950) transplanted the anterior hypophysis into the kidney capsule, injected estrogen and observed luteotrophic function by the grafts. His interpretation was that estrogen caused luteotropin secretion by a direct action on the hypOphyseal tissue. Subsequent studies on auto-transplantation of the anterior hypophysis to the renal capsule by Everett (1954) confirmed Desclin's (1950) finding, and further demonstrated that luteotrophin secretion was independent of estrogen treatment. TranSplantation of the pituitary graft to the kidney capsule of the hypophysectomized rat resulted in an increase of prolactin secretion, in a marked decrease of gonadotropin secretion (Nikitovitch- Winer and Everett, 1958a) and in the maintenance of the corpora lutea in a functional state for several months (Everett 1956; Desclin 1956; Quflligan and Rothchild, 1960). The pars distalis was autotransplanted to the renal capsule in porestrous or estrous rats and retransplanted via the transtemporal route in close contact with median eminence after several weeks (Nikitovitch~Winer and Everett, 1958b). This resulted in resumption of a normal rate of gonadotropin secretion, and thyroid and adrenal functions returned to normal. The animals showed cyclic ovarian function and some animals became pregnant when‘ mated with fertile males. 10 Muhlbock and Boot (1959) found that subcutaneous isografts of single or multiple pituitaries in mice were able to change the short estrous cycle of mice to repetitive pseudopregnancies. Ahren (1961) demonstrated increased prolactin secretion by heterotopic pars distalis grafts in male rats. Zeilmaker (1962) also studied the luteotrophic action of pituitary grafts on tranSplanted ovaries in male rats. The pituitary grafts contained predominantly small acidOphilhzcells, the ; so-called prolactin cells (Sanders and Rennels, 1957), recently con— firmed by electron micrographic study (Rennels, 1962). Cytologically, progressive disappearance of gonadotrophs occurred during the first three weeks after tranSplantation, and thereafter they were rare and very small (Nikitovitch-Winer and Everett, 1959). C. Prolactin secretion in vitro. The hypothalamic mechanism acts to enhance the secretion of all anterior pituitary hormones with the exception of prolactin, which is chronically depressed. Since the transplanted pituitary secreted increased amount of prolactin £3.3323 when hypothalamic influence was removed (Everett, 1954), it was of intereSt to several investigators to study pituitary prolactin secretion 13 vitro. Utilizing tissue or organ culture techniques, Meites et al., (1961) and Pasteels (19613) simultaneously demonstrated secretion of prolactin by rat pituitary explants. Nicoll and Meites (1962a) cultured anterior pituitary explants from rats, mice, guinea pigs, rabbits and monkeys and dem- onstrated increased amounts of prolactin secretion into the culture medium as compared with the amounts found in the fresh pituitary ll tissue. Pasteels (1962b) obtained similar results in cultures from human pituitaries. In extensive studies, Nicoll and Meites (1962b, 1963, 1964) found that oxytocin, vaSOpressin, epinephrine, acetylcholine, testosterone, progesterone, and cortisol had no direct action on pitu- itary prolactin release, whereas addition of estrodiol,thyroxine or triiodothyronine to the culture medium significantly increased pro- lactin release into the medium. D. Prolactin inhibiting factor (PIE) Recent studies £3 31532 have demonstrated the presence of pro- lactin inhibiting factor (PIF) in the hypothalamus. Talwalker et al., (1963) clearly demonstrated in a series of experiments the existence of this factor in rat hypothalamic extract which inhibited prolactin synthesis and release by the anterior pituitary. Schally et a1. (1965) reported that the addition of hypothalamic extracts of bovine, ovine or porcine origin inhibited the release of prolactin. Pasteels (1962a) showed that the addition of an aqueous hypothalamic extract to anterior pituitary organ culture decreased the quantity of prolactin released into the medium. A pituitary-hypothalamus co-culture incu- bated under the same conditions released only 50% of the amount of prolactin produced by the pituitary alone (Danon et al., 1963). Grosvenor et al., (1965) provided 32.3339 evidence for the existence of a hypothalamic inhibiting factor in the normal regulation of prolactin release in response to nursing or stress. However, their 13 3332 assay for PIF has not been demonstrated to exhibit a log-dose response relationship. The suckling stimulus and estradiol (Ratner -_ 12 and Meites, 1964) reserpine (Ratner et al., 1965) epinephrine and acetylcholine (Mittler and Meites, 1967), and perphenazine (Danon et al., 1963) were found to decrease the content of prolactin in- hibiting factor in the hypothalamus of rats. Gala and Reece (1965) reported that epinephrine acts directly on the pituitary to stimulate prolactin release, but their results were not confirmed by others. It was earlier suggested that the same hypothalamic mechanism stimulates LH secretion and inhibits the secretion of prolactin (Everett, 1956). Rothchild (1960) presented evidence indicating that they are brought about by separate agents. Everett and Quinn (1966) recently demonstrated by techniques of brain stimulation that the area controlling pseudopregnancy in rats differed from that con- trolling ovulation. PIF has not yet been purified, but purified LRF preparations (Schally et al., 1964) have been reported to be devoid of PIF activity. However, Dhariwal et al. (1965) indicated that PIF overlaps with LRF upon gel filtration on Sephadex. III. Influence of suckling stimulus on_prolactin secretion Selye (1934) first demonstrated that suckling can maintain secretion in the mammary glands of lactating rats, even when milk removal from these glands is prevented by tying the galactophores. He postulated that prolactin was released from the anterior pituitary in response to stimulation of the teats during suckling. Selye and McKeown (1934) also reported that suckling of female cyclic rats by young litters resulted in prolongation of cycles, maintenance of functional corpora lutea and development of the mammary glands. l3 Mammary gland involution following removal of litters has been shown to be retarded in mice (Hooker and Williams, 1940) and rats (Mixner and Turner, 1941) by application of turpentine to the nipples. The duration of lactation can be considerably extended by supplying a succession of fresh litters to rats (Bruce, 1958, Nicoll and Meites, 1959), and milk yield can be increased by more frequent suckling and by increasing the number of young (Reddy and Donker, 1964). Changes in nucleic acid and phospho-protein content of mammary glands of lactating rats resulting from arrest of suckling have been determined, using phOSphOprotein content as an index of the amount of milk stored in the gland (Ota, 1964). Ota and Yokoyama (1965) investigated the influence of the suckling stimulus on the resumption of lactation in lactating rats whose litters had been removed 3, 5 or 9 days previously. They observed restoration of lactation in all groups by renewed suckling within a relatively short period. The restoration of lactation became more difficult as the interval between the removal of the original litters and the application of foster litters increased. Stimuli associated with suckling or milking are essential for the maintenance of lactation, and the intensity of the suckling stimulus has a quantitative effect on maintenance and proliferation of the secretory epithelium (Moon, 1965). Folley and French (1949) reported that mammary involution after arrest of suckling resulted from both a depression of circu- lating levels of lactation maintaining hormones secreted from the anterior pituitary, and accumulation of milk in the mammary gland. 14 There is also considerable evidence that suckling stimulates pro- lactin secretion in lactating rats. The stimulus of suckling after a period of non-suckling reduced the prolactin content of the anterior pituitary of the rat (Reece and Turner, 1937). Regular suckling increased the pituitary prolactin content as compared with non-suckling in postpartum rabbits (Meites and Turner, 1948). A 90% reduction in pituitary prolactin content in lactating rats after 30 minutes suckling, following a 10 hour non—suckling period, was reported (Grosvenor and Turner, 1958a). However, other workers have been unable to confirm these results in other strains of rats (Meites and Nicoll, 1966). Mechanical stimulation of the cervix has been shown to deplete pituitary prolactin content in the rat (Herlyn et al., 1965). The suckling stimulus plays an important role in maintaining lactation by regulating the secretion of both anterior and posterior pituitary hormones. This stimulus induces release of prolactin, oxytocin and ACTH and perhaps other hormones favorable to lactation (Meites and Nicoll, 1959). This appears to be mediated through the suppression of an inhibitory hypothalamic center on the secretion of prolactin, and perhaps by activating another center to enhance the release of ACTH (see Meites, 1966). Evidence for increased ACTH secretion during Suckling is seen in the involution of the thymus in suckled female rats (Gregoire, 1947), in reduction of adrenal ascorbic acid (Tabachnick and Trentin, 1951) and by depletion of pituitary corticotropin (Denamur et al., 1965). Depletion of GH from the pituitary has also been reported 15 during suckling (Grosvenor, 1964a). Thyroid secretion rates are reported to be greater in lactating than in non-lactating rats (Grosvenor and Turner, 1958b), although no evidence is available for a suckling induced elevation in the rate of TSH secretion (Averill, 1966). There is evidence that Suckling Suppresses gonadotropin secretion and enhances prolactin secretion by depressing the gonadotropin releasing factors and PIF in the hypothalamus. Gona- dotropin secretion is depressed markedly during lactation and to a moderate extent during pseudopregnancy in the rat and mouse (Greenwald, 1962, Rothchild, 1960). Minaguchi and Meites (1967a) have recently demonstrated that LH content in the pituitary and the hypothalamic content of LRF are significantly decreased in lactating rats as compared with control non-lactating rats. Since suckling stimulates the nerve endings in the nipples, it is assumed that its action is mediated through a neural circuit ending in the central nervous system. The importance of neural pathways in the suckling stimulus of rats was emphasized by the work of aners and Baddeley (1957). The suckling stimulus has been shown to deplete hypothalamic PIF content in the rat (Ratner and Meites, 1964; Minaguchi and Meites, 1967a), thereby increases pituitary prolactin release. Using an organ culture technique Gala and Reece (1964b) reported that the PIF activity in aqueous hypothalamic extracts from rats killed in mid- lactation did not show any difference from that in extracts from non— lactating rats. However, they have not shown that PIF activity can 16 be quantitatively measured by their method. Grosvenor (1965), using an ig_yiyg method for assaying PIF activity in the hypothalamus, also did not find any alteration in hypothalamic prolactin inhibiting activity of rats during 30 minutes suckling as compared to nonsuckled controls. However, his in_yiyg method of assaying PIF has not been shown to be either specific or quantitative. He was unable to detect a 3-fold difference in concentration of PIF by his method. The evidence presented above favors the hypothesis that secretion of pro- lactin during lactation is maintained by a suckling induced depression of hypothalamic inhibition, i.e., of PIF production. IV. Effects of steroids on prolactin secretion A. Estrogen It has been established that estrogen can stimulate pituitary prolactin release and induce mammary development and lactation. In- jection of estrogen into female rats increases pituitary prolactin content (Reece, 1938) and promotes degranulation of acidophils. Desclin and Koulischer (1960) demonstrated that prolactin content in hetero- tropic tranSplanted pituitary increased by 31% over control values after injection of estrogen. More recently Strong evidence for a direct action of estrogen on the pituitary was reported by Nicoll and Meites, (1962b) in vitro studies of tissue culture of rat adenohypophysis slices. They found increased prolactin release into the medium by cultured pituitary when estradiol was incorporated. These results were confirmed by Ben-David et al. (1964), but both Gala and Reece (1964a) and Pasteels (1963) were unable to Show a direct action of 17 estrogen on the pituitary 13 vitro to increase prolactin secretion. Further evidence of a direct action of estrogen on prolactin release was presented by Mizuno et a1. (1964), who found that injection of estradiol increased prolactin release into the blood of rats bearing a pituitary "mammotropic tumor” known to secrete large amounts of prolactin. Ratner et al. (1963) reported that anterior pituitary from estrogen treated rats released greater amounts of prolactin into the medium than those of control untreated rats during a two-hour incubation. Kanematsu and Sawyer (1963) made implants of estradiol benzoate into posterior tuberal region of the hypothalamus and anterior pitu- iary of rabbits. They found that intra-hypothalamic implants of estrogen caused an increase in pituitary prolactin content whereas similar implants into the pituitary stimulated prolactin release. In rats, estrogen implants have been shown to promote prolactin re- lease (Ramirez et al., 1963). Ratner and Meites (1964) demonstrated that hypothalamic extract from estradiol-treated rats had no ability to inhibit pituitary prolactin release by rat pituitary upon incu- bation, whereas hypothalamic extracts from control cycling rats reduced prolactin release by the anterior pituitary. These authors concluded that estradiol or the suckling stimulus can completely deplete the PIF content in the hypothalamus. B. Testosterone (Androggns) The increased content of pituitary prolactin by the administration of progesterone, testosterone and cortisol is not mediated by a direct action of these steroids on prolactin release (Meites and l8 Nicoll, 1965). When normal cyclic female rats were injected with testosterone propionate daily for 10 days, large corpora lutea were observed (McKeown and Zuckerman, 1937). Wolfe and Hamilton (1937) reported that the vaginal epithelium was markedly mucified in female rats treated with male sex hormone. Deciduoma formation in rats during testosterone treatment has been shown to occur (Fluhmann and Laqueurgl942). These authors suggested that deciduoma formation is due to an increased production of prolactin by the anterior pituitary from testosterone administration. Their suggestion was further supported by the observation that testosterone induces mammary growth only in animals with an intact pituitary. Reece and Mixner (1939) demonstrated that daily injection of 200 ug of testosterone prOpionate for 15 days into mature castrated female rats produces a 40% augmentation of pituitary prolactin content, no change in pituitary weight and extensive development of the lobulo- alveolar system of the mammary gland and secretion. Pasteels (1961b) used pituitary cytology as a criterion, and compared the effects of an androgen with those of reserpine and found identical changes in the pituitary. He suggested a common controlling mechanism for both substances. Alloiteau (1959, 1960) demonstrated that the "estrogenic" action of testosterone on the corpora lutea of rats was maintained under the influence of prolactin. C. Progesterone In vivo experiments have shown that progesterone can increase pituitary prolactin content and initiate mammary secretion in rats. 19 Large doses of progesterone have been shown to produce a moderate increase in pituitary prolactin content in rats and guinea pigs (Meites and Turner, 1948) and initiate mammary secretion in ovariectomized mature femalerats (Reece and Bivins, 1942). Alloiteau and Vignal (1958) reported that a single treatment of progesterone can induce pseudopregnancy in rat. It has been Shown that a single injection of 10 mg progesterone on the day of estrus or 5 daily injections of 2 mg beginning on the day of estrus can induce pseudopregnancy of about 2 weeksduration (Everett, 1963). Daily injection of progesterone in amounts large enough to suppress estrus results in the persistence of large corpora lutea whose size and histologic appearance indicate functional Status (Phillips, 1937). Rothchild (1960) reported that increasing the daily dosage to 10 mg of progesterone does not produce any detectable damage to the corpora lutea. 0n the other hand, lactating rats after removal of their litters showed good lactation for at least 28 days with progesterone treatment (Rothchild, 1965). From these observations he suggested that progesterone prolongs the secretion of prolactin. Progesterone may inhibit galactopoiesis when given together with estrogen (Meites et al., 1963). Progesterone did not stimulate pituitary prolactin release 13.31539 (Meites and Nicoll, 1965) indicating no direct action on the pituitary. Rothchild (1960) proposed that the progesterone- prolactin relationship in the rat is that of a positive feedback mechanism, and that progesterone probably depresses CNS inhibition to stimulate pituitary prolactin secretion (Rothchild, 1965). Ben- 20 David et a1. (1964) reported that progesterone had no effect on hypothalamic inhibition of prolactin release by rat AP, when added to a pituitary-hypothalamus co-culture. There is some indirect evidence that progesterone influences prolactin secretion by acting through a hypothalamic mechanism (Barraclough and Cross, 1963). Ralph and Fraps (1960) reported that progesterone can influence gonadotropin release by acting though the hypothalamus. D. Cortisol_(§lucocorticoids) It has been reported that the secretion of both prolactin and ACTH are promoted under conditions of stress (Swingle et al., 1951; Nicoll et al., 1960), estrogen treatment (Gemzell, 1952; Meites and Turner, 1948) and the suckling stimulus (Gregoire, 1947). More recently Kitay (1964) has shown that administration of estradiol can overcome the depressing effects of cortisone on ACTH secretion as evidenced by its ability to prevent the reduction in pituitary ACTH concentration and inhibition of stress-induced ACTH release produced by cortisone. He further pointed out that a lesser degree of pituitary suppression is produced by cortisone in intact females than in males, and significantly greater inhibition can be achieved in female rats following ovariectomy. Although it was originally thought that the pituitary was the primary site of corticoid feedback inhibition, the more recent evidence suggests that corticoids act on the hypothalamus rather than on the pituitary gland itself (Vernikos-Danellis, 1965b). Smelik and Sawyer (1962) demonstrated that the greatest inhibition to the stress-induced 21 increase in blood corticoid levels was obtained with hydrocortisone implants into the median eminence and post-optic region, and to a lesser degree with implants between the supraoptic and paraventricular nuclei and the median eminence. They suggested that the blocking action of hydrocortisone is exerted on the production and release of corticotrOpin releasing factor (CRF). Their suggestion has been supported in the rat by the observation that a blocking dose of hydrocortisone which does not affect pituitary ACTH content leads to disappearance of the corticotrOpin releasing activity of the median eminence (Vernikos-Danellis, 1965b). There is some evidence that adrenalectomy reduces mammary growth in rats (Trentin and Turner, 1947). Adrenalectomy on the 4th day of lactation caused a marked inhibition of lactation (Cowie and Folley, 1947), but DCA or glucocorticoids could restore milk secretion to a considerable degree (Folley, 1952). Meiteset al. (1942) reported that the reduction in pituitary prolactin content following adrenalectomy may be due to a direct inhibitory effect on the pituitary and also to a reduction in food intake. ‘Turner and Meites (1947) observed that DCA did not increase pituitary prolactin content in female rats. However, Johnson and Meites (1955) reported that cortisone and hydrocortisone increased pituitary prolactin content by 23 and 41% reSpectively, and induced mammary growth and secretion in intact female rats. These authors suggested that the increase in pituitary prolactin content produced by glucocorticoids may have a role in the initiation and maintenance of lactation. Cortisol can stimulate mammary gland development in adren- 22 alectomized and ovariectomized rat (Selye, 1954). When hydrocortisone was given in combination with estradiol to these rats, marked mammary growth and secretion were observed. Nicoll and Meites (1964) cultured rat anterior pituitary slices with hydrocorticosterone and corticosterone to determine the direct action of these Steroids on the anterior pituitary. Cortisol markedly reduced the quantity of prolactin re- leased in the medium whereas corticosterone had no effect. The effects of cortisol were not considered to be of physiologic significance, since the doses used in vitro were very large. They suggested that glucocorticoids 32.3339 may stimulate prolactin secretion through the hypothalamus or by other mechanisms. H ~_.... _.._ Materials and Methods 1. Animals Mature male and female Sprague-Dawley rats from Spartan Animal Farms (Haslett, Michigan) and Holtzman Company (Madison, Wisconsin) were used in all experiments. The animals were housed in a temperature controlled (75 i 10F) and lighted (14 hrs/day) room. The animals were maintained on a diet of Wayne Lab Blox (Allied Mills, Chicago, Illinois),and tap water. Female hypophysectomized rats, 26-28 days olds, were obtained from Hormone Assay Labs, Chicago, Illinois, for growth hormone assays. Female Swiss-Webster albino mice (Spartan Animals Farms, Haslett, Michigan), approximately lO-15 g body weight were used for pituitary thyroid stimulating hormone (TSH) assay. White King-Squabs (Cascade Squab Farm, Grand Rapids, Michigan), 4 to 8 weeks old, were used for prolactin assays. 11. Incubation Technique Rats used in these experiments were killed quickly by guillotine, and the anterior pituitaries and the hypothalami were removed quickly. The posterior lobe was discarded and the anterior pituitary was weighed, frozen and stored at -200C until assayed. The hypothalamus and median eminence were collected in chilled 0.1N HCl (10 hypothalami/ 2 ml) and frozen at ~200C until used for incubation a few days later. A. Preparation of Acid Extract of Hypothalamus On the day of incubation the hypothalami were thawed and 23 24 homogenized in a ground glass homogenizer, and centrifuged at 12,000 g for 40 minutes at 40C. The supernatent was placed in protein free medium 199 (Difco Lab, Detroit, Michigan), and the pH was adjusted to 7.4 by adding drop by drop 1N NaOH and testing with glass electrodes. The hypothalamic extract was neutralized just before use. B. Incubation Adult male rats of the Sprague-Dawley strain were killed by guillotine and the posterior pituitaries were discarded and the anterior pituitaries were removed immediately and placed in a Petri dish over moistened filter paper. Each anterior pituitary was hemisected, one-half being placed in a control flask (25 ml Erlen- meyer) containing 2 ml of medium 199 and the other half in an ex- perimental flask. The equivalent of 2 or 3 anterior pituitaries (4 or 6 halves) was placed into each flask. The neutralized acid extract of hypothalami (equivalent of 1 or 2 hypothalami per in- cubated pituitary) was added rapidly to each flask. Incubations were carried out in a Dubnoff metabolic shaker (60 cycles/min) under constant gassing with 95% 02-5% CO2 at 37 : 0.50C for 4 hours. At the termination of the incubation, the pituitary halves were weighed and the medium was centrifuged at 2,500 g for 10 minutes and the supernatent was frozen at -ZOOC and stored until assayed. 25 III. Bioassays A. Prolactin The medium from incubation was assayed for prolactin activity by the intradermal pigeon-crop technique of Lyons (1937) as modified by Reece and Turner (1937). A dose of 0.1 or 0.2 ml of medium was injected daily for 4 days into each bird. A direct comparison was made between samples by injecting one sample over one side of the crop sac and the other sample over the other side of the crop of the same bird. Anterior pituitaries were homogenized in normal saline (0.9% NaCl Soln) and assayed for prolactin. Each injection was in a 0.1 ml volume and an equivalent of one fourth pituitary was injected daily into each bird for 4 days. The prolactin responses in each bird were expressed in Reece- Turner units (RTU) and in some cases these units were converted into International Units (IU) by use of a standard dose response curve established in the same breed of pigeons with NIH prolactin (Nicoll and Meites, 1963). B. Growth Hormone_(GH) Immature female Sprague—Dawley rats (Hormone Assay Labs, Chicago, Illinois) were hypophysectomized at 26-28 days of age and shipped to us 10 days later. Four days later they were used for assay of growth hormone content of the anterior pituitary by the standard tibia test of GreenSpan et al. (1950). The anterior pituitaries were homogenized with normal saline (0.9% NaCl) and the homogenate was adjusted to pH 9.6. Each rat was injected intraperitonially for 4 days in 0.2 m1 26 volume and the rats were killed on the 5th day. The diet of hypophysectomized rats was supplemented daily with orange slices, carrots and sugar cubes. C. Thyroid Stimulating Hormone_(TSH) The anterior pituitaries were assayed for TSH by the McKenzie assay (McKenzie, 1958) in Swiss-Webster albion female mice (Spartan Animal Farms, Inc., Haslett, Michigan). The mice were maintained on a low iodine diet for 10 days. Then 1.5 ug of 1131 was injected intraperitonially followed by subcutaneous injection of 10 pg L- thyroxine. The low iodine diet was replaced by a diet containing thyroid powder. Three days later the animals were available for assay. The animals were bled from the jugular instead of the tail vein. Two samples of blood, each 0.2 ml, were collected before and after the injection of the test material. In each experiment 2 doses of standard TSH (NIH-TSH-B3) and 2 doses of unknown samples were injected at the same time. The radio activity of each sample was determined by counting the sample for 10 minutes in a Mark 1 liquid scintillation counter (Nuclear:-Chicago). IV. Histological preparations The tissues were fixed in Bouin's fluid and sectioned at 6 u for histologic examination. The sections were stained with hematoxylin and eosin. The right inguinal mammary glands were removed and fixed in Bouin's fluid for whole mount preparation. The standard procedure of staining with Mayer's hematoxylin was followed and the glands were 27 rated for degree of growth as described previously (Talwalker and Meites, 1961): I. Few ducts; few or no end buds. II. Moderate duct growth; moderate number of end buds. III. Numerous ducts and branChes; many end buds. IV. Numerous ducts and branches; moderate lobulo- alveolar growth. V. Numerous ducts and branches with dense lobulo- alveolar growth as in mid or late pregnancy. V. Statistical Analysis Statistical analysis for prolactin was performed by the "t" test for paired observations. Bioassays for growth hormone and TSH were analysed according to Bliss (1952). Relative potencies, 95% confidence limits, parallelism and index of precision (A) were cal- culated as described by Bliss (1952). Combined standard deviations of all responses and the combined slope were considered in the analysis. EXPERIMENTAL EXP. I. CHANGES IN PITUITARY PROLACTIN RELEASE AND HYPOTHALAMIC PIF CONTENT DURING THE ESTROUS CYCLE OF RATS. Objective: In randomly cycling rats, considerable variability has been observed in pituitary prolactin content, (Nicoll and Meites, 1962b) pituitary prolactin release in yiggg, and in hypothalamic prolactin inhibiting factor (PIF) content (Talwalker et al., 1963; Ratner and Meites, 1964). These variations may be due to differences in secretion of the gonadal hormones during each stage of the estrous cycle. Changes during each phase of the estrous cycle of rats have been observed in pituitary LH concentration (Schwartz and Bartosik, 1962), plasma LH content (Ramirez and McCann, 1964a) and hypothalamic content of luteinizing hormone releasing factor (LRF)(Ramirez and Sawyer, 1965a). It was of interest therefore, to determine whether differences could be detected during each stage of the estrous cycle of rats in pitu- itary prolactin content, pituitary prolactin release 12 yiggg and in hypothalamic PIF content. Materials and Methods Mature female Sprague-Dawley rats (Spartan Animal Farms, Inc., Haslett, Michigan) were housed in a temperature (75 : 10F) and light (14 hrs/day) controlled room. Vaginal Smearswere taken each morning between 9-10 A.M. beginning one week after arrival of the rats. Only rats which had exhibited at least two regular cycles of 4 or 5 day 28 29 lengths were used in this study. At each stage of the estrous cycle (except metestrus), the rats were killed by guillotine, and the anterior pituitaries and hypothalami were quickly removed. The posterior lobe was discarded and the anterior pituitary was weighed, frozen and stored at -200C until assayed. The hypothalamus and median eminence were collected in chilled .lN HCl (10 hypothalami/2 ml) and frozen at -200C until assayed a few days later. Pituitaries were assayed for prolactin and hypothalami were assayed for PIF activity as described previously (p. 23). A 2 hour incubation was used to measure prolactin release by the anterior pituitaries of rats in different stages of the cycle. Results Pituitary prolactin content during the different phases of the estrous cycle are shown in Table l. Prolactin is expressed in Reece- Turner units (RTU) per pituitary gland in all experiments. In Exper- iments l and 2, pituitary prolactin content of proestrous and estrous rats were compared with pituitary prolactin content in diestrous rats. Prolactin in the pituitaries of the proestrous (Exp. 1) and estrous rats (Exp. 2) were significantly greater, by 64 and 71% reSpectively, than in the pituitaries of the diestrous rats. No significant difference in pituitary prolactin content was observed between the proestrous and estrous rats (Exp. 3). Prolactin release 13 vitro during a 2 hour incubation by the pituitaries of rats in different stages of the cycle are shown in Table 2. Prolactin release from the pituitaries of proestrous and estrous 3O sunglasses. “oz + Ho. V m new mo. V m «a >wmuflauwa uofiumucm\muwas umGuSH momma u m<\:Hm a ¢.o H w.q o H.m_H H.qHN monumm NH +m m> Co + me a ma H CNN 25:85 e m To H a: a BN H «HS .5388 2 acts: I m> «.0 + m.¢ o H.m + H.¢HN wsuumm NH N m6 H 5N A Wm H mama 3:88 *kqo .l .l we m.o + m.q m m.H + m.NNN monummoum o H ouamummmwn sAm4\:Hmv mcommam w mmmzm mumm .02 N fiwuomaoum MO .02 ufimeS hwom mfiumo wo .OZ .mxm mquwo mzomrwmm mm; UZHmDQ mMHm o.o + o.N o monummoum m H sam<\:eev monoummmma powwoamm mnowwem mmmsm mumm .02 N cwuomaoum mo .oz macho mo .02 .axm oeeH>.mm amendme anuauomm smaeneeaa zo maoso msomemm so Humane . N mqmfiH 32 rats was significantly greater, by 55 and 54% reSpectively, than from pituitaries of diestrous rats. The hypothalamic PIF contents in proestrous, estrous and diestrous rats are seen in Table 3. Anterior pituitary halves incu- bated with hypothalamic extract from proestrous rats released an average of 84% more prolactin than the corresponding anterior pitu- itary halves incubated with hypothalamic extract from diestrous rats (Exp. 1). Anterior pituitary halves incubated with hypothalamic ex- tract from estrous rats (Exp. 2) released an average of 42% more pro- lactin into the medium than by the corresponding pituitary halves incu- bated with hypothalamic extract from the diestrous rats. No differences in prolactin release were observed when anterior pituitary halves were incubated with hypothalamic extracts from proestrous and estrous rats (Exp. 3). Discussion These results Show that anterior pituitaries of proestrous and estrous rats contain significantly more prolactin than anterior pituitaries of diestrous rats, that the former release significantly more prolactin when incubated 12 vitro than the latter, and that proestrous and estrous rats have significantly less PIF in the hypothalamus than diestrous rats. The findings on pituitary prolactin content are in agreement with a previous study showing that the pituitaries of proestrous and estrous rats and guinea pigs contain more prolactin than the pituitaries of diestrous rats and guinea pigs (Reece and Turner, 1937; Reece, 1939). The Stimulus from ovarian 33 %HmanuHa woumssoaH ma commoaou aHuomHoum mo wEuou SH uamudoo mHm oHBmHm£uomhn mouonwnH H Hoo. 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