EFFECTS OF EXOGENOUS PROLACTIN ANDGRpwm ._ ~- :2. *- HORMONE ON THEIR SECRETION BYVTIHE * V j PITUITARY AND ON OTHER HORMO’NES I I Thesis for the Degree of Ph. D. I MICHIGAN STATE UNIVERSITY I JAMES LEONARD VOOGT 1970 L I B R A R Y Michigan State University I'Hf-‘Sik This is to certify that the thesis entitled EFFECTS OF EXOGENOUS PROLACTIN AND GROWTH HORMONE ON THEIR SECRETION BY THE PITUITARY AND ON OTHER HORMONES presented by James Leonard Voogt has been accepted towards fulfillment of the requirements for PhoDo degree in Ph231010gy Date 9/14/70 0-169 ABSTRACT EFFECTS OF EXOGENOUS PROLACTIN AND GROWTH HORMONE ON THEIR SECRETION BY THE PITUITARY AND ON OTHER HORMONES BY James Leonard Voogt 1. Changes in serum and anterior pituitary levels of prolactin were determined in female rats from age 21 days until maturity. Serum prolactin levels rose sharply on the day of vaginal opening whereas pituitary prolactin levels increased significantly soon after puberty. Daily in- jections of 0.01-5.0 ug of estradiol benzoate for 4 days beginning at 26 days of age (prior to puberty) significantly increased both serum and pituitary prolactin levels above those in similar rats injected with corn oil. These re- sults suggest that the sharp increase in serum prolactin levels on the day of vaginal opening and subsequent estrous phases of each cycle is due to estrogen secretion. 2. Serum and pituitary levels of prolactin and luteinizing hormone (LH) were measured during the estrous cycle in 3-month-old female rats and in old female rats in constant estrus or repeated pseudopregnancies. Serum pro- lactin and LH were low during metestrus and diestrus. A James Leonard Voogt sharp rise in serum LH and prolactin was noted during the late afternoon of proestrus. Thereafter serum LH decreased rapidly, but serum prolactin remained relatively high dur- ing the morning of estrus. Pituitary prolactin and LH decreased during the afternoon of proestrus. Serum pro— lactin levels in old constant estrous rats were similar to prolactin levels during estrus. Old rats showing repeated pseudopregnancies had serum prolactin levels similar to young mature rats in diestrus. Serum LH levels were about twice as high in constant estrous old rats as in old pseudopregnant rats. Both these levels in old rats are very low as compared to levels during proestrus in young mature rats. These results show that there is great fluctuation in serum and pituitary prolactin and LH during the estrous cycle, with peak levels of these hormones being found in the serum during the late afternoon of proestrus. The consistently high prolactin levels in the old constant estrous rats are probably due to persistent estrogen secretion, and may play a role in the onset of spontaneous mammary tumors. 3. The effect of an implant of prolactin in the median eminence (ME) of cycling female rats on serum pro- lactin and LH during the cycle, especially on the after- noon of proestrus, was measured. Serum prolactin was significantly lower during estrus after the ME implan- tation of prolactin. The proestrous prolactin peak in rats implanted with prolactin on the morning of proestrus James Leonard Voogt was completely inhibited as compared to controls. Serum LH was increased during both diestrus and estrus following the ME implantation of prolactin. These results support the hypothesis that prolactin acts back on the hypothalamus to inhibit its own release, and to stimulate LH release. 4. The effects of an implant of prolactin in ME of pseudopregnant (PP) rats on serum and pituitary LH, FSH, and prolactin was measured. In the PP rats implanted with prolactin, 28 of the 31 rats came into estrus 2 or 3 days later whereas 26 of 28 rats implanted with cocoa butter remained PP. Serum LH and FSH increased significantly following ME implantation of prolactin, whereas serum pro- lactin remained unaffected. Pituitary prolactin and FSH decreased significantly in the prolactin implanted rat. Mammary gland development was greatly decreased in these rats, and a definite stimulation of ovarian follicular development and uterine epithelial and endometrial layers was noted. These results demonstrate that ME implants of prolactin inhibit prolactin secretion and stimulate LH and FSH release. 5. The effects of ME implant of prolactin on serum prolactin following suckling in postpartum rats was studied. Control lactating rats with an ME implant of cocoa-butter had serum prolactin levels greater than 300 ng/ml serum following a 1-hour suckling period. Rats implanted with prolactin had serum prolactin levels less than 50 ng/ml serum following 1-hour of suckling. This large reduction James Leonard Voogt in serum prolactin was paralleled by decreased lactation, evidenced by reduced litter weight gain. These results indicate that the suckling stimulus is not capable of over- coming the inhibitory effect of an ME implant of prolactin on pituitary prolactin release. 6. ME implants of human and ovine growth hormone (GH) decreased pituitary GH concentration in cycling female rats. Rats implanted with either human or ovine GH also had reduced hypothalamic content of growth hormone releas- ing factor (GHRF), indicating that the mechanism of in- hibitory feedback of GH on its own secretion was by reduc- ing GHRF content in the hypothalamus. Serum prolactin levels were reduced in rats implanted with human GH and the mammary glands were atrOphied. Thus human GH inhibits both GH and prolactin release, presumably because it con- tains the activities of both hormones in its molecule. EFFECTS OF EXOGENOUS PROLACTIN AND GROWTH HORMONE ON THEIR SECRETION BY THE PITUITARY AND ON OTHER HORMONES BY James Leonard Voogt A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1970 Dedicated to my wife Mary ii ACKNOWLEDGMENTS The author wishes to express thanks to many pe0ple who collectively made it possible to execute these studies and prepare this thesis. Dr. Joseph Meites deserves utmost gratitude for his inspiration and guidance during the four years spent here. Most importantly, the author was able to observe first hand a scientific research philosophy that one could do well to emulate. The members of the guidance committee who willingly counseled the author in preparing this thesis and other matters deserve recognition: Drs. W. D. Collings, H. Hafs, T. W. Jenkins, E. P. Reineke, and A. J. Morris. Thanks are also extended to Drs. C. L. Chen and J. A. Clemens, both of whom collaborated with the author in some of these studies. The technical assistance from Mrs. Claire Twohy, Miss Janet Hoppough, and Mr. Eldon Cassell, especially in carrying out many radioimmunoassays, is deeply appreciated. The willingness of Miss Pam Duke to tyPe many letters, orders, and manuscripts, including the .initial draft of this thesis, is sincerely appreciated. The author also wishes to thank the Department of Physiology of Michigan State University and the National iii Institutes of Health for the predoctoral traineeship and fellowship which were granted from September, 1966 until the completion of these studies. The author offers many thanks to his wife Mary, who helped make these graduate years a period of time richer and more worthwhile than ever could have been experienced alone. iv LIST OF TABLES . . . . . . . . . . . TABLE OF CONTENTS LIST OF FIGURES. . . . . . . . . . . . . INTRODUCTION. . . . . . . . . . . . . . REVIEW OF LITERATURE O O O O C C O O O C O I. II. III. Anatomy of the Hypothalamo-Pituitary Axis . A. Anatomy of the Hypothalamus. . . . . B. Anatomy of the Hypothalamo-Pituitary Connections . . . . . . . . . . Physiology of the Hypothalamo-Pituitary AXiSo O O I O O O O O C C O O O A. Releasing and Inhibiting Factors of the Hypothalamus . . . . . . . . B. Feedback of Hormones on the Hypothalamo- Pituitary Axis . . . . . . . . . Prolactin, LH, and FSH Levels During Different Physiological States . . . . . A. Prolactin, LH, and FSH Levels Before and During Puberty. . . . . . . . B. Prolactin, LH, and FSH During the Rat Estrous Cycle . . . . . . . . . C. Prolactin, LH, and FSH During Pseudo- pregnancy in the Rat . . . . . . . D. Prolactin, FSH, and LH During Lactation. E. Prolactin, LH, and FSH Following Castration . . . . . . . . . . F. Prolactin, LH, and FSH in Old Female Rats . . . . . . . . . . . . G. Prolactin, FSH, and LH in Males . . . 21 22 23 24 25 26 27 29 MATERIALS AND METHODS . . . I. II. III. IV. V. VI. Animals . . . . . Stereotaxic Technique Preparation and Implantation of Hormones . I2_Vitro Incubation Technique. . . . . Assays of Pituitary Hormones . . . . . A. Bioassay . . . B. Radioimmunoassay. Methods of Statistical Analysis . . . . EXPERIMENTAL . . . . . . I. II. III. IV. Serum and Pituitary Prolactin Levels Before, During, and After Puberty in Female Rats . A. Objective . . B. Materials and Methods C. Results. . . . D. Discussion. . . Serum and Pituitary Prolactin and LH During the Estrous Cycle, Following and During Lactation. A. Objective . . B. Materials and Methods C. Results. . . . D. Discussion. . . Serum Prolactin and LH in A. Objective . . B. Materials and Methods C. Results. . . . D. Discussion. . . Effect of Median Eminence Prolactin on Serum Prolactin the Estrous Cycle. . A. Objective . . B. Materials and Methods C. Results. . . . D. Discussion. . . vi Ovariectomy, Implantation of and LH During Page 30 30 30 34 36 38 38 39 45 46 46 46 46 48 54 59 59 60 60 64 67 67 67 68 68 72 72 72 74 80 V. Effects of an Implant of Prolactin in Median Eminence of Pseudopregnant Rats on Serum and Pituitary LH, FSH, and Prolactin . . . . A. Objective . . . . . . . . . B. Materials and Methods . . . . . . C. Results . . . . . . . . . . . D. Discussion . . . . . . . . . . VI. Inhibition of Lactation and Serum Prolactin Postpartum Rats by Median Eminence Implan- tation of Prolactin . . . . . . . . A. Objective . . . . . . . . . B. Materials and Methods . . . . . . C. Results . . . . . . . . . . . D. Discussion . . . . . . . . . . VII. Pituitary GH and Hypothalamic GHRF After Median Eminence Implantation of Ovine and Human GH . . . . . . . . . . . . A. Objective . . . . . . . . . . B. Materials and Methods . . . . . . C. Results . . . . . . . . . . . D. Discussion . . . . . . . . . . GENERAL DISCUSSION . . . . . . . . . . . REFERENCES 0 O O O O O O O O O O O O 0 APPENDIX 0 O O O O O O O O O O O O 0 vii in Page 85 85 85 89 99 101 101 101 102 109 112 112 112 115 124 128 135 150 Table 10. 11. LIST OF TABLES Organ Weights and Serum and Pituitary Pro- lactin Levels Before, During, and After the Onset of Puberty . . . . . Organ Weights and Serum and Pituitary Pro- lactin Levels of 30-Day-Old Female Rats Following Estradiol Benzoate Administration Serum Prolactin and LH in Old Rats . Effect of ME Implant of Prolactin on Serum Prolactin During the Estrous Cycle Effect of ME Implant of Prolactin on Serum LH During the Estrous Cycle. . . . Effect of ME Implant of Prolactin on Prolactin During Proestrus . . . Effect of Median Eminence Implantation of Prolactin, FSH, or FSH/LH on Serum Prolactin in Ovariectomized Rats . . . . Effect of ME Implantation of Prolactin in PP Serum Rats on Serum Prolactin, LH, and FSH. Effect of ME Implantation of Prolactin in PP Rats on Pituitary Prolactin, LH, and FSH Concentration . . . . . . . Effect of ME Implantation of Prolactin in PP Rats on Hypothalamic PIF, LRF, and FSH—RF Content . . . . . . . . . Effect of ME Implantation of Prolactin in PP Rats on Organ Weights and Mammary Gland Ratings . . . . . . . . . viii Page 50 55 69 75 78 79 81 90 92 93 95 Table Page 12. Effect of Injecting Exogenous Ovine Prolactin on Serum Prolactin Levels Following Suckling . . . . . . . . . . . . 103 13. Effect of Exogenous Ovine Prolactin on Serum LH Levels Following Suckling . . . . . 105 14. Effect of a ME Implant of Ovine Prolactin in Lactating Rats on Daily Litter Weight Gain. 106 15. Effect of Median Eminence Implantation of Prolactin in Lactating Rats on Serum LH. . 107 16. Effect of GH Implant in the Median Eminence of Adult Female Rats for 7 Days on Pitui- tary GH Concentration. . . . . . . . 116 17. Effect of GH Implanted Into the Median Emi- nence of Adult Female Rats for 7 Days on Hypothalamic GHRF Activity . . . . . . 117 18. Effect of GH Implant in the Median Eminence of Adult Female Rats on Serum Prolactin Concentration . . . . . . . . . . 119 19. Effect of GH Implant in the Median Eminence on Body Growth of 21-Day-Old Female Rats . 120 20. Effect of GH Implant in the Median Eminence of Adult Female Rats on Organ Weights and Mammary Gland Development . . . . . . 122 ix Figure 1. 9A. QEL LIST OF FIGURES Stoelting, Stellar Model, Stereotaxic Instru- ment Used in These Studies to Place Hormones Into the Brain of Rats . . . . . . . View of a Rat Held in Place While Glass Tube Filled with a Hormone is Readied for Lower- ing Into the Brain Through Hole in Skull . Serum Prolactin Concentration Before, During, and After Onset of Puberty. VO = Vaginal Opening. *Indicates Rats with Ballooned Uteri. . . . . . . . . . . . . Pituitary Prolactin Concentration and Content Before, During, and After Onset of Puberty. VO = Vaginal Opening. *Indicates Rats with Ballooned Uteri . . . . . . . . . Serum and Pituitary Prolactin Levels of 30- Day-Old Female Rats Following Estradiol Benzoate Injections . . . . . . . . Serum and Pituitary Prolactin Concentration During the Estrous Cycle in the Rat. . . Serum and Pituitary LH Concentrations During the Estrous Cycle in the Rat . . . . . Effect of Median Eminence Implant of Prolactin on Serum Prolactin Concentration During Estrus in the Rat. . . . . . . . . Photomicrograph of Ovary from PP Rat Implanted with Cocoa Butter in ME. Note Predominance of Well-Developed Corpora Lutea. x15 . . Photomicrograph of Ovary from PP Rat Implanted with Prolactin. Note Numerous Follicles and Few Corpora Lutea. x15. . . . . . . Page 31 35 49 52 53 61 63 76 96 96 Figure 10A. Photomicrograph of Uterus from PP Rat Implanted with Cocoa Butter. Note Relative Lack of Estrogen Stimulation. x110 . . . . . . lOB. Photomicrograph of Uterus from PP Rat Implanted with Prolactin. Note Stimulation of Epithileum and Endometrium. x110 . . . . 11A. Well—Developed Mammary Gland from PP Rat Im- planted with Cocoa Butter, Showing Extensive Branching and Lobulo-Alveolar Development x10 0 O O O O O O O O O I O O 0 11B. Mammary Gland from PP Rat Implanted with Pro- lactin, Showing Branching and End Buds but Little Lobulo-Alveolar Development. x10 . . 12. Serum Prolactin Levels in Lactating Rats with ME Implants of Prolactin . . . . . . . 13A. Well-Developed Mammary Gland from Control Rat with Cocoa Butter Implant in ME, Showing Numerous Ducts, Branching, and Some Lubulo- Alveolar Growth . . . . . . . . . . 13B. Mammary Gland from Rat with Human GH Implant in ME, Showing Thin Atrophic Ducts and Few End Buds O O O O O O O O O O O O 0 13C. Mammary Gland from Rat with Ovine GH Implant in ME, Showing Numerous Ducts, Branching, and Slight Lobulo-Alveolar Growth . . . . xi Page 97 97 98 98 108 123 123 123 INTRODUCTION Since the birth of neuroendocrinology some 30-40 years ago, many large strides have been made on clarifying the nervous control of the anterior pituitary gland. Only the most hardened skeptics still doubt that the brain, primarily the hypothalamus, plays a very important role in regulating hormone secretion by the pituitary gland. The anterior pituitary hormone, prolactin, is uni- quely regulated by a prolactin inhibiting factor (PIF) in the hypothalamus. This factor, of unknown structure, is capable of inhibiting synthesis and release of prolactin from the pituitary. The other five anterior pituitary hormones are controlled, in large part, by substances pro- duced in the brain called releasing factors. These re- leasing factors stimulate the synthesis and release of all anterior pituitary hormones except prolactin. Each anterior pituitary hormone apparently has its own specific releasing factor. Very recently, a breakthrough of great dimensions cxzcurred in neuroendocrinology. The chemical structure of on£2 releasing factor, thyrotropin releasing factor (TRF) was determined to be a simple 3 amino acid compound, 1 pyro-glutamic acid, histidine, proline amide. It is easily synthesized and available commercially. This molecule is also called thyrotropin releasing hormone (TRH) by Dr. Schally and his collaborators. The laboratories of Guillemin and Schally, working independently, are responsi- ble for this advancement. It is hoped that the structures of the remaining hypothalamic factors, including PIF, will be forthcoming soon. There is no doubt that control of hypothalamic re- leasing factors, and therefore of anterior pituitary hor- mone secretion, is mediated in large part by the con- ventional long feedback 100p from target organ hormones. More recent evidence indicates that this classic negative (positive under some conditions) feedback mechanism of target organ hormones is not the sole internal control for secretion of anterior pituitary hormones. Anterior pituitary hormones may regulate their own secretion through the hypothalamus. This hypothesis is especially interest- ing for explaining control of prolactin and growth hormone since neither of these two hormones have classic target organs. Therefore, one aspect of this thesis is an in- vestigation of the role prolactin and growth hormone have in controlling their own secretion and possibly that of 113. In addi- tion, hypophysectomized or old female rats received a supplement of oranges, carrots, and sugar cubes. Old rats also were given monthly injections of Bicillin (Wyeth Labs, Philadelphia, Pa.), a wide spectrum antibiotic. All animals were housed in metal wire cages in temperature con- trolled rooms at 75:1 F with light from 7 AM to 9 PM and darkness from 9 PM to 7 AM. II. Stereotaxic Technique A Stoelting, Stellar model, stereotaxic instrument, especially designed for small animal use was used in all studies involving the placement of hormones into the brain of rats (Fig. 1). By using a stereotaxic instrument, one 30 31 Figure 1. Stoelting, stellar model, stereotaxic instrument used in these studies to place hormones into the brain of rats. 32 can fix a rat's head in a location which has all three cartesian planes in space as reference points. The instru- ment is so constructed as to hold the rat's head at a fixed position, and the relative location of the head is constant from rat to rat. The three planes used in stereotaxic work are the horizontal, vertical, and lateral (sagittal). The hori- zontal plane passes intracerebrally through the anterior and posterior commissures and is approximately parallel to the midportion of the corpus callosum. The vertical planes are perpendicular to the horizontal plane. The principal vertical plane is perpendicular to a line in the horizontal plane connecting the external auditory meatus, a line called the interaural line. The lateral or mid- sagittal plane is at a right angle to both the vertical horizontal planes and divides the brain into symmetrical halves. This plane passes through a reference landmark on the skull called bregma, the point where frontal and parietal bones meet at midline. In most of the present studies, rats weighing 180 to 220 grams were used for stereotaxic work. Since rats of this size have a fairly constant head size and shape from one individual rat to another, it was very easy to accurately place hormone implants in the brain. An atlas was used to locate the various areas of interest in the brain (deGroot, 1959). This atlas requires that the upper incisor bar is at a height of 5.0 mm above the interaural 33 line. This is necessary in order for the rat's brain to be fixed in a constant plane coinciding with that of the brain illustrated in the atlas. Rats were prepared for implantation by using ether as an anesthetic. The top of the head was shaved and washed with Nolvasan-S (Fort Dodge Laboratories, Inc., Fort Dodge, Iowa) a virucide. Ear plugs were inserted into each ear and seated against the rat's skull. The ear plugs were then placed into the ear bars and secured by tightening the set screws. The interaural line is the imaginary line connecting the tips of the ear plugs. If the head is properly placed, a line joining the two eyes must be parallel with the ear bar. This is very necessary to insure proper placement of the implant. The incisor bar was next positioned behind the upper incisor so that it was 5 mm above the interaural line. The head was checked to be immobile before work was begun on it. The top of the skull was exposed and dried to enable a clear and distinct view of bregma. The electrode carrier which held the hormone implant within a glass tube was positioned directly above bregma. All readings were made from this zero reference point. It was determined, mainly by trial and error that implants were placed in the median eminence of the tuber cinereum if the electrode carrier which held the glass tube for implantation was moved posteriorly 0.5-0.7 mm from bregma and the tube lowered to 1.0 mm above the base of the skull. 34 III. Preparation and Implantation of Hormones All hormones implanted into the rat were placed first inside a 23-gauge glass tube, approximately 2 cm in length. Hormones were prepared for implantation by mixing them thoroughly in equal quantities of cocoa butter, used as a vehicle. Cocoa butter alone was implanted into rats which served as controls. The hormone-cocoa butter mixture was tamped into the tip of the glass tube, which was weighed before and after addition of the mixture to determine the amount of hormone implanted. The end of the tube opposite the mixture was sealed with bone wax to prevent efflux of the hormone when lowered into the brain. The glass tube was placed into the chuck of the electrode carrier and lowered through a hole drilled in the skull until the tip of the tube touched the floor of the cranial cavity (Fig. 2). The tip was then raised 1.0 mm so that it was located in the anterior median eminence. Skull screws were placed into previously drilled holes adjacent to the tube. The entire area was then covered with dental cement, which hardened in 5-7 minutes. The glass tube was left in place, anchored by the cement, and the skull screws served to anchor the cement to the skull. At the end of each experiment, the rats implanted with hormones in the brain, were killed and the ventral surface of the brain was exposed under a dissecting micro- scope. Only those rats which had properly placed implants 35 Figure 2. View of a rat held in place while glass tube filled with a hormone is readied for lowering into the brain through hole in skull. 36 in the median eminence were used for collection of data. Approximately 90% of the rats used in these studies had properly located implants. IV. In Vitro Incubation Technique The hypothalamus, including the stalk-median emi- nence area to be assayed for releasing or inhibiting factors, was dissected out of the brain using a small scissors and curved forceps. All hypothalami from each group were placed immediately into cold 0.1 N HCl (1.0 hypothalamus/0.1 ml) and frozen at -20 C until used in the incubation. Just prior to incubation, which was always done within 3 weeks after collection of the hypothalami, the hypothalami were homogenized with a Sonifier Cell Dis- ruptor (Heat Systems, Inc., Plainview, New York). The homogenates were centrifuged at 12,000 g for 40 minutes at 4 C in a Sorvall RC 2-B centrifuge (Ivan Sorvall, Inc., Norwalk, Conn.). The volume of supernatant fluid was measured and the fluid was placed in protein free medium 199 (Difco Labs, Detroit, Michigan). The pH was adjusted to 7.4 with 1.0 N NaOH immediately prior to addition of the hypothalamic extract to 25 ml Erlenmeyer flasks for incubation. Adult male rats weighing 300-400 gms were used as donors of anterior pituitaries for the incubation. Males were chosen rather than females because of absence of 37 cyclic hormone changes. These rats were killed by rapid decapitation using a guillotine, and the anterior pitui- taries were removed quickly, separated from the posterior lobe, and placed in a petri dish on filter paper moistened with medium 199. The anterior pituitaries were halved with a razor blade, and one half was placed in the control Erlenmeyer flask and the other half in the experimental flask, each flask containing 2 ml of medium 199, pH-7.4. The number of pituitary halves which were placed into each flask depended on which releasing or inhibiting factor was being assayed. Incubations for LRF or PIF utilized 2 pituitary halves/flask. Incubations for GHRF and FSH-RF utilized 12 pituitary halves/flask. The incubation was done in a Dubnoff metabolic shaker at 60 cycles/minute under constant gassing with 95% 0 -5% 2 CO2 at 3710.5 C. A pre-incubation period of one-half hour was done for each incubation in which the anterior pitui- taries alone were incubated with the medium 199. At the end of this time period, the incubated medium was discarded and replaced by fresh medium. To this mixture of medium and anterior pituitary halves was added the neutralized hypothalamic extract. In general, control flasks received hypothalamic extracts from control animals and experimental flasks received hypothalamic extracts from experimental rats. Occasionally, additional control flasks received extract of cerebral cortex. 38 The incubation time depended on the particular re- leasing or inhibiting factor being assayed. The time for LRF or PIF was 2 hours, whereas the time for FSH-RF and GHRF was 6 hours. At the end of the incubation period, the pituitary halves were separated from the medium and weighed to allow one to equalize the relative amount of pituitary tissue found in each flask. The medium was centrifuged at 3,000 g for 20 minutes, and the supernatant was frozen at -20 C until assayed for pituitary hormones. V. Assays of Pituitary Hormones A. Bioassay Growth Hormone was measured in rat pituitaries and incubation medium by the standard tibia test of Greenspan 33,31, (1949). All bioassays included 2 doses of the con- trol pituitary tissue or incubation medium, and 2 doses of the experimental pituitary tissue or incubation medium. The reference standard was also assayed at 2 dose levels. Four female rats, hypophysectomized 17 days earlier at 26 days of age, were used for each assay point. Each rat was injected intraperitoneally with test material once daily for 4 days. On the 5th day the rats were killed, one tibia of each rat was split in the mid sagittal plane and stained with 2% silver nitrate. The width of the epiphysial cartilage was determined by taking the mean of 10 measure- ments on each tibia. 39 Pituitaries were bioassayed at 2 dose levels, 2 and 4 mg/assay rat/4 days. The 2 doses of incubation medium used were 0.3 and 1.2 equivalents of incubated anterior pituitary tissue. Thus, if a flask contained 12 pituitary halves weighing 60 mg in 4 ml of medium, .66 m1 and 2.64 ml was used for the 2 dose levels. Follicle stimulating hormone content in pituitaries was measured by the HCG ovarian augmentation method of Steelman and Pohley (1953) as modified by Parlow and Reichert (1963). Two dose levels of pituitary (15 mg and 30 mg) and standards were assayed, with 5 assay rats in each group. The FSH standard used was NIH-FSH-S4 with a stated potency of 36.44 IU/mg. B. Radioimmunoassay Radioimmunoassay techniques were used to measure serum, pituitary and incubation medium for prolactin, LH, and FSH. The radioimmunoassays for these 3 hormones all utilize a double antibody method widely used and accepted in other laboratories. The reader is directed to several books described below which have recently been published explaining the theories and application in basic and applied research of radioimmunoassays. A rather brief and understandable book on the subject is Radioisotopes in Medicine - In Vitro Studies by Hayes 31 31. (1968). A good book describing recent progress in immunoassay of gonadotropins is the First Karolinska Symposium on 40 Research Methods in Reproductive Endocrinology) edited by Diczfalusy (1969). Protein and Po1ypeptide Hormones, edited by Margoulies (1969), gives extensive coverage to radioimmunoassay theory and practical application, as well as many experimental results. 1. Radioimmunoassay of Rat Prolactin The radioimmunoassay used to measure prolactin in serum, pituitary, and incubation medium was developed by joint effort of Dr. Midgley's laboratory in the Department of Pathology of the University of Michigan at Ann Arbor, Michigan and this laboratory of Dr. Meites' at Michigan State University, largely through the efforts of Dr. Chao- ling Chen. A description of this method and its validation is found in a recent publication by Niswender, Chen 31 31. (1969). The Ph.D. dissertation by Dr. C. L. Chen (Michigan State University, 1969) describes the development of the assay, including the production of first and second anti- sera. The antibody against rat prolactin used in the present experiments, produced in rabbit #625, and used at a working dilution of 1:4000, consistently bound 40-50% of the radioiodinated rat prolactin that had been incubated. Purified rat prolactin (H1010 B and HIV-8-C) ob- tained from Dr. Ellis (NASA Research Center, Ames, Cali- fornia) was used for radioiodination with 1131 (high specific activity, carrier free, for protein iodination, purchased from Cambridge Nuclear, Cambridge, Massachusetts). 41 Two and one-half ug of rat prolactin was iodinated with 1 131 with 30 ug of Chloramine-T as the millicurrie of I oxidizing agent, for a time period of 2 minutes. After stopping the reaction with sodium metabisulfite, the mix- ture was layered on a 1x15 cm Bio-gel P 60 column. One milliliter fractions were collected, and tubes with peak labelled prolactin were retained. These tubes were diluted in 0.1% BSA-PBS (bovine serum albumin-phosphate buffered saline) to concentrations of .4-.8 ng prolactin per 100 ug, which gave counts of 60,000-120,000 CPM in a Nuclear-Chicago autogamma counter. It was this amount that was pipetted to each test tube during incubation. Production of sheep antiserum against rabbit gamma globulin (Anti-RGG) was done by immunizing mature female sheep with 3 subcutaneous injections of 100 mg rabbit gamma globulin (Nutritional Biochemical Corporation, Cleveland, Ohio), emulsified in Freund's complete adjuvant, at 3 week intervals. A blood volume of 800-1200 ml was obtained monthly, and the serum was titrated for the optimal dilution to precipitate the rabbit gamma globulin. The optimal dilution ranged from 1:3 to 1:8, dependent on time of booster injection and bleeding. The incubation procedure for assaying prolactin was carried out at 4 C. Disposable culture tubes, 12x75 mm (Kimble Owens, Illinois, Ohio) were used for all assays. On day 1, first antibody, unlabelled prolactin (known or unknown amount) and 1% BSA-PBS as diluent were added to 42 each tube. Twenty-four hours later, on day 2, 0.4-0.8 ng of labelled rat prolactin was added to each tube and the tubes were shaken. On day 3, the second antibody (anti- RGG) was added to each tube and each tube was shaken. On day 6, or 72 hours after addition of anti-RGG, 3 ml of PBS was added to each tube and the tubes were centrifuged in an International Centrifuge RP—2 at 2,000 rpm for 30 minutes. The supernatant of all tubes was decanted and the precipitate was counted in a Nuclear-Chicago autogamma counter. A standard curve was drawn on semi-logarithmic paper from 10 different doses of standard rat prolactin, each done in duplicate or triplicate. A range of 0.5-16.0 ng of rat prolactin (HIV-8-C with a relative potency of 0.66xNIH-P-Bl) usually comprised the straight part of the curve and was usable for obtaining data. Unknowns done in duplicate or triplicate were read out from the standard curve and expressed as ng of prolactin per m1 serum. It was possible to measure serum prolactin in adult female rats using 20-100 ml serum for assay. More serum, 75-150 ul, was required in immature female rats or male rats. Only 5-20 ul of a 0.25 mg/ml concentration of pituitary tissue was needed for assay. All pituitaries were prepared for assay by homogenization in cold phosphate buffered saline, pH-7.0, using a cell disruptor. 43 2. Radioimmunoassay of Rat LH Many of the procedures described for radioimmunoassay of rat prolactin are identical for assay of rat LH, and will not be repeated here. It is necessary to describe the various antisera and hormones used for radioiodination, however. In one experiment reported in this thesis, number V, a radioimmunoassay for rat LH utilizing anti-rat LH for antiserum and purified rat LH for iodination was used. This assay is described in the literature (Monroe 31 31., 1968). This assay utilized the reagents made available by the National Institute of Arthritis and Metabolic Diseases (NIAMD) of the National Institutes of Health (NIH). Rat LH for radioiodination labelled NIAMD-Rat LH-I-l, had a bio- logical potency of approximately 1.0 x NIH—LH-Sl and FSH contamination less than 0.04 x NIH-FSH-Sl. The antisera to rat LH, prepared in rabbits, and labelled NIAMD-Anti- Rat LH Serum-l, was used at a working dilution of l:32,000. This gave consistent binding of 35-45% with labelled LH. The same anti-RGG described previously in this section was used as the second antibody. The reference preparation used was crude pituitary extract labelled NIAMD—Rat LH- RP-l. This has a stated biological potency of 0.03 x NIH-LH-Sl and 0.54 x NIH-FSH-Sl. A second radioimmunoassay was used to obtain the rat serum and pituitary LH values found in experiments II, III, IV, and VI of this thesis. This assay, described 44 in detail in a recent publication (Niswender 33 31., 1968) utilizes ovine LH both for immunization and radioiodination. The antiserum to ovine LH was used at a dilution of 1:50,000 and bound 30-45% of the labelled hormone. The same refer- ence preparation was used for this LH assay as for the rat LH assay. In both of these LH radioimmunoassays, serum and pituitary samples were done in duplicate or triplicate. A similar procedure as described for radioimmunoassay of rat prolactin was used for calculation of actual levels of LH from a standard curve. All dilutions were done in egg white-PBS rather than BSA-PBS due to the LH contami- nation in BSA. 3. Radioimmunoassay of Rat FSH Many of the procedures described for radioimmunoassay of rat prolactin are identical for assay of rat FSH, and will not be repeated here. It is necessary to describe the various antisera and hormones used for radioiodination, however. The radioimmunoassay for rat FSH utilized anti-rat FSH serum and purified rat FSH for iodination. This assay was described at the Slst Endocrine Meetings (1969) by Parlow. This assay utilized the reagents made available by NIAMD. Rat FSH for radioiodination, labelled NIAMD- Rat FSH-I-l, had a biological potency of approximately lOOxNIH-FSH-Sl and LH contamination was less than 45 0.002xNIH-LH-Sl. The antisera to rat FSH, prepared in rabbits and labelled NIAMD-Anti-Rat-FSH-serum-1, was used at a working concentration of 1:625. This dilution bound 25-30% of the labelled FSH. The same anti-RGG described previously in this section was used as the second antibody. The reference preparation used was a crude pituitary ex- tract labelled NIAMD-Rat FSH-RP-l. This had a stated bio- logical potency of 2.1xNIH—FSH-Sl. All assays for FSH were done in duplicate and actual concentration values in ng/ml serum were obtained directly from a standard curve. VI. Methods of Statistical Analysis Each data point for serum and pituitary hormone con- centration obtained from radioimmunoassay was the mean of 2 or 3 assay values. Mean and the standard error of the mean were calculated from the average value of each sample for each group in the experiment. If only two groups of data, experimental and control, were obtained in one experiment, the "t" test was used to test for significance. If there were 3 or more groups, a test of significant differences was carried out by an "F" test for approxi- mation. If the "F" test revealed significant differences, the data were subjected to Duncan's multiple range test of significance (1955). Bioassays for GH and FSH were analyzed according to Bliss (1952). EXPERIMENTAL I. Serum and Pituitary Prolactin Levels Before, During, and After Puberty in Female Rats A. Objective Sexually immature female rats have a much lower pituitary prolactin content than mature female rats (Minaguchi 33 31., 1968), as measured by bioassay. By contrast, pituitary FSH and LH was reported to be high prepuberally (Kragt and Ganong, 1967; Ramirez and Sawyer, 1965). Pituitary prolactin levels, therefore, show an opposite trend from pituitary gonadotropins prepuberally. The purpose of this investigation was to assay serum as well as pituitary prolactin levels by radioimmunoassay before, during, and after vaginal opening, and after estro- gen injection. B. Materials and Methods 1. Experimental Animals A total of 156, 21-day-old female Sprague-Dawley rats (Spartan Research Animals, Haslett, Michigan) were used in these eXperiments. The rats were fed 33 libitum 46 47 Wayne Lab Blox (Allied Mills, Chicago, Illinois) and main- tained on a lighting schedule of 14 hr of light and 10 hr of darkness at 25:1 C. 2. Treatments All rats were examined for vaginal opening beginning at 21 days of age. Four groups of rats were killed at 21, 26, 31, and 33 days of age. Nineteen rats with closed vaginas were killed at 36 days of age and separated into 2 groups: one group had obviously ballooned uteri indi- cating proestrus, and the other group had small, non- ballooned uteri. A seventh group consisted of rats killed within 16 hours after vaginal opening, while the eighth and ninth groups consisted of rats killed 1-3 days after vaginal opening. The tenth group was killed during the first estrus following the estrus which accompanied vaginal opening. The eleventh group consisted of 3-month-old cycling females killed during estrus. In another experiment, 0.05 to 5.0 ug of estradiol benzoate (EB) dissolved in corn oil was injected once daily for 4 days into female rats, beginning at day 26 of life. These rats were killed at 30 days of age. All rats were anesthetized with ether, bled from the abdominal vena cava and decapitated with a guillotine be- tween 8 and 10 AM. The serum was separated and kept frozen at -20 C until the time of assay. The anterior pituitaries were individually weighed and frozen immediately at -20 C 48 until the time of assay. The ovaries and uteri were trimmed and weighed. 3. Prolactin Assay Prolactin of individual serum samples and pituitaries were measured by radioimmunoassay (Niswender 3£_31., 1969). Pituitaries were prepared in cold phosphate buffer saline by homogenizing with a Sonifier cell disruptor. Data from radioimmunoassay and organ weights were subjected to analy- sis of variance. Means of each group were analyzed further by Duncan's New Multiple Range Test (1955). C. Results 1. Prolactin Levels Serum prolactin levels were uniformly low (13-21 ng/ ml) from day 21 to day 36 (Fig. 3, Table 1). There was twice as much serum prolactin (not statistically signifi- cant) in the 36-day-old rats with ballooned uteri as in rats of similar age with non-ballooned uteri. A sharp 3-4 fold increase in serum prolactin (to 76 mug/m1) was noted on the day of vaginal opening, at which time all the rats were in estrus. A precipitous drop in serum prolactin occurred 1-3 days after vaginal opening, at which time most rats were in diestrus. There was a significant in- crease in serum prolactin at the next estrus (41-47 days of age), comparable to that seen during the estrus of 3-month-old virgin rats (70-80 mug/m1). 49 IOO r 1r ::-£3C>' E (9) (H8) \ ) o 5 1 60 " Z P— 8 _. 40+ 0 a: 0- , (IO) :2) 20 ~ '0’ (ll) (7) (1: I0) % (IO) (9) (a) 2) is in is 56 Sen; AGE (DAYS) 3 > I DAY POST VO - 2-3 DAYS POSTVO P ESTRUS(4I-47) " ESTRUS (3 mo) 1' Figure 3. Serum prolactin concentration before, during, and after onset of puberty. VO = Vaginal Open- ing. *Indicates rats with ballooned uteri. 50 .umcuo some Eouu Amo.o A no accumuuwc xauccoawwcoam uoc mum mudwuomuodsm mama ecu Sufi: acne: 358.53.?«5 .Auou: cocooaaen gun: was» eaousuenom .cnme ecu mo uouum pumvcmum H coo! N H mom.HHm~.mH oma.oumv.a vm.>HHm.mh na.mHm.Hh pn.omwm.nm mo.on.HH oHH.nHm.~h~ m Amcucoe mumv msuumm I I I I I I I I I I I I I I I I I I I I I I I I as“ m vum o+h~ m m.vmo o+mn «a vH+m an up om+H mv~ nu m+o mm 5v o+h o mN w+m mud m av H+m cw u m mmm.oHo~.~ o.mvo.ouav. mo.mmm.ma mm.nHm.mHH oa.mfla.om mm.oum.m mo.mum.oma m o> umom when mum o I o o I o o I o o I I o I o o I o o I o . Q % mmm o+mm a moo o+~m mm v+v ma mm ~H+o boa m.vm v+w vm mv o+~ v N1mm N+m wma n o> umo on H nma.o+vm.m v.mmo.o+mv. vm.va+o.oh oa.o+m.oma oo.~+~.mv o.m~.o+m.v no.~+m.ana oa anw o+o hm o> I I I I I I I I I I I I I I I I I I I I I % mam o+vm H mvo o+am no m+m mm mm n+9 «ma co H+o on mm o+v v up ~+m and ca mm on on I I I I I I I I I I I I I I I I I I I I I x mva o+vw a mmo o+~m mm H+o ma m.vm m+H mm an H+~ on mm o+m v no m+m and m m up on I I I I I I I I I I I I I I I I I I I I I % maa o+nm a mmo o+mm no m+v ma va n+a mp we H+m mm m.vH o+m m mm H+o mHH AH m on mm I I I I I I I I I I I I I I I I I O I I I h mmm o+ma m vmo o+mm mv N+h om v.mm 9+5 mm vs H+H on va o+m m mo H+H boa oa m on an o I O O I o O I c O I o o I o o I u 0 I 0 mi mva o+mv a vmo o+am mm ~+~ ma mm H+v on v.mm H+h MN ma o+v N va H+m as ea no cm I I I I I I I I I I I I I I I I I I I I I % mno o+HH H v.mmo o+vm mv H+o vH me H+H aw mu o+o ma ma o+a N .mm o+o ow OH m up am 0 me o Edumm one n 13 a 12 x a H E\ 35 195 H 3.5 was: We use mam>wa cwuomaoum sum»: mmwum>o Ad 3 xcom .oz .huuwnsm wo ummco on» umuwm can .mcwuso .mH0won mam>cd swuomaoum aumuwsuwm can Edumm can mucosa: cumu0I1.H manta 51 Pituitary prolactin concentration was slightly higher in 26- and 31—day-old rats than in 21-, 33-, and 36-day-old rats (Fig. 4, Table 1). It was not until the first estrus following vaginal opening, however, that pituitary pro- lactin concentration began to rise markedly. Three-month- old female rats killed during the morning of estrus had even higher pituitary prolactin levels than estrous rats 41-47 days old. A similar pattern was seen in pituitary prolactin content. Prolactin remained relatively low until the first estrus after vaginal opening, at which time a 2-3 fold increase was noted. There was another 3 fold increase in serum prolactin in 3-month-old estrous rats. Injections of 0.05 ug EB for 4 days beginning at 26 days of age did not increase serum prolactin levels above control rats injected with corn oil (Fig. 5). Rats injected with 0.10, 0.30, or 0.50 ug EB showed significant increments in serum prolactin concentration, whereas 5.0 ug was less effective than 0.30 or 0.50 ug in stimulating prolactin release. All of the rats injected with 0.10 ug or more EB had open vaginas and were in estrus at the time of killing. None of the controls or rats injected with 0.05 ug EB had open vaginas at the time of killing. Pituitary prolactin concentration and content increased similarly to serum levels, reaching a peak at a dose of 0.3 ug EB (Fig. 5). I50 125 IOO PITUITARY PROLACTIN CONCENTRATION (pg/mg) Figure 4. 52 AP PROLACTIN CONCENTRATION ' AP PR ACTIN CONTENT T Llllll‘gllfl‘i 2|263l3336365§g|§g AGE(DAYS) I; 5 5 3' a 88259 >-'DQ: <2Ei— 90mm mull-U o') co PITUITARY PROLACTIN CONTENT (pg/GLAND) Pituitary prolactin concentration and content before, during, and after onset of puberty. VO = Vaginal Opening. *Indicates rats with ballooned uteri. g/ml) R3 O 0': C) C) A a SERUM PROLACTIN (n on C) I D PITUITARY PROLACTIN CONCENTRATION (pg/mg) 1 I 1‘3 A Figure 5. i\) l 63 I '05 I 53 sssssssscsa 4 CORN 0.05 O.|O OIL EST RADIOL BE NZOATE \V \\ ‘3 \\\\\\\\\.~ \\\\\\\ as Z l 6'5 o 030 0.50 500 $0 C) as '0 PITUITARY PROLACTIN g CONTENT (pg/GLAND) ,0: C) Serum and pituitary prolactin levels of 30-day old female rats following estradiol benzoate injections. g/rnl_) m 0 RS (3 c1 PITUITARY PROLACTIN CONCENTRATION (pg/mg) R) E SERUM PROLACTIN (n 4s on o o Figure 5. N A 5 T '03 j 53 SS§§§§§§E‘FE Z """ 7,. . &\\\\\\t\\\\“§ IIII IIIII IIIII ouu‘ II-Q ..... 4-... l CORN 0.05 O.|O O30 0.50 500 OIL ESTRADIOL BE NZOATE (Jug) l IZO SO C) 03 'O PITUITARY PROLACTIN 3 CONTENT (pg/GLAND) p: (3 Serum and pituitary prolactin levels of 30-day old female rats following estradiol benzoate injections. 54 2. Organ Weights Body weight and anterior pituitary, ovarian and uterine weights increased significantly from day 21 to the day of vaginal opening at 35-39 days of age (Table l). The ballooned uteri of rats at 36 days of age were significantly heavier than the non-ballooned uteri of similar aged rats. However, ovarian weights of the 2 groups did not differ. There was a significant increase in ovarian weight on the day of vaginal opening compared to 36-day-old rats with closed vaginas. A significant increase in weight of the pituitary, ovaries, and uterus occurred at the first estrus following vaginal opening (41-47 days) as compared to rats at the onset of puberty. Injections of 0.10 ug EB for 4 days beginning at 26 days of age significantly increased uterine and anterior pituitary weights (Table 2). Doses of EB up to 5.0 ug further increased pituitary weight, but none of the doses affected body or ovarian weight. D. Discussion The present study shows that serum prolactin levels are low in prepubertal rats, and increase sharply at the time of vaginal opening. A sharp decline occurs for 1-3 days after vaginal opening and serum prolactin remains low until the next estrus, at which time it rises to the level observed during estrus in 3-month-old virgin rats. Both pituitary prolactin concentration and content are relatively 555 .uonuo coco scum .mo.o A no accumuwac aducdowuwcoam no: one nunwuomucmsm «Sam on» sud: acne: .ccoE on» no wouum cuuocdum + cam: m.v.m.~ H co.onma.o mme.onon.v v.n man.o~umm.ee ma.mum.~na ~o.~nm.qm mo~.onoa.m ~m.aHv.oa oH oo.m man.on~e.m nm°.om~a.o soa.o~u~m.mma nm.ous.awa ~m.awa.m~ .ma.ouan.v ~v.aun.moa ca om.o cam.owcm.m mwa.ouam.a sea.m~um¢.saa no.mua.nna ~a.mum.vn v-.ouam.v ~m.muo.aa o om.o ma~.o+aa.n «so.o+ao.a m.~ma v +mm.a¢ mo.m+s.aaa ~>.~+o.am man.o+mm.m -.n+m.aoa a oa.o «Ha.oum~.a ~mo.oumm.o -o.a Hem.s Na.oflm.um ~m.~us.- NmH.oum~.m -.muo.sa m mo.o ~Hd.o+em.fl ~mo.o+mn.o ~ao.a +~o.HH ~a.¢+m.sm -.H+m.e~ n.~oa.o+ q.m ~o.a+m.ao~ on oo.o pcuam\on m4 ma\o: Bunch HE\mse Away . . . m”. 3mm, :3. . 3... mm 33.... mao>oq cwuouaoum no . 0 ma as com u z Howcmuumm .cofiunnumwcwecc ouooncon Howcmuumo mcw3oHH0m nucu OHOEOM caonthIom no mHO>OH cavemaoua mucuwsuwm can asumm can nucmao3 camuonn.~ mqm<9 56 low before and at the time of puberty, and increase sig- nificantly soon after puberty, in agreement with Minaguchi 3£_31. (1968). However, these higher pituitary prolactin levels are not reflected by similar changes in serum pro- lactin, and may have no physiological significance. The lack of change in pituitary prolactin levels on the day of vaginal opening, when all the rats are in estrus, is in sharp contrast to the marked rise in prolactin. It is possible that a significant increase in serum prolactin does not necessarily require release of large quantities of prolactin from the pituitary. The total amount of pro- lactin in the blood appears to be low compared to the total in the pituitary, and therefore notable changes in serum prolactin may not always be reflected by comparable changes in pituitary prolactin levels. Several laboratories (Corbin and Daniels, 1967; Kragt and Ganong, 1967; Ramirez and McCann, 1963) reported that FSH and LH secretion increased as rats approached puberty. The increased gonadotropin secretion is believed to act on the ovaries to increase estrogen secretion, and estrogen is known to stimulate prolactin secretion. Estro- gen injections increase both pituitary and serum prolactin levels in mature ovariectomized rats (Chen and Meites, 1970; Niswender 33 31., 1969). The present study shows that estrogen also increases serum and pituitary prolactin in intact immature female rats. 57 The 36-day-old rats with closed vaginas but with ballooned, proestrous uteri, had serum prolactin levels of 26.9 mug/ml as compared to 13.6 mug/m1 serum in 36-day-old rats with non-ballooned uteri. Increased estrogen secretion by the ovaries of the former rats is believed to account for the 2 fold increase in serum prolactin in these rats. Injections of small doses of EB daily into immature rats have been shown to hasten puberty in rats (Corbin and Daniels, 1969; Ramirez and Sawyer, 1965). Ramirez and Sawyer (1966) observed a drop in LRF at the time of puberty and also following estrogen treatment in immature rats. Corbin and Daniels (1969) found a similar decline in FSH-RF and FSH after injections of estrogen into im— mature female rats. Both laboratories concluded that estrogen stimulated release of LRF and FSH-RF, respec- tively. We have recently reported that subcutaneous in- jections, or implantation of prolactin into the median eminence hastens puberty about 6-7 days in female rats (Clemens 31_31., 1969c), and that a prolactin implant in the median eminence increases release of FSH (Voogt 33 31., 1969b) and LH (Voogt 31 31., 1969a). It is possible therefore, that small doses of EB hastened puberty by stimulating prolactin secretion which in turn acted back on the hypothalamus to stimulate release of LRF and FSH-RF. This does not exclude the possibility that estrogen may 58 also act directly on the gonadotropin releasing factors of the hypothalamus. The low prolactin secretion in the immature rat is in direct contrast to the high gonadotropin secretion. A reciprocal pattern is frequently observed between pro- lactin and gonadotropin secretion in different repro- ductive states. However, a major exception to this pattern appears to be present on the day of vaginal open- ing, when increased serum LH levels are found as deter- mined by bioassay (Ramirez and Sawyer, 1965) and more recently by radioimmunoassay (A. R. Midgley 33 31., per- sonal communication). One cannot conclude from these results that pro- lactin has a definite role in the initiation of puberty in rats. It is apparent, however, that prolactin is released in large amounts on the day of vaginal opening, and that estrogen is a potent stimulator of prolactin secretion in the immature as well as in the adult female rat. The secretion of gonadotropins by the pituitary may, there- fore, be partly conditioned by prolactin secretion. 59 II. Serum and Pituitary Prolactin and LH During the Estrous Cycle! Following Ovariectomy, and During Lactation A. Objective Several reports have been published recently describ- ing changes in pituitary and serum prolactin, LH, and FSH during the rat estrous cycle and following castration. Reports on LH (Monroe 33 31., 1969), FSH (Parlow 33 31., 1969), and prolactin (Niswender 33 31., 1969) indicate that all 3 hormones were elevated during the late afternoon of proestrus. Serum LH and FSH were very low at all other stages of the cycle, whereas serum prolactin remained rela- tively high during estrus (Amenomori 33_31., 1970). Pitui- tary LH decreased in late proestrus (Monroe 33 31,, 1969). Following ovariectomy in the female rat, serum LH and FSH rose (Gay, 1970). Serum and pituitary prolactin levels declined significantly 3 weeks after ovariectomy (Amenomori 33 31., 1970). The measurement of serum and pituitary LH and pro- lactin during the cycle and following ovariectomy was done by this investigator to confirm the above reports. In addition, tabulation of these data in terms of definite concentrations in the blood and pituitaries established reference points. Thus during experimental manipulation of different physiological states, the experimenter can determine whether his results are within the physiological 60 range, and their possible relation to various physiological functions. B. Materials and Methods Animals used in this study were 3-month-old virgin female rats from Spartan Research Animals (Haslett, Michi- gan). Vaginal smears were taken for 2 weeks to be certain that all rats used in the study were cycling normally. Only rats which showed a definite 4-day cycle were used. Eight groups of rats were used for the study on the estrous cycle, with 6-10 rats/group. Light ether anesthesia was used, and all rats were bled from the abdominal vena cava. Pituitaries and sera were assayed for LH and pro- lactin. One group of rats was ovariectomized and bled 4 weeks later. The blood was pooled and assayed for prolactin and LH. Sera from adult male rats were pooled and assayed for LH and prolactin. Postpartum lactating rats, on the eighth day of lactation were also used as a source of serum for assay. C. Results In normally cycling female rats, serum prolactin was low during metestrus and diestrus (Fig. 6). A sharp rise in serum prolactin was noted during the late afternoon of proestrus, when values as high as 1000 ng/ml were recorded. Prolactin remained relatively high during the morning of 250 3 3 § SERUM PROLACTIN (ng/ml) 8 IOOm I Figure 6. 61 SERUM PROLACTIN [:1 PITUITARY PROLACTIN l .05 O “\‘\\'\\V “““w W / I 1 .04 O a) D a: . F... m LIJ l.- LL! 2 4; (’1 PITUITARY PROLACTIN CONCENTRATION (pg/mg) IOom 4pm 6pm 8pm lOan 4pm PROESTRUS ESTRUS Serum and pituitary prolactin concentration during the estrous cycle in the rat. 62 estrus (10 AM), and then decreased. Pituitary prolactin concentration reached a peak during early proestrus, and then decreased from 5.8 ug/mg gland at 10 AM of proestrus, to 3.71 ug/mg gland at 8 PM of proestrus (Fig. 6). This rapid decrease in pituitary prolactin occurred at the same time as the large increase in serum prolactin. Thereafter, pituitary prolactin began to rise. Serum LH showed a peak at the same time as the rise in serum prolactin during the late afternoon and evening of proestrus. However, at all other stages of the estrous cycle, serum LH was very low (Fig. 7). Pituitary LH did not fluctuate as greatly as pituitary prolactin (Fig. 7). A decrease from 4.7 ug/mg gland at 4 PM proestrus, to 3.8 ug/mg gland at 10 AM estrus was the largest change. Rats which were ovariectomized 4 weeks earlier had a pooled serum prolactin level of 20.4 ng/ml, very similar to diestrus levels. During lactation, serum prolactin averaged 213 ng/ml serum, considerably higher than during estrus, and comparable to late proestrus peak values. Male rats had serum prolactin levels very similar to ovariectomized female rats or female rats in diestrus (20.2 ng/ml). Female rats previously hypophysectomized had serum pro- lactin values less than 5.0 ng/ml. Serum LH levels following ovariectomy for 4 weeks rose to 686 ng/ml, comparable to peak proestrus levels. During lactation, serum LH was often undetectable unless IOOO " aoo - 2 I...” E C” s 600 +- I _l S a: 400 LL) (I) 200 ' 63 SERUM LH Cl PITUITARY LH .. ,0! O 1 .b o \\\\\\\\V K ““1““ \\ \\\\\\\ .\\\\\\\\\\\\\\\\ m o: '0 O PITUITARY LH CONCENTRATION (pg/mg) 6 Oam to D a: [.— (1) Lu '— Lu 2 Figure 7. 3.13.. I P S IOom lOom 4pm 6pm 8pm lOom 4pm (é) PROESTRUS ESTRUS g... (I) 9.1 C) Serum and pituitary LH concentrations during the estrous cycle in the rat. 64 as much as 200 ul of serum was assayed. Then values were calculated to be less than 100 ng/ml. Male serum LH levels were 87.0 ng/ml, somewhat higher than diestrus levels. Pituitary LH levels in ovariectomized rats were about 100 ug/mg, much higher than at any other stage measured. During lactation, pituitary LH concentration was reduced to less than 2 ug/mg. Male pituitaries had somewhat more LH. D. Discussion The changes in serum prolactin and LH during the estrous cycle are in good agreement with previously re- ported observations (Monroe 22 al., 1969; Niswender 3E.§l., 1969). In this study, high concentrations of serum LH and prolactin were noted during the late afternoon and early evening of proestrus. Serum prolactin remained high until the afternoon of estrus, in agreement with results reported by Amenomori gt 2£° (1970). Pituitary prolactin concentration decreased rapidly during late proestrus, during the time serum prolactin rose to high levels. This decrease in pituitary prolactin is probably the result of a large discharge of prolactin from the gland into the circulation at this time. Soon thereafter, prolactin concentration in the pituitary rose, indicating an increase in synthesis to replace the depleted hormone probably stimulated by increased estrogen. Pitui- tary LH also decreased during late proestrus, at the time 65 that serum LH rose. This is in good agreement with Monroe 22 El. (1969) using radioimmunoassay, and Schwartz and Bartosik (1962) using bioassay. Following ovariectomy, serum prolactin levels were considerably lower than during late proestrus or estrus. In this study serum prolactin levels ranged from 15-35 ng/ml, confirming reports by Niswender g£_al. (1969), and Amenomori gt El° (1970). Conversely, serum and pituitary LH rose considerably following ovariectomy, in agreement with Gay, 1970. Another physiological state, that of post- partum lactation, showed prolactin and LH to be recipro- cally related. Serum prolactin was high (162-403 ng/ml) during lactation, whereas serum and pituitary LH were low. Male rats were found to have low prolactin and rela— tively low LH in the serum. Serum prolactin values in male rats were similar to ovariectomized rats and cycling rats in diestrus, in agreement with Niswender et al. (1969). Serum LH in male rats was somewhat higher than during di- estrus in female rats, but not nearly as high as during proestrus. The results presented and discussed in this study not only confirmed previous reports in the literature, but made it possible for me to compare these results with other experiments in which I experimentally manipulated these "natural" states. These results yielded some refer- ence values, helpful not only to me, but hopefully, also to the reader. These confirmatory results also gave me 66 confidence in the radioimmunoassays themselves, and in my utilization of them. In addition, the rather large vari- ation in hormone levels in the serum during different physiological states noted in this experiment indicated the need for large numbers of animals per group during subsequent experiments. 67 III. Serum Prolactin and LH in Old Female Rats A. Objective Few data are available in the literature regarding prolactin and gonadotropin secretion in the aged animal. In the Ph.D. thesis of Clemens, 1968 (Michigan State Uni- versity, East Lansing, Michigan), it was reported that pituitary prolactin and FSH are higher in old constant estrous rats than in 3-month-old rats in estrus. Con- versely, pituitary LH was reported to be lower in old constant estrus rats. There are no reports of LH or prolactin levels in the serum of old female rats. The purpose of this study was to measure serum LH and prolactin in old female rats which were in constant estrus or experienced repeated pseudopregnancies. B. Materials and Methods The animals used in this study were discard Sprague- Dawley rats previously used for breeding by Spartan Re- search Animals (Haslett, Michigan). Daily vaginal smears were taken for 6 weeks before the rats were bled. Only rats which were 18-22 months old and showed either con- stant estrus or repeated pseudopregnancies were used in this study. Light ether anesthesia was used, and about 1 ml of blood was withdrawn via cardiac puncture from each rat. 68 Both prolactin and LH were measured by the radio- immunoassays described in the general Materials and Methods section. The LH assay used was that described by Niswender 2; El' (1968). C. Results Serum prolactin concentration, measured in ng/ml, averaged ll3.6:31.2 in 18 old rats in constant estrus (Table 3). This prolactin level is slightly higher than levels seen during the morning of estrus. Fourteen differ- ent rats were pseudopregnant when bled, and had a mean serum prolactin level of 28.2:3.l, which was significantly lower than the serum prolactin in constant estrus rats. This prolactin level of 28.2 ng/ml is comparable to di- estrous values in cycling rats. Serum LH concentration in 8 rats in constant estrus averaged 24.4 ng/ml (Table 3). This was significantly higher than the LH levels in 12 old rats pseudopregnant when bled, which averaged 13.2 ng/ml. These levels are comparable to cycling female rats in estrus or diestrus. However, the levels of serum LH in both groups are rela- tively low compared to levels in proestrus or following ovariectomy. D. Discussion The results presented here suggest that there are no changes in serum levels of prolactin or LH in constant 69 sec. v a.. mo. v a; .cme may mo mm H cmmza mumu m0 HmQEsz A v «IANHV m.HHm.mH Ame m.aflv.sm .Avav H.m H~.mm Away N.Hmum.mHH wocmcmmumoosmmm umommm msuumm ucmumcou HE\mc ma Esumm H HHE\mG GHMUMHOHA Edumm ummEm Hmcflmm> mo mama .mumu pHo 2H ma paw cwuomaoum Edummll.m mqm¢e 70 estrous old rats as compared to mature young rats on the day of estrus. Thus, the serum level of prolactin during estrus in normal mature rats, reported in experiment II of this thesis, is nearly the same as the serum prolactin level of old constant estrus rats reported in this experi- ment. However, the serum prolactin values of the old constant estrous rats are consistently high whereas the normal mature rats show cyclic fluctuation, with low values during each diestrous period (the latter usually about 48 hours duration during each cycle). These consistently high prolactin levels in the old constant estrous rats are believed to have a role in the onset of spontaneous mammary tumors. The persistent estrogen secretion is also believed to contribute to mammary as well as to pituitary tumoro- genesis in old female rats (Welsch gt al., 1970). The serum level of prolactin during diestrus of a cycling rat reported in experiment II of this thesis is comparable to the serum prolactin levels of old pseudopregnant rats. Clemens in his Ph.D. thesis (1968), reported an in- crease in pituitary FSH and a decrease in pituitary LH in old constant estrous rats. He also reported high FSH-RF levels in the hypothalamus, and suggested that the con- stant estrous state observed often in old rats is a result of a malfunction arising in the hypothalamus. Thus, high FSH is secreted which increases estrogen secretion, result- ing in constant estrus. Too little LH is secreted to cause ovulation, thus the rats remain in estrus. 71 Unfortunately serum FSH was not measured in the present experiment to determine whether this hypothesis is correct. The high serum prolactin levels found in constant estrous rats is well correlated with the high pituitary prolactin content reported earlier (Meites gt_§l., 1961; Clemens, Ph.D. Thesis, 1968). This increase in prolactin secretion observed during constant estrus is probably due to estrogen secretion. The pituitaries of old female rats in constant estrus are almost invariably enlarged, and occasionally these rats develop pituitary tumors (Clemens, Ph.D. Thesis, 1968). It is well established that estrogen is a potent stimulator of prolactin release (Chen and Meites, 1970). More work utilizing radioimmunoassays to measure serum levels of LH, prolactin and FSH is needed to deter- mine the nature of the changes in pituitary hormone secretion with age. Measures of hypothalamic hormone secretion are also needed. Only when these changes are clearly described, can exploration begin on the possible neural influence with age on the brain, particularly the hypothalamus. 72 IV. Effect of Median Eminence Implantation of Prolactin on Serum Prolactin and LH During the Estrous Cycle A. Objective Previously in this thesis I described variations in serum LH and prolactin levels during the estrous cycle in the rat. It has been noted in the literature review that implantation of prolactin into the median eminence de- creased pituitary prolactin concentration and may stimu- late LH release. Thus it was of interest to determine the effect of an implant of prolactin into the median eminence of cycling female rats on serum prolactin and LH, especially during the proestrous peak of these two hormones. The effects of an implant of prolactin, FSH, or FSH/LH in the median eminence of ovariectomized rats was also studied. B. Materials and Methods All rats used in this study were mature, cycling, virgin females obtained from Spartan Research Animals, Haslett, Michigan. Before the implantation of hormones, the cycles of all rats were followed by examining daily vaginal smears for at least 2 cycles, and only normally cycling rats were used for experimental purposes. Rats were also ovariectomized and used 3 weeks later. The design of this experiment is comprised of three distinct parts. The first 2 groups of rats received median eminence implants of either prolactin-cocoa butter 73 or cocoa butter alone at random times during the cycle. These rats were bled via cardiac puncture under light ether anesthesia immediately prior to implantation, and daily thereafter for 5 days. All bleedings (1 ml each) and implantations were done before ll AM, and vaginal smears were taken daily. After 5 days all rats were killed, and only those with properly placed implants were used. The second 2 groups of rats received implants in the median eminence of either prolactin-cocoa butter or cocoa butter alone during the morning of proestrus. These rats were bled via heart puncture under light ether anes- thesia 4 times on the same day that they were implanted. One ml of blood was removed each hour beginning at 4:30 PM. Vaginal smears were taken from all rats for several days after implantation, and only those rats which came into estrus the day after implantation were considered to be in proestrus when bled. A third part of the experiment used 4 groups of female rats which were ovariectomized and used for eXperi- ments 3 weeks later. These rats received median eminence implants of cocoa butter alone, prolactin, FSH or FSH/LH cocoa butter mixtures on day 0. Bleedings were done via heart puncture on day 2, 4, and 6. All serum samples were assayed for prolactin. The serum was separated from the blood and assayed for prolactin and LH. Prolactin and LH were measured by 74 the RIA described in the general Materials and Methods section. The reference preparations used were H 1010B pro- lactin and NIAMD-LH-RP-l. C. Results The effect of implanting prolactin into the median eminence of intact female rats during random stages of the cycle on serum prolactin is shown in Table 4. Only 2 stages, estrus and diestrus, are shown because these stages had large enough numbers of rats to give the data relia- bility. Prior to implantation, on day 0, serum prolactin during estrus was nearly the same in the two groups, 87.3 and 89.7 ng/ml. Following implantation of prolactin, the mean serum prolactin value during estrus during the 5 day post-implantation period was significantly lower (41.7 ng/ml) than on day 0. It also was lower than serum pro- lactin of control rats implanted with cocoa butter. There was no effect of the prolactin implant on serum prolactin during diestrus. Figure 8 illustrates the mean serum prolactin levels daily beginning on day 0, for rats in estrus at the time of bleeding. These results are the same as shown in Table 4 except they show the daily mean serum prolactin levels. This figure illustrates that as early as 1 day after implantation of prolactin into the median eminence, serum prolactin levels did not rise to pre-implantation levels. 75 sumo Eonm ucmummmwp >HHM0HumHumum you mum umfluomHmQSm mEmm may suflz mammz .Amo. A an umnuo n.m .cmme on“ mo mm H cmwz H mxmav m.m HR.H4 also m.v Hm.km msuumm «Ammo o.m Ho.mm mlvv m.m Hm.mm msuummflo lose cflpomaoum nioav s.mmfim.mm also m.maflk.mm msuumm mfiamv H.m Ho.mm mAmV m.m Ho.vm monummwo Aoav Hmuusn mooou DGMHQEH DGMHQEH umpmd mHOMmm macho msoupmm mwmm WM mo mmmum p ucmEummHB HAHE\ch cfiuomaoum Edumm .maomo msouumm may mcfiHso cfluomaonm Esumm co cfluomaoum mo ucmHmEH m: m0 uommmmll.v mamas 76 '1 [:3 Cocoa butter-implanted rats IOO - Prolactin-implanted rats ' ”'7 \l 0' (II C l \QWJ SERUM PROLACTIN (ng/ml) h) 0' _ i\\\\\ l Figure 8. Effect of median eminence implant of prolactin on serum prolactin concentration during estrus in the rat. 77 The effect of the median eminence implantation of prolactin on serum LH levels in cycling rats is shown in Table 5. Rats implanted with cocoa butter alone had similar serum LH levels during diestrus and estrus, and these serum LH levels were similar to pre-implantation LH levels. Following implantation of prolactin into the median eminence, serum LH levels increased from 53.4 ng/ml to 114.7 ng/ml during diestrus. Serum LH values during estrus increased from 42.6 ng/ml before implan- tation, to 97.3 ng/ml 1-5 days after implantation. These increases are significant (p < .05) when compared to pre- implantation levels of LH, and when compared to rats im- planted with cocoa butter. Prolactin-cocoa butter or cocoa butter alone was implanted into the median eminence during the morning of proestrus, and the rats were bled at l-hour intervals beginning at 4:30 in the afternoon. Rats implanted with prolactin had significantly lower serum prolactin levels compared to control rats implanted with cocoa butter (Table 6). Serum prolactin increased from 71.6 ng/ml at 4:30 PM to 186.7 ng/ml at 6:30 PM in controls. Rats given prolactin implants had less than 20 ng/ml prolactin in the serum at all bleeding times. This inhibition of serum prolactin was statistically significant at all times after prolactin implantation. Thirty-five adult female rats were ovariectomized 3 weeks prior to implantation with either cocoa butter 78 .Amo. A no umgno comm Eoum ucmumMMHp waamoflumflumum uoc mum umfluomummsm meow map nuwz mcmozn.m .cmmE on» NO mm H cmmza nxmav m.mmwm.nm mxmv s.mum.mv mnuumm nxmmv m.mmus.vaa mime H.muv.mm msuummfln “can cfiuomaonm was 5 «.4 Hm.mv mime v.wwa.av magnum mlmmv ~.m wk.mv mime m.4uk.mm msuummfla loan “munch moooo HCMHQEH DQMHQEH Hmumfi mnommm macho msouumm mumm mo .02 can mo mmmum ucwfiummua HAHE\ch mq Esumm .maowo msouumm may msflusp mg Edumm co nanomaoum mo ucmamaw m2 m0 pommmmll.m mamma 79 HOOD. V Q¥¥¥¥ mooo. v a... sec. v a.. No. v m* .cmmE msu mo mm H Gmmza .«.«m.a Hm.HH .«.m.m Ho.ma .«R.H Ho.HH m.vaum.ooa m.mmus.mma H.mmuo.vaa .m.m “H.4H «.mawo.ak m capomaoum m Hmubsm mooou 2m omuh Sm omuw Em omum Em omnv mumm unmanmmue HAHE\m:v :wuomaoum Edumm no .02 .msuummoum mcwuso cfluomaoum Edumm so cflpomaoum mo IA unmamafl m2 m0 uom«mmnl.m mqm¢e 80 alone, prolactin, FSH, or a FSH-LH mixture. All implants were placed in the median eminence area, and 200-250 ug of hormone was used in each implant. Serum prolactin levels were not affected by any of these hormone treatments as compared to cocoa-butter implanted rats (Table 7). Serum prolactin levels were low after ovariectomy, approximately the same as prolactin levels during diestrus. D. Discussion The results of these experiments show that implan- tation of prolactin into the median eminence of cycling female rats decreased serum prolactin during estrus, and increased serum LH during both diestrus and estrus for 5 days. Implantation of prolactin into the median eminence during the morning of proestrus inhibitied the rise in serum prolactin levels normally seen during the late after- noon and early evening of proestrus. This supports the hypothesis that prolactin acts back on the hypothalamus to inhibit its own release, and to stimulate LH release. Implantation of prolactin, FSH, or FSH-LH into the median eminence following ovariectomy had no effect on the already low serum prolactin concentrations. Several articles have recently appeared indicating that a short-loop feedback for prolactin exists. One of the first reports, by Clemens and Meites (1968), indicated that median eminence prolactin implants in cycling female rats inhibited mammary growth and luteal function, and 81 .cmmE may mo mm H cmmz H H.mHo.mm ~.mum.mm n.mus.ma v.muo.mm m.HH OH mq\mmm o.mHm.vm H.vus.ma n.4Hk.mm H.~Ho.- n.~a m mmm m.aum.ma m.mum.mH H.mum.mm o.4H«.mm N.HH m cfluomaoum 4.4Hm.mm R.HHo.mH m.mum.H~ n.4um.vm m.HH m umpusm moooo w awn v mmo N hmo o >mo Amav mumm ucmEummHB .p: m« mo .02 HAHE\ch cauomHoum Esumm .mumu pmNflEouomHHm>o ca :Huomaoum Esumm co mq\mmm Ho .mmm .cfluomaoum mo cofipmucmHQEH mocmcHEm cmHUmE mo pomMMMII.n mqmflh 82 decreased pituitary prolactin concentration. They also reported that rats with prolactin implants had a higher PIF content in the hypothalamus, indicating that the site of prolactin feedback action was at the hypothalamic level. Similar experiments in ovariectomized rats showed the same results as in intact rats. Sinha and Tucker (1968) in- jected female rats with ovine prolactin for 10 days, and found decreased pituitary prolactin content in these rats. Female rats with pituitary transplants under the kidney capsule also had reduced pituitary prolactin content. Ovariectomy did not alter these effects, indicating that the autofeedback control of prolactin can function in the absence of the ovary. Another report (Welsch gt 31., 1968a) indicated that pituitary prolactin was increased, and hypothalamic PIF reduced, following pituitary trans- plantation in intact rats. They also reported that ovariectomy plus pituitary transplantation reduced pitui- tary prolactin. The experiment reported here indicated that prolactin implants into the median eminence of ovariectomized rats did not decrease serum prolactin. It is important to note that serum prolactin levels after ovariectomy alone are already low. Similarly, implants of prolactin did not reduce serum prolactin during diestrus compared to controls. Thus it appears that when serum levels of prolactin are low, prolactin implantation into the median eminence does not lower them further. 83 As noted in eXperiment II of this thesis, serum prolactin increases sharply during the late afternoon of proestrus. Results of this experiment IV indicate that the prolactin implant in the median eminence completely suppressed this rise in serum prolactin. Thus the pro- lactin in the hypothalamus blocked whatever signal normally reaches the hypothalamo—pituitary axis to stimulate pro- 1actin release. It is possible that the signal is in- creased estrogen from the ovaries in response to LH and FSH. This increased estrogen may reach the hypothalamus and stimulate prolactin release by decreasing PIF. Nagasawa 33 31. (1969) showed that median eminence im- plants of estrogen significantly increased serum and pituitary prolactin levels. Evidence is already in the literature indicating that median eminence implants of prolactin can block the stimulatory effects of estrogen on pituitary prolactin (Welsch gt_al., 1968b). They re- ported that l.0 or 5.0 ug estradiol benzoate injected subcutaneously for 5 days was ineffective in increasing pituitary prolactin content and concentration in rats implanted with prolactin in the median eminence, whereas rats implanted with cocoa butter had increased pituitary prolactin levels following estrogen injection. The mechanism by which prolactin in the hypothalamus can block the stimulatory effect of estrogen on prolactin release is not known, but it is likely that this blocking action of prolactin explains the elimination of the serum 84 prolactin peak during proestrus following median eminence implantation of prolactin. Some indirect evidence has been published which suggested that prolactin may stimulate gonadotropin re- lease. Clemens EE.2$' (1969a), reported that implanting prolactin into the median eminence of lactating rats not only depressed lactation, but also stimulated ovulation. Stimulation of ovulation and termination of pregnancy also occurred in pregnant rats following implantation of pro- lactin into the median eminence (Clemens gt 31., 1969b). Implantation of prolactin into immature female rats hastened puberty (Clemens et 31., 1969c) and stimulated FSH (Voogt §£_al., 1969b), and LH (Voogt gt 31., 1969a) release as measured by FSH and LH bioassay of pituitary tissue. The study reported here gives evidence that serum LH is increased even on the days of diestrus and estrus when it is normally low, following implantation of pro- lactin into the median eminence. The mechanism(s) by which a prolactin implant in the median eminence stimu- lates LH release is not clear. 85 V. Effects of an Implant of Prolactin in Median Eminence of Pseudogregnant Rats on Serum and Pituitary LH, FSH, and Prolactin ' A. Objective A short loop inhibitory feedback by anterior pitui- tary (AP) hormones on the secretion of AP hormones has been reported, with indications that each AP hormone in- hibits only its own secretion (Corbin and Story, 1967). However this concept does not appear to apply to the "short 100p" feedback of prolactin. Thus implantation of pro- lactin in the median eminence (ME) reduced the duration of pseudopregnancy (Chen et 31., 1968) and pregnancy (Clemens et_§l., 1969b) by causing ovulation. None of the above work reported serum levels for LH, FSH, and prolactin after implanting prolactin into the ME. It was the purpose of the present investigation to deter- mine the effects of an implant of prolactin in the MB of pseudopregnant rats on serum and pituitary LH, FSH, and prolactin. LRF, FSH-RF and PIF levels in the hypothalamus also were measured. B. Materials and Methods 1. Animals Mature virgin female Sprague-Dawley rats weighing 200-250 g each were obtained from Spartan Research Ani- mals (Haslett, Michigan) and used for these experiments. Two estrous cycles were followed by taking vaginal smears 86 on all rats before the experiments began. Only rats with normal 4-5 day cycles were used. Pseudopregnancy (PP) was induced by mechanical stimulation of the cervix with a glass rod on the day(s) of vaginal cornification (day 0). The appearance of leucocytes in the vaginal smear was designated as day l of PP. The day a proestrous or estrous smear was found was designated as the final day of PP. 2. ME Implantation Prolactin (NIH-P-SB) supplied by the Endocrinology Study Section, NIH, was mixed with equal amounts of cocoa butter. The prolactin-cocoa butter mixture (approximately 250 ug prolactin) was tamped into one end of a 23-gauge glass tube, implanted into the ME with a Stoelting stereo- taxic instrument and left i2.§i£23 All rats were implanted on the fourth day of PP. Vaginal smears were taken daily and all rats were killed by guillotine on the morning 3 days after implantation (7 days after induction of PP). Only rats with proper ME implants, determined by exami- nation under a dissecting microscope, were used for obtain- ing data. 3: Collection and Preparation of .§§rum, PituitariesL and Hypo- .§_alami for Assay Blood was collected under ether anesthesia via the éflmdominal vena cava. Serum was separated and kept frozen at -20 C until assayed. AP's from the implanted rats were indiividually weighed, placed in cold .01 M phosphate buffer 87 in 0.14 M NaCl (pH=7.0), homogenized with a Sonifier cell disruptor and frozen until assayed. The hypothalami were removed immediately after killing the rats, homogenized in 0.1 N HCl, centrifuged at 12,000 g for 40 minutes at 4 C and neutralized with l N NaOH just prior to use. LRF (Piacsek and Meites, 1966), FSH-RF (Mittler and Meites, 1964), and PIF (Kragt and Meites, 1967) were measured by incubation of pituitary halves from mature male rats with pooled hypothalamic extracts. Incubation time for measur- ing LRF and PIF was 2 hours after one-half hr pre-incu- bation, whereas incubation time for FSH-RF was 6 hrs after one-half hr pre-incubation. The incubation medium 199 (Difco Labs, Detroit, Michigan) was stored at -20 C until assayed for LH, FSH, and prolactin. 4. Collection of Ovaries, Uteri, and Mammary Glands for Histological Examination Ovaries and uteri were removed, weighed and fixed in Bouin's fluid after killing the rats. They were stained with hemotoxylin and eosin and examined histologically. The left inguinal mammary gland was removed from each rat, fixed in Bouin's fluid and stained with Harris' hematoxylin. These glands were then rated for degree of development on a scale of 1-6. Ratings of 1-3 indicate progressive increases in ductal development and ratings of 4-6 indicate progressive increases in lobulo-alveolar development, as described previously (Welsch gt al., 1968a). 88 5. Radioimmunoassays (RIA) Prolactin from individual serum samples, pituitaries, and incubation media were measured at 3 dose levels by a double antibody RIA for rat prolactin described previously (Niswender 23 31., 1969). Purified rat prolactin was used as a reference preparation (HIV-8-C, biological potency = .77 x NIH-P-Bl, kindly supplied by Dr. S. Ellis, NASA, Ames Research Center, Moffett Field, California). LH and FSH from individual serum samples, pituitaries, and incubation medium were measured at 3 dose levels. LH was measured by a double antibody technique described by Monroe gt_al. (1968). The reference preparation for rat LH was NIAMD-LH-RF-l with a biological potency equal to 0.03 x NIH-LH-Sl. FSH was measured by a double antibody method described by Parlow gt El. (1969). The reference prepar- ation for rat FSH was NIAMD-FSH-RP-l with a biological potency equal to 2.1 x NIH-FSH-Sl. Pituitary FSH also was measured by the Steelman-Pohley method (Steelman and Pohley, 1953) as modified by Parlow and Reichert (1963). The reference standard used was NIH-FSH-S4, 36.44 IU/mg. Since all calculations compared only two means, controls and experimental groups, Student's "t" test was used to deter- mine significance of differences between groups. 89 C. Results 1. Effect of a ME Implant of Prolactin on Duration of PP Of a total of 28 rats implanted with cocoa butter alone (controls), 26 were still PP when they were killed 7 days after induction of PP. Among 31 rats implanted with prolactin, 28 came into estrus 2 or 3 days later. 2. Effect of a ME Implant of Prolactin on Serum LH, FSH, and Prolactin Serum prolactin concentration was unaffected by im- plantation of prolactin (Table 8). Duplicate measurements of serum prolactin by RIA indicated that during the seventh day of PP, prolactin values for control rats were 14-17 ng/ml serum, approximately equal to normal serum values during the diestrous phase of the estrous cycle. Similar serum prolactin values of 13-17 ng/ml were noted after prolactin implantation, at which time 93% of the rats were in estrus. Since normal serum prolactin values during the morning of estrus range from 80-120 ng/ml, it is evident that the prolactin implant blocked the increase in serum prolactin which normally occurs at estrus. Serum LH concentration increased (p < 0.005) from 109 ng/ml in the controls to 226 ng/ml following implan- ‘Uition of prolactin (Table 8). Serum FSH concentration Shcywed a rise similar to LH, increasing from 199 ng/ml in 90 mo. v m«« moo. v 6. .Hlmmlmmmlaz¢Hz u coflumummmum mocmmmwmfV .HummthIQZde u coHumHmmmHm mocmnmmmmm .OImI>Hm u cowumummmnm mocmummmmm .cmmE mg» mo mm H :mmZH o.~H¢.sH m «.n.mmfim.mmm *m.HmHH.omm m.HHm.mH H mm cfluomaoum o.HHm.~H m v.4muv.mma ~.m H~.moa o.Hum.¢H a «m umupsm moooo Esumm HE\m: . HEM” Ea new” 3.. M...” o mmm 0 ma cfluomHoum a m z .mmm cam .mq .GHUOMHOHQ Enumm GO mumu mm CH :HuomHonm mo coflumucmamfifl m2 mo ummmmMI|.m mqmda 91 the controls to 386 ng/ml in the rats implanted with pro- lactin (p < 0.05). 3. Effect of implant of Prolactin in the ME on Pituitary LH, FSH, and Prolactin Pituitary prolactin concentration decreased 47% following prolactin implantation, from 2.57 ug/mg AP in control rats to 1.35 ug/mg AP in rats implanted with pro- lactin (Table 9). Pituitary LH concentration, although slightly reduced, was not significantly affected by im- plantation of prolactin. Pituitary FSH concentration decreased from 5.87 ug/mg AP in the controls to 4.06 ug/mg AP after prolactin implantation (p < .001) (Table 9). Bio- assay of pituitary FSH revealed a similar decrease follow- ing prolactin implantation. 4. Effect of a ME Implant of Prolactin on Hypothalamic PIF, LRF, and FSH-RF Content Hypothalamic PIF content was increased only slightly following implantation of prolactin for 3 days, as indi- cated by decreased release of prolactin from the AP tissue during incubation with hypothalamic extract (Table 10). However this 14% decrease in prolactin release was not significant. The 21% decrease in LH release in response to hypothalamic extract from rats implanted with prolactin was not statistically significant. There was no effect on hypothalamic FSH-RF content after prolactin implantation. 92 sec. v m« .mump mammmofln HON HmlmmmlmHz 0: mm pmmmmumem .mumw «Hm mom HummnmmmuazaHz u coflnmumdmum mocmummmmv .Hnmmlmqlodez u coflumummmum mocmummmMm .UImI>Hm u cowumummmum mocmummmmm .cmmE may mo mm H cmmzH smmmMOHm Amo.m:¢~.mvso.m uuuuuuuuuu mm cfluomaoum smmmmoflm Am¢.muam.mvmm.q u uuuuuuuuu «N umuusm moooo «Hm «om.oumo.v wa.vHHm.mm «ea.onm.H mm :fluomHoum «Hm mm.ofism.m mv.mHmm.mm m~.owsm.m 4m umuusm moooo mammd . . m4 mE\ms . md mE\m: N.Hm¢ mE\ms mumm 0 mmm mmm Hocoo mmm mmH.ocoo ma mm .mcoo o .o usmEpmmHB w B cHuomHoum m z .coflumuucmocoo mmm cam .mq .cfluomHoum mumuflsuflm co mums mm QH GfluomHoum mo coflumucmamafl m2 m0 pomMMMII.m mqmdfi 93 .HlmmlmmmIQZHm u coflumummmum mocmumwmmm .cmmE mnu mo mm H cum:H mcawmom swaumwmm ovuavm mm :uuomHoum mmmwomofl vvmummmm «muomm 4m umuusm moooo v.HAm< mE\mcv m.afim¢ mE\mcv N.Hflm< mE\mcv mumm .pmz CH pmmmmamm .pmz ca pmmmmamm .pmz CH pmmmmamm mo .02 ucmfiummue 3mm mg cfluomfloum .ucmuaoo mmummm 6am .mmq .mHm onsmamguomss co mums an an cfluomaonm «0 coflumucmsmefl m: m0 pommmmuu.oa mamas 94 5. Effect of a ME Implant of Prolactin on Organ Weights and Mammary Gland Development Table 11 indicates that ovarian weight is unchanged following prolactin implantation. However, a distinct in- crease in follicular development and a decrease in luteal tissue was seen in ovaries from prolactin implanted rats (Fig. 9B) as compared with ovaries from cocoa butter im- planted controls which remained in PP (Fig. 9A). The dis- appearance of corpora lutea may be due not only to a reduction in prolactin release by the AP, but also to luteolytic activity resulting from increased LH release. The uterine weights of rats implanted with prolactin and cocoa butter were significantly greater (p < .00005) than in rats implanted with cocoa butter alone. Microscopic examination of the uterus from prolactin implanted rats (Fig. 10B) revealed a definite stimulation of the epithelial and endometrial layers when compared with the uterus of rats implanted with cocoa butter (Fig. 10A). AP weights did not differ 3 days after prolactin implantation. There was a significant atrophy of the mammary glands following prolactin implantation as seen by the mammary gland ratings (Table 11). The mammary glands of the control PP rats were given an average rating of 4.25 and showed a well developed lobulo-alveolar system (Fig. 11A), as compared with mammary glands from prolactin- implanted rats which were given an average rating of only 95 moooo. v 6*“ moo. v a. cam: man no mm H cams H .o~.ouoo.~ o~.ouoo.o .«o.-Hm.mam m.ouo.oo mm cflpomaoum o~.owmm.o m~.ouoo.o H.omuo.oflm o.~Ho.oo mm umuusm mooou mmcHumm AmEv uanmz H GQMHO HA95 uanmz H mcHHmuD H38V unmmmz mumm ucmEummHB wum 2 .>4 mfi .>< .>¢ GMHHM>O >¢ mo .02 .mmcHumu UCMHm humEEmE pom muanm3 cmmuo co mumu mm cH cHuOMHoum mo GOHumucm Hmsfl m2 m0 pommmmnu.aa mqmmH chUMHoum Enumm co cHuomHoum mcH>o msocmmoxm mcHuomth mo uomwmmll.mH mamme 104 compared to saline injected controls after 1 hour of suckling, the differences were not significant due to very large variations among rats. Effect of exogenous prolactin on serum LH levels following suckling is shown in Table 13. Serum LH levels following suckling were very low in both saline and prolactin injected rats at all 3 times measured. The second 2 groups of rats received either prolactin or cocoa butter implants in the MB on the fourth day of lactation, referred to as day 0 in Tables 14 and 15 and Fig. 12. The litters (8 pups/litter) of control lactating mother rats gained 10-21 gms/day for 6 days. Litters from mother rats implanted with prolactin gained much less, from 0.2-13.7 gms/day. Even 6 days after implantation of pro- lactin, these pups gained less than half as much as litters nursing control mother rats. Thus the prolactin implant was effective in reducing lactation for at least 6 days. All rats were bled 1 hour after suckling prior to ME implantation of prolactin or cocoa butter on day 0, the fourth day of lactation. Serum prolactin levels averaged 275 and 288 ng/ml in the 2 groups (Fig. 12). On day 2, ME prolactin completely inhibited the rise in serum pro- lactin usually observed following suckling. This inhi- bition lasted at least 6 days, similar to the period of time that lactation was decreased in rats with ME implants of prolactin. The effect of these ME implants of prolactin on serum LH following suckling is shown in Table 15. There 105 .cmmE map mo mm H ammzH m.mHo.mH o.auo.oo o.mum.am o afloomaonm H.mum.ma o.mHm.oH o.muo.om o meadow Ha moo o awn o soc mumm ems awn mo .02 u u a HAHE\maV mu Esumm .mCHonom maH3oHHom mHm>mH ma Edumm co aHuomHoum maH>o msoammoxm mo uommmmll.MH mamme 106 m.m m.OH H.NH h.MH N.o N.m mH aHuomHOHm m.am m.oa k.oa m.oa m.HH o.oa ma umuusm moooo o one m son o smo m son N moo H moo wwwuwwm pamfiummue AmEmv aHmw .uz Hmaqu mHHmo .>a .aHmm uamHm3 umuuHH mHHmp ao mama maHumuomH CH aHpUMHonm maH>o mo uamHQEH m: M NO ummmmmul.vH mamaa 107 .ammE map mo mm H ammz H o.muo.oH o.HHo.oH o.muo.oH H.muo.ma oH couomaoum H.muo.ma o.mHm.o o.muo.oa o.~Hm.oN ma umuusm «0006 o own o son m moo o smo mumm mo .02 pamfipmmue HAHE\mav ma Edumm a0 mama mCHpmuomH .mH Edumm aH aHuOMHoum mo COHumuamHmEH mmamaHEm CMHme mo pomMMMII.mH mamae 108 .cHuomHoum mo muamHmEH m2 auH3 mama maHuMHOMH aH mHm>mH aHuomHoum Edumm .NH mHsmHm m >40 4 >40 N >40 0 >40 lg. §o so? \\ a w 53%. 2.530%. g [I .. . com 0 53%.. $58 <88 D a 109 was no difference in serum LH levels following suckling in rats implanted with prolactin in the ME compared to cocoa-butter implanted controls. Serum LH was very low at all times in both groups. D. Discussion This study demonstrates that placement of prolactin into the median eminence of postpartum lactating rats significantly impairs lactation as measured by litter weight gains. This decrease in lactation is probably the result of an inhibition of the serum prolactin rise which normally occurs during suckling (Amenomori gp‘gl., 1970). As early as 2 days after implantation of prolactin into the ME, serum prolactin levels were less than 20 ng/ml after 1 hour of suckling, whereas control rats had serum prolactin levels of more than 300 ng/ml after 1 hour of suckling. This significant inhibition of serum prolactin continued for the duration of the experiment, 6 days. It appears that a glass tube filled with 250 ug pro- lactin and placed in the ME area of the hypothalamus, can release prolactin from its site for at least 6 days and inhibit prolactin secretion from the pituitary. About 40 ug of prolactin are calculated to be released daily, assuming that all of the prolactin is released in 6 days and the same amount is released each day. It is probable that lactating rats have an average serum prolactin level around 200 ng/ml serum, based on studies by Amenomori 110 pp Ei- (1970) and in this thesis. This means there may be about 2000 ng (200 ng/ml x 10 ml serum in lactating rats) of prolactin in the serum of a lactating rat. Since prolactin has a calculated half life of only 13 minutes (Gay ep'gl., 1970), it is possible that during lactation prolactin is secreted in much greater quantities per day than 40 ug, which may be the amount released/day from the glass tube. Thus this amount of prolactin acting on the ME may be within a physiological range. These results confirm work by Clemens §E_al. (1969a) who found that similar ME implants of prolactin decreased lactation. They also found decreased mammary gland weight and reduced secretory activity of the mammary gland. Similar to my results, they found that lactating rats im- planted with prolactin came into estrus and began to cycle. This was accompanied by ovaries which had large follicles and smaller corpora lutea than controls. These results and others from our laboratory de- scribed in the literature review suggest that an implant of prolactin in the ME not only inhibits prolactin re- lease, but also stimulates gonadotropin release. As described in experiments IV and V of this thesis, ME im- plants of prolactin increased serum LH and FSH levels. Similar serum LH increases were not found in the present experiment despite a return to estrus. One possible explanation is that the suckling stimulus temporarily blocks or inhibits LH release almost completely. As lll noted in the results, serum LH in control lactating rats is very low, almost undetectable, following suckling. The suckling stimulus is necessary to maintain post- partum lactation by evoking release of prolactin (Meites, 1959; Amenomori, 1970) and ACTH (Voogt gp_gl., 19690). It is believed that nerve impulses beginning at the nipples travel by way of the spinal cord to the brain and to the hypothalamus. These impulses are important for releasing prolactin and ACTH, presumably by inhibiting or lowering PIF levels, and stimulating CRF (corticotropin releasing factor) levels. The mechanism by which the pro- lactin implant in the ME interferes with this signal is unknown. It is possible that PIF activity is enhanced and prevents prolactin release during suckling, when prolactin is present in the ME. Clemens gp_al. (1968b) found a sig- nificant increase in hypothalamic PIF content and a sig- nificant decrease in pituitary prolactin concentration in cycling female rats implanted with prolactin in the ME. Minaguchi and Meites (1967) found that suckling reduced hypothalamic PIF content. It is possible that the con- stant presence of prolactin in the ME can overcome the stimulus evoked by suckling which normally would reduce PIF and increase prolactin release. 112 VII. PituitarinH and Hypothalamic GHRF After Median Eminence Implantation of Ovine and Human GH A. Objective This study attempts to elucidate the site where GH inhibits its own secretion by measuring the effects of im- plants of human or ovine GH into the median eminence area on pituitary GH and hypothalamic GHRF (growth hormone re- leasing factor) content. It was also of interest to deter- mine whether human GH, which has prolactin activity, could act to inhibit prolactin secretion, similar to the feedback properties of ovine prolactin. Serum prolactin, mammary development, pituitary weight, and body growth were measured in rats. B. Materials and Methods 1. Experimental Animals Immature 2l-day-old female rats and mature lO—lZ—week— old female rats of the Sprague-Dawley strain (Spartan Re- search Animals, Haslett, Michigan) were used for the im- plantation studies. Mature male rats weighing 350-400 gms were used as pituitary donors. For GH bioassays, immature female rats (Hormone Assay Labs, Chicago, Illinois) were hypophysectomized at 26 days of age and shipped 7 days later. Bioassays were begun 9-10 days later. All animals were maintained on a schedule of 14 hrs. of light and 10 hrs. of darkness at 2511 C. The rats were fed Wayne Lab 113 Blox pellets (Allied Mills, Chicago, Illinois). The diet of the hypophysectomized rats was supplemented with carrots, orange slices, and sugar cubes. 2. Treatments Immature 21-day-old female rats were implanted stereotaxically in the median eminence with a single pellet containing approximately 100 ug ovine GH mixed in choles- terol. A Stoelting stereotaxic instrument was used to- gether with DeGroot's atlas (1959). Control rats were implanted with an equivalent amount of cholesterol alone in pellet form. The animals were weighed and head to tail measurements were recorded daily for 2 weeks. In two separate experiments, mature cycling female rats were given a single stereotaxic implantation of approximately 250 ug ovine GH (OGH) or human GH (HGH) in cocoa butter in the median eminence area, while control rats received cocoa-butter (CB) alone. The GH-cocoa butter mixture was tamped into the tip of 23-gauge glass tubing, implanted and left lfl.§i§2} Implants were fixed in place by using dental cement and skull screws, as described previously by Clemens and Meites (1968b). Vaginal smears were taken daily prior to and after implantation. All rats were killed by guillotine 7 days after implantation and examined under a dissecting microscope for site of the implant. Only rats with properly located implants were used for obtaining data. 114 3. Preparation of Hypothalamic Extracts and Pituitaries The hypothalami were removed immediately after killing the rats, homogenized in 0.1 N HCl, centrifuged at 12,000 g for 40 minutes at 4 C and neutralized with 1 N NaOH just prior to use. GHRF was measured by incubating pituitary halves from male rats for 6 hours with hypothalamic ex- tract, as described previously by Dickerman 33 al, (1969a). The incubation medium was stored at -30 C until assayed for GH. Anterior pituitary glands (AP) from implanted rats were placed in cold saline, homogenized with a Sonifier cell disruptor and assayed for GH. 4. GH Bioassay Growth hormone activity was assayed by the standard tibia test of Greenspan g£_al. (1949). All bioassays in- cluded 2 doses of the control pituitary tissue or incu- bation medium, and 2 doses of the experimental pituitary tissue or medium. The reference standard (NIH-GH-S8) was also assayed at 2 dose levels. Four rats were used for each assay point. Statistical treatments included analy- sis of the symmetrical 4-point parallel line assays, calculated mean tibial responses, standards errors, analy- sis of variance, and relative potencies of experimental versus control test preparations. Student's "t" test was used to compare differences between 2 groups of organ weights, mammary gland ratings, etc., whereas one-way 115 analysis of variance was used when means of more than 2 groups were compared. C. Results 1. Pituitapy GH Concentration After Median Eminence GH Implantation Pituitaries were bioassayed at 2 dose levels, 2 and 4 mg/assay rat/4 days (Table 16). Pituitary GH decreased 25% in experiment I and 64% in experiment II, 7 days after HGH implantation in adult female rats, as compared to controls implanted with cholesterol or cocoa butter. Similar decreases in pituitary GH were observed following OGH implantation. The higher GH values in experiment I as compared to experiment II may be due to more sensitive bioassay rats. 2. Hypothalamic GHRF Following Median Eminence GH Implantation One dose (.5 hypothalamic equivalents/incubated pituitary) was used to assay hypothalamic content of GHRF ipjyipgp. Table 17 shows that implantation of HGH re- duced hypothalamic GHRF 48% in both experiments I and II. A similar decrease in hypothalamic GHRF was observed in experiment II following implantation of OGH. Hypothalamic GHRF was not measured in OGH implanted rats in experiment I. 116 .conHomum mo xmch o .muHEHH moampHmaoo wmm cam ammZM .ammE map mo mm H ammZN .mmumwlmHz mo muamHm>Hsmm ms nu Ummmmumme HNH. AoNo.nooH.ooNo. o.N Hm.oN oMoNN o o+moa N oH mo mca>o omN. Aaoo.nomo.oooN. o.N Hm.oa ‘ oMooN o I oowooa N oa no cmssm o.m +m.mv oHNoN o o+mmN N oH maouuqoo N moo. Ammo.nomm.onm. N.H Ho.oa mMmNH o n «Homo N oa mo mcfl>o o.a +o.oN NHHoN o m+th N m mHouuaou oNH. Ammo.umo4.ommo. o.v “N.oo oMmoN o I mHmvN N b m0 Gmfidm m.oa+o.mHH ouoam o o+NoN N o maouocoo a I mmmma s m m Amm+cmmzo loom . o4 m>wmwwmm N.Ham< mw\ so Ago gnome \m4 oso mwuwwz pamamsH .mmm . Nmanaa .>« mmoo .coHumuuammaoo aw humuHsuHm so mmmp n MOM mama mHmEmw uH3pm mo moamsHEm QMHme mau aH pamHQEH mm mo uommmmul.mH mqmda 117 .aonHomum mo xmpaH v .muHEHH moampHmaoo wmm paw ammZM .ammE map mo mm H cmmzN .mmImUImHz mo mucmHm>Hsvm ms nu pmmmmnmme moH. AoNo.uooH.ooom. o.ouo.o mMmoH N.N v+NNH m.o oH maH>o NMH. Amom.lnmm.0>vm. o.on.m mmHmH N.H I oHHoH m.o oH mo cassm m.H+v.HH mHOHN N.H h+omH m.o 0H mHoupcou N moo. lomo.noom.ooom. N.Hwo.oN oMooH N.H I mHmmH m.o n no amadm m.v+m.Nm mHmwN N.H ¢+mmH m.o m mHouucoo H AN. ma Ammwcmmzo umm momma . H MMWMMpMm mE\msv any apsz \muamHm>H:mm mwuwwz pamHQEH .mmm o . N oommmamm mo Hoanae .>« ma mmoo .pr>Hpom mmmw UHEmHmauomha co mama h HON mama mHmEmm uHSUM mo mocmaHEm cMHUmE map ouaH pmuamHmfiH mm m0 ummmwmII.NH mamas 118 3. Serum Prolactin Levels in Rats with Median Eminence HGH Implant Serum prolactin levels were measured by the radio- immunoassay of Niswender §E_gl. (1969). Blood samples were taken by heart puncture under light ether anesthesia just prior to and 3, 5, and 7 days after implantation of HGH in the median eminence. A significant (p < .002) decrease in serum prolactin was observed in rats in estrus following HGH implantation as compared to controls (Table 18). There also was a decrease in serum prolactin 3, 5, and 7 days after HGH implantation in rats in estrus as compared to pre-implantation estrous values (Day 0). There was no effect on serum prolactin in rats during diestrus. 4. Body Growth in Zl-Day-Old Female Rats Implanted with GH in the Median Eminence Table 19 shows the effect of an OGH implant into the median eminence of 21-day-old female rats on body weight and length. Body weight gain 7 days after implantation averaged 21.8 gms in OGH group and 14.7 gms in the choles- terol group, representing a significant increase in body weight. However, subcutaneous injections of 200 ug OGH had no effect on body weight as compared to saline in- jected controls. Three rats in this latter group failed to gain more than 2 gms during the first 7 days. By day 14, average body weight gains were equal in both groups. There were no differences in body length after 7 or 14 days. The average age at the time of vaginal opening was .amo. v 00 pamummep mHuamonHamHm mum mpmHuomHmQSm pamumMMHp auH3 mammz 119 a.m .cmmE map mo mm H ammzN .HmImImHzxnn.o u mmamuom HmonoHoHa pmumam m auHB OImI>Hm n GOHumummmHm moamummmmH ma.o Ho.Nm hm.omus.oo msupmm mo.m Ho.oa «H.N Hm.HN manummflo ma mo sass: am.omwo.mNN om.oNHo.No msuumm no.4 “N.mN wo.N Hm.oa manummflo oH maouucoo umm ucmHQEH mo .02 AHE\mav HaHuomHonm Esnmm mo mmmum .aoHumuuammaoo aHuomHoum Edumm co mumu mHmEmm uHspm Mo moamaHEm GMHUmE may CH uaMHQEH m0 m0 uommmmII.mH mqm4a 120 m0. v m« omumoumHzN .ammE map mo mm H ammZH m.owm.o m.ouo.o o.ouN.oH m.oHH.mm 4o.NHo.HN m.ouo.om oH Nmm mcH>o m.ouo.o m.oum.o m.oHo.oH o.NHm.om N.HHo.oH m.ouo.om oH HouopmmHoao HMMoowH wagowN HasooHHma HmooH moo Amos moo Hmo.uz whom a n an n on ommm H H H >a aHmo ma aHmo moom mo pamHmEH mmmmuoaH mmmmumaH .apmamq . >.o . w 0 ma Ho .0 sumcmq numcmH HmHuHcH us 6 m as o m H .u. H z .mumm mHmEmw UHOImmleN mo auzoum mpoa a0 moamaHEm amHUmE map aH uCMHQEH m0 m0 HomMMMII.mH mqm4fi 121 36.2:l.l days for the OGH group and 36.4:l.0 days for the cholesterol group. 5. Organ Weights and Mammapy Develgpment of Adult Female Rats 7 Days After OGH or HGH Implantation Table 20 summarizes data from 2 separate experiments. It shows that implantation of either OGH or HGH into the median eminence of adult cycling female rats had no effect on body, ovarian, or uterine weights by 7 days after im- plantation. A very significant decrease in AP weight in the HGH group was observed in both experiments. A less significant decrease in AP weight occurred in the OGH group. The mammary glands were fixed, stained, and rated for degree of development by a method described previously by Meites (1959). Mammary glands from control rats im- planted with cocoa butter showed well-developed ducts and some lobulo-alveolar development (Fig. 13A). Implantation of HGH resulted in marked atrophy of the mammary glands, with only ducts evident (Fig. 138). Implantation of OGH caused slight atrophy of the mammary glands when compared with the controls, but this was not nearly as significant as in the HGH group (Fig. 13C). Average mammary gland ratings (Table 20) of control rats were 3.810.l in experi- ment I and 3.710.2 in experiment II, whereas the HGH groups had ratings of 2.810.l and 2.310.l in experiments 1 and 2 respectively, representing a very significant 122 moooo. v a... mooo. V a.. No. v a. .N.oHH.m m.omHo.mmm o.HHo.HH o.oHo.o m.oHo.oNN «.mHo.moN oH no mcH>o ...H.on.N o.HNHH.ooN H.oHo.mo .m.oHo.o v.4Ho.HNN o.NHo.NoN oH so cuss: N.oHo.m o.omHH.ooN N.mHo.oo o.oHo.o N.¢Hm.omN o.oHH.ovN oH Houucoo N ..H.oHo.N In- In- ..m.on.o o.mHm.omN o.on.moN o no cues: H.oHo.m II. 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