w 0. .o 2}. . o r .. co .6 '5‘. . T;..'.'. o 0-. h..."‘ I A}?! - ';‘,"‘." N V n.‘ ‘ (A; ’ I -- x.-_. ‘... O ,. ..- - . .- - 0 Cu... 0. “fi. ”'0.‘ IE0... Fw... in. as“... flu. a u: .1. A EU 3. to. IMFHIS l. «=NJJJ“t i1 HMS & SIINS' 1 ,_ a 11 300K BRIBERY MB. ‘ mum _IINDIRS ABSTRACT LOCALIZATION OF GONADOTROPIN RELEASING HORMONE WITHIN THE BOVINE HYPOTHALAMUS BY Kerry S. Estes Fresh, frozen bovine hypothalami were cut in 500 micron sections in 3 planes and sections were extracted with 2N acetic acid. The LH releasing activity and gonadotropin releasing hormone (GnRH) concentrations of hypothalamic extracts were assessed by an in vitrg method using bovine 'pituitary cell monolayer cultures and radioimmunoassay specific for the releasing hormone. The endpoint of the i2_zit£g assay was LH concentration of incubation media 2 hr after introduction of hypothalamic extracts or synthetic GnRH to the cultures. Concentrations of LH were determined with radioimmunoassay. Concentrations of immunoreactive GnRH were expressed as ng GnRH/mg protein. Greatest concentrations of immunoreactive GnRH were found in the sections containing the medial pituitary stalk and the median eminence with smaller concentrations in the anterior hypothalamic region. Peak concentrations found in Kerry S. Estes the anterior hypothalamic region were about 25 percent of maximum concentrations found in the stalk-median eminence area. Biological releasing activity was confined to the region of the pituitary stalk and median eminence. The in yitrg LH response to hypothalamic extract from stalk con- taining sections was not parallel to the response for synthetic releasing hormone. Magnitude of biological response in sections from the stalk—median eminence region was greater than could be accounted for by immunological GnRH activity. These findings suggest that bovine pituitary cells in culture may respond to factors from extracts of bovine pituitary stalk and median eminence areas other than gonadotropin releasing hormone. This suggests that biologi- cal LH releasing activity and immunological GnRH are found in greatest concentrations in the areas of the bovine pituitary stalk and median eminence. LOCALIZATION OF GONADOTROPIN RELEASING HORMONE WITHIN THE BOVINE HYPOTHALAMUS BY 00’ Kerry S. Estes A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1976 ACKNOWLEDGMENTS I wish to express my graditude to a few individuals, without whose help and concern, this thesis would not have been possible. To Dr. Edward M. Convey, my major advisor, I am most grateful for the many hours he willingly devoted to careful and thoughtful direction of my graduate studies. The members of my graduate committee, Drs. Harold D. Hafs and Robert Echt, have been instrumental in guiding my studies and furthering my understanding of reproductive and cellular physiology. Dr. Roger Neitzel was most cheerful in his assis- tance with stubborn computer terminals and willingness to design and execute computer programs used to analyse these data. Dr. Vasantha Padmanabhan's help and expertise of cell culture techniques made feasible the bioassay. My student colleagues and especially, Jim Kesner, deserve recognition for their pleasant attitudes and assis- tance expressed at critical times. ii Appreciation is extended to the Dairy Department for the use of facilities and financial support. And finally, I thank my parents, whose continued love and support have given me courage to pursue my goals. iii TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . REVIEW OF LITERATURE. . . . . . . . . . Anatomy of the Hypothalamus and Hypophysis. . General. . . . . . . . . . Nuclear Distinctions within the Hypothalamus Neuroendocrine Intergrating Mechanisms- .An Historical Perspective. . . . . . . Role of the Adenohypophysis as "Master Gland". . . . . . . . . . Role of the Hypothalamus in Neurosecretory Control of AdenohypOphysis. . . . . . Evidence for Releasing Factors. . . . . . Initial Isolation and Characterization of Releasing Factors . . . . . . . . . Localization of the Sites of Releasing Hormone Production and Concentration. . . . . . MATERIALS AND METHODS . . . . . . . . . Experimental Design . . . . . . . . . Tissues . . . . . . . . . . . . . Collection. . . . . . . . . . . . Sectioning. . . . . . . . . . . Extraction Procedure . . . . . . . . iv Page vi vii 12 17 17 17 17 18 22 Radioimmunoassay (RIA) of Gonadotropin Releasing Hormone. . . . . . . . . . Bioassay for Gonadotropin Releasing Ho mone O O O O O O O O I O O O 0 Procedure for Gonadotropin Releasing Hormone Bioassay . . . . . . . . . Experimental Design for Gonadotropin Releasing Hormone Bioassay . . . . . . Experiment l--Dose Response of Gonadotropin Hormone and Hypothalamic Extract . . . Experiment 2--Bioassay of Sagittal Section of 5 Hypothalamic Areas . . . . . . . Experiment 3--Bioassay of Frontal Section Section Extracts of 6 Hypothalamic Areas. RESULTS AND DISCUSSION . . . . . . . . . Radioimmunoassay of Gonadotropin Releasing Hormone (GnRH). . . . . . . . . . . Standard Inhibition Curve for Gonadotropin Releasing Hormone . . . . . . . . . Gonadotropin Releasing Hormone in the Bovine Hypothalamus . . . . . . . . Bioassay of GonadotrOpin Releasing Hormone. . . . . . . . . . SUMMARY AND CONCLUSION . . . . . . . . . BIBLIOGRAPHY. . . . . . . . . . . . . APPENDICES . . . . . . . . . . . . . Appendix A. Procedure for Total Protein Determination . B. Composition of Reagents used in Radioimmunoassay. . . . . . . . . C. Iodination Procedure for GnRH . . . . . D. Composition of Culture Media . . . . . Page 23 28 28 32 32 33 33 35 35 35 39 51 65 68 76 76 78 81 82 LIST OF TABLES Table Page 1. Procedure for Radioimmunoassay of Gonadotropin Releasing Hormone. . . . . . . . . . . 27 2. Animal Variation in Number of Sections Cut and Total GnRH and Protein Content of Hypothalami in 3 Planes. . . . . . . . . 40 3. Effect of LH concentration of Pooled Hypothalamic Extracts on LH Response of Cultured Pituitary Cells . . . . . . . 58 vi LIST OF FIGURES Figure Page 1. Structure of gonadotr0pin releasing homone O O O O O O O O O O O O O O 11 2. Ventral surface of a bovine brain and a hypothalamic tissue block excised for sectioning O O O O O O I O O I 0 2 o 3. Typical elution profile separating iodinated hormone on an LH-20 column . . . . 25 4. Schematic representation of the preparation of bovine anterior pituitary cell cultures and design of in vitro experiments. . . . . 30 5. Inhibition curves for synthetic gonadotropin releasing hormone (GnRH) and bovine hypothalamic extract (B-HE) . . . 37 6. Multiple regression curve for immunological ‘ gonadotropin releasing hormone concentra- tions versus horizontal section. . . . . . 43 7. Multiple regression curve for immunological gonadotropin releasing hormone concentra- tions versus frontal section. . . . . . . 47 8. Multiple regression curve for immunological gonadotrOpin releasing hormone concentrations versus sagittal section . . . 49 9. Luteinizing hormone response of pituitary cell cultures to log dose of synthetic gonadotrOpin releasing hormone and hypothalamic extract . . . . . . . . . 53 vii Figure _ Page 10. Luteinizing hormone response of pituitary cell cultures to extract pools from 6 areas of 6 hypothalami cut in the frontal plane. . . . . . . . 60 ll. Luteinizing hormone response of pituitary cell cultures to extract pools from 5 areas of 6 hypothalami cut in the sagittal plane . . . . . . . . . . . 62 viii INTRODUCTION Numerous investigators have attempted to elucidate the mechanisms of neurohumoral control of the anterior pituitary. Early studies, using electrical stimulation, lesions and bioassay techniques, demonstrated the neurosec- retory centers which control the pituitary's release of several hormones are contained within the hypothalamus. With the structural identification of a neural hormone responsible for release of gonadotrOpins from the anterior pituitary, and subsequent development of specific and sensitive assays for the hormone, scientists are now able to localize areas within the hypothalamus which are responsible for regulation of hormones which control reproduction. Gonadotropin releasing hormones isolated from several species have identical structures. Further definition of discrete areas within the hypothalamus which secrete or produce various releasing hormones, will lead to more complete understanding of the neural organization and integrating mechanisms which regulate reproduction. The purpose of these experiments was to localize concentrations of gonadotropin releasing hormone activity \Within the bovine hypothalamus. Gonadotropin releasing activity measured by radioimmunoassay specific for this decapeptide and luteinizing hormone releasing activity determined by bioassay were compared in experiments designed to determine whether hypothalamic areas demonstrating immunoreactive hormone and biologically active releasing substance coincide. Elucidation of neural areas in which releasing hormones are concentrated will hopefully lead to further investigation of the mechanisms which control synthesis and stimulate secretion of releasing hormones. Understanding of the neural controls involved in reproduction, would lead to more effective and hopefully, safer methods to control fertility. REVIEW OF LITERATURE Anatomy of the Hypothalamus and Hypophysis General The hypothalamus is the most ventral portion of the diencephalon and is partially exposed on the ventral surface of the brain. Hypothalamic structures evident on the ventral surface (rostral to caudal) are: Optic chiasm, infundibulum, tuber cinereum and mammillary bodies. The hypophysis or pituitary, which lies within the sella tur- cica, is attached to the brain by the infundibulum or pituitary stalk. The base of the infundibulum together with the rostral portion of the tuber cinereum is often referred to as the median eminence. Functionally, the hypophysis is composed of two parts which reflect two different embryo- logical origins. The neurohypophysis or posterior pituitary, which secretes substances synthesized in the hypothalamus, arises from the embryonic infundibular process of the diencephalic floor. The adenohypophysis or anterior pitui- tary is glandular tissue which differentiates from a diverticulum of the stomodal roof called Rathke's pouch. Anatomically, the hypothalamus is defined dorsally by a groove in the wall of the third ventricle called the hypothalamic sulcus; rostrally by the optic chiasm; rostro- laterally by the first segment of the optic tract; caudolaterally by the groove formed by the cerebral pedun- cles; and caudally by the limiting border of the mammillary bodies. Nuclear Distinctions within the HypotHaIamus Three gray areas are contained within the hypothala- mus which are subdivided into specific nuclei. The supraoptic or rostral area lies above the Optic chiasm and fuses rostrally with the preOptic area which extends into the telencephalon. The most rostral area contains the supraoptic nuclei which are associated with the dorsal side of the optic tract, and the paraventricular nuclei which lie dorsomedially in close proximity to the wall of the third ventricle. Axons from these nuclei extended to the neurohypophysis. The periventricular nucleus is in the border of the third ventricle and includes fibers which extend to the thalamus. The tuberal area is dorsal to the tuber cinereum and just caudal to the supraoptic area. Within the tuberal area, the infundibulum attaches to the median eminence. Near this attachment, the ventral portion of the periventricular nucleus is histologically indistinguishable from the arcuate nucleus. For this reason, both names are often used to define the area enveloping the base of the third ventricle. Other nuclei of the tuberal area lie lateral to the periven- tricular nuclear border of the third ventricle and include the dorsomedial, ventromedial and lateral nuclei. The names of these nuclei define their relative position. However, no distinct separation exists for easy identification. The most caudal gray areas of the hypothalamus are the mammillary bodies which give rise to the mammillothala- mic tract which passes dorsolaterally to communicate with the thalamus. Dorsal to the mammillary bodies and between the diverging mammillothalamic fibers is the caudal hypothalamic nucleus. Neuroendocrine Intergrating Mechanisms- An Historical Perspective Role of the Adenghypophysis asIFMaster Gland" Greep (1974) extensively reviewed historical developments which established the adenohyp0physis as the major endocrine gland controlling hormonal secretion of the systemic endocrine system. Because of its major role in hormonal regulation, it is often referred to as the "Master Gland." Briefly, six hormones are secreted from the adeno- hypophysis; thyroid stimulating hormone (TSH), adrenocorti— cotroPic hormone (ACTH), follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH). The first four of these hormones have specific endocrine glands as their target tissues, while the last two hormones function to regulate metabolism at several target tissues. Role of the Hypothalamus in Neurosecretory Control of Adenohypophysis The hypothalamus is richly endowed with afferent and efferent nerve fibers connecting it to the higher nerve centers. These connections facilitate its functions which include control of the autonomic nervous system and regula- tion of appetite, water balance, body temperature, blood pressure, the sleep-walk cycle, and behavior. McCann and Porter (1969) reviewed develOpments leading to the concept of neurohumoral regulation of the adenohypophysis. As early as 1933, Hinsey and Markee suggested that hormonal transmission from hypothalamic neural cells could regulate the adenohypophysis. Credibil- ity was given to this suggestion with the discovery of a vascular plexus between the adenohypophysis and the hypothalamus. Green and Harris (1947) observed in living rats the flow of portal blood and concluded it was from the brain to the adenohypophysis. Numerous investigations have attempted to elucidate the mechanism of neurochemical con- trol of the adenohypophysis. McCann and Porter (1969) suggested criteria which should be met to prove the hypothe- sis that neurohumoral regulation of the adenohypophysis exists. They include: 1. demOnstration of secretory elements in the brain which produce substance within diffusible distance from the portal vessels; 2. identification and characterization using biologi- cal, physical and chemical criteria of substance originating from the hypothalamus within the portal vessels, and 3. demonstration that hypothalamic agents alter adenohypophyseal hormone release after entering the portal vessels. Evidence for Releasing Factors Halész, et a1. (1962) and Knigge (1962) transplanted adenohypophyseal tissue under a kidney capsule and into various regions of the brain including the hypothalamus in rats. They observed maintenance of adenohypophyseal func- tion only when the pituitary was implanted within the hypothalamus. From these observations, they concluded that the hypothalamus must function by secreting substances to maintain the adenohypophysis. Further studies with implanted tissue attempted to define areas within the hypothalamus essential for adenohyp0physeal maintenance. The hypophysiotrOpic area was defined as the area in which transplanted adenohypophyseal tissue was capable of main- taining integrity of the target tissues of the adenohypophy- seal hormones. The area included the arcuate nucleus, the ventral portion of the periventricular nucleus and the median eminence (Halasz, et a1., 1965 and Flament-Durand, 1965). Halasz and Pupp (1965) severed the hypophysiotropic area from all other brain connections and noted no change in adenohypOphyseal histology, thyroid weight or testicular weight. However, atrophy of the uterus and ovaries and hypertrophy of the adrenals were observed. These investiga- tors suggested that the hyp0physiotr0pic area secretes neurohumoral agents which control release of individual adenohypophyseal hormones. In addition, release of the substances can be modified by higher nerve centers. McCann et a1. (1960) and D6cke and Dorner (1965) lesioned the hypothalamus and concluded that destruction of the median eminence and more anterior regions of the hypothalamus interfered with LH secretion. By the late 19603, evidence was accumulating that the hypothalamus could be the regulating center for general endocrine control by its action on the adenohypophysis. Several investigators questioned the possibility that the hypothalamus was an important site of negative feedback inhibition on the adenohypOphysis. Ramirez, et a1. (1964) reported that estrogen implants into the median eminence prevented the post-castration rise of serum LH as well as lowering adenohypophyseal luteinizing hormone. Lisk (1965) demonstrated that estrogen implants in the arcuate- mammillary nuclei complex blocked pregnancy. Kato and Villee (1967) found that estrogen was selectively taken up by hypothalamic tissues. A classical approach in endocrinology when attempting to identify the origin of a hormone is to study effects of various types of extracts made from the tissue suspected of secreting the substance. McCann (1962) reported that crude acidic extracts of rat stalk median eminence caused increased plasma LH in ovariectomized, estrogen primed rats as measured by ovarian ascorbic acid depletion. The effect of the extract was negated when treated with trypsin, which indicated that the active agent was a protein or polypeptide. Thereafter, Guillemin and Schally (1963) demonstrated corticotropin releasing factor activity in acid extracts of sheep hypothalamus. Further investigations using bioassay systems demonstrated FSH, LH and TSH stimulating activity in hypothalamic extracts from rats, sheep, and pigs (Masao and McCann, 1964; Schally and Bowers, 1964; Schally et a1., 1967; Averill and Kennedy, 1967; Dhariwal et a1., 1967; Watanabe and McCann, 1968). Initial Isolation and Characterization of Releasing Factors Ovine and porcine hypothalamic tissue extracts proved to be a rich and readily available source of releas- ing factor activity. Investigations with these extracts were successful in isolation and identification of the substance responsible for thyrotropin releasing activity. The structure proved to be pyroglutamyl-histidyl-proline- amide and was capable of releasing TSH both in vivo and 10 in yitrg (Boler et a1., 1969). Two years later, the amino acid sequence of the releasing factor, isolated from porcine extracts (Matsuo et a1., 1971; Baba et a1., 1971) and ovine extracts (Amoss et a1., 1971; Burgus et a1., 1972), which has the ability to release both FSH and LH in ziyg and in yitgg, was reported. The structure of LH-RH/FSH-RH was determined to be (PYro)glu-his-trp-ser-tyr—gly-leu—arg-pro- gly-NH2 and was identical for ovine and bovine species (figure 1). This releasing hormone has been assigned a variety of names. For convenience, this writer will refer to it as gonadotropin releasing hormone (GnRH). Development of radioimmunoassay systems for the releasing hormones was begun shortly after their structural elucidation. Antibodies against conjugated TRH and GnRH were made in rabbits. Several laboratories reported assay systems for GnRH (Nett et a1., 1973; Jeffcoate et a1., 1973; Arimura et a1., 1973; Jutisz and Kerdelhué, 1974; Saito et a1., 1975; Sorrentino and Sundberg, 1975; Hendricks et a1., 1975; Root et a1., 1975; and Burger and Franchimont, 1974) and TRH (Bassiri and Utiger, 1974; Bryce, 1974) which are specific and sensitive in the picogram range. With the advent of radioimmunoassays, it became possible to measure both the hormonal fluctuation of GnRH and TRH in plasma and hypothalamic tissues as well as response of the releasing hormones under various experimentally induced conditions. 11 Gn-RH N—C M.W.-Hal H 0 CH2 CH2 CH CH: 2 H H H H H I I I I 2 I I HN—CH C-N- H -N-LH-C-N-CH-C -C~N-C~C-NH2 II II II II II I H O O O H 0 1 2 3 4 5 6 7 8 9 10 pGlu His Ttp S. Tyr Gly Lou Ara Pro Gly Figure 1. Structfire of Gonadotropin Releasing Hormone 12 Localization of the Sites of Releasing Hormone Production and Concentration Before direct and sensitive measurement of the releasing hormones with radioimmunoassay techniques, several investigators attempted to localize the site of neurohormone concentration within the hypothalamus by measuring ability of extracts from various regions to cause release of FSH, LH and TSH in 11352. McCann (1962) reported that extracts of the stalk-median eminence region caused more LH release than extracts of various other hypothalamic regions. Schneider et a1. (1969) found extracts of the suprachiasmatic area and median eminence were capable of releasing LH when incubated with adenohypophyseal tissue. These investigators further localized regions containing LH-releasing activity by sectioning the rat hypothalamus in three planes (Crighton et a1., 1970). Luteinizing hormone releasing activity was localized in the median eminence-arcuate region and the suprachiasmatic nucleus. On the basis of these data and data from previous lesion studies, these authors proposed the existence of two areas of releasing factor synthesis; (1) the suprachiasmatic region which was thought to be responsible for the LH ovulatory surge and, (2) the median eminence-arcuate region which was thought to maintain tonic control over LH release. Evidence gathered from investigations utilizing radioimmunoassay techniques for releasing factors, sub- stantiated and augmented the findings of previous reports 13 involving bioassay. Thyrotropin releasing hormone was found throughout the rat brain, but concentrated in the median eminence, dorsomedial hypothalamus and preoptic area (Winokur and Utiger, 1974: Brownstein et a1., 1974; Krulich et a1., 1974; Quijada et a1., 1974). Immunoreactive GnRH was found to be concentrated in the suprachiasmatic and median eminence regions (Quijada et a1., 1974; Palkovits et a1., 1974) and in the preoptic area (Wheaton et a1., 1974) . Palkovits et a1. (1974) assayed 16 rat hypothalamic nuclei for GnRH and found that the median eminence contains seven times more GnRH than the arcuate nucleus. The arcuate area had 73 percent of its GnRH concentrated in the central part of this nucleus. Araki et al. (1975) reported that GnRH concentration in the mid-hypothalamic region varied with the estrous cycle in rats. Concentrations were greatest prior to the pre-ovulatory LH surge. Immunohistological localization of GnRH has been reported by numerous investigators employing various techniques. GnRH has been observed in axons terminating in the median eminence close to the portal plexus (Mazzuca, 1974; Kordon et a1., 1974; Zimmerman et a1., 1974). Zimmerman et a1. (1974) reported staining in the perikarya of arcuate neurons and in tanycytes adjacent to the portal plexus in the mouse hypothalamus. Goldsmith and Ganong (1975) observed no staining of tanycytes or staining in the arcuate region. 14 The discrepancy in these reports was discussed by Baker et a1. (1975) in a comparison study of three immuno- histological techniques. These authors suggested that failure to consistantly observe immunoreactive sites in areas other than the median eminence could be accounted for by either relative sensitivities of the assay methods or removal of the relatively small quantities of GnRH present in the cell bodies during preparation of the sections. In addition, transport of GnRH away from the cell body may be so rapid that its concentration remains too low for immunocytochemical detection. Thus far, it appears from the literature that a general consensus exists on the presence of GnRH in the median eminence and arcuate nucleus. The preoptic- suprachiasmatic area has been suggested as the center for GnRH associated with the ovulatory surge of LH release. Evidence is by no means conclusive as studies have concen- trated on the rat as the experimental model with supporting evidence provided by studies utilizing mice, and guinea pigs. Gonadotropin releasing hormone does not appear to be species specific since it was originally isolated from ovine and porcine tissue and has been demonstrated effective in causing in yitgg and in yiyg LH and FSH release from a variety of species. In the past two years, a great deal of literature has appeared on the possible mode of action of the releasing factors and their potential clinical use. Several 15 investigators have suggested GnRH increases the rate of g3 2932 LH synthesis, as well as, release of LH from the adenohypophysis (Moguilevsky and Christot, 1973; Shiotani and Ban, 1974; Redding et a1., 1974). The kinetics of GnRH have also been investigated in experiments examining synthe- sis and degradation of the decapeptide. Kuhl and Taubert (1975a) isolated hypothalamic enzymes which decreased the biological activity of gonadotropin releasing hormone. These investigators subsequently demonstrated (Kuhl and Taubert, 1975b) that LH is capable of stimulating hypotha- lamic enzymes which inactivate gonadotropin releasing hormone. The mechanism for enzyme activation in these studies appeared to depend on the presence of elevated levels of plasma estrogen in female and testosterone in male rats. Kochman et al. (1975) measured a decrease of immunoreactive GnRH after incubation of synthetic hormone with hypothalamic and pituitary homogenates. Thin layer chromatography of the incubate indicated that GnRH was degraded into at least 3 fragments by hypothalamic and pituitary enzymes. Analogs to GnRH have been tested for their possible anti-ovulatory and pro-ovulatory properties by several laboratories (Corbin and Beattie, 1975; Schafer et a1., 1975; Kastin et a1., 1974; Vilchez—Martinez et a1., 1975). These researchers have suggested possible clinical uses of the decapeptide or related structures in fertility control. 16 The evidence conclusively indicates that the hypo- thalamus plays a major role in control of the adenohypophysis. However, sites of synthesis and mechanism for release of the releasing factors remains in question. To elucidate these questions, studies involving both rodents and other species are continuing in an attempt to meet the criteria proposed by McCann and Porter (1969). MATERIALS AND METHODS Experimental Design These experiments were designed to localize concen- trations of LH releasing factor activity within the bovine hypothalamus. Releasing hormone activity was initially quantified with a double antibody radioimmunoassay specific for gonadotropin releasing hormone. A bioassay of LH releasing activity was then performed on selected extracts of hypothalamic areas to compare areas of immunoreactive and biologically active LH releasing activity. Bioassay proce- dures involved in 21259 incubation of bovine pituitary primary cell cultures with hypothalamic extracts. Tissues Collection Bovine heads were collected from a local abbatoir with sex of the animal noted at slaughter. A block of tissue containing the hypothalamus together with the pitui- tary stalk was dissected from the exposed brain, cleaned of blood and meningeal membranes, and placed on Dry Ice within 30 min of slaughter. Dimensions of this tissue block were 17 18 approximately 4 cm in length (rostral to caudal), 3 cm in width, and 2 cm in depth, measured dorsally from the ventral tip of the stalk. Blocks of tissue were placed on Dry Ice with the ventral surface up to prevent flattening of the pituitary stalk. Frozen tissue blocks were trimmed utilizing the following landmarks: (l) posterior--the junction formed by the mammillary bodies and the pons; (2) lateral--the groove separating the cerebral peduncles and the hypothalamus; (3) anterior-~the rostral border of the optic chiasm and; (4) dorsal--7 mm dorsal to the plane formed by the median eminence (figure 2). Sectioning Frozen hypothalami were sectioned in frontal, horizontal or sagittal planes at -20°C in an IEC cryostat (Model CTD; Needham Heights, MA). Frozen hypothalami to be sectioned were afixed to brass grids by moistening the grid with glass distilled water, placing a frozen hypothala- mus on the grid and quickly freezing the water. Hypothalami to be sectioned in the horizontal plane were oriented for sectioning with the dorsal surface adhered to a brass grid. Sections were numbered from the most ventral tip of the pituitary stalk through the most dorsal portion of the hypothalamus. Similarly, hypothalami to be sectioned in the frontal plane were oriented with the optic chiasm adhered to a brass grid and cut sections were numbered from the Figure 1. 19 Ventral surface of a bovine brain and a hypothalamic tissue block excised for sectioning. on . ‘ I ’II II. I . . 9 0.5.1.0.”! I. I . J...- . . a u ' 1‘ C 'MIHH\ to .n I |‘ h 0.4 Fun ‘unuon Q. o .- . I . \ n II . I .n. “In. . 0 l ). sum.........u. r”... ._ .. I. __. 1} A. n. . ‘0 35"».t'. - .l I. a. u.- I a 1.... ‘ . v.9 .1" v a Q I v . C II- . I o I a fi 0.. . o n 6. O. 0" 0" a I I o '0 ~‘n I O .n a ' ' 'l - fun-‘l ' a 0-. . 9. .3 ‘ .. . h .c . .V o b n-‘°;‘7J . ~ 0‘ .-.', on u .I n ' . D u o ..u . . _ O '0. “I I Q‘- II 0 ‘ ‘r U... QI..I§.'Q I. ‘.t(0 s f .0. Co. “\v . 0| .. § . .5...- ..I0.h...ah K. ~ IMP. r\.-n‘.- .0 .80 .t -. Ono. u’i. 'wqf‘o WINK. .. . o 81 2.... 4 £4 a‘ (1;; . o 30.. U f.‘ any“.- - o. .or-c a ‘idl. ‘0‘ no. . a Q to. q .’ ..'_n u s '.O. O‘ o I. . Q . .i a} . 0 fl. H . . . no . 5 .. . u. .9 .11 ”0 l ..\l. u I... . 2 .4. . if)“. 4%.: \ undralfii. .Iniiu... ». H " ...- o. n 21 mammillary bodies rostrally. Sagittal sections were cut from one lateral border to the other. All sections were cut at 500 microns with manual operation of a microtome (IEC Custom Rotary Microtome; Needham Heights, MA). Each section was placed in a pre- labelled 15 x 85 mm disposable glass culture tube which was kept in Dry Ice. Overall dimensions of each block and sections containing the pituitary stalk were noted for each hypothalamus at the time of sectioning. Because of variation in size of each hypothalamus, sections were labelled according to the landmarks noted during sectioning. For the horizontal plane, sections were centered around the plane formed by the stalk of the pituitary and ventral base of the hypothalamus (plane of the median eminence, section 0). Sections dorsal to this plane were numbered 1 to 11, while sections ventral to this plane were numbered -1 to -8. Frontal hypothalamic sections were centered around the mid-point of the stalk of the pituitary. Sections from the mid-point (section 0) through the mammillary bodies were numbered -1 to -14, while those sections cut from the mid-point to the Optic chiasm were labelled 1 to 21. Similarily, sections cut in the lateral plane were centered around the mid-point of the pituitary stalk (section 0) with the lateral border, at the beginning of sectioning, labelled -15 and sections out after the mid- point designated with positive numbers to 15. 22 Extraction Procedure Each frozen tissue slice was transferred to a glass tissue homogenizer (Pyrex #7726) and homogenized in approxi- mately 0.5 m1 of 2N acetic acid at 4°C. Each homogenate was quantitatively transferred to a centrifuge tube using 2N acetic acid (4°C) to wash the homogenizer. Final volume was 3 milliliters. Aliquots from each sample were removed for protein determination (Miller, 1959; see Appendix 1). Total protein for each section was calculated in milligrams. Extraction efficiency was determined by adding 20,000 cpm 3H-GnRH (S.A. = 19.6 Ci/mMOl; Abbott Laboratories) in 100 ul distilled water to each homogenate. After vortexing, each sample was centrifuged at 10,000 x g for 30 min (Sorval RC-3 centrifuge) at 4°C. Two m1 of superna- tant were recovered and placed in a small vial. Extracted samples were rapidly frozen in ethanol cooled with Dry Ice, then lyophilized (Virtus Research Equipment; Gardiner, NY). All extracted samples from a given plane were lyophilized at the same time. Lyophilized extracts were reconstituted with 2 ml water double distilled in glass and frozen until assayed. At the time of assay, 3H was measured in a liquid scintillation spectrophotometer (Nuclear-Chicago) and utilized to calculate efficiency of extraction for each sample. 23 Radioimmunoassay (RIA) of GonadOtropin Releasing Hormone Principles of protein radioimmunoassays (RIA) have been discussed in detail by several investigators (Midgley et a1., 1969; Skelly, 1973). Specific antibody for GnRH was generously provided by Dr. Terry Nett (Colorado State University) and is the same R42 antibody reported by Nett et a1. (1973). GnRH was measured with modifications of the method described by these authors. Synthetic GnRH (3 ug, Abbott Laboratories) was labelled with sodium 125 I (l mCi, New England Nuclear) in the presence of chloramine-T (20 ug) using a modification of the method described by Greenwood and Hunter (1969; see Appendix III). The reaction was stopped after 45 sec by addition of sodium metabisulfate (500 ug, Appendix II.A.3.). With the aid of transfer solution (Appendix II.A.4.), the reaction mixture was layered onto a 1 x 15 cm Sephadex LH-20 column (Pharmacia Fine Chemicals, Appendix II.A.). Labelled GnRH was eluted with phosphate buffered saline (PBS, Appendix 11.3.1.) and fractions were collected at 3 min intervals in 12 x 75 mm disposable glass culture tubes con- taining 1 ml 0.1 percent pig skin gelatin in PBS (PSG-PBS, Appendix II.B.3.). This column separated labelled GnRH from free iodine. A typical elution profile is shown in figure 3. Labelled GnRH, found in the second peak, was diluted to approximately 20,000 Cpm/100 ul with 0.1 percent pig skin gelatin in phosphate buffered saline. Figure 3. 24 Typical elution profile separating iodinated hormone on Sephadex LH-20 column. Iodinated products were placed on a l x 15 cm Sephadex LH-20 column and eluted with phosphate buffered saline, and collected in 3 min fractions. Fractions 28 to 31 contained lZSI-GnRH as was evidenced from radioactivity in these fractions which bound to antibody for the hormone. 30 1 coum‘s x I03/20,.I /0.I min p k 25 IO 20 FRACTION NUMBER LH 20 COLUMN 30 4O 26 Each hypothalamic section extract was assayed at six dilutions. In addition, extracts of hypothalami sectioned in the same plane were assayed together to elimi- nate interassay variation. Four or five sets of standard synthetic GnRH concentrations (Appendix II.B.7.) were evenly distributed throughout each assay. Table 1 outlines the procedure followed for radioimmunoassay. The radioactivity of the precipitate was measured using an Automatic gamma counter (Nuclear-Chicago). Standard curves of counting time versus concentra- tion of synthetic GnRH added to each tube were obtained for each assay using a least squares third degree polynomial regression program on the Michigan State University 6500 Control Data Corporation computer (MSU CDC 6500). Gonado— tropin releasing hormone concentrations for each extract were calculated in ng/ml from the regression coefficients of the standard curves corrected for the dilution of extract added for each assay tube. Total GnRH content of each section was computed from hormone concentration of extract and percent of 3H-GnRH recovered. The ratio of total GnRH per mg of protein was calculated, resulting in hormone concentration per section. Multiple regression analysis of the hormone concen- tration versus section was chosen as the most appropriate analysis. The procedure was designed to indicate the areas where significant changes of hormone concentration occurred. Beginning with a ninth degree polynomial, a least squares 27 Table l.--Procedure for Radioimmunoassay of GonadotrOpin Releasing Hormone Time Procedure Day 1 Day 2 Day 3 Day 6 GnRH standards are dispensed in 500 ul aliquots to 10 x 75 mm polyvinyl culture tubes (Falcon Plastics Co.) 100 and 200 ul of undiluted extracts or extract diluted 1:5 or 1:25 with 0.1% PSG-PBS are dispensed into assay tubes and brought to 500 ul volume with 0.1% PSG-PBS. 200 ul R 42 anti-GnRH 1:40,000 (Appendix II.B.S.) are added and tubes vortexed. Assay is incubated 24 hr at 4°C. 100 ul 125I-GnRH diluted to about 20,000 cpm with 0.1% PSG-PBS is added and tubes vortexed. Assay is incubated 24 hr at 4°C. 200 ul sheep anti-rabbit gamma globulin (Appendix II.B.6.) is added and tubes vortexed. Assay is incubated 72 hr at 4°C. 2.5 ml PBS is added. Assay tubes are centrifuged at 1942 x g for 30 min, supernatants aspirated and the precipitates counted. step-wise deletion regression analysis was performed on the MSU CDC 6500 computer. This procedure resulted in a multiple regression curve of ng GnRH/mg protein versus section number using only those polynomial terms found to be significant at p< 0.05. 28 Bioassay for Gonadotropin ReleasingAHormone Procedure for Gonadotropin Releasinngormone BIOassay Steps in the preparation of bovine anterior pitui- tary cell cultures for GnRH bioassay are outlined in figure 4. For each culture, four bovine heads were obtained within 30 min after the animals were slaughtered at a local abba- toir. These heads were transported to the laboratory at room temperature. All laboratory culture procedures employed sterile technique. Within 90 min of death of animals, anterior pituitaries were dissected from other tissue and cut to approximately 1 mm cubes using a Stady- Riggs microtome (Thomas CO.) and scalpel. Tissue pieces were washed with preparation medium (Appendix IV.A.) until the wash medium no longer contained cellular debris. Tissue pieces were then pr0portioned to two 50 m1 Erlenmeyer flasks containing 25 ml 0.3 percent collagenase (150 u/mg, Sigma Chemical Co.) in preparation medium. Contents of each flask were stirred with a magnetic stirring bar for 45 min in a water bath at 37°C. The resulting cell suspension was filtered through cheesecloth into 50 m1 conical plastic tubes (Falcon Plastics Co.) and centrifuged at 267 x g for 5 min at room temperature. The supernatant was discarded and the cells resuspended in 30 ml of 0.25 percent Viokase (Grand Island Biological Co.) in preparation medium and stirred for 15 min at 37°C. This suspension was transferred 29 Figure 4. Schematic representation of the preparation of bovine anterior pituitary cell cultures and design of in vitro experiments. *Medium is preparation medium. ANTERIOR PITUITARY POSTERIOR PITUITARY COLLAGENASE (0.3%, 45 mIn.) VIOKASE (O. 25% , I5 min.) WASHED AND CENTRIFUGED. 5x GROWN TO MONOLAYER IN GROWTH MEDIUM BIOASSAY SCHE DU LE __\ILAS_H__5_:L_ IEBE‘IBEAIMENI . TREATMENT IEQSI'IBEAIMENII ? 2 hr. A 2 hr. f 2 hr. MEDIUM" TREATMENT MEDIUM * O MEDIUM' 31 to a 50 m1 conical plastic tube and centrifuged at 171 x g for 5 min at room temperature. The supernatant was dis- carded and the cells were resuspended in 50 m1 of prepara- tion medium and centrifuged at 171 x g for 4 minutes. This step was repeated 3 times to remove enzymes. After the final centrifugation, cells were suspended in 200 m1 growth medium (Appendix IV.B.) which resulted in a concentration of about 4 x 105 cells/milliliter. Four ml of this suspension were transferred to 30 m1 plastic culture flasks (Falcon Plastics Co.) and incubated at 37°C under constant gassing, 95 percent 02-5% C02. Medium was changed at 48, 72, and 96 hr after cells were added to the culture flasks. On day 6, after establishment of confluent monolay- ers of pituitary cells, each flask was washed with 4 m1 of preparation medium 5 times. Previous studies, in our laboratory with this culture system, indicated this step is necessary to obtain consistent pre-treatment LH concentra- tions between flasks. Pre-treatment medium was collected to establish initial hormone secretion from each flask after 2 hr of incubation. Treatment medium containing extracts of hypothalamus, synthetic GnRH, or vehicle was then added to each flask. Flasks were then incubated at 37°C for 2 hr after which the media was collected. Pre-treatment and treatment media for each flask were each collected in a 12 x 75 mm disposable glass culture tube and stored at -20°C until assayed for luteinizing hormone. 32 Each medium sample was thawed and assayed in dupli- cate for LH according to the procedure of Convey et al. (1976). Experimental Design for Gonadotropin ReIéasing Hormone Bioassay Experiment l--Dose Response of Gonadotropin Releasing Hormone and Hypothalamic Extract This experiment was designed to determine whether bovine pituitary cell cultures exhibit a similar dose- response of LH release when incubated with synthetic GnRH or hypothalamic extract. Extracted sections of 2 hypothalami cut in the frontal or sagittal plane were pooled as follows. A low extract pool calculated to contain 0.8 ng GnRH/ml was prepared by combining 400 ul of extract from each of 21 sections in which GnRH concentrations were determined with radioimmunoassay. A high extract pool, similarly formed from extracts of 17 sections, was calculated to contain 6.1 ng GnRH/milliliter. The total amount of tissue represented in the low and high pools of extract was similar (85 mg protein). On day 6 of culture, 65 culture flasks were random- ized into groups of five. Treatments were: 0, 0.1, 1.0, or 10 ng GnRH/ml media; or 50, 100, 200 ul high or low extract pool/flask; or 100 ul low extract pool/flask plus 0.1, 1.0, or 10 ng GnRH/ml media. GnRH was dissolved in 0.1 percent Knox gelatin in PBS and delivered at 40 ul/flask. 33 Linear regression analysis of LH response in treat- ment media was calculated on the log of dose for GnRH and hypothalamic extract concentrations. Experiment 2--Bioassay of Sagittal Section of 5 Hypothalamic Areas The objective of this experiment was twofold: (l) to compare areas of biological LH releasing activity and immunological GnRH in extracts from sagittally sectioned hypothalami, and (2) to determine whether immunoreactive GnRH measured in these extracts could be responsible for all biological LH releasing activity. Extract pools of 6 hypothalami cut in the sagittal plane were formed by adding 100 ul of extract from each section cut -5.5 to -4.5, -3 to -2, -0.5 to 0.5 to 0.5, 2 to 4 and 4.5 to 5.5 mm from the midpoint of the pituitary stalk. At the time of treatment, culture flasks were randomized and numbered in groups of six. Group treatment was; 0, 1.0, 10, or 100 ng GnRH/m1 media; 100 ul low extract pool/flask plus 0, 1.0, 10, or 100 ng GnRH/m1 media; or, 100 ul extract from one of the area pools/flask. Synthetic releasing hormone was dissolved in 0.1 percent Knox:PBS and delivered at 40 ul/flask. Experiment 3--Bioassay of Frontal Section Extracts of 6 Hypothalamic Areas This experiment was designed to compare areas of biological LH releasing activity and immunological GnRH within extracts from 6 hypothalami sectioned in the frontal 34 plane. Pools of extracts of hypothalami cut in the frontal plane were made as in Experiment 2 above. Areas pooled were from sections cut -6.5 to -5.5, -3.5 to -2.5, -0.5 to 0.5, 2.5 to 3.5, 5.5 to 6.5 and 8.5 to 9.5 mm from the mid-point of the pituitary stalk. Treatment regime and groups were the same as for bioassay of sagittal section extracts . RESULTS AND DISCUSS ION Radioimmunoassay of Gonadotropin ReIeasIng Hormone (GnRH) Standard Inhibition Curve for GonadotropIn Releasing Hormone The amount of radioactivity bound to antibody in the precipitate of assay tubes containing no unlabelled GnRH was set at 100 percent binding. Synthetic GnRH standards (Appendix II.B.7.) decreased the amount of 125I-GnRH bound to the antibody. Similarly, when 4 to 200 ul of extract from sections containing the stalk of the pituitary were added to assay tubes, a decrease in the amount of radioacti- vity bound to the antibody was observed. Typical inhibition curves of percent 125 I-GnRH bound versus log of the amount of synthetic GnRH and hypothalamic extract added per assay tube are shown in figure 5. The inhibition curve produced by hypothalamic extracts was parallel to the inhibition curve obtained with synthetic gonadotropin releasing hormone. The curves were parallel in the region where the quantity of 125I-GnRH bound to antibody was equal to 25 to 88 percent of that bound in assay tubes containing no unlabelled hormone. Minimum sensitivity, ie., the least 35 36 .mucwom m mo momma who O>HOO mmco now mucwom .Ammlmv uomuuxo OHEmamnuoman ocw>on Odo Ammsov chEuos mcfimmmamu chOHuOOmcom oauocucmw Mom mm>uso cowuwnfiscH .m ousmflm 37 CON ON. om 0? ET... .13 ._E\:m-ao 8 on .2w 0. N. mm m n; wzum Loo— ONnOB 1N3083d 38 amount of GnRH which resulted in significantly less binding of 125 I-GnRH than that in the buffer control tubes, ranged from 3 to 6 pg in 3 different assays. These results are in good agreement with those from other laboratories using the same antibody. Minimum sensi— tivities ranging from 1 to 12.5 pg and inhibition curves parallel to synthetic GnRH have been reported for rat, rhesus monkey, ovine and porcine stalk-median eminence area extracts; rat medial basal and preoptic-suprachiasmatic hypothalamic and pineal extracts; and, serum and/or plasma from humans, castrated sheep and rats (Nett et a1., 1973; Wheaton et a1., 1975; Clemens et a1., 1975; Araki et a1., 1975). Cross reaction with other hormones or fragments of the decapeptide has not been reported with this antibody, although some cross reaction has been reported for synthetic analogs of GnRH (Nett et a1., 1973). Reports from several laboratories using different antibodies to GnRH generated in guinea pigs or rabbits have shown minimum assay sensiti- vities ranging from 1 to 25 pg with varying degrees of cross reaction to fragments and analogs of releasing hormone (Kizer et a1., 1976; Jutisz and Kerdelhué, 1975; Root et a1., 1975; Sorrentino and Sundberg, 1975; Hendricks and Millar, 1975; Saito et a1., 1975; Burger and Franchimont, 1974; Bryce, 1974; Jeffcoate et a1., 1973). 39 Gonadotropin Releasing Hormone in the Bovine HypothaIamus Recovery of 3H-GnRH in extracts of tissue cut in the horizontal, frontal, and sagittal planes was 63.3 i 8.6 percent, 98.6 i 4.4 percent, and 92.9 i 12.1 percent (mean value i Standard Deviation) respectively. The difference in efficiency of extraction may be a result of more effec- tive homogenization of the later two planes since sections cut horizontally were the first tissues extracted. To our knowledge, other investigators have not reported extraction efficiency, therefore, comparison of the extraction proce- dure used here with those of others is not possible. Total GnRH content, protein content and number of sections obtained from each hypothalamus are shown in Table 2. Total mg of protein together with length and width measurements reflects the size of each hypothalamic block. The range in number of sections obtained from tissue blocks cut in the horizontal plane (15 to 19) resulted from variation in depth (ventral to dorsal) of the hypothalami. Similarily, number of sections cut in the frontal (33 to 40) and sagittal (25 to 27) planes resulted from variation in the length and width of these hypothalamic blocks. Total GnRH content ranged from 89.8 to 618.6 ng per hypothalamus. The variation is most evident when hormone values from hypothalami cut in the frontal and horizontal planes are compared. These tissues were obtained from mature cows. Standard errors of the mean for hypothalamic 40 Table 2.--Animal Variation in Number of Sections cut and Total GnRH and Protein Content of Hypothalami in 3 Planes Planea Animal, Sexb No. of Total Dimensions (mm) Sections GnRH (ng) Protein (ng’ Length Width H 1 F 15 184.5 117.3 21 15 H 2 F 18 228.1 138.0 23 15 H 3 F 18 152.0 138.8 20 14 H 4 F 19 618.6 121.6 20 16 H 5 F 19 99.4 105.4 18 14 H 6 F 19 241.7 120.0 20 15 M" 18 i .6 254.0 1 75.9 123.5 1 5.2 F 1 F 36 91.4 123.6 21 14 F 2 F 39 89.8 149.0 22 16 F 3 F 33 189.1 145.9 20 17 F 4 F 37 100.2 152.8 22 18 F 5 F 37 327.1 145.3 22 14 F 6 F 40 353.6 124.0 21 14 Av 37 1 1.0 191.9 i 49.4 140.1 5.3 S l M 25 412.0 136.6 22 15 S 2 M 27 459.3 171.3 20 16 S 3 M 26 539.0 130.2 22 14 S 4 M 26 310.6 144.0 22 15 S S M 26 370.0 139.3 19 15 S 6 S 26 474.0 158.9 21 16 Av 26 i .3 427.5 1 33.0 146.7 i 6.3 aSymbols are: H--horizonta1, F--frontals, S--sagitta1. bSymbols are: F--fema1e, M--ma1e, S--castrate male. cValues are mean 1 SE. 41 GnRH from bulls was smaller (33.0 ng) compared to those for cows (49.4 and 75.9 ng). These differences may reflect differences in reproductive states of the cows used as tissue donors. However, tissues were collected from slaugh— terhouse animals over a 5 month period and reproductive histories of the animals were not available. Araki et a1. (1975) monitored changes in GnRH content of the anterior and mid-hypothalamic regions of rats throughout the estrous cycle. These investigators found the anterior hypothalamic GnRH content to be highest during late diestrus and late proestrus while mid-hypothalamic content was greatest early in proestrus and during estrus. Comparison of hypothalamic GnRH content between bulls and cows indicate that males may have more hypothalamic GnRH relative to females. Total hypothalamic GnRH has been measured in other species with various RIA systems. Immunoreactive GnRH content of rat, guinea pig, and sheep hypothalami varied from 0.3 to 2.7 ng, 1.7 ng, and 34 to 52 ng, respectively in these reports (Burger and Franchimont, 1974; Root et a1. 1975; Araki et a1., 1975; Bryce, 1974; Jutisz and Kerdelhué, 1974; White et a1., 1974). Figures 6, 7, and 8 depict the multiple regression curves of GnRH concentration versus section for hypothalami cut in the horizontal, frontal, and sagittal planes. Figure 6 illustrates the curve derived from 5 of the 6 hypothalami assayed. When data from animal 4 was included in the analysis, the shape of the curve did not change, however, 42 Figure 6. Multiple regression curve for immunolog- ical gonadotrOpin releasing hormone concentration versus horizontal section. Analysis on hypothalamic sections from 5 animals. GnRH (ng)/Protein (mg) l5 l4 l3 l2 IO 1 -4 -3 43 DORSAL -———-> 1 l L I -2 -l 0 I 2 mm from Plane of ME 44 peak and baseline GnRH concentrations were increased more than fourfold. Total GnRH content for this animal was greater than that of any hypothalamus measured (618.6 ng). Lowest GnRH concentration (or baseline level for this hypothalamus) was greater than that of other hypothalamus sectioned in the horizontal plane. The multiple regression curve of section versus hormone concentration for the horizontal plane demonstrates that immunoreactive GnRH is contained in the region of the pituitary stalk (-4 to 0 mm) with greatest concentration in the mid-portion of the stalk (-3 to -2 mm). Gonadotropin releasing hormone 3 mm dorsal to the plane of the median eminence was less than 0.5 ng GnRH/mg protein. A slight increase in GnRH concentration in sections 4 mm dorsal to the plane of the median eminence was shown to be significant (p<0.035). Kizer et a1. (1976) dissected median eminence and pituitary stalks from 6 steers into 8 different regions. Highest concentrations of radioimmunoassayable GnRH were found in the middle external and anterior internal layers of the stalk-median eminence (4.0 and 4.1 ng GnRH/mg pro- tein). These areas are included in sections cut -3 to -2 mm which demonstrated greatest hormone concentration in this study. The discrepancy in peak concentration of hormone reported by these investigators (4.1 ng/mg) and data from this study (14.9 ng/mg) could be a result of animal varia- tion, RIA systems, correction for extraction efficiency, or method of sectioning. 45 The multiple regression curve of GnRH concentration versus section for those hypothalami cut in the frontal plane (figure 7) demonstrates that peak GnRH concentration resides at the mid-point of the pituitary stalk and median eminence with hormone concentrations decreasing anterior and posterior to this point for stalk contining sections (4.5 to -4.5 mm). Four mm anterior to the stalk a significant increase in hormone concentration was shown (p<0.035). The peak in GnRH concentration in the area between the pituitary stalk and the Optic chiasm (7.5 to 10.5 mm) was about 18 percent of the concentration peak found within the region of the stalk. Analysis of data obtained from hypothalami sectioned in the sagittal plane revealed a bilateral symmetry of hormone concentration about the mid-point of the stalk (0 mm) with the peak GnRH concentration localized at the mid-point (figure 8). Concentrations of releasing hormone significantly above baseline values of 1 ng GnRH/mg protein were found only in stalk containing sections (-3 to 3 mm). Although initial studies of releasing factor activity utilized extracts of bovine pituitary stalk-median eminence area (Schally and Bowers, 1969), reports subsequent to the development of specific assays for the releasing hormone have not attempted to localize the areas of GnRH concentration within the whole bovine hypothalamus. The results of this study indicate that immunological GnRH is 46 .mamsflcm m Eoum mcowuoom Owaoamnuommc co memHmcm .cofiuomm Hopcoum mamuo> cofluouucmocoo msoauon mcwmmmaou demon» Ioomcom amoemoHocOEEH How m>uoo OOMmmmHmmH mamfluasz .h wusmfim 47 -a> :00 -I- no I m0_mm._.z< {Rm 3255 .6 .350 So: EE 0 c n m . o _-~-n-¢- - q q d u q 1 1 “I m. h- l ‘0. o l 0. I I”. 1 0. N J '0. N 0. 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Fragments of GnRH in hypothalamic extract could possibly account for the increased magnitude of LH release when compared to predicted LH release on the basis of GnRH concentrations measured with radioimmunoassay. Graphs of LH release by hypothalamic extracts resulted in patterns similar to the multiple regression curves calculated from immunoreactive GnRH concentration versus section (compare figures 10 and 7, 11 and 8). Similar to results obtained with radioimmunoassay, maximum biological activity was demonstrated in extracts of sections which contained the mid-point of the pituitary stalk and median eminence. The smaller peak in immunologically reactive GnRH concentration measured in the anterior hypothalamic region of frontal sections was not seen with bioassay. It is possible that releasing hormone activity was not detected in the extract pool of this region because of limitation in the sensitivity of the bioassay. The culture used for bioassay of frontal section extracts probably had fewer LH secreting cells. Luteinizing hormone concentration of media in control flasks from this culture was about 10 per- cent of hormone concentration from control flasks in experiment 1. These results indicate that sensitivity of this culture was probably reduced. These results extend to the bovine and confirm the findings of Watanabe and McCann (1968), Crighton et a1. 64 (1968), Crighton et a1. (1970) and Quijada et a1. (1971) which localized gonadotropin releasing factor activity in the rat hypothalamus within the pituitary stalk and median eminence areas using in 31559 incubation of rat pituitary halves and extracts of hypothalamic areas. SUMMARY AND CONCLUS ION Six bovine hypothalami were cut in 500 micron sec- tions in the horizontal, frontal and sagittal planes and extracted with 2N acetic acid. Specific RIA for GnRH indicated that areas of immunoreactive releasing hormone concentration were confined to the pituitary stalk and median eminence region with a smaller concentration in the anterior hypothalamic region. Bioassay of extracted hypothalamic regions with monolayer cultures of bovine pituitary cells demonstrated LH releasing factor activity was confined within the areas of the pituitary stalk and median eminence. Magnitude of LH response from bovine pituitary cells exposed to hypothalamic extracts was greater than could be accounted for by releasing hormone measured immunologically. Comparison of lepes of the lines derived from linear regression analysis for LH response versus log of the dose of synthetic GnRH and hypothalamic extract indicated that the responses were not parallel (p<0.05). A steeper response curve was shown for hypothalamic extract than for synthetic hormone. These results indicate the possibility 65 66 of LH releasing activity or facilitation of LH release by substance in hypothalamic extract other than gonadotropin releasing hormone. It is also tenable that non-parallel dose responses were observed between extract and synthetic GnRH as a result of inhibition of LH release by the protein buffer used to introduce GnRH to the cultures. Further studies with monolayer cultures and their response to hypothalamic extract, synthetic GnRH, peptide fragments of GnRH, as well as other hypothalamic factors, could clarify whether cultured pituitary cells recognize molecules other than the decapeptide. It is possible that investigations such as these may suggest hypothalamic factors which facil- itate or inhibit the pituitary response to known hypothala- mic hormones. Factors which are found to potentiate LH release could lead to further investigation of cellular mechanisms involved in recognition of neural hormones and release of pituitary hormones. Through a combination of bioassay and radioimmuno- assay techniques, it was possible to extend to the bovine findings in other species which isolated areas of releasing hormone concentration. Previous reports have not stimultaniously demonstrated immunological and biological releasing activity concentration to reside in the median eminence and pituitary stalk and possibly, in the anterior hypothalamic region. Further investigation of hypothalamic organization, using techniques similar to those described herein, may 67 indicate that variations in regional neural hormone activity are responsible for the physiological changes associated with change in reproductive state throughout life in animals. B IBL IOGRAPHY BIBLIOGRAPHY Amoss, M., R. Burgus, R. Blackwell, W. Vale, F. Fellows, and R. Guillemin. 1971. Purification, amino acid composition and N-terminus of the hypothalamic luteinizing hormone releasing factor (LRF) of ovine origin. Biochem. BiOphys. Res. Comm. 44: 205. Araki, S., M. 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Alkaline copper reagent: 1 part 0.5 percent COpper sulphate in 1 percent sodium potassium tartrate to 10 parts 10 percent sodium carbonate in 0.5N sodium hydroxide. Stock Solutions: a. 1.0 percent copper sulphate stored at 4°C b. 2.0 percent sodium potassium tartrate stored at 4°C c. 10 percent carbonate in 0.5N sodium hydroxide stored at room temperature Not more than 30 min prior to assay, and in the following order: 5 ml solution a 5 ml solution b 100 ml solution c. 2. Folin and Ciocalteu Phenol Reagent 2N (Harleco Co.; Philadelphia, PA) stored at 4°C Not more than 30 min prior to assay, dilute 30 m1 of reagent with 300 ml double glass distilled water. 3. Bovine serum albumin -BSA- (Sigma Chemical Co.; St. Louis, MO) stock standard: 250 ul BSA/H2 0 m1. Stored at -20°C in 4 m1 aliquots. B. Method: 1. Dilute Stock BSA for triplicate standards of 0, 25, 50, 75, 125, and 250 ug/ml with glass distilled water. Dilute 50 ul of hypothalamic homogenate to be assayed to 1 ml with glass distilled water. 76 77 2. Add 1 ml of alkaline copper reagent to each tube. Let stand at room temperature for 10 minutes. 3. Add 3 ml diluted Folin and Ciocalteu Phenol Reagent as forcibily as practical. 4. Heat in water bath at 50°C for 10 minutes. 5. Cool to room temperature and read optical density at 540 mu with spectrophotometer (Gilford Model 2000: Oberlan, OH). Calculations: Calculate linear regression equation for optical density versus concentration of BSA standards. From this equation and Optical density of samples, calculate the protein in 50 ul of hypothalamic homogenate. Determine total protein in each section by multiplying protein per sample by 59. APPENDIX B APPENDIX B COMPOSITION OF REAGENTS USED IN RADIOIMMUNOASSAY Reagents for radioiodination l. 0.5 M sodium phosphate buffer, pH 7.5 Monobasic (0.5 M) Add 69.005 9 NaH2P04.H 0 to distilled water. Dissolve, dilute to 1 iter. Store at 4°C. Dibasic (0.5M) Add 70.98 9 Na HPO to distilled water. Heat to dissolae, dilute to 1 liter. Store at 4°C. Mix monobasic and dibasic to pH 7.5. Dispense in 1 ml portions, store at -20°C. 2. Chloramine-T Upon receiving chloramine-T, dispense into small, tightly sealed vials, cover with foil, and store at -20°C. Dilute 6 mg chloramine-T to 3 ml with PBS. Use within 30 minutes of preparation. 3. Sodium metabisulfite Dilute 30 mg NaZS O to 3 ml with PBS. Use within 30 minutes of pgegaration. 4. Transfer solution Sucrose -------------- 1.6 g KI ------------------- 0.1 g Dilute to 10 ml with distilled water. Dispense in 200 ul portions, store at -20°C. Reagents for Radioimmunoassay 1. 0.01 M phosphate buffered saline, pH 7.0 (PBS) NaCl ------------------ 143 g Monobasic phosphate---100 ml Dibasic phosphate ----- 260 m1 Merthiolate ----------- 1.75 g 78 2. 3. 4. 5. 6. 7. 79 Dissolve in distilled water and transfer to a large container. Dilute to 17.5 liters with distilled water. Adjust pH to 7.0, store at 4°C. disodium EDTA ---------- 18.612 9 Add approximately 950 ml PBS. Adjust pH to 7.0 with 5 N NaOH while stirring Dilute to 1 liter, store at 4°C. 0.1 percent pig skin gelatin - PBS (PSG-PBS) PSG (Kodac Co.) -------- 1.00 9 Add 1 liter distilled water. Heat and mix over magnetic mixer until dissolved Store at 4°C. 1:400 Normal Rabbit Serum (NRS) Obtain blood from rabbit that has not been used to develOp antibodies. Allow blood to clot at room temperature, Recover serum and store the serum inconvenient quantities at -20°C. Add 2.5 m1 of thawed serum to a 1 liter volumetric flask, dilute to 1 liter with 0.05 M PBS-EDTA, pH 7.0. Store at 4°C. Use within 2 weeks of preparation. Rabbit anti-GnRH l:40,000 Dilute lyophilized antisera with distilled water as directed on vial. to 1:400 Store in small vials in 500 ul aliquots at 20°C. On day of use, dilute thawed aliquot with 50 ml of 1:400 NRS. Store at 4°C. Anti-gamma globulin On day of use, dilute sheep serum containing anti-rabbit gamma globulin to the appropriate concentration with 0.05 M PBS-EDTA, pH 7.0. Store at 4°C. Hormone standards Weigh 200-400 ug GnRH (Abbott Labs) on Cahn Electrobalance. Dissolve with double distilled water to 3ug/20 ul Store at -20°C. Make stock solution of 1 ng GnRH/ml PSG-PBS. 80 Using Hamilton microliter syringes, add appropri- ate volumes of stock hormone solution to volumetic flasks and dilute with PSG-PBS to the following concentrations: 1.5, 3.0, 6.0, 9.0, 12, 15, 20, 30, 45, 60 and 120 pg/ml. Dispense each standard to small screw-cap vials in quantities suitable for 1 assay. Freeze in dry ice-ethanol, store at -20°C. Use within 2 weeks. APPENDIX C APPENDIX C IODINATION PROCEDURE FOR GnRH Column Preparation; Expand Sephadex LH 20 (Pharmacia Fine Chemicals; Piscataway, N.J.) in PBS and evacuate at room temperature overnight. Pour LH 20 into disposable glass column 1 x 15 cm. Rinse with 2 ml 0.1 percent PSG-PBS. Rinse with PBS for 2 hrs. Iodination: 125 To 1 mCi sodium Mass.) add: I (New England Nuclear; Cambridge, 25 ul 0.5 M phosphate buffer 3 ug GnRH/20 ul H O 10 ul Chloramine - T Mix 45 seconds with vigorous shaking. Add 50 ul NaZSZOS' Add 100 ul transfer solution. Gently transfer contents of reaction vial to prepared Sephadex column and fill with PBS. Collect 45 fractions at 3 minute intervals in 12 x 75 mm disposable glass culture tubes containing 1 m1 of 0.1 percent PSG-PBS. 81 APPENDIX D APPENDIX D COMPOSITION OF CULTURE MEDIA Preparation Medium Dubeccos Medium (Grand Island Biological Co.) powder 5 g/l water 3.7 g NaHCO3/liter 1 percent non-essential amino acids MEM (GIBCO) 1 percent essential amino acids MEM (GIBCO) 25 mM Hepes (GIBCO) 68 mg/l penicillin G 1595 u/mg (Sigma) 100 mg/l streptomycin SO4 (Sigma) 1 ml/l Fungizone (Amphotericin B, 250 mcg/ml, GIBCO) Adjusted to pH 7.4 with NaOH Filtered through 0.22 p membrane (Millipore) Growth Medium Preparation Medium + 1 percent L-glutamine + 10 percent post-partum cow serum 82 MICHIGAN STATE UNIVERSITY LIBRARIES III I 3 1293 03056 2296