G NlVERSlTY LIBRARIES n ll i1 Milli UJJl LIBRARY Michigan State University This is to certify that the thesis entitled Effects of Testosterone and Estradiol -17B On Growth Hormone Release From Dispersed Bovine Anterior Pituitary Cells In Vitro 1 presented by Hazem Abd Al-Rhman Hassan has been accepted towards fulfillment of the requirements for Master of SCidegree in Animal Science ' @fldfl 77% 915% Robert A . Merkel Major professor Date November BL 1989 0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution ll‘ Umomazz, PLACE ll RETURN BOX to romovo this checkout from your record. TO AVOID FINES Man on or baton duo om. DATE DUE DATE DUE DATE DUE usu In An Affirmdlvo Action/Em Opportunity Institution EFFECTS OF TESTOSTERONE AND ESTRADIOL -17B ON GROWTH HORMONE RELEASE FROM DISPERSED BOVINE ANTERIOR PITUITARY CELLS IN VITRO BY Hazem Abd Al-Rhman Hassan A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1989 ABSTRACT Effects of Testosterone and Estradiol-17B on Growth Hormone Release from Dispersed Bovine Anterior Pituitary Cells In Vitro BY Hazem Abd Al-Rhman Hassan The effect of estradiol-17B (E) and Testosterone (T) on secretion of growth hormone (GH) by primary cultures of bovine adenohypophyseal (bAP) cells was studied. Dispersed bAP cells (5X105 cells/well) from either calves, heifers, steers or cows were incubated for 24, 48 or 72 h with E (10'10, 10‘8 M ) or T ( 10‘7, 10’5 M ). At 24 h the cells were challenged for 1 h with 1-44 N84 growth hormone- releasing hormone (GRF). Fungizone (2.5ug/m1) reduced (P<.05) GH released after 6 d of plating. Phenol red (45uM) had no effect (P>.05) on GR released into the media for up to 6 d after plating. Preincubation of bAP cells with either E or T did not affect (P>.OS) basal GH released at 24, 48 or 72 h of treatment. Neither concentration of E and T (10'7M) had an effect (P>.05) on GRF-induced GH release. However, T (10's) increased (P<.05) the GH released in response to GRF challenge. These results indicate that E and T have no direct effect on basal GH release in Vitro, but T enhances the effect of GRF on GH release. ACKNOWLEDGEMENT In retrospect, it seems impossible to acknowledge all individuals contributing to the completion of this study. To those not mentioned here, I convey my heart-felt thanks. My first debt of sincere gratitude goes to Dr. Robert A. Merkel for sharing his wealth of knowledge, professional counseling, his patience and guidance during the course of this study. Further indebtedness and great appreciation is extended to Dr. H.A. Tucker and Dr. K.R. Refsal for serving on my Guidance Committee and for reviewing this thesis and their helpful suggestions. My heart-felt appreciation is extended to Dr. William J. Enright for being instrumental in the design and initiation of the study. Gratitude is also extended to Dr. Steven Zinn and Mr. Larry T. Chapin for teaching me and assisting with the GH radioimmunoassay. To Kristin F. Bronson for her patient and skillful teaching and assistance in tissue culture techniques, I extend my deepest thanks. Special thanks are given to Dr. J. Gill for his invaluable assistance in the statistical analysis of my data. Also, to Dr. A.L. Stinson for providing me with the histological slide of the bovine anterior pituitary and allowing me to use it. To the faculty, staff and graduate students of the Department of Food Science and Meat laboratory, especially Dr. Zain Saad, Mr. Tom Forton, Mr. Aubrey Schroeder, Mr. David Skjaerlund, Dr. Abdalla Babiker, Mr. Mustafa Farouk for unselfishly assisting in obtaining samples and endeared friendship, a simple thank you seems inadequate. For my dear brother and sisters, who have never really understood what I was doing here, but still ardently provided encouragement, words are totally inadequate. For my dear wife, I express with much reverence and appreciation her moral support and assistance through all my life. Without these people, neither the desire nor the opportunity to pursue advanced degrees would have been possible. iii TABLE 0? CONTENTS EASE List of Tables... ................................... . vii List of Figures................. ..................... viii Introduction........ ......... .. ...................... 1 Review of Literature................... .............. 4 A. Growth hormone secretion and regulation ........... 5 1- Growth hormone production and clearance ........ 5 2- Factors affecting GH production ................ 6 a. Ontogenesis of GH ........................... 6 b. Sex...... ................................... 7 c. Diet and metabolic substance ................ 8 d. Photoperiod and sleep ....................... 8 e. Stress and excercise ........................ 9 3- Regulation of GH secretion ..................... 10 a. Hypophysiotropic hormones ................... 11 i. Growth hormone releasing factor ......... 11 ii. Somatotropin release inhibiting factor... 12 b. Feedback regulation ......................... 14 i. Growth hormone autoregulation ............ 14 ii. Insulin like growth factor feedback control............ ..................... 15 4- Sexual dimorphism of GH secretion ............. 16 a. Plasma GH patterns in normal males and femaleSOOOOOO00.00.000.000...O 0000000000000 16 iv Page b. Influence of androgens on GR production.... 17 c. Influence of estrogens on GR production.....18 d. Gonadal steroids and GH regulation..........19 Materials and Methods................................21 A. Hormones and chemicals.........................21 B. Preparation of the cells.......... ............. 21 C. Cell dissociation and culture conditions.......22 D. Effect of phenol red and fungizone.............23 E. Incubation with test agents. ..... .... ....... ...24 F. Effect of cycloheximide......... ............... 25 G. Determination of intracellular GH ............. .25 H. Bovine GH-radioimmunoassay........ ............ .26 I. Statistical analysis............ ............... 27 Results and Discussion...................... ......... 28 A. Media composition and GH production.... ........ 28 1- Phenol red................... ............ ...29 2- Fungizone............... ......... . .......... 34 3- Cycloheximide....................... ...... .. 37 B. Selection of experimental conditions 1- Pattern of GH production in vitro........... 40 2- Cell culture architecture and intercellular communication............................... 45 3- Experimental conditions to be used.......... 48 C. Effect of steroids on GH released.............. 49 1- Effect of estradiol-17B and testosterone.... 49 Bags 2- Effect of testosterone metabolites ........ . 58 3- Effect of GRF challenge .................... 66 Summary and Conclusion .............................. 78 Literature Cited... ................................. 80 vi LIST OF TABLES Table Page 1 Effect of various concentrations of estrogen on GH released (ng/ml/24 h) from cow anterior pituitary cells in a static in Vitro incubation. so 2 Effect of GRF Challenge on GR released (ng/ml) from cells that incubated with different concentrations of estradiol—17B or testosterone for 24 h. 72 vii Pigure LIST OF BIGURES Photomicrograph of calf anterior pituitary cells (A) in primary culture 17 d after plating stained with Mallory stain ( magnification 7,560x ). (B)histological section of normal calf anterior pituitary cells stained with Orange 6 stain (magnification 18,9001”. Provided Courtesy of Dr. 3.1.. Stinson, Dept.of Anatomy. The effect of 45 uM phenol red on media an concentration from calf anterior pituitary cells in culture. Media was first changed 48 h after plating and every 24 h thereafter. Media were collected each 24 h from each well. The bar at 72 h after plating is the average GE concentration from all wells at the time the treatment was imposed;16n concentration in the media (ng. well .241) is shown on the left ordinate. GM concentration at 24, 48 and 72 h of treatment is expressed as a percentage of GM concentration at initiation of treatment (72 h after plating) and is shown on the right ordinate. Each point represents the mean of 6 replicate wells. Pooled standard error was 5 %. The effec g_8henol red (45uM) and estrogen (10-10, 10' , M) on media on concentration, expressed as“ a percentage of the GM concentration at initiation of treatment (72 h after plating): from calf anterior pituitary cells in culture. Each point represents the mean of 6 replicate wells. Pooled standard error was 4.7 9c. The effect of Pungisone (2.5 ug/ml) on media GH concentration from steer anterior pituitary cells in culture. The bar at 72 h after plating is the average on concentration in the media from all wells at the time the treatment was imposed. _§M concentration in the media (ng. well 24 h ordinate. GM concentration at 24, 48 and 72 h of treatment is expressed as a percentage of GE concentration at initiation of treatment (72 h viii is shown on the left gag 6 31 32 35 after plating) and is shown on the right ordinate. Each point represents the mean of 36 replicate wells. Pooled standard error was 2.3%. The effect of cycloheximide (10 or so ug/ml) on media on concentration from heifer anterior pituitary cells in culture. The bar at 72 h after plating is the average on concentration in the media from all wells at the time the treatment was imp osed. 3? concentration in the media (ng. well'yf . 24 h' ) is shown on the left ordinate. 83 concentration at 24, 48 and 72 h of treatment is expressed as a percentage of on concentration at initiation of treatment (72 h after plating) and is shown on the right ordinate. Each point represents the mean of 36 replicate wells. Pooled standard error was 3.2%. Pattern of as released from calf anterior pituitary cells. The bar at 0 time is the average cell content of on at plating. After plating, media were collected every 24 h from each well and assayed for GM concentration. Each bar represents the mean an concentration (ug/ml) of 12 replicates. Photomicrograph of calf anterior pituitary cells in culture (A) 24 h after plating (3,700X), (B) 72 h after plating (3,7003), (0) 96 h after plating (3,700!) and (D) histological section of normal bovine anterior pituitary cells (9,400X). Provided courtesy of Dr. A.L. Stinson, Dept. of Anatomy. The effect of estrogengom'1°,10'8 M) and testosterone (lo , 0' on media on concentration from calf anterior pituitary cells in culture. The bar at 72 h after plating is the average on concentration in the media from all wells at the time the treatment was i Tposed. Jill concentration in the media (ng. well' T. 24 ) is shown on the left ordinate. on concentration at 24, 48 and 72 h of treatment is expressed as a percentage of on concentration at initiation of treatment (72 h after plating) and is shown on the right ordinate. Each bar represents the mean of 72 replicate wells. Pooled standard error was 4.2%. The effect of estrogen 05:0: 10-8 M) and testosterone (10 on media on concentration from (A) steer anterior pituitary cells in culture and (8) heifer anterior ix 10 11 12 pituitary cells in culture. The bar at 72 h after plating is the average on concentration in the media from all wells at the time the treatment was imp osed. h"? concentration in the media (ng. wells? . ) is shown on the left ordinate. on concentration at 24, 48 and 72 h of treatment is expressed as a percentage of on concentration at initiation of treatment (72 h after plating) and is shown on the right ordinate. Each bar represents the mean of 12 replicate wells. Pooled standard error was 4.4 % for heifers and 6.1 % for steers. The effect of es ogen $10-10, 10'”8 M) and testosterone (10' , 0" M) on media on concentrations from 0(A) heifer anterior pituitary cells in culture and (B) cow anterior pituitary cells in culture. on concentration is expressed as a percentage of GB concentration at initiation of treatment (72 h after plating). Each bar represents the mean of 12 replicate wells. Pooled standard error was 5.8 % for heifers and 6.3 % for cows. The effect of testosterone metabolites (dihydrotestosterone and 3a-diol) on media on concentration from calf anterior pituitary cells in culture (A) the effect of low concentrations and (8) the effect of high concentrations. The bar at 72 h after plating is the average on concentration in the media from all wells at the time the treatment was imposrd.z Gnh _foncentration in the media (ng. well . ) is shown on the left ordinate. on concentration at 24, 48 and 72 h of treatment is expressed as a percentage of on concentration at initiation of treatment (72 h after plating) and is shown in th right ordinate. Each point represents the mean of 12 replicate wells. Pooled standard error was 4.2 % for low concentrations and 5.1% for high concentrations. Testosterone (T), dihydrotestosterone (DMT), 5a-androstane-3a,17D-diol (3a-DIOL). A) Basal on concentration (mg/ml) over time, 8) The effect 8f 1-44-MM growth hormone-releasing factor (10' M) on media on concentration from calf anterior pituitary cells in culture. GM concentration is expressed as a percentage of the basal on concentration. Each point represents the mean of 12 replicate wells. 63 13 The effect 8f 1-44-Mn growth hormone-releasing factor (10' M) chal enge for 1 h on media GB concentration (% of controls) from calf anterior pituitary cells which were preincubated wit estrggen (10-10, 10")” and testosterone (10' , ' M) for 24 h. Each bar represent the mean of 12 replicate wells. xi 73 INTRODUCTION Males of most vertebrate species, including humans, laboratory rodents (Wehrenberg et al., 1985) and ruminant animals (Irvine and Trenkle, 1971) are larger than females of the same species. Male compared with female cattle have slightly higher plasma concentrations of growth hormone (GH, Irvine and Trenkle, 1971). Sexual differences in pituitary content and release of GH first appear at the time of puberty (Hoeffler and Frawley, 1986). There are several lines of evidence to suggest that gonadal steroids, which increase at puberty, regulate this dimorphism (Davis et al., 1977). A number of studies had been conducted to examine the relationship of estradiol-17B (E) and testosterone (T) with secretion of GH (Cornin and Rogol, 1984; Jansson et al., 1985; Wehrenberg et al., 1985). Results of some of these studies have been contradictory (Davis et a1, 1977). For instance, it has been reported that E treatment increased plasma GH in humans (Goldzieher et al., 1976), sheep and cattle (Preston, 1975), while others have failed to show any effect of E on plasma GH in cattle (Beck et al., 1976). Androgen treatment stimulates GH secretion in humans (Illig and Prader, 1970) and in cattle (Convey et al., 1971; Anfinson et al., 1975; Johke, 1984). Possible explanations for the conflicting results may be due to differences in animal species, age of animals, and method and time of sample collection. In addition, differences in GM secretory pattern between adult male and female animals may play a physiological role in the rate of somatic growth (Isgaard et al., 1988). It is well documented that males have more muscle and protein and less fat than females (Schanbacher et al., 1980). In addition GB is known to have an anabolic effect on protein metabolism in muscle and a lipolytic effect on fat (Cryer and Daughaday, 1977; Trenkle and Topel, 1978). The pulsatile pattern of GH secretion in males has larger amplitudes with a low base line in between pulses, while in females GH secretion exhibits more frequent episodes with low amplitudes and a high base line between episodes (Eden et al., 1987). These observations suggest that the difference in growth and composition between male and female animals is associated with the sex steroids and they may have a role in modulating the GH secretory pattern. ’ Understanding the endogenous secretory pattern, of estrogen and testosterone and their effects on GH release, is of both theoretical and practical importance in order to take advantage of any potential growth—promoting effects these steroids might have on stimulating GH secretion and (or) synthesis. The overall objective of the present study I'K/"c (‘CI‘WCI ”1 9"" direct effects, or modulate the effect of growth hormone/ was to determine whether testosterone and estrogen have releasing factor (GRF), on GR production from dispersed bovine anterior pituitary cells in a static in vitro incubation. REVIEW 0! LITERATURE Growth is defined as the increase in the mass of an individual or the coordinated increase of all animal tissues over time (Beitz, 1985). The growth process involves interactions between nutrients, genotype, environment and the influence of a number of hormones on nutrient availability to cells and cell division (Spencer, 1986). Therefore, growth can be considered the most obvious long term process in animal production (Bauman et al., 1982). The primary goal of animal production is to supply high quality food for humans (Beitz, 1985). Thus, achieving maximum growth is a key objective in animal production (Young, 1987). Ruminants are important in human food production and animal ecology because of their unique digestive system for utilization of energy from plants with high cellulose content (Hood, 1982). Metabolic and endocrine manipulation is being used to improve the production of meat animals with the objective of attaining more protein and less fat deposition in their carcasses (Gopinath and Kitts, 1984). Implants of hormones (anabolic agents) have been widely used to improve the growth rate and the amount of meat produced from ruminants (Spencer and Garssen, 1983). These implants contain one or more of the sex steroids. It is generally agreed that these anabolic agents improve growth rate and alter metabolism favorably towards increased protein production and for decreased fat. The mechanisms of these actions have not been fully elucidated (Gopinath and Kitts, 1984). It has been suggested that the anabolic steroids act by altering the concentration of endogenous hormones, such as growth hormone (Buttery et al., 1978). Growth Hormone Secretion and Regulation Growth Hormone Production and Clearance. Growth hormone (GH) is secreted by specific anterior pituitary cells, the somatotrophs (Daughaday et al., 1975). The somatotropic cells predominate in the lateral portion of the pars distalis (Dellmann, 1981). The somatotropic granules average about 300 to 400 nm in diameter, have affinity for acidophillic stains and stain immunohistochem- ically for GH (Banks, 1981). CH is the most abundant hormone of all the active principles of the human anterior pituitary (Murad and Haynes, 1985). GB is synthesized within the endoplasmic reticulum and the nascent hormone is packed into secretory granules within the Golgi apparatus of the cell ( Phillips, 1987). Human GM is a single chain of 191 amino acids (Niall et al., 1971). It is a globular protein with a molecular weight of 22 Kdaltons, isoelecteric point of pH 4.9, and stable in pH 7.0 solution at 100 C (Li, 1987). In nonlactating Holstein cows, the GH secretion rate is 19.1 mg/d, and the serum half-life (tl/Z) of bovine GH (bGH) is 22.5 min ( Yousef et al., 1969). Gopinath and Kitts (1984) estimated the metabolic clearance rate of GM as 74.5 ml'1.kg body weight"':".h"1 and secretion rate as 0.91 ug'1.kg body weight"1.h'1 in steers. For calves, Trenkle (1976) reported the secretion rate as 2.6 ug'1.kg body weight-1.h'1. GM is secreted in a pulstile manner in all mammals” studied (Martin et al., 1978); including bulls and steers (Anfinson et al., 1975); and lactating cows (Vasilatos and Wangsness, 1981; Moseley et al., 1982). The frequency and amplitude of GH secretory episodes are determined by the balance between the two main hypothalamic peptides (growth hormone-releasing and -inhibiting factors, which regulate the secretion of GH (Baile, 1985). Factors Affecting GE Production. Several factors can alter GH concentrations in the blood such as age, sex, diet and metabolism, sleep, stress and exercise. Each of these factors will be discussed below. Ontogenesis of GM: Factors that determine GH production (synthesis and release) in developing animals are not fully understood (Walker et al., 1977). Pituitary responsiveness to the two hypothalamic peptides, growth hormone releasing factor (GRF) and somatotropin release- inhibiting factor (SRIF), and the presence of their receptors on somatotrophs are the major factors affecting GH secretion (Frohman et al., 1987). In cattle, circulating GM is higher in prepubertal calves than in pubertal animals ( Renyaert et a1.-, 1976; McCarthy et al., 1979; Johke et al., 1984 ). Somatotropic sensitivity to SRIF increases with age (Cuttler et al., 1986). The authors attributed the 'high plasma GH concentrations in young animals to a relative resistance to SRIF. In addition, GH released in response to GRF decreases with age (Johke et al., 1984). Sex of Animal and SH Secretion. Sex of animal is known to influence concentration of GH in the blood (McCarthy et al., 1979). Jones et al.(1965) was the first group to suggest that GM concentrations differed between sexes. They used acrylamide gel electrophoresis of rat pituitary extracts to quantitate GH. Birge et a1. (1967) demonstrated that anterior pituitaries from male rats 'contained more GH than those from female rats. It had been concluded that GH synthesis is greater in males as compared with pituitaries from females (McLeod et al., 1969; Burek and Frohman, 1970; Yamamoto et al., 1970). Using a reverse hemolytic plaque assay, the individual somatotrophs of male pituitaries were shown to have greater secretory capacities than those of females (Hoeffler and Frawley, 1986). Meanwhile, it was concluded that serum GH concentrations of males are higher than those of females at all ages studied (Renaert et al., 1976). Diet and Metabolic Substances and 8H Secretion. The three major classes of metabolic substrates (carbohydrate, protein, and fat) affect GH secretion (Martin and Reichlin, 1987). GH concentration is negatively correlated with both energy and nitrogen intake (Waghorn et al., 1987) . Also, increased fatty acids in the blood suppressed GH secretion (Imaki et al., 1985). In addition, insulin-induced hypoglycemia,as well as hypoglycemia from other causes, is a particularly potent stimulus to GH secretion (Murad and Haynes, 1985). Knopf et a1. (1968) reported that the individual amino acids differ in their capacity to evoke release of GB; for instance, arginine is a potent stimulus of GH release. Metabolic status is postulated to alter the effect of exogenous GH injection on nutrient partitioning (Sechen et al., 1989). Photoperiod and Sleep and SH Secretion: Exposure of animals to long daylights increased daily weight gain, food intake and metabolic efficiency (Schanbacher and Crouse, 1980). Barenton et al. (1988) demonstrated in sheep that GH as well as prolactin could be involved in mediating the growth-promoting effect of increasing photoperiod However, plasma GH and prolactin concentrations are affected differently by photoperiod. The authors suggested that photoperiodic stimulation of body weight in sheep is a consequence of light-induced GH secretion. A major burst of GH secretion in man is associated with the onset of sleep, as well as the electroencephalographic (EEG) pattern of slow-wave sleep (Takahashi, 1968). These results were confirmed by Knip et al. (1987) who found that the episodes of high GH release in rats were usually associated with slow-wave sleep. Also, it had been demonstrated by the same group that GRF was secreted in a pulsatile manner; however, there was no association between GRF peaks and slow-wave sleep. Stress and Exercise and SH Secretion. There is no doubt that a stress control mechanism operates in relation to the secretion of GH (Landon and Greenwood, 1968). A wide variety of stressful stimuli may cause an increase in the circulating concentrations of GH in a manner analogous to the release of adrenocorticotrophic hormone (ACTH) and the catecholamines (Brown and Reichlin, 1972). Martin and 10 Reichlin (1987) reported that physical and emotional stresses lead to an increase in GH secretion in humans. Also, moderate exercise may elevate plasma GH concentrations in man, independently from blood sugar concentrations (Roth, 1963). Regulation of on Secretion. Regulation of adenohypophyseal function is mediated mainly by hypothalamic regulatory peptides (Cryer and Daughaday, 1977). These peptides are transported from the hypothalamus to the anterior pituitary gland by the hypothalamo- adenohypophyseal-portal system, which originates from a capillary network in the median eminence (Murad and Haynes, 1985). The hypothalamic peptides are influenced by biogenic amines, ‘which. are 'transmitted from the central nervous system to the hypothalamus (Baile, 1981). Current evidence indicates that GM secretion, is under both stimulatory and inhibitory influences from hypothalamic and extra- hypothalamic sites (Reichlin, 1987). In addition, regulation of GH secretion is influence by the feedback mechanisms, as well as the neural signals, in regulating GH secretion (Tannenbaum, 1987). GRF and SRIF are the main hypothalamic peptides that regulate GH secretion (Jansson et al., 1987). SRIF withdrawal sets the timing of the episodic bursts of GH secretion (Kraicer et al., 1980), 11 while the magnitude of the bursts is set by the amount of GRF impinging upon the somatotrophs, before and during SRIF withdrawal ( Kraicer et al., 1988). Hypophysiotropic Hormones Growth Hormone-Releasing Factor. GRF was isolated and identified from pancreatic tumors (Guillemin et al., 1982; Rivier et al., 1982). Several GRF peptides have been isolated from these tumors: hpGRF-44, hpGRF-40, and hpGRF- 37 (Guillemin, 1984). The isolated peptides are the same major forms as those isolated from the human hypothalamus (Meister' et..al., 1987). Hypothalamic GRF isolated from other species differs from the human forms by at least three amino acids (Della-Fara et al., 1986). For instance, bovine GRF has been isolated and identified as a 44 amino acid peptide and differs from human forms in five amino acid residues (Esch, 1983). GRF is synthesized by cell bodies in the hypothalamus and transmitted through nerve endings to the median eminence where it' is secreted into the hypophyseal portal circulation (Meister et al., 1987). The majority of the GRF-immunoreactive cell bodies were found in the arcuate nucleus and medial perifornical region of the lateral hypothalamus (Merchenthaler et al., 1984) . The nerve fibers form a fan-like projection to the median ///I ,, 12 eminence where a dense accumulation of GRF-containing terminals are found (Martin and Reichlin, 1987). GRF has a high specificity for release of GH from dispersed pituitary cells ( Guillemin et al., 1984). Plasma GH concentrations increased proportionally to the log dose of hpGRF-44 and reached a peak at 5 to 10 min (iv injection) and 15 min (sc injection), although the peak sc injection was 37% of that of iv injection (Johke et al., 1984). The first 29 residues of hpGRF possess all the information required for full in vitro activity (Rivier et al., 1982). In addition, the effect of all GRF peptides on GH release showed identical dose-response curves (Guillmin, 1984). GRF is believed to be the major physiological secretagogue of GH, however the precise mechanism of action has yet to be established (Brazeau et al., 1982b). To date, GRF treatment increases 2+ concentration (Holl et al., 1987) the intracellular Ca and cyclic adenosine monophosphate (cAMP) synthesis and(or) stability (Tanner et al., 1988). GRF stimulates secretion, synthesis of GH, transcription of the GH gene, and proliferation of somatotrophs (Billestrup et al., 1987). Somatotropin Release-Inhibiting Factor (SRIF). SRIF had. been isolated. and. purified from, ovine hypothalamic tissue as a 14-amino acid containing peptide (Brazeau et al., 1973). SRIF had been detected in the brain and other 13 tissues in two forms, one is a 14- and another is a 28- amino acid containing peptide (Benoit et al., 1982). SRIF- 14 is less potent than SRIF-28 in its effect on GH- regulation ( Patel, 1987 ). Somatostatin may be categorized as a neurohormone in pituitary regulation, whereas in the nervous system it may be described as a neurotransmitter or neuromodulator (Reichlin, 1983). The SRIF molecule may inhibit some common cellular events in the pituitary and some other endocrine organs such as the pancreas, which results in inhibition of the release of some peptide hormones like insulin (Koeker et al., 1974). At the pituitary level, SRIF can inhibit basal release and GRF-stimulating effect of GH release (Wehrenberg et al., 1982). SRIF may be involved in short- loop feedback control of GH secretion (Martin and Reichlin, 1987). Incubation of hypothalamic tissue with GH (Berelowitz et al., 1981a) and SM-c (Berelowitz et al., 1981b) elevated the SRIF secretion into the media. SRIF reduces the calcium concentration in the somatotrophs (Thorner, 1987). He reported that concentration of calcium, amplitude and frequency of calcium spikes were associated with GH secretion . In addition, SRIF can reduce tissue cAMP (Borgeat et al., 1974) and it also inhibits release of stored and newly synthesized GH (Sheppard et al., 1979). 14 Feedback Regulation GH Autoregulation. GH itself might be capable of controlling its own signal via hypothalamic and (or) pituitary receptors (Martin and Reichlin, 1987). The concept of GH autoregulation was obtained by earlier studies that content or synthesis of pituitary GH was reduced by GH administration (Krulich and McCann, 1966; Muller and Pecile, 1966). Intravenous or intracerebroventricular injection of GH suppressed GH release independently from circulating SM-c (Abe et al., 1983). At the pituitary level, GH per se operates as a feedback inhibitor of basal GH release from bovine anterior pituitary cells in culture (Glenn, 1986). In addition, GH secretion from anterior pituitary cells in static in vitro incubation declines with time, whiLe it was shown to increased spontaneously in a perfusion system (Lapp et al., 1987). Furthermore, GH may increase the pituitary SRIF receptor binding capacity (Kata-Kami, 1985). At the hypothalamic level, GH: may stimulate SRIF release from incubated hypothalami in vitro (Berelowitz et al., 1981). Intercerebroventricular administration of GH increased SRIF released into the hypophyseal portal circulation (Chihara et al., 1981). It has been reported by Tennenbaum (1987) and Miki et al. (1988) that GH may decrease output of GRF concomitantly with increased release of SRIF. 15 Insulin-Like Growth Factor (IGF) Feedback control. IGF's are produced in many tissues and are believed to exert their action in a paracrine or autocrine fashion (Phillips, 1986) . High concentrations of immunoreactive IGF-1 and IGF-2 have been observed in the brain and all three lobes of the pituitary (Valentino et al., 1988). Evidence has indicated that the IGF peptides exert feedback control on SE secretion by acting at both the pituitary and the hypothalamic levels (Berelowitz et al., 1981b). Basal and stimulated GH release from rat anterior pituitary cells in vitro can be inhibited by administration of IGF-1 (Berelowitz et al., 1981b; Brazeau et al, 1982a; Goodyer et al., 1984). Furthermore, transcription of GH mRNA in rats was suppressed by IGF treatment (Fagin et al., 1988) and even after GRF challenge (Yamashita and Melmed, 1986). Increased circulating concentrations of GH increased IGF-1 gene expression in rat pituitaries, which may result in inhibiting GH gene expression (Fagin et al., 1988). It has been suggested that IGF-1 gene expression in the pituitary appears to be dependent on the circulating, and not the local, pituitary GH concentration. At the hypothalamic level , IGF stimulates SRIF secretion from rat hypothalamic fragments in vitro (Berelowitz et al., 1981a). Surprisingly, Glenn (1986) found no effect of IGF-1 on 16 basal or GRF-stimulated GH release from bovine anterior pituitary cells in vitro, while GH was suppressed in cultures of rat anterior pituitariy cells. a \ \“ a .- -*,_, --- (5 Sexual””D-imorphism of GH. Secretion. V_ _ .. A“! ..5) .__,___,. 9....41 -:~~—'“ w. - . ..r...~.—D-- ma“-~«.u—~~~M u"- “mu-- :4\_’»—‘.(‘~ 1 Males of most vertebrate species, including humans, laboratory rodents (Wehrenberg et al., 1985) and ruminant animals (Irvine and Trenkle, 1971) are larger than their female counterparts. Gonadal steroids, which constitute a M fl fundamental difference between the sexes, might mediate their effects on growth through regulating GH synthesis and release (Cornin and Rogol, 1984; Jansson et al., 1985; Wehrenberg et al., 1985). In cattle, intact males compared with females have consistently higher plasma GH 7 concentrations at all ages (Irvin and Trenkle, 1971; Reynaert et al., 1976). The difference in body weight gain between males and females becomes most pronounced near the / time of puberty (Wehrenberg‘iet a1, 1985). Also, the difference in pituitary GH content (Birge et al., 1967) and release (McLeod et al., 1969; Yamamoto et al., 1970; Burek and Frohman, 1970) appears around time of puberty, when concentrations for males exceed those of females. Plasma GH Patterns in Normal Males and Females. The episodic nature of GH secretion is influenced by animal age 17 and gonadal steroids (Eden et al., 1987) . GH secretory 5.x”, patterns are different between males and females (Ohlson et al., 1977). GB secretion in adult males has been described as high intermittent episodes with low or undetectable concentrations in between episodes in rats (Tannenbaum and Martin, 1976), sheep (Davis et al., 1977), and cattle (Anfinson et al., 1975). These large surges of GH release are often multiphasic with two or three smaller spikes of . GH within each surge (Millard et al., 1987). In adult J females, GH secretion occurs in more frequent peaks with lower amplitudes than in males, while GH concentrations / between pulses (baseline) are greater than in adult male rats (Saunders et al., 1976), pigs (Dubreuil et al., 1988), sheep (Davis and Borger, 1974), and cows (Vasilatos and Wangsness, 1981). Influence of Androgens on Plasma GH Pattern. Differences in growth rate between intact and castrated males have been known for many years (Dubreuil et al., 1989). The endocrine basis for this difference in growth rate has logically been attributed to testostegggne (Gortsema et al., 1974). However, the mechanism by which testosterone increases growth rate has not been fully explained (Anfinson et al., 1975). One possible explanation for the growth promoting effect of testosterone may be 18 through regulation of GH production (Jansson et al., 1985). It has been reported that increased testosterone concentrations are associated with increased circulating GH concentrations in human males (Illig and Prader, 1970) and in bulls (Convey et al, 1971). Gonadectomy of males resulted in partial feminization of the secretory pattern of GH in rats (Eden, 1987). In cattle, GH secretory spikes of bulls are greater in magnitude than in steers (Anfinson et al., 1975). Also, treatment of castrated male rats (Wehrenberg et al., 1985) and steers (Johke, 1983) with testosterone enhanced the pituitary GH response to GRF. Influence of Estradiol-178 on Plasma GH Pattern. Estrogenic compounds improve growth rate and meat production in cattle and sheep (Gopinath and Kitts, 1984). However, the underlying mechanism has not been clarified (Simard et al., 1986), although it has been suggested that estrogenic compounds may exert their effect on growth through regulation of GH production (Jansson et al., 1983). Treatment with these compounds increases weight of the pituitary gland (Elias and Weiner, 1987) and increases numbers of acidophillic cells in the pituitary gland (Clegg and Cole, 1954). Moreover, administration of estradiol-17B increases plasma GH concentrations (Breier et al., 1988). Estrogen seems to influence GH secretion during adult life 19 by increasing basal GH concentrations and GH pulse / frequency and by decreasing pulse amplitudes (Borger et al., 1973; Trenkle, 1976; Ohlson et al., 1977; Donaldson et al., 1981; Eden et al., 1987). It has been suggested that estrogen exerts a stimulatory effect on GH production via a direct action on the rat pituitary gland (Jansson et al., 1983; Simard et al., 1986. In addition, the increase in CH might be due to an increase in secretion rate, and not to a decrease in the clearance rate as suggested by Trenkle (1981) . Moreover the estrogen-induced increase in GH has been reported to be independent of circulating somatomedin concentrations (Weidemann et al., 1976). At the level of the hypothalamus estrogen treatment may increase some of the biogenic amines such as dopamine, opioid peptides, serotonin, and acetylcholine ( How and Pfaff, 1984; Elias and Weiner, 1987; Stumpf, 1988) which increase GH concentration in the blood (Martin and Reichlin, 1987; Armstrong et al., 1988). Gonadal Steroids and GH-Regulation. Male anterior pituitaries are capable of secreting more GH than those of females (Evans et al., 1985). These differences were attributed to ‘the secretory capacity of individual somatotrophs rather than to the difference in their number (Hoefler and Frawley, 1986). Also, it has been demonstrated s‘ .N 20 that the rate of GH synthesis by adult male pituitaries was greater than that of adult female pituitaries (Burek and Frohman, 1970). Individual somatotrophs in male pituitaries have a greater responsiveness and (or) sensitivity to GRF stimulation than those of females (Evans et al., 1985; Wehrenberg et al., 1985; Krieg et al., 1986). GRF induces cellular cyclic adenosine 3',5’-monophosphate (cAMP) accumulation and GH release in males to a greater extent than in females (Cronin and Rogol, 1984; Simard et al., 1986). Finally, it may be possible that the sex steroids exert an effect at the level of the hypothalamus to modulate neural signals, which may regulate the state of responsiveness of the somatotrophs (Evans et al., 1985). MATERIALS AND METHODS Hormones and Chemicals. Estradiol-17B, testosterone, 5a-Androstan-17B-OL-3-ONE, 5a—Androstane-3a,17B-Diol, N-2- Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) , sodium bicarbonate, penicillin and streptomycin solution, and collagenase type 1A, were purchased from Sigma Chemical Company, St. Louis, MO. Hanks’ balanced salt solution (HBSS), Dulbecco's Modified Eagle Medium with low glucose (DMEM) with and without phenol red , fungizone, pancreatin 4X N.F., trypan blue stain 0.4 % , and newborn calf serum lot# 4705 were purchased from GIBCO Laboratories, Grand Island, NY. Growth hormone releasing hormone 1-44 was generously donated by The Upjohn Company, Kalamazoo, MI. Preparation of Cells. Calf pituitaries were obtained from Wolverine Packing Company, Detroit, MI. Cow pituitaries were collected from Snyder’s slaughter house, Fowler, MI. Steer and heifer pituitaries were obtained from the Michigan State University Meat Laboratory. Pituitaries were removed as aseptically as possible and kept in a cold sterile oxygenated Hank’s Buffered Saline Solution (HBSS) until transferred to the laboratory. HBSS was supplemented with 10 mM HEPES, 10 U/ml penicillin, 10 ug/ml streptomycin and 2.5 'ug/ml fungizone. All subsequent procedures were performed under sterile conditions. 21 22 Cell Dissociation and Culture. Bovine pituitary cells were prepared following the same techniques as reported by Vale et al. (1972) as modified by Miller et al. (1977) and Padmanabhan et al. (1978). Briefly, posterior pituitaries were removed and anterior pituitaries were sliced into 1 mm thick slices using a Staddie Riggs microtome. Anterior pituitary slices were suspended in HBSS containing 0.3 96 collagenase, and the suspension was incubated at 37 C with gentle stirring. After 45 to 60 min the medium was replaced with HBSS containing 0.25 % pancreatin, and the incubation continued for 10 to 15 min. The resulting cell suspension was filtered through sterile gauze and the cells recovered by centrifugation of supernatant at 300 x g for 5 min. The average total cell yield was approximately 9.5 X 106 cells for each pituitary gland. Cells were suspended in culture medium (DMEM with phenol red). The medium was supplemented with penicillin (10 U/ml), streptomycin (10 ug/ml), HEPES (25mM), NaHC03, and 10 % newborn calf serum, at pH 7.4. Fungizone (2.5 ug/ml) was added during the first 48 h only. Cell viability as judged by trypan blue exclusion was greater than 90 % in all experiments. Suspended cells were plated at a denisity of 4 to 6 x 105 cells/ml. Cells were plated in Corning 24-well treated dishes (25820-Corning, NY). Cultures were maintained at 37C in a humidified 23 atmosphere of 5 % C02, 95 % 02; in a C02 incubator Model 3028, Forma Scientific (Marietta, OH). Medium was changed 48 h after plating, and then at 24-h intervals. All treatments were imposed 72 h after plating. Media were collected every 24 h and stored at -20 C until assayed for concentrations of GH. Effect of Phenol Red and Fungizone. The effect of phenol red on media concentration of GH was tested in anterior pituitary cells obtained from calves. Cells were plated at 5X105 cells.ml'1.well'1 using two 24dwell dishes. Cells were incubated with 0, 10-10, 10'8, 10'6M estradiol-17B (E) in the presence or absence of 45 uM phenol red. Treatments were started after 72 h of plating and continued for another 72 h with six replicate wells/treatment. Media were collected every 24 h and stored at -20 C until assayed for GH. Fungizone (Fz) effects were examined in anterior pituitary cells from steers. Three dishes (24 wells/dish) with plating density of 4X105 cells.ml'1.we11"1 were incubated with 0 or 2.5ug/ml Fz. Treatments were started after 72 h of plating. Fz was present for another 72 h with 36 replicate wells/treatment. Media were collected at 24 h intervals and stored at -20 C until assayed for GH. 24' Incubation with Test Agents. Estradiol-17B (E) or testosterone (T) were dissolved in absolute ethyl alcohol. Concentrations of 10’”, 10'8M of E and 10'”, 10'5M of T were prepared, with a final concentration of 0.01% or less of alcohol. The effects of E or T were studied in anterior pituitary cells from either calves, heifers, steers or cows. Anterior pituitary cells from calves were plated at 5X105 cells/well. Cells were incubated with respective steroids for 24 h with 24 replicate wells for each treatment. Each study was repeated in three independent experiments. After 24 h of incubation with the steroid, cells were washed four times with serum-free medium and then challenged with 10'8M GRF for 1 h. All media were collected and stored at -20 C until assayed for GH. Anterior pituitary cells from heifers, steers or cows were plated at 5x10S cells/well. The steroid was present for 72 h with 12 replicate wells for each treatment. This study was repeated one more time. Media were collected at 24 h intervals and stored at -20 C until assayed for GH. The effect of dihydrotestosterone (DHT) or 5a— Androstane-Ba,17B-diol (3a-Diol) on GH release was studied using calf anterior pituitary cells. Cells (5X105) were incubated with T (10'7,1o"5M), DHT (10‘3,1o'5n), 3A-Diol 25 (lo-10,10'8M) or control media. The respective steroids were present for 72 h with 12 replicate wells/treatment. Media were collected every 24 h and stored at -20 C until assayed for GH. Effect of Cycloheximide. Cycloheximide (CHX) is an inhibitor of protein synthesis. Anterior pituitary cells obtained from heifers were plated at 5X105 cells/ml and six dishes (24 wells/dish) were incubated with 0, 10 or 50 ug/ml CHX. Treatment was started after 72 h of plating and continued for another 72 h with 36 replicate wells for each treatment. Media were collected after each 24 h period and stored at —20 C until assayed for GH. Determination of Intracellular GH. Intracellular GH was determined using a modification to the procedure described by Jones et al. (1965) and Birge et al. (1967). Briefly, immediately after dispersion, an aliquot of calf anterior pituitary cells (5X105 cells/ml) was washed three times with phosphate buffer (0.05M, pH 7) and then resuspended in the same buffer. Cell membranes were disrupted using a Branson Sonifier Model 350 (Branson Sonic Power Company, Danbury, CT). Sonification was performed for 15 s at 50 % duty cycle and an output of 4. Then the tubes were centrifuged at 1800 x g for 30 min and the supernatant was stored at -20 C until assayed for GH. 26 Bovine GE Radioimmunoassay. Bovine GH (bGH) was measured in duplicate by double antibody radioimmunoassay (Purchas et al., 1970). All reagents used in this study were kindly provided by Dr. H.A.Tucker ( Department of Animal Science, Michigan State University). Briefly, bGH (NIH-GH) was labeled with 1251 on a Biogel P-60 column. The column yielded two radioactive peaks; the materials from the first peak (RSI-CH) was used in the assay. Rabbit anti-bovine gamma globulin (lst antibody, AB) was diluted 1:10 with rabbit control sera, which had been diluted 1:400 using phosphate buffer saline-EDTA (PBS-EDTA) 0.05M at pH 7. Sheep anti-rabbit gamma globulin (2nd AB) was diluted 1:15 using PBS-EDTA, 0.05M at pH 7. Standard tubes (one set /300 assay tubes) containing 0.1, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 5.0 ng standard NIH- GH were assayed with each experiment. Appropriate samples were prescreened and then all samples from one experiment were run in the same assay at relevant dilutions. Media were then incubated at 4 C with 1st AB for 24 h, followed by another 24 h incubation with the labeled hormone at 4 C. Finally, incubation was carried out for 72 h with the 2nd A8 at the same temperature . Then all tubes were centrifuged at 1800 x g for 30 min and the supernatants were decanted. The tops of the tubes were washed and left 27 to dry overnight and then all tubes were counted in a gamma counter (Gamma Trac 1290, Tm Analytic, Elk Grove Village, 11) for 1 min/tube. Radioimmunoassay standard curves were obtained from a mmltiple regression equation with linear, quadratic and cubic components. Statistical Analysis . In experiments conducted for 24 h, data were analyzed by a randomized complete block design to compare the effect of concentration of steroid with the control. In experiments in which the effect of time (72h) on GH production was studied data were analyzed by split plot design with repeat measurements (Gill,1978). The effect of GRF challenge on GH production was tested using an approximate t-test, which examines the ratio of GH released after GRF challenge for steroid treated and nontreated wells (Gill, personal communication). RESULTS AND DISCUSSION Media Composition and GH-Production Hormone production by dispersed anterior pituitary cells in culture can be altered by a variety of nonhumoral factors, such as medium pH, buffer composition, and medium formulation (Wilfinger et al., 1979 ). In the present study pH was adjusted to 7.4 and maintained throughout the experiment by using the COZ-NaHCO3 buffer system, in addition to 25 mM HEPES buffer. It is well known that serum supplements (15 to 20 %) can correct medium deficiencies and enhance in vitro anterior pituitary hormone production ( Wilfinger et al., 1979 ). However, serum components are highly variable and thus, become an important source of experimental error when used ix: high concentrations (Rayan, 1989). Ham (1974) indicated that serum supplements for primary cell cultures could be reduced by adjusting nutrient concentrations to optimum values. Under the experimental condition employed in the present experiments, all serum used was purchased as one lot of newborn calf serum ( lot No. 4705, GIBCO, NY ). Media, Dulbecco’s Modified Eagles Medium (DMEM), were supplemented with 10% serum as described by Padmanabhan et al. (1978), Simard et al. (1986) and Rayan (1989). Under 28 29 these conditions the cells were able to maintain a normal performance for more than 17 do. Figure 1a shows the anterior pituitary cells in culture after 17 d of plating. It is apparent from this figure that the cells have a normal appearance figure 1b, even 17 d after plating. This suggested that the conditions used in this experiment were sufficient to maintain the bovine anterior pituitary cells in a normal performance throughout the experimental period of approximately 7 d. I 9 Phenol Red. Phenol red, a pI-i indicator in tissue culture media, has estrogenic activity at concentrations of 15 to 45 uM (Berthois et al., 1986). The authors indicated that phenol red should be considered in any studies with estrogen and estrogen-responsive cells in cultures. Figure 2 illustrates the effect of 45 uM phenol red (concentration normally found in DMEM) on GH concentration found in the media after 24, 48, 72 h incubation of calf anterior pituitary cells in a static in vitro incubation. GH concentrations were not different (P>0.05) from controls (media without phenol red) at all periods studied. These results indicated that incubation of anterior pituitary cells with phenol red did not affect GH concentration in the media. The interaction between phenol red and various Figure 1 30 Photomicrograph of calf anterior pituitary cells (A)in primary culture 17 d after plating stained with Mallory stain ( magnification 7,560X ). (B)histological section of normal calf anterior pituitary cells stained with Orange G stain (magnification 18,900X). Provided Courtesy of Dr. A.L. Stinson, Dept.of Anatomy. Figure 2 32 The effect of 45 uM phenol red on media GH concentration from calf anterior pituitary cells in culture. Media was first changed 48 h after plating and every 24 h thereafter. Media were collected each 24 h from each well. The bar at 72 h after plating is the average GH concentration from all wells at the time the treatment was imposed;1 GH concentration in the media (ng. well .24 h 1) is shown on the left ordinate. GH concentration at 24, 48 and 72 h of treatment is expressed as a percentage of GH concentration at initiation of treatment (72 h after plating) and is shown on the right ordinate. Each point represents the mean of 6 replicate wells. Pooled standard error was 5 %. 33 [ z ] NouvalNaonoo HO b100 o o o o T1?.T.‘i‘m° e—e PHENOL RED * g STANDARD e—e CONTROL 0333333333:33333333333)3333333333333333331 3600 3000- 2400- 1800- 1200- 500- [I_H 172° TIBM‘ON] NouvalNzaoNoo HO [— 48 72 96 120 144 168 TIME AFTER PLATING 24 ' 2'4 TIME OF TREATMENT DO 34 concentrations of estrogen (10'10, 10'8, and 10'6M) on media GH concentration from calf anterior pituitary cells in culture is presented in figure 3. No differences (P>0.05) were observed between any of the treatment combinations and the controls. These observations indicate that there were no interactions between phenol red and all concentrations of estrogen used on GH released from anterior pituitary cells of heifers in culture for up to 72 h after plating. Fungizone. Long’ term incubation. with fungizone (Fz) , an antifungal agent, reduced GH secretion from the pituitary cell line, GH3 cells, (Lapp et al., 1987). Since this experiment dealt with a primary culture, the possibility for contamination exists even when great precaution is taken. Therefore, it was necessary to use antifungal agents to prevent fungal outgrowth. Fungizone is the most commonly used antifungal agent in tissue culture (Oosterom et al., 1983; Lapp et al., 1987). The apparent advantages of F2 have been: a) solubility in culture medium; b) stability in solutions at 37 C, and c) documented use with anterior pituitary cells in primary cultures ( Foord et al., 1983; Oosterom et al., 1983; Lapp et al., 1987 ). In the present study the effect of preincubation with Fz for 24, 48 and 72 h on GR Figure 3 35 The effec of phenol red (45uM) and estrogen (10-10, 10' , 10' M) on media GH concentration, expressed as a percentage of the GH concentration at initiation of treatment (72 h after plating), from calf anterior pituitary cells in culture. Each point represents the mean of 6 replicate wells. Pooled standard error was 4.7 %. 36 meDOI._ szfihfimh mmt< usE. INN obs s Im¢ I¢N 0? s obs KK KX KK K KKKKKK KKK XK [XXXXX KKX m zmlop E m iwlop N u Eo—lo— ”u JOEZOU “U K K K K XXX low cop [ z ] NOIlVHlNZ-IONOO H9 37 concentrations found in the media was observed. Figure 4 shows the effect of F2 (2.5 ug/ml, normal concentration used in cultured pituitary cells) on GH concentration from anterior pituitary cells of steers in culture. The data demonstrate no reduction (P>0.5) in GH concentration at 24 or 48 h after incubation with Fz. However, a reduction (P<0.05) of 25% in GH released was noted after 72 h of incubation with Fz. These results are in agreement with those of Lapp et al. (1987) who concluded that F2 is not an acceptable medium constituent for long term culture (more than 72 h) of somatotrophic cells. The data herein suggested that Fz may not be the best antifungal compound to be used in these tissue cultures. However , Fz can be used as an effective therapy, since the effect of F2 on GH secretion is reversible (Lapp et al., 1987). In the present study, Fz was added during the first 48 h only to prevent fungal outgrowth from contamination during pituitary collection and cell preparation. Cycloheximide. Cycloheximide (CHX) , an inhibitor of protein synthesis was investigated to determine whether cells in the present study and under the experimental conditions used were capable of synthesizing GH. Also of interest was whether the newly synthesized GH interferes with the amount of GH released. The effect of 10 and 50 38 Figure 4 The effect of Fungizone (2.5 ug/ml) on media GH concentration from steer anterior pituitary cells in culture. The bar at 72 h after plating is the average GH concentration in the media from all wells at the time the treatment was imposed. H concentration in the media (ng. well" . 24 h' ) is shown on the left ordinate. GH concentration at 24, 48 and 72 h of treatment is expressed as a percentage of GH concentration at initiation of treatment (72 h after plating) and is shown on the right ordinate. Each point represents the mean of 36 replicate wells. Pooled standard error was 2.3%. 39 [z] NOIlVBlNEIONOO HO Emmaoxm Eustace ...o us: «1 wt— — o% a ”$29; ozEfio more use. ON— L mm KP NB we VN L _ 16101026191016:€6161.101030101619101010101010319163:0101019.”)IO:t ’ Aflfidzflfiwnflu 3233.3 “23.028 I ..oEzoo olo noow room room room 1000- r comp [PH 172' IJ‘IBM'ON] NOIlVBlNZ-IONOO Ho 40 ug/ml CHM on the percentage of GH concentration found in the media after incubation of anterior pituitary cells from heifers for 24, 48, 72 h is presented in figure 5. Incubation with CHX for 24 h had no effect (P>0.05) on GH concentration but decreased (P<0.05) GH concentration after 48 and 72 h of incubation with CHX. These data suggest that CHX (10 and 50 ug/ml) had no effect on GH released at 24 h of incubation; however, it may effect viability of cells after 48 h when all protein synthesis was inhibited. Pattern of GH-Production In Vitro Figure 6 presents the pattern of GH production from calf anterior pituitary cells in vitro. The GH production from somatotropic cells declined from 24 to 96 h after plating as GH concentration in the media was measured at 24 h intervals. Media concentration of GH at 72 h of plating was 2.6 ug/ml and a further decrease of 42% of the later concentration was observed 96 h after plating. In a preliminary study using the same experimental conditions in culture, GH production was measured in cow anterior pituitary cells. A continuous decrease in OH production was noted after 17 d of plating which was about 80% of the media concentration at 72 h after plating. Similar findings were reported by Hoeffler and Frawley (1986) for anterior 41 Figure 5 The effect of cycloheximide (10 or 50 ug/ml) on media GH concentration from heifer anterior pituitary cells in culture. The bar at 72 h after plating is the average GH concentration in the media from all wells at the time the treatment was in osed. G concentration in the media (ng. well” . 24 h- ) is shown on the left ordinate. GH concentration at 24, 48 and 72 h of treatment is expressed as a percentage of GH concentration at initiation of treatment (72 h after plating) and is shown on the right ordinate. Each point represents the mean of 36 replicate wells. Pooled standard error was 3.2%. 42 [z] Nouvmnaonoo HO Hmmsoxa ._.zu§._.0.05) on GR concentration found in the media. This effect was confirmed in three independent replications utilizing cells from calf anterior pituitaries. These results are in agreement with the findings of Fukata and Martin (1986) and Wehrenberg et a1. (1985). These authors concluded that neither E nor T showed any apparent influence on basal GH released by rat anterior pituitary cells in culture. Simard et al. (1986) demonstrated that incubation with physiological concentrations of E for 72 h increased basal GH release as well as cellular GH content in rat anterior pituitary cells in primary culture. However, this effect was not observed at 24 h of incubation. The data herein demonstrate clearly that 24 h incubation with the concentrations of these steroids used 52 Figure 8 The effect of estgogen _(%0'10, 10'8 M) and testosterone (10 , 10 M) on media GH concentration from calf anterior pituitary cells in culture. The bar at 72 h after plating is the average GH concentration in the media from all wells at the time the treatment was imposed._fiH concentration in the media (ng. well . 24 h ) is shown on the left ordinate. GH concentration at 24, 48 and 72 h of treatment is expressed as a percentage of GH concentration at initiation of treatment (72 h after plating) and is shown on the right ordinate. Each bar represents the mean of 72 replicate wells. Pooled standard error was 4.2%. 53 [ z ] NouvalNaoNoo HO GNP .959: 82.2.... more us: ...stfifim ea c _ _ Nb ww m 0! em 0 KXXK KKK I KK DKKKKKrK—‘K—T ww>40.05) in GM concentration was noted after 24, 48 or 72 h of incubation with any of the mentioned concentrations of sex steroids. However, a significant (P<0.05) increase (33.6%) in GH release was noted after 72 h incubation with 10’5M T. Using the same experimental conditions, cow anterior pituitary cells were studied. Figure 10b shows the effect of various concentrations of E and T on GH released from cow anterior pituitary cells in culture. No differences (P>0.05) were observed between treatments at any time period studied. These data agree with the work of Wehrenberg et al. (1985) who found no significant differences in GB release at 24, 48, and 72 h of incubation of rat somatotrophs at physiological concentrations for either E or T. In contrast Simard et a1. (1986) reported a significant increase in GH released from rat anterior pituitary cells after 72 h of incubation with a physiological concentration of E. The present data suggest that neither E nor T had a direct effect on GH released from bovine anterior pituitary cells during static in vitro incubation. Testosterone Metabolites. The present study showed no direct effect of T on GH release from anterior pituitary cells in culture. Similarly, Wehrenberg et a1. (1985), Fukata and Martin (1986) and Simard et a1. (1988) 59 Figure 10 The effect of estsogen (go-10, 10"8 M) and testosterone (10' , 10" M) on media GH concentrations from (A) heifer anterior pituitary cells in culture and (B) cow anterior pituitary cells in culture. GH concentration is expressed as a percentage of GH concentration at initiation of treatment (72 h after plating). Each bar represents the mean of 12 replicate wells. Pooled standard error was 5.8 % for heifers and 6.3 % for cows. 6O "manor: ENE/DE more ”is: cm as me em 0 b F l— Q 19 non non ..o... ..om. 18 e 2012 .I. [on e 272 one ..om u zone. T. D 207.2 To .8 .6528 on... mamas: Q o? [GavoNVIS 3Alioadsaa :10 2] Ho 61 Hanoi Eustace Era m2: mm NB 9v .vN 0 _ _ _ 0 low ION Ton low Tom 10@ T. Ho. you ._. EWIOP III ._. EBIOF DID filow w EQIOP I w EOFIOF I j.Om JOKFZOU OIO MBOU Am [cavons 3N103ds38 .40 2] H0 62 also reported that T had no apparent influence on basal GH released by anterior pituitary cells in culture. Since the Sa-reduced metabolites act as intracellular mediators for many of the multiple actions that testosterone exerts on its target tissues (Martini,1982) study of the metabolites of testosterone is warranted. Moreover, the anterior pituitary cells (Cohen et al., 1980) and the hypothalamus (Sholiton et al., 1974) are able to metabolize T into 5a- androstane-17B-ol-3-one (dihydrotestosterone,DHT) and 5a- androstane-Ba,17B-diol (3a-diol). Meanwhile, Aakvaag and Haug 1979 reported that GH3 cells were able to convert T to E in culture. Therefore the effect of these 5a-reduced metabolites (DHT and 3a-diol) on GH released from anterior pituitary cells in culture was studied. In the present study anterior pituitary cells from calves were incubated with the following concentrations of T (10"5 and 10’7M), DHT (10'6 and 10'3M), and 3a-diol (10'8 and 10'10M, respectively). Figure 11a and 11b show the results of 72 h of incubation with these steroids on GM concentration. Incubation with either the low or high concentrations of each of these steroids had no effect (P>0.05) on GH concentration at either 24, 48, or 72 h. However, incubation with the higher concentrations of DHT and 3a- diol reduced (P<0.05) media concentration of GH after 72 h 63 Figure 11 The effect of testosterone metabolites (dihydrotestosterone and 3a-diol) on media GH concentration from calf anterior pituitary cells in culture (A) the effect of low concentrations and (B) the effect of high concentrations. The bar at 72 h after plating is the average GH concentration in the media from all wells at the time the treatment was imposed. GH _foncentration in the media (ng. well . 24 h ) is shown on the left ordinate. GH concentration at 24, 48 and 72 h of treatment is expressed as a percentage of GH concentration at initiation of treatment (72 h after plating) and is shown in th right ordinate. Each point represents the mean of 12 replicate wells. Pooled standard error was 4.2 % for low concentrations and 5.1% for high concentrations. Testosterone (T), dihydrotestosterone (DHT), 5a-androstane—3a,17B-diol (3a-DIOL). 64 [ z ] NOIlVHlNBONOO H0 o.» mm— 0 OP; ON: on: 3 on: e: on: own cm: 00—: OFF: Hmmsozg ._.zms:.0.05) the percentage (15.0:4.9, 3.511.1 and 14.714.7 ng/ml, respectively). However, GRF increased (P<0.05) the percentage by 38.2 % when incubated with 10'5M T. The nonsignificant effect of GRF following incubation with E is in agreement with data reported by Wehrenberg et al. (1985) and Fukata and Martin (1986) for rats. These 72 (Table 2) Effect of GR! challenge on an released from cells incubated with different concentrations of egtradiol-17E or testosterone for 24 h" ear . Treatment Unchallenged Challenged % 1 BEMc Testosterone 0 19.111.8 48.614.‘ 254.5 114.6 10'7 x 17.211.2 47.714.6 277.3 115.4 0 18.811.4 48.313.6 256.9 112.4 10'5 u 20.611.8 65.114.8 316.0‘117.1 Estrogen o 22.911.9 57.515.7 251.1 113.9 10'1° u 20.911.6 57.214.a 273.7 114.4 0 20.612.l 49.114.o 238.3 112.5 1.0.8 M 19.012.3 46.212.5 243.2 113.1 GR! (10'8 M) challenge for 1 h. a b Values are expressed as mean (ng/ml) of 24 wells 1 SEM. c (Challenged / unchallenged) % 1 SEE. * significantly different from controls (P<.05). 73 Figure 13 The effect_gf 1-44-NH growth hormone-releasing factor (10 M) chal enge for 1 h on media GH concentration (% of controls) from calf anterior pituitary cells which were preincubated wit estr en (10-10, 10’8M) and testosterone (10' , 10' M) for 24 h. Each bar represent the mean of 12 replicate wells. 74 mhzufiqu mt. [XXLX X1XXXXXXX7XX \ X \ \ 1\ m \ \ \ BL ._. Emlop nHU . ._. Ehlop a m in)? ”u . u ZOFIOF 8 405.200 nU . low ION, low. .fioom IO¢N IomN 10mm own [ z ] NouvaiNBONoo HO 75 authors demonstrated that incubation of rat anterior pituitary cells for 24 h with E (10'6 to 10'8M) showed no apparent influence on GRF-induced GH release. In contrast, Simard et a1. (1986, 1988) concluded that preincubation of rat anterior pituitary cells with E (lo'loM) for 72 h exerts a stimulatory effect on GRF-induced GH release. The data herein show that incubation of calf anterior pituitary cells with neither concentration of E affected GM release following GRF challenge. The increase in GM concentrations as a result of implantation with the estrogenic compounds in vivo (Breier et al., 1988) may be due to indirect mechanisms. For instance, E treatment can increase 61-! by altering serum somatomedins activity (Wiedemann, 1976). This may have a direct effect on GM production at the pituitary level (Yamashita and Melmed, 1986; Fagin et al., 1988) and (or) indirect effect at the hypothalamic level (Berelowitz et al., 1981a; Reichlin, 1983). Moreover, E can affect some biogenic amines in the brain (Stumpf, 1988; Kow and Pfaff, 1984) which has been reported to alter GH concentration in the blood (Martin and Reichlin, 1987: Armstrong et al, 1988). In the present study, incubation with (10'5M) T for 24 h enhanced the GRF-stimulated GI-I release in vitro. However, T (10'7M) had no effect on GRF-induced GM release. 76 Wehrenberg et a1. (1985) and Fukata and Martin (1986) observed no effect of the in vitro incubation with T (10"6 to 10'8 M) for 24 h on GRF-induced GH release in rats. The stimulating effect of T on GM production in vivo (Wehrenberg et al., 1985) may be, in part, due to modulating the somatotrophic response to GRF, which had been demonstrated in vitro in this study. The results of the present study agree with those of Cronin and Rogol (1984), Evans et al. (1985) and Ohlsson et al. (1987) who suggested that testicular androgen secretion increases the pituitary GH release in response to GRF in a static in vitro incubation. An additional observation worthy of comment is the difference in the GH released, from the same number of cells, between experiments. In heifers and calves the maximum media GH concentration for 24 h was in the range of 3000 ng/ml, while it was in the range of 2000 and 500 ng/ml in steers and cows, respectively. This observation agrees with data reported by Hoeffler and Frawley (1986) for rats, who concluded that the sexual differences in GM release are attributable to the secretory capacities of individual somatotrophs rather than to differences in the number of somatotrophic cells. In any case, it is conceivable that the GH secretion and the factors that alter GH production 77 are regulated mainly by long term changes in hypothalamic release of GRF and SRIF as well as sex steroids which may modulate their synthesis or release at the level of the hypothalamus. SUMMARY AND CONCLUB IONS The effect of estradiol-17B, testosterone and testosterone metabolites on growth hormone released from bovine anterior pituitary cells in a static in vitro incubation were studied. Anterior pituitary cells from either calves, heifers, steers or cows were dispersed and plated at 5X105 cells/ml in Corning 24-well dishes. Cells were allowed to attach for 48 h in a humidified atmosphere in a C02 incubator at 37 C. All treatments werev started after 72 h of plating and media were collected at 24 h intervals. Effects of some media components on GM were studied. Phenol red , a pH indicator which has been documented to have estrogenic activity in tissue cultures, had no influence (P>0.05) on GM released. Also, there was no interaction (P>0.05) between phenol red and added estrogen (10'10, 10's, 10"6 M) on GM released. Fungizone, an (antifungal agent. reduced (P<0.05) GH released (25%) after 72 h of incubation. Estradiol-17B (10'10, 10"8 M) or testosterone (10’7, 10'5M) was added to the cells for 24, 48 or 72 h. Neither estrogen nor testosterone had an effect (P>0.05) on the basal GH release from any of the pituitary cells irrespective of source (calves, heifers, steers, or cows). 78 79 Testosterone :metabolites, DHT (10-6, 1078M) and 3a-Diol (10’3, lo'loM), had no effect (P>0.05) on basal GH released at 24, 48 or 72 h, however the high concentrations of testosterone metabolites caused a decrease (P<0.05) in GM release. Calf anterior pituitary cells were also challenged with GRF (10"8 M) for 1 h. 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