FACTORS INFLUENCING BOVINE PROLACTIN AND GROWTH HORMONE Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY VALDIN G. SMITH 1974 III IIIIIIIIIIIIIQIIIIIIIIIIIII 3129 00 This is to certify that the thesis entitled Factors Influencing Bovine Prolactin and Growth Hormone Release presented by V . G . Smith has been accepted towards fulfillment of the requirements for Ph.D. degepin Dairy Science fizz“ Major professor E .M. Convey Date Se tember 25 1974 0-76” LIB RA .' " Michigan 5, "~13 University t1 4. APR 5&2001 . U j Exp. investig: (CH) rel: were ran: 5 m1 of j in 5 I1 0 effect of bovine pi Serum Pro the day b. 1h-15 rig/1 r13/1111 at 1 0n the 2 c' in cm tr and 17 and the start “usages f tine Wea t1 Within 2 m ABSTRACT morons runmcrnc BOVINE momcrm ms GROWTH HORMONE amuse By Valdin G. Smith Experiments were conducted in vivg and in m to investigate the control of prolactin and growth hormone (GH) release in the bovine. Ten lactating Holstein cows were randomly assigmed to receive subcutaneously either 5 ml of 50% ethanol (controls) or 80 mg of ergocryptine in 5 m1 of 50% ethanol on two consecutive days. The effect of ergocryptine on prolactin and GH release from bovine pituitary cell cultures was also investigated. Serum prolactin concentration in both groups of cows on the day before treatment, increased from an average of lit-16 ng/ml at 5 min before milking to approximately 30 ng/ml at 10 min after the start of milking (p < 0.05). On the 2 days of treatment prolactin concentration (ng/ml) in cows treated with ethanol averaged 20 and 35 (day 1) and 17 and 27 (day 2) at 5 min before and 10 min after the start of milking respectively (p < 0.05). Comparable averages for cows treated with ergocryptine were 1.3 and Lu (day l) and 1.1 and 1.1 (day 2). Following ergocryp- tine treatment serum prolactin concentration was decreased within 2 hr and remained suppressed for at least 5 days after tre (hug/m1) nest or a pituitary TC medium 60%. (p < concentra prolactin action ony Cell: cows, ate tr0115.11 re ledia pro following °f cows. 1926 and 1 TRH/ml To difference tent, Corr. cultfires f Th“from Media CH C but not H Valdin G. Smith after treatment. But average serum.CH concentration (h ng/ml) was not affected by either ergocryptine treat- ment or stimuli associated with milking. Incubation of pituitary cell cultures with 0.01 to 10 ug ergocryptine/ml TC medium 199, reduced prolactin concentration approximately 60%. (p < 0.001) but did not affect (p > 0.05) media on concentration. Therefore, ergocryptine decreases serum prolactin concentration in cattle perhaps by a direct action on the anterior pituitary. Cell cultures prepared from anterior pituitaries of cows. steers and a bull were incubated for 2 hr with thyro- tropin releasing hormone (TRH) at 72 hr or 96 hr of culture. Media prolactin concentration.was increased (p < 0.01) following addition of TRH to 72-hr pituitary cell cultures of cows. Prolactin concentration averaged -23. 799, 1966, 1926 and 1976 ng/ml after 0. 0.01. 0.1, 1.0 and 10 mg TRH/ml TC medium 199, respectively, when expressed as the difference in quantity released before and after TRH treat- ment. Comparable results were obtained with pituitary cell cultures from additional cows and from steers and a bull. Thyrotropin releasing hormone also increase1(p < 0.05) media GH concentration from 72-hr pituitary cell cultures but not from those treated at 96 hr. Neither baseline prolactin concentration nor IRR- induced prolactin release from cell cultures was affected by triiodothyronine (T3) or thyroxine (Tu) at concentrations of 0.1 or 1.0 ug/ml medium. But 5 and 50 ug Tu/ml medium decreased (p < 0.05) spontaneous release of prolactin and de to we Valdin G. Smith the quantity of prolactin released by TRH. Prolactin con- centration in the media averaged 161. 119.5 and 82.5 ng/ml after treatment with 0, 5 and 50 ug “In/m1 respectively. When these cultures were subsequently treated with TRH, prolactin released into the media averaged 261. 222 and 171.5 ng/ml respectively (p < 0.05). These results suggest that TRH releases prolactin in cattle at least in part by a direct action on the anterior pituitary and Ti; at high doses may inhibit spontaneous release of prolactin and the quantity releasable by TRH. The effect of concentrations of progesterone and estradiol, that approached physiological levels, on serum concentrations of prolactin and GH was also investigated. Serum progesterone concentration (ng/ml) increased (p < 0.05) from an average of 1.5 before. to 3-lt following placement of progesterone pessaries and remined elevated for at least 5 days following ovariectomr. Serum estradiol concentration (pg/m1) increased (p < 0.05) from an average of 8 before, to 21+ following placement of estradiOl im- plants and also remained elevated following ovariectonnr. Serum concentrations of these hormones decreased (p < 0.05) in untreated heifers within 24 hr after ovariectow. When depot steroids were removed the respective hormone concen- trations in serum decreased (p < 0.05) to levels comparable to concentrations in untreated ovariectomized heifers. There were no changes (p > 0.05) in serum concentrations of pro- lactin and GH attributable to steroid treatment. Neither did ova: (p > 0.C In into ova concentr any diff between serum GH bearing mater than in . lpproxim Prohctii lactin a. factors . ”341668 :‘ the 931m r°1e in . valdin G. Smith did ovariectomy or removal of depot steroids affect (p > 0.05) serum concentration of these hormones. In a subsequent study. placement of estradiol implants into ovariectomized heifers appeared to increase serum concentration of GH but not prolactin. Neither was there any difference (p > 0.05) in TRH-induced prolactin release between controls and estradiol-treated heifers. However. serum.GH concentration was 53% greater (p > 0.05) in heifers bearing h estradiol implants than in controls and 132% greater (p < 0.05) in heifers bearing 8 estradiol implants than in controls. Thus estradiol at concentrations which approximate those found at estrus in cattle do not influence prolactin concentration in serum. Hence, increase pro- lactin at or near estrus in cattle is probably due to factors other than increased serum estrogens. Similarly changes in serum progesterone of a magnitude expected during the estrous cycle of cows probably do not play a major role in control of prolactin and GH. FACTORS INFLUENCING BOVINE PROLACTIN AND GROWTH HORMONE BY Valdin G. Smith A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science 1974 Dr. C.A. for provi His appre. Hafs, W.D Bourne of and assist ful for t1- Drs. J, Me adviser, 1: tude for h Years of g ing this t ACKNOWLEDGEMENT The author wishes to express his gratitude to Dr. C.A. Lassiter, Chairman of the Dairy Science Department, for providing funds and facilities for his graduate studies. His appreciation is also extended to Drs. R.A. Tucker, H.D. Hafs, W.D. Oxender, J.H. Britt, R;W. Mellenberger and R.A. Bourne of the Dairy Physiology Department for their advice and assistance in his research program. He is also grate- ful for the advice and support of his committee members, Drs. J. Meites, R.S. Emery and W.G. Bergen. And to his advisor, Dr. Edward M. Convey, he extends a debt of grati- tude for his guidance and encouragement throughout five years of graduate studies and for his assistance in prepar- ing this thesis. To his co-graduate students he also expresses grati- tude for their help in different laboratory chores involved in this research. ii I- 191:2. tion I - receive I Vorke clan wi ”k 3.8. in 1361mml 'here I u— !l_ I - ‘ . _ BIOGRAPHICAL SKETCH of I was born in Jamaica, West Indies on December 12, 19%. After completion of elementary and secondary educa- tion I entered Jamaica School of Aaiculture in 1960 and received a diploma in agriculture in 1963. Thereafter I worked for 3 years as an artificial insemination techni- cian with the Ministry of Agriculture and Lands. In 1967 I entered Tuskegee Institute and received a 3.8. in animal science in 1969. I was accepted by the Department of Dairy Science, Michigan State University where I completed a M.S. in dairy physiology in 1971 under the guidance of Dr. E. M. Convey. Immediately thereafter I started working towards the Doctor of Philosophy degree which will be completed in Fall term 1971!. iii BIOGRAP] LIST OF LIST 0? LIST OF INTRODU< REVIEW ( A. B. TABLE OF CONTENTS BIOGRAPHCAL SKETCHIOOOOOOOOOOOOIOOOCIOIOOOOOOOOOOOOO LIST OF TABIIEOOIOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOO LIST OF FIGUREOOOOOOOOOOOOIOOOCCOOOOOOOOOOOIOOOOOOO. Page iii vii viii LIST OF APPNICEOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. x INTRODUCTIONCCCOOOCOO00....0......OOOOOCOOCOCCOCICOOO mm OF HMMEOOOOO.CCCOCCOOOOOO00.00.00.000... A. Prolactin and Growth Hormone (GH) requirement B. C. D. for hetationoooooooooooooooooooooooooooooooo Effect of Ergot Alkaloids on Prolactin and GH secretionOOOOOOOOOOOOOOOOOOOOOOO00.000.00.00. 1. In VLVQOOOOOOOOOOOOOOOIOOIOOOOOOOIOIOOOOOO 2.mm.................................. Thertropin Releasing Hormone (IRH)oooooooooo 1. GeneraJ-OIOOCOOOOOOICOOIOOOOOOCIOOOOCOOOOOO 2. Effect of TRH on Prolactin and GH concentrationaoooooooooooooooooooooooooooo 3. “Behanism Of TRH aetionooooooooooooooooooo h. Interaction of TRH with Thyroid Hormones.................................. Effect of Gonadal Steroids on Prolactin and GH concentrations............................ 1. EatrogenOOOOOOOOICOOOIOOOOOOOOOOOOIOOOOOOO 2. ProgGSteroneoooooooooooooooooooooooooooooo 3. Relationship of Gonadal Steroids and TRH on Prolactin and GH concentrations........ iv ‘0 “Q 0\ 4? ‘F 10 12 13 13 l? 20 MATERIAL: 01 0b. ”MILEANDMTHOD-GWMOOOOOOOOOOOOOOOOOOO0.... A. MiMJ-SOOO...O'COOOOIOOOCIOOOOOOO0.00.00.00.00 B. In Vitro ProcedllreSOOOOOOOOOOOOOOOOOOOOOOOOOOO C. E. Hormone Assays................................ 1. Protein Hormones........................... 2o SterOid Hormones..........................o Specific Objectives and Experimental Procedures.................................... Experiment 1. Effect of ergocryptine on Bovine Prolactin, GH, Cortisol and Milk Yield...... Objective 1: Effect of ergocryptine in vivg... Objective 2: Effect of ergocryptine in v1:: .. eriment 2. Thyrotropin Releasing Hormone TRH): Effect on Prolactin and GH release from Bovine Pituitary Cell Cultures......... Objective 1: Effect of TRH on Prolactin and GH Release m woooooooooooooo Objective 2: Prolactin Release in,xij:gu TRH VS GnRHoooooooooooooooooooooooooo Objective 3: Effect of triiodothyronine (T ) and thyroxine (T ) on TRH-indaced Prolactin Releasg in zit:g....... Experiment 3. Prolactin and Growth Hormone Release after Gonadal Steroids and TRH in Vivo and in VinOOIOOOOIOOOOOOOOOOI.0...... Objective 1: Serum Prolactin and GH after Gonadal Steroids in vivg......... Objective 2: Effect of Estradiol -l7a on TRH- induced prolactin and GH release in ViVOoooooooooooooooooooooooooo Objective 3: Effect of Estradiol -178 on base- line prolactin concentration and TRH—induced prolactin release in MOIOOOOOIOCOOIOOOOOOOOOOOOOOO Statis'tical ProcedureOOOOOOOOOOOOOOO0000...... V Page 21 21 22 2h 2h 25 26 26 26 27 27 27 28 28 29 29 30 31 32 REULT NH .n\ 3W BIBIJ Page REULTS AND DISCUSSIONOOOOOOOOOOOIOOOOOOOIOOOOOOOOOO. 33 hperiment 1. Effect of Ergocryptine on Bovine Prolactin. GH. Cortisol and Milk Yield........ 33 lo EffCCt Of ergocryptine in. Oooooooooooooooo 33 2. Effect of ergocryptineinv .............. 50 eriment 2. Thyrotropin Releasing Hormone TRH): Effect on Prolactin and GH release from Bovine Pituitary Cell Cultures................ 56 1. Effect of TRH on prolactin and GH release in Moose-0000000....oooooooooooooooooooooooo S6 2. Prolactin Release in m TRH vs GnRH........ 61 3. Effect of Triiodotrwronine (T3) and Thyroxine (T5!) on TRH-induced Prolactin Release in OOOIOOOCOOOOCOOOCOCOOOOOOCOOCOOOCOOOOCOO 6a Experiment 3. Prolactin and Growth Hormone Re- lease after Gonadal Steroids and TRH in 3119 andmMIOOOOOOOIOOOOOOOOOOOOOOOOOCOOOOOOO 71" 1. Serum Prolactin and GH after Gonadal ster°1d8mMIOIOOOOOOOIOCIOOOOOOO00...... 71‘ 2. Bovine serum Prolactin and GH Response to Betrad101 -179 and TRHOOOoooooooooooooooooooo 85 3. Prolactin Release in 211:2 in response to Estradiol -l7B and Tmrrotropin Releasing HormoneOIOOOOOO0.0..OOOOOOOOIOOOIOIOOCOOOO0.0 102 SUMY AND CONCLIISIONSOOOOOOOO00......OOOOOIIOOOOOO. 115 APPWICEOOOOOOOOOOOOOIOOOOOOO...OOOOOOOOOOOOOOOOOOC 119 BIBLIMRAPM0.00IOOOOOOOOOOOOOO...OOOOOOOIOOOIOOOOOOO 12]- vi 3. 5. 7. 9. 10 11 Taflole 1n 7. 8. 9. 10. 11. LIST OF TABLES Prolactin and growth hormone release from bovine pituitary cell cultures treated with ergocryptineOOOCOOOOOOOOOOOOOOOOO0.0.0.000...COO Prolactin release from bovine pituitary cell cultures treated with thyrotropin releasing hormonOOOOOO0..OOOOOCOOOOOOOIOOOIOOOOOOOOOOOOOOO Growth hormone release from.bovine pituitary cell cultures treated with thyrotropin releasing hormoneOOOIOOOOIOOICOOOOOOOIOOOOOOOOOOOOOOOOOOOO Prolactin release from bovine pituitary cell cultures treated with thyrotropin releasing hormoneOOOOOOOOOOOCOOOOCOOOOOOOO0.00.00.00.00... Growth hormone release from bovine pituitary cell cultures treated with thyrotropin releasing hormmeOOOIOOOOOOOIOOOOOOICOOCCIOOOOOOO00.00.... Prolactin release from.bovine pituitary cell cultures treated with gonadotropin releasing hormone or thyrotropin releasing hormone........ Prolactin release from bovine pituitary cell cultures treated with triiodothyronine. thyro- xine and thyrotropin releasing hormone.......... Prolactin release from.bovine pituitary cell cultures treated with triiodothyronine and thyrotropin releasing hormone................... Prolactin release from.bovine pituitary cell cultures treated with thyroxine and thyrotropin releasing hormoneOOOOOOCOOOOOOOOCIOOIOOIOIOOOOOI Effect of estradiol -l7p on basal and thyro- tropin releasing hormone-induced prolactin re- 51 57 57 59 62 63 65 66 68 lease from.bovine pituitary cell cultures....... 106 Prolactin release frommbowine pituitary cell cultures treated with estradiol -l7B and thyrotropin releasing hormone................... 107 vii Figure 1. cu cc Pin Pol 3. Pin le . In». 5 30.6 CEh Mme. . . . 6 7 Figure 1. 2. 3. 1+. 5. 6. 7. 8. 9. 10. ll. 12. LIST OF FIGURES Schematic representation of the preparation of anterior pituitary cell cultures and design Of mm experiments.-.................o.... Prolactin and growth hormone response to milk- ing on the day preceding ergocryptine treat- mntOOOCOCO00.00.000.000.0COOOCIICOCIOOOOOOCOOO Prolactin and growth hormone response to milk- ing on the first day of ergocryptine treatment. Prolactin and growth hormone response to milk- ing on the second day of ergocryptine treat- mntCOOOOOOOOOOIOO....0.0000COOOOOOOOOOCOOIOOOI Prolactin and growth hormone response to milk- ing on the day following ergocryptine treat- ment.....o.........................o........... Effect of ergocryptine and milking on serum cortisol concentration on the first day of er- gocryptine ueamntOOOOOII.OOOOOOIOOOOOOOIOOOI Chronic effect of two consecutive doses of ergocryptine on serum prolactin and growth hormone concentrations......................... ‘Milk yield in cows treated with ergocryptine on days 1 and 20.0.00000000000IOOOOOOOOIOOIOIOO Serum.progesterone concentration of heifers after placement of depot steroids and subse- quent ovarieCtmooooooooooooooooeoooooooooooooo Serum.estradiol concentration of heifers after placement of depot steroids and subsequent W19Ctmooooooooooooooooooooooooooooooooooooo Serum prolactin concentration of heifers after placement of depot steroids and subsequent WieCtOWooooooooeeoeooooooooooooooeeoococoons Serum growth hormone concentration of heifers after placement of depot steroids and subsequent omiectOWOOIIOOOOOOOOOOOOOOOCOCOOOOOOOIOOOOIO. viii Page 35 37 39 #2 "7 “9 75 79 82 8h (h I .- 15. ”1.11 16. Rachel 1?. sm.1 18. Tr“ 19. Thi 20, Figure 13. 1h. 15. 16. 17. 18. 19. 20. Page Serum estradiol concentration in ovariecto- mized heifers implanted with four estradiol- filled implmtaOOOOOOOOOIOOOOOOOOOOOOOOIOOI... Serum.prolactin concentration in ovariecto- mixed heifers implanted with four estradiol- filled iInPJ-antSOOOCIOOIOOOOCOO0.00IOOIIOOOOCI. Serum.growth hormone concentration in ovariec- tomized heifers implanted with four estradiol- filled implants...”oo......................... Thyrotropin releasing hormone-induced prolac- tin release from ovariectomized heifers im- planted with four estradiol-filled implants.... Thyrotropin releasing hormone-induced growth hormone release from ovariectomized heifers implanted with four estradiol-filled implants.. Serum estradiol concentration in ovariecto- mized heifers implanted with estradiol-filled inantSOOOOOOIOIOOOOOOOOOOOOOOOO0......00....C Thyrotropin releasing hormone-induced prolactin release from ovariectomized heifers implanted with eight estradiol-filled implants........... Thyrotropin releasing hormone-induced growth hormone release from ovariectomized heifers implanted with eight estradiol-filled implants. ix 87 89 91 9G 96 99 101 104 Appendix LIST OF APPENDICES Appendix 1. Preparation of media for cell culture. 2. Preparation of TC medium 199 . Page 119 119 Altho~ ascientif‘ has been a INTRODUCTION Although endocrinology did not attain importance as a scientific discipline until the twentieth century, much has been accomplished during this relatively short period toward understanding hormone-regulated mechanisms. Today we know that growth, reproduction and lactation of labora- tory and economically important animals appear to be endocrine regulated but the mechanism by which these events are controlled is not fully understood. In view of the importance of milk and dairy products, research has been directed toward finding a means of obtain- ing more milk from dairy cows. It appears that not only proper breeding. feeding and management influence milk pro- duction in dairy cows but hormones are also involved. Pro- lactin and growth hormone have beenimplicated as part of the lactogenic complex in certain laboratory species but their role in bovine lactation is not fully elucidated. Therefore the purpose of these studies was to investi- gate the physiological role and mechanisms by which prolactin and growth hormone are controlled in the bovine. An under- standing of the mechanism by which these hormones are con- trolled in the bovine may provide basic information required to manipulate hormones and achieve increase milk production. 1. A. Prolact Based induced in pituitary that anteri Grueter and Production With '1. m. 1933 executed to Bones for 12 in hYpOphySE prolact in . 1'1”“ (19m galactOphere initiation 0 Serve“ by th (1969) Sumna sir ”her prOla Could initia 2 ad E“h"enale :1. “ands . Pr°1act3 REVIEW OF LITERATURE A. Prolactin and Growth Hormone (GH) requirement for lactation Based on their observations that milk secretion was induced in psuedopregnant rabbits injected with anterior pituitary extracts, Stricker and Grueter (1928) suggested that anterior pituitary secretions were involved in lactation. Grueter and Stricker (1929) also obtained increase milk production in cows injected with ox pituitary extracts. With identification and isolation of prolactin (Riddle gt_§;. 1933) and GH (Li and Evans 19uu) many experiments were executed to demonstrate the requirements of these two hor- mones for lactation. Fredrikson (1939) induced milk secretion in hypophysectomized rabbits by daily injections of sheep prolactin. Subsequently, this result was confirmed by Lyons (l9h2) who injected sheep prolactin into a single galactophore of the mammary gland of rabbits and observed initiation of milk secretion in the segment of the gland served by that galactophore. In an excellent review. Cowie (1969) summarized his work and that ofcxhers that showed either prolactin or GH in combination with an adrenal corticoid could initiate lactation in hypOphysectomized. ovariectomized and adrenalectomized rats and mice with developed mammary glands. Prolactin was shown not to be galactopoietic in dairy 2. cows and 9‘ 19MB, Cotes _a_1. 1971). lactationa] lactin intc addition, 5 required fo prepartum 1‘ yield in th (1973) repo: tude of pro: duction in 1 released at reported for AlthOUgh alone Could Phb'sectomiZe acid and thy t° Wehypoph: of lactation! i’ield for Sex an lmediate 3 cows and ewes (Folley and Young l9h0, Sulman and Twersky 1998, Cotes et a1. l9h9, Wrenn and Sykes 1953 and Morag gt Q1. 1971). However, Gotsulenko (1968) observed increase lactational performance in goats following injection of pro- lactin into the arterial system of the mammary gland. In addition, Schams et a1. (1972) suggested that prolactin was required for lactogenesis in cattle since inhibition of the prepartum rise of serum prolactin resulted in decreased milk yield in the subsequent lactation. Koprowski and Tucker (1973) reported a significant correlation between the magni- tude of prolactin release to milking stimuli and milk pro- duction in the bovine which may suggest that prolactin released at milking may influence subsequent milk yield as reported for rats (Grosvenor and Mena 1973). Although. Cowie (1969) reported that sheep prolactin alone could induce traces of mammary secretion in the hypo- physectomized goat. administration of prolactin. GH. corti- coid and thyroid hormone was required to restore lactation to prehypophysectomy levels. However. following restoration of lactation, withdrawal of prolactin had no effect on milk yield for several weeks but cessation of GH treatment caused an immediate suppression of lactation. Administration of bovine OH to lactating cows has also been reported to enhance lactational performance (Cotes g1_§_. 1999, Donker and Peter- sen 1951, 1952, Chung g3_§l. 1953. Wrenn and Sykes 1953, Brumby and Hancock 1955, Hutton 1957 and Nachlin 1973). 8.211135— 1. w Sheles direct evi tion when rats suppr pregnancy a be reverse< concurrent: Similarly, rats bearir and when gi inhibited p effeCtS cou with ergoco: Yanai a] and Clemens that erect G ”men’u‘ati. Cryptine in: induced or ‘ inhibiting ] Q P01: 1971), fears 01') a TJrOléIC'Cin bu B. Effect of eggot alkalgids on Prolagtig and GH gegretign I-Iaxiae Shelesnyak (l95h, 1955, 1958) presented the first in- direct evidence that ergot drugs inhibited prolaction secre- tion when he observed that administration of ergotoxine to rats suppressed deciduoma formation and terminated psuedo- pregnancy and early pregnancy. many of these effects could be reversed with progesterone or prolactin administered concurrently or within Zh hr after ergotoxine treatment. Similarly, ergocornine blocked nidation in hypophysectomized rats bearing pituitary transplants (Varvuhidi §3_g;. 1966) and when given to rats on the morning after coitus it inhibited pregnancy (Carpent and Desclin 1969). These effects could be reversed with progesterone given concurrently with ergocornine. Yanai and Nagasawa (1970) Meites giggl. (1972), Shaar and Clemens (1972) and Wuttke and Meites (1972) demonstrated that ergot drugs suppressed serum and pituitary prolactin concentrations of rats. In addition. ergocornine and ergo- cryptine inhibited growth of either dimethylbenzanthrene- induced or spontaneous mammary tumors of rats. presumably by inhibiting prolactin secretion (Nagasawa and Meites 1970, Hanson g3_§1. 1970, Quadri and Meites 1971 and Cassell g1_g_. 1971). Following termination of treatment7growth of spon- taneous mammary tumors was resumed (Quadri and Meites 1971). Pearson g3_g1. (1969) also reported that exogenous bovine prolactin but not GH reactivated regressed mammary tumors of hmophy Inj proestr prolact estrus 1971; 1 inhibit rats 'm 1971). (Zielmai (Hagasau Varga fl a(1111111131: the Silpp in Nagas; basal 89] Medium), a inhibitec. in cows ( 351k Yiel Recen bearing p. hypophysectomized rats. Injection of ergocornine into rats on the morning of proestrus inhibited the characteristic increase in serum prolactin concentration observed on the afternoon of pro- estrus in this species (Yokoyama et a1. 1971, Wuttke et a1. 1971; 1972 and Yanai and Nagasawa 197“). Ergocornine also inhibited estrogen-induced prolactin release in ovariectomized rats in 2112 and from pituitary explants in 21529 (Lu.gj_g;& 1971). In addition ergot drugs suppressed lactation in rats. (Zielmaker and Carlson 1962, Shaar and Clemens 1972) mice (Nagasawa and Yanai 1972) and humans (del Pozo g3_g;. 1972, Varga g3_gl. 1972 and Lutterbeck §3_§_. 1971). In rats. administration of prolactin but not oxytocin could reverse the suppression of lactation induced by ergocornine (cited in Nagasawa and Yanai 1972). Although ergocryptine decreased basal serum.prolactin concentration of goats (Hart 1973. McMurty and Malven 1979) and cows (Karg g§__l. 1972) and inhibited prolactin release in response to milking stimuli in cows (Schams gj_g_. 1972a) this compound did not affect milk yield. Recently ergocryptine has been reported to suppress serum prolactin concentration in ewes (Niswender 1974) and in rats bearing pituitary homografts (Halven and Hoge 1971). Further- more, Schams giggg, (1972) demonstrated that when ergocryptine was administered to pregnant cows 3-h days before parturition it inhibited the prepartum rise in serum prolactin concentra- tion characteristic of this species (Ingalls g1_gl. 1973). In addition serum pr hormone 2. lull. Nessa acted di: of the g with erg pyknotic gland. comine . rat and cellular could be Similau-l cmissed n rather c forewer Miami prolifter Yahaj phoresis and rem mice SL1} With no 6 addition ergocryptine greatly diminished the magnitude of serum prolactin release in response to thyrotropin releasing hormone in cattle (Schams 1972). 2. In vitro Nassar gt a1. (1950) first suggested that ergot drugs acted directly on the anterior pituitary to cause necrosis of the gland. At autopsy. when pituitaries of rats treated with ergotoxine were examined microscopically a number of pyknotic nuclei were present in the anterior portion of the gland. However Pasteels gt_§_. (1971) reported that ergo- cornine and ergocryptine inhibited prolactin release from rat and human hypophyses in_yit;g without causing overt cellular destruction of the gland as prolactin secretion could be restored by washing the ergots from the explants. Similarly. Ectors g3__1. (1972) reported that ergocryptine caused no cellular destruction of the pituitary gland but rather caused an inhibition of exocytocis from prolactin cells. Moreover. explants treated with ergocryptine contained greater prolactin concentration than control explants as assayed by proliferation of the pigeon crop sac. Yanai and Nagasawa (1970a) used polyacrylamide gel electro- phoresis to determine pituitary prolactin and GH concentrations and reported that chronic administration of ergocryptine to mice suppressed pituitary prolactin content and concentration with no apparent effect on GH concentration. Similarly. when pituitaries from rats treated with ergocryptine were incubated in vitro with 1uC-leucine. prolactinrelease, but not its synth tary direc the p canno media: treat: explal decree use 0: ergocc synthesis. was inhibited while there was no effect on pitui- tary GH concentration (Yanai and Nagasawa 197“). Although it is apparent that ergot drugs can act directly on the adenohypophysis to inhibit prolactin release. the possibility of an additional effect on the hypothalamus cannot be excluded. When ergocornine was implanted into the median eminence of rats or when hypothalami from.ergocornine- treated rats were coincubated in yiigg_with normal pituitary explants. serum and pituitary prolactin concentrations were decreased (Wuttke g3_al. 1971). Hokfelt and Fuxe (1972) by use of fluorescence staining. reported that ergocryptine and ergocornine decreased the rate of disappearance of dopamine from the median eminence of lactating and pregnant rats. In general, these investigators suggest that ergocornine and ergocryptine may act at the hypothalamus to increase prolactin inhibiting factor. thus decreasing pituitary and serum pro- lactin levels. ‘n-R 1 a Hormo e l. ngeral Isolation of porcine TRH (Schally gt_g_. 1969) was follow- ed quickly with elucidation of its structure (Nair g5_g_, 1970) and synthesis of the tripeptide (Boler gj_g1. 1969). With synthesis and availability of TRH, many experiments have been conducted in different species to understand the physiology of TRH. Thyrotropin releasing hormone causes release of thyroid stimulating hormone (TSH) from pituitaries of several species (Pleisch 1971, Val to TSH t also inv changed b 1972). 2. 3.1122: Bowel reported ‘ Prolactin in humans Bowers & and N0el g Admin increased 8 (Fleischer §3_a_. 1970. Labella and Vivian 1971, Porter g3_a1. 1971. Vale g3_a_. 1972 and Haigler g3_§l. 1972). In addition to TSH the response of growth hormone and glucocorticoids was also investigated but only TSH concentration was consistently changed by TRH (Ormston et a1. 1971, 1971a and Gaul et al. 1972). 2. Effect of TRH on Prolactingnd GH concentrations Bowers et a1. (1971) and Jacobs et a1. (1971) first reported that administration of TRH to humans increased serum prolactin concentration. These initial reports were confirmed in humans (Friesen g§_g1. 1972, L'Hermite et a1. 1972, Bowers g;_a_, 1972, 1973. Jacobs §3_g1, 1973. Wilber 1973 and Noel g3_§1. 197“). Administration of TRH to lactating women not only increased serum prolactin concentration but caused breast engorgement. milk let down and increased milk fat and protein content of milk (Tyson et_g_. 1972, 1972a). Similarly. Convey,gt_§l. (1973a) reported that administration of TRH to 20 lactating dairy cows increased milk yield by 0.66 kg/cow/ day. but neither milk fat nor protein content of milk was affected. In contrast. Kelly g;_§_. (1973) observed no change in milk yield or its composition following administra- tion of TRH to four lactating cows and Adams g3_al. (1973) reported that administration of TRH to lactating rats (day 5- 21) had no effect on milk production as determined by litter weight gain at weaning. Within 2-15 min after injection. TRH increased serum prolactin Debeljuk and in ca l97h, Kel rats apper lactin ref released 1 release n. We 19?? injection serum prol 501‘ 6 days but caused iration. ‘ "33 increa- estrogen-p1 on the nor: Serum Drol. (3918 and . Pale rats Altho e: ‘ .1011 1n da prolactin concentration in sheep (Davis and Borger 1972, Debeljuk et a1. 1973, Fell et a1. 1973, Moseley et a1. 1973) and in cattle (Schams 1972, Convey 1973, Convey e: a . 1973. 197a, Kelly et a1. 1973 and Vines gt a1. 1973. 1979). But rats appeared to be the least responsive to TRH-induced pro- lactin release since doses of TRH (5-10 ug) which effectively released prolactin in man failed to stimulate prolactin release from rat pituitary explants ig,yitgg (Friesen and Hwang 1973). Lu et a1. (1972) also reported that a single injection of 5 or 7.5 ug TRH into male rats failed to increase serum prolactin concentration, and 50 ug TRH injected daily for 6 days greatly increased pituitary prolactin concentration lnrt caused only a slight increase in serum prolactin concen- tration. In contrast Mueller et a1. (1973) reported that TRii increased serum prolactin concentration in normal and estrogen-primed male rats and in normal female rats treated (ml the morning of proestrus. The tripeptide also increased sernxm prolactin concentration in proestrus and lactating rats (Deis and Alonso 1973, Blake 197a) and prolactin and on in male rats (Takahara et a1. 1971+). Although TRH consistently increased serum GH concentra- ticni in dairy heifers, (Vines gt_gl. l97h) lactating cows (Convey 1973, Convey M1. 1973) and in acromegalic humans (Saito M. 1971, Irie and Tsushima 1972, Schalch 21.3..- 1972, Cryder gt__a_. 1973 and Fagalia gj__a_.__. 1973) the effect 0f the tripeptide on GH release in normal humans is not clear. Irlnormal humans, Fleischer et a1. (1970), Bowers gt a1. (1971) a serum GH al- (197 failed t 3. m Sev nechanis tions. site of secretio my tun demonstr cell cu] Dibbet g lactin r Dannies thesis a ma A11 Stimuleu 10 (1971) and Torjesen g1_gl. (1973) reported that TRH increased serum GH concentration but Anderson gt_gl. (1971) Ormston g3 '51. (1971a). Saito gt_gl. (1971) and L'Hermite g3_g_. (1972) failed to confirm these results. 3. WW Several investigators have attempted to_elucidate the mechanism by which TRH can affect prolactin and GH concentra- tions. Tashjian g;_g1. (1971) suggested the pituitary as one site of action when they observed that TRH increased prolactin secretion and decreased GH secretion from cloned rat pitui- tary tumor cells in 31.312- Vale M. (1973) also demonstrated that TRH released prolactin from rat pituitary cell cultures and .hemi-pituitaries is 1132. Similarly, Dibbet giggl. (1973) demonstrated that TRH increased pro- lactin release from rat pituitary explants in 11529 and Dannies g3_g;. (1973) reported that TRH increased both syn- thesis and release of prolactin from.rat pituitary explants in 215922. Although Labella and Vivian (1971) reported that TRH stimulated prolactin and CH release from bovine pituitary explants in one of three experiments. Convey g1_g;. (1973) did not observe any TRH stimulation of prolactin release from steer pituitary explants in 21529. .Recently. machlin and Jacobs (1973) and Smith and Convey (1974) reported that TRH increased media prolactin and GH concentration from primary cell cultures of bovine pituitaries. Bourne g1_g_. (197k) also demonstrated TRH stimulation of prolactin that TRH pituitari that TRH on the pi ted that ‘ explants .- a‘lthOI‘s S increase messenger 11 release from bovine pituitary cell cultures. Thus TRH repeatedly caused prolactin release from bovine pituitary cells in culture but prolactin release from bovine pituitary explants is not demonstratable or at best variable. The reports of Labrie gj_gl. (1972) and Wilber (1973) that TRH selectively binds to plasma membrane of anterior pituitaries of rats and cattle also support the hypothesis that TRH can stimulate prolactin release by a direct action on the pituitary. Furthermore, Labrie gi_g1. (1973) demonstra- ted that within 2-6 min after addition of TRH to rat pituitary explants iglyitgg. cyclic AMP increased (loo-150%). These authors suggested that TRH might activate adenyl cyclase to increase cyclic AMP concentration which then served as the messenger for the action of TRH. Based on their observations that TRH was an anti- depressant agent. Kastin g$_g1. (1972) and Prange gt_g1. (1972) suggested that TRH can-act at sites other than the anterior pituitary. Bowers et a1. (1972), Noel et a1. (1973) and Jaffe g$_g1. (1973) reported that TRH-induced prolactin release was suppressed in humans treated with L—dopa 1-2 hr before TRH. These results were interpreted to mean that TRH acted on the hypothalamus, since L-dopa or its metabolite (dopamine) had been previously demonstrated to reduce serum prolactin concentration in rats, presumably by increasing hypothalamic prolactin inhibiting factor (Kamberi g3_g1. 1971. Lu and Meites 1972). But addition of dopamine to rat pitui- tary explant ip yifipg also inhibited prolactin secretion (Koch et a1. 1970) suggesting a dual site of action for dopamine . release b cholamine portal 53" of the pi' he Interaf and lieites 12 dopamine. Therefore L-dopa could inhibit TRH-induced prolactin release by increasing prolactin inhibiting factor or the cate- cholamine could be secreted into the hypothalamo-hypophyseal- portal system and counteracted the action of TRH at the level of the pituitary gland. h. Integaction of TRH with thygoid hormone Hollander et a1. (1972) reported that administration of TRH to humans increased serum thyroxine (Tu) and triiodo- thyronine (T3). Since Tu and T3 increased prolactin release from rat pituitary explants ip_vitro (Nicoll and Meites 1963) and increased rat pituitary prolactin content ipsyiyp_ (Chen and Meites 1969) the possibility existed that prolactin released in response to TRH was mediated via thyroid hormones. But in cattle this was unlikely since serum prolactin concen- tration increased within minutes after TRH injection and serum Tu increased only after several hours (Convey g3_g_. 1973). Results reported by Shaw g3_g1. (1972) lend credence to this hypothesis since it was demonstrated that feeding of thyroprotein to dairy cows increased serum Tu from 6 to 13 ug/lOO m1 without affecting serum prolactin concentration. Recently Vanjonack et a1. (197k) reported that administration of TRH to cows resulted in a biphasic response in serum Th' Within the first 30 min after injection, serum Th increased, then decreased to a nadir by 2 hr followed by another increase within 6 hr. Unfortunately these authors did not quantify serum prolactin concentration. In general, although the evidence is not conclusive. it appears that hypothyroidism stimulates while hyperthyroidism suppI‘ESSG (1972 an: the magn: rat pituz' These ob: e_t__a_l. 1‘. However, reported induced 5' Do Effflci exogenous rats, T}. (1962) a, increaSed % s Promoted and rabbi Pro] “plants fr“ esu “Wimh. Wthala ‘h’e . .I‘e ln‘te 13 suppresses the effect of TRH (Wilber 1973). Vale g3_g1. (1972 and 1973) demonstrated that thyroid hormones diminished the magnitude of TRH-induced prolactin and TSH release from rat pituitary explants and primary pituitary cell cultures. These observations were confirmed igfiyiyg in sheep (Debeljuk ,gt_al. 1973) and humans (Snyder et a1. 1973 and Yamaji 197M). However, Bowers et al. (1972) and Rapoport giggl. (1973) reported that administration of T3 to humans suppressed TRH- induced TSH release but not prolactin release. D. Effect of gonadal steroids on prolactin and GH concentra- tions 1. Egtrgggn As early as 1937 Reece and Turner reported that exogenous estrogen increased pituitary prolactin content of rats. These results were confirmed by Nicoll and Meites (1962) and Ben-David g3_g_. (1969) who reported that estrogen increased prolactin release from rat pituitary explants 1p 213:9. Similarly. intrahypophyseal implants of estrogen promoted prolactin release from rats (Ramirez and McCann l96h) and rabbits (Kanematsu and Sawyer 1963). Prolactin secretion was increased when rat pituitary explants were coincubated in giggg with hypothalamic extract from estrogen-primed rats (Ratner and Meites l96h), and enovid-treated rats (Minaguchi and Meites 1967). compared to hypothalamic extract from normal cycling rats. These results were interpreted to mean that hypothalami taken from rats exposed to estrogens. contained less PIP, therefore more pro- lactin was released from pituitaries coincubated with these hypothala thalami fr less PIF t In ma concentrai to diestrt Verhofstad In contra: apparent c' 1971. Jail Parently, dependent administer 1971, Free Althc are repord oral Cont] (Meites 15 1972), '1‘] inhibitor: StOOd’ bLI‘ suppreSS ] gen admin: prolac“tin hypothalami. Sar and Meites (1967) also reported that hypo- thalami from rats killed during proestrus and estrus contained less PIF than hypothalami from rats killed during diestrus. In many laboratory species pituitary and serum prolactin concentrations are greater at proestrus and estrus compared to diestrus (Reece 1939. Sar and Meites 1967, Kwa and Verhofstad 1967. Amenomori g3_§1. 1970 and Voogt et a1. 1970). In contrast no change in serum prolactin concentration was apparent during the menstrual cycle of women (Hwang gt_gl. 1971. Jaffe gj_gl. 1973 and Tyson and Friesen 1973). Ap- parently, the prolactin surge at proestrus in rats is estrogen- dependent since it could be eliminated with an antiestrogen administered on the day preceding proestrus (Neil g3_g_. 1971, Freeman et a1. 1972 and Yokoyama and Tomogane 1973). Although estrogens stimulate prolactin secretion there are reports that large doses of estrogens and estrogenic oral contraceptives can inhibit lactation in several species (Meites 1961, Cowie 1961. Morris 1967 and Koetsawang gjggg, 1972). The mechanism whereby large doses of estrogen are inhibitory to an established lactation is not clearly under- stood. but one possibility is that large doses of estrogen suppress prolactin secretion. However. large doses of estro- gen administered to ovariectomized rats did not inhibit serum prolactin concentration relative to non-treated ovariectomized rats (Chen and Meites 1970). Therefore Meites g3_§_. (1972) suggested that inhibition of lactation may be due to estrogen interference with the peripheral action of prolactin at the mammary gland. Bruce and Ramirez (1970) supported this hpothesi: the rammai hanced lat gland. G: of lactath ejection ll mcmary g2 lactation Changing ‘ genic com: Evide Concentrat Sinha and content in then 318111 They S‘dgge tent may I‘ we“ duI‘in DTOIactin i-J OI hypothesis when they observed that estrogen implanted into the mammary gland of rats inhibited lactation. but it en- hanced lactation when it was placed in the anterior pituitary gland. Griffith and Turner (1962) suggested that inhibition of lactation was due to estrogen interference with the milk ejection reflex since rats treated with estrogen had their mammary glands engorged with milk. Estrogen may also inhibit lactation by stimulating mammary gland growth. thereby changing the ability of the gland to respond to the lacto- genic complex. Evidence of the effect of gonadal steroids on prolactin concentration in farm animals is not conclusive. In heifers Sinha and Tucker (1969) reported that pituitary prolactin content increased from two days before to the day of estrus. then significantly decreased until two days after estrus. They suggested that the decrease in pituitary prolactin con- tent may reflect release of prolactin into the serum. How- ever during the estrous cycle there was no change in serum prolactin concentration of lactating and non-lactating cows (Schams and Karg 1970) and heifers (Wetteman and Hafs 1973). Similarly. serum prolactin concentration before. immediately after and 1 hr after milking dairy cows did not change sig- nificantly during the estrous cycle (Koprowski and Tucker 1973). Raud et a1. (1971) also failed to establish any re- lationship between the stage of the estrous cycle and serum prolactin concentration when blood was collected from cycling cows via jugular puncture. In contrast, when animals were bled via jugular cannulae serum prolactin concentration increase These 311 puncture concentr Swanson blood fi" reported precedin declined In and Davi concentr +rations eS'CI‘adio Prolacti: (1959) a Content Cha 16 increased at proestrus and estrus relative to diestrus. These authors suggested that stress associated with veni- puncture can cause erratic alterations in serum prolactin concentration to negate the response to other stimuli. But Swanson and Hafs (1971) and Swanson gt_gl. (1972) collected blood from heifers via venipuncture or jugular cannulae and reported increase serum prolactin concentrations 3-# days preceding estrus, it peaked at estrus then subsequently declined during diestrus. In ewes, Reeves et a1. (1970), Bryant et al. (1971) and Davis et al. (1971) demonstrated increase serum prolactin concentrations at proestrus and estrus relative to concen- trations during metestrus and diestrus. Injection of estradiol benzoate into anestrus ewes also increased plasma prolactin concentration (Fell gt_gl. 1972). Day g3_§l. (1959) also observed a linear increase in pituitary prolactin content of pigs between days 2-19 of the estrous cycle. Changes in estradiol concentration may also influence GH levels. In rats (Dickerman 1971) and mice (Sinha gt_g;. 1972) serum and pituitary GH concentrations were greater at proestrus and estrus than during diestrus. Spellacy g3_gl. (1969) reported increase GH concentration during the ovua- latory and pre-menstrual phases of women, a time when estrogen levels were elevated in urine (Brown 1960). Similarly, Unger (1965) demonstrated that fasting levels of serum GH were higher in women than men presumably due to higher levels of eStrogen in women. Koprowski and Tucker (1973) also observed increase serum GH concentration during the estrogenic phase of the estr namcy. cattle ( are repo 1973). Adn serum GE 3.3% ( efhylsti serum G}- diethyls increase 1965) ar Wining In Served 1 degDi‘te larly. ‘ Cycle 01 ,4 "J the estrous cycle of lactating cows, and during late preg- nancy. Increased serum GH concentration at parturition in cattle (Ingalls gt_gl. 1973) may be due to estrogens which are reported to be elevated in serum of heifers (Smith gt_g_. 1973). Administration of diethylstilbestrol to steers increased serum GH concentration (Trenkle 1970). Similarly, Lloyd gt_§l. (1971 and 1973) demonstrated that injection of di- ethylstilbestrol into rats increased pituitary weight and serum GH concentration. In humans, administration of ‘ diethylstilbestrol or estrogenic oral contraceptives also increased basal serum GH concentration (Frantz and Rabkin 1965) and increased the magnitude of GH release in response to arginine (Merimee et al. 1966 and Vela and Yen 1969). In contrast to these reports Ieiri §t_§l. (1971) ob- served no change in CH synthesis or release in rats at estrus despite marked increases in prolactin concentration. Simi- larly, Vines ej_a_. (197h) found no effect of the estrous cycle on basal serum GH concentration of dairy heifers. Neither did the stage of the estrous cycle affect the magni- tude of GH release in response to thyrotropin releasing hormone. . 2- Eregesterene Reece and Bivins (l9h2) demonstrated that administration of progesterone (15 mg) concurrently with estradiol benzoate (33 ug) to ovariectomized rats inhibited the increase in pituitary prolactin content normally associated with estrogen therapy. However. pituitary prolactin content was increased 58W.“ V Insane—’6 - i" - I . in. in rats r Chen and ported t} with estr prolactir given alc concentra 0'3 Proges mized I‘a‘t untreated PitUitari hYPOthala releaSed Cubated w Were inte or indire inhibitin one Can S (10898 to 196% and et\alz 19 centratio The imphence 93mg Wh terone co pI‘OIaCtin l arly' do: 18 in rats receiving 15 mg of progesterone but no estrogen. Chen and Meites (1970) confirmed these results when they re- ported that progesterone (0.5 - h mg) administered concurrently with estradiol benzoate (1.0 ug) inhibited estrogen-induced prolactin release in rats but progesterone (10 mg) when given alone resulted in nearly a doubling of serum prolactin concentration. Sar and Meites (1968) also reported that 10 mg of progesterone administered daily for 21 days to ovariecto- mized rats increased pituitary prolactin content relative to untreated ovariectomized rats. Furthermore. when hemi- pituitaries from normal rats were coincubated in 11329 with hypothalami from progesterone-treated-rats more prolactin was released into the media than when hemi-pituitaries were in- cubated with hypothalami from control rats. These results were interpreted to mean that progesterone either directly, or indirectly via conversion to estrogens, reduced prolactin inhibiting factor. Apparently, only high doses of progester- one can stimulate prolactin release since addition of low doses to rat pituitary explant in_zi§gg (Nicoll and Meites 196M) and to intact or hypothalamic-lesioned rats (Bishop gt a1. 1972) had no effect on media or serum prolactin con- centration. The ratio of estrogennprogesterone also appears to influence prolactin secretion in vivo since at proestrus and estrus when serum estrogens are elevated and serum proges- terone concentration is at a nadir, pituitary and serum prolactin concentrations are elevated in some species. Simi- larly, during pregnancy in cattle, serum prolactin ' . “WV-.fi- concentr parturit this coi concentra reites (: the incrv With est: ratios 0* Add: inhibit I chanisms Yoshinagg increase some Spec gesterone tel-one Co the ratio secretiOn Huh“ (196 Effective the fall trigger f 19 concentration was highest at approximately 2h hr before parturition (Schams and Karg 1970, Ingalls et al. 1973) and this coincided with high levels of serum estrogens and low concentration of serum progesterone (Smith e3_gl. 1973). Meites (1959) also reported that in rats and guinea pigs, the increase in pituitary prolactin concentration associated with estrogen therapy was inhibited with estrogensprogesterone ratios of 1:1000 to 132000. Additional evidence also suggests that progesterone can inhibit lactogenesis in certain species, although the me- chanisms by which this is accomplished are poorly understood. Yoshinaga et al. (1969) suggested that failure to observe increase prolactin concentration during most of pregnancy in some species may be attributed to the ratio of estrogenapro- gesterone. Towards the end of pregnancy the fall in proges- terone concentration and the increase in serum estrogens change the ratio of estrogennprogesterone which may stimulate prolactin secretion and precipitate lactation. In support of this view, Kuhn (1969) and Herrenkohl (1971) reported that progesterone effectively blocked lactogenesis in rats and suggested that the fall in serum progesterone near to parturition may be the trigger for lactogenesis. Furthermore, injection of prolactin into pregnant rabbits prevented the inhibitory effect of pro- gesterone on lactogenesis (Denamur and Delouis 1972). There is a paucity of information regarding the effect of progesterone on GH concentration. Administration of pro- gesterone (Bhatia gt al. 1972) or medroxyprogesterone acetate 1) f.” m o A reported hurrah fey levels in a. (1973 was augme 39“ and t (1973) de induced p: 20 (Simon et a1. 1967, Lawrence and Kirsteins 1970) to humans, suppressed GH release in response to arginine and hypogly- cemia. Malarkey and Daughaday (1971) also reported that administration of medroxyprogesterone acetate to acromegalic humans decreased serum GH concentration. But during preg- nancy, GH concentration was unchanged in serum of heifers (Oxender and Hafs 1971) and rats (Dickerman 1971). R lat onshi of onadal steroids and TRH o rolact’n and GH concentrations Bowers et a1. (1971), Friesen et al. (1972), Torjesen aLal- (1973). Jacobs 1131. (1973) and Noel et a1. (1971+) reported that TRH-induced prolactin release was greater in human females than males, presumably due to higher estrogen levels in the females. This view was supported by Jaffe gt al. (1973) who reported that TRH-induced prolactin release was augmented in early postpartum women, treated with estro- gen and testosterone to suppress lactation. Carlson et al. (1973) demonstrated that diethylstilbestrbl enhanced TRH- induced prolactin release but not GH release in humans. But Takahara et al. (1974) observed no difference in TRH-induced prolactin release between estradiol-treated rats and controls. Tyson et a1. (1972) also found no difference in the magnitude 'of prolactin released from women treated with TRH during the luteal or menstrual phase of the cycle. Neither did Vines §j_§_. (1970) observe any difference in the magnitude of pro- lactin or GH released from cows treated with TRH on different days of the estrous cycle. A- this. Lao Universi' at a loce heifers v nosed pre W0 month Michigan under 100 0n t dwelling New York) Cm of the MATERIALS AND METHOD - GENERAL A. Animals Lactating Holstein cows maintained in the Michigan State University herd and primiparous Holstein heifers, purchased at a local auction, were used in this study. All purchased heifers were palpated per rectum, and four which were diag- nosed pregnant were aborted by cesarean section approximately two months before they were involved in any experiment. At Michigan State University, these heifers were maintained under loose housing conditions with free access to pasture. 0n the day preceding each in 2122 experiment, an in- dwelling jugular cannula (Vinyl IV Tubing, Clay Adams Inc., New York) was inserted into each animal. Approximately #5 cm of the 2&0 cm cannula were inserted into one jugular vein and affixed to the neck and withers with tag cement (Nasco, Fort Atkinson, Wis.) on 7.6 x 12.? cm adhesive tape. Each cannula was flushed with 10 m1 of 3.5% sodium citrate and sealed until used for blood collection. At different time intervals, depending on the experi- mental design, 10 m1 of blood were collected according to the following procedure: (1) Approximately 5 m1 of blood which contained residual citrate used to keep the cannulae patent were collected and discarded. 21 A g I ' “u; g linger. (2) Ten i Prop (3) Afte fill and cl Blo serum wa:' 30 min. hormones Pre] and desi; Pimitar 1| 22 (2) Ten ml of blood were withdrawn and dispensed into poly- propylene centrifuge tubes (Sorval, Inc., Newton, Conn.). (3) After each blood sample was withdrawn, cannulae were filled with 3.5% sodium citrate which would be removed and discarded at the next collection period. Blood was allowed to clot at 4°C for 2h hr after which serum was obtained by centrifugation (2500 xg at 4°C) for 30 min. Serum samples were stored frozen until assayed for hormones. B- lnrzitze_eresedurea Preparation of bovine anterior pituitary cell cultures and design of experiments are shown in figure 1. Bovine pituitaries were collected within 30 min of death of the animals at a local abbattoir and transported at 37°C to the laboratory. Within 1 hr of death of animals, posterior pituitaries were discarded and anterior pituitaries were minced with scissors and washed four times with the medium that was used for culture (Appendix 1). Approximately 1.5 - 2.5 g of minced pituitary tissue were placed in 25 ml Erlenmeyer flasks. Ten ml of 0.2 - 0.3% collagenase (Type 1 - 135 u/mg Lot 1302u30, Sigma Chemical Co.) in culture medium were added to each flask, and the con- tents of each flask were incubated (37°C) with constant shaking in an Eberbach metabolic shaker at 180 ocillations/ min for #5 - 60 min. In some of the later experiments cell suspensions were obtained more quickly by stirring the erlen- meyer flasks containing tissue and collagenase on a Corning Figure 23 ANTERIOR P'“"”“* a POSTERIOR PITUITARY SCISSOR MINCED ‘ COLLAGENASE (0.3 %) DISPERSED CELLS WASH 4x; 375)! G ifig" A LFmiE—a' —-"~“:— -_ 5:; GROWN T0 MONOLAYER IN EAGLE'S MINIMUM ESSENTIAL MEDIUM WITH I096 cow SERUM "OR”ONE ASSAY HORMONE ASSAY I PRE-TREATMENT f POST-TREATMENT 9 I I MEDIUM MEDIUM l99 I99 PLUS TREATMENT Schematic representation of the preparation of anterior pituitary cell cultures and design of in vitro experiments. Figure 1 . . “Wan-cm- ‘n' as" magnetic The resc fi tered plastic fuged at discarde and cent collagen Fol pituitar cow seru' were traI. (25 cm2, inwbater '___ 2e magnetic mixer (Fisher Scientific Co., Pittsburg, Penn.). The resulting cell suspension obtained by either method was filtered through cheesecloth (2 layers) into 50 ml conical plastic tubes (Falcon Plastics, Oxnard, Cal.) and centri- fuged at 375 xg for 3 min at 25°C. The supernatant was discarded and the cells were washed with medium (Appendix 1) and centrifuged at least four times to remove residual collagenase. Following the final centrifugation cells from four pituitaries were suspended in 120 ml medium containing 10% cow serum (growth medium). Four ml of this cell suspension were transferred with serological pipettes to culture flasks (25 cm2, 30 ml tissue culture flasks, Falcon Plastics) and incubated at 37°C for 3-h days by which time confluent mono- layers were established. Medium was first replaced after #8 hr and thereafter at 2b hr intervals. The cells were used for experiments on day 3 or u depending on the time taken to establish confluent monolayers. For all in yigrg experi- ments, treatments were administered in h ml of Tissue Culture (TC) medium 199 (Appendix 2). C. Hormone Assays 1. Ergtein Hormones ' Prolactin, growth hormone and luteinizing hormone in sera and/or tissue culture media were quantified by double antibody radioimmunoassay (RIA) procedures previously described by Tucker (1971) Purchas g§_§l. (1970) and Oxender g3_§_. (1972), respectively. m‘b' “5'4"“! can“ 2. sea Sa RIA proz Wetteurr To viously lated f graphy fractio‘ m2 ml was add coid-fr a “Spa column °“ 11013) a Secon S°1Veni 1m3*.er CorticC fractic ing rad Dectrc has eh S'I‘Sates l“ractic prayiol Efficis f enmes- 2. Ster d Ho mones Serum progesterone and estradiol were quantified by RIA procedures previously reported (Louis et a1. 1973 and Wetteman gt a . 1972), respectively. Total corticoids were extracted from serum as pre- viously reported by Smith et al. (1972). Cortisol was iso- lated from the total corticoid-fraction by column chromato- graphy (Lin, 0xender and Hafs, unpublished). Briefly, the fractionation procedure was as follows. Approximately 0.2 m1 of chromatography solvent (chloroformzmethanolg 99:1) was added to each tube containing the isolated total corti- coid-fraction. The content of each tube was agitated with a disposable pippette then transferred to a LH-ZO sephadexn column (0.5 x 12 cm pippette: fitted with a 12-ml reservoir on top). Tubes containing the corticoid-fraction were rinsed a second time with an additional 0.2 ml of chromatography solvent which was also transferred to the columns. Approx- imately 8 ml of chromatography solvent were used to elute corticoids from the column which were collected in 1 m1- fractions. The elution profile was determined by quantify- ing radioactivity in each fraction in a liquid scintillation spectrophotometer (Nuclear Chicago Model, Mark 1). Cortisol was eluted in fractions 6, 7 and 8 and the fraction with the greatest amount of radioactivity or in some cases a pooled fraction was assayed for cortisol by protein binding procedures previously reported by Smith et al. (1972). Extraction efficiency and procedural losses were estimated from the dif- ference in radioactivity between 3H-cortisol added to the serum (before column-f D. Spec; Experime Objectiv m an avera nent. C stages 0 resPond Concentr ing the at 0500.. domly to 51111 of 5511 of administ dwelling every 2 j intemal: blood IVa: +vhe day ] follow“é from the 3am it a Nantifie at Each Ir. 26 (before extraction procedures) and radioactivity in the column-fraction assayed for cortisol. D. Specific Objectives and Experimental Procgdgzes Experiment 1.-- Effect of Er cr ti on Bov‘ e Prolactin GH Cortisol a d M 1k Yield Objective 1: Effect of Ergocrypting in vivo r me tal des' I Ten non-pregnant Holstein cows lactating an average of #2.8 days (range 10-97) were used in this experi- ment. Cows selected for this experiment were in the early stages of lactation since those in late lactation may not respond to milking stimuli with an increase in serum prolactin concentration (Johke 1970, Koprowski and Tucker 1973). Dur- ing the experimental period all cows were milked twice daily at 0500-0600 hr and 1700-1800 hr. Cows were assigned ran- domly to one of two groups to receive subcutaneously either 5 ml of 50% ethanol (controls) or 80 mg of ergocryptine in 5 ml of 50% ethanol on two consecutive days. Treatments were administered at 0700 hr and blood was collected via in- dwelling jugular cannulae according to the following schedule: every 2 hr following treatment until 1500 hr then at 30 min intervals to 1700 (initiation of milking). In addition, blood was collected via jugular cannulae at 1700-1800 hr on the day preceding treatment, each day of treatment and the day following treatment. Blood was also collected once daily from the coccygeal artery or vein by venipuncture on days 2, 3 and h after treatment. Prolactin, GH and cortisol were quantified in selected serum samples. Milk yield was recorded at each milking. Objecti for this was repl cubated : zen. The containir and was i decanted eJChthOl I: concentral and GH we 27 Objective 2I Effect of Ergocryptige in vitro e i e tal des' I Bovine anterior pituitary cell cultures which had grown to confluent monolayers by 96 hr were used for this study. 0n the day of the experiment growth medium was replaced with TC medium 199, (Appendix 2) cellswere in- cubated for 4 hr and the medium was decanted and stored fro- zen. Then each of four flasks received h ml of TC medium 199 containing either 0, 0.01, 1.0, or 10.0 ug ergocryptine/ml and was incubated for h hr after which the medium was again decanted and stored frozen. Ergocryptine was dissolved in ethanol prior to addition to TC medium 199 and the final concentration of ethanol in each flask was 0.1%. Prolactin and GH were quantified in the media. Experiment 2.--Thyrotzopin Releasing Hormone (TRH)I Effect Prolact‘n d GH R le m.B v n P' '- tgry Cell Cultures Objective lI Effect of TRH on Prolactin and GH release in v tro e ' ntal des' I Cell cultures prepared from pituitaries of four cows were in culture for 72 hr when they were in- cubated for 2 hr with TC medium 199. Thereafter, cell cul- tures (four flasks/treatment) were incubated for 2 hr with TC medium 199 containing either 0.0, 0.01, 0.1, 1.0 or 10.0 ng TRH/ml. In a second experiment, cell cultures prepared from pituitaries of four cows and three steers were in culture for 96 hr when they were incubated for 2 hr with TC medium 199. Then, cell cultures (four flasks/treatment) were incubated for 2 hr with either 0.0, 0.01, 0.1, 1.0 or 100.0 ng TRH/m1 medium. Po and etc: lbiee’til Ballets} hormone retained If there in respc TRH-indi mm of four ted for was incl. 0.1. 1 28 Following each incubation period the media were decanted and stored frozen until assayed for prolactin and GH. Objegtive 2I Ezolactin Release in vitEOI TRH vs GggH BgtignglgI The rationale for using gonadotropin releasing hormone (GnRH) was to test if bovine pituitary cell cultures retained their ability to distinguish different secretagogues. If there was no difference in the type of hormone released in response to different secretagogues one could argue that TRH-induced prolactin release was a non-specific response. r mental desi I Cell cultures prepared from pituitaries of four cows were in culture for 96 hr when they were incuba- ted for 2 hr with TC medium 199. Then each of four flasks was incubated for 2 hr with TC medium 199 containing either 0, 1, 10 or 100 ng GnRH/ml or 10 ng TRH/ml. Prolactin and luteinizing hormone (LH) were quantified in the media. Objective 3I fff-ct of f'odoth olige T aid t [[0 ’ne m on TR -;duced ' olact'n Releas v o Exgerimentgl designI Bovine pituitary cells were in culture for 96 hr when they were incubated for 2 hr with TC medium 199. Thereafter cells, (four flasks/treatment) were incubated for 2 hr with TC medium 199, containing thyroid hormone and/or TRH. The design of the experiment was a 2x3x2 factorial em- ploying 2 thyroid hormones (T3 and T4) at 3 levels (0, 0.1 and 1.0 ug/ml) and 2 doses of TRH (0 and 10 ng/ml). In a second experiment, bovine pituitary cells that were in culture for 96 hr were preincubated for 6 hr with TC medium 199 containing either 0 or 0.1 ug TB/ml. Then cells, (four flasks/treatment) were incubated for 2 hr with TC medium 199 contain' or 0.1 In cultures periods then 10 Til/ml th TRH/ml o‘ collecteI for prolz mm mm Materials tMEI-'3 day proEEStex estradiol tained ir ["65 X 5c in each s after Ste Via jugu] 29 containing either T3, (0 and 0.1 ug/ml) TRH (0 and 10 ng/ml) or 0.1 ug T3+10 ng TRH/ml. In the final study of this series, 96-hr pituitary cell cultures (5 flasks/treatment) were incubated for two 2-hr periods with TC medium 199 containing eitherI 1) 0 ug Tu/ml then 10 ng TRH/m1; 2) 5 ug Tu/ml then 10 ng TRH/m1; 3) 5 ug Tu/ml then 10 ng TRH + 5 ug Tu/mlz u) so ug Tu/ml then 10 ng TRH/ml or 5) so ug Tu/ml then 10 ng TRH + so ug Tu/ml. Media collected at the end of each incubation period were assayed for prolactin. gapegimegt 3.-- Prolaetin egd Growth Hormone geleaee efxe; G adal Ster 'd d T H ° V vo d V'tr Opjeetive lI Serug Pgolactig end GH afteg Geneeel Siezoide , i1; vivo EZEEZimfinI§l_Q£§152' Fifteen Holstein heifers (Section A of Materials and Method) were randomly assigned to receive at three days before ovariectomy either no steroids (n = 3): a progesterone pessary (n = h): estradiol -l7B (n = h) or both estradiol and progesterone (n = h). Estradiol -178 was con- tained in polydimethylsiloxane implants (I.D. 3.35, 0.D. n.65 x 50 mm). Four implants were placed subcutaneously, two in each ear. Heifers were ovariectomized on the third day after steroid treatment. Blood collection was accomplished via jugular vein puncture before steroid treatment: thereafter via jugular vein cannulae. A blood sample was collected from each heifer immediately before steroid treatment and just prior to ovariectomy. After ovariectomy blood samples were collected at 2 hr intervals for #8 hr then twice daily for 4 days. There- after depot steroids were removed and blood was collected every ten he steroi Follow at 2 h ment, every intrav was co 30 min Min. 30 2 hr for #8 hr then once daily for h days. Prolactin was quantified in all serum samples and GH, progesterone and estradiol were quantified only in selected serum samples. Objective 2I ffect of Estrad ol -1 TRH- d ced Prolact' end GH release in vivo ' tal des' I Approximately 60 days after ovariectomy, ten heifers were randomly assigned to receive either no steroid (control) or four implants containing estradiol -176. Following treatment with estradiol -l7B blood was collected at 2 hr intervals for 36 hr. Beginning at 72 hr after treat- ment, blood was collected at 30 min intervals for 90 min, then every 5 min for 30 min at which time all heifers received intravenously 33 ug TRH/100 kg body wt. Following TRH, blood was collected at h, 6, 8 and 10 min, at 5 min intervals until 30 min, at 15 min intervals until 60 min then at 90 and 120 min. Eight of these 10 heifers plus two additional ovariecto- mized heifers were used in a subsequent experiment. All heifers had just completed an experiment wherein they were treated with GnRH and all were bearing four implants contain- ing estradiol -17a. Implants were removed from five heifers and the remaining five received an additional four implants i.e. five heifers had no steroid (controls) and five had eight implants. Blood was collected at 2 hr intervals for 36 hr at which time all heifers received intravenously 33 ug TRH/100 kg body wt. Following TRH, the schedule for blood collection 'was similar to that used for heifers with four estradiol im- plants 0 31 Serum prolactin, GH and estradiol were quantified in samples selected from among those collected before TRH admin- istration, and after TRH treatment prolactin and GH were quantified in all serum samples. Objective 3I Effect of Estpadiol -17 on baseline prolactin concentration app TRH-induced_prolactin release in_xiree Experimental design: Cell cultures prepared from pituitaries of cows were 96 hr in culture when they were incubated for 2 hr with TC medium 199. Thereafter, cell cultures (four flasks/treatment) were incubated for 2 hr with TC medium 199 containing either 0, l, 10 or 100 pg estradiol/m1 or 10 ng TRH/ml. In a second experiment, pituitary cell cultures at 96 hr of culture were incubated for 2 hr with TC medium 199. Then cultures (four flasks/treatment) were incubated for 6 hr with TC medium 199 containing either 0, l, 10 or 100 pg estradiol/ml or 10 ng TRH/ml.' Following this incubation period, two flasks from each treatment group were incubated for 2 hr with TC medium 199 and the remaining two with 10 ng TRH/m1 TC medium 199. In the final study of this series, pituitary cell cul- tures were 96 hr in culture when they were incubated for 12 hr with TC medium 199 or TC medium 199 containing 10 ng estradiol ~17B/m1. Thereafter, each of four flasks was incubated for 2 hr with eitherI (1) TC medium 199 (2) 10 ng estradiol/m1 TC medium 199 (3) 10 ng TRH/ml TC medium 199 or (u) 10 ng estradiol + 10 ng TRH/ml TC medium 199. Prolactin was quanti- fied in the media. is m .( malt a; win: ' E- ital Th and Roh. to test I 32 E. Sgetistical Procedure The data were analysed by analysis of variance (Sokal and Rohlf 1969) and the procedure of Dunnett (1955) was used to test differences among means. RESULTS AND DISCUSSION Expegimentrle:Effect of Ergocryptine on Bovine ProlactinI CH, Cortisol and Milk Yield 1. Effect of Ergocryptine in vivo 0n the day preceding treatment, serum prolactin concen- tration in cows assigned to receive ethanol (control) avera- ged 16 and 33 ng/ml (figure 2) at 5 min before and 10 min after the start of milking respectively, and the difference between the means was significant (p < 0.05). Comparable averages for cows assigned to receive ergocryptine were 1H and 28 ng/ml and the difference between means was also sig- nificant (p < 0.05). But differences between treatment groups were not significant (p > 0.05). When GH was quantified in these same samples its concentration in serum.remained at approximately u ng/ml throughout the milking period (figure 2). Serum prolactin concentration (ng/ml) of control cows on days 1 and 2 of treatment averaged 20 and 17 at 5 min before and 35 and 27 at 5 min after the start of milking, respectively, (figures 3 and h) and the difference between means within day was significant (p < 0.05). Comparable averages for cows treated with 80 mg of ergocryptine were 1.3 and 1.1 at 5 min before and 1.4 and 1.1 at 5 min after milking, respectively, and differences between means were not significant (p > 0.05). On both days of treatment serum pro- 33 34 Figure 2. Prolactin and growth hormone response to milking on the day preceding ergocryptine treatment. 35 (Iw/BU) 3Nowa0H HlMOHS wnaas QFCDIDVION AEEV 0253:). 2 m>_.r<._mm m2; m. n n- ti o. _ IIOX ml _ _ mm _ 0253.2 mz_._.n_>moowmw d Dom—P200 0 10" L z_._.o...._._.m_000mw < .. I 401F200 o I I 0 Z c.0440?“ IIII S 3 no n o. W d ud m omv o H. onm U 5 w or“ ii lactin relati‘.‘ cryptir treatme SH conc nent GR ed at 3 Th Prolact: treatmel ing was with or; lactin I and 5 In differel parable and 1.1 out the Se] only in il‘téatmeI C I . I“mtlhe #0 lactin concentration was greater (p < 0.01) in control cows relative to comparable averages for cows treated with ergo- cryptine. In contrast to prolactin, neither ergocryptine treatment nor stimuli associated with milking affected serum GH concentration (figures 3 and b). On both days of treat- ment GH concentration in serum of both groups of cows remain- ed at 3-h ng/ml throughout the milking period. The effect of ergocryptine on suppression of serum prolactin was long lasting. Thus, on the day following treatment, prolactin in serum of blood collected around milk- ing was greater (p < 0.01) in control cows than cows treated with ergocryptine (figure 5). In control cows, serum pro- lactin concentration averaged 11 and 2h ng/ml at 5 min before and 5 min after the start of milking respectively, and the difference between means was significant (p < 0.05). Com- parable averages for cows treated with ergocryptine were 1.1 and 1.1 ng/ml. Serum GH concentration was unchanged through- out the milking period (figure 5). Serum cortisol concentration (figure 6) was determined only in samples collected around milking on the first day of treatment. There was no apparent effect (p > 0.05) of ergo- cryptine treatment on serum cortisol concentration. At 5 min before milking, serum cortisol concentration averaged 7.3 and h.l ng/ml in cows treated with ethanol (controls), and ergo- cryptine respectively, and was increased (p < 0.05) to 12.2 and 13.7 ng/ml respectively, at 10 min after the start of milking. Serum prolactin concentration decreased rather quickly ul Figure 5. Prolactin and growth hormone response to milking on the day following ergocryptine treatment. 42 (Iw/fw) 3Nowa0I-I HlMOHS 'wnuas AEEV 0233:). 2 m>_._._.h 0.05) on milk yield (figure 8). During the two days of treatment, average daily milk yields were 23.7 and 2h.0 kg for control- and ergocryptine-treated cows, respect- ively. By 10 days after treatment, control cows were produ- cing an average of 1 kg more milk daily than cows treated with ergocryptine but differences between means were not signifi- cant (p > 0.05). Considering the entire eXperimental period $ in 'u “ "' ,3 TT’T' #6 Figure 7. Chronic effect of two consecutive doses of ergo- cryptine on serum prolactin and growth hormone concentrations. 47 IIw/EIU) BNOWHOH HIMOHO wnaas oo I~ In I!) v ION o m e m N _ _. oIIII+IIII+IIII¢IIII¢IIII¢I ._ I mzfiaiooorw c re III / .5528 o 2:939: III / i wz _ha>mooomm O. 0. ON mm on on ow (NI/fin) NIlOV'IOUd wnaas . 1|“th I . " v" .‘ $334."? :1. . 'I " Ayuvw \"I. #8 Figure 8. Milk yield in cows treated with ergocryptine on days 1 and 20 49 Jombzoo di:-Id . -I~ In «I v —Io mz_ha>mooomw 0'0 L uz_ba>mooomu l _ Pm l 00 O N I III Inca-cu NNNN .LI 0 '0 (6x) mau )I‘IIIN A1070 oAv 50 following treatment, regression of milk yield on time after treatment revealed no difference (p > 0.05) in slope between the two treatments. 2. Effect of Ergocryptige in vitro Incubation of bovine pituitary cell cultures for h hr with ergocryptine in doses ranging from 0.01 to 10 ug/ml TC medium 199, resulted in approximately a 60% reduction in media prolactin concentration compared to prolactin concen- tration in media from pituitary cell cultures, not exposed to ergocryptine (table 1). Following h hr of incubation, media prolactin concentration averaged 57.8 and 32.9 ng/ml from control- and ergocryptine-treated cultures respectively, and the difference between means was significant (p < 0.001). However, differences due to dose of ergocryptine were not significant (p > 0.05). Although ergocryptine had no effect (p > 0.05) on media GH concentration the amount of GH released during the treat- ment incubation period appeared to be dependent upon pre- treatment media GH concentration. Hence GH concentrations of the treatment incubation period were adjusted by covariance 'based on pretreatment GH levels. These adjusted means are presented (table 1). Following # hr of incubation, media GH concentration averaged 29.7 ng/ml in cultures not exposed to ergocryptine and 25.1 ng/ml in cultures treated with ergocryp- tine but the difference between means was not significant (p > 0005). Lily; If» .L... avflaafw‘ . . .w. H. .o , F . . .4. a. 51. Table l. Prolactin and growth hormone release from bovine pituitary cell cultures treated with ergocryptine. Ergocr tine, tun Hormone in media ml Prolactin TEQQEL Growth hormone o 57.8 29.7 0.01 3u.o° 23.1 0.10 31.2° 26.2 1.0 35.2° 25.8 10.0 31.2° 25.3 samb 2.7 2.5 a'Mieans adjusted for variation in growth hormone release during pre-treatment incubation. bStandard error of mean calculated from error mean square (prglactin) and deviations mean square (growth hormone): n30 cLess than average of control flasks (0 ug/hl); p < 0.001. ._ ragga 4. ‘3 . 1.!» adUIIIIII. PI, .- 52 Results of this experiment clearly demonstrate that ergocryptine injected subcutaneously, significantly decreased resting concentrations of serum prolactin and prevented the increase in serum prolactin concentration that normally fol- lows milking in cows (Johke 1969, Tucker 1971, Koprowski and Tucker 1973). The action of this drug appears to be rapid, since 2 hr after administration serum prolactin concentration was significantly decreased and it remained suppressed for at least five days indicating a prolonged effect of ergocryptine on prolactin inhibition. The decline in serum prolactin con- centration caused by ergocryptine agrees with results for rats, (Nagasawa and Meites 1970, Brooks and Welsch l97h, Dohler and Wuttke 197k) humans, (del Pozo e3_e;. 1972, Varga fat—a... 1972) cows (Schams 2U}.- 1972, Karg 23.51.. 1972, Fell e3__;. l97h) and sheep (Niswender 1970). These data also confirm reports in cows (Karg.e34el. 1972) and goats (Hart 1973) that ergocryptine significantly decreased serum prolactin concentration without affecting milk yield. However, Fell e3_e1. (1974) observed a reduction in milk yield and protein content of milk following adminis- tration of ergocryptine to cows just prior to and after par- turition. Schams e3_el. (1972, 1973) and Karg and Schams (l97h) also reported that ergocryptine given to cows in late pregnancy inhibited the rise in serum prolactin concentration that occurs prior to parturition and suppressed milk yield in the subsequent lactation. Karg e1_el. (1972) had previously reported that ergocryptine suppressed bovine serum.prolactin 53 concentration to near 1 ng/ml, but failed to affect establish- ed lactations. Therefore these authors suggested that prolactin may be required for lactogenesis but not galactopoiesis in the bovine. Ergocryptine however, will inhibit established lac- tation in rats (Shaar and Clemens 1972) humans (del Pozo e3 al. 1973, Varga e3_el. 1972) and mice (Nagasawa and Yanai 1972) presumably due to inhibition of prolactin. Serum concentrations of GH and cortisol were not affect- ed by ergocryptine treatment, indicating a relative specifi- city of this drug with.regard to prolactin suppression. Failure of ergocryptine to suppress serum GH concentration confirms a previous report (Hart 1973) showing that ergocryp- tine suppressed serum prolactin but not GH concentration in lactating goats. Failure to detect an increase in serum GH concentration due to stimuli associated with milking corro- borates previous reports for cows (Tucker 1971, Reynaert and Posters 1972 and Koprowski and Tucker 1973a). But stimuli associated with milking or suckling increase serum.GH concen- tration in lactating goats (Hart and Flux 1973) and decreased pituitary GH concentration in lactating rats presumably by causing release of GH into the circulation (Grosvenor ej;_l, 1968 and Sar and Meites 1969). To my knowledge this is the first report which demon- strates that ergocryptine does not affect serum.glucocorticoid concentration. The increase in serum cortisol concentration in response to milking reported herein confirms previous results from our laboratory (Smith.e3_el. 1972 and Koprowski and Tucker 5h 1973a) and those of Wagner (1969) that showed increase total serum.glucocorticoids following milking in cows. The increase in serum.prolactin concentration of control cows, which began three days after treatment, might have re- sulted from stress associated with venipuncture which has been reported to increase serum prolactin concentration (Johke 1970, Raud t al. 1971 and Tucker 1971). If this is true. then failure of cows treated with ergocryptine to show increase serum.prolactin concentration in response to veni- puncture similar to cows treated with ethanol. indicates the effectiveness of ergocryptine to suppress prolactin release evoked by a stimulus other than milking. Although ergocryptine reduced serum prolactin concen- tration to approximately 1 ng/ml without affecting milk yield for 10 days after treatment, these results should not be interpreted as evidence that prolactin is not required for lactation in the bovine. Assuming a blood to milk ratio of #0031. a cow with serum prolactin concentration of 1 ng/ml and producing 25 kg of milk/day, the mammary gland would be ex- posed to approximately 20 mg of prolactin daily which may be adequate to sustain milk secretion. Conceivably. under normal conditions. far more prolactin may be present in bovine serum than is required to maintain milk secretion. Hence serum. prolactin levels may not reflect only lactational events and other physiological roles for this hormone should be considered. Results presented here also demonstrate that ergocryptine can act directly on bovine pituitary cell cultures in_zitgg 55 to inhibit prolactin release. These data confirm previous results that ergocryptine or ergocornine suppressed prolactin release in yitgg from.anterior pituitary explants of different species (Pasteels 91_a_. 1971, Lu 213;. 1971. Ectors §3_a__. 1972 and Yanai and Nagasawa 197%). Failure of this drug to affect GH release in 11322 is in agreement with results of Yanai and Nagasawa (1970a and l97h) which showed that ergo- cryptine suppressed prolactin release in xitgg from pituitary explants of rats and mice but had no effect on GR concentra- tion. Furthermore, failure of ergocryptine to affect GH re- lease despite significant suppression of prolactin release, suggests to us that its effect on prolactin release is not simply a noxious action. Ectors g3_g;. (1972) also provided evidence to refute any idea that the effect of ergocryptine on prolactin release was simply that of a noxious drug. They observed that ergocryptine caused no overt cellular destruct- ion of anterior pituitary explants in zitzg, but rather, suppressed prolactin release by inhibiting exocytosis from prolactin cells. In addition. when ergocryptine was rinsed from cultures previously treated. prolactin release was res- tored (Pasteels g3_§;. 1971). Although the results reported here would suggest that the 1p_yizg influence of ergocryptine on serum prolactin concentra- tion is at least in part via a direct action on the anterior pituitary, one cannot ignore the possibility that this drug inhibited prolactin release by acting at higher centers of the brain. or simply by affecting the metabolic clearance rate of I"! 56 prolactin. Experiment 2. o in Rel a i Hormo TRH : Eff c on o d GR Release from.Bov' e P Ce 1 C l e l. on Prolact n d GH el ase v Addition of TRH to pituitary cell cultures from.cows, at 72-hr of culture increased (p < 0.01) prolactin release into the media approximately 2-5 times relative to prolactin concentration for the 2-hr pretreatment period (table 2). Media prolactin concentration ranged from 508-587 ng/ml for all treatment groups during the 2-hr pretreatment period. Following 2 hr of incubation with TRH, average media prolac- tin concentration in control cultures (0 ng TRH/ml) showed a 9% decreased (p > 0.05) relative to comparable averages for the 2-hr pretreatment period. In contrast, media prolactin concentration was increased (p < 0.01) to approximately l.h ug/ml in cultures to which 0.01 ng TRH/ml was added and'to 2.5 ug/ml in cultures that received the higher doses of TRH. The difference between pre- and post-treatment prolactin concentration after 0, 0.01, 0.1, 1.0 and 10.0 ng TRH/ml was -23, 799, 1966, 1926 and 1976 ng/ml, respectively. Apparent- ly maximum prolactin release was achieved with 0.1 ng TRH/ml, as there were no differences (p > 0.05) in the magnitude of prolactin release among cell cultures, that received TRH at doses greater than 0.1 ng/ml medium. Growth hormone concentration in these same media is shown in table 3. Although TRH increased (p < 0.05) GH’re- lease, the magnitude of response was small compared to pro- lactin release. Media GH concentration from control cultures 57 Table 2. Prolactin release from'bovine pituitary cell cul es treated with thyrotropin releasing hormone (TRH). grolagtin in media e- Post- ¥E§7fil) tB- ’ng/ml) c . 0 576139 553379 43178 0.01 587338 1386320u 799317u 0.1 560152 2526359 19663u2 1.0 5&836 2u7u1116 19261120 10.0 508151 2h8u1157 1976:117 Table 3. Growth hormone release from bovine pituitary cell cultures treated with thyrotropin.releasing hormone (TRH). _______QI2lIhJMQDMXELjflLEEflLE______. Pre" b P 08 t- C (l§:m17 ‘ng/ml) o 9013 351:; -533 0.01 111312 12037. 925 0.1 102311 11u39 123a 1.0 10927 13015 21:“ 10.0 1011a 12213 2115 aValues are means 3 standard error. bIlean prolactin or growth hormone concentration of four flasks for the Z-hr period preceding treatment. cDifference between pre- and post-treatment. “a. . . I .. .Et 1 58 (0 ng TRH/m1) averaged 90 ng/ml for the 2-hr period prece- ding treatment and 85 ng/ml for the second 2-hr incubation period. But addition of TRH at levels as low as 0.01 ng/ml medium. increase media GH concentration as evidenced from the mean differences in GR concentration between pre and post- treatment incubation periods. These differences in GR concentration were -5, 9, 12, 21 and 21 ng/ml at 0.0, 0.01, 0.1, 1.0 and 10.0 ng TRH/m1 medium, respectively. maximum GH release was apparently achieved with 1.0 ng TRH/ml which is in contrast to prolactin where maximum release was apparent with 0.1 ng TRH/ml medium. Addition of TRH to pituitary cell cultures from cows, steers and a bull at 96-hr of culture increased (p < 0.05) prolactin release into the media (table h). Similar to 72-hr pituitary cell cultures there was a decrease in media prolactin concentration from all control cultures during the treatment incubation period relative to prolactin averages for the pretreatment period. In addition, media prolactin concentration from pituitary cell cultures of cows was 37% less than averages from the 72-hr cultures. But addition of TRH to cultures from cow pituitaries evoked prolactin release as evidenced from the mean difference in prolactin concen- tration before and after TRH. These differences in prolactin concentration were -27, 63, 109, 207, 226 and 137 ng/ml at O, 0.01, 0.1, 1.0, 10.0 and 100 ng TRH/ml respectively. Apparently, maximum prolactin release from these cultures was achieved with 1.0 ng TRH/ml which is in contrast to 72-hr cell cultures when maximum prolactin release was 59 Table 1}. Prolactin release from bovine pituitary cell cultures treated with thyrotropin releasing hormone (TRH) . TRH Sexb uggentc ugzmnt d (W (us/m1) o Cow (3) 1731'? MST-9 -271'1u 0.01 167321 236123 6333 0.1 193323 302% 109320 1.0 20119 #081121 207129 10.0 258315 neutuz 226127 100.0 230121 367337 137320 0 Steer (3) 123321 109312 -1u1'10 0.01 11039 123312 13313 0.1 lad-'1 176311 361’s 1.0 ind-'51!» 293326 153130 10.0 10939 2751'? 16636 100.0 11218 192111 80110 0 Bull (1) 57th 511's - 16 10.0 cute 78:10 1436 100.0 7236 1033.518 31312 a96-hr pituitary cell cultures. bNumber in parentheses equals n. cMean prolactin concentration of four flasks for the 2-hr period preceding treatment. dDifference betwen pre- and post-treatment. l! 60 obtained with 0.1 ng TRH/ml medium. Extending the dose of TRH to 100 ng/ml, apparently cause a reduction in prolactin release compared to that observed after 10 ng TRH/ml (p < 0.05). Thyrotropin releasing hormone also augmented (p < 0.05) prolactin release from pituitary cell cultures of steers (table h). The mean difference in prolactin concentration between the pre- and post-treatment incubation periods were ~11“ 13, 36. 153, 166 and 80 ng/ml for 0.0, 0.01. 0.1, 1.0. 10.0 and 100 ng TRH/m1 medium respectively. In 50% of the cultures that received 0.01 ng TRH/ml. prolactin concentra- tion was reduced during the treatment incubation period re- lative to the pretreatment period. and this accounted for the large standard error. Similar to results of 96-hr pituitary cell cultures from cows, maximum prolactin release was achieved with 1.0 ng TRH/m1 and extending the dose of TRH to 100 ng/ml resulted in a diminution (p < 0.05) in pro- lactin release compared to the response obtained with 10 ng TRH/ml. Addition of TRH to pituitary cell cultures of a bull also increased (p < 0.05) prolactin release into the media (table it»). The mean difference in prolactin concentration be- tween the pre- and post-treatment incubation periods was -6, 1“ and 31 ng/ml at 0, lo and 100 ng TRH/ml respectively. Re- lative to prolactin concentration after 10 ng TRH was added per ml of medium. there was a doubling in prolactin concentra- tion (14 vs 31 ng/ml) after 100 ng TRH/m1. Media prolactin °°ncentration was greater in pituitary cell cultures of cows 61 at 96-hr of culture, relative to prolactin concentration in cultures from pituitaries of steers and a bull. When GR concentration was determined in these media from 96-hr pituitary cell cultures. there was a decrease (p >'0.05) in GR concentration during the treatment incubation period. relative to GH averages for the pretreatment period (table 5). The decrease was observed among pituitary cell cultures of cows, steers and a bull and was independent of the dose of TRH (0.0 to 100 ng/ml medium). This reduction in GR concentration could not be attributed to TRH inhibition since the magnitude of decrease among TRH-treated cultures was not different (p > 0.05) from control cultures. 2. o c R ase in v on TRH G Addition of GnRH to 96-hr pituitary cell cultures of cows slightly stimulated prolactin release (table 6). Media prolactin concentration ranged from 326-360 ng/ml for all treatment groups during a 2-hr incubation period with T0 ‘medium 199. Following 2 hr of incubation with GnRH prolactin concentration decreased 13-38% relative to concentrations during the pretreatment period. The change in media pro- lactin concentration expressed as the difference in quantity of prolactin release before and after GnRH treatment averaged -133. -80. 4&7 and -51» ng/ml after 0, 1. 10 and 100 ng GnRH/ml respectively. Prolactin concentration was greater Cp‘< 0.05) in media from cultures treated with 10 and 100 ng GnRH/ml compare to prolactin concentration in control cultures (0 ng GnRH/ml). This increase however, was minimal relative to the comparable average 386 ng/ml from cultures treated with 62 Table 5. Growth hormone release from bovine pituitary cell cultures treated with thyrotropin releasing hor- mone (TRH) . W TRH Sexb gggmntc tiggtment d “E ml) rig/m1 o Cow (3) 10¢16 58111 -u6316 0.01 108112 71312 -3712 0.1 103315 53311 -5035 1.0 11233 6015 -52i7 10.0 146121 89310 -57316 100. 0 115112 511's -6u1' 5 o Steer (3) 96110 5835 ~38t8 0.01 100312 69310 -31311 0.1 1231'? 601’? -631’3 1.0 130312 7938 -51316 10.0 100312 8037 -20112 100.0 10612 6017 -4626 0 Bull (1) 5537 1:13 5 -1l+1' 5 10.0 6817 #738 -2136 100.0 7727 note -37t2 a96-hr pituitary cell cultures. bNumber in parentheses equals n. cMean prolactin concentration of four flasks for the 2-hr period preceding treatment. dDifference between pre- and post-treatment. 63” Table 6. Prolactin release from bovine pituitary cell cultures treated with gonadotropin releasingahormone (GnRH) or thyrotropin releasing hormone (TRH . Pre- : t Pgst c Treatment treatment treatment (E7m17 tag/ml GnRH o 3&738 214323 ~133t20 1 326121 2&6327 ~80318 10 3603 31312u -u7125d 100 auztio 288112 -5u111d TRH lo 3&91’31» 735110 38659" 3“Values are means 1' standard error. bMean prolactin concentration of four flasks for the 2-hr period preceding treatment. °Difference between pre- and post-treatment means. dGreater (p < 0.05) than average of control flasks (0 ng/ml). e”(greater than comparable means for flasks not treated with TRH p < 0.0 .' 6h 10 ng TRH/ml. When luteinizing hormone (LH) was quantified in these media, GnRH but not TRH increased (p < 0.05) media LH concen- tration. The difference between pre- and post-treatment LH concentration after 0, 1. 10 and 100 ng GnRH/ml and 10 ng TRH/ml was -6, 15, 18, 17 and -2 ng/ml respectively. 3. Effect of Triiodgthygonine (T3) and Thygoxine (Th) on TRH- induced Prolactin Release in vitrg Prolactin release from cell cultures was not affected by inclusion of either triiodothyronine (T3) or thyroxine (T4) at 0.0, 0.1 or 1.0 ug/ml in the incubation medium (table 7). Media prolactin concentration ranged from 23-29 ng/ml in cultures incubated for 2 hr with either TC medium 199 or thyroid hormones and was increased (p < 0.01) to uo-ns ng/ml in cultures incubated for 2 hr with either TRH or TRH and thyroid hormones. There was no difference (p > 0.05) in mean prolactin concentration between cultures exposed to TRH alone and those exposed to TRH and thyroid hormones. Pretreatment of cell cultures for 6 hr with 0.1 ug T3/n1 medium affected neither baseline prolactin concentration nor the amount of prolactin released in response to TRH during a subsequent 2-hr incubation period (table 8). After 2 hr of incubation with TC medium 199, prolactin released into the media averaged 21 and 26 ng/ml from cell cultures pretreated for 6 hr with 0 and 0.1 ug T3/m1 medium, respectively. Com- parable averages after 2 hr of incubation with either T3, TRH, or T3 + TRH were 25 and 23; 90 and 78 and 67 and 66 ng/ml, respectively. Media prolactin concentration after 65 Table 7. Prolactin release (ng/ml) rrom.bovine pituitary cell cultures heated with hiiodothyrcnine (T ) aghyroxine (Tu) and thyrohopin releasing hormone (TRH). Thyroid hormone Thyrohopin releasing (ug/ml) hormone ) 0 10 T3 0.0 2533 azizc 0.1 2312 #313c 1.0 2931 hats° Th 0.0 2&31 0011° 0.1 2031; u5i'uc 1.0 2013 uztuc a”Values are means 1' standard error. bMean prolactin concentration of four flasks for the 2-hr heatment period . cGreater than comparable means for flasks not heated with TRH (p < 0.01) .‘ 66 Table 8. Prolactin release from bovine pituitary cell cultures preheated for 6 hr with hiiodothyronine (T ) and then incubated for 2 as with T3 and thyroho in re- leasing hormone (TRH) . We: Treatment TC medium 199 TC medium 199 + if Non-treated control 2130 2515 T3d 2 5152 23:1 mu" 90:18 78:7 TRH" + TBd ‘ 67:13 55:5 8’Values are means 1' standard error. bMean prolactin concenhation of four flasks for the 2-hr heatment period . °Cell cultures preheated for 6 hr with T0 medium 199 or T0 medium 199 + T3. dConcenhation =3 0.1 ug/ml medium. eConcentration = 10 ng/ml medium. 67 2 hr of treatment with TRH was greater (p < 0.05) than that of cell cultures receiving no treatment or T3 alone. Although concurrent addition of T3 and TRH appeared to suppress TRH- induced prolactin release (90 vs 67) and 78 vs 66) ng/ml, differences between means were not significant (p > 0.05). In addition the presence of T3 for 2 hr or 8 hr (6 hr pre- treatment + 2 hr post-treatment) did not affect (p > 0.05) TRH-induced prolactin release (67 vs 66) ng/ml. Incubation of cell cultures with higher doses of Th resulted in a suppression of spontaneous prolactin release and the quantity of prolactin released by TRH (table 9). Fol- lowing a 2-hr incubation period with 0, 5 and 50 ug Tw/ml TC medium 199, media prolactin concentration averaged 161, 119.5 and 82.5 ng/ml respectively, and difference between means was significant (p < 0.05). When these cultures were further incubated for 2 hr with 10 ng TRH/ml medium. prolactin concentration averaged 261, 22h and 167 ng/ml in cultures previously exposed to 0, 5 and 50 ug Tu/ml medium.respect- ively. Comparable averages resulting from concurrent add- ition of TRH and Th during the second incubation period were 220 and 176 ng/ml in cultures previously exposed to 5 and 50 ug Tu/ml respectively. The increase in media prolactin con- centration after TRH was significant (p < 0.05) relative to averages before TRH treatment. Since the presence of Th did not appear to affect the magnitude of TRH-induced prolactin release (224 vs 220) and (167 vs 176) ng/ml, these averages were pooled to examine the main effects of Ta on the quantity of prolactin released by TRH. Overall treatment averages 68 Table 9. Prolactin release from'bovine pituitary cell cultures treated with hormone (TRH). ahyroxine (Tu) and thyrotropin releasing .___JflflflthJL 1222122:? Tu prgggitinp Tu TRH progggtin ug/ml ng/ml ug/ml ‘ng/ml 0 16131u 0 10 261110 127316 0 10 220113 11212 5 10 22013 Avg 119.538* 22232* 50 8&13 0 10 167311 50 8116 50 10 176111 Avg 82.512** 171,5tuao 8‘Values are means i standard error. b treatment periods. Less than the comparable average for period control. *=p<0.05 ** a p < 0.01 Mean prolactin concentration of five flasks for the 2-hr 69 for media prolactin concentration were 261, 222 and 171.5 ng/ml after TRH challenge of cultures previously exposed to 0, 5, or 50 ug Tu/ml respectively, and difference between means was significant (p < 0.05). Thus thyroxine did not appear to affect the action of TRH, but rather the releasable quantity of prolactin. Results reported here clearly demonstrate that TRH sti- mulates prolactin release from bovine pituitary cell cul- tures in vitro. These results agree with a previous report by Tashjian et al. (1971) showing that TRH stimulated prolac- tin release from cloned rat pituitary tumor cells 13 vitzo. More recently Vale et a1. (1973) also reported that TRH at 6 concentrations of 10'9 to 10' M enhanced prolactin release from rat pituitary cell cultures, and in a preliminary re- port Machlin and Jacobs (1973) observed increase prolactin release from calf pituitary cell cultures treated with TRH. In the present investigation prolactin release from bovine pituitary cell cultures was augmented with TRH at concentra- '11 to 3x10-7M. The decrease in tions of approximately 3x10 magnitude of prolactin release in response to 100 ng TRH/ml, has not to my knowledge been previously reported and may be due to toxicity of the tripeptide when used at high concentra- tions. Previously, Convey g$_a1. (1973) reported no stimula- tion of prolactin release from steer pituitary explants in- cubated in zitgg with TRH. Labella and Vivian (1971) using bovine pituitary explants reported that TRH caused only a 70 marginal stimulation of prolactin release in one of three experiments. Lu et a1. (1972) also failed to demonstrate prolactin release from rat hemipituitaries incubated with TRH. Vale et al. (1973) reported that TRH caused only a minimal stimulation of prolactin release from normal rat hemi- pituitaries in_yitgg but significantly increased prolactin release from hemipituitaries of hypothyroid rats. In the present investigation and those of Machlin and Jacobs (1973), TRH consistently stimulated prolactin release from bovine pituitary cell cultures, but release from bovine pituitary explants was inconsistent. Although the reasons for the different results are not apparent, several possibilities could be put forth. Perhaps enzymatic dispersion of bovine pituitary cells causes some cellular transformation that allows TRH to release prolactin from these cells but not from pituitary explants. Alternately excessive cutting of bovine pituitary explants may cause a great non-specific release of hormones which could mask smaller amounts released by a secretagogue. Holding pitui- tary cells in culture for 3-h days would allow damaged cells that would release hormone non-specifically, to be eliminated during media changes. But these views were not supported by Vale et al. (1973) who established that TRH stimulated pro- lactin release 13,21329 from both primary pituitary cell cultures and hemipituitaries of hypothyroid rats. Dibbet gt a1, (1973) also demonstrated TRH-induced prolactin re- lease from rat pituitary explants ig,yi§gg and recently 71 Porteus and Malven (197G) reported that TRH increased serum prolactin concentration in rats bearing pituitary homografts following hypOphysectomy and lesion of the median eminence. Although TRH unequivocally increased serum GH concentra- tion in cows (Convey 1973, Convey et al. 1973) and acromegalic humans (Irie and Tsushima 1972) the effect of the tripeptide on GR release in 31322 is not consistent. Tashjian g3_a;. (1971) reported that TRH inhibited GH release 1g 11:39 from the same tumor cells that secreted increase quantities of prolactin in response to TRH. But Lu et al. (1972) observed no change in media GH concentration following incubation of rat hemipituitaries with TRH. Labella and Vivian (1971) demonstrated that TRH enhanced GH release in 21339 from bo- vine pituitary explants in only one of three experiments. But recently Machlin and Jacobs (1973) and Carlson g3_a_, (l97h) reported that TRH significantly increased GH release in 313:9 from primary cell cultures and hemipituitaries of calves and rats respectively. Results of the present experiment do not clarify the effect of TRH on GR release ig,xi§gg. The reason why TRH stimulated GH release from 72-hr but not96-hr pituitary cell cultures is not clear. That prolactin release from 72-hr pituitary cell cultures was greater than from 96-hr cell cultures raises the possibility that the GH response may be attributed to cross reaction of the GH assay with high concen- trations of prolactin in the media. The possibility is improbable however, since the GH assay used cannot measure 72 NIH-bovine prolactin at levels less than 50 ng/tube (Koprow- ski and Tucker 1971) and the dilutions that were used in the GH assay would allow a maximum prolactin concentration of 25 ng/tube which should not interfere with the GH assay. The difference in baseline prolactin concentration and the magnitude of prolactin release in response to TRH by pituitary cell cultures of cows, steers and a bull cannot be attributed only to the physiological status of the donor animals. Differences could be due to age of the cell cultures, the number of viable cells present at the onset of the experi- ment and the sex of the pituitary donor. Likewise, differences 9 in TRH-induced prolactin release between 72- and 96-hr pitui- tary cell cultures of cows, could be due to age of the cultures, the number of viable cells present or the physiological status of the pituitary donor. The specificity of the response of pituitary cell cultures to TRH and GnRH was demonstrated by failure of TRH to increase media LH concentration, and the observation that GnRH only slightly stimulated prolactin release. Vale ejgah, (1972) reported that addition of TRH to rat pituitary cell cultures in yitgg stimulated release of prolactin and thyroid stimulating hormone (TSH) but not LH or follicle stimulating hormone (FSH). Luteinizing hormone releasing hormone (LH-RH) stimulated release of LH and FSH but not TSH or prolactin. In addition, Bowers g3_a_. (1971) observed an increase in serum concentration of prolactin but not LH following administration of TRH to humans and Kastin et al. (1973) reported that LH-RH 73 increased serum LH concentration but not prolactin in men. Thus similar to the pituitary iplsitu, cells in culture are also capable of discriminating between specific releasing hormones. Failure of T3 or T0 to increase baseline prolactin concentration from bovine pituitary cell cultures, supports previous results from our laboratory (Shaw et a1. 1972) which showed that feeding of thyroprotein to lactating cows had no effect on baseline serum prolactin concentration despite mar- ked increases in serum thyroxine. In view of these reports, it is unlikely that the galactopoietic effect of thyroxine and thyroactive substances in cattle (Blaxter et a1. l9h9) is attributable to stimulation of prolactin secretion but pre- sumably results from a general increase in body metabolism. In contrast to results obtained with the bovine cell cultures Meites (1963) demonstrated that both T3 and Th significantly increased prolactin release from rat pituitary explants in 113:9. In addition, Chen and Meites (1969) showed that T4 increased pituitary prolactin content of rats, presumably by a direct action on the anterior pituitary since there was no change in the activity of prolactin inhibiting factor. The reduction in magnitude of prolactin release from cell cultures treated with 5 or 50 ug Tw/ml medium,.confirms . a previous report by Vale et al. (1973) which showed that T3 or T”, in doses similar to those used in the present study, decreased spontaneous release of prolactin from hemipituita- ries of hypothyroid rats. The mechanism by which these doses Ii ‘8'! I 7'4 of Ta inhibit prolactin release in viho is equivocal but failure to affect LH release (Vale et al. 1973) suggests that the effect on prolactin release is not simply a noxious action. Results reported herein, suggest that Tu had no effect on the ac tion of TRH with regard to prolactin release, but might have affected the quantity of releasable prolactin. In contrast to results obtained _i_r_1 1i_t_r_o_, Bowers et a1, (1971) and Snyder et al. (1973) demonstrated that adminisha- tion of thyroid hormones to humans decreased the magnitude of prolactin release in response to TRH. These results were confirmed in sheep by Debeljuk et a1. (1973), but the mecha- nism by which this decrease was effected is equivocal. Ibcperiment 3: Prolactin and Growth Hormone Release after Gonadal Steroids and TRH in vivo and in vit_r_'_q 1 . Serum Prolactin and GH after Gonadal Steroids in vivo Serum progesterone concentration averaged 1.0, 1.8, 1.6, and 1.6 ng/ml (figure 9) in intact heifers immediately before they received no steroid heatment, eshadiol (E2), proges- terone (P) or E2+P, respectively, and differences among group means were not significant (p > 0.05). Progesterone concen- tration in serum of heifers was increased (p < 0.05) to 3~4 ng/ml at 72 hr following insertion of progesterone pessa- I‘ies but remained unchanged in heifers not receiving pessaries. BY 21+ hr after ovariectomy progesterone concenhation decreased (1) < 0.05) to approximately 0.2 ng/ml in heifers without Dessaries, but remained greater than 1.0 ng/ml for at least five days after ovariectomy in heifers bearing pessaries. 7 L n . 75 Figure 9. Serum progesterone concentration of heifers after placement of depot steroids and subsequent ovariectomy. Heifers were ovariectomized 3 days after placement of steroid implants. Implants were removed 6 days after ovariectomy. . A>05wm :40 Q :3 3 5.24.52. ._.2<._¢2_ P24152— x>o x> >0 a0&.‘l Wr' 1 .0. ' coo 76 ‘ VfiWflWfl «mg .55on D 9,3 8 5.24.55. ffflflflflffifiyflffififlflfio 93 N2 #24sz— 93 3 A Wflwrwmmrryy b222— x>0 C ._II 0 . 7 O O 93 n. ._.2<._a2_ .552. A WflflWflflififl-flo 5.05.2 _ Mar/KKK.) (um/bu) 3N083183908d wnsas 77 Progesterone concentration was greater (p < 0.05) the first three days after ovariectomy, in serum of heifers receiving both estradiol and progesterone than in serum of those receiving only progesterone. Within 2h hr after depot steroids were removed, progesterone concentration averaged 0.2 ng/ml in serum of all heifers regardless of treatment. Serum estradiol concentration averaged 8.2, 7.9, 12 and 6.h pg/ml (figure 10) in intact heifers immediately before they received no steroid treatment, E2, P or E2+P respect- ively (p > 0.05). Serum estradiol concentration then increased (p < 0.05) to an pg/ml at 72 hr, in heifers bearing four estradiol-filled implants but was unchanged (average 12 pg/ml) in heifers without estradiol implants. Estradiol concentra- tion was decreased (p < 0.05) to an average of 5 pg/ml at 2h hr after ovariectomy, in serum of heifers not receiving estradiol implants but remained greater than 20 pg/ml in heifers with estradiol implants during the six days after ovariectomy when the implants remained in place. Average serum estradiol concentration was 5 pg/ml at 2h hr after ’depot steroids were removed, and there was no difference (p > 0.05) between means for each treatment group. ' DeSpite marked increases in serum estradiol and proges- terone concentrations there were no significant changes in serum prolactin and GH concentrations attributable to steroid treatment. Serum.prolactin concentration prior to placement of depot steroids averaged 2a, 20, 19 and 30 ng/ml (figure 11) in heifers assigned to receive no steroid treatment, E2, Fm. ‘. h" 1‘5 78 Figure 10. Serum estradiol concentration of heifers after placement of depot steroids and subsequent ovariectomy. Heifers were ovariectomized 3 days after placement of steroid implants. Implants were removed 6 days after ovariectomy. >02mm #26312. x>o :40 Q A>o x>o. x>o x>o x>o x>0 ko 0.05). Comparable averages after 72 hr of exposure to exogenous gonadal steroids were 3h, 29, 23 and 2b ng/ml, and differences among means were neither different (p > 0.05) from one another nor from com- parable averages before steroid treatment. Considerable fluctuation in mean serum prolactin concentration was obser- ‘ved following ovariectomy but differences among treatment means or among these means and comparable averages before ovariectomy were not significant (p > 0.05). The decrease (p < 0.05) in serum prolactin concentration to a nadir at Lil h-lo hr.after ovariectomy, and the subsequent return to pre- ovariectomized levelshena characteristic of all treatment groups. Following removal of depot steroids serum prolactin concentration was unchanged (p > 0.05) relative to comparable averages before implants were removed. Average growth hormone concentration in serum of intact heifers prior to steroid treatment was not different (p > 0.05) among groups: average for all groups of heifers was 5 ng/ml (figure 12). At 72 hr after beginning of steroid treatment serum GH concentration averaged 6.5, 10.9, 5.8 and 7.h ng/ml in heifers that received no steroid, E2, P and E2+P respectively. The increase in mean serum GH concentration in heifers treated with estradiol alone, was due to one heifer whose serum GH concentration was ten times greater after estradiol treatment than before. Serum GH concentration was not affected (p > 0.05) by either ovariectomy or removal of depot steroids when com- pared with appr0priate averages before ovariectomy. 81 Figure 11. Serum prolactin concentration of heifers after .placement of depot steroids and subsequent ovariectomy. Heifers were ovariectomized 3 days after placement of steroid implants. Im- plants were removed 6 days after ovariectomy. 82 .3. 4055.5”. on .2523 53...... 50.. also: Eoeomigosemoa manor mm #W. ON.. mm thinW 0W ”N N.. W ¢W_40W..0amqqu:’mq¢ 0n VN N. Nmm .0. I; \/\/\. <<</ )\/\/\r I; L #8. 5b dduqqu-Juuq - 1 d - 405.200 On ow g (I‘ll/5“) MLOV'IOBd M1838 83 Figure 12. Serum growth hormone concentration of heifers after placement of depot steroids and subsequent ovariectomy. Heifers were ovariectomized 3 days after placement of steroid implants. In- plants were removed 6 days after ovariectomy. ml .6 252$ 55%: 50.. were: >zoeom_m<>o-emoa memo... mm 3.8. cm «a. : me cu r 3. cm. cm «a. (.me 3 c mm J .4 dean-—-- dud-ud-uuq1-u-qqqud E II\ {I g \s}? I m .7. m . "m .. .L .TIIIITIIIIIITTTTIITImtixIm ../\---........ K E. /’\/ 1 .858 \ O. 0. ON 0. 0. ON 0. n. O. n. om (aw/60) snowsow Mimosa wnsas 85 2. Bovine Serum Prolactin and Growth Hormone Response to Estradiol -l78 and TRH Estradiol concentration averaged 6 and 8 pg/ml in serum of ovariectomized heifers immediately before they received no steroid treatment (control) or four estradiol- filled implants respectively, (figure 13). Estradiol concen- ‘tration was increased (p < 0.01) to 55 pg/ml at 6 hr in sacrum of heifers receiving estradiol implants but decreased (p < 0.05) to 35 pg/ml by 18 hr after treatment. Serum (estradiol concentration was unchanged (p > 0.05) in serum cxf control heifers. By 72 hr after treatment, serum estra- diol concentration averaged 27 pg/ml in estradiol-treated liexifers which was greater (p < 0.05) than comparable average in controls. Serum prolactin concentration determined in blood collect- eeél at intervals during the first 36 hr after estradiol treat- nnenlt is shown in figure 1h. Analysis of treatment differences Irervealed that serum prolactin concentration was not affected (:1) > 0.05) by increased serum estradiol concentration. How- €?\rer, time by treatment interaction was significant (p < 0.05) €1r1d.was attributable to a difference in prolactin concentration between control and estradiol-treated heifers at 5 hr after 'tIPeatment: 31 vs 10 ng/ml for control and estradiol-treated heifers respectively. Growth hormone (figure 15) measured in Talese same samples, averaged 11 ng/ml in serum of heifers iJmmediately before estradiol treatment. After treatment, aVerage GH concentration was greater in serum of estradiol- treated heifers relative to controls, but the difference 86 Figure 13. Serum estradiol concentration in ovariectomized heifers implanted with four estradiol-filled- implants at time zero. Stigma-7.40.953 mmt< .5: ”is: we en mm on m. o. 0 cl d 405.200 I 544%: 40.05:.mw OIIIO q 1 Q O N ° 8 ¢ (um/Dd) 10|0V81$3 wnsas O D 0 CD 88 Figure 1h. Serum prolactin concentration in ovariectomized heifers implanted with four estradiol-filled- implants at time zero. 89 55%.. me. 305$..me rug 8:. m2:- 40¢h200 I ._.2<4A.Z. 40.0<¢._.mm I 0. 6. ON ON On an (um/6") NILGV‘IOBd wnsas £1.01. ’ . w . " 90 Figure 15. Serum growth hormone concentration in ovariec— tomized heifers implanted with four estradiol- filled implants at time zero. 91 ._.Z<4n..2_ Qt... 40_o 0.05). However, analysis of variance revealed a significant (p < 0.05) effect of time. Serum prolactin concentration increased in heifers following injection of TRH at 72 hr after estradiol treatment (figure 16). Immediately before TRH, serum prolactin concen- tration averaged 0.7 and 3.5 ng/ml in control and estradiol- E.) treated heifers respectively. Serum prolactin concentration I then increased (p < 0.01) to 75, 82, 69, 60 and 52 ng/ml at h, 6, 8, 10 and 15 min respectively, following TRH adminis- tration to control heifers. Comparable averages for estradiole £3 treated heifers were 62, 68, 61, 69 and 57 ng/ml. Serum prolactin concentration decreased (p < 0.05) towards pre- treatment averages by 2 hr after TRH was injected. Although TRH increased (p < 0.01) serum prolactin concentration re- lative to pretreatment averages, neither prolactin concentra- tion at the peak after TRH, nor the mean area under the prolactin response curve was different (p > 0.05) between controls and estradiol-treated heifers. Growth hormone concentration also increased in serum of ovariectomized heifers in response to TRH (figure 17). Imme- diately before TRH injection serum GH concentration averaged 7 and 15 ng/ml in control and estradiol-treated heifers re- spectively, but the difference between means was not signifi- cant (p > 0.05). Serum GH concentration then increased (p < 0.05) to 25, 26, 2a, 24 and 21 ng/ml at a. 6, 8, 10 and 15 mdn.respectively, following TRH injection into control It! at: .. IL fin! fii’fifli‘ .., i -| a . 93 Figure 16. Thyrotr0pin releasing hormone-induced prolactin release from ovariectomized heifers implanted with four estradiol-filled-implants. TRH (33 100 kg body wt) given at time zero, 72 hr after estradiol treatment. 94 02_m1... 0P w>_._.<4wm 2:2. mi... m202¢01 Ont. 0m. 00 00 d d m“ 40¢...200 I .mP2<4¢2.$ 40_oI... 0. m.>_...<4mm .EEvmsz... 0N. om 0.0 0..» 0m 0m 0_ 0 9. 0m- 1 . ..._.._..~. P J ' (F- 4om._.200 I .m...2<4n:2_ S 40_o 0.05) although after TRH injection serum GH concentration was 53% greater in estradiol-treated heifers than controls. Average serum estradiol concentration in ovariectomized heifers bearing eight estradiol-filled implants is shown in figure 18. At the beginning of this experiment all heifers had four estradiol implants and estradiol concentration avera- ged #2 and 33 pg/ml in serum of heifers immediately before estradiol implants were removed (controls) or heifers which received four additional implants, respectively. Estradiol concentration decreased (p < 0.05) to 17 pg/ml in serum of heifers at 6 hr after implants were removed (control) and averaged 10-15 pg/ml thereafter. In contrast, serum estradiol concentration increased (p < 0.05) to 71 pg/ml at 6 hr in those heifers receiving an additional four implants (total of 8) and remained elevated throughout the duration of the sampling period (36 hr). Immediately before TRH injection serum prolactin concen- tration averaged 7.3 and 8 ng/ml in control and estradiol- treated heifers respectively (figure 19). Serum prolactin then increased (p < 0.05) to 57, 52, 50, #7 and #6 ng/ml at h, 6, 8, 10 and 15 min following TRH administration to control . b .F .. .. . C .4 a i... t - 98 Figure 18. Serum estradiol concentration in ovariectomized heifers implanted with estradiol implants. At time zero all heifers were bearing four implants. Immediately thereafter, implants were removed from heifers in the control group and four addi- tional implants were placed in heifers in the estradiol-treated group. 99 0» T 55%.. Q: 1.555.me «wk... 3:. mac. ¢N 0. N. 0 405.28 I 05.24452. 405425.00 0 0'0 05.24452. 40.04c5.mu v 4 [T 1T 0. ON (um/66) 10I0vsisa mass 8 00 00 100 ‘. ~ - r :2 a 71mm” .aFF“ Figure 19. Thyrohopin releasing hormone-induced prolactin release from ovariectomized heifers implanted with eight eshadiol-filled-implants. TRH (33 ug/lOO kg body wt) given at time zero, 36 hr after estradiol heatment. 101 w202202 02—m4w4mm 2.50m....0¢>...5. 05. m>.5.44mm .2_2vu2.h 0N. Om 00 d d 35.24452. 0. 40.04m5hm OIIIIO 40:5.200 I m¢ 0» ON 0. 00- 0... 0m- 00 Om L (I‘ll/5") NILOV'IOUd WOHBS 102 heifers. Comparable averages for estradiol-treated heifers were 71, 6h, 75, 72 and 50 ng/ml. Similar to results obi tained with heifers bearing four estradiol implants, there was no difference in mean serum prolactin concentration after TRH injection between control heifers and those bearing eight estradiol implants. Serum GH also increased after TRH in- jection in these heifers (figure 20). Immediately before Tia TRH injection serum GH concentration averaged 2.7 and 0.6 ' 1 ng/ml in control and estradiol-treated heifers, respectively, (p > 0.05). Serum GH then averaged 6, 5, 6, 6 and 9 ng/ml “1‘ at h, 6, 8, 10 and 15 min respectively following TRH inject- ? ion into control heifers. Comparable averages for estradiol- treated heifers were 21, 17, 26, 23 and 17 ng/ml. The increase in serum GH concentration after TRH was 132% great- er in estradiol-treated heifers than controls and this in- crease was significant (p < 0.01). 3. '1 0 . 1 R ‘87‘ 1 V '10 .1 ReS-‘Oua 0 had 0 -l 3 an 1! 090-1Rl.s--:,_1;HM01‘ Media prolactin concentration averaged 138, 128, 1h? and 157 ng/ml following incubation of bovine pituitary cell cul- tures for 2 hr with 0, 1, 10 and 100 pg estradiol -17p/m1 TC medium 199, respectively, and difference between means was not significant (p > 0.05). In contrast, addition of 10 ng TRH/m1 medium stimulated prolactin release such that prolactin concentration in the media was 380 ng/ml which was greater (p < 0.01) than prolactin concentration for other treatment groups. Neither baseline prolactin concentration nor TRH-induced I I?" .1,“ w I, _ ._ ,9..." , ::._1 103 Figure 20. Thyrohopin releasing hormone-induced growth hormone release from ovariectomized heifers implanted with eight eshadiol-filled implants. TRH (33 ug/lOO kg body wt) given at time zero, 36 hr after eshadiol heatment. 104 020.220... 02.0404 sum. z_n.0m5.0m_>...5. 0. m>.5.44mm .58. 0.2.5. 0m. 00 JOE-zoo I 7324.55: 0. 40545.8 old 0.0 0.» On 0m 0. 0 q__-:fi-_ m.: on! m 0. v. 0. «N 0N (IWMBNOWHOH Hlmoae wnaas 105 ‘prolactin release was affected by pretreatment of cell cultures with estradiol -176. Media prolactin concentration ranged from 33-05 ng/ml during a 2 hr incubation period with TC medium 199 (table 10). Following 6 hr incubation with 0, l, 10 and 100 pg estradiol -17a/m1. prolactin concentra- tion in the media averaged 1&5, 163, 134 and 106 ng/ml, respectively, (p > 0.05). But media prolactin concentration averaged #02 ng/ml after 10 ng TRH/ml, and this was greater (p < 0.05) than all other group means. Thyrotropin-releasing hormone stimulated prolactin release from cell cultures 3 treated with estradiol -17a for 6 hr. However the mean E} difference in prolactin release from these cultures and com- parable cultures treated with TC medium 199 for 6 hr was not different (p > 0.05). The tripeptide however, did not stimulate prolactin release from cultures previously treated with TRH for 6 hr. Pretreatment of cell cultures for 12 hr with 10 ng estradiol -l7B/ml TC medium 199, also failed to affect prolactin release (table 11). Following incubation for 2 hr with TC medium 199 prolactin concentration averaged 09 and #9 ng/ml in media from cultures pretreated for 12 hr with 0 and 10 ng estradiol ~17B/ml, respectively. Comparable averages after incubation for 2 hr with 10 ng TRH/m1 medium, were 88 and 90 ng/ml and the difference between means was not significant (p > 0.05). The rise in serum concentrations of estradiol and pro- gesterone following placement of depot steroids into heifers, is in agreement with previous reports (Karsch gt al. 1973) 106 {Table 10. Effect of estradiol -l7a on basal and thyrotropin releasing hormone-induced prolactin release from bovine pituitary cell cultures. Prolactig in media 2 hr hr Treatment pretreatment treatment 2 hr TRH Estradiol + ’ng/Fi + c y (pg/m1) 0 33-2 105-17 39-h P1i 1061'11d 1 05:3 163319 08f6° 13331d J“ 10 3632 13418 #8220 L4 10738d 100 3532 11.617 012° 125115d (fig/m1) 10 3531 002300 361170 ultld aMean prolactin concentration of four flasks for the 2-hr preceding treatment. bMean prolactin concentration of four flasks for the 6-hr treatment period. cMean prolactin concentration of two flasks treated with 0 ng TRH/ml medium.after the 6-hr treatment period. Mean prolactin concentration of two flasks treated with 10 ng TRH/m1 medium after the 6-hr treatment period. d 107 Table 11. Prolactin release (ng/ml) from bovine pituitary cell cultures treated with estradiol '128 (E2) and thyrotropin releasing hormone (TRH). Prairea:mani.aadia§, Treatment TC medium 199 TC medium + Ezd Non-treated control 0912 #932 Fr} Ezd “5:2 5 0:3 ’b. TRHd 88:8 90:12 32d + TRHd 3029 9536 8‘Values are means 3 standard error. bMean prolactin concentration of four flasks for the 2-hr treatment period. cCell cultures pretreated for 12 hr with T0 medium 199 or TC medium 199 + estradiol ~17B. dConcentration = 10 ng/ml medium. 108 'which showed an increase of these hormones in serum of ovariec- tomized monkeys bearing silastic capsules containing estradiol -l7p or progesterone. A positive correlation between serum estradiol concentration and the number of estradiol-filled implants being administered was suggested by results of this experiment, as serum estradiol concentration in heifers bear- ing eight estradiol implants was twice that of heifers with four implants. Karsch gt_g;. (1973) also reported that serum estradiol concentration could be increased progressively in ovariectomized monkeys by increasing the number of estradiol -17B-filled capsules being administered. The concentration of serum estradiol following placement of four estradiol im- plants in these heifers, was approximately twice that normally found in heifers at estrus (Beal gt_g;. unpublished) when serum.was assayed by methods used in this study. However, after a single progesterone pessary was inserted, serum pro- gesterone concentration approximated concentration found in cows during the luteal phase of the estrous cycle (Kittok gt al. 1973) . The observation that estradiol and progesterone concen- trations in serum of these heifers were not different at 72 hr after beginning steroid treatment but before ovariectomy, and at four days afterovariectomy may indicate that exogenous steroids either partially or completely inhibited endogenous production of estradiol and progesterone. Therefore the con- centration of these hormones in heifers bearing implants may represent the quantity of hormone released from the implants. 109 Alternately, in the ovariectomized heifers there could be an increase in adrenal synthesis of estradiol and/0r progesterone or a change in the metabolic clearance rate of these hormones thus preventing any change in their concentration in serum. Serum estradiol concentrations reported herein for un- treated ovariectomized heifers, as well as concentrations re- ported by Short et a1. (1973) for ovariectomized cows, are “'3 high relative to estradiol concentrations in cattle during 1 the luteal phase of the estrous cycle or during the early post- partum period. Thus Wetteman et a1. (1972) reported average ‘ estradiol concentration of 3.6 pg/ml serum, in heifers during sJ the luteal phase of the estrous cycle and Echternkamp and Hansel (1973) reported 1-2 pg estradiol/ml serum, in cows during the early postpartum period and during the early luteal phase of the estrous cycle. Perhaps the high serum estradiol concentrations observed here were due to adrenal synthesis of the hormone. Similarly, the high serum concentration of estradiol (lo-l7 pg/ml) found in serum of ovariectomized heifers 36 hr following removal of depot estradiol could be due to residual estradiol or adrenal synthesis. The possibility that these high concentrations were due to assay contamination cannot be excluded, but this is improbable since serum from steers used as internal standards in all assays performed, averaged 2.8 pg estradiol/ml (n=8). Greater serum progesterone concentration in heifers treat- ed with both estradiol and progesterone relative to those treated with only progesterone may have resulted from vaginal 110 hyperemia. Estrogens are known to increase blood flow to the vagina and in these experiments the vulvas of estradiol-treated heifers were noticeably hyperemic. Increased blood flow could facilitate absorption of progesterone. Alternately the presence of estradiol might have caused a change in the metabolic clear- ance rate of progesterone. Failure of estradiol to alter baseline prolactin concentra- tion does not support previous results in cattle (Schams and Karg 1972, Schams and Reinardt 1973, Karg and Schams 1970 and Schams gt_gl. 197G) that showed increase plasma prolactin con- centration following infusion of 2-12 mg of estradiol-176 for 1-3 hr. These authors note a suppression of plasma prolactin concentration during the infusion period but an increase in plasma prolactin concentration when estradiol infusion was com- pleted. However, quantities of estradiol -17B infused would probably increase its concentration in serum to levels in excess of those reported in this study following placement of estradiol implants. Davis and Borger (1974) reported increase plasma prolactin concentration in ovariectomized ewes following a single injection of estradiol benzoate (0.5 ug/kg body wt). The increase in plasma prolactin was observed only at 8-10 pm (8-10 hr after estradiol treatment) and there were no appropriate controls. Thus, increase plasma prolactin concentration may be attributable to stimuli other than estradiol. In the present experiments, failure of estradiol at concentrations near those found during the normal estrous cycle to affect serum prolactin concentration in heifers suggest that changes in estradiol 111 concentration during the bovine estrous cycle do not in- fluence prolactin secretion. Thus reports of increased pro- lactin concentration near to or at estrus in the bovine (Raud 23.210 1971 and Swanson gt_gl. 1972) may have resulted from stimuli associated with the physical aspects of estrus (riding, butting, nervousness) rather than direct effects of estrogen on components of the prolactin control system. Increased serum prolactin concentration resulting from estrus activities could be expected since a variety of stimuli will increase serum prolactin in cows (Raud gt_gl. 1971, Tucker 1971 and Johke 1970). In contrast to results reported herein for heifers, Schams'gt_gl. (1970) observed increase plasma prolactin con- centration following infusion of 5, 10, #0 or 80 mg of pro- gesterone for 1 hr in bulls. Suppression of plasma prolactin concentration was observed during the infusion period but prolactin concentration was increased when infusion was com- pleted. However quantities of progesterone infused would probably increase its concentration in serum above quantities normally found during the luteal phase of the bovine estrous cycle or pregnancy. Unfortunately progesterone concentrations were not determined. Results reported herein however, agree with those of Nicoll and Meites (1960) that showed no change in media prolactin concentration following incubation of rat pituitary explants 13.21332, with l or 2 ug of progesterone/m1 medium. In addition, Bishop gt_gl. (1972) demonstrated no change in serum prolactin concentration following administration 112 of 1.5 mg of progesterone to rats with lesion in the hypotha- lamus. But higher doses of progesterone, (10 or 15 mg) will stimulate prolactin secretion in rats (Reece and Bivins 1902; Chen and Meites 1970). Whether progesterone per se or a metabolite, was the effective agent in stimulating prolactin secretion is not known. Failure of estradiol to affect baseline serum GH concen- tration in these heifers might have been due to the low concen- tration of estradiol obtained in serum following placement of implants. Serum GH concentration was increased in steers following daily administration of 10 mg diethylstilbestrol for 102 days (Trenkle 1970). Similar results were obtained in men (Carlson et al. 1973) with 2.5 mg diethylstilbestrol given twice daily for 5 days and in rats (Lloyd gt_g_. 1971 and 1973) given a single dose of l or 12 mg diethylstilbes- trol. In cycling dairy heifers Vines gt_gl. (unpublished) also failed to establish any relationship between serum GH concentration and days of the estrous cycle. But Koprowski and Tucker (1970a) observed a greater concentration of serum GH during the estrogenic phase of the estrous cycle of cows. Perhaps the GH control system of cows responds differently to estrogens than does that of heifers. The increase in serum prolactin concentration, the time to peak and the subsequent decline following TRH agree with previous results published from our laboratory (Convey 1973 and Convey gt_gl. 1973). Failure to observe any difference in TRH-induced prolactin release between control and estradiol- 113 treated heifers confirms a report by Vines gt_gl. (1970) which showed no difference in magnitude of prolactin release in response to TRH administered to cycling heifers on the day of estrus or on days 2, h, 7, 15 and 18 of the estrous cycle. Similar to results reported herein Tyson et a1. (1972) found no difference in TRH-induced prolactin release from women when the tripeptide was administered during the luteal or menstrual phase of the cycle. In contrast, Carlson gt_g;. (1973) and Jaffe gt_gl. (1973) demonstrated that estrogenic compounds would augment TRH-induced prolactin release in humans. The increase in TRH-induced GH release of heifers treated with estradiol is comparable to results in women showing that oral contraceptives with estrogenic activity would augment arginine-induced GH release (Vela and Yen 1969). The fact that TRH-induced GH release was signifi- cantly greater in heifers with eight estradiol implants rela- tive to their appropriate controls but not in heifers with four estradiol implants relative to their controls, suggests that the quantity of GH releasable by TRH was dependent on serum estradiol concentration. Vines et al. (1970) also failed to show any difference in magnitude of GH release in response to TRH, administered to dairy heifers on different days of the estrous cycle. Evidence that estradiol can act directly to stimulate prolactin release from.rat pituitary explants has been re- ported by others (Nicoll and Meites 1962, Ben-David gt_gl. 1960). Results presented here and those of Zolman (1973) 11” suggest that prolactin release by bovine pituitary cell cul- tures or pituitary explants in 21339 was not influenced by estradiol at concentrations used in these experiments. The response of cell cultures to TRH attests to the viability of these cells and that prolactin release could be stimulated by proper secretagogues. If one assumes that estradiol increased pituitary prolactin content but not release of the hormone, then after 6-12 hr of exposure to estradiol, intracellular prolactin content should increase in cell cultures and more prolactin should be available for release by TRH. Therefore, failure to observe any difference in TRH-induced prolactin release between estradiol-treated and control cultures, suggests that estradiol at these concentrations and for the time of exposure employed in this design did not affect the releasable source of pituitary prolactin content. These results lend credence to our earlier hypothesis that changes in estradiol concentration of a magnitude expected during the estrous cycle of the bovine do not appear to influence prolactin concentra- tion. Failure of bovine pituitary cell cultures to respond to a second challenge with TRH may have resulted from either a depletion of releaseable pituitary prolactin stores or the TRH- prolactin-releasing mechanism became refractory to TRH. In humans, Bowers g;l;_s_._l. (1971) observed a diminution in serum prolactin concentration after each of four consecutive inject- ions (3 hr intervals) with 000-800 ug TRH and Fell gt_gl. (1973) also demonstrated similar results in ewes with 20 ug TRH given at 1 hr intervals for 3 hr. SUMMARY AND CONCULSIONS Regulation of prolactin and growth hormone (GH) release in the bovine was studied by both in 2119 and in yitrg methods. Subcutaneous administration of 80 mg ergocryptine on two consecutive days to lactating Holstein cows decreased serum prolactin concentration to approximately 1 ng/ml for at least five days after beginning treatment, but neither milk yield nor serum concentrations of GH and cortisol was affected. In addition ergocryptine in doses of 0.01 to 10 ug/ml TC medium 199, decreased release of prolactin but not GH from bovine anterior pituitary cells in culture. It was concluded from these results that ergocryptine decreased serum prolactin concentration in cattle, at least in part by a direct action on the anterior pituitary. Failure to observe any change in milk yield despite marked reduction of serum prolactin concentration suggests that prolactin may not be required to maintain established lactations in cattle, or that far more prolactin is present in serum than is required for milk secretion. Prolactin release from bovine pituitary cell cultures in yitgg was consistently stimulated with thyrotropin releasing hormone (TRH) at doses of 0.01 to 100 ng/ml TC medium 199. The 115 116 effect on growth hormone release however, was equivocal, in that TRH stimulated GH release from cell cultures at 72 hr of culture but not at 96 hr. Gonadotropin releasing hormone (GnRH) at l, 10 and 100 ng/ml TC medium 199, slightly stimu- lated the release of both prolactin and luteinizing hormone (LH) from 96-hr pituitary cell cultures, but TRH had no effect on LH release from these cultures. Addition of triiodothyronine (T3) or thyroxine (T0) at 0.1 and 1.0 ug/ml TC medium 199 to cell cultures, affected neither baseline prolactin concentration nor the magnitude of prolactin release in response to TRH. However, 5 or 50 ug Tu/ml medium decreased spontaneous release of prolactin but not TRH-induced prolactin release. It was concluded from these results that: (l) TRH stimu- lates prolactin release in cattle at least in part by a direct action on the anterior pituitary: (2) The mechanism by which TRH causes prolactin release in cattle appears to be insensi- tive to inhibition by thyroid hormones and (3) Pituitary cells in culture are capable of discriminating between different releasing hormones similar to their observed effects in_yiy;, In order to investigate the effect of physiological con- centrations of estradiol -l7a and progesterone on serum pro- lactin and GH concentrations in cattle, progesterone pessaries and estradiol -l7B-fi11ed polydimethylsiloxane implants were placed into intact Holstein heifers that were subsequently ovariectomized. In addition the effect of estradiol -l7a on the magnitude of TRH-induced prolactin and GH release 1p 117 vivo and prolactin release ip vitro was investigated. Despite increase progesterone and/or estradiol concen- tration in serum of heifers bearing depot steroids there were ' no significant changes in baseline concentrations of serum prolactin and GH attributable to treatment. Thyrotropin releasing hormone administered to heifers bearing four or eight estradiol-filled implants increased serum prolactin concentration relative to pretreatment averages, but neither peak serum prolactin concentration nor area under the prolactin curves was affected by estradiol treatment. Serum GH concentration after TRH injection was 53% greater in heifers bearing four estradiol implants relative to their controls, but this increase was not signifcant. In contrast, serum GH concentration after TRH was 132% greater in heifers bearing eight estradiol implants relative to their controls and this increase was significant. Neither spontaneous release of prolactin nor TRH-induced prolactin release from pituitary cell cultures was affected by incubating cultures for 2-12 hr with TC medium 199, con: taining l, 10 and 100 pg or 10 ng/ml estradiol -l7a. Pituitary cell cultures chronically treated with TRH for 6 hr, failed to respond to a subsequent TRH challenge with increase prolactin release. It was concluded from these results that concentrations of serum estradiol and progesterone which approximate concen- trations found during the estrous cycle of cattle do not in- fluence significantly prolactin secretion. Hence, increased 118 serum prolactin concentration at or near estrus in cattle may [be due to physical stimuli associated with estrus activity, rather than a direct effect of estrogen on prolactin control mechanism. In contrast, the control of GH secretion may be associated with serum estradiol concentration. Failure of pituitary cell cultures to respond to consecutive challenges of TRH may be due to refractoriness of the TRH-prolactin re- leasing mechanism or depletion of releasable prolactin. APPENDICES 119 APPDIDIX 1 PREPARATION OF MEDIA FOR CELL CULTURE TC medium 199 10x concentration: 10.“ g/liter .TC Minimal Medium Eagle, Hanks ass“. 10.1. g/liter. Mix both solutions 50:50 v/v. Adjust ph to 7.2-7.0 with 10:: NaHCO3. Add 81111113103108 Fungizone (Amphotericin B): 5 ug/ml medium. Penicillin G: 100 units/ml medium Streptomycin sulfate: 100 ug/ml medium. Filter sterilize medium. Medium can be used up to 3 weeks after preparation. For growing cells add 10% cow serum (sterile) to the above medium = growth medium. aDIFCO Laboratories, Detroit, Michigan. bE.R. Squibb and Sons Inc., New York, N.Y. APPENDIX 2 PREPARATION 01“ TC MEDIUM 199 TC medium 199 (10x) concentration......................100 ml Sodium bicarbonate solution (2.85 NaHCoB).............. 80 ml Antibiotic solution (170 mg penicillin g/100 ml)....... 40 m1 Sterile glass double distilled H20.....................780 ml Total 1000 ml 120 BIBLIOGRAPHY 121 BIBLIOGRAPHY Adams, T.E., T.J. Rand and J.J. Reeves. 1973. Milk pro- duction in lactating rats treated with synthetic Egyrotropin releasing hormone. J. Dairy Sci. 56: Amenomori, Y., C.L. Chen and J. Meites. 1970. Serum pro- lactin levels in rats during different reproductive states. Endocrinology 86:506. Anderson, M.S., C.Y. Bowers, A.J. Kastin, D.S. Schalch, A.V. Schally, P.J. Snyder, R.D. Utiger, J.F. Wilber and A.J. Wise. 1971. Synthetic thyroprotein releas- ing hormone a potent stimulator of thyrotro in secretion in man. New Eng. J. Med. 285:1279. Ben-David, M., S. Dickstein and F.G. Sulman. 1964. 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