THE UNIVERSITY OF MANITOBA THE INTERRELATIONSHIP BETI,IEEN THYROID HORMONES AND OVARIAN STEROIDS IN RAINBOl.l TROUT, SALMO GAIRDNERI. C DANIEL GABRIEL CYR A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY DEPARTMENT OF ZOOLOGY WINNIPEG, MANITOBA DECEMBER,1987 Permission Ìras been granted L'autorisation a êté accordêe to the National Library of à la Bibliothèque r¡ationale Canada to microfil¡r this du Canada de mi crof i l-mer thesís and t,o lend or sell cette thèse et de prêter ou copies of the f,il¡n. de vendre des exemplaires du film " The author (copyright owner) L'auteur (tltuLaire du droit has reserved other d'auteur) se rêserve les pubJ-lcation rights, and autres droits de publícatÍon¡ nei ther the thes i s nor ni la thèse ni de longs extensive exÈracts f rom i.t extraits de cel-1e-ci ne may be printed or otherwíse doí vent être írnpr i mês ou reproduced wíthout hie/her autrement reproduits sana son writt,en permission. autorl.sat,lon écrÍte ø r,qEN t]-:r15-{Bt]?1-ü THE INTERRELATIONSHIP BET',{EEN TIIYROID I{OII}'ONES AND OVARIAI'I SEX STEROIDS IN RAINBOI^' TROUT, Salmo gairdneri tsY DANIEL GABRIEL CYR A thesis subnlitted to tlle. Facultl, of Gradt¡ate StL¡dies of the u'iversity of lvla'itoba in partial fulfillrnc¡rt of the require.rerrts of the desree of DOCTOR OF PHILOSOPHY o t98B Pe'urissio' has beer grarìred to trre LIBIìARy oF THE uNIVER- slrY oF i'lANITo'A ro rerrd or seil copies of- rhis rhesis. ro tire NATIONAL LtBRARy OF CANADA ro nricrofitnr rhis thesis ard ro re'd or seil copies of'rhe firm. and uNIVERsTTY N'l¡CROFlLivf S to publis| a¡r absrract of this thesis. The author reserves otrrer pubrication rights, a¡rd ¡leither rrle thesis nor extensive extracts frolu it may bc ¡rrirrted or other_ wise reproduced without tlìe author's rvritte¡l ¡rcrr:rissíon. TABLE OF CONTENTS Headi ng Page 48STR4CT......o................... o...o.......... o......... iV ACKNOWLEDGEMENTS.... o.. o o.. o..... o o........................ vi i LIST 0F TABLES.............. o................ o.... o........ Viii LIST 0F FIGURES....... o... o... o......... ...... o. xiv GLOSSARY 0F ABBREVIATIONS. o . .xxvi i CHAPTER 1: GENERAL INTR0DUCTION... o......... o o............. 1 SECTION I: EVIDENCE FOR THYROID-0VARIAN AXIS.. o... o........ 4 5 5 11 20 20 2L 22 23 24 27 27 29 30 40 SECTION II: EFFECTS OF THYROID HORMONES ON THE OVARY 49 CHAPTER 4: IPODATE INHIBITION 0F THYROID FUNCTION.. o o...... 50 IntrOduCti 0n... .. .... ... o . .. .. .. 50 Material s and Meth0ds.............................. o...... 51 ResultS...o.... ..... ......oo..... 54 Di SCuSSiOIì......................... o.......... o... Õ....... 58 CHAPTER 5: THE INFLUENCE OF THYROID STATUS ON OVARIAN ESTRADIOL SECRETI0N.......................oo......... 64 Introduction....... '........ r................... 64 Material s and Mêthods............ o.. o................... o o 65 Rgsul tS... o. o....... ........................ o... 69 Di SCUSSioIì................. o...... o....................... 79 CHAPTER 6: THYROID HORMONE INFLUENCE ON THE IN VITRO SECRETION OF ESTRADIOL BY OVARIAN FOLTTCTET........ B3 IntrOdUCtiOn.. ô. o. o. o.................................... o 83 Material s and Methods..... o o..... o o. o.... o o............... 84 ReSUI tS.............. o. o o....... o............... o......... 85 Di scussioh........ '.... .................. o........... 106 jj' 111 ABSTRACT The ínterrelationship between thyroid hormones (T4= L- thyroxine; T3= 3,5,3'-triiodo-L-thyronine) and ovarÍan Sex steroid hormones was assessed in rainbow trout. Both the effects of thyroid hormones on circu'lating'leveìs of l7B-estradiol (82) and sex steroid secretion by ovarian follicles and the effects of E2 on thyroid function were studied. Seasona'l variations in circulating levels of T3 and T4 were inversely correlated with tevels of E2 and testosterone (T) under control I ed I aboratory condi ti ons. l,lhi I e the spawni ng tíme coul d be altered by manipulating the photoperiod, the relationshÍp between serum thyroid hormoneS, E2 and T remained unchanged. In immature trout of the same age, serum E2 levels were low and serum T4 and T3 levels were higher than in mature trout. An experimentally-induced long-tem (22 days) physiologic elevation ín plasma T3 significantly increased the gonadosomatic index (GSI) of female trout. Combined treatment of T3 and salmon gonadotropin (GtH) caused the greatest increase in GSI and significantly increased plasma E2. Short-term (7 days) treatment with pharmacological doses of T3 did not alter the GSI and decreased the sensitivity of the ovary to GtH. PhysÍologic elevations of plasrna T3 for 7 days significantly increased the sensitivity of the ovary to GtH suggestÍng that T3 exerts a biphasic dose-dependant effect on the ovary. In vitro incubations of ovarian follicles (theca and granulosa) with GtH and T3 show that T3 acts upon the follicles to l_v amplify GtH-Índuced E2 secretion. T3 is more potent than T4 and the stimu'latÍon is independent of de novo proteÍn synthesÍs. Incubation of either thecal cel'ls or granulosa cells alone demonstrated that T3 amplÍfied GtH-induced T secretion by the theca and stimulated T to E2 conversion by the granulosa. T3 stÍmulation of GtH-induced E2 secretion by the follicles was blocked by theophylline, suggesting that T3 ma! inhibÍt phosphodi esterase acti vi ty. The administration of E2 to immature trout significantly decreased plasma T3 with no effects on totaì plasma T4 leveìs. This effect was largely the resuìt of a 10-fold decrease in the V¡1¿¡ of the hepatic 5'-monodeiodinase (5'D). Compartmental analysÍs of plasma T4 kinetÍcs showed a decrease in metabolic cìearance rate (MCR) and T4 degradation rate (DR) for E2-treated trout. Since plasma T4 leveis were unaltered by EZ, the decreased T4 DR indicates depressed T4 secretion by the E2-treated thyroid. This decrease in T4 DR is most reasonably explained by the decreased conversion of T4 to T3 which was supported by the depressed in vivo conversion of ¡1251110 ¡e ¡125111t ¿¡¿ 125r'. E2 had tittle influence on the T3 MCR but decreased the T3 DR and lowered bopth tissue and plasma T3 pools. To determine the effects of E2 on the properties of the plasma proteins that bind T4 and T3, immature trout were Ínjected with E2. E2 decreased the proportion of ¡125111t bound to plasma sites without signÍficant effect on [125¡110 binding. Separation of pìasma bindÍng proteins of control trout by agrose filtration on Bio-Gel A-1..5m revealed that both T3 and T4 bound to two main sites corresponding to presumed prealbumin andp-globu'lin fractions and that a sma'l'l portion of T4 aìso bound to lipoprotein. Addition of a calculated amount of unlabeìled T3 displaced [125¡11t from the 55 KDA (prealbumin) site indicating its role as the T3 high-affinity site. E2 treatment Ínitiated production of vitellogenin (VTG) which bound a very small proportion of T4 but did not bind T3. Kinetic analysis of [125111, or [125IJT4 binding conducted on miniature G-25 Sephadex columns in the presence of varying concentrations of the homologous ligand indicated that E2 increased the capacity of the ìow-affinity, high-capacity T3 binding site without effect on the high-affinity T3-binding site and without major effect on T4 binding to either its high-affinity or low-affinity sÍtes. In conclusion, a strong interrelationship exists between thyroid hormones and ovarian sex steroids in rainbow trout. The interactions between these two endocrine systems appears to regulate the partitioning of energy between somatic growth and ovarian development. T3 appears to stimulate growth and enhance early ovarian function. Once con¡nitted to vitellogenesis E2 depresses the thyroid system at several levels with presumed reduction in somatic Arowth processes. VI ACKNO}ILEDGEiIEiITS There are alurays a great many people who deserve to be thanked for their assistance and I hope not to forget any of them. I thank Dr. Geoff Eales for permitting me to pursue this project in his laboratory as well as for the guidance he offered. My thesÍs committee (Drs. E. Huebner, M. Paìmer, J. Klaverkamp, and N.R. Bromage) is thanked for their advice and their patience in listening to explanations of results duríng which I, more often than not, got caught up in ny enthusiasm and went on, and on, and or1...... DF. T. Dandy is thanked for reading the thesis and replacing Dr. E. Huebner on the fínal examination committee. Dr. N.R. Bromage There have been a number of sacrifices which were made in order for me to pursue my thesis work. I am grateful to my wife Mary for her understanding and patience and to my son David for providing much of the motivatÍon which at times appeared to be totalty drained away. I thank my parents for having taught me determination and supporting my many endeavors. I am extremely grateful to Mary Gregory for her continuous encouragement, advice, and suggestÍons which have strengthened this research project as a whole. The assistance of Ms. Shirìey Shostak in helping me with the many techniques which I have used as weìl as her boisterous laugh and sense of humor will always be remembered. I also thank Odd Bres, Bev Hemick, and Debbie MacLatchy for their assistance, stimulating conversations, and most of all for their friendship. The fínancial support from the Natural Sciences and Engineering Research Council in the form of a postgraduate scholarship is grateful ly acknowl edged. vtl LISÏ OF TABLES PAGE Table 4-1. Plasma T3 and T4 levels and hematocrit of trout injected every three days with saline or saline containing ipodate (5 mg/1009) and killed on days 1.,7 or 22. Two separate experiments (Trial I and II) are shown...oo.o...ô.................