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Pineal Gland Function During Tite Reproductive Cycle - a Multi Species Study

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Pineal Gland Function During Tite Reproductive Cycle - a Multi Species Study PINEAL GT,AND FUNCTION DURING THE REPRODUCTIVE CYCLE - A MULTI SPECIES STUDY by David John Kennaway B.Sc. (Hons). A thesis submitted to the University of Adelaide in fulfilment of the requirements for the degree of Doctor of Philosophy. Department of Obstetrics and Gynaecology University of Adelaide February, L97A i'¡v.'í'',i q lì,*,*,, i.,! ¡:i'd "'t This thesis ís dedicated to my wife Roslyn "Men who have excessive faith in their theories or ideas are not only iII prepared for making discoveries, they also make poor observations. They can see only a confirmation of their theory. " C la ude Bernard (c ited in Kitay and Altschule, L954) PINEAL GLAND FUNCTION DURING TITE REPRODUCTIVE CYCLE - A MULTI SPECIES STUDY TABLE OF CONTENTS Page DECI,ARATION PREFACE ACKNOWLEDGEMENTS SUMMARY. I GENERAL INTRODUCTION IV CHAPTER I LITERATURE REVIEW. I CHAPTER 2 GENERAL MATERIALS AND METHODS. 27 A. r) Indole Derivatives . 27 2) Steroids 27 3) Radioactive compounds . 27 4) Solvents 2B 5) Glassware. 2B 6) Scintillation Counter and Scintillation Fluids 29 B 1) Corticoid Assay. 29 2) oes tradiol Radioimmunoassay . 30 3) Progesterone Assays. 30 c. Plasma Tryptophan Assay. 3I CHAPTER 3 _ ASSESSMENT OF PINEAL FUNCTION. 33 A. PineaI Enzyme AssaYS. 33 f) Introduction 33 2) Assay of Hydroxyindole-O-Methyltransferase 34 3) Purification of HIOMT. 35 4) Michaelis-ltenten Kinetics. 36 5) PineaI Monoamine Oxidase 37 B. Radioimmunoassay of MeIa tonin 3B I) Introduction 3B 2 ) Synthesis of AntÍgen 39 3 ) tmmunizaLíon 40 4 ) Assay Method 4T 5 ) Plasma ExtracÈion. 4L 6 ) eineal Tissue Extraction 43 43 7 ) ereparation of Columns 43 B ) Sample Application 9 ) Immunoassay Procedure. 44 IO ) Sensitivity. 44 Page CIIAPTER 3 (cont'd) rr) Accuracy. 45 L2) Quality Control 45 13) Para lIe lism 45 L4) Spec ific ity 49 r5) Collaboration in World i¡Iide C ros s- ValidationStudy.. .. 52 16) Discussion.. 54 CHAPTER 4 PINEAL FUNCTION THROUGH THE SHEEP REPRODUCTIVE CYCLE 5B A. Diurnal Changes in Plasma Melatonin I Introduction 5B 2 Animals. 5B 3 Cannulation Procedure for Automated Blood Collection 5B 4) Blood Collection Configurations. 60 5) Diurnal Rhythm of Plasma Melatonin, Cortisol and tryptophan. 6t 6) Effect of Oim Red Light. on Diurnal Rhythm of Melatonin. 62 7) Effect of Chlorpromazine on Daytime Melatonin. 65 B) Discussion 66 B PineaI Function in Cycling and Ovariectomised Ewes. 69 1) Introduction 69 2) Melatonin During the Oestrous Cycle. 70 3) Protocol for the Ovariectomy Experiments 70 4) Effects of Sampling from the Right and Left JuguIar. 70 5) Effect of Ovariectomy on Daytime Levels of Plasma Melatonin 72 6) Effect of Ovariectomy on the Diurnal Rhythm of Plasma Melatonin 72 7) Effect of Depot Medroxy Progesterone Injection on Plasma Melatonin. 73 B) Effect of Oestradiol L7 ß Injection on Plasma Melatonin 73 9) Discussion 75 c PineaI Function During Pregnancy. 79 f) Introduction . 79 2) Animals. 80 3) Diurnal Rhythm of Plasma Melatonin in Pregnant Ewes. BO 4) FoetaI Sheep Pineal Gland Function - Enzymes BI 5) FoetaI Sheep PineaI and PIasma Melatonin 82 6) FoetaI Cannulation Procedure B4 7) Foetal PIasma Melatonin During NormaI Pregnancy - Chronic Studies. B6 B) FoetaI PIasma Melatonin During Adrenocorticotrophin Induced Parturition. BB e) Maternal-Foetal and Foetal-MaternaI Transfer of Melatonin. 90 r0) Discussion. 93 Page CHAPTER 5 - EFFECT OF PINEALECTOIVNT ON CIRCUI,ATING MEI,ATONfN IN SHEEP. 99 r) Introduction. 99 2) Operative Procedure 99 3) Plasma Melatonin Following Pinealectomy in Rams and Ewes . t0r 4) Diurnal Rhythm of Plasma Melatonin Following PinealectomY.. 10L 5) Plasma Melatonin LeveIs in Pregnant Sheep After PinealectomY. L02 6) Discussion. L02 CTIAPTER 6 PINEAL FUNCTION IN MALE AND FEMALE HUMANS r07 r) Introduction. LO7 2) Subjects. r07 3) Radíoimmunoassay of Human Plasma Melaton'in. t0B 4) Diurnat Rhythm of Plasrna Melatonin in Normal Volunteers. r09 5) Diurnal Rhythm of Plasma Melatonin tn Hospitalised Patients r09 6) Diurnal Rhythm of Melatonin During Pregnancy. t09 7) Daytime Plasma Melatonin LeveIs in Post Meno- pausal women. 113 B) Discussion. 113 CHAPTER 7 REPRODUCTIVE FUNCTION IN TV'IO PATIENTS WITH PINEAL TUMOURS. LI7 r) Introduction. IL7 2) Case History I S.D. IL7 3) Case History 2 E.S. r19 4) Blood Collection and Hormone Assays T20 5) Hormone Levels in Patient S.D. l-20 6) Hormone Levels in Patient E.S. L22 7) D iscuss ion . 122 CHAPTER B PINEAL FUNCTION IN THE SCINCID LIZARD TILTQUA RUGOSA. 129 1) Introduction. l.29 2) Animals Surgical Techniques L29 3) Diurnal Rhythm in Plasma Melatonin- ' 130 4) Effect of L2 hour Phase Shifts, Constant Light and Constant Dark . 13l 5) Effect of a 6 Hour Phase Shift', Parietalectomy and Eye Shielding 13l 6) Discuss ion. L32 CHAPTER 9 PTNEAL FUNCTION TN OTHER SPECTES. t3s f) The Role of the Pineal During the Period of Blastocyst Activation in the Tammar l¡laIIabY (Macropus eugenii) . 135 P39e CIIAPTER 9 (cont'd) 2) Studies on Pineal Function in the Rat. L37 3) Studies on Melatonin in Cattle, Donkeys, Chickens, Pig and Camel. L42 4) Plasma and Pineal Melatonin in the Brush-Tailed Possum (Trichosurus vulpecula) L44 CONCLUSION L4B REFERENCES t5r APPENDTX L7L DECI.ARATION I hereby declare that to the best of my knowledge and belief, that this thesis contains no material previously published by another person, except when due reference is made in the text. Oavid J. Kennaway B.Sc, (Uons) PREFACE During the course of this study various aspects were publ ished as follows. a) Papers 1. "The occurrence of hydroxyindole-O-methyltransferase (HIOMT) activity in foetal sheep pineal tissue and its relationship to preparturient endocrine changes" D J. Kennaway and R. F. Seamark J Reprod. Fert. 45 529-53L (1975) 2 "A specific radioimmunoassay for melatonin in biological tissue and fluids and its validation by gas chromatography nìass spectrome try" D. J. Kennaway, R. c. Frith, G. Phillipou, C. D. Matthews and R. F. Seamark Endocrinology I01, LL9-L27 (Le77) 3 "On the presence of melatonin in pineal glands and plasma of foetal sheep" D. J. KennawaY, C D. Matthews, R. F. Seamark, c. Phillipou and M. Schilthuis J Steroid Biochem B 559-563 (Le7 7 ) 4 "Changtes in plasma tryptophan and melatonin content in penned sheep" D. J. Kennaway, K. J. Porter and R" F Seamark Aust. J. eiol. Sci. (1978) In Press. b) Abstracts 1. "PineaI function in foetal and neonatal lambs" D. J. Kennaway, C. D. Matthews, K. Umapathysivam and R. F. Seamark J. Reprod. Fert 36 46A-46e (Le74) 2 "Pineal changes during the period of blastocyst activation in the Tammar wallaby macropus eugenii. D. J. Kenna\^ray and R. F. Seamark J. Reprod. Fert 46 5O3-5O4 (1976) 3 "Diurnal rhythm in plasma melatonin persists into late pregnancy in humans" D" J. Kennaway, A. Lecornu, C. D. Matthews, A. Slavotinek and R. F. Seamark Proc. Endocrine. Soc. Aust. L9 28 (1976) 4 'rfncrease in melatonin content of foetal sheep pineal tissue approaching term" D. J. KennavJay and R. F. Seamark Theriogenology 6 626 (L976) 5 "Plasma melatonin: an index of pineal function?" D. J. Kennaway, C. D. Matthews and R F. Seamark Proc. Endocrine Soc Aust 20 (L977) 6 "The occurrence and synthesis of melatonin in the foetal sheep pineal gland" D. J. Kennaway and R. F. Seamark Proc. fnt. Symp. PineaI Gland, Jerusalem p. 19 (1977) 7 "Pineal function in pregnant sheep" c. D. Matthews, I. C. Kowanko, R. F. Seamark and D. J. Kennaway Aust. Nz J. obsr. L6 25o-25L (1976) . B. "Melatonin in pregnant h.umans and sheep" C. D. Matthews, D. J. Kennaway, A. LeCornu and R. F Seamark. Aust. NZ J. obst. 16 252 (1976) . 9. "Melatonin Ín foetal and maternal ovine plasma" C. D. Matthews, I. C. Kowanko, D. J. Kennaway and R. F. Seamark J. Reprod. EerL. 46 49A-499 (L976) . 10. "Pineal gland function in pregnancy" C. D. Matthews, D. J. Kennaway, M. Schilthuis and R. F. Seamark Proc. VI International Cong. Endocrinology, Hamburg, p. 7 (Le76) . II. "Plasma melatonin values Ín man and some domestic animals; initial observations on the effects of pregnancy in man and pinealectomy in sheep" c D. Matthews, D. J. Kennaway, R. G. Frith, G. Phittipou, A Le Cornu and R. F. seamark. .T Endocrinology 73 4L P - 42 P (L977) ACKNOWLEDGEMENTS My sincere thanks go to my supervisor Dr. R. F. Seamark for his encouragement and enthusiasm throughout the course of this work. I should also like to thank Dr. c. D. Matthews for his help and the many valuable discussions with him. A special thanks goes to my fellow postgraduate candidates and the staff of the endocrine laboratories who toleratediny obsession with the pineal gland. Many aspects of my work would have been impossible with- out the help and enthusiasm of my collaborators. In particular I wish to thank Mr. Ken Porter for his excellent surgery and surgical teaching and his staff for their care of the animals. To Ann Le Cornu, Brenda BeII and Meg Kaethner, I extend my appreciation for their valuable assistance with some of the melatonin and steroid assays. I am indebted to Dr. c. Phillipou and Mr. G. Frith for their help with the validation of the radio- immunoassay. For providing samples and access to their patients and animals I wish to thank Drs. J. M. Obst, E. Dunstctn, c. H. Tyndale-Biscoe, G. McCuIIoch, W. G. Breed, R. S. Tulsi, B. Firth, M. lvellby and Prof . w. v. McFarlane. r wish to thank also Mr. T. lJodaway and his staff in the QEH clinical photography department for some of the photographs in this thesis. ]- SUMMARY f) The thesis concerns a study of pineal gland function during the reproductive cycle of several species including the sheep, human and a scincid Lizard Tiliqua rugosa.
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