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Neurohypophysial principles of the western brook lamprey (Lampetra richardsoni) and the Pacific hagfish… Rurak, Danny William 1971

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THE NEUROHYPOPHYSIAL PRINCIPLES OF THE WESTERN BROOK LAMPREY (Lampetra richardsoni) AND THE PACIFIC HAGFISH (Polistotrema stoutii) by DANNY WILLIAM RURAK B.Sc, University of British Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1971 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 a g r e e tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f *f The U n i v e r s i t y o f B r i t i s h Co lumb ia Vancouve r 8 , Canada Date . S, /?7/ i ABSTRACT Pharmacologic and chromatographic methods were employed to identify the neurohypophysial peptides present in, two species of cyslostomes, the western brook lamprey, Lampetra richardsoni, and the Pacific hagfish, Polistotrema  s t o u t i i . The evidence obtained indicated the presence of low amounts of 8-arginine -oxytocin or arginine vasotocin in both animals. No evidence was provided to suggest the existence of a second biologically active posterior pituitary principle in either species, and with the lamprey, i t appeared unlikely that more than one percent of the total a c t i v i t y extracted from the neurohypophysial tissues could have been due to a second peptide without being detected. The results of the study thus supported previous claims that cyclo-stomes were unique amoung vertebrates in the possession of a single active neurohypophysial principle. The evolutionary significance of this feature of lamprey and hagfish is discussed, as well as the possibility of the existence, in the pituitary of lower vertebrates, of "intermediate" neurohypophysial peptides with low biological a c t i v i t y . Pharmacologic and chromatographic investigations of midbrain and hind-brain "control" material from the western brook lamprey suggested that small amounts of arginine vasotocin may have been present in these tissues. The possible explanations for this extra-hypothalamic location of a neurohypophy-s i a l peptide are discussed, in light of similar findings in other vertebrates. Subsequent to the demonstration of the presence of arginine vasotocin in Lampetra richardsoni, the levels of the peptide were studied in animals of various ages and in larvae kept under various photoperiods. Determinations of the amounts of AVT present in the lamprey during its l i f e history were made in i i an attempt to corroborate published h i s t o l o g i c a l observations which suggested that i n Lampetra p l a n e r i a marked dep l e t i o n of the p r i n c i p l e occurred at metamorphosis. Although the i n t e r p r e t a t i o n of r e s u l t s was complicated by the presence of s u b s t a n t i a l amounts of contaminating substances i n the p i t u i t a r y e x t r a c t s , i t appeared u n l i k e l y that the l e v e l s of AVT were d r a s t i c a l l y reduced a t transformation i n L. r l c h a r d s o n l . Rather, the data indicated that the amounts of the peptide, as we l l as the q u a n t i t i e s of other a c t i v e substances i n both p i t u i t a r y and hindbrain t i s s u e s , were augmented when larvae trans-formed i n t o a d u l t s . This increase i n r a t uterus a c t i v i t y extracted from the t i s s u e s was c o r r e l a t e d with an increase i n dry weight of the b r a i n , and i t appeared l i k e l y that the two occurrences were c a u s a l l y l i n k e d . No s i g n i f i c a n t deviations were noted i n the r a t uterus a c t i v i t y extracted from groups of l a r v a l lamprey kept under d i f f e r e n t photoperiods. The v a r i a -tions that occurred amoung the neurohypophysial t i s s u e s were p a r a l l e l e d by di f f e r e n c e s amoung the c o n t r o l t i s s u e s ; t h i s suggested that photoperiod had e f f e c t s on other b i o l o g i c a l l y a c t i v e subatances i n the c e n t r a l nervous system besides AVT. i i i TABLE OF CONTENTS Page Abstract 1 List of Tables i x List of Figures x i 1 1 Definitions and Abbreviations xviii Acknowledgements xix Introduction • 1 I. Background Studies 1 A. The hypothalamo-hypophysial system of the mammals 1 B. The pharmacology and biochemistry of the mammalian neurohypophysial principles..... 3 C. Evolutionary and phylogenetic considerations 5 II. The Hypothalamo-Neurohypophysial System of Cyclostomes 6 A. The neurohypophysis of the lamprey •• 6 1. Anatomy • 6 2. Active principles in the neurohypophysis of the lamprey.. 9 B. The neurohypophysis of the hagfish 11 1. Anatomy 11 2. Active principles in the neurohypophysis of the hagfish.. 14 III. Statement of the Problem 15 General Materials and Methods 17 I. Collection of Material 17 A. Lamprey 17 1. Collection 17 2. Identification 18 iv Page 3. Dissection 19 B. Hagfish 2 0 1. Collection 20 2. Dissection.... • 20 II. Lyophilization and Storage 22 III. Extraction » 22 IV. Estimation of Biological Activities 23 A. Individual assay methods 23 1. Isolated rat uterus assay....'. 23 2. Rat vasopressor assay 26 3. Rat antidiuretic assay 27 4. Rat milk ejection assay.. 30 5. Frog bladder assay , 31 6. Natriferic assay 33 B. Calculation of potencies and statistics 35 V. Purification Methods 39 A. Partial purification of crude extracts 39 1. Ultrafiltration 39 2. Trichloroacetic acid treatment 40 B. Separation methods 41 1. Paper chromatography 41 2. Ion-exchange chromatography 42 a. Precycling and equilibration of the resin 42 b. Building of the column 43 c. Loading 43 d. Elution 44 e. pH and conductivity measurements 44 V Page VI. Chemical Methods 44 A, Lowry peptide determination. 44 B. Thioglycollate inactivation 45 Section I. Histological Observations of the Hypothalamo-Hypophysial System of Lampetra richardsoni 47 A. Introduction 47 B. Methods.......... 47 C. Results 48 1. The appearance of the hypothalamo-hypophysial system in in s i t u brains 48 2. The appearance of the hypothalamo-hypophysial system in dissected brains.. 54 D. Discussion and conclusions 54 Section II. The Isolation and Identification of the Neurohypophysial Principles of the Lamprey, Lampetra richardsoni 59 A. Studies of the ammocoete 59 1. The oxytocic a c t i v i t i e s of crude extracts and the effects of sodium thioglycollate on the ac t i v i t y . . . . . . . 59 2. Purification of ammocoete extracts by paper chromatography 63 a. Purification of crude extracts 63 i . Pooled samples B and C 63 i i . Ammocoete extracts from section III 67 b. Paper chromatography of partially purified extracts.... 74 i . Trichloroacetic acid treatment of crude extracts 74 i i . Paper chromatography of the partially purified extracts 76 i i i . Pharmacology of the oxytocic peak from the pituitary chromatogram 76 3. Purification of ammocoete neurohypophysial material by ion-exchange chromatography....... 80 v i Page a. Pituitary extract 81 i . Purification 81 i i . Pharmacological characterization of the active peak (peak II) from the column 84 b. Hindbrain extract 87 B. Studies of the adult 92 1. The oxytocic a c t i v i t i e s of crude extracts and the effects of sodium thioglycollate on the activity 92 2. Purification and pharmacology of the adult tissues dis-sected in a more precise manner (sample G) 94 a. Pharmacological characterization of the crude neuro-hypophysial extract 94 b. Partial purification of the neurohypophysial extract of sample G by u l t r a f i l t r a t i o n . . 97 c. Paper chromatography of the partially purified neuro-hypophysial extract of sample G.... 98 3. Purification and pharmacology of adult sample H 101 a. Purification and pharmacology of the pituitary extract of sample H 102 i . Partial purification with trichloroacetic acid 102 i i . Column chromatography of the partially purified pituitary extract 102 i i i . Pharmacology of the eluted peaks 104 b. Purification and pharmacology of the hindbrain extract of adult sample H 108 i . Purification 108 i i . Pharmacology of the eluted peaks 110 C. Discussion 113 Section III. Changes in the Oxytocic Activity of the Neurohypophysis of the Lampery During Development and the Variation in Photoperiod 124 A. Introduction 124 v i i Page B. The oxytocic activity of the neurohypophysis during the l i f e history of richardsoni 125 1. Methods 125 2. Results and discussion.. , 127 C. The effects of photoperiod alterations on the oxytocic act-ivity in the neurohypophysis 134 1. Methods 134 2. Results and discussion 135 Section IV. The Isolation and Identification of the Neurohypophysial Principles of the Hagfish,, Polistotrerna stoutii 141 A. The oxytocic activities of the crude extracts and the effects of sodium thioglycollate on the activity 141 B. Preliminary purification of the crude extracts v?ith t r i -chloroacetic acid 143 C. Column chromatography of the partially purified neurohy-pophysial extract... 144 D . Pharmacological characterization of the active fraction from the CMC column. 148 E. Discussion and conclusions 151 General Discussion 152 I. Evaluation of the Methods Used for Purification and Identif-ication of the Cyclostome Neurohypophysial Principles 152 A. Methods for purification 152 1 . Preliminary purification procedures 154 2. Chromatographic techniques 155 B. Methods for the identification of the neurohypophysial peptides 157 1. Inactivation with sodium thioglycollate 157 2. Chromatographic methods 159 3. Pharmacological methods 162 a. Effectiveness of the biological assays in characterizing AVT 162 v i i i Page b. Effectiveness of the biological assays in detecting a second neurohypophysial hormone in cyclostomes. 165 II. The Neurohypophysial Hormones of Cyclostomes 167 Literature Cited 172 Append icies 183 ix LIST OF TABLES Table Page I. Amino acid sequences of some known natural neurohyp-ophysial principles 4 II. Oxytocic a c t i v i t i e s of crude extracts of ammocoete neuro-hypophysial and control tissues and their resistance to thioglycollate. 60 III. Purification of sample D with trichloroacetic acid 75 IV. Pharmacological comparison of eluates from the standard and pituitary chromatograms of sample D (5 and 6 year old ammocoetes) 78 V. Purification of the pituitary extract of sample E with trichloroacetic acid 81 VI. Biological a c t i v i t i e s of the AVT peak (peak II) resulting from ion-exchange chromatography of the pituitary extract of sample E 85 VII. Purification of the hindbrain extract of sample E with trichloroacetic acid..... 88 X Table Page VIII. Biological activities of the partially purified hind-brain extract of sample E 8 8 IX. Oxytocic activities of srude extracts of adult lamprey neurohypophysial and control tissues and their resistance to thioglycollate. - 93 X. Biological activities of a crude extract of neurohypo-physial and diencephalic tissues from adult lamprey (sample G) 95 XI. Partial purification of crude extracts of adult lamprey neurohypophysial tissues by ultrafiltration (sample G) • and by treatment with trichloroacetic acid (sample H)... 99 XII. Biological activities of the AVT peak (peak c) resulting from ion-exchange chromatography of a pituitary extract from adult lamprey (sample H) 105 XIII. Biological activities of fractions resulting from ion-exchange chromatography of a partially purified hind-brain extract from adult lamprey (sample II) I l l XIV. Summary of the chromatographic and pharmacologic inves-tigations carried out on pituitary and control extracts of the lamprey, Lampetra richardsoni 114 x i Table Page XV. Amounts of oxytocic activity extracted from neurohy-pophysial and hindbrain tissues of Lampetra richardsoni at various stages of i t s l i f e history 128 XVI. Oxytocic levels in neurohypophysial and control extracts from lamprey kept under different photoperiods 136 XVII. Oxytocic a c t i v i t i e s of crude extracts of hagfish neuro-hypophysial and control tissues and their reseistance to thioglycollate 142 XVIII. Purification of the hagfish pituitary and control extracts with T.C.A 144 XIX. Biological a c t i v i t i e s of the AVT fractions (peaks A and B) resulting from ion-exchange chromatography of a pit-uitary extract of adult hagfish 149 XX. Chromatographic behaviour of synthetic AVT and the AVT-like principle from lamprey neurohypophysial tissues in the solvent system butanolracetic acid:water (4:1:5).... 161 XXI. Buffer conductivities at which AVT and the AVT-like principle from cyclostomes were eluted from CMC ion-exchange columns. 161 x i i Table Page XXII. Purification of solutions of albumin and synthetic neurohypophysial peptides using u l t r a f i l t r a t i o n 187 XXIII. Purification of solutions of albumin and synthetic neuro-hypophysial peptides using trichloroacetic acid......... 188 x i i i LIST OF FIGURES Figure Facing Page 1 The brain and pituitary of the ammocoete larva of the lamprey, Petromyzon marinus, sagittal section.. 8 2 The hypothalamus and pituitary of the hagfish, Myxine glutinosa, sagittal section 13 3 Lateral view of the brain of a lamprey, showing the regions that were dissected for pharmacological studies 21 4 Diagram of the recording apparatus used in the rat anti-diuretic assay 29 5 Diagram of the apparatus used to carry out the natriferic assay.... 34 6 Sample records from rat uterus, antidiuretic, rat milk ejection, vasopressor, na t r i f e r i c and frog bladder assays.. 36 7 Sagittal section through the head of a lamprey showing the brain and pituitary regions. Section stained with chrome-haetnatoxylin-phloxine 49 xiv Figure Facing Page 8 Sagittal section through the head of a larval lamprey showing the brain and pituitary regions. Section stained with aldehyde-fuchsin. 52 9 Sagittal section through the mesencephalon of a larval lamprey showing several large Muller c e l l s 53 10 Sagittal section through the head of a larval lamprey from which the brain has been removed 53 11 Serial horizonatal sections through the dissected pituitary component of a brain dissected from a larval lamprey • -55 12 Sagittal section through the hindbrain, control component o f a brain dissected from a larval lamprey 56 13 Sagittal section through a larval lamprey showing the mesencephalic and metencephalic regions of the brain...... 56 14 Paper chromatogarphy of crude extracts of ammocoete neuro-hypophysial and control tissues (pooled samples B and C) in butanolracetic acidzwater (4:1:5) 64 15 Paper chromatography of crude extracts of ammocoete neuro-hypophysial and control tissues (larvae from section III) in butanol:acetic acid:water (4:1:5) 68 XV Figure Facing Page 1 6 Effects of synthetic AVT and an eluate (Rf 0 . 1 - 0 . 2 ) from a paper chromatogram of crude ammocoete neurohypophysial material (larvae from section III) on urine flow and blood pressure in the ethanol-anaesthetized rat 70 17 Paper chromatography of 5-hydroxytryptamine, acetylcholine, and epinephrine in butanolracetic acidrwater ( 4 : 1 : 5 ) 73 18 Paper chromatography of pa r t i a l l y purified extracts of ammocoete neurohypophysial and control tissues (sample D) in butanol:acetic acid:water ( 4 : 1 : 5 ) 77 19 Purification of par t i a l l y purified ammocoete neurohypophy-s i a l material (sample E) by ion-exchange chromatography on CMC cellulose 83 2 0 Potencies of purified ammocoete neurohypohysial material (peak II, sample E) on several preparations when assayed against synthetic arginine vasotocin 8 6 2 1 Effects of the specific antagonists, atropine and 2-brom lysergic acid diethylamide, on the oxytocic a c t i v i t y of par t i a l l y purified ammocoete hindbrain material (sample E). 9 0 2 2 Biological a c t i v i t i e s of a crude extract of adult lamprey neurohypophysial material (sample G) 96 xv i Figure Facing Page .23 Paper chromatography of a partially purified extract of adult lamprey neurohypophysial tissue (sample G) in butanol: ace tic acid:water (4:1:5) - 100 24 Purification of a partially purified extract of adult lamprey neurohypophysial tissue (sample H) by ion-exchange chromatography on CMC cellulose 103 25 Potencies of purified neurohypophysial material (peak c) from adult lamprey (sample K) on several biological assays using synthetic arginine vasotocin as the standard 106 26 Separation of the active substances present in a partially purified extract of adult lamprey control tissue (sample H) using ion-exchange chromatography on CMC cellulose - 109 27 Growth curve of Lampetra richardsoni from the Salmon River . 126 28 Oxytocic activity and dry weight of neurohypophysial tissues from larval, metamorphosing and adult lamprey... 129 29 Oxytocic activity and dry weight of hindbrain tissues from larval, metamorphosing and adult lamprey 130 xv i i Figure Facing Page .30 Oxytocic levels in neurohypophysial and hindbrain tissues from larval lamprey kept under different photoperiods 137 31 Purification of an extract of hagfish neurohypophysial tissue by ion-exchange chromatography on CMC cellulose.. 146 32 Potencies of purified neurohypophysial material (peak B) from hagfish on several biological assays using synthetic arginine vasotocin as the standard 150 33 Ion-exchange chromatography of synthetic arginine vaso-tocin on CMC cellulose resin 190 34 Ion-exchange chromatography of albumin, synthetic oxy-tocin and synthetic arginine vasotocin on CMC cellulose resin 191 x v i i i DEFINITIONS AND ABBREVIATIONS potency of arginirte vasotocin - throughout this study, the amounts of -3 AVT are expressed in terms of milliunits ( 10 I. U.) of rat uterus activity -9 (magnesium absent) or nanograms (10 g ) of synthetic AVT equivalent. The specific a c t i v i t y of the hormone was taken to be 100 I. U./mg of 0.1 mU/ng. Ach • acetylcholine ADH » rat antidiuretic assay (see pp. 27-30). AVP a 3-phenylalanine, 8-arginine - oxytocin or arginine vasopressin. AVT « 8-arginine -oxytocin or arginine vasotocin. 2-brom LSD •» 2-brom lysergic acid diethylamide. CMC «= carboxymethyl cellulose cation-exchange resin. FB o frog bladder assay (see pp. 31-33). 5-KT • 5-hydroxytryptamine. ME m rat milk ejection or galactobolic assay (see pp. 30-31). Na = nat r i f e r i c assay or sodium transport assay (see pp. 33-35). RU(-Mg44), RU o both terms refer to the isolated rat uterus assay with magnesium absent from the bathing solution. It is also refered to as the oxy-tocic assay.(see pp. 23-25). RU(+Mg++), RUMg " both terms refer to the isolated rat uterus assay with magnesium present in the bathing solution (see pp. 25-26). T.C.A. = trichloroacetic acid. xix ACKNOWLEDGEMENTS A great many people within the Department of Zoology freely offered me help and advice during the course of this study. In particular, I wish to thank Dr. A. M. Perks for the suggestion of the research topic and for his advice and interest throughout the period of research and in the preparation of the manuscript. In addition, I am grateful to Dr. D. J. Randall, Dr. J. E. Phill i p s and Dr. H. D. Fisher for their c r i t i c a l evaluation of the manuscript. Thanks are also extended to Mrs. Elizabeth Vizsolyi for her help with some of the biological assays, and to Dr. J. E. Phil l i p s and Mr. Fred McConnell for the loan of various pieces of apparatus. The histological material used in this study was prepared by Miss Daphne Hardsj the competence and interest she showed throughout the work are greatly appreciated. Of the many persons who aided me in the collection of the lamprey used in this investigation, I would like to mention Miss L i i s Mirk and Mr. Jim Bryan as especially deserving of thanks. I am also grateful to the Master and crew of CNAV Laymore and to Dr. J. E. Mclnerney for their cooperation in obtaining hagfish. Personal financial support and the costs of the research were borne by the National Research Council of Canada, to whom I am grateful. 1 INTRODUCTION I. Background Studies A. The Hypothalalamo-Hypophysial System of the Mammals The vertebrate pituitary is a composite gland consisting of two components readily separable on embryological, morphological and functional grounds (see Ball and Baker, 1969). The anterior portion or adenohypophysis, in mammals consisting of the pars distal i s , the pars intermedia and the pars tuberalis, develops early in embryogenesis from an upgrowth of the stomadeum (Rathke's pouch). Concomittant with th i s , there is a neural downgrowth from the diencephalon which is termed the infundibulum; eventually i t forms the , neurohypophysis, which in mammals consists of the median eminence, the infundibular stalk and the pars nervosa. As the adenohypophysial and neuro-hypophysial anlages develop into the adult gland, connection with the buccal cavity is l o s t . Continuity with the ventral diencephalon (hypothalamus) is retained, however, by way of the infundibular stalk. This link has led many workers to speculate on brain-pituitary relationships (see Harris et a l , 1966), but i t is only in the last twenty years that the true nature of such relation-ships have been elucidated. Whereas i t was once thought that the adenohypo-physis operated relatively independently of central nervous regulation, and that the i n t r i n s i c endocrine activity of the pars nervosa was controlled by hypothalamic innervation; i t is now recognized that neural participation in hypophysial physiology is of a much greater magnitude, and extends to both . anterior and posterior portions of the gland. Indeed the neurohypophysis is not an endocrine gland in i t s e l f , but merely a site for storage and release 2 of hormones synthesized by neurons in the hypothalamus, and transported down thei r axons to capillaries in the pars nervosa. The neurosecretory nature of the hypothalatno-neurohypophysial system was f i r s t demonstrated clearly by Bargmann and his associates in the late 1940*s (see Sloper, 1966); subsequent investigations have added a mass of evidence to support this concept (see Scharrer and Scharrer, 1954; Bern and Khowles, 1966; Sloper, 1966). Biosyn-thesis of the neurohypophysial hormones appears to take place in the supra-optic and paraventricular nuclei (Sachs and Takabatke, 1964; Sachs, 1967). It i s thought that the synthesis is ribosomal ( i . e . involves messenger and transfer R.N.A.).and results i n i t i a l l y in an inactive precursor molecule. .Precursor molecules are then combined, perhaps by the Golgi apparatus, to form stainable neurosecretory granules about 1000 A in diameter. These granules are transported down the neurosecretory c e l l axons to the pars nervosa, where the hormones are stored and eventually released. Hypothalamic control of adenohypophysial function appears to be effected by neurons whose axons terminate on or near capillaries in the median eminence. These capillary beds are drained by portal vessels which end d i s t a l l y i n sinusoids in the pars d i s t a l i s . Many lines of evidence support the hypothesis that hormones (releasing or inhibiting factors) released from the axons into the portal system modify the int r i n s i c endocrine m c t i v i t y of the adenohypophysis (Harris et a l , 1966). Thus, both anterior and posterior portions of the pituitary receive products which are neurosecretory i n nature. Neurohormones reach the adenohypophysis through the hypophysial portal system; once there, they act locally to regulate the synthesis or release of anterior lobe hormones. The neurohypophysis receives i t s products by direct innervation, and i t serves only as a storage site for these hormones, which are released into the systemic circulation. B. The Pharmacology and Biochemistry of the Mammalian Neurohypophysial  Principles While the nature of brain-pituitary relationships has come to light only in the last two decades, the existence of biologically active substances in the neurohypophysis has been known since the turn of the century. During th i s period the major pharmacological actions (oxytocic, milk ejecting, vasopressor, avian depressor, and antidiuretic) of mammalian neurohypophysial extracts were demonstrated and shown not to be due to other active substances such as epinephrine or acetylcholine (Kamm et al_, 1928; Perks, 1969). It was not until 1928, however, that Kamm and his co-workers clearly demonstrated the presence of two active principles, one causing uterine contractions and the other active in raising blood pressure. This line of research culminated in the elucidation of the structures of the two compounds by du Vigneaud and hi s co-workers (see du Vigneaud, 1956). Synthesis of compounds with biological properties identical to those of the natural hormones provided conclusive proof for the proposed structures. Both principles are cyclic octapeptides consisting of a five membered ring attached to a linear tripeptide, as shown i n Table I. The ring is joined by a disulphide bridge between the cysteine residues at positions 1 and 6. The two hormones di f f e r in the amino acids at positions 3 and 8. One peptide, oxytocin, contains an isoleucyl residue at position 3 find a leucyl at position 8; the other, arginine vasopressin, possesses phenylalanine and arginine i n the same positions. The presence of arginine at position 8 in vasopressin has prompted the use of the term "basic neurohypophysial hormone** to denote i t and other analogues with basic amino acids in this position (e.g. lysine vasopressin, arginine vasotocin). Oxytocin and oxytocin-like peptides, on the other hand, are termed "neutral" since they have a neutral residue at position 8. Oxytocin has strong oxytocic* Table I. Amino acid sequences of some known natural neurohypophysial principles.* 1. ARGININE VASOTOCIN C^s-Tyr-Ileu-Gln-Asn-Cys-Pro-Arg-Gly-NH« 1 2 3 4 5 6 7 8 9 2. ARGININE VASOPRESSIN Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NHg 3. OXYTOCIN Cys-Tyr-Ileu-Gln-Asn-Cys-Pro-Leu-Gly-NHg 4. ISOTOCIN ((4-serine, 8-isoleucine] -oxytocin) Cys-Tyr-lieu-Ser-Asn-Cy*s-Pro-Ileu-Gly-NHg 5. GLUMTTOCIN (^-serine, 8-glutamine]-oxytocin) Cys-Tyr-Ileu-Ser-Asn-Cys-Pro-Gln-Gly-NHg * Amino acids differing from those in the corresponding positions in the arginine vasotocin molecule are underlined. galactobolic and avian depressor a c t i v i t i e s . In mammals i t appears to function in the ejection of milk in the lactating female, and perhaps to aid in the expulsion of the foetus at birth (Gorbman and Bern, 1962). No functions have yet been found in the male. Arginine vasopressin accounts for the vasopressor and antidiuretic a c t i v i t i e s of mammalian neurohypophysial extracts. However, only the latter action appears to have any physiological role, since large doses of the hormone are needed to cause a significant rise in blood pressure (Gorbman and Bern, 1962). C. Evolutionary and Phylogenetic Considerations Since a l l vertebrates possess a pituitary gland, ideas on the earliest evolutionary origins of the hypophysis are highly speculative. However, since there are variations in the morphology and physiology of the gland, many workers have investigated the lower vertebrates in an attempt to gain an insight into the evolution of the hypothalamo-hypophysial system. Studies have been made on the phyletic variations in the gland i t s e l f and in the hormones i t produces. Included in the latter line of research are investi-gations on the distribution of neurohypophysial hormones throughoutl the vertebrate series, and successful identification of the peptides has been possible because of the sensitive biological assays which detect and characterize the principles by their pharmacology. The small size of the molecules makes structure elucidation relatively easy when compared to the formidable task of identifying the large protein hormones of the adenohypo-physis, although this asset is largely offset by the small amounts of material present. Studies have been remarkably successful to date, with the identification of at least nine natural neurohypophysial analogues. Evidence that the biosynthesis of the posterior lobe hormones occurs ribosomally and thus is under direct genetic control (see Sachs, 1967) has prompted various workers to propose evolutionary schemes for the peptides based on point mutations i n the genetic code (Sawyer, 1961a, 1965b; Acher, 1963; F o l l e t t and Heller, 1964; Vliegenthart and Versteeg, 1967; Pickering and Heller, 1969). However, each proposal has eventually been made obsolete by the identification of the principles of further species. It is l i k e l y that this trend w i l l continue u n t i l a much larger group of vertebrates are examined. II. The Kypothalamo-Keurohypophysial System of Cyclostomes The importance of cyclostomes in any consideration of hypophysial evolution is clear. As the only l i v i n g representatives of the Agnatha, lamprey and hagfish must be placed at the bottom of any phyletic sequence for neurohypophysial hormones. Opinions appear to be divided as to whether the cyclostomes are a monophyletic or diphyletic group (see Heintz, 1963), and since the hypothalamo-hypophysial systems of the two forms are di s t i n c t l y different, they w i l l be considered separately. A. The Neurohypophysis of the Lamprey 1. Anatomy The embryological development of the lamprey pituitary differs somewhat from that seen in other vertebrates (Leach, 1951; van der Kamer and Schreurs, 1959; Gorbman and Bern, 1962; Wingstrand, 1966). As in other species, the adenohypophysial analage arises from ectoderm under the forebrain. But in the lampreys, i t becomes associated with the olfactory placode to form a so l i d nasopharyngeal stalk, extending caudally and ventrally from the nasopharyngeal p i t to about the level of the developing infundibulum. The posterior portion of the stalk develops into the adenohypophysis, while the anterior region remains during embryonic and larval l i f e as an undifferen-tiated cord of c e l l s connecting the pituitary and the nasopharyngeal p i t . At metamorphosis, a lumen appears in the cord and grows under and behind the pituitary as a sac, the nasopharyngeal pouch. In spite of i t s unique embryological development the lamprey pituitary i s similar to that of higher vertebrates, although much more primitive; this can be seen in figure 1. The neurohypophysis is represented merely by the thickened floor of the third ventricle, there being no infundibular stalk. The adenohypophysis is a flattened structure situated midventrally beneath the diencephalon. The anterior portion or pars d i s t a l i s is embedded in connective tissue which separates i t from both the posterior region, or pars intermedia, and the neurohypophysis. The pars intermedia is closely apposed to the posterior portion of the neurohypophysis. During larval development, the pars d i s t a l i s becomes divided by connective tissue septa in rostral and proximal portions, and each of these in turn become divided into several lobules. The pars intermedia, on the other hand, remains undivided ( van der Kamer and Schreurs, 1959). Although a number of workers have studied the adenohypophysial cytology of Petromyzoniformes, l i t t l e is known about the functions of the different c e l l typed (see Ball and Baker, 1969). The evidence suggests, however, that the pars d i s t a l i s elaborates a gonadotrophic hormone (Ball and Baker, 1969), and i t has been shown that the pars inter-media secretes a melanophore stimulating hormone (Young, 1935; Lanzing, 1954; Larsen, 1968). The hypothalamo-neurohypophysial tract of the lamprey has i t s origins i n neurons located in the preoptic nuclei, which are considered to be analo-gous to both the supraoptic and paraventricular nuclei of tetrapods (Scharrer and Scharrer, 1954). The nuclei are situated on either side of the third ventricle; their neurons extend in an arc beginning just anterior to the optic chiasma and swing dorsally and caudally. The axons of these c e l l s 8 Figure 1 The brain and pituitary of the ammocoete larva of the lamprey, Petromyzon marlnus, sagittal section,(after Oztan and Gorbman, 1960). (1) third ventricle; (2) subcommissural organ; (3) choroid plexus; (4) habenula; (5) pineal body; (6) preoptic recess; (7) naso-pharyngeal stalk; (8) blood vessel; (9) optic chiasma; (10) adeno-hypophysis (rostral zone); (11) preoptic neurosecretory axons ending close to the adenohypophysis; (12) adenohypophysis (proximal zone); (13) preoptico-hypophysial tract (ventral division); (14) preoptico-hypophysial tract (lateral division); (15) pars intermedia; (16) pars nervosa; (17) infundibular cavity; (18) axons from posterior hypothal-amic nucleus; (19) posterior hypothalamic nucleus (neurosecretory); and (20) preoptic axons connecting to the midbrain. CO extend in several directions (Oztan and Gorbman, I960). The majority extend from the ventral part of the nuclei, then travel ventrally and posteriorly along the floor of the diencephalon to terminate in the posterior regions of the neurohypophysis or pars nervosa. A second group of axons follows the same i n i t i a l course as the above-mentioned fibres, but seems to end in the connective tissue between the rostral pars d i s t a l i s and the brain. The pars nervosa is innervated by a second set of axons, which originate in the posterolateral portions of the preoptic nuclei. These fibres pass f i r s t l a t e r a l l y , then in a postero-ventral direction and approach the neurohypo-physis l a t e r a l l y . Oztan and Gorbman (I960) have described one f i n a l group of preoptic neurons, the axons of which travel posteriorly from the dorsal and caudal aspects of the nuclei through the mesencephalon into the hindbrain. The terminations of these fibres have not yet been located. The preoptic nuclei supply the bulk of the innervation to the neurohypo-physis, and they have been noted in a l l studies of the lamprey hypothalamo-neurohypophysial system (see Green, 1951; van der Kamer and Schreurs, 1959; J^rgenson and Larsen, 1966; Wingstrand, 1966; Ferks, 1969). Oztan and Gorbman (1960), however, have described another group of neurosecretory-like c e l l s which send axons to the pars nervosa in the larvae of Petromyzon  marinus and Lampetra lamottei. The paired nuclei l i e just dorsal to the posterior recess and are perhaps analogous to the nucleus l a t e r a l i s tuberis of teleosts. Some of their axons are directed anteriorly to terminate in the pars nervosa, but the majority end near blood vessels close to the ventral surface of the posterior diencephalon-anterior mesencephalic junction-(see figure 1). 2. Active Principles i n the Neurohypophysis of the Lamprey Research into the nature of lamprey neurohypophysial hormones has been 10 rel a t i v e l y scarce, and as yet the principle(s) has not been chemically identified. Early work by Herring (1913) demonstrated that pituitary extracts from Petromyzon f l u v i a t i l u s (= Lampetra f l u v i a t i l u s ?) had a weak antidiuretic effect in cats. It was not until 40 years later, however, that Herring's results were extended by Lanzing (1954). He found that forebrain extracts (including the pituitary) of Lampetra f l u v i a t i l u s possessed oxytocic and frog water-balance a c t i v i t i e s . But the results were only qualitative and offered no real evidence as to the identity of the active principle(s). Moreover, although the oxytocic activity was shown not to be due to histamine or acetylcholine, i t was not destroyed by incubation with 0.1 N NaOH, a .treatment that inactivates neurohypophysial peptides. Therefore, i t appears that not a l l of the acti v i t y was due to a posterior pituitary principle. A more thorough study was carried out on the marine lamprey, Petromyzon marinus, by Sawyer and his associates (Sawyer, 1955, 1965a; Sawyer et a l , 1959, I960, 1961). Extracts of ventral forebrain (including the neurohypophysis) possessed vasopressor, antidiuretic, oxytocic, galactobolic, hen oviduct and frog bladder stimulating a c t i v i t i e s . The potencies of the extract on these assays were consistent with those of [8-arginine]-oxytocin or arginine vasotocin. This peptide possesses the ring of oxytocin and the " t a i l " of arginine vasopressin (see Table I".) and has been found in a l l vertebrates so far examined, except for mammals. Recent work by Vizsolyi and Perks (1969) has provided evidence that i t also exists in foetal mammals. Purification of the lamprey extract by gel f i l t r a t i o n (Sephadex G-25) and ion-exchange chromatography (carboxymethyl cellulose) resulted in one active fraction, and this had the pharmacological properties of arginine vasotocin (AVT). No ac t i v i t y was found in the fractions expected to contain a neutral oxytocin-l i k e principle. On the basis of this evidence, Sawyer concluded that the lamprey neurohypophysis contained only one active principle, arginine vasotocin. However, biological assays of the crude extracts may not have detected small amounts of another hormone, and this small amount could have broken down during the prolonged period of storage which occurred prior to chromatography in Sawyer's experiments. Sawyer's hypothesis of a single principle in the lamprey was also reached by Follett and Heller (1964) in a study of Lampetra f l u v i a t i l u s . Crude extracts of fresh pituitary tissue contained oxytocic and vasopressor a c t i v i t i e s in a ratio similar to that found by Sawyer for Petromyzon. A high na t r i f e r i c acvitity relative to • ... oxytocic potency was also detected, and i t was shown that this could be completely abolished by incubation with sodium thioglycollate. Paper .chromatography using the solvent system butanol:acetic acid:water (4:1:5) yielded biological activity between Rf's 0.25 and 0.35. An eluate of this region possessed vasopressor, oxytocic and antidiuretic a c t i v i t i e s in a ratio similar to that obtained for arginine vasotocin. To detect possible small amounts of ac t i v i t y in other regions of the chromatogram, Follett and Heller assayed the eluates for guinea pig milk ejecting activity, since this assay was highly sensitive to oxytocin. The pressor-oxytocic peak contained the bulk of this a c t i v i t y , but traces were found up to Rf 0.8. In several chrom-atograms a small peak of activity in the region Rf*s 0.5 to 0.6 was noted; this region also contained rat antidiuretic a c t i v i t y . However, Follett and Heller dismissed this activity as an artifact resulting from t a i l i n g of the major AVT peak. Thus evidence to date suggests that the hypothalamo-neuro-hypophysial system of the lampreys elaborates only one active principle, arginine vasotocin. B. The Neurohypophysis of the Hagfish 1) Anatomy The embrylogical development of the hagfish pituitary appears to be similar to that of the lamprey, with the adenohypophysis being formed from the epithelium of the nasopharyngeal duct (Fernholm, 1969). However, most authorites consider the hagfish pituitary to be more primitive than that of the Petromyzoniformes (Matty, 1960; Gorbman, 1965; Ball and Baker, 1969; Perks, 1969). This conclusion is based mainly on the appearance of the adenohypophysis, which consists of f o l l i c l e s and clusters of c e l l s embedded in connective tissue beneath the neurohypophysis, as illustrated in figure 2. No clear division into pars d i s t a l i s and pars intermedia exists. The adeno-hypophysis is separated from the brain by a layer of connective tissue along i t s entire length, except in larger (older?) specimens, where the caudal region is in intimate contact with the infundibulum (Matty, 1960; Adam, 1963; Fernholm and Olsson, 1970). Although several different c e l l types have been described in the hagfish adenohypophysis (Olsson, 1959; Olsson et a l , 1965; Fernholm and Olsson, 1970), l i t t l e is known of their functions or of their relationships to the adenohypophysial c e l l types of higher vertebrates (Adam, 1963; Ball and Baker, 1969; Fernholm and Olsson, 1970). The hypothalamo-neurohypophysial system of the Myxiniformes shows some marked variations from that of the lamprey. Again the neurohypophysis is represented by the thickened floor of the diencephalon, but the posterior region is eveaginated to form a hollow sac, the infundibular process or neural lobe. The sac is directed caudally and is flattened so that i t possesses dorsal and ventral surfaces (Olsson, 1959; Perks, 1969). Most of the neuro-secretory axons reaching the neural lobe terminate in the dorsal area, close to a surface network of blood vessels; this region thus seems to be analogous to the pars nervosa of more advanced pituitaries (Gorbman et a l , 1963). The neurosecretory fibres take their origins from neurons in the dorsal regions of a pair of ill-defined preoptic nuclei (Olsson, 1959; Adam, 1963). Fine 13 Figure 2 The hypothalamus and pituitary of the hagfish, Myxine glutinosa, sagittal section (after Olsson, 1959). (1) optic tract (poorly developed); (2) preoptic nucleus, pars parvocellularis or anterior region; (3) preoptic nucleus, pars magnocellularis; (4) primordium hippocampi; (5) postoptic nucleus; (6) postoptic commissure; (7) possible median eminence, with a few neurosecretory endings (Herring bodies); (8) postoptic recess; (9) areas of remnants of buried ependymal c e l l s ; (10) third ventricle; (11) infundibular process; (12) adenohypophysis; and (13) preoptico-hypophysial tract. neurosecretory granules can be identified in the perikaryon of these c e l l s with the performic acid-Astra blue technique, a method which is more sensitive than the classic chromium-haematoxylin-phioxin stain of Gomori (Olsson, 1959). The neurosecretory neurons are unipolar and do not appear to send processes to the third ventricle. Their axons pass ventrally and posteriorally over a wide area of the diencephalic walls and do not converge to form bundles u n t i l they have reached the floor of the hypothalamus. Just behind the optic chiasma some of the fibres pass near to an elaborate vascular network located in this region and accumulations of neurosecretory material ("Herring bodies") are seen. The majority of the axons continue into the infundibular process and •terminate near the dorsal surface. L i t t l e or no neurosecretory material is found in the ventral region closest to the adenohypophysis (Olsson, 1959; Adam, 1963). Matty (I960) reports some stainable substance in the infundibular canal; this finding has also been noted for Myxine embryos (Fernholm, 1969). Olsson (1959) describes neurosecretory accumulations in the well vascularized hypothalamic floor just anterior to the infundibular process, and suggests that this area is comparable to the median eminence of tetrapods. However, Gorbman et a l (1963) disputed this suggestion, claiming that the area in question is nothing more than the anterior portion of the pars nervosa. 2. Active Principles in the Neurohypophysis of the Hagfish Virtually nothing is known about the neurohypophysial principle(s) in hagfish, aside from a few scattered reports in the literature. Herring (1913) reported the presence of a vasopressor principle in pituitary extracts of Myxine glutinosa; this was followed almost 50 years later by Adam's demon-stration (1961) of a frog water-balance a c t i v i t y in the same species. Fol l e t t and Heller (1964) found oxytocic and natriferic effects in extracts of Myxine, but they were not able to detect any vasopressor activity. Although each 15 pituitary appeared to contain about 0,5 mU of oxytocic a c t i v i t y , no more than one quarter of this proved labile to treatment with sodium thioglycollate. F o l l e t t and Heller concluded that the hagfish pituitary contains no more than about 0.13 mU of oxytocic a c t i v i t y , a disappointingly low amount. The a c t i v i t i e s detected to date are consistent with the presence of arginine vasotocin, but there is no quantitative evidence that this is indeed so. There is no evidence for or against the presence of a second hormone. III. Statement of the Problem The identity of the neurohypophysial principles of cyclostomes is .extremely important i n any considerations of neurohypophysial hormone evolution. Present evidence suggests that the lamprey elaborates only one active agent, arginine vasotocin, and i t has been assumed, rather than proved that the hagfish are similar in this respect. Therefore, cyclostomes would appear to be unique i n the possession of only one posterior pituitary principle, since a l l other vertebrates have at least two. If this view is correct, then AVT appears to be a strong candidate for the ancestor of a l l neurohypophysial hormones, and the presence of two agents in higher vertebrates may have resulted from a gene duplication. But the number and identity of cyclostome neurohypophysial peptides is far from settled. So l i t t l e is known about the pharmacology of the hagfish neural lobe that even the suggestion that AVT is present seems speculative. Certainly nothing can be said about the presence or absence of other active peptides. Although lampreys have been looked at in greater detail, the possibility that they elaborate another hormone in addition to AVT cannot be completely discounted. As mentioned e a r l i e r , the methods employed by previous workers may have f a i l e d to detect small amounts of a second peptide. Moreover, a l l studies to date have been 16 carried out on spawning or prespawning adults, and i t is possible that at other stages of i t s l i f e the lamprey may have greater amounts of hormone. This p o s s i b i l i t y is supported by the 1959 observation of van der Kamer and Schreurs that the amount of neurosecretory material in the pituitary of Lampetra planeri is highest in ammocoetes, just prior to metamorphosis. The adult stage of the lamprey's l i f e represents no more than one quarter of i t s total existence (Fletcher, 1963), the remainder of which is spent as a burrowing, f i l t e r feeding ammocoete larva. It may be possible to regard the ammocoete as the most primitive l i v i n g vertebrate, and therefore the identity of i t s neurohypophysial principles seems of considerable importance in any consideration of the evolution of posterior pituitary hormones (Sawyer, 1966). It was the purpose of the work described here to use sensitive chromato-graphic and pharmacological methods to characterize the neurohypophysial peptides in representatives of both groups of cyclostomes; f i r s t l y , in larvae and adults of the western brook lamprey, Lampetra richardsoni (Vladykov and F o l l e t t ) , and, secondly, in adults of the Pacific hagfish, Polistotrema  st o u t i i (Lockington). Neither species has been previously examined. Attempts were also made to determine the amounts of neurohypophysial material present in Lampetra during i t s l i f e , and to see i f the amount was altered by changes in the external environment. 17 GENERAL MATERIALS AND METHODS I. Collection of Material * A. Lamprey 1. Collection Adult and larval western brook lamprey, Lampetra richardsoni (formerly Lampetra planeri, see Vladykov and Fo l l e t t , 1965) were caught in the following streams in the lower Fraser Valley : the Salmon River, Bertrand Creek and Marshall Creek. Most adult lamprey were taken in the spring and early summer of 1968 and 1969, during spawning. At these times the animals were engaged in nest building and mating, and could be located easily in the shallow water at the head of r i f f l e s . As communal spawning occurs in Lj_ richardsoni (Fletcher, 1963), as many as a dozen animals could be present in one nest. Capture was effected by setting a pole seine net ( 4 x 6 feet) 10 to 20 feet downstream from the nests, and then driving the lampreys into i t from an upstream direction; forcing the animals to move downstream was accomplished by disturbing the stream bottom (1968 season) or by use of an electric shocker (1969 season). Ammocoetes from the Salmon River and Bertrand Creek were captured in a manner similar to that used for adults. The net was set downstream from the mudbank in which the larvae were burrowed. The mud was then disturbed by walking through i t , causing many ammocoetes to move downstream into the net. This method resulted in the capture of larvae of a l l sizes. In Marshall Creek, the creek width and current were not great enough to allow use of the seine net. Instead the elec t r i c shocker was u t i l i z e d . The electrode was pushed into - i 18 the mud, and as the ammocoetes emerged they were scooped up with a long handled dip net. Only large specimens were caught by this procedure. Captured lamprey were transported back to the laboratory in a styrofoam ice chest f i l l e d with stream water. Until dissected they were kept unfed under natural photoperiod in 15 gallon aquaria f i l l e d with f i l t e r e d , aerated, dechlorinated fresh water. Cooling coils kept the water temperature more or o less constant at about 15 C. Most animals were k i l l e d and dissected within a week after, capture, although some were kept for as long as a month. 2. Identification As the anadromous, parasitic Pacific lamprey, Entosphenous tridentatus - (Gairdner) and Lampetra richardsoni are both present in most streams in the Fraser Valley (Vladykov and F o l l e t t , 1965; Carl et a l , 1967), i t was necessary to c l a s s i f y and separate the two species. In adults this was accomplished on the basis of tooth development, the non-parasitic Lampetra having blunt, degenerate teeth in comparison to the well-formed type found in Entosphenous (Pletcher, 1963; Carl et a l , 1967). Ammocoetes were identified by myotome counts. Two parallel cuts were made along the l e f t side of the body from the last g i l l opening to just posterior to the vent. The skin was pulled away with forceps and the myotomes between the last g i l l pouch and the anterior edge of the vent were counted. Individuals with counts between 57 and 66 were identified as Lampetra, while those with myotome numbers between 68 and 74 were c l a s s i f i e d as Entosphenous. The identification of ammocoetes with -overlapping myotome counts (i.e. between 66 and 68) was accomplished by noting the pattern of melanophore distribution on the head and t a i l (Pletcher, 1963; Vladykov and F o l l e t t , 1965). Lampetra larvae captured in the Salmon River were divided into age classes using a growth curve formulated for this stream by Pletcher (1963) (see Section III of Results). 19 3. Dissection The lamprey was decapitated at about the level of the third g i l l c l e f t and the head was pinned ventral side down to a small piece of styrofoam board under a dissecting microscope. The dorsal surface of the brain and spinal cord was exposed by removing the overlying muscle and cutting through the roof of the braincase. Ey gently l i f t i n g the olfactory lobes, the olfactory nerves were exposed and cut. The forebrain was then l i f t e d further dorsally and posteriorly to uncover the optic nerves which were severed. The pituitary gland was now v i s i b l e and was l i f t e d gently from its depression in the floor of the chondocranium. This operation exposed the oculomotor nerves, which were cut. Next, attention was turned to the caudal regions of the brain. The cut end of the spinal cord was l i f t e d dorsally, while at the same time the spinal nerves were cut. This operation was continued rostrally u n t i l a l l the spinal and cranial nerves were severed, and the brain was freed from the \ chondocranium. It was then placed ventral surface up on the styrofoam block. Since there is no infundibular process in the Petromyzoniformes, the neuro-hypophysis is represented merely by the thickened floor of the diencephalon (van der Kamer and Schreurs, 1959; Cztan and Gorbman, 1960; Perks, 1969). Thus i t was not possible to be as precise in dissecting out the pituitary as i t is in higher vertebrates where there i s a definite pituitary stalk and lobe. In most cases the brain was transected at the diencephalic-mesencephalic border, caudal to the pituitary. The cerebral lobes were removed, and the remaining tissue was kept as the neurohypophysial component of the brain. The posterior portion of the brain (midbrain, hindbrain and a small amount of spinal cord) was retained as control tissue. In one group of adult lamprey (those caught in 1968), a more precise dissection was carried out. A wedge of tissue was removed from the ventral forebrain; i t s base was the floor of the diencephalon from the o p t i c chiasma to the po s t e r i o r edge of the p i t u i t a r y , ahd i t s apex extended i n an antero-dorsal d i r e c t i o n into the telencephalon, t o include the preoptic n u c l e i . Spinal cord was used as a c o n t r o l t i s s u e . The regions of the b r a i n taken i n each of the di s s e c t i o n s are i l l u s t r a t e d i n fi g u r e 3. Throughout t h i s study, the f o r e b r a i n component of the b r a i n , i n c l u d i n g the neurohypophysis, was denoted by the terms "neurohypophysial" and " p i t u i t a r y " (e.g. neurohypophysial e x t r a c t ) , although i t was r e a l i z e d that much o f the diencephalon and telencephalon were included with the neurohypophysis i n these d i s s e c t i o n s . The midbrain, hindbrain and sp i n a l cord ti s s u e components were r e f e r r e d to as hindbrain or con t r o l m a t e r i a l . A l l dissected material was immediately placed i n small polythene v i a l s (Beem o capsules, F i s h e r S c i e n t i f i c ) , and then frozen and stored i n dry ice (-78.5 C) i m t i l l y o p h i l i z e d . B. Hagfish 1. C o l l e c t i o n Hagfish, Polistotrema s t o u t i i (Lockington), were captured i n Mayne Bay, Barkley Sound from CNAV Laymore, i n October, 1968. Traps c o n s i s t i n g of f i v e g a l l o n p a i l s with holes punched i n the sides were baited with f i s h e n t r a i l s and set on the bottom at about 20 fathoms depth. A few hours l a t e r the traps were hauled up and the h a g f i s h removed. They were kept i n an open salt-water tank u n t i l dissected. 2. D i s s e c t i o n Hagfish brains were dissected i n a manner s i m i l a r to that described f o r the lamprey. The animal was decapitated and the b r a i n exposed d o r s a l l y . S t a r t i n g f i r s t from the r o s t r a l end, and then from a p o s t e r i o r d i r e c t i o n , the b r a i n was freed from the chondocranium and placed v e n t r a l surface up. The f o r e b r a i n minus the o l f a c t o r y bulbs and the dorsal portions of the 21 Figure 3 Lateral view of the brain of a lamprey, shewing the regions that were dissected for pharmacological studies. Heavy stippling delimits the pituitary component of the brain; vertical hatching indicates the control component; and Roman numerals refer to the appropriate cranial nerves. A General dissection that was carried out for a l l ammocoete samples, and for one adult group (sample H). B A more precise dissection that was performed on one group of adult lamprey (sample G). J / A 22 telencephalic hemispheres was taken as the neurohypophysial tissue; throughout the study, i t was referred to as neurohypophysial or pituitary material. Control or hindbrain tissue consisted i f midbrain, hindbrain and a small amount of spinal cord. Samples were placed in Beem capsules and stored in . dry ice un t i l freeze-dried. II. Lyophilization and Storage Lyophilization was performed using a high-vacuum pump (Welch Scienti f i c Co.) and stainless steel trap ( V i r t i s Co.) f i l l e d with a dry ice-methanol mixture. The vi a l s containing the dissected tissues were placed in a round-bottomed flask which was connected to the trap, and a vacuum of approximately 0.01 mm Hg was maintained for 12 to 18 hours. After this time, the capsules o were removed and stored in vacuo over phosphorous pentoxide at 4 C in glass dessicators (Jensen's Co.) unti l extracted. III. Extraction Extraction of homogenized, lyophilized tissue was carried out as described in the Brit i s h Pharmacopoea. Tissue was homogenized in 0.25% acetic acid at a concentration of 20 mg/ml using a Thomas tissue extractor (Thomas Co., Philadelphia). When the tissue was completely disrupted and dispersed, the extract solution was transferred to a tapered glass centrifuge tube (5 or 15 ml), which was then stoppered lig h t l y with cotton wool and placed in a boil i n g water bath for three minutes. The tube was removed from the bath, cooled i n cold water, and then centrifuged at 1470 g's for 30 minutes in a c l i n i c a l centrifuge (International Equipment Co., Mass.). After centrifuga-o t i o n , the supernatant was removed and stored In a screw-cap v i a l at 4 C; the precipitate was discarded. Occassionally the precipitate was reextracted in 23 one quarter of the i n i t i a l volume of acetic acid. However, as this procedure yielded only about five percent of the biological a c t i v i t y present in the f i r s t extract, i t was not carried out as a routine method. IV. Estimation of Biological Activities A. Individual Assay Methods 1. Isolated Rat Uterus Assay This bioassay was routinely performed for the detection and estimation of biological a c t i v i t y in crude and purified tissue extracts. It is based on the procedure described by Holton (1948) and modified by Munsick (I960). Female Wistar rats weighing about 220 g in f u l l oestrus (determined by vaginal smear) were used. The animal was stunned by a blow on the head and a ventral incision was made in throat region to sever the spinal cord. Longitud-inal and lateral incisions were made in the abdomen and the uterus was exposed. Beginning at the ovaries, both horns were freed gently from fat and connective tissue. The vagina was transected, and the freed uterus was placed in a Petri dish containing the modified van Dyke-Hasting's solution (Munsick, I960) described below. The uterine horns were separated by a longitudinal cut through the vagina, and one horn was kept for later use or discarded. A s i l k thread was passed through the t i p of the ovary of the other horn and tied securely. The vaginal end was tied to a fine glass hook drawn from 9 mm glass tubing. The hook, and i t s attached uterus, were placed in a five ml muscle bath containing van Dyke-Hasting*s solution. A supply of 95% 02~57, C02 was connected to the muscle hook, and the gas mixture was allowed to bubble through the bath. The thread tied to the ovary was attached to one end of a balanced isotonic muscle lever (C.F. Palmer and Sons, London) which lig h t l y pressed upon a smoked kymograph drum. 24 The muscle bath was connected through a glass c o i l to a two l i t e r Erlenmeyer flask containing van Dyke-Hastings solution. The whole apparatus was immersed in a five gallon aquarium f i l l e d with fresh water kept at 32°C by a Bromwell Thermonix pump. Injections were given into the muscle bath every five minutes with a microliter syringe (Hamilton Co., Whittier, C a l i f . ) . When the uterus had ceased contracting in response to an injection, the muscle bath was flushed through from below with bathing solution from the resevoir flask. Extracts of unknown potency were assayed against standardized neuro-hypophysial peptides, either synthetic oxytocin (Syntocinon, Sandoz Pharma-ceuticals) or synthetic arginine vasotocin (Sandoz), supplied through the kindness of Dr. B. Berde. Assays were carried out by the 4-point method (Holton, 1948). Injections were given in groups of four, consisting of a high and low dose of standard, and a high and low dose of unknown, in random order. The ratio of high dose to low was the same for both standard and unknown. Four to s i x of these groups normally comprised a complete group. Often, when chromatographic eluates were being assayed, potency estimations were based on a single 4-point group. Less frequently, a response to a dose of unknown was bracketed by two standard doses.to give an estimate of biological a c t i v i t y . The modified van Dyke-Hastings solution used as the bathing solution in this assay was developed by Munsick (I960). Two stock solutions were used to make the fi n a l bath solution : Stock Solution A NaCl 120.67 g combined and made up to NaHC03 46.62 g 18 1 with d i s t i l l e d water. KC1 8.275 g phenolsulphonephthalein 0.054 g 25 Stock Solution B Na^HP04 22.714 g made up to one 1 with d i s t i l l e d water. NaHgPO^  :H20 6.349 g made up to one 1 with d i s t i l l e d water. Approximately equal volumes of the above solutions were combined to obtain a buffered solution of pH 7.4. The f i n a l mixture in the resevoir contained : • glucose 1.0 g CaCl 2 1.0 ml of a 1.0 M solution Stock solution B 20 ml These were combined and made up to 2 1 with stock solution A. When a fresh batch of bathing solution was prepared, 957. 02-5% C02 was bubbled through i t unti l the phenol red changed from red to orange in color. This indicated that the pH of the solution had dropped from about 8.0 to 7.4. Throughout the assay the gas mixture was bubbled through the resevoir flask to keep the solution oxygenated and to maintain a correct pH (7.4). The presence of magnesium' in the bathing solution increases the sensit-i v i t y of the isolated rat uterus to neurohypophysial peptides (Munsick, I960). This property was used both to increase the sensitivity of the uterus when assaying weakly active chromatographic eluates, and as the basis of a separate bioassay for the pharmacological characterization of purified extracts. The same stock solutions were used as for the van Dyke-Hastings solution described above. The f i n a l mixture consisted of : glucose 1.0 g CaCl 2 1.0 ml of a 1.0 M solution . 2 6 MgCl2 1.0 ml of a 1.0 M solution Stock Solution B 20 ml These were combined and made up to 2 1 with stock solution A. 2. Rat Vasopressor Assay Vasopressor a c t i v i t i e s of extracts were assayed on male Wistar rats of about 300 g weight using a procedure adopted from Dekanski (1952). The rat was anesthetized with 175 mg/100 g body weight of urethane (Matheson, Coleman and Bell Co.) injected subcutaneously. Phenoxybenzamine hydrochloride (Dibenzyline, Smith, French and Cline Co.), at 0.5 mg/100 g body weight, was • injected along with the urethane. This e<-adrenergic blocking agent lowered the rat's blood pressure and abolished the pressor effects of hypertensive amines such as epinephrine, 5-hydroxytryptamine and nicotine (Dekanski, 1952; Goodman and Gilman, 1965). After 20 to 40 minutes, the anesthetized rat was placed with i t s ventral surface upwards on a styrofoam operating platform. A median longitudinal incision was made in the skin of the throat region and the right external jugular vein was exposed by blunt dissection. The vessel was cleared of connective tissue over about 1 cm of i t s length, and two s i l k ligatures were placed around i t . The distal ligature was tied off. Using a 22 gauge hypodermic needle (Becton, Dickinson and Co.), the vein was punctured between the two ligatures and a piece of PE 10 polythene tubing (Clay-Adams Co.), with a bevelled t i p , was inserted. The tubing was pushed into the vessel towards the heart for a short distance and then securely tied with the two ligatures. The dis t a l end of the catheter was flared and connected to a 22 gauge needle attached to a one ml glass tuberculin syringe (Becton, Dickinson and Co.); the whole system was f i l l e d with a 0.9% NaCl solution which contained heparin 27 (Sigma Chemicals), 0.5 mg/100 ml, as an anticoagulant. When the jugular cannulation was completed, the right carotid artery was exposed and separated from the right vagus nerve and any adhering connec-tive tissue. It was cannulated with PE 50 tubing in a manner similar to that described for the jugular vein, except that the artery was clamped proximal to both ligatures u n t i l the catheterization was completed. Blood pressure was recorded from the carotid cannula with a Statham P23AA strain gauge transducer coupled to a Beckman Dynograph recorder. The transducer dome and cannula were f i l l e d with heparinized saline. Upon completion of surgery, heparin, at 0.5 mg /100 g body weight (in 0.9% saline) was injected intravenously to prevent c l o t formation during the assay. The 4-point assay method described earlier was employed, with doses spaced at ten minute intervals. Injections were given v i a the jugular vein, and they were washed in with 0.2 ml of 0.9% NaCl. Unknown solutions were assayed against Pitressin (Parke-Davis Co.), a mixture of purified natural lysine and arginine vasopressin, or against synthetic arginine vasotocin. The respone measured was the increase in systolic carotid pressure caused by the injected substance. 3. Rat Antidiuretic Assay The assay employed to estimate antidiuretic activity was modified from procedures described by Dicker (1953) and Sawyer (1961*>). Male Wistar rats weighing about 250 g were given an oral dose of 12% ethanol (5 ml/100 g body weight) using a number 8 urethral catheter (Rusch, W. Germany) as a stomach tube. After 30 to 40 minutes, the animal was usually ready for surgery. The skin on the throat was cut and the right external jugular vein was exposed and cannulated as described for the vasopressor assay. In a similar manner, the trachea was catheterized with PE 240 tubing. Next, a small hole was cut in the abdominal wall about two cm anterior to the penis. The bladder was •- . • "28 located with forceps and gently pulled through the opening. A small incision was made in an avascular region of the bladder, and a length of PE 200 tubing with a flared end was inserted and tied in place with s i l k ligatures. To insure against any loss of urine, a ligature was placed around the penis and securely tied. The rat was then loaded to 108 percent of it s body weight with a hydrating solution containing 1.57. ethanol and 0.5% NaCl, by stomach tube. This level of water load was maintained throughout the assay by replacing the f l u i d lost as urine by an equal volume of hydrating solution. When urine flow reached a constant, high level, the assay was begun. Again, the 4-point assay method was employed. The standards used were Pitressin and arginine vasotocin. Injections were given via the jugular cannula and washed in with 0.2 ml of saline. A response normally lasted 15 to 30 minutes; the next injection was administered only when urine flow had returned to a baseline level. Antidiuretic responses were monitored in two ways, as shown in figure 4. The bladder was connected to a drop counter (C.F. Palmer) which was coupled to : 1) a motorized writing lever and smoked kymograph drum (both from C.F. Palmer), and 2) an electric timer (Precision S c i e n t i f i c Co.) and an impulse counter (modified from a Beckman fraction collector control unit). The writing lever and timer were in operation only when a drop of urine was between the two electrodes of the drop counter. Decreased urine flow (i.e. an antidiuresis) produced high excursions of the writing lever and large counts on the timer. Conversely, diuresis resulted in low lever deflections and smaller time counts. When the drop f e l l from between the electrodes, the writing arm resumed i t s resting position and the timer stopped, but the time remained registered on the recorder. Therefore, the timer recorded the cumulative length of time during which successive drops were in contact with the electrodes. The total number of drops was 29 Figure 4 Diagram of the recording apparatus used in the rat antidi r e t i c assay. < ,9; WRITING LEVER DROP COUNTER SWITCH RELAY INE CORD TO BLADDER ELECTRODES ( 30 registered by the impulse counter, and the record on the kymograph gave the time course of the response, and showed in particular the.time of onset and completion of the antidiuresis. The magnitude of the response was calculated by dividing the total time registered during the antidiuresis by the total number of drops; this quotient was in turn divided by a similar number obtained from the baseline preceding the injection. The resulting value represented, i n arbitrary units, the degree of reduction of urine flow as a factor of the resting rate. • > 4. Rat Milk Ejection Assay The assay of galactobolic a c t i v i t y was accomplished by the method of Bisset et a l (1967). Lactating female rats (250 to 350 g) of the Wistar strain were used from 8 to 16 days after parturition. The number of young varied from 8 to 12. It was found that better preparations resulted i f the rat was removed from i t s l i t t e r the night before the assay was performed. The animal was anesthetized with either urethane (175 mg/100 g body weight) given subcutaneously, or by sodium pentabarbital (Nembutal, Abbott Chemical Co., 4.5 mg/100 g body weight) injected intraperitoneally. The jugular vein was cannulated as described above and normally used as the injection route. An increased senstivity could be achieved by giving doses v i a the saphenous branch of the femoral artery (Bisset et a l , 1967). This method was t r i e d i n i t i a l l y , but only small volumes (less than 50 ^1) could be injected, and the preparation was found to react readily to buffers. Hence, i t was unsuitable for the assaying of buffered chromatogram eluates of weak ac t i v i t y , and was not used routinely. Milk ejection pressure was recorded from the two most dist a l pairs of teats (lower and upper inguinal). The teat was grasped with forceps, a l i t t l e xylocaine ( Astra Pharmaceuticals, Mississauga, Ont.) applied, and the t i p 31 of the teat excised. A piece of PE 60 tubing was inserted about 5 mm into the primary duct of the gland and tied securely in place with a s i l k ligature around the teat. The catheter was clamped in a position to maintain the natural alignment of the teat and to apply a slight tension. A Statham P23BB strain gauge transducer was connected to the distal end of the cannula, and the whole system was f i l l e d with a 3.87. solution of sodium citrate to prevent c l o t t i n g of the milk. Recordings were made with a Beckman Dynograph recorder. When the cannulation was completed, a small amount of citrate solution (about 0.2 ml) was injected into the gland through the transducer to clear milk from the t i p of the cannula. The assay was carried out by the 4-point • method. Injections were given every five minutes and were washed in with 0.1 ml of 0.97. saline. Synthetic oxytocin and synthetic arginine vasotocin were used as standards. If the sensitivity of the preparation f e l l during the course of the assay, 0.2 ml of citrate solution was injected into the gland to increase the intramammary pressure and restore the sensitivity. 5. Frog Bladder Assay The assay of neurohypophysial hormones by their effect on water transport across the isolated urinary bladder of the bullfrog, Rana cates-beina, was carried out according to the method of Sawyer (1960g). Large bullfrogs (Carolina Biological Supply) were l i g h t l y anesthetized in ether and double pithed. The abdomen was opened by longitudinal and lateral incisions, and the bladder was carefully removed. The freed bladder was placed in a Petri dish containing the Ringer's solution described below and divided into i t s two lobes. Next, each hemibladder was pulled over the flared end of an open glass tube and securely tied with s i l k thread. The tube and bladder were f i l l e d with about five ml of d i s t i l l e d water and suspended in a 50 ml round-bottomed glass tube containing 25 ml of Ringer's solution. Air was bubbled through the outer bath by means of a glass tube fused to the base of the round-bottomed vessel. The rate of water loss from the bladder was determined throughout the assay by weighing the glass tube and it s attached bladder every 15 minutes on a heavily damped balance. The bladder was ligh t l y blotted prior to weighing. When the rate of water loss reached a steady, low value (about 2-3 mg/min.), an injection of standard or unknown was given to the outer bath. After 45 minutes (three weighings) the response was considered to be over, and the Ringer's solution in the outer bath was replaced with fresh solution. The bladder was then allowed 45 minutes to recover, after which time the rate of water loss usually had returned to a baseline level. The response was calculated by averaging the weight losses over the last two 15 minute periods (30 and 45 minutes) of the response; from this value was subtracted a similar value obtained for the baseline preceding the response. Four-point assays were normally carried out. One group (four injections) was performed on each bladder; four bladders comprised a complete assay. The standards used were synthetic oxytocin and AVT. The Ringer's solution used in the assay was prepared from two stock solutions : Stock Solution A NaCl 94.0 g combined and made up to one 1 KC1 3.7 g with d i s t i l l e d water. CaCl :H 0 5.3 g Stock Solution B NaH2P04 :2H20 2.0 g NaHC03 40.0 g with d i s t i l l e d water. combined and made up to one 1 glucose 4.0 g 33 phenolsulphonephthalein 0.012 g To make the f i n a l Ringer's solution 50 ml of A and 50 ml of B vere mixed and made up to one 1 with d i s t i l l e d water. 6. Natriferic Assay Neurohypophysial principles, especially AVT, promote the active transport of sodium by the isolated skin of anuran amphibians (Morel et a l , 1958). This feature was used as the basis of a biological assay following the method of Sawyer (1960§ and employing apparatus described by Packer (1967). Large bullfrogs (Rana catesbeiana) were used for the assay. The spinal cord was cut in the neck region and the animal double pithed. Then a section of skin three to four cm across was removed from the abdomen and placed in aerated Ringer's solution. The apparatus used to hold the skin during the assay is shown in figure 5. The frog skin chamber consisted of two identical plexiglass blocks, which were clamped together to form the complete apparatus. Into the apposing surface of each block was d r i l l e d a 5/8 inch hole ten degrees off the horizontal. Three ver t i c a l holes (3/8 inch) were d r i l l e d into each block to connect with the horizontal chamber; these served for the introduction of electrodes and an a i r supply, and for the addition of reagents. The frog skin was placed between the two blocks so as to separate the two chambers. The apparatus was then tightly clamped in a vise and both sides were f i l l e d quickly with Ringer's solution. To measure the potential d i f f e r -ence across the skin, two calomel electrodes (type K401, Radiometer, Copen-hagen) were placed i n the pair of holes closest to the skin and connected to a Radiometer pH meter (type PHM22r) set on the m i l l i v o l t mode. The potential was set to zero by introducing an equal voltage of opposite polarity across the skin with s i l v e r - s i l v e r chloride electrodes inserted in the pair of holes most distal to the skin. The current (short-circuit current, sodium current) 34 Figure 5 Diagram of the apparatus used to carry out the natriferic assay (effect of neurohypophysial hormones on the rate of sodium transport across the isolated skin of the bullfrog, Rana catesbel-ana). AMMETER VOLTMETER CALOMEL ELECTRODE PLEXIGLASS CHAMBER OUTLET PORT FROG SKIN 35 necessary to cause this voltage drop across the membrane was measured with a P h i l l i p s type PM2400 volt-amp-ohmeter. The current and hence the opposing voltage could be altered by means of a variable resistance to compensate for changes in the membrane voltage. Injections of standards and extracts were added to the solution bathing the serosal side of the skin. PE 10 tubing, inserted through the middle hole of each block, and connected to an a i r supply, kept the solutions mixed and aerated. After an injection was given, the potential across the frog skin was readjusted to zero every five minutes and the short-circuit current recorded. A response was considered to be over when the current ceased to r i s e , at : which time the Ringer 1 s solution from both sides of the chamber was removed through the exit ports and replaced with fresh solution. The next injection was given only when the current had returned to a baseline level. A response and the return to baseline normally lasted about 90 minutes. Four-point assays were carried out, the standard being AVT • The Ringer*s solution used had the following composition (from Hoar, 1967) : NaCt 6.50 g KC1 0.14 g combined and made up to one 1 CaCl 2 0.12 g with d i s t i l l e d water. NaHC03 0.20 g B. Calculation of Potencies and Statistics Figure 6 gives representative responses from each of the biological assays described above. Potencies of the unknown solutions assayed were calculated in the following manner (Holton, 1948) : a c t i v i t y of unknown (mU/ml) = (R)(c)  volume of d where : R •» antilog M 36 Figure 6 Sample records from rat uterus, antidiuretic, rat milk ejection, vasopressor, natriferic and frog bladder assays. Rat uterus assay a - 0.006 ml of purified ammocoete neurohypophysial extract. b - 0.006 ml of 1:500 synthetic arginine vasotocin (AVT, 10 I. U./ml). c - 0.008 ml of 1:500 synthetic AVT. d - 0.008 ml of purified ammocoete neurohypophysial extract. Rat antidiuretic assay - arrows indicate time of injections. a • 0.020 ml of 1:10 purified ammocoete neurohypophysial , b - 0.015 ml of 1:5000 synthetic AVT. c - 0.030 ml of 1:5000 synthetic AVT. d - 0.040 ml of 1:10 purified ammocoete neurohypophysial extract. Rat milk ejection assay - arrows indicate time of injections. a • 0.006 ml of purified ammocoete neurohypophysial extract, b - 0.015 ml of 1:2000 synthetic AVT. c - 0.020 ml of 1:2000 synthetic AVT. d » 0.008 ml of purified ammocoete neurohypophysial extract. Rat vasopressor assay - arrows indicate time of injections. a • 0.10 ml of purified ammocoete neurohypophysial extract, b - 0.10 ml of 1:500 synthetic AVT. c - 0.20 ml of 1:500 synthetic AVT. d - 0.20 ml of purified ammocoete neurohypophysial extract. Natriferic assay - large arrows indicate the time of injections. - small arrows indicate the time of replacement of the bathing solution. - uA » short c i r c u i t current in microamperes. a - 0.025 ml of purified ammocoete neurohypophysial extract. b - 0.025 ml of 1:500 synthetic AVT. c " 0.050 ml of 1:500 synthetic AVT. d •» 0.050 ml of purified ammocoete neurohypophysial extract. - large arrows indicate the time of injections. - small arrows indicate the time of replacement of the bathing solution. - uL - water loss from bladder in milligrams/15 minute period, a - 0.015 ml of purified ammocoete neurohypophysial extract. b - 0.0125 ml of 1:500 synthetic AVT. c - 0.025 ml of 1:500 synthetic AVT. d - 0.030 ml of purified ammocoete neurohypophysial extract. extract. Frog bladder assay RAT UTERUS ASSAY ANTIDIURETIC ASSAY NATRIFERIC ASSAY FROG BLADDER ASSAY 160r i 2 HR. I HR. 1 37 and M - ((A+D) - (B+C)] (log c-log b) (C+D) - (A+B) the symbols denote the following : A « sum of the responses to low dose of unknown. B •» sum of the responses to low dose of standard. C «= sum of the responses to high dose of standard. D " sum of the responses to high dose of unknown. b = low dose of standard. c «=> high dose of standard. d = high dose of unknown. 957. f i d u c i a l limits were calculated by the analysis of variance method of Holton (1948). Pharmacological characterization of purified cyclostome extracts was accomplished by a method developed from Gaddum*s (1955) ratio of discrimina-tion. The extract was assayed against standard arginine vasotocin on several different preparations, the hypothesis being that extract and standard were identical ( i . e . both were AVT). If this hypothesis was correct, then the : potencies of the extract obtained from the various bioassays would always be equal, and the ratio of one potency to another would always equal 1. In calculating these a c t i v i t y ratios, the potency of the extract on the rat uterus assay (magnesium absent) was divided into a l l other potencies to obtain a series of ratios, e.g. vasopressor to rat uterus, VP/RU. If extract and standard were indeed identical, then a l l : the ratios would not be significantly different from 1; i f they were different, one or more ratios might d i f f e r from unity. Clearly, the success of this method depends on the choice of bioassays and on the pharmacological properties of the unknown and standard. It is necessary to use assay methods with different s e n s i t i v i t i e s for the various neurohypophysial analogues. While the technique can never conclusively -- . 38 prove the identity of an unknown principle, i t can discriminate between peptides with different biological properties, for example, oxytocin and AVT. Occasionally, with cyclostome extracts of low a c t i v i t y , a less compre-hensive pharmacological characterization than the one described above was carried out. The procedure employed was the "classical method" of character-ization (see Sawyer, 1968), in which the unknown was assayed against mammalian principles (oxytocin and vasopressin) on several assays. The a c t i v i t y ratios . obtained were then compared with published values for AVT and other analogues; a similarity between the ratios of the unknown and those of a particular analogue (usually AVT) was taken as evidence that the active agent in the extract was identical to the analogue. 95% fiducial limits for the potency ratio (a/b) were calculated by the following formula : confidence limits •» + (t)y§n? "+ 3m^  where : Sm0 = standard error of M (see p. 37) in the calculation of potency a. Sn^ » standard error of M in the calculation of potency b. t «=» Student's t for F degrees of freedom, 2 2_2 with F - RSm0) + (Smb) 3 Sn£ + Smf . r o ——b Ra Rb and ; Ra «• degrees of freedom for the error of the potency estimation a. Rb • «• degrees of freedom for the error of the potency estimation b. 39 V. Purification Methods A. Partial Purification of Crude Extracts As a preliminary step to separation of cyclostome active principles, crude extracts were partially purified by one of two procedures : 1) u l t r a f i l t r a t i o n . 2) treatment with trichloroacetic acid. 1. U l t r a f i l t r a t i o n U l t r a f i l t r a t i o n is a process in which the rate of transport of small molecules is preferentially enhanced by inducing a solvent flow through a membrane (Morris and Morris, 1964). Liquid flow is usually produced by applying a pressure differential across the membrane. In the method employed here, a positive pressure was applied to the solution side of the membrane by means of a syringe. As the purpose of the preliminary purification was to separate the neurohypophysial peptide(s) from contaminants of larger molecular weight, i t necessary to use u l t r a f i l t r a t i o n membranes with a pore size large enough to allow the passage of neurohypophysial material of low molecular weight (about 1000), but small enough to exclude the large components present in the crude extracts. Two such membranes were employed j Diaflo UM-10 with a molecular weight cut-off range of 10,000, and Diaflo UM-2 with a cut-off of 1,000 (both from Amicon Corp., Mass.). The solution to be u l t r a f i l t e r e d was taken up in a one ml u l t r a f i l t r a -tion syringe (Amicon Corp., Mass.), which was attached to a metal holder containing the membrane. The syringe and holder were then clamped in a ver t i c a l position and the screw plunger was slowly turned to build up pressure within the syringe. The u l t r a f i l t r a t e was collected as i t issued from the exit port of the membrane holder. When no solution remained in the syringe, 40 the holder was detached and disassembled. The small amount of liquid retained in the holder (about 0.1 ml) was taken up in a Hamilton syringe and mixed with a small amount (about .3 to .5 ml) of 0.257. acetic acid. This solution was treated as described above and the two f i l t r a t e s were combined. 2. Trichloroacetic Acid Treatment The general protein precipitating agent, trichloroacetic acid (T.C.A.) was used to remove large molecular weight protein components from crude extracts. The method followed was based on procedures described by Roy (1969) and Warberg and Thorn (1969). T.C.A. (50% w/v solution) was added to the solution to give a f i n a l concentration of 10% T.C.A. The solution was then o allowed to stand at 4 C for 30 minutes to allow for precipitation of protein, after which time i t was centrifuged at 1470 g*s for one half hour. The supernatant was transferred to a 60 ml separatory funnel and extracted three times with five volumes of diethyl ether to remove T.C.A. After each extrac-tion, the upper ether layer was discarded and the pH of the lower aqueous phase was checked with pHydrion indicator strips (Micro Essential Laboratory, N.Y.). Three washings with ether raised the pH from less than 1 to 3.5 to 4.0. o The extract was then placed in a water bath at 37 C and bubbled with nitrogen to remove any traces of ether. During i n i t i a l experiments with this method, the centrifuged precipitate resulting from the T.C.A. treatment was resuspended in a small amount of 0.25% acetic acid. The procedure described above was then repeated on this residual solution. However, only small amounts of ac t i v i t y (about 5% of the total) were recovered by this process, and in later purifications i t was not carried out. 41 B. Separation Methods 1. Paper Chromatography Descending paper chromatography was carried out as described by Heller and Pickering (1961) and Perks (1966). Butanol, acetic acid and water in a volume ratio of 4:1:5 were placed in a separatory funnel and shaken for 15 minutes. The shaking was repeated for five minutes every half hour for 90 minutes. At the end of this time, the emulsion was allowed to separate into two layers. The lower phase was run into four Petri dishes in the bottom of a 12" x 12" x 24" glass chromatography tank. The l i d of the tank was sealed with Lubriseal (Thomas Co., Philadelphia), and the system was equilibrated at room temperature for one hour. Solutions to be chromatographed were spotted on a 24 cm x 54 cm sheet of Whatman 3MM chromatography paper. Three solutions (usally two unknowns and a mixture of synthetic oxytocin and synthetic AVT) were applied to each sheet using Hamilton microliter syringes. Origins were 2.5 cm wide, 2.5 cm apart and located 5.2 cm from the antisiphon rod. To speed the drying of the spots, a portable hair dryer was used to blow a stream of cool a i r over the paper. When spotting was completed, the sheet was placed in the chromatography tank and suspended from the solvent trough. The l i d was replaced, and the tank was le f t for one hour to re-equilibrate. The upper phase of the solvent mixture was then added to the solvent trough through holes in the tank l i d ; the holes were stoppered and the chromatogram was l e f t to develop for 12 to 14 hours at room temperature. At the end of this time, the chromatogram was removed and the solvent front marked. Solvent remaining on the sheet was removed by drying in a stream of cool a i r . Three longitudinal strips were cut from the chromatogram, one beneath each origin. Each strip was 0.5 cm wider than the origin on each side, to include any lateral diffusion. The strips 42 were cut into ten equal pieces between the origin and the solvent front, each piece corresponding to 0.1 Rf units. The resulting squares were folded and placed in separate, numbered, five ml beakers containing one ml of 0.257. acetic acid. At the end of one to two hours,each square was removed and and the eluate was squeezed back into the appropriate beaker using a five ml polythene syringe. The beaker was then sealed with Parafilm (American Can Co.) o and stored at 4 C until the eluate was assayed for biological a c t i v i t y . 2. Ion-Exchange Chromatography Column chromatography with carboxymethyl cellulose resin was carried out using procedures described by Sawyer and van Dyke (1963) and Wilson (1968). a. Precycling and Equilibration of the Resin Precycling was performed according to the manufacturer's instructions. The dry exchanger (usually about 10 g) was added to 15 volumes of 0.5 N NaOH; the mixture was stirred with a magnetic s t i r r e r for one half hour and f i l t e r e d on a Buchner funnel. The resin was then stirred into 150 ml of d i s t i l l e d water and l e f t for five minutes, after which time the solution was fi l t e r e d and the pH of the f i l t r a t e checked. This process was repeated until the pH of the f i l t r a t e f e l l to approximately 8. The exchanger was then added to 15 volumes of 0.5 N HCl, stirred for 30 minutes, f i l t e r e d and washed with d i s t i l l e d water un t i l the pH of the washings rose above 5. This acid treatment was repeated once. Equilibration of the resin with the appropriate buffer (sodium acetate or ammonium acetate) was carried out at 0.2 M concentration and pH 5. The exchanger was added to 200 ml of buffer, stirred and f i l t e r e d . This process was repeated until the conductivity and pH of successive f i l t r a t e s were constant and equal to the buffer pH and conductivity. In a similar manner, partial equilibration with the starting buffer was accomplished by s t i r r i n g the resin into 200 ml of starting buffer (0.02 M)» leaving for 5 minutes and 43 f i l t e r i n g . This process was repeated once, b. Building of the Column The equilibrated resin was packed into a glass burette (15 or 25 ml) using an LKB pe r i s t a l t i c chromatography pump (LKB Produckter-AB, Stockholm). A small wad of glass wool was placed in the bottom of the burette to prevent clogging of the stopcock with resin beads. Starting buffer was added to a depth of two to three cm and the stopcock was closed. Then a relatively thick slurry of the resin was pumped into the column and the stopcock was opened. To speed the flow of buffer through the column, and the settling of the exchanger, pressure was increased inside the burette by passing a i r through the pump. When the column was built to the desired height, the pump was connected to a resevoir of starting buffer and the flow rate adjusted to 15 ml/hour. Starting buffer was passed through the.column overnight to complete equilibration of the resin. C. Loading The solution to be chromatographed was adjusted to the proper pH (5) with NaOH or NH4CH and diluted to the conductivity of the starting buffer with d i s t i l l e d water. Following this procedure, the column was drained of buffer u n t i l the meniscus reached the surface of the gel. The extract was carefully added with a 500 ul Hamilton syringe so as not to disturb the surface of the gel. The extract was then allowed to flow into the resin, and to speed up this process, a i r pressure inside the burette was increased by using the pe r i s t a l t i c pump. The walls of the burette were then washed with a small amount of starting buffer, which in turn was passed into the column bed. At no time was the liqu i d level in the column permitted to go below the surface of the resin. The column was then f i l l e d with starting buffer. A pe r i s t a l t i c pump was connected to the inflow at the top of the column, and the flow rate was i 44 adjusted to 12 to 15 ml/hour. I n i t i a l l y , a volume of starting buffer equal to three to four void volumes (10 to 15 ml) was passed through the column, to elute any substances not bound to the exchange groups, and then the gradient was started. d. Elution Development of the column was accomplished by increasing the concentra-tion of the buffer from 0.02 M to 0.20 M. No increase in buffer pH was employed. An exponential gradient was used to reduce t r a i l i n g of the eluted peaks. The mixing chamber consisted of a 100 ml beaker stirred with a magnetic s t i r r e r , and i n i t i a l l y f i l l e d with 0.02 M buffer. To this was connected a .150 ml Erlenmeyer flask f i l l e d with 0.20 M buffer. e. pH and Conductivity Measurements At a l l stages of column chromatography, the pH was checked with a Radiometer type PHM22R pH meter, and the conductivity was measured with a Radiometer type CDM 2d conductivity meter. VI. Chemical Methods A. Lowry Peptide Determination Total peptide concentration in crude and purified extracts was measured by the modified Folin phenol method of Lowry et a l (1953). The reagents used were as follows : A 2% Na 2C0 3 in 0.1 N NaOH. B 0.57. CuS04:5H20 in 17. potassium tartrate. C alkaline copper solution «= 50 ml of reagent A + 1 ml of reagent B. D - 1 N Folin-Ciocolteau phenol reagent. - 45 Fractionated bovine serum albumin (Armour and Armour Co.) was used as the standard. A series of standard solutions : 25, 50, 100, 200, 300, 400 and 500 ug/ml was prepared by dilution of a 0.5 mg/ml stock solution. To measure peptide concentration, 0.2 ml of the unknowns and standards were pipetted into a series of five ml test tubes. One ml of reagent C was added to each tube and the tubes were shaken and allowed to stand for 10 to 15 minutes. Then, 0.1 ml of reagent D was added and mixed in immediately with a Vortex-genie agitator (Fisher S c i e n t i f i c ) . The solutions were l e f t for 30 to 60 minutes, after which time the samples were read against a blank on a Unicam SP600 spectrophotometer at a wavelength of 750 millimicrons. Peptide concentrations for unknown solutions were calculated form a curve plotted for the standards. B. Thioglycollate Inactivation The destruction of oxytocic activity by sodium thioglycollate was used as a means of distinguishing the neurohypophysial principles of cyclostome extracts from biologically active contaminating substances. The method of Vogt (1953) was followed. Equal volumes of 0.2 M thioglycollic acid and 0.2 M sodium bicarbonate were mixed to yield 0.1 M sodium thioglcollate. This was added to the extract in a volume ratio of one to nine, and the pH of the resulting solution was adjusted to 7 with solid NaliC03 . Incubations were carried out at room temperature for from 30 minutes to as much as 48 hours. Parallel incubations consisting of thioglycollate-treated synthetic oxytocin and an untreated extract control were also run. At the end of the incubation time, a l l solutions were tested on the isolated rat uterus. Treated extract material was usually assayed against both synthetic oxytocin and the untreated extract control. Occassionally, 0.1 M thioglycollate and 0.257. acetic acid were mixed in a one to nine ratio and added to the muscle bath with an 46 injection, of standard. In no instance did this result in a diminuation of the response evoked by the standard, indicating that lack of a c t i v i t y in treated material was not due to inhibition of uterine contractions by the presence of sodium thioglycollate. 47 SECTION I Histological Observations of the Hypothalamo-Hypophysial System of Lampetra richardsoni A. Introduction A preliminary examination of the hypothalamo-hypophysial system of L. richardsoni was carried out for two reasons. The f i r s t was that the microan-atomy of the neurohypophysis in this species had not been previously described •and i t seemed of interest to compare i t with published results on other lampreys. Secondly, i t was advisable to study the histology of the pituitary and adjacent brain in an attempt to gauge the effectiveness of the techniques employed in dissecting the tissues used for pharmacological studies. As mentioned earlier (p.19), the absence of an infundibular stalk in the lamprey did not allow a precise dissection of the neurohypophysis to be accomplished. By observing the hypothalamo-hypophysial system in both in in s i t u brains and in brains which had been dissected as described previously (p.19), i t seemed possible to insure that a l l the neurohypophysis was included in the dissected pituitary component. B. Methods Only ammocoetes were given histological study, because adult lamprey were not readily available when the work was carried out (September). The larvae used were five and six years old, and were captured in Marshall Creek. Both in situ and dissected brains were examined. To obtain the former tissue, the lamprey was decapitated, and a mid-dorsal incision was made in the head 48 to expose the brain. The head was then placed in Bouin's fixative for 12 hours. Prior to sectioning, much of the ventral and lateral aspects of the head were cut away, in order to reduce the size of the specimens. Dissected neurohypophysial and control tissue components were obtained using the technique described in Materials and Methods (p.19)1 The brain was removed from the chondocranium and transected at the diencephalic-mesencephalic border; the rostral half minus the cerebral lobes was taken as the pituitary component, and the caudal portion was used as control tissue. Tissues were fixed in Bouin's solution for 12 hours and then transferred to 707. alcohol. The samples were dehydrated in an ascending series of alcohols, ending with four changes of absolute ethanol. The alcohol was removed by three changes of xylene and the tissues were then transferred to paraffin wax (m.p. o «=• 56 to 57 C.) in which they were embedded. The embedded samples were sectioned at 8 microns and dried for 24 hours at C. Subsequently, they were deparaffinized in xylene and run through a descending series of alcohols to water. Two staining techniques were employed, both of which have been shown to demonstrate hypothalamic neurosecretory material. The chrome-haema-toxylin-phloxine stain of Gomori (1941) was used i n i t i a l l y ; later samples were stained by the aldehyde-fuchsin-haematoxylin-light green-orange G technique of Halmi (1951) as modified by Dawson (1953). Details of the staining methods are given in Appendix A. C. Results 1. The Appearance of the Hypothalamo-Kypophysial System in In Situ Brains The pituitary gland of Lampetra richardsoni ammocoetes is shown in figure 7. Its appearance was similar to that of other lampreys, especially Lampetra planeri (van der Kamer and Schreurs, 1959; Gorbman, 1965). The 49 Figure 7 Sagittal section, through the head of a lamprey showing the brain and pituitary regions. Section stained with chrome-haematoxylin-phloxine. Anterior end to the l e f t . a. magnification = 50 x. b. magnification •» 200 x. b.«* blood sinus; c.p.= choroid plexus; h.« habenular region; i.e.- infundibular cavity; mes.«= mesencephalon; met.- metancephalon; n.» notocord; n.p.« region of preoptic nucleus; n= nasopharyngeal stalk; o.c.« optic chiasma; o.t.« optic tectum; p.= pineal; p.d.= pars d i s t a l i s ; p.i.« pars intermedia; p.n.« pars nervosa; p.p.d.= proximal pars dis-t a l i s ; p.r.= posterior recess of infundibular cavity. 50 adenohypophysis was clearly divided into pars d i s t a l i s and pars intermedia. The former component was separated from the ventral surface of the brain by a layer of connective tissue which also divided i t into rostral and proximal portions. No connective tissue septum was present between the pars intermedia and the pars nervosa; these parts of the pituitary were more or less closely apposed, except where large cavities separated them. Since these cavities contained erythrocytes, they appeared to be blood sinuses. Ventral to the adenohypophysis, the nasopharyngeal canal extended caudally to about the level of the proximal pars d i s t a l i s . In common with other Petromyzoniformes, there was no infundibular process below the brain in the brook lamprey larvae. The glandular portion of the neurohypophysis was represented by the thin floor of the diecephalon, dorsal to the pars d i s t a l i s , and by the thickened pars nervosa which overlay the pars intermedia, and extended caudally to the level of the posterior recess. Using Gomori's chrome-haematoxylin-phloxine method, the intensity of staining of the neurosecretory material was not remarkable, even when the sections were stained for longer than the recommended time (25 instead of 15 minutes). A sooty-gray color was obtained for the pars nervosa, while the corresponding region of a control teleost (guppy, Poecilia sp.) stained dense black when treated in the same way. The neurohypophysial region rostral to the pars nervosa did not stain appreciably greater than indifferent brain. These results were similar to those obtained by van der Kamer and Schreurs' (1959), who also used the Gomori method to stain the neurohypophysis of L. planeri. The preoptic nuclei did not appear to be affected by the Gomori reagent, and no neurosecretory fibres were seen leading to the neurohypophysis. In view of the slight staining of the pars nervosa, i t seemed l i k e l y that the chrome-haematoxylin-phloxine method was not sensitive enough to stain any neurosecretory material present in the preoptic soma or axons. Likewise, no evidence was obtained for the presence of preoptic fibres running into the hindbrain, or for the existence of neurosecretory neurons dorsal to the posterior recess 9 although both have been reported by Oztan and Gorbman (I960) in ammocoetes of Petromyzon marinus and Lampetra lamottei. However, the illustrations of the neurohypophysis in Oztan 1s and Gorbman,s paper (I960) are based on sections stained with aldehyde-fuchsin, and i t appeared possible the neurosecretory substances in the preoptic nuclei, the region of the posterior recess and the hindbrain could be clearly differentiated only by the aldehyde-fuchsin procedure. However, in the studies carried out here, •sections treated with this stain did not delimit the preoptic nuclei or show neurosecretion around the posterior recess (figure 8). Nevertheless, the pars nervosa was strongly stained, and neurosecretory material was observed in the neurohypophysis dtsrsal to the pars distal i s . It seemed unlikely that the failure to demonstrate neurosecretory granules in the preoptic neurons was the result of failure of the staining methods employed, since both the chrome-haematoxylin-phloxine stain and the aldehyde-fuchsin reagent clearly stained the substances in the pars nervosa. It appeared more probable that, for some unknown reason, the neurosecretory neurons and axons were depleted of stainable material and therefore were not stained. Aldehyde-fuchsin'positive granules were also noted i n the large c e l l s of Muller in the mes- and rhomb-encephaIon (figure 9), but these granules did not appear to be stained by the Gomori reagent. Similar results have been reported by Sterba (1961, 1962, 1969) in Lampetra planeri; he also observed granules in c e l l s of the trigeminus and vagus nuclei and several mesencephalic nuclei, as well as in various c e l l s in the spinal cord. These latter granules were not demonstrated in the present investigation. 52 Figure 8 Sagittal section through the head of a larval lamprey showing the brain and pituitary regions. Section stained with aldehyde-fuchsin. Anterior end to the right. a. magnification «•» 50 x. b. magnification » 200 x. b.«= blood sinus; c.p.™ choroid plexus; h.= habenular region; i.c=> infundibular cavity; mes."* mesencephalon; met.= metancephalon; n.«= notocord; n.p.™ region of preoptic nucleus; n.s.m.1" neurosecretory material; o.c.= optic chiasma; o.e.« olfactory epithelium; o.t.«= optic tectum; p.= pineal; p.i.«» pars intermedia; p.n.= pars nervosa; p.p.d.= proximal pars d i s t a l i s ; r.p.d.= rostral pars d i s t a l i s . 53 Figure 9 Sagittal section through the mesencephalon of a larval lamprey showing several large Muller c e l l s . Section staines with aldehyde-fuchsin. Anterior end to the right. Magnification «= 800 x. A.F. a aldehyde-fuchsin-positive granules. Figure 10 Sagittal section through the head of a larval lamprey from which the brain has been removed. Section stained with haematoxylin-eosin. Anterior end to the right. Magnification = 50 x. n.«= notocord; n.s.» nasopharyngeal stalk; o.e," olfactory epi-thelium; p.d.» pars d i s t a l i s ; p.i.= pars intermedia; s.=» nasal sac. 54 2. The Appearance of the Hypothalamo-Hypophysial System in Dissected  Brains Histological examination of the dissected pituitary and control tissues from five ammocoetes made several points clear. The f i r s t was that a l l portions of the neurohypophysis were dissected from the chondocranium while most or a l l of the adenohypophysis was l e f t behind. This can be seen in figure 10, which is a median sagittal section through the head of an ammocoete whose brain had been removed. Both the pars distal is and pars intermedia remained embedded in the connective tissue of the floor of the chondocranium, but l i t t l e i f any nervous tissue was retained within the brain cavity. Thus, i t seemed l i k e l y .that the extracts of the neurohypophysial tissue used for pharmacological studies were not contaminated by any anterior pituitary hormones, and that a l l of the neurohypophysis was included with the dissected brain. It was also apparent that the stainable substances in the neurohypophysis were located ros t r a l to the level at which the brain was transected, and, therefore, that a l l of the hypothalamo-hypophysial system was contained in the pituitary component of the dissected tissues. This can be seen in figures 11 and 12, which are sections of pituitary and control tissues. Comparison of the latter figure with sections of in si t u brain (figure 13) suggested that the transection of the central nervous system was made at a level rostral to the mesencephalic-metancephalic border, and not at the diencephalic-mesencephalic junction as had been thought. No evidence was obtained for the presence of any neurosecretory material in the control tissues. D. Discussion and Conclusions The hypothalamo-hypophysial system of Lampetra richardsoni ammocoetes appeared quite similar to that of other lampreys, especially to the closely 55 Figure 11 Serial horizontal sections through the dissected pituitary com-ponent of a brain dissected from a larval lamprey. Sections stained with chrome-haematoxylin-phloxine. Anterior end to the l e f t . Magnification » 75 x. a = most dorsal section d => most ventral section. c.h.= cerebral hemisphere; c.v.= cerebral ventricle; i.c.» infun-dibular cavity; M.» Miiller c e l l ; p.n.= pars nervosa; t.v.= third ventricle. 56 Figure 12 Sagittal section through the hindbrain, control component of a brain dissected from a larval lamprey. Section stained with chrome-haematoxylin-phloxine. Anterior end to the right. Magnification => 50 x. c.p.>=» choroid plexus; f,V. d fourth ventricle; m," medulla oblong-ata. Figure 13 Sagittal section through a larval lamprey showing the mesenceph-a l i c and metencephalic regions of the brain. Section stained with chrome-haematoxylin-phloxine. Anterior end to the l e f t . Magnification =50 x. c.= cerebellum; c.p.= choroid plexus; f.v.=» fourth ventricle; m.=» medulla oblongata; M.»» Muller c e l l ; n.<= notochord; o.t.= optic tectum. 57 related L. planeri. Material located within the pars nervosa was stained by the two techniques employed, both of which have been shown to stain hypothal-amic neurosecretory material (Sloper, 1955). However, these methods did not indicate the presence of any neurosecretory material in the preoptic neurons, which have been shown by many workers to be neurosecretory in nature (Green, 1951; van der Kamer and Schreurs, 1959; Oztan and Gorbman, I960; Perks, 1969; Sterba, 1969). It appeared possible that the neurons and their axons were depleted of their stainable neurosecretory granules, perhaps as a result of stress. Leatherland and Dodd (1969) reported that capture of Anguilla anguilla with an electric shocker reduced the amounts of neurosecretory material in the preoptic neurons and axons within four hours of capture. In the present study, the lampreys were obtained simply by digging up the stream bottom with a shovel and picking out the burrowed ammocoetes. They were transported to the laboratory in a styrofoam ice chest f i l l e d with stream water, and were then immediately k i l l e d and dissected. Conceivably, stress, resulting from the capture and subsequent transportation, could have depleted the stainable material in the preoptic neurons, and the immediate sacrifice of the animals perhaps would not have allowed time for a replenishment of the neurosecretory substances. This explanation would also account for the failure to demonstrate neurosecretory-like c e l l s outside the hypothalamo-hypophysial system. Such eells have been reported,:'-%o occur around the posterior recess by Oztan and Gorbman (1960), and in various other regions of the brain by Sterba (1961, 1962, 1969). Examination of the pituitary and control tissues offered some indications as to the portions of the brain which were taken during the dissection of the material used in the pharmacological investigations to be described in the following sections. It appeared that the neurohypophysial component did not 58 contain appreciable amounts of adenohypophysial tissue, but that substantial amounts of the forebrain and midbrain were included. The inclusion of mesen-cephalon with the neurohypophysis was somewhat surprising, since i t had been thought that the brain was transected at the diencephalic-mesencephalic border; however, i t was apparent that the division was made at a somewhat more caudal level. While this undoubtedly increased the levels of the contam-inants in the neurohypophysial extracts, i t v i r t u a l l y insured that a l l portions of the hypothalamo-neurohypophysial system were contained within the dissected pituitary component of the brain. 59 SECTION II The Isolation and Identification of the Neurohypophysial Principles of  the Lamprey, Lampetra richardsoni A. Studies of the Ammocoete 1. The Oxytocic Activities of Crude Extracts and the Effects of Sodium  Thioglycollate on the Activity, Rat uterus potencies of the ammocoete tissues extracted in the course of this study are l i s t e d in Table II. Extracts of a l l pituitary and control tissues possessed detectable oxytocic a c t i v i t y , and in a l l cases save one (sample E), the a c t i v i t y of the former tissue was greater than that of the latter. A c t i v i t i e s of a l l extracts were exceedingly low, however, even when compared to other fishes (see Fol l e t t and Heller, 1964; Perks,1966). The low potencies of the extracts and the similarity between the rat uterus levels of pituitary and hindbrain extracts made i t uncertain as to whether a l l of the ac t i v i t y extracted from the neurohypophysial tissues was due to posterior pituitary hormones. In an attempt to determine the proportion of the total a c t i v i t y due to neurohypophysial principles, the extracts were treated with 0.01 M sodium thioglycollate. Although the precise mechanism is not known, incubation of posterior pituitary hormones with sodium thioglycol-late results in a destruction of biological a c t i v i t y (Rudinger and Krejci, 1968). With ammocoete neurohypophysial extracts, no more than 59% of the total oxytocic a c t i v i t y was abolished by thioglycollate, even with incubation times of up to 24 hours. Synthetic oxytocin, on the other hand, was completely inactivated after 30 to 60 minutes of treatment. There appeared to be l i t t l e Table I I . Oxytocic a c t i v i t i e s of crude extracts of ammocoete neurohypophysial and con t r o l t i s s u e s and t h e i r resistance to t h i o g l y c o l l a t e . Neurohypophysial Extracts Control (hindbrain) Extracts Sample * A c t i v i t y Resistance to t h i o g l y c o l l a t e A c t i v i t y Resistance to T h i o g l y c o l l a t e mU/mg mU/gland incubation 7. a c t i v i t y mU/mg mU/gland incubation % a c t i v i t y time remaining destroyed time remaining destroyed A-U2 year olds (95) 3.05 0.17 2 hr. 72 28 0.86 0.05 2 hr. 104 0 3 41 59 3 108 0 B-3 year olds (123) 3.89 0.36 14 45 55 2.21 0.27 14 91 9 C-4 year olds (154) 4.14 0.55 8 45 55 2.90 0.50 8 121 0 D-5&6 year olds (171) 2.84 0.52 5 106 0 1.31 0.41 7 117 0 24 76 24 24 248 0 E-mature ammocoetes 1.75 0.48 1.75 0.54 (372) ** * values i n parentheses indicate the number of ammocoetes i n each sample. ** Sample E was taken from Marshall Creek; a l l other samples are from the Salmon River 61 r e l a t i o n s h i p between the length of the incubation and the amount of p i t u i t a r y a c t i v i t y destroyed (see Table I I ) , s i n c e samples A, B and C, with incubation times ranging from 3 to 14 hours, showed about the same l e v e l s of i n a c t i v a t i o n . However, with treatments l a s t i n g l e s s than three hours, the r e a c t i o n of t h i o -g l y c o l l a t e with the neurohypophysial material d i d not appear to be complete, as suggested by the 727. r e t e n t i o n o f a c t i v i t y shown by sample A a f t e r two hours of incubation. Sample D, which had l o s t no a c t i v i t y a f t e r f i v e hours and showed only a 24% i n a c t i v a t i o n a f t e r 24 hours, apparently e i t h e r reacted with the t h i o g l y c o l l a t e at a slower rate than the other samples, or had a smaller proportion of neurohypophysial hormone. The former p o s s i b i l i t y was hard to e x p l a i n , since a l l extracts were treated with the same concentration of t h i o g l y c o l l a t e under approximately the same conditions. Differences i n pH and temperature undoubtedly occurred amoung the d i f f e r e n t samples, and i t i s known that these parameters can a f f e c t the r e a c t i o n of t h i o g l y c o l l a t e with neurohypophysial hormones.(Rudinger and K r e j c i , 1968). However, i t seemed u n l i k e l y that the v a r i a t i o n s i n pH and/or temperature would have been large enough t o cause pronounced d i f f e r e n c e s i n the degree of i n a c t i v a t i o n , and thus to e x p l a i n the low los s of a c t i v i t y exhibited by sample D a f t e r t h i o g l y c o l l a t e treatment. I t has been suggested (Perks, 1966; Sawyer, 1968) that disulphide c o n t a i n i n g p r o t e i n contaminants present i n crude extracts of neurohypophysial t i s s u e may compete with the p o s t e r i o r p i t u i t a r y hormones f o r t h i o g l y c o l l a t e and thereby reduce the rate of i n a c t i v a t i o n of the l a t t e r compounds. The presence of large amounts of these contaminants conceivably could even r e s u l t i n the expenditure of the t h i o g l y c o l l a t e before a l l of the neurohypophysial material was i n a c t i v a t e d . This hypothesis could p o s s i b l y e x p l a i n the apparently lower r e a c t i o n rates of the p i t u i t a r y extracts as compared to that of oxytocin (at l e a s t 3 hours f o r maximum i n a c t i v a t i o n compared to one hour). However, i t _ 62 seems unable to account f o r the low degree of i n a c t i v a t i o n of sample D, since - the amounts of Lowry p o s i t i v e m a t e r i a l , although r e l a t i v e l y high (1500 to 1900 ug/ml), were approximately the same i n a l l samples. Thus, no explanation f o r the low i n a c t i v a t i o n of sample D was r e a d i l y apparent. It should be noted, however, that evidence to be presented l a t e r (p.8o) suggested that approxi-mately 507. of the t o t a l potency of t h i s sample was due to a neurohypophysial p r i n c i p l e , and t h a t , therefore, sample D reacted with the t h i o g l y c o l l a t e to a lower degree than d i d the other e x t r a c t s . The e f f e c t s of t h i o g l y c o l l a t e on hindbrain extracts were quite d i f f e r e n t from the e f f e c t s on p i t u i t a r y m a t e r i a l , as can be seen from Table I I . In only .one instance (sample B) was there an apparent los s of a c t i v i t y ; t h i s was only 9%, which was almost c e r t a i n l y w i t h i n the e r r o r of the potency estimation. With a l l other samples, there was an increase i n oxytocic potency; t h i s was most s t r i k i n g i n the case of sample D. A f t e r f i v e hours incubation time, the t r e a t e d material possessed 117% of the a c t i v i t y of the untreated c o n t r o l ; while a f t e r 24 hours the a c t i v i t y was 248% that of the c o n t r o l , representing almost a 2.5 f o l d increase i n potency. This augmentation of potency was not due t o s t i m u l a t i o n of the uterus by the t h i o g l y c o l l a t e , since n e i t h e r t h i o -g l y c o l l a t e alone, nor t h i o g l y c o l l a t e treated oxytocin potentiated the responses of the uterus. The increase i n a c t i v i t y conceivably could have been caused by t h i o g l y c o l l a t e induced l i b e r a t i o n of uterotonic substances from the hindbrain e x t r a c t s . T h i o g l y c o l l a t e , l i k e other t h i o l compounds i s capable of reducing d i s u l p h i d e bonds ( B a i l e y , 1967). Conceivably, the release of a c t i v e agents could have occurred through such a mechanism, with the t h i o g l y c o l l a t e breaking d i s u l p h i d e bonds which bound the oxytocic materials i n an i n a c t i v e complex. Such an e f f e c t of t h i o g l y c o l l a t e does not appear to have been reported i n the l i t e r a t u r e , however, and i t i s d i f f i c u l t to even speculate as to the nature 63 of these supposed disulphide-bound active substances. In a l l of the experiments with thioglycollate inactivation, the amounts of active materials available were limited, and estimates of biological a c t i v i t y were made on the basis of single 4-point groups. Therefore, the results are subject to a rather large degree of error. However, the differences noted in the effects of thioglycollate on the pituitary extracts on one hand and on the hindbrain extracts on the other seem striking and consistent enough to be considered genuine. They also suggest that the pituitary and control extracts contained different active substances. 2. Purification of ammocoete extracts by paper chromatography a. Purification of crude extracts The results described above indicated that not a l l of the activity present in pituitary extracts was due to neurohypophysial hormones. Therefore, i t was decided to separate the contaminating substances from the neurohypo-physial material prior to i t s pharmacological characterization. Paper chrom-atography of the extracts, using the solvent system butanoltacetic acid:water (4:1:5) seemed the simplest way of purifying the small amounts of material available. Two samples were treated in this manner : 1) a pooled extract consisting of samples B and C 2) an extract of lamprey material described in Section III. i . Pooled samples B and C Figure 14 indicates the results of the chromatogram of this sample. Chromatography of the pituitary extract yielded two regions of oxytocic a c t i v i t y . The faster moving spot (Rf»s 0.3 to 0.5) ran in the same area as synthetic AVT, while the slower component barely migrated from the origin. No oxytocic ac t i v i t y was detected i n the Rf's (0.6 to 0.8) where oxytocin or any other neutral principle would have run. To determine whether the two 64 Figure 14 Paper chromatography of crude extracts of ammocoete neuro-hypophysial and control tissues (pooled samples B and C) in buta-nol :acetic acid:water (4:1:5). white columns - rat uterus ac t i v i t y (magnesium absent) in mU/tnl. cross-hatched columns =» rat antidiuretic a c t i v i t y in mU/ml. numbers above columns « antidiuretic ratio of the eluate = rat antidiuretic a c t i v i t y rat uterus activity (-Mg^. Standard chromatogram load - 500 ng (50 mU) synthetic AVT, 25 mU synthetic oxytocin recovery - 507.. Pituitary chromatogram load " 43 mU oxytocic a c t i v i t y , 956 pg Lowry peptide. recovery • 477. of "oxytocic a c t i v i t y . Control chromatogram  load « 29 mU oxytocic a c t i v i t y , 1390 pg Lowry peptide. recovery - 3777. of oxytocic a c t i v i t y . 50 STANDARD Rf - 65 oxytocic regions contained d i f f e r e n t substances or whether the r e t e n t i o n of a c t i v i t y at the o r i g i n was merely an a r t i f a c t r e s u l t i n g from overloading of the chromatogram, the eluates were tested f o r r a t a n t i d i u r e t i c a c t i v i t y . The majority of the a n t i d i u r e t i c a c t i v i t y was located i n the f a s t e r moving peak, but small amounts extended back to Rf's 0-0.1. In t h i s connection, i t may be noted that the a n t i d i u r e t i c assay was much more s e n s i t i v e to AVT than the r a t uterus, and thus detected amounts of the hormone below the s e n s i t i v i t y threshold of the oxytocic assay. The eluate from Rf«s 0.3-0.4 possessed a n t i d i u r e t i c and r a t uterus a c t i v i t i e s i n a r a t i o of about 3.7 to one, while the r a t i o of i t s potency on the r a t uterus with magnesium present to i t s . a c t i v i t y with magnesium absent was 3.9. The corresponding r a t i o s f o r AVT are 1.55 and 1.9 (Sawyer, 1965), but considering the errors involved i n the s i n g l e group potency estimations of the eluate, the agreement obtained between the r a t i o s f o r the eluate and f o r AVT were considered to be f a i r l y c l o s e ; t h i s w i l l be discussed i n more d e t a i l l a t e r (pp.163 - 164 ). Incubation of the eluate with 0.01 M sodium t h i o g l y c o l l a t e r e s u l t e d i n a more than 92% loss of r a t uterus a c t i v i t y , which indicated that the a c t i v e substance present was prob-ab l y a neurohypophysial hormone. The slower moving region of oxytocic a c t i v i t y possessed l i t t l e a n t i d i u r e t i c a c t i v i t y and thus appeared to d i f f e r from the m a t e r i a l i n the "AVT" peak at Rf's 0.3-0.4. A f t e r several days, no r a t uterus a c t i v i t y could be detected i n the eluates of t h i s region, and i t appeared that the a c t i v e substances, whatever t h e i r i d e n t i t y , were l a b i l e , and d i f f e r e d from neurohypophysial p r i n c i p l e s . Because of t h i s r a p i d breakdown, i t was not p o s s i b l e to t e s t the e f f e c t s of sodium t h i o g l y c o l l a t e . Remarkably high amounts of oxytocic a c t i v i t y were recovered from the chromatogram of the b r a i n e x t r a c t , much more than was a p p l i e d . This obser-v a t i o n , although unusual, i s not unique, since s i m i l a r changes have been seen by Pickering (private communication) in chromatograms of crude neurointermed-iate lobes of elasmobranchs; they have been tentatively explained as a result of the removal of inhibitors. Most of the a c t i v i t y was eluted from the origin and Rf's 0-0.1, and, as with the material present at the origin of the pituitary chromatogram, the activity appeared to be very labile and was not detectable after a few days storage at 4° C. This slow moving material caused small antidiuretic effects; however, the ac t i v i t y was much less than the oxytocic potency of the eluates. The time course of the antidiuresis was different from that effected by synthetic AVT, in that i t had a longer latent period. Oxytocic a c t i v i t y was also found in Rf's 0.3-0.6, running between the positions typical of AVT and oxytocin. This region possessed antidiuretic a c t i v i t y as well, but again the antidiuresis was quite different from that caused by neurohypophysial principles, in that i t exhibited a long latent period, prolonged antidiuresis and gradual return to normal urine flow. To determine the chromatographic behaviour of the Lowry-positive material present in the ammocoete extracts, Lowry peptide determinations were made on the eluates from the chromatograms of the extracts. To conserve material, 0.05 ml of each eluate was diluted to 0.20 ml and this solution was used in the determination. Lowry material was detected only at the origin and in Rf's 0-0.1 in both the pituitary and hindbrain chromatograms; the amounts of reacting material in the higher Rf's were presumably lower than the sensitivity of the method. Approximately 50% of the Lowry-positive substances loaded on to the chromatogram were recovered from the origin and Rf 0-0.1; this indicated that most of the Lowry-positive material did not migrate appreciably in the solvent system employed. Swiatkiewcz (1967) found a similar distribution of Lowry reacting substances on chromatograms of crude neurolntermediate lobe extracts of Squalus acanthias. It seems li k e l y that the substances at the 67 origin, and Rf 0-0.1 were relatively high molecular weight proteins, since molecules of this type would have had relatively low s o l u b i l i t i e s in the mobile organic phase of the solvent system (White et a l , 1968). Also the binding of large amphoteric proteins to the cellulose matrix of the chromato-gram by hydrogen bonding, ionic interactions and van der Waals forces would have hindered their migration (Smith, 1969). i i . Ammocoete extracts from section III The material used in this chromatogram resulted from experiments dealing with the effects of photoperiod on the hormone content of the ammocoete neurohypophysis (section III, pp.134-140 ). As no marked effects of light conditions were noted, i t was decided to use the extracts resulting from the experiments in pharmacologic and chromatographic studies. The chromatograms of the pituitary and hindbrain extracts of this material were very similar to those described above (p.63), as can be seen in figure 15. In this case, however, the a c t i v i t i e s applied to and recovered from the chromatograms were lower, and, therefore, not as many tests could be performed on the eluates. Chromatography of the neurohypophysial extract yielded two regions of oxytocic ac t i v i t y . The peak (Rf 0.2-0.3) of the faster moving spot ran in the same area as synthetic AVT, and possessed RUMg/RU and ADH/RU ratios of 1.5 and 7.7 respectively. Even considering the errors involved i n the potency estimations, the latter ratio seemed somewhat high for AVT, which has an ADH/RU ratio of 1.55 (Sawyer, 1965a). The small amount of material available did not allow testing the eluate for l a b i l i t y to thio-glycollate. The rat uterus a c t i v i t y at the origin and Rf 0-0.I again appeared to be very labile and the i n i t i a l a c t i v i t y was so low that insufficient material was available for the assay of antidiuretic a c t i v i t y . The chromato-gram of the control extract yielded trace amounts of oxytocic ac t i v i t y in 6 8 Figure 15 Paper chromatography of crude extracts of ammocoete neuro-hypophysial and control tissues (larvae from section III) in buta-nol racetic acid:water (4:1:5). white columns - rat uterus ac t i v i t y (magnesium absent) in mU/ml. stippled columns » rat uterus a c t i v i t y (magnesium present) . i n mU/tnl. cross-hatched columns « rat antidiuretic a c t i v i t y in mU/ml. Standard chromatogram load = 500 ng (50 mU) synthetic AVT, 25 mU synthetic oxytocin. recovery « 397.. Pituitary chromatogram load «• 27 mU oxytocic act i v i t y , 845 ug Lowry peptide. recovery » 267.-of oxytocic ac t i v i t y . Control chromatogram load » 20 mU oxytocic act i v i t y , 960 ug Lowry peptide. recovery - 117. of oxytocic a c t i v i t y . T 0 T .1 i . V . V , • V . V.I V . V . ' . ' . V . ' . ' I V . V . V . V X M • • • • • • • o • •! • • • • • • • • • • • • • » • , • • • • • • e o • • • • • • •••••• • ••••• • ••••< o • • . . . . . . •*•*** # * ' • • • o » o o * • • • • dj STANDARD • • • • i • • • ( • • • 0 |0*0*0*0*» ^ • • • ' I • • • I • • • • t • • • I • • • • t> • • • • • • • • _ 'WML mi v . v . i 1 1.0 .8 8886 PITUITARY > 9 • • • k * * * " r • * " I .3 .4 T 1 1 1 1 6 .7 .8 .9 1.0 CONTROL T 0 .2 .5 .7 & .9 1 1.0 Rf 69 three regions, but again the levels were so low that few eluates could be checked for antidiuretic a c t i v i t y . However, the eluates from Rf 0.1-0.2 of the pituitary chromatogram and Rf 0.2-0.3 of the control chromatogram produced antidiuretic effects similar to those found in eluates of the hind-brain chromatogram of the pooled sample of extracts B and C. The antidiureses were pronounced and, with larger doses, very prolonged. Bisset and Lewis (1962) e l l i c i t e d comparable responses in rats with large injections of bradykinin and substance P. Although these substances are not normally classed as anti-diuretics, they are potent hypotensive agents. Doses which caused a f a l l in a r t e r i a l blood pressure of 40 mm Hg or more also resulted in antidiuretic responses which persisted long after the blood pressure had returned to normal. Bisset and Lewis postulated that the pronounced f a l l in blood pressure evoked the release of endogenous arginine vasopressin and that this was responsible for the antidiuresis. Although the ethanol anesthesia used here and by Bisset and Lewis has been claimed to supress the release of endogenous vasopressin (Ames and van Dyke, 1952; Dicker, 1953), Ginsburg and Brown (1956) reported that severe haemorrhage and subsequent marked hypotension resulted in increased amounts of vasopressin in the blood of ethanol-anesthetised rats. To check whether the antidiuretic responses evoked by the chromatogram eluates were due to a f a l l in blood pressure, the l e f t carotid artery was cannulated i n several rats used in antidiuretic assays and blood pressure was monitored during the responses. A typical result is shown in figure 16. While an injection of 0.375 ng of AVT caused a pronounced antidiuresis with no marked f a l l in blood pressure, 100 ul of the eluate of Rf 0.1-0.2 from the pituitary chromatogram produced an antidiuresis accompanied by a prolonged hypotensive response. There appeared to be a correlation between the level--of blood pressure and the antidiuretic effect. Although urine flow approached 70 Figure 16 Effects of synthetic AVT and an eluate (Rf 0.1-0.2) from a paper chromatogram of crude ammocoete neurohypophysial material (larvae from section III) on urine flow and blood pressure in the ethanol-anaesthetized rat. upper trace •= relative urine flow. lower trace «• systolic blood pressure (in mm Hg) in the le f t carotid artery. TIME, MINUTES TIME , MINUTES 71 the baseline level approximately one half way through the depressor response, i t was not un t i l the blood pressure had returned to normal that the antidiu-resis ended. Injections of the eluate of Rf 0.2-0.3 from the hindbrain chrom-atogram produced similar effects on both urine flow and blood pressure. The substances responsible appeared to be potent hypotensive agents in that their effects persisted for over 30 minutes. Follett and Heller (1964) have reported that crude neurohypophysial extracts from Myxine glutinosa also contained a substance that caused a marked f a l l i n blood pressure. In contrast to those e l l i c i t e d by the Lampetra extracts, the hypotensive effects of bradykinin and substance P lasted only two to three minutes, even in cases of large reductions in blood pressure (greater than 50 mm Hg)-(Bisset and Lewis, 1962). It may have been that the low arterial pressure resulting from injection of the eluates was pronounced enough to cause some reduction in the glomerular f i l t -ration rate and thereby reduce urine flow. However, the lack of a complete parallelism between the changes in blood pressure and urine flow suggested that direct renal effects of hypotension did not play a major role in the antidiu-r e t i c response. The curves in- figure 16 are better explained by assuming that a release of endogenous vasopressin occurred only as a consequence of the f a l l i n blood pressure at the beginning of the of the response, and not as a result of the persisting hypotension. This explanation is supported by the work of Ginsburg and Brown (1956) which indicated that vasopressin release evoked by haemorrhage in rats occurred only during periods when the blood pressure was actually f a l l i n g , and therefore that the stimulus e l l i c i t i n g release was the change and not the actual level of pressure. Lack of material prevented the testing the eluates of other Rf's for similar effects, but i t seemed possible, i f not l i k e l y , that Rf's adjacent to the active regions also contained small amounts of the hypotensive substances. 72 If this were the case i t would explain the high antidiuretic potency of Rf 0.2-0.3 of the pituitary chromatogram, higher than was consistent with the presence of AVT alone. In addition to the synthetic oxytocin and AVT used as standards in the chromatography of this sample, several other biologically active substances known to be present in brain tissue were applied to another sheet and chrom-atographed along with the s t r i p containing the unknowns. This was done in an attempt to provide evidence as to the identities of the contaminating substances eluted from the chromatograms of the ammocoete extracts. Knowledge of their identities would certainly have been useful in effecting their removal from the neurohypophysial material. It can be seen from figure 17 that the peaks of 5-hydroxytryptamine, epinephrine and acetylcholine a l l ran in approximately the same region, Rf 0.3-0.6. Lederis (1961) reported that the Rf of substance P (which also i s known to be present in brain tissue) in the same solvent system was 0.29-0.39, while that of oxytocin was 0.55-0.65. Thus none of these substances were responsible for the activity eluted from the origin and Rf 0-0.1 of the extract chromatograms. However, the faster moving components on these chromatograms could have contained traces of these compounds, and the presence of substance P or a substance P-like peptide would certainly have explained the potent hypotensive a c t i v i t i e s of eluates of this region. The results of this section suggested the possible presence of arginine vasotocin in ammocoete neurohypophysial extracts; no evidence was obtained for the existence of a second posterior pituitary principle. However, the data were certainly not conclusive on either count. A variety of other active substances were also detected, and, although the identities of these compounds were not determined, their marked l a b i l i t y suggested that they were not neuro-hypophysial peptides. Nonetheless, their presence served as a hindrance in the 73 Figure 17 Paper chromatography of 5-hydroxytryptamine, acetylcholine, and epinephrine in butanol:acetic acidrwater (4:1:5). - amounts of the active substances recovered from the chrom-chromatograms were estimated on the rat uterus assay (5-HT Ach) or on the vasopressor assay (epinephrine). 5-Hydroxytryptamine load » 100 ug. recovery « 847.. Acetycholine load = 100 ug. recovery •» 747.. Epinephrine load - 100 pg. recovery •» 567.. 7 0 r 5-HYDROXYTRYPTAMINE x i in 35 T" 0 .2 T -.5 .6 .7 .8 .9 1.0 ACETYLCHOLINE 70| x o < 35 0L o .4 T -.6 .7 .8 1 1.0 70r EPINEPHRINE UJ cr x | 35| ID .4 T " .7 .8 1 1.0 Rf 74 identification of the neurohypophysial principles and therefore i t was desire--able to effect their.removal. For several reasons i t seemed unlikely that paper chromatography of crude extracts would provide further evidence as to the identity of the ammocoete neurohypophysial hormone; neither was i t probabl that the method could effectively remove contaminating substances. The high Lowry peptide concentrations of the crude extracts limited the amount of material that could be loaded on to the chromatogram. Overloading probably would have interfered with the migration of the neurohypophysial principle(s) (Swiatkiewicz, 1967; Saith, 1969). Removal of at least a portion of the Lowry-positive material from the crude extracts prior to chromatography would allow greater amounts of the active substances to be chromatographed; this in turn would permit a more complete pharmacological characterization of the biolog-i c a l l y active eluates. Also, at least some of the non-neurohypophysial active material (that remaining at the origin) was associated with the Lowry-positive substances, and i t seemed possible that removal of the latter substances from the crude extracts would also result in removal of the active compounds. There fore, i t was decided to purify the crude extracts prior to chromatography, b. Paper chromatography of pa r t i a l l y purified extracts i . Trichloroacetic acid treatment of crude extracts Although preliminary purification of neurohypophysial extracts is often accomplished by gel f i l t r a t i o n (Sawyer, 1968), this method seemed unsuitable for the present work, since i t usually results in dilution of the applied sample, especially when loading volumes are small, as in this case (Pharmacia, 1969). Dilution of the sample was disadvantageous because i t was desireable to keep the volume for paper chromatography as small as possible (Morris and Morris, 1964). Pilot experiments with trichloroacetic acid purification (see Appendix B) indicated that this method was quite satisfactory for purifying 75 small amounts of material. Treatment of solutions of albumin and oxytocin or AVT removed the bulk of the Lowry-positive material (albumin) with l i t t l e loss of biological a c t i v i t y . Table III gives the results of treatment of the pituitary and hindbrain extracts of ammocoete sample D (5 and 6 year olds) with T.C.A. The amount of Lowry-positive material removed was less than with the albumin solutions. Perhaps this was because some of the Lowry substances in the crude extracts were low molecular weight proteins and peptides and thus were resistant to T.C.A. treatment (White et a l , 1968). Approximately one half of the oxytocic a c t i v i t y was removed from both extracts, and since the t r i a l runs with synthetic hormones indicated that T.C.A. did not inactivate neurohypophysial peptides, i t seemed l i k e l y that the act i v i t y removed from the crude extracts was non-neurohypophysial i n nature. Table III. Purification of sample D with trichloroacetic acid. Extract Oxytocic a c t i v i t y Xowry peptide Factor of purification * mU/ml 7o recovery pg/ml 7 . removed  pituitary extract before T.C.A 63.5 1500 49 69 3.3 after T.C.A. 33.1 460 hindbrain extract before T.C.A. 26.4 1370 64 58 2.4 after T.C.A. 19.3 580 * calculated by dividing the total amount of Lowry peptide material present in the extract prior to treatment with T.C.A. by the amount present after treament. 76 i i . Paper chromatography of the p a r t i a l l y purified extracts Figure 18 shows the results of paper chromatography of the T.C.A. purified pituitary and control extracts of sample D. A l l of the oxytocic activity recovered from the neurohypophysial chromatogram ran in one region (Rf 0.2-0.4), which was the same as synthetic AVT. Since the eluates of both Rf»s possessed antidiuretic a c t i v i t i e s consistent with the presence of AVT, i t appeared that the peak was homogeneous. No rat uterus a c t i v i t y was detected at the origin and at Rf 0-0.1, and no evidence was provided for the existence of a neutral neurohypophysial principle running ahead of the AVT peak. Lowry-positive material was detected in a l l ten Rf 1 s of the pituitary chromatogram, and, in contrast to the chromatograms of crude extracts (see pp. 66-67), only a small portion (187.) was eluted from the origin and Rf 0-0.1. The peak of proteinaceous material running ahead of the oxytocic peak was certainly not due to any AVT present, since the maximum amount of hormone that could have been recovered from the chromatogram (approximately 200 ng, calculated from the specific a c t i v i t y for AVT given by Sawyer, 1965a) was much too low to give the Lowry reading obtained. It is l i k e l y that the substances in the peak were small peptides or peptide fragments, since larger protein molecules probably would not migrate in the solvent system employed. Very l i t t l e a c t i v i t y was recovered from the chromatogram of the hindbrain extract, and a l l of this was located in Rf 0.2-0.3. The active material appeared rather l a b i l e , as the ac t i v i t y disappeared after a few days. As a result, no further tests could be carried out on the eluate. However, the l a b i l i t y of the active material in i t s e l f was an indication that the substance present was not a neurohypophysial peptide. i i i . Pharmacology of the oxytocic peak from the pituitary chromatogram Table IV l i s t s the potencies of the most active eluate (Rf 0.2-0.3) 77 Figure 18 Paper chromatography of partially purified extracts of ammo-coete neurohypophysial and control tissues (sample D) in butanol: acetic acid:water (4:1:5). stippled columns « rat uterus a c t i v i t y (magnesium present) in mU/ml. white columns = Lowry peptide concentration in ug/ml. Standard chromatogram load - 500 ng (50 mU) synthetic AVT, 50 mU synthetic oxytocin. recovery «=» 787.. Pituitary chromatogram load = 28 mU oxytocic a c t i v i t y , 390 a^g Lowry peptide. recovery - 827. of oxytocic a c t i v i t y . Control chromatogram load » 24 mU oxytocic act i v i t y , 730 ^ig Lowry peptide. recovery « 217. of oxytocic ac t i v i t y . lOOr STANDARD 50 t « • • • • • • • i • • • • - • • • 4 • • • • • • • p • • • • • • • • • I • • • • J • • • • a 1 i.o i 1 r o .1 .3 .4 .5 .8 .9 50r \zs\ i ( i 1 r r I i ' i i ii i iii • • • • • > • • • • 11 "'"I PITUITARY "I i 1200 100 .8 -Ho 1.0 10 i 5 i 1 r o .1 I I I u • • • « • • • • • * • « • • • • • • • « • • • • • • • « • • • • « • • • • • 0 CONTROL 1 .4 Rf .8 1 1.0 Table IV. Pharmacological comparison of eluates from the standard and pituitary chromatograms of sample D< (5 and 6 year old ammocoetes). Biological assay rat uterus (Mg + +absent) rat uterus (Mg + +present) frog bladder antidiuretic rat milk ejection Biological activity of puri-fled ammocoete material * mU/ml ratio to RU-Mg"1"* 18.7 34.6 1.9 5560 297 53.8 2.9 22.0 1.2 Biological a c t i v i t y of synthetic AVT ** mU/ml ratio to RU-Mg++ 52.9 81.0 1.5 7567 144 Biological a c t i v i t y of synthetic oxytocin *** mU/ml ratio to RU-Mg++ 19.2 19.0 0.99 0 **** o **** 125.0 2.4 32.0 1.7 * eluate of Rf 0.2-0.3 from pituitary chromatogram. ** eluate of Rf 0.2-0.3 from standard chromatogram. *** eluate of Rf 0.5-0.6 from standard chromatogram. **** no effect was noted with the dose given (2.0 mU rat uterus a c t i v i t y ) . - 79 from the pituitary chromatogram on a variety of biological preparations. The High frog bladder to rat uterus ratio of the eluate (297) clearly differentiated the active substance from oxytocin. But this r a t i o , as well as the other potency ratios of the eluate were similar to those of the AVT standard, consid-ering the errors involved in the single 4-point group estimations of biological a c t i v i t y . These single group determinations were necessitated by the low levels of a c t i v i t y in the eluate. Arginine vasotocin has a much higher a c t i v i t y on the amphibian bladder than do other neurohypophysial analogues (Pickering, 1970). the various published values for i t s FB/RU ratio include 910 (Sawyer, 1965a), 270 (Sawyer, 1960a) and 210 (Sawyer, 1960b). In this study, direct tests on ^ synthetic AVT gave the FB/RU ratio as consistently within the range of 150 to 300, and this is in good agreement with the value of 297 found for the ammo-coete eluate. AVT is also characterized by an ADH/RU ratio of approximately 1.5 (Sawyer, 1965a), which is quite different from the ratios for other posterior pituitary hormones, and again this is f a i r l y close to the value of 2.9 f o r the ammocoete material. Incubation of the eluate with 0.01 M sodium thio-glycollate completely abolished the rat uterus a c t i v i t y , suggesting that the active substance was indeed a neurohypophysial peptide. The pharmacological data presented in Table IV suggested that the ammo-coete neurohypophysis possessed AVT, but no evidence was provided for the existence of a second posterior pituitary principle. Since a l l of the ac t i v i t y recovered from the pituitary chromatogram was apparently due to AVT, i t would seem that the T.C.A. was very effective in removing the contaminating active substances present in the crude extract. It w i l l be recalled that treatment of tl i i s same pituitary extract (sample D) with sodium thioglycollate resulted in only a 24% reduction in biological a c t i v i t y (p. 61). Even assuming a 100% recovery of AVT from the chromatogram, the amounts of the hormone eluted - 80 accounted for approximately 40% of the total oxytocic activity that was treated with T.C.A., and therefore 407. of the crude extract a c t i v i t y . Taking into account the losses of AVT that undoubtedly occurred during the purification, the proportion of the crude activity due to AVT was closer to 507.. Thus sample D was similar to the other ammocoete samples in that only about one half of the oxytocic ac t i v i t y extracted from the pituitary tissue was apparently due to a neurohypophysial principle. 3. Purification of ammocoete neurohypophysial material by ion-exchange  chromatography Further pharmacological characterization of the ammocoete neurohypophysial material clearly required more purified hormone than could be made available using paper chromatography as the main purification method. Although prelim-inary purification of the crude extracts with T.C.A. allowed more material to be loaded on to the paper chromatograms, an upper limit of about 1.5 ml was set on the volume of extract that could be applied. Volumes greater than this required excessive amounts of time (greater than 12 hours) to apply to the paper, and resulted in a broad origin and poorer resolution. Consequently, ion-exchange chromatography using a cation-exchange resin was considered as a method for purifying the neurohypophysial material. Whatman microgranular carboxymethyl cellulose resin was chosen because i f i t s high capacity for proteins (Morris and Morris, 1964); this high capacity would allow greater amounts of material to be applied to the column without overloading the resin with the proteinaceous substances present in the extracts. Preliminary experiments with synthetic AVT and oxytocin (see Appendix C) indicated, that with a starting buffer concentration of 0.02 M and pH of 5, AVT was bound to the resin and could be eluted as a sharp peak by increasing the buffer concen-tration to 0.20 M. Oxytocin, on the other hand, was not bound at a buffer 81 concentration of 0.02 M, but passed through the column in the loading volume, Published data (Sawyer, 1965a; Wilson, 1969) indicated that oxytocin would bind to the resin i f the buffer concentration was lowered to 0.002 M. However, this was not possible here, as i t would have necessitated a large dilution (50 to 100 fold) of the sample, and i t seemed unlikely that adequate resolution could be obtained with such a large loading volume (Morris and Morris, 1964). Hence, i t seemed that any neutral principle present in the ammocoete neuro-hypophysial extracts would probably pass through the column in the loading volume; however, i t was possible that i t would s t i l l be detected as long as the loading volume was kept low. On the basis of the pi l o t experiments, i t was decided to purify ammocoete sample E (mature animals from Marshall Creek) on a CMC column after preliminary purification with T.C.A. Since the pituitary and control extracts were treated differently, they w i l l be considered separately, a. Pituitary extract i . Purification Preliminary treatment of the crude pituitary extract with T.C.A. removed about 707. of the Lowry-positive substances and 657. of the total oxytocic a c t i v i t y (Table V); this represented a 3.5 fold purification. Table V. Purification of the pituitary extract of sample E with trichloro-although not necessarily as a single peak (see Appendix C, figure ). acetic acid. Oxytocic ac t i v i t y Lowry peptide Factor of purification * Extract mU/ml 7. recovery pg/ml 7. removed before T.C.A. 36.1 1970 65 71 3.5 after T.C.A. 28.8 568 * calculated by dividing the total amount of Lowry peptide material present in the extract prior to treatment with T.C.A. by the amount present after treatment. 82 The resulting p a r t i a l l y purified extract had an oxytocic activity/Lowry peptide ratio (-specific a c t i v i t y , Wilson, 1968) of 0.052 mU/ug. After adjust-ment to the proper pH and conductivity, the solution was loaded on to a 0.5 cm x 14.0 cm column of CMC resin. Development of the column with a gradient of increasing buffer concentration (0.02 M to 0.20 M) resulted in two fractions of oxytocic a c t i v i t y (figure 19). The f i r s t active fraction, peak I, contained only 1.4%. of the total a c t i v i t y recovered from the column. No antidiuretic a c t i v i t y was detected in t h i s fraction, and the oxytocic a c t i v i t y was totally resistant to the action of sodium thioglycollate. Indeed, i t appeared that incubation of the eluate with thioglycollate resulted in a two-fold increase .in a c t i v i t y , a situation reminiscent of the behaviour of hindbrain extracts towards the thi o l reagent (see p.62). On the basis of this evidence i t was concluded that the small amount of ac t i v i t y in peak I was probably not neuro-hypophysial in nature. The bulk of the a c t i v i t y (98.6%) removed from the column came off as a sharp peak, peak II, i n tubes 28 to 30. The conductivity at which the material came off was comparable to that needed to elute synthetic AVT from a similar column (see Appendix C). The elution volume of the peak was greater than for most of the Lowry-positive material, with the result that the specific activity of tube 29 was 1.57 raU/ug, representing a 30 fold purification of the loading solution. It is probable that the purification was far from complete, however, since synthetic oxytocin was found to have an oxytocic/Lowry r a t i o of 292 mU/ ug, and pure AVT would be expected to give a value of the same order of magni-tude. No oxytocic a c t i v i t y was detected in the void volume of the column, which is where a neutral hormone would be expected to run. With the level of sensit-i v i t y of the bioassays used, an ac t i v i t y of at least 0.35 mU/ml would have been 83 Figure 19 Purification of pa r t i a l l y purified ammocoete neurohypophysial material (sample E) by ion-exchange chromatography on CMC cellulose. a. column =» 0.5 cm x 14.0 cm of Whatman CM-22 carboxymethyl cellulose. b. loading solution - 8.8 ml, at pH 5, conductivity 0.93, containing 100 mU oxytocic a c t i v i t y and 1990 ug Lowry peptide. Specific a c t i v i t y - 0.052 mU/ug. c. starting buffer = 0.02 M, pH 5 sodium acetate (conductivity •. 1.04), to A. d. gradient » 0.02 M, pH 5 sodium acetate to 0.20 M, pH 5 sodium acetate (conductivity » 7.8), A to B. e. flow rate •» 12 ml/hr. f . volume collected/tube - 3 ml. g. recovery = 827. of oxytocic a c t i v i t y . open c i r c l e s - rat uterus ac t i v i t y (magnesium present) of the eluted fractions in mU/tube. closed circles •= Lowry peptide concentration of the eluted fractions in ug/ml. open triangles = specific conductivity of the eluted fractions in millimhos. 84 detected. If i t is assumed that any neutral peptide that may have been present would have passed through the column without being either diluted or concen-trated, then the peptide would have been eluted in the loading volume of 8.8 ml (see figure 19). Therefore, no more than 3.1 mU (0.35 mU/ml x 8.8 ml) or about 3% of the total a c t i v i t y could have been present in the region l i k e l y to contain a neutral hormone without being located. In actual fact, the minimum possible amount was probably less than this, since the material in peak I with an acti v i t y of 0.10 mU/ml was detected. Hence i t is improbable that the pituitary extract contained more than 0.9 mU (0.10 mU/ml x 8.8 ml) of an oxytocin-like principle; this represents less than 17, of the total a c t i v i t y present in the extract prior to chromatography. i i . Pharmacological characterization of the active peak (peak II) from the column The pharmacological characterization of peak II was accomplished by assaying the most active fraction from the peak (tube 29) against synthetic AVT on a variety of biological preparations. The eluate was acid i f i e d to about pH 4.5 with galcial acetic acid and was then assayed against an AVT standard of 200 ng/ml concentration made up in ammonium acetate at the same pH and conductivity of the unknown. The acidic pH of the solutions was considered advisable to reduce inactivation of the hormone (Ryle and Sanger, 1955; Wilson, 1968). The same standard was used throughout the series of bioassays, the assumption being that the rates of decay of standard and unknown would be identical. The results of the pharmacological comparison between tube 29 and synthetic AVT are given in Table VI and figure 20. By way of contrast, figure 20 also gives the potencies of equivalent amounts (as assayed on the rat uterus with no magnesium) of oxytocin and arginine vasopressin on the same preparations with AVT as the standard. From the data, 85 table VI. Biological a c t i v i t i e s of the AVT peak (peak II) resulting from ion-exchange chromatography of the pituitary extract of sample E. Biological assay Potency of peak II, Ratio to potency on ng AVT/ml * rat uterus (-Mg*-1) * rat uterus (-M^ -*) 162.4 (127.5-206.9) 1.00 rat uterus (+Mg+H) 170.8 (154.0-189.4) 1.05 (0.82-1.35) frog bladder 246.2 (174.5-347.4) 1.51 (1.02-2.24) antidiuretic 176.5 (115.2-270.5) 1.08 (0,68-1.72) na t r i f e r i c 190.6 (120.0-302.8) 1.17 (0.72-1.91) vasopressor 151.2 (121.4-188.4) 0.93 (0.68-1.26) * the values i n parentheses refer to 957. fiducial limits. 86 Figure 20 Potencies of purified ammocoete neurohypophysial material (peak II, sample E) on several preparations when assayed against synthetic arginine vasotocin. Upper histogram - columns indicate the biological a c t i v i t i e s of peak II on the various preparations, expressed in ng AVT/ml. - v e r t i c a l lines through columns indicate 957. confidence limits. Middle histogram - columns indicate the calculated potencies of oxytocin on the various preparations expressed in ng AVT/ml. Lower histogram - columns indicate the potencies of arginine vasopressin on the various preparations expressed in ng AVT/ml. * values were calculated from data given by Sawyer (1965a). • values were calculated from data given by Pickering (1970). 400r-300 -< 200 o z 100 PEAK II RU(-Mg) RU(+Mg^ FB ADH NA VP > < 200r-100 0«-RU(-Mg^ RU(fMg"1* FB OXYTOCIN ADH * NA + V P * 5000p 4500 -4000 -3500 -> < O 200 I00 I — \ ARGININE VASOPRESSIN vWV WW 1 I RU (-Mg*) RU (+Mg+t* FB ADH * NA • V P * 87 i t was evident that none of the potency ratios of tube 29 differed s i g n i f i -cantly from one, and that therefore the active material in the peak was indistinguishable from arginine vasotocin, at least with those bioassays employed. On the other hand, figure 20 indicates that oxytocin and vasopressin would have been differentiated from AVT by pharmacological comparison. The former hormone has low antidiuretic and vasopressor a c t i v i t i e s when compared to its oxytocic potency, while vasopressin exhibits low oxytocic a c t i v i t i e s . Furthermore, both of the mammalian principles have l i t t l e effect on the frog skin and bladder. Additional evidence supporting the view that the activity in peak II was due to a neurohypophysial hormone came from treatment of a .sample from the peak with sodium thioglycollate. Incubation of the aliquot with the th i o l regeant resulted in a 977. reduction in the rat uterus potency; this compared favorably with a 967. inactivation of oxytocin treated in the same manner. b. Hindbrain extract During the period that the pituitary extract was being purified and assayed, the control extract was stored at 4° C in a stoppered v i a l . Over this time (13 days) a 437. reduction in oxytocic potency occurred. In the course of this study, crude extracts of neurohypophysial tissue were stored for comparable lengths of time, but they underwent only slight or no inactiv-ation. It is possible that that the hindbrain a c t i v i t y that was destroyed was due to the labile substances detected on the paper chromatograms of crude extracts (see pp. 63-67). After 13 days storage, the control material was treated with 10% T.C.A.; the purification achieved was similar to that obtained with the pituitary extract, as can be seen in Table VII. A sample of the T.C.A. purified extract was then incubated for 12 hours with 0.01 M sodium thiogly-collate and assayed against control material that had been treated in exactly 88 Table VII. Purification of the hindbrain extract of sample E v i t h t r i -chloroacetic acid. Oxytocic a c t i v i t y Lowry peptide Factor of Extract purification * • mU/ml % recovery ug/ml % removed •  before T.C.A. 20.0 1750 70 79 4.7 after T.C.A. 14.1 370 * calculated by dividing the total amount of Lowry peptide material present in the extract prior to treatment with T.C.A. by the amount present after treatment. Table VIII. Biological a c t i v i t i e s of the pa r t i a l l y purified hindbrain extract of sample E. Potency of extract *  Biological assay mU/ml rat i o to total ratio to thioglycol-__ rat uterus (-Mg*"!) late-labile rat uterus rat uterus (-Mg+ )^ 14.1 rat uterus (^Mg^ 14.1 1.0 3.6 vasopressor 9.8 0.7 2.5 rat milk ejection 3.8 0.3 1.0 * the extract was assayed against mammalian oxytocin and vasopressin, and not against arginine vasotocin. . . • - 89 the same way except that no thioglycollate had been added. A complete assay (4 groups) was performed and a mean loss of ac t i v i t y of 28.1% (95% confidence interval «• 13.8%-40.1%) was found to have occurred in the thioglycollate treated material. Since thioglycollate is thought to be f a i r l y specific in Its effects on neurohypophysial principles (Rudinger and Krejci, 1968), this result suggested the possible presence of a posterior pituitary-like hormone in the hindbrain extract. This pos s i b i l i t y was increased by assay of the remaining T.C.A.-treated control solution against oxytocin and vasopressin on several different preparations (Table VIII). In addition to Its effects on the rat uterus, the extract exhibited vasopressor and milk ejecting a c t i v i t i e s . The presence of the latter a c t i v i t y was particularly surprising, since Bisset et a l , 1967) have reported that this assay was quite specific for neurohypoph-y s i a l hormones, with other compounds such as 5-HT, acetylcholine and substance P having effects only at high dose levels. When the potencies of the extract on the various bioassays were divided by the portion of the total oxytocic a c t i v i t y destroyed by thioglycollate (28.1% or 4.0 mU/ml), the ratios obtained were consistent with the presence of AVT. The VP/RU ratio (2.5) was especially suggestive of AVT, since no other natural neurohypophysial analogues possess a value of the same order of magnitude (Sawyer, 1965a). The data were far from conclusive however. It is possible that assay of the extract on the isolated frog bladder might have c l a r i f i e d the situation, but insufficient material remained to allow this. It seems possible, however, that a portion (about 8%) of the total oxytocic a c t i v i t y extracted from the hindbrain tissues of sample E was due to arginine vasotocin. The identity of the other 92% of the activity could not be ascertained. Addition of atropine (1 ug/ml) and 2-brom lysergic acid diethylamide (0.5 ^ig/ml) to the rat uterus muscle bath in no way ditntn-ished the response of the uterus to the T.C.A.-treated extract (see figure 21). 90 Figure 21 Effects of the specific antagonists, atropine and 2-brom-lysergic acid diethylamide, on the oxytocic activity of partially purified ammocoete hindbrain material (sample E). arrows - times of addition of atropine (1 ug/ml) and 2-brom LSD ( 0 . 5 ug/ml) to the bathing solution, s o l i d triangles - responses to synthetic oxytocin. Doses are expressed in mU. so l i d c i r c l e s - responses to the sample E extract. Doses are expressed in microliters, open squares - responses to acetylcholine. Doses are expressed in micrograms. open c i r c l e s - responses to 5-hydroxytryptamine. Doses are expressed in micrograms. A A © a • B aos cos 5.o 3.75 a. ,7o lo"o M 3.75 aw i°o ° A ATROPINE 2-BROM LSD 91 Atropine is a specific antagonist to acetylcholine, while 2-brom LSD completely abolishes the uterine responses to 5-HT (Brocklehurst and Z e i t l i n , 1967). Thus no significant amount of the oxytocic ac t i v i t y of the extract could have been due to these substances. 92 B. Studies of the Adult 1. The oxytocic a c t i v i t i e s of crude extracts and the effects of sodium  thioglycollate on the activity. Two separate lots of adult tissues were extracted and assayed for rat uterus a c t i v i t y ; the results are given in Table IX. In one l o t , sample G, an effort was made to dissect neurohypophysial tissue i n a more precise manner than in any other sample studied in this research, so that there would be less contamination from brain tissue. The biological a c t i v i t i e s measured would then reflect more closely the true values for the pars nervosa. Only a wedge of tissue containing the neurohypophysis and a portion of the diencephalon was taken as the pituitary component for sample G; spinal cord was used as a control (see pp. 19-20). The dissection of the other lot of adults (sample H) was the same as that carried out on the ammocoete samples.(see p. 19). Although the activity/mg of the sample G neurohypophysial material was significantly greater than that of sample H, the t o t a l activity extracted/animal was about the same in both groups, even though the amount of tissue taken/animal was greater in sample H. This suggested that most of the ac t i v i t y extracted from the lamprey forebrain was localized i n the ventral diencephalon, and that l i t t l e was present in the telencephalon; although the poss i b i l i t y of a difference between the two groups of animals could not be eliminated. Similarly, i t appeared that the majority of the a c t i v i t y extracted from the control tissue of sample H was located i n the mesencephalon and metancephalon, since the spinal cord control of sample G contained only small amounts of rat uterus a c t i v i t y . Treatment of the neurohypophysial extract of sample G with sodium thio-glycollate resulted in a 50 to 70% reduction in oxytocic potency (Table IX). Table IX. Oxytocic a c t i v i t i e s of crude extracts of adult lamprey neurohypophysial and control tissues and their resistance to thioglycollate. Neurohypophysial Extracts Control Extracts ** Sample * mU/mg+ mU/animal+ 7. after thio- mU/mgt mU/animal+ % after thio-glycol late ' glycol late 2.00 (1.63-2.48) 30-50 0.27 0.046 131 1.88 (1.63-2.16) — 1.39 (1.19-1.62) 1.69 (1.44-1.97) — * values in parentheses indicate, the number of animals i n each sample. ** the control tissue for sample G was spinal cord, for sample H i t was spinal cord, metancephalon and mesen--cephalon. •f values in parentheses refer to 95% fiducial limits. G-adults from Salmon 5.45 (4.41-6.73) River (146) H«adults from Salmon River, Bertrand 1.83 (1.59-2.11) Creek and Marshall Creek (141) vO 94 An aliquot of the control extract, on the other hand, possessed 131% of the act i v i t y of untreated material after incubation with thioglycollate. Both results were similar to those obtained from treatment of the ammocoete extracts with the t h i o l reagent, and they suggested that signifigant amounts of b i o l -ogically active contaminants were present in the pituitary extracts. 2. Purification and pharmacology of the adult tissues dissected in a more  precise manner (sample G) a. Pharmacological characterization of the crude neurohypophysial extract Although the results of the thioglycollate treatment of the pituitary extract of sample G had indicated that not a l l of the oxytocic a c t i v i t y present was due to a neurohypophysial principle, i t was decided to assay the crude extract on several different preparations. Sawyer and his co-workers (Sawyer e£ al_, 1961) had carried out a similar characterization of a crude neurohypophysial extract from Petromyzon marinus , and i t was considered of interest to compare their results with the potencies of the Lampetra extract. Synthetic AVT was used as the standard. It was expected that i f not a l l of the rat uterus a c t i v i t y of the extract were due to AVT, the extract would show lower potencies on the other assays. The results are presented in Table X and figure 22; the figure also gives the a c t i v i t i e s of the extract when assayed against oxytocin and vasopressin (Pitressin) by the •'classical" method of assay. Sawyer's extract was also assayed against oxytocin and vasopressin, and his results were similar to those obtained here. He concluded that the bulk of the neurohypophysial a c t i v i t y in Petromyzon was due to AVT, since the pharmacological profile of the extract was similar to the one obtained for AVT. Assay of the lampetra neurohypophysial extract directly against AVT indicated that other active substances besides AVT were present. As expected, the potency of the extract on the rat uterus assay (magnesium 95 Table X. Biological a c t i v i t i e s of a crude extract of neurohypo-physial and diencephalic tissues from adult lamprey (sample G). Biological assay rat uterus (-Mg4"4) rat uterus (•Mg4"') antidiuretic frog bladder Potency of extract, ng AVT/ml * 769.2 (724.0-817.3) 501.7 (452.2-556.7) 695.7 (510.3-948.1) 317.7 (149.4-673.0) Ratio to potency on rat uterus (-Mg * 1.00 0.65 (0.58-0.73) 0.91 (0.67-1.23) 0.41 (0.19-0.88) * the values in parentheses refer to 95% fiducial limits. Figure 22 Biological a c t i v i t i e s of a crude extract of adult lamprey neurohypophysial material (sample G). Upper histogram - potencies of the extract when assayed against the mammalian ' principles, oxytocin and arginine vasopressin. Lower histogram - potencies of the extract when assayed against synthetic arginine vasotocin. - v e r t i c a l lines indicate 957. confidence limits. 9000 8000-ASSAYS OF EXTRACT AGAINST OXYTOCIN AND VASOPRESSIN 600 v 400 3 E 200 0 L RU(-Mgt RU(+Mg) ADH FB 1200 ASSAYS OF EXTRACT AGAINST AVT 1000 800 > < 600 400 200 RU <-MgY RUtWgt ADH FB 97 absent) was greater than i t s potency on the other preparations. It seemed possible that the extra rat uterus a c t i v i t y could be explained in two ways : 1) by the presence of non-neurohypophysial, biologically active, oxytocic contaminants such as substance P or 5-HT. 2) by the presence of small amounts of ^ second neurohypophysial principle which exhibited a strong oxytocic potency, but which had low a c t i v i t i e s on the other biological assays. Of the naturally occurring posterior pituitary analogues studied, only AVT shows high frog bladder a c t i v i t y , but strong antidiuretic a c t i v i t i e s are possessed by other basic principles such as lysine and arginine vasopressin. Oxytocin-like analogues have relatively low potencies on both assays. While the rat uterus act i v i t y of a l l neurohypophysial hormones is potentiated by the presence of magnesium in the bathing solution, that of oxytocin is enhanced less than for the others* The RU (+Mg4"*)/RU (-Mg*4) ratio for oxytocin is usually given as one or slightly less than one, since most biological assays are carried out using either oxytocin or the USP Reference Posterior Pituitary Powder as the standard {Munsick, 1968). Thus, oxytocin, or an oxytocin-like principle, seemed the only po s s i b i l i t y for a second hormone in Lampetra. b. Partial purification of the neurohypophysial extract of sample G by  u l t r a f i l t r a t i o n To resolve the question of a second neurohypophysial principle in the pituitary extract, the remaining volume of sample G was subjected to paper chromatography in butanol:acetic acid:water. Preliminary removal of the Lowry-positive contaminants was accomplished by u l t r a f i l t r a t i o n , using an appropriate syringe f i t t e d with an u l t r a f i l t r a t i o n membrane (Amicom Diaflo UM-10 membrane, molecular weight cutoff range « 10,000). This method was employed as a possible alternative to treatment of the extract with T.C.A. It seemed preferable to precipitation of the proteinaceous substances with T.C.A. because i t was less 98 time consuming, i t avoided the addition of extra reagents, and i t seemed less l i k e l y to produce arti f a c t s by the possible cleavage of protein materials. Table XI. gives the results of the purification. It appeared that u l t r a f i l -tration and T.C.A. treatment possessed about equal merits as purification methods, although s l i g h t l y less Lowry-positive material was removed by the former procedure..However, t r i a l runs using solutions of albumin and oxytocin or AVT (see Appendix B) indicated a considerable v a r i a b i l i t y in the effective-ness of individual u l t r a f i l t r a t i o n membranes, and the method was discarded in favor of the use of T.C.A. c * ^pe*" chromatography of the p a r t i a l l y purified neurohypophysial  extract of sample G Separation of the active principles in the sample G pituitary extract was accomplished by paper chromatography in butanol:acetic acid:water (4:1:5). Synthetic oxytocin and AVT were run separately as standards. As the control extract of sample G contained very small amounts of oxytocic a c t i v i t y , i t was not chromatographed. The separation of the neurohypophysial extract that was achieved in shown in figure 23. Oxytocic a c t i v i t y was detected between Rf*s 0.2 and 0.9; this encompassed the entire region occupied.by the AVT and oxytocin standards. Since no ac t i v i t y was found at the origin, i t would seem that the ultrafiltratiom method was as effective as T.C.A. in removing the slow-running, biologically active contaminants from the crude extract. The bulk of the a c t i v i t y eluted from the pituitary chromatogram was present in Rf 0.3-0.4, as was most of the AVT recovered from the standard chromatogram. Assay of the extract eluates for antidiuretic a c t i v i t y indicated that Rf's between 0.2 and 0.5 possessed potencies more or less consistent with the presence of AVT (see figure 23). However, only a trace of antidiuretic a c t i v i t y was found in Rf 0.5-0.6, and none was detected in Rf 0.7-0.8. Unfortunately, 99 Table XI. Partial purification of crude extracts of adult lamprey neurohypo-physial tissues by u l t r a f i l t r a t i o n (sample G) and by treatment with trichloroacetic acid (sample H). Extract Oxytocic a c t i v i t y Lowry peptide Factor of purification * mU/ml % recovery jig/ml 7, removed  sample G before u l t r a f i l t r a t i o n 109.1 1380 47 67 3.2 after u l t r a f i l t r a t i o n 51.4 460 sample H before T.C.A. 36.6 1433 90 68 3.2 after T.C.A. 32.9 450 * calculated by dividing the total amount of Lowry peptide material present in the extract prior to purification by the amount present after purification. 100 Figure 23 -Paper chromatography of a part i a l l y purified extract of adult lamprey neurohypophysial tissue (sample G) in butanoltacetic acid: water (4:1:5). white columns •» rat uterus a c t i v i t y (magnesium absent) in mU/ml. . ; cross-hatched columns = rat antidiuretic a c t i v i t y in mU/ml. numbers above columns «» antidiuretic ratio of the eluate •= rat antidiuretic a c t i v i t y rat uterus activity (-Mg**"). AVT chromatogram load - 500 ng (50 mU). recovery «= 237.. Oxytocin chromatogram load = 25 mU. recovery = 207.. Pituitary chromatogram load «= 50 mU oxytocic a c t i v i t y , 490 ug Lowry peptide. recovery - 357. of oxytocic ac t i v i t y . Rf 101 there was insufficient material for the antidiuretic assay of the eluate of Rf 0.6-0.7. Nonetheless, the data suggested that only a portion of the rat uterus a c t i v i t y eluted from Rf 0.5-0.8 was due to AVT, since not enough anti-diuretic a c t i v i t y was present. Moreover, AVT does not normally run in this region of the chromatogram; rather i t is the area in which neutral hormones migrate. A l l naturally occurring neutral neurohypophysial analogues exhibit extremely low antidiuretic potencies (ADH/RU ratio for oxytocin « 0.0024, Sawyer, 1965$; thus, i t seemed possible that the fast moving substance was an oxytocin-like principle. Unfortunately, lack of material did not allow treatment of the eluates with thioglycollate, which would have been of great help in determining whether the ac t i v i t y eluted from Rf 0.5-0.8 was neurohypo-physial i n nature. 3. Purification and pharmacology of adult sample H Since the results obtained for the neurohypophysial extract of sample G had suggested the presence of a second active neurohypophysial hormone in adult Lampetra, i t was decided to investigate this p o s s i b i l i t y further with sample H. As with the ammocoete material, separation of the active principles by paper chromatography did not seem adequate to handle the relatively large amounts of material necessary to carry out a pharmacological characterization of the hormones. The success encountered with column chromatography of ammo-coete sample E (pp. 81-84) prompted the use of this method to purify sample H. Again, preliminary purification of the extracts with T.C.A. seemed advisable to lessen the pos s i b i l i t y of overloading the column with contaminating sub-stances. Both pituitary and control extracts were purified by the same methods, as i t was f e l t that separation of the active substances in the latter extract would settle the question of the possible presence of AVT in hindbrain tissues (see pp. 87-89). 102 a. Purification and pharmacology of the pituitary extract of sample H i . Preliminary purification with trichloroacetic acid. Treatment of the pituitary extract with 10% T.C.A. resulted in a 68% removal of the Lowry-positive material, as shown in Table XI. This degree of purification was similar to that obtained with the ammocoete extracts; however, a greater recovery of oxytocic activity was obtained with the adult extract. Whether this higher recovery represented a greater proportion of AVT present in the adult extract, or whether i t was due to the presence of greater amounts of T.C.A.-resistant active contaminants, could not be ascertained. i i . Column chromatography of the part i a l l y purified pituitary extract Chromatography of the part i a l l y purified extract on Whatman CMC resin yielded three fractions containing 97% of the oxytocic ac t i v i t y loaded on to the column (figure 24). Eighty-eight percent of the recovered a c t i v i t y was present in the last fraction to leave the column, peak c which was eluted at about the same conductivity as synthetic AVT (see Appendix C). The bulk of the Lowry-positive material was removed before the elution of peak c, with the result that the purification of the fraction was 66.4 fo l d . The specific a c t i v i t y of the peak (4.85 mU/pg) was much less than that of naturally occurr-ing neurohypophysial peptides (e.g. synthetic oxytocin •» 292 raU/ug), suggesting that the purification was not complete. Two other regions of oxytocic activity were eluted from the column. Peak a, containing only 7 mU, or 3% of the total a c t i v i t y , came off the column in the loading volume, which also contained most of the Lowry-positive substances. Peak b, with 17 mU, or 9% of the i n i t i a l a c t i v i t y , was eluted from the column just in front of peak c; its position corresponded^closely with the f i r s t peak eluted from the ammocoete material that was subjected to ion-exchange chromatography (peak I, sample E; see pp. 81-83). 103 Figure 24 Purification of a part i a l l y purified extract of adult lamprey neurohypophysial tissue (sample H) by ion-exchange chromatography on CMC cellulose. a. column - 0.5 cm x 14.0 cm of Whatman CM-22 carboxymethyl cellulose. b. loading solution « 10.9 ml, at pH 5, conductivity 1.01, containing 193 mU oxytocic a c t i v i t y and 2640 ug Lowry peptide. Specific a c t i v i t y = 0.073 yug/ml. c. starting buffer - 0.02 M, pH 5 ammonium acetate (conductivity » 1.02), to A. d. gradient = 0.02 M, pH 5 ammonium acetate to 0.20 M, pll 5 ammonium acetate (conductivity => 7.8), A to B. e. flow rate = 12 ml/hr. f. volume collected/tube «=» 3 ml. g. recovery = 977. of oxytocic activity. open ci r c l e s «=• rat uterus activity (magnesium present) of the eluted fractions in mU/tube. closed circles » Lowry peptide concentration of the eluted fractions in ug/ml. open triangles «= specific conductivity of the eluted fractions in millimhos. mU/TUBE -fc CD ro cn o o o o -T 1 1 1 104 i i i . Pharmacology of the eluted peaks In order to pharmacologically characterize peak c, the pooled material from tubes 40, 41 and 42 was assayed against synthetic AVT (200 ng/ml) made up in ammonium acetate buffer at the same pH and conductivity as the peak (pH"»5, conductivity^.5 mmho). Since the presence of AVT in adult lamprey had been demonstrated previously (Sawyer, 1965a; Foll e t t and Heller, 1964), the number of bioassays by which the eluate material was tested was less than had been employed in the pharmacological characterization of the ammocoete material. The potencies of the peak by the various assays are l i s t e d in Table XII and figure 25, and can be compared with the pharmacological profile of the crude extract of adult lamprey neurohypophysial tissue (sample G, Table X and figure 22). While the RU («J-Mg+VRU (-Mg4) and VP/RU ratios were not significantly different from one, the FB/RU ratio was greater than unity. This latter dis-crepancy was most lik e l y an a r t i f a c t , due to the method in which this partic-ular assay was carried out. With most assays, the doses comprising a group were given in random order. In this case, however, standards were given f i r s t ; this was because individual bladders would often begin to leak part way through a group, thereby wasting the doses given prior to the onset of leaking. Since material from peak c was relatively scarce, injections of the unknown were not given un t i l the standard responses had been obtained, so as to lessen the po s s i b i l i t y of wastage of peak material. However, with v i r t u a l l y a l l bladders that lasted for a complete group, the sensitivity to neurohypophysial hormones increased during the course of the assay. Thus the unknown was assayed on the bladder when the sensitivity to posterior pituitary hormones was greater than when the standard was tested, and potencies above the true value were obtained. This explanation seems quite plausible in accounting for the high frog bladder a c t i v i t y of the peak. Since AVT is the most active neurohypophysial analogue Table XII. Biological a c t i v i t i e s of the AVT peak (peak c) resulting from ion-exchange chromatography of a . pituitary extract from adult lamprey (sample H). Biological Assay rat uterus (-Mg*4) rat uterus (+Mg4+) vasopressor frog bladder Potency of peak c, ng AVT/ml * 108.7 (95.3-123.9) 111.2 (102.0-121.1) 103.8 (73.4-146.5) 177.2 (127.9-245.6) Ratio to potency on rat uterus (-Mgr4) * 1.00 1.02 (0.88-1.18) 0.96 (0.67-1.36) 1.62 (1.21-2.19) * the values in parentheses refer to 95% fiducial limits. 106 Figure 25 Potencies of purified neurohypophysial material (peak c) from adult lamprey (sample H) on several biological assays using synthetic arginine vasotocin as the standard. - the vertical lines through the columns indicate 95% confi-dence limits. 250 200 150 > < 100 50 RU (-Mg+) RU (fMg"+) VP FB • 107 in promoting water movement across the amphibian bladder (Pickering, 1970), i t seems extremely unlikely that the.enhanced frog bladder potency was due to the presence of contaminating substances* It i s of interest to compare the results obtained from peak c with the pharmacological profile of the crude extract of the other adult group (sample G, Table X and figure 22). It w i l l be recalled that the crude extract of sample G contained more rat uterus ac t i v i t y than could be accounted for by the presence of AVT alone. The actual excess of oxytocic a c t i v i t y (compared to the rat uterus potency with magnesium present in the bathing solution) was about 357.. The amount of rat uterus ac t i v i t y in peak c, on the other hand, was entirely consistent with AVT, indicating that the contaminating substances had been removed by the purification methods. A portion (about 12%) of the extra oxytocic ac t i v i t y in the crude extract of sample G could be accounted for by peaks a and b of sample H, assuming, of course, that the two samples contained the same types of contaminants. The remainder of the non-neurohypo-physial rat uterus a c t i v i t y present in sample G was probably removed from the sample H extract by treatment with T.C.A. About 163 mU of AVT were recovered from the column; this represents approximately 737. of the total activity present in the extract prior to puri-fication with T.C.A. and column chromatography. Thus, i t seems l i k e l y that the high recovery of activity from the T.C.A. purification step (Table XI) was due i n part to the high proportion of the ac t i v i t y that was due to AVT. The small amounts of ac t i v i t y present in peaks a and b did not allow an extensive investigation of these fractions. The oxytocic ac t i v i t y of the peaks did not prove labile to sodium thioglycollate, indicating that the active substances were probably not neurohypophysial in nature. Thus, although peak a was eluted in the region where a neutral hormone would be expected to come off, 108 i t is unlikely that i t contained any such substance. This fraction contained 3% of the total a c t i v i t y applied to the column, so i t is improbable that the neurohypophysis of adult Lampetra possessed signifigant amounts of a neutral principle. Nothing else was done with either fraction. The elution character-i s t i c s of peak b suggested that i t was identical to peak I of the chromato-graphed ammocoete pituitary extract (sample E, p.-82). It is also possible that the active substances In both peaks a and b were the same as the material eluted from Rf 0.5-0.8 of the paper chromatogram of the other adult pituitary extract (sample G, pp. 98-101). But without further pharmacological data, neither hypothesis can be stated with any degree of certainty. b. Purification and pharmacology of the hindbrain extract of adult ' s a r n P l e H i . Purification During the course of purification of the hindbrain material, the fate of the Lowry-positive substances was not followed. Treatment of the extract with T.C.A. resulted in removal of 61% of the oxytocic a c t i v i t y ; this was sl i g h t l y greater than the losses obtained in the T.C.A.-treatment of other lamprey extracts. The p a r t i a l l y purified extract was placed on a CMC column and the active substances were eluted by a two-step buffer gradient : 1) 0.02 M to 0.20 M, pH 5 ammonium acetate; 2) 0.20 M to 1.0 M, pH 7 ammonium acetate. The latter gradient was employed to elute any substances with a high binding a f f i n i t y for the resin. Four regions of oxytocic a c t i v i t y were eluted from the column, a l l of which came off before completion of the f i r s t step of the gradient (figure 26). Peak 1, with 17% of the total a c t i v i t y , came off in the loading volume. Peak 2 contained 8% of the ac t i v i t y recovered from the column and was eluted at the beginning of the gradient. Peaks 3 and 4, accounting for 43% and 32% 109 Figure 26 Separation of active substances present in a p a r t i a l l y pur-i f i e d extract of adult lamprey control tissue (sample K) using ion-exchange chromatography on CMC cellulose. a. column «= 0.5 cm x 14.0 cm Whatman CM-22 carboxymethyl cellulose. b. loading solution » 21.0 ml, at pH 5, conductivity 1.0, containing 74 mU oxytocic a c t i v i t y . c. starting buffer » 0.02 M, pH 5 ammonium acetate (conductivity « 1.04), to A. d. gradient « i ) 0.02 M, pH 5 ammonium acetate to 0.20 M, pH 5 ammonium acetate (conductivity = 7.8), A to B. » i i ) 0.20 M, pH 5 ammonium acetate to 1.0 M, pH 7 ammonium acetate (conductivity => 37.2), B to C. e. flow rate «=» 15 ml/hr. f. volume collected/tube = 3 ml. g. recovery «* 76% of oxytocic activity. closed c i r c l e s •= rat uterus a c t i v i t y (magnesium present) of the eluted fractions in mU/tube. soli d line •» specific conductivity of the eluted fractions in millimhos. LOADING VOLUME A B C I H 1 I TUBE NUMBER 110 of the recovered activity respectively, came off at intermediate positions in the 0.02 to 0.20 M gradient. Only 76% of the ac t i v i t y loaded on the column vas recovered; this was a somewhat lower recovery than was obtained with chromatography of the pituitary extract of sample H. No other active fractions were detected, however, even when the buffer concentration was raised above 0.20 M. It seems unlikely that any active material was retained on the column, unless the substances were highly basic in nature or were bound by non-ionic interactions. i i . Pharmacology of the eluted peaks The results of pharmacological and chemical tests on the active fractions eluted from the column are given in Table XIII. It was apparent that peak 4 had many pharmacological a c t i v i t i e s in common with AVT; the presence of milk ejecting and strong frog bladder a c t i v i t i e s was particularly interesting, since the former effect is rather specific to neurohypophysial hormones (Bisset et a l , 1967), while the latter was strongly suggestive of AVT. The l a b i l i t y of the oxytocic ac t i v i t y to sodium thioglycollate and the position of the peak in the elution profile also hinted at the presence of AVT in peak 4. On the basis of a l l these data, i t seemed probable that small amounts of arginine vasotocin were present in the hindbrain tissues of adult Lampetra richardsoni. The amount of AVT eluted represented 32% of the activity recovered from the column and about 9% of the total rat uterus a c t i v i t y extracted from the hind-brain tissues of sample H. From the results presented in Table XIII, i t would seem that the activ-i t i e s contained in peaks 1, 2 and 3 were not due to neurohypophysial principles; the lack of milk ejecting a c t i v i t y in the eluates and their resistance to thioglycollate were both strongly suggestive of this. From its position in the elution p r o f i l e , peak 3 may have been the same as peak I of the chromatographed Table XIII. Biological a c t i v i t i e s of fractions resulting from ion-exchange chromatography of a p a r t i a l l y purified hindbrain extract from adult lamprey (sample H). The peaks were assayed against oxytocin and vasopres-s i n (Pitressin). Potency of peak 1, Potency of peak 2, Potency of peak 3, Potency of peak 4 Biological assay mU/ml mU/ml mU/ml tnU/ml ratio to potency on rat uterus (-Mg*7 * rat uterus (-Mg ) -- — 1.6 1.5 1.0 (1.0) rat uterus (+Mg ) 0.4 0.5 — 3.5 2.3 (1.9) antidiuretic 0.8 0 0 2.3 1.5 (1.6) vasopressor -- — 0 5.6 3.7 (2.0) frog bladder 0 — — 369.5 246.3 (150-300) rat milk ejection 0 0 0 0.6 0.4 (2.0) l a b i l i t y of rat uterus a c t i v i t y to sodium t h i - resistant resistant resistant destroyed oglycollate — the fraction was not tested for the a c t i v i t y . 0 no acti v i t y was detected. * the values in parentheses give the potency ratios for AVT. A l l but the FB/RU and ME/RU ratios were taken f ? T T T i ?• <>-r O .96 r"0; these two exec- Tons were determined during the present investigations • 112 ammocoete pituitary extract (sample E, p. 82), and also peak b of the neuro-hypophysial extract of sample H (p. 102). Also, materials in peak 1 may have been present in peak a of the pituitary extract of sample H (p. 102). Nothing can be said about the substance(s) contained in peak 2. 113 C. Discussion (Section II) A summary of the work described. In t h i s s e c t i o n i s presented i n Table XTV. A l l the extracts of lamprey neurohypophysial t i s s u e s appeared to possess a neurohypophysial p r i n c i p l e , and f o r most samples the evidence suggested the presence of arg i n i n e vasotocin. This was p a r t i c u l a r l y true f o r ammocoete samples D and E and f o r both adult groups (samples G and H). Thus i t appears l i k e l y that L« richardsoni' elaborates AVT at a l l stages of i t s l i f e . No evidence was provided f o r the presence of a second neurohypophysial peptide, and the methods used precluded the p o s s i b i l i t y of more than about |7. of the t o t a l a c t i v i t y extracted from the t i s s u e s being due to a second a c t i v e hormone. The l e v e l s of AVT present i n the lamprey were very low. Data r e s u l t i n g from treatment of the p i t u i t a r y extracts with sodium t h i o g l y c o l l a t e and T.C.A., and from chromatography its&cated that no more than 507. of the t o t a l a c t i v i t y was due to the hormone. When the l e v e l of contaminating a c t i v i t y was subtracted from the t o t a l , i t was evident that the hypothalamo-neurohypophysial system of adult western brook lamprey contained about 1 mU of oxytocic a c t i v i t y or approximately 10 ng of AVT. Ammocoetes possessed even les s hormone (the changes i n hormone content during the l i f e h i s t o r y of the lamprey w i l l be dealt with i n se c t i o n I I I ) . In comparison with other vertebrates t h i s amount was extremely low. For example, the p o s t e r i o r p i t u i t a r y of the sheep (Oris  a r l e s ) possesses about 30,000 mU of a c t i v i t y ( V i z s o l y i , 1968), that of the domestic fowl (Gallus domesticus) contains 268 mU , that of the trout (Salmo  i r l d e u s ) 81 raU (H e l l e r and Pi c k e r i n g , I960), and that of the dogfish (Squalus  acanthlas), 147 mU (Perks, 1966). Of the two species of lamprey reported on i n the l i t e r a t u r e , Lampetra f l u v i a t l l u s possessed 1.2 mU of oxytocic a c t i v i t y per neurohypophysis ( F o l l e t t and H e l l e r , 1964), while f o r e b r a i n e x t r a c t s of Table XIV. Summary of the chromatographic and pharmacologic investigations carried out on pituitary and control extracts of the lamprey, Lampetra richardsoni. * Crude Extract Purified Extract Evidence for the Presence of AVT Sample A » 1 & 2 year old ammocoetes pituitary extract control extract B «• 3 year old ammocoetes pituitary extract control extract C " 4 year old ammocoetes pituitary extract control extract ammocoetes from Section I I I pituitary extract control extract Biological assays carried out RU RU RU RU RU RU RU RU Results of thiogly-collate treatment p a r t i a l l y l a b i l e resistant p a r t i a l l y l a b i l e resistant p a r t i a l l y l a b i l e resistant Methods of iBiological assays purification carried out pituitary paper control paper paper paper RU, RUMg, ADH RU, ADH !RU, RUMg, ADH RU, ADH Results of thiogly-collate treatment completely labile RU, thio i n crude extract, none. RU, thio i n crude extract. Rf, RUMg/RU, ADH/RU, thio from purified material. none. RU, thio from crude extract. ADH/RU, RUMg/RU, Rf from purified material. none. Table XIV. Cont'd. Crude Extract Sample D - 5 & 6 year old ammocoetes pituitary extract control extract E «•> mature ammocoetes pituitary extract control extract G » adults pituitary extract-control extract H - adults pituitary extract control extract Biological assay Results of thiogly-carried out collate treatment Purified Extract Evidence for the Presence of AVT par t i a l l y l a b i l e resistant RU RU RU RU RU, RUMg, FB, ADH p a r t i a l l y labile RU resistant RU RU Methods of purification T.C.A., paper T.C.A., paper T.C.A., CMC UF, paper T.C.A.,CMC T.C.A., CMC * Abbreviations used : paper « paper chromatography of extract in butanol:acetic acid water (4:1:5). thio •» destruction of oxytcocic a c t i v i t y in extract as a result of incubation £ with 0.01 M sodium thioglycollate. m Rf " paper chromatographic behaviour of extract a c t i v i t y i n butanol:acetic acid: water (4:li5). EC " buffer conductivity at which a c t i v i t y was eluted from a CMC column. A l l other abbreviations are explained on p. ** Samples B and C were pooled prior to purification and pharmacological characterization. Biological assays carried out RU, RUMg, FB, ADH, i ME. RU, RUMg, FB, ADH, VP, Na. |RU, RUMg, VP, ME. IRU, ADH. RU, RUMg, VP, FB. , RU, RUMg, ADH, FB, -| VP, ME. Results of thiogly-collate treatment completely destroyed completely destroyed p a r t i a l l y destroyed completely destroyed RU,'thio from.crude extract. Rf, RU/RUMg, FB/RU, ADH/RU, ME/RU, thio from purified material, none. EC, RUMg/RU, FB/RU, ADH/RU, VP/RU, Na/RU, thio from purified material. RUMg/RU, VP/RU, ME/RU, thio from T.C.A.-purified extract. RUMg/RU, ADH/RU, FB/RU, thio from crude extract, Rf, ADH/RU from purified material. none. EC, RUMg/RU, VP/RU, FB/RU from purified material. EC, RUMg/RU, ADH/RU, FB/RU, VP/RU, ME/RU, thio from purified material. 116 Petromyzon. marlnus yielded 47 to 68 mU/brain (Sawyer, 1955). The actlvity/mg (specific a c t i v i t y ) of the latter species was 5.3 mU/mg, which is comparable to the level of a c t i v i t y (5.45 mU/mg) of sample G, the adult group that was dissected more precisely than the other lamprey samples. It would seem that the higher hormone content of Petromyzon was simply the result of the larger size of this animal compared to the brook lamprey. Lampetra f l u v i a t i l u s is also larger than L. richardsoni (50 g as compared to about 10 g; the former value was calculated from data given by Folle t t and Heller, 1964), yet both species possessed about the same level of a c t i v i t y per pituitary, suggesting that the specific a c t i v i t y for the river lamprey was lower than for L. . richardsoni. A similar situation has been reported for elasmobranchs by Perks (1966); the neurointermediate lobe of larger species of sharks examined contained l i t t l e more or even less oxytocic activity than did the smaller forms. ' The presence of oxytocic a c t i v i t y in the midbrain and hindbrain of lamprey in amounts comaprable to the neurohypophysial tissues was somewhat surprising i n view of the published data. Sawyer (1955) reported that extracts o f ventral hindbrain of Petromyzon contained only about 10% of the rat uterus a c t i v i t y of ventral forebrain; while Perks and Dodd (1963) found that in elasmo-branchs the oxytocic potency of indifferent brain (mixed telencephalon and medulla) was never more than 3.5% of the potency of the corresponding extracts o f neurointermediate lobe. However, the discrepancies i n the levels of ac t i v i t y o f control extracts between this study and the published data may be the result of differences i n the composition of the control tissues. Data from the present investigation suggested that the bulk of the oxytocic a c t i v i t y extracted from lamprey forebrain tissues (including the neurohypophysis) was localized i n the diencephalon, v i t h l i t t l e occurring i n the telencephalon. . 117 A non-homogenous distribution of acti v i t y also appeared to exist in the control tissues, since spinal cord possessed l i t t l e a c t i v i t y . A similar situation i n elasmobranchs would account for the low oxytocic potencies of indifferent brain from these animals (Perksand Dodd, 1963); this explanation assumes a low level of ac t i v i t y i n the medulla as well as the spinal cord. The low oxytocic a c t i v i t i e s of Sawyer*s control extracts from Petromygon could also be accounted for in this way. What was more surprising than the level of ac t i v i t y i n the eontrol extracts, was the indication that AVT was present in the midbrain and hind-brain tissues. The evidence for its presence was f a i r l y strong, both in ammocoetes (sample E, pp. 87-89) and adults (sample H, pp. 110-111). The fact that the peptide was detected only in purified hindbrain extracts was probably the result of the small amounts of the hormone available. Certainly, i t s destruction by thioglycollate i n the crude extracts would have been masked by the increase i n ac t i v i t y that resulted from treatment of the hindbrain extracts with the t h i o l reagent. However, the material present in peak 4 of the CMC column eluates of the adult sample H control extract showed every indication of being AVT, and the data suggest that small amounts of the hormone were present i n the T.C.A.-purified hindbrain extract of ammocoete sample E. There would seem to be three possible explanations for the presence of AVT in the hindbrain tissues. One is that the occurrence was an ar t i f a c t resulting from accidental inclusion of small amounts of neurohypophysis in the control material. This possibility is diminished by the results from section I (pp. 54-58) which indicated that, in the dissection of lamprey tissues for pharmacological studies, the brain was transected caudal to the diencephalic-mesencephalic border and thus well posterior to the pituitary. It seems unlikely, then, that portions of the neurohypophysis would have been included -_. 118 i n the control tissues, so that the pars nervosa as the source for the hind-brain AVT seems improbable. A second explanation for the presence of AVT in the control extracts is that the hormone vas synthesized in regions outside the preoptic nuclei, in a more posterior area of the brain. Granules staining with chrome-haematoxylin-phloxine and aldehyde-fuchsin have been reported to occur in nerve c e l l s and their processes located in various regions of the lamprey brain; these areas include nuclei dorsal to the posterior recess in Petromyzon marinus and Lampetra lammottei, which have been described by Oztan and Gorbman (1960). Sterba (1961, 1962, 1969) has reported that c e l l s which contain aldehyde-fuchsin- positive granules are found in various areas of the lamprey brain (mesencephalon, metancephalon and spinal cord); however, the c e l l s were not affected by the Gomori reagent. In the present investigation, no notice was made of any of the above-mentioned, neurosecretory-like c e l l s , but, as - discussed e a r l i e r (section I, pp. 50-51, 57), this may have been due to the depletion of neurosecretory material from the c e l l s as a result of stress. The presence of "neurosecretory granules" ( i . e . those stained with classical neurosecretory stains such as chrome-haematoxylin-phloxine and aldehyde-fuchsin) i n neurons is not, of course, absolute proof that the c e l l s elaborate neuro-hypophysial hormones, nor, indeed, can i t even be considered as conclusive proof that they are neurosecretory in nature (Sloper, 1966). Pharmacological investigations, as well as further histological studies are necessary to resolve the question as to whether extra-hypothalamic elaboration of AVT occurs in lamprey. Txi this regard, i t is of interest to note that Lacanilao, (1969,1970) has presented evidence for the existence of arginine vasotocin or an arginine vasotocin-like principle in the urophysis of the mudsucker, Glllichthys mlrabilis; this suggests that in teleosts, at least, AVT is 119 synthesized in neurosecretory systems outside of the hypothalamus. A f i n a l possible explanation for AVT in the hindbrain tissues is that the hormone was synthesized i n the preoptic nuclei and was then transported to the mid- and hind- brain* Two routes of transport appear to exist : 1) the axons of the preoptic neurons and 2) the cerebrospinal f l u i d . Nerve fibres containing stainable neurosecretory material appear to travel from the preoptic neurons to the hindbrain in P. marlnus and L. lamottei (Oztan and Gorbman, 1960). Preoptic neurosecretory processes have also been observed to terminate in the optic tectum of metamorphosed lamprey (Sterba, 1969). It seems possible that these axons could have contained arginine vasotocin and thus accounted for the presence of the hormone in the control tissues. Evidence to support this view comes from investigations of higher vertebrates. The non-hypophysial termination of hypothalamic neurosecretory neurons has been observed in : - 1 amphibian's, reptiles, birds and mammals (Legait, 1956; Legait and Legait, 1956, 1957, 1958); in these species, the fibres run to the habenular nuclei and the septum. In some forms, especially the reptiles, other processes travel rostrally into the telencephalon and caudally into the ventral aspects of the mesencephalon and the floor of the fourth ventricle. Legait and Legait (1957) have also investigated the tench (Tinea tinea); they found that the extra-hypophysial neurosecretory fibres were much less well developed in this species than in the higher vertebrates. Extracts of the regions of the brain containing the fibre terminations in bats, dogs, pigs and turtles were found to possess oxytocic a c t i v i t y , a portion of which proved labile to sodium thioglycollate (Grignon and Lamarche, 1959; Ruckebusch and Ruckebusch, I960). Further evidence for the presence of neurohypophysial-like principles outside the hypothalamo-neurohypophysial system resulted from work by Legait and Legait (1958) who demonstrated a toad water-balance effect in extracts of the 120 habenular nuclei of the chicken. later pharmacological and chromatographic investigations of beef and pig pineals (Mileu et a l , 1962; Pavel, 1965) suggested, the presence of arginine vasotocin in the former species and lysine vasotocin (8-lysine oxytocin) in the lat t e r . Milcu and his co-workers (1962) hypothesized that the AVT present in the beef pineal was synthesized in the habenular nuclei and then transported to the pineal v i a the epithalamic neurosecretory system described by Barry (1958). However, i t also seems possible that the hormone was produced i n hypothalamic (paraventricular) nuclei and reached the habenula by the neurosecretory-like fibres described above. Although some doubt is cast on these results by the failure of Ebels ei: a l , ( 1 9 6 5 ) to repeat the observations of Milcu et a l (1962) on beef pineals, they suggest the possibility of neurohypophysial peptides occurring i n extra-hypophysial regions of the central nervous system. However, the presence of AVT in the habenulae or pineal of the lamprey would not explain the detection of the hormone in the control extracts, since these areas of the brain were excluded from the hindbrain tissues during dissection. The preoptic axons reported by Oztan and Gorbman (1960) to travel back into the hindbrain cannot be ruled out as the source of the AVT in the control tissues. : Transport of AVT from the preoptic nuclei into the mesen- and metan-cephalon v i a the cerebrospinal f l u i d is suggested by the termination of short ("dendritic") preoptic fibres at the third ventricle and by the appearance of stainable neurosecretory material i n the ventricle (Oztan and Gorbman,,1960; Perks, 1969; Sterba, 1969). Early work on mammalian cerebro-spinal f l u i d had suggested the presence of neurohypophysial peptides, but later investigations failed to substantiate this p o s s i b i l i t y (Vogt, 1953; Heller and Ginsburg, 1966; Fitzpatrick and Bentley, 1968), and i t appears unlikely that the f l u i d in mammals, at least, contains significant amounts 121 of neurohypophysial hormones (greater than 2.5 mU/ml, Vogt, 1953). However, low levels of the peptides may be present; the histological demonstration of stainable material within the third ventricle of submammalian vertebrates, and also i n some mammals (see Heller and Ginsburg, 1966), suggests this p o s s i b i l i t y . Of the three p o s s i b i l i t i e s discussed above, none can be completley ruled out as an explanation for the presence of AVT in the hindbrain extracts. Certainly, the first-mentioned alternative, the inclusion of portions of the neurohypophysis in the control tissues, is rendered improbable by the histo-logical results presented i n section I. Elimination of one of the other two explanations is less easy however, and would not possible without further research into the neurosecretory centres present in the central nervous system of lampreys. L i t t l e was ascertained concerning the nature of the non-neurohypophysial active substances i n the pituitary and control extracts, since i t was not the purpose of this investigation to catalogue the pharmacologically active compounds present in the lamprey brain. Nonetheless, on the basis of the scanty data available, several general comments may be made as to the identity of these substances. Of the biologically active agents that could have been present in the brain tissue, the following are possible candidates : histamine, epinephrine, norepinephrine, dopamine, acetylcholine, 5-hydroxytryptamine, substance P and the prostaglandins (see Vogt, 1953; Amin et a l , 1954; Lederis, 1961; E l l i o t et a l , 1962; Lerabeck and Zetler, 1965; Friede, 1966; Ramwell et a l , 1966a and b; von Euler, 1966). Not a l l of these substances have been demonstrated to occur i n cyclostomes, but their presence in the brains of higher vertebrates makes this appear possible. Of these compounds, the cate-cholamines and histamine inhibit the rat uterus (Jensen and Sund, 1960a and b; 122 Rudinger and Krejci, 1968), and therefore they could not have contributed to the non-neurohypophysial oxytocic a c t i v i t y . A l l the other agents are active in contracting the rat uterus (Horton and Main, 1966; Sturmer, 1968), and could have accounted for some of the a c t i v i t y . However, i t is unlikely that the rat uterus a c t i v i t y which was recovered from the origin of the paper chromatograms of crude extracts, and which was removed by T.C.A. was due to any of these latter substances. 5-HT, Ach and substance P migrate from the origin of paper chromatograms in the solvent system employed (see p. 72 and Lederis, 1961). Also, they are not destroyed by T.C.A. and are insoluble in ether (Pemow, 1953), so that they would not have been removed by the T.C.A. treatment. Prostaglandins, on the other hand, are soluble in ether at low pH's (Ambache, 1966) and thus would have been extracted by the ether. Their chromatographic behaviour in butanoljacetic acidrwater does not appear to have been reported in the literature; however, as unsaturated, long-chain fatty acids they are soluble in butanol (White et a l , 1968), and therefore they would most l i k e l y migrate at least some distance from the origin. Thus, prostaglandins could have contributed to the faster moving active components of the crude extract chromatograms; moreover, as these substances are hypo-tensive agents (Horton and Main, 1966), they could have been responsible for the a b i l i t y of the eluates to lower blood pressure (see pp. 69-71). It is unlikely, however, that they accounted for the a c t i v i t y that remained at the origin; these active substances would appear to have been either large protein molecules that were precipitated by the T.C.A., or lipoidal compounds that were removed by the ether extraction, or substances that were bound to these molecules. Of the small amount of non-neurohypophysial a c t i v i t y that was not removed by the T.C.A. treatment, i t seems possible that at least a portion of this could have been due to Ach, 5-HT or substance P. It w i l l be recalled that 123 ion-exchange chromatography of the T.C.A.-purified extracts yielded several peaks of a c t i v i t y besides the AVT fraction, one of which came off in the gradient. Both Ach and 5-HT are basic molecules and probably would have been eluted during the gradient; indeed, Ach is extremely basic and may not have come off at a l l (White et a l , 1968). Substance P is an ampholyte with an iso-e l e c t r i c point of about 7 (Lembeck and Zetler, 1962). With the concentration and pH of the starting buffer used in chromatography in this study, i t is unlikely that any substance P present would have been bound to the resin, rather i t would have passed through the column in the loading volume. Thus the po s s i b i l i t y that some of the above-mentioned active substances could have accounted for a significant portion of the non-neurohypophysial a c t i v i t y extracted from the lamprey tissues i s unlikely, but the presence of small amounts of the compounds cannot be dismissed. More data would have to be obtained to establish the nature of the active contaminants. 124 SECTION III Changes In the Oxytocic Activity of the Neurohypophysis of the Lamprey  During Development and the Variation of Photoperiod A. Introduction The purpose of this investigation was to determine whether the hormone content of the neurohypophysis was altered during the development of L. richardsoni, and whether the amount of ac t i v i t y present could be affected by changes in the photoperiod in which the animals were held. Published obser-vations on the histology of the lamprey neurohypophysis suggested that both these p o s s i b i l i t i e s were l i k e l y . Van der Kamer and Schreurs (1959) reported a gradual increase in stainable neurosecretory material in the posterior pituitary of larval Lampetra planeri as the animals matured. The amount was maximal just prior to metamorphosis; during transformation to the adult form the quantity decreased, and the material remaining was located near the third ventricle and around c a p i l l a r i e s . After metamorphosis, there was l i t t l e neuro-secretory material l e f t in the neurohypophysis, and even this was lost after spawning. Van der Kamer and Schreurs postulated that in the transforming and adult brook lamprey, large quantities of AVT were necessary to maintain a proper water balance, and that this explained the low amounts of neurosecretory material i n the pars nervosa. In a study of the ammocoetes of Petromyzon marinus, Oztan and Gorbman (1960) described changes in the stainability of the hypothalamo-hypophysial system in response to alterations of external illumination. Exposure of the larvae to continuous light for three weeks resulted i n a depletion of Gomori-125 and aldehyde-fuchsin-positive granules in the soma and axons of neurons in the ventral and anterior aspects of the preoptic nuclei. No difference in stai n a b i l i t y of the neurohypophysis was noted however, even after three weeks of treatment. An opposite effect was observed i n ammocoetes kept in constant darkness : there was an increase in stainable material in the neurons and axons of the preoptic nuclei, but again no marked changes were noted in the degree of stainability of the pars nervosa. The return of both groups of ammocoetes to a natural diurnal illumination resulted in a return to the "control" type of distribution of stainable neurosecretory material. Oztan and Gorbman noted that the axons of the affected neurons appeared to terminate over the rostral pars d i s t a l i s , which suggested that ultimately changes in photoperiod may have had effects on the adenohypophysis. Til view of these published reports, i t was decided to look for changes in the oxytocic ac t i v i t y of the entire hypothalamo-neurohypophysial system of Lampetra richardsoni during i t s l i f e history and in response to changes in photoperiod. B. The oxytocic a c t i v i t y of the neurohypophysis during the l i f e history of  L. richardsoni 1. Methods The ammocoetes used in this study were taken from the Salmon River and separated into different age classes by means of a growth curve formulated by Pletcher (1963) for the brook lamprey i n the stream. The growth curve is presented in figure 27 and indicates that the larval l i f e of L. richardsoni lasts at least six years. Pletcher made no allowances for a decrease in length before or during metamorphosis, or for the presence of a rest period prior to transformation. Both these events are thought to occur in the l i f e histories 126 Figure 27 Growth curve of Lampetra richardsoni from the Salmon River (after Pletcher, 1963). 127 of other species of lamprey (Leach, 1940; Pletcher, 1963); thus, the duration of larval l i f e in the brook lamprey may be longer than six years. Metamor-phosing lampreys were captured in both the Salmon River and Marshall Creek, and were identified on the basis of the morphological c r i t e r i a given by Leach (1940) and Pletcher (1963) - In particular by the appearance of the mouth, eyes and f i n s . The adult lamprey used in this study (sample H) were collected in Marshall Creek instead of the Salmon River. This sample was used because the adults taken from the Salmon River (sample G) were dissected in a more precise manner than the ammocoete samples, so sthat comparisons of the levels of a c t i v i t y i n the adults and ammocoetes would have had l i t t l e meaning (pp. .19-20, 92-93). Sample H, on the other hand, was dissected in the same manner as the larvae, and since the total activity/animal extracted from the neuro-hypophysial tissues of the two adult samples were about the same (see p.93), i t seemed j u s t i f i a b l e to use the results from the adult specimens of sample H, even though they were not taken from the same stream as the ammocoetes. 2. Results and Discussion The oxytocic a c t i v i t i e s of the various samples are given i n Table XV and figures 28 and 29. Activity was detected in a l l extracts, and treatment of the samples with sodium thioglycollate suggested that at least a portion of the ac t i v i t y (approximately 507.) in the pituitary extracts was due to a neurohyp-ophysial principle (see Section II). No evidence was obtained for a marked f a l l i n neurohypophysial act i v i t y at metamorphosis, as had been suggested on histological grounds (van der Kamer and Schreurs, 1959); i n fact, a marked increase i n the total a c t i v i t y extracted per pituitary was noted at metamor-phosis. A similar situation existed in the hindbrain tissues, suggesting that the changes in the neurohypophysial extract a c t i v i t y were not primarily due to alterations i n the amounts of AVT. The results from Section II strengthened Table XV. Amounts of oxytocic ac t i v i t y extracted from neurohypophysial and hindbrain tissues of lampetra  richardsoni at various stages of i t s l i f e history. Neurohypophys i a l Extracts * Hindbrain Extracts * Sample mU/mg mU/animal mU/mg mU/animal 1 & 2 year old ammocoetes 3.05 (2.51-3.42) 0.17 (0.15-0.19) 0.86 0.05 3 year old ammocoetes 3.89 (3.47-4.47) 0.36 (0.32-0.42) 2.21 (1.88-2.61) 0.27 (0.23-0.32) 4 year old ammocoetes 4.14 (3.67-4.67) 0.55 (0.48-0.62) 2.90 (2.11-3.98) 0.50 (0.36-0.69) 5 & 6 year old ammocoetes 2.84 (2.22-3.64) 0.52 (0.41-0.67) 1.31 (1.12-1.52) 0.41 (0.35-0.47) metamorphosing ammocoetes 2.13 (1.89-2.39) 1.75 (1.56-1.97) 1.70 (1.44-2.00) 1.28 (1.09-1.51) adults 1.83 (1.59-2.11) 1.88 (1.63-2.16) 1.39 (1.19-1.62) 1.69 (1.44-1.97) * values in parentheses refer to 95% fiducial limits. to 03 129 Figure 28 Oxytocic activity and dry weight of neurohypophysial tissues from larval, metamorphosing and adult lamprey. META. • lamprey undergoing metamorphosis. mU «=» rat uterus activity (magnesium absent). - vertical lines through activity columns give 957. confi-dence limits. 6 r ACTIVITY/MG E 3 r ACTIVITY/ANIMAL =3 E WEIGHT l.0r 0.5l I <*2 YEARS i — • I I 3 YEARS 4 YEARS 5*6 YEARS META. ADULT 130 Figure 29 Oxytocic ac t i v i t y and dry weight of hindbrain tissues from la r v a l , metamorphosing and adult lamprey. META. «• lamprey undergoing metamorphosis. mU •» rat uterus a c t i v i t y (magnesium absent). - v e r t i c a l lines through activity columns give 957. confi-dence limits. ACTIVITY/MG ACTIVITY/ANIMAL 1.0 WEIGHT e> 0.5-il I 1 |<*2 YEARS 3 YEARS 4 YEARS 5*6 YEARS META. ADULT 131 t h i s p o s s i b i l i t y , since they indicated that no more than 507. of the oxytocic a c t i v i t y of neurohypophysial tissues was due to AVT, while l i t t l e or no AVT was present in hindbrain. The increase in total a c t i v i t y that occurred in both pituitary and control extracts at metamorphosis was not accompanied by an increase i n activity/mg; on the contrary, there was a gradual f a l l in activity/mg, beginning in the latter stages of larval l i f e and persisting into the adult stage. The most l i k e l y explanation for these results was an increase in the size of the brain, or of a portion of the brain, with no concomittant r i s e in the specific a c t i v i t y of the tissues. No increase in body length appears to occur during transformation; indeed, there seems to be a slight .reduction i n length from that of the ammocoetes (Leach, 1940; Pletcher, 1963). However, an increase in brain weight did appear to take place during metamor-phosis, as shown in figures 28 and 29. These figures il l u s t r a t e the dry weights of pituitary and hindbrain tissues of the lamprey samples examined. The values were obtained by dividing the total weight of tissue in a sample by the number of animals i n the sample. It appeared that a striking increase in brain weight occurred at metamorphosis and continued on into adult l i f e . This increase in brain size does not appear to have been reported in the liter a t u r e , and the reasons for i t s occurrence are obscure. Perhaps i t was the result of the elaboration of olfactory and visual areas of the central nervous system in conjunction with the increased development of the eyes and olfactory apparatus that occurs at transformation (Young, 1962). At any rate, the enlargement of the brain seems to account for the rise in total oxytocic a c t i v i t y that occurred at metamorphosis. The f a l l in activity/mg is harder to explain. The parallelism between the ac t i v i t i e s of the forebrain and hind-brain suggests that the decrease was not due to a reduction in the specific a c t i v i t y of one particular active substance, such as AVT. It seems more 132 l i k e l y that the decrease in potency /mg was due to a greater enlargement of areas of the brain with low oxytocic a c t i v i t i e s as compared to the increase in size that occurred in the portions of the brain with high biological a c t i v i t y . In section II (p. 92), i t was indicated that the majority of the oxytocic a c t i v i t y extracted from the pituitary component of the brain was located in the diencephalon, with l i t t l e occurring in the telencephalon. An increase in size of the latter area, in association wlh elaboration of the olfactory apparatus, with a smaller enlargement of the diencephalon, would result in an increase in the total a c t i v i t y of the tissues, and also In a lower specific a c t i v i t y . A similar situation could have occurred in the hindbrain samples. The preceding discussion, while offering explanations for the changes in biological a c t i v i t y that occurred i n the pituitary and control samples, says nothing about possible alterations in the amounts of AVT during the l i f e history of the lamprey. The presence of a large amount of contaminating a c t i v i t y i n the extracts makes interpretations of the results in terms of AVT levels d i f f i c u l t . However, i t seems l i k e l y that there was no marked depletion of AVT from the neurohypophysis at metamorphosis. A large reduction in even 50% of the a c t i v i t y of the pituitary extract ( i . e . that due to AVT) certainly would have been reflected by a f a l l in both total oxytocic a c t i v i t y and in activity/mg. In actual fact, a slight reduction in activity/mg did occur, but there was a large increase i n total a c t i v i t y . Thus, i t appears unlikely that a depletion of stainable neurosecretory material from the neurohypophysis at transformation reported by van der Kamer and Schreurs (1959) was accompanied by a reduction in the content of AVT. Indeed, since completion of this study, doubt had been cast on the ear l i e r histological observations which suggested that this change might take place. Unpublished data by Sage (personal 133 communication.) suggests that there was no marked decrease in sta i n a b i l i t y of the neurohypophysis at metamorphosis in Lampetra planer1. In a study involving 50 animals, some kept in the laboratory and some captured i n the wild, there was an increase i n the intensity of staining and apparent amount of neurosecretory material in the neurohypophysis throughout the period between metamorphosis and spawning* No reduction i n stainability was seen to to occur at transformation, but the stainable material was markedly decreased after spawning. Sage's data seem to be at variance with those published by van der Kamer and Schreurs (1959). However, the latter study involved 14 animals, only 7 of which were metamorphosing or mature adults, and the v a l i d i t y of results obtained with these few specimens seems questionable. In view of t h i s , Sage's data seem more reli a b l e . Moreover, they are supported by published observations on teleosts which indicate a reduction in either stain-able material of biological a c t i v i t y present in the neurohypophysis at spawning (Sawyer and Pickford, 1963; Sokol, 1961; Tamura and Honma, 1969). In the study reported here, adults were not separated into pre- and post-spawning classes, so i t was not possible to determine whether the decrease i n stain-a b i l i t y reported by Sage was accompanied by a reduction in oxytocic a c t i v i t y . However, the majority (677.) of the adults collected were in the pre-spawning condition, so that any decrease i n neurohypophysial a c t i v i t y that did occur in the post-spawning lamprey may well have been masked by the higher potency of the pre-spawning individuals. Indeed, i f the spavned-out animals had low levels of a c t i v i t y , i t would mean that the pre-spawned individuals possessed greater amounts of AVT than was indicated by the present study. 134 C. The effects of photoperiod alterations on the oxytocic ac t i v i t y in the  neurohypophys is 1. Methods To carry out this study, approximately 200 ammocoetes (4 to 6 years old) were taken from the Salmon River and divided into four groups of approximately 50 animals each. The groups were placed In separate 15 gallon aquaria which were f i l l e d with f i l t e r e d , aerated fresh water kept at 16° C. Prior to addition of the ammocoetes, mud from the Salmon River was placed on the bottom of one tank to a depth of about 15 cm; the other three tanks were l e f t bare. Fine netting was then placed over the tops of the tanks to prevent escape of the lampreys, and the tanks were covered with plywood'boxes which allowed no penetration of external light. A small door was located in the top of each container i n order to allot/ removal of the ammocoetes at the end of the experiment. Illumination of the tanks was provided by 14 watt fluorescent bulbs attached to the lids of the boxes. The following photoperiod regimes were maintained for the tanks : tank 1 - natural photoperiod (11 hours light/13 hours dark) tank 2 - natural photoperiod, mud present on tank bottom tank 3 - 2 4 hours light tank 4 - 2 4 hours dark. The animals i n this tank escaped through a small hole in the netting covering the tank, with the result that this experimental group was lost. The lamprey were kept under these conditions for two weeks. Periodic inspec-tions were made on tanks 1, 2 and 3 during the lighted phase of their photo-period; this was not possible with tank 4, which was therfore not opened over the experimental period. Survival in the former tanks was 1007.. At the end of the two weeks, the lamprey were quickly removed from the tanks and immediately k i l l e d and dissected as described in Materials and Methods (pp. 19-20). 135 2. Results and Discussion The results obtained from the. three groups of ammocoetes l e f t at the end of the experiment are given in Table XVI and figure 30. Included with the results of the present experiment are the extract a c t i v i t i e s of the 5 and 6 year old ammocoetes used in Sections II and III (sample D). These animals were kept under natural photoperiod in bare tanks for about one week between capture and dissection; they were thus held under conditions similar to those experienced by group 1 of the photoperiod study, and can be used as a "control" for the latter ammocoetes. There were minor differences between the oxytocic a c t i v i t i e s of the various groups, but overlap of their confidence limits suggested that they could not be considered significant. Nevertheless, several interesting points emerged from the results. It appeared that the differences in pituitary a c t i v i t i e s of the three groups were more or less paralleled by variations in the potencies of the hindbrains extracts. This was reminiscent of the results of part A of this section, and suggested that any photoperiod effects on brain oxytocic ac t i v i t y were the result of alterations in the levels of biologically active substances other than AVT. Extracts of group 1 had a c t i v i t i e s similar to those of sample D; this was perhaps to be expected since both groups of ammocoetes were maintained under v i r t u a l l y identical conditions. The two other groups (2 and 3) exhibited apparently greater potencies than group 1. Ammocoetes kept under the "most natural" conditions (group 2) possessed the highest neurohypophysial activ-i t y , although i t was not much greater than the ac t i v i t y of group 3, which was comprised of animals held under to the most "unatural" environment (24 hours l i g h t , no mud on tank bottom). Therefore, i t would seem that stress, resulting from maintainance of the lamprey under unatural conditions, did not cause a marked depletion of active substances from the neurohypophysis Table XVI. Oxytocic levels in neurohypophysial and control extracts from lamprey kept under different photoperiods. Neurohypophysial Extracts * Hindbrain Extracts * Sample mU/mg mU/animal mU/mg mU/animal sample D, from Section II nat* 2.84 (2.22-3.64) 0.52 (0.41-0.67) 1.31 (1.12-1.52) 0.41 (0.35-0.47) ural photoperiod. group 1, natural photoperiod. 2.29 (1.72-3.05) 0.68 (0.51-0.90) 1.63 (0.65-4.05) 0.51 (0.21-1.28) group 2, natural photoperiod, 4.21 (2.85-6.20) 1.28 (0.87-1.89) 4.32 1.27 mud on tank bottom. group 3, 24 hours l i g h t . 3.72 (2.39-5.78) 1.20 (0.77-1.87) 5.53 (3.55-8.62) 1.66 (1.06-2.59) * values i n parentheses refer to 95% fiducial limits. o> 137 Figure 30 Oxytocic levels in neurohypophysial and hindbrain tissues from larval lamprey kept under different photoperiods. r white columns - rat uterus a c t i v i t y (magnesium absent)/mg lyophilized tissue, in mU. cross-hatched columns - rat uterus activity/animal, in mU. - v e r t i c a l lines through the columns give 95% confidence limits. NEUROHYPOPHYSIS 3 SAMPLE D GROUP I GROUP 2 GROUP 3 NATURAL NATURAL NATURAL 24 HOURS PHOTOPERIOD PHOTOPERIOD PHOTOPERIOD LIGHT MUD 138 over the time involved. It has been shown that other forms of stress result in histological changes in the teleost neurohypophysis, with either a depletion or accumulation of neurosecretory material. Leatherland and Dodd (1969) demonstrated that in the eel (Angullla anguilla) changes of background color and sudden alterations in water temperature resulted in a.depletion of neurosecretory material from the neurohypophysis. However, the effects lasted Only a few hours, after which time, the appearance of the pituitary gland had returned to normal. Carlson and Holmes (1962) reported that the handling of rainbow trout was followed by an Increase in the oxytocic and antidiuretic a c t i v i t i e s of the neurohypophysis. The results of this study did not suggest that any decrease in AVT occurred i n the ammocoetes kept under constant Humiliation. The differences that did exist amoung the three experimental groups were not s t a t i s c a l l y significant, and could not be explained on the basis of the histological data of Cztan and Gorbman (1960). Perhaps this was not surprising, since the latter results indicated that only the stainable material in the neurons of the preoptic nuclei were affected by alterations in photoperiod, while the pars nervosa was unchanged. This suggests the pos s i b i l i t y of a transference of neurosecretory substances between the two regions with no resulting overall changes in the hormone content of the neurohypophysis. Moreover, the histo-logical observations of Oztan and Gorbman (1960) demonstrated that only a portion of the preoptic neurons responded to changes in external illumination and that these supplied neurosecretory material to the region of the neuro-hypophysis above the rostral pars d i s t a l i s . It seems possible that the neurons were supplying releasing or inhibiting factors to the adenohypophysis, so that alterations in their synthetic or secretory activity may not have been detected by biological assay of neurohypophysial extracts. Thus, i t was not 139 possible to state whether the hypothalamo-neurohypophysial system of ammo-coetes was sensitive to changes in photoperiod. In tetrapods, various workers have demonstrated histological changes in the hypothalamic neurosecretory system in response to alterations in external illumination; in some cases, i t has been shown that the changes are correlated with the reproductive a c t i v i t i e s of the animals, suggesting that photoperiod may influence repro-ductive function by way of the hypothalamo-hypophysial system (Fiske and Greep, 1959; Oksche et a l , 1959; Critchlow and Wilson, 1963). Seasonal variation in the content of stainable neurosecretory material in the neurohypophysis has been reported to occur in several species of teleosts, and the changes have been correlated with either the reproductive or migratory cycle (Arvy et a l , 1959; Polenov, 1959; Leatherland and Dodd, 1960; Sokol, 1961; Honma and Tamura, 1969; Honma and Suzuki, 1968; Tamura and Honma, 1969). Honma and Suzuki (1968) have shown that altering the photoperiod affected the timing of the neuro-secretory and reproductive cycles of the teleost f i s h , Pleoglossus al t i v e l u s . However, in none of these studies were parallel estimations of biological a c t i v i t y and stainability of the neurohypophysis carried out. Sokol (1961), working with the k i l l i f i s h , Fundulus hetreoclitus, found that the density of neurosecretion in the pars nervosa decreased progressively in the spring and was lowest after spawning; this depletion occurred in both sexes. Sawyer and Pickford (1963) determined the hormone content in the neurohypophysis of the same species at different times of the year. At spawning they observed a depletion of only one hormone,[4-serine, 8-isoleucine] -oxytocin (isotocin), and this occurred only in females. In males, no reduction in the amounts of isotocin were observed, even though Sokol (1961) had reported a reduction in sta i n a b i l i t y . A similar situation has been reported in rats by Moses and his co-workers (1963). They noted a depletion of stainable material from the 140 pituitary of rats in response to injections of formalin; however, this was not accompanied by any change in the vasopressin content. Conversely, Lederis (1964) reported that exposure of rainbow trout (Salmo irideus) to seawater resulted in a 50% f a l l in the AVT content of the neurohypophysis, but there was no appreciable depletion of neurosecretory granules. A l l these data emphasize that a reduction in stainable neurosecretory material from the posterior pituitary is not necessarily accompanied by a f a l l in the levels of the neurohypophysial hormones, and vice-versa. The discrepancy between the published data on histological changes in the lamprey neurohypophysis and the pharmacological results obtained here may be another example of this lack of association. 141 SECTION IV The Isolation and Identification of the Neurohypophysial Principles  of the Hagfish, Polistotrema s t o u t i i A. The Oxytocic a c t i v i t i e s of the crude extracts and the effects of sodium  thioglycollate on the ac t i v i t y This investigation of hagfish neurohypophysial principles was carried out on a total of 132 animals. They were dissected as described previously (Materials and Methods, pp. 20-22), and the neurohypophysial and control tissues were extracted at a concentration of 40 mg/ml instead of 20 mg/ml as was used for the lamprey extracts. This higher tissue concentration was employed because of the anticipated lower specific a c t i v i t y of the hagfish neurohypophysis when compared to that of the lamprey. Table XVII gives the crude a c t i v i t i e s of the pituitary and hindbrain tissues of the hagfish. The neurohypophysial extract contained rat uterus a c t i v i t y equivalent to 1.57 mU/gland; this was somewhat higher than the value of 0.50 mU/gland reported for Myxine glutlnosa by Folle t t and Heller (1964). After 18 hours of incubation of the extract with 0.01 M sodium thioglycollate, only 24.5% of the rat uterus a c t i v i t y was abolished (Table XVII), suggesting that the hagfish neurohypophysis contained only about 0.39 mU of neurohypoph-s i a l hormone. Follett and Heller (1964) found a similar level of thioglycol-late l a b i l i t y i n neurohypophysial extracts of Myxine. However, as discussed i n Section II (pp. 61-62), i t seems possible that the presence of interfering substances in the hagfish crude extracts could have reduced the rate of inactivation of the neurohypophysial hormones by the thioglycollate, and Table XVII. Oxytocic a c t i v i t i e s of crude extracts of hagfish neurohypophysial and control tissues and their resistance to thioglycollate. Rat Uterus Activity * Resistance to Thioglycollate Sample incubation 7. ac t i v i t y ____________ mU/mg mU/animal time remaining destroyed Neurohypophysis 0.23 (0.20-0.28) 1.57 (1.32-1.86) 18 hr. 75 25 Hindbrain 0.13 (0.10-0.16) 0.68 (0.55-0.82) 18 hr. 0 217 * the values in parentheses refer to 957. fiducial limits. 4> to 143 thereby prevented the complete destruction of neurohypophysial a c t i v i t y during the time of incubation. As with the lamprey, the crude extract of hindbrain tissues from the hagfish contained significant amounts of oxytocic a c t i v i t y as compared to the pituitary extract. However, treatment of the material with sodium thioglycol-late yielded almost a 2 . 2 fold increase in potency. This situation was simi-la r to that encountered in treatment of the lamprey control extracts with thioglycollate, and i t suggested that the neurohypophysial and control extracts contained different active substances. B. Preliminary purification of the crude extracts with trichloroacetic acid It was expected that purification of the hagfish extracts would involve the same problems that were faced i n working with the lamprey extracts : the removal of large proportions of proteinaceous substances and non-neurohypoph-y s i a l biologically active materials. The success encountered with T.C.A. and ion-exchange chromatography i n purifying the lamprey extracts prompted their use here. . Lowry peptide determinations of the hagfish crude extracts indicated that they did not contain as much proteinaceous material as the lamprey extracts (Table XVIII), which is surprising when i t is recalled that the extracts were made at twice the concentration as were those of the lamprey. Moreover, i t appeared that the T.C.A. was not very effective in purifying the hagfish samples, since i t removed very l i t t l e of the Lowry-positive material, and v i r t u a l l y none of the (non-neurohypophysial) oxytocic ac t i v i t y from the pituitary and control extracts (Table XVIII). The reasons for this failure were uncertain. Certainly, i f the bulk of the Lowry-positive substances was comprised of small, T.C.A.-resistant peptides, l i t t l e would have been 144 accomplished by treating the extracts with the T.C.A. Similarly, i f most of the uterotonic substances present in the extracts were not precipitated by the T.C.A. or extracted by the ether, the purification method would have had l i t t l e effect on the biological activity of the samples. As discussed in Section II (pp. 121-123), most of the substances known to be active on the rat uterus are resistant to T.C.A. and are non-1ipoidal in nature, so that t h e i r presence i n the hagfish extracts would explain the relative ineffec-tiveness of the method in purifying the crude extracts. Table XVIII. Purification of the hagfish pituitary and control extracts with T.C.A. Extract pituitary extract before T.C.A. after T.C.A. control extract before T.C.A. after T.C.A. Oxytocic ac t i v i t y Lowry peptide mU/ml 7. recovery jjg/ml 7. removed 9.4 9.0 5.3 5.3 96 100 720 680 1760 1136 36 Factor of purification * 1.1 1.6 * calculated by dividing the total amount of Lowry peptide material present i n the extract prior to treatment with T.C.A. by the amount present after treatment. C. Column chromatography of the pa r t i a l l y purified neurohypophysial extract The failure of T.C.A. to effectively purify the hagfish neurohypophysial extract presented a serious problem in the further purification of the active substances. The total amount of Lowry-positive material i n the extract 145 (13,600 ug) was much greater than had been present in the lamprey extracts which had been purified by ion-exchange chromatography. It seemed possible the hagfish extract would overload the column, and this would result in a poor resolution of the neurohypophysial principle(s). However, other methods for a preliminary purification were of dubious value, or were impossible in the present investigation, because of such problems as low recoveries or excessive dilution of the active material. Therefore i t was decided to proceed directly with an attempt to purify the hagfish neurohypophysial extract by column chromatography on Whatman CM-22 carboxymethyl cellulose resin. Accord-ingly, the T.C.A.-purifled solution was adjusted to the proper pH and conduc-t i v i t y , and loaded on to a 14.0 x 0.5 cm column of the resin. Development of the column by a gradient of increasing buffer concentration yielded only one fraction of oxytocic a c t i v i t y , and this came off in the gradient (figure 31). The active material came off the column somewhat earli e r than was anticipated for AVT,'but this may have resulted from overloading of the column, a situation i n which competition from impurities might have reduced the a b i l i t y of the peptide to attach to the binding s i t e s . Certainly, i t is l i k e l y that the long " t a i l " of activity behind the main peak was due to overloading, and i t also seems possible that the presence of two peaks in the active region could be attributed to the application of too great a solute load to the resin (Heinicke, unpublished observations). However, although the elution profile of the active fractions was far from ideal, the purification obtained was satisfactory, since the 40.7 fold degree of purification of peak B compared very favorably with the purification resulting from chromatography of the lamprey extracts. No other active regions were detected in the column eluates, and no evidence was obtained for the presence of a second neurohypophysial principle 146 Figure 31 Purification of an extract of hagfish neurohypophysial tissue by ion-exchange chromatography on CMC cellulose. a . column » 0.5 cm x 14.0 cm Whatman CM-22 carboxymethyl cellulose. b. loading solution - 50.0 ml, at pH 5, conductivity 1.0, containing 180 mU oxytocic a c t i v i t y and 13,600 ug Lowry peptide. Specific a c t i v i t y = 0.013 mU/^ g. c. starting buffer - 0.02 M, pH 5 ammonium acetate (conductivity -1.04), to A. d. gradient - 0.02 M, pH 5 ammonium acetate to 0.20 M, pH 5 ammonium acetate (conductivity «=• 7.8), A to B. e. flow rate ° 15 ml/hr. f. volume collected/tube « 3 ml. g. recovery - 47% of oxytocic activity. open ci r c l e s - rat uterus activity (magnesium present) of the eluted fractions in mU/tube. closed-circles - Lowry peptide concentration of the eluted fractions in^ig/ml. open triangles - specific conductivity of the eluted fractions in millimhos. mU/TUBE _ _ ro CJ -f* Q o o o o I 1 1 : 1 1 ro A o> oo CONDUCTIVITY, mMHO o w w o o o o o /JG LOWRY/ML 147 in. the extract. However, this failure to demonstrate the presence of other active substances (either neurohypophysial or non-neurohypophysial) could possibly be explained by the relatively large loading volume (50 ml) of the sample which resulted from dilution of the T.C.A.-purified extract to the proper conductivity prior to application to the column. With the level of sensitivity of the assays used in checking the eluates, approximately 40 mU, or 227. of the applied a c t i v i t y , could have passed undetected through the column in the loading volume, assuming an homogeneous distribution of active substances. This large amount of ac t i v i t y that might not have been noticed in the column eluates may also explain the low recovery (477.) of oxytocic substances from the column; the recovery was much lower than those obtained during chromatography of the lamprey extracts. It seems possible that the calculated recovery was not a true reflection of the amount of a c t i v i t y that was either destroyed during development of the column, or not eluted, since a substantial proportion of the "missing" ac t i v i t y could have passed through the column undetected. The pos s i b i l i t y that some of this undetected a c t i v i t y was due to a second neurohypophysial hormone cannot be completely discounted. The total amount of ac t i v i t y recovered from the column was 84 mU, and a l l of this was apparently due to AVT (see below). This represents approxi- . mately 407. of the total a c t i v i t y treated with T.C.A., i.e., 407. of the a c t i v i t y in the crude extract. Taking into account the losses of AVT that must have occurred during purification, the actual portion of the crude extract activity attributable to the hormone was probably closer to 50%. It w i l l be recalled that incubation of the crude neurohypophysial extract with thioglycollate suggested that only 257. of the total a c t i v i t y was due to a posterior pituitary hormone. The same situation was seen in some of the lamprey samples (pp. 61-62, 79-80), and thus i t seemed that treatment of • 148 crude extracts of cyclostome pituitary tissues with sodium thioglycollate could yield low estimates of the neurohypophysial contribution to the total potency. D. Pharmacological characterization of the active fraction from the CMC  column Pharmacological characterization of the active substance eluted from the column was accomplished by assaying the eluates against synthetic AVT on several preparations. The AVT standard had a concentration of 50 ng/ml, and was made up i n ammonium acetate buffer at the same concentration as the eluates. Both the standard and unknowns were acidified with glacial acetic acid to reduce inactivation of the active principles, but were neutralized immediately prior to assay. Although i t was f e l t that the presence of two peaks in the active region was an a r t i f a c t , and did not represent the exist-ence of two active substances, peaks A and B were assayed separately. The results of the assays are presented in Table XIX and figure 32. The limited amount of material in peak A did not permit a complete series of assays to be performed, and no estimate was made of the potency of the peak on the rat uterus in the absence of magnesium. Accordingly, for peak A, activity ratios were calculated using the oxytocic potency with magnesium present as the denominator. The results obtained suggested that the bulk of the ac t i v i t y present in the peak was due to AVT. Admittedly, the ADH/RU ratio for peak A is far from unity, but this value was estimated on the basis of a single injection of unknown In the antidiuretic assay, and is thus subject to a large degree of error. Peak B was characterized more thoroughly, and, as none of. the a c t i v i t y ratios differed significantly from one, i t appeared highly probable that the active substance present was AVT. In addition to the a c t i v i t i e s l i s t e d in Table XIX, peak B also possessed rat vasopressor 149 Table XLX. Biological a c t i v i t i e s of the AVT fractions (peaks A and B) resulting from ion-exchange chromatography of a pituitary extract of adult hagfish. Major Peak (peak B) Biological Assay rat uterus (-Mg?"*) rat uterus (+Mg+'*) rat milk ejection antidiuretic frog bladder Potency of peak B, ng AVT/ml * 23.0 (19.7-27.0) 20.5 (17.1-24.7) 27.4 (20.8-36.1) 32.6 (21.5-49.4) 20.3 (9.2-44.4) Ratio to potency on rat uterus (-Mg1"*)* 1.00 0.89 (0.71-1.12) 1.19 (0.88-1.59) , 1.41 (0.92-2.16) 0.88 (0.40-1.93) Minor peak (peak A) Biological Assay rat uterus (+Mg+"*) rat milk ejection antidiuretic ** frog bladder ** Potency of peak A, ng AVT/ml * 24.1 (21.1-27.6) 26.3 (24.0-28.8) 66.5 19.3 Ratio to potency on rat uterus (-fMg4"1)* 1.00 1.07 (0.94-1.25) 2.76 0.80 * the values in parentheses refer to 957. fiducial limits. ** potency estimates were made on the basis of single injections of unknown. 150 Figure 32 Potencies of purified neurohypophysial material (peak B) from hagfish on several biological assays using synthetic arginine vaso-tocin as the standard. - the v e r t i c a l lines through the columns indicate 957. confi-dence limits. 50 f 40 30 > < CD 2 20 10 0 RU(-Mg ++ RlN+Mg"*) ME ADH FB 151 a c t i v i t y , but Interference effects from the buffer made a quantitative estimation of the potency impossible. Treatment of peaks A and B with 0.01 M sodium thioglycollate completely abolished the oxytocic a c t i v i t y , thereby strengthining the view that the activity eluted from the column was due to AVT. E. Discussion and conclusions Since much of the discussion presented in Section II is also pertinent i n considering the results of this section, l i t t l e more w i l l be said here. The findings presented above suggest that the neurohypophysis of the hagfish elaborates arginine vasotocin, and therefore they support and extend the scanty and indecisive data published on the hagfish neurohypophysial princ-i p l e s . The amount of AVT in the hagfish posterior pituitary was no more than about 0.75 mU, assuming that 50% of the ac t i v i t y of the neurohypophysial extract was due to the hormone. This value, while very low, was nearly s i x times the hormone content of the neurohypophysis of Myxine glutinosa (0.13 mU, F o l l e t t and Heller, 1964). However, Foll e t t and Heller arrived at their value by assuming that only the portion of the crude extract activity destroyed by sodium thioglycollate was due to AVT, and i t seems possible that, as was the case here, thioglycollate treatment of the crude extract gave a low estimate of the neurohypophysial contribution to the total crude extract potency. The neurohypophysial hormone content of Pollstotrema was s l i g h t l y less than that of L. richardsoni, and, as discussed in Section II (p. 113), the amounts in the lamprey were extremely low when compared to other vertebrates. No evidence was obtained for the presence of a second posterior pituitary principle in the hagfish; although, as mentioned e a r l i e r , the low a c t i v i t y of the sample extract and the relatively large volume that was applied to the CMC column limited the p o s s i b i l i t i e s of detecting small amounts of a second active agent. 152 GENERAL DISCUSSION Discussions concerned with individual experiments and particular aspects of this study have been presented previously in the appropriate section, and therefore this discussion w i l l be limited mainly to general considerations. Nevertheless, some points would benefit by further emphasis, and others arise out of consideration of the results as a whole. I. Evaluation of the Methods Used for Purification and Identification of the Cyclostome Neurohypophysial Principles. The isolation and identification of the neurohypophysial principles of Lampetra richardsoni and Polistotrema stout11 involved methods which could not prove conclusively the identity of the active agents, but they could provide strong evidence that the principles were not different from particular known hormones. It seems important to consider the methods in some detail and to comment on their effectiveness and r e l i a b i l i t y in characterizing the cyclostome peptides. A. Methods of purification The main object of the research described in the preceding sections was the identification of the neurohypophysial hormones of certain species of cyclostomes. This required at least partial purification of the hormones in order to reduce the p o s s i b i l i t y of interference from contaminating substances. A number of different methods for the purification of posterior pituitary hormones have been used in different laboratories; these include salt precip-itatio n , counter-current distribution, partition chromatography, gel 153 f i l t r a t i o n , paper chromatography and ion-exchange chromatography (Acher, 1966; Fitzpatrick and Bentley, 1968; Sawyer, 1968). However, the purification of the neurohypophysial principles of cyclostomes presented particular problems which were of much less importance in studies of the hormones of other vertebrates. These problems stemmed primarily from the extremely low biolog-i c a l a c t i v i t i e s present in the cyclostome neurohypophysis, a c t i v i t i e s (per pituitary) which were lower than those of any other vertebrate on record (see Section II, p. 113; Sawyer, 1968). This serious d i f f i c u l t y made i t necessary to collect large numbers of animals, and even then the biological a c t i v i t i e s available were much less than those used in the purification of the principles of other vertebrates (see, for example, purification schemes described by Acher et a l , 1958, 1964; Sawyer and van Dyke, 1963; Pickering and Heller, 1969). Consequently, this investigation required methods which were capable of handling small amounts of hormones without large losses of material during the stage of the purification. Many of the techniques employed by other workers seemed unsatisfactory for such small scale operations. More-over, the low activity of the cyclostome neurohypophysis required that the materials be extracted at a high tissue concentration ( 20 and 40 mg lyophilized tissue/ml) in order to provide a solution of sufficient activity to be assayed. Even at these concentrations, the ac t i v i t y in the crude extracts was minimal and care had to be taken not to dilute the samples to the point of extinction at any step of the purification. Therefore, methods such as gel f i l t r a t i o n , which often result in an increase in sample volume (Pharmacia, 1969), could not be u t i l i z e d . The high ratio of tissue to acetic acid used during extraction also increased the concentration of contaminating substances present in the extracts; this, coupled with the unavoidable inclusion of much brain tissue in the. cyclostome neurohypophysial extracts, resulted in large amounts of contaminants. These impurities had to be removed 154 i f a clear identification of the neurohypophysial hormones was to be achieved. After consideration of the above-mentioned factors, four purification methods were employed in this study : u l t r a f i l t r a t i o n , treatment with t r i -chloroacetic acid, paper chromatography and cation-exchange chromatogrpahy. Of these, the f i r s t two methods were used as preliminary purification proce-dures, which were followed by one of the more refined chromatographic tech-niques. 1. Preliminary purification procedures The main purpose of the f i r s t stage of purification was the gross removal of proteinaceous contaminants; this was necessary to prevent any over-loading or interference with the chromatograms. T.C.A. appeared to be s l i g h t l y more effective than u l t r a f i l t r a t i o n in purifying the lamprey extracts, but, for unknown reasons, i t was completely ineffective in removing contaminants from the hagfish material. U l t r a f i l t r a t i o n had the disadvantage of a consid-erable variation in the effectiveness of individual membranes, so that i t was not possible to estimate beforehand the degree of purification that would be obtained. One possible reason for this variation may have been the known technical d i f f i c u l t y of ensuring a proper mixing of the extract during ultra-f i l t r a t i o n : i f the solution is not mixed, material may build up at the membrane surface so that the properites of the molecular f i l t e r are affected (Amicon, 1969). Such mixing was not possible in the apparatus available for u l t r a f i l t r a t i o n of the small quantities of extract available in these exper-iments. With T.C.A., on the other hand, the degree of purification of the lamprey extracts f e l l within f a i r l y narrow limits. Other workers (Wilson, 1968; Perks, personal communication) have also concluded that u l t r a f i l t r a t i o n with Amicon membranes is unreliable. T.C.A. has been used by various workers to p a r t i a l l y purify neurohypophysial hormones, and in particular i t has been 155 employed in the extraction of posterior pituitary hormones from blood (Fitz-patrick and Bentley, 1968). It is an effective agent for denaturing and removing large molecular weight protein contaminants from solution, and i t has the advantage of being easily removed from solution by procedures such as extraction with ether. In pilot studies and in investigations of the lamprey, the T.C.A. removed large amounts of Lowry-positive material with l i t t l e effect on the levels of AVT, so that the factors of purification obtained (approxi-mately 3) compared favorably with those achieved by gel f i l t r a t i o n of neuro-hypophysial extracts in other species (Swiatkiewicz, 1967; V i s z c y l i , 1968; Wilson, 1968). It is important to note that the method did not result i n any dilution of the cyclostome extracts. 2. Chromatographic techniques One of two chromatographic methods was employed in the second stage of purification of the cyclostome extracts; the f i r s t method was paper chrom-atography in butanoltacetic acidrwater (4:1:5), and the second was cation-exchange chromatography on carboxymethylcellulose resin. Both techniques have been employed by other workers in the purification of neurohypophysial peptide and they have been found particularly useful in the resolution of mixtures of basic peptides (Ward and Guilleman, 1957; Pickering and Heller, 1969; Heller and Pickering, 1961; Sawyer and van Dyke, 1963; Sawyer, 1968; Wilson, 1968). In this study, paper chromatography was not suitable for purifying the amounts of material necessary for a comprehensive pharmacological characterization of the active substances. The main reason for this was the low concentration of AVT in the cyclostome extracts; this increased the volumes which had to be applied to the paper, so that the time needed for application became excessive and the contaminants present became sufficient to cause artifacts due to over-loading. The sample volumes used in paper chromatography in this work were 156 often as much as one m i l l i l i t e r ; in contrast, Morris and Morris (1964) suggest that sample volumes as low as two to four microliters are preferable for maximum resolution during paper chromatography. Another disadvantage of paper chromatography was that the recoveries of applied materials, although highly variable, were generally lower than those obtained in column chromatography. Although paper chromatography was unsuitable for the purification of rela- -t i v e l y large amounts of a c i t i v i t y , i t was useful when materials were in short supply, as was often the case; in addition, i t acted as a method for charac-terization of the active principles by their chromatographic behaviour. In contrast, cation exchange chromatography was not a particularly useful method for characterizing the AVT, since the buffer conductivities at which the hormone was eluted varied with different columns. However, the technique was particularly effective in purifying AVT and purifications as high as 30 to 40 fold were obtained. The columns were eluted with a gradient of increas-ing buffer concentration. No rise i n buffer pH was u t i l i z e d , as carried out by other workers (Sawyer and van Dyke, 1963; Sawyer, 1965a), since increasing the pH of the eluates might have augmented the rate of inactivation of the AVT by disulphide interchange.(Ryle and Sanger, 1955; Wilson, 1968). The purification sequence that proved most effective in this study was a two stage procedure, comprising a preliminary purification with T.C.A., followed by chromatography of the extract on CMC. This method yielded AVT of sufficient purity for phramacological studies. Relatively small amounts of hormone were processed by this method (no more than 200 ng at a time), so that i t appears the technique is well suited to the purification of neuro-hypophysial material from sub-mammalian vertebrates, in which the levels of hormones are generally low. 157 B. Methods for the identification of the neurohypophysial peptides Three lines of evidence were used to identify the neurohypophysial hormones present in the cyclostomes examined in this study. These were : 1 ) the l a b i l i t y of the extracted a c t i v i t y to sodium thioglycollate, 2) chrom-atographic behaviour of the active substances, 3) pharmacological character-i s t i c s of the active principles. Of these, the latter data were by far the most important. Of course, the ultimate proof of the identity of the hormones would require the elucidation of their structure by amino acid analysis and sequence determination. This would then have to be followed by synthesis of compounds with the proposed structures, and the demonstration that their bidlogical a c t i v i t i e s were identical to those of the natural peptides. This procedure requires large amounts of starting material, and this was not possible here. However, as an alternative to chemical studies, there exist a f a i r l y large group of biological assays which exhibit differential sensitiv-i t i e s towards the different posterior pituitary hormone analogues. The responses involved in these assays are readily quantifiable and the potency estimations obtained lend themselves to s t a t i s t i c a l analysis. By using several of these assays, i t is possible to discriminate between different peptides. These pharmacological data can be combined with the results from chromatographic studies and from inactivation tests to provide strong evidence for the identity of an unknown peptide. 1 . Inactivation with sodium thioglycollate Treatment of cyclostome extracts with sodium thioglycollate was used extensively throughout this study to determine whether the rat uterus ac t i v i t y was attributable to neurohypophysial peptides. The s p e c i f i c i t y of the thio-glycollate test for posterior pituitary hormones was assumed during these experiments; however, at least one authority has questioned the v a l i d i t y of 158 t h i s assumption (Sawyer, 1966). Sawyer suggested that other biologically active tissue components could contain disulphide groups, and therefore they might be suseptible to reduction by thioglycollate, with a consequent loss of a c t i v i t y . As yet, however, no such tissue components appear to have been identified. Sawyer also noted that many of the non-neurohypophysial utertonic substances (e.g. Ach, 5-KT, bradykinin, substance P) are unstable in crude .tissue extracts and therefore would become inactivated over the time of Anoubation necessary for the destruction of neurohypophysial biological a c t i v i t y by the thioglycollate. Even i f this had occurred in the cyclostome incubates, i t would not have been a source of error, since the inactivation of the non-neurohypophysial active agents would have taken place both in the thioglycollate-treated aliquots and i n the control samples against which the former solutions were assayed. Sawyer's other criticism of the method - that •other sulphydryl-containing tissue components could protect the neurohypophy-s i a l peptides from reduction - would seem to be suported by the results of the present study. In several instances (see pp. 79-80, 147-148), estimations, resulting from thioglycollate treatment,of neurohyophysial hormone present i n crude extracts were lower than values obtained after further purification of the extracts. As discussed earlier (pp.61-62), one possible explanation for this discrepancy is that contaminating substances in the extracts competed with the AVT for thioglycollate and thereby reduced the rate of inactivation of the hormone. It would seem, therefore, that thioglycollate estimations are of only limited value in studies of crude extracts, and that such tests should be used only to give a qualitative demonstration of the presence of neurohypophysial hormones, rather than a quantitative estimation of their proportion. On the other hand, the r e l i a b i l i t y of results obtained by treating purified preparations with thioglycollate would seem to be satisfactory, since 159 no significant levels of competitive or inhibiting contaminants would be present, and the existence of non-neurohypophysial active substances, labile to the thiol reagent, has not yet been demonstrated. To the warnings voiced by Sawyer (1966) on the worth of sodium thio-glycollate as a test for the presence of neurohypophysial peptides must be added one more criticism, which has come to light in this study. This is the p o s s i b i l i t y of the liberation of active substances as a result of incubation of tissue extracts with thioglycollate. Such a release of uterotonic substances apparently occurred during treatment of the control, hindbrain extracts with the t h i o l reagent. The identity of these active agents is a mystery, but i t is clear that their presence was a possible source of error, in that they could have masked the destruction of neurohypophysial a c t i v i t y . Indeed, this probably occurred in the hindbrain extracts, since later purification suggested that small amounts of AVT were present in at least some of the control extracts. Whether the destruction of neurohypophysial a c t i v i t y in the crude pituitary extracts was masked by a similar liberation of active substances could not be determined. Certainly the low estimates of the amounts of AVT i n thioglycollate treated extracts could be explained on this basis. However, as discussed above, the low estimates could also be accounted for on the basis of competition between the neurohypophysial peptides and other sulph-ydryl-containing tissue components for the thioglycollate. Whatever the explanation, i t is apparent that treatment of impure tissue extracts with sodium thioglycollate as a test for the presence of neurohypophysial peptides, involves severally potentially serious sources of error. 2. Chromatographic methods The main purpose of chromatography in this study was to purify the neurohypophysial hormones present in the crude extracts. However, the 160 chromatographic methods also provided data which were useful in the character-ization of the active substances. This was particularly true of paper chrom-atography in butanol:acetic acidtwater, a method which clearly separates neutral and basic posterior pituitary hormones (Heller and Lederis, 1958). Table XX gives the Rf's of synthetic AVT and the AVT-like principle from the lamprey extracts studied in this investigation. It can be seen that despite variations in the migration of AVT on the various chromatograms, the Rf's of "standard" and "unknown" AVT run on the same chromatograms corresponded closely. These data, by themselves, were certainly not convincing evidence for the presence of AVT in the lamprey neurohypophysis, since other biologi-c a l l y active substances present in brain tissue exhibited similar chromato-graphic behaviour (see p. 72). However, taken in conjunction with the data resulting from pharmacological and chemical methods, the results obtained from paper chromatography lent useful support to the evidence that AVT constitutes the main neurohypophysial principle in Lampetra richardsoni. It can be seen from Table XXI that a f a i r l y wide variation existed in the buffer concentrations at which AVT was eluted from the different ion-exchange columns. Part of this variation may have been due to differences in the packing of the resin in the various columns (Morris and Morris, 1964), and i t is possible that the early elution of the AVT-like peptide during chromatography of the hagfish extract resulted from the presence of large amounts of contaminating substances. Although ion-exchange chromatography was not as precise as paper chromatography in characterizing the cyclostome material, i t was s t i l l useful, since i t clearly differentiated AVT from other active substances present in the extracts. However, the main purpose of cation-exchange chromatography in this study was the purification of the neurohypoph-y s i a l material prior to pharmacological characterization. 161 Table XX. Chromatographic behaviour of synthetic AVT and the AVT-like principle from lamprey neurohypophysial tissues in the solvent system butanol:acetic acid:water (4:1:5). Chromatogram - Active substance Total Rf Rf of peak 3 & 4 year ammocoetes synthetic AVT 0.3-0.6 0.4-0.5 - extract AVT 0.4-0.6 0.4-0.5 ammocoetes from section synthetic AVT 0.3-0.5 0.3-0.4 III extract AVT 0.3-0.6 0.3-0.4 5 & 6 year ammocoetes synthetic AVT 0.3-0.4 0.3-0.4 extract AVT 0.3-0.5 0.3-0.4 adult lamprey synthetic AVT 0.3-0.6 0.4-0.5 extract AVT . 0.3-0.6 0.4-0.5 Table XXI. Buffer conductivities at which AVT and the AVT-like principle from cyclostomes were eluted from carboxymethylcellulose ion-exchange columns. Loading solution synthetic AVT AVT, oxytocin, albumin ammocoete neuro-hypophysial extract adult neurohypo-physial extract adult hindbrain extract hagfish neuro-hypophys i a l extract Column Gradient Conductivity, mMho 0.5x20 cm CMC 0.02 M NaAc - 0.20 M NaAc 4.29-4.78 0.9x10 cm CMC 0.02 M NaAc - 0.20 M NaAc 0.5x14 cm CMC 0.02 M NaAc - 0.20 M NaAc 0.5x14 cm CMC 0.02 M NH^Ac-0.20 M NH^Ac 0.5x14 cm CMC 0.02 M NH^Ac-0.20 M NIfyAc 0.5x14 cm CMC 0.02 M NH4Ac-0.20 M N H 4 A C 5.11-5.14 3.60-4.18 3.92-5.00 3.53-3.95 2.31-3.98 162 3. Pharmacological methods The identification of the cyclostome neurohypophysial principles involved two separate problems; these were : 1) the isolation and character-ization of arginine vasotocin, which already had been tentatively identified in cyclostomes, 2) the possible detection and identification of a second and probably neutral hormone : although there was no evidence in the literature for i t s presence, i t remained a possi b i l i t y since a neutral peptide is found in a l l other vertebrate p i t u i t a r i e s . The two lines of research presented different demands on the methods employed. Characterization of the AVT required bioassays which were capable of differentiating this particular hormone from other posterior pituitary principles; while demonstration of a second neurohypophysial agent necessitated methods with a high degree of sensitivity, a. Effectiveness of the biological assays in characterizing AVT The method used in the pharmacological characterization of the purified material obtained from the cyclostomes was a modification of Gaddum*s (1955) index of discrimination (see Heller and Pickering, 1961). This technique involved assaying the purified extracts against the hormone which they were thought to contain, i.e. arginine vasotocin. Previous studies of cyclostome neurohypophysial hormones had employed the "classical method" of pharmacological characterization : in this method the unknowns were assayed by many diverse techniques either directly or indirectly against posterior pituitary reference standard, i.e. against mammalian oxytocin and vasopressin (see Sawyer et a l , 1961; Foll e t t and Heller, 1964; Sawyer, 1965a). The profile of a c t i v i t y obtained for the unknown was then compared with the profiles for synthetic analogues, and, on the basis of this comparison, a tentative ident-i f i c a t i o n of the unknown substance could be made. There are seversT-problems 163 which can arise in this method : 1) Since the standard and unknown are often different substances, variations can occur in the sensitivities to standard and unknown from one preparation to another; this has been illustrated in a striking manner by the work of Heinicke and Perks (1969) on magnesium ratios of neurohypophysial extracts from elasmobranchs. 2) If the active peptides present in the unknown and standard prepar-ations are different, the regressions between log-dose and response . may not always be parallel for the two materials, and errors are l i k e l y to arise-(Sawyer, 1966; Berde and Boissanis, 1968). 3) The development of tachyphylaxis to one peptide but not to the other during an assay is also possible, and can lead to errors in the potency estimation (Sawyer, 1966; Berde and Boissanis, 1968). In the method emplyed in this study, unknown and standard were presumably identical, and therefore the assay preparations would be unable to d i f f e r -entiate them in any way; consequently, the above problems would not arise. As mentioned earl i e r (p. 37), the usefulness of pharmacological methods in distinguishing one particular hormone from other analogues depends both on the pharmacological characteristics of the peptide and on the choice of the biological assays, employed. In this study, the majority of the prepar-ations were sensitive to most neurohypophysial peptides, and some would react to non-neurohypophysial substances. However, this lack of s p e c i f i c i t y was largley offset by the notable quantitative differences in the responses of different assays to different peptides; differences which result in a unique spectrum of biological a c t i v i t y exhibited by arginine vasotocin. This charac-t e r i s t i c profile of a c t i v i t i e s is due to the structure of AVT, which a molecular hybrid between oxytocin and arginine vasopressin (Acher et a l , 1964). 164 AVT possesses the ring structure of oxytocin and the side chain of AVP, and as a result exhibits both oxytocic effects (rat uterus, avian depressor and galactobolic assays) and the ac t i v i t i e s possessed by the vasopressins (vaso-pressor and antidiuretic a c t i v i t i e s ) . Although the potencies of AVT on these assays are sl i g h t l y less than those of the mammalian principles, a l l of it s activites are of the same order of magnitude. This is reflected in the VP/RU and ADH/RU ratios of AVT (1.55 and 1.96 respectively, Sawyer, 1965a), which are close to unity. The corresponding ratios for neutral hormones are very low; for example, [4-serine, 8-isoleucine] - oxytocin has VP/RU and ADH/RU ratios of 0.005 and less than 0.0003 respectively. On the other hand, the naturally occurring vasopressins have high vasopressor and antidiuretic a c t i v i t i e s ; the VP/RU and ADH/RU ratios for arginine vasopressin are both 44 (Sawyer, 1965a). It is clear that the above-listed differences between AVT, on one hand, and the neutral hormones and vasopressins, on the other, are probably great enough to allow a tentative identification of AVT in neurohy-pophysial extracts (Fitzpatrick and Bentley, 1968). However, the effects of AVT on water and sodium transport across the skin and urinary bladder of certain anuran amphibians are even more useful in distinguishing i t from other posterior pituitary analogues. Virtually a l l neurohypophysial hormones increase the rate of transport of sodium and water across these membranes; but AVT is much more active than a l l other analogues that have been examined (Pickering, 1970). [8-lysine!]-oxytocin (lysine vasotocin) also has appreciable frog bladder a c t i v i t y , but i t is only about one eighth as active as AVT (Sawyer, 1965a). Moreover, lysine vasotocin can be differentiated from AVT on the basis of it s relatively low antidiuretic activity (Sawyer, 1965a). Other biological assays, such as the isolated hen oviduct (Munsick et a l , 1960) and the amphibian oviduct (Heller et a l , 1967), also exhibit a relatively high 165 sensitivity for AVT, but they are either more d i f f i c u l t to perform or are less v e i l standardized than the frog bladder and frog skin assays. It would seem that assay of an unknown substance on a number of the above biological assays can provide strong evidence either for or against the presence of AVT; particularly useful in this regard are the rat uterus, antidiuretic, vasopressor, frog bladder and natriferic assays. When the unknown is assayed directly against AVT, as done in this study, the technique would appear to be the most powerful method available for the pharmacological characterization of arginine vasoticin. b. Effectiveness of the biological assays in detecting a second  neurohypophysial hormone in cyclostomes In the search for a second posterior pituitary hormone in the lamprey and hagfish, two assumptions were made. The f i r s t was that the second principle, i f i t existed, would be a neutral, oxytocin-like analogue, since this is the situation that occurs in a l l other vertebrates so far investigated (Sawyer, 1970). The second assumption was that the principle would possess at least some of the pharmacological a c t i v i t i e s common to other neutral analogues, and i n particular, i t would exhibit appreciable rat uterus activity. The f i r s t assumption implied that the hormone would behave chromatographically the same as other oxytocin-like peptides; that i s , that i t would run in the region Rf 0.6-0.8 during paper chromatography, and that i t would be eluted in the loading volume during ion-exchange chromatography (Perks, 1966; Sawyer et a l , 1967). Therefore, these regions of the chromatograms were carefully checked for the presence of active substances. The assay used to check these areas was the isolated rat uterus with magnesium present. This preparation was chosen because of i t s high sensitivity, its ease to perform and because the a b i l i t y to contract the rat uterus is exhibited by a l l naturally occurring 166 neutral hormones. The high sensitivity of this method was of particular importance; with magnesium present, the threshold dose for the uterus varied from approximately 0.04 to 0.40 mU, and i t was normally below 0.10 mU. However, this is not as sensitive as some other bioassays for neutral peptides (see Ryden and Sjoholm, 1962; van Dongen and Hays, 1966; Bissett et a l , 1967; van Dongen and Marshall, 1967; F i e l i t z et al_, 1970). These assays posed various problems (low order of precision, sensitivity to buffers, d i f f i c u l t y in performing) that were not encountered with the rat uterus assay. The one major disadvantage of the rat uterus assay as a method for dectecting a second neurohypophysial peptide was Its lack of s p e c i f i c i t y . In - the course of this study, the method detected several fractions, which on the basis of their chromatographic behaviour, could have contained a neutral posterior pituitary principle (see for example, pp. 98-101, 107-108); but, in every case, evidence obtained by other techniques suggested that the active substances were non-neurohypophysial in nature. These latter techniques included testing the a c t i v i t y for l a b i l i t y to sodium thioglycollate, and assaying the active substances for rat milk ejecting a c t i v i t y . Both of these methods are considered to be f a i r l y specific for neurohypophysial hormones (Bissett et a l , 1967; Fitzpatrick and Bentley, 1968). It is f e l t that the pharmacological methods employed in this study ' were f a i r l y effective in providing evidence against the presence of a second biologically active neurohypophysial hormone in L. richardsoni. In both adults and larvae, no more than about one percent of the total neurohypophysial act i v i t y could have been due to a second hormone without being detected. With P . stoutit, the evidence against the existence of a second peptide was not as strong as with the lamprey, since significant amounts of a neutral principle (22% of the total activity) could have passed through the ion-exchange column 167 without being detectd by the rat uterus assay. The i n a b i l i t y to detect this large amount of ac t i v i t y was primarily the result of the low activity/ml and large volume of the hagfish extract. Since any neutral peptide that may have been present in the hagfish extracts would not have been bound to the cation-exchange resin with the buffer concentrations used, i t would not have been concentrated during this purification step. Of course, concentration of the hormone would have increased the likelihood of finding i t in the eluted fractions, but as discussed ear l i e r (p. 81), binding of a neutral hormone to the resin would have required a dilution of the sample that was deemed unacceptable for adequate recovery of the AVT that was present In the extract. II. The Neurohypophysial Hormones of Cyclostomes Arginine vasotocin has now been tentatively identified in three species of lamprey : Petromyzon marinus (Sawyer et a l , 1961; Sawyer, 1965a), Lampetra f l u v i a t i l u s (Follett and Heller, 1964), and, in the work presented here, Lampetra richardsoni. Also, i t appears to be present in two species of hagfish : Myxine glutinosa (Follett and Heller, 1964) and Polistotretna s t o u t l l . The present study, which used direct comparisons with the synthetic peptide, has provided the strongest evidence yet for the presence of AVT in cyclostomes. Nevertheless, f i n a l proof awaits chemical identification of the hormone, and i t i s of interest to note that Acher (1968) has reported that he is working towards this end. As yet, however, i t has been impossible to obtain products of sufficient quantity and purity for chemical analysis. In the absence of this chemical information, i t seems reasonable to accept the pharmacological and chromatographic data, and to assume that the hypothalamo-neurohypophysial systems of lamprey and hagfish elaborate arginine vasotocin. The results of the present study suggest that AVT was present in larval as well as adult 168 L. richardsoni. Therefore the hormone is present in what may be considered the most primitive vertebrates; this observation supports the view held by several authorities that AVT is the antecedent of a l l vertebrate neurohypo-physial hormones (Geshwind, 1967; Vliegenthart and Versteeg, 1967; Sawyer, 1968; Heller and Pickering, 1969). It is present in a l l non-mammalian verte-brates so far examined, and has recently been found in foetal sheep and seals (Vizsolyi and Perks, 1969). Therefore, i t would seem probable that represent-atives of a l l vertebrates possess the capacity to synthesize AVT at some stage of their l i f e histories, and that arginine vasotocin, for reasons not yet clear, has exhibited a remarkable s t a b i l i t y during vertebrate evolution. No evidence was provided in this study for the presence of a second neurohypophysial principle in cyclostomes. Admittedly, with hagfish, the evidence was not very conclusive, since substantial amounts of another hormone could have escaped detection. With lamprey, however, the maximum amount of a neutral hormone that could have been present represented less than one percent of the total a c t i v i t y in the neurohypophysis. If one assumes that this second hormone, i f indeed present, was identical to one of the naturally-occurring neutral peptides (oxytocin, isotocin, mesotocin, glumitocin), then the molar rat i o of AVT/neutral peptide would have been greater than 99. Indeed, i f glumitocin were present, the ratio would have been considerably greater than 99, since this hormone has a much lower absolute potency than AVT (Sawyer et a l , 1969). Mole ratios of the neurohypophysial hormones in the neural lobe of other vertebrates have been calculated by Follett (1963). While the amount of the basic peptide was greater than the amount of neutral in many of the species examined, the highest ratio of basic/neutral was 10.2, this being in the herring g u l l . In teleosts the ratio was approximately three, and in the Chondrichthyes i t was less than one (Sawyer et a l , 1967; Pickering and Heller, 169 1969). Only in the Chondrostei does there appear to much larger amounts of AVT than neutral peptide, since the amount of the latter principle is not more than 3.97. of the former (Follett and Heller, 1964). If, therefore, the lamprey does contain small amounts of one of the natural neutral peptides that have been identified to date, the proportion of the neutral would be much less than occurs in any other vertebrates except the Chondrosteans. In considering the possibility of the existence of a second neurohy-pophysial principle in cyclostomes, i t is necessary to emphasize that the methods employed both in this study and in previous investigations would only have detected the presence of peptides active on the pharmacological prepar-ations used. The possibility that lower vertebrates may possess neurohypophy-s i a l hormones which are not active on the existing biological assays has been suggested by various workers, mainly as a result of consideration of the evolution of the hormones (Geshwind, 1967; Vliegenthart and Versteeg, 1967; Heller and Pickering, 1969; Sawyer, 1970). Recent attempts at proposing evolutionary schemes for the peptides have involved consideration of the genetic code, and have attempted to explain the phylogenetic distribution of the neurohypophysial hormones on the basis of gene duplication and single base mutations in the cistron of an ancestral molecule (perhaps AVT). A l l naturally-occurring posterior pituitary peptides d i f f e r from each other only in the identities of the amino acid moieties in the third, fourth, and eighth positions (Sawyer, 1970). Most of the differences in the residues present at these positions can be accounted for by single base changes in the appropriate cod oris, and therefore, i t is possible to propose schemes in which one hormone "evolves" into another simply as the result of a single base change. However, problems arise in considering the origins of glumitocin ([4-serine, 8-glutamine -oxytoeiiij),which has been identified in certain elasmobranchs, and of isotocin 170 ([4-serine, 8-isoleucine]-oxytocin), which is found in teleosts (Perks, 1969). Both these peptides possess serine in position 4, in contrast to the glutamine possessed by other analogues. Conversion of glutamine to serine (or vice-versa) cannot occur through a single base change in any of the t r i p l e t s coding for glutamine. At least two mutations are required, with a 4-proline as the most probable intermediate (Sawyer et a l , 1969). These speculations have led two groups to synthesize 4-proline analogues of the neutral neurohypophysial hormones (Rudinger et al_, 1969; Sawyer et a l , 1969). A number of these peptides have been synthesized and characterized pharmacologically; these include [4-proline, 8-isoleucine]-oxytocin, [4-proline, 8-glutamine]-oxytocin and [4-proline, 8-leucine]-oxytocin. A l l three analogues possessed rat uterus and milk ejecting a c t i v i t i e s . However, the potencies were extremely low, as for example in [4-proline, 8-leucine]-oxytocin which possessed an RU (+Mg"*) value of 0.007 U/mg as compared to 450 U/mg for oxytocin,(Sawyer et a l , 1969). It is clear that, as a result of these low a c t i v i t i e s , the peptides could have been present in species already studied without being noticed, and the possi--b i l i t y of their existence in cyclostomes certainly cannot be excluded. However, most authorities feel that these intermediates would have appeared later in vertebrate evolution, perhaps during the development of the placoderms; the l i v i n g species most l i k e l y to possess the peptides would appear to be the primitive actinopterygians, especially .the Chondrostei, and the primitive selachian elasmobranchs (Sawyer, 1965b; Vliegenthart and Versteeg, 1967; Geshwind, 1967; Heller and Pickering, 1969). It would seem worthwhile to further investigate lower vertebrates, especially the cyclostomes and primitive fishes, for the presence of possible "intermediate" neurohypophysial analogues with low biological a c t i v i t y . Clearly, the pharmacological methods in current use would be of l i t t l e value in such 171 investigations, since they are capable of detecting only massive amounts of the 4-proline analogues* It has been suggested that attempts be made to design new bioassay procedures with a high sensitivity for these analogues (Sawyer et al_, 1969). However, this approach would seem to involve a large degree of ri s k , since there is no guarantee that tissues which are sensitive to the 4-proline peptides do indeed exist.It seems possible that radioimmunological techniques may be useful in the detection of these principles in lower vertebrates. 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Subsequent to running the sections through alcohol to water, the tissues were refixed in Bouin 1s solution for from 12 to 24 hours; they were then washed thoroughly in tap water to remove picric acid. Next the sections were oxidized for about one minute in a solution containing 0.3 percent each of potassium permanganate and sulphuric acid; this was followed by decolorization with a two to five percent solution of sodium bisulphite, and washing in tap water. The tissues were then stained for from 15 to 25 minutes with the chrome-haematoxylin stain given below, and differentiated in a one percent hydrochloric acid-alcohol solution for about one minute. The sections were washed with tap water until they were clear blue, and counterstained with an 0.5 percent aqueous solution of phloxine for five minutes. Next, they were immersed in a five percent phosphotungstic acid solution for one minute and washed with tap water for five minutes. The stained sections were f i n a l l y differentiated in 95 percent alcohol, transferred to absolute ethanol, cleared in xylol and mounted in balsalm. Chrome-Haematoxylin Solution 1% aqueous haematoxylin solution 50 ml combine to form sol-37. chromium alum solution 50 ml ution A. 57. potassium dichromate solution 2 ml combine and add to 0.2 N sulphuric acid 2 ml solution A. 184 The stain was ripe after 48 hours and was f i l t e r e d before use. B. Aldehyde-Fuchsin-Light Green-Orange G Stain (Halmi, 1952, as modified by  Dawson, 1953). The deparafinnized sections were run through descending alcohols to water, and then treated for about one minute with a solution containing 0.3 percent potassium permanganates and 0.3 percent sulphuric acid. Subsequent to this oxidation, the tissues were decolorized in a 2.5 percent solution of sodium bisulphite, and stained with the aldehyde-fuchsin stain given below. Staining time varied from two to ten minutes; the slides were checked periodically under a microscope and were removed when the proper degree of staining had been reached. The treated sections were rinsed in two changes of 95 percent ethanol, and then l e f t in a third change for five to ten minutes. Following th i s , they were rinsed in 70 percent alcohol and d i s t i l l e d water, and counterstained for 45 seconds with the Light green-Orange G solution described below. Next, the stained material was rinsed b r i e f l y in 0.2 percent acetic acid and 95 percent alcohol; this was followed by two changes of absolute ethanol of two minutes each. The slides were then blotted carefully, subjected to two changes of xylol (two and five minutes, respectively) and mounted in c l a r i t e . Aldehyde-Fuchsin Solution 607. alcohol 100 ml basic fuchsin 0.5 g paraldehyde^ 1 ml concentrated HC1 1.5 ml The solution was made up two to three days in advance. 185 Light Green-Orange G Solution d i s t i l l e d water 100 ml light green SF yellowish 0.2 g orange G 1.0 g chromatrope 2R 0.5 g phosphotungstic acid 0.5 g glacial acetic acid 1 ml 186 APPENDIX B Pilot Experiments with Trichloroacetic Acid and U l t r a f i l t r a t i o n Prior to the employment of T.C.A. and u l t r a f i l t r a t i o n as methods for the partial purification of cyclostome extracts, t r i a l runs were performed on solutions of albumin and synthetic neurohypophysial peptides. The procedures followed were the same as those given in Materials and Methods (pp. 39-40). The results of the pilot experiments are given in Tables XXXII and XXXIII. Treatment of the solutions with T.C.A. generally resulted in a greater recovery of biological activity than was obtained using u l t r a f i l t r a t i o n . Although the recoveries by the latter method were sometimes high, they varied widely; a similar variation existed in the amounts of albumin removed, so that i t was not possible to predict beforehand the degreee of purification that would be obtained. On the basis of these results, i t appeared that, of the two methods, T.C.A. offered the greater potential for purifying the cyclostome extracts. Table XXII. Purification of solutions of albumin and synthetic neurohypophysial peptides using u l t r a f i l t r a t i o n . Solution Volume treated Membrane used 7. recovery of oxytocic ac t i v i t y 7. removal of albumin Factor of purification * 2600 pg/ml albumin, 100 mU/ml oxytocin. 1.0 ml 1.0 ml UM 2 ** UM 10*** 72 7. 79 7. 47 7. ' 32 7. 1.9 1.5 2700 ug/ml albumin, 500 ng/ml AVT, 35 mU/ml oxytocin. 2.0 ml UM 10 49 7. 80 7. 5.1 2800 ug/ml albumin, 100 ng/ml AVT. 2.0 ml 2.0 ml UM 10 UM 10 62 7. 67 7. 90 7. 99 7. 10.2 95.0 2.0 ml UM 10 95 7. 100 7. — * calculated by dividing the total amount of Lowry peptide material present in the extract prior to treatment with the amount present after treatment. ** UM 2 membranes had a molecular weight cutoff range of approximately 1000. *** UM 10 membranes had a molecular weight cutoff range of approximately 10,000. Table XXIII. Purification of solutions of albumin and synthetic neurohypophysial peptides using trichloroacetic acid. Solution Volume 7. recovery of 7. removal Factor of treated oxytocic activity of albumin purification * 1750 ug/ml albumin, 5.0 ml 108 7. 92 7. 12.2 100 mU/ml oxytocin. 1750 ug/ml albumin, 3.0 ml 89 7. 100 7. 1000 ng/ml AVT. * calculated by dividing the amount of Lowry peptide material present in the solution prior to treatment with T.C.A. by the amount present after treatment. 189 APPENDIX C Pilot Experiments with Ion-Exchange Chromatography T r i a l runs using Whatman carboxymethyl cellulose cation-exchange resin and synthetic neurohypophysial peptides were carried out to determine the elution characteristics of the peptides, especially arginine vasotocin. The procedure followed was the same as that given in Materials and Methods (pp. 42-44), and the results are given i n figures 33 and 34. They indicated that with a starting buffer concentration of 0.02 M and pH of 5, AVT was bound to the resin and could be eluted by employing a gradient of increasing buffer concentration. Oxytocin (and presumably oxytocin-like analogues), on the other hand, was not bound, and passed through the column in the loading volume. 190 Figure 33 Ion-exchange chromatography of synthetic argenine vasotocin on CMC cellulose resin. a. column = 0.5 cm x 20.0 cm of Whatman CM-22 carboxymethyl cellulose. b. loading solution •-•=• 5.0 ml, at pH 5, conductivity 1.04, containing 1000 ng AVT. c. starting buffer « 0.02 M, pH 5 sodium acetate (conductivity = 1.04), to A. d. gradient = 0.02 M, pH 5 sodium acetate to 0.20 M, pH 5 sodium acetate (conductivity » 8.2), A to B. e. flow rate = 12 ml/hr. f. volume collected/tube • 1 ml. g. recovery = 1007. of oxytocic a c t i v i t y . open ci r c l e s «= rat uterus activity (magnesium present) of the eluted fractions in mU/tube. open triangles - specific conductivity of the eluted fractions in millimhos. OHWW 'AllAllOnaNCO CO r -co "T~ CVJ T O 1 CD * < < < < < < < < O - O O < —a. < < < < < < < < < < < < < < < < < < < < < < < o CO o cr. UJ m U J m 3 h-O CM Jo L_ O CD O o 191 Figure 34 Ion-exchange chromatography of albumin, synthetic oxytocin and synthetic arginine vasotocin on CMC cellulose resin. a. column » 0.9 cm x 10.0 cm of Whatman CM-22 carboxymethyl cellulose. b. loading: solution - 4.6 ml, at pH 5, conductivity 1.04, containing 2000 ng AVT, 100 mU oxytocin and 1400 ug Lowry peptide (albumin). c. starting buffer • 0.02 M, pH 5 sodium acetate (conductivity » 1.04), to A. d. gradient «= 0.02 M, pH 5 sodium acetate to 0.20 M, pH 5 sodium acetate (conductivity = 8.2), A to B. e. flow rate - 15 ml/hr. f. volume collected/tube = 3 ml. g. recovery - oxytocin => 1007.. - AVT - 637.. open ci r c l e s = rat uterus a c t i v i t y (magnesium present) of the eluted fractions in mU/tube. closed circles » Lowry peptide concentration of the eluted fractions in ug/ml. open triangles «= specific conductivity of the eluted fractions in millimhos. mU/TUBE 

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