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Role of prolactin in osmotic and ionic regulation of the marine form (trachurus) of the threespine stickleback,… Lam, Toong Jin 1969

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THE ROLE OF PROLACTIN IN OSMOTIC AND IONIC REGULATION OF THE MARINE FORM (TRACHURUS) OF THE THREESPINE STICKLEBACK, GASTEROSTEUS ACULEATUS L.v IN FRESH WATER by « TOONG JIN LAM B.Sc.(Hons), U n i v e r s i t y of B r i t i s h Columbia, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY, i n the Department of Zoology We accept t h i s thesis' as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1969 In p r e s e n t i n g t h i s t h e s i s i n 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 a t the U n i v e r s i t y o f B r i t i s h C olumbia, I ag r e e t h a t t h e 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 Study. I f u r t h e r a g r e e 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 purposes 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 . I t 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 . The U n i v e r s i t y o f B r i t i s h Columb Vancouver 8, Canada Department of i i A b s t r a c t The r o l e of p r o l a c t i n i n osmotic and i o n i c r e g u l a t i o n . of the marine threespine . s t i c k l e b a c k (Gasterosteus ;:,aculeatus L., form trachurus) i n f r e s h water has-been i n v e s t i g a t e d i n win t e r (or l a t e autumn) and. s p r i n g . S t i c k l e b a c k s i n l a t e autumn or e a r l y w i n t e r , when t r a n s f e r r e d from sea water to f r e s h water, s u f f e r e d a high m o r t a l i t y which could be" reduced by p r o l a c t i n treatment. The--f i s h a l s o d i s p l a y e d a greater f a l l i n plasma o s m o l a l i t y and a sm a l l e r f a l l i n u r i n e o s m o l a l i t y than l a t e - s p r i n g f i s h t r a n s f e r r e d to f r e s h water i n the same way; t h i s seasonal d i f f e r e n c e was apparently t r i g g e r e d by ph o t o p e r i o d i c changes and c o u l d be e l i m i n a t e d by p r o l a c t i n treatment of the l a t e -autumn or e a r l y - w i n t e r f i s h . S i m i l a r l y , a seasonal d i f f e r e n c e e x i s t s i n the h i s t o l o g i c a l p i c t u r e of the g l o m e r u l i of late-autumn and l a t e - s p r i n g s t i c k l e b a c k s , and t h i s d i f f e r e n c e c o u l d be e l i m i n a t e d by p r o l a c t i n treatment of the former f i s h . The f a l l i n plasma o s m o l a l i t y i n late-autumn and winter f i s h a f t e r t r a n s f e r to f r e s h water was p a r a l l e l e d by a r a p i d drop i n plasma sodium and c h l o r i d e , which could be c o r r e c t e d by a s i n g l e i n j e c t i o n of p r o l a c t i n given 24 hr before the t r a n s f e r . Plasma potassium, however, seemed un a f f e c t e d by p r o l a c t i n treatment. The evidence suggests th a t p r o l a c t i n i s e s s e n t i a l f o r freshwater s u r v i v a l of s t i c k l e b a c k s and that p r o l a c t i n undergoes seasonal changes i n s e c r e t i o n a s s o c i a t e d w i t h p h o t o p e r i o d i c i i i changes; the s e c r e t i o n : i s minimal i n the autumn and winter when the f i s h l i v e i n sea water or b r a c k i s h water, and maximal i n the s p r i n g and summer when the f i s h migrate to (or are i n ) f r e s h water, to breed. Thus p r o l a c t i n may be i n v o l v e d i n the.freshwater m i g r a t i o n of s t i c k l e b a c k s . Next, the mechanism of a c t i o n of p r o l a c t i n was s t u d i e d . P r o l a c t i n seems to e x e r t i t s e f f e c t s ( i n f r e s h water) on the three recognized organs of osmotic and i o n i c r e g u l a t i o n i n t e l e o s t s , v i z . kidneys, g i l l s and gut. In the kidneys, p r o l a c t i n i n c r e a s e d u r i n e flow, apparently as a r e s u l t of an i n c r e a s e d GFR. p r o l a c t i n reduced the apparent i n c r e a s e i n i n t r a c a p s u l a r space i n the g l o m e r u l i of the late-autumn and winter s t i c k l e -backs, and, consequently, i n c r e a s e d the percentage frequency o f g l o m e r u l i w i t h no evident i n t r a c a p s u l a r space. The data are i n t e r p r e t e d to mean that p r o l a c t i n rendered g l o m e r u l i more f u n c t i o n a l o r more g l o m e r u l i f u l l y f u n c t i o n a l a n d , hence, i n c r e a s e d G F R . Since the i n c r e a s e i n u r i n e f l o w and G F R was p a r a l l e l e d by a decrease i n u r i n e o s m o l a l i t y and u r i n e c o n c e n t r a t i o n s of sodium and c h l o r i d e , p r o l a c t i n must a l s o i n c r e a s e r e n a l t u b u l a r r e a b s o r p t i o n of sodium and c h l o r i d e ( A ) and/or decrease water r e a b s o r p t i o n ( B ) ; and s i n c e the t o t a l r e n a l l o s s of sodium and c h l o r i d e d i d not appear to be s i g n i f i c a n t l y i n c r e a s e d d e s p i t e an i n c r e a s e i n G F R , A must occur w i t h or without B . P r o l a c t i n , however, apparently i n c r e a s e d the t o t a l r e n a l l o s s of potassium and d i d not a f f e c t the t u b u l a r potassium r e a b s o r p t i o n , although there was a suggestion t h a t p r o l a c t i n a c t u a l l y decreased tubu l a r r e a b s o r p t i o n of potassium. i v In the g i l l s (or other regions around the .head), p r o l a c t i n reduced the net osmotic i n f l u x of water and the net 14 14 l o s s o f sodium, c h l o r i d e and C (from i n j e c t e d C - i n u l i n ) ; the l a t t e r was probably because p r o l a c t i n reduced the o u t f l u x . These changes were accompanied by the behaviour of the g i l l mucous c e l l s , which were i n c r e a s e d i n d e n s i t y by p r o l a c t i n treatment, suggesting a c a u s e - o r - e f f e c t r e l a t i o n s h i p . In the gut, p r o l a c t i n reduced water a b s o r p t i o n and, a t the same time, seemed.to reduce the freshwater d r i n k i n g r a t e . Thus, i t appears t h a t p r o l a c t i n was able to reduce or prevent osmotic f l o o d i n g of s t i c k l e b a c k s i n f r e s h water by reducing e x t r a r e n a l osmotic i n f l u x of water and i n c r e a s i n g r e n a l l o s s of water v i a an i n c r e a s e i n GFR and u r i n e flow, and a l s o , p o s s i b l y , by reducing d r i n k i n g r a t e and water abs o r p t i o n by the gut; a t the same time, p r o l a c t i n reduced e x t r a r e n a l l o s s of sodium and c h l o r i d e but d i d not apparently a f f e c t r e n a l l o s s of the i o n s , which was s m a l l compared to the e x t r a r e n a l l o s s . By these mechanisms, p r o l a c t i n maintained plasma o s m o l a l i t y and sodium and c h l o r i d e l e v e l s a f t e r t r a n s f e r of the f i s h to f r e s h water, and, consequently, was able to promote freshwater s u r v i v a l of the f i s h i n the autumn and w i n t e r . V T a b l e o f Contents page LIST OF TABLES "'. v i i i LIST OF FIGURE'S i x ACKNOWLEDGMENTS \ x i i PREFACE x i i i INTRODUCTION 1 PART I EFFECTS OF PROLACTIN 5 CHAPTER 1 GENERAL MATERIALS AND METHODS 6 I n t r o d u c t i o n 6 F i s h 6 E x p e r i m e n t a l Procedures 7 CHAPTER 2 PROLACTIN AND SEASONAL CHANGES OF FRESHWATER OSMOREGULATION 10 I n t r o d u c t i o n 10 M a t e r i a l s and Methods > 10 R e s u l t s 12 E f f e c t s o f p r o l a c t i n , on plasma o s m o l a l i t y 12 E f f e c t s o f p r o l a c t i n on u r i n e o s m o l a l i t y 15a D i s c u s s i o n 17 CHAPTER 3 PROLACTIN AND FRESHWATER SURVIVAL 21 I n t r o d u c t i o n 21 M a t e r i a l s and Methods 21 R e s u l t s 22 D i s c u s s i o n \ 22 CHAPTER 4 PROLACTIN AND IONIC REGULATION 25 I n t r o d u c t i o n 25 M a t e r i a l s and Methods 25 R e s u l t s 26 Plasma i o n s 26 Urine, ions. 28 Urine/Plasma (U/P) r a t i o 37 D i s c u s s i o n 41 Plasma i o n s 41. U r i n e i o n s 42 v i Page PART I I MECHANISM (S) OF ACTION OF PROLACTIN ... 47 CHAPTER 5 PROLACTIN AND THE KIDNEYS 48' I n t r o d u c t i o n 48 M a t e r i a l s a n d Methods 48 .Urine c o l l e c t i o n . 4 8 Kidney p r e p a r a t i o n s 53 Glomerular- measurements 53 Experimental procedure ( h i s t o l o g i c a l study) 53 S t a t i s t i c a l method 54 R e s u l t s 54 U r i n e f l o w 54 Diameters of Bowman's capsule and glomerular t u f t 54 Percentage change of glomerular diameters 57 Glomerular r a t i o -t u f t diameter/capsule diameter 59 Percentage frequency of g l o m e r u l i . w i t h no i n t r a c a p s u l a r space 62 D i s c u s s i o n Urine f l o w 62 H i s t o l o g i c a l f i n d i n g s 65 Renal mechanism of a c t i o n of p r o l a c t i n 70 CHAPTER 6 PROLACTIN AND THE HEAD REGION (GILLS) 75 I n t r o d u c t i o n 75 M a t e r i a l s and Methods 76 14 14 Net l o s s of C (from i n j e c t e d C - i n u l i n ) , sodium and c h l o r i d e v i a head r e g i o n 76 In v i t r o weight i n c r e a s e of g i l l s 78 R e s u l t s Net i o n l o s s v i a head r e g i o n 81 Net c!4 l o s s v i a head r e g i o n 83 Weight i n c r e a s e of i s o l a t e d g i l l s i n f r e s h water 85 D i s c u s s i o n * 85 v i i Page CHAPTER 7 PROLACTIN AND GILL MUCOUS CELLS .•. ~ 95 I n t r o d u c t i o n 95 M a t e r i a l s and Methods 96 G i l l p r e p a r a t i o n s . 97 E s t i m a t i o n o f g i l l mucous c e l l d e n s i t y 97 R e s u l t s 99 Anatomy and morphology o f the g i l l arches 99 E f f e c t o f p r o l a c t i n on the maintenance o f mucous c e l l s on the g i l l f i l a m e n t s o f seawater-adapted s t i c k l e b a c k s 101 E f f e c t o f p r o l a c t i n on the maintenance o f mucous c e l l s on the g i l l f i l a m e n t s o f seawater-adapted s t i c k l e b a c k s f o l l o w i n g a change i n ambient s a l i n i t y from sea to f r e s h water 103 D i s c u s s i o n 106 CHAPTER 8 PROLACTIN AND THE GUT 112 I n t r o d u c t i o n 112 M a t e r i a l s and Methods . 113 D r i n k i n g r a t e 113 In v i t r o gut water t r a n s p o r t 114 R e s u l t s 116 D r i n k i n g r a t e 116 In v i t r o gut water a b s o r p t i o n 116 D i s c u s s i o n 116 CHAPTER 9 SYNOPSIS AND GENERAL DISCUSSION 124 I n t r o d u c t i o n 124 Synopsis 124 Gene r a l d i s c u s s i o n 128, LITERATURE CITED 137 v i i i L i s t of Tables TABLE - Page 2.1 E f f e c t s of p r o l a c t i n on plasma and u r i n e o s m o l a l i t i e s of winter and l a t e - s p r i n g s t i c k l e b a c k s ' f o l l o w i n g t r a n s f e r from sea water, to fresh, water. 13 4. I P r o l a c t i n and u r i n e i o n l e v e l s and U/P r a t i o s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. "31 4.II E u r y h a l i n e t e l e o s t s i n f r e s h water (FW) and . sea water (SW) , comparing GFR, u r i n e f l o w and u r i n e composition a f t e r adaptation to f r e s h or sea water. 46 5. I P r o l a c t i n and u r i n e f l o w of s t i c k l e b a c k s w i t h the head r e g i o n i n c o n t i n u o u s l y -oxygenated d e c h l o r i n a t e d f r e s h water and the remaining body i n p a r a f f i n o i l . 55 5. I I P r o l a c t i n and glomerular h i s t o l o g y of G. a c u l e a t u s , form trachurus, i n sea water and a f t e r t r a n s f e r to f r e s h water. 56 5 . I l l P r o l a c t i n and r e n a l e x c r e t i o n of sodium, potassium and c h l o r i d e i n f r e s h water. 71 6. I Composition of Ringer S o l u t i o n 80 6. I I P r o l a c t i n and net i o n l o s s v i a the head r e g i o n of s t i c k l e b a c k s ( i n autumn or e a r l y w i n t e r ) . 82 14 6 . I l l P r o l a c t i n and plasma C a c t i v i t y of s t i c k l e b a c k s w i t h the- head r e g i o n i n f r e s h water. 86 7.1 E f f e c t of changes of ambient s a l i n i t y and of p r o l a c t i n on the d e n s i t y of mucous c e l l s on the g i l l f i l a m e n t s of autumn marine s t i c k l e b a c k s . 102 8. I p r o l a c t i n and the d r i n k i n g r a t e of s t i c k l e -backs during the f i r s t 24 hr i n f r e s h water (head region) 117 8. I I p r o l a c t i n and i n v i t r o gut water absorption of s t i c k l e b a c k s . 118 8 . I l l D r i n k i n g r a t e s of t e l e o s t s 120 i x L i s t of F i g u r e s FIGURE Page 2.1 E f f e c t s of p r o l a c t i n on plasma o s m o l a l i t y of s t i c k l e b a c k s f o l l o w i n g t r a n s f e r from . sea water to f r e s h water. 14 2.2 E f f e c t s of p r o l a c t i n on u r i n e o s m o l a l i t y of s t i c k l e b a c k s f o l l o w i n g t r a n s f e r from sea water to f r e s h water. 16 3.1 p r o l a c t i n and freshwater s u r v i v a l of G. acu l e a t u s, form tr a c h u r u s . v 23 4.1 P r o l a c t i n and plasma sodium f o l l o w i n g t r a n s f e r of f i s h from sea water to f r e s h water. 27 4.2 P r o l a c t i n and plasma potassium f o l l o w i n g t r a n s f e r of f i s h from sea water to f r e s h water. 29 -4.3 P r o l a c t i n and plasma c h l o r i d e f o l l o w i n g t r a n s f e r of f i s h from sea water to f r e s h water. 30 4.4 P r o l a c t i n and u r i n e sodium l e v e l s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. 32 4!.5 P r o l a c t i n and u r i n e potassium l e v e l s o f s t i c k l e b a c k s , i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. 34 4.6 P r o l a c t i n and u r i n e c h l o r i d e l e v e l s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. 36" 4.7 P r o l a c t i n and sodium u/p r a t i o s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. 38 4.8 p r o l a c t i n and potassium U/P r a t i o s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. 39' 4„9 p r o l a c t i n and c h l o r i d e U/P r a t i o s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water 40 5.1 The apparatus used f o r u r i n e c o l l e c t i o n and the study of exchanges of m a t e r i a l s v i a the head r e g i o n of s t i c k l e b a c k s . 50 X FIGURE Page 5.2 P r o l a c t i n and percentage change of the Bowman's capsule and the glomerular t u f t i n s t i c k l e b a c k s a f t e r t r a n s f e r from sea water to f r e s h water. 58 5.3 (a)' G l o m e r u l i w i t h l i t t l e " or no i n t r a -capsular space, more f r e q u e n t l y found i n p r o l a c t i n - i n j e c t e d than s o l v e n t -i n j e c t e d s t i c k l e b a c k s . (b) G l o m e r u l i . w i t h l a r g e i n t r a c a p s u l a r space, more f r e q u e n t l y found i n s o l v e n t -i n j e c t e d than p r o l a c t i n - i n j e c t e d s t i c k l e b a c k s . 60 5.4 P r o l a c t i n and glomerular r a t i o ( t u f t diameter/capsule diameter) o f s t i c k l e -backs i n sea water and a f t e r t r a n s f e r to f r e s h water. 61 5.5 P r o l a c t i n and percentage frequency o f g l o m e r u l i w i t h no (evident) i n t r a -capsular space of s t i c k l e b a c k s i n sea water and a f t e r t r a n s f e r to f r e s h water. 63 14 14 6.1 P r o l a c t i n and net l o s s of C (C -i n u l i n or i t s breakdown products) v i a the head r e g i o n ( i n f r e s h water) of s t i c k l e b a c k s . 84 6.2 P r o l a c t i n and weight in c r e a s e of i s o l a t e d g i l l s of s t i c k l e b a c k s a f t e r d i f f e r e n t period's o f i n c u b a t i o n i n f r e s h water. 87 7.1 a and b G i l l f i l a m e n t s of second g i l l arch; d e n s i t y category IV (maximum c o n d i t i o n ) , A F s t a i n . c and d G i l l f i l a m e n t of second g i l l arch; d e n s i t y category I , AF s t a i n . 98 7.2 E f f e c t of p r o l a c t i n and change of ambient s a l i n i t y on d e n s i t y o f mucous c e l l s on g i l l f i l a m e n t s of s t i c k l e b a c k s , measured by percent o f g i l l p r e p a r a t i o n s i n each of - four d e n s i t y c a t e g o r i e s ( I , I I , I I I and I V ) . 100 x i FIGURE 7.3 7.4 9.1 Page P r o l a c t i n and g i l l mucous c e l l d e n s i t y o f s t i c k l e b a c k s t r a n s f e r r e d to f r e s h water. 104 Histogram showing the d e n s i t y o f g i l l mucous: c e l l s , o f u n i n j e c t e d s t i c k l e b a c k s i n sea. water and a f t e r 1 and 4 days o f t r a n s f e r to f r e s h water. 108 Schematic diagram summarizing the f i n d i n g s and hypotheses o f t h i s t h e s i s . 125 XI1 Acknowledgments I would l i k e to express my s i n c e r e a p p r e c i a t i o n and g r a t i t u d e to my s u p e r v i s o r Dr. W.S. Hoar who suggested t h i s problem and who has guided and encouraged me s i n c e my undergraduate days. H i s help i n the p r e p a r a t i o n o f the manuscri and i n pers o n a l matters i s . a l s o g r a t e f u l l y a p p r e c i a t e d . I would a l s o l i k e to thank the f o l l o w i n g : Dr. J.F. Le a t h e r l a n d f o r h i s help and c o l l a b o r a t i o n i n some areas of the t h e s i s ; Dr. P.A. Dehnel, Dr. G.H. Dixon, Dr. J.E. P h i l l i p s and Dr. D. McPhail f o r t h e i r c r i t i c a l r e a d i n g of the manuscript and t h e i r help d u r i n g the i n v e s t i g a t i o n ; my l a b o r a t o r y companions, Dr. P.H. Johansen, Dr. M. Weisbart, Dr. M. Ogawa, Dr. J.F. Leatherland, Dr. s. Pandey and Dr. J.C. Fenwick, f o r many i n t e r e s t i n g and f r u i t f u l d i s c u s s i o n s ; Mr. F.P. McConnell. f o r some t e c h n i c a l help; Miss L. Bogstie f o r t y p i n g the manuscript; and a l l those f r i e n d s , who helped me to c o l l e c t s t i c k l e b a c k s a t one time or another. I am g r a t e f u l to the Malaysian Government f o r g i v i n g me the o p p o r t u n i t y to study abroad and to the Canadian Government (the E x t e r n a l A i d O f f i c e ) f o r the award of a Colombo P l a n S c h o l a r s h i p . x i i i P r e f a c e I t has been s a i d t h a t preface i s w r i t t e n more f o r the. b e n e f i t of the w r i t e r than f o r the reader. I t i s c e r t a i n l y t r u e i n t h i s p r e f a c e where I hope to j u s t i f y the w r i t i n g of t h i s t h e s i s i n chapters, a format not u s u a l l y adopted. In the f i r s t place,, s i n c e the t h e s i s covers a number of areas i n c h r o n o l o g i c a l . o r d e r , separate i n t r o d u c t i o n and d i s c u s s i o n would seem necessary f o r each area i f the o b j e c t i v e s of the v a r i o u s s t u d i e s are to be made c l e a r . Secondly, i f the more usu a l format of w r i t i n g i n s e c t i o n s i s adopted, i t would e n t a i l a number of subsections, subheadings, e t c , which would make w r i t i n g , d i f f i c u l t . T h i r d l y , most p a r t s of the work have been p u b l i s h e d or submitted f o r p u b l i c a t i o n and the v a r i o u s manuscripts can be c o n v e n i e n t l y organized w i t h some m o d i f i c a t i o n s as chapters i n t h i s t h e s i s . L a s t l y , t h i s format of w r i t i n g w i l l not n e c e s s a r i l y l o s e the coherence of the t h e s i s because the f i n d i n g s may be summarized and g e n e r a l l y discussed i n a concluding chapter. INTRODUCTION The marine form (trachurusV of the threespine s t i c k l e b a c k , Gast.eros.teus- aculeatus i s an anadromous t e l e o s t . The f i s h migrate to f r e s h water i n the s p r i n g , spawn, and . r e t u r n to the sea i n the autumn; the o f f s p r i n g a l s o move to the sea i n the autumn. Concomitant w i t h the seasonal c y c l e of" m i g r a t i o n , there are" a s s o c i a t e d : changes i n s a l i n i t y preference (Baggerman, 1957) and freshwater s u r v i v a l (Smith, 1962). During the w i n t e r , marine s t i c k l e b a c k s c o n s i s t e n t l y p r e f e r s a l t water when placed i n sharp s a l i n i t y g r a d i e n t s and show high m o r t a l i t y i f p l a c e d i n f r e s h waters of low m i n e r a l ( e s p e c i a l l y calcium) content. In the s p r i n g , they develop a freshwater preference and a c a p a c i t y to l i v e i n waters.with very low d i s s o l v e d s o l i d s . There are good reasons to b e l i e v e t h a t such seasonal changes are r e g u l a t e d through the h y p o t h a l a m i c - p i t u i t a r y system (Hoar, 1965). The p i t u i t a r y f a c t o r (s) r e s p o n s i b l e has not yet been e s t a b l i s h e d although t h y r o t r o p i c hormone (TSH) has been i m p l i c a t e d i n the s a l i n i t y preference changes (Baggerman, 1957). In recent years, the p i t u i t a r y hormone p r o l a c t i n has been shown to be e s s e n t i a l f o r the s u r v i v a l , i n f r e s h water of s e v e r a l species of e u r y h a l i n e f i s h : Fundulus h e t e r o c l i t u s ( P i c k f o r d , e t a l . , 1965), P o e c i l i a l a t i p i n n a ( B a l l and O l i v e r e a u , 1964) , P o e c i l i a formosa ( B a l l , e_t a l . . , 1965) , i Xiphophorus maculatus, X. h e l l e r i i (Schreibman and Kallman, 1966), T i l a p i a mossambica (Dharmamba, et. a l . , 1967), and 2 G a m b u s i a ( C h a m b o l l e , 1 9 6 6 ) . T h e s e t e l e o s t s , when-h y p o p h y s e c t o m i z e d , s u r v i v e w e l l i n d i l u t e s e a w a t e r b u t d i e i n f r e s h w a t e r w i t h i n a f e w d a y s . When i n j e c t e d w i t h m a m m a lian p r o l a c t i n , h o w e v e r , t h e h y p o p h y s e c t o m i z e d f i s h s u r v i v e w e l l i n f r e s h w a t e r . No o t h e r hormones ( p i t u i t a r y , a d r e n o c o r t i c a l a n d o t h e r s ) y e t t e s t e d a r e e f f e c t i v e i n r e p l a c e m e n t t h e r a p y ( p i c k f o r d , e t a l . , 1 965; S c h r e i b m a n a n d K a l l m a n , 1 9 6 6 ) . " I t a p p e a r s l i k e l y t h a t p r o l a c t i n ..... i s a m o s t i m p o r t a n t hormone f o r o s m o r e g u l a t i o n i n t e l e o s t s . , I n d e e d , i t may b e t h e o n l y hormone f o r w h i c h t h e r e i s a n i n c o n t r o v e r t i b l e p h y s i o l o g i c — a s o p p o s e d t o p h a r m a c o l o g i c — r o l e i n o s m o r e g u l a t i o n i n f i s h e s " ( B e r n , 1 9 6 7 ) . F i s h p r o l a c t i n o r p a r a l a c t i n ( B a l l , 1965) i s p r o d u c e d b y a g r o u p o f e r y t h r o s i n o p h i l i c c e l l s ( e t a c e l l s ) i n t h e r o s t r a l r e g i o n o f t h e p i t u i t a r y c a l l e d r o s t r a l p a r s d i s t a l i s o r p r o a d e n o h y p o p h y s i s . ( O l i v e r e a u a n d B a l l , 1 9 6 4 ) . I n F u n d u l u s . h e t e r o c l i t u s , t h e s e c e l l s show p r e c i p i t i n r e a c t i o n s w i t h a n t i b o d y t o o v i n e p r o l a c t i n , t h u s d e m o n s t r a t i n g t h e p r e s e n c e o f a p r o l a c t i n - l i k e s u b s t a n c e i n t h e s e c e l l s (Emmart, e t a l . , 1 9 66; Emmart a n d M o s s a k o w s k i , 1 9 6 7 ) . The c e l l s a l s o show c h a n g e s c o n s i s t e n t w i t h a r o l e o f p r o l a c t i n p r o d u c t i o n , i n . a n i m a l s m a i n t a i n e d i n f r e s h w a t e r . The r o s t r a l p a r s d i s t a l i s e n l a r g e s a n d t h e c e l l s a p p e a r a c t i v e d u r i n g f r e s h w a t e r a d a p t a t i o n i n F u n d u l u s ( B a l l a n d P i c k f o r d , 1 9 6 4 ) , P o e c i l i a ( B a l l a n d O l i v e r e a u , 1964) a n d T i l a p i a (Dharmamba a n d N i s h i o k a , 1 9 6 8 ) , w h i l s t i t r e d u c e s i n a r e a a n d t h e c e l l s a p p e a r i n a c t i v e d u r i n g s e a w a t e r a d a p t a t i o n . C h a n g e s o f t h e c e l l s 3 w i t h c h a n g e s i n a m b i e n t s a l i n i t y h a v e a l s o b e e n d e t e c t e d a t t h e u l t r a s t r u c t u r a l l e v e l (Dharmamba a n d N i s h i o k a , 1 9 6 8 ) . F u r t h e r m o r e , i n t h e t r a n s p l a n t a t i o n o f t h e f i s h ' s own p i t u i t a r y t o a n o t h e r s i t e o f t h e b o d y ( d o r s a l m u s c u l a t u r e ) , i n P o e c i l i a . , i t i s t h e . r o s t r a l r e g i o n , a n d n o t t h e c a u d a l r e g i o n , o f t h e p i t u i t a r y t h a t i s e f f e c t i v e i n p r o m o t i n g f r e s h w a t e r s u r v i v a l o f t h e f i s h ( B a l l , 1 9 6 5 ) . F i s h p r o l a c t i n h a s n o t b e e n p u r i f i e d t o t h e e x t e n t t h a t i t c a n b e u s e d e c o n o m i c a l l y i n i n v e s t i g a t i o n s o f t h e r o l e o f p r o l a c t i n i n f i s h e s ; i n s t e a d m ammalian p r o l a c t i n ( p a r t i c u l a r l y o v i n e p r o l a c t i n ) h a s u s u a l l y b e e n u s e d . M a m m a l i a n p r o l a c t i n , - h o w e v e r , a p p e a r s to* b e d i f f e r e n t i n c e r t a i n a s p e c t s f r o m f i s h p r o l a c t i n , , a l t h o u g h i t c a n r e p l a c e , f i s h p r o l a c t i n i n p r o m o t i n g f r e s h w a t e r s u r v i v a l o f h y p o p h y s e c t o m i z e d e u r y h a l i n e - t e l e o s t s . M a m m a l i a n p r o l a c t i n h a s a number o f b i o l o g i c a l e f f e c t s (See B e r n , 1 9 6 7 ) , t h r e e o f w h i c h a r e r e c o g n i z e d a s b e i n g s u f f i c i e n t l y r e l i a b l e t o s e r v e a s m e t h o d s o f b i o a s s a y ( z a r r o w , et, a l . , 1964; M e i t e s a n d N i c o l l , 1 9 6 6 ) : (a) t h e i n c r e a s e i n i n t r a d u c t a l h y d r o s t a t i c p r e s s u r e f o l l o w i n g i n t r o d u c t i o n o f ' t h e hormone i n t o t h e m a j o r mammary d u c t o f t h e e s t r o g e n - a n d p r o g e s t e r o n e - p r i m e d r a b b i t ( mammotropic a c t i v i t y ) , (b) s t i m u l a i t i o n of. t h e c r o p g l a n d s o f t h e p i g e o n , a n d ( c ) t h e b e h a v i o r a l r e s p o n s e ( w a t e r - d r i v e ) o f t h e h y p o p h y -s e c t o m i z e d n e w t D j e m i c t y l u s v i r i d e s c e n s . P i t u i t a r i e s o f t e l e o s t f i s h show o n l y m i n i m a l m a m m o t r o p i c a n d p i g e o n c r o p - s t i m u l a t i n g a c t i v i t i e s ; s e c r e t i o n i n t h e two g l a n d s i s n o t s t i m u l a t e d a l t h o u g h t h e a c t i v i t y 4 r e s p o n s i b l e f o r the p r o l i f e r a t i o n o f the e p i t h e l i u m i s . , demonstrable ( N i c o l l , e t a_l. ,1966; Chadwick, 19.66; N i c o l l and Bern, 1968). There i s good evidence, on the other hand, t h a t the b i o l o g i c a l a c t i v i t y which induces water d r i v e i n the. e f t stage, o f D i e m i c t y l u s i s p r e s e n t i n a l l t e l e o s t s (Grant and P i c k f o r d , 1959). I t appears, t h e r e f o r e , t h a t f i s h p r o l a c t i n has some, bu t not a l l , o f the p r o p e r t i e s o f mammalian-, p r o l a c t i n ; ; a t the" same time', mammalian p r o l a c t i n w i l l r e g u l a r l y induce the responses c h a r a c t e r i s t i c o f the c o r r e s p o n d i n g substance i n the p i t u i t a r i e s o f lower v e r t e b r a t e s . Working Hypothesis I t seems p o s s i b l e t h a t a p r o l a c t i n - l i k e substance ( p a r a l a c t i n ) i s r e s p o n s i b l e f o r the p h y s i o l o g i c a l changes t h a t enable marine s t i c k l e b a c k s t o . t o l e r a t e the freshwater h a b i t a t i n the s p r i n g and summer. The r o l e o f p r o l a c t i n i n osmotic and i o n i c r e g u l a t i o n o f marine s t i c k l e b a c k s i n f r e s h water was t h e r e f o r e i n v e s t i g a t e d a t d i f f e r e n t seasons. 5 P a r t I EFFECTS OF PROLACTIN 6 CHAPTER 1 General M a t e r i a l s and Methods I n t r o d u c t i o n In t h i s chapter, m a t e r i a l s and methods which are- common to the v a r i o u s s t u d i e s are described, i n order to a v o i d unnecessary r e p e t i t i o n s . These w i l l o n l y be r e f e r r e d to i n subsequent chapters. M a t e r i a l s and methods s p e c i f i c to the i n d i v i d u a l s t u d i e s , however, w i l l be given l a t e r i n the a p p r o p r i a t e chapters. F i s h The marine form of the threespine s t i c k l e b a c k , Gasterosteus ac u l e a t u s , form trachurus, was used throughout the s t u d i e s . C h a r a c t e r i s t i c a l l y , the f i s h i s covered on both s i d e s by l a r g e number of bony plates, extending from the head r i g h t down to the caudal peduncle, and has well-developed k e e l s . Only f i s h i n the s i z e range of approximately 1-2 gm were used. Source of f i s h A l l f i s h were - c o l l e c t e d i n the c o a s t a l waters around CbaT Harbour', Vancouver, Canada. C o l l e c t i o n s were" made, i n the months of October to December ( r e f e r r e d to subsequently as the late-autumn or e a r l y - w i n t e r s t i c k l e b a c k s ) , and i n May and June ( l a t e - s p r i n g s t i c k l e b a c k s ) . F i s h c o l l e c t e d i n t h i s area are o f t e n p a r a s i t i z e d w i t h the cestode, Schistocephalus, which l i e s i n the abdominal c a v i t y and causes a v i s i b l e d i s t e n s i o n of the abdomen. F i s h 7 which showed a distended abdomen, t h e r e f o r e , w e r e n o t used. Furthermore,-many f i s h showed a reduced number of l a t e r a l p l a t e s extending o n l y up to the mid-flank. These f i s h may be hybrids between the marine form and the freshwater form ( l e i u r u s ) , and were a l s o not used i n the s t u d i e s . .Maintenance of f i s h ' H o l d i n g . c o n d i t i o n s . The f i s h were h e l d i n sea water (12-14% 0 c h l o r i n i t y ) , cooled to a temperature of 10° t 1°C by c o i l s of running tap water. The sea water was aerated and f i l t e r e d c o n t i n u o u s l y . Photoperiod c o n t r o l . The late-autumn or e a r l y - w i n t e r s t i c k l e b a c k s were maintained under c o n t r o l l e d short photo-periods (8 hours of l i g h t a l t e r n a t i n g w i t h 16 hours of darkness). The l a t e - s p r i n g s t i c k l e b a c k s , on the other hand, were maintained under c o n t r o l l e d long photoperiods (16 hours of l i g h t a l t e r n a t i n g w i t h 8 hours of darkness). The i n t e n s i t y of l i g h t was i n each case about 3 0 foot-candles a t the water s u r f a c e . Feeding. The f i s h were f e d f r o z e n b r i n e shrimp on a l t e r n a t e d a y s . u n t i l , commencement of experimental treatments.. Experimental Procedures A f t e r a t l e a s t two weeks i n the stock tanks under the above c o n d i t i o n s , groups of f i s h were t r a n s f e r r e d to experimental tanks or aquaria under s i m i l a r c o n d i t i o n s . 8 A f t e r a t l e a s t one more week i n the experimental tanks the v a r i o u s treatments were i n i t i a t e d . I n j e c t i o n Before i n j e c t i o n , the f i s h were anaesthesized one a t a time i n 0.02% s o l u t i o n of MS 222 ( t r i c a i n e methanesulphonate). This was done i n e a r l i e r s t u d i e s (Chapters 2 and 4) but was d i s c o n t i n u e d s i n c e i t i s time-consuming and appears unne ce S'S a-r y-." A dose of 20 p.q of ovine p r o l a c t i n (#3 0409, F e r r i n g , Malmo, Sweden) d i s s o l v e d i n 0.02 ml or 0.03 ml of the s o l v e n t (0.1% sodium c a r b o x y m e t h y l c e l l u l o s e i n 0.6% NaCl s o l u t i o n ) was i n j e c t e d i n t o each f i s h ( " ^ l gm) i n t r a p e r i t o n e a l l y , u sing a 0.25 ml t u b e r c u l i n s y r i n g e equipped w i t h a #30 gauge needle. The needle was passed through the h y p a x i a l muscle between two l a t e r a l p l a t e s d o r s a l to the u r i n o g e n i t a l and a n a l apertures. By d i r e c t i n g the needle a n t e r i o v e n t r a l l y , i t was p o s s i b l e to i n j e c t i n t o the coelom w i t h the muscle a c t i n g as a s e a l to prevent leakage. S i m i l a r l y , t h e c o n t r o l f i s h were i n j e c t e d w i t h 0.02 ml or 0.03 ml o f the s o l v e n t . In cases- where the' f i s h used were a l l approximately 1 gm weight, a dose of 10 /ag i n 0.01 ml was used. The dosage used was adapted from t h a t of 10 jug per gm weight found to be e f f e c t i v e i n promoting freshwater s u r v i v a l of a number of hypophysectomized t e l e o s t s ( p i c k f o r d , e t a l . , 1965; Schreibman and Kallman, 1966; Dharmamba, et a_l., 1967, and reviews by these a u t h o r s ) . A f t e r i n j e c t i o n s , the f i s h were re t u r n e d to sea water. The f i s h r e c e i v e d e i t h e r a s i n g l e i n j e c t i o n or m u l t i p l e i n j e c t i o n s on a l t e r n a t e days as i n d i c a t e d i n the subsequent chapters. 10 CHAPTER 2 P r o l a c t i n ; a n d seasonal changes of freshwater, osmoregulation., 1 . I n t r o d u c t i o n In t h i s chapter, the hypothesis that seasonal changes of freshwater osmoregulation of the marine s t i c k l e b a c k (G. a c u l e a t u s , form trachurus) occur, and t h a t these changes, are due to seasonal changes of p r o l a c t i n s e c r e t i o n , was t e s t e d . Thus i t was p o s t u l a t e d t h a t p r o l a c t i n s e c r e t i o n i s absent or minimal i n the autumn or e a r l y winter when the f i s h l i v e i n sea water, but maximal i n the s p r i n g when the f i s h prepare f o r m i g r a t i o n i n t o f r e s h water. I f t h i s hypothesis i s c o r r e c t , p r o l a c t i n treatment would i n c r e a s e the c a p a c i t y of autumn or winter f i s h to osmoregulate i n f r e s h water to the l e v e l of s p r i n g f i s h , w h i l e i t would have no s i g n i f i c a n t e f f e c t i n s p r i n g fisfcu Osmoregulatory c a p a c i t y i n f r e s h water was determined by stu d y i n g the changes of plasma and u r i n e o s m o l a l i t i e s f o l l o w i n g t r a n s f e r of the f i s h from sea water to f r e s h water. M a t e r i a l s and Methods Late-autumn and e a r l y - w i n t e r s t i c k l e b a c k s were used i n one experiment, and l a t e - s p r i n g s t i c k l e b a c k s i n another but s i m i l a r experiment. Both groups of f i s h were h e l d i n sea water a t a temperature of 14.5 - 0.5°C. This temperature was used r a t h e r than the usual 10 t 1°C (See Chapter 1) so t h a t the i T h i s chapter has been p u b l i s h e d (Can.J.Zool. 45,509-516,1967) 11 two groups o f f i s h had the same temperature; i n the l a t e s p r i n g , i f the sea water was c o o l e d by c o i l s o f running tap water a l o n e as i n the e a r l y w i n t e r , the- temperature would be g r e a t e r than 10 t 1°C. The f i s h were g i v e n a s i n g l e i n j e c t i o n o f e i t h e r -p r o l a c t i n or the s o l v e n t 24 hours b e f o r e t r a n s f e r i n t o f r e s h water. The o s m o l a l i t i e s o f the u r i n e and plasma were determined a t i n t e r v a l s (0/ 1, 4, 8, 12, 24 hr) f o l l o w i n g t r a n s f e r to f r e s h water a t a temperature o f 14.0 - 0.5°C. A s i n g l e u r i n e c o l l e c t i o n was made from any one f i s h . U r i n e was c o l l e c t e d by applying a c a p i l l a r y tube to the- u r i n o - g e n i t a l a p e r t u r e which had f i r s t been b l o t t e d dry, and e x e r t i n g g e n t l e p r e s s u r e . The drop o f u r i n e trapped i n the center o f the tube was immediately f r o z e n on dry i c e and the tubes were l a t e r s e a l e d w i t h plasticine-.,and s t o r e d a t -10°C u n t i l the analysis-was done. Blood was c o l l e c t e d from the same f i s h f o l l o w i n g u r i n e c o l l e c t i o n by severance o f the caudal peduncle. Blood f l o w i n g from the caudal a r t e r y was c o l l e c t e d i n h e p a r i n i z e d tubes. The tube s wore c e n t r i f u g e d as soon as p o s s i b l e (2000 r.p.m. f o r 10 min). The plasma samples were c o l l e c t e d i n c a p i l l a r y tubes which were s e a l e d and s t o r e d i n the same manner as the u r i n e samples. F i v e i n d i v i d u a l f i s h were sampled a t each i n t e r v a l and the average v a l u e was used i n making comparisons. O s m o l a l i t y was determined by a m e l t i n g - p o i n t method d e s c r i b e d by Gross (1954) and o t h e r s (Hickman, 1959). 12 R e s u l t s E f f e c t s o f P r o l a c t i n on Plasma O s m o l a l i t y The data-are summarized i n Ta b l e 2.1. F o l l o w i n g t r a n s f e r from sea water to f r e s h water, t h e r e was an abrupt drop o f plasma o s m o l a l i t y i n a l l f i s h d u r i n g the f i r s t hour ( F i g . 2.1.)-.. A f t e r 4 hr i n f r e s h water, wi n t e r f i s h i n j e c t e d with., the. s o l v e n t of ...prolactin ( c o n t r o l ) showed, a f u r t h e r d e c l i n e o f plasma o s m o l a l i t y . Subsequently the f i s h showed some osmotic adjustment, but were o n l y a b l e to r a i s e the plasma o s m o l a l i t y to a v a l u e some 50 mOsmol below the seawater v a l u e by the end o f the f i r s t 24 hr. Winter f i s h i n j e c t e d w i t h p r o l a c t i n , however, were a b l e t o r e s t o r e the plasma o s m o l a l i t y c l o s e to the normal seawater v a l u e a f t e r 4 hr i n f r e s h water. Subsequently the plasma o s m o l a l i t y showed a s l i g h t drop and seemed to approach e q u i l i b r i u m . The drop i n plasma o s m o l a l i t y a f t e r 24 hr i n f r e s h water i s b e l i e v e d to be due to the d e p l e t i o n o f p r o l a c t i n through i t s metabolism, s i n c e the f i s h r e c e i v e d o n l y a s i n g l e i n j e c t i o n o f p r o l a c t i n 48 hr e a r l i e r . The above e f f e c t of p r o l a c t i n on plasma., o s m o l a l i t y adds support to the work o f P i c k f o r d , pang and Sawyer (1966) who have found t h a t p r o l a c t i n enableshypophy-sectomized F. h e t e r o c l i t u s to m a i n t a i n normal serum o s m o l a l i t y i n f r e s h water.. More r e c e n t l y , s i m i l a r r e s u l t s have been found i n g o l d f i s h , C a r a s s i u s auratus (Donaldson,et a l . , 1968), and T. mossambica (Dharmamba, e t a l . , 1967), a l t h o u g h i n _T. mossambica, p r o l a c t i n o n l y p a r t i a l l y p r e vents the drop i n plasma o s m o l a l i t y f o l l o w i n g the t r a n s f e r o f hypophysectomized C O H Table 2 . 1 E f f e c t s of p r o l a c t i n on plasma and urine osmolalities of winter and late-spring sticklebacks following transfer from sea water to fresh water. Time i n fresh water (hr) Winter Late spring Solvent(control) P r o l a c t i n Solvent(control) P r o l a c t i n Osmolality Osmolality Osmolality (mosmol/kg H 2 0 ) (mosmol/kg H 2 O ) (mosmol/kg H 2 0 ) M.F.L. — M.P.Li — M.F.L. M.F.L. (mm) Plasma Urine (mm) Plasma Urine (mm) Plasma Urine (mm) Plasma Urine Osmolality (mosmol/kg H 2 O ) 0 62±1 344±3 295± 9 60±1 342±17 295±16 55±1 339±2 254± 9 54±1 3 51+5 247± 8 1. 61+1 305+8 3 0 l i 9 61+1 3 04+ 3 216+13 54±1 3 06^2 211+ 5 ** 185+17 55±1 320+5 156± 9 4 60+2 264+8 272 + 6 i 61+1 342± * * 4. * 5 2063 9 55+1 33 012 55+1 346+7 . ** 1661 4 8 63+1 282+5 263± 6 62+1 325± 6 182+18 54+1 326+6 161+11 55+1. 324+4 180±12 12 62+1 296+3 233 + 9 63+1 326+ 7 198+10 56-1 316+3 182^ 6 57+1 315+4 184+17 24 62+1 293+2 23 0+12 61±2 303 + 1 208+16 56+1 315+3 164+13* 58+1 313+4 163+15* NOTE: A l l values are means (Istandard error); single asterick, s i g n i f i c a n t l y d i f f e r e n t from corresponding winter control at P <C.05; double asterisk, s i g n i f i c a n t l y d i f f e r e n t from corresponding winter control at P^.01; a l l other possible crosswise paired comparisons not s i g n i f i c a n t ; Tukey's W (Steel and To r r i e 1960) for urine, W.05=61 and W. 01= 69; for plasma, W.05 =31 and W.oi=35; M.F.L., mean fork length (+standard e r r o r ) . 14 F i g . 2.1 E f f e c t s of p r o l a c t i n on plasma osmolality of sticklebacks following transfer from sea water to fresh water; abscissa represents hr a f t e r transfer to fresh water. - W I N T E R S P R I N G O C O N T R O L O L T H A C O N T R O L A L T H J 15 f i s h to f r e s h water. I t i s i n t e r e s t i n g t h a t i n T. mossambica, the e f f e c t of p r o l a c t i n u s u a l l y disappeared a f t e r 48 hr i r r e s p e c t i v e of whether the hormone was administered i n a s i n g l e dose or as d a i l y i n j e c t i o n s (Dharmamba, e t a l . , 1967). S o l v e n t - i n j e c t e d f i s h i n l a t e s p r i n g (June), a f t e r being t r a n s f e r r e d to f r e s h water, showed a p a t t e r n of changes of plasma o s m o l a l i t y not s i g n i f i c a n t l y d i f f e r e n t from the p r o l a c t i n -t r e a t e d winter f i s h ( F i g . 2.1). In the s p r i n g f i s h , p r o l a c t i n treatment d i d not produce any s i g n i f i c a n t e f f e c t s . The winter f i s h appeared to reach an e q u i l i b r i u m plasma o s m o l a l i t y i n f r e s h water a t about 29 5 mosmol/kg and the s p r i n g f i s h , a t about 315 mosmol/kg H2O. Koch and Heuts (1943) have r e p o r t e d e s s e n t i a l l y s i m i l a r f i n d i n g s i n Gasterosteus ac u l e a t u s , form trachurus, the form used i n the present s t u d i e s . As f a r as can be made out from t h e i r graph (p. 260), the serum o s m o l a l i t y i n f r e s h water of s e x u a l l y immature s t i c k l e b a c k s (equivalent to the winter f i s h here) appeared to be i n the r e g i o n of 308 mosmol/kg H2O (when converted from 9 g NaCl per l i t e r ) , and t h a t of s e x u a l l y mature f i s h ( e q u i v a l e n t to the s p r i n g f i s h here), i n the r e g i o n of 325 mosmol/kg H 20. In 11% 0 Nacl, the serum o s m o l a l i t y of s e x u a l l y immature f i s h appeared to be i n the r e g i o n of 349 mosmol/kg H2O (10.2%0 NaCl) and t h a t of s e x u a l l y mature f i s h , i n the r e g i o n of 342 mosmol/kg H2O (10.0%o N a c l ) . The tr e n d i n both f r e s h water and sea water i s thus s i m i l a r to t h a t of the present s t u d i e s although there . appears to be some q u a n t i t a t i v e d i f f e r e n c e s which may be due to d i f f e r e n t techniques of measurement of o s m o l a l i t y . 15a In the predominantly freshwater form of the threespine s t i c k l e b a c k , Gasterosteus acu l e a t u s , form gymnurus, on the other hand, Koch and Heuts (1942, 1943) have found the serum o s m o l a l i t y i n f r e s h water to be much higher i n s e x u a l l y immature f i s h (10.00 ± 0.05 g N a d per l i t e r or 342 + 2 mosmolAg H2O) than i n s e x u a l l y mature f i s h (8.90 - 0.19 g Nacl per l i t e r or. 304 * 6 mosmol/kg H2O) . In sea water, the immature f i s h were able to m a i n t a i n the serum o s m o l a l i t y constant but the s e x u a l l y mature f i s h c o uld not. E f f e c t s , o f P r o l a c t i n on Urine. O s m o l a l i t y The data are summarized i n Table 2.1. In gene r a l , - v a r i a b i l i t y was greater among u r i n e o s m o l a l i t i e s than among plasma o s m o l a l i t i e s . The v a r i a b i l i t y may i n p a r t be due to the c o l l e c t i o n technique (see M a t e r i a l s and Methods) s i n c e u r i n e samples may sometimes be contaminated w i t h gut f l u i d s . As seen i n F i g . 2,2, a f t e r t r a n s f e r from sea water to f r e s h water, winter f i s h which had been i n j e c t e d w i t h the s o l v e n t o f p r o l a c t i n ( c o n t r o l ) showed l i t t l e a b i l i t y to produce a more d i l u t e u r i n e although i t would be expected on the b a s i s of the usual osmoregulatory mechanism encountered i n freshwater t e l e o s t s (parry, 1966; Hickman and Trump, 1969). Comparing the r e s p e c t i v e plasma and u r i n e o s m o l a l i t i e s , i t can be seen t h a t the f i s h , though producing a s l i g h t l y hypoosmotic u r i n e i n sea water, produced approximately i s o s m o t i c u r i n e i n the f i r s t 4 hr i n f r e s h water. Subsequently, the f i s h produced hypoosmotic u r i n e but i t s c a p a c i t y to do so seemed l i m i t e d . Winter f i s h i n j e c t e d w i t h p r o l a c t i n , however, produced h i g h l y hypoosmotic u r i n e soon a f t e r being t r a n s f e r r e d to f r e s h water 16 F i g . 2.2 E f f e c t s of p r o l a c t i n on u r i n e o s m o l a l i t y o f s t i c k l e b a c k s f o l l o w i n g t r a n s f e r from sea water to f r e s h water; a b s c i s s a r e p r e s e n t s hr a f t e r t r a n s f e r to f r e s h water. 17 and continued to do so ( F i g . 2.2). A f t e r 24 hr i n f r e s h water, there was a s u b s t a n t i a l r i s e i n u r i n e o s m o l a l i t y . As mentioned e a r l i e r , the corresponding plasma o s m o l a l i t y showed a drop from the previous value ( F i g . 2.1) and i t i s b e l i e v e d to be due to t h e - d e p l e t i o n of i n j e c t e d p r o l a c t i n . L a t e - s p r i n g (June) f i s h , when i n j e c t e d w i t h s o l v e n t and t r a n s f e r r e d to f r e s h water, r a p i d l y produced h i g h l y hypo-osmotic u r i n e ( F i g . 2.2). Although the u r i n e o s m o l a l i t y appeared to be, a t every time i n t e r v a l , lower than t h a t o f the p r o l a c t i n - t r e a t e d w i n ter f i s h , the d i f f e r e n c e was not s i g n i f i c a n t and the p a t t e r n of changes i n the two groups of f i s h was s i m i l a r ( F i g . 2.2). The g r e a t e s t d i f f e r e n c e (although not s i g n i f i c a n t ) o c curred a f t e r 24 hr i n f r e s h water and t h i s was the time when i t i s b e l i e v e d t h a t the p r o l a c t i n i n j e c t e d i n t o the winter f i s h had been metabolized or otherwise depleted. P r o l a c t i n treatment i n the s p r i n g f i s h d i d not produce any s i g n i f i c a n t changes. In sea water there appeared to be a d i f f e r e n c e i n u r i n e o s m o l a l i t y of winter and s p r i n g f i s h , p r o l a c t i n - or s o l v e n t -i n j e c t e d ( F i g . 2.2, 0' h r ) ; the d i f f e r e n c e , however, was not s i g n i f i c a n t . D i s c u s s i o n In t h i s study, mammalian p r o l a c t i n a l t e r e d the f r e s h -water osmoregulation of the marine form of the threespine s t i c k l e b a c k a t c e r t a i n seasons. In wi n t e r , when s t i c k l e b a c k s normally l i v e i n sea water, they showed a l i m i t e d c a p a c i t y f o r osmoregulation i f t r a n s f e r r e d to f r e s h water, p r o l a c t i n 18 i n j e c t i o n s enhanced the freshwater osmoregulatory, c a p a c i t y so t h a t , f o l l o w i n g t r a n s f e r to f r e s h water, the f i s h promptly exc r e t e d h i g h l y hypoosmotic u r i n e and showed a r e l a t i v e l y s m a l l drop i n plasma o s m o l a l i t y . In l a t e s p r i n g , when the f i s h have al r e a d y migrated or are s t i l l m i g r a t i n g to f r e s h water, they are able to osmoregulate w e l l i n f r e s h water as evidenced by t h e i r plasma and u r i n e o s m o l a l i t i e s a f t e r t r a n s f e r to t h i s medium. P r o l a c t i n i n j e c t i o n s i n these f i s h d i d not produce s i g n i f i c a n t e f f e c t s . This f i n d i n g , together w i t h the f i n d i n g that p r o l a c t i n treatment brought the winter f i s h c l o s e to the l a t e - s p r i n g c o n d i t i o n of freshwater osmoregulation, suggests t h a t the d i f f e r e n c e i n freshwater, osmoregulatory a b i l i t y between the winter and l a t e - s p r i n g f i s h i s due to p r o l a c t i n . Thus p r o l a c t i n s e c r e t i o n seems to be absent or minimal i n the winter (or late-autumn) s t i c k l e b a c k s but maximal i n the l a t e - s p r i n g f i s h . For t h i s reason the winter (or late-autumn) f i s h may be regarded as " p h y s i o l o g i c a l l y hypophysectomized", a t l e a s t as regards p r o l a c t i n . Lam (1965) has found th a t r e s u l t s comparable to those obtained here f o r u r i n e o s m o l a l i t i e s of s t i c k l e b a c k s can be brought about by p h o t o p e r i o d i c manipulations. Autumn f i s h which had been maintained under sh o r t photoperiod (8L 16D) f o r over a month were unable to produce very d i l u t e u r i n e when t r a n s f e r r e d d i r e c t l y i n t o f r e s h water, although autumn f i s h which had been maintained under long photoperiod (16L 8D) f o r over a month could do so. P r o l a c t i n treatment i n the s h o r t -photoperiod f i s h enabled i t to produce d i l u t e u r i n e i n f r e s h 19 water j u s t as w e l l as the long-photoper.iod f i s h . Some of these r e s u l t s were summarized by Hoar (1965). I t seems, t h e r e f o r e , t h a t the seasonal v a r i a t i o n s i n p r o l a c t i n s e c r e t i o n are c o n t r o l l e d by the photoperiod. I t i s i n t e r e s t i n g t h a t G o u r d j i and T i x i e r - V i d a l (1966) have found that continuous exposure to l i g h t i n Peking ducks i n November enhances g r e a t l y t h e i r p r o l a c t i n s e c r e t i o n . The mechanism of c o n t r o l of p r o l a c t i n s e c r e t i o n by the" photoperiod i s ••'•a.?matter of s p e c u l a t i o n . A number of mechanisms are p o s s i b l e (See Lam, 1965). Sage (1966, 1968) has presented evidence which suggests t h a t p r o l a c t i n s e c r e t i o n i n Xiphophorus hybrids may be due to a d i r e c t response to changes of osmotic pressure of the b l o o d r e s u l t i n g from changes i n the environment. Probably, t h e r e f o r e , i n s t i c k l e b a c k s , i n c r e a s i n g or long photoperiod s t i m u l a t e s p r o l a c t i n s y n t h e s i s r a t h e r than p r o l a c t i n r e l e a s e . P r o l a c t i n may o n l y be r e l e a s e d when the f i s h enters e s t u a r i n e waters. Evidence from s t u d i e s of e c t o p i c p i t u i t a r y t r a n s p l a n t a t i o n i n a few t e l e o s t s suggests t h a t p r o l a c t i n s y n t h e s i s and/or r e l e a s e i n t e l e o s t s may be e i t h e r independent of hypothalamic mediation or under i n h i b i t o r y hypothalamic c o n t r o l ( B a l l , 1965; B a l l and O l i v e r e a u , 1965; B a l l e t a l _ . , 1965; O l i v e r e a u and B a l l , 1966; Schreibman and Kallman, 1964)- l a c k of q u a n t i t a t i v e data on r a t e s of s e c r e t i o n of the hormone i n g r a f t e d f i s h compared w i t h normal f i s h prevents a d e c i s i o n i n t h i s regard. I f we assume tha t the i n h i b i t o r y hypothalamic c o n t r o l of p r o l a c t i n s e c r e t i o n occurs i n t e l e o s t s as i n mammals and p o s s i b l y other 20 v e r t e b r a t e s except b i r d s (Meites and N i c o l l , 1966), i n c r e a s i n g or long'photoperiod would a c t by removing t h i s hypothalamic i n h i b i t i o n v i a i n h i b i t i n g the r e l e a s e o f a p r o l a c t i n ^ i n h i b i t i n g f a c t o r , e i t h e r d i r e c t l y or i n d i r e c t l y through other systems. A more t a n g i b l e suggestion cannot be made without more i n v e s t i g a t i o n s i n t h i s v i r t u a l l y unknown area i n t e l e o s t s . 21 CHAPTER 3 P r o l a c t i n and .freshwater s u r v i v a l I n t r o d u c t i o n : • • Smith (1962) has shown t h a t marine s t i c k l e b a c k s show, hi g h m o r t a l i t y i n the autumn and e a r l y winter when placed i n f r e s h w a t e r of low m i n e r a l ( e s p e c i a l l y calcium) content, but s u r v i v e w e l l i n the same medium i n the s p r i n g . This seasonal d i f f e r e n c e i n freshwater s u r v i v a l of s t i c k l e b a c k s may be e x p l a i n e d by the seasonal p r o l a c t i n s e c r e t i o n suggested by evidence i n Chapter 2. Thus the f a i l u r e of the autumn or e a r l y - w i n t e r s t i c k l e b a c k s to s u r v i v e i n d e m i n e r a l i z e d f r e s h water may be because p r o l a c t i n s e c r e t i o n i s absent or minimal i n these f i s h . This hypothesis was t e s t e d i n the f o l l o w i n g experiments. M a t e r i a l s and Methods E a r l y - w i n t e r s t i c k l e b a c k s were used., Three groups of f i s h (22-24 f i s h i n each group, each f i s h weighing about 1 gm); were.removed from the stock tank, and p l a c e d s e p a r a t e l y i n Permascreen (McLennan, McFeely and p r i o r Ltd.) b a s k e t s - f l o a t e d by means of p o l y s t y r e n e b l o c k s i n the same aquarium of sea water (temperature 10° t 1°C). A f t e r one week i n these c o n d i t i o n s , one group was i n j e c t e d w i t h p r o l a c t i n , a second group w i t h the s o l v e n t , w h i l s t the- t h i r d group was l e f t i n t a c t . I n j e c t i o n s were continued on 1 T h i s Chapter i s i n press i n Gen. Comp. E n d o c r i n o l . 22 a l t e r n a t e days. Twenty-four hours a f t e r the t h i r d i n j e c t i o n , the baskets c o n t a i n i n g the f i s h were removed from sea water and p l a c e d i n d i s t i l l e d water f o r 1-2 minutes (to f a c i l i t a t e the removal of ions from the baskets and body surfaces o f the f i s h ) before t r a n s f e r to the same aquarium c o n t a i n i n g f r e s h water of low m i n e r a l content (Calcium <" 1 ppm; c o n d u c t i v i t y = 19.0 mho) a l s o a t a temperature of 10 1" l ° c I n j e c t i o n s of p r o l a c t i n and s o l v e n t were continued on a l t e r n a t e days. The dead f i s h i n each group were removed every morning and the number recorded. R e s u l t s The r e s u l t s obtained are g r a p h i c a l l y presented i n F.ig. 3.1. I t i s evident t h a t p r o l a c t i n i n j e c t i o n s enhanced s u r v i v a l i n f r e s h water of the e a r l y - w i n t e r s t i c k l e b a c k s (p <0.005). The u n i n j e c t e d f i s h appeared to succumb to f r e s h water as r e a d i l y as d i d the s o l v e n t - i n j e c t e d , f i s h . The time to reach a 50% m o r t a l i t y f o r these two groups was 3-5 days, whereas the p r o l a c t i n - i n j e c t e d group d i d not reach a 50% m o r t a l i t y during the course of the experiment (9 days). These r e s u l t s confirm s e v e r a l s i m i l a r p r e l i m i n a r y experiments of longer d u r a t i o n ( >2 weeks) i n which again a 50% m o r t a l i t y f o r the p r o l a c t i n -i n j e c t e d f i s h was never reached. D i s c u s s i o n The r o l e of p r o l a c t i n i n freshwater s u r v i v a l of a number of e u r y h a l i n e t e l e o s t s f o l l o w i n g hypophysectomy has been 23 F i g . 3.1. P r o l a c t i n and freshwater s u r v i v a l of G. acu l e a t u s , form trachurus. A b s c i s s a represents the days a f t e r t r a n s f e r from sea.to f r e s h water. 24 w e l l e s t a b l i s h e d . The s t i c k l e b a c k , however, seems unable to • l i v e i n f r e s h water during the e a r l y winter (or l a t e autumn), even w i t h i t s p i t u i t a r y i n t a c t , and p r o l a c t i n i n j e c t i o n s were able to promote s u r v i v a l . This i s f u r t h e r evidence t h a t the e a r l y - w i n t e r (or late-autumn) s t i c k l e b a c k i s ' p h y s i o l o g i c a l l y hypophysectomized 1 w i t h regards to p r o l a c t i n and supports the e a r l i e r suggestion of p r o l a c t i n involvement i n the freshwater m i g r a t i o n of the f i s h . 25 CHAPTER 4 P r o l a c t i n and i o n i c regulation"*" Introduction, So f a r p r o l a c t i n i n j e c t i o n s enable the autumn or e a r l y - w i n t e r s t i c k l e b a c k s to osmoregulate b e t t e r when t r a n s f e r r e d from sea water to f r e s h water (Chapter 2) and to s u r v i v e longer i n f r e s h water of low m i n e r a l ( e s p e c i a l l y calcium) content (Chapter 3) . Recent evidence i n d i c a t e s an i o n - r e g u l a t o r y r o l e f o r p r o l a c t i n i n c e r t a i n t e l e o s t s ( B a l l and Ensor, 1965/ 1967; P o t t s and Evans, 1966; Maetz, et^ a l . , 1967a/b). The r o l e of p r o l a c t i n i n the r e g u l a t i o n of plasma and u r i n e sodium, potassium and c h l o r i d e was t h e r e f o r e examined i n the e a r l y - w i n t e r s t i c k l e b a c k s f o l l o w i n g t r a n s f e r from sea to -fresh water. M a t e r i a l s and Methods Late-autumn and e a r l y - w i n t e r s t i c k l e b a c k s were used. One group of f i s h was i n j e c t e d w i t h p r o l a c t i n , a second group w i t h the s o l v e n t of p r o l a c t i n , w h i l s t a t h i r d group was l e f t i n t a c t . Twenty-four hours a f t e r the i n j e c t i o n s , the three groups were t r a n s f e r r e d s e p a r a t e l y to three tanks c o n t a i n i n g d e c h l o r i n a t e d Vancouver tap water a t temperature 10 ± 1°C. At i n t e r v a l s f o l l o w i n g t r a n s f e r , u r i n e and bl o o d were c o l l e c t e d from 6-8 f i s h from each group; these f i s h were then k i l l e d - . ^"Part of t h i s chapter (plasma e l e c t r o l y t e s ) has been p u b l i s h e d (Can. J . Zool. 46, 1095-1097, 1968) 26 The plasma samples and u r i n e samples from these 6-8 f i s h were s e p a r a t e l y pooled, and s t o r e d i n wax-sealed polyethylene tubings (PE 50) a t -10°C u n t i l analyzed. The above was repeated so t h a t two pooled plasma samples and urines samples were obtained f o r each group a t each time i n t e r v a l . The experiment was repeated to o b t a i n a t l e a s t three pooled samples f o r each group a t each time i n t e r v a l . Intact* c o n t r o l f i s h were sampled only a t 0, 1 and 24 hours". For sodium a n a l y s i s , 2 u l a l i g u o t s of the pooled samples were taken, u s i n g Drummond Microcaps (Drummond S c i e n t i f i c Co., Broomall, pa.), and added to 5 ml of g l a s s - d i s t i l l e d water; f o r potassium a n a l y s i s , 5 u l a l i q u o t s were added to 3 ml of a s o l u t i o n c o n t a i n i n g 100 ppm sodium. Determinations of sodium and potassium i n the d i l u t e d samples were made by emission flame photometry us i n g a Unicam SP 900A Spectro-photometer (Unicam Instruments L t d . , Cambridge, England). For c h l o r i d e a n a l y s i s , plasma and u r i n e samples from i n d i v i d u a l f i s h , r a t h e r than pooled samples were used. Determinations were made p o t e n t i o m e t r i c a l l y on 1 u l samples d i l u t e d i n 20 u l of 50% a c e t i c a c i d and t i t r a t e d a g a i n s t s i l v e r n i t r a t e (^O.OIN). Re s u l t s Plasma ions Mean data on plasma sodium + 9 5 % confidence i n t e r v a l (t Qr,Sx; S t e e l and T o r r i e , 1960) are presented i n F i g . 4.1. Both the s o l v e n t - i n j e c t e d and i n t a c t c o n t r o l s showed r a p i d 27 F i g . 4.1. P r o l a c t i n and plasma sodium f o l l o w i n g t r a n s f e r of f i s h from sea water to f r e s h water. The a b s c i s s a represents hr a f t e r t r a n s f e r . Each p o i n t represents mean of a t l e a s t 3 pooled plasma samples (+95% confidence i n t e r v a l ) . h u n i n j e c t e d f i s h . 28 d e c l i n e of plasma sodium f o l l o w i n g t r a n s f e r from _sea water to f r e s h water. A f t e r 24 hr, the l e v e l s i n these groups f e l l by about 25%. A s i n g l e p r o l a c t i n i n j e c t i o n , 24 hr p r i o r to t r a n s f e r , reduced the f a l l s i g n i f i c a n t l y (p 4. 0.01) and appeared to maintain the l e v e l constant a f t e r 4 hr. I t i s apparent from F i g . 4.2 t h a t p r o l a c t i n had no e f f e c t on plasma potassium. The three groups of f i s h showed considerable' v a r i a b i l i t y and no s i g n i f i c a n c e was found. F i g u r e 4.3 shows mean c h l o r i d e values ( + 95% confidence i n t e r v a l ) . I t i s evident t h a t p r o l a c t i n i n j e c t i o n reduced s i g n i f i c a n t l y the f a l l of plasma c h l o r i d e which occurred when the f i s h were t r a n s f e r r e d from sea to f r e s h water, p r o l a c t i n , however, .did not appear to maintain the c h l o r i d e l e v e l constant a f t e r 4 hr as i t d i d f o r sodium. The d i f f e r e n c e may be r e a l or due to the f a c t t h a t the sodium and c h l o r i d e data were obtained i n two d i f f e r e n t experiments. Moreover, determinations of sodium were made on pooled plasma samples w h i l s t c h l o r i d e s were determined on i n d i v i d u a l samples. U r i n e ions Mean data on u r i n e sodium - standard e r r o r are presented i n Table 4.1 and F i g . 4.4. Two-way a n a l y s i s of variance, of the data (time' vs s o l v e n t and p r o l a c t i n treatments) showed a highly s i g n i f i c a n t treatment e f f e c t (p <^  0.005) . Thus-, p r o l a c t i n treatment, i n ge n e r a l , s i g n i f i c a n t l y lowered u r i n e sodium c o n c e n t r a t i o n . 29 F i g . 4.2. p r o l a c t i n and plasma potassium f o l l o w i n g t r a n s f e r of f i s h from sea water to f r e s h water. The a b s c i s s a represents hr a f t e r t r a n s f e r . Each p o i n t represents mean of a t l e a s t 3 pooled plasma samples (± stand-ard e r r o r ) . 9 -8 -7 -J 6 s = . 5 -4 -3 -T 0 Ll P L A S M A K" O 2 4 6 8 S O L V E N T P R O L A C T I N U N T R E A T E D H O U R 30 F i g . 4.3. p r o l a c t i n and plasma c h l o r i d e , f o l l o w i n g t r a n s f e r of f i s h from sea water to f r e s h water. The a b s c i s s a represents hr a f t e r t r a n s f e r . Each p o i n t represents mean of 5 i n d i v i d u a l plasma samples from 5 f i s h (t 95% confidence i n t e r v a l ) . Table 4. I P r o l a c t i n and u r i n e i o n l e v e l s and U/P r a t i o s to f r e s h water. Values given are means -U/P r a t i o s were c a l c u l a t e d from plasma and 2 l e a s t s i g n i f i c a n t d i f f e r e n c e ; * P<0.05; TIME I N ; 0 ' „ 1 Ions' Treatment meq/l+Sx(n) U/p meq/l+Sx(n) U/P Solvent 53.3+4.9- (3) 0.31 55.3+4.1 (3) 0. 36' Na (pooled) p r o l a c t i n 49.0+6.5 (3) 0. 29 "kit 34.0*4.9 (4) 0. 21(1) u n i n j e c t e d 50.0*5.0 (3) 0.29 50.0*4.5 (3) 0. 34 l s d 2 (p = 0.05) 13.5 12.6 Solvent 2 . 0 4 ± 0 . 5 1 ( 4 ) 0.37 1.80*0.33 (3) 0. 28 K (pooled) p r o l a c t i n 1 . 8 9 ± 0 . 3 3 ( 4 ) 0.31 1.64*0.38(4) 0. 28 u n i n j e c t e d 1 . 9 7 ± 0 . 5 0 ( 3 ) 1.73*0.36(3) 0. 28 l s d 2 (p = 0.05) 0.90 0.97 Solvent 68.0*0.7 (5) 0.49 72.0*9.8 (5) 0. 61 C l (un- p r o l a c t i n 66.0*1.7 (5) 0.46 65.0*3.3 (5) 0o 47 pooled) l s d 2 (p = 0.05) 10.1 1 0 „ 1 31 of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r ± standard e r r o r (Sx); n = number of samples; u r i n e means. from two-way a n a l y s i s of variance; ** P < 0 . 0 1 ; *** P < 0 .005 . Treatment F-*-(degree of freedom of treatment, FRESH WATER (hr) 12 24 meg/l±Sx(n) U/P meqA+Sx (n) U/P meq/l+Sx (n) U/P ^ . f . e r r o r ) 55.0+6.6 (3) 0.37 32> 0±4*5 (4) 0.20(6) 12 .6 57.0+5.0 (3) 0.46 47.5+1.4 (3) 0.37 * 40.0+4.0 (3) 0.26 35.0+2.5 (3) 0.23 . 43.0+2.5 (3) 0.34 13.5 13.5 3 2.49 (1,22) *** 1.03+0.15(3) 0.18 1 . 0 1 ± 0 . 0 7 ( 4 ) 0.17 0.97 1.70+0.29 (3) 0.28 1.49+0.07 (3) 0.44 v 1.90+0.43(3) 0.42 2.00+0.15(3) 0.55 -1.50+0.10(3), 0.37 1.04 1.04 0.08 (1,24) 68.0+4.3 (5) 0.59 3 8 . 0 - 2 . 1 (5) 0.29 10 .1 26.0+0.8 (5) 0.30 20.0+0.7 (5) 0.23 ^ 24.30 20.0+1.4 (5) 0.18 16.0+0.6 (5) 0.16 10 .1 1 0 o l "| .: J (1-*** 32 F i g . 4.4. p r o l a c t i n and u r i n e sodium l e v e l s of s t i c k l e b a c k s i n sea water and .following t r a n s f e r to f r e s h water. Each p o i n t represents mean + standard e r r o r . A b s c i s s a represents hr i n f r e s h water. A u n i n j e c t e d f i s h . HOUR 33 The s o l v e n t - i n j e c t e d f i s h d i d not show s i g n i f i c a n t changes i n u r i n e sodium f o l l o w i n g t r a n s f e r from sea water to f r e s h water, nor d i d they d i f f e r s i g n i f i c a n t l y from the unin j e c t e d f i s h a t 0, 1 and 24 hr. The p r o l a c t i n - i n j e c t e d . . f i s h d i d not d i f f e r s i g n i f i c a n t l y from the s o l v e n t - i n j e c t e d nor the u n i n j e c t e d f i s h i n sea water, but showed a s i g n i f i c a n t r e d u c t i o n of u r i n e sodium co n c e n t r a t i o n 1 hr a f t e r t r a n s f e r to f r e s h water (P ^ 0.05) and was s i g n i f i c a n t l y d i f f e r e n t from the solven.t-inj.ected (P<0.01) or the u n i n j e c t e d (P./0.05) c o n t r o l s a t t h i s time. A f t e r t h i s i n i t i a l d e c l i n e , the p r o l a c t i n - i n j e c t e d f i s h d i d not show any f u r t h e r s i g n i f i c a n t change during the 24-hr study p e r i o d , but maintained a s i g n i f i c a n t l y lower u r i n e sodium c o n c e n t r a t i o n than the s o l v e n t - i n j e c t e d f i s h up to 12 hr (4 hr: p<0.01; 12 hr:, P<0.05). There was no s i g n i f i c a n t d i f f e r e n c e among the p r o l a c t i n -i n j e c t e d , s o l v e n t - i n j e c t e d , and u n i n j e c t e d f i s h a t 24 hr. This might i n d i c a t e that the e f f e c t of p r o l a c t i n on u r i n e sodium was t r a n s i e n t or e l s e the p r o l a c t i n i n j e c t e d 48 hr p r e v i o u s l y (a s i n g l e i n j e c t i o n ) had been metabolized. Data on u r i n e potassium are summarized i n Table 4.1 and F i g . 4.5. Two-v/ay a n a l y s i s of variance of the data showed t h a t s o l v e n t and p r o l a c t i n treatments were not s i g n i f i c a n t l y d i f f e r e n t . Both s o l v e n t - i n j e c t e d and p r o l a c t i n - i n j e c t e d f i s h showed a f a l l of u r i n e potassium c o n c e n t r a t i o n a f t e r 4 hr i n f r e s h water, and subsequently recovered; the u n i n j e c t e d f i s h s i m i l a r l y showed a f a l l a t 1 hr. A t 24 hr the p r o l a c t i n -34 F i g . 4.5. P r o l a c t i n and u r i n e potassium l e v e l s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. Each p o i n t represents mean - standard e r r o r . A b s c i s s a represents hr i n f r e s h water. A u n i n j e c t e d f i s h . PROLACTIN SOLVENT J. 24 HOUR 35 i n j e c t e d f i s h appeared to excrete a higher c o n c e n t r a t i o n of potassium i n the u r i n e than e i t h e r the s o l v e n t - i n j e c t e d or the u n i n f e c t e d c o n t r o l s but the d i f f e r e n c e s were not s i g n i f i c a n t . Table 4.1 and F i g . 4.6 show mean data of u r i n e c h l o r i d e - standard e r r o r . Two-way a n a l y s i s of variance of the data showed t h a t p r o l a c t i n treatment s i g n i f i c a n t l y lowered u r i n e c h l o r i d e c o n c e n t r a t i o n (P <C 0.005). In the f i r s t 4 hr f o l l o w i n g t r a n s f e r from sea water to f r e s h water, the s o l v e n t - i n j e c t e d f i s h d i d not s i g n i f i c a n t l y change the p a t t e r n of u r i n a r y c h l o r i d e e x c r e t i o n . The p r o l a c t i n - i n j e c t e d f i s h , however, excreted a s i g n i f i c a n t l y lower u r i n e c h l o r i d e c o n c e n t r a t i o n a t 4 hr, when compared to e i t h e r t h e i r i n i t i a l seawater value or the s o l v e n t - i n j e c t e d f i s h (p < 0.01) . At 12 hr the u r i n e c h l o r i d e c o n c e n t r a t i o n of s o l v e n t -i n j e c t e d f i s h d e c l i n e d markedly (p<0.01); that of p r o l a c t i n -i n j e c t e d f i s h a l s o showed a f u r t h e r s i g n i f i c a n t f a l l (p<0.05). From 12 hr to 24 hr, there was no s i g n i f i c a n t change i n both the s o l v e n t - i n j e c t e d and p r o l a c t i n - i n j e c t e d f i s h . The-p r o l a c t i n - i n j e c t e d and s o l v e n t - i n j e c t e d f i s h were not s i g n i f i c a n t l y d i f f e r e n t a t 12 hr or 24 hr when the t e s t was based on e r r o r variance of a l l the data of the experiment ( l s d t e s t ; S t e e l and T o r r i e , 1960) but were s i g n i f i c a n t l y d i f f e r e n t (p<^ 0.05) when the t e s t was based on e r r o r variance of the 12-hr data or. the 24-hr data alone ( t - t e s t ) . This i s 36 Fig..4.6. p r o l a c t i n and u r i n e c h l o r i d e l e v e l s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. Each p o i n t represents mean - standard e r r o r . A b s c i s s a represents hr i n f r e s h water. A u n i n j e c t e d f i s h . • • Q PROLACTIN 12 24 H O U R 37 because the 12-hr or 24-hr data had less v a r i a b i l i t y than those of e a r l i e r time i n t e r v a l s . U r i n e , U x . . • pTasW < P > r a t l ° U/P r a t i o s f o r the ions were c a l c u l a t e d from the means (Table 4.1). .Figure 4.7 shows changes of U/P r a t i o of sodium a f t e r t r a n s f e r of f i s h to f r e s h water. Apparently the U/P r a t i o of" the s o l v e n t - i n j e c t e d or the u n i n j e c t e d f i s h i n c r e a s e d i n f r e s h water whereas th a t of p r o l a c t i n - i n j e c t e d f i s h decreased. I t i s apparent t h a t p r o l a c t i n treatment lowered the U/P r a t i o of sodium. E f f e c t of p r o l a c t i n treatment on U / P r a t i o of potassium i s shown i n F i g . 4.8. Both s o l v e n t - i n j e c t e d and p r o l a c t i n -i n j e c t e d f i s h i n f r e s h water showed an i n i t i a l f a l l of U/P r a t i o and a subsequent r i s e ; the u n i n j e c t e d f i s h appeared to show s i m i l a r response (1 hr, 24 h r ) . The p r o l a c t i n - i n j e c t e d f i s h , however, appeared to have a higher U/P r a t i o a t 12 hr and 24 hr than the s o l v e n t - i n j e c t e d f i s h or the u n i n j e c t e d f i s h (24 h r ) . F i g u r e 4.9 shows the e f f e c t of p r o l a c t i n treatment on U/P r a t i o of c h l o r i d e . The U/P r a t i o of the s o l v e n t - i n j e c t e d f i s h i n f r e s h water in c r e a s e d i n i t i a l l y , f o l l o w e d by an abrupt decrease, p r o l a c t i n treatment appeared to prevent the , inc r e a s e and reduce the r a t i o . 38 F i g . 4.7. P r o l a c t i n and sodium U/P r a t i o s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. Each p o i n t represents a s i n g l e value c a l c u l a t e d from u r i n e and plasma means. A b s c i s s a represents hr a f t e r t r a n s f e r to f r e s h water. A u n i n j e c t e d f i s h . 39 F i g . 4.8. P r o l a c t i n and potassium U/P r a t i o s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. Each p o i n t represents a s i n g l e value c a l c u l a t e d from u r i n e and plasma means. A b s c i s s a represents hr a f t e r t r a n s f e r to f r e s h water. © solvent O prolactin A uninjected 40 F i g . 4.9. P r o l a c t i n and c h l o r i d e U/P r a t i o s of s t i c k l e b a c k s i n sea water and f o l l o w i n g t r a n s f e r to f r e s h water. Each p o i n t represents a s i n g l e value c a l c u l a t e d from u r i n e and plasma means. A b s c i s s a represents hr a f t e r t r a n s f e r to f r e s h water. PROLACTIN • " — O . i 2 4 HOUR 41 D i s c u s s i o n P l a s m a i o n s E v i d e n c e shows t h a t p r o l a c t i n p r e v e n t e d t h e r a p i d d e c l i n e o f p l a s m a s o d i u m a n d c h l o r i d e w h i c h o c c u r r e d i n t h e e a r l y - w i n t e r G. a c u l e a t u s t r a c h u r u s f o l l o w i n g t r a n s f e r f r o m s e a w a t e r t o f r e s h w a t e r , b u t a p p e a r e d t o h a v e no e f f e c t o n p l a s m a p o t a s s i u m . B a l l a n d E n s o r ( 1 9 6 5 , 1967) h a v e shown s i m i l a r r e s u l t s ' f o r p l a s m a s o d i u m a n d p o t a s s i u m i n f r e s h w a t e r h y p o p h y s e c t o m i z e d P o e c i l i a l a t i p i n n a . The p l a s m a s o d i u m l e v e l i n t h i s f i s h f a l l s b y a b o u t 2 5 % , 24 h r a f t e r h y p o p h y s e c t o m y . I t i s i n t e r e s t i n g t h a t t h e e a r l y - w i n t e r G. a . t r a c h u r u s showed t h e same p e r c e n t a g e d e c l i n e o f p l a s m a s o d i u m , e v e n w i t h t h e p i t u i t a r y i n t a c t , when t h e f i s h w e r e t r a n s f e r r e d f r o m s,ea. t o f r e s h w a t e r . T h i s may b e f u r t h e r e v i d e n c e t h a t G. a... t r a c h u r u s i s " p h y s i o l o g i c a l l y h y p o p h y s e c t o m i z e d " i n t h e f a l l a n d e a r l y w i n t e r . The p r e s e n t w o r k i s t h e f i r s t r e p o r t o f p r o l a c t i n h a v i n g a s o d i u m - m a i n t a i n i n g e f f e c t i n a n i n t a c t f i s h i n f r e s h w a t e r . I n b o t h i n t a c t F u n d u l u s k a n s a e ( S t a n l e y a n d F l e m i n g , 1967) a n d A n g u i l l a a n g u i l l a ( O l i v e r e a u a n d C h a r t i e r - B a r a d u c , • 1 9 6 6 ) , h e l d i n f r e s h w a t e r , p r o l a c t i n t r e a t m e n t p r o d u c e s no e f f e c t o n s e r u m o r p l a s m a s o d i u m , a l t h o u g h i n t h e s e c a s e s f i s h h a v e b e e n a c c l i m a t e d i n f r e s h w a t e r r a t h e r t h a n t r a n s f e r r e d f r o m s e a t o f r e s h w a t e r a s i n t h e p r e s e n t w o r k . W h i l s t p r o l a c t i n h a s no e f f e c t e v e n i n h y p o p h y s e c t o m i z e d F. k a n s a e , h e l d i n f r e s h w a t e r , i t r e d u c e s s i g n i f i c a n t l y t h e f a l l i n p l a s m a 42 sodium and potassium i n freshwater hypophysectomized A. a n g u i l l a , i f i n j e c t e d immediately a f t e r hypophysectomy. The r o l e of p r o l a c t i n on plasma c h l o r i d e r e g u l a t i o n has not been p r e v i o u s l y r e p o r t e d . Burden (1956) found t h a t hypophysectomy i n freshwater Fundulus h e t e r o c l i t u s r e s u l t e d i n hypochloremia. Since p r o l a c t i n treatment has been shown to be e f f e c t i v e replacement therapy f o r freshwater s u r v i v a l i n hypophysectomized F_. h e t e r o c l i t u s ( p i c k f o r d et a l . , 1965), p r o l a c t i n would be expected to prevent hypochloremia. In the e a r l y - w i n t e r G. a_. trachurus, p r o l a c t i n appears to have such an e f f e c t , a t l e a s t to a c e r t a i n extent. U r i n e ions Evidence shows t h a t p r o l a c t i n treatment reduced u r i n e sodium l e v e l s but had no s i g n i f i c a n t e f f e c t on potassium l e v e l s o f s t i c k l e b a c k s f o l l o w i n g t r a n s f e r from sea water to f r e s h water. S i m i l a r r e s u l t s have been found f o r sodium i n both i n t a c t and hypophysectomized Fundulus kansae (Stanley and Fleming, 1967) and f o r sodium and potassium i n i n t a c t Salmo  g a i r d n e r i (Enomoto, 1965); both f i s h were maintained i n f r e s h water. P r o l a c t i n lowered the U/P r a t i o of sodium; t h i s suggests, t h a t p r o l a c t i n may i n c r e a s e r e n a l sodium r e a b s o r p t i o n and/or decrease water r e a b s o r p t i o n . This w i l l be f u r t h e r d iscussed i n Chapter 5. P r o l a c t i n however appeared to r a i s e the U/P r a t i o of potassium i n f r e s h water a f t e r an i n i t i a l l a c k of e f f e c t . This may not be s t a t i s t i c a l l y s i g n i f i c a n t s i n c e p r o l a c t i n d i d not 43 a f f e c t plasma and u r i n e potassium l e v e l s s i g n i f i c a n t l y . On the other, hand, i t , i s p o s s i b l e t h a t p r o l a c t i n may inc r e a s e r e n a l sodium r e a b s o r p t i o n and, a t the same time, decrease potassium r e a b s o r p t i o n , s i m i l a r to the r e n a l a c t i o n of aldosterone i n mammals. There i s some evidence t h a t a c t i v e sodium t r a n s p o r t v i a sodium- and potassium-activated adenosine t r i p h b sphatase (ATPase) i s i n v o l v e d i n r e n a l sodium r e a b s o r p t i o n i n mammals (Katz and E p s t e i n , 1966); i f t h i s occurred here, the above suggestion would not be too f a r - f e t c h e d s i n c e an inc r e a s e i n sodium r e a b s o r p t i o n ( i n one d i r e c t i o n ) would by t h i s mechanism be accompanied by an inc r e a s e i n potassium s e c r e t i o n ( i n the opposite d i r e c t i o n ) ( S e e post, e t a l . , 1960; Dunham and Glynn,. 1961),. which would r e s u l t i n a decrease i n net.,, potassium r e a b s o r p t i o n . p r o l a c t i n a l s o reduced u r i n e c h l o r i d e l e v e l s and the c h l o r i d e U/P r a t i o of s t i c k l e b a c k s i n f r e s h water. S i m i l a r l y , p r o l a c t i n reduced u r i n e c h l o r i d e l e v e l s i n S_. g a i r d n e r i maintained i n f r e s h water (Enomoto, 1965). Thus p r o l a c t i n may in c r e a s e r e n a l c h l o r i d e r e a b s o r p t i o n and/or decrease water r e a b s o r p t i o n ; t h i s may occur d i r e c t l y or i n d i r e c t l y as- a r e s u l t of the a c t i o n of p r o l a c t i n on r e n a l sodium r e a b s o r p t i o n . Thus p r o l a c t i n may in c r e a s e r e n a l sodium r e a b s o r p t i o n , c r e a t i n g an e l e c t r o p o t e n t i a l g r a d i e n t favourable to in c r e a s e d passive c h l o r i d e r e a b s o r p t i o n . I f , however, p r o l a c t i n a l s o decreased potassium r e a b s o r p t i o n as suggested above, the e l e c t r o p o t e n t i a l g r a d i e n t would be e l i m i n a t e d and the p r o l a c t i n e f f e c t on u r i n e c h l o r i d e would then be d i r e c t . 44 The p a t t e r n of c h l o r i d e e x c r e t i o n of both_.the s o l v e n t - , i n j e c t e d and p r o l a c t i n - i n j e c t e d . f i s h appeared d i f f e r e n t from that of e i t h e r sodium 'or potassium e x c r e t i o n . In sea water and during the i n i t i a l p e r i o d i n f r e s h water, c h l o r i d e u r i n e l e v e l s and U/P r a t i o s were higher than- those of e i t h e r sodium or potassium (Figs. 4.4,.4.5, 4.6, 4.7, 4.8, 4.9); t h i s d i f f e r e n c e might be a s s o c i a t e d w i t h r e n a l s e c r e t i o n of d i v a l e n t ions such as magnesium and sulphate. In sea water, t e l e o s t s s e c r e t e d i v a l e n t ions i n t o u r i n e , and s i n c e more d i v a l e n t c a t i o n s apparently are se c r e t e d than d i v a l e n t anions (Hickman, 1968 b ) , a net u r i n e e l e c t r o p o s i t i v i t y would r e s u l t , c r e a t i n g an e l e c t r o p o t e n t i a l g r a d i e n t which would reduce net c h l o r i d e (or other anions) r e a b s o r p t i o n and/or enhance r e a b s o r p t i o n o f sodium and/or potassium; hence more c h l o r i d e would be excreted than sodium or potassium. The r e n a l s e c r e t i o n of d i v a l e n t ions may p e r s i s t f o r a b r i e f p e r i o d f o l l o w i n g t r a n s f e r of f i s h to f r e s h water; t h i s might e x p l a i n why the u r i n e c h l o r i d e l e v e l s remained higher than sodium or potassium l e v e l s during the f i r s t 4 hr i n f r e s h water. The s e c r e t i o n of d i v a l e n t i o n s , however, soon ceases i n f r e s h water (Hickman, unpublished). When t h i s occurred, r e d u c t i o n of net c h l o r i d e r e a b s o r p t i o n and/or s t i m u l a t i o n of r e a b s o r p t i o n of sodium and/or potassium would be removed, causing, an in c r e a s e i n net c h l o r i d e r e a b s o r p t i o n (or a f a l l i n c h l o r i d e U/P r a t i o ) , and/or a decrease i n r e a b s o r p t i o n of sodium and/or potassium (or a r i s e i n sodium and/or potassium U/P r a t i o s ) . F i g u r e 4.9 shows a marked f a l l of c h l o r i d e U/P r a t i o from 4 hr i n f r e s h water 45 onwards, w h i l s t F i g s . 4.7, 4.8 show corresponding r i s e . i n . U/P r a t i o s of sodium and potassium r e s p e c t i v e l y . Thus i t appears t h a t the u r i n a r y e x c r e t i o n of c h l o r i d e , sodium and potassium was a f f e c t e d by r e n a l s e c r e t i o n o f d i v a l e n t i o n s . I t i s of i n t e r e s t t h a t e u r y h a l i n e t e l e o s t s adapted to sea water show higher u r i n e l e v e l s of c h l o r i d e than sodium, w h i l s t the same f i s h adapted to f r e s h water show s i m i l a r or lower l e v e l s o f c h l o r i d e than sodium (see Table 4. I I ) . I t must be p o i n t e d out, however, t h a t , i n t h i s study, the c h l o r i d e data were obtained i n a d i f f e r e n t experiment from th a t which provided the sodium and potassium data. More-over, the c h l o r i d e values were determined from i n d i v i d u a l samples whilst., sodium and potassium values were determined from pooled samples, although i n p r e l i m i n a r y t e s t s , p o o l i n g of samples was not found to s i g n i f i c a n t l y a f f e c t the r e s u l t s . The d i f f e r e n c e s between the p a t t e r n of c h l o r i d e e x c r e t i o n and t h a t of e i t h e r sodium or potassium may, t h e r e f o r e , be due to d i f f e r e n c e s of experimental, c o n d i t i o n s r a t h e r than s e c r e t i o n of d i v a l e n t i o n s . Table 4. II E u r y h a l i n e t e l e o s t s in f r e s h water (FW) and sea water (SW) , comparing GFR, u r i n e f l o w and u r i n e composition a f t e r a daptation to f r e s h or sea water (Adapted from Hickman and Trump, 1969). H a b i t a t . GFR ml/hr/kg Urine f l o w ml/hr/kg Na mEq/1 C l " mEq/1 K mEq/1 References A n g u i l l a a n g u i l l a (European eel) FW SW 4.60*0.53 3.52±0.41 18.9±3.0 n i l 1.03+0.21 0.63+0.09 6.5+1.0 119+5.7 0.65+0.16. S h a r r a t t et a l . , 2.08+0.9 1964. P l a t i c h t h y s FW .f iesus' (flounder) SW 4.16±0.22 1.78+0.09 16.4+3.6 2.4 ±0.27 0.60±0.05 35.8±8.9 16.3±1.1 103.0±13.0 1.55±0.88 2.82±0.47 Lahlou, 1967 p a r a l i c h t h y s  l e thostigma (southern flounder) FW SW 3.8 (3.4-4.1) 1.69 (1.4-2.1) 2.9 (2.6-3.0) 0.22 (0.11-0.31) 27.9 (23-31) 17.1 (0.1-32.0) 6.4 (3.2-7.4) 120.6 (51-148) 2.1 (1.9-2.2) 1.42 (0.47-3.6) Hickman (Unpublished observations) Hickman, 1968a, b A n g u i l l a FW paponica (Japanese eel) SW 3.1330.78 2.8030.26 2.2630.17 7.46±1.16 2.68^0.55 0.3830.04 20.634.86 41.331.47 Oide and U t i d a , 1968 P a r t I I MECHANISM (S) OF ACTION OF PROLACTIN 48 CHAPTER 5 P r o l a c t i n and the K i d n e y s 1 I n t r o d u c t i o n .  In previous chapters, p r o l a c t i n has been shown to reduce u r i n e o s m o l a l i t y and u r i n e sodium and c h l o r i d e l e v e l s of autumn or e a r l y - w i n t e r s t i c k l e b a c k s f o l l o w i n g ' t r a n s f e r from -sea -water to 'fresh water. In t h i s chapter, the r e n a l mechanism(s) of a c t i o n of p r o l a c t i n i s examined i n greater d e t a i l . The e f f e c t s of p r o l a c t i n on u r i n e f l o w and the h i s t o l o g y o f the g l o m e r u l i of autumn or early-^winter s t i c k l e b a c k s were i n v e s t i g a t e d . From the h i s t o l o g i c a l f i n d i n g s , deductions are made re g a r d i n g the p h y s i o l o g i c a l a c t i o n of p r o l a c t i n a t the l e v e l of the g l o m e r u l i . The v a r i o u s f i n d i n g s are f i n a l l y s y n t h e s i z e d i n a d i s c u s s i o n of a p o s s i b l e r e n a l mechanism,of a c t i o n of p r o l a c t i n . M a t e r i a l s .and Methods Late-autumn and e a r l y - w i n t e r s t i c k l e b a c k s were. used. In the u r i n e - f l o w study, l a r g e r (~ 2 gm) f i s h were used, and a s i n g l e i n j e c t i o n of p r o l a c t i n or the s o l v e n t was- g i v e n . In the h i s t o l o g i c a l study, approximately 1 gm f i s h were used, and m u l t i p l e i n j e c t i o n s on a l t e r n a t e days were given. U rine c o l l e c t i o n Conventional techniques of u r i n e c o l l e c t i o n i n v o l v i n g c a t h e t e r i z a t i o n of the u r i n a r y bladder have not been f e a s i b l e Mparts of t h i s chapter ( h i s t o l o g y , and technique f o r u r i n e c o l l e c t i o n ) are i n press i n Can. J . z o o l . 49 i n the s t i c k l e b a c k due to the smallness of the u r i n o g e n i t a l aperture and u r i n a r y bladder. For t h i s reason, a new technique of u r i n e c o l l e c t i o n was introduced. In t h i s technique, the f i s h was suspended by means of a rubber diaphragm w i t h the head r e g i o n i n continuously-oxygenated water and the remainder of i t s body i n p a r a f f i n o i l . U rine was c o l l e c t e d i n the p a r a f f i n o i l . Apparatus. F i g u r e 5.1 shows c o n s t r u c t i o n d e t a i l s of the apparatus. P l e x i - g l a s s tubings and p l a t e s were used; j o i n t s were cemented w i t h p l e x i - g l a s s chips d i s s o l v e d i n ethylene d i c h l o r i d e . The apparatus c o n s i s t s of a trunk chamber (#4 i n F i g . 5.1) Which has a continuous groove (down one s i d e , along the bottom and up the other side) c l o s e to one end, so t h a t another, chamber, the head chamber (3), can s l i d e i n and out of i t along the groove. The trunk chamber i s about 80% f i l l e d w i t h p a r a f f i n o i l c ooled to a temperature of 10 - 1°C. The head chamber i s shaped l i k e a pipe w i t h a "stem" and a "bowl" and w i t h the attached p l a t e used as the s l i d e . Before the experiment, the "bowl" of the head chamber i s f i t t e d w i t h a rubber diaphragm having a s m a l l o v a l s l i t i n the middle; molten p a r a f f i n wax i s then a p p l i e d to the r i m of the diaphragm (7), which, on s o l i d i f i c a t i o n , f a s t e n s the diaphragm to the "bowl". A.short l e n g t h of the c l o s e d end of a b a l l o o n i s used as the diaphragm; a. s m a l l s l i t i s cut on the t i p of the c l o s e d end. When the b a l l o o n i s s t r e t c h e d and f i t t e d on the head chamber, i t leaves a depression around the s l i t ; t h i s i s u s e f u l because, a f t e r 50 P i g . 5.1. The apparatus used f o r u r i n e c o l l e c t i o n and the study o f exchanges of m a t e r i a l s v i a the head r e g i o n of s t i c k l e b a c k s . Keys to numerals:. 1. Water 2. p a r a f f i n , o i l 3. .Head chamber 4. Trunk chamber 5. Rubber diaphragm 6. Second double-membrane diaphragm 7. Wax f o r h o l d i n g 5 8. S i l i c o n grease 9. Urine 10. PE 50 tubing 11. Hypodermic needle 12. Tygon tubing 13. Groove 14. 02 source 15. S u r g i c a l t h r e a d f o r h o l d i n g anus cannula i n p l a c e . 16. Anus cannula ( P E 10 tubing w i t h open f l a r e d end which was i n s e r t e d i n t o the anus). 17. Hematocrit tube 18. V i a l 19. Sand f o r h o l d i n g v i a l Note: P a r a f f i n o i l l e v e l was higher than the water l e v e l ; anus cann u l a t i o n was done o n l y when f u n c t i o n s of the head r e g i o n alone were studied(Chapter 6). 51 the head r e g i o n of the f i s h has been i n s e r t e d through the s l i t , the depression can be f i l l e d up w i t h non-toxic s i l i c o n grease (8) to minimize leakage,. Procedure. Each f i s h was f i r s t weighed i n f r e s h water ^(weight of beaker+water+f i s h ) - (weight of beaker +waterj] and then subjected to the f o l l o w i n g procedures: 1. The head end of the f i s h was i n s e r t e d through the s l i t of the diaphragm i n t o the head chamber u n t i l the opercula had joist c l e a r e d the diaphragm ( F i g . 5.1). 2. Approximately 12 ml of d e c h l o r i n a t e d tap water a t a temperature of 10 ± l ° c were poured down the "stem" of the - head chamber; t h i s f i l l e d the "bowl" and a s m a l l p o r t i o n of the "stem". The. f i s h was now able to "breathe" i n water although the trunk ( p o s t e r i o r to the opercula) was suspended i n the a i r . The next four procedures were c a r r i e d out whi l e the f i s h was i n t h i s c o n d i t i o n . 3. The depression on the diaphragm around the f i s h was f i l l e d t i g h t l y w i t h s i l i c o n grease. 4. A second double-membrane diaphragm (open p o r t i o n of the same b a l l o o n w i t h a p p r o p r i a t e s l i t s (6) was f i t t e d on the f i s h j u s t a n t e r i o r to the u r i n o g e n i t a l and anal pores which are c l o s e together. This diaphragm helped to l o c a l i z e , on the a n t e r i o r s i d e , water which might leak out along the, underside of the f i s h and, on the p o s t e r i o r s i d e , u r i n e which might t r a v e l along the underside a n t e r i o r l y . 5. Grease was smeared around the s l i t s of the second diaphragm. 52 6. The water i n the head chamber was poured out and r e p l a c e d by approximately the same amount of f r e s h d e c h l o r i n a t e d water a t 10 i 1°C. This was to avo i d excessive accumulation of metabolic wastes i n the water. 7. The chamber w i t h the f i s h suspended was then lowered i n t o the trunk chamber c o n t a i n i n g p a r a f f i n o i l (temperature 10 t 1°C). "The p a r a f f i n o i l was a t a higher l e v e l than the water i n the head chamber. 8. The apparatus was placed i n a bath of running water a t a temperature of 10 ± 1°C. 9. Oxygen was co n t i n u o u s l y bubbled through the water i n the head chamber by means of a short PE 50 polyethylene tube f i t t e d over a hypodermic needle (11) ; the needle was connected to a oxygen c y l i n d e r by a piece of tygon tubing (12). Note: a i r was inadequate f o r t h i s purpose. 10. The apparatus was covered w i t h a b l a c k p l a s t i c sheet to a v o i d disturbance of the f i s h u n t i l i t was time f o r sampling. 11. A f t e r 24 hr, the u r i n e excreted during the p e r i o d was seen as drop(s) i n the p a r a f f i n o i l (9). To f a c i l i t a t e r e c o g n i t i o n of u r i n e , the f i s h were i n j e c t e d before the experiment ( 5 hr a f t e r s o l v e n t or p r o l a c t i n i n j e c t i o n s ) w i t h amaranth (a. r e d dye) i n 0.6% NaCl s o l u t i o n ; the u r i n e excreted i n t h i s case was coloured red. 12. The u r i n e was sucked i n t o a PE 100 polyethylene tubing by means of a medicine dropper; the tubing had been c a l i b r a t e d to give volume i n ml (or p i ) per cm so that by 53 measuring the length(s) of u r i n e column(s) i n the- tub i n g , the u r i n e volume was determined. Kidney p r e p a r a t i o n s F i s h were k i l l e d by d e c a p i t a t i o n and the kidneys removed and f i x e d i n Bouin's f i x a t i v e . A f t e r dehydration i n ethanol and embedding i n p a r a f f i n wax (m.p. 56°C), the t i s s u e s were se c t i o n e d a t 5 u and s t a i n e d w i t h haematoxylin and eo s i n . Glomerular measurements A t l e a s t s i x f i s h were used i n each experimental group. The means of the measurements of diameters of Bowman's capsules and the glomerular t u f t s of the s i x f i s h are given i n Table 5. IT. For each f i s h , e i g h t kidney s e c t i o n s from two s l i d e s were examined, i n c l u d i n g the f i r s t kidney s e c t i o n and every nth s e c t i o n on the two s l i d e s (n = t o t a l number of sections- on the two s l i d e s / 7 ) . The f i r s t two g l o m e r u l i observed i n each s e c t i o n were taken and the diameters of the Bowman's capsule and the glomerular t u f t measured along the a x i s of the f i x e d c a l i b r a t e d micrometer ( m a g n i f i c a t i o n 1.25 x 450). Experimental procedure ( h i s t o l o g i c a l study) S t i c k l e b a c k s (~ 1 g body weight) were maintained i n the experimental tanks of sea water f o r 3-4 days under c o n d i t i o n s of l i g h t and temperature s i m i l a r to the stock tanks. The f i s h were d i v i d e d i n t o three groups, w h i l s t s t i l l i n sea water; one group was i n j e c t e d w i t h p r o l a c t i n , another w i t h s o l v e n t , and a t h i r d group was l e f t u n i n j e c t e d . Twenty-four hours a f t e r the t h i r d i n j e c t i o n (6th day), a group each of p r o l a c t i n - and 54 s o l v e n t - i n j e c t e d f i s h was k i l l e d (0 day) and the remainder of both groups t r a n s f e r r e d to d e c h l o r i n a t e d Vancouver tap water a t a temperature of 9.0 1 1.0°c. I n j e c t i o n s were continued on a l t e r n a t e days. Groups of p r o l a c t i n - and s o l v e n t - i n j e c t e d f i s h were k i l l e d a f t e r 1, 4 and 7 days i n f r e s h water. The u n i n j e c t e d group was not t r a n s f e r r e d to f r e s h water, but was k i l l e d a f t e r 6 days i n sea water. S t a t i s t i c a l Method ( h i s t o l o g i c a l study) The data were subjected to two-way a n a l y s i s of variance (treatment ( p r o l a c t i n and solvent) vs time i n f r e s h water); glomerular t u f t and Bowman's capsule were considered s e p a r a t e l y and the means compared by Tukey's W ( S t e e l and T o r r i e , 1960). The seawater data were used again i n the one-way a n a l y s i s of v a r i a n c e w i t h data on the u n i n j e c t e d f i s h so t h a t the means of the s o l v e n t - i n j e c t e d , u n i n j e c t e d , and p r o l a c t i n - i n j e c t e d f i s h may be compared by Tukey's W. R e s u l t s U r i n e f l o w The r e s u l t s are summarized i n Table 5.1. I t i s evident th a t p r o l a c t i n i n c r e a s e d the u r i n e f l o w of s t i c k l e b a c k s during the 24 hr i n the apparatus ( F i g . 5.1). Unpaired t-test-showed a s i g n i f i c a n c e a t P ^ 0.005. Diameters of Bowman's capsule and glomerular t u f t (Table 5 . I I ) . In sea water (0 day), p r o l a c t i n - i n j e c t e d f i s h possessed s i g n i f i c a n t l y smaller Bowman's capsules and glomerular t u f t s Table 5.1 P r o l a c t i n and u r i n e f l o w of s t i c k l e b a c k s w i t h the head r e g i o n i n continuously-oxygenated d e c h l o r i n a t e d f r e s h water and the remaining body i n p a r a f f i n o i l . Mean* u r i n e f l o w i Sx (ul/gm/24 hr) (number of f i s h ) Solvent (Control) P r o l a c t i n 31.8 J 1.7 (7) 59.0 ± 8.0 (5) (= 1.33 ml / k g/hr) (= 2.46 m l A g / h r ) *** P ^ 0.005 ( t - t e s t ) in Table 5. I I P r o l a c t i n arid glomerular h i s t o l o g y of G. aculea t u s , form trachurus, i n sea water and a f t e r t r a n s f e r to f r e s h water. Time i n f r e s h water (days) Mean f o r k l e n g t h (range) (mm) Bowman1s Capsule Diameter (u) Glomerular Tuft Diameter (u) Solvent U n i n j e c t e d P r o l a c t i n Solvent U n i n j e c t e d P r o l a c t i n Solvent U n i n j e c t e d P r o l a c t i n 0 (seawater) 44(39-52) 47 (41-52) 45 (39-52) 4 4 . 4 ± 0 o 9 42.8+1.1 * * 38.1+0.9 (**) 40. 38. 1+0.8 4+0.9 35.8+0.9 (*) 1 43 (36-54) 43(37-48) 3 5 . 4 - 0 . 6 38 .430 .9 32. 4 - 0 . 6 + * * 3 6 . 4 - 0 . 8 4 44(38-52) 40(36-45) 42.0+0.8 4 0 . 6 ± 0 . 6 38. 9 - 0 . 6 3 8 . 8 - 0 . 8 7 41(35-46) 41(35-45) 43 .810 .8 39.6+0.6** 40. 0 ± 0 . 8 39.1+0.6 Note: values given are means of 6 f i s h x 8 kidney s e c t i o n s x 2 g l o m e r u l i (= 96 glomeruli) + standard e r r o r ; u n i n j e c t e d s t i c k l e b a c k s were sampled o n l y a t 0 day (sea water); ** s i g n i f i c a n t l y d i f f e r e n t from s o l v e n t - i n j e c t e d f i s h a t p<0.01; (**) s i g n i f i c a n t l y d i f f e r e n t from u n i n j e c t e d f i s h a t P<0.01; (*) s i g n i f i c a n t l y d i f f e r e n t from u n i n j e c t e d f i s h a t P<0.05; f o r Bowman's capsule, Tukey's W.05 = 3 . 5 , W.QI = 4 . 1 ; f o r glomerular t u f t , W.05 = 3 . 1 , W.QI = 3 . 7 ; f o r comparison between p r o l a c t i n -i n j e c t e d , s o l v e n t - i n j e c t e d and u n i n j e c t e d f i s h a t 0 day, capsule: W.05 = 3 . 0 , W.QI=3.5 t u f t : W.05 = 2 . 5 , W.01=3.7 57 than e i t h e r the s o l v e n t - i n j e c t e d c o n t r o l s (p<£0.01) or the u n i n j e c t e d c o n t r o l s (p < 0.01 f o r capsules; p<0.05 f o r t u f t s ) . One day a f t e r t r a n s f e r to f r e s h water, the p r o l a c t i n -i n j e c t e d f i s h showed s i g n i f i c a n t l y l a r g e r glomerular t u f t diameters than the s o l v e n t - i n j e c t e d c o n t r o l s (p< 0»01); the Bowman's capsule diameters were not s i g n i f i c a n t l y d i f f e r e n t i n the two groups a t t h i s time. Four days a f t e r t r a n s f e r to f r e s h water, the diameters of Bowman's capsules and glomerular t u f t s d i d not d i f f e r s i g n i f i c a n t l y between the s o l v e n t - and p r o l a c t i n - i n j e c t e d f i s h . A f t e r one week i n f r e s h water, the s o l v e n t - i n j e c t e d f i s h showed enlarged Bowman's capsule diameters, s i g n i f i c a n t l y l a r g e r than thoseof p r o l a c t i n - i n j e c t e d f i s h (P<0.01). The diameters of the glomerular t u f t s , however, did- not. d i f f e r s i g n i f i c a n t l y between the two groups. Percentage change- of glomerular diameters Since the s o l v e n t - i n j e c t e d and p r o l a c t i n - i n j e c t e d f i s h d i f f e r e d s i g n i f i c a n t l y i n glomerular diameters i n sea water-p r i o r to t r a n s f e r to f r e s h water, the changes of glomerular diameters i n f r e s h water may be best seen as percentage changes of the i n i t i a l seawater (0 day) values of the r e s p e c t i v e groups. The r e s u l t s are presented i n F i g . 5.2. Whereas the s o l v e n t -i n j e c t e d f i s h showed a sharp percentage f a l l from the i n i t i a l value a f t e r one day i n f r e s h water (p <.0.01), the p r o l a c t i n -i n j e c t e d f i s h showed a s l i g h t r i s e . The solvent-in-jeefeed f i s h made a marked recovery (p^O.01), but were only a b l e - a t b e s t to r e s t o r e the i n i t i a l seawater c o n d i t i o n (no s i g n i f i c a n t 58 F i g . 5.2 P r o l a c t i n and percentage change of the Bowman1s capsule and the glomerular t u f t i n s t i c k l e b a c k s , _G. acule a t u s , form. trachurus, a f t e r t r a n s f e r from sea water to f r e s h water. O A Keys: ^ Bowman's capsule; ^ glomerular t u f t 59 percentage change). The p r o l a c t i n - i n j e c t e d f i s h , _ however, showed a percentage in c r e a s e i n the diameters of Bowman's capsules and glomerular t u f t s ; the percentage increase i n t u f t diameters was greater than t h a t of the capsular diameters. A f t e r four days i n f r e s h water, the glomerular t u f t had co n s i d e r a b l y i n c r e a s e d from the i n i t i a l seawater c o n d i t i o n (P < 0.05) and a f t e r one week, markedly in c r e a s e d (P 0.01) ; however, the Bowman's capsule d i d not inc r e a s e s i g n i f i c a n t l y during the whole p e r i o d i n f r e s h water. On the other hand, i n the so-l v e h t - i n j e c t e d f i s h , the Bowman's capsules and the glomerular t u f t s d i d not d i f f e r s i g n i f i c a n t l y i n t h e i r changes from the r e s p e c t i v e i n i t i a l seawater c o n d i t i o n during the whole p e r i o d i n f r e s h water. Glomerular r a t i o - t u f t diameter/capsule diameter The p r o l a c t i n - i n j e c t e d f i s h showed l i t t l e i n t r a c a p s u l a r space i n the g l o m e r u l i , much l e s s than the s o l v e n t - i n j e c t e d c o n t r o l s ( F i g . 5.3 a, b ) . As a q u a n t i t a t i v e measure of t h i s d i f f e r e n c e , the glomerular r a t i o ( t u f t diameter/capsule diameter) was c a l c u l a t e d . The means of the data t 95% confidence i n t e r v a l s (t Q 5 Sx; S t e e l and T o r r i e , 1960), are presented i n F i g . 5.4. In sea water, both the u n i n j e c t e d and s o l v e n t - i n j e c t e d f i s h had a low glomerular r a t i o i n d i c a t i v e of a- r e l a t i v e l y l a r g e i n t r a c a p s u l a r space. P r o l a c t i n i n j e c t i o n s increased, the r a t i o s i g n i f i c a n t l y (p ^  0..05) . A f t e r t r a n s f e r to f r e s h water, the s o l v e n t - i n j e c t e d f i s h d i d not show s i g n i f i c a n t changes. P r o l a c t i n - i n j e c t e d f i s h , however, showed a s i g n i f i c a n t i n c r e a s e a f t e r one week (p< 0.01), 60 F i g . 5.3 (a) Glomeruli w i t h l i t t l e or no i n t r a -capsular space, more f r e q u e n t l y found i n p r o l a c t i n - i n j e c t e d than s o l v e n t -i n j e c t e d s t i c k l e b a c k s , (b) G l o m e r u l i w i t h l a r g e i n t r a c a p s u l a r space, more f r e q u e n t l y found i n s o l v e n t - i n j e c t e d than p r o l a c t i n - i n j e c t e d s t i c k l e b a c k s . Keys: GT - glomerular t u f t ; BC - Bowman's capsule; IS - i n t r a c a p s u l a r space; NS - neck segment 61 F i g . 5.4. P r o l a c t i n and glomerular r a t i o / T u f t diameter s t i c k l e b a c k s . v Capsule diameter } or s t i c K i e b a c K s , G.,aculeatus, form trachurus, i n sea water and a f t e r t r a n s f e r to f r e s h water. A b s c i s s a represents days i n f r e s h water. Each p o i n t i s mean ± 9 5 % confidence i n t e r v a l . A u n i n j e c t e d 62 and were s i g n i f i c a n t l y d i f f e r e n t from the s o l v e n t - i n j e c t e d c o n t r o l s a t each time i n t e r v a l [j? ^  0.05 (1, 4 days); P < 0.01 (7 daysjj. Percentage, frequency of g l o m e r u l i w i t h no i n t r a c a p s u l a r space The data were based on the expression: number of g l o m e r u l i w i t h no i n t r a c a p s u l a r space y 100% t o t a l number of g l o m e r u l i examined The means ±95% confidence i n t e r v a l s are presented i n F i g . 5.5. In sea water, both the u n i n j e c t e d and s o l v e n t - i n j e c t e d s t i c k l e b a c k s had a low mean percentage frequency of g l o m e r u l i w i t h no i n t r a c a p s u l a r space ( F i g . 5.3 a ) ; t h i s percentage frequency d i d not change f o l l o w i n g t r a n s f e r (of the s o l v e n t -i n j e c t e d f i s h ) to f r e s h water. The p r o l a c t i n - i n j e c t e d f i s h i n sea water, however, had a higher mean percentage frequency of g l o m e r u l i w i t h no i n t r a c a p s u l a r space (P<0.05); the value i n c r e a s e d f o l l o w i n g t r a n s f e r to f r e s h water, e s p e c i a l l y one week.after t r a n s f e r (p 40.01), and was s i g n i f i c a n t l y d i f f e r e n t from t h a t of the s o l v e n t - i n j e c t e d c o n t r o l s a t each time i n t e r v a l Jp< 0.05 (1, 4 days); P<£0.01 (7 days]]. D i s c u s s i o n Urine, f l o w P r o l a c t i n i n c r e a s e d u r i n e f l o w of autumn or e a r l y - w i n t e r s t i c k l e b a c k s when the f i s h were place d s i n g l y i n the apparatus. ( F i g . 5.1) w i t h the head r e g i o n i n continuously-oxygenated d e c h l o r i n a t e d f r e s h water and the remaining body i n p a r a f f i n 63 F i g . 5.5. P r o l a c t i n and percentage frequency of g l o m e r u l i w i t h no (evident) i n t r a -capsular space of s t i c k l e b a c k s , G. aculeatus, form trachurus, i n sea water and a f t e r t r a n s f e r to f r e s h water. A b s c i s s a represents days i n f r e s h water. Each p o i n t i s mean t 95% confidence i n t e r v a l . A U n i n j e c t e d f i s h 64 o i l . I t i s assumed t h a t s o l v e n t i n j e c t i o n s d i d not produce any s i g n i f i c a n t e f f e c t on u r i n e flow; the i n j e c t i o n s had not s i g n i f i c a n t l y a f f e c t e d freshwater s u r v i v a l and plasma and u r i n e i o n l e v e l s (chapters 3 and 4).. The technique of u r i n e c o l l e c t i o n used here, however, has a t l e a s t three l i m i t a t i o n s ; (1) the o p e r a t i v e manipulations and handling may cause " l a b o r a t o r y d i u r e s i s " (Forster and Berglund, 1956), (2) i t i s not known whether p a r a f f i n o i l produces any p h y s i o l o g i c a l e f f e c t ( s ) , although i t i s not t o x i c to the f i s h , and (3) the u r i n e c o l l e c t e d may be contaminated w i t h e x c r e t i o n s from the anus (faeces and p o s s i b l e s m a l l amounts of f l u i d s ) . The f i r s t l i m i t a t i o n i s e q u a l l y a p p l i c a b l e to c o n v e n t i o n a l c a t h e t e r i z a t i o n techniques. The s t i c k l e b a c k , d i d not apparently show any r e s i s t a n c e except f o r a b r i e f i n i t i a l p e r i o d , when subjected t o the c o n d i t i o n s of the technique. The t h i r d l i m i t a t i o n may be e l i m i n a t e d a t l e a s t w i t h regards to faeces s i n c e the f i s h had been s t a r v e d f o r a t l e a s t three days p r i o r to the experiment and no faeces were evident i n the u r i n e . Furthermore, the i n h e r e n t e f f e c t s ( i f any) of the technique would have been c o n t r o l l e d i n the s o l v e n t - i n j e c t e d f i s h . Thus, whether or not the q u a n t i t a t i v e data obtained by t h i s technique represent 'normal' u r i n e f l o w of s t i c k l e b a c k s , the e f f e c t of p r o l a c t i n may s t i l l h o l d . I t i s p e r t i n e n t to note t h a t the mean u r i n e f l o w of p r o l a c t i n - i n j e c t e d f i s h compared fav o u r a b l y w i t h those of other e u r y h a l i n e t e l e o s t s maintained i n f r e s h water (See Table 4 . I I ) . 65 P r o l a c t i n has a l s o been shown to i n c r e a s e u r i n e f l o w i n hypophysectomized .Fundulus kansae maintained i n f r e s h water (Stanley and Fleming, 1967), but not i n i n t a c t F_. kansae (Stanley and Fleming, 1967) nor i n i n t a c t salmo g a i r d n e r i (Enomoto, 1965) maintained i n f r e s h water. That p r o l a c t i n i n c r e a s e d u r i n e f l o w of i n t a c t autumn or e a r l y - w i n t e r s t i c k l e b a c k s may -be f u r t h e r evidence th a t these f i s h were " p h y s i o l o g i c a l l y hypophysectomized" w i t h regards to p r o l a c t i n (See Chapters 2 and 3). H i s t o l o g i c a l F i n d i n g s The r e s u l t s of the h i s t o l o g i c a l study have provided (a) clues to the e f f e c t of p r o l a c t i n on glomerular f u n c t i o n s , and (b) confirmatory evidence of seasonal p r o l a c t i n s e c r e t i o n . These two p o i n t s w i l l now be discussed s e p a r a t e l y . The p h y s i o l o g i c a l a c t i o n of p r o l a c t i n . In the s o l v e n t -i n j e c t e d late-autumn or e a r l y - w i n t e r s t i c k l e b a c k , i n both sea and f r e s h water, many g l o m e r u l i showed m i l d to marked increas e s i n the s i z e of the i n t r a c a p s u l a r space ( F i g . 5.3 b ) . S i m i l a r glomerular h i s t o l o g y has been found i n the u n i n j e c t e d s t i c k l e b a c k s (sea water), i n the u n i n j e c t e d late-autumn s t i c k l e b a c k s of another study (Ogawa, 1968), and i n a marine t e l e o s t , the daddy s c u l p i n , Myoxocephalus s c o r p i u s , ( G r a f f l i n , 1933). G r a f f l i n regarded such changes as a " p h y s i o l o g i c a l i n v o l u t i o n ! ' (rather than a " p a t h o l o g i c a l process") rendering g l o m e r u l i incompetent and ensuring o l i g u r i a i n marine t e l e o s t s . This view was shared by Smith (193o) and M a r s h a l l and Smith (19.3 0) . 66 P r o l a c t i n i n j e c t i o n s appeared to reduce such glomerular changes i n the s t i c k l e b a c k s i n c e the i n j e c t i o n s i n c r e a s e d the glomerular r a t i o ( t u f t diameter/capsule diameter) and the percentage frequency of g l o m e r u l i without (evident) i n t r a -capsular space. Thus, i f G r a f f l i n ' s hypothesis i s c o r r e c t , p r o l a c t i n should render g l o m e r u l i more f u n c t i o n a l or more g l o m e r u l i f u l l y f u n c t i o n a l ; t h i s would i n c r e a s e the glomerular f i l t r a t i o n r a t e (Hickman., 1965). Hickman (1965) has f u r t h e r proposed t h a t the greater the r a t i o of the g l o m e r u l i p a r t i a l l y f u n c t i o n a l to those f u l l y f u n c t i o n a l , the greater i s the water r e a b s o r p t i o n . I f such i s the case i n s t i c k l e b a c k s , p r o l a c t i n would a l s o decrease water r e a b s o r p t i o n . In sea water, p r o l a c t i n appeared a l s o to cause a r e d u c t i o n of i n t r a c a p s u l a r space ( F i g s . 5.4 and 5.5, 0 day), but the p h y s i o l o g i c a l m a n i f e s t a t i o n of t h i s a c t i o n ( i . e . the i n c r e a s e i n GFR) may have been p a r t i a l l y or f u l l y prevented by the r e d u c t i o n i n s i z e of the glomerular t u f t s , which appeared a l s o to be the a c t i o n of p r o l a c t i n (Table 5. I I ) . Thus:. i t appears t h a t , w h i l e p r o l a c t i n reduced i n t r a c a p s u l a r space i n both sea water and f r e s h water, i t reduced-the glomerular t u f t - s i z e i n sea water but not i n f r e s h water. I t seems l i k e l y , however, tha t the r e d u c t i o n of t u f t s i z e i n sea water was a secondary e f f e c t of p r o l a c t i n . v i a other hormone(s) (normally f u n c t i o n a l i n seawater adaptation) a c t i n g to. overcome the primary, e f f e c t of ' p r o l a c t i n i n reducing i n t r a c a p s u l a r space. J u s t how p r o l a c t i n reduced the i n t r a c a p s u l a r space i s a matter of s p e c u l a t i o n . P r o l a c t i n might i n c r e a s e the r e n a l 67 blood flow, as i n the cat (Lockett, 1965), and hence i n c r e a s e the s i z e o f the glomerular t u f t ; t h i s would reduce the i n t r a c a p s u l a r space. A l t e r n a t e l y , p r o l a c t i n may reduce the i n t r a c a p s u l a r space by i n c r e a s i n g i n some way the drainage of the tubula r o u t l e t (neck segment) of the g l o m e r u l i . - I t i s of i n t e r e s t t h a t i n M. sc o r p i u s the i n c r e a s e i n s i z e of the i n t r a c a p s u l a r space i s i n v a r i a b l y a s s o c i a t e d w i t h marked c o n s t r i c t i o n of the neck segment. Thus, i f t h i s a l s o occurred i n the s t i c k l e b a c k , p r o l a c t i n might f u n c t i o n by 'opening' the neck segment d i r e c t l y or i n d i r e c t l y . In g e n e r a l , the data appear to favour the a l t e r n a t e hypothesis s i n c e p r o l a c t i n d i d not in c r e a s e the t u f t s i z e except a f t e r one day i n f r e s h water, when compared w i t h the-s o l v e n t - i n j e c t e d c o n t r o l s (Table 5. I I ; 0, 4, 7 days) although i t reduced the i n t r a c a p s u l a r space ( F i g s . 5.4 and 5.5). This i s p a r t i c u l a r l y c l e a r i n the 7-day data; p r o l a c t i n - i n j e c t e d f i s h showed s i g n i f i c a n t l y s maller diameters of the Bowman's capsules than the s o l v e n t - i n j e c t e d c o n t r o l s , but the glomerular t u f t diameters d i d not d i f f e r s i g n i f i c a n t l y between the two groups. The seawater data are a l s o more c o n s i s t e n t w i t h t h i s hypothesis i f we assume tha t the r e d u c t i o n of the t u f t s i z e i s due to other hormone(s) as suggested e a r l i e r . On the other hand, the one-day data favour the f i r s t hypothesis s i n c e , i n t h i s case, p r o l a c t i n i n c r e a s e d s i g n i f i c a n t l y the t u f t s i z e without s i g n i f i c a n t l y a f f e c t i n g the s i z e of the Bowman's capsules, when compared w i t h the s o l v e n t - i n j e c t e d c o n t r o l s , perhaps, i n f r e s h water, p r o l a c t i n e x e r t s o n l y a 68 t r a n s i e n t i n c r e a s e i n r e n a l blood f l o w {as i n the cat (Lockett, 1965JJ; t h i s a c t i o n then leads to the 'opening' of the neck segment. No matter what the mechanism may be, i t may be concluded t h a t p r o l a c t i n i n c r e a s e d the glomerular f i l t r a t i o n r a t e i n the s t i c k l e b a c k . Seasonal p r o l a c t i n s e c r e t i o n . Ogawa (1968) has r e p o r t e d seasonal d i f f e r e n c e s i n glomerular h i s t o l o g y of marine s t i c k l e b a c k s (G. aculeatus, form trachurus) i n l a t e autumn and l a t e s p r i n g f o l l o w i n g t r a n s f e r from sea water to f r e s h water. The hypothesis presented here i s tha t the seasonal d i f f e r e n c e s are due to s e c r e t i o n of p r o l a c t i n or inc r e a s e d p r o l a c t i n s e c r e t i o n d u r i n g the l a t e s p r i n g . I f t h i s hypothesis i s c o r r e c t , p r o l a c t i n i n j e c t i o n s i n the late-autumn or e a r l y - w i n t e r s t i c k l e b a c k s should b r i n g about glomerular changes s i m i l a r to those found by Ogawa i n the l a t e - s p r i n g s t i c k l e b a c k s . The f i n d i n g s support the.theory. In sea water, the p r o l a c t i n - i n j e c t e d s t i c k l e b a c k s i n l a t e autumn or. e a r l y winter possessed s i g n i f i c a n t l y s maller g l o m e r u l i than e i t h e r the s o l v e n t - i n j e c t e d or the u n i n j e c t e d c o n t r o l s . S i m i l a r l y , i n Ogawa's work, the l a t e - s p r i n g f i s h (uninjected) had s i g n i f i c a n t l y smaller g l o m e r u l i than the late-autumn f i s h ( u n i n f e c t e d ) , although, i n t h i s case, the d i f f e r e n c e may. be because the l a t e - s p r i n g f i s h were smaller i n s i z e (smaller mean standard body length) than the late-autumn f i s h . Ogawa (1968) found th a t a f t e r t r a n s f e r to f r e s h water, the l a t e - s p r i n g (uninjected) f i s h showed the f o l l o w i n g 69 glomerular changes when compared w i t h the late-autumn f i s h ( u n i n j e c t e d ) : (1) Greater percentage i n c r e a s e s i n glomerular s i z e from the i n i t i a l seawater c o n d i t i o n . (2) Greater percentage i n c r e a s e s i n s i z e of the glomerular t u f t than of the Bowman's capsule w h i l e t h i s d i f f e r e n t i a l i n c r e a s e was not evident or even rev e r s e d i n d i r e c t i o n apparently (capsule > t u f t ) i n the late-autumn f i s h . (This' i s not d i s c u s s e d by Ogawa, but i s evident from the data.) (3) No apparent evidence of swollen ,'Bowman' s capsules w i t h expanded i n t r a c a p s u l a r space which were observed i n the late-autumn f i s h . P r o l a c t i n i n j e c t i o n s i n t h i s study induced s i m i l a r changes i n the late-autumn or e a r l y - w i n t e r s t i c k l e b a c k s . The evidence suggests t h a t the glomerular changes i n the l a t e - s p r i n g s t i c k l e b a c k s d e s c r i b e d by Ogawa are brought about by p r o l a c t i n , s e c r e t i o n or in c r e a s e d p r o l a c t i n s e c r e t i o n i n the s p r i n g ; t h i s confirms previous evidence t h a t the seasonal d i f f e r e n c e i n kidney f u n c t i o n (as i n d i c a t e d by u r i n e o s m o l a l i t y ) i s due to seasonal p r o l a c t i n s e c r e t i o n (Chapter 2 ) . There appear, however, some q u a n t i t a t i v e d i f f e r e n c e s between the c o n t r o l s i n the two s t u d i e s (the s o l v e n t - i n j e c t e d late-autumn or e a r l y - w i n t e r s t i c k l e b a c k s i n this- study and the u n i n j e c t e d late-autumn s t i c k l e b a c k s , of Ogawa,'s st u d y ) . The d i f f e r e n c e s are u n l i k e l y to be due to so l v e n t i n j e c t i o n s , s i n c e the i n j e c t i o n s d i d not produce s i g n i f i c a n t e f f e c t s i n sea water i n t h i s study and i n both sea water and f r e s h water 70 i n e a r l i e r s t u d i e s (Chapters 3 and 4). The d i f f e r e n c e s may, however, be due to the d i f f e r e n t experimental c o n d i t i o n s used i n the two s t u d i e s e s p e c i a l l y w i t h regards to temperature, and photoperiod.' Renal mechanism of a c t i o n of p r o l a c t i n With the. data on the e f f e c t of p r o l a c t i n on u r i n e flow, the u r i h e data of Chapter 4 may be f u r t h e r i n t e r p r e t e d . The two s e t s of data may be c o r r e l a t e d as shown i n Table 5. I I I . The u r i n e i o n l e v e l s used are the means of values of 1, 4, 12 and 24 hr i n f r e s h water (chapter 4 ) . This i n d i r e c t method was o n l y used because u r i n e i o n l e v e l s were not determined i n the u r i n e - f l o w study; some of the u r i n e samples were contaminated when they were i n a d v e r t e n t l y sucked s l i g h t l y beyond the c a l i b r a t e d PE 100 tubing i n t o the improperly-cleaned but dry medicine dropper during the sampling of u r i n e volume. I f the above e x t r a p o l a t i o n s of data are c o r r e c t , p r o l a c t i n apparently i n c r e a s e d r e n a l l o s s of i o n s ; the i n c r e a s e i n the case of sodium-and c h l o r i d e may not be s i g n i f i c a n t i n view of the v a r i a b i l i t y of the data. Thus, although p r o l a c t i n reduced u r i n e sodium and c h l o r i d e l e v e l s (Chapter 4), i t d i d not appear to s i g n i f i c a n t l y a f f e c t r e n a l l o s s of the ions but i n c r e a s e d r e n a l water l o s s . S i m i l a r r e s u l t s have been shown in. hypophysectomized Fundulus kansae maintained i n f r e s h water; p r o l a c t i n increases u r i n e f l o w without a f f e c t i n g r e n a l sodium l o s s although i t reduces u r i n e sodium l e v e l (Stanley and Fleming, 1967). 71 Table 5. I l l P r o l a c t i n and r e n a l e x c r e t i o n of sodium, potassium and c h l o r i d e i n f r e s h water. The u r i n e l e v e l s are the means of values of 1, 4, 12 and 24 hr i n f r e s h water (Chapter 4 ) . Ion Treatment Urine l e v e l Urine volume Renal e x c r e t i o n meq/1 .pl/gm/24 hr ueq/gm/24 hr Na Solvent P r o l a c t i n 55. 0 35.3 31.8+1.7 59.0+8.0 1.75 2.08 K Solvent P r o l a c t i n 1.51 1.64 31.811.7 59.0+8.0 0.05 0.10 C l Solvent P r o l a c t i n 46.5 34.8 31.8+1.7 59.0+8.0 1.48 2.05 72 S i n c e p r o l a c t i n a l s o i n c r e a s e d p l a s m a s o d i u m a n d c h l o r i d e l e v e l s ( C h a p t e r 4) a n d may i n c r e a s e g l o m e r u l a r f i l t r a t i o n r a t e (GFR) a s s u g g e s t e d e a r l i e r i n t h e C h a p t e r , t h e amount o f s o d i u m a n d c h l o r i d e f i l t e r e d i n t o t h e r e n a l t u b u l e s i n p r o l a c t i n - i n j e c t e d f i s h w o u l d b e much g r e a t e r t h a n i n t h e c o n t r o l s . To e x c r e t e t h e same amount o r e v e n s l i g h t l y g r e a t e r amount o f s o d i u m a n d c h l o r i d e t h a n t h e c o n t r o l s w o u l d t h e r e f o r e mean t h a t p r o l a c t i n i n c r e a s e d t u b u l a r r e a b s o r p t i o n o f s o d i u m a n d c h l o r i d e , a c o n c l u s i o n a l s o s u g g e s t e d b y t h e p r o l a c t i n e f f e c t o n U/P r a t i o s (See C h a p t e r 4 ) . T h i s p r o l a c t i n a c t i o n m i g h t o c c u r i n a n y o n e , two o r a l l t h r e e o f t h e f o l l o w i n g r e g i o n s o f t h e u r i n a r y s y s t e m s i m u l t a n e o u s l y : (1) C o l l e c t i n g t u b u l e s a n d d u c t s . S i n c e t h i s r e g i o n may b e r e l a t i v e l y i m p e r m e a b l e t o w a t e r ( H i c k m a n a n d Trump, 1 9 6 9 ) , p r o l a c t i n w o u l d i n c r e a s e i o n r e a b s o r p t i o n w i t h r e l a t i v e l y l i t t l e o s m o t i c a c c o m p a n i m e n t o f w a t e r . I n t h i s c a s e , p r o l a c t i n m i g h t o r m i g h t n o t r e d u c e w a t e r r e a b s o r p t i o n f u r t h e r . (2) p r o x i m a l t u b u l e s . T h i s r e g i o n i s , r e l a t i v e l y p e r m e a b l e t o w a t e r ( H i c k m a n a n d Trump, 1 9 6 9 ) . P r o l a c t i n m u s t t h e r e f o r e i n c r e a s e i o n r e a b s o r p t i o n , a n d c o n c o m i t t a n t l y r e d u c e w a t e r r e a b s o r p t i o n ( e i t h e r h e r e o r e l s e w h e r e ) i n o r d e r t h a t i t r e d u c e d u r i n e i o n l e v e l s ( C h a p t e r 4 ) . (3) U r i n a r y b l a d d e r . I n t h e e u r y h a l i n e E u r o p e a n f l o u n d e r , P l a t i c h t h y s f l e s u s , . t h e u r i n e c o n c e n t r a t i o n o f s o d i u m , a n d p o t a s s i u m , a n d t h e u r i n e o s m o l a l i t y , a r e s i g n i f i c a n t l y r e d u c e d i n t h e u r i n a r y b l a d d e r ( L a h l o u , 1 9 6 7 ) . Thus p r o l a c t i n m i g h t a l s o i n c r e a s e i o n r e a b s o r p t i o n i n t h i s r e g i o n w h i c h may a l s o b e r e l a t i v e l y i m p e r m e a b l e t o w a t e r , l i k e t h e a m p h i b i a n b l a d d e r . 73 I t may be p o i n t e d out t h a t s t i c k l e b a c k s l a c k d i s t a l tubules (Ogawa, 1968). I t i s of i n t e r e s t t h a t O l i v e r e a u and Lamoine (1968) have found h i s t o l o g i c a l changes i n the r e n a l tubules of the e e l , A n g u i l l a a n g u i l l a , f o l l o w i n g p r o l a c t i n treatment, p r o l a c t i n apparently i n c r e a s e s the a c t i v i t y of the tubule c e l l s (based on h i s t o l o g i c a l c r i t e r i a ) , p a r t i c u l a r l y that of the c o l l e c t i n g tubules and ducts. The above d i s c u s s i o n argues f o r a dual r e n a l r o l e of p r o l a c t i n : (1) i t i n c r e a s e s GFR, hence u r i n e flow, and (2) i t i n c r e a s e s t u b u l a r r e a b s o r p t i o n of sodium and c h l o r i d e ; t h i s may or may not be accompanied by a r e d u c t i o n i n water r e a b s o r p t i o n as discussed above. The net r e s u l t of these two a c t i o n s i s that p r o l a c t i n i n c r e a s e s water e x c r e t i o n without a f f e c t i n g e x c r e t i o n of sodium and c h l o r i d e . I t i s evident, however, th a t p r o l a c t i n i n c r e a s e d r e n a l potassium e x c r e t i o n . This i n c r e a s e may be due e n t i r e l y to. the i n c r e a s e i n GFR and u r i n e f l o w s i n c e p r o l a c t i n d i d not s i g n i f i c a n t l y a f f e c t u r i n e potassium l e v e l s (Chapter 4 ) . That p r o l a c t i n may a l s o reduce t u b u l a r potassium r e a b s o r p t i o n i s a p o s s i b i l i t y t h a t cannot be r u l e d out without f u r t h e r evidence (Chapter 4 ) . In c o n t r a s t , p r o l a c t i n has been shown to reduce r e n a l e x c r e t i o n of sodium but has no e f f e c t on u r i n e f l o w i n i n t a c t Fundulus kansae maintained i n f r e s h water (Stanley and Fleming, 1967). S i m i l a r r e s u l t s have a l s o been shown i n i n t a c t Salmo  g a i r d n e r i i n f r e s h water; p r o l a c t i n a l s o reduces r e n a l c h l o r i d e 74 e x c r e t i o n i n t h i s f i s h . The d i f f e r e n c e between i n t a c t F:. kansae and S_. g a i r d n e r i on the one hand, and i n t a c t s t i c k l e -backs on the other, may be t h a t the former f i s h are capable of p r o l a c t i n s e c r e t i o n w h i l s t . s t i c k l e b a c k s ( i n l a t e autumn or e a r l y winter) apparently s e c r e t e l i t t l e or no p r o l a c t i n ( " p h y s i o l o g i c a l l y hypophysectomized"). I t i s i n t e r e s t i n g v t h a t i n hypophysectomized F. kansae, as mentioned before, p r o l a c t i n produces r e s u l t s s i m i l a r to those found i n s t i c k l e b a c k s . As a p o s s i b l e e x p l a n a t i o n of the above d i f f e r e n c e i n p r o l a c t i n a c t i o n ( s ) between i n t a c t and hypophysectomized ( s u r g i c a l or " p h y s i o l o g i c a l " ) f i s h , i t may be supposed t h a t i n i n t a c t f i s h the glomerular e f f e c t of p r o l a c t i n i s somehow suppressed w h i l e the t u b u l a r e f f e c t of p r o l a c t i n remains u n i n h i b i t e d . Thus u r i n e f l o w would be u n a f f e c t e d while r e n a l sodium and c h l o r i d e l o s s was reduced. Analogy may be drawn here to the e f f e c t of p r o l a c t i n on mammalian kidney. P r o l a c t i n i n c r e a s e s GFR i n i s o l a t e d cat kidneys perfused w i t h blood from headless donors. (Lockett, 1965) but. not i n i n t a c t , rats.. (Lockett and N a i l , 1965). L o c k e t t (1965) a t t r i b u t e d t h i s f a i l u r e of response i n i n t a c t r a t s to "the r e f r a c t o r i n e s s of the r e n a l v a s c u l a r bed which develops on repeated exposure to l a c t o g e n i c hormone" (= p r o l a c t i n ) . This might s i m i l a r l y be the case: i n i n t a c t P . kansae and S_. g a i r d n e r i . A l t e r n a t e l y , the e x p l a n a t i o n may be t h a t the glomerular, e f f e c t of. p r o l a c t i n i s an a l l - o r - n o n e phenomenon whi l e the t u b u l a r e f f e c t of p r o l a c t i n i s not; yet other explanations are e q u a l l y p o s s i b l e (Stanley and Fleming, 1967). 75 CHAPTER 6 P r o l a c t i n and the Head Region ( g i l l s ) I n t r o d u c t i o n In Chapter 4, p r o l a c t i n has been .shown to prevent or a t l e a s t reduce the r a p i d f a l l of plasma sodium, and c h l o r i d e l e v e l s i n autumn or e a r l y - w i n t e r s t i c k l e b a c k s f o l l o w i n g t r a n s f e r from sea water to f r e s h water. Since p r o l a c t i n d i d not apparently a f f e c t or might even s l i g h t l y i n c r e a s e r e n a l l o s s of the ions (chapter 5), the p r o l a c t i n e f f e c t on plasma sodium and c h l o r i d e l e v e l s must be due to e x t r a r e n a l mechanism(s) of i o n cons e r v a t i o n and/or due to r e d u c t i o n of hemodilution of the i o n s ; (hemodilution i s used here and subsequently to mean d i l u t i o n of the ions due to the i n c r e a s e i n blood volume a f t e r osmotic i n f l u x of water) . In hypophysectomized F_. h e t e r o c l i t u s , A. a n g u i l l a and P_. l a t i p i n n a , p r o l a c t i n reduces sodium o u t f l u x , a pparently l a r g e l y e x t r a r e n a l (Potts and Evans, 1966; Maetz, e t a l . , 1967 a, b; Ensor and B a l l , 1968). The r o l e of p r o l a c t i n on the l o s s of sodium and c h l o r i d e v i a the head r e g i o n of s t i c k l e b a c k s was t h e r e f o r e i n v e s t i g a t e d . Since i n v i t r o techniques have been s u c c e s s f u l l y used i n the study of net water i n f l u x or o u t f l u x of g i l l s by weighing (Bellamy, 1961; Kamiya, 1967), the e f f e c t o f p r o l a c t i n on i n v i t r o net water i n f l u x of s t i c k l e b a c k g i l l s was a l s o i n v e s t i g a t e d . Lam (1968) has demonstrated l o s s of C " ^ - i n u l i n (at l e a s t 14 14 C from the i n j e c t e d C - i n u l i n ) v i a the head r e g i o n of s t i c k l e b a c k s . I t was t h e r e f o r e o f i n t e r e s t to i n v e s t i g a t e the 76 e f f e c t of p r o l a c t i n on t h i s l o s s of C - i n u l i n . .Although the relevance of t h i s i n v e s t i g a t i o n to the problems of t h i s t h e s i s may not be immediately apparent,the data from t h i s i n v e s t i g a t i o n were used i n the c a l c u l a t i o n of d r i n k i n g r a t e s (See Chapter 8 ) . M a t e r i a l s and Methods Late-autumn or e a r l y - w i n t e r s t i c k l e b a c k s were used. 14 14 Net l o s s o f ,,C (from, inj.ected C - i n u l i n ) , sodium and, c h l o r i d e y j j j head r e g i o n . W h i l s t i n sea water, s t i c k l e b a c k s ( fv 2 gm) were i n j e c t e d w i t h p r o l a c t i n or the s o l v e n t on a l t e r n a t e days. Twenty-four hr a f t e r the t h i r d i n j e c t i o n , the f i s h were s t u d i e d using the technique d e s c r i b e d i n the previous chapter f o r u r i n e c o l l e c t i o n . In t h i s case, however, the water i n the head chamber (See F i g . 5.1) was sampled a t p e r i o d i c i n t e r v a l s . The use of p a r a f f i n o i l , besides the diaphragms, ensured better* i s o l a t i o n of u r i n e from the water i n the head chamber than h i t h e r t o achieved w i t h diaphragms alone (Wikgren, 1953; B e n t l e y and F o l l e t t , 1963; post, e t a l . , 1965). F u r t h e r -more the p a r a f f i n o i l was kept a t a higher l e v e l than the water l e v e l ( F i g . 5.1), so t h a t the pressure on the p a r a f f i n s i d e of the diaphragm was s l i g h t l y greater than t h a t on the water side;- any tendancy of water to leak from the head r e g i o n was probably prevented by the net pressure of the o i l . U r i n e , however, might t r a v e l along the underside of the f i s h from the u r i n o g e n i t a l opening i n t o the head chamber. As 77 mentioned b e f o r e , t h i s was minimized, i f not e l i m i n a t e d , by the double-membrane diaphragm. As an a d d i t i o n a l p r e c a u t i o n , the anus was cannulated; the cannula helped to l o c a l i z e u r i n e c o l l e c t e d a t the u r i n o g e n i t a l opening j u s t p o s t e r i o r to the anus. In f a c t , i n many cases, anus can n u l a t i o n appeared to p a r t i a l l y block, u r i n e r e l e a s e i n s t i c k l e b a c k s , and thus f u r t h e r helped to prevent Urine contamination of the'head chamber. Anus c a n n u l a t i o n i s i l l u s t r a t e d i n the i n s e r t of P i g . 5.1 (15, 16); gut f l u i d s were c o l l e c t e d ' b y the arrangement as shown (16, 17, 18, 19), although the gUt f l u i d s never reached the hematocrit tube (17), but f i l l e d o n l y a sh o r t l e n g t h of the PE 10 polye t h y l e n e cannula (16). 14 C l o s s . Fxsh were a l s o i n j e c t e d w i t h 0.025 ml of 14 C - i n u l i n m 0.6% N a d (0.5 uc per f i s h ) , 5 hr a f t e r the l a s t ( t h i r d ) i n j e c t i o n of p r o l a c t i n or the s o l v e n t . Before being put i n the apparatus ( F i g . 5.1), each f i s h was p l a c e d i n running d e c h l o r i n a t e d f r e s h water (same temperature) 14 f o r about 5 minutes, to wash C - i n u l i n o f f the e x t e r n a l , s u r f a c e , and then weighed i n f r e s h water. The water surrounding the head of the f i s h i n the apparatus was sampled i n i t i a l l y and a f t e r 2, 4 and 24 hours. The volumes sampled and the f i n a l remaining volume were determined by weighing. The sample (30 p i volume) was counted on a Nuclear Chicago l i q u i d s c i n t i l l a t i o n counter (Mark 1 Model 6860) us i n g the s o l v e n t system of Bray (1960). The t o t a l a c t i v i t y (counts per min) i n the medium a t each time i n t e r v a l was c a l c u l a t e d w i t h c o r r e c t i o n s made f o r the volume and 78 a c t i v i t y o f the sample or samples removed, and f o r the background a c t i v i t y . A t the end of the experiment, the f i s h was removed from the apparatus, the t a i l r e g i o n wiped clean of p a r a f f i n o i l w i t h .Kleenex f a c i a l t i s s u e s , and blood c o l l e c t e d by severance of the caudal peduncle. One u l of plasma d i l u t e d i n 3 0 u l d i s t i l l e d water was then counted as above. Ion l o s s . F i s h were weighed ( i n f r e s h water) before being p l a c e d i n the apparatus. The water i n the head chamber was sampled i n i t i a l l y and a f t e r 24 hours. The volumes sampled and the f i n a l remaining volume were determined by weighing. Sodium and c h l o r i d e l e v e l s were determined on 50 u l samples d i l u t e d i n 3 ml d i s t i l l e d water (for sodium) or i n 50 u l of 50% a c e t i c a c i d ( f o r c h l o r i d e ) . D e t a i l s of q u a n t i t a t i v e determination of the ions are given i n Chapter 4. The t o t a l amounts of the ions i n the medium i n i t i a l l y and a f t e r 24 hr were c a l c u l a t e d w i t h c o r r e c t i o n s made f o r the volume and the amount of the ions of the sample or samples removed. The i n i t i a l amount of the. i o n s s u b t r a c t e d from the f i n a l (24 hr) amount gave net l o s s of the i o n s . Jn_yJLtro weight i n c r e a s e of g i l l s The s t i c k l e b a c k (~2 gm) was k i l l e d by t r a n s e c t i o n of the s p i n a l cord i n the neck r e g i o n and immediately d i s s e c t e d to expose the heart and the g i l l s v e n t r a l l y . w i t h the exception of the v e n t r a l a o r t a , the blood v e s s e l s ( i n c l u d i n g the .dorsal aorta) i n the r e g i o n of the heart were cut, and blood which flowed out was mopped up by cotton wool. Leakage 79 of blood, however, soon subsided or ceased completely, probably as a r e s u l t of formation of c l o t s a t the cut ends of the v e s s e l s . Ovine p r o l a c t i n was i n j e c t e d v i a the v e n t r i c l e and v e n t r a l a o r t a i n t o the g i l l s i n a dose of 10 ug d i s s o l v e d i n 0.02 ml of 0.6% NaCl 5 ug/gm body weight). The c o n t r o l s were i n j e c t e d . s i m i l a r l y w i t h 0.02 ml of 0.6% NaCl. Immediately, •after the i n j e c t i o n , the e n t i r e g i l l arches w i t h the heart ( s t i l l beating) attached ( h e a r t - g i l l preparation) were removed i n t a c t and placed i n a mod i f i e d marine Ringer s o l u t i o n of composition shown i n Table 6 . 1 . The h e a r t - g i l l p r e p a r a t i o n was l e f t i n the Ringer s o l u t i o n f o r 3 0 min to a l l o w p r o l a c t i n to e q u i l i b r a t e throughout the g i l l s and a l s o to a l l o w f o r any p o s s i b l e l a g p e r i o d i n p r o l a c t i n a c t i o n ; p r e l i m i n a r y experiments showed t h a t , without t h i s step, r e s u l t s were more v a r i a b l e among the i n d i v i d u a l g i l l arches of the same p r o l a c t i n - i n j e c t e d animal, which may be a t t r i b u t e d to d i f f e r e n t i a l amounts of p r o l a c t i n i n the g i l l arches due to inadequate e q u i l i b r a t i o n . During t h i s p e r i o d i n . the. Ringer,,, the hearts i n some pre p a r a t i o n s may cease to beat; these were d i s c a r d e d i n order to ensure u n i f o r m i t y of the p r e p a r a t i o n s . At the end of the p e r i o d , i n d i v i d u a l g i l l s were, removed by c u t t i n g through each g i l l bar a t two p o i n t s , v e n t r a l l y and d o r s a l l y where the g i l l s f i l a m e n t s ended. As each g i l l was removed i t was placed on cotton wool soaked i n Ringer. . Surface blood c l o t s were removed from the g i l l s by drawing them across wet cotton wool. Table 6. I Composition of Ringer s o l u t i o n ; adapted and m o d i f i e d from House and Green (1965). rtiM/1 gm/1 NaCl 170.0 9.937 KC1 6.0 0.447 C a c l 2 1.6 0.596 MgCl 2 1.0 0.203 NaHC0 3 2.3 0.193 KH 2P0 4 0.5 0.068 Glucose 2.8 0.504 81 I s o l a t e d g i l l s were then incubated i n d i v i d u a l l y i n 100 ml of d e c h l o r i n a t e d f r e s h water a t room temperature (20 t l °c ) contained i n g l a s s bowls. Oxygen was c o n t i n u o u s l y bubbled through the medium by means of PE 50 tubings (See F i g . 5.1, 10, 11, 12). Each g i l l was weighed on a M e t t l e r (Type H5, Cap. 160 g, No 543 01) balance ( s e n s i t i v i t y : 1 mg) before and a f t e r i n c u b a t i o n f o r 15, 30 and 60 minutes. Before weighing, the su r p l u s water on the surface of the g i l l was drained o f f by r a p i d l y p l a c i n g the g i l l on a dry Kleenex f a c i a l t i s s u e , f i r s t on one s i d e and then on the other using a p a i r of f o r c e p s . A f t e r weighing the g i l l was r e p l a c e d i n the medium f o r f u r t h e r i n c u b a t i o n . A t the end of the experiment, each g i l l was d r i e d i n an oven a t 110°c f o r 24 hours and then weighed (dry weight) on a Cenco (cap. 160 g, No 34359) balance ( s e n s i t i v i t y : 0.1 mg). The experiment was repeated f i v e times. The t o t a l number of g i l l arches used i n the p r o l a c t i n - i n j e c t e d - o r the. s o l v e n t - i n j e c t e d group was 40 (5 f i s h x 8 g i l l a r c h e s ) . R e s u l t s Net i o n l o s s v i a head r e g i o n • I t i s evident from Table 6. I I th a t p r o l a c t i n reduced the net l o s s of sodium and c h l o r i d e v i a the head r e g i o n of s t i c k l e b a c k s i n f r e s h water (P< 0.05; t - t e s t ) . There appears to be s l i g h t l y g r e ater l o s s of c h l o r i d e than sodium. The 82 Table 6. I I p r o l a c t i n and net i o n l o s s v i a the head r e g i o n of s t i c k l e b a c k s ( i n autumn or e a r l y w i n t e r ) ; f i s h were s t u d i e d i n the apparatus de s c r i b e d i n Chapter 5 w i t h d e c h l o r i n a t e d f r e s h water i n the head chambers. * p<0.05 ( t - t e s t ) Ions Mean net i o n l o s s t Sx (number of f i s h ) ( peg/gm/24 hr) Solvent ( c o n t r o l ) P r o l a c t i n Na 20„9 + 4.1 (8) 11.1 t 2.7* (8) C l 22.2 I 1.2 (8) 14.3 t 0.7 (8) 83 d i f f e r e n c e may be due to l o s s of potassium which would s e t up an e l e c t r o p o t e n t i a l g r a d i e n t causing f u r t h e r l o s s of c h l o r i d e . 14 14 Net C (G - i n u l i n or i t s breakdown products) l o s s vjLa.  head r e g i o n 14 F i g . 6.1 shows the e f f e c t of p r o l a c t i n on net l o s s of C. v i a the head r e g i o n of s t i c k l e b a c k s i n f r e s h water, values (% 1-ul-plasma a c t i v i t y per gm body weight) given are means.of. - 14 12 f i s h ± standard e r r o r (Sx) . Since the amount of >C present. i n the medium a t any one time would a f f e c t subsequent l o s s of 14 C , the r e s u l t s a t the d i f f e r e n t time i n t e r v a l s of e i t h e r of the two groups o f f i s h were not independent and a n a l y s i s o f v a r i a n c e o f the r e s u l t s cannot be performed. Comparison between the two groups of f i s h a t each i n t e r v a l was made by t - t e s t . The p r o l a c t i n - i n j e c t e d f i s h showed a s i g n i f i c a n t l y 14 sma l l e r net l o s s of C than the s o l v e n t - i n j e c t e d f i s h a t 2, 4-and 24 hr (P<0.G5). I t appears t h e r e f o r e t h a t p r o l a c t i n . 14 i n j e c t i o n s had reduced the net l o s s of C v i a the head r e g i o n ( i n f r e s h water) of s t i c k l e b a c k s . 14 14 The C a c t i v i t y was probably C - i n u l i n s i n c e t h i n - l a y e r chromatography showed t h a t the a c t i v i t y was n e i t h e r f r u c t o s e nor what was represented" by a c o n c e n t r a t e d - s u l p h u r i c - a c i d 14 14 d i g e s t of C. - i n u l i n , but appeared to be C - i n u l i n (Lam, 1968) . I t was noted that the t e r m i n a l plasma a c t i v i t y i n these experiments and some p r e l i m i n a r y experiments was f r e q u e n t l y lower i n p r o l a c t i n - i n j e c t e d f i s h than i n s o l v e n t - i n j e c t e d f i s h 84 F i g . 6.1. P r o l a c t i n and net l o s s of C (C - i n u l i n or i t s breakdown products) v i a the head r e g i o n ( i n f r e s h water) of s t i c k l e b a c k s . Each p o i n t represents mean of 12 f i s h I standard e r r o r . 85 although the two-groups of f i s h were- i n j e c t e d w i t h approximately 14 the same amount of C - i n u l i n p r i o r to the experiments. The means of the data ± standard e r r o r are given i n Table 6. I I I . Using t - t e s t , the p r o l a c t i n values were s i g n i f i c a n t l y lower than the s o l v e n t values (P < 0.05). Weight i n c r e a s e of i s o l a t e d g i l l s i n f r e s h water The. mean data (gm wt i n c r e a s e per gm dry wt of g i l ' l ) ± 9 5 % confidence i n t e r v a l (Sx X t.05) are presented i n F i g . 6.2. I t i s evident t h a t p r o l a c t i n s i g n i f i c a n t l y reduced the weight i n c r e a s e of i s o l a t e d g i l l s incubated i n f r e s h water. -Comparison between the two groups of f i s h a t each time i n t e r v a l by t - t e s t ( a n a l y s i s of v a r i a n c e cannot be performed f o r s i m i l a r reasons as above) showed a l e v e l of s i g n i f i c a n c e of a t l e a s t P <0.0'1. I t may seem t h a t a dose of 10 ug of p r o l a c t i n (5 ug per gm body weight) was r a t h e r high f o r an i n t r a v a s c u l a r i n j e c t i o n . However, the experimental procedures e n t a i l e d some, l o s s of the i n j e c t e d p r o l a c t i n s i n c e i t may leak out of cut ends of blood v e s s e l s before they were completely s e a l e d by b l o o d c l o t s . A smaller dose of 5 ug was a l s o e f f e c t i v e but apparently to a l e s s e r degree. Furthermore Sage (1968) has shown th a t p i t u i t a r i e s of Xiphophorus (comparable i f not s m a l l e r i n s i z e than s t i c k l e b a c k s ) may r e l e a s e approximately 20 ug/gm/24 hr of p r o l a c t i n when c u l t u r e d on d i l u t e media. D i s c u s s i o n Evidence showed t h a t p r o l a c t i n reduced the net l o s s of sodium, c h l o r i d e and C 1 4 - i n u l i n (at l e a s t C 1 4 from i n j e c t e d Table 6. I l l p r o l a c t i n and plasma C a c t i v i t y of s t i c k l e b a c k s w i t h the head r e g i o n i n f r e s h water (* 0.05); CPM = counts per min. 14 Plasma C a c t i v i t y per u l i n CPM {means ± Sx (number of fish}] Solvent p r o l a c t i n 4031 - 501 (16) 2736 t 454* ( i 6) 87 F i g . 6. 2. p r o l a c t i n and weight i n c r e a s e of i s o l a t e d g i l l s of s t i c k l e b a c k s a f t e r d i f f e r e n t periods of i n c u b a t i o n i n f r e s h water. Each p o i n t represents mean of 40 g i l l arches +9 5 % confidence i n t e r v a l . 0 15 3 0 4 5 6 0 MIN. 88 C - i n u l i n ) v i a the head r e g i o n ( i n f r e s h water) of s t i c k l e b a c k s . To produce t h i s e f f e c t , p r o l a c t i n may: (1) reduce the a c t u a l . l o s s of the s o l u t e s , (2) inc r e a s e the uptake (active) of the solutes," and (3) inc r e a s e d r i n k i n g . The l a s t p o s s i b i l i t y may be e l i m i n a t e d s i n c e l a t e r evidence (Chapter .8) suggested th a t p r o l a c t i n may decrease d r i n k i n g r a t h e r than in c r e a s e i t . The data, however, do not permit any co n c l u s i o n regarding the f i r s t and second p o s s i b i l i t i e s . In hypophysectomized F_.~ h e t e r o c l i t u s and A. a n g u i l l a r i n f r e s h water, p r o l a c t i n has been shown to reduce sodium out-flux, which i s l a r g e l y e x t r a r e n a l , but not to a f f e c t sodium i n f l u x (Potts and Evans, 1966; Maetz e t a l . , 1967 a, b ) . In hypophysectomized P. l a t i p i n n a , too, p r o l a c t i n reduces sodium o u t f l u x i n f r e s h water, presumably- l a r g e l y e x t r a r e n a l (Ensor and B a l l , 1968). However, i n hypophysectomized F_. kansae, p r o l a c t i n a t higher doses seems to s t i m u l a t e the a c t i v e uptake of sodium i n f r e s h water whi l e having no e f f e c t on the o u t f l u x (Fleming, and B a l l , 1967) . Energy-wise ( a l b e i t t e l e o l o g i c a l ) - , . i t seems more l i k e l y t h a t p r o l a c t i n should reduce the o u t f l u x r a t h e r than i n c r e a s e the a c t i v e i n f l u x . Furthermore i t seems 14 u n l i k e l y t h a t s t i c k l e b a c k s would a c t i v e l y absorb C - i n u l i n or i t s breakdown products. Thus i t may be t e n t a t i v e l y concluded tha t p r o l a c t i n reduced the a c t u a l l o s s of the s o l u t e s v i a the head region- ( i n f r e s h water) of s t i c k l e b a c k s . 14 14 There are s e v e r a l ways by which C - (C - i n u l i n or i t s break-down product (s.)) and the ions may have been l o s t v i a the head r e g i o n of the f i s h , and p r o l a c t i n may reduce l o s s by 89 any one or more of the ways. F i r s t l y , the s o l u t e s may have d i f f u s e d out or been r e g u r g i t a t e d from the gut. Since the gut f l u i d contained r e l a t i v e l y low a c t i v i t y (< 5% of ambient medium a c t i v i t y around the head) which may be accounted f o r by d r i n k i n g , and was con t i n u o u s l y c o l l e c t e d by means of a cannula, 14 t h i s , pathway of C l o s s may not have been s i g n i f i c a n t . A l s o , s i n c e the f i s h had been s t a r v e d f o r a t l e a s t 3 days p r i o r to the experiment, the gut may not contain s u f f i c i e n t amounts of sodium and c h l o r i d e to c o n t r i b u t e s i g n i f i c a n t l y to the l o s s of the ions by t h i s pathway. Secondly, the s o l u t e s may be l o s t as a r e s u l t of t i s s u e damage and/or hemorrage, the l a c k o f which, however, e l i m i n a t e d t h i s p o s s i b i l i t y . T h i r d l y , the s o l u t e s may have been l o s t v i a the g i l l s and/or b u c c a l c a v i t y and pharynx. S e v e r a l means of l o s s by t h i s pathway are p o s s i b l e . P i n o c y t o s i s or revers e p i n o c y t o s i s 14 may have been i n v o l v e d , a t l e a s t i n the case of C . S c o t t e t a l . , (1964) have demonstrated ab s o r p t i o n of i n u l i n . and. albumin from the proximal tubule i n Necturus and suggested involvement of p i n o c y t o s i s . D i f f u s i o n through aqueous pores of the g i l l s and/or b u c c a l c a v i t y and pharynx i s another p o s s i b l e method. Although i n u l i n i s a r e l a t i v e l y l a r g e molecule, i t i s a long-chain molecule and might be moved through aqueous pores i n a lengthwise o r i e n t a t i o n ( S o i l , 1967). A l t e r n a t e l y , the s o l u t e s may d i f f u s e through temporary holes l e f t as c e l l s , such as mucous and " c h l o r i d e c e l l s " , are 90 shed p r i o r to being r e p l a c e d . Clarkson (1967) has shown i n h i s model of the t r a n s p o r t of s a l t and water across i s o l a t e d r a t ileum t h a t the holes l e f t by extruded e p i t h e l i a l c e l l s determine the p a s s i v e p e r m e a b i l i t y of the t i s s u e ; a t the same time the e x f o l i a t i o n of e p i t h e l i a l c e l l s f u n c t i o n s as a s e l e c t i v e b a r r i e r e x c l u d i n g substances bound to them. The 14 l a t t e r may a l s o be a means of l o s s of C and the ions v i a the head r e g i o n . 14 Since death of f i s h d i d not appear to a f f e c t C - l o s s (Lam, 1968), l o s s by simple d i f f u s i o n through aqueous pores or v i a the gut seems a more p l a u s i b l e e x p l a n a t i o n than the other a l t e r n a t i v e t h e o r i e s . S i m i l a r l y , l o s s of the ions i s l i k e l y to be due to simple d i f f u s i o n although the. sit.e(s) of 14 l o s s may d i f f e r from th a t of C . The above d i s c u s s i o n suggests t h a t p r o l a c t i n may reduce 14 the. p a s s i v e o u t f l u x of sodium, c h l o r i d e and C , probably v i a the g i l l s . No matter what the a c t u a l mechanism was, the f a c t i s t h a t p r o l a c t i n reduced net l o s s of the s o l u t e s v i a the head, r e g i o n . This a c t i o n of p r o l a c t i n may account, a t l e a s t i n p a r t , f o r i t s a c t i o n i n m a i n t a i n i n g plasma sodium and c h l o r i d e l e v e l s of s t i c k l e b a c k s t r a n s f e r r e d to f r e s h water (See Chapter 4 ) . Since the e x t r a r e n a l l o s s of the ions was much greater than the r e n a l l o s s (compare Table 5. I l l and Table 6. I I ) , r e d u c t i o n of the e x t r a r e n a l l o s s would seem of great importance as an e f f i c i e n t means of conservation of the i o n s . I t i s i n t e r e s t i n g t h a t p r o l a c t i n reduced the e x t r a r e n a l l o s s r a t h e r than the r e n a l l o s s (see Chapter 5). 91 Despite the f i n d i n g t h a t p r o l a c t i n reduced .the e x t r a r e n a l 14 l o s s of C i n f r e s h water, the p r o l a c t i n - i n j e c t e d f i s h showed 14 a lower plasma C a c t i v i t y per u l than the s o l v e n t - i n j e c t e d f i s h , although the two groups of f i s h had been i n j e c t e d w i t h 14 approximately the same amount of C - i n u l i n p r i o r to the experiments. This suggests three p o s s i b i l i t i e s . F i r s t l y , p r o l a c t i n i n c r e a s e d the r e n a l clearance or at l e a s t e x c r e t i o n 14 14 of C (probably C - i n u l i n ) , much more than i t reduced the e x t r a r e n a l l o s s . Thus i t may be f u r t h e r evidence t h a t p r o l a c t i n i n c r e a s e d GFR, as suggested by the h i s t o l o g i c a l data i n Chapter 14 5. Secondly, p r o l a c t i n decreased the uptake of C - i n u l i n 14 i n t o the b l o o d from the coelom where the C - i n u l i n had been i n j e c t e d i n t r a p e r i t o n e a l l y . T h i r d l y , p r o l a c t i n i n c r e a s e d the b l o o d volume (hemodilution);•evidence, however, showed th a t the op p o s i t e was more l i k e l y to be t r u e , i . e . p r o l a c t i n decreased , hemodilution (as d i s c u s s e d e a r l i e r ) . The l a s t p o s s i b i l i t y may, t h e r e f o r e , be e l i m i n a t e d . The f i r s t p o s s i b i l i t y s e e m s . l i k e l y although the second p o s s i b i l i t y cannot be r u l e d out. The i n v i t r o g i l l experiments showed th a t p r o l a c t i n reduced the weight i n c r e a s e of i s o l a t e d g i l l s of s t i c k l e b a c k s , incubated i n f r e s h water. The weight i n c r e a s e was l i k e l y to be due to net osmotic i n f l u x of water, the g i l l s being hyperosmotic to the i n c u b a t i o n medium. Assuming t h i s to be true and t h a t the i n i t i a l osmotic g r a d i e n t was the same i n the s o l v e n t -i n j e c t e d and p r o l a c t i n - i n j e c t e d g i l l s , the evidence suggests t h a t p r o l a c t i n may reduce the net osmotic i n f l u x of water v i a the g i l l s of s t i c k l e b a c k s i n f r e s h water. 92 There are, however, two p o s s i b i l i t i e s which might a f f e c t the r e s u l t s of the experiments. F i r s t l y , the i n j e c t e d p r o l a c t i n molecules may e x e r t some osmotic e f f e c t s . In f r e s h water, the osmotic e f f e c t s of p r o l a c t i n molecules i n the g i l l s , i f any, would o n l y i n c r e a s e the osmotic g r a d i e n t and hence the osmotic i n f l u x of water; y e t , p r o l a c t i n reduced the osmotic i n f l u x of water, a t l e a s t as evidenced by the r e d u c t i o n i n weight i n c r e a s e . Thus the osmotic e f f e c t s of p r o l a c t i n molecules would e i t h e r be non-existent o r , i f present, o n l y reduce the hormonal e f f e c t s . Furthermore, i n a few s i m i l a r experiments, i n j e c t i o n s of a s i m i l a r dose of p u r i f i e d bovine growth hormone (donated by N a t i o n a l I n s t i t u t e s of Hea l t h , Bethesda, Maryland, U.S.A; NIH-GH-B6), a p r o t e i n hormone s t r u c t u r a l l y r a t h e r s i m i l a r to p r o l a c t i n , had not produced the above p r o l a c t i n e f f e c t s b u t appeared to enhance the weight i n c r e a s e o f the g i l l s i n s t e a d , although t h i s l a t t e r p o i n t needs to be confirmed. Secondly, the osmotic g r a d i e n t i n the two groups of g i l l s , might have. been, a l t e r e d , d i f f e r e n t l y d u r i n g the half-hour p e r i o d i n the Ringer. The blood i n the g i l l might have been s l i g h t l y d i l u t e d by the s o l v e n t (0.6% NaCl) or p r o l a c t i n i n j e c t i o n s so tha t i t would be s l i g h t l y hyposmotic to the Ringer. I f so, the f l u x e s of ions and water i n the Ringer might be a f f e c t e d by the osmotic and/or hormonal e f f e c t s of p r o l a c t i n . T h i s , p o s s i b i l i t y , however, might not have been s i g n i f i c a n t s i n c e p r e l i m i n a r y experiments c a r r i e d out without t h i s i n c u b a t i o n i n the Ringer, had a l s o shown a s i m i l a r p r o l a c t i n e f f e c t on the g i l l s i n f r e s h water, although, i n t h i s case, the 93 r e s u l t s were more-.variable, which may be a t t r i b u t e d to inadequate e q u i l i b r a t i o n pf p r o l a c t i n i n the g i l l s . Moreover, p r o l a c t i n has not been shown to a f f e c t sodium turn-over r a t e s of F_. h e t e r o c l i t u s i n hyperosmotic environment (sea water) (Maetz e t a l . , 1967 b; p o t t s and Evans, 1966). Thus i t may be concluded t h a t p r o l a c t i n reduced the net osmotic water i n f l u x of i s o l a t e d s t i c k l e b a c k g i l l s , incubated i n f r e s h water, not as a r e s u l t of a reduced osmotic g r a d i e n t , but as a r e s u l t of a r e d u c t i o n i n the p e r m e a b i l i t y of the g i l l s to water. Chan, e t al.(1968) have s i m i l a r l y suggested t h a t p r o l a c t i n renders the g i l l s and/or s k i n of A. a n g u i l l a l e s s permeable to water, as an e x p l a n a t i o n of t h e i r f i n d i n g t h a t p r o l a c t i n prevents the muscle hyperhydration of hypophysectomized A. a n g u i l l a i n f r e s h water. The above a c t i o n of p r o l a c t i n on osmotic water i n f l u x v i a the g i l l s , together w i t h the p r o l a c t i n a c t i o n of i n c r e a s i n g u r i n e flow, may reduce hemodilution of sodium and c h l o r i d e and thus c o n t r i b u t e , a t l e a s t i n p a r t , to the maintenance, of. the plasma sodium and c h l o r i d e l e v e l s i n s t i c k l e b a c k s t r a n s f e r r e d to f r e s h water. B a l l (1969) has argued: "... when hypophy-sectomized P_. l a t i p i n n a f a i l i n f r e s h water the 25% or greater f a l l i n plasma sodium ( l e v e l s ) occurs i n the presence o f normal potassium l e v e l s , i n d i c a t i n g t h a t hemodilution i s not a-major f a c t o r i n f a i l u r e " . Since p r o l a c t i n d i d not a f f e c t plasma potassium l e v e l s , although i t r a i s e d the plasma sodium and c h l o r i d e l e v e l s (chapter 4), the same argument may a l s o hold. On the other hand, leakage of potassium from r e d blood 94 c e l l s i n t o plasma might occur during hemodilution, and, a l s o , p r o l a c t i n may i n c r e a s e r e n a l l o s s of potassium (see Chapter 5); e i t h e r or both of these e f f e c t s would mask any e f f e c t of hemodilution on plasma potassium l e v e l s . Thus i t may be argued t h a t p r o l a c t i n d i d not a f f e c t plasma potassium l e v e l s d e s p i t e an apparent i n c r e a s e i n l o s s of potassium because of the concomittant r e d u c t i o n i n the hemodilution of the i o n which occurred' i n the c o n t r o l s . 95 CHAPTER 7 P r o l a c t i n and G i l l Mucous C e l l s I n t r o d u c t i o n The r o l e of the p i t u i t a r y gland i n the maintenance of the mucous c e l l s i n t e l e o s t s has been i n v e s t i g a t e d w i t h c o n t r o v e r s i a l r e s u l t s . The number of g i l l or s k i n mucous c e l l s was found to be reduced f o l l o w i n g hypophysectomy i n Fundulus h e t e r o c l i t u s (Burden, 1956), Betta splendens (Schreibman and Kallman, 1965) and Carassius auratus (Ogawa and Johansen, 1967). On the other hand, attempts to demonstrate an e f f e c t of hypophysectomy on mucous c e l l s of T i l a p i a  mossambica (Bern, 1967) and P o e c i l i a l a t i p i n n a ( B a l l , 1969) have so f a r been u n s u c c e s s f u l . In s e v e r a l species of i n t a c t c i c h l i d s , p r o l a c t i n has-been shown to cause p r o l i f e r a t i o n of s k i n mucous c e l l s (Egami and I s h i i , 1962; Blum and F i e d l e r , 1966). In hypophysectomized g o l d f i s h (C_. auratus) , the s k i n mucous c e l l s are maintained by e c t o p i c p i t u i t a r y t r a n s p l a n t s (Ogawa and Johansen, 1967), p o s s i b l y by f i s h p r o l a c t i n s ecreted by the t r a n s p l a n t s (cf B a l l e_t a_l., 1965) . The above works suggest an i n f l u e n c e of p r o l a c t i n on mucous c e l l s , but again t h i s e f f e c t does not appear to occur i n T_. mossambica and P. l a t i p i n n a (Bern, 1967; B a l l , 1969). In view.of these c o n f l i c t i n g data, the mucus-maintaining r o l e of p r o l a c t i n has been examined i n the s t i c k l e b a c k , which seems to be p a r t i c u l a r l y a p p r o p r i a t e f o r the study. In the f i r s t p l a c e , s u r g i c a l hypophysectomy does not appear to be 96 necessary to c o n t r o l p r o l a c t i n s e c r e t i o n . As mentioned before, the s t i c k l e b a c k seems to secrete l i t t l e or no p r o l a c t i n i n the autumn and e a r l y w i n t e r , and consequently, as f a r as p r o l a c t i n i s concerned, may be considered ' p h y s i o l o g i c a l l y hypophy-sectomized' a t t h i s time. Secondly, p r o l a c t i n has been shown to improve osmotic and i o n i c r e g u l a t i o n i n the autumn and e a r l y winter when seawater-adapted s t i c k l e b a c k s were t r a n s f e r r e d to f r e s h water (Chapters 2 and 4). Any e f f e c t of p r o l a c t i n on g i l l mucous c e l l d e n s i t y a f t e r t r a n s f e r of the f i s h from sea to f r e s h water may t h e r e f o r e be compared w i t h the osmotic and i o n i c r e g u l a t i n g e f f e c t s of the hormone to determine i f any c o r r e l a t i o n e x i s t s . M a t e r i a l s and Methods Late-autumn and e a r l y - w i n t e r s t i c k l e b a c k s were used. F i s h were maintained i n the experimental tanks (sea water) f o r 3-4 days. One group of f i s h was then i n j e c t e d w i t h p r o l a c t i n , another w i t h the s o l v e n t and a t h i r d group was l e f t i n t a c t . A f t e r three i n j e c t i o n s on a l t e r n a t e days and 24. hr a f t e r the l a s t i n j e c t i o n (6 days a f t e r the f i r s t i n j e c t i o n ) , a group each of the s o l v e n t - i n j e c t e d , p r o l a c t i n - i n j e c t e d and u n i n j e c t e d f i s h was k i l l e d f o r g i l l p r e p arations (see below); the remaining . u n i n j e c t e d f i s h were t r a n s f e r r e d to f r e s h water, w h i l s t the remaining groups of s o l v e n t - and p r o l a c t i n - i n j e c t e d f i s h were each d i v i d e d i n t o two groups: a l a r g e r group was t r a n s f e r r e d to f r e s h water, and the other remained i n sea water. I n j e c t i o n s were continued on a l t e r n a t e days i n both sea water and f r e s h 97 water. Groups of p r o l a c t i n - and s o l v e n t - i n j e c t e d f i s h were k i l l e d a f t e r 5 hr, 1, 4 and 8 days i n f r e s h water; the two groups of i n j e c t e d f i s h which remained i n sea water were k i l l e d a f t e r 8 days (14 days i n sea water a f t e r the f i r s t . i n j e c t i o n ) , and the u n i n j e c t e d f i s h were sampled a f t e r 1 and 4 days i n f r e s h water. G i l l p r e p a r a t i o n s The f i s h were k i l l e d by d e c a p i t a t i o n and the i n t a c t g i l l arches immediately d i s s e c t e d and f i x e d i n Bouin's f i x a t i v e . The i n d i v i d u a l g i l l arches from one s i d e were removed and s t a i n e d f o r 1 hr i n Gabe's (1953) aldehyde f u c h s i n (AF). A f t e r washing i n 70%, and dehydration i n 95% and 100% ethanol under standard c o n d i t i o n s , they were c l e a r e d ; i n methyl s a l i c y l a t e and mounted on s l i d e s . E s t i m a t i o n of g i l l mucous c e l l . d e n s i t y The g i l l p r e p a r a t i o n s were examined by means of a b i n o c u l a r microscope and graded according to the f o l l o w i n g c r i t e r i a : a. d e n s i t y of mucous c e l l s on the g i l l f i l a m e n t s , b. r e p r o d u c i b i l i t y of the mucous c e l l d e n s i t y from f i l a m e n t to f i l a m e n t and between g i l l s , c. i n t e n s i t y of s t a i n a b l e m a t e r i a l w i t h i n the mucous c e l l s and the i n t a c t n e s s of these c e l l s , and d. c o n t i n u i t y of the arrangement of mucous c e l l s i n p a r a l l e l d i a g onal rows across the f i l a m e n t s . Four c a t e g o r i e s of mucous c e l l d e n s i t y ( I , I I , I I I and IV) were recognised and standard p r e p a r a t i o n s s e l e c t e d (extreme cases of mucous c e l l d e n s i t y are i l l u s t r a t e d i n F i g . 7.1 a, b, c, and d. The percentage of g i l l p r e p a r a t i o n s w i t h i n each of the c a t e g o r i e s 98 F i g . 7.1 a and b G i l l f i l a m e n t s of second g i l l arch; d e n s i t y category IV (maximum c o n d i t i o n ) , AF s t a i n . Note numerous mucous c e l l s (mc) on g i l l f i l a m e n t (gf) i n s p i r a l arrangement. Note a l s o how f i l a m e n t s are f r e e of each other. F i g . 7.1 c and d G i l l f i l a m e n t of second g i l l arch; d e n s i t y category I , AF s t a i n . Note almost absence of mucous c e l l s (mc) apart from few at f i l a m e n t t i p s ( g f ) . Note a l s o how f i l a m e n t s are clumped together. Key to F i g . 7.1 f c : f i l a m e n t c a r t i l a g e , ga: g i l l a rch, gf: g i l l f i l a m e n t , 1: l a m e l l a e , mc: mucous c e l l , me: melanophore. 9'9 f o r each of the c o n t r o l and experimental groups of f i s h was determined and compared by means of histograms ( F i g . 7.2) and the Kilmogorov-Smirnov two-sample t e s t ( S i e g e l , 1956). As a mean value of the mucous c e l l d e n s i t y of each group of f i s h , a "percentage estimate of mucous c e l l d e n s i t y " was made as f o l l o w s : -(1 x n-j.) + (2.x n Z I ) + (3 x nllx) + (4 x n i y ) x 1 Q Q 4 (n-r + nlx + n l x l + n I V ) where 1, 2, 3 and 4 are the 'scores' assigned to the c a t e g o r i e s I, I I , I I I and IV r e s p e c t i v e l y , and n j , n I I # n j I I # and n I V are. the number of p r e p a r a t i o n s w i t h i n the r e s p e c t i v e c a t e g o r i e s . Using t h i s "percentage estimate", the mucous c e l l d e n s i t y i n the d i f f e r e n t c o n d i t i o n s of the experiment may be r e a d i l y compared. I t should be noted, however, that the t e s t of s i g n i f i c a n c e of the data was based on comparisons of the a c t u a l percentages w i t h i n c a t e g o r i e s between any two groups of f i s h and not on the "percentage estimates". R e s u l t s 1. Anatomy and morphology of the g i l l arches The b r a n c h i a l complex of the s t i c k l e b a c k i s composed of four p a i r s of g i l l arches. The a n t e r i o r arch ranges i n l e n g t h from 3 to 4 mm i n f i s h over 50 mm i n l e n g t h , and has 25-45 p a i r s of f i l a m e n t s of about 1 mm i n l e n g t h . The a n t e r i o r border of the g i l l arch bears long (0.5 mm) a n t e r i o - v e n t r a l l y p r o j e c t i n g g i l l r a k e r s ; the g i l l r a k e r s of the p o s t e r i o r 100 F i g . 7.2 E f f e c t of p r o l a c t i n and change of ambient s a l i n i t y on d e n s i t y of mucous c e l l s on g i l l f i l a m e n t s of s t i c k l e b a c k s , measured by percent of g i l l p r eparations i n each of four d e n s i t y c a t e g o r i e s ( I , I I , I I I and I V ) . K i l l e d a f t e r 5 hours i n f r e s h water. 50 . 40 £ 30 <D u c_ o. 20 10 . 0 p r o l a c t i n | | s o l v e n t n n c a t e g o r i e s 101 border are s m a l l ( £ 0.1 mm) . G i l l arches two and. three are s i m i l a r i n l e n g t h to the f i r s t arch, but the g i l l r a k e r s of both the a n t e r i o r and p o s t e r i o r borders are minute. The f o u r t h g i l l J : ; a r c h i s sh o r t e r (2.5 - 3.0 mm), but the length of the f i l a m e n t s i s s i m i l a r ; the g i l l r a kers are again minute. S e v e r a l melanophores are found along the le n g t h and a t the base of each f i l a m e n t . Each f i l a m e n t i s composed of a s p i r a l s e r i e s of sm a l l l a m e l l a e one to three c e l l s i n thickness supported by a c a r t i l a g i n o u s r o d ( F i g . 7.1 a-d). The mucous c e l l s on the f i l a m e n t s are con f i n e d to the bases of the lamellae so t h a t they appear to l i e w i t h i n depressions on the f i l a m e n t s i n s p i r a l s . S c a t t e r e d mucous c e l l s are a l s o found on the g i l l r a k e r s , but they d i d not vary c o n s i s t e n t l y and only the c e l l s o f the f i l a m e n t s were considered. The mucous c e l l s s t a i n r e a d i l y w i t h AF and a f t e r prolonged s t a i n i n g w i t h aqueous a l c i a n b l u e . P r e p a r a t i o n s s t a i n e d w i t h a l c i a n blue show an int e n s e background s t a i n making mucous c e l l r e c o g n i t i o n d i f f i c u l t . Mucin m a t e r i a l w i t h i n the c e l l s and w i t h i n the depressions of the g i l l f i l a m e n t s s t a i n s deep v i o l e t w i t h AF, as does mucus scraped from the f l a n k s and g i l l chamber of the f i s h and a i r - d r i e d on a s l i d e . 2. E f f e c t of. p r o l a c t i n on the maintenance of mucous c e l l s on  the g i l l f i l a m e n t s of seawater-adapted s t i c k l e b a c k s The r e s u l t s are summarised i n Table 7. I . The "percentage estimate of mucous c e l l d e n s i t y " showed l i t t l e change i n the s o l v e n t - and p r o l a c t i n - i n j e c t e d f i s h over the Table 7. I E f f e c t of changes of ambient s a l i n i t y and. of p r o l a c t i n on the d e n s i t y of H mucous c e l l s on the g i l l f i l a m e n t s of autumn marine threespihe s t i c k l e -backs; ''"based on comparisons of percentages w i t h i n c a t e g o r i e s between groups (Kilmogorov-Smirnov two-sample t e s t ) % estimate of mucous c e l l d e n s i t y S i g n i f i c a n c e " (see text) Experimental C o n d i t i o n # of Treatment f i s h Length mm % of g i l l p r e p a r a t i o n s i n each d e n s i t y category I I I I I IV 6 days Sea ( j u s t water before transfer") p r o l a c t i n s o l v e n t none 11 11 14 37-45 36- 46 37- 52 9.1 45.5 27.3 45.5 18.2 27.3 9.1 18.2 14.3 42.9 35.7 7.1 66 55 + 59 1 1 p > 0.05 14 days p r o l a c t i n 24 37-52 33.3 29.2 20.8 16.7 s o l v e n t 22 36-52 50.0 31.8 13.6 4.6 55 43 p > 0. 05 5 hr p r o l a c t i n 10 . 37-52 10.0 20.0 40.0 30.0 so l v e n t 14 39-55 28.6 50.0 7.1 14.3 72 45 p < 0.05 Fr e s h water 1 day p r o l a c t i n 11 37-48 so l v e n t 9 36-54 none 15 36-50 27.3 27.3 44.4 ' 44.4 40.0 33.3 36.3 9.1 11.2 0 26.7 0 57 42 i 47 p > 0.05 4 days p r o l a c t i n 16 35-50 so l v e n t 14 38-52 none 12 35-51 43.8 31.3 18.8 6.1 28.6 50.0 21.4 0 66.7 16.7 16.7 0 47 48 38 p "70.05 8 days p r o l a c t i n 11 ' 35-45 9.1 81.8 9.1 0 so l v e n t 9 35-46 77.8 22.2. 0 0 103 14-day p e r i o d compared w i t h one another or w i t h the 6-day u n i n j e c t e d c o n t r o l s . Comparisons of percentages w i t h i n c a t e g o r i e s between the s o l v e n t - and p r o l a c t i n - i n j e c t e d f i s h o f e i t h e r the 6-. or 14-day groups d i d not r e v e a l any s i g n i f i c a n c e (Kilmogorov-Sminov two-sample t e s t ) , although i n both cases there appears to be a greater percentage of g i l l p r e p a r a t i o n s of the p r o l a c t i n - i n j e c t e d f i s h i n the two higher d e n s i t y c a t e g o r i e s compared w i t h the s o l v e n t - i n j e c t e d f i s h ; the two groups of i n j e c t e d f i s h of 6 or 14 days a l s o d i d not d i f f e r s i g n i f i c a n t l y from the 6-day u n i n j e c t e d f i s h . 3. E f f e c t of p r o l a c t i n on the maintenance of mucous c e l l s on  the g i l l f i i a m e n t s of seawater-adapted s t i c k l e b a c k s  f o l l o w i n g a change i n ambient s a l i n i t y from sea to f r e s h water. The r e s u l t s are summarized i n Table 7.1 and the "percentage estimates of mucous c e l l d e n s i t y " are g r a p h i c a l l y presented i n F i g . 7.3. A S mentioned i n the preceding s e c t i o n , there was no s i g n i f i c a n t d i f f e r e n c e between the mucous c e l l d e n s i t y of the g i l l f i l a m e n t of the p r o l a c t i n - and s o l v e n t -i n j e c t e d f i s h i n sea water immediately before t r a n s f e r to f r e s h water, nor d i d the p r o l a c t i n - and s o l v e n t - i n j e c t e d groups d i f f e r s i g n i f i c a n t l y from the u n i n j e c t e d c o n t r o l s (See 6-day seawater values i n Table 7. I ) . A f t e r 5 hr i n f r e s h water, however, the p r o l a c t i n - i n j e c t e d f i s h showed a r i s e , and the s o l v e n t - i n j e c t e d f i s h a f a l l i n the "percentage estimate of mucous c e l l d e n s i t y " when compared w i t h t h e i r r e s p e c t i v e seawater c o n t r o l s or the u n i n j e c t e d seawater c o n t r o l s (before 104 F i g . 7.3 P r o l a c t i n and g i l l mucous c e l l d e n s i t y of s t i c k l e b a c k s t r a n s f e r r e d to f r e s h water. Measured by changes i n the "percentage estimate of g i l l mucous c e l l d e n s i t y " (see t e x t ) . 8 0 ,-• .v . ••./^•f::&^-;-'r-- D A Y S > 105 t r a n s f e r ) . Although these d i f f e r e n c e s from the seawater c o n t r o l s were not s i g n i f i c a n t , those between the mucous c e l l d e n s i t y of the p r o l a c t i n - and s o l v e n t - i n j e c t e d f i s h a t t h i s time i n f r e s h water were (P = 0.05) (See a l s o F i g . 7.2). A f t e r 24.hr i n f r e s h water, a l l the three groups of f i s h showed a f a l l i n mucous c e l l d e n s i t y compared w i t h t h e i r r e s p e c t i v e seawater c o n t r o l s (before t r a n s f e r ) , b u t the d i f f e r e n c e s were not s i g n i f i c a n t . The p r o l a c t i n - i n j e c t e d f i s h appeared to maintain a higher mucous c e l l d e n s i t y than e i t h e r the s o l v e n t - i n j e c t e d or the u n i n j e c t e d c o n t r o l s a t t h i s time, but the d i f f e r e n c e s were again not s i g n i f i c a n t , nor' was the d i f f e r e n c e between the s o l v e n t - i n j e c t e d and u n i n j e c t e d f i s h s i g n i f i c a n t . . A f t e r 4 days i n f r e s h water, the mucous c e l l d e n s i t y of the s o l v e n t - and p r o l a c t i n - i n j e c t e d f i s h was s i m i l a r , w h i l s t t h a t of the u n i n j e c t e d f i s h was lower, but not s i g n i f i c a n t l y d i f f e r e n t . Although n e i t h e r the p r o l a c t i n -i n j e c t e d nor the s o l v e n t - i n j e c t e d f i s h showed any s i g n i f i c a n t change i n mucous c e l l d e n s i t y a t t h i s time when compared w i t h t h e i r r e s p e c t i v e or the u n i n j e c t e d seawater c o n t r o l s (before t r a n s f e r ) , the u n i n j e c t e d f i s h had a s i g n i f i c a n t l y lower mucous c e l l d e n s i t y than the u n i n j e c t e d seawater c o n t r o l s (P ^~ 0.05; =2=0.025) . A f t e r 8 days i n f r e s h water, w h i l s t the mucous c e l l d e n s i t y of the p r o l a c t i n - i n j e c t e d f i s h appeared to be maintained a t about the same l e v e l as the 4-day value, that of the s o l v e n t -i n j e c t e d f i s h showed a drop (P — 0.05); the d i f f e r e n c e 106 between the two groups of f i s h a t t h i s time was again s i g n i f i c a n t (p< 0.01). The mucous c e l l s of the s o l v e n t -i n j e c t e d f i s h appeared depleted when compared w i t h the seawater c o n t r o l s before transfer- to f r e s h water (6-day values i n Table 7. I ) ; the mucous c e l l d e n s i t y was s i g n i f i c a n t l y lower, than t h a t of the s o l v e n t - i n j e c t e d seawater c o n t r o l s (P< 0.05) or t h a t of the u n i n j e c t e d seawater c o n t r o l s (p< 0.025) or t h a t of the above two groups of seawater c o n t r o l s together (P 0.025). However, t h i s mucous c e l l d e n s i t y of the s o l v e n t - i n j e c t e d f i s h was hot s i g n i f i c a n t l y d i f f e r e n t from the 14-day s o l v e n t -i n j e c t e d seawater f i s h , although i t appeared to be lower. D i s c u s s i o n The f i n d i n g s described above show that the d e n s i t y of mucous c e l l s on the g i l l f i l a m e n t s of the threespine s t i c k l e b a c k maintained i n sea water was not s i g n i f i c a n t l y a f f e c t e d by continuous i n j e c t i o n of p r o l a c t i n . The s l i g h t , but not s i g n i f i c a n t , d e p l e t i o n of mucus i n the 14-day f i s h compared w i t h the 6-day samples may be a r e s u l t of the repeated handling. A f t e r t r a n s f e r to f r e s h water there appeared to. be a d e p l e t i o n of the g i l l mucous c e l l s i n the s o l v e n t - i n j e c t e d and the u n i n j e c t e d f i s h . However, the evidence seems i n c o n c l u s i v e t h a t the- d e p l e t i o n was the r e s u l t of the change of ambient s a l i n i t y from sea water to f r e s h water s i n c e the mucous c e l l d e n s i t y of the s o l v e n t - i n j e c t e d f i s h a f t e r 8 days i n f r e s h water was not s i g n i f i c a n t l y d i f f e r e n t from that of the s o l v e n t - i n j e c t e d f i s h which remained i n sea water f o r the same 107 p e r i o d of time (14-day sea water i n Table 7. I ) . Repeated , handling d u r i n g i n j e c t i o n s or the p h y s i c a l i n j e c t i o n s themselves c o u l d have caused the d e p l e t i o n . On the other hand, the data of the u n i n j e c t e d f i s h i n d i c a t e t h a t the t r a n s f e r . t o f r e s h water could cause d e p l e t i o n of mucous c e l l s . The f i s h showed a p r o g r e s s i v e lowering of mucous c e l l d e n s i t y a f t e r t r a n s f e r to f r e s h water so t h a t a f t e r 4 days i n f r e s h water, the mucous c e l l d e n s i t y was s i g n i f i c a n t l y lower than the seawater value (see a l s o F i g . 7.4 ). Whatever the cause of d e p l e t i o n of mucous c e l l s a f t e r t r a n s f e r of the f i s h to f r e s h water, p r o l a c t i n i n j e c t i o n s s i g n i f i c a n t l y reduced t h i s d e p l e t i o n both i n i t i a l l y and a f t e r 8 days. The p o i n t of i n t e r e s t i s t h a t p r o l a c t i n a f f e c t e d the g i l l mucous c e l l s i n f r e s h water and not i n sea water. This seems to c o r r e l a t e w i t h the f i n d i n g s t h a t p r o l a c t i n a f f e c t e d the osmotic and i o n i c r e g u l a t i o n of s t i c k l e b a c k s i n f r e s h water and not i n sea water (see Chapters 2 and 4). The changes i n the mucous c e l l d e s h i t y of the g i l l s of the p r o l a c t i n - and s o l v e n t -i n j e c t e d f i s h between the time of t r a n s f e r to f r e s h water and 1 day a f t e r , a l s o c o r r e l a t e d c l o s e l y w i t h the changes i n plasma o s m o l a l i t y of the late-autumn f i s h s i m i l a r l y t r e a t e d (see F i g . 2.1). S o l v e n t - i n j e c t e d autumn s t i c k l e b a c k s showed a f a l l i n plasma o s m o l a l i t y w i t h i n the f i r s t 4 hr of t r a n s f e r to f r e s h water, and a p a r t i a l recovery a f t e r 24 hr; the p r o l a c t i n - i n j e c t e d f i s h maintained t h e i r plasma o s m o l a l i t y over a p e r i o d o f 12 hr but showed a f a l l a f t e r 24 hr. This may i n d i c a t e a c a u s e - o r - e f f e c t r e l a t i o n s h i p i . e . the e f f e c t 108 F i g . 7.4 Histogram showing the d e n s i t y of g i l l mucous c e l l s of u n i n j e c t e d s t i c k l e b a c k s i n sea water and a f t e r 1 and 4 days of t r a n s f e r to f r e s h water; measured by percent of g i l l p r e p a r a t i o n s i n each of four d e n s i t y c a t e g o r i e s ( I , I I , I I I and I V ) . I n m iv categories 109 of p r o l a c t i n on the mucous c e l l s may be the cause or the r e s u l t of the p r o l a c t i n e f f e c t on plasma o s m o l a l i t y . The r o l e of mucus i n osmotic and i o n i c r e g u l a t i o n i n t e l e o s t s i s not c l e a r , although such a f u n c t i o n has been suggested by i n d i r e c t evidence. Removal of the mucous covering of the s k i n or damage to the s k i n by severe handling r e s u l t s i n a lowering of plasma i o n i c content i n s e v e r a l species of t e l e o s t s ( G r a f f l i n , 1931; F i r l y , 1932; Raffy, 1949; D r i l h o n , 1952). A l s o , Burden (1956) r e p o r t s a higher number of mucous c e l l s i n the g i l l s of freshwater-adapted Fundulus h e t e r o c l i t u s , compared w i t h seawater-adapted animals. Mucous c e l l s are found a t a l l the most obvious s i t e s of osmotic and i o n i c i n t e r a c t i o n s between the t e l e o s t and i t s environment (namely the s k i n , g i l l s , i n t e s t i n e and k i d n e y ) , and a t some or a l l o f these s i t e s they may p l a y a r o l e i n decreasing the p e r m e a b i l i t y of the e p i t h e l i u m to ions (van Oosten, 1957; P o t t s and Evans, 1966) and/or water. I t has been suggested t h a t p r o l a c t i n may r e g u l a t e mucus s e c r e t i o n , which may i n tu r n be a means by which the osmotic and/or i o n i c r e g u l a t i n g p r o p e r t i e s of p r o l a c t i n are e f f e c t e d ( p i c k f o r d e_t a_l., 1966; P o t t s and Evans, 1966). Although i n t h i s study, p r o l a c t i n . d i d maintain a r e l a t i v e l y high g i l l mucous c e l l d e n s i t y i n f r e s h water, which seems, to c o r r e l a t e w i t h i t s e f f e c t on plasma-o s m o l a l i t y , the evidence i s i n s u f f i c i e n t f o r any c o n c l u s i o n to be made regarding the above suggestion. There are, however, a few p o i n t s which argue a g a i n s t the importance of mucous c e l l s as the primary locus of a c t i o n 110 of p r o l a c t i n . In the f i r s t p l a c e , the changes of-mucous c e l l d e n s i t y i n f r e s h water d i d not seem to c o r r e l a t e w i t h the r e s u l t s on freshwater s u r v i v a l of s t i c k l e b a c k s (see Chapter 3 ). S t i c k l e b a c k s showed a 50% m o r t a l i t y w i t h i n 3-5 days a f t e r t r a n s f e r to f r e s h water and p r o l a c t i n i n j e c t i o n s s i g n i f i c a n t l y reduced t h i s m o r t a l i t y . P r o l a c t i n i n j e c t i o n s , however, d i d not a f f e c t the g i l l mucous c e l l d e n s i t y a t t h i s time i n f r e s h water (4 days). Secondly, a s i g n i f i c a n t e f f e c t of the t r a n s f e r to f r e s h water on the mucous c e l l s has not been unquestionably shown i n t h i s study. T h i r d l y , p r o l a c t i n ' has not as yet been shown to a f f e c t the mucous c e l l s of T. mossambica (Bern, 1967) and P_. l a t i p i n n a ( B a l l , 1969) although these f i s h are dependent on p r o l a c t i n for- s u r v i v a l i n f r e s h water (see Int r o d u c t i o n ) and f o r plasma sodium maintenance, a t l e a s t i n the case of P_. l a t i p i n n a ( B a l l and Ensor, 1965, 1967). Before any c o n c l u s i o n can be made i n t h i s regard, the r o l e of mucous c e l l s i n osmotic and i o n i c r e g u l a t i o n i n t e l e o s t s needs to be more p r e c i s e l y defined; f o r example, the e f f e c t of mucus on membrane p e r m e a b i l i t y of t e l e o s t s needs to be. i n v e s t i g a t e d , i f not e x p e r i m e n t a l l y , a t l e a s t t h e o r e t i c a l l y . With reg a r d to the t h e o r e t i c a l aspect of the problem, P a t t l e (1967 ) has t h i s to say: "We do not y e t know how to c a l c u l a t e the r a t e a t which water w i l l d i f f u s e through a very d i l u t e j e l l y , such as- mucus, when a c o n c e n t r a t i o n g r a d i e n t i s present. The o r d i n a r y I l l d i f f u s i o n c o e f f i c i e n t s — the " t r a c e r d i f f u s i o n c o e f f i c i e n t s " — of s m a l l amounts of gas across a mucous b a r r i e r are f a i r l y w e l l understood. When the m a t e r i a l t h a t i s d i f f u s i n g i s water, the a c t u a l s w e l l i n g agent i n the j e l l y , we do not know how to l i n k up the r a t e of d i f f u s i o n of the water w i t h the d i f f u s i o n c o e f f i c i e n t s of gases i n the j e l l y . The measurement of the r e l a t i o n s h i p between the co n c e n t r a t i o n g r a d i e n t and the a c t i v i t y ^ g r a d i e n t i s a l s o very d i f f i c u l t indeed.". 112 CHAPTER 8 P r o l a c t i n and .the Gut I n t r o d u c t i o n The importance of the gut i n the osmoregulation of marine t e l e o s t s was f i r s t r ecognized and demonstrated by Homer Smith (1930). Since then, evidence has i n c r e a s i n g l y confirmed i t (see Reviews by Par r y , 1966; P o t t s , 1968). I t i s now c l e a r t h a t marine t e l e o s t s d r i n k sea water and absorb water from the gut, wh i l e freshwater t e l e o s t s d r i n k water to a much l e s s e r extent (Smith, 1930; M u l l i n s , 1950; P o t t s , e t a l . , 1967; Evans, 1967; P o t t s and Evans, 1967) s i n c e d r i n k i n g o n l y aggravates t h e i r osmotic problems. The water t r a n s p o r t i n the gut has been s t u d i e d •in v i t r o i n s e v e r a l t e l e o s t s (House and Green, 1963, 1965; Sha-rratt e t a l . , 1964; Smith, 1964; A u l l , 1966; Hirano, 1967; Hirano, et a l . , , 1967; U t i d a , e t a l . , 1967; Oide, 1967; Oide and U t i d a , 1967). This t r a n s p o r t i s greater i n gut sacs from f i s h adapted to sea water than i n those from f i s h i n f r e s h water ( S h a r r a t t , e t . a l . , 1964; U t i d a , e t a l . , 1967; Oide and U t i d a , 1967). The endocrine c o n t r o l of the osmoregulatory r o l e of the gut i n t e l e o s t s has on l y j u s t begun to be s t u d i e d (Green and Matty, 1966; Hirano, e t a l . , 1967; Hirano, 1967; Hirano and U t i d a , 1968). Such a c o n t r o l seems necessary i n eu r y h a l i n e t e l e o s t s which have to a d j u s t the f u n c t i o n a l r o l e of the gut to changing environmental s a l i n i t i e s . In the Japanese e e l ( A n g u i l l a j a p o n i c a ) , the p i t u i t a r y - i n t e r r e n a l ( a d r e n o c o r t i c a l ) 113 a x i s has been shown to "be i n v o l v e d i n the. c o n t r o l , of the gut water t r a n s p o r t during seawater adaptation (Hirano, e t a l . , 1967; Hirano, 1967; Hirano and U t i d a , 1968). In the present study, the r o l e of p r o l a c t i n i n the c o n t r o l of d r i n k i n g and i n v i t r o gut water t r a n s p o r t during freshwater adaptation has been i n v e s t i g a t e d i n the s t i c k l e b a c k . M a t e r i a l s and Methods p r i n k i n g ; : r a t e . The d r i n k i n g r a t e s were i n d i r e c t l y determined from the experiments de s c r i b e d i n Chapter 6 f o r the study of the e f f e c t 14 of p r o l a c t i n on l o s s of C - i n u l i n v i a the head r e g i o n , s i n c e i n these experiments, gut f l u i d s were a l s o c o l l e c t e d . The volumes of the gut f l u i d s c o l l e c t e d were measured. The gut cannula had been c a l i b r a t e d to gi v e volume i n ml (or jul) per cm so th a t the len g t h of f l u i d column i n the cannula was a measure of the volume of the gut f l u i d . One u l of the 14 f l u i d was taken and the C a c t i v i t y determined i n the same 14 way as plasma C a c t i v i t y (see Chapter 7 ) . The t o t a l a c t i v i t y i n the gut f l u i d was then c a l c u l a t e d . Assuming t h a t the a c t i v i t y i n the gut f l u i d (x) came v i a d r i n k i n g of the e x t e r n a l medium i n the head chamber, the d r i n k i n g r a t e may be c a l c u l a t e d i f the e x t e r n a l c o n c e n t r a t i o n 14 . x o f C a c t i v i t y (y) was a l s o known, i . e . d r i n k i n g r a t e = — 14 u l per 24 hr. Since the e x t e r n a l C c o n c e n t r a t i o n was 14 dependent on net l o s s of C v i a the head r e g i o n and v a r i e d w i t h time, the mean co n c e n t r a t i o n f o r the 24-hr p e r i o d was 114 estimated f o r each f i s h and used as the e x t e r n a l . c o n c e n t r a t i o n 14 (y) i n the c a l c u l a t i o n of the d r i n k i n g r a t e . The C con c e n t r a t i o n a t 12 hr was estimated as the mean s i n c e the concentrations appeared to vary w i t h time as a r e c t a n g u l a r -h y p e r b o l i c f u n c t i o n (see F i g . 6.1). In v i t r o gut water t r a n s p o r t S t i c k l e b a c k s (<-^  2 gm) were i n j e c t e d w h i l s t i n sea water w i t h e i t h e r p r o l a c t i n or the s o l v e n t on a l t e r n a t e days f o r three i n j e c t i o n s . Twenty-four hr a f t e r the l a s t i n j e c t i o n , each f i s h was k i l l e d by t r a n s e c t i o n of the s p i n a l cord i n the neck r e g i o n and d i s s e c t e d to expose the gut. The gut c o n s i s t s of oesophagus, stomach ( f a i r l y - w e l l d i f f e r e n t i a t e d ) and i n t e s t i n e ; the stomach i s separated from the i n t e s t i n e by a p y l o r u s . A polyethylene tube (PE 50) f i l l e d w i t h pre-oxygenated d e c h l o r i n a t e d f r e s h water and connected v i a a hypodermic needle to a 0.25 ml sy r i n g e c o n t a i n i n g the water, was lowered through the mouth i n t o the i n t e s t i n e . Water was then i n j e c t e d i n t o the i n t e s t i n e to wash the contents o f f v i a the anus; t h i s was done s e v e r a l times. ( I t was necessary to i n j e c t water i n t o the i n t e s t i n e r a t h e r than the stomach because i f i n j e c t e d i n t o the stomach the water would be dammed to a l a r g e extent a t the pylorus by the p y l o r i c s p h i n c t e r . ) A f t e r emptying the i n j e c t e d f l u i d by gen t l e pressure along the gut, the gut was t i e d o f f c l o s e to the anus by two c l o s e l y - a p p l i e d thread l i g a t u r e s . Two other l i g a t u r e s were p u l l e d t i g h t over the PE 50 tube (which was s t i l l i n s e r t e d i n the i n t e s t i n e ) i n the r e g i o n of the oesophagus j u s t a n t e r i o r 115 to the stomach. Approximately 0.04 ml of pre-oxygenated d e c h l o r i n a t e d f r e s h water was then i n j e c t e d i n t o the gut, and as the tube was withdrawn, the l i g a t u r e s were t i g h t l y t i e d . The distended sac was removed, b l o t t e d on a Kleenex f a c i a l t i s s u e , and weighed ( i n i t i a l weight) on a M e t t l e r balance (Type H5, cap. 160g, No 54301).. The sac was then incubated a t room temperature (20 - 1°C) i n 5 ml of continuously-oxygenated Ringer (see Table 6. I f o r composition) contained i n a t e s t -tube. A f t e r 1 hr of i n c u b a t i o n , the sac was removed, b l o t t e d , and weighed ( f i n a l w eight). The sac was then opened by c u t t i n g b oth ends as c l o s e to the l i g a t u r e s as p o s s i b l e and the f l u i d g e n t l y squeezed out onto a Kleenex f a c i a l t i s s u e . The sac t i s s u e proper and the l i g a t u r e d end t i s s u e s were then d r i e d i n an oven a t 110°C. A f t e r 24 hr, the sac t i s s u e proper was weighed (dry weight A) s e p a r a t e l y from the l i g a t u r e d end t i s s u e s (dry weight B). The d i f f e r e n c e between the i n i t i a l and f i n a l weights was taken as the net t r a n s p o r t of water from the sac to the ambient Ringer, and the d i f f e r e n c e between the i n i t i a l weight and the t o t a l dry weight (A + B), as the i n i t i a l water content of the sac. Surface area of the sac was assumed to be 2/3 p r o p o r t i o n a l to (dry weight A) . The net water t r a n s p o r t was then expressed as % i n i t i a l water content per (mg dry weight A ) 2 ' 3 . 116 R e s u l t s . D r i n k i n g r a t e The data were expressed as % body weight per 24 hr.. The means ± standard e r r o r are given i n Table 8. I . The p r o l a c t i n value was s i g n i f i c a n t l y lower than the s o l v e n t value (P< 0.005; t - t e s t ) . Thus i t appeared t h a t p r o l a c t i n reduced the d r i n k i n g r a t e of s t i c k l e b a c k s i n f r e s h water under the c o n d i t i o n s d e s c r i b e d i n Chapters 5 and 6. tIn v i t r o gut water a b s o r p t i o n The means of the data of 13 f i s h ± standard e r r o r are given i n Table 8. I I . The p r o l a c t i n value was s i g n i f i c a n t l y lower than the s o l v e n t value (P<0.05). Thus p r o l a c t i n appeared a l s o to reduce water a b s o r p t i o n i n the gut, i f the change i n weights of the gut sacs before and a f t e r i n c u b a t i o n was due to water movement across the gut, and any i n t r i n s i c weight changes of the .gut t i s s u e s themselves were n e g l i g i b l e . D i s c u s s i o n The f i n d i n g t h a t p r o l a c t i n reduced the drinking^ r a t e of s t i c k l e b a c k s during the 24 hr i n f r e s h water (head region) i s a r a t h e r s u r p r i s i n g one. I t i s , however, not unequivocal,' s i n c e the evidence was i n d i r e c t and based on two important 14 assumptions. F i r s t l y , i t was assumed th a t a l l the C a c t i v i t y i n the gut came v i a d r i n k i n g . The a c t i v i t y , however, could 14 have entered the gut from the coelom, i n t o which C - i n u l i n had been i n j e c t e d , d i r e c t l y or v i a the bloo d . This was assumed 117 Table 8. I P r o l a c t i n and the d r i n k i n g r a t e of s t i c k l e b a c k s during the f i r s t 24 hr i n f r e s h water (head r e g i o n ) ; values i n parentheses are c a l c u l a t e d r a t e s per hour (for comparison w i t h values given i n Table 8. I l l ) ; *** P<^ 0.005. Mean d r i n k i n g r a t e _ Sx {% body weight per 24 hr) Solvent p r o l a c t i n 20.5 - 1.7 (0.85 to.07) + *** 7.8 - 2.3 (0.33- 0.1) 118 Table 8. I I P r o l a c t i n and i n v i t r o gut water ab s o r p t i o n of s t i c k l e b a c k s ; * P < 0.05. In v i t r o gut water a b s o r p t i o n ; means of 13 f i s h t Sx:» 2/3 % i n i t i a l water content per(mg dry weight) ' Solvent P r o l a c t i n 24.0 t 1.8 18.4 t 1.3* 119 to be n e g l i g i b l e s i n c e the f i s h gut has been shown to be r e l a t i v e l y impermeable to C - i n u l i n ( A u l l , 1966; Evans, 1967). Secondly, i t was assumed t h a t there was no movement of 14 the i n g e s t e d C across the gut i n t o the blood. This absorption 14 of C i s u n l i k e l y not o n l y because of the r e l a t i v e 14 i m p e r m e a b i l i t y of f i s h gut to C - i n u l i n , which the a c t i v i t y was most probably (Lam, 1968), but because the movement would 14 have to be u p h i l l s i n c e the b l o o d contained a higher C 14 c o n c e n t r a t i o n than the gut f l u i d . A c t i v e absorption of C , however, must remain a p o s s i b i l i t y . Nevertheless, the d r i n k i n g r a t e s of s t i c k l e b a c k s obtained here seem reasonable when compared to those of other t e l e o s t s which have been s t u d i e d (see Table 8. I l l ) . p a r t i c u l a r l y , the r a t e s compare fav o u r a b l y w i t h those of T. mossambica and F. h e t e r o c l i t u s , which are more i n the s i z e range of s t i c k l e b a c k s than the other t e l e o s t s (see Table 8. I l l ) , In f a c t the s o l v e n t value i s very s i m i l a r to the freshwater 14 d r i n k i n g r a t e of F. h e t e r o c l i t u s determined using C - i n u l i n as a marker. The l a t t e r value, however, may be higher than the t r u e v a l u e s i n c e the determination was based on counting the 14 whole animal f o r C - i n u l i n , p a r t of which might have entered v i a the head r e g i o n (Lam, 1968) and s i n c e the value i s much 35 higher than the value determined u s i n g S -sulphate as a marker and counting o n l y the gut f o r the a c t i v i t y . I f so, i t may be noted t h a t the p r o l a c t i n value of s t i c k l e b a c k s i s c l o s e r to the freshwater d r i n k i n g r a t e s of T. mossambica and F. h e t e r o c l i t u s Table 8. I l l D r i n k i n g Rates of T e l e o s t s Species S i z e range % body weight p er hr (gm) Sea Water Fresh Water References Gasterosteus aculeatus i 1-2 4.0 M u l l i n s , 1950 <^ 0.85 (Solvent) 0.33 ( P r o l a c t i n ) Present i n v e s t i g a t i o n (See t e x t f o r c o n d i t i o n s of experiments) T i l a p i a mossambica <3 1.1110.17 0.26 ±0. 04 P o t t s , e t _ a l _ . , 1967 Fundulus h e t e r o c l i t u s 2-5 2.3 0+0.27 ( C 1 4 l n u l i n ) 1 1.54i0.21(S 3 5 S u l p h a t e ) 2 0 . 8 3 + 0 . 3 9 ( C 1 4 l n u l i n ) 1 0.14+0.05(S 3 5Sulphate) P o t t s and Evans, 2 1967 Blennius p h o l i s 6.0 House, 1963 P l a t i c h t h y s >50 1.0 Motais and Maetz, 1964 0.5 Hickman, 1968 c Serranus s c r i b a 0.5 Motais and Maetz, 1965 X i p h i s t e r atropurpureus 7-40 0.03.4*0.006 0.007+0.002 ( i n 10% sea water) Evans, 1967 A n g u i l l a >50 0.3 • Smith, 1930 Myoxocephalus >50 0.3 14 2 35 Using C - i n u l i n as a marker; Using S -Sulphate as a marker. 121 than the s o l v e n t value. This i s i n t e r e s t i n g because these two f i s h have been shown to r e q u i r e p r o l a c t i n f o r s u r v i v a l i n f r e s h water (see I n t r o d u c t i o n ) . Furthermore, the d r i n k i n g r a t e s found here are lower than the seawater r a t e of s t i c k l e b a c k s estimated by M u l l i n s (1950), as would be expected s i n c e the r a t e s were determined here w i t h the head regio n s of the f i s h i n f r e s h water. Thus i t appears t h a t i n the 24 hr i n f r e s h water, the d r i n k i n g r a t e of s t i c k l e b a c k s d e c l i n e d and p r o l a c t i n was able to reduce the r a t e more r a p i d l y . I t i s d i f f i c u l t to v i s u a l i z e the mechanism by which p r o l a c t i n c o u l d reduce the d r i n k i n g r a t e , i f indeed i t d i d . I t seems more p l a u s i b l e t h a t d r i n k i n g i n t e l e o s t s i s under nervous c o n t r o l v i a a hypothalamic t h i r s t center, as i n mammals, than under hormonal c o n t r o l . Nevertheless, p r o l a c t i n may a f f e c t the nervous c o n t r o l ( i f indeed the nervous c o n t r o l i s the case) e i t h e r d i r e c t l y or i n d i r e c t l y ; thus p r o l a c t i n may i n some way i n h i b i t the hypothalamic centre or the t r a n s m i s s i o n of impulse, or may modify the s t i m u l u s . The i n v i t r o s t u d i e s suggest t h a t p r o l a c t i n a l s o reduced the a b s o r p t i o n of water from the gut f i l l e d w i t h d e c h l o r i n a t e d f r e s h water. Thus p r o l a c t i n may decrease the p e r m e a b i l i t y of the gut to water as i n the g i l l (see Chapter 6). Since the gut i s r i c h l y s u p p l i e d w i t h mucous c e l l s , p r o l a c t i n might a l s o e x e r t i t s e f f e c t on gut p e r m e a b i l i t y v i a the mucous c e l l s (see Chapter 7). 122 I t has been shown th a t the i n v i t r o gut water a b s o r p t i o n i s g r eater i n seawater-adapted e e l s (A n g u i l l a ) • than i n freshwater-adapted e e l s ( S h a r r a t t , e t a l . , 1964; U t i d a e t a l . , 1967; Oide and U t i d a , 1967). This d i f f e r e n c e between the two groups of f i s h c o uld be e l i m i n a t e d by hypophysectomy of seawater-adapted e e l s (Hirano e t a l . , 1967) or treatment of freshwater-adapted e e l s w i t h ACTH or C o r t i s o l (Hirano and U t i d a , 1968), and co u l d be r e s t o r e d i n the hypophysectomized seawater-adapted e e l s by C o r t i s o l treatment (Hirano, 1967). This suggests t h a t during seawater adaptation ACTH i s secreted which i n c r e a s e s the water a b s o r p t i o n by the gut v i a s t i m u l a t i o n , of C o r t i s o l secretion by the i n t e r r e n a l and that during freshwater adaptation, ACTH i s e i t h e r not sec r e t e d or secreted i n amounts inadequate to cause an e f f e c t . Hirano and U t i d a (1968) a l s o found that i n j e c t i o n of p r o l a c t i n i n freshwater-adapted e e l s was without e f f e c t on the i n v i t r o gut water t r a n s p o r t . However, the work of Hirano and co-workers d i f f e r s from the present work i n two aspects besides a p o s s i b l e species d i f f e r e n c e . F i r s t l y , whereas i n the work of Hirano and co-workers, p r o l a c t i n i n j e c t i o n was done i n freshwater-adapted eels.which probably has endogenous s e c r e t i o n of p r o l a c t i n , i n the present work, i t was done i n seawater-adapted s t i c k l e b a c k s which were apparently " p h y s i o l o g i c a l l y hypophysectomized" as regards p r o l a c t i n . Secondly, i n the work of Hirano and co-workers, the i s o l a t e d guts were bathed on both mucosal and s e r o s a l s i d e s by i d e n t i c a l Ringer s o l u t i o n , whereas i n the present work, the 123 guts were bathed on the mucosal s i d e by d e c h l o r i n a t e d f r e s h water and on the s e r o s a l s i d e by Ringer; the mechanism ..of water t r a n s p o r t from the mucosa to the serosa must, t h e r e f o r e , be d i f f e r e n t i n the two cases. In the former case water t r a n s p o r t (net) would e i t h e r be secondary to a c t i v e t r a n s p o r t o f s o l u t e s as i n the mammalian gut (Curran and Solomon, 1957; Curran, 1960) or i n v o l v e an " a c t i v e water pump" (House and Green, 1965); thus, ACTH (or C o r t i s o l ) may a c t i n t h i s case v i a s t i m u l a t i o n of a c t i v e t r a n s p o r t of s o l u t e s (e.g. sodium). I t i s i n t e r e s t i n g t h a t the augmentative e f f e c t of C o r t i s o l on the water t r a n s p o r t i n the e e l gut was p a r a l l e l e d by an i n c r e a s e i n sodium t r a n s p o r t (Hirano and U t i d a , 1968). On the other hand, i n the present work, water t r a n s p o r t i n the i s o l a t e d gut would o n l y i n v o l v e p a s s i v e osmosis. Under t h i s c o n d i t i o n , ACTH (or C o r t i s o l ) which, on the b a s i s of the work of Hirano and co-workers, would be expected to be se c r e t e d i n the seawater-adapted s t i c k l e b a c k s used i n the s t u d i e s , may be i n e f f e c t i v e on gut water t r a n s p o r t (since i t may o n l y a c t v i a a c t i v e t r a n s p o r t as d i s c u s s e d above). P r o l a c t i n , however, may f u n c t i o n under t h i s c o n d i t i o n . Prom the above d i s c u s s i o n / i t i s c l e a r that the endocrine c o n t r o l of d r i n k i n g and gut water abs o r p t i o n i n t e l e o s t s r e q u i r e s more i n v e s t i g a t i o n s . The f i n d i n g s r e p o r t e d here merely suggest a p o s s i b l e r o l e of p r o l a c t i n i n t h i s regard. They need to be confirmed and s u b s t a n t i a t e d . 124 CHAPTER 9 Synopsis and General D i s c u s s i o n I n t r o d u c t i o n w, Since the f i n d i n g s and hypotheses i n t h i s t h e s i s have al r e a d y been discussed i n the preceding chapters, i t o n l y remains i n t h i s concluding chapter to present' a synopsis and a general d i s c u s s i o n of c e r t a i n p e r t i n e n t questions a r i s i n g from the t h e s i s . • Synopsis The f i n d i n g s and hypotheses i n t h i s t h e s i s are s c h e m a t i c a l l y summarized i n F i g . 9.1. The p i t u i t a r y gland of the s t i c k l e b a c k , Gasterosteus.  aculeatus (form trachurus) seems unable during the l a t e autumn and winter (at l e a s t e a r l y and mid-winter) to se c r e t e p r o l a c t i n i n amounts adequate f o r s u r v i v a l i n f r e s h water of low m i n e r a l content, although competent to do so i n s p r i n g and summer, a seasonal d i f f e r e n c e t r i g g e r e d by p h o t o p e r i o d i c changes (Lam, 1965). . In nature, the f i s h l i v e s i n sea water or b r a c k i s h water during the l a t e autumn and wi n t e r , and migrates to f r e s h water f o r breeding i n s p r i n g or e a r l y summer. Thus, p r o l a c t i n may be i n v o l v e d i n the freshwater m i g r a t i o n of the f i s h . The evidence may be summarized as f o l l o w s : -S t i c k l e b a c k s i n l a t e autumn or e a r l y w i n t e r , when t r a n s f e r r e d from sea water to f r e s h water, s u f f e r e d a high m o r t a l i t y which c o u l d be reduced by p r o l a c t i n treatment. The f i s h a l s o d i s p l a y e d a greater f a l l i n plasma o s m o l a l i t y and 125 F i g . 9.1 Schematic diagram summarizing the f i n d i n g s and hypotheses of t h i s t h e s i s ; f i n d i n g s r e g a r d i n g potassium are not i n c l u d e d . Keys: S.W. = sea water F.W. = f r e s h water ENVIR = environmental PROL SECRN = p r o l a c t i n s e c r e t i o n - = l i t t l e or no p r o l a c t i n s e c r e t i o n + = p r o l a c t i n s e c r e t i o n ' ? .= i n d i r e c t or suggestive evidence - = w i t h or without SEASON NATURAL ENVIR. PROL. RBBUMS AND MECHANISMS) OF ACTION AUTUMN WINTER S.W. Short or decreasing photo-p e r i o d F a i l u r e to osrao- and i o n - r e g u l a t e and hence, to su r v i v e i n F.W. -SPRING SUMMER F.W. (or Long or migrat- i n c r e a s i n g m g to F.W.) photo-p e r i o d r-Reduces l o s s of ions and^-C 1 4 - ( i n u l i n ? ) • Head -( g i l l s ? ) Reduces osmotic i n f l u x of H2O*--" 1 Increases d e n s i t y of g i l l raucous<.-J. L c e l l s -Increases GFR(?) —» increas e s u r i n e f l o w + 4>kidneys-| ^ c r e a s e s reabsorption); ^ 2 of Na and C l ± r e d u c t i o n of H2O -reabsorption(?) Decreases Urine osmol-a l i t y and N a + and C l ~ l e v e l s Increases H2O excre-t i o n without a f f e c t i n g N a + and C l " e x c r e t i o n (Net e f f e c t ) l»Gut rReduces d r i n k i n g r a t e ( ? ) [.Reduces r e a b s o r p t i o n of H2O I? I maintains plasma ^ o s m o l a l i t y and- N a + and C l " l e v e l s FW S u r v i v a l 126 a s m a l l e r f a l l i n u r i n e o s m o l a l i t y than l a t e - s p r i n g f i s h t r a n s f e r r e d to f r e s h water i n the same way, and t h i s seasonal d i f f e r e n c e could be e l i m i n a t e d by p r o l a c t i n treatment of the late-autumn or e a r l y - w i n t e r f i s h . S i m i l a r l y , a seasonal d i f f e r e n c e e x i s t s i n the h i s t o l o g i c a l p i c t u r e o f the g l o m e r u l i of late-autumn and l a t e - s p r i n g s t i c k l e b a c k s (Ogawa, 1968), and t h i s d i f f e r e n c e c o u l d be e l i m i n a t e d by p r o l a c t i n treatment of the former f i s h . . The f a l l i n plasma o s m o l a l i t y i n late-autumn and winter f i s h a f t e r t r a n s f e r to f r e s h water was p a r a l l e l e d by a r a p i d drop i n plasma sodium and c h l o r i d e , which c o u l d be c o r r e c t e d by a s i n g l e i n j e c t i o n of p r o l a c t i n given 24 hr before the t r a n s f e r , plasma potassium, however, seemed u n a f f e c t e d by p r o l a c t i n treatment. I t w i l l be seen t h a t although not s u r g i c a l l y hypophysectomized, the late-autumn and winter s t i c k l e b a c k behaved remarkably l i k e hypophysectomized P_. l a t i p i n n a ( B a l l and Ensor, 1965, 1967). Next, the mechanism of a c t i o n of p r o l a c t i n was s t u d i e d . P r o l a c t i n seems to e x e r t i t s e f f e c t s ( i n f r e s h water) on the three r e c o g n i z e d organs of osmotic and i o n i c r e g u l a t i o n i n t e l e o s t s , v i z . kidneys, g i l l s and gut. In the kidneys, p r o l a c t i n i n c r e a s e d u r i n e flow, a p p a rently as a r e s u l t of an i n c r e a s e d GFR. P r o l a c t i n reduced the apparent i n c r e a s e i n i n t r a c a p s u l a r space i n the g l o m e r u l i o f the late-autumn and winter s t i c k l e -backs, and, consequently, i n c r e a s e d the percentage.frequency of g l o m e r u l i w i t h no evident i n t r a c a p s u l a r space. The data are i n t e r p r e t e d to mean tha t p r o l a c t i n rendered g l o m e r u l i more 127 f u n c t i o n a l or more g l o m e r u l i f u l l y f u n c t i o n a l and,, hence i n c r e a s e d GFR (Hickman, 1965). Since the i n c r e a s e i n u r i n e f l o w and GFR was p a r a l l e l e d by a decrease i n u r i n e o s m o l a l i t y and u r i n e concentrations of sodium and c h l o r i d e , p r o l a c t i n must a l s o i n c r e a s e r e n a l t u b u l a r r e a b s o r p t i o n of sodium and c h l o r i d e (A) and/or decrease water r e a b s o r p t i o n (B); and s i n c e the t o t a l r e n a l l o s s of sodium and c h l o r i d e d i d not appear to be s i g n i f i c a n t l y i n c r e a s e d d e s p i t e an i n c r e a s e i n GFR, A must occur w i t h or without B. p r o l a c t i n , however; apparently i n c r e a s e d the t o t a l r e n a l l o s s of potassium and d i d not a f f e c t the t u b u l a r potassium r e a b s o r p t i o n , although there, was a suggestion t h a t p r o l a c t i n a c t u a l l y decreased t u b u l a r r e a b s o r p t i o n of potassium, an.action r e m i n i s c e n t of the r e n a l a c t i o n of aldosterone i n mammals. In the g i l l s (or other regions around the head), p r o l a c t i n reduced- the net osmotic i n f l u x of water and the net l o s s of 14 14 sodium, c h l o r i d e and C (from i n j e c t e d C - i n u l i n ) ; the l a t t e r was probably because p r o l a c t i n reduced the o u t f l u x , as has been shown f o r sodium i n F. h e t e r o c l i t u s , A. a n g u i l l a and P_. l a t i p i n n a (Potts and Evans, 1966; Maetz ejt a l . , 1967 a, b; Ensor and B a l l , 1968). These changes were accompanied by the behayiour of the g i l l mucous c e l l s , which were in c r e a s e d i n . d e n s i t y by p r o l a c t i n treatment, suggesting a c a u s e - o r - e f f e c t r e l a t i o n s h i p . In the gut, p r o l a c t i n reduced water' ab s o r p t i o n arid, a t the same time, seemed to reduce the freshwater d r i n k i n g rate.; These r e s u l t s however, are r a t h e r p r e l i m i n a r y and need to be confirmed. 128 Thus, i t appears th a t p r o l a c t i n was able 1 to reduce or prevent osmotic f l o o d i n g of s t i c k l e b a c k s i n f r e s h water by-reducing e x t r a r e n a l osmotic i n f l u x of water and i n c r e a s i n g r e n a l l o s s of water v i a an i n c r e a s e i n GFR and u r i n e flow, and a l s o , p o s s i b l y , by reducing d r i n k i n g r a t e and water abs o r p t i o n by the gut; a t the same time, p r o l a c t i n reduced e x t r a r e n a l l o s s of sodium and c h l o r i d e but d i d not apparently a f f e c t r e n a l l o s s of the i o n s , which was s m a l l compared to the e x t r a r e n a l l o s s . By these mechanisms, p r o l a c t i n maintained plasma o s m o l a l i t y and sodium and c h l o r i d e l e v e l s a f t e r t r a n s f e r of the f i s h to f r e s h water, and, consequently, was able to promote freshwater s u r v i v a l of the f i s h i n the autumn and w i n t e r . General D i s c u s s i o n I t i s evident, t h a t p r o l a c t i n plays an e s s e n t i a l r o l e i n the c o n t r o l of freshwater osmotic and i o n i c r e g u l a t i o n of the marine s t i c k l e b a c k , Gasterosteus acu l e a t u s , form trachurus and, hence, i s l i k e l y to be involved, i n the freshwater m i g r a t i o n of the f i s h , a c o n c l u s i o n supported by the apparent s e c r e t i o n or i n c r e a s e i n s e c r e t i o n of p r o l a c t i n i n the s p r i n g preparatory to the freshwater m i g r a t i o n . I t i s u n c e r t a i n , however, whether the r o l e of p r o l a c t i n i s primary or secondary v i a s t i m u l a t i o n or i n h i b i t i o n of other hormone(s). In A. a n g u i l l a , p r o l a c t i n was found to. s t i m u l a t e the. t h y r o t r o p i n (TSH) c e l l s and the t h y r o i d (Olivereau, 1966). Thus p r o l a c t i n may a c t v i a TSH and t h y r o x i n e , which have been 129 i m p l i c a t e d i n osmoregulation o f s t i c k l e b a c k s . Koch and Heuts (1942, 1943) and Heuts ...(1943, 1945) found t h a t feeding of d e s i c c a t e d t h y r o i d gland i n f l u e n c e d the osmoregulation i n both sea water and f r e s h water o f two species ..of s t i c k l e b a c k s . The species s t u d i e d , however, are the freshwater form (gymnurus or leiur.us) of G. acule a t u s , and Pygosteus p u n q i t i u s . I t was shown by the same Workers th a t the p a t t e r n of blood c h l o r i d e changes at. low s a l i n i t y ( f r e s h water) i n the marine form (trachurus) of G. acu l e a t u s , which was the form used i n the present s t u d i e s , was d i f f e r e n t from t h a t i n gymnurus -but s i m i l a r to t h a t i n P y g o s t e u s p u n q i t i u s . I t was, t h e r e f o r e , thought t h a t the e f f e c t o f t h y r o i d feeding i n trachurus, though not t e s t e d , would be s i m i l a r to tha t i n P. pungit-i-us; P_. p u n q i t i u s , when f e d t h y r o i d m a t e r i a l w h i l e i n f r e s h Water, showed an i n c r e a s e i n blood c h l o r i d e s (Heuts, 1943), an e f f e c t a l s o shown by p r o l a c t i n i n the present s t u d i e s (Chapter 4 ) . Furthermore, Baggerman (1957, 1959) showed that treatment of marine s t i c k l e b a c k s (G. aculeatus trachurus) w i t h thyroxine produced a preference f o r f r e s h water while a n t i - t h y r o i d drugs induced a s a l t - w a t e r preference. The r o l e of TSH and/or thyr o x i n e i n osmotic and i o n i c r e g u l a t i o n of t e l e o s t s , however, has not been e s t a b l i s h e d . The l i t e r a t u r e - o f f e r s a r a t h e r confused and d i f f u s e d p i c t u r e . For example, w h i l e Hoar (1951) found t h a t thyroxine treatment d i d not a f f e c t the s a l i n i t y t o l e r a n c e of young coho salmon, Smith (1956) found t h a t thyroxine r a i s e d the s a l i n i t y t o l e r a n c e of brown t r o u t , and t h i o u r e a and t h i o u r a c i l reduced t h i s t o l e r a n c e ; 130 these two works are themselves c o n t r a d i c t e d by the works on G. aculeatus , form gymnurus, and P. pungitius. (Koch and Heuts, 1942, 1943; Heuts, 1943, 1945). In these f i s h , feeding of t h y r o i d gland seemed to reduce t h e i r s a l i n i t y t o l e r a n c e , a t l e a s t t h e i r c a p a c i t y to osmo (io n o ) r e g u l a t e i n sea-water. Furthermore, the t h y r o i d a c t i v i t y of s e v e r a l e u r y h a l i n e t e l e o s t s f o l l o w i n g changes of ambient s a l i n i t y i s not c o n s i s t e n t w i t h a r o l e of thyroxine i n freshwater adaptation. Thus the t h y r o i d a c t i v i t y has been shown to i n c r e a s e a t higher ambient s a l i n i t i e s i n the f l o u n d e r , P l a t i c h t h y s s t e l l a t u s (Hickman, 1959), the t r o u t , Salmo g a i r d n e r i (Eales, 1963) and the p r i c k l y s c u l p i n , Cottus asper (Bonn and Hoar, 1965). Although s t i c k l e b a c k s have an a c t i v e t h y r o i d gland a t the time of m i g r a t i o n to f r e s h water (Koch and Heuts, 1942), Wiggs (1962) showed, using r a d i o l o g i c a l methods, th a t t h y r o i d a c t i v i t y i n c r e a s e d i n s t i c k l e b a c k s t r a n s f e r r e d i n the l a b o r a t o r y from f r e s h to sea water. Further c o n t r a d i c t o r y r e s u l t s have been shown f o r t h y r o i d a c t i v i t y a t the time o f ' m i g r a t i o n of a number of migratory f i s h e s (see reviews by Baggerman, 1957, 1959, 1962; and Hoar, 1959, 1963). For example, i n the P a c i f i c salmon, some species migrate to the sea w i t h quiescent glands w h i l e others have more a c t i v e t h y r o i d s a t t h a t time (Hoar and B e l l , 1950). The.various c o n t r a d i c t i o n s demonstrated f o r the r o l e of the t h y r o i d gland and thy r o x i n e make i t d i f f i c u l t to conceive of a mechanism f o r the e f f e c t of t h y r o i d hormone on osmotic and i o n i c r e g u l a t i o n and/or migratory behavior. Most 131 authors seem to f e e l that the r o l e of the t h y r o i d hormone i s an i n d i r e c t one (e.g. Hoar and B e l l / 1950; Hoar, 1952); i t a f f e c t s c e l l u l a r metabolism which i n t u r n a f f e c t s a number of processes. As osmotic r e g u l a t i o n r e q u i r e s an'increase i n metabolic energy, th y r o x i n e might a f f e c t osmoregulation by s t i m u l a t i n g c e l l u l a r metabolism ( j e n k i n , 1962). In t h i s connection, i t i s i n t e r e s t i n g to note t h a t Graetz (1931) has shown t h a t Gasterosteus has a higher r e s p i r a t o r y r a t e i n f r e s h water than i n i s o s m o t i c sea water. In the f l o u n d e r , P l a t i c h t h y s Stellatus, Hickman (1959) has found higher t h y r o i d a c t i v i t y i n sea water than i n f r e s h water, which i s c o r r e l a t e d w i t h a higher standard metabolic r a t e . He suggested that t h i s , may be a demonstration of c a l o r i g e n i c a c t i o n of t h y r o i d not p r e v i o u s l y obtained i n work v/ith t e l e o s t s . To complicate matters f u r t h e r , i t i s p o s s i b l e t h a t the r e v e r s e s i t u a t i o n to t h a t of p r o l a c t i n s t i m u l a t i o n of TSH and t h y r o x i n e s e c r e t i o n may occur, s i n c e t h y r o i d hormones have been shown to s t i m u l a t e d i r e c t l y p r o l a c t i n s e c r e t i o n i n mammals (see review by Meites and N i c o l l , 1966). Thus, the suggestion of p r o l a c t i n a c t i o n v i a s t i m u l a t i o n of TSH and t h y r o x i n e s e c r e t i o n i s a p o s s i b l e but h i g h l y improbable one. Another p o s s i b i l i t y i s p r o l a c t i n a c t i o n v i a s t i m u l a t i o n of the i n t e r r e n a l (the homolog of* the mammalian adrenal c o r t e x ) . P r o l a c t i n seems to have ACTH-like effects., on e l e c t r o l y t e r e g u l a t i o n i n F. kansae ( B a l l and Fleming, unpublished, c i t e d by B a l l , 1969; B a l l and Ensor, 1968) and Myxine (Chester Jones e t a l . , 1962). These authors have suggested th a t the 132 i n j e c t e d p r o l a c t i n i n these f i s h acted as a mimic of endogenous ACTH and s t i m u l a t e d i n t e r r e n a l s e c r e t i o n . F u r t h e r -more, Chambolle (1967) claimed t h a t ACTH was as e f f e c t i v e as p r o l a c t i n i n the freshwater s u r v i v a l of hypophysectomized Gambusia. However, p r o l a c t i n has not been shown to s t i m u l a t e the i n t e r r e n a l i n F_. h e t e r o c l i t u s ( P i c k f o r d and Kosto, 1957), l a t i p i n n a ( B a l l and Ensor, 1968), and A. a n g u i l l a (Chan e t a l . , 1968). By f a r the greater amount of evidence shows th a t p r o l a c t i n i s h i g h l y s p e c i f i c i n i t s a c t i o n s . Thus i n F_. h e t e r o c l i t u s , the a b i l i t y to promote freshwater s u r v i v a l a f t e r hypophysectomy i s unique to p r o l a c t i n ; i t i s not shared by th y r o x i n e , ACTH, TSH, growth hormone, ACTH-TSH-growth hormone combination, p o s t e r i o r lobe e x t r a c t , a r g i n i n e v a s o t o c i n , i s o t o c i n , urophyseal e x t r a c t s , DOC, C o r t i s o l , a l d o s t e r o n e , , c o r p u s c l e s of Stannius. e x t r a c t s , hog r e n i n and p a r a t h y r o i d hormone ( p i c k f o r d e t a l . , 1965). S i m i l a r l y , i n hypophy-sectomized X. maculatus, the f r e s h w a t e r - s u r v i v a l property of p r o l a c t i n i s not shared by va s o p r e s s i n , o x y t o c i n , growth hormone, TSH and ACTH (Schreibman and Kallman, 1966). A l s o , i n _T. mossambica, p r o l a c t i n , but not ACTH, w i l l ensure freshwater s u r v i v a l a f t e r hypophysectomy. (Handin et a l . ( 1964; Dharmamba e t a l . , 1967). Following, the f i n d i n g t h a t ovine p r o l a c t i n prevents the r a p i d f a l l i n plasma sodium i n hypophysectomized P_. l a t i p i n n a i n f r e s h water ( B a l l and Ensor, 1965), t h i s a c t i o n was shown to be a s p e c i f i c p r o p e r t y of p r o l a c t i n . I n e f f e c t i v e hormones, 133 each t e s t e d a t a low and a h i g h dose, were o x y t o c i n , v a s o p r e s s i n , a r g i n i n e v a s o t o c i n , i s o t o c i n , ACTH, growth hormone, TSH and alpha MSH. Gonadotrophins were excluded, s i n c e n a t u r a l . o r experimental a l t e r a t i o n s i n the p i t u i t a r y - o v a r y a x i s were found not to a f f e c t s u r v i v a l i n f r e s h water ( B a l l and Ensor, 1967). F u r t h e r evidence o f the s p e c i f i c i t y of the sodium-conserving a c t i v i t y of p r o l a c t i n , t h i s time of the f i s h ' s own p r o l a c t i n i t s e l f , comes from e c t o p i c p i t u i t a r y transplants- i n P_. formosa. In these .preparations, some f u n c t i o n s pass i n t o t o t a l or p a r t i a l obeyance (gonadotrophins, ACTH, growth hormone, MSH), but the s e c r e t i o n of p r o l a c t i n and TSH p e r s i s t s and so. does the sodium-conserving a c t i v i t y ( B a l l e t a l . , 1965 and unpublished, c i t e d by B a l l , 1969). One would not expect these r e s u l t s i f the sodium-conserving a c t i v i t y were due to ACTH, gonadotrophins, MSH or growth hormone, alone or i n combination; and the involvement of endogenous TSH .is u n l i k e l y , s i n c e treatment w i t h thiourea, d i d not a f f e c t the a c t i v i t y ( B a l l , 1969). The p i t u i t a r y h i s t o p h y s i o l o g y of F_. h e t e r o c l i t u s , £.* l a t i p i n n a and T_. mossambica, i n r e l a t i o n to freshwater a d a p t a t i o n , a l s o a t t e s t s to the e x c l u s i v e involvement i n t h i s r e g a r d of the e t a c e l l s which have been shown to s e c r e t e p r o l a c t i n (see I n t r o d u c t i o n and a l s o B a l l , 1968, 1969). Thus i t seems l i k e l y t h a t the p r o l a c t i n e f f e c t s i n the s t i c k l e b a c k are a l s o s p e c i f i c , i f not a l l of the e f f e c t s , a t 134 l e a s t the sodium-conserving e f f e c t which, as mentioned i n Chapter 4 and i n the Synopsis, was remarkably s i m i l a r to t h a t i n hypophysectomized P_. l a t i p i n n a . The i n v i t r o g i l l e x p e r i -ments a l s o showed t h a t the p r o l a c t i n a c t i o n i n reducing osmotic i n f l u x of water was a d i r e c t one. This i s supported by the work of Chan e_t a l . (1968) who showed that p r o l a c t i n , and not ACTH nor C o r t i s o l , was able to prevent the hyperhydration of muscle i n hypophysectomized freshwater A. a n g u i l l a . Two p o s s i b i l i t i e s , however, must remain, a t l e a s t f o r the s t i c k l e b a c k . In the f i r s t p l a c e , w h i l e some p r o l a c t i n e f f e c t s (presumably the more important ones) may be d i r e c t and s p e c i f i c ; others may be i n d i r e c t . In the second p l a c e , w h i l e some p r o l a c t i n e f f e c t s (the h i g h l y s p e c i f i c ones.) may not r e q u i r e a d d i t i o n a l hormonal f a c t o r (s) as s y n e r g i s t ( s ) , others may. In T. mossambica, p r o l a c t i n o n l y p a r t i a l l y prevented the drop i n plasma o s m o l a l i t y f o l l o w i n g the t r a n s f e r of hypophysectomized f i s h to f r e s h water, which could not be e x p l a i n e d i n terms of dose-response r e l a t i o n s h i p (Dharmamba, e t aly 1967). The authors suggested t h a t a d d i t i o n a l hormonal f a c t o r (s) may a c t as s y n e r g i s t ( s ) w i t h p r o l a c t i n i n t h i s r e g a r d i n the f i s h . S i m i l a r l y , the f a c t t h a t p r o l a c t i n has o n l y so f a r been shown to produce an e f f e c t on mucous c e l l s i n i n t a c t f i s h (see Chapter 7) suggests the p o s s i b i l i t y t h a t synergism of p r o l a c t i n w i t h other hormone(s) i s r e q u i r e d f o r the r e a l i z a t i o n of t h i s e f f e c t . Analogy may be drawn here to the c o n t r o l of l a c t a t i o n i n mammals, which requires the 135 s y n e r g i s t i c a c t i o n of p r o l a c t i n w i t h a number of other hormones (see review by Meites and N i c o l l , 1966). The s t i c k l e b a c k has been shown to be " p h y s i o l o g i c a l l y hypophysectomized" i n the autumn and winter o n l y w i t h regards to p r o l a c t i n (present s t u d i e s ) and gonadotropins (Baggerman, 1957; Hoar, 1962; Ahsan and Hoar, 1963); synergism of the exogenous p r o l a c t i n w i t h other p i t u i t a r y hormones besides gonadotropins must t h e r e f o r e remain a p o s s i b i l i t y . Conclusions Whether i t s e f f e c t s were d i r e c t or i n d i r e c t , or whether synergism w i t h other hormones was r e q u i r e d , p r o l a c t i n was shown i n these- studies, to be e s s e n t i a l f o r the freshwater osmotic and i o n i c r e g u l a t i o n of the s t i c k l e b a c k , Gasterosteus aculeatus, form trachurus. The evidence suggests the involvement of t h i s hormone i n the anadromous m i g r a t i o n of the f i s h i n the f o l l o w i n g manner:-During the autumn and winter when s t i c k l e b a c k s l i v e i n sea water or b r a c k i s h water, l i t t l e or no p r o l a c t i n i s secreted, even when the f i s h are subjected to freshwater c o n d i t i o n s ; thus, a t t h i s time of the year s t i c k l e b a c k s cannot l i v e - i n f r e s h water. As s p r i n g i s approached, due to the i n c r e a s i n g daylength (or in c r e a s e d daylength), i n c r e a s i n g q u a n t i t i e s of p r o l a c t i n are secreted, or can be secreted under appropriate environmental s t i m u l i , preparatory to freshwater m i g r a t i o n i n the s p r i n g . The l e v e l of s e c r e t i o n f i n a l l y reaches i t s optimum i n l a t e s p r i n g and summer when the f i s h are i n f r e s h 136 water. W h i l s t i n f r e s h water, p r o l a c t i n enables the f i s h to osrao- and i o n - r e g u l a t e e f f i c i e n t l y and consequently promotes s u r v i v a l of the f i s h i n t h i s medium. When the f i s h r e t u r n s to sea water i n the autumn, s e c r e t i o n of p r o l a c t i n ceases or i s reduced to a minimal l e v e l , probably because of the decreasing daylength (or decreased daylength). The d e t a i l s of the above s t o r y may have to be m o d i f i e d as r e s e a r c h proceeds, but the essence of i t i s b e l i e v e d to be c o r r e c t . 137 L i t e r a t u r e C i t e d Ahsan, S.N. and Hoar, W.S. (1963). Some e f f e c t s of gonadotropic hormones on the three-spine s t i c k l e b a c k , Gasterosteus  a c u l e a t u s . Can. J . Zool., 41, 1045-1053. A u l l , F. (1966). Absorption of f l u i d from i s o l a t e d i n t e s t i n e of the t o a d f i s h , Opsanus tau, Comp. Biochem. P h y s i o l . , ... 17, 867-870. Baggerman, B. 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