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Studies of the sheep neurophypophysis during pregnancy and foetal development Vizolyi, Elizabeth 1968

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STUDIES OF THE SHEEP NEUROHYPOPHYSIS DURING PREGNANCY AND FOETAL DEVELOPMENT by ELIZABETH VIZSOLYI B.Sc, Eotvos Lorand University, Budapest, 1954 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1968 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 a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and 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 p u rposes may be g r a n t e d by the Head o f my Department or 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 of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date ^QHfAXH ABSTRACT The b i o l o g i c a l a c t i v i t i e s of the neurohypophyses of pregnant and embryonic sheep, Ovis a r i e s were s t u d i e d . The l e v e l s of hormonal a c t i v i t i e s of the neurohypophyses v a r i e d w i t h the stage of pregnancy, and were always lower than l e v e l s i n the c o n t r o l (non-pregnant) glands. Chemical s t u d i e s of the p u r i f i e d o x y t o c i c and vasopressor moieties of the pregnant sheep have i n d i c a t e d t h a t these agents are o x y t o c i n and a r g i n i n e v a s o p r e s s i n . An increase of b i o l o g i c a l l y a c t i v e agents of the p o s t e r i o r p i t u i t a r y was detected concomitant w i t h embryonic development. The amount of vasopressor a c t i v i t y g r e a t l y surpassed the o x y t o c i c a c t i v i t y i n the neurohypophyses of f o e t a l sheep a l l through g e s t a t i o n , but the d i f f e r e n c e became sm a l l e r w i t h the advancement of i n t r a u t e r i n e l i f e . Pharmacological and chemical s t u d i e s i n d i c a t e d o x y t o c i n and a r g i n i n e v a s o p r e s s i n as the a c t i v e peptides of the f o e t a l neurohypophysis. In a d d i t i o n to these two neurohypophysial peptides c h a r a c t e r i s t i c of the mammals, the presence of a t h i r d p e p t i d e was i n d i c a t e d . Pharmacological s t u d i e s i n d i c a t e t h a t t h i s t h i r d neurohypophysial peptide of the foetuses may be a r g i n i n e v a s o t o c i n , the a n t i d i u r e t i c p r i n c i p l e of lower v e r t e b r a t e s . i i TABLE OP CONTENTS Page A b s t r a c t i L i s t of Tables v L i s t of P l a t e s and Figures v i Abb r e v i a t i o n s Used i x Acknowledgements x i I n t r o d u c t i o n 1 The Hypothalamo-Hypophyseal System of the Ad u l t Mammal 1 The Neurohypophysis d u r i n g Pregnancy and L a c t a t i o n 4 The Hypothalamo-Hypophysial System of the Mammalian Foetus 5 Statement of the Problem 7 M a t e r i a l s and Methods '.. 9 C o l l e c t i o n and Storage of M a t e r i a l s 9 E x t r a c t i o n 10 E s t i m a t i o n of B i o l o g i c a l A c t i v i t i e s 10 a. I s o l a t e d r a t uterus o x y t o c i c assay 11 b. Rat vasopressor assay ; 13 c. Rat a n t i d i u r e t i c assay 15 d. Frog waterbalance assay 16 Standards 18 Methods of P u r i f i c a t i o n 18 a. Gel f i l t r a t i o n 18 b. Paper chromatography 20 Chemical Methods f o r the A n a l y s i s of Peptides . 21 a. E s t i m a t i o n of t o t a l peptide c o n c e n t r a t i o n 21 b. H y d r o l y s i s of the peptides 22 Page i i i c. Amino a c i d a n a l y s i s . 7 23 S t a t i s t i c a l Methods 25 Se c t i o n I Studies of the Pregnant Sheep 27 1. B i o l o g i c a l a c t i v i t i e s of the crude e x t r a c t s . . 27 2. P u r i f i c a t i o n and amino a c i d a n a l y s i s of the a c t i v e p r i n c i p l e s of the pregnant sheep 32 a. P u r i f i c a t i o n by g e l f i l t r a t i o n 35 b. Amino a c i d a n a l y s i s 40 D i s c u s s i o n (Section I) 47 S e c t i o n I I .Studies of the B i o l o g i c a l A c t i v i t i e s and the Chemical Nature of the A c t i v e Agents i n the Neurohypophysis of the F o e t a l and Newborn sheep ... 52 1. B i o l o g i c a l a c t i v i t i e s of the f o e t a l neurohypophysis 52 a. Acetone d r i e d t i s s u e 53 b. L y o p h i l i s e d t i s s u e 55 c. Comparison of b i o l o g i c a l a c t i v i t i e s i n acetone d r i e d and l y o p h i l i s e d glands 57 2. P u r i f i c a t i o n and amino a c i d a n a l y s i s of the a c t i v e agents i n the neurohypophyses of sheep foetuses 61 a. P a r t i a l p u r i f i c a t i o n of f o e t a l neurohypophysial p r i n c i p l e s by paper chromatography 63 b. Gel f i l t r a t i o n of crude e x t r a c t s from l a t e foetuses 69 c. Amino a c i d a n a l y s i s of the p u r i f i e d peptides of the l a t e foetuses 73 D i s c u s s i o n (Section I I ) «, 77 General D i s c u s s i o n 82 1. Changes i n the hormonal l e v e l of the neurohypophysis d u r i n g pregnancy 83 iv Page 2. The possible r o l e of the a n t i d i u r e t i c p r i n c i p l e during embryonic development 86 3. The importance of the presence of arginine vasotocin i n the foetus 89 Summary 95 L i t e r a t u r e Cited 98 V LIST OF TABLES Table Page I Potencies and the r a t i o s of the potencies i n the acetone d r i e d t i s s u e s of pregnant sheep 30 I I Potencies and the r a t i o s o f the potencies i n the l y o p h i l i s e d t i s s u e s of pregnant sheep 31 I I I Potencies and the r a t i o s of potencies i n the acetone d r i e d t i s s u e s of sheep foetuses and lambs 54 TV Potencies and the r a t i o s of the potencies i n the l y o p h i l i s e d t i s s u e s of sheep foetuses and lambs 56 v i LIST OF PLATES AND FIGURES Facing P l a t e Page 1 Diagram of the p i t u i t a r y of the sheep x F i g u r e 1 Sample records from r a t uterus, vasopressor-and a n t i d i u r e t i c assays 28 2 Weighted means of o x y t o c i c , vasopressor and a n t i d i u r e t i c a c t i v i t i e s i n the acetone d r i e d neurohypophyses of c o n t r o l and pregnant sheep 33 3 Weighted means of o x y t o c i c , vasopressor and a n t i d i u r e t i c a c t i v i t i e s i n the l y o p h i l i s e d neurohypophyses of c o n t r o l and pregnant sheep 34 4 Comparison between the b i o l o g i c a l a c t i v i t i e s of the acetone d r i e d and l y o p h i l i s e d neuro-hypophyses of c o n t r o l and pregnant sheep .... 36 5 R a t i o s of b i o l o g i c a l a c t i v i t i e s i n acetone d r i e d and l y o p h i l i s e d neurohypophyses of c o n t r o l and pregnant sheep 37 6 P u r i f i c a t i o n of the a c t i v e neurohypophysial agents of pregnant sheep by the use of 100 cm Sephadex G-15 column 39 v i i F a c i ng F i g u r e Page 7 P u r i f i c a t i o n of the b i o l o g i c a l l y a c t i v e p r i n c i p l e s from the crude e x t r a c t s of pregnant sheep neurohypophyses by the use of 200 cm Sephadex G-15 column 41 8 Sample p l a t e of the t h i n l a y e r chromatography of dansyl d e r i v a t i v e s of standard amino acid s and the o x y t o c i c and vasopressor moieties from sheep p o s t e r i o r p i t u i t a r y e x t r a c t s 43 .9 Graphed values of dansylated standard amino acid s and the o x y t o c i c and vasopressor mo i e t i e s of the sheep neurohypophysis 45 10 Weighted means of b i o l o g i c a l a c t i v i t i e s i n acetone d r i e d neurohypophyses of sheep foetuses and lambs 58 11 Ratios of b i o l o g i c a l a c t i v i t i e s i n the acetone d r i e d neurohypophyses of sheep foetuses and lambs . 60 12 Weighted means of b i o l o g i c a l a c t i v i t i e s i n the l y o p h i l i s e d neurohypophyses of sheep foetuses and lambs 0 62 13 Ratios of b i o l o g i c a l a c t i v i t i e s i n the l y o p h i l i s e d neurohypophyses of sheep foetuses and lambs 64 v i i i Facing Figure Page 14 Comparison of r a t i o s of b i o l o g i c a l a c t i v i t i e s between the acetone dried and l y o p h i l i s e d tissue of sheep foetuses and lambs .. 66 15 Paper chromatogram of crude extracts of the neurohypophyses of sheep foetuses at 89-91 days of gestational age 68 16 P u r i f i c a t i o n of crude extracts of sheep foetuses at 138-141 days of gestational age, on 200 cm Sephadex G-15 column 70 17 Sample plate of t h i n layer chromatography of the dansyl derivatives of standard amino acids and of the oxytocic moiety of the adult pregnant sheep and the foetus 72 18 Graphed values of dansylated standard amino acids, and the dansylated hydrolysate of the f o e t a l vasopressor moiety 74 i x ABBREVIATIONS USED a l a - a l a n i n e l e u - l e u c i n e a r g - a r g i n i n e l y s - • l y s i n e asp - a s p a r t i c a c i d met - methionine cyS - c y s t i n e p ro - p r o l i n e Cyt - c y s t e i c a c i d phe - phenylalanine g l u - glutamic s e r - s e r i n e g l y - g l y c i n e t y r - t y r o s i n e h i s - h i s t i d e i n e t h r e - threonine i l e u - i s o l e u c i n e v a l - v a l i n e X PLATE I Diagram of the p i t u i t a r y of the sheep Ovis a r i e s . Mid s a g i t t a l plane. A f t e r : P.M. D a n i e l and M a r j o r i e M.L. P i c k f o r d , 1957. Infundibular stem T bird ventricle Optic chiasma Pituitary sta lk Neural (posterior) ! obe Pars intermedia Pars distaiis x i ACKNOWLEDGEMENTS Among the many people who aided me d u r i n g my s t u d i e s and research, I would l i k e to mention o n l y a few whose c o n t r i b u t i o n s were e s p e c i a l l y v a l u a b l e . Dr. A.M. PerTcs, my su p e r v i s o r , who not o n l y suggested the research t o p i c and aided i n i t s progress w i t h h i s advice, but who a l s o c o l l e c t e d a l l the t i s s u e s used i n the course of t h i s i n v e s t i g a t i o n . Dr. G.S. Dawse of the N u f f i e l d I n s t i t u t e , who k i n d l y made the m a t e r i a l a v a i l a b l e . Miss L i i s Mirk, who c a r r i e d out the t h i n l a y e r chromatography; Mr. Steve Borden, who helped w i t h the s t a t i s t i c a l a n a l y s i s of the r e s u l t s ; Mr. E.J. McCulloch and Mr. D. Anderson f o r t h e i r t e c h n i c a l advice. Research C o u n c i l Grant No. 680646 covered a l l the costs of the research. I wish to thank a l l the above people f o r t h e i r c o n t r i b u t i o n t o the completion of t h i s work. 1 INTRODUCTION The Hypothalamo-Hypophyseal System of the Adult Mammal Scharrer (1944, 1954) and Bargman (1951) u s i n g Gomori's chrom-alum haematoxylin p h l o x i n s t a i n , demonstrated the presence of neurosecretory granules i n the sup r a o p t i c and p a r a - v e n t r i c u l a r n u c l e i of the hypothalamus. These same granules-were d i s t r i b u t e d along the axons of nerve f i b e r s , l e a d i n g from the n u c l e i to the pars nervosa, and w i t h i n the pars nervosa i t s e l f . F u r ther h i s t o l o g i c a l s t u d i e s f o l l o w i n g s e c t i o n s of the t r a c t from the hypothalamus to the p o s t e r i o r lobe, showed accumulation of s e c r e t o r y m a t e r i a l r o s t r a l to the cut, i n d i c a t i n g the d i r e c t i o n of movement of the neurosecretory m a t e r i a l . These workers formulated the g e n e r a l l y accepted hypothalamo-hypophyseal concept of the p o s t e r i o r p i t u i t a r y , which pointed to the hypothalamus as the p l a c e of s y n t h e s i s , and the pars nervosa as the storage organ f o r the neurohypophyseal hormones. The p h y s i o l o g i c a l a c t i o n s of p o s t e r i o r lobe e x t r a c t s were discovered j u s t before the t u r n of the century. I n j e c t i o n s of the e x t r a c t s caused e l e v a t i o n of blood pressure ( O l i v e r and Shafer, 1895), c o n t r a c t i o n s of the uterus (Dale, 1906) and e j e c t i o n of mi l k from the mammary gland (Ott and Scott, 1910). The a c t i v e agents were separated i n 1928 by Kamm, A l d r i c h , Grote, Rowe and Bugbee, i d e n t i f i e d and sy n t h e t i s e d by 2 du Vigneaud, R e s s l e r , Swan, Roberts, Katsoyannis and Gordon i n 1953. The a c t i v e p r i n c i p l e s were i d e n t i f i e d as two c y c l i c o ctapeptides, o x y t o c i n and v a s o p r e s s i n ; they shared a molecular weight of approximately 1,000, but d i f f e r e d from one another i n two amino a c i d s . The primary a c t i o n o f o x y t o c i n i s on•the mammary gland d u r i n g l a c t a t i o n , where i t i s r e s p o n s i b l e f o r the e j e c t i o n of milk, as a response to s u c k l i n g by the young. I t s a b i l i t y to c o n t r a c t the uterus i s a l s o w e l l demonstrated, and i t appears to cause rythmic c o n t r a c t i o n s d u r i n g labour. Various secondary a c t i o n s can be e l i c i t e d by the a d m i n i s t r a t i o n of o x y t o c i n , and among them are the f o l l o w i n g : d i l a t i o n of blood v e s s e l s i n the e x t r e m i t i e s , increased glomerular f i l t r a t i o n r a t e i n the kidney, and a p o s s i b l e c a p a c i t y to enhance e l e c t r o l y t e e x c r e t i o n by the kidneys ( P i c k f o r d , 1960, 1964). These secondary responses were e l i c i t e d by pharmacolog-i c a l dose l e v e l s of the hormone; consequently, t h e i r importance to the organism under p h y s i o l o g i c a l c o n d i t i o n s i s f a r from proven. The f i r s t a c t i o n of vasopressin to be discovered was i t s pharmacological a c t i o n i n causing c o n s t r i c t i o n of blood v e s s e l s ( O l i v e r and Shafer, 1895); t h i s p roperty of v a s o p r e s s i n i s s t i l l commonly used to d e t e c t and assay the p e p t i d e i n e x t r a c t s of the p o s t e r i o r lobe. The p h y s i o l o g i c a l s i g n i f i c a n c e of vasopressin, however, l i e s i n i t s very potent a n t i - d i u r e t i c e f f e c t . I t promotes water reabsorption by a c t i n g on the d i s t a l tubules and c o l l e c t i n g ducts of the 3 kidney. Besides i t s a c t i o n on membranes i n the kidney, i t i s a t t r i b u t e d w i t h the c a p a c i t y to a c t s i m i l a r l y on membranes of other organs. Wakim (1966) has summarized the a v a i l a b l e evidence f o r some of the e x t r a r e n a l a c t i o n s e l i c i t e d by the hormone: a f t e r i n j e c t i o n s of v a sopressin there i s a marked f a l l i n the e x c r e t i o n of f l u i d from the pancreas, from the l i v e r , and from the sweat glands. Water absorption through the i s o l a t e d f r o g s k i n i s a l s o increased i n the presence of t h i s hormone (Ussing and Anderson, 1957). The accumulated data shows t h a t v a s o p r e s s i n acts on the membranes of w i d e l y v a r i e d organs, w i t h the same end r e s u l t - waterconservation. The r a t i o of v a s o p r e s s i n / o x y t o c i n (V/0) i s approximately one i n the p o s t e r i o r lobe of most mammals. The hormones are stored attached t o , and probably r e l e a s e d accompanied by, a l a r g e p r o t e i n molecule (M.W. 25000) named neurophysine ( H e l l e r and Ginsburg, 1966). On each neurophysine molecule there i s one b i n d i n g s i t e f o r o x y t o c i n and another f o r v a s o p r e s s i n . The s i t e s are s p e c i f i c f o r the t e r m i n a l amino acids of the r e s p e c t i v e octapeptide (Ginsburg, 1964). B i n d i n g of the t e r m i n a l amino acids does not i n a c t i v a t e the hormones, the complex e x h i b i t s both vasopressor and o x y t o c i c a c t i v i t i e s (van Dyke, _et a l . , 1954). The e x i s t a n c e of a common c a r r i e r f o r the two octapeptides, r a i s e s one of the most i n t e r e s t i n g , and s t i l l unresolved questions, concerning the mechanism of r e l e a s e of the hormones. The r e l e a s e of one neurohypophyseal peptide i s u s u a l l y accompanied by the r e l e a s e of the other, i n accord w i t h the e x i s t a n c e of a common c a r r i e r , however, 4 under many circumstances one of the hormones i s relea s e d i n much greater q u a n t i t i e s than the other. F o l l o w i n g haemorrhage i n anaesthetised r a t s ( H e l l e r , 1961), or the severe dehydration of dogs and r a t s ( P i c k f o r d , 1964), a p r e f e r e n t i a l r e l e a s e of a n t i d i u r e t i c hormone occurs. On the other hand, l a c t a t i o n appears to p r e f e r e n t i a l l y deplete the gland of i t s s t o r e d o x y t o c i n . The mechanism by which t h i s d i f f e r e n t i a l r e l e a s e i s accomplished i s under extensive i n v e s t i g a t i o n (Ginsburg, 1964; Dicker, 1966), but remains unresolved at the present. The Neurohypophysis d u r i n g Pregnancy and L a c t a t i o n During pregnancy, an a d d i t i o n a l burden i s placed on the water r e g u l a t o r y system, and i t i s r a t h e r c r u c i a l to keep the uterus quiescent, t o prevent e x p u l s i o n of the foetus. C o n s i d e r i n g t h a t the main a c t i o n s of the neurohypophysial hormones are on water r e g u l a t i o n and on the uterus, a change i n the l e v e l of stored neurohypophysial hormones would not be unexpected. However, d i r e c t s t u d i e s on r a t neuro-hypophyses by Acher, Chauvet and O l i v r y (1956) and by H e l l e r and L e d e r i s (1959), f a i l e d to show any changes i n e i t h e r the mean hormonal content of the gland, or i n the vasopressor/ o x y t o c i c (V/0) r a t i o s at any stage of pregnancy. Their s t u d i e s d i d not i n c l u d e animals s h o r t l y before, or at p a r t u r i t i o n . Although no changes i n the neurohypophysial hormone l e v e l s were detected d u r i n g pregnancy i n the r a t , other evidence ( H e l l e r , 1957) i n d i c a t e s an i n f l u e n c e of the rep r o d u c t i v e c y c l e on the l e v e l of the p o s t e r i o r p i t u i t a r y 5 hormones. In the r a t the amounts of vasopressin and o x y t o c i n were found to r i s e d u r i n g the f o l l i c u l a r phase and to drop a t metestrous. In the same species, the d i u r e t i c response to. water was a l s o impaired immediately a f t e r c e s s a t i o n of estrous (Ginsburg, 1950 as quoted by H e l l e r , 1961). L a c t a t i o n depletes the p o s t e r i o r p i t u i t a r i e s of dogs and r a t s (van Dyke, Adamson and Engel, 1955; H e l l e r and L e d e r i s , 1959) of i t s o x y t o c i c and vasopressor a c t i v i t i e s . Some authors have reported t h a t the V/0 r a t i o r i s e s due to a p r e f e r e n t i a l d e p l e t i o n of o x y t o c i n (Acher, Chauvet, O l i v r y , 1956; van Dyke et a l . , 1955) but others have found i t u n a l t e r e d , (Maculay, Landgrebe and Waring 1950). H e l l e r (1961) have pointed out t h a t those workers who u t i l i z e d acetone d r i e d glands observed a r i s e i n V/0 r a t i o , but those who worked on f r e s h glands found the r a t i o u n a l t e r e d . He has suggested t h a t the apparent c o n t r a d i c t i o n i n these r e s u l t s can be explained on the b a s i s of a p r e f e r e n t i a l s o l u b i l i t y o f o x y t o c i n i n acetone. The Hypothalamo-Hypophysial System of the Mammalian Foetus H i s t o l o g i c a l s t u d i e s of the embryonic development of the hypothalamo-hypophysial system are f a r more numerous than s t u d i e s of i t s hormonal content. Bernischke and McKay (1953) and Meitner (1961), working i n the human, found the f i r s t n eurosecretory m a t e r i a l i n the hypothalamic n u c l e i i n the f o u r t h month of g e s t a t i o n . Only a f t e r a f u r t h e r three weeks of development, could s e c r e t o r y m a t e r i a l be detected i n the 6 p o s t e r i o r lobe of the p i t u i t a r y . These observations are confirmed by s t u d i e s i n the cow ( K i v a l o and T a l a n t i , 1957), mouse, r a t and dog embryos (Yakovleva, 1966). In a l l the mammalian foetuses so f a r s t u d i e d , some of the workers a l s o noted an e a r l i e r appearance and greater abundance of s e c r e t o r y m a t e r i a l i n the supra-optic than i n the para-v e n t r i c u l a r nucleus (Yakovleva, 1966). These f i n d i n g s p o i n t t o an e a r l i e r embryonic development of the hypothalamic n u c l e i than the p o s t e r i o r lobe of the p i t u i t a r y , and to an e a r l i e r appearance of the su p r a - o p t i c than the para-v e n t r i c u l a r nucleus. There i s l i t t l e evidence a v a i l a b l e concerning the b i o l o g i c a l a c t i v i t i e s present i n the f o e t a l neurohypophysis. Oxytocic and vasopressor a c t i v i t i e s have been detected by Dic k e r and T y l e r (1953) i n the p o s t e r i o r p i t u i t a r i e s of dog, cat and human foetuses. They found a r a t i o of vasopressor to o x y t o c i c a c t i v i t y as h i g h as 28 to 1 i n the human foetus at 112 days of g e s t a t i o n . At 190 days of i n t r a u t e r i n e l i f e the r a t i o f a l l s t o 5 and reaches u n i t y at b i r t h . In the cat where the V/0 r a t i o i n the a d u l t i s 1.2, the r a t i o at 54 days of g e s t a t i o n a l age i s 4.7, and a t 21 days of post partum i s s t i l l as high as 3.3. These r e s u l t s i l l u s t r a t e the pre-dominance of vasopressor a c t i v i t y over the o x y t o c i c a c t i v i t y d u r i n g i n t r a u t e r i n e l i f e . The vasopressor a c t i v i t y remains higher u n t i l b i r t h i n human and even long a f t e r b i r t h i n the ca t . I t i s suggested t h a t the r e l a t i v e m a t u r i t y of the animal at b i r t h determines how near to u n i t y the r a t i o between 7 these two a c t i v e p r i n c i p l e s i s at t h i s time. Although i n the human the s t u d i e s of the neruo-hypophysis d u r i n g embryonic development, performed by Di c k e r and T y l e r , are r e l a t i v e l y complete, human t i s s u e u s u a l l y i s a v a i l a b l e o n l y a considerable time a f t e r death. In view of the very r a p i d d e p l e t i o n of the p o s t e r i o r p i t u i t a r y of i t s a c t i v e agents f o l l o w i n g death, a general breakdown of t i s s u e s post mortem could have c o n t r i b u t e d to the low potencies and high r a t i o s reported by these authors i n the human foetuses. Further doubt i s cas t on these r e s u l t s by H e l l e r (1961), who again suggests, t h a t these high r a t i o s , at l e a s t i n p a r t , could be a t t r i b u t e d to the e f f e c t of acetone, used i n c o l l e c t -i n g the glands: "The p r o p o r t i o n of vasopressor to o x y t o c i c a c t i v i t y i n immature p o s t e r i o r p i t u i t a r y glands have been reported t o be very h i g h . But H e l l e r and Le d e r i s could r e c e n t l y show, t h a t i n i n f a n t r a t s such high r a t i o s were obtained o n l y when glands were t r e a t e d w i t h acetone before the usual e x t r a c t i o n i n d i l u t e a c i d . " No attempts have been made p r e v i o u s l y , to i d e n t i f y the a c t i v e agents i n the f o e t a l neurohypophysis, by e i t h e r pharmacological or chemical means. The only i n d i c a t i o n to the p o s s i b l e nature of these a c t i v e peptides are the st u d i e s of H e l l e r (1959), performed i n immature r a t s . By paper-chroma to graphic s t u d i e s he i d e n t i f i e d the a c t i v e agents i n the immature r a t neurohypophysis as o x y t o c i n and vasop r e s s i n . Statement of the Problem Previous work on the pharmacology of the neuro-hypophysis d u r i n g pregnancy, i s l i m i t e d to one species only, the r a t . The neurohypophyses of very few species have been s t u d i e d d u r i n g t h e i r f o e t a l development. In the foetus, o n l y the r e s u l t s obtained i n the human are a t a l l extensive, but they are of u n c e r t a i n value, s i n c e the t i s s u e s may w e l l have been obtained a considerable time a f t e r death. Only the s t u d i e s i n the cat are c l e a r l y r e l i a b l e , and these are l i m i t e d to one g e s t a t i o n a l age o n l y . In the work performed here, i n v e s t i g a t i o n s were extended to a f u r t h e r species, the sheep, Ovis a r i e s . A l l t i s s u e s were d i s s e c t e d immediately a f t e r death and at known stages of pregnancy. Healthy foetuses were d e l i v e r e d by c a e s a r i a n s e c t i o n , and t h e i r neurohypophyses were compared d i r e c t l y w i t h those of t h e i r mothers. Due to the considerable doubt c a s t on r e s u l t s obtained i n acetone d r i e d glands, p a r a l l e l s t u d i e s were c a r r i e d out on acetone d r i e d and l y o p h i l i s e d t i s s u e s . Since there i s no previous data on the chemical nature of the a c t i v e agents i n the foetus, p r e l i m i n a r y e f f o r t s were a l s o d i r e c t e d towards the i d e n t i f i c a t i o n of these agents. 9 MATERIALS AND METHODS C o l l e c t i o n and Storage of M a t e r i a l s P o s t e r i o r p i t u i t a r i e s from sheep Ovis a r i e s of the Clunrose breed were c o l l e c t e d i n the 1964 and 1965 breeding seasons. The samples were taken from 3 year o l d pregnant females and from t h e i r foetuses, at known g e s t a t i o n a l ages, together w i t h c o n t r o l s from non-breeding 3 year o l d females. The animals were anaesthetised by intravenous i n j e c t i o n of C h l o r a l o s e (30'mg/Kg as a 40 mg/ml s o l u t i o n ) , an a n a e s t h e t i c reported to be without any e f f e c t on the hormone content of the p o s t e r i o r p i t u i t a r y (Ginsburg and Brown, 1956). The foetuses were d e l i v e r e d by Caesarian s e c t i o n and the p l a c e n t a t i e d o f f and cut. The p o s t e r i o r lobe of the p i t u i t a r y was removed from the pregnant female and the foetus i n the f o l l o w i n g manner: The s u p e r f i c i a l s k i n was removed w i t h a s c a l p e l , the cranium cut w i t h a Desouter Saw, t o expose the e n t i r e b r a i n . The f r o n t a l end of the b r a i n was l i f t e d up, and proceeding c a u d a l l y the o p t i c nerves, the i n f u n d i b u l a r stem, the c r a n i a l nerves, and a l l other connections to the c r a n i a l f l o o r were severed, exposing the p i t u i t a r y . Then the e n t i r e p i t u i t a r y was removed from the s e l l a t u r c i c a , and separated i n t o the a n t e r i o r and p o s t e r i o r lobes. The p o s t e r i o r lobes 10 were d r i e d e i t h e r i n acetone (1964 season) or l y o p h i l i s e d (1965 season). The acetone d r i e d t i s s u e s were d i s s e c t e d i n t o acetone which had been d r i e d over CaC^. The acetone was decanted and exchanged at l e a s t three times du r i n g the f i r s t 24 hours. The glands were then subjected to prolonged acetone e x t r a c t i o n by being stored f o r 12-14 months i n dry acetone at room temperature. A f t e r removal from the f l u i d , the glands were exposed to a i r t o remove r e s i d u a l acetone, and then sealed i n t o g l a s s tubes and sto r e d a t 4°C u n t i l the time of e x t r a c t i o n . L y o p h i l i s e d t i s s u e s were d i s s e c t e d i n t o g l a s s tubes submerged i n a d r y - i c e methanol mixture. The frozen samples were placed on a vacuum pump, evacuated at 0.1 mm Hg pressure f o r 24 hours, and sto r e d at room temperature over P2O5 u n t i l the end of the season. The samples were sealed a t the end of the season and stored at 4°C u n t i l e x t r a c t i o n . E x t r a c t i o n S i n g l e glands of a d u l t s or foetuses were homogenized i n 0.25% a c e t i c a c i d at 2 mg/ml w i t h a Thomas E x t r a c t o r . The homogenate was heated f o r 3 minutes i n a b o i l i n g water-bath, cooled, and f i l t e r e d on Whatman #1 f i l t e r p a p e r . The e x t r a c t s were immediately assayed and the remaining s o l u t i o n s t o r e d i n the r e f r i g e r a t o r at 4°C. Es t i m a t i o n of B i o l o g i c a l A c t i v i t i e s The b i o l o g i c a l a c t i v i t y of crude e x t r a c t s , and 11 p u r i f i e d products, were estimated by means of four assay methods. a. I s o l a t e d r a t uterus assay f o r o x y t o c i c a c t i v i t y . b. Rat Vasopressor assay c. Rat a n t i d i u r e t i c assay d. Frog waterbalance assay. In a l l types of b i o a s s a y Holton's (1948) "4 p o i n t " method was used f o r more accurate determination of potency (and s t a t i s t i c a l e s t i m a t i o n of p o s s i b l e e r r o r s ) . a. I s o l a t e d r a t uterus o x y t o c i c assay Method, as described by Holton (1948) and modified by Munsick, 1960: V i r g i n a l b i n o females of the Wistar s t r a i n were s e l e c t e d i n f u l l or pro-oestrous, by microscopic examination of t h e i r v a g i n a l smears. The r a t s were stunned w i t h a blow on the head and k i l l e d by d e c a p i t a t i o n . The uterus, exposed by a midventral and two s a g i t t a l i n c i s i o n s , was c a r e f u l l y d i s s e c t e d , w i t h the o v a r i e s i n t a c t and p a r t o f the vagina attached. The d i s s e c t e d uterus was t r a n s -f e r r e d i n t o a p e t r i - d i s h c o n t a i n i n g van Dyke-Hastings s o l u t i o n (composition f o l l o w s ) , a l l remaining f a t and connective t i s s u e removed, and the uterus d i v i d e d i n t o i t s two horns. The v a g i n a l end of one horn was t i e d w i t h s i l k thread to a g l a s s hook, p u l l e d from an a i r supply tube. A longer p i e c e of thread was sewn through the ovary. The g l a s s hook w i t h the uterus was then immersed i n a 5 ml muscle bath c o n t a i n i n g van Dyke-Hastings s o l u t i o n , and ( connected to a supply of 5% C0 2-95% 0 2. The f r e e end of the thread sewn to the ovary was secured to a c a r e f u l l y balanced w r i t i n g l e v e r , which i n t u r n r e s t e d a g a i n s t the smoked drum of a Palmer Kymograph. The muscle bath was connected to a r e s e r v o i r system f o r van Dyke-Hastings s o l u t i o n , and the whole complex was kept at constant temperature (32°C) i n a water-bath by a Bromwell Thermomix pump. At 5 minute i n t e r v a l s , doses of standard (Syntocinon, 10 IU/ml, Sandoz) a l t e r n a t e d w i t h doses of unknown e x t r a c t were p i p e t t e d i n t o the muscle bath. A f t e r the response reached i t s maximum, the van Dyke-Hastings s o l u t i o n i n the muscle bath was exchanged w i t h f r e s h s o l u t i o n from the r e s e r v o i r , by means of a rubber bulb attached to the r e s e r v o i r . Two types of van Dyke-Hastings s o l u t i o n were used to support the uterus, d i f f e r i n g from one another o n l y i n the presence or absence of Mg + + (Munsick, 1960). The van Dyke-Hastings s o l u t i o n was prepared f r e s h d a i l y by the use of two stock s o l u t i o n s . (1) van-Dyke Hastings s o l u t i o n without Mg4"1" Stock S o l u t i o n A NaCl 132.466g NaHC0 3 51.170g KC1 9.082g Phenol red ^ 0.054g Na s a l t Combined, made up t o 2 l i t e r s i n d i s t H 0 2 13 Stock Solution B Na 2HP0 4 22.714g up to 1 l i t e r with d i s t H 20 NaH2P04 5.520g up to 1 l i t e r with d i s t H 20 Approximately equal volumes of NaB^PG^ and Na2HPO^ were mixed, and the pH adjusted to 7.4 The f i n a l mixture in the reservoir contained: 200 ml of stock solution A 20 ml of stock solution B 2 ml of 0.5 M Ca C l 2 1 g dextrose Combined, and made up to 2 l i t e r s with d i s t H 0 2 ++ van-Dyke-Hastings solution with Mg Preparation of the van Dyke-Hastings solution with Mg + + i s i d e n t i c a l to the one described for van Dyke-Hastings solution without Mg + +, with the exception that the f i n a l mixture i n the reservoir contains 200 ml of stock A 20 ml of stock B 2 ml of 0.5 M CaCl 2 1 g dextrose And an addi t i o n a l : 2 ml of 0.5 M MgCl 2 Combined, made up to 2 l i t e r s i n d i s t H 20 Rat vasopressor assay The pressor a c t i v i t y of posterior lobe extracts was 14 determined as described by van Dyke, Adamson and Engel, 1955. Wistar r a t s were anesthetised by subcutaneous i n j e c t i o n s of urethane (175 mg/lOOg body wt) and Dibenzylene (0.5 mg/lOOg). Dibenzylene, an adrenergic b l o c k i n g agent was used to lower and s t a b i l i z e the blood pressure, p r e v e n t i n g i n t e r f e r e n c e w i t h the pre s s o r assay by v a r i o u s adrenergic pressor and depressor agents. The r a t s were ready f o r surgery i n 30-45 minutes, f o l l o w i n g the a d m i n i s t r a t i o n of the an a e s t h e t i c . The s k i n on the t h r o a t was cut, the j a g u l a r v e i n l o c a t e d and freed from connective t i s s u e . Two l i g a t u r e s were placed around the v e i n , and the one d i s t a l to the heart t i e d . The v e i n was punctured w i t h a 22 guage needle, a cannula (PE 10 Intermedic) i n s e r t e d , and secured w i t h the two l i g a t u r e s . The c a r o t i d a r t e r y and trachea, were cannulated i n the same manner, us i n g PE 50 Intermedic tubing f o r the a r t e r y and PE 250 f o r the trachea. To prevent formation of blood c l o t s during the assay 0.5 mg Heparin s o l u t i o n ( i n 0.9% NaCl) was i n j e c t e d per 100 g body wt. i n t r a v e n o u s l y . I n j e c t i o n s of standard and unknown s o l u t i o n s were administered through the cannula i n the j a g u l a r v e i n . The blood pressure was recorded from the c a r o t i d cannula by means of a Stratham 23AA transducer, coupled to a Beckman dynograph recorder. The assay was c a r r i e d out by i n j e c t i o n s of two doses o f standard ( P i t r e e s i n 20 IU/ml, Parke Davies Co.) matched f o r magnitude of responses by two doses unknown s o l u t i o n , a t 10 minute i n t e r v a l s . For e s t i m a t i o n of potency Holton's "4 p o i n t " s t a t i s t i c a l method was a p p l i e d . c. Rat a n t i d i u r e t i c assay The a n t i d i u r e t i c potency o f p o s t e r i o r p i t u i t a r y e x t r a c t s was estimated i n a n e s t h e t i s e d r a t s by the method of Sawyer, (1961) . Male a l b i n o r a t s of W i s t a r s t r a i n were l o c a t e d to 5% of t h e i r body weight w i t h 12% ethanol, by means of a stomach tube. Anesthesia occurred between 25-40 minutes. To ensure complete i n s e n s i t i v i t y to the s u r g i c a l procedure, X y l o c a i n e Hydrochloride (2% s o l u t i o n , without epinephrine, A s t r a Pharmaceuticals) was a p p l i e d l o c a l l y to the areas of i n c i s i o n . The j u g u l a r v e i n and trachea were cannulated as d e s c r i b e d f o r the p r e s s o r assay. The bladder was cannulated i n the f o l l o w i n g manner: ^ inch midventral i n c i s i o n was made through the s k i n and abdominal muscle approximately h inch a n t e r i o r to the p e n i s . The bladder was eased through the i n c i s i o n and cut i n a r e l a t i v e l y avascular area. The end of a PE 200 Intermedic cannula, which had been f l a r e d by heat, was i n s e r t e d through the i n c i s i o n and t i e d i n t o p l a c e w i t h a l i g a t u r e c l o s e to the u r e t h r a l e x i t . To ensure no l o s s of u r i n e d u r i n g the assay, the penis was t i e d w i t h another l i g a t u r e . The i n c i s i o n i n the abdominal w a l l was sutured. The prepared r a t was hydrated up to 8% of i t s body weight, and the l o s t u r i n e replaced throughout the assay w i t h 16 a s o l u t i o n of 1.5% ethanol i n 0.5% NaCl, by way of a stomach tube. The cannula from the bladder was connected to a Palmer drop counter and recorder. Responses to standard ( P i t r e s s i n 20 IU/ml Parke Davies Co.) and unknown s o l u t i o n s were recorded on the smoked drum of a Palmer Kymograph, as a decrease i n the number of drops per u n i t time. I n j e c t i o n s were administered through the cannula l e a d i n g to the j u g u l a r v e i n at times when the u r i n e flow had returned to the b a s e l i n e l e v e l . For e s t i m a t i o n of potency, the 4 p o i n t s t a t i s t i c a l a n a l y s i s of Holton was employed. d. Frog waterbalance assay Frog waterbalance assay was c a r r i e d out according to Sawyer 1960. The apparatus f o r the assay c o n s i s t e d of an open g l a s s tube, f l a r e d a t one end. Near the other end a g l a s s rod was sealed t o the tube to form a handle. This glas s tube was suspended i n a l a r g e r outer container i n t o which a g l a s s tube was p u l l e d near the base, to be connected to an a i r supply. To c a r r y out the assay, frogs of the species Rana  catesbiana were p i t h e d , the bladder exposed and removed. The d i s s e c t e d bladder was d i v i d e d i n t o i t s two lobes, the cut end of a h a l f bladder was p u l l e d over the f l a r e d end of the gla s s tube, and secured onto i t w i t h a s i l k thread. The g l a s s tube was f i l l e d w i t h 5 ml of d i s t ^ 0 and suspended i n 25 ml f r o g Ringers s o l u t i o n contained i n the outer bath (composition f o l l o w s ) . The bladders were weighed every 15 minutes and were considered adequate f o r assay when they showed a very slow steady l o s s of weight. or unknown s o l u t i o n were administered i n t o the outer bath. The r a t e o f wa t e r - l o s s from the tube through the bladder was measured by weighing the gl a s s tubes w i t h the attached bladders every 15 minutes throughout the assay. The weight l o s s d u r i n g the p e r i o d of 15-45 minutes f o l l o w i n g the additon of the standard or the p i t u i t a r y e x t r a c t was considered the response. A f t e r 45 minutes the f r o g Ringer's s o l u t i o n i n the outer bath was exchanged w i t h f r e s h s o l u t i o n and the bladder was allowed 45 minutes to recover i n which time the r a t e of wat e r - l o s s returned to the b a s e l i n e l e v e l . The Ringer's s o l u t i o n used i n the f r o g waterbalance assay was prepared from two stock s o l u t i o n s . Stock, s o l u t i o n A Doses of standard (Syntocinon 10 IU/ml, Sandoz) NaCl 94 .'0 g KC1 3.7 g CaC1.2H 20 5.3 g Stock s o l u t i o n B 2.0 g NaHCO 3 40.0 g glucose 4.0 g phenol red 12.0 mg 50 ml of stock s o l u t i o n A and stock s o l u t i o n B were mixed and made up to 1 l i t e r i n d i s t i l l e d H 20 Standards The s y n t h e t i c o x y t o c i n p r e p a r a t i o n , Syntocinon (Sandoz) used as standard i n the r a t uterus and f r o g water-balance assays, was assayed against the U.S. I n t e r n a t i o n a l Standard. The potency was i n good agreement w i t h t h a t s t a t e d by the company and no adjustment of the results.was necessary. The commercial vasopressin p r e p a r a t i o n , P i t r e s s i n , used as standard i n the r a t vasopressor and a n t i d i u r e t i c assays i s a p a r t i a l l y p u r i f i e d mixture of a r g i n i n e and l y s i n e v a s o p r e s s i n . The two peptides d i f f e r i n t h e i r r e s p e c t i v e vasopressor and a n t i d i u r e t i c p otencies, and no i n f o r m a t i o n i s a v a i l a b l e from the company as to the r a t i o of the two peptides present i n a p a r t i c u l a r p r e p a r a t i o n of P i t r e s s i n . P i t r e s s i n , assayed against the U.S. I n t e r n a t i o n a l Standard was found to have a lower a n t i d i u r e t i c than vasopressor a c t i v i t y . C o r r e c t i o n i n the r e s u l t s were made to compensate for t h i s discrepancy. Methods of P u r i f i c a t i o n For the s e p a r a t i o n of b i o l o g i c a l l y a c t i v e p r i n c i p l e s from the crude e x t r a c t s two methods were a p p l i e d : a. Gel f i l t r a t i o n b. Paper chromatography a. Gel F i l t r a t i o n P u r i f i c a t i o n s of the a c t i v e p r i n c i p l e s was achieved by p a s s i n g the p o s t e r i o r lobe e x t r a c t s through a (Dextran) column of Sephadex G 15 (Pharmacia, Uppsala). 19 The procedure used was as f o l l o w s : i . P r e p a r a t i o n of the Sephadex A 40-120 u p a r t i c l e s i z e Sephadex G-15, w i t h water r e g a i n value of 1.5 +0.2 g/g and bed volume of 3 ml/g dry g e l , was used. The dry g e l was added to a l a r g e volume of 0.2 M a c e t i c a c i d . The s l u r r y thus formed was mixed f o r one hour on a magnetic s t i r r e r , allowed to s e t t l e f o r 30 minutes, and the a c e t i c a c i d decanted and discarded along w i t h the f i n e n o n - s e t t l i n g f r a c t i o n . The process was repeated four times. A f t e r the f o u r t h washing, the remaining Sephadex was taken up i n enough 0.2 M a c e t i c a c i d to form a t h i c k s l u r r y , and used to b u i l d the column. i i . P r e p a r a t i o n of the column A Kontes chromaflex (100 cm x 25 mm volume 450 ml) column was used e i t h e r by i t s e l f , o r extended w i t h a Kontes chromaflex (100 cm x 25 mm volume 450 ml) extender to double the h e i g h t and volume. The column was f i x e d v e r t i c a l l y onto a stand, w i t h the help of a plumb l i n e , and f i l l e d w i t h 0.2 M a c e t i c a c i d . A ^ inch t h i c k l a y e r o f g l a s s wool was placed a t the bottom, followed by a s i m i l a r l a y e r of small g l a s s beeds. A l a r g e funnel was f i t t e d to the top of the column by means of a rubber stopper, and f i l l e d w i t h 0.2 M a c e t i c a c i d . The Sephadex G-15 s l u r r y was added to the a c e t i c a c i d i n the fun n e l , and was s t i r r e d c o n t i n u o u s l y w i t h an e l e c t r i c mixer. A d d i t i o n a l s l u r r y was added i n t o the funnel at i n t e r v a l s . The Sephadex g e l was'packed i n t o a s i n g l e column over a p e r i o d of 18 hours, and i n t o a double column oyer a p e r i o d of 36 hours. i i i . P u r i f i c a t i o n p r o c e d u r e B e f o r e u s i n g t h e column, 0.2 M a c e t i c a c i d was a l l o w e d t o r u n t h r o u g h i t f o r a p e r i o d o f 6 h o u r s . A t t h e c o m p l e t i o n o f t h e a c e t i c a c i d w ashing, t h e l i q u i d above t h e g e l s u r f a c e was a l l o w e d t o r u n down u n t i l t h e meniscus r e a c h e d t h e s u r f a c e . The p o s t e r i o r l o b e e x t r a c t s (see e x t r a c t i o n ) were c a r e f u l l y a p p l i e d t o t h e top o f t h e column, and p e r m i t t e d t o r u n i n t o t h e g e l . The column was f i l l e d w i t h a c e t i c a c i d (0.2 M) and c o n n e c t e d t o a r e s e r v o i r c o n t a i n i n g t h e same. The s o l u t i o n w h i c h had p a s s e d t h r o u g h t h e column was c o l l e c t e d i n 2.8 ml f r a c t i o n s , b y t h e use o f an LKB U l t r o r a c Type 7000 f r a c t i o n c o l l e c t o r , b. Paper chromatography The p a p e r chromatography t e c h n i q u e s used, were as d e s c r i b e d b y H e l l e r and P i c k e r i n g (1961) and P e r k s (1966). B u t a n o l , a c e t i c a c i d , w a t e r i n a 4:1:5 volume were shaken i n a s e p a r a t o r y f u n n e l f o r 15 minutes i m m e d i a t e l y a f t e r m i x i n g , and e v e r y 15 m i n u t es t h e r e a f t e r f o r a p e r i o d o f 1% h o u r s . The s o l v e n t system t h e n was a l l o w e d t o s e p a r a t e i n t o two l a y e r s . The aqueous l a y e r was p l a c e d i n t o f o u r P e t r i d i s h e s i n t h e b o t t o m o f a 12" x 12" x 24" g l a s s chromatography t a n k . The l i d o f t h e tank was s e a l e d i n t o p l a c e w i t h " L u b r i s e a l " , and t h e system was e q u i l i b r a t e d a t 20°C f o r one h o u r . S t a n d a r d s and unknown s o l u t i o n were a p p l i e d t o a 24 x 54 cm s h e e t o f Whatman 3MM chromatography paper, w i t h t h e use o f 1 ml t u b e r c u l i n s y r i n g e s . C o l d a i r from a p o r t a b l e h a i r d r y e r was allowed to pass through the o r i g i n , to speed up the d r y i n g of the spots. The chromatogram was then placed i n the e q u i l i b r a t e d chromatographic tank, and the system was allowed to e q u i l i b r a t e f o r a f u r t h e r hour. At the end of the e q u i l i b r a t i o n p e r i o d the b u t a n o l l a y e r of the s o l v e n t mixture was added to the s o l v e n t bath through openings i n the g l a s s l i d . The opening i n the l i d was c l o s e d w i t h the rubber stopper i t h e l d p r e v i o u s l y . The system was allowed to run f o r 12 hours. At the completion of the run, the chromatogram was removed, the s o l v e n t f r o n t marked, and the remaining s o l v e n t d r i v e n o f f by a c o o l flow of a i r . L o n g i t u d i n a l s t r i p s of the dry chromatograms were cut at the width of the o r i g i n s p l u s an e x t r a 0.5 cm to a l l o w f o r d i f f u s i o n on each s i d e . The lengthwise s t r i p s were d i v i d e d i n t o ten equal p a r t s between the o r i g i n and the s o l v e n t f r o n t , each r e s u l t i n g square corresponding to 0.1 Rf u n i t . The squares were numbered, f o l d e d and e l u t e d i n d i v i d -u a l l y i n 5 ml beakers, which contained 1 ml o f 0.25% a c e t i c a c i d . The e l u t i o n was c a r r i e d out f o r a p e r i o d of 6 hours at 4°C. The e l u a t e was then recovered from the paper, by pressure, and s t o r e d at 4°C u n t i l analysed f o r b i o l o g i c a l a c t i v i t i e s . Chemical Methods f o r the A n a l y s i s of Peptides a. E s t i m a t i o n of t o t a l peptide c o n c e n t r a t i o n The e s t i m a t i o n of t o t a l peptide c o n c e n t r a t i o n was c a r r i e d out by the c o l o r i m e t r i c method of Lowry, Rosebrough, F a r r and R a n d a l l (1951) as f o l l o w s : i . Reagents A - 2% N a 2C0 3 i n 0.1 N NaOH B - 0.5% CuSO 5H 0 i n 1% K t a r t a r a t e 4 2 C - a l k a l i n e copper s o l u t i o n 50:1 mixture o f reagent A + B D - 1 N F o l i n - C i o c a l t e a u Phenol reagent i i . Standards A s e r i e s o f standards 10,25,50,100,150,200 and 300 ug were prepared by d i l u t i o n from a 50 mg % Bovine Serum Albumin Stock ( f r a c t i o n v Armour P h a r m a c e u t i c a l s ) . i i i . Measurement of p e p t i d e content 0.2 ml o f the unknown and the standards were p i p e t t e d i n t o a s e r i e s o f 2 ml t e s t u b e s . To each sample 1 ml o f reagent C was added, shaken and allowed to stand f o r 15-30 minutes. 0.1 ml o f F o l i n C i o c a l t e a u reagent was then added v e r y r a p i d l y and mixed immediately. A f t e r a p e r i o d of 40-60 minutes the c o l o u r was f u l l y developed. The samples were read a g a i n s t a b l a n k on a Unicam SP 600 spectrophotometer a t a wavelength o f 750 m i l l i m i c r o n s , u s i n g a red f i l t e r p h o t o c e l l . b. H y d r o l y s i s o f the p e p t i d e s F r a c t i o n s w i t h i d e n t i c a l b i o l o g i c a l a c t i v i t i e s , o b t a i n e d from the Sephadex column, were pooled and l y o p h i l i s e d . The r e s i d u e was d i s s o l v e d i n d i s t H 20 and l y o p h i l i s e d a gain. The procedure was repeated a t l e a s t t h r e e times i n order to get r i d o f a l l a c e t i c a c i d remaining i n the r e s i d u e . With the l a s t washing, the samples were t r a n s f e r r e d i n t o a g l a s s 23 b u l b . 4 ml of t r i p l e d i s t i l l e d HCl was added to the d r y resi d u e , and N 2 gas bubbled through the l i q u i d f o r 15 minutes i n order to remove trapped a i r . The samples were then frozen i n l i q u i d . n i t r o g e n and evacuated f o r 30 minutes a t 10 u Hg pressure on a vacuum pump. At the end of the 30 minutes, the glas s bulb was sealed w i t h a blowtorch, under vacuum, and t r a n s f e r r e d i n t o an oven at 108°C (Fish e r Isotemp) f o r 22 hours. At the end of the 22 hours, the samples were removed from the-oven, allowed to c o o l and t r a n s f e r r e d i n t o 50 ml f l a s h evaporator f l a s k s . F u r t h e r c o o l i n g , f o r 15 minutes, f o l l o w e d i n a deep-freeze. The f l a s k s , w h i l s t s t i l l c o l d , were attached to a f l a s h evaporator, and allowed t o warm s l o w l y under vacuum and w i t h continuous t u r n i n g of the r o t o r . When the samples were at approximately room temperature, they were g r a d u a l l y lowered i n t o a 37°C waterbath, and d r i e d . The d r y powder was d i s s o l v e d i n i c e d , d i s t H 20 and evaporated again, to wash out the remaining HCl. The washing was repeated at l e a s t three times u n t i l no t r a c e s of HCl remained, c. Amino a c i d a n a l y s i s The amino a c i d a n a l y s i s was performed by t h i n l a y e r chromatography of the dansyl d e r i v a t i v e s of the amino a c i d s , as described by Levison, 1967. i . P r e p a r a t i o n of the p l a t e s One p a r t of the s i l i c a g e l was s t i r r e d i n t o a s l u r r y i n 2 p a r t s (w/v) of g l a s s d i s t i l l e d water, f o r approximately three minutes. The s l u r r y was'spread i n 250 micron thickness onto 20" x 20" g l a s s p l a t e s w i t h the a i d of a Warner-Chilcott spreader. The p l a t e s were allowed t o dry at room temperature, u n t i l the s i l i c a became opaque (10-20 minutes). They were then warmed i n a stream o f hot a i r f o r 10 minutes, u n t i l the s i l i c a became dry, and placed f o r 30 minutes i n a d r y i n g oven at 110°C. i i . D a n s y l a t i o n of the hy d r o l y s a t e s F i v e m i c r o l i t e r s of 0.2N NaHCX^, and f i v e micro-l i t e r s of standard dansyl c h l o r i d e * were added to one m i l i -micromole h y d r o l y s a t e . The r e a c t i o n was allowed to proceed f o r three hours. Amino a c i d s were dansylated i n the same manner to p r o v i d e standards f o r chromatographic comparisons. i i i . Chromatography of the hydrolysates One m i c r o l i t r e of the h y d r o l y s a t e s , c o n t a i n i n g 1 muM of peptide was a p p l i e d to the p l a t e s , beside one milimicromole samples of the standard amino a c i d s . c The p l a t e s were placed i n t h i n l a y e r chromatographic tanks which contained a small amount of the appropriate s o l v e n t system, and the system was allowed to e q u i l i b r a t e f o r 30 minutes. At the end of the 30 minutes e q u i l i b r a t i o n p e r i o d , the pan at the bottom of the tank was f i l l e d w i t h the s o l v e n t to be i n contact w i t h the edge of the p l a t e . The s o l v e n t was allowed t o run up the p l a t e s u n t i l i t reached w i t h i n an inch from the top. The p l a t e s were then removed from the tanks, and i n a few minutes, when they appeared dry, the spots f o r the standards and the unknowns were l o c a t e d under u l t r a v i o l e t l i g h t and * 1 di-methylaminonaphtaline 5 s u l p h a n y l c h l o r i d e marked. The Rf values were read, and d i r e c t comparisons w i t h the standards permitted i d e n t i f i c a t i o n of the amino acid s i n the h y d r o l y s a t e s . Four s o l v e n t systems were s e l e c t e d f o r t h e i r a b i l i t y t o separate v a r i o u s groups of amino a c i d s . Solvent system 1; chloroform: b e n z y l a l c o h o l : a c e t i c a c i d , 70:30:3 Solvent system 2; benzyl a l c o h o l : p y r i d i n e : a c e t i c a c i d , 16:4:1 Solvent system 3; b u t a n o l : a c e t i c a c i d : water, 1:5:4 Solvent system 4; chloroform: b u t a n o l : a c e t i c a c i d , 6:3:1 S t a t i s t i c a l Methods Mean hormonal potencies are c a l c u l a t e d by the use of weighted means according to K.A. Brownlee (1965) u s i n g the formula: k k . X = ^ w x / ^ w j j j j i 2 where w =1/6' i i and confidence l i m i t s at the 5% p r o b a b i l i t y l e v e l were assigned u s i n g the f o l l o w i n g formula: k where V- = 1/ f w x ^ i The r a t i o s of vasopressor or a n t i d i u r e t i c a c t i v i t y to o x y t o c i c a c t i v i t y were c a l c u l a t e d by the formula ( B l i s s , 1956): 26 M = ab ~2 2 2 b-6"bt b Confidence l i m i t s were assig n e d a t the 5% p r o b a b i l t l y l e v e l a c c o r d i n g to the formula: X- = M + M - (M) -2 S2 t 2a - a a 2_ r-2 2 b 5 b f c b SECTION I STUDIES ON THE PREGNANT SHEEP P o s t e r i o r p i t u i t a r y glands from pregnant sheep a t two stages of pregnancy, and from c o n t r o l non-pregnant sheep were c o l l e c t e d throughout two breeding seasons. The b i o l o g i c a c t i v i t i e s of the crude e x t r a c t s were determined. Crude e x t r a c t s were p u r i f i e d , and the p u r i f i e d product was analysed f o r amino a c i d composition. The two stages of pregnancy, represented i n the samples c o l l e c t e d , were 89-91 days and 138-141 days. Since the d u r a t i o n of pregnancy i n the sheep i s 145-147 days, these stages correspond to about mid-pregnancy, and to j u s t a few days p r i o r , t o p a r t u r i t i o n , r e s p e c t i v e l y . 1. B i o l o g i c a l a c t i v i t i e s of the crude e x t r a c t s . The b i o l o g i c a l a c t i v i t i e s of three to f i v e i n d i v i d u a l glands, at 89-91 days, and 138-141 days of g e s t a t i o n were estimated from both acetone d r i e d and l y o p h i l i s e d m a t e r i a l . To complete each s e r i e s , two c o n t r o l s from non-pregnant animals were in c l u d e d . In the acetone d r i e d s e r i e s , one gland, the onl y one a v a i l a b l e d u r i n g the process of n a t u r a l d e l i v e r y , i s a l s o i n c l u d e d . Representative r e s u l t s from these bio-assays are shown i n P i g . 1. -A l l glands e x h i b i t e d o x y t o c i c , vasopressor and a n t i -d i u r e t i c a c t i v i t i e s . The r e s u l t s are presented i n Table I f o r acetone d r i e d , and i n Table I I f o r l y o p h i l i s e d t i s s u e . 28 F i g u r e 1 Sample records from r a t uterus, vasopressor and a n t i d i u r e t i c assays. Rat uterus assay A = 0.08 ml of 1:250, 2 mg/ml p o s t e r i o r p i t u i t a r y e x t r a c t of pregnant sheep. B = 0.08 ml of 1:1000 s y n t h e t i c o x y t o c i n (Syntocinon, 10 I.U./ml) . C = 0.12 ml of 1:1000 s y n t h e t i c o x y t o c i n . D = 0.12 ml of 1:250 of 2 mg/ml e x t r a c t Rat vasopressor assay A = 0.1 ml of 1:60, 2 mg/ml p o s t e r i o r p i t u i t a r y e x t r a c t of pregnant sheep. B = 0.06 ml of 1:400 standard vasopressin ( P i t r e s s i n , 20 I.U./mg) . C = 0.12 ml of 1:400 standard v a s o p r e s s i n . D = 0.2ml of 1:60 p o s t e r i o r p i t u i t a r y e x t r a c t Rat a n t i d i u r e t i c assay A = 0.05 ml of 1:40,000 standard v a s o p r e s s i n . B = 0.05 ml of 1:2,000, 2 mg/ml p o s t e r i o r p i t u i t a r y e x t r a c t . C = 0.1 ml of 1:40,000 standard vasopressin D = 0.1 ml of 1:2,000 p o s t e r i o r p i t u i t a r y e x t r a c t . Rat Uterus A s s a y Weighted means were c a l c u l a t e d f o r corresponding stages of g e s t a t i o n by the s t a t i s t i c a l method described on p. 25-in Methods. The mean o x y t o c i c , vasopressor and a n t i d i u r e t i c a c t i v i t i e s i n the acetone d r i e d glands are presented i n F i g . 2. At 90 days of pregnancy the o x y t o c i c a c t i v i t y was 60% below t h a t i n the non-pregnant a d u l t sheep; t h i s f a l l i s f o l lowed by a r e l a t i v e r i s e at 140 days, but the potency remained below the c o n t r o l l e v e l throughout g e s t a t i o n . The changes i n vasopressor and a n t i d i u r e t i c a c t i v i t i e s p a r a l l e l e d those o f the o x y t o c i c a c t i v i t y . The one gland taken at deli-very revealed a marked increase i n o x y t o c i c potency, and i n terms of o x y t o c i c a c t i v i t y , i t was by f a r the most a c t i v e p i t u i t a r y t i s s u e encountered i n the course of t h i s i n v e s t i g a t i o n . The apparent increase i n o x y t o c i c a c t i v i t y was accompanied by a much l e s s e r increase i n the vasopressor and a n t i d i u r e t i c a c t i v i t i e s . The e s t i m a t i o n of the b i o l o g i c a l a c t i v i t i e s of the l y o p h i l i s e d glands o f pregnant females showed changes i n hormonal content, s i m i l a r to those found i n acetone d r i e d t i s s u e s . The mean potencies are shown i n F i g . 3. Again, a decrease i n the stored l e v e l of the oxytocic, vasopressor and a n t i d i u r e t i c a c t i v i t i e s was found at 90 days of pregnancy, f o l l o w e d by a r e l a t i v e r i s e i n a l l three a c t i v i t i e s at 140 days of g e s t a t i o n . In F i g . 4, the hormonal l e v e l s d u r i n g g e s t a t i o n are compared d i r e c t l y i n acetone d r i e d and l y o p h i l i s e d glands. 30 TABLE I POTENCIES AND THE RATIOS OF THE POTENCIES IN THE ACETONE-DRIED TISSUE OF PREGNANT SHEEP No. Gest. Days RU mU/mg Pressor mU/mg ADH mU/mg V/0 R a t i o ADH/0 R a t i o 2 90 505.0 + 32.2 467.5 + 44.0 402.0 + 28.6 0.92 + 0.10 0.80 + 0.55 4 89 677.5 + 96.0 341.3 + 37.5 479.8 +101.6 0.50 +0.11 0.70 + 0.12 7 91 1191.6 +150.0 373.6 + 33.0 568.2 + 82.5 0.31 +0.05 0.47 + 0.08 8 138 748.8 +120.0 478.9 + 74.1 542.0 +321.0 0.64 + 0.14 0.74 + 0.12 9 138 1536.2 +252.8 1086.1 +148.8 0 1275.7 +384.3 0.71 + 0.14 0.83 + 0.11 13 138 1080.1 + 82.5 664.5 + 49.1 567.0 +133.0 0.62 + 0.06 0.55 + 0.36 27 Term 2454.4 +802.0 1015.9 +188.4 , 910.8 +159.0 0.41 +0.14 . 0.37 + 0.13 1 C o n t r o l 1954.4 +134.9 1220.0 +140.5 1480.6 +461.1 0.62 + 0.04 0.76 + 0.17 2 Co n t r o l 1350.5 +205.6 1416.8 +430.0 1.05 + 0.34 TABLE I I POTENCIES AND THE RATIOS OF THE POTENCIES IN THE LYOPHILISED TISSUE OF PREGNANT SHEEP NO. Gest. Days RU mU/mg Press o r mU/mg ADH mU/mg V/0 R a t i o A/0 R a t i o 2 87 1706.0 +296.0 1273.0 +243.5 1115.4 +543.9 0.75 +0.18 0.65 + 0.3 3 90 1405.8 +106.5 963.5 + 83.6 1565.1 +937.3 0.70 +0.07 1.1 +0.64 16 90 1231.0 +117.0 1005.5 +216.4 999.0 +682.6 0.82 +0.18 0.81 + 0. 50 4 90 1220.6 +101.0 893.0 + 74.9 0.73 + .141 13 89 1213.4 +109.0 850.0 +265.0 0.70 +0.23 19 141 1261.2 +171.0 926.4 +242.8 963.4 +208.8 0.73 + 0.31 0.76 + 0.79 22 143 1689.6 +101.9 1069.2 +344.1 1526.1 +643.0 0.63 +0.15 0.90 + 0.38 23 141 1662.2 +117.5 1041.5 + 88.0 915.9 +248.8 0.63 +0.06 0.54 +0.31 10 140 1520.1 +135.5 1388.6 +139.6 0.91 +0.11 17 137 1483.5 +180.5 750.1 + 49.6 0.50 +0.08 1 C o n t r o l 1224.6 +231.0 1297.4 +272.4 1050.4 +205.1 1.05 + 0. 28 0.86 +0.35 2 C o n t r o l 1568.9 +127.2 1464.1 +215.7 1267.6 +458.1 0.93 +0.25 0.81 + 0.29 3 Co n t r o l 2094.2 +306.9 1502.0 +299.8 1083.9 +366.9 0.73 + 0.21 0.59 +0.35 32 The s i n g l e value obtained i n the acetone d r i e d s e r i e s i s omitted here, s i n c e there was no l y o p h i l i s e d sample f o r comparison. The graph i l l u s t r a t e s the s i m i l a r i t y i n the p a t t e r n of change seen d u r i n g pregnancy; t i s s u e s taken i n two d i f f e r e n t breeding seasons, and subjected to two d i f f e r e n t d r y i n g procedures, gave e s s e n t i a l l y s i m i l a r r e s u l t s . The f a c t t h a t the o v e r a l l changes are very s i m i l a r i n the two d i f f e r e n t groups of sheep suggests t h a t the a l t e r a t i o n s i n the l e v e l of sto r e d hormones are meaningful, and t h a t they are not due to random v a r i a b i l i t y . I t appears probable t h a t acetone treatment does not obscure the changes which occur i n the neurohypophysis d u r i n g g e s t a t i o n , but o n l y magnifies them. The r a t i o s of vasopressor to o x y t o c i c a c t i v i t y (V/0) and of a n t i d i u r e t i c to o x y t o c i c a c t i v i t y (A/0), were c a l c u l a t e d f o r i n d i v i d u a l glands, and confidence l i m i t s a t P<0.05 assigned according to the method of B l i s s (1956). The r e s u l t s are included i n Table I and Table I I . Ratios of the mean potencies were c a l c u l a t e d by the same s t a t i s t i c a l method (see p.25 Methods) and the r e s u l t s f o r both acetone d r i e d and l y o p h i l i s e d t i s s u e are given i n F i g . 5. Comparison between the r a t i o s of glands from c o n t r o l non-pregnant sheep, and those from pregnant animals, f o l l o w i n g e i t h e r l y o p h i l i s a t i o n or acetone treatment, r e v e a l s no s t a t i s t i c a l l y s i g n i f i c a n t change i n r a t i o s . 2. P u r i f i c a t i o n and amino a c i d a n a l y s i s of the a c t i v e  p r i n c i p l e s of pregnant sheep Samples of the neurohypophysial e x t r a c t s of pregnant 33 Figure 2 Weighted means of oxytocic, vasopressor and a n t i d i u r e t i c a c t i v i t i e s in the acetone dried neuro-hypophyses of control and pregnant sheep. mU/mg = m i l l i u n i t b i o l o g i c a l a c t i v i t y / mg dry tissu e P R E G N A N T S H E E P ( A C E T O N E DRIED) Oxytocic Vasopressor Antidiuretic • • • control term control term control term i . . t I . f i • , I 0 50 100 147 0 50 100 147 0 50 100 147 Days Gestation Days Gestation Days Gestation 34 Figure 3 Weighted means of oxytocic, vasopressor and a n t i d i u r e t i c a c t i v i t i e s i n the l y o p h i l i s e d neuro-hypophyses of control and pregnant sheep. mU/mg = m i l l i u n i t b i o l o g i c a l activity/mg dry tissue P R E G N A N T S H E E P (LYOPHILISED) Oxytocic Vasopressor Antidiuretic 2 5 0 0 r 2 0 0 0 1500 6 o E 1000 500 control term J 0 50 100 147 Days Gestation control term j 1 50 100 147 Days Gestation 0 50 100 Days Gestation 147 sheep were pooled f o r chemical s t u d i e s . Because of the i n s e n s i t i v i t y of chemical methods as compared to pharmacological an a l y s i s , i t was necessary to mix the e x t r a c t s w i t hout regard to the stage of pregnancy a t which they were taken, or to the method of d r y i n g . The mixed samples were regarded merely as examples of p o s t e r i o r p i t u i t a r y e x t r a c t s from sheep i n a pregnant s t a t e , and they l a t e r served as c o n t r o l s f o r the s t u d i e s of the f o e t a l neurohypophysial agents The pooled samples were p u r i f i e d by a new m o d i f i c a t i o n of the g e l f i l t r a t i o n method, and the e l u a t e s from the column were analysed f o r p r o t e i n content (Lowry p e p t i d e ) , and f o r b i o l o g i c a l a c t i v i t i e s ( o x ytocic and vasopressor assay). D e t a i l s o f these estimations are given i n the s e c t i o n on methods. The p u r i f i e d agents were p a r t i a l l y analysed f o r t h e i r chemical s t r u c t u r e by the use of t h i n l a y e r chromatography. a. P u r i f i c a t i o n by g e l f i l t r a t i o n 15 ml of neurohypophysial e x t r a c t at a c o n c e n t r a t i o n of 2 mg/ml, and which contained a t o t a l of 27.0 +0.9 I.U. ( I n t e r n a t i o n a l U n i t ) of r a t uterus and 24.0 + 3.0 I.U. of vasopressor a c t i v i t y , was a p p l i e d to a 100 cm column of Sephadex G-15 dextran g e l which was suspended i n 0.2 M a c e t i c a c i d (see Methods). A r e s e r v o i r f i l l e d w i t h 0.2 M a c e t i c a c i d was connected to the column, and the a c e t i c a c i d was allowed to flow through at a r a t e of 16 ml/hr. The f i r s t 100 ml of e l u a t e represented the v o i d volume of the column and was d i s c a r d e d . The next 150 f r a c t i o n s of 2.8 ml each were then c o l l e c t e d i n t o t e s t tubes i n a f r a c t i o n c o l l e c t o r . Figure 4 Comparison between the b i o l o g i c a l a c t i v i t i e s of the acetone dried and l y o p h i l i s e d neurohypophyses of control and pregnant sheep. mU/mg = milliunit/mg dry tissue Oxytocic P R E G N A N T S H E E P o lyophilised ® acetone dried Vasopressor Antidiuretic Days Gestation Days Gestation 37 Figure 5 Ratios of b i o l o g i c a l a c t i v i t i e s in acetone dried and l y o p h i l i s e d neurohypophyses of control and pregnant sheep. Ratio of V/0 = m i l l i u n i t of vasopressor a c t i v i t y per mg dry t i s s u e / m i l l i u n i t of oxytocic a c t i v i t y per mg dry tissue Ratio of A/0 = m i l l i u n i t of a n t i d i u r e t i c a c t i v i t y per mg dry t i s s u e / m i l l i u n i t oxytocic a c t i v i t y per mg dry tissue 1.5 1.0 o > o o +z a rr 0.5 50 100 Days Gestation RATIO O F ACTIVITIES. P R E G N A N T S H E E P 38 The f r a c t i o n s were analysed f o r p r o t e i n content, vasopressor and o x y t o c i c a c t i v i t y , and the r e s u l t s are given i n F i g . 6. The mass of p r o t e i n , as determined by the Lowry p e p t i d e determination, came o f f the column i n the f i r s t 50 f r a c t i o n s and showed a remarkable s e p a r a t i o n of the a c t i v e p r i n c i p l e s . The a n a l y s i s f o r b i o l o g i c a l a c t i v i t e s revealed t h a t the vasopressor and o x y t o c i c a c t i v i t e s were a l s o p a r t i a l l y separated i n the e l u a t e . The recovery f o r o x y t o c i c a c t i v i t y was j u s t above 100%; the apparent increase i n the t o t a l o x y t o c i c moiety i s undoubtedly an a r t i f a c t , due to the inherent e r r o r of the e s t i m a t i o n . The recovery f o r vasopressor a c t i v i t y was approximately 80%. The p a r t i a l r e s o l u t i o n o f the a c t i v e p r i n c i p l e s by the G-15 column, suggested the p o s s i b i l i t y t h a t an increase i n the lengt h o f the column, might r e s u l t i n a complete r e s o l u t i o n of the two b i o l o g i c a l l y a c t i v e agents. Therefore, an extender was used to b u i l d a 200 cm column. 15 ml of e x t r a c t at 2 mg/ml, re p r e s e n t i n g 27.0 + 0.9 I.U. t o t a l o x y t o c i c and 24.0 + 3.0 I.U. t o t a l vasopressor a c t i v i t y , was subjected to the same procedure as used p r e v i o u s l y f o r the sh o r t column. The f r a c t i o n s were analysed as before, and the r e s u l t s are presented i n F i g . 7. There was a complete s e p a r a t i o n of the two a c t i v e p r i n c i p l e s . Large p r o t e i n s were e l u t e d f i r s t , w i t h o x y t o c i c and vasopressor a c t i v i t i e s f o l l o w i n g , i n t h a t order. The increased length of the column d i d not a l t e r the recovery f o r e i t h e r o x y t o c i n or va s o p r e s s i n ; the recovery of o x y t o c i n was again approximately 100% and t h a t of vasopressin 3 9 Figure 6 P u r i f i c a t i o n of the active neurohypophysial agents of pregnant sheep by the use of 100 cm Sephadex G-15 column. 180 Purification of Pregnant Sheep Extract on 100 cm. Sephadex G-15 column -, 1800 150 a> • o S 100 a >> s o 50 Lowry. oxytocic —•-x i \ I X vasopressor A. 20 40 60 T u b e N u m b e r 80 100 120 (I t u b e = 2.8 ml) 140 1500 < a in o n -i n Ul in O 1000 500 3 c \ 3 X >< —*• o o 160 180 200 220 40 approximately 80%. No increase i n c o n d u c t i v i t y , above t h a t o f the supporting 0.2 M a c e t i c a c i d , was detected i n any of the f r a c t i o n s , so t h a t no e l e c t r o l y t e peak could be r e s o l v e d ; t h i s was probably because of the low s a l t content o f the crude e x t r a c t . The peaks of b i o l o g i c a l a c t i v i t i e s showed chemical evidence f o r t h e i r peptide content. In the o x y t o c i c peak ( F i g . 7), 5 ug/ml Lowry peptide content, corresponded t d 600 mU/ml o x y t o c i c a c t i v i t y , or 120 I.U. o x y t o c i c a c t i v i t y / mg Lowry pep t i d e . This value compares favourably w i t h the 133 I.U./mg peptide obtained f o r pure s y n t h e t i c o x y t o c i n by Perks and Sawyer (1965) . On the b a s i s of t h i s measure-ment, the o x y t o c i c peak i n F i g . 7 appears to be almost, or perhaps completely, pure. The Lowry peptide content of the vasopressor peak i s even s m a l l e r than t h a t of the o x y t o c i c peak. Again, t h i s suggests a pure or almost pure pe p t i d e . The o x y t o c i c a c t i v i t y i n the vasopressor peak can be completely accounted f o r by the 3-5% i n t r i n s i c o x y t o c i c a c t i v i t y of vas o p r e s s i n (Adamson, Engel, van Dyke, 1954). b. Amino a c i d a n a l y s i s A f t e r v a r i o u s estimations of the d i f f e r e n t f r a c t i o n s were completed, those eluates which contained i d e n t i c a l b i o l o g i c a l a c t i v i t i e s were pooled, and the pooled samples were hydrolysed i n 6 N HCI. The hyd r o l y s a t e s were then mixed w i t h dansyl c h l o r i d e , to form dansyl d e r i v a t i v e s of t h e i r c o n s t i t u e n t amino a c i d s . The reacted h y d r o l y s a t e s 41 Figure 7 P u r i f i c a t i o n of the b i o l o g i c a l l y active p r i n c i p l e s from the crude extracts of pregnant sheep neurohypophyses, by the use of 200 cm Sephadex G-15 column. Tube Number (I tube = 2.8ml ) were a p p l i e d t o t h i n l a y e r ( s i l i c a ) chromatographic p l a t e s , alongside standard dansylated amino a c i d s (see Methods). The p l a t e s were developed i n four s o l v e n t systems which were s e l e c t e d to giv e optimal separations of the 16 amino a c i d s used as standards. The so l v e n t systems were: (1) chloroform: b e n z y l a l c o h o l : a c e t i c a c i d , (70:30:3) (2) b e n z y l a l c o h o l : p y r i d i n e : a c e t i c a c i d , (16:4:1) (3) b u t a n o l : a c e t i c a c i d : water (1:5:4) (4) chloroform: b u t a n o l : a c e t i c a c i d , (6:3:1) As yet, no s o l v e n t system has been found to separate mono-dansylated l y s i n e from d a n s y l - a r g i n i n e . A f t e r chromatography was complete, the spots were made v i s i b l e under u l t r a v i o l e t l i g h t and t h e i r p o s i t i o n marked. A r e p r e s e n t a t i v e p l a t e i s shown i n F i g . 8. Because of the known v a r i a b i l i t y of the R f values of dansyl d e r i v a t i v e s between d i f f e r e n t experiments (Perks, p e r s o n a l communication), an e f f o r t was made to employ d i r e c t comparison of the c o n s t i t u e n t amino acids of the hydrolysates w i t h the standards on the same p l a t e s . However, the values of the standards were a l s o c a l c u l a t e d on each p l a t e and compared d i r e c t l y to the ^ £~ s °f the amino acids of the unknown ( F i g . 9 ) . In the eluates which contained the o x y t o c i c 43 Figure 8 Sample pl a t e of the t h i n layer chromatography of dansyl derivatives of standard amino acids and the hydrolysates of the oxytocic and vasopressor moieties of the adult pregnant sheep, and of the hydrolysate of the f o e t a l vasopressor moiety DNS = dansyl s o l v e n t f ron t o o 1>NS T>WS Ueu LfcU O •pNS o i DNS hydrolysates Oxyt. Vasopr. Vasopr. Adult Adult Foetal o o o o HNS D N S - Q -0 0 0 o o o Q Q . 0 -o o T>NS o r i g i n J moiety, the e i g h t c o n s t i t u e n t amino acid s of o x y t o c i n were i d e n t i f i e d ; these were - t y r o s i n e , i s o l e u c i n e , glutamic a c i d j a s p a r t i c a c i d , p r o l i n e , l e u c i n e , g l y c i n e and c y s t e i c a c i d . A c i d h y d r o l y s i s would have caused the l o s s of ammonia from glutamine and asparagine to form the ( i d e n t i f i e d ) glutamic and a s p a r t i c a c i d s . In a d d i t i o n to the e i g h t amino ac i d s l i s t e d , the hy d r o l y s a t e s contained l y s i n e , a l a n i n e , and a f u r t h e r amino a c i d which could have been e i t h e r monodansylated l y s i n e or a r g i n i n e . L y s i n e i s a known contaminant of hydr o l y s a t e s of neurohypophysial peptides (Perks and Sawyer, 1965), and a l a n i n e may be generated by the breakdown of c y s t e i n (Perks, p e r s o n a l communication). Due to the s i m i l a r m o b i l i t y of mono-dansylated l y s i n e and a r g i n i n e , i t cannot be determined which of the two amino ac i d s was present i n the h y d r o l y s a t e . The presence of mono-dansylated l y s i n e , however, i s the most l i k e l y p o s s i b i l i t y , s i n c e t h i s amino a c i d was seen to be present i n i t s d i -dansylated form, and both dansyl d e r i v a t i v e s are f r e q u e n t l y formed simultaneously. A n a l y s i s of the vasopressor peak showed the e i g h t c o n s t i t u e n t amino acid s of a r g i n i n e vasopressin, t h a t i s to say - t y r o s i n e , phenylalanine, glutamic a c i d , a s p a r t i c a c i d , p r o l i n e , a r g i n i n e , and c y s t e i c a c i d i n p l a c e of c y s t e i n . Again, l y s i n e and a l a n i n e were detected i n a d d i t i o n t o the above amino a c i d s . I t may be concluded t h a t the amino acid s known to be present i n o x y t o c i n and vasopressin were i d e n t i f i e d i n 45 F i g u r e 9 Graphed v a l u e s o f d a n s y l a t e d standard amino a c i d s , and the d a n s y l a t e d h y d r o l y s a t e s o f the o x y t o c i c and v a s o p r e s s o r m o i e t i e s from the p u r i f i e d e x t r a c t s o f the neurohypophyses o f a d u l t pregnant sheep. d. NH = d a n s y l a t e d ammonia 2 d i - l y s = d i - d a n s y l d e r i v a t i v e o f l y s i n e € -Lys = mono- d a n s y l d e r i v a t i v e o f l y s i n e Amino a c i d s o f the unknowns are l a b e l l e d o n l y where i d e n t i f i c a t i o n o f t h e i r R v a l u e s correspond f c l o s e l y t o the R o f a standard amino a c i d . • S O L V E N T S Y S T E M 1 S O L V E N T S Y S T E M 2 S O L V E N T S Y S T E M 3 S O L V E I* I T S Y S T E 1 : M 4 vasopressor stondord oxytocic vasopressor standard oxytocic vasopressor standard oxytocic vasopressor Stondord oxytocic • • Pro • Ala • • Glu _«-_.. A lltu b d. NHj • L«u • Phe ° Vol • Pro • Ala • Gly • di-Lys • Tyr • His • Glu • Thr • Ser e Asp • CyS • r.yt. Ara • peptide • • Pro • Ala • • Glu • • • • Phe • • Tyr • • d. NHj • • Leu • Pro, Vol • Phe • Ala • di-Lys • Tyr 0 Gly • His « Glu t> Set,Thr 0 0 lieu 0 Leu 0 0 • Tyf 0 0 0 0 0 0 Arg 0 Cyt • Vol • dl-Lys 0 Ala, Ptw 0 His 0 d. NH2 0 Leu, lieu 0 Tyr 0 Pro, Glu 0 Gly.Ser • Thr.CyS 0 Asp 0 C Lys 0 Arg 0 0 0 0 0 6 Lys 0 Cyt • peptide 0 0 0 Tyr, Lys • Gly,Glu 0 Asp 0 * Pro • lieu 0 Leu 0 d.NHj 0 Tyr 0 di-Lys 0 Gly 0 Glu 0 Asp 0 peptide 0 0 0 Tyr, Lys 0 Gly.Glu 0 Asp 0' the f r a c t i o n s e x h i b i t i n g the appropriate b i o l o g i c a l a c t i v i t i e s . The presence of a d d i t i o n a l amino acid s i n both h y d r o l y s a t e s i s probably due t o the breakdown of c o n s t i t u e n t amino a c i d s , together w i t h contamination from glassware. The extent of p u r i f i c a t i o n ( i . e . the p r o p o r t i o n o f the a d d i t i o n a l amino a c i d s present) could not be determined, s i n c e the t h i n l a y e r s e p a r a t i o n of dansyl amino a c i d s i s a d i f f i c u l t method t o make a c c u r a t e l y q u a n t i t a t i v e . However, the l i m i t e d amount of m a t e r i a l a v a i l a b l e made i t impossible to use any other q u a n t i t a t i v e method of amino a c i d a n a l y s i s . Nevertheless, as f a r as i t could be determined, the p u r i f i c a t i o n procedure appeared t o be very good f o r such a simple system, s i n c e contaminant amino acid s were few, and t h e i r dansyl d e r i v i t i v e s n e i t h e r t r a i l e d nor overloaded the chromatograms. On the b a s i s of t h i s mainly q u a l i t a t i v e system, the p u r i f i e d product from the G-15 dextran columns appeared to be almost pure. 47 DISCUSSION (Section I) The r e s u l t s reported here have shown a r e d u c t i o n i n the l e v e l of the p o s t e r i o r p i t u i t a r y hormones d u r i n g the course of pregnancy. The l e v e l of both o x y t o c i c and vasopressor a c t i v i t i e s were lowest at 90 days of g e s t a t i o n . Although the amount of the s t o r e d hormones remained below t h a t of the c o n t r o l non-pregnant animal throughout g e s t a t i o n , a trend towards increased storage could be seen a t 140 days. The s i n g l e gland taken d u r i n g n a t u r a l labour appeared to show a remarkable increase i n the o x y t o c i c a c t i v i t y . The same p a t t e r n i n the changing hormonal content of the p i t u i t a r y d u r i n g pregnancy was observed i n two d i f f e r e n t breeding seasons. Therefore, the f a l l i n the peptide content of the neurohypophysis d u r i n g mid-pregnancy would appear to be s i g n i f i c a n t s i n c e i t i s d i f f i c u l t to a t t r i b u t e i t to a chance v a r i a b i l i t y which might occur w i t h i n one breeding season. Further, s i n c e i t occurred i n both l y o p h i l i s e d and acetone d r i e d t i s s u e s , i t i s u n l i k e l y to be an a r t i f a c t caused by the method of d r y i n g . No s i g n i f i c a n t changes i n the r a t i o of vasopressor to o x y t o c i c a c t i v i t y were detected a t any stage of pregnancy.. The c l o s e l i n k between o x y t o c i n and reproduction, and the importance of a n t i d i u r e t i c hormone i n water metabolism, which must change d u r i n g pregnancy, would suggest t h a t 4 8 changes i n the hormonal content of the neurohypophysis d u r i n g the course of pregnancy might be a n t i c i p a t e d , and the r e s u l t s presented here demonstrated that such changes do, i n f a c t , occur. Previous attempts, l i m i t e d to the r a t , f a i l e d to f i n d any such changes i n e i t h e r the l e v e l or the r a t i o of the neurohypophysial peptides d u r i n g pregnancy ( H e l l e r and L e d e r i s , 1956; Acher et a l . , 1956). However, v a r i a t i o n s have been seen to occur i n connection w i t h other aspects of reproduction, such as i n the estrous c y c l e , or i n l a c t a t i o n . H e l l e r (1958) detected phasic changes i n the amount of s t o r e d peptides i n the p o s t e r i o r p i t u i t a r y of r a t s , and these changes corresponded w i t h p a r t i c u l a r stages i n the estrous c y c l e . L e v e l s of vasopressor and o x y t o c i c a c t i v i t i e s were found to be higher i n the f o l l i c u l a r phase than i n metestrous. A l o s s of the a c t i v e agents from the p o s t e r i o r p i t u i t a r y of dogs and r a t s during l a c t a t i o n was a l s o observed (Dicker and T y l e r , 1953; H e l l e r and L e d e r i s , 1957; Acher et _ a l . , 1956) . A f a l l i n the content of both hormones was found by a l l i n v e s t i g a t o r s , but there was a disagreement concern-i n g the nature of the changes i n the V/0 r a t i o s . H e l l e r (1961), i n reviewing these r e s u l t s , noted t h a t changes i n r a t i o s were found by workers using acetone d r i e d glands, but not by those working w i t h f r e s h glands. He a t t r i b u t e d the r i s e i n r a t i o t o a p r e f e r e n t i a l s o l u b i l i t y of o x y t o c i n i n acetone. The p o s s i b l e p r e f e r e n t i a l s o l u b i l i t y of o x y t o c i n i n acetone from glands taken from animals under c e r t a i n p h y s i o l o g i c a l c o n d i t i o n s , r a i s e s doubt concerning those r e s u l t s obtained from acetone d r i e d t i s s u e s . The s t u d i e s i n pregnant sheep presented here do not confirm such p r e f e r e n t i a l s o l u b i l i t y of o x y t o c i n , s i n c e the r a t i o s of the hormones were s i m i l a r i n both l y o p h i l i s e d and acetone d r i e d t i s s u e s , even when acetone treatment was prolonged. However, e s t i m a t i o n of the absolute hormonal potencies, f o l l o w i n g acetone treatment, showed a l o s s o f both o x y t o c i c and vasopressor a c t i v i t i e s from glands taken d u r i n g pregnancy, but not from the c o n t r o l s . The experiments reported here were designed p r i m a r i l y to evaluate changes i n b i o l o g i c a l a c t i v i t i e s d u r i n g the course of pregnancy, but the g r e a t e r l o s s of hormonal a c t i v i t y f o l l o w i n g acetone treatment i s an i n t e r e s t i n g problem i n i t s e l f . P o s s i b l y , the answer to t h i s problem l i e s i n an a l t e r e d b i n d i n g of the a c t i v e p r i n c i p l e s d u r i n g pregnancy. Sachs (1967) i n h i s s t u d i e s of neurohypophysial response to haemorrhage, noted an i n i t i a l i ncrease i n the amount of v a s o p r e s s i n r e l e a s e d i n t o the bloodstream, u n t i l approximately 10-20% of the t o t a l a n t i d i u r e t i c hormone content of the p o s t e r i o r p i t u i t a r y was depleted. However, f u r t h e r haemorrhage could not e l i c i t any a d d i t i o n a l r e l e a s e o f the a n t i d i u r e t i c hormone from the neurohypophysis. Sachs suggests t h a t the stored v a s o p r e s s i n i s heterogenous, c o n s i s t i n g of a q u i c k l y and a s l o w l y r e l e a s a b l e component, and w i t h the two components d i f f e r i n g from one another i n t h e i r b i n d i n g w i t h i n the gland. /An increase i n the q u i c k l y r e l e a s a b l e , or l o o s e l y bound component of the neurohypophysial hormones, 50 d u r i n g pregnancy, might account f o r the greater l o s s of a c t i v i t y which occurred d u r i n g acetone treatment of the glands of pregnant animals. In p r evious work, two b a s i c methods were u t i l i s e d f o r the p u r i f i c a t i o n of neurohypophysial p e p t i d e s . The f i r s t , used by Acher (1955). and Chauvet (1960), made use of the s p e c i f i c a f f i n i t y of the peptides f o r the c a r r i e r p r o t e i n , neurophysine. In t h i s method, the complex formed between the neurohypophysial octapeptides and neurophysine was p r e c i p i t a t e d by NaCl, and d i a l y s e d to e l i m i n a t e contaminating p e p t i d e s . The neurophysine was then p r e c i p i t a t e d w i t h t r i c h l o r a c e t i c a c i d , and the a c t i v e agents were l e f t i n s o l u t i o n . The a c t i v e agents were separated from one another on i o n exchange columns. The r e l a t i v e s i m p l i c i t y of t h i s method makes i t a t t r a c t i v e ; however, i t came under c r i t i c i s m because of the danger of contaminating the p u r i f i e d products w i t h bovine neurohypophysial peptides which remained unremoved d u r i n g the o r i g i n a l p r e p a r a t i o n of the bovine neurophysine. The other more g e n e r a l l y used p u r i f i c a t i o n method i n v o l v e s simple g e l f i l t r a t i o n of crude e x t r a c t s , f o l l o w e d by the use of ion-exchange columns f o r the r e s o l u t i o n of the two neurohypophysial peptides (Porath and F l o d i n , 1959; Yamashiro, 1964). In 1966, Frankland and h i s co-workers combined the two methods, and subjected neurophysine-peptide complexes, obtained by Acher's method, t o g e l f i l t r a t i o n on G-25 Sephadex supported i n 1 N a c e t i c a c i d and performed the chromato-graphic separation w i t h 0.1 M formic a c i d as the suspension f l u i d . They r e p o r t t h a t t h i s two step method r e s u l t e d i n a complete s e p a r a t i o n o f the two a c t i v e peptides from the c a r r i e r p r o t e i n . The p u r i f i c a t i o n procedure used i n the present i n v e s t i g a t i o n was designed to s i m p l i f y and improve c u r r e n t l y accepted methods of g e l f i l t r a t i o n , as a p p l i e d to neurohypophysial p e p t i d e s . The use o f formic a c i d was avoided, s i n c e i t i s a potent reducing agent. I t i s p o s s i b l e t h a t i t could e f f e c t b i o l o g i c a l a c t i v i t y , b y causing chemical changes i n the neurohypophysial peptides, and, f u r t h e r , i t might have d e t r i m e n t a l e f f e c t s on the p r e p a r a t i o n s used f o r b i o l o g i c a l assays. In c o n t r a s t , the a c e t i c a c i d which was used i n the p u r i f i c a t i o n procedure reported here, i s a weak a c i d , which has no reducing potency, and i s a normal component of m e t a b o l i s i n g systems. The procedure a l s o e l i m i n a t e d such severe steps as treatment w i t h t r i c h l o r a c e t i c a c i d , and steps which might add a r t i f a c t s such as the p o s s i b l e a d d i t i o n of f o r e i g n peptides w i t h neurophysine. I t was a simple and r e l a t i v e l y m i l d system, yet i t r e s o l v e d h i g h l y p u r i f i e d neurohypophysial peptides i n a s i n g l e step. The r e s u l t s of the amino a c i d a n a l y s i s were i n good agreement w i t h those of Acher and Fromageot (1957), who i d e n t i f i e d o x y t o c i n and a r g i n i n e vasopressin as the a c t i v e peptides of sheep neurohypophysis of unknown reproductive c o n d i t i o n . 52 SECTION I I STUDIES OF THE BIOLOGICAL ACTIVITIES AND CHEMICAL  NATURE OF THE ACTIVE AGENTS IN THE NEUROHYPOPHYSIS  OF THE FOETAL AND NEWBORN SHEEP In t h i s p a r t of the i n v e s t i g a t i o n , p o s t e r i o r p i t u i t a r i e s of sheep embryos at d i f f e r e n t g e s t a t i o n a l ages, and of newborn lambs, were assayed f o r t h e i r b i o l o g i c a l a c t i v i t i e s . Samples at corresponding stages of i n t r a u t e r i n e development were pooled and subjected to p u r i f i c a t i o n procedures; the neurohypophyses o f e a r l y foetuses were p u r i f i e d by paper chromatography, and of the l a t e (140 days) foetuses by g e l f i l t r a t i o n . The d u r a t i o n of g e s t a t i o n i s 145-147 days i n the sheep. Foetuses at 89-91 days of g e s t a t i o n a l age weighed o n l y 500-600 grams, lacked f u r , and showed the d i s p r o p o r t i o n a t e head measurement of the e a r l y foetus. By 140 days o f g e s t a t i o n a l age, which i s only a few days p r i o r to p a r t u r i t i o n , the foetuses weighed 4-5 kgs, were covered w i t h f u r , and g e n e r a l l y appeared to be f u l l y developed. 1. B i o l o g i c a l a c t i v i t i e s of the f o e t a l neurohypophysis S i n g l e glands of foetuses a t 89-91 days, and a t 138-141 days of g e s t a t i o n a l age were c o l l e c t e d immediately a f t e r death. The glands were i n d i v i d u a l l y e x t r a c t e d and assayed f o r o x y t o c i c , vasopressor and a n t i d i u r e t i c a c t i v i t i e s . S i m i l a r 53 s t u d i e s were made on glands taken from lambs at b i r t h and at 13-15 days of p o s t - n a t a l age, i n order t o determine whether trends seen d u r i n g f o e t a l development might continue i n t o e a r l y p o s t - n a t a l l i f e . C o l l e c t i o n s were made i n two d i f f e r e n t breeding seasons. The glands i n the f i r s t b r eeding season were acetone d r i e d and allowed t o stand i n acetone up to an a d d i t i o n a l 14 months. P o s t e r i o r p i t u i t a r i e s i n the second breeding season were immediately l y o p h i l i s e d and sto r e d over P2°5 f ° r a s i m i l a r p e r i o d (see Methods). Vasopressor, a n t i d i u r e t i c and o x y t o c i c a c t i v i t i e s were detected i n a l l glands. The r e s u l t s o f the b i o l o g i c a l assays are presented i n Table I I I f o r acetone d r i e d , and i n Table IV f o r l y o p h i l i s e d t i s s u e s . Weighted means were c a l c u l a t e d f o r groups of t i s s u e s a t the same developmental stage, and confidence l i m i t s assigned at the 5% p r o b a b i l i t y l e v e l as desc r i b e d i n the Methods, a. Acetone d r i e d t i s s u e The weighted means of o x y t o c i c , vasopressor and a n t i d i u r e t i c a c t i v i t i e s f o r corresponding developmental stages are p l o t t e d i n F i g . 10. The average o x y t o c i c a c t i v i t y a t 90 days of g e s t a t i o n was low, and amounted to o n l y 10 mU/mg dry powder. Since the average weight of the glands at the e a r l y stage of g e s t a t i o n a l age i s o n l y 0.8 mg, the t o t a l o x y t o c i c a v t i v i t y i n the e a r l y f o e t a l sheep p i t u i t a r y d i d not exceed 8 mU/gland i n the acetone d r i e d t i s s u e . In the same glands the vasopressor and a n t i d i u r e t i c a c t i v i t i e s were nota b l y higher and averaged 120 mU/mg, or a t o t a l of 96 mU/ 54 TABLE I I I POTENCIES AND THE RATIOS OF THE POTENCIES IN THE ACETONE-DRIED TISSUES OF SHEEP FOETUSES AND LAMBS No. Gest. Days RU mU/mg Pressor mU/mg ADH mU/mg V/0 R a t i o A/0 Ra t i o 4b 89 14.0 113.0 + 37.5 149.6 + 21.3 10.0 13.6 2 90 8.3 170.6 + 32.5 144.9 + 19.4 20.0 17.0 6a 91 10.2 122.8 + 59.0 109.0 + 10.0 12.0 10.0 29 87 31.8 + 2.0 557.0 +102.0 769.1 +135.0 18.0 + 2.0 24.2 + 6.8 8 138 157.0 + 12.2 1237.0 +191.4 2004.0 +1022.0 7.8 + 0.95 12.7 + 6.0 9b 138 93.3 + 16.9 713.4 +124.2 879.6 +284.0 7.6 + 1.83 9.4 + 3.3 13 138 59.9 + 7.6 653.6 +116.4 591.1 +193.8 11.0 + 1.8 10.0 + 5.0 27c Term 196.0 + 35.1 901.2 +199.9 921.0 +212.5 4.6 + 1.24 4.7 + 1.24 1 15 p.n. 63.0 + 7.8 263.0 + 53.6 201.6 + 47.0 4.2 + 0.93 3.2 + 0.08 2 13 p.n. 101.7 + 7.6 342.9 + 74.8 164.3 + 55.'2 3.3 + 0.76 1.6 + 0.15 55 gland. At 140 days, a 10 f o l d increase i n the o x y t o c i c and an approximately 8 f o l d increase i n the vasopressor and a n t i -d i u r e t i c potencies were detected. At the same time, the weight of the glands increased from 0.8 mg to 3.5 mg, making the t o t a l i ncrease i n the amount of stored peptides 45 f o l d . A f u r t h e r r i s e i n potency was detected at p a r t u r i t i o n i n the o x y t o c i c and vasopressor, but not the a n t i d i u r e t i c a c t i v i t i e s . This discrepancy between the vasopressor and a n t i d i u r e t i c a c t i v i t i e s i s probably not s i g n i f i c a n t i n view of the overlapping c o n f i d -ence l i m i t s . A f t e r b i r t h , a l l three b i o l o g i c a l a c t i v i t i e s appeared t o be s i g n i f i c a n t l y lower than at l a t e g e s t a t i o n . The V/0 and A/0 r a t i o s , at a high value i n the e a r l y g e s t a t i o n a l age, e x h i b i t e d a very steady change throughout g e s t a t i o n , and are presented i n P i g . 11. At 90 days of g e s t a t i o n the r a t i o s approximated 14:1, which r e f l e c t e d the preponderance of the vasopressor and a n t i d i u r e t i c a c t i v i t i e s a t t h i s e a r l y stage of g e s t a t i o n . The p r o p o r t i o n a l l y greater accumulation of o x y t o c i c a c t i v i t y i n the neurohypophysis w i t h the advancement of g e s t a t i o n and e a r l y p o s t - n a t a l development, r e s u l t e d i n the steady f a l l of the r a t i o s throughout g e s t a t i o n . At 13-15 days of p o s t - n a t a l age, the r a t i o of vasopressor to o x y t o c i c a c t i v i t y was s t i l l f a r from the a d u l t r a t i o of approximately 1, and was found to be 3:1 i n favour of the vasopressor moiety. b. L y o p h i l i s e d t i s s u e The weighted means f o r o x y t o c i c , vasopressor and a n t i d i u r e t i c a c t i v i t i e s at corresponding g e s t a t i o n a l stages i 56 TABLE IV POTENCIES AND THE RATIOS OF THE POTENCIES IN THE LYOPHILISED TISSUES OF SHEEP FOETUSES AND LAMBS NO. Gest Days . RU mU/mg Pressor mU/mg ADH mU/mg V/0 R a t i o ADH/O Ra t i o 2a 87 52.0 738.0 +156.0 1058.5 +601.0 14.0 12.0 3 90 70.9 + 13.4 897.1 + 83.6 1517.0 +269.3 12.6 + 2.16 21.0 + 7.0 . 16a 90 53.9 + 7.0 1126.0 +269.4 1265.0 +225.8 20.8 . + 7.5 23.4 +4.6 4 90 50.3 + 18.0 899.8 +101.4 17.8 + 5.2 13a 89 74.0 + 13.6 937.0 +117.8 12.6 + 3.3 17b 137 104.2 + 12.8 746.8 +132.5 7.2 +0.8 10 140 145.6 + 15.8 490.0 + 41.3 3.4 +0.65 19b 141 110.5 + 16.6 2126.3 +323.4 2734.3 +1001.6 19.0 + 3.38 24.0 + 7.8 23c 141 202.0 + 15.0 1472.6 +293.0 2405.8 +858.0 7.3 + 1.50 12.0 + 3. 27 22 143 176.6 + 24.0 1524.5 +514.8 1428.0 +530.3 8.6 + 3.0 8.1 + 3.2 2 Terra 142.3 + 25.0 776.6 + 97.8 1333.7 +243.1 5.4 +0.8 9.4 + 3.1 1 12 d.p.n. 154.9 + 9.3 801.1 + 79.5 820.3 +177.9 5.2 + 0.58 5.3 + 1.1 57 were p l o t t e d i n F i g . 12. The average o x y t o c i c a c t i v i t y a t 90 days of g e s t a t i o n a l age was low, and amounted to on l y 60 mU/mg dry powder. The average weight of a l y o p h i l i s e d gland at t h i s stage was approximately 0.6 mg and, t h e r e f o r e , the t o t a l o x y t o c i c a c t i v i t y i n the e a r l y stage of g e s t a t i o n d i d not exceed a t o t a l of 40 mU/mg. In the same glands, the vasopressor a c t i v i t y was approximately 900 mU/mg, and the a n t i d i u r e t i c a c t i v i t y 1200 mU/mg; t h i s corresponds t o a t o t a l of 550 mU/gland vasopressor and 720 mU/gland a n t i d i u r e t i c a c t i v i t y . At 140 days of ge s t a t i o n , an increase i n a l l b i o l o g i c a l a c t i v i t i e s was detected, but the increase d i d not appear t o be. as great as was detected i n the acetone d r i e d t i s s u e s . A f a l l i n the p o s t e r i o r p i t u i t a r y content of the a c t i v e agents was again demonstrated at 13-15 days of p o s t - n a t a l age, but i n c o n t r a s t to the acetone d r i e d t i s s u e s , a f a l l i n potency a l s o occurred at the time of n a t u r a l d e l i v e r y . The V/0 and A/0 r a t i o s , presented i n F i g . 12, e x h i b i t e d a s i m i l a r p a t t e r n of s t e a d i l y decreasing values as was alr e a d y noted i n the acetone d r i e d t i s s u e s . c. Comparison of b i o l o g i c a l a c t i v i t i e s i n acetone d r i e d and l y o p h i l i s e d glands The two s e r i e s of t i s s u e s were s i m i l a r i n showing,an increased accumulation of the a c t i v e agents w i t h the advance-ment of g e s t a t i o n , a predominance of vasopressor and a n t i -d i u r e t i c a c t i v i t i e s over the o x y t o c i c a c t i v i t y d u r i n g embryonic development, and an apparent r e d u c t i o n i n the 58 Figure 10 Weighted means of b i o l o g i c a l a c t i v i t i e s in acetone dried neurohypophyses of sheep foetuses and lambs. mU/mg = m i l l i u n i t per mg dry gland S H E E P F O E T U S ( A C E T O N E DRIED) 2 0 0 0 r 1500 cn E \ E 1000 -5 0 0 h pressor 10 30 5 0 70 9 0 110 Days Gesta t ion 130 147 T term 2 5 Post Nata l l e v e l of a c t i v e peptides a f t e r b i r t h . The changes i n r a t i o s d u r i n g i n t r a u t e r i n e l i f e and e a r l y p o s t - n a t a l development have shown e s s e n t i a l l y the same p a t t e r n i n both s e r i e s , as i l l u s t r a t e d i n F i g . 13. However, the two s e r i e s d i f f e r e d from one another i n s e v e r a l other r e s p e c t s . The d i f f e r e n c e between the acetone d r i e d and l y o p h i l i s e d glands was very pronounced i n the absolute hormonal p o t e n c i e s . At 90 days of g e s t a t i o n o x y t o c i c and vasopressor a c t i v i t i e s were 80-90% lower i n the acetone d r i e d than i n the l y o p h i l i s e d glands. At 140 days, the l o s s of a c t i v i t y f o r both b i o l o g i c a l a c t i v i t i e s amounted to approximately 30%, and a f t e r b i r t h again a g r e a t e r l o s s of a c t i v i t y was detected, approximately 50-60%. From these values i t appeared t h a t the extent of acetone e x t r a c t i o n was c l o s e l y s i m i l a r f o r the o x y t o c i c and vasopressor a c t i v i t i e s , but v a r i e d from one stage of develop-ment to the other. Acetone e x t r a c t e d a high p r o p o r t i o n of both a c t i v e agents at the e a r l y stage of embryonic and at p o s t - n a t a l development, but e x h i b i t e d r e l a t i v e l y l i t t l e i n f l u e n c e on the hormonal content of the p i t u i t a r y i n l a t e g e s t a t i o n . In the l y o p h i l i s e d s e r i e s , a c o n s i s t e n t l y h igher a n t i d i u r e t i c than vasopressor a c t i v i t y was detected, but i n the acetone d r i e d s e r i e s a good correspondance between the two a c t i v i t i e s was found. In view of the f a c t t h a t vasopressor and a n t i d i u r e t i c assays supposedly measure the R a t i o s of b i o l o g i c a l a c t i v i t i e s i n the acetone d r i e d neurohypophyses of sheep foetuses and lambs. R a t i o of V/0 = weighted mean of vasopressor a c t i v i t y per mg dry gland/weighted mean of o x y t o c i c a c t i v i t y per mg dry gland R a t i o of A/0 = weighted mean of a n t i d i u r e t i c a c t i v i t y per mg dry gland/weighted mean of o x y t o c i c a c t i v i t y per mg dry gland Ratio of V / O b i o l o g i c a l a c t i v i t i e s of the same peptide, t h i s f i n d i n g was unexpected. F i n a l l y , i n the acetone d r i e d s e r i e s a f a l l i n potency of the n e u r a l lobe was detected a f t e r d e l i v e r y , w h i l e i n the l y o p h i l i s e d glands the o x y t o c i c , vasopressor and a n t i d i u r e t i c a c t i v i t i e s were found to be already lower at the time of d e l i v e r y . Since these estimations are based on s i n g l e glands i n both cases, the discrepancy could be due to i n d i v i d u a l v a r i a t i o n s . However, i t could a l s o represent a d e p l e t i o n of the p o s t e r i o r p i t u i t a r y of the lamb d u r i n g d e l i v e r y . Although both glands were taken i n the l a t e phase of the f i r s t stage of d e l i v e r y , i t i s p o s s i b l e t h a t labour had been more arduous i n the case o f the lamb i n which the p o s t e r i o r p i t u i t a r y was consequently l y o p h i l i s e d ; e x t r a d e p l e t i o n could have occurred as a r e s u l t of e x t r a s t r e s s . 2. P u r i f i c a t i o n and amino a c i d a n a l y s i s of the a c t i v e agents  i n the neurohypophyses of sheep foetuses In view of the high V/0 and A/0 r a t i o s detected i n the neurohypophyses of sheep foetuses, a p o s s i b l e s e p a r a t i o n and i d e n t i f i c a t i o n of the a c t i v e p r i n c i p l e s appeared to be e s p e c i a l l y important. A r g i n i n e v a s o p r e s s i n has an i n t r i n s i c r a t uterus a c t i v i t y as w e l l as vasopressor a c t i v i t y , and these are i n an approximate V/0 r a t i o of 20:1 (Berde, Boissonnas, 1966). Since the r a t i o s recorded i n the i n d i v i d u a l glands o f sheep foetuses o f t e n approached t h i s f i g u r e , i t seemed p o s s i b l e t h a t the e a r l y sheep foetus possessed a r g i n i n e 62 Figure 12 Weighted means of b i o l o g i c a l a c t i v i t i e s in the l y o p h i l i s e d neurohypophyses of sheep foetuses and lambs. mU/mg = m i l l i u n i t b i o l o g i c a l activity/mg dry gland t SHEEP F O E T U S (LYOPHILISED) 63 v a s o p r e s s i n alone, and was f r e e of o x y t o c i n . However, the low b i o l o g i c a l a c t i v i t i e s and l i m i t e d amount of m a t e r i a l i n the e a r l y foetuses made p u r i f i c a t i o n by g e l f i l t r a t i o n i mpossible. Therefore, the pooled sample of p o s t e r i o r p i t u i t a r i e s of e a r l y foetuses, which contained both acetone d r i e d and l y o p h i l i s e d glands, was subjected t o paper chromatography i n order to determine whether an a c t i v i t y peak, t y p i c a l of o x y t o c i n i t s e l f could be r e s o l v e d from the mixture. Neurohypophysial e x t r a c t s from the l a t e r , 140 days o l d foetuses were subjected to g e l f i l t r a t i o n , and the p u r i f i e d p r i n c i p l e s were anlaysed f o r t h e i r amino a c i d composition. a. P a r t i a l p u r i f i c a t i o n of f o e t a l neurohypophysial  p r i n c i p l e s by paper chromatography The p o s t e r i o r p i t u i t a r i e s of 22 foetuses at 89-91 days of g e s t a t i o n a l age, w i t h a t o t a l d r y weight of 14 mg, were ex t r a c t e d i n 1 ml of 0.25% a c e t i c a c i d (see Methods). 0.84 ml of e x t r a c t , which represented a t o t a l of 350 mU of o x y t o c i c and 7250 mU of vasopressor a c t i v i t y , was a p p l i e d to Whatmann #3MM chromatography paper. 500 mU s y n t h e t i c o x y t o c i n ("Syntocinon") and 12500 mU commercial vasopressin p r e p a r a t i o n ( " P i t r e s s i n " ) were run alongside the e x t r a c t s as standards. The chromatogram was developed i n b u t a n o l / a c e t i c acid/water (4:1:5) f o r a p e r i o d of 13 hours at room temperature. A f t e r completion of the development, the chromatogram was d r i e d i n a c o o l stream of a i r , and when'dry, cut i n t o squares, F i g u r e 13 Rati o s of b i o l o g i c a l a c t i v i t i e s i n the l y o p h i l i s e d neurohypophyses of sheep foetuses and lambs. R a t i o of V/0 = weighted mean of vasopressor a c t i v i t y / mg dry tissue/weighted mean of o x y t o c i c a c t i v i t y per mg dry t i s s u e . R a t i o of A/0 = weighted mean of a n t i d i u r e t i c a c t i v i t y per mg dry tissue/weighted mean of o x y t o c i c a c t i v i t y per mg dry t i s s u e . Ratio of V / O 65 of which each one represented 0.1 of an uni t (see Methods). Each square was eluted in 0.25% acetic acid and analysed for b i o l o g i c a l a c t i v i t i e s . The results are presented i n F i g . 15, and show a successful separation of oxytocin from vasopressin. Synthetic oxytocin was located at R^ 0.5-0.7, and arginine/lysine vasopressin (Pitressin) at Rf 0.3-0.4, with a trace (15 mU) over-lapping into R^ 0.5. From the 500 mU synthetic oxytocin applied, a t o t a l of 163 mU was recovered, representing a 32% recovery. The 4000 mU P i t r e s s i n recovered was again a 32% recovery. Both oxytocic and vasopressor a c t i v i t i e s were detected in the chromatogram of the f o e t a l p o s t e r i o r p i t u i t a r y extracts. Oxytocic a c t i v i t y was found from Rf 0.2 to 0.7, and vasopressor a c t i v i t y in Rf 0.2 to 0.4, with traces of a c t i v i t y in Rf 0.5 and 0.6. The rat uterus a c t i v i t y in R^ 0.5-0.7 corresponds to the location of synthetic oxytocin, and the r a t uterus a c t i v i t y in Rf units corresponding to those of the vasopressor a c t i v i t y can be p a r t i a l l y explained by the i n t r i n s i c oxytocic a c t i v i t y of vasopressin. In view of the r a t uterus a c t i v i t y in Rf 0.5-0.7, which runs ahead of vasopressin, and corresponds well to the location of the synthetic oxytocin, i t i s reasonably certain that the neurohypophyses of early foetuses contain oxytocin i t s e l f . The oxytocic a c t i v i t y recovered i n R f 0.5-0.7 i s approximately 32% of the rat uterus a c t i v i t y detected in the crude extracts, 66 Figure 14 Comparison of ra t i o s of b i o l o g i c a l a c t i v i t i e s between the acetone dried and l y o p h i l i s e d tissue of sheep foetuses and lambs. Ratio of V/0 = weighted means of vasopressor a c t i v i t y per mg dry tissue/weighted means of oxytocic a c t i v i t y per mg dry tis s u e . Ratio of A/0 = weighted means of a n t i d i u r e t i c a c t i v i t y per mg dry tissue/weighted means of oxytocic a c t i v i t y per mg dry tissue. i RATIO OF ACTIVITIES, FOETAL SHEEP O lyophilised o > o o o rr 50 Doys Gestation 100 147 12 25 r term Post Notal 50 100 Days Gestation 147 12 25 T term Post Natal which gives a recovery, s i m i l a r to the recovery found f o r the standards. Vasopressor a c t i v i t y , from the f o e t a l e x t r a c t s was l o c a t e d at R f 0.2-0.4 w i t h t r a c e s i n 0.5 and 0.6 as was seen a l s o i n the case of standard v a s o p r e s s i n . This i s i n good agreement w i t h the R_^  values of a r g i n i n e / l y s i n e v a s o p r e s s i n ( P i t r e s s i n ) and i n d i c a t e s the probable presence o f a r g i n i n e v a s o p r e s s i n i n the e a r l y f o e t u s . In the chromatogram of e a r l y foetuses, r a t uterus a c t i v i t y was detected i n the R^ u n i t s corresponding to the vasopressor a c t i v i t y as w e l l as i n those which corresponded to the R^ values of o x y t o c i n i t s e l f . As pointed out e a r l i e r , the presence of r a t uterus a c t i v i t y i n the vasopressor peak was not e n t i r e l y s u r p r i s i n g , i n view of the i n t r i n s i c o x y t o c i c a c t i v i t y possessed by vaso p r e s s i n . However, the magnitude of t h i s a c t i v i t y was found to be 5 times too great t o be accounted f o r by the amount of vasopressin present. This discrepancy suggested the p o s s i b l e presence of a slow-moving p r i n c i p l e , w i t h chromatographic behaviour s i m i l a r to t h a t of a r g i n i n e vasopressin, but which contrasted w i t h i t i n possessing a r e l a t i v e l y higher i n t r i n s i c o x y t o c i c a c t i v i t y . The o n l y known n a t u r a l l y o c c u r i n g peptide w i t h the p r o p e r t i e s approximating to these, i s a r g i n i n e v a s o t o c i n . A r g i n i n e v a s o t o c i n i s d i s t i n g u i s h e d from the other neurohypophysial peptides, by i t s very high a c t i v i t y i n promoting water t r a n s p o r t through the i s o l a t e d f r o g bladder. Therefore, i n order to determine the p o s s i b l e presence of a r g i n i n e v a s o t o c i n 68 Figure 15 Paper chromatogram of crude extract of the neurohypophyses of sheep foetuses at 89-91 days of gestational age. Black = rat uterus a c t i v i t y Striped = vasopressor a c t i v i t y White — frog waterbalance a c t i v i t y . R f. 0 .1 . .2 3 .4 -5 .6 .7 .8 .9 !.0 2500 2000 r 1 i . 1 i "f • - 1 — * - — " — I 1 — r t . S y n t h e t i c O x y t o c i n R a t U t e r u s bssssssssa — . ! - ' ?rd V a s o p r e s s i n V a s o p r e s s o r 38000 36000 h 69 w i t h i n the vasopressor peak, f r o g bladder assay was c a r r i e d out. As shown i n F i g . 16, a very h i g h f r o g bladder a c t i v i t y was detected at the peak.of the vasopressor a c t i v i t y , and a l e s s e r q u a n t i t y was found i n the two u n i t s on e i t h e r s i d e . The f r o g bladder a c t i v i t y , which amounted to 38,000 mU i n the r e g i o n between R^ 0.2-0.3, i s 450 times too great to be a t t r i b u t a b l e t o the amount ov~vasopressin present. I t i s 100 times too great to be e l i c i t e d by the amount of r a t uterus a c t i v i t y i n t h i s r e g i o n , i f t h i s r a t uterus a c t i v i t y was the r e s u l t of a p o s s i b l e t r a i l i n g of o x y t o c i n i n t o the vasopressor peak. In any case, such t r a i l i n g i s most u n l i k e l y i n view of the s m a l l amount of o x y t o c i n present. Therefore, there i s a strong suggestion t h a t the neurohypophyse of e a r l y foetuses c o n t a i n a r g i n i n e v a s o t o c i n i n a d d i t i o n to a r g i n i n e v a s o p r e s s i n and o x y t o c i n . b. Gel f i l t r a t i o n o f crude neurohypophysial e x t r a c t s from  l a t e foetuses Since s e p a r a t i o n and a h i g h degree of p u r i f i c a t i o n was achieved w i t h maternal e x t r a c t s by the use of 200 cm G-15 Sephadex columns, the same method was a p p l i e d to the p u r i f i c a t i o n of neurohypophysial e x t r a c t s of l a t e foetuses. Acetone d r i e d and l y o p h i l i s e d neurohypophyses of sheep foetuses a t 138-141 days of g e s t a t i o n a l age were pooled, and e x t r a c t e d i n 0.25% a c e t i c a c i d . A t o t a l of 15 ml e x t r a c t , r e p r e s e n t i n g 4.4 + 0.48 I.U. o x y t o c i c and 34.0 + 8.0 I.U. vasopressor a c t i v i t y , were a p p l i e d to a 200 cm G-15 Sephadex column. The e l u a t e from the column was c o l l e c t e d i n 70 Figure 16 P u r i f i c a t i o n of crude extract of sheep foetuses at 138-141 days of gestational age, on 200 cm Sephadex G-15 column. Purification of Foetal Sheep Ext rac t 150 a> •a g" 100 C L 5 E \ 3 50 0 20 on 2 0 0 cm. Sephadex 6 -15 column x n I J - J L 4 0 6 0 •Lowry vasopressor. oxytocic 1500 1000 5 0 0 80 100 120 140 160 180 2 0 0 2 2 0 Tube Number ( I tube = 2. 8ml) 2.8 ml f r a c t i o n s as p r e v i o u s l y described f o r the maternal e x t r a c t s and the f r a c t i o n s were analysed f o r b i o l o g i c a l a c t i v i t i e s , p r o t e i n content and c o n d u c t i v i t y . The r e s u l t s are shown i n F i g . 17. The f i r s t 150 ml of e l u a t e represented the v o i d volume and was disc a r d e d . The mass o f p r o t e i n s , as determined by the Lowry peptide method, were concentrated i n the f i r s t 40-70 f r a c t i o n s . The o x y t o c i c a c t i v i t y was w e l l separated from the Lowry peptide s e c t i o n by 70 f r a c t i o n s . A f u r t h e r 20 f r a c t i o n s d i v i d e d the vasopressin from the o x y t o c i c peak. The marked s e p a r a t i o n of the a c t i v e agents from the p r o t e i n f r a c t i o n s , and the complete r e s o l u t i o n of the o x y t o c i c and vasopressor a c t i v i t i e s i n d i c a t e d t h a t a good p u r i f i c a t i o n of the hormones was achieved. The 2.0 I.U. o x y t o c i c a c t i v i t y detected i n the o x y t o c i c peak represented a 100% recovery, i f the c o n t r i b u t i o n of the l a r g e amount of vasopressin t o the o x y t o c i c a c t i v i t y of the crude e x t r a c t i s taken i n t o account. The recovery f o r vasopressin was again approximately 80%. The pooled f r a c t i o n s of the o x y t o c i c peak were assayed f o r t h e i r o x y t o c i c a c t i v i t y both i n the presence and absence of 0.5 mM MgCl. The r a t i o of o x y t o c i c a c t i v i t y w i t h Mg + +/ without Mg + + was found to be 0.84, which i s i n good agreement w i t h the value of 0.86 reported by Munsick (1960) f o r the p o t e n t i a t i o n of s y n t h e t i c o x y t o c i n . Since o x y t o c i n i s the on l y known neurohypophysial p r i n c i p l e i n which t h i s r a t i o i s l e s s than 1, t h i s r e s u l t s t r o n g l y suggests t h a t the r a t uterus 72 Figure 17 Sample pla t e of t h i n layer chromatography of the dansyl derivatives of standard amino acids, and of the hydrolysate of the oxytocic moiety of the adult pregnant sheep and the foetus. DNS = dansyl s o l v e n t f r o n t o D N S L e u 0 DNS I l eu 0 O N S G l y 0 DNS Tyr 1 0 D N S P r o - i *-DNS C y t -0-D N S A s p D N S h y d r o l y s a t e s 0 xytocl c Adul t F o e t a l ft o 0 o o O O o 0 D N S NH 0 o o o D N S Gfu 0 di DNS| Lys r o r i g i n 0 (al c o n t r a c t i n g p r i n c i p l e of the l a t e foetus i s o x y t o c i n i t s e l f . The e s t i m a t i o n of the o x y t o c i c a c t i v i t y throughout the vasopressor peak, showed t h a t one f r a c t i o n (#187 i n F i g . 17) contained an u n u s u a l l y h i g h o x y t o c i c a c t i v i t y , which could not be accounted f o r by the i n t r i n s i c o x y t o c i c a c t i v i t y of va s o p r e s s i n . Frog waterbalance assay was c a r r i e d out on t h i s f r a c t i o n t o determine whether i t might c o n t a i n a r g i n i n e v a s o t o c i n , as the work presented above had suggested t h a t t h i s peptide might be present i n the young foetuses. The t o t a l f r o g waterbalance a c t i v i t y i n the 2.8 ml of e l u a t e present i n t h i s tube was found to be 5,600 mU. This value i s 80 times greater than could be expected from a r g i n i n e v a s o p r e s s i n alone, i f i t were present i n the q u a n t i t y i n d i c a t e d by the pre s s o r assay. Again, t h i s suggests the presence of a peptide other than vasopressin, a peptide possessing u n u s u a l l y h i g h f r o g bladder a c t i v i t y . The same arguments given p r e v i o u s l y f o r the s i m i l a r a c t i v i t y found i n the e a r l y foetus suggest t h a t t h i s peptide might be a r g i n i n e v a s o t o c i n . c. Amino a c i d a n a l y s i s of the p u r i f i e d peptides of the l a t e foetuses The o x y t o c i c and vasopressor f r a c t i o n s of 140 days o l d foetuses, obtained from the G-15 Sephadex column were pooled, l y o p h i l i s e d and hydrolysed i n 6N HCl (see p. 22 Methods). The hy d r o l y s a t e s were reacted w i t h the f l u o r e s c e n t 74 F i g u r e 18 Graphed R^ values of dansylated standard amino a c i d s , and the dansylated h y d r o l y s a t e o f the f o e t a l vasopressor moiety. d. NH^ = dansyl ammonia d i - Lys = d i - dansyl d e r i v a t i v e o f l y s i n e l y s i n e = mono-dansylated d e r i v a t i v e of l y s i n e /Amino a c i d s of the unknowns are l a b e l l e d o n l y where i d e n t i f i c a t i o n of t h e i r R^ values correspond c l o s e l y t o the R of a standard amino a c i d . f S O L V E N T SYSTEM I S O L V E N T S Y S T E M 2 standard vasopressor standard vasopressor 0.2 — 0.1 -S lleu d. N H2 • Leu 2 Ph e ° Vol • Pro Ala Gly di- Lys • Tyr • His # Glu • Thr O Ser • Asp * CyS, « Cvi. Arg • peptide • Pro Gly # Glu ? • peptide d. NH, lieu Leu Pro, Vol • Phe • Ala • di-Lys Tyr G ly His Glu Ser, Thr • Phe • Ala, di-Lys • Tyr S O L V E N T SYSTEM 3 ta n dor d vo s o p res s o r S O L V E N T S Y S T E M 4 s tondard vasopressor e> e Vol di-Lys Ala, Piie His d. NH2 Leu, lieu Tyr Pro, Glu Gly, Ser Thr, CyS Asp € Lys Arg • Cyt • Arg • Cyt • Pro * lieu Leu d. NH2 Tyr di-Lys Gly Glu • Asp peptide • Gly, Gib • Asp • Cyt 7 5 marker, d a n s y l c h l o r i d e , as d e s c r i b e d i n d e t a i l on p. 23 The r e s u l t i n g amino a c i d complexes were a p p l i e d t o t h i n l a y e r s i l i c a p l a t e s , b e s i d e d a n s y l a t e d amino a c i d standards. The h y d r o l y s a t e s from the fo e t u s were developed on the p l a t e s p a r a l l e l t o h y d r o l y s a t e s from the a d u l t , to p r o v i d e a d i r e c t comparison between the amino a c i d c omposition b f the two. The p l a t e s were run i n f o u r d i f f e r e n t s o l v e n t systems f o r o p t i m a l r e s o l u t i o n o f the standard amino a c i d s . The c o n s t i t u e n t amino a c i d s i n the h y d r o l y s a t e s o f the maternal and f o e t a l o x y t o c i c m o i e t i e s had shown an i d e n t i c a l chromatographic behaviour, as i l l u s t r a t e d by the sample p l a t e g i v e n i n F i g . 17. The more complete a n a l y s i s o f the maternal m a t e r i a l had shown the e i g h t c o n s t i t u e n t amino a c i d s o f o x y t o c i n , as a l r e a d y d i s c u s s e d i n S e c t i o n I, and s i n c e the f o e t a l o x y t o c i c p r i n c i p l e e x h i b i t e d the same chromatographic p a t t e r n , t h i s i n d i c a t e s t h a t the amino a c i d composition o f the f o e t a l o x y t o c i c p e p t i d e i s the same as t h a t o f the a d u l t . ' When taken i n c o n j u n c t i o n w i t h the p h a r m a c o l o g i c a l data, t h i s s t r o n g l y suggests t h a t o x y t o c i n i t s e l f i s the o x y t o c i c agent o f the l a t e f o e t u s e s . The much g r e a t e r amount o f v a s o p r e s s o r agent i n the l a t e f o e t u s e s allowed a more complete a n l a y s i s o f the c o n s t i t u e n t amino a c i d s o f t h i s p e p t i d e . The v a l u e s o f the standard amino a c i d s and the c o n s t i t u e n t amino a c i d s o f the h y d r o l y s a t e are shown i n F i g . 18. The c o n s t i t u e n t amino acids i d e n t i f i e d were: phenylalanine, arginine, aspartic acid, proline, glycene, glutamin acid, tyrosine, and c y s t e i c acid. In addition to the eight constituent amino acids of arginine vasopressin, l y s i n e and alanine were also detected. It i s assumed that these two amino acids were introduced into the hydrolysates during proces ing either by the breakdown of constituent amino acids (as i n the case of alanine), or as contamination from the glassware. 77 DISCUSSION (Section I I ) The e s t i m a t i o n of b i o l o g i c a l a c t i v i t i e s i n sheep foetuses and lambs have shown a s t e a d i l y i n c r e a s i n g accumulation of the b i o l o g i c a l l y a c t i v e p r i n c i p l e s through-out g e s t a t i o n . I n c r e a s i n g amounts of hormonal a c t i v i t i e s w i t h the advancement of embryonic development were c h a r a c t e r i s t i c of both the acetone d r i e d and l y o p h i l i s e d glands. Around the time of b i r t h a secondary f a l l i n both o x y t o c i c and vasopressor a c t i v i t i e s were found. This f a l l i n potency appeared to occur at the time of n a t u r a l d e l i v e r y i n the l y o p h i l i s e d s e r i e s , but occurred o n l y a f t e r b i r t h i n the acetone d r i e d s e r i e s . The r a t i o of vasopressor to o x y t o c i c and of a n t i d i u r e t i c to o x y t o c i c a c t i v i t i e s were high at the e a r l y g e s t a t i o n a l age (90 days), and i n some cases approached the value of 20. During the course of embryonic development and e a r l y p o s t - n a t a l l i f e , these very high r a t i o s decreased s t e a d i l y . The r e s u l t s i n the sheep foetuses and newborn lambs agree g e n e r a l l y w i t h those reported by Dicker and T y l e r (1953 a, b) f o r human and dog foetuses, w i t h respect to the o r i g i n a l l y high, and s t e a d i l y d e c l i n i n g V/0 r a t i o s d u r i n g g e s t a t i o n . The h i g h r a t i o between the vasopressor and o x y t o c i c a c t i v i t i e s i n the f o e t a l neurohypophysis i s the r e s u l t of the ( 78 very low o x y t o c i c a c t i v i t i e s during embryonic development. As before, i n connection w i t h various other aspects of the work reported here, the question a r i s e s as to whether the low o x y t o c i c content of the immature p o s t e r i o r p i t u i t a r y i s a product of depressed hypothalamic s e c r e t i o n , or t o an increased d e p l e t i o n of the gland of i t s o x y t o c i c moiety. The l a t t e r p o s s i b i l i t y appears to be an u n l i k e l y one. On the basis of the primary a c t i o n s of o x y t o c i n i n the a d u l t organism, where i t i s connected w i t h reproduction and the n u r s i n g of the young, i t i s hard to envisage a p o s s i b l e f u n c t i o n f o r j t h i s agent i n the f o e t a l organism. 1 The more l i k e l y p o s s i b i l i t y appears to be a depressed hypothalamic pr o d u c t i o n o f the o x y t o c i c agent. H i s t o l o g i c a l evidence supports t h i s hypothesis. S t a i n i n g w i t h Gomori's chrome haematoxylin p h l o x i n s t a i n shows an e a r l i e r embryonic appearance of s t a i n a b l e m a t e r i a l i n the supra-optic nucleus, than i n the bodies of the p a r a v e n t r i c u l a r c e l l s of human foetuses (Meitner, 1959). Since the p a r a v e n t r i c u l a r nucleus i s mainly r e s p o n s i b l e f o r the p r o d u c t i o n of vasopressin ( H e l l e r , 1966), t h i s o b s e r v a t i o n p o i n t s to a l a t e r embryonic development of the o x y t o c i n producing center. A l a t e r embryonic development of the p a r a v e n t r i c u l a r nucleus has been demonstrated i n other species as w e l l , such as the mouse, r a t and c a l f (Yakovleva, 1966). I t i s necessary t o p o i n t out, t h a t h i s t o l o g i c a l evidence, f o r the presence of neurohypophysial hormones i s o f t e n u n r e l i a b l e , and b i o l o g i c a l a c t i v i t i e s can o f t e n be 79 detected b e f o r e the appearance of s e c r e t o r y m a t e r i a l ( H e l l e r , 1961). Further, a disappearance of these granules can occur without a l o s s i n potency ( F a r r e l l , 1967). However, the l a r g e number of s i m i l a r r e p o r t s , which give h i s t o l o g i c a l evidence f o r a time d i f f e r e n c e between the appearance of s e c r e t o r y m a t e r i a l i n the two hypothalamic n u c l e i , seem to make a st r o n g argument i n favour of a l a t e r embryonic appearance of the o x y t o c i n s y n t h e t i s i n g s t r u c t u r e s . In the r e s u l t s concerning the neurohypophysial hormone content o f the sheep foetuses reported here, a c o n s i s t e n t l o s s of b i o l o g i c a l a c t i v i t y was found f o l l o w i n g prolonged exposure of the glands to acetone. This problem has already been discussed i n S e c t i o n I, i n connection w i t h the same phenomena i n pregnant females. The s t u d i e s on f o e t a l neurohypophyses o n l y emphasised f u r t h e r the f a c t t h a t acetone leaches out both a c t i v e agents to approximately the same degree. However, the extent of t h i s removal v a r i e s w i t h d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s . In the f o e t a l glands the g r e a t e s t l o s s of a c t i v i t y was observed at 90 days of g e s t a t i o n , and a lower l o s s occurred d u r i n g the e a r l y post-n a t a l age. On the other hand, there was o n l y a s l i g h t l o s s of a c t i v i t y i n l a t e g e s t a t i o n , when the foetuses had been i n u t e r o f o r 140 days. The comparison of potencies i n the l y o p h i l i s e d and acetone d r i e d glands of sheep foetuses have a l s o shown a ^ c o n s i s t e n t l y and s i g n i f i c a n t l y higher a n t i - d i u r e t i c than vasopressor a c t i v i t y i n the l y o p h i l i s e d but not i n the acetone d r i e d t i s s u e s . Again, o n l y suggestions can be made as to the p o s s i b l e f a c t o r s c o n t r i b u t i n g to the greater a n t i d i u r e t i c , than vasopressor a c t i v i t i e s recorded i n the l y o p h i l i s e d glands o f foetuses. Since these two bio-assays supposedly measure the potency of the same hormone; the higher a n t i d i u r e t i c potency could be due to e i t h e r an a d d i t i o n a l a n t i d i u r e t i c agent i n the hypophysis of the foetuses or p o s s i b l y to the presence of a vasodepressor agent. Due to l a c k of m a t e r i a l , no t e s t s could be c a r r i e d out to determine the p o s s i b l e presence of e i t h e r agent. In view of the f a c t , however, t h a t other workers have come across the same phenomena ( H i l d and Z e t t l e r , 1953), and t h a t v a r i o u s r e p o r t s c l a i m the s e p a r a t i o n of the vasopressor from the a n t i d i u r e t i c p r i n c i p l e (Kammen and De Wield, 1960) the problem warrants f u r t h e r i n v e s t i g a t i o n . The p u r i f i c a t i o n and a n a l y s i s of the a c t i v e p r i n c i p l e s of the f o e t a l sheep neurohypophysis have shown the presence of an o x y t o c i c and vasopressor p r i n c i p l e . The values of the b i o l o g i c a l l y a c t i v e f r a c t i o n s i n the p o s t e r i o r p i t u i t a r y e x t r a c t s of the foetuses at 90 days of g e s t a t i o n a l age suggest t h a t these a c t i v e agents are: o x y t o c i n and a r g i n i n e va s o p r e s s i n . Again, i n the o l d e r , 140 days o l d embryos, both the pharmacological evidence and the amino a c i d a n a l y s i s p o i n t to o x y t o c i n and a r g i n i n e v a s o p r e s s i n being the a c t i v e agents. In a d d i t i o n to the two nexirohypophysial peptides t y p i c a l of the mammal, the presence of a t h i r d peptide was detected. The chemical behaviour and pharmacological properties of t h i s t h i r d peptide suggest that i t might be arginine vasotocin, the a n t i d i u r e t i c p r i n c i p l e of lower vertebrates. The amount of t h i s peptide appears to decline with the advancement of gestation. Calculations, which converted the b i o l o g i c a l a c t i v i t i e s to mg peptide present, indicated that i n early gestation (90 days) arginine vasopressin and arginine vasotocin are present i n an approximately 1:1 r a t i o . By 140 days of intrauterine l i f e the r a t i o of the two peptides was approximately 120 i n favour of arginine vasopressin. 82 GENERAL DISCUSSION The data obtained in the sheep during pregnancy and embryonic development, have shown that changes occur in the neurohypophysis of both the foetus and the mother during the course of pregnancy. About mid-pregnancy, the neurohypophysis of the mother contains a reduced l e v e l of i t s active agents, and although there i s an increase i n potencies towards l a t e gestation, the hormonal leve l s do not return to the control, non-pregnant l e v e l throughout the duration of pregnancy. During embryonic development, an increasing accumulation of b i o l o g i c a l l y active agents were detected. The presence of both peptides, c h a r a c t e r i s t i c of the adult mammal, oxytocin and vasopressin have been shown to be present in the foetus. In addition to these active p r i n c i p l e s , the presence of a t h i r d neurohypophysial peptide, probably arginine vasotocin, has been indicated. The neurohypophysis of the embryo i s characterised by a pre-dominance of vasopressor over oxytocic a c t i v i t y . The r e s u l t s in both the pregnant adult and i n the foetus have been discussed b r i e f l y i n the l i g h t of the l i m i t e d l i t e r a t u r e , d i r e c t l y concerned with these topics. However, the most important questions have not yet been touched on; these concern the r e l a t i o n s h i p of the changes i n 83 the neurohypophysis to the more general p i c t u r e of pregnancy and f o e t a l development, and to the p o s s i b l e p h y s i o l o g i c a l r o l e of the a c t i v e agents i n the metabolism o f the embryo. This general d i s c u s s i o n w i l l attempt to develop some of these problems. However, because of the s c a r c i t y of information, many of the f o l l o w i n g c o n s i d e r a t i o n s are p u r e l y s p e c u l a t i v e . 1. Changes i n hormonal l e v e l of the neurohypophysis d u r i n g  pregnancy The lowered a c t i v i t y of the neurohypophysis d u r i n g pregnancy r a i s e s the question of whether t h i s d e p l e t i o n i s due to a depressed hypothalamic p r o d u c t i o n of hormones, or to a greater r e l e a s e i n t o the bloodstream. E i t h e r change might be r e l a t e d to changes i n other endocrine glands, and t h e r e f o r e , a b r i e f d e s c r i p t i o n of the primary endocrine m o d i f i c a t i o n s which take p l a c e must be given. The main endocrine changes d u r i n g pregnancy are the elevated estrogen and progesterone l e v e l s , which may o r i g i n a t e to a l a r g e extent from the corpus luteum, of from the p l a c e n t a . In the sheep, where the p l a c e n t a takes over the pro d u c t i o n of these s t e r o i d s at 50-60 days of g e s t a t i o n (M a r s h a l l , 1959), the progesterone l e v e l r i s e s to 6 ug/ml i n the p e r i p h e r a l blood of the pregnant female at 40 days of g e s t a t i o n and remains at t h i s l e v e l u n t i l 100 days. At about 100 days the blood progesterone l e v e l r i s e s and reaches a second p l a t e a u at 12 ug/ml blood, and t h i s i s maintained up to 36 hrs post p a r t u r i t i o n (Neher and Zarrow, 1958). Although 84 there i s no information concerning the estrogen l e v e l s in the sheep during pregnancy, i n the goat and the cow, which are s i m i l a r to the sheep i n t h e i r hormonal control of gestation, estrogen l e v e l was reported to r i s e s t e a d i l y in the second h a l f of pregnancy and reached a maximum value at term (Barrie, Patterson and Underhill, 1935; Bromskov, 1939). Evidence concerning the possible action of the stero i d hormones and the posterior p i t u i t a r y peptides i s suggestive, but sketchy. At the l e v e l of the target organs, progesterone has been shown to decrease the s e n s i t i v i t y of the uterus to oxytocin, w h i l s t estrogen enhances the action of t h i s peptide (F i t z p a t r i c k , 1966). The target organs of vasopressin react i n a s i m i l a r manner to progesterone and estrogen; s e n s i t i v i t y i s lessened i n the presence of the former and increased by the l a t t e r s t e r o i d . Cobo (1967) reported a high threshold for a n t i d i u r e t i c responses to vasopressin i n pregnant women, and Pickford (1966) found a s i m i l a r e f f e c t of progesterone on the s a l t excretion and vascular response to the same p r i n c i p l e . At the l e v e l of the hyopothalamic nuclei, Cross and S i l v e r (1965) reported depressed a c t i v i t y of both the supra-opti c and paraventricular nuclei, following administration of progesterone. Besides evidence for the influence of steroid hormones on the production and effectiveness of the neurohypophysial 85 peptides, these peptides may i n t u r n e f f e c t the s t e r o i d l e v e l s themselves d i r e c t l y or i n d i r e c t l y , through the mediation of the gonadotrophic hormones of the adeno-hypophysis. The r o l e of the neurohypophysial hormones as r e l e a s i n g f a c t o r s f o r the gonadotrophic hormones has been reviewed by M a r t i n i (1966), and o n l y one example w i l l be given here. I t has been shown t h a t l a r g e doses of o x y t o c i n w i l l b r i n g c a t t l e i n t o premature estrous, and i t i s presumed t h a t the peptide i n h i b i t s progesterone production, so t h a t the consequent f a l l i n blood l e v e l unleashes the r e l e a s e of gonadotrophic hormones from the adenohypophysis (Labsethwar et a l . , 1964) These fragmentary p i e c e s of evidence encourage the s p e c u l a t i o n t h a t the elevated progesterone l e v e l renders the t a r g e t organs l e s s s e n s i t i v e to the neurohypophysial hormones at 90 days of pregnancy. This i n tu r n might r e s u l t i n an increased r e l e a s e from the p o s t e r i o r p i t u i t a r y of i t s a c t i v e agents, through secondary mechanisms, only p a r t l y understood a t the present. At the same time, the ele v a t e d progesterone l e v e l might a l s o p a r t i a l l y i n h i b i t hypothalamic production of the pe p t i d e s . The combined e f f e c t of an increased r e l e a s e and p a r t i a l l y i n h i b i t e d p r o d u c t i o n could r e s u l t i n the lowered a c t i v i t y of the neurohypophysis as found i n the s t u d i e s of pregnant sheep at 90 days o f g e s t a t i o n . In the l a t t e r p a r t of pregnancy, the r i s i n g estrogen l e v e l , which reaches i t s maximum at term, could p o s s i b l y counteract the a c t i o n of progesterone, both at the l e v e l of the t a r g e t organ and i n the hypothalamus. The r i s i n g estrogen l e v e l would r e e s t a b l i s h a balance nearer t o t h a t of the c o n t r o l , where the r e l e a s e df the neurohypophysial peptides w i l l be decreased and the production by the hypothalamus increased. This would r e s u l t i n a greater accumulation of neurosecretory m a t e r i a l i n the neuro-hypophysis and would e x p l a i n the secondary r i s e i n a c t i v i t i e s found at 140 days or pregnancy i n the sheep. 2 . The p o s s i b l e r o l e of the a n t i d i u r e t i c p r i n c i p l e , d u r i n g  embryonic development The r e l a t i v e abundance of the vasopressor or a n t i -d i u r e t i c p r i n c i p l e i n the neurohypophysis of the foetus, represents one of the most i n t e r e s t i n g and s t i l l unresolved problems of p o s t e r i o r p i t u i t a r y f u n c t i o n d u r i n g embryonic development. I t i s not c e r t a i n whether t h i s predominance of the a n t i d i u r e t i c a c t i v i t y d u r i n g i n t r a u t e r i n e l i f e represents an e a r l i e r appearance of the s y n t h e t i c mechanism f o r the pr o d u c t i o n of the a n t i d i u r e t i c agent or whether i t r e f l e c t s the greater use of t h i s agent by the organism. However, t h i s b r i e f d i s c u s s i o n w i l l attempt t o show a p o s s i b l e use f o r the a n t i d i u r e t i c p r i n c i p l e by the f o e t a l organism. General evidence i n d i c a t e s a r o l e f o r the a n t i -d i u r e t i c a c t i v i t y i n the volume r e g u l a t i o n of the amniotic f l u i d . B e r n i s c k e and McKay (1953) c o r r e l a t e d h i s t o l o g i c a l 87 s t u d i e s of the p o s t e r i o r p i t u i t a r y of human foetuses w i t h the volume of amniotic f l u i d present, and found t h a t foetuses w i t h v e r y l i t t l e s t a i n a b l e m a t e r i a l i n the p i t u i t a r y have a great excess of amniotic f l u i d , and conversely, an abundance of s e c r e t o r y m a t e r i a l was a s s o c i a t e d w i t h smaller amniotic f l u i d volume. On the b a s i s of these observations i t might appear t h a t the f o e t a l neurohypophysis p l a y s some p a r t i n the r e g u l a t i o n o f the amniotic f l u i d volume, but very l i t t l e i s known about the p o s s i b l e t a r g e t organs which might be concerned. The primary a c t i o n o f the a n t i d i u r e t i c hormone i n the a d u l t would suggest t h a t the f o e t a l kidney could be a p o s s i b l e t a r g e t organ f o r the a c t i o n of the embryonic a n t i d i u r e t i c p r i n c i p l e . However, H e l l e r (1949) working w i t h both newborn humans and r a t s , f a i l e d to e l i c i t e i t h e r a d i u r e t i c response t o waterload, or an a n t i d i u r e t i c response to dehydration. On the other hand, P a u l i n e (1962) recorded t h a t h y d r a t i o n of the f o e t a l sheep at 130 days of g e s t a t i o n r e s u l t e d i n an increased glomerular f i l t r a t i o n r a t e and elevated u r i n e flow. I n j e c t i o n s of vasopressin brought both the elevated f i l t r a t i o n r a t e and u r i n e flow back to a c o n t r o l l e v e l . However, vas o p r e s s i n was found to be without e f f e c t by both the above workers i n animals where f i l t r a t i o n r a t e and u r i n e flow have not been p r e v i o u s l y elevated. Vasopressin at no time promoted the formation o f hypertonic u r i n e i n immature animals. On the b a s i s of these and s e v e r a l other experiments P a u l i n e concluded t h a t the immature kidney i s unable to concentrate i t s u r i n e , and a t t r i b u t e s t h i s f a i l u r e 8 8 t o an e l e c t r o l y t e r e a b s o r p t i o n by the immature kidney, which exceeds the r e a b s o r p t i o n of water. However, i n passing, i t should be po i n t e d out t h a t the f a i l u r e t o produce hy p e r t o n i c u r i n e by the foetus i s not s u r p r i s i n g , s i n c e i t i s p r a c t i c a l l y impossible t o produce a shortage of water i n the foetus, which can draw a l l i t needs from-the maternal organism. Furthermore, the discharge of such a h y p e r t o n i c u r i n e i n t o the embryonic f l u i d compartments would appear to be questionable b e n e f i t f o r both the mother and the foet u s . However, the prod u c t i o n of hy p e r t o n i c u r i n e i s not the o n l y form of water r e g u l a t i o n a v a i l a b l e t o an animal. For example, the amphibian may u t i l i s e water t r a n s p o r t through the s k i n and the bladder (Sawyer, 1967). Nor are the membranes of the kidney the o n l y s t r u c t u r e s i n an a d u l t mammal where v a s o p r e s s i n can promote water r e a b s o r p t i o n ; t h i s i s shown by the a c t i o n of the peptide on the membranes of the pancreas, l i v e r and sweat glands, where i t reduces the volume of s e c r e t i o n (Wakim, 1967). In the f o e t a l organism too, there are many p l a u s i b l e a l t e r n a t e t a r g e t organs, f o r a p o s s i b l e e f f e c t of the a n t i -d i u r e t i c p r i n c i p l e , besides the kidneys. A b r i e f d e s c r i p t i o n of the prod u c t i o n of the amniotic f l u i d , and the compartments i n which i t i s contained suggest s e v e r a l p o s s i b l e p o i n t s o f a c t i o n f o r the a n t i d i u r e t i c agent. In f o e t a l sheep of l e s s than 90 days of g e s t a t i o n a l age, u r i n e from the bladder passes i n t o the a l l a n t o i c sac. Beyond t h i s age, the u r i n e passes i n t o the amniotic sac i n p r o g r e s s i v e l y greater amounts (Pauline, Nixon, 1962). The r a t e o f f o e t a l u r i n e production i s more than adequate to account f o r the volume of embryonic f l u i d , but i t i s by no means t o t a l l y r e s p o n s i b l e f o r the p r o d u c t i o n and maintenance of the f l u i d environment of the f o e t u s . In the sheep, the b u c c a l c a v i t y and the f o e t a l lungs can s e c r e t e up to 15 ml of f l u i d per hour (Reynolds, 1953) . The swallowing of amniotic f l u i d i s a l s o known to occur, and absorption from the g a s t r o i n t e s t i n a l t r a c t a l s o c o n t r i b u t e s to the exchange of water and e l e c t r o l y t e s between the foetus and the embryonic f l u i d s . Therefore, the p o s s i b l e t a r g e t organs of the embryonic neurohypophysial peptides could i n c l u d e the a l l a n t o i c and/or the amniotic membranes ou t s i d e the foetus, or the lungs and b u c c a l c a v i t y w i t h i n the f o e t a l organism. There i s no evidence i n the l i t e r a t u r e to i n d i c a t e whether any of these membranes could respond to v a s o p r e s s i n or to f o e t a l neurohypophysial e x t r a c t . More research w i l l have to be done before the great abundance of a n t i d i u r e t i c p r i n c i p l e reported here can be f i t t e d i n t o the general p i c t u r e of the embryonic development, and the p h y s i o l o g y of the foetus. However, the great number of p o t e n t i a l t a r g e t organs and the p o s s i b l e need of the foetus to r e g u l a t e i t s aqueous environment suggests t h a t t h i s hormone may w e l l be important to the organism dur i n g i t s i n t r a u t e r i n e l i f e . 3. The importance of the presence of a r g i n i n e v a s o t o c i n i n the foetus 90 The high f r o g waterbalance a c t i v i t y detected i n the e a r l y sheep foetuses, w i t h i t s p o s s i b l e i m p l i c a t i o n t h a t the e a r l y f o e t a l neurohypophysis contains a t h i r d a c t i v e peptide, a r g i n i n e v a s o t o c i n , i s important f o r more than one reason. F i r s t l y , i t i s the f i r s t time t h a t t h i s a n t i d i u r e t i c agent, t y p i c a l of lower v e r t e b r a t e s , has been found i n a mammalian organism. Secondly, i t r a i s e s the p o s s i b i l i t y t h a t the t a r g e t organs of the foetus at the e a r l i e r stages of embryonic development could be responsive t o a r g i n i n e v a s o t o c i n , r a t h e r than to a r g i n i n e 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 promotes water re a b s o r p t i o n i n the amphibian kidney, and a l s o r e g u l a t e s water uptake through t h e s k i n and bladder of c e r t a i n amphibians (Sawyer, 1967). I t might be speculated t h a t the a c t i o n of a r g i n i n e v a s o t o c i n on e x t r a r e n a l membranes could be of s p e c i a l relevance to the foetus, where i t might promote water movement through the embryonic membranes, through the lungs or through the b u c c a l c a v i t y . F u r ther, i t may be of some s i g n i f i c a n c e , t h a t a r g i n i n e v a s o t o c i n increases the e l e c t r o l y t e e x c r e t i o n and i n f l u e n c e s the glomerular f i l t r a t i o n r a t e of l u n g f i s h and c e r t a i n other s p e c i e s . These two l a t t e r a c t i o n s of a r g i n i n e v a s o t o c i n might be important to the f o e t a l organism, c o n s i d e r i n g t h a t e l e c t r o l y t e r e a b s o r p t i o n i n the foetus exceeds water rea b s o r p t i o n , and t h a t r e g u l a t i o n o f the volume of the produced u r i n e could be demonstrated on the l e v e l of changes i n the glomerular f i l t r a t i o n r a t e o n l y . A r g i n i n e v a s o t o c i n t h a t could p o s s i b l y a i d i n the r e g u l a t o r y processes i n v o l v e d 91 i n the amniotic f l u i d formation, and the e l e c t r o l y t e balance o f the e a r l y f o e t u s . However, the demonstration o f the presence of a r g i n i n e v a s o t o c i n i n mammalian foetuses, has another s i g n i f i c a n c e . I t i s important i n c o n s i d e r a t i o n s of the phyl o g e n e t i c d i s t r i b u t i o n of the neurohypophysial a n t i -d i u r e t i c p r i n c i p l e . The neurohypophysis o f most v e r t e b r a t e c l a s s e s c o n t a i n an a c t i v e r a t uterus c o n t r a c t i n g p r i n c i p l e , and an a n t i -d i u r e t i c p r i n c i p l e . In the o x y t o c i n l i k e r a t uterus c o n t r a c t i n g p r i n c i p l e s , a great many mutations appear to have occurred d u r i n g e v o l u t i o n . The s t r u c t u r e of the a c t i v e o x y t o c i c p r i n c i p l e can vary not o n l y from one c l a s s of v e r t e b r a t e s to another, but a l s o between species w i t h i n the same c l a s s (Sawyer, 1967). On the other hand a r g i n i n e v a s o t o c i n e x h i b i t s a remarkable s t a b i l i t y throughout the phylogenetic t r e e of v e r t e b r a t e s . There i s evidence f o r i t s e x i s t a n c e i n a l l v e r t e b r a t e s except f o r the Mammals ( V l i e g e n t h a r t and Versteeg, 1967; Sawyer, 1967). V l i e g e n t h a r t and Versteeg (1967) have reviewed the e v o l u t i o n a r y aspects of the neurohypophysial peptides and they have po i n t e d out tha t the Cyclostomes possess o n l y one peptide, a r g i n i n e v a s o t o c i n , but a l l other v e r t e b r a t e c l a s s e s s y n t h e t i s e two a c t i v e p e p t i d e s . They suggest t h a t the two a c t i v e peptides evolved by a doubling of the v a s o t o c i n gene. Vasoto c i n remained unchanged, but f u r t h e r mutations took p l a c e 92 i n the n e u t r a l peptide, and produced a s e r i e s of o x y t o c i n analogs. A p o i n t mutation i n the v a s o t o c i n gene produced a r g i n i n e v a s o p r e s s i n , the a n t i d i u r e t i c hormone of most mammals. In the Suina, a f u r t h e r mutation replaced a r g i n i n e w i t h l y s i n e to produce l y s i n e v a s o p r e s s i n . The probable presence of a r g i n i n e v a s o t o c i n i n mammalian foetuses, as found here, does not f i t the p i c t u r e of neurohypophysial e v o l u t i o n which i s accepted at present. The presence of three a c t i v e agents i n the foetus, c o n t r a d i c t s the theory t h a t a p o i n t mutation i n the v a s o t o c i n gene produced a r g i n i n e v a s o p r e s s i n . Such a p o i n t mutation would suggest the complete disappearance of the parent gene. A p a r a l l e l presence of v a s o p r e s s i n and v a s o t o c i n would c a l l f o r a f u r t h e r doubling of the v a s o t o c i n gene, and i f such doubling of the gene indeed occurred, some neurons of the mammalian neurohypophysis might r e t a i n the c a p a c i t y to produce a r g i n i n e v a s o t o c i n i n l a t e r l i f e a l s o . The decrease i n the t o t a l amount of a r g i n i n e v a s o t o c i n and i n the r a t i o of a r g i n i n e 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 , toward the end of i n t r a u t e r i n e l i f e , as was found i n the sheep and discussed e a r l i e r , might be the r e s u l t of the f o l l o w i n g p o s s i b i l i t i e s . F i r s t l y , i n e a r l y embryonic l i f e , there may be no c a l l f o r the r e l e a s e of e i t h e r a n t i d i u r e t i c p r i n c i p l e , and the product o f the more p r i m i t i v e gene a r g i n i n e v a s o t o c i n dominates. With the advancement of g e s t a t i o n , p o s s i b l y the gene producing the a n t i d i u r e t i c agent of the. a d u l t mammalian neurohypophysis could become dominant. Secondly, the f o e t a l 9 3 membranes could be more s e n s i t i v e t o a r g i n i n e v a s o t o c i n i n e a r l y g e s t a t i o n , and t h e r e f o r e p h y s i o l o g i c a l s t i m u l i might be i n favour of the p r o d u c t i o n of t h i s agent. In l a t e r embryonic l i f e , and a f t e r b i r t h , the mammalian t a r g e t organs may become s e n s i t i v e to vasopressin, and the p h y s i o l o g i c a l s t i m u l i could promote the production of a r g i n i n e v a s o p r e s s i n . The v a s o t o c i n producing neurons could consequently e i t h e r degenerate, or they could produce such s m a l l q u a n t i t i e s t h a t the presence of a r g i n i n e v a s o t o c i n might not be d e t e c t a b l e by conventional s t u d i e s . In p a s s i n g i t i s worthwhile t o p o i n t out t h a t the presence of three neurohypophysial peptides was a l s o demonstrated i n some of the Suina, where some species produce both a r g i n i n e and l y s i n e v asopressin, i n a d d i t i o n to o x y t o c i n ( H e l l e r , 1966). However, t h i s s i t u a t i o n i s not analogous to the simultanous presence of a r g i n i n e vasopressin and a r g i n i n e v a s o t o c i n i n the foetus, s i n c e the presence of the two vasopressins i n the p i g f a m i l y can be explained by c r o s s -breeding between species e l a b o r a t i n g one or the other of the vasopressins, and producing o f f s p r i n g s where both the a r g i n i n e and l y s i n e v a s o p ressin producing genes s u r v i v e . In the case of the foetus the presence of the two a n t i d i u r e t i c p r i n c i p l e s could not be accounted f o r i n t h i s manner, s i n c e there i s no known a d u l t mammal t h a t has a r g i n i n e v a s o t o c i n as the a c t i v e a n t i d i u r e t i c agent. Although o n l y suggestions can be made at the present as to the p o s s i b l e genetic i m p l i c a t i o n s o f the presence of a r g i n i n e v a s o t o c i n i n the p i t u i t a r y of a mammalian foetus, i t could become an important aid to the understanding of the evolution of the neurohypophysial peptides. 9 5 SUMMARY The p o s t e r i o r p i t u i t a r i e s o f pregnant sheep were c o l l e c t e d throughout two breeding seasons, and were e i t h e r d r i e d i n acetone, or l y o p h i l i s e d . E x t r a c t s of the p o s t e r i o r p i t u i t a r i e s were analyses f o r b i o l o g i c a l a c t i v i t i e s , p u r i f i e d , and the p u r i f i e d agents examined f o r t h e i r amino a c i d composition. The r e s u l t s of these s t u d i e s have shown: (1) A f a l l i n potency f o r the o x y t o c i c , vasopressor and a n t i d i u r e t i c a c t i v i t i e s occurred i n the neurohypophyses d u r i n g pregnancy. The l o s s of a c t i v i t y was found t o be more marked i n the e a r l i e r (90 days) stage of pregnancy, than i n the l a t e r (140 days) stage. At the time of n a t u r a l d e l i v e r y , a very marked increase i n the o x y t o c i c a c t i v i t y of the p o s t e r i o r p i t u i t a r y of the mother was detected. (2) The p a t t e r n of changes i n the b i o l o g i c a l a c t i v i t i e s throughout pregnancy were s i m i l a r i n the acetone d r i e d and l y o p h i l i s e d t i s s u e s , but acetone treatment r e s u l t e d i n a g r e a t e r l o s s of the b i o l o g i c a l a c t i v i t i e s from the neurohypophyses of pregnant sheep. (3) In the foetus, w i t h the advancement of g e s t a t i o n , an accumulation of a l l a c t i v e p r i n c i p l e s of the neuro-hypophyses was found. At around the time of b i r t h a secondary f a l l i n potency was detected. (4) The vasopressor and a n t i d i u r e t i c a c t i v i t i e s 9 6 greatly surpassed the oxytocic a c t i v i t y a l l through gestation as was shown by the large V/0 and A/0 r a t i o s . The r e l a t i v e l y greater accumulation of the oxytocic moiety, during the l a t t e r h a l f of embryonic development, resulted in the d e c l i n i n g V/0 and A/0 r a t i o s at the l a t e r stages of intrauterine l i f e , and early post natal age. (6 ) In the acetone treated glands, b i o l o g i c a l a c t i v i t i e s were found to be lower f o r oxytocic vasopressor and antidiuret a c t i v i t i e s , than i n the l y o p h i l i s e d glands. The loss of a c t i v i t y from the acetone treated tissue was of the same magnitude for both oxytocic and vasopressor a c t i v i t i e s , but varied from one stage of development to the other. (7) A new modification of the gel f i l t r a t i o n method was employed, to achieve a one step p u r i f i c a t i o n of the b i o l o g i c a l l y active p r i n c i p l e s from the crude neurohypophyseal extracts of the pregnant sheep, and sheep foetuses. (8) The amino acid analysis of the p u r i f i e d neurohypophyseal agents have indicated that these are oxytocin and arginine vasopressin. (9 ) Paper chromatographic studies of the neuro-hypophyseal extracts of early (90 days) foetuses again indicated oxytocin and arginine vasopressin as the active agents of these glands. (10) An unusually high frog waterbalance a c t i v i t y i n the p u r i f i e d neurohypophyseal extracts of sheep foetuses, indicated the presence of a t h i r d peptide i n these extracts. 97 I t i s suggested that t h i s t h i r d peptide i s arginine vasotocin, the a n t i d i u r e t i c p r i n i c p l e of lower vertebrates. i 98 LITERATURE CITED Acher, R., Chauvet, T. and O l i v r y , G., 1956. Sur 1'existence e v e n t u e l l e d'une hormone unique neurohypophysaire. R e l a t i o n entre l ' o x y t o c i n e l a vasopressine et l a p r o t e i n e de van Dyke e x t r a i t e s de l a neurohypophyse du boeut. Biochim. Biophys. Acta, 22: 421-427. 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