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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 U n i v e r s i t y , 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 a c c e p t t h i s t h e s i s as conforming t o the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1968  In p r e s e n t i n g an  this  thesis  in partial  advanced degree a t the U n i v e r s i t y  the  Library  I further for  shall  make i t f r e e l y  agree that  permission  f u l f i l m e n t of the requirements f o r of B r i t i s h  available  Columbia,  I agree  that  f o r r e f e r e n c e and S t u d y .  f o rextensive  copying of this  thesis  s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e Head o f my D e p a r t m e n t o r  by  h i s representatives.  of  this  written  thesis  It i s understood  forfinancial  gain  permission.  Department o f The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  Date  ^QHfAXH  Columbia  shall  that  copying or p u b l i c a t i o n  n o t be a l l o w e d w i t h o u t  my  ABSTRACT  The b i o l o g i c a l a c t i v i t i e s o f t h e neurohypophyses o f p r e g n a n t and embryonic  sheep, O v i s a r i e s were s t u d i e d .  The l e v e l s o f hormonal a c t i v i t i e s o f t h e neurohypophyses v a r i e d w i t h t h e s t a g e o f pregnancy, than l e v e l s i n the c o n t r o l  and were always  (non-pregnant)  glands.  lower Chemical  s t u d i e s o f t h e p u r i f i e d 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 o f t h e pregnant  sheep have i n d i c a t e d t h a t t h e s e 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 i n c r e a s e o f b i o l o g i c a l l y a c t i v e agents o f t h e p o s t e r i o r p i t u i t a r y was d e t e c t e d c o n c o m i t a n t w i t h development.  embryonic  The amount o f v a s o p r e s s o r a c t i v i t y g r e a t l y  s u r p a s s e d t h e o x y t o c i c a c t i v i t y i n t h e neurohypophyses o f f o e t a l sheep a l l t h r o u g h g e s t a t i o n , b u t the d i f f e r e n c e became s m a l l e r w i t h t h e advancement o f i n t r a u t e r i n e  life.  P h a r m a c o l o g i c a l and c h e m i c a l 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 t h e a c t i v e p e p t i d e s o f t h e f o e t a l neurohypophysis.  I n a d d i t i o n t o t h e s e two  neurohypophysial  p e p t i d e s c h a r a c t e r i s t i c o f t h e mammals, the p r e s e n c e o f a t h i r d p e p t i d e was  indicated.  Pharmacological studies  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 f o e t u s e s may be a r g i n i n e v a s o t o c i n , t h e a n t i d i u r e t i c p r i n c i p l e o f lower v e r t e b r a t e s .  ii  TABLE OP CONTENTS Page Abstract  i  L i s t o f Tables  v  L i s t o f P l a t e s and F i g u r e s  v i  A b b r e v i a t i o n s Used  ix  Acknowledgements  x i  Introduction The Hypothalamo-Hypophyseal A d u l t Mammal  1 System o f t h e 1  The Neurohypophysis d u r i n g Pregnancy and Lactation The Hypothalamo-Hypophysial  4  System o f t h e  Mammalian F o e t u s  5  Statement o f t h e Problem  7  M a t e r i a l s and Methods  '..  C o l l e c t i o n and S t o r a g e o f M a t e r i a l s  9 9  Extraction  10  Estimation of Biological A c t i v i t i e s a. I s o l a t e d r a t u t e r u s o x y t o c i c a s s a y b. R a t v a s o p r e s s o r a s s a y ; c. R a t a n t i d i u r e t i c a s s a y d. F r o g w a t e r b a l a n c e a s s a y Standards  10 11 13 15 16 18  Methods o f P u r i f i c a t i o n a. G e l f i l t r a t i o n b . Paper chromatography  18 18 20  C h e m i c a l Methods f o r t h e A n a l y s i s o f P e p t i d e s .  21  a. E s t i m a t i o n o f t o t a l p e p t i d e 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 o f t h e p e p t i d e s  22  Page c. Amino a c i d a n a l y s i s  .  7  S t a t i s t i c a l Methods Section  23 25  I  S t u d i e s o f t h e P r e g n a n t Sheep  27  1. B i o l o g i c a l a c t i v i t i e s o f t h e 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 o f t h e a c t i v e p r i n c i p l e s o f t h e p r e g n a n t sheep  32  a. P u r i f i c a t i o n b y 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  Discussion  (Section  I)  47  Section I I . S t u d i e s o f t h e B i o l o g i c a l A c t i v i t i e s and t h e C h e m i c a l N a t u r e o f t h e A c t i v e Agents i n t h e Neurohypophysis o f t h e F o e t a l and Newborn sheep ... 1. B i o l o g i c a l a c t i v i t i e s o f t h e f o e t a l neurohypophysis a. Acetone d r i e d t i s s u e b. L y o p h i l i s e d t i s s u e c. Comparison o f b i o l o g i c a l a c t i v i t i e s i n a c e t o n e d r i e d and l y o p h i l i s e d g l a n d 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 o f t h e a c t i v e agents i n t h e neurohypophyses o f sheep f o e t u s e s a. P a r t i a l p u r i f i c a t i o n o f f o e t a l n e u r o h y p o p h y s i a l p r i n c i p l e s b y paper chromatography b . G e l f i l t r a t i o n o f crude e x t r a c t s foetuses  from l a t e  c. Amino a c i d a n a l y s i s o f t h e p u r i f i e d peptides of the l a t e foetuses Discussion  (Section  II)  «,  General Discussion 1. Changes i n t h e hormonal l e v e l o f t h e n e u r o h y p o p h y s i s d u r i n g pregnancy  52 52 53 55 57  61 63 69 73 77 82 83  iii  iv Page 2. The p o s s i b l e r o l e o f 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  86  3. The importance o f the presence o f a r g i n i n e v a s o t o c i n i n the f o e t u s  89  Summary  95  Literature Cited  98  V  LIST OF TABLES Table I  Page P o t e n c i e s and t h e r a t i o s o f t h e p o t e n c i e s i n the acetone d r i e d  tissues  of pregnant  sheep II  30  P o t e n c i e s and t h e r a t i o s o f t h e p o t e n c i e s i n the l y o p h i l i s e d t i s s u e s  of pregnant  sheep III  31  P o t e n c i e s and t h e r a t i o s o f p o t e n c i e s i n t h e acetone d r i e d  tissues  o f sheep f o e t u s e s and  lambs  TV  54  P o t e n c i e s and t h e r a t i o s o f t h e p o t e n c i e s i n the l y o p h i l i s e d t i s s u e s and lambs  o f sheep f o e t u s e s 56  vi LIST OF PLATES AND  FIGURES  Facing  Plate 1 Figure 1  2  Page Diagram o f t h e p i t u i t a r y o f t h e sheep  x  Sample r e c o r d s from r a t u t e r u s , vasopressorand a n t i d i u r e t i c a s s a y s Weighted means o f o x y t o c i c , v a s o p r e s s o r 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 t h e acetone neurohypophyses  of control  dried  and p r e g n a n t  sheep 3  28  33  Weighted means o f o x y t o c i c , v a s o p r e s s o r 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 control  and p r e g n a n t  sheep 4  34  Comparison between t h e b i o l o g i c a l o f t h e acetone d r i e d hypophyses  5  and l y o p h i l i s e d  neuro-  and p r e g n a n t sheep  ....  36  R a t i o s o f b i o l o g i c a l a c t i v i t i e s i n acetone dried  and l y o p h i l i s e d neurohypophyses  control 6  of control  activities  of  and p r e g n a n t sheep  37  P u r i f i c a t i o n of the a c t i v e neurohypophysial agents o f p r e g n a n t sheep b y t h e use o f 100 cm Sephadex G-15  column  39  vii Facing Page  Figure 7  P u r i f i c a t i o n o f the b i o l o g i c a l l y a c t i v e principles  from t h e c r u d e e x t r a c t s o f  p r e g n a n t sheep neurohypophyses b y t h e use o f 200 cm Sephadex G-15 column 8  41  Sample p l a t e o f t h e t h i n l a y e r chromatography o f d a n s y l d e r i v a t i v e s o f s t a n d a r d amino a c i d s and t h e 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 sheep p o s t e r i o r pituitary extracts  .9  Graphed  43  values of dansylated  standard  amino a c i d s and t h e 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 o f t h e sheep n e u r o h y p o p h y s i s 10  45  Weighted means o f b i o l o g i c a l a c t i v i t i e s i n a c e t o n e d r i e d neurohypophyses o f sheep f o e t u s e s and lambs  11  58  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 a c e t o n e d r i e d neurohypophyses o f sheep f o e t u s e s and lambs  12  .  60  Weighted means o f b i o l o g i c a l a c t i v i t i e s i n t h e l y o p h i l i s e d neurohypophyses o f sheep f o e t u s e s and lambs  13  0  62  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 o f sheep f o e t u s e s and lambs  64  viii Facing Page  Figure 14  Comparison o f r a t i o s o f b i o l o g i c a l a c t i v i t i e s between  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 o f sheep f o e t u s e s and lambs 15  ..  66  Paper chromatogram  o f crude e x t r a c t s o f the  neurohypophyses o f sheep foetuses a t 89-91 days o f g e s t a t i o n a l age 16  68  P u r i f i c a t i o n o f crude e x t r a c t s o f sheep f o e t u s e s a t 138-141 days o f g e s t a t i o n a l age, on 200 cm Sephadex G-15  17  column  70  Sample p l a t e o f t h i n l a y e r chromatography o f the d a n s y l d e r i v a t i v e s o f standard  amino  a c i d s and o f the o x y t o c i c moiety o f the a d u l t pregnant sheep and the f o e t u s 18  Graphed  values of dansylated  amino a c i d s , and the d a n s y l a t e d o f the f o e t a l v a s o p r e s s o r moiety  72  standard hydrolysate 74  ix  ABBREVIATIONS USED  ala  -  alanine  leu  -  leucine  arg  -  arginine  lys  - • lysine  asp  -  aspartic acid  met  -  methionine  cyS  -  cystine  pro  -  proline  Cyt  -  cysteic acid  phe  -  phenylalanine  glu  -  glutamic  ser  -  serine  gly  -  glycine  tyr  -  tyrosine  his  -  histideine  thre  -  threonine  ileu  -  isoleucine  val  -  valine  X  PLATE I Diagram o f t h e p i t u i t a r y o f t h e sheep Ovis  aries.  Mid  s a g i t t a l plane.  After: 1957.  P.M.  D a n i e l and M a r j o r i e M.L.  Pickford,  Infundibular stem T bird ventricle  Neural (posterior) ! obe  Optic chiasma  Pituitary stalk  Pars  distaiis  Pars intermedia  xi  ACKNOWLEDGEMENTS  Among t h e many p e o p l e who a i d e d me d u r i n g my s t u d i e s and r e s e a r c h , I would l i k e t o 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 s u p e r v i s o r , who n o t o n l y suggested t h e r e s e a r c h t o p i c and a i d e d i n i t s p r o g r e s s w i t h h i s a d v i c e , b u t who a l s o c o l l e c t e d a l l t h e t i s s u e s used i n t h e c o u r s e o f this investigation. Dr. G.S. Dawse o f t h e 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 t h e m a t e r i a l a v a i l a b l e . M i s s L i i s M i r k , who c a r r i e d o u t t h e t h i n l a y e r chromatography; Mr. S t e v e Borden, who h e l p e d w i t h t h e statistical  a n a l y s i s o f t h e r e s u l t s ; Mr. E . J . M c C u l l o c h and  Mr. D. Anderson f o r t h e i r t e c h n i c a l a d v i c e . R e s e a r c h C o u n c i l G r a n t No. 680646 covered  a l l the  costs o f the research. I w i s h t o thank a l l t h e above p e o p l e f o r t h e i r c o n t r i b u t i o n t o the completion  o f t h i s work.  1  INTRODUCTION The Hypothalamo-Hypophyseal  System o f t h e A d u l t Mammal  S c h a r r e r (1944, 1954) and Bargman (1951) u s i n g Gomori's  chrom-alum h a e m a t o x y l i n p h l o x i n s t a i n ,  demonstrated  the presence o f neurosecretory granules i n t h e supraoptic and p a r a - v e n t r i c u l a r n u c l e i o f t h e hypothalamus.  These same  g r a n u l e s - w e r e d i s t r i b u t e d a l o n g t h e axons o f n e r v e f i b e r s , l e a d i n g from t h e n u c l e i t o t h e p a r s n e r v o s a , and w i t h i n t h e pars nervosa i t s e l f .  Further h i s t o l o g i c a l studies  s e c t i o n s o f t h e t r a c t from t h e hypothalamus  following  to the posterior  l o b e , showed a c c u m u l a t i o n o f s e c r e t o r y m a t e r i a l r o s t r a l t o t h e c u t , i n d i c a t i n g t h e d i r e c t i o n o f movement o f t h e neurosecretory material.  These w o r k e r s f o r m u l a t e d t h e  g e n e r a l l y a c c e p t e d hypothalamo-hypophyseal  concept o f t h e  p o s t e r i o r p i t u i t a r y , w h i c h p o i n t e d t o t h e hypothalamus as t h e p l a c e o f s y n t h e s i s , and t h e p a r s n e r v o s a as t h e s t o r a g e o r g a n f o r t h e neurohypophyseal hormones. The p h y s i o l o g i c a l a c t i o n s o f p o s t e r i o r l o b e e x t r a c t s were d i s c o v e r e d j u s t b e f o r e t h e t u r n o f t h e c e n t u r y .  Injections  o f t h e e x t r a c t s caused e l e v a t i o n o f b l o o d p r e s s u r e ( O l i v e r and S h a f e r , 1895), c o n t r a c t i o n s o f t h e u t e r u s (Dale, 1906) and e j e c t i o n o f m i l k from t h e mammary g l a n d (Ott and S c o t t ,  1910).  The a c t i v e agents were s e p a r a t e d i n 1928 by Kamm, A l d r i c h , G r o t e , Rowe and Bugbee, i d e n t i f i e d and s y n t h e t i s e d by  2  du V i g n e a u d , R e s s l e r , Swan, R o b e r t s , i n 1953.  The  octapeptides, molecular one  Katsoyannis  and  Gordon  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 o x y t o c i n and v a s o p r e s s i n ;  weight of approximately  a n o t h e r i n two  cyclic  t h e y shared a  1,000, b u t d i f f e r e d  from  amino a c i d s .  The p r i m a r y a c t i o n o f o x y t o c i n i s on•the mammary g l a n d 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 t h e e j e c t i o n o f m i l k , as a r e s p o n s e t o s u c k l i n g by t h e young. I t s a b i l i t y t o c o n t r a c t the u t e r u s and  i s a l s o w e l l demonstrated,  i t appears t o cause r y t h m i c c o n t r a c t i o n s d u r i n g  Various  s e c o n d a r y a c t i o n s can be e l i c i t e d by t h e a d m i n i s t r a t i o n  o f o x y t o c i n , and  among them are t h e f o l l o w i n g : d i l a t i o n  blood v e s s e l s i n the e x t r e m i t i e s , increased f i l t r a t i o n r a t e i n the k i d n e y ,  of  glomerular  and a p o s s i b l e c a p a c i t y t o  enhance e l e c t r o l y t e e x c r e t i o n by the k i d n e y s 1964).  labour.  (Pickford,  1960,  These secondary r e s p o n s e s were e l i c i t e d by pharmacolog-  i c a l dose l e v e l s o f t h e hormone; c o n s e q u e n t l y ,  t h e i r importance  t o the o r g a n i s m 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  t o be d i s c o v e r e d  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  vessels  Shafer,  ( O l i v e r and  vasopressin peptide  is s t i l l  1895); t h i s p r o p e r t y  commonly used t o d e t e c t and  i n e x t r a c t s of the p o s t e r i o r lobe.  was  blood  of assay t h e  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 o f v a s o p r e s s i n , however, l i e s i n i t s v e r y potent a n t i - d i u r e t i c effect.  I t promotes w a t e r  reabsorption  by a c t i n g on the d i s t a l t u b u l e s and c o l l e c t i n g d u c t s o f  the  3  kidney.  Besides  i t s a c t i o n on membranes i n t h e k i d n e y , i t  i s a t t r i b u t e d w i t h t h e c a p a c i t y t o a c t s i m i l a r l y on membranes of other organs.  Wakim (1966) has  summarized t h e a v a i l a b l e  e v i d e n c e f o r some o f 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 hormone: fall  the  a f t e r i n j e c t i o n s o f v a s o p r e s s i n t h e r e i s a marked  i n t h e e x c r e t i o n o f f l u i d from t h e p a n c r e a s , from the  l i v e r , and  from t h e sweat g l a n d s .  Water a b s o r p t i o n t h r o u g h  t h e i s o l a t e d f r o g s k i n i s a l s o i n c r e a s e d i n the p r e s e n c e o f t h i s hormone ( U s s i n g and Anderson, 1957).  The  accumulated  d a t a shows t h a t v a s o p r e s s i n a c t s on t h e membranes o f 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 The  waterconservation.  r a t i o of vasopressin/oxytocin  approximately  one  (V/0)  is  i n the p o s t e r i o r l o b e o f most mammals.  The hormones a r e s t o r e d a t t a c h e d t o , and p r o b a b l y accompanied by,  a l a r g e p r o t e i n m o l e c u l e (M.W.  named n e u r o p h y s i n e ( H e l l e r and G i n s b u r g ,  released  25000)  1966).  On  each  n e u r o p h y s i n e m o l e c u l e t h e r e 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 a n o t h e r 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 a c i d s o f the r e s p e c t i v e o c t a p e p t i d e 1964).  B i n d i n g o f t h e t e r m i n a l amino a c i d s does not  i n a c t i v a t e the hormones, the complex e x h i b i t s b o t h and o x y t o c i c a c t i v i t i e s The  unresolved  (van Dyke, _et a l . ,  vasopressor  1954).  e x i s t a n c e o f a common c a r r i e r f o r the  octapeptides,  two  r a i s e s one o f the most i n t e r e s t i n g , and  questions, concerning  o f the hormones. peptide  (Ginsburg,  The  still  t h e mechanism of r e l e a s e  r e l e a s e o f one  neurohypophyseal  i s u s u a l l y accompanied by the r e l e a s e o f the o t h e r ,  i n a c c o r d 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  u n d e r many c i r c u m s t a n c e s  one o f t h e hormones i s r e l e a s e d i n  much g r e a t e r q u a n t i t i e s than t h e o t h e r .  F o l l o w i n g haemorrhage  i n a n a e s t h e t i s e d r a t s ( H e l l e r , 1961), o r t h e s e v e r e dehydration  o f 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 o f a n t i d i u r e t i c hormone o c c u r s .  On t h e o t h e r hand,  l a c t a t i o n appears t o p r e f e r e n t i a l l y d e p l e t e t h e g l a n d o f i t s stored oxytocin.  The mechanism b y w h i c h t h i s  differential  r e l e a s e i s a c c o m p l i s h e d i s under e x t e n s i v e i n v e s t i g a t i o n (Ginsburg, the  1964; D i c k e r , 1966), b u t remains u n r e s o l v e d a t  present.  The Neurohypophysis d u r i n g Pregnancy and L a c t a t i o n D u r i n g pregnancy, an  a d d i t i o n a l burden i s p l a c e d on  t h e w a t e r 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 t o keep the uterus quiescent, t o prevent  expulsion o f the foetus.  C o n s i d e r i n g t h a t t h e main a c t i o n s o f t h e n e u r o h y p o p h y s i a l hormones a r e on w a t e r r e g u l a t i o n and on t h e u t e r u s , a change i n the l e v e l o f stored neurohypophysial be unexpected.  However, d i r e c t s t u d i e s on r a t neuro-  hypophyses b y Acher, Chauvet and O l i v r y and L e d e r i s  hormones would n o t  (1959),  (1956) and b y H e l l e r  f a i l e d t o show any changes i n e i t h e r t h e  mean hormonal c o n t e n t o f t h e g l a n d , o r i n t h e v a s o p r e s s o r / oxytocic  (V/0) r a t i o s a t any s t a g e o f pregnancy.  d i d n o t i n c l u d e animals Although  Their studies  s h o r t l y before, o r at p a r t u r i t i o n .  no changes i n t h e n e u r o h y p o p h y s i a l  hormone  l e v e l s were d e t e c t e d d u r i n g pregnancy i n t h e r a t , o t h e r e v i d e n c e ( 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 o f t h e r e p r o d u c t i v e c y c l e on t h e l e v e l o f t h e p o s t e r i o r p i t u i t a r y  5 hormones.  I n t h e r a t t h e amounts o f v a s o p r e s s i n and o x y t o c i n  were found t o r i s e d u r i n g t h e f o l l i c u l a r phase and t o drop at metestrous. to. w a t e r was  I n t h e same s p e c i e s , t h e d i u r e t i c  response  a l s o impaired immediately a f t e r c e s s a t i o n of  e s t r o u s ( G i n s b u r g , 1950 as quoted b y H e l l e r ,  1961).  L a c t a t i o n d e p l e t e s the p o s t e r i o r p i t u i t a r i e s o f dogs and r a t s  (van Dyke, Adamson and E n g e l , 1955; H e l l e r and  L e d e r i s , 1959) o f i t s o x y t o c i c and v a s o p r e s s o r a c t i v i t i e s . Some a u t h o r s have r e p o r t e d t h a t t h e V/0 r a t i o r i s e s due t o a p r e f e r e n t i a l depletion of oxytocin 1956; van Dyke e t a l . , unaltered,  (Acher, Chauvet,  Olivry,  1955) b u t o t h e r s have found i t  (Maculay, Landgrebe  and Waring  1950). H e l l e r  (1961) have p o i n t e d o u t t h a t t h o s e w o r k e r s who acetone d r i e d g l a n d s observed a r i s e i n V/0  utilized  r a t i o , but those  who worked on f r e s h g l a n d s found t h e r a t i o u n a l t e r e d .  He  has s u g g e s t e d t h a t the a p p a r e n t c o n t r a d i c t i o n i n t h e s e r e s u l t s can be e x p l a i n e d on t h e b a s i s o f a p r e f e r e n t i a l  solubility  o f o x y t o c i n i n acetone. The Hypothalamo-Hypophysial  System o f t h e Mammalian F o e t u s  H i s t o l o g i c a l s t u d i e s o f t h e embryonic development  of  t h e h y p o t h a l a m o - h y p o p h y s i a l system a r e f a r more numerous t h a n s t u d i e s o f i t s hormonal  content.  B e r n i s c h k e and McKay  (1953)  and M e i t n e r (1961), w o r k i n g i n t h e human, found the f i r s t neurosecretory 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 o f g e s t a t i o n . o f development,  O n l y a f t e r a f u r t h e r t h r e e weeks  c o u l d s e c r e t o r y m a t e r i a l be d e t e c t e d i n t h e  6  p o s t e r i o r lobe o f the p i t u i t a r y .  These o b s e r v a t i o n s a r e  c o n f i r m e d b y s t u d i e s i n t h e cow ( K i v a l o and T a l a n t i , mouse, r a t and dog embryos (Yakovleva, 1966).  1957),  In a l l the  mammalian f o e t u s e s s o f a r s t u d i e d , some o f t h e w o r k e r s a l s o noted an e a r l i e r appearance and g r e a t e r abundance o f s e c r e t o r y m a t e r i a l i n t h e s u p r a - o p t i c than i n t h e p a r a v e n t r i c u l a r nucleus  ( Y a k o v l e v a , 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 o f t h e h y p o t h a l a m i c n u c l e i than t h e p o s t e r i o r lobe o f t h e p i t u i t a r y ,  and t o an  e a r l i e r appearance o f t h e s u p r a - o p t i c than t h e p a r a v e n t r i c u l a r nucleus. There i s l i t t l e e v i d e n c e a v a i l a b l e c o n c e r n i n g t h e biological  a c t i v i t i e s present i n the f o e t a l  neurohypophysis.  O x y t o c i c and v a s o p r e s s o r a c t i v i t i e s have been d e t e c t e d by D i c k e r and T y l e r (1953) i n t h e p o s t e r i o r p i t u i t a r i e s o f dog, cat  and human f o e t u s e s . They found a r a t i o o f v a s o p r e s s o r  t o o x y t o c i c a c t i v i t y as h i g h as 28 t o 1 i n t h e human f o e t u s a t 112 days o f g e s t a t i o n .  A t 190 days o f i n t r a u t e r i n e  t h e r a t i o f a l l s t o 5 and reaches u n i t y a t b i r t h .  life  In the c a t  where t h e V/0 r a t i o i n t h e a d u l t i s 1.2, t h e r a t i o a t 54 days o f g e s t a t i o n a l age i s 4.7, and a t 21 days o f p o s t partum i s s t i l l as h i g h as 3.3.  These r e s u l t s i l l u s t r a t e t h e p r e -  dominance o f v a s o p r e s s o r a c t i v i t y o v e r t h e o x y t o c i c a c t i v i t y during intrauterine l i f e .  The v a s o p r e s s o r a c t i v i t y remains  h i g h e r u n t i l b i r t h i n human and even l o n g a f t e r b i r t h cat.  I t i s suggested  i n the  that the r e l a t i v e maturity of the  animal a t b i r t h determines  how near t o u n i t y t h e r a t i o between  7  t h e s e two a c t i v e p r i n c i p l e s i s a t t h i s Although  time.  i n t h e human t h e s t u d i e s o f t h e neruo-  h y p o p h y s i s d u r i n g embryonic development, performed b y D i c k e r and T y l e r , a r e 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 c o n s i d e r a b l e time a f t e r d e a t h .  I n view o f  the very r a p i d d e p l e t i o n of t h e p o s t e r i o r p i t u i t a r y o f i t s a c t i v e agents f o l l o w i n g death,  a g e n e r a l breakdown o f t i s s u e s  p o s t mortem c o u l d have c o n t r i b u t e d t o t h e low p o t e n c i e s and h i g h r a t i o s reported by these authors  i n t h e human f o e t u s e s .  F u r t h e r doubt i s c a s t on t h e s e r e s u l t s b y H e l l e r (1961), who again suggests,  t h a t these high r a t i o s , a t l e a s t i n p a r t ,  c o u l d be a t t r i b u t e d t o t h e e f f e c t o f 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 o f v a s o p r e s s o r  to oxytocic  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 r e p o r t e d t o be v e r y h i g h .  B u t H e l l e r and L e d e r i s c o u l d  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 h i g h r a t i o s were o b t a i n e d o n l y when glands were t r e a t e d w i t h acetone b e f o r e 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 a t t e m p t s have been made p r e v i o u s l y , t o i d e n t i f y t h e a c t i v e agents i n t h e f o e t a l n e u r o h y p o p h y s i s , b y e i t h e r pharmacological  o r c h e m i c a l means.  The o n l y i n d i c a t i o n t o  the p o s s i b l e nature o f these a c t i v e peptides are the studies o f H e l l e r (1959), performed i n immature r a t s .  By p a p e r -  chroma t o g r a p h i c s t u d i e s he i d e n t i f i e d t h e a c t i v e agents i n t h e immature r a t n e u r o h y p o p h y s i s as o x y t o c i n and v a s o p r e s s i n . Statement o f t h e Problem P r e v i o u s work on t h e pharmacology o f t h e neuro-  h y p o p h y s i s d u r i n g pregnancy, i s l i m i t e d t o one the r a t .  The  species only,  neurohypophyses o f v e r y few s p e c i e s 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. o n l y the r e s u l t s obtained  In t h e f o e t u s ,  i n t h e human are a t a l l e x t e n s i v e ,  b u t t h e y a r e o f u n c e r t a i n v a l u e , s i n c e the t i s s u e s may have been o b t a i n e d a c o n s i d e r a b l e t i m e a f t e r d e a t h .  well  Only  t h e s t u d i e s i n the c a t a r e c l e a r l y r e l i a b l e , and t h e s e l i m i t e d t o one  g e s t a t i o n a l age  only.  In t h e work p e r f o r m e d h e r e ,  i n v e s t i g a t i o n s were  extended t o a f u r t h e r s p e c i e s , the sheep, O v i s a r i e s . t i s s u e s were d i s s e c t e d i m m e d i a t e l y s t a g e s o f pregnancy.  Healthy  are  All  a f t e r d e a t h and a t known  f o e t u s e s 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 t h o s e o f t h e i r mothers. doubt c a s t on r e s u l t s o b t a i n e d  Due  t o the  considerable  i n acetone d r i e d g l a n d s ,  p a r a l l e l s t u d i e s were c a r r i e d o u t on acetone d r i e d and lyophilised tissues. S i n c e t h e r e i s no p r e v i o u s d a t a on t h e  chemical  n a t u r e o f t h e a c t i v e agents i n t h e f o e t u s , 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 t h e i d e n t i f i c a t i o n o f these  agents.  9  MATERIALS AND METHODS  C o l l e c t i o n and S t o r a g e o f 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 O v i s a r i e s o f t h e C l u n r o s e b r e e d were c o l l e c t e d i n t h e 1964 and 1965 b r e e d i n g seasons.  The samples were t a k e n from 3 year o l d pregnant  females and from t h e i r f o e t u s e s , a t known g e s t a t i o n a l t o g e t h e r w i t h c o n t r o l s from n o n - b r e e d i n g  ages,  3 year o l d females.  The a n i m a l s were a n a e s t h e t i s e d b y i n t r a v e n o u s i n j e c t i o n o f 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 r e p o r t e d t o be w i t h o u t any e f f e c t on t h e hormone c o n t e n t o f t h e p o s t e r i o r p i t u i t a r y ( G i n s b u r g and Brown,  1956). The f o e t u s e s were d e l i v e r e d b y C a e s a r i a n s e c t i o n and  t h e p l a c e n t a t i e d o f f and c u t . The p o s t e r i o r l o b e o f t h e p i t u i t a r y was removed from t h e p r e g n a n t  female and t h e f o e t u s  i n t h e 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 , t h e cranium c u t w i t h a Desouter Saw, t o expose t h e e n t i r e b r a i n . The f r o n t a l end o f t h e b r a i n was l i f t e d up, and p r o c e e d i n g c a u d a l l y t h e o p t i c n e r v e s , t h e i n f u n d i b u l a r stem, t h e c r a n i a l n e r v e s , and a l l o t h e r c o n n e c t i o n s t o t h e c r a n i a l f l o o r were s e v e r e d , e x p o s i n g t h e p i t u i t a r y .  Then t h e e n t i r e  p i t u i t a r y was removed from t h e s e l l a t u r c i c a , and s e p a r a t e d i n t o t h e a n t e r i o r and p o s t e r i o r l o b e s .  The p o s t e r i o r l o b e s  10 were d r i e d e i t h e r i n acetone  (1964 season) o r l y o p h i l i s e d  (1965 s e a s o n ) . The acetone d r i e d t i s s u e s were d i s s e c t e d a c e t o n e w h i c h had been d r i e d o v e r C a C ^ .  into  The acetone was  decanted and exchanged a t l e a s t t h r e e times d u r i n g t h e f i r s t 24 h o u r s .  The g l a n d s were t h e n s u b j e c t e d t o p r o l o n g e d  acetone e x t r a c t i o n b y b e i n g s t o r e d a c e t o n e a t room t e m p e r a t u r e .  f o r 12-14 months i n d r y  A f t e r removal from t h e f l u i d ,  t h e g l a n d s were exposed t o a i r t o remove r e s i d u a l and t h e n s e a l e d  i n t o g l a s s tubes and s t o r e d  acetone,  a t 4°C u n t i l t h e  time o f e x t r a c t i o n . Lyophilised  t i s s u e s were d i s s e c t e d  submerged i n a d r y - i c e methanol m i x t u r e .  into glass The f r o z e n  tubes samples  were p l a c e d on a vacuum pump, evacuated a t 0.1 mm Hg p r e s s u r e for  24 h o u r s , and s t o r e d  t h e end o f t h e season. o f t h e season and s t o r e d  a t room t e m p e r a t u r e o v e r P2O5 u n t i l The samples were s e a l e d a t 4°C u n t i l  a t t h e end  extraction.  Extraction S i n g l e g l a n d s o f a d u l t s o r f o e t u s e s were homogenized i n 0.25% a c e t i c a c i d a t 2 mg/ml w i t h  a Thomas E x t r a c t o r .  The homogenate was h e a t e d f o r 3 minutes  i n a b o i l i n g water-  b a t h , c o o l e d , 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 i m m e d i a t e l y assayed and t h e r e m a i n i n g s o l u t i o n stored  i n t h e r e f r i g e r a t o r a t 4°C.  Estimation  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 o f crude e x t r a c t s , and  11 p u r i f i e d p r o d u c t s , were e s t i m a t e d by means o f f o u r a s s a y 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 activity.  b.  Rat V a s o p r e s s o r  assay  c.  Rat a n t i d i u r e t i c  d.  Frog waterbalance  assay assay.  In a l l t y p e s o f b i o a s s a y H o l t o n ' s method was  used f o r more a c c u r a t e d e t e r m i n a t i o n o f  (and s t a t i s t i c a l a.  (1948) "4 p o i n t " potency  estimation of p o s s i b l e e r r o r s ) .  I s o l a t e d r a t uterus o x y t o c i c assay Method, as d e s c r i b e d by H o l t o n  b y Munsick, 1960:  (1948) and m o d i f i e d  V i r g i n a l b i n o females o f the W i s t a r  strain  were s e l e c t e d i n f u l l o r p r o - o e s t r o u s , by m i c r o s c o p i c examination  o f 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 b l o w on the head and k i l l e d by d e c a p i t a t i o n . u t e r u s , exposed by a m i d v e n t r a l and two s a g i t t a l was  The  incisions,  c a r e f u l l y d i s s e c t e d , w i t h t h e o v a r i e s i n t a c t and p a r t  o f the v a g i n a a t t a c h e d .  The d i s s e c t e d u t e r u s was  trans-  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 solution  ( c o m p o s i t i o n f o l l o w s ) , a l l r e m a i n i n g f a t and  c o n n e c t i v e t i s s u e removed, and t h e u t e r u s d i v i d e d i n t o i t s two h o r n s .  The v a g i n a l end o f one h o r n was  tied with s i l k  t h r e a d t o a g l a s s hook, p u l l e d from an a i r s u p p l y tube. l o n g e r p i e c e o f t h r e a d was  sewn through t h e o v a r y .  g l a s s hook w i t h t h e u t e r u s was  t h e n immersed i n a 5 ml  muscle b a t h c o n t a i n i n g van D y k e - H a s t i n g s s o l u t i o n , (  The  and  A  c o n n e c t e d t o a s u p p l y o f 5% C 0 - 9 5 % 0 . 2  2  The f r e e end o f  t h e t h r e a d sewn t o t h e o v a r y was s e c u r e d t o 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  against the  smoked drum o f a Palmer Kymograph. The muscle b a t h was connected t o a r e s e r v o i r  system  f o r v a n D y k e - H a s t i n g s s o l u t i o n , and t h e whole complex was k e p t a t c o n s t a n t t e m p e r a t u r e (32°C) i n a w a t e r - b a t h b y a Bromwell Thermomix pump. A t 5 minute i n t e r v a l s , doses o f s t a n d a r d ( S y n t o c i n o n , 10 IU/ml, Sandoz) a l t e r n a t e d w i t h doses o f unknown e x t r a c t were p i p e t t e d  i n t o t h e muscle b a t h .  A f t e r the response  reached i t s maximum, t h e van Dyke-Hastings s o l u t i o n i n t h e muscle b a t h was exchanged w i t h f r e s h s o l u t i o n from t h e r e s e r v o i r , b y means o f a r u b b e r b u l b a t t a c h e d t o t h e reservoir. Two t y p e s o f van D y k e - H a s t i n g s s o l u t i o n were used t o s u p p o r t t h e u t e r u s , d i f f e r i n g from one a n o t h e r o n l y i n t h e p r e s e n c e o r absence o f M g  + +  (Munsick, 1960).  The van D y k e - H a s t i n g s s o l u t i o n was p r e p a r e d f r e s h d a i l y b y t h e use o f two s t o c k s o l u t i o n s . (1)  van-Dyke H a s t i n g s s o l u t i o n w i t h o u t Mg " " 4  1  Stock S o l u t i o n A NaCl NaHC0  132.466g 3  51.170g  KC1  9.082g  Phenol r e d  ^ 0.054g  Na  salt  Combined, made up t o 2 liters  indist H 0 2  13 Stock S o l u t i o n B Na HP0  22.714g  up t o 1 l i t e r w i t h d i s t  H 0  NaH P04  5.520g  up t o 1 l i t e r w i t h d i s t  H 0  Approximately  equal volumes o f NaB^PG^ and  2  4  2  2  Na HPO^ were mixed, and the pH a d j u s t e d t o 7.4 2  The  final  mixture  i n the r e s e r v o i r  contained:  200 ml o f s t o c k s o l u t i o n A 20 ml o f s t o c k s o l u t i o n B 2 ml o f 0.5 M C a C l  2  1 g dextrose Combined, and made up t o 2 l i t e r s with d i s t H 0 2 ++ van-Dyke-Hastings s o l u t i o n w i t h Mg P r e p a r a t i o n o f t h e van Dyke-Hastings s o l u t i o n w i t h Mg  ++  i s identical  t o t h e one d e s c r i b e d f o r van Dyke-  H a s t i n g s s o l u t i o n without M g , w i t h the e x c e p t i o n ++  t h a t t h e f i n a l mixture  i n the r e s e r v o i r contains  200 ml o f s t o c k A 20 ml o f stock B 2 ml o f 0.5 M CaCl 2 1 g dextrose And  an a d d i t i o n a l :  2 ml o f 0.5 M M g C l  2  Combined, made up t o 2 liters Rat v a s o p r e s s o r assay The p r e s s o r a c t i v i t y o f p o s t e r i o r  in dist  H0 2  lobe e x t r a c t s was  2  14 determined  as d e s c r i b e d b y van Dyke, Adamson and E n g e l ,  1955. W i s t a r r a t s were a n e s t h e t i s e d b y subcutaneous i n j e c t i o n s o f urethane Dibenzylene  (175 mg/lOOg body wt) and  (0.5 mg/lOOg).  Dibenzylene,  an a d r e n e r g i c  b l o c k i n g agent was used t o lower and s t a b i l i z e t h e b l o o d p r e s s u r e , 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 t h e p r e s s o r assay b y v a r i o u s a d r e n e r g i c p r e s s o r and d e p r e s s o r  agents.  The r a t s were r e a d y f o r s u r g e r y 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 anaesthetic. The s k i n on t h e t h r o a t was c u t , t h e j a g u l a r v e i n l o c a t e d and f r e e d from c o n n e c t i v e t i s s u e .  Two  ligatures  were p l a c e d around t h e v e i n , and t h e one d i s t a l t o t h e h e a r t tied. cannula  The v e i n was p u n c t u r e d w i t h a 22 guage n e e d l e , a (PE 10 I n t e r m e d i c )  two l i g a t u r e s .  i n s e r t e d , and secured w i t h t h e  The c a r o t i d a r t e r y and trachea, were c a n n u l a t e d  i n t h e same manner, u s i n g PE 50 I n t e r m e d i c t u b i n g f o r t h e a r t e r y and PE 250 f o r t h e t r a c h e a . To p r e v e n t f o r m a t i o n o f b l o o d c l o t s d u r i n g t h e a s s a y 0.5 mg H e p a r i n s o l u t i o n  ( i n 0.9% NaCl) was i n j e c t e d p e r 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 o f s t a n d a r d and unknown s o l u t i o n s were a d m i n i s t e r e d through t h e c a n n u l a i n t h e j a g u l a r v e i n .  The  b l o o d p r e s s u r e was r e c o r d e d from t h e c a r o t i d c a n n u l a b y means o f a Stratham  23AA t r a n s d u c e r , c o u p l e d t o a Beckman dynograph  recorder. The assay was c a r r i e d o u t b y i n j e c t i o n s o f two doses  of  standard  ( P i t r e e s i n 20 IU/ml, Parke D a v i e s Co.)  matched  f o r magnitude o f responses by two doses unknown s o l u t i o n , 10 minute i n t e r v a l s .  For e s t i m a t i o n of potency  at  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 The  assay  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  e s t i m a t e d i n a n e s t h e t i s e d r a t s by t h e method o f  Sawyer, (1961) . Male a l b i n o r a t s o f W i s t a r s t r a i n were l o c a t e d t o 5% of  t h e i r body w e i g h t w i t h 12% e t h a n o l , by means o f a stomach  tube.  A n e s t h e s i a o c c u r r e d between 25-40 m i n u t e s .  complete  To  ensure  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  H y d r o c h l o r i d e (2% s o l u t i o n , w i t h o u t e p i n e p h r i n e , A s t r a P h a r m a c e u t i c a l s ) was The  a p p l i e d l o c a l l y to the areas of  incision.  j u g u l a r v e i n and t r a c h e a were c a n n u l a t e d as  d e s c r i b e d f o r the p r e s s o r assay. i n the f o l l o w i n g manner:  The b l a d d e r was  cannulated  ^ inch midventral i n c i s i o n  was  made through t h e s k i n and abdominal muscle a p p r o x i m a t e l y h inch a n t e r i o r t o the p e n i s .  The b l a d d e r was  eased  through  t h e i n c i s i o n and c u t i n a r e l a t i v e l y a v a s c u l a r a r e a .  The  end  of  a PE 200 I n t e r m e d i c c a n n u l a , which had been f l a r e d by h e a t ,  was  i n s e r t e d through t h e 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 t o the u r e t h r a l e x i t .  To ensure no l o s s o f  u r i n e d u r i n g t h e assay, t h e p e n i s was  tied with  ligature.  The  another  i n c i s i o n i n t h e abdominal w a l l was  sutured.  The p r e p a r e d r a t was h y d r a t e d up t o 8% o f i t s body weight,  and the l o s t u r i n e r e p l a c e d throughout  the assay w i t h  16 a s o l u t i o n o f 1.5% e t h a n o l i n 0.5% N a C l , by way o f a stomach t u b e . The c a n n u l a from t h e b l a d d e r was Palmer drop c o u n t e r and r e c o r d e r .  connected t o a  Responses t o s t a n d a r d  ( P i t r e s s i n 20 IU/ml P a r k e D a v i e s Co.)  and unknown s o l u t i o n s  were r e c o r d e d on t h e smoked drum o f a Palmer Kymograph, as a d e c r e a s e i n t h e number o f drops p e r u n i t t i m e . I n j e c t i o n s were a d m i n i s t e r e d through t h e c a n n u l a l e a d i n g t o t h e j u g u l a r v e i n a t t i m e s when t h e u r i n e f l o w had r e t u r n e d t o t h e b a s e l i n e l e v e l .  F o r e s t i m a t i o n o f potency,  t h e 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 o f H o l t o n was d.  employed.  Frog waterbalance assay F r o g w a t e r b a l a n c e assay was  t o Sawyer 1960.  The a p p a r a t u s f o r the assay c o n s i s t e d o f an  open g l a s s tube, f l a r e d a t one end. g l a s s r o d was g l a s s tube was  c a r r i e d out a c c o r d i n g  Near the o t h e r end a  s e a l e d t o t h e tube t o form a h a n d l e .  This  suspended i n a l a r g e r o u t e r c o n t a i n e r i n t o  w h i c h a g l a s s tube was p u l l e d n e a r t h e base, t o be  connected  t o an a i r s u p p l y . To c a r r y out t h e assay, f r o g s o f the s p e c i e s Rana c a t e s b i a n a were p i t h e d , t h e b l a d d e r exposed and removed. The d i s s e c t e d b l a d d e r was d i v i d e d i n t o i t s two l o b e s , t h e cut  end o f a h a l f b l a d d e r was p u l l e d o v e r the f l a r e d end o f  t h e g l a s s tube, and s e c u r e d onto i t w i t h a s i l k t h r e a d . g l a s s tube was  f i l l e d w i t h 5 ml o f d i s t ^ 0  The  and suspended i n  25 ml f r o g R i n g e r s s o l u t i o n c o n t a i n e d i n the o u t e r b a t h (composition f o l l o w s ) . The b l a d d e r s were weighed e v e r y 15 minutes and were  c o n s i d e r e d adequate f o r a s s a y when t h e y showed a v e r y slow steady l o s s o f weight. Doses o f s t a n d a r d  ( S y n t o c i n o n 10 IU/ml, Sandoz)  o r unknown s o l u t i o n were a d m i n i s t e r e d i n t o t h e o u t e r b a t h . The r a t e o f w a t e r - l o s s from t h e tube through t h e b l a d d e r was measured b y w e i g h i n g t h e g l a s s tubes w i t h t h e a t t a c h e d b l a d d e r s e v e r y 15 minutes t h r o u g h o u t t h e a s s a y .  The w e i g h t  l o s s d u r i n g t h e p e r i o d o f 15-45 minutes f o l l o w i n g t h e a d d i t o n o f t h e s t a n d a r d o r t h e p i t u i t a r y e x t r a c t was c o n s i d e r e d t h e response.  A f t e r 45 minutes t h e f r o g R i n g e r ' s  s o l u t i o n i n t h e o u t e r b a t h was exchanged w i t h f r e s h  solution  and t h e b l a d d e r was a l l o w e d 45 minutes t o r e c o v e r i n w h i c h time t h e r a t e o f w a t e r - l o s s returned t o t h e b a s e l i n e l e v e l . The R i n g e r ' s s o l u t i o n used i n t h e f r o g  waterbalance  a s s a y was p r e p a r e d from two s t o c k s o l u t i o n s . Stock, s o l u t i o n A 94 .'0 g  NaCl KC1  3.7 g  CaC1.2H 0 2  5.3 g  Stock s o l u t i o n B 2.0 g NaHCO3 glucose phenol r e d  40.0  g  4.0 g 12.0  mg  50 ml o f s t o c k s o l u t i o n A and s t o c k s o l u t i o n B were mixed and made up t o 1 l i t e r i n d i s t i l l e d H 0 2  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 , S y n t o c i n o n (Sandoz) used as s t a n d a r d i n t h e r a t u t e r u s and f r o g w a t e r b a l a n c e a s s a y s , was Standard.  assayed a g a i n s t t h e U.S.  The p o t e n c y was  International  i n good agreement w i t h t h a t  s t a t e d by t h e company and no adjustment  of the results.was  necessary. The commercial  vasopressin preparation, P i t r e s s i n ,  used as s t a n d a r d i n the r a t v a s o p r e s s o r and  antidiuretic  a s s a y s i s a p a r t i a l l y p u r i f i e d m i x t u r e o f a r g i n i n e and vasopressin.  lysine  The two p e p t i d e s d i f f e r i n t h e i r r e s p e c t i v e  v a s o p r e s s o r and a n t i d i u r e t i c p o t e n c i e s , 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 t h e company as t o t h e r a t i o o f t h e  two  peptides present i n a p a r t i c u l a r preparation of P i t r e s s i n . P i t r e s s i n , assayed was  a g a i n s t t h e U.S.  International  Standard  found t o have a l o w e r a n t i d i u r e t i c than v a s o p r e s s o r  activity.  C o r r e c t i o n i n the r e s u l t s were made t o compensate  for t h i s discrepancy. Methods o f 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.  a.  Gel  filtration  b.  Paper chromatography  Gel F i l t r a t i o n P u r i f i c a t i o n s o f t h e a c t i v e p r i n c i p l e s was  b y p a s s i n g t h e p o s t e r i o r l o b e e x t r a c t s through a column o f Sephadex G 15  (Pharmacia,  Uppsala).  achieved (Dextran)  19 The p r o c e d u r e used was i.  as f o l l o w s :  P r e p a r a t i o n o f t h e Sephadex  A 40-120 u p a r t i c l e s i z e Sephadex G-15, r e g a i n v a l u e o f 1.5 gel, 0.2  was  used.  +0.2  g/g  and bed volume o f 3 ml/g  The d r y g e l was  M acetic acid.  The  w i t h water dry  added t o a l a r g e volume o f  s l u r r y t h u s formed was  mixed f o r  one  h o u r on a magnetic s t i r r e r , a l l o w e d t o s e t t l e f o r 30 minutes, and t h e a c e t i c a c i d d e c a n t e d and d i s c a r d e d a l o n g w i t h  the  fine non-settling fraction.  four  times.  The p r o c e s s was  repeated  A f t e r the f o u r t h washing, t h e r e m a i n i n g  t a k e n up i n enough 0.2  Sephadex  was  M a c e t i c a c i d t o form a t h i c k s l u r r y ,  and used t o b u i l d t h e column. ii.  P r e p a r a t i o n o f the column  A Kontes c h r o m a f l e x  (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  t h e h e i g h t and volume.  volume 450 ml)  The  column was  extender to double  f i x e d v e r t i c a l l y onto  a s t a n d , w i t h the h e l p o f a plumb l i n e , and acetic acid.  f i l l e d w i t h 0.2  A ^ i n c h t h i c k l a y e r o f g l a s s w o o l was  M  placed  a t the bottom, f o l l o w e d by a s i m i l a r l a y e r o f s m a l l g l a s s beeds.  A l a r g e f u n n e l was  f i t t e d t o the top o f the column by  means o f a r u b b e r s t o p p e r , and The  Sephadex G-15  f u n n e l , and was  s l u r r y was  M acetic acid.  added t o t h e a c e t i c a c i d i n t h e  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 m i x e r .  A d d i t i o n a l s l u r r y was The  f i l l e d w i t h 0.2  added i n t o the f u n n e l a t  intervals.  Sephadex g e l was'packed i n t o a s i n g l e column o v e r a p e r i o d  o f 18 h o u r s , and  i n t o a d o u b l e column o y e r a p e r i o d o f 36 h o u r s .  iii.  P u r i f i c a t i o n procedure  B e f o r e u s i n g the allowed to run  c o l u m n , 0.2  through i t f o r a period  completion of the g e l s u r f a c e was  allowed t o run The  down u n t i l  surface.  extraction)  were c a r e f u l l y a p p l i e d  p e r m i t t e d to run  with  acetic acid  containing  the  The collected  posterior  i n t o the  (0.2  M)  and  was  of 6 hours.  a c e t i c a c i d washing, the  reached the  and  M acetic acid  liquid the  At above  gel.  The  top  (see  of the  c o l u m n was  connected to a  the  meniscus  lobe extracts to the  the  column, filled  reservoir  same.  s o l u t i o n w h i c h had i n 2.8  ml  passed through the  f r a c t i o n s , by  the  use  of  column  an LKB  was  Ultrorac  Type 7000 f r a c t i o n c o l l e c t o r , b.  Paper The  chromatography  paper chromatography techniques used, were  d e s c r i b e d by  Heller  Butanol,  and  Pickering  a f t e r mixing,  and  e v e r y 15  of  The  solvent  hours.  i n t o two  layers.  The  chromatography tank.  the  The  immediately  s y s t e m t h e n was  a 12"  l i d of the  and  the  for a  allowed to  placed  x 12"  separate  into  x 24"  t a n k was  s y s t e m was  period  four  glass  sealed  into  equilibrated  at  hour.  S t a n d a r d s and 24  minutes  a q u e o u s l a y e r was  "Lubriseal",  20°C f o r one  f o r 15  (1966).  volume were  minutes t h e r e a f t e r  P e t r i d i s h e s i n the bottom of  place with  Perks  a c e t i c a c i d , w a t e r i n a 4:1:5  shaken i n a s e p a r a t o r y funnel  1%  (1961) and  as  unknown s o l u t i o n w e r e a p p l i e d  x 54  cm  s h e e t o f Whatman 3MM  use  of  1 ml  tuberculin  to  chromatography paper,  syringes.  Cold a i r from a  a with portable  h a i r d r y e r was  allowed  up t h e d r y i n g o f the The  t o p a s s t h r o u g h t h e o r i g i n , t o speed spots.  chromatogram was  then placed  c h r o m a t o g r a p h i c tank, and t h e system was  i n the e q u i l i b r a t e d allowed  e q u i l i b r a t e f o r a f u r t h e r hour.  A t t h e end o f  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 o f the  m i x t u r e was  the solvent  added t o the s o l v e n t b a t h through openings i n  the glass l i d .  The  rubber stopper  o p e n i n g i n t h e l i d was  i t held previously.  t o run f o r 12 h o u r s . chromatogram was remaining  to  The  At the completion  closed with  system was  the  allowed  o f t h e run,  the  removed, the s o l v e n t f r o n t marked, and  the  s o l v e n t d r i v e n o f f by a c o o l f l o w o f a i r .  L o n g i t u d i n a l s t r i p s o f the d r y chromatograms were c u t a t t h e w i d t h o f the o r i g i n s p l u s an e x t r a 0.5  cm  a l l o w f o r d i f f u s i o n on each s i d e .  strips  The  lengthwise  to  were d i v i d e d i n t o t e n e q u a l p a r t s between the o r i g i n and s o l v e n t f r o n t , each r e s u l t i n g square c o r r e s p o n d i n g unit.  The  acid.  The  a t 4°C.  e l u t i o n was  The  pressure,  t o 0.1  squares were numbered, f o l d e d and e l u t e d  u a l l y i n 5 ml b e a k e r s , w h i c h c o n t a i n e d  e l u a t e was  and  the  1 ml o f 0.25%  Rf  individacetic  c a r r i e d out f o r a p e r i o d o f 6 h o u r s then recovered  s t o r e d a t 4°C  from t h e paper, by  u n t i l analysed  for biological  activities. C h e m i c a l Methods f o r the A n a l y s i s o f a.  Estimation of t o t a l peptide The  Peptides concentration  estimation of t o t a l peptide concentration  was  c a r r i e d out by t h e c o l o r i m e t r i c method o f Lowry, Rosebrough,  Farr  and  Randall i.  (1951) as  follows:  Reagents A - 2% N a C 0 2  B  - 0.5%  C  - alkaline  CuSO  50:1  i n 0.1  3  4  5H 0 2  i n 1% K  copper  mixture  N NaOH tartarate  solution  of reagent A +  B  D - 1 N F o l i n - C i o c a l t e a u Phenol ii.  Standards  A series 300  Albumin Stock iii.  into  of standards  ug were p r e p a r e d b y  0.2  dilution  from  Measurement o f p e p t i d e ml  o f t h e unknown and  r e a g e n t C was  added,  minutes.  ml  0.1  very rapidly  read  10,25,50,100,150,200  and  a 50 mg % B o v i n e  Serum  ( 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 ) .  a s e r i e s o f 2 ml  minutes  reagent  testubes. s h a k e n and  of F o l i n  and  mixed  a t a wavelength  To  each  allowed  to stand  C i o c a l t e a u r e a g e n t was After  a U n i c a m SP  600  millimicrons,  pipetted  sample 1 ml  f u l l y developed.  on  o f 750  the s t a n d a r d s were  immediately.  t h e c o l o u r was  against a blank  content  of  for  15-30  then  added  a p e r i o d o f 40-60  The  samples were  spectrophotometer  u s i n g a red  filter  photocell. b.  H y d r o l y s i s of the peptides Fractions with  obtained  from  identical biological  activities,  t h e Sephadex column, w e r e p o o l e d  The  r e s i d u e was  The  procedure  dissolved  was  repeated  get r i d of a l l a c e t i c the l a s t washing,  acid  in dist  H 0 2  at least remaining  and  and  lyophilised.  lyophilised  t h r e e times  i n order  i n the r e s i d u e .  the samples were t r a n s f e r r e d  again.  into  to  With a glass  23 bulb.  4 ml o f t r i p l e d i s t i l l e d H C l was added t o t h e d r y  r e s i d u e , and N minutes  2  gas b u b b l e d t h r o u g h t h e l i q u i d f o r 15  i n o r d e r t o remove t r a p p e d a i r . The samples were  t h e n f r o z e n 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 p r e s s u r e on a vacuum pump.  A t t h e end o f t h e 30  m i n u t e s , t h e g l a s s b u l b was s e a l e d w i t h a b l o w t o r c h , under vacuum, and t r a n s f e r r e d i n t o an oven a t 108°C ( F i s h e r Isotemp) f o r 22 h o u r s . A t t h e end o f t h e 22 h o u r s , t h e samples were removed from the-oven,  a l l o w e d t o 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,  followed 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 a t t a c h e d t o a  f l a s h e v a p o r a t o r , and a l l o w e d t o warm s l o w l y under vacuum and with continuous t u r n i n g o f the r o t o r .  When t h e samples were  a t a p p r o x i m a t e l y room t e m p e r a t u r e , t h e y were g r a d u a l l y lowered i n t o a 37°C w a t e r b a t h , 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 0 and evaporated a g a i n , t o 2  wash o u t t h e r e m a i n i n g H C l .  The washing was r e p e a t e d a t  l e a s t t h r e e times u n t i l no t r a c e s o f H C l remained, c.  Amino a c i d  analysis  The amino a c i d a n a l y s i s was performed b y t h i n  layer  c h r o m a t o g r a p h y o f t h e d a n s y l d e r i v a t i v e s o f t h e amino a c i d s , as d e s c r i b e d by L e v i s o n , 1967. i.  Preparation of the plates  One p a r t o f t h e 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) o f g l a s s d i s t i l l e d w a t e r , f o r a p p r o x i m a t e l y t h r e e minutes.  The s l u r r y was'spread i n 250 m i c r o n t h i c k n e s s onto  20" x 20" g l a s s p l a t e s w i t h t h e a i d o f a W a r n e r - C h i l c o t t spreader. The p l a t e s were a l l o w e d t o d r y a t room u n t i l t h e s i l i c a became opaque (10-20 m i n u t e s ) .  temperature, They were  t h e n warmed i n a stream o f h o t a i r f o r 10 minutes,  u n t i l the  s i l i c a became d r y , and p l a c e d f o r 30 minutes i n a d r y i n g oven at  110°C. ii.  Dansylation of the hydrolysates  F i v e m i c r o l i t e r s o f 0.2N NaHCX^, and f i v e m i c r o liters  o f s t a n d a r d d a n s y l c h l o r i d e * were added t o one m i l i -  micromole h y d r o l y s a t e . for  three hours.  The r e a c t i o n was a l l o w e d t o p r o c e e d  Amino a c i d s were d a n s y l a t e d i n t h e same  manner t o p r o v i d e s t a n d a r d s f o r chromatographic iii.  comparisons.  Chromatography o f t h e h y d r o l y s a t e s  One m i c r o l i t r e o f t h e h y d r o l y s a t e s , c o n t a i n i n g 1 muM o f p e p t i d e was a p p l i e d t o t h e p l a t e s , b e s i d e one m i l i m i c r o m o l e samples o f t h e s t a n d a r d amino a c i d s . The p l a t e s were p l a c e d c  i n t h i n l a y e r chromatographic  tanks which contained a s m a l l  amount o f t h e a p p r o p r i a t e s o l v e n t system, and t h e system was a l l o w e d t o e q u i l i b r a t e f o r 30 m i n u t e s .  A t t h e end o f t h e 30  minutes e q u i l i b r a t i o n p e r i o d , t h e pan a t t h e bottom o f t h e t a n k was f i l l e d w i t h t h e s o l v e n t t o b e i n c o n t a c t w i t h t h e edge o f t h e p l a t e .  The s o l v e n t was a l l o w e d t o r u n up t h e  p l a t e s u n t i l i t reached w i t h i n an i n c h from t h e t o p .  The  p l a t e s were then removed from t h e t a n k s , and i n a few minutes, when t h e y appeared d r y , t h e s p o t s f o r t h e s t a n d a r d s and t h e 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 sulphanylchloride  marked.  The R f v a l u e s were read, and d i r e c t comparisons w i t h  t h e s t a n d a r d s p e r m i t t e d i d e n t i f i c a t i o n o f t h e amino a c i d 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 s e p a r a t e v a r i o u s groups o f amino a c i d s . S o l v e n t system 1;  chloroform: benzyl alcohol: a c e t i c acid, 70:30:3  S o l v e n t system 2;  benzyl alcohol: pyridine: acetic acid, 16:4:1  S o l v e n t system 3;  b u t a n o l : a c e t i c a c i d : w a t e r , 1:5:4  S o l v e n t system 4;  c h l o r o f o r m : 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 p o t e n c i e s a r e c a l c u l a t e d b y t h e use o f w e i g h t e d means a c c o r d i n g t o K.A. Brownlee  (1965) u s i n g t h e  formula: k k. X = ^ w x / ^ w j j j j where w  i  i  2 =1/6' i  and c o n f i d e n c e l i m i t s a t t h e 5% p r o b a b i l i t y l e v e l were a s s i g n e d u s i n g t h e 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 o f v a s o p r e s s o r o r a n t i d i u r e t i c a c t i v i t y t o 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 b y t h e f o r m u l a  (Bliss,  1956):  26  M = ~2  ab 2 2  b-6" t b b  C o n f i d e n c e l i m i t s were a s s i g n e d level  a c c o r d i n g to the  X- = M + M -  a t the  formula:  (M)  -  a  2  Sa t a 2  2_ b  5  2  r-2 b f  c  2 b  5%  probabiltly  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 g l a n d s from p r e g n a n t sheep a t two s t a g e s o f pregnancy, and from c o n t r o l non-pregnant were c o l l e c t e d t h r o u g h o u t two b r e e d i n g seasons.  sheep  The b i o l o g i c  a c t i v i t i e s o f t h e crude e x t r a c t s were d e t e r m i n e d .  Crude  e x t r a c t s were p u r i f i e d , and t h e p u r i f i e d p r o d u c t was a n a l y s e d f o r amino a c i d c o m p o s i t i o n . The two s t a g e s o f pregnancy, r e p r e s e n t e d i n t h e samples c o l l e c t e d , were 89-91 days and 138-141 d a y s .  Since  the d u r a t i o n o f pregnancy i n t h e sheep i s 145-147 days, t h e s e s t a g e s c o r r e s p o n d t o about mid-pregnancy, days p r i o r , t o p a r t u r i t i o n , 1.  and t o j u s t a few  respectively.  B i o l o g i c a l a c t i v i t i e s o f t h e crude  extracts.  The b i o l o g i c a l a c t i v i t i e s o f t h r e e t o f i v e i n d i v i d u a l g l a n d s , a t 89-91 days, and 138-141 days o f g e s t a t i o n e s t i m a t e d from b o t h acetone d r i e d and l y o p h i l i s e d To complete each s e r i e s , two c o n t r o l s a n i m a l s were i n c l u d e d .  from  material.  non-pregnant  In the acetone d r i e d s e r i e s ,  g l a n d , the o n l y one a v a i l a b l e d u r i n g the p r o c e s s o f delivery,  i s also included.  diuretic activities. f o r acetone d r i e d ,  one natural  R e p r e s e n t a t i v e r e s u l t s from  t h e s e b i o - a s s a y s a r e shown i n P i g . 1. A l l glands e x h i b i t e d  were  oxytocic,  v a s o p r e s s o r and a n t i -  The r e s u l t s a r e p r e s e n t e d i n T a b l e I  and i n T a b l e I I f o r l y o p h i l i s e d t i s s u e .  28  Figure 1 Sample r e c o r d s from r a t u t e r u s , v a s o p r e s s o r and a n t i d i u r e t i c Rat u t e r u s A  =  assays.  assay 0.08 ml o f 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 o f pregnant  B  =  sheep.  0.08 ml o f 1:1000 s y n t h e t i c o x y t o c i n ( S y n t o c i n o n , 10 I.U./ml) .  C  =  0.12 ml o f 1:1000 s y n t h e t i c o x y t o c i n .  D  =  0.12 m l o f 1:250 o f 2 mg/ml e x t r a c t  Rat v a s o p r e s s o r A  =  assay  0.1 ml o f 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 o f pregnant  B  =  sheep.  0.06 ml o f 1:400 s t a n d a r d v a s o p r e s s i n ( P i t r e s s i n , 20 I.U./mg) .  C  =  0.12 ml o f 1:400 s t a n d a r d v a s o p r e s s i n .  D  =  0.2ml o f 1:60 p o s t e r i o r p i t u i t a r y  Rat a n t i d i u r e t i c  extract  assay  A  =  0.05 ml o f 1:40,000 s t a n d a r d v a s o p r e s s i n .  B  =  0.05 ml o f 1:2,000, 2 mg/ml p o s t e r i o r pituitary  extract.  C  =  0.1 ml o f 1:40,000 s t a n d a r d v a s o p r e s s i n  D  =  0.1 ml o f 1:2,000 p o s t e r i o r p i t u i t a r y  extract.  Rat Uterus  Assay  Weighted means were c a l c u l a t e d f o r c o r r e s p o n d i n g s t a g e s o f g e s t a t i o n b y t h e s t a t i s t i c a l method d e s c r i b e d on p. 2 5 - i n Methods. The mean o x y t o c i c , v a s o p r e s s o r 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 t h e acetone d r i e d g l a n d s a r e p r e s e n t e d i n F i g . 2.  A t 90 days o f pregnancy  t h e o x y t o c i c a c t i v i t y was  60% b e l o w t h a t i n t h e non-pregnant a d u l t sheep; t h i s  fall  i s f o l l o w e d b y a r e l a t i v e r i s e a t 140 days, b u t t h e p o t e n c y remained below t h e c o n t r o l l e v e l throughout  gestation.  The changes i n v a s o p r e s s o r 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 g l a n d t a k e n a t d e l i - v e r y r e v e a l e d a marked i n c r e a s e i n o x y t o c i c p o t e n c y , and i n terms o f o x y t o c i c a c t i v i t y , b y f a r t h e most a c t i v e p i t u i t a r y t i s s u e encountered course o f t h i s i n v e s t i g a t i o n .  i t was i n the  The apparent i n c r e a s e i n  o x y t o c i c a c t i v i t y was accompanied b y a much l e s s e r i n c r e a s e i n t h e v a s o p r e s s o r and a n t i d i u r e t i c  activities.  The e s t i m a t i o n o f t h e b i o l o g i c a l a c t i v i t i e s o f t h e l y o p h i l i s e d g l a n d s o f p r e g n a n t females showed changes i n hormonal c o n t e n t , s i m i l a r t o t h o s e found i n acetone  dried  tissues.  Again,  The mean p o t e n c i e s a r e shown i n F i g . 3.  a decrease i n the s t o r e d l e v e l o f 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 a t 90 days o f pregnancy, f o l l o w e d b y a r e l a t i v e r i s e i n a l l t h r e e a c t i v i t i e s a t 140 days o f g e s t a t i o n . In F i g . 4, t h e hormonal l e v e l s d u r i n g g e s t a t i o n a r e 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 g l a n d s .  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 Ratio  ADH/0 Ratio  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  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  Control  1954.4 +134.9  1220.0 +140.5  1480.6 +461.1  0.62 + 0.04  0.76 + 0.17  2  Control  1350.5 +205.6  1416.8 +430.0  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  Pressor mU/mg  ADH mU/mg  V/0 Ratio  A/0 Ratio  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  22  143  1689.6 +101.9  23  141  10 17  963.4 +208.8  0.73 + 0.31  0.76 + 0.79  1069.2 +344.1  1526.1 +643.0  0.63 +0.15  0.90 + 0.38  1662.2 +117.5  1041.5 + 88.0  915.9 +248.8  0.63 +0.06  0.54 +0.31  140  1520.1 +135.5  1388.6 +139.6  0.91 +0.11  137  1483.5 +180.5  750.1 + 49.6  0.50 +0.08  1  Control  1224.6 +231.0  1297.4 +272.4  1050.4 +205.1  1.05 0.86 + 0. 28 +0.35  2  Control  1568.9 +127.2  1464.1 +215.7  1267.6 +458.1  0.93 +0.25  0.81 + 0.29  3  Control  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 v a l u e o b t a i n e d i n t h e acetone d r i e d s e r i e s i s o m i t t e d h e r e , s i n c e t h e r e 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 t h e s i m i l a r i t y i n t h e  p a t t e r n o f 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 b r e e d i n g seasons, and s u b j e c t e d t o two d i f f e r e n t d r y i n g p r o c e d u r e s , gave e s s e n t i a l l y s i m i l a r  results.  The f a c t t h a t t h e o v e r a l l changes a r e v e r y s i m i l a r i n t h e two d i f f e r e n t groups o f sheep s u g g e s t s t h a t t h e a l t e r a t i o n s i n t h e l e v e l o f s t o r e d hormones a r e m e a n i n g f u l , and t h a t t h e y a r e n o t due t o random v a r i a b i l i t y .  I t appears p r o b a b l e t h a t  acetone t r e a t m e n t does n o t o b s c u r e t h e changes w h i c h o c c u r i n t h e n e u r o h y p o p h y s i s d u r i n g g e s t a t i o n , b u t o n l y m a g n i f i e s them. The r a t i o s o f v a s o p r e s s o r t o o x y t o c i c a c t i v i t y (V/0) and o f a n t i d i u r e t i c t o 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 g l a n d s , and c o n f i d e n c e l i m i t s a t P < 0 . 0 5 a s s i g n e d a c c o r d i n g t o t h e method o f B l i s s  (1956).  The r e s u l t s  a r e i n c l u d e d i n T a b l e I and T a b l e I I . R a t i o s o f t h e mean p o t e n c i e s were c a l c u l a t e d b y t h e same s t a t i s t i c a l method (see p.25 Methods) and t h e r e s u l t s f o r b o t h acetone d r i e d and l y o p h i l i s e d t i s s u e a r e g i v e n i n F i g . 5.  Comparison  t h e r a t i o s o f g l a n d s from c o n t r o l non-pregnant  between  sheep, and  t h o s e from p r e g n a n t a n i m a l s , f o l l o w i n g e i t h e r  lyophilisation  o r acetone t r e a t m e n t , r e v e a l s no s t a t i s t i c a l l y  significant  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 o f t h e a c t i v e p r i n c i p l e s o f p r e g n a n t sheep Samples o f t h e n e u r o h y p o p h y s i a l e x t r a c t s o f p r e g n a n t  33  Figure 2 Weighted means o f o x y t o c i c , antidiuretic activities  v a s o p r e s s o r and  i n the acetone d r i e d neuro-  hypophyses o f c o n t r o l and pregnant sheep.  mU/mg  =  milliunit biological activity/ mg d r y t i s s u e  PREGNANT  SHEEP  Oxytocic  ( A C E T O N E DRIED) Vasopressor  •  control  t  term  i  .  .  0  50  100  Days Gestation  147  •  f  control  I 0  Antidiuretic  term  . 50  100  Days Gestation  147  •  control  i 0 Days  term  •  ,  I  50  100  147  Gestation  34  Figure 3 Weighted means o f o x y t o c i c , v a s o p r e s s o r and antidiuretic  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 o f c o n t r o l 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 / m g d r y tissue  PREGNANT  SHEEP  Antidiuretic  Vasopressor  Oxytocic 2500  (LYOPHILISED)  r  2000  1500 6  o E 1000  500  term J  control 0  50 Days  100  Gestation  term  control j  50  147 Days  100  Gestation  1  147  0  50 Days  100  Gestation  147  sheep were p o o l e d f o r c h e m i c a l s t u d i e s .  Because o f t h e  i n s e n s i t i v i t y o f c h e m i c a l methods as compared t o p h a r m a c o l o g i c a l a n a l y s i s , i t was n e c e s s a r y t o mix t h e e x t r a c t s w i t h o u t r e g a r d t o t h e s t a g e o f pregnancy t a k e n , o r t o t h e method o f d r y i n g .  a t w h i c h t h e y were  The mixed samples were  r e g a r d e d m e r e l y as examples 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 from sheep i n a p r e g n a n t s t a t e , and t h e y l a t e r s e r v e d as c o n t r o l s f o r the s t u d i e s o f the f o e t a l neurohypophysial  agents  The p o o l e d 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 o f t h e g e l f i l t r a t i o n method, and t h e e l u a t e s from t h e column were a n a l y s e d f o r p r o t e i n c o n t e n t (Lowry p e p t i d e ) , and f o r biological activities  ( o x y t o c i c and v a s o p r e s s o r a s s a y ) .  D e t a i l s o f t h e s e e s t i m a t i o n s a r e g i v e n i n t h e 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 a n a l y s e d f o r  t h e i r c h e m i c a l s t r u c t u r e by t h e use o f t h i n  layer  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 o f n e u r o h y p o p h y s i a l e x t r a c t a t a c o n c e n t r a t i o n  of  2 mg/ml, and which c o n t a i n e d a t o t a l o f 27.0 + 0 . 9  ( I n t e r n a t i o n a l U n i t ) o f r a t u t e r u s and 24.0 + 3.0 v a s o p r e s s o r a c t i v i t y , was Sephadex G-15 acetic acid  a c e t i c a c i d was was  I.U.  of  a p p l i e d t o a 100 cm column o f  d e x t r a n g e l w h i c h was (see Methods).  I.U.  suspended i n 0.2  M  A r e s e r v o i r f i l l e d w i t h 0.2  connected t o t h e column, and the a c e t i c  a l l o w e d t o f l o w t h r o u g h a t a r a t e o f 16 m l / h r .  The  M acid first  100 ml o f e l u a t e r e p r e s e n t e d t h e v o i d volume o f the column and was d i s c a r d e d .  The n e x t 150 f r a c t i o n s o f 2.8 ml each were  t h e n 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  collector.  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 o f the acetone d r i e d and l y o p h i l i s e d neurohypophyses o f c o n t r o l and pregnant sheep.  mU/mg = m i l l i u n i t / m g  dry tissue  PREGNANT  SHEEP  o lyophilised ® acetone Oxytocic  dried  Vasopressor  Days  Gestation  Antidiuretic  Days  Gestation  37  Figure 5 Ratios of b i o l o g i c a l d r i e d and l y o p h i l i s e d  a c t i v i t i e s i n acetone  neurohypophyses o f c o n t r o l and  pregnant sheep.  R a t i o o f V/0  =  m i l l i u n i t of vasopressor a c t i v i t y per mg d r y 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 p e r mg d r y t i s s u e R a t i o o f A/0  =  m i l l i u n i t of a n t i d i u r e t i c mg d r y t i s s u e / m i l l i u n i t mg d r y t i s s u e  a c t i v i t y per  o x y t o c i c a c t i v i t y per  RATIO  1.5  1.0 o >  o o  +z a  rr  0.5  50  100 Days Gestation  OF  ACTIVITIES.  PREGNANT  SHEEP  38 The f r a c t i o n s were a n a l y s e d f o r p r o t e i n c o n t e n t , v a s o p r e s s o r and o x y t o c i c a c t i v i t y , and t h e r e s u l t s a r e g i v e n i n F i g . 6.  The mass o f p r o t e i n , as d e t e r m i n e d b y t h e  Lowry p e p t i d e d e t e r m i n a t i o n , came o f f t h e column i n t h e f i r s t 50 f r a c t i o n s and showed a remarkable active principles.  separation of the  The a n a l y s i s f o r b i o l o g i c a l  activites  r e v e a l e d t h a t t h e v a s o p r e s s o r 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 r e c o v e r y 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%; t h e apparent i n c r e a s e i n t h e t o t a l o x y t o c i c moiety i s undoubtedly to  the inherent error of the estimation.  an a r t i f a c t , due  The r e c o v e r y f o r  v a s o p r e s s o r a c t i v i t y was a p p r o x i m a t e l y 80%. The p a r t i a l r e s o l u t i o n o f t h e a c t i v e p r i n c i p l e s b y t h e G-15 column, suggested  t h e p o s s i b i l i t y t h a t an i n c r e a s e i n t h e  l e n g t h o f t h e column, might r e s u l t i n a complete of  t h e two b i o l o g i c a l l y a c t i v e a g e n t s .  was used t o b u i l d a 200 cm column.  resolution  T h e r e f o r e , an e x t e n d e r  15 ml o f e x t r a c t a t 2 mg/ml,  r e 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 v a s o p r e s s o r a c t i v i t y , was s u b j e c t e d t o t h e same procedure  as used p r e v i o u s l y f o r t h e s h o r t column.  The  f r a c t i o n s were a n a l y s e d as b e f o r e , and t h e r e s u l t s a r e p r e s e n t e d i n F i g . 7. two a c t i v e p r i n c i p l e s .  There was a complete  separation of the  L a r g e p r o t e i n s were e l u t e d f i r s t ,  with  o x y t o c i c and v a s o p r e s s o r 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 o r d e r . The  i n c r e a s e d l e n g t h o f t h e column d i d n o t a l t e r t h e r e c o v e r y  for  e i t h e r oxytocin or vasopressin; the recovery o f oxytocin  was a g a i n a p p r o x i m a t e l y 100% and t h a t o f v a s o p r e s s i n  39  Figure 6 P u r i f i c a t i o n o f the a c t i v e neurohypophysial agents o f pregnant sheep by the use o f 100 Sephadex G-15  column.  cm  Purification of  180  Pregnant  Sheep Extract  -,  on 100 cm. Sephadex G-15 column  150  1800  1500 < a  in  o n  oxytocic —•-  a> •o  S a  -i n Ul  in  vasopressor  O  100  1000  >> s  o  Lowry.  x  3 c \ 3  i\  I X  50  500 X >< —*•  o o  A.  20  40  Tube  60  Number  80  100  120  (I t u b e = 2.8 ml)  140  160  180  200  220  40 a p p r o x i m a t e l y 80%.  No i n c r e a s e i n c o n d u c t i v i t y , above  t h a t o f t h e s u p p o r t i n g 0.2 M a c e t i c a c i d , was d e t e c t e d i n any o f t h e 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 c o u l d be r e s o l v e d ; t h i s was p r o b a b l y b e c a u s e o f t h e low s a l t c o n t e n t o f t h e crude  extract.  The peaks o f b i o l o g i c a l a c t i v i t i e s showed evidence f o r t h e i r peptide content.  chemical  I n t h e o x y t o c i c peak  ( F i g . 7 ) , 5 ug/ml Lowry p e p t i d e c o n t e n t , corresponded t d 600 mU/ml o x y t o c i c a c t i v i t y , o r 120 I.U. o x y t o c i c a c t i v i t y / mg Lowry p e p t i d e .  T h i s v a l u e compares f a v o u r a b l y w i t h t h e  133 I.U./mg p e p t i d e o b t a i n e d f o r p u r e s y n t h e t i c o x y t o c i n b y P e r k s and Sawyer (1965) .  On t h e b a s i s o f t h i s measure-  ment, t h e o x y t o c i c peak i n F i g . 7 appears t o be almost, o r perhaps c o m p l e t e l y , p u r e . The Lowry p e p t i d e c o n t e n t o f t h e v a s o p r e s s o r peak i s even s m a l l e r than t h a t o f t h e o x y t o c i c peak. t h i s s u g g e s t s a p u r e o r almost p u r e p e p t i d e .  Again,  The o x y t o c i c  a c t i v i t y i n t h e v a s o p r e s s o r peak can be c o m p l e t e l y accounted  f o r b y t h e 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 o f  v a s o p r e s s i n (Adamson, E n g e l , van Dyke, b.  Amino a c i d  1954).  analysis  After various estimations of the d i f f e r e n t were completed,  those e l u a t e s which contained  fractions  identical  b i o l o g i c a l a c t i v i t i e s were p o o l e d , and t h e p o o l e d samples were h y d r o l y s e d i n 6 N HCI.  The h y d r o l y s a t e s were then  mixed w i t h d a n s y l c h l o r i d e , t o form d a n s y l d e r i v a t i v e s o f t h e i r c o n s t i t u e n t amino a c i d s .  The r e a c t e d 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 o f 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 e x t r a c t s o f pregnant  sheep  neurohypophyses,  G-15  column.  by the use o f 200 cm Sephadex  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 ) c h r o m a t o g r a p h i c p l a t e s , a l o n g s i d e s t a n d a r d d a n s y l a t e d amino a c i d s (see Methods). The p l a t e s were d e v e l o p e d i n f o u r s o l v e n t systems w h i c h were s e l e c t e d t o g i v e o p t i m a l s e p a r a t i o n s o f t h e 16 amino a c i d s used as s t a n d a r d s . (1)  The s o l v e n t systems were:  chloroform: benzyl a l c o h o l : a c e t i c acid, (70:30:3)  (2)  benzyl alcohol: pyridine: acetic acid, (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: butanol: a c e t i c acid, (6:3:1)  As y e t , no s o l v e n t system has been found t o s e p a r a t e mono-dansylated  l y s i n e from d a n s y l - a r g i n i n e .  After  chromatography was complete, t h e s p o t s 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 o f t h e known v a r i a b i l i t y o f t h e R d a n s y l d e r i v a t i v e s between d i f f e r e n t e x p e r i m e n t s  f  values of  (Perks,  p e r s o n a l communication), an e f f o r t was made t o employ d i r e c t comparison o f t h e c o n s t i t u e n t amino a c i d s o f t h e h y d r o l y s a t e s w i t h t h e s t a n d a r d s on t h e same p l a t e s .  However, t h e  v a l u e s o f t h e s t a n d a r d s 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 t o t h e ^ £ ~ °f t h e amino a c i d s o f t h e s  unknown ( F i g . 9 ) . I n t h e e l u a t e s w h i c h c o n t a i n e d t h e o x y t o c i c  43  Figure 8 Sample p l a t e o f the t h i n l a y e r chromatography o f d a n s y l d e r i v a t i v e s o f standard  amino a c i d s and  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  the  moieties  o f the a d u l t pregnant sheep, and o f the h y d r o l y s a t e o f the f o e t a l v a s o p r e s s o r  DNS  =  dansyl  moiety  solvent  front  i DNS Oxyt. Adult  hydrolysates Vasopr. Adult  Vasopr. Foetal  o o  o  T>WS  o  o  1>NS  Ueu  HNS  LfcU  O  o  o  0  o  0 0  o o  •pNS  o  o  T>NS  DNS - Q -  Q  Q  .  0  -  origin J  moiety, the eight 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  i d e n t i f i e d ; t h e s e were - t y r o s i n e ,  isoleucine,  were  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 acid.  A c i d h y d r o l y s i s would have caused t h e l o s s o f  ammonia from g l u t a m i n e and a s p a r a g i n e t o form t h e ( i d e n t i f i e d ) g l u t a m i c and a s p a r t i c a c i d s . acids l i s t e d , and  I n a d d i t i o n t o t h e e i g h t amino  the hydrolysates  contained l y s i n e ,  alanine,  a f u r t h e r amino a c i d w h i c h c o u l d have been e i t h e r  monodansylated l y s i n e o r a r g i n i n e . contaminant o f hydrolysates  Lysine  i s a known  of neurohypophysial peptides  (Perks and Sawyer, 1965), and a l a n i n e may be g e n e r a t e d by t h e breakdown o f c y s t e i n  (Perks, p e r s o n a l communication).  Due t o t h e s i m i l a r m o b i l i t y o f mono-dansylated l y s i n e and arginine,  i t cannot be d e t e r m i n e d which o f t h e two amino  a c i d s was p r e s e n t i n t h e h y d r o l y s a t e .  The p r e s e n c e o f mono-  d a n s y l a t e d l y s i n e , however, i s t h e 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 t o be p r e s e n t i n i t s d i d a n s y l a t e d form, and b o t h d a n s y l d e r i v a t i v e s a r e f r e q u e n t l y formed s i m u l t a n e o u s l y . A n a l y s i s o f t h e v a s o p r e s s o r peak showed t h e e i g h t constituent  amino a c i d s o f a r g i n i n e v a s o p r e s s i n ,  that i s to  say - t y r o s i n e , p h e n y l a l a n i n e , g l u t a m i c 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 o f c y s t e i n . A g a i n , l y s i n e and a l a n i n e were d e t e c t e d i n a d d i t i o n t o t h e above amino a c i d s . I t may be c o n c l u d e d t h a t t h e amino a c i d s known t o be p r e s e n t i n o x y t o c i n  and v a s o p r e s s i n  were i d e n t i f i e d i n  45  Figure  9 Graphed  acids, and  values  of dansylated  standard  amino  and t h e d a n s y l a t e d h y d r o l y s a t e s o f t h e o x y t o c i c  vasopressor  moieties  from t h e p u r i f i e d  the neurohypophyses o f a d u l t pregnant  d . NH  extracts of  sheep.  =  dansylated  ammonia  =  d i -dansyl derivative of lysine  =  mono- d a n s y l d e r i v a t i v e  2  di-  lys  € -Lys  of lysine  Amino a c i d s o f t h e unknowns a r e l a b e l l e d where  identification  of their R  values  only correspond  f closely  to the R  o f a standard  amino  acid.  1  • SOLVENT  1  SYSTEM  stondord  vasopressor  SOLVENT  oxytocic  SYSTEM  standard  vasopressor  2  oxytocic  S O L V E NT  vasopressor  standard  Vol • dl-Lys 0 Ala, Ptw 0 His 0 d. NH 0 Leu, lieu 0 Tyr 0 Pro, Glu 0 Gly.Ser • Thr.CyS  oxytocic  lltu d. NHj • L«u • Phe ° Vol  b  A  •  •  Pro  •  Pro  0  peptide  0  •  • Pro  0  • 0 • d. NHj  •  • Ala  • Ala  • Ala  • Gly • di-Lys • Tyr  •  • • •  • •  Phe  •  •  • Tyr  • His  •  Glu  _«-_..  • Glu • Thr • Ser e Asp • CyS • r.yt. Ara  • •  Glu  Leu Pro, Vol Phe  • Ala • di-Lys • Tyr Gly • His « Glu t> Set,Thr  0  •  2  0  0 Asp  0  0  0  0  Arg  0  Cyt  0 peptide  * •  Pro lieu  0  Leu  0  d.NHj  0  Tyr  0  0  0  0 C Lys 0 Arg 0  oxytocic  0  0 lieu 0 Leu  0  Stondord  • peptide  •  •  vasopressor  4  S Y S T E: M  S O L V E I*I T  3  SYSTEM  0  6 Lys  0  Tyr, Lys  •  Gly,Glu  0  Tyr, Lys  0 di-Lys 0 Gly 0  Glu  0  Asp  0 Gly.Glu  0 • Tyf  0 Cyt  0  Asp  0 Asp  0  0  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 activities.  The p r e s e n c e o f a d d i t i o n a l amino a c i d s i n  b o t h h y d r o l y s a t e s i s p r o b a b l y due t o t h e breakdown o f c o n s t i t u e n t amino a c i d s , t o g e t h e r w i t h c o n t a m i n a t i o n glassware.  The e x t e n t o f p u r i f i c a t i o n  o f t h e a d d i t i o n a l amino a c i d s p r e s e n t )  from  ( i . e . the proportion c o u l d n o t be  d e t e r m i n e d , s i n c e t h e t h i n l a y e r s e p a r a t i o n o f d a n s y l 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, t h e l i m i t e d amount o f m a t e r i a l a v a i l a b l e made i t i m p o s s i b l e t o use any o t h e r q u a n t i t a t i v e method o f amino acid analysis.  Nevertheless,  as f a r as i t c o u l d b e  d e t e r m i n e d , t h e p u r i f i c a t i o n p r o c e d u r e appeared t o be v e r y good f o r such a s i m p l e system, s i n c e contaminant amino a c i d s were few, and t h e i r d a n s y l 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 n o r overloaded  t h e chromatograms.  On t h e b a s i s o f t h i s  q u a l i t a t i v e system, t h e p u r i f i e d p r o d u c t  mainly  from t h e G-15  d e x t r a n columns appeared t o be a l m o s t p u r e .  47  DISCUSSION ( S e c t i o n I)  The  r e s u l t s r e p o r t e d h e r e have shown a r e d u c t i o n  i n t h e l e v e l o f 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 c o u r s e o f pregnancy. vasopressor Although  The  l e v e l of both oxytocic  the  and  a c t i v i t i e s were l o w e s t a t 90 days o f g e s t a t i o n .  the amount o f t h e s t o r e d hormones remained below  t h a t o f t h e c o n t r o l non-pregnant a n i m a l t h r o u g h o u t g e s t a t i o n , a t r e n d towards i n c r e a s e d s t o r a g e c o u l d be seen a t 140 The  days.  s i n g l e g l a n d t a k e n d u r i n g n a t u r a l l a b o u r appeared t o show  a r e m a r k a b l e i n c r e a s e 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 t h e c h a n g i n g hormonal c o n t e n t o f t h e p i t u i t a r y d u r i n g p r e g n a n c y was  o b s e r v e d i n two d i f f e r e n t  seasons.  t h e f a l l i n the p e p t i d e c o n t e n t o f  Therefore,  breeding the  n e u r o h y p o p h y s i s d u r i n g mid-pregnancy w o u l d appear t o be s i g n i f i c a n t since 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 w h i c h m i g h t o c c u r w i t h i n one season.  Further, since i t occurred  i n both l y o p h i l i s e d  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 t o be an caused b y the method o f d r y i n g . the r a t i o of vasopressor  No  breeding and  artifact  s i g n i f i c a n t changes i n  t o o x y t o c i c a c t i v i t y were d e t e c t e d  a t any s t a g e o f pregnancy.. The and  c l o s e l i n k between o x y t o c i n and  reproduction,  the importance o f a n t i d i u r e t i c hormone i n w a t e r metabolism,  w h i c h must change d u r i n g pregnancy, would suggest t h a t  48  changes i n t h e hormonal c o n t e n t o f the  neurohypophysis  d u r i n g t h e c o u r s e o f pregnancy might be a n t i c i p a t e d , t h e r e s u l t s p r e s e n t e d h e r e demonstrated do,  i n fact, occur.  and  t h a t such changes  Previous attempts, l i m i t e d to the r a t ,  f a i l e d t o f i n d any such changes i n e i t h e r t h e l e v e l o r t h e r a t i o o f t h e n e u r o h y p o p h y s i a l p e p t i d e s d u r i n g pregnancy ( H e l l e r and L e d e r i s , 1956; Acher e t a l . ,  1956). However,  v a r i a t i o n s have been seen t o o c c u r i n c o n n e c t i o n w i t h o t h e r a s p e c t s o f r e p r o d u c t i o n , such as i n t h e e s t r o u s c y c l e , o r in lactation.  Heller  (1958) d e t e c t e d p h a s i c changes i n t h e  amount o f s t o r e d p e p t i d e s i n t h e p o s t e r i o r p i t u i t a r y o f r a t s , and t h e s e changes c o r r e s p o n d e d w i t h p a r t i c u l a r s t a g e s i n the estrous c y c l e .  L e v e l s o f v a s o p r e s s o r and o x y t o c i c  a c t i v i t i e s were found t o be h i g h e r i n t h e f o l l i c u l a r phase than i n metestrous.  A l o s s o f t h e a c t i v e agents from t h e  p o s t e r i o r p i t u i t a r y o f dogs and r a t s d u r i n g l a c t a t i o n was observed  ( D i c k e r and T y l e r , 1953; H e l l e r and L e d e r i s ,  Acher e t _ a l . , 1956) . found b y a l l  A fall  also  1957;  i n t h e c o n t e n t o f b o t h hormones was  i n v e s t i g a t o r s , b u t t h e r e was  i n g t h e n a t u r e o f t h e changes i n t h e V/0  a disagreement ratios.  Heller  concern(1961),  i n r e v i e w i n g t h e s e r e s u l t s , noted t h a t changes i n r a t i o s were found b y w o r k e r s u s i n g acetone d r i e d g l a n d s , b u t n o t by those working w i t h fresh glands.  He a t t r i b u t e d t h e 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 o f 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 o f o x y t o c i n i n acetone from g l a n d s t a k e n from a n i m a l s 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 c o n c e r n i n g t h o s e  r e s u l t s o b t a i n e d from acetone d r i e d t i s s u e s .  The  studies  i n p r e g n a n t sheep p r e s e n t e d h e r e do n o t c o n f i r m 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 , since the r a t i o s of t h e hormones were s i m i l a r i n b o t h l y o p h i l i s e d and d r i e d t i s s u e s , even when acetone t r e a t m e n t was  acetone  prolonged.  However, e s t i m a t i o n o f t h e a b s o l u t e hormonal p o t e n c i e s , f o l l o w i n g acetone t r e a t m e n t , showed a l o s s o f b o t h o x y t o c i c and v a s o p r e s s o r a c t i v i t i e s from g l a n d s t a k e n d u r i n g pregnancy, b u t n o t from t h e c o n t r o l s . The e x p e r i m e n t s r e p o r t e d h e r e were d e s i g n e d p r i m a r i l y to  e v a l u a t e 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 t h e  c o u r s e o f pregnancy, b u t t h e g r e a t e r l o s s o f hormonal a c t i v i t y f o l l o w i n g acetone t r e a t m e n t i s an problem i n i t s e l f .  interesting  P o s s i b l y , t h e answer t o 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 o f t h e 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 o f n e u r o h y p o p h y s i a l  r e s p o n s e t o haemorrhage, n o t e d an i n i t i a l  increase i n the  amount o f v a s o p r e s s i n r e l e a s e d i n t o t h e b l o o d s t r e a m ,  until  a p p r o x i m a t e l y 10-20% o f t h e t o t a l a n t i d i u r e t i c hormone c o n t e n t of  t h e p o s t e r i o r p i t u i t a r y was d e p l e t e d .  haemorrhage c o u l d n o t e l i c i t  However, f u r t h e r  any a d d i t i o n a l r e l e a s e o f t h e  a n t i d i u r e t i c hormone from t h e n e u r o h y p o p h y s i s . t h a t the s t o r e d v a s o p r e s s i n i s heterogenous,  Sachs s u g g e s t s  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 a n o t h e r i n t h e i r b i n d i n g w i t h i n the gland.  /An i n c r e a s e i n t h e q u i c k l y r e l e a s a b l e , o r  l o o s e l y bound component o f t h e n e u r o h y p o p h y s i a l hormones,  50  d u r i n g pregnancy,  might a c c o u n t f o r t h e g r e a t e r l o s s o f  a c t i v i t y which o c c u r r e d d u r i n g acetone t r e a t m e n t o f t h e glands o f pregnant  animals.  In p r e v i o u s work, two b a s i c methods were u t i l i s e d for the p u r i f i c a t i o n of neurohypophysial peptides. used b y Acher  The f i r s t ,  (1955). and Chauvet (1960), made u s e o f t h e  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.  I n t h i s method, t h e complex formed between  t h e n e u r o h y p o p h y s i a l o c t a p e p t i d e s and n e u r o p h y s i n e was p r e c i p i t a t e d b y N a C l , and d i a l y s e d t o e l i m i n a t e c o n t a m i n a t i n g peptides.  The n e u r o p h y s i n e 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 t h e a c t i v e agents were l e f t i n solution.  The a c t i v e agents were s e p a r a t e d from one a n o t h e r  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 o f 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 b e c a u s e o f t h e danger o f c o n t a m i n a t i n g t h e p u r i f i e d w i t h b o v i n e n e u r o h y p o p h y s i a l p e p t i d e s which  products  remained  unremoved d u r i n g t h e o r i g i n a l p r e p a r a t i o n o f t h e b o v i n e neurophysine. The o t h e r 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 s i m p l e g e l f i l t r a t i o n o f crude e x t r a c t s , f o l l o w e d b y t h e use o f ion-exchange  columns f o r t h e r e s o l u t i o n o f t h e  two n e u r o h y p o p h y s i a l p e p t i d e s ( P o r a t h and F l o d i n , Yamashiro,  1959;  1964).  I n 1966, F r a n k l a n d and h i s co-workers  combined t h e two  methods, and s u b j e c t e d n e u r o p h y s i n e - p e p t i d e complexes, b y Acher's method, t o g e l f i l t r a t i o n on G-25 Sephadex  obtained  supported  i n 1 N a c e t i c a c i d and performed the chromato-  g r a p h i c s e p a r a t i o n w i t h 0.1 fluid.  M f o r m i c a c i d as the  They r e p o r t t h a t t h i s two  suspension  s t e p 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 p e p t i d e s from t h e carrier protein. The  p u r i f i c a t i o n p r o c e d u r e used i n t h e  i n v e s t i g a t i o n was accepted  t o s i m p l i f y and  methods o f g e l f i l t r a t i o n ,  neurohypophysial avoided,  designed  peptides.  present  improve c u r r e n t l y  as a p p l i e d t o  The use o f f o r m i c a c i d  s i n c e i t i s a p o t e n t r e d u c i n g agent.  was  I t is possible  that 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 changes i n t h e n e u r o h y p o p h y s i a l  p e p t i d e s , and,  chemical  further, i t  m i g h t have d e t r i m e n t a l e f f e c t s on t h e 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  used i n t h e p u r i f i c a t i o n p r o c e d u r e r e p o r t e d h e r e , a c i d , w h i c h has no r e d u c i n g p o t e n c y , and component o f m e t a b o l i s i n g systems.  was  i s a weak  i s a normal  The p r o c e d u r e a l s o  e l i m i n a t e d such s e v e r e s t e p s as t r e a t m e n t w i t h  trichloracetic  a c i d , and s t e p s w h i c h 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 with neurophysine. was  It  a s i m p l e and r e l a t i v e l y m i l d system, y e t 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 The  peptides  i n a s i n g l e step.  r e s u l t s o f t h e amino a c i d a n a l y s i s were i n good  agreement w i t h those o f 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 v a s o p r e s s i n as the a c t i v e p e p t i d e s o f sheep n e u r o h y p o p h y s i s o f unknown r e p r o d u c t i v e condition.  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 part 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 o f sheep embryos a t d i f f e r e n t g e s t a t i o n a l ages, and o f newborn lambs, were a s s a y e d f o r t h e i r b i o l o g i c a l activities.  Samples a t c o r r e s p o n d i n g s t a g e s o f i n t r a u t e r i n e  development were p o o l e d and s u b j e c t e d t o p u r i f i c a t i o n p r o c e d u r e s ; t h e neurohypophyses o f e a r l y f o e t u s e s were p u r i f i e d b y p a p e r chromatography, and o f t h e 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 o f g e s t a t i o n i s 145-147 days i n t h e sheep. F o e t u s e s a t 89-91 days o f g e s t a t i o n a l age weighed o n l y 500-600 grams, l a c k e d f u r , and showed t h e d i s p r o p o r t i o n a t e head measurement o f t h e e a r l y f o e t u s .  B y 140 days o f g e s t a t i o n a l  age, w h i c h i s o n l y a few days p r i o r t o p a r t u r i t i o n , t h e f o e t u s e s weighed 4-5 k g s , were c o v e r e d w i t h f u r , and g e n e r a l l y appeared t o be f u l l y d e v e l o p e d . 1.  B i o l o g i c a l a c t i v i t i e s o f the f o e t a l neurohypophysis S i n g l e g l a n d s o f f o e t u s e s a t 89-91 days, and a t 138-  141 days o f g e s t a t i o n a l age were c o l l e c t e d i m m e d i a t e l y a f t e r death.  The g l a n d s 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 , v a s o p r e s s o r and a n t i d i u r e t i c a c t i v i t i e s .  Similar  53  s t u d i e s were made on g l a n d s t a k e n from lambs a t b i r t h and a t 13-15 days o f p o s t - n a t a l age, i n o r d e r t o d e t e r m i n e whether t r e n d s seen d u r i n g f o e t a l development m i g h t c o n t i n u e into early post-natal 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 b r e e d i n g seasons.  The g l a n d s i n t h e f i r s t b r e e d i n g  season were acetone d r i e d and a l l o w e d t o s t a n d i n acetone up t o an a d d i t i o n a l 14 months.  Posterior p i t u i t a r i e s i n the  second b r e e d i n g season were i m m e d i a t e l y l y o p h i l i s e d and s t o r e d o v e r P2°5 f °  r a  Vasopressor,  s i m i l a r p e r i o d (see Methods). 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 d e t e c t e d i n a l l g l a n d s .  The r e s u l t s o f t h e b i o l o g i c a l  a s s a y s a r e p r e s e n t e d i n T a b l e I I I f o r acetone d r i e d , and i n T a b l e 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 o f t i s s u e s a t t h e same  developmental  s t a g e , and c o n f i d e n c e l i m i t s a s s i g n e d a t t h e 5% p r o b a b i l i t y l e v e l as d e s c r i b e d i n t h e Methods, a.  Acetone d r i e d  tissue  The w e i g h t e d means o f o x y t o c i c , v a s o p r e s s o r 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 s t a g e s a r e p l o t t e d i n F i g . 10.  developmental  The average o x y t o c i c a c t i v i t y  a t 90 days o f g e s t a t i o n was low, and amounted t o o n l y 10 mU/mg d r y powder.  S i n c e t h e average w e i g h t o f t h e glands a t t h e  e a r l y s t a g e o f g e s t a t i o n a l age i s o n l y 0.8 mg, t h e t o t a l o x y t o c i c a v t i v i t y i n t h e e a r l y f o e t a l sheep p i t u i t a r y d i d n o t exceed  8 mU/gland i n t h e acetone d r i e d t i s s u e .  In the  same glands t h e v a s o p r e s s o r and a n t i d i u r e t i c a c t i v i t i e s were n o t a b l y h i g h e r and averaged  120 mU/mg, o r a t o t a l o f 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 Ratio  A/0 Ratio  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  138  59.9 + 7.6  653.6 +116.4  591.1 +193.8  11.0 + 1.8  10.0 + 5.0  196.0 + 35.1  901.2 +199.9  921.0 +212.5  4.6 + 1.24  4.7 + 1.24  13  27c  Term  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.  A t 140 days, a 10 f o l d i n c r e a s e i n t h e o x y t o c i c and  an a p p r o x i m a t e l y 8 f o l d i n c r e a s e i n t h e v a s o p r e s s o r and a n t i d i u r e t i c p o t e n c i e s were d e t e c t e d .  A t t h e same time, t h e w e i g h t  o f t h e g l a n d s i n c r e a s e d from 0.8 mg t o 3.5 mg, making t h e t o t a l i n c r e a s e i n t h e amount o f s t o r e d p e p t i d e s 45 f o l d . r i s e i n p o t e n c y was d e t e c t e d a t p a r t u r i t i o n and v a s o p r e s s o r , b u t n o t t h e a n t i d i u r e t i c  A further  i n the o x y t o c i c  activities.  d i s c r e p a n c y between t h e v a s o p r e s s o r and a n t i d i u r e t i c i s probably not s i g n i f i c a n t ence l i m i t s .  This activities  i n view o f t h e o v e r l a p p i n g c o n f i d -  A f t e r b i r t h , a l l three b i o l o g i c a l  activities  appeared t o be s i g n i f i c a n t l y l o w e r than a t 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 ,  a t 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 v e r y s t e a d y change g e s t a t i o n , and a r e p r e s e n t e d i n P i g . 11. gestation the ratios  approximated  A t 90 days o f  14:1, which r e f l e c t e d t h e  p r e p o n d e r a n c e o f t h e v a s o p r e s s o r and a n t i d i u r e t i c at t h i s e a r l y stage o f g e s t a t i o n .  throughout  activities  The p r o p o r t i o n a l l y g r e a t e r  a c c u m u l a t i o n o f o x y t o c i c a c t i v i t y i n t h e neurohypophysis  with  t h e advancement o f 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 t h e s t e a d y f a l l o f t h e r a t i o s throughout g e s t a t i o n . A t 13-15 days o f p o s t - n a t a l age, t h e r a t i o o f v a s o p r e s s o r t o 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 t h e a d u l t r a t i o o f  a p p r o x i m a t e l y 1, and was found t o be 3:1 i n f a v o u r o f t h e vasopressor b.  moiety.  Lyophilised  tissue  The w e i g h t e d means f o r o x y t o c i c , v a s o p r e s s o r and antidiuretic  i  a c t i v i t i e s a t corresponding gestational  stages  56  TABLE I V 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 Ratio  ADH/O Ratio  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  23c  141  202.0 + 15.0  1472.6 +293.0  2405.8 +858.0  7.3 + 1.50  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  19.0 + 3.38  24.0 + 7.8 12.0 + 3. 27  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 o f g e s t a t i o n a l age was low, and amounted t o o n l y 60 mU/mg d r y powder.  The average w e i g h t o f a l y o p h i l i s e d  g l a n d a t t h i s s t a g e was a p p r o x i m a t e l y 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 o f g e s t a t i o n did  n o t exceed a t o t a l o f 40 mU/mg.  I n t h e same g l a n d s , t h e  v a s o p r e s s o r a c t i v i t y was a p p r o x i m a t e l y 900 mU/mg, and t h e 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 c o r r e s p o n d s t o a t o t a l o f 550 mU/gland v a s o p r e s s o r and 720 mU/gland antidiuretic activity. in did  A t 140 days o f g e s t a t i o n , an i n c r e a s e  a l l b i o l o g i c a l a c t i v i t i e s was d e t e c t e d , b u t t h e i n c r e a s e n o t appear t o be. as g r e a t as was d e t e c t e d i n t h e acetone  dried tissues.  A fall  i n the p o s t e r i o r p i t u i t a r y content o f  t h e a c t i v e agents was a g a i n demonstrated  a t 13-15 days o f  p o s t - n a t a l age, b u t i n c o n t r a s t t o t h e acetone d r i e d a fall  tissues,  i n p o t e n c y a l s o o c c u r r e d a t t h e time o f n a t u r a l  delivery. The V/0 and A/0 r a t i o s , p r e s e n t e d 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 o f s t e a d i l y d e c r e a s i n g v a l u e s as was a l r e a d y n o t e d i n t h e acetone d r i e d c.  tissues.  Comparison o f b i o l o g i c a l a c t i v i t i e s i n acetone and l y o p h i l i s e d  dried  glands  The two s e r i e s o f t i s s u e s were s i m i l a r i n showing,an i n c r e a s e d a c c u m u l a t i o n o f t h e a c t i v e agents w i t h t h e advancement o f g e s t a t i o n , a predominance o f v a s o p r e s s o r 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 t h e  58  F i g u r e 10 Weighted means o f b i o l o g i c a l  activities in  acetone d r i e d neurohypophyses o f sheep f o e t u s e s and lambs.  mU/mg = m i l l i u n i t p e r mg d r y gland  SHEEP  2000  FOETUS  (ACETONE  DRIED)  r  1500  cn E \ E  1000  -  500  h  pressor  10  30  50  Days  70 Gestation  90  110  130  147  T term  25 Post Natal  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 t h e same p a t t e r n i n b o t h 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, t h e two s e r i e s d i f f e r e d from one in several other respects.  another  The d i f f e r e n c e between t h e  acetone d r i e d and l y o p h i l i s e d g l a n d s was t h e a b s o l u t e hormonal p o t e n c i e s .  v e r y pronounced i n  A t 90 days o f g e s t a t i o n  o x y t o c i c and v a s o p r e s s o r a c t i v i t i e s were 80-90% lower i n t h e acetone d r i e d than i n t h e l y o p h i l i s e d g l a n d s . days, t h e l o s s o f a c t i v i t y f o r b o t h b i o l o g i c a l  At  140  activities  amounted t o a p p r o x i m a t e l y 30%, and a f t e r b i r t h a g a i n a g r e a t e r l o s s o f a c t i v i t y was  d e t e c t e d , a p p r o x i m a t e l y 50-60%.  From t h e s e v a l u e s i t appeared t h a t the e x t e n t o f e x t r a c t i o n was  acetone  c l o s e l y s i m i l a r f o r t h e o x y t o c i c and  v a s o p r e s s o r a c t i v i t i e s , b u t v a r i e d from one s t a g e o f d e v e l o p ment t o t h e o t h e r .  Acetone e x t r a c t e d a h i g h p r o p o r t i o n o f  b o t h a c t i v e agents a t t h e e a r l y s t a g e o f embryonic and p o s t - n a t a l development, b u t e x h i b i t e d r e l a t i v e l y  at  little  i n f l u e n c e on t h e hormonal c o n t e n t o f the p i t u i t a r y i n l a t e gestation. In t h e 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 i g h e r a n t i d i u r e t i c than v a s o p r e s s o r a c t i v i t y was d e t e c t e d , b u t i n t h e acetone d r i e d s e r i e s a good correspondance two a c t i v i t i e s was  found.  between t h e  I n view o f the f a c t t h a t  v a s o p r e s s o r and a n t i d i u r e t i c a s s a y s supposedly measure t h e  Ratios of b i o l o g i c a l d r i e d neurohypophyses  R a t i o o f V/0  =  a c t i v i t i e s i n t h e acetone  o f sheep f o e t u s e s and lambs.  w e i g h t e d mean o f v a s o p r e s s o r a c t i v i t y p e r mg d r y g l a n d / w e i g h t e d mean o f o x y t o c i c a c t i v i t y p e r mg d r y g l a n d  R a t i o o f A/0  =  w e i g h t e d mean o f a n t i d i u r e t i c  activity  p e r mg d r y g l a n d / w e i g h t e d mean o f o x y t o c i c a c t i v i t y p e r mg d r y g l a n d  Ratio  of  V/O  b i o l o g i c a l a c t i v i t i e s o f t h e same p e p t i d e , t h i s was  finding  unexpected. Finally,  i n t h e acetone d r i e d s e r i e s a f a l l i n  p o t e n c y o f t h e n e u r a l l o b e was d e t e c t e d 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 t o be a l r e a d y lower at the time of d e l i v e r y .  S i n c e t h e s e e s t i m a t i o n s a r e based  on s i n g l e g l a n d s i n b o t h c a s e s , t h e d i s c r e p a n c y c o u l d be to i n d i v i d u a l v a r i a t i o n s .  due  However, i t c o u l d a l s o r e p r e s e n t  a d e p l e t i o n o f t h e p o s t e r i o r p i t u i t a r y o f the lamb d u r i n g delivery.  A l t h o u g h b o t h g l a n d s were taken i n t h e l a t e phase  o f t h e f i r s t s t a g e o f d e l i v e r y , i t i s p o s s i b l e t h a t l a b o u r had been more arduous i n t h e case o f t h e lamb i n which 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 ;  the extra  d e p l e t i o n c o u l d have o c c u r r e d as a r e s u l t o f 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 o f t h e a c t i v e  agents  i n t h e neurohypophyses o f sheep f o e t u s e s I n v i e w o f t h e h i g h V/0  and A/0  r a t i o s detected i n  t h e neurohypophyses o f sheep f o e t u s e s , 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 o f t h e a c t i v e p r i n c i p l e s appeared 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  t o be intrinsic  r a t u t e r u s a c t i v i t y as w e l l as v a s o p r e s s o r a c t i v i t y , and a r e i n an approximate V/0 1966).  r a t i o o f 20:1  (Berde, B o i s s o n n a s ,  Since the r a t i o s recorded i n the i n d i v i d u a l  o f sheep f o e t u s e s o f t e n approached  these  glands  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 f o e t u s p o s s e s s e d  arginine  62  Figure  12 Weighted means o f 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 o f 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 a c t i v i t y / m g d r y gland  t  SHEEP  FOETUS  (LYOPHILISED)  63 v a s o p r e s s i n a l o n e , 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  limited  amount o f m a t e r i a l i n the e a r l y f o e t u s e s 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  impossible.  T h e r e f o r e , the p o o l e d  sample  o f 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 b o t h acetone d r i e d and l y o p h i l i s e d g l a n d s , was  subjected to  p a p e r chromatography i n o r d e r t o d e t e r m i n e whether an a c t i v i t y peak, t y p i c a l o f o x y t o c i n i t s e l f c o u l d be r e s o l v e d from the m i x t u r e . Neurohypophysial old  e x t r a c t s from the l a t e r , 140 days  f o e t u s e s were s u b j e c t e d 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 a n l a y s e d 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 b y paper chromatography The p o s t e r i o r p i t u i t a r i e s o f 22 f o e t u s e s a t 89-91 o f 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 w e i g h t o f 14 mg, e x t r a c t e d i n 1 ml o f 0.25%  acetic acid  o x y t o c i c and 7250 mU  o f v a s o p r e s s o r a c t i v i t y , was  Whatmann #3MM chromatography p a p e r . ("Syntocinon") and 12500 mU  500 mU  acid/water  0.84  of applied to  synthetic oxytocin  commercial v a s o p r e s s i n p r e p a r a t i o n  ( " P i t r e s s i n " ) were run a l o n g s i d e t h e e x t r a c t s as The chromatogram was  were  (see Methods).  ml o f e x t r a c t , w h i c h r e p r e s e n t e d a t o t a l o f 350 mU  days  developed  standards.  in butanol/acetic  (4:1:5) f o r a p e r i o d o f 13 hours a t room  temperature.  A f t e r c o m p l e t i o n o f t h e development, the chromatogram  was  d r i e d i n a c o o l stream o f a i r , and when'dry, c u t i n t o  squares,  F i g u r e 13 Ratios o f 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 o f sheep f o e t u s e s and lambs.  R a t i o o f V/0  =  w e i g h t e d mean o f v a s o p r e s s o r  activity/  mg d r y t i s s u e / w e i g h t e d mean o f o x y t o c i c a c t i v i t y p e r mg d r y t i s s u e . R a t i o o f A/0  =  w e i g h t e d mean o f a n t i d i u r e t i c  activity  p e r mg d r y t i s s u e / w e i g h t e d mean o f o x y t o c i c a c t i v i t y p e r mg d r y t i s s u e .  Ratio of  V/O  65  o f which each one r e p r e s e n t e d 0.1 o f an  unit  (see  Methods). Each square was analysed f o r b i o l o g i c a l presented  eluted i n 0.25% acetic acid activities.  The r e s u l t s  are  i n F i g . 15, and show a s u c c e s s f u l s e p a r a t i o n  of o x y t o c i n from v a s o p r e s s i n . l o c a t e d a t R^ 0.5-0.7,  Synthetic oxytocin  l a p p i n g i n t o R^ 0.5.  From the 500 mU  a p p l i e d , a t o t a l o f 163 mU was The 4000 mU  was  and 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 ) a t R f 0.3-0.4, w i t h a t r a c e (15 mU)  recovery.  and  over-  synthetic oxytocin  recovered, r e p r e s e n t i n g a 3 2 %  Pitressin  recovered was  again a 3 2 %  recovery. Both o x y t o c i c and v a s o p r e s s o r a c t i v i t i e s were d e t e c t e d i n the chromatogram of the f o e t a l  posterior  pituitary  found  R f 0.2  extracts.  t o 0.7,  O x y t o c i c a c t i v i t y was  and v a s o p r e s s o r a c t i v i t y  traces of a c t i v i t y  i n R f 0.5  i n R^ 0.5-0.7 corresponds and  and 0.6.  from  i n R f 0.2  t o 0.4,  The r a t uterus  with  activity  t o the l o c a t i o n of s y n t h e t i c o x y t o c i n ,  the r a t u t e r u s a c t i v i t y  i n R f u n i t s corresponding to  those o f the v a s o p r e s s o r a c t i v i t y can be p a r t i a l l y e x p l a i n e d 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 v a s o p r e s s i n . of the r a t u t e r u s a c t i v i t y  In view  i n R f 0.5-0.7, which runs ahead  of v a s o p r e s s i n , and corresponds w e l l to the l o c a t i o n o f the s y n t h e t i c o x y t o c i n , i t i s r e a s o n a b l y c e r t a i n t h a t the neurohypophyses o f e a r l y f o e t u s e s c o n t a i n o x y t o c i n The o x y t o c i c a c t i v i t y recovered i  n  R  f  itself.  0.5-0.7 i s approximately  3 2 % of the r a t uterus a c t i v i t y d e t e c t e d i n the crude  extracts,  66  F i g u r e 14 Comparison o f r a t i o s o f b i o l o g i c a l a c t i v i t i e s between 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 o f sheep  f o e t u s e s and lambs.  R a t i o o f V/0  =  weighted means o f v a s o p r e s s o r mg d r y t i s s u e / w e i g h t e d  a c t i v i t y per  means o f o x y t o c i c  a c t i v i t y p e r mg d r y t i s s u e . Ratio of A/0  =  weighted means o f a n t i d i u r e t i c a c t i v i t y p e r mg d r y t i s s u e / w e i g h t e d  means o f o x y t o c i c  a c t i v i t y p e r mg d r y t i s s u e .  i  RATIO OF ACTIVITIES,  FOETAL O  SHEEP  lyophilised  o > o o o rr  50 Doys  100 Gestation  147 12  r  25  term Post Notal  50 Days  100 Gestation  147  T  term  12  25  Post Natal  w h i c h g i v e s a r e c o v e r y , s i m i l a r t o t h e r e c o v e r y found f o r the  standards. Vasopressor  was l o c a t e d a t R  f  a c t i v i t y , from t h e f o e t a l e x t r a c t s  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 t h e case o f s t a n d a r d v a s o p r e s s i n .  This i s  i n good agreement w i t h t h e R_^ v a l u e s o f 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 t h e p r o b a b l e  presence  of arginine vasopressin i n the early foetus. I n t h e chromatogram o f e a r l y f o e t u s e s , r a t u t e r u s a c t i v i t y was d e t e c t e d i n t h e R^ u n i t s c o r r e s p o n d i n g t o t h e v a s o p r e s s o r a c t i v i t y as w e l l as i n those w h i c h t o t h e R^ v a l u e s o f o x y t o c i n i t s e l f . the presence  corresponded  As p o i n t e d o u t e a r l i e r ,  o f r a t u t e r u s a c t i v i t y i n t h e v a s o p r e s s o r peak  was n o t e n t i r e l y s u r p r i s i n g , i n v i e w o f t h e 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 v a s o p r e s s i n .  However, t h e  magnitude o f t h i s a c t i v i t y was found t o be 5 times t o o g r e a t t o be accounted  f o r b y t h e amount o f v a s o p r e s s i n p r e s e n t .  T h i s d i s c r e p a n c y suggested  the p o s s i b l e presence  moving p r i n c i p l e , w i t h c h r o m a t o g r a p h i c  o f a slow-  behaviour s i m i l a r t o  t h a t o f a r g i n i n e vasopressin, b u t which contrasted w i t h i t in possessing a r e l a t i v e l y higher i n t r i n s i c oxytocic 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 p e p t i d e w i t h t h e p r o p e r t i e s approximating  t o these, i s a r g i n i n e v a s o t o c i n .  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 t h e o t h e r  neurohypophysial  p e p t i d e s , by i t s v e r y h i g h a c t i v i t y i n promoting t r a n s p o r t through t h e i s o l a t e d f r o g b l a d d e r . order t o determine  the p o s s i b l e presence  Arginine  water  Therefore, i n  of arginine vasotocin  68  F i g u r e 15 Paper chromatogram o f crude e x t r a c t o f the neurohypophyses o f sheep f o e t u s e s a t 89-91 gestational  days o f  age.  Black  =  r a t uterus  activity  Striped  =  vasopressor  White  —  f r o g waterbalance  activity activity  .  0  .1 .  .2  r  i .  1  . Synthetic  1  3  .4  i  "f •  R . f  -5  .6  - 1 — * - — " — I  .7  .8  1—  r  Rat  Oxytocin  -' ?rd  Vasopressin  Vasopressor  38000  36000 h  !.0  Uterus  bssssssssa  2500 2000  .9 t  —.  !  69 w i t h i n the v a s o p r e s s o r peak, f r o g b l a d d e r assay was  carried  out.  activity  was  As shown i n F i g . 16, a v e r y h i g h f r o g b l a d d e r  d e t e c t e d a t t h e p e a k . o f t h e v a s o p r e s s o r a c t i v i t y , and  l e s s e r q u a n t i t y was The  found  i n t h e two  a  u n i t s on e i t h e r s i d e .  f r o g b l a d d e r a c t i v i t y , w h i c h amounted t o 38,000 mU  in  t h e r e g i o n between R^ 0.2-0.3, i s 450 times too g r e a t t o be a t t r i b u t a b l e t o t h e amount o v ~ v a s o p r e s s i n p r e s e n t .  It  i s 100 t i m e s too g r e a t t o be e l i c i t e d by the amount o f r a t uterus a c t i v i t y i n t h i s region, i f t h i s r a t uterus was  activity  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  v a s o p r e s s o r peak.  I n any case, such t r a i l i n g i s most  u n l i k e l y i n v i e w o f t h e s m a l l amount o f o x y t o c i n p r e s e n t . T h e r e f o r e , t h e r e i s a s t r o n g s u g g e s t i o n t h a t t h e neurohypophyse of e a r l y foetuses contain a r g i n i n e vasotocin 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.  G e l f i l t r a t i o n o f crude n e u r o h y p o p h y s i a l late  e x t r a c t s from  foetuses S i n c e s e p a r a t i o n and a h i g h degree o f p u r i f i c a t i o n  was G-15  achieved w i t h maternal  e x t r a c t s b y the use o f 200  Sephadex columns, t h e same method was  p u r i f i c a t i o n of neurohypophysial  cm  a p p l i e d to the  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 o f sheep f o e t u s e s a t 138-141 days o f g e s t a t i o n a l age were p o o l e d , and e x t r a c t e d i n 0.25%  acetic acid.  e x t r a c t , r e p r e s e n t i n g 4.4 + 0.48 8.0  A t o t a l o f 15 ml  I.U. o x y t o c i c and 34.0  I.U. v a s o p r e s s o r a c t i v i t y , were a p p l i e d t o a 200 cm  Sephadex column.  The  e l u a t e from the column was  + G-15  collected in  70  Figure  16 P u r i f i c a t i o n o f crude e x t r a c t o f sheep  a t 138-141 days o f g e s t a t i o n a l age, on 200 cm G-15  column.  foetuses Sephadex  Purification on  150  200  of  Foetal Sheep  cm. Sephadex  6-15  Extract column  1500  x  n  a> •a  g"  100  •Lowry  CL  vasopressor.  1000  5 E \ 3  50  500  oxytocic  I  J-JL  0  20  40 Tube  60 Number  80  100  120  ( I tube = 2. 8ml)  140  160  180  200  220  2.8 ml f r a c t i o n s as p r e v i o u s l y d e s c r i b e d f o r t h e m a t e r n a l e x t r a c t s and t h e f r a c t i o n s were a n a l y s e d 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 c o n t e n t and c o n d u c t i v i t y .  The r e s u l t s  a r e shown i n F i g . 17. The f i r s t 150 ml o f e l u a t e r e p r e s e n t e d t h e v o i d volume and was d i s c a r d e d .  The mass o f p r o t e i n s , as determined  b y t h e Lowry p e p t i d e method, were c o n c e n t r a t e d i n t h e 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  from t h e Lowry p e p t i d e s e c t i o n b y 70 f r a c t i o n s .  separated 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 t h e v a s o p r e s s i n from t h e o x y t o c i c peak. The marked s e p a r a t i o n o f t h e a c t i v e agents from t h e p r o t e i n f r a c t i o n s , and t h e complete  r e s o l u t i o n o f t h e o x y t o c i c and  v a s o p r e s s o r 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 o f t h e hormones was a c h i e v e d .  The 2.0 I.U. o x y t o c i c a c t i v i t y  d e t e c t e d i n t h e o x y t o c i c peak r e p r e s e n t e d a 100% r e c o v e r y , i f t h e c o n t r i b u t i o n o f t h e l a r g e amount o f v a s o p r e s s i n t o t h e o x y t o c i c a c t i v i t y o f t h e crude e x t r a c t i s taken i n t o  account.  The r e c o v e r y f o r v a s o p r e s s i n was a g a i n a p p r o x i m a t e l y 80%. The p o o l e d f r a c t i o n s o f t h e o x y t o c i c peak were for of  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 0.5 mM MgCl.  without Mg  + +  assayed  and absence  The r a t i o o f o x y t o c i c a c t i v i t y w i t h M g / + +  was found t o be 0.84, which  i s i n good agreement  w i t h t h e v a l u e o f 0.86 r e p o r t e d b y Munsick (1960) f o r t h e potentiation of synthetic oxytocin.  Since oxytocin i s the  o n l y known n e u r o h y p o p h y s i a l 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 t h a n 1, t h i s r e s u l t s t r o n g l y s u g g e s t s t h a t t h e r a t u t e r u s  72  Figure  17 Sample p l a t e o f t h i n l a y e r  chromatography  o f the d a n s y l d e r i v a t i v e s o f standard amino a c i d s , and o f the h y d r o l y s a t e o f the o x y t o c i c moiety o f the a d u l t pregnant sheep and the f o e t u s .  DNS  =  dansyl  solvent  1  front  DNS  hydrolysates  0 xytocl c Adult  o DNS  ft o  0  0 DNS  DNS  I leu  Pro  0 o  o OO o  Leu  DNS  0  0  0  DNS Tyr  ONS Gly  *-  NH  0oo  DNS Cyt  -i  Foetal  -0-  DNS Asp  r  origin  0(al  o DNS Gfu  0  di DNS| Lys  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 oxytocin itself. The  e s t i m a t i o n o f t h e o x y t o c i c a c t i v i t y throughout  the vasopressor 17) c o n t a i n e d  peak, showed t h a t one f r a c t i o n  (#187 i n F i g .  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 , w h i c h  c o u l d n o t be accounted f o r b y t h e 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 vasopressin.  F r o g w a t e r b a l a n c e a s s a y was c a r r i e d  o u t on t h i s f r a c t i o n t o d e t e r m i n e whether i t m i g h t 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 t h e work p r e s e n t e d  above had  suggested t h a t t h i s p e p t i d e m i g h t be p r e s e n t foetuses.  i n t h e young  The t o t a l f r o g w a t e r b a l a n c e a c t i v i t y i n t h e 2.8  ml o f e l u a t e p r e s e n t  i n t h i s tube was found t o be 5,600 mU.  T h i s v a l u e i s 80 times g r e a t e r t h a n c o u l d be expected from a r g i n i n e vasopressin alone,  i f i t were p r e s e n t  q u a n t i t y i n d i c a t e d b y t h e p r e s s o r assay.  i n the  Again, t h i s  suggests  the presence o f a peptide other than vasopressin, a peptide possessing unusually high frog bladder a c t i v i t y .  The same  arguments g i v e n p r e v i o u s l y f o r t h e s i m i l a r a c t i v i t y found i n the e a r l y f o e t u s s u g g e s t t h a t t h i s p e p t i d e m i g h t be a r g i n i n e vasotocin. c.  Amino a c i d a n a l y s i s o f t h e p u r i f i e d p e p t i d e s o f t h e late  foetuses The  o x y t o c i c and v a s o p r e s s o r  old foetuses, obtained pooled,  from t h e G-15 Sephadex column were  l y o p h i l i s e d and h y d r o l y s e d  Methods).  f r a c t i o n s o f 140 days  i n 6N H C l (see p. 22  The h y d r o l y s a t e s were r e a c t e d w i t h t h e f l u o r e s c e n t  74  F i g u r e 18 Graphed R^ v a l u e s o f d a n s y l a t e d s t a n d a r d amino a c i d s , and t h e d a n s y l a t e d h y d r o l y s a t e o f t h e f o e t a l vasopressor moiety.  d. NH^  = d a n s y l ammonia  d i - Lys  = d i - dansyl derivative  of lysine  l y s i n e = mono-dansylated d e r i v a t i v e  of lysine  /Amino a c i d s o f t h e unknowns a r e 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 c o r r e s p o n d c l o s e l y t o the R  o f a s t a n d a r d amino a c i d . f  SOLVENT  standard  SYSTEM  I  vasopressor  •  SOLVENT  standard  SYSTEM  2  vasopressor  SOLVENT  ta n dor d  °  lleu d. N H Leu Ph e Vol  •  Pro  •  2  •  Pro  peptide  Ala  0.2  0.1  Tyr  •  His  Gly  —  -  #  Glu  • O •  Thr Ser Asp  «  Cvi. Arg  *  CyS,  #  s tondard  SYSTEM  4  vasopressor  Vol di-Lys Ala, Piie His d. NH Leu, lieu Tyr Pro, Glu Gly, Ser Thr, CyS  peptide  Glu ?  • *  Pro lieu Leu d. NH  2  Tyr di-Lys  lieu Leu Pro, Vol  Gly di- Lys  SOLVENT  Asp  d. NH,  •  vo s o p res s o r  2  2  •  3  peptide e> e  S  SYSTEM  € Lys Arg  •  Phe  •  Ala  •  di-Lys  •  Ala, di-Lys  Tyr G ly His Glu Ser, Thr  •  Tyr  •  Phe  •  Gly •  Arg  •  Cyt  Glu  •  Gly, Gib  Cyt •  Asp  •  Cyt  •  Asp  75  marker,  in detail  on p . 23  The r e s u l t i n g amino a c i d c o m p l e x e s w e r e a p p l i e d  to thin  layer  dansyl  silica  standards. on  chloride,  plates, beside  dansylated  The h y d r o l y s a t e s  the p l a t e s p a r a l l e l  provide  as d e s c r i b e d  amino  acid  from t h e f o e t u s were  to hydrolysates  from the a d u l t ,  a d i r e c t c o m p a r i s o n b e t w e e n t h e amino  c o m p o s i t i o n b f t h e two. solvent  developed to  acid  The p l a t e s w e r e r u n i n f o u r d i f f e r e n t  systems f o r optimal  r e s o l u t i o n o f the standard  amino  acids. The c o n s t i t u e n t  amino a c i d s  t h e m a t e r n a l and f o e t a l identical  i n F i g . 17.  since  of oxytocin,  the f o e t a l  oxytocic  chromatographic pattern,  this  composition of the f o e t a l  pharmacological data, itself  i s the oxytocic  late  foetuses  constituent of  the standard  of  the hydrolysate  i n Section  I,  t h e same  t h e amino  acid  i s t h e same as  i n conjunction  strongly  amount  allowed  amino a c i d s  peptide  suggests  agent o f the l a t e  The much g r e a t e r  constituent  discussed  indicates that  oxytocic  this  analysis  p r i n c i p l e exhibited  t h a t o f t h e a d u l t . ' When t a k e n  the  as i l l u s t r a t e d b y t h e  The more c o m p l e t e  as a l r e a d y  of  h a d shown an  t h e m a t e r n a l m a t e r i a l h a d shown t h e e i g h t  amino a c i d s and  moieties  chromatographic behaviour,  sample p l a t e g i v e n of  oxytocic  i n the hydrolysates  with the that  oxytocin  foetuses.  of vasopressor  agent i n  a more c o m p l e t e a n l a y s i s o f t h e  of this peptide.  amino a c i d s  The  values  and t h e c o n s t i t u e n t  a r e shown i n F i g . 18.  The  amino  acids  constituent  amino a c i d s i d e n t i f i e d were:  phenylalanine,  a s p a r t i c a c i d , p r o l i n e , glycene, and  cysteic acid.  glutamin a c i d , t y r o s i n e ,  In a d d i t i o n t o the e i g h t  amino a c i d s o f a r g i n i n e v a s o p r e s s i n , were a l s o d e t e c t e d . a c i d s were i n t r o d u c e d  arginine,  constituent  l y s i n e and a l a n i n e  I t i s assumed t h a t these two amino i n t o the h y d r o l y s a t e s  during  proces  i n g e i t h e r by the breakdown o f c o n s t i t u e n t amino a c i d s (as i n the case o f a l a n i n e ) , o r as contamination from the glassware.  77  DISCUSSION (Section I I )  The e s t i m a t i o n o f b i o l o g i c a l a c t i v i t i e s i n sheep f o e t u s e s 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 active p r i n c i p l e s  out g e s t a t i o n .  through-  I n c r e a s i n g amounts o f hormonal a c t i v i t i e s  w i t h t h e advancement o f embryonic development were c h a r a c t e r i s t i c o f b o t h t h e acetone d r i e d and l y o p h i l i s e d glands.  Around t h e time o f b i r t h a secondary  fall  o x y t o c i c and v a s o p r e s s o r a c t i v i t i e s were found. potency  i n both This f a l l i n  appeared t o o c c u r a t t h e time o f 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 o f v a s o p r e s s o r t o o x y t o c i c  and o f a n t i d i u r e t i c t o o x y t o c i c a c t i v i t i e s were h i g h a t t h e e a r l y g e s t a t i o n a l age (90 d a y s ) , and i n some cases t h e v a l u e o f 20.  D u r i n g t h e c o u r s e o f embryonic development  and e a r l y p o s t - n a t a l l i f e , steadily.  approached  these very high r a t i o s  decreased  The r e s u l t s i n t h e sheep f o e t u s e s and newborn  lambs agree g e n e r a l l y w i t h t h o s e r e p o r t e d b y D i c k e r and Tyler to  (1953 a, b) f o r human and dog f o e t u s e s , w i t h r e s p e c t  t h e o r i g i n a l l y h i g h , 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  during gestation. The h i g h r a t i o between t h e v a s o p r e s s o r and o x y t o c i c a c t i v i t i e s i n t h e f o e t a l neurohypophysis  (  i s the r e s u l t of the  78  v e r y low o x y t o c i c a c t i v i t i e s d u r i n g embryonic development. As b e f o r e ,  i n connection w i t h various other aspects of  the  work r e p o r t e d here, t h e q u e s t i o n a r i s e s as t o whether t h e low o x y t o c i c c o n t e n t o f the immature p o s t e r i o r p i t u i t a r y i s a p r o d u c t o f d e p r e s s e d h y p o t h a l a m i c s e c r e t i o n , o r 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. l a t t e r p o s s i b i l i t y appears t o be an u n l i k e l y one. basis of the primary  On  The  the  a c t i o n s o f o x y t o c i n i n t h e a d u l t organism,  where i t i s c o n n e c t e d w i t h r e p r o d u c t i o n and t h e n u r s i n g of t h e young, i t i s h a r d t o e n v i s a g e  a possible function for j  t h i s agent i n t h e f o e t a l o r g a n i s m .  1  The more l i k e l y p o s s i b i l i t y appears t o be a d e p r e s s e d h y p o t h a l a m i c p r o d u c t i o n o f t h e o x y t o c i c agent. evidence supports  t h i s hypothesis.  chrome h a e m a t o x y l i n  Histological  S t a i n i n g w i t h Gomori's  p h l o x i n s t a i n shows an e a r l i e r embryonic  appearance o f s t a i n a b l e m a t e r i a l i n t h e s u p r a - o p t i c  nucleus,  t h a n i n t h e b o d i e s o f the p a r a v e n t r i c u l a r c e l l s o f human foetuses  ( M e i t n e r , 1959).  Since the p a r a v e n t r i c u l a r nucleus  i s m a i n 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 o f (Heller,  vasopressin  1966), t h i s o b s e r v a t i o n p o i n t s t o a l a t e r embryonic  development o f the o x y t o c i n p r o d u c i n g  center.  A  later  embryonic development o f the p a r a v e n t r i c u l a r n u c l e u s been demonstrated i n o t h e r s p e c i e s as w e l l , mouse, r a t and c a l f  (Yakovleva,  I t i s necessary evidence,  such as  the  1966).  t o p o i n t out, t h a t h i s t o l o g i c a l  f o r the p r e s e n c e o f n e u r o h y p o p h y s i a l  often unreliable,  has  hormones i s  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 d e t e c t e d b e f o r e t h e appearance ( H e l l e r , 1961).  of secretory material  Further, a disappearance of these granules  can o c c u r w i t h o u t a l o s s i n p o t e n c y  ( F a r r e l l , 1967).  However,  t h e l a r g e number o f s i m i l a r r e p o r t s , which g i v e h i s t o l o g i c a l e v i d e n c e f o r a t i m e d i f f e r e n c e between t h e appearance  of  s e c r e t o r y m a t e r i a l i n t h e two h y p o t h a l a m i c n u c l e i , seem t o make a s t r o n g argument i n f a v o u r o f a l a t e r appearance  o f the o x y t o c i n s y n t h e t i s i n g  embryonic  structures.  In the r e s u l t s concerning the neurohypophysial hormone c o n t e n t o f t h e sheep f o e t u s e s r e p o r t e d h e r e , a c o n s i s t e n t l o s s o f b i o l o g i c a l a c t i v i t y was  found  p r o l o n g e d exposure o f the g l a n d s t o acetone.  following  This problem  has a l r e a d y been d i s c u s s e d i n S e c t i o n I , i n c o n n e c t i o n w i t h the same phenomena i n p r e g n a n t f e m a l e s . f o e t a l neurohypophyses o n l y emphasised  The s t u d i e s on f u r t h e r the f a c t t h a t  acetone l e a c h e s out b o t h a c t i v e agents t o a p p r o x i m a t e l y t h e same degree.  However, t h e e x t e n t o f t h i s removal v a r i e s w i t h  different physiological conditions.  I n the f o e t a l  glands  t h e g r e a t e s t l o s s o f a c t i v i t y was o b s e r v e d a t 90 days o f g e s t a t i o n , and a l o w e r l o s s o c c u r r e d d u r i n g t h e e a r l y p o s t n a t a l age.  On t h e o t h e r hand, t h e r e was o n l y a s l i g h t  loss  o f a c t i v i t y i n l a t e g e s t a t i o n , when t h e f o e t u s e s had been i n u t e r o f o r 140  days.  The comparison o f p o t e n c i e s i n t h e l y o p h i l i s e d  and  acetone d r i e d g l a n d s o f sheep f o e t u s e s 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 h i g h e r a n t i - d i u r e t i c t h a n 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 .  A g a i n , o n l y s u g g e s t i o n s can be made  as t o t h e 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 t o t h e  greater  a n t i d i u r e t i c , than v a s o p r e s s o r a c t i v i t i e s r e c o r d e d i n t h e l y o p h i l i s e d glands o f f o e t u s e s .  S i n c e t h e s e two b i o - a s s a y s  s u p p o s e d l y measure t h e p o t e n c y o f t h e same hormone; t h e h i g h e r a n t i d i u r e t i c p o t e n c y c o u l d be due t o 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 t h e h y p o p h y s i s o f t h e foetuses or p o s s i b l y to the presence of a agent.  Due  vasodepressor  t o l a c k o f m a t e r i a l , no t e s t s c o u l d be  o u t t o determine  carried  t h e p o s s i b l e p r e s e n c e o f e i t h e r agent.  In  view o f t h e f a c t , however, t h a t o t h e r w o r k e r s have come a c r o s s t h e 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 the a n t i d i u r e t i c p r i n c i p l e problem w a r r a n t s f u r t h e r  (Kammen and De W i e l d , 1960)  from the  investigation.  The p u r i f i c a t i o n and a n a l y s i s o f t h e a c t i v e p r i n c i p l e s o f t h e f o e t a l sheep neurohypophysis  have shown t h e  o f an o x y t o c i c and v a s o p r e s s o r p r i n c i p l e .  The  presence 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 o f t h e f o e t u s e s a t 90 days o f g e s t a t i o n a l age  suggest  t h a t t h e s e a c t i v e agents a r e : o x y t o c i n and a r g i n i n e vasopressin.  A g a i n , i n the o l d e r , 140 days o l d embryos,  b o t h the p h a r m a c o l o g i c a l e v i d e n c e and the amino a c i d  analysis  p o i n t t o 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 b e i n g t h e a c t i v e agents. I n a d d i t i o n t o t h e two n e x i r o h y p o p h y s i a l p e p t i d e s t y p i c a l o f t h e mammal, t h e p r e s e n c e o f a t h i r d p e p t i d e  was  detected.  The chemical b e h a v i o u r  and  pharmacological  p r o p e r t i e s o f t h i s t h i r d p e p t i d e suggest t h a t i t might 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 o f vertebrates.  The amount o f t h i s p e p t i d e appears  w i t h the advancement o f g e s t a t i o n .  lower to d e c l i n e  C a l c u l a t i o n s , which  converted the b i o l o g i c a l a c t i v i t i e s t o mg p e p t i d e p r e s e n t , i n d i c a t e d t h a t i n e a r l y g e s t a t i o n (90 days) a r g i n i n e v a s o p r e s s i n and a r g i n i n e v a s o t o c i n are p r e s e n t i n an approximately  1:1  ratio.  By 140 days o f i n t r a u t e r i n e  the r a t i o o f the two p e p t i d e s was favour o f a r g i n i n e v a s o p r e s s i n .  approximately  120 i n  life  82  GENERAL DISCUSSION  The data o b t a i n e d i n the sheep d u r i n g pregnancy and embryonic development, have shown t h a t changes occur i n the neurohypophysis  o f both the f o e t u s and the mother d u r i n g  the course of pregnancy. neurohypophysis  About mid-pregnancy, the  o f the mother c o n t a i n s a reduced l e v e l o f  i t s a c t i v e agents, and although t h e r e i s an i n c r e a s e i n p o t e n c i e s towards l a t e g e s t a t i o n , the hormonal l e v e l s do not r e t u r n to the c o n t r o l , non-pregnant l e v e l throughout  the  d u r a t i o n o f pregnancy. During embryonic development, an  increasing  accumulation o f b i o l o g i c a l l y a c t i v e agents were d e t e c t e d . The presence of both p e p t i d e s , c h a r a c t e r i s t i c o f the a d u l t mammal, o x y t o c i n and v a s o p r e s s i n have been shown to be p r e s e n t i n the f o e t u s . principles,  In a d d i t i o n t o these a c t i v e  the presence of a t h i r d neurohypophysial p e p t i d e ,  p r o b a b l y a r g i n i n e v a s o t o c i n , has been i n d i c a t e d . neurohypophysis  The  o f the embryo i s c h a r a c t e r i s e d by a p r e -  dominance of v a s o p r e s s o r over o x y t o c i c a c t i v i t y . The r e s u l t s i n both the pregnant  a d u l t and i n the  f o e t u s have been d i s c u s s e d 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 t o p i c s . However, the most important q u e s t i o n s have not y e t been touched on; these concern the r e l a t i o n s h i p o f the changes i n  83  t h e n e u r o h y p o p h y s i s t o t h e more g e n e r a l p i c t u r e o f pregnancy and f o e t a l development,  and t o t h e 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 o f t h e a c t i v e agents i n t h e m e t a b o l i s m o f t h e embryo. T h i s g e n e r a l d i s c u s s i o n w i l l attempt t o d e v e l o p some o f t h e s e problems.  However, because o f t h e s c a r c i t y o f i n f o r m a t i o n ,  many o f t h e f o l l o w i n g c o n s i d e r a t i o n s a r e 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 o f t h e n e u r o h y p o p h y s i s d u r i n g pregnancy The l o w e r e d a c t i v i t y o f t h e n e u r o h y p o p h y s i s d u r i n g  pregnancy r a i s e s t h e q u e s t i o n o f whether t h i s d e p l e t i o n i s due t o a d e p r e s s e d h y p o t h a l a m i c p r o d u c t i o n o f hormones, o r 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 t o changes i n o t h e r e n d o c r i n e g l a n d s , and therefore, 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 w h i c h t a k e p l a c e must be  given.  The main e n d o c r i n e changes d u r i n g pregnancy a r e t h e e l e v a t e d e s t r o g e n and p r o g e s t e r o n e l e v e l s , w h i c h may  originate  t o a l a r g e e x t e n t from t h e corpus luteum, o f from t h e placenta.  I n t h e sheep, where t h e p l a c e n t a t a k e s o v e r t h e  p r o d u c t i o n o f t h e s e s t e r o i d s a t 50-60 days o f g e s t a t i o n ( M a r s h a l l , 1959), t h e p r o g e s t e r o n e l e v e l r i s e s t o 6 ug/ml i n t h e p e r i p h e r a l b l o o d o f t h e p r e g n a n t female a t 40 days o f g e s t a t i o n and remains a t t h i s l e v e l u n t i l 100 d a y s .  A t about  100 days t h e b l o o d p r o g e s t e r o n e l e v e l r i s e s and r e a c h e s a second p l a t e a u a t 12 ug/ml b l o o d , and t h i s i s m a i n t a i n e d up t o 36 h r s p o s t p a r t u r i t i o n  (Neher and Zarrow, 1958).  Although  84  t h e r e i s no i n f o r m a t i o n c o n c e r n i n g the e s t r o g e n  levels  i n the sheep d u r i n g pregnancy, i n the goat and the  cow,  which are s i m i l a r t o the sheep i n t h e i r hormonal c o n t r o l o f g e s t a t i o n , estrogen l e v e l was  r e p o r t e d to r i s e  i n the second h a l f o f pregnancy and reached a t term  ( B a r r i e , P a t t e r s o n and U n d e r h i l l ,  steadily  a maximum v a l u e  1935;  Bromskov,  1939). Evidence c o n c e r n i n g the p o s s i b l e a c t i o n o f the s t e r o i d hormones and the p o s t e r i o r p i t u i t a r y p e p t i d e s i s s u g g e s t i v e , but  sketchy. A t the l e v e l o f the t a r g e t organs,  progesterone  has  been shown t o decrease the s e n s i t i v i t y o f the u t e r u s t o o x y t o c i n , w h i l s t estrogen enhances the a c t i o n o f t h i s p e p t i d e (Fitzpatrick,  1966).  The  t a r g e t organs of v a s o p r e s s i n  r e a c t i n a s i m i l a r manner t o progesterone  and  estrogen;  s e n s i t i v i t y i s l e s s e n e d i n the presence o f the former i n c r e a s e d by the l a t t e r s t e r o i d .  Cobo (1967) r e p o r t e d a h i g h  t h r e s h o l d f o r a n t i d i u r e t i c responses pregnant women, and P i c k f o r d  and  to vasopressin i n  (1966) found a s i m i l a r  o f progesterone on the s a l t e x c r e t i o n and v a s c u l a r  effect response  t o the same p r i n c i p l e . At the l e v e l o f the hyopothalamic Silver  (1965) r e p o r t e d depressed  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 ,  n u c l e i , Cross  and  a c t i v i t y o f both the suprafollowing administration of  progesterone. B e s i d e s evidence f o r the i n f l u e n c e o f s t e r o i d hormones on the p r o d u c t i o n and e f f e c t i v e n e s s o f the  neurohypophysial  85  p e p t i d e s , t h e s e p e p t i d e s may i n t u r n e f f e c t t h e s t e r o i d l e v e l s themselves d i r e c t l y o r i n d i r e c t l y , through t h e m e d i a t i o n o f t h e g o n a d o t r o p h i c hormones o f t h e adenohypophysis.  The r o l e o f t h e n e u r o h y p o p h y s i a l hormones as  r e l e a s i n g f a c t o r s f o r t h e g o n a d o t r o p h i c hormones has been reviewed  by M a r t i n i  given here.  (1966), and o n l y one example w i l l be  I t h a s been shown t h a t l a r g e doses o f 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  e s t r o u s , and i t i s presumed  t h a t t h e p e p t i d e i n h i b i t s p r o g e s t e r o n e p r o d u c t i o n , so t h a t t h e consequent  fall  i n blood l e v e l unleashes the r e l e a s e o f  g o n a d o t r o p h i c hormones from t h e adenohypophysis  (Labsethwar  e t a l . , 1964) These f r a g m e n t a r y p i e c e s o f e v i d e n c e encourage t h e s p e c u l a t i o n t h a t the elevated progesterone l e v e l renders t h e t a r g e t organs l e s s s e n s i t i v e t o t h e n e u r o h y p o p h y s i a l hormones a t 90 days o f pregnancy.  T h i s i n t u r n might  result  i n an i n c r e a s e d r e l e a s e from t h e p o s t e r i o r p i t u i t a r y o f i t s a c t i v e agents, t h r o u g h secondary mechanisms, o n l y p a r t l y understood a t the p r e s e n t .  A t t h e same t i m e , t h e e l e v a t e d  p r o g e s t e r o n e 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 h y p o t h a l a m i c production of the peptides.  The combined e f f e c t o f an  i n c r e a s e d 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 c o u l d r e s u l t i n t h e lowered a c t i v i t y o f t h e neurohypophysis  as found  i n t h e s t u d i e s o f p r e g n a n t sheep a t 90 days o f g e s t a t i o n . In  t h e l a t t e r p a r t o f pregnancy,  the r i s i n g estrogen  l e v e l , w h i c h reaches i t s maximum a t term, c o u l d p o s s i b l y  c o u n t e r a c t t h e a c t i o n o f p r o g e s t e r o n e , b o t h a t the l e v e l of  t h e t a r g e t organ and i n t h e hypothalamus.  The  rising  e s t r o g e n l e v e l would r e e s t a b l i s h a b a l a n c e n e a r e r t o t h a t of  t h e c o n t r o l , where t h e r e l e a s e d f t h e n e u r o h y p o p h y s i a l  p e p t i d e s w i l l be d e c r e a s e d and t h e p r o d u c t i o n b y t h e hypothalamus i n c r e a s e d .  T h i s would r e s u l t i n a g r e a t e r  accumulation o f n e u r o s e c r e t o r y m a t e r i a l i n the neuroh y p o p h y s i s and would e x p l a i n t h e s e c o n d a r y r i s e i n a c t i v i t i e s found a t 140 days o r pregnancy i n t h e sheep. 2.  The p o s s i b l e r o l e o f t h e a n t i d i u r e t i c p r i n c i p l e ,  during  embryonic development The r e l a t i v e abundance o f t h e v a s o p r e s s o r o r 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 o f the foetus, r e p r e s e n t s one o f t h e most i n t e r e s t i n g and s t i l l  unresolved  problems o f 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. of  I t i s n o t c e r t a i n whether t h i s  predominance  the a n t i d i u r e t i c a c t i v i t y during i n t r a u t e r i n e  life  r e p r e s e n t s an e a r l i e r appearance o f t h e s y n t h e t i c mechanism for  t h e p r o d u c t i o n o f t h e a n t i d i u r e t i c agent o r whether i t  r e f l e c t s t h e g r e a t e r use o f t h i s agent b y t h e o r g a n i s m . 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 t h e a n t i d i u r e t i c p r i n c i p l e b y t h e 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 t h e volume r e g u l a t i o n o f t h e a m n i o t i c fluid.  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 o f t h e p o s t e r i o r p i t u i t a r y o f human f o e t u s e s the volume o f a m n i o t i c with very l i t t l e  f l u i d present,  found t h a t  foetuses  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 o f s e c r e t o r y m a t e r i a l was f l u i d volume.  and  with  fluid,  and c o n v e r s e l y ,  an abundance  associated with smaller  amniotic  On t h e b a s i s o f t h e s e o b s e r v a t i o n s  i t might  appear t h a t t h e f o e t a l n e u r o h y p o p h y s i s 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 t h e a m n i o t i c  f l u i d volume, b u t v e r y  little  i s known about t h e p o s s i b l e t a r g e t organs w h i c h might be concerned.  The p r i m a r y a c t i o n o f the a n t i d i u r e t i c hormone  i n t h e a d u l t would s u g g e s t t h a t t h e f o e t a l k i d n e y c o u l d  be  a p o s s i b l e t a r g e t o r g a n f o r t h e a c t i o n o f the embryonic antidiuretic principle.  However, H e l l e r (1949) w o r k i n g  w i t h b o t h newborn humans and  r a t s , f a i l e d to e l i c i t  a d i u r e t i c response t o waterload, to dehydration.  On  o r an a n t i d i u r e t i c r e s p o n s e  the o t h e r hand, P a u l i n e  (1962) r e c o r d e d  t h a t h y d r a t i o n o f the f o e t a l sheep a t 130 days o f r e s u l t e d i n an i n c r e a s e d elevated urine flow. the elevated level.  either  glomerular  gestation  f i l t r a t i o n rate  and  I n j e c t i o n s of vasopressin brought both  f i l t r a t i o n r a t e and u r i n e f l o w b a c k t o a c o n t r o l  However, v a s o p r e s s i n was  found t o be w i t h o u t e f f e c t  by b o t h the above w o r k e r s i n a n i m a l s where f i l t r a t i o n and u r i n e f l o w have not been p r e v i o u s l y e l e v a t e d . a t no t i m e promoted the f o r m a t i o n immature a n i m a l s .  of hypertonic  On t h e b a s i s o f t h e s e and  rate  Vasopressin  urine i n  several  other  e x p e r i m e n t s P a u l i n e c o n c l u d e d t h a t t h e immature k i d n e y i s unable to concentrate  i t s u r i n e , and  attributes this failure  88  to  an e l e c t r o l y t e r e a b s o r p t i o n b y t h e immature k i d n e y ,  exceeds t h e r e a b s o r p t i o n o f w a t e r .  which  However, i n p a s s i n g , i t  s h o u l d b e p o i n t e d o u t t h a t t h e f a i l u r e t o produce h y 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 i m p o s s i b l e t o produce a s h o r t a g e o f w a t e r i n t h e f o e t u s , w h i c h can draw a l l i t needs from-the m a t e r n a l organism.  F u r t h e r m o r e , t h e d i s c h a r g e o f such a h y p e r t o n i c  u r i n e i n t o t h e embryonic f l u i d compartments would appear t o be q u e s t i o n a b l e b e n e f i t f o r b o t h t h e mother and t h e f o e t u s . However, t h e p r o d u c t i o n o f h y p e r t o n i c u r i n e i s n o t t h e o n l y form o f w a t e r r e g u l a t i o n a v a i l a b l e t o an a n i m a l . F o r example,  t h e amphibian may u t i l i s e w a t e r t r a n s p o r t t h r o u g h  t h e s k i n and t h e b l a d d e r  (Sawyer, 1967).  Nor a r e t h e  membranes o f t h e k i d n e y t h e 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 w a t e r r e a b s o r p t i o n ;  this i s  shown b y t h e a c t i o n o f t h e p e p t i d e on t h e membranes o f t h e p a n c r e a s , l i v e r and sweat g l a n d s , where i t reduces t h e volume o f s e c r e t i o n (Wakim, 1967). In t h e f o e t a l o r g a n i s m t o o , t h e r e a r e 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 o f t h e 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 brief description  o f t h e p r o d u c t i o n o f t h e a m n i o t i c f l u i d , and t h e 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 t h e a n t i d i u r e t i c agent. In  f o e t a l sheep o f l e s s than 90 days o f g e s t a t i o n a l  age, u r i n e from t h e b l a d d e r p a s s e s i n t o t h e a l l a n t o i c s a c . Beyond t h i s age, t h e u r i n e p a s s e s i n t o t h e a m n i o t i c s a c i n  p r o g r e s s i v e l y g r e a t e r amounts ( P a u l i n e , Nixon,  1962).  The  r a t e o f f o e t a l u r i n e p r o d u c t i o n i s more than adequate t o a c c o u n t f o r the volume o f embryonic f l u i d , b u t i t i s b y  no  means t o t a l l y r e s p o n s i b l e f o r t h e p r o d u c t i o n and maintenance o f t h e f l u i d environment o f the f o e t u s .  In the sheep, t h e  b u c c a l c a v i t y and the f o e t a l l u n g s can s e c r e t e up t o 15 ml f l u i d p e r h o u r (Reynolds, fluid  1953) .  The  of  swallowing of amniotic  i s a l s o known t o o c c u r , and a b s o r p t i o n from t h e  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 t o t h e exchange o f w a t e r and e l e c t r o l y t e s between t h e f o e t u s and t h e embryonic fluids. Therefore, neurohypophysial  t h e p o s s i b l e t a r g e t organs o f t h e embryonic  p e p t i d e s c o u l d i n c l u d e the a l l a n t o i c and/or  t h e a m n i o t i c membranes o u t s i d e t h e f o e t u s , o r t h e l u n g s 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 o r g a n i s m . evidence  and  There i s no  i n t h e l i t e r a t u r e t o i n d i c a t e whether any o f  these  membranes c o u l d respond t o v a s o p r e s s i n o r t o f o e t a l neurohypophysial  extract.  More r e s e a r c h w i l l have t o be done  b e f o r e t h e g r e a t abundance o f a n t i d i u r e t i c p r i n c i p l e r e p o r t e d h e r e can be f i t t e d  i n t o t h e g e n e r a l p i c t u r e o f the embryonic  development, and t h e p h y s i o l o g y o f t h e f o e t u s .  However, the  g r e a t number o f 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 o f the f o e t u s t o 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 i m p o r t a n t t o the o r g a n i s m d u r i n g its intrauterine 3.  life.  The  importance o f the p r e s e n c e o f 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 f o e t u s e s , 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 t h e 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, arginine vasotocin, Firstly,  i s i m p o r t a n t f o r more than one r e a s o n .  i t i s t h e f i r s t t i m e 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 o f l o w e r 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 o f t h e f o e t u s a t t h e e a r l i e r s t a g e s o f embryonic development c o u l d be r e s p o n s i v e vasotocin,  r a t h e r than t o a r g i n i n e  to arginine  vasopressin.  A r g i n i n e v a s o t o c i n promotes w a t e r r e a b s o r p t i o n i n t h e amphibian k i d n e y , the  s k i n and b l a d d e r  and a l s o r e g u l a t e s w a t e r uptake t h r o u g h o f c e r t a i n amphibians (Sawyer, 1967).  I t m i g h t be s p e c u l a t e d  that the action of arginine  vasotocin  on e x t r a r e n a l membranes c o u l d be o f s p e c i a l r e l e v a n c e  to the  f o e t u s , where i t m i g h t promote w a t e r movement through t h e embryonic membranes, t h r o u g h t h e l u n g s o r t h r o u g h t h e b u c c a l cavity.  Further,  arginine vasotocin  i t may be o f some s i g n i f i c a n c e , t h a t increases the e l e c t r o l y t e excretion  influences the glomerular c e r t a i n other species.  f i l t r a t i o n r a t e o f l u n g f i s h and  These two l a t t e r a c t i o n s o f a r g i n i n e  v a s o t o c i n might be i m p o r t a n t t o t h e f o e t a l organism, that e l e c t r o l y t e reabsorption reabsorption,  and  considering  i n t h e f o e t u s exceeds w a t e r  and t h a t r e g u l a t i o n o f t h e volume o f t h e  produced u r i n e c o u l d be demonstrated on t h e l e v e l o f changes i n the glomerular  f i l t r a t i o n rate only.  Arginine  vasotocin  that could p o s s i b l y a i d i n the regulatory processes  involved  91  i n the amniotic f l u i d formation,  and t h e e l e c t r o l y t e  balance  of the e a r l y foetus. However, t h e d e m o n s t r a t i o n  o f the presence o f  a r g i n i n e v a s o t o c i n i n mammalian f o e t u s e s , has a n o t h e r significance.  I t i s important  i n considerations ofthe  phylogenetic d i s t r i b u t i o n o f the neurohypophysial  anti-  diuretic principle. The n e u r o h y p o p h y s i s 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 u t e r u s 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 diuretic principle.  In the oxytocin 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 g r e a t many m u t a t i o n s appear t o have occurred during evolution.  The s t r u c t u r e o f t h e a c t i v e o x y t o c i c  p r i n c i p l e can v a r y n o t o n l y from one c l a s s o f v e r t e b r a t e s t o a n o t h e r , b u t a l s o between s p e c i e s w i t h i n t h e same c l a s s (Sawyer, 1967). On t h e o t h e r 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 o f vertebrates.  There i s e v i d e n c e 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 e x c e p t f o r t h e 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 V e r s t e e g  (1967) have reviewed t h e  e v o l u t i o n a r y aspects o f the neurohypophysial  p e p t i d e s and t h e y  have p o i n t e d o u t t h a t t h e C y c l o s t o m e s p o s s e s s o n l y one p e p t i d e , arginine vasotocin, but a l l other vertebrate classes 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 s u g g e s t t h a t t h e two  a c t i v e p e p t i d e s e v o l v e d b y a d o u b l i n g o f t h e v a s o t o c i n gene. V a s o t o c i n remained unchanged, b u t f u r t h e r m u t a t i o n s took p l a c e  92  i n t h e n e u t r a l p e p t i d e , and produced a s e r i e s o f 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 , t h e a n t i d i u r e t i c hormone o f most mammals. I n the S u i n a , a f u r t h e r m u t a t i o n  replaced arginine with  lysine  to p r o d u c e l y s i n e v a s o p r e s s i n . The p r o b a b l e p r e s e n c e o f a r g i n i n e v a s o t o c i n i n mammalian f o e t u s e s , as found h e r e , does not f i t the p i c t u r e of neurohypophysial  e v o l u t i o n w h i c h i s accepted  at present.  The p r e s e n c e o f t h r e e a c t i v e agents i n t h e f o e t u s , c o n t r a d i c t s the theory t h a t a p o i n t mutation arginine vasopressin.  i n the v a s o t o c i n gene produced  Such a p o i n t m u t a t i o n would s u g g e s t the  c o m p l e t e d i s a p p e a r a n c e o f the p a r e n t  gene.  A p a r a l l e l presence  o f 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 d o u b l i n g o f t h e v a s o t o c i n gene, and occurred,  i f such d o u b l i n g o f t h e gene  indeed  some neurons o f t h e mammalian n e u r o h y p o p h y s i s m i g h t  r e t a i n the c a p a c i t y t o 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 also. The d e c r e a s e i n t h e t o t a l amount o f 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 t h e end o f i n t r a u t e r i n e l i f e ,  as was  found i n t h e sheep  and d i s c u s s e d e a r l i e r , might be t h e r e s u l t o f t h e f o l l o w i n g possibilities.  Firstly,  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 o f 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 t h e p r o d u c t 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.  W i t h the advancement o f g e s t a t i o n , p o s s i b l y t h e  gene p r o d u c i n g  t h e a n t i d i u r e t i c agent o f the. a d u l t mammalian  n e u r o h y p o p h y s i s c o u l d become dominant.  Secondly, the  foetal  93  membranes c o u l d 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 m i g h t be i n f a v o u r o f t h e p r o d u c t i o n o f t h i s agent. embryonic l i f e , organs may  In l a t e r  and a f t e r b i r t h , t h e mammalian t a r g e t  become s e n s i t i v e t o v a s o p r e s s i n , and  the  p h y s i o l o g i c a l s t i m u l i c o u l d promote t h e p r o d u c t i o n arginine vasopressin. consequently  The v a s o t o c i n p r o d u c i n g  of  neurons c o u l d  e i t h e r d e g e n e r a t e , o r t h e y c o u l d produce such  s m a l l q u a n t i t i e s t h a t the p r e s e n c e o f a r g i n i n e v a s o t o c i n m i g h t n o t be d e t e c t a b l e by c o n v e n t i o n a l s t u d i e s . In passing i t i s worthwhile presence of three neurohypophysial  t o p o i n t out t h a t t h e p e p t i d e s was  also  d e m o n s t r a t e d i n some o f the S u i n a , where some s p e c i e s produce b o t h 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 , i n a d d i t i o n t o o x y t o c i n ( H e l l e r , 1966). the simultanous  However, t h i s s i t u a t i o n i s n o t analogous t o p r e s e n c e o f a r g i n i n e v a s o p r e s s i n and  v a s o t o c i n i n t h e f o e t u s , s i n c e the p r e s e n c e o f the vasopressins  arginine two  i n t h e p i g f a m i l y can be e x p l a i n e d by c r o s s -  b r e e d i n g between s p e c i e s e l a b o r a t i n g one or t h e o t h e r o f vasopressins,  and p r o d u c i n g  the  o f f s p r i n g s where b o t h the 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 p r o d u c i n g  genes s u r v i v e .  I n t h e case o f  t h e f o e t u s t h e p r e s e n c e o f t h e two a n t i d i u r e t i c p r i n c i p l e s c o u l d not be accounted f o r i n t h i s manner, s i n c e t h e r e i s no known a d u l t mammal t h a t has a n t i d i u r e t i c agent.  a r g i n i n e v a s o t o c i n as t h e a c t i v e  Although  only suggestions  can be made  a t t h e p r e s e n t as t o the p o s s i b l e g e n e t i c i m p l i c a t i o n s o f p r e s e n c e o f 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 o f a  the  mammalian foetus,  i t c o u l d become an important a i d t o the  understanding o f the e v o l u t i o n o f the neurohypophysial peptides.  95  SUMMARY  The p o s t e r i o r collected dried  p i t u i t a r i e s o f p r e g n a n t sheep were  t h r o u g h o u t two b r e e d i n g seasons, and were  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  either posterior  p i t u i t a r i e s were a n a l y s e s 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 t h e p u r i f i e d agents examined f o r t h e i r amino acid composition. (1)  A fall  The r e s u l t s o f t h e s e s t u d i e s have shown: 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 o c c u r r e d i n t h e d u r i n g pregnancy.  The l o s s o f a c t i v i t y was  neurohypophyses found t o be more  marked i n t h e e a r l i e r (90 days) s t a g e o f pregnancy, t h a n i n the  later  (140 days) s t a g e .  A t t h e time o f n a t u r a l  delivery,  a v e r y marked i n c r e a s e i n t h e o x y t o c i c a c t i v i t y o f t h e posterior  p i t u i t a r y o f t h e mother was d e t e c t e d . (2)  The p a t t e r n o f changes i n the b i o l o g i c a l  a c t i v i t i e s t h r o u g h o u t pregnancy were s i m i l a r i n t h e acetone dried  and l y o p h i l i s e d t i s s u e s , b u t acetone t r e a t m e n t  resulted  i n a g r e a t e r l o s s o f the b i o l o g i c a l a c t i v i t i e s from  the neurohypophyses (3)  of pregnant  sheep.  I n t h e f o e t u s , w i t h t h e advancement o f g e s t a t i o n ,  an a c c u m u l a t i o n o f a l l a c t i v e p r i n c i p l e s o f t h e neurohypophyses  was  secondary f a l l (4)  found.  A t around t h e t i m e o f b i r t h a  i n p o t e n c y was d e t e c t e d .  The v a s o p r e s s o r and a n t i d i u r e t i c a c t i v i t i e s  96  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 a l l through g e s t a t i o n as was  shown b y the l a r g e V/0 and A/0 r a t i o s .  The  relatively  g r e a t e r accumulation o f the o x y t o c i c moiety, d u r i n g the l a t t e r h a l f o f embryonic development,  r e s u l t e d i n the d e c l i n i n g  V/0  and A/0 r a t i o s a t the l a t e r stages o f i n t r a u t e r i n e l i f e ,  and  early post natal (6)  age.  In the acetone t r e a t e d glands, b i o l o g i c a l  a c t i v i t i e s were found t o be lower f o r o x y t o c i c v a s o p r e s s o r and a n t i d i u r e t 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 g l a n d s .  The l o s s o f a c t i v i t y from the acetone t r e a t e d t i s s u e was  o f the  same magnitude f o r both o x y t o c i c and v a s o p r e s s o r a c t i v i t i e s , b u t v a r i e d from one stage o f development (7) was  to the o t h e r .  A new m o d i f i c a t i o n o f the g e l f i l t r a t i o n method  employed, t o a c h i e v e a one s t e p p u r i f i c a t i o n o f 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 t h e crude neurohypophyseal e x t r a c t s of the pregnant sheep, and sheep (8)  foetuses.  The amino a c i d a n a l y s i s o f the p u r i f i e d  neurohypophyseal agents have i n d i c a t e d t h a t these 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 . (9)  Paper chromatographic s t u d i e s o f the neuro-  hypophyseal e x t r a c t s o f e a r l y  (90 days)  f o e t u s e s again  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 agents o f these glands. 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