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Melanocyte-stimulating activity in the pituitary, blood plasma and amniotic fluid of the fetal guinea… Bryant, Harold Norman William 1978

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MELANOCYTE-STIMULATING ACTIVITY IN THE PITUITARY, BLOOD PLASMA AND AMNIOTIC FLUID OF THE FETAL GUINEA PIG by HAROLD NORMAN WILLIAM BRYANT B . S c , U n i v e r s i t y o f Guelph, 197^ A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department o f Zoology We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Oc t o b e r , 1978 © H a r o l d Norman W i l l i a m B r y a n t , 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoology The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V5T 1W5 Date October 18. 1978 i i ABSTRACT M e l a n o c y t e - s t i m u l a t i n g a c t i v i t i e s were measured i n the f e t a l g u i n e a p i g i n o r d e r t o determine whether m e l a n o c y t e -s t i m u l a t i n g hormone (MSH) was p r e s e n t over the l a t t e r h a l f o f g e s t a t i o n . A c t i v i t y was measured by b i o a s s a y u s i n g s k i n of the l i z a r d , A n o l i s c a r o l i n e n s i s , i n v i t r o . 'The a b i l i t y of unknown samples t o darken the s k i n was measured i n e q u i v -a l e n t amounts o f s y n t h e t i c alpha-MSH. P i t u i t a r y a c t i v i t y was measured i n b o t h the p o s t e r i o r and a n t e r i o r l o b e s i n f e t u s e s from 39 t o 70 days o f g e s t a t i o n . P o s t e r i o r p i t u i t a r y v a l u e s ranged from 18.0 ng/lobe at 39 days t o 1 0 8 l ng/lobe at 70 days and from 288 ng/mg a t 39 days t o 2962 ng/mg a t 70 days. A n t e r i o r lobe a c t i v i t y ranged from 10.5 ng/lobe a t 42 days t o 403 ng/lobe a t 70 days and from 42.4 ng/ mg a t 55 days t o 523 ng/mg at 70 days. Whole p i t u i t a r y a c t i -v i t y ranged from 28.9 ng/gland at 39 days t o 1484 ng/gland a t 70 days and from 143 ng/mg a t 55 days t o 1308 ng/mg a t 70 days. I n a l l c a s e s , t h e r e was a c l e a r l i n e a r r e l a t i o n s h i p between age i n days and the l o g o f the measured a c t i v i t y b o t h i n ng/ g l a n d and i n ng/mg. The average r a t i o o f p o s t e r i o r lobe t o a n t e r i o r lobe a c t i v i t y was 3-0 (per l o b e ) or 6.7 (per w e i g h t ) . A c t i v i t y i n a l i t t e r of newborns agreed c l o s e l y w i t h a c t i v i t i e s i n f e t u s e s o f the same p o s t - c o i t u m age. The average p i t u i t a r y a c t i v i t y i n f o u r a d u l t s was 8620 ± 1300 ng/gland o r 2600 ± 590 ng/mg (mean ± s t a n d a r d e r r o r o f the mean). A c t i v i t y i n f e t a l b r a i n t i s s u e was 1.36 ± 0.24 ng/mg and i i i i n one sample of a d u l t b r a i n t i s s u e was 0.75 ng/mg. M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y was measured i n 1 3 p o o l e d samples o f f e t a l plasma. Age. ranged from 45 t o 70 days of g e s t a t i o n . Average plasma a c t i v i t y was 1 0 . 0 ± 1 . 6 ng/ml and a c t i v i t y d i d not appear t o change w i t h i n c r e a s i n g g e s t a t i o n a l age. A c t i v i t y i n the plasma i n f o u r out of f i v e pregnant g u i n e a p i g s was 1 2 . 1 ± 2.8 ng/ml. I n the f i f t h a n i m a l no measurable a c t i v i t y was p r e s e n t . A c t i v i t y i n normal a d u l t females was 7.9 ± 2.2 ng/ml. There was no s i g n i f i c a n t d i f -f e r e n c e between the a c t i v i t y i n the two a d u l t groups ( S t u d e n t ' s t t e s t , u n p a i r e d o b s e r v a t i o n s , v a r i a n c e s e q u a l ) . There was a l s o no s i g n i f i c a n t d i f f e r e n c e between e i t h e r a d u l t group and the plasma a c t i v i t y I n the f e t u s . Plasma a c t i v i t y i n newborns was 1.92 ± 0.63 ng/ml. Thus an apparent drop i n a c t i v i t y o c c u r s j u s t a f t e r b i r t h . However, the a c t i v i t y was o n l y s i g n i f i c a n t l y d i f f e r e n t from l e v e l s i n e i t h e r a d u l t s o r the f e t u s a t P<0.1. A m n i o t i c f l u i d m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y was meas-ured i n 30 samples t a k e n from f e t u s e s r a n g i n g i n age from 42 to 67 days of g e s t a t i o n . A c t i v i t y was d e t e c t e d i n 10 samples, but was o n l y measurable i n e i g h t . A c t i v i t y was 79-4 ± 1 4 . 3 pg/ml. No changes o c c u r r e d w i t h i n c r e a s i n g g e s t a t i o n a l age. I n c u b a t i o n of samples w i t h t r y p s i n i n d i c a t e d t h a t a p r o t e i n was i n v o l v e d . F o u r t e e n o t h e r a m n i o t i c f l u i d samples had a l i g h t e n i n g e f f e c t on the A n o l i s s k i n . The substance r e s p -o n s i b l e f o r t h i s a c t i v i t y was not i d e n t i f i e d . M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i s found i n h i g h l e v e l s i v i n the g u i n e a p i g f e t u s . T h i s s t u d y p r o v i d e s the f i r s t comp-r e h e n s i v e measurement of the dynamics of m e l a n o c y t e - s t i m u l -a t i n g a c t i v i t y i n the f e t u s over the l a t t e r p a r t o f g e s t a t i o n i n b o t h the p i t u i t a r y and the plasma. A c t i v i t y i s a l s o p r e s -ent i n newborns and i n a d u l t s . I d e n t i f i c a t i o n o f s p e c i f i c p e p t i d e s must await f u r t h e r s t u d y . T h i s study s u p p o r t s e v i -dence c o l l e c t e d i n o t h e r s p e c i e s t h a t MSH or r e l a t e d p e p t i d e s may be i m p o r t a n t i n f e t a l p h y s i o l o g y and/or pregnancy i n mammals. V TABLE OF CONTENTS Page LIST OF FIGURES i x ACKNOWLEDGEMENTS x INTRODUCTION 1 1. S t r u c t u r e of mammalian m e l a n o c y t e - s t i m u l a t i n g hormones 1 2. S t r u c t u r a l r e l a t i o n s h i p t o o t h e r p e p t i d e s 4 3. A n a t o m i c a l c o n s i d e r a t i o n s 5 4. MSH i n the p o i k i l o t h e r m s 9 5. E f f e c t s o f MSH i n the Mammalia 11 a. I n the a d u l t 11 b. I n the f e t u s 14 STATEMENT OF THE PROBLEM 17 MATERIALS AND METHODS 18 1. Animals 18 a. Guinea p i g s ( C a v i a p r o c e l l u s ) 18 b. A n o l i s c a r o l i n e n s i s 18 2. Sample c o l l e c t i o n and p r e p a r a t i o n 18 a. P i t u i t a r y and b r a i n t i s s u e 18 b. B l o o d plasma samples 19 c. A m n i o t i c f l u i d 20 3. B i o a s s a y f o r m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y 20 a. P r e p a r a t i o n o f the s k i n 21 b. Measurement o f t h e r e f l e c t i o n q u o t i e n t 21 c. C a l c u l a t i o n o f r e l a t i v e p otency and s t a t i s t i c a l methods 22 d. S p e c i a l i z e d p r o c e d u r e s f o r a m n i o t i c f l u i d .... 25 v i Page 4 . S t a n d a r d p r e p a r a t i o n 26 5 . S i l i c i c a c i d p r e t r e a t m e n t 26 6 . T r y p s i n i n c u b a t i o n 26 7 . S a l i n e s 27 a. L i z a r d R i n g e r ' s s o l u t i o n 27 b. Sodium phosphate b u f f e r 27 SECTION I : MELANOCYTE-STIMULATING ACTIVITY IN FETAL AND ADULT TISSUES 28 1. I n t r o d u c t i o n • 28 2 . R e s u l t s 29 a. M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n the f e t a l p i t u i t a r y 29 b. M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n the p i t u i t a r i e s of newborns 36 c. M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n a d u l t p i t u i t a r i e s 39 d. M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n b r a i n t i s s u e 39 3 . D i s c u s s i o n 40 a. S i g n i f i c a n c e of m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n f e t a l p i t u i t a r i e s 40 b. S i g n i f i c a n c e of m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n the b r a i n 43 SECTION I I : MELANOCYTE-STIMULATING ACTIVITY IN FETAL, ADULT AND NEWBORN BLOOD PLASMA 45 1 . I n t r o d u c t i o n 45 2 . R e s u l t s 47 a. Assay p r o c e d u r e s 47 b. M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n f e t a l plasma 47 v i i Page c. M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n the plasma of a d u l t s 50 d. M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n the plasma of newborns 53 3. D i s c u s s i o n 53 a. S i g n i f i c a n c e of m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n plasma 53 b. Source of m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n f e t a l plasma 56 c. Comments on m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y and plasma . : 57 SECTION I I I : A PRELIMINARY ESTIMATION OP MELANOCYTE-STIMULATING ACTIVITY IN AMNIOTIC FLUID ... 59 1. I n t r o d u c t i o n 59 2. R e s u l t s 60 a. E s t i m a t e d m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y of a m n i o t i c f l u i d 60 b. T r y p s i n i n c u b a t i o n of a m n i o t i c f l u i d samples . 62 3. D i s c u s s i o n 62 a. E v a l u a t i o n of the assay method 62 b. Nature and s i g n i f i c a n c e of me l a n o c y t e -s t i m u l a t i n g a c t i v i t y i n a m n i o t i c f l u i d 63 c. Source of m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n a m n i o t i c f l u i d 64 d. Source of m e l a n o c y t e - a g g r e g a t i o n response t o a m n i o t i c f l u i d 65 GENERAL DISCUSSION 68 1. Choice of assay 68 2. S i g n i f i c a n c e of p e p t i d e s r e l a t e d t o MSH 69 a. S i g n i f i c a n c e of p a r s i n t e r m e d i a ACTH 70 b. S i g n i f i c a n c e of p i t u i t a r y LPH 71 v i i i Page 3. Comparison of m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y l e v e l s 72 a. I n the g u i n e a p i g 72 b. Among d i f f e r e n t s p e c i e s 72 4. MSH i n f e t a l p h y s i o l o g y 73 5. C o n c l u s i o n 77 BIBLIOGRAPHY 78 i x LIST OF FIGURES F i g u r e F a c i n g Page 1. Comparison of the. s t r u c t u r e s of alpha-MSH and v a r i o u s forms of beta-MSH i n mammals 2 2. Comparison of the r e l a t e d s t r u c t u r e s of MSH, ACTH, CLIP and LPH p e p t i d e s 6 3. R e l a t i o n s h i p between the l o g a r i t h m of the dose of s t a n d a r d MSH and the c o l o u r change of A n o l i s s k i n i n v i t r o as measured by the r e f l e c t i o n ' q u o t i e n t . . 23 4. The r e l a t i o n s h i p between the age of the g u i n e a p i g f e t u s i n days of g e s t a t i o n and m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n the p o s t e r i o r p i t u i t a r y measured i n ng of a l p h a -MSH (per lobe or per mg) and p l o t t e d on a l o g s c a l e 30 5. The r e l a t i o n s h i p between the age of the g u i n e a p i g f e t u s i n days of g e s t a t i o n and m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n the a n t e r i o r p i t u i t a r y measured i n ng of a l p h a -MSH (per lobe or p e r mg) and p l o t t e d on a l o g s c a l e 32 6. The r e l a t i o n s h i p between the age of the g u i n e a p i g f e t u s i n days of g e s t a t i o n and the m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n the whole p i t u i t a r y g l a n d as measured i n ng of a l p h a -MSH ( p e r g l a n d o r per mg) and p l o t t e d on a l o g s c a l e 34 7. The r e l a t i o n s h i p between the age of the g u i n e a p i g f e t u s i n days of g e s t a t i o n and the dry weight of the p o s t e r i o r , a n t e r i o r and whole p i t u i t a r y i n mg 37 8. A comparison of the m e l a n o c y t e - s t i m u l a t i n g a c t -' i v i t i e s (ng of alpha-MSH/ml) i n the plasma of the normal female a d u l t , pregnant a d u l t , f e t u s and newborn g u i n e a p i g 48 9. The r e l a t i o n s h i p between the age of the g u i n e a p i g f e t u s , i n days of g e s t a t i o n , and the m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y of the plasma as measured i n ng of alpha-MSH per ml 51 X ACKNOWLEDGEMENTS I w i s h t o t h a n k D r . A.M. P e r k s f o r much a d v i c e a n d g u i -d a nce b o t h i n r e s e a r c h m e t h o d o l o g y a n d i n t h e w r i t i n g o f t h i s t h e s i s a n d f o r g e n e r o u s f i n a n c i a l s u p p o r t o f t h e r e s e a r c h i t s e l f . F i n a n c i a l s u p p o r t was s u p p l i e d i n p a r t by t h e N a t i o n a l R e s e a r c h C o u n c i l o f C a n a d a i n t h e f o r m o f a P o s t g r a d u a t e S c h o l a r s h i p f o r 1 9 7 4 - 7 5 -I am g r a t e f u l t o D r . P.A. D e s a u l l e s a n d D r . K. S c h e i b l i o f C i b a - G e i g y , B a s l e , S w i t z e r l a n d , f o r t h e k i n d g i f t o f s y n -t h e t i c a l p h a - M S H . Many members o f t h e D e p a r t m e n t o f Z o o l o g y w e re h e l p f u l i n many ways. I n p a r t i c u l a r : D r . H.D. F i s h e r , D r . W.S. H o a r and D r . J . E . P h i l l i p s f o r c r i t i c i s m o f t h e t h e s i s ; B i l l H o l t f o r h i s u n i q u e i n s i g h t ; a n d R a c h e l l e B o u f f a r d , M a r g a r e t M c D o n a l d a n d C a r o l i n e S m i t h f o r m o r a l s u p p o r t . I am a l s o most g r a t e f u l t o C o l l e e n H o l l i d a y f o r t h e t y -p i n g o f t h e t h e s i s and t o b o t h h e r and S t e v e De B e e r f o r a d -d i t i o n a l m o r a l s u p p o r t . 1 INTRODUCTION 1. S t r u c t u r e o f Mammalian M e l a n o c y t e - S t l m u l a t i n g Hormones M e l a n o c y t e - s t i m u l a t i n g hormones (MSH) a r e s m a l l s i n g l e c h a i n p o l y p e p t i d e s w i t h h i g h melanosome d i s p e r s i n g p o t e n c y a s m e a s u r e d by f r o g o r l i z a r d s k i n b i o a s s a y s . M e l a n o c y t e - s t i m -u l a t i n g a c t i v i t y i s p r e s e n t i n t h e p i t u i t a r y e x t r a c t s o f a l l mammalian s p e c i e s s t u d i e d . T h e r e a r e two b a s i c t y p e s o f MSH w h i c h h a v e b e e n d e s i g n a t e d t h e a l p h a and b e t a f o r m s . They d i f f e r b o t h i n p o t e n c y and i n m o l e c u l a r w e i g h t . W i t h one e x c e p t i o n , a l l mammalian MSH p e p t i d e s i s o l a t e d t o d a t e c o n t a i n t h e h e p t a p e p t i d e s e q u e n c e M e t - G l u - H i s - P h e - A r g -T r p - G l y , t h e c o r e s e q u e n c e r e q u i r e d f o r b i o l o g i c a l a c t i v i t y ( L a n de & L e r n e r , 1 9 6 7 ; R a m a c h a n d r a n & L i , 1 9 6 7 ) . A s i n g l e c hange o c c u r s i n a beta-MSH i n t h e c a m e l ( L i e_t a l , 1 9 7 5 ) • T h e i r s t r u c t u r e s a r e shown i n F i g u r e 1. Alpha-MSH i s a t r i d e c a p e p t i d e w i t h a n a c e t y l g r o u p b l o c k -i n g t h e amino t e r m i n a l s e r i n e , a n d a n a m i n a t e d v a l i n e r e s i d u e a t t h e c a r b o x y l t e r m i n a l . The a m i n o a c i d s e q u e n c e i s i d e n t i c a l i n a l l mammalian s p e c i e s s t u d i e d t o d a t e . I t i s a l s o t h e most p o t e n t o f a l l known m e l a n o c y t e - s t i m u l a t i n g s u b s t a n c e s (Ramachan-d r a n & L i , 1 9 6 7 ) . Alpha-MSH was i n i t i a l l y i s o l a t e d f r o m h o g ( S u s ) p i t u i t a r y g l a n d s ( L e r n e r & L e e , 1 9 5 5 ) . The p e p t i d e was s u b s e q u e n t l y i s o l a t e d f r o m p i t u i t a r i e s o f t h e cow ( B o s ) ( H a r r i s & L e r n e r , 1 9 5 7 ) , t h e s h e e p ( O v i s ) ( L e e et_ a l , 1 9 6 3 ) a n d t h e h o r s e ( E q u u s ) ( D i x o n & L i , i 9 6 0 ) . Alpha-MSH has n o t b e e n d e f -i n i t e l y i d e n t i f i e d i n any s i g n i f i c a n t amount i n a d u l t human p i t -u i t a r i e s ( H a r r i s , 1 9 5 9 ; Abe e t a l , 1 9 6 7 ) e x c e p t p o s s i b l y d u r i n g p r e g n a n c y ( V i s s e r & Swaab, 1 9 7 7 ) . R e c e n t l y , a l p h a - M S H has b e e n 2 F i g u r e 1: Comparison of the s t r u c t u r e s of alpha-MSH and v a r i o u s forms of beta-MSH i n mammals. Alpha-MSH i s s t r u c t u r a l l y c o n s i s t e n t i n a l l s p e c i e s from which i t has been i s o l a t e d . The h e p t a p e p t i d e core sequ-ence r e q u i r e d f o r b i o l o g i c a l a c t i v i t y i s u n d e r l i n e d . B e t a -MSH p e p t i d e s are named a c c o r d i n g t o the s p e c i e s from which they..were i n i t i a l l y i s o l a t e d . M u l t i p l e forms of beta-MSH have been i s o l a t e d from some s p e c i e s . The o c c u r r e n c e of g l u t a m i n e i n p o s i t i o n 8 of the second camel beta-MSH i s the on l y v a r i a t i o n observed t o date i n the h e p t a p e p t i d e core sequence. Alpha-MSH: CH -Ser--Try--Ser--Met--Glu--His--Phe- -Arg -Trp--Gly--Lys--Pro--Val--NH2 Beta-MSH P e p t i d e s : Human: A l a - G l u - L y s -Lys-Asp--Glu- Gly--Pro--Tyr--Arg--Met--Glu--His--Phe- -Arg -Trp--Gly--Ser--Pro--Pro- -Lys--Asp Monkey: Asp- -Glu- Gly--Pro--Tyr--Arg--Met--Glu--His--Phe- -Arg -Trp--Gly--Ser--Pro--Pro- -Lys--Asp P o r c i n e : Asp- -Glu-•Gly--Pro--Tyr--Lys--Met--Glu--His--Phe- -Arg -Trp--Gly--Ser--Pro--Pro- -Lys--Asp Equine: Asp- -Glu-•Gly--Pro--Tyr--Lys--Met--Glu--His--Phe- -Arg -Trp--Gly--Ser--Pro--Arg--Lys--Asp B o v i n e : Asp- -Ser-•Gly--Pro--Tyr--Lys--Met--Glu--His--Phe- -Arg -Trp--Gly--Ser--Pro--Pro- -Lys--Asp Camel (1) : Asp- -Gly-•Gly--Pro--Tyr--Lys--Met--Glu--His--Phe- -Arg -Trp--Gly--Ser--Pro--Pro- -Lys--Asp Camel (2) : Asp- -Gly-•Gly--Pro--Tyr--Lys--Met--Gln--His--Phe- -Arg -Trp--Gly--Ser--Pro--Pro- -Lys--Asp 4 i s o l a t e d from camel (Camelus) p i t u i t a r i e s ( L i e_t a l , 1 9 7 5 ) . Beta-MSH was i n i t i a l l y i s o l a t e d and c h a r a c t e r i z e d from p o r c i n e p i t u i t a r i e s (Geschwind e_t a l , 1 9 5 6 ) . T h i s p e p t i d e was found t o be 18 amino a c i d r e s i d u e s i n l e n g t h . I n comparison t o alpha-MSH, the amino t e r m i n a l was extended by t h r e e r e s i d u e s and the c a r b o x y l t e r m i n a l was extended by two. A l s o , c e r t a i n amino a c i d d i f f e r e n c e s were found i n the remainder of the pep-t i d e sequence. However, when beta-MSH was i s o l a t e d from b o v i n e p i t u i t a r i e s , a s e r i n e r e s i d u e was l o c a t e d i n p o s i t i o n 2 r a t h e r t h a n the glutamate found i n the p o r c i n e p e p t i d e (Geschwind et_ a l , 1 9 5 7 ) (amino a c i d r e s i d u e s are numbered from the amino term-i n a l end). Both of these p e p t i d e s were s u b s e q u e n t l y i s o l a t e d from o v i n e p i t u i t a r i e s (Lee e_t a l , 1 9 6 3 ) • Two beta-MSH pep-t i d e s were i s o l a t e d from equine g l a n d s ( D i x o n & L i , 1 9 6 1 ) ; one was unique t o t h i s s p e c i e s w h i l e the second was i d e n t i c a l t o p o r c i n e beta-MSH. E l e c t r o p h o r e t i c s t u d i e s have r e v e a l e d m u l t -i p l e forms of beta-MSH i n most mammals thus f a r s t u d i e d ( B u r g e r s , 1 9 6 3 ; H o e k s t r a & Van der Wal, 1 9 7 1 ) ; o n l y some of these p e p t i d e s appear t o be s i m i l a r t o those i s o l a t e d above. Human beta-MSH was o r i g i n a l l y i s o l a t e d as a s i d e f r a c t i o n d u r i n g the e x t r a c t -i o n of growth hormone ( D i x o n , I960). Four e x t r a r e s i d u e s were found at the amino t e r m i n a l r e s u l t i n g i n a t o t a l of 22 i n t h i s s p e c i e s . The m e l a n o c y t e - s t i m u l a t i n g p o t e n c y , p e r u n i t w e i g h t , of beta-MSH p e p t i d e s v a r i e s from about 12% t o about 97$ o f alpha-MSH potency (Ramachandran & L i , 1 9 6 7 ) . 2. S t r u c t u r a l R e l a t i o n s h i p t o Other P e p t i d e s The m e l a n o c y t e - s t i m u l a t i n g hormones b e l o n g t o a f a m i l y of n o n - c y s t e i n e c o n t a i n i n g p i t u i t a r y p e p t i d e s of s i m i l a r p r i m a r y 5 s t r u c t u r e a n d p o s s i b l y o f r e l a t e d b i o s y n t h e t i c o r i g i n ( L o w r y & S c o t t , 1 9 7 7 ) - The f a m i l y i n c l u d e s a d r e n o c o r t i c o t r o p i n (ACTH), c o r t i c o t r o p i n - l i k e i n t e r m e d i a t e l o b e p e p t i d e ( C L I P ) , b e t a -l i p o t r o p i n ( b e t a - L P H ) a nd g a m m a - l i p o t r o p i n (gamma-LPH). On d e t a i l e d e x a m i n a t i o n o f p r i m a r y s t r u c t u r e t h e y n a t u r a l l y f a l l i n t o two g r o u p s , e a c h w i t h an a p p a r e n t p r e c u r s o r m o l e c u l e . T h e s e s t r u c t u r a l r e l a t i o n s h i p s a r e shown i n F i g u r e 2. • The p o s s i b l e p a r e n t m o l e c u l e f o r one g r o u p i s ACTH. Alpha-MSH i s i d e n t i c a l t o r e s i d u e s 1 - 13 o f ACTH i n a l l v e r t -e b r a t e s p e c i e s s t u d i e d . C L I P i s i d e n t i c a l t o r e s i d u e s 18 - 39> t h e C - t e r m i n a l p o r t i o n o f t h e ACTH s e q u e n c e . B e t a - L P H I s t h e p a r e n t m o l e c u l e f o r t h e s e c o n d g r o u p . T h i s p e p t i d e c o n t a i n s 91 r e s i d u e s ; gamma-LPH r e p r e s e n t s r e s i -d ues 1 - 5 8 w h i l e beta-MSH i s i d e n t i c a l t o r e s i d u e s 41 - 5 8 . R e c e n t l y , e n k e p h a l i n ( b e t a - L P H 61 - 6 5 ) , a l p h a - e n d o r p h i n ( b e t a -LPH. 61 - 7 6 ) , g a m m a - e n d o r p h i n ( b e t a - L P H 6 l - 77) and b e t a -e n d o r p h i n ( b e t a - L P H 61 - 91) h a v e b e e n i s o l a t e d f r o m p i t u i t a r y e x t r a c t s ; t h e s e p e p t i d e s a r e a g o n i s t s o f o p i a t e r e c e p t o r s i n t h e b r a i n ( s e e r e v i e w by G u i l l e m i n et_ a l , 1 9 7 7 ) • The p h y s i o l o g i c a l r o l e o f ACTH i n t h e c o n t r o l a n d f u n c t i o n o f t h e a d r e n a l c o r t e x i s w e l l e s t a b l i s h e d . The l i p o t r o p i n s a r e named f o r t h e i r a b i l i t y t o m o b i l i z e s t o r e d l i p i d b u t t h i s a c t i -v i t y has n o t b e e n shown t o be p h y s i o l o g i c a l . B o t h LPH a n d ACTH h a v e a s m a l l d e g r e e o f m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y ( L i e_t a l , 1 9 6 5 ; L e e e t a l , 1 9 6 3 ) • 3. A n a t o m i c a l C o n s i d e r a t i o n s I n most mammals t h e p i t u i t a r y c o n t a i n s a d i s c r e t e p a r s i n t e r m e d i a ; i n t h i s c a s e MSH i s f o u n d p r e d o m i n a n t l y i n t h a t 6 F i g u r e 2: Comparison of the r e l a t e d s t r u c t u r e s of MSH, ACTH, CLIP and LPH p e p t i d e s . Through a comparison of p r i m a r y s t r u c t u r e , these p e p t i d e s n a t u r a l l y f a l l i n t o two groups. I n the f i r s t group, the sequence of alpha-MSH i s i d e n t i c a l t o r e s i d u e s 1 - 13 o f ACTH; CLIP i s i d e n t i c a l t o r e s i d u e s 18 - 39 of ACTH. I n the second group, gamma-LPH i s i d e n t i c a l t o r e s i d u e s 1 -58 of beta-LPH; beta-MSH i s i d e n t i c a l t o r e s i d u e s 41 -48 of b o t h gamma and beta-LPH. I t has been suggested t h a t ACTH and beta-LPH may a c t as p r e c u r s o r s f o r the o t h e r p e p t i d e s ( S c o t t & Lowry, 1 9 7 7 ) . ACTH Ser V a l Arg 1 13 18 Alpha-MSH Ser V a l 1 13 CLIP Arg 18 Beta-LPH GlU' 1 Gamma-LPH G l u 1 Beta-MSH Phe 39 Phe 39 -Asp : Asp-41 58 -Asp Asp 41 58 Asp Asp 41 58 -Glu 91 8 t i s s u e . ACTH i s f o u n d l a r g e l y i n t h e p a r s d i s t a l i s . The p a r s i n t e r m e d i a a n d p a r s d i s t a l i s a r e l o c a t e d i n t h e p o s t e r i o r a n d a n t e r i o r l o b e s o f t h e p i t u i t a r y r e s p e c t i v e l y . The l a r g e s t p a r t o f t h e p o s t e r i o r l o b e , h o w e v e r , i s p a r s n e r v o s a t i s s u e w h i c h i s d e v o i d o f e i t h e r hormone. The o r i g i n a l v i e w t h a t MSH and ACTH were s y n t h e s i z e d i n and r e l e a s e d f r o m s o l e l y t h e p a r s i n t e r m e d i a a n d p a r s d i s t a l i s r e s p e c t i v e l y i s t o o s i m p l i s t i c a t b e s t . B a k e r and Drummond ( 1 9 7 2 ) f o u n d t h a t p a r s i n t e r m e d i a c e l l s i n t h e r a t s t a i n e d i m m u n o c h e m i c a l l y w i t h a n t i - 1 7 - 3 9 ACTH a s w e l l a s a n t i - 1 - 2 4 ACTH. S i n c e c r o s s r e a c t i o n w i t h a l p h a - M S H c o u l d o n l y a c c o u n t f o r t h e l a t t e r r e a c t i o n , t h e r e s u l t s u g g e s t e d t h a t ACTH m i g h t be p r e s e n t i n p a r s i n t e r m e d i a c e l l s . A l s o , O r t h et_ a l ( 1 9 7 3 ) , f o u n d i m m u n o l o g i c a l b e t a - M S H - l i k e a c t i v i t y i n a c e l l l i n e o f an a n t e r i o r p i t u i t a r y t u m o u r i n t h e mouse. W h i l e s p e c i f i c s t u d i e s on a v a r i e t y o f s p e c i e s show much v a r i a t i o n , a g e n e r a l c o n s e n s u s s u g g e s t s t h a t b i o a c t i v e ACTH i s p r e s e n t i n b o t h t h e p a r s d i s t a l i s a n d p a r s i n t e r m e d i a w h i l e a l p h a - M S H a n d C L I P a r e r e s t r i c t e d t o t h e p a r s i n t e r m e d i a . B o t h p a r s i n t e r m e d i a c e l l s a n d c o r t i c o t r o p h s o f t h e p a r s d i s t a l i s c o n t a i n beta-MSH and b e t a - L P H i m m u n o r e a c t i v i t y ( L o w r y & S c o t t , 1 9 7 7 ) . P e p t i d e d i s t r i b u t i o n i n human p i t u i t a r i e s i s c o n s i s t e n t w i t h t h i s g e n e r a l scheme. W h i l e t h e human f e t u s p o s s e s s e s a d i s c r e t e p a r s i n t e r m e d i a , t h i s g l a n d r e g r e s s e s a t b i r t h a n d , i n t h e a d u l t , i t i s r e p r e s e n t e d by i s o l a t e d c e l l s t h a t i n v a d e t h e p a r s n e r v o s a . T h u s , i n t h e a d u l t p i t u i t a r y , a l p h a - M S H a c t i v i t y h a s b e e n n o t e d o n l y i n a s m a l l number o f A C T H - i m m u n o r e a c t i v e c e l l s t h a t i n v a d e d t h e p o s t e r i o r l o b e , o r were l o c a t e d a l o n g i t s b o r d e r ( N I e u w e n h u i j z e n - K r u s e m a n & S c h r o d e r - v a n d e r E l s t , 1 9 7 6 ) . Beta-MSH i m m u n o r e a c t i v e m a t e r i a l i s f o u n d s o l e l y i n t h e c o r t i c o t r o p h s o f t h e p a r s d i s t a l i s ( P h i f e r e_t a l , 1 9 7 4 ) . I n t h e f e t a l p i t u i t a r y , h o w e v e r , t h e p a r s i n t e r m e d i a c o n t a i n s a s i g n i f i c a n t amount o f a l p h a - M S H a c t i v i t y ( S i l m a n et_ a l , 1 9 7 6 ; V i s s e r & Swaab, 1 9 7 7 ) . I n t h e g u i n e a p i g , t h e s p e c i e s u s e d i n t h i s s t u d y , t h e p a r s i n t e r m e d i a i s a d i s c r e t e t i s s u e i n t h e a d u l t a n d a d h e r e s c l o s e l y t o t h e p a r s n e r v o s a i n t h e p o s t e r i o r l o b e ( A r o n & P e t r o v i c , 1 9 5 9 ) . The p a r s i n t e r m e d i a o f t h e g u i n e a p i g c o n t -a i n s b o t h i m m u n o r e a c t i v e ACTH and a l p h a - M S H , a p p a r e n t l y i n t h e same c e l l s (Tramu & D u b o i s , 1 9 7 7 ) . T h u s , a s i n o t h e r mammals, MSH i s n o t t h e o n l y m e l a n o c y t e - s t i m u l a t i n g p e p t i d e f o u n d i n t h e p a r s i n t e r m e d i a o f t h e p i t u i t a r y . I n a d d i t i o n , s i g n i f i c a n t am-o u n t s o f MSH may be p r e s e n t i n t h e p a r s d i s t a l i s . 4. MSH i n t h e P o i k i l o t h e r m s The most h i g h l y d o c u m e n t e d e f f e c t o f mammalian m e l a n o c y t e -s t i m u l a t i n g p e p t i d e s i s t h a t o f d i s p e r s i o n o f p i g m e n t g r a n u l e s ( m e l a n o s o m e s ) i n t h e m e l a n o p h o r e s o f t h e s k i n o r s c a l e s o f v a r -i o u s l o w e r v e r t e b r a t e s . D i s p e r s a l o f t h e melanosomes r e s u l t s i n a d a r k e n i n g o f s k i n hue a n d o f t e n a n a c t u a l c o l o u r c h a n g e . I n 1 9 1 9 , A t w e l l f i r s t f o u n d t h a t h y p o p h y s e c t o m i z e d t a d p o l e s i m -m e r s e d i n b o v i n e e x t r a c t s o f p a r s i n t e r m e d i a q u i c k l y d a r k e n e d . P e r m a n e n t m o d i f i c a t i o n o f t h e p i g m e n t a r y s y s t e m by r e m o v a l o f t h e p i t u i t a r y g l a n d was d e m o n s t r a t e d by S m i t h ( 1 9 2 0 ) . Hogben a n d Slome ( 1 9 3 6 ) showed t h a t a d a p t i v e d a r k e n i n g i n r e s p o n s e t o d a r k b a c k g r o u n d was d e p e n d e n t on r e t i n a l s t i m u l a t i o n . I t was p r o p o s e d t h a t r e t i n a l s t i m u l a t i o n c a u s e d t h e b r a i n t o r e l e a s e 10 a hormone f r o m t h e p a r s i n t e r m e d i a , l a t e r t e r m e d m e l a n o c y t e -s t i m u l a t i n g hormone, w h i c h d i r e c t l y m e d i a t e d a d a p t i v e c o l o u r c h a n g e s v i a d i s p e r s i o n o f p i g m e n t i n t h e m e l a n o p h o r e s . A s i m i l a r s i t u a t i o n e x i s t s i n f i s h . H y p o p h y s e c t o m y r e s u l t s i n b l a n c h i n g i n t h e d o g f i s h a n d r e - d a r k e n i n g o c c u r s w i t h p i t u i t a r y e x t r a c t t r e a t m e n t ( L u n d s t r o m & B a r d , 1 9 3 2 ) . W h i l e c o l o u r change i s l a r g e l y u n d e r h u m o u r a l c o n t r o l i n t h e e l a s m o b r a n c h s , n e u r a l c o n t r o l i s a l s o i n v o l v e d i n many t e l e o s t s ( L a n d g r e b e & M i t c h e l l , 1 9 6 6 ; B a k e r & B a l l , 1 9 7 5 ) . S p e c i e s s p e c i f i c d i f f e r e n c e s h ave a l s o b e e n o b s e r v e d i n t h e R e p t i l i a . C o l o u r c h a n g e i n A n o l i s c a r o l i n e n s i s i s e x c l -u s i v e l y r e g u l a t e d by h o r m o n a l f a c t o r s ( K l e i n h o l z , 1 9 3 8 ) w h i l e t h e c h a n g e s s e e n i n C h a maeleo a r e u n d e r c o m p l e t e n e u r a l c o n t -r o l (Hogben & M i r v i s h , 1 9 2 8 ) . P r o l o n g e d MSH s t i m u l a t i o n r e s u l t s i n a n i n c r e a s e i n b o t h t h e m e l a n i n c o n t e n t a n d t h e a b s o l u t e number o f m e l a n o p h o r e s i n Xenopus ( L a n d g r e b e & W a r i n g , 1 9 4 4 ) . L o s s o f t h e n e u r o i n t e r -m e d i a t e l o b e , i n t h e d o g f i s h , S c y l i o r h i n u s c a n i c u l u s , r e s u l t s i n f e w e r m e l a n o p h o r e s a n d , i n some c a s e s , a t o t a l l o s s o f p i g m e n t ( W i l s o n & Dodd, 1 9 7 3 ) . T h r e e m e l a n o c y t e - s t i m u l a t i n g c o mponents were s e p a r a t e d by e l e c t r o p h o r e s i s on s t a r c h f r o m t h e p i t u i t a r y e x t r a c t s o f b o t h t h e b u l l f r o g ( Rana c a t e s b e i a n a ) and t h e l i z a r d ( A n o l i s  c a r o l i n e n s i s ) ( B u r g e r s , 1 9 6 3 ) • A t r i d e c a p e p t i d e , v e r y s i m i l a r i n s e q u e n c e t o mammalian a l p h a - M S H , has b e e n i s o l a t e d f r o m p i t u i t a r y g l a n d e x t r a c t s o f t h e d o g f i s h S q u a l u s a c a n t h i a s ( L o w r y & C h a d w i c k , 1 9 7 0 ; B e n n e t t e t a l , 1 9 7 4 ) . A b e t a - M S H -l i k e p e p t i d e has a l s o b e e n i s o l a t e d f r o m t h i s s p e c i e s ( B e n n e t t 11 e t a l , 1 9 7 4 ) a n d f r o m S c y l i o r h i n u s c a n l c u l u s ( L o v e & P i c k e r i n g , 1 9 7 4 ) . The s e q u e n c e s o f t h e s e m e l a n o c y t e - s t i m u l a t i n g p e p t i d e s a r e r e m a r k a b l y s i m i l a r t o mammalian p e p t i d e s , e s p e c i a l l y i n t h e c a s e o f t h e a l p h a - M S H - l i k e p e p t i d e . 5. E f f e c t s o f MSH i n t h e Mammalia a. I n t h e A d u l t A l a r g e amount o f e v i d e n c e has a c c u m u l a t e d w i t h r e s p e c t t o t h e p o s s i b l e t r o p h i c e f f e c t s o f MSH i n t h e Mammalia b u t no c l e a r p h y s i o l o g i c a l f u n c t i o n f o r t h e s e p e p t i d e s has b e e n e s t a b l i s h e d t o d a t e . S e v e r a l d e t a i l e d r e v i e w s h a v e a p p e a r e d f a i r l y r e c e n t l y (Howe, 1 9 7 3 ; N o v a l e s , 1 9 7 4 ) . A number o f c l i n i c a l c o n d i t i o n s o f h y p e r p i g m e n t a t i o n a r e a s s o c i a t e d w i t h h i g h MSH l e v e l s a n d l a r g e i n j e c t i o n s o f p e p t i d e c a n c a u s e s k i n d a r k e n i n g i n man ( M c G u i r e & L e r n e r , 1 9 6 3 ) • The c h l o a s m a o f p r e g n a n c y may be a s s o c i a t e d w i t h t h e e l e v a t e d beta-MSH c o n c e n t r a t i o n s o b s e r v e d t o w a r d s t e r m ( S h i z u m e & L e r n e r , 1 9 5 4 ) . I n t h e w e a s e l ( M u s t e l a e r m i n e a b a n g s i ) , MSH a p p e a r s t o p l a y a r o l e i n t h e n o r m a l r e g u l a t i o n o f c o l o u r p e l a g e c y c l e s ( R u s t , 1 9 6 5 ) and c o a t c o l o u r d a r k e n i n g i s o b s e r v e d i n m i c e b e a r i n g M S H - s e c r e t i n g p i t u i t a r y t u m o u r s ( G e s c h w i n d & H u s e b y , 1 9 6 8 ) . However i t i s u n l i k e l y t h a t t h e i s o l a t e d and s p o r a d i c p i g m e n t a r y e f f e c t s due t o MSH n o t e d i n mammals a r e i n d i c a t i v e o f g e n e r a l p h y s i o l o g i c a l f u n c t i o n i n g . C e r t a i n e x t r a p i g m e n t a r y a c t i o n s o f MSH a r e r e l a t e d t o i t s s t r u c t u r a l s i m i l a r i t y t o o t h e r h o r m o n e s , most n o t a b l y ACTH. Th e s e i n c l u d e i t s s t e r o i d o g e n i c a c t i o n , l i p o l y t i c a c t i o n , h y p o c a l c e m i c a c t i o n , a q u e o u s f l a r e r e s p o n s e , p o s i t i v e c h r o n o -t r o p i c a c t i o n , i n d u c t i o n o f s t r e t c h i n g and y a w n i n g and a c t i o n s 12 on c o n d i t i o n e d r e f l e x e s (see N o v a l e s , 1 9 7 4 ) . S t u d i e s r e g a r d i n g a p o s s i b l e s t e r o i d o g e n i c a c t i o n of MSH a r e ' c o n t r a d i c t o r y (see Howe, 1 9 7 3 ) . D e s a u l l e s e t a l ( 1 9 6 6 ) found t h a t s y n t h e t i c alpha-MSH had a s i g n i f i c a n t e f f e c t on c o r t i c o s t e r o n e p r o d u c t i o n i n v i t r o but had on l y 0.25$ of the a c t i v i t y o f ACTH. I n v i v o , alpha-MSH, a t doses o f 3 ug/kg, d i d have a s i g n i f i c a n t e f f e c t on the r e l e a s e of c o r t i c o s t e r o n e i n the hypophysectomized r a t but had no e f f e c t on a d r e n a l a s c o r b i c a c i d l e v e l s . MSH appears t o e x h i b i t c e r t a i n t h y r o t r o p i c a c t i o n s . MSH I n c r e a s e s the r a t e of i n c o r p o r a t i o n of i o d i n e i n t o the t h y r o i d and h i s t o l o g i c a l e v i d e n c e I n d i c a t e s t h a t t h y r o i d a c t i v i t y i s i n c r e a s e d . I t i s u n c l e a r whether MSH a c t s d i r e c t l y or by s t i m -u l a t i n g TSH s e c r e t i o n (see C e h o v i c , 1 9 6 7 ) . Cehovic ( 1 9 6 5 ) a l s o observed t h a t i n j e c t i o n s of s y n t h e t i c alpha-MSH or n a t u r a l beta-MSH produced a s i g n i f i c a n t r e d u c t i o n i n the weight of the t e s t e s and c e r t a i n a c c e s s o r y organs i n the r a t . Alpha-MSH has a l s o been shown t o i n c r e a s e human growth hormone l e v e l s i n normal s u b j e c t s but the response was not dose-dependent ( B i r k h a u s e r e t a l , 1 9 7 5 ) . O r i a s and McCann ( 1 9 7 2 ) r e p o r t e d a n a t r i u r e t i c e f f e c t of MSH i n the r a t ; t o t a l sodium e x c r e t e d , sodium c o n c e n t r a t i o n and the sodium t o p o t a s s i u m r a t i o i n the u r i n e were a l l i n c r e a s e d . I n c o n t r a s t , Page e_t a l ( 1 9 7 4 ) found t h a t alpha-MSH, i n comb-i n a t i o n w i t h growth hormone, i n c r e a s e d a l d o s t e r o n e output and hence decre a s e d n a t r i u r e s i s i n sodium r e s t r i c t e d r a t s . 13 The most l i k e l y a r e a s where MSH may e x h i b i t a t r u e p h y s i o -l o g i c a l f u n c t i o n a r e 1) n e u r a l t r a n s m i s s i o n a n d / o r p s y c h o -p h y s i o l o g y , 2) s e b a c e o u s g l a n d p r o d u c t i o n and s e c r e t i o n o r 3) f e t a l p h y s i o l o g y . The t h i r d p o s s i b i l i t y w i l l be d i s c u s s e d i n t h e n e x t s e c t i o n . K r i v o y a n d G u i l l e m i n ( 1 9 6 1 ) showed t h a t MSH i n c r e a s e s t h e a m p l i t u d e and d u r a t i o n o f m o n o - s y n a p t i c p o t e n t i a l s i n t h e s p i n a l c o r d o f t h e c a t . B r a i n e x t r a c t s f r o m t h e cow, monkey and man h a v e s i g n i f i c a n t amounts o f m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y (Rudman et_ a l , 1 9 7 3 ) • I m m u n o r e a c t i v e a l p h a - M S H h a s b e e n l o c a t e d i n v a r i o u s r e g i o n s o f t h e r a t b r a i n ( O l i v e r & P o r t e r , 1 9 7 8 ) and m e l a n o t r o p i c p e p t i d e s i n c r e a s e p r o t e i n s y n -t h e s i s i n t h e mouse b r a i n (Rudman et_ a l , 1 9 7 4 b ) . Beta-MSH a c t i v i t y i s p r e s e n t i n t h e c e r e b r o s p i n a l f l u i d o f man i n s i g n i f i c a n t l y h i g h e r amounts t h a n t h a t f o u n d i n p l a s m a ( S m i t h & S h u s t e r , 1 9 7 6 ) . K a s t i n e_t a l ( 1 9 7 1 ) made t h e f i r s t s y s t e m a t i c s t u d y o f t h e b e h a v i o u r a l e f f e c t s o f a l p h a - M S H i n human s u b j e c t s . MSH imp-r o v e d v i s u a l b u t n o t v e r b a l r e t e n t i o n and h a d a p o s i t i v e e f f e c t on s p a t i a l p e r f o r m a n c e . T h i s a c t i v i t y i s c e n t r e d i n t h e MSH/ ACTH 4 - 1 0 r e g i o n ( s e e De W i e d , 1 9 7 7 ) a n d i t a p p e a r s t h a t t h i s s e q u e n c e may h a v e a p r e d o m i n a n t i n f l u e n c e on a t t e n t i o n a l p r o c -e s s e s and p e r c e p t u a l f u n c t i o n i n g i n man (Sandman et_ a l , 1 9 7 7 ) • I n t h e r a t , ACTH and r e l a t e d p e p t i d e s d e l a y e x t i n c t i o n o f a c t i v e a v o i d a n c e b e h a v i o u r and f a c i l i t a t e p a s s i v e a v o i d a n c e b e h a v i o u r (Sandman et_ a l , • 1 9 7 3 ) . A l s o , t h e r e was e v i d e n c e f o r an i n c r e a s e i n e m o t i o n a l i t y as i n d e x e d by d e f e c a t i o n ( D a t t a & K i n g , 1 9 7 7 ) . Alpha-MSH a p p e a r s t o e n h a n c e t h e r e l e a s e o f 14 a g g r e s s i o n - p r o m o t i n g pheromone i n t o the u r i n e of dominant mice ( N o w e l l & Wouters, 1 9 7 5 ) . Thody and S h u s t e r ( 1 9 7 1 a , b ) showed t h a t b o t h beta-MSH and beta-LPH p o s s e s s e d s e b o t r o p h i c a c t i v i t y . Removal of the n e u r o i n t e r m e d i a t e lobe i n r a t s l e d t o a decrease i n sebum s e c -r e t i o n t o a l e v e l almost as low as complete hypophysectomy (Thody & S h u s t e r , 1 9 7 5 ) . Alpha-MSH a c t s s y n e r g i s t i c a l l y w i t h t e s t o s t e r o n e i n i n c r e a s i n g sebum p r o d u c t i o n i n sebaceous g l a n d s , and i n c r e a s i n g the organ weight of the p r o s t a t e , p r e -p u t i a l g l a n d s and s e m i n a l v e s i c l e s i n h y p o p h y s e c t o m i z e d - c a s t r -a t e d r a t s ( E b l i n g e t a l , 1 9 7 5 ) . I t appears t h a t t e s t o s t e r o n e and alpha-MSH may have d i f f e r e n t a c t i o n s on the sebaceous g l a n d s ; alpha-MSH seems t o a c t l a r g e l y on l i p o g e n e s i s (the g r e a t e s t e f -f e c t s were seen on the b i o s y n t h e s i s of wax e s t e r s and s qualene) w h i l e t e s t o s t e r o n e a f f e c t s c e l l u l a r p r o l i f e r a t i o n and t u r n o v e r (Thody et_ a l , 1 9 7 6 ) . Treatment w i t h drugs t h a t promote MSH r e l -ease r e s u l t e d i n i n c r e a s e d sebum s e c r e t i o n , w h i l e r e l e a s e - i n h i -b i t i n g drugs had the o p p o s i t e e f f e c t (Thody & S h u s t e r , 1 9 7 7 ) . Thus i t appears t h a t the normal f u n c t i o n i n g o f c e r t a i n g l a n d s i n the r a t may depend on p i t u i t a r y MSH or r e l a t e d p e p t i d e s , b. I n the F e tus While the work r e p o r t e d here was i n p r o g r e s s , s e v e r a l r e p o r t s appeared i n the l i t e r a t u r e t h a t suggested t h a t MSH may be of p h y s i o l o g i c a l importance i n the f e t u s and/or development. Recent e v i d e n c e suggests t h a t alpha-MSH may have a c o r t i s o l - r e l e a s i n g f u n c t i o n on the a d r e n a l of the f e t u s . I n man, a d u l t p i t u i t a r i e s l a c k s i g n i f i c a n t amounts of alpha-MSH (Abe e t a l , 1 9 6 7 ) . However, S i l m a n e_t a l ( 1 9 7 6 ) a demonstrated t h a t human f e t a l p i t u i t a r i e s c o n t a i n l a r g e amounts of p e p t i d e s 15 r e s e m b l i n g alpha-MSH and CLIP and o n l y r e l a t i v e l y s m a l l amounts of i n t a c t ACTH. J u s t b e f o r e p a r t u r i t i o n t h e r e i s a sharp i n c r e a s e i n ACTH r e l a t i v e , t o the s m a l l e r p e p t i d e s . Thus, s i g n i f i c a n t amounts of alpha-MSH may occur i n the human p i t u i -t a r y o n l y d u r i n g f e t a l l i f e . E v i d e n c e i n the r a b b i t s u g gests t h a t these o b s e r v a t i o n s may have a f u n c t i o n a l b a s i s . At days 22 and 28 p o s t - c o i t u m , alpha-MSH caused a marked i n c r e a s e i n b l o o d C o r t i s o l c o n c e n t r a t i o n s w h i l e ACTH had no e f f e c t ( C h a l l i s & T o r o s i s , 1 9 7 7 ) . However i n newborns, w h i l e the response t o alpha-MSH was s t i l l s i g n i f i c a n t , the response t o ACTH was much l a r g e r . Alpha-MSH may be i n v o l v e d i n the " i n t r a u t e r i n e growth s p u r t " i n the r a t . T h i s sudden a c c e l e r a t i o n i n the r a t e of f e t a l body weight I n c r e a s e o c c u r s at day 19 of g e s t a t i o n . While alpha-MSH s t i m u l a t e d f e t a l growth, o t h e r h y p o p h y s e a l hormones i n c l u d i n g A C T H , A C T H ^ _ ^ Q and growth hormone had no e f f e c t (Swaab & V i s s e r , 1 9 7 7 ) . S i n c e growth i n h i b i t i o n f o l l o w e d s p e c i f i c alpha-MSH a n t i b o d y a d m i n i s t r a t i o n a p h y s i o l o g i c a l f u n c t i o n of endogenous hormone I s s t r o n g l y s uggested. These a u t h o r s have a l s o l o c a t e d alpha-MSH i n p i t u i t a r i e s of human f e t u s e s between 15 and 26 weeks g e s t a t i o n ( V i s s e r & Swaab, 1 9 7 7 ) . Thody ( 1 9 7 7 ) suggests t h a t MSH may s t i m u l a t e f e t a l sebac-eous g l a n d s i n the p r o d u c t i o n of the vernex t h a t p r o t e c t s f e t a l s k i n from the a m n i o t i c f l u i d . A d m i n i s t r a t i o n of alpha-MSH f o r the f i r s t 5 weeks of l i f e i n the r a t produced a permanent i n c r e a s e i n sebum s e c r e t i o n ; s i m i l a r t r e a t m e n t i n the a d u l t had no s u s t a i n e d e f f e c t s . The e v i d e n c e p r e s e n t e d above c e r t a i n l y s u g g e s t s t h a t MSH may have a p h y s i o l o g i c a l r o l e i n the f e t u s and p o s s i b l y i n the newborn. STATEMENT OF THE PROBLEM 17 At t h e o u t s e t o f t h i s s t u d y , t h e r e was some e v i d e n c e f o r t h e p r e s e n c e , b u t no e v i d e n c e f o r t h e s i g n i f i c a n c e , o f MSH i n t h e f e t u s . W h i l e t h e 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 MSH i n t h e a d u l t r e m a i n e d u n r e s o l v e d , t h e p o s s i b l e i m p o r t a n c e o f t h e hormone i n f e t a l p h y s i o l o g y h a d n o t b e e n p r o p e r l y i n v e s t i g a t e d . As a r e s u l t t h i s s t u d y was u n d e r t a k e n t o i n v e s t i g a t e c h a n g e s i n MSH l e v e l s i n f e t a l t i s s u e s o v e r a s i g n i f i c a n t p o r t i o n o f g e s t a t i o n . S t u d i e s h a d shown t h a t MSH and r e l a t e d p e p t i d e s were p r e s e n t i n t h e f e t a l p i t u i t a r y a t v a r i o u s a g e s d u r i n g m i d t o l a t e g e s t a t i o n i n c e r t a i n s p e c i e s . To o b t a i n a more c o m p r e h e n s i v e v i e w o f t h e d y n a m i c s o f MSH p o t e n c y b o t h p i t -u i t a r y and p l a s m a a c t i v i t y was m e a s u r e d i n g u i n e a p i g f e t u s e s r a n g i n g i n age f r o m a b o u t 40 d a y s g e s t a t i o n up t o a n d i n c l u d i n g t e r m . MSH was a l s o m e a s u r e d i n t h e a m n i o t i c f l u i d . A c t i v i t y was m e a s u r e d i n newborns t o o b t a i n a p r e l i m i n a r y i n d i c a t i o n o f p o s s i b l e c h a n g e s a t p a r t u r i t i o n . L e v e l s were a l s o c o m p a r e d t o t h o s e i n a d u l t s . M easurement o f c h a n g e s i n hormone l e v e l s i n v a r i o u s t i s s u e s and f l u i d s i s a l o g i c a l f i r s t s t e p i n i n v e s t i g a t i n g w h e t h e r t h e s e p e p t i d e s may i n f a c t be f u n c t i o n a l b e f o r e b i r t h . MATERIALS & METHODS 1 8 1. Animals a. Guinea P i g s ( C a v l a p r o c e l l u s ) Pregnant and nonpregnant ani m a l s were o b t a i n e d from the Department of Zoology v i v a r i u m , U.B.C. Except f o r the exper-iments u s i n g samples t a k e n from newborns, most a n i m a l s were o b t a i n e d on the day of s a c r i f i c e . G e s t a t i o n a l age of the f e t u s e s was determined u s i n g the weight and l e n g t h d a t a of Draper ( 1 9 2 0 ) and I b s e n ( 1 9 2 8 ) . G e s t a t i o n l a s t s a p p r o x i m a t e l y 68 days i n the g u i n e a p i g ( I l l i n g -w orth e t a l , 1 9 7 4 ) . b. A n o l i s c a r o l i n e n s i s A n o l i s l i z a r d s were o b t a i n e d from a l o c a l p e t s t o r e and kept i n the l a b u n t i l used i n the b i o a s s a y . They were f e d Te n e b r i o l a r v a e and s u p p l i e d w i t h water ad l i b i t u m . 2• Sample C o l l e c t i o n and P r e p a r a t i o n a. P i t u i t a r y and B r a i n T i s s u e Pregnant g u i n e a p i g s were l i g h t l y a n a e s t h e t i z e d w i t h e t h e r and the u t e r u s was removed. Both f e t a l and a d u l t p i t u i t a r y g l a n d s were c o l l e c t e d . Newborns were k i l l e d by c o n c u s s i o n . A l l animals were d e c a p i t a t e d and d i s s e c t e d i m m e d i a t e l y . The p i t -u i t a r y g l a n d was exposed by f i r s t removing the p o s t e r i o r d o r s a l r e g i o n of the cranium and then l i f t i n g the b r a i n from the back w h i l e c u t t i n g the c r a n i a l n e r v e s . The a n t e r i o r and p o s t e r i o r l o b e s were s e p a r a t e d u s i n g f i n e f o r c e p s and the lobe s were d e p o s i t e d i n d i v i d u a l l y i n t o Beam c a p s u l e s and f r o z e n i m m e d i a t e l y on dry i c e . Samples of b r a i n t i s s u e t a k e n from the h y p o t h a l a m i c 19 r e g i o n were a l s o c o l l e c t e d . S a m p l e s were l y o p h i l i z e d u n d e r l o w p r e s s u r e ( 0 . 2 mm Hg) f o r 24 h o u r s u s i n g a h i g h vacuum pump ( W e l c h S c i e n t i f i c Co.-, S k o k i e , I l l i n o i s ) . S a m p l e s n o t e x t r a c t e d a n d a s s a y e d i m m e d i -a t e l y w e r e s t o r e d i n v a c u o o v e r P„Cv a t 4°C. — 2 o The e x t r a c t i o n p r o c e d u r e f o l l o w e d t h e m e t h o d o f S c o t t a n d L o w r y ( 1 9 7 4 ) f o r ACTH and r e l a t e d p e p t i d e s . I n d i v i d u a l o r p o o l e d t i s s u e s a m p l e s were w e i g h e d and t h e n h o m o g e n i z e d b r i e f l y i n 5$ a c e t i c a c i d u s i n g a P o t t e r E l v e h j e m . T i s s u e G r i n d e r (Wheaton S c i e n t i f i c , M i l l v i l l e , N.J.) and b l e n d e r ( C o l e P a r m e r Co., C h i c a g o , I l l i n o i s ) . The homogenate was b o i l e d f o r t h r e e m i n u t e s , a l l o w e d t o c o o l a n d t h e n c e n t r i f u g e d a t 2500 g f o r 20 m i n u t e s . The s u p e r n a t a n t was r e m o v e d a nd u s u a l l y a s s a y e d imm-e d i a t e l y . O t h e r w i s e s a m p l e s were f r o z e n a n d a s s a y e d t h e f o l -l o w i n g d a y . P r i o r t o a s s a y , s a m p l e s were n e u t r a l i z e d w i t h NaOH. N e u t r a l i z e d s a m p l e s w ere d i l u t e d w i t h R i n g e r ' s s o l u t i o n a n d a p-p r o p r i a t e c o n c e n t r a t i o n s were a s s a y e d as d e s c r i b e d b e l o w , b. B l o o d P l a s m a S a m p l e s A T I a d u l t a n d n e w b o r n a n i m a l s were k i l l e d by c o n c u s s i o n . B l o o d was c o l l e c t e d by c a r d i a c p u n c t u r e u s i n g s y r i n g e s m o i s t -e n e d w i t h a 10 mg$ s o l u t i o n o f s o d i u m h e p a r i n i n j e c t i o n ( 1 0 m g / m l ; 1 0 0 0 I . U . ( U . S . P . ) / m l ) . I n b o t h t h e f e t u s e s a n d t h e new-b o r n s , b l o o d o b t a i n e d f r o m l i t t e r m a t e s was p o o l e d t o o b t a i n s a m p l e s o f a d e q u a t e v o l u m e f o r p r o p e r a s s a y p r o c e d u r e s . Whole b l o o d was i m m e d i a t e l y i m m e r s e d i n i c e a n d c e n t r i f u g e d i n p o l y -e t h y l e n e t u b e s a t 2 5 0 0 g a t 4°C f o r 10 m i n u t e s . The p l a s m a was a s p i r a t e d and i m m e d i a t e l y e x t r a c t e d , o r f r o z e n a t -30°C u n t i l e x t r a c t i o n . 20 M e l a n o c y t e - s t i m u l a t i n g p e p t i d e s were e x t r a c t e d u s i n g the s i l i c i c a c i d method of Orth e_t a l (1976). Each ml of plasma was mixed w i t h 0.5 ml of a s u s p e n s i o n of 200 mg s i l i c i c a c i d per ml of 0.05M sodium phosphate b u f f e r , pH 7-5, w i t h NaCl added t o 77mM; tubes were capped and mixed f o r 30 minutes at room temperature on a v o r t e x m ixer ( F i s h e r S c i e n t i f i c ) . Tubes were c e n t r i f u g e d at 3000 g f o r 7 minutes and the s u p e r n a t e s were a s p i r a t e d . The s i l i c i c a c i d cake was washed t w i c e w i t h 2.5 ml o f d i s t i l l e d w a t e r ; a f t e r c e n t r i f u g i n g , each wash was a s p i r a t e d and d i s c a r d e d . To e l u t e adsorbed p e p t i d e , the s i l -i c i c a c i d cake was resuspended i n 1.25 ml of a c e t i c a c i d - a c e t o n e -water (1:20:80) per ml of o r i g i n a l plasma sample and t h i s s us-p e n s i o n was mixed f o r 30 minutes on the v o r t e x m i x e r . A f t e r c e n t r i f u g i n g a t 3000 g f o r 7 m i n u t e s , the s u p e r n a t a n t was t r a n s f e r r e d t o another tube and the m i x t u r e was e v a p o r a t e d and d r i e d under N£ at 40°C. Tubes were s t o r e d at -30°C u n t i l a ssayed. The d r i e d e x t r a c t was r e c o n s t i t u t e d i n 8 t o 10 ml of R i n g e r ' s s o l u t i o n and a p p r o p r i a t e amounts were added t o the s o l -u t i o n b a t h i n g the t e s t s k i n (see assay p r o c e d u r e s b e l o w ) , c. A m n i o t i c F l u i d Pregnant a n i m a l s were k i l l e d by c o n c u s s i o n . A f t e r e x p o s i n g the u t e r u s an i n c i s i o n was made i n the muscle w a l l and a m n i o t i c f l u i d was removed d i r e c t l y from the a m n i o t i c sac w i t h a s y r i n g e . Samples were e i t h e r assayed i m m e d i a t e l y or more o f t e n were f r o z e n u n t i l . a s s a y . 3. B i o a s s a y f o r M e l a n o c y t e - S t i m u l a t i n g A c t i v i t y The b i o a s s a y f o r MSH a c t i v i t y i s based on the hormone's a b i l i t y t o d i s p e r s e pigment i n the melanophores of the s k i n of 21 v a r i o u s lower v e r t e b r a t e s . When a dose of s t a n d a r d MSH or unknown i s a d m i n i s t e r e d e i t h e r i n v i v o or i n v i t r o , the degree of r e s u l t a n t c o l o u r change can be q u a n t i f i e d . I n i t i a l l y t h i s was done s u b j e c t i v e l y , as by Hogben and Winton ( 1 9 2 2 ) u s i n g f r o g s i n v i v o . Shizume e_t a l ( 1 9 5 4 ) i n c r e a s e d the a c c u r a c y and s e n s i t i v i t y of the assay by s e c u r i n g p i e c e s of f r o g s k i n on a m e t a l frame and measuring the c o l o u r change as changes i n l i g h t r e f l e c t a n c e u s i n g a r e f l e c t o m e t e r . The assay i n the p r e s e n t study i s based on the method of B j o r k l u n d et_ a l ( 1 9 7 4 ) u s i n g the l i z a r d A n o l i s c a r o l i n e n s i s as the t e s t a n i m a l . A l l s k i n r e q u i r e d f o r one assay can be a c q u i r e d from one a n i m a l ( i n the f r o g a s s a y , up t o f o u r a n i m a l s are r e q u i r e d ) and the s k i n w i l l f l o a t on the t e s t medium making the use of m e t a l frames u n n e c e s s a r y . Some m o d i f i c a t i o n s were made i n measurement t e c h n i q u e and s t a t i s t i c a l a n a l y s i s . a. P r e p a r a t i o n of the S k i n A n o l i s were p l a c e d on a w h i t e background c a u s i n g the s k i n t o t u r n p a l e green due t o the i n h i b i t i o n of MSH r e l e a s e . A n i -mals were then k i l l e d by d e c a p i t a t i o n u s i n g sharp s c i s s o r s . A f t e r the removal of l e g s and t a i l the s k i n was s l i t v e n t r a l l y and the t o r s o was s k i n n e d . E i g h t round s k i n p i e c e s , 7 mm i n d i a m e t e r , were cut out u s i n g a sharpened s t e e l tube and f l o a t e d i n d i v i d u a l l y on l i z a r d R i n g e r ' s s o l u t i o n i n s m a l l p l a s t i c b e a k e r s . Only the homogeneous, l i g h t green d o r s a l s k i n was used. S k i n p i e c e s were e q u i l i b r a t e d on R i n g e r ' s s o l u t i o n f o r one hour b e f o r e b e i n g used i n the assay. b. Measurement of the R e f l e c t i o n Q u o t i e n t R e f l e c t i o n was measured w i t h a P h o t o v o l t P h o t o e l e c t r i c R e f l e c t i o n and Glo s s m e t e r 6 7 1 ( P h o t o v o l t Corp., New Y o r k ) . The 22 p o r t of the r e f l e c t o m e t e r s e a r c h u n i t f i t over the l i p of 6 ml p o l y e t h y l e n e beakers c o n t a i n i n g p i e c e s of l i z a r d s k i n f l o a t i n g on the t e s t medium so t h a t no e x t r a n e o u s l i g h t c o u l d r e a c h the p h o t o c e l l and b i a s the measurement. Test beakers were p a i n t e d b l a c k f o r the same r e a s o n . The response of the s k i n t o e i t h e r a s t a n d a r d of unknown p r e p a r a t i o n i s termed the r e f l e c t i o n q u o t i e n t and i s d e f i n e d as T/B. B i s the I n i t i a l b a s e l i n e r e f l e c t i o n measured from the s k i n w h i l e i t f l o a t s on c o n t r o l R i n g e r ' s s o l u t i o n ; the s k i n i s then t r a n s f e r r e d t o the t e s t s o l u t i o n and T i s the r e f -l e c t i o n measured a f t e r an exposure time of 15 minutes.• Samples c o n t a i n i n g m e l a n o c y t e - s t i m u l a t i n g potency cause the s k i n t o t u r n brown r e s u l t i n g i n a lower r e a d i n g on the r e f l e c t i o n meter. A f t e r t e s t i n g , s k i n p i e c e s were r e t u r n e d t o R i n g e r ' s s o l u t i o n f o r a 45 minute wash. S i n c e the s k i n response i s temperature s e n s i t i v e beakers o c o n t a i n i n g s k i n p i e c e s were kept i n a water b a t h at 23 C f o r the d u r a t i o n of the experiment. c. C a l c u l a t i o n of R e l a t i v e Potency and S t a t i s t i c a l Methods When r e f l e c t i o n q u o t i e n t s , measured u s i n g s k i n p i e c e s t a k e n from a s i n g l e a n i m a l , are p l o t t e d w i t h r e s p e c t t o the l o g of the dose, a normal s i g m o i d a l curve r e s u l t s . The l i n e a r p o r t i o n s of the dose-response c u r v e s , of the s t a n d a r d and unknown, are used f o r the c a l c u l a t i o n of the r e l a t i v e potency of the unknown p r e p a r a t i o n as compared t o the s t a n d a r d . A sample s t a n d a r d curve i s shown i n F i g u r e 3. The potency of the unknown was c a l c u l a t e d u s i n g a 2x2 f a c t o r i a l d e s i g n u t i l i z i n g a h i g h and low dose of b o t h s t a n d a r d 23 F i g u r e 3: R e l a t i o n s h i p between the l o g a r i t h m of the dose of s t a n d a r d MSH and the c o l o u r change of A n o l i s s k i n i n v i t r o as measured by the r e f l e c t i o n q u o t i e n t . The curve shows the l i n e a r p o r t i o n of the dose-response curve of A n o l i s s k i n t o v a r y i n g c o n c e n t r a t i o n s o f syn-t h e t i c alpha-MSH. A b s c i s s a : Log of the dose of s t a n d a r d MSH a p p l i e d t o the t e s t s k i n i n pg/ml. O r d i n a t e : Response of the s k i n as measured by the r e f l -e c t i o n q u o t i e n t . The r e f l e c t i o n q u o t i e n t i s the r a t i o of the r e f l e c t i o n measured from the t e s t s k i n a f t e r a 15 minute exposure t o the t e s t s o l u t i o n , d i v i d e d by the r e f l e c t i o n measured from the s k i n w h i l e on c o n t r o l R i n g e r ' s s o l u t i o n j u s t b e f o r e the s k i n i s t r a n s f e r r e d t o the t e s t s o l u t i o n . 2 4 ±N3LLOnO NOI1031d3d 25 and unknown ( B l i s s , 1 9 5 2 ) . The h i g h dose was f r o m two t o f o u r t i m e s t h e c o n c e n t r a t i o n o f t h e l o w d o s e ; h i g h d o s e p o t e n c y o f s t a n d a r d r a n g e d f r o m 200 t o 1 0 0 0 pg/ml d e p e n d i n g on t h e s e n s i -t i v i t y o f t h e i n d i v i d u a l t e s t s k i n . Doses were c h o s e n so t h a t r e s u l t i n g r e s p o n s e s were l o c a t e d on t h e l i n e a r p o r t i o n o f t h e l o g d o s e - r e s p o n s e c u r v e . The e i g h t s k i n p i e c e s u s e d a l l o w e d f o r t h e measurement o f two r e s p o n s e s f o r e a c h t r e a t m e n t g r o u p a t one t i m e . F o r e a c h unknown s a m p l e t h e t e s t p r o c e d u r e was r e p e a t e d so t h a t f o u r r e s p o n s e s were o b t a i n e d f o r e a c h g r o u p . B e c a u s e , on o c c a s i o n , t h e s e n s i t i v i t y o f t h e s k i n p i e c e s c h a n g e d on t h e s e c o n d r e s p o n s e a r a n d o m i z e d b l o c k d e s i g n was u s e d i n t h e a n a l y s i s o f v a r i a n c e . I f t h e e f f e c t was n o t s i g n i f i c a n t a c o m p l e t e l y r a n d o m i z e d d e s i g n was u s e d . The s t a n d a r d e r r o r was c a l c u l a t e d as d e s c r i b e d by B l i s s ( 1 9 5 2 ) . Two a n d some-t i m e s t h r e e unknowns c o u l d be a s s a y e d u s i n g t h e s k i n f r o m a s i n g l e l i z a r d . The a s s a y p r e c i s i o n r a n g e d f r o m 0.010 t o 0.284 w i t h a mean o f 0.097-d. S p e c i a l i z e d P r o c e d u r e s f o r A m n i o t i c F l u i d B e c a u s e o f t h e l o w a c t i v i t y i n a m n i o t i c f l u i d , t e s t s k i n was f l o a t e d d i r e c t l y on a m n i o t i c f l u i d s a m p l e s . S a m p l e s w i t h a v o l u m e u n d e r 6 m l were d i l u t e d t o t e s t v o l u m e . S i n c e t h e r e f l e c t i v e p r o p e r t i e s o f a m n i o t i c f l u i d d i f f e r e d f r o m t h o s e o f R i n g e r ' s s o l u t i o n , t h e b a s e l i n e measurement was t a k e n i m m e d i a t -e l y a f t e r t r a n s f e r r i n g t h e s k i n p i e c e s t o t h e t e s t s o l u t i o n . B e c a u s e o f t h e 6 m l v o l u m e r e q u i r e d f o r s a m p l e measurement a n d t h e l o w a c t i v i t i e s p r e s e n t , o n l y t h e c o n c e n t r a t e d a m n i o t i c f l u -i d s a m p l e c o u l d be m e a s u r e d . T h e r e f o r e t h e 2x2 f a c t o r i a l d e s i g n was u n s u i t a b l e f o r c a l c u l a t i n g unknown a c t i v i t y . I n s t e a d , t h e a v e r a g e r e s p o n s e o f t h e unknown was f i t t e d t o t h e s t a n d a r d c u r v e . 26 I f the a c t i v i t y was measurable, the sample was i n c u b a t e d w i t h t r y p s i n (see below) and measured a g a i n . Assay p r e c i s i o n by t h i s method ranged from 0.067 t o 0.611 w i t h an average of 0 . 2 6 0 . The r e s u l t has l e s s v a l i d i t y but i s adequate as a f i r s t a p p r o x i m a t i o n . 4. S t a n d a r d P r e p a r a t i o n Alpha-MSH was used as the s t a n d a r d because i t i s the most p o t e n t m e l a n o c y t e - s t i m u l a t i n g p e p t i d e and because i t i s the most commonly used s t a n d a r d at the p r e s e n t t i m e . S y n t h e t i c alpha-MSH was o b t a i n e d from C i b a - G e i g y , B a s e l , S w i t z e r l a n d and was r e f r i g e r a t e d at 4°C. S t o c k s o l u t i o n s of 1 0 - 2 0 pg/ml were p r e p a r e d i n a c i d a l b u m i n s a l i n e ( 0 . 6 ml 12N H C l , 1.0 g b o v i n e albumin and 8.6 g NaCl per l i t r e of H 2 0 ) (Abe & N i c h o l s o n , 1 9 7 5 ) and s t o r e d i n 1-2 ml a l i q u o t s i n p o l y e t h y l e n e tubes at -30°C. One a l i q u o t was used t o p r e p a r e the s t a n d a r d s o l u t i o n on the day of each assay. 5. S i l i c i c A c i d P r e t r e a t m e n t S i l i c i c a c i d ( 1 0 0 mesh; M a l l i n c k r o d t Chem., M o n t r e a l ) was d r i e d I n an oven ( F i s h e r S c i e n t i f i c ) at 110°C f o r at l e a s t 8 hours t o remove the undetermined q u a n t i t y of absorbed H^O. To m i n i m i z e permanent b i n d i n g of the p e p t i d e t o the adsorbent and t o o b t a i n r e p r o d u c i b l e r e t e n t i o n p r o p e r t i e s , d i s t i l l e d water was re-added t o the d r i e d s i l i c i c a c i d ( 2 5 $ of the s i l i c i c a c i d by w e i g h t ) . The m i x t u r e was a g i t a t e d v i g o r o u s l y and a l l o w e d t o e q u i l i b r a t e f o r 8 t o 16 hours (Snyder, 1 9 7 5 ) . 6. T r y p s i n I n c u b a t i o n 5.5 ml of s t a n d a r d or unknown p r e p a r a t i o n p l u s 0.5 ml of t r y p s i n s o l u t i o n were i n c u b a t e d f o r 30 minutes a t . 3 0 C i n a 27 water b a t h shaker (Eberbach Corp., Ann A r b o r , M i c h i g a n ) . The t r y p s i n s o l u t i o n c o n s i s t e d of 1 mg of pure t r y p s i n (two times c r y s t a l l i z e d , Sigma Co., S t . L o u i s ) , 10 ml sodium phosphate b u f f e r (pH 7-35) and 40 ml of d i s t i l l e d w a t e r . 7. S a l i n e s a. L i z a r d R i n g e r ' s S o l u t i o n 6.5 g N a C l , 0.14 g KC1, 0.24 g anhydrous C a C l 2 and 0.5 g NaHCO^ were d i s s o l v e d i n d i s t i l l e d water t o a f i n a l volume of 1 l i t r e . b. Sodium Phosphate B u f f e r The b u f f e r c o n s i s t s of p r o p o r t i o n s of the f o l l o w i n g s o l u t i o n s : (1) 22.714 g Na^PO^ made t o 1 l i t r e and (2) 6.349 g NaH^PO^ made t o 1 l i t r e . (2) i s added t o (1) u n t i l the d e s i r e d pH i s m a i n t a i n e d . 28 SECTION I : MELANOCYTE-STIMULATING A C T I V I T Y  I N FETAL AND ADULT TISSUES 1. I n t r o d u c t i o n B o t h ACTH a n d MSH a c t i v i t y h a v e b e e n r e p o r t e d i n w h o l e p i -t u i t a r i e s o f human f e t u s e s a s e a r l y as e l e v e n weeks ( K a s t i n e_fc a l , 1 9 6 8 ) . MSH a c t i v i t y ( p e r mg o f t i s s u e ) d i d n o t cha n g e w i t h d e v e l o p m e n t ( t o 23 w e e k s ) w h i l e ACTH a c t i v i t y d e c r e a s e d . I n t h e p a r s d i s t a l i s , ACTH a c t i v i t y a p p e a r s a t s e v e n weeks ( D u b o i s e t a l , 1 9 7 3 ; B a k e r & J a f f e , 1 9 7 5 ) . D u b o i s e t a l ( 1 9 7 3 ) f o u n d no e v i d e n c e f o r a l p h a - M S H o r t h e l i p o t r o p i n s i n t h e p a r s d i s t a l i s b u t showed t h a t as i n t h e a d u l t , b o t h beta-MSH a nd ACTH a c t i -v i t y c o u l d be l o c a t e d i n t h e same c e l l s . However a r e c e n t s t u -dy r e p o r t e d t h a t a l p h a - M S H was l o c a t e d i n s c a t t e r e d c e l l s i n t h e a n t e r i o r l o b e i n t h e human f e t u s ( V i s s e r & Swaab, 1 9 7 7 ) . M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y h a s a l s o b e e n i d e n t i f i e d i n t h e p i t u i t a r y o f f e t a l r o d e n t s . U s i n g t i s s u e i m p l a n t s i n t h e f r o g Rana t e m p o r a r i a , Enemar ( 1 9 6 3 ) f o u n d m e l a n o c y t e -s t i m u l a t i n g a c t i v i t y i n t h e p a r s d i s t a l i s o f 1 5 - d a y mouse em-b r y o s a n d i n t h e n e u r o i n t e r m e d i a t e l o b e o f t h e 1 6 - d a y embryo. G r a n u l a r i n c l u s i o n s f i r s t a p p e a r i n t h e c e n t r a l p a r t o f t h e e p i t h e l i u m o f t h e p a r s i n t e r m e d i a i n t h e 1 7 - d a y embryo ( E u r e n i u s & J a r s k a r , 1 9 7 5 ) . U s i n g i m m u n o f l u o r e s c e n c e s t u d i e s i n t h e r a t f e t u s , b e t a -MSH c e l l s were l o c a t e d on day 16 i n t h e p a r s d i s t a l i s a n d on day 17 i n t h e p a r s i n t e r m e d i a ; a l p h a - M S H c e l l s a p p e a r on day 18 and a r e r e s t r i c t e d t o t h e p a r s i n t e r m e d i a (Dupouy & D u b o i s , 1 9 7 5 ) . ACTH c e l l s a r e e v i d e n t on day 17 i n t h e p a r s d i s t a l i s 29 and on day 18 i n the p a r s i n t e r m e d i a . S e c r e t a r y g r a n u l e s appear i n the p a r s i n t e r m e d i a at the 14.5-day stage (Negm, 1 9 7 0 ) . M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y has not been measured i n the p i t u i t a r y of the f e t a l g u i n e a p i g . A study of m e l a n o c y t e -s t i m u l a t i n g a c t i v i t y i n r e l a t i o n t o g e s t a t i o n a l age might g i v e some i d e a as t o i t s f u n c t i o n a l s i g n i f i c a n c e , i f any, d u r i n g f e t a l l i f e . A l i t t e r of newborns was a l s o assayed t o see i f any s i g n i f i c a n t changes occur at p a r t u r i t i o n . F e t a l a c t i v i t y was a l s o compared t o l e v e l s found i n the a d u l t . S e v e r a l •.. :. . e x t r a c t s of b r a i n t i s s u e were a l s o measured. S i n c e MSH p e p t i d e s have not been i s o l a t e d and sequenced i n t h i s s p e c i e s , t h e i r s t r u c t u r e may d i f f e r from known p e p t i d e s . T h e r e f o r e , b i o l o g i -c a l a c t i v i t y was measured, r a t h e r than more s p e c i f i c immunolo-g i c a l a c t i v i t i e s (see g e n e r a l d i s c u s s i o n ) . 2. R e s u l t s a. M e l a n o c y t e - s t i m u l a t i n g A c t i v i t y i n the F e t a l P i t u i t a r y The a n t e r i o r and p o s t e r i o r l o b e s were c o l l e c t e d and assayed s e p a r a t e l y . Some i n d i v i d u a l l o b e s were assayed but i n most cases whole l i t t e r s were p o o l e d b e f o r e assay. Thus most p o i n t s on F i g u r e s 4 t o 6 r e p r e s e n t the average t i s s u e a c t i v i t y of from 2 t o 6 l i t t e r m a t e s . Glands were t a k e n from an i m a l s between 39 and 70 days of g e s t a t i o n . M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n c r e a s e s b o t h i n t o t a l a c t i v i t y p e r g l a n d and a c t i v i t y p e r u n i t dry weight i n b o t h p o r t i o n s of the g l a n d as g e s t a t i o n p r o g r e s s e s . P o s t e r i o r p i t u i t a r y a c t i v i t i e s are shown i n F i g u r e 4. P o s t e r i o r p i t u i t a r y v a l u e s ranged from 18.0 ng/lobe at 39 days 30 F i g u r e 4: The r e l a t i o n s h i p between the age of the g u i n e a p i g f e t u s i n days of g e s t a t i o n and the melano-c y t e - s t i m u l a t i n g a c t i v i t y i n the p o s t e r i o r p i t u i t a r y measured i n ng o f alpha-MSH (per lobe or per mg) and p l o t t e d on a l o g s c a l e . Each p o i n t r e p r e s e n t s the average b i o l o g i c a l a c t i v i t y of t i s s u e c o l l e c t e d from one t o s i x l i t t e r m a t e s and p o o l e d b e f o r e e x t r a c t i o n . The l i n e of b e s t f i t i s shown on each graph and i s d e f i n e d as: (a) y = 0.0491x - 0 .4668, ?r 0.9304; and (b) y = 0.0244x + 1.543, v = 0.5438 ..(r -c o e f f i c i e n t of d e t e r m i n a t i o n ) . A b s c i s s a : Age of the f e t u s i n days of g e s t a t i o n as c a l c u l a t e d u s i n g the d a t a of Draper ( 1 9 2 0 ) and I b s e n (1928) . O r d i n a t e : M e l a n o c y t e - s t i m u l a t i n g . a c t i v i t y i n e q u i v a l e n t amounts of s y n t h e t i c alpha-MSH, as measured by b i o a s s a y i n v i t r o , p l o t t e d on a l o g s c a l e . 31 1000 posterior pituitary f (a) ng/lobe 300 100 x i a. (0 c > I -O < z !5 I - 3000 (/) LLI I ->-o o 30 (b) ng/mg 1000 300 + 40 50 60 _ i I 70 GESTATIONAL AGE - DAYS 32 F i g u r e 5'• The r e l a t i o n s h i p between the age of the g u i n e a p i g f e t u s i n days of g e s t a t i o n and the melano-c y t e - s t i m u l a t i n g a c t i v i t y i n the a n t e r i o r p i t u i t a r y measured i n ng of alpha-MSH ( p e r lobe or p er mg) and p l o t t e d on a l o g s c a l e . Each p o i n t r e p r e s e n t s the average b i o l o g i c a l a c t i v i t y of t i s s u e c o l l e c t e d from one t o s i x l i t t e r m a t e s and p o o l e d b e f o r e e x t r a c t i o n . The l i n e of b e s t f i t i s shown on each graph and i s d e f i n e d as: (a) y = 0 . 0 4 9 7 x - 0 . 9 4 0 9 , r 2 = 0 . 8 9 3 4 ; and.(b) y = 0 . 0 2 7 3 x + 0 . 5 7 8 4 , r 2 = 0 . 5 2 9 4 ( r 2 = c o e f f i c i e n t of d e t e r m i n a t i o n ) . A b s c i s s a : Age o f the f e t u s i n days o f g e s t a t i o n as c a l c u l a t e d u s i n g the d a t a of Draper ( 1 9 2 0 ) and I b s e n ( 1 9 2 8 ) . O r d i n a t e : M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n e q u i v a l e n t amounts of s y n t h e t i c alpha-MSH, as measured by b i o a s s a y i n v i t r o , p l o t t e d on a l o g s c a l e . 33 (/) (3 (0 c >-> O < < anterior pituitary (a) ng/lobe 300 .& 100 30 10 (b) ng/mg i UJ > a o 300 < 100 30h 40 50 60 i I 70 G E S T A T I O N A L A G E - D A Y S 34 F i g u r e 6 : The r e l a t i o n s h i p between the age of the g u i n e a p i g f e t u s i n days of g e s t a t i o n and the melano-c y t e - s t i m u l a t i n g a c t i v i t y i n the whole p i t u i t a r y g l a n d as measured i n ng of alpha-MSH (per g l a n d or per mg) and p l o t t e d on a l o g s c a l e . Each p o i n t r e p r e s e n t s the average b i o l o g i c a l a c t i v i t y of the g l a n d s as c a l c u l a t e d by combining the r e s u l t s o b t a i n -ed f o r the p o s t e r i o r and a n t e r i o r l o b e s ( F i g u r e s 4 and 5 ) . T i s s u e was c o l l e c t e d from one t o s i x l i t t e r m a t e s and p o o l e d b e f o r e e x t r a c t i o n . The l i n e of b e s t f i t i s shown on each graph and i s d e f i n e d as: (a) y = 0.0505x - 0.3982, r2 = 0 . 9 4 3 6 ; and (b) y = 0.0265x + 1 . 0 4 4 3 , r 2 = 0.6136 ( r 2 = c o e f f i c i e n t of d e t e r m i n a t i o n ) . A b s c i s s a : Age of the f e t u s i n days of g e s t a t i o n as c a l c u l a t e d u s i n g the d a t a of Draper (1920) and I b s e n (1928) . O r d i n a t e : M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n e q u i v a l e n t amounts of s y n t h e t i c alpha-MSH, as measured by b i o a s s a y i n v i t r o , p l o t t e d on a l o g s c a l e . 1000 300h 100h 30 1000r 300h 100W/ (b) ng/mg 40 50 60 70 G E S T A T I O N A L A G E - D A Y S 36 t o 1 0 8 1 n g / l o b e a t 70 d a y s a n d f r o m 288 ng/mg a t 39 d a y s t o 2962 ng/mg a t 70 d a y s . A n t e r i o r p i t u i t a r y a c t i v i t y shows a s i m i l a r t r e n d b u t m e a s u r e d a c t i v i t y i s l o w e r ( F i g u r e 5 ) . A c t i v i t y r a n g e d f r o m 1 0 . 5 n g / l o b e a t 42 d a y s t o 403 n g / l o b e a t 70 d a y s a n d f r o m 4 2 . 4 ng/mg a t 55 d a y s t o 523 ng/mg a t 70 d a y s . T o t a l p i t u i t a r y g l a n d a c t i v i t y i s shown i n F i g u r e 6 . A c t i v i t y i n t h e w h o l e g l a n d r a n g e s f r o m 2 8 . 9 n g / g l a n d a t 39 d a y s t o 1484 n g / g l a n d a t 70 d a y s a n d f r o m 143 ng/mg a t 55 d a y s t o 1308 ng/mg a t 70 d a y s . R e g r e s s i o n a n a l y s i s showed t h a t t h e l i n e a r r e l a t i o n s h i p b e t w e e n g e s t a t i o n a l age and t h e l o g o f t h e a c t i v i t y was s i g n i -f i c a n t a t P< . 0 0 5 i n a l l c a s e s . T h e r e i s a c l e a r l i n e a r r e l a t -i o n s h i p b e t w e e n age i n d a y s a n d t h e l o g o f t h e . m e a s u r e d a c t i v i t y i n n g / l o b e . W i t h a c t i v i t y m e a s u r e d i n ng/mg t h e l i n e a r r e l a t -i o n s h i p d i s p l a y s more s c a t t e r b u t i s s t i l l h i g h l y s i g n i f i c a n t . The c o e f f i c i e n t o f d e t e r m i n a t i o n r a n g e d f r o m 0.529 t o 0 . 9 4 4 . The r a t i o o f p o s t e r i o r l o b e t o a n t e r i o r l o b e a c t i v i t y was much l o w e r t h a n e x p e c t e d ( s e e d i s c u s s i o n ) . The a v e r a g e r a t i o f o r p e r l o b e a c t i v i t y was 3 - 0 w i t h i n d i v i d u a l a s s a y r a t i o s r a n -g i n g f r o m 1 . 7 t o 4 . 7 - P e r w e i g h t a c t i v i t y r a t i o s a v e r a g e d 6 . 7 a n d r a n g e d f r o m 3 -4 t o 1 1 . 7 . No t r e n d s were o b s e r v e d w i t h i n -c r e a s i n g g e s t a t i o n a l a g e . F i g u r e 7 shows t h e i n c r e a s e i n d r y w e i g h t o f t h e p o s t e r i o r , a n t e r i o r a n d w h o l e p i t u i t a r y as g e s t a t i o n p r o g r e s s e s . b. M e l a n o c y t e - S t i m u l a t i n g A c t i v i t y i n t h e P i t u i t a r i e s o f  Newborns P i t u i t a r i e s were c o l l e c t e d f r o m . a l i t t e r o f f o u r n e wborns 37 F i g u r e 7' The r e l a t i o n s h i p between the age of the g u i n e a p i g f e t u s i n days of g e s t a t i o n and the dry weight of the p o s t e r i o r , a n t e r i o r and whole p i t u i t a r y i n mg. Each p o i n t r e p r e s e n t s the average dry weight of t i s s u e c o l l e c t e d from one t o s i x l i t t e r m a t e s and p o o l e d b e f o r e w e i g h i n g . A b s c i s s a : Age of the f e t u s i n days of g e s t a t i o n as c a l c u l a t e d u s i n g the d a t a of Draper (1920) and I b s e n (1928) . O r d i n a t e : Average dry weight o f the lobe or g l a n d i n mg. 38 0.6 <X2r posterior lobe -J 1 1 1 i i_ E CO CO o I -X o >-a: Q 14r 1.0h 0.6 0.2h anterior lobe -lh -•jjL whole pituitary 14h 1.0h 0.6H 02\-_ i i_ J L 40 50 60 GESTATIONAL AGE - DAYS 70 w i t h i n a few h o u r s o f d e l i v e r y . M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n t h e p o s t e r i o r l o b e was 986 n g / l o b e o r 4430.ng/mg and i n t h e a n t e r i o r l o b e was 348 n g / l o b e o r 589 ng/mg. By c o m p a r i s o n w i t h f e t a l w e i g h t s , b i r t h was j u d g e d t o have o c c u r r e d a b o u t 67 d a y s p o s t c o i t u m . M e l a n o c y t e - s t i m u l a t i n g a c t i v i t i e s a g r e e c l o s e l y w i t h t h o s e t a k e n f r o m f e t a l p i t u i t a r i e s o f t h e same p o s t c o i t u m a g e , a l t h o u g h ng/mg a c t i v i t y i n t h e p o s t e r i o r l o b e a p p e a r s t o be s l i g h t l y h i g h e r . H o w e v e r , a v e r a g e p o s t e r i o r l o b e w e i g h t was ap-p r o x i m a t e l y 50$ o f t h a t o f f e t u s e s o f t h e same p o s t c o i t a l a g e . c. M e l a n o c y t e - s t i m u l a t i n g A c t i v i t y i n A d u l t P i t u i t a r i e s M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y was m e a s u r e d i n w h o l e g l a n d s t a k e n f r o m f o u r a d u l t f e m a l e g u i n e a p i g s . The a v e r a g e a c t i v i t y was 8620 ± 1300 n g / g l a n d o r 2600 ± 590 ng/mg (mean + s t a n d a r d e r r o r o f t h e mean). I n one a n i m a l 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 were m e a s u r e d s e p a r a t e l y . The r a t i o o f p o s -t e r i o r t o a n t e r i o r l o b e a c t i v i t y (ng/mg) was 4.0; t h i s i s w i t h i n t h e r a n g e o f v a l u e s f o u n d i n t h e f e t u s . d. M e l a n o c y t e - S t i m u l a t i n g A c t i v i t y i n B r a i n T i s s u e B e c a u s e o f t h e p o s s i b l e e f f e c t s o f MSH on n e u r a l t i s s u e a nd r e p o r t s o f m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n t h e b r a i n o f o t h e r s p e c i e s (Rudman et_ a l , 1973) , s e v e r a l t i s s u e s a m p l e s f r o m t h e h y p o t h a l a m i c r e g i o n were a s s a y e d . M e l a n o c y t e - s t i m u l a -t i n g a c t i v i t y i n f o u r f e t u s e s (two a s s a y s e a c h o f two p o o l e d . s a m p l e s ) a v e r a g e d I.36 ± 0.24 ng/mg. Lower a c t i v i t y (1.12 n g / mg) was f o u n d i n y o u n g f e t u s e s (39 a n d 40 d a y s ) a n d s l i g h t l y h i g h e r a c t i v i t y ( 1 . 5 9 ng/mg) o c c u r r e d i n l a t e r f e t u s e s (60 and 61 d a y s ) . B r a i n a c t i v i t y was o n l y 0.35$ a n d 0.15$, r e s p e c t i v e -l y , o f t h e a c t i v i t y i n t h e p o s t e r i o r p i t u i t a r y a t t h e same s t a g e 40 o f g e s t a t i o n . One s a m p l e t a k e n f r o m a f e m a l e a d u l t m e a s u r e d 0.75 ng/mg. 3. D i s c u s s i o n a. S i g n i f i c a n c e o f M e l a n o c y t e - s t i m u l a t i n g A c t i v i t y i n  F e t a l P i t u i t a r i e s M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n t h e g u i n e a p i g p i t u i t a r y a p p e a r s t o be i n c r e a s i n g r a p i d l y t h r o u g h o u t t h e l a t t e r h a l f o f g e s t a t i o n . T h i s r e p r e s e n t s b o t h a n i n c r e a s e i n t o t a l a c t i v i t y due t o g l a n d g r o w t h and a n i n c r e a s e i n a c t i v i t y p e r u n i t d r y w e i g h t . However a t t e r m , a c t i v i t y p e r u n i t d r y w e i g h t i s s t i l l o n l y a b o u t one h a l f o f t h a t .found i n t h e a d u l t . Thus t h e i n -c r e a s e i n a c t i v i t y l a t e i n g e s t a t i o n may be p a r t o f t h e d e v e l o p -m e n t a l p r o c e s s r a t h e r t h a n a change r e l a t i n g t o f e t a l s t a t u s p e r  s e . C e r t a i n l y i n t h e r a t , w h i c h i s b o r n i n a more i m m a t u r e s t a t e , p i t u i t a r y MSH a c t i v i t y d o e s n o t show a r a p i d i n c r e a s e u n t i l f i v e weeks p o s t p a r t u m (Howe, 1 9 7 3 ) • No o b v i o u s c h a n g e s were e v i d e n t w i t h p a r t u r i t i o n s i n c e hormone l e v e l s i n t h e p i t u i -t a r i e s o f a l i t t e r o f n ewborns c o r r e l a t e d w e l l w i t h t h e a c t i v i t y o f f e t u s e s o f t h e same s i z e a n d w e i g h t . P o s t e r i o r p i t u i t a r y l e v e l s (ng/mg) i n t h e newborns were s l i g h t l y h i g h e r b u t t h i s may be an a r t i f a c t . The w e i g h t o f t h e c o l l e c t e d t i s s u e was l o w e r t h a n e x p e c t e d . S t a l k t i s s u e , w h i c h c o n s i s t s o f l i t t l e o r no p a r s i n t e r m e d i a t i s s u e b u t c o n t r i b u t e s t o t h e o v e r a l l w e i g h t o f t h e p o s t e r i o r l o b e , may h a v e b e e n l e f t b e h i n d d u r i n g d i s s e c t i o n . The l o w e r w e i g h t o f t h e r e m a i n i n g t i s s u e w o u l d r e s u l t i n an a p p a r e n t i n c r e a s e i n a c t i v i t y . G u i n e a p i g p i t u i t a r i e s c o n t a i n a r e l a t i v e l y l a r g e amount o f m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y . Of t e n s p e c i e s f r o m v a r i o u s 4 1 v e r t e b r a t e c l a s s e s , a s s a y e d by S h a p i r o e t a l (1972), g u i n e a p i g p i t u i t a r i e s c o n t a i n e d t h e h i g h e s t b i o l o g i c a l MSH a c t i v i t y (900 -1500 ng/mg). A d u l t v a l u e s m e a s u r e d i n t h e c u r r e n t s t u d y w e re h i g h e r , a v e r a g i n g 2600 ng/mg. T h i s was e x p e c t e d s i n c e a c t i v i t y i n t h e f o r m e r s t u d y was m e a s u r e d p e r wet w e i g h t . I n c o n t r a s t , l e v e l s i n t h e r a t r a n g e d f r o m 100 t o 300 ng/mg a n d were u n d e r 200 ng/mg i n t h e cow (wet w e i g h t s ) . H o w e v e r , T i l d e r s e_t a l (1975) f o u n d o v e r 2000 ng/mg o f b i o l o g i c a l a c t i v i t y i n r a t p i -t u i t a r i e s . W h i l e o t h e r s t u d i e s g i v e s i m i l a r v a l u e s , a f a i r amount o f v a r i a t i o n i s e v i d e n t i n r e p o r t e d a c t i v i t i e s . The r a t i o o f p o s t e r i o r t o a n t e r i o r l o b e . a c t i v i t y was l o w e r t h a n e x p e c t e d . C o n c e n t r a t i o n s i n ng/mg w e r e , on a v e r a g e , o n l y 6.7 t i m e s g r e a t e r i n t h e p o s t e r i o r l o b e ; t o t a l p o s t e r i o r l o b e a c t i v i t y was o n l y 3-0 t i m e s g r e a t e r . Thus a s i g n i f i c a n t p o r t i o n o f t h e h i g h o v e r a l l a c t i v i t y i n t h e g u i n e a p i g p i t u i -t a r y i s due t o t h e h i g h a c t i v i t y f o u n d i n t h e a n t e r i o r l o b e . S h a p i r o et_ a l (1972) d i d n o t m e a s u r e t h e i n d i v i d u a l l o b e s o f t h e g u i n e a p i g p i t u i t a r y s e p a r a t e l y b u t , i n t h e r a t t h e y f o u n d a r a t i o o f a c t i v i t i e s o f a b o u t 30 (ng/mg). B j o r k l u n d et_ a l (1972) f o u n d o n l y . 1 . 0 % o f t h e t o t a l g l a n d u l a r m e l a n o c y t e - s t i m u -l a t i n g a c t i v i t y i n t h e a n t e r i o r l o b e o f t h e r a t . O t h e r s t u d i e s i n d i c a t e t h a t a b o u t 10$ o f t h e t o t a l m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n t h e cow i s p r e s e n t i n t h e a n t e r i o r l o b e (Rudman e t a l , 1973)- E v e n c o m p a r e d t o t h e l a s t s t u d y . t h e a n t e r i o r l o b e o f t h e g u i n e a p i g p i t u i t a r y c o n t a i n s a v e r y l a r g e p e r c e n t a g e o f t h e t o t a l b i o l o g i c a l a c t i v i t y i n t h e w h o l e g l a n d . T h i s c o n t r i b u t i o n may e x p l a i n i n p a r t t h e r e l a t i v e l y h i g h o v e r a l l a c t i v i t y f o u n d i n t h i s s p e c i e s . The c o m p a r i s o n s a b o v e a l s o 42 show t h a t a l a r g e amount of v a r i a t i o n i n the c o n t r i b u t i o n of the pars d i s t a l i s t o o v e r a l l a c t i v i t y e x i s t s among d i f f e r e n t s p e c i e s . As i n o t h e r r o d e n t s , alpha-MSH appears t o be the predom-i n a n t MSH p e p t i d e i n the g u i n e a p i g ( H o e k s t r a & Van der Wal, 1 9 7 1 ) . I n bo t h the r a t and the mouse s i g n i f i c a n t amounts of alpha-MSH have been found i n the pa r s d i s t a l i s (Mains & E i p p e r , 1 9 7 5 ; U s a t e g u i e t a l , 1 9 7 6 ) . S i n c e alpha-MSH i s the most p o t e n t of a l l m e l a n o c y t e - s t i m u l a t i n g p e p t i d e s , s i g n i f i c a n t conc-e n t r a t i o n s of t h i s p e p t i d e i n the pa r s d i s t a l i s might account f o r the h i g h a c t i v i t y measured i n the g u i n e a p i g . S i n c e ACTH has o n l y one t o t h r e e p e r c e n t of the m e l a n o c y t e - s t i m u l a t i n g potency of alpha-MSH, q u a n t i t i e s i n excess of 10 ug/mg would be r e q u i r e d t o account f o r the m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y measured i n the a d u l t . C e r t a i n l y i n the r a t ACTH c o n c e n t r a t i o n s i n the pa r s d i s t a l i s are under 1 ug/mg ( U s a t e g u i e_fc a l , 1 9 7 6 ) and i n the g u i n e a p i g the whole p i t u i t a r y c o n t a i n s under 0.5 ug/ mg of b i o l o g i c a l ACTH a c t i v i t y ( S h a p i r o e t a l , 1 9 7 2 ) . Thus the r e s u l t s s t r o n g l y suggest t h a t the g u i n e a p i g pa r s d i s t a l i s may c o n t a i n s i g n i f i c a n t amounts of alpha-MSH. P o s t e r i o r lobe m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i s a l s o h i g h e r i n the g u i n e a p i g as compared t o the r a t . P e t a l l e v e l s l a t e i n g e s t a t i o n are a c t u a l l y h i g h e r t h a n l e v e l s measured i n a d u l t r a t s . A g a i n t h i s h i g h a c t i v i t y s uggests t h a t l a r g e con-c e n t r a t i o n s of alpha-MSH are p r e s e n t . I n g e n e r a l , s i g n i f i c a n t q u a n t i t i e s of alpha-MSH are p r o b a b l y p r e s e n t i n g u i n e a p i g p i t u i t a r i e s ; j u s t how much a c t i v i t y i s .43 due t o beta-MSH, ACTH o r L P H , w h i c h a r e a l s o l i k e l y t o be p r e s e n t , r e m a i n s t o be a n s w e r e d . b. S i g n i f i c a n c e o f M e l a n o c y t e - s t i m u l a t i n g A c t i v i t y  i n t h e B r a i n B i o l o g i c a l m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y h a s b e e n l o -c a t e d i n many r e g i o n s o f t h e b r a i n i n s e v e r a l s p e c i e s i n c l u d i n g t h e cow, t h e monkey, man (Rudman e_t a l , 1 9 7 3 ) and t h e r a t (Rudman e_t a l , 1 9 7 4 a ) . A c t i v i t y was h i g h e s t i n t h e h y p o t h a l a m u s b u t s i g n i f i c a n t amounts were p r e s e n t i n t h e c h o r o i d p l e x u s a n d i n t h e p i n e a l . I n t h e r a t , O l i v e r a n d P o r t e r ( 1 9 7 8 ) f o u n d 0 . 1 6 3 ± 0.044 ng/mg o f alpha-MSH a c t i v i t y i n t h e p i n e a l a n d 0.143 ± 0.022 ng/mg o f alpha-MSH a c t i v i t y i n t h e h y p o t h a l a m u s . S i n c e t h e s e a c t i v i t i e s r e p r e s e n t o n l y i m m u n o r e a c t i v e a l p h a - M S H , a n d as ACTH a n d LPH a r e a l s o p r e s e n t i n b r a i n t i s s u e ( K r i e g e r e_t a l , 1 9 7 7 ) , t h e b i o l o g i c a l m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y may d i f f e r somewhat. I n t h e p r e s e n t s t u d y a c t i v i t y i n one a d u l t m e a s u r e d O.76 ng/mg ( a p p r o x i m a t e l y f i v e t i m e s t h e i m m u n o r e a c t i v e a c t i -v i t y i n t h e h y p o t h a l a m u s o f t h e r a t ) a n d i n f o u r f e t u s e s a v e r -a g e d I . 3 6 ± 0.24 ng/mg. A l t h o u g h t h e a c t i v i t y m e a s u r e d i n b r a i n t i s s u e i s much l o w e r t h a n t h a t f o u n d i n t h e p i t u i t a r y , l e v e l s a r e r e l a t i v e l y h i g h as c o m p a r e d t o a c t i v i t y i n t h e b r a i n t i s s u e o f t h e r a t . The s i g n i f i c a n c e o f e v e n h i g h e r a c t i v i t y i n t h e g u i n e a p i g f e t u s must a w a i t f u r t h e r s t u d y . The p r e s e n c e o f MSH a n d r e l a t e d p e p t i d e s i n b r a i n t i s s u e i s i n t e r e s t i n g , e s p e c i a l l y c o n s i d e r i n g t h e b e h a v i o u r a l e f f e c t s o f t h e s e p e p t i d e s b o t h i n man a n d o t h e r s p e c i e s . A c t i v i t y m e a s u r e d i n h y p o p h y s e c t o m i z e d a n i m a l s s u g g e s t s t h a t t h e p i t u i - v t a r y may n o t be t h e p r i m a r y s o u r c e o f alpha-MSH i n t h e ' b r a i n . The presence of MSH i n v e s i c l e s of s m a l l s i z e suggests t h a t the p e p t i d e may a c t as a n e u r o t r a n s m i t t e r ( P e l l e t i e r & Dube, 1 9 7 7 ) . S i n c e LPH a c t s as a p r e c u r s o r f o r a g o n i s t s of o p i a t e r e c e p t o r s i n the b r a i n , t h i s whole f a m i l y of p e p t i d e s may be i m p o r t a n t i n v a r i o u s areas of n e u r a l f u n c t i o n i n g . 45 SECTION I I : MELANOCYTE-STIMULATING A C T I V I T Y IN. FETAL, ADULT AND NEWBORN BLOOD PLASMA 1. I n t r o d u c t i o n As i n t h e a d u l t , s u b s t a n t i a l q u a n t i t i e s o f m e l a n o c y t e -s t i m u l a t i n g a c t i v i t y a r e p r e s e n t i n t h e p i t u i t a r y o f t h e g u i n e a p i g f e t u s . I n i t s e l f , t h i s s i t u a t i o n may o n l y r e p r e s e n t a s y n t h e s i s a n d s t o r a g e o f p e p t i d e w i t h o u t c o n c o m i t a n t r e l e a s e i n t o t h e b l o o d s t r e a m . D e m o n s t r a t i o n o f s i g n i f i c a n t c i r c u l -a t i n g l e v e l s o f a c t i v e p e p t i d e w o u l d l e n d f u r t h e r s u p p o r t t o t h e p o s s i b i l i t y t h a t MSH c o u l d be t r o p h i c i n t h e f e t u s . D a t a on c i r c u l a t i n g m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n t h e f e t u s a r e a l m o s t t o t a l l y l a c k i n g . A n c e s and P o m e r a n t z ( 1 9 7 4 ) f o u n d s i g n i f i c a n t l y h i g h e r l e v e l s o f i m m u n o r e a c t i v e beta-MSH i n c o r d v e n o u s b l o o d s a m p l e s f r o m n e w b o r n i n f a n t s as com p a r e d t o m a t e r n a l l e v e l s ( 4 4 9 - 5 ± 81 . 1 a n d 246.7 ± 15-8 p g / m l r e -s p e c t i v e l y ) . N o r m a l l e v e l s i n n o n p r e g n a n t women were a b o u t 50 p g / m l o f p l a s m a o r l e s s . E a r l i e r e v i d e n c e s u g g e s t e d t h a t m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n m a t e r n a l b l o o d was e l e v a t e d i n human p r e g n a n c y and t h a t l e v e l s i n c r e a s e d t o w a r d t e r m ( S h i z u m e & L e r n e r , 1 9 5 4 ) . The l a t e r s t u d y s u p p o r t s t h e e a r l i e r o b s e r v a t i o n b u t s i n c e S h i z u m e a nd L e r n e r u s e d a r b i t r a r y u n i t s i n m e a s u r i n g a c t i v i t y a d i r e c t c o m p a r i s o n i s n o t p o s s i b l e . The p e r c e n t a g e i n c r e a s e , h o w e v e r , does seem s i m i l a r . Thody e_t a l ( 1 9 7 4 ) f o u n d n o r m a l p l a s m a l e v e l s o f 6 0 . 7 ± 3-8 p g / m l beta-MSH ( b o t h s e x e s ) . W h i l e t h e s e r e s u l t s a g r e e w i t h t h o s e o f A n c e s a n d P o m e r a n t z ( 1 9 7 4 ) , a much s m a l l e r i n c r e a s e was f o u n d i n p r e g n a n t women ( 1 0 1 . 9 ± 1 1 . 3 pg/ml) and a l l v a l u e s w e re w i t h i n t h e same r a n g e as t h e m a l e a nd f e m a l e c o n t r o l s . W h i l e S i l m a n et_ a l ( 1 9 7 5 ) f o u n d no i m m u n o r e a c t i v e a l p h a - M S H d u r i n g l a t e p r e g n a n c y , C l a r k e t a l ( 1 9 7 8 ) r e p o r t e d d e t e c t a b l e l e v e l s o f p l a s m a immuno-r e a c t i v e a l p h a - M S H i n 50$ o f s u b j e c t s i n t h e t h i r d t r i m e s t e r o f p r e g n a n c y (mean o f 108 ± 5 p g / m l ) . P l a s m a ACTH l e v e l s c o n t r i b u t e t o m e a s u r e d m e l a n o c y t e - s t i n m u l a t i n g a c t i v i t y . . P l a s m a a c t i v i t y i n t h e f e t u s h a s b e e n s t u -d i e d i n t h e sheep a n d i n t h e human. I n t h e s h e e p , p l a s m a l e v e l s i n c r e a s e o v e r t h e s e c o n d h a l f o f p r e g n a n c y f r o m 1 1 7 ± 19 p g / m l t o 2 86 ± 63 p g / m l . M a t e r n a l p l a s m a ACTH was u n c h a n g e d o v e r t h e same t i m e p e r i o d (64 ± 9 pg/ml) ( J o n e s et_ a l , 1 9 7 7 ) . I n c o n -t r a s t , hormone l e v e l s i n t h e human f e t u s p e a k much e a r l i e r i n g e s t a t i o n (249 ± 6 5 . 7 p g / m l , 1 2 - 1 9 w e e k s ) a n d a v e r a g e 143 ± 7 p g / m l j u s t b e f o r e t e r m ( W i n t e r s e t a l , 1 9 7 4 ) . Women i n l a b o u r e x h i b i t e d h i g h e r p l a s m a l e v e l s ( 1 9 4 ± 2 8 . 3 pg/ml) t h a n t e r m i n -f a n t s . S i n c e ACTH was p r e s e n t i n s u c h s m a l l amounts ( p g l e -v e l s ) , m e a s u r a b l e m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y p r e s e n t i n t h e p l a s m a o f t h e sh e e p o r man c o u l d n o t be a t t r i b u t a b l e t o i t . E v i d e n c e s u g g e s t s t h a t MSH l e v e l s may be s i g n i f i c a n t l y e l e -v a t e d i n m a t e r n a l p l a s m a d u r i n g p r e g n a n c y i n man. Immunoreac-t i v e MSH i s a l s o e l e v a t e d i n t h e p l a s m a o f . t h e human a t b i r t h . However,' no a t t e m p t has b e e n made t o m e a s u r e m e l a n o c y t e - s t i m u l a -t i n g a c t i v i t y i n f e t a l p l a s m a d u r i n g g e s t a t i o n . T h e r e f o r e , p l a s m a m e l a n o c y t e - s t i m u l a t i n g a c t i v i t i e s w e r e m e a s u r e d i n t h e f e t a l g u i n e a p i g o v e r t h e l a s t t r i m e s t e r o f p r e g n a n c y . T h i s a c -t i v i t y c o u l d t h e n be c o m p a r e d t o t h e s i g n i f i c a n t p i t u i t a r y l e ^ v e l s m e a s u r e d o v e r t h e same t i m e p e r i o d . A c t i v i t y was a l s o meas-u r e d i n n e w b o r n p l a s m a and i n b o t h c o n t r o l a n d p r e g n a n t f e m a l e s . 47 2. R e s u l t s a. Assay Procedure I n i t i a l l y plasma samples were assayed by s i m p l y d i l u t i n g crude plasma w i t h R i n g e r ' s s o l u t i o n so t h a t the plasma made up no more tha n 10$ of the f i n a l s o l u t i o n (Abe & N i c h o l s o n , 1 9 7 5 ) . However, when t h i s method was used, t h e r e was o f t e n a p a r t i a l i n h i b i t i o n of the normal response of the A n o l i s s k i n t o m e l a n o c y t e - s t i m u l a t i n g m a t e r i a l p r e s e n t . The r e s u l t a n t dose-response curves were not p a r a l l e l t o the s t a n d a r d curve and were o f t e n o p p o s i t e i n s l o p e . T h e r e f o r e subsequent samples were e x t r a c t e d w i t h s i l i c i c a c i d as d e s c r i b e d i n the methods. When s t a n d a r d MSH was added t o the plasma, and e x t r a c t e d i n t h i s manner, r e c o v e r y averaged 80%. I n c o n t r a s t , the r e c o v e r y o f s t a n d a r d MSH when e x t r a c t e d from R i n g e r ' s s o l u t i o n a l o n e , u s i n g the same method, was never more than 5 0 $ . The average a c t i v i t i e s measured i n a d u l t , f e t a l and new-born, plasma are compared i n F i g u r e 8. S i g n i f i c a n t d i f f e r e n c e s between sample means were t e s t e d u s i n g S t u d e n t ' s t t e s t f o r u n p a i r e d o b s e r v a t i o n s w i t h e q u a l v a r i a n c e s , ( S t e e l & T o r r i e , I 9 6 0 ) . b. M e l a n o c y t e - s t i m u l a t i n g A c t i v i t y i n F e t a l Plasma T h i r t e e n p o o l e d samples, each c o l l e c t e d from a s i n g l e l i t t e r of from two t o f i v e f e t u s e s ( t o t a l number of f e t u s e s = 45) were assayed u s i n g the s i l i c i c a c i d method. Samples were t a k e n from f e t u s e s between 45 and 70 days of g e s t a t i o n and sample volumes ranged from 0.4 t o 4.0 ml. Assay v a l u e s were c o r r e c t e d f o r the measured l o s s i n a c t i v i t y due t o e x t r a c t i o n as c a l c u l a t e d u s i n g s t a n d a r d MSH (see above). Plasma v a l u e s averaged 10.0 ± 48 F i g u r e 8: A comparison of the m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y (ng of alpha-MSH/ml) i n the plasma of the normal female a d u l t , pregnant a d u l t , f e t u s and newborn g u i n e a p i g . A c t i v i t i e s r e p r e s e n t the sample mean ± the s t a n d a r d e r r o r of the mean. The number of measurements i s g i v e n f o r each group. F o r the f e t u s and newborn, each measurement r e p r e s e n t s a p o o l e d sample c o l l e c t e d from a s i n g l e l i t t e r ( t o t a l number of an i m a l s r e p r e s e n t e d = 45 and 10 r e s p e c t -i v e l y ) . The a c t i v i t y i n the plasma of newborns was s i g n i -f i c a n t l y d i f f e r e n t from t h a t measured i n o t h e r groups but o n l y a t P<0.1 ( S t u d e n t ' s t t e s t f o r the comparison of two means, u n p a i r e d o b s e r v a t i o n s , e q u a l v a r i a n c e s ) . O r d i n a t e : Average m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n e q u i v a l e n t amounts of s y n t h e t i c alpha-MSH, as. measured by b i o a s s a y in. v i t r o , i n ng/ml of plasma. MELANOCYTE - STIMULATING ACTIVITY ng of alpha-MSH/ml plasma 50 1.6 ng/ml and ranged from 3.66 ng/ml t o 18.4 ng/ml. Over the p e r i o d o f g e s t a t i o n measured, no c l e a r i n c r e a s e or decrease i n plasma a c t i v i t y was observed (see F i g u r e 9 ) . The v a r i a t i o n i n measured a c t i v i t y i s q u i t e h i g h , a t l e a s t from 57 days u n t i l term. c. M e l a n o c y t e - s t i m u l a t i n g A c t i v i t y i n the Plasma o f A d u l t s Plasma samples c o l l e c t e d from f i v e pregnant g u i n e a p i g s were assayed as above. Two plasma samples of lower a c t i v i t y were t a k e n from mothers e a r l y i n g e s t a t i o n ( w e l l b e f o r e 45 days) and two samples of h i g h e r a c t i v i t y were t a k e n from mothers c a r r y i n g f e t u s e s of 66 days g e s t a t i o n . A c t i v i t y i n these f o u r samples ranged from 6.1 t o 1 8 . 5 ng/ml and averaged 1 2 . 1 ± 2.8 ng/ml. The v a r i a t i o n observed was s i m i l a r t o t h a t found i n the f e t u s and a l t h o u g h the average a c t i v i t y i n a d u l t s i s h i g h e r , the d i f f e r e n c e was not s t a t i s t i c a l l y s i g n i f i c a n t . I n the f i f t h case the a c t i v i t y was v e r y low and c o u l d be accounted f o r by the e r r o r i n the assay. There were no obvious d i f f e r e n c e s i n e i t h e r the a n i m a l or sample i n v o l v e d t h a t might account f o r t h i s d i s c r e p a n c y . T h i s a n i m a l was c a r r y i n g f e t u s e s of 65 days g e s t a t i o n . Plasma samples c o l l e c t e d from t h r e e nonpregnant females were a l s o assayed. Measured a c t i v i t y ranged from 3.8 t o 19.0 ng/ml and averaged 7-9 ± 2.2 ng/ml. The average i s over 4 ng/ml l e s s than t h a t found i n pregnant a n i m a l s b u t , due t o the v a r i -a t i o n p r e s e n t and the s m a l l sample s i z e s , the d i f f e r e n c e i s not s i g n i f i c a n t . There i s a l s o no s i g n i f i c a n t d i f f e r e n c e as comp-are d t o f e t a l plasma l e v e l s . 51 F i g u r e 9: The r e l a t i o n s h i p between the age of the g u i n e a p i g f e t u s , i n days of g e s t a t i o n , and the melano-c y t e - s t i m u l a t i n g a c t i v i t y o f the plasma as measured i n ng of alpha-MSH p e r ml. Each p o i n t r e p r e s e n t s the average b i o l o g i c a l a c t i v i t y of plasma c o l l e c t e d from l i t t e r m a t e s and p o o l e d b e f o r e e x t r -a c t i o n . The t o t a l number of f e t u s e s r e p r e s e n t e d i s g i v e n f o r each sample. V a l u e s are c o r r e c t e d f o r the c a l c u l a t e d l o s s i n a c t i v i t y due t o the s i l i c i c a c i d e x t r a c t i o n p r o c -edure . A b s c i s s a : Age of the f e t u s i n days of g e s t a t i o n as c a l c u l a t e d u s i n g the d a t a of Draper (1920) and I b s e n (1928) . O r d i n a t e : M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y p e r ml of plasma i n e q u i v a l e n t amounts of s y n t h e t i c alpha-MSH, as measured by b i o a s s a y i n v i t r o . > < E 51 25 20 15 *3 ' 5 ' 5 O c LU 2 10 hp A//>l i 0"40 *5 •3 *2 •4 *4 *2 *3 J _ J 50 60 70 GESTATIONAL AGE - DAYS IV) 53 d. M e l a n o c y t e - S t l m u l a t l n g A c t i v i t y I n the Plasma of  Newborns Plasma samples were c o l l e c t e d from two l i t t e r s o f new-borns ( t o t a l of 10 pups) and assayed as d e s c r i b e d above. The ages of the pups I n the two l i t t e r s were judged t o be approx-i m a t e l y two and between f o u r and s i x hours r e s p e c t i v e l y . Cor-r e c t e d plasma a c t i v i t y i n the younger l i t t e r was 1.29 ng/ml; i n the o l d e r l i t t e r the a c t i v i t y was almost t w i c e as h i g h ( 2 . 5 5 ng/ml). The average a c t i v i t y was 1.92 ± 0.63 ng/ml. T h i s v a l u e I s s i g n i f i c a n t l y d i f f e r e n t from the a c t i v i t y found i n b o th the f e t u s and i n normal and pregnant a d u l t females but o n l y a t the P<0.1 l e v e l . 3. D i s c u s s i o n a. S i g n i f i c a n c e of M e l a n o c y t e - S t i m u l a t i n g A c t i v i t y i n  Plasma , S i g n i f i c a n t amounts of m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y are p r e s e n t i n the plasma of f e t a l , a d u l t and newborn g u i n e a p i g s . The s p e c i f i c hormone or hormones i n v o l v e d cannot be p o s i t i v e l y i d e n t i f i e d from the p r e s e n t r e s u l t s . However, the h i g h a c t i -v i t y measured i n p i t u i t a r y t i s s u e , c o u p l e d w i t h the e l e c t r o -p h o r e t i c s t u d i e s of o t h e r workers i n c l u d i n g H o e k s t r a and Van der Wal ( 1 9 7 1 ) , suggests t h a t s u b s t a n t i a l c o n c e n t r a t i o n s of a l p h a -MSH are p r o b a b l y p r e s e n t . Thus, s u b s t a n t i a l amounts of a l p h a -MSH may be p r e s e n t i n the plasma. Indeed, r e l a t i v e l y h i g h a c t i -v i t i e s were measured i n t h i s s t u d y . I f a l a r g e p e r c e n t a g e of the a c t i v i t y measured i n the plasma, of e i t h e r the a d u l t or f e t u s , were due t o l e s s p o t e n t p e p t i d e s , c o n c e n t r a t i o n s of up t o 100 times would be r e q u i r e d t o account f o r the a c t i v i t y meas-ur e d . For example, ACTH c o n c e n t r a t i o n s of w e l l over 1.0 ug/ml o f p l a s m a w o u l d be r e q u i r e d when ACTH i s u s u a l l y m e a s u r e d a t pg l e v e l s i n t h e p l a s m a o f most s p e c i e s i n b o t h t h e a d u l t a n d t h e f e t u s ( s e e i n t r o d u c t i o n , t h i s s e c t i o n ) . Thus w h i l e ACTH and f o r m s o f LPH may a c c o u n t f o r some o f t h e a c t i v i t y m e a s u r e d , a l p h a - , a n d p o s s i b l y beta-MSH p r o b a b l y a c c o u n t f o r t h e l a r g e s t p e r c e n t a g e . As w i t h t h e p i t u i t a r y r e s u l t s , m e l a n o c y t e - s t i m u l a t i n g a c -t i v i t i e s i n t h e p l a s m a ' o f b o t h a d u l t and f e t a l g u i n e a p i g s a r e , f o r t h e most p a r t , h i g h e r t h a n l e v e l s r e p o r t e d i n o t h e r s p e c i e s . A l t h o u g h v a l u e s g i v e n f o r t h e a d u l t r a t v a r y , t h e y a r e c o n s i s -t e n t l y l o w e r t h a n t h e a v e r a g e o f 7-93 ng/ml m e a s u r e d i n n o n -p r e g n a n t g u i n e a p i g s . U s i n g b i o a s s a y t e c h n i q u e s B j o r k l u n d et_ a l ( 1 9 7 2 ) f o u n d 1.2 ng alp h a - M S H / m l o f r a t p l a s m a , w h i l e T i l d e r s e t a l ( 1 9 7 5 ) m e a s u r e d o n l y 0.4 n g / m l . C e l i s ( 1 9 7 5 ) h o w e v e r , f o u n d up t o 8 ng/ml o f b i o l o g i c a l MSH i n r a t serum d u r i n g p r o -e s t r u s ; l e v e l s i n a l l o t h e r p a r t s o f t h e , c y c l e were b e l o w 2 n g / m l . V a r i a t i o n i n t h e p l a s m a a c t i v i t y i n f e m a l e g u i n e a p i g s s e e n i n t h i s s t u d y may be due t o s i m i l a r c h a n g e s . By r a d i o i m m u n o -a s s a y U s a t e g u i et_ a l ( 1 9 7 7 ) m e a s u r e d l e s s t h a n 0.1 ng/ml o f alpha-MSH i n t h e p l a s m a o f u n t r e a t e d r a t s . I m m u n o r e a c t i v e b e t a -MSH l e v e l s a r e v e r y . l o w i n human p l a s m a a v e r a g i n g b e t w e e n 15 and 60 p g / m l ( T a n a k a e t a l , 1 9 7 8 ) . P l a s m a m e l a n o c y t e - s t i m u l a t i n g a c t i v i t i e s m e a s u r e d i n t h e * f e t a l g u i n e a p i g show some v a r i a b i l i t y , b u t o v e r a l l , l e v e l s d i d n o t a p p e a r t o change m a r k e d l y f r o m 45 d a y s o f g e s t a t i o n u n t i l t e r m . A l t h o u g h a c t i v i t y was h i g h e r t h a n t h a t f o u n d i n n o n p r e g -n a n t a d u l t s and l o w e r t h a n t h e a v e r a g e a c t i v i t y i n m a t e r n a l s a m p l e s , t h e d i f f e r e n c e s were n o t s i g n i f i c a n t ( s e e F i g u r e 8 ) . 55 More a d u l t s a m p l e s w o u l d have t o be a s s a y e d i n o r d e r t o d e t e r -mine w h e t h e r t h i s r e s u l t r e l a t e d t o t h e s m a l l s a m p l e s i z e s o r a r e a l s i m i l a r i t y i n p l a s m a a c t i v i t i e s . As s e e n i n t h e human, p l a s m a m e l a n o c y t e - s t i m u l a t i n g hormone l e v e l s may be h i g h e r i n p r e g n a n c y i n t h e g u i n e a p i g . A l t h o u g h t h e d a t a s u g g e s t t h a t a r e a l i n c r e a s e may be p r e s e n t , t h e d i f f e -r e n c e i n t h e means was n o t s i g n i f i c a n t ( S t u d e n t ' s t t e s t ) . A s i m i l a r i n s i g n i f i c a n t i n c r e a s e o c c u r r e d i n human p r e g n a n c i e s (Thody e t a l , 1 9 7 4 ) . A l s o t h e l i m i t e d d a t a a v a i l a b l e s u g g e s t t h a t l e v e l s may be r i s i n g p r o g r e s s i v e l y t h r o u g h o u t g e s t a t i o n ; t h i s has a l s o b e e n o b s e r v e d i n t h e human ( S h i z u m e & L e r n e r , 1 9 5 4 ) . C e r t a i n l y t h e p a r a l l e l i s i n t e r e s t i n g a n d may c h a r a c t e r -i z e some common e l e m e n t of. mammalian p r e g n a n c y . The a b s e n c e o f m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n one p r e g n a n t a d u l t i s p u z z -l i n g . No i r r e g u l a r i t i e s o c c u r r e d i n t h e c o l l e c t i o n o r e x t r a c -t i o n o f t h e p l a s m a and t h e a n i m a l i n v o l v e d seemed h e a l t h y , as were t h e f e t u s e s . M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y m e a s u r e d i n t w o p l a s m a sam-p l e s c o l l e c t e d f r o m two l i t t e r s o f n e wborns was l o w e r t h a n a c t i -v i t y i n e i t h e r a d u l t o r f e t a l p l a s m a . W h i l e F i g u r e 8 s u g g e s t s t h a t t h e d i f f e r e n c e i n a c t i v i t y may be r e a l , t h e d i f f e r e n c e was o n l y s i g n i f i c a n t a t t h e P<0.1 l e v e l a n d must be a c c e p t e d w i t h r e s e r v a t i o n a t t h i s t i m e . P l a s m a t a k e n f r o m t h e y o u n g e s t l i t t e r ( a b o u t two h o u r s oldO h a d t h e l o w e s t a c t i v i t y ( 1 . 2 9 ng/ml) o f any s a m p l e m e a s u r e d i n t h i s s t u d y . A l t h o u g h a c t i v i t y m e a s u r e d i n t h e s e c o n d l i t t e r ( f o u r t o s i x h o u r s o l d ) was a l m o s t d o u b l e t h a t o f t h e f i r s t ( 2 . 5 5 n g / m l ) , i t i s s t i l l o v e r 1 ng/ml b e l o w t h e l o w e s t a c t i v i t i e s m e a s u r e d i n e i t h e r t h e f e t u s o r a d u l t . 56 The r e s u l t s suggest t h a t a l a r g e drop I n hormone l e v e l s may occur at b i r t h , or j u s t a f t e r , f o l l o w e d by a l a t e r r e c o v -e r y . More work i s needed t o i n v e s t i g a t e these changes i n d e t a i l . A s i m i l a r s i t u a t i o n i s seen i n g l u c o c o r t i c o i d con-c e n t r a t i o n s i n newborn humans ( S i p p e l l e_t a l , 1978). L e v e l s f e l l s i g n i f i c a n t l y between two and s i x hours a f t e r b i r t h but i n c r e a s e d a g a i n by 12 h o u r s . There i s some e v i d e n c e t h a t ACTH may f o l l o w a s i m i l a r p a t t e r n . The a u t h o r s suggested t h a t these changes r e p r e s e n t e d a d a p t a t i o n phenomena seen i n newborns d u r i n g the f i r s t few hours a f t e r b i r t h . Comparisons w i t h o t h e r s p e c i e s may not be r e l e v a n t , however. Recent e v i d e n c e suggests t h a t c o r t i c o s t e r o n e l e v e l s i n the r a t show a s l i g h t , but s i g n i -f i c a n t , i n c r e a s e at b i r t h f o l l o w e d by a l a t e r decrease ( C o r b i e r & R o f f i , 1978). b. Source of M e l a n o c y t e - S t i m u l a t i n g A c t i v i t y i n F e t a l  Plasma A l t h o u g h the f e t u s appears t o be g e n e r a l l y autonomous I n the c o n t r o l of i t s e n d o c r i n e f u n c t i o n , i t I s s t i l l p o s s i b l e t h a t m a t e r n a l hormones may c r o s s the p l a c e n t a . T h i s must be kept i n mind when one c o n s i d e r s the s i g n i f i c a n c e of any h o r -mone l e v e l s measured i n f e t a l plasma. Q u e s t i o n s i n v o l v i n g p l a c -e n t a l t r a n s f e r of m a t e r n a l p e p t i d e hormones are l a r g e l y unans-wered, c e r t a i n l y i n the g u i n e a p i g . P l a c e n t a l t r a n s p o r t of t h y r o x i n e from mother t o f e t u s i s m e a s urable, but c o n s i d e r e d t o be l i m i t e d i n the g u i n e a p i g (London e_t a l , 1963). O x y t o c i n , however, r e a d i l y c r o s s e s the g u i n e a p i g p l a c e n t a i n b o t h d i r e c t i o n s , a t l e a s t between 6 l and 65 days of g e s t a t i o n ( B u r t o n e t a l , 1974). I t has been 57 suggested t h a t ACTH may c r o s s the p l a c e n t a and s t i m u l a t e the f e t a l a d r e n a l d u r i n g the i n i t i a t i o n of p a r t u r i t i o n (see I l l i n g w o r t h e_t a l , 1 9 7 4 ) . S i n c e f e t a l and m a t e r n a l plasma m e l a n o c y t e - s t i m u l a t i n g a c t i v i t i e s are v e r y s i m i l a r i n the p r e s e n t s t u d y , p l a c e n t a l t r a n s f e r between mother and f e t u s may o c c u r . However, the l i m i t e d d a t a a v a i l a b l e s u g gests t h a t m a t e r n a l plasma m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y may be i n c -r e a s i n g as g e s t a t i o n p r o g r e s s e s . I f p l a c e n t a l t r a n s f e r of these p e p t i d e s was s u b s t a n t i a l l a t e i n g e s t a t i o n , one would expect a p a r a l l e l i n c r e a s e i n plasma l e v e l s i n the f e t u s . S i n c e no I n c r e a s e was ob s e r v e d , m a t e r n a l plasma a c t i v i t y may not con-t r i b u t e t o a c t i v i t y measured i n the f e t u s . E v i d e n c e suggests t h a t t h e r e i s l i t t l e p l a c e n t a l t r a n s f e r of ACTH and MSH i n the human. No r e l a t i o n s h i p was found b e t -ween the ACTH c o n c e n t r a t i o n s i n p a i r e d samples of m a t e r n a l and f e t a l b l o o d ( A l l e n e t a l , 1 9 6 3 ; W i n t e r s e t a l , 1 9 7 4 ) . MSH c o n c e n t r a t i o n s i n c o r d b l o o d were s i g n i f i c a n t l y h i g h e r than those found i n the m a t e r n a l c i r c u l a t i o n (Ances & Pomerantz, 1 9 7 4 ) . While the g u i n e a p i g p l a c e n t a i s s i m i l a r i n s t r u c t u r e t o t h a t of the human, the t r a n s f e r of p e p t i d e s between mother and f e t u s may not be comparable I n the two s p e c i e s . F u r t h e r s t u d i e s are r e q u i r e d t o determine whether m e l a n o c y t e - s t i m u l a t i n g a c t -i v i t y i n g u i n e a p i g f e t a l plasma i s of f e t a l o r i g i n . c. Comments on M e l a n o c y t e - S t i m u l a t i n g A c t i v i t y and Plasma The s i l i c i c a c i d e x t r a c t i o n p r o c e d u r e was used because crude plasma, even d i l u t e d by a f a c t o r of 10 w i t h R i n g e r ' s s o l -u t i o n , had an i n h i b i t o r y e f f e c t on the response of l i z a r d s k i n . 58 I s l a n d e t a l (1965) r e p o r t e d t h e same e f f e c t a t a d i l u t i o n f a c t o r o f 5) h u t n o t 10, u s i n g a f r o g s k i n b i o a s s a y . H o w e v e r , K a s t i n et_ a l (1969) s o m e t i m e s o b t a i n e d i n h i b i t o r y e f f e c t s a t a d i l u t i o n f a c t o r o f 10 i n h i s i n v i t r o f r o g " s k i n b i o a s s a y . B o t h o f t h e s e r e p o r t s r e f e r t o human p l a s m a . I n t h e p r e s e n t s t u d y t h i s i n h i b i t o r y e f f e c t p r o d u c e d a v e r y f l a t l o g d o s e -r e s p o n s e c u r v e w h i c h , a t t i m e s , was o p p o s i t e i n s l o p e t o t h a t o f t h e s t a n d a r d . V a r i a b i l i t y i n t h e s l o p e o f t h e r e s p o n s e c u r v e s u g g e s t s t h a t v a r y i n g c o n c e n t r a t i o n s o f two s u b s t a n c e s . e a c h h a v i n g an o p p o s i t e e f f e c t on l i z a r d s k i n , c o u l d be p r e s e n t i n t h e p l a s m a . No s u g g e s t i o n s h a v e b e e n made as t o t h e n a t u r e o f t h e i n h i b i t o r y s u b s t a n c e . Human p l a s m a does n o t n o r m a l l y c o n t a i n t h e q u a n t i t i e s o f i n h i b i t i n g a g e n t s , such as C o r t i s o l o r c a t e c h o l a m i n e s , r e q u i r e d t o i n t e r f e r e w i t h t h e MSH b i o a s s a y (Abe & N i c h o l s o n , 1975)- P o s s i b l y human and/or g u i n e a p i g p l a s m a c o n t a i n s some o t h e r f a c t o r w h i c h i n h i b i t s t h e normal-r e s p o n s e o f m e l a n o p h o r e s t o m e l a n o c y t e - s t i m u l a t i n g s u b s t a n c e s . U s i n g t h e s i l i c i c a c i d e x t r a c t i o n p r o c e d u r e , b e t t e r r e c o v -e r y was o b s e r v e d when s t a n d a r d MSH was a d d e d t o p l a s m a r a t h e r t h a n t o R i n g e r ' s s o l u t i o n . T h i s s u g g e s t s t h a t , a t l e a s t d u r i n g t h i s p r o c e d u r e , MSH i n p l a s m a may f o r m p a r t o f a p e p t i d e -p r o t e i n c o m p l e x . w h i c h i s more e a s i l y b o u n d t o t h e s i l i c i c a c i d . T h i s may i n d i c a t e t h a t t h e p e p t i d e g e n e r a l l y f o r m s s u c h comp-l e x e s i n t h e p l a s m a . MSH c o v a l e n t l y b ound t o a l b u m i n seems t o h ave a l m o s t i d e n t i c a l p o t e n c y t o t h a t o f f r e e p e p t i d e ( E b e r l e e t a l , 1977). 59 SECTION I I I : A PRELIMINARY ESTIMATION OF  MELANOCYTE-STIMULATING ACTIVITY IN AMNIOTIC FLUID 1. I n t r o d u c t i o n The o c c u r r e n c e of r e l a t i v e l y h i g h m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n the p i t u i t a r y and e s p e c i a l l y i n the plasma, of f e t a l and a d u l t g u i n e a p i g s , r a i s e s the q u e s t i o n of whether any of t h i s a c t i v i t y f i n d s i t s way i n t o the a m n i o t i c f l u i d . A m n i o t i c f l u i d i s a heterogenous system: a s o l u t i o n I n which u n d i s s o l v e d m a t e r i a l i s suspended. As a s o l u t i o n , amni-o t i c f l u i d c o n s i s t s of o n l y 1 - 2 $ s o l u t e s and 98 - 99$ w a t e r . Compared t o serum, the p r o t e i n c o n t e n t i s e l e c t r o p h o r e t i c a l l y and immunochemically s i m i l a r , but o n l y 5$ as c o n c e n t r a t e d (Bonsnes, 1 9 6 6 ) . The c o m p o s i t i o n of a m n i o t i c f l u i d changes markedly as pregnancy p r o g r e s s e s . D u r i n g the e a r l i e s t weeks, the f l u i d i s an u l t r a f i l t r a t e of m a t e r n a l plasma ( L i n d e_t a l , 1 9 6 9 ) . To-wards m i d - g e s t a t i o n the c o n c e n t r a t i o n s of the more d i f f u s i b l e s o l u t e s are c l o s e r t o those of f e t a l , r a t h e r t h a n m a t e r n a l , serum s u g g e s t i n g t h a t the f l u i d becomes an e x t e n s i o n of the f e t a l e x t r a c e l l u l a r f l u i d s . I n the second h a l f of pregnancy, f e t a l s k i n becomes i n c r e a s i n g l y l e s s permeable and the a m n i o t i c f l u i d c o m p o s i t i o n becomes i n c r e a s i n g l y dominated by the c o n t r -i b u t i o n of f e t a l u r i n e ( L i n d , 19-75). I n g e n e r a l , the importance and o r i g i n of p r o t e i n hormones i n the a m n i o t i c f l u i d are not w e l l u n d e r s t o o d . Hormones o r i g -i n a t i n g i n the p l a c e n t a and the mother are p r e s e n t i n the amn-i o t i c f l u i d i n v a r y i n g amounts. I n the human, these i n c l u d e 60 c h o r i o n i c g o n a d o t r o p i n , c h o r i o n i c somatomammotropin, p r o l a c t i n and g l u c a g o n . Growth hormone i s p r e s e n t i n s u b s t a n t i a l amounts but i s p r o b a b l y of f e t a l o r i g i n (Dawood, 1 9 7 7 ) • Ances and Pomerantz ( 1 9 7 4 ) found immunoreactive beta-MSH a c t i v i t y i n human a m n i o t i c f l u i d from 30 weeks of g e s t a t i o n . L e v e l s i n c r e a s e d from about 300 pg/ml at j u s t over 30 weeks t o up t o 2000 pg/ml at 44 weeks. Tuim a l a e t a l ( 1 9 7 6 ) found a s i g n i f i c a n t l y h i g h e r l e v e l of ACTH i n a m n i o t i c f l u i d , a t 26 t o 30 weeks i n the human ( 4 2 9 ± 9 7 . 4 pg/ml) as compared t o e a r l y ( 2 0 8 . 7 ± 90.6 pg/ml) or l a t e ( 1 7 2 . 7 ± 97.4 pg/ml) p r e g -nancy . There are no r e p o r t e d measurements of e i t h e r MSH or ACTH l e v e l s i n the a m n i o t i c f l u i d of the g u i n e a p i g . While b o t h these hormones appear t o be p r e s e n t i n s i g n i f i c a n t amounts i n human a m n i o t i c f l u i d , t h e s e measurements were made u s i n g r a d i o -immunoassay methods. T h e r e f o r e , a f i r s t attempt was made t o measure b i o l o g i c a l m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n the amn-i o t i c f l u i d of the g u i n e a p i g . Measured a c t i v i t y c o u l d then be compared w i t h plasma a c t i v i t y . 2. R e s u l t s a. E s t i m a t e d M e l a n o c y t e - S t i m u I a t i n g A c t i v i t y of  A m n i o t i c F l u i d Due t o the low a c t i v i t y measured i n a m n i o t i c f l u i d , samples were measured w i t h o u t d i l u t i o n wherever p o s s i b l e . S m a l l samples were d i l u t e d t o t e s t volume. S i n c e o n l y one unknown c o n c e n t r a t i o n was measured, r e l a t i v e potency c o u l d not be measured u s i n g the 2x2 f a c t o r i a l d e s i g n (see M a t e r i a l s and Methods). R e p l i c a t e measurements of the samples were t a k e n 61 and the average v a l u e was f i t t e d t o the s t a n d a r d curve t o c a l -c u l a t e the unknown a c t i v i t y . R e s u l t i n g v a l u e s have p o o r e r v a l i d i t y as compared t o those c a l c u l a t e d u s i n g the f a c t o r i a l method (see d i s c u s s i o n ) . T h e r e f o r e , measured a c t i v i t y l e v e l s s h o u l d be t r e a t e d as p r e l i m i n a r y e s t i m a t e s . The 30 a m n i o t i c f l u i d samples measured were t a k e n from f e t u s e s r a n g i n g i n age from 42 t o 67 days g e s t a t i o n . Sample volume averaged 5-0 ml w i t h a range of 1.1 t o 6.0 ml. Of these 30 samples, 10 had d e t e c t a b l e m e l a n o c y t e - s t i m u l a t i n g a c t -i v i t y but o n l y i n t r a c e amounts i n two c a s e s . A c t i v i t y i n the e i g h t samples w i t h measurable a c t i v i t y averaged 79-4 ± 14.3 pg/ml w i t h a range of a c t i v i t i e s of 3 5 . 8 t o 166 pg/ml. These samples were t a k e n from f e t u s e s r a n g i n g i n age from 42 t o 65 days. From these few measurements no r e l a t i o n s h i p b e t -ween a c t i v i t y and g e s t a t i o n a l age was e v i d e n t . S e v e r a l o f the r e m a i n i n g a m n i o t i c f l u i d samples caused l i t t l e change i n the c o l o u r of A n o l i s s k i n but a g r e a t e r number (14) produced an o p p o s i t e l i g h t e n i n g e f f e c t . S k i n p l a c e d on a m n i o t i c f l u i d became a v e r y p a l e homogeneous green c o l o u r ; t h i s was i n c o n t r a s t t o the hue of s k i n p l a c e d on R i n g e r ' s s o l -u t i o n where the o v e r a l l c o l o u r was a d a r k e r green w i t h some v a r i a t i o n over p o r t i o n s of i t s s u r f a c e . On v a r i o u s o c c a s i o n s t h i s l i g h t e n i n g e f f e c t o c c u r r e d i n samples t h a t a l s o c o n t a i n e d m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y . The brown c o l o u r produced was much l i g h t e r than the normal response t o m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y and, as a r e s u l t , the r e f l e c t i o n r e a d i n g would go up d e n o t i n g a n e g a t i v e or op-p o s i t e e f f e c t . Thus, s e v e r a l of the n e g a t i v e samples may 62 a c t u a l l y h a v e p o s s e s s e d m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y . I n two o r t h r e e o f t h e s a m p l e s w i t h m e a s u r e d a c t i v i t y t h e r e s p o n s e n o t e d v i s u a l l y was g r e a t e r t h a n i n d i c a t e d by t h e r e a d i n g r e c -o r d e d , p r o b a b l y due t o t h i s i n t e r f e r i n g a c t i v i t y . The o c c u r -r e n c e o f t h i s s e c o n d a c t i v i t y d i d n o t seem t o c o r r e l a t e i n any way w i t h g e s t a t i o n a l a g e . T h u s , w h i l e t h e s e r e s u l t s seem t o i n d i c a t e t h a t m e l a n o c y t e -s t i m u l a t i n g a c t i v i t y i s p r e s e n t on o c c a s i o n i n t h e a m n i o t i c i f l u i d o f t h e g u i n e a p i g , t h e l e v e l s m e a s u r e d a r e a p p r o x i m a t e a t b e s t due t o t h e a s s a y m e t h o d a n d t o t h e p r o b a b l e o c c u r r e n c e o f a s e c o n d s u b s t a n c e o f o p p o s i t e e f f e c t . b. T r y p s i n I n c u b a t i o n o f A m n i o t i c F l u i d S a m p l e s I n o r d e r t o d e t e r m i n e w h e t h e r t h e m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n t h e a m n i o t i c f l u i d was due t o a p r o t e i n , s i x o f t h e p o s i t i v e s a m p l e s w ere i n c u b a t e d w i t h t r y p s i n f o r 30 m i n u t e s a t 37°C. I n f i v e o u t o f s i x c a s e s , t h e m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y was d e s t r o y e d by t h e t r y p s i n . The a c t i v i t y i n c r e a s e d ' s l i g h t l y i n t h e s i x t h s a m p l e . A l l p r e p a r a t i o n s o f s t a n d a r d MSH i n c u b a t e d w i t h t r y p s i n l o s t a t l e a s t 90$ o f t h e i r a c t i v i t y . T r y p s i n a p p e a r e d t o h a v e no e f f e c t on t h e s a m p l e s w i t h l i g h t e n -i n g a c t i v i t y . 1 T h i s w o u l d i n d i c a t e t h a t t h e m e l a n o c y t e - s t i m u l a t -i n g a c t i v i t y i n a m n i o t i c f l u i d i s due t o a p r o t e i n o r p e p t i d e . 3. D i s c u s s i o n a. E v a l u a t i o n o f t h e A s s a y M e t h o d T h i r t y - t h r e e p e r c e n t o f t h e a m n i o t i c f l u i d s a m p l e s t e s t e d p o s s e s s e d some m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y as m e a s u r e d by b i o a s s a y . B e c a u s e o f t h e l o w a c t i v i t i e s p r e s e n t , p o t e n c y c o u l d n o t be m e a s u r e d u s i n g a p r o p e r f o u r - p o i n t a s s a y w i t h h i g h and l o w d o s e s o f unknown and s t a n d a r d . I n s t e a d , t h e a v e r a g e r e s p -o nse o f t h e f l u i d h a d t o be f i t t e d t o t h e MSH s t a n d a r d c u r v e . S i n c e a d o s e - r e s p o n s e c u r v e was u n a v a i l a b l e f o r t h e unknown, p a r a l l e l i s m w i t h t h e s t a n d a r d c o u l d n o t be t e s t e d . T h e r e f o r e i t i s p o s s i b l e t h a t some o t h e r a c t i v e c o n s t i t u e n t , u n r e l a t e d t o MSH a n d r e l a t e d p e p t i d e s , was b e i n g m e a s u r e d . A l s o , a c o m b i n e d s l o p e c o u l d n o t be c a l c u l a t e d r e s u l t i n g i n p o o r e r a s s a y p r e c i s i o n ( B l i s s , 1 9 5 2 ) . S i n c e t h e unknown v a l u e s f e l l t o one end o f t h e s t a n d a r d l i n e , f u r t h e r e r r o r i s i n t r o d u c e d i n t h e c a l c u l a t i o n o f i n d i v i d u a l s a m p l e a c t i v i t y . T h e r e f o r e r e s u l t s g i v e n h e r e must be c o n s i d e r e d as e s t i m a t e s o n l y . F u r -t h e r s t u d i e s s h o u l d i n v o l v e p u r i f i c a t i o n a nd c o n c e n t r a t i o n o f s a m p l e s t o a v o i d i n t e r f e r e n c e f r o m s p u r i o u s e f f e c t s a nd t o e n -s u r e t h a t a c t i v i t i e s c a n be m e a s u r e d w i t h b e t t e r a c c u r a c y . b. N a t u r e and S i g n i f i c a n c e o f M e l a n o c y t e - S t i m u l a t i n g  A c t i v i t y i n A m n i o t i c F l u i d . M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y was p r e s e n t i n 33$ o f t h e g u i n e a p i g a m n i o t i c f l u i d s a m p l e s t e s t e d . B e c a u s e t h e s l o p e o f t h e unknown c o u l d n o t be c o m p a r e d t o t h a t o f s t a n d a r d MSH, t h e a s s a y , i t s e l f , gave no i n d i c a t i o n o f t h e n a t u r e o f t h e a c t i v e s u b s t a n c e . I n f i v e o u t o f s i x c a s e s i n c u b a t i o n - o f a m n i o t i c f l u i d s a m p l e s w i t h a n e x c e s s o f t r y p s i n d e s t r o y e d any m e l a n o c y t e -s t i m u l a t i n g a c t i v i t y p r e s e n t , s u g g e s t i n g t h a t a p e p t i d e was i n -v o l v e d - The p e p t i d e c o u l d be MSH as s t a n d a r d p r e p a r a t i o n s i n c u b a t e d w i t h t r y p s i n l o s t a t l e a s t 90$ o f t h e i r a c t i v i t y when a s -s a y e d . B e c a u s e o f t h e l o w a c t i v i t i e s m e a s u r e d , t h e m e l a n o c y t e -s t i m u l a t i n g r e s p o n s e c o u l d be due t o nanogram/ml q u a n t i t i e s o f ACTH o r e v e n LPH. S i n c e i m m u n o r e a c t i v e ACTH- l e v e l s i n human a m n i o t i c f l u i d r a n g e f r o m 173 t o 429 p g / m l ( T u i m a l a et_ a l , 1 9 7 6 ) , a c t i v i t y i n t h e g u i n e a p i g w o u l d h a v e t o be somewhat h i -g h e r t o a c c o u n t f o r t h e m e a s u r e d m e l a n o c y t e - s t i m u l a t i n g a c t i v i -t y . S i n c e t h e e x a c t c o m p o s i t i o n o f g u i n e a p i g a m n i o t i c f l u i d h a s n o t b e e n w e l l s t u d i e d , some o t h e r p e p t i d e c o u l d be i n v o l v e d . Any s p e c u l a t i o n on t h e s i g n i f i c a n c e o f m e l a n o c y t e - s t i m u l a -t i n g a c t i v i t y i n g u i n e a p i g a m n i o t i c f l u i d i s p r o b a b l y p r e m a -t u r e . M e a s u r a b l e l e v e l s may s i m p l y r e f l e c t t h e s i g n i f i c a n t l e -v e l s f o u n d i n f e t a l a n d m a t e r n a l p l a s m a . H o w e v e r , a c t i v i t y was much l o w e r t h a n t h a t f o u n d I n p l a s m a o f e i t h e r t h e m o t h e r o r t h e f e t u s . I n c o n t r a s t , beta-MSH l e v e l s i n human a m n i o t i c f l u i d f r o m 30 weeks o f g e s t a t i o n onwards a r e a t l e a s t as h i g h as l e -v e l s f o u n d i n t h e p l a s m a o f newborns ( A n c e s & P o m e r a n t z , 1 9 7 4 ) . T h i s d i f f e r e n c e may s i m p l y r e f l e c t t h e measurement p r o b l e m s c a u s e d by t h e i n t e r f e r i n g m e l a n o c y t e - p a l i n g r e s p o n s e . From, v i -s u a l o b s e r v a t i o n h o w e v e r , i t seemed u n l i k e l y t h a t g u i n e a p i g am-n i o t i c f l u i d c o n t a i n e d t h e h i g h a c t i v i t y m e a s u r e d i n t h e p l a s m a . I n t h e human, l e v e l s i n c r e a s e a s g e s t a t i o n p r o g r e s s e s b u t t h e r e was no e v i d e n c e f o r t h i s i n t h e g u i n e a p i g . Due t o d i f f e r e n c e s i n a s s a y t e c h n i q u e s , h o w e v e r , t h e a c t i v e p e p t i d e s b e i n g m e a s u r e d may be d i f f e r e n t i n t h e two, s p e c i e s . N o n e t h e l e s s , t h e r e s u l t s s u g g e s t t h a t t h e d y n a m i c s o f a m n i o t i c f l u i d m e l a n o c y t e - s t i m u l a -t i n g a c t i v i t y ' m a y be q u i t e d i f f e r e n t i n t h e two s p e c i e s , c. S o u r c e o f M e l a n o c y t e - s t i m u l a t i n g A c t i v i t y i n  A m n i o t i c F l u i d A m n i o t i c f l u i d hormones o r i g i n a t e i n e i t h e r t h e f e t u s , 'or t h e m o t h e r . T h o s e o r i g i n a t i n g i n t h e .mother e n t e r t h e f l u i d e i -t h e r v i a t h e p l a c e n t a , o r v i a d i f f u s i o n t h r o u g h t h e f e t a l mem-65 b r a n e s . Human c h o r i o n i c g o n a d o t r o p i n a n d c h o r i o n i c somatomammotro-p i n r e a c h t h e a m n i o t i c f l u i d v i a p a s s a g e t h r o u g h t h e c h o r i o n and a m n i o n ( J o s i m o v i c h , 1 9 7 3 ; Dawood, 1 9 7 7 ) . ACTH a n d MSH f o u n d i n human a m n i o t i c f l u i d p r o b a b l y o r i g i n a t e i n t h e f e t u s . The ACTH l e v e l i n t h e m a t e r n a l c i r c u l a t i o n o f a p a t i e n t w i t h N e l s o n ' s syndrome was a b o u t 100 t i m e s h i g h e r t h a n t h a t f o u n d i n t h e a m n i -o t i c f l u i d ( A l l e n et_ a l , 1 9 7 3 ) ; t h i s s u g g e s t e d t h a t s i m p l e f i l -t r a t i o n o f ACTH t h r o u g h t h e a m n i o t i c membrane f r o m t h e m a t e r n a l c i r c u l a t i o n was u n l i k e l y . I n v i v o d e g r a d a t i o n o f ACTH i n t h e f l u i d i s a l s o a p o s s i b i l i t y . MSH c o n c e n t r a t i o n s i n b o t h c o r d b l o o d a n d a m n i o t i c f l u i d w ere s i g n i f i c a n t l y h i g h e r t h a n t h o s e f o u n d i n t h e m a t e r n a l c i r c u l a t i o n ( A n c e s & P o m e r a n t z , 1 9 7 4 ) . I n t h e g u i n e a p i g , a m n i o t i c f l u i d a c t i v i t y i s much l o w e r t h a n a c t i v i t y i n e i t h e r f e t a l o r m a t e r n a l p l a s m a . The s i m i -l a r i t y i n t h e p l a s m a l e v e l s p r o v i d e s l i t t l e i n f o r m a t i o n as t o t h e s o u r c e o f t h e a m n i o t i c f l u i d a c t i v i t y s i n c e t h e c o n c e n t r a -t i o n g r a d i e n t s f r o m m a t e r n a l a n d f e t a l p l a s m a t o t h e a m n i o t i c f l u i d w o u l d be t h e same. The a c t i v i t y p r e s e n t may be due s o l e l y t o p e p t i d e e n t e r i n g t h e f l u i d v i a t h e f e t a l u r i n e . T h e r e i s a l s o t h e p o s s i b i l i t y t h a t MSH and r e l a t e d p e p t i d e s a r e d e g r a d e d i n a m n i o t i c f l u i d b u t t h i s was n o t e v i d e n t o v e r t h e t i m e p e r i o d i n v o l v e d i n t h e b i o a s s a y p r o c e d u r e s . F u r t h e r s t u d i e s a r e t h e r e -f o r e r e q u i r e d b e f o r e a n y t h i n g d e f i n i t e c a n be s t a t e d r e g a r d i n g t h e s o u r c e o f m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i n a m n i o t i c f l u i d , d. S o u r c e o f t h e M e l a n o c y t e - A g g r e g a t i o n R e s p o n s e t o  A m n i o t i c F l u i d A l m o s t h a l f o f t h e a m n i o t i c f l u i d s a m p l e s h a d a s i g n i f i c a n t a g g r e g a t i o n e f f e c t on l i z a r d melanophores r e s u l t i n g i n p a l i n g of the s k i n and lower r e f l e c t i o n v a l u e s . I n some cases t h i s a c t i v i t y was p r e s e n t i n samples which appeared t o c o n t a i n m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y . A g r e y i s h brown c o l o u r , q u i t e u n l i k e the normal response t o MSH, would r e s u l t . The r e s u l t i n g r e f l e c t i o n v a l u e s were h i g h and, as a r e s u l t , the c a l c u l a t e d a c t i v i t y was lower than e x p e c t e d from v i s u a l o b s e r v -a t i o n . Thus the m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y c a l c u l a t e d may be much lower than the a c t u a l a c t i v i t y p r e s e n t i n the s ample s . Compared t o the s p o r a d i c i n h i b i t o r y e f f e c t s of plasma on s k i n r e s p o n s e , t h i s e f f e c t was more c o n s i s t e n t . The plasma r a r e l y caused an a c t u a l p a l i n g r e s p o n s e . The melanocyte aggr-e g a t i o n e f f e c t may be caused by some o t h e r substance i n the a m n i o t i c f l u i d . C a techolamines w i l l b l o c k MSH-induced c o l o u r changes i n A n o l i s . Doses of from 0.05 t o 5-0 ng/ml w i l l b l o c k the response t o a 0.2 ng/ml dose of alpha-MSH ( T i l d e r s e t a l , 1 9 7 5 ) . Nor-e p i n e p h r i n e i s the most p o t e n t i n t h i s r e g a r d . Ben-Jonathan and Maxson ( 1 9 7 8 ) found s i g n i f i c a n t l e v e l s of n o r e p i n e p h r i n e (about 1 ng/ml) and dopamine (up t o 5 ng/ml) i n the a m n i o t i c f l u i d of the r a t d u r i n g the l a s t f i v e days b e f o r e term. H o r o w i t z ( 1 9 5 8 ) showed t h a t e p i n e p h r i n e not o n l y b l o c k s the response t o MSH but a l s o causes a d d i t i o n a l b l a n c h i n g of A n o l i s s k i n t r a n s f e r r e d from normal R i n g e r ' s . Other c a t e c h o l a m i n e s may a c t i n a s i m i l a r manner. I f g u i n e a p i g a m n i o t i c f l u i d a l s o c o n t a i n s h i g h c a t e c h o l a m i n e c o n c e n t r a t i o n s , t h i s may e x p l a i n the p a l i n g response observed. 67 C o r t i s o l may have an i n h i b i t o r y e f f e c t on the MSH-induced response i n the f r o g , Rana p i p i e n s (Abe & N i c h o l s o n , 1 9 7 5 ) . Murphy et_ a l ( 1 9 7 5 ) found t h a t C o r t i s o l l e v e l s i n human amni-o t i c f l u i d c o r r e l a t e d w e l l w i t h f e t a l plasma l e v e l s and t h a t l e v e l s i n the f l u i d i n c r e a s e d from 5 ng/ml at 1 0 - 1 5 weeks t o 22 ng/ml j u s t b e f o r e term. Plasma C o r t i s o l l e v e l s appear t o be h i g h i n the g u i n e a p i g at b i r t h (up t o 1 ug/ml) ( I l l i n g w o r t h e t a l , 1 9 7 4 ) and t h e r e f o r e may a l s o be h i g h i n a m n i o t i c f l u i d , a t l e a s t l a t e i n g e s t a t i o n . Assuming t h a t A n o l i s s k i n r e s -ponds i n the same manner as f r o g s k i n , i t i s p o s s i b l e t h a t C o r t i s o l l e v e l s may be h i g h enough i n g u i n e a p i g a m n i o t i c f l u i d t o i n t e r f e r e w i t h the m e l a n o c y t e - s t i m u l a t i n g r e s p o n s e . I n c o n t r a s t , Shizume e_t a l ( 1 9 5 4 ) found no i n h i b i t o r y e f f e c t w i t h e i t h e r C o r t i s o l or t e s t o s t e r o n e , e s t r a d i o l and c o r t i s o n e , even at c o n c e n t r a t i o n s of 0.1 mg/ml. Shizume et_ aJL ( 1 9 5 4 ) a l s o found t h a t i n c r e a s i n g the o s m o l a r i t y of the R i n g e r ' s s o l u t i o n b a t h i n g the f r o g s k i n would decrease the MSH-induced r e s p o n s e . I n g e n e r a l , osmo-l a r i t y c o r r e l a t e s v e r y s t r o n g l y w i t h the sodium c o n c e n t r a t i o n i n m i l l i e q u i v a l e n t s (meq.) ( L i n d e_t a l , 1 9 6 9 ) - The sodium c o n c e n t r a t i o n of g u i n e a p i g a m n i o t i c f l u i d e q u a l s about 125 meq/1 ( V i z s o l y i & P e r k s , 1 9 7 4 ) w h i l e the c o n c e n t r a t i o n of the l i z a r d R i n g e r ' s s o l u t i o n used was o n l y 111 meq/1. T h i s i n c r e a s e i n o s m o l a r i t y may have had a n e g a t i v e e f f e c t on MSH-induced re s p o n s e . GENERAL DISCUSSION 68 1. Choice of Assay M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y was measured by b i o a s s a y even though radioimmunoassays (RIA) have c e r t a i n advantages i n s e n s i t i v i t y and p r e c i s i o n . The b i o a s s a y used i n the p r e s e n t study was s e n s i t i v e enough f o r measuring a c t i v i t y i n the b l o o d plasma of the g u i n e a p i g . When c e r t a i n t e c h n i c a l problems are overcome, the assay may a l s o be s e n s i t i v e enough f o r measuring a m n i o t i c f l u i d a c t i v i t y p r o p e r l y . I n the c u r r e n t c a s e , the g r e a t e r s p e c i f i c i t y i n h e r e n t i n RIA methods may not be an advantage s i n c e the s p e c i f i c sequ-ences of m e l a n o c y t e - s t i m u l a t i n g p e p t i d e s i n the g u i n e a p i g are s t i l l unknown. Four e l e c t r o p h o r e t i c a l l y d i s t i n c t MSH p e p t i d e s o c c u r i n the g u i n e a p i g p i t u i t a r y ( H o e k s t r a & Van der Wal, 1 9 7 1 ) . The s t r o n g e s t a c t i v i t y i s a s s o c i a t e d w i t h a p e p t i d e e l e c t r o -p h o r e t i c a l l y I d e n t i c a l t o alpha-MSH. S i n c e alpha-MSH i s s t r u c t u r a l l y i d e n t i c a l i n a l l s p e c i e s where i t has been sequ-enced, i t i s l i k e l y t o be the predominant MSH i n the g u i n e a p i g . However, w h i l e two out of the r e m a i n i n g t h r e e MSH p e p t i d e s are e l e c t r o p h o r e t i c a l l y s i m i l a r t o known beta-MSH p e p t i d e s , they may not be i d e n t i c a l . A l t h o u g h equine beta-MSH has i d e n t -i c a l e l e c t r o p h o r e t i c p r o p e r t i e s t o b o v i n e beta-MSH, the sequ-ence d i f f e r s i n two p o s i t i o n s (Dixon & L i , 1 9 6 1 ) . S h a p i r o e_t a l ( 1 9 7 2 ) i n d i c a t e d t h a t known MSH p e p t i d e s may o n l y account f o r 5$ of the b i o a c t i v e MSH a c t i v i t y i n the g u i n e a p i g . These a u t h o r s came t o s i m i l a r c o n c l u s i o n s i n some o t h e r s p e c i e s . While p e r c e n t a g e s quoted f o r the r a t were lower than those g i v e n i n o t h e r s t u d i e s ( U s a t e g u i e t a l , 1 9 7 6 ) , i t i s g e n e r a l l y a c c e p t e d t h a t known p e p t i d e s do not account f o r a l l the b i o l -o g i c a l a c t i v i t y found i n v a r i o u s s p e c i e s . Thus measuring the immunoreactive a c t i v i t y of known MSH p e p t i d e s may not have p r o v i d e d a complete assessment of the a c t i v i t y o f a t i s s u e or f l u i d . B i o a s s a y methods p r o b a b l y g i v e a b e t t e r i n d i c a t i o n o f the f u n c t i o n a l a c t i v i t y p r e s e n t . S i l m a n et_ a l ( 1 9 7 5 ) found s i g n i -f i c a n t b i o l o g i c a l MSH a c t i v i t y i n the plasma of pregnant women but found no a c t i v i t y u s i n g radioimmunoassays f o r known MSH and r e l a t e d p e p t i d e s . Immunoassays may measure p r e c u r s o r m o l e c u l e s which are not b i o l o g i c a l l y a c t i v e . C e r t a i n l y i n the f e t a l g u i n e a p i g where MSH had not been measured p r e v i o u s l y , a meas-ure of the t o t a l b i o l o g i c a l a c t i v i t y s h o u l d g i v e a c l e a r e r i d e a of t r o p h i c p o t e n t i a l . A measure of immunoreactive alpha-MSH would have been i d e a l f o r comparative purposes and s h o u l d be done i n f u t u r e s t u d i e s . Accuracy i n the p r e s e n t r e s u l t s i s dependent upon the f u l l p otency of the s t a n d a r d MSH p r e p a r a t i o n . P r e p a r a t i o n and use of the s y n t h e t i c alpha-MSH f o l l o w e d s t a n d a r d p r o c e d u r e s i n a l l c a s e s . The average s e n s i t i v i t y of A n o l i s s k i n t o alpha-MSH was s i m i l a r t o v a l u e s g i v e n by o t h e r authors- ( B j o r k l u n d et_ a l , 1972 T i l d e r s ej: a l , 1 9 7 5 ) . Some s e a s o n a l v a r i a t i o n i n s e n s i t i v i t y was e v i d e n t over the course of the st u d y . 2. S i g n i f i c a n c e of P e p t i d e s R e l a t e d t o MSH The h i g h l e v e l s of m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y found i n b o t h the a n t e r i o r and p o s t e r i o r l o b e s of the p i t u i t a r y 70 s u g g e s t t h a t s i g n i f i c a n t amounts o f alpha-MSH may be p r e s e n t i n t h e p a r s d i s t a l i s , as w e l l as i n t h e p a r s i n t e r m e d i a . H o w e v e r , b o t h p o r t i o n s o f t h e g l a n d may a l s o c o n t a i n b eta-MSH, ACTH a n d LPH ( L o w r y & S c o t t , 1977). S i n c e t h e s e p e p t i d e s w o u l d a c c o u n t f o r some of'' t h e m e l a n o c y t e - s t i m u l a t i n g a c t i v i t y m e a s u r e d , t h e o c c u r r e n c e and s i g n i f i c a n c e o f t h e s e p e p t i d e s must be c o n s i d e r -ed. The o c c u r r e n c e o f a p e p t i d e i n a n e n d o c r i n e t i s s u e i s n o t n e c e s s a r i l y c o n c o m i t a n t w i t h s e c r e t i o n o f t h a t p e p t i d e i n t o t h e b l o o d s t r e a m . The p r e s e n c e o f ACTH and LPH i n t h e p a r s i n t e r m e d i a r e p r e s e n t s i n i t s e l f o n l y a p o t e n t i a l s e c r e t o r y s o u r c e o f t h e s e hormones. a. S i g n i f i c a n c e o f P a r s I n t e r m e d i a ACTH The s i g n i f i c a n c e o f p a r s i n t e r m e d i a ACTH has b e e n t h e s u b -j e c t o f much s t u d y r e c e n t l y . W h i l e c o r t i c o t r o p h i c c e l l s o f t h e p a r s d i s t a l i s d i s p l a y e d h y p e r t r o p h y a f t e r a d r e n a l e c t o m y a nd r e g r e s s i o n a f t e r c o r t i c o s t e r o i d a d m i n i s t r a t i o n , c o r t i c o t r o p h i c c e l l s o f t h e p a r s i n t e r m e d i a were o n l y m i n i m a l l y a f f e c t e d ( B a k e r & Drummond, 1972). S t u d i e s i n t h e r a t s u g g e s t t h a t p a r s i n t e r -m e d i a ACTH may be t r o p h i c i n a r e a s u n r e l a t e d t o a d r e n o c o r t i c a l f u n c t i o n i n g . S t u d i e s w i t h c a t e c h o l a m i n e s s u g g e s t e d t h a t t h e r e l e a s e o f p a r s i n t e r m e d i a ACTH may be u n d e r n e u r a l c o n t r o l a n d a r e d u c t i o n i n t h e c o n c e n t r a t i o n o f ACTH h a s b e e n o b s e r v e d a f t e r n e u r o g e n i c s t r e s s ( F i s h e r & M o r i a r t y , 1977)• H o w e v e r , G r e e r e t a l (1975) showed t h a t t h e s i z e o f t h e ACTH p o o l was p r o b a b l y i n s u f f i c i e n t t o s u p p l y a t r o p h i c r e s p o n s e t o s t r e s s . S i n c e n e u r a l c o n t r o l l i n g m e c h anisms h a v e b e e n i m p l i c a t e d i n p a r s i n t e r m e d i a f u n c t i o n i n g i n g e n e r a l , c o n t r o l o f MSH a n d ACTH r e l e a s e may r e l a t e more s p e c i f i c a l l y t o t h e p o r t i o n o f 71 the g l a n d i n q u e s t i o n , r a t h e r t h a n t o the i n d i v i d u a l p e p t i d e s . C e r t a i n l y i n man, beta-MSH l e v e l s p a r a l l e l those o f ACTH b o t h i n the pars d i s t a l i s and i n plasma (Abe et_ a l , 1 9 6 7 ) . Pars i n t e r m e d i a ACTH may be the p r e c u r s o r of alpha-MSH ( S c o t t & Lowry, 1 9 7 7 ) • C o n c e n t r a t i o n s of c a r b o x y l t e r m i n a l ACTH i m m u n o a c t i v i t y were s i g n i f i c a n t l y h i g h e r t h a n ACTH-^^g a c t i v i t y i n the p o s t e r i o r p i t u i t a r y of the p i g , cow and g u i n e a p i g ( S c o t t e_t a l , 1 9 7 2 ) . T h i s i n d i c a t e d t h a t CLIP was i n h i g h e r c o n c e n t r a t i o n than i n t a c t ACTH. T h i s suggests t h a t ACTH i s s p l i t e n z y m a t i c a l l y t o r e l e a s e b o t h CLIP and alpha-MSH. Thus ACTH l o c a t e d i n the pars i n t e r m e d i a of the g u i n e a p i g may not a c t u a l l y e n t e r the b l o o d s t r e a m . b. S i g n i f i c a n c e of P i t u i t a r y LPH Evid e n c e o b t a i n e d from work w i t h the p i t u i t a r i e s of s e v e r a l s p e c i e s i n c l u d i n g the p i g , sheep and man s t r o n g l y s u g g ests t h a t beta-LPH I s the p r e c u r s o r of beta-MSH, as w e l l as b e t a - e n d o r p h i n and o t h e r o p i o i d a g o n i s t s . The beta-LPH p r e c u r s o r model has r e c e n t l y been r e v i e w e d by C h r e t i e n e_t a l ( 1 9 7 7 ) . C r i n e e_t a l ( 1 9 7 7 ) i s o l a t e d o n l y s m a l l amounts of beta-LPH from beef p i t u i t a r i e s but r e l a t i v e l y l a r g e r amounts of gamma-LPH and b e t a - e n d o r p h i n . T h i s suggests t h a t beta-LPH syn-t h e s i z e d i n the t i s s u e i s c l e a v e d , r e s u l t i n g i n h i g h e r l e v e l s of the c o n s t i t u e n t p e p t i d e s . The h i g h a c t i v i t y found i n the a n t e r i o r lobe of the g u i n e a p i g p i t u i t a r y c o u l d be due p a r t l y t o beta-MSH formed from enzy-m a t i c c l e a v a g e of beta-LPH i n the c o r t i c o t r o p h s . I t i s u n l i k e l y t h a t beta-LPH i s b e i n g b r o k e n down i n the e x t r a c t i o n p r o c e d u r e s to y i e l d beta-MSH s i n c e beta-LPH i s h i g h l y r e s i s t a n t t o • 7 2 d e g r a d a t i o n i n v a r i o u s i s o l a t i o n p r o c e d u r e s . Because of the r e l a t i v e l y low a c t i v i t y of LPH however, these p e p t i d e s c o u l d v e r y w e l l be p r e s e n t i n h i g h c o n c e n t r a t i o n i n bo t h the p i t u i t a r y and plasma w i t h o u t a f f e c t i n g the measured MSH a c t i v i t y s i g n i f i -c a n t l y . 3. Comparison of M e l a n o c y t e - s t i m u l a t i n g A c t i v i t y L e v e l s a. I n the Guinea P i g The p r e s e n t r e s u l t s i n d i c a t e t h a t the p i t u i t a r y and plasma of the f e t a l g u i n e a p i g c o n t a i n u n u s u a l l y h i g h l e v e l s of melano-c y t e - s t i m u l a t i n g p e p t i d e s . A c t i v i t y i s a l s o h i g h i n the a d u l t female. I n c o n t r a s t , a m n i o t i c f l u i d r e s u l t s were i n c o n s i s t e n t ; o n l y a s m a l l amount of a c t i v i t y was p r e s e n t and o n l y I n some samples. Over the l a s t 15 days of g e s t a t i o n p i t u i t a r y a c t i v i t y i s I n c r e a s i n g r a p i d l y w h i l e plasma a c t i v i t y i s r e l a t i v e l y c o n s t -ant. Assuming t h a t the r a t e of p e p t i d e d e g r a d a t i o n and c l e a r -ance remains r e l a t i v e l y c o n s t a n t , s e c r e t i o n from the p i t u i t a r y would appear t o be i n c r e a s i n g i n p a r a l l e l w i t h i n c r e a s i n g b l o o d volume. I n which ca s e , h o m e o s t a t i c feedback mechanisms a c t i n g on the p i t u i t a r y i n the c o n t r o l of plasma p e p t i d e l e v e l s may be f u n c t i o n a l at t h i s p o i n t i n development. The drop i n plasma • a c t i v i t y j u s t a f t e r b i r t h may i n d i c a t e t h a t h o m e o s t a t i c mechan-isms are t e m p o r a r i l y upset at p a r t u r i t i o n . Due t o measurement prob l e m s , l i t t l e can be s a i d about a m n i o t i c f l u i d a c t i v i t i e s at t h i s time (see S e c t i o n I I I , d i s c u s s i o n ) . b. Among D i f f e r e n t S p e c i e s C i r c u l a t i n g m e l a n o c y t e - s t i m u l a t i n g a c t i v i t i e s v a r y con-s i d e r a b l y from s p e c i e s t o s p e c i e s (see S e c t i o n I I , d i s c u s s i o n ) . L e v e l s range from t r a c e amounts i n normal man t o ng/ml q u a n t i -t i e s i n g u i n e a p i g plasma. P i t u i t a r y l e v e l s a l s o d i s p l a y 73 much v a r i a t i o n , p a r t i c u l a r l y i n t h e r a t i o o f p o s t e r i o r t o a n -t e r i o r l o b e a c t i v i t y . T h i s s u g g e s t s t h a t t h e r o l e o f MSH a n d r e l a t e d p e p t i d e s may v a r y i n d i f f e r e n t mammalian s p e c i e s . Com-p a r i s o n o f b i o a s s a y a n d r a d i o i m m u n o a s s a y a c t i v i t i e s s u g g e s t s t h a t d i f f e r e n c e s i n m e t h o d o l o g y may a c c o u n t f o r some o f t h e d i s c r e p a n c i e s . A l s o , v a r i a t i o n i n t h e s e n s i t i v i t y o f t a r g e t t i s s u e s i n d i f f e r e n t s p e c i e s may a c c o u n t f o r t h i s w i d e r a n g e i n p l a s m a a c t i v i t i e s . 4. MSH i n F e t a l P h y s i o l o g y No c l e a r r o l e f o r MSH has b e e n e s t a b l i s h e d i n a d u l t mam-m a l s ; p o s s i b l y t h i s p e p t i d e i s o n l y t r o p h i c i n t h e f e t u s . As i n t h e human ( A n c e s & P o m e r a n t z , 1 9 7 4 ) , a c t i v i t y may be h i g h e r i n t h e f e t a l p l a s m a o f t h e g u i n e a p i g t h a n i n t h e p l a s m a o f n o n -p r e g n a n t a d u l t s . A c t i v i t y was a l s o h i g h e r i n p r e g n a n t a n i m a l s i n b o t h t h e human and t h e g u i n e a p i g . Thus MSH and r e l a t e d p e p -t i d e s may be i m p o r t a n t i n p r e g n a n c y o r f e t a l p h y s i o l o g y . C e r t a i n s t u d i e s i n a d u l t humans h a v e s u g g e s t e d t h a t p i t u -i t a r y MSH may n o t o c c u r i n s i g n i f i c a n t amounts i n n o r m a l man. Alpha-MSH a c c o u n t s f o r b u t a v e r y s m a l l f r a c 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 y f o u n d i n t h e a d u l t p i t u i t a r y (Abe e t a l , 1 9 6 7 ) . S i n c e a l p h a - M S H i s f o u n d l a r g e l y i n t h e p a r s i n t e r m e d i a i n most mammals, t h i s l a c k o f a c t i v i t y may be a d i r e c t c o n s e q u e n c e o f t h e r e g r e s s i o n o f t h a t t i s s u e a t b i r t h . A l s o , i t h a s b e e n s u g g e s t e d t h a t t h e beta-MSH f o u n d i n e x t r a c t s o f human p i t u i -t a r i e s may be a d e g r a d a t i v e p r o d u c t o f LPH f o r m e d i n t h e e x t r a c -t i o n p r o c e d u r e ( B l o o m f i e l d e t a l , 1 9 7 4 ) . S i n c e MSH p e p t i d e s may be s y n t h e s i z e d i n t h e b r a i n , beta-MSH m e a s u r e d i n c e r e b r o -s p i n a l f l u i d ( S m i t h & S h u s t e r , 1 9 7 6 ) and i n n o r m a l p l a s m a would be o f n e u r a l o r i g i n . These views are not h e l d by some o t h e r w o r k e r s , however. C h r e t i e n e_fc a l ( 1 9 7 7 ) f e e l t h a t the low l e v e l s of beta-MSH i s o l a t e d from human t i s s u e r e s u l t from the c o m p l e x i t y and r e l a t i v e l y slow l i b e r a t i o n of t h i s p e p t i d e from the beta-LPH p r e s u r s o r . I t may be s i g n i f i c a n t however, t h a t S i l m a n e_t a l ( 1 9 7 6 ) found s i g n i f i c a n t q u a n t i t i e s of alpha-MSH i n the human f e t a l p i t u i t a r y a t 24 weeks of g e s t a t i o n . V i s s e r and Swaab ( 1 9 7 7 ) found alpha-MSH I n the p a r s I n t e r m e d i a of human f e t u s e s b e t -ween 15 and 26 weeks g e s t a t i o n ; s i m i l a r a c t i v i t y was a l s o r e p -o r t e d i n one a d u l t p i t u i t a r y d u r i n g pregnancy. S i n c e plasma beta-MSH l e v e l s a r e e l e v a t e d i n human pregnancy (Ances & Pomerantz, 1 9 7 4 ) , i t may be p o s s i b l e t h a t beta-MSH i s o n l y r e l -eased from the p i t u i t a r y d u r i n g g e s t a t i o n . P o s s i b l y beta-MSH i s r e l e a s e d from the p l a c e n t a . C e r t a i n l y a r e l a t i o n s h i p b e t -ween pregnancy or f e t a l development and MSH l e v e l s i n man i s s t r o n g l y s u g g e s t e d . MSH c o u l d be i n v o l v e d i n the r e g u l a t i o n of s t e r o i d s e c r -e t i o n from the f e t a l a d r e n a l . S t u d i e s i n sheep suggest t h a t a n o t h e r p i t u i t a r y f a c t o r i n a d d i t i o n t o ACTH may be i m p o r t a n t I n c o n t r o l l i n g C o r t i s o l r e l e a s e from the a d r e n a l c o r t e x i n the f e t u s . I t was apparent i n the sheep t h a t the prepartum a c c e l -e r a t i o n i n a d r e n a l growth and g l u c o c o r t i c o i d s e c r e t i o n was not preceded by e l e v a t e d ACTH i n f e t a l plasma (Jones e_t a l , 1 9 7 7 ) . I n c h r o n i c a l l y c a t h e t e r i z e d f e t u s e s no i n c r e a s e i n f e t a l plasma ACTH was observed b e f o r e the r i s e i n plasma C o r t i s o l p r i o r t o p a r t u r i t i o n ( N a t h a n i e l s z , 1 9 7 6 ) . A l s o , i n f u s i o n of even h i g h doses of ACTH ( 1 0 ug/hr) produced a s m a l l e r immediate response i n C o r t i s o l s e c r e t i o n i n f e t u s e s of e a r l y g e s t a t i o n a l age as 75 compared t o the term f e t u s . An i n c r e a s e d s e n s i t i v i t y of the a d r e n a l c o r t e x demonstrated i n the l a s t t r i m e s t e r c o u l d account f o r o n l y a p o r t i o n of the i n c r e a s e d C o r t i s o l c o n c e n t r a t i o n s . While ACTH l e v e l s r i s e s l o w l y as g e s t a t i o n p r o g r e s s e s i n the f e t a l sheep ( N a t h a n i e l s z , 1 9 7 6 ) , l e v e l s i n the human f e t u s are a c t u a l l y d r o p p i n g i n the l a s t t r i m e s t e r d u r i n g the p e r i o d of g r e a t e s t a d r e n a l c o r t i c a l growth ( W i n t e r s et_ a l , 1 9 7 4 ) . T h i s may s i m p l y r e f l e c t the e s t a b l i s h m e n t of a feedback e f f e c t of C o r t i s o l on ACTH s e c r e t i o n . However, S i l m a n e t a l ( 1 9 7 6 ) found t h a t ACTH was not produced i n l a r g e q u a n t i t i e s by the f e t a l p i t u i t a r y over a l a r g e p a r t of g e s t a t i o n ; i n c o n t r a s t alpha-MSH was produced i n s i g n i f i c a n t amounts. C i r c u m s t a n t i a l e v i d e n c e suggests t h a t alpha-MSH may c o n t r o l f e t a l C o r t i s o l s e c r e t i o n e a r l y i n g e s t a t i o n . At days 22 and 28 p o s t - c o i t u m i n the r a b b i t , alpha-MSH caused a marked i n c r e a s e i n b l o o d C o r t i s o l c o n c e n t r a t i o n s w h i l e ACTH had no e f f e c t ( C h a l l i s & T o r o s i s , 1 9 7 7 ) . There may be a t e m p o r a l r e l a t i o n s h i p between the development of a f u n c t i o n a l p a r s i n t e r -media and measurable C o r t i s o l l e v e l s i n f e t a l plasma. C o r t i -cotropins appear i n the pa r s d i s t a l i s i n the human f e t u s a t 7 weeks (Baker & J a f f e , 1 9 7 5 ) ; a t 8.5 weeks no C o r t i s o l i s p r e s -ent i n f e t a l plasma but t o t a l a d r e n a l c o r t i c o i d l e v e l s were e x t r e m e l y h i g h . Plasma C o r t i s o l l e v e l s were d e t e c t e d at 11.weeks (Murphy & D i e z d'Aux, 1 9 7 2 ) . The f i r s t appearance of f u n c t i o n a l melanotrophs i n the human p i t u i t a r y I s a l s o r e p o r t e d t o o c c u r at about 11 weeks ( K a s t i n e_t a l , 1 9 6 8 ) a l t h o u g h r e p o r t s i n the l i t e r a t u r e v a r y on t h i s p o i n t . I n the monkey ACTH d i d not i n c r -ease C o r t i s o l r e l e a s e from the f e t a l zone of the a d r e n a l c o r t e x • 76 but d i d from the d e f i n i t i v e zone ( J a f f e et_ a l , 1 9 7 8 ) . S i n c e the f e t a l zone of the human a d r e n a l r e g r e s s e s a t about the same time t h a t alpha-MSH a c t i v i t y d e c r e a s e s ( a p p a r e n t l y due to r e g r e s s i o n of the p a r s i n t e r m e d i a ) , t h e r e may be a f u n c t i o n a l r e l a t i o n s h i p . How r e l e v a n t these d i s c u s s i o n s a re t o f e t a l p h y s i o l o g y i n the g u i n e a p i g i s unknown as l i t t l e work has been done i n t h i s s p e c i e s . While o v e r a l l t r e n d s i n plasma m e l a n o c y t e -s t i m u l a t i n g a c t i v i t i e s are s i m i l a r t o those i n the human, d i f -f e r e n c e s are apparent. F o r example, the i n c r e a s e i n plasma a c t i v i t y seen i n pregnancy i s much s m a l l e r t h a n t h a t r e p o r t e d i n humans (Ances & Pomerantz, 1 9 7 4 ) , p r o b a b l y due t o the h i g h l e v e l s measured i n the normal a d u l t . I n the g u i n e a p i g f e t u s , a d r e n a l weight I n c r e a s e s s t e a d -i l y t hroughout g e s t a t i o n and plasma c o r t i c o s t e r o i d l e v e l s i n c r e a s e towards term, r e a c h i n g v e r y h i g h l e v e l s (up t o 1 ug/ ml) at p a r t u r i t i o n ( I l l i n g w o r t h et_ a l , 1 9 7 4 ) . Plasma MSH l e v e l s are q u i t e v a r i a b l e but f a i r l y s teady over the l a s t 15 days of g e s t a t i o n . More s t u d i e s are needed t o i n v e s t i g a t e whether the s i g n i f i c a n t l e v e l s o f these hormones i n l a t e g e s t -a t i o n are i n any way i n t e r r e l a t e d . V i s s e r and Swaab ( 1 9 7 7 ) suggested t h a t alpha-MSH may be i n v o l v e d i n p r e - n a t a l growth i n the r a t . The f e t a l g u i n e a p i g grows r a p i d l y l a t e i n g e s t a t i o n at a time when plasma MSH l e v e l s are h i g h ; however, d e m o n s t r a t i o n of a cause and e f f e c t r e l a t i o n -s h i p must a w a i t f u r t h e r s t u d y . M e l a n o c y t e - s t i m u l a t i n g a c t i v i t y i s a l s o p r e s e n t i n b r a i n t i s s u e i n b o t h the a d u l t and f e t a l g u i n e a p i g . Thus MSH may 77 have a n e u r a l f u n c t i o n i n b e h a v i o u r a l m o d i f i c a t i o n , or n e u r a l t r a n s m i s s i o n , as suggested i n o t h e r s p e c i e s (see S e c t i o n I , d i s c u s s i o n ) . 5. C o n c l u s i o n The p r e s e n t study has shown t h a t s i g n i f i c a n t l e v e l s of m e l a n o c y t e - s t i m u l a t i n g p e p t i d e s are p r e s e n t i n both the p i t -u i t a r y , and the b l o o d , i n the f e t a l g u i n e a p i g . P i t u i t a r y l e v e l s i n c r e a s e d s t e a d i l y from about 40 days of g e s t a t i o n u n t i l term; plasma l e v e l s were r e l a t i v e l y c o n s t a n t over t h i s p e r i o d . P r e l i m i n a r y i n v e s t i g a t i o n s u g gests t h a t plasma l e v e l s show a t r a n s i e n t drop i n a c t i v i t y j u s t a f t e r b i r t h . T h i s phenomenon s h o u l d be s t u d i e d i n d e t a i l . Low but measurable a c t i v i t y was a l s o p r e s e n t i n 33$ of the a m n i o t i c f l u i d samples assayed. R e c e n t l y s e v e r a l p o s s i b i l i t i e s f o r a p h y s i o l o g i c a l r o l e f o r MSH i n the f e t u s have been proposed. S i n c e no c l e a r f u n c -t i o n f o r these p e p t i d e s has been e s t a b l i s h e d i n the a d u l t , f u r t h e r s t u d i e s s h o u l d I n v e s t i g a t e p o s s i b l e e f f e c t s of MSH i n the f e t u s . 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"The S t a t i s t i c s o f B i o a s s a y , w i t h s p e c i a l r e f e r e n c e t o t h e v i t a m i n s " , A c a d e m i c P r e s s , New Y o r k , 6 2 8 p. B l o o m f i e l d , G.A., S c o t t , A.P., L o w r y , P . J . , G i l k e s , J.J.H., and R e e s , L.H. 1 9 7 4 . A r e a p p r a i s a l o f human /S-MSH. N a t u r e 2 5 2 , 4 9 2 - 4 9 3 . B o n s n e s , R. 1 9 6 6 . C o m p o s i t i o n o f a m n i o t i c f l u i d . C l i n . O b s t e t . G y n e c o l . 9, 440-448. B u r g e r s , A . C . J . 196 3- M e l a n o p h o r e s t i m u l a t i n g hormones i n v e r t e b r a t e s . Ann. N.Y. A c a d . S c i . 1 0 0 , 6 6 9 - 6 7 7 . B u r t o n , A.M., I l l i n g w o r t h , D.V., C h a l l i s , J.R.G., and M c N e i l l y , A.S. 1 9 7 4 . P l a c e n t a l t r a n s f e r o f o x y t o c i n i n t h e g u i n e a - p i g and i t s r e l e a s e d u r i n g p a r t u r i t i o n . J . E n d o c r i n o l . 6 0 , 4 9 9 - 5 0 6 . C e h o v i c , G. 1 9 6 5 . A c t i o n du t r a i t e m e n t p r o l o n g e a v e c l e s hormones m e l a n o p h o r i q u e s (MSH) s u r l e s g o n a d e s des s o u r i s m a l e s . C.R. A c a d . S c i . (D) ( P a r i s ) 2 6 1 , 1 4 0 5 - 1 4 0 8 . C e h o v i c , G.. 1 9 6 7 . E f f e t de MSH s u r l ' a x e h y p o t h a l a m u s -h y p o p h y s e - t h y r o i d e . Rev. E u r . E n d o c r i n o l . 4_, 1 2 5 - 1 3 8 . C e l l s , M.E. 1 9 7 5 . Serum MSH l e v e l s and t h e h y p o t h a l a m i c enzymes i n v o l v e d i n t h e f o r m a t i o n o f MSH-RF d u r i n g t h e e s t r o u s c y c l e , i n t h e r a t . N e u r o e n d o c r i n o l o g y 1 8 , 2 5 6 - 2 6 2 . C h a l l i s . J.R.G., and T o r o s i s , J.D. 1 9 7 7 . I s *-MSH a t r o p h i c hormone t o a d r e n a l f u n c t i o n i n t h e f o e t u s ? N a t u r e 2 6 9 , 8 1 8 - 8 1 9 . 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