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11 B [i.e. Eleven beta] - Hydroxysteroid NADP Oxidoreductase in mouse foetal tissues 1976

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116-HYDROXYSTEROID:NADP OXIDOREDUCTASE IN MOUSE FOETAL TISSUES by NICOLE JOCELYNE MICHAUD B . S c , U n i v e r s i t y o f O t t a w a , 1974 A T h e s i s S u b m i t t e d i n P a r t i a l F u l f i l m e n t o f The R e q u i r e m e n t s f o r t h e D e g r e e o f MASTER OF SCIENCE i n t h e D e p a r t m e n t o f B i o c h e m i s t r y We a c c e p t t h i s t h e s i s a s 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 B R I T I S H COLUMBIA S e p t e m b e r , 1976 (<3) N i c o l e J o c e l y n e M i c h a u d , 197 6 In p resent ing t h i s t he s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree tha t permiss ion fo r ex ten s i ve copying o f t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r ep re sen ta t i ve s . It i s understood that copying or p u b l i c a t i o n o f t h i s t he s i s f o r f i n a n c i a l gain s h a l l not be a l lowed without my w r i t t e n permi s s ion . Department of Biochemistry The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date October 28, 1976 i ABSTRACT C o r t i c o s t e r o n e i n f o e t a l t i s s u e s a f t e r i n j e c t i o n o f t h e 14 m o t h e r w i t h C - c o r t i c o s t e r o n e was d e t e r m i n e d by a c e t y l a t i o n 3 . w i t h H - a c e t i c a n h y d r i d e a nd c r y s t a l l i z a t i o n t o c o n s t a n t s p e c i f i c a c t i v i t y . The c o r t i c o s t e r o n e c o n t e n t o f w h o l e f o e t a l t i s s u e v a r i e d b e t w e e n g e s t a t i o n a l d a y s 13 and 17 f r o m 641 t o 300 ng/g r e s p e c t i v e l y . The s p e c i f i c a c t i v i t y o f f o e t a l hormone r e c o v e r e d r e m a i n e d e s s e n t i a l l y c o n s t a n t ; a f t e r a 1 5 - m i n u t e p u l s e t h i s was a s much a s o n e - f o u r t h t h a t o f m a t e r n a l hormone. However, p l a c e n t a , h e a d and l i v e r showed d i s t i n c t l y d i f f e r e n t p a t t e r n s o f m e t a b o l i s m , w h i c h c h a n g e d g r e a t l y d u r i n g t h i s t i m e i n h e a d and l i v e r , w i t h a d e c r e a s e i n t h e c o n v e r s i o n o f c o r t i - c o s t e r o n e t o 1 1 - d e h y d r o c o r t i c o s t e r o n e and a r i s e i n f o e t a l l i v e r 1 1 3 - h y d r o x y s t e r o i d : N A D P o x i d o r e d u c t a s e a c t i v i t y . T h i s m i t o c h o n d r i a l enzyme, Km=33yM, pH opt i m u m 6, w h i c h r e d u c e s t h e 1 1 - d e h y d r o m e t a b o l i t e t o t h e b i o l o g i c a l l y a c t i v e 116-OH compound, i n c r e a s e d s h a r p l y , r a i s i n g t h e r e l a t i v e amount o f t h e l a t t e r i n f o e t a l t i s s u e s f r o m 15 t o 9 1 % d u r i n g t h i s p e r i o d . One d a y a f t e r r e m o v a l o f m a t e r n a l a d r e n a l s , f o e t a l c o r t i c o s t e r o n e was n o r m a l and m a t e r n a l l e v e l s c l o s e t o n o r m a l , i n d i c a t i n g a b i l i t y o f f o e t a l a d r e n a l s t o f u n c t i o n . M a t e r n a l hormone, h o w e v e r , c r o s s e d t o t h e f o e t u s r e a d i l y a n d i t was c o n s i d e r e d most l i k e l y t h a t , n o r m a l l y , t h e m a t e r n a l s o u r c e p r e d o m i n a t e s . R e g a r d l e s s o f o r i g i n , f o e t a l o r m a t e r n a l , h o w e v e r , t h e hormone i s m a i n - t a i n e d i n d i f f e r e n t f o e t a l t i s s u e s i n a d i s t i n c t and d i f f e r e n t manner. ACKNOWLEDGEMENTS I wish to express my deep a p p r e c i a t i o n t o Dr. A.F. Burton f o r h i s guidance, j u d i c i o u s a d v i c e and p a t i e n c e throughout the course of my r e s e a r c h and f o r h i s encouragement and con- c e r n d u r i n g the w r i t i n g of my t h e s i s . I a l s o wish to thank my s i s t e r , Miss F r a n c i n e Michaud f o r her t e c h n i c a l a s s i s t a n c e d u r i n g my l a s t summer of r e s e a r c h . i i i TABLE OF CONTENTS ABSTRACT i ACKNOWLEDGEMENTS i i TABLE OF CONTENTS i i i L I S T OF TABLES v i L I S T OF FIGURES v i i L I S T OF ABBREVIATIONS v i i i INTRODUCTION 1 MATERIALS 10 1. A n i m a l s 10 2. B u f f e r s 10 3. S o l v e n t s 10 4. TLC 11 5. R a d i o a c t i v e s t e r o i d s 11 6. N o n - r a d i o a c t i v e r e a g e n t s 12 7. S c i n t i l l a t i o n s o l v e n t 12 8. L i q u i d s c i n t i l l a t i o n s p e c t r o m e t e r 12 9. T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y 12 10. A u t o r a d i o g r a p h y 13 i v METHODS 14 1. A n i m a l s 14 (a) I n j e c t i o n o f m i c e (b) A d r e n a l e c t o m y 2. T i s s u e s 15 (a) M i n c e d t i s s u e s (b) C e l l f r a c t i o n a t i o n 3. I n c u b a t i o n 16 (a) F^nzymatic s y n t h e s i s o f 1 1 - d e h y d r o c o r t i c o s t e r o n e (b) M i n c e d t i s s u e s (c) Coenzyme s p e c i f i c i t y (d) pH (e) K i n e t i c a n a l y s i s 4. E x t r a c t i o n o f t i s s u e s 13 (a) B l o o d a n d t i s s u e s f r o m m i c e (b) I n c u b a t e d m i n c e d t i s s u e s a n d c e l l f r a c t i o n s 5. C h r o m a t o g r a p h y 18 6. A c e t y l a t i o n 19 7. C r y s t a l l i z a t i o n 20 8. S t a n d a r d s f o r e f f i c i e n c y o f a c e t y l a t i o n 22 9. Quench c u r v e a nd c a l c u l a t i o n o f t h e d o u b l e l a b e l * 2 2 10. T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y 22 V EXPERIMENTAL RESULTS A. I n v i v o e x p e r i m e n t s 25 1. C o r t i c o s t e r o n e c o n t e n t o f t i s s u e s 25 2. E f f e c t o f a d r e n a l e c t o m y o f t h e m o t h e r on t h e t o t a l amount o f c o r t i c o s t e r o n e 28 14 3. M e t a b o l i s m o f C - c o r t i c o s t e r o n e i n t h e p l a c e n t a and f o e t a l t i s s u e s 31 4. C o r t i c o s t e r o n e a n d i t s 1 1 - d e h y d r o m e t a b o l i t e i n . f o e t a l t i s s u e s 33 B. I n v i t r o e x p e r i m e n t s 3 5 1. I n v i t r o m e t a b o l i s m o f c o r t i c o s t e r o n e a n d 1 1 - d e h y d r o . c o r t i c o s t e r o n e 35 2. L o c a l i z a t i o n o f t h e r e d u c t a s e a c t i v i t y 37 3. M e t a b o l i s m o f c o r t i c o s t e r o n e i n t h e f o e t a l h e a d 39 C. Enzyme k i n e t i c s 41 1. C e l l f r a c t i o n a t i o n a n d c o enzyme s p e c i f i c i t y 41 2. D e v e l o p m e n t o f t h e r e d u c t a s e a c t i v i t y i n t h e f o e t a l l i v e r 44 3. pH o p t i m u m 4 6 4. Km 4 6 5. C o n t e n t and s p e c i f i c a c t i v i t y o f r e c o v e r e d 14 C - c o r t i c o s t e r o n e i n t h e f o e t a l b o d i e s 52 6. H i s t o l o g i c a l s t u d i e s 54 DISCUSSION 59 BIBLIOGRAPHY 6 6 VI L I S T OF TABLES I D e t e r m i n a t i o n o f t h e p u r i t y o f c r y s t a l l i z e d " ^ C - c o r t i c o s t e r o n e " ^ H - a c e t a t e 21 I I T o t a l amount o f c o r t i c o s t e r o n e i n t h e m o t h e r and t h e f o e t u s e s on d a y s 13 and 17 26 14 I I I S p e c i f i c a c t i v i t y o f r e c o v e r e d C - c o r t i - c o s t e r o n e 27 I V E f f e c t o f a d r e n a l e c t o m y o f t h e m o t h e r on t h e l e v e l s o f c o r t i c o s t e r o n e i n t h e m o t h e r 29 X 30 V C o n t e n t a n d s p e c i f i c a c t i v i t y o f r e c o v e r e d • ^ C - c o r t i c o s t e r o n e i n t h e f o e t u s a f t e r a d r e n a l e c t o m y o f t h e m o t h e r V I R e l a t i v e amount o f " C - c o r t i c o s t e r o n e a n d i t s 1 1 - d e h y d r o m e t a b o l i t e i n t h e p l a c e n t a a n d f o e t a l t i s s u e s ^ A V I I C o r t i c o s t e r o n e a n d i t s 1 1 - d e h y d r o m e t a b o l i t e i n f o e t a l t i s s u e s V I I I I n v i t r o m e t a b o l i s m o f c o r t i c o s t e r o n e a n d 1 1 - d e h y d r o c o r t i c o s t e r o n e b y f o e t a l t i s s u e s 36 I X R e d u c t i o n o f 1 1 - d e h y d r o c o r t i c o s t e r o n e b y t h e f o e t a l l i v e r and r e s i d u a l c a r c a s s 38 Enzyme a c t i v i t y a n d coenzyme s p e c i f i c i t y i n t h e c r u d e s u b c e l l u l a r f r a c t i o n s 42 X I Enzyme a c t i v i t y a n d coenzyme s p e c i f i c i t y i n w a s h e d f r a c t i o n s a n d t h e i r w a s h e s 43 X I I D e v e l o p m e n t o f t h e r e d u c t a s e a c t i v i t y i n f o e t a l l i v e r 45 X I I I C o n t e n t and s p e c i f i c a c t i v i t y o f r e c o v e r e d • ' - ^ C - c o r t i c o s t e r o n e i n t h e f o e t a l b o d y 53 V l l L I S T OF FIGURES Quench c u r v e f o r H and C I n v i t r o m e t a b o l i s m o f * C - c o r t i c o s t e r o n e by t h e f o e t a l h e a d a t d a y s 14 and 15 pH d e p e n d e n c e o f r e d u c t a s e a c t i v i t y E f f e c t o f c o n c e n t r a t i o n o f 1 1 - d e h y d r o c o r t i - c o s t e r o n e on r e d u c t a s e a c t i v i t y L i n e w e a v e r - B u r k p l o t f o r r e d u c t i o n o f 1 1 - d e h y d r o c o r t i c o s t e r o n e E f f e c t o f c o n c e n t r a t i o n o f c o r t i c o s t e r o n e on d e h y d r o g e n a s e a c t i v i t y L i n e w e a v e r - B u r k p l o t f o r d e h y d r o g e n a t i o n o f c o r t i c o s t e r o n e S e c t i o n s o f a d r e n a l s f r o m f o e t a l m i c e t r e a t e d w i t h d e x amethasone TEM o f a d r e n a l s f r o m f o e t a l m i c e t r e a t e d w i t h d e x a methasone v i i i L I S T OF ABBREVIATIONS A d r x : a d r e n a l e c t o m y , a d r e n a l e c t o m i z e d AR : a u t o r a d i o g r a m , a u t o r a d i o g r a p h y CBG : c o r t i c o s t e r o i d b i n d i n g g l o b u l i n ( T r a n s c o r t i n ) c p d .A : 1 1 - d e h y d r o c o r t i c o s t e r o n e cpd.B : c o r t i c o s t e r o n e DMP : 2 , 4 , 6 - t r i - ( d i m e t h y l ) - a m i n o m e t h y l p h e n o l DDSA : d o d e c y l s u c c i n i c a n h y d r i d e LM : l i g h t m i c r o s c o p y L/S : l e c i t h i n / s p h i n g o m y e l i n r a t i o MDA : m e t h y l n a d i c a n h y d r i d e PO : p r o p y l e n e o x i d e RDS : r e s p i r a t o r y d i s t r e s s syndrome SEM : s t a n d a r d e r r o r o f t h e mean TEM : t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e TLC : t h i n l a y e r c h r o m a t o g r a p h y UV : u l t r a v i o l e t 1 INTRODUCTION The e n d o c r i n e c o n t r o l o f pregnancy i s p r o v i d e d b o t h by the mother and t h e f o e t o - p l a c e n t a l u n i t . I n most s p e c i e s , t h e p e p t i d e hormones; e.g., ACTH, and serum p r o t e i n s do n o t c r o s s the p l a c e n t a and s e r v e t o r e g u l a t e t h e l e v e l s o f s t e r o i d s on t h e m a t e r n a l s i d e . On t h e o t h e r hand, t h e s t e r o i d hormones can c r o s s t h e p l a c e n t a . The c o r t i c o s t e r o i d s c r o s s i n s i g n i f i c a n t amounts t o t h e f o e t a l s i d e i n some s p e c i e s , n o t a b l y mouse and man. Oe s t r o g e n s and androgens, as w e l l as t h e c a t e c h o l a m i n e s and t h y r o n i n e s a r e s u b j e c t t o a v e r y r i g o u r o u s m e t a b o l i c c o n t r o l , m a i n l y by s u l f u r y l a t i o n i n f o e t a l t i s s u e s ( 1 ) . Whereas i n t h e p l a c e n t a o e s t r o g e n s and o t h e r s t e r o i d s hormones a r e p r e s e n t p r e d o m i n a n t l y i n an u n c o n j u g a t e d " f r e e " form, t h e y a r e e x t e n s i v e l y and r a p i d l y c o n j u g a t e d i n t h e f o e t u s . One o f t h e i m p o r t a n t f a c t o r s w h i c h i s t h o u g h t t o i n f l u - ence t h e growth and s e c r e t o r y a c t i v i t y o f t h e f o e t a l a d r e n a l i s t h e s e c r e t i o n on ACTH by t h e f o e t a l p i t u i t a r y . A r e v i e w by J o s t (2) summarized e x p e r i m e n t a l e v i d e n c e on t h a t a s p e c t i n t he mouse, r a t , sheep, r a b b i t and g u i n e a p i g . Absence o f t h e p i t u i t a r y , d e s t r u c t i o n o f t h e heads by X-ray beams o r s u r g i c a l d e c a p i t a t i o n o f f o e t u s e s a l l r e s u l t i n a d e c r e a s e i n s i z e o f t h e a d r e n a l c o r t e x a t b i r t h , a t r o p h y o f t h e c o r t i - c a l c e l l s and m o d i f i c a t i o n i n t h e d i s t r i b u t i o n o f l i p i d s , w h i c h can be r e v e r s e d by g i v i n g ACTH t o t h e f o e t u s e s a t t h e time o f s u r g e r y . 2 In the human anencoephalic foetus, the f o e t a l zone of the f o e t a l adrenal regresses prematurely and the d e f i n i t i v e cortex, while hypoplastic, undergoes premature d i f f e r e n t i a - t i o n . Hence i t seems reasonable to believe that ACTH d e f i - ciency i s responsible for the adrenal abnormalities i n the anencoephalic infant. Administration of the synthetic c o r t i c o s t e r o i d , dexa- methasone, to pregnant Rhesus monkeys (3) resulted i n atrophy of f o e t a l adrenal gland and an appreciable regression of the f o e t a l zone. Dexamethasone i s believed to suppress p i t u i t a r y ACTH s e l e c t i v e l y . If t h i s assumption can be c o r r e c t l y applied to the foetus, these experiments provide strong support for the b e l i e f that ACTH i s an important trophic factor for the f o e t a l zone of the adrenal i n utero. That the foetus can synthesize c o r t i c o s t e r o i d s i s well established. Dupouy et a l (4, 5) have shown in the rat f o l - lowing adrenalectomy on the mother on day 18, that the l e v e l of corticosterone i n maternal blood was the same as i n the untreated, i n d i c a t i n g that not only the f o e t a l rat adrenal has the a b i l i t y to synthesize c o r t i c o s t e r o i d s , but also that the hormones can cross the placenta from the foetus to the mother, i n contrast to the sheep ( 6 , 7 ) . The lev e l s of corticosteroids are also regulated on the maternal side by transcortin. This protein, also c a l l e d 3 c o r t i c o s t e r o i d b i n d i n g g l o b u l i n ( C B G ) , b i n d s s p e c i f i c a l l y C o r t i s o l , c o r t i c o s t e r o n e , t h e i r 1 1 - d e o x y m e t a b o l i t e s a n d p r o - g e s t e r o n e , b u t n o t a n y o f t h e i r o t h e r m e t a b o l i t e s ( 8 ) . I t i s p r e s e n t i n m o s t s p e c i e s e x a m i n e d a n d i s s y n t h e s i z e d i n t h e l i v e r . The o n l y f a c t o r s t h a t seem t o i n f l u e n c e t h e l e v e l s o f t r a n s c o r t i n a r e p r e g n a n c y a n d t h e l e v e l o f o e s t r o - g e n s . O n l y a b o u t 1 0 % o f t h e t o t a l c o r t i c o s t e r o i d i s f r e e , t h e r e s t b e i n g b o u n d t o t r a n s c o r t i n . G a l a a n d W e s t p h a l (9) h a v e r e p o r t e d a m a r k e d i n c r e a s e d u r i n g p r e g n a n c y i n t h e c o r t i c o s t e r o i d - b i n d i n g a c t i v i t y i n t h e s e r u m o f t h e mouse, r a b b i t a n d g u i n e a p i g , f o l l o w e d b y a d e c l i n e t o t h e l e v e l o f n o n - p r e g n a n t a n i m a l s d u r i n g l a c t a t i o n . F r e e s e r u m c o r t i c o s t e r o i d c o n c e n t r a t i o n s p a r a l l e l e d t h e b o u n d s t e r o i d b u t w e r e o n l y a f r a c t i o n o f t h e t o t a l . T h i s i n c r e a s e o c c u r s a l s o i n human a n d o t h e r s p e c i e s , t h e l e v e l o f t o t a l c o r t i c o s t e r o i d i n c r e a s i n g s e v e r a l f o l d , t h e f r e e s t e r o i d s i n c r e a s i n g p r o p o r t i o n a t e l y . The t r a n s c o r t i n - b o u n d s t e r o i d i s n o t a b l e t o e n t e r t i s s u e s , a n d d o e s n o t c r o s s t h e p l a c e n t a a n d b i o l o g i c a l a c t i v i t y i s a t t r i b u t e d o n l y t o t h e f r e e s t e r o i d ( 8 ) . The p a s s a g e o f c o r t i c o s t e r o i d s a c r o s s t h e p l a c e n t a shows a p a t t e r n w h i c h v a r i e s w i t h s p e c i e s a n d w h i c h i s a t l e a s t p a r t i a l l y d e t e r m i n e d b y t h e t y p e o f p l a c e n t a . L i g g i n s (6) h a s shown i n t h e ewe t h a t c o r t i c o s t e r o i d s c r o s s t h e p l a c e n t a 4 i n the d i r e c t i o n f oetus to mother, but not i n the r e v e r s e d i r e c t i o n . On the other hand, adrenalectomy of r a t s (4) and i n mice (2) has shown t h a t , when the maternal source of s t e r o i d s y n t h e s i s i s removed, the maternal s t e r o i d l e v e l s are r e s t o r e d to normal w i t h i n a few hours by s y n t h e s i s i n f o e t a l a d r e n a l s . Measurements of cord blood C o r t i s o l i n the human (10, 11, 12, 13) suggest t h a t c o r t i c o s t e r o i d s a l s o c r o s s the p l a c e n t a i n both d i r e c t i o n s . Comparison of the p l a c e n t a i n v o l v e d (14) shows t h a t the sheeps possess the syndesmochorial type, which i s c h a r a c t e r i z e d by c h o r i o n i c v i l l i occupying deep p i t s i n the u t e r i n e l i n i n g , w i t h some l o c a l d e s t r u c t i o n of the u t e r i n e e p i t h e l i u m which allows the c h o r i o n i c ectoderm to come i n t o d i r e c t c o n t a c t w i t h the v a s c u l a r maternal c o n n e c t i v e t i s s u e . The rodents and the human possess a haemochorial type of p l a c e n t a which i s c h a r a c t e r i z e d by a more thorough e r o s i o n of the s u p e r f i c i a l u t e r i n e mucosa and the branching c h o r i o n i c v i l l i are d i r e c t l y bathed by maternal blood i s s u i n g from opened v e s s e l s . The a c t i v e s t e r o i d i s a l s o metabolized i n the f o e t o - p l a c e n t a l u n i t . An enzyme, 11B-hydroxysteroid:NADP o x i d o r e - ductase has been i d e n t i f i e d i n mouse and i n human f o e t a l t i s s u e s (15, 16). T h i s enzyme dehydrogenates the 113- hydroxyl group which i s e s s e n t i a l f o r b i o l o g i c a l a c t i v i t y of the hormone, forming 11-keto m e t a b o l i t e s . T h i s i s con- s i d e r e d to be r e s p o n s i b l e f o r the high l e v e l s of c o r t i s o n e i n hum- an cord blood (17) . Burton e t a l (18) noted i n the mouse an 5 i n v e r s e r e l a t i o n s h i p between the r a t e of conversion of c o r t i c o s t e r o n e to 11-dehydrocorticosterone i n f o e t a l t i s s u e s and the c a p a c i t y of c o r t i c o s t e r o n e to e l i c i t an increase i n l i v e r glycogen. A sharp increase i n glycogen d e p o s i t i o n occurred i n f o e t a l l i v e r p a r a l l e l i n g a decrease i n the r a t e 14 14 of conversion of C - c o r t i c o s t e r o n e to C - l l - d e h y d r o c o r t i - costerone. This was thought to be due, not to a decrease i n dehydrogenase a c t i v i t y , but r a t h e r to the appearance of an enzyme i n f o e t a l l i v e r which reduced the m e t a b o l i t e back to c o r t i c o s t e r o n e . This a c t i v i t y developed only l a t e i n gesta- t i o n and r e s u l t e d i n a r i s e i n c o r t i c o s t e r o n e at the expense of the m e t a b o l i t e . C o r t i c o s t e r o i d s have been shown to play major r o l e s i n f o e t a l development. Experimental work has shown t h a t func- t i o n a l maturation of f o e t a l lamb lungs can be a c c e l e r a t e d by s t i m u l a t i o n of the f o e t a l adrenal c o r t e x or by a d m i n i s t r a t i o n of g l u c o c o r t i c o i d s (6). L i g g i n s suggested t h a t g l u c o c o r t i - coids caused l i b e r a t i o n of s u r f a c t a n t i n t o the a l v e o l i , perhaps by i n d u c t i o n of an enzyme concerned w i t h the b i o - s y n t h e s i s of s u r f a c t a n t . Gluck (19) has devised a biochemical method to assess the m a t u r i t y of the f o e t a l lung. A f t e r having presented evidence t h a t there i s c o n t i n u i t y between the f o e t a l lung and amniotic f l u i d , i t was suggested t h a t the amniotic f l u i d l e c i t h i n / s p h i n g o m y e l i n (L/S) r a t i o would r e f l e c t the degree 6 o f f o e t a l l u n g m a t u r i t y . I t h a s b e e n shown t h a t c o r t i - c o s t e r o i d s i n d u c e t h e enzyme c h o l i n e p h o s p h o t r a n s f e r a s e , i n c r e a s i n g t h e s y n t h e s i s o f p h o s p h a t i d y l c h o l i n e (20), w h i c h i s t h e c r i t i c a l f a c t o r i n t h e s u r f a c t a n t w h i c h l o w e r s s u r f a c e t e n s i o n i n t h e a l v e o l i . I n v e s t i g a t i o n s by Murphy on human i n f a n t s (21) h a v e r e v e a l e d t h a t u m b i l i c a l c o r d b l o o d C o r t i s o l and c o r t i - s o ne l e v e l s w e r e l o w e r i n i n f a n t s w i t h r e s p i r a t o r y d i s t r e s s s yndrome (RDS) t h a n i n t h e n o r m a l c o n t r o l s . A l s o , i n t h e c a s e o f p r e m a t u r e r u p t u r e o f membrane, a l o w C o r t i s o l l e v e l was a c c o m p a n i e d by RDS, w h e r e a s a h i g h e r C o r t i s o l l e v e l was n o t . The C o r t i s o l l e v e l was a l w a y s a t l e a s t d o u b l e i n t h o s e i n f a n t s w i t h o u t RDS. T h e s e f i n d i n g s p r o v i d e a p h y s i o l o g i c j u s t i f i c a t i o n f o r a t t e m p t e d p r e v e n t i o n o f d i s e a s e r e s u l t i n g f r o m p u l m o n a r y i m m a t u r i t y i n t h e human by t h e p r e n a t a l a d m i n i s t r a t i o n o f g l u c o c o r t i c o i d s . C o n t r o l l e d t r i a l s o f b e t a m e t h a s o n e t h e r a p y h a v e b e e n c a r r i e d o u t (22) i n m o t h e r s i n whom p r e m a t u r e d e l i v e r y was t h r e a t e n e d . The r e s p i r a t o r y d i s t r e s s syndrome o c c u r r e d l e s s o f t e n i n t r e a t e d b a b i e s and t r e a t m e n t h a s become w i d e l y a p p l i e d i n t h e l a s t two y e a r s . The i m p o r t a n c e o f t h e f u n c t i o n a l f o e t a l h y p o t h a l a m u s - p i t u i t a r y - a d r e n a l a x i s on t h e o n s e t o f l a b o u r h a s b e e n d e m o n s t r a t e d i n t h e s h e e p ( 7 ) . L i g g i n s h a s shown t h a t i n f u s i o n o f a d r e n o c o r t i c o t r o p i c hormone (ACTH) o r C o r t i s o l i n t o t h e f o e t a l l a m b s i n d u c e d p r e m a t u r e p a r t u r i t i o n , w h e r e a s 7 i n f u s i o n o f t h e s e hormones i n t o t h e ewes d i d n o t . I n the same a n i m a l , D r o s t and Holm (23) have shown t h a t pregnancy can be p r o l o n g e d by a d r e n a l e c t o m y o f t h e f o e t a l lamb. I n t h e r a b b i t , i f a d r e n a l c o r t i c o s t e r o i d s p l a y a r o l e i n i n i t i a t i n g l a b o u r , N a t h a n i e l s z and A b e l (24) d i d not succeed i n showing whether t h e y a r e s e c r e t e d by t h e m a t e r n a l o r t h e f o e t a l a d r e n a l s . However, i n t h e f o e t a l r a t , t h e plasma c o r t i c o s t e r o n e c o n c e n t r a t i o n was shown t o be as h i g h as s i x t i m e s m a t e r n a l v a l u e s a t day 19 o f g e s t a t i o n ( g e s t a t i o n p e r i o d : 22 days) and t o be a p p r o x i m a t e l y e q u a l t o m a t e r n a l v a l u e s from day 20 t o term ( 2 5 ) . The d i r e c t p a r t i c i p a t i o n i n t h e o n s e t o f l a b o u r o f t h e f o e t u s has not been prov e n i n t h e human b u t some e v i d e n c e s u g g e s t s i t ; e.g., t h e p r o l o n g a t i o n o f pregnancy a s s o c i a t e d w i t h anencoephaly (the absence o f p i t u i t a r y f o r m a t i o n (26) ). C o r t i s o l l e v e l s a r e t h r e e t i m e s h i g h e r i n f o e t u s a t 32 weeks t h a n a t 18 weeks ( 1 0 ) . However, premature i n f a n t s w i t h RDS sometimes have v e r y low C o r t i s o l l e v e l s , s u g g e s t i n g t h a t w h i l e C o r t i s o l i s e s s e n t i a l f o r normal development, i t i s not t h e p r i m a r y f a c t o r i n i t i a t i n g l a b o u r , a t l e a s t when t h i s o c c u r s p r e m a t u r e l y . I n j e c t i o n o f dexamethasone i n t o Rhesus monkeys d i d not i n d u c e p a r t u r i t i o n ( 3 ) . So, p o s s i b l y i n p r i m a t e s , t h e c o r t i c o s t e r o i d s a r e n o t t h e p r i m a r y f a c t o r i n d u c i n g p a r t u r i t i o n . 8 Because of the s i m i l a r i t y of the p l a c e n t a of the human and the mouse (14), which i s of the haemochorial type, i t was reasoned t h a t the mouse p r o v i d e s a model f o r s t e r o i d t r a n s p o r t and metabolism i n mammals i n those i n s t a n c e s where the hormone has been shown to c r o s s the p l a c e n t a e a s i l y and i s m e t a b o l i z e d e x t e n s i v e l y to the 11-keto d e r i v a t i v e . S ince there i s a l r e a d y a p o o l of s t e r o i d i n the animal a t the moment of the experiment, the a d m i n i s t r a t i o n of r a d i o - a c t i v e hormone r e s u l t s i n a d i l u t i o n of the i s o t o p e , the e x t e n t of which can be used to estimate changes i n the r e l a - t i v e s i z e of the p o o l . To determine a c c u r a t e l y s m a l l amounts of c o r t i c o s t e r o i d s i n the maternal b l o o d and/or i n the f o e t a l t i s s u e s , s e v e r a l methods have been used. Murphy (28) has developed a compe- t i t i v e p r o t e i n - b i n d i n g r a d i o - a s s a y . Burton (16) used a f l u o r o m e t r i c method which has the l i m i t a t i o n of d e t e c t i n g o n l y the a c t i v e form of the hormone and of r e q u i r i n g l a r g e r volumes of b l o o d . Values o b t a i n e d agree w e l l w i t h o t h e r methods (28, 29). Although r e d u c i n g e n z y m a t i c a l l y the i n a c t i v e m e t a b o l i t e to c o r t i c o s t e r o n e made i t p o s s i b l e to measure the former f l u o r o m e t r i c a l l y , i t would be d i f f i c u l t to use w i t h the mouse because of the s m a l l q u a n t i t i e s of blood and t i s s u e . 9 An even more s e n s i t i v e method has been developed by 14 H i l l m a n and Giroud (29) whereby a t r a c e amount of C- 3 l a b e l l e d hormone i s a c e t y l a t e d w i t h a c e t i c - H anhydride and p u r i f i e d by s e v e r a l steps of paper chromatography t o a con- 3 14 s t a n t r a t i o o f H/ C. The method used i n t h i s work i s a m o d i f i c a t i o n i n which a f t e r a c e t y l a t i o n r a d i o i n e r t s t e r o i d a c e t a t e was added and the mixture was c o c r y s t a l l i z e d . Con- 3 14 stancy of i s o t o p e r a t i o H/ C a f t e r the t h i r d and f o u r t h c r y s t a l l i z a t i o n was accepted as the c r i t e r i o n o f p u r i t y o f the compound. 10 MATERIALS A n i m a l s UBC S w i s s m i c e w e r e o b t a i n e d f r o m t h e z o o l o g y v i v a r i u m a t t h i s u n i v e r s i t y . The g e s t a t i o n p e r i o d was 19 d a y s . Day 1 was d e f i n e d a s t h e d a y f o l l o w i n g t h e m a t i n g n i g h t . To d e t e r m i n e t h e d a y o f p r e g n a n c y a c c u r a t e l y , t h e m a l e s w e r e l e f t w i t h t h e f e m a l e s f r o m 9 p.m. t o 9 a.m. B u f f e r s K r e b s - E g g l e s t o n p h o s p h a t e b u f f e r c o n t a i n s : 0.9% N a C l , 1.15% K C I , 3.82% MgS0 4.7 H 2 0 , 0.IM p h o s - p h a t e b u f f e r , pH 7.4 ( 3 0 ) . P h o s p h a t e a n d a c e t a t e b u f f e r s f r o m pH 4 t o 9 w e r e p r e p a r e d f o l l o w i n g t h e p r o c e d u r e o f Hawk, O s e r a n d Sum- m e r s o n ( 3 1 ) . S o l v e n t s E v e r y s o l v e n t u t i l i z e d was d i s t i l l e d : c h l o r o f o r m , c a r b o n t e t r a c h l o r i d e , m e t h a n o l , e t h a n o l , n - h e x a n e , e t h y l a c e t a t e , t o l u e n e , d i c h l o r o m e t h a n e a n d a c e t o n e . 11 4. TLC O n l y E a s t m a n S i l i c a g e l TLC p l a t e s w i t h f l u o r e s - c e n t i n d i c a t o r w e r e u s e d . They were washed w i t h d i s - t i l l e d m e t h a n o l p r i o r t o u s e . 5. R a d i o a c t i v e compounds 3 A c e t i c - H a n h y d r i d e ( s p e c i f i c a c t i v i t y 400 Ci/mmole) comes i n s e a l e d a m p u l e s t o be p r o t e c t e d a g a i n s t h u m i d i t y . I t was p u r c h a s e d i n b a t c h e s o f 100 m C i . f r o m New E n g l a n d N u c l e a r C o r p . I t was d i l u t e d 10 t i m e s w i t h n o n - r a d i o a c t i v e a c e t i c a n h y d r i d e . A n h y d r o u s b e n z e n e was t h e n a d d e d t o g i v e a f i n a l c o n c e n t r a t i o n o f 10% a c e t i c a n h y d r i d e i n d r y b e n z e n e ( v / v ) . The r e a g e n t was s t o r e d a t room tem- p e r a t u r e i n a d e s i c c a t o r c o n t a i n i n g D R I E R I T E . 3 1, 2- H c o r t i c o s t e r o n e ( s p e c i f i c a c t i v i t y 51.6 C i / 14 mmole) and 4- C - c o r t i c o s t e r o n e ( s p e c i f i c a c t i v i t y 57.3 mCi/mmole) w e r e p u r c h a s e d f r o m New E n g l a n d N u c l e a r C o r p . E a c h was s p o t t e d o n t o TLC t o p u r i f y a n d was 14 e l u t e d w i t h a c e t o n e . C - l l - d e h y d r o c o r t i c o s t e r o n e was 14 s y n t h e s i z e d e n z y m a t i c a l l y f r o m C - c o r t i c o s t e r o n e ( c f . M e t h o d s ) . 12 6. N o n - r a d i o a c t i v e reagents The coenzymes NAD, NADP, NADH and NADPH were bought from Sigma Chemical Company, St. L o u i s , Mo. C o r t i c o s t e r o n e , 1 1 - d e h y d r o c o r t i c o s t e r o n e and c o r t i - c osterone a c e t a t e were ob t a i n e d from S t e r a l o i d s , Pawling, N.Y. 7. S c i n t i l l a t i o n s o l v e n t The s c i n t i l l a t i o n mixture was made of 4 g of 2,5-diphenyloxazole (PPO) and 0.1 g of l , 4 - b i s - 2 - ( 5-phenyloxazolyl) benzene (POPOP) d i s s o l v e d i n 1 l i t r e of t o l u e n e . 8. L i q u i d s c i n t i l l a t i o n spectrometer The l i q u i d s c i n t i l l a t i o n spectrometer was a U n i l u x IIA, Nuclear Chicago. The raw data were reduced w i t h a Hewlett-Packard 65 c a l c u l a t o r . 9. T r a n s m i s s i o n e l e c t r o n microscopy (a) Phosphate b u f f e r B u f f e r A: 0.IM sodium c a c o d y l a t e and 0.5% CaCl i n d i s t i l l e d water 13 B u f f e r B: t o e v e r y f o u r p a r t s o f b u f f e r A, one p a r t o f 3.5% N a C l . F o r pH 7.2, 143 m l o f b u f f e r A a n d 57 m l o f b u f f e r B. (b) E pon 812 The e m b e d d i n g r e s i n c o n s i s t e d o f 28.62 g Epon 8 1 2 , 21.0 g DDSA, 10.53 g MNA a n d 1% DMP 30. (c) S e c t i o n i n g S e c t i o n i n g was p e r f o r m e d w i t h a m a n u a l u l t r a - m i c r o t o m e S o r v a l l MT-I " P o r t e r - B l u m " . 10. A u t o r a d i o g r a p h y M e d i c a l X - r a y f i l m s (NS2T) w e r e o b t a i n e d f r o m K o d a k . 14 METHODS 1. Animals (a) I n j e c t i o n of mice Pregnant mice were i n j e c t e d subcutaneously a t the back of the neck w i t h 0.9% NaCI c o n t a i n i n g 14 0.5 yCi of C - c o r t i c o s t e r o n e . Unless otherwise s t a t e d , they were s a c r i f i c e d 15 min. l a t e r w i t h an overdose of CO,,, the u t e r u s removed from the mother and kept on i c e i n P e t r i d i s h e s c o n t a i n i n g a f i l t e r paper wet w i t h s a l i n e . Then the b l o o d was drawn from the mother w i t h a h e p a r i n i z e d s y r i n g e to prevent c o a g u l a t i o n . The f o e t u s e s and p l a c e n t a e were removed from the uterus - u s u a l l y t h r e e were pooled - weighed and ground i n 5 ml of s a l i n e . (b) Adrenalectomy Pregnant mice on day 16 were a n a e s t h e t i z e d w i t h 0.1 ml/10 g body weight of a s o l u t i o n c o n t a i n i n g 6.7% nembutal and 0.9% e t h a n o l . The a d r e n a l s were removed and the mice were kept under normal c o n d i - t i o n s with water and food f o r the next 24 h r s . 15 2. T i s s u e s (a) Minced t i s s u e s F o e t a l t i s s u e s were pooled (three heads, t h r e e c a r c a s s e s , three l i v e r s ) , minced on a wet f i l t e r paper, put i n t o 1 ml i n c u b a t i o n mixture and kept on i c e u n t i l the time of i n c u b a t i o n . (b) C e l l f r a c t i o n a t i o n The l i v e r s from 18-day foe t u s e s from two or three mothers were pooled, homogenized i n 6 ml Krebs b u f f e r c o n t a i n i n g 0.25M sucrose and c e l l f r a c t i o n a - t i o n was performed as f o l l o w s : A f t e r removing 1 ml, the whole homogenate was c e n t r i f u g e d a t 1,200 x g f o r 10 min. The n u c l e a r sediment was homogenized a second time to break the unbroken c e l l s and was r e - c e n t r i f u g e d . The mitoc h o n d r i a were c e n t r i f u g e d at 15,000 x g f o r 20 min and the microsomes a t 100,000 x g f o r 30 min. Each f r a c t i o n was r e s u s - pended i n the same b u f f e r and washed twice. The f i n a l volume was made up to the e q u i v a l e n t o f 50 mg t i s s u e / m l b u f f e r . Incubations (a) Enzymatic s y n t h e s i s o f 11- d e h y d r o c o r t i c o s t e r o n e Guinea p i g l i v e r microsomes were ob t a i n e d by c e l l f r a c t i o n a t i o n as d e s c r i b e d f o r mouse f o e t a l l i v e r s and were d i l u t e d to the e q u i v a l e n t of 100 mg t i s s u e / m l b u f f e r . They c o u l d be s t o r e d f r o z e n without s i g n i f i c a n t l o s s of a c t i v i t y . The i n c u b a t i o n mixture c o n s i s t e d of 0.1 ml microsome .14 p r e p a r a t i o n , 0.5 uCi C - c o r t i c o s t e r o n e , 0.1 ml Krebs-sucrose b u f f e r , 1 mg NADP i n a 2 5-ml erlenmeyer f l a s k . The i n c u b a t i o n was c a r r i e d out a t 37°C f o r 30 min. and the f l a s k s were shaken a t 120 c y c l e s / m i n . (16). (b) Minced t i s s u e s Minced t i s s u e s were incubated w i t h 1 ml 0.9% s a l i n e o r Krebs b u f f e r c o n t a i n i n g 20,000 cpm 3 . 14 H - c o r t i c o s t e r o n e o r 2,000 cpm C - l l - d e h y d r o c o r - t i c o s t e r o n e . In order to make p o s s i b l e the develop- ment of autoradiograms, the b r a i n t i s s u e s were 14 incubated w i t h 10,000 cpm C - c o r t i c o s t e r o n e . Unless otherwise s t a t e d , the i n c u b a t i o n s were c a r r i e d a t 37°C i n 25-ml erlenmeyer f l a s k s , stop- pered w i t h a s i l i c o n e stopper and shaken a t 120 c y c l e s / m i n . f o r 15 min. The r e a c t i o n was 17 stopped by p u t t i n g the f l a s k s on i c e , f o l l o w e d by e x t r a c t i o n w i t h s o l v e n t s . Coenzyme s p e c i f i c i t y One ml from each f r a c t i o n was incubated w i t h 0.5 mg coenzyme (NADH or NADPH) and 2,000 cpm 14 C - l l - d e h y d r o c o r t i c o s t e r o n e . pH optimum Incubations were c a r r i e d out i n 1 ml b u f f e r at d i f f e r e n t pHs (4 to 9) and 2,000 cpm 1 4 C - 1 1 - d e h y d r o c o r t i c o s t e r o n e . The r e d u c t i o n o f 11- d e h y d r o c o r t i c o s t e r o n e was c a r r i e d out i n f l a s k s gassed w i t h ^ • K i n e t i c a n a l y s i s The o x i d a t i o n r e a c t i o n was c a r r i e d out wit h 14 v a r i o u s s u b s t r a t e c o n c e n t r a t i o n s , 2,000 cpm C- c o r t i c o s t e r o n e , 0.5 mg NADP a t pH 8.0. The 14 r e d u c t i o n r e a c t i o n was c a r r i e d out wit h C - l l - d e h y d r o c o r t i c o s t e r o n e , 0.5 mg NADPH at pH 5.5. 18 E x t r a c t i o n of t i s s u e s (a) Blood and t i s s u e s from mice Samples were e x t r a c t e d i n 6 v o l . of n-hexane by shaking v i g o u r o u s l y 20 times. The upper phase, c o n t a i n i n g much n e u t r a l l i p i d , was d i s c a r d e d . S t e r o i d s were e x t r a c t e d by a d d i t i o n of 6 v o l . of dichloromathane (CH 2C1 2), shaking 20 times v i g o u r - o u s l y . CH 2C1 2 was evaporated down to dryness a t 4 0°C under N 2 gas to a v o i d o x i d a t i o n of the s t e r o i d s . B efore d r y i n g CH 2C1 2 down, 10 yg of c a r r i e r s t e r o i d s were added to each tube, o n e - f i f t h of each sample was removed f o r s p o t t i n g onto TLC to determine the 14 amount of C - s t e r o i d b e f o r e a c e t y l a t x o n w i t h t r i t i u m . The remaining f o u r - f i f t h s were pr o c e s s e d through to c r y s t a l l i z a t i o n . (b) Incubated minced t i s s u e s and c e l l f r a c t i o n s In these cases, the e n t i r e CH 2C1 2 f r a c t i o n was s p o t t e d onto TLC. Chromatography 10 yg of the n o n - r a d i o a c t i v e compounds to be d e t e c t e d ( c o r t i c o s t e r o n e and 1 1 - d e h y d r o c o r t i c o s t e r o n e , 19 o r c o r t i c o s t e r o n e and c o r t i c o s t e r o n e a c e t a t e ) w e r e a d d e d t o e a c h s a m p l e as a c a r r i e r f o r l o c a t i o n on t h e TLC. A f t e r e v a p o r a t i o n , t h e e x t r a c t s w e r e d i s s o l v e d i n two t o t h r e e d r o p s o f c h l o r o f o r m - m e t h a n o l (3:1) a n d s p o t t e d o n t o E a s t m a n TLC p l a t e s a t room t e m p e r a t u r e i n two d i f f e r e n t s y s t e m s : (a) n - h e x a n e : e t h y l a c e t a t e (4:1) t o r emove r e s i d u a l f a t s ; (b) t o l u e n e : c h l o r o f o r m : m e t h a n o l : w a t e r ( 1 2 0 : 6 0 : 2 0 : 1 ) t o s e p a r a t e s t e r o i d s . The z o n e s w e r e l o c a t e d u n d e r s h o r t w a v e UV, c u t o u t a n d e l u t e d w i t h a c e t o n e . 6. A c e t y l a t i o n F o l l o w i n g t h e p r o c e d u r e o f H i l l m a n and G i r o u d (29) 3 a n d K l i m a n ( 3 2 ) , 1 v o l . o f a c e t i c - H a n h y d r i d e and 9 v o l . o f r a d i o i n e r t a c e t i c a n h y d r i d e w e r e m i x e d and made t o 12% a c e t i c a n h y d r i d e i n b e n z e n e , w i t h s p e c i f i c a c t i v i t y 3 o f 40 C i / m m o l e . T h i r t y \il a c e t i c - H a n h y d r i d e and 20 y l d r y p y r i d i n e , t o k e e p t h e r e a c t i o n m i x t u r e i n s o l u t i o n , w e r e a d d e d t o e a c h s a m p l e a f t e r d r y i n g . 20 The a c e t i c - " H a n h y d r i d e a n d p y r i d i n e w e r e m e a s u r e d f r o m m i c r o v o l u m e t r i c p i p e t t e s s t o r e d i n a d e s i c c a t o r o v e r D R I E R I T E u n t i l r e a d y f o r u s e . A f t e r t h o r o u g h m i x i n g by g e n t l e s h a k i n g , t h e t u b e s w e r e s t o p p e r e d VERY t i g h t l y a n d i n c u b a t e d f o r 24 h r s . a t 3?°C ( 3 3 ) . 0.5 m l 2 5 % e t h a n o l was a d d e d t o h y d r o l y z e t h e 3 e x c e s s a c e t i c - H a n h y d r i d e t o a c e t i c a c i d . The s a m p l e s w e r e t h e n e x t r a c t e d w i t h 6 v o l . C C l ^ b y s h a k i n g 20 t i m e s v i g o u r o u s l y t o s e p a r a t e t h e s t e r o i d s . The w a t e r l a y e r was r e m o v e d a n d t h e s a m p l e was w a s h e d w i t h a n o t h e r 3 0.5 m l o f w a t e r t o g e t r i d o f r e s i d u a l H - a c e t a t e , w h i c h was d i s c a r d e d . 7. C r y s t a l l i z a t i o n A f t e r e v a p o r a t i o n o f C C l ^ , t h e s a m p l e s c o n t a i n i n g r a d i o a c t i v e c o r t i c o s t e r o n e a c e t a t e w e r e d i s s o l v e d i n 1 m l o f m e t h a n o l c o n t a i n i n g 10 mg o f n o n - r a d i o a c t i v e c o r t i c o s t e r o n e a c e t a t e . C o l d d i s t i l l e d w a t e r was a d d e d d r o p w i s e t o t h e c o l d s a m p l e s t o a l l o w c r y s t a l l i z a t i o n t o b e g i n . A f t e r f o u r t o s i x h o u r s a t 4°C, t h e c r y s t a l s w e r e r e d i s s o l v e d i n 1 m l o f m e t h a n o l . O n e - f i f t h o f e a c h s a m p l e was r e m o v e d a f t e r t h e t h i r d c r y s t a l l i z a t i o n a n d c o u n t e d . T h i s p r o c e d u r e was r e p e a t e d t h r e e t o f o u r i 3 14 t i m e s o r u n t i l a c o n s t a n t H/ C r a t i o was o b t a i n e d ( T a b l e I ) . 21 TABLE I. DETERMINATION OF THE PURITY OF CRYSTALLIZED 1 4C-CORTICOSTERONE - 21- 3H-ACETATE Sample 1: Mother 3 a t day 14 C r y s t a l l i z a t i o n 3 4 5 3H 11,625 9,943 1 4 c 671 573 3 H / 1 4 C 17.33 17 . 35 Sample 2: Foetus 32 a t day 18 3H 7,827 3,696 2,784 1 4 c 212 128 103 3 H / 1 4 C 36.95 28.93 27.08 A d i f f e r e n c e s m a l l e r than 10% between i s o t o p e r a t i o H/ "C a f t e r two s u c c e s s i v e c r y s t a l l i z a t i o n s was accepted as the c r i t e r i o n o f p u r i t y of the compound (29). 22 8. Standards f o r e f f i c i e n c y o f a c e t y l a t i o n 14 Approximately 10,000 dpm of C - c o r t i c o s t e r o n e was a c e t y l a t e d a t the same time as the samples. The c o r t i - c o s terone zone was cut out of the TLC, e l u t e d w i t h acetone and counted to determine the amount of cpd.B t h a t had not been a c e t y l a t e d . The c o r t i c o s t e r o n e a c e t a t e zone was a l s o c u t out and e l u t e d . Before pro- ceeding t o the c r y s t a l l i z a t i o n of the l a t t e r , o n e - f i f t h of each standard was counted; the f o u r - f i f t h s remaining were c r y s t a l l i z e d . 9. Quench curve and c a l c u l a t i o n o f the double l a b e l A quench curve f o r the d e t e r m i n a t i o n of the e f f i - 3 14 c i e n c y of c o u n t i n g f o r H and C i s shown i n F i g u r e 1. I t was checked a t l e a s t every o t h e r month. The c a l c u l a - t i o n s f o r the double i s o t o p e a n a l y s i s were done a c c o r d i n g t o the method o f Kobayashi and Maudsley (34). 10. T r a n s m i s s i o n e l e c t r o n microscopy F i x a t i o n was performed i n g l u t a r a l d e h y d e r p h o s p h a t e b u f f e r (4:1) f o r 1 h. The t i s s u e s were q u i c k l y t r a n s - f e r r e d i n t o a r i n s e phosphate b u f f e r f o r 15 min. F I G U R E 1, Q U E N C H C U R V E F O R 3H A N D '"C 1 2 3 4 5 E X T E R N A L S T A N D A R D R A T I O 2 4 P o s t - o s m i f i c a t i o n was done i n an osmium t e t r a o x i d e : p h o s p h a t e b u f f e r (1:1) f o r 40 m i n . A f t e r w a r d s t h e t i s s u e s w e r e k e p t i n p h o s p h a t e b u f f e r u n t i l d e h y d r a t i o n . M e t h a n o l was u s e d f o r d e h y d r a t i o n b e c a u s e i t i s t h e b e s t known t o r e d u c e c h a n g e s i n v o l u m e o f t h e t i s s u e . I t h a s a l s o s m a l l e r m o l e c u l a r s i z e a n d c a n p e n e t r a t e t h e t i s s u e more e a s i l y . The t i s s u e s w e r e l e f t f o r 5 m i n . i n 3 0 % , 5 0 % , 70% a n d 90% m e t h a n o l , t h e n 2 x 30 m i n . i n p r o p y l e n e o x i d e ( P O ) . The t i s s u e s w e r e d e h y d r a t e d i n PO:Epon 812 3:1, 1:1, and 1:3 f o r 1 h. e a c h . They w e r e embedded i n 1 0 0 % E p o n 812 and t h e r e s i n was a l l o w e d t o h a r d e n a t 60°C f o r 24 h. The t h i n s e c t i o n s w e r e s t a i n e d w i t h 0.2% l e a d c i t r a t e f o r 20 s e c o n d s . E l e c t r o n m i c r o g r a p h s w e r e made a t o r i g i n a l m a g n i f i c a t i o n s o f 3,000 t o 12,000 w i t h a P h i l i p s P-75 e l e c t r o n m i c r o s c o p e . EXPERIMENTAL RESULTS I n v i v o e x p e r i m e n t s 1. C o r t i c o s t e r o n e c o n t e n t o f t i s s u e s The t o t a l amount o f c o r t i c o s t e r o n e i n t h e m o t h e r an d i n t h e f o e t u s e s on d a y s 13 and 17 a r e shown i n T a b l e I I . B e t w e e n t h e s e two d a y s , t h e l e v e l o f c o r t i c o s t e r o n e i n c r e a s e d i n t h e m o t h e r b u t d e c r e a s e d i n t h e f o e t u s e s . W h i l e t h e t o t a l amount d e c r e a s e d on t h e f o e t a l s i d e ( T a b l e I I ) ( p < . 0 1 ) , i t s s p e c i f i c a c t i v i t y a c t u a l l y i n c r e a s e d ( T a b l e I I I ) , i n d i c a t i n g no c h a n g e i n t h e r e l a t i v e r a t e o f t r a n s f e r a c r o s s t h e p l a c e n t a . On d a y 17 t h e s p e c i f i c a c t i v i t y o f f o e t a l c o r t i c o s t e r o n e was a b o u t o n e - f o u r t h t h a t i n m a t e r n a l b l o o d . 26 TABLE I I . TOTAL AMOUNT OF CORTICOSTERONE IK THE MOTHER AND FOETUSES ON GESTATIONAL DAYS 13 AND 17 G e s t a t i o n a l Day M o t h e r . ng cpd.B/ml. b l o o d ± SEM F o e t u s e s ng c p d . B / g t i s s u e ± SEM 13 720.3 ±139.3 ( 5 ) * 641.9 ±185.4 (3) 17 1,145.4 ±103.5 (6) 300.9 ±48.6 (3) * number o f o b s e r v a t i o n s i n p a r e n t h e s e s 27 TABLE I I I . S P E C I F I C A C T I V I T Y OF RECOVERED C-CORTICOSTERONE G e s t a t i o n a l Day ^ M o t h e r # C-dpm/ng cpd.B 1 . F o e t u s e s # C-dpm/ng cpd . B 13 24.52 ±3.6 0.99 ±0.22 17 12.04 ±1.77 3.44 ±0.47 28 E f f e c t o f a d r e n a l e c t o m y o f t h e m o t h e r on t h e t o t a l amount o f c o r t i c o s t e r o n e The e f f e c t o f a d r e n a l e c t o m y o f t h e m o t h e r on d a y 16 on t h e amount o f c o r t i c o s t e r o n e i n t h e m o t h e r 24 h r s . l a t e r i s shown i n T a b l e I V . The m a t e r n a l s o u r c e o f s t e r o i d s y n t h e s i s h a s b e e n r e m o v e d , y e t t h e m o t h e r ' s b l o o d c o n t a i n e d a p p r o x i - m a t e l y n o r m a l l e v e l o f hormone ( T a b l e I V ) . The f o e t a l l e v e l s o f c o r t i c o s t e r o n e w e r e a l s o e s s e n - t i a l l y n o r m a l ( T a b l e V ) . T h i s i n d i c a t e s t h a t , i n t h e a b s e n c e o f m a t e r n a l hormone, t h e f o e t a l a d r e n a l i s c a p a b l e o f f u n c t i o n i n g a n d o f m a i n - t a i n i n g s u b s t a n t i a l p r o d u c t i o n o f hormone. 29 TABLE I V . EFFECT OF ADRENALECTOMY OF THE MOTHER ON THE LEVELS OF CORTICOSTERONE I N THE MOTHER ng c p d . B / g t i s s u e 14 C-dpm/ng cpd . B S a m p l e ±SEM ±SEM C o n t r o l s (6) 1,145.4 +103.5 12.04 ±1.77 A d r x (2) 1,586.0 14.92 312. 3 31.60 30 TABLE V. CONTENT AND S P E C I F I C A C T I V I T Y OF RECOVERED 1 4C-CORTICOSTERONE I N THE FOETUS AFTER ADRENALECTOMY OF THE MOTHER Sample ng c p d . B / g t i s s u e ± SEM "^C-dpm/ng c p d . B ± SEM C o n t r o l s 300.9 ±48.6 3.44 ±0.47 A d r x 437.2 0 .58 1,344.2 0.23 181.4 2.64 219.4 2 . 02 31 3. M e t a b o l i s m o f C - c o r t i c o s t e r o n e i n t h e p l a c e n t a and t h e f o e t a l t i s s u e s T a b l e V I shows t h e r e l a t i v e amount o f c o r t i - c o s t e r o n e a nd 1 1 - d e h y d r o c o r t i c o s t e r o n e i n t h e p l a c e n t a a n d f o e t a l t i s s u e s r e c o v e r e d i n t h e two 14 t i s s u e s 15 m i n . a f t e r i n j e c t i o n o f C - c o r t x - c o s t e r o n e i n t o t h e m o t h e r . The a c c u m u l a t i o n i n t h e p l a c e n t a o f more t h a n 7 5% o f t h e t o t a l c o u n t s r e c o v e r e d was c o n s t a n t f o r d a y s 12 a n d 14. M o s t o f i t was i n t h e f o r m o f c o r t i c o s t e r o n e . I n c o n t r a s t , t h e f o e t a l t i s s u e s c o n t a i n e d l e s s t h a n 20% o f t h e r a d i o a c t i v i t y , a n d m o s t o f i t was i n t h e f o r m o f 1 1 - d e h y d r o c o r t i c o s t e r o n e , s h o w i n g a n e x t e n s i v e m e t a b o l i s m o f t h e a c t i v e hormone i n t h e f o e t a l t i s s u e s . 32 TABLE V I . RELATIVE AMOUNT OF C-CORTICOSTERONE AND ITS 11-DEHYDROMETABOLITE I N THE PLACENTA AND FOETAL TISSUES % o f T o t a l C o u n t s R e c o v e r e d ± SEM G e s t a t i o n a l Day P l a c e n t a F o e t u s e s c p d . B c p d . A cpd. B c p d .A 12 (7) 75.6 ±1.5 12.4 ±1.8 1.9 ±0.2 10.2 ±0.5 14 (7) 76.0 +1.1 3.1 ±0.5 3.5 ±0.2 16.6 ±1.3 16 (8) 55.1 +1.9 1.4 ±0.2 21.7 ±1.0 22.8 ±1.3 C o r t i c o s t e r o n e a n d i t s 1 1 - d e h y d r o m e t a b o l i t e i n t h e f o e t a l t i s s u e s The c o n v e r s i o n o f c o r t i c o s t e r o n e t o i t s 1 1 - d e h y d r o m e t a b o l i t e was d e t e r m i n e d by t h e p e r - 14 c e n t a g e o f C - l a b e l l e d hormone o v e r t h e t o t a l c o u n t s r e c o v e r e d i n t h e f o e t u s e s 15 m i n . a f t e r 14 i n j e c t i o n o f 0.5 y C i C - c o r t i c o s t e r o n e i n t o t h e m o t h e r . T a b l e V I I shows t h e m e t a b o l i s m o f c o r t i - c o s t e r o n e b y t h e f o e t a l t i s s u e s d u r i n g 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 . I n g o o d a g r e e m e n t w i t h t h e r e s u l t s i n T a b l e V I , a v e r y h i g h p e r c e n t a g e o f i n j e c t e d r a d i o a c t i v e c o r t i c o s t e r o n e was f o u n d i n t h e i n a c t i v e 1 1 - d e h y d r o f o r m f r o m d a y s 12 t o 14. T h e n , a d r a m a t i c c h a n g e i n t h e m e t a b o l i s m o c c u r r e d a r o u n d d a y 16, w h e r e m o s t o f t h e hormone was now r e c o v e r e d i n t h e a c t i v e f o r m . A t r a n s i e n t c h a n g e o c c u r r e d on d a y 13 i n t h e r e l a t i v e amount o f c o r t i c o s t e r o n e (p=.02) a n d 1 1 - d e h y d r o c o r t i c o s t e r o n e ( p < . 0 1 ) , w h e r e a s d a y 14 was v i r t u a l l y i d e n t i c a l t o d a y 12. 34 TABLE V I I . CORTICOSTERONE AND ITS 11-DEHYDRO METABOLITE IN FOETAL TISSUES % o f T o t a l C o u n t s i n F o e t a l T i s s u e s ± SEM G e s t a t i o n a l Day c p d . B c p d . A 12 (7) 15.5 ±1.6 84.6 ±1. 5 13 (5) 24.5 ±3.9 73.5 ±3. 1 1 4 ( 7 ) 16.9 ±0*. 3 83.1 ±0. 3 16 (8T 49 .1 ±1.1 52.3 ±2. 3 17 (8) 90.9 ±1.1 9.1 ±1. 1 35 B. In v i t r o experiments 1. In v i t r o metabolism of c o r t i c o s t e r o n e and 1 1 - d e h y d r o c o r ticosterone The r e l a t i o n s h i p between the dehydrogenation of c o r t i c o s t e r o n e to 11- d e h y d r o c o r t i c o s t e r o n e and the r e d u c t i o n of the l a t t e r to c o r t i c o s t e r o n e can have a gre a t e f f e c t upon the b i o l o g i c a l a c t i v i t y of c o r t i c o s t e r o n e . ' In order t o study the m e t a b o l i c f a t e of the two compounds, minced p l a c e n t a l and f o e t a l t i s s u e s were incubated w i t h 3 14 H - c o r t i c o s t e r o n e and C - l l - d e h y d r o c o r t i c o s t e r o n e . G e s t a t i o n a l days 13, 14 and 15 were chosen because around these days, a g r e a t change i n the conver- s i o n of c o r t i c o s t e r o n e to 11 - d e h y d r o c o r t i c o s t e r o n e o c c u r r e d , as i n d i c a t e d i n Table V I I . The r e s u l t s i n Table V I I I show t h a t 1) the p l a c e n t a was the most a c t i v e t i s s u e f o r the reduc- t i o n to c o r t i c o s t e r o n e and t h i s a c t i v i t y remained constant f o r the 3-day p e r i o d ; 2) the head was the most a c t i v e i n dehydrogenating c o r t i c o s t e r o n e ; and 3) the body showed reductase a c t i v i t y i n c r e a s i n g w i t h time of g e s t a t i o n . TABLE V I I I . I N VITRO METABOLISM OF CORTICOSTERONE AND 11-DEHYDROCORTICOSTERONE BY FOETAL TISSUES % o f D e h y d r o g e n a t i o n o f C o r t i c o s t e r o n e ±SEM % R e d u c t i o n o f 1 1 - D e h y d r o - c o r t i c o s t e r o n e ±SEM G e s t a t i o n a l Day P l a c e n t a Head Body P l a c e n t a Head Body 13 (12) 20.7 ±1.0 62.6 ±1.5 61.1 ±1.9 83.4 ±2.5 8.8 ±1.4 8.1 ±1.2 14 (8) 16.2 ±1.0 61.0 ±1.3 49.0 ±4.7 88.0 ±1.6 7.3 ±0.6 12.0 ±1.4 15 (8) 18.5 ±1.8 46.5 ±1.2 27.9 ±1.1 86.9 ±2.0 14.0 ±0.8 37.5 ±1.9 r e d u c t i o n d e h y d r o g e n a t i o n 13 4.1 0.14 0.13 14 5.4 0.12 0.24 15 4.7 0.30 1.34 37 L o c a l i z a t i o n o f t h e r e d u c t a s e a c t i v i t y S i n c e t h e w e i g h t o f t h e f o e t a l l i v e r i n c r e a s e s a g r e a t d e a l d u r i n g 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 (20 mg a t d a y 14; 70 mg a t d a y 18) an d s i n c e i t i s a t a r g e t t i s s u e f o r c o r t i c o s - t e r o n e ( 1 8 ) , i t was s u s p e c t e d t h a t t h e r e d u c t a s e a c t i v i t y shown by t h e b o d y was due t o t h e l i v e r . I n o r d e r t o t e s t t h i s , t h e f o e t a l l i v e r s f r o m g e s t a t i o n a l d a y 15 f o e t u s e s w e r e r e m o v e d f r o m t h e c a r c a s s e s a nd l i v e r a n d c a r c a s s w e r e i n c u b a t e d s e p a r a t e l y . I t i s e v i d e n t , a c c o r d i n g t o T a b l e I X , t h a t t h e l i v e r e f f e c t e d a n e t r e d u c - t i o n a n d t h e c a r c a s s e x h i b i t e d a s i g n i f i c a n t n e t d e h y d r o g e n a t i o n . 38 TABLE I X . REDUCTION OF 11-DEHYDROCORTICOSTERONE BY THE FOETAL L I V E R AND RESIDUAL CARCASS R e a c t i o n % C o n v e r s i o n o f 1 1 - d e h y d r o c o r t i c o s t e r o n e ±SEM L i v e r C a r c a s s D e h y d r o g e n a t i o n (8) R e d u c t i o n (8) „ , . r e d u c t i o n R a t i o , , d e h y d r . 21.6 +1.3 48.4 ±1.4 2.24 29.9 ±2.7 12.5 ±1.3 0.42 39 3. M e t a b o l i s m o f c o r t i c o s t e r o n e i n t h e f o e t a l h e a d D u r i n g t h e i n v i v o e x p e r i m e n t s , a u t o r a d i o - grams (AR) w e r e made o f t h e TLC b e f o r e e l u t i o n t o • s e e i f t h e r e w e r e m e t a b o l i t e s o f c o r t i c o s t e r o n e o t h e r t h a n 1 1 - d e h y d r o c o r t i c o s t e r o n e i n s i g n i f i c a n t a m o u n t s . Whereas t h e AR o f t h e TLC f r o m t h e b o d i e s d i d n o t show any d e t e c t a b l e b a n d , t h o s e f r o m t h e h e a d s showed many b a n d s . They w e r e f a i n t , p r o b a b l y 14 due t o t h e l o w amount o f C a c c u m u l a t e d i n t h e h e a d . 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 s e b a n d s c o u l d be r e p r o d u c e d , m i n c e d f o e t a l h e a d s w e r e i n c u b a t e d w i t h 10,000 cpm o f c h r o m a t o g r a p h i c a l l y p u r e ^ C - c o r t i c o s t e r o n e a n d t h e TLC w e r e e x p o s e d t o an X - r a y f i l m . F i g u r e 2 shows t h a t i n d e e d c o r t i - c o s t e r o n e i s m e t a b o l i z e d e x t e n s i v e l y i n t o s e v e r a l compounds by t h e f o e t a l h e a d . The i n v i t r o m e t a b o l i s m was s i m i l a r t o t h a t i n v i v o . The AR shows t h a t b e t w e e n d a y s 14 and 1 5 , t h e r e i s a d r a m a t i c c h a n g e i n t h e m e t a b o l i s m o f c o r t i c o s t e r o n e . A t d a y 15 t h e r e i s a d e c r e a s e i n t h e r a t e o f m e t a b o l i s m o f c o r t i - c o s t e r o n e t o 1 1 - d e h y d r o c o r t i c o s t e r o n e . O n l y t h e TLC z o n e s f r o m c o r t i c o s t e r o n e a n d 1 1 - d e h y d r o c o r t i c o s t e r o n e h a v e b e e n e l u t e d . F i g u r e 2 f o o t - 14 n o t e shows t h e p e r c e n t a g e C o f t h e t o t a l f o r e a c h z o n e . The r e m a i n i n g z o n e s h a v e n o t y e t b e e n i d e n t i f i e d . 40 FIGURE 2. METABOLISM OF CORTICOSTERONE BY THE FOETAL HEAD The s t r a i g h t d a r k b a n d a t t h e t o p o f t h e r i g h t - h a n d c h r o m a t o g r a m i s an a r t i f a c t F o o t n o t e : G e s t a t i o n a l Day % o f T o t a l 1 4 C - d p m ±SEM c p d . B c p d . A 14 (8) 15 (4) 15.7 +0.9 52.8 ±3.0 84.4 ±0.9 47.3 ±3.0 41 C. Enzyme k i n e t i c s 1. C e l l f r a c t i o n a t i o n and coenzyme s p e c i f i c i t y I n o r d e r t o l o c a l i z e t h e s u b c e l l u l a r f r a c t i o n r e s p o n s i b l e f o r t h e r e d u c t a s e a c t i v i t y a n d t o d e t e r - m i n e t h e coenzyme r e q u i r e m e n t , f o e t a l l i v e r s w e r e h o m o g e n i z e d a nd t h e n u c l e a r , m i t o c h o n d r i a l a n d m i c r o s o m a l , a s w e l l a s t h e c r u d e h o m o g e n a t e a nd t h e f i n a l s u p e r n a t a n t , w e r e t e s t e d f o r r e d u c t a s e a c t i v i t y i n t h e p r e s e n c e o f NADH o r NADPH. Wh e r e a s t h e enzyme a c t i v i t y seemed r a t h e r e v e n l y d i s t r i b u t e d i n t h e c r u d e u nwashed f r a c t i o n s ( T a b l e X ) , NADPH a p p e a r e d t o be t h e p r e f e r r e d c o e n z y m e . S i n c e i t was f e l t t h a t w a s h i n g t h e s u b c e l l u l a r f r a c t i o n s w o u l d i m p r o v e t h e l o c a l i z a t i o n o f t h e , enzyme a c t i v i t y , t h e f r a c t i o n s w e r e w a s h e d i n t h e same b u f f e r a n d t e s t e d f o r a c t i v i t y . A s T a b l e X I i n d i c a t e s , t h e h i g h e s t r e d u c t i o n was l o c a l i z e d i n t h e m i t o c h o n d r i a . The p r e f e r e n c e f o r NADPH was v e r y m a r k e d . The n u c l e a r f r a c t i o n showed a s i g - n i f i c a n t r e d u c t a s e a c t i v i t y , b u t i t a l s o h a d a s u b s t a n t i a l amount o f b r o k e n c e l l s t h a t s e d i m e n t e d w i t h t h e n u c l e i . R e - h o m o g e n i z a t i o n o f t h e " n u c l e a r " p e l l e t y i e l d e d a more p u r e f r a c t i o n , w h i c h a c t i v i t y d e c r e a s e d w i t h w a s h i n g . 42 TABLE X. ENZYME A C T I V I T Y AND COENZYME S P E C I F I C I T Y I N THE CRUDE SUBCELLULAR FRACTIONS S u b c e l l u l a r F r a c t i o n % S u b s t r a t e C o n v e r t e d +SEM +NADH +NADPH Homogenate (3) N u c l e i (3) M i t o c h o n d r i a (3) M i c r o s o m e s (3) S u p e r n a t a n t (3) 46.7 ±6.3 41.3 ±0.8 56.5 ±3.4 37.9 ±3.2 26.9 ±2.1 66.0 ±4.8 53.5 ±3.7 63.3 ±4.6 59.4 ±3.6 86.3 ±3.2 E a c h i n c u b a t i o n m i x t u r e c o n t a i n e d t h e e q u i v a l e n t o f 50 mg t i s s u e 43 TABLE X I . ENZYME A C T I V I T Y AND COENZYME S P E C I F I C I T Y I N WASHED FRACTIONS AND THEIR WASHES o, o S u b s t r a t e C o n v e r t e d S u b c e l l u l a r F r a c t i o n s F r a c t i o n s Washes NADH NADPH NADH NADPH N u c l e i 8.2 77.5 11.6 79.5 R e - h o m o g e n i z e d n u c l e i 7.4 29.0 — — M i t o c h o n d r i a 8.6 95.2 12 . 6 85.1 M i c r o s o m e s 5.0 11.1 7.3 11.2 S u p e r n a t a n t 6.9 30.3 — — E a c h f i g u r e . , i s an a v e r a g e o f d e t e r m i n a t i o n s o n two s e p a r a t e b a t c h e s o f enzyme 44 When m i t o c h o n d r i a w e r e k e p t i n t h e b u f f e r f o r more t h a n 30 m i n . , enzyme a c t i v i t y was r e l e a s e d i n t o t h e w a s h . I n a l l c a s e s , t h e coenzyme s p e c i - f i c i t y f o r NADPH was v e r y c l e a r . 2. D e v e l o p m e n t o f t h e r e d u c t a s e a c t i v i t y i n t h e f o e t a l l i v e r H a v i n g l o c a l i z e d t h e l i v e r a s a s i t e o f r e d u c - t i o n , e x p e r i m e n t s w e r e c a r r i e d o u t t o d e t e r m i n e w h e t h e r o r n o t t h e p r e s e n c e o f coenzyme was t h e l i m i t i n g f a c t o r t o t h e enzyme r e a c t i o n . I t was f o u n d t h a t t h e a d d i t i o n o f t h e coe n z y m e NADPH i n c r e a s e d o n l y s l i g h t l y t h e r e d u c t i o n o f 11- d e h y d r o c o r t i c o s t e r o n e t o c o r t i c o s t e r o n e b y m i n c e d f o e t a l l i v e r ( T a b l e X I I ) . 45 TABLE X I I . DEVELOPMENT OF THE REDUCTASE A C T I V I T Y I N TEE FOETAL L I V E R G e s t a t i o n a l Day % S u b s t r a t e R e d u c e d ±SEM W i t h o u t NADPH W i t h NADPH 14 11.9 ±0.9 (5) 14.0 ±0.8 (6) 15 39.4 ±2.2 (4) 56.8 ±3.5 (5) 46 3. pH o p t i m u m The pH o p t i m u m f o r r e d u c t i o n was d e t e r m i n e d by i n c u b a t i n g a c r u d e p r e p a r a t i o n a t pH v a r y i n g f r o m 4.5 t o 9. I t was e s t a b l i s h e d t o be b e t w e e n 5.5 and 6 ( F i g u r e 3 ) . T h e s e two pH v a l u e s g a v e t h e same a c t i v i t y . 4. Km The r e d u c t i o n o f 1 1 - d e h y d r o c o r t i c o s t e r o n e ( F i g u r e 4) showed a s u b s t r a t e i n h i b i t i o n o c c u r r i n g b e t w e e n 8 0 a n d 100 yM. The d e h y d r o g e n a t i o n o f c o r t i c o s t e r o n e ( F i g u r e 6) showed s u b s t r a t e i n h i b i t i o n a t a l o w e r c o n c e n t r a t i o n ( b e t w e e n 15 and 20 yM). The Km a n d Vmax f o r b o t h r e d u c t i o n a n d d e h y d r o - g e n a t i o n r e a c t i o n s w e r e d e t e r m i n e d f r o m t h e L i n e w e a v e r - B u r k p l o t . F o r t h e r e d u c t i o n o f 1 1 - d e h y d r o c o r t i c o s t e r o n e t o c o r t i c o s t e r o n e , t h e Km was 3 3 yM and Vmax, 0.4 y m o l e s / m i n . / m l ( F i g u r e 5 ) . F o r t h e r e v e r s e r e a c t i o n , Km=10 yM and Vmax=0.21 y m o l e s / m i n . / m l . PH U N I T S FIGURE 4, EFFECT OF CONCENTRATION OF 11-DEHYDROCORTICOSTERONE ON REDUCTASE ACTIVITY 0 5 10 20 30 40 60 . 80 100 11-DEHYDROCORTICOSTERONE (VM) FIGURE 5. LINEWEAVER-BURK PLOT FOR REDUCTION 11-DEHYDROCORTICOSTERONE 0.1 0.2 0.3 0-4 1/yM 11-DEHYDROCORTICOSTERONE KM = 33 nM VMAX = C M UMOLES/MIN/ML F IGURE 6 . E F F E C T OF. CONCENTRATION OF C O R T I C O S T E R O N E ON • DEHYDROGENASE A C T I V I T Y CORT ICOSTERONE (yM) FIGURE 7, LINEWEAVER-BURK PLOT FOR DEHYDROGENATION OF CORTICOSTERONE -0.5 0 0.5 1.0 1.5 2.0 1/yfi OF CORTICOSTERONE KM = 10 KM VMAX = 0 , 2 1 UMOLES/MIN/ML 52 5. C o n t e n t a n d s p e c i f i c a c t i v i t y o f r e c o v e r e d 14 C - c o r t i c o s t e r o n e i n t h e f o e t a l b o d y S i n c e t h e m e t a b o l i c a c t i v i t y o f t h e h e a d was g r e a t l y d i f f e r e n t o f t h a t o f t h e b o d y ( T a b l e V I I I ) , 14 t h e f a t e o f C - c o r t i c o s t e r o n e was e x a m i n e d i n t h e f o e t u s e s w i t h t h e h e a d r e m o v e d d u r i n g t h e c r i t i c a l p e r i o d w h e r e t h e m e t a b o l i s m o f c o r t i - c o s t e r o n e i n t h e b o d y c h a n g e d d r a s t i c a l l y . As shown i n T a b l e X I I I , t h e r e i s a d r a m a t i c i n c r e a s e i n s p e c i f i c a c t i v i t y a t d a y 1 5 , i n d i c a t i n g a n e n r i c h m e n t o f t h e c o r t i c o s t e r o n e f o e t a l p o o l b y t h e i s o t o p e . T h i s i s assumed t o be due t o 14 t h e r e d u c t i o n o f C - l l - d e h y d r o c o r t i c o s t e r o n e . 53 TABLE X I I I . CONTENT AND S P E C I F I C A C T I V I T Y OF CORTICOSTERONE RECOVERED I N THE FOETAL BODY G e s t a t i o n a l Day ng c p d . B / g t i s s u e ±SEM 14 C-dpm/ng cpd . B ±SEM 13 (5) 812.9 ±157.1 0.71 ±0.12 15 (6) 564.0 ±66.3 8.44 ±1.44 54 6. H i s t o l o g i c a l s t u d i e s C o r t i c o s t e r o i d s a r e r e g u l a t e d i n a c o m p l e x manner i n v a r i o u s f o e t a l t i s s u e s . The e f f e c t o f a s y n t h e t i c s t e r o i d w h i c h , b e c a u s e o f i t s s t r u c - t u r a l m o d i f i c a t i o n s , e s c a p e s t h i s m e t a b o l i c r e g u - l a t i o n h a s b e e n r e p o r t e d ( 4 0 ) . D e x a m e t h a s o n e w h i c h i s n o t b o u n d by s e r u m t r a n s c o r t i n , a c cumu- l a t e d more e x t e n s i v e l y i n f o e t a l t i s s u e a n d u n d e r - w e n t l e s s m e t a b o l i c a l t e r a t i o n t h a n c o r t i c o s t e r o n e , t h e n a t u r a l hormone i n t h e mouse. A l t h o u g h h i g h d o s e s o f c o r t i c o s t e r o n e i n j e c t e d i n t o p r e g n a n t m o t h e r s h a d l i t t l e e f f e c t , d e x a m e t h a s o n e was l e t h a l t o mouse f o e t u s e s . An a t t e m p t was made t o f i n d a b a s i s f o r t h i s t o x i c i t y . F o e t a l t i s s u e s a nd p l a c e n t a w e r e e x a m i n e d 4 a n d 20 h r s . a f t e r t h e 1 7 - d a y - p r e g n a n t m o t h e r h a d b e e n i n j e c t e d w i t h 2 00 yg o f d e x a m e t h a s o n e . T h e r e was no e v i d e n c e o f h i s t o l o g i c a l c h a n g e s . D e x a m e t h a s o n e h a s b e e n r e p o r t e d t o c a u s e i n v o l u t i o n o f f o e t a l a d r e n a l c o r t e x b u t t o s t i m u - l a t e t h e d e v e l o p m e n t o f t h e m e d u l l a a n d c a t e - c h o l a m i n e s y n t h e s i s (2). So f o e t a l a d r e n a l s w e r e e x a m i n e d u n d e r LM ( F i g u r e 8) and TEM ( F i g u r e 9 ) . No s i g n i f i c a n t d i f f e r e n c e was o b s e r v e d i n t r e a t e d a d r e n a l s , e x c e p t p o s s i b l y f o r a s l i g h t r e d u c t i o n i n s i z e . o f t h e g l a n d i t s e l f . FIGURE 8. LIGHT MICROGRAPHS OF FOETAL ADRENAL GLANDS OF NORMAL MICE (UPPER) AND THOSE FROM MOTHERS TREATED 2 0 HO PREVIOUSLY WITH 200 yg DEXAMETHASONE (LOWER) X 400 56 FIGURE 9. TRANSMISSION ELECTRON MICROGRAPHS OF FOETAL ADRENAL GLANDS OF NORMAL MICE (UPPER) AND THOSE FROM MOTHERS TREATED 20 HOURS PREVIOUSLY WITH 200 yg DEXA- METHASONE (LOWER) X12000 59 DISCUSSION The t r a n s p o r t a c r o s s t h e p l a c e n t a o f r a d i o a c t i v e c o r t i - c o s t e r o n e r e v e a l e d some i n t e r e s t i n g f e a t u r e s o f c o r t i c o s t e r o n e m e t a b o l i s m i n t h e f o e t u s . F r o m g e s t a t i o n a l d a y s 13 t o 1 7 , t h e m a t e r n a l c o r t i c o s t e r o n e l e v e l i n c r e a s e d , w h e r e a s on t h e f o e t a l s i d e , i t d e c r e a s e d by h a l f ( T a b l e I I ) . T h i s c a n p r o b a b l y be a t t r i b u t e d t o t h e i n c r e a s e w h i c h o c c u r s a t t h e t i m e i n t h e t r a n s c o r t i n c o n t e n t o f m a t e r n a l b l o o d ( 8 ) . T h i s p r o t e i n b i n d s c o r t i c o s t e r o n e s p e c i f i c a l l y a nd r e d u c e s i t s p a s s a g e i n t o t i s s u e s . On t h e o t h e r h a n d , t h e s p e c i f i c a c t i - v i t y o f i n j e c t e d c o r t i c o s t e r o n e i n t h e f o e t u s a c t u a l l y i n c r e a s e d somewhat ( T a b l e I I I ) . T h i s s u g g e s t s t h a t t h e r e i s no c h a n g e i n t h e f o e t a l p o o l ; e . g . , by f o e t a l a d r e n a l p r o d u c t i o n o f c o r t i c o s t e r o n e , b e t w e e n d a y s 13 and 17. The c o n t e n t o f f o e t a l c o r t i c o s t e r o n e on d a y s 13 and 17 i s s i m i l a r t o t h a t r e p o r t e d o n d a y 16 a n d d e t e r m i n e d b y f l u o r o m e t r i c a n a l y s i s ( 3 5 ) . The r o l e o f t h e p l a c e n t a a s a b a r r i e r was d r a m a t i c a l l y shown by t h e l a r g e a c c u m u l a t i o n o f c o u n t s r e c o v e r e d f r o m t h e p l a c e n t a and t h e f o e t a l t i s s u e s ( T a b l e V I ) . From d a y 12 t o 14, t h e r e was more t h a n . 7 5 % o f t h e l a b e l l e d c o r t i c o s t e r o i d s r e c o v e r e d i n t h e two t i s s u e s , i n t h e p l a c e n t a . T h e r e was v e r y l i t t l e o f t h e 1 1 - d e h y d r o m e t a b o l i t e o f c o r t i c o s t e r o n e i n t h e p l a c e n t a , w h e r e a s i n t h e f o e t u s , t h e hormone was e x t e n s i v e l y m e t a b o l i z e d t o 1 1 - d e h y d r o c o r t i c o s t e r o n e . F rom 60 t h e s e e x p e r i m e n t s ( T a b l e s V I and V I I I ) , i t a p p e a r s t h a t t h e p l a c e n t a c o n t r o l l e d t h e f l o w o f hormone i n t o t h e f o e t u s , w h e r e a s t h e f o e t a l t i s s u e s assumed d i f f e r e n t m e t a b o l i c f u n c t i o n s . Whether o r n o t t h e hormone f o u n d i n t h e f o e t u s i s o f f o e t a l o r m a t e r n a l o r i g i n c a n n o t be d e t e r m i n e d d e f i n i t e l y f r o m t h e p r e s e n t d a t a ( T a b l e IV and V ) . Removal o f m a t e r n a l a d r e n a l s r e s u l t e d i n n o r m a l f o e t a l l e v e l s o f c o r t i c o s t e r o n e . However, t h e s p e c i f i c a c t i v i t y o f f o e t a l c o r t i c o s t e r o n e 15 m i n u t e s a f t e r i n j e c t i o n was as h i g h as o n e - q u a r t e r t h a t i n m a t e r n a l b l o o d , i n d i c a t i n g r a p i d and e x t e n s i v e t r a n s f e r f r o m m o t h e r t o f o e t u s ( T a b l e s I I I and X I I I ) . D u r i n g 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 , t h e m e t a b o l i s m o f c o r t i c o s t e r o n e on t h e f o e t a l s i d e c h a n g e d g r e a t l y ( T a b l e V I I ) . A t t h e b e g i n n i n g o f t h e t r i m e s t e r ; i . e . , f r o m day 12 t o 14, m e t a b o l i s m was e x t e n s i v e i n t h e d i r e c t i o n o f f o r m a t i o n o f t h e 1 1 - d e h y d r o m e t a b o l i t e o f c o r t i c o s t e r o n e , w h i c h r e p r e s e n t e d a r o u n d 80% o f a l l c o u n t s r e c o v e r e d i n t h e f o e t a l t i s s u e s . Then a f t e r day 14, t h e r e was a d r a s t i c c h a n g e i n t h e d e h y d r o - g e n a t i o n o f c o r t i c o s t e r o n e and by day 17, o v e r 90% o f t h e hormone was r e c o v e r e d i n t h e a c t i v e f o r m . Hence, t h e r e i s e v i d e n c e t h a t t h e f o e t u s c a n m e t a b o l i z e c o r t i c o s t e r o n e a c t i v e l y f o l l o w i n g a d e f i n i t e p a t t e r n d u r i n g 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 . 61 Not o n l y c a n t h e f o e t u s m e t a b o l i z e c o r t i c o s t e r o n e a c t i v e l y , b u t t h i s m e t a b o l i s m i s c o m p a r t m e n t a l i z e d ( T a b l e V I I I ) . The i n v i t r o m e t a b o l i s m by t h e p l a c e n t a , t h e f o e t a l b o d i e s o r t h e f o e t a l h e a d s f r o m g e s t a t i o n a l d a y s 13, 14 and 15 showed t h a t t h e p l a c e n t a p l a y e d a c o n s t a n t and v e r y a c t i v e r o l e i n r e d u c i n g 1 1 - d e h y d r o c o r t i c o s t e r o n e . From t h e s e d a y s , t h e meta- b o l i s m by t h e h e a d was d e f i n i t e l y t o w a r d s t h e i n a c t i v a t i o n o f c o r t i c o s t e r o n e , w h e r e a s t h e body e x h i b i t e d a v e r y s t r o n g d e h y d r o g e n a s e a c t i v i t y a t day 13; t h i s d e c r e a s e d by o n e - h a l f by day 15. I t a p p e a r s t h e n t h a t w i t h i n t h e f o e t u s i t s e l f , c o r t i c o s t e r o i d s a r e m e t a b o l i z e d q u i t e d i f f e r e n t l y i n d i f f e r e n t t i s s u e s . The r e d u c t i o n o f 1 1 - d e h y d r o c o r t i c o s t e r o n e became t h e c e n t r e o f i n t e r e s t o f t h i s work, so e f f o r t s were made t o l o c a l i z e and s t u d y t h e d e v e l o p m e n t o f t h e r e d u c t a s e a c t i v i t y . I t i s e v i d e n t , a c c o r d i n g t o T a b l e IX, t h a t t h e f o e t a l l i v e r was t h e m a j o r s i t e o f r e d u c t i o n . The r e s i d u a l a c t i v i t y e f f e c t e d by t h e c a r c a s s m i g h t be a t t r i b u t e d m o s t l y t o t h e f o e t a l l u n g s w h i c h a r e a n o t h e r t a r g e t t i s s u e f o r c o r t i - c o s t e r o n e and s i n c e r e d u c t a s e ' a c t i v i t y h as b e e n o b s e r v e d i n t h e human l u n g ( 3 6 ) . I n t h e same s y s t e m , t h e d e h y d r o g e n a t i o n o f c o r t i c o s t e r o n e was h i g h e r i n t h e r e s i d u a l c a r c a s s , p r o b a b l y due t o t h e f o e t a l k i d n e y s . When f o e t a l l i v e r on d a y s 14 and 15 was i n c u b a t e d w i t h 1 1 - d e h y d r o c o r t i c o s t e r o n e , a d d i t i o n o f t h e coenzyme NADPH 62 i n c r e a s e d o n l y s l i g h t l y the c o n v e r s i o n t o c o r t i c o s t e r o n e (Table X I I ) , i n d i c a t i n g t h a t t h e coenzyme was n o t l i k e l y t h e l i m i t i n g f a c t o r t o t h e r e a c t i o n , b u t r a t h e r an i n c r e a s e i n enzyme c o n c e n t r a t i o n was r e s p o n s i b l e f o r t h e c o n v e r s i o n t o t h e a c t i v e hormone. S i n c e i t has been shown t h a t t h e r e was a d e f i n i t e change i n c o r t i c o s t e r o i d m e t a b o l i s m i n t h e f o e t u s d u r i n g t h e t h i r d t r i m e s t e r o f pregnancy (Table V I I ) and t h a t t h e l i v e r was t h e major s i t e o f r e d u c t i o n , i t was d i f f i c u l t t o e x p l a i n t h a t t h e s p e c i f i c a c t i v i t y o f i n j e c t e d c o r t i c o s t e r o n e changed o n l y s l i g h t l y from day 13 t o 17 i n whole f o e t a l t i s s u e s . An e x p e r i m e n t was d e s i g n e d t o s t u d y the f a t e o f i n j e c t e d 14 C - c o r t i c o s t e r o n e i n t h e h e a d l e s s body, where t h e r e d u c t i o n i s p r o m i n e n t . T a b l e X I I I shows t h a t t h e r e was an 1 1 - f o l d i n c r e a s e i n s p e c i f i c a c t i v i t y a t day 15. T h i s c o u l d not be due t o an i n c r e a s e i n t r a n s p o r t o f t h e hormone from th e m a t e r n a l s i d e , s i n c e t h e s p e c i f i c a c t i v i t y would have remained c o n s t a n t . I t c o u l d n o t be a t t r i b u t e d t o a change i n f o e t a l p o o l s i z e o f c o r t i c o s t e r o n e e i t h e r , s i n c e t h e amount o f hormone i n t h e body was i n t h e same o r d e r o f magnitude as t h a t o f t h e whole f o e t u s . I t i s p o s t u l a t e d t h a t t h i s s harp r i s e i n s p e c i f i c a c t i v i t y was due t o t h e e n z y m a t i c r e d u c t i o n by the f o e t a l l i v e r o f l a b e l l e d 1 1 - d e h y d r o c o r t i c o s t e r o n e w h i c h was i t s e l f t h e p r o d u c t o f t h e d e h y d r o g e n a t i o n o f t h e 14 i n j e c t e d l a b e l l e d c o r t i c o s t e r o n e ; i . e . , i n j e c t e d C- c o r t i c o s t e r o n e c 1 e h y d r o g e n a t i o n > l d c - l l - d e h y d r o c o r t i c o s t e r o n e 63 reduction 14 > C-corticosterone, enriching considerably the f o e t a l pool of l a b e l l e d corticosterone, The r e s u l t s from the incubation i n v i t r o of the f o e t a l brain (Figure 2) give add i t i o n a l evidence of the compart- mentalization of c o r t i c o s t e r o i d metabolism i n the foetus. Many un i d e n t i f i e d metabolites have been detected by AR of TLC from the head, which were not present on those from the body. No attempt has yet been made to i d e n t i f y these zones, but here too there i s evidence of a s t r i k i n g change between days 14 and 15, with a decrease i n the formation of the unknown metabolites as well as i n the dehydrogenation of corticosterone. The s p e c i f i c i t y for NADPH as coenzyme agrees well with reports i n the l i t e r a t u r e (37, 38) for a s i m i l a r enzyme i n other tissues. The pH optimum was found to be on the acid side; i . e . , between 5.5 and 6. A dip i n the reductase a c t i - v i t y was observed at pH 7 (p<.02), the same pH at which Wong (39) also observed a dip for the dehydrogenation of corticosterone by adult mouse l i v e r and placenta. This work has pointed out the importance of the 113- hydroxysteroid:NADP oxidoreductase i n the f o e t a l l i v e r during the t h i r d trimester of pregnancy i n the mouse. The f i r s t outstanding feature i s the compartmentalization of the oxidoreductase a c t i v i t y . Not only was the foetus capable of 64 m e t a b o l i z i n g c o r t i c o s t e r o i d s , but there was a d e f i n i t e p a t t e r n of metabolism i n d i f f e r e n t organs. The p l a c e n t a metabolized the s t e r o i d hormone and a l s o appeared to assume a r e g u l a t o r y c o n t r o l over the flow of s t e r o i d from the maternal to the f o e t a l s i d e . In the human, the major s i t e of dehydrogenation of c o r t i c o s t e r o i d s i s the p l a c e n t a , where maternal C o r t i s o l i s l a r g e l y converted to c o r t i s o n e , the predominant c o r t i - c o s t e r o i d i n f o e t a l and cord blood (11, 16, 29). In the mouse the head appears to be the most a c t i v e s i t e (Table V I I I ) . In both, the l i v e r i s a s i t e of r e d u c t i o n of the me t a b o l i t e to the 11B-0H form of the a c t i v e hormone (16, 18 and Table I X ) . Burton has found t h i s reductase a c t i v i t y i n human f o e t a l l i v e r as e a r l y as 20 weeks (16). In f o e t a l mouse l i v e r the appearance of glycogen d e p o s i t i o n c o i n c i d e d w i t h the appear- ance of reductase a c t i v i t y (13). In the human lung, another " t a r g e t " organ of c o r t i c o s t e r o i d s , reductase a c t i v i t y has a l s o been demonstrated (36). An a l t e r n a t e e x p l a n a t i o n can be o f f e r e d f o r the obser- v a t i o n s of Murphy (10, 11), who found c o n s i s t e n t l y increased C o r t i s o l l e v e l s i n human f o e t a l a r t e r i a l blood as compared w i t h u m b i l i c a l venous blood e n t e r i n g the fo e t u s . She a t t r i - buted t h i s to production by the f o e t a l a d r e n a l . However, t h i s could as e a s i l y be due to reductase a c t i v i t y i n f o e t a l l i v e r and lung, which reduces a p o r t i o n of the abundant 65 c o r t i s o n e w h i c h i s p r e s e n t . T h u s , t h e c h a n g e s i n t h e amount o f a c t i v e hormone c o u l d be d e t e r m i n e d n o t b y t h e f o e t a l g l a n d s b u t r a t h e r b y c h a n g e s i n t h e m e t a b o l i s m o f m a t e r n a l s t e r o i d s , w i t h e a c h t i s s u e s h o w i n g i t s own d i s t i n c t p a t t e r n o f m e t a b o l i s m . BIBLIOGRAPHY DICZFALUZY, E. P r o c . 2nd I n t . C o n g r . H o r m o n a l S t e r o i d s , I n t . C o n g r . S e r i e s 1 3 2 , 82-95 ( 1 9 6 7 ) . JOST, A. a n d P. PICON. A d v a n c e s i n M e t a b o l i c D i s o r d e r s , 4, 123-184 (1970) . C H A L L I S , J.R.C., I . J . DAVIES, K. BENIRCHKE, A.G. HENRICKX, and K . J . KYAN. 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