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The regulation and role of corticosteroids during fetal development Tye, Lesley Margaret 1979

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THE REGULATION AND ROLE OF CORTICOSTEROIDS DURING FETAL DEVELOPMENT by LESLEY MARGARET TYE B.Sc, University of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF BIOCHEMISTRY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1979 (S) L e s l e y Margaret Tye, 197 9 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l -able for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written per-mission . Department of Biochemistry The University of B r i t i s h Columbia Vancouver, B r i t i s h Columbia, Canada Date: A p r i l , 1979 ( i i ) ABSTRACT 14 Fe t a l mouse txssues were incubated with C-corticosterone 3 together with H-ll-dehydrocorticosterone and the steroids were extracted and separated chromatographically to determine the r a t i o , u ^ f ^ C t l ° l 1 - . Between gestational days 14 and 19, the dehydrogenation ^ J ' r a t i o i n placenta increased from 6.3 to 23.4; i n brain from 0.06 to 0.63; i n gut from 0.1 to 0.5; and i n l i v e r from 0.29 to 8.3. In lung, the r a t i o rose from 0.16 to 6.7 only a f t e r day 16, two days l a t e r . Treatment of mothers 16 h e a r l i e r with 200 ug dexamethasone increased the r a t i o i n lung and placenta on day 16 3 but not e a r l i e r . After i n j e c t i o n of H-corticosterone into 3 mothers, the amount of H-ll-dehydrocorticosterone, which was 98% of the t o t a l on day 14, declined, while unchanged corticosterone increased at least tenfold i n a l l tissues examined between days 14 and 19. A receptor has been found i n f e t a l brain with K^= 3 8.3 nM which binds H-dexamethasone to the extent of 0.14 pmoles/mg protein on day 14. The number of receptor s i t e s did not increase with gestational age, indicating that these are not c r i t i c a l i n i n i t i a t i n g steroid-dependent processes. In f e t a l brain and 3 placenta, receptors bound both H-corticosterone and 11-dehydro-corticosterone. In cytosol and nucleus, both l a b e l l e d steroids were displaced competitively by each other. The H-ll-dehydro-corticosterone-receptor complex not only entered the nucleus but was bound to chromatin s l i g h t l y more than was the hormone corticosterone. Of parameters r e f l e c t i n g changing l e v e l s of active hormone i n f e t a l tissues, the i n v i t r o incorporation 14 3 3 of C-leucine, H-uridine and H-thymidine into acid-insoluble ( i i i ) components of tissues were the most sen s i t i v e . The incorpor-ation of these precursors decreased between days 14 and 19 by as much as 90%. The deposition of glycogen varied i n d i f f e r e n t f e t a l tissues but did not r e f l e c t hormonal changes. By a l l parameters, the i n j e c t i o n of mothers with 200 ug dexamethasone 16 h e a r l i e r resulted i n acceleration of the normal pattern, producing values on day 14 which were normally observed on days 15-19. Fetuses which had been treated with dexamethasone i n utero were born and appeared normal. Other steroid-induced processes included increased amino acid content of f e t a l brain and the conversion of glucose to fructose i n f e t a l l i v e r and gut. It i s concluded that the regulation of corticosteroids i s accomplished not simply by a c t i v a t i o n of the f e t a l pituitary-adrenal axis, but by the interconversion i n in d i v i d u a l tissues of corticosterone and i t s 11-dehydro metabolite. The 11-dehydrocorticosterone i s not only an abundant metabolite, but serves as a reservoir of p o t e n t i a l hormone and can compete with corticosterone for both cytosol and nuclear receptor s i t e s . Since i t binds to chromatin, i t i s possible that 11-dehydro-corticosterone exerts actions at the t r a n s c r i p t i o n a l l e v e l . Corticosteroids exert t h e i r e f f e c t s on f e t a l development at an e a r l i e r stage than has been hitherto reported. (iv) TABLE OF CONTENTS Page ABSTRACT ( i i ) TABLE OF CONTENTS (iv) LIST OF TABLES (vii) LIST OF FIGURES (ix) LIST OF APPENDICES (xii) ABBREVIATIONS USED ( x i i i ) ACKNOWLEDGEMENTS (xv) INTRODUCTION 1 H i s t o r i c a l Review 1 Present Problem 13 MATERIALS 15 Animals 15 Buffers 15 Chemicals 15 Radioactive Chemicals 16 Solvents 17 Chromatography 17 Autoradiography 17 S c i n t i l l a t i o n Supplies 18 METHODS 19 Injection of Mice 19 Preparation of Tissues 19 Enzymatic Synthesis of 11-dehydrocorticosterone (cpd. A) 20 In V i t r o Steroid Incubations 21 (v) Page Extraction Procedure 21 Chromatography 22 In Vivo Metabolism of Steroids 23 Characterization of Steroids 23 C r y s t a l l i z a t i o n 23 Isolation of Glycogen from Tissues 24 Anthrone Assay for Glycogen 24 Incorporation of Leucine, Uridine and Thymidine 25 Ornithine Decarboxylase Assay .26 Glucose Metabolism i n Fetal Tissue 2 7 Glucose and Fructose-6-P0 4 Metabolism i n Cytosol Preparations 2 7 Incorporation of Glucosamine 28 STEROID RECEPTOR ASSAYS 28 Cytosol Receptor Preparation and Assay 28 Nuclear Preparation and Assay 29 Assay of Radioactivity 30 EXPERIMENTAL RESULTS 32 1 4 1. Metabolism of C-labelled Corticosterone and 11-dehydrocorticosterone i n Fetal Tissues 32 2. The Metabolism of Corticosterone and 11-dehydrocorticosterone i n Fetal Tissues on Different Gestational Days 38 3. Recovery of cpd.^A and B afte r Injection of Mothers with H-cpd. B 46 4. Glycogen Deposition i n Fetal Tissues 46 5. The ln_ V i t r o Incorporation of Leucine, Uridine and Thymidine into Fetal Tissues 54 6. Ornithine Decarboxylase A c t i v i t y i n Placenta 67 (vi) 7. Amino Acid Analysis of Fetal Brain 8 . Investigation of Glycoprotein Synthesis 9. Variation i n the Acid I n s o l u b i l i t y bf DNA from Fetal Tissues 10. Studies on Corticosteroid Receptors in Fetal Tissues DISCUSSION BIBLIOGRAPHY APPENDICES PUBLICATIONS ( v i i ) LIST OF TABLES Page 1 SUBSTRATE INCORPORATION INTO FETAL BRAIN WITH TIME 26 2 CHARACTERIZATION OF 11-DEHYDROCORTICOSTERONE (AS THE C-21 ACETATE) FROM BRAIN 35 3 EFFECT OF DEXAMETHASONE INJECTION ON THE ACTIVITY OF STEROID C - l l OXIDOREDUCTASE ON GESTATIONAL DAY 14 44 4 EFFECT OF DEXAMETHASONE INJECTION ON THE ACTIVITY OF STEROID C - l l OXIDOREDUCTASE ON GESTATIONAL DAY 16 45 5 EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON GLYCOGEN DEPOSITION ON GESTATIONAL DAY 16 5 3 6 EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON LEUCINE INCORPORATION IN FETAL TISSUES 6 3 7 EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON URIDINE INCORPORATION IN FETAL TISSUES 64 8 EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON THYMIDINE INCORPORATION IN FETAL TISSUES 65 9 SUMMMARY OF FETAL TISSUE REDUCTASE (R) AND DEHYDROGENASE (D) ACTIVITIES 6 6 10 INCORPORATION OF 14C-GLUCOSAMINE INTO FETAL LIVER AND GUT 73 11 THE EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS GIVEN 3H-THYMIDINE ON THE PRECIPITATION OF RADIOACTIVE MATERIAL FROM FETAL TISSUES TREATED WITH PCA 82 12 PROPERTIES OF CORTICOSTEROID RECEPTORS(S) IN FETAL MOUSE BRAIN 84 13 EFFECT OF VARIOUS FACTORS ON BINDING OF 3H-CPD. B IN CYTOSOL 86 L4 COMPETITIVE DISPLACEMENT OF LABELLED STEROID IN CYTOSOL 88 15 COMPETITIVE DISPLACEMENT OF STEROIDS BOUND IN NUCLEAR FRACTIONS 9 7 16 CHARACTERIZATION OF STEROID FROM RECEPTOR-COMPLEXES 98 ( v i i i ) EFFECT OF TIME ON THE EFFICIENCY OF GLUCOSE ACETYLATION EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON THE SPECIFIC ACTIVITY OF MATERNAL AND FETAL BLOOD GLUCOSE ( i x ) LIST OF FIGURES Page QUENCH CURVE FOR 3H AND 1 4 C 31 AUTORADIOGRAM OF CHROMATOGRAPHED EXTRACTS:OF FETAL BRAIN INCUBATED WITH 14C-CORTICOSTERONE 34 3 AUTORADIOGRAM OF CHROMATOGRAPHED EXTRACTS OF MOUSE FETAL LIVER AND PLACENTA INCUBATED WITH 1 4C-11-DEHYDROCORTICOSTERONE 37 4 IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE IN FETAL BRAIN AND GUT 39 5 IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE IN PLACENTA 4 0 6 IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE IN FETAL LIVER AND LUNG 41 7 RECOVERY OF CPD. A AND B IN FETAL BRAIN, GUT AND HEART AFTER INJECTION OF 3H-CPD. B 4 8 8 RECOVERY OF CPD. A AND B IN FETAL LIVER, LUNG AND PLACENTA AFTER INJECTION OF 3H-CPD. B 48 9 STANDARD CURVE FOR THE DETERMINATION OF GLYCOGEN BY THE ANTHRONE METHOD 50 10 GLYCOGEN CONTENT OF FETAL BRAIN,GUT AND HEART ON GESTATIONAL DAYS 14 TO 19 51 11 GLYCOGEN CONTENT OF FETAL LIVER, LUNG AND PLACENTA ON GESTATIONAL DAYS 14 TO 19 52 (x) Page 12 IN VITRO INCORPORATION OF 1 4C-LEUCINE INTO FETAL BRAIN, GUT AND HEART 55 13 J_N VITRO INCORPORATION OF 1 4C-LEUCINE INTO FETAL LIVER, LUNG AND PLACENTA 56 14 IN VITRO INCORPORATION OF 3H-URIDINE INTO FETAL BRAIN, GUT AND HEART 57 15 IN VITRO INCORPORATION OF 3H-URIDINE INTO FETAL LIVER, LUNG AND PLACENTA 58 16 IN VITRO INCORPORATION OF 3H-THYMIDINE INTO FETAL BRAIN, GUT AND HEART 6 0 17 IN VITRO INCORPORATION OF THYMIDINE INTO FETAL LIVER, LUNG AND PLACENTA 61 18 ORNITHINE DECARBOXYLASE ACTIVITY ON GESTATIONAL DAYS 14 TO 19 IN PLACENTA AND THE EFFECT OF DEXAMETHASONE INJECTION 68 19 AMINO ACID ANALYSIS OF FETAL BRAIN ON GESTATIONAL DAYS 14, 15 AND DAY 14 DEX-TREATED 72 20 AUTORADIOGRAM OF CHROMATOGRAPHED EXTRACTS OF FETAL GUT 7 6 21 THE PERIODATE-RESORCINOL REACTION FOR BOUND SIALIC ACID PERFORMED ON PCA PRECIPITATES OF FETAL GUT, LUNG, LIVER AND PLACENTA 78 22 UV SCAN OF PCA PRECIPITATE REDISSOLVED IN DILUTE BASE 80 (xi) 23 PRECIPITATION OF RADIOACTIVITY BY PCA FROM TISSUES OF FETUSES REMOVED FROM MOTHERS INJECTED WITH 3H-THYMIDINE 24 ISOLATION OF RECEPTOR COMPLEXES FROM PLACENTA ON SEPHADEX G-25 25 AUTORADIOGRAM OF CHROMATOGRAPHED EXTRACTS OF 14 PLACENTA INCUBATED WITH C-CPD. A AND 11-KETOPROGESTERONE 26 NUCLEI ISOLATED FROM FETAL BRAIN BY HYPOTONIC SHOCK WITH DILUTE M g C l 2 27 ISOLATION OF RECEPTOR COMPLEXES FROM FETAL BRAIN ON SEPHADEX G-25 28 AUTORADIOGRAMS OF CHROMATOGRAPHED EXTRACTS OF THE ACETYLATED DERIVATIVES OF 1 4C-GLUCOSE RECOVERED FROM MATERNAL AND FETAL MOUSE BLOOD ( x i i ) LIST OF APPENDICES Page APPENDIX I 12 3 APPENDIX I I 124 ( x i i i ) ABBREVIATIONS USED cpd. A 21-Hydroxy-4-pregnene-3,11,20-trione, 11-dehydrocorticosterone (see Appendix I) cpd. B 113,21-Dihydroxy-4-pregnene-3,20-dione, corticosterone (see Appendix I) Dex 9aFluoro ,113,1 7a., 21 -Trihydroxy-1 ,4-pregnadiene-3,20-dione, Dexamethasone (see Appendix I) DNA deoxyribonucleic acid DNAse deoxyribonuclease dpm disintegrations per minute EDTA (e t h y l e n e d i n i t r i l o ) - t e t r a a c e t i c acid, tetrasodium s a l t 11-Epi B 11a,21-Dihydroxy-4-pregnene-3,20-dione, 11-Epicorticosterone (see Appendix I) Kd di s s o c i a t i o n constant KEB Krebs-Eggleston bicarbonate buffer KEP Krebs-Eggleston phosphate buffer (xiv) 11-KP 4-Pregnene-3,11,20-Trione, 11-ketoprogesterone (see Appendix I) min minute PCA perchloric acid RNA ribonucleic acid RNAse ribonuclease SEM standard error of the mean TCA { t r i c h l o r a c e t i c acid TLC thin layer chromatography TRIS 2-amino-2-hydroxymethyl-1,3,propanediol. xg r e l a t i v e c e n t r i f u g a l force C-11 steroi d oxidoreductase w i l l be used for the reversible enzyme 113-hydroxysteroid dehydrogenase, EC 1.1.1.146 corticosterone 11-dehydrocortico-sterone (xv) ACKNOWLEDGMENTS I would l i k e to express my gratitude to the many people who assisted me during my research. In t h i s Department, I am grateful to Dr. J.F. Richards for the ornithine decarboxylase determinations and Mr. Joe Durgo for the amino acid analysis. Thanks are due to Dr. M.E. Todd for her microscopic examination of f e t a l tissues, and to Dr. N. Auersperg for photographing the nuclei in F i g . 26. Both are members of the Department of Anatomy, University of B r i t i s h Columbia. I am also indebted to Mr. C. Remey of the Department of Pathology for assistance with the periodate-resorcinol data. I also wish to thank my family for t h e i r support and under-standing, p a r t i c u l a r l y i n the l a s t s i x months of my research. F i n a l l y , I have great pleasure i n expressing my indebtness to Dr. A.F. Burton for his guidance, encouragement and sense of humour which were invaluable throughout my studies. I was a r e c i p i e n t of the University of B r i t i s h Columbia Scholarship-Fellowship award (1977-78). 1 INTRGDUCTI CN The f e t u s during i n t r a u t e r i n e l i f e i s dependent upon mater-n a l sources f o r a l l the n u t r i e n t s and energy supply necessary f o r growth and d i f f e r e n t i a t i o n . These n u t r i e n t s are r e c e i v e d by the f e t u s v i a the p l a c e n t a and the u m b i l i c a l c o r d . They c r o s s the p l a c e n t a to the f e t u s , and waste products pass i n the o p p o s i t e d i r e c t i o n . As e a r l y as 19 31, Bohr suggested t h a t an observed f e t a l r e s p i r a t o r y q u o t i e n t of 1.0 i m p l i e d t h a t carbohydrate was the s o l e metabolic f u e l f o r the f e t u s (1). Measurements of r a t e s of glucose uptake i n f e t a l lambs suggest t h a t i t i s f a s t enough to account f o r the t o t a l f e t a l oxygen consumption (2); I f most of the glucose i s i n f a c t o x i d i z e d , i t may be the p r i n c i p a l metabolic f u e l o f the f e t u s . T h i s b e l i e f i s strengthened by the r e l a t i v e i m p e r m e a b i l i t y of the p l a c e n t a of d i v e r s e s p e c i e s to f r e e f a t t y a c i d s and ketone bodies (3), and i n many s p e c i e s , i n c l u d i n g man and mouse, glucose i s c o n s i d e r e d to be the major f e t a l f u e l . There i s an i n t e r e s t i n g s p e c i e s p e c u l i a r i t y found i n ungu-l a t e s (4). The p l a c e n t a has an e f f i c i e n t mechanism f o r con-v e r t i n g maternal glucose to f r u c t o s e , which i s then s e c r e t e d i n t o the f e t a l c i r c u l a t i o n . The same c a p a b i l i t y has been shown to e x i s t i n the human p l a c e n t a (5, 6), but the amount of f r u c t o s e s y n t h e s i z e d i s n e g l i g i b l e . 2 The role of glucose as the predominant f e t a l energy source has been challenged recently i n f e t a l sheep ( 7 ) , but any a l t e r -native metabolic f u e l has not been defined as yet. The placental transport of glucose has been well charac-te r i z e d . Transport has been shown to be by " f a c i l i t a t e d d i f -fusion" i n sheep (8) and human (9) placenta. Transport i s i n the same d i r e c t i o n as for a d i f f u s i o n gradient, but the rate exceeds that which would be predicted from d i f f u s i o n alone. Since f e t a l blood glucose i s usually one-half that of the mother, glucose normally passes from mother to fetus. Glucose transport i s mediated by a c a r r i e r , presumably protein i n nature, which can bind glucose rev e r s i b l y and trans-port i t across the placental membrane. This c a r r i e r exhibits saturation k i n e t i c s at high glucose concentrations and shows some preference for glucose, although other sugars may act as competitive i n h i b i t o r s (10). I t also shows a degree of stereo-s p e c i f i c i t y , as the b i o l o g i c a l l y important D-sugars are trans-ported much faster than t h e i r corresponding L-isomers (11). Transport i s unlike l y an active process since no e f f e c t of metabolic poison has been demonstrated (10). Other possible sources of fuel for the fetus are amino acids and l i p i d s . The fetus, although i t receives maternal amino acids, l i k e l y conserves them for protein synthesis, since l i t t l e transamination or deamination occurs during f e t a l l i f e (12) and i n several species gluconeogenesis has been shown to 3 commence onl y a f t e r b i r t h (13). Phosphopyruvate carboxykinase (EC 4.1.1.32) i s co n s i d e r e d t o be the l i m i t i n g enzyme i n t h i s pathway (14). The amount of l i p i d made a v a i l a b l e t o the f e t u s i s very s m a l l . C h o l e s t e r o l t r a n s f e r i s slow, and p h o s p h o l i p i d s and t r i g l y c e r i d e s have not been shown to c r o s s the p l a c e n t a l bar-r i e r i n t a c t i n any s p e c i e s i n v e s t i g a t e d so f a r (15). Thus, t r a n s f e r i s v i r t u a l l y r e s t r i c t e d t o f r e e f a t t y a c i d s , and the amount of f a t t y a c i d t r a n s f e r r e d v a r i e s c o n s i d e r a b l y among s p e c i e s . For example, t r a n s f e r i s high i n guinea p i g (16), but low i n sheep (17) and human (18). Since the mammalian f e t u s has been shown to o x i d i z e f a t t y a c i d s very s l o w l y (19), i t i s presumed t h a t l i p i d i s not l i k e l y a major f u e l f o r the f e t u s . The p l a c e n t a l t r a n s f e r of n u c l e i c a c i d p r e c u r s o r s has been demonstrated (20) , but t h e i r r o l e i n the f e t a l economy remains as y e t undefined, s i n c e i t has been observed t h a t the f e t a l r a t i s able t o s y n t h e s i z e the g r e a t e r p a r t of i t s n u c l e i c a c i d de novo (21). The p l a c e n t a , as w e l l as s e r v i n g a t r a n s p o r t f u n c t i o n , i s a l s o i n v o l v e d i n hormone s y n t h e s i s and can be c o n s i d e r e d an endocrine organ. I t has been observed t h a t i n animals w i t h s h o r t g e s t a t i o n times ( l e s s than two months), removal of the o v a r i e s d u r i n g pregnancy lead s t o a b o r t i o n . But i n animals w i t h l o n g e r g e s t a t i o n p e r i o d s , the p l a c e n t a l endocrine func-t i o n s are s u f f i c i e n t l y w e l l developed e a r l y i n pregnancy t o 4 permit maintenance of g e s t a t i o n a f t e r ovariectomy of mothers (22). The e a r l i e s t time a t which ovariectomy can be performed without t e r m i n a t i o n of pregnancy v a r i e s with d i f f e r e n t s p e c i e s , but i n man i t i s known to be a t f i v e weeks. This suggests t h a t , w h i l s t p i t u i t a r y hormones are necessary f o r o v u l a t i o n and i m p l a n t a t i o n , p l a c e n t a l hormones can p l a y an important r o l e i n the c o n t i n u a t i o n of pregnancy. In s p e c i e s of s h o r t g e s t a t i o n a l p e r i o d s , maternal hormones predominate; i . e . , progesterone and estrogens are maternal i n o r i g i n . Three major pep t i d e hormones are s y n t h e s i z e d by the primate p l a c e n t a . These are c h o r i o n i c gonadotrophin, p l a c e n t a l l a c t o g e n and c h o r i o n i c t h y r o t r o p i n . The r o l e these p l a y i n pregnancy i s s t i l l not c l e a r l y d e f i n e d . C e r t a i n l y t h e i r e f f e c t would appear to be p r i m a r i l y on the maternal s i d e , s i n c e the p l a c e n t a forms a r e l a t i v e l y impermeable b a r r i e r and the c o n c e n t r a t i o n o f these hormones i n the maternal blood i s many times h i g h e r than t h a t observed i n the f e t u s (23). The p l a c e n t a i s a l s o i n v o l v e d i n the s y n t h e s i s of s t e r o i d hormones i n c l u d i n g progesterone and estrogens (24). In g e n e r a l , compounds of sma l l molecular s i z e d i f f u s e more r a p i d l y a c r o s s the p l a c e n t a l membrane than l a r g e r m olecular weight. Uncharged molecules c r o s s more r a p i d l y than i o n i z e d , and f a t - s o l u b l e substances more r a p i d l y than those o f low l i p i d s o l u b i l i t y . These are g e n e r a l p r o p e r t i e s observed f o r membrane systems. 5 Compounds of molecular weight exceeding 1000 d a l t o n s c r o s s the p l a c e n t a only by processes as y e t undefined. The p l a c e n t a , t h e r e f o r e , though f r e e l y permeable t o smal l maternal hormones, i s r e l a t i v e l y impermeable t o maternal p e p t i d e hormones. Although wi t h s u f f i c i e n t l y s e n s i t i v e techniques some t r a n s f e r of maternal hormones can u s u a l l y be demonstrated, i t i s g e n e r a l l y h e l d t h a t the t r a n s f e r i s too slow to a f f e c t the f e t u s s i g n i f i -c a n t l y , and c l i n i c a l and experimental s i t u a t i o n s i n d i c a t e the inadequacy of such t r a n s f e r . For example, the occurrence of adren a l h y p o p l a s i a i n anencephalic f e t u s e s (25) and the f a i l u r e of normal t h y r o i d development shown i n d e c a p i t a t e d f e t u s e s (26) i n d i c a t e the u n a v a i l a b i l i t y of maternal a d r e n o c o r t i c o t r o p h i c hormone (ACTH) and t h y r o t r o p h i c hormone (TSH), and t h e r e f o r e the i n a b i l i t y of the maternal hormones to c r o s s the p l a c e n t a l membrane and compensate f o r f e t a l i n s u f f i c i e n c y . I t would t h e r e f o r e appear t h a t the f e t a l endocrine system matures e a r l y and f u n c t i o n s autonomously. In f a c t , ACTH (27), TSH (28) , i n s u l i n (29) and growth hormone (30) have been shown to be present i n the human f e t a l c i r c u l a t i o n as e a r l y as the t e n t h week of g e s t a t i o n . In some s p e c i e s , c o r t i c o s t e r o i d s do not c r o s s the p l a c e n t a r e a d i l y ; e.g., sheep (31). In o t h e r s , n o t a b l y man (24) and mouse (32), maternal c o r t i c o s t e r o i d s c r o s s r e a d i l y to the f e t u s . What a c t u a l l y determines the amount of c o r t i c o s t e r o i d c r o s s i n g the p l a c e n t a i s not known, but t r a n s c o r t i n i s b e l i e v e d to p l a y a r o l e . T h i s 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 i s known to i n c r e a s e d u r i n g pregnancy:or a f t e r a d m i n i s t r a t i o n of estrogens (33), and t h i s i s c o n s i s t e n t w i t h an observed i n c r e a s e d maternal 6 l e v e l of hormone i n humans and o t h e r s p e c i e s . Gala and Westphal (34) have shown t h a t t r a n s c o r t i n l e v e l s i n c r e a s e d u r i n g preg-nancy i n r a b b i t , r a t and mouse. In the mouse, t r a n s c o r t i n l e v e l s were shown to i n c r e a s e r a p i d l y a f t e r g e s t a t i o n a l day .11. Both the mouse and man have a hemochorial type of p l a c e n t a (35) , the p l a c e n t a being e n t i r e l y f e t a l i n o r i g i n w i t h o n l y a s i n g l e l a y e r of c a p i l l a r y endothelium s e p a r a t i n g the maternal and f e t a l c i r c u l a t i o n s . Hormones which can c r o s s the p l a c e n t a such as androgens, estrogens, catecholamines and, i n some s p e c i e s , c o r t i c o s t e r o i d s (24, 32), are e x t e n s i v e l y metabolized by the f e t u s . A predomi-nant mechanism i n v o l v e d i s s u l f u r y l a t i o n , s i n c e the f e t a l a d r e n a l c o n t a i n s a c t i v e s u l f o k i n a s e a c t i v i t y (24). These s u l f a t e d com-pounds are known to be b i o l o g i c a l l y i n a c t i v e . The p l a c e n t a has a c t i v e s u l f a t a s e so the s u l f a t e group can be c l e a v e d and the unconjugated hormone r e l e a s e d i n t o the maternal blood. The con-jugated form i s too p o l a r t o c r o s s the p l a c e n t a l b a r r i e r (36). The f e t u s and p l a c e n t a form a f u n c t i o n u n i t , the " f e t o -p l a c e n t a l " u n i t (24) i n primates. T h i s u n i t c a r r i e s out s t e r o i d b i o s y n t h e t i c r e a c t i o n s which n e i t h e r the p l a c e n t a nor f e t u s alone c o u l d p o s s i b l y complete; f o r example, estrogen b i o s y n -t h e s i s . T h i s concept does not h o l d i n other s p e c i e s , f o r example the mouse, where estrogens and progesterone are o f maternal o r i g i n . 7 A f e a t u r e s i m i l a r i n b o t h man a n d mouse i s 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 i d s . I n b o t h t h e r e i s e x t e n s i v e c o n v e r s i o n o f t h e hormones t o t h e 1 1 - d e h y d r o m e t a b o l i t e , w h i c h i s b i o l o -g i c a l l y i n a c t i v e . H owever, w h e r e a s i n man t h e p l a c e n t a h a s b e e n shown t o be t h e m a j o r s i t e o f d e h y d r o g e n a t i o n o f C o r t i s o l ( 3 7 ) , i n t h e mouse 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 o c c u r s i n t h e h e a d ( 3 8 ) . I n s e v e r a l s p e c i e s , i t h a s b e e n e s t a b l i s h e d t h a t t h e f e t u s c a n s y n t h e s i z e c o r t i c o s t e r o i d s . F o r e x a m p l e , i t h a s b e e n shown t h a t 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 m a t e r n a l b l o o d o n day 18 f o l l o w i n g m a t e r n a l a d r e n a l e c t o m y , was t h e same a s i n t h e i n t a c t m o t h e r s , i n d i c a t i n g t h a t t h e f e t a l r a t a d r e n a l h a s t h e a b i l i t y t o s y n t h e s i z e c o r t i c o s t e r o i d s a n d t h a t t h e hormone c a n c r o s s t h e p l a c e n t a t o t h e m o t h e r f r o m t h e f e t u s ( 3 9 ) . M i c h a u d a n d B u r t o n showed t h a t t h e f e t a l mouse a d r e n a l c o u l d s y n t h e s i z e c o r t i c o s t e r o i d s by g e s t a t i o n a l d a y 1 6 - 17 ( 3 8 ) . A f t e r i n j e c t i o n o f l a b e l l e d c o r t i c o s t e r o n e i n t o a d r e n a l e c t o m i z e d m o t h e r s , t h e d i s t r i b u t i o n o f hormone i n m a t e r n a l a n d f e t a l t i s s u e was t h e same a s i n i n t a c t m i c e , i n d i c a t i n g t h a t , r e g a r d -l e s s o f w h e t h e r t h e hormone i s o f m a t e r n a l o r f e t a l o r i g i n , t h e movement a c r o s s t h e p l a c e n t a i n b o t h d i r e c t i o n s i s r a p i d ( 3 8 ) . Murphy and L e o n g (40) h a v e shown t h a t C o r t i s o l i s h i g h e r i n b l o o d l e a v i n g t h e human f e t u s i n t h e u m b i l i c a l a r t e r y t h a n t h a t e n t e r i n g b y t h e u m b i l i c a l v e i n , s u g g e s t i n g s y n t h e s i s o f C o r t i s o l i n t h e f e t u s . I n v i e w o f t h e l a r g e amount o f c o r t i s o n e 8 p r e s e n t and the e x i s t e n c e of an enzyme capable of r e d u c i n g t h i s t o C o r t i s o l i n f e t a l l i v e r and f e t a l lung (41) , i t i s p o s s i b l e t h a t a small change i n the r e d u c t i o n of c o r t i s o n e t o C o r t i s o l i n f e t a l t i s s u e s r a t h e r than demovo s y n t h e s i s c o u l d account f o r t h i s increment. The s i g n i f i c a n c e of t h i s p r o p o s a l i s t h a t i t suggests p o s s i b l e q u a n t i t a t i v e changes i n the c o n c e n t r a t i o n of hormone i n f e t a l t i s s u e s , r e g a r d l e s s of whether the s t e r o i d s are of maternal or f e t a l o r i g i n , without involvement of the f e t a l p i t u i t a r y - a d r e n a l a x i s . D e t a i l s o f the mechanism of a c t i o n of s t e r o i d hormones have been e s t a b l i s h e d o n l y i n r e c e n t y e a r s . F o l l o w i n g c e l l u l a r up-take i n " t a r g e t " t i s s u e , the s t e r o i d i s bound to a c y t o p l a s m i c p r o t e i n r e c e p t o r . In a temperature-dependent r e a c t i o n , the cy t o p l a s m i c s t e r o i d - r e c e p t o r complex moves i n t o the nucleus and binds to a s p e c i f i c s i t e on chromatin. This b i n d i n g a l t e r s the DNA template a c t i v i t y and the s t e r o i d then e x e r t s i t s e f f e c t on the c e l l by m o d i f i c a t i o n a t the l e v e l of t r a n s c r i p t i o n (42). Since a v a r i e t y of f e t a l t i s s u e s have been observed t o have g l u c o c o r t i c o i d r e c e p t o r s (4 3), these t i s s u e s c o u l d p o s s i b l y have the c a p a c i t y t o respond t o g l u c o c o r t i c o i d s and the p r e n a t a l a d m i n i s t r a t i o n o f these hormones c o u l d c o n c e i v a b l y a l t e r the developmental p r o c e s s . S p e c i f i c g l u c o c o r t i c o i d r e c e p t o r s have been shown by B a l l a r d and B a l l a r d t o be prese n t e a r l y i n f e t a l l i f e i n l i v e r , s m a l l i n t e s t i n e , kidney, heart.,., muscle, s k i n and lung of human 9 f e t u s e s (43). Both B a l l a r d and B a l l a r d (44), and Giannopoulos, Mulay .and Solomon ..(4 5) have shown the presence of cy t o p l a s m i c g l u c o c o r t i c o i d r e c e p t o r s from day 18 of g e s t a t i o n i n f e t a l r a b b i t l u n g . These o b s e r v a t i o n s i n d i c a t e t h a t the presence of an a c t i v e r e c e p t o r system i s not a l i m i t i n g f a c t o r i n the onset of g l u c o c o r t i c o i d responsiveness i n , f o r example, the f e t a l lung, which undergoes s t e r o i d - i n d u c e d changes a t t h i s time. The s i g n i f i c a n c e of the r e c e p t o r i n terms of the development of the t i s s u e has onl y been w e l l e s t a b l i s h e d i n f e t a l l u n g . C o r t i c o s t e r o i d s have been shown to p l a y a r o l e i n f e t a l development i n the l a t t e r stages of g e s t a t i o n . One e f f e c t i s t h a t on f e t a l l i v e r glycogen d e p o s i t i o n . More than a century ago, Claude Bernard (46) demonstrated t h a t glycogen i s pr e s e n t i n mammalian f e t a l l i v e r , and t h a t t h i s p o l y s a c c h a r i d e makes i t s appearance i n t h i s t i s s u e a t a r e l a t i v e l y l a t e stage o f g e s t a -t i o n . In a l l mammalian s p e c i e s i n v e s t i g a t e d so f a r there i s a marked and r a p i d i n c r e a s e i n f e t a l l i v e r glycogen l e v e l s s h o r t l y before b i r t h (47). Although f e t a l l i v e r glycogen d e p o s i t i o n i s known to be s t i m u l a t e d prematurely by c o r t i c o s t e r o i d i n j e c t i o n (48, 49), i t i s not the onl y f a c t o r i n v o l v e d . J o s t has shown t h a t i f d e c a p i -t a t e d r a t f e t u s e s o f adrenalectomized mothers are gi v e n C o r t i s o l , the l i v e r glycogen content i s i n c r e a s e d . In r a b b i t s , c o r t i -c o s t e r o i d s were found to have no e f f e c t on glycogen storage even when extremely l a r g e doses were g i v e n . Only when a p i t u i t a r y p r o l a c t i n - l i k e substance was gi v e n w i t h the c o r t i c o s t e r o i d s was 10 any g l y c o g e n d e p o s i t i o n apparent„ ( 2 5 ) . A d u a l h o r m o n a l c o n -t r o l e f f e c t e d by c o r t i c o s t e r o i d s and a p i t u i t a r y hormone h a s b e e n p o s t u l a t e d f o r d e p o s i t i o n o f g l y c o g e n . H i s t o l o g i c a l s t u d i e s by A r o n (50) i n d i c a t e a c o r r e l a t i o n b e t w e e n t h e a p p e a r -a n c e o f t h e i s l e t s o f L a n g e r h a n s and t h e i n c r e a s e o f f e t a l l i v e r g l y c o g e n , w h i c h i m p l i e s a p o s s i b l e r o l e f o r i n s u l i n . L i v e r i s n o t t h e o n l y f e t a l s t o r e o f g l y c o g e n . S i z a b l e s t o r e s h a v e b e e n o b s e r v e d i n l u n g , h e a r t a n d s k e l e t a l m u s c l e ( 4 7 ) . The p a t t e r n o f d e p o s i t i o n i n e a c h d i f f e r s , a n d t h e a p p e a r a n c e o f i n s u l i n a n d a d r e n o c o r t i c a l hormones i n t h e f e t a l c i r c u l a t i o n a t a l a t e s t a g e o f g e s t a t i o n i n r o d e n t s d o e s n o t e x p l a i n d e p o s i t i o n o f g l y c o g e n i n l u n g , h e a r t a n d m u s c l e a t a n e a r l i e r t i m e . No h o r m o n a l i n f l u e n c e s h a v e b e e n r e c o g n i z e d s o f a r f o r m u s c l e and l u n g d e p o s i t s ( 4 7 ) , b u t P i c o n a n d B o u h n i k h a v e shown t h a t h e a r t g l y c o g e n may be i n f l u e n c e d by a d r e n o -c o r t i c a l hormones ( 5 1 ) . F e t a l g l y c o g e n s t o r e s d e c r e a s e r a p i d l y , r e a c h i n g 10% o r l e s s o f t h e i r i n i t i a l v a l u e s w i t h i n two t o t h r e e h o u r s o f b i r t h (4 7 ) . T h e s e s t o r e s r e p r e s e n t a r e a d i l y a v a i l a b l e s o u r c e o f e n e r g y d u r i n g t h e e a r l y n e o n a t a l p e r i o d , a s t h e b r a i n i s d e p e n -d e n t upon c i r c u l a t i n g g l u c o s e f o r e n e r g y ( 5 2 ) . The n e w b o r n c a n e x i s t o n i t s c a r b o h y d r a t e s t o r e s f o r t h r e e t o f o u r h o u r s a f t e r b i r t h , b u t b e f o r e t h e s e s t o r e s a r e e x h a u s t e d t h e c o n c e n t r a t i o n o f f r e e f a t t y a c i d i n t h e p l a s m a r i s e s , r e a c h i n g a p e a k s i x t o 24 h o u r s a f t e r b i r t h ( 5 3 ) . 11 During l a t e r stages of f e t a l growth, t r i g l y c e r i d e s are synthesized from glucose and deposited i n the l i v e r and f a t depots. It i s these stores which are u t i l i z e d by the newborn aft e r carbohydrate stores are exhausted. A further type of l i p i d storage i s found i n brown adipose tissue. This store seems to have a function i n r e l a t i o n to postpartum thermo-regulation (54). Lachance and Page have shown that c o r t i c o -s t e r o i d administration leads to an increase i n brown adipose tissue fat content (55). Enzymes which are known to be stimulated by c o r t i c o s t e r o i d s antenatally include: mitochondrial 1-alanine-glyoxylate aminotranferase (EC 2.6.1.2) (56); glycogen synthetase (EC 2.4.1.11) (57) and phenylethanolamine-N-methyltransferase (EC 2.1.1.28) (58). The development of the adrenal medulla was retarded and the content of epinephrine was low i n decapitated rat., fetuses, but could be restored by administration of ACTH to fetuses or of C o r t i s o l to either mother or fetuses (25). Experimental work has shown that the maturation of f e t a l lamb lungs can be accelerated either by stimulation of the f e t a l adrenal cortex or by administration of adrenal c o r t i c o -steroids ' (59) . Liggins (59) suggested that these steroids caused l i b e r a t i o n of surfactant into the a l v e o l i , perhaps by induction of an enzyme involved i n surfactant biosynthesis. I t was reported by F a r r e l l and Zachman (60) that i n j e c t i o n of rabbit -fetuses with a synthetic c o r t i c o i d increased the a c t i v i t y of choline phosphotransferase (EC 2.7.8.2) in the lung, but as 12 a c t i v i t y of t h i s enzyme declines normally i n late gestation, i t seems unlikel y that t h i s could be a c r i t i c a l step (61). More recently, the a c t i v i t y of phosphatidic acid phosphatase (EC 3.3.1.4) has been found to increase i n the f e t a l lung (62) and was stimulated by treatment with the synthetic c o r t i c o i d , betamethasone (63). I t appears, therefore, that t h i s enzyme i s the regulatory factor for surfactant synthesis during lung development. Liggins has reported on the c l i n i c a l use of betamethasone in a series of patients who threatened to d e l i v e r prematurely (64). This study indicated that infant f e t a l lungs can appa-rently be induced to mature s u f f i c i e n t l y when treated i n t h i s way, since the incidence of respiratory d i s t r e s s syndrome f o l -lowing these premature d e l i v e r i e s was decreased d r a s t i c a l l y from what would be expected. In sheep, i t has been demonstrated that a functional pituitary-adrenal axis i s necessary for the onset of labor, since an infusion of ACTH or of c o r t i c o s t e r o i d s into f e t a l lambs induced premature p a r t u r i t i o n (65). During the week before normal b i r t h there i s an increase i n the f e t a l plasma c o r t i c o -steroid concentration, unrelated to that i n the maternal c i r c u -l a t i o n . Pregnancy i n the sheep can be prolonged by adrenalectomy of the fetus (66) . This d i r e c t p a r t i c i p a t i o n of the fetus i n the onset of labor has been demonstrated i n several species, but not i n primates, although human anencephaly i s associated with prolonged gestation (67). Normal concentrations of C o r t i s o l 13 have been observed i n cord blood and amniotic f l u i d i n anen-cephalic cases (68). Therefore, some factor other than C o r t i -sol must be responsible for f a i l u r e of these pregnancies to terminate. Observations on rhesus monkeys showed that neither f e t a l adrenalectomy (6 9) nor i n j e c t i o n of synthetic c o r t i c o i d s (70) could induce p a r t u r i t i o n prematurely. Thus, i n primates, corticosteroids are not alone a s u f f i c i e n t factor for the induction of p a r t u r i t i o n . Corticosteroids, thus, have been shown to play roles i n several aspects of f e t a l development. They appear to be res-ponsible for the induction of various enzymes, for maturation of f e t a l lung and adrenal medulla, for deposition of glycogen in f e t a l l i v e r and, i n some species, for the i n i t i a t i o n of par-t u r i t i o n . These e f f e c t s are a l l observed in'the l a t e stages of gestation, and the only corticosteroid-induced e f f e c t that has been documented i n e a r l i e r pregnancy i s the induction of c l e f t palate, which was attributed to excess c o r t i c o i d s , i n several species including the mouse (71). Beyond t h i s , l i t t l e else i s known of the role of these hormones i n early f e t a l development. The purpose of t h i s study was to explore the metabolism of cort i c o s t e r o i d s i n f e t a l mouse tissues at various stages of gestation and to examine the p o s s i b i l i t y that d i f f e r e n t tissues might regulate the l e v e l of active c o r t i c o s t e r o i d independently of the f e t a l pituitary-adrenal axis. The mouse was chosen as an experimental model since i t , l i k e man, has a hemochorial placenta across which cort i c o s t e r o i d s are transferred r e a d i l y 14 and i n which c o r t i c o s t e r o i d s are 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 m e t a b o l i t e . The r e s u l t s to be d e s c r i b e d show t h a t each f e t a l t i s s u e has i t s own p a r t i c u l a r p a t t e r n of c o r t i c o s t e r o i d metabolism and can r e g u l a t e i t s c o r t i c o s t e r o n e content independently. T h e r e f o r e , the p i t u i t a r y - a d r e n a l a x i s , although f u n c t i o n a l , i s not a major f a c t o r i n f e t a l c o r t i c o s t e r o n e r e g u l a t i o n . V a r i o u s b i o c h e m i c a l parameters such as l e u c i n e , u r i d i n e and thymidine i n c o r p o r a t i o n i n t o f e t a l t i s s u e s were shown to be r e g u l a t e d by c o r t i c o s t e r o i d s a t a stage i n development much e a r l i e r than most other c o r t i c o s t e r o i d - i n d u c e d events which have been r e p o r t e d to occur p r e n a t a l l y . F i l i a l l y , i t was found t h a t 1 1 - d e h y d r o c o r t i c o s t e r o n e , a major m e t a b o l i t e of c o r t i c o s t e r o n e which i s c o n s i d e r e d b i o l -o g i c a l l y i n a c t i v e , a c t u a l l y competed f o r r e c e p t o r s i t e s a t both c y t o s o l and n u c l e a r l e v e l s . T h i s compound was a l s o observed to b i n d to the chromatin f r a c t i o n of the nucleus. T h i s r a i s e s the p o s s i b i l i t y t h a t 11-dehydrocorticosterone may serve a more p o s i t i v e r o l e i n f e t a l development than has h i t h e r t o been c o n s i d e r e d . 15 MATERIALS Animals Mice wereoof the non-inbred UBC Swiss s t r a i n raised at the Department of Zoology Vivarium at t h i s u n i v ersity. The gesta-t i o n period was 19 days. Day 1 was defined as the day following mating, on which a plug was observed. Matings were made between 1800 and 0700 h. Buffers Krebs-Eggleston buffers contain 0.154M solutions of the following s a l t s , mixed i n the r e l a t i v e proportions indicated: NaCl-100; KC1-4; MgSC>4. 7H20-1. For phosphate buffer these s a l t s were, mixed with 0.2 vol of 0. IM Na2'HPO . 2H20. f i n a l pH 7.4. For bicarbonate buffer, 0.21 ;.vol of 0.154M NaHCC>3 was added i n place of the phosphate and the bicarbonate buffer gassed with CC>2 immediately before use. A small amount of phenol red indicator was included i n the bicarbonate buffer, approximately 5 mg/1 (72). T r i s buffer used i n the nuclear receptor experiments contained: 0.6M KC1, 10 mM Tris-HCl and 1.5 mM EDTA, f i n a l pH 8.0. Chemicals Dexamethasone (Decadron powder) was a g i f t from Merck and Company, Rahway, N.J. The 11-epicorticosterone was kindly 16 supplied by Dr. J.C. Babcock, The Upjohn Company, Kalamazoo, MI. Corticosterone, 11-dehydrocorticosterone, the free alcohols and the C-21 acetates as well as 11-ketoprogesterone were obtained from Steraloids (Wilton, NH). T r i s , EDTA, d i t h i o -t h r e i t o l , glycogen (grade V),RNase containing 50-75 Kunitz units/mg protein and DNase containing 2,000 Kunitz units/mg protein were purchased from Sigma Chemical Corp., St. Louis, MO. Pronase, 45,000 PKU units/mg, was obtained from Calbiochem, Sephadex G-25 was obtained from Pharmacia, Uppsala, Sweden. Anthrone was from British.Drug Houses Ltd., Poole, England. Bovine serum albumin was obtained from Mann Research Labs, and N-ethyl maleimide from Dajac Labs, Philadelphia, PA. The sesame o i l used was obtained from Lifestream Natural Foods, Richmond, B.C. (a l o c a l supplier)„ and was free of additives including anti-oxidants. Charcoal (Norit A) and a l l other chemicals were obtained from either Fisher S c i e n t i f i c or Canlab, i n Vancouver. Radioactive Chemicals Radioactive compounds purchased from New England Nuclear 3 Corp. (Montreal) included: H-acetic anhydride (400 Ci/mmole); 3 14 corticosterone-1,2,6,7- H (82.1 Ci/mmole); corticosterone-4- C 3 (57.3 mCi/mmole); dexamethasone-1,2,4- H (21.2 Ci/mmole). 14 3 C- or H-labelled 11-dehydrocorticosterone were prepared enzymatically from l a b e l l e d corticosterone, as w i l l be described l a t e r . Compounds obtained from Amersham/Searle, Arlington Heights, IL included: D-f ructose-6-phosphate- 4C (u) (268 mCi/ 17 mmole); D-glucosamine HCl-l-'^C (60-68 mCi/mmole); D-glucose-14 3 C(u) (331 mCi/mmole); thymidine methyl- H (26 Ci/mmole); 3 uridine-5- H (30 Ci/mmole). Solvents A l l solvents used were obtained from Fisher Chemical or Canlab, Vancouver. These included: n-butanol, chloroform, dichloromethane, ethanol, ethyl acetate, ethyl ether, n-hexane, methanol, petroleum ether, pyridine and toluene. A l l solvents were freshly d i s t i l l e d before use. Chromatography Eastman s i l i c a gel TLC sheets with fluorescent indicator were used. These were washed with d i s t i l l e d methanol p r i o r to use. Whatman No. 1 paper was used for separation of sugars and for p u r i f i c a t i o n of steroids, since quantitative recovery of steroids from paper was superior. Autoradiography Medical X-ray films (NS2T) were obtained from Kodak and I l f e x 90 X-ray f i l m was purchased from I l f o r d Ltd. through l o c a l suppliers. 18 S c i n t i l l a t i o n S u p p l i e s E c o n o f l u o r , a premixed s c i n t i l l a t i o n s o l u t i o n , was pur-chased from New England Nuclear Corp., Montreal. S o l u b i l i z e r s used i n c l u d e d P r o t o s o l and a l s o B i o - s o l v e BBS-3 o b t a i n e d from Beckman Instruments, F u l l e r t o n , CA. 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. Quenching was determined by the method of e x t e r n a l standard r a t i o s (73) . 19 METHODS Injection of Mice Mice which were injected with dexamethasone received sub-cutaneously 0.4 ml of a suspension i n sesame o i l prepared as follows: a solution of dexamethasone, 2 mg/ml i n ethanol, was layered over sesame o i l and approximately three-quarters of the ethanol evaporated under a stream of nitrogen, u n t i l the steroi d was beginning to come out of solution. The o i l and ethanol were then mixed thoroughly to give a suspension of 0.5 mg/ml. This solution was mixed well again just p r i o r to use. The injected mice were k i l l e d 16 hours l a t e r . 3 Mice injected with H-corticosterone received a solution of the ste r o i d i n saline subcutaneously, 1 v>Ci/0.5 ml, 15 minutes pr i o r to removal of f e t a l tissues. 3 Mice injected with H-thymidine received subcutaneously 0.5 ml of a saline solution containing 5 yCi, 30 minutes p r i o r to removal of f e t a l t issues. Preparation of'".Tissues Mice were k i l l e d with excess C0 2 and the u t e r i removed quickly from the mother and placed on ice i n P e t r i dishes con-taining saline-moistened f i l t e r paper. The fetuses were then removed and the appropriate tissues dissected out and pooled 20 for incubation. Tissues, up to 300 mg, were placed i n a 25 ml Erlenmeyer f l a s k and minced f i n e l y with s c i s s o r s . Buffer and substrates were then added and the fl a s k stoppered with a s i l i c o n e stopper. Flasks were then placed i n a water bath shaking at 120 cycles/min at 37°C for the length of time as required for incubation purposes. Unless stated otherwise, a l l incubations were performed under these conditions. Flasks were kept c h i l l e d on ice during preparation. Enzymatic Synthesis of 11-dehydrocorticosterone (cpd. A) 3 14 Either H- or C-ll-dehydrocorticosterone was prepared by incubating the appropriately l a b e l l e d corticosterone (cpd. B) with a guinea pig l i v e r microsome preparation f o r t i f i e d with NADP (37). The incubation mixture was extracted with CH 2C1 2, which was evaporated and the residue spotted onto Whatman No. 1 paper impregnated with methanol:formamide, (1:1; v:v) . The chromatogram was developed i n benzene saturated with formamide. The steroid zones were eluted with chloroformrmethanol (1:1; v:v), which was f i l t e r e d through sintered glass to remove p a r t i c l e s of ce l l u l o s e , then evaporated under a stream of N 2, and the residue redissolved i n ethanol. Corticosterone contains as an impurity, 11-oxygenated progesterone intermediates, which c o - c r y s t a l l i z e with c o r t i c o -sterone and can be removed only by chromatography. This amounts to approximately 15% of the UV-absorbance of a sample of cpd. B (74). Accordingly, a l l cpd. B was p u r i f i e d by chromatography before use. 21 Because of the i n s t a b i l i t y of free 11-dehydrocorticosterone (cpd. A), t h i s compound, both the radioactive and non-radioactive, was freshly p u r i f i e d by chromatography immediately before use. The concentration of non-radioactive compounds was determined by UV absorbance of solutions at 237-240nm i n a Unicam SP 800A spectrophotometer. In V i t r o Steroid Incubations Minced tissues were incubated i n 1 ml of Krebs-Eggleston i A phosphate buffer (KEP) containing either 5 nCi of G-labelled cpd. A or B when required for autoradiography. Otherwise, 14 3 2.1 nCi C-cpd. B and 36 nCi H-cpd. A were incubated together in order to determine both dehydrogenase and reductase a c t i v i t y simultaneously. A l l incubations were carried out for 15 min and the reaction stopped by c h i l l i n g the sample on i c e , f o l -lowed by extraction. Extraction Procedure The samples were f i r s t extracted with 6 vol n-hexane, by shaking vigorously 20 times. The upper phase, containing neutral l i p i d , was removed by aspiration and discarded. The steroids were then extracted i n 6 vol i c e - c o l d CP^C^, shaking 20 times. The CH 2C1 2 was removed and evaporated to dryness under a stream of N 2. The residue was dissolved i n chloroform: methanol (1:1; v:v) and spotted onto a s i l i c a gel TLC sheet. The tube containing the residue was rinsed several times with 22 s o l v e n t to ensure e f f i c i e n t t r a n s f e r of s t e r o i d . Chromatography N o n - r a d i o a c t i v e cpd. A and B, 10 yg each, were added to each sample p r i o r t o e x t r a c t i o n to a c t as c a r r i e r s , and a l s o t o serve as markers, a l l o w i n g l o c a t i o n o f the zones under s h o r t -wave UV l i g h t (24 0nm). The TLC sheets were developed succes-s i v e l y i n two s o l v e n t systems: 1) n-hexane:ethyl a c e t a t e (4:1; v : v ) , which c a r r i e s r e s i d u a l f a t s w i t h the s o l v e n t f r o n t , 2) toluene:chloroform:methanol:water (120:60:20:1; v:v) to r e s o l v e the s t e r o i d zones. In t h i s system, the C-21 a c e t a t e s move ahead of the f r e e s t e r o i d s , w h i l e other m e t a b o l i t e s move more sl o w l y . The zones were marked, cut out and scraped i n t o v i a l s to which 10 ml of Econo-f l u o r s c i n t i l l a t i o n f l u i d was added. The v i a l s were mixed w i t h a v o r t e x mixer and counted. Most samples c o n t a i n e d a t l e a s t s e v e r a l hundred counts. In V i v o Metabolism of S t e r o i d s Upon removal of t i s s u e from mice which had been i n j e c t e d w i t h l a b e l l e d s t e r o i d , 1 ml KEP was added to each t i s s u e p l u s 10 yg each of cpd. A and B c a r r i e r . The sample was c h i l l e d on i c e , and homogenized q u i c k l y i n a S o r v a l l Omnimixer (Ivan S o r v a l l Inc., 23 Newtown, CT) . The sample was then e x t r a c t e d and chromato-graphed and the zones e l u t e d and counted as d e s c r i b e d above. C h a r a c t e r i z a t i o n o f S t e r o i d s In a d d i t i o n t o chromatography on paper or TLC, charac-t e r i z a t i o n by d e r i v a t i v e formation and c o - c r y s t a l l i z a t i o n w i t h a u t h e n t i c added c a r r i e r was performed. A c e t i c anhydride and dry p y r i d i n e , 0.3 ml each, were added to the sample a f t e r d r y -i n g . The samples were mixed, t i g h t l y stoppered and incubated at 37°C f o r 1 h. E t h a n o l , 0.5 ml o f 25% aqueous s o l u t i o n , was added t o hy d r o l y z e excess a c e t i c anhydride. The samples were then e x t r a c t e d w i t h 6 v o l CH 2C1 2, the aqueous l a y e r removed and the samples r e - e x t r a c t e d w i t h another 0.5 ml water. C r y s t a l l i z a t i o n The CH 2C1 2 l a y e r was evaporated and the r e s i d u e r e d i s s o l v e d i n 1 ml methanol c o n t a i n i n g 10 mg 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 or 11-dehydrocorticosterone a c e t a t e . I c e - c o l d d i s t i l l e d water was added dropwise t o the samples t o the p o i n t of i n c i p i -ent c r y s t a l l i z a t i o n , and the samples were s t o r e d a t 0-4°C to permit slow c r y s t a l l i z a t i o n t o occur. A f t e r 6 t o 24 h, the c r y s t a l s were packed by c e n t r i f u g a t i o n , the mother l i q u o r de-canted and the c r y s t a l s r e d i s s o l v e d i n 2 ml methanol. An a l i -quot was removed f o r d e t e r m i n a t i o n o f UV a b s o r p t i o n and another a l i q u o t was assayed f o r r a d i o a c t i v i t y . To the remaining s o l u -t i o n water, was added t o repeat the c r y s t a l l i z a t i o n procedure. 24 Isolation of Glycogen from Tissues Mice were k i l l e d and tissues removed as described pre-viously. Each tissue was weighed quickly and placed i n a 15 ml centrifuge tube to which 2 ml 2N NaOH was added. The sample was then placed i n a water bath at 50-60°C to dissolve the tissues. Two v ol ethanol containing 0.1% L i C l were then added and the sample mixed and stood at room temperature, which i s more favourable for the p r e c i p i t a t i o n of glycogen. The sample was then centrifuged at 1200 xg for 5 min and the supernatant discarded. The p r e c i p i t a t e was redissolved i n 1.8 ml water and 0.2 ml 3N PCA added. This procedure pr e c i p i t a t e s glycoprotein which could react with the anthrone reagent. The sample was centrifuged and the supernatant transferred. The p e l l e t was washed with 1 ml 0.3N PCA and the washing added to the super-natant. Two vol ethanol-LiCl were added, and a white flocculent p r e c i p i t a t e of glycogen resulted. The sample was centrifuged, and the p e l l e t was redissolved i n 1 ml d i s t i l l e d water (75). Anthrone Assay for Glycogen To 280 ml d i s t i l l e d water, 720 ml concentrated H 2S0 4 were added. The contents were cooled to 35°C, and 500 mg anthrone dissolved therein. Then 3 0 g thiourea were added and dissolved, ahdtthe mixture t i g h t l y stoppered and stored at 0-4°C. Five ml anthrone reagent was added to 1 ml of glycogen sample and the solution mixed well. A marble was placed over 25 t h e t u b e , a n d t h e t u b e p u t i n a b o i l i n g w a t e r b a t h f o r 10 m i n . The s a m p l e was t h e n c o o l e d t o room t e m p e r a t u r e f o r 15 m i n a n d a b s o r b a n c e was d e t e r m i n e d a t 62 0 nm i n a B a u s c h a n d Lomb S p e c t r o n i c 70 s p e c t r o p h o t o m e t e r . I n c o r p o r a t i o n o f L e u c i n e , U r i d i n e a n d T h y m i d i n e M i n c e d t i s s u e s w e r e i n c u b a t e d i n 1 m l K r e b s - E g g l e s t o n b i c a r -14 b o n a t e b u f f e r (KEB) c o n t a i n i n g e i t h e r 0.14 p C i l e u c i n e - C, 3 3 0.46 p C i u r i d i n e - H o r 0.46 y C i t h y m i d i n e - H. I n c u b a t i o n s w e r e c a r r i e d o u t f o r 1 h , t h e n t h e s a m p l e s w e r e c h i l l e d o n i c e a n d h o m o g e n i z e d i n a S o r v a l l O m n i m i x e r . An a l i q u o t o f 0.1 m l was t h e n r e m o v e d f o r p r o t e i n d e t e r m i n a t i o n by t h e m e t h o d o f L o w r y , R o s e -b r o u g h , F a r r a n d R a n d a l l ( 7 6 ) , t h e n 0.1 m l 30% TCA was a d d e d . The s a m p l e was c e n t r i f u g e d , t h e p r e c i p i t a t e w a s h e d 4x w i t h 3 m l 3% TCA a n d s o l u b i l i z e d i n BBS-3 s o l u b i l i z e r . E c o n o f l u o r s c i n -t i l l a t i o n f l u i d , 10 m l , was t h e n a d d e d a n d t h e s a m p l e a s s a y e d f o r r a d i o a c t i v i t y . The i n c o r p o r a t i o n o f l e u c i n e , u r i d i n e a n d t h y m i d i n e w i t h t i m e . i s . i l l u s t r a t e d i n t h e ; c a s e • o f f e t a l . b r a i n J i n T a b l e 1. I t c a n be s e e n t h a t t h e i n c o r p o r a t i o n o f l e u c i n e i s l i n e a r w i t h t i m e t h r o u g h o u t t h e p e r i o d o f i n v e s t i g a t i o n . I n c o r p o r a t i o n o f u r i d i n e i s m a x i m a l a t 30 m i n , w h i l e t h a t o f t h y m i d i n e i s i n -c r e a s i n g b u t n o t n e c e s s a r i l y l i n e a r . The p u l s e t i m e c h o s e n f o r t h e s e e x p e r i m e n t s was 1 h , d u r i n g w h i c h t i m e t h e i n c o r p o r a t i o n o f t h e t h r e e i s o t o p e s was o f s u f f i c i e n t m a g n i t u d e t o p e r m i t c o m p a r i s o n o f v a r i o u s t i s s u e s u n d e r t h e s e c o n d i t i o n s . 26 Table 1 SUBSTRATE INCORPORATION INTO FETAL BRAIN WITH TIME I n c o r p o r a t i o n , dpm/mg p r o t e i n o f : Time, min Leucine U r i d i n e Thymidine 30 4176 + 730 1146 + 82 10217 + 1121 ' 60 9058 •+ .564 : 1109:+ 48 ,134 03 r+229 8 90 13410 + 1116 — 21175 + 2454 120 — 1110 + 38 — Each f i g u r e i s a mean + SEM o f fou r samples. O r n i t h i n e Decarboxylase'Assay T i s s u e was homogenized i n 5 v o l of 5 0 mM T r i s - H C l b u f f e r c o n t a i n i n g 0.1 mM EDTA, 5 mM d i t h i o t h r e i t o l , pH 7.3, and the homogenate c e n t r i f u g e d a t 20,0 00 xg f o r 2 0 min. An a l i q u o t of the supernatant was incubated'with a r e a c t i o n mixture c o n t a i n i n g 0.2 mM p y r i d o x a l phosphate, 5 mM d i t h i o t h r e i t o l ,and;..L-ornithine a t c o n c e n t r a t i o n s of 0.03-0.3 mM, i n c l u d i n g e i t h e r 50 n C i of 14 14 . C-D,L o r n i t h i n e or 25 n C i of C-L-ormthme. The t o t a l r e a c -t i o n mixture volume was 0.1 ml. Samples were incubated a t 37°C f o r 30 min i n tubes f i t t e d w i t h c e n t r e w e l l s c o n t a i n i n g 0.1 ml hyamine. The r e a c t i o n was terminated by i n j e c t i n g 0.1 ml of 2 M c i t r i c a c i d through the stopper, and the l a b e l l e d CO^ c o l l e c t e d f o r 30 min. The c e n t r e w e l l s were then removed and p l a c e d i n t o s c i n t i l l a t i o n v i a l s 5 ml A q u a s o l added and assayed f o r r a d i o -a c t i v i t y (77) . 27 Glucose Metabolism i n F e t a l T i s s u e 14 Glucose- C (u) , 0.86 y C i , was incubated w i t h minced t i s s u e and homogenized as d e s c r i b e d f o r l e u c i n e s t u d i e s . A f t e r a d d i -t i o n of TCA, the p r e c i p i t a t e was d i s c a r d e d and the a c i d - s o l u b l e supernatant was e x t r a c t e d 4x with 1 ml p o r t i o n s of e t h y l ether to remove TCA. An a l i q u o t of t h i s s o l u t i o n was reduced i n volume and s p o t t e d onto a , s i l i c a g e l TLC sheet which was developed i n two dimensions: 1) chloroform:methanol:17% NH^OH (2:2:1; v : v ) ; 2) b u t a n o l : a c e t i c acid:water (3:1:1; v : v ) . Autoradiograms of the sheets were prepared, a f t e r which the sheets were sprayed w i t h n i n h y d r i n to d e t e c t amino a c i d s . An a l i q u o t was removed f o r amino a c i d a n a l y s i s i n a Beck-man amino a c i d a n a l y z e r system w i t h UR-30 r e s i n , 0.9 x 52 cm, w i t h N a - c i t r a t e b u f f e r s . Glucose and Fructose-6-PO^ Metabolism i n C y t o s o l P r e p a r a t i o n s F e t a l l i v e r and gut were homogenized i n 0.14M KC1 i n a P o t t e r - E l v e h j e m T e f l o n homogenizer, and the sample was c e n t r i -fuged a t 105,000 xg f o r 30 min. The supernatant was mixed w i t h an 14 14 equal volume of KEB c o n t a i n i n g e i t h e r C-glucose (u) or C-fructose-6-PO^ (u), 0.1 yCi, and incubated f o r 30 min a t 37°C. At the end of t h i s p e r i o d , 0.1 v o l 3 0% TCA was added and the 28 p r e c i p i t a t e removed by centrifugation. The supernatant was extracted 4x with ethyl ether to remove TCA and was evaporated down and spotted onto Whatman No. 1 f i l t e r paper and the chromatogram developed i n butanol:acetic acidrwater (40:4:10; v:v) for 96 h or more. Zones were v i s u a l i z e d by spraying with 2% a n i l i n e i n ethyl ether-and.Cheating the chromatograms at 100°C for 10 min. Incorporation of Glucosamine The tissue was incubated and homogenized as described for leucine studies. After homogenization, 0.4 ml 5N PCA was added. The tissue was incubated for 1 h i n 1.6 ml KBB containing 14 0.2 yCi C-glucosamine. The p r e c i p i t a t e was then washed 4x with 3 ml 0.5N PCA, 3x with methanol:chloroform:ethyl ether (1:1:1; v:v) and s o l u b i l i z e d and counted, as described above. STEROID RECEPTOR ASSAYS Cytosol Receptor Preparation and Assay Fe t a l brain was removed, minced and washed 3x with i c e - c o l d KEP. The tissue was homogenized i n 5 ml cold KEP and centrifuged f i r s t at 1200 xg, to remove much debris, then recentrifuged at 105,000 xg i n a Beckman L-5 65 model ultracentrifuge for 1 h. The cytosol obtained was used i n subsequent assays. To 0.5 ml cytosol was added 1.5 ml of a solution containing 8 nM 29 lab e l l e d steroid. In some instances, t h i s solution contained 2 yM 11-epicorticosterorie which has been found to eliminate much of the non-specific binding (78, 79). The assays were carri e d out at 0°C for 30 min and stopped by the addition of 40 mg dextran-coated charcoal to remove unbound steroi d . Samples were centrifuged at 1200 xg to pack the charcoal, and an aliquot was removed, mixed with BBS-3 s o l u b i l i z e r and Econo-f l u o r , and assayed for r a d i o a c t i v i t y . Protein determinations were made on the cytosol and the r e s u l t s were expressed as dpm steroid bound/mg protein. Nuclear Preparation and Assay Nuclei from f e t a l brain and placenta were prepared by f i r s t homogenizing the tissue i n a Potter-Elvehjem Teflon homogenizer i n 2 ml KEP containing 2.8 mM glucose. The radio-active steroid, 2 yCi, dissolved i n no more than 20 y l ethanol, was added and the sample l e f t on i c e for 15 min with occasional ag i t a t i o n . The sample was then incubated at 37°C for 10 min i n a shaking water bath. At the end of t h i s time, the sample was c h i l l e d on i c e and 50 ml cold 1.5 mM MgCl 2 added. This pro-cedure has been found to shatter c e l l s , leaving nuclei i n t a c t i n many tissues. The nuclei were harvested by centrifugation at 1200 xg at 0-4°C and washed 2x with MgCl 2 solution. The i s o l a t e d nuclei were then disrupted by homogenizing i n 2 ml cold T r i s buffer, pH 8.0, by grinding i n a glass Ten Broeck homogenizer.' The preparation was then centrifuged at 105,000 xg for 15 min. An aliquot of supernatant, usually 0.5' ml, was placed on a column 30 of Sephadex G-25, 11 x 180 mm, and eluted using T r i s buffer, pH 8.0, at 4°C. Fractions of 1 ml were c o l l e c t e d using an LKB f r a c t i o n c o l l e c t o r , monitored by absorption at 280 nm. Assay of Radioactivity A quench curve for the determination of the e f f i c i e n c y of 3 14 counting for H and C i s shown i n F i g . 1. I t was checked at least once every month. The calculations for the double isotope analysis were done according to the method of Kobayashi and Maudsley (73). 31 FIGURE 1. QUENCH CURVE FOR AND 1 4 C 1.0 2.0 3.0 4.0 5.0 EXTERNAL STANDARD RATIO EXPERIMENTAL RESULTS 1 4 Metabolism of C-labelled Corticosterone  and 11-dehydrocorticosterone i n Fetal Tissues The metabolism of cpd. A and B i n f e t a l head, l i v e r and placenta on various gestational days was examined. 14 Tissues were incubated with either C-labelled cpd. A or B and C t ^ C ^ extracts of tissues were spotted onto s i l i c a gel TLC sheets and developed i n , " f i r s t , hexane:ethyl acetate (4:1) and, second, toluene:chloroform:methanol:water (120:60:20:1). Autoradiograms were then prepared. In F i g . 2 the pattern of corticosterone metabolism i n f e t a l head changes remarkably with gestational age. There i s a decreased conversion of the active cpd. B to the inactive 11-dehydro metabolite; i . e . , there i s a decrease in dehydrogenation with increasing gestational age. On day 14, a large number of metabolites can be observed which have disappeared by day 15. These metabolic changes were found to occur i n the brain, rather than i n adjoining structures of the s k u l l . The zones have been i d e n t i f i e d as follows: the fastest moving zone was i d e n t i f i e d by c o - c r y s t a l l i z a t i o n with authen-t i c added c a r r i e r as 11-dehydrocorticosterone acetate (Table 2) and the zone d i r e c t l y behind i t as corticosterone acetate. The slower moving derivatives have been i d e n t i f i e d F i g . 2: Autoradiogram of chromatographed extracts of f e t a l brain 14 incubated with C-corticosterone. .1. and 2: Gestational day 14; 3 and 4: day 15; 5 and 6: day 17. The zones and the average counts recovered i n each on day 14 are: AAc: 11-dehydrocorticosterone acetate 364 dpm BAc: corticosterone acetate 44 3 A: 11-dehydrocorticosterone 3631 B: corticosterone 5 35 20aA: 20a-dihydro-11-dehydrocorticosterone 666 20aB: 20a-dihydrocorticosterone 344 Note the increase i n size of the B zone and decrease i n size of the A zone i n 3 to 6, as compared with 1 and 2. 34 35 t e n t a t i v e l y as the 20a-dihydro derivatives of 11-dehydro-corticosterone and corticosterone (the slowest zone). Reference 2 0ct-dihydro compounds were prepared by incubating non-radioactive corticosterone with a dialyzed 105,000 xg supernatant of mouse l i v e r f o r t i f i e d with NADPH, and separating the products chromatographically, as described by Krehbiel, Burton and Darrach ( 8 0 ) . The r a d i o a c t i v i t y of the metabolites and t h e i r acetate derivatives coincided during chromatography with the UV absorbance of the added reference compounds and could be distinguished from t h e i r 20 3 isomers. Table 2 CHARACTERIZATION OF 11-DEHYDROCORTICOSTERONE (AS THE C-21-ACETATE) FROM BRAIN BY CO-CRYSTALLIZATION WITH AUTHENTIC CARRIER Dpm/mg Second c r y s t a l s 4 49 Fourth c r y s t a l s 541 Fourth mother liqu o r 4 84 The reduction of cpd. A to cpd. B i n f e t a l mouse l i v e r and placenta on gestational days 14 and 17 i s i l l u s t r a t e d i n Fig. 3. Although the reducing capacity of the placenta remains high throughout gestation, the l i v e r appears to al t e r from a dehydrogenating tissue on day 14 to a reducing tissue by day 17. It i s of in t e r e s t to note here that i n man the placenta has been shown to be the s i t e of dehydro-genation ( 3 9 ) , whereas i n f e t a l mouse the placenta i s F i g . 3: Autoradiogram of chromatographed e x t r a c t s of mouse 14 f e t a l l i v e r and p l a c e n t a xncubated w i t h C-11-dehydrocorti-costerone . A: 1 1 - d e h y d r o c o r t i c o s t e r o n e : B: c o r t i c o s t e r o n e . 1, f e t a l l i v e r , day 14; 2, f e t a l l i v e r , day 17; 3, p l a c e n t a , day 14; 4, p l a c e n t a , day 17. Note the i n c r e a s e i n the s i z e of B zone i n l i v e r on g e s t a t i o n a l day 17. 37 38 d e f i n i t e l y a s i t e of reduction, and the . brain appears to be the major s i t e of dehydrogenation. 2 . .The Metabolism of Corticosterone and 11-dehydrocorticosterone i n Fetal Tissues on Different Gestational Days" The rela t i o n s h i p between the dehydrogenation of cpd. B to cpd. A and the reduction of the l a t t e r to cpd. B can •:. .. have great e f f e c t upon the t o t a l amount of b i o l o g i c a l l y active hormone i n a p a r t i c u l a r f e t a l t i s s u e . In order to examine the metabolic fate of these two compounds, placenta 14 and f e t a l tissues were incubated simultaneously with ' i C-3 l a b e l l e d cpd. B and H-labelled cpd. A. Tissues were examined on gestational days 1 4 , 16 and 19 to observe the a c t i v i t y of the reversi b l e C-11 s t e r o i d oxidoreductase. 3 14 H-cpd. A and C-cpd. B were incubated simultaneously. The products were separated chromatographically, eluted and 1 4 assayed for r a d i o a c t i v i t y . The amount of C l a b e l i n the cpd. A zone would indicate dehydrogenase a c t i v i t y , and con-3 versely the amount of H la b e l i n the cpd. B zone indicates reductase a c t i v i t y . From these data the percentage of reduc-t i o n and dehydrogenation could be calculated, and the net , ,. reduction _, reaction expressed as a r a t i o of -, , , rr- • The c dehydrogenation results for placenta and various f e t a l tissues are presented i n Figs. 4 to 6 . 39 FIGURE 4, IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE IN FETAL BRAIN AND GUT, 1.0 u Each p o i n t i s the mean of a t l e a s t f i v e d e t e r m i n a t i o n s + SEM. The d i f f e r e n c e between day 14 and day 16 i s , f o r gut P<.05, f o r b r a i n P<.01. Values f o r day 16 vs day 19 d i f f e r s i g n i f i c a n t l y , P<.01. — b r a i n — A ; gut 40 FIGURE 5 , IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE IN PLACENTA. 3 0 O CD — o i - cn u Q ZJ > a x L U L U 2 0 < cm 10 14 16 18 GESTATIONAL DAY P n r K S JSh ^ P O i n , ± S t h e m e a n o f a t l e a s t e i 9 h t va lues + SEM. Trl s ^ n i r i L n t ^ f o L 1 6 ' d a y 1 6 ^ d ^ 1 9 t h e d i f f e r e n c e s 41 FIGURE 6. IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE IN FETAL LIVER AND LUNG, 10 Q UJ cd=3 LU IS Q >-X LU < / l AY 14 16 GESTATIONAL DAY 18 Each p o i n t i s the mean o f a t l e a s t f i v e v a l u e s . Some SEM are shown. For both day 14 vs day 16, and f o r day 16 1 vs day 19, the d i f f e r e n c e s are s i g n i f i c a n t , P<.01. l i v e r - — — ^ ; l u n g o • 42 Fig. 4 shows that i n both f e t a l brain and gut, dehydrogenation predominates. This i s indicated by a .3 r f d u c t l ° " r a t i o of less than 1.0. Both tissues, show dehydrogenatxon a decrease i n dehydrogenase a c t i v i t y with..increasing.^ .. gestational age, as indicated, by a r a t i o approaching 1.0. In F i g . 5, placenta i s shown to be an active s i t e of reduction of cpd. A. This reducing capacity increases with gestational age. Fig . 6 shows that i n both l i v e r and lung on day 14, dehydrogenation p r e v a i l s . The dotted l i n e indicates a reduction r a t i o of 1.0, below which dehydrogenation dehydrogenation predominates and above which r e d u c t i o n predominates. By day 16, the l i v e r i s a c t i v e l y r e d u c i n g , while the lung i s s t i l l dehydrogenating. But by day 19, both are r e d u c i n g t i s s u e s . T h e r e f o r e , l i v e r and lung have d i f f e r e n t t i m i n g i n the development of reductase a c t i v i t y , the lung being l a t e r . The h e a r t was a l s o examined f o r C-11 s t e r o i d oxido-reductase a c t i v i t y , but none was observed. Smith, Torday and Giroud (81) have r e p o r t e d i n d u c t i o n of C-11 s t e r o i d oxidoreductase a c t i v i t y w i t h C o r t i s o l i n lung c e l l s taken from a human f e t u s a t m i d g e s t a t i o n . T h e r e f o r e , i t was an i n t e r e s t i n g p o s s i b i l i t y t h a t i n j e c t i o n 43 of dexamethasone might stimulate enzyme a c t i v i t y i n the f e t a l mouse. Table 3 shows that no e f f e c t on enzyme a c t i v i t y i n any tissue was observed when mothers were injected on day 13.5, 16 hours p r i o r to the i n v i t r o assay. When animals were treated on day 15.5 and assayed on day 16 (Table 4), s i g n i f i c a n t e f f e c t s were found i n both placenta (P<.01) and lung (P<.05). Therefore, i n these tissues c o r t i c o -sterone would appear to influence i t s own formation by the enzyme C-11 steroi d oxidoreductase and can be considered to be "autocatalytic" i n these tissues. I t should be noted here that the synthetic s t e r o i d dexamethasone was used in these experiments because i t does not bind to maternal transcortin and also escapes metabolic pathways i n the fetus (82). Therefore, a more e f f e c t i v e dose can cross the placenta and exert a b i o l o g i c a l e f f e c t . 44 Table 3 EFFECT OF DEXAMETHASONE INJECTION ON THE ACTIVITY OF STEROID C - l l OXIDOREDUCTASE ON GESTATIONAL DAY 14 Reduction Ratio Dehydrogenation Gestational Day Tissue Day 14 Control Day 14 Dex Placenta 6. 30±1.."0 8.26±1. 9 (8) (8) Brain 0.06±0.003 0.07±0. 003 (10) (10) Gut 0.10±0.01 0.12±0. 02 (5) (5) Liver 0.29±0.07 0.34±0. 09 (5) (5) Lung 0.16±0.02 0.25±0. 06 (5) (5) Each figure i s a mean + SEM of the number of samples shown i n parentheses. None of the above treated d i f f e r e d s i g n i f i c a n t l y from control values. 45 Table 4 EFFECT OF DEXAMETHASONE INJECTION ON THE ACTIVITY OF STEROID C - l l OXIDOREDUCTASE ON GESTATIONAL DAY 16 Reduction ~ .. ^ . , , . Ratio Dehydrogenation Gestational Day Tissue Day 16 Control Day 16 Dex Placenta 16.7±0.82 24.5+2.0 (8) (11) Brain 0.30±0.04 ."0.23+0.01 (8) (8) Gut 0.17+0.03 0.13+0.05 (6) (7) Liver 3.54+0.65 2.99±0.45 (6) (9) Lung 0.29±0.02 0.51±0.08 (6) (6) Each figure i s the mean ± SEM of the number of samples shown i n parentheses. The difference between treated and control was s i g n i f i c a n t i n the case of placenta (P<0.01) and lung (P<0.05). 46 3. Recovery of cpd. A and B a f t e r Injection 3 of Mothers with H-cpd. B 3 H-cpd. B was injected into the mother mice on gesta-t i o n days 14, 16 and 19, and 15 min l a t e r the placenta and f e t a l tissues were removed, extracted, chromatographed and the r a d i o a c t i v i t y i n both cpd. A and B zones assayed. Figs. 7 and 8 show that the percentage of cpd. B found i n a l l f e t a l tissues increases with gestational age. Since the in. v i t r o studies (Figs. 4-6) show that i n some tissues there was a decrease i n dehydrogenase a c t i v i t y and i n others an increase i n reducing capacity, with increasing gestational age, an increase i n cpd. B content would be expected. Therefore, a tissue might control the hormone content either by a c t i v e l y reducing the 11-dehydro meta-b o l i t e to cpd. B, or by decreasing the rate of conversion of cpd. B to i t s inactive 11-dehydro metabolite. 4. Glycogen Deposition in Fetal Tissues It would seem reasonable that, since cpd. B i s regu-lated so that the amount present i n each tissue varies considerably with gestational age, the hormone might have s i g n i f i c a n t metabolic e f f e c t s i n these tissues. In order to examine t h i s p o s s i b i l i t y , parameters were sought which might be sensitive indicators of corticosterone action. One p o s s i b i l i t y considered was glycogen deposition i n FIGURE 7, RECOVERY OF CPD A AND B IN FETAL BRAIN/ GUT AND HEART AFTER INJECTION OF CPD B~ H. 75 L C D C D 50 CQ a. Q + < 25 16 GESTATIONAL DAY 18 shown 1 / ^ 1 the mean of f o u r v a l u e s . Some SEM are 1 6 ^ £ H f S t h G d i f f e r e n c e between day 14 vs day 16, and day 16 vs day 19 are s i g n i f i c a n t , P<.01 ~ h e a r t i b r a i n ^ ; g U t •-48 FIGURE 8, RECOVERY OF CPD A AND B IN FETAL LIVER, LUNG AND PLACENTA AFTER INJECTION OF CPD B~^H. GESTATIONAL DAY Each p o i n t i s the mean of fo u r v a l u e s . Some SEM are shown. The d i f f e r e n c e between day 14 and day 16 i n the case of l i v e r and lung are s i g n i f i c a n t , P<.01; p l a c e n t a shows no s i g n i f i c a n t d i f f e r e n c e . Values f o r day 16 vs day 19 d i f f e r s i g n i f i c a n t l y i n a l l c a s e s , P<.01. l i v e r A — — ._; l ung • o ; p l a c e n t a 49 f e t a l tissues, since t h i s i s the only parameter thought to be influenced by corticosteroids at t h i s early stage of gestation. Fi g . 9 shows the standard curve for the anthrone reaction. Although i t deviates s l i g h t l y from l i n e a r i t y above 0.6 mg, the determination on samples was made i n the l i n e a r range. Figs. 10 and 11 show that the pattern of deposition does not r e f l e c t the reductase pattern of deposition observed previously (Figs. 4-6) and therefore glycogen cannot be considered to represent a useful parameter of steroid action. The e f f e c t of i n j e c t i o n of dexamethasone 16 h e a r l i e r on the deposition of glycogen i n various tissues on gesta-t i o n a l day 16 i s shown i n Table 5. Only l i v e r glycogen was stimulated s i g n i f i c a n t l y (P<.01); placenta showed a de-crease (P<.01). Injections on day 13.5 followed by assays on day 14 showed no s i g n i f i c a n t increase i n l i v e r . This would imply that glycogen deposition i n l i v e r might depend on factors other than c o r t i c o s t e r o i d s . During the glycogen i s o l a t i o n procedure, removal of glycoprotein i s accomplished by PCA p r e c i p i t a t i o n as described by Roe and Dailey (75). Since the amount of th i s p r e c i p i t a t e appeared to vary with both tissue and FIGURE 9. STANDARD CURVE FOR THE DETERMINATION OF GLYCOGEN BY THE ANTHRONE METHOD. 2.0 1.5 1.0 0.5 MG GLYCOGEN 51 FIGURE 10. GLYCOGEN CONTENT OF FETAL BRAIN, GUT AND HEART ON GESTATIONAL DAYS 14 TO 19. GESTATIONAL DAY Each p o i n t i s the mean o f a t l e a s t f o u r d e t e r m i n a t i o n s . Some SEM are shown. he a r t » •« ; b r a i n i ^ - — — A ; gut • 52 FIGURE 11, GLYCOGEN CONTENT OF FETAL LIVER, LUNG AND PLACENTA ON GESTATIONAL DAYS 14 TO 19, 6 0 i _ 2 4 0 LU LU LU 13 O U >-_J (3 £ 2 0 j 16 GESTATIONAL DAY 18 Each p o i n t i s the mean of a t l e a s t four determinations, Some SEM are shown. l i v e r * ; lung o ci ; p l a c e n t a o 53 Table 5 EFFECT ON GLYCOGEN DEPOSITION IN FETAL TISSUES OF DEXAMETHASONE INJECTIONS INTO MOTHERS Tis s u e Day 16 C o n t r o l Day 16 Dex P l a c e n t a 12 82+.83 9. 02+. 33,: .:. (6) (14) Lung 8 .51+.17 8. 39+. 81 (4) (7) L i v e r 0 .32+.05 3. 64 + . 64 (7) (8) Gut 1 .93+.20 2 51 + . '36 (9) (5) B r a i n 4 .16+.30 4 76+. 10 (4) (11) Heart 21 .74+.76 22 .59+3 .52 (6) (5) Each i s the mean + SEM of the number of samples shown i n parentheses. The d i f f e r e n c e i n t r e a t e d and unt r e a t e d was s i g n i f i c a n t i n the case of l i v e r and p l a c e n t a (P<.01). 54 gestational age, further characterization w i l l be dealt with l a t e r . 5. The In V i t r o Incorporation of Leucine, Uridine  and Thymidine into Fetal Tissues Several groups have examined c o r t i c o s t e r o i d e f f e c t s on f e t a l lung and have reported a decrease i n the rate of c e l l d i v i s i o n induced by c o r t i c o s t e r o i d s ( 8 3 , 8 4 ) . This implies that the action of c o r t i c o s t e r o i d s on tissue might be r e f l e c t e d i n c e l l d i v i s i o n , synthesis and d i f f e r e n t i a -t i o n . It has been shown that action of c o r t i c o s t e r o i d s on lymphocytes resulted in decreased incorporation of radio-active leucine, uridine and thymidine ( 8 5 ) . As these are parameters of protein RNA and DNA synthesis, the i n v i t r o incorporation of these substrates was therefore examined on gestational days 14 to.19 i n placenta and f e t a l t i s s u e s . As can be observed i n Figs. 12 and 1 3 , there occurred i n a l l tissues a s i g n i f i c a n t decrease i n leucine incorpora-t i o n from day 14 to day 1 9 . However, heart (Fig. 12) and lung (Fig. 13) remained constant between days 14 and 16 (P value not s i g n i f i c a n t ) , but decreased s i g n i f i c a n t l y a f t e r day 1 6 . Uridine incorporation (Figs. 1 4 - 1 5 ) i n most tissues showed a decline between days 14 and 1 9 , although heart (Fig. 14) increased s i g n i f i c a n t l y from day 14 to 16 ( P < . 0 1 ) . FIGURE BRAIN/ 12, GUT IN VITRO INCORPORATION OF 1 4C-LEUCINE INTO FETAL AND HEART. GESTATIONAL DAY Each p o i n t i s the mean o f f i v e t o twelve d e t e r m i n a t i o n s + SEM. The d i f f e r e n c e i s s i g n i f i c a n t f o r b r a i n and gut day 14 vs day 16, P<.01; f o r day 16 vs day 19 b r a i n and gut P<.01 and h e a r t P<.05. b r a i n s ™ t ; g u t * — — * ; h e a r t " * 56 FIGURE 13. IN VITRO INCORPORATION OF -^C-LEUCINE INTO FETAL LIVER, LUNG AND PLACENTA. 125 Q LU I-< C s l cn I o c: CL r — cc O LU o ZD LU I I x 5 5 75 LU \— o cn CL. CL. 2 5 16 GESTATIONAL DAY 18 Each p o i n t i s the mean of f i v e to twelve d e t e r m i n a t i o n s + SEM. The d i f f e r e n c e i s s i g n i f i c a n t f o r p l a c e n t a and l i v e r day 14 vs day 16, P<.01; f o r day 16 vs day 19 lung and l i v e r , P<.01 and p l a c e n t a i s not s i g n i f i c a n t . l i v e r A — — p l a c e n t a o o; lungo^ FIGURE IN VITRO INCORPORATION OF ^H-URIDINE INTO FETAL BRAIN, GUT AND HEART. 8 0 Q LU I - CN) < I cm o o i—I CL. CC X O C_> Z Z —• i — i LU LU Q I—« a: 2 4 0 CL 5 ^ •i-14 16 GESTATIONAL DAY 18 Each p o i n t i s the mean of f i v e t o twelve de t e r m i n a t i o n s + SEM. The d i f f e r e n c e i s s i g n i f i c a n t f o r day 14 vs day 16 b r a i n and gut P<.01 and h e a r t P<.02; f o r day 16 vs day 19 gut and h e a r t P<.01. b r a i n / * - — g u t * • ; h e a r t • 58 FIGURE 15. IN VITRO INCORPORATION OF ^H-URIDINE INTO FETAL LIVER, LUNG AND PLACENTA. GESTATIONAL DAY Each p o i n t i s the mean of f i v e to twelve d e t e r m i n a t i o n s + SEM. The d i f f e r e n c e i s s i g n i f i c a n t f o r day 14 vs day 16 l i v e r P<.05 and p l a c e n t a P<.01; f o r day 16 vs day 19 l i v e r P<.01 and lung P<.02. l i v e r A — — p l a c e n t a o o; lung D a 59 Thymidine incorporation i n most instances showed a decreasing pattern of incorporation with increasing gesta-t i o n a l age (Figs. . 1 6 - 1 7 ) . Again, lung was an exception--as was the case i n leucine i n c o r p o r a t i o n — t h e values for day 14 to 16 did not differ... s i g n i f i c a n t l y , but the incor-poration between days 16 and 19 did (P< . 0 1 ) . The decline i n incorporation of the three substrates after day 14 i n brain, l i v e r , placenta and gut, and after day 16 i n heart and lung, -could well be interpreted as r e f l e c t i n g increased corticosterone i n these tissues at these times (Figs. 7 - 8 ) . These parameters appear to o f f e r a much better i n d i -cation of e f f e c t s which are taking place which might r e f l e c t changes i n hormone l e v e l i n these tissues. To confirm t h i s , the synthetic c o r t i c o i d dexamethasone was injected on gestational day 1 3 . 5 and 16 h l a t e r the tissues were removed and assayed for isotope incorporation. Leucine values (Table 6) were decreased s i g n i f i c a n t l y (P<.0.1.) i n a l l tissues except lung and heart. This f o l -lows the pattern observed for lung and heart which was shown not to a l t e r between gestational days 14 and 1 6 . I t should be noted that the Dex e f f e c t appears to be stimula-t i n g natural development prematurely; i . e . , the same general trends as would normally occur i n each tissue are followed but are accelerated. 60 FIGURE 16. IN VITRO INCORPORATION OF 3H-THYMIDINE INTO FETAL BRAIN, GUT AND HEART. 4 0 0 Q LU I-< DC CM O I CL O DC i—I O O X LU LU 2: I— — o Q CC —> CL > - CD a. 3 0 0 2 0 0 100 14 16 18 GESTATIONAL DAY Each p o i n t i s the mean o f f i v e t o t w e l v e d e t e r m i n a t i o n s + SEM. The d i f f e r e n c e i s s i g n i f i c a n t f o r day 14 vs day 16 a l l t i s s u e s , P<.01; f o r day 16 vs day 19 b r a i n ancTgut P<.01 and h e a r t P<.05. — h e a r t ; b r a i n A - — — A ; gut 61 FIGURE 17. IN VITRO INCORPORATION OF 3H-THYMIDINE INTO FETAL LIVER, LUNG AND PLACENTA. Q UJ t -< cn CM 0 I 01 CD cn i — i o LU LU —' o Q cn <-> C L C L 4 0 0 3 0 0 2 0 0 100 HI-A \ \ \ \ .....y \ \ i \ \ 14 16 18 GESTATIONAL DAY Each p o i n t i s the mean of f i v e to twelve d e t e r m i n a t i o n s + SEM. The d i f f e r e n c e i s s i g n i f i c a n t f o r day 14 vs day 16 i n a l l t i s s u e s P<.01; f o r day 16 vs day 19 lung and l i v e r P<.01 and p l a c e n t a P<.05. l i v e r s — — p l a c e n t a o — o ; lung » « 62 Uridine values (Table 7) were decreased s i g n i f i c a n t l y i n l i v e r , placenta and brain (P<.01). No s i g n i f i c a n t e f f e cts were observed i n the other tissues, although i n gut a s l i g h t decrease was observed. The incorporation of thymidine (Table 8) showed s i g -n i f i c a n t decreases i n a l l tissues (P<.01). Thymidine appears to be the most sensitive parameter, since i n a l l instances Dex i n j e c t i o n resulted i n s i g n i f i c a n t l y decreased incorporation. Liver appears to be the most sensitive tissue, since i t was q u a n t i t a t i v e l y the most affected by Dex i n j e c t i o n by a l l parameters' (Tables 6-8) . Table 9 summarizes the change i n the r a t i o of reduction: dehydrogenation with increasing gestational age i n various f e t a l tissues. I t should be noted that either by a decrease i n dehydrogenation (Type 1), or an increase i n reduction (Type 2) an increased corticosterone content can be produced i n a p a r t i c u l a r tissue. The relationship i n time between the changes i n the incorporation of various substrates and the r e l a t i v e amount of corticosterone i s evident. 63 Table 6 EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON LEUCINE INCORPORATION IN FETAL TISSUES Leucine Incorporation Dpm/mg protein Tissue Day 14 Control Day 14 Dex Placenta 759±96 465±40 Lung 6,6741546 6,947±1,074 Liver 10,810±639 6,693±568 Gut 14,62711,017 9,248±900 Brain 9,900±451 5,083±559 Heart 3,057±401 3,3431553 Each value i s the mean of at least f i v e determinations + SEM. Values for treated placenta, l i v e r , gut and brain d i f f e r s i g n i f i c a n t l y from controls (P<.01); lung and heart do not. 64 Table 7 EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON URIDINE INCORPORATION IN FETAL TISSUES Uridine Incorporation Dpm/mg protein Tissue Day 14 Control Day 14 Dex Placenta 1017+44 725+105 Lung 4310+879 4150+628 Liver 3138+481 1758+99 Gut 4578+610 3245+497 Brain 1668+81 1213+107 Heart 6383+290 4857+686 Each value i s the mean of at least f i v e determinations + SEM. Values for treated placenta, brain and l i v e r d i f f e r s i g n i f i c a n t l y (P<.01) from controls; gut, lung and heart do not. 65 Table 8 EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON THYMIDINE INCORPORATION IN FETAL TISSUES Thymidine Incorporation Dpm/mg protein Tissue Day 14 Control Day 14 Dex Placenta 1,040+132 641+55 Lung 18,137+941 12,258+1,297 Liver 36,603+2,210 12,244+2,504 Gut 41,177+3,215 18,138+1,853 Brain 19,283+557 16,009+511 Heart 11,909+953 4,746+993 Each value i s the mean of at lea s t f i v e determinations - SEM. In a l l cases the difference between treated and control values i s s t a t i s t i c a l l y s i g n i f i c a n t (P<.01). 66 Table 9 SUMMARY OF FETAL TISSUE REDUCTASE (R) AND DEHYDROGENASE (D) ACTIVITIES TYPE 1 TYPE 2 TYPE 3 G e s t a t i o n a l Day Ti s s u e Day 14 Day 16 Day 19-B r a i n D + D Gut D •fD + + D L i v e r D R + R Lung D + D R P l a c e n t a R + R + + R + denotes decrease; f denotes i n c r e a s e T Y P E 1 T Y P E 2 BEAD) DEHYDROGENATION RATIO< 1 DAY lk BLOOD \ DAY 14 REDUCTION RATIO > 1 A DAY 19 DAY 16 L I V E R See t e x t p. 6 2 6.7 6 . Ornithine Decarboxylase A c t i v i t y i n Placenta The a c t i v i t y of the enzyme ornithine decarboxylase has been reported to be a sensitive indicator of the response of tissues to various, ^ hormonal s t i m u l i . An increase i n a c t i v i t y of t h i s enzyme invariably accompanies increased c e l l d i v i s i o n ( 7 7 ) . Ornithine decarboxylase a c t i v i t y has been shown to decline during development i n f e t a l r a t l i v e r (86) and placenta (77) . I t was of i n t e r e s t , therefore, to examine a c t i v i t y during gestation i n the mouse. The a c t i v i t y decreased i n mouse placenta with increasing gestational age (Fig. . 18) . The i n j e c t i o n of dexamethasone also produced prematurely on day 14 values normally observed between days 1 7 - 1 8 (Fig. 18) . . Ornithine decarboxylase was then assayed i n f e t a l brain, but low a c t i v i t y was observed. No further studies were made, since i t did not appear that t h i s would be as useful a parameter for the tissues under inves t i g a t i o n . However, as indicated by the data on placenta, the d e c l i n -ing rates of several processes, including the incorporation of leucine (Fig. 13), uridine (Fig. 1 5 ) , thymidine (Fig. 17) and ornithine decarboxylase a c t i v i t y (Fig. 1 8 ) , a l l appear to be mediated by c o r t i c o s t e r o i d . 68 FIGURE 18, ORNITHINE DECARBOXYLASE ACTIVITY GESTATIONAL DAYS 14 TO 19 IN PLACENTA AND THE EFFECT OF DEXAMETHASONE INJECTION. Each p o i n t i s the mean o f f i v e t o e i g h t d e t e r m i n a t i o n s + SEM. The Dex va 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 day 14 c o n t r o l (P<.01). 69 7. Amino Acid Analysis of Fetal Brain At the outset of t h i s investigation, i t was a n t i c i -pated that, since glucose i s the major organic substrate reaching the fetus, changes i n i t s d i s p o s i t i o n might r e f -l e c t events i n a tissue which are..influenced by c o r t i c o -steroids. In fact, t h i s expectation was not r e a l i z e d i n the case of glycogen deposition (Figs. 10-11); however, a number of i n t e r e s t i n g observations were made which lend credence to the concept that c o r t i c o s t e r o i d s play an impor-tant r o l e i n f e t a l development at t h i s early stage of gestation. Fetal brain on gestational day 14, with and without p r i o r treatment with dexamethasone, and on day 15 were 4 incubated with C-glucose (u), and the acid-soluble con-stituents separated on TLC sheets, developed i n two dimen-sions. Six samples of each were examined, and autoradio-grams were prepared. These indicated decreased conversion of glucose to a number of other constituents i n the treated samples and those on day 15. When sprayed with ninhydrin, a substantial increase i n several ninhydrin-staining com-ponents were also observed on these sheets. This was examined further using an amino acid analyzer, the r e s u l t s of which are i l l u s t r a t e d i n F i g . 19. S i g n i f i c a n t increases are evident on day 15 as compared with day 14, i n the a c i d i c amino acids glutamic, aspartic arid alanine, threonine and tyrosine. Few basic amino acids were present, and the 70 l e v e l of l e u c i n e was e q u a l l y low i n a l l . Treatment with Dex produced i n samples of g e s t a t i o n a l day 14 a p a t t e r n i n t e r m e d i a t e between t h a t of day 14 and day 15. 8. I n v e s t i g a t i o n of G l y c o p r o t e i n S y n t h e s i s During the i s o l a t i o n of glycogen ( F i g . 10-11), one step i n the procedure r e q u i r e d the p r e c i p i t a t i o n of g l y c o -p r o t e i n s w i t h a c i d . An unexpectedly l a r g e p r e c i p i t a t e was sometimes observed, which appeared to change with g e s t a t i o n a l age i n some d e f i n i t e p a t t e r n i n each t i s s u e . T h i s suggested p o s s i b l e s t i m u l a t i o n of g l y c o p r o t e i n syn-t h e s i s . To t e s t t h i s , the c a p a c i t y o f f e t a l t i s s u e s to s y n t h e s i z e g l y c o p r o t e i n s was examined. As seen i n Table .10, f e t a l l i v e r and gut both have the r e q u i s i t e 14 enzymes f o r s y n t h e s i z i n g g l y c o p r o t e i n s from C-gluco-samine, and the r e l a t i v e amount i n c o r p o r a t e d was s i m i l a r on g e s t a t i o n a l day 14 and l a t e r i n f e t a l gut. The f i r s t step i n g l y c o p r o t e i n s y n t h e s i s , which i s known to be the r e g u l a t o r y step, i s the s y n t h e s i s of g l u c o -samine. The c a p a c i t y of f e t a l t i s s u e s to c a r r y out t h i s r e a c t i o n was examined i n c y t o s o l p r e p a r a t i o n s of gut and 14 l i v e r incubated w i t h C-fructose-6-PO^, which i s the sub-s t r a t e f o r the enzyme ' Glutarnine-fructose-6-P amino-t r a n s f e r a s e (EC.5.3.1.19). • A c i d - s o l u b l e e x t r a c t s were chromatographed, and autoradiograms were prepared, which i n d i c a t e d no d i f f e r e n c e i n the c a p a c i t y of f e t a l F i g . 19: Amino a c i d a n a l y s i s of 50 v l of samples from f e t a l b r a i n on g e s t a t i o n a l day 14 (1), day 14 Dex-treated (2) and day 15 (3). Cpd.X i n d i c a t e s an u n i d e n t i f i e d c o n s t i t u e n t . 72 < c _ - 1 73 Table 10 INCORPORATION OF 14C-GLUCOSAMINE INTO FETAL LIVER AND GUT Tissue Incorporation, dpm/mg Protein Liver, day 14 Gut, day 14 Gut, days 17-19 768+130 828+54 969+161 Each point i s a mean of at least four determinations + the SEM. 74 t i s s u e s o f d i f f e r e n t g e s t a t i o n a l age t o m e t a b o l i z e t h e s u b -s t r a t e t o a number o f i n t e r m e d i a t e s . When c y t o s o l p r e p a r a t i o n s o f l i v e r a n d g u t w e r e 14 i n c u b a t e d w i t h C - g l u c o s e and a u t o r a d i o g r a m s o f t h e c h r o m a t o g r a p h e d e x t r a c t s w e r e p r e p a r e d ( F i g . 2 0 ) , a s t r i k -i n g d i f f e r e n c e was e v i d e n t b e t w e e n s a m p l e s o f g e s t a t i o n a l day 14 a n d d a y 16 i n t h e c o n v e r s i o n o f g l u c o s e t o f r u c t o s e . L i t t l e f r u c t o s e was f o r m e d on g e s t a t i o n a l day 14, w h e r e a s m o s t o f t h e g l u c o s e was c o n v e r t e d i n s a m p l e s o f l a t e r g e s t a t i o n a l a g e . T r e a t m e n t w i t h Dex p r o d u c e d a p a t t e r n on day 14 s i m i l a r t o s a m p l e s o f l a t e r a g e , a g a i n i n d i c a t i n g t h e i n v o l v e m e n t o f c o r t i c o s t e r o i d i n t h e s e e v e n t s . I n some s a m p l e s , t h e c o n v e r s i o n a p p e a r e d t o p r o c e e d w i t h o u t t h e f o r m a t i o n o f . p h o s p h o r y l a t e d i n t e r m e d i a t e s , w h i c h a p p e a r as t h e more p o l a r c o n s t i t u e n t s . The l a t t e r d i d i n c r e a s e , h o w e v e r , i n D e x - t r e a t e d s a m p l e s on d a y 14; f r o m 1196 t o 2790 dpm. I t i s i n t h i s a r e a t h a t i n t e r m e d i a t e s n e c e s s a r y f o r g l y c o p r o t e i n s y n t h e s i s o c c u r . A t t h i s p o i n t , h o w e v e r , e x a m i n a t i o n o f t h e p r e c i p i -t a t e s o b t a i n e d as " g l y c o p r o t e i n " d u r i n g t h e i s o l a t i o n o f g l y c o g e n , r e v e a l e d t h a t m o s t o f t h e m a t e r i a l p r e s e n t was n o t i n f a c t g l y c o p r o t e i n . The p e r i o d a t e - r e s o r c i n o l r e a c -t i o n f o r b o und s i a l i c a c i d , a m e a s u r e o f g l y c o p r o t e i n c o n -t e n t i n t i s s u e s (87) showed t h a t , w h i l e t h e r e was some g l y c o p r o t e i n p r e s e n t , t h e r e w e r e much l a r g e r q u a n t i t i e s o f m a t e r i a l r e s e m b l i n g d e o x y r i b o n u c l e i c a c i d ( F i g . 2 1 ) . F i g . 20: Autoradiogram of chromatographed e x t r a c t s of f e t a l gut. 1 and 2, day 14; 3 and 4, day 16. Note the i n c r e a s e d amount of f r u c t o s e on day 16. F: f r u c t o s e G: glucose 76 F i g . 21: The p e r i o d a t e - r e s o r c i n o l r e a c t i o n f o r bound s i a l i c a c i d performed on PCA p r e c i p i t a t e s of f e t a l t i s s u e s : 1. B r a i n 2. L i v e r 3. Gut 4. Lung 5. S i a l i c A c i d Standard 78 T 1 r A. (nm) 79 Examination of the absorption pattern of the p r e c i p i t a t e (Fig. 22) also indicated that i t was DNA. 9. Variation i n the Acid I n s o l u b i l i t y of DNA from Fetal Tissues Experiments were ca r r i e d out i n which mothers were 3 injected with H-thymidine and k i l l e d 30 min l a t e r . The tissues were processed as for glycogen determinations, and the acid-insoluble p r e c i p i t a t e was redissolved i n d i l u t e NH^OH and assayed for r a d i o a c t i v i t y . Fetal brain, l i v e r , gut, lung, heart and placenta were included and the re s u l t s expressed as the percentage of t o t a l counts i n the tissue digest which were recovered i n the pr e c i p i t a t e (Fig. 23). The material which did not pr e c i p i t a t e at room temperature was stored at 0-4°C, and upon standing, the remaining DNA eventually precipitated. Treatment of mice with Dex produced changes which indicated that the trend i n each tissue with increasing gestational age was accelerated. The only two tissues which did not show a decrease, placenta and lung, were the two which showed an increase between days 14 and 16, before declining thereafter (Table 11). These observations suggest some difference i n the properties of these macromolecules, the significance of which remains obscure, but which i l l u s t r a t e yet another phenomenon which appears to be influenced at t h i s stage of 8 0 W A V E L E N G T H ( N M ) 81 F I G U R E 2 3 . P R E C I P I T A T I O N OF R A D I O A C T I V I T Y B Y P C A FROM T I S S U E S OF F E T U S E S R E M O V E D FROM M O T H E R S I N J E C T E D W I T H 3 R - T H Y M I D I N E . G E S T A T I O N A L D A Y h e a r t l i v e r ; b r a i n — — ; gut A — — — - A ; p l a c e n t a o o ; l u n g o — — — o 82 Table 11 3 THE EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS GIVEN H-THYMIDINE ON THE PRECIPITATION OF RADIOACTIVE MATERIAL FROM FETAL TISSUES TREATED WITH PCA % of t o t a l r a d i o a c t i v i t y p r e c i p i t a t e d by PCA addition Tissue Day 14 Control Day 14 Dex Liver 100 69 Gut 100 81 Lung 75 78 Heart 100 74 Brain 92 67 Placenta 67 47 Each value i s an average of two to s i x determinations. 83 gestation at least i n part as a r e s u l t of c o r t i c o s t e r o i d action. Studies on Corticosteroid Receptors i n Fetal Tissues While a number of observations have been made i n t h i s investigation which appear worthy of further exploration, the primary aim of t h i s work was to examine the fate of corti c o s t e r o i d s and t h e i r i n t e r a c t i o n with constituents of f e t a l t issues. Attention was therefore redirected to a search for s p e c i f i c c o r t i c o s t e r o i d receptors i n f e t a l tissues and an examination of some of t h e i r properties. Most workers investigating c o r t i c o s t e r o i d receptors have found that endogenous steroids present a problem i n attempting to lab e l receptor s i t e s . This has been approached by adrenalectomizing animals p r i o r to the assay. This i s c l e a r l y not feasible i n the case of f e t a l mice. Also, nearly a l l workers i n t h i s area use synthetic co r t i c o s t e r o i d s with a 9afluoro substituent which i s claimed to bind more e f f e c t i v e l y to receptors. The use of 3 such a compound, H-dexamethasone, has enabled the demon-st r a t i o n of a s p e c i f i c receptor i n f e t a l brain, and calcu-l a t i o n of a constant for the i n t e r a c t i o n (Table 12) . The value of 8.3 nM i s consistent with those reported for such receptors i n other tissues, for a h i g h - a f f i n i t y , low-capacity receptor. The number of binding s i t e s did not increase with increasing gestational age, and, i n fact, 84 Table 12 PROPERTIES OF CORTICOSTEROID RECEPTOR(S) IN FETAL MOUSE BRAIN K d 8 . 3 nM T o t a l s t e r o i d bound 0 .14 pmoles/mg p r o t e i n .{.day 14) R e l a t i v e b i n d i n g (dpm/mg p r o t e i n ) On g e s t a t i o n a l day 14: 6433 + 1109 (6) On g e s t a t i o n a l day 1 7 : 1551 + 345 (5) 85 the concentration per mg protein declined. This was true even when d i t h i o t h r e i t o l , which has been reported to s t a b i l i z e receptors i n some tissues, was included. Because of the abundance of 11-dehydrocorticosterone i n f e t a l t issues, i t was of i n t e r e s t to examine the i n t e r -action of t h i s metabolite and the active hormone with s p e c i f i c c e l l u l a r receptors. I t was found that rapid methods of separating bound and unbound steroid, by gel f i l t r a t i o n on Sephadex G-25 or using dextran-coated charcoal, were s a t i s f a c t o r y , whereas d i a l y s i s for 16 h resulted i n loss of s p e c i f i c binding of these two steroids. Repeated attempts to obtain a Scatchard plo t using cpd. A and B were unsuccessful. I t was concluded that the con-centration of endogenous steroid was probably so high that i t was not possible to achieve s u f f i c i e n t l y low concentra-tions for these determinations. Repeated washing of tissue p r i o r to assay had no s i g n i f i c a n t e f f e c t . It was possible to l a b e l receptor s i t e s , however, and carry out various tests on the r e l a t i v e binding of cpd. A and B. Variation i n the actual counts bound from one experiment to the next was attributed to the fact that the endogenous pool might vary, thereby influencing available binding s i t e s . How-ever, minimum values of around 25,000 dpm were obtained, and r e s u l t s were very consistent within any experiment. This i s i l l u s t r a t e d i n Table 13, which shows the influence 3 of various factors on the binding of H-cpd. B. 86 Table 13 EFFECT OF VARIOUS FACTORS ON BINDING OF 3H-CPD. B IN CYTOSOL Dpm/mg Protein % Control 781801 100 + 1 mM D i t h i o t h r e i t o l 766270 98 + 1 mM N-ethyl maleimide 775356 99 + RNase (1 mg/ml) 950614 122 + DNase (.25 mg/ml) 758290 97 + Pronase (1.5 mg/ml) 552760 71 87 As can be seen, the binding of the stero i d was un- . affected by nucleases, but diminished by pronase. The incomplete e f f e c t of the l a t t e r was attributed to the low a c t i v i t y of the preparation which was used. Dithio-t h r e i t o l had no e f f e c t , nor did N-ethyl maleimide. The competitive i n t e r a c t i o n of cpd. A and-.B-Lisoevident in Table 14. It should be noted that what appears to be a large excess of unlabelled stero i d i n r e l a t i o n to the small amount of l a b e l l e d compound—about 8 nM--is mis-leading. The endogenous pool of both cpd. A and B i n f e t a l tissue preparations i s probably of the order 100-200 nM (74, 88). Accordingly, the displacement which was observed was only modest, but consistent. Since cpd. A and B appear to compete i n the cytosol for the same receptor, i t was of i n t e r e s t to observe whether or not the cpd. A-receptor complex could enter the nucleus. Previous work i n t h i s laboratory has demonstrated a s p e c i f i c receptor for co r t i c o s t e r o i d s i n mouse placenta (78) i n both cytosol and n u c l e i . Nuclei could be prepared from placenta by hypotonic shock, and experiments were carri e d out on t h i s tissue to ascertain whether or not a cpd. A-receptor complex could be i s o l a t e d . The nuclear sap was fractionated on a Sephadex G-25 column and the r e s u l t s are shown in F i g . 24. Both cpd. A and B are demonstrable i n a...protein:-bound form. However, 8 8 Table 1 4 COMPETITIVE DISPLACEMENT OF LABELLED STEROID IN CYTOSOL Dpm Bound/mg Protein % Control, 3H-cpd. B 7 8 1 9 0 1 + 1 8 2 2 6 1 0 0 + 8 0 0 nM cpd. A 6 4 0 6 4 1 + 4 3 9 4 2 8 2 Control, 3 H-cpd. A 8 6 3 9 + 9 4 7 1 . 0 0 i + 8 0 0 nM cpd. B 4 4 8 9 + 1 0 6 8 5 4 Each figure i s a mean + SEM of four values. The ""H-cpd. B 3 . and H-cpd. A were examined i n d i f f e r e n t experiments. The binding varied considerably from one experiment to the next, which was attributed to the influence of endo-genous steroids. The differences here do not i l l u s t r a t e r e l a t i v e a f f i n i t i e s for the two steroids, since i n the same experiment the binding of cpd. A and B was of the same magnitude. The decrease observed i n the presence of unlabelled competitor i s s i g n i f i c a n t i n each case, P < . 0 2 . 89 F I G U R E 2 4 . . IS O L A T I O N OF R E C E P T O R C O M P L E X E S FROM P L A C E N T A ON S E P H A D E X G - 2 5 . VOLUME ( M L ) OD ^ <\ ; Dpm cpd. A — — — • ; Dpm cpd. B o 90 due to the reducing capacity of placenta (Fig. 5), which can reduce cpd. A to B, i t was thought possible that some 3 conversion of the H-cpd. A might be responsible for the apparent nuclear binding. Therefore, the use of 11-ketoprogesterone, a steroid capable of blocking reductase a c t i v i t y (84) was considered. 14 Fig . 25 shows that when C-cpd. A was incubated i n the presence of 6 pM 11-ketoprogesterone, the conversion to cpd. B was s u b s t a n t i a l l y decreased. The inc l u s i o n of 11-ketoprogesterone i n nuclear binding assays, however, did not diminish the binding of cpd. A. A photograph of nuclei isolated"from f e t a l brain by hypotonic shock i s shown i n F i g . 26. Fetal brain has strong dehydrogenase a c t i v i t y (Fig. 4), which would prevent reduction of the l a b e l l e d cpd. A. Fig. 2 7 indicates that both cpd. A and B enter the 3 nucleus. There i s , however, a larger amount ofv H-cpd. A unbound compared with cpd. B, and, since the number of counts of each added was the same, thi s consistent d i f f e r -ence i s thought to be r e a l . These data demonstrate that cpd. A can enter the nucleus either free of protein-bound. The p o s s i b i l i t y was then considered that cpd. A could be more than a competi-tor, and might i t s e l f bind to chromatin, as has been shown F i g . 25: Autoradiogram of chromatographed extracts of placenta-1 4 incubated with C—1 1-dehydrocorticosterone and 1 1-ketopro-gesterone. 1 + 2 controls 3 + 4 containing ;6 y.M 11-ketoprogesterone A: 11-dehydrocorticosterone B: corticosterone 92 1 2 3 4 F i g . 26: N u c l e i i s o l a t e d from f e t a l b r a i n by hypotonic shock wi t h d i l u t e MgCl 9. x80 0 . m a g n i f i c a t i o n . 94 F I G U R E 2 7 . I S O L A T I O N OF R E C E P T O R C O M P L E X E S FROM F E T A L B R A I N ON S E P H A D E X G - 2 5 . 96 for those steroids which exert b i o l o g i c a l a c t i v i t y (42). The i s o l a t e d nuclei were therefore extracted with high s a l t , 0.6M KC1 i n T r i s buffer, pH 8.0, and centrifuged at 105,000 xg for 15 min (89). The:rsteroid i n the super-natant was separated into protein-bound and unbound f r a c -tions by dextran-coated charcoal and the r a d i o a c t i v i t y i n these f r a c t i o n s , as well as that t i g h t l y bound to the p e l l e t , which contains 98% of tissue DNA (90), were assayed. Both cpd. A and B bind to the.chromatin-fraction. Competitive displacement of each steroid by 12 yM of the other was observed (Table 15). I t might be noted that i n these experiments the tissue was minced and not diluted by homogenization p r i o r to the assay. The endogenous steroids would therefore by r e l a t i v e l y more concentrated, making i t d i f f i c u l t to domonstrate competition with added steroid. The i d e n t i t y of the steroids bound to receptors i s of c r i t i c a l importance. Accordingly, characterization was accomplished by chromatography and by c o - c r y s t a l l i z a t i o n with authentic added c a r r i e r compounds of steroids is o l a t e d from receptor complexes. As shown i n Table 16, the steroid recovered from each f r a c t i o n proved to be the one which had been added in each case. 97 Table 15 COMPETITIVE DISPLACEMENT OF STEROIDS BOUND IN NUCLEAR FRACTIONS % of Counts i n F r a c t i o n Protein-bound Unbound P e l l e t 3H-cpd. A 100 100 100 + cpd. A 59 67 59 + cpd. B 66 69 92 3H-cpd. B 100 100 100 + cpd. A 53 100 81 + cpd. B 60 92 74 Values are expressed as percentages i n order t o combine f o u r d i f f e r e n t experiments. The n u c l e a r r a d i o a c t i v i t y i s expressed as t h a t which was e x t r a c t e d by s a l t , protein-bound and unbound, and t h a t which was bound t o the p e l l e t . 98 Table 16 CHARACTERIZATION OF STEROID FROM RECEPTOR-COMPLEXES (a) C o - c r y s t a l l i z a t i o n S t e r o i d T i s s u e Crys dpm, t a l s /yg % Recc jvery 1 s t 2nd x l s counts cpd. B cpd. B cpd. A B r a i n , c y t o s o l P l a c e n t a , c y t o s o l P l a c e n t a , n u c l e i 8.43 42.93 1.66 8.39 4 9.54: 1.46 34 41 53 I 3 1 : 46 55 Recovery r e f e r s to the f i n a l c r y s t a l crop determined by UV absorbance 2 37-24 0 nm and the counts a s s o c i a t e d w i t h those c r y s t a l s of C-21 a c e t a t e d e r i v a t i v e s (p. 23). (b) Chromatography Recovery of counts i n cpd. A zone % Recovered Unbound 73 Bound 75 P e l l e t 74 The s t e r o i d n u c l e i with was e x t r a c t e d from each f r a c t i o n o f b r a i n CH 2C1 2 and chromatographed on TLC sheets. 99 DISCUSSION U n t i l g e s t a t i o n day 14, the amount of 1 1 - d e h y d r o c o r t i -costerone i n f e t a l t i s s u e s i s g r e a t e r than the amount of a c t i v e hormone, c o r t i c o s t e r o n e . T h i s was a t t r i b u t e d i n e a r l i e r work (32) to dehydrogenase a c t i v i t y i n f e t a l t i s s u e s and p l a c e n t a . Subsequently, however, i t was found t h a t the r a t i o of r e d u c t i o n : dehydrogenation in'mouse p l a c e n t a was 4.0 or more (38). In t h i s r e s p e c t , the p l a c e n t a of mouse d i f f e r s from other s p e c i e s i n c l u d i n g man (37). AutoradiogramS of f e t a l b r a i n , p l a c e n t a and l i v e r ( F i g s . 2-3) i n d i c a t e d t h a t each t i s s u e m etabolizes c o r t i c o s t e r o n e d i f f e r e n t l y . B r a i n was observed to be a s i t e of dehydrogenation, p l a c e n t a of r e d u c t i o n and l i v e r developed r e d u c i n g c a p a c i t y , changing from a dehydrogenating s i t u a t i o n on day 14 to a r e d u c i n g one day 17. These d i f f e r n c e s l e d to the concept t h a t each f e t a l t i s s u e might r e g u l a t e i t s own c o r t i c o s t e r o n e content by the a c t i v i t y of the r e v e r s i b l e enzyme C - l l s t e r o i d oxidoreductase, r a t h e r than by simple a c t i v a t i o n of the f e t a l p i t u i t a r y - a d r e n a l a x i s . In b r a i n many m e t a b o l i t e s of c o r t i c o s t e r o n e were observed on day 14, which almost disappeared by day 15. These were i d e n t i f i e d as the c o r t i c o s t e r o n e - 2 1 - a c e t a t e , 1 1 - d e h y d r o c o r t i -costerone-21-acetate, the f r e e cpd. A and B and the C-2 0ctdihydro d e r i v a t i v e s of both cpd. A and B. The C-20adihydro d e r i v a t i v e s are known to be b i o l o g i c a l l y i n a c t i v e , whereas the a c e t a t e con-jugates and the 11-dehydrocorticosterone can be converted to 10 0 c o r t i c o s t e r o n e i n t i s s u e s which c o n t a i n the a p p r o p r i a t e enzymes (91). The formation of a c e t a t e e s t e r s i s not a common f i n d i n g , but has been r e p o r t e d i n the b r a i n of two o t h e r s p e c i e s (92). The c o n v e r s i o n of C o r t i s o l t o c o r t i s o n e has a l s o been r e p o r t e d i n a d u l t r a t i b r a i n (93). Since the t i s s u e s examined appeared to metabolize c o r t i -costerone and the 11-dehydro m e t a b o l i t e d i f f e r e n t l y , these and other f e t a l t i s s u e s were examined u s i n g a double i s o t o p e method, such t h a t both dehydrogenation and r e d u c t i o n c o u l d be examined si m u l t a n e o u s l y . Both b r a i n and gut ( F i g . 4) are s i t e s o f dehydrogenation, although t h i s a c t i v i t y d e c l i n e d a f t e r day 14 so_ t h a t by day 19 the r a t i o of reduction:dehydrogenation i n c r e a s e d t e n -f o l d i n b r a i n and f i v e f o l d i n gut. T h i s would mean a decreased 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 e n t e r i n g the t i s s u e to i t s i n -a c t i v e 11-dehydro m e t a b o l i t e , and hence i n c r e a s e d amounts of unchanged a c t i v e hormone found i n these t i s s u e s . T h i s i s con-s i s t e n t with the i n c r e a s i n g amount of unchanged c o r t i c o s t e r o n e found on day 17 i n b r a i n ( F i g . 2) and i n b r a i n and gut ( F i g . 7). The p l a c e n t a ( F i g . 5) i s an a c t i v e s i t e o f r e d u c t i o n throughout g e s t a t i o n , and the r e d u c i n g c a p a c i t y i n c r e a s e s s i g -n i f i c a n t l y w i t h g e s t a t i o n a l age. Conceivably, the p l a c e n t a f u n c t i o n s to reduce the i n a c t i v e 11-dehydro m e t a b o l i t e formed i n f e t a l t i s s u e s , before t u r n i n g i t t o the maternal compartment. By c o n t r a s t , f e t a l l i v e r i s a s i t e of dehydrogenation on day 14, 10 1. but by day 16 has developed a reducing capacity, the r a t i o being increased to 3.5. It seems l i k e l y that increased reductase a c t i v i t y i n l i v e r and placenta i s responsible for any increased hormone r e s u l t i n g from reduction of the 11-dehydro metabolite between gestational days 14 and 16 (Figs. 7-8). The lung has been investigated more intensively than other f e t a l tissues i n recent years. The r i s e i n reductase a c t i v i t y towards the end of gestation i s well documented (84, 94, 95, 96). The mouse resembles other species i n t h i s respect, as s i g n i f i -cant reducing capacity does not develop u n t i l a f t e r day 16 of gestation (Fig. 6). It i s also of i n t e r e s t to note the d i f -ference i n the time of development of the lung as compared with changes i n l i v e r , brain and gut (Figs. ,4-6) . I t can be concluded that the presence of the enzyme C-11 steroid oxidoreductase can modify the l e v e l of corticosterone i n a tissue so that, where dehydrogenation predominates, the content of active corticosterone i n that tissue and i n blood leaving i t w i l l be lower than i n blood entering. On the other hand, where reductase a c t i v i t y predominates, the corticosterone (or Cortisol) w i l l be augmented by reduction of 11-dehydro-corticosterone (or cortisone) which i s abundant i n f e t a l l i f e (32). Whatever the l e v e l of active hormone i n a tissue, that l e v e l can be increased by a r i s i n g concentration i n the blood entering the tissue, by a decrease i n the rate of dehydrogena-t i o n of the hormone within the tissue or by an enhanced rate of reduction of the 11-dehydro metabolite (Table 9). 102 The f e t a l tissues lung, l i v e r and placenta are active s i t e s of reduction (Figs. 5-6), which increases with gesta-t i o n a l age. I t was of inte r e s t therefore to examine the e f f e c t of dexamethasone i n j e c t i o n on the C-11 steroid oxidoreductase a c t i v i t y . As indicated i n Table 4, both lung and placenta showed s i g n i f i c a n t l y increased reductase a c t i v i t y on day 16, . 16 h following dexamethasone i n j e c t i o n into mothers. The i n j e c -t i o n increased the l e v e l i n placenta to that normally found on gestational day 19, whereas i n lung i t increased i t s i g n i f i -cantly (P<.05), but not as high as the day 19 l e v e l . This may be interpreted as in d i c a t i n g that the l e v e l of active hormone in these tissues can influence the tate of i t s own production. No e f f e c t was observed i n other tissues on day 16 (Table 4) and none of the tissues responded when injected e a r l i e r (Table 3). Smith, Torday and Giroud (81) have shown induction of C-11 steroid oxidoreductase i n f e t a l human lung c e l l s incubated i n v i t r o with C o r t i s o l . This finding would support present data suggesting that maternal i n j e c t i o n of synthetic c o r t i c o i d induces reductase a c t i v i t y i n the mouse lung on day 16. The net r e s u l t of increased reductase a c t i v i t y i n placenta, l i v e r and lung, and of decreased dehydrogenase a c t i v i t y i n brain and gut i s r e f l e c t e d i n the increased proportion of unchanged hormone which can be recovered from these tissues with increas-ing gestational age aft e r day 14 (Figs. 7-8). 103 This i s .also seen i n heart which, although having no detectable C-11 s t e r o i d oxidoreductase a c t i v i t y i t s e l f , i s probably influenced by the hormone concentration i n blood from l i v e r and lung (Fig. 7 ) . Smith (84) has shown that f e t a l lung contributes s i g n i f i c a n t l y to c i r c u l a t i n g c o r t i c o s t e r o i d s i n the rat. :, It can be concluded from t h i s work that the regulation of corticosteroids i n f e t a l tissue i s not determined by the f e t a l pituitary-adrenal axis, but rather by the interconversion of the hormone and i t s 11-dehydro metabolite. The large pool of the l a t t e r serves as a reservoir of potential hormone. The usefulness of glycogen deposition i n a tissue as a possible parameter of corticosterone action was examined i n f e t a l tissues on various gestational days. Sizable amounts of glycogen were observed i n l i v e r , lung, heart and placenta, with l e s s e r quantities i n gut and brain (Figs. 1 0 - 1 1 ) . F i g . 11 shows that there i s l i t t l e glycogen i n l i v e r early in gestation but that from day 17 the content increases rapidly and reaches a concentration of 4 6 mg/g by day 1 9 . This sharp r i s e i n glycogen accumulation i n the l i v e r i s observed i n species where the gestational period i s short and usually pro-ceeds in the l a s t f i f t h of gestation ( 4 7 ) . Since the glycogen content of l i v e r i s low on day 16 and does not appear to increase s i g n i f i c a n t l y u n t i l day 1 7 , the 10 4 development of l i v e r reductase a c t i v i t y does not appear to be the l i m i t i n g factor contributing to glycogen deposition, as on day 16 the reductase capacity i s high (Fig. 6) and the c o r t i -costerone l e v e l cannot be considered l i m i t i n g . Therefore, factors other than c o r t i c o s t e r o i d might also be involved i n f e t a l l i v e r glycogen deposition i n mouse. The presence of high l e v e l s of i n s u l i n i n the rat fetus during hepatic glycogen deposition has implicated i n s u l i n i n glycogen storage (97). Plas and Numez (98), using cultured f e t a l hepatocytes,,.. have shown a marked glycogenic e f f e c t of i n s u l i n only i n hepatocytes previously exposed to C o r t i s o l . This suggests that corticosteroids are responsible for the onset of glycogen storage and that i n s u l i n only potentiates t h i s e f f e c t . Therefore, corticosteroids might induce some step necessary for i n s u l i n action, and one p o s s i b i l i t y i s the induc-t i o n of the r a t e - l i m i t i n g enzyme, glycogen.synthetase, as i t has been shown that C o r t i s o l might be responsible for the synthesis of t h i s enzyme during development.(56, 99). Placental glycogen (Fig. 11) r i s e s during gestation to a peak concentration of 13 mg/g on day 1 6 . A decline i n glycogen i s observed thereafter, at the same time the l i v e r glycogen appears to accumulate. Bernard (100) and V i l l e e (101) postu-lated that placental glycogen i s a storage depot for f e t a l use and that i t serves to regulate f e t a l blood glucose l e v e l s . But i t has been shown that under conditions of f e t a l stress, glucose i s not l i b e r a t e d from the placenta and glycogen l e v e l s are not 105 reduced (102, 103). A l s o , enzyme s t u d i e s have shown t h a t the p l a c e n t a throughout g e s t a t i o n l a c k s the enzyme glucose-6 phosphatase (EC 3.1.3.9), which i s necessary f o r the f i n a l r e l e a s e of glucose from glycogen (104, 105).' The p l a c e n t a l glycogen i s t h e r e f o r e more l i k e l y to be s t o r e d f o r l o c a l use and might p l a y a r o l e as an emergency energy r e s e r v e f o r vasomotor f u n c t i o n (103). The amount of p l a c e n t a l glycogen found v a r i e s c o n s i d e r a b l y w i t h the s p e c i e s (47). The d e p o s i t i o n of lung glycogen ( F i g . 11) i n the mouse d i f f e r s a p p r e c i a b l y from the p a t t e r n observed i n other s p e c i e s (47). Glycogen c o n c e n t r a t i o n i n mouse lung r i s e s to a l e v e l o f 10 mg/g and remains c o n s t a n t from day 16 to 19 of g e s t a t i o n . T h i s i s a p p a r e n t l y a s p e c i e s d i f f e r e n c e . The accumulation of lung glycogen does not correspond to the lung C - l l s t e r o i d o x idoreductase p a t t e r n ( F i g . 6), s i n c e lung c o n t a i n s l a r g e glycogen d e p o s i t s on day 16, a time a t which t h i s enzyme i n lung i s predominately dehydrogenating. Heart glycogen i n c r e a s e s from g e s t a t i o n a l day 14 and reaches a peak c o n c e n t r a t i o n of 22 mg/g on day 16. There-a f t e r , the glycogen content decreases to a value of 8 mg/g on day 19. T h i s p a t t e r n corresponds to that•observed i n o t h e r s p e c i e s (47); however, the amount of glycogen found p r i o r to b i r t h , i n h e a r t , i s thought to vary i n v e r s e l y w i t h m a t u r i t y arid hence a l a r g e r glycogen c o n c e n t r a t i o n might have been expected, s i n c e the newborn mouse i s r e l a t i v e l y very immature. But t h i s r e l a t i o n s h i p i s not r i g i d , and the d e c l i n e i n glycogen appears to be a s s o c i a t e d w i t h a p a r t i c u l a r stage 106 i n h e a r t development which might occur mainly i n utero i n the mouse. Since no d e t e c t a b l e C-11 s t e r o i d oxidoreductase a c t i v i t y was observed i n h e a r t , t h i s i n c r e a s e i n glycogen cannot be con-s i d e r e d d i r e c t l y a t t r i b u t a b l e to reductase a c t i v i t y , but might r e f l e c t the i n c r e a s e o f c o r t i c o s t e r o n e i n the b l o o d due t o the l i v e r reductase a c t i v i t y d e v e l o p i n g between days 14 and 16 ( F i g s . 6-7). B r a i n and gut showed much lower c o n c e n t r a t i o n of glycogen with v a l u e s o f 4 and 2 mg/g, r e s p e c t i v e l y . These d e p o s i t s were not g r e a t l y a l t e r e d with i n c r e a s i n g g e s t a t i o n a l age, and cannot be a t t r i b u t e d t o a d e c r e a s i n g dehydrogenase a c t i v i t y ( F i g . 4). In an attempt to c o r r e l a t e glycogen d e p o s i t i o n i n f e t a l t i s s u e s w i t h c o r t i c o s t e r o i d s , mothers were i n j e c t e d 16 h p r i o r to t i s s u e examination, with the s y n t h e t i c c o r t i c o i d , dexametha-sone. F e t a l l i v e r was the o n l y t i s s u e i n which glycogen i n -creased s i g n i f i c a n t l y (P<.01) (Table 5). Animals assayed two days e a r l i e r showed no i n c r e a s e i n l i v e r glycogen. T h i s r e i n -f o r c e s the concept o f a d u a l c o n t r o l of glycogen s y n t h e s i s i n f e t a l l i v e r (25), and i n d i c a t e s t h a t f a c t o r s other than c o r t i -c o s t e r o i d s are i n v o l v e d i n the e a r l i e r glycogen d e p o s i t i o n i n h e a r t and lung. The decrease i n t r e a t e d p l a c e n t a (Table 5) suggests t h a t dexamethasone i s a c c e l e r a t i n g the t r e n d which occurs normally. Glycogen d e p o s i t i o n does not appear t o be a good parameter r e f l e c t i n g s t e r o i d m e tabolic p a t t e r n s . 10.7 The c h o i c e o f o t h e r p a r a m e t e r s m e a s u r e d - - t h e i n c o r p o r a t i o n o f l e u c i n e , u r i d i n e , t h y m i d i n e - - w a s b a s e d o n t h e a s s u m p t i o n t h a t , s i n c e t h e s e a r e s u b s t r a t e s f o r p r o t e i n and n u c l e i c a c i d s y n t h e s i s , t h e i r u t i l i z a t i o n m i g h t r e a s o n a b l y be e x p e c t e d t o r e f l e c t m a j o r c h a n g e s i n s y n t h e t i c a c t i v i t y i n a t i s s u e , a n d t h u s a c t a s a p o s s i b l e i n d i c a t o r o f a n y m e t a b o l i c e f f e c t s p r o d u c e d by c o r t i c o s t e r o i d s . I n f a c t , a c l o s e r e l a t i o n s h i p was o b s e r v e d b e t w e e n c h a n g e s 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 and s u b -s t r a t e u t i l i z a t i o n 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 . I n c r e a s e d c o r t i c o s t e r o n e ( F i g s . 7 -8 ) r e s u l t e d i n d e c r e a s e d i n c o r p o r a t i o n o f l e u c i n e ( F i g s . 1 2 - 1 3 ) , u r i d i n e ( F i g s . 1 4 - 1 5 ) and t h y m i d i n e ( F i g s . 1 6 - 1 7 ) . The s i g n i f i c a n c e o f a d e c l i n e i n s u b s t r a t e i n c o r p o r a t i o n a f t e r g e s t a t i o n a l d a y 14 i n b r a i n , g u t , p l a c e n t a a n d l i v e r ( F i g s . 1 2 - 1 7 ) i s n o t known. A l t h o u g h t h e a n t e n a t a l e f f e c t s o f c o r t i c o s t e r o n e on r a t l u n g (84) a n d o f C o r t i s o l o n f e t a l r a b b i t l u n g (83) h a v e b e e n r e p o r t e d t o i n v o l v e d e c r e a s e d g r o w t h a n d c e l l d i v i s i o n , i t h a s a l s o b e e n o b s e r v e d t h a t C o r t i s o l s t i m u -l a t e s l u n g g r o w t h a t a n e a r l i e r s t a g e o f g e s t a t i o n ( 4 1 ) . The f e t a l mouse g r o w s f r o m a b o u t 2 00 mg on g e s t a t i o n a l day 14 t o o v e r 1 g o n d a y 1 9 , a n d t h i s g r o w t h i s h y p e r p l a s t i c ; i . e . , t h e r e i s a s y n c h r o n o u s i n c r e a s e i n p r o t e i n a n d n u c l e i c a c i d i n t i s s u e s ( 1 0 6 ) . F i g . 19 shows t h a t t h e amount o f l e u c i n e p r e s e n t i n f e t a l b r a i n d o e s n o t i n c r e a s e f r o m g e s t a t i o n a l d a y 14 t o day 15 n o r i s i t a f f e c t e d by d e x a m e t h a s o n e i n j e c t i o n . T h e r e f o r e , t h i s 108 would rule out the p o s s i b i l i t y that these e f f e c t s are due to an increased leucine pool, d i l u t i n g the l a b e l l e d precursor causing a decrease i n incorporation of the l a b e l . This implies that the decline i n incorporation of the substrates might be i n t e r -preted as a decrease i n the rate of synthesis of the macro-molecules into which they are incorporated. The decline i n leucine, uridine and thymidine incorpora-t i o n i n l i v e r , placenta, brain and gut (Figs. 12-17) between gestational days 14 and 16 coincides with increased reductase a c t i v i t y i n placenta (Fig. 5) and l i v e r (Fig. 6) and decreased dehydrogenase a c t i v i t y i n brain and gut (Fig. 4) both of which increase the active c o r t i c o s t e r o i d i n these tissues at that time (Figs. 7-8). Dexamethasone i n j e c t i o n s i g n i f i c a n t l y decreased the leucine, thymidine and, i n a l l but gut, uridine incorporated (Tables 6-8). This indicates that these e f f e c t s are steroid-induced. The pattern of leucine, uridine and thymidine incorpora-tion into heart and lung i s more d i f f i c u l t to interpret. Lung, for a l l parameters measured, maintained constant isotope incor-poration between gestational days 14 and 16 (Figs. 13, 15, 17). Only af t e r day 16 did the incorporation pattern decline. This decline correlates well with the increased reductase a c t i v i t y found i n lung after day 16 (Fig. 6). Heart appeared to follow the pattern of lung, at l e a s t a f t e r day 16, when in a l l 109 parameters the isotope incorporation declined (Figs. 12, 14, 16) . Uridine incorporation increased s i g n i f i c a n t l y i n heart from day 14 to 16 (Fig. 14) and the thymidine pattern (Fig. 16) declined steadily from day 14 onward. Therefore, the heart might be influenced not only by corticosteroids i n lung, but c i r c u l a t i n g steroids contributed by other tissues, notably l i v e r (Figs. 6-7) as well. The i n j e c t i o n of dexamethasone had no s i g n i f i c a n t e f f e c t on leucine (Table 6) or uridine incorpora-t i o n i n either tissue, but s i g n i f i c a n t l y (P<.01) lowered the thymidine (Table 8). Possibly the stimulus was applied too early to e l i c i t the response which i s normally observed a f t e r day 16; for instance, dexamethasone was found to increase lung reductase a c t i v i t y only on day 16 (Table 4), but not e a r l i e r (Table 3). I t would appear, however, that the incorporation of thymi-dine i s the most sensitive parameter r e f l e c t i n g changes i n cor-t i c o s t e r o i d action (Figs. 16-17); t h i s was found to be the case also i n lymphocytes treated with corticosteroids (85) . Liver was the most responsive tissue by a l l parameters (Figs. 13, 15, 17) . These events, the significance of which remain: to be established, occur at an e a r l i e r stage i n gestation than most of the c o r t i c o s t e r o i d e f f e c t s which have been hitherto recorded. Ornithine Decarboxylase (EC 4.1.1.17) i s an indicator of change i n metabolic or p r o l i f e r a t i v e a c t i v i t y of tissues induced by hormones (77). The a c t i v i t y of t h i s enzyme was observed to decline with gestational age i n mouse placenta 110 (Fig. 18), and has also been observed to decline with gesta-t i o n a l age i n both rat l i v e r (86) and placenta (77) . This decline corresponds to the observed decrease i n the incorpora-tion of leucine (Fig. 13), uridine (Fig. 15) and thymidine (Fig. 17), and also the increased reductase a c t i v i t y after day 14 i n placenta (Fig. 5). The i n j e c t i o n of dexamethasone into mothers, day 13.5, 16 h pr i o r to assay, resulted i n a s i g n i f i c a n t l y decreased (P<.01) enzyme a c t i v i t y (Fig. 18), i n d i c a t i n g that the c o r t i c o s t e r o i d e f f e c t could be induced prematurely. Thymidine kinase a c t i v i t y (EC 2.7.1.75) has also been observed to decline i n rat placenta (77), with increasing ges-t a t i o n a l age. Its a c t i v i t y i s related to the rate of p r o l i f e -r a t i o n of a population of c e l l s and c o r r e l a t i o n with growth rate has been demonstrated (107, 108). Thymidine kinase a c t i v i t y has been shown to decline a f t e r treatment with c o r t i c o s t e r o i d (109). These data support, the contention that c o r t i c o s t e r o i d s are a f f e c t i n g the rate of growth i n f e t a l tissues, possibly inducing d i f f e r e n t i a t i o n . H i s t o l o g i c a l examination of tissues from treated animals has not revealed any s i g n i f i c a n t differences evident by l i g h t microscopy. Therefore, these biochemical changes would appear to occur p r i o r to morphological a l t e r a t i o n s . I l l F i g . 19 shows t h a t between days 14 and 15 the r e i s an apparent i n c r e a s e i n amino a c i d content of f e t a l b r a i n . The f e t a l b r a i n c o n t a i n s s i g n i f i c a n t amounts of a s p a r t a t e , g l u t a -mate t h r e o n i n e , s e r i n e , g l y c i n e , a l a n i n e , t y r o s i n e and some v a l i n e . Very few b a s i c amino a c i d s were noted and are not i n c l u d e d i n the data presented. A l l of the above-mentioned amino a c i d s , e x c l u d i n g perhaps v a l i n e , i n c r e a s e d s i g n i f i c a n t l y from day 14 t o day 15. There was a l s o an apparent i n c r e a s e i n the t h r e o n i n e l e v e l with r e s p e c t to s e r i n e , and a l a n i n e w i t h r e s p e c t to g l y c i n e . These appeared t o i n c r e a s e , which might i n d i c a t e some d i f f e r e n c e i n the s y n t h e t i c a b i l i t y o f the b r a i n to produce a p a r t i c u l a r amino a c i d on d i f f e r e n t g e s t a t i o n a l days. Dexamethasone i n j e c t e d 16 h e a r l i e r produced a p a t t e r n i n t e r m e d i a t e between these observed days 14 and 15. Th i s c o u l d r e p r e s e n t an i n c r e a s e by the s t e r o i d of t r a n s -aminase a c t i v i t y , which has not been demonstrated i n most f e t a l t i s s u e s (12). Glucosamine was i n c o r p o r a t e d i n t o f e t a l l i v e r and gut on day 14 as r e a d i l y as l a t e r , i n d i c a t i n g t h a t the t i s s u e s c o n t a i n the r e q u i s i t e enzymes f o r g l y c o p r o t e i n s y n t h e s i s (Table 10). Since fructose-6-PO^ u t i l i z a t i o n was not d i f f e r e n t , i t appears p o s s i b l e t h a t the formation of f r u c t o s e from glucose c o u l d be a l i m i t i n g step ( F i g . 20). While no evidence was ob t a i n e d which i n d i c a t e d t h a t g l y c o p r o t e i n s y n t h e s i s was i n f a c t being so r e g u l a t e d , t h i s remains an a t t r a c t i v e p o s s i b i l i t y f o r f u t u r e i n v e s t i g a t i o n . The c o n v e r s i o n o f glucose t o f r u c t o s e , a p p a r e n t l y 112 without formation o f phosphorylated i n t e r m e d i a t e s , has been shown i n sheep (4) and human (5, 6) p l a c e n t a , and i s known t o proceed i n two s t e p s : f i r s t , c o n v e r s i o n of glucose to s o r b i -t o l ; and, secondly, of s o r b i t o l t o f r u c t o s e (5). The i n c r e a s -i n g c o r t i c o s t e r o i d i n these t i s s u e s ( F i g s . 7-8) and s t i m u l a t i o n by i n j e c t i o n o f dexamethasone i n d i c a t e t h a t t h i s might be another process mediated by c o r t i c o s t e r o i d s . The t r a n s f e r o f n u c l e i c a c i d p r e c u r s o r s across the p l a c e n t a has been observed i n the r a t (2). T r a n s f e r of thymidine across mouse p l a c e n t a was r e p o r t e d not to occur u n t i l g e s t a t i o n a l day 11 (110); t h i s has a l s o been r e p o r t e d i n the r a t e (111). 3 The e a r l i e s t a t which mice were i n j e c t e d w i t h H-thymidme i n the p r e s e n t work was day 14, and high counts were observed i n the a c i d - i n s o l u b l e p r e c i p i t a t e of a l l f e t a l t i s s u e s . T h i s i n d i c a t e s t h a t thymidine can indeed c r o s s the p l a c e n t a r e a d i l y i n the mouse. I t has been suggested t h a t p l a c e n t a l growth must reach a c e r t a i n stage and the t r a n s f e r mechanism developed before thymidine t r a n s f e r can occur (110). The v a r i a t i o n i n the amount of DNA which p r e c i p i t a t e s i n a c i d a t room temperature i s shown i n F i g . 23. The s i g n i f i c a n c e o f these changes i s not known, but might i n d i c a t e some a l t e r a -t i o n i n molecular a g g r e g a t i o n a s s o c i a t e d w i t h m e t a b o l i c changes i n these t i s s u e s . As seen i n Table 11, as w i t h a l l other parameters determined i n t h i s w o r k — t h e i n c o r p o r a t i o n of l e u c i n e , u r i d i n e and thymidine, and the d e p o s i t i o n of g l y c o g e n — w h a t e v e r 113 trend there appeared to be i n a tissue was nearly always. accelerated when dexamethasone was injected. Since a l t e r a t i o n i n the metabolism of corticosterone has been shown to have profound e f f e c t s on parameters including the incorporation of leucine, uridine and thymidine; ornithine decarboxylase a c t i v i t y ; the amino acid content of brain; glucose metabolism i n l i v e r and gut; i t seems probable that the tissues in which these changes occur are "target" tissues of the stero i d and therefore would be expected to have s p e c i f i c s t e r o i d recep-tors. The question arises, then, do these receptors have a regulatory role i n mediating s t e r o i d action? F e t a l brain was examined on gestational days 14 and 17 (Table 12). Ba l l a r d and Ballard (4 3) have demonstrated the presence of s t e r o i d receptors early i n gestation i n various human f e t a l t i s s u e s . The for dexamethasone reported by them for human lung was 8.9 nM, very sim i l a r to the value 8.3 nM found i n t h i s work for f e t a l mouse brain (Table 12). The receptor concentration appeared to decrease i n f e t a l brain between gestational days 14 and 17 (Table 12), but t h i s can be attributed to an increase i n brain protein content with the number of receptors remaining e s s e n t i a l l y constant. Since the metabolic alterations i n f e t a l brain occur a f t e r day 14 (Figs. 2, 4), i t i s concluded that, while receptors are essen-t i a l for mediating stero i d e f f e c t s , they are not the c r i t i c a l factor i n i t i a t i n g changes. This role i s attributed s o l e l y to the a c t i v i t y of the enzyme C-11 s t e r o i d oxidoreductase. 114 Due to the high l e v e l of endogenous steroi d present i n f e t a l tissues, which was determined to be about 200-300 ng/g tissue (38, 88), i t was d i f f i c u l t to obtain a Scatchard p l o t or to observe competitive i n t e r a c t i o n of added steroids. Repeated attempts were made to determine d i s s o c i a t i o n constants for both corticosterone and 11-dehydrocorticosterone without success. Table 14 shows that cpd. A can displace l a b e l l e d cpd. B from receptor s i t e s , and vice versa. Examination of cytosol receptor c h a r a c t e r i s t i c s (Table 1.3) showed that the binding of cpd. B was not affected by the nucleases, RNase and DNase; nor by d i t h i o t h r e i t o l or N-ethyl maleimide. The receptor was sensitive to pronase action, i n d i c a t i n g that i t i s l i k e l y a protein molecule with no nucleic acid, s i m i l a r to the findings of others (43, 112). Lack of s e n s i t i v i t y to sulfhydryl reagents confirms the observation of Wong and Burton (78) on binding i n mouse placenta. Since cpd. B may be displaced by i t s 11-dehydro metabolite and considering the high l e v e l of the l a t t e r , cpd. A, present in f e t a l tissues, i t seems l i k e l y that cpd. A might prevent binding of cpd. B and hence block i t s metabolic e f f e c t s . The fetus, therefore, might not only be protected from maternal corticosterone by active metabolism (Figs. 4-6), but also by ef f e c t i v e blocking of corticosterone receptor s i t e s . Cpd. A was found i n the nucleus and was protein-bound, although i n contrast to cpd. B, which was almost e n t i r e l y 115 bound, there was a l s o a l a r g e amount of unbound cpd. A (F i g . 27). These data suggest t h a t not o n l y i s the 11-dehydro compound able to b i n d to a r e c e p t o r i n the c y t o s o l , but t h a t the s t e r o i d - r e c e p t o r complex can move from the c y t o s o l i n t o the nucleus. Cortexolone, the 11-deoxy analog of C o r t i s o l , i s known to form a c y t o s o l - r e c e p t o r complex which i s t r a n s -l o c a t e d i n t o the nucleus, and which competes wi t h C o r t i s o l , b l o c k i n g the e f f e c t s induced by the l a t t e r (89, 113, 114). Cortexolone, however, u n l i k e C o r t i s o l , does not b i n d to chromatin, and i t i s thought t h a t t h i s i s why i t does not i t s e l f e l i c i t a response (89). The 11-dehydro m e t a b o l i t e of C o r t i s o l , c o r t i s o n e , binds o n l y very weakly to the C o r t i s o l r e c e p t o r i n human lung (115) (see Appendix I ) . S u r p r i s i n g l y , cpd. A a c t u a l l y bound to the chromatin f r a c t i o n a t l e a s t as w e l l as the a c t i v e hormone, cpd. B. Again, the i d e n t i t y ' o f the bound s t e r o i d was e s t a b l i s h e d as f r e e cpd. A (Table 16) . These f i n d i n g s .indicate; .that;fcpd. 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Plas, C. and Nunez, J. J. B i o l . Chem. 251: 1431 (1976). 99. C a h i l l , G.F. , J r . In: The Human Adrenal Cortex . . (Christy, N.P., ed.), Harper and Row, New York. = p.205 (1971). 100. Bernard, C. Journal de Physiologies de 11homme et des animaux 2: 336 (1859). 101. V i l l e e , C.A. J. B i o l . Chem. 205: 113 (1953). 102. Huggett, A. St. C. J. Physiol. 67_: 360 (1929). 103. Robb, S.A. and Hytten, F.E. B r i t . J. Obst. Gynaec. 8_3: 43 (1976). 104. Walker, D.G., Lea, M.A., R o s s i t e r , G . and Addison, M. Arch. Biochem. Biophys. 120: 646 (1967). 105. Gennser, G., Lundquist, I. and Nilsson, E. B i o l . Neonat. 21: 148 (1972). 106. Winick, M.: C e l l u l a r Growth of the Fetus and Placenta. In Fetal Growth and Development. (Waisman, H.A. and Kerr, G.R., eds.) McGraw-Hill, New York. p.19 (1970). 107. Bukovsky, J. and Roth, J.S. Advances i n Enzyme Regula-t i o n 2 : 371 (1964) . 108. Garland, M.R., Ng, T.W. and Richards, J.F. Cancer Res. 31: 1348 (1971). 109. Greengard, 0. and Machovich, R. Biochem. Biophys. Acta 286: 382 (1972). 110. Nanda, R. Arch. Oral B i o l . 16: 435 (1971). 111. A t l a s , M., Bond, V.P. and Cronkite, E.P. J. Histochem. Cytochem. 8: 171 (1960). 112. Agarwal, M.K. FEBS Let. 85_: 1 (1978). 113. Munck, A. and Brinck-Johnsen, T. J. B i o l . Chem. 243: 5556 (1968). 122 114. Mosher, K.M., Young, D.A. and Muck, A. J . B i o l . Chem. 246: 654 (1971). 115. B a l l a r d , P.L., C a r t e r , J.E., Graham, B.S. and Baxter, J.D. J . C l i n . E n d o c r i n o l . Metab. 41: 290 (1975). 116. Wong, M.D. and Burton, A.F. B i o l . Neonate 18: 146 (1971). 123 APPENDIX I Structures of the steroids used 113-hydroxysteroid dehydrogenase CH2OH CH2OH corticosterone, cpd. B 11-dehydrocorticosterone, cpd. A CH_OH I 2 --0H 3* llg-hydr'oxysteroid dehydrogenase NADP NADPH CH o0H C o r t i s o l cortisone LEAF 123a OMITTED IN PAGE NUMBERING, 11 - k e t o p r o g e s t e r o n e 124 APPENDIX II At the o u t s e t of t h i s i n v e s t i g a t i o n , i t was a n t i c i p a t e d t h a t the metabolism of glucose would be of major i n t e r e s t , and a method was t h e r e f o r e developed which pe r m i t t e d the determina-t i o n of glucose i n s m a l l samples o f mouse f e t a l blood and 14 d e t e r m i n a t i o n of the t r a n s f e r of C-glucose from the mother. Although the major i n t e r e s t s h i f t e d , the method was thought to be of i n t e r e s t and of p o s s i b l e u s e f u l n e s s , and i s d e s c r i b e d below. P r e p a r a t i o n of the Glucose T e t r a a c e t a t e C a r r i e r . Glucose, 1 g, 5 ml a c e t i c anhydride and 10 ml p y r i d i n e were added to a 50 ml Erlenmeyer ; f l a s k , which was then s t o p -pered and p l a c e d i n a 37°C water bath, shaking a t 120 c y c l e s / min f o r 2 h. At the end of the i n c u b a t i o n , the s o l u t i o n was c o o l e d and added to 70 ml of d i s t i l l e d water, on i c e , i n c e n t r i -fuge tubes. The tubes were then covered w i t h p a r a f i l m and allowed to stand o v e r n i g h t a t 0-4°C to promote c r y s t a l forma-t i o n . The c r y s t a l s were c o l l e c t e d by c e n t r i f u g a t i o n , the water removed and the c r y s t a l s d i s s o l v e d i n as small an amount of e t h y l a c e t a t e as p o s s i b l e . The samples were c r y s t a l l i z e d by dropwise a d d i t i o n of petroleum e t h e r (bp 80-100°C). At the f i r s t s i g n of c r y s t a l s , a few drops of d i e t h y l e t h e r were added to prevent formation of an amorphous form. C r y s t a l s were allowed to form o v e r n i g h t at 0-4°C, c o l l e c t e d by c e n t r i f u g a t i o n and the mother l i q u o r removed. The c r y s t a l s were then d r i e d , weighed 125 and r e - d i s s o l v e d i n e t h y l a c e t a t e such t h a t there was 20 mg of c o l d c a r r i e r per 50 y l . T h i s s o l u t i o n was s t o r e d i n the f r e e z e r w e l l stoppered with a s i l i c o n e stopper and covered with p a r a f i l m . The p y r i d i n e used i n a l l experiments was d i s t i l l e d j u s t p r i o r to use and 1/16" molecular s i e v e added to remove any water t r a c e s . A c e t i c anhydride was a l s o s t o r e d i n t h i s way. Mass spectroscopy o f the compound confirmed i t s i d e n t i t y as glucose t e t r a a c e t a t e . (Courtesy of Dr. L.S. W e i l e r , Dept. of Chemistry, U n i v e r s i t y o f B.C.) Chromatographic I d e n t i f i c a t i o n of Glucose T e t r a a c e t a t e Zone A small amount of the prepared compound was s p o t t e d onto a TLC sheet and developed twice i n hexanerethyl a c e t a t e (4:1; v : v ) . Simply by h o l d i n g the chromatogram up to a l i g h t source, the zone c o u l d be observed by a change i n the d e n s i t y o f the TLC sheet. Upon standing, the compound was o x i d i z e d and the t e t r a a c e t a t e zone became c l e a r l y v i s i b l e , being a brown c o l o u r . TLC sheets were sprayed with a glucose s t a i n i n g reagent ( S t a h l Reagent No. 134) and these zones were shown to correspond. 14 A c e t y l a t i o n of C - l a b e l l e d Glucose Standards C-glucose (u), 10,000 dpm, 10 yg of n o n - r a d i o a c t i v e glucose, 0.5 ml 5% TCA were mixed and e x t r a c t e d s e v e r a l times 126 with d i e t h y l ether. The aqueous layer was removed and evapo-rated under N 2 with several additions of ethanol to remove f i n a l traces of water. The sample was then stored i n an evacuated desiccator for several days u n t i l dryness was ensured. Ninety y l of non-radioactive acetic anhydride and pyridine were added and the tube stoppered and sealed with parafilm. The tube was placed i n a shaking water bath under conditions previously described and incubated for the required length of time, 1/2, 1, 2, 4 and 8 h. After the appropriate time, the samples were removed, placed on i c e , 500 yg cold c a r r i e r added plus 0.5 ml of d i s t i l l e d water to hydrolyze remaining acetic anhydride. The samples were then extracted with 2.5 ml of i c e - c o l d ethyl acetate to remove the glucose-acetate. The ethyl acetate layer was removed and the aqueous f r a c t i o n re-extracted with another 2.5 ml of ethyl acetate. This f r a c t i o n was added to the previous and the ethyl acetate re-extracted with 0.5 ml of d i s t i l l e d water to remove remaining acetic acid. The ethyl acetate layer was evaporated under N 2, spotted onto a TLC sheet and developed twice i n hexane:ethyl acetate (4:1; v:v). The sheet was then autoradiographed i n order to show any products formed throughout the incubation. An aliquot of the aqueous f r a c t i o n was counted so that the percentage of acetylation could be calculated. As can be seen from F i g . 28, the major portion of the r a d i o a c t i v i t y was i n the glucose t e t r a -acetate zone, In fact, the o v e r a l l e f f i c i e n c y df acetylation was such that the tetraacetate accounted for 72 - 76 percent (Table 17). The time of incubation i n studies following was a r b i t r a r i l y selected as 2 h. 127 Table 17 EFFECT OF TIME ON THE EFFICIENCY OF GLUCOSE ACETYLATION Time (h) % Ace.tyla.ti.on. % Glucose .Te.tra. Acetate 1/2 78.2 72.8 .1 84.0 76 .2 2 79.3 72.1 4 83.1 75.2 8 83.9 74.9 Each value i s a mean of four determinations. Injection of Mice and Processing of Blood Control mice on gestational day 18-19 were injected with 14 . 5 yCi of C-glucose (u) 15 mm prxor to k i l l i n g . The mouse was anesthetized with 1:6 nembutal d i l u t e d with 0.9% s a l i n e . The fetuses were then removed, decapitated and the blood co l l e c t e d on a piece of parafilm kept on i c e . The blood was then mixed with a drop of saline and transferred into a 5 ml t e s t -tube. Maternal blood was c o l l e c t e d by severing the a r t e r i e s near the heart. One drop of 6 0% TCA was added to each sample and the r e s u l t i n g p r e c i p i t a t e centrifuged at 0-4°C and d i s -carded. The remaining acid-soluble f r a c t i o n was frozen u n t i l further use. 128 Dexamethasone-treated mice received 2 00 yg Dex solution i n t r a p e r i t o n e a l l y 1 h p r i o r to the subcutaneous i n j e c t i o n 14 of C-labelled glucose; the animals were processed as pre-viously mentioned. Acetylation of Maternal and Fetal Blood Samples Maternal and f e t a l blood samples from mice injected with 14 . . C-glucose were acetylated with non-radioactive acetic anhydride, 500 pg c a r r i e r glucose tetraacetate was added, and the samples chromatographed as described. Autoradiograms indicated that the r a d i o a c t i v i t y recovered i n maternal blood was predominantly glucose tetraacetate, whilst i n f e t a l samples about half was i n t h i s zone (Fig. 28). The maternal and f e t a l blood samples were divided i n h a l f . One-half of the sample was acetylated with non-radioactive 3 acetic anhydride and the other with H-acetic anhydride d i l u t e d 14 to 1 yCi/ymole. This allowed c a l c u l a t i o n of C-glucose i n the sample. This was necessary since upon acetylation with l a b e l l e d acetic anhydride 10,000 dpm of glucose was added to these samples 14 3 . . in order to allow reasonable C to H counts for r e - c r y s t a l l i z a t i o n 14 studies. The contribution of t h i s C-glucose l a b e l was corrected for i n c a l c u l a t i o n . The sample was extracted with dieth y l ether several times and the aqueous f r a c t i o n evaporated to dryness under N 2. The sample was placed i n a desiccator and stored evacuated over DRIERITE for several days to ensure complete dryness before acetylation as described previously. F i g . 28: Autoradiograms o f chromatographed e x t r a c t s of the 14 a c e t y l a t e d d e r i v a t i v e s of C-glucose recovered from maternal and f e t a l mouse blood. A c i d - s o l u b l e e x t r a c t s of a few micro-l i t r e s o f blood were chromatographed on s i l i c a g e l TLC sheets developed i n hexane:ethyl a c e t a t e (4:1; v : v ) . 1. maternal blood 2. f e t a l blood 3. glucose standard GA: glucose t e t r a a c e t a t e 130 131 T a b l e 18 EFFECT OF DEXAMETHASONE INJECTION INTO:'MOTHERS ON THE S P E C I F I C ACTIVITY OF FETAL BLOOD GLUCOSE 3 S p e c i f i c A c t i v i t y x l O C o n t r o l D e x - t r e a t e d 1. 6.4 2. 3.5 3. 2.9 4. 8.2 1> 10.0 2. 7.6 3. 6.7 4. 1.1 X = 5.25 X = 6.35 132 Wong and Burton (116) have observed that i n j e c t i o n of mothers with dexamethasone followed by i n j e c t i o n of l a b e l l e d glucose resulted i n a decline i n uptake of the label by the fetus. Since the mouse placenta has c o r t i c o s t e r o i d receptors (78, 79) the p o s s i b i l i t y that the c o r t i c o s t e r o i d could control the f e t a l f u e l supply was considered. The data of Wong and Burton (116) does not di s t i n g u i s h between the p o s s i b i l i t y of decrease i n lab e l i n fetuses being due to d i l u t i o n caused by maternal hyperglycemia, or i f t h i s i n fact represents an actual decrease i n t o t a l glucose crossing the placenta. To determine which alternative occurs the s p e c i f i c a c t i v i t y of glucose i n dexamethasone treated and control f e t a l blood was measured. As observed i n Table 18, the s p e c i f i c a c t i v i t y of the f e t a l blood remained e s s e n t i a l l y the same i n both instances. This implies that the observed decrease caused by p r i o r dexa-methasone i n j e c t i o n was a true r e f l e c t i o n of glucose crossing the placenta. The purpose of such a mechanism remains unclear but one might speculate that under stressvumaternal hyperglycemia r e s u l t s , causing a hyperglycemic state i n the fetus which w i l l ultimately promote growth. This would c l e a r l y be* a disadvantage for su r v i v a l . Therefore, a mechanism to dampen t h i s e f f e c t might increase the chance of su r v i v a l , e s p e c i a l l y of the mother. Multiple injections of dexamethasone have been shown to cause f e t a l death and reabsorption (116) and increased f e t a l loss was observed during very hot weather. This suggests that 133 p r o l o n g e d s t r e s s o f t h e mother might r e s u l t i n f e t a l l o s s t h e r e b y i n c r e a s i n g t h e chance o f s u r v i v a l o f t h e mother, and t h a t t h i s i s mediated by c o r t i c o s t e r o i d s . 

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