Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1979_A1 T94.pdf [ 6.84MB ]
Metadata
JSON: 831-1.0094847.json
JSON-LD: 831-1.0094847-ld.json
RDF/XML (Pretty): 831-1.0094847-rdf.xml
RDF/JSON: 831-1.0094847-rdf.json
Turtle: 831-1.0094847-turtle.txt
N-Triples: 831-1.0094847-rdf-ntriples.txt
Original Record: 831-1.0094847-source.json
Full Text
831-1.0094847-fulltext.txt
Citation
831-1.0094847.ris

Full Text

THE  REGULATION AND ROLE OF CORTICOSTEROIDS DURING FETAL DEVELOPMENT by LESLEY MARGARET TYE B.Sc,  U n i v e r s i t y 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  t h e s i s as conforming  to the r e q u i r e d  THE  UNIVERSITY OF BRITISH April,  (S)  standard  Lesley  COLUMBIA  1979  M a r g a r e t T y e , 197 9  In p r e s e n t i n g  this thesis i n p a r t i a l  requirements f o r an advanced degree a t the  f u l f i l m e n t of U n i v e r s i t y of  Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y able  for reference  and  study.  the  I f u r t h e r agree t h a t  British avail-  permission  f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes be granted by the  Head of my  Department or by h i s  may  representatives.  I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r financial  gain s h a l l not  be allowed without my  mission .  Department of B i o c h e m i s t r y  The  U n i v e r s i t y of B r i t i s h  Columbia  Vancouver, B r i t i s h Columbia, Canada  Date:  April,  1979  written  per-  (ii)  ABSTRACT 14  F e t a l mouse txssues were incubated w i t h  C-corticosterone  3  together w i t h  H - l l - d e h y d r o c o r t i c o s t e r o n e and the s t e r o i d s were  e x t r a c t e d and separated c h r o m a t o g r a p h i c a l l y t o determine , u^f^Ctl°l1. dehydrogenation  ratio  Between g e s t a t i o n a l days 14 and ^ J  r a t i o i n p l a c e n t a i n c r e a s e d from 6.3 0.06  t o 0.63;  8.3.  i n gut from  0.1  t o 0.5;  t o 23.4;  19, '  the  from  and i n l i v e r from 0.29  In lung, the r a t i o rose from 0.16  two days l a t e r .  i n brain  the  to 6.7  to  o n l y a f t e r day  16,  Treatment of mothers 16 h e a r l i e r w i t h 200 ug  dexamethasone i n c r e a s e d the r a t i o i n lung and p l a c e n t a on day 3 but not e a r l i e r . A f t e r i n j e c t i o n of H - c o r t i c o s t e r o n e i n t o  16  3  mothers, the amount of  H - l l - d e h y d r o c o r t i c o s t e r o n e , which  was  98% of the t o t a l on day 14, d e c l i n e d , w h i l e unchanged c o r t i c o s t e r o n e i n c r e a s e d a t l e a s t t e n f o l d i n a l l t i s s u e s examined between days 14 and  19.  A r e c e p t o r has been found i n f e t a l b r a i n w i t h  K^=  3  8.3  nM which binds  p r o t e i n on day 14.  H-dexamethasone t o the extent of 0.14  The number of r e c e p t o r s i t e s d i d not i n c r e a s e  with g e s t a t i o n a l age, initiating  pmoles/mg  i n d i c a t i n g t h a t these are not c r i t i c a l i n  steroid-dependent p r o c e s s e s .  In f e t a l b r a i n  and  3  p l a c e n t a , r e c e p t o r s bound both corticosterone.  H - c o r t i c o s t e r o n e and  In c y t o s o l and nucleus, both l a b e l l e d  were d i s p l a c e d c o m p e t i t i v e l y by each o t h e r .  The  bound to chromatin  corticosterone.  s l i g h t l y more than was  Of parameters r e f l e c t i n g changing  steroids  H-ll-dehydro-  c o r t i c o s t e r o n e - r e c e p t o r complex not o n l y e n t e r e d the but was  11-dehydro-  nucleus  the hormone l e v e l s of  a c t i v e hormone i n f e t a l t i s s u e s , the i n v i t r o i n c o r p o r a t i o n 14 3 3 of C - l e u c i n e , H - u r i d i n e and H-thymidine i n t o a c i d - i n s o l u b l e  (iii)  components of t i s s u e s were the most s e n s i t i v e . a t i o n of these p r e c u r s o r s by as much as 90%.  The  The  incorpor-  decreased between days 14 and  19  d e p o s i t i o n of glycogen v a r i e d i n  d i f f e r e n t f e t a l t i s s u e s but d i d 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 w i t h 200  ug  dexamethasone 16 h e a r l i e r r e s u l t e d i n a c c e l e r a t i o n of normal p a t t e r n , producing values observed on days 15-19.  on day  steroid-induced  been t r e a t e d w i t h  appeared normal.  processes i n c l u d e d i n c r e a s e d  of f e t a l b r a i n and f e t a l l i v e r and  14 which were normally  Fetuses which had  dexamethasone i n u t e r o were born and  the c o n v e r s i o n  gut.  the  Other  amino a c i d content  of glucose to f r u c t o s e i n  I t i s concluded t h a t the r e g u l a t i o n of  c o r t i c o s t e r o i d s i s accomplished not  simply  by 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 , but by the i n t e r c o n v e r s i o n i n i n d i v i d u a l t i s s u e s of c o r t i c o s t e r o n e and  i t s 11-dehydro  The  11-dehydrocorticosterone i s not o n l y an abundant  but  serves  as a r e s e r v o i r of p o t e n t i a l hormone and  w i t h c o r t i c o s t e r o n e f o r both c y t o s o l and Since  nuclear  can  metabolite.  metabolite, compete  receptor  sites.  i t binds to chromatin, i t i s p o s s i b l e t h a t 11-dehydro-  c o r t i c o s t e r o n e e x e r t s a c t i o n s at the t r a n s c r i p t i o n a l l e v e l . C o r t i c o s t e r o i d s e x e r t t h e i r e f f e c t s on f e t a l development a t an e a r l i e r stage than has been h i t h e r t o  reported.  (iv)  TABLE OF CONTENTS  Page ABSTRACT  (ii)  TABLE OF CONTENTS  (iv)  LIST OF TABLES  (vii)  LIST OF FIGURES  (ix)  LIST OF APPENDICES  (xii)  ABBREVIATIONS USED  (xiii)  ACKNOWLEDGEMENTS  (xv)  INTRODUCTION  1  H i s t o r i c a l Review  1  Present  13  Problem  15  MATERIALS Animals  15  Buffers  15  Chemicals  15  R a d i o a c t i v e Chemicals  16  Solvents  17  Chromatography  17  Autoradiography  17  Scintillation  18  Supplies  19  METHODS I n j e c t i o n of Mice  19  Preparation of Tissues  19  Enzymatic S y n t h e s i s o f (cpd. A)  11-dehydrocorticosterone  In V i t r o S t e r o i d Incubations  20 21  (v)  Page E x t r a c t i o n Procedure  21  Chromatography  22  In V i v o Metabolism o f S t e r o i d s  23  Characterization of Steroids  23  Crystallization  23  I s o l a t i o n o f Glycogen from T i s s u e s  24  Anthrone Assay f o r Glycogen  24  I n c o r p o r a t i o n o f Leucine,  Uridine  and Thymidine  25  O r n i t h i n e Decarboxylase Assay Glucose Metabolism i n F e t a l T i s s u e Glucose and F r u c t o s e - 6 - P 0  4  .26 27  Metabolism  i n Cytosol Preparations  27  I n c o r p o r a t i o n o f Glucosamine  28  STEROID RECEPTOR ASSAYS  28  C y t o s o l Receptor P r e p a r a t i o n and Assay  28  Nuclear P r e p a r a t i o n and Assay  29  Assay o f R a d i o a c t i v i t y  30  EXPERIMENTAL RESULTS 14 1. Metabolism o f C - l a b e l l e d C o r t i c o s t e r o n e and 11-dehydrocorticosterone i n F e t a l Tissues 2. The Metabolism o f C o r t i c o s t e r o n e and 11-dehydrocorticosterone i n F e t a l Tissues on D i f f e r e n t G e s t a t i o n a l Days 3. Recovery o f cpd.^A and B a f t e r I n j e c t i o n of Mothers with  H-cpd. B  32 32 38 46  4.  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  46  5.  The ln_ V i t r o I n c o r p o r a t i o n o f Leucine, U r i d i n e and Thymidine i n t o F e t a l T i s s u e s O r n i t h i n e Decarboxylase A c t i v i t y i n P l a c e n t a  54 67  6.  (vi)  7.  Amino A c i d A n a l y s i s o f F e t a l B r a i n  8.  Investigation of Glycoprotein  9.  V a r i a t i o n i n the A c i d I n s o l u b i l i t y bf DNA from F e t a l T i s s u e s  10.  Studies on C o r t i c o s t e r o i d Receptors in F e t a l Tissues  DISCUSSION BIBLIOGRAPHY APPENDICES PUBLICATIONS  Synthesis  (vii) LIST OF TABLES  SUBSTRATE INCORPORATION WITH TIME  2  CHARACTERIZATION OF 11-DEHYDROCORTICOSTERONE (AS THE C-21 ACETATE) FROM BRAIN  35  EFFECT OF DEXAMETHASONE INJECTION ON THE ACTIVITY OF STEROID C - l l OXIDOREDUCTASE ON GESTATIONAL DAY 14  44  EFFECT OF DEXAMETHASONE INJECTION ON THE ACTIVITY OF STEROID C - l l OXIDOREDUCTASE ON GESTATIONAL DAY 16  45  EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON GLYCOGEN DEPOSITION ON GESTATIONAL DAY 16  53  EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON LEUCINE INCORPORATION IN FETAL TISSUES  63  EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON URIDINE INCORPORATION IN FETAL TISSUES  64  EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON THYMIDINE INCORPORATION IN FETAL TISSUES  65  9  SUMMMARY OF FETAL TISSUE REDUCTASE DEHYDROGENASE (D) ACTIVITIES  66  10  INCORPORATION OF C-GLUCOSAMINE INTO FETAL LIVER AND GUT  3  4  5 6 7 8  11  INTO FETAL BRAIN  Page  1  (R) AND  26  14  73  THE EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS GIVEN H-THYMIDINE ON THE PRECIPITATION OF RADIOACTIVE MATERIAL FROM FETAL TISSUES TREATED WITH PCA 82 3  12 13 L4 15 16  PROPERTIES OF CORTICOSTEROID RECEPTORS(S) IN FETAL MOUSE BRAIN  84  EFFECT OF VARIOUS FACTORS ON BINDING OF H-CPD. B IN CYTOSOL  86  COMPETITIVE DISPLACEMENT OF LABELLED STEROID IN CYTOSOL  88  COMPETITIVE DISPLACEMENT OF STEROIDS BOUND IN NUCLEAR FRACTIONS  97  CHARACTERIZATION OF STEROID FROM RECEPTORCOMPLEXES  98  3  (viii)  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  (ix)  LIST OF FIGURES  Page QUENCH CURVE FOR H AND 3  AUTORADIOGRAM  1 4  C  31  OF CHROMATOGRAPHED EXTRACTS:OF FETAL  BRAIN INCUBATED WITH C-CORTICOSTERONE  34  14  3  AUTORADIOGRAM  OF CHROMATOGRAPHED EXTRACTS OF MOUSE  FETAL LIVER AND PLACENTA INCUBATED WITH  14  C-11-  DEHYDROCORTICOSTERONE 4  37  IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE IN FETAL BRAIN AND GUT  5  39  IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE IN PLACENTA  6  40  IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE IN FETAL LIVER AND LUNG  7  41  RECOVERY OF CPD. A AND B IN FETAL BRAIN, GUT AND HEART AFTER INJECTION OF H-CPD. B  48  3  8  RECOVERY OF CPD. A AND B IN FETAL LIVER, LUNG AND PLACENTA AFTER INJECTION OF H-CPD. B 3  9  STANDARD CURVE FOR THE DETERMINATION OF GLYCOGEN BY THE ANTHRONE METHOD  10  50  GLYCOGEN CONTENT OF FETAL BRAIN,GUT AND HEART ON GESTATIONAL DAYS 14 TO 19  11  48  51  GLYCOGEN CONTENT OF FETAL LIVER, LUNG AND PLACENTA ON GESTATIONAL DAYS 14 TO 19  52  (x)  Page 12  IN VITRO INCORPORATION BRAIN, GUT AND  13  14  15  16  17  18  ORNITHINE  C - L E U C I N E INTO F E T A L 56  OF H-URIDINE 3  INTO F E T A L 57  OF H-URIDINE 3  INTO F E T A L  PLACENTA  58  OF H-THYMIDINE INTO F E T A L 3  HEART  60  IN VITRO INCORPORATION LIVER, LUNG AND  1 4  HEART  IN VITRO INCORPORATION BRAIN, GUT AND  OF  PLACENTA  IN VITRO INCORPORATION LIVER, LUNG AND  C - L E U C I N E INTO F E T A L 55  IN VITRO INCORPORATION BRAIN, GUT AND  1 4  HEART  J_N VITRO INCORPORATION LIVER, LUNG AND  OF  OF THYMIDINE  INTO F E T A L  PLACENTA  DECARBOXYLASE  61 ACTIVITY ON  DAYS 14 TO 19 IN PLACENTA AND  GESTATIONAL  THE E F F E C T OF  DEXAMETHASONE INJECTION 19  AMINO ACID ANALYSIS OF F E T A L BRAIN ON DAYS 14, 15 AND  20  68  DAY  GESTATIONAL  14 DEX-TREATED  72  AUTORADIOGRAM OF CHROMATOGRAPHED EXTRACTS  OF  F E T A L GUT 21  76  THE PERIODATE-RESORCINOL  REACTION FOR BOUND  S I A L I C ACID PERFORMED ON PCA PRECIPITATES OF FETAL GUT, 22  LUNG, LIVER AND  PLACENTA  UV SCAN OF PCA P R E C I P I T A T E REDISSOLVED BASE  78 IN DILUTE 80  (xi)  23  PRECIPITATION OF F E T U S E S 3  24  BY P C A FROM  REMOVED FROM MOTHERS  INJECTED  TISSUES  WITH  H-THYMIDINE  ISOLATION  OF R E C E P T O R C O M P L E X E S FROM  ON S E P H A D E X  25  OF R A D I O A C T I V I T Y  G-25  AUTORADIOGRAM PLACENTA  PLACENTA  OF CHROMATOGRAPHED 14  I N C U B A T E D WITH  EXTRACTS  OF  C - C P D . A AND 1 1 -  KETOPROGESTERONE 26  NUCLEI  I S O L A T E D FROM F E T A L  SHOCK WITH D I L U T E  27  ISOLATION  OF R E C E P T O R  ON S E P H A D E X  28  BY H Y P O T O N I C  2  C O M P L E X E S FROM F E T A L  BRAIN  G-25  AUTORADIOGRAMS ACETYLATED  MgCl  BRAIN  OF CHROMATOGRAPHED  DERIVATIVES  FROM MATERNAL AND  OF  1 4  EXTRACTS  OF T H E  C - G L U C O S E RECOVERED  F E T A L MOUSE  BLOOD  (xii)  L I S T OF APPENDICES  Page APPENDIX I  12 3  APPENDIX I I  124  (xiii)  ABBREVIATIONS USED  cpd. A  21-Hydroxy-4-pregnene-3,11,20-trione, 11-dehydrocorticosterone  cpd. B  113,21-Dihydroxy-4-pregnene-3,20-dione, corticosterone  Dex  (see Appendix I)  (see Appendix I)  9aFluoro ,113,1 7a., 21 -Trihydroxy-1 ,4-pregnadiene3,20-dione, Dexamethasone  (see Appendix I)  DNA  deoxyribonucleic  DNAse  deoxyribonuclease  dpm  d i s i n t e g r a t i o n s per minute  EDTA  (ethylenedinitrilo)-tetraacetic tetrasodium  11-Epi B  acid  acid,  salt  11a,21-Dihydroxy-4-pregnene-3,20-dione, 11-Epicorticosterone  (see Appendix I)  Kd  dissociation  constant  KEB  Krebs-Eggleston  bicarbonate  KEP  Krebs-Eggleston  phosphate  buffer  buffer  (xiv)  11-KP  4-Pregnene-3,11,20-Trione, 11-ketoprogesterone  (see Appendix I)  min  minute  PCA  perchloric  RNA  ribonucleic  RNAse  ribonuclease  SEM  standard e r r o r of the mean  TCA  {  acid  acid  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  relative centrifugal  force  C-11 s t e r o i d oxidoreductase w i l l be used f o r the r e v e r s i b l e enzyme  113-hydroxysteroid dehydrogenase, EC 1.1.1.146  corticosterone  11-dehydrocorticosterone  (xv) ACKNOWLEDGMENTS I would l i k e t o express my g r a t i t u d e t o the many people who a s s i s t e d me d u r i n g my r e s e a r c h .  In t h i s Department, I  am g r a t e f u l t o Dr. J.F. R i c h a r d s f o r the o r n i t h i n e decarboxylase determinations and Mr. Joe Durgo f o r the amino a c i d  analysis.  Thanks are due t o Dr. M.E. Todd f o r her m i c r o s c o p i c examination of f e t a l t i s s u e s , and t o Dr. N. Auersperg f o r photographing the n u c l e i i n F i g . 26.  Both are members of the Department o f  Anatomy, U n i v e r s i t y o f B r i t i s h Columbia.  I am a l s o i n d e b t e d  to Mr. C. Remey o f the Department o f Pathology f o r a s s i s t a n c e w i t h the p e r i o d a t e - r e s o r c i n o l data. I a l s o wish t o thank my f a m i l y f o r t h e i r support and unders t a n d i n g , p a r t i c u l a r l y i n the l a s t s i x months o f my r e s e a r c h . Finally,  I have g r e a t p l e a s u r e i n e x p r e s s i n g my indebtness  to Dr. A.F. Burton f o r h i s guidance, encouragement and sense o f humour which were i n v a l u a b l e throughout my s t u d i e s . I was a r e c i p i e n t of the U n i v e r s i t y o f B r i t i s h S c h o l a r s h i p - F e l l o w s h i p award  (1977-78).  Columbia  1  INTRGDUCTI CN  The  fetus  nal  sources  for  g r o w t h and  by  the  cross the  during  f o r a l l the  placenta  opposite  As  e a r l y as  of  fuel  the  of  i n c l u d i n g man  of  the and  implied  f o r the  total  the  acids and  necessary  are  received  u m b i l i c a l cord.  They  waste p r o d u c t s pass  in  fetus lambs  fetal  and  that  (1).  an  observed  Measurements o f  of  the  i t may belief  rates  (2);  enough I f most  the p r i n c i p a l  i s strengthened  placenta  ketone bodies  mouse, g l u c o s e  be  the  i t is fast  oxygen consumption  This  fetal  c a r b o h y d r a t e was  suggest that  oxidized,  fetus.  that  of d i v e r s e  ( 3 ) , and  i s considered  i n many t o be  by  species species,  the  major  fuel.  T h e r e i s an (4).  The  i n t e r e s t i n g species placenta  verting maternal glucose into  1.0  impermeability  free fatty  lates  and  Bohr s u g g e s t e d  i s in fact  fuel  relative  fetal  energy supply  These n u t r i e n t s  fetus,  in fetal  f o r the  glucose  metabolic  to  19 31,  glucose uptake  the  to the  quotient  sole metabolic  to account  placenta  and  i s d e p e n d e n t upon m a t e r -  direction.  respiratory  of  nutrients  differentiation.  f e t u s v i a the the  intrauterine l i f e  the  fetal  to e x i s t i n the synthesized  has to  circulation.  an  The  i n ungu-  e f f i c i e n t mechanism f o r c o n -  f r u c t o s e , which  human p l a c e n t a  is negligible.  p e c u l i a r i t y found  i s then  same c a p a b i l i t y has (5, 6 ) , b u t  the  secreted been  amount o f  shown fructose  2  The r o l e of glucose as the predominant f e t a l energy has been c h a l l e n g e d r e c e n t l y i n f e t a l sheep  ( 7 ) , but any  source alter-  n a t i v e m e t a b o l i c f u e l has not been d e f i n e d as y e t .  The p l a c e n t a l t r a n s p o r t of glucose has been w e l l characterized.  Transport has been shown t o be by " f a c i l i t a t e d  f u s i o n " i n sheep (8) and human (9) p l a c e n t a .  dif-  Transport i s i n  the same d i r e c t i o n as f o r a d i f f u s i o n g r a d i e n t , but the r a t e exceeds t h a t which would be p r e d i c t e d from d i f f u s i o n  alone.  Since f e t a l blood glucose i s u s u a l l y one-half t h a t of the mother, glucose normally passes  from mother t o f e t u s .  Glucose t r a n s p o r t i s mediated by a c a r r i e r ,  presumably  p r o t e i n i n n a t u r e , which can b i n d glucose r e v e r s i b l y and t r a n s p o r t i t a c r o s s the p l a c e n t a l membrane.  This c a r r i e r  exhibits  s a t u r a t i o n k i n e t i c s at h i g h glucose c o n c e n t r a t i o n s and shows some p r e f e r e n c e f o r g l u c o s e , although other sugars may competitive i n h i b i t o r s  (10).  a c t as  I t a l s o shows a degree of s t e r e o -  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 t r a n s ported much f a s t e r than t h e i r corresponding L-isomers  (11).  Transport i s u n l i k e l y an a c t i v e process s i n c e no e f f e c t of metabolic p o i s o n has been demonstrated ( 1 0 ) .  Other p o s s i b l e sources of f u e l f o r the f e t u s are amino a c i d s and l i p i d s .  The  f e t u s , although i t r e c e i v e s maternal  amino a c i d s , l i k e l y conserves them f o r p r o t e i n s y n t h e s i s , s i n c e little (12)  t r a n s a m i n a t i o n or deamination  occurs d u r i n g f e t a l  and i n s e v e r a l s p e c i e s gluconeogenesis  life  has been shown t o  3  commence (EC  only  after birth  (13). Phosphopyruvate  4.1.1.32) i s c o n s i d e r e d  t o be t h e l i m i t i n g  carboxykinase  enzyme i n t h i s  pathway ( 1 4 ) .  The small.  amount o f l i p i d Cholesterol  triglycerides rier  made a v a i l a b l e t o t h e f e t u s  t r a n s f e r i s s l o w , and p h o s p h o l i p i d s  have n o t been shown t o c r o s s  i n t a c t i n any s p e c i e s  transfer  i s virtually  amount o f f a t t y species.  restricted  i n sheep  (17) and human  has  b e e n shown t o o x i d i z e lipid  r a t i s able  placenta,  involved  (20) , b u t t h e i r  gestation  ovaries  during  with longer tions  since  among  i n guinea p i g (16), but t h e mammalian  very  slowly  a major f u e l  to synthesize  fetus  (19), i t i s  f o r the fetus.  r o l e i n the f e t a l  has  economy  i t has b e e n o b s e r v e d t h a t t h e the greater  as w e l l  as s e r v i n g  i n hormone s y n t h e s i s  endocrine organ. short  a c i d s , and t h e  part  of i t s n u c l e i c  de novo ( 2 1 ) .  The also  acids  i s not l i k e l y  r e m a i n s as y e t u n d e f i n e d ,  acid  fatty  Since  bar-  Thus,  placental transfer of nucleic acid precursors  been d e m o n s t r a t e d  fetal  so f a r ( 1 5 ) .  to free fatty  (18).  and  the placental  acid transferred varies considerably  low  The  investigated  F o r example, t r a n s f e r i s h i g h  presumed t h a t  i s very  a transport  and c a n be c o n s i d e r e d  I t h a s been o b s e r v e d t h a t times  gestation  are s u f f i c i e n t l y  i n animals  ( l e s s t h a n two m o n t h s ) ,  pregnancy leads periods, well  function, i s  to abortion.  with  removal o f the But i n animals  the placental endocrine  developed  an  func-  e a r l y i n pregnancy t o  4  permit maintenance o f g e s t a t i o n a f t e r (22).  The e a r l i e s t  without but  time  a t which ovariectomy  t e r m i n a t i o n o f pregnancy  i n man  ovariectomy  implantation,  c a n be  performed  varies with d i f f e r e n t  i t i s known t o be a t f i v e weeks.  that, whilst p i t u i t a r y  o f mothers  This  hormones a r e n e c e s s a r y  species,  suggests  f o r o v u l a t i o n and  p l a c e n t a l hormones c a n p l a y an i m p o r t a n t r o l e i n  the c o n t i n u a t i o n o f pregnancy. periods, maternal  In species o f s h o r t g e s t a t i o n a l  hormones p r e d o m i n a t e ;  estrogens are maternal  i . e . , progesterone  and  in origin.  T h r e e m a j o r p e p t i d e hormones a r e s y n t h e s i z e d by t h e p r i m a t e placenta.  These a r e c h o r i o n i c  and c h o r i o n i c t h y r o t r o p i n . still  not c l e a r l y  t o be p r i m a r i l y a relatively  The r o l e  defined.  impermeable b a r r i e r  i n the fetus  lactogen  side,  their  e f f e c t would  is  appear  s i n c e t h e p l a c e n t a forms  and t h e c o n c e n t r a t i o n o f t h e s e  b l o o d i s many t i m e s h i g h e r t h a n  (23).  the s y n t h e s i s o f s t e r o i d  placental  these p l a y i n pregnancy  Certainly  on t h e m a t e r n a l  hormones i n t h e m a t e r n a l observed  gonadotrophin,  The p l a c e n t a i s a l s o  that  involved i n  hormones i n c l u d i n g p r o g e s t e r o n e a n d  estrogens (24).  I n g e n e r a l , compounds rapidly weight. and  a c r o s s t h e p l a c e n t a l membrane t h a n l a r g e r Uncharged m o l e c u l e s  fat-soluble  solubility. systems.  of small molecular size  c r o s s more r a p i d l y  s u b s t a n c e s more  rapidly  diffuse molecular  than  ionized,  t h a n t h o s e o f low  These a r e g e n e r a l p r o p e r t i e s o b s e r v e d  more  lipid  f o r membrane  5  Compounds o f m o l e c u l a r cross  the placenta  placenta,  only  therefore,  by p r o c e s s e s  1000  as y e t u n d e f i n e d .  sufficiently  s e n s i t i v e techniques  t h a t t h e t r a n s f e r i s t o o slow t o a f f e c t  cantly,  and c l i n i c a l  and e x p e r i m e n t a l  inadequacy o f such t r a n s f e r . adrenal  hypoplasia  hormone the  i t i s generally  the fetus  fetuses  and  hormone  (25) and t h e f a i l u r e  appear t h a t the f e t a l  f u n c t i o n s autonomously.  insulin  (29) and g r o w t h hormone  present  i n t h e human f e t a l  (26)  (TSH), and t h e r e f o r e the p l a c e n t a l  insufficiency.  endocrine  In f a c t ,  fetuses  adrenocorticotrophic  o f t h e m a t e r n a l hormones t o c r o s s  and compensate f o r f e t a l  therefore  signifi-  situations indicate the  of maternal  (ACTH) and t h y r o t r o p h i c  membrane  hormones.  F o r example, t h e o c c u r r e n c e o f  i n anencephalic  the u n a v a i l a b i l i t y  inability  maternal  some t r a n s f e r  o f n o r m a l t h y r o i d d e v e l o p m e n t shown i n d e c a p i t a t e d indicate  The  impermeable t o m a t e r n a l p e p t i d e  o f m a t e r n a l hormones c a n u s u a l l y be d e m o n s t r a t e d , held  daltons  though f r e e l y permeable t o s m a l l  hormones, i s r e l a t i v e l y Although with  weight exceeding  ACTH  I t would  system matures ( 2 7 ) , TSH  early  (28) ,  (30) have b e e n shown t o be  circulation  as e a r l y as t h e t e n t h  week o f g e s t a t i o n .  In readily; mouse  some s p e c i e s , e . g . , sheep  c o r t i c o s t e r o i d s do n o t c r o s s  (31).  In others,  notably  (32), maternal c o r t i c o s t e r o i d s c r o s s  man  the placenta (24) and  r e a d i l y to the fetus.  What a c t u a l l y d e t e r m i n e s t h e amount o f 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 n o t known, b u t t r a n s c o r t i n i s b e l i e v e d  a role.  This  increase  during  (33),  and t h i s  to play  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 t o pregnancy:or a f t e r a d m i n i s t r a t i o n i s consistent with  an o b s e r v e d  of estrogens  increased  maternal  6  level (34)  o f hormone i n humans and  species.  have shown t h a t t r a n s c o r t i n l e v e l s  n a n c y i n r a b b i t , r a t and  mouse.  l e v e l s were shown t o i n c r e a s e  Both the (35) , t h e single and  other  mouse and  placenta  being  estrogens,  are  cross  the  known t o be  s u l f a t a s e so  f e t u s and unit  biosynthetic could  only  a  maternal  activity  fetus.  since  the  be  A  the  i n primates.  does n o t  fetal  The  cleaved  and  maternal blood.  The  nor  i n other  species,  con-  (36).  "feto-  u n i t c a r r i e s out placenta  has  the  placental barrier  This  adrenal  placenta  f o r example, e s t r o g e n hold  predomi-  These s u l f a t e d com-  inactive.  mouse, where e s t r o g e n s and  origin.  androgens,  form a f u n c t i o n u n i t , the  p o s s i b l y complete; concept  the  (24).  i n t o the  polar to cross  (24)  by  s u l f a t e group can  placenta  s u c h as  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  r e a c t i o n s which n e i t h e r the  This  example t h e maternal  the  .11.  placenta  i n o r i g i n with  placenta  biologically  the  form i s too  placental"  thesis.  fetal  is sulfurylation,  u n c o n j u g a t e d hormone r e l e a s e d  alone  preg-  mouse, t r a n s c o r t i n  endothelium separating  active sulfokinase  pounds a r e  The  during  have a h e m o c h o r i a l t y p e o f  extensively metabolized  n a n t mechanism i n v o l v e d  jugated  Westphal  r a p i d l y a f t e r g e s t a t i o n a l day  c a t e c h o l a m i n e s and,  32),  active  and  circulations.  Hormones w h i c h can  contains  increase  In the  entirely  l a y e r of c a p i l l a r y  fetal  (24,  man  Gala  steroid  fetus biosynfor  progesterone are  of  7  A feature of  corticosteroids.  In both there  gically been  inactive.  i s extensive  However, whereas  shown t o be t h e m a j o r  (37),  site  i n man  conversion  which  i s biolo-  the placenta  of dehydrogenation  i n t h e mouse d e h y d r o g e n a t i o n  of  of  of corticosterone  has Cortisol  occurs i n  head ( 3 8 ) .  the  In can  several  the level  of corticosterone  i n maternal blood  maternal  placenta  Michaud  t o t h e mother  and Burton  synthesize  After  injection  mothers, tissue  the fetal  of labelled corticosterone  Cortisol  mouse  adrenal  and  fetal  or fetal  regard-  origin, the  i n both d i r e c t i o n s i s rapid (38).  fetus  Cortisol  i s higher  i n the umbilical artery  by t h e u m b i l i c a l v e i n ,  i n the fetus.  adrenalectomized  indicating that,  (40) h a v e shown t h a t  t h e human  into  o f hormone i n m a t e r n a l  the placenta  Murphy and Leong  entering  cross  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 day 16-17 ( 3 8 ) .  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  leaving  has t h e a b i l i t y  (39).  the fetal  w a s t h e same a s i n i n t a c t m i c e ,  blood  shown  o n d a y 18  t h e hormone c a n  from t h e fetus  showed t h a t  the distribution  movement a c r o s s  i thas been  r a t adrenal  c o r t i c o s t e r o i d s and t h a t  could  the fetus  a d r e n a l e c t o m y , w a s t h e same a s i n t h e i n t a c t  indicating that  synthesize  that  that  F o r example,  mothers,  less  i t has been e s t a b l i s h e d  corticosteroids.  following  the  species,  synthesize  that  in  a n d mouse i s t h e m e t a b o l i s m  hormones t o t h e 11-dehydro m e t a b o l i t e ,  the  to  s i m i l a r i n b o t h man  suggesting  In view of the large  synthesis  amount o f  than of  cortisone  8  present  and t h e e x i s t e n c e  to Cortisol that in  a small  fetal  for this it  in fetal  o f an enzyme c a p a b l e  liver  and f e t a l  change i n t h e r e d u c t i o n  t i s s u e s r a t h e r than demovo increment.  are  of cortisone to Cortisol synthesis  The s i g n i f i c a n c e o f t h i s  tissues, regardless  of maternal or f e t a l  fetal  origin,  could  been e s t a b l i s h e d o n l y  proposal  i s that  concentration  o f whether t h e s t e r o i d s  without  involvement of the  protein  receptor.  cytoplasmic  of action of steroid  i n recent  i n "target" tissue,  binds  account  pituitary-adrenal axis.  D e t a i l s o f t h e mechanism  take  this  (41) , i t i s p o s s i b l e  s u g g e s t s p o s s i b l e q u a n t i t a t i v e changes i n the  o f hormone i n f e t a l  years.  the s t e r o i d  i s bound  In a temperature-dependent  to a s p e c i f i c  template  the  cell  site  activity  on c h r o m a t i n .  and t h e s t e r o i d  cellular  to a  have up-  cytoplasmic  r e a c t i o n , the  a v a r i e t y of f e t a l  glucocorticoid  receptors  This binding then e x e r t s  by m o d i f i c a t i o n a t t h e l e v e l  Since  Following  hormones  s t e r o i d - r e c e p t o r c o m p l e x moves i n t o t h e n u c l e u s  DNA  the  lung  of reducing  alters  i t s effect  and the on  of t r a n s c r i p t i o n (42).  t i s s u e s have been o b s e r v e d t o have  (4 3 ) , t h e s e  tissues could  p o s s i b l y have  c a p a c i t y t o r e s p o n d t o g l u c o c o r t i c o i d s and t h e p r e n a t a l  a d m i n i s t r a t i o n o f these developmental  Specific Ballard small  hormones  could  conceivably  a l t e r the  process.  glucocorticoid receptors  and B a l l a r d t o be p r e s e n t  intestine,  kidney,  have b e e n shown by  early i n fetal  life  heart.,., m u s c l e , s k i n and l u n g  in liver, o f human  9  fetuses  (43). Both B a l l a r d  M u l a y .and Solomon ..(4 5) glucocorticoid rabbit  lung.  and B a l l a r d  have shown t h e p r e s e n c e o f c y t o p l a s m i c  receptors  f r o m day 18 o f g e s t a t i o n i n f e t a l  These o b s e r v a t i o n s  an  active receptor  of  glucocorticoid  ( 4 4 ) , and G i a n n o p o u l o s ,  i n d i c a t e t h a t the presence o f  system i s n o t a l i m i t i n g responsiveness  factor  i n the onset  i n , f o r example, t h e f e t a l  l u n g , which undergoes s t e r o i d - i n d u c e d changes a t t h i s The  significance  the t i s s u e  of the receptor  t h a t on f e t a l  liver  Claude Bernard  i n mammalian  stages  glycogen  of gestation.  deposition.  (46) d e m o n s t r a t e d  and t h a t t h i s  appearance i n t h i s  tissue  at a relatively  stimulated  the  fetal  liver  prematurely  not the only  tated  century  i s present  p o l y s a c c h a r i d e makes i t s late  stage  of gesta-  liver  glycogen  levels  is a shortly  b i r t h (47).  Although  is  a  i n v e s t i g a t e d so f a r t h e r e  marked a n d r a p i d i n c r e a s e i n f e t a l before  One e f f e c t i s  that glycogen  liver,  I n a l l mammalian s p e c i e s  in fetal  More t h a n  fetal  tion.  lung.  have b e e n shown t o p l a y a r o l e  development i n the l a t t e r  ago,  i n terms o f t h e development o f  has o n l y been w e l l e s t a b l i s h e d i n f e t a l  Corticosteroids  time.  factor  glycogen  d e p o s i t i o n i s known t o be  by c o r t i c o s t e r o i d involved.  glycogen  content  c o s t e r o i d s were f o u n d  (48, 4 9 ) ,  i t  J o s t h a s shown t h a t i f d e c a p i -  r a t fetuses of adrenalectomized  liver  injection  mothers a r e g i v e n  i s increased.  t o have no e f f e c t  In r a b b i t s ,  Cortisol,  corti-  on g l y c o g e n  storage  pituitary  when e x t r e m e l y  l a r g e d o s e s were g i v e n .  O n l y when a  prolactin-like  substance  the c o r t i c o s t e r o i d s  was g i v e n w i t h  even  was  10  any  glycogen  trol  deposition  e f f e c t e d by  apparent„ ( 2 5 ) .  c o r t i c o s t e r o i d s and  been p o s t u l a t e d  for deposition  studies  by  (50)  ance of  the  glycogen,  Aron  islets  which  Liver  of  the  Langerhans  only  have been observed  (47).  The  appearance of circulation  a  late  explain  deposition  earlier  time.  far  No  f o r muscle  have  of  heart  (4 7 ) .  These  energy  during  on  birth,  stores  of  free  24  hours  before  fatty  hormone  increase for  of  heart  stage  of  of  glycogen  i n lung,  appear-  fetal  liver  insulin.  glycogen. and  Sizable  skeletal  i n each d i f f e r s ,  gestation  has  Histological  and  a d r e n o c o r t i c a l hormones  the  i n the  i n rodents heart  muscle  and  fetal  does  not  muscle  at  i n f l u e n c e s have been r e c o g n i z e d  deposits  (47), but  g l y c o g e n may  be  decrease  values  Picon  and  i n f l u e n c e d by  an so  Bouhnik adreno-  a  glucose  a c i d i n the  after birth  (53).  period,  f o r energy  stores  stores  to  three  10%  hours of  r e a d i l y a v a i l a b l e source  early neonatal  these  rapidly, reaching  w i t h i n two  represent  i t s carbohydrate  but  role  store  i n lung,  stores  d e n t upon c i r c u l a t i n g exist  and  the  con-  (51).  initial  the  and  deposition  lung  glycogen  their  fetal  hormonal  hormones  Fetal less  of  and  shown t h a t  cortical  of  insulin  at  hormonal  a pituitary  glycogen.  a possible  stores  pattern  dual  i n d i c a t e a c o r r e l a t i o n between the  implies  i s not  of  A  the  (52).  for three  are  plasma  as  to  exhausted  rises,  brain The  four the  reaching  is  or birth of depen-  newborn hours  can  after  concentration a peak  six  to  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 synthesized depots.  from glucose and  deposited  are  i n the l i v e r and  fat  I t i s these s t o r e s which are u t i l i z e d by the newborn  a f t e r carbohydrate s t o r e s are exhausted. l i p i d storage  A f u r t h e r type of  i s found i n brown adipose t i s s u e .  This  store  seems to have a f u n c t i o n i n r e l a t i o n t o postpartum thermoregulation  (54).  Lachance and Page have shown t h a t  steroid administration t i s s u e f a t content  leads to an i n c r e a s e  antenatally include:  (EC 2.1.1.28) (58). and  stimulated  mitochondrial  by c o r t i c o s t e r o i d s  1-alanine-glyoxylate  (EC 2.6.1.2) (56); glycogen synthetase  (EC 2.4.1.11) (57)  retarded  i n brown adipose  (55).  Enzymes which are known to be  aminotranferase  cortico-  and  phenylethanolamine-N-methyltransferase  The  development of the a d r e n a l medulla  the content of epinephrine  was  low  in  decapitated  rat., f e t u s e s , but c o u l d be r e s t o r e d by a d m i n i s t r a t i o n  of ACTH  to f e t u s e s or of C o r t i s o l t o e i t h e r mother or f e t u s e s  Experimental work has  (25).  shown t h a t the maturation o f  lamb lungs can be a c c e l e r a t e d e i t h e r by s t i m u l a t i o n of f e t a l adrenal  c o r t e x or by a d m i n i s t r a t i o n  s t e r o i d s ' (59) .  Liggins  (59)  was  of a d r e n a l  fetal the  cortico-  suggested t h a t these s t e r o i d s caused  l i b e r a t i o n of s u r f a c t a n t i n t o the a l v e o l i , perhaps by  induction  of an enzyme i n v o l v e d i n s u r f a c t a n t b i o s y n t h e s i s .  was  reported  by F a r r e l l and  Zachman (60)  t h a t i n j e c t i o n of r a b b i t -  fetuses with a synthetic c o r t i c o i d increased c h o l i n e phosphotransferase  It  the a c t i v i t y  (EC 2.7.8.2) i n the lung, but  of as  12  a c t i v i t y o f t h i s enzyme d e c l i n e s normally i n l a t e g e s t a t i o n , i t seems u n l i k e l y t h a t t h i s c o u l d be a c r i t i c a l r e c e n t l y , the a c t i v i t y of p h o s p h a t i d i c (EC 3.3.1.4) has and was  betamethasone the r e g u l a t o r y  (63).  (61).  More  a c i d phosphatase  been found t o i n c r e a s e  stimulated  step  i n the f e t a l lung  by treatment w i t h the s y n t h e t i c  (62)  corticoid,  I t appears, t h e r e f o r e , t h a t t h i s enzyme i s  f a c t o r f o r surfactant synthesis during  lung  development.  L i g g i n s has  reported  on the c l i n i c a l  i n a s e r i e s of p a t i e n t s who (64).  threatened  use of betamethasone  t o d e l i v e r prematurely  T h i s study i n d i c a t e d t h a t i n f a n t f e t a l lungs can  appa-  r e n t l y be induced to mature s u f f i c i e n t l y when t r e a t e d i n t h i s way,  s i n c e the i n c i d e n c e  of r e s p i r a t o r y d i s t r e s s syndrome  lowing these premature d e l i v e r i e s was what would be  fol-  decreased d r a s t i c a l l y  from  expected.  In sheep, i t has been demonstrated t h a t a f u n c t i o n a l p i t u i t a r y - a d r e n a l a x i s i s necessary f o r the onset of  labor,  s i n c e an i n f u s i o n of ACTH or of c o r t i c o s t e r o i d s i n t o f e t a l lambs induced premature p a r t u r i t i o n (65). normal b i r t h there  i s an i n c r e a s e  steroid concentration, lation. of the  unrelated  During the week before  i n the f e t a l plasma c o r t i c o to t h a t i n the maternal  Pregnancy i n the sheep can be prolonged by fetus  (66) .  adrenalectomy  This d i r e c t p a r t i c i p a t i o n of the f e t u s i n  the onset o f l a b o r has  been demonstrated i n s e v e r a l s p e c i e s ,  not i n primates, although human anencephaly i s a s s o c i a t e d prolonged g e s t a t i o n  circu-  (67).  Normal c o n c e n t r a t i o n s  of  but  with  Cortisol  13  have been observed i n c o r d b l o o d and amniotic f l u i d i n anenc e p h a l i c cases sol  (68).  T h e r e f o r e , some f a c t o r other than  Corti-  must be r e s p o n s i b l e f o r f a i l u r e o f these pregnancies to  terminate.  Observations on rhesus monkeys showed t h a t n e i t h e r  f e t a l adrenalectomy  (6 9)  nor i n j e c t i o n o f s y n t h e t i c  (70) c o u l d induce p a r t u r i t i o n prematurely.  corticoids  Thus, i n primates,  c o r t i c o s t e r o i d s are not alone a s u f f i c i e n t f a c t o r f o r the induction of p a r t u r i t i o n .  C o r t i c o s t e r o i d s , thus, have been shown t o p l a y r o l e s i n s e v e r a l aspects o f f e t a l development.  They appear t o be  res-  p o n s i b l e f o r the i n d u c t i o n of v a r i o u s enzymes, f o r maturation of  f e t a l lung and a d r e n a l medulla, f o r d e p o s i t i o n of glycogen  in  f e t a l l i v e r and,  turition.  i n some s p e c i e s , f o r the i n i t i a t i o n of par-  These e f f e c t s are a l l observed i n ' t h e l a t e stages o f  g e s t a t i o n , and the o n l y c o r t i c o s t e r o i d - i n d u c e d  e f f e c t that  has  been documented i n e a r l i e r pregnancy i s the i n d u c t i o n of c l e f t p a l a t e , which was  a t t r i b u t e d t o excess c o r t i c o i d s , i n s e v e r a l  s p e c i e s i n c l u d i n g the mouse (71).  Beyond t h i s , l i t t l e  else i s  known o f the r o l e of these hormones i n e a r l y f e t a l development.  The purpose  o f t h i s study was t o e x p l o r e the metabolism  of  c o r t i c o s t e r o i d s i n f e t a l mouse t i s s u e s a t v a r i o u s stages of g e s t a t i o n and t o examine the p o s s i b i l i t y t h a t d i f f e r e n t might r e g u l a t e the l e v e l of a c t i v e c o r t i c o s t e r o i d 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 .  independently  The mouse was  an experimental model s i n c e i t , l i k e man,  tissues  chosen  as  has a hemochorial  p l a c e n t a across which c o r t i c o s t e r o i d s are t r a n s f e r r e d  readily  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  results  has  i t s own  and  can  particular  regulate  Therefore, not  t o be  d e s c r i b e d show t h a t e a c h f e t a l pattern of c o r t i c o s t e r o i d  i t s corticosterone content  the p i t u i t a r y - a d r e n a l  a major f a c t o r  in fetal  axis,  thymidine  t o be  incorporation into  r e g u l a t e d by  much e a r l i e r  metabolism  independently.  although  functional,  corticosterone regulation.  Various biochemical parameters and  s u c h as  fetal  corticosteroids  leucine,  t i s s u e s were  a t a stage  w h i c h have been r e p o r t e d t o o c c u r i t was  found  uridine shown  i n development  than most o t h e r c o r t i c o s t e r o i d - i n d u c e d  Filially,  tissue  events  prenatally.  that 11-dehydrocorticosterone,  major m e t a b o l i t e o f c o r t i c o s t e r o n e which i s c o n s i d e r e d ogically both  cytosol  observed This  inactive, and  actually  nuclear levels.  t o b i n d to the  raises  chromatin  the p o s s i b i l i t y  may  s e r v e a more p o s i t i v e  has  hitherto  been  competed  that  role  considered.  biol-  for receptor sites  T h i s compound was fraction  o f the  at  also  nucleus.  11-dehydrocorticosterone  in fetal  development  a  than  is  15  MATERIALS  Animals  Mice wereoof the non-inbred UBC  Swiss s t r a i n r a i s e d at  Department of Zoology V i v a r i u m at t h i s u n i v e r s i t y . t i o n p e r i o d was  19 days.  Day  mating, on which a plug was 1800  and  1 was  defined  observed.  The  as the day  the  gestafollowing  Matings were made between  0700 h.  Buffers  Krebs-Eggleston b u f f e r s c o n t a i n  0.154M s o l u t i o n s of  f o l l o w i n g s a l t s , mixed i n the r e l a t i v e p r o p o r t i o n s NaCl-100; KC1-4; MgSC> . 7H 0-1. 4  were, mixed w i t h 0.2  2  of the phosphate and  included  For phosphate b u f f e r these 2  ;.vol of 0.154M NaHCC> was 3  KC1,  7.4.  added i n p l a c e  A small amount of phenol red i n d i c a t o r  i n the b i c a r b o n a t e b u f f e r , approximately 5 mg/1  10 mM  For  2  T r i s b u f f e r used i n the n u c l e a r 0.6M  2  salts  the b i c a r b o n a t e b u f f e r gassed w i t h CC>  immediately before use. was  indicated:  v o l of 0. IM Na 'HPO . 2H 0. f i n a l pH  b i c a r b o n a t e b u f f e r , 0.21  the  T r i s - H C l and  1.5  mM  receptor  (72).  experiments c o n t a i n e d :  EDTA, f i n a l pH  8.0.  Chemicals  Dexamethasone (Decadron powder) was Company, Rahway, N.J.  The  a g i f t from Merck  11-epicorticosterone  was  kindly  and  16  s u p p l i e d by Dr. J.C. Babcock, The Upjohn Company, Kalamazoo, MI.  Corticosterone,  and the C-21  1 1 - d e h y d r o c o r t i c o s t e r o n e , the f r e e  alcohols  a c e t a t e s as w e l l as 11-ketoprogesterone were  o b t a i n e d from S t e r a l o i d s  (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 c o n t a i n i n g 50-75 K u n i t z units/mg p r o t e i n and DNase c o n t a i n i n g 2,000 K u n i t z units/mg p r o t e i n were purchased from Sigma Chemical Corp., S t . L o u i s , Pronase, 45,000 PKU Sephadex G-25 Anthrone was  was  units/mg, was  MO.  o b t a i n e d from Calbiochem,  o b t a i n e d from Pharmacia, Uppsala, Sweden.  from B r i t i s h . D r u g Houses L t d . , Poole, England.  Bovine serum albumin was N-ethyl maleimide  o b t a i n e d from Mann Research Labs, and  from Dajac Labs, P h i l a d e l p h i a , PA.  sesame o i l used was  o b t a i n e d from L i f e s t r e a m N a t u r a l  Richmond, B.C. (a l o c a l supplier)„ and was including anti-oxidants.  The Foods,  free of a d d i t i v e s  C h a r c o a l ( N o r i t A) and a l l o t h e r  chemicals were o b t a i n e d from e i t h e r F i s h e r S c i e n t i f i c or Canlab, in  Vancouver.  R a d i o a c t i v e Chemicals  R a d i o a c t i v e compounds purchased from New  England Nuclear  3  Corp.  (Montreal) i n c l u d e d : H - a c e t i c anhydride (400 Ci/mmole); 3 14 c o r t i c o s t e r o n e - 1 , 2 , 6 , 7 - H (82.1 Ci/mmole); c o r t i c o s t e r o n e - 4 C 3  (57.3 mCi/mmole); dexamethasone-1,2,4- H 14  (21.2  Ci/mmole).  3 C- or  H - l a b e l l e d 1 1 - d e h y d r o c o r t i c o s t e r o n e were prepared  e n z y m a t i c a l l y from l a b e l l e d c o r t i c o s t e r o n e , as w i l l be d e s c r i b e d later.  Compounds o b t a i n e d from Amersham/Searle, A r l i n g t o n  Heights, IL i n c l u d e d :  D-f ructose-6-phosphate- C (u) (268 4  mCi/  17  mmole); D-glucosamine 14 C(u)  H C l - l - ' ^ C (60-68 mCi/mmole); D-glucose-  3 (331 mCi/mmole); thymidine methyl- H (26 3  Ci/mmole);  u r i d i n e - 5 - H (30 Ci/mmole).  Solvents A l l s o l v e n t s used were o b t a i n e d from F i s h e r Chemical or Canlab, Vancouver.  These i n c l u d e d :  n-butanol, c h l o r o f o r m ,  dichloromethane, e t h a n o l , e t h y l a c e t a t e , e t h y l e t h e r , n-hexane, methanol, petroleum e t h e r , p y r i d i n e and t o l u e n e .  A l l solvents  were f r e s h l y d i s t i l l e d b e f o r e use. Chromatography  Eastman s i l i c a g e l TLC sheets w i t h f l u o r e s c e n t were used.  indicator  These were washed w i t h d i s t i l l e d methanol p r i o r t o  use.  Whatman No. 1 paper was used f o r s e p a r a t i o n o f sugars and f o r p u r i f i c a t i o n of s t e r o i d s , s i n c e q u a n t i t a t i v e r e c o v e r y o f s t e r o i d s from paper was  superior.  Autoradiography  M e d i c a l X-ray f i l m s  (NS2T) were o b t a i n e d from Kodak and  I l f e x 90 X-ray f i l m was purchased from I l f o r d L t d . through local  suppliers.  18  Scintillation  Supplies  Econofluor, chased  f r o m New  a premixed  scintillation  England N u c l e a r Corp.,  s o l u t i o n , was  Montreal.  Solubilizers  u s e d 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 Beckman I n s t r u m e n t s ,  The  liquid  Nuclear/Chicago. external  Fullerton,  scintillation  (73) .  from  CA.  s p e c t r o m e t e r was a U n i l u x I I A  Q u e n c h i n g was d e t e r m i n e d  standard r a t i o s  pur-  by t h e method o f  19  METHODS  I n j e c t i o n o f Mice  Mice which were i n j e c t e d w i t h dexamethasone r e c e i v e d subcutaneously 0.4 ml o f a suspension i n sesame o i l prepared as follows:  a s o l u t i o n o f dexamethasone, 2 mg/ml i n e t h a n o l , was  l a y e r e d over sesame o i l and approximately t h r e e - q u a r t e r s of the e t h a n o l evaporated under a stream o f n i t r o g e n , u n t i l the s t e r o i d was beginning t o come out o f s o l u t i o n .  The o i l and e t h a n o l were  then mixed thoroughly t o g i v e a suspension of 0.5 mg/ml.  This  s o l u t i o n was mixed w e l l again j u s t p r i o r t o use. The i n j e c t e d mice were k i l l e d 16 hours  later.  3 Mice i n j e c t e d w i t h  H-corticosterone received a solution  of the s t e r o i d i n s a l i n e subcutaneously, 1 v>Ci/0.5 ml, 15 minutes p r i o r t o removal  of f e t a l  tissues.  3 Mice i n j e c t e d with  H-thymidine r e c e i v e d subcutaneously  0.5 ml o f a s a l i n e s o l u t i o n c o n t a i n i n g 5 y C i , 30 minutes p r i o r to removal  of f e t a l  tissues.  P r e p a r a t i o n of'".Tissues  Mice were k i l l e d w i t h excess C 0  2  and the u t e r i  removed  q u i c k l y from the mother and p l a c e d on i c e i n P e t r i d i s h e s cont a i n i n g s a l i n e - m o i s t e n e d f i l t e r paper.  The f e t u s e s were then  removed and the a p p r o p r i a t e t i s s u e s d i s s e c t e d out and pooled  20  for incubation.  T i s s u e s , up t o 300 mg, were p l a c e d i n a 25 ml  Erlenmeyer f l a s k and minced f i n e l y w i t h s c i s s o r s .  B u f f e r and  s u b s t r a t e s were then added and the f l a s k stoppered  with a s i l i c o n e  stopper. 120  F l a s k s were then p l a c e d i n a water bath  cycles/min  a t 37°C f o r t h e l e n g t h o f time as r e q u i r e d f o r  i n c u b a t i o n purposes.  Unless  s t a t e d otherwise,  were performed under these c o n d i t i o n s . on i c e d u r i n g  a l l incubations  F l a s k s were kept  chilled  preparation.  Enzymatic S y n t h e s i s o f 11-dehydrocorticosterone 3 Either  shaking a t  (cpd. A)  14  H- o r  C - l l - d e h y d r o c o r t i c o s t e r o n e was prepared by  i n c u b a t i n g the a p p r o p r i a t e l y l a b e l l e d c o r t i c o s t e r o n e (cpd. B) with a guinea NADP (37).  p i g l i v e r microsome p r e p a r a t i o n f o r t i f i e d  The i n c u b a t i o n mixture was e x t r a c t e d w i t h  which was evaporated  CH C1 , 2  chromatogram was developed i n benzene s a t u r a t e d w i t h steroid  The formamide.  zones were e l u t e d w i t h chloroformrmethanol  which was f i l t e r e d through cellulose,  2  and the r e s i d u e s p o t t e d onto Whatman No. 1  paper impregnated w i t h methanol:formamide, (1:1; v:v) .  The  with  then evaporated  sintered  (1:1; v : v ) ,  g l a s s to remove p a r t i c l e s o f  under a stream o f N , and the r e s i d u e 2  redissolved i n ethanol. C o r t i c o s t e r o n e c o n t a i n s as an i m p u r i t y , progesterone  11-oxygenated  i n t e r m e d i a t e s , which c o - c r y s t a l l i z e with  sterone and can be removed o n l y by chromatography. to approximately (74).  cortico-  T h i s amounts  15% o f the UV-absorbance o f a sample o f cpd. B  A c c o r d i n g l y , 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 f r e e  11-dehydrocorticosterone  (cpd. A), t h i s compound, both the r a d i o a c t i v e and n o n - r a d i o a c t i v e , was  f r e s h l y p u r i f i e d by chromatography immediately  The c o n c e n t r a t i o n of n o n - r a d i o a c t i v e compounds was  before  use.  determined  by  UV absorbance of s o l u t i o n s at 237-240nm i n a Unicam SP 800A spectrophotometer.  In  Vitro Steroid  Incubations  Minced t i s s u e s were incubated i n 1 ml of phosphate b u f f e r (KEP)  Krebs-Eggleston i A c o n t a i n i n g e i t h e r 5 nCi of G-labelled  cpd. A or B when r e q u i r e d f o r autoradiography. 2.1  nCi  14  C-cpd. B and  i n order to determine simultaneously.  36 nCi  3  Otherwise,  H-cpd. A were incubated t o g e t h e r  both dehydrogenase and reductase  activity  A l l i n c u b a t i o n s were c a r r i e d out f o r 15  min  and the r e a c t i o n stopped by c h i l l i n g the sample on i c e , f o l lowed by  extraction.  Extraction  The  Procedure  samples were f i r s t e x t r a c t e d w i t h 6 v o l n-hexane, by  shaking v i g o r o u s l y 20 times. neutral l i p i d ,  was  The upper phase, c o n t a i n i n g  removed by a s p i r a t i o n and d i s c a r d e d .  The  s t e r o i d s were then e x t r a c t e d i n 6 v o l i c e - c o l d C P ^ C ^ , shaking 20 times.  The C H C 1 2  under a stream of N . 2  methanol  (1:1; v:v)  2  was The  removed and evaporated r e s i d u e was  dryness  dissolved i n chloroform:  and s p o t t e d onto a s i l i c a  The tube c o n t a i n i n g the r e s i d u e was  to  g e l TLC  sheet.  r i n s e d s e v e r a l times  with  22  solvent  t o ensure e f f i c i e n t  transfer  of steroid.  Chromatography  N o n - r a d i o a c t i v e c p d . A a n d B, 10 yg e a c h , were a d d e d t o e a c h sample p r i o r  to extraction  serve as markers, a l l o w i n g wave UV l i g h t sively  (24 0nm).  i n two s o l v e n t  1)  2)  In t h i s  systems:  (4:1; v : v ) , w h i c h  toluene:chloroform:methanol:water the steroid  carries  front, (120:60:20:1; v : v )  zones.  s y s t e m , t h e C-21 a c e t a t e s move a h e a d o f t h e f r e e  w h i l e o t h e r m e t a b o l i t e s move more s l o w l y . marked, c u t o u t and s c r a p e d fluor  short-  The TLC s h e e t s were d e v e l o p e d s u c c e s -  fats with the solvent  to resolve  and a l s o t o  l o c a t i o n o f t h e zones under  n-hexane:ethyl acetate residual  t o a c t as c a r r i e r s ,  scintillation  fluid  into vials  was a d d e d .  a v o r t e x m i x e r and c o u n t e d . s e v e r a l hundred  Most  steroids,  The z o n e s were  t o w h i c h 10 ml o f E c o n o The v i a l s were m i x e d  samples c o n t a i n e d  with  at least  counts.  In V i v o M e t a b o l i s m o f S t e r o i d s  Upon r e m o v a l o f t i s s u e with l a b e l l e d  steroid,  from mice w h i c h had been  1 m l KEP was added t o e a c h t i s s u e p l u s  e a c h o f c p d . A and B c a r r i e r . homogenized  quickly  injected  The sample was c h i l l e d  i n a S o r v a l l Omnimixer  10 y g  on i c e , and  (Ivan S o r v a l l I n c . ,  23  Newtown, CT) .  The sample was t h e n  e x t r a c t e d and c h r o m a t o -  graphed and t h e zones e l u t e d and counted  as d e s c r i b e d above.  Characterization of Steroids  In  addition  terization authentic  by d e r i v a t i v e  at  f o r m a t i o n and c o - c r y s t a l l i z a t i o n  a d d e d c a r r i e r was p e r f o r m e d .  dry p y r i d i n e , ing.  t o c h r o m a t o g r a p h y on p a p e r o r TLC, c h a r a c -  0.3 ml e a c h ,  Acetic  the  stoppered  dry-  and i n c u b a t e d  E t h a n o l , 0.5 ml o f 25% aqueous s o l u t i o n ,  added t o h y d r o l y z e e x c e s s then  a n h y d r i d e and  were added t o t h e sample a f t e r  The s a m p l e s were m i x e d , t i g h t l y 37°C f o r 1 h .  with  extracted with  acetic  anhydride.  The s a m p l e s were  6 v o l C H C 1 , t h e aqueous l a y e r 2  samples r e - e x t r a c t e d w i t h  2  another  was  removed and  0.5 ml w a t e r .  Crystallization  The in  CH C1 2  2  l a y e r was e v a p o r a t e d  and t h e r e s i d u e  redissolved  1 ml m e t h a n o l 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  acetate or 11-dehydrocorticosterone  acetate.  Ice-cold  distilled  w a t e r was a d d e d d r o p w i s e t o t h e s a m p l e s t o t h e p o i n t o f ent c r y s t a l l i z a t i o n , permit  a n d t h e s a m p l e s were s t o r e d a t 0-4°C t o  slow c r y s t a l l i z a t i o n  to occur.  c r y s t a l s were p a c k e d by c e n t r i f u g a t i o n , canted quot  and t h e c r y s t a l s  redissolved  After  6 t o 24 h , t h e  t h e mother l i q u o r de-  i n 2 ml m e t h a n o l .  An  ali-  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 a n d a n o t h e r  a l i q u o t was a s s a y e d tion  incipi-  for radioactivity.  To t h e r e m a i n i n g  water, was added t o r e p e a t t h e c r y s t a l l i z a t i o n  solu-  procedure.  24 I s o l a t i o n of Glycogen from T i s s u e s  Mice were k i l l e d and t i s s u e s removed as d e s c r i b e d p r e viously.  Each t i s s u e was weighed q u i c k l y and p l a c e d i n a 15 ml  c e n t r i f u g e tube t o which 2 ml 2N NaOH was added.  The sample  was then p l a c e d i n a water bath a t 50-60°C t o d i s s o l v e the tissues.  Two v o l e t h a n o l c o n t a i n i n g 0.1% L i C l were then added  and the sample mixed and stood at room temperature, which i s more f a v o u r a b l e f o r the p r e c i p i t a t i o n of glycogen.  The  sample  was then c e n t r i f u g e d a t 1200 xg f o r 5 min and the supernatant discarded.  The p r e c i p i t a t e was r e d i s s o l v e d i n 1.8 ml water and  0.2 ml 3N PCA added.  T h i s procedure p r e c i p i t a t e s  glycoprotein  which c o u l d r e a c t w i t h the anthrone reagent.  The sample  was  c e n t r i f u g e d and the supernatant t r a n s f e r r e d .  The p e l l e t  was  washed with 1 ml 0.3N PCA and the washing added t o the supernatant.  Two v o l e t h a n o l - L i C l were added, and a white f l o c c u l e n t  p r e c i p i t a t e of glycogen r e s u l t e d . and the  The sample was  centrifuged,  p e l l e t was r e d i s s o l v e d i n 1 ml d i s t i l l e d water (75).  Anthrone Assay f o r Glycogen  To 280 ml d i s t i l l e d water, 720 ml c o n c e n t r a t e d H S 0 2  added.  4  were  The contents were c o o l e d to 35°C, and 500 mg anthrone  dissolved therein.  Then 3 0 g t h i o u r e a were added and d i s s o l v e d ,  ahdtthe mixture t i g h t l y stoppered and s t o r e d a t 0-4°C.  F i v e ml anthrone reagent was added t o 1 ml o f glycogen sample and the s o l u t i o n mixed w e l l .  A marble was p l a c e d over  25  the  tube,  and  The  s a m p l e was  the  then  a b s o r b a n c e was Spectronic  70  Incorporation  Minced  tube put  in a boiling  cooled  to  determined  at  water bath  room t e m p e r a t u r e 62 0 nm  for  for 15  i n a B a u s c h and  10  min.  min  and  Lomb  spectrophotometer.  of  Leucine,  t i s s u e s were  Uridine  and  incubated  Thymidine  in  1 ml  Krebs-Eggleston  bicar-  14 bonate 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 pCi 3 3 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.  leucine  0.46  Incubations  pCi  carried  out  for  homogenized  1 h,  in a  then  the  samples were c h i l l e d  S o r v a l l Omnimixer.  removed  for protein determination  brough,  Farr  The 3%  s a m p l e was TCA  and  tillation for  and  Randall  centrifuged,  solubilized fluid,  10  by  then  the  a l i q u o t of  the 0.1  method of ml  30%  ml,  was  t h e n added and  on  ice  0.1  ml  were and was  Lowry,  TCA  was  p r e c i p i t a t e washed  i n BBS-3 s o l u b i l i z e r .  C,  4x  then  Rose-  added. with  Econofluor  the  sample  and  thymidine  3  ml  scin-  assayed  radioactivity.  The  incorporation  time.is. i l l u s t r a t e d can  (76),  An  -  be  seen t h a t  uridine  but  not  period 30  necessarily 1 h,  of  was  three  comparison of  of  min,  t h e s e e x p e r i m e n t s was the  uridine  incorporation  i s maximal at  creasing  leucine,  i n the ;case•of  the  time throughout the  of  isotopes various  of  f e t a l .brainJin Table  1.  of  with  leucine  investigation. while linear.  during  with  that  of  The  which  sufficient  is linear  Incorporation  thymidine  pulse  time  the  time  of  is inchosen  for  incorporation  magnitude to  t i s s u e s under these  It  conditions.  permit  26  Table 1 SUBSTRATE  INCORPORATION  INTO FETAL BRAIN WITH TIME  Incorporation, Time, m i n 30  Uridine  Thymidine  4176  1146  10217  13410  90  Each f i g u r e  : 1109:+ 48  + 1116  was h o m o g e n i z e d 0.1 mM  pyridoxal  at concentrations  —  + 2454  samples.  EDTA, 5 mM  dithiothreitol,  Tris-HCl  pH 7.3, and t h e  a t 20,0 00 x g f o r 2 0 m i n .  phosphate,  5 mM  o f 0.03-0.3 mM,  buffer  An a l i q u o t o f  dithiothreitol  containing  ,and;..L-ornithine  including either  50 n C i o f  14  . C-D,L o r n i t h i n e  o r 25 n C i o f  m i x t u r e volume was  0.1 m l .  30 m i n i n t u b e s f i t t e d  hyamine. citric for  1110 + 38  i n 5 v o l o f 5 0 mM  14  for  21175  s u p e r n a t a n t was i n c u b a t e d ' w i t h a r e a c t i o n m i x t u r e  0.2 mM  + 1121  ,134 03 r+229 8  —  i s a mean + SEM o f f o u r  homogenate c e n t r i f u g e d  tion  + 82  Decarboxylase'Assay  Tissue  the  + 730  —  120  containing  Leucine  9058 •+ .564  ' 60  Ornithine  dpm/mg p r o t e i n o f :  The t o t a l  Samples were i n c u b a t e d  with centrewells  containing  reac-  a t 37°C 0.1 ml  The r e a c t i o n was t e r m i n a t e d by i n j e c t i n g 0.1 ml o f 2 M  a c i d through the stopper,  30 m i n .  The c e n t r e w e l l s  scintillation activity  C-L-ormthme.  (77) .  vials  a n d t h e l a b e l l e d CO^ c o l l e c t e d  were t h e n removed and p l a c e d  5 ml A q u a s o l  added and a s s a y e d  into  for radio-  27  Glucose Metabolism  in Fetal  Tissue  14 Glucoseand  C  (u) , 0.86  y C i , was  h o m o g e n i z e d as d e s c r i b e d f o r l e u c i n e  t i o n o f TCA,  t h e p r e c i p i t a t e was e x t r a c t e d 4x w i t h  to  An  remove TCA.  v o l u m e and developed  i n two  After  the  g e l TLC  1)  chloroform:methanol:17%  2)  b u t a n o l : a c e t i c acid:water  acid-soluble  reduced  sheet which  a l i q u o t was  amino a c i d  ether in  was  of the  NH^OH  (2:2:1;  (3:1:1;  v:v);  v:v).  s h e e t s were p r e p a r e d ,  after  s h e e t s were s p r a y e d w i t h n i n h y d r i n t o d e t e c t amino  An  addi-  p o r t i o n s of e t h y l  s o l u t i o n was  tissue  dimensions:  Autoradiograms  man  1 ml  aliquot of t h i s  s p o t t e d onto a , s i l i c a  studies.  d i s c a r d e d and  s u p e r n a t a n t was  the  i n c u b a t e d w i t h minced  removed f o r amino a c i d  a n a l y z e r system  analysis  w i t h UR-30 r e s i n ,  which acids.  i n a Beck-  0.9  x 52  cm,  with Na-citrate buffers.  Glucose  and  Fetal  Fructose-6-PO^  liver  and  a t 105,000 xg  e q u a l volume o f KEB fructose-6-PO^ A t t h e end  i n Cytosol Preparations  g u t were h o m o g e n i z e d i n 0.14M  Potter-Elvehjem Teflon fuged  Metabolism  h o m o g e n i z e r , and  f o r 30 m i n .  The  y C i , and  o f t h i s p e r i o d , 0.1  sample was  supernatant  containing either  ( u ) , 0.1  the  14  KC1  C-glucose  was  TCA  was  centri-  m i x e d w i t h an  (u) o r  i n c u b a t e d f o r 30 min v o l 3 0%  in a  at  added and  14  C-  37°C. the  28  p r e c i p i t a t e removed by c e n t r i f u g a t i o n .  The  e x t r a c t e d 4x with e t h y l ether to remove TCA down and  spotted onto Whatman No.  chromatogram developed for  96 h or more.  1 filter  10  and was  was  evaporated  paper and  i n butanol:acetic acidrwater  the (40:4:10;  Zones were v i s u a l i z e d by s p r a y i n g w i t h  a n i l i n e i n e t h y l ether-and.Cheating for  supernatant  v:v)  2%  the chromatograms a t 100°C  min.  I n c o r p o r a t i o n of Glucosamine  The t i s s u e was for  incubated and homogenized as d e s c r i b e d  leucine studies.  added.  A f t e r homogenization,  The t i s s u e was  0.4  incubated f o r 1 h i n 1.6  ml  5N PCA  was  ml KBB c o n t a i n i n g  14 0.2  yCi  C-glucosamine.  w i t h 3 ml 0.5N (1:1:1; v:v)  PCA,  The p r e c i p i t a t e was  then washed 4x  3x w i t h methanol:chloroform:ethyl  and s o l u b i l i z e d and counted,  ether  as d e s c r i b e d above.  STEROID RECEPTOR ASSAYS  C y t o s o l Receptor  P r e p a r a t i o n and  F e t a l b r a i n was KEP.  The t i s s u e was  first  a t 1200  at  removed, minced and washed 3x w i t h homogenized i n 5 ml c o l d KEP  ice-cold  and c e n t r i f u g e d  xg, to remove much d e b r i s , then r e c e n t r i f u g e d  105,000 xg i n a Beckman L-5  1 h.  Assay  The c y t o s o l obtained was  0.5 ml c y t o s o l was  65 model u l t r a c e n t r i f u g e f o r used  i n subsequent assays.  To  added 1.5 ml of a s o l u t i o n c o n t a i n i n g 8 nM  29  labelled steroid.  In some i n s t a n c e s ,  this solution  contained  2 yM 1 1 - e p i c o r t i c o s t e r o r i e which has been found to e l i m i n a t e much of the n o n - s p e c i f i c b i n d i n g  (78, 79).  The assays were  c a r r i e d out a t 0°C f o r 30 min and stopped by the a d d i t i o n of 40 mg dextran-coated c h a r c o a l Samples were c e n t r i f u g e d  t o remove unbound s t e r o i d .  a t 1200 xg to pack the c h a r c o a l ,  and  an a l i q u o t was removed, mixed w i t h BBS-3 s o l u b i l i z e r and Econof l u o r , and assayed f o r r a d i o a c t i v i t y .  Protein determinations  were made on the c y t o s o l and the r e s u l t s were expressed as dpm  s t e r o i d bound/mg p r o t e i n .  Nuclear P r e p a r a t i o n  and Assay  N u c l e i from f e t a l b r a i n and p l a c e n t a were prepared by f i r s t homogenizing the t i s s u e i n a P o t t e r - E l v e h j e m homogenizer i n 2 ml KEP c o n t a i n i n g  2.8 mM  Teflon  glucose.  The r a d i o -  a c t i v e s t e r o i d , 2 y C i , d i s s o l v e d i n no more than 20 y l e t h a n o l , was added and the sample l e f t on i c e f o r 15 min w i t h agitation.  occasional  The sample was then incubated a t 37°C f o r 10 min i n  a shaking water bath.  At the end o f t h i s time, the sample was  c h i l l e d on i c e and 50 ml c o l d 1.5 mM M g C l cedure has been found to s h a t t e r c e l l s , many t i s s u e s .  2  added.  T h i s pro-  leaving nuclei i n t a c t i n  The n u c l e i were harvested by c e n t r i f u g a t i o n a t  1200 xg a t 0-4°C and washed  2x w i t h M g C l  2  solution.  The i s o l a t e d  n u c l e i were then d i s r u p t e d by homogenizing i n 2 ml c o l d  Tris  b u f f e r , pH 8.0, by g r i n d i n g i n a g l a s s Ten Broeck homogenizer.' The p r e p a r a t i o n  was then c e n t r i f u g e d  at 105,000 xg f o r 15 min.  a l i q u o t of supernatant, u s u a l l y 0.5' ml, was p l a c e d  on a column  An  30 of Sephadex G-25, 11 x 180 mm, pH 8.0, a t 4°C.  Fractions  and e l u t e d u s i n g  Tris buffer,  of 1 ml were c o l l e c t e d u s i n g an  LKB f r a c t i o n c o l l e c t o r , monitored by a b s o r p t i o n  a t 280 nm.  Assay of R a d i o a c t i v i t y  A quench curve f o r the d e t e r m i n a t i o n of the e f f i c i e n c y of 3 counting f o r  H and  14 C i s shown i n F i g . 1.  l e a s t once every month.  I t was checked a t  The c a l c u l a t i o n s f o r the double  isotope  a n a l y s i s were done a c c o r d i n g t o the method of Kobayashi and Maudsley (73).  31  FIGURE 1.  1.0  2.0  QUENCH CURVE FOR  3.0  4.0  EXTERNAL STANDARD RATIO  AND  1  4  C  5.0  EXPERIMENTAL RESULTS  14 Metabolism of C-labelled Corticosterone and 11-dehydrocorticosterone i n F e t a l T i s s u e s  The metabolism of cpd. A and B i n f e t a l head, and p l a c e n t a on v a r i o u s g e s t a t i o n a l days was  liver  examined.  14 T i s s u e s were incubated w i t h e i t h e r B and C t ^ C ^ gel  TLC  e x t r a c t s of t i s s u e s were s p o t t e d onto  sheets and developed  (4:1) and,  second,  (120:60:20:1). In  Autoradiograms were then  hexane:ethyl  acetate  prepared.  F i g . 2 the p a t t e r n of c o r t i c o s t e r o n e metabolism i n  i s a decreased  w i t h g e s t a t i o n a l age.  There  c o n v e r s i o n of the a c t i v e cpd. B to the  11-dehydro m e t a b o l i t e ; i . e . , there i s a  i n dehydrogenation day  in,"first,  silica  toluene:chloroform:methanol:water  f e t a l head changes remarkably  inactive  C - l a b e l l e d cpd. A or  decrease  with i n c r e a s i n g g e s t a t i o n a l age.  On  14, a l a r g e number of m e t a b o l i t e s can be observed which  have disappeared by day  15.  These metabolic changes were  found t o occur i n the b r a i n , r a t h e r than i n a d j o i n i n g s t r u c t u r e s of the  The  zones have been i d e n t i f i e d as f o l l o w s :  moving zone was tic  skull.  the  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 w i t h  added c a r r i e r as 11-dehydrocorticosterone  fastest authen-  acetate  (Table 2) and the zone d i r e c t l y behind i t as c o r t i c o s t e r o n e acetate.  The  slower moving d e r i v a t i v e s have been i d e n t i f i e d  F i g . 2:  Autoradiogram  of chromatographed e x t r a c t s of f e t a l  brain  14 incubated w i t h day  14; 3 and 4:  C-corticosterone. day  15; 5 and 6:  .1. and 2: day  average counts recovered i n each on day  17.  Gestational The  zones and  the  dpm  14 a r e :  AAc:  11-dehydrocorticosterone a c e t a t e  364  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 i n c r e a s e i n s i z e of the B zone and decrease i n s i z e of the A zone i n 3 t o 6, as compared w i t h 1 and  2.  34  35  t e n t a t i v e l y as the 2 0 a - d i h y d r o d e r i v a t i v e s of 11-dehydroc o r t i c o s t e r o n e and c o r t i c o s t e r o n e (the slowest Reference 2 0ct-dihydro compounds were prepared  zone). by i n c u b a t i n g  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 w i t h a d i a l y z e d 1 0 5 , 0 0 0 xg supernatant  o f mouse l i v e r f o r t i f i e d w i t h NADPH, and  s e p a r a t i n g t h e products  chromatographically,  by K r e h b i e l , Burton and Darrach of the m e t a b o l i t e s  as d e s c r i b e d  ( 8 0 ) . The r a d i o a c t i v i t y  and t h e i r a c e t a t e d e r i v a t i v e s c o i n c i d e d  d u r i n g chromatography w i t h the UV absorbance of the added r e f e r e n c e compounds and c o u l d be d i s t i n g u i s h e d 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 l i q u o r  The  4 84  r e d u c t i o n o f cpd. A t o cpd. B i n f e t a l mouse  liver  and p l a c e n t a on g e s t a t i o n a l days 14 and 17 i s i l l u s t r a t e d i n F i g . 3.  Although the reducing  c a p a c i t y o f the p l a c e n t a  remains h i g h throughout g e s t a t i o n , the l i v e r appears t o alter  from a dehydrogenating t i s s u e on day 14 t o a r e d u c i n g  t i s s u e by day 1 7 .  I t i s o f i n t e r e s t t o note here t h a t i n  man the p l a c e n t a has been shown t o be the s i t e o f dehydrogenation  ( 3 9 ) , whereas i n f e t a l mouse the p l a c e n t a i s  Fig.  3:  Autoradiogram  o f c h r o m a t o g r a p h e d e x t r a c t s o f mouse 14  fetal  liver  and  p l a c e n t a xncubated  with  C-11-dehydrocorti-  costerone . A:  11-dehydrocorticosterone:  liver,  day  14;  2,  4,  p l a c e n t a , day  in  liver  on  fetal 17.  liver,  Note t h e  gestational  day  17.  B:  corticosterone.  day  17;  1,  fetal  3, p l a c e n t a , day  i n c r e a s e i n the  size  of B  14; zone  37  38  d e f i n i t e l y a s i t e of r e d u c t i o n , and be the major s i t e of  2.  the . b r a i n appears t o  dehydrogenation.  .The Metabolism of C o r t i c o s t e r o n e and 1 1 - d e h y d r o c o r t i c o s t e r o n e i n F e t a l T i s s u e s on D i f f e r e n t G e s t a t i o n a l Days"  The r e l a t i o n s h i p between the dehydrogenation  of cpd. B  to cpd. A and the r e d u c t i o n of the l a t t e r t o cpd. B can •:. .. have g r e a t 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 a c t i v e 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 . examine the metabolic  In order t o  f a t e of these two compounds, p l a c e n t a 14  and f e t a l t i s s u e s were incubated s i m u l t a n e o u s l y w i t h 3 l a b e l l e d cpd. B and  H - l a b e l l e d cpd. A.  T i s s u e s were  examined on g e s t a t i o n a l days 1 4 , 16 and 19 t o observe a c t i v i t y of the r e v e r s i b l e C-11 s t e r o i d  'i C-  the  oxidoreductase.  3 14 H-cpd. A and C-cpd. B were incubated  simultaneously.  The products were separated c h r o m a t o g r a p h i c a l l y , e l u t e d and 14  assayed  for radioactivity.  The amount of  C l a b e l i n the  cpd. A zone would i n d i c a t e dehydrogenase a c t i v i t y , and con3 v e r s e l y the amount o f reductase a c t i v i t y .  H l a b e l i n the cpd. B zone i n d i c a t e s From these data the percentage  of reduc-  t i o n and dehydrogenation c o u l d be c a l c u l a t e d , and the net , ,. reduction _, r e a c t i o n expressed as a r a t i o of -, , , rr- • The dehydrogenation c  r e s u l t s f o r p l a c e n t a and v a r i o u s f e t a l t i s s u e s are presented i n Figs. 4 to 6 .  39 FIGURE 4,  IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE  FETAL BRAIN AND  IN  GUT,  1.0 u  E a c h p o i n t i s t h e mean o f 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. T h e d i f f e r e n c e between d a y 14 and d a y 16 i s , f o r g u t P<.05, f o r b r a i n P<.01. V a l u e s f o r d a y 16 v s d a y 19 d i f f e r s i g n i f i c a n t l y , P<.01. —  brain  — A ; gut  40  FIGURE 5,  IN VITRO ACTIVITY OF STEROID C - l l OXIDOREDUCTASE IN  PLACENTA.  30  20  O CD  —  o  u  Q  i - cn ZJ  >  a x LU L U  10  <  cm  14  18  16 GESTATIONAL DAY  SJS ^ h  P n r  Trl  K  P O i  n,  ±  S  t  h  e  s^niriLnt^foL  m  e  1 6  a  n  '  o  f  a  t  d  l  a  e  y  a  s  1  6  t  e  ^  i  9  h  d  ^  t  1  v a l u e s + SEM. differences 9  t  h  e  41  FIGURE 6.  IN VITRO ACTIVITY OF STEROID C - l l  FETAL LIVER AND  OXIDOREDUCTASE  IN  LUNG,  10  / l  LU  IS  Q Q >X UJ LU cd=3  <  AY  14  16 GESTATIONAL  18 DAY  E a c h p o i n t i s t h e mean o f a t l e a s t f i v e v a l u e s . Some SEM a r e shown. F o r b o t h d a y 14 v s d a y 16, a n d f o r d a y 16 v s day 19, t h e d i f f e r e n c e s a r e s i g n i f i c a n t , P<.01. 1  l i v e r - — — ^ ; lungo  •  42  F i g . 4 shows t h a t i n both f e t a l b r a i n and dehydrogenation predominates.  gut,  This i s i n d i c a t e d by a  .3 f °" r a t i o of l e s s than 1.0. Both t i s s u e s , show dehydrogenatxon a decrease i n dehydrogenase a c t i v i t y with..increasing.^ .. r  d  u  c  t  l  g e s t a t i o n a l age,  as i n d i c a t e d , by a r a t i o approaching  1.0.  In F i g . 5, p l a c e n t a i s shown t o be an a c t i v e s i t e of r e d u c t i o n of cpd. A.  This reducing capacity increases  with g e s t a t i o n a l age.  F i g . 6 shows t h a t i n both l i v e r and dehydrogenation p r e v a i l s . reduction  dehydrogenation  r  a  p r e d o m i n a t e s and day  16,  still  the  io  the  14,  dotted l i n e i n d i c a t e s a  of 1.0, below which dehydrogenation  above w h i c h r e d u c t i o n p r e d o m i n a t e s .  liver  is actively  dehydrogenating.  tissues. in  t  The  lung on day  Therefore,  But liver  reducing,  by and  day  19,  while both  the  are  lung  activity,  the  is  reducing  l u n g have d i f f e r e n t  development of reductase  By  lung  timing being  later.  The  h e a r t was  reductase  a l s o examined  activity,  but  S m i t h , T o r d a y and o f C-11  steroid  none was  Giroud  oxidoreductase  lung c e l l s  taken  Therefore,  i t was  f o r C-11  (81)  have r e p o r t e d  activity  interesting  oxido-  observed.  f r o m a human f e t u s a t an  steroid  with  induction  Cortisol i n  midgestation.  possibility  that  injection  43  of dexamethasone might  s t i m u l a t e enzyme a c t i v i t y i n the  f e t a l mouse.  Table 3 shows t h a t no e f f e c t on enzyme a c t i v i t y  i n any  t i s s u e was observed when mothers were i n j e c t e d on day 13.5, 16 hours p r i o r t o the i n v i t r o assay.  When animals were  t r e a t e d 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 p l a c e n t a (P<.01) and lung  (P<.05).  T h e r e f o r e , i n these t i s s u e s  cortico-  sterone would appear t o i n f l u e n c e i t s own formation by the enzyme C-11 s t e r o i d o x i d o r e d u c t a s e and can be c o n s i d e r e d to be " a u t o c a t a l y t i c " i n these t i s s u e s .  I t should be noted here t h a t t h e s y n t h e t i c dexamethasone was used i n these experiments  steroid  because  i t does  not b i n d t o maternal t r a n s c o r t i n and a l s o escapes m e t a b o l i c pathways i n the f e t u s  (82). T h e r e f o r e , a more e f f e c t i v e  dose can c r o s s the p l a c e n t a and e x e r t a b i o l o g i c a l  effect.  44  Table 3 EFFECT OF DEXAMETHASONE INJECTION ON THE ACTIVITY OF STEROID C - l l OXIDOREDUCTASE ON GESTATIONAL DAY 14  Reduction Ratio Dehydrogenation G e s t a t i o n a l Day Tissue  Day 14 C o n t r o l  Day 14 Dex  Placenta  6. 30±1.."0  8.26±1. 9  (8)  (8)  0.06±0.003  0.07±0. 003  (10)  (10)  Brain  Gut  Liver  Lung  0.10±0.01  0.12±0. 02  (5)  (5)  0.29±0.07  0.34±0. 09  (5)  (5)  0.16±0.02  0.25±0. 06  (5)  (5)  Each f i g u r e i s a mean + SEM o f the number o f samples shown i n parentheses.  None o f the above t r e a t e d  s i g n i f i c a n t l y from c o n t r o l  values.  differed  45  Table 4 EFFECT OF DEXAMETHASONE INJECTION ON THE ACTIVITY OF STEROID C - l l OXIDOREDUCTASE ON GESTATIONAL DAY 16  Reduction ~ .. ^ . , ,. Ratio Dehydrogenation G e s t a t i o n a l Day Tissue  Day 16 C o n t r o l  Day 16 Dex  Placenta  16.7±0.82  24.5+2.0  (8)  (11)  Brain  0.30±0.04 (8)  Gut  (8)  0.17+0.03 (6)  Liver  3.54+0.65  2.99±0.45 (9)  0.29±0.02 (6)  0.13+0.05 (7)  (6) Lung  ."0.23+0.01  0.51±0.08 (6)  Each f i g u r e i s the mean ± SEM o f the number o f samples shown i n parentheses.  The d i f f e r e n c e between t r e a t e d and  c o n t r o l was s i g n i f i c a n t i n the case of p l a c e n t a and  lung  (P<0.05).  (P<0.01)  46  3.  Recovery of cpd.  A and  B after Injection  3  of Mothers w i t h  H-cpd. B  3 H-cpd. B was t i o n days 14,  i n j e c t e d i n t o the mother mice on  16 and  19,  15 min  and  l a t e r the p l a c e n t a  f e t a l t i s s u e s were removed, e x t r a c t e d , the  r a d i o a c t i v i t y i n both cpd. F i g s . 7 and  A and  in. v i t r o s t u d i e s  t i s s u e s there was  expected.  B zones assayed.  ( F i g s . 4-6)  age.  show t h a t i n some  i n reducing capacity,  an i n c r e a s e  i n cpd.  with  and  increasing  B content would  be  T h e r e f o r e , a t i s s u e might c o n t r o l the hormone  b o l i t e t o cpd.  4.  B  with g e s t a t i o n a l  content e i t h e r by a c t i v e l y r e d u c i n g the  of cpd.  and  a decrease i n dehydrogenase a c t i v i t y  i n others an i n c r e a s e g e s t a t i o n a l age,  and  chromatographed  8 show t h a t the percentage of cpd.  found i n a l l f e t a l t i s s u e s i n c r e a s e s Since the  gesta-  B, or by d e c r e a s i n g the  11-dehydro metar a t e of c o n v e r s i o n  B to i t s i n a c t i v e 11-dehydro m e t a b o l i t e .  Glycogen D e p o s i t i o n  in Fetal  Tissues  I t would seem reasonable t h a t , s i n c e cpd. l a t e d so t h a t the considerably  amount present i n each t i s s u e  w i t h g e s t a t i o n a l age,  B i s reguvaries  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 t i s s u e s .  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 One  s e n s i t i v e i n d i c a t o r s of c o r t i c o s t e r o n e  p o s s i b i l i t y c o n s i d e r e d was  action.  glycogen d e p o s i t i o n  in  FIGURE 7,  RECOVERY OF CPD A AND  HEART AFTER  75  B IN FETAL BRAIN/  GUT  INJECTION OF CPD B~ H.  L  CD CD  50 CQ  a. Q  + <  25  16  18  GESTATIONAL  DAY  shown 1/^1 t h e mean o f f o u r v a l u e s . Some SEM a r e 1 6 ^ £ H f between d a y 14 v s d a y 16, a n d d a y 16 v s d a y 19 a r e s i g n i f i c a n t , P<.01 ~ S  hearti  t  h  G  d  i  f  f  e  r  e  n  c  brain  e  ^  ; g t •U  AND  48  FIGURE AFTER  8,  RECOVERY OF CPD A AND  B IN FETAL LIVER, LUNG AND  PLACENTA  INJECTION OF CPD B ~ ^ H .  GESTATIONAL  DAY  E a c h p o i n t i s t h e mean o f f o u r v a l u e s . Some SEM a r e shown. The d i f f e r e n c e between day 14 and d a y 16 i n t h e c a s e o f l i v e r and l u n g a r e 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 . V a l u e s f o r d a y 16 v s 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 — — ._;  lung  •  o ; placenta  49  f e t a l t i s s u e s , s i n c e t h i s i s the o n l y parameter  thought t o  be i n f l u e n c e d by c o r t i c o s t e r o i d s a t t h i s e a r l y stage o f gestation.  F i g . 9 shows the standard curve f o r the anthrone reaction.  Although i t d e v i a t e s s l i g h t l y from  linearity  above 0.6 mg, the d e t e r m i n a t i o n on samples was made i n the linear  range.  Figs.  10 and 11 show t h a t the p a t t e r n o f d e p o s i t i o n  does not r e f l e c t the reductase p a t t e r n o f d e p o s i t i o n observed p r e v i o u s l y  ( F i g s . 4-6) and t h e r e f o r e glycogen  cannot be c o n s i d e r e d t o r e p r e s e n t a u s e f u l parameter o f steroid  action.  The e f f e c t o f i n j e c t i o n o f dexamethasone 16 h e a r l i e r on the d e p o s i t i o n o f glycogen i n v a r i o u s t i s s u e s on g e s t a t i o n a l day 16 i s shown i n Table 5. was s t i m u l a t e d s i g n i f i c a n t l y crease  (P<.01).  Only l i v e r  glycogen  (P<.01); p l a c e n t a showed a de-  I n j e c t i o n s on day 13.5 f o l l o w e d by assays  on day 14 showed no s i g n i f i c a n t i n c r e a s e i n l i v e r .  This  would imply t h a t glycogen d e p o s i t i o n i n l i v e r might  depend  on f a c t o r s 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 o f g l y c o p r o t e i n i s accomplished by PCA p r e c i p i t a t i o n as d e s c r i b e d by Roe and D a i l e y  (75). Since the amount of  t h i s p r e c i p i t a t e appeared t o vary w i t h both t i s s u e 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 1 0 .  GLYCOGEN CONTENT OF FETAL BRAIN, GUT AND HEART ON  GESTATIONAL DAYS 1 4 TO 1 9 .  GESTATIONAL DAY  E a c h p o i n t i s t h e mean o f a t l e a s t Some SEM a r e shown. heart  »  •« ; b r a i n i ^ - — —  four  A  determinations.  ; gut •  52  FIGURE 1 1 ,  GLYCOGEN CONTENT OF FETAL LIVER, LUNG AND PLACENTA  ON GESTATIONAL DAYS 1 4 TO 1 9 ,  60i_  j 2  LU  40  LU  LU 13  O U >_J (3  £  20  16  18  GESTATIONAL DAY  E a c h p o i n t i s t h e mean o f a t l e a s t Some SEM a r e shown. liver  * ; lung o  four  determinations,  ci ; placenta o  53  Table  5  EFFECT ON GLYCOGEN DEPOSITION IN FETAL TISSUES OF DEXAMETHASONE INJECTIONS INTO MOTHERS  Tissue Placenta  Day 12  16  Control  82+.83 (6)  Lung  8 .51+.17  Day  16  9. 02+. 33,: .:. (14) 8. 39+. 81 (7)  (4) Liver  0 .32+.05  3. 64 + . 64 (8)  (7) Gut  1 .93+.20  2 51 + .'36 (5)  (9) Brain  4 .16+.30 (4)  Heart  21 .74+.76  Dex  4 76+. 10 (11) 22 .59+3 .52  (6)  (5)  E a c h i s t h e mean + SEM o f t h e number o f s a m p l e s shown i n parentheses. was s i g n i f i c a n t (P<.01).  The d i f f e r e n c e i n t r e a t e d and u n t r e a t e d i n the case o f l i v e r  and p l a c e n t a  54  g e s t a t i o n a l age, with  5.  The and  dealt  later.  In V i t r o I n c o r p o r a t i o n of Leucine, Thymidine i n t o F e t a l T i s s u e s  Several on  f u r t h e r c h a r a c t e r i z a t i o n w i l l be  groups have examined c o r t i c o s t e r o i d e f f e c t s  f e t a l lung and  cell  Uridine  have r e p o r t e d  a decrease i n the r a t e of  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 ) .  implies  t h a t the a c t i o n o f c o r t i c o s t e r o i d s on t i s s u e 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 , s y n t h e s i s tion.  This  I t has  and  differentia-  been shown t h a t a c t i o n of c o r t i c o s t e r o i d s on  lymphocytes r e s u l t e d i n decreased i n c o r p o r a t i o n of a c t i v e l e u c i n e , u r i d i n e and parameters of p r o t e i n RNA  thymidine  and  DNA  i n c o r p o r a t i o n of these s u b s t r a t e s  (85).  As these  synthesis, was  g e s t a t i o n a l days 14 t o . 1 9 i n p l a c e n t a  the  therefore and  As can be observed i n F i g s . 12 and  lung  14 to day  19.  examined  1 3 , there  However, heart  a f t e r day  s i g n i f i c a n t ) , but decreased  incorporation  (Figs. 14-15)  showed a d e c l i n e between days 14 and (Fig.  occurred incorpora-  ( F i g . 12)  14) i n c r e a s e d  (P<.01).  and 16  significantly  16.  Uridine  on  fetal tissues.  ( F i g . 13) remained c o n s t a n t between days 14 and  (P value not  are  in vitro  i n a l l t i s s u e s a s i g n i f i c a n t decrease i n l e u c i n e t i o n from day  radio-  i n most t i s s u e s  1 9 , although  s i g n i f i c a n t l y from day  heart  14 to 16  FIGURE  12,  BRAIN/  GUT AND  IN VITRO  INCORPORATION  OF  1 4  C - L E U C I N E INTO FETAL  HEART.  GESTATIONAL DAY E a c h p o i n t i s t h e 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 b r a i n and g u t day 14 v s d a y 16, P<.01; f o r d a y 16 v s day 19 b r a i n and g u t P<.01 and h e a r t P<.05. brains™t;  gut*——*;  heart"  *  56  FIGURE 1 3 . LUNG AND  IN VITRO  INCORPORATION  OF -^C-LEUCINE  INTO FETAL LIVER,  PLACENTA.  125  Q LU  I<  cn  o  CL  cc O  5 LU  Csl I  c: r—  x  5 75  LU  \—  o  cn  o  CL.  ZD LU I  I CL.  25  18  16 GESTATIONAL DAY  E a c h p o i n t i s t h e 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 p l a c e n t a and l i v e r day 14 v s d a y 16, P<.01; f o r d a y 16 v s d a y 19 l u n g and l i v e r , P<.01 and p l a c e n t a i s n o t s i g n i f i c a n t . liver A — — p l a c e n t a o  o;  lungo^  IN  FIGURE  VITRO INCORPORATION  OF ^H-URIDINE INTO FETAL BRAIN,  GUT AND HEART.  80  Q LU  I - CN) < I cm o  o i—I CL.  CC  X  C_> Z  Z —• LU  O i—i  LU  2  40  Q I—«  a: CL  5 ^  •ii- 14  18  16 GESTATIONAL DAY  E a c h p o i n t i s t h e 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 d a y 14 v s d a y 16 b r a i n and g u t P<.01 a n d h e a r t P<.02; f o r d a y 16 v s d a y 19 g u t and h e a r t P<.01. b r a i n / * - — g u t *  • ; heart •  58  FIGURE 1 5 .  IN VITRO INCORPORATION  LIVER, LUNG AND  OF ^H-URIDINE INTO FETAL  PLACENTA.  GESTATIONAL DAY E a c h p o i n t i s t h e 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 d a y 14 v s 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 v s d a y 19 l i v e r P<.01 and l u n g P<.02. liver A — — p l a c e n t ao  o; l u n g D  a  59  Thymidine i n c o r p o r a t i o n i n most i n s t a n c e s decreasing  showed a  p a t t e r n of i n c o r p o r a t i o n w i t h i n c r e a s i n g  t i o n a l age ( F i g s . . 1 6 - 1 7 ) .  Again, lung was an  exception--  as was the case i n l e u c i n e i n c o r p o r a t i o n — t h e day  gesta-  values f o r  14 t o 16 d i d not differ... s i g n i f i c a n t l y , but the i n c o r -  p o r a t i o n between days 16 and 19 d i d ( P < . 0 1 ) .  The  d e c l i n e i n i n c o r p o r a t i o n o f the t h r e e  a f t e r day 14 i n b r a i n , l i v e r , day  16 i n h e a r t  reflecting these times  placenta  and gut,  substrates and a f t e r  and l u n g , -could w e l l be i n t e r p r e t e d as  increased  c o r t i c o s t e r o n e i n these t i s s u e s at  (Figs. 7 - 8 ) .  These parameters appear t o o f f e r a much b e t t e r  indi-  c a t i o n o f e f f e c t s which a r e t a k i n g p l a c e which might r e f l e c t changes i n hormone l e v e l confirm  i n these t i s s u e s .  To  t h i s , the s y n t h e t i c c o r t i c o i d dexamethasone was  i n j e c t e d on g e s t a t i o n a l day 1 3 . 5 and 16 h l a t e r the t i s s u e s were removed and assayed f o r i s o t o p e  Leucine values  incorporation.  (Table 6 ) were decreased  (P<.0.1.) i n a l l t i s s u e s except lung and h e a r t .  significantly This  fol-  lows the p a t t e r n observed f o r lung and h e a r t which was shown not t o a l t e r between g e s t a t i o n a l days 14 and 1 6 . I t should  be noted t h a t the Dex e f f e c t appears t o be s t i m u l a -  t i n g n a t u r a l development prematurely; i . e . , the same general  trends  as would normally occur i n each t i s s u e are  f o l l o w e d but are a c c e l e r a t e d .  60  FIGURE 1 6 . GUT AND  IN VITRO INCORPORATION  OF H-THYMIDINE INTO FETAL BRAIN, 3  HEART.  400  Q LU I<  DC  O CL  DC  O O  LU 2:  —  300 CM I  O i—I X LU I—  o  Q —>  CC  >-  CD  200  CL  a.  100  14  16  18  GESTATIONAL DAY E a c h p o i n t i s t h e 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 d a y 14 v s d a y 16 a l l t i s s u e s , P<.01; f o r d a y 16 v s d a y 19 b r a i n a n c T g u t P<.01 and h e a r t P<.05. — heart  ;  brain  A-——A;  gut  61  FIGURE 1 7 . LUNG AND  IN VITRO  INCORPORATION OF H-THYMIDINE INTO FETAL LIVER, 3  PLACENTA.  400  A Q UJ t<  cn 0  300 CM I  01  CD  LU  LU  —'  o cn  cn o  Q  <->  i—i  CL  \ \ \ \  200  .....y  CL  \ \  i  \ \  100  HI-  14  16  18  GESTATIONAL DAY E a c h p o i n t i s t h e 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 d a y 14 v s day 16 i n a l l t i s s u e s P<.01; f o r d a y 16 v s day 19 l u n g and l i v e r P<.01 and p l a c e n t a P<.05. livers— —  placenta o — o ;  lung »  «  62  U r i d i n e values  (Table 7) were decreased  i n l i v e r , p l a c e n t a and b r a i n  (P<.01).  No  significantly  significant  e f f e c t s were observed i n the o t h e r t i s s u e s , although gut a s l i g h t decrease was  The  in  observed.  i n c o r p o r a t i o n of thymidine  (Table 8) showed s i g -  n i f i c a n t decreases i n a l l t i s s u e s (P<.01).  Thymidine  appears to be the most s e n s i t i v e parameter, s i n c e i n a l l i n s t a n c e s Dex  injection resulted in significantly  incorporation.  L i v e r appears to be the most s e n s i t i v e  t i s s u e , s i n c e i t was Dex  decreased  q u a n t i t a t i v e l y the most a f f e c t e d by  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 r e d u c t i o n :  dehydrogenation with i n c r e a s i n g g e s t a t i o n a l age fetal tissues.  I t should be noted t h a t e i t h e r by a decrease  i n dehydrogenation  (Type 1), or an i n c r e a s e i n r e d u c t i o n  (Type 2) an i n c r e a s e d c o r t i c o s t e r o n e content in a particular  The  i n various  can be produced  tissue.  r e l a t i o n s h i p i n time between the changes i n the  i n c o r p o r a t i o n o f v a r i o u s s u b s t r a t e s and the r e l a t i v e amount of c o r t i c o s t e r o n e i s e v i d e n t .  63  Table 6 EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON LEUCINE INCORPORATION IN FETAL TISSUES  Leucine I n c o r p o r a t i o n Dpm/mg p r o t e i n Tissue Placenta Lung  Day 14 C o n t r o l  Day 14 Dex  759±96  465±40  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 o f a t l e a s t + SEM.  determinations  Values f o r t r e a t e d p l a c e n t a , l i v e r , gut and b r a i n  differ significantly do n o t .  five  from c o n t r o l s (P<.01); lung and heart  64  Table 7 EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON URIDINE INCORPORATION IN FETAL TISSUES  Uridine Incorporation Dpm/mg p r o t e i n Tissue  Day  14 C o n t r o l  Day  14 Dex  Placenta  1017+44  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  725+105  Each value i s the mean of a t l e a s t f i v e + SEM.  Values f o r t r e a t e d p l a c e n t a , b r a i n and l i v e r d i f f e r  significantly do n o t .  determinations  (P<.01) from c o n t r o l s ; gut, lung and heart  65  Table 8 EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS ON THYMIDINE INCORPORATION IN FETAL TISSUES  Thymidine I n c o r p o r a t i o n Dpm/mg p r o t e i n Tissue Placenta  Day 14 C o n t r o l 1,040+132  Day 14 Dex 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 v a l u e i s the mean of a t l e a s t f i v e - SEM.  determinations  In a l l cases the d i f f e r e n c e between t r e a t e d and  control values i s s t a t i s t i c a l l y  significant  (P<.01).  66  Table 9 SUMMARY OF FETAL TISSUE REDUCTASE (R) AND DEHYDROGENASE (D) ACTIVITIES  Gestational Tissue  TYPE 1  TYPE 2  TYPE 3 3  Day 14  Day  Day 16  Day 19-  Brain  D  +D  Gut  D  •fD  ++D  Liver  D  R  +R  Lung  D  +D  R  Placenta  R  +R  ++R  + denotes decrease; f denotes  increase  BEAD)  A  DEHYDROGENATION  TYPE 1 RATIO< 1  DAY lk  DAY 19  DAY 14  DAY 16  BLOOD  REDUCTION  \  TYPE 2 RATIO > 1 LIVER  See  t e x t p. 6 2  6.7  6.  Ornithine  The has  Decarboxylase A c t i v i t y i n P l a c e n t a  a c t i v i t y o f the enzyme o r n i t h i n e  been r e p o r t e d  decarboxylase  to be a s e n s i t i v e i n d i c a t o r of the  response o f t i s s u e s t o various, ^hormonal s t i m u l i . increase  i n a c t i v i t y o f t h i s enzyme i n v a r i a b l y accompanies  increased  cell division (77).  Ornithine  a c t i v i t y has been shown t o d e c l i n e d u r i n g fetal rat liver therefore, mouse.  An  ( 8 6 ) and p l a c e n t a  (77) .  t o examine a c t i v i t y d u r i n g  decarboxylase development i n I t was o f i n t e r e s t ,  gestation  i n the  The a c t i v i t y decreased i n mouse p l a c e n t a  increasing  g e s t a t i o n a l age ( F i g . . 1 8 ) .  with  The i n j e c t i o n o f  dexamethasone a l s o produced prematurely on day 14 v a l u e s normally observed between days 1 7 - 1 8 ( F i g .  Ornithine  18)..  decarboxylase was then assayed i n f e t a l  b r a i n , but low a c t i v i t y was observed.  No f u r t h e r  studies  were made, s i n c e i t d i d not appear t h a t t h i s would be as u s e f u l a parameter f o r the t i s s u e s under i n v e s t i g a t i o n . However, as i n d i c a t e d by the data on p l a c e n t a ,  the d e c l i n -  i n g r a t e s o f s e v e r a l p r o c e s s e s , i n c l u d i n g the i n c o r p o r a t i o n of l e u c i n e and  ( F i g . 13), u r i d i n e  ( F i g . 1 5 ) , thymidine  o r n i t h i n e decarboxylase a c t i v i t y  to be mediated by c o r t i c o s t e r o i d .  (Fig. 17)  ( F i g . 1 8 ) , a l l appear  68  FIGURE 1 8 ,  ORNITHINE DECARBOXYLASE  1 4 TO 1 9 IN PLACENTA AND  ACTIVITY GESTATIONAL  THE EFFECT OF DEXAMETHASONE  DAYS  INJECTION.  E a c h p o i n t i s t h e 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 v a l u e i s s i g n i f i c a n t l y d i f f e r e n t f r o m t h e day 14 c o n t r o l (P<.01).  69  7.  Amino A c i d A n a l y s i s of F e t a l B r a i n  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 pated t h a t , s i n c e glucose i s the major o r g a n i c reaching  the  anticisubstrate  f e t u s , 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 t i s s u e which are..influenced by c o r t i c o steroids.  In f a c t , t h i s e x p e c t a t i o n  the case of glycogen d e p o s i t i o n  was  not r e a l i z e d i n  ( F i g s . 10-11); however, a  number of i n t e r e s t i n g o b s e r v a t i o n s  were made which l e n d  credence to the concept t h a t c o r t i c o s t e r o i d s p l a y an  impor-  t a n t r o l e i n f e t a l development a t t h i s e a r l y stage of gestation.  F e t a l b r a i n on g e s t a t i o n a l day  14, w i t h and  p r i o r treatment w i t h dexamethasone, and  on day  without  15 were  4 incubated  with  C-glucose  s t i t u e n t s separated sions.  (u), and  on TLC  sheets,  the a c i d - s o l u b l e condeveloped i n two  S i x samples o f each were examined, and  grams were prepared.  These i n d i c a t e d decreased  of glucose to a number of other samples and  those on day  a substantial increase  15.  constituents  of which are i l l u s t r a t e d i n F i g . 19.  tyrosine.  Few  aspartic  was  the r e s u l t s  S i g n i f i c a n t increases  15 as compared w i t h day  a c i d i c amino a c i d s glutamic, and  treated  ninhydrin,  This  examined f u r t h e r u s i n g an amino a c i d a n a l y z e r ,  on day  conversion  i n s e v e r a l n i n h y d r i n - s t a i n i n g com-  ponents were a l s o observed on these sheets.  are e v i d e n t  autoradio-  i n the  When sprayed w i t h  dimen-  14,  i n the  arid a l a n i n e ,  b a s i c amino a c i d s were present,  threonine and  the  70  level Dex  of  l e u c i n e was  produced  intermediate  8.  i n samples o f between t h a t  I n v e s t i g a t i o n of  During the step  i n the  proteins was  equally  low  day  Glycoprotein  of  An  14  and  14  day  a  pattern  15.  glycogen the  ( F i g . 10-11),  p r e c i p i t a t i o n of  This  age  i n some d e f i n i t e p a t t e r n  suggested p o s s i b l e  thesis.  To  synthesize Table  .10,  test this,  liver  glyco-  with  i n each t i s s u e .  s t i m u l a t i o n of glycoprotein the  glycoproteins fetal  one  unexpectedly large p r e c i p i t a t e  sometimes o b s e r v e d , w h i c h a p p e a r e d t o change  gestational  with  Synthesis  procedure required  with acid.  Treatment  g e s t a t i o n a l day of  isolation  in all.  capacity  was  and  of  fetal  examined.  gut  As  tissues  seen  b o t h have t h e  synto  in  requisite 14  enzymes f o r s y n t h e s i z i n g  glycoproteins  s a m i n e , and  amount i n c o r p o r a t e d  on  the  relative  g e s t a t i o n a l day The  first  known t o be samine.  The  r e a c t i o n was  the  14  step  and  later  in fetal  in glycoprotein  regulatory  capacity  of  step,  fetal  from  C-glucowas  gut.  synthesis,  i s the  similar  which i s  synthesis  t i s s u e s to carry  examined i n c y t o s o l p r e p a r a t i o n s  of  out  of  glucothis  gut  and  14 liver strate  incubated f o r the  transferase  with  C-fructose-6-PO^, which i s the  enzyme ' G l u t a r n i n e - f r u c t o s e - 6 - P  (EC.5.3.1.19). • A c i d - s o l u b l e  were c h r o m a t o g r a p h e d , and w h i c h i n d i c a t e d no  amino-  extracts  a u t o r a d i o g r a m s were  d i f f e r e n c e i n the  sub-  capacity  prepared, of  fetal  Fig.  19:  brain day  15  Amino a c i d a n a l y s i s  on g e s t a t i o n a l ( 3 ) . Cpd.X  day 14  indicates  o f 50 v l o f s a m p l e s  from  ( 1 ) , day 14 D e x - t r e a t e d an u n i d e n t i f i e d  fetal  (2) and  constituent.  72  <c_  -1  73  Table 10 INCORPORATION OF C-GLUCOSAMINE INTO FETAL LIVER AND GUT 14  Tissue  Incorporation, dpm/mg P r o t e i n  L i v e r , day 14  768+130  Gut, day 14  828+54  Gut,  969+161  days 17-19  Each p o i n t i s a mean of a t l e a s t + the SEM.  four  determinations  74  tissues strate  of to  d i f f e r e n t g e s t a t i o n a l age a number o f  to metabolize  the  sub-  intermediates.  When c y t o s o l p r e p a r a t i o n s  of  liver  and  gut  were  14 incubated  with  C-glucose  and  autoradiograms  chromatographed e x t r a c t s were p r e p a r e d ing  d i f f e r e n c e was  day  14  and  Little  day  the  day  the  14  i n the  was  f o r m e d on  age.  Treatment with  similar  to  involvement of  samples of  the  the  of.phosphorylated  as  the  however,  At tates  this  glycogen, not  revealed  f o r bound  tent  in tissues  glycoprotein material  to  The  fructose.  14,  whereas later pattern  again  indicating  day  14;  sialic (87)  most of The  during the  resembling  which  did from  of  appear  increase, 1196  the  the  to  necessary  precipi-  isolation  of  present  was  material  periodate-resorcinol  showed t h a t , w h i l e there  without  intermediates  a c i d , a measure of  present,  In  occur.  "glycoprotein" that  proceed  latter  however, examination  in fact glycoprotein.  tion  of  as  to  intermediates,  area that  synthesis  point,  obtained  age,  appeared  s a m p l e s on  It i s in this  glycoprotein  glucose  produced a  later  strik-  gestational  i n samples of Dex  constituents.  i n Dex-treated  2 7 9 0 dpm. for  conversion  more p o l a r  a  c o r t i c o s t e r o i d i n these events.  some s a m p l e s , formation  of  g e s t a t i o n a l day  converted  the  (Fig. 20),  between samples of  conversion  g l u c o s e was  gestational on  16  fructose  most of  evident  of  glycoprotein there  w e r e much l a r g e r  deoxyribonucleic  reac-  acid  was  con-  some  quantities (Fig.  21).  Fig. gut.  20:  Autoradiogram o f chromatographed  1 and 2, day  amount o f f r u c t o s e  14;  3 and 4, day  on day  F:  fructose  G:  glucose  16.  16.  extracts  Note  the  of  fetal  increased  76  Fig.  21:  acid  performed  The p e r i o d a t e - r e s o r c i n o l  reaction  on PCA p r e c i p i t a t e s o f f e t a l  1.  Brain  2.  Liver  3.  Gut  4.  Lung  5.  Sialic  Acid  Standard  f o r bound tissues:  sialic  78  T  1  A. (nm)  r  79  Examination o f the a b s o r p t i o n p a t t e r n ( F i g . 22) a l s o i n d i c a t e d t h a t  9.  Variation  i n the A c i d  o f the p r e c i p i t a t e  i t was DNA.  I n s o l u b i l i t y o f DNA from F e t a l T i s s u e s  Experiments were c a 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  t i s s u e s were processed as f o r glycogen d e t e r m i n a t i o n s , and the  acid-insoluble  p r e c i p i t a t e was r e d i s s o l v e d  NH^OH and assayed f o r r a d i o a c t i v i t y . gut,  i n dilute  Fetal brain,  lung, h e a r t and p l a c e n t a were i n c l u d e d  liver,  and the r e s u l t s  expressed as the percentage o f t o t a l counts i n the t i s s u e d i g e s t which were recovered i n the p r e c i p i t a t e The  material  ( F i g . 23).  which d i d not p r e c i p i t a t e a t room temperature  was s t o r e d  a t 0-4°C, and upon s t a n d i n g , the remaining DNA  eventually  precipitated.  Treatment o f mice w i t h Dex  produced changes which i n d i c a t e d t i s s u e with increasing  t h a t the t r e n d  gestational  age was  i n each  accelerated.  The  only two t i s s u e s which d i d not show a decrease, p l a c e n t a  and  lung, were the two which showed an i n c r e a s e  days 14 and 16, before d e c l i n i n g t h e r e a f t e r  (Table 11).  These o b s e r v a t i o n s suggest some d i f f e r e n c e properties  between  i n the  o f these macromolecules, the s i g n i f i c a n c e of  which remains obscure, but which i l l u s t r a t e y e t another phenomenon which appears t o be i n f l u e n c e d  a t t h i s stage o f  80  WAVELENGTH  (NM)  81 FIGURE OF  23.  FETUSES  PRECIPITATION REMOVED  FROM  OF  RADIOACTIVITY  MOTHERS  INJECTED  GESTATIONAL  heart liver  ; brain A — — — - A  WITH  PCA 3  FROM  TISSUES  R-THYMIDINE.  DAY  — — ;  ; placenta o  BY  gut  o ; lung  o — — — o  82  Table 11 3  THE EFFECT OF DEXAMETHASONE INJECTION INTO MOTHERS GIVEN HTHYMIDINE 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 by PCA a d d i t i o n Tissue  Day  14 C o n t r o l  Day 14 Dex  Liver  100  69  Gut  100  81  75  78  Heart  100  74  Brain  92  67  Placenta  67  47  Lung  precipitated  Each value i s an average o f two t o s i x d e t e r m i n a t i o n s .  83  g e s t a t i o n a t l e a s t i n p a r t as a r e s u l t o f c o r t i c o s t e r o i d action.  Studies  on C o r t i c o s t e r o i d Receptors i n F e t a l  While a number of o b s e r v a t i o n s  Tissues  have been made i n t h i s  i n v e s t i g a t i o n which appear worthy o f f u r t h e r e x p l o r a t i o n , the primary aim o f t h i s work was to examine the f a t e o f c o r t i 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 c o n s t i t u e n t s o f fetal tissues. search  A t t e n t i o n was t h e r e f o r e r e d i r e c t e d t o a  f o r s p e c i f i c c o r t i c o s t e r o i d receptors  t i s s u e s and an examination o f some o f t h e i r  in fetal properties.  Most workers i n v e s t i g a t i n g c o r t i c o s t e r o i d r e c e p t o r s have found t h a t endogenous s t e r o i d s present attempting t o l a b e l r e c e p t o r  sites.  approached by a d r e n a l e c t o m i z i n g assay. mice.  a problem i n  T h i s has been  animals p r i o r t o the  T h i s i s c l e a r l y not f e a s i b l e i n the case o f f e t a l A l s o , n e a r l y a l l workers i n t h i s area use s y n t h e t i c  c o r t i c o s t e r o i d s with a 9 a f l u o r o s u b s t i t u e n t which i s claimed  t o bind more e f f e c t i v e l y t o r e c e p t o r s .  The use o f  3  such a compound,  H-dexamethasone, has enabled the demon-  s t r a t i o n of a s p e c i f i c receptor l a t i o n o f a constant value  i n f e t a l b r a i n , and c a l c u -  f o r the i n t e r a c t i o n (Table  12) .  The  of 8.3 nM i s c o n s i s t e n t with those r e p o r t e d f o r  such r e c e p t o r s  i n other  capacity receptor.  t i s s u e s , f o r a h i g h - a f f i n i t y , low-  The number o f b i n d i n g s i t e s d i d not  i n c r e a s e w i t h i n c r e a s i n g g e s t a t i o n a l age, and, i n f a c t ,  84  T a b l e 12 PROPERTIES OF CORTICOSTEROID RECEPTOR(S) IN FETAL MOUSE BRAIN  K  8.3  d  Total  steroid  bound  0.14  nM pmoles/mg  .{.day 14)  Relative binding (dpm/mg p r o t e i n ) On g e s t a t i o n a l day 1 4 :  6433  + 1109 (6)  day 1 7 :  1551  + 345  On g e s t a t i o n a l  (5)  protein  85  the c o n c e n t r a t i o n per mg  protein declined.  T h i s was  even when d i t h i o t h r e i t o l , which has been r e p o r t e d s t a b i l i z e r e c e p t o r s i n some t i s s u e s , was  Because of the abundance of i n f e t a l t i s s u e s , i t was a c t i o n of t h i s m e t a b o l i t e  to  included.  11-dehydrocorticosterone  of i n t e r e s t to examine the and  true  inter-  the a c t i v e hormone w i t h  s p e c i f i c c e l l u l a r receptors.  I t was  found t h a t r a p i d  methods of s e p a r a t i n g bound and unbound s t e r o i d , by g e l f i l t r a t i o n on Sephadex G-25  or u s i n g  dextran-coated  c h a r c o a l , were s a t i s f a c t o r y , whereas d i a l y s i s f o r 16 h r e s u l t e d i n l o s s of s p e c i f i c b i n d i n g of these two s t e r o i d s . Repeated attempts to o b t a i n a Scatchard and B were u n s u c c e s s f u l .  I t was  concluded  c e n t r a t i o n of endogenous s t e r o i d was i t was  not p o s s i b l e to achieve  t i o n s f o r these d e t e r m i n a t i o n s .  p l o t u s i n g cpd. t h a t the con-  probably  so h i g h t h a t  s u f f i c i e n t l y low  concentra-  Repeated washing of t i s s u e  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 . to l a b e l r e c e p t o r s i t e s , however, and  I t was  c a r r y out  t e s t s on the r e l a t i v e b i n d i n g of cpd. A and B. i n the a c t u a l counts bound from one was  A  possible  various Variation  experiment to the next  a t t r i b u t e d to the f a c t t h a t the endogenous p o o l might  vary, thereby  influencing available binding s i t e s .  ever, minimum values of around 25,000 dpm  were  and r e s u l t s were very c o n s i s t e n t w i t h i n any T h i s i s i l l u s t r a t e d i n Table  How-  obtained,  experiment.  13, which shows the i n f l u e n c e 3  of v a r i o u s f a c t o r s on the b i n d i n g of  H-cpd. B.  86  Table 13 EFFECT OF VARIOUS FACTORS ON BINDING OF H-CPD. B IN CYTOSOL 3  Dpm/mg P r o t e i n  %  781801  100  766270  98  + 1 mM N-ethyl maleimide  775356  99  + RNase  (1 mg/ml)  950614  122  + DNase  (.25 mg/ml)  758290  97  552760  71  Control + 1 mM  Dithiothreitol  + Pronase  (1.5 mg/ml)  87  As can be seen, the b i n d i n g of the s t e r o i d was  un- .  a f f e c t e d by nucleases, but d i m i n i s h e d by pronase. incomplete  e f f e c t of the l a t t e r was  The  a t t r i b u t e d to the  low a c t i v i t y of the p r e p a r a t i o n which was  used.  Dithio-  t h r e i t o l had no e f f e c t , nor d i d N-ethyl maleimide.  The i n Table  c o m p e t i t i v e i n t e r a c t i o n of cpd. A 14.  and-.B-Lisoevident  I t should be noted t h a t what appears to be a  l a r g e excess o f u n l a b e l l e d s t e r o i d i n r e l a t i o n t o the small amount of l a b e l l e d compound—about 8 nM--is misleading.  The  endogenous pool of both cpd. A and B i n f e t a l  t i s s u e p r e p a r a t i o n s i s probably of the order (74, 88). was  A c c o r d i n g l y , the displacement  100-200 nM  which was  observed  o n l y modest, but c o n s i s t e n t .  Since cpd. A and B appear to compete i n the c y t o s o l f o r the same r e c e p t o r , i t was  of i n t e r e s t to  observe  whether or not the cpd. A - r e c e p t o r complex c o u l d e n t e r the nucleus.  P r e v i o u s work i n t h i s l a b o r a t o r y has  demonstrated  a s p e c i f i c r e c e p t o r f o r c o r t i c o s t e r o i d s i n mouse p l a c e n t a (78) i n both c y t o s o l and n u c l e i . from p l a c e n t a by hypotonic  N u c l e i c o u l d be  prepared  shock, and experiments were  c a r r i e d out on t h i s t i s s u e to a s c e r t a i n whether or not a cpd. A-receptor  complex c o u l d be  The n u c l e a r sap was  isolated.  f r a c t i o n a t e d on a Sephadex  column and the r e s u l t s are shown i n F i g . 24. and B are demonstrable i n a...protein:-bound  G-25  Both cpd. A  form.  However,  8 8  Table 1  4  COMPETITIVE DISPLACEMENT OF LABELLED STEROID IN CYTOSOL  Dpm Bound/mg Control, +  8  0  0  80 0  H-cpd. B  nM cpd. A  Control, +  3  3  Protein  7 8 1 9 0 1 + 1 8 2 2 6  1 0 0  6 4 0 6 4 1 + 4 3 9 4 2  8 2  8 6 3 9 + 9 4 7  H-cpd. A  nM cpd. B  4 4 8 9 + 1 0 6 8  Each f i g u r e i s a mean + SEM of four v a l u e s . and  3  %  1 . 0 0 i 5 4  The ""H-cpd. B  .  H-cpd. A were examined i n d i f f e r e n t experiments.  The b i n d i n g v a r i e d c o n s i d e r a b l y  from one experiment t o  the next, which was a t t r i b u t e d t o the i n f l u e n c e o f endogenous s t e r o i d s .  The d i f f e r e n c e s here do not i l l u s t r a t e  relative affinities  f o r the two s t e r o i d s , s i n c e i n the  same experiment the b i n d i n g same magnitude.  o f cpd. A and B was o f the  The decrease observed i n the presence o f  u n l a b e l l e d competitor i s s i g n i f i c a n t i n each case, P < . 0 2 .  89  FIGURE  24..  ISOLATION OF RECEPTOR  COMPLEXES  FROM  PLACENTA  SEPHADEX G - 2 5 .  VOLUME  OD  ^  <\ ; Dpm  cpd.  (ML)  A  — — — •  ; Dpm  cpd.  B  o  ON  90  due  to the r e d u c i n g c a p a c i t y of p l a c e n t a  can reduce cpd. A t o B, i t was  ( F i g . 5), which  thought p o s s i b l e t h a t some  3  c o n v e r s i o n of the  H-cpd. A might be r e s p o n s i b l e f o r the  apparent n u c l e a r b i n d i n g .  T h e r e f o r e , the use of  ketoprogesterone,  a s t e r o i d capable of b l o c k i n g  activity  considered.  (84) was  11reductase  14 Fig.  25 shows t h a t when  C-cpd. A was  the presence of 6 pM 11-ketoprogesterone, to cpd. B was  s u b s t a n t i a l l y decreased.  incubated i n  the The  conversion  i n c l u s i o n of  11-ketoprogesterone i n n u c l e a r b i n d i n g assays, however, d i d not d i m i n i s h the b i n d i n g of cpd.  A.  A photograph of n u c l e i i s o l a t e d " f r o m f e t a l b r a i n by hypotonic  shock i s shown i n F i g . 26.  dehydrogenase a c t i v i t y of the l a b e l l e d cpd.  Fig.  F e t a l b r a i n has  ( F i g . 4), which would prevent  strong reduction  A.  2 7 i n d i c a t e s t h a t both cpd. A and B e n t e r  the  3  nucleus.  There i s , however, a l a r g e r amount ofv H-cpd. A  unbound compared w i t h cpd. B, and, counts of each added was ence i s thought to be  s i n c e the number of  the same, t h i s c o n s i s t e n t d i f f e r -  real.  These data demonstrate t h a t cpd. A can e n t e r nucleus  e i t h e r f r e e of protein-bound.  the  The p o s s i b i l i t y  was  then c o n s i d e r e d t h a t cpd. A c o u l d be more than a competitor,  and might i t s e l f b i n d to chromatin,  as has been shown  F i g . 25:  Autoradiogram 1  incubated w i t h  of chromatographed e x t r a c t s of placenta-  4 C — 1 1 - d e h y d r o c o r t i c o s t e r o n e and  gesterone. 1+2  controls  3 + 4 c o n t a i n i n g ;6 y.M A: B:  11-ketoprogesterone  11-dehydrocorticosterone corticosterone  11-ketopro-  92  1  2  3  4  Fig.  26:  Nuclei  with  dilute  isolated  MgCl . 9  from  fetal  brain  x800.magnification.  by  hypotonic  shock  94  FIGURE  27.  SEPHADEX  ISOLATION  G-25.  OF  RECEPTOR  COMPLEXES  FROM  FETAL  BRAIN  ON  96  for  those s t e r o i d s which e x e r t b i o l o g i c a l a c t i v i t y  The  i s o l a t e d n u c l e i were t h e r e f o r e e x t r a c t e d w i t h  high s a l t , at  (42).  0.6M  KC1  i n T r i s b u f f e r , pH 8.0,  105,000 xg f o r 15 min  natant was  (89).  and c e n t r i f u g e d  The:rsteroid i n the  separated i n t o protein-bound  super-  and unbound f r a c -  t i o n s by dextran-coated c h a r c o a l 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 w e l l as t h a t t i g h t l y bound to the p e l l e t , which c o n t a i n s 98% of t i s s u e DNA  (90), were  assayed.  Both cpd. A and B bind to t h e . c h r o m a t i n - f r a c t i o n . Competitive displacement other was  observed  these experiments homogenization  of each s t e r o i d by 12 yM of the  (Table 15). the t i s s u e was  I t might be noted t h a t i n minced and not d i l u t e d  p r i o r to the assay.  by  The endogenous s t e r o i d s  would t h e r e f o r e by r e l a t i v e l y more c o n c e n t r a t e d , making it difficult  The critical  to domonstrate c o m p e t i t i o n w i t h added  steroid.  i d e n t i t y of the s t e r o i d s bound to r e c e p t o r s i s of importance.  accomplished  Accordingly, characterization  by chromatography and by  co-crystallization  w i t h a u t h e n t i c added c a r r i e r compounds of s t e r o i d s from r e c e p t o r complexes.  As shown i n Table 16, the  recovered from each f r a c t i o n proved had been added i n each case.  was  isolated steroid  to be the one which  97  Table  15  COMPETITIVE DISPLACEMENT OF STEROIDS BOUND IN NUCLEAR FRACTIONS  % o f Counts i n F r a c t i o n Unbound  100  100  100  + cpd. A  59  67  59  + cpd. B  66  69  92  100  100  100  + cpd. A  53  100  81  + cpd. B  60  92  74  3  3  H-cpd. A  H-cpd. B  Values  a r e e x p r e s s e d as p e r c e n t a g e s  four d i f f e r e n t is  Pellet  Protein-bound  experiments.  i n o r d e r t o combine  The n u c l e a r  radioactivity  e x p r e s s e d a s t h a t w h i c h was e x t r a c t e d by s a l t ,  p r o t e i n - b o u n d a n d unbound, a n d t h a t w h i c h was bound t o the  pellet.  98  Table CHARACTERIZATION  (a)  16  OF STEROID FROM  RECEPTOR-COMPLEXES  Co-crystallization Crys t a l s dpm,/yg 1st  Steroid  Tissue  cpd. B  Brain,  cpd. B  Placenta,  cytosol  cpd. A  Placenta,  nuclei  cytosol  8.43 42.93 1.66  % Reccj v e r y  2nd  xls  counts  8.39  34  I  4 9.54:  41  :  1.46  53  3  1  46 55  R e c o v e r y r e f e r s t o t h e f i n a l c r y s t a l c r o p d e t e r m i n e d by UV a b s o r b a n c e 2 37-24 0 nm and t h e c o u n t s a s s o c i a t e d w i t h t h o s e c r y s t a l s o f C-21 a c e t a t e d e r i v a t i v e s (p. 2 3 ) .  (b)  Chromatography Recovery o f counts i n cpd. A %  zone  Recovered  Unbound  73  Bound  75  Pellet  74  The s t e r o i d was e x t r a c t e d f r o m e a c h f r a c t i o n o f b r a i n a n d c h r o m a t o g r a p h e d on TLC s h e e t s . nuclei with CH C1 2  2  99  DISCUSSION  Until costerone  g e s t a t i o n day 14, t h e amount o f 1 1 - d e h y d r o c o r t i -  in fetal  tissues  i s g r e a t e r than  t h e amount o f 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  to  in fetal  dehydrogenase a c t i v i t y  Subsequently,  however, i t was f o u n d  dehydrogenation respect,  tissues  in earlier  t h e p l a c e n t a o f mouse d i f f e r s  4.0 o r more from  (32)  and p l a c e n t a .  t h a t the r a t i o  in'mouse p l a c e n t a was  work  other  of reduction: (38).  In  this  species  i n c l u d i n g man ( 3 7 ) .  AutoradiogramS of f e t a l indicated  b r a i n , p l a c e n t a and l i v e r  t h a t each t i s s u e metabolizes  B r a i n was o b s e r v e d reduction  dehydrogenating  situation  These d i f f e r n c e s  the r e v e r s i b l e  than  by s i m p l e  In on  t h a t each f e t a l  enzyme C - l l s t e r o i d  identified  a  tissue  by t h e a c t i v i t y  oxidoreductase,  rather  of the f e t a l p i t u i t a r y - a d r e n a l  b r a i n many m e t a b o l i t e s o f c o r t i c o s t e r o n e were  day 14, w h i c h a l m o s t  from  on day 14 t o a r e d u c i n g one day 17.  l e d t o the concept  activation  placenta of  reducing c a p a c i t y , changing  m i g h t 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 c o n t e n t of  2-3)  corticosterone differently.  t o be a s i t e o f d e h y d r o g e n a t i o n ,  and l i v e r d e v e l o p e d  (Figs.  disappeared  by day 15.  axis.  observed  T h e s e were  as t h e 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, d e r i v a t i v e s of both  the f r e e  c p d . A and B and t h e C-2 0ctdihydro  c p d . A and B.  a r e known t o be b i o l o g i c a l l y  The C - 2 0 a d i h y d r o  inactive,  derivatives  whereas t h e a c e t a t e  j u g a t e s and t h e 1 1 - d e h y d r o c o r t i c o s t e r o n e  con-  c a n be c o n v e r t e d t o  10 0 corticosterone (91). but  i n t i s s u e s which c o n t a i n  The f o r m a t i o n  h a s been r e p o r t e d  The c o n v e r s i o n in  of acetate  the appropriate  e s t e r s i s n o t a common  i n t h e b r a i n o f two o t h e r  of Cortisol  enzymes finding,  species (92).  t o c o r t i s o n e has a l s o been  reported  adult r a t i b r a i n (93).  Since costerone other  t h e t i s s u e s examined appeared t o m e t a b o l i z e a n d t h e 11-dehydro m e t a b o l i t e  fetal  t i s s u e s were e x a m i n e d  using  differently, a double  such t h a t b o t h d e h y d r o g e n a t i o n and r e d u c t i o n  corti-  t h e s e and  isotope  could  method,  be e x a m i n e d  simultaneously.  B o t h b r a i n and g u t ( F i g . 4) a r e s i t e s although day  this  activity  19 t h e r a t i o  fold  declined after  active  i n gut.  a c t i v e hormone f o u n d  s i s t e n t with  T h i s w o u l d mean a  and hence i n c r e a s e d i n these  tissues.  t h e i n c r e a s i n g amount o f u n c h a n g e d  f o u n d on day 17 i n b r a i n  The p l a c e n t a  fetal  decreased  with  amounts o f This  ( F i g . 5) i s an a c t i v e s i t e  g e s t a t i o n a l age.  t i s s u e s , before  By c o n t r a s t ,  fetal  liver  i s con-  corticosterone  capacity increases  Conceivably,  sig-  the placenta  t u r n i n g i tt o the maternal i s a site  7).  of reduction  f u n c t i o n s t o r e d u c e t h e i n a c t i v e 11-dehydro m e t a b o l i t e in  ten-  ( F i g . 2) a n d i n b r a i n and g u t ( F i g .  throughout g e s t a t i o n , and the r e d u c i n g nificantly  increased  of corticosterone entering the t i s s u e to i t s i n -  11-dehydro m e t a b o l i t e ,  unchanged  day 14 s o _ t h a t b y  of reduction:dehydrogenation  i n b r a i n and f i v e f o l d  conversion  o f dehydrogenation,  formed  compartment.  o f d e h y d r o g e n a t i o n on day 14,  10 1.  but by day increased  16 has  developed a r e d u c i n g  to 3.5.  I t seems l i k e l y that i n c r e a s e d  a c t i v i t y i n l i v e r and p l a c e n t a  i s responsible  hormone r e s u l t i n g from r e d u c t i o n of the between g e s t a t i o n a l days 14 and  The  lung has  The  gestation ference  reductase  f o r any  11-dehydro  (Figs.  increased  metabolite  7-8).  The  r i s e i n reductase  of g e s t a t i o n i s w e l l documented  mouse resembles other  cant reducing  16  being  been i n v e s t i g a t e d more i n t e n s i v e l y than  f e t a l t i s s u e s i n recent years. towards the end  c a p a c i t y , the r a t i o  species  (84,  i n t h i s respect,  activity 94,  as  95,  I t i s a l s o of i n t e r e s t to note the  16  of  dif-  i n the time of development of the lung as compared w i t h  changes i n l i v e r , b r a i n and  gut  ( F i g s . ,4-6) .  I t can be concluded t h a t the presence of the enzyme s t e r o i d oxidoreductase can modify the l e v e l of in a tissue  so  t h a t , where dehydrogenation predominates,  l e a v i n g i t w i l l be lower than i n blood  entering.  On  hand, where reductase a c t i v i t y predominates, the (or C o r t i s o l ) w i l l be augmented by r e d u c t i o n of corticosterone  C-11  corticosterone  content of a c t i v e c o r t i c o s t e r o n e i n t h a t t i s s u e and  (32).  96).  signifi-  c a p a c i t y does not develop u n t i l a f t e r day  ( F i g . 6).  other  the  i n blood the  other  corticosterone 11-dehydro-  (or c o r t i s o n e ) which i s abundant i n f e t a l  life  Whatever the l e v e l o f a c t i v e hormone i n a t i s s u e , t h a t  l e v e l can be i n c r e a s e d  by a r i s i n g c o n c e n t r a t i o n  i n the  blood  e n t e r i n g the t i s s u e , by a decrease i n the r a t e of dehydrogenat i o n of the hormone w i t h i n the t i s s u e or by an enhanced r a t e of r e d u c t i o n of the  11-dehydro m e t a b o l i t e  (Table  9).  102  The  f e t a l t i s s u e s l u n g , l i v e r and p l a c e n t a are  active  s i t e s of r e d u c t i o n ( F i g s . 5-6), which i n c r e a s e s w i t h g e s t a t i o n a l age.  I t was  of i n t e r e s t t h e r e f o r e to examine the  of dexamethasone i n j e c t i o n on the C-11 activity.  steroid  effect  oxidoreductase  As i n d i c a t e d i n Table 4, both lung and p l a c e n t a  showed s i g n i f i c a n t l y i n c r e a s e d reductase a c t i v i t y on day 16 h f o l l o w i n g dexamethasone i n j e c t i o n i n t o mothers.  16, .  The  injec-  t i o n i n c r e a s e d the l e v e l i n p l a c e n t a t o t h a t normally found g e s t a t i o n a l day cantly  on  19, whereas i n lung i t i n c r e a s e d i t s i g n i f i -  (P<.05), but not as h i g h as the day  19 l e v e l .  This  may  be i n t e r p r e t e d as i n d i c a t i n g t h a t the l e v e l of a c t i v e hormone i n these t i s s u e s can i n f l u e n c e the t a t e of i t s own No e f f e c t was  observed  i n o t h e r t i s s u e s on day  16  production. (Table 4)  none of the t i s s u e s responded when i n j e c t e d e a r l i e r  Smith, Torday and Giroud C-11  (Table 3).  (81) have shown i n d u c t i o n of  s t e r o i d oxidoreductase i n f e t a l human lung c e l l s  i n v i t r o with C o r t i s o l .  and  incubated  T h i s f i n d i n g would support present  data s u g g e s t i n g t h a t maternal  i n j e c t i o n of s y n t h e t i c 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 i n c r e a s e d reductase a c t i v i t y i n p l a c e n t a , l i v e r and l u n g , and of decreased dehydrogenase a c t i v i t y i n b r a i n and gut i s r e f l e c t e d i n the i n c r e a s e d p r o p o r t i o n of unchanged hormone which can be recovered from these t i s s u e s with i n c r e a s i n g g e s t a t i o n a l age a f t e r day  14  (Figs.  7-8).  103  T h i s i s .also seen i n h e a r t which, although having no C - 1 1 s t e r o i d oxidoreductase a c t i v i t y  detectable  itself, is  probably i n f l u e n c e d by the hormone c o n c e n t r a t i o n l i v e r and lung contributes the  (Fig. 7 ) .  Smith  i n blood  from  ( 8 4 ) has shown t h a t f e t a l  lung  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  rat. , :  I t can be concluded from t h i s work t h a t the r e g u l a t i o n of c o r t i c o s t e r o i d s i n f e t a l t i s s u e i s not determined by 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 , but r a t h e r by the i n t e r c o n v e r s i o n of the hormone and i t s 11-dehydro m e t a b o l i t e .  The l a r g e pool of  the l a t t e r serves as a r e s e r v o i r o f p o t e n t i a l hormone.  The  usefulness  of glycogen d e p o s i t i o n  i n a t i s s u e as a  p o s s i b l e parameter of c o r t i c o s t e r o n e a c t i o n was examined i n f e t a l t i s s u e s on v a r i o u s  g e s t a t i o n a l days.  were observed i n l i v e r , lung, q u a n t i t i e s i n gut and b r a i n  Fig.  heart  (Figs.  11 shows t h a t there  S i z a b l e amounts of glycogen and p l a c e n t a ,  with l e s s e r  10-11).  is little  glycogen i n l i v e r  i n g e s t a t i o n but t h a t from day 17 the content i n c r e a s e s and  reaches a c o n c e n t r a t i o n  o f 4 6 mg/g by day 1 9 .  This  early  rapidly sharp  r i s e i n glycogen accumulation i n the l i v e r i s observed i n s p e c i e s where the g e s t a t i o n a l p e r i o d i s short and u s u a l l y proceeds i n the l a s t  Since  f i f t h of gestation  (47).  the glycogen content o f l i v e r i s low on day 16 and  does not appear t o i n c r e a s e  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 o f l i v e r reductase a c t i v i t y does not appear t o be the l i m i t i n g f a c t o r c o n t r i b u t i n g t o glycogen d e p o s i t i o n , as on day  16 the reductase c a p a c i t y i s h i g h  costerone l e v e l cannot  ( F i g . 6) and the c o r t i -  be c o n s i d e r e d l i m i t i n g .  Therefore,  f a c t o r s other than c o r t i c o s t e r o i d might a l s o be i n v o l v e d i n f e t a l l i v e r glycogen d e p o s i t i o n i n mouse.  The presence  of high l e v e l s of i n s u l i n i n the r a t f e t u s  d u r i n g h e p a t i c glycogen d e p o s i t i o n has i m p l i c a t e d i n s u l i n i n glycogen storage  (97). P l a s and Numez (98), u s i n g c u l t u r e d  f e t a l hepatocytes,,.. have shown a marked g l y c o g e n i c e f f e c t o f i n s u l i n o n l y i n hepatocytes p r e v i o u s l y exposed t o C o r t i s o l . T h i s suggests t h a t c o r t i c o s t e r o i d s are r e s p o n s i b l e f o r the onset of glycogen storage and t h a t i n s u l i n o n l y p o t e n t i a t e s t h i s effect.  T h e r e f o r e , c o r t i c o s t e r o i d s might induce some step  necessary f o r i n s u l i n a c t i o n , and one p o s s i b i l i t y  i s the induc-  t i o n o f the r a t e - l i m i t i n g enzyme, glycogen.synthetase,  as i t has  been shown t h a t C o r t i s o l might be r e s p o n s i b l e f o r the s y n t h e s i s of t h i s enzyme d u r i n g development.(56,  P l a c e n t a l glycogen  99).  ( F i g . 11) r i s e s d u r i n g g e s t a t i o n t o a  peak c o n c e n t r a t i o n of 13 mg/g on day 1 6 . A d e c l i n e i n glycogen i s observed appears  t h e r e a f t e r , a t the same time the l i v e r  to accumulate.  Bernard  ( 1 0 0 ) and V i l l e e  l a t e d t h a t p l a c e n t a l glycogen i s a storage depot  glycogen  (101) postuf o r f e t a l use  and t h a t i t serves t o r e g u l a t e f e t a l b l o o d glucose l e v e l s . i t has been shown t h a t under c o n d i t i o n s o f f e t a l s t r e s s ,  But  glucose  i s not l i b e r a t e d from the p l a c e n t a and glycogen l e v e l s are not  105 reduced  (102,  placenta  103).  Also,  throughout  phosphatase  gestation  and  play a role  vasomotor f u n c t i o n found v a r i e s  The  (47).  (104, t o be  (103).  The  deposition of  and  stored  day  i s predominately  Heart glycogen  a time  glycogen  i n t h e mouse  i n other species  16  to a  t o 19 o f  The  gestation. of  lung contains large enzyme i n  dehydrogenating.  i n c r e a s e s from g e s t a t i o n a l  after,  the glycogen content decreases  on day  19.  This pattern corresponds  on  day  day  to b i r t h , i n  been e x p e c t e d ,  since  immature.  this relationship  found  to vary i n v e r s e l y  with  c o n c e n t r a t i o n might  t h e newborn mouse i s r e l a t i v e l y  t o be  mg/g  to that•observed i n  prior  m a t u r i t y arid hence a l a r g e r g l y c o g e n  and  There-  to a value of 8  s p e c i e s ( 4 7 ) ; however, t h e amount o f g l y c o g e n heart, i s thought  14  16.  other  i n glycogen appears  level  accumulation  a t which t h i s  a peak c o n c e n t r a t i o n o f 22 mg/g  But  use  to the lung C - l l s t e r o i d  oxidoreductase p a t t e r n ( F i g . 6), since  reaches  for local  ( F i g . 11)  i s apparently a species difference.  16,  placental  reserve for  the p a t t e r n observed  c o n s t a n t from  g l y c o g e n d e p o s i t s on day  final  s p e c i e s (47).  lung glycogen  l u n g g l y c o g e n does n o t c o r r e s p o n d  lung  The  c o n c e n t r a t i o n i n mouse l u n g r i s e s remains  the  glucose-6  amount o f p l a c e n t a l  c o n s i d e r a b l y w i t h the  Glycogen  105).'  as an emergency e n e r g y  a p p r e c i a b l y from  o f 10 mg/g This  glycogen  i s t h e r e f o r e more l i k e l y  might  differs  l a c k s t h e enzyme  (EC 3.1.3.9), w h i c h i s n e c e s s a r y f o r t h e  r e l e a s e o f g l u c o s e from glycogen  enzyme s t u d i e s have shown t h a t  i s not r i g i d ,  and  have  very  the  associated with a particular  decline stage  106  i n h e a r t development which might occur mainly  i n utero  i n the  mouse.  S i n c e no was  d e t e c t a b l e C-11  observed  i n heart, this  sidered d i r e c t l y reflect liver  attributable  activity  and  gut  greatly  altered  attributed  I n an  cannot  activity,  be  but  d e v e l o p i n g between d a y s  con-  might to  14 and  to t i s s u e sone.  the  16  costeroids h e a r t and  increasing  glycogen  T h e s e d e p o s i t s were  gestational  age,  glycogen  and  liver  was  the  (Table 5).  increase i n l i v e r  indicates  lung.  Animals  The  decrease  Glycogen  good p a r a m e t e r r e f l e c t i n g  This  rein-  corti-  deposition in  in treated placenta the  two  synthesis i n  other than  glycogen  t h a t dexamethasone i s a c c e l e r a t i n g  occurs normally.  dexametha-  assayed  glycogen.  that factors  i n v o l v e d i n the e a r l i e r  prior  i n which glycogen i n -  o f a dual c o n t r o l of glycogen  ( 2 5 ) , and  fetal  16 h  synthetic corticoid,  the o n l y t i s s u e (P<.01)  showed no  are  with  cannot  (Fig. 4).  deposition in  c o r t i c o s t e r o i d s , m o t h e r s were i n j e c t e d  f o r c e s the concept liver  respectively.  to correlate  significantly  days e a r l i e r  suggests  with  examination,  Fetal  creased  2 mg/g,  concentration of  t o a d e c r e a s i n g dehydrogenase a c t i v i t y  attempt  tissues with  fetal  to reductase  showed much l o w e r  w i t h v a l u e s o f 4 and  be  increase i n glycogen  activity  6-7).  Brain  not  oxidoreductase  t h e 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 t h e b l o o d due  reductase  (Figs.  steroid  (Table  5)  t r e n d which  d e p o s i t i o n d o e s n o t a p p e a r t o be steroid metabolic  patterns.  a  10.7  The of  choice  leucine,  that,  these  synthesis,  thus  other  uridine,  since  reflect  of  parameters measured--the  thymidine--was  are  substrates  their utilization  major changes a possible  produced  by  c o r t i c o s t e r o i d s . In  observed  between changes  utilization  corticosterone of  leucine  The  12-17)  nucleic  activity any  be  in a  close  7-8)  tissue,  uridine  and  effects  age.  i n decreased 14-15)  (Figs.  to  relationship  gestational  resulted  acid  expected  metabolic  fact, a  increasing  12-13),  day  i s not  corticosterone  on  lung  (83)  cell  division,  a  14  decline  in brain,  known.  was  and  sub-  Increased  incorporation and  thymidine  rat  (84)  lung  have been r e p o r t e d i t has  growth at  also  fetal  mouse grows f r o m a b o u t 1 g on  19,  is  synchronous  day  and  to  earlier  this  increase  incorporation  gut,  and  and  placenta  the  of  2 00  growth  effects  decreased  growth  stage of on  antenatal  fetal  involve  mg  liver  C o r t i s o l on  been observed  lung  over  an  in substrate  Although  lates  a  and  i n c o r t i c o s t e r o i d metabolism  s i g n i f i c a n c e of  gestational  (Figs.  for protein  16-17).  (Figs.  after  (Figs.  assumption  i n d i c a t o r of  with  (Figs.  the  in synthetic  as  strate  b a s e d on  might reasonably  act  incorporation  that  Cortisol  gestational  day  is hyperplastic;  in protein  and  nucleic  rabbit and  stimu-  (41).  gestation  of  The 14  to  i.e.,  there  acid  in  tissues  present  in  fetal  (106).  Fig. brain is  19  shows t h a t  does not  i t affected  increase by  the  amount of  leucine  from g e s t a t i o n a l  dexamethasone  day  injection.  14  to  day  Therefore,  15  nor  this  108  would r u l e out the p o s s i b i l i t y t h a t these e f f e c t s are due increased  l e u c i n e p o o l , d i l u t i n g the l a b e l l e d p r e c u r s o r  a decrease i n i n c o r p o r a t i o n of the l a b e l .  to  an  causing  This implies  that  the d e c l i n e i n i n c o r p o r a t i o n of the s u b s t r a t e s might be  inter-  p r e t e d as a decrease i n the r a t e o f s y n t h e s i s of the macromolecules i n t o which they are  The  incorporated.  d e c l i n e i n l e u c i n e , u r i d i n e and  t i o n i n l i v e r , p l a c e n t a , b r a i n and g e s t a t i o n a l days 14 and a c t i v i t y i n placenta  gut  thymidine  ( F i g s . 12-17) between  16 c o i n c i d e s with  ( F i g . 5) and  liver  dehydrogenase a c t i v i t y i n b r a i n and  incorpora-  gut  increased  reductase  ( F i g . 6) and  decreased  ( F i g . 4) both of which  i n c r e a s e the a c t i v e c o r t i c o s t e r o i d i n these t i s s u e s a t t h a t time  ( F i g s . 7-8).  Dexamethasone i n j e c t i o n  decreased the l e u c i n e , thymidine and, incorporated are  (Tables 6-8).  significantly  i n a l l but gut,  uridine  T h i s i n d i c a t e s t h a t these e f f e c t s  steroid-induced.  The  p a t t e r n of l e u c i n e , u r i d i n e and  t i o n i n t o h e a r t and  thymidine  lung i s more d i f f i c u l t to i n t e r p r e t .  f o r a l l parameters measured, maintained constant p o r a t i o n between g e s t a t i o n a l days 14 and Only a f t e r day  incorpora-  16  isotope i n c o r -  ( F i g s . 13,  15,  16 d i d the i n c o r p o r a t i o n p a t t e r n d e c l i n e .  d e c l i n e c o r r e l a t e s w e l l with found i n lung a f t e r day  16  the i n c r e a s e d reductase  ( F i g . 6).  Lung,  17). This  activity  Heart appeared to f o l l o w  the p a t t e r n of lung, at l e a s t a f t e r day  16, when i n a l l  109  parameters the 16) .  isotope  incorporation declined  Uridine incorporation  from day  14 to 16  ( F i g . 14)  d e c l i n e d s t e a d i l y from day might be  increased and  14,  s i g n i f i c a n t l y i n heart  the thymidine p a t t e r n  14 onward.  Therefore,  the  (Fig.  ( F i g s . 6-7)  as w e l l .  by o t h e r t i s s u e s ,  The  t i o n i n e i t h e r t i s s u e , but thymidine  (Table  8).  but  notably  i n j e c t i o n of dexamethasone  no s i g n i f i c a n t e f f e c t on l e u c i n e  (Table  significantly  P o s s i b l y the  6) or u r i d i n e  was  had  incorpora-  (P<.01) lowered  stimulus  16)  heart  i n f l u e n c e d not only by c o r t i c o s t e r o i d s i n lung,  c i r c u l a t i n g steroids contributed liver  ( F i g s . 12,  the  applied  too  e a r l y 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  reductase a c t i v i t y only on day (Table  16  (Table  found to i n c r e a s e 4), but  not  lung  earlier  3).  I t would appear, however, that the  i n c o r p o r a t i o n of  thymi-  dine i s the most s e n s i t i v e parameter r e f l e c t i n g changes i n c o r ticosteroid action  ( F i g s . 16-17); t h i s was  found to be the  a l s o i n lymphocytes t r e a t e d w i t h c o r t i c o s t e r o i d s (85) . was  the most r e s p o n s i v e t i s s u e by a l l parameters  17) .  These events, the  case  Liver  ( F i g s . 13,  s i g n i f i c a n c e of which remain: to  15,  be  e s t a b l i s h e d , occur at an e a r l i e r stage i n g e s t a t i o n 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 h i t h e r t o r e c o r d e d .  Ornithine  Decarboxylase  (EC 4.1.1.17) i s an i n d i c a t o r 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 t i s s u e s induced by hormones (77).  The  a c t i v i t y o f t h i s enzyme  observed to d e c l i n e with g e s t a t i o n a l age  i n mouse  was  placenta  110 ( F i g . 18), and has a l s o been observed t i o n a l age i n both r a t l i v e r d e c l i n e corresponds  to decline with gesta-  (86) and p l a c e n t a (77) .  t o the observed  t i o n o f l e u c i n e ( F i g . 13), u r i d i n e  decrease  i n the i n c o r p o r a -  ( F i g . 15) and thymidine  ( F i g . 17), and a l s o the i n c r e a s e d reductase a c t i v i t y day  This  after  14 i n p l a c e n t a ( F i g . 5 ) .  The i n j e c t i o n o f dexamethasone i n t o mothers, day 13.5, 16 h p r i o r t o assay, r e s u l t e d i n a s i g n i f i c a n t l y decreased enzyme a c t i v i t y  (P<.01)  ( F i g . 18), i n d i c a t i n g t h a t the c o r t i c o s t e r o i d  e f f e c t c o u l d be induced  prematurely.  Thymidine kinase a c t i v i t y  (EC 2.7.1.75) has a l s o been  observed t o d e c l i n e i n r a t p l a c e n t a (77), w i t h i n c r e a s i n g t a t i o n a l age.  ges-  I t s a c t i v i t y i s r e l a t e d t o the r a t e of p r o l i f e -  r a t i o n o f a p o p u l a t i o n o f c e l l s and c o r r e l a t i o n w i t h growth r a t e has been demonstrated  (107, 108).  Thymidine kinase a c t i v i t y has been shown t o d e c l i n e a f t e r treatment w i t h c o r t i c o s t e r o i d  (109). These data support, the  c o n t e n t i o n t h a t 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 r a t e o f growth i n f e t a l t i s s u e s , p o s s i b l y i n d u c i n g 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  o f t i s s u e s from t r e a t e d animals  has not r e v e a l e d any s i g n i f i c a n t d i f f e r e n c e s e v i d e n t by l i g h t microscopy.  T h e r e f o r e , these b i o c h e m i c a l changes would appear  to occur p r i o r t o m o r p h o l o g i c a l  alterations.  Ill  Fig.  19 shows t h a t between d a y s  apparent increase fetal  i n amino a c i d c o n t e n t  brain contains  mate t h r e o n i n e ,  significant  Very  few b a s i c  included  i n the data  indicate  amounts o f a s p a r t a t e ,  A l l o f t h e above-mentioned significantly  respect  t o s e r i n e , and a l a n i n e  These appeared t o i n c r e a s e , which  some d i f f e r e n c e i n t h e s y n t h e t i c a b i l i t y  Dexamethasone i n j e c t e d 16 h e a r l i e r  intermediate  This  between t h e s e o b s e r v e d d a y s  could  represent  aminase a c t i v i t y ,  gluta-  t y r o s i n e a n d some  t o p r o d u c e 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 days.  The  T h e r e was a l s o an a p p a r e n t i n c r e a s e i n  l e v e l with  to glycine.  brain.  perhaps v a l i n e , i n c r e a s e d  f r o m d a y 14 t o d a y 15.  respect  i s an  amino a c i d s were n o t e d a n d a r e n o t  presented.  amino a c i d s , e x c l u d i n g  threonine  of fetal  serine, glycine, alanine,  valine.  the  14 and 15 t h e r e  an i n c r e a s e  with might  of the b r a i n  gestational  produced  a pattern  14 and 15.  by t h e s t e r o i d o f t r a n s -  which has n o t been d e m o n s t r a t e d  i n most  fetal  t i s s u e s (12).  G l u c o s a m i n e was i n c o r p o r a t e d  into fetal  day  14 a s r e a d i l y a s l a t e r ,  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  Since  fructose-6-PO^  that the formation  limiting  step  regulated,  (Fig. 20).  that this  investigation.  and g u t on  i n d i c a t i n g that the tissues synthesis  of fructose  from g l u c o s e  i t appears c o u l d be a  W h i l e no e v i d e n c e was o b t a i n e d  glycoprotein  synthesis  was i n f a c t  r e m a i n s an a t t r a c t i v e p o s s i b i l i t y The c o n v e r s i o n  o f glucose  contain  (Table 10).  u t i l i z a t i o n was n o t d i f f e r e n t ,  possible  indicated  liver  being  which so  f o r future  to fructose,  apparently  112  without  formation of phosphorylated  shown i n s h e e p  (4) and  human  proceed  steps:  first,  tol;  i n two  and,  secondly,  ing c o r t i c o s t e r o i d by  injection  another  to fructose (Figs.  transfer  of nucleic  acid  i n the  (2).  rat  r e p o r t e d not  day  has  this  to  The  sorbiincreas-  and s t i m u l a t i o n  that this  might  be  corticosteroids.  mouse p l a c e n t a was (110);  (5). 7-8)  been  i s known t o  conversion of glucose  o f dexamethasone i n d i c a t e  been o b s e r v e d  11  p l a c e n t a , and  tissues  p r o c e s s m e d i a t e d by  The has  (5, 6)  of s o r b i t o l i n these  i n t e r m e d i a t e s , has  p r e c u r s o r s across the p l a c e n t a T r a n s f e r of thymidine  to occur u n t i l  across  gestational  a l s o been r e p o r t e d i n the r a t e  (111).  3  The  earliest  a t w h i c h m i c e were i n j e c t e d w i t h  t h e p r e s e n t work was the a c i d - i n s o l u b l e indicates  I t has  reach a c e r t a i n  tion in  indeed  in  tissues.  that placental  transfer  can  occur  variation  As  i s shown i n F i g . 23.  and  thymidine,  seen  developed  The  i n Table  11,  in  significance some  altera-  associated with metabolic  in this work—the and  g r o w t h must  which p r e c i p i t a t e s  c h a n g e s i s n o t known, b u t m i g h t i n d i c a t e  tissues.  readily  (110).  i n t h e amount o f DNA  aggregation  This  cross the p l a c e n t a  t r a n s f e r mechanism  parameters determined uridine  of a l l f e t a l  were o b s e r v e d  the  i n molecular  these  can  high counts  been s u g g e s t e d  a t room t e m p e r a t u r e  of these  and  in  s t a g e and  before thymidine  acid  14,  precipitate  t h a t thymidine  i n t h e mouse.  The  day  H-thymidme  changes  as w i t h a l l o t h e r i n c o r p o r a t i o n of  the d e p o s i t i o n of  leucine,  glycogen—whatever  113  trend t h e r e appeared  to be i n a t i s s u e was  a c c e l e r a t e d when dexamethasone was  n e a r l y always.  injected.  Since a l t e r a t i o n i n the metabolism  of c o r t i c o s t e r o n e has  been shown t o have profound e f f e c t s on parameters 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;  i n c l u d i n g the ornithine  decarboxylase a c t i v i t y ; the amino a c i d content of b r a i n ; glucose metabolism  i n l i v e r and gut; i t seems probable t h a t the t i s s u e s  i n which these changes occur are " t a r g e t " t i s s u e s of the  steroid  and t h e r e f o r e would be expected t o have s p e c i f i c s t e r o i d  recep-  tors.  The q u e s t i o n a r i s e s , then, do these r e c e p t o r s have a  r e g u l a t o r y r o l e i n mediating s t e r o i d a c t i o n ? examined on g e s t a t i o n a l days 14 and Ballard  (4 3) have demonstrated  17  Fetal brain  (Table 12).  Ballard  dexamethasone r e p o r t e d by them f o r human lung was  brain  nM  and  the presence of s t e r o i d r e c e p t o r s  e a r l y i n g e s t a t i o n i n v a r i o u s human f e t a l t i s s u e s .  s i m i l a r to the v a l u e 8.3  was  The 8.9  nM,  for very  found i n t h i s work f o r f e t a l mouse  (Table 12).  The r e c e p t o r c o n c e n t r a t i o n appeared b r a i n between g e s t a t i o n a l days 14 and  17  t o decrease i n f e t a l (Table 12), but  this  can be a t t r i b u t e d t o an i n c r e a s e i n b r a i n p r o t e i n content w i t h the number of r e c e p t o r s remaining e s s e n t i a l l y c o n s t a n t .  Since  the m e t a b o l i c a l t e r a t i o n s i n f e t a l b r a i n occur a f t e r day  14  ( F i g s . 2, 4), i t i s concluded t h a t , w h i l e r e c e p t o r s are essential  f o r mediating s t e r o i d e f f e c t s , they are not the  f a c t o r i n i t i a t i n g changes.  critical  T h i s r o l e i s a t t r i b u t e d 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 t o the h i g h l e v e l o f endogenous s t e r o i d present i n f e t a l t i s s u e s , which was determined tissue  t o be about 200-300 ng/g  (38, 88), i t was d i f f i c u l t t o o b t a i n a Scatchard p l o t o r  to observe attempts  c o m p e t i t i v e i n t e r a c t i o n o f added s t e r o i d s .  were made t o determine  d i s s o c i a t i o n constants f o r both  c o r t i c o s t e r o n e and 11-dehydrocorticosterone without  Table  Repeated  success.  14 shows t h a t cpd. A can d i s p l a c e l a b e l l e d cpd. B  from r e c e p t o r s i t e s , and v i c e v e r s a . receptor c h a r a c t e r i s t i c s  Examination  of cytosol  (Table 1.3) showed t h a t the b i n d i n g of  cpd. B was not a f f e c t e d by the n u c l e a s e s , RNase and DNase; nor by d i t h i o t h r e i t o l o r N-ethyl maleimide. s e n s i t i v e t o pronase  The r e c e p t o r was  action, i n d i c a t i n g that i t i s l i k e l y a  p r o t e i n molecule w i t h no n u c l e i c a c i d , s i m i l a r t o the f i n d i n g s of others  (43, 112). Lack o f s e n s i t i v i t y t o s u l f h y d r y l reagents  confirms the o b s e r v a t i o n of Wong and Burton  (78) on b i n d i n g i n  mouse p l a c e n t a .  Since cpd. B may be d i s p l a c e d by i t s 11-dehydro m e t a b o l i t e and c o n s i d e r i n g the h i g h l e v e l o f the l a t t e r , cpd. A, present i n f e t a l t i s s u e s , i t seems l i k e l y t h a t cpd. A might prevent b i n d i n g o f cpd. B and hence block i t s m e t a b o l i c e f f e c t s . f e t u s , t h e r e f o r e , might not o n l y be p r o t e c t e d from c o r t i c o s t e r o n e by a c t i v e metabolism  The  maternal  ( F i g s . 4-6), but a l s o by  e f 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 c o n t r a s t t o cpd. B, which was almost  entirely  115  bound, t h e r e was (Fig.  also  a l a r g e amount o f unbound c p d .  A  27).  These d a t a  suggest  t h a t not o n l y i s the  11-dehydro  compound a b l e t o b i n d t o a r e c e p t o r i n t h e c y t o s o l , the  s t e r o i d - r e c e p t o r complex c a n move f r o m  the nucleus. is  Cortexolone,  known t o f o r m  located  into  blocking  effects  Cortexolone, chromatin, itself  elicit  i t i s thought a response  Surprisingly,  Again, free  at least  the  cpd.  and  induced  i n human l u n g  fraction  of  w h i c h competes w i t h by  the  latter  that this  (89).  (115)  cpd.  The  Cortisol,  (89,  113,  114).  does not  bind  to  i s why  i t does  not  11-dehydro m e t a b o l i t e  of  Cortisol  (see A p p e n d i x I ) .  A a c t u a l l y bound t o t h e  as w e l l as  identity'of A  into  Cortisol,  c o r t i s o n e , binds o n l y very weakly to the  Cortisol,  receptor  11-deoxy a n a l o g  however, u n l i k e C o r t i s o l ,  and  the c y t o s o l  that  a c y t o s o l - r e c e p t o r complex w h i c h i s t r a n s -  the nucleus,  the  the  but  the  active  chromatin  hormone, c p d .  t h e bound s t e r o i d was  B.  e s t a b l i s h e d as  ( T a b l e 16) .  These f i n d i n g s . i n d i c a t e ; .that;fcpd. A m i g h t be more t h a n a competitor, thus  as  illustrated  far i s insufficient  abolite sibility  to determine  14-15, a l t h o u g h whether or not  i s bound e v e n i n p a r t t o a d i f f e r e n t exists  a biologically  p l a y a more p o s i t i v e  role  inactive  a t the l e v e l  evidence  t h e met-  receptor.  that 11-dehydrocorticosterone,  been c o n s i d e r e d t o be fact  i n Tables  of  ju  The  w h i c h has steroid,  pos  always  might i n  transcription.  116  BIBLIOGRAPHY 1.  Bohr, C. In: Chemical Embryology.- (Needham, J . , edi) Macmillan Co., New York. p.728 (1931).  2.  Alexander, D.P., B r i t t o n , H.G., Cohen, M.M. and Nixon, D.A. In: F o e t a l Autonomy (Wolstenholme, G.E.W. and O'Connor, M., eds.) C h u r c h i l l , London. p.95 (1969).  3.  S h e l l e y , H.J.  4.  Huggett, A. S t . G., Warren, F.L. and Warren, J . P h y s i o l . 113: 258 (1951).  5.  Hagerman, D.D. and V i l l e e , C.A. 911 (1952).  6.  Chinard, F.P., Danesino, V., Hartman, W.L., St. G., P a u l , W. and Reynolds, S.R.M. 132: 289 (1956).  7.  James, E . J . , Raye, J.R., Gresham, E.L., Makowski, E.L., Meschia, G. and B a t t a g l i a , F.C. P e d i a t r i c s 50: 361 (1972).  8.  Widdas, W.F.  9.  R i c e , P.A.,  Proc. Nutr. Soc. 28_ 42  J . P h y s i o l . 118,  (1969).  J. Clin.  23  N.V.  I n v e s t . 31: Huggett, A. J. Physiol.  (1952).  Rourke, J.E., N e s b i t t , R.E.L., J r .  Invest. 7:  213  Gynecol.  (1976).  10.  E l y , P.A.  J . P h y s i o l . 184:  255  (1966).  11. 12.  F o l k a r t , G.R., Dancis, J . and Money, W.L. Amer. J . Obstet. Gynec. 8£: 221 (1960). Hahn, P. and K i r b y , L. I n : P e r i n a t a l Pharmacology (Dancis, J . and Hwang, J.C. , eds.) ..Raven: P r e s s , New York. p.79 (1974).  13.  Hahn, P. and S k a l a , J .  C l i n . Obstet. Gynecol. 14_:  655  (1971). 14.  Yeung, D. and O l i v e r , I.R.  15.  Robertson, A.F. and Sprecher, H. 183: 3 (1968). H e r s h f i e l d , M.S. and Nemeth, A.M. (1968).  16.  J . Biochem. 103:  744  A c t a Ped. Scand.  (1967). Suppl.  J . L i p i d Res. 9_:  17.  James, E., Meschia, G. and B a t t a g l i a , F.C. Exper. B i o l . Med. 138: 823 (1971).  18.  Dancis, J . , Jansen, V., Dayden, H.J., L e v i t z , M. P e d i a t . Res. 7: 192  460  Proc. Soc.  S c h e i d e r , H. (1973).  and  Hahn, P., Vavrouskova, E., J i r a s e k , J . and Uher, J . Neonat. 7: 348 (1964).. Hayashi, T.T., S h i n , D.H. and Wiand, S. Gynec. 102: 1144 (1968).  Biol.  Amer. J . Obstet.  Dancis, J . and B a l i s , M.E. J . B i o l . Chem. 2 07: (1954) .  367  Thau, R.B. and Lanman, J.T. i n The P l a c e n t a and i t s Maternal Supply L i n e : E f f e c t s o f I n s u f f i c i e n c y on the Fetus (Gruenwald, P., e d . ) , B a l t i m o r e U n i v e r s i t y Park P r e s s . p.125 (1975). Kaplan, S.L. and Grumbach, M.M. 25: 1370 (1965).  C l i n . Endocrinol.  Metabol  D i c f a l u s y , E. Proc. 2nd I n t . Congr. Hormonal S t e r o i d s , I n t . Congr. S e r i e s 132: 82 (1967). J o s t , A. and P i c o n , P. 4: 123 (1970).  Advances i n M e t a b o l i c D i s o r d e r s  J o s t , A. I n : F o e t a l Autonomy (Wolstenholme, G.E.W. and O'Connor, M. ) . C h u r c h i l l , London,., p.79 (1969). T a y l o r , N.R.W., L o r a i n e , J.A. and Robertson, H.A. J . Endocr. 9: 334 (1953). F i s h e r , D.A., Hobel, C.J., Garza, R. and P i e r c e , C.A. P e d i a t r i c s 46_: 208 (1970). Robb, P. Quart. J . Exper. P h y s i o l . Cog. Med. S c i . 46: 335 (1961). Pavlova, E.G., Pronina, T.S. and Skebelskaya, Y.B. Gen. Comp. E n d o c r i n o l . 10_: 269 (1968). L i g g i n s , G.C. J . E n d o c r i n o l . £5:  515 (1969).  Burton, A.F. and Jeyes, C L . Can. J . Biochem. 46_: 15 (1968) . Slaunwhite, W.R., J r . and Sandberg, I n v e s t . 38_: 384 (1959) . Gala, R.R. and Westphal, U. 47 (1967). Amoroso, E.C.  A.A.  J. Clin.  A c t a Endocr. Copenh. 55:  B r i t . Med. B u l l . 17_: 81 (1961).  L e v i t z , M. and Dancis, J . (1963).  C l i n . Obstet. Gynecol. (5: 62  1 1 8  37.  Burton, A.F. , M c C l e l l a n , D.J., Drummond, M.R., Mah, T . C , Thomson, M.J., Wong, W. and T u r n e l l , R.W. J. Clin. Endocr. Metab. 3 9 : 9 5 0 ( 1 9 7 4 ) .  38.  Michaud, N.J. and Burton, A.F.  B i o l . Neonate 3 2 _ :  1 3 2  ( 1 9 7 7 ) .  39.  Dupouy, J.P., G o f f i g n y , H. and Magre, S. 3 4 7  40.  J . Endocr. 6 5 :  ( 1 9 7 5 ) .  Leong, M.K.H. and Murphy, B.E.P. 1 2 4  :  4 7 1  ( 1 9 7 6 )  Am. J . Obstet. Gynec.  .  41.  Smith, B.T., Torday, J.S. and Giroud, C.J.P. Invest. 5 3 : 1 5 1 8 ( 1 9 7 4 ) .  42.  O'Malley, B.W. and B u l l e r , R.E. 1  43.  J. Clin.  J . I n v e s t . Dermat. 6 8 _ :  ( 1 9 7 7 ) .  B a l l a r d , P.L. and B a l l a r d , R.A. 4 7 7  J . C l i n . Invest. 5 3 :  ( 1 9 7 4 ) .  44.  B a l l a r d , P.L. and B a l l a r d , R.A. U.S.A. 6 9 : 2 6 6 8 ( 1 9 7 2 ) .  45.  Giannopoulos, G., Mulay, S. and Solomon, S. Biophys. Res. Commun. 4 J 7 : 4 1 1 ( 1 9 7 2 ) .  46.  Bernard, C  Proc. N a t l . Acad. S c i . Biochem.  Comp. Rend. Acad. S c i . (Paris) 4 J 3 : 8 9  ( 1 8 5 9 ) .  4 7 .  S h e l l e y , H.J.  B r i t . Med. B u l l .  1/7:  1 3 7  ( 1 9 6 1 ) .  48.  J o s t , A. I n : Recent Progress i n Hormone Research, V o l . XXII. (Pincus, G., ed.) Academic Press Inc., New York;. p . 5 4 1 ( 1 9 6 6 ) .  49.  Burton, A.F., G r e e n a l l , R.M. and T u r n e l l , R.W. Biochem.  £ 8 _ :  ( 1 9 7 0 )  5 0 .  Aron, M.  51.  P i c o n , L. and Bouhnik, J . 1 6 0 :  52.  Bull.  1 7 8  2 8 8  Can. J .  .  Soc. Chem. B i o l .  (Paris)  1 4 8 :  1 4 8 3  Comp. Rend. Soc. B i o l .  ( 1 9 2 2 )  (Paris)  ( 1 9 6 6 ) .  Himwich, H.E., B e r n s t e i n , A.O., H e r r l i c h , H., C h e s l e r , A. and Fazekas, J.F.  Am. J . P h y s i o l .  5 3 .  Heim, T. and H u l l , D.  J . Physiol.  5 4 .  H u l l , D.  55.  Lachance, J.P. and Page, E.  B r i t . Med. B u l l .  ( 1 9 5 3 ) .  2 2 ^  9 2  1 8 7 :  1 3 5 :  2 7 1  3 8 7  ( 1 9 4 2 ) .  ( 1 9 6 6 ) .  ( 1 9 6 6 ) .  Endocrinology 5 2 _ : 5 7  119  Snell,  K. and Walker, D.G. (1972) .  Biochem. J . 128:  Greengard, 0. and Dewey, H.K. (1970). M a r g o l i s , F.L., R o f f i , 275 (1966).  403  Dev. B i o l . 1:  J . and J o s t , A.  4 52  Science 154:  De hLemos, R.A., Shermeta, D.W., K r e l s o n , J.H., Kotas, R and Avery, M.E. Am. Rev. Resp. D i s . 102: 4 59 (1970). F a r r e l l , P.M. and Zachman, R.D. (1973) .  Science 179:  297  Rooney, S.A., Wai-Lee, T.S., Gobran, L. and Motoyama, B Biochem. Biophys. Acta 431: 447 (1976). S c h u l t z , P.M., Jimenez, J.M. , MacDonald,' P.C. and Johnston, J.M. Gynecol. I n v e s t . 5: 222 (1974). B r e h i e r , A., Benson, B.J., W i l l i a m s , M.C, Mason, R.J. and B a l l a r d , P.L. Biochem. Biophys. Res. Commun. 77: 883 (1977). L i g g i n s , G.C. (1972). L i g g i n s , G.C.  and Howie, M.B. J . Endocr. 4_2:  Drost, M. and Holm, L.W.  P e d i a t r i c s 5_0: 323  515  (1968).  J . Endocr. 40:  293  (1968).  Anderson, A.B. , Lawrence, K.M. and Turnbull., A.C.. J . Obstet. Gynec. Br. Cwlth. 76_: 196 (1969). Pokoly, T.B.  Amer. J . Obstet. Gynec. 117:  549  (1973).  Mueller-Heubach, E., Myers, R.E. and Adamsons, K. Am. J . Obstet. Gynec. 112: 221 (1972). C h a l l i s , J.R.C., Davies, I . J . , Benirchke, K., Henrichx, A.G. and Ryan, K.J. E n d o c r i n o l o g y 95_: 1300 (1974) . Pinsky, L. and DiGeorge, A.M. Krebs, H.A. and E g g l e s t o n , L.V. (1940).  Science 147:  402  (1965)  Biochem. J . 3_4_:  442  Kobayashi, Y. and Maudsley, D.V. In: B i o l o g i c a l Appli c a t i o n s o f L i q u i d S c i n t i l l a t i o n Counting, Academic P r e s s , New York./. p. 78 (1974). Michaud, N.J. Columbia  M.Sc. T h e s i s , U n i v e r s i t y of B r i t i s h (1976).  120  Roe, J.H. and D a i l e y , R.E. (1966).  A n a l . Biochem. 15:  24 5  Lowry, O.H., Rosebrough, N.J., F a r r , A.L. and R a n d a l l , R.R. J . B i o l . Chem. 193: 265 (1951). Thomson, M.J. and R i c h a r d s , J.F. L i f e S c i . 22_: 337 (1978). Wong, M.D. and Burton, A.F. Biochem. Biophys. Res. Commun. 50: 71 (1973) . Wong, M.D. and Burton, A.F. (1974).  Can. J . B i o c . 52_: 190  K r e h b i e l , R.E., Burton, A.F. and Darrach, M. Biochem. 4(3: 1797 (1962) .  Can. J .  Smith, B.T., Torday, J.S. and Giroud, C.T.P. 22: 515 (1973).  Steroids  Wong, M.D., Thomson, M.J. and Burton, A.F. 28: 12 (1976).  Biol.  Carson, S.H., Taeusch, H.W. and Avery, M.E. P h y s i o l . 34: 660 (1973). Smith, B.T.  P e d i a t . Res. 12_:  Neonate  J . Appl.  12 (1978).  Makman, M.H., Dvorkin, B. and White, A. S c i . 68: 1269 (1971) . R u s s e l l , D.H. and McVicker, T.A. 259: 247 (1972).  Proc. Nat. Acad.  Biochem. Biophys. A c t a  Reid, P.E., C u l l i n g , C.F.A., Ramey, C.W., Dunn, W.L. and C l a y , M.G. Can. J . Biochem. 55_: 493 (1977). Nguyen-Trong-Tuan, Rekdal, D.J. and Burton, A.F. Neonate 18_: 78 (1971).  Biol.  T u r n e l l , R.W., K a i s e r , N., K i l h o l l a n d , R.J. and Rosen, F. J . B i o l . Chem. 24_9: 1133 (1974). I s c h i i , D.N., P r a t t , W.B. and Aronow, L. 3896 (1972).  B i o c h e m i s t r y 11  F i e s e r , L.F. and F i e s e r , M. I n : S t e r o i d s , Reinhold P u b l i s h i n g Corp., New York (1959). p.600. Grosser, B.I. and A x e l r o d , L.R. Grosser, B.I. and B l i s s , E.L.  S t e r o i d s 9_:  S t e r o i d s 8_:  229 (1967).  915 (1966).  N i c o l a s , T.E., Johnson, R.G., Lugg, M.A. and Kim, P.A. L i f e S c i . 22: 1517 (1978).  121  95.  Smith, B.T. and Giroud, C.J.P. Can. J . P h y s i o l . Pharmacol. 53; 1037 (1975).  96.  Torday, J.S., Smith, B.T. and Giroud, C.J.P. nology 96: 1462 (1975).  97.  Manns, J.G. and Brockman, R.P. 47:  Endocri-  Can. J . P h y s i o l .  Pharmacol,  917 (1969).  98.  P l a s , C. and Nunez, J .  J . B i o l . Chem. 251:  1431 (1976).  99.  C a h i l l , G.F. , J r . I n : The Human Adrenal Cortex . . ( C h r i s t y , N.P., ed.), Harper and Row, New York. p.205 (1971). Bernard, C. J o u r n a l de P h y s i o l o g i e s de 1 homme e t des =  100.  1  animaux 2:  336 (1859).  101.  V i l l e e , C.A.  102.  Huggett, A. S t . C.  103.  Robb, S.A. and Hytten, F.E. B r i t . J . Obst. Gynaec. 8_3: 43 (1976). Walker, D.G., Lea, M.A., R o s s i t e r , G . and Addison, M. A r c h . Biochem. Biophys. 120: 646 (1967).  104.  J . B i o l . Chem. 205:  113 (1953).  J . P h y s i o l . 67_:  360 (1929).  105.  Gennser, G., Lundquist, I . and N i l s s o n , E. 21: 148 (1972).  106.  Winick, M.: C e l l u l a r Growth of the Fetus and P l a c e n t a . In F e t a l 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 Regulat i o n 2 : 371 (1964) .  108.  Garland, M.R., Ng, T.W. and R i c h a r d s , J.F. 31: 1348 (1971).  109.  Greengard, 0. and Machovich, 286:  R.  Biol.  Neonat.  Cancer Res.  Biochem. Biophys. A c t a  382 (1972).  110.  Nanda, R.  Arch. O r a l B i o l . 16:  435 (1971).  111.  A t l a s , M., Bond, V.P. and C r o n k i t e , E.P.  J . Histochem.  Cytochem. 8 : 171 (1960). 112.  Agarwal, M.K.  FEBS L e t . 85_: 1 (1978).  113.  Munck, A. and Brinck-Johnsen, T. 5556 (1968).  J . B i o l . Chem. 243:  122  114.  Mosher, K.M., Young, D.A. 246: 654 ( 1 9 7 1 ) .  and Muck, A.  115.  B a l l a r d , P.L., C a r t e r , J . E . , Graham, B.S. and B a x t e r , J . C l i n . E n d o c r i n o l . Metab. 4 1 : 290 ( 1 9 7 5 ) .  116.  Wong, M.D.  and B u r t o n , A . F .  Biol.  J. Biol.  N e o n a t e 18:  Chem.  146  J.D.  (1971).  123  APPENDIX I  Structures  o f the s t e r o i d s used  113-hydroxysteroid dehydrogenase  CH OH  CH OH  2  corticosterone,  cpd. B  2  11-dehydrocorticosterone,  cpd. A  llg-hydr'oxysteroid dehydrogenase  CH_OH I  CH 0H  2  o  NADP  --0H  3* Cortisol  NADPH  cortisone  LEAF 123a OMITTED IN PAGE NUMBERING,  11-ketoprogesterone  124  APPENDIX I I  At the that  outset of t h i s  investigation,  the metabolism of glucose  a method was t i o n of  w o u l d be  t h e r e f o r e developed  glucose  i n small  i t was  anticipated  o f major i n t e r e s t ,  which permitted  the  s a m p l e s o f mouse f e t a l  and  determina-  blood  and  14 determination Although be  of  of the  t r a n s f e r of  the major i n t e r e s t  interest  and  C-glucose  shifted,  from the  t h e method was  o f p o s s i b l e u s e f u l n e s s , and  mother.  thought  to  i s described  below. P r e p a r a t i o n of the  Glucose,  Glucose  1 g,  5 ml  were added t o a 50 ml pered min  and  placed  f o r 2 h.  cooled  and  allowed tion.  removed and ethyl  the  added t o The  anhydride  Erlenmeyer ; f l a s k ,  end  of the  70 ml  of d i s t i l l e d  overnight  at  acetate  crystals  at  with  then 120  by  stop-  cycles/  solution  was  ice, in  parafilm  0-4°C t o promote c r y s t a l  The  dropwise a d d i t i o n of petroleum  pyridine  w h i c h was  w a t e r , on  centri-  and forma-  c e n t r i f u g a t i o n , the  d i s s o l v e d i n as  as p o s s i b l e .  10 ml  shaking  covered  c r y s t a l s were c o l l e c t e d the  and  i n c u b a t i o n , the  t u b e s were t h e n  to stand The  acetic  Carrier .  i n a 37°C w a t e r b a t h ,  At  fuge tubes.  Tetraacetate  s m a l l an  amount  of  s a m p l e s were c r y s t a l l i z e d ether  (bp  80-100°C).  At  a few  drops of d i e t h y l  e t h e r were added  prevent  o f an  amorphous f o r m .  C r y s t a l s were  to form o v e r n i g h t mother l i q u o r  a t 0-4°C, c o l l e c t e d  removed.  The  by  by  the  sign of c r y s t a l s , formation  water  to  allowed  c e n t r i f u g a t i o n and  c r y s t a l s were t h e n  first  the  d r i e d , weighed  125  and  re-dissolved  cold  carrier  per  freezer well with  in ethyl 50  yl.  a c e t a t e such  that  T h i s s o l u t i o n was  stoppered with  a silicone  t h e r e was  prior  pyridine  t o use  and  water t r a c e s .  s t o r e d i n the  s t o p p e r and  used  1/16"  i n a l l experiments molecular  Acetic  anhydride  was  covered  distilled  s i e v e added t o remove was  also  as g l u c o s e t e t r a a c e t a t e . of Chemistry,  ( C o u r t e s y o f Dr.  University of  of Glucose  A s m a l l amount o f t h e p r e p a r e d  Simply  by  c o u l d be  developed  by  twice  visible,  Dept.  s p o t t e d onto  acetate  to a l i g h t  oxidized  and  b e i n g a brown c o l o u r .  source, the  staining  z o n e s were shown t o  reagent  sheet.  of  C-labelled  ( S t a h l R e a g e n t No.  134)  correspond.  C-glucose glucose,  0.5  ml  5%  Glucose  ( u ) , 10,000 dpm,  Standards  10  TCA were m i x e d and  zone  s h e e t s were  14 Acetylation  zone  the t e t r a a c e t a t e TLC  a  (4:1; v : v ) .  a change i n t h e d e n s i t y o f t h e TLC  sprayed with a glucose these  Weiler,  T e t r a a c e t a t e Zone  i n hexanerethyl  Upon s t a n d i n g , t h e compound was became c l e a r l y  i t s identity  compound was  h o l d i n g t h e c h r o m a t o g r a m up observed  way.  B.C.)  Chromatographic I d e n t i f i c a t i o n  s h e e t and  L.S.  just  any  stored in this  Mass s p e c t r o s c o p y o f t h e compound c o n f i r m e d  and  of  parafilm.  The  TLC  20 mg  yg o f n o n - r a d i o a c t i v e extracted several  times  126  with d i e t h y l ether. r a t e d under N final  2  The  removed and evapo-  w i t h s e v e r a l a d d i t i o n s of e t h a n o l to remove  t r a c e s of water.  evacuated  aqueous l a y e r was  The  sample was  then s t o r e d i n an  d e s i c c a t o r f o r s e v e r a l days u n t i l dryness was  N i n e t y y l of n o n - r a d i o a c t i v e a c e t i c anhydride added and the tube stoppered and tube was  ensured.  and p y r i d i n e were  sealed with parafilm.  The  p l a c e d i n a shaking water bath under c o n d i t i o n s  p r e v i o u s l y d e s c r i b e d and incubated f o r the r e q u i r e d l e n g t h of  time, 1/2,  1, 2, 4 and 8 h.  A f t e r the a p p r o p r i a t e time,  the samples were removed, p l a c e d on i c e , 500 yg c o l d added p l u s 0.5 ml of d i s t i l l e d water to h y d r o l y z e a c e t i c anhydride. of  The  remaining  samples were then e x t r a c t e d w i t h 2.5  i c e - c o l d e t h y l a c e t a t e to remove the g l u c o s e - a c e t a t e .  e t h y l a c e t a t e l a y e r was e x t r a c t e d w i t h another was  carrier  The  removed and the aqueous f r a c t i o n r e 2.5 ml of e t h y l a c e t a t e .  This f r a c t i o n  added to the p r e v i o u s and the e t h y l a c e t a t e r e - e x t r a c t e d  with 0.5  ml of d i s t i l l e d water to remove remaining a c e t i c  The e t h y l a c e t a t e l a y e r was onto a TLC sheet and developed (4:1; v : v ) .  The  sheet was  evaporated under N , 2  twice i n hexane:ethyl  then autoradiographed  acetate  i n order to  the i n c u b a t i o n .  of  so t h a t the percentage  the aqueous f r a c t i o n was  counted  a c e t y l a t i o n c o u l d be c a l c u l a t e d .  arbitrarily  aliquot of  i n the glucose  tetra-  In f a c t , the o v e r a l l e f f i c i e n c y df a c e t y l a t i o n  such t h a t the t e t r a a c e t a t e accounted  (Table 17).  An  As can be seen from F i g . 28,  the major p o r t i o n of the r a d i o a c t i v i t y was a c e t a t e zone,  acid.  spotted  show any products formed throughout  was  ml  The  f o r 72 - 76 p e r c e n t  time o f i n c u b a t i o n i n s t u d i e s f o l l o w i n g was  s e l e c t e d as 2 h.  127  Table EFFECT OF TIME ON THE  Time  Each  (h)  17  EFFICIENCY OF GLUCOSE ACETYLATION  % Ace.tyla.ti.on.  % Glucose .Te.tra. A c e t a t e  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  value  i s a mean of f o u r d e t e r m i n a t i o n s .  I n j e c t i o n of Mice and P r o c e s s i n g of Blood  C o n t r o l mice on g e s t a t i o n a l day 5 y C i of was  14  C-glucose  (u) 15 mm  a n e s t h e t i z e d w i t h 1:6  18-19  were i n j e c t e d w i t h  . prxor to k i l l i n g .  The mouse  nembutal d i l u t e d w i t h 0.9%  saline.  The f e t u s e s were then removed, d e c a p i t a t e d and the blood c o l l e c t e d on a p i e c e of p a r a f i l m kept on i c e .  The blood  was  then mixed w i t h a drop of s a l i n e and t r a n s f e r r e d i n t o a 5 ml tube.  Maternal blood was  near the h e a r t .  One  c o l l e c t e d by s e v e r i n g the  drop of 6 0% TCA was  arteries  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 c e n t r i f u g e d a t 0-4°C and carded.  The  further  use.  remaining  test-  a c i d - s o l u b l e f r a c t i o n was  dis-  frozen u n t i l  128  Dexamethasone-treated mice r e c e i v e d 2 00 yg Dex intraperitoneally  1 h p r i o r to the subcutaneous  solution  injection  14 of  C - l a b e l l e d glucose; the animals were processed as p r e -  v i o u s l y mentioned. A c e t y l a t i o n of Maternal and F e t a l Blood Samples  Maternal and f e t a l blood samples from mice i n j e c t e d w i t h 14  . . C-glucose were a c e t y l a t e d w i t h n o n - r a d i o a c t i v e a c e t i c  anhydride,  500  pg c a r r i e r glucose t e t r a a c e t a t e was  the samples chromatographed as d e s c r i b e d .  added, and  Autoradiograms  i n d i c a t e d t h a t the r a d i o a c t i v i t y recovered i n maternal was  predominantly  about h a l f was  glucose t e t r a a c e t a t e , w h i l s t i n f e t a l  i n t h i s zone  The maternal  blood samples  ( F i g . 28).  and f e t a l blood samples were d i v i d e d i n h a l f .  One-half o f the sample was  a c e t y l a t e d with non-radioactive 3 a c e t i c anhydride and the o t h e r w i t h H - a c e t i c anhydride d i l u t e d 14 to 1 yCi/ymole. sample.  T h i s was  T h i s allowed c a l c u l a t i o n of  C-glucose  i n the  necessary s i n c e upon a c e t y l a t i o n w i t h  labelled  a c e t i c anhydride 10,000 dpm of glucose was added t o these samples 14 3 . . i n order t o a l l o w reasonable C t o H counts f o r r e - c r y s t a l l i z a t i o n 14 studies.  The c o n t r i b u t i o n o f t h i s  corrected for i n c a l c u l a t i o n .  C-glucose  The sample was  label  extracted with  d i e t h y l e t h e r s e v e r a l times and the aqueous f r a c t i o n to dryness under N . 2  and s t o r e d evacuated  The  sample was  was  evaporated  placed i n a desiccator  over DRIERITE f o r s e v e r a l days to  ensure  complete dryness before a c e t y l a t i o n as d e s c r i b e d p r e v i o u s l y .  Fig.  28:  Autoradiograms o f chromatographed e x t r a c t s o f  the  14 acetylated and  fetal  litres  derivatives  of  mouse b l o o d .  C-glucose  recovered  maternal  A c i d - s o l u b l e e x t r a c t s o f a few  o f b l o o d were c h r o m a t o g r a p h e d on  developed  from  i n hexane:ethyl  acetate  1.  maternal  blood  2.  fetal  3.  glucose  GA:  glucose tetraacetate  blood  standard  (4:1;  silica v:v).  g e l TLC  microsheets  130  131  Table  18  E F F E C T OF DEXAMETHASONE I N J E C T I O N INTO:'MOTHERS ON S P E C I F I C A C T I V I T Y OF F E T A L BLOOD G L U C O S E  3 Specific Control  A c t i v i t y xlO Dex-treated  1.  6.4  1>  10.0  2.  3.5  2.  7.6  3.  2.9  3.  6.7  4.  8.2  4.  1.1  X = 5.25  X = 6.35  THE  132  Wong and Burton  (116) have observed  that i n j e c t i o n of  mothers w i t h dexamethasone f o l l o w e d by i n j e c t i o n o f l a b e l l e d glucose r e s u l t e d i n a d e c l i n e i n uptake o f t h e l a b e l by the fetus.  Since the mouse p l a c e n t a has c o r t i c o s t e r o i d r e c e p t o r s  (78, 79) the p o s s i b i l i t y t h a t the c o r t i c o s t e r o i d c o u l d c o n t r o l the f e t a l f u e l supply was c o n s i d e r e d . Burton  The data o f Wong and  (116) does not d i s t i n g u i s h between t h e p o s s i b i l i t y  of decrease i n l a b e l i n f e t u s e s being due t o d i l u t i o n by maternal hyperglycemia, a c t u a l decrease determine  caused  o r i f t h i s i n f a c t r e p r e s e n t s an  i n t o t a l g l u c o s e c r o s s i n g the p l a c e n t a .  To  which a l t e r n a t i v e occurs the s p e c i f i c a c t i v i t y o f  glucose i n dexamethasone t r e a t e d and c o n t r o l f e t a l blood was measured.  As observed  i n Table 18, t h e s p e c i f i c a c t i v i t y o f  the f e t a l blood remained e s s e n t i a l l y the same i n both i n s t a n c e s . T h i s i m p l i e s t h a t the observed  decrease caused by p r i o r dexa-  methasone i n j e c t i o n was a t r u e r e f l e c t i o n o f glucose c r o s s i n g the p l a c e n t a .  The purpose o f such a mechanism remains u n c l e a r but one might s p e c u l a t e t h a t under stressvumaternal hyperglycemia causing a hyperglycemic promote growth. survival.  s t a t e i n the f e t u s which w i l l  results,  ultimately  T h i s would c l e a r l y be* a disadvantage f o r  T h e r e f o r e , a mechanism t o dampen t h i s e f f e c t might  i n c r e a s e the chance o f s u r v i v a l , e s p e c i a l l y o f the mother.  M u l t i p l e i n j e c t i o n s of dexamethasone have been shown t o cause f e t a l death and r e a b s o r p t i o n (116) and i n c r e a s e d f e t a l l o s s was observed  d u r i n g very hot weather.  T h i s suggests t h a t  133  prolonged stress  o f t h e mother might r e s u l t i n f e t a l  thereby increasing  loss  the chance o f s u r v i v a l o f the mother,  t h a t t h i s i s m e d i a t e d by  corticosteroids.  and  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094847/manifest

Comment

Related Items