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

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116-HYDROXYSTEROID:NADP IN  OXIDOREDUCTASE  MOUSE F O E T A L T I S S U E S  by N I C O L E J O C E L Y N E MICHAUD B.Sc,  A Thesis The  University  Submitted  o f Ottawa,  i n Partial  1974  Fulfilment of  Requirements f o r t h e Degree o f  MASTER  in  OF  SCIENCE  t h e Department of Biochemistry  We  accept to  this  thesis  the required  as  standard  THE U N I V E R S I T Y OF B R I T I S H September,  conforming  COLUMBIA  1976  (<3) N i c o l e J o c e l y n e M i c h a u d , 197 6  In p r e s e n t i n g t h i s t h e s i s  in p a r t i a l f u l f i l m e n t o f the requirements  for  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the  L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and  I f u r t h e r agree t h a t p e r m i s s i o n for  f o r e x t e n s i v e copying o f t h i s  study. thesis  s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r  by h i s  representatives.  It  i s understood that copying o r p u b l i c a t i o n  o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my written  permission.  Department of  Biochemistry  The U n i v e r s i t y o f B r i t i s h Columbia 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Date  O c t o b e r 28,  1976  i  ABSTRACT  Corticosterone  i n foetal  tissues  after  injection  of the  14 mother w i t h  C-corticosterone  3 with  specific tissue  anhydride  activity.  varied  recovered  between g e s t a t i o n a l The s p e c i f i c  remained  this  essentially  patterns head  and l i v e r ,  with  foetal  sharply,  a decrease  tissues  and a r i s e  oxidoreductase  6, w h i c h active  and  close  adrenals  crossed that, of  to the foetus  normally,  origin,  tained manner.  to function. readily  the maternal  foetal  source  foetal  reduces the  hormone,  was  ability  predominates.  i n a distinct  after  normal of  however, most  likely  Regardless  however, t h e hormone  tissues  compound,  One d a y  a n d i t was c o n s i d e r e d  or maternal,  i n different  period.  indicating  Maternal  This  amount o f t h e l a t t e r i n  corticosterone  foetal  time  corti-  116-OH  adrenals,  t o normal,  of  activity.  removal o f maternal levels  this  i n foetal  this  foetal  hormone. different  during  15 t o 9 1 % d u r i n g  maternal  from  15-minute  i n the conversion  the relative  641 t o 300  of maternal  greatly  to the biologically  raising  a  foetal  hormone  showed d i s t i n c t l y  e n z y m e , Km=33yM, p H o p t i m u m  11-dehydro m e t a b o l i t e increased  after  that  to 11-dehydrocorticosterone  mitochondrial  13 a n d 17 f r o m  constant;  changed  113-hydroxysteroid:NADP  o f whole  a c t i v i t y of foetal  and l i v e r  of metabolism, which  costerone liver  head  content  days  was a s much a s o n e - f o u r t h  However, p l a c e n t a ,  in  acetylation  and c r y s t a l l i z a t i o n t o c o n s t a n t  The c o r t i c o s t e r o n e  respectively.  pulse  by  . H-acetic  ng/g  was d e t e r m i n e d  and  i s maindifferent  ACKNOWLEDGEMENTS  I wish t o express for  h i sguidance,  the  course  cern during  my deep a p p r e c i a t i o n t o D r . A . F . B u r t o n  judicious advice  o f my r e s e a r c h  and p a t i e n c e  and f o r h i s e n c o u r a g e m e n t and c o n -  t h e w r i t i n g o f my  thesis.  I a l s o w i s h t o t h a n k my s i s t e r , for  throughout  Miss Francine  h e r t e c h n i c a l a s s i s t a n c e d u r i n g my l a s t  Michaud  summer o f r e s e a r c h .  i i i  TABLE  OF  CONTENTS  ABSTRACT  i  ACKNOWLEDGEMENTS  TABLE  i  OF C O N T E N T S  i  LIST  OF T A B L E S  LIST  OF F I G U R E S  LIST  OF A B B R E V I A T I O N S  i  i  i  v i  v i i  v i i i  INTRODUCTION  1  MATERIALS  10  1.  Animals  10  2.  Buffers  10  3.  Solvents  10  4.  TLC  11  5.  Radioactive  6.  Non-radioactive  7.  Scintillation  8.  Liquid  9.  Transmission  10.  steroids  11  reagents  solvent  scintillation electron  Autoradiography  12 12  spectrometer  12  microscopy  12 13  iv  METHODS 1.  2.  3.  4.  14 Animals  14  (a)  Injection  of  (b)  Adrenalectomy  mice  Tissues  15  (a)  Minced  (b)  Cell  tissues  fractionation  Incubation  16  (a)  F^nzymatic s y n t h e s i s corticosterone  (b)  Minced  (c)  Coenzyme  (d)  pH  (e)  Kinetic  Extraction  of  11-dehydro  tissues specificity  analysis  of tissues  (a)  B l o o d and t i s s u e s  (b)  Incubated minced cell fractions  13 from  mice  tissues  and  5.  Chromatography  18  6.  Acetylation  19  7.  Crystallization  20  8.  Standards  22  9.  Quench c u r v e and c a l c u l a t i o n o f t h e double l a b e l *  22  Transmission electron  22  10.  f o r efficiency of acetylation  microscopy  V  EXPERIMENTAL  RESULTS  A.  experiments  In  vivo  1.  Corticosterone  2.  E f f e c t of the t o t a l  3. 4. B.  of  tissues  25  adrenalectomy of the mother amount o f c o r t i c o s t e r o n e in  on 28  the 31  C o r t i c o s t e r o n e and i t s 1 1 - d e h y d r o metabolite in.foetal tissues  33  experiments  3 5  In v i t r o metabolism of c o r t i c o s t e r o n e 11-dehydro.corticosterone  2.  L o c a l i z a t i o n of  3.  Metabolism foetal  C.  content  14 Metabolism of C-corticosterone p l a c e n t a and f o e t a l t i s s u e s  In v i t r o 1.  25  of  the  reductase  corticosterone  and  activity in  39  Enzyme k i n e t i c s  41  1.  Cell  2.  Development of  fractionation the  and  coenzyme  reductase  the  3.  pH  optimum  4. 5.  Km C o n t e n t and s p e c i f i c a c t i v i t y o f 14 C-corticosterone i n the f o e t a l Histological  DISCUSSION BIBLIOGRAPHY  specificity  41  activity  in  6.  37  the  head  foetal  35  liver  44 4 6  studies  46 recovered bodies  52 54 59 66  VI  L I S T OF  I  Determination  of the purity  "^C-corticosterone II  TABLES  of  crystallized  "^H-acetate  21  T o t a l amount o f c o r t i c o s t e r o n e i n t h e m o t h e r a n d t h e f o e t u s e s o n d a y s 1 3 a n d 17  26  14 III  Specific activity costerone  IV  Effect levels  V  VI  of recovered  C-corti-  o f a d r e n a l e c t o m y o f t h e m o t h e r on of c o r t i c o s t e r o n e i n the mother  C o n t e n t and s p e c i f i c a c t i v i t y o f •^C-corticosterone i n the foetus adrenalectomy o f the mother  27 the 29  recovered after  R e l a t i v e amount o f "C-corticosterone 11-dehydro metabolite i n the p l a c e n t a foetal tissues  and i t s and ^  A  VII  C o r t i c o s t e r o n e and in foetal tissues  VIII  I n v i t r o m e t a b o l i s m o f c o r t i c o s t e r o n e and 11-dehydrocorticosterone by f o e t a l t i s s u e s  36  Reduction of 11-dehydrocorticosterone f o e t a l l i v e r and r e s i d u a l c a r c a s s  38  IX X  XI XII  XIII  i t s 11-dehydro  30  metabolite  by  the  Enzyme a c t i v i t y and coenzyme s p e c i f i c i t y the crude s u b c e l l u l a r f r a c t i o n s  i n  Enzyme a c t i v i t y and coenzyme s p e c i f i c i t y washed f r a c t i o n s and t h e i r washes  i n  Development of the reductase foetal liver C o n t e n t and  specific  •'-^C-corticosterone  activity  activity  42 43  i n 45  of  i n the foetal  recovered body  53  V l l  Quench  curve  LIST  OF  for  H  FIGURES  and  In v i t r o m e t a b o l i s m o f the f o e t a l head at days  pH  dependence  Effect of costerone  of  C  *C-corticosterone 14 a n d 15  reductase  by  activity  concentration of 11-dehydrocortion r e d u c t a s e a c t i v i t y  Lineweaver-Burk p l o t for reduction 11-dehydrocorticosterone  E f f e c t of concentration of dehydrogenase a c t i v i t y  Lineweaver-Burk corticosterone  plot  c o r t i c o s t e r o n e on  for dehydrogenation  Sections of adrenals w i t h dexamethasone  from  TEM o f a d r e n a l s dexamethasone  foetal  from  of  foetal  mice  mice  of  treated  treated  with  v i i i  LIST  OF  ABBREVIATIONS  Adrx  :  adrenalectomy,  adrenalectomized  AR  :  autoradiogram,  autoradiography  CBG  :  corticosteroid  c p d .A  :  cpd.B  :  DMP  :  2,4,6-tri-(dimethyl)-amino  DDSA  :  dodecyl  LM  :  light  L/S  :  lecithin/sphingomyelin ratio  MDA  :  methyl  PO  :  propylene  RDS  :  respiratory  SEM  :  standard  TEM  :  transmission electron  TLC  :  thin  UV  :  ultraviolet  binding  globulin  (Transcortin)  11-dehydrocorticosterone corticosterone  succinic  methyl  anhydride  microscopy  nadic  anhydride  oxide distress  error  layer  syndrome  o f t h e mean microscope  chromatography  phenol  1  INTRODUCTION  The  endocrine  c o n t r o l o f pregnancy i s p r o v i d e d b o t h by  t h e mother and t h e f o e t o - p l a c e n t a l u n i t .  I n most s p e c i e s ,  t h e p e p t i d e h o r m o n e s ; e . g . , ACTH, a n d s e r u m p r o t e i n s do n o t c r o s s t h e p l a c e n t a and s e r v e t o r e g u l a t e t h e l e v e l s o f s t e r o i d s on t h e m a t e r n a l  side.  On t h e o t h e r h a n d , t h e  s t e r o i d hormones c a n c r o s s t h e p l a c e n t a .  The c o r t i c o s t e r o i d s  c r o s s i n s i g n i f i c a n t a m o u n t s t o t h e f o e t a l s i d e i n some s p e c i e s , n o t a b l y mouse a n d man. as w e l l as t h e c a t e c h o l a m i n e s a very rigourous metabolic in and  foetal tissues  (1).  O e s t r o g e n s and a n d r o g e n s ,  and t h y r o n i n e s a r e s u b j e c t t o  c o n t r o l , mainly  Whereas i n t h e p l a c e n t a  oestrogens  o t h e r s t e r o i d s hormones a r e p r e s e n t p r e d o m i n a n t l y  unconjugated conjugated  "free"  form,  they a r e e x t e n s i v e l y and r a p i d l y  f a c t o r s which i s thought  ence t h e g r o w t h and s e c r e t o r y a c t i v i t y  (2)  summarized e x p e r i m e n t a l  to influ-  of the foetal  t h e s e c r e t i o n o n ACTH b y t h e f o e t a l p i t u i t a r y .  by J o s t  i n an  i n the foetus.  One o f t h e i m p o r t a n t  is  by s u l f u r y l a t i o n  evidence  i n t h e mouse, r a t , s h e e p , r a b b i t a n d g u i n e a  adrenal  A  review  on t h a t  aspect  pig.  Absence  o f t h e p i t u i t a r y , d e s t r u c t i o n o f t h e h e a d s b y X - r a y beams or s u r g i c a l decapitation of foetuses a l l r e s u l t  in a  in size of the adrenal cortex at b i r t h ,  of the c o r t i -  atrophy  decrease  c a l c e l l s and m o d i f i c a t i o n i n t h e d i s t r i b u t i o n o f l i p i d s , w h i c h c a n be r e v e r s e d b y g i v i n g ACTH t o t h e f o e t u s e s a t t h e time o f surgery.  2  In the human anencoephalic  f o e t u s , the f o e t a l zone of  the f o e t a l adrenal r e g r e s s e s prematurely and  the  definitive  c o r t e x , while h y p o p l a s t i c , undergoes premature d i f f e r e n t i a tion.  Hence i t seems reasonable  to b e l i e v e t h a t ACTH d e f i -  c i e n c y i s r e s p o n s i b l e f o r the a d r e n a l a b n o r m a l i t i e s anencoephalic  i n the  infant.  A d m i n i s t r a t i o n of the s y n t h e t i c c o r t i c o s t e r o i d , dexamethasone, to pregnant Rhesus monkeys (3) r e s u l t e d i n of f o e t a l adrenal gland and f o e t a l zone.  atrophy  an a p p r e c i a b l e r e g r e s s i o n of  the  Dexamethasone i s b e l i e v e d to suppress p i t u i t a r y  ACTH s e l e c t i v e l y .  I f t h i s assumption can be c o r r e c t l y a p p l i e d  to the f o e t u s , these experiments p r o v i d e the b e l i e f t h a t ACTH i s an important f o e t a l zone of the adrenal  s t r o n g support  for  t r o p h i c f a c t o r f o r the  i n utero.  That the foetus can s y n t h e s i z e c o r t i c o s t e r o i d s i s w e l l established. lowing  Dupouy e t a l (4, 5) have shown i n the r a t f o l -  adrenalectomy on the mother on day  of c o r t i c o s t e r o n e i n maternal blood was untreated, has  18,  t h a t the  level  the same as i n the  i n d i c a t i n g t h a t not only the f o e t a l r a t a d r e n a l  the a b i l i t y to s y n t h e s i z e c o r t i c o s t e r o i d s , but a l s o t h a t  the hormones can c r o s s the p l a c e n t a from the foetus to the mother, i n c o n t r a s t to the sheep  The  (6,7).  l e v e l s of c o r t i c o s t e r o i d s are a l s o r e g u l a t e d on  maternal s i d e by t r a n s c o r t i n .  This p r o t e i n , also c a l l e d  the  3  corticosteroid  binding  globulin  corticosterone, their  C o r t i s o l ,  gesterone,  b u t n o t any o f t h e i r  is  i n most  present  the  liver.  species  The o n l y  of transcortin  gens.  Only  rest being  Gala during the  Free  level  steroid  (9) h a v e  increase  and t h e l e v e l corticosteroid  reported  a marked  of oestroi s free,  increase  a fraction  occurs  also  t o enter  and b i o l o g i c a l  p i g , f o l l o w e d by a  animals  concentrations  corticosteroid  i s not able  during  paralleled  lactation. t h e bound  of the total.  i n human a n d o t h e r  species, the  increasing severalfold, The  tissues,  activity  the free  transcortin-bound  and does  not cross the  i s attributed  only  to the  steroid (8).  The  passage  of corticosteroids  a pattern which v a r i e s with partially has  seem t o i n f l u e n c e t h e  increasing proportionately.  placenta  (8). I t  and i s s y n t h e s i z e d i n  o f non-pregnant  corticosteroid  of total  pro-  i n the corticosteroid-binding activity i n  b u t were o n l y  steroids  metabolites  and  to transcortin.  to the level  This  specifically  metabolites  o f t h e mouse, r a b b i t and g u i n e a  serum  steroid  examined  a r e pregnancy  and Westphal  serum  other  10% o f t h e t o t a l  bound  pregnancy  decline  free  about  11-deoxy  factors that  levels  the  (CBG), b i n d s  species  determined by t h e type  across  the placenta  and which  i s at least  of placenta.  s h o w n i n t h e ewe t h a t c o r t i c o s t e r o i d s  shows  cross  L i g g i n s (6) the placenta  4  in  the  direction  foetus  direction.  On  i n mice  (2)  has  steroid  synthesis  are  the  t o mother, but  other  shown t h a t , when t h e m a t e r n a l i s removed, t h e m a t e r n a l  adrenals.  human  (10,  cross  the  placenta  11,  12,  13)  placenta  occupying  local  d e s t r u c t i o n of  shows t h a t t h e  deep p i t s the  i n the  tissue.  the branching  active steroid unit.  d u c t a s e has tissues hydroxyl of the  Cortisol  sheeps p o s s e s s  An  16).  in in  the  the  with  contact The  with  rodents  some  superficial  chorionic v i l l i  are  i n mouse and  i n the  i n human  group which i s e s s e n t i a l f o r b i o l o g i c a l 11-keto m e t a b o l i t e s .  responsible  an c o r d  blood  (17) .  directly  vessels.  foeto-  f o r the  high  levels  Burton et a l  (18)  noted  113activity  This of  oxidore-  foetal  T h i s enzyme d e h y d r o g e n a t e s t h e  t o be  the  of p l a c e n t a which i s  i s also metabolized  sidered  the  the  and  enzyme, 1 1 B - h y d r o x y s t e r o i d : N A D P  hormone, f o r m i n g  the  chorionic  lining,  i s s u i n g f r o m opened  been i d e n t i f i e d  (15,  synthesis  a more t h o r o u g h e r o s i o n o f t h e  b a t h e d by m a t e r n a l b l o o d  placental  levels  Comparison of  uterine  human p o s s e s s a h a e m o c h o r i a l t y p e  u t e r i n e mucosa and  and  of  u t e r i n e e p i t h e l i u m which allows  vascular maternal connective  by  source  w h i c h i s c h a r a c t e r i z e d by  e c t o d e r m t o come i n t o d i r e c t  characterized  (4)  suggest that c o r t i c o s t e r o i d s a l s o  (14)  villi  The  h o u r s by  i n both d i r e c t i o n s .  syndesmochorial type,  reverse  steroid  Measurements o f c o r d b l o o d  involved  chorionic  i n the  hand, a d r e n a l e c t o m y o f r a t s  r e s t o r e d t o n o r m a l w i t h i n a few  foetal  not  i s con-  c o r t i s o n e i n hum-  i n t h e mouse  an  5  i n v e r s e r e l a t i o n s h i p between the r a t e o f c o n v e r s i o n c o r t i c o s t e r o n e to 11-dehydrocorticosterone and  of  in foetal tissues  the c a p a c i t y of c o r t i c o s t e r o n e t o e l i c i t an i n c r e a s e  l i v e r glycogen.  A sharp i n c r e a s e i n g l y c o g e n  in  deposition  occurred  i n f o e t a l l i v e r p a r a l l e l i n g a d e c r e a s e i n the r a t e o f c o n v e r s i o n of 14 C - c o r t i c o s t e r o n e t o 14 C - l l - d e h y d r o c o r t i costerone.  T h i s was  thought t o be due,  not t o a d e c r e a s e i n  dehydrogenase a c t i v i t y , but r a t h e r t o the appearance o f enzyme i n f o e t a l l i v e r w h i c h reduced the m e t a b o l i t e corticosterone. t i o n and of the  an  back t o  This a c t i v i t y developed only l a t e i n gesta-  r e s u l t e d i n a r i s e i n c o r t i c o s t e r o n e a t the expense metabolite.  C o r t i c o s t e r o i d s have been shown t o p l a y major r o l e s i n f o e t a l development.  E x p e r i m e n t a l work has  shown t h a t  func-  t i o n a l m a t u r a t i o n o f f o e t a l lamb l u n g s can be a c c e l e r a t e d s t i m u l a t i o n of the f o e t a l a d r e n a l of g l u c o c o r t i c o i d s ( 6 ) .  c o r t e x or by  by  administration  L i g g i n s suggested t h a t g l u c o c o r t i -  c 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  alveoli,  perhaps by i n d u c t i o n of an enzyme concerned w i t h the b i o synthesis of  surfactant.  G l u c k (19) has d e v i s e d the m a t u r i t y  a biochemical  o f the f o e t a l l u n g .  method t o a s s e s s  A f t e r having presented  e v i d e n c e t h a t t h e r e i s c o n t i n u i t y between the f o e t a l and  amniotic  f l u i d , i t was  lecithin/sphingomyelin  suggested t h a t the a m n i o t i c  (L/S)  lung fluid  r a t i o would r e f l e c t the degree  6  of  f o e t a l lung maturity.  costeroids  induce  increasing  the  is  the  the  synthesis factor  i n the  alveoli.  Investigations revealed sone  that  syndrome case of was  (RDS)  premature  The  by  justification  i n the  for  administration  of  distress treatment  The  pituitary-adrenal demonstrated  axis  i n the  the on  sheep  the  f o e t a l lambs  often  (7).  induced  onset  a  disease the  out  in  Liggins  level  in  was those  resulting  in  The  last  of  mothers  respiratory babies two  and  years.  hypothalamus-  labour has  the  level  trials  (22)  foetal  of  hormone  distress  prenatal  in treated  i n the  corti-  physiologic  Controlled  functional  i n f u s i o n of a d r e n o c o r t i c o t r o p i c into  of  human by  applied  the  Cortisol  threatened.  less  and  Cortisol  provide  prevention  surface  have  l e a s t double  have been c a r r i e d  become w i d e l y  importance of  at  findings  d e l i v e r y was  (21)  Also,  low  which  lowers  respiratory  controls.  always  i n the  syndrome o c c u r r e d has  with  membrane, a  was  (20),  Cortisol  glucocorticoids.  therapy  whom p r e m a t u r e  of  attempted  immaturity  in  blood  normal  These  from pulmonary  betamethasone  which  whereas a higher  level  RDS.  choline  human i n f a n t s  in infants  RDS,  Cortisol  infants without  cord  corti-  phosphotransferase,  surfactant  Murphy on  rupture  shown t h a t  phosphatidyl  i n the  by  lower  than  accompanied  not.  of  umbilical  l e v e l s were  been  enzyme c h o l i n e  critical  tension  I t has  has  shown  (ACTH) o r  been that Cortisol  premature p a r t u r i t i o n , whereas  7  infusion  o f t h e s e hormones i n t o t h e ewes d i d n o t .  same a n i m a l , D r o s t c a n be p r o l o n g e d the r a b b i t , initiating in  and  Holm  (23)  o f t h e f o e t a l lamb.  i f adrenal corticosteroids l a b o u r , N a t h a n i e l s z and  play a role  Abel  (24)  d i d not  However, i n t h e f o e t a l r a t , t h e  c o r t i c o s t e r o n e c o n c e n t r a t i o n was  period: values  v a l u e s a t day  22 d a y s ) and f r o m day  The  succeed  approximately  i t ; e.g.,  the primary  occurs  of labour of some  the  evidence  (the absence of p i t u i t a r y f o r m a t i o n  (10).  factor  with  l e v e l s , suggesting  initiating  RDS  that  l a b o u r , a t l e a s t when t h i s  of dexamethasone i n t o  parturition  primates, the c o r t i c o s t e r o i d s inducing p a r t u r i t i o n .  ).  f o r normal development, i t i s  Injection  induce  (26)  h i g h e r i n f o e t u s a t 32 w e e k s  However, p r e m a t u r e i n f a n t s  i s essential  prematurely.  monkeys d i d n o t  maternal  the p r o l o n g a t i o n of pregnancy a s s o c i a t e d  sometimes have v e r y low C o r t i s o l while Cortisol  as h i g h as s i x  (25).  l e v e l s are three times  t h a n a t 18 w e e k s  the  plasma  equal to  n o t b e e n p r o v e n i n t h e human b u t  w i t h anencoephaly Cortisol  to term  or  19 o f g e s t a t i o n ( g e s t a t i o n  d i r e c t p a r t i c i p a t i o n i n the onset  f o e t u s has suggests  20  t o be  shown t o be  In  in  s h o w i n g w h e t h e r t h e y a r e s e c r e t e d by t h e m a t e r n a l  times maternal  the  h a v e shown t h a t p r e g n a n c y  by a d r e n a l e c t o m y  foetal adrenals.  not  In  (3).  So,  possibly  are not the primary  Rhesus in  factor  8  Because o f the and  t h e mouse  reasoned  similarity  (14), which i s of  metabolism  has  b e e n shown t o c r o s s  extensively  Since  the  the  t h a t t h e mouse p r o v i d e s  and  at  of  to the  there  11-keto  haemochorial  a pool  the  hormone r e s u l t s  in a dilution  extent  o f w h i c h can  used  size  To in  of  the  blood  and/or  Murphy  a c t i v e form of the  volumes o f b l o o d .  Values  methods  Although  (28,  29).  inactive metabolite  (28)  the  obtained  metabolized  i n the  animal radio-  the rela-  tissue.  corticosteroids  tissues,  developed (16)  reducing  several a  compe-  used  a  of d e t e c t i n g  of r e q u i r i n g l a r g e r  agree w e l l with enzymatically  other the  t o c o r t i c o s t e r o n e made i t p o s s i b l e t o  t h e mouse b e c a u s e o f t h e  and  has  hormone and  to  blood  hormone  changes i n the  limitation  former  with  is  was  transport  isotope,  Burton  measure the use  of the  foetal  protein-binding radio-assay.  the  i t  administration of  amounts o f  i n the  f l u o r o m e t r i c method w h i c h has only  type,  pool.  methods have b e e n u s e d . titive  and  steroid  to estimate  determine a c c u r a t e l y small  the maternal  easily  of  active  tive  human  derivative.  experiment,  be  the  i n s t a n c e s where t h e  placenta  i s already  t h e moment o f t h e  of  a model f o r s t e r o i d  i n mammals i n t h o s e the  placenta  fluorometrically,  i t w o u l d be  difficult  small q u a n t i t i e s of  9  An e v e n more s e n s i t i v e Hillman  and G i r o u d  method h a s been d e v e l o p e d by  labelled  hormone i s a c e t y l a t e d  purified  by s e v e r a l 3  stant  ratio of  modification a c e t a t e was  H/  steps o f paper chromatography t o a con-  14 C.  The method u s e d  i n which a f t e r  acetylation  i n t h i s work i s a radioinert  steroid  added and t h e m i x t u r e was c o c r y s t a l l i z e d . 3  stancy of isotope crystallization the  14 amount o f C3 w i t h a c e t i c - H a n h y d r i d e and  (29) whereby a t r a c e  compound.  ratio  Con-  14 H/  was a c c e p t e d  C after  t h e t h i r d and  fourth  as t h e c r i t e r i o n o f p u r i t y  of  10  MATERIALS  Animals  UBC S w i s s vivarium 19  at this  days.  mating  were o b t a i n e d  university.  Day 1 was d e f i n e d  night.  accurately, 9 p.m.  mice  To d e t e r m i n e  t h e males were  to 9  from  the zoology  The g e s t a t i o n p e r i o d  was  as t h e day f o l l o w i n g t h e t h e day o f left  pregnancy  with  the females  buffer  contains:  from  a.m.  Buffers  Krebs-Eggleston 0.9%  NaCl,  phate  phosphate  1.15% K C I , 3.82% MgS0 .7 H 0 , 4  2  0.IM  phos-  b u f f e r , p H 7.4 ( 3 0 ) .  Phosphate prepared  and acetate  buffers  f o l l o w i n g the procedure  from  pH 4 t o 9 w e r e  o f Hawk, O s e r  a n d Sum-  merson ( 3 1 ) .  Solvents  Every carbon ethyl  solvent  utilized  tetrachloride, acetate,  was d i s t i l l e d :  methanol,  toluene,  ethanol,  dichloromethane  chloroform,  n-hexane, and acetone.  11  4.  TLC  Only cent  indicator  tilled  5.  Eastman S i l i c a were used.  methanol  Radioactive  g e l TLC p l a t e s w i t h  prior  fluores-  They were washed w i t h  dis-  t o use.  compounds  3 Acetic-  H anhydride  comes i n s e a l e d It  was  acetic give dry  Corp.  i n b a t c h e s o f 100 m C i .  I t was  anhydride.  a final benzene  perature  activity  a m p u l e s t o be p r o t e c t e d  purchased  Nuclear  (specific  diluted  Ci/mmole)  against  humidity.  f r o m New  10 t i m e s w i t h  A n h y d r o u s b e n z e n e was  concentration (v/v).  400  The  r e a g e n t was  i n a desiccator containing  non-radioactive  then  o f 10% a c e t i c  England  added t o  anhydride i n  stored  a t room  tem-  DRIERITE.  3 1, 2- H c o r t i c o s t e r o n e  (specific  activity  51.6 C i /  14 m m o l e ) a n d 4-  C-corticosterone  57.3 mCi/mmole) w e r e p u r c h a s e d Corp.  Each  eluted  with  synthesized (cf.  (specific f r o m New  activity  England  Nuclear  was  s p o t t e d o n t o TLC t o p u r i f y a n d was 14 acetone. C-ll-dehydrocorticosterone 14 enzymatically  Methods).  from  C-corticosterone  was  12  6.  Non-radioactive  The bought  coenzymes NAD, NADP, NADH and NADPH were  from  Sigma C h e m i c a l  Corticosterone, costerone Pawling,  7.  Liquid  solvent  scintillation  mixture  was made o f 4 g o f  (PPO) and 0.1 g o f l , 4 - b i s - 2 benzene  scintillation  liquid  IIA, Nuclear  (POPOP) d i s s o l v e d  in 1 litre  Chicago.  B u f f e r A:  spectrometer  was a U n i l u x  The raw d a t a were r e d u c e d  with  65 c a l c u l a t o r .  Transmission electron  Phosphate  spectrometer  scintillation  a Hewlett-Packard  (a)  Steraloids,  toluene.  The  9.  from  N.Y.  (5-phenyloxazolyl)  8.  S t . L o u i s , Mo.  1 1 - d e h y d r o c o r t i c o s t e r o n e and c o r t i -  2,5-diphenyloxazole  of  Company,  a c e t a t e were o b t a i n e d  Scintillation  The  reagents  microscopy  buffer  0.IM s o d i u m c a c o d y l a t e and 0.5% C a C l in  distilled  water  13  Buffer  For  pH  buffer  (b)  Epon  B:  7.2,  one  o f 3.5%  part  143 m l  of buffer  A,  NaCl.  of buffer  embedding r e s i n  8 1 2 , 21.0  A and  consisted  g DDSA, 1 0 . 5 3  57 m l  of  g MNA  o f 28.62  g  and  30.  Sectioning  Sectioning microtome  10.  parts  812  1% DMP  (c)  four  B.  The Epon  to every  was  Sorvall  performed  MT-I  with  a manual  ultra-  "Porter-Blum".  Autoradiography  Medical Kodak.  X-ray  films  (NS2T) w e r e  obtained  from  14  METHODS  1.  Animals  (a)  Injection  of  mice  Pregnant  m i c e were i n j e c t e d  the back o f the neck w i t h  0.9%  subcutaneously  at  NaCI c o n t a i n i n g  14 0.5  yCi of  stated,  C-corticosterone.  t h e y were s a c r i f i c e d  overdose  o f CO,,,  and  on  kept  p a p e r wet from  ice in Petri  with  and (b)  saline.  the mother w i t h  coagulation. from  the uterus  the  The  uterus  ground  15 min. removed  otherwise  later from  with  mother  dishes containing a  filter  drawn  a heparinized syringe to  f o e t u s e s and  of  an  the  T h e n t h e b l o o d was  - usually  i n 5 ml  Unless  p l a c e n t a e were  t h r e e were p o o l e d  prevent removed  - weighed  saline.  Adrenalectomy  Pregnant 0.1 6.7%  ml/10  m i c e on  day  16  were a n a e s t h e t i z e d  with  g body w e i g h t o f a s o l u t i o n c o n t a i n i n g  nembutal  removed and  and  0.9%  ethanol.  t h e m i c e were k e p t  t i o n s w i t h w a t e r and  food  The  adrenals  under normal  f o r the  next  24  were  condi-  hrs.  15  2.  Tissues  (a)  Minced  tissues  Foetal carcasses,  t i s s u e s were p o o l e d three  (three heads,  three  l i v e r s ) , m i n c e d on a wet f i l t e r  p a p e r , p u t i n t o 1 ml i n c u b a t i o n m i x t u r e a n d k e p t on  (b)  i c euntil  t h e time o f i n c u b a t i o n .  Cell fractionation The  livers  from  18-day f o e t u s e s  t h r e e m o t h e r s were p o o l e d , buffer  containing  homogenized  0.25M s u c r o s e  t i o n was p e r f o r m e d a s f o l l o w s :  a  10 m i n .  The n u c l e a r  fractiona-  A f t e r removing 1 ml, a t 1,200 x g  s e d i m e n t was h o m o g e n i z e d  second time t o break t h e unbroken c e l l s  re-centrifuged. at  i n 6 ml K r e b s  and c e l l  t h e w h o l e homogenate was c e n t r i f u g e d for  f r o m two o r  The m i t o c h o n d r i a  were  a n d was  centrifuged  15,000 x g f o r 20 m i n a n d t h e m i c r o s o m e s a t  100,000 x g f o r 30 m i n .  E a c h f r a c t i o n was  pended i n t h e same b u f f e r a n d washed t w i c e . final  volume was made up t o t h e e q u i v a l e n t  tissue/ml  buffer.  resusThe o f 50 mg  Incubations  (a)  Enzymatic  synthesis  of  Guinea p i g l i v e r cell  11-dehydrocorticosterone  m i c r o s o m e s were o b t a i n e d  f r a c t i o n a t i o n as d e s c r i b e d  f o r mouse  livers  and were d i l u t e d t o t h e e q u i v a l e n t  100 mg  tissue/ml  frozen without  buffer.  They c o u l d be  significant  by  foetal of  stored  loss of a c t i v i t y .  The  i n c u b a t i o n m i x t u r e c o n s i s t e d o f 0.1 ml m i c r o s o m e .14 preparation,  0.5  uCi  Krebs-sucrose b u f f e r , erlenmeyer f l a s k . at  Minced  1 mg NADP i n a 2 5-ml  The i n c u b a t i o n  was  c a r r i e d out  (16).  tissues  M i n c e d t i s s u e s were i n c u b a t e d  with  0.9% s a l i n e o r K r e b s b u f f e r c o n t a i n i n g 3  0.1 ml  37°C f o r 30 m i n . and t h e f l a s k s were s h a k e n a t  120 c y c l e s / m i n . (b)  C-corticosterone,  . H-corticosterone  ticosterone.  o r 2,000 cpm  In order  14  1 ml 20,000  cpm  C-ll-dehydrocor-  t o make p o s s i b l e t h e d e v e l o p -  ment o f a u t o r a d i o g r a m s , t h e b r a i n t i s s u e s were 14 incubated  with  10,000 cpm  Unless otherwise carried  stated,  C-corticosterone. the incubations  a t 37°C i n 25-ml e r l e n m e y e r  pered with  a silicone  120 c y c l e s / m i n .  stopper  f o r 15 m i n .  were  flasks,  stop-  and s h a k e n a t The r e a c t i o n  was  17  stopped by  by p u t t i n g t h e f l a s k s  e x t r a c t i o n with  Coenzyme  on i c e , f o l l o w e d  solvents.  specificity  One m l f r o m e a c h f r a c t i o n was i n c u b a t e d 0.5 mg coenzyme  with  (NADH o r NADPH) a n d 2,000 cpm  14  C-ll-dehydrocorticosterone.  pH  optimum Incubations  at d i f f e r e n t  were c a r r i e d  pHs (4 t o 9) a n d 2,000 cpm  dehydrocorticosterone. dehydrocorticosterone gassed with  Kinetic  The  o u t i n 1 ml b u f f e r 1 4  C-11-  The r e d u c t i o n o f 11was c a r r i e d  out i n flasks  ^ •  analysis  o x i d a t i o n r e a c t i o n was c a r r i e d  out with 14  various  substrate concentrations,  corticosterone,  2,000 cpm  0.5 mg NADP a t pH 8.0.  C-  The 14  r e d u c t i o n r e a c t i o n was c a r r i e d dehydrocorticosterone,  out with C - l l -  0.5 mg NADPH a t pH 5.5.  18  Extraction  (a)  of  Blood  tissues  and  tissues  from  mice  Samples were e x t r a c t e d i n 6 v o l . o f by  shaking  v i g o u r o u s l y 20  times.  c o n t a i n i n g much n e u t r a l l i p i d , S t e r o i d s were e x t r a c t e d by dichloromathane ously. 4 0°C  CH C1 2  under N  Before  2  was gas 2  2  shaking  upper  phase,  discarded. of  20  6 v o l . of  times  vigour-  down t o d r y n e s s  to avoid o x i d a t i o n of the 2  down, 10  to each tube,  removed  was  addition  evaporated  drying CH C1  were added was  2  2  (CH C1 ),  The  n-hexane  for spotting  onto  steroids.  yg o f c a r r i e r  one-fifth TLC  of each  at  steroids sample  to determine  the  14 amount o f tritium. through (b)  The to  Incubated  In  C-steroid before remaining  acetylatxon with  f o u r - f i f t h s were  processed  crystallization. minced  these  s p o t t e d onto  tissues  cases,  the  and  cell  entire  fractions  CH C1 2  2  fraction  was  TLC.  Chromatography  10 detected  yg o f t h e n o n - r a d i o a c t i v e compounds t o ( c o r t i c o s t e r o n e and  be  11-dehydrocorticosterone,  19  or  corticosterone  added  to  TLC.  After  in  two  to  spotted two  each  corticosterone  sample  as  a  evaporation,  three  onto  drops  carrier the  of  acetate)  for  extracts  plates  at  were  location were  on  the  dissolved  chloroform-methanol  E a s t m a n TLC  different  (a)  and  (3:1)  and  room t e m p e r a t u r e  in  systems:  n-hexane:  ethyl  acetate  (4:1)  to  remove  residual  fats;  (b)  toluene: to  The and  6.  chloroform:  separate  with  water  (120:60:20:1)  steroids.  zones were  eluted  methanol:  located  under  shortwave  UV,  cut  out  acetone.  Acetylation  Following  the  procedure  of  Hillman  and  Giroud  (29)  3  and of  Kliman  (32),  radioinert  12%  acetic  1 vol.  acetic  anhydride  of  acetic-  H  anhydride  anhydride were mixed i n benzene, with  and  specific  and made  9  vol.  to  activity  3  of  40  dry were  Ci/mmole.  pyridine, added  to  to  Thirty keep  each  \il a c e t i c -  the  reaction  sample a f t e r  H  anhydride  mixture  drying.  in  and  20  yl  solution,  20  The  acetic-"H anhydride  from microvolumetric over  DRIERITE u n t i l  mixing  by g e n t l e  VERY t i g h t l y  and p y r i d i n e were  pipettes stored  i n a desiccator  ready  f o ruse.  After  shaking,  the tubes  were  and i n c u b a t e d  0.5 m l 2 5 % e t h a n o l  measured  thorough stoppered  f o r 24 h r s . a t 3?°C ( 3 3 ) .  was a d d e d  t o hydrolyze the  3 excess  acetic-  were then  H anhydride  extracted with  acid.  t o separate  was r e m o v e d  a n d t h e s a m p l e was w a s h e d w i t h 3  w h i c h was  The  6 v o l . C C l ^by shaking  vigourously  0.5 m l o f w a t e r  7.  to acetic  the steroids.  samples 20  The w a t e r  to get r i d of residual  times  layer  another  H-acetate,  discarded.  Crystallization  After  evaporation  radioactive  corticosterone  to  c o n t a i n i n g 10 mg  acetate.  to the cold  begin.  t h e samples  After  four  Cold  of non-radioactive  distilled  samples t o a l l o w t o s i xhours  and i  s a m p l e was removed counted.  times  or until  (Table I ) .  This  after  procedure  a constant  3  H/  w a t e r was  crystallization  One-fifth of  the third  crystallization  was r e p e a t e d 14  added  a t 4°C, t h e c r y s t a l s  were r e d i s s o l v e d i n 1 ml o f methanol. each  containing  c o r t i c o s t e r o n e a c e t a t e were d i s s o l v e d i n  1 ml o f methanol  dropwise  of CCl^,  C ratio  was  three  to four  obtained  21  TABLE I .  DETERMINATION  OF THE PURITY  OF CRYSTALLIZED C-CORTICOSTERONE 14  Sample  1:  H  1 4  3  1 4  3  H  1 4  3  c  H/  1 4  C  4  671  573 17 . 35  17.33  Foetus  32 a t d a y 18  7,827  3,696  2,784  212  128  103  36.95  28.93  27.08  A difference smaller  than  after  crystallizations  two s u c c e s s i v e  criterion  5  9,943  C  Sample 2:  3  11,625  c  H/  3  M o t h e r 3 a t d a y 14  Crystallization 3  - 21- H-ACETATE  10% b e t w e e n i s o t o p e  ratio  was a c c e p t e d  o f p u r i t y o f t h e compound (29).  H/ "C as t h e  22  8.  Standards  for efficiency  of  acetylation  14 Approximately acetylated costerone acetone  10,000 dpm  a t t h e same t i m e zone was  of  as t h e samples.  9.  and c o u n t e d  to determine  also  zone was  with  t h e amount o f cpd.B  c u t o u t and e l u t e d .  ceeding  to the c r y s t a l l i z a t i o n  of each  s t a n d a r d was  counted;  corti-  The c o r t i c o s t e r o n e  acetate  Before  pro-  of the l a t t e r ,  one-fifth  the f o u r - f i f t h s  remaining  crystallized.  Quench c u r v e  and c a l c u l a t i o n  A quench c u r v e  o f the double  of counting f o r  H and  I t was  checked  e v e r y o t h e r month.  at least  f o r the double  Transmission electron  i n F i g u r e 1. The  calcula-  buffer  (4:1) f o r 1 h .  ferred  into  and M a u d s l e y ( 3 4 ) .  microscopy  was p e r f o r m e d  a rinse  C i s shown  i s o t o p e a n a l y s i s were done a c c o r d i n g  t o t h e method o f K o b a y a s h i  Fixation  effi-  14  ciency  tions  label  f o r the determination o f the 3  10.  The  c u t o u t o f t h e TLC, e l u t e d  t h a t had n o t been a c e t y l a t e d .  were  C - c o r t i c o s t e r o n e was  i n glutaraldehyderphosphate  The t i s s u e s were q u i c k l y  phosphate b u f f e r  f o r 15 m i n .  trans-  FIGURE  1  1,  QUENCH  2 EXTERNAL  CURVE  3 STANDARD  FOR  3  H  4 RATIO  AND  '"C  5  24  Post-osmification phosphate tissues  buffer  best It the in  known  was  used  smaller  t i s s u e more  i n an osmium  The  changes  i t i s the  i n volume o f the t i s s u e .  m o l e c u l a r s i z e and can p e n e t r a t e The  tissues  were  then  left  f o r 5 min.  2 x 30 m i n . i n  o x i d e (PO).  t i s s u e s were dehydrated  1:3  and  t h e r e s i n was  f o r 1 h. e a c h .  thin  magnifications  i n PO:Epon  They were embedded  allowed t o harden  sections  20 s e c o n d s .  electron  until dehydration.  f o r d e h y d r a t i o n because  easily.  and  The  tetraoxide:  Afterwards the  buffer  30%, 50%, 70% and 90% m e t h a n o l ,  propylene  for  i n phosphate  t o reduce  has a l s o  done  ( 1 : 1 ) f o r 40 m i n .  were kept  Methanol  was  were  Electron  stained  3:1,  1:1,  i n 100% Epon  812  a t 60°C f o r 24  h.  with  citrate  micrographs  o f 3,000 t o 12,000 w i t h  microscope.  812  0.2%  lead  w e r e made a t a Philips  original  P-75  EXPERIMENTAL  In  1.  vivo  experiments  Corticosterone  content  The t o t a l and  II.  Between  corticosterone  the foetuses.  on  the foetal  no c h a n g e  side  actually  these  While  maternal  blood.  the level  of  i n the mother b u t decreased the total  (Table  II)  increased  (p<.01),  (Table  rate  amount  was  decreased i t s  specific  I I I ) , indicating  of transfer  On d a y 17 t h e s p e c i f i c  corticosterone  i n the mother  13 a n d 1 7 a r e s h o w n i n  two d a y s ,  i n the r e l a t i v e  placenta.  foetal  on days  increased  in  activity  of tissues  amount o f c o r t i c o s t e r o n e  i n the foetuses  Table  the  RESULTS  across  activity  about one-fourth  of  that i n  26  TABLE I I . IK  T O T A L AMOUNT OF  CORTICOSTERONE  THE MOTHER AND F O E T U S E S ON G E S T A T I O N A L DAYS 1 3 AND 1 7  Mother . ng cpd.B/ml. b l o o d ± SEM G e s t a t i o n a l Day  ng  Foetuses cpd.B/g t i s s u e ± SEM  13  720.3 ±139.3 (5)*  641.9  ±185.4 (3)  17  1,145.4 ±103.5 (6)  300.9  ±48.6 (3)  * number o f o b s e r v a t i o n s  i n parentheses  27  TABLE  III.  S P E C I F I C A C T I V I T Y OF R E C O V E R E D  ^ Gestational  Day  #  Mother C-dpm/ng  1  cpd.B  #  .  C-CORTICOSTERONE  Foetuses C-dpm/ng c p d . B  13  24.52  ±3.6  0.99  ±0.22  17  12.04  ±1.77  3.44 ±0.47  28  Effect on t h e  of adrenalectomy of the mother t o t a l amount o f c o r t i c o s t e r o n e  The day  e f f e c t of  16  on  the  mother  24  hrs.  maternal  amount o f later  source  removed, yet  of  the  mately  normal  foetal  l e v e l s of  tially  normal  the  adrenalectomy  absence of  of  the  corticosterone  i s shown i n T a b l e  steroid synthesis  mother's  level  of  blood  hormone  corticosterone  (Table  V).  maternal of  This  i s capable  taining  substantial production  in IV.  has  (Table were  The  been approxi-  IV). also  The essen-  indicates that,  functioning of  and  foetal of  on  the  contained  hormone, the  adrenal  mother  main-  hormone.  in  29  TABLE I V .  E F F E C T OF A D R E N A L E C T O M Y  ON THE L E V E L S OF C O R T I C O S T E R O N E  Sample C o n t r o l s (6) A d r x (2)  ng cpd.B/g ±SEM 1,145.4  tissue  +103.5  OF THE MOTHER  I N THE MOTHER  14 C-dpm/ng ±SEM  cpd.B  12.04 ±1.77  1,586.0  14.92  312. 3  31.60  30  T A B L E V. 1 4  CONTENT AND  C-CORTICOSTERONE  S P E C I F I C A C T I V I T Y OF  I N THE F O E T U S A F T E R  RECOVERED  ADRENALECTOMY  OF THE MOTHER  Sample Controls Adrx  ng cpd.B/g t i s s u e ± SEM 300.9  ±48.6  "^C-dpm/ng c p d . B ± SEM 3.44  ±0.47  437.2  0 .58  1,344.2  0.23  181.4  2.64  219.4  2 . 02  31  3.  Metabolism of and t h e f o e t a l  Table costerone placenta  C-corticosterone i n the placenta tissues  V I shows t h e r e l a t i v e  amount o f  and 1 1 - d e h y d r o c o r t i c o s t e r o n e and f o e t a l  tissues  recovered  corti-  i n the i n t h e two  14 tissues  15 m i n . a f t e r  costerone the  into  the mother.  p l a c e n t a o f more  recovered  was  of  i n t h e form  i t was  contrast,  injection  than  constant  the foetal  C-cortx-  The a c c u m u l a t i o n 75% o f t h e t o t a l  f o r days  tissues  of the radioactivity,  the  form  contained  tissues.  counts Most In  less  than  and m o s t o f i t was i n  of 11-dehydrocorticosterone,  extensive metabolism  i n  12 a n d 1 4 .  of corticosterone.  20%  foetal  of  of the active  showing  hormone  an  i n the  32  TABLE V I .  R E L A T I V E AMOUNT OF  C-CORTICOSTERONE  AND  I T S 1 1 - D E H Y D R O M E T A B O L I T E I N THE P L A C E N T A  AND  FOETAL TISSUES  % of Total  Counts Recovered  Day  cpd. B  SEM  Foetuses  Placenta Gestational  ±  cpd. A  cpd.B  c p d .A  12 ( 7 )  75.6  ±1.5  12.4  ±1.8  1.9  ±0.2  10.2  ±0.5  14 ( 7 )  76.0  +1.1  3.1  ±0.5  3.5  ±0.2  16.6  ±1.3  16 ( 8 )  55.1  +1.9  1.4  ±0.2  21.7  ±1.0  22.8  ±1.3  C o r t i c o s t e r o n e and i t s 1 1 - d e h y d r o i n the f o e t a l t i s s u e s  The  conversion  of  11-dehydro m e t a b o l i t e  metabolite  corticosterone to i t s was  determined  by  the  per-  14 centage counts  of  C - l a b e l l e d hormone o v e r  recovered  i n the  foetuses  15  the  total  min.  after  14 injection the  of  0.5  yCi  C-corticosterone  mother. Table  VII  shows t h e  costerone  by  the  trimester  of  pregnancy.  the  results  of  injected  in  the  14.  Then,  now  A  foetal  i n Table  VI,  tissues  a very  of  corti-  during  In good  11-dehydro  a dramatic  around  day  recovered  form  change  the  agreement high  third with  percentage  16,  i n the  amount o f  days  where most of active  identical  found 12  the  hormone  form.  on  day  13  in  12.  the  and  (p<.01), whereas day t o day  to  metabolism  c o r t i c o s t e r o n e (p=.02)  11-dehydrocorticosterone virtually  from  i n the  t r a n s i e n t change o c c u r r e d  relative  was  metabolism  r a d i o a c t i v e c o r t i c o s t e r o n e was  inactive  occurred was  into  14  34  TABLE V I I . IN  CORTICOSTERONE  AND  I T S 11-DEHYDRO M E T A B O L I T E  FOETAL TISSUES  % of Gestational  Day  T o t a l Counts cpd. B  i n Foetal  Tissues cpd. A  12  (7)  15.5  ±1.6  84.6  ±1. 5  13  (5)  24.5  ±3.9  73.5  ±3.1  16.9  ±0*. 3  83.1  ±0. 3  49 .1 ±1.1  52.3  ±2. 3  14(7) 16  (8T  17 ( 8 )  90.9  ±1.1  9.1  ±1. 1  ±  SEM  35  B.  In v i t r o  1.  experiments  In v i t r o metabolism o f 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  The of  r e l a t i o n s h i p between t h e d e h y d r o g e n a t i o n  corticosterone  the  reduction  can  have a g r e a t  activity the  3  of the l a t t e r  and f o e t a l  Gestational  The placenta  and  14  t o study minced with  C-ll-dehydrocorticosterone.  d a y s 13, 14 and 15 were c h o s e n  because  change i n t h e c o n v e r -  to 11-dehydrocorticosterone  as i n d i c a t e d i n T a b l e V I I .  r e s u l t s i n Table VIII was  show t h a t  1) t h e  t h e most a c t i v e t i s s u e f o r t h e r e d u c -  to corticosterone  constant  In order  t i s s u e s were i n c u b a t e d  sion of corticosterone  tion  upon t h e b i o l o g i c a l  these days, a g r e a t  occurred,  to corticosterone  f a t e o f t h e two compounds,  H-corticosterone  around  effect  of corticosterone.'  metabolic  placental  t o 1 1 - d e h y d r o c o r t i c o s t e r o n e and  and t h i s  f o r t h e 3-day p e r i o d ;  activity  2) t h e head  the  most a c t i v e i n d e h y d r o g e n a t i n g  and  3) t h e body showed r e d u c t a s e a c t i v i t y  w i t h time o f g e s t a t i o n .  remained was  corticosterone; increasing  TABLE V I I I . AND  I N V I T R O M E T A B O L I S M OF  11-DEHYDROCORTICOSTERONE  CORTICOSTERONE  BY F O E T A L  TISSUES  % Reduction o f 11-Dehydrocorticosterone ±SEM  % of Dehydrogenation of Corticosterone ±SEM Gestational  Day  Placenta  Head  Body  Placenta  Head  Body  20.7  ±1.0  62.6  ±1.5  61.1  ±1.9  83.4  ±2.5  8.8  ±1.4  8.1  ±1.2  14 ( 8 )  16.2  ±1.0  61.0  ±1.3  49.0  ±4.7  88.0  ±1.6  7.3  ±0.6  12.0  ±1.4  15  18.5  ±1.8  46.5  ±1.2  27.9  ±1.1  86.9  ±2.0  14.0  ±0.8  37.5  ±1.9  13  (12)  (8)  reduction dehydrogenation 13  4.1  0.14  0.13  14  5.4  0.12  0.24  15  4.7  0.30  1.34  37  Localization  Since increases of  the weight a great  pregnancy  and  of the reductase  of the foetal  deal  ( 2 0 mg  during  a t day 14;  ( 1 8 ) , i t was  suspected  activity  shown b y t h e b o d y was  In order  to test  gestational carcasses  d a y 15  70 mg  a t day  18)  for corticos-  that the  reductase  due t o t h e  the foetal  and c a r c a s s  I t i s evident,  IX, that the l i v e r and t h e c a r c a s s  dehydrogenation.  trimester  livers  liver. from  f o e t u s e s were removed from t h e  and l i v e r  separately. Table  this,  liver  the third  since i t i s a target tissue  terone  tion  activity  were  according  incubated to  effected a net  reduc-  exhibited a significant  net  38  TABLE I X .  R E D U C T I O N OF  BY THE F O E T A L L I V E R AND  11-DEHYDROCORTICOSTERONE RESIDUAL CARCASS  % Conversion of 11-dehydrocorticosterone Reaction  Liver  ±SEM  Carcass  Dehydrogenation (8)  21.6  +1.3  29.9  ±2.7  Reduction  48.4  ±1.4  12.5  ±1.3  „ ,. Ratio  (8)  reduction , , dehydr.  2.24  0.42  39  3.  Metabolism of c o r t i c o s t e r o n e i n t h e f o e t a l head  During grams  the i n vivo experiments,  (AR) w e r e m a d e o f t h e T L C b e f o r e  • see i f t h e r e were m e t a b o l i t e s other  than  amounts. did  elution  11-dehydrocorticosterone  i n significant  W h e r e a s t h e AR o f t h e T L C f r o m  s h o w e d many b a n d s .  to  of corticosterone  n o t show a n y d e t e c t a b l e b a n d ,  heads  autoradio-  those  They were  the bodies from the  faint,  probably  14 due  t o t h e l o w amount o f  head. could  In order  pure an  t o determine  be r e p r o d u c e d ,  incubated  with  whether  minced  foetal  1 0 , 0 0 0 cpm o f  i nthe  these  heads  bands  were  chromatographically  ^ C - c o r t i c o s t e r o n e and t h e TLC w e r e e x p o s e d t o  X-ray  costerone  film.  Figure  2 shows t h a t i n d e e d  i s metabolized  compounds by t h e f o e t a l was  C accumulated  similar  to that  between days  the metabolism  is  a decrease  Only  to  head.  i n vivo.  several  The i n v i t r o T h e AR shows  14 a n d 1 5 , t h e r e  in  costerone  extensively into  corti-  metabolism that  i s a dramatic  of corticosterone.  change  A t d a y 15  i n the rate of metabolism  of  there  corti-  11-dehydrocorticosterone.  t h e TLC z o n e s  dehydrocorticosterone  from  c o r t i c o s t e r o n e and 11-  have been  eluted.  Figure  2  foot-  14 note zone.  shows t h e p e r c e n t a g e The r e m a i n i n g  zones  C of the total  f o r each  have n o t y e t been  identified.  40  F I G U R E 2.  M E T A B O L I S M OF C O R T I C O S T E R O N E  BY THE F O E T A L  The s t r a i g h t d a r k b a n d a t t h e t o p o f t h e r i g h t - h a n d chromatogram i s an a r t i f a c t  Footnote:  % of Total Gestational  Day  cpd. B  1 4  C-dpm  ±SEM  cpd. A  14  (8)  15.7  +0.9  84.4  ±0.9  15  (4)  52.8  ±3.0  47.3  ±3.0  HEAD  41  C.  Enzyme  1.  kinetics  Cell  fractionation  In  order  responsible mine  and coenzyme  to localize  specificity  the subcellular  f o rt h e reductase  activity  t h e coenzyme r e q u i r e m e n t ,  foetal  fraction  and t o d e t e r livers  were  homogenized  and t h e n u c l e a r , m i t o c h o n d r i a l and  microsomal,  as w e l l  the  final  activity the in  supernatant,  activity  t h e crude  rather evenly  fractions  fractions  i t was f e l t would  that washing  improve  activity,  indicates,  very  the subcellular  the localization  the fractions  marked.  were washed  amount  the nuclei.  pellet  yielded  decreased  The p r e f e r e n c e  The n u c l e a r  reductase  substantial with  of the , i n the  As Table  XI  t h e h i g h e s t r e d u c t i o n was l o c a l i z e d i n  mitochondria.  nificant  distributed  ( T a b l e X ) , NADPH  same b u f f e r a n d t e s t e d f o r a c t i v i t y .  the  Whereas  t o be t h e p r e f e r r e d coenzyme.  Since  enzyme  o f NADH o r N A D P H .  seemed  unwashed  homogenate and  were t e s t e d f o r reductase  i n t h e presence  enzyme  appeared  as t h e crude  with  f o r NADPH w a s  fraction  showed  a  sig-  activity,  b u t i t a l s o had a  o f broken  cells  that  Re-homogenization  a more  pure  washing.  fraction,  sedimented  of the "nuclear" which  activity  42  T A B L E X.  ENZYME A C T I V I T Y AND COENZYME  I N THE CRUDE S U B C E L L U L A R  FRACTIONS  % Substrate Subcellular Fraction  SPECIFICITY  Converted  +NADH  +SEM  +NADPH  H o m o g e n a t e (3)  4 6 . 7 ±6.3  6 6 . 0 ±4.8  Nuclei  41.3  ±0.8  53.5  ±3.7  56.5  ±3.4  63.3  ±4.6  37.9  ±3.2  59.4  ±3.6  26.9  ±2.1  86.3  ±3.2  (3)  Mitochondria Microsomes  (3)  Supernatant  Each  (3)  (3)  incubation mixture contained the  equivalent  o f 50 mg  tissue  43  TABLE X I .  ENZYME A C T I V I T Y AND COENZYME  I N WASHED F R A C T I O N S  AND T H E I R  o, o  SPECIFICITY  WASHES  Substrate  Converted Washes  Fractions Subcellular Fractions  NADH  NADPH  NADH  NADPH  Nuclei  8.2  77.5  11.6  79.5  Re-homogenized nuclei  7.4  29.0  —  —  Mitochondria  8.6  95.2  12 . 6  85.1  Microsomes  5.0  11.1  7.3  11.2  Supernatant  6.9  30.3  Each on  —  figure., i s an average o f d e t e r m i n a t i o n s  two s e p a r a t e b a t c h e s  o f enzyme  —  44  When m i t o c h o n d r i a w e r e k e p t for  more  into  30 m i n . , e n z y m e  t h e wash.  ficity  2.  than  In a l l cases,  f o r NADPH w a s v e r y  tion,  experiments  whether  factor  that  increased  dehydrocorticosterone foetal  liver  as a s i t e  of reduc-  out t o determine  o f coenzyme was t h e  t o t h e enzyme  only slightly  speci-  activity  were c a r r i e d  the addition  was r e l e a s e d  t h e coenzyme  the liver  o r not the presence  limiting found  localized  activity  clear.  Development o f t h e reductase in the foetal liver  Having  i n the buffer  reaction.  o f t h e coenzyme  I t was NADPH  t h e r e d u c t i o n o f 11-  to corticosterone  (Table X I I ) .  by minced  45  TABLE X I I . ACTIVITY  D E V E L O P M E N T OF THE R E D U C T A S E  I N TEE FOETAL L I V E R  % Substrate Gestational  Day  Without  NADPH  Reduced With  ±SEM NADPH  14  1 1 . 9 ±0.9 (5)  1 4 . 0 ±0.8 (6)  15  3 9 . 4 ±2.2 (4)  5 6 . 8 ±3.5 (5)  46  3.  pH  optimum  The by  pH o p t i m u m  incubating  from  the  4.  a crude  4.5 t o 9.  5.5 a n d 6  f o rreduction  was  preparation  a t pH  I t was e s t a b l i s h e d  (Figure  same  activity.  The  reduction  3).  These  determined varying  t o be  between  t w o pH v a l u e s  gave  Km  (Figure between  4) s h o w e d  11-dehydrocorticosterone  a substrate  8 0 a n d 1 0 0 yM.  corticosterone inhibition and  of  6) s h o w e d  substrate  concentration  (between  15  20 y M ) .  The genation  Km a n d Vmax reactions  Lineweaver-Burk  f o rboth  plot.  Km w a s 3 3 yM a n d V m a x , (Figure  5).  Vmax=0.21  reduction  were determined  11-dehydrocorticosterone  and  occurring  The d e h y d r o g e n a t i o n o f  (Figure  a t a lower  inhibition  and  dehydro-  from t h e  For the reduction  of  to corticosterone, the 0.4  ymoles/min./ml  For the reverse ymoles/min./ml.  reaction,  Km=10 yM  PH  UNITS  FIGURE 4,  EFFECT OF CONCENTRATION OF 11-DEHYDROCORTICOSTERONE  ON REDUCTASE ACTIVITY  0  5  10  20  30  40  60  .  11-DEHYDROCORTICOSTERONE (VM)  80  100  FIGURE 5.  LINEWEAVER-BURK PLOT FOR REDUCTION 11-DEHYDROCORTICOSTERONE  0.1  0.2  1/yM  0.3  0-4  11-DEHYDROCORTICOSTERONE  KM = 3 3  nM  VMAX = C M  UMOLES/MIN/ML  FIGURE  6.  EFFECT  OF. C O N C E N T R A T I O N  • DEHYDROGENASE  ACTIVITY  CORTICOSTERONE  (yM)  OF  CORTICOSTERONE  ON  FIGURE 7,  LINEWEAVER-BURK  PLOT FOR DEHYDROGENATION  OF CORTICOSTERONE  -0.5  0  0.5  1.0  1/yfi OF CORTICOSTERONE  KM  =  10 KM  VMAX = 0 , 2 1  UMOLES/MIN/ML  1.5  2.0  52  5.  C o n t e n t and s p e c i f i c a c t i v i t y o f 14 C-corticosterone i n the f o e t a l  Since greatly  the metabolic  different  recovered body  a c t i v i t y o f the head  of that  o f t h e body  (Table  was VIII),  14 the  fate  the  foetuses  critical  of  C-corticosterone with  period  costerone  in  specific  enrichment  where  XIII,  examined i n during  the metabolism of  there  corti-  i s a dramatic  As  increase  15, i n d i c a t i n g  of the corticosterone foetal  reduction  the  changed d r a s t i c a l l y .  a c t i v i t y a t day  by t h e i s o t o p e . the  t h e head removed  i n the body  shown i n T a b l e  was  an  pool  T h i s i s assumed t o be due t o 14 of C-ll-dehydrocorticosterone.  53  TABLE X I I I .  CONTENT AND  OF C O R T I C O S T E R O N E  Gestational  Day  SPECIFIC ACTIVITY  RECOVERED  I N THE F O E T A L BODY  ng cpd.B/g ±SEM  tissue  14 C-dpm/ng ±SEM  13  (5)  812.9  ±157.1  0.71  ±0.12  15  (6)  564.0  ±66.3  8.44  ±1.44  cpd.B  54  6.  Histological studies  Corticosteroids manner a  i n various  synthetic  tural  are regulated  foetal tissues.  s t e r o i d which,  modifications,  lation  has been  reported  i s n o t bound  lated  more e x t e n s i v e l y  the  by  less metabolic natural  to  mouse f o e t u s e s .  basis  for this  placenta  were  of  i n t h e mouse.  examined  An  a t t e m p t was Foetal  u n d e r LM  size.of  lethal  made t o f i n d tissues  a  and  20 h r s . a f t e r t h e injected  T h e r e was  has been  reported  of f o e t a l adrenal  significant  high  no  with  evidence  changes.  synthesis  adrenals, in  Although  cortex  the development of the medulla  cholamine  No  corticosterone,  e f f e c t , d e x a m e t h a s o n e was  examined 4 and  Dexamethasone  late  under-  injected into pregnant  yg o f dexamethasone.  involution  accumu-  i n f o e t a l t i s s u e and  toxicity.  histological  regu-  Dexamethasone  transcortin,  17-day-pregnant mother had been 2 00  effect of  metabolic  a l t e r a t i o n than  hormone  little  (40).  serum  doses of c o r t i c o s t e r o n e mothers had  The  complex  because of i t s s t r u c -  escapes t h i s  which  went  i n a  (2). (Figure  possibly  the gland  but to and  stimu-  cate-  So  f o e t a l adrenals  were  8)  a n d TEM  9).  d i f f e r e n c e was  except  to cause  (Figure  observed  for a slight  itself.  i n treated reduction  FIGURE  8.  NORMAL M I C E  L I G H T M I C R O G R A P H S OF F O E T A L A D R E N A L GLANDS OF ( U P P E R ) AND THOSE FROM MOTHERS  P R E V I O U S L Y WITH 2 0 0 y g DEXAMETHASONE  T R E A T E D 2 0 HO  (LOWER) X 4 0 0  56  F I G U R E 9.  T R A N S M I S S I O N E L E C T R O N M I C R O G R A P H S OF F O E T A L  A D R E N A L GLANDS OF NORMAL M I C E MOTHERS  ( U P P E R ) AND THOSE  T R E A T E D 20 HOURS P R E V I O U S L Y WITH 200  METHASONE  (LOWER)  FROM  y g DEXA-  X12000  59  DISCUSSION  The  transport  costerone  revealed  metabolism the  time  be  foetus.  i t decreased  level  by  a t t r i b u t e d to  i n the  placenta  the  transcortin content  passage  into tissues.  vity  injected corticosterone  is  no  corticosterone  somewhat  change  On  i n the  of  III).  foetal pool;  of  content  of  foetal corticosterone  to  reported  analysis  The  corticosterone,  on  r o l e of  the  the  large  placenta  and  the  there  recovered very in  was  16  blood  the  (8).  This  specific  acti-  actually that  foetal 13  13  determined  at  the  suggests  days  the  reduces i t s  This  on  17,  can  occurs  foetus  by  to  This  i n the  e.g.,  13  and  between days  and  of  as  accumulation  foetal tissues  two  the  placenta,  extensively  placenta  more t h a n . 7 5 %  i n the  little  the  corticosterone  adrenal  and  and  by  there  17.  17  The  is similar  fluorometric  (35).  shown by  14,  day  hand,  corti-  w h e r e a s on  which  maternal  other  production  that  of  II).  specifically  the  (Table  (Table  increase  binds  increased  radioactive  increased,  half  protein  of  of  From g e s t a t i o n a l days  corticosterone  side,  probably  the  some i n t e r e s t i n g f e a t u r e s  i n the  maternal  foetal  across  of  a of  b a r r i e r was counts  (Table the  VI).  metabolized  to  from  From day  placenta.  11-dehydro m e t a b o l i t e i n the  recovered  12  the to  labelled corticosteroids  t i s s u e s , i n the  whereas  dramatically  foetus,  of the  There  was  corticosterone hormone  11-dehydrocorticosterone.  was From  60  these  experiments  placenta whereas  (Tables  c o n t r o l l e d the the  foetal  VI  and  flow  tissues  of  VIII),  i t appears  hormone  assumed  into  the  different  that  the  foetus,  metabolic  functions.  Whether foetal from  or  the  or  not  maternal present resulted  However,  the  minutes  in  maternal  from  mother  to  During  the  third  corticosterone  At  the  beginning was  on of  Then  80%  of  after  genation hormone  day  of was  a l l counts  recovered  that  actively  following  of  there  in  the  the  V).  foetal  as  high and  extensive  a  and  changed  direction  in  the  drastic by  day  17,  form.  foetus  can  metabolize pattern  that  of  metabolism  greatly day  12  (Table to  formation  foetal in  over  14, of  the  tissues. the  90%  Hence,  dehydro-  of  the  there  is  corticosterone  during  VII).  represented  change  active  definite  maternal  transfer  the  c o r t i c o s t e r o n e , which  was  of  one-quarter  i . e . , from  recovered  definitely  corticosterone.  pregnancy,  side  of  XIII).  the  pregnancy.  of  is  corticosterone  as  and  of  foetus  Removal  of  in  a  the  levels  trimester;  corticosterone  evidence  mester  of  and  III  foetal  in  determined  rapid  trimester  extensive  14,  was  (Tables  the  be  foetal  activity  the  11-dehydrometabolite around  IV  indicating  foetus  of  metabolism  (Table  injection  blood,  found  cannot  i n normal  specific  after  hormone  origin  data  adrenals  15  the  the  last  t r i -  61  Not actively, The or  only  can the foetus  but this  i n vitro  that  heads  the placenta  reducing bolism  by t h e p l a c e n t a ,  from  gestational  played  by t h e head  dehydrogenase day 15.  was  a constant  definitely  whereas  activity  I t appears  corticosteroids  corticosterone  i s compartmentalized  11-dehydrocorticosterone.  corticosterone,  by  metabolism  metabolism  the foetal  metabolize  t h e body  From  active  these  towards  quite  showed  role i n  days,  t h e meta-  the inactivation  exhibited a very decreased  VIII).  bodies  1 3 , 14 a n d 15  and v e r y  that within  are metabolized  the foetal  days  a t d a y 13; t h i s then  (Table  of  strong by  the foetus  differently  one-half itself,  i n different  tissues.  The centre  reduction of 11-dehydrocorticosterone  of interest  localize  and study  It  i s evident,  was  the major  effected foetal  of this  according site  which  the  lung  due  might  reductase  to the foetal  When  foetal  made t o  of the reductase IX, t h a t  target tissue 'activity  activity.  the foetal  The r e s i d u a l  to the  for corti-  the  i n the residual  liver  activity  mostly  has been  ( 3 6 ) . I n t h e same s y s t e m ,  c o r t i c o s t e r o n e was h i g h e r  were  be a t t r i b u t e d  are another  and s i n c e  of  to Table  of reduction.  costerone human  so e f f o r t s  the development  by t h e c a r c a s s  lungs  work,  became t h e  observed  i n  dehydrogenation  carcass,  probably  kidneys.  liver  on days  11-dehydrocorticosterone,  14 a n d 15 w a s  addition  incubated  o f t h e coenzyme  with  NADPH  62  increased (Table  only  slightly  conversion  X I I ) , i n d i c a t i n g t h a t the  limiting  f a c t o r to the  enzyme c o n c e n t r a t i o n the  the  corticosterone  coenzyme was  r e a c t i o n , but  was  to  responsible  rather  not  an  f o r the  likely  the  increase  in  conversion  to  a c t i v e hormone.  Since  i t has  b e e n shown t h a t t h e r e  i n c o r t i c o s t e r o i d metabolism i n the t r i m e s t e r of pregnancy  (Table  major s i t e of r e d u c t i o n ,  VII)  i t was  was  foetus and  a d e f i n i t e change during  t h a t the  difficult  third  l i v e r was  activity  of  injected corticosterone  slightly  f r o m day  13  t o 17  changed  f a t e of  the  only  i n whole f o e t a l t i s s u e s .  d e s i g n e d to study the  the  to e x p l a i n that  specific  e x p e r i m e n t was  the  An  injected  14 C-corticosterone i s prominent. increase due  i n the  T a b l e X I I I shows t h a t t h e r e was  in specific  t o an  increase  activity  pool  I t could  not  hormone i n t h e b o d y was  rise  be  15.  the  This  an  reduction 11-fold  could  hormone f r o m  not  be  the  a c t i v i t y would have  remained  a t t r i b u t e d t o a change i n f o e t a l either, since  i n the  the whole f o e t u s .  in specific  of  specific  s i z e of c o r t i c o s t e r o n e  that of  a t day  i n transport  maternal s i d e , s i n c e the constant.  h e a d l e s s body, where the  same o r d e r  the  of magnitude  I t i s postulated  a c t i v i t y was  due  to the  amount o f  that this  enzymatic  as sharp  reduction  by t h e f o e t a l l i v e r o f l a b e l l e d 11-dehydrocorticosterone w h i c h was i t s e l f t h e p r o d u c t o f t h e d e h y d r o g e n a t i o n o f t h e 14 injected  labelled corticosterone;  corticosterone  c 1  ehydrogenation >  i.e., injected l d  C-  c-ll-dehydrocorticosterone  63  reduction  >  14  C - c o r t i c o s t e r o n e , e n r i c h i n g c o n s i d e r a b l y the  f o e t a l p o o l of l a b e l l e d c o r t i c o s t e r o n e ,  The r e s u l t s from the i n c u b a t i o n i n v i t r o of the brain  foetal  (Figure 2) g i v e a d d i t i o n a l evidence of the compart-  m e n t a l i z a t i o n of c o r t i c o s t e r o i d metabolism i n the f o e t u s . Many u n i d e n t i f i e d m e t a b o l i t e s have been d e t e c t e d by AR TLC  of  from the head, which were not p r e s e n t on those from  body.  No attempt  has y e t been made to i d e n t i f y these  the  zones,  but here too t h e r e i s evidence of a s t r i k i n g change between days 14 and  15, w i t h a decrease  i n the f o r m a t i o n of the  unknown m e t a b o l i t e s as w e l l as i n the dehydrogenation  of  corticosterone.  The  specificity  f o r NADPH as coenzyme agrees w e l l w i t h  r e p o r t s i n the l i t e r a t u r e other t i s s u e s .  (37, 38)  The pH optimum was  found to be on the  acid  s i d e ; i . e . , between 5.5  and  v i t y was  7 (p<.02), the same pH a t which  observed  a t pH  Wong (39) a l s o observed  6.  f o r a s i m i l a r enzyme i n  A d i p i n the r e d u c t a s e  a d i p f o r the dehydrogenation  acti-  of  c o r t i c o s t e r o n e by a d u l t mouse l i v e r and p l a c e n t a .  T h i s work has p o i n t e d out the importance hydroxysteroid:NADP  oxidoreductase  of the  i n the f o e t a l  113-  liver  d u r i n g the t h i r d t r i m e s t e r of pregnancy i n the mouse.  The  f i r s t o u t s t a n d i n g f e a t u r e i s the c o m p a r t m e n t a l i z a t i o n  of the  oxidoreductase a c t i v i t y .  Not o n l y was  the f o e t u s capable of  64  m e t a b o l i z i n g c o r t i c o s t e r o i d s , b u t t h e r e was a d e f i n i t e p a t t e r n of metabolism i n d i f f e r e n t organs. metabolized  The p l a c e n t a  t h e s t e r o i d hormone and a l s o appeared t o assume  a r e g u l a t o r y c o n t r o l o v e r t h e f l o w o f s t e r o i d from t h e maternal  to the f o e t a l side.  In t h e human, t h e major s i t e o f d e h y d r o g e n a t i o n o f c o r t i c o s t e r o i d s i s t h e p l a c e n t a , where m a t e r n a l i s l a r g e l y converted  Cortisol  t o c o r t i s o n e , t h e predominant c o r t i -  c o s t e r o i d i n f o e t a l and c o r d b l o o d  (11, 16, 2 9 ) . I n t h e  mouse t h e head appears t o be t h e most a c t i v e s i t e  (Table  VIII).  In both, the l i v e r i s a s i t e of reduction of the metabolite t o t h e 11B-0H form o f t h e a c t i v e hormone (16, 18 and T a b l e I X ) . Burton  has found t h i s r e d u c t a s e  l i v e r as e a r l y as 20 weeks (16). appearance o f g l y c o g e n ance o f r e d u c t a s e  a c t i v i t y i n human f o e t a l I n f o e t a l mouse l i v e r t h e  d e p o s i t i o n c o i n c i d e d w i t h t h e appear-  activity  (13).  I n t h e human l u n g , a n o t h e r  " t a r g e t " organ o f c o r t i c o s t e r o i d s , r e d u c t a s e  a c t i v i t y has  a l s o been demonstrated ( 3 6 ) .  An a l t e r n a t e e x p l a n a t i o n can be o f f e r e d f o r t h e o b s e r v a t i o n s o f Murphy (10, 1 1 ) , who found c o n s i s t e n t l y i n c r e a s e d C o r t i s o l l e v e l s i n human f o e t a l a r t e r i a l b l o o d as compared w i t h u m b i l i c a l venous b l o o d e n t e r i n g t h e f o e t u s . b u t e d t h i s t o p r o d u c t i o n by t h e f o e t a l a d r e n a l . t h i s c o u l d as e a s i l y be due t o r e d u c t a s e  She a t t r i However,  activity i n foetal  l i v e r and l u n g , which reduces a p o r t i o n o f t h e abundant  65  cortisone of  i s present.  a c t i v e hormone  glands  but rather  steroids, of  which  with  metabolism.  could  the changes  be d e t e r m i n e d  by changes  each  Thus,  n o t by t h e f o e t a l  i n the metabolism  t i s s u e showing  i n t h e amount  i t s own  of maternal  distinct  pattern  BIBLIOGRAPHY  D I C Z F A L U Z Y , E. P r o c . 2nd I n t . C o n g r . H o r m o n a l I n t . C o n g r . S e r i e s 132, 82-95 (1967).  JOST,  A. a n d P. 4, 1 2 3 - 1 8 4  CHALLIS, and  Steroids,  PICON. Advances i n M e t a b o l i c D i s o r d e r s , (1970) .  J . R . C . , I . J . D A V I E S , K. B E N I R C H K E , A.G. H E N R I C K X , K . J . KYAN. E n d o c r i n o l o g y 9 5 ( 5 ) , 1300-1305 (1974)  DUPOUY, J . P . , H. 65, 347-352  GOFFIGNY, and (1975) .  S. MAGRE.  J.Endocrinol.  M I L K O V I C , K., J . P A U N O V I C , Z. K N I E W A L D , a n d S. E n d o c r . 93, 115-118 (1973).  MILKOVIC.  LIGGINS,  G.C.  J . E n d o c r i n o l . 45, 515-523  (1969)  LIGGINS,  G.C.  J . E n d o c r i n o l . 42,  (1968)  323-329  W E S T P H A L , U. "Steroid-Protein Interaction", o n E n d o c r i n o l o g y , V o l . 4, N.Y. (1971). G A L A , R.R. a n d U. (1967) .  WESTPHAL.  Acta  Endocr.  MURPHY, B . E . P .  Amer.J.Obst.Gynec.  115,  MURPHY, B . E . P .  Amer.J.Obst.Gynec.  124(5),  P O L O K Y , T.B.  Amer.J.Obst.Gynec.  117,  55,  47-61  521-525  (1973).  471-473  549-553  J O L I V E T , A., H. B L A N C H I E R , a n d J . P . G A U T R A Y . Q b s t . G y n e c . 119, 775-783 (1974). AREY, L.B. "Developmental Anatomy". P h i l a d e l p h i a (1965) .  Monographs  (1976)  (1973).  Amer.J,  S a u n d e r s C o . , 7 t h ed.  67  P A S Q U A L I N I , J . R . , N. W I Q V I S T , a n d E. D I C Z F A L U Z Y . Endoc. 60, 237-248 (1969).  Acta  BURTON, A . F . , D . J . M c C L E L L A N , M.R. DRUMMOND, T.G. MAH, M . J . THOMSON, W. WONG, a n d R.W. TURNELL. J.Clin. E n d o c r . M e t a b . 395, 950-954 ( 1 9 7 4 ) . MURPHY, B . E . P . (1973) .  J.Clin.Endocr.Metab.  B U R T O N , A . F . , R.M. G R E E N A L L , a n d R.W. Biochem. 48, 178-183 (1970).  36, 1037-103!  TURNELL.  Can.J.  G L U C K , L . , M.V. K U L O V I C H , R.C. B O R E R , a n d W.N. KEIDAL. Amer.J.Obst.Gynec. 1 2 1 ( 1 ) , 142-155 (1974). G L U C K , L . a n d M.V. K U L O V I C H . Symposium on R e s p i r a t o r y D i s o r d e r s i n t h e Newborn. Ped.Clin.N.Amer. 20(2), 367-379 (1973). MURPHY, B . E . P . (1974) . L I G G I N S , G.C. (1972).  DROST, M.  Amer.J.Obst.Gynec. 119(8),  a n d M.B.  a n d L.W.  NATHANIELSZ,  P.W.  HOWIE.  HOLM.  a n d M.  Pediatrics  J.Endocr.  ABEL.  H O L T , P.G. a n d I . T . O L I V E R . (1968) .  1112-1120  50(4),  40, 293-296  J.Endocr.  Biochem.J.  515-525  (1968).  57, 47-54  108,  339-341  A N D E R S O N , A . B . , K.M. L A W R E N C E , a n d A . C . T U R N B U L L , J.Obst.Gynec. B r . C w l t h . 76, 196-199 (1969). NWOSU, U.C., E . E . W A L L A C H , a n d R . J . BOLOGNESE, Obst.Gynec. 4 7 ( 2 ) , 137-142 (1976). MURPHY, B . E . P .  J . C l i n . E n d o c r . 27, 973-990  (1967).  (1973  68  H I L L M A N , D.A. a n d C . J . P . 243-248 (1965).  GIROUD.  J.Clin.Endocr. 25,  K R E B S , H.A. a n d L . V . E G G L E S T O N . B i o c h e m . J . 3 4 , 4 4 2 - 4 5 9 (1940) . OSER, L . , e d . Hawk's P h y s i o l o g i c a l N.Y. ( 1 9 6 5 ) , p p . 1 2 2 1 - 1 2 2 2 . K L I M A N , B. a n d R . E . P E T E R S O N . 1654 (1960) . S T A C H E N K O , J . , C. L A P L A N T E , Biochem. 4 2 , 1275-1291  Chemistry.  14th ed.,  J.Biol.Chem. 235,  and C.J.P. (1964).  GIROUD.  1639-  Can.J. >  K O B A Y A S H I , Y. a n d D.V. M A U D S L E Y . Biological Applications of 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 , N.Y., 1 9 7 4 , 2 2 - 2 5 . N G U Y E N , T . T . , D . J . R E K D A L , a n d A . F . BURTON. 18, 78-84 ( 1 9 7 1 ) . S M I T H , B . T . , J . S . TORDAY, a n d C . J . P . 308 ( 1 9 7 3 ) . Abstract.  K O E R N E R , D.R.  E n d o c r . 7 9 , 935-938  GROSSER, B . I . a n d E . L . B L I S S . (1966) .  WONG, W.T.Y.  GIROUD.  Biol.Neonate  Ped.Res.  7,  (1966)  S t e r o i d s 8 ( 6 ) , 915-92!  B . S c . T h e s i s , U.B.C.  (1970)  WONG, M.D., M . J . THOMSON, a n d A . F . BURTON. 12-17 (1976).  B i o l . N e o n a t e 28  

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