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The effects of arginine vasopressin and arginine vasotocin on the movement of water across the isolated… Pisani, Sheilnin B. 1986

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c. / THE EFFECTS OF ARGININE VASOPRESSIN AND ARGININE VASOTOCIN ON THE MOVEMENT OF WATER ACROSS THE ISOLATED AMNION AND SKIN OF THE FETAL GUINEA-PIG  by SHEILNIN B. PISANI B.Sc,  Simon F r a s e r  U n i v e r s i t y , B r i t i s h Columbia, 1980  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Department of Zoology)  Me accept t h i s t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA April  ©  1986  S h e i l n i n B. P i s a n i , 1986  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  of B r i t i s h Columbia, I agree t h a t  the L i b r a r y s h a l l make  it  and  f r e e l y a v a i l a b l e f o r reference  study.  I  further  agree t h a t p e r m i s s i o n 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 may  be  department o r by h i s o r her understood t h a t  granted by  representatives.  s h a l l not  be  It is  allowed without my  permission.  Department o f  ^oo-OfrV  The U n i v e r s i t y of B r i t i s h 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  STTW  my  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 f i n a n c i a l gain  Date  the head o f  APRIL ,  t^frk  Columbia  written  ii  ABSTRACT  T h i r t y - n i n e amniotic between  30  and  membranes from guinea-pig  68 days of g e s t a t i o n  were  set up in  flow  c o u l d be measured g r a v i m e t r i c a l l y .  and  vitro,  (0.44  in  osmotic g r a d i e n t s  maternal  side  conditions. transfer the  of  the  vasopressin  of  (AVP)  membrane;  this  f e t a l surface  slowed or reversed  of term).  this  response  epithelium.  complex and  low,  net  arginine  activity)  to  declined,  the  and  of term) showed only  10 membranes between 28 and  showed p a r a l l e l  was  of  time, the e f f e c t  microscopy of  AVP  a  gestation  The  changes in the epithelium  simple s t r u c t u r e e a r l y in to  vivo  u n t i l about 58 days of  weak response i n 13 experiments.  relatively  was  the  response  one  the amniotic  there  flow.  l o s t ; membranes over 64 days (0.94  of  f e t a l to  The  After this  days of g e s t a t i o n  water  reproduced in  Addition  was  Electron  net  Small h y d r o s t a t i c  l00mU/ml (vasopressor  with f e t a l age,  of term)  f e t a l to the maternal s i d e , in  preparations. at  which  were maintained from the  water from the  majority  (0.85  in  In the absence of hormone,  of  increased  apparatus  - 1.00  fetuses  to one  70  structure  changed from a  gestation,  when  the  that appeared to be more  p o s s i b l y more s p e c i a l i z e d  in f u n c t i o n by  about  50 days (0.75  of term).  There was  an apparent degeneration  of e p i t h e l i a l  c e l l s between 62 and  64 days, when the amnion  ceased a  to respond to AVP.  membrane  intercellular  at  38 spaces  days to  E l e c t r o n microscopic AVP  studies  revealed  that  caused  dilate.  Morphometric  on the  analysis  showed that the dimensions of the spaces i n the AVP t r e a t e d e p i t h e l i u m were s i g n i f i c a n t l y g r e a t e r preparation  of  76  guinea-pig  amniotic  fluid  fetuses  its  roughly  maximal  structure fluid  then  showed  that  declined.  to  AVP,  and  appeared t o be compatible  the  The  peak  with  the  time i t s  with an a c t i v e r o l e i n  A f t e r approximately  56 -  58  days,  both  f l u i d volume and the response of the amnion t o AVP  declined.  These  physiological amniotic  control  with the time at which the amnion showed  response  transport.  amniotic  a  i n c r e a s e d d u r i n g the course of  g e s t a t i o n , reached a peak, and coincided  in  (p<0.001).  S t u d i e s on volume  than  results  role  of  AVP  sac i n the f i r s t  situation  in  most  are in  80%  species,  consistent  supplying  fluid  of • g e s t a t i o n . the  with  t o the  Unlike  amniotic  a  the  fluid  volume  just  before  i n c r e a s e d again, and reached i t s maximum value delivery. Skin from 35 mid-term f e t a l guinea-pigs  (0.49 -  0.70  of  term) was s e t up i n the same g r a v i m e t r i c apparatus used  in  the  amnion  gradients  in  experiments. hydrostatic  or  However, osmotic  absence of hormones, there was l i t t l e water  in  either  direction.  arginine vasopressin 5  -  There  was  were  pressure.  no  In the  or no net t r a n s f e r of  Arginine vasotocin  (AVT) or  (AVP) added to the s e r o s a l s u r f a c e  lOOmU/ml (vasopressor  of water towards the  there  at  a c t i v i t y ) produced a net uptake  serosal  side  (towards  the  fetus).  a l i n e a r r e l a t i o n s h i p between the log.dose, and  iv  the AVT  rate was  of  u p t a k e of  more t h a n  3.9mU/ml;  AVP,  t w i c e as  skin  before  compatible  with  an  may  periderm  be  the  It  and  partially  of  t o AVT  fetal  skin  potent  response  of  fetal  (thresholds:  fine  structure appeared  of  the  to  be  transport.  The  the  amniotic  epithelium;  it  neurohypophysial  the  gestational  to  AVP  The  AVT  AVT,  in f l u i d  of  the  i n the fetal  more r e a d i l y t h a n that  However,  role  f o r changes  gestation.  AVP  keratinization  resembled  that  as  The  active  action  reponsible  during  responds the  layer  i s concluded  structure  fluid  site  f o r both p e p t i d e s .  10.4mU/ml).  guinea-pig  outer  water,  finds  changes  in  the  amnion  may  be  volume of  skin,  t o AVP.  hormones.  like  amniotic frog  Perhaps  i t s true  skin,,  i t is  on  physiological  role.  Supervisor  V  TABLE OF CONTENTS  L I S T OF TABLES L I S T OF FIGURES ACKNOWLEDGEMENTS  v i i viii X  GENERAL INTRODUCTION The  1  Fetal Fluids  Structures  1  Involved  in Intra-Uterine Fluid  Exchange  ..  . 2  The  F e t a l Kidney  2  The  Fetal Urinary  The  F e t a l Lung  5  The  Fetal Gastro-Intestinal Tract  7  The  Amniotic  9  The  F e t a l Skin  11  The  U m b i l i c a l Cord  13  Bladder  Membrane  The  Amniotic  Fluid  The  O r i g i n o f I n t r a - U t e r i n e Water  Hormonal C o n t r o l  4  14  of I n t r a - U t e r i n e F l u i d s  STATEMENT OF THE PROBLEM  18 19 21  GENERAL METHODS  24  1 . N e t Water Flow E x p e r i m e n t s  24  (a)  The I s o l a t e d Amnion P r e p a r a t i o n  24  (b)  The I s o l a t e d S k i n  27  Preparation  2.  Salines  28  3.  Hormone S o l u t i o n s  29  4. E l e c t r o n M i c r o s c o p y  o f t h e Amnion  30  5.  of the F e t a l Skin  31  E l e c t r o n Microscopy  vi  SECTION I  32  THE EFFECTS OF ARGININE VASOPRESSIN ON THE BULK FLOW OF WATER ACROSS THE ISOLATED AMNION OF THE FETAL GUINEA-PIG AT DIFFERENT STAGES OF GESTATION .. 32 INTRODUCTION  32  RESULTS  33  1 . The E f f e c t s of AVP on the Amnion  33  2. The Fine Epithelium E a r l y Amniotic Mature Amniotic  Structure  the  Amniotic 38  Epithelium Epithelium  Near-Term Amniotic 3. F i n e After  of  Structure Incubation  (28, 30, 35, 38 days)  (50, 58, 62 days) ... 43  Epithelium  (64, 68, 70 days)  of the Amniotic With AVP  4. The Volume of Amniotic  40  46  Epithelium 50  Fluid  52  DISCUSSION  56  SECTION II  65  THE EFFECTS OF ARGININE VASOTOCIN AND ARGININE VASOPRESSIN ON WATER TRANSPORT ACROSS THE SKIN OF THE FETAL GUINEA-PIG  65  INTRODUCTION  65  RESULTS  67  1. The I s o l a t e d  F e t a l Skin  2. The E f f e c t s of AVT and AVP Transport Across F e t a l Skin  67 on  Net  Water 67  3. F i n e S t r u c t u r e of the F e t a l Epidermis  74  DISCUSSION  81  GENERAL DISCUSSION  88  REFERENCES  100  vi i  LIST OF TABLES  Table I  II  Page The Response of the Amnion Course of G e s t a t i o n  The Volume of Amniotic  to AVP During the 36  F l u i d During the  Course of G e s t a t i o n  55  III  The Response of the F e t a l Skin To AVT  70  IV  The Response of the F e t a l Skin to AVP  71  vi i i  LIST OF FIGURES Figure  1.  2.  3.  4.  5.  6,7.  8,9.  10.  11.  Page  The Gravimetric A p p a r a t u s Used f o r t h e in vitro S t u d y of Net Water Movement Through t h e I s o l a t e d Amnion of t h e G u i n e a - P i g  The E f f e c t s of AVP on Net Water Movement A c r o s s the I s o l a t e d Amnion o f t h e G u i n e a - P i g  25  ..  35  The Changes i n t h e R e s p o n s e o f the Amnion t o AVP T h r o u g h t h e C o u r s e o f G e s t a t i o n  39  The Fine Structure E p i t h e l i u m Through the  41  of the Amniotic C o u r s e of G e s t a t i o n ....  E l e c t r o n M i c r o g r a p h s of Epithelium  the  The Fine Epithelium  the Mature  Structure  Amniotic 42  Amniotic 44,45  Electron Micrographs Amniotic E p i t h e l i u m  Structure Days (0.91 term)  of  Early  of of  of  Near-term 47,48  the Amniotic term) and 64  Effect of AVP on t h e Amniotic Epithelium  the  E p i t h e l i u m at Days (0.94  62 of 51  Fine  Structure  of  the 53  ix  LIST OF FIGURES  (continued)  Figure 12.  13.  14.  15.  Page The Volume of Amniotic Stages of G e s t a t i o n  Fluid  at  Various 54  G e s t a t i o n a l Changes i n the S t r u c t u r e of the Amniotic E p i t h e l i u m , the Response to AVP, and the Volume of Amniotic F l u i d i n the Guinea-Pig  64  The E f f e c t s of D i f f e r e n t Doses of AVT and AVP on the Net Movement of Water Through the Skin of the F e t a l Guinea-Pig (at 0.49 0.70 of term)  68  Log Dose-Response Curves f o r the E f f e c t s of AVT and AVP on Net Water Movement Through the F e t a l Skin (at 0.49 - 0.70 of term)  73  16,17. Fine S t r u c t u r e of the F e t a l Epidermis  at 35  Days  18.  The F e t a l Periderm a t 35 Days  19.  Fine S t r u c t u r e of the F e t a l Epidermis Days  75,76  77  at 52 79  X  ACKNOWLEDGEMENT S I thank t h e N a t u r a l S c i e n c e s a n d E n g i n e e r i n g Council  o f Canada  postgraduate I for  am i n d e b t e d  to  Mr.  electron and  J.  microscope.  Veto  financial  forhelpful  advice,  and  f o r h i s invaluable help with the  I thank Mr. Tsun Takman  of the guinea-pigs  Bruce M c G i l l i v r a y  generous  Perks,  t o D r . Dan W. R u r a k , D r . J o h n  a n d D r . H.D. F i s h e r  Laszlo  breeding  grateful  and  of a  1981-82.  t o my s u p e r v i s o r , D r . A n t h o n y M.  encouragement,  I am a l s o  E. P h i l l i p s ,  a s s i s t a n c e i n t h e form  s c h o l a r s h i p f o r the year  guidance,  support.  for financial  Research  used  in this  f o r typing the manuscript.  f o r the study,  care  a n d Mr.  1  GENERAL INTRODUCTION  The F e t a l During  fetal  Fluids  development,  a l a r g e q u a n t i t y of water  accumulates w i t h i n the uterus and fetal,  placental,  (Kerpel-Fronius, these  and  1970).  compartments  i s distributed  In  amniotic  the  normally  human  compartments  fetus  contain  between  near  an average of about  2800 ml, 400 ml, and 800 ml of water, r e s p e c t i v e l y 1974).  An  additional  fluid  should be i n c l u d e d i n some pregnancy,  water  space,  the  species.  accumulation  Over  from  the  (Kerpel-Fronius, large the  amount means  1970).  of  by  mother  (Seeds,  a l l a n t o i c sac, the  whole  of  i n the human i s concurrent  with a net t r a n s f e r of 380 mEq of sodium potassium  term,  to  the  and  173  mEq  intra-uterine  The mechanisms  by  which  of  cavity such  a  f l u i d accumulates w i t h i n the uterus, and  which  i t s volume  and  composition  are  c o n t r o l l e d , a r e not yet f u l l y understood. I s o t o p i c s t u d i e s have r e v e a l e d that there i s a dynamic exchange  and  electrolytes  amniotic f l u i d and mother  (Vosburgh et  et  al. ,  of  water  1959;  Hutchinson  et  between al. ,  al. ,  the f e t u s ,  1948;  1955;  Friedman  1959).  The  s t r u c t u r e s i n v o l v e d i n t h i s exchange, besides the p l a c e n t a , are the  the  umbilical  c o r d , e x t r a - p l a c e n t a l membranes (e.g.,  amnion), and f e t a l  organs such as the kidney,  lung, g a s t r o - i n t e s t i n a l t r a c t and s k i n . a  role  in  regulating  the  volume  bladder,  A l l appear to p l a y  and  composition  of  2  intra-uterine fluids. amnion  and  of  In t h i s  the  w i l l be c o n s i d e r e d .  fetal  thesis,  the  role  of the  skin in r e g u l a t i n g f e t a l  Before  this  i s done,  a  fluids  review  of  i n t r a - u t e r i n e water metabolism i s i n order.  S t r u c t u r e s Involved  The  i n I n t r a - U t e r i n e F l u i d Exchange  F e t a l Kidney The  fetal  kidneys begin  life.  to  function  relatively  early  Cameron and Chambers (1938) demonstrated that  the r e n a l tubule c e l l s of the three month o l d human are  capable  of  transporting  phenol  medium i n t o the t u b u l a r lumen in found  in  in  fetus  red from the o u t s i d e  vitro.  Urine  has  been  the human f e t a l bladder as e a r l y as eleven weeks  of*gestation  (Abramovich,  weeks),  human f e t u s v o i d s approximately  the  u r i n e per day (Abramovich,  1968).  1973).  By  mid-gestation  (20  7 t o 14 ml of  In the f e t a l  lamb  (which  has a g e s t a t i o n p e r i o d of 145 days), glomerular  and t u b u l a r  f u n c t i o n are present  and  1963).  However,  by  days  (Alexander  Nixon,  the immature kidney does not f u n c t i o n as  e f f i c i e n t l y as the a d u l t In  60  kidney.  a l l mammals, the glomerular  i s lower  i n the f e t u s than  Barnes,  1976).  This  i n the  i s partly  filtration  adult a  r a t e (GFR)  (Kleinman,  result  1975;  of the lower  a r t e r i a l blood pressure of the f e t u s , but f a c t o r s such as a low  renal  glomerular  blood  flow  and  c e l l s may a l s o be  structural  important  immaturity  (Kleinman,  of  1975).  3  Likewise,  tubular  gestation, et  al.  is  transport,  relatively  for  sodium in the proximal  than  in more mature animals.  of  filtrate  at  from  early  i n the f e t u s .  intrinsic  transport  Alexander  and  60  percent to  90  of  the  percent c l o s e to term.  sheep  lower  about m i d - g e s t a t i o n , 28 percent Due ability  of that  than  in  i s only  filtered. the f e t a l  excrete excess  water and  sodium.  t u b u l e s to lower the r e a b s o r p t i o n of f i l t e r e d kidney  to respond to dehydration  to concentrate As  fetal  a  by  of  the  inability  i s also limited  lower  (Kleinman,  et  al.  , 1977;  of  Abramovich el  urine  and  its  1975).  hypotonic  al.  per  day  (van  , 1979).  The  fetal  sheep v o i d s amounts of s i m i l a r magnitude (200 day),  in  C l o s e to term, the human f e t u s  v o i d s an average of about 500 ml Otterlo  sodium  t u b u l a r r e a b s o r p t i o n of  water, the normal f e t u s e x c r e t e s l a r g e amounts of u r i n e , d e s p i t e the low GFR.  of  as i t i s not as w e l l able  urine as the a d u l t kidney  result  the  in i t s  E x c r e t i o n of  distal  The  hampered  is limited  sodium i s  ability  further  kidney  excess  (Kleinman, 1975).  young  sodium a b s o r p t i o n ; at  the amount of water reabsorbed  to the low GFR, to  (1961)  glomerular  that water r e a b s o r p t i o n  even  capacity  Nixon  These i n v e s t i g a t o r s found is  Capek  sheep, the t u b u l a r r e a b s o r p t i o n  mid-gestation  fetuses  in  tubule of newborn r a t s i s lower  that i n f e t a l  sodium i n c r e a s e s  evident  inefficient  (1968) showed that the  have observed  though  - 500  ml  per  these high r a t e s of u r i n e flow are in excess  a d u l t r e s t i n g v a l u e s , when expressed  on the b a s i s  of  of  flow  4  per  unit  body  osmolality 144  weight  of f e t a l  urine  mEq/litre and  (see  chloride  reabsorption fetal  thereafter amniotic  of the ions with  maturation  i t passes  sac (Alexander  1971).  In s p e c i e s  human,  urine  1965).  However, u r i n e  fluid,  as w i l l  In water  80  electrolyte  days  increasing  an a l l a n t o i c  66  more  41  sodium  effective  of r e n a l  of  to  in urine  function.  sac v i a  the  gestation,  and  amounts  into  1961; M e l l o r sac,  into the amniotic  i s not the only  source  the  and S l a t e r ,  such  as the  cavity  (Seeds,  of  amniotic  later. is  the  metabolism.  the  relatively  immature  by  placental  regulation,  fetal  90  and Nixon,  passes only  be seen  -  in  without  a  from  into the a l l a n t o i c  the a d u l t , the kidney and  The d r o p  at  about  from  suggests  about  from  from 68 t o 44 m E q / l i t r e  1979).  flows  The  the  concentrations  until  1958).  term;  of c h l o r i d e drops  Abramovich,  sheep, u r i n e  urachus  kidney as  prime  regulator  of  In t h e f e t u s , however,  i s assisted in this  function  w e l l a s r e g u l a t i o n by o t h e r  structures.  F e t a l Urinary It  Boylan  Bladder  h a s been o b s e r v e d  produced  the  ,  i n t h e human d e c r e a s e s  o f sodium d r o p s  m i d - t e r m t o t e r m and t h a t  The  al.  mOsm/kg a t 17 - 21 weeks t o 137 mOsm/kg  concentration  In  et  (Alexander  by t h e k i d n e y  et  fetal  al.  the  composition  c a n be a l t e r e d i n t h e f e t a l  (1958) f o u n d guinea-pig  that  that  has  a  the urine higher  of  urine  bladder.  i n the bladder of  osmolarity  than  that  5  formed water  by  the  into  the f e t a l et  Alexander fetal  al.  sheep;  close  to  samples  kidney.  (1958)  an h o u r , were  of  same. fetal  renal  Stanier  pigs  i n bladder  urine  suggesting  play  from  fetuses  but subsequent  i n the bladder  f o r up t o  o f t h e b l a d d e r may n o t a l w a y s  lower  the bladder  that  in  some sodium  the  transporting  to that  concentration  than  from t h e b l a d d e r .  demonstrated actively  plasma  i s always markedly hypoosmotic much  circulation  taken  (1971) o b s e r v e d t h a t  A  pelvis,  lumen.  t h e same phenomenon i n  to  w h i c h had s t a y e d  bladder  hypertonic.  pelvis.  present  the  samples  hypotonic  However, t h e e f f e c t s the  from  observed  bladder  were  of u r i n e ,  suggests a r e a b s o r p t i o n of  circulation  initial  term  This  urine  et  France  fetal  of the  the  into  al.  sheep b l a d d e r  urine  o f sodium i s  from  reabsorption  be  renal  the f e t a l  (1972)  have  i s capable of  sodium.  These  observations  suggest that  a role  i n osmoregulation.  the f e t a l  bladder  may  The F e t a l Lung The exchange,  lungs  of  this  and a r e i n s t e a d intra-uterine  the  function  fetus being  thought t o p l a y water  and  are  not  a role  ions.  i n the  From  formed,  is  secreted  t h e l u n g s and w h i c h  for  their  the  they a r e f i l l e d  normal development  in  gas  p e r f o r m e d by t h e p l a c e n t a ,  airways are f i r s t in  involved  (Olver  et  with  exchange  of  t i m e when t h e fluid,  which  i s probably important al.  , 1973; A l c o r n  et  6  al. of  , 1977).  The  gestation  l u n g s of  contain  body w e i g h t , and ml/kg  per  1974;  Scarpelli  would  amount  al.  al.  to  per  hour  , 1975).  about  i n the  (1985) have o b s e r v e d the  fetal  The  100  found  hydrogen,  lower  and  in  fetal  and  fetal  the  liquid  , 1971; a ml  fluid  Olver 4  kg  per  third  per  kg  of  1  to  3  and  Strang,  fetus,  day.  this et  Setnikar 1  to  P e r k s and  lung  fluid  they  liquid,  7  Cassin  secretion  by  investigated  ion t r a n s p o r t  concluded  generated  by  the  alveolar  liquid,  transport  of  the  that  C l ~ ions  and  of  the  of  lung but  Olver  secretion  the  the  of ions  to  these  effects  differences  the  and  concentrations  plasma  across  of  bicarbonate  simply  that  (1969)  higher,  According  not to  al.  from  concentrations  in ionic  active transport across  the  ions  lung.  the  Adamson et  plasma.  suggest  elaborated  markedly  c h l o r i d e ions are  due  ultrafiltrate  differs  plasma.  from p l a s m a a r e  concentrations;  and  al.  guinea-pig.  differences  Gibbs-Donnan e q u i l i b r i u m  an  fluid  a r a t e of about  r a t e s of  fetal  alveolar  in  investigators,  merely  of  at  For  phosphates, calcium  than  alveolar  ml  last  s e c r e t i o n r a t e s of a b o u t  similar  potassium  proteins,  t o 40  - 300  (or a l v e o l a r )  fluid  that  lamb i n t h e  goat.  lung  amniotic  30  el  (Normand et  fetal  fluid  (1959) have o b s e r v e d  ml/kg  in  about  secrete  hour  the  and  in  of  a  protein  liquid is  of  i s not  actively  Strang  (1974)  alveolar  epithelium  of  lung  liquid  is  from  plasma  to  CI  alveolar  i s i n e x c e s s of H C 0  epithelium; 3  transport  this in  the  7  opposite d i r e c t i o n . electrical  gradient  Na  moves p a s s i v e l y  set  up  by  i n response to the  C l ~ movement,  and  the  r e s u l t a n t osmotic f o r c e of NaCl causes the net t r a n s f e r water i n t o the a l v e o l a r Fluid and  space.  s e c r e t e d i n the lungs leaves  v i a the  trachea,  most of i t e n t e r s the amniotic c a v i t y , though  some may  en route (Adams et  be swallowed 1984).  Lung  fluid  may  volume of amniotic f l u i d Cassin  and  Perks  al. , 1963; Harding et  contribute in  (1982)  some  significantly  species.  along  with  found that  the  p l a c e n t a and kidney.  i n f u s i o n of 0.9% NaCl  f e t a l goat r e s u l t e d  al. , to the  According  to  the lungs of the f e t u s may a l s o  serve an important r o l e i n c o n t r o l l i n g  the  of  into  fetal  body  These the  fluids,  investigators  circulation  of  i n an i n c r e a s e i n the s e c r e t i o n of +  lung f l u i d ,  i n c l u d i n g Na , and CI .  the  kidney to e x c r e t e a sodium or water load may be  fetal  The l i m i t e d a b i l i t y of  compensated by e x c r e t i o n v i a the lungs.  The F e t a l G a s t r o - I n t e s t i n a l There  cells, human  i s abundant evidence in  swallows  Tract  utero.  to  show  that  the  E a r l y i n v e s t i g a t o r s found  epithelial  lanugo h a i r s , and v e r n i x caseosa i n the gut fetus,  substances Abramovich,  to  and the  1979;  attributed swallowing 1981).  the of  Since  presence  amniotic then,  fetus  of  of  fluid  radiography  the these (see and  i s o t o p i c t r a c e r s t u d i e s have confirmed these  observations.  From  movement  experiments  which  followed  the  of  8  erythrocytes  labelled  amniotic f l u i d  i n t o the g a s t r o - i n t e s t i n a l t r a c t ,  (1966)  estimated  with  that  radioactive  chromium  Pritchard  the 16 week o l d normal human f e t u s  swallows about 7 ml of amniotic f l u i d per day (2 and  the  28  ml/kg/hr). magnitude  week  o l d fetus  Abramovich  about  120  (1970) obtained  ml  ml/kg/hr)  per  values  of  day (4 similar  as those observed by P r i t c h a r d f o r mid-term  f e t u s e s , using r a d i o a c t i v e c o l l o i d a l g o l d . amount  At  human  term,  the  of amniotic f l u i d swallowed by a normal human f e t u s  averages about 200 t o 500 Abramovich  et  al . ,  ml  per  1979).  day  (Pritchard, et  Harding  al.  1965;  (1984) have  found s i m i l a r r a t e s of swallowing i n the f e t a l lamb late  from  during  gestation. According to Abramovich  human  fetus  (1970), gut p e r i s t a l s i s  i s established  i n the  by m i d - g e s t a t i o n ; s t u d i e s ' o n  f e t a l swallowing at t h i s stage have shown that about 78 93  percent  of  the  radioactive  tracer  amniotic f l u i d accumulates i n  the  that  actively  amniotic  fluid  is  lower  g a s t r o - i n t e s t i n a l t r a c t , with water being its  passage.  Wright  and  a b s o r p t i o n of amniotic f l u i d Their  experiments  swallowed amniotic circulation.  These  Nixon  into  is  that  passed  down  absorbed  the  during  (1961) have s t u d i e d the  perhaps  reabsorbed  investigators  the  gut, suggesting  i n the gut of the f e t a l  indicate fluid  given  to  into  lamb.  a l l of the the  fetal  found that sodium  ions  from swallowed amniotic f l u i d are a c t i v e l y t r a n s p o r t e d from the  gut lumen towards the f e t a l e x t r a c e l l u l a r  fluid  (i.e.,  9  from mucosa to serosa) and suggested that t h i s t r a n s p o r t of sodium  probably  reabsorption.  accounts An  for  H  +  The  and C l  Wright,  i n the f e t a l  actively  1974).  from the  The  serosa  active  fetal  extracellular  f e t u s e s of sheep (Wright and rabbit  and  guinea-pig  to  rabbit  the  transport  -  to  the  water  (Wright,  f e t a l gut, l i k e that of the a d u l t , t r a n s p o r t s  accounts f o r t h e negative p o t e n t i a l respect  of  a c t i v e r e a b s o r p t i o n of sodium ions from  the gut has a l s o been observed 1974).  much  of  mucosa  (see  ions probably  i n the gut lumen, fluid,  Nixon,  observed  1961),  (see M e l l o r ,  with i n the  goat,  human,  1969; 1970; M e l l o r et  al. , 1969).  The Amniotic Membrane The amnion i s the  innermost  of  the  extra-embryonic  membranes, coming i n d i r e c t c o n t a c t with the amniotic (Bourne,  1962).  considerably Morris, appears  The  between  method  of  species  amnion (Biggers,  1975) but the s t r u c t u r e of essentially  the  0.50  definitive  mm  I t has no  thick,  blood  amnion  The t y p i c a l  of a s i n g l e l a y e r of e p i t h e l i u m o v e r l y i n g  l a y e r s of c o n n e c t i v e t i s s u e .  varies  1972: Steven and  the same i n a l l mammals.  mammalian amnion i s from 0.02 to composed  formation  fluid  and i s several  vessels  in  most s p e c i e s , and i s not i n n e r v a t e d (Wynn, 1974). There  i s much evidence  to  suggest  that  the  amnion  serves as a pathway f o r f l u i d exchange between the amniotic f l u i d and the maternal  circulation.  Paul  et  al.  (1956)  10  found  that i n f e t a l r a b b i t s at 0.75 of term,  half  of  the  amniotic and - al.  water  exchange  f l u i d occurs a c r o s s  between the  approximately  the mother and the  fetal-placental  h a l f occurs a c r o s s the f e t a l membranes. (1959) observed  gestation  in  that throughout  the  the  complex  Hutchinson  entire  period  et of  human, there e x i s t s a d i r e c t pathway of  exchange between the amniotic  fluid  and  mother,  via  the  chorioamnion. The  cells  of  the  amniotic  epithelium  d e s c r i b e d as being s e c r e t o r y by T a u s s i g and H u l l et  al.  (1958),  the human amnion,  Armstrong  p i n o c y t o s i s i n l i v i n g c e l l s of  suggesting  fluid.  been  (1927) and Danforth  based on h i s t o l o g i c a l evidence.  (1968) have observed  transporting  have  that  Electron  they  are  capable  of  m i c r o s c o p i c s t u d i e s of the  amniotic e p i t h e l i u m have r e v e a l e d that  its  ultrastructure  i s s i m i l a r to that of other t r a n s p o r t i n g e p i t h e l i a , such as those which l i n e the mammalian g a l l b l a d d e r , the and  the  amphibian  urinary  bladder  1978; Hoang-Ngoc Minh et  Herendael,  al.  intestine,  (Hoyes,  1968a;  , 1980;  1981;  van King  1978; 1980). The amnion i s f r e e l y permeable al.  (1969)  obtained  a  permeability  d i f f u s i o n of t r i t i a t e d water a c r o s s about 3X10~ of 0.5X10-" obtained  a  cm/sec, and Page et to  4X10-"  values  guinea-pig amnion.  of  to  cm/sec.  the  same  Lloyd  coefficient the  al.  water.  human  f o r the  amnion  (1974) observed Holt  and  Perks  et  of  values (1975)  order of magnitude f o r the  The p e r m e a b i l i t y  coefficient  for  the  11  net  transfer  o f water a c r o s s  about a hundred water by  times greater  by s i m p l e d i f f u s i o n ,  a  non-diffusional  t h e membrane  (Seeds,  According placental be  a  to  disc  site  Seeds  the a m n i o t i c  paucity  of b l o o d  intercellular  is  to  also  cavity  in  of  fluid  term)  the  in  extends  with  the  due  ions  to  the  in proximity  that  the  amniotic  and  to  the  arrangement maternal  uterine  decidua  been  likened  to  fluid,  chorionic uterine  facilitate  plasma;  the  the  structure  cytotrophoblast  (which b o t h p e r s i s t a  et  epithelium,  the  can  to  complex  parietal  of t h e p r i m i t i v e  parietal  has  to  However, Hoang-Ngoc M i n h  chorion  formed by t h e a s s o c i a t i o n and  pass  w a t e r and  t h e f l o w of l a r g e amounts of  d e c i d u a , where t h e v a s c u l a r exchange  can  away f r o m t h e  mother,,  observed  the  and  the  of  i n maternal tissue  have  facilitate  cytotrophoblast,  isotopic  amnion) i s u n l i k e l y  exchange  and  system p r e s e n t  found  water  t h e amnion  the r e f l e c t e d  vessels  1981)  that  of  through porous channels i n  (1980)  l a e v e and amnion.  (1980;  thought  flow  significant  between  al.  than the t r a n s f e r  suggesting  bulk  by o s m o s i s i s  1967).  (i.e.,  for  the c h o r i o n  t h e human amnion  placenta  by  till these  investigators.  The F e t a l  Skin  Prior skin  is  t o the development permeable  significant  fluid  to  o f t h e s t r a t u m corneum,  water,  exchange  and  between  may  be  the f e t a l  a and  site  fetal of  amniotic  12  compartments. of  tritiated  the  human  between  Permeability water a c r o s s  fetus  Seeds,  prior  The  t o m i d - t e r m have been  1970).  uppermost the  Unkeratinized ( L i n d et  al . ,  l a y e r of  periderm,  periderm  has  transporting 1968b). early  and  tubular These  epithelia  skin  epithelium  will  varying  the  After  permeability can perform  and  fetus,  P a r m l e y and Seeds  keratinization  rate  et of  al.  in  common  skin  to  that  to  the  renal  young  corneum  there  diminishes.  fetuses  sodium  diffusion  volume by  s l o w s down.  in fetal  In t h e human decrease  in  skin  with  i t s skin.  with  a  well  at a l l .  thickens,  Hoyes  in  which e a r l y  was no d i f f u s i o n  (1972) f o u n d t h a t a s f e t a l  fetus,  tubules  was d e m o n s t r a b l e , a n d i n s k i n  stratum  renal  of v a s o p r e s s i n .  (1970) f o u n d a l a r g e in  other  1965; H o y e s ,  to keratinize, i t s role  water  i n the  with  resemblance  the  is  (1972), the c e l l s of  influence  i o n exchange p r o b a b l y  permeability  developed  of  freely  epithelium,  t h e same f u n c t i o n w i t h  the skin begins  water  Lind  the  (Parmley  studies  the amniotic  striking  under  values  al.so  eventually control i t s extracellular  vasopressin,  skin  like  t o L i n d and Hytten a  to  fetal  ultrastructural  i n v e s t i g a t o r s have s u g g e s t e d  which  is  of  f o u n d t o be  laeve  ( B r e a t h n a c h and W y l l i e ,  bear  skin  1972).  characteristics  According  fetal  skin  unkeratinized  human f e t u s have shown t h a t , the  unkeratinized  t h e human amnion and c h o r i o n  permeable t o sodium  called  isolated  f o r the d i f f u s i o n  0.45X10"" and 0.86X10"" cm/sec, s i m i l a r  measured a c r o s s and  coefficients  the  (1968b) h a s  13  observed  that  between  human, t h e c e l l s signs shed  of and  layers  a definitive  The U m b i l i c a l The  vessels  the  layer fetal  fetus  to  important  site  amniotic  fluid  to  (i.e., to  when t h e  periderm does region  the  t o be  functional  jelly)  c e n t e r , and  formed  from  is the  of g e l a t i n o u s  which which  supports  in  important  the  at the  to the  -  1968b).  into  like  20  becomes with  keratinization  which  The the  (Hoyes,  and  in  is  from  resembles  human,  a  time  poorly  the  fetal  however,  fetal  epidermis except  thus  can  continue  an  begins  the  cord epithelium,  1969).  of  the  It i s roughly at t h i s  one  f e t u s , and  skin  ion-transfer.  materials  weeks  an  epithelial  1969),  cord  differentiates  1969).  term  of  the  the periderm  Hoyes,  the  s t a g e when s k i n  epithelium  keratinize  until  (1973),  17  The  i n water and  exchange  between Hoyes,  1965;  the  i s c o v e r e d by  H i e r s c h , 1969).  Leeson,  Abramovich  (see Hoyes,  close  corneum,  (Wharton's  structure  not  show  weeks, t h e p e r i d e r m  t h e a m n i o t i c e p i t h e l i u m and  cord  developed  26  periderm  i s composed m a i n l y  (Thomsen and  thought  according  stratum  ( L e e s o n and  According  occur  by  the  i s c o n t i n u o u s w i t h t h e amnion and  resembles  likewise  and  cord  in  that  skin  beneath  i n the  Cord  tissue  epithelium  weeks of g e s t a t i o n  i s evident.  umbilical  connective  of  i n the l a y e r s  keratinization  beneath,  blood  17 t o 20  .to  in a be  14  According unexpectedly Wharton's  to  after  human amniotic c a v i t y ,  cord.  between  Plentl in  cord  al.  (1959),  the  l a r g e c o n c e n t r a t i o n of i s o t o p i c water found i n  jelly  transfer  et  Hutchinson  vitro  injection  of  the t r a c e r  i n d i c a t e s that  amniotic  fluid  much  of  i n t o the  the  water  and f e t u s i s a c r o s s the  (1961) p e r f u s e d the i s o l a t e d human  umbilical  and based on the c o n c e n t r a t i o n s of t r i t i a t e d  water i n Wharton's j e l l y , estimated that between 40 and ml  of  water  (1973) with in  Abramovich between  3  across  vivo  the  cord  experiments  and 59 ml of water i s absorbed  hour a f t e r  mid-gestation.  diffusional Although  i s exchanged  In  50  per  hour.  suggested  that  v i a the cord per  a l l these  studies,  only  f l u x e s of water a c r o s s the cord were measured.  there appears t o be a l a r g e amount of  diffusional  exchange of i s o t o p i c water a c r o s s the c o r d , i t i s not known i f there a r e s i g n i f i c a n t net t r a n s f e r s of  fluid  Clearly,  are  studies  measuring  assess the r e l a t i v e fetal  bulk  and  a l l mammals  advancing  of  the  accumulation  i t s volume  increases  g e s t a t i o n , and i n most  prior  as  a  Fluid of amniotic  e a r l y , c o i n c i d i n g with the outgrowth of the  thereafter  just  r e q u i r e d to  structure.  The Amniotic  begins  vivo.  importance of the u m b i l i c a l cord  osmoregulatory  In  flows  in  to term (Adolph,  species  1967).  fluid  amnion,  p r o g r e s s i v e l y with reaches  a  peak  In the human, volumes  up t o 10 ml have been r e p o r t e d as e a r l y as  eight  weeks  15  of  gestation  a  maximum volume a t 38 weeks, a v e r a g i n g  then  (Seeds,  1965).  noted a r a p i d d e c l i n e as term In  nearly  the f i r s t isotonic  with  maternal  i n composition  higher  chloride  concentration  more  According  the  human  1972).  data  from a p p r o x i m a t e l y  in  of  osmolality  sodium  concentration  of  - 140 m E q / l i t r e  to  about  that in  120  occur  and  concentration  of the  fetus  a t term  fluid  uric  Pitkin,  of f e t a l acid  and fluid  16  weeks,  of hydrogen  ions  g e s t a t i o n , when f e t a l fluid  during  of amniotic  fluid increase  such as  levels  increase  urea,  (Benzie in  the e a r l i e s t  tissues are poorly  o r i g i n a t e s as a  about  1969; L i n d ,  (Seeds and H e l l e g e r s ,  that  the  progressively  (Gillibrand,  an  The  i n the  averages  declines  above plasma and  10 - 16  40 w e e k s ) .  waste p r o d u c t s  1974),  et  i n the  by c h a n g e s  i n the composition  h a s been p o s t u l a t e d  amniotic  10  i n amniotic  mEq/litre  becomes matures.  as the f e t u s matures a r e a p r o g r e s s i v e  1974;  It  i t  (1969) a n d B e n z i e  paralleled  and t h e r e a f t e r  the concentration  ,  the  for a  protein  273 mOsm/kg between  Between  sodium  Other changes  creatinine al.  lower  (approximately  are  ions.  135  1981).  a  and i s  except  However, as  fluid is  plasma,  fluid,  of G i l l i b r a n d  weeks t o 250 mOsm/kg a t t e r m  level  1000 m l , and  the average o s m o l a l i t y of amniotic  drops  changes  fetal  and  hypotonic  found  approached.  and  concentration  progressively  (1974),  (1961)  about  to extracellular  (Seeds,  to  a n d Inman  h a l f of g e s t a t i o n , the a m n i o t i c  similar  al.  Elliot  et the  1968). weeks  differentiated,  dialysate  of  maternal  1 6  plasma a c r o s s al.  ,  1967).  begin  to  of  As  play  production half  t h e p l a c e n t a and t h e c h o r i o a m n i o n  an  and  increasingly  important  d i s p o s a l of a m n i o t i c  gestation,  significantly by  the f e t u s matures, v a r i o u s  fetal  urine  to the amniotic  begins  fluid,  the i n c r e a s i n g c o n c e n t r a t i o n s of  uric  acid  kidneys  and c r e a t i n i n e ,  the  i n c r e a s i n g h y p o t o n i c i t y of amniotic  be  due  (Seeds,  1972).  human  (van  O t t e r l o et  fetal  sheep  amniotic  i s reflected fluid  urea,  by t h e f e t a l  of plasma  I t h a s been  (Lind  suggested  fluid  could  providing contribute  fetal  lungs  in  et  are  the f e t a l  an  may  that  in  part  al. , 1971). fluid have  at least pathway  et a  al.  the  some f l u i d  1979).  magnitude  source  of  active  i n the f e t a l  lungs  of  the  to the amniotic  tract.  cavity  (1971) o b s e r v e d  that  lecithin lung,  human  thus fetus  compartment.  f o r t h e d i s p o s a l of a m n i o t i c  gastro-intestinal  The  (Alexander  the amniotic  surface  o r i g i n a t e d only that  ,  s h e e p a s much a s 300 ml p e r  Gluck  contains  al.  500 ml p e r day  additional  may be s e c r e t e d i n t o  evidence  important  i s approximately  , 1977; A b r a m o v i c h  The  fluid  et  term  v o i d s amounts o f s i m i l a r  human a m n i o t i c  the  al.  fluid;  of lung  (Normand  An  1972).  contribute  The a v e r a g e volume o f u r i n e v o i d e d by  f e t u s near  al. , 1958).  which  In t h e second  t o the a d d i t i o n o f i n c r e a s i n g volumes of h y p o t o n i c  the  day  i n the  to  those  Hytten,  et  role  amniotic  and  urine  tissues  and t h i s  than  et  fetal  which a r e e x c r e t e d  i n concentrations higher 1970; S e e d s ,  fluid.  (Behrman  fluid  The amount o f  is fluid  s w a l l o w e d by t h e mature human and s h e e p f e t u s p e r day i s o f  17  the same order of magnitude as the amount of u r i n e produced (Pritchard, Harding  et  1965;  al. , 1984).  The  reabsorbed  into  presumably  1976; Abramovich et  Barnes,  swallowed  amniotic  the f e t a l  i n t e s t i n a l e p i t h e l i u m (Wright  al. , 1979; fluid  is  c i r c u l a t i o n v i a the  and Nixon, 1961;  Abramovich,  1970), as d i s c u s s e d p r e v i o u s l y . Although  fetal  volume  of amniotic  volume  is  swallowing fluid  probably et  Abramovich  al.  and  voiding  achieved (1979)  fluid  (scanty  by  have  other  observed  amniotic  structures. that  amniotic  fluid),  even  fluid)  the  fluid  volumes  study, Minei and Suzuki late-term  fetal  are  rhesus  swallowing  polyhydramnios,  with  normal range w i t h i n readjustment pathways.  in  f l u i d production  monkey  the  In  and  amount of  and  found  only  there  of  was  amniotic  i n r e n a l agenesis, other  (e.g., f e t a l  lack of c o n t r i b u t i o n by f e t a l occasionally  esophagus  a  that the transient  an  apparent  f l u i d by other pathways  u r i n e ; normal amniotic  observed  when there i s no u r i n e flow i n t o the amniotic  amniotic  even  The  polyhydramnios  fluid  and  1979).  of  lungs) may compensate-for the  volumes  Abramovich,  in  f l u i d volumes r e t u r n i n g t o  days;  volume  an i n t e r e s t i n g  the  resulted in  amniotic three  Likewise,  normal.  (1976) occluded  absence of f e t a l  (see  in  swallowed and voided by the f e t u s i s the same as when  amniotic  the  the  i n normal f e t u s e s , the c o n t r o l of  polyhydramnios ( e x c e s s i v e volumes of oligohydramnios  influence  volume  have been sac  and composition of  f l u i d c o u l d be c o n t r o l l e d by bulk movement  the amnion, the u m b i l i c a l c o r d , and the f e t a l  skin.  across  18  The In within this  the  human, a b o u t  daily  (Seeds,  or  net  fetal  increase  must  Fluid  can  either  extraplacental  membranes.  these  be  and  1965;  o s m o t i c and several  are  solute  fetal  plasma  1977), and water  in  been f o u n d  that  bulk of  accumulation  flow  in  osmotic that  and  mother  by water  can  response  to  hydrostatic  in  across  the in  w h i c h water  an  (see colloid  placenta  in  attempt  to  accumulates.  i n m a t e r n a l and  fetal  The plasma  higher  in maternal  than  (Kerpel-Fronius,  1970;  Armentrout  et  via  provide  the  are  lower  in f e t a l  the  placenta.  ( w h i c h depend p r i m a r i l y on  may  vivo  i n osmotic',  measured  the  such g r a d i e n t s  o f water  Gradients  been  t h e r e f o r e cannot  plasma)  and  slightly  accumulation  pressures  growth  fetal  I t has  1 976).  same or  of  or  concentrations  e i t h e r the  large part  placenta  by  t h e mechanisms by  a  originate  the  gradients  have  which c o u l d  since  e n t r y ' i n t o the i n t r a - u t e r i n e  hydrostatic pressure  species  elucidate total  Barnes,  accumulates  p r e g n a n c y , and  for  been s u g g e s t e d  f o r the  fluid  t r a n s f e r r e d from t h e  gain  via  created i t has  responsible  Seeds,  be  barriers  experimentally pressure,  of  sources,  required  ultimately  1965).  that  metabolic  compartments  cross  liters  i s f a r i n e x c e s s of  uterine  survival  four  i n t r a - u t e r i n e cavity during  amount  from the  the  O r i g i n of I n t r a - U t e r i n e Water  driving  al.  force  Colloid  protein  in ,  for  osmotic  concentrations  t h a n m a t e r n a l p l a s m a and  so  19  favour  a  movement  direction,  as do  pressure,  and  a bulk  of  flow  Thus  present  osmotic fluid  net  explained  fluid  of  of  and  fetal  by  Likewise,  the  amnion  gradients  fluid  (Garby,  1957).  mother t o  gestation in  favour  amniotic  from t h e  differences  hydrostatic  1965).  mother  during  maternal  hydrostatic  from t h e  the  to  in  across  water  compartments  simply  osmotic,  the  (Seeds,  gradients  transfer  intra-uterine  in  in a direction  extracellular  the  water  trans-placental gradients  when  hydrostatic  into, the  of  cannot  osmotic,  between  the be  colloid  the  fluid  compartments.  Hormonal C o n t r o l of Recently, the  evidence  accumulation  achieved first  and  through  has  c o n t r o l of  the  vasotocin  fetuses.  This  vertebrates, during  fetal  hormone,  is  present  life.  In  the  osmoregulatory  function,  the  the  skin  and  Therefore,  amnion, thought  a  found  and  involved  Perks  system.  (1969)  pituitaries all  mammalian  affecting  bladder  Perks  The  mammalian  sub-mammalian  pituitaries  fluid  AVT  (Sawyer, Holt  only  has  transport  (1974) and  be  discovered of  anuran amphibians,  restricted in  endocrine  in  e f f e c t s o f AVT  structure  t o be  and  urinary  Vizsolyi  (1977a) t e s t e d t h e  own  i n the  in  that  i n t r a - u t e r i n e f l u i d s may  fetus's  (AVT)  Fluids  accumulated which suggests  c l u e came when V i z s o l y i  arginine  Intra-Uterine  an  across 1970).  and  Perks  and  other  hormones on  the  to  fetal  life,  was  intra-uterine  water  which  transport.  20  Vizsolyi  and Perks  (1974)  found  that  vasopressin  (AVP) c a u s e d an i n c r e a s e  water  the  from  isolated and  guinea-pig  were  (1975)  transfer  increased  across that  the  permeability  guinea-pig Tyson,  vivo  would  the  amniotic  favour  water  and  vasopressin  vasopressin from  the  the  amnion.  prolactin,  the  These  results  aided  was f o u n d  the  al.  to  maternal  to  the  and  (Leontic  Cortisol,  transfer  of  that  sodium  of the guinea-pig  c a u s e d an i n c r e a s e  and  that  (I977a,b) found  surface  The  fluid in  Prolactin  sodium  flux  in  vasopressin  amnion.  unidirectional  t o the f e t a l  vasopressin  reduce  observed  influence  and P e r k s  direction.  by  amnion o f t h e  human  (1975)  be  circulation  amnion,  the guinea-pig  Holt  increased  opposite  n e t water  , 1979); s u c h an e f f e c t  et  a l s o appear  amnion, w h e r e a s p r o l a c t i n in  on  of the i s o l a t e d  al.  et  requirements could  the a c t i o n s of p r o l a c t i n  transfer across  the  water  1975) and  Manku  found  Manku  AVP  amnion.  via  et  the  i n osmotic  (1977a)  from t h e m a t e r n a l  water  of  t h e r e t e n t i o n o f any a c c u m u l a t e d  sac.  reversed  of  Prolactin  1977; L e o n t i c  on  across  guinea-pig  ( H o l t and P e r k s ,  and  Cortisol  to  gradients Perks  effects  hormones.  surface  vasopressin.  cavity  arginine  f l u x e s o f water a c r o s s t h e  with  of f l u i d  amniotic  fetal  and  intra-uterine  by t h e t r a n s p o r t  and  i n t h e n e t movement o f  against  Holt  the  the  neurohypophysial the  amnion,  confirmed  suggested  to  the  maternal-fetal diffusional  amnion  met  to  hydrostatic pressure.  that  al.  maternal  AVT  i n sodium  combined and  flux  actions  perhaps  of  other  21  hormones, c o u l d the  amniotic The  fluid  fetal  regulating  ensure that  t h e volume and  remain c o n s t a n t  endocrine  fetal  lungs,  s k i n and u r i n a r y b l a d d e r .  found  that  secretion  of lung  mechanism  that  overhydration probably At  absorption  vasopressin et  and  and al.  Perks  ,  the  have f o u n d t h a t across  the  their of  Perks  (1982)  increase  fluid  is  (Perks  that  (1976)  unidirectional bladder  during are  Cassin,  by  1982;  experiments,  Holt  facilitates  u r i n a r y bladder  and F r a n c e  a  such s t r e s s .  vasopressin  et  f l u x e s of water  l i k e w i s e a f f e c t e d by n e u r o h y p o p h y s i a l  goat,  facilitated  and  of f e t a l  i n the  kidneys  to handle  t h e s k i n a n d .the  and  on t h e  the  preliminary  observed  France  skin  acting  to the fetus  since  ability  lung  In  across  guinea-pig.  and  loading,  1982).  water a b s o r p t i o n  Cassin  of importance  epinephrine  (1977a)  by  +  be  in  levels.  N a , and C l ~ i n t h e f e t a l  salt  limited  birth,  Walters  or  fluid  caused a s i g n i f i c a n t  fluid, may  narrow  of  s y s t e m may a l s o be r e s p o n s i b l e f o r  extracellular  prolactin  within  composition  of  al.  (1976)  and  sodium  s h e e p and p i g s a r e  hormones.  STATEMENT OF THE PROBLEM The further  present the  regulated.  was u n d e r t a k e n  mechanisms Attention  neurohypophysial across  study  by w h i c h was  hormones  to elucidate  intra-uterine fluids are  focused on  i n order  the  on net  the  effects  of  t r a n s f e r of water  t h e amnion and t h e s k i n o f t h e f e t a l  guinea-pig.  22  The that of  s t u d i e s of V i z s o l y i  b o t h AVT  water  and  from  guinea-pig  AVP  may  amnion.  be  the  physiological which  of  AVP  on  bulk  the  studied  changes  water  The  amnion  the  fine  structure  through  gestation. the  vasopressin  amnion, (France,  i n the  the  o n l y changes  flux  across  water  Therefore, studies  to  important this  i t seemed changes  to t e s t  peptide  gestation, and  has  the  fetal  is  bulk  effects  amnion were  amnion  of  to  AVP  Changes i n  compared  with  amniotic  skin  Holt  and  skin  flow.  Perks,  plentiful  i n the  fetal  keratinizes on  1977a).  measured.  preliminary-  In a d d i t i o n , the  fetal  pituitary  (see  Perks,  water movement  A c c o r d i n g l y , the e f f e c t s  to  diffusional  these  on  fluid  responds  have been  to extend  p r e v i o u s l y been t e s t e d  skin.  the  were  volume  fetal  AVP  the amniotic e p i t h e l i u m ,  of AVT  skin  the  in unidirectional  important in  of  the e f f e c t s  b e f o r e the  not  the  AVP  fetal  1976;  However, t o d a t e of  to  possible  to preliminary  study  the  AVP  with  s t a g e s of g e s t a t i o n .  the  as w i t h c h a n g e s  a  studies  limited  of  the  and t h e r e f o r e , in  across  response  of  as w e l l  Like  of t h i s ,  transfer  of  investigators,  peptide  f l o w s have been  shown  movement  surface  response,  important  at d i f f e r e n t  in  fetal  inducing this  further.  response  the  In s p i t e  have  of c a u s i n g a n e t  T h e r e f o r e , i n the p r e s e n t net  investigated was  at  system.  observations.  to  (1974)  According to these  more  measure  Perks  are capable  the maternal  i s more e f f e c t i v e  and  of AVP  and  it  was  skin; at  mid  1977), across AVT  on  23  net  water  investigated.  transport  across  the  fetal  skin  were  24  GENERAL METHODS  1.  Net Water Flow  (a) The  I s o l a t e d Amnion P r e p a r a t i o n  Net was  on  water  determined  Perks  (1974),  fetal  lengths,  The  ages  the  at  removed and cm  2  and  from  faced  tied  i . e . , the  the  Ibsen  inside  out  stages from  were  horns  29).  an  The  area  t h e end area).  surface  of  bathed  outer bath c o n t a i n i n g  The  the  oxygenated  50 ml was  placenta  with intact,  t u b e was  of  bath  was  surface  filled  (p. 28)  and  maternal  maintained  placed.  was  s u p p o r t i n g tube of in with  The  w i t h a i r by means of a s i d e arm  saline  throughout  outer bath which  (1 the  vivo, 2.5  suspended  by means o f a c o n s t a n t - t e m p e r a t u r e chamber outer  data  amnion  by a m n i o t i c f l u i d  an  which  u s i n g the  into aerated maternal  fetal  in  experiment  crown-rump  anaesthetised  of a g l a s s The  of the tube.  temperature  the  reflected  of warmed, a e r a t e d a m n i o t i c s a l i n e  This  gestation.  o u t e r y o l k - s a c membrane  ml  37°C.  of  fetuses,  w i t h membranes and  ( s e e p.  over  S t u d i e s were c a r r i e d  (1928).  the u t e r i n e  o f f , and  and  determined  number of t h e  g u i n e a - p i g amnion  method o f V i z s o l y i  various  guinea-pigs  cross-sectional  amnion,  at  fetuses,  37°C  stripped  isolated  i n F i g u r e 1.  were  pregnant  were d i s s e c t e d  was  shown  (1920) and  and  the  u s i n g the g r a v i m e t r i c  weights,  of D r a p e r  saline  flow through  guinea-pigs  Gestational  ether,  Experiments  at the into was  prevented  25  Figure  1.  The G r a v i m e t r i c A p p a r a t u s u s e d f o r t h e in vitro Study of Net Water Movement Through the Isolated Amnion of the Guinea-Pig (from V i z s o l y i and P e r k s , 1974).  25A  B a l a n c e Lever To Recorder  Differential Transformer  Supporting tube 2 cm  Water bath (37°C)  Amniotic saline  Fetal_ side" Maternal I side" " "  Maternal saline  Amniotic membrane  \  Outer container  Compressed air  26  bubbles from d i s t u r b i n g the membrane. not  float  but  The inner  was supported by a weighted balance l e v e r ,  and the weight was a d j u s t e d so that the f l u i d inner  tube  difference of  the  was  2  cm  vivo  1974).  thread  The  l e v e r passed  transducer.  a  coil  in  fluid  of  the  transfer  to  formed  the  of  movable  differential  the  inner  tube  a c r o s s the amnion produced concomitant  changes  in  Beckman  RS  Dynograph  recorder.  system was c a l i b r a t e d by adding known amounts of f l u i d the inner s u p p o r t i n g tube. In  l a t e r experiments on the amnion, the apparatus was  m o d i f i e d as f o l l o w s :  rather  than  measuring  caused  by  changes  of the inner tube were measured.  was  Perks,  which were c o n t i n u o u s l y recorded using  e i t h e r a S o l t e c 220 or a The  and  a Trans-Tek  weight  movement of the balance l e v e r and output,  sides  the s u p p o r t i n g tube t o the  r o d , which  f o r the  from  (see V i z s o l y i  attaching  Changes  The  the 2 cm h y d r o s t a t i c p r e s s u r e head  conditions  through  core  transducer  i n the  above that i n the outer bath.  and  reproduced in  resulting  level  i n o s m o l a r i t i e s of the s a l i n e s on the two  membrane,  central  tube d i d  f l u i d t r a n s f e r a c r o s s the amnion, a c t u a l weight  suspended  hanger  The  The  inner  tube  i n the outer bath by a t h r e a d a t t a c h e d to a  f o r bottom weighing on a M e t t l e r AE  balance.  displacement  system  was  a d d i t i o n of known amounts  163  electronic  c a l i b r a t e d as b e f o r e , with the of  fluid  Weights were recorded at three minute  to  the  inner  intervals.  tube.  27  After  the  amnion  was  e q u i l i b r a t e f o r 30 - 45 salines  in  place,  minutes,  i t was  during  were changed at approximately  which  15 minute  A f t e r the e q u i l i b r a t i o n p e r i o d , both s a l i n e s to  allowed time  each  test  vasopressin  period.  was  were  each  salines, values per cm  of  test,  the inner and  changed  l00mU/ml  2  s a l i n e , at a  (vasopressor  activity).  solutions  the  membrane  were was  replaced  allowed  with  fresh  to recover.  All  as  mg  of membrane per minute.  I s o l a t e d Skin  Skin  Preparation  from the back region of f e t u s e s between 33 and  days of g e s t a t i o n (0.49  - 0.70  sharp  set  dissection  apparatus However,  used there  and for  the  were  no  of  up  both  s a l i n e , and the  inner  tube  term) in  the  initial  and  outer  were the same. i n s i d e of the  The  i n the  same  48 by  gravimetric experiments.  or osmotic  bath contained l e v e r was  inner tube and  s e r o s a l s u r f a c e of the s k i n  inner tube.  isolated  serosal surfaces  the weight on the balance l e v e l s of f l u i d  was  amnion  hydrostatic  g r a d i e n t s between the mucosal and  that  arginine  f o r the net movement of water were expressed  (b) The  skin;  change,  start  which l a s t e d f o r up to 30 minutes, both  outer  and  this  i n c l u d e d i n the inner amniotic  f i n a l concentration After  During  the  intervals.  ensure that i o n i c c o n d i t i o n s were i d e n t i c a l at the  of  to  pressure of  the  maternal  adjusted  so  outer  bath  faced  the  28  After  the  skin  was  set up  allowed to e q u i l i b r a t e 30 changed  at  about  15  i n the  - 45 minutes, with s a l i n e s  minute i n t e r v a l s .  experiments, the  s a l i n e s on both s i d e s  changed  the  after  change, a r g i n i n e included surface  in of  l00mU/ml  equilibration  vasopressin  the  inner  the  skin,  or  final  to 30 minutes, the  and  was  were done. in  the  arginine  After  were  During  this  vasotocin  was  the  salines  being  skin  concentrations  serosal of  Whenever p o s s i b l e ,  p a r a l l e l , using s k i n  the  two  from fetuses  5  -  treatment,  were  changed,  allowed to recover before f u r t h e r  tests  hormones were t e s t e d from the  same mother,  of  the  hormones  in  salines  were  designed  by  reproduce  the  i n order to compare the e f f e c t i v e n e s s i n f l u e n c i n g water flow through the  2.  of  was  in the amnion  period.  activity).  which l a s t e d up skin  As  s a l i n e which c o n t a c t e d the at  (vasopressor  the  apparatus, i t  skin.  Salines Maternal  and  amniotic  V i z s o l y i and  Perks (1974)  present  maternal  in  respectively.  to  (or f e t a l ) plasma and  They were composed of the  a) Amniotic  electrolytes  amniotic  fluid,  following:  saline  NaCl KC1 CaCl -2H 0 MgCl -6H 0 Glucose 2  2  2  2  g r a m s / l i t r e of d i s t i l l e d 7.31 0.46 0.33 0.23 1 .00  water  29  A phosphate b u f f e r at  (0.2M N a H P O „ - N a H P O ; pH 7.8) was added 2  10- ml b u f f e r per l i t r e  2  ft  of s a l i n e .  s a l i n e was 7.4, and i t s o s m o l a r i t y  The f i n a l pH of the  286 mOsm/litre  (Advanced  Instruments osmometer). b) Maternal s a l i n e g r a m s / l i t r e of d i s t i l l e d 8.76 0.41 0.32 0.26 1 .00  NaCl KC1 CaCl -2H 0 MgCl -6H 0 Glucose A  2  2  2  2  phosphate  The  final  buffer pH  water  was added as f o r the amniotic  was  7.4,  and  the  osmolarity  saline.  was  314  mOsm/litre.  3.  Hormone The  (AVP)  Solutions hormones used were.synthetic a r g i n i n e  and  Chemicals,  synthetic St.  arginine  Louis,  Mo.).  vasotocin Each  was  vasopressin  (AVT)  (Sigma  dissolved  in a  s o l u t i o n of 0.25% a c e t i c a c i d i n 0.9% NaCl, t o give a f i n a l concentration  of 100 IU/ml (of vasopressor a c t i v i t y ) , a t a  pH of 3.5. 0.5% c h l o r o b u t o l was added as a p r e s e r v a t i v e . Just diluted or  prior  the  i n e i t h e r amniotic  skin  experiments,  concentrations in  to  osmolarity  experiments,  or maternal s a l i n e respectively)  from 5 to 100 mU/ml. or  hormones were added.  pH  of  the  the  to  hormones were ( f o r amnion give  No measurable  salines  occured  final changes  when the  30  For c o n t r o l experiments, 0.25%  acetic  diluted  acid  and  solution  gestation  dissection,  guinea-pig  were  fixed  saline.  samples  for . 1  of the f o l l o w i n g composition  hour  at  room  temperature fixative  (see G l a u e r t , 1975):  3.31 33.71 40.00 1000.00 2.5% 320 mOsm/litre 7.4  2  2  reflected  glutaraldehyde  NaH PO -H 0 (g) N a H P O „ - 7 H 0 (g) 25% g l u t a r a l d e h y d e i n water (ml) D i s t i l l e d water t o make (ml) F i n a l c o n c e n t r a t i o n of g l u t a r a l d e h y d e Osmolarity of phosphate b u f f e r pH of f i x a t i v e ft  of  f e t u s e s between 28 and 70 days of  (22°C) i n a 2.5% phosphate-buffered  2  only  of the Amnion  Immediately a f t e r from  containing  0.5% c h l o r o b u t o l i n 0.9% NaCl was  i n t o the amniotic or maternal  4 . E l e c t r o n Microscopy  amnion  a  2  In a d d i t i o n , 1 mM of MgCl was added t o the f i x a t i v e f o r b e t t e r p r e s e r v a t i o n of the o r g a n e l l e s . 2  F o l l o w i n g t h i s primary  f i x a t i o n , the t i s s u e was washed  overnight at 4°C i n phosphate b u f f e r and then p o s t - f i x e d i n 2%  phosphate-buffered  at 22°C. through  osmium t e t r o x i d e (OsO„) f o r one hour  The t i s s u e was subsequently increasing  concentrations  dehydrated of  by passage  ethanol,  propylene  oxide, a c c o r d i n g t o a schedule  (1975).  F o l l o w i n g d e h y d r a t i o n , the t i s s u e was embedded i n  an epoxy  resin  50-60nm)  were  (Epon  c u t , placed  s t a i n e d with l e a d examination  812).  citrate  given  and then  Ultrathin on  and  sections  uncoated uranyl  by  (about  copper g r i d s and  acetate  under a Z e i s s EM 10 microscope.  Glauert  prior  to  31  Thick with  sections  t o l u i d i n e blue  f-^r c o n f i r m a t i o n seen u n d e r the  5.  of  of  epon-embedded  t i s s u e were  and  examined under t h e  the  general  electron  light  m o r p h o l o g y of  the  stained  microscope amnion  as  microscope.  E l e c t r o n Microscopy of the F e t a l Skin Skin  and  52  electron those  from t h e days  of  back  gestation  microscope.  described  region  The  f o r the  of  fetal  was  also  methods u s e d  amnion.  guinea-pigs studied were  the  at  35  under  the  same  as  32  SECTION I  THE EFFECTS OF ARGININE VASOPRESSIN ON THE BULK FLOW OF WATER ACROSS THE ISOLATED AMNION OF THE FETAL GUINEA-PIG AT DIFFERENT STAGES OF GESTATION  INTRODUCTION Vizsolyi vasotocin  (AVT)  influence  the  isolated the  and Perks and  rate  amnion  (1974) d e m o n s t r a t e d arginine  of  net  the  (i.e.,  maternal  towards  hydrostatic  to  the  the amniotic  pressure  hormones  activity, likely  were  t o be i n v o l v e d  fetus,  for  the  one t h a t transport  from t h e m o t h e r . seemed  more  observations. observed amniotic  across  the  found  that  of  water  o f t h e membrane  against  osmotic  and  The amnion was f o u n d t o be  t o AVT ( t h r e s h o l d d o s e s  This  suggested  in a possible  could  f o r the  o f water  effective, addition,  that  more  extraplacental  to the amniotic  were  AVP was  p h y s i o l o g i c a l system i n  c o n t r o l an  a large v a r i a b i l i t y  with  Vizsolyi  although  to  and  i t  preliminary  Perks  i n r e s p o n s e between that  route  sac and f e t u s ,  AVP,  limited  membranes, a n d i t was p o s s i b l e  to d i f f e r e n c e s  both  They  surface  cavity),  However, s t u d i e s  In  (AVP)  1.0 mU/ml a n d 6.4 mU/ml o f v a s o p r e s s o r  respectively).  the  arginine  a net uptake  fetal  gradients.  more s e n s i t i v e t o AVP t h a n  transfer  guinea-pig.  hormones a r e c a p a b l e o f c a u s i n g  from  two  vasopressin  water  of the f e t a l  that  this  (1974)  different was  i n t h e g e s t a t i o n a l age of t h e membranes.  due  33  The studies the  on  if  of  of AVP  of  changes  epithelium  from  g e s t a t i o n was  microscopic  morphological  across  guinea-pigs  i n an the  effort  at  to  see  amnion t o  AVP  the  to  the  guinea-pigs out.  The  the  fluid  at  activity.  see  if  epithelium  out  the  various  p u r p o s e of  functional  volume of  of  u l t r a s t r u c t u r e of  the  on  hormone  this which  Electron a  membrane  caused  any  amniotic, e p i t h e l i u m . amniotic  gestation.  fluid  T h i s was  be  was  measured  at  to  see  if  s t r u c t u r e of  the  done  t r a n s p o r t p r o p e r t i e s and  amnion t h r o u g h g e s t a t i o n m i g h t amniotic  earlier  transport  f e a t u r e s of  changes i n the  stages in  fetal  carried  its  AVP  Finally,  changes  to  of  s t u d i e s were a l s o c a r r i e d  with  different  water  i n r e s p o n s e of  investigation  related  treated  net  membranes f r o m f e t a l  t o examine c y t o l o g i c a l be  on  to extend the  age.  amniotic  may  undertaken  g e s t a t i o n were t e s t e d  systematic  stages  was  Amniotic  were any  fetal A  study  effects  stages  there  with  was  the  amnion.  various  the  present  r e l a t e d to changes  volume.  in  the  _  RESULTS  1. The  E f f e c t s of AVP  Thirty-nine 0.44 up  of in  t e r m and  the  methods.  on the Amnion  amniotic term  gravimetric Upon  membranes  (30  - 68  of  fetuses  d a y s of g e s t a t i o n )  apparatus,  immersion  from  the  as  between were  set  in  the  described  supporting  tubes  in  the  34  outer  baths,  23  p a s s a g e o f water saline, osmotic side the  of from  presumably  t h e membranes the amniotic  in  of  t h e amnion.  fetal  t o maternal  (range=0.07  to  of  direction  2.46  prevailing  water  mg/cm  net  the  the  the f e t a l resting  t o the maternal f l o w o f water i n  was 0.69 mg/cm per  2  minute).  of f l u i d ,  and  5  are  flow  (100 mU/ml) a d d e d  showed  saline,  of  initial  fluid  in  the maternal  to  a resting  i n agreement w i t h However, t o vary  responses  are  membranes  were  preparations saline. rates,  a  11 slow  against the  cases  flow.  the results  of  shown i n F i g u r e 2. tested which  with  the t e s t s performed.  as  the  average  rate  before  flow  fetus.  The r e s t i n g of  flow  treatment  in  recovered, observations and  Perks t o AVP  Examples  of  o f 32 d i f f e r e n t  There  obtained  rates,  reversing  the response  AVP was s u b s t i t u t e d  I shows t h e v a l u e s  all  Vizsolyi  A total  AVP.  direction,  When t h e AVP was  These  of  t h e age o f t h e  AVP-influenced  immediately  rate.  t h e magnitude  with  in  Table  flow  saline  of causing a  to fetal  g r a d i e n t s , i n most  fetal-maternal resting  returned  appeared  minute  to the amniotic  s u r f a c e o f t h e amnion was c a p a b l e  the p r e v a i l i n g  (1974).  per  However,  removed a n d t h e s a l i n e s c h a n g e d , most membranes and  maternal  gradients.  the f e t a l  against  2  to  steady  t h e h y d r o s t a t i c and  towards t h e a m n i o t i c  Vasopressin on  from  to  The a v e r a g e  membranes showed no f l o w uptake  saline  response  gradients maintained  showed a slow  were  seven  with a control  for resting  flow  and t h e r e s p o n s e s , f o r flow  r a t e was d e f i n e d  the  12 m i n u t e p e r i o d  w i t h AVP.  The A V P - i n f l u e n c e d  35  Figure  2.  The E f f e c t s of AVP on Net Water Movement t h e I s o l a t e d Amnion of t h e G u i n e a - P i g .  Across  Membranes were t a k e n from fetuses at various stages of g e s t a t i o n , as indicated on each graph. H o r i z o n t a l b a r s show p e r i o d s of c o n t a c t with lOOmU/ml AVP or with control saline. O r d i n a t e s : r a t e of n e t w a t e r movement (mg/cm per minute); the dotted l i n e represents zero c h a n g e ; v a l u e s above t h i s l i n e indicate water uptake towards t h e f e t a l s i d e of t h e membrane ( i . e . , towards the amniotic cavity); values below t h e l i n e i n d i c a t e water l o s s t o w a r d s t h e maternal side of the membrane. Abscissae: time, i n minutes. 2  35A  0.5h 0.0  S\  0.5  control  1.0  c E CM  18  9  1.0-  co E  0.5-  1.0  —  0.5  x  0.5-  o  1.0 <-  O  L  AVP  18  27  1.0  36  9  18  27  36  68 days  2.5  2.5  2.0h  2.0  1.5h  1.5-  1.0  1.0-  05  0.5-  0.0  0.0  0.5  05  AVP  18  AVP  0.5  •— 56 days  1.0  c _i  0.0  c E  I  36  I  - 40 days  0.0  3  27  ,— 32 days  E  o >  _ J  27  36  1.0  Time ; minutes  —^*  V AVP  18  27  36  Table Response Number  Gestational Age  I:  The  Rate  (days)  Response  of  (mg/cm  Resting 2  per  of  the  Amnion  Flow  to  Rate  minute)  AVP D u r i n g of  the  Course  of  A V P - I n f l u e n c e d Flow  (mg/cm  2  per  minute)  Gestation Response: of  AVP  Increase  3  (mg/cm  2  per  1  30  0  +0 . 1 0  0 . 10  2  32  0  +0 . 15  3  32  0 . , 15 0. .37  4  33  5  38  - 0 . . 34  - 0 . 14  6  40  +0, . 0 3  +0 . 7 0  0, .20 0. .67  +0 . 17  - 0 .20  0  0  7  48  +0, . 0 6  +0 .21  0 . . 15  48  + 0 , , 10  +0 . 4 2  0 . . 32  9  50  +0. ,21  +0. . 3 4  0 . . 13  10  50  +0. . 4 8  + 1 .37  0. 89  1 1  52  +0. .51  0 . 51  12  54  - 0 . .92  13  55  - 0 . .30  +0. . 3 3 +0. .37  0. 67  14  55  - 0 . .60  +0. . 2 0  0. 80  15  56  - 0 . 32  16  58  +0. .95 +0. . 76  0  0  1 .2 7  +0. ,04  0. 76 1 .55  + 0 . . 18  0 . 18  +0. ,09  0 . 92  - 0 . ,56 - 0 . 73  0  58  18  60  19  62  -o.8 3  20 21  65  - 0 . 31  65  - 0 . 70  22  66  0  0  0  23  66  0  0  0  24  66  - 0 . 07  +0. 0 6  0 . 13  67  - 0 . 75  - 0 . 80 - 0 . 49  0  26  68  27  68  28  68  29  68,  30 31  68'  32 Negat1ve mother).  0  - 0 . 49  O  0  0  0  0 0  0 -2 . 46  0 - 2 . 46  0  68  - 0 . 29 -0. 1 1  - 0 . 48 - 0 . 13  0 0  68  - 0 . 59  - 0 . 66  0  values  (-)  Pos1t1ve  indicate values  flow (+)  of  fluid  Indicate  from  flow  the  from  amniotic  maternal  continued  on  next  to  to  the  maternal  amnlot1c  page  . . . .  sal1ne  sal1ne  ( i . e . .  (towards  the  towards fetus).  In  Rate  with  minute)  1 .25  17  '  - 1 . 51  Flow  0  8  25  Note:  The  Maternal-Fetal  the  Table  1.  The  rate  of  resting  2.  The  rate  of  AVP-1nf1uenced  3.  The  response  flow as  In  addition  gestation;  and  zero  to none  1s  the  flow  Is  the  flow  increase  AVP-1nfluenced  1n  flow.  average  Is  the  the No  rate  I  of  average  rate change  of 1n  (continued)  flow  rate  flow  in  of  the  12  minutes  flow  In  the  towards  flow,  or  an  the  15  fetus,  Increase  before  minutes  and In  Is  treatment  with AVP.  Immediately  the  difference  flow  towards  the  from  fetuses  at  after  AVP a d d i t i o n .  between  maternal  the  saline  response.  the  tests  listed  above,  of  these  showed  any  there  Increase  were 1n  7  control  preparations,  maternal-fetal  fluid  flow  taken with  control  saline.  various  stages  of  resting Is  taken  38  flow  rate  was  the average r a t e of flow i n £he 15 minutes  a f t e r AVP a d d i t i o n . in  the rate  of  The response was d e f i n e d as the change flow with AVP.  i n c r e a s e i n the net flow saline,  was  taken  increased gestation was  an 2  mg/cm  with  outwards,  as  quantified graphically  No change of flow, or an  zero  towards  response.  i n F i g u r e 3.  fetal  age, up  (0.85 ot term).  The  The  until  and was l o s t .  about  experiments.  2  the f e t u s of 1.55 declined  per minute)  i n 13  i n the a b i l i t y t o respond t o AVP  appeared  t o be abrupt, o c c u r r i n g between  and  days.  66  days,  (0.94 of term) showed  (0.13 mg/cm  loss  days of  58  the e f f e c t  Membranes over 64 days  The  to AVP  58  The peak response, a t  A f t e r t h i s time,  only one weak response  r e s u l t s are  response  i n c r e a s e d water movement towards  per minute.  the maternal  approximately  62  None of the seven c o n t r o l membranes, taken  from f e t u s e s of  different  gestational  i n c r e a s e i n maternal t o f e t a l  fluid  ages,  showed any  flow.  2. The F i n e S t r u c t u r e of the Amniotic E p i t h e l i u m The  structure  of  the epithelium  from ten amniotic  membranes taken from g u i n e a - p i g f e t u s e s between 28 days  of  gestation  (term  =  approximately  s t u d i e d under  the e l e c t r o n microscope.  description,  the membranes  groups: e a r l y  ( c o n s i s t i n g of amnions  have  68  and 70  days) was  For convenience  been d i v i d e d at  28,  of  i n t o three  30,  days),  mature (50, 58, 62 d a y s ) , and near-term  days).  There were marked changes i n the e p i t h e l i u m  35, 38  (64, 68, 70 during  39  Figure  3.  The Changes i n t h e R e s p o n s e o f t h e Amnion AVP T h r o u g h t h e C o u r s e o f G e s t a t i o n .  to  Each point represents a separate membrane (n=32). O r d i n a t e : Increase i n the r a t e of net water transport i n the maternal to fetal direction (i.e., t h e r e s p o n s e ) , i n mg/cm p e r minute. Abscissa: Gestational age o f the f e t u s , in days. C u r v e o f b e s t f i t e s t i m a t e d by computer ( l e a s t s q u a r e s f i t t i n g to B-splines; program d e s i g n e d by C. Moore, U.B.C. C o m p u t i n g Centre). 2  40  the course an  of g e s t a t i o n , as seen i n F i g u r e 4,  epithelium  which  from each of the three groups.  Within  shows each  group, no c l e a r d i f f e r e n c e s i n s t r u c t u r e were e v i d e n t .  E a r l y Amniotic The  Epithelium  amniotic  gestation  was  (28, 30, 35, 38 days)  epithelium  found  to  in  the  early  4A, 5A, 5D).  basement membrane  The average width of the e p i t h e l i u m  was 2 . 7 6 ± 0 . 1 4 M m (iS.E.M.; n = 40: 10 readings membranes).  There was no c l e a r evidence  type of c e l l ,  i n t h i s or any other  There  was  rarely  c e l l s , and i n t e r c e l l u l a r 5A,  5D).  The  desmosomes.  any  spaces were  S t r u c t u r e s resembling  amniotic  fluid  microvilli,  evident  A  5B).  the  (Figures  4A,  had a few conspicuous junctions,  which  surface,  which  faces  the  The a p i c a l s u r f a c e had few number  with  advancing  ( g l y c o c a l y x ) , composed of f i n e  filaments p r o j e c t i n g in  narrow  tight  increased in  s u r f a c e coat  between adjacent  spaces over short areas, were  apical  (Figure  but these  gestation. branching  the  f o r more than one  overlapping  o b l i t e r a t e d the i n t e r c e l l u l a r  f o r each of 4  stage.  l a t e r a l c e l l borders  o f t e n observed at  of  be composed of a s i n g l e l a y e r of  f l a t t e n e d c e l l s u n d e r l a i n by a "continuous (Figures  stages  from  the  microvilli,  was  o l d e r specimens of t h i s group, but i t d i d  not appear t o be p a r t i c u l a r l y w e l l developed. The  epithelial  n u c l e o l i were endoplasmic  often  cell  n u c l e i were l a r g e and o v a l , and  observed.  reticulum  were  Large  amounts  scattered  of  throughout  rough the  41  Figure  4.  The Fine S t r u c t u r e of t h e A m n i o t i c Through the Course of G e s t a t i o n .  A. B. C.  28 d a y s 50 d a y s 64 d a y s  Epithelium  ( E a r l y Amnion). (Mature Amnion). (Near Term A m n i o n ) .  ac: amniotic n: n u c l e u s ;  c a v i t y ; mv: microvilli; c: c o n n e c t i v e t i s s u e . X  5333.  41 A  42  F i g u r e 5.  Electron Micrographs Epithelium.  of  the  Early  Amniotic  A.  E p i t h e l i u m at 35 days of g e s t a t i o n (0.51 of term). The cells are flattened, and the i n t e r c e l l u l a r space (ICS) i s narrow. Several desmosomes (D) are e v i d e n t . The a p i c a l s u r f a c e of the c e l l s has a few m i c r o v i l l i (MV), which project into the amniotic c a v i t y . A basement membrane (BM) separates the e p i t h e l i u m from the underlying connective t i s s u e . A number of o r g a n e l l e s are seen i n the cytoplasm. N: nucleus; M: mitochondrion; ER: rough endoplasmic r e t i c u l u m ; Go: g o l g i apparatus. X 16,000.  B.  Intercellular j u n c t i o n s at the a p i c a l s u r f a c e of e p i t h e l i a l c e l l s at 35 days. T: t i g h t j u n c t i o n ; D: desmosome. X 44,000. Epithelial cell at 38 days of g e s t a t i o n (0.56 of term). The membrane-bound v e s i c l e s (Ve) beneath the a p i c a l s u r f a c e appear to be coated. MV: m i c r o v i l l i ; G l y : glycogen; Go: g o l g i apparatus; N: nucleus; M: mitochondrion. X 28,000.  D.  Micrograph of e p i t h e l i a l cell at 38 days showing the narrow intercellular space characteristic of early amnion. Several desmosomes (D) are evident. Cytoplasmic f i l a m e n t s (F) are observed i n the v i c i n i t y of desmosomes. Clusters of particles which resemble glycogen (Gly) are seen in the cytoplasm. MV: m i c r o v i l l i ; Ve: membrane-bound vesicles; ER: rough endoplasmic r e t i c u l u m ; M: mitochondrion; BM: basement membrane. X 40,000.  4 2 A  43  cytoplasm, (Figures the  and  number  5A, 5C, 5 D ) .  nuclei  and  Membrane-bound especially seen  a  vesicles  near  appeared amounts  of  cytoplasm.  of  loosely evident  _the i n t e r c e l l u l a r and b a s a l  substance  to  mitochondria  appeared  with  glycogen-like Cytoplasmic  in association  near  cisternae.  the  spaces.  evident seen  packed in  surfaces  (Figure  increase  were  c o m p l e x e s were o f t e n  were  of these v e s i c l e s  filamentous  except  Golgi  consisted  at the a p i c a l  number  of  cytoplasm,  Some were  of  the  also  cells.  t o be c o a t e d w i t h a  5C).  The number  advancing particles  filaments  were  fine  vesicles  gestation. were  w i t h desmosomes  of  A  seen  Small in  rarely  the  observed,  ( F i g u r e 5D).  Mature Amniotic E p i t h e l i u m (50, 58, 62 days) The in  shape,  (Figures was  epithelial and  cells  the  epithelium  4B, 6A, 7 A ) .  5.l9±0.24Mm  at this  The a v e r a g e  (±S.E.M.; n=30:  s t a g e were more appeared width  to  cuboidal  be  of the  thicker  epithelium  10 r e a d i n g s f o r e a c h  of 3  membranes). There The  was now  intercellular  dilated  than  microvi11i-1ike Numerous of  these  though  c o n s i d e r a b l e o v e r l a p p i n g between  spaces  in  the  folds  convoluted  epithelium,  vesicles  into  them  were seen  were  still  were much more a b u n d a n t  in  more  they  (Figure  had 6C).  i n the v i c i n i t y  present. these  and  and  Desmosomes were numerous a t  tight-junctions  microvilli  early  more  projecting,  membrane-bound spaces.  were  cells.  this The than  stage, apical in  the  44  F i g u r e 6.  The Fine Structure Epithelium.  of  the  Mature  Amniotic  Micrograph of e p i t h e l i a l c e l l s a t 50 days of gestation (0.74 of term) i l l u s t r a t i n g the d i l a t e d i n t e r c e l l u l a r spaces (ICS) and numerous a p i c a l m i c r o v i l l i (MV) c h a r a c t e r i s t i c of the mature amniotic e p i t h e l i u m . N: nucleus; ER: rough endoplasmic reticulum; M: mitochondrion; D: desmosomes; Ve: membrane-bound vesicles; G l y : glycogen; BM: basement membrane. X 13,000.  B:  A p i c a l p o r t i o n of e p i t h e l i a l c e l l a t 50 days showing the c l u s t e r s of microvilli (MV). Projecting from the t i p s of the m i c r o v i l l i are the f i n e branching f i l a m e n t s which make up the glycocalyx. The cytoplasm c o n t a i n s bundles of f i l a m e n t s (F) and glycogen ( G l y ) . X 26,000.  C.  I n t e r c e l l u l a r region of e p i t h e l i a l c e l l at 50 days. Microvilli-like f o l d s p r o j e c t i n t o the intercellular spaces (ICS). Numerous membrane-bound v e s i c l e s (Ve) are evident i n the v i c i n i t y of the spaces. D: desmosomes; F: cytoplasmic filaments; G l y : glycogen. X 26,000.  45  F i g u r e 7.  The F i n e Structure Epithelium.  of  the  Mature  Amniotic  A.  E p i t h e l i u m at 62 days of g e s t a t i o n (0.91 of term). Microvilli-like folds are present i n the i n t e r c e l l u l a r spaces (ICS). MV: m i c r o v i l l i ; F: cytoplasmic f i l a m e n t s ; ER: rough endoplasmic r e t i c u l u m ; D: desmosomes; N: nucleus. X 24,000.  B.  Microvilli (MV) on the a p i c a l s u r f a c e of an e p i t h e l i a l c e l l at 62 days. X 34,000.  C.  Micrograph of the apical region of an epithelial c e l l at 62 days i l l u s t r a t i n g a w e l l developed glycocalyx, composed of fine, branching filaments projecting from the m i c r o v i l l i (MV). D: desmosomes; G l y : glycogen. X 26,000.  ^5 A  46  earlier  specimens, and  in  longitudinal  sections  of  the  epithelium,  appeared as e l a b o r a t e networks in some regions  (Figures 6B,  7B).  more  The  prominent,  (Figures 6B,  s u r f a c e coat  with  7C).  longer and more branching  The  filaments  beyond  the t i p s of m i c r o v i l l i  As  the  in  early  present  in  change.  Deposits  still  the  cytoplasm,  present  in  numerous  and  particles the  extended  now  to  0.5/xni  of the amnion.  organelles  the n u c l e i showed resembling  mature  was  filaments  up  i n some areas  epithelium,  of  (glycocalyx)  were little  glycogen  epithelium.  were  Cytoplasmic  f i l a m e n t s were more abundant in the mature e p i t h e l i u m in the e a r l y e p i t h e l i u m , and throughout the c e l l  Near-Term Amniotic As the end cells  ( F i g u r e s 6B,  Epithelium  of  appeared  they o f t e n occurred  The  f l a t t e n e d , as seen in F i g u r e s 4C, of  the  readings As  epithelium  The  c e l l s were once more 8B;  the average width  (S.E.M.;  n = 30:  10  intercellular  spaces  were  dilated,  but  were none of the i n f o l d i n g s of the l a t e r a l membranes  There  of the mature amnion (Figures  the  8A,  8B,  9A,  were wide areas of open communication between  the i n t e r c e l l u l a r beneath  2.57±0.09Mtn  epithelial  i n the mature amnion, the c e l l s showed c o n s i d e r a b l e  characteristic 9B).  8A,  the  from each of 3 membranes).  overlapping. there  was  70 days)  approached,  degenerate.  i n bundles  7A).  (64, 68,  gestation  to  6C,  than  spaces and  epithelium  the  connective  ( F i g u r e s 8B,  9B).  tissue Tight  lying  junctions  47  F i g u r e 8.  E l e c t r o n Micrographs of the Near-term Epithelium.  Amniotic  A.  E p i t h e l i u m at 68 days of g e s t a t i o n (term). The e p i t h e l i a l c e l l i s f l a t t e n e d and appears to be degenerating. Note the disintegrating, abnormally flattened nucleus (N). The cytoplasm is filled with randomly-arranged f i l a m e n t s . Large v a c u o l e s (V) i n the cytoplasm may be the remains of o r g a n e l l e s . There are a few s h o r t , t h i c k m i c r o v i l l i (MV) on the apical surface. The f i l a m e n t s of the g l y c o c a l y x are shorter than i n the mature amnion. The intercellular spaces (ICS) are d i l a t e d but do not have m i c r o v i l l i - l i k e f o l d s of the lateral membrane projecting into them. There are numerous desmosomes (D), and tight junctions are never observed. BM: basement membrane.. X 20,000.  B.  E p i t h e l i u m at 70 days of g e s t a t i o n (judged to be slightly over-due). In the near-term amnion, the intercellular spaces (ICS) are often grossly dilated near the base of the e p i t h e l i a l c e l l and are i n open communication with the u n d e r l y i n g c o n n e c t i v e t i s s u e . MV: m i c r o v i l l i ; D: desmosomes; V: vacuole; BM: basement membrane. X 20,000.  <r7A  0 5Mm  48  Figure  9.  E l e c t r o n Micrographs Epithelium.  of the Near-term  Amniotic  A.  Micrograph of an epithelial c e l l at 68 days showing the randomly arranged 1Onm cytoplasmic filaments ( F ) . The a p i c a l c e l l membrane has a l a y e r of e l e c t r o n - d e n s e m a t e r i a l about 20nm t h i c k on i t s c y t o p l a s m i c s u r f a c e . ICS: i n t e r c e l l u l a r space; D: desmosomes; MV: m i c r o v i l l i . X 50,000.  B.  Micrograph showing the d i s i n t e g r a t i n g nucleus (N) of e p i t h e l i u m at 68 days. The nuclear matrix c o n t a i n s dense masses of h e a v i l y s t a i n e d heterochromatin and very l i t t l e of the lightly s t a i n e d , more d i s p e r s e d euchromatin. ICS: i n t e r c e l l u l a r space; BM: basement membrane. X 56,000. Desmosomes at the intercellular j u n c t i o n of epithelial cells at 68 days. Cytoplasmic filaments (F) appear to converge on the desmosomes. Note the t h i c k e n i n g of the apical c e l l membrane. X 110,000.  48 A  49  were never observed between c e l l s , and desmosomes were more numerous'now than at any membrane its  other  stage.  had an e l e c t r o n - d e n s e l a y e r  cytoplasmic  reminiscent  surface  of  (Figures  The  9A,  9C); (see  Montagna  and  The m i c r o v i l l i  were  less  abundant  now  the mature amnion, and appeared A g l y c o c a l y x was  still  evident but the  f l a t t e n e d and appeared  to be d e g e n e r a t i n g .  condensed  of  lighter seen  to be s h o r t e r and  not as long as i n the mature amnion.  masses  (heterochromatin)  intensely  ( F i g u r e s 4C, 8A,  stained,  more  to  euchromatin  Fawcett in  metabolically  active  contained  stained  chromatin  little  cells  those  of  the  (euchromatin)  (e.g., see F i g u r e s  nuclei  than  They  chromatin  (1981),  their  filaments  The n u c l e i were  9B) but  dispersed  i n the younger e p i t h e l i a  According  was  skin  than  were  this  keratinizing  1974).  thicker.  cell  (about 20nm t h i c k ) on  Parakkal, in  apical  5A,  with  6A).  abundant  are  generally  with  coarse  more  masses of  heterochromat i n . The  feature  that most d i s t i n g u i s h e d the e p i t h e l i a i n  t h i s group from the younger ones was the  cytoplasmic  observed  which  (Figures 8A, amnion.  may  8B).  have  and  (Figures 9A,  in  appeared 9C).  degenerating  No glycogen was  lOnm  almost  all  O c c a s i o n a l l y , vacuoles were  been  The cytoplasm was  approximately arranged  organelles.  a l o s s of  organelles  present i n the  near-term  f i l l e d with f i l a m e n t s which were diameter.  to  converge  These on  were the  randomly desmosomes  50  Epithelial  degeneration  appeared  c e l l s of the e p i t h e l i u m at 62 days of term)  (Figure  10A)  to be sudden. gestation  The  (0.91  of  were l a r g e , with a l l t h e i r o r g a n e l l e s  unchanged from t h e i r mature s t a t e , but by 64 days (0.94 term), c e l l degeneration was  3.  Fine  Structure  Incubation With A sample gestation  amnion  fixed  a f t e r a net water surface  of  the  c o n t a i n i n g AVP minutes.  the  Amniotic  from  for  flow amnion  at a  was  T h i s treatment  in  exposed  A control  at  38  of  days  of  immediately  which  the  fetal  to  amniotic  saline  100  mU/ml  for  15  sample  from  the  same  t r e a t e d in the same manner, except that a c o n t r o l  NaCl) i n s t e a d of AVP  s a l i n e f o r the 15 minute hydrostatic  pressure  a c e t i c a c i d and 0.5% was  test  period.  gradients  i n both p r e p a r a t i o n s .  diluted  during Electron  chlorobutol  i n t o the amniotic The  osmotic  and  incubation  were  micrographs  of  a m n i o t i c e p i t h e l i u m incubated with c o n t r o l s a l i n e were  compared with those of the AVP-treated amnion, as with  After  f o l l o w e d a 30 minute i n c u b a t i o n i n  in  the  Epithelium  fetus  concentration  ( c o n t a i n i n g 0.25%  identical  10B).  e l e c t r o n microscopy  solution 0.9%  a  experiment,  s a l i n e without hormone. f e t u s was  (Figure  AVP  of  was  of  complete  of  those  well  as  of an unincubated membrane taken from the same  f e t u s from which the other samples were o b t a i n e d . In  the  control  amnion, the e p i t h e l i a l  spaces were s l i g h t l y more d i l a t e d than i n  the  intercellular unincubated  51  0  Figure  10.  S t r u c t u r e of the Amniotic E p i t h e l i u m at 62 days (0.91 of term) and 64 days (0.94 of term). These micrographs show the sudden change from normal t o degenerating c e l l s , which appears t o occur between (A) 62 and (B) 64 days of gestation. MV: m i c r o v i l l i ; Nu: nucleus; BM: basement membrane. X 17,500.  SI A  M V  52  amnion.  However,  dramatic Figures out  increase 11A  and  using  cells  a  from  results:  11B).  MOP  t o the  right  space  AVP-stimulated  spaces  constituted  cells  by  hormone difference  and  fetuses  intact  the  fluid  Figure during  fluid  between  The  12  Table I I .  30  within shows t h e  Ten  from  the  following  intercellular cell  micograph.  the  In  intercellular the  equivalent  T h i s r e p r e s e n t d an i n c r e a s e  trtest No  260%  showed other  in  the  that  the  differences  between t h e u n i n c u b a t e d ,  control  epithelia.  Fluid  was  collected  from  days of g e s t a t i o n  a m n i o t i c sac was  gestation.  variation  cells,  A  AVP-stimulated  (see  carried  cells  electron  (p<0.00O.  4. The Volume of Amniotic Amniotic  ten  (S.E.M.) of  amnion.  were a p p a r e n t  a  image a n a l y s e r .  a r e a of a p p r o x i m a t e l y  is significant  structure  incubated,  i n the  19.04±2.68%  treated  was  (S.E.M.) o f t h e a r e a o f t h e  epithelial  space  was  spaces  compared, w i t h t h e  same p a r a m e t e r s .  intercellular  of the  e p i t h e l i u m , t h e mean  5.26±1.81%  of the  the  Videoplan  were  there  analysis  e p i t h e l i u m and  In t h e c o n t r o l  amnion,  dimensions  Morphometric  epithelium  a r e a was  in  the  Kontron  space  in  in  the c o n t r o l  AVP-treated  the  i n the A V P - t r e a t e d  punctured  emptied  rate  of a c c u m u l a t i o n  at the d i f f e r e n t  mean  guinea-pig  term  (68  with a needle  was  The  and  76  into a graduated  volumes  and a l l  cylinder.  of a m n i o t i c and  s t a g e s of g e s t a t i o n  the are  days).  fluid  range  of  shown i n  53  Figure  11.  E f f e c t of AVP on the Amniotic E p i t h e l i u m .  Fine  Structure  of the  A.  Amniotic epithelium at 38 days of g e s t a t i o n (0.56 of term) t r e a t e d with c o n t r o l s a l i n e f o r 15 minutes. The i n t e r c e l l u l a r space (ICS) i s not g r o s s l y d i l a t e d . MV: m i c r o v i l l i ; T: t i g h t j u n c t i o n ; D: desmosome; BM: basement membrane. X 28,000.  B.  Amniotic e p i t h e l i u m at 38 days t r e a t e d with l00mU/ml AVP f o r 15 minutes. There is a dramatic i n c r e a s e i n i n t e r c e l l u l a r space (ICS) area. MV: m i c r o v i l l i ; T: t i g h t j u n c t i o n ; D: desmosome; BM: basement membrane. X 28,000.  54  Figure  12.  The Volume o f A m n i o t i c of G e s t a t i o n .  Fluid  at Various  Stages  E a c h p o i n t r e p r e s e n t s a d i f f e r e n t f e t u s (n=76). Curve o f b e s t f i t e s t i m a t e d by computer ( l e a s t squares f i t t i n g t o B - s p l i n e s ) .  Gestational  age  ;  days  55  Table I I : The Volume of Amniotic F l u i d During • the Course of G e s t a t i o n  Gestational Age (days) 30 34 38 40 44 45 47 48 50 51 52 56 58 62 64 65 66 67 68  Volume of Amniotic F l u i d (ml) (Mean ± S.E.M.) 1.0 2.6 4.5 ± 4.4 5.9 4.7 + 6.5 ± 6.5 ± 6.4 ± 5.9 ± 6.7 ± 6.8 ± 3.7 ± 3.5 ± 1 .4 ± 5.5 ± 4.2 ± 7.1 + 6.3 ±  i  The mean volume between  30  0.3  4.0 - 5.0 -  0.2 0.5 0.2 0.3 0.5 0.4 0.7 0.6 0.3 0.5 0.3 0.5 0.8 0.5  4.2 6.0 5.7 5.0 4.7 5.1 5.0 3.1 3.0 0.5 5.0 3.5 5.3 3.6  of  fluid  Number of Observat ions  Range (ml)  was  -  1 1 3 1 1 5 2 7 6 4 8 4 2 3 3 4 3 4 14  5.0 7.0 7.4 7.0 6.8 8.0 8.0 4.3 4.0 2.0 6.0 5.0 9.0 10.5  1 .8±0.8  ml  (S.E.M.)  - 34 days and then rose g r a d u a l l y to a mean of  6.8±0.7 ml at 56 days.  Between 47 and 56 days , the  lay  range of 4.7 to 8.0 ml  within  a  narrow  time, there was a sharp d e c l i n e .  At  about  58  volume  After  this  days,  the  average volume was down to 3.7±0.6 ml and by 64 days i t had dropped to 1.4±0.5 ml. prior  to  term,  the  However, volume  after  64  days,  shortly  again i n c r e a s e d s h a r p l y , and  reached i t s maximum average value (7.1±0.8 ml) at 67 At  term,  the  average volume was s t i l l  Between 65 and 68  days  observed, with volumes  a  wide  range  high of  days.  (6.3±0.5 m l ) . variation  ranging from 3.6 t o 10.5 ml.  was  56  DISCUSSION The and  present  Perks  study c o n f i r m s the o b s e r v a t i o n by V i z s o l y i  (1974)  that  AVP  i s capable of causing a net  uptake of water a c r o s s the amnion, i n the maternal direction,  against  pressure.  gradients  Of p a r t i c u l a r  in  h y d r o s t a t i c and osmotic  importance i n t h i s study  d i s c o v e r y that the response  was found  the response  completely In  brief  vasopressin  had  through  vitro  magnitude  of  of  gestation).  After  this  of the amnion d e c l i n e d r a p i d l y , and was  statement,  Seeds  no  as  measureable  (1967) commented that  1000  mU/ml  effect  on  p r e p a r a t i o n s of  human  (vasopressor net water  amnion.  flow  However,  membranes used i n h i s study were o b t a i n e d a f t e r normal  delivery. is  days  i n doses as h i g h  activity)  the  The  l o s t by term.  a  in  the  t o i n c r e a s e with f e t a l age, up u n t i l  about 0.85 of term (58 time,  was  of the guinea-pig amnion t o AVP  changes d u r i n g the course of g e s t a t i o n . the response  to f e t a l  In l i g h t of the o b s e r v a t i o n s recorded  possible  that  the  species  it  n e g a t i v e r e s u l t s of Seeds may have  been due t o l o s s of response however,  here,  of the human amnion  differences  Whether the human amnion at  might  earlier  also  be  stages  at  term;  important.  of  gestation  responds to AVP remains t o be seen. The  changes i n the magnitude of the  guinea-pig  amnion  to  AVP  epithelium.  of the  during the course of g e s t a t i o n  were p a r a l l e l e d by changes i n the amniotic  response  The  fine  epithelium  structure changed  of the from  a  57  relatively  simple  r e s p o n s e t o AVP complex 0.75  and  of  with  structure  was  low,  possible  term  (50  microvilli,  and  glycocalyx. increase  filamentous  the  i n some o t h e r  toad  urinary  role is  that  amnion by the  has King  King,  1982).  guinea-pig lateral into  surface the  like  the  component  function (see  one  epithelia,  Another  gall  have  a  in epithelia  1981).  reported  i s a l s o present  1980)  and  change  the  the  borders.  The  microvilli-like  area sites  of  active solute  such  as  In  as  the  and  the  an  acid  1981).  It i s  specific  what t h i s  transport.  mammalian  that  guinea-pig  i n the  amnion  (Hoyes,  of  1968a;  in  the of  the  folds projecting  these In  an  i n some  colon  role  filamentous  i n the  further  addition,  (1980) o b s e r v e d the  such  increasing complexity  s p a c e s would  for transport.  is also  occurred  was  intercellular  here,  A  human  that  highly  rather but  greatly A  with 1965;  of  filamentous  bladder  charged  may  occurred  to  described  (Fawcett,  the  proliferation  for transport.  Fawcett,  (King,  changes t h a t  served  mammalian  ( 1 9 7 8 ) , and  study, Kashgarian tissues  about  epithelium  cell  the  by  probably  been p r e v i o u s l y  r h e s u s monkey  more  highly  negatively  remains obscure  be  to  a  area  glycocalyx  in transport  glycocalyx  be  the  appeared  the  of  transporting  is  mucopolysaccharide  were  changes  bladder,  and  when  striking  a p i c a l surface  found  believed  Two  development  glycocalyx  intestine,  that  gestation  These  the  t o one  in gestation,  more s p e c i a l i z e d i n f u n c t i o n  days).  advancing  early  and  increase folds  the may  interesting transporting renal  distal  58  tubules,  there  membrane  surface  complexity  of  increases that may  the  the  increase  increases  sites.  intercellular ion transport  High  region.  The  related  t o an i n c r e a s e  advancing  course  degeneration AVP.  Just  localization  changes  in  the  the  cytoplasmic membrane)  plasma  in  this  it  also  epithelium  i s not s u r p r i s i n g that the found t o i n c r e a s e term, of  during  epithelial  response  the  to a degenerated guinea-pig  epithelium.  amnion a p p e a r e d t o  62 and 64 d a y s o f g e s t a t i o n , and t h e l o s s  of  also occurred  cytoplasmic  filaments, are  to  i n r e s p o n s e t o AVP was a b r u p t , so  from a h e a l t h y  AVP  be  In l i g h t o f  guinea-pig  at roughly  i n the s t r u c t u r e of the e p i t h e l i u m  loss  detected  1974), and  may  function.  loss  between  changes  gestation  a total  occur  density  available  occurring  coincided  in  of  (Wynn,  before  degeneration  surface  at the l a t e r a l  Shortly  with  suggested  o f membrane-bound v e s i c l e s  in transport  gestation,  Cell  He  number  epithelium  advancing  as the d e c l i n e  to  activity.  of g e s t a t i o n .  was t h e change  response  with  o f ATPase have been  r e s p o n s e o f t h e amnion t o AVP was the  spaces, concomitant  of a c t i v e t r a n s p o r t  with  these maturational with  levels  i n c r e a s i n g number  the cytoplasm  and  i n the  i n t h e human a m n i o t i c  i s suggestive  i n the length  i n b a s o l a t e r a l membrane  electron microscopic  this  i n the base-lateral  a n d an i n c r e a s e  be r e l a t e d t o an i n c r e a s e  membrane  in  marked  density  in active  transport by  were  organelles,  and t h e  this near  the  thickening  s i m i l a r t o changes observed  of  time. term  The  (e.g.,  increase the  of  in  plasma  in keratinizing  59  skin  (Parakkal  and A l e x a n d e r ,  1972; Montagna and  Parakkal,  1974). Differentiation course  of  pregnancy,  guinea-pig, Hoyes, in  the  has  has  1968a;  apparent  cow  similar  to  been r e p o r t e d  1975),  keratinization  that  been  1982).  Cell  i n the guinea-pig reported  by K i n g  although  the  at  are  degenerating  numbers  of  term a p p r o a c h e s , but a u n i f o r m l y seen  in  and  amnion n e a r  term  two  human  weeks  amnion, cells  fetuses  (Hoyes,  appears  of  r h e s u s monkey amnion  1980).  s e v e r a l days before  took  term  the p e r m e a b i l i t y  w a t e r movement a c r o s s place  gradients.  against One way  coupling  of  solutes.  Holt  increased  water  the  flux  1979). _  same (King,  sloughs  on t h e amnion was n o t m e r e l y  of the e p i t h e l i u m  this  (1977a) sodium  in  and  transport ' to  of  The  (Tiedemann,  t h e amnion  i n which  1975).  epithelium  osmotic  and P e r k s  not observed except i n  amniotic  a c t i o n of v a s o p r e s s i n  increase  net  the  In t h e c a t , t h e e n t i r e  The to  true  as  e p i t h e l i u m as  membranes f r o m o v e r d u e be  there  evident  study,  to  of  degeneration  degenerating was  studied  present  off  the  degeneration  which  In the  the  1980), and  ( 1 9 7 8 ) , who  commenced was n o t m e n t i o n e d . increasing  in  ( B o u r n e , 1962;  (King,  i n the l a s t  time  during the  observed  i n t h e human  morphology of the e p i t h e l i u m  gestation,  epithelium  i n t h e r h e s u s monkey  (Tiedmann,  previously  the  of the amniotic  t o water, as  response  hydrostatic  could  occur  to  AVP  pressure is  by  a  the a c t i v e t r a n s p o r t of found  that  ions across  vasopressin  the guinea-pig  60  amnion,  in  time-frame that  the  i n which  water  vasopressin  the  t r a n s f e r of  may  transport  present  in higher  fetal  and  Slater  (1971),  is possible  It osmotic to  (see is  not  transepithelial that  The  (Diamond and  grossly  dilated  collapse  the  under  the  spaces  i s not  in  to M e l l o r  and pump  gallbladder, and  travel closely  sites  amniotic  epithelium,  transport  against  might  be  thought such  fluid  necessary prevailing is  channels,  with  are  as  1977;  local  It  t o be  tubule,  Oschman,  of  located.  intercellular  rabbit gallbladder,  conditions  in  electrogenic,  anatomical  tissues  when maximal  than  chloride  ions  an  1977a).  are  1967;  to  are  fluid  a  ions;  ions  mammalian  chloride  renal proximal  Bossert,  In  i n the  sodium  Chloride  of  effect  secondary  f o l d s p r o j e c t i n g i n t o them,  the  1980).  result  tortuous  coupling  gallbladder,  Perks,  on  according  amnion  gradients,  intercellular  water-solute  As  water  the  microvilli-like  and  the  the  within  net  tansport.  in amniotic  sodium and and  sodium  (1977a), the  ions.  same  suggested  have been  c l e a r where t h e  gradients  possible  be  across  Holt  effect  plasma  amnion.  that  Perks  to  the  this  have been d i r e c t l y  concentration  the  sodium t r a n s p o r t  and  within  flux;  coupled  chloride  t h i s may  and  water  ions could  of  maternal  in  be  Holt  not  sodium  active  together  could  s u g g e s t e d by  of  it  direction,  i t increased  transport  However, as  located  same  the  the  and  involved.  the  sites  mammalian intestine  Diamond,  intercellular transport  i n which t r a n s p o r t  spaces  occurs, is  of  1971; are but  inhibited  61  (Tormey  and  Diamond, 1967).  S p r i n g and Hope ( 1978, ,1979)  have observed the same phenomenon i n the l i v i n g of Necturus  gallbladder.  Treatment of  term)  of the amnion at 38 days of g e s t a t i o n  with  intercellular control  AVP  resulted  incubated  expansion of i n t e r c e l l u l a r observed  in  one at term  in  a  marked  (0.56  dilation  spaces i n the e p i t h e l i u m when compared  specimen  absence  epithelium  without  hormone.  spaces with  AVP  guinea-pig amnion at 0.76  to  structural  changes  in  treatment  term  i s c o n s i s t e n t with the l o s s of  hormone  near  intercellular  These  spaces may  was  of term, but not i n  treatment  term.  a  A similar  (Goh and Perks, unpublished o b s e r v a t i o n s ) .  of  of  amnion with response  results  suggest  p l a y a major r o l e  in  The AVP  to  the  that  the  AVP-mediated  flow of f l u i d a c r o s s the amniotic e p i t h e l i u m . King fracture  (1982) and B a r t e l s and analysis,  epithelial  cells  has  found  been  have  that  via  gap  amnion, which mechanism  c y c l i c AMP,  rapidly  junctions  (see  Fawcett,  nerves  or  to  neighbouring  1981).  blood  supply,  In the such  p l a y an important r o l e i n c o - o r d i n a t i n g  a m p l i f y i n g the response of groups AVP  freeze  r e l e a s e d i n response to  pass  no  by  In other t i s s u e s , i t  can  has  may  (1983),  i d e n t i f i e d gap j u n c t i o n s between  i n the human amnion.  hormonal s t i m u l a t i o n , cells  Wang  of  epithelial  cells  a and to  stimulation. The  changes  vasopressin  in  the  structure  and  of the amniotic membrane may  response  to  be r e l a t e d to the  62  long-term occur of  changes  term  fluid  approaches  adequate  without  early,  fluid  reaches  again  at  is vital  in relative  movement i n  utero  normal  growth  development  of  possible volume  o n l y when t h e u t e r u s of  fluid  p r o t e c t s t h e f e t u s from  and  from  mechanical  insulation  (Seeds,  Kerpel-Fronius uterus  by t h e a m n i o t i c  placental, also  (1970),  o f t h e volume by  stages  of  gestation  a d e q u a t e volume the  now  effects  less  f o r normal  necessary tissues,  for  and i s  1972).  uterine  the  1965). amnion  probably fetal  wall  thermal  According  fluid  to  of the  f o r normal  The a m n i o t i c  fetal  fluid  fetal,  fluid  may  imbalance  by  Vasopressin-mediated during necessary  the  earlier  t o ensure  g r o w t h , whereas n e a r  an  term,  v u l n e r a b l e f e t u s does n o t s u f f e r d e l e t e r i o u s  resulting  degenerating  (Seeds,  is  Reynolds,  The a m n i o t i c  i s necessary  f u n c t i o n as a b u f f e r a g a i n s t  control  fluid  develop  progressive distension  and u t e r i n e d e v e l o p m e n t .  as a r e s e r v o i r  can  The  a n d may a l s o p r o v i d e  the  serving  fetal  1983).  Reynolds,  fluid  term  1965;  a b r a s i o n by t h e  shock,  1965;  in early  i n which t o  is  An  i s d i s t e n d e d w i t h an a d e q u a t e  (Moessinger,  also  life  1967).  (Seeds,  Active fetal and  accumulation  approaching  1972).  that  1967).  to f e t a l  environment  weightlessness  fluid  a peak, and t h e n a s  a l l (Adolph,  spacious  the  (Adolph,  though the f e t u s any  provides a moist,  of a m n i o t i c  In most mammals,  begins  of  mid-gestation,  move  volume  diminishes  volume  survive  and  the  during gestation.  amniotic  and  in  from  amniotic  less  stringent  epithelium.  control  by  the  63  In amniotic then  the g u i n e a - p i g ,  as  in other  fluid  to  increase,  was  decline.  found  The  most  sensitive  time  i t s s t r u c t u r e appeared  role  in f l u i d  transport.  gestation,  of  the  consistent fluid  both  amnion  to  with  t h e volume increase  obscure. the  the in  sac  i n the  also  1935).  situation  again been  in  time  r a t e of u r i n e f l o w and  rapidly amniotic  fluid  reflects  this  stage;  lost. this  in  volume. amniotic  less  control  between  62  and  days the  13 g i v e s a summary of t h e  section.  species, Such  an  domestic  cat  increase  of  lung  fluid  i n an  that  volume j u s t  64  gestation.  is a relative  t h e volume by  and  are  remains  which r e s u l t s  fluid  response  for this  secretion  e p i t h e l i u m degenerates,  Figure  the  It i s possible  of  of  term.  reason  there  enlarging fetus,  term  amniotic  the  days  in supplying  i n most o t h e r  observed  active  results  80%  around  the  - 58  the  of AVP  just  At p r e s e n t ,  Perhaps at t h i s  first  at  with  56  These  and  time  w i t h an  volume and  role  the  and  approximately  fluid  peak,  with  t o AVP,  declined.  u n l i k e the  has  variability  in  After  a  compatible  physiological  increased  (Wislocki,  in  a  t o be  amniotic AVP  to the a m n i o t i c  Interestingly,  reach  peak c o i n c i d e d r o u g h l y  w h i c h t h e amnion was  of  s p e c i e s , t h e volume of  of  prior the  from  increase  the  great  t o and amnion  at at  g e s t a t i o n the  response  t o AVP  is  results  discussed  64  Figure  13.  G e s t a t i o n a l Changes i n the S t r u c t u r e of the Amniotic Epithelium, the Response to AVP, and the Volume of Amniotic F l u i d i n the Guinea-Pig  A.  Diagrammatic representation of the amniotic e p i t h e l i u m at 28, 50, and 64 days of g e s t a t i o n . S t r i p e d regions represent nuclei. The upper s u r f a c e faces the amniotic f l u i d .  B.  The changes i n the reponse of the amnion to AVP (100 mU/ml) from 30 to 68 days of gestation (0.44 to 1.00 of term).  C.  The volume of amniotic f l u i d between 30 and 68 days of g e s t a t i o n (0.44 to 1.00 of term).  Gestational  age  ; days  65  SECTION  II  THE EFFECTS OF ARGININE VASOTOCIN AND ARGININE VASOPRESSIN ON WATER TRANSPORT ACROSS THE SKIN OF THE FETAL GUINEA-PIG  INTRODUCTION The  fetal  epithelium, germ  and  layer  studies fetal  to  of  (1976) fluxes The  periderm, 1968b).  Holt  the  observed  that vasopressin  of  skin  transport  across  amphibian  increasing  bulk  to  the  s k i n of  of  the  rather  than  its  amniotic  the  skin  found  is  more  France  bi-directional the  fetal  fetal  s k i n to changes  water  effective  movement of  i t seemed i m p o r t a n t in bulk  the  However,  to vasopressin  of  lamb.  on  vasotocin  studies  that  skin.  s k i n of a m p h i b i a n s  Therefore,  on  mucosal-serosal  In a d d i t i o n , t h e  1965).  is  experiments  effects  diffusional  than  extra  water movement a c r o s s  of  vasopressin  same  the  s k i n of a n u r a n a m p h i b i a n s .  Bentley,  the  on  fetal  increased  the  reminiscent  skin,  1968).  responsive  water a c r o s s vasopressin  were  the  the  of  (1977a) i n the  amniotic  In a d d i t i o n ,  amnion,  increase  through  from  cells  those  Perks  water  isotopic  the  In p r e l i m i n a r y  and  the  1973).  resemble Like  with  derived  Rhodes,  tritiated  of  (Maetz,  and  c a u s e d a marked  effects  fetal  s t r u c t u r e s are  vasopressin.  guinea-pig,  flux  in  the  (Hoyes,  vasopressin  i s continuous  human have shown t h a t  layer,  responsive the  both  (Saunders  i n the  epithelium  epidermis  at water  i s more  (Heller to flow,  and  extend and  to  66  comparisons studies  AVT  presented  investigation skin.  of  of  w i t h AVP. below,  the  fine  This i s carried  together  with  a  out  i n the  preliminary  s t r u c t u r e of g u i n e a - p i g  fetal  67  RESULTS  1. The  Isolated Fetal  Skin of  from  term) was  In t h e  35 m i d - t e r m  of  or  fetal  vitro,  hormones,  preparations serosal  2.  The  E f f e c t s of AVT  the  with  skin  activity)  to  Figure  AVT at  and AVP  the  majority  of  additional in  of  the  water  in  preparations  the  mucosal  to  fetus),  and  in 2  water  from  the  of  on Net Water Transport  14  III  side  of  illustrates  and  a p p l i e d to the of  these net  serosal  base-line  doses  i n the  responses obtained Tables  of  surface.  o r AVP  showed t h a t  increase the  methods.  gradients  7  towards  0.70  no  movement  water  to  Across  Skin  Tests  an  with  a slow p a s s a g e  t o the mucosal  Fetal  of  (i.e.,  t h e r e was  i n the  net  However,  direction  described  pressure,  showed a s p o n t a n e o u s u p t a k e of serosal  (0.49  and  (n=26) showed no  direction.  guinea-pigs  as  hydrostatic  preparations either  in  s e t up  absence  osmotic  Skin  5  to  peptides  serosal  100  mU/ml  are  capable  t r a n s f e r of w a t e r the the  t o the  skin  show  all  (resting)  flow  rate,  causing  from the  mucosal  the  the  fetus).  (±S.E.M.) f o r a l l the  v a r i o u s doses  IV  (vasopressor of  (towards  averages  surface  of  values  AVT  and  obtained  hormone-influenced  AVP. for flow  68  Figure  14.  The E f f e c t s o f D i f f e r e n t D o s e s o f AVT and AVP on t h e Net Movement o f Water T h r o u g h the Skin of the F e t a l Guinea-pig ( a t 0.49 - 0.70 o f term).  2  Ordinates: t h e r a t e s o f w a t e r movement (mg/cm per minute). The d o t t e d l i n e represents zero change; v a l u e s above t h i s l i n e r e p r e s e n t w a t e r uptake towards the s e r o s a l ( f e t a l ) s i d e of the skin, whereas v a l u e s below t h i s l i n e r e p r e s e n t water flow towards t h e mucosal s u r f a c e of the skin (towards the a m n i o t i c f l u i d ) . Abscissae: time, i n minutes. Arrows indicate time of addition o f hormone. A l l values a r e expressed as t h e mean ± t h e s t a n d a r d e r r o r o f t h e mean (S.E.M.).  Rate  of  Water  Movement • mg/cm o  in  o  O b  O In  in  2  min O b  o  O  »3 c  \ /  /  /  i  \  I  \  3 t» 3  5  5  o Ul  \  •o  3 c  •H  o b  3  3  c  o  en  o  b  O  —  I\J  bi  ui  b  o Ul  o b  o  ui  — b  / /  Ul  o o  o  ui  — b  O Ul  o b  o  — b  ui  1  JT"  (O -  o  3 c > <  \  <  u  O Ul  o b  o Ul  3 c  5  \  I  \ H  /  < 00 >  69  rate, for as  and t h e i n c r e a s e  AVT and AVP, the average  water  flow  i n flow  respectively.  increase  in  in  before  rate  of  flow  of  25  AVT was  the  a  total  of  rate  mucosal-serosal  was  of 22 t e s t s w i t h  mU/ml.  but these  In t h r e e  replaced with  a  the a d d i t i o n of defined  as  the  immediately  resting  Although  a large v a r i a b i l i t y  hormone  general  existed  trend with  occurred  within  average,  the  doses  original  14).  Most  complete  appeared  to  hours a f t e r  III).  fresh  within  10  f o r more t h a n to continue be s e n s i t i v e  the  that  the  30 m i n u t e s  for  t o hormone  dissection.  In  period.  of  a the  addition.  On  sooner  with  returned  after  response  to  of AVT by  recovery  periods.  was  hormone did  The  f o r approximately  most  dose  was  i n experiments  longer  no  R e s p o n s e t o AVT  the removal  minutes  were  there  preparations  and  or  i n which  test  was a c h i e v e d  salines,  at  to a given  hormone  rate after  I t was o b s e r v e d  last  (Table  (Figure  usually  the  i n the magnitude  of flow  to  doses  preparations,,  rate  the skin  were a l l o w e d  doses  flow  showed no  there  in sensitivity  peak  resting  five  to  during  3 to 9 minutes a f t e r  returning  withdrawal.  rate  between  higher  were  saline,  t o w a r d s an i n c r e a s e  response  usually  flow  AVT,  control preparations,  control  in  the  rate  i n t h e 12 m i n u t e p e r i o d  changes  higher  response),  The r e s p o n s e was c a l c u l a t e d  flow  r e s p o n s e t o t h e hormone,  of  the  t h e a d d i t i o n of hormone.  Out  below  (i.e.,  t h e 15 m i n u t e p e r i o d a f t e r  hormone, a n d t h e r e s t i n g average  rate  preparations,  not  which skin two two  Table I I I : The Response of the F e t a l  Dose of AVT (ntU/ml of vasopressor act 1 v i t y )  Rate of RestIng Flow (mg/cm per minute)  Rate of AVTInfluenced Flow (mg/cm per minute) 2  2  S k i n t o AVT  Response: The Increase i n Rate of Mucosal-Serosal Flow with AVT (mg/cm per minute)  Average Response (± S.E.M.) (mg/cm per minute)  p-values  4  2  3  2  C o n t r o l s (n=3) 5  10  25  50  100  Note:  0  0  0  +0..48 0 +0., 1 1 +0..47  0 .48 0 0 0  0 0 0 +0.. 28  +0..26 +0. 32 0 +0. 56  0..26 0,. 32 0 0..28  0 +0. 07 0 +0. 21 0 0 0  +0. 34 +0. 17 +0. 72 0 +0. 42 +0. 33 +0. 56  -0. 14 0 0 +0. 20 0 0 0  0 0 +0 .22 +0,.59  1  0  0.12±0.12  p>0.05  0.21+0.07  p>0.05  0..34 0,. 10 0.,72 0 0.,42 0. 33 0..56  0.35±0.09  p<0.01  +0. 33 +0. 50 +0. 42  0. 47 0. 50 0. 42  0.46±0.02  p<0.01  + 1 .17 +0. 95 +0. 75 +0. 77  0. 97 0. 95 0. 75 0. 77  0.8610.06  p<0.001  P o s i t i v e values (+) I n d i c a t e flows 1n the mucosal-serosal d i r e c t i o n (towards the f e t u s ) . Negative values I n d i c a t e flows 1n the serosa 1-mucosa 1 d i r e c t i o n (towards the amniotic f l u i d ) . For an e x p l a n a t i o n of terms (1 - 4 ) , see the notes at the bottom of Table IV.  (-)  ^  Table IV:  Dose of AVP (mU/ml of vasopressor act 1v1ty)  Rate of Resting Flow (mg/cm per minute) 1  2  The Response of the F e t a l  Rate of AVPInfluenced F l o w (mg/cm per mlnute) 2  2  Skin t o AVP  Response: The Increase i n Rate of Mucosal-Serosal Flow with AVP (mg/cm per minute)  Average Response ( + S.E.M.) (mg/cm per minute)  p-values  4  2  3  2  10  25  50  100  Note: 1. 2. 3. 4.  0 0 0  0 0 0  0 0 0  -0.08 0 +0.11 0 0 +0.24 0  0 +0.21 +0.50 +0.45 0 +0.28 +0.13  0.08 0.21 0.39 0.45 0 0.04 0.13  0 +0.09 0 +0.24  +0.56 +0.35 +0.24 +0.47  0.56 0.26 0.24 0.23  +0.47 +0.55 +0.36 +0.66  0.47 0.55 0.36 0.66  0 0 0 0  o  0.19±0.07  p<0.05  0.32+0.08  p<0.02  0.51±0.06  p<0.001  P o s i t i v e values (+) I n d i c a t e flows 1n the mucosal-serosal d i r e c t i o n (towards the f e t u s ) . Negative values (-) I n d i c a t e flows i n the serosal-mucosal d i r e c t i o n (towards the a m n i o t i c f l u i d ) . The r a t e of r e s t i n g flow 1s the average r a t e of flow d u r i n g the 12 minute p e r i o d Immediately p r i o r to treatment with hormone. The r a t e of hormone-1nf1uenced flow 1s the average r a t e of flow In the 15 minutes Immediately a f t e r hormone addi t ion. The response i s the i n c r e a s e i n the r a t e of mucosal-serosal water flow; i t i s the d i f f e r e n c e between 1 and 2. No change, or a decrease 1n mucosal-serosal flow 1s taken as zero response. The p values r e f e r t o Student's t - t e s t s ; they are based on the Increase 1n flow r a t e In the mucosal-serosal direction.  72  clear  responses  period.  However,  limited the  to f i r s t  addition  before III),  water  tests  a  as  (Student's  estimates  were  flow  higher  t-test;  were  performed  with  in  after  than  those  see  Table  with  AVP.  dose  of  AVP.  IV).  Of  of  the  rate of 10 mU/ml  response  The p a t t e r n o f r e s p o n s e f o r AVT  treatment;,  10  minutes  withdrawal.  a s seen  in Figure  t o AVP was i n g e n e r a l  with was t h e  the  skin  hormone a d d i t i o n , a n d  within  the response  mU/ml.  As w i t h AVT, t h e r e was  w i t h i n 3 t o 9 minutes a f t e r  However,  25  w i t h AVP a t d o s e s above  magnitude  described  below  i n c r e a s e i n the average  see T a b l e  the  doses  as w i t h AVT, r e c o v e r e d  of  hour  10 mU/ml.  were  treatment  t-test,  that  responded  two  The mean r a t e s o f  significant  flow a f t e r  increasing  the  d i d n o t show any i n c r e a s e i n m u c o s a l - s e r o s a l  t r a n s p o r t ; these was  within  AVT were s i g n i f i c a n t l y  o f 18  increase  same  responses.  of  four  (Student's an  obtained  i n q u a n t i f y i n g the data,  w i t h a l l d o s e s above  these,  There  be  t h e hormone was added  A total  water  could  after  hormone  14, t h e m a g n i t u d e  lower  than  that f o r  AVT. Despite considerable v a r i a b i l i t y in  their  a linear  sensitivity  t o a g i v e n dose of e i t h e r  l o g dose-response  peptides  (Figure  mU/ml a n d  f o r AVP  activity).  There  between  relationship  existed  1 5 ) . The t h r e s h o l d d o s e i t was appeared  the magnitude of t h e response a g i v e n dose of e i t h e r  10.4  mU/ml  preparations AVT o r AVP, f o r t h e two  f o r AVT was 3.9 (of  vasopressor  t o be no r e l a t i o n s h i p  between  a n d t h e age o f t h e f e t u s f o r  AVT o r AVP.  73  Figure  15.  Log Dose-Response Curves f o r the E f f e c t s of AVT and AVP on Net Water Movement Through the F e t a l Skin (at 0.49 - 0.70 of term).  A l l v a l u e s are expressed as the mean ± standard e r r o r of the mean (see T a b l e s I I I and I V ) . Ordinate: the increase in the r a t e of mucosal-serosal water flow during treatment with e i t h e r AVT or AVP ( i n mg/cm per minute). Abscissa: Dose of AVT or AVP (in mU/ml, vasopressor a c t i v i t y ) , p l o t t e d on a l o g s c a l e . L i n e s of best f i t computed by the method of least squares. (AVT: y = 0.518X - 0.305, r = 0.94, p<0.05; AVP: y = 0.501X - 0.510, r = 0.99, p<0.05). 2  73A  74  3. Fine S t r u c t u r e of the F e t a l Epidermis Skin at  35  taken  days  gestation  from t h e  of  (0.76  back  gestation of  region (0.51  term) was  of of  fetal  guinea-pigs  t e r m ) and  examined  under  52  the  days  of  electron  microscope. At  35  days,  germinative outer  periderm  i n the  were  projecting generally the  apical  surface  projecting thin  microvilli. structures observed contained  basal  and The  coat  of  r e s t of  had  (Figure  18A).  mitochondria  The and  cavity  all  the  the  and  the  signs  of  layers  the  folds were  surface.  18A,  The  18B).  surface  the  of  microvilli  of  A the  periderm,  and  occasionally periderm  membrane-bound v e s i c l e s .  layers.  the  periderm  of  were  reticulum  of  c e n t r a l part  i n the  c y t o p l a s m of  appeared  basal  throughout  (Figures  junctions  rough endoplasmic  intermediate  no  a number  o b s e r v e d on  tight  a  microvilli-like  cells  above t h e  was  resembling  in  i n some a r e a s t h e  amniotic  of  Large q u a n t i t i e s  spaces  had  periderm  These o r g a n e l l e s and  stage.  Desmosomes were p r o m i n e n t  c o m p l e x e s and seen.  the  i n t o the  filamentous  this  were o b s e r v e d  them.  elevated of  T h e r e were  Intercellular  f l a t t e n e d , but was  consisted  intermediate-layers,  17).  skin at  dilated,  into  cell  two  16,  glycogen  epidermis.  epidermis  or  (Figures  intracellular  the  epidermis  l a y e r , one  keratinization of  the  cells Golgi  were o c c a s i o n a l l y  t o be  more abundant  in  the  Nuclei  were o b s e r v e d  in a l l  75  Figure  16.  Fine Structure Days.  of  the  Fetal  Epidermis  at  35  The e p i d e r m i s c o n s i s t s of a b a s a l l a y e r ( B ) , 1 or 2 i n t e r m e d i a t e l a y e r s ( I ) and a s u p e r f i c i a l layer, the periderm (P). On the apical (mucosal) surface of the periderm are m i c r o v i l l i (mv) w h i c h p r o j e c t i n t o t h e a m n i o t i c cavity. There are l a r g e d e p o s i t s of g l y c o g e n (gly) in all layers of the epidermis. I n t e r c e l l u l a r s p a c e s (ICS) a r e d i l a t e d and have infoldings of the plasma membranes. All layers, including the periderm, have large nuclei (n). X 18,000.  75 A  76  Figure  17.  Fine Structure Days.  of  the  Fetal  Epidermis  a t 35  This micrograph shows t h e l a r g e d e p o s i t s of glycogen ( g l y ) c h a r a c t e r i s t i c of early fetal skin. B: basal layer; I: intermediate layer; P: p e r i d e r m ; ICS: intercellular spaces; mv: microvilli. X 18,000.  77  Figure  18.  The F e t a l Periderm at 35 Days. The cytoplasm c o n t a i n s membrane-bound v e s i c l e s (Ve), n u c l e i (N) , mitochondria (M), and glycogen (gly). Glycogen i s a l s o observed i n the l a y e r beneath the periderm (micrograph B ) . The m i c r o v i l l i (mv) have a t h i n filamentous c o a t . T: t i g h t j u n c t i o n ; D: desmosomes; ICS: i n t e r c e l l u l a r spaces; F: f i l a m e n t s . A. X 33,000 B. X 37,000  77 A  78  the  layers,  including  the periderm  f i l a m e n t s were o b s e r v e d cells,  (Figure 16).  i n the cytoplasm  b u t were r a r e i n t h e o t h e r  were seen  mainly  i n the v i c i n i t y  of  layers.  Bundles  the  of  periderm  These f i l a m e n t s  of t h e desmosomes  (Figures  18A, 18B). By  52  days,  keratinization spaces  the  skin  (Figures  were l e s s d i l a t e d ,  declined.  There  showed  19A,  definite  19B).  The  and t h e amount  was e v i d e n c e  uppermost o f t h e i n t e r m e d i a t e l a y e r s . electron-dense bundles with  of c y t o p l a s m i c f i l a m e n t s . the  precursors  associated of k e r a t i n  definitive of  stratum  flattened  cells  in close  and  (1959);  they  with c l o s e l y (Figure  thought  to  Parakkal,  was e v i d e n t b e n e a t h  mammalian  1974).  The  h a d now  periderm  was  disappeared.  to those  (Figure  filaments  (about  did  show  not  The  "keratin  filled  embedded  1Onm  defined  A  layers  the periderm.  as  the  were d e v o i d o f a l l o r g a n e l l e s a n d were fibrils  skin,  i n an amorphous  flattened  The c e l l s  matrix  and t h e a p i c a l  of the  periderm  o f t h e s t r a t u m corneum i n t h a t  t h i c k e n e d p l a s m a membranes,  organelles  be  Brody  packed  similar  together  by  19B).  microvilli  i n the  proximity to  These g r a n u l e s , are  had  contained  o f t h e s t r a t u m corneum d i s p l a y e d t h e t y p i c a l of a d u l t  had  (Montagna  formation  corneum, composed o f one o r two  cells,  pattern"  were  fibrils,  glycogen  This layer  keratohyalin granules  of  intercellular  of  of k e r a t i n  signs  19B).  The  and  lost  cytoplasm  in diameter).  the k e r a t i n  had  was  However,  p a t t e r n observed  they  a l l .their filled  the  with  periderm  i n the stratum  79  Figure  19.  F i n e S t r u c t u r e of the Days.  Fetal  Epidermis  at  52  A.  Survey micrograph of the keratinizing epidermis. P: periderm; SC: stratum corneum; I: intermediate layer; B: b a s a l l a y e r ; De: dermis; K: k e r a t o h y a l i n granule.- X 9,000.  B.  Higher magnification micrograph of the epidermal surface. P: periderm; SC: stratum corneum; I : intermediate l a y e r ; f: filaments; K: k e r a t o h y a l i n granule. X 42,000.  80  corneum; embedded cells  the  filaments  were l o o s e l y  i n an amorphous g r o u n d  appeared  t o be s e p a r a t i n g  corneum, and i n some r e g i o n s There  were  desmosomes granules  no  tight  appeared were  s t r a t u m corneum.  not  substance. from  had  junctions to  have  packed,  a n d were n o t The  the u n d e r l y i n g  disappeared  periderm stratum  altogether.  i n t h e p e r i d e r m , and t h e  degenerated.  observed'in either  Keratohyalin  the periderm or the  81  DISCUSSION The and  AVP  results  presented  here  a r e c a p a b l e of c a u s i n g  a  have shown t h a t net  across  the  skin  of t h e  fetal  serosal  direction  (i.e.,  towards the  the  potential  absence  amniotic  o f any  apparent  both  movement  of  AVT  water  g u i n e a - p i g i n the mucosal  cavity).  fetal  circulation  This  occurred  g r a d i e n t s i n osmotic  or  to  from  i n the  hydrostatic  pressure. A linear  relationship  and  the  i n c r e a s e i n the  for  both p e p t i d e s .  unlikely  to  the  (Heller  of  and  of  was  the  guinea-pig Perks,  t o AVP.  similar  consistently The  log  dose,  responses log  here  have  also  exist  peptides  addition, been  amnion  were  dose-effect  on  b l a d d e r of a n u r a n In  flow,  for water  amphibians linear  observed  for  diffusional  (Vizsolyi  log  and  the water  Perks,  1977a). response to  (1974) f o r t h e a m n i o n . skin  Linear  1965).  p a t t e r n of the  remarkably  Perks  the  v a s o p r e s s i n on b o t h b u l k and  H o l t and The  and  relationships  flow a c r o s s the 1974;  that  neurohypophysial  Bentley,  the  o f m u c o s a l - s e r o s a l water  artefacts.  a c r o s s the s k i n  dose-response effects  between  s u c h as t h o s e o b s e r v e d  actions  absorption  rate  T h i s suggested  be  relationships  existed  of the s k i n  that  observed  t o AVT  and  AVP  by V i z s o l y i  and  However, u n l i k e  showed h i g h e r  t h r e s h o l d s o f t h e amnion  the  responses t o AVP  and  amnion,  t o AVT AVT  than were  82  1.0  and 6.4 mU/ml (of vasopressor a c t i v i t y ) ,  while 3.9  the corresponding values  mU/ml.  for the s k i n were 10.4 and  The f e t a l s k i n i s s i m i l a r t o the s k i n of anuran  amphibians,  i n that i t responds more r e a d i l y t o v a s o t o c i n  than t o v a s o p r e s s i n that  ( H e l l e r and Bentley,  the r e c e p t o r s  i n the  fetus  d i s t i n g u i s h between two p e p t i d e s amino  acid;  AVP  octapeptide,  has  whereas  Sawyer, 1970).  AVT  At present,  skin  are not  identical  solutions  suggests that France are  reabsorption  isoleucine  instead  on net water t r a n s p o r t across the  of  but the f a c t that water  any  bathing  apparent  both  stimulating  across  (see  the mechanisms of a c t i o n of the  gradients,  surfaces  has observed that v a s o p r e s s i n of  by one  a t p o s i t i o n 3 of the  an  the s k i n  of  with  transport.  and v a s o t o c i n  increase fetal  flow  of the s k i n ,  i t may be coupled t o a c t i v e s o l u t e  (1976)  capable  I t appears  which d i f f e r only  has  known,  occurs i n the absence  1965).  are complex enough t o  phenylalanine  neurohypophysial peptides fetal  respectively,  in  sheep  sodium from  an  i s o t o n i c or d i l u t e e x t e r n a l medium, and thus i t i s p o s s i b l e that  water t r a n s p o r t  or a c o t r a n s p o r t  i s coupled t o e i t h e r sodium  system.  Unlike  transport  the s i t u a t i o n i n amphibian  s k i n , no s i g n i f i c a n t p o t e n t i a l d i f f e r e n c e e x i s t s across the s k i n of the f e t u s , even i n the presence transport  ( M e l l o r , 1970; L i n d et  In t h i s r e g a r d , such  uphill  al. , 1972; France,  the s k i n resembles other  as the g a l l - b l a d d e r e p i t h e l i u m ,  transport  of  occurs without an apparent  leaky  sodium 1976).  membranes,  i n which a c t i v e Na/Cl potential  difference  83  (Diamond,  1971).  Studies the  in  human have  arginine  sheep,  shown t h a t  vasotocin  (Vizsolyi Skowsky  and and  increases  steadily  exists  fetal  gestation. around  In  and  Perks  and  1977).  AVP  is  present  concentration  in  fetal  towards  the  fetal  Perks,  the  and  1973).  discovery  i n t h e mammalian  the  similar  role  during  relic  present  fetal  highest before  of  study  life,  peptide  has  of AVT  s k i n , and  evidence  seal,  AVT  is  lost  physiological  role  on  as  term  adult  (Perks  It i s p o s s i b l e that  and  and  Perks;  Fisher  human f e t u s . was  see  Perks  (1977)  have  Prior  thought Whether  o r whether  this  to i t s to  AVT  be  has  i t i s merely  i s not  yet  an but  across  fact  the the  a possible clue.  known,  a  i n i n f l u e n c i n g water t r a n s p o r t  the  in  plentiful  decline i n the  of  higher  skin keratinizes, that  vasotocin  The  q u a n t i t i e s in f e t a l the  pituitaries  portion  evolution  provided  throughout  a  f e t u s , AVT  fetal  1973;  only  (Kontor  i n the  neurohypophysis  However,  t o sub-mammalian v e r t e b r a t e s .  effectiveness the  trend  and  Vizsolyi,  levels  1977).  Skowsky  a  inactive  its  guinea-pig  observed  specific  lamb and  but  i s a l s o t r u e of  restricted  for  i t i s completely  1973;  Vizsolyi,  birth.  pituitaries  and  Vizsolyi,  in  and  vasopressin  1969;  mid-gestation,  approaches,  arginine  guinea-pig,  fetal  Fisher, its  the  the  Perks,  and  fur seal,  both  exist  gestation,  in  the  that  this  peptide  pituitaries gives fetus,  skin.  prior  strong AVT  i s found to  in  mid-term,  circumstantial finds  its  true  84  During secretes saline  the l a s t  AVP  in  and d e x t r a n  Weitzman  et  observed  that  rhesus  al.  in  to  infusions  (Alexander  1978).  results  of  and P e r k s AVP  water  et  early  gestation, from  stress;  i t may  re.absorption  a marked  The  from  doses to  However,  across  into  the  sensitivity  in  reasons  sensitive and  from  vitro  like  the  that  study,  to  In l i g h t of  postulate  skin  cause  to  However, a t  that  AVT i s  to  similar water  present  i n the f e t u s . affect  the  skin  be c o n s i d e r e d p h a r m a c o l o g i c a l . to  suggest  that  of t r u e p h y s i o l o g i c a l  well  environment  have c a u s e d  Vasopressin-induced  the toad bladder  1981; D a v i s  et  the  effects: and i t s  a decline in water  flow  i s known t o be h i g h l y  t o t h e c o n c e n t r a t i o n o f sodium a n d Ausiello,  AVP  response  which  i t s natural may  to  the  t o the osmotic  amnion.  fluid.  exist  reflections  t o hormones.  tissues  on  in  ( a n d AVP)  be h i g h and must  were  maintenance  act  fetal  keratinization,  o f AVT s e c r e t i o n  o f AVT  Removal o f t h e s k i n  present  (1973) have  i n c r e a s e i n serum AVP  the  to skin  the amniotic  several  responses  across  al.  i n response  pituitary  then  lamb  1974; 1976;  (1974) have s u g g e s t e d  prior  the f e t a l  i s no e v i d e n c e  appear  al . ,  infusion  secretion  movement  secreted  (Roy  et  o b s e r v a t i o n s , i t seems r e a s o n a b l e  there  fetal  haemorrhage, or h y p e r t o n i c  Skowsky  in  the  may l i e i n t h e need t o t r a n s p o r t s u f f i c i e n t  control these  gestation,  response  ,  Vizsolyi  significance stimuli  of  hypertonic saline  monkey  levels.  third  al.  calcium  ions  , 1981), a n d i n t h e  t h e s a l i n e s may have been l e s s  than  ideal.  85  In  a d d i t i o n , a l l hormone m o l e c u l e s  their in  target  vivo  sites  situation,  effectively  carry  Finally,  in  a  effects Brown,  of  It  AVT  water  be  has  perfusing to  may in  the  are  reached  skin;  in  skin  would  site  of  an  action.  situation,  the  in synergism with  other  adult  California (ACTH) and  thought  transfer across situation  the the  its  act  interesting net  been  various  vertebrate  reduces the  fluid  found  may  to  the also  Newt  thyroid  enhance  skin  the  (Brown  exist  transfer  most  (Hirano,  (Brown and it  and  in  the  discussed  previously,  1982; the  bladder  of  amniotic  Perks,  1975;  Leontic  it  s i m i l a r e f f e c t s on  et  across  al.  it  For  fetal  water in  reduces example,  skin  to  et  Brown  (see  Hirano,  the  fetal  all  the  membrane  in  some  actions  the  of In  gills, As  reduce  the  (Holt  and  i t is possible  that  appears  1979), and  movement  1980).  membrane t o ,  skin.  al . , 1983). of  of  prolactin  water  permeability  prolactin  permeability  effects  a p p e a r s t o oppose t h e  reduces  urinary  cases  the  Brown,  and  the  of  and  intestine,  the  surfaces  1980).  permeability  fish,  study  t o a f f e c t i o n and  in  amphibians,  vasotocin  to  osmoregulatory  classes;  permeability  has  of  physiological  (TSH)  Such a  on  Prolactin  teleost  thickness  have  fetus.  prolactin  urodele  hormone  not  adrenocorticotrophin  on  would  across  the  hormone  1982).  mammalian  blood  example,  torosa),  stimulating  the  peptides  For  (Taricha  t o the  true  neurohypophysial hormones.  due  may  to water  skin.  86  The  structure  keratinization transport.  of  is  compatible  In the a d u l t  epidermis  (the  dehydrated  c e l l s , and  being  virtually  Parakkal,  1974).  active.  intercellular were  it  probably  the  site  were  present  suggested  that  glycogen, 1954).  throughout between found  sloughed in  tight  in f l u i d  layer  study,  on  Montagna  in a similar  of AVT. a l l  the  fetal  glycogen  Large  the  observed  skin  liver  vesicles,  dilated  Adjacent  may  cells.  also The  cells  desmosomes.  quantities  I ) , and  of  I t has  fetal  and  function  wide  off.  the a m n i o t i c  keratinization  It displayed similar  of  The  energy spaces  e p i t h e l i u m b e f o r e term.  The  fluid  periderm  before  changes as  for  (Rothman,  intercellular  a m n i o t i c compartments.  t o undergo p a r t i a l  been  s e r v e s as a s t o r a g e organ  represent a source  be  glycogen  t h e e p i d e r m i s c o u l d f a c i l i t a t e e x c h a n g e of  the  The  I t might  layers.  assumes t h i s  the  (amniotic)  fashion.  epidermal  and  layer,  apical  and  other  physiologically  mitochondria. junctions  dead,  most  epidermal  their  the  function,  i t was  t o be  of  of  the a m n i o t i c e p i t h e l i u m ( s e c t i o n  in  epidermal  role  and  1972;  membrane-bound  and  b e f o r e the  The  water  which appeared  functions  of a c t i o n  before  composed  t h e uppermost  nuclei,  resembled  to  present  microvilli  by  is  Alexander,  the  spaces,  attached  periderm  was  and  cells  large  active  skin  serves a purely p r o t e c t i v e  In  They had  surface,  for  corneum)  i n t h e young f e t u s , had  w i t h an  impermeable  (Parakkal  periderm,  guinea-pig  mammal, t h e uppermost  stratum  substances  that  fetal  those  being seen  disappearance  87  of  the periderm  corneum  was p r e c e d e d  resembling  that  periderm has p r e v i o u s l y level  in  the  ( B r e a t h n a c h and W y l l i e , Hashimoto, and  1966;  Alexander,  resemble  that  of t h e a d u l t  been  human,  by t h e f o r m a t i o n o f a  studied  rhesus 1965;  Bonneville,  1972).  (Brody,  1959).  and  1968; Hoyes,  mouse,  and  Larsson,  rat 1965;  1968b; P a r a k k a l .  The g u i n e a - p i g p e r i d e r m was  o f t h e o t h e r mammals.  The  at the u l t r a s t r u c t u r a l  monkey, Brody  stratum  found t o  88  GENERAL DISCUSSION  The the not  experiments d e s c r i b e d  ability  of the guinea-pig  I have shown that  amnion to respond to AVP  the same throughout g e s t a t i o n ; there appears  progressive AVP  in Section  increase  in  to  the  is  followed  of  observation amnion  hormones  on  the  Future  studies  early  in  amnion  must  s p e c i e s may  that  not  does  term,  take  the this  in  the  g e s t a t i o n , c l o s e to term, or a f t e r  normal d e l i v e r y in other amnion  a  term.  of  i n t o c o n s i d e r a t i o n ; a lack of response  very  the  of  by an abrupt d e c l i n e , such that by  response i s reduced to z e r o .  effects  be  the magnitude of the response to  with advancing g e s t a t i o n , up u n t i l about 0.85  This  is  not  necessarily  mean  respond to the hormone at a l l .  Membranes from f e t u s e s at a l l stages of g e s t a t i o n  must  be  t e s t e d to a s c e r t a i n the true s i t u a t i o n . Although the guinea-pig studied 1968;  at  the  ultrastructural  King, ^978), there has  investigation gestation. amnion days), amnion  The  between has  epithelium  of  changes  present 28  and  been  level no  occurring  study 70  on days  shown that the changes i n  to respond to AVP  has  been p r e v i o u s l y  (Wynn and  French,  detailed  systematic  at v a r i o u s  stages of  the  structure  of g e s t a t i o n the  of . the  (term = 68  ability  of  the  are c l o s e l y p a r a l l e l e d by changes  i n the s t r u c t u r e of the amniotic the  s t r u c t u r e of the e p i t h e l i u m  AVP  treatment.  epithelium. appears to be  These o b s e r v a t i o n s  would  In  addition,  influenced  agree  with  by the  89  suggestion the  of G a r b y  underlying  structure In  of  tissues  earliest  dialysate  of p l a s m a  amnion  of  transport amnion  early  role  more  As  the  i n c r e a s e s and  results  fluid of  the  complexity  of  ability  present the  to respond  is  a  (Adolph,  study  regulation, studies  as m e n t i o n e d  of H o l t  prolactin  is  water a c r o s s retain  water  capable  of  amniotic  and  Perks  capable the  e f f e c t s may  As  of  the  as  p l a y s a more  composition  Seeds,  that  the  to  of  amniotic in The  increasing  i t s structure  w e l l be  and  related  to  fluid.  general  amniotic  fluid  introduction.  1977b)  indicate  The that  f e t a l - m a t e r n a l flow  amnion,  by  the  1972).  slowing  suggested  contribute  passive  1967;  thereby  helping  compartment;  i n c r e a s i n g t h e movement of sac.  a  a  simple  decline  (1975;  guinea-pig  within  t o be  also involved in amniotic i n the  is  progressive  t o v a s o p r e s s i n , may  i s probably  allow  volume of  i n t e r m s of  i n c r e a s i n g volume of a m n i o t i c Prolactin  and  suggest  amnion,  fluid  However,  the  fetal  relatively  probably  t h e volume  there  when  thought  i t .  f e t u s matures,  osmolarity  is The  it  not  controlling  amniotic  could easily  across  and  amnion.  the and  complex,  in controlling  rate  gestation,  1967).  stages  filtrate  the  of  plasma  (Behrman,  of p l a s m a  becomes  amniotic  the  maternal  these  fluid.  fluid  stages  epithelium, i s the  are poorly d i f f e r e n t i a t e d , with  the  tissue,  transport across  isotonic  active  t h a t the  connective  fluid  the  (1957),  these  amniotic  sodium  of to  i t is also of  the  investigators,  such  fluid  out  hypotonicity.  90  There  i s some e v i d e n c e  amnion  to  t o suggest  prolactin  and  Perks  in  fetal-maternal  that  the response of  a l s o changes d u r i n g  (1975) f o u n d  pregnancy.  water  flow  gestation.  across  However,  t h e amnion  advancing  only  r o u g h , and f u r t h e r e x p e r i m e n t s a r e r e q u i r e d . In a d d i t i o n t o p r o l a c t i n hormones t h a t  amniotic see  fluid.  Perks,  similar but  detected  fluid  which  vasopressin  to  relationship  was  t h e r e may be  (unpublished  the had  of  observations;  p r e s e n c e o f an a g e n t i n an  antidiuretic  effect  in the ethanol-anaesthetised r a t ,  which d i d not appear  According  increased  i n f l u e n c e t h e volume a n d c o m p o s i t i o n  1977),  to  the  and v a s o p r e s s i n ,  P l a t h and P e r k s  human a m n i o t i c  Holt  that the p r o l a c t i n - i n d u c e d reduction  with  other  the  t o be a n e u r o h y p o p h y s i a l  Perks  (1977),  this  agent  evidence  a s y e t , and f u r t h e r s t u d i e s a r e n e e d e d .  of  protein  adrenocorticotropin, estriol, in  amniotic  fluid  Belisle  could  affect  (see  and T u l c h i n s k y , t h e amnion.  guinea-pig  (Manku et  particular  interest  al.  et  1980),  Cortisol  and  and p r o l a c t i n  , 1975).  and d e s e r v e  vasopressin-induced  al . ,  .no A  (e.g., C o r t i s o l ,  a r e found  1971; Dawood,  some  of  found  t o oppose  on w a t e r  transport  h a s been  amnion, and t h i s  importance  influence  angiotensin,  Carretero  is  hormones  as w e l l as p r o s t a g l a n d i n s  a c t i o n s of v a s o p r e s s i n  across  steroid  thyroxine,  progesterone),  1977;  the  and  there  act  on- t h e  variety  however,  may  physiologically of t h i s  amnion;  new  peptide.  these  may be o f p h y s i o l o g i c a l  The p r o s t a g l a n d i n s study water  as they flow  a r e of  a r e known t o  in  the  toad  91  u r i n a r y bladder and the mammalian c o l l e c t i n g tubule ( K i n t e r et  al. ,  1981).  In a d d i t i o n , the amnion has been found t o  s y n t h e s i z e p r o s t a g l a n d i n s (see K i e r s e , al. ,  1979;  1982; Olson et  al. , 1983).  Holt and Perks  (1977b) have observed that  Mitchell  the  et  sodium  p e r m e a b i l i t y of the guinea-pig amnion i s low throughout the course of g e s t a t i o n , but r i s e s days",  just  prior  to  term,  dramatically and  at  observations)  Goh and Perks  have observed a s i m i l a r marked  i n c r e a s e i n the p e r m e a b i l i t y of the c h l o r i d e ions at term.  65  i s p a r t i c u l a r l y high i n  membranes from f e t u s e s judged t o be overdue. (unpublished  about  guinea-pig  amnion  to  In a d d i t i o n , North and Segal (1976)  have found that the p e r m e a b i l i t y of the  guinea-pig  to  urea and acetamide  certain  non-electrolytes  such  as  i n c r e a s e s j u s t a few days b e f o r e term. consistent  with  the  not  in  the  unreasonable  degeneration  ( i . e . , the  apparent  present study j u s t p r i o r to term. to  of  are  degeneration)  suggest  the  that  amniotic  the  sudden  epithelium,  concomitant abrupt changes i n the t r a n s p o r t the  changes  dramatic changes i n the s t r u c t u r e of  the amniotic e p i t h e l i u m observed  These  amnion  membrane, are hormonally induced.  It i s uniform  and  physiology  been  suggested  that  l o c a l c o n t r o l of f e t a l  maturation i n humans may produce a labor  (Olson  et  al. , 1983).  of  Such changes may i n  f a c t be a s s o c i a t e d with the i n i t i a t i o n of p a r t u r i t i o n . has  the  signal  that  membrane initiates  A c c o r d i n g t o Schwarz et  (1975) a c e n t r a l r o l e f o r the f e t a l membranes  It  al.  (the amnion  92  and  chorion)  plausible to the  in light  fetal  exposure onset  i n the  which  the  membrane i n t e g r i t y  Recent  to  labor  labor in  and  the  increase  fetal  mammals  1984).  is  and  a s s o c i a t e d w i t h a m n i o t i c membrane  epithelium 1977;  and  McCoshen  i s probably et  concentrations pregnancies compared t o Ron  i n the  al. in  the  amnion  al.  rupture  by  amnion,  through  (1982),  changing a  the  are  (Ron  et  prolactin  of  to  effect  is  al . ,  Mitchell at  the  thought  to  onset  of  suggest  the  that  integrity.  human  amniotic  (Healy  et  al.  ,  prolactin  particularly  high in  membranes  when  al . ,  1982).  According  may  cause  premature  viscoelastic  possible  1980).  et  Interestingly,  r u p t u r e of  t o normal pregnancy et  to  synthesized there  1982).  with premature  evidence  cytoplasm  ,  of w h i c h i s  the  prolactin  i s present  a  c o u l d s t i m u l a t e the  There  Prolactin  Such  withdrawal  parturition. is  some  Once  progesterone  According  contractility is  substance  (Mitchell  progesterone  or  i n the  (Ryan,  that  membranes a t t e r m  myometrial  a  ensue.  i n p r o s t a g l a n d i n p r o d u c t i o n which  generate  results  the withdrawal  chorion  Powell, local  rupture  contains  indicates  i n t h e amnion and  his associates, of  commonly  could  many  i n t h e human  Mitchell  level  premature  is  injury  a g a i n s t premature l a b o r .  is lost,  initiate  synthesized  and  as  c o u l d be p r o g e s t e r o n e ,  evidence  1982;  fetus  parturition  observations that  P e r h a p s t h e amnion  protects  thought  such  human  hypertonic solutions,  labor.  substance  of  of t h e c l i n i c a l  membranes,  to  of  initiation  on  p r o p e r t i e s of its  water  the and  93  e l e c t r o l y t e content. events  leading  These changes c o u l d  to  the  initiation  p o s s i b l e t h a t the changes i n the properties  of the guinea-pig  then  result  of p a r t u r i t i o n .  structure  and  in  It i s  transport  amnion observed c l o s e t o term  were mediated by p r o l a c t i n . Hormone-mediated  fluid  transport  provides a possible explanation retention amniotic  of  large  f o r the  g r a d i e n t s tending  this cavity. composition  the  fluid  f l u i d w i t h i n the  to cause f l u i d in  to flow out of  the  (e.g.,  ability  to  respond  perhaps p r o l a c t i n ) of the amnion. outer  yolk-sac  amnion,  also  membrane, responds  maternal-fetal Perks,  supplying  This  water  to  fluid  tissue  i f the  transported  via  the f e t a l  The  f e t u s r e q u i r e s an ever  growth,  as  structure  and  v a s o p r e s s i n and guinea-pig,  vasopressin  the  also  increasing  be  important  compartment.  yolk-sac  into  by  the usual g r a d i e n t s (see  may  amnion, changes during the course The  and  which i s c l o s e l y apposed to the  to the amniotic  i n t e r e s t i n g t o see  to  In the  water flow a g a i n s t  1977).  volume  that occur during the course of  g e s t a t i o n may be a r e s u l t of changes i n the activity  and  i n s p i t e of p h y s i c a l and  The p r o g r e s s i v e changes of  the amnion  accumulation  amounts of hypotonic  compartment, which occurs  biochemical  across  in  I t would be  membrane,  like  the  of g e s t a t i o n . the  amniotic  compartment  membranes i s now made a v a i l a b l e t o the f e t u s .  70-95%  (Kleinman, 1975).  of  i n c r e a s i n g supply  i t s total  body  of  water  weight  The d a i l y requirements c o u l d be  for  i s water met  at  94  least  partly  by  In  addition,  sac  during  that  uptake of the  fetal  when f e t a l  volume of  fetal  fetal  body  occur  during  amniotic fetus  transfer  water;  (Bruns  such  et  body w a t e r , a t  have  skin  may  flow  i n the  occurs.  capable  isolated  hormones;  in  were m e a s u r e d water  water  that  fetal  across previous  (France,  happening  through  the  requirements  the  and  this to  i n the  the  human,  concentration,  significant  effect  in  fluid  amniotic  upon volume  t h a t n e t movements  a mechanism  of  f o r p r o t e c t i n g the  in regulating  first  half  The  results  shown  that  causing  a net  guinea-pig  of  in  neurohypophysial transfer  skin,  in  of  of  the  i s the  first  evidence  the  fetal  skin  in  H o l t and  Perks,  may the  fluid  mucosal of  bulk  response  studies, only u n i d i r e c t i o n a l  vivo.  skin  experiments  This  1976;  fetal  of g e s t a t i o n , b e f o r e  f l o w e x p e r i m e n t s g i v e a more a c c u r a t e  m i g h t be fluid  of  direction.  occurring  found  amniotic  electrolyte  have a r o l e  are  serosal  ( 1 963)  , 1963).  hormones  to  no  constitute  i n S e c t i o n I I have  the  amniotic  from t h e  suggesting  described  across  from t h e  sharply,  been o b s e r v e d  alterations  least  keratinization  fluid  w a t e r and  stress,  al.  fetal  cavity.  al .  declined  of  I t has  limits,  fluid  The  fluid  in maternal  certain  amniotic  r a b b i t s were e x p e r i m e n t a l l y d e h y d r a t e d ,  compartment.  within  fluid  B r u n s et  dehydration.  a net  imbalances  from the  f e t u s c o u l d draw  the a m n i o t i c  suggested  fluid  to  fluxes  1977a). p i c t u r e of  Net what  Hormone-mediated a b s o r p t i o n a i d in supplying growing  fetus.  the  of  day-to-day  During  fetal  95  dehydration,  such  replenishing  fetal  the a m n i o t i c limited  effects  on  This  of t h e f e t a l  would  kidney  Such an e x t r a - p l a c e n t a l mechanism the  fetus  during  importance the  i n response  surfaces. in  fetal et  ,  drive  imposing  compartment Between  across ten  gradients  from  fetal  related  weight  and  the  twenty  (or f e t a l  Hytten,  weeks fluid  of g e s t a t i o n , the composition fetal  1970).  In  extracellular  c o n c e n t r a t i o n s of - d i f f u s a b l e  maternal  amniotic plasma  to  and  the  fetal  i n the absence and  serosal  exist  (Lind  which to  vivo  f l u i d and  i n composition  would  the f e t a l  et  al.  g e s t a t i o n i n the closely  .addition,  related  gestation  i n these  of amniotic  substances  ,  of  well  early  fluid  is  f l u i d ; most n o t a b l y , t h e  f l u i d are closer  (Lind  of  or to the length  stages  in  water  t o t h e f e t u s in  i s very  and  urea  of  amniotic  (Lind  to  urine.  s u r f a c e a r e a ) and much l e s s  t o p l a c e n t a l weight  similar  f o r the  the s k i n .  human, t h e volume o f a m n i o t i c to  the s k i n  f l u i d are similar  passively  from  the s t r e s s .  t o hormones o c c u r r e d  1969; 1972), a n d no fluid  up  o f g e s t a t i o n , when t h e a m n i o t i c  extracellular  al.  transfer  supplying  g r a d i e n t s between t h e m u c o s a l  half  in  would be o f p a r t i c u l a r  T h i s may be o f s i g n i f i c a n c e  the f i r s t  make  aid  s t r e s s came from t h e m o t h e r ,  net uptake of f l u i d a c r o s s  any a p p a r e n t  could  to concentrate  for  dehydration  i f the osmotic  guinea-pig of  fetal  p l a c e n t a were t h e a g e n t The  skin  body w a t e r , by n e t w a t e r  compartment.  ability  the  such  t o those  a s sodium  and  of f e t a l  than  1969; 1972; L i n d and  Hytten,  96  1972). to  T h e s e o b s e r v a t i o n s have l e d L i n d  suggest  that  during  this  r e g a r d e d a s an e x t e n s i o n that two  the f e t a l  skin,  time, the amniotic  of f e t a l  which  composition  space.  After  of  i s the only  fluid  when t h e s k i n  relationship  weight  b r e a k s down, and t h e c o m p o s i t i o n  (Lind  between a m n i o t i c  progressively  and H y t t e n ,  1972).  is  fluid after  consistent  Section  fetus  ability  influence the  skin  this  in regulating  extracellular  amniotic  fluid  of f e t a l  plasma  that  t h e now  the  amnion  "externalized" This  suggestion  resembles anuran amphibians w i t h  t o a b s o r b water of  through  neurohypophysial  a p p e a r s t o be a m a j o r  may be t r u e  appears suggested AVT  plentiful  to  be  i n the fetus,  more  i n Section finds in  mid-gestation, be  volume  i n t h e amnion r e p o r t e d i n  i t s skin  hormones.  before  responsive  mammalian  osmoregulatory.  fetal  the  role.  the  i t s levels  fetus  t o AVP. fetal  This  skin  and  keratinizes.  As skin  peptide i s  pituitaries  t h e t i m e when f e t a l However,  organ,  of  AVT t h a n  I I , i t i s p e r h a p s on  i t s physiological  at  skin  to  to  In t h e a n u r a n s ,  the s k i n  the  respect  when under t h e  osmoregulatory  Remarkably, as i n t h e amphibians,  that  of  keratinizes.  the changes  the  I.  The its  with  between t h e  f l u i d volume and f e t a l  I t i s possible  the skin  and  k e r a t i n i z e s , the  from t h e c o m p o s i t i o n  becomes more i m p o r t a n t amniotic  barrier  extended  linear  departs  fluid,  in regulating  in this  mid-gestation,  f l u i d may be  extracellular  c o m p a r t m e n t s , may be i n v o l v e d  and  and h i s a s s o c i a t e s  before  i s believed  decline  to  i n the  97  advanced (Perks,  fetus,  and i t i s n o t p r e s e n t  1977; Skowsky and F i s h e r ,  physiologicl plasma  role  i n the fetus,  after  in adult  1977).  I f AVT h a s a t r u e  i t should appear  appropriate  osmotic  However, a t p r e s e n t , t h e r e  AVT  by t h e f e t a l  fetuses  s u b j e c t e d t o osmotic  In t h e a d u l t  fetus,  the  some  infants  relatively  born  without  water and e l e c t r o l y t e other  mechanisms  important  osmoregulatory  gestation.  Abramovich  kidneys  take  over  of  the  at  about  fluid  the time  exchange.  epithelium  periderm.  There  i s no e v i d e n c e  on  transfer  conservation the  limited  that  of r e n a l  probably  first that  half after  to  with  the f e t a l  as y e t of hormonal  the  need  arises  that  f e t u s , the  resemble  The f e t a l  of  c o r d may  concerned  i n t h e human  an  the  I t may be s i g n i f i c a n t  begins  when  i n the fetus, ability  the  is  of the u m b i l i c a l  across the cord.  be o s m o r e g u l a t o r y ;  (1975),  suggesting  f o r lack  skin  in  of k e r a t i n i z a t i o n  cord  also  at birth,  functions  umbilical  fluid  Kleinman  (1973) has s u g g e s t e d surface  does not  have no a b n o r m a l i t i e s i n  organ  the  However, i n  kidney  to  the f e t a l  keratinizes,  fetal-amniotic  fluid.  adequately  skin  some  i s the prime r e g u l a t o r  According  compensate  As we have s e e n ,  i s no e v i d e n c e o f  inefficient  composition  function.  (e.g.,  and s t u d i e s on young  of e x t r a c e l l u l a r  a p p e a r t o be a s i m p o r t a n t .  fetal  stress are required.  mammal, t h e k i d n e y  of volume and t o n i c i t y the  pituitary,  in  stimuli  dehydration). secretion  pituitaries  effects  bladder for  may  water  t h e b l a d d e r c o u l d compensate f o r  of r e n a l  tubules to  concentrate  urine  98  by  reabsorbing  aided  by  water v i a t h e  vasopressin;  increase mucosal-serosal the  fetal  guinea-pig  inefficiency be  of  compensated  by  bladder  a p p e a r s t o be  fetal  pig  fetal  of  the et  (France  swallowed  hormonal to  mechanism  of  Morley  i n the  osmoregulation.  site  for  With similar and  al.  ( C a s s i n and regard to  also  of  the  this Perks,  an  et  sodium  the  in  the  involved  in  fetal  s o d i u m and  water  and  Nixon,  i s under  have been in  the  (see  1961;  found  gastro-  Lam,  a similar  1972;  hormonal  additional site lung  could  be  may  for  be  a  overhydration mediated  by  1982).  to osmoregulation,  The  sheep  reduce  transport  fetal  the  sub-mammalian v e r t e b r a t e s  amphibians.  the  The  ions during  effect  could  (France  bladder.  fish  of  ion v i a  to  to  Likewise,  fetal  be  Cortisol  , 1981), and  Finally,  found  this  some s p e c i e s et  the  the  transport  e x c r e t i o n of w a t e r and  l o a d i n g , and  prolactin  ion  of  (Wright  and  found  of. sodium  reabsorbs  fluid  perhaps  bladder  vasopressin  the  f e t u s would p r o v i d e  fetal  salt  actively  and  the  w a t e r and  a c t i n g on  Prolactin  water  tract  1980;  to  been  1977a).  in  been  , 1976), may by  tract  by  It i s p o s s i b l e that  influence  Hirano,  or  bladder al.  control.  intestinal  across  reabsorption  w h i c h has  amniotic  1974).  has  Perks,  facilitated  osmoregulation  Wright,  flux  reabsorption  Prolactin,  gastro-intestinal from  water  ( H o l t and  Sodium  permeability  hormone  active reabsorption  wall.  , 1976).  epithelium,  this  renal tubular  bladder  al.  bladder  a c t i o n s of  mammalian such as  fetus  teleost  neurohypophysial  is fish  hormones  99  on  w a t e r and  and  the  ion transport across  u r i n a r y bladder  these  peptides  on  al.  1978);  The  ,  bladder  and  et of  al.  ,  1978;  fetal  outlined of the  other  are  1980).  Further  hormones  no  add  on  metabolism.  the  the  those (Lam,  control  f e t u s on  effects  of  (Perks  et  fetal  lung,  observed 1972;  in Perks  mechanisms  (e.g.,  our  the  than  effects  in osmoregulation  thyroxine, to  of  amnion,  doubt more complex  s t u d i e s i n the  vertebrates  skin,  bladder  teleosts  which p l a y a r o l e  may  water  to  Hormonal  catecholamines,  1976)  intra-uterine  similar  metabolism are  sub-mammalian  Bentley,  and  a c t i o n s of p r o l a c t i n  Hirano,  here.  angiotensin,  skin  s t r u c t u r e s i n the  fluid  fetal  reminiscent  the amphibian  amnion  osmoregulatory  are  the  in  aldosterone, Cortisol  —  understanding  see of  100  REFERENCES  A b r a m o v i c h , D.R. (1968) The volume o f a m n i o t i c fluid in early pregnancy. /. Obstet. Gynaec. Brit. Cwlt h. 75:728-731 . Abramovich, D.R. (1970) Fetal factors influencing the volume a n d c o m p o s i t i o n o f l i q u o r a m n i i . /. Obstet. Gynaec. 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