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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 Fraser University, 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 thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1986 © Sheilnin B. Pisani, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^oo-OfrV  The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date S T T W A P R I L , t^frk i i ABSTRACT Thirty-nine amniotic membranes from guinea-pig fetuses between 30 and 68 days of gestation (0.44 - 1.00 of term) were set up in vitro, in apparatus in which net water flow could be measured gravimetrically. Small hydrostatic and osmotic gradients were maintained from the f e t a l to the maternal side of the membrane; thi s reproduced in vivo conditions. In the absence of hormone, there was a net transfer of water from the f e t a l to the maternal side, in the majority of preparations. Addition of arginine vasopressin (AVP) at l00mU/ml (vasopressor a c t i v i t y ) to the f e t a l surface slowed or reversed t h i s flow. The response increased with f e t a l age, u n t i l about 58 days of gestation (0.85 of term). After t h i s time, the effect declined, and was l o s t ; membranes over 64 days (0.94 of term) showed only one weak response in 13 experiments. Electron microscopy of 10 membranes between 28 and 70 days of gestation showed p a r a l l e l changes in the structure of the amniotic epithelium. The epithelium changed from a r e l a t i v e l y simple structure early in gestation, when the response to AVP was low, to one that appeared to be more complex and possibly more specialized in function 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 to respond to AVP. Electron microscopic studies on a membrane at 38 days revealed that AVP caused the i n t e r c e l l u l a r spaces to d i l a t e . Morphometric analysis showed that the dimensions of the spaces in the AVP treated epithelium were s i g n i f i c a n t l y greater than in a control preparation (p<0.001). Studies on 76 guinea-pig fetuses showed that the volume of amniotic f l u i d increased during the course of gestation, reached a peak, and then declined. The peak coincided roughly with the time at which the amnion showed i t s maximal response to AVP, and with the time i t s structure appeared to be compatible with an active role in f l u i d transport. After approximately 56 - 58 days, both amniotic f l u i d volume and the response of the amnion to AVP declined. These results are consistent with a physiological role of AVP in supplying f l u i d to the amniotic sac in the f i r s t 80% of • gestation. Unlike the situa t i o n in most species, the amniotic f l u i d volume increased again, and reached i t s maximum value just before delivery. Skin from 35 mid-term f e t a l guinea-pigs (0.49 - 0.70 of term) was set up in the same gravimetric apparatus used in the amnion experiments. However, there were no gradients in hydrostatic or osmotic pressure. In the absence of hormones, there was l i t t l e or no net transfer of water in either d i r e c t i o n . Arginine vasotocin (AVT) or arginine vasopressin (AVP) added to the serosal surface at 5 - lOOmU/ml (vasopressor a c t i v i t y ) produced a net uptake of water towards the serosal side (towards the fetus). There was a linear r e l a t i o n s h i p between the log.dose, and i v the r a t e of uptake of water, f o r both p e p t i d e s . However, AVT was more than twice as potent as AVP ( t h r e s h o l d s : AVT, 3.9mU/ml; AVP, 10.4mU/ml). The f i n e s t r u c t u r e of the g uinea-pig s k i n before k e r a t i n i z a t i o n appeared to be compatible with an a c t i v e r o l e i n f l u i d t r a n s p o r t . The outer periderm l a y e r resembled the amniotic e p i t h e l i u m ; i t may be the s i t e of a c t i o n of neurohypophysial hormones. I t i s concluded that the g e s t a t i o n a l changes i n the s t r u c t u r e and response to AVP of the amnion may be p a r t i a l l y r e p o n s i b l e f o r changes in the volume of amniotic f l u i d during g e s t a t i o n . The f e t a l s k i n , l i k e f r o g skin,, responds to AVT more r e a d i l y than to AVP. Perhaps i t i s on the f e t a l s k i n that f e t a l AVT f i n d s i t s true p h y s i o l o g i c a l r o l e . S u p e r v i s o r V TABLE OF CONTENTS LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS X GENERAL INTRODUCTION 1 The F e t a l F l u i d s 1 S t r u c t u r e s Involved i n I n t r a - U t e r i n e F l u i d Exchange . 2 The F e t a l Kidney .. 2 The F e t a l U r i n a r y Bladder 4 The F e t a l Lung 5 The F e t a l G a s t r o - I n t e s t i n a l T r a c t 7 The Amniotic Membrane 9 The F e t a l Skin 11 The U m b i l i c a l Cord 13 The Amniotic F l u i d 14 The O r i g i n of I n t r a - U t e r i n e Water 18 Hormonal C o n t r o l of I n t r a - U t e r i n e F l u i d s 19 STATEMENT OF THE PROBLEM 21 GENERAL METHODS 24 1 . Net Water Flow Experiments 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 Skin P r e p a r a t i o n 27 2. S a l i n e s 28 3. Hormone S o l u t i o n s 29 4. E l e c t r o n Microscopy of the Amnion 30 5. E l e c t r o n Microscopy of the F e t a l Skin 31 v i 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 Effects of AVP on the Amnion 33 2. The Fine Structure of the Amniotic Epithelium 38 Early Amniotic Epithelium (28, 30, 35, 38 days) 40 Mature Amniotic Epithelium (50, 58, 62 days) ... 43 Near-Term Amniotic Epithelium (64, 68, 70 days) 46 3. Fine Structure of the Amniotic Epithelium After Incubation With AVP 50 4. The Volume of Amniotic F l u i d 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 Isolated Fetal Skin 67 2. The Effects of AVT and AVP on Net Water Transport Across Fetal Skin 67 3. Fine Structure of the Fetal Epidermis 74 DISCUSSION 81 GENERAL DISCUSSION 88 REFERENCES 100 v i i LIST OF TABLES Table Page I The Response of the Amnion to AVP During the Course of Gestation 36 II The Volume of Amniotic F l u i d During the Course of Gestation 55 III The Response of the Fetal Skin To AVT 70 IV The Response of the Fetal Skin to AVP 71 v i i i LIST OF FIGURES F i g u r e Page 1. The G r a v i m e t r i c Apparatus Used f o r the in vitro Study of Net Water Movement Through the I s o l a t e d Amnion of the Guinea-Pig 25 2. The E f f e c t s of AVP on Net Water Movement Across the I s o l a t e d Amnion of the Guinea-Pig .. 35 3. The Changes i n the Response of the Amnion to AVP Through the Course of G e s t a t i o n 39 4. The Fine S t r u c t u r e of the Amniotic E p i t h e l i u m Through the Course of G e s t a t i o n .... 41 5. E l e c t r o n Micrographs of the E a r l y Amniotic E p i t h e l i u m 42 6,7. The Fine S t r u c t u r e of the Mature Amniotic E p i t h e l i u m 44,45 8,9. E l e c t r o n Micrographs of the Near-term Amniotic E p i t h e l i u m 47,48 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) 51 11. E f f e c t of AVP on the Fine S t r u c t u r e of the Amniotic E p i t h e l i u m 53 ix LIST OF FIGURES (continued) Figure Page 12. The Volume of Amniotic F l u i d at Various Stages of Gestation 54 13. Gestational Changes in the Structure of the Amniotic Epithelium, the Response to AVP, and the Volume of Amniotic F l u i d in the Guinea-Pig 64 14. The E f f e c t s of Different Doses of AVT and AVP on the Net Movement of Water Through the Skin of the Fetal Guinea-Pig (at 0.49 -0.70 of term) 68 15. Log Dose-Response Curves for the Eff e c t s of AVT and AVP on Net Water Movement Through the Fetal Skin (at 0.49 - 0.70 of term) 73 16,17. Fine Structure of the Fe t a l Epidermis at 35 Days 75,76 18. The Fetal Periderm at 35 Days 77 19. Fine Structure of the Fe t a l Epidermis at 52 Days 79 X ACKNOWLEDGEMENT S I thank the N a t u r a l S c i e n c e s and En g i n e e r i n g Research C o u n c i l of Canada f o r f i n a n c i a l a s s i s t a n c e i n the form of a postgraduate s c h o l a r s h i p f o r the year 1981-82. I am indebted to my s u p e r v i s o r , Dr. Anthony M. Perks, f o r guidance, encouragement, and generous f i n a n c i a l support. I am a l s o g r a t e f u l to Dr. Dan W. Rurak, Dr. John E. P h i l l i p s , and Dr. H.D. F i s h e r f o r h e l p f u l a d v i c e , and to Mr. L a s z l o J . Veto f o r h i s i n v a l u a b l e h e l p with the e l e c t r o n microscope. I thank Mr. Tsun Takman f o r the care and breeding of the guinea-pigs used i n t h i s study, and Mr. Bruce M c G i l l i v r a y f o r ty p i n g the manuscript. 1 GENERAL INTRODUCTION The Fetal Fluids During f e t a l development, a large quantity of water accumulates within the uterus and i s di s t r i b u t e d between f e t a l , placental, and amniotic compartments (Kerpel-Fronius, 1970). In the human fetus near term, these compartments normally contain an average of about 2800 ml, 400 ml, and 800 ml of water, respectively (Seeds, 1974). An additional f l u i d space, the a l l a n t o i c sac, should be included in some species. Over the whole of pregnancy, water accumulation in the human i s concurrent with a net transfer of 380 mEq of sodium and 173 mEq of potassium from the mother to the intra-uterine cavity (Kerpel-Fronius, 1970). The mechanisms by which such a large amount of f l u i d accumulates within the uterus, and the means by which i t s volume and composition are controlled, are not yet f u l l y understood. Isotopic studies have revealed that there i s a dynamic exchange of water and e l e c t r o l y t e s between the fetus, amniotic f l u i d and mother (Vosburgh et al. , 1948; Friedman et al. , 1959; Hutchinson et al. , 1955; 1959). The structures involved in thi s exchange, besides the placenta, are the umbilical cord, extra-placental membranes (e.g., the amnion), and f e t a l organs such as the kidney, bladder, lung, g a s t r o - i n t e s t i n a l tract and skin. A l l appear to play a role in regulating the volume and composition of 2 intra-uterine f l u i d s . In t h i s thesis, the role of the amnion and of the f e t a l skin in regulating f e t a l f l u i d s w i l l be considered. Before t h i s i s done, a review of intra-uterine water metabolism i s in order. Structures Involved in Intra-Uterine F l u i d Exchange The Fetal Kidney The kidneys begin to function r e l a t i v e l y early in f e t a l l i f e . Cameron and Chambers (1938) demonstrated that the renal tubule c e l l s of the three month old human fetus are capable of transporting phenol red from the outside medium into the tubular lumen in vitro. Urine has been found in the human f e t a l bladder as early as eleven weeks of*gestation (Abramovich, 1968). By mid-gestation (20 weeks), the human fetus voids approximately 7 to 14 ml of urine per day (Abramovich, 1973). In the f e t a l lamb (which has a gestation period of 145 days), glomerular and tubular function are present by 60 days (Alexander and Nixon, 1963). However, the immature kidney does not function as e f f i c i e n t l y as the adult kidney. In a l l mammals, the glomerular f i l t r a t i o n rate (GFR) is lower in the fetus than in the adult (Kleinman, 1975; Barnes, 1976). This i s partly a result of the lower a r t e r i a l blood pressure of the fetus, but factors such as a low renal blood flow and structural immaturity of glomerular c e l l s may also be important (Kleinman, 1975). 3 Likewise, tubular transport, though evident early in gestation, i s r e l a t i v e l y i n e f f i c i e n t in the fetus. Capek et al. (1968) showed that the i n t r i n s i c transport capacity for sodium in the proximal tubule of newborn rats i s lower than in more mature animals. Alexander and Nixon (1961) have observed that in f e t a l sheep, the tubular reabsorption of sodium increases from 60 percent of the glomerular f i l t r a t e at mid-gestation to 90 percent close to term. These investigators found that water reabsorption in young sheep fetuses i s even lower than sodium absorption; at about mid-gestation, the amount of water reabsorbed i s only 28 percent of that f i l t e r e d . Due to the low GFR, the f e t a l kidney i s l i m i t e d in i t s a b i l i t y to excrete excess water and sodium. Excretion of excess sodium i s further hampered by the i n a b i l i t y of d i s t a l tubules to lower the reabsorption of f i l t e r e d sodium (Kleinman, 1975). The f e t a l kidney i s also limited in i t s a b i l i t y to respond to dehydration as i t i s not as well able to concentrate urine as the adult kidney (Kleinman, 1975). As a result of the lower tubular reabsorption of water, the normal fetus excretes large amounts of hypotonic urine, despite the low GFR. Close to term, the human fetus voids an average of about 500 ml of urine per day (van Otterlo et al. , 1977; Abramovich el al. , 1979). The f e t a l sheep voids amounts of similar magnitude (200 - 500 ml per day), and these high rates of urine flow are in excess of adult resting values, when expressed on the basis of flow 4 per u n i t body weight (Alexander et al. , 1958). The o s m o l a l i t y of f e t a l u r i n e in the human decreases from about 144 mOsm/kg at 17 - 21 weeks to 137 mOsm/kg at term; the c o n c e n t r a t i o n of sodium drops from 68 to 44 m E q / l i t r e from mid-term to term and that of c h l o r i d e drops from 66 to 41 m E q / l i t r e (see Abramovich, 1979). The drop in u r i n e sodium and c h l o r i d e c o n c e n t r a t i o n s suggests a more e f f e c t i v e r e a b s o r p t i o n of the ions with maturation of r e n a l f u n c t i o n . In f e t a l sheep, u r i n e flows i n t o the a l l a n t o i c sac v i a the urachus u n t i l about 80 - 90 days of g e s t a t i o n , and t h e r e a f t e r i t passes i n i n c r e a s i n g amounts i n t o the amniotic sac (Alexander and Nixon, 1961; M e l l o r and S l a t e r , 1971). In s p e c i e s without an a l l a n t o i c sac, such as the human, u r i n e passes only i n t o the amniotic c a v i t y (Seeds, 1965). However, u r i n e i s not the only source of amniotic f l u i d , as w i l l be seen l a t e r . In the a d u l t , the kidney i s the prime r e g u l a t o r of water and e l e c t r o l y t e metabolism. In the f e t u s , however, the r e l a t i v e l y immature kidney i s a s s i s t e d i n t h i s f u n c t i o n by p l a c e n t a l r e g u l a t i o n , as w e l l as r e g u l a t i o n by other f e t a l s t r u c t u r e s . The Fetal Urinary Bladder I t has been observed that the composition of u r i n e produced by the kidney can be a l t e r e d i n the f e t a l b ladder. Boylan et al. (1958) found that the u r i n e i n the bladder of the f e t a l g u i n e a - p i g has a higher o s m o l a r i t y than that 5 formed by the kidney. T h i s suggests a r e a b s o r p t i o n of water i n t o the f e t a l c i r c u l a t i o n from the bladder lumen. Alexander et al. (1958) observed the same phenomenon i n f e t a l sheep; i n i t i a l bladder samples taken from f e t u s e s c l o s e to term were hypotonic to plasma but subsequent samples of u r i n e , which had stayed i n the bladder f o r up to an hour, were h y p e r t o n i c . However, the e f f e c t s of the bladder may not always be the same. S t a n i e r (1971) observed that the bladder u r i n e of f e t a l p i g s i s always markedly hypoosmotic to that of the r e n a l p e l v i s . A much lower c o n c e n t r a t i o n of sodium i s present i n bladder u r i n e than i n u r i n e from the r e n a l p e l v i s , suggesting some sodium r e a b s o r p t i o n i n t o the f e t a l c i r c u l a t i o n from the bladd e r . France et al. (1972) have demonstrated that the f e t a l sheep bladder i s capable of a c t i v e l y t r a n s p o r t i n g sodium. These o b s e r v a t i o n s suggest that the f e t a l bladder may play a r o l e i n osmoregulation. The Fetal Lung The lungs of the f e t u s are not i n v o l v e d i n gas exchange, t h i s f u n c t i o n being performed by the p l a c e n t a , and are i n s t e a d thought to p l a y a r o l e i n the exchange of i n t r a - u t e r i n e water and i o n s . From the time when the airways are f i r s t formed, they are f i l l e d with f l u i d , which i s s e c r e t e d i n the lungs and which i s probably important fo r t h e i r normal development (Olver et al. , 1973; A l c o r n et 6 al. , 1977). The lungs of the f e t a l lamb i n the l a s t t h i r d of g e s t a t i o n c o n t a i n about 30 to 40 ml of f l u i d per kg of body weight, and s e c r e t e f l u i d at a r a t e of about 1 to 3 ml/kg per hour (Normand el al. , 1971; Olver and Strang, 1974; S c a r p e l l i et al. , 1975). For a 4 kg f e t u s , t h i s would amount to about 100 - 300 ml per day. S e t n i k a r et al. (1959) have observed s e c r e t i o n r a t e s of about 1 to 7 ml/kg per hour i n the f e t a l g u i n e a - p i g . Perks and C a s s i n (1985) have observed s i m i l a r r a t e s of lung f l u i d s e c r e t i o n i n the f e t a l goat. The lung (or a l v e o l a r ) f l u i d d i f f e r s markedly from amniotic f l u i d and f e t a l plasma. Adamson et al. (1969) found t h a t i n a l v e o l a r l i q u i d , the c o n c e n t r a t i o n s of hydrogen, potassium and c h l o r i d e ions are h i g h e r , and of p r o t e i n s , phosphates, c a l c i u m ions and b i c a r b o n a t e ions lower than i n f e t a l plasma. A c c o r d i n g to these i n v e s t i g a t o r s , the d i f f e r e n c e s i n i o n i c c o n c e n t r a t i o n s of a l v e o l a r l i q u i d from plasma are not simply the e f f e c t s of a Gibbs-Donnan e q u i l i b r i u m due to d i f f e r e n c e s i n p r o t e i n c o n c e n t r a t i o n s ; they suggest that the lung l i q u i d i s not merely an u l t r a f i l t r a t e of plasma but i s a c t i v e l y e l a b o r a t e d by the lung. Olver and Strang (1974) i n v e s t i g a t e d ion t r a n s p o r t a c r o s s the a l v e o l a r e p i t h e l i u m and concluded that the s e c r e t i o n of lung l i q u i d i s generated by the a c t i v e t r a n s p o r t of CI from plasma to a l v e o l a r l i q u i d , a c r o s s the a l v e o l a r e p i t h e l i u m ; t h i s t r a n s p o r t of C l ~ ions i s i n excess of HC0 3 t r a n s p o r t i n the 7 opposite d i r e c t i o n . Na moves passively in response to the e l e c t r i c a l gradient set up by C l ~ movement, and the resultant osmotic force of NaCl causes the net transfer of water into the alveolar space. F l u i d secreted in the lungs leaves via the trachea, and most of i t enters the amniotic cavity, though some may be swallowed en route (Adams et al. , 1963; Harding et al. , 1984). Lung f l u i d may contribute s i g n i f i c a n t l y to the volume of amniotic f l u i d in some species. According to Cassin and Perks (1982) the lungs of the fetus may also serve an important role in c o n t r o l l i n g f e t a l body f l u i d s , along with the placenta and kidney. These investigators found that infusion of 0.9% NaCl into the c i r c u l a t i o n of the f e t a l goat resulted in an increase in the secretion of lung f l u i d , including Na +, and CI . The limited a b i l i t y of the f e t a l kidney to excrete a sodium or water load may be compensated by excretion via the lungs. The Fetal Gastro-Intestinal Tract There i s abundant evidence to show that the fetus swallows in utero. Early investigators found e p i t h e l i a l c e l l s , lanugo hairs, and vernix caseosa in the gut of the human fetus, and attributed the presence of these substances to the swallowing of amniotic f l u i d (see Abramovich, 1979; 1981). Since then, radiography and isotopic tracer studies have confirmed these observations. From experiments which followed the movement of 8 erythrocytes la b e l l e d with radioactive chromium from amniotic f l u i d into the gas t r o - i n t e s t i n a l t r a c t , Pritchard (1966) estimated that the 16 week old normal human fetus swallows about 7 ml of amniotic f l u i d per day (2 ml/kg/hr) and the 28 week old fetus about 120 ml per day (4 ml/kg/hr). Abramovich (1970) obtained values of similar magnitude as those observed by Pritchard for mid-term human fetuses, using radioactive c o l l o i d a l gold. At term, the amount of amniotic f l u i d swallowed by a normal human fetus averages about 200 to 500 ml per day (Pritchard, 1965; Abramovich et al . , 1979). Harding et al. (1984) have found similar rates of swallowing in the f e t a l lamb during late gestation. According to Abramovich (1970), gut p e r i s t a l s i s in the human fetus i s established by mid-gestation; studies'on f e t a l swallowing at thi s stage have shown that about 78 to 93 percent of the radioactive tracer given into the amniotic f l u i d accumulates in the lower gut, suggesting that amniotic f l u i d i s a c t i v e l y passed down the gas t r o - i n t e s t i n a l t r a c t , with water being absorbed during i t s passage. Wright and Nixon (1961) have studied the absorption of amniotic f l u i d in the gut of the f e t a l lamb. Their experiments indicate that perhaps a l l of the swallowed amniotic f l u i d i s reabsorbed into the f e t a l c i r c u l a t i o n . These investigators found that sodium ions from swallowed amniotic f l u i d are a c t i v e l y transported from the gut lumen towards the f e t a l e x t r a c e l l u l a r f l u i d ( i . e . , 9 from mucosa to serosa) and suggested that t h i s transport of sodium probably accounts for much of the water reabsorption. An active reabsorption of sodium ions from the gut has also been observed in the f e t a l rabbit (Wright, 1974). The f e t a l gut, l i k e that of the adult, transports H + and Cl act i v e l y from the serosa to the mucosa (see Wright, 1974). The active transport of ions probably accounts f o r - t h e negative potential in the gut lumen, with respect to f e t a l e x t r a c e l l u l a r f l u i d , observed in the fetuses of sheep (Wright and Nixon, 1961), goat, human, rabbit and guinea-pig (see Mellor, 1969; 1970; Mellor et al. , 1969). The Amniotic Membrane The amnion is the innermost of the extra-embryonic membranes, coming in direc t contact with the amniotic f l u i d (Bourne, 1962). The method of amnion formation varies considerably between species (Biggers, 1972: Steven and Morris, 1975) but the structure of the d e f i n i t i v e amnion appears e s s e n t i a l l y the same in a l l mammals. The t y p i c a l mammalian amnion is from 0.02 to 0.50 mm thick, and i s composed of a single layer of epithelium overlying several layers of connective tissue. It has no blood vessels in most species, and is not innervated (Wynn, 1974). There i s much evidence to suggest that the amnion serves as a pathway for f l u i d exchange between the amniotic f l u i d and the maternal c i r c u l a t i o n . Paul et al. (1956) 10 found that in f e t a l rabbits at 0.75 of term, approximately half of the water exchange between the mother and the amniotic f l u i d occurs across the fe t a l - p l a c e n t a l complex and half occurs across the f e t a l membranes. Hutchinson et - al. (1959) observed that throughout the entire period of gestation in the human, there exists a d i r e c t pathway of exchange between the amniotic f l u i d and mother, via the chorioamnion. The c e l l s of the amniotic epithelium have been described as being secretory by Taussig (1927) and Danforth and Hull (1958), based on h i s t o l o g i c a l evidence. Armstrong et al. (1968) have observed pinocytosis in l i v i n g c e l l s of the human amnion, suggesting that they are capable of transporting f l u i d . Electron microscopic studies of the amniotic epithelium have revealed that i t s ultrastructure is similar to that of other transporting e p i t h e l i a , such as those which l i n e the mammalian gallbladder, the intestine, and the amphibian urinary bladder (Hoyes, 1968a; van Herendael, 1978; Hoang-Ngoc Minh et al. , 1980; 1981; King 1978; 1980). The amnion i s freely permeable to water. Lloyd et al. (1969) obtained a permeability c o e f f i c i e n t for the d i f f u s i o n of t r i t i a t e d water across the human amnion of about 3X10~a cm/sec, and Page et al. (1974) observed values of 0.5X10-" to 4X10-" cm/sec. Holt and Perks (1975) obtained values of the same order of magnitude for the guinea-pig amnion. The permeability c o e f f i c i e n t for the 11 net t r a n s f e r of water a c r o s s the human amnion by osmosis i s about a hundred times g r e a t e r than the t r a n s f e r of i s o t o p i c water by simple d i f f u s i o n , suggesting that water can pass by a n o n - d i f f u s i o n a l bulk flow through porous channels i n the membrane (Seeds, 1967). According to Seeds (1980) the amnion away from the p l a c e n t a l d i s c ( i . e . , the r e f l e c t e d amnion) i s u n l i k e l y to be a s i t e f o r s i g n i f i c a n t exchange of water and ions between the amniotic c a v i t y and the mother,, due to the p a u c i t y of blood v e s s e l s i n maternal t i s s u e i n p r o x i m i t y to the c h o r i o n laeve and amnion. However, Hoang-Ngoc Minh et al. (1980; 1981) have observed that the complex i n t e r c e l l u l a r system present i n the amniotic e p i t h e l i u m , thought to f a c i l i t a t e the flow of l a r g e amounts of f l u i d , i s a l s o found i n the c h o r i o n and the c h o r i o n i c c y t o t r o p h o b l a s t , and extends to the p a r i e t a l u t e r i n e decidua, where the v a s c u l a r arrangement can f a c i l i t a t e the exchange of f l u i d with maternal plasma; the s t r u c t u r e formed by the a s s o c i a t i o n of the p r i m i t i v e c y t o t r o p h o b l a s t and the p a r i e t a l u t e r i n e decidua (which both p e r s i s t t i l l term) has been l i k e n e d to a p l a c e n t a by these i n v e s t i g a t o r s . The Fetal Skin P r i o r to the development of the stratum corneum, f e t a l s k i n i s permeable to water, and may be a s i t e of s i g n i f i c a n t f l u i d exchange between the f e t a l and amniotic 12 compartments. P e r m e a b i l i t y c o e f f i c i e n t s f o r the 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 i s o l a t e d u n k e r a t i n i z e d s k i n of the human f e t u s p r i o r to mid-term have been found to be between 0.45X10"" and 0.86X10"" cm/sec, s i m i l a r to values measured a c r o s s the human amnion and c h o r i o n laeve (Parmley and Seeds, 1970). U n k e r a t i n i z e d s k i n i s al.so f r e e l y permeable to sodium (Lind et al . , 1972). The uppermost l a y e r of u n k e r a t i n i z e d f e t a l s k i n i s c a l l e d the periderm, and u l t r a s t r u c t u r a l s t u d i e s i n the human f e t u s have shown t h a t , l i k e the amniotic e p i t h e l i u m , the periderm has c h a r a c t e r i s t i c s i n common with other t r a n s p o r t i n g e p i t h e l i a (Breathnach and W y l l i e , 1965; Hoyes, 1968b). According to L i n d and Hytten (1972), the c e l l s of e a r l y f e t a l s k i n bear a s t r i k i n g resemblance to r e n a l t u b u l a r e p i t h e l i u m under the i n f l u e n c e of v a s o p r e s s i n . These i n v e s t i g a t o r s have suggested that the young f e t u s , which w i l l e v e n t u a l l y c o n t r o l i t s e x t r a c e l l u l a r volume by v a r y i n g the p e r m e a b i l i t y of the r e n a l tubules with v a s o p r e s s i n , can perform the same f u n c t i o n with i t s s k i n . A f t e r the s k i n begins to k e r a t i n i z e , i t s r o l e i n f e t a l water and ion exchange probably d i m i n i s h e s . In the human f e t u s , Parmley and Seeds (1970) found a l a r g e decrease i n s k i n p e r m e a b i l i t y to water i n f e t u s e s i n which e a r l y k e r a t i n i z a t i o n was demonstrable, and i n s k i n with a w e l l developed stratum corneum there was no d i f f u s i o n at a l l . L i n d et al. (1972) found that as f e t a l s k i n t h i c k e n s , the ra t e of sodium d i f f u s i o n slows down. Hoyes (1968b) has 13 observed that between 17 to 20 weeks of g e s t a t i o n i n the human, the c e l l s i n the l a y e r s beneath the periderm show si g n s of k e r a t i n i z a t i o n and by 26 weeks, the periderm i s shed and a d e f i n i t i v e stratum corneum, formed from the l a y e r s beneath, i s e v i d e n t . The Umbilical Cord The u m b i l i c a l c o r d i s composed mainly of g e l a t i n o u s co n n e c t i v e t i s s u e (Wharton's j e l l y ) which supports the blood v e s s e l s i n the c e n t e r , and which i s covered by an e p i t h e l i u m that i s continuous with the amnion and the s k i n of the f e t u s (Thomsen and H i e r s c h , 1969). The e p i t h e l i a l l a y e r resembles the amniotic e p i t h e l i u m and the periderm of f e t a l s k i n (Leeson and Leeson, 1965; Hoyes, 1969), and i s l i k e w i s e thought to be important i n water and i o n - t r a n s f e r . According to Abramovich (1973), the c o r d becomes an important s i t e i n the exchange of m a t e r i a l s with the amniotic f l u i d at the stage when s k i n k e r a t i n i z a t i o n begins to occur ( i . e . , between 17 - 20 weeks i n the human, ac c o r d i n g to Hoyes, 1968b). I t i s roughly at t h i s time when the c o r d e p i t h e l i u m d i f f e r e n t i a t e s from a p o o r l y developed s t r u c t u r e i n t o one which resembles the f e t a l periderm (see Hoyes, 1969). The c o r d e p i t h e l i u m , however, does not k e r a t i n i z e l i k e the f e t a l epidermis except i n a region c l o s e to the f e t u s , and thus can continue .to be f u n c t i o n a l u n t i l term (Hoyes, 1969). 14 According to Hutchinson et al. (1959), the unexpectedly large concentration of isotopic water found in Wharton's j e l l y after i n j e c t i o n of the tracer into the human amniotic cavity, indicates that much of the water transfer between amniotic f l u i d and fetus i s across the cord. P l e n t l (1961) perfused the isolated human umbilical cord in vitro and based on the concentrations of t r i t i a t e d water in Wharton's j e l l y , estimated that between 40 and 50 ml of water i s exchanged across the cord per hour. Abramovich (1973) with in vivo experiments suggested that between 3 and 59 ml of water i s absorbed via the cord per hour after mid-gestation. In a l l these studies, only d i f f u s i o n a l fluxes of water across the cord were measured. Although there appears to be a large amount of d i f f u s i o n a l exchange of isotopic water across the cord, i t i s not known i f there are s i g n i f i c a n t net transfers of f l u i d in vivo. C l e a r l y , studies measuring bulk flows are required to assess the r e l a t i v e importance of the umbilical cord as a f e t a l osmoregulatory structure. The Amniotic F l u i d In a l l mammals the accumulation of amniotic f l u i d begins early, coinciding with the outgrowth of the amnion, and thereafter i t s volume increases progressively with advancing gestation, and in most species reaches a peak just p r i o r to term (Adolph, 1967). In the human, volumes of up to 10 ml have been reported as early as eight weeks 1 5 of g e s t a t i o n (Seeds, 1965). E l l i o t and Inman (1961) found a maximum volume at 38 weeks, averaging about 1000 ml, and then noted a r a p i d d e c l i n e as term approached. In the f i r s t h a l f of g e s t a t i o n , the amniotic f l u i d i s ne a r l y i s o t o n i c with maternal and f e t a l plasma, and i s s i m i l a r i n composition to e x t r a c e l l u l a r f l u i d , except f o r a higher c h l o r i d e c o n c e n t r a t i o n and a lower p r o t e i n c o n c e n t r a t i o n (Seeds, 1972). However, i t becomes p r o g r e s s i v e l y more hypotonic as the f e t u s matures. According to the data of G i l l i b r a n d (1969) and Benzie et al. (1974), the average o s m o l a l i t y of amniotic f l u i d i n the human drops from approximately 273 mOsm/kg between 10 - 16 weeks to 250 mOsm/kg at term (approximately 40 weeks). The changes i n o s m o l a l i t y are p a r a l l e l e d by changes i n the l e v e l of sodium i o n s . Between 10 and 16 weeks, the c o n c e n t r a t i o n of sodium i n amniotic f l u i d averages about 135 - 140 m E q / l i t r e and t h e r e a f t e r d e c l i n e s p r o g r e s s i v e l y to about 120 m E q / l i t r e at term ( G i l l i b r a n d , 1969; L i n d , 1981). Other changes i n the composition of amniotic f l u i d that occur as the f e t u s matures are a p r o g r e s s i v e i n c r e a s e in the c o n c e n t r a t i o n of f e t a l waste products such as urea, c r e a t i n i n e and u r i c a c i d above plasma l e v e l s (Benzie et al. , 1974; P i t k i n , 1974), and an in c r e a s e i n the c o n c e n t r a t i o n of hydrogen ions (Seeds and H e l l e g e r s , 1968). I t has been p o s t u l a t e d t h a t d u r i n g the e a r l i e s t weeks of g e s t a t i o n , when f e t a l t i s s u e s are p o o r l y d i f f e r e n t i a t e d , the amniotic f l u i d o r i g i n a t e s as a d i a l y s a t e of maternal 1 6 plasma a c r o s s the p l a c e n t a and the chorioamnion (Behrman et al. , 1967). As the f e t u s matures, v a r i o u s f e t a l t i s s u e s begin t o play an i n c r e a s i n g l y important r o l e i n the pro d u c t i o n and d i s p o s a l of amniotic f l u i d . In the second h a l f of g e s t a t i o n , f e t a l u r i n e begins to c o n t r i b u t e s i g n i f i c a n t l y to the amniotic f l u i d , and t h i s i s r e f l e c t e d by 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 amniotic f l u i d urea, u r i c a c i d and c r e a t i n i n e , which are e x c r e t e d by the f e t a l kidneys i n c o n c e n t r a t i o n s higher than those of plasma ( L i n d and Hytten, 1970; Seeds, 1972). I t has been suggested that 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 f l u i d may i n pa r t be due to the a d d i t i o n of i n c r e a s i n g volumes of hypotonic u r i n e (Seeds, 1972). The average volume of u r i n e voided by the human f e t u s near term i s approximately 500 ml per day (van O t t e r l o et al. , 1977; Abramovich et al. , 1979). The f e t a l sheep v o i d s amounts of s i m i l a r magnitude (Alexander et al. , 1958). The lungs are an a d d i t i o n a l source of amniotic f l u i d ; in the f e t a l sheep as much as 300 ml per day of lung f l u i d may be s e c r e t e d i n t o the amniotic c a v i t y (Normand et al. , 1971). Gluck et al. (1971) observed that human amniotic f l u i d c o n t a i n s a s u r f a c e a c t i v e l e c i t h i n which c o u l d have o r i g i n a t e d only i n the f e t a l lung, thus p r o v i d i n g evidence that the lungs of the human f e t u s c o n t r i b u t e at l e a s t some f l u i d to the amniotic compartment. An important pathway f o r the d i s p o s a l of amniotic f l u i d i s the f e t a l g a s t r o - i n t e s t i n a l t r a c t . The amount of f l u i d swallowed by the mature human and sheep f e t u s per day i s of 1 7 the same order of magnitude as the amount of urine produced (Pritchard, 1965; Barnes, 1976; Abramovich et al. , 1979; Harding et al. , 1984). The swallowed amniotic f l u i d i s presumably reabsorbed into the f e t a l c i r c u l a t i o n via the i n t e s t i n a l epithelium (Wright and Nixon, 1961; Abramovich, 1970), as discussed previously. Although f e t a l swallowing and voiding influence the volume of amniotic f l u i d in normal fetuses, the control of volume i s probably achieved by other structures. Abramovich et al. (1979) have observed that even in polyhydramnios (excessive volumes of amniotic f l u i d ) and oligohydramnios (scanty amniotic f l u i d ) , the amount of f l u i d swallowed and voided by the fetus i s the same as when amniotic f l u i d volumes are normal. In an interesting study, Minei and Suzuki (1976) occluded the esophagus in the late-term f e t a l rhesus monkey and found that the absence of f e t a l swallowing resulted in only a transient polyhydramnios, with amniotic f l u i d volumes returning to normal range within three days; there was an apparent readjustment in the volume of amniotic f l u i d by other pathways. Likewise, in renal agenesis, other pathways of f l u i d production (e.g., f e t a l lungs) may compensate-for the lack of contribution by f e t a l urine; normal amniotic f l u i d volumes and occasionally even polyhydramnios have been observed when there i s no urine flow into the amniotic sac (see Abramovich, 1979). The volume and composition of amniotic f l u i d could be controlled by bulk movement across the amnion, the umbilical cord, and the f e t a l skin. 18 The Origin of Intra-Uterine Water In the human, about four l i t e r s of f l u i d accumulates w i t h i n the i n t r a - u t e r i n e c a v i t y d u r i n g pregnancy, and s i n c e t h i s amount i s f a r i n excess of that which c o u l d o r i g i n a t e from u t e r i n e or f e t a l metabolic sources, a l a r g e p a r t of the d a i l y net i n c r e a s e r e q u i r e d f o r f e t a l growth and s u r v i v a l must u l t i m a t e l y be t r a n s f e r r e d from the mother (Seeds, 1965). F l u i d can gain e n t r y ' i n t o the i n t r a - u t e r i n e compartments e i t h e r v i a the p l a c e n t a or by the e x t r a p l a c e n t a l membranes. I t has been found that water can c r o s s these b a r r i e r s by bulk flow i n response to e x p e r i m e n t a l l y c r e a t e d g r a d i e n t s of osmotic and h y d r o s t a t i c p r e s s u r e , and i t has been suggested that such g r a d i e n t s may be r e s p o n s i b l e f o r the accumulation of water in vivo (see Seeds, 1965; Barnes, 1 976). G r a d i e n t s i n osmotic', c o l l o i d osmotic and h y d r o s t a t i c pressure a c r o s s the p l a c e n t a i n s e v e r a l s p e c i e s have been measured in an attempt to e l u c i d a t e the mechanisms by which water accumulates. The t o t a l s o l u t e c o n c e n t r a t i o n s i n maternal and f e t a l plasma are e i t h e r the same or s l i g h t l y higher i n maternal than in f e t a l plasma ( K e r p e l - F r o n i u s , 1970; Armentrout et al. , 1977), and t h e r e f o r e cannot provide the d r i v i n g f o r c e f o r water accumulation v i a the p l a c e n t a . C o l l o i d osmotic p r e s s u r e s (which depend p r i m a r i l y on p r o t e i n c o n c e n t r a t i o n s in plasma) are lower in f e t a l than maternal plasma and so 19 favour a movement of water i n the f e t a l to maternal d i r e c t i o n , as do t r a n s - p l a c e n t a l g r a d i e n t s i n h y d r o s t a t i c p r e s s u r e , when present (Seeds, 1965). Likewise, h y d r o s t a t i c and osmotic g r a d i e n t s a c r o s s the amnion favour a bulk flow of f l u i d i n a d i r e c t i o n from the amniotic f l u i d into, the e x t r a c e l l u l a r f l u i d of the mother (Garby, 1957). Thus the net t r a n s f e r of water from the mother to the i n t r a - u t e r i n e compartments d u r i n g g e s t a t i o n cannot be e x p l a i n e d simply by d i f f e r e n c e s i n osmotic, c o l l o i d osmotic, and h y d r o s t a t i c g r a d i e n t s between the f l u i d compartments. Hormonal Control of Intra-Uterine Fluids Recently, evidence has accumulated which suggests that the accumulation and c o n t r o l of i n t r a - u t e r i n e f l u i d s may be achieved through the f e t u s ' s own endocrine system. The f i r s t c l u e came when V i z s o l y i and Perks (1969) d i s c o v e r e d a r g i n i n e v a s o t o c i n (AVT) i n the p i t u i t a r i e s of mammalian f e t u s e s . T h i s hormone, found i n a l l sub-mammalian v e r t e b r a t e s , i s present i n mammalian p i t u i t a r i e s only d u r i n g f e t a l l i f e . In the anuran amphibians, AVT has an osmoregulatory f u n c t i o n , a f f e c t i n g f l u i d t r a n s p o r t across the s k i n and the u r i n a r y bladder (Sawyer, 1970). T h e r e f o r e , V i z s o l y i and Perks (1974) and H o l t and Perks (1977a) t e s t e d the e f f e c t s of AVT and other hormones on the amnion, a s t r u c t u r e r e s t r i c t e d to f e t a l l i f e , which was thought to be i n v o l v e d i n i n t r a - u t e r i n e water t r a n s p o r t . 20 V i z s o l y i and Perks (1974) found that AVT and a r g i n i n e v a s o p r e s s i n (AVP) caused an i n c r e a s e i n the net movement of water from the maternal to the f e t a l s u r f a c e of the i s o l a t e d guinea-pig amnion, a g a i n s t g r a d i e n t s i n osmotic and h y d r o s t a t i c p r e s s u r e . H o l t and Perks (1977a) found that m a t e r n a l - f e t a l d i f f u s i o n a l f l u x e s of water a c r o s s the amnion were i n c r e a s e d with v a s o p r e s s i n . Manku et al. (1975) confirmed the e f f e c t s of AVP on net water t r a n s f e r a c r o s s the gu i n e a - p i g amnion. These r e s u l t s suggested that i n t r a - u t e r i n e water requirements c o u l d be met by the t r a n s p o r t of f l u i d from the maternal c i r c u l a t i o n to the amniotic c a v i t y v i a the amnion, a i d e d by neurohypophysial hormones. P r o l a c t i n was found to reduce the p e r m e a b i l i t y to water of the i s o l a t e d amnion of the guin e a - p i g (Holt and Perks, 1975) and the human ( L e o n t i c and Tyson, 1977; L e o n t i c et al. , 1979); such an e f f e c t in vivo would favour the r e t e n t i o n of any accumulated f l u i d i n the amniotic sac. Manku et al. (1975) observed that C o r t i s o l r e v e r s e d the a c t i o n s of p r o l a c t i n and v a s o p r e s s i n on water t r a n s f e r a c r o s s the gu i n e a - p i g amnion. P r o l a c t i n and v a s o p r e s s i n a l s o appear to i n f l u e n c e sodium t r a n s f e r a c r o s s the amnion. Ho l t and Perks (I977a,b) found that v a s o p r e s s i n i n c r e a s e d the u n i d i r e c t i o n a l f l u x of sodium from the maternal to the f e t a l s u r f a c e of the guinea - p i g amnion, whereas p r o l a c t i n caused an i n c r e a s e i n sodium f l u x in the opp o s i t e d i r e c t i o n . The combined a c t i o n s of p r o l a c t i n , v a s o p r e s s i n and C o r t i s o l , and perhaps other 21 hormones, c o u l d ensure that the volume and composition of the amniotic f l u i d remain constant w i t h i n narrow l e v e l s . The f e t a l endocrine system may a l s o be r e s p o n s i b l e f o r r e g u l a t i n g f e t a l e x t r a c e l l u l a r f l u i d by a c t i n g on the lungs, s k i n and u r i n a r y bladder. C a s s i n and Perks (1982) found that p r o l a c t i n caused a s i g n i f i c a n t 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 , Na +, and C l ~ i n the f e t a l goat, a mechanism that may be of importance to the f e t u s d u r i n g o v e r h y d r a t i o n or s a l t l o a d i n g , s i n c e the kidneys are probably l i m i t e d i n t h e i r a b i l i t y to handle such s t r e s s . At b i r t h , a b s o r p t i o n of lung f l u i d i s f a c i l i t a t e d by v a s o p r e s s i n and epinephrine (Perks and C a s s i n , 1982; Walters et al. , 1982). In p r e l i m i n a r y experiments, H o l t and Perks (1977a) observed that v a s o p r e s s i n f a c i l i t a t e s water a b s o r p t i o n a c r o s s the s k i n and .the u r i n a r y bladder of the g u i n e a - p i g . France (1976) and France et al. (1976) have found that u n i d i r e c t i o n a l f l u x e s of water and sodium across the sk i n and bladder of f e t a l sheep and pigs are l i k e w i s e a f f e c t e d by neurohypophysial hormones. STATEMENT OF THE PROBLEM The present study was undertaken i n order to e l u c i d a t e f u r t h e r the mechanisms by which i n t r a - u t e r i n e f l u i d s are r e g u l a t e d . A t t e n t i o n was focused on the e f f e c t s of neurohypophysial hormones on the net t r a n s f e r of water acr o s s the amnion and the s k i n of the f e t a l g u i n e a - p i g . 22 The s t u d i e s of V i z s o l y i and Perks (1974) have shown that both AVT and AVP are capable of causing a net movement of water from the maternal to the f e t a l s u r f a c e of the gui n e a - p i g amnion. According to these i n v e s t i g a t o r s , AVP i s more e f f e c t i v e at i n d u c i n g t h i s response, and t h e r e f o r e , may be the more important p e p t i d e i n a p o s s i b l e p h y s i o l o g i c a l system. In s p i t e of t h i s , s t u d i e s with AVP which measure bulk flows have been l i m i t e d to p r e l i m i n a r y o b s e r v a t i o n s . T h e r e f o r e , i n the present study the e f f e c t s of AVP on net water t r a n s f e r a c r o s s the amnion were i n v e s t i g a t e d f u r t h e r . The response of the amnion to AVP was s t u d i e d at d i f f e r e n t stages of g e s t a t i o n . Changes in the response of the amnion to AVP were compared with changes i n 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 , as w e l l as with changes i n the volume of amniotic f l u i d through g e s t a t i o n . L i k e the amnion, the f e t a l s k i n responds to v a s o p r e s s i n (France, 1976; H o l t and Perks, 1977a). However, to date only changes in u n i d i r e c t i o n a l d i f f u s i o n a l f l u x of water ac r o s s the f e t a l s k i n have been measured. T h e r e f o r e , i t seemed important to extend these preliminary-s t u d i e s to changes i n bulk flow. In a d d i t i o n , i t was important to t e s t the e f f e c t s of AVT on the f e t a l s k i n ; t h i s p e p t i d e i s p l e n t i f u l i n the f e t a l p i t u i t a r y at mid g e s t a t i o n , before the s k i n k e r a t i n i z e s (see Perks, 1977), and has not p r e v i o u s l y been t e s t e d on water movement across the f e t a l s k i n . A c c o r d i n g l y , the e f f e c t s of AVP and AVT on 23 net water t r a n s p o r t a c r o s s the f e t a l s k i n were i n v e s t i g a t e d . 24 GENERAL METHODS 1 . Net Water Flow Experiments (a) The Isolated Amnion Preparation Net water flow through the i s o l a t e d g uinea-pig amnion was determined using the g r a v i m e t r i c method of V i z s o l y i and Perks (1974), shown i n F i g u r e 1. S t u d i e s were c a r r i e d out on f e t a l g u inea-pigs at v a r i o u s stages of g e s t a t i o n . G e s t a t i o n a l ages were determined from the crown-rump le n g t h s , weights, and number of the f e t u s e s , using the data of Draper (1920) and Ibsen (1928). The pregnant guinea-pigs were a n a e s t h e t i s e d with ether, and the f e t u s e s , with membranes and p l a c e n t a i n t a c t , were d i s s e c t e d from the u t e r i n e horns i n t o a e r a t e d maternal s a l i n e at 37°C (see p. 29). The outer y o l k - s a c membrane was s t r i p p e d o f f , and an area of r e f l e c t e d amnion was removed and t i e d over the end of a g l a s s supporting tube (1 cm 2 c r o s s - s e c t i o n a l a r e a ) . The f e t a l s u r f a c e of the amnion, i . e . , the s u r f a c e bathed by amniotic f l u i d in vivo, faced the i n s i d e of the tube. The tube was f i l l e d with 2.5 ml of warmed, a e r a t e d amniotic s a l i n e (p. 28) and suspended in an outer bath c o n t a i n i n g 50 ml of maternal s a l i n e at 37°C. T h i s temperature was maintained throughout the experiment by means of a constant-temperature chamber i n t o which the outer bath was p l a c e d . The outer bath was oxygenated with a i r by means of a s i d e arm which prevented 25 F i g u r e 1. The G r a v i m e t r i c Apparatus used f o r the in vitro Study of Net Water Movement Through the I s o l a t e d Amnion of the Guinea-Pig (from V i z s o l y i and Perks, 1974). 25A To -Recorder Supporting tube 2 cm Water bath ( 3 7 ° C ) Fetal_ side" Maternal I side" " " Balance Lever Differential Transformer Amniotic membrane \ Outer container Compressed air Amniotic saline Maternal saline 26 bubbles from disturbing the membrane. The inner tube did not f l o a t but was supported by a weighted balance lever, and the weight was adjusted so that the f l u i d l e v e l in the inner tube was 2 cm above that in the outer bath. The difference in osmolarities of the salines on the two sides of the membrane, and the 2 cm hydrostatic pressure head reproduced in vivo conditions (see V i z s o l y i and Perks, 1974). The thread attaching the supporting tube to the lever passed through a rod, which formed the movable central core for the c o i l of a Trans-Tek d i f f e r e n t i a l transducer. Changes in the weight of the inner tube resulting from f l u i d transfer across the amnion produced movement of the balance lever and concomitant changes in transducer output, which were continuously recorded using either a Soltec 220 or a Beckman RS Dynograph recorder. The system was cal i b r a t e d by adding known amounts of f l u i d to the inner supporting tube. In l a t e r experiments on the amnion, the apparatus was modified as follows: rather than measuring displacement caused by f l u i d transfer across the amnion, actual weight changes of the inner tube were measured. The inner tube was suspended in the outer bath by a thread attached to a hanger for bottom weighing on a Mettler AE 163 electronic balance. The system was ca l i b r a t e d as before, with the addition of known amounts of f l u i d to the inner tube. Weights were recorded at three minute i n t e r v a l s . 27 After the amnion was in place, i t was allowed to e q u i l i b r a t e for 30 - 45 minutes, during which time the salines were changed at approximately 15 minute in t e r v a l s . After the e q u i l i b r a t i o n period, both salines were changed to ensure that ionic conditions were i d e n t i c a l at the start of each test period. During t h i s change, arginine vasopressin was included in the inner amniotic saline, at a f i n a l concentration of l00mU/ml (vasopressor a c t i v i t y ) . After each test, which lasted for up to 30 minutes, both the inner and outer solutions were replaced with fresh salines, and the membrane was allowed to recover. A l l values for the net movement of water were expressed as mg per cm2 of membrane per minute. (b) The Isolated Skin Preparation Skin from the back region of fetuses between 33 and 48 days of gestation (0.49 - 0.70 of term) was isolated by sharp diss e c t i o n and set up in the same gravimetric apparatus used for the i n i t i a l amnion experiments. However, there were no hydrostatic or osmotic pressure gradients between the mucosal and serosal surfaces of the skin; both inner tube and outer bath contained maternal saline, and the weight on the balance lever was adjusted so that the lev e l s of f l u i d in the inner tube and outer bath were the same. The serosal surface of the skin faced the inside of the inner tube. 28 After the skin was set up in the apparatus, i t was allowed to equilibrate 30 - 45 minutes, with salines being changed at about 15 minute i n t e r v a l s . As in the amnion experiments, the salines on both sides of the skin were changed after the e q u i l i b r a t i o n period. During t h i s change, arginine vasopressin or arginine vasotocin was included in the inner saline which contacted the serosal surface of the skin, at f i n a l concentrations of 5 -l00mU/ml (vasopressor a c t i v i t y ) . After the treatment, which lasted up to 30 minutes, the salines were changed, and the skin was allowed to recover before further tests were done. Whenever possible, the two hormones were tested in p a r a l l e l , using skin from fetuses from the same mother, in order to compare the effectiveness of the hormones in influencing water flow through the skin. 2 . Salines Maternal and amniotic salines were designed by V i z s o l y i and Perks (1974) to reproduce the e l e c t r o l y t e s present in maternal (or f e t a l ) plasma and amniotic f l u i d , respectively. They were composed of the following: a) Amniotic saline grams/litre of d i s t i l l e d water NaCl KC1 CaCl 2-2H 20 MgCl 2-6H 20 Glucose 7.31 0.46 0.33 0.23 1 .00 29 A phosphate buffer (0.2M NaH 2PO„-Na 2HPO f t; pH 7.8) was added at 10- ml buffer per l i t r e of sa l i n e . The f i n a l pH of the saline was 7.4, and i t s osmolarity 286 mOsm/litre (Advanced Instruments osmometer). b) Maternal saline A phosphate buffer was added as for the amniotic s a l i n e . The f i n a l pH was 7.4, and the osmolarity was 314 mOsm/litre. 3 . Hormone Solutions The hormones used were.synthetic arginine vasopressin (AVP) and synthetic arginine vasotocin (AVT) (Sigma Chemicals, St. Louis, Mo.). Each was dissolved in a solution of 0.25% acetic acid in 0.9% NaCl, to give a f i n a l concentration of 100 IU/ml (of vasopressor a c t i v i t y ) , at a pH of 3.5. 0.5% chlorobutol was added as a preservative. Just prior to the experiments, the hormones were di l u t e d in either amniotic or maternal saline (for amnion or skin experiments, respectively) to give f i n a l concentrations from 5 to 100 mU/ml. No measurable changes in osmolarity or pH of the salines occured when the hormones were added. grams/litre of d i s t i l l e d water NaCl KC1 CaCl 2-2H 20 MgCl 2-6H 20 Glucose 8.76 0.41 0.32 0.26 1 .00 30 For control experiments, a solution containing only 0.25% acetic acid and 0.5% chlorobutol in 0.9% NaCl was dil u t e d into the amniotic or maternal saline. 4. Electron Microscopy of the Amnion Immediately after dissection, samples of refl e c t e d amnion from guinea-pig fetuses between 28 and 70 days of gestation were fixed for . 1 hour at room temperature (22°C) in a 2.5% phosphate-buffered glutaraldehyde f i x a t i v e of the following composition (see Glauert, 1975): NaH2POft-H20 (g) 3.31 Na 2HPO„-7H 20 (g) 33.71 25% glutaraldehyde in water (ml) 40.00 D i s t i l l e d water to make (ml) 1000.00 F i n a l concentration of glutaraldehyde 2.5% Osmolarity of phosphate buffer 320 mOsm/litre pH of f i x a t i v e 7.4 In addition, 1 mM of MgCl 2 was added to the f i x a t i v e for better preservation of the organelles. Following this primary f i x a t i o n , the tissue was washed overnight at 4°C in phosphate buffer and then post-fixed in 2% phosphate-buffered osmium tetroxide (OsO„) for one hour at 22°C. The tissue was subsequently dehydrated by passage through increasing concentrations of ethanol, and then propylene oxide, according to a schedule given by Glauert (1975). Following dehydration, the tissue was embedded in an epoxy resin (Epon 812). Ul t r a t h i n sections (about 50-60nm) were cut, placed on uncoated copper grids and stained with lead c i t r a t e and uranyl acetate prior to examination under a Zeiss EM 10 microscope. 31 Thick s e c t i o n s of epon-embedded t i s s u e were s t a i n e d with t o l u i d i n e blue and examined under the l i g h t microscope f-^r c o n f i r m a t i o n of the general morphology of the amnion as seen under the e l e c t r o n microscope. 5. Electron Microscopy of the Fetal Skin Skin from the back region of f e t a l guinea-pigs at 35 and 52 days of g e s t a t i o n was a l s o s t u d i e d under the e l e c t r o n microscope. The methods used were the same as those d e s c r i b e d f o r the amnion. 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 V i z s o l y i and Perks (1974) demonstrated that a r g i n i n e v a s o t o c i n (AVT) and a r g i n i n e v a s o p r e s s i n (AVP) both i n f l u e n c e the ra t e of net water t r a n s f e r a c r o s s the i s o l a t e d amnion of the f e t a l g u i n e a - p i g . They found that the hormones are capable of ca u s i n g a net uptake of water from the maternal to the f e t a l s u r f a c e of the membrane ( i . e . , towards the amniotic c a v i t y ) , a g a i n s t osmotic and h y d r o s t a t i c pressure g r a d i e n t s . The amnion was found to be more s e n s i t i v e to AVP than to AVT ( t h r e s h o l d doses f o r the two hormones were 1.0 mU/ml and 6.4 mU/ml of vasopressor a c t i v i t y , r e s p e c t i v e l y ) . T h i s suggested that AVP was more l i k e l y to be i n v o l v e d i n a p o s s i b l e p h y s i o l o g i c a l system i n the f e t u s , one that c o u l d c o n t r o l an e x t r a p l a c e n t a l route f o r the t r a n s p o r t of water to the amniotic sac and f e t u s , from the mother. However, s t u d i e s with AVP, although i t seemed more e f f e c t i v e , were l i m i t e d to p r e l i m i n a r y o b s e r v a t i o n s . In a d d i t i o n , V i z s o l y i and Perks (1974) observed a l a r g e v a r i a b i l i t y i n response between d i f f e r e n t amniotic membranes, and i t was p o s s i b l e that t h i s was due to d i f f e r e n c e s i n the g e s t a t i o n a l age of the membranes. 33 The present study was undertaken to extend the e a r l i e r s t u d i e s on the e f f e c t s of AVP on net water t r a n s p o r t a c r o s s the amnion. Amniotic membranes from f e t a l guinea-pigs at v a r i o u s stages of g e s t a t i o n were t e s t e d i n an e f f o r t to see i f there were any changes i n response of the amnion to AVP with f e t a l age. A systematic i n v e s t i g a t i o n of the u l t r a s t r u c t u r e of the amniotic e p i t h e l i u m from f e t a l guinea-pigs at v a r i o u s stages of g e s t a t i o n was c a r r i e d out. The purpose of t h i s was to examine c y t o l o g i c a l f e a t u r e s of the e p i t h e l i u m which may be r e l a t e d to i t s f u n c t i o n a l a c t i v i t y . E l e c t r o n m i c r o s c o p i c s t u d i e s were a l s o c a r r i e d out on a membrane t r e a t e d with AVP to see i f the hormone caused any morphological changes i n the amniotic, e p i t h e l i u m . F i n a l l y , the volume of amniotic f l u i d was measured at d i f f e r e n t stages of g e s t a t i o n . T h i s was done to see i f changes i n the t r a n s p o r t p r o p e r t i e s and s t r u c t u r e of the amnion through g e s t a t i o n might be r e l a t e d to changes in the amniotic f l u i d volume. _ RESULTS 1. The E f f e c t s of AVP on the Amnion T h i r t y - n i n e amniotic membranes from f e t u s e s between 0.44 of term and term (30 - 68 days of g e s t a t i o n ) were set up i n the g r a v i m e t r i c apparatus, as d e s c r i b e d i n the methods. Upon immersion of the s u p p o r t i n g tubes in the 34 outer baths, 23 of the membranes showed a slow steady passage of water from the amniotic s a l i n e to the maternal s a l i n e , presumably i n response to the h y d r o s t a t i c and osmotic g r a d i e n t s maintained from the f e t a l to the maternal s i d e of the amnion. The average r e s t i n g flow of water i n the f e t a l to maternal d i r e c t i o n was 0.69 mg/cm2 per minute (range=0.07 to 2.46 mg/cm2 per minute). However, 11 membranes showed no flow of f l u i d , and 5 showed a slow uptake of water towards the amniotic s a l i n e , a g a i n s t the p r e v a i l i n g g r a d i e n t s . V a s o p r e s s i n (100 mU/ml) added to the amniotic s a l i n e on the f e t a l s u r f a c e of the amnion was capable of causing a net flow of f l u i d i n the maternal to f e t a l d i r e c t i o n , a g a i n s t the p r e v a i l i n g g r a d i e n t s , i n most cases r e v e r s i n g the i n i t i a l f e t a l - m a t e r n a l r e s t i n g flow. When the AVP was removed and the s a l i n e s changed, most membranes recovered, and returned to a r e s t i n g flow r a t e . These o b s e r v a t i o n s are i n agreement with the r e s u l t s of V i z s o l y i and Perks (1974). However, the magnitude of the response to AVP appeared to vary with the age of the f e t u s . Examples of responses are shown i n F i g u r e 2. A t o t a l of 32 d i f f e r e n t membranes were t e s t e d with AVP. There were seven p r e p a r a t i o n s in which AVP was s u b s t i t u t e d with a c o n t r o l s a l i n e . Table I shows the value s obtained f o r r e s t i n g flow r a t e s , AVP-influenced flow r a t e s , and the responses, f o r a l l the t e s t s performed. The r e s t i n g flow r a t e was d e f i n e d as the average r a t e of flow i n the 12 minute p e r i o d immediately before treatment with AVP. The AVP-influenced 35 F i g u r e 2. The E f f e c t s of AVP on Net Water Movement Across the I s o l a t e d Amnion of the Guinea-Pig. Membranes were taken from f e t u s e s at v a r i o u s stages of g e s t a t i o n , as i n d i c a t e d on each graph. H o r i z o n t a l bars show p e r i o d s of c o n t a c t with lOOmU/ml AVP or with c o n t r o l s a l i n e . O r d i n a t e s : r a t e of net water movement (mg/cm2 per minute); the d o t t e d l i n e r e presents zero change; values above t h i s l i n e i n d i c a t e water uptake towards the f e t a l s i d e of the membrane ( i . e . , towards the amniotic c a v i t y ) ; v a l u e s below the l i n e i n d i c a t e water l o s s towards the maternal s i d e of the membrane. A b s c i s s a e : time, i n minutes. 35A 0.5h c E CM E co E c E o > o 3 I O 0.0 0.5 1.0 S\ 9 18 ,— 32 days 1 . 0 -0 . 5 -0.0 0 . 5 -1.0L<-— x A V P 18 27 3 6 •— 56 days 2.5 2.0h 1.5h 1.0 0 5 0.0 0.5 1.0 A V P c o n t r o l _ J I 27 36 - 40 days 18 27 36 1.0 0.5 0.0 0.5 1.0 2.5 2.0 1 . 5 -1 . 0 -0 . 5 -0.0 0 5 1.0 c _ i A V P 9 18 27 3 6 68 days —^* V A V P 18 27 3 6 Time ; m i n u t e s T a b l e I : T h e R e s p o n s e o f t h e A m n i o n t o A V P D u r i n g t h e C o u r s e o f G e s t a t i o n R e s p o n s e G e s t a t i o n a l R a t e o f R e s t i n g F l o w R a t e o f A V P - I n f l u e n c e d F l o w R e s p o n s e : T h e I n c r e a s e I n R a t e N u m b e r A g e ( d a y s ) ( m g / c m 2 p e r m i n u t e ) ( m g / c m 2 p e r m i n u t e ) o f M a t e r n a l - F e t a l F l o w w i t h A V P 3 ( m g / c m 2 p e r m i n u t e ) 1 3 0 0 +0 . 10 0 . 10 2 32 0 +0 . 15 0 . , 15 3 32 - 0 . 2 0 +0 . 17 0 . . 3 7 4 33 0 0 0 5 38 - 0 . . 34 - 0 . 14 0 , . 2 0 6 4 0 +0, . 0 3 +0 . 7 0 0 . . 6 7 7 48 +0, . 0 6 +0 .21 0 . . 15 8 48 +0, , 10 +0 . 4 2 0 . . 32 9 5 0 +0. ,21 +0. . 3 4 0 . . 13 10 5 0 +0. . 4 8 + 1 . 3 7 0 . 8 9 1 1 52 0 +0. .51 0 . 51 12 54 - 0 . . 92 +0. . 3 3 1 . 2 5 13 55 - 0 . . 3 0 +0. . 3 7 0 . 6 7 14 55 - 0 . . 6 0 +0. . 2 0 0 . 8 0 15 56 - 0 . 32 +0. . 9 5 1 . 2 7 16 58 0 +0. . 76 0 . 76 17 58 - 1 . 51 +0. , 04 1 . 5 5 18 6 0 0 +0. . 18 0 . 18 19 62 -o. 8 3 +0. , 0 9 0 . 92 2 0 6 5 - 0 . 31 - 0 . , 56 0 21 6 5 - 0 . 7 0 - 0 . 73 O 22 66 0 0 0 2 3 66 0 0 0 24 66 - 0 . 0 7 +0 . 0 6 0 . 13 2 5 ' 67 - 0 . 75 - 0 . 8 0 0 26 68 - 0 . 4 9 - 0 . 4 9 0 2 7 68 0 0 0 28 68 0 0 0 2 9 6 8 , - 2 . 46 - 2 . 46 0 3 0 6 8 ' - 0 . 29 - 0 . 48 0 31 68 - 0 . 1 1 - 0 . 13 0 32 68 - 0 . 5 9 - 0 . 66 0 N o t e : N e g a t 1 v e v a l u e s ( - ) i n d i c a t e f l o w o f f l u i d f r o m t h e a m n i o t i c t o t h e m a t e r n a l s a l 1 n e ( i . e . . t o w a r d s t h e m o t h e r ) . P o s 1 t 1 v e v a l u e s (+) I n d i c a t e f l o w f r o m m a t e r n a l t o a m n l o t 1 c s a l 1 n e ( t o w a r d s t h e f e t u s ) . c o n t i n u e d o n n e x t p a g e . . . . T a b l e I ( c o n t i n u e d ) 1. T h e r a t e o f r e s t i n g f l o w I s t h e a v e r a g e r a t e o f f l o w i n t h e 12 m i n u t e s b e f o r e t r e a t m e n t w i t h A V P . 2 . T h e r a t e o f A V P - 1 n f 1 u e n c e d f l o w I s t h e a v e r a g e r a t e o f f l o w I n t h e 15 m i n u t e s I m m e d i a t e l y a f t e r A V P a d d i t i o n . 3 . T h e r e s p o n s e 1s t h e i n c r e a s e 1n t h e r a t e o f f l o w t o w a r d s t h e f e t u s , a n d I s t h e d i f f e r e n c e b e t w e e n t h e r e s t i n g f l o w a n d A V P - 1 n f l u e n c e d f l o w . No c h a n g e 1n f l o w , o r a n I n c r e a s e I n f l o w t o w a r d s t h e m a t e r n a l s a l i n e I s t a k e n a s z e r o r e s p o n s e . I n a d d i t i o n t o t h e t e s t s l i s t e d a b o v e , t h e r e w e r e 7 c o n t r o l p r e p a r a t i o n s , t a k e n f r o m f e t u s e s a t v a r i o u s s t a g e s o f g e s t a t i o n ; n o n e o f t h e s e s h o w e d a n y I n c r e a s e 1n m a t e r n a l - f e t a l f l u i d f l o w w i t h c o n t r o l s a l i n e . 38 flow rate was the average rate of flow in £he 15 minutes after AVP addition. The response was defined as the change in the rate of flow with AVP. No change of flow, or an increase in the net flow outwards, towards the maternal saline, was taken as zero response. The results are quantified graphically in Figure 3. The response to AVP increased with f e t a l age, up u n t i l about 58 days of gestation (0.85 ot term). The peak response, at 58 days, was an increased water movement towards the fetus of 1.55 mg/cm2 per minute. After t h i s time, the ef f e c t declined and was l o s t . Membranes over 64 days (0.94 of term) showed only one weak response (0.13 mg/cm2 per minute) in 13 experiments. The loss in the a b i l i t y to respond to AVP appeared to be abrupt, occurring between approximately 62 and 66 days. None of the seven control membranes, taken from fetuses of di f f e r e n t gestational ages, showed any increase in maternal to f e t a l f l u i d flow. 2. The Fine Structure of the Amniotic Epithelium The structure of the epithelium from ten amniotic membranes taken from guinea-pig fetuses between 28 and 70 days of gestation (term = approximately 68 days) was studied under the electron microscope. For convenience of description, the membranes have been divided into three groups: early (consisting of amnions at 28, 30, 35, 38 days), mature (50, 58, 62 days), and near-term (64, 68, 70 days). There were marked changes in the epithelium during 39 F i g u r e 3. The Changes i n the Response of the Amnion to AVP Through the Course of G e s t a t i o n . Each p o i n t r e p r e s e n t s a separate membrane (n=32). O r d i n a t e : Increase i n the r a t e of net water t r a n s p o r t i n the maternal to f e t a l d i r e c t i o n ( i . e . , the response), i n mg/cm2 per minute. A b s c i s s a : G e s t a t i o n a l age of the f e t u s , in days. Curve of best f i t estimated by computer ( l e a s t squares f i t t i n g to B - s p l i n e s ; program designed by C. Moore, U.B.C. Computing C e n t r e ) . 40 the course of gestation, as seen in Figure 4, which shows an epithelium from each of the three groups. Within each group, no clear differences in structure were evident. Early Amniotic Epithelium (28, 30, 35, 38 days) The amniotic epithelium in the early stages of gestation was found to be composed of a single layer of flattened c e l l s underlain by a "continuous basement membrane (Figures 4A, 5A, 5D). The average width of the epithelium was 2.76±0 . 1 4 M m (iS.E.M.; n = 40: 10 readings for each of 4 membranes). There was no clear evidence for more than one type of c e l l , in th i s or any other stage. There was rarely any overlapping between adjacent c e l l s , and i n t e r c e l l u l a r spaces were narrow (Figures 4A, 5A, 5D). The l a t e r a l c e l l borders had a few conspicuous desmosomes. Structures resembling tight junctions, which oblit e r a t e d the i n t e r c e l l u l a r spaces over short areas, were often observed at the apical surface, which faces the amniotic f l u i d (Figure 5B). The apical surface had few m i c r o v i l l i , but these increased in number with advancing gestation. A surface coat (glycocalyx), composed of fine branching filaments projecting from the m i c r o v i l l i , was evident in the older specimens of th i s group, but i t did not appear to be p a r t i c u l a r l y well developed. The e p i t h e l i a l c e l l nuclei were large and oval, and nuc l e o l i were often observed. Large amounts of rough endoplasmic reticulum were scattered throughout the 41 F i g u r e 4. 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 Through the Course of G e s t a t i o n . A. 28 days ( E a r l y Amnion). B. 50 days (Mature Amnion). C. 64 days (Near Term Amnion). ac: amniotic c a v i t y ; mv: m i c r o v i l l i ; n: nucleus; c: c o n n e c t i v e t i s s u e . X 5333. 41 A 42 Figure 5. Electron Micrographs of the Early Amniotic Epithelium. A. Epithelium at 35 days of gestation (0.51 of term). The c e l l s 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 evident. The apical surface 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 cavit y . A basement membrane (BM) separates the epithelium from the underlying connective tissue. A number of organelles are seen in the cytoplasm. N: nucleus; M: mitochondrion; ER: rough endoplasmic reticulum; Go: golgi apparatus. X 16,000. B. I n t e r c e l l u l a r junctions at the apical surface of e p i t h e l i a l c e l l s at 35 days. T: tight junction; D: desmosome. X 44,000. E p i t h e l i a l c e l l at 38 days of gestation (0.56 of term). The membrane-bound vesicl e s (Ve) beneath the apical surface appear to be coated. MV: m i c r o v i l l i ; Gly: glycogen; Go: golgi apparatus; N: nucleus; M: mitochondrion. X 28,000. D. Micrograph of e p i t h e l i a l c e l l at 38 days showing the narrow i n t e r c e l l u l a r space c h a r a c t e r i s t i c of early amnion. Several desmosomes (D) are evident. Cytoplasmic filaments (F) are observed in the v i c i n i t y of desmosomes. Clusters of p a r t i c l e s which resemble glycogen (Gly) are seen in the cytoplasm. MV: m i c r o v i l l i ; Ve: membrane-bound ve s i c l e s ; ER: rough endoplasmic reticulum; M: mitochondrion; BM: basement membrane. X 40,000. 4 2 A 43 cytoplasm, and a number of mitochondria were evident ( F i g u r e s 5A, 5C, 5D). G o l g i complexes were o f t e n seen near the n u c l e i and c o n s i s t e d of l o o s e l y packed c i s t e r n a e . Membrane-bound v e s i c l e s were evident i n the cytoplasm, e s p e c i a l l y near _the i n t e r c e l l u l a r spaces. Some were a l s o seen at the a p i c a l and b a s a l s u r f a c e s of the c e l l s . A number of these v e s i c l e s appeared to be coated with a f i n e f ilamentous substance (Figure 5C). The number of v e s i c l e s appeared to i n c r e a s e with advancing g e s t a t i o n . Small amounts of g l y c o g e n - l i k e p a r t i c l e s were seen i n the cytoplasm. Cytoplasmic f i l a m e n t s were r a r e l y observed, except i n a s s o c i a t i o n with desmosomes ( F i g u r e 5D). Mature Amniotic Epithelium (50, 58, 62 days) The e p i t h e l i a l c e l l s at t h i s stage were more c u b o i d a l i n shape, and the e p i t h e l i u m appeared to be t h i c k e r ( F i g u r e s 4B, 6A, 7A). The average width of the e p i t h e l i u m was 5.l9±0.24Mm (±S.E.M.; n=30: 10 readings f o r each of 3 membranes). There was now c o n s i d e r a b l e o v e r l a p p i n g between c e l l s . The i n t e r c e l l u l a r spaces were more con v o l u t e d and more d i l a t e d than i n the e a r l y e p i t h e l i u m , and they had m i c r o v i 1 1 i - 1 i k e f o l d s p r o j e c t i n g , i n t o them ( F i g u r e 6C). Numerous membrane-bound v e s i c l e s were seen i n the v i c i n i t y of these spaces. Desmosomes were numerous at t h i s stage, though t i g h t - j u n c t i o n s were s t i l l p r e s e n t . The a p i c a l m i c r o v i l l i were much more abundant i n these than i n the 44 Figure 6. The Fine Structure of the Mature Amniotic Epithelium. Micrograph of e p i t h e l i a l c e l l s at 50 days of gestation (0.74 of term) i l l u s t r a t i n g the di l a t e d i n t e r c e l l u l a r spaces (ICS) and numerous apical 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 epithelium. N: nucleus; ER: rough endoplasmic reticulum; M: mitochondrion; D: desmosomes; Ve: membrane-bound v e s i c l e s ; Gly: glycogen; BM: basement membrane. X 13,000. B: Apical portion of e p i t h e l i a l c e l l at 50 days showing the c l u s t e r s of m i c r o v i l l i (MV). Projecting from the t i p s of the m i c r o v i l l i are the fine branching filaments which make up the glycocalyx. The cytoplasm contains bundles of filaments (F) and glycogen (Gly). 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. M i c r o v i l l i - l i k e folds project into the i n t e r c e l l u l a r spaces (ICS). Numerous membrane-bound v e s i c l e s (Ve) are evident in the v i c i n i t y of the spaces. D: desmosomes; F: cytoplasmic filaments; Gly: glycogen. X 26,000. 45 Figure 7. The Fine Structure of the Mature Amniotic Epithelium. A. Epithelium at 62 days of gestation (0.91 of term). M i c r o v i l l i - l i k e folds are present in 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 filaments; ER: rough endoplasmic reticulum; D: desmosomes; N: nucleus. X 24,000. B. M i c r o v i l l i (MV) on the a p i c a l surface 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 e p i t h e l i a l c e l l at 62 days i l l u s t r a t i n g a well developed glycocalyx, composed of fin e , branching filaments projecting from the m i c r o v i l l i (MV). D: desmosomes; Gly: glycogen. X 26,000. ^5 A 46 e a r l i e r specimens, and in longitudinal sections of the epithelium, appeared as elaborate networks in some regions (Figures 6B, 7B). The surface coat (glycocalyx) was now more prominent, with longer and more branching filaments (Figures 6B, 7C). The filaments extended up to 0.5/xni beyond the t i p s of m i c r o v i l l i in some areas of the amnion. As in the early epithelium, numerous organelles were present in the cytoplasm, and the nuclei showed l i t t l e change. Deposits of p a r t i c l e s resembling glycogen were s t i l l present in the mature epithelium. Cytoplasmic filaments were more abundant in the mature epithelium than in the early epithelium, and they often occurred in bundles throughout the c e l l (Figures 6B, 6C, 7A). Near-Term Amniotic Epithelium (64, 68, 70 days) As the end of gestation approached, the e p i t h e l i a l c e l l s appeared to degenerate. The c e l l s were once more flattened, as seen in Figures 4C, 8A, 8B; the average width of the epithelium was 2.57±0.09Mtn (S.E.M.; n = 30: 10 readings from each of 3 membranes). As in the mature amnion, the c e l l s showed considerable overlapping. The i n t e r c e l l u l a r spaces were d i l a t e d , but there were none of the infoldings of the l a t e r a l membranes ch a r a c t e r i s t i c of the mature amnion (Figures 8A, 8B, 9A, 9B). There were wide areas of open communication between the i n t e r c e l l u l a r spaces and the connective tissue l y i n g beneath the epithelium (Figures 8B, 9B). Tight junctions 47 Figure 8. Electron Micrographs of the Near-term Amniotic Epithelium. A. Epithelium at 68 days of gestation (term). The e p i t h e l i a l c e l l i s flattened and appears to be degenerating. Note the disi n t e g r a t i n g , abnormally flattened nucleus (N). The cytoplasm i s f i l l e d with randomly-arranged filaments. Large vacuoles (V) in the cytoplasm may be the remains of organelles. There are a few short, thick m i c r o v i l l i (MV) on the apical surface. The filaments of the glycocalyx are shorter than in the mature amnion. The i n t e r c e l l u l a r 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 folds of the l a t e r a l membrane projecting into them. There are numerous desmosomes (D), and tight junctions are never observed. BM: basement membrane.. X 20,000. B. Epithelium at 70 days of gestation (judged to be s l i g h t l y over-due). In the near-term amnion, the i n t e r c e l l u l a r spaces (ICS) are often grossly d i l a t e d near the base of the e p i t h e l i a l c e l l and are in open communication with the underlying connective tissue. 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. A. B. Electron Micrographs of the Near-term Amniotic Epithelium. Micrograph of an e p i t h e l i a l c e l l at 68 days showing the randomly arranged 1Onm cytoplasmic filaments (F). The apical c e l l membrane has a layer of electron-dense material about 20nm thick on i t s cytoplasmic surface. 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. Micrograph showing the di s i n t e g r a t i n g nucleus (N) of epithelium at 68 days. The nuclear matrix contains dense masses of heavily stained heterochromatin and very l i t t l e of the l i g h t l y stained, more dispersed 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 i n t e r c e l l u l a r junction of e p i t h e l i a l c e l l s at 68 days. Cytoplasmic filaments (F) appear to converge on the desmosomes. Note the thickening of the apical c e l l membrane. X 110,000. 48 A 4 9 were never observed between c e l l s , and desmosomes were more numerous'now than at any other stage. The apical c e l l membrane had an electron-dense layer (about 20nm thick) on i t s cytoplasmic surface (Figures 9A, 9C); t h i s was reminiscent of keratinizing skin (see Montagna and Parakkal, 1974). The m i c r o v i l l i were less abundant now than in the mature amnion, and appeared to be shorter and thicker. A glycocalyx was s t i l l evident but the filaments were not as long as in the mature amnion. The nuclei were flattened and appeared to be degenerating. They contained condensed masses of intensely stained chromatin (heterochromatin) (Figures 4C, 8A, 9B) but l i t t l e of the l i g h t e r stained, more dispersed chromatin (euchromatin) seen in the younger e p i t h e l i a (e.g., see Figures 5A, 6A). According to Fawcett (1981), c e l l s with abundant euchromatin in their nuclei are generally more metabolically active than those with coarse masses of heterochromat i n . The feature that most distinguished the e p i t h e l i a in t h i s group from the younger ones was a loss of almost a l l the cytoplasmic organelles. Occasionally, vacuoles were observed which may have been degenerating organelles (Figures 8A, 8B). No glycogen was present in the near-term amnion. The cytoplasm was f i l l e d with filaments which were approximately lOnm in diameter. These were randomly arranged and appeared to converge on the desmosomes (Figures 9A, 9C). 50 E p i t h e l i a l degeneration appeared to be sudden. The c e l l s of the epithelium at 62 days of gestation (0.91 of term) (Figure 10A) were large, with a l l their organelles unchanged from their mature state, but by 64 days (0.94 of term), c e l l degeneration was complete (Figure 10B). 3 . Fine Structure of the Amniotic Epithelium After Incubation With AVP A sample of amnion from a fetus at 38 days of gestation was fixed for electron microscopy immediately after a net water flow experiment, in which the f e t a l surface of the amnion was exposed to amniotic saline containing AVP at a concentration of 100 mU/ml for 15 minutes. This treatment followed a 30 minute incubation in saline without hormone. A control sample from the same fetus was treated in the same manner, except that a control solution (containing 0.25% acetic acid and 0.5% chlorobutol in 0.9% NaCl) instead of AVP was diluted into the amniotic saline for the 15 minute test period. The osmotic and hydrostatic pressure gradients during incubation were i d e n t i c a l in both preparations. Electron micrographs of the amniotic epithelium incubated with control saline were compared with those of the AVP-treated amnion, as well as with those of an unincubated membrane taken from the same fetus from which the other samples were obtained. In the control amnion, the e p i t h e l i a l i n t e r c e l l u l a r spaces were s l i g h t l y more d i l a t e d than in the unincubated 51 0 Figure 10. Structure of the Amniotic Epithelium at 62 days (0.91 of term) and 64 days (0.94 of term). These micrographs show the sudden change from normal to degenerating c e l l s , which appears to 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, in the AVP-treated amnion, there was a dramatic i n c r e a s e i n the dimensions of the spaces (see F i g u r e s 11A and 11B). Morphometric a n a l y s i s was c a r r i e d out using a Kontron MOP Videoplan image a n a l y s e r . Ten c e l l s from the c o n t r o l e p i t h e l i u m and ten c e l l s from the AVP-treated e p i t h e l i u m were compared, with the f o l l o w i n g r e s u l t s : In the c o n t r o l e p i t h e l i u m , the mean i n t e r c e l l u l a r space area was 5.26±1.81% (S.E.M.) of the area of the c e l l t o the r i g h t of the space i n the e l e c t r o n micograph. In the AVP-stimulated e p i t h e l i a l c e l l s , the i n t e r c e l l u l a r spaces c o n s t i t u t e d 19.04±2.68% (S.E.M.) of the e q u i v a l e n t c e l l s by the same parameters. T h i s representd an i n c r e a s e i n i n t e r c e l l u l a r space area of approximately 260% i n the hormone t r e a t e d amnion. A t r t e s t showed that the d i f f e r e n c e i s s i g n i f i c a n t (p<0.00O. No other d i f f e r e n c e s i n s t r u c t u r e were apparent between the unincubated, c o n t r o l incubated, and AVP-stimulated e p i t h e l i a . 4. The Volume of Amniotic F l u i d Amniotic f l u i d was c o l l e c t e d from 76 g u i n e a - p i g f e t u s e s between 30 days of g e s t a t i o n and term (68 days). The i n t a c t amniotic sac was punctured with a needle and a l l the f l u i d w i t h i n was emptied i n t o a graduated c y l i n d e r . F i g u r e 12 shows the rate of accumulation of amniotic f l u i d d u r i n g g e s t a t i o n . The mean volumes and the range of v a r i a t i o n at the d i f f e r e n t stages of g e s t a t i o n are shown i n Table I I . 53 Figure 11. Effe c t of AVP on the Fine Structure of the Amniotic Epithelium. A. Amniotic epithelium at 38 days of gestation (0.56 of term) treated with control saline for 15 minutes. The i n t e r c e l l u l a r space (ICS) i s not grossly d i l a t e d . MV: m i c r o v i l l i ; T: tight junction; D: desmosome; BM: basement membrane. X 28,000. B. Amniotic epithelium at 38 days treated with l00mU/ml AVP for 15 minutes. There is a dramatic increase in 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: tight junction; D: desmosome; BM: basement membrane. X 28,000. 54 F i g u r e 12. The Volume of Amniotic F l u i d at V a r i o u s Stages of G e s t a t i o n . Each 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 of best f i t estimated by computer ( l e a s t squares f i t t i n g to B - s p l i n e s ) . G e s t a t i o n a l a g e ; d a y s 55 Table I I : The Volume of Amniotic F l u i d During • the Course of Gestation Gestational Volume of Amniotic Range (ml) Number of Age (days) F l u i d (ml) Observat ions (Mean ± S.E.M.) 30 1.0 1 34 2.6 1 38 4.5 ± 0.3 4.0 - 5.0 3 40 4.4 - 1 44 5.9 1 45 4.7 + 0.2 4.2 - 5.0 5 47 i 6.5 ± 0.5 6.0 - 7.0 2 48 6.5 ± 0.2 5.7 - 7.4 7 50 6.4 ± 0.3 5.0 - 7.0 6 51 5.9 ± 0.5 4.7 - 6.8 4 52 6.7 ± 0.4 5.1 - 8.0 8 56 6.8 ± 0.7 5.0 - 8.0 4 58 3.7 ± 0.6 3.1 - 4.3 2 62 3.5 ± 0.3 3.0 - 4.0 3 64 1 .4 ± 0.5 0.5 - 2.0 3 65 5.5 ± 0.3 5.0 - 6.0 4 66 4.2 ± 0.5 3.5 - 5.0 3 67 7.1 + 0.8 5.3 - 9.0 4 68 6.3 ± 0.5 3.6 - 10.5 14 The mean volume of f l u i d was 1 .8±0.8 ml (S.E.M.) between 30 - 34 days and then rose gradually to a mean of 6.8±0.7 ml at 56 days. Between 47 and 56 days , the volume lay within a narrow range of 4.7 to 8.0 ml After t h i s time, there was a sharp decline. At about 58 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. However, after 64 days, shortly prior to term, the volume again increased sharply, and reached i t s maximum average value (7.1±0.8 ml) at 67 days. At term, the average volume was s t i l l high (6.3±0.5 ml). Between 65 and 68 days a wide range of variation was observed, with volumes ranging from 3.6 to 10.5 ml. 56 DISCUSSION The present study confirms the observation by V i z s o l y i and Perks (1974) that AVP i s capable of causing a net uptake of water across the amnion, in the maternal to f e t a l d i r e c t i o n , against gradients in hydrostatic and osmotic pressure. Of pa r t i c u l a r importance in thi s study was the discovery that the response of the guinea-pig amnion to AVP changes during the course of gestation. The magnitude of the response was found to increase with f e t a l age, up u n t i l about 0.85 of term (58 days of gestation). After this time, the response of the amnion declined rapidly, and was completely l o s t by term. In a brief statement, Seeds (1967) commented that vasopressin in doses as high as 1000 mU/ml (vasopressor a c t i v i t y ) had no measureable effect on net water flow through in vitro preparations of human amnion. However, the membranes used in his study were obtained after normal delivery. In l i g h t of the observations recorded here, i t is possible that the negative results of Seeds may have been due to loss of response of the human amnion at term; however, species differences might also be important. Whether the human amnion at e a r l i e r stages of gestation responds to AVP remains to be seen. The changes in the magnitude of the response of the guinea-pig amnion to AVP during the course of gestation were p a r a l l e l e d by changes in the fine structure of the amniotic epithelium. The epithelium changed from a 57 r e l a t i v e l y simple s t r u c t u r e e a r l y i n g e s t a t i o n , when the response to AVP was low, to one that appeared to be more complex and p o s s i b l e more s p e c i a l i z e d in f u n c t i o n by about 0.75 of term (50 days). Two s t r i k i n g changes that occurred with advancing g e s t a t i o n were the p r o l i f e r a t i o n of m i c r o v i l l i , and the development of a h i g h l y filamentous g l y c o c a l y x . These changes probably served to g r e a t l y i n c r e a s e the a p i c a l s u r f a c e area f o r t r a n s p o r t . A h i g h l y filamentous g l y c o c a l y x l i k e the one d e s c r i b e d here, i s a l s o found i n some other t r a n s p o r t i n g e p i t h e l i a , such as the toad u r i n a r y bladder, mammalian g a l l bladder and the i n t e s t i n e , and i s n e g a t i v e l y charged with an a c i d mucopolysaccharide component (Fawcett, 1965; 1981). It i s b e l i e v e d t h a t the g l y c o c a l y x may have a r a t h e r s p e c i f i c r o l e i n t r a n s p o r t f u n c t i o n i n e p i t h e l i a but what t h i s r o l e i s remains obscure (see Fawcett, 1981). A filamentous g l y c o c a l y x has been p r e v i o u s l y r e p o r t e d i n the guinea-pig amnion by King (1978), and i s a l s o present i n the amnion of the rhesus monkey (King, 1980) and the human (Hoyes, 1968a; King, 1982). Another change that o c c u r r e d i n the guinea-pig e p i t h e l i u m was the i n c r e a s i n g complexity of the l a t e r a l c e l l borders. The m i c r o v i l l i - l i k e f o l d s p r o j e c t i n g i n t o the i n t e r c e l l u l a r spaces would f u r t h e r i n c r e a s e the s u r f a c e area f o r t r a n s p o r t . In a d d i t i o n , these f o l d s may be the s i t e s of a c t i v e s o l u t e t r a n s p o r t . In an i n t e r e s t i n g study, Kashgarian (1980) observed that in some t r a n s p o r t i n g t i s s u e s such as the mammalian colon and r e n a l d i s t a l 58 t u b u l e s , there were marked i n c r e a s e s i n the base-lateral membrane s u r f a c e d e n s i t y and an i n c r e a s e i n the le n g t h and complexity of the i n t e r c e l l u l a r spaces, concomitant with i n c r e a s e s i n a c t i v e ion t r a n s p o r t a c t i v i t y . He suggested that the i n c r e a s e i n b a s o l a t e r a l membrane s u r f a c e d e n s i t y may be r e l a t e d to an i n c r e a s e i n the number of a v a i l a b l e t r a n s p o r t s i t e s . High l e v e l s of ATPase have been d e t e c t e d by e l e c t r o n m i c r o s c o p i c l o c a l i z a t i o n at the l a t e r a l plasma membrane i n the human amniotic e p i t h e l i u m (Wynn, 1974), and t h i s i s sug g e s t i v e of a c t i v e t r a n s p o r t o c c u r r i n g i n t h i s r e g i o n . The i n c r e a s i n g number of membrane-bound v e s i c l e s in the cytoplasm with advancing g e s t a t i o n may a l s o be r e l a t e d to an i n c r e a s e i n t r a n s p o r t f u n c t i o n . In l i g h t of these m a t u r a t i o n a l changes i n the g u i n e a - p i g e p i t h e l i u m with advancing g e s t a t i o n , i t i s not s u r p r i s i n g that the response of the amnion to AVP was found to i n c r e a s e d u r i n g the course of g e s t a t i o n . S h o r t l y before term, e p i t h e l i a l degeneration c o i n c i d e d with a t o t a l l o s s of response to AVP. J u s t as the d e c l i n e i n response to AVP was abrupt, so was the change from a h e a l t h y to a degenerated e p i t h e l i u m . C e l l degeneration i n the guinea-pig amnion appeared to occur between 62 and 64 days of g e s t a t i o n , and the l o s s of response to AVP a l s o o c c u r r e d at roughly t h i s time. The changes i n the s t r u c t u r e of the e p i t h e l i u m near term (e.g., the l o s s of cy t o p l a s m i c o r g a n e l l e s , the i n c r e a s e i n cyt o p l a s m i c f i l a m e n t s , and the t h i c k e n i n g of the plasma membrane) are s i m i l a r to changes observed i n k e r a t i n i z i n g 59 s k i n (Parakkal and Alexander, 1972; Montagna and Parak k a l , 1974). D i f f e r e n t i a t i o n of the amniotic e p i t h e l i u m during the course of pregnancy, s i m i l a r to that observed in the guin e a - p i g , has been r e p o r t e d i n the human (Bourne, 1962; Hoyes, 1968a; 1975), i n the rhesus monkey (King, 1980), and in the cow (Tiedmann, 1982). C e l l degeneration and apparent k e r a t i n i z a t i o n i n the guinea - p i g amnion near term has p r e v i o u s l y been r e p o r t e d by King (1978), who s t u d i e d the morphology of the e p i t h e l i u m i n the l a s t two weeks of g e s t a t i o n , although the time at which degeneration commenced was not mentioned. In the human amnion, there are i n c r e a s i n g numbers of degenerating c e l l s evident as term approaches, but a u n i f o r m l y degenerating e p i t h e l i u m as seen i n the present study, was not observed except i n membranes from overdue f e t u s e s (Hoyes, 1975). The same appears to be true of the rhesus monkey amnion (King, 1980). In the c a t , the e n t i r e amniotic e p i t h e l i u m sloughs o f f s e v e r a l days before term (Tiedemann, 1979). _ The a c t i o n of v a s o p r e s s i n on the amnion was not merely to i n c r e a s e the p e r m e a b i l i t y of the e p i t h e l i u m to water, as net water movement across the amnion i n response to AVP took p l a c e a g a i n s t osmotic and h y d r o s t a t i c pressure g r a d i e n t s . One way i n which t h i s c o u l d occur i s by a c o u p l i n g of water t r a n s p o r t ' to the a c t i v e t r a n s p o r t of s o l u t e s . H o l t and Perks (1977a) found that v a s o p r e s s i n i n c r e a s e d the f l u x of sodium ions across the guinea-pig 60 amnion, i n the same d i r e c t i o n , and w i t h i n the same time-frame in which i t i n c r e a s e d water f l u x ; t h i s suggested that water t r a n s p o r t c o u l d be coupled to sodium t a n s p o r t . However, as suggested by H o l t and Perks (1977a), the e f f e c t of v a s o p r e s s i n may not have been d i r e c t l y on sodium ions; the t r a n s f e r of sodium ions c o u l d have been secondary to an a c t i v e t r a n s p o r t of c h l o r i d e i o n s . C h l o r i d e ions are present i n higher c o n c e n t r a t i o n i n amniotic f l u i d than i n f e t a l and maternal plasma and a c c o r d i n g to M e l l o r and S l a t e r (1971), t h i s may be the r e s u l t of a c h l o r i d e pump l o c a t e d i n the amnion. As i n the mammalian g a l l b l a d d e r , sodium t r a n s p o r t a c r o s s the amnion i s not e l e c t r o g e n i c , and i t i s p o s s i b l e that sodium and c h l o r i d e ions t r a v e l c l o s e l y together (see Holt and Perks, 1977a). I t i s not c l e a r where the anatomical s i t e s of l o c a l osmotic g r a d i e n t s w i t h i n the amniotic e p i t h e l i u m , necessary to e f f e c t net water t r a n s p o r t a g a i n s t p r e v a i l i n g t r a n s e p i t h e l i a l g r a d i e n t s , might be l o c a t e d . I t i s p o s s i b l e that the tortuous i n t e r c e l l u l a r channels, with m i c r o v i l l i - l i k e f o l d s p r o j e c t i n g i n t o them, are i n v o l v e d . The i n t e r c e l l u l a r spaces are thought to be the s i t e s of w a t e r - s o lute c o u p l i n g i n t i s s u e s such as the mammalian g a l l b l a d d e r , the r e n a l proximal tubule, and the i n t e s t i n e (Diamond and B o s s e r t , 1967; Oschman, 1977; Diamond, 1971; 1980). In the r a b b i t g a l l b l a d d e r , i n t e r c e l l u l a r spaces are g r o s s l y d i l a t e d when maximal f l u i d t r a n s p o r t occurs, but c o l l a p s e under c o n d i t i o n s i n which t r a n s p o r t i s i n h i b i t e d 61 (Tormey and Diamond, 1967). Spring and Hope ( 1978, ,1979) have observed the same phenomenon in the l i v i n g epithelium of Necturus gallbladder. Treatment of the amnion at 38 days of gestation (0.56 of term) with AVP resulted in a marked d i l a t i o n of i n t e r c e l l u l a r spaces in the epithelium when compared to a control specimen incubated without hormone. A similar expansion of i n t e r c e l l u l a r spaces with AVP treatment was observed in guinea-pig amnion at 0.76 of term, but not in one at term (Goh and Perks, unpublished observations). The absence of s t r u c t u r a l changes in term amnion with AVP treatment i s consistent with the loss of response to the hormone near term. These results suggest that the i n t e r c e l l u l a r spaces may play a major role in AVP-mediated flow of f l u i d across the amniotic epithelium. King (1982) and Bartels and Wang (1983), by freeze fracture analysis, have i d e n t i f i e d gap junctions between e p i t h e l i a l c e l l s in the human amnion. In other tissues, i t has been found that c y c l i c AMP, released in response to hormonal stimulation, can pass rapidly to neighbouring c e l l s via gap junctions (see Fawcett, 1981). In the amnion, which has no nerves or blood supply, such a mechanism may play an important role in co-ordinating and amplifying the response of groups of e p i t h e l i a l c e l l s to AVP stimulation. The changes in the structure and response to vasopressin of the amniotic membrane may be related to the 62 long-term changes i n the volume of amniotic f l u i d that occur d u r i n g g e s t a t i o n . In most mammals, the accumulation of amniotic f l u i d begins e a r l y , reaches a peak, and then as term approaches d i m i n i s h e s again (Adolph, 1967). An adequate volume of f l u i d i s v i t a l to f e t a l l i f e i n e a r l y and m i d - g e s t a t i o n , though the f e t u s approaching term can s u r v i v e without any at a l l (Adolph, 1967). The f l u i d p r o v i d e s a moist, spacious environment i n which to develop and move i n r e l a t i v e w e i g h t l e s s n e s s (Seeds, 1965; Reynolds, 1972). A c t i v e f e t a l movement i n utero i s necessary f o r normal growth and development of f e t a l t i s s u e s , and i s p o s s i b l e only when the uterus i s d i s t e n d e d with an adequate volume of f l u i d (Moessinger, 1983). The amniotic f l u i d a l s o p r o t e c t s the f e t u s from a b r a s i o n by the u t e r i n e w a l l and from mechanical shock, and may a l s o p r o v i d e thermal i n s u l a t i o n (Seeds, 1965; Reynolds, 1972). According to K e r p e l - F r o n i u s (1970), the p r o g r e s s i v e d i s t e n s i o n of the uterus by the amniotic f l u i d i s necessary f o r normal f e t a l , p l a c e n t a l , and u t e r i n e development. The amniotic f l u i d may a l s o f u n c t i o n as a b u f f e r a g a i n s t f e t a l f l u i d imbalance by s e r v i n g as a r e s e r v o i r (Seeds, 1965). Vasopressin-mediated c o n t r o l of the volume by the amnion duri n g the e a r l i e r stages of g e s t a t i o n i s probably necessary to ensure an adequate volume f o r normal f e t a l growth, whereas near term, the now l e s s v u l n e r a b l e f e t u s does not s u f f e r d e l e t e r i o u s e f f e c t s r e s u l t i n g from l e s s s t r i n g e n t c o n t r o l by the degenerating amniotic e p i t h e l i u m . 63 In the g u i n e a - p i g , as i n other s p e c i e s , the volume of amniotic f l u i d was found to i n c r e a s e , reach a peak, and then d e c l i n e . The peak c o i n c i d e d roughly with the time at which the amnion was most s e n s i t i v e to AVP, and with the time i t s s t r u c t u r e appeared to be compatible with an a c t i v e r o l e i n f l u i d t r a n s p o r t . A f t e r approximately 56 - 58 days of g e s t a t i o n , both amniotic f l u i d volume and the response of the amnion to AVP d e c l i n e d . These r e s u l t s are c o n s i s t e n t with a p h y s i o l o g i c a l r o l e of AVP i n s u p p l y i n g f l u i d to the amniotic sac i n the f i r s t 80% of g e s t a t i o n . I n t e r e s t i n g l y , u n l i k e the s i t u a t i o n i n most other s p e c i e s , the volume i n c r e a s e d again j u s t around term. Such an i n c r e a s e has a l s o been observed i n the domestic c a t ( W i s l o c k i , 1935). At present, the reason f o r t h i s remains obscure. Perhaps at t h i s time there i s a r e l a t i v e i n c r e a s e i n the r a t e of u r i n e flow and s e c r e t i o n of lung f l u i d from the r a p i d l y e n l a r g i n g f e t u s , which r e s u l t s i n an i n c r e a s e in amniotic f l u i d volume. I t i s p o s s i b l e that the great v a r i a b i l i t y i n amniotic f l u i d volume j u s t p r i o r to and at term r e f l e c t s l e s s c o n t r o l of the volume by the amnion at t h i s stage; between 62 and 64 days of g e s t a t i o n the amniotic e p i t h e l i u m degenerates, and the response to AVP i s l o s t . F i g u r e 13 g i v e s a summary of the r e s u l t s d i s c u s s e d i n t h i s s e c t i o n . 64 Figure 13. Gestational Changes in the Structure of the Amniotic Epithelium, the Response to AVP, and the Volume of Amniotic F l u i d in the Guinea-Pig A. Diagrammatic representation of the amniotic epithelium at 28, 50, and 64 days of gestation. Striped regions represent nuclei. The upper surface faces the amniotic f l u i d . B. The changes in 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 gestation (0.44 to 1.00 of term). G e s t a t i o n a l a g e ; d a y s 65 SECTION II THE EFFECTS OF ARGININE VASOTOCIN AND ARGININE VASOPRESSIN ON WATER TRANSPORT ACROSS THE SKIN OF THE FETAL GUINEA-PIG INTRODUCTION The f e t a l epidermis i s continuous with the amniotic e p i t h e l i u m , and both s t r u c t u r e s are d e r i v e d from the same germ l a y e r (Saunders and Rhodes, 1973). In a d d i t i o n , s t u d i e s i n the human have shown that the c e l l s of the e x t r a f e t a l l a y e r , the periderm, resemble those of the amniotic e p i t h e l i u m (Hoyes, 1968b). L i k e the amnion, the s k i n i s responsive to v a s o p r e s s i n . In p r e l i m i n a r y experiments on the g u i n e a - p i g , H o l t and Perks (1977a) found that v a s o p r e s s i n caused a marked i n c r e a s e i n the mucosal-serosal f l u x of t r i t i a t e d water through the f e t a l s k i n . France (1976) observed that v a s o p r e s s i n i n c r e a s e d b i - d i r e c t i o n a l f l u x e s of i s o t o p i c water a c r o s s the s k i n of the f e t a l lamb. The e f f e c t s of v a s o p r e s s i n on water movement acr o s s the f e t a l s k i n were re m i n i s c e n t of i t s e f f e c t s on water t r a n s p o r t a c r o s s the s k i n of anuran amphibians. However, in amphibian s k i n , v a s o p r e s s i n i s more e f f e c t i v e at i n c r e a s i n g bulk r a t h e r than d i f f u s i o n a l movement of water (Maetz, 1968). In a d d i t i o n , the s k i n of amphibians i s more respons i v e to v a s o t o c i n than to v a s o p r e s s i n ( H e l l e r and Bentley, 1965). T h e r e f o r e , i t seemed important to extend s t u d i e s of the f e t a l s k i n to changes in bulk flow, and to 66 comparisons of AVT with AVP. T h i s i s c a r r i e d out i n the s t u d i e s presented below, together with a p r e l i m i n a r y i n v e s t i g a t i o n of the f i n e s t r u c t u r e of g u i n e a - p i g f e t a l s k i n . 67 RESULTS 1. The Isolated Fetal Skin Skin from 35 mid-term f e t a l guinea-pigs (0.49 to 0.70 of term) was set up in vitro, as d e s c r i b e d i n the methods. In the absence of hormones, and with no g r a d i e n t s of osmotic or h y d r o s t a t i c p r e s s u r e , the m a j o r i t y of p r e p a r a t i o n s (n=26) showed no net movement of water i n e i t h e r d i r e c t i o n . However, 7 a d d i t i o n a l p r e p a r a t i o n s showed a spontaneous uptake of water i n the mucosal to s e r o s a l d i r e c t i o n ( i . e . , towards the f e t u s ) , and i n 2 p r e p a r a t i o n s there was a slow passage of water from the s e r o s a l to the mucosal s u r f a c e . 2 . The Effects of AVT and AVP on Net Water Transport Across Fetal Skin T e s t s with AVT or AVP a p p l i e d to the s e r o s a l s u r f a c e of the s k i n at doses of 5 to 100 mU/ml (vasopressor a c t i v i t y ) showed that these p e p t i d e s are capable of c a u s i n g an i n c r e a s e i n the net t r a n s f e r of water from the mucosal to the s e r o s a l s i d e of the s k i n (towards the f e t u s ) . F i g u r e 14 i l l u s t r a t e s the averages (±S.E.M.) f o r a l l the responses obtained to the v a r i o u s doses of AVT and AVP. Tables III and IV show a l l the v a l u e s obtained f o r b a s e - l i n e ( r e s t i n g ) flow r a t e , hormone-influenced flow 68 F i g u r e 14. 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). O r d i n a t e s : the r a t e s of water movement (mg/cm2 per minute). The do t t e d l i n e r e p r e s e n t s zero change; val u e s above t h i s l i n e represent water uptake towards the s e r o s a l ( f e t a l ) s i d e of the s k i n , whereas values below t h i s l i n e represent water flow towards the mucosal s u r f a c e of the s k i n (towards the amniotic f l u i d ) . A b s c i s s a e : time, in minutes. Arrows i n d i c a t e time of a d d i t i o n of hormone. A l l val u e s are expressed as the mean ± the standard e r r o r of the mean (S.E.M.). 2 R a t e o f W a t e r M o v e m e n t • m g / c m min 3 o in O b o i n • H I \ / 3 c 5 O In O b o / • o »3 c 5 O o b \ \ o Ul 3 t» 3 i \ / 3 c o en JT" (O -o O b bi / I \ H / — I\J ui b o Ul o b o — ui b / \ \ < > < o o Ul o 1 o — ui b 3 c < O Ul o b o — ui b u 3 c 5 O Ul o b o Ul 00 > 69 r a t e , and the in c r e a s e i n flow rate ( i . e . , the response), f o r AVT and AVP, r e s p e c t i v e l y . The response was c a l c u l a t e d as the average i n c r e a s e i n the r a t e of mucosal-serosal water flow i n the 15 minute p e r i o d a f t e r the a d d i t i o n of hormone, and the r e s t i n g flow r a t e was d e f i n e d as the average r a t e of flow i n the 12 minute p e r i o d immediately before the a d d i t i o n of hormone. Out of a t o t a l of 22 t e s t s with AVT, f i v e showed no response to the hormone, but these were to doses at or below 25 mU/ml. In three c o n t r o l p r e p a r a t i o n s , i n which AVT was r e p l a c e d with a c o n t r o l s a l i n e , there were no changes i n r e s t i n g flow r a t e d u r i n g the t e s t p e r i o d . Although a l a r g e v a r i a b i l i t y i n s e n s i t i v i t y to a given dose of hormone e x i s t e d between p r e p a r a t i o n s , , there was a general t r e n d towards an i n c r e a s e i n the magnitude of the response with higher doses (Table I I I ) . Response to AVT occurred w i t h i n 3 to 9 minutes a f t e r hormone a d d i t i o n . On average, the peak r a t e of flow was achieved sooner with higher doses (Figure 14). Most p r e p a r a t i o n s returned to the o r i g i n a l r e s t i n g flow r a t e a f t e r the removal of AVT by r e t u r n i n g the s k i n to f r e s h s a l i n e s , and recovery was u s u a l l y complete w i t h i n 10 minutes a f t e r hormone withdrawal. I t was observed that the response d i d not u s u a l l y l a s t f o r more than 30 minutes i n experiments which were allowed to continue f o r longer p e r i o d s . The s k i n appeared to be s e n s i t i v e to hormone f o r approximately two hours a f t e r the d i s s e c t i o n . In most p r e p a r a t i o n s , two Table III: The Response of the Fetal Skin to AVT Dose of AVT (ntU/ml Rate of RestIng Rate of AVT- Response: The Average Response p-values 4 of vasopressor Flow (mg/cm per Influenced Flow 2 Increase in Rate (± S.E.M.) (mg/cm2 act 1vity) minute) (mg/cm2 per of Mucosal-Serosal per minute) minute) Flow with AVT 3 (mg/cm2 per minute) Controls (n=3) 0 0 0 0 5 0 +0. .48 0 .48 0 0 0 0.12±0.12 p>0.05 +0 .22 +0. , 1 1 0 +0, .59 1 +0. .47 0 10 0 +0. .26 0. .26 0 +0. 32 0, . 32 0.21+0.07 p>0.05 0 0 0 +0. . 28 +0. 56 0. .28 25 0 +0. 34 0. .34 +0. 07 +0. 17 0, . 10 0 +0. 72 0. ,72 +0. 21 0 0 0.35±0.09 p<0.01 0 +0. 42 0. ,42 0 +0. 33 0. 33 0 +0. 56 0. .56 50 -0. 14 +0. 33 0. 47 0 +0. 50 0. 50 0.46±0.02 p<0.01 0 +0. 42 0. 42 100 +0. 20 + 1 . 17 0. 97 0 +0. 95 0. 95 0.8610.06 p<0.001 0 +0. 75 0. 75 0 +0. 77 0. 77 Note: Positive values (+) Indicate flows 1n the mucosal-serosal d i r e c t i o n (towards the fetus). Negative values (-) Indicate flows 1n the serosa 1-mucosa 1 direc t i o n (towards the amniotic f l u i d ) . For an explanation of terms (1 - 4), see the notes at the bottom of Table IV. ^ Table IV: The Response of the Fetal Skin to AVP Dose of AVP (mU/ml Rate of Resting Flow 1 (mg/cm2 per Rate of AVP- Response: The Average Response p-values 4 of vasopressor Influenced Flow 2 Increase in Rate ( + S.E.M.) (mg/cm2 act 1v1ty) minute) (mg/cm2 per mlnute) of Mucosal-Serosal Flow with AVP3 (mg/cm2 per minute) per minute) 10 0 0 0 0 0 0 o 0 0 0 25 -0.08 0 0.08 0 +0.21 0.21 +0.11 +0.50 0.39 0.19±0.07 p<0.05 0 +0.45 0.45 0 0 0 +0.24 +0.28 0.04 0 +0.13 0.13 50 0 +0.56 0.56 +0.09 +0.35 0.26 0.32+0.08 p<0.02 0 +0.24 0.24 +0.24 +0.47 0.23 100 0 +0.47 0.47 0 +0.55 0.55 0.51±0.06 p<0.001 0 +0.36 0.36 0 +0.66 0.66 Note: Positive values (+) Indicate flows 1n the mucosal-serosal d i r e c t i o n (towards the fetus). Negative values (-) Indicate flows in the serosal-mucosal d i r e c t i o n (towards the amniotic f l u i d ) . 1. The rate of resting flow 1s the average rate of flow during the 12 minute period Immediately prio r to treatment with hormone. 2. The rate of hormone-1nf1uenced flow 1s the average rate of flow In the 15 minutes Immediately a f t e r hormone addi t ion. 3. The response is the increase in the rate of mucosal-serosal water flow; i t is the difference between 1 and 2. No change, or a decrease 1n mucosal-serosal flow 1s taken as zero response. 4. The p values refer to Student's t-tests; they are based on the Increase 1n flow rate In the mucosal-serosal d i r e c t i o n . 72 c l e a r responses c o u l d be obtained w i t h i n the two hour p e r i o d . However, i n q u a n t i f y i n g the data, estimates were l i m i t e d to f i r s t responses. The mean r a t e s of flow a f t e r the a d d i t i o n of AVT were s i g n i f i c a n t l y higher than those before the hormone was added (Student's t - t e s t ; see Table I I I ) , with a l l doses above 10 mU/ml. A t o t a l of 18 t e s t s were performed with AVP. Of these, four d i d not show any i n c r e a s e i n mucosal-serosal water t r a n s p o r t ; these were with doses below 25 mU/ml. There was a s i g n i f i c a n t i n c r e a s e i n the average r a t e of water flow a f t e r treatment with AVP at doses above 10 mU/ml (Student's t - t e s t , see Table I V ) . As with AVT, there was an i n c r e a s e i n the magnitude of the response with i n c r e a s i n g dose of AVP. The p a t t e r n of response was the same as th a t d e s c r i b e d f o r AVT treatment;, the s k i n responded w i t h i n 3 to 9 minutes a f t e r hormone a d d i t i o n , and as with AVT, recovered w i t h i n 10 minutes a f t e r hormone withdrawal. However, as seen i n F i g u r e 14, the magnitude of the response to AVP was i n gene r a l lower than that f o r AVT. D e s p i t e c o n s i d e r a b l e v a r i a b i l i t y between p r e p a r a t i o n s in t h e i r s e n s i t i v i t y to a given dose of e i t h e r AVT or AVP, a l i n e a r l o g dose-response r e l a t i o n s h i p e x i s t e d f o r the two pep t i d e s ( F i g u r e 15). The t h r e s h o l d dose f o r AVT was 3.9 mU/ml and f o r AVP i t was 10.4 mU/ml (of vasopressor a c t i v i t y ) . There appeared to be no r e l a t i o n s h i p between the magnitude of the response and the age of the f e t u s f o r a given dose of e i t h e r AVT or AVP. 73 Figure 15. Log Dose-Response Curves for the E f f e c t s of AVT and AVP on Net Water Movement Through the Fetal Skin (at 0.49 - 0.70 of term). A l l values are expressed as the mean ± standard error of the mean (see Tables III and IV). Ordinate: the increase in the rate of mucosal-serosal water flow during treatment with either AVT or AVP (in mg/cm2 per minute). Abscissa: Dose of AVT or AVP (in mU/ml, vasopressor a c t i v i t y ) , plotted on a log scale. Lines 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). 73A 74 3. Fine Structure of the Fetal Epidermis Skin taken from the back region of f e t a l guinea-pigs at 35 days of g e s t a t i o n (0.51 of term) and 52 days of g e s t a t i o n (0.76 of term) was examined under the e l e c t r o n microscope. At 35 days, the epidermis c o n s i s t e d of a b a s a l germinative l a y e r , one or two i n t e r m e d i a t e - l a y e r s , and the outer periderm ( F i g u r e s 16, 17). There were no s i g n s of k e r a t i n i z a t i o n i n the s k i n at t h i s stage. Large q u a n t i t i e s of i n t r a c e l l u l a r glycogen were observed in a l l l a y e r s of the epidermis. I n t e r c e l l u l a r spaces throughout the epidermis were d i l a t e d , and had m i c r o v i l l i - l i k e f o l d s p r o j e c t i n g i n t o them. The c e l l s of the periderm were g e n e r a l l y f l a t t e n e d , but i n some areas the c e n t r a l p a r t of the c e l l was e l e v a t e d above the r e s t of the s u r f a c e . The a p i c a l s u r f a c e of the periderm had a number of m i c r o v i l l i p r o j e c t i n g i n t o the amniotic c a v i t y ( F i g u r e s 18A, 18B). A t h i n filamentous coat was observed on the s u r f a c e of the m i c r o v i l l i . Desmosomes were prominent i n the periderm, and s t r u c t u r e s resembling t i g h t j u n c t i o n s were o c c a s i o n a l l y observed ( F i g u r e 18A). The cytoplasm of the periderm c e l l s c o n t a i n e d mitochondria and membrane-bound v e s i c l e s . G o l g i complexes and rough endoplasmic r e t i c u l u m were o c c a s i o n a l l y seen. These o r g a n e l l e s appeared to be more abundant i n the b a s a l and intermediate l a y e r s . N u c l e i were observed i n a l l 75 F i g u r e 16. F i n e S t r u c t u r e of the F e t a l Epidermis at 35 Days. The epidermis 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 rmediate l a y e r s (I) and a s u p e r f i c i a l l a y e r , the periderm (P). On the a p i c a l (mucosal) s u r f a c e of the periderm are m i c r o v i l l i (mv) which p r o j e c t i n t o the amniotic c a v i t y . There are l a r g e d e p o s i t s of glycogen ( g l y ) i n a l l l a y e r s of the epidermis. I n t e r c e l l u l a r spaces (ICS) are d i l a t e d and have i n f o l d i n g s of the plasma membranes. A l l l a y e r s , i n c l u d i n g the periderm, have l a r g e n u c l e i ( n ) . X 18,000. 75 A 76 F i g u r e 17. Fine S t r u c t u r e of the F e t a l Epidermis at 35 Days. T h i s micrograph shows the 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 e a r l y f e t a l s k i n . B: ba s a l l a y e r ; I: intermediate l a y e r ; P: periderm; ICS: i n t e r c e l l u l a r spaces; mv: m i c r o v i l l i . X 18,000. 77 Figure 18. The Fetal Periderm at 35 Days. The cytoplasm contains membrane-bound vesicles (Ve), nuclei (N) , mitochondria (M), and glycogen ( g l y ) . Glycogen is also observed in the layer beneath the periderm (micrograph B). The m i c r o v i l l i (mv) have a thin filamentous coat. T: tight junction; D: desmosomes; ICS: i n t e r c e l l u l a r spaces; F: filaments. A. X 33,000 B. X 37,000 77 A 78 the l a y e r s , i n c l u d i n g the periderm (Figure 16). Bundles of f i l a m e n t s were observed i n the cytoplasm of the periderm c e l l s , but were rare in the other l a y e r s . These f i l a m e n t s were seen mainly i n the v i c i n i t y of the desmosomes ( F i g u r e s 18A, 18B). By 52 days, the s k i n showed d e f i n i t e s i g n s of k e r a t i n i z a t i o n ( F i g u r e s 19A, 19B). The i n t e r c e l l u l a r spaces were l e s s d i l a t e d , and the amount of glycogen had d e c l i n e d . There was evidence of k e r a t i n formation i n the uppermost of the in t e r m e d i a t e l a y e r s . T h i s l a y e r c o n t a i n e d e l e c t r o n - d e n s e k e r a t o h y a l i n granules i n c l o s e p r o x i m i t y to bundles of c y t o p l a s m i c f i l a m e n t s . These g r a n u l e s , together with the a s s o c i a t e d f i b r i l s , are thought to be the p r e c u r s o r s of k e r a t i n (Montagna and P a r a k k a l , 1974). A d e f i n i t i v e stratum corneum, composed of one or two l a y e r s of f l a t t e n e d c e l l s , was e v i d e n t beneath the periderm. The c e l l s of the stratum corneum d i s p l a y e d the t y p i c a l " k e r a t i n p a t t e r n " of a d u l t mammalian s k i n , as d e f i n e d by Brody (1959); they were devoid of a l l o r g a n e l l e s and were f i l l e d with c l o s e l y packed f i b r i l s embedded i n an amorphous matrix (Figure 19B). The periderm was f l a t t e n e d and the a p i c a l m i c r o v i l l i had now disappeared. The c e l l s of the periderm were s i m i l a r to those of the stratum corneum i n that they had t h i c k e n e d plasma membranes, and had l o s t a l l . t h e i r o r g a n e l l e s ( F i g u r e 19B). The cytoplasm was f i l l e d with f i l a m e n t s (about 1Onm i n d i a m e t e r ) . However, the periderm d i d not show the k e r a t i n p a t t e r n observed i n the stratum 79 Figure 19. Fine Structure of the Fetal Epidermis at 52 Days. A. Survey micrograph of the kerati n i z i n g epidermis. P: periderm; SC: stratum corneum; I: intermediate layer; B: basal layer; De: dermis; K: keratohyalin granule.- X 9,000. B. Higher magnification epidermal surface. P: corneum; I: intermediate K: keratohyalin granule. micrograph of the periderm; SC: stratum layer; f: filaments; X 42,000. 80 corneum; the f i l a m e n t s were l o o s e l y packed, and were not embedded i n an amorphous ground substance. The periderm c e l l s appeared to be s e p a r a t i n g from the u n d e r l y i n g stratum corneum, and i n some regions had disappeared a l t o g e t h e r . There were no t i g h t j u n c t i o n s i n the periderm, and the desmosomes appeared to have degenerated. K e r a t o h y a l i n granules were not observed'in e i t h e r the periderm or the stratum corneum. 81 DISCUSSION The r e s u l t s presented here have shown that both AVT and AVP are capable of c a u s i n g a net movement of water a c r o s s the s k i n of the f e t a l g u i n e a - p i g i n the mucosal to s e r o s a l d i r e c t i o n ( i . e . , towards the f e t a l c i r c u l a t i o n from the p o t e n t i a l amniotic c a v i t y ) . T h i s o c c u r r e d i n the absence of any apparent g r a d i e n t s i n osmotic or h y d r o s t a t i c p r e s s u r e . A l i n e a r r e l a t i o n s h i p e x i s t e d between the l o g dose, and the i n c r e a s e i n the r a t e of mucosal-serosal water flow, f o r both p e p t i d e s . T h i s suggested that the responses were u n l i k e l y to be a r t e f a c t s . L i n e a r l o g d o s e - e f f e c t r e l a t i o n s h i p s such as those observed here a l s o e x i s t f o r the a c t i o n s of neurohypophysial p e p t i d e s on water a b s o r p t i o n a c r o s s the s k i n and bladder of anuran amphibians ( H e l l e r and B e n t l e y , 1965). In a d d i t i o n , l i n e a r l o g dose-response r e l a t i o n s h i p s have been observed f o r the e f f e c t s of v a s o p r e s s i n on both bulk and d i f f u s i o n a l water flow a c r o s s the g u i n e a - p i g amnion ( V i z s o l y i and Perks, 1974; H o l t and Perks, 1977a). The p a t t e r n of the response of the s k i n to AVT and AVP was remarkably s i m i l a r to that observed by V i z s o l y i and Perks (1974) f o r the amnion. However, u n l i k e the amnion, the s k i n c o n s i s t e n t l y showed higher responses to AVT than to AVP. The t h r e s h o l d s of the amnion to AVP and AVT were 82 1.0 and 6.4 mU/ml (of vasopressor a c t i v i t y ) , respectively, while the corresponding values for the skin were 10.4 and 3.9 mU/ml. The f e t a l skin i s similar to the skin of anuran amphibians, in that i t responds more readily to vasotocin than to vasopressin (Heller and Bentley, 1965). It appears that the receptors in the fetus are complex enough to disti n g u i s h between two peptides which d i f f e r only by one amino acid; AVP has phenylalanine at position 3 of the octapeptide, whereas AVT has isoleucine instead (see Sawyer, 1970). At present, the mechanisms of action of the neurohypophysial peptides on net water transport across the f e t a l skin are not known, but the fact that water flow occurs in the absence of any apparent gradients, with i d e n t i c a l solutions bathing both surfaces of the skin, suggests that i t may be coupled to active solute transport. France (1976) has observed that vasopressin and vasotocin are capable of stimulating an increase in sodium reabsorption across the skin of f e t a l sheep from an isotonic or d i l u t e external medium, and thus i t i s possible that water transport i s coupled to either sodium transport or a cotransport system. Unlike the situ a t i o n in amphibian skin, no s i g n i f i c a n t potential difference exists across the skin of the fetus, even in the presence of u p h i l l sodium transport (Mellor, 1970; Lind et al. , 1972; France, 1976). In t h i s regard, the skin resembles other leaky membranes, such as the gall-bladder epithelium, in which active Na/Cl transport occurs without an apparent potential difference 83 (Diamond, 1971). S t u d i e s i n sheep, the f u r s e a l , the g u i n e a - p i g , and the human have shown that both a r g i n i n e v a s o p r e s s i n and a r g i n i n e v a s o t o c i n e x i s t i n the f e t a l neurohypophysis ( V i z s o l y i and Perks, 1969; Perks and V i z s o l y i , 1973; Skowsky and F i s h e r , 1977). AVP i s present throughout g e s t a t i o n , and i t s c o n c e n t r a t i o n i n f e t a l p i t u i t a r i e s i n c r e a s e s s t e a d i l y towards b i r t h . However, v a s o t o c i n e x i s t s i n f e t a l p i t u i t a r i e s f o r only a p o r t i o n of g e s t a t i o n . In the f e t a l lamb and s e a l , AVT i s p l e n t i f u l around m i d - g e s t a t i o n , but i t s l e v e l s d e c l i n e as term approaches, and i t i s completely l o s t i n the a d u l t (Perks and V i z s o l y i , 1973; Perks, 1977). I t i s p o s s i b l e that t h i s i s a l s o t r u e of the guinea-pig (Kontor and Perks; see Perks and V i z s o l y i , 1973). Skowsky and F i s h e r (1977) have observed a s i m i l a r t rend i n the human f e t u s . P r i o r to i t s d i s c o v e r y i n the mammalian f e t u s , AVT was thought to be r e s t r i c t e d to sub-mammalian v e r t e b r a t e s . Whether AVT has a s p e c i f i c r o l e d u r i n g f e t a l l i f e , or whether i t i s merely an i n a c t i v e r e l i c of peptide e v o l u t i o n i s not yet known, but the present study has p r o v i d e d a p o s s i b l e c l u e . The higher e f f e c t i v e n e s s of AVT i n i n f l u e n c i n g water t r a n s p o r t a c r o s s the f e t a l s k i n , and the f a c t that t h i s peptide i s found i n h i g h e s t q u a n t i t i e s i n f e t a l p i t u i t a r i e s p r i o r to mid-term, before the s k i n k e r a t i n i z e s , g i v e s strong c i r c u m s t a n t i a l evidence that i n the f e t u s , AVT f i n d s i t s true p h y s i o l o g i c a l r o l e on the s k i n . 84 During the l a s t t h i r d of g e s t a t i o n , the f e t a l lamb s e c r e t e s AVP i n response t o haemorrhage, or hyp e r t o n i c s a l i n e and dextran i n f u s i o n s (Alexander et al . , 1974; 1976; Weitzman et al. , 1978). Skowsky et al. (1973) have observed that h y p e r t o n i c s a l i n e i n f u s i o n i n t o the f e t a l rhesus monkey r e s u l t s i n a marked i n c r e a s e i n serum AVP l e v e l s . V i z s o l y i and Perks (1974) have suggested that the s i g n i f i c a n c e of AVP s e c r e t i o n i n response to the osmotic s t i m u l i may l i e i n the need to t r a n s p o r t s u f f i c i e n t AVP to c o n t r o l water movement a c r o s s the amnion. In l i g h t of these o b s e r v a t i o n s , i t seems reasonable to p o s t u l a t e that i n early g e s t a t i o n , p r i o r to s k i n k e r a t i n i z a t i o n , AVT i s se c r e t e d from the f e t a l p i t u i t a r y i n response to s i m i l a r s t r e s s ; i t may then a ct on the ski n to cause water re.absorption from the amniotic f l u i d . However, at present there i s no evidence of AVT s e c r e t i o n i n the f e t u s . The doses of AVT (and AVP) which a f f e c t the s k i n appear to be hig h and must be co n s i d e r e d p h a r m a c o l o g i c a l . However, s e v e r a l reasons e x i s t to suggest that the responses were r e f l e c t i o n s of true p h y s i o l o g i c a l e f f e c t s : Removal of the s k i n from i t s n a t u r a l environment and i t s maintenance in vitro may w e l l have caused a d e c l i n e i n s e n s i t i v i t y to hormones. V a s o p r e s s i n - i n d u c e d water flow a c r o s s t i s s u e s l i k e the toad bladder i s known t o be h i g h l y s e n s i t i v e to the c o n c e n t r a t i o n of sodium and c a l c i u m ions (Roy and A u s i e l l o , 1981; Davis et al. , 1981), and i n the present study, the s a l i n e s may have been l e s s than i d e a l . 85 In a d d i t i o n , a l l hormone molecules may not have reached t h e i r t a r g e t s i t e s due to the t h i c k n e s s of the s k i n ; i n an in vivo s i t u a t i o n , the blood p e r f u s i n g the s k i n would e f f e c t i v e l y c a r r y the hormone to i t s s i t e of a c t i o n . F i n a l l y , i n a true p h y s i o l o g i c a l s i t u a t i o n , the neurohypophysial peptides may act i n synergism with other hormones. For example, i n the a d u l t C a l i f o r n i a Newt (Taricha torosa), a d r e n o c o r t i c o t r o p h i n (ACTH) and t h y r o i d s t i m u l a t i n g hormone (TSH) are thought to enhance the e f f e c t s of AVT on water t r a n s f e r a c r o s s the s k i n (Brown and Brown, 1982). Such a s i t u a t i o n may a l s o e x i s t i n the mammalian f e t u s . I t would be i n t e r e s t i n g to study the e f f e c t s of p r o l a c t i n on net f l u i d t r a n s f e r a c r o s s f e t a l s k i n . P r o l a c t i n has been found to a f f e c t ion and water movement acr o s s v a r i o u s osmoregulatory s u r f a c e s i n a l l the v e r t e b r a t e c l a s s e s ; i n most cases i t reduces membrane p e r m e a b i l i t y (Hirano, 1980). For example, p r o l a c t i n reduces the p e r m e a b i l i t y of the s k i n to water i n some urodele amphibians, and appears to oppose the a c t i o n s of v a s o t o c i n (Brown and Brown, 1982; Brown et al . , 1983). In t e l e o s t f i s h , i t reduces the p e r m e a b i l i t y of the g i l l s , i n t e s t i n e , and u r i n a r y bladder (see Hirano, 1980). As d i s c u s s e d p r e v i o u s l y , p r o l a c t i n appears to reduce the p e r m e a b i l i t y of the amniotic membrane to water (Holt and Perks, 1975; L e o n t i c et al. , 1979), and i t i s p o s s i b l e that i t has s i m i l a r e f f e c t s on the f e t a l s k i n . 86 The s t r u c t u r e of f e t a l g u i n e a - p i g s k i n before k e r a t i n i z a t i o n i s compatible with an a c t i v e r o l e i n f l u i d t r a n s p o r t . In the a d u l t mammal, the uppermost l a y e r of the epidermis (the stratum corneum) i s composed of dead, dehydrated c e l l s , and serves a p u r e l y p r o t e c t i v e f u n c t i o n , being v i r t u a l l y impermeable to water and most other substances (Parakkal and Alexander, 1972; Montagna and Pa r a k k a l , 1974). In the present study, i t was observed that i n the young f e t u s , the uppermost epidermal l a y e r , the periderm, had c e l l s which appeared to be p h y s i o l o g i c a l l y a c t i v e . They had m i c r o v i l l i on t h e i r a p i c a l (amniotic) s u r f a c e , l a r g e n u c l e i , membrane-bound v e s i c l e s , d i l a t e d i n t e r c e l l u l a r spaces, and m i t o c h o n d r i a . Adjacent c e l l s were a t t a c h e d by t i g h t j u n c t i o n s and desmosomes. The periderm resembled the amniotic e p i t h e l i u m ( s e c t i o n I ) , and i t probably f u n c t i o n s i n a s i m i l a r f a s h i o n . I t might be the s i t e of a c t i o n of AVT. Large q u a n t i t i e s of glycogen were present i n a l l the epidermal l a y e r s . I t has been suggested that the f e t a l s k i n serves as a storage organ f o r glycogen, before the l i v e r assumes t h i s f u n c t i o n (Rothman, 1954). The glycogen may a l s o represent a source of energy f o r epidermal c e l l s . The wide i n t e r c e l l u l a r spaces throughout the epidermis c o u l d f a c i l i t a t e exchange of f l u i d between the f e t a l and amniotic compartments. The periderm was found to undergo p a r t i a l k e r a t i n i z a t i o n before being sloughed o f f . I t d i s p l a y e d s i m i l a r changes as those seen i n the amniotic e p i t h e l i u m before term. The disappearance 87 of the periderm was preceded by the formation of a stratum corneum resembling that of the a d u l t (Brody, 1959). The periderm has p r e v i o u s l y been s t u d i e d at the u l t r a s t r u c t u r a l l e v e l i n the human, rhesus monkey, mouse, and r a t (Breathnach and W y l l i e , 1965; Brody and La r s s o n , 1965; Hashimoto, 1966; B o n n e v i l l e , 1968; Hoyes, 1968b; Parakkal. and Alexander, 1972). The guinea - p i g periderm was found to resemble t h a t of the other mammals. 88 GENERAL DISCUSSION The experiments described in Section I have shown that the a b i l i t y of the guinea-pig amnion to respond to AVP i s not the same throughout gestation; there appears to be a progressive increase in the magnitude of the response to AVP with advancing gestation, up u n t i l about 0.85 of term. This i s followed by an abrupt decline, such that by term, the response i s reduced to zero. Future studies of the e f f e c t s of hormones on the amnion must take t h i s observation into consideration; a lack of response in the amnion very early in gestation, close to term, or after normal delivery in other species may not necessarily mean that the amnion does not respond to the hormone at a l l . Membranes from fetuses at a l l stages of gestation must be tested to ascertain the true s i t u a t i o n . Although the guinea-pig epithelium has been previously studied at the u l t r a s t r u c t u r a l l e v e l (Wynn and French, 1968; King, ^978), there has been no detailed systematic investigation of changes occurring at various stages of gestation. The present study on the structure of . the amnion between 28 and 70 days of gestation (term = 68 days), has shown that the changes in the a b i l i t y of the amnion to respond to AVP are c l o s e l y p a r a l l e l e d by changes in the structure of the amniotic epithelium. In addition, the structure of the epithelium appears to be influenced by AVP treatment. These observations would agree with the 89 suggestion of Garby (1957), that the e p i t h e l i u m , and not 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 , i s the r a t e c o n t r o l l i n g s t r u c t u r e of f l u i d t r a n s p o r t a c r o s s the amnion. In the e a r l i e s t stages of g e s t a t i o n , when f e t a l t i s s u e s are p o o r l y d i f f e r e n t i a t e d , the amniotic f l u i d i s i s o t o n i c with maternal plasma and i s thought to be a d i a l y s a t e of plasma (Behrman, 1967). The r e l a t i v e l y simple amnion of these e a r l y stages c o u l d e a s i l y allow a p a s s i v e t r a n s p o r t of plasma f i l t r a t e a c r o s s i t . However, as the amnion becomes more complex, i t probably p l a y s a more a c t i v e r o l e i n c o n t r o l l i n g the volume and composition of the f l u i d . As the f e t u s matures, the volume of amniotic f l u i d i n c r e a s e s and there i s a p r o g r e s s i v e d e c l i n e i n amniotic f l u i d o s m o l a r i t y (Adolph, 1967; Seeds, 1972). The r e s u l t s of the present study suggest that the i n c r e a s i n g complexity of the amnion, i n terms of i t s s t r u c t u r e and a b i l i t y to respond to v a s o p r e s s i n , may w e l l be r e l a t e d to the i n c r e a s i n g volume of amniotic f l u i d . P r o l a c t i n i s probably a l s o i n v o l v e d i n amniotic f l u i d r e g u l a t i o n , as mentioned in the g e n e r a l i n t r o d u c t i o n . The s t u d i e s of H o l t and Perks (1975; 1977b) i n d i c a t e that p r o l a c t i n i s capable of slowing f e t a l - m a t e r n a l flow of water a c r o s s the g u i n e a - p i g amnion, thereby h e l p i n g to r e t a i n water w i t h i n the amniotic compartment; i t i s a l s o capable of i n c r e a s i n g the movement of sodium out of the amniotic sac. As suggested by these i n v e s t i g a t o r s , such e f f e c t s may c o n t r i b u t e to amniotic f l u i d h y p o t o n i c i t y . 90 There i s some evidence to suggest that the response of the amnion to p r o l a c t i n a l s o changes duri n g pregnancy. Holt and Perks (1975) found that the p r o l a c t i n - i n d u c e d r e d u c t i o n i n f e t a l - m a t e r n a l water flow a c r o s s the amnion i n c r e a s e d with advancing g e s t a t i o n . However, the r e l a t i o n s h i p was only rough, and f u r t h e r experiments are r e q u i r e d . In a d d i t i o n to p r o l a c t i n and v a s o p r e s s i n , there may be other hormones that i n f l u e n c e the volume and composition of amniotic f l u i d . P l a t h and Perks (unpublished o b s e r v a t i o n s ; see Perks, 1977), d e t e c t e d the presence of an agent i n human amniotic f l u i d which had an a n t i d i u r e t i c e f f e c t s i m i l a r to v a s o p r e s s i n in the e t h a n o l - a n a e s t h e t i s e d r a t , but which d i d not appear to be a neurohypophysial p e p t i d e . A c c o r d i n g to Perks (1977), t h i s new agent may a c t p h y s i o l o g i c a l l y on- the amnion; however, there i s .no evidence of t h i s as y e t , and f u r t h e r s t u d i e s are needed. A v a r i e t y of p r o t e i n and s t e r o i d hormones (e.g., a d r e n o c o r t i c o t r o p i n , t h y r o x i n e , a n g i o t e n s i n , C o r t i s o l , e s t r i o l , p r o g e s t e r o n e ) , as w e l l as p r o s t a g l a n d i n s are found i n a mniotic f l u i d (see C a r r e t e r o et al . , 1971; Dawood, 1977; B e l i s l e and T u l c h i n s k y , 1980), and some of these c o u l d a f f e c t the amnion. C o r t i s o l has been found to oppose the a c t i o n s of v a s o p r e s s i n and p r o l a c t i n on water t r a n s p o r t a c r o s s g u i n e a - p i g amnion, and t h i s may be of p h y s i o l o g i c a l importance (Manku et al. , 1975). The p r o s t a g l a n d i n s are of p a r t i c u l a r i n t e r e s t and deserve study as they are known to i n f l u e n c e v a s o p r e s s i n - i n d u c e d water flow i n the toad 91 urinary bladder and the mammalian c o l l e c t i n g tubule (Kinter et al. , 1981). In addition, the amnion has been found to synthesize prostaglandins (see Kierse, 1979; M i t c h e l l et al. , 1982; Olson et al. , 1983). Holt and Perks (1977b) have observed that the sodium permeability of the guinea-pig amnion is low throughout the course of gestation, but ris e s dramatically at about 65 days", just prior to term, and i s p a r t i c u l a r l y high in membranes from fetuses judged to be overdue. Goh and Perks (unpublished observations) have observed a similar marked increase in the permeability of the guinea-pig amnion to chloride ions at term. In addition, North and Segal (1976) have found that the permeability of the guinea-pig amnion to certain non-electrolytes such as urea and acetamide increases just a few days before term. These changes are consistent with the dramatic changes in the structure of the amniotic epithelium ( i . e . , the apparent degeneration) observed in the present study just prior to term. It i s not unreasonable to suggest that the sudden uniform degeneration of the amniotic epithelium, and the concomitant abrupt changes in the transport physiology of the membrane, are hormonally induced. Such changes may in fact be associated with the i n i t i a t i o n of p a r t u r i t i o n . It has been suggested that l o c a l control of f e t a l membrane maturation in humans may produce a signal that i n i t i a t e s labor (Olson et al. , 1983). According to Schwarz et al. (1975) a central role for the f e t a l membranes (the amnion 92 and chorion) i n the i n i t i a t i o n of human p a r t u r i t i o n i s p l a u s i b l e i n l i g h t of the c l i n i c a l o b s e r v a t i o n s that i n j u r y to the f e t a l membranes, such as premature rupture or exposure to h y p e r t o n i c s o l u t i o n s , commonly r e s u l t s i n the onset of l a b o r . Perhaps the amnion c o n t a i n s a substance which p r o t e c t s the f e t u s a g a i n s t premature l a b o r . Once membrane i n t e g r i t y i s l o s t , l a b o r c o u l d ensue. Such a substance c o u l d be progesterone, the withdrawal of which i s thought to i n i t i a t e l a b o r i n many mammals (Ryan, 1980). Recent evidence i n the human i n d i c a t e s t hat progesterone i s s y n t h e s i z e d i n the amnion and c h o r i o n ( M i t c h e l l et al . , 1982; M i t c h e l l and Powell, 1984). Acc o r d i n g to M i t c h e l l and h i s a s s o c i a t e s , l o c a l progesterone withdrawal at the l e v e l of the f e t a l membranes at term c o u l d s t i m u l a t e the i n c r e a s e 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 i s thought to generate myometrial c o n t r a c t i l i t y and the onset of p a r t u r i t i o n . There i s some evidence to suggest that p r o l a c t i n i s a s s o c i a t e d with amniotic membrane i n t e g r i t y . P r o l a c t i n i s present i n the cytoplasm of human amniotic e p i t h e l i u m and i s probably s y n t h e s i z e d there (Healy et al. , 1977; McCoshen et al. , 1982). I n t e r e s t i n g l y , p r o l a c t i n c o n c e n t r a t i o n s i n the amnion are p a r t i c u l a r l y high i n pregnancies with premature rupture of the membranes when compared to normal pregnancy (Ron et al . , 1982). According to Ron et al. (1982), p r o l a c t i n may cause premature rupture by changing the v i s c o e l a s t i c p r o p e r t i e s of the amnion, through a p o s s i b l e e f f e c t on i t s water and 93 e l e c t r o l y t e content. These changes could then result in events leading to the i n i t i a t i o n of p a r t u r i t i o n . It i s possible that the changes in the structure and transport properties of the guinea-pig amnion observed close to term were mediated by p r o l a c t i n . Hormone-mediated f l u i d transport across the amnion provides a possible explanation for the accumulation and retention of large amounts of hypotonic f l u i d within the amniotic compartment, which occurs in spite of physical and biochemical gradients tending to cause f l u i d to flow out of t h i s cavity. The progressive changes in the volume and composition of the f l u i d that occur during the course of gestation may be a result of changes in the structure and a c t i v i t y (e.g., a b i l i t y to respond to vasopressin and perhaps prolactin) of the amnion. In the guinea-pig, the outer yolk-sac membrane, which i s closely apposed to the amnion, also responds to vasopressin by increasing maternal-fetal water flow against the usual gradients (see Perks, 1977). This tissue may also be important in supplying water to the amniotic compartment. It would be interes t i n g to see i f the yolk-sac membrane, l i k e the amnion, changes during the course of gestation. The f l u i d transported into the amniotic compartment via the f e t a l membranes i s now made available to the fetus. The fetus requires an ever increasing supply of water for growth, as 70-95% of i t s t o t a l body weight i s water (Kleinman, 1975). The dai l y requirements could be met at 94 l e a s t p a r t l y by uptake of f l u i d from the amniotic c a v i t y . In a d d i t i o n , the f e t u s c o u l d draw f l u i d from the amniotic sac d u r i n g f e t a l d e h y d r a t i o n . Bruns et al . ( 1 963) found that when f e t a l r a b b i t s were e x p e r i m e n t a l l y dehydrated, the volume of the amniotic f l u i d d e c l i n e d s h a r p l y , and t h i s suggested a net t r a n s f e r of f l u i d from the amniotic to the f e t a l compartment. I t has been observed t h a t i n the human, imbalances i n maternal water and e l e c t r o l y t e c o n c e n t r a t i o n , w i t h i n c e r t a i n l i m i t s , have no s i g n i f i c a n t e f f e c t upon f e t a l body water; a l t e r a t i o n s i n amniotic f l u i d volume occur d u r i n g such s t r e s s , s u g g e s t i n g that net movements of amniotic f l u i d c o n s t i t u t e a mechanism f o r p r o t e c t i n g the f e t u s (Bruns et al. , 1963). The f e t a l s k i n may have a r o l e i n r e g u l a t i n g f e t a l body water, at l e a s t i n the f i r s t h a l f of g e s t a t i o n , before k e r a t i n i z a t i o n o c c u r s . The r e s u l t s of experiments d e s c r i b e d i n S e c t i o n II have shown that neurohypophysial hormones are capable of c a u s i n g a net t r a n s f e r of f l u i d a c r o s s the i s o l a t e d f e t a l g u i n e a - p i g s k i n , i n the mucosal to s e r o s a l d i r e c t i o n . T h i s i s the f i r s t evidence of bulk flow o c c u r r i n g across the f e t a l s k i n i n response to hormones; i n p r e v i o u s s t u d i e s , o n l y u n i d i r e c t i o n a l f l u x e s were measured (France, 1976; H o l t and Perks, 1977a). Net water flow experiments g i v e a more ac c u r a t e p i c t u r e of what might be happening in vivo. Hormone-mediated a b s o r p t i o n of f l u i d through the s k i n may a i d i n s u p p l y i n g the day-to-day water requirements of the growing f e t u s . During f e t a l 95 d e h y d r a t i o n , such e f f e c t s on the s k i n c o u l d a i d i n r e p l e n i s h i n g f e t a l body water, by net water t r a n s f e r from the amniotic compartment. T h i s would make up f o r the l i m i t e d a b i l i t y of the f e t a l kidney to conc e n t r a t e u r i n e . Such an e x t r a - p l a c e n t a l mechanism f o r s u p p l y i n g water to the f e t u s d u r i n g f e t a l d e h y d r ation would be of p a r t i c u l a r importance i f the osmotic s t r e s s came from the mother, and the p l a c e n t a were the agent imposing the s t r e s s . The net uptake of f l u i d a c r o s s the s k i n of the f e t a l g u inea-pig i n response to hormones occu r r e d i n the absence of any apparent g r a d i e n t s between the mucosal and s e r o s a l s u r f a c e s . T h i s may be of s i g n i f i c a n c e to the f e t u s in vivo i n the f i r s t h a l f of g e s t a t i o n , when the amniotic f l u i d and f e t a l e x t r a c e l l u l a r f l u i d are s i m i l a r i n composition ( L i n d et al. , 1969; 1972), and no g r a d i e n t s e x i s t which would d r i v e f l u i d p a s s i v e l y from the amniotic to the f e t a l compartment ac r o s s the s k i n . Between ten and twenty weeks of g e s t a t i o n i n the human, the volume of amniotic f l u i d i s very c l o s e l y r e l a t e d to f e t a l weight (or f e t a l s u r f a c e area) and much l e s s w e l l r e l a t e d to p l a c e n t a l weight or to the l e n g t h of g e s t a t i o n (Lind and Hytten, 1970). In . a d d i t i o n , i n these e a r l y stages of g e s t a t i o n , the composition of amniotic f l u i d i s s i m i l a r to f e t a l e x t r a c e l l u l a r f l u i d ; most notably, the 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 substances such as sodium and urea i n amniotic f l u i d are c l o s e r to those of f e t a l than maternal plasma (Lind et al. , 1969; 1972; L i n d and Hytten, 96 1972). These o b s e r v a t i o n s have l e d L i n d and h i s a s s o c i a t e s to suggest that d u r i n g t h i s time, the amniotic f l u i d may be regarded as an extension of f e t a l e x t r a c e l l u l a r f l u i d , and that the f e t a l s k i n , which i s the only b a r r i e r between the two compartments, may be i n v o l v e d i n r e g u l a t i n g the volume and composition of f l u i d i n t h i s extended e x t r a c e l l u l a r space. A f t e r m i d - g e s t a t i o n , when the s k i n k e r a t i n i z e s , the l i n e a r r e l a t i o n s h i p between amniotic f l u i d volume and f e t a l weight breaks down, and the composition of amniotic f l u i d departs p r o g r e s s i v e l y from the composition of f e t a l plasma (L i n d and Hytten, 1972). I t i s p o s s i b l e that the amnion becomes more important i n r e g u l a t i n g the now " e x t e r n a l i z e d " amniotic f l u i d a f t e r the s k i n k e r a t i n i z e s . T h i s suggestion i s c o n s i s t e n t with the changes i n the amnion r e p o r t e d i n S e c t i o n I. The f e t u s resembles anuran amphibians with respect to i t s a b i l i t y to absorb water through i t s s k i n when under the i n f l u e n c e of neurohypophysial hormones. In the anurans, the s k i n appears to be a major osmoregulatory organ, and t h i s may be true i n the f e t u s , before the s k i n k e r a t i n i z e s . Remarkably, as i n the amphibians, the s k i n of the f e t u s appears to be more respon s i v e to AVT than to AVP. As suggested i n S e c t i o n I I , i t i s perhaps on the f e t a l s k i n that AVT f i n d s i t s p h y s i o l o g i c a l r o l e . T h i s peptide i s p l e n t i f u l i n mammalian f e t a l p i t u i t a r i e s before m i d - g e s t a t i o n , at the time when f e t a l s k i n i s b e l i e v e d to be osmoregulatory. However, i t s l e v e l s d e c l i n e i n the 97 advanced f e t u s , and i t i s not present i n a d u l t p i t u i t a r i e s (Perks, 1977; Skowsky and F i s h e r , 1977). I f AVT has a true p h y s i o l o g i c l r o l e i n the f e t u s , i t should appear i n f e t a l plasma a f t e r a p p r o p r i a t e osmotic s t i m u l i (e.g., d e h y d r a t i o n ) . However, at pr e s e n t , there i s no evidence of AVT s e c r e t i o n by the f e t a l p i t u i t a r y , and s t u d i e s on young f e t u s e s s u b j e c t e d to osmotic s t r e s s are r e q u i r e d . In the a d u l t mammal, the kidney i s the prime r e g u l a t o r of volume and t o n i c i t y of e x t r a c e l l u l a r f l u i d . However, i n the f e t u s , the r e l a t i v e l y i n e f f i c i e n t kidney does not appear to be as important. According to Kleinman (1975), some i n f a n t s born without kidneys have no a b n o r m a l i t i e s i n water and e l e c t r o l y t e composition at b i r t h , suggesting that other mechanisms compensate adequately f o r lack of r e n a l f u n c t i o n . As we have seen, the f e t a l s k i n i s probably an important osmoregulatory organ i n the f i r s t h a l f of g e s t a t i o n . Abramovich (1973) has suggested that a f t e r the sk i n k e r a t i n i z e s , the s u r f a c e of the u m b i l i c a l c o r d may take over some of the f u n c t i o n s concerned with f e t a l - a m n i o t i c f l u i d exchange. I t may be s i g n i f i c a n t that at about the time of k e r a t i n i z a t i o n i n the human f e t u s , the u m b i l i c a l c o r d e p i t h e l i u m begins to resemble the f e t a l periderm. There i s no evidence as yet of hormonal e f f e c t s on f l u i d t r a n s f e r a c r o s s the c o r d . The f e t a l bladder may a l s o be osmoregulatory; when the need a r i s e s f o r water c o n s e r v a t i o n i n the f e t u s , the bladder c o u l d compensate f o r the l i m i t e d a b i l i t y of r e n a l t u b u l e s to concentrate u r i n e 98 by reabsorbing water v i a the bladder e p i t h e l i u m , perhaps ai d e d by v a s o p r e s s i n ; t h i s hormone has been found to i n c r e a s e mucosal-serosal water f l u x a c r o s s the bladder of the f e t a l guinea-pig (Holt and Perks, 1977a). Likew i s e , i n e f f i c i e n c y of r e n a l t u b u l a r r e a b s o r p t i o n of. sodium c o u l d be compensated by a c t i v e r e a b s o r p t i o n of the ion v i a the bladder w a l l . Sodium r e a b s o r p t i o n i n the f e t a l sheep bladder appears to be f a c i l i t a t e d by v a s o p r e s s i n (France et al. , 1976). P r o l a c t i n , which has been found to reduce the p e r m e a b i l i t y of the bladder to water and sodium i n the f e t a l p i g (France et al. , 1976), may a l s o be i n v o l v e d i n f e t a l osmoregulation by a c t i n g on the b l a d d e r . The f e t a l g a s t r o - i n t e s t i n a l t r a c t a c t i v e l y reabsorbs sodium and water from swallowed amniotic f l u i d (Wright and Nixon, 1961; Wright, 1974). I t i s p o s s i b l e that t h i s t r a n s p o r t i s under hormonal c o n t r o l . P r o l a c t i n and C o r t i s o l have been found to i n f l u e n c e water and ion t r a n s p o r t i n the g a s t r o -i n t e s t i n a l t r a c t of some s p e c i e s of f i s h (see Lam, 1972; Hirano, 1980; Morley et al. , 1981), and a s i m i l a r hormonal mechanism i n the f e t u s would provide an a d d i t i o n a l s i t e f o r f e t a l osmoregulation. F i n a l l y , the f e t a l lung may be a s i t e f o r e x c r e t i o n of water and ions d u r i n g o v e r h y d r a t i o n or s a l t l o a d i n g , and t h i s e f f e c t c o u l d be mediated by p r o l a c t i n ( C a s s i n and Perks, 1982). With regard to osmoregulation, the mammalian f e t u s i s s i m i l a r to sub-mammalian v e r t e b r a t e s such as t e l e o s t f i s h and amphibians. The a c t i o n s of neurohypophysial hormones 99 on water and ion t r a n s p o r t a c r o s s the f e t a l s k i n , amnion, and the u r i n a r y bladder are r e m i n i s c e n t of the e f f e c t s of these p e p t i d e s on the amphibian s k i n and bladder (Perks et al. , 1978); The a c t i o n s of p r o l a c t i n on the f e t a l lung, bladder and amnion are s i m i l a r to those observed i n osmoregulatory s t r u c t u r e s i n the t e l e o s t s (Lam, 1972; Perks et al. , 1978; Hirano, 1980). 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