/. Embryol. exp. Morph., Vol. U, Part I, pp. 167-174, March 1963 Printed in Great Britain
Electrical Potential Differences Across the Foetal Membranes of the Rabbit1
by v. KRESPI and j. DAVIES2 From the Departments of Physiology and Anatomy, Washington University Medical School
THE chemical composition of the foetal fluids in the rabbit was studied by Davies & Routh (1957). These investigators showed that the amniotic and exocoelomic fluids resembled extracellular fluid in their ionic composition whereas the allantoic fluid more closely resembled intracellular fluid. These findings were confirmed by Dickerson & McCance (1957). An experimental study of the trans-membrane potentials in foetal rabbits was undertaken in order to gain some knowledge of their relative permeabilities to certain ions. The process by which a substance is transported across a membrane against its electrochemical gradient is called 'active transport' and requires the expenditure of metabolic energy. If the cell membrane separates ions to which it is differentially permeable, then an electric potential gradient also exists across the membrane. The equilibrium potential for any ion distributed unequally across a membrane may be calculated from the Nernst equation and compared with the actual trans-membrane potential obtained by direct measurement. The membrane is most permeable to the ion whose equilibrium potential best approximates that determined empirically. For example, the Nernst equation for potassium leads to the following expression (25°C):
RT [K+]_ ^ln581og where E = the electric potential difference across the membrane in millivolts and [K?] and [K+] = the concentration of potassium inside and outside the cell, respectively. The observed potential difference between body extracellular fluid, on the one hand, and amniotic or allantoic fluid, on the other, was com- pared with the calculated equilibrium values for the various ions. Since there is no ionic concentration gradient between amniotic and body extracellular fluids, no membrane potential should be detectable across it. The
1 Aided by Grants No. AMO4394 and B-937 of the National Institutes of Health, U. S.P.H.S., and by a grant from the Josiah Macy, Jr. Foundation. 2 Author's address: Department of Anatomy, Washington University Medical School, Scott and Euclid Avenues, St. Louis 10, Missouri, U.S.A. 168 V. KRESPI AND J. DAVIES allantois, on the other hand, separates fluids containing different concentrations of sodium, potassium and chloride. If it is more permeable to one of these ions than to another, a membrane potential should exist across it.
MATERIAL AND METHODS New Zealand white rabbits were used and were studied from about the twentieth to the twenty-third day of pregnancy, at which time the allantoic fluid is conspicuous and easily identifiable by its opalescence. The animals were anesthetized with intravenous sodium nembutal and the uterine horn delivered through a mid-line abdominal incision. A small incision was made along the
uterine lumen
amnion visceral yolk sac exocoelom
sinus terminalis
paraplacental chorion allantois
TEXT-FIG. 1. Schematic cross section of the rabbit uterus about the twenty-first day of pregnancy based on an actual section showing the disposition of the foetal membranes and the chemical composition of the amniotic and allantoic fluids with respect to Na+ and Cl~, based on the findings of Davies & Routh (1957). anti-mesometrial side of the uterus over a uterine swelling and the membranes allowed to bulge out. The yolk sac and paraplacental chorion (see Fig. 1) were carefully removed, allowing the exocoelomic fluid to escape and exposing the surface of the amnion and the allantois. The active electrode was a glass micro- electrode filled with concentrated potassium chloride into which a fine tungsten wire was introduced. The indifferent electrode was a silver wire attached to the POTENTIALS ACROSS FOETAL MEMBRANES 169 peritoneal surface of the abdominal wall or to some abdominal viscus. The true zero point was obtained by placing both electrodes in the peritoneal cavity and balancing out any junction potentials. The micro-electrode was then inserted successively into each fluid cavity, using a micro-manipulator, and the change in potential between the two electrodes was measured at the instant that the micro-electrode penetrated a membrane. The electrical potential difference between the two electrodes was led through a Medistor pre-amplifier and displayed on a Tektronix oscilloscope.
RESULTS Nine rabbits with a total of forty-two foetuses were studied. Two rabbits (nine foetuses) were at the twenty-first day of pregnancy, five rabbits (eighteen foetuses) at the twenty-second day, and two rabbits (fourteen foetuses) were at the twenty-third day. The mean allantoic membrane potential values and their standard errors were as follows (Table I): -43±4-4 mv at 21 days
TABLE 1 Electrical potential differences across the rabbit allantois 21 days 22 days 23 days
Rabbit Foetus Rabbit Foetus Rabbit Foetus No. No. — mv No. No. — mv No. No. — mv 1 1 30 1 1 40 1 1 40 2 25 2 40 2 + 8 3 50 3 40 3 40 4 40 4 40 4 20 5 40 5 50 5 20 6 50 6 50 6 20 2 1 70 2 1 70 7 30 2 30 2 12 2 1 50 3 50 3 80 2 0 3 1 60 3 50 4 1 20 4 50 2 20 5 50 3 20 6 0 4 50 7 80 5 50 5 1 60 2 28 3 24
(-25 to -70 mv), -42±4-4 mv at 22 days (-12 to -70mv), and -32±6-9 mv at 23 days (+ 8 to - 80 mv), the allantoic fluid being negative with respect to the peritoneal cavity. In one foetus, a positive potential of 8 mv was recorded. Amniotic membrane potentials were recorded in thirty-eight foetuses. In thirty-six, the average was 0 mv (- 8 to +8 mv); in one foetus, the amniotic fluid was 20 mv positive with respect to the peritoneal cavity, in another 40 mv 170 V. KRESPI AND J. DAVIES positive. All values refer to those recorded on first puncturing the membranes with the recording electrode, since it was observed that the potential difference declined rapidly with successive punctures. There is some evidence that this was not a result of the deterioration of the membranes with time due to exposure to air, for in one case in which the experiment had to be interrupted for 2 hr. after removal of the yolk sac, an allantoic membrane potential of —40 mv was recorded from that foetus (Table 1: 22 days, Rabbit No. 1, Foetus No. 4). Deterioration of the electrode could not be responsible for the decline in potential difference either, since the same electrode used on the next foetus gave results appropriate to that foetus. In five rabbits, several foetuses were left undisturbed, the animals killed with nembutal, and the foetal membrane potentials measured after varying lengths of time. After 1 hour the allantoic membrane potential of one foetus was found to be - 30 mv in a rabbit in which the average had been - 43 mv (- 40 to - 50 mv) before killing; after 2 hours, it had declined to 0 and - 20 mv in two embryos respectively and to —10 mv in a foetus in which it had previously been -40 mv (second puncture for this embryo). After 3 hours, the average allantoic membrane potential declined to -9 mv (0 to -14 mv) in a rabbit in which it had been - 39 mv (- 25 to - 50 mv). Four hours after death, no membrane potentials could be recorded from any of sixteen foetuses. The amniotic membrane potentials were 0 mv at all the above intervals after death.
DISCUSSION At 22 days, the allantoic fluid concentrations of K+, Na+, and Cl~ are 59-6, 24 and 68 m equiv. per 1., respectively; the exocoelomic fluid concentrations of the same ions are 4-8, 129 and 98 m equiv. per 1., respectively (Davies & Routh, 1957). From these figures (assuming that the ionic composition of the intra- peritoneal fluid is similar to that of the exocoelomic fluid) and the Nernst equation, the calculated equilibrium potential across the allantois is - 64 mv for K+, + 43 mv for Na+ and - 9 mv for Cl~. The measured average allantoic membrane potential at 22 days is - 42 mv. This value most closely approximates the equilibrium potential calculated for K+. Similarly, in agreement with the ionic gradient across it, the amnion of the rabbit foetus has zero membrane potential. The results indicate that the allantois is much more permeable to potassium than it is to sodium or chloride, and more permeable to chloride than it is to sodium. The question arises as to whether the proteins in solution in the allantoic fluid might play a role in maintaining the potassium gradient across the allantois. Is this a Donnan potential, or active transport? In the absence of information regarding the protein content of foetal extracellular fluid, Donnan equilibria cannot be calculated. According to Davies & Routh (1957), the amniotic and allantoic fluids of the rabbit contain 335 and 38 mg. protein per 100 ml., respectively. Both these figures are much lower than the POTENTIALS ACROSS FOETAL MEMBRANES 171 adult extracellular fluid protein content of approximately 3 • 3 per cent. There is no justification for assuming the same value for foetal extracellular fluid. On the other hand, since the high protein content of amniotic fluid, in com- parison to allantoic fluid, does not appear to have led to a trans-amnion electrical potential difference, it seems reasonable to assume that the allantoic membrane potential is not maintained by the allantoic fluid protein content. The fact that the allantoic membrane potential declines to zero with time after death, makes it likely that the ionic concentration gradients across it are maintained by an active metabolic process. Widdas (1961) reported measurements of electrical potentials across sheep allantoic and cat amniotic membranes. While his results on the allantoic membrane are qualitatively similar to those reported here for the rabbit, his measurements on the amniotic membrane differ considerably in that they showed an appreciable negative potential with respect to the mother's peri- toneum. A discussion of this difference must await a knowledge of the con- centrations of ions across the sheep and cat amnion. The chemical composition of the foetal fluids of the pig in the first third of pregnancy has been described by Dickerson & McCance (1957). Active transport of sodium across the isolated chorio-allantoic and allantoic membranes of the pig from the foetal to the maternal side was shown by Crawford & McCance (1960). The results reported here on the rabbit amnion are in accordance with those of Garby (1957) who studied the human amniotic membrane in vitro and showed that (1) it is per- meable to all the substances studied, (2) the composition of the amniotic fluid is similar to that of plasma with respect to Cl~, (3) the Na+ and K+ and the total molecular and ionic concentrations are slightly less than those of plasma, and (4) there is no measurable electrical potential across it. It was suggested, on the basis of morphological studies, that the most likely source of the allantoic fluid in the rabbit was the mesonephros (Davies & Routh, 1957). However, it was shown by Stanier (1960) that the chemical composition of the bladder urine at about the eighteenth day in the rabbit resembled that of the foetal serum and not that of the allantoic fluid. Thus, the characteristic composition of the allantoic fluid is probably to be attributed to the activity of the allantoic membrane rather than to foetal renal activity. At a later stage (about 23 days), when the metanephros is well differentiated, the composition of the bladder urine indicates that there is considerable absorption of chlorides and phosphates during the passage of the urine through the kidney but little absorption of water (Crawford & McCance, 1960). There is probably an overlap of function of the metanephros and mesonephros between the eighteenth and the twenty-third day (Davies & Routh, 1957): the allantoic fluid has largely disappeared, however, by the twenty-third day. The foetal membranes of the rabbit are histologically complex and have been described by light and electron microscopy by Falck Larsen & Davies (1962). The amnion and allantois are essentially three-layered. The inner layer in each 172 V. KRESPI AND J. DAVIES case consists of a singly layer of flattened cells of ectodermal (amnion) or endo- dermal (allantois) origin. The free surfaces of these cells are thrown up into microvilli and show other evidences of active absorptive activity. Adjoining cell membranes are interlocked and connected at several points by desmosomes. The basal plasma membrane is simple and rests on a basement membrane. The cytoplasm contains Gogli vacuoles and membranes, relatively little ergasto- plasm and a few scattered mitochondria, some of which are swollen. The outer layer of each membrane is identical with the lining of the exocoelomic cavity (see Text-fig. 1) and consists of irregularly flattened mesemchymal cells with no basement membrane. These cells contain a few mitochondria and extensive ergastoplasmic membranes and granules. The cells form a dis- continuous layer, the underlying connective tissue being exposed in places. The intermediate layer of the amnion and allantois consists of a relatively thick layer of mesenchyme composed of scattered fibroblastic cells, bundles of collagen and abundant intercellular ground substance, possibly mucopolysac- charide in nature. Thus in both the amnion and the allantois there is only one uninterrupted single cell layer, which is in each case the inner one and most probably the seat of the membrane potential. This situation may be compared with that proposed for the skin of the frog by Koefoed-Johnsen & Ussing (1958). The entire wall of the amnion is avascular. That of the allantois is avascular where it faces the amnion and the exocoelomic cavity (Text-fig. 1): where it lies on the chorio-allantoic placental disc it is vascularized by umbilical (allantoic) vessels. At the interface between the amnion and the allantois the opposing layers are fused and the two mesenchymal layers are in contact with no inter- vening exocoelomic epithelium. The allantois may be loculated and subdivided by incomplete (?) septa. Irregular spaces unconnected with the general exocoelomic cavity are found lateral to the edge of the placental disc (Text-fig. 1). An in vivo system, such as that presented by the fluid compartments of the foetal rabbit, seems to be an ideal one for the study of the ionic fluxes across the membranes involved and for the elucidation of the role of these membranes in the physiology of development.
SUMMARY 1. Electrical potentials measured across the allantoic membrane of the foetal rabbit averaged - 43 mv at the twenty-first day, - 42 mv at the twenty- second day and - 32 mv at the twenty-third day of pregnancy. Potentials across the amniotic membrane were 0 mv except in one case in which + 20 mv and one case in which +40 mv were observed. The allantoic membrane potentials approximate those predicted for K+ by the Nernst equation using previous chemical measurements of Davies & Routh (1957). The amniotic membrane potential is also consistent with the chemical findings. POTENTIALS ACROSS FOETAL MEMBRANES 173 2. Four hours following death, no potential difference can be measured across the allantois. The results point to a greater permeability of the allantois to K+ than to Na+ or Cl~ and to a metabolic process maintaining the ionic gradient across it, this situation being perhaps similar to that observed by others in the isolated allantois and chorio-allantois of the pig. 3. A brief summary of the fine structure and disposition of the foetal mem- branes involved is also given.
RESUME Differences de potentiel electrique a travers les annexes embryonnaires du lapin 1. Les potentiels electriques mesures a travers la membrane allantoi'dienne du foetus de lapin etaient en moyenne de —43 mv le 21e jour, —42 mv le 22e jour, et - 32 mv le 23e jour de la gestation. Les potentiels a travers la membrane amniotique etaient de 0 mv, sauf dans un cas ou Ton a observe + 20 mv, et un autre ou Ton a observe +40 mv. Les potentiels de la membrane allantoidienne sont approximativement ceux prevus pour le K+ per l'equation de Nernst a partir de mesures chimiques anterieures de Davies et Routh (1957). Le potentiel de la membrane amniotique est egalement en accord avec les donnees chimiques. 2. Quatre heures apres la mort, on ne peut mesurer de difference de potentiel a travers l'allantoide. Les resultats indiquent une plus grande permeabilite de l'allantoide au K+ qu'au Na+ ou au Cl~, et un processus metabolique maintenant le gradient ionique a travers elle, cette situation etant peut-etre semblable a celle observee par d'autres auteurs sur l'allantoide et l'allanto- chorion isoles du pore. 3. On donne egalement un bref resume de la structure fine et de la disposition des annexes embryonnaires.
ACKNOWLEDGEMENT Grateful acknowledgement is made for technical help and advice to Dr W. W. Sleator and to Dr E. W. Dempsey, also to Susan Philpott and Birgit Schmelling for assistance with the experiments.
REFERENCES
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KOEFOED-JOHNSEN, V. & USSING, H. H. (1958). The nature of the frog skin potential. Ada physiol. scand. 42, 298-308. MCCANCE, R. A. & STANIER, M. (1960). The function of the metanephros of foetal rabbits and pigs. J. Physiol. 151, 479-83. STANIER, W. (1960). The function of the mammalian mesonephros. /. Physiol. 151,472-8. WIDDAS, W. F. (1961). Transport mechanisms in the foetus. Brit. med. Bull. 17, 107-11.
{Manuscript received 17th July 1962)