Phy8iol. (1978), 279, pp. 385-394 With 5 text-ftgure8 Printed in Great Britain

385

EVIDENCE OF ACTIVE SODIUM TRANSPORT IN THE VISCERAL YOLK SAC OF THE RAT IN VITRO

BY S. T. H. CHAN AND P. Y. D. WONG From the Department of Phy8iology and Zoology, University of Hong Kong, Hong Kong

(Received 20 December 1977) SUMMARY

1. Na transport has been studied in the isolated rat visceral yolk sac from day 17-5 of gestation to term. 2. The transepithelial potential difference (p.d.) and the short circuit current (s.c.c.) in the isolated yolk sac were found to vary with gestational age, with peak values at day 19-5. The maximal p.d. and s.c.c. were 3-85 + 0-32 mV (the fetal side positive) and 19-5 + 53 ,uA cm-2 respectively. 3. Simultaneous determination of the two-way Na+ flux and the s.c.c. revealed a preferential active movement of Na in the maternal to fetal direction. The net flux was found to be 50% higher than the s.c.c. 4. Both the p.d. and the s.c.c. were found to be reduced by cooling and by the uncoupling agent 2,4-dinitrophenol. 5. The s.c.c. altered in a curvilinear fashion with the Na+ concentration in the bathing solution, with an apparent Km of about 20 mm-Na+. Removing Cl ions from the bathing solutions had no effect on the p.d. and s.c.c. 6. Addition of amiloride (10-4 M) to either side of the visceral yolk sac had no effect on the s.c.c. but application of ouabain (10-5 M) to the fetal side caused a profound fall in the s.c.c. 7. The possible physiological role of this active Na transport by the visceral yolk sac in the formation of amniotic fluid is discussed. INTRODUCTION

The importance of the embryonic membranes in normal fetal development in mammals has long been recognized since they provide not only specific extraembryonic compartments and structures essential for the nutrition, excretion and mechanical protection of the fetus but also some distinct membranous (epithelial) structures which probably play some functional roles in the selective transfer of various substances at different stages of gestation. Although the morphology and function of the embryonic membranes in various mammals, including the yolk sacs of rat (Everett, 1935; Jollie, 1968; Payne & Deuchar, 1972; Jensh, Koszalka, Jensen & Brent, 1974; Jensh, Koszalka, Jensen, Biddle & Brent, 1977), the chorioallantoic membrane of pig (Crawford & McCance, 1960), and the amniotic membrane of guinea-pig (North & Segal, 1976) have been studied, there is still very little informa13

PHY 279

S. T. H. CHAN AND P. Y. D. WONG 386 tion concerning their secretary function and their possible regulatory role in the preferential transport of biological materials. In the rat, the Na concentration in the extraembryonic fluid is comparatively higher than those in the fetal plasma and maternal plasma in the later half of gestation particularly at day 18-5 (Tam & Chan, 1977). It is of interest to investigate whether this accumulation of Na is a result of an active transport across the embryonic membranes during this period. The present study therefore aims to elucidate the transport of sodium across the visceral yolk sac and the amniotic membrane in rat from day 17-5 of gestation to term by measurements of the two-way Na flux and the potential difference (p.d.) and short circuit current (s.c.c.) across these membranes in vitro. It is hoped that our investigation will provide a working model for the study of the secretary and absorptive functionss, if any, of the embryonic membranes in the rat. METHODS

Female Wistar albino rats were placed with males of proven fertility and the morning when sperms were found in the vaginal smear was considered to be day 0 5 of gestation. Rats were killed at various stages of gestation, the uteri with the conceptus in 8itU were removed and immediately placed in chilled Krebs bicarbonate solution. The uterine wall was dissected open and the conceptuses were isolated and placed in individual petri-dish containing fresh Krebs bicarbonate solution. The capsular portion of the visceral yolk sac and the amnionwere carefully cut free with a pair of fine scissors and separated from the chorioallantoic placenta and the fetus. The visceral yolk sac and the amnion were then ready for mounting on to the Perspex chamber specifically designed for the presentstudy (see below). Membranes used were always from freshly killed rats. No study was made before day 16-5 of gestation. The Krebs bicarbonate solution used had the following composition: NaCl, 118 mm; KCl, 4.7 mi; CaCl2, 2-56 mM; MgSO4, 1-17 mM; NaH2PO4, 1-13 mM; NaHCO3, 25 mM; glucose, 111 mm. This solution when bubbled with 95 % 02 and 5 % CO2 had a pH 7-4. In the Na-free solution, NaCl was replaced by choline Cl (140 mM) and NaHCO2 was omitted. The solution was buffered by Tris (5 mm, pH 7.4) and gassed with pure 02. In the Cl-free solution, NaCl (118 mM) was substituted by an equivalent amount of Na isethionate. Solution of amiloride (10 mM; Merck, Sharp & Dohme) in distilled water was prepared freshly for each experiment. Ouabain (Sigma) and 2,4-dinitrophenol (B.D.H.) were made up in distilled water in a concentration of1 mM. Measurement of the p.d. and 8.c.c. The Perspex chamber specially constructed for measuring the p.dA and the s.c.c. of the isolated rat embryonic membranes is shown diagrammatically in Fig. 1. The visceral yolk sac or the

amniotic membrane was clamped horizontally

between the two chambers. The lower chamber

(volume 0 5 ml.) could be perfused with Krebs bicarbonate solution through tubes with taps at the base of the chamber. The upper chamber (volume 2 ml.), which was open to the atmosphere, was surrounded by a circulation of water prewarmed to 37 0C in a thermostatic waterbath. The medium in this chamber was gassed with 95 % 0° and 5 %C02 and could be withdrawn and replaced within a few seconds. The mixing of applied drugs in this chamber is virtually instantaneous. The embryonic membrane was made to bulge upwards into hemisphere by slight application of pressure in the lower chamber (this was not found to affect the p.d. and s.c.c.). In most experiments, the maternal side of the visceral yolk sac (or the somatic mesoderm of the amnion) faced upwards so that the shape resembled that in vivo and in utero. The surface area of the membrane between the two chambers was estimated to be about 1-57cm2. Transepithelial potentials were measured using conventional KCl-agar bridges which led via calomel cells to a digital milli-voltmeter (Weston). The potential could also be switched to a short-circuiting device consisting of two operational amplifier feedback circuits which delivered current to the epithelium through two AgCl electrodes and two 0-9 % saline-agar bridges. a

ACTIVE Na TRANSPORT BY RAT YOLK SAC

387

The current delivered to the epithelium was monitored on a potentiometric recorder (Devices) as the potential developed across an accurately known resistor.

Measurement of Na fluxes The two-way Na flux was measured on two pieces of visceral yolk sac taken from the same embryo using 22Na as marker. The procedure was as follows: the visceral yolk sac from one embryo was dissected into halves. To measure the maternal to fetal flux, one half was mounted with the maternal side facing upwards. The epithelium was short circuited as described and the current was continuously monitored on a chart recorder. After 20 min of equilibration, 22Na (1 juc, Amersham, specific activity 300 mc/m-mole) was added to the maternal bathing solution

.4- 02/C02(95% :5%)

37'C water from thermostatic water-bath

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Fig. 1. Diagram of the chambers used to measure p.d. and s.e.c. in the rat embryonic membranes. The visceral yolk sac or the amnion is clamped horizontally between two Perspex chambers with the maternal side of the membrane facing upwards. See text for explanation. e = potential recording electrode, ¢ = current passing electrode.

(upper chamber). A 'hot' sample was taken to determine the specific activity of 22Na in the maternal bathing solution. After 30 min, the fetal bathing solution was flushed out from the lower chamber and counted for 22Na activity. To measure the fetal to maternal flux, the other half of the visceral yolk sac was clamped in a similar manner. After 20 min, the lower chamber was filled with Krebs bicarbonate solution containing 22Na (1 /zc/ml.). The maternal facing solution was taken from the upper chamber and measured for 22Na activity at the end of 30 min. 22Na was counted on a gamma counter (Nuclear Chicago, Model 1195R). The s.c.c.s from the two halves of the same yolk sac were found to have similar values and the mean current was taken.

13-2

388

S. T. H. CHAN AND P. Y. D. WONG RESULTS

The basal p.d. and s.x.c. in the amniotic membrane and the visceral yolk sac The transepithelial potential across the amniotic membrane was measured as 0 13 + 0-03 mV (mean+ s.E., n = 7); the fetal side was positively charged with respect to the maternal side. The yolk sac, however, developed a potential of a few millivolts across the two surfaces with the fetal side positively charged. The variation of the p.d. and s.c.c. in relation to experimental time in the day 19-5 yolk sac is shown in Table 1. The p.d. was found unchanged during the first 40 min of the measurement period. The s.c.c., on the other hand, was below 10 UA cm-2 soon after setting up of the membrane but gradually stabilized at about 20 1tA cm-2 after 15 min of equilibration. Since the p.d. across the amniotic membrane was small when compared with that of the visceral yolk sac, subsequent studies of the p.d. and s.c.c. were therefore concentrated mainly on the yolk sac. 25

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Fig. 2 Fig. 3 Fig. 2. Effect of gestational age on the p.d. (open circles) and s.c.c. (filled circles) of the day 19-5 rat yolk sac. Each point represents the mean + S.E. of four to six experiments. Fig. 3. Effect of temperature on the p.d. (open circles) and s.c.c. (filled circles) of the day 19-5 rat yolk sac. Each point represents the mean + S.E. of four experiments.

Variation of p.d. and s.c.c. in visceral yolk sacs of different gestational age Measurements of p.d. and s.c.c. of visceral yolk sacs from day 17-5 to day 21-5 of gestation were made; membranes from earlier stages were not included because of their small size and the presence of the parietal yolk sac. Results showed that both p.d. and s.c.c. across the membrane increased from day 17-5 and reached a peak value at day 19-5 (Fig. 2). The p.d. and s.c.c. then declined from day 19-5 to day 21-5. Accordingly, the day 19-5 yolk sac was studied in all subsequent experiments. Determination of Na fluxes To see whether the s.c.c. is a measurement of the net transport of sodium across the visceral yolk sac, the bidirectional Na flux was measured. Table 2 gives the Na fluxes in both directions and the s.c.c. of four day 19-5 yolk sacs obtained from four rats. Both Na flux and s.c.c. were expressed in mc cm-2 30 min-'. The maternal to

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S. T. H. CHAN AND P. Y. D. WONG 390 fetal flux was found to be 2-06 times higher than the fetal to maternal flux (P < 0*05, Student's t test). The net flux amounted to 147 % of the s.c.c. (the net Na flux and the s.c.c. are not significantly different). It can be inferred that a net transport of Na from the maternal to fetal side is the main source of p.d. and s.c.c. across the membrane. The s.c.c. is therefore taken as an indication of the rate of Na transport across the visceral yolk sac. Effect of cooling and 2,4-dinitrophenol on the p.d. and s.c.c. across the yolk sac Both the p.d. and s.c.c. showed a marked dependence on the temperature of the experimental chamber (Fig. 3). Lowering the temperature from 37 to 33 0C caused a reduction in the p.d. of 1P25 mV and in the s.c.c. of 7-5 1tA cm-2. At 23 0C both the p.d. and s.c.c. were reduced by about 80%. From an Arrhenius plot of the data over the range 23-37 0C0 it was calculated that the activation energy for the reactions to maintain the p.d. and s.c.c. at normal values is approximately 13 kWal per mole. 25-

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The effect of the uncoupling agent 2,4-dinitrophenol is shown in Table 3. Application of the drug (10-5 M) to the maternal side caused a profound fall in the s.c.c. Inhibition was about 82 % and was completed within 15 min.

Effect of varying Na concentration on the s.c.c. In this experiment the membranes of the visceral yolk sac were washed on both sides with solutions containing different sodium concentrations and the s.c.c. was measured at each Na concentration. The relationship between the s.c.c. and the Na concentration is shown in Fig. 4. At zero Na, no s.c.c. was detected and when Na concentration was increased in a stepwise manner; the s.c.c. also increased in a curvilinear manner exhibiting saturation kinetics with the apparent Km of about 20 mm. The s.c.c. was hence dependent on the Na concentration in the incubation medium.

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S. T. H. CHAN AND P. Y. D. WONG

Effect of removal of C1 ions When Cl ions were removed from the incubation medium in both chambers, the p.d. and s.c.c. across the yolk sac were respectively 2-98 + 0-48 mV and 16-5 + 14,1UA cm-2 (mean + s.E., n = 4). These values were not significantly different from the control values of 2-95 + 0-86 mV (mean + s.E., n = 4) for p.d. and 18-8 + 1.1 tUA cm-' (mean + S.E., n = 4) for s.c.c.

Effect of amiloride and ouabain The effect of amiloride (10-4M), a drug which impairs the passive Na transport across epithelia (for review see Cuthbert, 1974) is shown in Fig. 5. It was found that addition of the drug (t0-4 M) to either side of the membrane had no effect on the s.c.c. In other experiments, ouabain (10-5 M) was also added to either chamber to study whether a Na+-K+ pump is involved in the s.c.c. and hence the rate of Na transport across the visceral yolk sac. It was found that addition of the drug to the maternal side did not affect the s.c.c., whereas addition to the foetal side caused a profound fall. Inhibition was completed after 20 min of application (Table 4). DISCUSSION

Since the composition of the extraembryonic fluid of the rat fetus is consistently different from those of the maternal and fetal blood (Tam & Chan, 1977), it is most likely that the extraembryonic membranes serve not only as structures that provide the mechanical support and protection for the developing fetus but also as some effective barriers to diffusion with secretary and absorptive functions. Most epithelia secrete or absorb fluid and active Na transport across the membrane barrier appears to play an important part in the mechanisms by which this is achieved (Diamond & Wright, 1969). In our present study, the p.d. and s.c.c. measurements have been confined to the visceral yolk sac and the amniotic membrane since at the gestational period included in the present investigation only these two membranes remain intact and functional. As our results showed that the p.d. across the amnion was small and negligible, the amniotic membrane was probably passive at this stage and was unlikely to be involved in any active transport process. In this context, North & Segal (1976) have also shown that the guinea-pig amnion was unlikely to be the site of formation of amniotic fluid during day 50 to 70 gestation. The visceral yolk sac, however, had a p.d. of a few millivolts with the fetal side positive with respect to the maternal side. Simultaneous determination of the s.c.c. and the Na flux has shown that under short-circuit conditions the maternal to fetal flux was 2 times higher than the fetal to maternal flux and the net flux was close to the s.c.c. although 50% higher. The s.c.c. was likely to be contributed by the net maternal to fetal transport of Na ions. This view was further supported by the dependence of the s.c.c. on the Na concentration (Fig. 4). The p.d. and s.c.c. were inhibited by cooling and by the uncoupling agent, 2,4dinitrophenol, suggesting that they are indices and measurements of an active process. The s.c.c. alters in a-curvilinear fashion with the Na concentration in the bathing solution, exhibiting saturation kinetics with the apparent K,, of about

393 ACTIVE Na TRANSPORT BY RAT YOLK SAC 20 mM-Na+. The kinetics of sodium transport across the rat yolk sac are therefore similar to those observed in amphibian skin (Cereijido, Herrera, Flanigan & Curran, 1964), amphibian bladder (Leaf, 1965; Cuthbert & Wong, 1971), rat ileum (Curran & Solomon, 1957), gall-bladder (Diamond, 1962), kidney proximal tubule (Gyory, Lingard & Young, 1973) and rat cauda epididymidis (Wong & Yeung, 1978). The s.c.c. on the other hand, was not affected by the removal of Cl ions from the bathing solution, indicating that Cl transport could not have contributed to the s.c.c. Amiloride, a drug which has been found to inhibit Na transport in some epithelia by impeding Na+ entry into cells (see Cuthbert, 1974), had no effect on the s.c.c. in the rat visceral yolk sac. In this respect the characteristics of Na transport across the yolk sac differ from that of other epithelia. Ouabain inhibits Na transport when added to the fetal side of the membrane. It is pertinent that a Na+-K+ pump located in the fetal side extrudes Na+ out of the cells into the fetal fluid. The active transport of sodium across the rat visceral yolk sac may play a role in the formation of amniotic fluid. The peak values of the p.d. and s.c.c. were at days 18-5 and 19-5 and coincided with the time when the volume of the extraembryonic fluid and the Na content therein were found to be maximal (Tam & Chan, 1977). The active sodium transport appears to be a developmental process in the later part of gestation. The functional significance and the biochemical implication of such an event at day 18-5 to 20*5 gestation is unknown. However, since water transport across epithelia is generally accepted to be a passive osmotic process secondary to the active Na transport across the epithelium (Diamond & Tormey, 1966), it is very likely that the visceral yolk sac could be triggered (by the rupture of sac or by some factors yet to be identified) to undergo a process of active Na transport as a natural ontogenetic event following the rupture of the sac and the Reichart's membrane and thereby increase formation of extraembryonic fluid. Mellor (1969) has measured the potential difference between mother and the amniotic fluid in the rat and found a potential of 15 mV with the amniotic fluid positive with respect to the mother. Na transport across the visceral yolk sac in vivo is therefore carried out against this potential difference. As a result of these events, the fetus is provided with a large 'private pool' of amniotic fluid with adequate free volume for the more vigorous fetal movement and the more rapid development and growth of the fetus near term. The observed decline in fluid volume, and the Na concentration therein, after day 21-5 was found to coincide with the drop in p.d. and s.c.c. and hence the disappearance of the differential transport mechanism or properties of the visceral yolk sac. This decline might be due to the possibly 'aged' and 'leaky' nature of the sac, which at day 21-5 becomes highly expanded and greatly stretched with obvious morphological transformation and signs of cell autolysis in both the endodermal and the splanchnic mesodermal components (S. T. H. Chan & P. Y. D. Wong, unpublished observations). We are grateful to Merck, Sharp & Dohme for a gift of amiloride.

394

S. T. H. CHAN AND P. Y. D. WONG REFERENCES

CEREIJIDO, M., HERRERA, F. C., FLANIGAN, W. J. & CURRAN, P. F. (1964). The influence of Na concentration on Na transport across frog skin. J. gen. Physiol. 47, 879-893. CRAWFORD, J. D. & MCCANCE, R. A. (1960). Na+ transport by the chorioallantoic membrane of the pig. J. Phy8iol. 151, 458-471. CuTRRAN, P. F. & SOLOMON, A. K. (1957). Ion and water fluxes in the ileum of rats. J. gen. Phy8iol. 41, 143-168. CUTHBERT, A. W. (1974). Characteristics of sodium channels in transporting epithelia. In Drugs and Transport Proce88es, ed. CALLINGHAM, B. A., pp. 173-184. London: Macmillan. CUTHBERT, A. W. & WONG, P. Y. D. (1971). The effect of metal ions and antidiuretic hormone on oxygen consumption in toad bladder. J. Phydiol. 219, 39-56. DIAMOND, J. M. (1962). The reabsorptive function of the gall bladder. J. Physiol. 101, 442-473. DIAMOND, J. M. & TORMEY, J. McD. (1966). Studies on the structural basis of water transport across epithelial membranes. Fedn Proc. 25, 1458-1463. DIAMOND, J. M. & WRIGHT, E. H. (1969). Biological membranes: the physical basis of ion and nonelectrolyte selectivity. A. Rev. Phy8iol. 31, 581-646. EVERETT, J. W. (1935). Morphological and physiological studies of the placenta in the albino rat. J. exp. Zool. 70, 243-284. GYORY, A. Z., LINGARD, J. M. & YOUNG, J. A. (1973). Kinetics of Na+ reabsorption in rat proximal tubules perfused in vivo. Proc. Au8t. Phy8iol. Pharmacol. Soc. 4, 52-53. JENSH, R. P., KoszALicA, T. R., JENSEN, M. & BRENT, R. L. (1974). Morphologic evolution of the rat parietal yolk sac from the 12th to 19th day of gestation. Teratology 9, A-23, 1974. JENSH, R. P., KoszALKA, T. R., JENSEN, M., BIDDLE, L. & BRENT, R. L. (1977). Morphologic alterations in the parietal yolk-sac of the rat from the 12th to the 19th day of gestation. J. Embryol. exp. Morph. 39, 9-21. JOLLIE, WV. P. (1968). Changes in the fine structure of the parietal yolk-sac of the rat placenta with increase in age. Am. J. Anat. 122, 513-532. LEAF, A. (1965). Transepithelial transport and its hormonal control in toad bladder. Ergebn. Phy8iol. 56, 216-263. MELLOR, D. J. (1969). Potential differences between mother and foetus at different gestational ages in the rat, rabbit and guinea-pig. J. Physiol. 204, 395-405. NORTH, P. M. & SEGAL, M. B. (1976). A study of the transport and permeability properties of the guinea-pig amniotic membrane. J. Physiol. 256, 245-256. PAYNE, G. S. & DEUCHAR, E. M. (1972). An in vitro study of the functions of embryonic membranes in the rat. J. Embryol. exp. Morph. 27, 533-542. TAM, P. P. L. & CHAN, S. T. H. (1977). Changes in the composition of maternal plasma, fetal plasma and fetal extraembryonic fluid during gestation in the rat. J. Reprod. Fert. 51, 41-51. WONG, P. Y. D. & YEUNG, C. H. (1978). Absorptive and secretary functions of the perfused rat cauda epididymidis. J. Physiol. 275, 13-26.

Evidence of active sodium transport in the visceral yolk sac of the rat in vitro.

Phy8iol. (1978), 279, pp. 385-394 With 5 text-ftgure8 Printed in Great Britain 385 EVIDENCE OF ACTIVE SODIUM TRANSPORT IN THE VISCERAL YOLK SAC OF T...
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