J. Phyeiul. (1978), 280, pp. 319-330 With 1 text-figure Printed in G(reat Britain
319
OUABAIN-SENSITIVE FLUID ACCUMULATION AND ION TRANSPORT BY RABBIT BLASTOCYSTS By JOHN D. BIGGERS, RAYMOND M. BORLAND AND CLAUDE P. LECHENE From the Laboratory of Human Reproduction and Reproductive Biology and Biotechnology Resource in Electron Probe Microanalysis, Department of Physiology, Harvard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115, U.S.A.
(Received 9 November 1977) SUMMARY
1. By incubating 6-day post-coitum (p.c.) blastocysts in medium supplemented with either 20 mm-sucrose or 10 mM-KCl it was possible to show that the trophectoderm of the blastocyst prevents the concentration of K in the blastocoele fluid rising to external concentrations. 2. The concentrations of K in the blastocoele fluid are maintained predominantly by leakage of K from the trophoblast cells into the blastocoele and by ouabainsensitive transport of K into the trophoblast cells from the blastocoele fluid. 3. Exposure of blastocysts to ouabain on the juxtacoelic, but not abcoelic, surface of the trophectoderm may inhibit blastocoele fluid accumulation. INTRODUCTION
Borland, Biggers & Lechene (1976) showed that K accumulates in the blastocoele fluid of the rabbit, and that its rate of accumulation increases from 5 to 31 nm cm-2 hr-1 between the 4th and 6th day of development. Petzoldt (1971), using K+ selective electrodes, however, found that the K+ activities in the blastocoele fluid and uterine fluid of rabbits 6 or 7 days pregnant were 10-12 mm and 20 mm respectively. Thus, although there is a net transfer of K into the blastocoele cavity, mechanisms prevent its concentration from rising to that found in the external uterine fluid. At least three mechanisms could limit the accumulation of K in rabbit blastocoele fluid: 1. Limited diffusion of K from uterine fluid into the blastocoele through the trophoblast junctional complexes and intercellular spaces. 2. Limited leakage and transport of K from the trophoblast cells into the blastocoele fluid. 3. Significant leakage of K into the blastocoele cavity coupled with the active transport of K from the cavity back into the trophoblast cells. Borland et al. (1976) also showed that 6-day rabbit blastocysts in vitro respond to a lowering of the chemical potential of water in the external medium by an increase in the concentrations of Na and Cl in blastocoele fluid, and by the continued accumulation of fluid with no change in the concentration of K. The accumulation of Na,
J. D. BIGGERS, R. M. BORLAND AND C. P. LECHENE 320 C1 and water by the rabbit blastocyst appears to be regulated, in part by Na+-K+ ATPase (E.C. 3.6. 1.3) since these processes are inhibited by ouabain in the external medium (Smith, 1970; Gamow & Daniel, 1970). However, in these experiments the concentrations of ouabain that were used were high, and no attempt was made to differentiate between abcoelic and juxtacoelic effects of this steroidal glycoside. Using X-ray spectrometry by electron probe excitation, we have estimated the concentrations of Na, Cl and K in picolitre samples of blastocoele fluid after exposing the blastocyst to ouabain in the external medium (abcoelic ouabain exposure) or in the blastocoele fluid (juxtacoelic ouabain exposure), and have demonstrated that K is actively transported out of the blastocoele cavity. From these experiments and other evidence analysed in the Discussion it is suggested that K accumulates in the blastocoele cavity by leakage from the trophoblast cells, but is prevented from rising to high levels by being actively transported back into the cells by pumps located on the juxtacoelic surface of the trophectoderm. The concomitant transport of Na into the blastocoele is responsible for fluid accumulation in the blastocyst. METHODS
Collection of blastocyst8 Eight to ten lb New Zealand white rabbits were naturally mated to mature males. Ovulation occurs approximately 10 hr after coitus. At 6 days post-coitum (p.c.) the female rabbits were sacrificed by chloroform anaesthesia. The uterine horns were cannulated and the blastocysts flushed from
the uteri with Krebs Ringer bicarbonate containing 0-1 I% glucose (KRBG) warmed at 370C. KRBG contains (mM): NaCl, 119; KCl, 4-74; CaCl2, 1-71; KH2PO4, 1-19; MgSO4. 7H20, 1-19; NaHCO3, 25; and glucose, 5-55. The embryos were then incubated in a modified F10 medium (MF10) (Van Blerkom & Manes, 1974) until transfer to experimental media. The modified medium (MF10) consisted of Ham medium (Microbiological Associates) buffered with 0-02 N-2-hydroxyethylpiperazine-N'-2-ethane sulphonic acid (HEPES, Calbiochem) adjusted to pH 7-4 with N-NaOH and containing 20% foetal calf serum (Microbiological Associates). Rabbit embryos cultured from the two-cell stage to the blastocyst stage in medium supplemented with 20 % foetal calf serum are morphologically indistinguishable from freshly collected embryos at the electron microscopic level (Van Blerkom, Manes & Daniel, 1973) and show quantitative patterns of protein synthesis identical to freshly collected embryos as measured using sodium dodecyl sulphate polyacrylamide gel electrophoresis (Van Blerkom. & Manes, 1974). Furthermore, 5-day rabbit blastocysts cultured in vitro for 8-16 hr in medium supplemented with serum can be subsequently transferred to suitable recipient does and readily develop into neonates (Staples, 1967). It is thus assumed that if significant transtrophectoderm electrolyte gradients are required for normal rabbit blastocyst development, the diffusion and transport processes establishing such gradients would be apparent in blastocysts incubated in vitro under similar conditions. The embryos were incubated in 1-2 ml. medium in Falcon no. 3033 tissue culture tubes at 37-50C in a high humidity atmosphere consisting of 5 % C02 in air.
F1O
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Effect of external K concentration
on blastocoele fluid The effect of external K concentration on 6-day p.c. rabbit blastocysts was determined by incubating the blastocysts in normal medium containing 6-9 mm-K or MF10 medium in which the K concentration had been raised to 16-9 mm with KCL. Twenty mm sucrose was added to the control unsupplemented medium, to equalize the osmolalities of both media. In the first experiment the blastocysts were incubated for 4 hr and in the second for 12 hr. Five rabbits were used in each experiment, the blastocysts from each rabbit providing one replicate. Since the number of blastocysts recovered varied from rabbit to rabbit, the number of blastocysts allotted to each treatment varied between replicates. The diameter of each blastocyst was measured at the beginning and end of the incubation period using a calibrated eye-piece graticule, and the
MF10
TRANSPORT BY RABBIT BLASTOCYSTS
321
volumes calculated assuming sphericity of the embryos. At the end of the incubation period samples of blastocoele fluid from each blastocyst were analysed for Na, Cl, K, Ca, Mg, S and P with the electron probe.
Effect of ouabain on blawtocoele fluid The volume and elemental composition of the blastocoele fluid in blastocysts from rabbits 6 days p.c. was determined after incubating blastocysts in control medium without any ouabain, after exposing the juxtacoelic surface of the trophectoderm to ouabain by microinjection into the blastocoele cavity, and after exposing the abcoelic surface of the trophectoderm to the drug by adding it to the culture medium. In the control blastocysts 156 nl. MF10 medium was drawn into a siliconized, oil-filled micropuncture pipette (6-8 jsm o.d.). The pipette, connected to an air syringe, was inserted into the rabbit blastocoele under a stereomicroscope using a de Fonbrune micromanipulator. The fluid was then injected into a blastocoele and the pipette removed. The 156 nl. fluid injected into the blastocoele was less than 2 % of the volume of the blastocoele cavity. Leakage from the puncture site was minimal or absent and the site appeared to heal immediately after pipette removal. The blastocysts, exposed to ouabain in the blastocoele, were microinjected with 156 nl. serum-free medium containing 1 mM ouabain. The concentration of ouabain intially present in the ouabain injected blastocysts was calculated from the initial volume measurement of each blastocyst. In the first experiment the embryos were incubated for 4 hr. Eight blastocysts were microinjected with ouabain and contained an initial ouabain concentration of 15-4 .!Sm, standard deviation 4-53. Those blastocysts exposed to ouabain externally were microinjected with 156 nl. serum-free medium and then incubated in a medium containing 10 /Im of the drug. Twelve blastocysts injected with ouabain and incubated for 12 hr contained an initial mean ouabain concentration of 12-9 FM, standard deviation 6-1. Five rabbits were used in each experiment, the blastocysts from each rabbit providing one replicate. The number of blastocysts allotted to each of the three treatments varied between rabbits. The diameter of each blastocyst was measured at the beginning and end of the incubation period and the volumes calculated. At the end of the incubation period samples of blastocoele fluid were obtained from each blastocyst and analysed for Na, Cl, K, Ca, Mg, S and P with the electron probe.
Collection of blawtocoele fluid At the end of the incubation periods the blastocysts were micropunctured according to the techniques of Borland et al. (1976) and the samples stored frozen under mineral oil until sample preparation for electron probe analysis.
Sample preparation and electron probe microanalyaia The techniques of sample preparation and electron probe microanalysis of picolitre samples have been previously described (see Lechene & Warner, 1977, for a review; Borland et al. 1976, 1977a). Briefly, the unknown solutions and a series of standard solutions were placed with calibrated picolitre volumetric pipettes (20-80 pl.) onto the surface of a beryllium support covered with mineral oil. The pipettes were made on a de Fonbrune microforge and calibrated with tritiated water. The oil on the beryllium block was removed and the sample preparation freeze dried under vacuum (1.33 mPa, -70 0C). The freeze dried microdroplets were analysed for Na, Cl, K, Ca, Mg, S and P using a Cameca MS/46 microprobe with an automated stage and interfaced with a Hewlett-Packard 2100A computer and Tektronix 4012 display terminal (Moher & Lechene, 1975). X-ray emissions from Na and Mg were analysed using potassium acid phthalate diffracting crystals and emissions from P, S, Cl, K and Ca were analysed using pentaerythritol diffracting crystals. The concentrations of the seven elements in blastocoele fluid were determined with respect to the counts obtained from identical volumes of fluid containing known concentrations of the seven elements. These calculations were performed with the HewlettPackard computer and Tektronix display terminal.
II
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322
J. D. BIGGERS, R. M. BORLAND AND C. P. LECHENE
Statietical analy8e8 Each experiment was analysed for initial and final volumes, changes in volume, and for each element assuming an unbalanced two-way crossed classification with interaction using the computational procedures described by Searle (1971). All computations were done on a timesharing system connected to a Hewlett-Packard 4000 computer. In unbalanced designs a nonsignificant interaction mean square in the analysis of variance does not necessarily imply that interaction effects are negligible. Since in the present data the interaction mean square was considerably larger than the residual mean square in many cases, the interaction mean squares have been used throughout to provide conservative estimates of error and significance tests. The probabilities shown in Tables 1 and 2 were calculated from the t values for the difference between unpaired means. The probabilities shown in Tables 3, 4 and 5 were calculated from F values using the formula for testing the homogeneity of means given by Searle (1971)(formula (130), p. 326). RESULTS
Effect of external K concentration on bla8tocoele fluid The mean initial, mean final and mean increase in volume after 4 and 12 hr incubation in control and high K media are shown in Tables 1 and 2. There was no significant effect of K concentration in the external medium on the volume of fluid accumulated. After 4 hr the volume increased on average by 4-2 /1d., S.E. of mean 1-98, d.f. 9, and after 12 hr by 12-5 Asl., S.E. of mean 2-48, d.f. 9. The mean concentrations of Na, Cl and K in the blastocoele fluids after 4 and 12 hr incubation are also shown in Tables 1 and 2. Analyses of variance showed that no significant differences in the concentrations of Na and Cl developed as the result of incubation in the two media. Other results, not shown, also fail to demonstrate any effects of external K on the concentrations of Ca, Mg, S and P. The concentration of K in the medium, however, did slightly increase the K concentration in the blastocoele fluid after both 4 and 12 hr incubations. Raising the external concentration of K by 10 mm increased the concentration of K in the blastocoele fluid by 1-9 mM, S.E. of mean 1-2, d.f. 9 after 4 hr incubation, and 2-3 mm, s.E. of mean 1-8, d.f. 9 after 12 hr incubation. The increases, which are not statistically significant, are relatively small compared with the gradient created experimentally across the trophectoderm. Effect of ouabain on the blastocoele fluid The initial and final volumes, and the increase in volume of blastocysts cultured for 4 hr in control medium and in the presence of internally and externally administered ouabain are shown in Table 3. The control blastocysts accumulated 3-3 ,ul. fluid. Blastocysts exposed to ouabain on the abcoelic surface of the trophectoderm accumulated only an average of 0-4 pl. fluid, while the ouabain injected blastocysts decreased in volume. Despite the large trends the differences just fail to reach statistical significance because of the large sampling errors. The mean concentrations of Na, Cl, K, Ca, Mg, S and P in the blastocoele fluid after incubation for 4 hr in control medium and in the presence of internally and externally administered ouabain are shown in Table 4. Analyses of variance show that ouabain administered by the juxtacoelic route produced significant changes in the composition of blastocoele fluid: K and Ca concentrations increased, while P concentration decreased. The concentrations of Na, Cl, Mg and S did not change significantly. Blastocysts exposed to ouabain via the abcoelic route experienced a
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J. D. BIGGERS, R. M. BORLAND AND C. P. LECHENE significant decrease in P. Na, Cl, K, Ca, Mg and S concentrations are not significantly changed by the treatment. The mean concentrations of Na, Cl, K, Ca, Mg, S and P in the blastocoele fluid after incubation for 12 hours in control medium and in the presence of internally and externally administered ouabain are shown in Table 5. Analyses of variance show that ouabain administered by the juxtacoelic route produced significant changes in the composition of blastocoele fluid: K, Ca and Mg concentrations increased while P decreased. Na and Cl concentrations also decreased but both changes are only of borderline statistical significance. S concentration did not significantly change. Blastocysts exposed to ouabain via the abcoelic route experienced a significant increase in blastocoelic fluid Ca and S and a decrease in P. Na, Cl, K and Mg concentrations were not significantly changed by the treatment.
324
TABLE 3. The mean initial and final volumes and increases in volume of 6-day p.c. rabbit blastocysts cultured for 4 hr in control medium without ouabain, ouabain injected into the blastocoele cavity (juxtacoelic route) and ouabain in the medium (abcoelic route). All S.E. of mean have been estimated from the interaction mean squares with 14 degrees of freedom. The probabilities have been calculated from F-tests with 2 and 14 degrees of freedom, respectively Increase Initial volume Final volume in volume No. Route of ouabain (gl.) administration blastocysts (Q1.) (A1.) 7 3-3±2-0 15-1±3-3 None (control)*t 11-8+2±0 8-4 ± 3-0 -1-2 + 1-8 9-6 ± 1-8 9 Juxtacoelic (15-4 IM)t 12-8± 3-1 0-4+1 9 8 12-4±1-8 Abcoelic (10 #M)t Probability, all means 0-514 are equal 0-102 0-334 * Modified F10 supplemented with 20 % calf serum. t Microinjected with 156 nl. serum-free MF1O. t Microinjected with 156 nl. serum free MF1O containing 1 mm ouabain; final concentration
of ouabain estimated from the initial volume estimate of each blastocyst. DISCUSSION
Gradient of K across the trophectoderm Blastocysts incubated in media containing either 6-9 or 16-9 mM-K accumulate the same amount of blastocoele fluid (Tables 1 and 2) and appear grossly morphologically normal. Samples of these blastocoele fluids were analysed for their concentrations of Na, Cl, K, Ca, Mg, S and P (Tables 1 and 2). Only the concentration of K was slightly affected by supplementing the bathing medium with K. When the external concentration of K was 6-9 mm, the K concentration in the blastocoele fluid was approximately the same. In contrast, when the external concentration of Kwas 16-9 mm the K concentration was higher but only to a small extent (K gradient 7 mm). These results are quantitatively consistent with the previous observations of Petzoldt (1971), demonstrating that the concentration of K in rabbit blastocoele fluid in vivo is less than that in uterine fluid. Thus, the rabbit trophectoderm can maintain a significant K gradient both in vitro and in vivo.
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Influx of K into the blastocoele cavity Diffusion of K from uterine fluid into the blastocoele through the membrane junctions and lateral intercellular spaces is probably very limited for several reasons. The trophectoderm of the rabbit blastocyst which is a simple squamous epithelium has been reported to have a higher electrical resistance ( > 2500 Q cm2) (Cross & Brinster, 1970; Powers, Borland & Biggers, 1977) than the extremely tight frog skin with a resistance of 2000 Q cm2 (Ussing & Zerahn, 1951), where conductance through the intercellular spaces is less than 40 % of the total conductance across the epithelium (Ussing & Windhager, 1964). Furthermore, the rabbit blastocyst can generate and maintain large Na and Cl concentration differences across the trophectoderm when the embryo is incubated in medium containing the impermeant sucrose (Borland et al. 1976). Also ouabain injected blastocysts are capable of maintaining large K concentration differences across the trophectoderm (Tables 4 and 5). K accumulation in the blastocoele must therefore occur largely by a two step process: K movement into the trophoblast cell from the bathing medium and from the trophoblast cell into the blastocoele. Each step would be by active transport if K moves against its electrochemical gradient. The electrical gradients across the trophoblast have been measured in 5-, 6- and 7-day p.c. rabbit blastocysts incubated in MF1O medium (Powers et al. 1977). The membrane potential of the trophoblast cell is - 46-7 mV (cell interior negative to medium) on days 6-7. The transtrophectodermal potential difference in 4- and 5-day p.c. blastocysts is negative (- 3-1 and - 44 mV, respectively), but changes polarity and increases in magnitude to + 14 5 mV by 6-5 days p.c. and reaches + 21-0 mV on day 7. Movement of K into the trophoblast cell at all stages is, therefore, down an electrical gradient, while K movement into the blastocoele from the cell is up an electrical gradient. The movement of K into the trophoblast cell is probably also up a concentration gradient, and the movement into the blastocoele cavity down a concentration gradient. The K activities inside the trophoblast cell, however, have not been measured and our conclusions about the mechanism of K transport into or out of the trophoblast cell assumes that the intercellular K concentration is higher than the extracellular levels. Recent histochemical studies have demonstrated Na+-K+ ATPase activity on both the abcoelic and juxtacoelic surfaces of the rabbit trophectoderm on the 6th day p.c. (Moon Kim & J. D. Biggers, unpublished; reviewed by Borland, 1977). The presence of this enzyme on both surfaces of the trophoblast cell could suggest that K is actively transported up its concentration gradient into the cell. K movement into the blastocoele from the trophoblast cell may occur by either a passive or an active process. Borland et al. (1976) obtained evidence that argues against active K transport into the blastocoele. Rabbit blastocysts were incubated in culture medium supplemented with increasing concentrations of sucrose, to which the trophectoderm is relatively impermeable. Up to a sucrose concentration of 80 mM significant blastocyst expansion continued, although at a diminishing rate. For the expansion to occur against the osmotic gradient induced by sucrose, the various substances that are actively transported into the blastocoele fluid must increase the osmolality of the blastocoele fluid above that of the external medium. In these experiments only the concentrations of Na and Cl in the blastocoele fluid increased
TRANSPORT BY RABBIT BLASTOCYSTS 327 to a major extent. In contrast, blastocoele fluid K concentrations were not affected by the presence of sucrose in the medium. The data, therefore, suggest that K movement from the trophoblast cell into the blastocoele is via passive, rather than active processes. Blastocoele
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Fig. 1. A, B, C. A hypothetical model demonstrating the possible relationship between Na+-K+ ATPase and rabbit trophectodermal Na and K transport. In Fig. 1 A, Na+-K+ ATPase is shown to be present on both the juxtacoelic (blastocoelic) and abcoelic surfaces ofthe trophoblast cell. The trophectoderm is exposed to ouabain on the juxtacoelic surface (Fig. 1 B) and on the abcoelic surface (Fig. 1C). Changes in the concentrations of elements are indicated by upward and downward facing arrows.
328
J. D. BIGGERS, R. M. BORLAND AND C. P. LECHENE
Efflux of K from the blastocoele cavity Assuming that the Na+-K+ ATPase present on both the abcoelic and juxtacoelic surfaces of the trophoblast cells is necessary for homeostasis by transporting Na+ and K+ across the plasma membrane, as in a large number of cells (Katz & Epstein, 1968), a model for these normal relationships can be proposed (Fig. 1 A). A constant leakage of K from the trophoblast cells is shown as the predominant mode of K influx into the blastocoele. Both abcoelic and juxtacoelic Na+-K+ ATPase (Na pumps) are presumed to regulate the Na and K levels of intracellular fluid. In addition to their role in cellular homeostasis, sodium pumps on the juxtacoelic surface are shown to influence blastocoele fluid K levels by pumping K from the blastocoele into the intracellular space with the co-ordinant pumping of Na from the intracellular space into the blastocoele. Fig. 1 B and C show the predicted response of blastocysts to ouabain on their juxtacoelic and abcoelic surfaces if it is assumed that the membrane junctions and intercellular spaces are highly impermeable to K, and that the Na+-K+ ATPase activity that is located on both surfaces of the trophoblast cell is inhibited by ouabain. If the basic assumptions of the model are correct juxtacoelic ouabain should cause blastocoele K levels to increase and Na levels to decrease. The results presented in Table 4 show that after 4 hr incubation the blastocoelic K concentration is elevated by 5.30 mM, S.E. of mean 2-01, P = 0020 while the concentration of Na is unaffected. After 12 hr incubation the blastocoelic K concentration is elevated by 13-86 mM, S.E. of mean 5'78, P = 0031. The blastocoelic concentration of Na is also lowered by 16 mM, S.E. of mean 7-81, P = 0060 and the Cl concentration by 10 mM, S.E. of mean 5-66, P = 0099. Thus the predictions of model 1B are confirmed, thereby providing strong evidence that a ouabain-sensitive transport system, presumably Na+-K+ ATPase, located on the juxtacoelic surface of the trophoblast cells, is involved in the regulation of blastocoele K levels. The model shown in Fig. 1C predicts that abcoelic ouabain may increase and decrease the concentrations of Na and K, respectively in the trophoblast cells. A decrease in intracellular K could then, in turn, decrease the K influx into the blastocoele. An increase in intracellular sodium could also stimulate the juxtacoelic Na/K pumps, thus producing a compensating rise in cell K concentration and an increment in blastocoele fluid Na concentration. The model thus predicts that blastocoele fluid K levels should fall if abcoelic Na/K pumps exist and are inhibited by ouabain. The results presented in Tables 4 and 5 fail to demonstrate that abcoelic ouabain causes a significant fall in the K concentration in the blastocoele fluid. Possibly the rate of leakage of K through the abcoelic surface of the trophoblast cell is too slow for a significant drop in K concentration to occur during the experimental period.
Ouabain-sensitive fluid transport The primary role of Na and Cl in the transport of fluid across the trophectoderm has been demonstrated by studies on the effect of lowering the chemical activity of water in the bathing medium below that of the blastocoele fluid with sucrose (Borland et al. 1976). Active NaCl transport into the blastocoele causes water to move passively. Rabbit blastocoele fluid is nearly isotonic to medium even when the medium osmol-
TRANSPORT BY RABBIT BLASTOCYSTS 329 ality is varied from 230 to 370 m-osmole by varying the NaCl concentration (Borland, Biggers & Lechene, 1977b). These results are consistent with blastocyst expansion occurring by local osmosis and fluid transport into closed channels, such as the intercellular spaces of the trophectoderm. If local osmosis in forward-facing channels does occur, the active transport sites should line these channels and be accessible to pump inhibitors that are microinjected into the blastocoele. The Na+K+ ATPase on the juxtacoelic surface of the trophectoderm, particularly the enzyme located on membranes lining the lateral intercellular channels, could be the site of Na+ transport and concomitant fluid movement. This would be true if the Na/K pump ratio is greater than one and is therefore rheogenic. A rheogenic Na+ transport system that is amiloride sensitive is acquired by the 6-7 day p.c. rabbit blastocyst (Powers et al. 1977), and may be related to the Na/K pumps demonstrated by the present studies. As shown in Table 3, after 4 hr incubations, ouabain injected blastocysts decreased in volume, while control blastocysts and blastocysts exposed to ouabain on the abcoelic surface of the trophectoderm both accumulated fluid. These large trends though not quite statistically significantly different (P = 0*107) strongly suggest that the activity of juxtacoelic ouabain-sensitive transport sites is required for blastocoele fluid accumulation. The hydrostatic pressure of blastocoele fluid in 6-day p.c. rabbit blastocysts is approximately 10 mmHg (T. H. Strunk, R. M. Borland, J. D. Biggers and C. P. Lechene, unpublished). This positive blastocoele pressure could cause fluid loss from the cavity if transtrophectoderm Na transport and concomitant passive water movement into the blastocoele is appreciably blocked by juxtacoelic ouabain. The concentrations of juxtacoelic ouabain used in these experiments may not have been sufficient to completely inhibit Na and water transport. The trends suggest, however, that the effect of ouabain on fluid accumulation by the rabbit blastocyst is similar to the effect of this drug on amphibian blastulas in which ouabain in the medium does not affect blastula formation, whereas ouabain injected into the blastocoele causes blastula collapse (Slack & Warner, 1973). Thus, juxtacoelic Na pumps in the blastocyst are probably important in both transepithelial ion transport and fluid movement. The mechanisms involved appear to be similar to those already demonstrated in the kidney (Katz & Epstein, 1967; Torretti, Hendler, Weinstein, Longnecker & Epstein, 1972; Silva, Hayslett & Epstein, 1973), toad bladder (Herrera, 1966), and rabbit ileum (Schultz & Zalusky, 1964). The work reported in this paper has been supported by grants from the Ford Foundation (720-0369), the Rockefeller Foundation (RF-65040), the National Institutes of Health (RO1HL-15552-03 and PO 7-RR00679-02) and the National Institute of Child Health and Human Development (HD-06919-O1A1). Dr Borland performed the experiments reported here while supported by a post-doctoral fellowship from the Ford Foundation Program. We are indebted to Dr Eddington Y. Lee for help with the preparation of the programmes used to analyse the data. A preliminary account of these results has been given by Borland (1977).
330
J. D. BIGGERS, R. M. BORLAND AND C. P. LECHENE
REFERENCES BORLAND, R. M. (1977). Transport processes in the mammalian blastocyst. In Development in Mammals, vol. 1, ed. JOHNSON, M. H., pp. 31-69. Elsevier-North Holland Biomedical Press. BORLAND, R. M., BIGGERS, J. D. & LECHENE, C. P. (1976). Kinetic aspects of rabbit blastocoele fluid accumulation: an application of electron probe microanalysis. Devl Biol. 50, 201-211. BORLAND, R. M., BIGGERS, J. D. & LECHENE, C. P. (1977a). Studies on the composition and formation of mouse blastocoele fluid using electron probe microanlysis. Devl Biol. 55, 1-8. BORLAND, R. M., BIGGERS, J. D. & LECHENE, C. P. (1977b). Fluid transport by rabbit preimplantation blastocysts in vitro. J. Reprod. Fert. 51, 131-135. CRoss, M. H. & BRINSTER, R. L. (1970). Influence of ions, inhibitors and anoxia on transtrophoblast potential of rabbit blastocyst. Expl Cell Res. 62, 303-309. GAMOW, E. & DANIEL, J. C. (1970). Fluid transport in the rabbit blastocyst. Wilhelm Roux Arch. EntwMech. Org. 164, 261-278. HERRERA, F. C. (1966). Action of ouabain on sodium transport in the toad urinary bladder. Am. J. Physiol. 210, 980-986. KATZ, A. I. & EPSTEIN, F. H. (1967). The role of sodium-potassium-activated adenosine triphosphatase in the reabsorption of sodium by kidney. J. clin. Inve8t. 46, 1999-2011. KATZ, A. I. & EPSTEIN, F. H. (1968). Physiologic role of Na-K ATPase in the transport of cations across biologic membranes. New Engl. J. Med. 278, 253-261. LECHENE, C. P. & WARNER, R. R. (1977). Ultramicroanalysis: X-ray spectrometry by electron probe excitation. A. Rev. Biophys. Bioeng. 6, 57-85. MOHER, T. & LECHENE, C. (1975). Automated electron probe analysis of biological samples. Process control and data reduction by interactive graphic display. Biosci. Commun. 1, 314-329. PETZOLDT, V. U. (1971). Untersuchung uber das anorganische Milieu in Uterus und Blastozyste des Kaninchens. Zool. Jb. Physiol. 75, S 547-593. PowERs, R. D., BORLAND, R. M. & BIGGERS, J. D. (1977). Acquisition of amiloride-sensitive, rheogenic Na+ transport in the rabbit blastocyst. Nature, Lond. 270, 603-604. SCHULTZ, S. G. & ZALUsKY, R. (1964). Ion transport in isolated rabbit ileum. I. Short-circuit current and Na fluxes. J. gen. Physiol. 47, 567-584. SEARLE, S. R. (1971). Linear Models. New York: Wiley. SILVA, P., HAYSLETT, J. P. & EPSTEIN, F. H. (1973). The role of Na-K activated adenosine triphosphatase in potassium adaptation. Stimulation of enzymatic activity by potassium loading. J. clin. Invest. 52, 2665-2671. SLACK, C. & WARNER, A. E. (1973). Intracellular and intercellular potentials in the early amphibian embryo. J. Physiol. 232, 313-330. SMITH, M. W. (1970). Active transport in the rabbit blastocyst. Experentia 26, 736-738. STAPLES, R. E. (1967). Development of 5 day rabbit blastocysts after culture at 37 'C. J. Reprod. Fert. 13, 369-372. ToRREErI, J., HENDLER, E., WEINSTEIN, E., LONGNECKER, R. E. & EPSTEIN, F. H. (1972). Functional significance of Na-K-ATPase in the kidney: effects of ouabain inhibition. Am. J. Physiol. 222, 1398-1405. UssrNG, H. H. & WINDHAGER, E. E. (1964). Nature of shunt path and active sodium transport path through frog skin epithelium. Acta physiol. 8cand. 61, 484-504. USSING, H. H. & ZERAHN, K. (1951). Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta physiol. 8cand. 23, 110-127. VAN BLERKOM, J. & MANES, C. (1974). Development of preimplantation rabbit embryos. II. A comparison of qualitative aspects of protein synthesis. Devl Biol. 40, 40-51. VAN BLERKOM, J., MANEs, C. & DANIEL, J. C. (1973). Development of preimplantation rabbit embryos in vivo. I. An ultrastructural comparison. Devl Biol. 35, 262-282.
319
OUABAIN-SENSITIVE FLUID ACCUMULATION AND ION TRANSPORT BY RABBIT BLASTOCYSTS By JOHN D. BIGGERS, RAYMOND M. BORLAND AND CLAUDE P. LECHENE From the Laboratory of Human Reproduction and Reproductive Biology and Biotechnology Resource in Electron Probe Microanalysis, Department of Physiology, Harvard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115, U.S.A.
(Received 9 November 1977) SUMMARY
1. By incubating 6-day post-coitum (p.c.) blastocysts in medium supplemented with either 20 mm-sucrose or 10 mM-KCl it was possible to show that the trophectoderm of the blastocyst prevents the concentration of K in the blastocoele fluid rising to external concentrations. 2. The concentrations of K in the blastocoele fluid are maintained predominantly by leakage of K from the trophoblast cells into the blastocoele and by ouabainsensitive transport of K into the trophoblast cells from the blastocoele fluid. 3. Exposure of blastocysts to ouabain on the juxtacoelic, but not abcoelic, surface of the trophectoderm may inhibit blastocoele fluid accumulation. INTRODUCTION
Borland, Biggers & Lechene (1976) showed that K accumulates in the blastocoele fluid of the rabbit, and that its rate of accumulation increases from 5 to 31 nm cm-2 hr-1 between the 4th and 6th day of development. Petzoldt (1971), using K+ selective electrodes, however, found that the K+ activities in the blastocoele fluid and uterine fluid of rabbits 6 or 7 days pregnant were 10-12 mm and 20 mm respectively. Thus, although there is a net transfer of K into the blastocoele cavity, mechanisms prevent its concentration from rising to that found in the external uterine fluid. At least three mechanisms could limit the accumulation of K in rabbit blastocoele fluid: 1. Limited diffusion of K from uterine fluid into the blastocoele through the trophoblast junctional complexes and intercellular spaces. 2. Limited leakage and transport of K from the trophoblast cells into the blastocoele fluid. 3. Significant leakage of K into the blastocoele cavity coupled with the active transport of K from the cavity back into the trophoblast cells. Borland et al. (1976) also showed that 6-day rabbit blastocysts in vitro respond to a lowering of the chemical potential of water in the external medium by an increase in the concentrations of Na and Cl in blastocoele fluid, and by the continued accumulation of fluid with no change in the concentration of K. The accumulation of Na,
J. D. BIGGERS, R. M. BORLAND AND C. P. LECHENE 320 C1 and water by the rabbit blastocyst appears to be regulated, in part by Na+-K+ ATPase (E.C. 3.6. 1.3) since these processes are inhibited by ouabain in the external medium (Smith, 1970; Gamow & Daniel, 1970). However, in these experiments the concentrations of ouabain that were used were high, and no attempt was made to differentiate between abcoelic and juxtacoelic effects of this steroidal glycoside. Using X-ray spectrometry by electron probe excitation, we have estimated the concentrations of Na, Cl and K in picolitre samples of blastocoele fluid after exposing the blastocyst to ouabain in the external medium (abcoelic ouabain exposure) or in the blastocoele fluid (juxtacoelic ouabain exposure), and have demonstrated that K is actively transported out of the blastocoele cavity. From these experiments and other evidence analysed in the Discussion it is suggested that K accumulates in the blastocoele cavity by leakage from the trophoblast cells, but is prevented from rising to high levels by being actively transported back into the cells by pumps located on the juxtacoelic surface of the trophectoderm. The concomitant transport of Na into the blastocoele is responsible for fluid accumulation in the blastocyst. METHODS
Collection of blastocyst8 Eight to ten lb New Zealand white rabbits were naturally mated to mature males. Ovulation occurs approximately 10 hr after coitus. At 6 days post-coitum (p.c.) the female rabbits were sacrificed by chloroform anaesthesia. The uterine horns were cannulated and the blastocysts flushed from
the uteri with Krebs Ringer bicarbonate containing 0-1 I% glucose (KRBG) warmed at 370C. KRBG contains (mM): NaCl, 119; KCl, 4-74; CaCl2, 1-71; KH2PO4, 1-19; MgSO4. 7H20, 1-19; NaHCO3, 25; and glucose, 5-55. The embryos were then incubated in a modified F10 medium (MF10) (Van Blerkom & Manes, 1974) until transfer to experimental media. The modified medium (MF10) consisted of Ham medium (Microbiological Associates) buffered with 0-02 N-2-hydroxyethylpiperazine-N'-2-ethane sulphonic acid (HEPES, Calbiochem) adjusted to pH 7-4 with N-NaOH and containing 20% foetal calf serum (Microbiological Associates). Rabbit embryos cultured from the two-cell stage to the blastocyst stage in medium supplemented with 20 % foetal calf serum are morphologically indistinguishable from freshly collected embryos at the electron microscopic level (Van Blerkom, Manes & Daniel, 1973) and show quantitative patterns of protein synthesis identical to freshly collected embryos as measured using sodium dodecyl sulphate polyacrylamide gel electrophoresis (Van Blerkom. & Manes, 1974). Furthermore, 5-day rabbit blastocysts cultured in vitro for 8-16 hr in medium supplemented with serum can be subsequently transferred to suitable recipient does and readily develop into neonates (Staples, 1967). It is thus assumed that if significant transtrophectoderm electrolyte gradients are required for normal rabbit blastocyst development, the diffusion and transport processes establishing such gradients would be apparent in blastocysts incubated in vitro under similar conditions. The embryos were incubated in 1-2 ml. medium in Falcon no. 3033 tissue culture tubes at 37-50C in a high humidity atmosphere consisting of 5 % C02 in air.
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Effect of external K concentration
on blastocoele fluid The effect of external K concentration on 6-day p.c. rabbit blastocysts was determined by incubating the blastocysts in normal medium containing 6-9 mm-K or MF10 medium in which the K concentration had been raised to 16-9 mm with KCL. Twenty mm sucrose was added to the control unsupplemented medium, to equalize the osmolalities of both media. In the first experiment the blastocysts were incubated for 4 hr and in the second for 12 hr. Five rabbits were used in each experiment, the blastocysts from each rabbit providing one replicate. Since the number of blastocysts recovered varied from rabbit to rabbit, the number of blastocysts allotted to each treatment varied between replicates. The diameter of each blastocyst was measured at the beginning and end of the incubation period using a calibrated eye-piece graticule, and the
MF10
TRANSPORT BY RABBIT BLASTOCYSTS
321
volumes calculated assuming sphericity of the embryos. At the end of the incubation period samples of blastocoele fluid from each blastocyst were analysed for Na, Cl, K, Ca, Mg, S and P with the electron probe.
Effect of ouabain on blawtocoele fluid The volume and elemental composition of the blastocoele fluid in blastocysts from rabbits 6 days p.c. was determined after incubating blastocysts in control medium without any ouabain, after exposing the juxtacoelic surface of the trophectoderm to ouabain by microinjection into the blastocoele cavity, and after exposing the abcoelic surface of the trophectoderm to the drug by adding it to the culture medium. In the control blastocysts 156 nl. MF10 medium was drawn into a siliconized, oil-filled micropuncture pipette (6-8 jsm o.d.). The pipette, connected to an air syringe, was inserted into the rabbit blastocoele under a stereomicroscope using a de Fonbrune micromanipulator. The fluid was then injected into a blastocoele and the pipette removed. The 156 nl. fluid injected into the blastocoele was less than 2 % of the volume of the blastocoele cavity. Leakage from the puncture site was minimal or absent and the site appeared to heal immediately after pipette removal. The blastocysts, exposed to ouabain in the blastocoele, were microinjected with 156 nl. serum-free medium containing 1 mM ouabain. The concentration of ouabain intially present in the ouabain injected blastocysts was calculated from the initial volume measurement of each blastocyst. In the first experiment the embryos were incubated for 4 hr. Eight blastocysts were microinjected with ouabain and contained an initial ouabain concentration of 15-4 .!Sm, standard deviation 4-53. Those blastocysts exposed to ouabain externally were microinjected with 156 nl. serum-free medium and then incubated in a medium containing 10 /Im of the drug. Twelve blastocysts injected with ouabain and incubated for 12 hr contained an initial mean ouabain concentration of 12-9 FM, standard deviation 6-1. Five rabbits were used in each experiment, the blastocysts from each rabbit providing one replicate. The number of blastocysts allotted to each of the three treatments varied between rabbits. The diameter of each blastocyst was measured at the beginning and end of the incubation period and the volumes calculated. At the end of the incubation period samples of blastocoele fluid were obtained from each blastocyst and analysed for Na, Cl, K, Ca, Mg, S and P with the electron probe.
Collection of blawtocoele fluid At the end of the incubation periods the blastocysts were micropunctured according to the techniques of Borland et al. (1976) and the samples stored frozen under mineral oil until sample preparation for electron probe analysis.
Sample preparation and electron probe microanalyaia The techniques of sample preparation and electron probe microanalysis of picolitre samples have been previously described (see Lechene & Warner, 1977, for a review; Borland et al. 1976, 1977a). Briefly, the unknown solutions and a series of standard solutions were placed with calibrated picolitre volumetric pipettes (20-80 pl.) onto the surface of a beryllium support covered with mineral oil. The pipettes were made on a de Fonbrune microforge and calibrated with tritiated water. The oil on the beryllium block was removed and the sample preparation freeze dried under vacuum (1.33 mPa, -70 0C). The freeze dried microdroplets were analysed for Na, Cl, K, Ca, Mg, S and P using a Cameca MS/46 microprobe with an automated stage and interfaced with a Hewlett-Packard 2100A computer and Tektronix 4012 display terminal (Moher & Lechene, 1975). X-ray emissions from Na and Mg were analysed using potassium acid phthalate diffracting crystals and emissions from P, S, Cl, K and Ca were analysed using pentaerythritol diffracting crystals. The concentrations of the seven elements in blastocoele fluid were determined with respect to the counts obtained from identical volumes of fluid containing known concentrations of the seven elements. These calculations were performed with the HewlettPackard computer and Tektronix display terminal.
II
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322
J. D. BIGGERS, R. M. BORLAND AND C. P. LECHENE
Statietical analy8e8 Each experiment was analysed for initial and final volumes, changes in volume, and for each element assuming an unbalanced two-way crossed classification with interaction using the computational procedures described by Searle (1971). All computations were done on a timesharing system connected to a Hewlett-Packard 4000 computer. In unbalanced designs a nonsignificant interaction mean square in the analysis of variance does not necessarily imply that interaction effects are negligible. Since in the present data the interaction mean square was considerably larger than the residual mean square in many cases, the interaction mean squares have been used throughout to provide conservative estimates of error and significance tests. The probabilities shown in Tables 1 and 2 were calculated from the t values for the difference between unpaired means. The probabilities shown in Tables 3, 4 and 5 were calculated from F values using the formula for testing the homogeneity of means given by Searle (1971)(formula (130), p. 326). RESULTS
Effect of external K concentration on bla8tocoele fluid The mean initial, mean final and mean increase in volume after 4 and 12 hr incubation in control and high K media are shown in Tables 1 and 2. There was no significant effect of K concentration in the external medium on the volume of fluid accumulated. After 4 hr the volume increased on average by 4-2 /1d., S.E. of mean 1-98, d.f. 9, and after 12 hr by 12-5 Asl., S.E. of mean 2-48, d.f. 9. The mean concentrations of Na, Cl and K in the blastocoele fluids after 4 and 12 hr incubation are also shown in Tables 1 and 2. Analyses of variance showed that no significant differences in the concentrations of Na and Cl developed as the result of incubation in the two media. Other results, not shown, also fail to demonstrate any effects of external K on the concentrations of Ca, Mg, S and P. The concentration of K in the medium, however, did slightly increase the K concentration in the blastocoele fluid after both 4 and 12 hr incubations. Raising the external concentration of K by 10 mm increased the concentration of K in the blastocoele fluid by 1-9 mM, S.E. of mean 1-2, d.f. 9 after 4 hr incubation, and 2-3 mm, s.E. of mean 1-8, d.f. 9 after 12 hr incubation. The increases, which are not statistically significant, are relatively small compared with the gradient created experimentally across the trophectoderm. Effect of ouabain on the blastocoele fluid The initial and final volumes, and the increase in volume of blastocysts cultured for 4 hr in control medium and in the presence of internally and externally administered ouabain are shown in Table 3. The control blastocysts accumulated 3-3 ,ul. fluid. Blastocysts exposed to ouabain on the abcoelic surface of the trophectoderm accumulated only an average of 0-4 pl. fluid, while the ouabain injected blastocysts decreased in volume. Despite the large trends the differences just fail to reach statistical significance because of the large sampling errors. The mean concentrations of Na, Cl, K, Ca, Mg, S and P in the blastocoele fluid after incubation for 4 hr in control medium and in the presence of internally and externally administered ouabain are shown in Table 4. Analyses of variance show that ouabain administered by the juxtacoelic route produced significant changes in the composition of blastocoele fluid: K and Ca concentrations increased, while P concentration decreased. The concentrations of Na, Cl, Mg and S did not change significantly. Blastocysts exposed to ouabain via the abcoelic route experienced a
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324
TABLE 3. The mean initial and final volumes and increases in volume of 6-day p.c. rabbit blastocysts cultured for 4 hr in control medium without ouabain, ouabain injected into the blastocoele cavity (juxtacoelic route) and ouabain in the medium (abcoelic route). All S.E. of mean have been estimated from the interaction mean squares with 14 degrees of freedom. The probabilities have been calculated from F-tests with 2 and 14 degrees of freedom, respectively Increase Initial volume Final volume in volume No. Route of ouabain (gl.) administration blastocysts (Q1.) (A1.) 7 3-3±2-0 15-1±3-3 None (control)*t 11-8+2±0 8-4 ± 3-0 -1-2 + 1-8 9-6 ± 1-8 9 Juxtacoelic (15-4 IM)t 12-8± 3-1 0-4+1 9 8 12-4±1-8 Abcoelic (10 #M)t Probability, all means 0-514 are equal 0-102 0-334 * Modified F10 supplemented with 20 % calf serum. t Microinjected with 156 nl. serum-free MF1O. t Microinjected with 156 nl. serum free MF1O containing 1 mm ouabain; final concentration
of ouabain estimated from the initial volume estimate of each blastocyst. DISCUSSION
Gradient of K across the trophectoderm Blastocysts incubated in media containing either 6-9 or 16-9 mM-K accumulate the same amount of blastocoele fluid (Tables 1 and 2) and appear grossly morphologically normal. Samples of these blastocoele fluids were analysed for their concentrations of Na, Cl, K, Ca, Mg, S and P (Tables 1 and 2). Only the concentration of K was slightly affected by supplementing the bathing medium with K. When the external concentration of K was 6-9 mm, the K concentration in the blastocoele fluid was approximately the same. In contrast, when the external concentration of Kwas 16-9 mm the K concentration was higher but only to a small extent (K gradient 7 mm). These results are quantitatively consistent with the previous observations of Petzoldt (1971), demonstrating that the concentration of K in rabbit blastocoele fluid in vivo is less than that in uterine fluid. Thus, the rabbit trophectoderm can maintain a significant K gradient both in vitro and in vivo.
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J. D. BIGGERS, R. M. BORLAND AND C. P. LECHENE
Influx of K into the blastocoele cavity Diffusion of K from uterine fluid into the blastocoele through the membrane junctions and lateral intercellular spaces is probably very limited for several reasons. The trophectoderm of the rabbit blastocyst which is a simple squamous epithelium has been reported to have a higher electrical resistance ( > 2500 Q cm2) (Cross & Brinster, 1970; Powers, Borland & Biggers, 1977) than the extremely tight frog skin with a resistance of 2000 Q cm2 (Ussing & Zerahn, 1951), where conductance through the intercellular spaces is less than 40 % of the total conductance across the epithelium (Ussing & Windhager, 1964). Furthermore, the rabbit blastocyst can generate and maintain large Na and Cl concentration differences across the trophectoderm when the embryo is incubated in medium containing the impermeant sucrose (Borland et al. 1976). Also ouabain injected blastocysts are capable of maintaining large K concentration differences across the trophectoderm (Tables 4 and 5). K accumulation in the blastocoele must therefore occur largely by a two step process: K movement into the trophoblast cell from the bathing medium and from the trophoblast cell into the blastocoele. Each step would be by active transport if K moves against its electrochemical gradient. The electrical gradients across the trophoblast have been measured in 5-, 6- and 7-day p.c. rabbit blastocysts incubated in MF1O medium (Powers et al. 1977). The membrane potential of the trophoblast cell is - 46-7 mV (cell interior negative to medium) on days 6-7. The transtrophectodermal potential difference in 4- and 5-day p.c. blastocysts is negative (- 3-1 and - 44 mV, respectively), but changes polarity and increases in magnitude to + 14 5 mV by 6-5 days p.c. and reaches + 21-0 mV on day 7. Movement of K into the trophoblast cell at all stages is, therefore, down an electrical gradient, while K movement into the blastocoele from the cell is up an electrical gradient. The movement of K into the trophoblast cell is probably also up a concentration gradient, and the movement into the blastocoele cavity down a concentration gradient. The K activities inside the trophoblast cell, however, have not been measured and our conclusions about the mechanism of K transport into or out of the trophoblast cell assumes that the intercellular K concentration is higher than the extracellular levels. Recent histochemical studies have demonstrated Na+-K+ ATPase activity on both the abcoelic and juxtacoelic surfaces of the rabbit trophectoderm on the 6th day p.c. (Moon Kim & J. D. Biggers, unpublished; reviewed by Borland, 1977). The presence of this enzyme on both surfaces of the trophoblast cell could suggest that K is actively transported up its concentration gradient into the cell. K movement into the blastocoele from the trophoblast cell may occur by either a passive or an active process. Borland et al. (1976) obtained evidence that argues against active K transport into the blastocoele. Rabbit blastocysts were incubated in culture medium supplemented with increasing concentrations of sucrose, to which the trophectoderm is relatively impermeable. Up to a sucrose concentration of 80 mM significant blastocyst expansion continued, although at a diminishing rate. For the expansion to occur against the osmotic gradient induced by sucrose, the various substances that are actively transported into the blastocoele fluid must increase the osmolality of the blastocoele fluid above that of the external medium. In these experiments only the concentrations of Na and Cl in the blastocoele fluid increased
TRANSPORT BY RABBIT BLASTOCYSTS 327 to a major extent. In contrast, blastocoele fluid K concentrations were not affected by the presence of sucrose in the medium. The data, therefore, suggest that K movement from the trophoblast cell into the blastocoele is via passive, rather than active processes. Blastocoele
Medium
A
B Na +
Ouabain K#
Na
C K
Ouabain -,a
Fig. 1. A, B, C. A hypothetical model demonstrating the possible relationship between Na+-K+ ATPase and rabbit trophectodermal Na and K transport. In Fig. 1 A, Na+-K+ ATPase is shown to be present on both the juxtacoelic (blastocoelic) and abcoelic surfaces ofthe trophoblast cell. The trophectoderm is exposed to ouabain on the juxtacoelic surface (Fig. 1 B) and on the abcoelic surface (Fig. 1C). Changes in the concentrations of elements are indicated by upward and downward facing arrows.
328
J. D. BIGGERS, R. M. BORLAND AND C. P. LECHENE
Efflux of K from the blastocoele cavity Assuming that the Na+-K+ ATPase present on both the abcoelic and juxtacoelic surfaces of the trophoblast cells is necessary for homeostasis by transporting Na+ and K+ across the plasma membrane, as in a large number of cells (Katz & Epstein, 1968), a model for these normal relationships can be proposed (Fig. 1 A). A constant leakage of K from the trophoblast cells is shown as the predominant mode of K influx into the blastocoele. Both abcoelic and juxtacoelic Na+-K+ ATPase (Na pumps) are presumed to regulate the Na and K levels of intracellular fluid. In addition to their role in cellular homeostasis, sodium pumps on the juxtacoelic surface are shown to influence blastocoele fluid K levels by pumping K from the blastocoele into the intracellular space with the co-ordinant pumping of Na from the intracellular space into the blastocoele. Fig. 1 B and C show the predicted response of blastocysts to ouabain on their juxtacoelic and abcoelic surfaces if it is assumed that the membrane junctions and intercellular spaces are highly impermeable to K, and that the Na+-K+ ATPase activity that is located on both surfaces of the trophoblast cell is inhibited by ouabain. If the basic assumptions of the model are correct juxtacoelic ouabain should cause blastocoele K levels to increase and Na levels to decrease. The results presented in Table 4 show that after 4 hr incubation the blastocoelic K concentration is elevated by 5.30 mM, S.E. of mean 2-01, P = 0020 while the concentration of Na is unaffected. After 12 hr incubation the blastocoelic K concentration is elevated by 13-86 mM, S.E. of mean 5'78, P = 0031. The blastocoelic concentration of Na is also lowered by 16 mM, S.E. of mean 7-81, P = 0060 and the Cl concentration by 10 mM, S.E. of mean 5-66, P = 0099. Thus the predictions of model 1B are confirmed, thereby providing strong evidence that a ouabain-sensitive transport system, presumably Na+-K+ ATPase, located on the juxtacoelic surface of the trophoblast cells, is involved in the regulation of blastocoele K levels. The model shown in Fig. 1C predicts that abcoelic ouabain may increase and decrease the concentrations of Na and K, respectively in the trophoblast cells. A decrease in intracellular K could then, in turn, decrease the K influx into the blastocoele. An increase in intracellular sodium could also stimulate the juxtacoelic Na/K pumps, thus producing a compensating rise in cell K concentration and an increment in blastocoele fluid Na concentration. The model thus predicts that blastocoele fluid K levels should fall if abcoelic Na/K pumps exist and are inhibited by ouabain. The results presented in Tables 4 and 5 fail to demonstrate that abcoelic ouabain causes a significant fall in the K concentration in the blastocoele fluid. Possibly the rate of leakage of K through the abcoelic surface of the trophoblast cell is too slow for a significant drop in K concentration to occur during the experimental period.
Ouabain-sensitive fluid transport The primary role of Na and Cl in the transport of fluid across the trophectoderm has been demonstrated by studies on the effect of lowering the chemical activity of water in the bathing medium below that of the blastocoele fluid with sucrose (Borland et al. 1976). Active NaCl transport into the blastocoele causes water to move passively. Rabbit blastocoele fluid is nearly isotonic to medium even when the medium osmol-
TRANSPORT BY RABBIT BLASTOCYSTS 329 ality is varied from 230 to 370 m-osmole by varying the NaCl concentration (Borland, Biggers & Lechene, 1977b). These results are consistent with blastocyst expansion occurring by local osmosis and fluid transport into closed channels, such as the intercellular spaces of the trophectoderm. If local osmosis in forward-facing channels does occur, the active transport sites should line these channels and be accessible to pump inhibitors that are microinjected into the blastocoele. The Na+K+ ATPase on the juxtacoelic surface of the trophectoderm, particularly the enzyme located on membranes lining the lateral intercellular channels, could be the site of Na+ transport and concomitant fluid movement. This would be true if the Na/K pump ratio is greater than one and is therefore rheogenic. A rheogenic Na+ transport system that is amiloride sensitive is acquired by the 6-7 day p.c. rabbit blastocyst (Powers et al. 1977), and may be related to the Na/K pumps demonstrated by the present studies. As shown in Table 3, after 4 hr incubations, ouabain injected blastocysts decreased in volume, while control blastocysts and blastocysts exposed to ouabain on the abcoelic surface of the trophectoderm both accumulated fluid. These large trends though not quite statistically significantly different (P = 0*107) strongly suggest that the activity of juxtacoelic ouabain-sensitive transport sites is required for blastocoele fluid accumulation. The hydrostatic pressure of blastocoele fluid in 6-day p.c. rabbit blastocysts is approximately 10 mmHg (T. H. Strunk, R. M. Borland, J. D. Biggers and C. P. Lechene, unpublished). This positive blastocoele pressure could cause fluid loss from the cavity if transtrophectoderm Na transport and concomitant passive water movement into the blastocoele is appreciably blocked by juxtacoelic ouabain. The concentrations of juxtacoelic ouabain used in these experiments may not have been sufficient to completely inhibit Na and water transport. The trends suggest, however, that the effect of ouabain on fluid accumulation by the rabbit blastocyst is similar to the effect of this drug on amphibian blastulas in which ouabain in the medium does not affect blastula formation, whereas ouabain injected into the blastocoele causes blastula collapse (Slack & Warner, 1973). Thus, juxtacoelic Na pumps in the blastocyst are probably important in both transepithelial ion transport and fluid movement. The mechanisms involved appear to be similar to those already demonstrated in the kidney (Katz & Epstein, 1967; Torretti, Hendler, Weinstein, Longnecker & Epstein, 1972; Silva, Hayslett & Epstein, 1973), toad bladder (Herrera, 1966), and rabbit ileum (Schultz & Zalusky, 1964). The work reported in this paper has been supported by grants from the Ford Foundation (720-0369), the Rockefeller Foundation (RF-65040), the National Institutes of Health (RO1HL-15552-03 and PO 7-RR00679-02) and the National Institute of Child Health and Human Development (HD-06919-O1A1). Dr Borland performed the experiments reported here while supported by a post-doctoral fellowship from the Ford Foundation Program. We are indebted to Dr Eddington Y. Lee for help with the preparation of the programmes used to analyse the data. A preliminary account of these results has been given by Borland (1977).
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REFERENCES BORLAND, R. M. (1977). Transport processes in the mammalian blastocyst. In Development in Mammals, vol. 1, ed. JOHNSON, M. H., pp. 31-69. Elsevier-North Holland Biomedical Press. BORLAND, R. M., BIGGERS, J. D. & LECHENE, C. P. (1976). Kinetic aspects of rabbit blastocoele fluid accumulation: an application of electron probe microanalysis. Devl Biol. 50, 201-211. BORLAND, R. M., BIGGERS, J. D. & LECHENE, C. P. (1977a). Studies on the composition and formation of mouse blastocoele fluid using electron probe microanlysis. Devl Biol. 55, 1-8. BORLAND, R. M., BIGGERS, J. D. & LECHENE, C. P. (1977b). Fluid transport by rabbit preimplantation blastocysts in vitro. J. Reprod. Fert. 51, 131-135. CRoss, M. H. & BRINSTER, R. L. (1970). Influence of ions, inhibitors and anoxia on transtrophoblast potential of rabbit blastocyst. Expl Cell Res. 62, 303-309. GAMOW, E. & DANIEL, J. C. (1970). Fluid transport in the rabbit blastocyst. Wilhelm Roux Arch. EntwMech. Org. 164, 261-278. HERRERA, F. C. (1966). Action of ouabain on sodium transport in the toad urinary bladder. Am. J. Physiol. 210, 980-986. KATZ, A. I. & EPSTEIN, F. H. (1967). The role of sodium-potassium-activated adenosine triphosphatase in the reabsorption of sodium by kidney. J. clin. Inve8t. 46, 1999-2011. KATZ, A. I. & EPSTEIN, F. H. (1968). Physiologic role of Na-K ATPase in the transport of cations across biologic membranes. New Engl. J. Med. 278, 253-261. LECHENE, C. P. & WARNER, R. R. (1977). Ultramicroanalysis: X-ray spectrometry by electron probe excitation. A. Rev. Biophys. Bioeng. 6, 57-85. MOHER, T. & LECHENE, C. (1975). Automated electron probe analysis of biological samples. Process control and data reduction by interactive graphic display. Biosci. Commun. 1, 314-329. PETZOLDT, V. U. (1971). Untersuchung uber das anorganische Milieu in Uterus und Blastozyste des Kaninchens. Zool. Jb. Physiol. 75, S 547-593. PowERs, R. D., BORLAND, R. M. & BIGGERS, J. D. (1977). Acquisition of amiloride-sensitive, rheogenic Na+ transport in the rabbit blastocyst. Nature, Lond. 270, 603-604. SCHULTZ, S. G. & ZALUsKY, R. (1964). Ion transport in isolated rabbit ileum. I. Short-circuit current and Na fluxes. J. gen. Physiol. 47, 567-584. SEARLE, S. R. (1971). Linear Models. New York: Wiley. SILVA, P., HAYSLETT, J. P. & EPSTEIN, F. H. (1973). The role of Na-K activated adenosine triphosphatase in potassium adaptation. Stimulation of enzymatic activity by potassium loading. J. clin. Invest. 52, 2665-2671. SLACK, C. & WARNER, A. E. (1973). Intracellular and intercellular potentials in the early amphibian embryo. J. Physiol. 232, 313-330. SMITH, M. W. (1970). Active transport in the rabbit blastocyst. Experentia 26, 736-738. STAPLES, R. E. (1967). Development of 5 day rabbit blastocysts after culture at 37 'C. J. Reprod. Fert. 13, 369-372. ToRREErI, J., HENDLER, E., WEINSTEIN, E., LONGNECKER, R. E. & EPSTEIN, F. H. (1972). Functional significance of Na-K-ATPase in the kidney: effects of ouabain inhibition. Am. J. Physiol. 222, 1398-1405. UssrNG, H. H. & WINDHAGER, E. E. (1964). Nature of shunt path and active sodium transport path through frog skin epithelium. Acta physiol. 8cand. 61, 484-504. USSING, H. H. & ZERAHN, K. (1951). Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta physiol. 8cand. 23, 110-127. VAN BLERKOM, J. & MANES, C. (1974). Development of preimplantation rabbit embryos. II. A comparison of qualitative aspects of protein synthesis. Devl Biol. 40, 40-51. VAN BLERKOM, J., MANEs, C. & DANIEL, J. C. (1973). Development of preimplantation rabbit embryos in vivo. I. An ultrastructural comparison. Devl Biol. 35, 262-282.
