DEVELOPMENTAL

Kinetic

(1976)

Aspects of Rabbit Blastocoele Fluid Accumulation: Application of Electron Probe Microanalysis

RAYMOND Biotechnology Human

50, 201-211

BIOLOGY

M. BORLAND,

Resource Reproduction

JOHN D. BIGGERS,

AND CLAUDE

An

P. LECHENE

in Electron Probe Microanalysis, Department of Physiology, and Laboratory and Reproductive Biology, Harvard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115 Accepted

January

of

2, 1976

X-ray spectrometry by electron probe excitation was used to analyze the content of Na, Cl, K, Ca, Mg, S, and P in picoliter samples of blastocoele fluid obtained from single rabbit blastocysts. There are significant changes in the concentrations of these elements between stages of development of blastocysts in uiuo. The concentration of K increases approx 40% between 110-135 hr postcoitum (p.c.) and does not increase further by 159 hr. The concentrations of Cl at 110 and 135 hr p.c. are significantly lower than the Cl level at 159 hr p.c. The concentrations of magnesium are very low (less than 0.4 mAf) at each stage of development. Ca and S concentrations do not change during development, whereas P decreases from 110-135 hr p.c. The rates of accumulation of the solutes in the rabbit blastocoele in uiuo have been estimated from the measured concentrations and from previously published rates of fluid accumulation. These analyses indicate that each trophoblast cell transports an ever increasing quantity of certain solutes which is closely correlated with the inward movement of water. Rabbit blastocysts raised in vitro from 128-140 hr p.c. can accumulate fluid in the presence of increasing concentrations of sucrose (40, 80, 120 mA4). Electron probe microanalysis of the blastocoele fluid under these conditions shows that the blastocoele concentrations of both Na and Cl (but not K, Ca, Mg, S, and P) increase 1 m&f in response to every 2 mA4 of sucrose gradient across the trophectoderm. These results directly indicate that the accumulation of fluid in the blastocoele is secondary to the active transport of NaCl. INTRODUCTION into a squamous epithelium with characOne of the early events in the develop- teristics similar to the skin of the frog ment of the preimplantation embryo is the (Ussing, Erlij, and Lasser, 1974). Furtherformation of a fluid-filled cavity, the blas- more, it has been shown by the use of the tocoele. Recent evidence obtained by the short-circuit technique of Ussing and Zerfreeze fracture technique, and by transahn (1951) that the blastocyst of the 6-day mission electron microscopy, have demon- rabbit can actively transport Na+, Cl-, strated unequivocally that the outer troand HCO,- (Cross, 1973, 1974). phoblast cells of rabbit and mouse blastoTo understand the quantitative aspects cysts become associated by tight junctions of blastocyst development, it is necessary (Ducibella et al., 1975; Hastings and to know the elemental composition of the Enders, 1975). Earlier, Cross (1973) blastocoele fluid at different developshowed that the resistance of the trophecmental stages, the rates at which the diftoderm (defined by Gardner and Papaioanferent elements accumulate in the blastonou, 1975) of the g-day rabbit blastocyst coele, and to understand the mechanisms has the high value of 2647 ohms cm2 and a involved in the movement of the elements transtrophectoderm electrical potential and water across the trophectoderm difference of -13.4 mV, blastocoele nega- boundary into the blastocoele cavity (see tive to culture medium. Thus, the outer Biggers, 1972, for a review). By the comcells of the blastocyst become organized bined use of micropuncture techniques and 201 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

202

DEVELOPMENTAL

BIOLOGY

X-ray spectrometry by electron probe excitation, it has been possible to analyze the elemental composition of the rabbit blastocoele fluid at earlier stages than done hitherto, to estimate the rates of elemental accumulation in the blastocoele, and to demonstrate that the movement of fluid into the blastocoele of the 5liz-day rabbit blastocyst is coupled to the accumulation of Na and Cl. The results are presented in this paper.

VOLUME

50, 1976

of IlO-hr p.c. embryos collected either with KRBG or mineral oil was identical. Sample preparation and electron probe microanalysis. The techniques of sample preparation and electron probe microanalysis of picoliter samples have been previously described (Lechene, 1970, 1974). Briefly, the unknown solutions and a series of standard solutions are placed with calibrated picoliter volumetric pipets (2080 pl) onto the surface of a beryllium support covered with mineral oil. These pipets MATERIALS AND METHODS were made on a de Fonbrune microforge Source of blastocysts and collection of and calibrated with tritiated H,O. The oil blastocoele fluid. Eight to ten pound New on the beryllium block is removed with a Zealand white rabbits were mated natum-xylene wash and the sample prepararally to mature males. Ovulation was as- tion on the beryllium block immediately sumed to occur 10 hr after mating. At 110, frozen in isopentane at -150°C. The mi135, and 159 hr postcoitum (p.c.), the fe- crodroplets are then freeze-dried under male rabbits were sacrificed. At 135 and vacuum (lo-” Torr, -70°C) so that very 159 hours p.c. the embryos were flushed small crystals (less than 1 pm diameter) from the uterine horns with mineral oil are obtained. The beryllium block is rewarmed at 37°C. Due to the low recovery warmed under vacuum to avoid water conrate of IlO-hr p.c. embryos using mineral densation when the block is returned to oil flushings, Kreb’s Ringer bicarbonate atmospheric pressure. The analyses were with 0.1% glucose (KRBG) warmed at 37°C performed with a Cameca MS/46 microwas used to collect most of the embryos at probe. The electron accelerating potential this stage. KRBG contains (r&f): NaCl, was 11 keV and the beam current was 119; KCl, 4.74; CaCl,, 1.71; KH2P04, 1.19; fixed at 200 nA on the beryllium. The MgSO,*7H,O, 1.19; NaHCO,, 25; and glu- beam was kept static and the beam diamecose, 5.55. The embryos flushed with ter adjusted to excite the entire dried samKRBG were placed immediately under ple. X-ray emissions from Na and Mg were mineral oil and the KRBG withdrawn analyzed using potassium acid phthalate with a Pasteur pipet. In this manner the diffracting crystals and emissions from P, Cl, K, and Ca using pentaerythritol difembryos at all three stages were totally surrounded by mineral oil and were then fracting crystals. The concentrations of the immobilized by suction applied through a unknowns were determined with respect pipet with a micrometer syringe. Micro- to the counts obtained from identical volumes of five standard solutions that conpuncture techniques of renal physiology (Lechene et al., 1969) were used to collect tain known amounts of the seven elethe blastocoele fluid. Micropuncture pipets ments. These calculations were performed (9-15 pm o.d.) filled with mineral oil were with a Hewlett-Packard 2100 A computer inserted into the blastocoele with a de Fon- and Tektronix 4012 display terminal. Incubation of blastocysts in the presence brune micromanipulator and the samples removed by suction. The samples were fro- of sucrose. Blastocysts were flushed from the uterine horns of rabbits 128 hr p.c. zen ( - 20°C) under mineral oil equilibrated with warm (37°C) Kreb’s Ringer bicarbonwith 0.9% saline until sample preparation the same day. The elemental composition ate containing 0.1% glucose (KRBG). The

BORLAND,

BIGGERS

AND LECHENE

embryos were washed with KRBG and then incubated for I2 hr in a modified FlO medium (Van Blerkom and Manes, 1974) [FlO (Microbiological Associates) buffered with 0.02 M N-2-hydroxyethylpiperazineN’-2-ethanesulfonic acid (HEPES, Calbiochem) adjusted to pH 7.4 with 1 N NaOH and containing 20% fetal calf serum (Microbiological Associates)]. Each embryo was incubated in 2 ml of medium in a Falcon #3033 tissue culture tube at 37.5”C in a high humidity atmosphere in 5% CO, in air. Where sucrose (Sigma Chemical Co.) was added to culture medium, the osmolality of the resulting media was measured on a Model 3L osmometer (Advanced Instruments, Inc.) at the end of the 12-hr culture period. To collect blastocoele fluid, the rabbit blastocysts were placed under light weight paraffin oil (Fisher 125/ 135) and the culture medium withdrawn with a Pasteur pipet. The blastocysts were micropunctured and samples analyzed with the electron probe as described above. The diameter of each blastocyst at the beginning and end of the incubation period was measured using a calibrated microscope eye-piece gaticule and the volumes calculated assuming sphericity of the embryos. Permeability of rabbit blastocysts to sucrose. The permeability of rabbit blastocysts to sucrose was determined by incubating four embryos for 12 hr in culture medium containing 44.4 miJ4 (n[‘4Clsucrose (U) (NEC-100, New England Nuclear). At the end of the incubation period, samples of blastocoele fluid were obtained by micropuncture. Aliquots of the incubation medium and blastocoele fluid were counted in a Beckman LS-250 liquid scintillation counter. The results showed that the concentration of sucrose in the blastocoele fluid was 7% of the medium concentration. Statistical analyses. The results comprise two main sections. The first are observations made on the elemental compo-

Rabbit

Blastocoele

Fluid

203

sition of blastocysts obtained 110, 135, and 159hr p.c. Seven rabbits were used at each time. Analyses of variance showed that the within mother variances were considerably smaller than the between mother variances for all elements. The means and standard errors were therefore calculated as weighted means of the means for individual mothers, allotting a weight equal to the number of blastocysts. Test of significance between the weighted means corresponding to each element were done using Duncan’s multiple range test (Duncan, 1955). The results were similar when analyzed after transformation of the data to logarithms. The results are therefore presented after analysis of the untransformed data. The second section is an experiment demonstrating the effect of different external concentrations of sucrose in the medium on the elemental composition of the blastocoele fluid. The experiment was designed to make comparisons on blastocysts obtained from the same mother exploiting the uniformity of blastocoele fluid within mothers. Since it is impossible to control the number of blastocysts obtained per mother, the experiment has yielded unbalanced data. The experiment has been analyzed for the increase in volume and for each element assuming an unbalanced 2way crossed classification with interaction using the computational procedures given by Searle (1971). In all cases the interaction term in the analyses of variance was not significant indicating that the responses within rabbits were consistent. Weighted means, and their variances, of the treatment effects were therefore computed. A linear regression of these means on the concentration of sucrose and its standard error was then calculated using formulae given by Hatheway and Williams (1958). The significance of this regression coefficient and departures from linearity were tested by a partitioning of xs. Since the adjusted means in this partic-

204

DEVELOPMENTAL

BIOLOGY

ular experimental design are independent because all covariances are zero (Searle, 1971), the formulae used were a simplification of the general ones given by Hatheway and Williams (1958). All computations were done on a timesharing system connected to a HewlettPackard 2000F computer.

Effect of External Sucrose on the Expansion of the Rabbit Blastocyst and the Composition of Blastocoele Fluid Experimental design. Five-and-one-half day rabbit blastocysts were incubated in a modified FlO medium containing 0, 40, 80, and 120 mM sucrose for 12 hr. Fluid accumulation does not reach a steady state in 12 hr, but blastocysts continue to expand if cultured for longer time intervals. The osmolalities of the media at the end of the 12hr incubation period are shown in Table 2. Five rabbits were used to provide blastocysts. The diameters of each blastocyst were measured at the beginning and end of the incubations, with the exception of the second replicate. A sample of blastocoele fluid was obtained from each blastocyst by micropuncture at the end of the incubation and analyzed for Na, Cl, K, Ca, Mg, S, and P by electron probe microanalysis. Those blastocysts from the first two rabbits were pooled and distributed at random between the four treatments. The blastocysts from the remaining three rab-

Elemental Composition of Rabbit Blastocoele Fluid 110, 135, and 159 Hr Postcoitum The weighted mean concentrations of Na, Cl, K, Ca, Mg, S, and P in uiuo in the blastocoele fluid of the rabbit 110, 135, and 159 hr p.c. are shown in Table 1. There are significant changes in the concentrations of several elements during this period. The concentration of potassium increases significantly by about 40% between 110-135 hr and does not increase further by 159 hr. The concentration of sodium increases slightly by about 6.9% between 135-159 hr whereas the concentration of Cl increases 39% during this period. The concentrations of phosphorus decrease significantly between 110-159 hr of development. The concentration of magnesium is low at each stage of development, decreases from 110 TABLE

1

(m&I f SEM) OF SODIUM, IN THE BLASTOCOELIC FLUID HR POSTCOITUM)

Element

Rabbit

110

op *, ’ For each element level.

with

different

fluid

postcoitum 135

132 ‘- 2 78.7 + 2.4 6.81 t 0.33” 1.38 % 0.088 0.32 r 0.044= 3.44 2 0.27 1.80 k 0.14= 7 Rabbits 41 Blastocysts DF = 6 the means

CHLORIDE, POTASSIUM, CALCIUM, OF THE RABBIT (AT 110, 135, AND 159

blastocoelic

Hours

Sodium Chloride Potassium Calcium Magnesium Sulphur Phosphorus

50, 1976

to 135 hr p.c., and increases from 135 to 159 hr p.c. In contrast, there are no major changes in the concentrations of calcium or sulphur.

RESULTS

THE WEIGHTED MEAN CONCENTRATIONS MAGNESIUM, SULPHUR, AND PHOSPHORUS

VOLUME

131 k 2 82.1 k 1.9 9.61 f 0.28 1.25 ? 0.14 0.089 + O.Olb 2.77 k 0.23 0.32 + 0.27 7 Rabbits 36 Blastocysts DF = 6 superscripts

are significantly

159 140 + 2” 111 ? 4a 9.71 2 0.27 1.52 ? 0.14 0.18 + 0.022 2.96 2 0.30 0.24 + 0.23 7 Rabbits 42 Blastocysts DF = 6 different

at the P = 0.05

BORLAND, TABLE

BIGGERS

AND LECHENE

2

THE OSMOLALITY

OF MODIFIED FlO INCUBATION MEDIUM CONTAINING DIFFERENT CONCENTRATIONS OF SUCROSE, AND THE ADJUSTED MEAN INITIAL VOLUME AND ADJUSTED MEAN INCREASE IN VOLUME OF 5%DAY RABBIT BLASTOCYSTS INCUBATED IN THE SOLUTIONS FOR 12 HR~ Sucrose concentration (mM)

Osmo&al-

Adjusted

mean volume (pl SEM (DF = 21)

(mOsm) Initial

0

40 80 120

+

312 350 387 430

2.62 2.42 3.09 2.37

2 t i2

Increase in volume in 12 hr

0.75 0.71 0.75 0.65

4.72 3.01 1.56 0.94

n The adjusted means resulted from the model assumed in the analysis of variance.

t t f f

0.82 0.77 0.82 0.71 linear

bits were kept separate and randomized between treatments. The experiment thus had four replicates. Since the number of blastocysts recovered varied from rabbit to rabbit, the number of blastocysts allotted to each treatment varied both within and between replicates. Effect of External Sucrose on the Increase in Volume of the Blastocyst

Rabbit

Blastocoele

Fluid

205

incubation medium. The concentrations of K, Ca, and Mg were not significantly affected by the presence of sucrose in the incubation medium. A linear regression has been fitted between the mean concentrations of Na, Cl, S, and P in the blastocoele fluid and the concentrations of sucrose in the medium, corrected for a 7% uptake of sucrose into the blastocoele cavity. The significance of the regression coefficients, and the significance of departures from linearity were tested by a partitioning of xZ. The results are summarized in Table 4. There is no linear regression of the concentration of S on sucrose concentration. The linear regression for P is significant but is small. In contrast the slopes of the regressions of Na and Cl concentrations on sucrose concentration are high and are not significantly different from a value of 0.5. DISCUSSION

Composition of Rabbit Blastocoele Fluid In Vivo

This is the first report of (1) the joint analysis of seven elements present in indiThe increase in volume of each blasto- vidual samples of blastocoele fluid from cyst was calculated from the diameters at single blastocysts, and (2) the elemental the beginning and end of the incubation composition of rabbit blastocoele fluid 4-6 period. The increase in volume that oc- days p.c. Prior to this time, the only inforcurred in the presence of different concen- mation available on the ionic composition trations of sucrose is also shown in Table 2, of mammalian blastocoele fluid concerned together with the initial volumes. The in- the Na+, Cl-, and K+ concentrations in hibitory effect of sucrose on the increase in rabbit blastocysts 6 days p.c. and older volume is highly significant; the effect is (Lewis and Lutwak-Mann, 1954). The conconcentration dependent and appears to centrations of Na and K, determined by fall off between SO-120 m&f sucrose. electron probe microanalysis in 159-hr p.c. rabbit blastocoele fluid (Table 11, are very Effect of External Sucrose on the Elemen- similar to the values found by flame photal Composition of Blastocoele Fluid tometry in this earlier study. The concenThe weighted mean concentrations of tration of Cl in 159-hr p.c. blastocoele fluid Na, Cl, K, Ca, Mg, S, and P in the blasto- obtained by electron probe microanalysis coele fluid after incubation in media con- (111 mM) is higher than the previously taining different concentrations of sucrose published value of 74.3 mM in 6l/2-day p.c. are shown in Table 3. Analyses of variance blastocoele fluid estimated by a titrimetric show that significant differences in con- method from a pooled sample from a small centration of Na, Cl, S, and P occur with number of embryos (Lewis and Lutwakdifferent concentrations of sucrose in the Mann, 1954).

206

DEVELOPMENTAL

BIOLOGY

VOLUME

TABLE ADJUSTED THEIR

MEAN

STANDARD

Sucrose concentrations (m&f) 0 40 80 120

COMPOSITION ERRORS (DF F10 MEDIUM Na

160 180 194 217

OF RABBIT

CONTAINING

Cl

f f -e +

2 2 2 1

132 156 169 191

-t + ” k

DIFFERENT

K

2 2 2 2

5.93 5.64 6.43 6.04

+ 2 + +

REGRESSION

OF CONCENTRATIONS CONCENTRATION

(d/liter)

BLASTOCOELE

FLUID

0.40 0.44 0.42 0.41

0.83 0.54 0.89 1.03

(b)

OF Na, OF SUCROSE

OF Na, Cl, K, Ca, Mg, AFTER 12 HR INCUBATION

CONCENTRATIONS

Ca

TABLE THE

3

OF THE CONCENTRATIONS = 24),

50, 1976

-t f + f

0.33 0.21 0.22 0.41

k k 2 k

IN MODIFIED

OF SUCROSE

S

Mg

0.048 0.052 0.050 0.049

S, AND P, AND

0.070 0.076 0.075 0.071

3.22 2.72 3.48 3.99

-t T k f

P

0.25 0.27 0.27 0.26

2.59 2.48 3.43 3.62

+ + + f

0.18 0.19 0.19 0.18

4 Cl, S, AND IN THE

P IN THE BLASTOCOELE INCUBATION MEDIUM

FLUID

ON THE

Element

Linear

regression

(b)

Sb’l

Tests of Significance x’,,,: linear regression x~,~,: departures from regression a s,,, Standard

error

Cl

0.500 0.031

0.509 0.038

255*** 1.34

linear

of regression

Na

coefficient.

183*** 2.31

** 0.01 > P > 0.001;

The concentrations of both Na and Cl at 110 and 135 hr p.c. (132-131 and 78-82 mM liter, respectively) are smaller than previously published rabbit serum values (142150 and 100-110 mM/liter, respectively) (Dittmer, 1961). At 159 hr p.c., both Na and Cl concentrations in the blastocoele fluid approach serum levels. The concentration of K in the blastocoele fluid is only slightly larger at 110 hr p.c. (6.81 mM/ liter) than previously reported serum levels (5.5-6.0 &/liter) (Dittmer, 1961), but is approx twofold greater than serum levels at 135-159 hr p.c. It is more meaningful to compare the blastocoele K+ concentrations to the K+ concentration that has been reported in rabbit uterine estrous fluid (10.7 mM) (Lutwak-Mann, 1962a). Based on this concentration of K+ in rabbit uterine fluid and the electronegativity of the rabbit blastocoele (Cross, 1973), there is currently no evidence for active K+ transport into the blastocoele. The concentrations of Ca and Mg at all three stages of blastocyst development are less than rab-

S

***

P

0.009 0.006

0.011 0.004

2.51 1.65

7.94** 1.70

P c: 0.001.

bit serum levels (2.6-5.0 and 0.84-1.22 n&f/liter, respectively) (Dittmer, 1961). It would be more appropriate to compare the composition of rabbit blastocoele fluid to that of the uterine fluid in the microenvironment of the embryo. However, because of the paucity of fluid in this microenvironment, it has not yet been possible to obtain adequate samples for probe analysis. The concentrations of the major cations (Na+ and K+) in rabbit blastocoele fluid in utero are much greater than the concentration of the major anion (Cl-) that was measured (Table 1). This apparent electrical charge imbalance is probably due to the high concentrations of HCO,- in rabbit blastocoele fluid. Cross (1974) demonstrated active HCO,- accumulation in 6 day p.c. rabbit blastocysts. He hypothesized that the high HCO,- content of uterine secretions (see Biggers and Bellve, 1974, for a review) may represent the source for the high concentrations of HCO,accumulated in the blastocoele. The concentration of HCO,,- in blastocoele

BORLAND,

BIGGERS

AND LECHENE

fluid is approx three times greater than rabbit serum HCO,- on Day 5 p.c., but progressively decreases to plasma levels by Day 8 p.c. (Lutwak-Mann, 196213). The decrease is paralleled by an increase in Clconcentration in blastocoele fluid between 135 and 159 hr p.c. (Table 1). Therefore, the concentrations of both Cl- and HCO,tend to balance the concentrations of Na+ and K+ as the blastocyst develops. Kinetics Fluid

of the Formation In Vivo

of Blastocoele

The analyses of rabbit blastocoele fluid at several developmental stages enable us to evaluate the kinetics of blastocyst formation by considering the total movement of solutes into the blastocoele cavity (Biggers, 1972). If C and V are the concentration of a particular solute and the volume of the blastocoele fluid at a specific time during development, the total accumulation of substance by that time is given by: A = CV. The rate of accumulation then given by: dA/dt

= C.dVldt

of the solute is

+ V.dC/dt.

(9

C.dV/dt is the rate of accumulation attributable to the rate of change in the volume of fluid transported (assuming the concentration of the solute in the fluid remains constant in the infinitesimal interval dt); V.dC/dt is the rate of accumulation attributable to the rate of change of the composition of fluid transported (asTABLE THE VOLUME

Age

Volume

(days P.C.)

(~1)

RATE

OF ACCUMULATION

Average area

0.0157 0.566 11.5 66.1

5 6

7 8

345

Daniel

(1964).

78.9 238.0

207

Fluid

5 OF RABBIT

surface (mm’)

0.300 2.61 24.8

Blastocoele

suming the volume of the blastocoele remains constant in the infinitesimal interval dt). Estimates of C for Na, Cl, K, Ca, Mg, S, and P are given in Table 1, and other estimates for Na, Cl and K on Days 7 and 8 p.c. can be obt.ained from the literature (Lewis and Lutwak-Mann, 1954). In addition, estimates of V and dVldt are also available in the literature (Table 5) (Daniel, 1964). The data in Table 1 show that no significant change occurs in the concentration of calcium and sulphur, and therefore dCldt is assumed to be zero. The other elements change significantly in concentration but the data are insufficient to determine dCldt precisely. However, if the rate of change of volume is very much greater than the rate of change of concentration of an element, the second term of (i) can be ignored, and the locus of points [In (dAl dt), In (dVldtI1 will fall on a straight line of unit slope. This relationship can be used as a graphical test of the relative magnitudes of dV/dt and dC/dt. Deviations from a straight line of unit slope indicate that the calculation of the rate of accumulation cannot ignore the second term of (i). The results in Fig. 1 indicate that satisfactory estimates of the rates of accumulation can be calculated for Na, Cl, K, Ca, and S using only the first term of (i). The rates of accumulation of Na and Cl in the rabbit blastocoele increase lO,OOOfold between Days 4-8 p.c. from 0.0002 pmole hr-‘/embryo to 2 pmole hr-‘lembryo (Fig. 1). The validity of these calculated rates of accumulation is supported by BLASTOCOELE

Fluid dVldt/embryo (~1 hr-I)

4

U From

AND

Rabbit

0.0023 0.068 0.8 5.6 18

accumulation

FLU@ rate

dvidtlarea W&

dVldtlcel1 (pl hr-‘)

7.57 26.05

2.29

32.26

70.98 75.63

7.61

9.96 22.0 23.4

208

DEVELOPMENTAL

BIOLOGY DAYS

VOLUME

50, 1976

POST-COITUM

I

RATE

OF

FLUID

ACCUMULATION

(pl/hr/embryo)

FIG. 1. The log of the rates of accumulation of S, Ca, Na, Cl, K, Mg, and P (mmole/hr/embryo) (ordinate), calculated from the formula dAldt = CdVldt, are plotted against the log of the rates of fluid accumulation (&hr/embryo) (abscissa). The ages of the embryos, expressed in the number of days postcoiturn (p.c.), are shown at the top of each figure. The deviation of the Mg and P curves from straight lines of unit slopes indicates that omission of the second term of equation (i) is unjustified for these two elements and that the calculated rates of accumulation for both Mg and P are grossly overestimated.

a comparison of the rates of accumulation of Na and Cl we have calculated for Day 6 blastocysts (0.12 pmole hr-‘/embryo and 0.08 pmole hr-‘/embryo, respectively) with measured rates of accumulation of Na and Cl (0.17 pmole hr-‘/embryo for each) in short-circuited rabbit embryos (Cross, 1973). The discrepancies in values, although small, may be due to differences in the exact age of the embryos and the fact that ion fluxes in short-circuited blastocyst preparations were measured in vitro using culture medium whose Na and Cl content may be very unlike that of uterine fluid. The rate of accumulation of Na, Cl, K, Ca, and S through the surface of the rabbit blastocyst can be calculated from the data in Fig. la, b, and the surface area of the blastocyst shown in Table 5. The results, shown in Table 6 demonstrate that the rate of accumulation per unit area of all these elements increases with development. This passage occurs through a squamous epithelium held together by tight junctions (Ducibella et al., 1975; Hastings and Enders, 1975), with a high electrical

TABLE 6 THE RATE OF ACCUMULATION OF Na, Cl, K, Ca, AND S PER UNIT AREA IN 4-6 DAYS POSTCOITUM RABBIT BLASTOCYSTS Element

Rate of accumulation (pmole crne2 hr-9 Days postcoitum 4

Na Cl K Ca s

0.0978 0.0582 0.0050 0.00102 0.00253

5 0.342 0.214 0.0250 0.00326 0.00721

6 0.452 0.358 0.0313 0.00490 0.00955

resistance (Cross, 1973). All elements must pass through the trophoblast cells and/or their intercellular junctions. Since the area of the individual trophoblast cells of the rabbit blastocyst remains approximately constant (0.31 pm2) at all ages (Daniel, 1964), the rate of passage of Na, Cl, K, Ca, and S through a trophoblast cell and surrounding intercellular junctions must increase with development. It must be emphasized, however, that these calculations are based on the assumption that the rabbit blastocyst is composed of a homogeneous population of cells and ignores

BORLAND,

BIGGERS

the possibility that the endoderm and cells of the primitive streak in embryos 6-8 days p.c. could have different fluid and solute transporting capacities than those of the trophoblast cells. Transport of Water into the Blastocoele The effects of incubating 5%day rabbit blastocysts in media containing sucrose support the hypothesis that the transport of water into the blastocoele cavity is coupled to the active transport of Na+ and Cl-, as suggested by Cross (19731, who demonstrated the active transport of both Na+ and Cl- into the 6-day p.c. rabbit blastocoele. Let it be assumed that in the absence of sucrose in the incubation media the fluids on either side of the trophectoderm are isosmotic, so that the chemical activities of water on both sides are equal. The effect of adding sucrose to the bathing medium is to lower the chemical activity of water outside. In the absence of other forces water should move out of the blastocyst to its environment, and the blastocyst ;! ,n

Rabbit Blastocoele Fluid

AND LECHENE

209

should shrink. The results presented in Table 2 and Fig. 2, however, show that up to a sucrose concentration of 80 mA4 significant blastocyst expansion continues, although at a diminished rate. For this expansion to occur against a chemical activity gradient either the movement of water involves the direct expenditure of metabolic energy for active water transport as proposed by Tuft and Boving (19701, or some other substances must be actively transported into the blastocyst to lower the chemical activity of water in the blastocoele fluid below that in the external medium. The results presented in Tables 3 and 4 show that only the concentrations of Na and Cl in the blastocoele fluid increase to a major extent in the presence of external sucrose. Moreover these concentrations are above those found in the bathing medium. Thus, the active transport of Na and Cl into the blastocoele fluid is the major factor in lowering the chemical activity of water in this fluid. The fact that 1 mA4 NaCl is transported for every 2 mM of

70

r

0 II

80 Sucrose

med,umy-

[Sucrose

blos,ocoe~

m mrnol.~):~

FIG. 2. Effects of sucrose concentration gradients (x-axis) on the concentration gradients of Na, Cl, and K (y-axis) across rabbit trophectoderm. The increases in rabbit blastocyst volumes (~1/12 hr) in the presence of increasing sucrose concentrations are shown by the dotted line. Rabbit blastocysts were incubated from 128-140 hr postcoitum in Ham’s FlO buffered with 0.02 M HEPES containing 20% fetal calf serum in the presence or absence of sucrose (0, 40, 80, and 120 m&f). The sucrose gradient was calculated assuming that the concentration of sucrose in the blastocoele fluid at the end of the 12-hr incubation is 7% of the medium sucrose concentration.

210

DEVELOPMENTAL

BIOLOGY

sucrose gradient suggests that the active transport of NaCl is of sufficient magnitude to account for water movement. Thus, there is no need to postulate the active transport of water. Also, the data do not support the view of Gamow and Daniel (1970) that there is transport of a hypotonic fluid since such a mechanism would require less than 1 mM NaCl transported for every 2 mM sucrose gradient. Similar coupling of water movement to the active transport of Na and Cl has been previously shown in “unilateral” rabbit gall bladder preparations in which the same quantitative relations were found (Diamond, 1964). In conclusion, we postulate that in the development of the blastocyst of the rabbit, each trophoblast cell transports an ever increasing quantity of certain solutes and that the large amount of water that accumulates is moved by being coupled to the accumulation of Na and Cl. The changes in the accumulation rates of solutes and in the composition of rabbit blastocoele fluid are probably regulated by (a) the activation or synthesis of additional transport systems with development, (b) permeability changes in the trophoblast cells, (c) changes in the elemental composition of the uterine fluid in the microenvironment of the blastocyst, and/or (d) permeability changes in the intercellular pathways between trophoblast cells. The work reported in this paper has been supported by grants from the Ford Foundation (7200369), the Rockefeller Foundation (RF-65040), the Population Council (M74.331, the National Institutes of Health (ROl-HL-15552-03 and PO 7RR00679-02), and the National Institute of Child Health and Human Development (HD-06916-OlAl). We are indebted to Dr. Eddington Y. Lee for help with the preparation of programs used to analyze the data. Dr. Borland is a postdoctoral fellow supported by the Ford Foundation. REFERENCES BIGGERS, J. D. (1972). Mammalian blastocyst and amnion formation. In “The Water Metabolism of the Fetus” (A. C. Barnes and A. E. Seeds, eds.). Charles C Thomas, Springfield, Ill.

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BIGGERS, J. D., and BELLV~, A. R. (1974). Carbon dioxide in developmental processes. In “Carbon Dioxide and Metabolic Regulation” (G. Nahas and K. E. Schaefer, eds.). Springer, Berlin. CROSS, M. H. (1973). Active sodium and chloride transport across the rabbit blastocoele wall. Biol. Reprod. 8, 566-575. CROSS, M. H. (1974). Rabbit blastocoele bicarbonate: Accumulation rate. Biol. Reprod. 11, 654-662. DANIEL, J. C. (1964). Early growth of rabbit trophoblast. Amer. Naturalist 98, 85-98. DIAMOND, J. M. (1964). The mechanism of isotonic water transport. J. Gen. Physiol. 48, 15-42. DITTMER, D. S. (1961). “Blood and Other Body Fluids,” pp. 35-37. Federation of American Societies for Experimental Biology, Washington, D.C. DUCIBELLA, T., ALBERTINI, D. F., ANDEKSON, E., and BIGGERS, J. D. (1975). The preimplantation mammalian embryo: Characterization of intercellular junctions and their appearance during development. Develop. Biol. 45, 231-250. DUNCAN, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, l-42. GAMOW, E., and DANIEL, J. C. (1970). Fluid transport in the rabbit blastocyst. Wilhelm Roux’ Archiu. 164, 261-278. GARDNER, R. L., and PAPAIOANNOU, V. E. (1975). Differentiation in the trophectoderm and inner cell mass. In “The Early Development of Mammals” (N. Balls and A. E. Wild, eds.), pp. 107-132. Cambridge University Press, London. HASTINGS, R. A., and ENDERS, A. C. (1975). Junctional complexes in the preimplantation rabbit embryo. Anat. Rec. 181, 17-34. HATHEWAY, W. H., and WILLIAMS, E. J. (1958). Effcient estimation of the relationship between plot size and the variability of crop yields. Biometrics 14, 207-222. LECHENE, C. P. (1970). The use of the electron microprobe to analyze very minute amounts of liquid samples. In “Proceedings of the 5th National Conference on Electron Probe Analysis” (New York), 32A-32C. LECHENE, C. P. (1974). Electron probe microanalysis of picoliter liquid samples. In “Microprobe Analysis as Applied to Cells and Tissues” (T. Hall, P. Echlin, and R. Kaufmann, eds.). Academic Press, New York. LECHENE, C. P., MOREL, F., GUINNEBAULT, M., and DE ROUFFIGNAC, D. (1969). Etude par microponction de l’elaboration de l’urine. I. Chez le rat dans differents etats de diurese. Nephron 6, 457-477. LEWIS, P. R., and LUTWAK-MANN, C. (1954). The content of sodium, potassium and chloride in rabbit blastocysts. Biochim. Biophys. Acta 14, 589590. LUTWAK-MANN, C. (1962a). Some properties of uterine and cervical fluid in the rabbit. Biochim. Biophys. Acta 58, 637-639.

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C. (1962b). Glucose, lactic acid and bicarbonate in rabbit blastocyst fluid. Nature (London) 193, 653-654. SEARLE, S. R. (1971). “Linear Models.” Wiley, New York. TUFT, P. H., and B~VING, B. G. (1970). The forces involved in water uptake by the rabbit blastocyst. J. Erp. Zool. 174, 165-172. USSING, H. H., ERLIJ, D., and LASSER, U. (1974). Transport pathways in biological membranes.

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Rev. Physiol. 36, 17-49. H. H., and ZERAHN, K. (19511. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol. Scandinau. 23, 110-127. VAN BLERKOM, J., and MANES, C. (1974). Development of preimplantation rabbit embryos. II. A comparison of qualitative aspects of protein synthesis. Devel. Biol. 40. 40-51. USSING,