Influence of ions, inhibitors and anoxia on transtrophoblast potential of rabbit blastocyst

Experimental Cell Research 62 (1970) 303-309

INFLUENCE

OF IONS, INHIBITORS

ON TRANSTROPHOBLAST

POTENTIAL

AND ANOXIA

OF RABBIT

BLASTOCYST

M. H. CROSS and R. L. BRINSTER Laboratory of Reproductive Physiology, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pa 19104, USA

SUMMARY The influence of ions, metabolic inhibitors and anoxia on the transtrophoblast potential difference (TPD) of the 6 day rabbit blastocyst was studied in vitro. TPD measurements were made using glass microelectrodes with Ag-AgC1 wire at the liquid-metal junction. When Cl- is replaced by SO*= in the external medium (Krebs Ringer bicarbonate glucose), the steady negative TPD was changed to a positive value. Replacement of Cl- by Br-, NO*- or I- decreased the TPD to less than 50% of the normal value. In a medium in which Na+ was replaced by lithium the TPD increased by about 30%. K+ free Ringer had no effect on the TPD. The TPD was greatly reduced or abolished by anoxia, sodium cyanide, 2&dinitrophenol and iodoacetic acid while ouabain increased the TPD by 30%. This TPD is also temperature dependent (Q10of 1.7), dropping suddenly at 44°C. These results suggest that active ionic transport processes may play a role in determining the TPD.

Measurements of the electrical potential difference across the trophoblast layer of the expanding rabbit blastocyst have been performed in vitro using glass microelectrodes [5]. It was shown that the negative transtrophoblast potential difference (TPD), present in the 5 day (postcoitum) rabbit blastocyst, increased on day 6 of development and then decreased almost to zero on day 7, just prior to implantation. The purposes of the present study on the 6 day rabbit blastocyst were: (a) to determine the ions involved in maintaining the TPD of the rabbit blastocyst; and (b) to determine the dependency of TPD on metabolic processes. 20

-

701818

METHODS Six-day rabbit embryos were obtained by natural mating of New Zealand white rabbits. Eagle medium containing 20% calf serum was used to flush the embryos from the uterus and for storing the embryos, prior to use, under culture conditions as previously described [5]. For measurements of the TPD in vitro, a specially constructed plexiglass chamber was used (fig. 1). The chamber was mounted under a binocular microscope and consists of an inner chamber for maintenance of the blastocyst and an outer water jacket kept at 37°C by circulating heated water. The inner chamber holds up to 5 ml of medium which can be changed without removing the blastocyst. Electrical measurements were recorded with glass microelectrodes. The microelectrodes, obtained by pulling glass tubing (Pyrex, o. d. 1 mm) to tip diameters of less than 1 fi with a vertical puller (Kopf), were filled with 3 M KCl. Only microelectrodes with resistances of 5-20 megohms and tip potentials less than 5 mV were used. The microelectrodes were led via Ag-AgCl wire to a high input impedance potentioExptl Cell Res 6.2

304 M. H. Cross & R. L. Brinster MICROSCOPE

\

/

Fig. 1. Plexiglass chamber. A and B are the outlet and

inlet to water jacket (C) for water heated to 37°C. D, inner medium chamber. The fluid in chamber D can be drained off at the tap (E) and replaced at F.

Gas enters at G, a polyethylene tube, and may be bubbled through the culture medium if desired. The blastocyst sits on a polyethylene sucker (ZZ) (PE 10, flared as its tip to form a seat) housed in a glass capillary tube (I). The sucker is connected to a negative pressure device and the glass capillary tube may be freely raised or lowered to any level in the chamber. Four ports enter the chamber 90” apart (only two ports shown, K and J) for the entrance of microelectrodes, individually carried in a micromanipulator, or agar bridge connections to reference electrodes. M, 6-day rabbit blastocyst.

meter (Radiometer, Copenhagen). The medium was connected to ground by-an A&A&l electrode with a medium-agar bridge. When the effects of substituting the various experimental solutions for Krebs Ringer were tested on the electrode system, a change in potential of less than 1.5 mV was detected. Details of the methods and precautions used to achieve accurate measurements were nreviouslv described 151. The normal Krebs- Ringer Solution used in all experiments contained (mM): NaCl, 119; KCl, 4.78; CaC&, 1.71; KH,PO*, 1.19; MgSO,, 1.19; NaHCO*, 25; glucose, 5.55. When chloride was replaced by the anions Br-, I-, SO,- or NOI-, the chloride present as NaCl and KC1 was replaced. In the preparation of the sulphate solution CaCll was also replaced by C&O& and sucrose was added so that the final osmolarity was equal to that of the other solutions (308 mosmols). In the low sodium solution NaCl was replaced by LiCl and in the sodium-free solution KHCO, was also substituted for NaHCO,. Potassium-free solution was prepared by replacing KC1 with NaCl and KHsPOl with NaH,PO,. All solutions Exptl Cell Res 62

were kept at pH 7.4 by continuous gassing with humidified 5% CO, in air. To test the effect of anoxia, 5% CO2 in N, was bubbled through the medium before the experiment (for at least 30 min) and over the medium throughout the experiment. The temperature in the chamber was 37°C except in the experiment to test the effects of temperature on TPD. In this case, different temperatures were obtained by adjusting the thermostat of the circulating water bath, and the chamber temperature was monitored by a small thermistor connected to a calibrated meter. Temperature changes were made at a rate of 0.6”C/min. Inhibitors weremade up with the normal Krebs Ringer medium in the following concentrations: sodium cyanide, 10-8M; iodoacetic 10-SM; 2.4dinitronhenol (DNP). acid (IAA), ’ ” 2 x lo-4M; and ouabain, lO+M. At the beginning of the experiment the embryos were removed from the incubator and placed in the experimental chamber containing normal Krebs Ringer medium for 5 min equilibration. After this equilibrium period the microelectrode was inserted into the blastocoele cavity and the steady TPD was recorded 5 min later. The microelectrode was then removed from the blastocyst, the medium drained, and the experimental solution added. After a period of equilibration with the new medium, the microelectrode was reinserted into the blastocoele cavity and remained there throughout the experimental treatment period. Following the experimental period the external medium was again changed back to normal Krebs Ringer, the embryo was allowed to equilibrate and then penetrated by the microelectrode. In this way each blastocyst served as its control before and after being exposed to the experimental medium. The changing of solutions consisted of lowering the blastocyst (by lowering the glass capillary tube; fig. 1, I) to the lowest level of the chamber. emptying the solution to the top of the blastocyst, washing th; chamber once with the new medium using this same procedure, and then adding the new medium to the original level. Thus the external medium could be changed in a short period of time (2-3 min) and at no time would the embryo be directly exposed to the atmosphere. It was calculated that by using this method of changing media, the final control medium would contain approx. 1% of the treatment medium. Between withdrawal and insertion of the microelectrode, during the solution change, zero readings were recorded to check the stability of the microelectrode. The washing procedure had little effect by itself on the TPD (see last column, table 2). Only embryos that conformed to the following criteria were used in this study: (1) the trophoblast layer of cells did not shrink from the zona pellucida during any phase of the experiment; (2) a steady TPD was maintained during the initial control period; and (3) the blastocysts were normal as judged from their general gross appearance (i.e. absence of degenerated cells, tears, etc.). Each mean reported in the data represents the results obtained from a number of embrvos each of which was utilized only for one experimental determination. Each blastocyst from a rabbit was used for a different treatment. Student’s t test was employed for comparison of treatments.

Transtrophoblast potential

Table 1. Changes in the transtrophoblast potential difference (mV) following

305

replacement of

chloride ions in the external medium with other anions Anion replacement for ClSolutions Initial control (5)* 5 min’ Expt (10) 5 min Final control (5) 5 min

- 10.4+0.71a + 3.5kO.74 - 6.5kO.58

Br(PI= 5)

NO,(n=6)

-10.8k1.46 - 5.3kO.77 - 7.OkO.81

-10.6+1&l - 3.8k1.02 - 7.5k1.14

-9.451.69 - 4.5 kO.61 -5.Ok1.13

a Mean +_S.E.M. * Numbers in parentheses represent period of equilibration before insertion of microelectrode. ’ Time TPD was recorded after insertion of microelectrode. n, number of determinations; control solution, normal Krebs Ringer. Each blastocvst served as its control before (initial control) and after being exposed (final control) to the experimental medium.

tion of the I- potential appeared to be returning to the control values after 5 min. Table 1 shows the results obtained when the Replacement of the external NaCl with different anions replaced chloride in the LiCl (leaving the external [Cl-] unchanged) external medium. The TPD was decreased resulted in an increase in the TPD (table 2). significantly (P
Table 2. Effect on the transtrophoblast potential difference (mv) of changing the external Na+ and K+ concentration Solutions Initial control (5)* 5 min” Expt (10) 5 min 10 rein Final control (5) 5 min

Low Na+ (25 mM; x=6)

Naf Free (n=6)

K+ Free (n=5)

Control

-7.Ok0.83’ -9.3k1.05

- 9.6k0.68 -11.621.18 - 12.5 f0.84 - 9.7kO.50

- 10.9 f 1.94 - 9.722.46

-1l.li1.47 - 9.2k1.08

- 9.5t2.23

- 9.0+0.90

-6.5kl.00

(n=5)

a Mean +_S.E.M. * Numbers shown in parentheses represent period of equilibration before insertion of microelectrode. ’ Time TPD was recorded after insertion of microelectrode. n, number of determinations; control solution, normal Krebs Ringer;*LiCl replaced NaCl. Each blastocyst served as its control before (initial control) and after being exposed (final control) to the experimental medium. Exptl Cd Res 62

306 44. H. Cross & R. L. Brinster -M-IS-12 -II -

-12-II -10 -9 -

-IO-

-6 -

-9-

-7 -

-6 -

-6 -

-7 -

-5 -

-6 : t 0

-4-

Figs 2-4. Abscissa: Time (min); ordinate: TPD (mV). Fig. 2. Effect of ouabain (10-4M) on the TPD. The

vertical lines represent the S.E.M. Control group maintained in normal Krebs Ringer (Kr) after washing.

approx. a 30% increase over the control value in both the low Na+ and Na+ free solutions. When the control medium was returned, the TPD returned to values similar to those obtained during the initial control period. Experiments not included in table 2, performed to determine the effect of the increased K+ concentration used in the Na+ free experiments (where KHCO, was substituted for NaHCO,, 25 mM), showed no appreciable effect on the TPD of the elevated K+ concentration. When Kf free medium was used (table 2), the values recorded for the TPD did not differ from those obtained during the control period. Table 2 also shows a set of control values recorded from embryos carried through the same experimental procedure. In this case, all solution changes were made with the normal Krebs Ringer. These results indicate that the experimental procedure used to change the solution surrounding the embryos had no appreciable effect on the TPD. The addition of 10-4M ouabain to the medium surrounding the blastocyst caused the TPD to increase steadily for at least thirty min (fig. 2). Although the TPD value after 30 min was 37% greater than the initial control value, it was not significantly different from this control value. However, when the Exptl Cd Res 62

-3 -2 -I 0

5

0

5

IO

15

20

e5

30

35

40

0

5

Fig. 3. Effect of DNP (2 x lO+M) and IAA (10-8M)

on the TPD. Vertical lines reoresent the S.E.M. Control group maintained in normal Krebs Ringer (Kr) after washing.

TPD recorded at 30 min of exposure was compared to the 30 min TPD recorded for a separate control group of blastocysts (fig. 2), it was significantly different (P < 0.05). Fig. 3 shows the effects of inhibitors of aerobic and anaerobic metabolism on the TPD. 2,4-dinitrophenol, a respiratory inhibitor which uncouples oxidative phosphorylation and IAA, an inhibitor of the Embden-Meyerhof pathway, both caused similar depressions in TPD. IAA decreasedthe TPD to 16% (P
Transtrophoblast potential

Fig. 4. Effect of anoxia (normal Krebs Ringer bubbled

with 5% CO2 in N,). Vertical lines represent S.E.M. Kr, normal Krebs Ringer (5% CO* in air).

caused a rapid, almost instantaneous decrease of the control TPD from an initial value of - 12-J 1.6 (mean+ S.E.M.) to zero. When the embryos were returned to the control medium, the potential recovered over a 5 min period to -9.l& 1.7 which was lower but not significantly different than the initial control value. The effect of anoxia (95% Nz and 5% CO,) on the TPD was tested in five experiments (fig. 4). The TPD decreasedto zero after 35 min, and became positive after 40 min. In two experiments continued past 40 min the TPD reached values of + 3.0 to i-4.0 mV. The curve representing the effect of anoxia

-14 -13 -12 -II -10 -9 -8 -7-6 *I

21 24

28

31

34

37

40

44

“C; ordinate: TPD (mv). TPD is shown as a function of the temperature. n, 4.

Fig. 5. Abscissa:

307

on the TPD (fig. 4) appears similar to those representing the effects of DNP and IAA except for the initial level period of the potential seen at the beginning of exposure and the appearance of a positive potential after 40 min. The anoxia effect was rapidly reversed (3 min) by the addition of air. In four experiments the change in TPD with temperature was measured between 24” and 44°C at 3” intervals. The TPD increased between 24” and 34°C and was 1.7 times greater at 34” than at 24°C (fig. 5). The TPD remained constant between 34” and 37°C started to decrease at 40”, and dropped suddenly to zero at 44°C. DISCUSSION When the Cl- was replaced by SO6 in the external medium, without changing the Na+ and K+ concentrations, the TPD rapidly changed to a positive value. When Cl- was replaced by other anions, the degree of depolarization of the membranes was in the order of SO,= > NO,= > I- > Br-. Removal of the external Naf ions, without changing the K+ and Cl- concentrations, resulted in an increase in the TPD. When K+ was removed from the medium, the TPD was not affected. Since the removal of Na+ and Cl- did influence the TPD, it is likely, therefore, that these ions play an important role in maintaining the TPD of the rabbit blastocyst. The inhibition of the TPD produced by metabolic inhibitors (DNP, CN-, IAA) and anoxia demonstrate that metabolic energy must be supplied in order to maintain the TPD. From the present experiments the relative proportions of energy derived from either aerobic or anaerobic metabolism, for the maintenance of TPD, cannot be determined. DNP and IAA, tested individually, caused similar depressions in the TPD. Similar observations have been made in other Exptl Cell Res 62

308 M. H. Cross & R. L. Brinster

tissues [7]. For example, there is ample evidence that the frog skin, the toad bladder and various mucosal epithelia are sensitive to glycolytic and oxidative inhibitors [6]. These investigations suggest that most active transport processes across epithelial membranes derive energy from both aerobic and anaerobic sources; other membranes such as the mammalian erythrocyte are wholly anaerobic [13]. At the time of blastocyst formation in the rabbit, glycolytic activity and Krebs cycle activity are markedly increased [lo, Ill, and glucose seems to be converted to lactic acid and carbon dioxide in roughly equal molar quantities on day six

moved from outside to inside by the trophoblast cells. Although it is not possible from the present experiments to establish the nature of the mechanisms producing the TPD in the rabbit blastocyst, the results obtained from the treatments with inhibitors and anoxia do suggest the presence of active ionic transport processes. The increased negative TPD seen in the ouabain experiment may indicate that the TPD is related to active Na+ transport. Ouabain is known to be a specific inhibitor of membrane Na-K ATPase [3] and has become a generally accepted test for active Na+ transport [14]. The combined changes in TPD caused by ouabain and replacement of ions in the external medium [l, 121. Other temperature-dependent potentials could be interpreted to support the idea of have been reported [4, 171 and have been active movement of Na+ and Cl- into the shown to result from metabolically linked blastocoele cavity. However, this has not processes[17]. A Q10of 1.7, obtained for the been established, and further experimentaTPD of the rabbit blastocyst, is comparable tion using combined ionic flux and short to the Q10reported for active ion transport circuit current techniques is needed to conin other tissues [19] and is larger than that for firm this view. The rapid accumulation of fluid which free diffusion (Q1,,= 1.3). Thus the Q10 reported here is compatible with the idea that occurs in the preimplanted rabbit blastocoele the TPD of the 6 day rabbit blastocyst is cavity probably results from the active movedependent upon chemical processes. There ment of ions across the blastocyst wall. The appears to be a range of temperatures (34” to coupling of water movement to ion movement 37”) in which an optimum TPD can be main- has been shown in many other tissues (i.e. tained. Reconstitution of rabbit blastocysts gallbladder, stomach, small intestines and has also been shown to occur throughout a in vivo frog skin). Observations from these similar range of temperature [9]. This also preparations form the basis of the widely held appears to be the case for the developmental view that the driving force for the isosmotic processes of other mammalian embryos. flow of water across epithelial membranes Brinster [2] has shown that the development arises from physical coupling of the water to of two-cell mouse embryos into blastocysts, net NaCl transport [8]. Freezing point dein vitro, takes place between 33” and 39°C. terminations on the blastocoele fluid of the In vivo isotope studies 1161have presented expanding blastocyst [18] and chemical deevidence to support a concept of active terminations of this fluid [15] indicate that transport of ions into the blastocoele cavity the blastocoele fluid is hypo-osmotic, which of the 6 day rabbit blastocyst. A higher up- does not seem conducive to blastocyst extake of labeled Na+ and I- as compared to the pansion. It seemsmuch more likely that exother ions tested (K+, PO,-, SO,=) suggest pansion of the blastocyst would result from that Naf and possibly Cl- may be actively active transport of ions from outside to inside Exptl CeN Res 62

Tramtrophoblast potential

with subsequent passive diffusion of water. It therefore seems that the blastocoele fluid would have to be iso- or hypertonic and that under these conditions the difference in ionic content and freezing point depression between fluid inside and outside the blastocoele may be too small to measure. Observations in our laboratory (unpublished) that rabbit blastocysts will not expand in vitro in medium deficient in Cl- ion further indicate the importance of the external ionic content to the mechanism of fluid accumulation. This work was supported by the National Institutes of Health contract no. NIH 69-2141 and training grant 00239.

REFERENCES 1. Brinster, R L, Exptl cell res 54 (1969) 205. 2. - Pathways to conception (ed A Sherman). Thomas, Springfield, Ill. In press.

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Caldwell, P C, J physiol 152 (1960) 545.

: Carpenter, D b, J gen physidl 50 (1967) 1467. 5: Cross, M H & Brinster, R L, Exptl cell res 58 (1969) 125. 6. Csaky, C Z, Ann rev physiol 27 (1965) 415. 7. Curran, P F, J gen physiol43 (1960) 1137. 8. Curran; P F & McIntosh, J R, Nature 193 (1962) 347. Daniel, J C, J exptl zoo1 154 (1963) 231. 1;: Fridhandler, L, Exptl cell res 22 (1961) 303. 11. Fridhandler, L, Hafez, E S E & Pincus, G, Exptl cell res 13 (1957) 132. 12. Fridhandler. L. Wastila. W B & Palmer. W M. Fertility & sterility 18 (1967) 819. 13. Glynn, I M, J physiol 134 (1956) 278. 14. - Pharmacol rev 16 (1964) 381. 15. Lutwak-Mann. C. Imolantation of ova fed P Eckstein). Cambridge University Press, London (1959). 16. Lutwak-Mann, C, Boursnell, J C & Bennett, J P, J reprod fertil 1 (1960) 169. Senft, J P, J gen physiol 50 (1967) 1835. ii: Tuft,.P H &-B&&g, B G, ‘Carnegie inst Wash yearbook 67 (1967-1968) 455. 19. Ussing, H H, Handbuch der experimentellen Pharmakologie (ed 0 Eichler & A Farah) .n. 63. Springer, Berlin‘(1963). Received February 2, 1970

Exptl Cell Res 62