Surface contraction of dividing newt eggs induced by lowering the external ionic strength or ph, or by trypsinization

During studies on the contractility of amphibian eggs, I observed that when dividing newt eggs were transferred from a saline to a sucrose medium, their newly formed, unpigmented egg surface shrank extensively. This shrinkage was rapid and non-lethal and was found to be due to active contraction of the unpigmented region. Similar shrinkage was observed on lowering the extracellular pH or on trypsinizing the eggs.

Contractile events in amphibian eggs are related to the actomyosin system (Perry et al. 1971; Clark & Merriam, 1978; Meeusen et al. 1980) and are inhibited by cytochalasin B (Luchtel et al. 1976; Selman et al. 1976; Merriam & Christensen, 1983; but see Manes et al. 1978; Merriam et al. 1983). The presence of external Ca²⁺ is necessary for contraction following cortical pricking (Holtfreter, 1943), or treatment with detergent or polycation (Gingell, 1970), although not in normal cleavage (Baker & Warner, 1972), or in contraction due to exposure to Ca ionophore (Schroeder & Strickland, 1974), or injection of detergent (Kubota, 1979). In the above studies, the contraction induced experimentally in living eggs has been observed in only the pigmented surface.

The contractile response reported here occurs in only the unpigmented surface and without exogenous Ca²⁺. From the present findings, a new mechanism inducing contraction is suggested.

Fertilized, uncleaved eggs of the newt, Cynopus (Tritrus) pyrrhogaster, were obtained by ovulation induced with the chorion gonadotropin hormone, and their capsules were removed surgically.

In this study, Steinberg solution (SS; 59mM-NaCl, 0·67mM-KCl, 0·83mM-MgSCh, 0·34mM-Ca(NO3)2, and buffer) was used and was adjusted to various pH values, with 5 mM-citric acid at pH 4·0-6×00B7;0, 5 mM-PIPES at pH 6·5-7·4, 5 mM-HEPES at pH 8·0, and 5 mM-boric acid at pH 9·0 and 10·0. These solutions showed no appreciable harmful effect during the 5·10 min observation periods. Eggs were bathed in SS at pH 7#x00B7;4 until cleavage started and then transferred to a Petri dish containing SS at pH 7’0, unless otherwise noted. The Petri dish was 6 cm in diameter and its surface was coated with Paraplast (Monoject). The individual eggs were deprived of their vitelline membrane with watch-makers’ forceps, and placed on a concave paraffin surface (about 5 mm in diameter, 0’7 mm deep). As cleavage progressed, new, unpigmented surface became exposed to the medium.

The bathing medium was changed during the period between when the boundary of the pigmented and unpigmented egg surfaces had been established and when the furrow bottom became invisible by apposition of the deeper part of the unpigmented surfaces. During medium change, mechanical injury to the egg was prevented by placing a glass tube over the egg on the paraffin bed; then the old medium was removed, 20-30ml of new medium was introduced, except when enzyme solutions were used, and the tube was then removed. The time when the new medium came in contact with the tube was taken as the time of medium change. In practice, about 0·2 ml of solution remained in the tube during medium change. It was found that washing the dish with soap immediately before introducing an egg was necessary to maintain a strong repulsive force of the paraffin surface against water.

In experiments on the effect of CO₂, a modified SS containing 20 mM-NaCl and 40 mM-NaHCO₃ as CO2 buffer was equilibrated with 100% CO₂, and mixed with SS of pH 6·5 in different ratios just before use. In this case, the dividing eggs studied had been cultured in SS of pH 6·5. The pH value of the 100% CO₂ solution introduced into the dish was initially 6·4—6·5, but increased gradually during the 10 min observation period.

Stock solutions of trypsin (Sigma, type III) and trypsin inhibitor (from soy bean; Sigma) were prepared every day during the experiment and kept cool. Individual cleaving eggs were bathed in SS at pH 7·4 before trypsin treatment and then in 0·2, 2 and 15-20 ml of SS at pH 7·4 containing trypsin at 2500, 250 and 250·25 ·g ml⁻¹, respectively. A stock solution of 1 mg of cytochalasin B (Aldrich) in 1 ml of dimethyl sulphoxide (DMSO) was prepared, and diluted with SS to 10 μg ml⁻¹ for use. Solutions of polylysine (M_r 55 000; Sigma), sialic acid (Wako), concanavalin A (ConA; Sigma), Ricinis communis agglutinin (RCA; Boehringer-Mannheim), wheat germ agglutinin (WGA; Vector) and Pronase E (Riken) were also prepared just before use.

When the external medium of dividing newt eggs was changed from SS to 0·1 M-sucrose solution containing 1 mM-CaCl₂ and buffered with 1 mM-Na-PIPES at pH7·0 (sucrose medium, SM), the unpigmented surface shrank extensively (Fig. 1). This response was accompanied by: (1) transient lifting of the furrow bottom at an early stage of contraction; (2) rounding of the whole egg, which in amphibian eggs shows increase in tension of the cell surface (Harvey & Fankhauser, 1933); and (3) compression of a papilla with a slender neck present on the unpigmented surface into a small darkish spot. These changes indicate that the primary cause of change in area was active contraction of the unpigmented surface itself, not passive shrinkage resulting from relaxation of the adjacent pigmented layer. Thus, the decrease in area of the unpigmented layer is hereafter called contraction.

The higher density of SM should round-up dipped eggs by decreasing their relative weight and thus should reduce the furrow width, resulting in an apparent increase in contraction. But in fact this density effect was minute, because, when eggs (n = 3) were exposed to SS containing Ficol of the same density as SM, their average furrow width deviated only 3% in 5 min from the value in normal SS. This small deviation was determined from photographs taken at 1-min intervals before and after exposure to Ficol solution.

Eggs that had contracted or had begun to contract during brief exposure to SM showed increase in furrow width when returned to SS. After an interval, these eggs could respond to SM again (Fig. 2). Since in intact control eggs at the same cleavage stage the furrow width increased about 6% in 10 min, the early quick reversal of furrow width seen here seemed to be due mainly to relaxation of the contracted surface, although the extent of this reversal was not large. The extent of early reversal decreased with the time of exposure to SM within treatment times of 1·5-3×00B7;5 min. Eggs kept continuously in SM cleaved without relaxation and. entered the second division and, the following day, some of them formed compact masses of cell sizes comparable to those of late morulae or early blastulae under unsterilized conditions.

Dividing eggs were bathed in two types of Ca-free SM, one with 1 mM-Na-EGTA at pH 7-0 and the other with 3mM-Na-EGTA at pH 8·0, to lower the Ca²⁺ concentration of the medium. Extensive contraction occurred in both media, although its greatest extent was less in 3 mM-EGTA medium (Fig. 3). These results show that external Ca²⁺ is not absolutely essential for the contraction. The following procedures also had little or no effect on the contraction : change in the sucrose concentration from 100 mM to 50 or 150 mM to make the SM hypotonic or hypertonic; replacement of sucrose by an equimolar concentration of sorbitol; addition of the minor components of SS, 0·67mM-KCl and, or, 0·83 mM-MgS0₄, to SM. However, Ca²⁺ and sucrose were added to the medium in this study, because in their absence local membrane fracture often occurred during stretching of the polar egg surface far from the contracting unpigmented region.

On replacement of sucrose by NaCl in a molar ratio to sucrose of 59:100, the degree of contraction decreased with increase in NaCl concentration to 29-5 mM (Fig. 4). This means that removal of NaCl by medium change triggered surface contraction. This effect was not specific to NaCl, as demonstrated by the fact that when 59 mM-NaCl in SS was replaced by an equimolar concentration of KC1, choline chloride, or NaCHjCOO (each n = 10), eggs showed only weak responses: the minimum mean CI value in the first 5 min was —15% in choline medium. Moreover, subsequent exposure to SM induced marked contraction. Thus the surface response in SM seemed to be due to decrease in the extracellular ionic strength.

Eggs were treated with SS containing 10/rgml⁻¹ cytochalasin B (and 1% DMSO) for 8-10 min until the cleavage furrow had almost disappeared, and were then exposed to SM containing cytochalasin B at 10μgml⁻¹. During the subsequent 5 min, little contraction (final mean CI, —7%) was observed in 4 of 7 eggs, while the unpigmented surface ruptured in the other 3. In another experiment with less-drastic results, soon after the appearance of a primordial unpigmented surface, eggs with their vitelline membrane (n = 10) were transferred to SS with 10/tgml⁻¹ cytochalasin B and, 15-20 min later when the furrow had regressed and the boundary of the unpigmented surface became definite, they were transferred to cytochalasin-free SM. All the eggs showed much weaker contraction in 5 min (final mean CI, —18%) than that of controls not previously exposed to cytochalasin B (—70%; n = 10). Addition of 1% DMSO to normal SS and SM did not affect progress of cleavage and contraction, respectively. These results demonstrate that the surface response is affected by cytochalasin B.

Contraction was also induced when eggs were bathed in SS at pH 6·0, 5·0 and 4-0 in the presence (Fig. 5) and absence of Ca²⁺, but not when they were exposed to alkaline solutions at pH 8·0, 9·0 and 10·0: in the latter solutions the furrow width increased slightly. In acidic solutions the response increased with decrease in the pH value. The contraction induced in acidic medium, especially at pH 4·0, resembled that at low ionic strength in both the rapidity of CI change and the absence of requirement for exogenous Ca²⁺. Eggs treated with acidic SS retained responsiveness to SM, and eggs incubated overnight in acidic SS cleaved into many blastomeres, although the adhesion of adjacent cells became incomplete to various degrees.

The possibility that acidification of the medium lowered the intracellular pH, and that this induced a surface response was examined by experiments on the effect of CO₂. Results showed that eggs exposed to different CO₂ concentrations did not show significant responses (Fig. 6A): the minimal CI value observed after 10 min (—13% in 20% CO₂ solution; see Fig. 1e gend) was very different from the values observed after 5 min in media at pH4·0 and 5·0 (about —40%). After exposure to CO₂, eggs responded more slowly to SM, their rates of response decreasing with increase of CO₂ concentration, indicating that under certain conditions intracellular acidification even has an inhibitory effect (Fig. 6B). It is very unlikely that an increase in tension in the pigmented area exposed to CO₂ hindered contraction of the unpigmented area since, as shown in Fig. 6A, during exposure to CO₂ the diameters of eggs were decreased in the first 1·5-5 min, indicating rounding-up, but did not decrease during the later period from 6 to 10 min.

The effects of various reagents dissolved in SS at pH 7·4 on contraction of eggs in 5min were examined with the following results: 0·1-1 mgml”¹ WGA, lmgml⁻¹ ConA, and lmgml⁻¹ RCA had little or no effect (minimum mean CI values, >—10%); 0·7-5mgml⁻¹ sialic acid and lmgml⁻¹ polylysine had slight effects (> —20%) ; proteolytic enzymes had marked effects.

On treatment with trypsin at 0·25-2500μg ml⁻¹, contraction in dividing eggs increased with the trypsin concentration (Fig. 7); the minimal CI values in 5 min (— 34 to —40%) were observed with trypsin at 25-2500 μg ml⁻¹. Contraction was also observed with 0·05-1% Pronase E. Exposure to trypsin at 2500 μg ml⁻¹ caused breakage of the egg membrane in 10-20 min, sometimes with furrow regression, but many eggs exposed to trypsin at 250μgml⁻¹ for 20 min and subsequently to SS showed neither cytolysis nor marked retardation of cleavage by the following day. The time course of CI change was similar to those at low ionic strength and low pH, and the response occurred again without external Ca²⁺. Addition of trypsin inhibitor to trypsin solutions of 2500 (n = 10), 25 (n = 4) and 2-5 (n = 4) μgml⁻¹ consistently suppressed the response completely, indicating that the effect of trypsin is due to its proteolytic action.

Similar results were obtained with trypsin at 2500 μg ml⁻¹ in modified SS in which 59 mM-NaCl was replaced by an equimolar concentration of KC1, choline chloride or NaCH₃COO (Fig. 8). Thus K⁺ efflux, if any, does not mediate the response and the contraction induced by trypsin is possible in Na⁺-free and Cl⁻-free conditions as in SM.

In the present experiments, the new unpigmented surface of dividing newt eggs showed extensive contraction after three different kinds of treatment : with decreased extracellular ionic strength or pH, and with proteases. The responses to these three treatments were similar in their localization, rapid time course, and absence of absolute requirement for external Ca²⁺, suggesting that they were due to the same contractile mechanism.

In amphibian eggs, microfilaments or an actomyosin system are involved in contractile events such as cleavage (Bluemink, 1970; Selman & Perry, 1970; Meeusen et al. 1980) and wound healing (Bluemink, 1972), and cytochalasin blocks both cleavage (Luchtel et al. 1976) and wound healing (Merriam & Christensen, 1983), and also weakens and spreads the unpigmented surface (Selman et al. 1976). The present experiment showed that contraction of the unpigmented surface was also inhibited by cytochalasin B. From these findings it is highly probable that microfilament organization is involved in the contractile responses.

Holtfreter (1943) reported that Ca²⁺ is required for wound closing of amphibian eggs. Gingell (1970) proposed that the polycation- and detergent-induced contraction of the pigmented surface of Xenopus eggs is mediated by Ca²⁺ influx, but the response reported here occurred in the absence of external Ca²⁺ : the requirement for Ca²⁺ seemed to be met internally, as in normal cleavage (Baker & Warner, 1972) and in contraction induced by treatment with a Ca ionophore (Schroeder & Strickland, 1974) or injection of detergent (Kubota, 1979).

Ion currents through the membrane have been detected in dividing amphibian eggs: there is an outward current caused by K⁺ efflux in the unpigmented surface (e.g. see Woodward, 1968; de Laat et al. 1974), and an inward current possibly due to Na⁺ influx, Ca²⁺ influx and Cl⁻ efflux, which is at its maximum in the pigmented region bordering the unpigmented region (Kline et al. 1983). However, K⁺, Na⁺ and Cl⁻ fluxes as well as Ca²⁺ influx, if they occur during contraction, are not essential for initiation or mediation of the response, since contraction induced by trypsin occurred normally when these possible fluxes were prevented by changing ion diffusion gradients across the membrane. With regard to the effect of H⁺, acidification of the bathing medium induced contraction, but acidification of the egg cytoplasm with CO₂ did not cause a marked response, and rather inhibited subsequent contraction in SM. These results suggest that the response is not due to decrease in intracellular pH, a possibility that is compatible with the observations in Xenopus eggs that the furrow regresses when the cytoplasm is acidified 0·65 pH unit below normal (Lee & Steinhardt, 1981) and that the internal pH is little affected by the external pH (Webb & Nuccitelli, 1981). Thus the present findings, together with previous reports, suggest that the response induced by lowering the pH of the medium is mediated via a surface change.

Many studies have shown that, in culture, spreading mammalian cells rapidly round-up and become detached on treatment with trypsin. This rounding-up is associated with simultaneous reorganization of the cytoskeleton (Lazarides, 1976; Sobel, 1983), although it is uncertain whether this change in cell shape is due to active surface contraction, because it also occurs on treatment with cytochalasin D alone (Miranda et al. 1974), and the discontinuous distribution of subsurface microfilaments in trypsinized cells (Furcht & Wendelschafer-Crabb, 1978) is not compatible with generation of a force. Vogel (1978) proposed that reorganization of the cytoskeleton on protease treatment may be mediated by an initial cleavage of surface glycoprotein. In the present case, this transmembrane process is supported by the fact that the unpigmented surface is coated with a thin layer of glycocalyx staining with Ruthenium Red (Kalt, 1971; Bluemink & de Laat, 1973) and Lanthanum Red (Singal & Sanders, 1974); the response began after a short delay (about 30 s) on exposure to trypsin, and this lag time was the same as that of the response to low pH (4-0), which was probably mediated by a surface change.

A mechanism that would probably lead to contraction in the unpigmented surface is as follows. Since, in general, reduction of the external ionic strength or pH changes the surface charges of cells, these treatments may modify the conformation of surface molecules containing glycoprotein by altering the balance of their positive and negative charges, as suggested in the case of red blood cells (Donath & Gingell, 1983; Wolf & Gingell, 1983). Trypsinization could also cause structural changes by cleavage of molecules and subsequent electrostatic equilibration. These conformational changes may in turn cause changes in organization and activation of the underlying cytoskeleton, leading to contraction (cf. Geiger, 1983).