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vegetal polarity in the amphibian egg
DEVELOPMENTAL
BIOLOGY
62, 334-343 (1978)
The Plasma-Membrane IMP Pattern as Related to AnimalNegetal Polarity in the Amphibian Egg JOHN Hubrecht
G. BLUEMINK
Laboratory,
International
Received June 6,1977;
AND
LEON
Embryological
G. J. TERTOOLEN Institute,
Utrecht,
The Netherlands
accepted in revised form September 16,1977
Freeze-fracture electron microscopy of the plasma membrane of the fertilized, uncleaved Xenopus egg shows that intramembranous particles (IMPS) range in size from ca. 50 to 200 A and that more IMPS are attached to the E-face than to the P-face. The overall IMP densities of the animal and the vegetal hemisphere do not differ significantly. IMP-free regions (4, ca. 0.1 Frn) on the tips of surface protrusions were irregularly distributed in the animal and the vegetal half (E-face) occupying ca. 8.5 and 2%, respectively of the free area. The relative densities for 16 different IMP sizes have been compared, on the basis of seven animal and seven vegetal halves, counting (E-faces only) ca. 10,000 IMPS in each hemisphere. For IMP sizes of 581 A, a significant difference (P < 0.0005) was found, more small IMPS being present in the animal half. Some evidence for IMP-associated thin elements was found. These findings are discussed in relation to plasma membrane anisotropy and the morphogenetic role of the egg cortex. INTRODUCTION
Experimental evidence exists that the distribution of cytoplasmic components in the uncleaved, fertilized egg may depend on the cortex, i.e., the plasma membrane and its associated structures (Dollander, 1961; Pasteels, 1964; Arnold and WilliamsArnold, 1974). According to the selective gene activity theory of differentiation, the differential distribution of cytoplasmic components among embryonic cells elicits different patterns of gene activity. Hence it can be assumed that information relevant to the pattern of gene action is contained in an anisotropic arrangement of plasma-membrane constituents. Accepting the idea that a cortical pattern of information can be set up and maintained for some time in the form of an anisotropic organization of the plasma membrane, one must assume that such a membrane is static/rigid rather than fluid/dynamic (see Singer and Nicolson, 1972). Topographical heterogeneity in the plane of a continuous membrane can presumably exist by virtue of cross-linking cytoplasmic elements, i.e., microfilaments and microtubules. 334 0012-1606/78/0622-0334$02.00/O Copyright All rights
0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
Using freeze-fracture electron microscopy, it is possible to analyze local differences in plasma membrane composition. The aim of the present study was to compare the animal and vegetal hemispheres of the fertilized, uncleaved Xenopus egg (stage 1; Nieuwkoop and Faber, 1967). Lack of differences in the distribution pattern of intramembranous particles (IMPS) between the two membrane regions would provide evidence against the concept that animal/vegetal polarity may reside in an anisotropic arrangement of plasma-membrane components. MATERIALS
AND
METHODS
Eggs of Xenopus laevis were obtained and prepared as described earlier (Bluemink et al., 1976). Membrane fragments obtained from uncleaved, fertilized eggs were used for freeze-fracturing. The membrane fragments were isolated from the animal or the vegetal hemisphere as follows. Small squares (5 x 8 mm) of glass cleaned overnight in chromic acid were rinsed in distilled water and air-dried. The clean squares were etched for ca. 1
BLUEMINK
AND TERTOOLEN
Membrane
min in a 3:l mixture of barium sulfate and ammonium fluoride/sulfuric acid (l:l), thoroughly rinsed, and air-dried. They were then briefly exposed to a solution of 10 mg/ml of poly-L-lysine (Sigma Chemical Co., St. Louis, MO.) in water, thoroughly rinsed with distilled water, and air-dried (Mazia et al., 1975). For obtaining isolated pieces of membrane, the vegetal or animal hemisphere of a decapsulated egg (without vitelline membrane) was brought into contact with the polylysine-coated surface in water. ARer 10 min of contact, the decapsulated egg sticks to the glass. Redundant parts of the egg can be removed with a small pipet, leaving a fragment of the plasma membrane together with a thin layer of cytoplasm attached to the glass. Such specimens, obtained from eggs at about 30 min before the first cleavage, were fixed for lo-30 min in 2.50% glutaraldehyde in Steinberg solution (Steinberg, 1957) at room temperature, rinsed, and equilibrated against 20% glycerol in Steinberg solution for at least 2 hr. A thin perforated metal (Vacon 12) plate (2 x 6 mm) was put on top, and the sandwiched specimen was rapidly immersed in a thawed layer of nitrogen and pressed against the solid cold nitrogen underneath. For fracturing, the frozen sample was inserted into a fracture tool for sandwiched specimens, a moditication of the device described by Pfenninger and Rinderer (1975). Fracturing of the membrane was achieved by prying the metal plate off the glass support. Fracturing, etching, and coating were carried out at -100°C in a vacuum of better than lo+ Torr in a Denton instrument (type D.F.E.-3). The platinum/carbon-coated replicas were cleaned by exposing them to 1:l sodium hypochlorite in water for at least 2 hr at room temperature. They were then rinsed in distilled water and collected on single-hole grids covered with a carbon-coated supporting film of Pioloform F (Stockem, 1970). The replicas were inspected in a Zeiss EM-10 electron microscope at an
Structure
of Xenopw
Egg
335
acceleration voltage of 80 kV using a 60m objective aperture. A carbon difTraction grating replica of 54,864 lines per inch was used to obtain accurate magnification of specimen negatives at identical microscope settings. For reproducibility of the microscope setting, a selected grid holder was used for all EM recordings. Hysteresis effects were eliminated by using the Zeissmonostable device to switch the lens current control to maximum before setting at the selected magnification step. This makes for a relative reproducibility of the magnification of better than 1.5%. Specimen negatives (Kodak electron microscope film 4489, estar base) at a magnification of x40,185 were enlarged 7.5 times on Kodak Veribrom (plastic base). A square of 10 x 10 cm was outlined on the micrographs, and all IMPS within the square were measured. The area of the measured region and the dimensions of the IMPS were estimated with a Wang digitizing system (type 2262-l; point resolution, 0.25 mm) in combination with a Wang 2200 calculator. IMP sizes were determined by measuring the widest part of the shadow at right angles to the direction of the shadow. The freeze-fracture nomenclature is as proposed by Branton et al. (1975). RESULTS
Plasma membrane regions obtained from seven animal and seven vegetal hemispheres were used. The isolated membrane fragments (area, ca. 0.2-0.6 mm’) were taken invariably in a region within a radius of ca. 0.4 mm from the center of either the animal or the vegetal pole. The membrane fragments attached to a polylysine-coated glass surface gave rise to large membrane areas exposing the inner aspect of the external leaflet that remains attached to the glass (E-face), whereas in the same replicas inwardly folded edges of the membrane fragments gave rise to Pfaces. The exposed P-face area was always far less than the E-face area. It is generally accepted that in freezefracture replicas of split membranes the
336
DEVELOPMENTAL BIOLOGY
IMPS represent protein complexes, while the smooth interface represents the cleaved lipid bilayer (Marchesi et al., 1972; Pinto Da Silva, 1972; Pinto Da Silva et al., 1973; Singer and Nicolson, 1972; Bullivant, 1973; Tourtellotte and Zupnik, 1973). The primary aim of the present study was to find a qualitative difference in IMP population of the animal versus the vegetal hemisphere. To discriminate different IMP sizes in a range of ca. 20-200 A, electron micrographs of high magnification (x 301,400) are required. Exclusively IMP-populated areas of E-faces were used for making the counts; IMP-free areas (4, ca. 0.1 pm) were not involved. Most IMPS are attached to the E-face (Figs. l-4). The mean numbers of IMPS per square micrometer amount in the animal hemisphere to 1934 f 262 and in the vegetal hemisphere to 1872 -+ 229. AccordTABLE
VOLUME 62, 1978
ing to Student’s t test, using the 0.95 confidence interval for the mean, these densities are not significantly different (P > 0.10). IMP-free regions (4, ca 0.1 pm) on the tips of surface protrusions (Fig. 3; see also Bluemink et al., 1976a) were irregularly distributed in the animal (Fig. 3) and the vegetal (Fig. 4) half occupying ca. 8.5 and 2%, respectively, of the free area. Presumably because in these preparations the plasma membrane has been firmly attached to the polylysine-coated substrate, many protrusions show hardly any depth. The IMPS vary greatly in size. For the measurement of IMP sixes 110 electron x 301,387) micrographs (magnification, were used, covering 58 x 0.1 pm2 of the animal hemisphere (seven eggs, 12,216 IMPS) and 52 x 0.1 pm2 of the vegetal hemisphere (seven eggs, 9734 IMPS). The 1
FREQUENCIES OF IMPS, SUBDIVIDED INTO SIZE CLASSES, ON THE PLASMA-MEMBRANE E-FACES OF THE ANIMAL AND THE VEGETAL HEMISPHERE, RESPECTIVELY, TOGETHER WITH STATISTICAL ANALYSIS IMP size Animal hemisphere Vegetal hemisphere PC (A) N” SEM c1* CI N SEM 56 64 72 81 89 97 105 113 121 129 137 145 154 162 170 178
t
39 59 76 115 113 165 194 195 221 194 168 147 94 61 35 20
5 6 8 9 10 11 9 10 10 10 11 12 10 8 5 4
29-49 48-70 60-92 98-132 95-131 144-186 176-212 175-215 201-241 174-214 147-189 123-171 75-113 46-76 25-45 13-27
18 34 43 70 93 142 225 223 245 225 201 145 91 48 31 18
3 4 4 5 7 10 13 10 9 10 13 10 7 6 4 3
13-23 27-41 35-51 60-80 80-106 121-161 199-251 203-243 228-262 208-244 176-226 125-161 76-106 37-59 22-42 12-24
u N = average number of IMPS per square micrometer. ’ CI = variance at 0.95 confidence interval for the mean (7’ factor = 1.982 x SEM value). c P values are for the two-tailed probability estimated for 108 degrees of freedom according test.
<0.0005 <0.0005 <0.0005 <0.0005 >0.05 >O.lO >0.05 >0.05 >0.05 -co.05 co.05 z-o.5 >0.5 >O.l >0.5 >0.5
to Student’s
FIG. 1. E-face, animal hemisphere. EF, external face; arrows, IMPS of spiny appearance; arrowhead, cluster of ca. seven small IMPS. x 320,000. This and all following micrographs show the plasma membrane of the Xenopus egg. Encircles arrowheads indicate direction of shadowing in micrographs. FIG. 2. E-face, vegetal hemisphere. Arrow, IMPS of spiny appearance. x 320,000.
338
DEVELOPMENTAL BIOLOGY
IMPS, ranging in size between 56 and 178 A, were subdivided into 16 classes (8-A intervals). The results of these measurements together with the statistical values are given in Table 1. Figure 5 represents the same data in graphical form. Only for IMP sizes of 581 A are the plasma membranes of the animal and the vegetal halves significantly different (P < 0.0005; see Table 1); that is, more small IMPS are present in the plasma membrane of the animal hemisphere. For IMP sizes of >81 A, the relative frequencies for both membranes are not significantly different, except for the 129- and 137-A size classes (P < 0.005, see Table 1); the graph does not illustrate this because the shaded areas overlap slightly. In the animal hemisphere, IMPS of different sizes were not evenly mixed. Small IMPS distributed among larger ones occasionally form aggregates of ca. 4-10 IMPS having almost no interspace between them (Fig. 1). No such clustering of medium or large IMPS has been observed in either the animal or the vegetal half. In the replicas, inwardly folded edges of the membrane fragments gave rise to Pfaces. Invariably the exposed P-face area was far less than the exposed E-face area. The P-faces had many small pits and less IMPS were attached to the P-face (ca. 100 IMPs/pm2) than to the E-face, just as has been reported before (Bluemink et al., 1976a,b). P-faces from the animal and the vegetal hemisphere showed no obvious difference in IMP density. In comparing the IMP population in the animal half versus the vegetal half, the P-faces have not been involved because of the intrinsic low IMP density. Close scrutiny reveals that the IMPS are not simple globules. Filament-like extensions give a spiny appearance to the profiles of many IMPS (Figs. 1 and 2). FIG. 3. E-face, animal hemisphere. 38,500. FIG. 4. E-face, vegetal hemisphere.
VOLUME 62, 1978
Occasionally longer “filaments” are seen to extend from an IMP or, more rarely, to run from one IMP to another (Figs. 8 and 9). In replicas where the direction of shadowing happens to be perpendicular to the direction of the “filaments,” the shadows conform to those of the IMPS suggesting that they exist as ridges on the matrix. Stereomicrographs (not illustrated) have provided conclusive evidence that the “filaments” exist as elevations in the profile of the replica. In the replica in areas of clean ice, or in IMP-free areas of the cleaved membrane bilayer, single Xlaments,” i.e., not associated with IMPS, have not been observed. DISCUSSION
The plasma membrane of the amphibian egg is peculiar in several respects. The IMPS are larger than usual (Bluemink et al., 1976; Sanders and DiCaprio, 1976a,b; Smith et al., 1976; Decker and Friend, 1974). The replicas of the polylysine-attached membrane fragments have provided irrefutable evidence that the IMPS attached to the E-face outnumber the IMPS seen on the P-face. This finding conforms to our earlier observations (Bluemink et al., 1976a,b) based on replicas of whole eggs prepared with a different technique. More IMPS attached to the Eface than to the P-face have been found also in the plasma membrane of early embryos of another amphibian (Smith et al., 1976). Apart from amphibian embryos a reversal of asymmetry in IMP distribution has been reported for a few other biological systems (Allen, 1976; Wade et al., 1975). The overall IMP densities of the animal (1934 -+ 262 IMPS/pm*) and the vegetal hemisphere (1872 ? 229 IMPs/~m2) do not differ significantly (P > 0.10). When a correction is made for the 8.5% IMP-free
Stars, particle-poor
regions corresponding
Stars, particle-poor
regions.
x 38,500.
to surface protrusions.
x
340
DEVELOPMENTALBIOLOGY 300
VOLUME 62, 1978
-
200-
100
-
I 40
/ 56
I 72
I
/ 69
,
1 105
I
I 121
I
I 137
I 154
I
I 170
I
I
5 FIG. 5. Frequency distributions of IMPS subdivided into 8-8 classes. Vertical, number of IMPS per square micrometer; horizontal, IMP size in angstrom units; solid dots, animal hemisphere; open squares, vegetal hemisphere. The shaded areas indicate the variance at the 0.95 confidence interval for the mean (T-factor = 1.982 x SEM).
area in the animal and the 2% IMP-free area in the vegetal hemisphere, the estimated overall densities of the animal (1770 f 262 IMPs/pm2) and the vegetal hemisphere (1834 + 229 IMPs/pm2) are still not significantly different. Comparing the IMP-populated E-faces, it could be shown that the IMP pattern in the animal half differs significantly from that seen in the vegetal half, more small IMPS (581 A) being present in the animal hemisphere. In addition a small difference in density of the 129- to 137-A IMPS was found. In the animal half, the small (181 A) IMPS partly occur as aggregates, but the possibility cannot be excluded that aggregation is an artifact produced during fixation. Plasma membrane anisotropy is known to exist in the mouse egg (Johnson et al., FIG. FIG. FIG. FIG.
6. 7. 8. 9.
P-face, animal hemisphere. P-face, vegetal hemisphere. E-face, animal hemisphere. Same as for Fig. 8.
1975), the fucoid egg (Robinson and Jaffe, 1975; Nuccitelli and Jaffe, 1976), and the ascidian egg (Ortolani et al., 1977). So far only one other report has provided evidence for plasma-membrane anisotropy related to egg polarity as studied by freezefracture electron microscopy (Peng, 1976). An estimate of the overall IMP density during polarization in fucoid eggs (Peluetia) has shown that at 6 hr after fertilization more IMPS per square micrometer are found at the thallus end (1460 f 150) than at the rhizoid end (1010 f 60). This difference becomes more prominent at 8 hr after fertilization. The functional implication of plasma membrane anisotropy as related to egg polarity is that the pattern of membrane components may exert a regulatory effect on the distribution of cytoplasmic constit-
Small number of IMPS and small pits (arrow). x 160,000. Small number of IMPS and small pits (arrow). x 160,000. IMP-associated thin filaments (arrows). x 504,000.
342
DEVELOPMENTAL BIOL~CY
uents. The local differences in IMP pattern may constitute a form of “macromolecular braille” coding for positional information (McMahon and West, 1976). Membraneassociated elements of the cytoskeleton, i.e., microfilaments and microtubules (see Morrill and Perkins, 1973), may transmit the effect of local differences in the plasma membrane to the cytoplasm. The question whether the IMP-associated filaments are artifacts or structural components is open to discussion. It could be suggested that they represent cracks in the fracture face which are related to the direction of fractioning or that they arise artificially by plastic deformation of membrane-associated proteins. In the replicas there was a fixed angle between the direction of membrane splitting and the direction of shadowing. Using the direction of shadowing an an indicator, one can determine that the orientation of the IMP-associated filaments has no fixed angle to the direction of membrane splitting (see Fig. 9). It has been reported that plastic deformation of actin occasionally may give rise to “filaments” on the E-face of freeze-fractured membranes (McNutt, 1976). Filamentous actin (Perry et al., 1971), as well as myosin, occur in large amounts in the cortical cytoplasm of the amphibian egg (Franke et al., 1976; see this reference for further literature). Thus it is conceivable that during freeze-fracturing filaments drawn out from the cortical cytoplasm have appeared on the E-face. However, actin or myosin filaments would measure 50 h; or more (see Franke et al., 1976), whereas the filaments on the E-face obviously are much thinner. The 20-A IMP-associated filaments could represent the thin elements of the cortical anastomosing network (Franke et al., 1976) drawn out from the cortical cytoplasm during freeze-fracturing. It is conceivable that the IMPS are anchored to the elements of the cortical network in the same way as the IMPS in the erythro-
V~LIJME 62, 1978
cyte membrane (Elgsaeter and Branton, 1974; Elgsaeter et al., 19761, which are anchored to the membrane-associated spectrin network. In the amphibian egg, structural continuity with the cortical anastomosing network would provide the delicate plasma membrane the tough support needed to withstand stress forces during morphogenesis (Beloussov et al., 1975) and to define cell shape. The topographical differences in IMP distribution could rely on a spacing mechanism for which the elements of the cortical network form the anchorage sites. However, stronger evidence than that presented in this report will be necessary to prove the existence of such a mechanism. Grateful acknowledgment is made to Dr. J. Faber for editorial help and to Dr. S. W. de Laat for making the computer program to evaluate IMP sizes. REFERENCES R. D. (1976). Freeze-fracture evidence for intramembrane changes accompanying membrane recycling in Paramecium. Cytobiologie 1% 254-273. ARNOLD, J. M., and WILLIAMS-ARNOLD, L. D. (1974;. Cortical-nuclear interactions in cephalopod development: Cytochalasin B effects on the informational pattern in the cell surface. J. Embryol. Exp. Morphol. 31, l-25. BELOUSSOV, L. V., DORFMAN, J. G., and CHERDANTZEV, V. G. (1975). Mechanical stresses and morphological patterns in amphibian embryos. J. Embryol. Exp. Morphol. 34, 559-574. BLUEMINK, J. G., TEBTOOLEN, L. G. J., VERVERGAERT, P. H. J. Th., and VERKLEIJ, A. J. (1976a). Freeze-fracture electron microscopy of preexisting and nascent cell membrane in cleaving eggs of Xenopus laevis. Biochim. Biophys. Acta 443, 143155. BLUEMINK, J. G., TERTOOLEN, L. G. J., VERVERGAERT, P. H. J. Th., and VERKLEIJ, A. J. (1976b). Membrane biogenesis in dividing eggs of Xenopus Zocvis analysed by freeze-fracture electron microscopy. In “Proceedings of the Sixth European Congress on Electron Microscopy (Jerusalem),” Vol. 2, pp. 348-350. TAL International Publishing Company, Israel. BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY, M. J., MOOR, H., M~~HIJETHALER, K., NORTHCOTE, D. H., PACKER, L., SATIR, B., SATIR, P., SPETH, V., STAEHELIN, L. A., STEERE, R. L.,
ALLEN,
BLUEMINK
AND TERTOOLEN
Me ‘mbrune Structure
and WEINSTEIN, R. (19’75). Freeze etching nomenclature. Science 190, 54-56. BULLIVANT, S. (1973). Freeze-etching and freezefracturing. In “Advanced Techniques in Biological Electron Microscopy” (J. K. Koehler,,ed.), pp. 67112. Springer-Verlag, Berlin. DECKER, R. S., and FRIEND, D. S. (1974). Assembly of gap junctions during amphibian neurulation. J. Cell Biol. 62, 32-47. DOLLANDER, A. (1961). Le cortex de l’oeuf d’amphibien. Anat. Anz. 109 (Ergiinzung), 274306. Verhandl. 1 Eur. Anat. Kongr. Strassbourg, 1960. ELGSAETER, A., and BRANTON, D. (1974). Intramembrane particle aggregation in erythrocyte ghosts. I. The effects of protein removal. J. Cell Biol. 63, 1018-1030. ELGSAETER, A., SHOTTON, D. M., and BRANTON, D. (1976). Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation. B&him. Biophys. Actu 426,101-122. FRANKE, W. W., RATHKE, P. C., SEIB, E., TRENDELENBURG, M. F., OSBORN, M., and WEBER, K. (1976). Distribution and mode of arrangement of microfilamentous structures and actin in the cortex of the amphibian oocyte. Cytobiologie 14, lll130. JOHNSON, M. H., EAGER, D., and MUGGLETON-HARRIS, A. (1975). Mosaicism in organisation of concanavalin A receptors on surface membrane of mouse egg. Nature (London) 257, 321-322. MARCHESI, V. T., TILLACK, T. W., JACKSON, R. L., SEGREST, J. P., and SCOTT, R. E. (1972). Chemical characterization and surface orientation of the major glycoprotein of the human erythrocyte membrane. Proc. Nat. Acad. Sci. USA 69, 14451449. MAZIA, D., SCHATTEN, G., and WINFIELD, S. (1975). Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy. J. Cell Biol. 66, 198-200. MCMAHON, D., and WEST, C. (1976). Transduction of positional information during development. In “The Cell Surface in Animal Embryogenesis and Development” (G. Poste, and G. L. Nicolson, ElsevierlNorth-Holland eds.1, PP. 449-493, Biomedical Press, Amsterdam. MCNUTT, N. S. (1976). Plastic deformation of actin in the production of filaments on the E-face of freeze-fractured plasma membranes. J. Cell Biol. 70, Ma. MORRILL, J. B., and PERKINS, F. 0. (1973). Microtubules in the cortical region of the egg of Lymnaeu during cortical segregation. Develop. Biol. 33, 206-212. NIEUWKOOP, P. D., and FABER, J. (1967). “Normal table of Xenopus luevis (Daudin),” 2nd ed. NorthHolland, Amsterdam.
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NUCCITELLI, R., and JAFFE, L. F. (1976). The ionic components of the current pulses generated by developing fucoid eggs. Develop. Biol. 49,518-531. ORTOLANI, G., O’DELL, D. S., and MONROY, A. (1977). Localized binding of Dolichos lectin to the early Ascidiu embryo. Exp. Cell Res. 106, 402404. PASTEEIS, J. J. (1964). The morphogenetic role of the cortex of the amphibian egg. Advan. Morphog. 3, 363-390. PENG, H. B. (1976). “Polarization of the Fucoid Egg: Galvanotropism and Ultrastructure.” Ph.D. Thesis, Perdue University. PERRY, M. M., JOHN, H. A., and THOMAS, N. S. T. (1971). Actin-like filaments in the cleavage furrow of the newt egg. Exp. Cell Res. 65, 249-253. PFENNINGER, K. H., and RINDERER, E. R. (1975). Methods for freeze-fracturing of nerve tissue cultures and cell monolayers. J. Cell Biol. 65, 15-28. PINTO DA SILVA, P. (1972). Translational mobility of membrane intercalated particles of human erythrocyte ghosts. J. Cell Biol. 53, 777-787. PINTO DA SILVA, P., Moss, S., and FUDENBERG, H. H. (1973). Anion sites on the membrane intercalated particles of human erythrocyte ghost membranes. Freeze-etch localization. Exp. Cell Res. 81, 127-138. ROBINSON, K. R., and JAFFE, L. F. (1975). Polarizing fucoid eggs drive a calcium current through themselves. Science 187, 70-72. SANDERS, E. J., and DICAPRIO, R. A. (1976a). A freeze-fracture and concanavalin A-binding study of the membrane of cleaving Xenopus embryos. Differentiation 7, 13-21. SANDERS, E. J., and DICAPRIO, R. A. (1976b). Intracellular junctions in the Xenopus embryo prior to gastrulation. J. Exp. 2001. 197, 415-421. SINGER, S. J., and NICOLSON, G. L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175, 720-731. SMITH, L. C., JOHNSON, R. G., and SHERIDAN, J. D. (1976). Gap junctions and tight junctions in early amphibian embryos: Freeze-fracture study. J. Cell Biol. 70, 255a. STEINBERG, M. (1957). Carnegie Inst. Washington Yeurb. 56, 347. STOCKEM, W. (1970). Die Eignung von Pioloform fiir die Herstellung elektronenmikroskopischer Trlgerfllme. Mikroskopie 26, 185-189. TOURTELLOTTE, M. E., and ZUPNIK, J. S. (1973). Freeze-fractured Acholeplusmu Zaidluwii membranes: Nature of particles observed. Science 179, 84-86. WADE, J. B., DISCALA, V. A., and Karnovsky, M. J. (1975). Membrane structural specialization of the toad urinary bladder revealed by the freezefracture technique. I. The granular cell. J. Membrane Biol. 22, 385-402.
BIOLOGY
62, 334-343 (1978)
The Plasma-Membrane IMP Pattern as Related to AnimalNegetal Polarity in the Amphibian Egg JOHN Hubrecht
G. BLUEMINK
Laboratory,
International
Received June 6,1977;
AND
LEON
Embryological
G. J. TERTOOLEN Institute,
Utrecht,
The Netherlands
accepted in revised form September 16,1977
Freeze-fracture electron microscopy of the plasma membrane of the fertilized, uncleaved Xenopus egg shows that intramembranous particles (IMPS) range in size from ca. 50 to 200 A and that more IMPS are attached to the E-face than to the P-face. The overall IMP densities of the animal and the vegetal hemisphere do not differ significantly. IMP-free regions (4, ca. 0.1 Frn) on the tips of surface protrusions were irregularly distributed in the animal and the vegetal half (E-face) occupying ca. 8.5 and 2%, respectively of the free area. The relative densities for 16 different IMP sizes have been compared, on the basis of seven animal and seven vegetal halves, counting (E-faces only) ca. 10,000 IMPS in each hemisphere. For IMP sizes of 581 A, a significant difference (P < 0.0005) was found, more small IMPS being present in the animal half. Some evidence for IMP-associated thin elements was found. These findings are discussed in relation to plasma membrane anisotropy and the morphogenetic role of the egg cortex. INTRODUCTION
Experimental evidence exists that the distribution of cytoplasmic components in the uncleaved, fertilized egg may depend on the cortex, i.e., the plasma membrane and its associated structures (Dollander, 1961; Pasteels, 1964; Arnold and WilliamsArnold, 1974). According to the selective gene activity theory of differentiation, the differential distribution of cytoplasmic components among embryonic cells elicits different patterns of gene activity. Hence it can be assumed that information relevant to the pattern of gene action is contained in an anisotropic arrangement of plasma-membrane constituents. Accepting the idea that a cortical pattern of information can be set up and maintained for some time in the form of an anisotropic organization of the plasma membrane, one must assume that such a membrane is static/rigid rather than fluid/dynamic (see Singer and Nicolson, 1972). Topographical heterogeneity in the plane of a continuous membrane can presumably exist by virtue of cross-linking cytoplasmic elements, i.e., microfilaments and microtubules. 334 0012-1606/78/0622-0334$02.00/O Copyright All rights
0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
Using freeze-fracture electron microscopy, it is possible to analyze local differences in plasma membrane composition. The aim of the present study was to compare the animal and vegetal hemispheres of the fertilized, uncleaved Xenopus egg (stage 1; Nieuwkoop and Faber, 1967). Lack of differences in the distribution pattern of intramembranous particles (IMPS) between the two membrane regions would provide evidence against the concept that animal/vegetal polarity may reside in an anisotropic arrangement of plasma-membrane components. MATERIALS
AND
METHODS
Eggs of Xenopus laevis were obtained and prepared as described earlier (Bluemink et al., 1976). Membrane fragments obtained from uncleaved, fertilized eggs were used for freeze-fracturing. The membrane fragments were isolated from the animal or the vegetal hemisphere as follows. Small squares (5 x 8 mm) of glass cleaned overnight in chromic acid were rinsed in distilled water and air-dried. The clean squares were etched for ca. 1
BLUEMINK
AND TERTOOLEN
Membrane
min in a 3:l mixture of barium sulfate and ammonium fluoride/sulfuric acid (l:l), thoroughly rinsed, and air-dried. They were then briefly exposed to a solution of 10 mg/ml of poly-L-lysine (Sigma Chemical Co., St. Louis, MO.) in water, thoroughly rinsed with distilled water, and air-dried (Mazia et al., 1975). For obtaining isolated pieces of membrane, the vegetal or animal hemisphere of a decapsulated egg (without vitelline membrane) was brought into contact with the polylysine-coated surface in water. ARer 10 min of contact, the decapsulated egg sticks to the glass. Redundant parts of the egg can be removed with a small pipet, leaving a fragment of the plasma membrane together with a thin layer of cytoplasm attached to the glass. Such specimens, obtained from eggs at about 30 min before the first cleavage, were fixed for lo-30 min in 2.50% glutaraldehyde in Steinberg solution (Steinberg, 1957) at room temperature, rinsed, and equilibrated against 20% glycerol in Steinberg solution for at least 2 hr. A thin perforated metal (Vacon 12) plate (2 x 6 mm) was put on top, and the sandwiched specimen was rapidly immersed in a thawed layer of nitrogen and pressed against the solid cold nitrogen underneath. For fracturing, the frozen sample was inserted into a fracture tool for sandwiched specimens, a moditication of the device described by Pfenninger and Rinderer (1975). Fracturing of the membrane was achieved by prying the metal plate off the glass support. Fracturing, etching, and coating were carried out at -100°C in a vacuum of better than lo+ Torr in a Denton instrument (type D.F.E.-3). The platinum/carbon-coated replicas were cleaned by exposing them to 1:l sodium hypochlorite in water for at least 2 hr at room temperature. They were then rinsed in distilled water and collected on single-hole grids covered with a carbon-coated supporting film of Pioloform F (Stockem, 1970). The replicas were inspected in a Zeiss EM-10 electron microscope at an
Structure
of Xenopw
Egg
335
acceleration voltage of 80 kV using a 60m objective aperture. A carbon difTraction grating replica of 54,864 lines per inch was used to obtain accurate magnification of specimen negatives at identical microscope settings. For reproducibility of the microscope setting, a selected grid holder was used for all EM recordings. Hysteresis effects were eliminated by using the Zeissmonostable device to switch the lens current control to maximum before setting at the selected magnification step. This makes for a relative reproducibility of the magnification of better than 1.5%. Specimen negatives (Kodak electron microscope film 4489, estar base) at a magnification of x40,185 were enlarged 7.5 times on Kodak Veribrom (plastic base). A square of 10 x 10 cm was outlined on the micrographs, and all IMPS within the square were measured. The area of the measured region and the dimensions of the IMPS were estimated with a Wang digitizing system (type 2262-l; point resolution, 0.25 mm) in combination with a Wang 2200 calculator. IMP sizes were determined by measuring the widest part of the shadow at right angles to the direction of the shadow. The freeze-fracture nomenclature is as proposed by Branton et al. (1975). RESULTS
Plasma membrane regions obtained from seven animal and seven vegetal hemispheres were used. The isolated membrane fragments (area, ca. 0.2-0.6 mm’) were taken invariably in a region within a radius of ca. 0.4 mm from the center of either the animal or the vegetal pole. The membrane fragments attached to a polylysine-coated glass surface gave rise to large membrane areas exposing the inner aspect of the external leaflet that remains attached to the glass (E-face), whereas in the same replicas inwardly folded edges of the membrane fragments gave rise to Pfaces. The exposed P-face area was always far less than the E-face area. It is generally accepted that in freezefracture replicas of split membranes the
336
DEVELOPMENTAL BIOLOGY
IMPS represent protein complexes, while the smooth interface represents the cleaved lipid bilayer (Marchesi et al., 1972; Pinto Da Silva, 1972; Pinto Da Silva et al., 1973; Singer and Nicolson, 1972; Bullivant, 1973; Tourtellotte and Zupnik, 1973). The primary aim of the present study was to find a qualitative difference in IMP population of the animal versus the vegetal hemisphere. To discriminate different IMP sizes in a range of ca. 20-200 A, electron micrographs of high magnification (x 301,400) are required. Exclusively IMP-populated areas of E-faces were used for making the counts; IMP-free areas (4, ca. 0.1 pm) were not involved. Most IMPS are attached to the E-face (Figs. l-4). The mean numbers of IMPS per square micrometer amount in the animal hemisphere to 1934 f 262 and in the vegetal hemisphere to 1872 -+ 229. AccordTABLE
VOLUME 62, 1978
ing to Student’s t test, using the 0.95 confidence interval for the mean, these densities are not significantly different (P > 0.10). IMP-free regions (4, ca 0.1 pm) on the tips of surface protrusions (Fig. 3; see also Bluemink et al., 1976a) were irregularly distributed in the animal (Fig. 3) and the vegetal (Fig. 4) half occupying ca. 8.5 and 2%, respectively, of the free area. Presumably because in these preparations the plasma membrane has been firmly attached to the polylysine-coated substrate, many protrusions show hardly any depth. The IMPS vary greatly in size. For the measurement of IMP sixes 110 electron x 301,387) micrographs (magnification, were used, covering 58 x 0.1 pm2 of the animal hemisphere (seven eggs, 12,216 IMPS) and 52 x 0.1 pm2 of the vegetal hemisphere (seven eggs, 9734 IMPS). The 1
FREQUENCIES OF IMPS, SUBDIVIDED INTO SIZE CLASSES, ON THE PLASMA-MEMBRANE E-FACES OF THE ANIMAL AND THE VEGETAL HEMISPHERE, RESPECTIVELY, TOGETHER WITH STATISTICAL ANALYSIS IMP size Animal hemisphere Vegetal hemisphere PC (A) N” SEM c1* CI N SEM 56 64 72 81 89 97 105 113 121 129 137 145 154 162 170 178
t
39 59 76 115 113 165 194 195 221 194 168 147 94 61 35 20
5 6 8 9 10 11 9 10 10 10 11 12 10 8 5 4
29-49 48-70 60-92 98-132 95-131 144-186 176-212 175-215 201-241 174-214 147-189 123-171 75-113 46-76 25-45 13-27
18 34 43 70 93 142 225 223 245 225 201 145 91 48 31 18
3 4 4 5 7 10 13 10 9 10 13 10 7 6 4 3
13-23 27-41 35-51 60-80 80-106 121-161 199-251 203-243 228-262 208-244 176-226 125-161 76-106 37-59 22-42 12-24
u N = average number of IMPS per square micrometer. ’ CI = variance at 0.95 confidence interval for the mean (7’ factor = 1.982 x SEM value). c P values are for the two-tailed probability estimated for 108 degrees of freedom according test.
<0.0005 <0.0005 <0.0005 <0.0005 >0.05 >O.lO >0.05 >0.05 >0.05 -co.05 co.05 z-o.5 >0.5 >O.l >0.5 >0.5
to Student’s
FIG. 1. E-face, animal hemisphere. EF, external face; arrows, IMPS of spiny appearance; arrowhead, cluster of ca. seven small IMPS. x 320,000. This and all following micrographs show the plasma membrane of the Xenopus egg. Encircles arrowheads indicate direction of shadowing in micrographs. FIG. 2. E-face, vegetal hemisphere. Arrow, IMPS of spiny appearance. x 320,000.
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DEVELOPMENTAL BIOLOGY
IMPS, ranging in size between 56 and 178 A, were subdivided into 16 classes (8-A intervals). The results of these measurements together with the statistical values are given in Table 1. Figure 5 represents the same data in graphical form. Only for IMP sizes of 581 A are the plasma membranes of the animal and the vegetal halves significantly different (P < 0.0005; see Table 1); that is, more small IMPS are present in the plasma membrane of the animal hemisphere. For IMP sizes of >81 A, the relative frequencies for both membranes are not significantly different, except for the 129- and 137-A size classes (P < 0.005, see Table 1); the graph does not illustrate this because the shaded areas overlap slightly. In the animal hemisphere, IMPS of different sizes were not evenly mixed. Small IMPS distributed among larger ones occasionally form aggregates of ca. 4-10 IMPS having almost no interspace between them (Fig. 1). No such clustering of medium or large IMPS has been observed in either the animal or the vegetal half. In the replicas, inwardly folded edges of the membrane fragments gave rise to Pfaces. Invariably the exposed P-face area was far less than the exposed E-face area. The P-faces had many small pits and less IMPS were attached to the P-face (ca. 100 IMPs/pm2) than to the E-face, just as has been reported before (Bluemink et al., 1976a,b). P-faces from the animal and the vegetal hemisphere showed no obvious difference in IMP density. In comparing the IMP population in the animal half versus the vegetal half, the P-faces have not been involved because of the intrinsic low IMP density. Close scrutiny reveals that the IMPS are not simple globules. Filament-like extensions give a spiny appearance to the profiles of many IMPS (Figs. 1 and 2). FIG. 3. E-face, animal hemisphere. 38,500. FIG. 4. E-face, vegetal hemisphere.
VOLUME 62, 1978
Occasionally longer “filaments” are seen to extend from an IMP or, more rarely, to run from one IMP to another (Figs. 8 and 9). In replicas where the direction of shadowing happens to be perpendicular to the direction of the “filaments,” the shadows conform to those of the IMPS suggesting that they exist as ridges on the matrix. Stereomicrographs (not illustrated) have provided conclusive evidence that the “filaments” exist as elevations in the profile of the replica. In the replica in areas of clean ice, or in IMP-free areas of the cleaved membrane bilayer, single Xlaments,” i.e., not associated with IMPS, have not been observed. DISCUSSION
The plasma membrane of the amphibian egg is peculiar in several respects. The IMPS are larger than usual (Bluemink et al., 1976; Sanders and DiCaprio, 1976a,b; Smith et al., 1976; Decker and Friend, 1974). The replicas of the polylysine-attached membrane fragments have provided irrefutable evidence that the IMPS attached to the E-face outnumber the IMPS seen on the P-face. This finding conforms to our earlier observations (Bluemink et al., 1976a,b) based on replicas of whole eggs prepared with a different technique. More IMPS attached to the Eface than to the P-face have been found also in the plasma membrane of early embryos of another amphibian (Smith et al., 1976). Apart from amphibian embryos a reversal of asymmetry in IMP distribution has been reported for a few other biological systems (Allen, 1976; Wade et al., 1975). The overall IMP densities of the animal (1934 -+ 262 IMPS/pm*) and the vegetal hemisphere (1872 ? 229 IMPs/~m2) do not differ significantly (P > 0.10). When a correction is made for the 8.5% IMP-free
Stars, particle-poor
regions corresponding
Stars, particle-poor
regions.
x 38,500.
to surface protrusions.
x
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DEVELOPMENTALBIOLOGY 300
VOLUME 62, 1978
-
200-
100
-
I 40
/ 56
I 72
I
/ 69
,
1 105
I
I 121
I
I 137
I 154
I
I 170
I
I
5 FIG. 5. Frequency distributions of IMPS subdivided into 8-8 classes. Vertical, number of IMPS per square micrometer; horizontal, IMP size in angstrom units; solid dots, animal hemisphere; open squares, vegetal hemisphere. The shaded areas indicate the variance at the 0.95 confidence interval for the mean (T-factor = 1.982 x SEM).
area in the animal and the 2% IMP-free area in the vegetal hemisphere, the estimated overall densities of the animal (1770 f 262 IMPs/pm2) and the vegetal hemisphere (1834 + 229 IMPs/pm2) are still not significantly different. Comparing the IMP-populated E-faces, it could be shown that the IMP pattern in the animal half differs significantly from that seen in the vegetal half, more small IMPS (581 A) being present in the animal hemisphere. In addition a small difference in density of the 129- to 137-A IMPS was found. In the animal half, the small (181 A) IMPS partly occur as aggregates, but the possibility cannot be excluded that aggregation is an artifact produced during fixation. Plasma membrane anisotropy is known to exist in the mouse egg (Johnson et al., FIG. FIG. FIG. FIG.
6. 7. 8. 9.
P-face, animal hemisphere. P-face, vegetal hemisphere. E-face, animal hemisphere. Same as for Fig. 8.
1975), the fucoid egg (Robinson and Jaffe, 1975; Nuccitelli and Jaffe, 1976), and the ascidian egg (Ortolani et al., 1977). So far only one other report has provided evidence for plasma-membrane anisotropy related to egg polarity as studied by freezefracture electron microscopy (Peng, 1976). An estimate of the overall IMP density during polarization in fucoid eggs (Peluetia) has shown that at 6 hr after fertilization more IMPS per square micrometer are found at the thallus end (1460 f 150) than at the rhizoid end (1010 f 60). This difference becomes more prominent at 8 hr after fertilization. The functional implication of plasma membrane anisotropy as related to egg polarity is that the pattern of membrane components may exert a regulatory effect on the distribution of cytoplasmic constit-
Small number of IMPS and small pits (arrow). x 160,000. Small number of IMPS and small pits (arrow). x 160,000. IMP-associated thin filaments (arrows). x 504,000.
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DEVELOPMENTAL BIOL~CY
uents. The local differences in IMP pattern may constitute a form of “macromolecular braille” coding for positional information (McMahon and West, 1976). Membraneassociated elements of the cytoskeleton, i.e., microfilaments and microtubules (see Morrill and Perkins, 1973), may transmit the effect of local differences in the plasma membrane to the cytoplasm. The question whether the IMP-associated filaments are artifacts or structural components is open to discussion. It could be suggested that they represent cracks in the fracture face which are related to the direction of fractioning or that they arise artificially by plastic deformation of membrane-associated proteins. In the replicas there was a fixed angle between the direction of membrane splitting and the direction of shadowing. Using the direction of shadowing an an indicator, one can determine that the orientation of the IMP-associated filaments has no fixed angle to the direction of membrane splitting (see Fig. 9). It has been reported that plastic deformation of actin occasionally may give rise to “filaments” on the E-face of freeze-fractured membranes (McNutt, 1976). Filamentous actin (Perry et al., 1971), as well as myosin, occur in large amounts in the cortical cytoplasm of the amphibian egg (Franke et al., 1976; see this reference for further literature). Thus it is conceivable that during freeze-fracturing filaments drawn out from the cortical cytoplasm have appeared on the E-face. However, actin or myosin filaments would measure 50 h; or more (see Franke et al., 1976), whereas the filaments on the E-face obviously are much thinner. The 20-A IMP-associated filaments could represent the thin elements of the cortical anastomosing network (Franke et al., 1976) drawn out from the cortical cytoplasm during freeze-fracturing. It is conceivable that the IMPS are anchored to the elements of the cortical network in the same way as the IMPS in the erythro-
V~LIJME 62, 1978
cyte membrane (Elgsaeter and Branton, 1974; Elgsaeter et al., 19761, which are anchored to the membrane-associated spectrin network. In the amphibian egg, structural continuity with the cortical anastomosing network would provide the delicate plasma membrane the tough support needed to withstand stress forces during morphogenesis (Beloussov et al., 1975) and to define cell shape. The topographical differences in IMP distribution could rely on a spacing mechanism for which the elements of the cortical network form the anchorage sites. However, stronger evidence than that presented in this report will be necessary to prove the existence of such a mechanism. Grateful acknowledgment is made to Dr. J. Faber for editorial help and to Dr. S. W. de Laat for making the computer program to evaluate IMP sizes. REFERENCES R. D. (1976). Freeze-fracture evidence for intramembrane changes accompanying membrane recycling in Paramecium. Cytobiologie 1% 254-273. ARNOLD, J. M., and WILLIAMS-ARNOLD, L. D. (1974;. Cortical-nuclear interactions in cephalopod development: Cytochalasin B effects on the informational pattern in the cell surface. J. Embryol. Exp. Morphol. 31, l-25. BELOUSSOV, L. V., DORFMAN, J. G., and CHERDANTZEV, V. G. (1975). Mechanical stresses and morphological patterns in amphibian embryos. J. Embryol. Exp. Morphol. 34, 559-574. BLUEMINK, J. G., TEBTOOLEN, L. G. J., VERVERGAERT, P. H. J. Th., and VERKLEIJ, A. J. (1976a). Freeze-fracture electron microscopy of preexisting and nascent cell membrane in cleaving eggs of Xenopus laevis. Biochim. Biophys. Acta 443, 143155. BLUEMINK, J. G., TERTOOLEN, L. G. J., VERVERGAERT, P. H. J. Th., and VERKLEIJ, A. J. (1976b). Membrane biogenesis in dividing eggs of Xenopus Zocvis analysed by freeze-fracture electron microscopy. In “Proceedings of the Sixth European Congress on Electron Microscopy (Jerusalem),” Vol. 2, pp. 348-350. TAL International Publishing Company, Israel. BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY, M. J., MOOR, H., M~~HIJETHALER, K., NORTHCOTE, D. H., PACKER, L., SATIR, B., SATIR, P., SPETH, V., STAEHELIN, L. A., STEERE, R. L.,
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BLUEMINK
AND TERTOOLEN
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