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Cortical granules remaining after fertilization in Xenopus laevis
DEVELOPMENTAL
Cortical
BIOLOGY
30, 228- 232 (1973)
Granules
Remaining
after Fertilization
MINORU KOTANI, KOHJI IKENISHI’ Department
in Xenopus
laevis
AND KAZUYUKI TANABE
of Biology, Faculty of Science, Osaka City University,
Sugimoto-cho,
Sumiyoshi-ku,
Osaka 558, Japan Accepted July 31, 1972 egg, l-cell, Eggs of Xenopus lueuis were examined in an electron microscope at unfertilized 2-cell, 32-cell, and blastula stages. Granules closely resembling cortical granules were observed within the “germinal plasm” as well as in the peripheral cytoplasm of all the eggs examined. A staining method was developed that makes it easier to count cortical granules in thick Epon sections. Light and electron microscope examinations revealed that granules remaining after fertilization possessed morphological characteristics wholly consistent with those of cortical granules of unfertilized eggs. These granules were confirmed to be true cortical granules. INTRODUCTION
It has so far been generally accepted that, in anuran eggs, cortical granules found in the cortical cytoplasm of unfertilized eggs disappear soon after fertilization, extruding the inclusions into the perivitelline space (Balinsky, 1966; Van Gansen, 1966; see Kemp and Istock, 1967). In the case of Xenopus laevis, breakdown of cortical granules starts about 3 min after fertilization and the overwhelming majority of the granules disappear within about 10 min after fertilization (Balinsky, 1966). Kemp (1962) has reported that some granules remain intact throughout the wave of breakdown following pricking in eggs of Ranu pipiens, but he has not referred to the fate of these granules in the subsequent developmental processes. As far as we know, no observation has so far been reported in anuran eggs, which claims the presence of cortical granules in embryos at 2-cell (about 90 min after fertilization at room temperature in Xenopus laeuis) or at more advanced stages. In the course of the study on the ultraplasm” in structure of the “germinal Xenopus laevis, granules indistinguishable in electron microscope morphology from ‘Present address: Laboratory of Biology, Gifu College of Dentistry, Takano 1851, Hozumi-cho, Motosu-gun, Gifu Prefecture, Japan. 228 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
the cortical granules were incidentally observed in the peripheral cytoplasm as well as in the region of the “germinal plasm” of cleaving eggs. In this short communication it will be asserted that one should consider possible functions of cortical granules in developing embryos other than their function at the time of fertilization. MATERIALS
AND
METHODS
Freshly laid fertilized eggs were obtained from Xenopus laevis by injecting 200 and 300 units of gonadotropic hormone, respectively, into sexually matured male and female. Unfertilized eggs were obtained by injecting the hormone into a solitary female. Embryos were staged after Nieuwkoop and Faber (1956). Electron microscopy. Eggs were fixed at the following stages; prior to fertilization, stages 1, 2- (beginning of the first cleavage), 2, 6, and somewhat earlier than stage 9 (designated as stage 9-). They were fixed with 5% glutaraldehyde in 0.08 M or 0.1 A4 phosphate buffer (pH 7.3) in an ice bath (0 - 4°C) for 5-7 hr; jelly coats were removed by forceps within the first 2 hr. Eggs were then transected equatorially with a razor blade, and the vitelline membrane was removed. Both animal and vegetal hemispheres were washed overnight with 0.08 M or 0.1 M
BRIEF NOTES
229
contain finely granular materials. The contents, however, are rather inhomogenous and usually consist of 3 components, a denser one, a lighter one, and an intermediate one. The distribution of these components varies from granule to granule. In Fig. 2 a granule on the left has all 3 components; an intermediate component showing some network distribution, a lighter one corresponding to the meshes, and a denser one forming a patch near the surface: in a granule on the right are 2 components, a denser one forming patches in places and an intermediate one uniformly distributed. Granules having similar morphology to the cortical granules are found mainly in the peripheral cytoplasm facing the outer surface or certain cleavage furrows of eggs at all the stages examined, both in animal and vegetal halves (Fig. 3). They have diameters of approximately from l-2 pm. These granules were also found to be localized in the region of the “germinal plasm” and other subperipheral areas of the cytoplasm in embryos of stages 6 and 9- (Fig. 4). At stage 9- the granules were also found in blastomeres facing the blastocoel. Even at a stage as late as 18 %, a granule closely resembling those elucidated in this study has been observed in a bottle cell of neural ectoderm in Xenopus laevis (Schroeder, 1970, Fig. 13), although it was not indicated by the author as being a cortical granule. Light microscope examinations also revealed that, in cleaving eggs, granules having the same staining properties as those of cortical granules are found frequently in peripheral parts and sometimes in subperipheral parts of the cytoplasm, such as the “germinal plasm,” or in interior parts of blastomeres, among which are included blastomeres facing the blastocoel (Figs. 5-7). These granules showed similar distribution and frequency RESULTS AND DISCUSSION of appearance as those of the granules obCortical granules of unfertilized eggs served in electron microscopy. Unpubare bounded by an unit membrane and lished data showed that these granules phosphate buffer, pH 7.3, at 4” C. They were postfixed with 1.0% osmium tetroxide in the same buffer in the ice bath for 3 hr. After washing in cold distilled water, the fixed materials were stained in a saturated solution of uranyl acetate for 3 hr and were embedded in Epon (Luft, 1961). Sections were cut on a PorterBlum MT-2 ultramicrotome, stained with lead citrate, and examined in a JEM-7 electron microscope at 80 kV. The “germinal plasm” was located in a thick section stained with 0.5% toluidine blue in 0.5% borax, with reference to faintly staining cytoplasmic islands surrounded by smaller yolk platelets. These islands were demonstrated to correspond to the blue cytoplasmic islands after Azan staining in paraffin sections (Czolowska, 1969). Light microscopy. Sections, about 0.5 p in thickness, were deprived of Epon by a slight modification of Lane and Europa’s technique (Lane and Europa, 1965): i.e., treating them for 10 min with potassium hydroxide saturated in absolute ethanol. Sections were then hydrated through an ethanol series to distilled water, immersed in 0.1% toluidine blue in 0.2 M acetate buffer (pH 4.0) for a second, washed with distilled water, stained with 0.5% aniline blue and 1.9% orange G in 1.9% oxalic acid and 0.05% phosphotungstic acid solution for 10 min, rinsed briefly with distilled water, dried in a hot air stream, and mounted directly with balsam. Cortical granules of unfertilized eggs were stained a distinctive dark blue, yolk platelets light yellow, mitochondria and other cytoplasmic inclusions pale blue, and pigment granules black (Fig. 1). This technique was applied to discriminate residual cortical granules under magnification of 1500-fold.
230
DEVELOPMENTALBIOLOGY
VOLUME 30, 1973
Key to abbreuiations: B, blastocoel; CG, cortical granule; GP, island of “germinal plasm”; IS, intercellular space; A4, mitochondrion; P, pigment granule; Y, yolk platelet. FIG. 1. Light micrograph of cortical granules. Thick section was deprived of Epon by KOH treatment and stained with toluidine blue and aniline blue-orange G. In actual sections cortical granules were stained dark blue and pigment granules black, so that it is easy to distinguish them from each other. Cortical granules (arrows) form a row in cortical cytoplasm of unfertilized egg. x 967.
231
BRIEF Noms
were stained purplish-red by PAS reaction in thick Epon sections, as were cortical granules of unfertilized eggs. Therefore it may be concluded that granules whose morphological characteristics are wholly consistent with those of cortical granules of unfertilized eggs are genuine cortical granules. As to distribution and frequency of appearance of these residual cortical granules, the animal hemisphere does not seem to exhibit any markedly different features from those of the vegetal hemisphere. In one of the islands of the “germinal plasm,” however, 50 or more cortical granules were counted on serial thick sections. These granules seem originally to be situated in the cortical cytoplasm, from which they are incorporated into the islands of the “germinal plasm” during early cleavage stages. It is estimated from the numbers of cortical granules found in thick sections that one whole egg at cleavage stages may carry approximately 1% of the cortical granules in unfertilized eggs. Cortical granules have been found in all of nearly 100 cleaving eggs so far examined which were obtained from more than 10 different females. What is more, they were found in eggs and embryos derived from a batch in which 96.5% (170/ 176) of the eggs developed normally into morulae. These facts indicate that cortical granules persisting into cleavage stages is not an abnormal phenomenon, but a
normal and common one. The contents of residual cortical granules in Xenopus might be released into the cytoplasm as in a unique case of the bivalve mollusc Barnes candida (Pasteels and de Harven, 1962), although this remains uncertain, since we have so far been unable to observe an actual process of breakdown of cortical granules in the cytoplasm of cleaving eggs. On the other hand, the fact that neutral and acid mucopolysaccharides, and probably some proteins are contained in cortical granules (see Kemp and Istock, 1967) leads us to speculate that these substances of residual cortical granules, after being extruded, might function as intercellular materials which seem to concern cell movement (Bell, 1963) or as a “carpet” which appears to be indispensable for cell division (Yaoi and Kanaseki, 1972). We are grateful to Dr. Tadashi Fujiwara, Medical School, Osaka City University, for his kind permission to use the electron microscope and other laboratory equipment, Dr. Kaoru Takamoto, Kyoto Prefectural University of Medicine, for valuable advice, and Dr. Marina Sohkawa-Dan, for critical reading of the manuscript. We are also thankful to Mr. Tsutomu Iwamoto for technical advice in electron microscopy, and Mr. Taizo Kimura for help in the preparation of electron micrographs. REFERENCES B. I. (1966). Changes in the ultrastructure of amphibian eggs following fertilization. Acta Embryol. Morphol. 9, 132-154.
BALINSKY,
FIG. 2. Cortical granules found in cortical cytoplasm of vegetal hemisphere of unfertilized egg, showing heterogeneity of the contents. Contents of a granule on the left consist of 3 components; a component of intermediate density showing some network distribution, a lighter one corresponding to the meshes, and a denser one forming a patch near surface. A granule on the right possesses a few patches of denser component within an intermediate component which is uniformly distributed throughout granule. x 19,500. FIG. 3. A cortical granule remaining in cortical layer of vegetal blastomere at stage 6, with 2 patches of denser component. x 26,000. FIG. 4. Two cortical granules in the region of “germinal plasm” at stage 6. Germinal granule is not illustrated in this figure. Main bodies of small yolk platelets clearly shows crystalline structure under higher magnification. x 26,000. FIGS. 5-7. Light micrographs of cortical granules. Fig. 5: cortical granules (arrows) in peripheral part of animal blastomere at stage 6, distributed among numerous pigment granules. Very small yolk platelets are seen to be dispersed. Fig. 6: cortical granule (arrow) in interior part of animal blastomere facing blastocoel, at stage 9-. Nuclei are not seen in this section. Fig. 7: cortical granules (arrows) are located within 2 different islands of “germinal plasm” between which an intercellular space is seen, at stage 6. Fig. 5. x 865; Fig. 6, x 763; Fig. 7, x 744.
232
DEVELOPMENTALBIOLOGY
BELI, L. G. E. (1963). Some observations concerning cell movement and cell cleavage. “Cell Growth and Cell Division; Symp. Int. Sot. Cell Biol.” Vol. 2, pp. 215-228. Academic Press, New York. CZOLOWSK~ R. (1969). Observations on the origin of the ‘germinal cytoplasm’ in Xenopus laeois. J. Embryol. Exp. Morphol. 22,229-251. KEMP, N. E. (1962). Rupture of cortical granules in eggs of Ranu pipiens activated by pricking. Anut. Rec. 142, 247 (Abstract). KEMP, N. E., and ISTOCK, N. L. (1967). Cortical changes in growing oocytes and in fertilized or pricked eggs of Rana pipiens. J. Cell Biol. 34, 111-122. LANE, B. P., and EUROPA, D. L. (1965). Differential staining of ultrathin sections of Epon-embedded tissues for light microscopy. J. Histochem. Cytothem. 13, 579-582. LUFT, J. H. (1961). Improvements in epoxy resin embedding method. J. Biophys. Biochem. Cytol. 9,
VOLUME 30. 1973
469414. NIEUWKOOP, P. D., and FABER, J. (1956). “Normal Table of Xenopus laeois (Daud.).” North Holland Publ., Amsterdam. PASTEELS,J. J., and DE HARVEN, E. (1962). Etude au microscope electronique du cortex de l’oeuf de Barnes candidu (mollusque bivalve), et son evolution au moment de la fecondations, de la maturation, et de la segmentation. Arch. Biol. 73, 465490. SCHROEDER, T. E. (1970). Neurulation in Xenopus laeois. An analysis and model based upon light and electron microscopy. J. Embryol. Exp. Morphol. 23, 427-462. VAN GANSEN, P. (1966). Effect de la fecondation sur l’ultrastructure du cytoplasme peripherique de l’oeuf de Xenopus lnevis. J. Embryol. Exp. Morphol. 15, 365-369. YAOI, Y., and KANASEKI, T. (1972). Role of microexudate carpet in cell division. Nature (London) 237, 283-285.
Cortical
BIOLOGY
30, 228- 232 (1973)
Granules
Remaining
after Fertilization
MINORU KOTANI, KOHJI IKENISHI’ Department
in Xenopus
laevis
AND KAZUYUKI TANABE
of Biology, Faculty of Science, Osaka City University,
Sugimoto-cho,
Sumiyoshi-ku,
Osaka 558, Japan Accepted July 31, 1972 egg, l-cell, Eggs of Xenopus lueuis were examined in an electron microscope at unfertilized 2-cell, 32-cell, and blastula stages. Granules closely resembling cortical granules were observed within the “germinal plasm” as well as in the peripheral cytoplasm of all the eggs examined. A staining method was developed that makes it easier to count cortical granules in thick Epon sections. Light and electron microscope examinations revealed that granules remaining after fertilization possessed morphological characteristics wholly consistent with those of cortical granules of unfertilized eggs. These granules were confirmed to be true cortical granules. INTRODUCTION
It has so far been generally accepted that, in anuran eggs, cortical granules found in the cortical cytoplasm of unfertilized eggs disappear soon after fertilization, extruding the inclusions into the perivitelline space (Balinsky, 1966; Van Gansen, 1966; see Kemp and Istock, 1967). In the case of Xenopus laevis, breakdown of cortical granules starts about 3 min after fertilization and the overwhelming majority of the granules disappear within about 10 min after fertilization (Balinsky, 1966). Kemp (1962) has reported that some granules remain intact throughout the wave of breakdown following pricking in eggs of Ranu pipiens, but he has not referred to the fate of these granules in the subsequent developmental processes. As far as we know, no observation has so far been reported in anuran eggs, which claims the presence of cortical granules in embryos at 2-cell (about 90 min after fertilization at room temperature in Xenopus laeuis) or at more advanced stages. In the course of the study on the ultraplasm” in structure of the “germinal Xenopus laevis, granules indistinguishable in electron microscope morphology from ‘Present address: Laboratory of Biology, Gifu College of Dentistry, Takano 1851, Hozumi-cho, Motosu-gun, Gifu Prefecture, Japan. 228 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
the cortical granules were incidentally observed in the peripheral cytoplasm as well as in the region of the “germinal plasm” of cleaving eggs. In this short communication it will be asserted that one should consider possible functions of cortical granules in developing embryos other than their function at the time of fertilization. MATERIALS
AND
METHODS
Freshly laid fertilized eggs were obtained from Xenopus laevis by injecting 200 and 300 units of gonadotropic hormone, respectively, into sexually matured male and female. Unfertilized eggs were obtained by injecting the hormone into a solitary female. Embryos were staged after Nieuwkoop and Faber (1956). Electron microscopy. Eggs were fixed at the following stages; prior to fertilization, stages 1, 2- (beginning of the first cleavage), 2, 6, and somewhat earlier than stage 9 (designated as stage 9-). They were fixed with 5% glutaraldehyde in 0.08 M or 0.1 A4 phosphate buffer (pH 7.3) in an ice bath (0 - 4°C) for 5-7 hr; jelly coats were removed by forceps within the first 2 hr. Eggs were then transected equatorially with a razor blade, and the vitelline membrane was removed. Both animal and vegetal hemispheres were washed overnight with 0.08 M or 0.1 M
BRIEF NOTES
229
contain finely granular materials. The contents, however, are rather inhomogenous and usually consist of 3 components, a denser one, a lighter one, and an intermediate one. The distribution of these components varies from granule to granule. In Fig. 2 a granule on the left has all 3 components; an intermediate component showing some network distribution, a lighter one corresponding to the meshes, and a denser one forming a patch near the surface: in a granule on the right are 2 components, a denser one forming patches in places and an intermediate one uniformly distributed. Granules having similar morphology to the cortical granules are found mainly in the peripheral cytoplasm facing the outer surface or certain cleavage furrows of eggs at all the stages examined, both in animal and vegetal halves (Fig. 3). They have diameters of approximately from l-2 pm. These granules were also found to be localized in the region of the “germinal plasm” and other subperipheral areas of the cytoplasm in embryos of stages 6 and 9- (Fig. 4). At stage 9- the granules were also found in blastomeres facing the blastocoel. Even at a stage as late as 18 %, a granule closely resembling those elucidated in this study has been observed in a bottle cell of neural ectoderm in Xenopus laevis (Schroeder, 1970, Fig. 13), although it was not indicated by the author as being a cortical granule. Light microscope examinations also revealed that, in cleaving eggs, granules having the same staining properties as those of cortical granules are found frequently in peripheral parts and sometimes in subperipheral parts of the cytoplasm, such as the “germinal plasm,” or in interior parts of blastomeres, among which are included blastomeres facing the blastocoel (Figs. 5-7). These granules showed similar distribution and frequency RESULTS AND DISCUSSION of appearance as those of the granules obCortical granules of unfertilized eggs served in electron microscopy. Unpubare bounded by an unit membrane and lished data showed that these granules phosphate buffer, pH 7.3, at 4” C. They were postfixed with 1.0% osmium tetroxide in the same buffer in the ice bath for 3 hr. After washing in cold distilled water, the fixed materials were stained in a saturated solution of uranyl acetate for 3 hr and were embedded in Epon (Luft, 1961). Sections were cut on a PorterBlum MT-2 ultramicrotome, stained with lead citrate, and examined in a JEM-7 electron microscope at 80 kV. The “germinal plasm” was located in a thick section stained with 0.5% toluidine blue in 0.5% borax, with reference to faintly staining cytoplasmic islands surrounded by smaller yolk platelets. These islands were demonstrated to correspond to the blue cytoplasmic islands after Azan staining in paraffin sections (Czolowska, 1969). Light microscopy. Sections, about 0.5 p in thickness, were deprived of Epon by a slight modification of Lane and Europa’s technique (Lane and Europa, 1965): i.e., treating them for 10 min with potassium hydroxide saturated in absolute ethanol. Sections were then hydrated through an ethanol series to distilled water, immersed in 0.1% toluidine blue in 0.2 M acetate buffer (pH 4.0) for a second, washed with distilled water, stained with 0.5% aniline blue and 1.9% orange G in 1.9% oxalic acid and 0.05% phosphotungstic acid solution for 10 min, rinsed briefly with distilled water, dried in a hot air stream, and mounted directly with balsam. Cortical granules of unfertilized eggs were stained a distinctive dark blue, yolk platelets light yellow, mitochondria and other cytoplasmic inclusions pale blue, and pigment granules black (Fig. 1). This technique was applied to discriminate residual cortical granules under magnification of 1500-fold.
230
DEVELOPMENTALBIOLOGY
VOLUME 30, 1973
Key to abbreuiations: B, blastocoel; CG, cortical granule; GP, island of “germinal plasm”; IS, intercellular space; A4, mitochondrion; P, pigment granule; Y, yolk platelet. FIG. 1. Light micrograph of cortical granules. Thick section was deprived of Epon by KOH treatment and stained with toluidine blue and aniline blue-orange G. In actual sections cortical granules were stained dark blue and pigment granules black, so that it is easy to distinguish them from each other. Cortical granules (arrows) form a row in cortical cytoplasm of unfertilized egg. x 967.
231
BRIEF Noms
were stained purplish-red by PAS reaction in thick Epon sections, as were cortical granules of unfertilized eggs. Therefore it may be concluded that granules whose morphological characteristics are wholly consistent with those of cortical granules of unfertilized eggs are genuine cortical granules. As to distribution and frequency of appearance of these residual cortical granules, the animal hemisphere does not seem to exhibit any markedly different features from those of the vegetal hemisphere. In one of the islands of the “germinal plasm,” however, 50 or more cortical granules were counted on serial thick sections. These granules seem originally to be situated in the cortical cytoplasm, from which they are incorporated into the islands of the “germinal plasm” during early cleavage stages. It is estimated from the numbers of cortical granules found in thick sections that one whole egg at cleavage stages may carry approximately 1% of the cortical granules in unfertilized eggs. Cortical granules have been found in all of nearly 100 cleaving eggs so far examined which were obtained from more than 10 different females. What is more, they were found in eggs and embryos derived from a batch in which 96.5% (170/ 176) of the eggs developed normally into morulae. These facts indicate that cortical granules persisting into cleavage stages is not an abnormal phenomenon, but a
normal and common one. The contents of residual cortical granules in Xenopus might be released into the cytoplasm as in a unique case of the bivalve mollusc Barnes candida (Pasteels and de Harven, 1962), although this remains uncertain, since we have so far been unable to observe an actual process of breakdown of cortical granules in the cytoplasm of cleaving eggs. On the other hand, the fact that neutral and acid mucopolysaccharides, and probably some proteins are contained in cortical granules (see Kemp and Istock, 1967) leads us to speculate that these substances of residual cortical granules, after being extruded, might function as intercellular materials which seem to concern cell movement (Bell, 1963) or as a “carpet” which appears to be indispensable for cell division (Yaoi and Kanaseki, 1972). We are grateful to Dr. Tadashi Fujiwara, Medical School, Osaka City University, for his kind permission to use the electron microscope and other laboratory equipment, Dr. Kaoru Takamoto, Kyoto Prefectural University of Medicine, for valuable advice, and Dr. Marina Sohkawa-Dan, for critical reading of the manuscript. We are also thankful to Mr. Tsutomu Iwamoto for technical advice in electron microscopy, and Mr. Taizo Kimura for help in the preparation of electron micrographs. REFERENCES B. I. (1966). Changes in the ultrastructure of amphibian eggs following fertilization. Acta Embryol. Morphol. 9, 132-154.
BALINSKY,
FIG. 2. Cortical granules found in cortical cytoplasm of vegetal hemisphere of unfertilized egg, showing heterogeneity of the contents. Contents of a granule on the left consist of 3 components; a component of intermediate density showing some network distribution, a lighter one corresponding to the meshes, and a denser one forming a patch near surface. A granule on the right possesses a few patches of denser component within an intermediate component which is uniformly distributed throughout granule. x 19,500. FIG. 3. A cortical granule remaining in cortical layer of vegetal blastomere at stage 6, with 2 patches of denser component. x 26,000. FIG. 4. Two cortical granules in the region of “germinal plasm” at stage 6. Germinal granule is not illustrated in this figure. Main bodies of small yolk platelets clearly shows crystalline structure under higher magnification. x 26,000. FIGS. 5-7. Light micrographs of cortical granules. Fig. 5: cortical granules (arrows) in peripheral part of animal blastomere at stage 6, distributed among numerous pigment granules. Very small yolk platelets are seen to be dispersed. Fig. 6: cortical granule (arrow) in interior part of animal blastomere facing blastocoel, at stage 9-. Nuclei are not seen in this section. Fig. 7: cortical granules (arrows) are located within 2 different islands of “germinal plasm” between which an intercellular space is seen, at stage 6. Fig. 5. x 865; Fig. 6, x 763; Fig. 7, x 744.
232
DEVELOPMENTALBIOLOGY
BELI, L. G. E. (1963). Some observations concerning cell movement and cell cleavage. “Cell Growth and Cell Division; Symp. Int. Sot. Cell Biol.” Vol. 2, pp. 215-228. Academic Press, New York. CZOLOWSK~ R. (1969). Observations on the origin of the ‘germinal cytoplasm’ in Xenopus laeois. J. Embryol. Exp. Morphol. 22,229-251. KEMP, N. E. (1962). Rupture of cortical granules in eggs of Ranu pipiens activated by pricking. Anut. Rec. 142, 247 (Abstract). KEMP, N. E., and ISTOCK, N. L. (1967). Cortical changes in growing oocytes and in fertilized or pricked eggs of Rana pipiens. J. Cell Biol. 34, 111-122. LANE, B. P., and EUROPA, D. L. (1965). Differential staining of ultrathin sections of Epon-embedded tissues for light microscopy. J. Histochem. Cytothem. 13, 579-582. LUFT, J. H. (1961). Improvements in epoxy resin embedding method. J. Biophys. Biochem. Cytol. 9,
VOLUME 30. 1973
469414. NIEUWKOOP, P. D., and FABER, J. (1956). “Normal Table of Xenopus laeois (Daud.).” North Holland Publ., Amsterdam. PASTEELS,J. J., and DE HARVEN, E. (1962). Etude au microscope electronique du cortex de l’oeuf de Barnes candidu (mollusque bivalve), et son evolution au moment de la fecondations, de la maturation, et de la segmentation. Arch. Biol. 73, 465490. SCHROEDER, T. E. (1970). Neurulation in Xenopus laeois. An analysis and model based upon light and electron microscopy. J. Embryol. Exp. Morphol. 23, 427-462. VAN GANSEN, P. (1966). Effect de la fecondation sur l’ultrastructure du cytoplasme peripherique de l’oeuf de Xenopus lnevis. J. Embryol. Exp. Morphol. 15, 365-369. YAOI, Y., and KANASEKI, T. (1972). Role of microexudate carpet in cell division. Nature (London) 237, 283-285.