Cortical differentiation of the animal pole during maturation division in fertilized eggs of Tubifex (Annelida, Oligochaeta)

DEVELOPMENTAL

BIOLOGY

85, ‘77-88 (1981)

Cortical Differentiation of the Animal Pole during Maturation Division in Fertilized Eggs of Tubifex (Annelida, Oligochaeta) II. Polar Body Formation TAKASHI Zoological

Institute,

Faculty

SHIMIZU

of Science, Hokkaido

University,

Sapporo, 060, Japan

Received June 6 1980; accepted in revised form December 22, 1980

Changes in the cortical organization at the animal pole are examined by scanning and transmission electron microscopy in the Tubifeez egg undergoing second polar body formation. At very early anaphase of the second meiosis, the egg surface overlying the meiotic apparatus is undulated, but its neighboring surface appears to be smooth. Although a microfilamentous cortical layer is found in the smooth area, the cortical layer of the undulating area is thin and devoid of filamentous structures except for its central part where some filaments are observed. This local differentiation takes place normally in colchicine-treated eggs where the meiotic apparatus is destroyed. Along with the progression of the anaphase movement, the egg surface of the undulating area is, first, uplifted into a cone-shaped cytoplasmic bulge (presumptive polar body); then the height and surface area of the bulge gradually increase. The distal surface of the growing bulge appears to be undulated whereas the sides of the bulge are relatively smooth. Transmission electron microscopy reveals that a thick microfilamentous cortical layer is always localized at the proximal region of this bulge; other regions of the bulge are characterized by a thin cortical layer which is devoid of filamentous structure except for the apical portion of the bulge. Microfilaments at the base of the bulge are perpendicular or oblique to the egg surface. The cortical layer of the egg which is continuous to that of the proximal region of the bulge comprises microfilaments running parallel to the surface. The attainment of the bulge to its full size is followed by the development of the cleavage furrow along its base. The cleavage furrow appears to bisect the spindle midway between its poles. In cytochalasin B-treated eggs, where some cortical microfilaments are detected at the animal pole, a cytoplasmic bulge lower in height and wider in the diameter of its base than the normal one forms at the animal pole; however, it is subsequently resorbed into the egg. The formation of a cleavage furrow is not observed in these eggs. The mechanism of the polar body formation is discussed in the light of the present observations. INTRODUCTION

bulge. However, although there are some articles concerning the fine structure of polar body formation (Long0 and Anderson, 1969, 1970; Longo, 1972; Burgess, 1977; Meijer, 1979), fine structural details of the process of the bulge formation appear to be scanty. The previous paper (Shimizu, 1981) described the cortical organization at the animal pole in the Tubifex egg undergoing formation of the second meiotic apparatus (second metaphase). Extending the previous observations to the advanced meiotic phases, we have investigated the fine structural features of the surface and cortical cytoplasmic layer of the animal pole in eggs undergoing the second polar body formation. In this communication, we describe the changes in the microfilament organization in the cortical layer, and the relationship to the surface morphology and formation of the cytoplasmic bulge. The results obtained appear to exemplify Chambers’ or Wolpert’s speculation of local differentiation of the egg cortex. The process of the polar

The primary importance of the animal egg relates to its large size which guarantees its development into a complex multicellular embryo. The large egg size is retained by means of two extremely unequal meiotic divisions which produce one large egg and two or three small cells, i.e., polar bodies. Thus, the formation of polar bodies is a precise mechanism to reduce the double set of chromosomes to a single set without disturbing developmental potential of the egg (Wilson, 1925). Polar body formation consists of two steps (Longo, 1972): (i) formation of a cytoplasmic bulge for the polar body, and (ii) the subsequent development of cleavage furrow at the base of this bulge. The second step has been revealed to be a microfilament-dependent process (Longo, 1972; Peaucellier et al., 1974). As for the first step, Chambers (1917) and Wolpert (1960) postulated that local differentiation of the egg cortex at the animal pole is responsible for formation of the cytoplasmic 77

0012-1606/81/0900’7’7-12.$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

DEVELOPMENTALBIOLOGYVOLUME85,1981

78

body formation in the Tubifex egg has been briefly described elsewhere (Shimizu, 1979). MATERIALSANDMETHODS

Fertilized eggs of the freshwater oligochaete, Tubifex huttai NOMURA, were obtained as previously described (Shimizu, 1981). For experiments, they were all freed from the cocoon with a pair of watchmaker’s forceps in the culture medium for Tubifex embryos (Inase, 1960). Unless otherwise stated, all experiments were performed at room temperature (19-ZO’C). For scanning and transmission electron microscopy, eggs of various phases of the second meiosis were fixed and processed according to the method described previously (Shimizu, 1981). Meiotic phases were recognizable by the changes of the egg shape under the dissecting microscope (Shimizu, 1979). If eggs were present in an intact cocoon during the time of polar body formation, the gross shape of polar bodies appear to vary; conceivably dense packing of eggs in the cocoon seems to relate to this variation. To eliminate such a variation as much as possible, eggs subjected to electron microscopy were all taken out of cocoons at least 30 min ahead of the initiation of anaphase movement. Cytochalasin B (Sigma) was dissolved in dimethyl sulfoxide (10 mg/ml) and diluted with the culture medium immediately before use. Control eggs were immersed in the culture medium containing identical concentration of dimethyl sulfoxide. Colchicine (Merck) was dissolved in deionized water (10 mglml) and diluted with the culture medium before use. RESULTS

Tubifex eggs remain at metaphase of the second meiosis for about 60 min after completion of the second meiotic apparatus; during this period, there is no change in the structure of the meiotic apparatus and the cortical organization at the animal pole (Shimizu, 1981). Initiation of the anaphase movement of the second meiosis is recognizable by the appearance of meridionally running shallow grooves on the equatorial surface of the egg (onset of the second deformation movement; see Shimizu, 1979). Initial

Phase

At very early anaphase of the second meiosis, no significant alteration in the form of the second meiotic apparatus is detected, though the splitting of chromosomes arranged on the spindle is just initiated (inset in Fig. 1). At this meiotic phase, some changes in the surface morphology of the animal pole become apparent. The outer surface of the pole is classified into two areas: circular undulating area and its surrounding smooth area (Figs. 1

and 2a). The undulating area bears a number of short blebs arranged in a bouquet (Fig. 2a). In sections, the meiotic apparatus is found just beneath the undulating area whose diameter is comparable to the width of the spindle (Fig. 1). Corresponding to the surface morphology, the fine structure of the cortical layer is also locally differentiated. Except for the bleb-bearing portion (Fig. 2b), the cortical layer of the undulating area is devoid of filamentous structures and so thin that membraneous organelles are located very near the surface (Fig. 2~). Although the filamentous cortical layer of the bleb-bearing portion is closely associated with microtubules of the meiotic apparatus similar to that in metaphase eggs, its thickness appears to have decreased (cf. Shimizu, 1981). As Fig. 2c illustrates, the thick electron-dense cortical layer in the smooth area contrasts with the thin layer in the undulating area. It comprises microfilaments running parallel to the surface. Microtubules, probably emanated from the peripheral aster of the meiotic apparatus, run parallel to the surface, and are located near the filamentous cortical layer (Fig. 2~). Effects of colchicine. To determine the extent to which appearance of microfilaments in a specific area depends on the meiotic apparatus and other microtubular system, cortical organization was examined in eggs treated with colchicine which disrupts microtubules. Eggs at early metaphase of the second meiosis, i.e., about 30 min after the first polar body formation, were exposed to 1.5 mg/ml colchicine for 60-70 min, and fixed when the control eggs from the same cocoon initiated anaphase movement of chromosomes. The outer surface of the animal pole in these eggs is similar to that in the intact control eggs whereas the meiotic apparatus is completely destroyed at the animal pole. A circular undulating area and its surrounding smooth area are clearly discernible (Fig. 3a). Filamentous cortical layer underlies selectively the smooth surface, but not the undulating area (Figs. 3b and c). This result suggests that the appearance of filamentous cortical layer in a specific area at the initial phase of the polar body formation is independent of the presence of the meiotic apparatus. Later, a cytoplasmic bulge forms at the animal pole surface in these colchicinetreated eggs; however, it is subsequently resorbed into the egg. Formation of Cytoplasmic Bulge (Presumptive Polar Body) and Development of Cleavage Furrow As the anaphase movement proceeds, a cytoplasmic bulge for the second polar body is formed at the animal pole. Figure 4 depicts the process of formation of cytoplasmic bulge. At 15-20 min after initiation of anaphase movement, the undulated egg surface is found to be uplifted in a cone-shaped cytoplasmic bulge (Fig. 4a); this is midanaphase of the second meiosis. Along with the

TAKASHI SHIMIZU

Polar Body Formation

79

FIG. 1. Animal pole of an egg at very early anaphase of the second meiosis. One of chromosomes found in the spindle is illustrated in the inset; it shows initial sign of its splitting. The egg surface indicated by arrowheads is undulated and is associated with the peripheral aster (PA) of the meiotic apparatus. Note the regional difference in thickness of the cortical layer. B, bleb; L, lipid droplet; M, mitochondria; Y, yolk granule. X4000; inset, X20,000.

progression of the meiotic division, the bulge increases in height and surface area (Fig. 4b); then at early telophase, i.e., about 30 min after midanaphase, the cleavage furrow appears at the proximal region of the bulge (Fig. 4~). The distal region of the bulge has a rugged surface whereas other regions are relatively smooth (Figs. 4b and c). Judging from the external structure and the dimension, it may be thought that the rugged surface of the distal region of the bulge corresponds to the undulating area at the initial phase. Although a thin filamentous cortical layer is found at the apical portion of the bulge during anaphase, a thick filamentous layer is always localized at the proximal region of the bulge during the increase in its surface area. In other regions of the bulge, the cortical layer is thin and does not contain filamentous material; vesicles in the subcortical region look as if they are directly exposed to the inner aspect of the plasma membrane, but their fusion with the surface membrane does not appear to take place. Adjacent to the bulge, the cortical layer of the egg comprises microfilaments. At early or midtelophase, the

filamentous cortical layer is observed exclusively at the proximal region of the bulge, i.e., the leading margin of the cleavage furrow; this layer is continuous with that of the egg (Fig. 5a). At the apical portion of the bulge, no filamentous cortical layer is detected (Fig. 5b). Microfilaments at the proximal region of the bulge are arranged perpendicularly or obliquely to the surface (Fig. 5~); however, those in the egg proper run parallel to the surface (Fig. 5d). During the growth of the cytoplasmic bulge, the asters located at both poles of the apparatus become indistinct (Fig. 5a). The outer pole of the spindle is found in the cytoplasmic bulge; the distal cytoplasm of the bulge is rich in mitochondria and lipid droplets, but microtubules are few in number (Fig. 5b). The diameter of the bulge is almost comparable to the width of the spindle and the cleavage furrow appears to bisect the spindle approximately midway between its poles (Fig. 5a). As the cleavage furrow becomes deeper during telophase, chromosomes undergo decondensation first in the egg then in the bulge (Figs. 5a and b).

80

DEVELOPMENTALBIOLOGYVOLUME85,1981

FIG. 2. Scanning (a) and transmission (b and c) electron micrographs of the animal pole of eggs at very early anaphase of the second meiosis. (b and c) are enlarged views of Fig. 1. (a) Two surfaces are discernible: undulating area (U) and relatively smooth area (S). A bouquet ofblebs (B) is located in the undulating area. x4300. (b) Bleb (B&bearing portion. Note thin filamentous cortical layer (arrow) with which some microtubules are associated. ~40,000. (c) Cortical organization of the smooth area (S) and the undulating area (U). An arrowhead indicates the boundary of these areas. The smooth area is characterized by thick filamentous cortical layer (arrow). ~34,000.

TAKASHI

SHIMIZU

Polar Body Fomation

81

FIG. 3. Scanning (a) and transmission (d and c) electron micrographs of eggs treated with 1.5 mg/ml colchicine for 60 min and fixed at early anaphase of the second meiosis. (a) Surface architecture of the animal pole. An undulating area (U) with a bouquet of blebs and a smooth area (S) are discernible. x3500. (b) Cortical region of the undulating area showing a thin layer apposed to the surface membrane. M, mitochondea. ~40,000. (c) Cortical region of the smooth area. Note thick filamentous cortical layer (arrow) apposed to the surface membrane. M, mitochondria. x40,000.

Effects of cytochalasin B. To know the role of microfilaments in the formation of the cytoplasmic bulge and cleavage furrow, eggs were treated with 50 pg/ml cytochalasin B for 60 min before the initiation of the second polar body formation; after rinsing thoroughly in the culture medium without this drug, they were allowed to develop in this medium for another 40 or 80 min and fixed for electron microscopy. Polar body formation occurs normally in the control eggs. In cytochalasin-treated eggs fixed 40 min after treatment when the control eggs are at late anaphase or early telophase, a cytoplasmic bulge is found at the animal pole whereas the deformation movement is completely inhibited (Fig. 6a; also see Shimizu, 1978b). This bulge is, however, not only lower in height but also wider in diameter of its base compared with that of the control eggs. The periphery of the bulge is covered with numerous blebs; they are also seen on its apical portion (Fig. 6b). The egg surface around the bulge is complicated with a number of blebs and pronounced indentations (Fig. 6~). The cortical layer mostly comprises granular material; however, in some places, bundles of microfilaments can be seen (inset in Fig. 6~). Since the granular material is not observed in the cortical layer of the control eggs and intact eggs, they may represent residues of microfilaments disrupted by cytochalasin B. With the lapse of time, the cytoplasmic bulge is resorbed into the egg without attaining the maximal height observed in the in-

tact eggs. A cleavage furrow is not observed. The suppression of polar body formation may come about because of the disruption of the microfilaments in the cortical layer of the animal pole. Anaphase movement of chromosomes and their ensuing decondensation takes place normally in these cytochalasin-treated eggs. Terminal

Phase

About 70 min following the initiation of second polar body formation, a cytoplasmic bridge with a midbody connecting the polar body and the egg is found at the animal pole (Fig. 7). Decondensed chromosomes (karyomeres) in the egg fuse one another (inset in Fig. 7), then form a female pronucleus. Those in the bulge also undergo similar morphogenesis. At this phase of the meiosis, the meiotic apparatus is no longer present. This is the terminal phase of the second polar body formation (very late telophase). Subsequently the separation of the polar body from the egg takes place. At the terminal phase, the electron-dense cortical layer is confined to the vicinity of the cytoplasmic bridge (inset in Fig. 7). The cortical layer of the egg in other places becomes thin resulting from the disappearance of filamentous material which is observed at preceding midanaphase (compare Fig. 4 with Fig. 5a). Within a short time after formation of the second polar body, such a filamentous cortical layer seems to disap-

82

DEVELOPMENTAL BIOLOGY

VOLUME 85, 1981

FII G. 4. A series of scanning electron micrographs at the same magnification ( x 2750) illustrating the cytoplasmic bulge at the animal pole of eggs at 15 (a), 35 (b), and 50 (c) min after initiation of the anaphase movement of the second meiosis. Eggs were all derived from the same cocoon. A .t the early , the cytoplasmic bulge has a rugged surface and a clump of small bulbous projections (asterisk) is located on the bulge (a). Arrowf leads in (b and c) point to the boundary between the distal and side regions of the bulge. Note the difference in the surface morphology of these ! two regio Ins. (c) clearly shows development of cleavage furrow (CF) at the base of the bulge.

TAKASHI SHIMIZU

Polar Body Formation

83

FIG. 5. (a) Animal pole of an egg at 50 min after initiation of anaphase movement. A cleavage furrow (arrowheads) is evident at the base of the cytoplasmic bulge (CB). Note the thin cortical layer of the bulge in contrast to the thick electron-dense layer of the egg proper. Although chromosomes (arrow) in the bulge still remain in condensed state, those (double arrow) in the egg proper appear to undergo decondensation. VM, vitelline membrane. x 1300. (b-d) Higher-magnification views of (a). (b) Apical portion of the bulge. Mitochondria (M), lipid droplets (L), and yolk granules (Y) are located near the surface. Note rugged surface and thin cortical layer. Arrows point to chromosomes. The inset shows a decondensing chromosome found in the egg proper. VM, vitelline membrane. x5000; inset, x 10,000. (c) Cortical layer of the leading margin of the cleavage furrow. Note the cortical microfilaments (arrow) arranged obliquely to the surface. B, bleb; V, vesicle. ~44,000. (d) Cortical layer of the egg near the cytoplaemic bulge, A bundle of microfilaments (arrow) is seen running parallel to the surface. M, mitochondria. ~44,000.

pear; neither the filamentous layer nor the cytoplasmic mic bulge and subsequent development of cleavage furbridge is detected at the animal pole at the time of syn- row along its base (Longo, 1973; Burgess, 197’7);the first gamy, i.e., about 20 min after termination of the second step has been postulated to depend on “local differentiapolar body formation. tion” of the egg cortex at the animal pole (Chambers, 1917; Wolpert, 1960). The present study reveals that DISCUSSION local differentiation of the animal pole surface of the TuIn several animal species so far studied, polar body b@x egg is detected as early as the initiation of the anaformation consists of two steps, formation of a cytoplas- phase movement of chromosomes. The surface overlying

DEVELOPMENTALBIOLOGYVOLUME85.1981

FIG. 6. Scanning (a and b) and transmission (c) electron micrographs showing cytoplasmic bulge formation in cytochalasin B-treated eggs. Eggs at metaphase of the second meiosis were treated with 50 pg/ml cytochalasin B for 60 min and fixed 40 min later when the control eggs were at early teiophase. (a) An egg viewed from the side. Note a low cytoplasmic bulge (arrows) at the animal pole. VP, vegetal pole. x 130. (b) Overhead view of the animal pole of the egg shown in (a). The cytoplasmic bulge (CB) is surrounded by numerous blebs. x 1000. (c) Section of a portion in the vicinity of the bulge. Numerous blebs (B) are seen over granular cortical layer. Note intricate surface contour. In some places, microfilamentous structures are found (arrows in the inset). ~21,500.

the meiotic apparatus becomes undulated but the neighboring area remains relatively smooth. During the period up to midanaphase, the egg surface of this undulating area sprouts to form a cytoplasmic bulge. Then along with the increase in surface area of the bulge, smooth surface membrane is found to cover the sides of the bulge. Thus, two surface regions are discernible on the bulge: an undulating distal region and relatively smooth sides of the bulge. After bulge reaches full size, a cleavage furrow develops at its base. These findings not only suggest that polar body formation in the Tubifex egg is a two-step process as in other animal species, but also

allow us to probe into the mechanism of the formation of the cytoplasmic bulge. On the basis of the surface morphology revealed by the present study, it may be possible to dissect the process of bulge formation into two parts: (i) jutting out of the animal pole until midanaphase, and (ii) the ensuring increase in surface area of the bulge. Initial

Sur$ace Change

Corresponding to the surface differentiation at the initiation of the second polar body formation, the structure

TAKASHI SHIMIZU

of the cortical layer is also regionally different. It is thin and does not contain filamentous material at the undulating area except for the portion bearing a bouquet of blebs whereas a filamentous cortical layer is found apposed to the surface of the smooth area. Considering that microfilaments possess a contractile property (Wessells et al., 1971; Shimizu, 1978b), the two-ply structure created by the apposition of microfilaments to the plasma membrane could endow the surface with rigidity and render it smooth. On the other hand, the surface of the undulating area devoid of filamentous material appears to be pliant so that the jutting out of this area may be induced by the internal pressure of the egg (Chambers, 1917; Wolpert, 1960; Hamaguchi and Hiramoto, 1978). The surface of the undulating area is subsequently found to cover the distal region of the cytoplasmic bulge. Though the roughness of the surface is less intensive than before, the surface undulation persists during the bulge growth. Presently, how this surface undulation is brought about at early anaphase and maintained during the formation of the second polar body is unknown. No special structures other than endoplasmic reticulum are found near the inner aspect of the undulating surface membrane; therefore, it is plausible that the intrinsic molecular organization of the plasma membrane gives rise to this surface undulation (Sheetz and Singer, 1974). On the other hand, we cannot eliminate the possibility that the surface undulation observed in the present study is an artifact resulting from a possible shrinkage of eggs produced during the preparation for scanning electron microscopy (cf. Schroeder, 1979). At metaphase, the circular wavy area which corresponds to the present undulating area possesses patches of microfilaments apposed to the inner aspect of the surface membrane, and the surface of its central portion bearing a bouquet of blebs is underlain with a continuous filamentous cortical layer; however, no microfilaments are detected in the cortical layer around this wavy area (Shimizu, 1980). Thus, it is thought that concurrent with the initiation of the second polar body formation the wavy area and its neighboring area undergo opposite changes in the cortical structures. While the polymerization of microfilaments may be triggered by calcium ions released from intracellular stores (Shimizu, 1978a), either their appearance or specific location in the cortical layer around the undulating area takes place independently of the meiotic apparatus and other microtubular system. Alternatively, in view of the idea that polymerization of microfilaments is initiated on the plasma membrane (Schroeder, 1975; Lin and Lin, 1979), it is conceivable that positional information for specific localization of microfilaments is stored in the plasma membrane or its associated structures; in other words, initiation sites of microfilament -polymerization are distributed in a specific pattern at the animal pole.

85

Polar Body Formation

It has been postulated that the peripheral aster of the meiotic apparatus plays an important role in “weakening” of the egg cortex (Chambers, 1917). The present study indicates that such a weakening of the cortex may relate to the disappearance of microfilaments from the cortical layer. Although a number of microtubules are found associated with the cortical layer of the undulating area, it is presently unclear whether this disappearance is controlled by the meiotic apparatus; even when the meiotic apparatus is destroyed by colchicine, the filamentous cortical layer disappears from the undulating area. Increase in Surface Area of Cytoplasmic

Bulge

The growth of cytoplasmic bulge is characterized by its increase in surface area and height. Figure 8 is a summary of changes in microfilament distribution in growing bulge; it is indicated that the increase in surface area of the sides of the bulge solely contributes to the bulge growth. There are two possible mechanisms to explain this increase. (i) New plasma membrane originating from membraneous organelles is inserted into the preexisting surface membrane, as seen in cases of cleavage furrow development in various animal eggs (Bluemink and de Laat, 1973; Sanders, 1975). (ii) Reorganization of the cortical layer takes place (Bluemink, 1970; Opas and Soltynska, 1978): Microfilaments disappear from a specific part of the cortical layer, then the surface membrane there slopes upward forming the sides of the bulge. In the present study, no fusion of the plasma membrane with membraneous organelles can be found at any phase of the bulge growth. Insertion of membranes into the surface of the bulge, if any, does not seem significant for the increase in its surface area. Thus, as the second possibility indicates, the surface membrane covering the bulge might be mostly derived from the preexisting surface membrane of the animal pole. Therefore, changes of the microfilament domain (Fig. 8) result from a precisely programmed depletion of filaments. However, it is unlikely that their simple breakdown is solely responsible for such a depletion, because the physiological condition of the cytoplasm in the Tubifex egg at anaphase of meiosis is favorable for microfilament-polymerization (Shimizu, 1978a). Rather, the distribution of components in the plasma membrane on which microfilaments anchor may change during the bulge formation. Recently, Hoessli et al. (1980) have shown in mouse lymphocytes that a-actinin which represents a necessary element to join the plasma membrane and microfilaments moves along the membrane concurrently with redistribution of surface receptors. There is evidence that the organization of the plasma membrane is altered during mitosis in mammalian cells (Blanquet et al., 1977; Berlin et al., 1978) and polar body formation in Spisula eggs (Longo, 1979). However, it remains to be determined

86

DEVELOPMENTALBIOLOGY

VOLUME85, 1981

FIG. 7. Animal pole of an egg ‘70min after initiation of anaphase movement. A cytoplasmic bridge with a midbody (MB) is evident. However, direct exposure of microtubules of its apical portion to the outside indicates that the bridge fractured during preparation for electron microscopy; thereby cytoplasmic bulge might be lost. Numerous microtubules are seen in the cytoplasmic bridge and in its vicinity of the egg. Around these fascicles of microtubules (Mt) are found a number of mitochondria (RI) and Golgi complex (G). Note the filamentous cortical layer apposed to the smooth egg surface (arrows) in the vicinity of the bridge. Arrows in the inset indicate the distribution of filamentous cortical layer; fusing karyomeres (K) with some nucleoli are located far from the animal pole surface. x 13,300; inset, x 1000.

TAKASHI SHIMIZU

whether such a reorganization of the plasma membrane in dividing cells relates to changes in distribution of a-actinin which reportedly concentrates in cleavage furrow (see Fujiwara et al., 1978). To ensure the increase in bulge height, there should be a mechanism which prevents an unlimited increase in the diameter of the base of the bulge during its increase in surface area. Microfilaments found at the proximal region of the bulge, especially those arranged perpendicularly to the surface, may play such a role as pulling the egg surface inward. In fact, a bulge formed in eggs whose microfilaments are mostly disrupted by cytochalasin B is low and broad compared with that in the intact eggs, as in Spisula eggs (Longo, 1972). Nevertheless, cytochalasin treatment fails to abrogate bulge formation. It may be supposed that remnants of microfilaments which probably escape the disruptive effect of cytochalasin B play a skeletal role endowing the surface with rigidity and that the surface circumscribed by this rigid area would swell out by the increased internal pressure of the egg.

Polar Body Formation

87

FIG. 8. Distribution of microfilaments (MF) in the cortical layer at various phases of the second meiotic division. (A) Early anaphase, (B) late anaphase, (C) early telophase. The areas presented correspond to those indicated by two arrows in the right row. In order to be proportioned with (A) and for convenience of the comparison, the contour in (B) and (C) is simplified by eliminating the shape of the cytoplasmic bulge. Arrowheads in (B) and (C) point to the regions corresponding to the proximal regions of the bulge. FCL, filamentous cortical layer.

margin of the forthcoming cleavage furrow is brought close to the meiotic spindle along with the bulge growth. Supposing that the meiotic spindle retains the capacity to induce cleavage furrow at late anaphase or early telophase, it is conceivable that the cortical layer brought Furrowing close to the spindle is stimulated to form a cleavage furrow. Perpendicularly arranged microfilaments at the The attainment of the cytoplasmic bulge to its full size proximal region of the growing bulge might play an imis followed by development of cleavage furrow along its portant role in making surface -spindle interaction possibase, which is known to be microfilament dependent ble. (Longo, 1972; Peaucellier et al., 1974). In the Tubifex egg, the cleavage furrow appears to bisect the spindle The author wishes to thank Professor T. S. Yamamoto for his invaluapproximately midway between its poles (Fig. 5a). Iniable advice. He is also grateful to Dr. T. E. Schroeder of University of tiation of furrowing may be a process dependent on the Washington for reading and criticizing the manuscript. This work was function of the meiotic apparatus (Shimizu, 1979). Al- supported in part by Grant-in-Aid for Scientific Research (474325)from though, during early or midanaphase, some microtubules the Ministry of Education of Japan. which probably emanated from the peripheral aster are observed beneath the filamentous cortical layer underlyREFERENCES ing the smooth surface area, they do not appear to associC. F., and SCHROEDER,T. E. (1979). Cell cleavage. Ultraate with the cortical layer. Furthermore, these microtu- ASNES, structural evidence against equatorial stimulation by aster microtubules all run parallel to the surface, suggesting that no bules. Exp. Cell Res. 122, 327-338. tubules from the inner aster reach the egg periphery BERLIN, R. D., OLIVER, J. M., and WALTER, R. J. (1978). Surface functions during mitosis I: phagocytosis, pinocytosis and mobility of there. Therefore, the establishment of the position of the surface-bound Con A. Cell 15.327-341. cleavage furrow may be carried out by some process P. R., DECAESTECKER,A.-M., and COLLYN-D’HOOGHE, other than such a direct stimulation of the egg cortex by BLANQUET, M. (1977). Cell-cycle dependent changes in the surface membrane araster microtubules as postulated by Rappaport (1971) chitecture of BHK 21/C13 cells. Cytobiologie 16, 27-51. (cf. Burgess, 1977; Asnes and Schroeder, 1979). BI.UEMINK, J. G. (1970). The first cleavage of the amphibian egg. An electron microscope study of the onset of cytokinesis in the egg of This negative evidence suggests an alternative mechaAmbystoma mexicanum. J. Ultrastruct. Res. 32, 142-166. nism, i.e., that the spindle, another component of the BLUEMINK,J. G., and DELAAT, S. W. (1973). New membrane formation meiotic apparatus, is involved in furrow establishment. during cytokinesis in normal and cytochalasin B-treated eggs of Rappaport and Rappaport (1974) showed in cultured Xenopus laevis. I. Electron microscope observations. J. Cell Biol. newt kidney cells that the mitotic spindle also has the ca59, 89-108. pacity of the furrow establishment. Furthermore, such a BURGESS,D. R. (1977). Ultrastructure of meiosis and polar body formation in the egg of the mud snail, Zlyanassa obsoleta. In “Cell capacity of the spindle in echinoderm eggs is reportedly Shape and Surface Architecture” (J. R. Revel, U. Henning, and F. retained as late as telophase of the mitosis when the Fox, eds.), pp. 569-579. Alan R. Liss, New York. asters are no longer distinct (Rappaport, 1975). In the CHAMBERS,R. (1917). Microdissection studies. II. The cell aster: A reTubifex egg, the surface which would become the leading versal gelation phenomenon. J. Exp. 2001. 23, 483-505.

DEVELOPMENTALBIOLOGYVOLUME85.1981

88

FUJIWARA,K., PORTER,M. E., and POLLARD, T. D. (1978). Alpha-actinin localization in the cleavage furrow during cytokinesis. J. Cell Biol.

79, 268-275.

112,55-62. HOESSLI, D., RUNGGER-BRANDLE, E., JOCKUSH, B. M., BIANI, G. (1980). Lymphocyte cr-actinin. Relationship to brane and co-capping with surface receptors. J. Cell Biol. 314. INASE, M. (1960). The culture solution of the eggs of Tubifex. Tohoku Univ. Ser. IV (Biol.)

and GABcell mem84, 305-

Sci. Rep.

26, 65-67.

LIN, D. C., and LIN, S. (1979). Actin polymerization induced by a mobility-related high-affinity cytochalasin binding complex from human erythrocyte membrane. Proc. Nat. Acad. Sci. USA 76, 2245-2349. LONGO,F. J. (1972). The effects of cytochalasin B on the events of fertilization in the surf clam Spisula solidissima. I. Polar body formation. J. Exp. Zool. 182,321-344. LONGO, F. J. (1973). Fertilization: A comparative ultrastructural review. Biol. Reprod. 9, 149-215. LONGO,F. J. (1979). Surface alterations of fertilized surf clam (Spisula solidissima) eggs induced by concanavalin A. Develop. Biol. 68,422439.

LONGO,F. J., and ANDERSON,E. (1969). Cytological aspects of fertilization in the lamellibranch, Mytilus edulis. I. Polar body formation and development of the female pronucleus. J. Exp. Zool. 172,69-96. LONGO,F. J., and ANDERSON,E. (1970). An ultrastructural analysis of fertilization in the surf clam, Spisula solidissima. I. Polar body formation and development of the female pronucleus. J. Ultrustruct. Res. 33, 495-514. MEIJER, L. (1979). Hormonal control of oocyte maturation in Arenicola marina L. (Annelida, Polychaeta). II. Maturation and fertilization. Growth Diner.

L. (Polychaete Annelid). J. Em-

RAPPAPORT,R. (1971). Cytokinesis in animal cells. Znt. Rev. Cytol. 31,

HAMAGUCHI, M. S., and HIRAMOTO, Y. (1978). Protoplasmic movement during polar-body formation in starfish oocytes. Exp. Cell Res.

Develop.

vated eggs of Sabellaria alveolata bryol. Exp Morphol. 31, 61-74.

21, 315-329.

OPAS,J., and SOLTYNSKA,M. S. (1978). Reorganization of the cortical layer during cytokinesis in mouse blastomeres. Exp. Cell Res. 113, 208-211. PEAUCELLIER, G., GUERRIER, P., and BERGERARD,J. (1974). Effects of cytochalasin B on meiosis and development of fertilized and acti-

169-213.

RAPPAPORT,R. (1975). Establishment and organization of the cleavage mechanism. In “Molecules and Cell Movement” (S. Inoue and R. E. Stephens, eds.), pp. 287-304. Raven Press, New York. RAPPAPORT, R., and RAPPAPORT, B. N. (1974). Establishment of cleavage furrow by the mitotic spindle. J. Exp. Zool. 189, 189-196. SANDERS,E. J. (1975). Aspects of furrow membrane formation in the cleaving Drosophila embryo. Cell Tiss. Res. 166, 463-474. SCHROEDER,T. E. (1975). Dynamics of the contractile ring. In “Molecules and Cell Movement” (S. Inoue and R. E. Stephens, eds.), pp. 305-334. Raven Press, New York. SCHROEDER,T. E. (1979). Surface area change at fertilization: resorption of the mosaic membrane. Develop. Biol. 70, 306-326. SHEETZ, M. P., and SINGER, S. J. (1974). Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions. Proc. Nat. Acad. Sci. USA 71, 4457-4461. SHIMIZU, T. (1978a). Deformation movement induced by divalent ionophore A23187 in the Tubifex egg. Develop. Growth Di,ffer. 20,27-33. SHIMIZU, T. (1978b). Mode of microfilament-arrangement in normal and cytochalasin-treated eggs of Tubifex (Annelida, Oligochaeta). Acta Embryol.

Exp.

1978, 59-74.

SHIMIZU, T. (1979). Surface contractile activity of the Tubifex egg: its relationship to the meiotic apparatus functions. J. Exp. Zool. 208, 361-378. SHIMIZU,T. (1981). Cortical differentiation of the animal pole during maturation division in fertilized eggs of Tub$ex (Annelida, Oligochaeta). I. Meiotic apparatus formation. Develop. Biol. 85,65-76. WES~ELLES, N. K., SFQONER,B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A., TAYLOR, E. L., WRENN, J. T., and YAMADA, K. M. (1971). Microfilaments in cellular and developmental processes. Science 171, 135-143. WILSON, E. B. (1925). “The Cell in Development and Heredity.” Macmillan, New York. WOLPERT, L. (1960). The mechanics and mechanisms of cleavage. Znt. Rev. Cytol.

10, 163-216.