- Email: [email protected]
A subcortical, pigment-containing structure in Xenopus eggs with contractile properties
DEVELOPMENTAL
BIOLOGY
95,
4:?9-446
(1983)
A Subcortical, Pigment-Containing .Xenopus Eggs with Contractile R. W. MERRIAM, Department
of NewobioLogy Received
R. A. SAUTERER,
and Behavior, June
State
llniversity
19, 1981; accepted
Structure in Properties
AND K. CHRISTENSEN of New York, Stay
in revised
fm
October
Brook,
NW
York
1179.4
7, 1982
An accumulation of insoluble, finely granular material has been observed under the pigmented surface of Xenopus eggs by a specialized “dry fracture” technique and scanning electron microscopy. Cortical granules and pigment granules can be recognized with the techniques and can be seen to be embedded in the material. Thin sections show that the region also contains mitoehondria and membranous vesicles or reticula. Yolk platelets are largely excluded from the heaviest accumulations of the material. The substance is most dense just under the cortex and grades off gradually into the more diffuse, yolk-containing network of the endoplasm. The accumulation of material is much thicker in the animal hemisphere of the egg than in the vegetal hemisphere, and the pigment embedded in it defines the pigmented area of the animal hemisphere. In the pigmented area the material excludes yolk for a thickness of 3-7+ pm from the surface. In the vegetal hemisphere there is no such accumulation and yolk platelets can be found almost touching the plasmalemma. Cortical contractions have been experimentally induced in eggs. Their relative strength correlates with the relative thickness of the finely granular, subcortical material. During contraction the material accumulates to much greater thicknesses, excluding yolk from thicknesses of 15-30+ pm from the surface. The contracting entity is, or is in, the finely granular material. Injection of cytochalasins into the eggs inhibits cleavage furrow operation but does not inhibit the induced cortical contractions. The induced contractions thus do not seem to be dependent on actin microfilamentogenesis as is the operation of the contractile ring of the cleavage furrow. The differential sensitivity to cytochalasins of the contractile ring and the system responding in the induced cortical contractions, suggests a two-component system for cortical contractions in the egg. A model is presented which accomodates the available data.
accompanied by an accumulation of pigment as the surface coat condensed in that region (see also Perry and In two notable papers, Holtfreter (1943a,b) described Waddington, 1966). In these reports Holtfreter strongly a “surface coat” on the surface of the early embryos of implicated the pigmented ‘*surface coat” with contracseveral species of amphibians. This surface coat was tions and movements at the surface of the egg and early marked by its content of pigment. Not only could the embryo. presence of the coat be detected by the presence of pigSince those observations were reported, a number of studies have been made of the surface cytoplasm of ment, but there was seen to be a direct relationship between the thickness of the coat and the amount of amphibian eggs and embryos by thin sectioning and observation with the electron microscope (e.g., Franke pigment. et al., 1976; Grey et al., 1974; Selman and Perry, 1970; In a series of experiments Holtfreter showed that this coat was intimately associated with the movement of Singal and Sanders, 1974). These investigations have established that the “surface coat” of Holtfreter is not pigmented ectodermal cells over the surface of the extracellular. Rather, a dense felt-like layer about 0.1 cleaving embryo. In fact, the spreading of the pigpm thick, just under the plasmalemma, overlays a mented coat of the unfertilized, uncleaved egg occurred thicker layer which contains the pigment. This latter in overaged eggs of Rana Ipilriens. This showed that the epibolic movement of thle pigmented coat was a phe- layer is reported to be 3-5 pm thick in the animal hemisphere of amphibian eggs. It contains the cortical grannomenon intrinsic to the egg coat itself and was not ules, membranous vesicles, and mitochondria. Yolk tends dependent on cellular movement per se in the normally cleaving embryo. The same observation had been made to be excluded by the matrix of this peripheral subon overaged eggs of C~ae~:o~~e~ (Liltie, 1906) and eggs cortical cytoplasm. Curtis (1960) found that pieces of the pigmented corof other species as well (ffor reviews see Brachet, 197’7; Holtfreter, 1943b). tex of Xenopus eggs could be removed with needles. The Holtfreter also showed that when an ectoderm cell excised cortex was tough and described as being about changes shape in wound healing or at the blastopore of 0.5-3.0 pm thick. It was sufficiently coherent to allow a gastrula, a constriction of one end of the cell was transplantation and still retain biological activity. When INTRODUCTION
439 0012-1606/83/020439-08$03.00/O Copyright All tights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
440
DEVELOPMENTAL
BIOLOGY
fixed and sectioned for electron microscopy, the isolated cortex could be seen to include embedded pigment, mitochondria, and other granules underneath a thin hyaline layer associated with the plasmalemma. The cortex from the unpigmented vegetal hemisphere was too fragile to allow such experimental handling. Puncture wounds made in the pigmented hemisphere of amphibian eggs heal by active closure in the presence of calcium ion (Bluemink, 1972; Gingell, 1970; Luckenbill, 1971; Schroeder and Strickland, 1974). This process also seems to involve the cortical layers under the plasmalemma. The superficial felt-like layer becomes thicker around the wound and extends from the surface in blebs or outfoldings as the wound is constricted. A ring of filamentous material develops in or under the felt-like layer around the hole. A zone of vesicles underlies the felt-like layer and under that a massive accumulation of pigment and yolk occurs around the edge of the wound. The thickening of the felt-like layer and the accumulation of pigment have led to suggestions that these layers contract during the hole closure process. Bluemink (1972) noted a functional distinction between two contractile phenomena associated with the amphibian egg cortex. Vinblastine at loo-250 pg/ml inhibited cleavage furrow formation, but had no effect on wound healing. In this paper we induce contractions at the surface of Xenows eggs and utilize a dry-fracture technique for observing the fine structure of the cortical regions with the scanning electron microscope. The dense, subcortical cytoplasmic material, in which pigment is embedded, is shown to thicken and become more dense during contraction. We distinguish the operation of the contractile ring of the cleavage furrow from that of the induced surface contractions by showing that cytochalasins inhibit the furrow but not the induced surface contractions. This distinction leads us to propose a model in which two different contractile systems are envisioned near the surface of the egg. METHODS
AND
MATERIALS
Oocytes were obtained from the ovaries of adult female Xenopus laevis frogs. The excised ovaries were vigorously swirled for 20 min at 30°C in a solution containing 2 mg/ml of collagenase, Type II (Sigma Chemical Co., St. Louis, MO.) and 0.1 M phosphate buffer, pH 7.5, after the method of Schorderet-Slatkine and Drury (1973). The largest oocytes, thus freed of mesothelial and connective tissue components, were then washed in Ringer’s solution. Egg laying was induced by injection of human chorionic gonadotropin. Freshly stripped eggs were dejellied by treatment with a solution of 40 mM /3-mercaptoethanol, pH 8.9, followed by washings in 0.1 strength
VOLUME
95, 1983
Ringer’s solution. The vitelline membrane was then removed manually with forceps. Cells to be tested were transferred by pipet into the test solution. Shape changes were recorded photographically through a Leitz macro lens. Microinjections were done essentially by the methods of Gurdon (1973). Eighty nanoliters was injected into each cell from a calibrated micropipet. This represented about 8% of total cell volume. The injection carrier solution contained 88 mM NaCl; 1 mM KCl; 15 mM TrisHCl, pH 7.2. Cytochalasins B (Aldrich Chemical Co., Milwaukee, Wise.) or D (Sigma) were dissolved in dimethyl sulfoxide (DMSO) at 10 mg/ml and diluted with carrier solution to give about 10 pg/ml of cell water, assuming that half of the cell volume was water. Control injections always were made with the same concentration of DMSO in injection carrier. Oocyte or egg internal structures were observed by scanning electron microscopy (SEM) after “dry fracturing” the cells (Leverah et al., 1980). The cells were either fixed in 3% glutaraldehyde, buffered to pH 7.4 by 0.1 Mphosphate buffer, for 3 hr at 5”C, or were quickfrozen in liquid propane at liquid nitrogen temperatures and the ice was substituted by acetone at -40°C. In both cases, when water had been replaced by acetone, the cells were split open with fine needles in acetone. After critical-point drying, the fracture faces were coated with gold-palladium (60/40) for observation in a JOEL JSM035C scanning electron microscope (SEM). For thin sections, eggs were fixed 3 hr in 3% glutaraldehyde and buffered to neutral pH with 0.1 M phosphate buffer at 5°C. After washing, eggs were postfixed in 1% osmium tetroxide and embedded in Polybed. Sections were stained with lead citrate and uranyl acetate before observation in a JOEL 1OOB electron microscope. RESULTS
In previous work we had found that Xenopus eggs could be induced to undergo shape changes that involved contractions at or near the surface (Merriam et al., 1978). When placed into a buffered medium containing 0.9 M sucrose and 4% ethanol, the pigmented surface would isometrically contract in surface area, become cone-shaped, and finally contract into a small wrinkled nipple on top of a distended unpigmented hemisphere (Fig. 1). Ethanol by itself would induce contraction of the pigmented hemisphere into a wrinkled nipple without the intervening cone shape. Sucrose by itself produced no observable response. When a hole was made in the responding egg, the yolky endoplasm was squeezed out, relieving the internal pressure. In this case both animal and vegetal hemispheres contracted simultaneously (Fig. 2). These observations were inter-
MERRIAM,
SAUTERER,
AND
CHRISTENSEN
Subwrtical
Cmtractile
Structure
FIG. 1. Unfertilized Xenopus eggs with the pigmented cortex contracted into a wrinkled nipple by exposure to 0.9 Msucrose and 4% ethanol. The yolky endoplasm has been squeezed out of the animal hemisphere into a distended vegetal hemisphere. X25. FIG. 2. Unfertilized Xenopus eggs (arrows) induced to contract by exposure to 0.9 M sucrose and 4% ethanol. Holes have been made in the eggs so that the yolky endoplasm (Y) is being squeezed out. Note the isometric decrease in surface area of both pigmented and unpigmented hemispheres. X25.
preted to mean that both pigmented and unpigmented surfaces could respond by contracting, but that the contractions of the pigmented surface were much stronger. We were interested in visualizing those subsurface structures which produced strong contractions in the pigmented hemisphere and weak contraptions in the unpigmented hemisphere. Thin sectioning and observations with transmitted electrons produced an image which has been observed many times (Fig. 3). A thin felt-like layer can be seen under the plasmalemma which is devoid of large organellles and largely devoid of granular materials. It tills projections extending from the surface. Sometimes microfilaments can be seen in microvilli, embedded in the feelt-like matrix. Below the feltlike layer is a much thicker layer which is characterized primarily by the absence of yolk but does include cortical granules, pigment granules, some mitoehondria, and many membrane-limited vesicles or reticula. When the peripheral cytoplasm of the pigmented hemisphere is dry-fractured and the fracture face is observed by scanning electron microscopy, a vague distinction can again be made between a subcortical layer of cytoplasm and the deeper endoplasm. Such an image of an egg in the relaxed condition is seen in Fig. 4. The distinction in this type of image rests on two things: the outer 3-7 pm of cytoplasm is largely devoid of yolk and the background cytoplasmic material is more dense. This dense accumulation of material may account for the exclusion of large yolk platelets from the region. At
higher magnifications the accumulated materials can be seen in the dry-fractured state to be finely granular in texture (Fig. 8). The extreme cortical layer, between the cortical granules and the plasmalemma (arrowhead in Fig. 8). does not show this granular texture and probably corresponds to the felt-like layer seen in Fig. 3. Abundant pigment granules can be seen to be embedded in the granular subcortical cytoplasm (Figs. 3 and 8). The dense, pigment-containing, subcortieal material seen in the animal hemisphere is absent in the unpigmented vegetal hemisphere (Fig. 5). Yolk platelets and other organelles can be found in close approximation to the plasmalemma. The dense, granular cytoplasmic material which seems to exclude yolk under the pigmented surface is largely gone. Instead, one sees a more lacy, vesiculated reticulum of material in which all organelles are suspended (Figs. 5 and 7). The transition between the pigment-containing cytoplasmic material of the animal hemisphere and the more delicate cytoarchitecture of the vegetal hemisphere surface is sharp. It is clear that the presence of surface pigment is defined by the extent of the dense subcortical material in which it is embedded. The cortical structures of the pigmented surface are strong enough to isolate in buffered saline solutions. The egg is cut in half and a stream of buffer blown across the cut surface. The endoplasm washes away leaving an insoluble cortex. Electron microscopy of the isolate shows embedded pigment and cortical granules
442
DEVELOPMENTAL BIOLOGY
VOLUME 95,1983
MERRIAM,
SAUTERER,
AND
CHRISTENSEN
in a cohesive matrix, plus adherent yolk on the cytoplasmic surface (unpublished observations). The structure is 5-15 pm thick and will keep its coherency indefinitely. This shows that the cytoplasmic matrix of the cortical region is an insoluble structure in which organelles are embedded. The unpigmented cortex is too fragile to be isolated in this manner. Contractions induced in the egg surface were much stronger in the pigmented surface than in the unpigmented surface. The contracting entity also contained the surface pigment because it was carried into the wrinkled nipple during the contraction (Fig. 1). We considered the possibility that the dense, pigment-containing subcortical material was the contractile element that responded to our induction. If this were so, we might expect it to become thickened or more dense during contraction because the surface area of the pigmented hemisphere became much reduced during contraction. To test this possibility we induced contraction and fixed the eggs in the contracted state. The resultant pigmented nipples were very tough and hard to fracture in the dried state, but such fractures were obtained. As can be seen in Fig. 6, the dense, pigment-containing subcortical material of the cytoplasm has become much thicker. In highly contracted cases, the entire yolky endoplasm has been squeezed out into the vegetal hemisphere and the entire nipple is filled with the dense material. We conclude that the matrix contains, or is, the responding contractile element. Actin has been shown to be a prominent component of the cortical cytoplasm of Xenqimcs eggs and oocytes (Franke et al., 1976). We wondered if the contracting cytoplasmic matrix might be operating through the involvement of actin microfilaments. If this were the case, we might expect the system to be sensitive to the cy-
SuhwrticaZ
Ccmtractile
TABLE THE EFFECT OF CYTOCHALASIN CO~RA~ION
Frog
Injected
1
None DMSO CB
lO/lO 1307
None
12112
2
Contraction
417"
DMSO
7/10
CB
802”
443
Structure 1 D ON INDUCED IN EGGS
Cleavage furrow
Percentage contract
CORTICAL
Percentage cleaved
23/26 20/24 3/w
100
76 57
88 83 25
13/17 7112 O/12"
100 70 67
76 58 0
“Many cells went bad after injection ment distribution looked good enough
of CB. Only were scored.
cells whose
pig-
tochalasins. To test this possibility we injected cytochalasin D into the animal hemisphere of fertilized eggs. The amount injected was sufficient to bring the intracellular concentration to at least 10 pg/ml of cell water. The injected eggs were placed for 15 min into an appropriate medium and then divided into two groups. One group was allowed to develop in 0.1 Amphibian Ringer’s solution so that the initiation of a normal cleavage furrow could be observed. The other group was challenged with 4% ethanol to see if a contraction of the pigment of the animal hemisphere could be observed. The results with eggs of two frogs can be seen in Table 1. In the eggs of frog 2 which had been injected with CB, it was observed that the accumulation of pigment at the animal pole and even the movement of pigment into the region of the future cleavage furrow occurred normally. Yet in no case did a cleavage furrow form. We conclude from this and from the data of Table 1 that an internal concentration of CB, sufficient to block
FIG. 3. A transmission elect.ron micrograph of a thin section of the pigmented, animal hemisphere cortical region of a relaxed Xenopus egg. A cortical granule (cg) and pigment granule (p) may be seen embedded in a matrix which shows granular material and an extensive membranous reticulum. Note the finely textured matrix just under the plasmalemma (arrow) which is concentrated within the surface projections. X36,830. FIG. 4. A scanning electron micrograph of the fracture face of a dry-fractured Xenqms egg. The egg was fixed in the relaxed state. The location of the plasmalemma of the animal, pigmented surface is marked by an arrow. Note the accumulation of dense matrix 3-7 pm thick beneath the plasmalemma and the less dense, yolky endoplasm under it. X2450. FIG. 5. A scanning electron micrograph of the fracture face of a dry-fractured Xenopus egg. The location of the plasmalemma of the vegetal, unpigmented surface is marked by an arrow. Note the lack of any appreciable accumulation of dense matrix under the plasmalemma, as seen in Fig. 4. Yolk (Y) can be found right up to the surface in a more delicate supporting meshwork. X2450. FIG. 6. A scanning electron micrograph of the fracture face of a dry-fractured Xenopus egg. The location of the plasmalemma of the animal, pigmented surface is marked by an arrow. This egg was induced to contract with 4% ethanol. The micrograph shows the deep accumulation of dense matrix, 22 pm thick, beneath the plasmalemma. This matrix should be compared with that of the egg in the relaxed state of Fig.
4. X2450. FIG. 7. A scanning electron micrograph of the fracture face of a dry-fractured Xenopus egg. The unpigmented surface is marked by an arrow. At this higher magnification the delicate supporting be seen to be vaguely granular in texture. X13,720. FIG. 8. A scanning electron micrograph of the fracture face of a dry-fractured Xenopus egg. The pigmented surface is marked by an arrow. Microvilli may be seen projecting from the surface pigment granules (p) can be recognized. The texture of the dense matrix immediately under the head) while the deeper matrix has a vaguely granular appearance. The smallest granules seen granules seen in the thin section of Fig. 3. X13,720.
location of the plasmalemma of the vegetai, meshwork under the plasmalemma can location of the plasmalemma of the animal, of the egg and cortical granules (cg) and plasmalemma often appears hyaline (arrow by this technique are larger than the dark
444
DEVELOPMENTAL
BIOLOGY
even the initiation of the cleavage furrow, still allows an induced contraction of the pigmented cortex and normal precleavage movements of pigment. DISCUSSION
The developmental importance of contractile systems near the egg surface has been repeatedly emphasized in numerous publications. Holtfreter (1943a,b), showed that epibolic movements of the animal hemisphere were accompanied by movements of superficial pigment. Superficial pigment concentrations were also associated with cellular shape changes in wound healing and gastrulation (see also Perry and Waddington, 1966). In animal eggs, the cortex has been shown to be actively engaged in the engulfment of the fertilizing sperm by the formation or extension of microvilli around the sperm head. The microvilli contain actin microfilamentous cores and they subsequently fuse to form a fertilization cone (Longo, 1980; Picheral, 1911; Shalgi and Phillips, 1980; Tilney and Jaffe, 1980; Vacquier, 1981). Morphologically, the fertilization cone consists of the superficial, felt-like layer of the cortex (e.g., Hylander et al., 1981; Kudo, 1980). The cone and the enclosed sperm nucleus are withdrawn into the deeper cortical cytoplasm. ~ytochalasin B prevents fertilization cone formation and sperm entry in Marine animal eggs (GouldSomero et al., 1977; Longo, 1978; Schatten and Schatten, 1980). Since the fertilization cone involves the formation of microfilamentous actin arrays, it is not surprising that CB prevents its formation and the entry of the sperm (e.g., Brown and Spudich, 1981; Grumet et al., 1979). It is noteworthy, however, that a roughly concomitant cortical function, cortical granule exocytosis, is not inhibited by CB (Gould-Somero et al., 1977; Longo, 1978; Manes et al., 1978). The cortical granules lie beneath the felt-like outer cortex and in amphibian eggs are embedded in the dense, pigment-containing material. Their exoeytosis is an active process, involving both the movement to the plasmalemma and the extrusion of their contents in a short period of time (Grey et al., 1974). The lack of sensitivity to CB by this process suggests that exocytosis does not involve aetin microfilamentogenesis. Additional types of cortical contractions occur at the surface of amphibian eggs between fertilization and first cleavage. The pigmented surface contracts isometrically in area within 5-6 min after sperm entry (Elinson, 1975). This event could still occur after egg activation in the presence of CB (Manes et al., 1978). In fertilized Discoglossus eggs (Klag and Ubbels, 1975) and Xenopus eggs another isometric contraction of the pigmented surface occurs at about 70 min postfertilization. In Xen-
VOLUME
95, 1983
opus eggs this contraction relaxes as the first cleavage furrow forms and is completely insensitive to CB, even when injected into the egg (Christensen and Merriam, 1981). Still other types of cortical contractions have been recorded in fertilized animal eggs (Hara et al., 1980; for a review see Vacquier (1981)). Cortical contractions, more sharply localized in the superficial cortex as contractile rings, occur during cleavage (e.g., Perry et al., 1971; Schroeder, 1973), in meiosis (Longo, 1972), and in the polar lobe formation of certain molluscan eggs (Schmidt et al., 1980). All of these events can be shown to be inhibitable by CB. A hole, made in the surface of amphibian eggs, will heal in the presence of calcium ions. This process is accompanied by the appearance of a ring of a&in-like microfilaments in the superficial cortical region around the wound (Bluemink, 1972). The hole apparently closes by a circumferential constriction of the cortex. Superficial pigment is moved toward the hole during closure (Bluemink, 1972; Gingell, 1970; Luckenbill, 1971). The inclusion of CB in the medium during healing completely blocks wound closure in Xenom eggs (Christensen and Merriam, 1981). A final type of cortical movement in amphibian eggs may be mentioned. A grey crescent is a cortical region which forms opposite to the point of sperm entry in normal fertilization (for a review see Brachet (1977)). It is marked by the movement of pigmented cortex away from the area, and is associated with the establishment of the future embryonic axes. It is still not clear whether the grey crescent is formed by a rotation of the pigmented cortex over the underlying yolky endoplasm (Elinson and Manes, 1978; Klag and Ubbels, 1975) or by cortical contractions initiated at the site of sperm entry (Palacek et al., 1978). What is clear, however, is that again cortical movements are involved and that a grey crescent-like region can form in the presence of CB in the cytoplasm {Manes et al., 1978). Other workers have demonstrated that the amphibian egg cortex can be experimentally induced to contract. Most of these inductions have implicated calcium ion as the trigger. For example, the localized application of polyvalent ions to the surface of frog eggs will induce movement of surface pigment toward the point of application if exogenous calcium ions are present (Gingell, 1970). The calcium ionophore, A23187, will also induce localized contractions in the same manner (Schroeder and Strickland, 1974). Total immersion of an egg in A23187 will induce a total cortical contraction (Schroeder and Strickland, 1974) that resembles the total contraction obtained in this study with ethanol, or by the direct injection of 1 gg of calcium into the cytoplasm (Hollinger and Schuetz, 1976). It is likely that the CBinsensitive cortical contractions, as demonstrated in this
MERRIAM,
FIG. 9. A schematic description.
interpretation
SAUTERER,
of the structure
AND
CHRISTENSEN
of the contractile
study at least, are triggered by calcium. The operation of a CB-sensitive contraction, the constriction of the contractile ring in cytokinesis, is also probably dependent on calcium (Baker and Warner, 1972). Our observations, coupled with those of many other workers, has suggested to us that a two-component contractile system exists near the surface of amphibian eggs. We present a model for such a concept in Fig. 9. The most superficial, “true cortex” is probably represented by the felt-like or hyaline layer seen just under the plasmalemma in thin sections. This element is about 0.1 pm thick and would correspond to the cortex of somatic cells. Through reversible actin filamentogenesis, it would be involved with the formation and operation of sperm engulfment, the contractile ring in cytokinesis, microvilli formation, endocytosis, etc. Its functioning would be sensitive to CB inhibition. The underlying dense pigment and cortical granulecontaining cytoplasm, whose ground material is apparently dense enough to exclude yolk, could possibly be a specialized structure for egg cell functions or perhaps be more universal in a less massive form. In the pigmented hemisphere of Xenopus eggs it is 3-V pm thick in the relaxed state. For want of a better term we will refer to it here as a subcortical matrix. Its functions might involve cortical granule exocytosis, establishment of the grey crescent, and the embryonic axes of symmetry. It might be involved in later embryonic events such as epibolic movements of ectoderm cells and cellular shape changes associated with gastrulation. It is probably the “tough surface coat” of Holtfreter (1943a). Pieces of it are probably what Curtis (1960) transplanted in his classical work on the embryonic organizer. Since its contractile mechanism is not sensitive to the cytochalasins, its molecular makeup and mechanism of action would presumably be different to some degree from that of the outer true cortex. Both may operate on the basis of calcium stimulation. Although this model seems to fit most of the available data, it should be obvious that other interpretations are possible and that much work needs to be done to establish the final mechanisms and structures involved.
Subcortical
surface
Contractile
in the pigmented
445
Structure
hemisphere
of a Xenqpus
egg. See text
for
We are grateful for the excellent technical assistance of Mrs. Joanne Kruse. It is a pleasure also to acknowledge the cooperation of Dr. Benjamin Walcott and his technician Cissy McKeon in doing the transmission electron microscopy work. This study was supported by Grant PCM 7824591 from the National Science Foundation. REFERENCES BAKER, P. F., and WARNER, A. E. (1972). Intracellular calcium and cell cleavage in early embryos of Xenqpus Levis. J. Cell BioL 53, 579-581. BLUEMINK, J. G. (1971). Cytokinesis and cytochalasin-induced furrow regression in the first cleavage zygote of Xenopus luevis Ztitich Zellfm-sch. 121, 102-126. BLUEMINK, J. G. (1972). Cortical wound healing in the amphibian egg: an electron microscopical study. .I Ultrastruct. Res. 41, 95-114. BRACHET, J. (1977). An old enigma: The grey crescent of amphibian eggs. Cm-. Top. Dev. BioL 11,133-186. BROWN, S. S., and SPUDICH, J. A. (1981). Mechanism of action of cytochalasin: Evidence that it binds to actin filament ends. J. Cell BioL 88, 487-491. CHRISTENSEN, K., and MERRIAM, R. W. (1981). Unpublished observations. CURTIS, A. S. G. (1960). Cortical grafting in Xenopus laevis. J. EmbryoL Exp. Mwph.oL 8, 163-173. ELINSON, R. P. (1975). Site of sperm entry and a cortical contraction associated with egg activation in the frog Rana pipiens. DOT. BioL 47, 257-268. ELINSON, R. P., and MANES, M. E. (1978). Morphology of the site of sperm entry on the frog egg. Dev. BioL 63, 67-75. FRANKE, 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. Cytobiolcgy 14, 111-130. GINGELL, D. (1970). Contractile responses at the surface of an amphibian egg. J. EmbryoL Exp. MwphoL 23, 583-609. GOULD-SOMERO, M., HOLLAND, L., and PAUL, M. (1977). Cytochalasin B inhibits sperm penetration into eggs of Urechis caupo (Echiura). Dev. BioL 58, 11-22. GREY, R. D., WOLF, D. P., and HEDRICK, J. L. (1974). Formation and structure of the fertilization envelope in Xenopus luevis. Dew. BioL 36, 44-61. GRUMET, M., FLANAGAN, M. D., LIN, D. C., and LIN, S. (1979). Inhibition of nuclei-induced actin polymerization by cytochalasins. J. Cell BioL 83, 316a. GURDON, J. B. (1973). “The Control of Gene Expression in Animal Development.” Oxford and Harvard Univ. Presses, London/New York/Cambridge, Mass. HARA, K., TYDEMAN, P., and KIRSCHNER, M. (1980). A cytoplasmic clock with the same period as the division cycle in Xen0pu.s eggs. Proc Nat. Acad. Sci. USA 77, 462-466.
446
DEVELOPMENTAL
BIOLOGY
HOLLINGER, T. G., and SCHKJETZ, A. W. (1976). “Cleavage” and cortical granule breakdown in Rana pipens oocytes induced by direct microinjection of calcium. J. Cell Bid 71, 395-401. HOLTFRETER, J. (1943a). Properties and functions of the surface coat in amphibian embryos. J. Exp. Zoo!. %3,251-323. HOLTFRETER, J. (1943b). A study of the mechanisms of gastrulation. J. Exp. Zd 94, 261-317. HYLANDER, B. L., ANSTROM, J., and SUMMERS, R. G. (1981). Premature sperm incorporation into the primary oocyte of the polychaet Pectinaria: Male pronuclear formation and oocyte maturation. Item. BioL
82,382-387.
KLAG, J. J., and UBBELS, G. A. (1975). Regional morphological and eytochemical differentiation in the fertilized egg of ~coglosaus p&us (anuran). ~~ent~a~~~ 3,15-20. KUDO, S. (1980). Sperm penetration and the formation of a fertilization cone in the common carp egg. Dew. Growth D$eren 22, 403414. LEVERAH, J., MERRIAM, R. W., and SAUTERER, R. (1980). The amphibian egg cortex as revealed by dry fracture and scanning electron microscopy. J. CeU Bio2l 87, 90a. LILLIE, R. R. (1906). Observations and experiments concerning the elementary phenomena of development in Chaetopterus J. Exp Zool 3,153-269.
LONGO, F. J. (1973). Fertilization: a comprehensive ultrastructural review. Biol. Reproduct. 9, 149-215. LONGO, F. J. (1978). Effects of cytochalasin B on sperm-egg interactions. L&I. Biol. 67, 249-265. LONGO, F. J. (1980). Organization of microfilaments in sea urchin (A+ bacia punctulata) eggs at fertilization: Effects of cytochalasin B. Dev. Bio2l 74, 422-433. LUCHTEL, D., BLUEMINK, J. G., and DE LAAT, S. W. (1976). The effect of injected cytochalasin B on filament organization in the cleaving egg of Xeaopus laevis. J. L%rastruct. Res. 54, 406-419. LUCKENBILL, L. M. (1971). Dense material associated with wound closure in the axolotl egg. Exp. Cell Res. 66, 263-267. MANES, M. E., ELINSON, R. P., and BARBIERI, F. D. (1978). Formation of the amphibian grey crescent: Effects of colehicine and cytochalasin B. Wilhelm Rot&a Arch. 18%,99-104. MERRIAM, R. W., NOWINSKK, M., and HAAS, K. (1978). The demonstration of localized contractile programs in living oocytes and eggs of Xenopus lo&s. A paper presented at “The Cytoskeleton and Con-
VOLUME
95, 1983
tractile Networks of Nonmuscle Cells,” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. May 1’7-21, 1978. PALACEK, J., UBBELS, G. A., and RZEHAK, K. (1978). Changes of the external and internal pigment pattern upon fertilization in the egg of Xenopus la&u&. J. Em&r& Ezp. ~~~1 45, 203-214. PICKERAL, B. (1977). La fecondation chez le triton Pleurodele II. La penetration des spermatozoides et al reaction locale de l’oeuf. J. iXtrastmLct. Res. 60, 181-202. 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.
PERRY, M. M., and WADDINGTON, C. H. (1966). Ultrastructure of the blastopore cells in the newt. f. Embryol. Exp. Morphol. 15,317-330. SCHATTEN, H., and SCHA~EN, G. (1980). Surface activity at the egg plasma membrane during sperm incorporation and its cytochalasin I3 sensitivity. Dev. Biol 78, 435-449. SCHMIDT, B. A., KELLY, P. T., MAY, M. C., DAVIS, S. E., and CONRAD, G. W. (1980). Characterization of actin from fertilized eggs of IZlyanassa obsolete during polar body formation and cytokinesis. Deu. Biol 76, 126-140. SCHORDERET-SLATKINE, S., and DRURY, K. C. (19’73). Progesterone-induced maturation in oocytes of Xentrpus lo&s. Appearance of a maturation-promoting factor in enucleated oocytes. Cell L?$eren. 2,247-254.
SCHROEDER, T. E. (1973). Actin in dividing cells: Contractile ring filaments bind heavy meromyosin. Proc. Nat. Acad Sci. USA 70,16881692. SCHROEDER, T. W., and STRICKLAND, D. L. (1974). Ionophore A23287, calcium and contractility in frog eggs. Exp. Cell Res. 83, 139-142. SELMAN, G. G., and PERRY, M. (1970). Ultrastructure changes in the surface layers of the newt’s egg in relation to the mechanism of its cleavage. J. Cell sei 6, 207-227. SHALGI, R., and PHILLIPS, D. (1980). Mechanics of sperm entry in cycling hamsters. J. UZtra&wct. Res. 71, 154-161. SINGAL, P. K., and SANDERS, E. J. (1974). An ultrastructural study of the first cleavage of Xenopus embryos. J. Ultra&m&. Res. 47,433451.
TILNEY, L. G., and JAFFE, L. A. (1980). Actin, microvilli and the fertilization cone of sea urchin eggs. J. CeU Biol. 87, 771-782. VACBUIER, V. D. (1981). Dynamic changes of the egg cortex. Dev. BioL 84, l-26.
BIOLOGY
95,
4:?9-446
(1983)
A Subcortical, Pigment-Containing .Xenopus Eggs with Contractile R. W. MERRIAM, Department
of NewobioLogy Received
R. A. SAUTERER,
and Behavior, June
State
llniversity
19, 1981; accepted
Structure in Properties
AND K. CHRISTENSEN of New York, Stay
in revised
fm
October
Brook,
NW
York
1179.4
7, 1982
An accumulation of insoluble, finely granular material has been observed under the pigmented surface of Xenopus eggs by a specialized “dry fracture” technique and scanning electron microscopy. Cortical granules and pigment granules can be recognized with the techniques and can be seen to be embedded in the material. Thin sections show that the region also contains mitoehondria and membranous vesicles or reticula. Yolk platelets are largely excluded from the heaviest accumulations of the material. The substance is most dense just under the cortex and grades off gradually into the more diffuse, yolk-containing network of the endoplasm. The accumulation of material is much thicker in the animal hemisphere of the egg than in the vegetal hemisphere, and the pigment embedded in it defines the pigmented area of the animal hemisphere. In the pigmented area the material excludes yolk for a thickness of 3-7+ pm from the surface. In the vegetal hemisphere there is no such accumulation and yolk platelets can be found almost touching the plasmalemma. Cortical contractions have been experimentally induced in eggs. Their relative strength correlates with the relative thickness of the finely granular, subcortical material. During contraction the material accumulates to much greater thicknesses, excluding yolk from thicknesses of 15-30+ pm from the surface. The contracting entity is, or is in, the finely granular material. Injection of cytochalasins into the eggs inhibits cleavage furrow operation but does not inhibit the induced cortical contractions. The induced contractions thus do not seem to be dependent on actin microfilamentogenesis as is the operation of the contractile ring of the cleavage furrow. The differential sensitivity to cytochalasins of the contractile ring and the system responding in the induced cortical contractions, suggests a two-component system for cortical contractions in the egg. A model is presented which accomodates the available data.
accompanied by an accumulation of pigment as the surface coat condensed in that region (see also Perry and In two notable papers, Holtfreter (1943a,b) described Waddington, 1966). In these reports Holtfreter strongly a “surface coat” on the surface of the early embryos of implicated the pigmented ‘*surface coat” with contracseveral species of amphibians. This surface coat was tions and movements at the surface of the egg and early marked by its content of pigment. Not only could the embryo. presence of the coat be detected by the presence of pigSince those observations were reported, a number of studies have been made of the surface cytoplasm of ment, but there was seen to be a direct relationship between the thickness of the coat and the amount of amphibian eggs and embryos by thin sectioning and observation with the electron microscope (e.g., Franke pigment. et al., 1976; Grey et al., 1974; Selman and Perry, 1970; In a series of experiments Holtfreter showed that this coat was intimately associated with the movement of Singal and Sanders, 1974). These investigations have established that the “surface coat” of Holtfreter is not pigmented ectodermal cells over the surface of the extracellular. Rather, a dense felt-like layer about 0.1 cleaving embryo. In fact, the spreading of the pigpm thick, just under the plasmalemma, overlays a mented coat of the unfertilized, uncleaved egg occurred thicker layer which contains the pigment. This latter in overaged eggs of Rana Ipilriens. This showed that the epibolic movement of thle pigmented coat was a phe- layer is reported to be 3-5 pm thick in the animal hemisphere of amphibian eggs. It contains the cortical grannomenon intrinsic to the egg coat itself and was not ules, membranous vesicles, and mitochondria. Yolk tends dependent on cellular movement per se in the normally cleaving embryo. The same observation had been made to be excluded by the matrix of this peripheral subon overaged eggs of C~ae~:o~~e~ (Liltie, 1906) and eggs cortical cytoplasm. Curtis (1960) found that pieces of the pigmented corof other species as well (ffor reviews see Brachet, 197’7; Holtfreter, 1943b). tex of Xenopus eggs could be removed with needles. The Holtfreter also showed that when an ectoderm cell excised cortex was tough and described as being about changes shape in wound healing or at the blastopore of 0.5-3.0 pm thick. It was sufficiently coherent to allow a gastrula, a constriction of one end of the cell was transplantation and still retain biological activity. When INTRODUCTION
439 0012-1606/83/020439-08$03.00/O Copyright All tights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
440
DEVELOPMENTAL
BIOLOGY
fixed and sectioned for electron microscopy, the isolated cortex could be seen to include embedded pigment, mitochondria, and other granules underneath a thin hyaline layer associated with the plasmalemma. The cortex from the unpigmented vegetal hemisphere was too fragile to allow such experimental handling. Puncture wounds made in the pigmented hemisphere of amphibian eggs heal by active closure in the presence of calcium ion (Bluemink, 1972; Gingell, 1970; Luckenbill, 1971; Schroeder and Strickland, 1974). This process also seems to involve the cortical layers under the plasmalemma. The superficial felt-like layer becomes thicker around the wound and extends from the surface in blebs or outfoldings as the wound is constricted. A ring of filamentous material develops in or under the felt-like layer around the hole. A zone of vesicles underlies the felt-like layer and under that a massive accumulation of pigment and yolk occurs around the edge of the wound. The thickening of the felt-like layer and the accumulation of pigment have led to suggestions that these layers contract during the hole closure process. Bluemink (1972) noted a functional distinction between two contractile phenomena associated with the amphibian egg cortex. Vinblastine at loo-250 pg/ml inhibited cleavage furrow formation, but had no effect on wound healing. In this paper we induce contractions at the surface of Xenows eggs and utilize a dry-fracture technique for observing the fine structure of the cortical regions with the scanning electron microscope. The dense, subcortical cytoplasmic material, in which pigment is embedded, is shown to thicken and become more dense during contraction. We distinguish the operation of the contractile ring of the cleavage furrow from that of the induced surface contractions by showing that cytochalasins inhibit the furrow but not the induced surface contractions. This distinction leads us to propose a model in which two different contractile systems are envisioned near the surface of the egg. METHODS
AND
MATERIALS
Oocytes were obtained from the ovaries of adult female Xenopus laevis frogs. The excised ovaries were vigorously swirled for 20 min at 30°C in a solution containing 2 mg/ml of collagenase, Type II (Sigma Chemical Co., St. Louis, MO.) and 0.1 M phosphate buffer, pH 7.5, after the method of Schorderet-Slatkine and Drury (1973). The largest oocytes, thus freed of mesothelial and connective tissue components, were then washed in Ringer’s solution. Egg laying was induced by injection of human chorionic gonadotropin. Freshly stripped eggs were dejellied by treatment with a solution of 40 mM /3-mercaptoethanol, pH 8.9, followed by washings in 0.1 strength
VOLUME
95, 1983
Ringer’s solution. The vitelline membrane was then removed manually with forceps. Cells to be tested were transferred by pipet into the test solution. Shape changes were recorded photographically through a Leitz macro lens. Microinjections were done essentially by the methods of Gurdon (1973). Eighty nanoliters was injected into each cell from a calibrated micropipet. This represented about 8% of total cell volume. The injection carrier solution contained 88 mM NaCl; 1 mM KCl; 15 mM TrisHCl, pH 7.2. Cytochalasins B (Aldrich Chemical Co., Milwaukee, Wise.) or D (Sigma) were dissolved in dimethyl sulfoxide (DMSO) at 10 mg/ml and diluted with carrier solution to give about 10 pg/ml of cell water, assuming that half of the cell volume was water. Control injections always were made with the same concentration of DMSO in injection carrier. Oocyte or egg internal structures were observed by scanning electron microscopy (SEM) after “dry fracturing” the cells (Leverah et al., 1980). The cells were either fixed in 3% glutaraldehyde, buffered to pH 7.4 by 0.1 Mphosphate buffer, for 3 hr at 5”C, or were quickfrozen in liquid propane at liquid nitrogen temperatures and the ice was substituted by acetone at -40°C. In both cases, when water had been replaced by acetone, the cells were split open with fine needles in acetone. After critical-point drying, the fracture faces were coated with gold-palladium (60/40) for observation in a JOEL JSM035C scanning electron microscope (SEM). For thin sections, eggs were fixed 3 hr in 3% glutaraldehyde and buffered to neutral pH with 0.1 M phosphate buffer at 5°C. After washing, eggs were postfixed in 1% osmium tetroxide and embedded in Polybed. Sections were stained with lead citrate and uranyl acetate before observation in a JOEL 1OOB electron microscope. RESULTS
In previous work we had found that Xenopus eggs could be induced to undergo shape changes that involved contractions at or near the surface (Merriam et al., 1978). When placed into a buffered medium containing 0.9 M sucrose and 4% ethanol, the pigmented surface would isometrically contract in surface area, become cone-shaped, and finally contract into a small wrinkled nipple on top of a distended unpigmented hemisphere (Fig. 1). Ethanol by itself would induce contraction of the pigmented hemisphere into a wrinkled nipple without the intervening cone shape. Sucrose by itself produced no observable response. When a hole was made in the responding egg, the yolky endoplasm was squeezed out, relieving the internal pressure. In this case both animal and vegetal hemispheres contracted simultaneously (Fig. 2). These observations were inter-
MERRIAM,
SAUTERER,
AND
CHRISTENSEN
Subwrtical
Cmtractile
Structure
FIG. 1. Unfertilized Xenopus eggs with the pigmented cortex contracted into a wrinkled nipple by exposure to 0.9 Msucrose and 4% ethanol. The yolky endoplasm has been squeezed out of the animal hemisphere into a distended vegetal hemisphere. X25. FIG. 2. Unfertilized Xenopus eggs (arrows) induced to contract by exposure to 0.9 M sucrose and 4% ethanol. Holes have been made in the eggs so that the yolky endoplasm (Y) is being squeezed out. Note the isometric decrease in surface area of both pigmented and unpigmented hemispheres. X25.
preted to mean that both pigmented and unpigmented surfaces could respond by contracting, but that the contractions of the pigmented surface were much stronger. We were interested in visualizing those subsurface structures which produced strong contractions in the pigmented hemisphere and weak contraptions in the unpigmented hemisphere. Thin sectioning and observations with transmitted electrons produced an image which has been observed many times (Fig. 3). A thin felt-like layer can be seen under the plasmalemma which is devoid of large organellles and largely devoid of granular materials. It tills projections extending from the surface. Sometimes microfilaments can be seen in microvilli, embedded in the feelt-like matrix. Below the feltlike layer is a much thicker layer which is characterized primarily by the absence of yolk but does include cortical granules, pigment granules, some mitoehondria, and many membrane-limited vesicles or reticula. When the peripheral cytoplasm of the pigmented hemisphere is dry-fractured and the fracture face is observed by scanning electron microscopy, a vague distinction can again be made between a subcortical layer of cytoplasm and the deeper endoplasm. Such an image of an egg in the relaxed condition is seen in Fig. 4. The distinction in this type of image rests on two things: the outer 3-7 pm of cytoplasm is largely devoid of yolk and the background cytoplasmic material is more dense. This dense accumulation of material may account for the exclusion of large yolk platelets from the region. At
higher magnifications the accumulated materials can be seen in the dry-fractured state to be finely granular in texture (Fig. 8). The extreme cortical layer, between the cortical granules and the plasmalemma (arrowhead in Fig. 8). does not show this granular texture and probably corresponds to the felt-like layer seen in Fig. 3. Abundant pigment granules can be seen to be embedded in the granular subcortical cytoplasm (Figs. 3 and 8). The dense, pigment-containing, subcortieal material seen in the animal hemisphere is absent in the unpigmented vegetal hemisphere (Fig. 5). Yolk platelets and other organelles can be found in close approximation to the plasmalemma. The dense, granular cytoplasmic material which seems to exclude yolk under the pigmented surface is largely gone. Instead, one sees a more lacy, vesiculated reticulum of material in which all organelles are suspended (Figs. 5 and 7). The transition between the pigment-containing cytoplasmic material of the animal hemisphere and the more delicate cytoarchitecture of the vegetal hemisphere surface is sharp. It is clear that the presence of surface pigment is defined by the extent of the dense subcortical material in which it is embedded. The cortical structures of the pigmented surface are strong enough to isolate in buffered saline solutions. The egg is cut in half and a stream of buffer blown across the cut surface. The endoplasm washes away leaving an insoluble cortex. Electron microscopy of the isolate shows embedded pigment and cortical granules
442
DEVELOPMENTAL BIOLOGY
VOLUME 95,1983
MERRIAM,
SAUTERER,
AND
CHRISTENSEN
in a cohesive matrix, plus adherent yolk on the cytoplasmic surface (unpublished observations). The structure is 5-15 pm thick and will keep its coherency indefinitely. This shows that the cytoplasmic matrix of the cortical region is an insoluble structure in which organelles are embedded. The unpigmented cortex is too fragile to be isolated in this manner. Contractions induced in the egg surface were much stronger in the pigmented surface than in the unpigmented surface. The contracting entity also contained the surface pigment because it was carried into the wrinkled nipple during the contraction (Fig. 1). We considered the possibility that the dense, pigment-containing subcortical material was the contractile element that responded to our induction. If this were so, we might expect it to become thickened or more dense during contraction because the surface area of the pigmented hemisphere became much reduced during contraction. To test this possibility we induced contraction and fixed the eggs in the contracted state. The resultant pigmented nipples were very tough and hard to fracture in the dried state, but such fractures were obtained. As can be seen in Fig. 6, the dense, pigment-containing subcortical material of the cytoplasm has become much thicker. In highly contracted cases, the entire yolky endoplasm has been squeezed out into the vegetal hemisphere and the entire nipple is filled with the dense material. We conclude that the matrix contains, or is, the responding contractile element. Actin has been shown to be a prominent component of the cortical cytoplasm of Xenqimcs eggs and oocytes (Franke et al., 1976). We wondered if the contracting cytoplasmic matrix might be operating through the involvement of actin microfilaments. If this were the case, we might expect the system to be sensitive to the cy-
SuhwrticaZ
Ccmtractile
TABLE THE EFFECT OF CYTOCHALASIN CO~RA~ION
Frog
Injected
1
None DMSO CB
lO/lO 1307
None
12112
2
Contraction
417"
DMSO
7/10
CB
802”
443
Structure 1 D ON INDUCED IN EGGS
Cleavage furrow
Percentage contract
CORTICAL
Percentage cleaved
23/26 20/24 3/w
100
76 57
88 83 25
13/17 7112 O/12"
100 70 67
76 58 0
“Many cells went bad after injection ment distribution looked good enough
of CB. Only were scored.
cells whose
pig-
tochalasins. To test this possibility we injected cytochalasin D into the animal hemisphere of fertilized eggs. The amount injected was sufficient to bring the intracellular concentration to at least 10 pg/ml of cell water. The injected eggs were placed for 15 min into an appropriate medium and then divided into two groups. One group was allowed to develop in 0.1 Amphibian Ringer’s solution so that the initiation of a normal cleavage furrow could be observed. The other group was challenged with 4% ethanol to see if a contraction of the pigment of the animal hemisphere could be observed. The results with eggs of two frogs can be seen in Table 1. In the eggs of frog 2 which had been injected with CB, it was observed that the accumulation of pigment at the animal pole and even the movement of pigment into the region of the future cleavage furrow occurred normally. Yet in no case did a cleavage furrow form. We conclude from this and from the data of Table 1 that an internal concentration of CB, sufficient to block
FIG. 3. A transmission elect.ron micrograph of a thin section of the pigmented, animal hemisphere cortical region of a relaxed Xenopus egg. A cortical granule (cg) and pigment granule (p) may be seen embedded in a matrix which shows granular material and an extensive membranous reticulum. Note the finely textured matrix just under the plasmalemma (arrow) which is concentrated within the surface projections. X36,830. FIG. 4. A scanning electron micrograph of the fracture face of a dry-fractured Xenqms egg. The egg was fixed in the relaxed state. The location of the plasmalemma of the animal, pigmented surface is marked by an arrow. Note the accumulation of dense matrix 3-7 pm thick beneath the plasmalemma and the less dense, yolky endoplasm under it. X2450. FIG. 5. A scanning electron micrograph of the fracture face of a dry-fractured Xenopus egg. The location of the plasmalemma of the vegetal, unpigmented surface is marked by an arrow. Note the lack of any appreciable accumulation of dense matrix under the plasmalemma, as seen in Fig. 4. Yolk (Y) can be found right up to the surface in a more delicate supporting meshwork. X2450. FIG. 6. A scanning electron micrograph of the fracture face of a dry-fractured Xenopus egg. The location of the plasmalemma of the animal, pigmented surface is marked by an arrow. This egg was induced to contract with 4% ethanol. The micrograph shows the deep accumulation of dense matrix, 22 pm thick, beneath the plasmalemma. This matrix should be compared with that of the egg in the relaxed state of Fig.
4. X2450. FIG. 7. A scanning electron micrograph of the fracture face of a dry-fractured Xenopus egg. The unpigmented surface is marked by an arrow. At this higher magnification the delicate supporting be seen to be vaguely granular in texture. X13,720. FIG. 8. A scanning electron micrograph of the fracture face of a dry-fractured Xenopus egg. The pigmented surface is marked by an arrow. Microvilli may be seen projecting from the surface pigment granules (p) can be recognized. The texture of the dense matrix immediately under the head) while the deeper matrix has a vaguely granular appearance. The smallest granules seen granules seen in the thin section of Fig. 3. X13,720.
location of the plasmalemma of the vegetai, meshwork under the plasmalemma can location of the plasmalemma of the animal, of the egg and cortical granules (cg) and plasmalemma often appears hyaline (arrow by this technique are larger than the dark
444
DEVELOPMENTAL
BIOLOGY
even the initiation of the cleavage furrow, still allows an induced contraction of the pigmented cortex and normal precleavage movements of pigment. DISCUSSION
The developmental importance of contractile systems near the egg surface has been repeatedly emphasized in numerous publications. Holtfreter (1943a,b), showed that epibolic movements of the animal hemisphere were accompanied by movements of superficial pigment. Superficial pigment concentrations were also associated with cellular shape changes in wound healing and gastrulation (see also Perry and Waddington, 1966). In animal eggs, the cortex has been shown to be actively engaged in the engulfment of the fertilizing sperm by the formation or extension of microvilli around the sperm head. The microvilli contain actin microfilamentous cores and they subsequently fuse to form a fertilization cone (Longo, 1980; Picheral, 1911; Shalgi and Phillips, 1980; Tilney and Jaffe, 1980; Vacquier, 1981). Morphologically, the fertilization cone consists of the superficial, felt-like layer of the cortex (e.g., Hylander et al., 1981; Kudo, 1980). The cone and the enclosed sperm nucleus are withdrawn into the deeper cortical cytoplasm. ~ytochalasin B prevents fertilization cone formation and sperm entry in Marine animal eggs (GouldSomero et al., 1977; Longo, 1978; Schatten and Schatten, 1980). Since the fertilization cone involves the formation of microfilamentous actin arrays, it is not surprising that CB prevents its formation and the entry of the sperm (e.g., Brown and Spudich, 1981; Grumet et al., 1979). It is noteworthy, however, that a roughly concomitant cortical function, cortical granule exocytosis, is not inhibited by CB (Gould-Somero et al., 1977; Longo, 1978; Manes et al., 1978). The cortical granules lie beneath the felt-like outer cortex and in amphibian eggs are embedded in the dense, pigment-containing material. Their exoeytosis is an active process, involving both the movement to the plasmalemma and the extrusion of their contents in a short period of time (Grey et al., 1974). The lack of sensitivity to CB by this process suggests that exocytosis does not involve aetin microfilamentogenesis. Additional types of cortical contractions occur at the surface of amphibian eggs between fertilization and first cleavage. The pigmented surface contracts isometrically in area within 5-6 min after sperm entry (Elinson, 1975). This event could still occur after egg activation in the presence of CB (Manes et al., 1978). In fertilized Discoglossus eggs (Klag and Ubbels, 1975) and Xenopus eggs another isometric contraction of the pigmented surface occurs at about 70 min postfertilization. In Xen-
VOLUME
95, 1983
opus eggs this contraction relaxes as the first cleavage furrow forms and is completely insensitive to CB, even when injected into the egg (Christensen and Merriam, 1981). Still other types of cortical contractions have been recorded in fertilized animal eggs (Hara et al., 1980; for a review see Vacquier (1981)). Cortical contractions, more sharply localized in the superficial cortex as contractile rings, occur during cleavage (e.g., Perry et al., 1971; Schroeder, 1973), in meiosis (Longo, 1972), and in the polar lobe formation of certain molluscan eggs (Schmidt et al., 1980). All of these events can be shown to be inhibitable by CB. A hole, made in the surface of amphibian eggs, will heal in the presence of calcium ions. This process is accompanied by the appearance of a ring of a&in-like microfilaments in the superficial cortical region around the wound (Bluemink, 1972). The hole apparently closes by a circumferential constriction of the cortex. Superficial pigment is moved toward the hole during closure (Bluemink, 1972; Gingell, 1970; Luckenbill, 1971). The inclusion of CB in the medium during healing completely blocks wound closure in Xenom eggs (Christensen and Merriam, 1981). A final type of cortical movement in amphibian eggs may be mentioned. A grey crescent is a cortical region which forms opposite to the point of sperm entry in normal fertilization (for a review see Brachet (1977)). It is marked by the movement of pigmented cortex away from the area, and is associated with the establishment of the future embryonic axes. It is still not clear whether the grey crescent is formed by a rotation of the pigmented cortex over the underlying yolky endoplasm (Elinson and Manes, 1978; Klag and Ubbels, 1975) or by cortical contractions initiated at the site of sperm entry (Palacek et al., 1978). What is clear, however, is that again cortical movements are involved and that a grey crescent-like region can form in the presence of CB in the cytoplasm {Manes et al., 1978). Other workers have demonstrated that the amphibian egg cortex can be experimentally induced to contract. Most of these inductions have implicated calcium ion as the trigger. For example, the localized application of polyvalent ions to the surface of frog eggs will induce movement of surface pigment toward the point of application if exogenous calcium ions are present (Gingell, 1970). The calcium ionophore, A23187, will also induce localized contractions in the same manner (Schroeder and Strickland, 1974). Total immersion of an egg in A23187 will induce a total cortical contraction (Schroeder and Strickland, 1974) that resembles the total contraction obtained in this study with ethanol, or by the direct injection of 1 gg of calcium into the cytoplasm (Hollinger and Schuetz, 1976). It is likely that the CBinsensitive cortical contractions, as demonstrated in this
MERRIAM,
FIG. 9. A schematic description.
interpretation
SAUTERER,
of the structure
AND
CHRISTENSEN
of the contractile
study at least, are triggered by calcium. The operation of a CB-sensitive contraction, the constriction of the contractile ring in cytokinesis, is also probably dependent on calcium (Baker and Warner, 1972). Our observations, coupled with those of many other workers, has suggested to us that a two-component contractile system exists near the surface of amphibian eggs. We present a model for such a concept in Fig. 9. The most superficial, “true cortex” is probably represented by the felt-like or hyaline layer seen just under the plasmalemma in thin sections. This element is about 0.1 pm thick and would correspond to the cortex of somatic cells. Through reversible actin filamentogenesis, it would be involved with the formation and operation of sperm engulfment, the contractile ring in cytokinesis, microvilli formation, endocytosis, etc. Its functioning would be sensitive to CB inhibition. The underlying dense pigment and cortical granulecontaining cytoplasm, whose ground material is apparently dense enough to exclude yolk, could possibly be a specialized structure for egg cell functions or perhaps be more universal in a less massive form. In the pigmented hemisphere of Xenopus eggs it is 3-V pm thick in the relaxed state. For want of a better term we will refer to it here as a subcortical matrix. Its functions might involve cortical granule exocytosis, establishment of the grey crescent, and the embryonic axes of symmetry. It might be involved in later embryonic events such as epibolic movements of ectoderm cells and cellular shape changes associated with gastrulation. It is probably the “tough surface coat” of Holtfreter (1943a). Pieces of it are probably what Curtis (1960) transplanted in his classical work on the embryonic organizer. Since its contractile mechanism is not sensitive to the cytochalasins, its molecular makeup and mechanism of action would presumably be different to some degree from that of the outer true cortex. Both may operate on the basis of calcium stimulation. Although this model seems to fit most of the available data, it should be obvious that other interpretations are possible and that much work needs to be done to establish the final mechanisms and structures involved.
Subcortical
surface
Contractile
in the pigmented
445
Structure
hemisphere
of a Xenqpus
egg. See text
for
We are grateful for the excellent technical assistance of Mrs. Joanne Kruse. It is a pleasure also to acknowledge the cooperation of Dr. Benjamin Walcott and his technician Cissy McKeon in doing the transmission electron microscopy work. This study was supported by Grant PCM 7824591 from the National Science Foundation. REFERENCES BAKER, P. F., and WARNER, A. E. (1972). Intracellular calcium and cell cleavage in early embryos of Xenqpus Levis. J. Cell BioL 53, 579-581. BLUEMINK, J. G. (1971). Cytokinesis and cytochalasin-induced furrow regression in the first cleavage zygote of Xenopus luevis Ztitich Zellfm-sch. 121, 102-126. BLUEMINK, J. G. (1972). Cortical wound healing in the amphibian egg: an electron microscopical study. .I Ultrastruct. Res. 41, 95-114. BRACHET, J. (1977). An old enigma: The grey crescent of amphibian eggs. Cm-. Top. Dev. BioL 11,133-186. BROWN, S. S., and SPUDICH, J. A. (1981). Mechanism of action of cytochalasin: Evidence that it binds to actin filament ends. J. Cell BioL 88, 487-491. CHRISTENSEN, K., and MERRIAM, R. W. (1981). Unpublished observations. CURTIS, A. S. G. (1960). Cortical grafting in Xenopus laevis. J. EmbryoL Exp. Mwph.oL 8, 163-173. ELINSON, R. P. (1975). Site of sperm entry and a cortical contraction associated with egg activation in the frog Rana pipiens. DOT. BioL 47, 257-268. ELINSON, R. P., and MANES, M. E. (1978). Morphology of the site of sperm entry on the frog egg. Dev. BioL 63, 67-75. FRANKE, 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. Cytobiolcgy 14, 111-130. GINGELL, D. (1970). Contractile responses at the surface of an amphibian egg. J. EmbryoL Exp. MwphoL 23, 583-609. GOULD-SOMERO, M., HOLLAND, L., and PAUL, M. (1977). Cytochalasin B inhibits sperm penetration into eggs of Urechis caupo (Echiura). Dev. BioL 58, 11-22. GREY, R. D., WOLF, D. P., and HEDRICK, J. L. (1974). Formation and structure of the fertilization envelope in Xenopus luevis. Dew. BioL 36, 44-61. GRUMET, M., FLANAGAN, M. D., LIN, D. C., and LIN, S. (1979). Inhibition of nuclei-induced actin polymerization by cytochalasins. J. Cell BioL 83, 316a. GURDON, J. B. (1973). “The Control of Gene Expression in Animal Development.” Oxford and Harvard Univ. Presses, London/New York/Cambridge, Mass. HARA, K., TYDEMAN, P., and KIRSCHNER, M. (1980). A cytoplasmic clock with the same period as the division cycle in Xen0pu.s eggs. Proc Nat. Acad. Sci. USA 77, 462-466.
446
DEVELOPMENTAL
BIOLOGY
HOLLINGER, T. G., and SCHKJETZ, A. W. (1976). “Cleavage” and cortical granule breakdown in Rana pipens oocytes induced by direct microinjection of calcium. J. Cell Bid 71, 395-401. HOLTFRETER, J. (1943a). Properties and functions of the surface coat in amphibian embryos. J. Exp. Zoo!. %3,251-323. HOLTFRETER, J. (1943b). A study of the mechanisms of gastrulation. J. Exp. Zd 94, 261-317. HYLANDER, B. L., ANSTROM, J., and SUMMERS, R. G. (1981). Premature sperm incorporation into the primary oocyte of the polychaet Pectinaria: Male pronuclear formation and oocyte maturation. Item. BioL
82,382-387.
KLAG, J. J., and UBBELS, G. A. (1975). Regional morphological and eytochemical differentiation in the fertilized egg of ~coglosaus p&us (anuran). ~~ent~a~~~ 3,15-20. KUDO, S. (1980). Sperm penetration and the formation of a fertilization cone in the common carp egg. Dew. Growth D$eren 22, 403414. LEVERAH, J., MERRIAM, R. W., and SAUTERER, R. (1980). The amphibian egg cortex as revealed by dry fracture and scanning electron microscopy. J. CeU Bio2l 87, 90a. LILLIE, R. R. (1906). Observations and experiments concerning the elementary phenomena of development in Chaetopterus J. Exp Zool 3,153-269.
LONGO, F. J. (1973). Fertilization: a comprehensive ultrastructural review. Biol. Reproduct. 9, 149-215. LONGO, F. J. (1978). Effects of cytochalasin B on sperm-egg interactions. L&I. Biol. 67, 249-265. LONGO, F. J. (1980). Organization of microfilaments in sea urchin (A+ bacia punctulata) eggs at fertilization: Effects of cytochalasin B. Dev. Bio2l 74, 422-433. LUCHTEL, D., BLUEMINK, J. G., and DE LAAT, S. W. (1976). The effect of injected cytochalasin B on filament organization in the cleaving egg of Xeaopus laevis. J. L%rastruct. Res. 54, 406-419. LUCKENBILL, L. M. (1971). Dense material associated with wound closure in the axolotl egg. Exp. Cell Res. 66, 263-267. MANES, M. E., ELINSON, R. P., and BARBIERI, F. D. (1978). Formation of the amphibian grey crescent: Effects of colehicine and cytochalasin B. Wilhelm Rot&a Arch. 18%,99-104. MERRIAM, R. W., NOWINSKK, M., and HAAS, K. (1978). The demonstration of localized contractile programs in living oocytes and eggs of Xenopus lo&s. A paper presented at “The Cytoskeleton and Con-
VOLUME
95, 1983
tractile Networks of Nonmuscle Cells,” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. May 1’7-21, 1978. PALACEK, J., UBBELS, G. A., and RZEHAK, K. (1978). Changes of the external and internal pigment pattern upon fertilization in the egg of Xenopus la&u&. J. Em&r& Ezp. ~~~1 45, 203-214. PICKERAL, B. (1977). La fecondation chez le triton Pleurodele II. La penetration des spermatozoides et al reaction locale de l’oeuf. J. iXtrastmLct. Res. 60, 181-202. 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.
PERRY, M. M., and WADDINGTON, C. H. (1966). Ultrastructure of the blastopore cells in the newt. f. Embryol. Exp. Morphol. 15,317-330. SCHATTEN, H., and SCHA~EN, G. (1980). Surface activity at the egg plasma membrane during sperm incorporation and its cytochalasin I3 sensitivity. Dev. Biol 78, 435-449. SCHMIDT, B. A., KELLY, P. T., MAY, M. C., DAVIS, S. E., and CONRAD, G. W. (1980). Characterization of actin from fertilized eggs of IZlyanassa obsolete during polar body formation and cytokinesis. Deu. Biol 76, 126-140. SCHORDERET-SLATKINE, S., and DRURY, K. C. (19’73). Progesterone-induced maturation in oocytes of Xentrpus lo&s. Appearance of a maturation-promoting factor in enucleated oocytes. Cell L?$eren. 2,247-254.
SCHROEDER, T. E. (1973). Actin in dividing cells: Contractile ring filaments bind heavy meromyosin. Proc. Nat. Acad Sci. USA 70,16881692. SCHROEDER, T. W., and STRICKLAND, D. L. (1974). Ionophore A23287, calcium and contractility in frog eggs. Exp. Cell Res. 83, 139-142. SELMAN, G. G., and PERRY, M. (1970). Ultrastructure changes in the surface layers of the newt’s egg in relation to the mechanism of its cleavage. J. Cell sei 6, 207-227. SHALGI, R., and PHILLIPS, D. (1980). Mechanics of sperm entry in cycling hamsters. J. UZtra&wct. Res. 71, 154-161. SINGAL, P. K., and SANDERS, E. J. (1974). An ultrastructural study of the first cleavage of Xenopus embryos. J. Ultra&m&. Res. 47,433451.
TILNEY, L. G., and JAFFE, L. A. (1980). Actin, microvilli and the fertilization cone of sea urchin eggs. J. CeU Biol. 87, 771-782. VACBUIER, V. D. (1981). Dynamic changes of the egg cortex. Dev. BioL 84, l-26.