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Electron microscope studies on eggs of Mytilus edulis
J. U L T R A S T R U C T U R E R E S E A R C H
7, 467-487 (1962)
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Electron Microscope Studies on Eggs of Mytilus edulis 1 WALTER J. HUMPHREYS
Department of Zoology, University of California, Berkeley, California Received May 10, 1962 Normal eggs of the bay mussel, Mytilus edulis, centrifuged eggs, eggs treated with sperm extract, plasmolyzed eggs, and homogenates of whole eggs, were surveyed by electron microscopy. Microvilli increase the egg surface area about fifteenfold. They extend through the vitelline membrane, and delicate fibrils extend from their tips into a jelly coat. Hypertonic sea water detaches the microvilli from the egg and they are retained in the vitelline membrane. Sperm extract causes the vitelline membrane, an intercellular substance, and cortical granules of the cortex to become invisible. The endoplasm of the centrifuged egg stratified into (1) a lipid layer, (2) a clear layer, (3) a mitochondrial layer, and (4) a yolk layer. The constituents of these layers and of the cortex are described. Many of these constituents are recognizable in thin sections of pellets obtained by centrifugation of egg homogenates. The findings are discussed. Eggs of Mytilus edulis have been shown to have an early determination and segregation of morphogenic factors (25) some of which, as in the egg of Dentalium (36), are transferred to the CD cell by the end of the first cleavage. Quantitative comparisons of AB and CD blastomeres and isolated polar lobes have been made with regard to oxygen consumption (7), phosphate uptake (8), content of peptidase (5), and the uptake rate of amino acids (1). Recently, cytochemical studies have also been employed to compare these cytoplasms (24). The present electron microscope survey of the constituents of the Mytilus egg was intended to serve as a basis for subsequent studies on the distribution of constituents in early cleavage stages, which could then be compared with the studies cited above. Before the completion of this survey, two reports on the fine structure of Mytilus eggs appeared (16, 29). The present study is more extensive, and different techniques of fixation and embedding have yielded additional information. MATERIALS AND METHODS Gametes of Mytilus edulis collected from San Francisco Bay were obtained by allowing the animals to spawn in individual dishes of sea water. Eggs were not used unless a control 1 Supported in part by United States Public Health Service Grant RG 6025 to Dr. W. E. Berg.
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sample showed 90% cleavage or better. Most of the material was fixed overnight at 1°C in 2% osmium tetroxide buffered with Veronal-acetate at pH 7.4. Fixation was improved somewhat when the tonicity of the fixative was adjusted to that of the bay water by preparation in diluted sea water, and when fixation times of 1-3 hours at room temperature were used. Fixed materials were washed in distilled water, rapidly dehydrated in ethanol, and embedded in Araldite (12), butyl methacrylate (18), or Epon (I1). The epoxy resins were cured in thin flat sheets from which individual specimens with the desired orientation were cut out, trimmed, and cemented onto larger blocks for sectioning. Sections were cut with a Porter-Blum ultramicrotome and viewed with an RCA EMU-2D or EMU-3E microscope. Centrifuged eggs were subjected to 12,000 g for 20 minutes at 10-12°C and fixed immediately. Homogenates 2 of eggs were prepared in half-molar sucrose by vigorously sucking and squirting the eggs in and out of a fine-tipped pipette, the tip of which had been drawn to a diameter smaller than that of the eggs. Most of the heavier inclusions were removed by centrifuging the homogenate at 3000 g for 10 minutes. The supernatant was drawn off and recentrifuged at 15,000 g for 20 minutes at 1-2°C. The resulting pellets, which were deposited at the tips of small centrifuge tubes, were fixed, washed, and embedded without being removed from the tubes. The tips of the tubes were circumscribed by a glass-cutting wheel until they could be detached and pulled free of the underlying plastic. A tube was then mounted in the microtome chuck and the exposed plastic sectioned. Since an angle-head centrifuge was used, the stratification of inclusions in the pellet was at an angle to the long axis of the centrifuge tube. The angle was increased by appropriate tilting of the specimen in the microtome chuck so that sections were cut in a plane passing from the centrifugal to the centripetal side of the pellet. RESULTS
The egg surface Surface layers of the egg are shown in Fig. 1. The vitelline m e m b r a n e is a b o u t 0.65 # thick. It is traversed by n u m e r o u s evenly spaced microvilli which first pass t h r o u g h a n electron-lucent layer a b o u t 0.2 # thick. The tips of the microvilli, some of which bifurcate, extend just b e y o n d tile outer border of the vitelline m e m b r a n e . The surface of a microvillus represents a p r o t r u s i o n of the plasma m e m b r a n e which encloses a slender c o l u m n of cytoplasm from the egg. Delicate filaments extending from the tip of each microvillus interlace with similar filaments from adjacent micro2 Methods for obtaining, fixing, and embedding homogenate pellets were devised by Dr. W. E. Berg. FIG. 1. The egg surface and cortex showing cortical granules (cg), small vesicles (v), the relationship of the plasma membrane (pm) to the microvilli (my), and the relationship of the microvilli to the vitelline membrane (vm). Delicate fibrils (f) extend from the tips of the microvilli into the jelly coat (jc). An electron-lucent layer (t) lies under the vitelline membrane, x 39,000. FIG. 2. Surface of a fertilized egg. The outer limit of the jelly coat (jc), peripheral to the microvilli (rnv), is defined by an electron-dense line (l), probably an artifact, which is sometimes found over a small region of an egg. pbl, pb2, polar bodies. × 3000. FIG. 3. Microvilli of a section cut in a plane semitangential to the egg surface; lheir lateral surfaces appear as evenly spaced ovals, x 15,000.
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villi to form a network in the substance of the faintly electron-dense jelly coat peripheral to the vitelline membrane. Some sections show this coat to be as much as four times the thickness of the vitelline membrane (Fig. 2); in others it varies and may not be seen at all, although filaments are found extending from the tips of the microvilli. Infrequently, sections show a part of the peripheral coat confined within an electron-dense line (Fig. 2). Such lines are not found in electron micrographs of unspawned oocytes of mature size (unpublished observations), and they are regarded as artifacts probably resulting from an accumulation of debris on the jelly coat after spawning. In sections cut in a plane semitangential to the egg surface the microvilli appear as evenly spaced ovals or circles (Fig. 3), depending on the plane of sectioning and the degree of compression. Inspection of many electron micrographs like those of Figs. 1 and 3 gives the impression that filaments of the type which extend from the tips of the microvilli to form a loose network in the jelly coat material also extend from the lateral surfaces of the microvilli to form a dense network within the substance of the vitelline membrane. As the cleavage furrow of the first division forms, the vitelline membrane does not advance with it (Fig. 4), an observation previously reported by Mancuso (16). Microvilli in this region become detached from the egg and remain in the substance of the vitelline membrane, the furrow beneath them containing an intercellular substance which has a granular texture similar to that of the hyaloplasm. An unfertilized egg treated with sperm extract, prepared by the method of Berg (4), loses its irregular shape and becomes spherical. The vitelline membrane disappears, and the microvilli appear longer, smaller in diameter, and without regularity of spacing, a predictable consequence of removing the membrane, which normally maintains an even spacing of microvilli from their bases to their tips (Fig. 7). These effects are evident in Fig. 5, which shows the first cleavage furrow of an egg cleaving in sperm extract. Microvilli are detached in the region of the furrow as they are when the vitelline membrane is present (compare with Fig. 4). Some of the detached microvilli are in the jelly layer, which sperm extract does not remove (33). Other effects of sperm extract are the disappearance of the intercellular substance normally found in the cleavage furrow, and the disappearance of cortical granules. Hypertonic sea water causes the main cytoplasmic mass of the egg to shrink away from the vitelline membrane, the latter exhibiting no osmotic properties. As the cytoplasm shrinks, the microvilli are detached from the egg and retained within the vitelline membrane (Fig. 6).
The cortex A well-defined cortical region extends from the bases of the microvilli into the egg for a distance of 1.5-2.0 # (Fig. 7). It is characterized by cortical granules, small
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FIG. 4. Microvilli (my) stripped away f r o m the egg surface as the first cleavage furrow advances t o w a r d the interior. A n intercellular s u b s t a n c e (is) occupies the space between the p l a s m a m e m b r a n e s (pro). vm, vitelline m e m b r a n e , x 9400. Fro. 5. T h e furrow of a n egg cleaving in a s p e r m extract. T h e vitelline m e m b r a n e h a s been dissolved, a n d the intercellular s u b s t a n c e is n o t seen in the space of t h e furrow (fr). Microvilli (my) are stripped a w a y f r o m t h e inbuckling surface. C o m p a r e with Fig. 4. × 2700. FIG. 6. P o r t i o n of the egg surface fixed before plasmolysis h a d completely stripped t h e microvilli f r o m t h e egg. D e t a c h e d microvilli (my) are within the s u b s t a n c e of the vitelline m e m b r a n e (vm). T h e p l a s m a m e m b r a n e (pro) f r o m which t h e microvilli are detached s h o w s little if a n y d a m a g e , cg, cortical granule, x 31,000.
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vesicles, and a sparsity of such inclusions as lipid, yolk, and mitochondria. The cortical granule (Fig. 8) is a moderately electron-dense, spherical to oval-shaped body (depending probably on the degree of compression that results from sectioning) with a diameter of about 0.8 #. At high magnifications it appears as a mass of small (10 m/x) particles, incompletely confined within a membrane, so that many of the particles are in free contact with cortical cytoplasm. An electron-lucent space is often seen between the cortical granule membrane and the mass of particles within it, although this is probably an artifact of fixation or subsequent embedding procedures.
The endoplasm A study of unfertilized centrifuged eggs sectioned along the axis of stratification afforded a systematic means of enumerating and describing the components of the egg. Fig. 9 illustrates how the major inclusions stratify. A well-defined cortical layer practically free of yolk, lipid, and mitochondria extends from the bases of the microvilli into the egg for a distance of well over 1 ,u and surrounds the endoplasm, which is separated into four major layers. Proceeding from the centripetal end of the egg, they are: (1) a lipid layer, (2) a wide layer of hyaloplasm, (3) a layer of mitochondria, and (4) the yolk layer. Mitochondria in contact with lipid droplets are found in considerable numbers in the lipid layer. Mitochondria in the yolk layer were apparently trapped there by centrifugally moving yolk granules. Some yolk granules are scattered throughout the layer of mitochondria. The wide band of hyaloplasm is rather sharply divided into two layers of about equal width, the centripetal half containing many vesicles which are not found in the centrifugal half. Constituents of the centripetal cap are shown at a higher magnification in Fig. 10. The lipid droplets displaced against the cortex are identifiable by their size (1.5 #), a high degree of osmiophilia, and a characteristic wrinkled, splotched appearance, a distortion of their natural spherical shape caused probably by fixation or subsequent treatment. The presence of a membrane reported by Reverberi and Mancuso (29) to enclose the lipid droplets could not be confirmed. Scattered among the lipid droplets and the associated mitochondria are vesicles with diameters measuring 20-400 m#. Membranes of the larger vesicles are sparsely studded with electron-dense granules about 15 m# in diameter. These with similar-appearing granules among the inclusions of this region probably correspond to Palade granules of mammalian cells (20). FIG. 7. Low power electron micrograph of an unfertilized egg. The cortical region is characterized by the presence of cortical granules (cg), small vesicles, and a sparsity of yolk (y), lipid (l), and mitochondria (rn). The lipid droplets tend to be clumped into clusters, x 3000. F~G. 8. Cortical region of an unfertilized egg showing 15-m# particles (p), small vesicles (v), and the granular texture of the cortical granules (cg). The relationship of the yolk granule (y) to the yolk membrane (yrn), and the yolk vesicle membrane (yvm) is shown, yv, yolk vesicle, x 66,000.
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The vesicles displaced to the centripetal half of the clear zone are uniform in size (ca. 400 m#) and general appearance (Fig. 11). As in the lipid layer, 15-m/z particles are distributed on and among them. This system of vesicles and granules probably represents part of the endoplasmic reticulum, as this region is high in RNA content (24). Small vesicles (20-60 m#) are scattered among the larger vesicles singly or in clusters (Fig. 12). The only structures visible at higher magnifications of the centrifugal half of the clear zone are the 15-m# particles and microvesicles (Fig. 13). The mitochondrial layer (Figs. 9, 13) contains a few yolk granules and a scattering of vesicles 20-400 m# in diameter, some of which were probably trapped there by mitochondria and yolk. The vesicles associated with yolk particles will be discussed below. The mitochondrial profiles are spherical, oval, or often rod shaped, a finding which suggests that most of the mitochondria are cylindrical rods about 0.35 # wide and up to 2.7 # long. Where the mitochondrial layer merges with the yolk layer, inclusions are found which look either like atypical yolk or atypical mitochondria. Fig. 14 shows a number of these inclusions. They are intermediate in size between yolk and mitochondria, some containing whorls of concentric or spiraling membranes, others having paired internal membranes resembling cristae of mitochondria. Indeed, a spectrum of bodies transitional in appearance between yolk and mitochondria is present. Often, clusters of 20-60-m# vesicles are found near these bodies. Occasionally found at this level of stratification are large, very complex systems of vesicles, small granules, dense globules with less dense loci, vesicles within vesicles, etc., all confined, at least partially, within a double membrane. Fig. 15 shows such a system which measures over 2#. Clusters of 20-60-m# vesicles are usually found near such structures. Also found at the centrifugal side of the mitochondrial layer are agglomerations of small vesicles which, seen in profile, may or may not have a surrounding membrane (Fig. 16). Resembling the multivesicular bodies of rat oocytes (32), these units measure about 0.9/z and the contained vesicles measure 15-16 m#. No dense nuclear body was found within the group of vesicles as in the case of the rat egg (32). The yolk layer is shown at a higher magnification in Fig. 17. In addition to yolk granules, it contains mitochondria, vesicles 20-400 m# in diameter (those 20-60 m# occurring singly or in clusters), multivesicular bodies, subcortical cortical granules, 15-m# granules, and large electron-dense globules of unknown classification. No section contained an empty zone at the centrifugal end of the egg, as was reported by Reverberi and Mancuso (29). A yolk particle (1 #) consists of a moderately electron-dense, granular material surrounded by a membrane (Fig. 8). It is typically found in association with a large vesicle which may surround a greater part of its surface. This type of vesicle is found only in association with yolk particles, and its appearance is unique when compared with other vesicles in the egg. It is probably an artifact of fixation, but for convenience it will be referred to as the "yolk vesicle,"
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FIG. 9. Low power electron micrograph of a section cut along the axis of stratification of a centrifuged unfertilized egg. The arrow indicates the direction of centrifugal force. Cortical granules (cg) are n o t displaced from the cortex. Inclusions of the endoplasm have migrated to form four main layers: (1) a lipid layer, (2) a clear layer, (3) a mitochondrial layer, and (4) a yolk layer. The clear layer is stratified into a vesicular layer (2v) and a granular layer (2g). l, lipid; m, mitochondria; y, yolk; v, vesicles. × 3000.
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and the membrane delimiting it will be called the "yolk vesicle membrane". The latter is irregular in contour, assuming a characteristic rippled or fluted appearance wherever in contact with the yolk membrane (Figs. 8, 13). Material within the yolk vesicle is electron lucent with a sparse suspension of granular- to fibrous-appearing particles. The globoid inclusions of u n k n o w n significance found in the yolk layer are somewhat larger than yolk granules, of lower electron density, unsymmetrical, irregularly contoured, and partially surrounded by a membrane (ub, Fig. 17). They are not associated with vesicles of the type found with yolk particles Within these bodies are found whorls of membranes and vesicles ranging from a b o u t 20 to 100 m/~ in diameter.
Homogenates of unfertilized eggs Centrifugation of egg homogenates causes the suspended inclusions to be laid down in layers that form a pellet. Stratification is the same as in the centrifuged egg. Vesicles with 15-m# particles on their surfaces, corresponding to the vesicles in the vesicular half of the clear zone of the centrifuged egg, are found in the centripetal layer of the pellet. Next is found a layer of mitochondria, and the centrifugal layer consists mostly of yolk. Cortical granules (most of which are in the cortex of the centrifuged egg) and multivesicular bodies are sometimes found in the mitochondrial and vesicular layers of the pellet. Yolk particles in the pellet do not have vesicles in contact with their surfaces, the absence of which indicates that the "yolk vesicles" in the fixed egg referred to above m a y be artifacts. Fig. 18 shows part of the mitochondrial layer of the pellet; although swollen, the mitochondria are clearly recognizable. Vesicles in the centripetal layer are shown in a different part of the same section in Fig. 19. Electron micrographs of thin sections of embedded microsomal fractions of homogenized m a m m a l i a n liver made by Palade and Siekevitz (21) are similar in appearance to Fig. 19. The vesicles in Fig. 19 have fewer granules on their membranes than the vesicles of the liver homogenate. This would be expected, however, since the vesicle membranes in the fixed egg of Mytilus have fewer granules than the membranes of the endoplasmic reticulum of m a m m a l i a n liver. FIG. 10. Centripetal end of a centrifuged unfertilized egg. Lipid droplets (1) with attached mitochondria (m) are displaced against the cortex, the latter containing faintly electron-dense cortical granules (cg). Scattered among the inclusions are vesicles (v) whose membranes are sparsely studded with small particles (p) that also occur freely scattered in the cytoplasm, my, microvilli, x 14,000. FIG. 11. Vesicular half of the clear layer of a centrifuged unfertilized egg. 15-m# particles (p) are on the membranes of the vesicles (v) and scattered among them. x 29,000. Fic. 12. Cluster of small vesicles (v) found in the vesicular half of the clear layer of a centrifuged unfertilized egg. × 15,000. FI6. 13. Junction of the granular half of the clear layer with the mitochondrial layer of a centrifuged unfertilized egg. The granular zone contains many 15-m# particles (p) and small vesicles (v). m, mitochondria; y, yolk; yvm, yolk vesicle membrane; yv, yolk vesicle, x 15,000.
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F~c. 14. Junction of the mitochondrial layer with the yolk layer. Yolk particles (y), particles intermediate in appearance between yolk and mitochondria (i), and systems of small vesicles (v) are present, m, mitochondria; p, 15-m# particles, x 27,000. Fro. 15. Same region as Fig. 14. A large inclusion is shown which is a complex system of membranes, vesicles, and granules. A system of small vesicles is also present, x 12,000. FI~. 16. A muttivesicular body (mvb) in the mitochondrial layer of a centrifuged unfertilized egg x 23,000.
Normal distribution of inclusions Constituents of the normal unfertilized egg fixed a few minutes after spawning have the same appearance, dimensions, and associations as in the centrifuged egg. N o t counting the mitotic apparatus of the first maturation division, which is present shortly after spawning (10), there is an essentially uniform distribution of the subcortical cytoplasmic constituents with the exception of the free cytoplasmic vesicles (as distinguished from yolk vesicles), and possibly the lipid droplets. Sections of only two of the eggs studied cut through the spindle area and the rest of the egg in a definite animal-vegetal plane; these had a gradient of lipid in a vegetal-animal direction. The lipid droplets tend to be grouped in clusters that favor peripheral positions in the egg (Fig. 7). In profile the clusters appear as groups of f r o m two to eight droplets in close contact with one another. The droplets resist coalescence at this stage: even when crowded together in the lipid cap of the centrifuged egg they
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FIG. 17. Centrifugal end of a centrifuged unfertilized egg. Among the yolk particles (y) and their associated vesicles
:jr) are mitochondria (m), unsymmetrical bodies (ub), multivesicular bodies (mvb), 15-m# particles (p), subcortical cortical granules (cg-s), and vesicles (v) of a wide size range cg, cortical granules of the cortex, x 12,000.
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FtG. 18. The mitochondrial layer of a pellet obtained by centrifugation of a homogenate of unfertilized eggs. × 18,000. FIG. 19. A different part of the same section used for Fig. 18 showing the centripetal layer of the pellet. The membranes of the vesicles are sparsely studded with small particles (p). × 18,000. Fro. 20. Section of a mature unfertilized egg. The endoplasm has vesicle-packed regions distributed in channeMike patterns. × 27,000.
do not coalesce to f o r m droplets larger than those found in the uncentrifuged egg (compare with Fig. 9). As in the centrifuged egg the free vesicles of the cytoplasm vary in size from 20 to 400 m # in diameter. Proceeding from the plasma m e m b r a n e medially, the size of the vesicles increases. Only the smaller vesicles appear in the cortex, and for a distance of about 5 # below the plasma m e m b r a n e the vesicles are no larger than 300 m#. The larger vesicles are found deeper within the egg (Fig. 7). This occurrence of large free vesicles in the center of the unfertilized egg, and a decrease in their size toward the periphery, was f o u n d without exception in sections of over twenty different eggs embedded in three different media, methacrylate, Epon, and Araldite. Often the large vesicles (over 300 m/z) are aggregated in regions that exclude other cytoplasmic constituents such as yolk, lipid, and mitochondria. These vesicle-packed regions often appear in sections as channels in the cytoplasm (Fig. 20).
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DISCUSSION
The egg surface The vitelline membrane of Mytilus was described by Hertwig (14) and by Meres (17) as an extremely thin membrane overlying a thicker membrane that envelops the egg. Wada, Collier, and Dan (33) were in agreement with this description, and they identified the thicker membrane as the hyaline layer, the "greater part of which adheres to the vitelline membrane when the cytoplasmic surface shrinks in hypertonic solutions" (p. 174). They stated that when Field (10) described the vitelline membrane of the Mytilus egg as being at least 1 # thick he probably erred by including the width of the hyaline layer in his estimate. They suggested that the intercellular cement makes up the "hyaline layer" which underlies the vitelline membrane. In addition, these investigators demonstrated by the use of an India ink suspensions that a jelly coat about 8 # thick lies outside the other two layers. Electron microscopy reveals that the structure of the vitelline membrane is more complex than had been supposed. In Mancuso's (16) study of the egg surface of Mytilus the limited resolution of the .electron micrographs and the quality of fixation made his results difficult to evaluate. He proposed, for example, that the spawned egg normally has its microvilli detached from the cortical layer, the microvilli contributing "transverse structures" to the egg membrane. These structures were without doubt microvilli detached by the fixation procedures he used. More conventional fixation does not detach microvilli from the eggs of Mytilus or other invertebrate eggs (9, 28). Electron microscopy does not reveal the presence of an extremely thin vitelline membrane of the type described by Meres (17), and by Wada et al. (33) either on freshly shed eggs or on oocytes of mature size within the ovary. Fixation and embedding might cause the loss of such a thin membrane from shed eggs, but its loss from oocytes surrounded by ovarian tissue would be very unlikely. The electron-dense lines sometimes found in electron micrographs located over small regions of the jelly layer of shed eggs (Fig. 2) are interpreted as being artifacts caused either by a precipitation of the jelly layer or by an accumulation of debris on the jelly coat surface. Since lines of this type are not found on the jelly coats of unspawned eggs, accumulation of debris on the jelly coats of shed eggs is the probable cause. In light microscopy a thin layer of this type might logically be identified as a raised portion of a very thin vitelline membrane. The maximal thickness of the layer identified in electron micrographs as the jelly layer is about 4 #, about half the thickness of the jelly coat of the living egg, as demonstrated by Wada et al. (33). The reduced thickness may be due to shrinkage resulting from fixation or subsequent treatment of the egg. The thicker membrane described by Meres, and considered by Wada et al. to
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be the hyaline layer, is actually the layer containing the substance of the vitelline membrane, having a thickness of about 0.65 /~. The disappearance of this layer following exposure to sperm extract of Mytilus confirms this identification, since sperm extract is known to dissolve the vitelline membrane (4). Osmotic indifference of the vitelline membrane is indicated by the fact that when it is completely detached from the egg surface by plasmolysis, its original dimensions are retained. Strong adherence of the surfaces of the microvilli to the substance of the viteltine membrane is indicated by the retention of the microvilli within the vitelline membrane of a plasmolyzed egg. The surface of the Mytilus egg has the same components as the surface of the egg of Spisula, described by Rebhun (28), and they bear the same relationships to one another. Rebhun's electron micrographs do not show the jelly coat nor the fibrils that extend from the tips of the microvilli into it. In Spisula the substance of the vitelline membrane through which the microvilli extend is of different densities, whereas in Mytilus the material is of uniform density. In Mytilus there is no suggestion of spacing of the microvilli in hexagonal arrays as is the case in Spisula. The electronlucent layer medial to the extracellular material of the vitelline membrane of Spisula is referred to by Rebhun as a perivitelline space that increases in width following fertilization of the egg. In the Mytilus egg this layer does not change in width following fertilization (compare Figs. 2 and 7). Differences in the histological techniques of electron microscopy may account for some, if not all, of these differences. This study is in agreement with Rebhun's conclusion that the microvilli and the extracellular substance through which they project are intimately associated. Plasmolysis of the Mytilus egg will detach the vitelline membrane from the entire surface of the egg, stripping it of virtually all the microvilli. However, since sperm extract removes the extracellular material through which the microvilli project, and leaves them attached to the egg surface, it would seem inappropriate to include them as part of the vitelline membrane as proposed by Rebhun. Microvilli have been observed on the surface of several invertebrate eggs including the closely related lamellibranch Spisula (28) and the annelid Hydroides (9). Among the vertebrates they have been found on oocytes of the frog (15) and on oocytes of several mammals including the mouse (38), rat (19), guinea pig (2), and man (34). Microvilli are usually found at sites of active secretion or absorption. The extension of delicate fibrils from the tips of the microvilli of Mytilus eggs suggests that part of the jelly coat and vitelline membrane may be secreted by the microvilli, a point that could be clarified by an electron microscope study on oogenesis. The gain in surface area of the Mytilus egg due to the microvilli could not be precisely determined because the average diameter of the microvilli varied with fixation. Spacing of the microvilli, however, was always quite regular. Therefore, if a conservative estimate of the
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average diameter of the microvilli is used, a rough approximation of surface area increase is easily obtained by following the method of Kemp (15) for computing surface area increase due to microvilli. Assuming the egg has a diameter of 65 # and that one-third the area of the egg surface is occupied by the bases of microvilli, each being a cylinder 0.08 # in diameter and 0.85 # long, the total gain in surface area due to the microvilli is about 15 times that of a simple sphere of the same size. This considerable increase in surface area is an important factor in the interpretation of studies which involve rates of uptake of substances into the egg cytoplasm. Berg and Prescott (8) found the uptake of phosphate into isolated AB and CD blastomeres of Mytilus to correlate with surface areas (which were calculated as though microvilli were not present). Isolated polar lobes and D blastomeres, however, accumulated phosphate at a lower rate than AB and D blastomeres. It was suggested that the limiting factor in phosphate uptake might be due to properties of the cell surface rather than the internal cellular metabolism, and that microvilli on the surface of the egg might be involved. The lower rate of phosphate uptake by isolated polar lobes might be due to a relatively smaller number of microvilli, or lack of microvilli, in the constricted region of the polar lobe. The lower rate of uptake of glycine, methionine, and adenine into isolated polar lobes compared to the rate of uptake into the other egg cytoplasm, as determined by autoradiographic methods (1), might also be due to the absence or reduced number of microvilli on a considerable portion of the isolated polar lobe rather than to a lower metabolic rate of the polar lobe cytoplasm. The coFtcx
The present study is in agreement with the observations by Field (10) that the cortex is sharply differentiated from the endoplasm in the egg of Mytilus edulis. The cortex of the Mytilus californianus egg is similarly well defined, as has been shown by Worley (37), who in describillg the cortex, however, made the following statement: "All the positively identified mitochondria are rod-shaped, two to four times as long as wide. A few of these are scattered in the endoplasm, but the majority are lined up in tremendous numbers perpendicular to the egg's surface either within, or just below the egg cortex" (p. 80). There can be little doubt that the bodies Worley identified as mitochondria were cortical granules. The distribution of cortical granules in the egg of the closely related M. edulis is exactly the same as the distribution of Worley's "mitochondria" in the egg of M. californianus. Most are found closely packed together in and just below the cortex, although a few are scattered in the endoplasm. They occur in such large numbers in the cortex of the unfertilized egg that they exclude other inclusions, even most of the mitochondria. Since centrifugation does not displace them, they must be relatively well anchored in the cortex.
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Their disappearance following treatment of the egg with sperm extract suggests that, in addition to the lytic activities described by Berg (4), dissolution of the egg membrane and the intercellular cement, a third lytic activity is also present in the extract, which causes dissolution of cortical granules. The latter activity could be due to the same proteinlike substance, or substances responsible for the other lytic activities of sperm extract, or it could be due to another type of substance. A type of low-molecular weight egg surface lysin (Androgamone Ill) has been found in methanol extracts of sea urchin sperm by RunnstrSm et al. (31) which according to present evidence (30) is an unsaturated eighteen-carbon fatty acid. A lytic agent with similar chemical properties may be present in the sperm extract of Mytilus that could cause the cortical granules to disappear. The fact that the irregularly shaped eggs of Mytilus become spherical following treatment with sperm extract may relate to this third lytic activity.
The endoplasm Attachment of mitochondria to the surfaces of lipid droplets in the centripetal oil cap of centrifuged eggs has been observed in eggs of echinoderms (22), ascidians (6), and in annelids (35). This affinity has been cited by Pasteels et al. (22) as evidence that the enzymes for lipid metabolism are located in the structure of the mitochondrion. This study is in agreement with the findings of Reverberi and Mancuso (29) that the vesicles which make up the endoplasmic reticulum of the Mytilus egg are displaced by centrifugation to a layer just below the lipid cap. Likewise, Rebhun (27) found in electron microscope studies of centrifuged unfertilized Spisula eggs, that the vesicles consistently stratified in a single layer just below the lipid cap, at the centripetal end of the clear zone. Electron micrographs of centrifuged Arbacia eggs made by Gross et al. (13) showed the vesicles of the clear zone to be smooth surfaced; they failed to stratify into a layer, and there was great variability in their size and number. These findings led the authors to view many of these vesicles as artifacts of fixation produced in response to chemical injury. In Mytilus eggs the vesicles consistently stratify as a layer in the centripetal half of the clear zone, they have particles on their surfaces, and they show no significant variability in size, number, or distribution. In short, using the criteria of Gross et al., nothing about the appearance or distribution of these vesicles suggests that they are artifacts. Perhaps the most direct and convincing evidence for the existence of these vesicles in the normal cytoplasm of the egg would be their isolation from homogenates of the egg. In essence, this was done by centrifugally stratifying the components of whole egg homogenates into pellets. Yolk, mitochondria, and vesicles migrated to discrete layers just as they do in the centrifuged egg, and these inclusions were clearly recognizable in thin sections of the pellets. The vesicles were about the same size as those in the egg, and
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their membranes had small particles on their surfaces. The essentially unchanged appearance of these vesicles in the pellet is strong evidence for their existence, as such, in the normal unfertilized egg. The occurrence of bodies transitional in appearance between that of yolk and mitochondria has been reported in descriptions of the fine structure of other eggs. Odor (19) reported them to be present in the unilaminar follicles of rat ova and described them as atypical mitochondria with dense matrices containing relatively few cristae. She described how these bodies decrease in size and become more uniform in shape as the oocytes reach full maturity. Wartenberg and Stegner (34) concluded from their study of the fine structure of human ova that oval bodies in the cytoplasm are transformed into yolk granules, mitochondria, and multivesicular bodies. Bellairs (3) found mitochondria inside yolk particles of chick blastoderm, and offered as one of several possible explanations of this fact that the circular vesicles which form within the yolk particle as it breaks down, may have been converted to form mitochondria. Further electron microscope studies on oogenesis and the breakdown of yolk are needed to clarify the significance of the particles reported here as intermediate in appearance between yolk and mitochondria. Worley (37) described the presence of numerous bodies 1 # or less in diameter within the cytoplasm of eggs of M. ealifornianus which were osmiophilic, chromophobic interiorly, chromophilic exteriorly, and vitally stainable with methylene blue. These inclusions he called "Golgi bodies." Mistaken identification of cortical granules for mitochondria (see above) led him to believe that the minute bodies of the interior cytoplasm which stained with methylene blue were not mitochondria but "reserve Golgi elements." Golgi apparatuses are rarely encountered in electron micrographs of mature unfertilized Mytilus eggs, and what Worley described as "reserve Golgi bodies" were probably mitochondria. The unsymmetrical bodies found in the yolk layer of the centrifuged egg (Fig. 17, ub) look very much like the "compound vesicular Golgi bodies" described by Worley, since they contain osmiophobic centers which look like vesicles. They are relatively few in number, however, and would not account for the large number of "Golgi bodies" which Worley described as being present in the egg. The only other particles besides the unsymmetrical bodies and lipid droplets that are numerous and close to 1 # in diameter are the yolk particles, the staining characteristics of which may have led to their being identified as Golgi bodies. Multivesicular bodies, which are probably homologous to the multivesicular bodies described by Sotelo and Porter (32) in the oocyte of the rat, have been described in the eggs of the surf clam, Spisula, by Rebhun (26) and identified as being the metachromatically staining/3-granules of Pasteels and Mulnard (23). Rebhun found aggregations of small, smooth-surfaced vesicles concentrated into regions up to 1/z in diameter, and approximately spherical in shape, in the upper hyaline layer of the 3 2 - 621823 J . Ultrastructure Researct~
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centrifuged egg, the zone in which the metachromatically staining fl-granules are found. Similar aggregations of small vesicles are found just below the lipid layer in centrifuged Mytilus eggs (Fig. 12), and some are also found in the mitochondrial layer in the region just centripetal to the yolk (Fig. 16). Membrane-bound multivesicular bodies are found in the centripetal part of the yolk layer (Fig. 17), although they may have been trapped there by moving yolk particles during centrifugation. Evidence from electron microscopy of Spisula eggs indicates that the fl-granules are multivesicular bodies, and the distribution of multivesicular bodies in centrifuged Mytilus eggs is in agreement with this. The metachromatically staining a-particles of Pasteels and Mulnard (23) and of Rebhun (26) have not been identified by means of electron microscopy. These particles go to the centrifugal end of the egg with the yolk in centrifuged eggs. Thin sections of centrifuged Mytilus eggs do not reveal any particles that might obviously be regarded as the c~-particles. Mitochondria in the yolk layer are in the same size range as a-particles, but Rebhun found that in centrifuged Spisula eggs the mitochondrial layer did not stain metachromatically, thus eliminating the possibility that mitochondria might have been misidentified as ~.-particles. Vesicles ranging from a few millimicrons in diameter to the large size of the yolk vesicles are present in the centrifugal layer (Fig. 17), and it is possible that a particular type of vesicle of the proper size is selectively, metachromatically stainable. Identification of the a-particles at the submicroscopic level will require further, more specifically oriented studies. Changes in the fine structure of the Mytilus egg following fertilization, and a survey of the distribution of the major inclusions during the first cleavage, will be reported in a later paper. The author wishes to thank Dr. W. E. Berg for suggesting this investigation and for his unfailing generous help and advice. Grateful acknowledgment is made to the Donner Laboratory of Medical Physics for the use of its electron microscope and to Dr. D. R. Pitelka and Dr. T. L. Hayes for their advice on techniques of electron microscopy. This investigation was submitted in partial fulfillment of requirements for the degree of Doctor of Philosophy, Department of Zoology, University of California, Berkeley, California.
REFERENCES 1. ABD-EL-WAHAB,A. and PANTELOURIS,E. M., Exptl. Ceil Research 13, 78 (1957).
2. 3. 4. 5. 6. 7.
ANDERSON,E. and BEAMS,H. W., J. Ultrastructure Research 3, 432 (1960). BELLA~RS,R., J. Embryol. ExptL MorphoL 6, 149 (1958). BERG, W. E., Biol. Bull. 98, 128 (1950). -Proc. Soe. Exptl. Biol. Med. 85, 606 (1954). BERG, W. E. and HUMPHREYS,W. J., Developmental Biol. 2, 42 (1960). BERG, W. E. and KUTSKY,P. B., Biol. Bull. 101, 47 (1951).
ELECTRON MICROSCOPYOF MYTILUS EGGS 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
37. 38.
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BERG, W. E. and PRESCOTT,D. M., Exptl. Cell Research 14, 402 (1958). COLWIN, A. L. and COLWIN, L. H., J. Biophys. Biochem. Cytol. 7, 321 (1960). FIELD, I. A., Bull. U. S. Bur. Fisheries 38, 127 (1921-1922). FINCK, H., J. Biophys. Biochem. Cytol. 7, 27 (1960). GLAUERT,A. M. and GLAUERT, R. I-I., or. Biophys. Biochem. Cytol. 4, 191 (1958). GROSS, P. R., PHILPOTT, D. E. and NAss, S., J. Biophys. Biochem. Cytol. 7, 135 (1960). HERTWIG, O., Morphol. Jahrb. 4, 177 (1878). K~MV, N. E., J. Biophys. Biochem. Cytol. 2, 281 (1956). MANCUSO,V., Rend. 1st. super, sanith 23, 793 (1960). MEVES,F., Arch. mikroskop. Anat. 78, 47 (1915). NEWMAN,S. B., BORYSKO,E. and SWERDLOW,M., J. Research Natl. Bur. Standards 43, 183 (1949). ODOR, D. L., J. Biophys. Biochem. Cytol. 7, 567 (1960). PALADE,G. E., J. Biophys. Biochem. Cytol. 1, 59 (1955). PALADE, G. E. and SIEKEVITZ,P. J., J. Biophys. Biochem. Cytol. 2, 171 (1956). PASTEELS,J. J., CASTIAUX,P. and VANDERMEERSSCHE,G., Arch. biol. (Li@e) 69, 627 (1959). PASTEELS,J. J. and MULNARO, J., Arch. biol. (Li@e) 68, 115 (1957). PufcI, I., Acta Embryol. Morphol. ExptI. 4, 96 (1961). RATTENBURY,J. C. and BERG, W. E., J. Morphol. 95, 393 (1954). REBHUN, L. I., Ann. N. I1. Acad. Sci. 90, 357 (1960). -J. Ultrastructure Research 5, 208 (1961). -ibid. 6, 107 (1962). REVERBERI,G. and MANCUSO, V., Acta. Embryol. Morphol. Exptl. 4, 102 (1961). RUNNSTR~3M,J., Advances in Enzymol. 9, 241 (1949). RUNNSTR6M, J., LINDVALL,S. and TISELIUS,A., Nature 153, 285 (1944). SOTELO,J. R. and PORTER, K. R., .]. Biophys. Bioehem. CytoI. 5, 327 (1959). WADA, S. K., COLLIER,J. R. and DAN, J. C., Exptl. Cell Research 10, 168 (1956). WARTENBERG,H. and STEGNER,H., Z. Zellforseh. 52, 450 (1960). WEBER, R., Wilhelm Roux" Arch. Entwicklungsmeeh. Organ 150, 542 (1958). WILSON, E. B., J. Exptl. Zool. 1, 1 (1904). WORLEY,L. G., J. Morphol. 75, 77 (1944). YAMADA,E., MUTA, T., MOTOMURA,A. and KOGA, H., Kurume Med. J. 4, 148 (1957).
7, 467-487 (1962)
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Electron Microscope Studies on Eggs of Mytilus edulis 1 WALTER J. HUMPHREYS
Department of Zoology, University of California, Berkeley, California Received May 10, 1962 Normal eggs of the bay mussel, Mytilus edulis, centrifuged eggs, eggs treated with sperm extract, plasmolyzed eggs, and homogenates of whole eggs, were surveyed by electron microscopy. Microvilli increase the egg surface area about fifteenfold. They extend through the vitelline membrane, and delicate fibrils extend from their tips into a jelly coat. Hypertonic sea water detaches the microvilli from the egg and they are retained in the vitelline membrane. Sperm extract causes the vitelline membrane, an intercellular substance, and cortical granules of the cortex to become invisible. The endoplasm of the centrifuged egg stratified into (1) a lipid layer, (2) a clear layer, (3) a mitochondrial layer, and (4) a yolk layer. The constituents of these layers and of the cortex are described. Many of these constituents are recognizable in thin sections of pellets obtained by centrifugation of egg homogenates. The findings are discussed. Eggs of Mytilus edulis have been shown to have an early determination and segregation of morphogenic factors (25) some of which, as in the egg of Dentalium (36), are transferred to the CD cell by the end of the first cleavage. Quantitative comparisons of AB and CD blastomeres and isolated polar lobes have been made with regard to oxygen consumption (7), phosphate uptake (8), content of peptidase (5), and the uptake rate of amino acids (1). Recently, cytochemical studies have also been employed to compare these cytoplasms (24). The present electron microscope survey of the constituents of the Mytilus egg was intended to serve as a basis for subsequent studies on the distribution of constituents in early cleavage stages, which could then be compared with the studies cited above. Before the completion of this survey, two reports on the fine structure of Mytilus eggs appeared (16, 29). The present study is more extensive, and different techniques of fixation and embedding have yielded additional information. MATERIALS AND METHODS Gametes of Mytilus edulis collected from San Francisco Bay were obtained by allowing the animals to spawn in individual dishes of sea water. Eggs were not used unless a control 1 Supported in part by United States Public Health Service Grant RG 6025 to Dr. W. E. Berg.
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sample showed 90% cleavage or better. Most of the material was fixed overnight at 1°C in 2% osmium tetroxide buffered with Veronal-acetate at pH 7.4. Fixation was improved somewhat when the tonicity of the fixative was adjusted to that of the bay water by preparation in diluted sea water, and when fixation times of 1-3 hours at room temperature were used. Fixed materials were washed in distilled water, rapidly dehydrated in ethanol, and embedded in Araldite (12), butyl methacrylate (18), or Epon (I1). The epoxy resins were cured in thin flat sheets from which individual specimens with the desired orientation were cut out, trimmed, and cemented onto larger blocks for sectioning. Sections were cut with a Porter-Blum ultramicrotome and viewed with an RCA EMU-2D or EMU-3E microscope. Centrifuged eggs were subjected to 12,000 g for 20 minutes at 10-12°C and fixed immediately. Homogenates 2 of eggs were prepared in half-molar sucrose by vigorously sucking and squirting the eggs in and out of a fine-tipped pipette, the tip of which had been drawn to a diameter smaller than that of the eggs. Most of the heavier inclusions were removed by centrifuging the homogenate at 3000 g for 10 minutes. The supernatant was drawn off and recentrifuged at 15,000 g for 20 minutes at 1-2°C. The resulting pellets, which were deposited at the tips of small centrifuge tubes, were fixed, washed, and embedded without being removed from the tubes. The tips of the tubes were circumscribed by a glass-cutting wheel until they could be detached and pulled free of the underlying plastic. A tube was then mounted in the microtome chuck and the exposed plastic sectioned. Since an angle-head centrifuge was used, the stratification of inclusions in the pellet was at an angle to the long axis of the centrifuge tube. The angle was increased by appropriate tilting of the specimen in the microtome chuck so that sections were cut in a plane passing from the centrifugal to the centripetal side of the pellet. RESULTS
The egg surface Surface layers of the egg are shown in Fig. 1. The vitelline m e m b r a n e is a b o u t 0.65 # thick. It is traversed by n u m e r o u s evenly spaced microvilli which first pass t h r o u g h a n electron-lucent layer a b o u t 0.2 # thick. The tips of the microvilli, some of which bifurcate, extend just b e y o n d tile outer border of the vitelline m e m b r a n e . The surface of a microvillus represents a p r o t r u s i o n of the plasma m e m b r a n e which encloses a slender c o l u m n of cytoplasm from the egg. Delicate filaments extending from the tip of each microvillus interlace with similar filaments from adjacent micro2 Methods for obtaining, fixing, and embedding homogenate pellets were devised by Dr. W. E. Berg. FIG. 1. The egg surface and cortex showing cortical granules (cg), small vesicles (v), the relationship of the plasma membrane (pm) to the microvilli (my), and the relationship of the microvilli to the vitelline membrane (vm). Delicate fibrils (f) extend from the tips of the microvilli into the jelly coat (jc). An electron-lucent layer (t) lies under the vitelline membrane, x 39,000. FIG. 2. Surface of a fertilized egg. The outer limit of the jelly coat (jc), peripheral to the microvilli (rnv), is defined by an electron-dense line (l), probably an artifact, which is sometimes found over a small region of an egg. pbl, pb2, polar bodies. × 3000. FIG. 3. Microvilli of a section cut in a plane semitangential to the egg surface; lheir lateral surfaces appear as evenly spaced ovals, x 15,000.
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villi to form a network in the substance of the faintly electron-dense jelly coat peripheral to the vitelline membrane. Some sections show this coat to be as much as four times the thickness of the vitelline membrane (Fig. 2); in others it varies and may not be seen at all, although filaments are found extending from the tips of the microvilli. Infrequently, sections show a part of the peripheral coat confined within an electron-dense line (Fig. 2). Such lines are not found in electron micrographs of unspawned oocytes of mature size (unpublished observations), and they are regarded as artifacts probably resulting from an accumulation of debris on the jelly coat after spawning. In sections cut in a plane semitangential to the egg surface the microvilli appear as evenly spaced ovals or circles (Fig. 3), depending on the plane of sectioning and the degree of compression. Inspection of many electron micrographs like those of Figs. 1 and 3 gives the impression that filaments of the type which extend from the tips of the microvilli to form a loose network in the jelly coat material also extend from the lateral surfaces of the microvilli to form a dense network within the substance of the vitelline membrane. As the cleavage furrow of the first division forms, the vitelline membrane does not advance with it (Fig. 4), an observation previously reported by Mancuso (16). Microvilli in this region become detached from the egg and remain in the substance of the vitelline membrane, the furrow beneath them containing an intercellular substance which has a granular texture similar to that of the hyaloplasm. An unfertilized egg treated with sperm extract, prepared by the method of Berg (4), loses its irregular shape and becomes spherical. The vitelline membrane disappears, and the microvilli appear longer, smaller in diameter, and without regularity of spacing, a predictable consequence of removing the membrane, which normally maintains an even spacing of microvilli from their bases to their tips (Fig. 7). These effects are evident in Fig. 5, which shows the first cleavage furrow of an egg cleaving in sperm extract. Microvilli are detached in the region of the furrow as they are when the vitelline membrane is present (compare with Fig. 4). Some of the detached microvilli are in the jelly layer, which sperm extract does not remove (33). Other effects of sperm extract are the disappearance of the intercellular substance normally found in the cleavage furrow, and the disappearance of cortical granules. Hypertonic sea water causes the main cytoplasmic mass of the egg to shrink away from the vitelline membrane, the latter exhibiting no osmotic properties. As the cytoplasm shrinks, the microvilli are detached from the egg and retained within the vitelline membrane (Fig. 6).
The cortex A well-defined cortical region extends from the bases of the microvilli into the egg for a distance of 1.5-2.0 # (Fig. 7). It is characterized by cortical granules, small
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FIG. 4. Microvilli (my) stripped away f r o m the egg surface as the first cleavage furrow advances t o w a r d the interior. A n intercellular s u b s t a n c e (is) occupies the space between the p l a s m a m e m b r a n e s (pro). vm, vitelline m e m b r a n e , x 9400. Fro. 5. T h e furrow of a n egg cleaving in a s p e r m extract. T h e vitelline m e m b r a n e h a s been dissolved, a n d the intercellular s u b s t a n c e is n o t seen in the space of t h e furrow (fr). Microvilli (my) are stripped a w a y f r o m t h e inbuckling surface. C o m p a r e with Fig. 4. × 2700. FIG. 6. P o r t i o n of the egg surface fixed before plasmolysis h a d completely stripped t h e microvilli f r o m t h e egg. D e t a c h e d microvilli (my) are within the s u b s t a n c e of the vitelline m e m b r a n e (vm). T h e p l a s m a m e m b r a n e (pro) f r o m which t h e microvilli are detached s h o w s little if a n y d a m a g e , cg, cortical granule, x 31,000.
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vesicles, and a sparsity of such inclusions as lipid, yolk, and mitochondria. The cortical granule (Fig. 8) is a moderately electron-dense, spherical to oval-shaped body (depending probably on the degree of compression that results from sectioning) with a diameter of about 0.8 #. At high magnifications it appears as a mass of small (10 m/x) particles, incompletely confined within a membrane, so that many of the particles are in free contact with cortical cytoplasm. An electron-lucent space is often seen between the cortical granule membrane and the mass of particles within it, although this is probably an artifact of fixation or subsequent embedding procedures.
The endoplasm A study of unfertilized centrifuged eggs sectioned along the axis of stratification afforded a systematic means of enumerating and describing the components of the egg. Fig. 9 illustrates how the major inclusions stratify. A well-defined cortical layer practically free of yolk, lipid, and mitochondria extends from the bases of the microvilli into the egg for a distance of well over 1 ,u and surrounds the endoplasm, which is separated into four major layers. Proceeding from the centripetal end of the egg, they are: (1) a lipid layer, (2) a wide layer of hyaloplasm, (3) a layer of mitochondria, and (4) the yolk layer. Mitochondria in contact with lipid droplets are found in considerable numbers in the lipid layer. Mitochondria in the yolk layer were apparently trapped there by centrifugally moving yolk granules. Some yolk granules are scattered throughout the layer of mitochondria. The wide band of hyaloplasm is rather sharply divided into two layers of about equal width, the centripetal half containing many vesicles which are not found in the centrifugal half. Constituents of the centripetal cap are shown at a higher magnification in Fig. 10. The lipid droplets displaced against the cortex are identifiable by their size (1.5 #), a high degree of osmiophilia, and a characteristic wrinkled, splotched appearance, a distortion of their natural spherical shape caused probably by fixation or subsequent treatment. The presence of a membrane reported by Reverberi and Mancuso (29) to enclose the lipid droplets could not be confirmed. Scattered among the lipid droplets and the associated mitochondria are vesicles with diameters measuring 20-400 m#. Membranes of the larger vesicles are sparsely studded with electron-dense granules about 15 m# in diameter. These with similar-appearing granules among the inclusions of this region probably correspond to Palade granules of mammalian cells (20). FIG. 7. Low power electron micrograph of an unfertilized egg. The cortical region is characterized by the presence of cortical granules (cg), small vesicles, and a sparsity of yolk (y), lipid (l), and mitochondria (rn). The lipid droplets tend to be clumped into clusters, x 3000. F~G. 8. Cortical region of an unfertilized egg showing 15-m# particles (p), small vesicles (v), and the granular texture of the cortical granules (cg). The relationship of the yolk granule (y) to the yolk membrane (yrn), and the yolk vesicle membrane (yvm) is shown, yv, yolk vesicle, x 66,000.
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The vesicles displaced to the centripetal half of the clear zone are uniform in size (ca. 400 m#) and general appearance (Fig. 11). As in the lipid layer, 15-m/z particles are distributed on and among them. This system of vesicles and granules probably represents part of the endoplasmic reticulum, as this region is high in RNA content (24). Small vesicles (20-60 m#) are scattered among the larger vesicles singly or in clusters (Fig. 12). The only structures visible at higher magnifications of the centrifugal half of the clear zone are the 15-m# particles and microvesicles (Fig. 13). The mitochondrial layer (Figs. 9, 13) contains a few yolk granules and a scattering of vesicles 20-400 m# in diameter, some of which were probably trapped there by mitochondria and yolk. The vesicles associated with yolk particles will be discussed below. The mitochondrial profiles are spherical, oval, or often rod shaped, a finding which suggests that most of the mitochondria are cylindrical rods about 0.35 # wide and up to 2.7 # long. Where the mitochondrial layer merges with the yolk layer, inclusions are found which look either like atypical yolk or atypical mitochondria. Fig. 14 shows a number of these inclusions. They are intermediate in size between yolk and mitochondria, some containing whorls of concentric or spiraling membranes, others having paired internal membranes resembling cristae of mitochondria. Indeed, a spectrum of bodies transitional in appearance between yolk and mitochondria is present. Often, clusters of 20-60-m# vesicles are found near these bodies. Occasionally found at this level of stratification are large, very complex systems of vesicles, small granules, dense globules with less dense loci, vesicles within vesicles, etc., all confined, at least partially, within a double membrane. Fig. 15 shows such a system which measures over 2#. Clusters of 20-60-m# vesicles are usually found near such structures. Also found at the centrifugal side of the mitochondrial layer are agglomerations of small vesicles which, seen in profile, may or may not have a surrounding membrane (Fig. 16). Resembling the multivesicular bodies of rat oocytes (32), these units measure about 0.9/z and the contained vesicles measure 15-16 m#. No dense nuclear body was found within the group of vesicles as in the case of the rat egg (32). The yolk layer is shown at a higher magnification in Fig. 17. In addition to yolk granules, it contains mitochondria, vesicles 20-400 m# in diameter (those 20-60 m# occurring singly or in clusters), multivesicular bodies, subcortical cortical granules, 15-m# granules, and large electron-dense globules of unknown classification. No section contained an empty zone at the centrifugal end of the egg, as was reported by Reverberi and Mancuso (29). A yolk particle (1 #) consists of a moderately electron-dense, granular material surrounded by a membrane (Fig. 8). It is typically found in association with a large vesicle which may surround a greater part of its surface. This type of vesicle is found only in association with yolk particles, and its appearance is unique when compared with other vesicles in the egg. It is probably an artifact of fixation, but for convenience it will be referred to as the "yolk vesicle,"
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FIG. 9. Low power electron micrograph of a section cut along the axis of stratification of a centrifuged unfertilized egg. The arrow indicates the direction of centrifugal force. Cortical granules (cg) are n o t displaced from the cortex. Inclusions of the endoplasm have migrated to form four main layers: (1) a lipid layer, (2) a clear layer, (3) a mitochondrial layer, and (4) a yolk layer. The clear layer is stratified into a vesicular layer (2v) and a granular layer (2g). l, lipid; m, mitochondria; y, yolk; v, vesicles. × 3000.
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and the membrane delimiting it will be called the "yolk vesicle membrane". The latter is irregular in contour, assuming a characteristic rippled or fluted appearance wherever in contact with the yolk membrane (Figs. 8, 13). Material within the yolk vesicle is electron lucent with a sparse suspension of granular- to fibrous-appearing particles. The globoid inclusions of u n k n o w n significance found in the yolk layer are somewhat larger than yolk granules, of lower electron density, unsymmetrical, irregularly contoured, and partially surrounded by a membrane (ub, Fig. 17). They are not associated with vesicles of the type found with yolk particles Within these bodies are found whorls of membranes and vesicles ranging from a b o u t 20 to 100 m/~ in diameter.
Homogenates of unfertilized eggs Centrifugation of egg homogenates causes the suspended inclusions to be laid down in layers that form a pellet. Stratification is the same as in the centrifuged egg. Vesicles with 15-m# particles on their surfaces, corresponding to the vesicles in the vesicular half of the clear zone of the centrifuged egg, are found in the centripetal layer of the pellet. Next is found a layer of mitochondria, and the centrifugal layer consists mostly of yolk. Cortical granules (most of which are in the cortex of the centrifuged egg) and multivesicular bodies are sometimes found in the mitochondrial and vesicular layers of the pellet. Yolk particles in the pellet do not have vesicles in contact with their surfaces, the absence of which indicates that the "yolk vesicles" in the fixed egg referred to above m a y be artifacts. Fig. 18 shows part of the mitochondrial layer of the pellet; although swollen, the mitochondria are clearly recognizable. Vesicles in the centripetal layer are shown in a different part of the same section in Fig. 19. Electron micrographs of thin sections of embedded microsomal fractions of homogenized m a m m a l i a n liver made by Palade and Siekevitz (21) are similar in appearance to Fig. 19. The vesicles in Fig. 19 have fewer granules on their membranes than the vesicles of the liver homogenate. This would be expected, however, since the vesicle membranes in the fixed egg of Mytilus have fewer granules than the membranes of the endoplasmic reticulum of m a m m a l i a n liver. FIG. 10. Centripetal end of a centrifuged unfertilized egg. Lipid droplets (1) with attached mitochondria (m) are displaced against the cortex, the latter containing faintly electron-dense cortical granules (cg). Scattered among the inclusions are vesicles (v) whose membranes are sparsely studded with small particles (p) that also occur freely scattered in the cytoplasm, my, microvilli, x 14,000. FIG. 11. Vesicular half of the clear layer of a centrifuged unfertilized egg. 15-m# particles (p) are on the membranes of the vesicles (v) and scattered among them. x 29,000. Fic. 12. Cluster of small vesicles (v) found in the vesicular half of the clear layer of a centrifuged unfertilized egg. × 15,000. FI6. 13. Junction of the granular half of the clear layer with the mitochondrial layer of a centrifuged unfertilized egg. The granular zone contains many 15-m# particles (p) and small vesicles (v). m, mitochondria; y, yolk; yvm, yolk vesicle membrane; yv, yolk vesicle, x 15,000.
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F~c. 14. Junction of the mitochondrial layer with the yolk layer. Yolk particles (y), particles intermediate in appearance between yolk and mitochondria (i), and systems of small vesicles (v) are present, m, mitochondria; p, 15-m# particles, x 27,000. Fro. 15. Same region as Fig. 14. A large inclusion is shown which is a complex system of membranes, vesicles, and granules. A system of small vesicles is also present, x 12,000. FI~. 16. A muttivesicular body (mvb) in the mitochondrial layer of a centrifuged unfertilized egg x 23,000.
Normal distribution of inclusions Constituents of the normal unfertilized egg fixed a few minutes after spawning have the same appearance, dimensions, and associations as in the centrifuged egg. N o t counting the mitotic apparatus of the first maturation division, which is present shortly after spawning (10), there is an essentially uniform distribution of the subcortical cytoplasmic constituents with the exception of the free cytoplasmic vesicles (as distinguished from yolk vesicles), and possibly the lipid droplets. Sections of only two of the eggs studied cut through the spindle area and the rest of the egg in a definite animal-vegetal plane; these had a gradient of lipid in a vegetal-animal direction. The lipid droplets tend to be grouped in clusters that favor peripheral positions in the egg (Fig. 7). In profile the clusters appear as groups of f r o m two to eight droplets in close contact with one another. The droplets resist coalescence at this stage: even when crowded together in the lipid cap of the centrifuged egg they
ELECTRON MICROSCOPY OF MYTILUS EGGS
479
FIG. 17. Centrifugal end of a centrifuged unfertilized egg. Among the yolk particles (y) and their associated vesicles
:jr) are mitochondria (m), unsymmetrical bodies (ub), multivesicular bodies (mvb), 15-m# particles (p), subcortical cortical granules (cg-s), and vesicles (v) of a wide size range cg, cortical granules of the cortex, x 12,000.
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FtG. 18. The mitochondrial layer of a pellet obtained by centrifugation of a homogenate of unfertilized eggs. × 18,000. FIG. 19. A different part of the same section used for Fig. 18 showing the centripetal layer of the pellet. The membranes of the vesicles are sparsely studded with small particles (p). × 18,000. Fro. 20. Section of a mature unfertilized egg. The endoplasm has vesicle-packed regions distributed in channeMike patterns. × 27,000.
do not coalesce to f o r m droplets larger than those found in the uncentrifuged egg (compare with Fig. 9). As in the centrifuged egg the free vesicles of the cytoplasm vary in size from 20 to 400 m # in diameter. Proceeding from the plasma m e m b r a n e medially, the size of the vesicles increases. Only the smaller vesicles appear in the cortex, and for a distance of about 5 # below the plasma m e m b r a n e the vesicles are no larger than 300 m#. The larger vesicles are found deeper within the egg (Fig. 7). This occurrence of large free vesicles in the center of the unfertilized egg, and a decrease in their size toward the periphery, was f o u n d without exception in sections of over twenty different eggs embedded in three different media, methacrylate, Epon, and Araldite. Often the large vesicles (over 300 m/z) are aggregated in regions that exclude other cytoplasmic constituents such as yolk, lipid, and mitochondria. These vesicle-packed regions often appear in sections as channels in the cytoplasm (Fig. 20).
E L E C T R O N M I C R O S C O P Y OF M Y T I L U S E G G S
481
DISCUSSION
The egg surface The vitelline membrane of Mytilus was described by Hertwig (14) and by Meres (17) as an extremely thin membrane overlying a thicker membrane that envelops the egg. Wada, Collier, and Dan (33) were in agreement with this description, and they identified the thicker membrane as the hyaline layer, the "greater part of which adheres to the vitelline membrane when the cytoplasmic surface shrinks in hypertonic solutions" (p. 174). They stated that when Field (10) described the vitelline membrane of the Mytilus egg as being at least 1 # thick he probably erred by including the width of the hyaline layer in his estimate. They suggested that the intercellular cement makes up the "hyaline layer" which underlies the vitelline membrane. In addition, these investigators demonstrated by the use of an India ink suspensions that a jelly coat about 8 # thick lies outside the other two layers. Electron microscopy reveals that the structure of the vitelline membrane is more complex than had been supposed. In Mancuso's (16) study of the egg surface of Mytilus the limited resolution of the .electron micrographs and the quality of fixation made his results difficult to evaluate. He proposed, for example, that the spawned egg normally has its microvilli detached from the cortical layer, the microvilli contributing "transverse structures" to the egg membrane. These structures were without doubt microvilli detached by the fixation procedures he used. More conventional fixation does not detach microvilli from the eggs of Mytilus or other invertebrate eggs (9, 28). Electron microscopy does not reveal the presence of an extremely thin vitelline membrane of the type described by Meres (17), and by Wada et al. (33) either on freshly shed eggs or on oocytes of mature size within the ovary. Fixation and embedding might cause the loss of such a thin membrane from shed eggs, but its loss from oocytes surrounded by ovarian tissue would be very unlikely. The electron-dense lines sometimes found in electron micrographs located over small regions of the jelly layer of shed eggs (Fig. 2) are interpreted as being artifacts caused either by a precipitation of the jelly layer or by an accumulation of debris on the jelly coat surface. Since lines of this type are not found on the jelly coats of unspawned eggs, accumulation of debris on the jelly coats of shed eggs is the probable cause. In light microscopy a thin layer of this type might logically be identified as a raised portion of a very thin vitelline membrane. The maximal thickness of the layer identified in electron micrographs as the jelly layer is about 4 #, about half the thickness of the jelly coat of the living egg, as demonstrated by Wada et al. (33). The reduced thickness may be due to shrinkage resulting from fixation or subsequent treatment of the egg. The thicker membrane described by Meres, and considered by Wada et al. to
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be the hyaline layer, is actually the layer containing the substance of the vitelline membrane, having a thickness of about 0.65 /~. The disappearance of this layer following exposure to sperm extract of Mytilus confirms this identification, since sperm extract is known to dissolve the vitelline membrane (4). Osmotic indifference of the vitelline membrane is indicated by the fact that when it is completely detached from the egg surface by plasmolysis, its original dimensions are retained. Strong adherence of the surfaces of the microvilli to the substance of the viteltine membrane is indicated by the retention of the microvilli within the vitelline membrane of a plasmolyzed egg. The surface of the Mytilus egg has the same components as the surface of the egg of Spisula, described by Rebhun (28), and they bear the same relationships to one another. Rebhun's electron micrographs do not show the jelly coat nor the fibrils that extend from the tips of the microvilli into it. In Spisula the substance of the vitelline membrane through which the microvilli extend is of different densities, whereas in Mytilus the material is of uniform density. In Mytilus there is no suggestion of spacing of the microvilli in hexagonal arrays as is the case in Spisula. The electronlucent layer medial to the extracellular material of the vitelline membrane of Spisula is referred to by Rebhun as a perivitelline space that increases in width following fertilization of the egg. In the Mytilus egg this layer does not change in width following fertilization (compare Figs. 2 and 7). Differences in the histological techniques of electron microscopy may account for some, if not all, of these differences. This study is in agreement with Rebhun's conclusion that the microvilli and the extracellular substance through which they project are intimately associated. Plasmolysis of the Mytilus egg will detach the vitelline membrane from the entire surface of the egg, stripping it of virtually all the microvilli. However, since sperm extract removes the extracellular material through which the microvilli project, and leaves them attached to the egg surface, it would seem inappropriate to include them as part of the vitelline membrane as proposed by Rebhun. Microvilli have been observed on the surface of several invertebrate eggs including the closely related lamellibranch Spisula (28) and the annelid Hydroides (9). Among the vertebrates they have been found on oocytes of the frog (15) and on oocytes of several mammals including the mouse (38), rat (19), guinea pig (2), and man (34). Microvilli are usually found at sites of active secretion or absorption. The extension of delicate fibrils from the tips of the microvilli of Mytilus eggs suggests that part of the jelly coat and vitelline membrane may be secreted by the microvilli, a point that could be clarified by an electron microscope study on oogenesis. The gain in surface area of the Mytilus egg due to the microvilli could not be precisely determined because the average diameter of the microvilli varied with fixation. Spacing of the microvilli, however, was always quite regular. Therefore, if a conservative estimate of the
ELECTRON MICROSCOPY OF MYTILUS EGGS
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average diameter of the microvilli is used, a rough approximation of surface area increase is easily obtained by following the method of Kemp (15) for computing surface area increase due to microvilli. Assuming the egg has a diameter of 65 # and that one-third the area of the egg surface is occupied by the bases of microvilli, each being a cylinder 0.08 # in diameter and 0.85 # long, the total gain in surface area due to the microvilli is about 15 times that of a simple sphere of the same size. This considerable increase in surface area is an important factor in the interpretation of studies which involve rates of uptake of substances into the egg cytoplasm. Berg and Prescott (8) found the uptake of phosphate into isolated AB and CD blastomeres of Mytilus to correlate with surface areas (which were calculated as though microvilli were not present). Isolated polar lobes and D blastomeres, however, accumulated phosphate at a lower rate than AB and D blastomeres. It was suggested that the limiting factor in phosphate uptake might be due to properties of the cell surface rather than the internal cellular metabolism, and that microvilli on the surface of the egg might be involved. The lower rate of phosphate uptake by isolated polar lobes might be due to a relatively smaller number of microvilli, or lack of microvilli, in the constricted region of the polar lobe. The lower rate of uptake of glycine, methionine, and adenine into isolated polar lobes compared to the rate of uptake into the other egg cytoplasm, as determined by autoradiographic methods (1), might also be due to the absence or reduced number of microvilli on a considerable portion of the isolated polar lobe rather than to a lower metabolic rate of the polar lobe cytoplasm. The coFtcx
The present study is in agreement with the observations by Field (10) that the cortex is sharply differentiated from the endoplasm in the egg of Mytilus edulis. The cortex of the Mytilus californianus egg is similarly well defined, as has been shown by Worley (37), who in describillg the cortex, however, made the following statement: "All the positively identified mitochondria are rod-shaped, two to four times as long as wide. A few of these are scattered in the endoplasm, but the majority are lined up in tremendous numbers perpendicular to the egg's surface either within, or just below the egg cortex" (p. 80). There can be little doubt that the bodies Worley identified as mitochondria were cortical granules. The distribution of cortical granules in the egg of the closely related M. edulis is exactly the same as the distribution of Worley's "mitochondria" in the egg of M. californianus. Most are found closely packed together in and just below the cortex, although a few are scattered in the endoplasm. They occur in such large numbers in the cortex of the unfertilized egg that they exclude other inclusions, even most of the mitochondria. Since centrifugation does not displace them, they must be relatively well anchored in the cortex.
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Their disappearance following treatment of the egg with sperm extract suggests that, in addition to the lytic activities described by Berg (4), dissolution of the egg membrane and the intercellular cement, a third lytic activity is also present in the extract, which causes dissolution of cortical granules. The latter activity could be due to the same proteinlike substance, or substances responsible for the other lytic activities of sperm extract, or it could be due to another type of substance. A type of low-molecular weight egg surface lysin (Androgamone Ill) has been found in methanol extracts of sea urchin sperm by RunnstrSm et al. (31) which according to present evidence (30) is an unsaturated eighteen-carbon fatty acid. A lytic agent with similar chemical properties may be present in the sperm extract of Mytilus that could cause the cortical granules to disappear. The fact that the irregularly shaped eggs of Mytilus become spherical following treatment with sperm extract may relate to this third lytic activity.
The endoplasm Attachment of mitochondria to the surfaces of lipid droplets in the centripetal oil cap of centrifuged eggs has been observed in eggs of echinoderms (22), ascidians (6), and in annelids (35). This affinity has been cited by Pasteels et al. (22) as evidence that the enzymes for lipid metabolism are located in the structure of the mitochondrion. This study is in agreement with the findings of Reverberi and Mancuso (29) that the vesicles which make up the endoplasmic reticulum of the Mytilus egg are displaced by centrifugation to a layer just below the lipid cap. Likewise, Rebhun (27) found in electron microscope studies of centrifuged unfertilized Spisula eggs, that the vesicles consistently stratified in a single layer just below the lipid cap, at the centripetal end of the clear zone. Electron micrographs of centrifuged Arbacia eggs made by Gross et al. (13) showed the vesicles of the clear zone to be smooth surfaced; they failed to stratify into a layer, and there was great variability in their size and number. These findings led the authors to view many of these vesicles as artifacts of fixation produced in response to chemical injury. In Mytilus eggs the vesicles consistently stratify as a layer in the centripetal half of the clear zone, they have particles on their surfaces, and they show no significant variability in size, number, or distribution. In short, using the criteria of Gross et al., nothing about the appearance or distribution of these vesicles suggests that they are artifacts. Perhaps the most direct and convincing evidence for the existence of these vesicles in the normal cytoplasm of the egg would be their isolation from homogenates of the egg. In essence, this was done by centrifugally stratifying the components of whole egg homogenates into pellets. Yolk, mitochondria, and vesicles migrated to discrete layers just as they do in the centrifuged egg, and these inclusions were clearly recognizable in thin sections of the pellets. The vesicles were about the same size as those in the egg, and
ELECTRON MICROSCOPY OF MYTILbS EGGS
485
their membranes had small particles on their surfaces. The essentially unchanged appearance of these vesicles in the pellet is strong evidence for their existence, as such, in the normal unfertilized egg. The occurrence of bodies transitional in appearance between that of yolk and mitochondria has been reported in descriptions of the fine structure of other eggs. Odor (19) reported them to be present in the unilaminar follicles of rat ova and described them as atypical mitochondria with dense matrices containing relatively few cristae. She described how these bodies decrease in size and become more uniform in shape as the oocytes reach full maturity. Wartenberg and Stegner (34) concluded from their study of the fine structure of human ova that oval bodies in the cytoplasm are transformed into yolk granules, mitochondria, and multivesicular bodies. Bellairs (3) found mitochondria inside yolk particles of chick blastoderm, and offered as one of several possible explanations of this fact that the circular vesicles which form within the yolk particle as it breaks down, may have been converted to form mitochondria. Further electron microscope studies on oogenesis and the breakdown of yolk are needed to clarify the significance of the particles reported here as intermediate in appearance between yolk and mitochondria. Worley (37) described the presence of numerous bodies 1 # or less in diameter within the cytoplasm of eggs of M. ealifornianus which were osmiophilic, chromophobic interiorly, chromophilic exteriorly, and vitally stainable with methylene blue. These inclusions he called "Golgi bodies." Mistaken identification of cortical granules for mitochondria (see above) led him to believe that the minute bodies of the interior cytoplasm which stained with methylene blue were not mitochondria but "reserve Golgi elements." Golgi apparatuses are rarely encountered in electron micrographs of mature unfertilized Mytilus eggs, and what Worley described as "reserve Golgi bodies" were probably mitochondria. The unsymmetrical bodies found in the yolk layer of the centrifuged egg (Fig. 17, ub) look very much like the "compound vesicular Golgi bodies" described by Worley, since they contain osmiophobic centers which look like vesicles. They are relatively few in number, however, and would not account for the large number of "Golgi bodies" which Worley described as being present in the egg. The only other particles besides the unsymmetrical bodies and lipid droplets that are numerous and close to 1 # in diameter are the yolk particles, the staining characteristics of which may have led to their being identified as Golgi bodies. Multivesicular bodies, which are probably homologous to the multivesicular bodies described by Sotelo and Porter (32) in the oocyte of the rat, have been described in the eggs of the surf clam, Spisula, by Rebhun (26) and identified as being the metachromatically staining/3-granules of Pasteels and Mulnard (23). Rebhun found aggregations of small, smooth-surfaced vesicles concentrated into regions up to 1/z in diameter, and approximately spherical in shape, in the upper hyaline layer of the 3 2 - 621823 J . Ultrastructure Researct~
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centrifuged egg, the zone in which the metachromatically staining fl-granules are found. Similar aggregations of small vesicles are found just below the lipid layer in centrifuged Mytilus eggs (Fig. 12), and some are also found in the mitochondrial layer in the region just centripetal to the yolk (Fig. 16). Membrane-bound multivesicular bodies are found in the centripetal part of the yolk layer (Fig. 17), although they may have been trapped there by moving yolk particles during centrifugation. Evidence from electron microscopy of Spisula eggs indicates that the fl-granules are multivesicular bodies, and the distribution of multivesicular bodies in centrifuged Mytilus eggs is in agreement with this. The metachromatically staining a-particles of Pasteels and Mulnard (23) and of Rebhun (26) have not been identified by means of electron microscopy. These particles go to the centrifugal end of the egg with the yolk in centrifuged eggs. Thin sections of centrifuged Mytilus eggs do not reveal any particles that might obviously be regarded as the c~-particles. Mitochondria in the yolk layer are in the same size range as a-particles, but Rebhun found that in centrifuged Spisula eggs the mitochondrial layer did not stain metachromatically, thus eliminating the possibility that mitochondria might have been misidentified as ~.-particles. Vesicles ranging from a few millimicrons in diameter to the large size of the yolk vesicles are present in the centrifugal layer (Fig. 17), and it is possible that a particular type of vesicle of the proper size is selectively, metachromatically stainable. Identification of the a-particles at the submicroscopic level will require further, more specifically oriented studies. Changes in the fine structure of the Mytilus egg following fertilization, and a survey of the distribution of the major inclusions during the first cleavage, will be reported in a later paper. The author wishes to thank Dr. W. E. Berg for suggesting this investigation and for his unfailing generous help and advice. Grateful acknowledgment is made to the Donner Laboratory of Medical Physics for the use of its electron microscope and to Dr. D. R. Pitelka and Dr. T. L. Hayes for their advice on techniques of electron microscopy. This investigation was submitted in partial fulfillment of requirements for the degree of Doctor of Philosophy, Department of Zoology, University of California, Berkeley, California.
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BERG, W. E. and PRESCOTT,D. M., Exptl. Cell Research 14, 402 (1958). COLWIN, A. L. and COLWIN, L. H., J. Biophys. Biochem. Cytol. 7, 321 (1960). FIELD, I. A., Bull. U. S. Bur. Fisheries 38, 127 (1921-1922). FINCK, H., J. Biophys. Biochem. Cytol. 7, 27 (1960). GLAUERT,A. M. and GLAUERT, R. I-I., or. Biophys. Biochem. Cytol. 4, 191 (1958). GROSS, P. R., PHILPOTT, D. E. and NAss, S., J. Biophys. Biochem. Cytol. 7, 135 (1960). HERTWIG, O., Morphol. Jahrb. 4, 177 (1878). K~MV, N. E., J. Biophys. Biochem. Cytol. 2, 281 (1956). MANCUSO,V., Rend. 1st. super, sanith 23, 793 (1960). MEVES,F., Arch. mikroskop. Anat. 78, 47 (1915). NEWMAN,S. B., BORYSKO,E. and SWERDLOW,M., J. Research Natl. Bur. Standards 43, 183 (1949). ODOR, D. L., J. Biophys. Biochem. Cytol. 7, 567 (1960). PALADE,G. E., J. Biophys. Biochem. Cytol. 1, 59 (1955). PALADE, G. E. and SIEKEVITZ,P. J., J. Biophys. Biochem. Cytol. 2, 171 (1956). PASTEELS,J. J., CASTIAUX,P. and VANDERMEERSSCHE,G., Arch. biol. (Li@e) 69, 627 (1959). PASTEELS,J. J. and MULNARO, J., Arch. biol. (Li@e) 68, 115 (1957). PufcI, I., Acta Embryol. Morphol. ExptI. 4, 96 (1961). RATTENBURY,J. C. and BERG, W. E., J. Morphol. 95, 393 (1954). REBHUN, L. I., Ann. N. I1. Acad. Sci. 90, 357 (1960). -J. Ultrastructure Research 5, 208 (1961). -ibid. 6, 107 (1962). REVERBERI,G. and MANCUSO, V., Acta. Embryol. Morphol. Exptl. 4, 102 (1961). RUNNSTR~3M,J., Advances in Enzymol. 9, 241 (1949). RUNNSTR6M, J., LINDVALL,S. and TISELIUS,A., Nature 153, 285 (1944). SOTELO,J. R. and PORTER, K. R., .]. Biophys. Bioehem. CytoI. 5, 327 (1959). WADA, S. K., COLLIER,J. R. and DAN, J. C., Exptl. Cell Research 10, 168 (1956). WARTENBERG,H. and STEGNER,H., Z. Zellforseh. 52, 450 (1960). WEBER, R., Wilhelm Roux" Arch. Entwicklungsmeeh. Organ 150, 542 (1958). WILSON, E. B., J. Exptl. Zool. 1, 1 (1904). WORLEY,L. G., J. Morphol. 75, 77 (1944). YAMADA,E., MUTA, T., MOTOMURA,A. and KOGA, H., Kurume Med. J. 4, 148 (1957).