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Electron microscope studies on the vitelline membrane of the surf clam, Spisula solidissima
J. ULTRASTRUCTURERESEARCH6, 107-122 (1962)
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Electron Microscope Studies on the Vitelline Membrane of the Surf Clam, Spisula solidissima 1 LIONEL I. REBHUN
Department of Biology, Princeton University, Princeton, New Jersey, and the Marine Biological Laboratory, Woods Hole, Massachusetts Received June 26, 1961 The vitelline membrane of the egg of Spisula is described. It consists of a hexagonal array of microvilli embedded in two layers of material, the outer and inner fibrillar layers. These layers, each about k/~ thick, are separated from the egg proper by an irregular perivitelline space approximately {- /* thick. After fertilization, the perivitelline space increases in thickness to a variable extent, but never more than double that prior to fertilization. The other layers show no change visible with the electron microscope. The vitelline membrane may be isolated from the egg, in which case both the fibrillar layers and the microvilli are removed together. The vitelline membrane lifts from the egg surface over cleavage furrows and again the fibrillar layers and microvilli act as a unit. The evidence suggests that the term vitelline membrane should be applied in this egg to the complex of microvilli (cytoplasmic extensions) and fibrillar layers (extracellular), since they act in mechanical processes as a unitary object.
T h e surfaces of eggs, especially those of vertebrates a n d higher invertebrates, generally consist of a c o m p l e x series of extra- a n d intracellular layers. The i n t e r a c t i o n of ripe eggs w i t h s p e r m m a y involve m a n y successively activated processes such as s p e r m " i n a c t i v a t i o n " b y i n t e r a c t i o n with j e l l y - c o a t fertilizin (14, 23), release of lysins in initial s p e r m p e n e t r a t i o n of egg m e m b r a n e s (6, 7, 23), fertilization cone f o r m a t i o n (3, 20), a n d a c t i v a t i o n of a b l o c k to p o l y s p e r m y , to n a m e a few. To u n d e r s t a n d these reactions in a wide series of animals it is clear t h a t a detailed picture of the structures involved m u s t be available, especially so since the sea urchin, a m u c h used form, m a y be a t y p i c a l in its m o d e of f e r t i l i z a t i o n - m e m b r a n e f o r m a t i o n . Indeed, in a variety of molluscs a n d annelids we (and m a n y others) have studied, a fertilizat i o n m e m b r a n e as such is n o t f o r m e d , n o r do cortical granules (which are present) b r e a k d o w n on fertilization. I n Spisula soIidissima, the o r g a n i s m used in this work, little visible change o t h e r t h a n a slight thickening in the vitelline m e m b r a n e occurs on z This work supported by grants from the National Science Foundation and the American Cancer Society.
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fertilization, and with the electron microscope no obvious alteration of the component parts can be seen. Nevertheless, a block to polyspermy does develop, which thus occurs in the absence of formation of a major new morphological dement, i.e., a fertilization membrane. A chemical change in the vitelline membrane does appear to occur shortly after fertilization, however, as will be disussed in the following paper, although this may be more related to germinal vesicle breakdown than to fertilization per se. This paper will discuss the morphology of the vitelline membrane of the surf clam, S. solidissima, in the unfertilized egg, and through the early cleavage stages.
MATERIALS AND METHODS Oocytes of the surf clam, S. solidissima, were obtained, handled, and fertilized following Allen (2). Fixation and embedding procedures for most of this material were described in 17. Briefly, eggs were fixed as suspensions in ice-cold isotonic or hypotonic Dalton's or Palade's fixative and vacuum embedded in prepolymerized methacrylate at about 70°C. In addition, some of the material was embedded in Araldite 502 according to the methods of Finck (9), and some of it was prepared by freeze-substitution. Techniques for the latter procedure are described in 18. Briefly, these consisted of rapid freezing in Freon 12 (dichlorodifluoromethane) at -150°C, dehydration in acetone containing 1% osmium tetroxide (8) at -80°C, embedding in Araldite 502, and subsequent staining of mounted sections with potassium permanganate (12). Eggs were collected by gentle hand centrifugation at various times in the cleavage cycle, from immediately prior to fertilization to after second cleavage, and prepared by one of the above methods. In addition, pieces of ovary were also prepared as above. Sections were cut with a Porter-Blum microtome and observed with an RCA EMU2d microscope.
OBSERVATIONS A . VITELLINE MEMBRANE OF THE UNFERTILIZED EGG
1. Normal structure
The vitelline membrane appears to be a complex, approximately 1/~ thick, consisting of cytoplasmic extensions associated with fibrillar extracellular material. The cytoplasmic extensions (microvilli) have dimension of 1 # (Figs. 1, 2 and 4) in length, and approximately 300-400 A in diameter, tapering to approximately 100 A at the tips (Fig. 3). The latter dimensions are clearly averages since slices perpendicular to the axes of the microvilli show them to be somewhat angular in cross-section (Fig. 3), possibly owing to distortion during preparation. They are arranged in a somewhat irregular hexagonal array, with typical distances between nearest neighbors (center to center) of 500-600 A, and hexagon diameters of 900-1100 A. The bases of
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FIG. 1. Perpendicular section of vitelline membrane of an unfertilized Spisula oocyte. The microvilli (Mi), outer (OF) and inner (IF) fibrillar layers, perivitelline space (P), cortical granules (C), and small cytoplasmic vesicles (Ve) are clearly visible. Note the branching of microvilli at the unlabeled arrows. Isotonic Dalton fixative, embedded in methacrylate, x 29,000. FIG. 2. Vitelline membrane of an unfertilized oocyte. Features similar to those in Fig. 1 can be seen, except that the inner fibrillar layer is not clear and microvilli appear distorted. Fixation by freezesubstitution, Araldite 502 embedding, permanganate staining. Note the double membrane complex at the arrows, x 25,000.
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several microvilli may fuse near the main body of the egg so that branching occurs. The branching always takes place in the perivitelline space (see below), i.e., never more than k # from the egg proper. No normal formed elements of the cytoplasm can be found in the microvilli beyond the branch points, i.e., the microvilli appear be devoid of particles even down to the 150-,~ particles so abundant in free form in the cytoplasm of these eggs (17). The plasmalemma (cell membrane) bounding the microvilli appears single, and about 75-100 & thick in ordinary osmium preparations. In frozen-substituted eggs (Fig. 2) the plasmalemma often appears double, two 25-A layers separated by a 25-A space, similar to the membranes of the ergastoplasm and Golgi bodies of frozen-substituted preparations in these same eggs (18). The tips of the villi in many preparations appear to be somewhat solid rather than tubular. The extracellular fibrillar material associated with the microvilli is arranged in two layers; an outer dense layer about ~-# thick, and an inner, paler layer of the same thickness. The fibrillar material of these layers is probably the same in both, but organized with different density. After fixation with hypotonic fixatives, the fibrillar material appears less dense than after fixation with isotonic fixatives, probably owing to extraction of material. No accurate estimate of the length of these fibrils, or of uniformity of this length, can be obtained from these micrographs because the fibrils, which are oriented parallel to the egg surface, interweave. The fibrils in the inner layer are considerably more randomly oriented. Many of these features can be seen to better advantage in sections which have been stained with KMnO~, since the fibrils avidly bind this substance under the conditions we have employed (see especially the following paper). The final "component" of the surface seen in vertical sections of the vitelline membrane is a space of quite variable thickness, but on the average about k # thick, lying between the egg surface (that which is not extended into microvilli) and the inner border of the inner fibrillar layer. This space appears devoid of electron-dense material in our preparations. We consider it to be homologous to the perivitelline space of other eggs (e.g., Arbacia) and shall so designate it in subsequent disscussion.
2. Association of fibrils and microvilli There are three lines of evidence which indicate that the complex of microvilli and fibrils acts as a unit in many mechanical and chemical processes. One of these stems from the behavior of these elements during cleavage, and will be described below. A second concerns events occurring in the membrane during chemically induced dissolution, and will be described in the following paper. The strongest evidence of a complex comes from the simple observation that the entire vitelline membrane may be removed as a whole, and examination shows what we interpret to be the components described in Section A-1 composing the isolated entity. More specif-
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FIG. 3. Tangential section of a ripe ovarian egg. The section intersects the outer and inner fibrillar layers but appears to be just above the perivitelline space. Note the hexagonal arrangement of microvilli, the angular cross-sections of the individual microvilli, and the beginning of the branching of the microvilli (arrows). The hexagonal array is probably distorted by conditions of fixation and embedding, x 45,000.
ically, we noticed d u r i n g experiments involving particle separations f r o m egg h o m o genates m a d e in isotonic, buffered KC1 m e d i a t h a t the h o m o g e n a t e s a n d the " d e b r i s " layer o b t a i n e d b y centrifugation at 1000 × g for 10 minutes c o n t a i n e d w h a t a p p e a r e d to be t r a n s p a r e n t " e n v e l o p e s , " often with m a n y particles associated, b u t often with no visible a t t a c h e d debris (visible in the phase microscope). Pellets of this m a t e r i a l were fixed in o s m i u m in sea water, a n d r u n up a n d e m b e d d e d in m e t h a c r y l a t e in a
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fashion similar to that of whole eggs. An electron micrograph of a structure which we believe to represent the vitelline membrane appears in Fig. 5. Differences with the vitelline membrane of the intact cell quite clearly exist in that the region of fibrillar material is now occupied by what apears to be considerably denser material. Nevertheless, the microvilli are still identifiable as is the fibrillar material. It is clear then that these light and electron microscope observations indicate that the vitelline membrane may be isolated as a unit, and that this unit is composed of both microvilli and fibrillar material, albeit in somewhat modified form. The microvilli are thus not to be imagined as merely projecting into layers of fibrils, but as intimately associated with those layers.
3. Development of the vitelline membrane Our observations of developing oocytes in the ovary are by no means complete, hut do allow us to make some statements concerning the formation of the microvilli. Specifically, the oocytes appear to develop from columnar-like cells lining tubules of the ovary. Microvilli first appear on the lumenal surface of the cell and such cells, except for the large and distally placed nucleus, appear to resemble absorptive epithel~ ial cells. As development proceeds, these cells enlarge and bulge into the ovarian lumen, and as they do so the microvilli begin to develop over the free surface, finally forming also on surfaces between adjacent cells (see Fig. 6). Our observations are not extensive enough to decide at what stage the fibrillar material forms, but it can certainly be seen after the cells have begun to enlarge and round up.
B. VITELLINE MEMBRANE D U R I N G EARLY CLEAVAGE
1. Precleavage In many eggs (e.g., sea urchins (3, 10)) the vitelline membrane undergoes extensive modification as the result of fertilization, forming a so-called fertilization membrane. In Spisula there are no startling changes which result from fertilization, either as seen with the light or the electron microscope. With the former technique the vitelline membrane appears to increase somewhat in thickness in some eggs, and in some batches of eggs, more than in others. However, in no eggs that we have seen has this increase been more than of the order of ½#, and this in only a rough estimate. In the electron microscope the only difference that we can see after fertilization is an increase in the average width of the perivitelline space, rarely more than about k # in extent, and actually quite difficult to measure accurately because of the irregular shape of the perivitelline space, this irregularity being due to the microvilli projecting into the space. For a comparison of a fertilized and an unfertilized egg in approximately the same orientation see Figs. 1 and 7. That considerable differences exist
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FIG. 4. Vitelline m e m b r a n e of an unfertilized oocyte sectioned somewhat tangentially compared to that in Fig. 1. The granular material (Gr) just external to the tips of the microvilli may represent a precipitated jelly-coat. It appears in many, but n o t all, of our micrographs, x 18,500. FIG. 5. Isolated vitelline membrane. The swollen remnants of microvilli (arrows) can be seen as well as the considerably darkened fibrillar material, x 9500. FIG. 6. Unripe, but nearly fully grown ovarian oocytes. The lower oocyte has not yet completed formation of its vitelline m e m b r a n e (see arrows). Where the m e m b r a n e is complete, the normal components can be seen, except for an (irregularly) decreased inner fibrillar layer. × 18,500. 8
62173313 J. Ultrastrueture Research
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Fit. 7. Vitelline membrane of an egg at approximately first cleavage. The outer and inner fibrillar layers do not appear to have changed. The perivitelline space has increased in width by an irregular amount. Compare with Fig. 1, taken from the same batch of eggs. x 29,500.
in the vitelline membrane proper prior to and subsequent to germinal vesicle breakdown with respect to response to a chemical agent, will be seen in the following paper, However, no structural differences appear to exist with the microscopic techniques so far used (compare Figs. 1-4 and 6 with Figs. 7-11).
2. Vitelline membrane over the cleavage furrow An interesting relationship between the vitelline membrane and the cleavage furrow appears at the time of cytokinesis, which supports the contention (Section A2) that the microvilli and fibrillar material act as a unit. Specifically, as the furrow pinches in, the vitelline membrane separates from the cell surface in the furrow, and remains spanning the deepening cleft. With the light microscope this appears as a layer about 1 # thick, continuous with the vitelline membrane over the rest of the egg, which continues over the furrow, closing it off from the surrounding sea water, but spanning and not dipping in with it (see Fig. 1 of the following paper). In the electron micrographs it can be seen that the spanning membrane is composed of microvilli still embedded in the fibrillar layers, both the dense outer one and the lighter inner one (Figs. 8, 9 and 11). Their bases are now closed over by membranes continuous with
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Fro. 8. First polar body almost completely separated from the egg proper (remaining connection can be seen between arrows). Note that the vitelline membrane (V) remains attached by microvilli to the polar body proper, but that over the furrow the vitelline membrane itself (microvilli, outer and inner fibrillar layers) is detached from the egg surface and spans furrow. Remnants (R) of microvillar connections with the surface can be seen. × 20,000. the walls of the microvilli. In addition, the dimensions a n d spacing of the microvilli d o n o t a p p e a r to change in the s p a n n i n g m e m b r a n e . I n some electron m i c r o g r a p h s , microvilli n e a r the p r o x i m a l b o r d e r s of the f u r r o w a p p e a r to have their bases stretched a l m o s t twice their length (i.e., the t o t a l microvillus is n o w up to 2 # long), as if they were being stretched between the deepening f u r r o w a n d the u n b e n d i n g m e m b r a n e . 8 " - - 62173313
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FIG. 9. The intact vitelline membrane can here be seen spanning the deepening first-cleavage furrow (F). What appear to be remmants of the surface are attached to the membrane (arrows). They would appear to have been pulled off the deepening furrow. Note that the furrow surface itself is intact so that new membrane has probably been formed to compensate for that remaining with vitellline membrane. Note also the subsurface vesicles (Ve). x 13,500. These stretched microvilli m a y c o r r e s p o n d to the " s t r i a t i o n s " which can often be seen with the p h a s e m i c r o s c o p e at these p o i n t s in the cleaving egg. Similar relationships, t h o u g h on a smaller scale, occur over the furrows of the p o l a r bodies (Fig. 8). A g a i n , however, no structural change in terms of the details o f fibrillar a n d microvillar o r g a n i z a t i o n can be seen. C. MISCELLANEOUS OBSERVATIONS
1. Subsurface vesicles I n m a n y eggs small vesicles, 100-500 • in diameter, m a y be seen i m m e d i a t e l y (within 1000 A) b e n e a t h the surface. T h e y resemble, a n d m a y be, m i c r o p i n o c y t o s i s
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Fie. 10. Partially tangential section of fertilized egg over the cleavage furrow (F) where the vitelline membrane is sometimes somewhat puckered. Cross-sections of microvilli can be seen, and the approximately hexagonal arrangement of microvilli seen in unfertilized eggs is seen still to persist (arrow, lower left). × 19,000. vacuoles, such as occur in a b s o r p t i v e epithelia (see Disussion), a l t h o u g h they are by no means as n u m e r o u s in these eggs as in some other cells. T h e y are m o r e easily seen in " s u b t a n g e n t " sections of the surface where the perivitelline space is " s p r e a d o u t " (see Figs. 1, 9 a n d i3). 2. Fertilization cones
W e have on two occasions seen fertilization cones with s p e r m heads e m b e d d e d in them. Fig. 12 shows such a cone, a n d shows it as p r o t r u d i n g a p p r o x i m a t e l y 2 # f r o m
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FIG. 11. An egg entering second cleavage. Note spindle (SP) in late anaphase in the large (CD) cell. Cleavage in the smaller (AB) cell takes place approximately 1-2 minutes after that in the CD cell. The polar bodies (PB) can be seen beneath the vitelline membrane, and the furrow from the previous cleavage with spanning vitelline membrane, can be seen. x 3000.
the outer surface of the vitelline membrane. The cone appears to protrude through a large gap in the vitelline membrane.
3. Jelly-coat A jelly-coat 1-2/z thick surrounds the egg as can be seen if eggs are put into a suspension of Chinese ink in sea water. The ink marks the jelly-coat exterior. The only thing in electron micrographs which may possibly correspond to this coat is the granular layer seen in Fig. 4. This most commonly occurs on ovarian eggs, unfertilized shed eggs, and recently fertilized eggs.
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F~o. 12. A fertilization cone containing a s p e r m h e a d c a n be seen projecting t h r o u g h a n d b e y o n d t h e vitelline m e m b r a n e in this egg at a b o u t 8 m i n u t e s after fertilization, x 25,000.
DISCUSSION The evidence presented above suggests that the vitelline membrane of S. solidissima, which in the light microscope looks like a transparent 1-# layer, is actually an organized complex composed of cytoplasmic projections (microvilli), tightly associated with extracellular fibrillar material. Indeed, in the conditions in which we have observed them, the two components are not separated by mechanical means and further, in experiments to be described in the following paper, the first response of the membrane to chemical dissolution is again unitary. The binding is thus tight enough to cause the microvilli to pinch off at their bases during mechanical isolation, and also during the natural process of cleavage-furrow formation. What the nature of this binding is we do not know. That the fibrillar layer itself is not merely jelly-like, but actually possesses a tension-resisting organization, is indicated by experimental evidence offered by Schechter (21). He showed that the vitelline membrane resists stretching during osmotic swelling. The fibrils in the membrane are in the correct orientation for exerting such a resistance. Microvilli have been described in a number of absorptive epithelia, e.g., proximal
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Fie_,. 13. A tangential view of the egg surface (5 minutes after fertilization) showing the outer and inner fibrillar layers, perivitelline space, etc. The section includes pieces of the egg proper and arrows point to small vesicles in the egg which may possibly represent micropinocytosis vesicles, x 29,500.
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.convoluted tubules of the kidney (19), intestinal epithelial cells (15, 16), and gall bladder (24), to name a few. Their function is generally assumed to be related to increase in surface area involved in absorption processes (11, 15). Relatively constant structures accompanying microvillated borders are tubular and vesicular invaginations extending into the cellular cytoplasm from the bases of some of the microvilli (15, 16, 19, 24). In eggs, microvilli have been reported in a number of forms, e.g., in sea urchins (1), in annelids (5, 7), amphibia (11), and in rodents (4, 22), and would appear to be a constant feature of oocytes, which may, however, disappear at certain times in development (22) (the oocyte of the enteropneust Saccoglossus may be an exception (5)). In addition, jelly-like layers usually accompany these microvilli, e.g., the zona pellucida material in mammals, and the middle and inner border layers in Hydroides (5-7). The existence of pocketings at the bases of microvilli which might indicate micropinocytosis in eggs has been pointed out by Anderson and Beams (4) in guinea pig oocytes, and the results we have presented above do not contradict these suggestions. It should be carefully pointed out, however, that actual pinocytosis has not been shown in any oocyte system, and the participation of microvilli in absorption not demonstrated (see also 16). In our material, however, the oocytes develop as part of an epithelium and such an absorptive process may play a role in early oogenesis. A micropinocytosis mechanism in ripe eggs may be "left over," as it were, from earlier processes. Tubular invaginations which may be related to micropinocytosis have been seen in cleaving sea urchin eggs (13), where their occurrence and distribution suggests a deep involvement with the process of furrow formation. However, no such structures have as yet been seen in Spisula eggs. Tyler (23) has suggested a process of "specific" pinocytosis as being involved in sperm penetration into sea urchin (and presumably other) eggs. The pinocytosis is postulated to be a reaction of possibly expanded tips of microvilli projecting through the vitelline membrane. The block to polyspermy occurs in part through a subsequent withdrawal of microvilli through the vitellinc membrane, and fertilization membrane elevation consequent on cortical granule breakdown. However these hypotheses may fare for sea urchins, they would appear to be inapplicable to Spisula, since (a) it would be difficult to interpret Fig. 12 as an expanded microvillus, and (b) no evidence exists that the microvilli themselves change in any way during feritlization except, possibly, to increase in length owing to perivitelline-space enlargement. It is clear, therefore, that for Spisula the block to polyspermy involves a delicate, rather than grossly obvious change, in the vitelline membrane, and that pinocytosis, if it exists in these eggs, is probably not tied to sperm penetration, as attractive as such a hypothesis might be.
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1. AFZELIUS,B. A., ExptL Cell Research 10, 257 (1956). 2. ALLEN, R. D., Biol. Bull. 105, 213 (1953). 3. - in MCELROY, W. D. and GLASS, B, (Eds.), The Chemical Basis of Development, p. 17. The Johns Hopkins Press, Baltimore, 1958. 4. ANDERSON,E. and BEAMS, H. W., 9". Ultrastructure Research 3, 432 (1960). 5. COLWlN, A. L., COLWlN, L. H. and P~mVOTT, D. E., d. Biophys. Biochem. CytoL 3, 489 (1957). 6. COLWIN,A. L. and COLWlN, L. H., aT.)3io?hys. Biochem. Cytol. 7, 321 (1960). 7. COLWlN, L. H. and COLWlN,A. L., J. Biophys. Biochem. CytoI. 7, 315 (1960). 8. F~DER, N. and SIDMAN,R. L., J. Biophys. Biochem. Cytol. 4, 593 (1958). 9. FINCK, H., Y. Biophys. Biochem. Cytol. 7, 27 (1960). 10. HARVEY, E. B., The American Arbacia and Other Sea Urchins. Princeton University Press, Princeton, 1956. 11. KEMP, N. E., ,1. Biophys. Biochem. Cytol. 2, 281 (1956). 12. LAWN, A. M., J. Biophys. Biochem. Cytol. 7, 197 (1960). 13. MERCER, E. H. and WOLPERT, L., Exptl. Cell Research 14, 629 (1958). 14. METZ, C. B., in TYLER, A., YON BORSTEL,R. C. and METZ, C. B. (Eds.), The Beginnings of Embryonic Development, p. 23. A.A.A.S., Washington, D. C., 1957. 15. PALAY, S. and KARLIN, L. J., J. Biophys. Biochem. Cytol. 5, 363 (1959). 16. - ibid. 3, 373 (1959). 17 REBrIUN, L. I., J. Biophys. Biochem. Cytol. 9, 785 (1961). 18~ - J. Ultrastructure Research 5, 208 (1961). 19.1RHODIN, J., Intern. Rev. Cytol. 7, 485 (1958). 20. ROTHSCHILD,LORD, Fertilization. Methuen & Co., London, 1956. 21. SCHECHTER, V., Exptl. Cell Research 10, 619 (1956). 22. SOTELO,J. R. and PORTER, K. R., J. Biophys. Biochem. Cytol. 5:327 (1959). 23. TYLER, A., Exptl. Cell Research, Suppl. 7, 183 (1959). 24. YAMADA,E., J. Biophys. Biochem. CytoL 1,445 (1955).
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Electron Microscope Studies on the Vitelline Membrane of the Surf Clam, Spisula solidissima 1 LIONEL I. REBHUN
Department of Biology, Princeton University, Princeton, New Jersey, and the Marine Biological Laboratory, Woods Hole, Massachusetts Received June 26, 1961 The vitelline membrane of the egg of Spisula is described. It consists of a hexagonal array of microvilli embedded in two layers of material, the outer and inner fibrillar layers. These layers, each about k/~ thick, are separated from the egg proper by an irregular perivitelline space approximately {- /* thick. After fertilization, the perivitelline space increases in thickness to a variable extent, but never more than double that prior to fertilization. The other layers show no change visible with the electron microscope. The vitelline membrane may be isolated from the egg, in which case both the fibrillar layers and the microvilli are removed together. The vitelline membrane lifts from the egg surface over cleavage furrows and again the fibrillar layers and microvilli act as a unit. The evidence suggests that the term vitelline membrane should be applied in this egg to the complex of microvilli (cytoplasmic extensions) and fibrillar layers (extracellular), since they act in mechanical processes as a unitary object.
T h e surfaces of eggs, especially those of vertebrates a n d higher invertebrates, generally consist of a c o m p l e x series of extra- a n d intracellular layers. The i n t e r a c t i o n of ripe eggs w i t h s p e r m m a y involve m a n y successively activated processes such as s p e r m " i n a c t i v a t i o n " b y i n t e r a c t i o n with j e l l y - c o a t fertilizin (14, 23), release of lysins in initial s p e r m p e n e t r a t i o n of egg m e m b r a n e s (6, 7, 23), fertilization cone f o r m a t i o n (3, 20), a n d a c t i v a t i o n of a b l o c k to p o l y s p e r m y , to n a m e a few. To u n d e r s t a n d these reactions in a wide series of animals it is clear t h a t a detailed picture of the structures involved m u s t be available, especially so since the sea urchin, a m u c h used form, m a y be a t y p i c a l in its m o d e of f e r t i l i z a t i o n - m e m b r a n e f o r m a t i o n . Indeed, in a variety of molluscs a n d annelids we (and m a n y others) have studied, a fertilizat i o n m e m b r a n e as such is n o t f o r m e d , n o r do cortical granules (which are present) b r e a k d o w n on fertilization. I n Spisula soIidissima, the o r g a n i s m used in this work, little visible change o t h e r t h a n a slight thickening in the vitelline m e m b r a n e occurs on z This work supported by grants from the National Science Foundation and the American Cancer Society.
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fertilization, and with the electron microscope no obvious alteration of the component parts can be seen. Nevertheless, a block to polyspermy does develop, which thus occurs in the absence of formation of a major new morphological dement, i.e., a fertilization membrane. A chemical change in the vitelline membrane does appear to occur shortly after fertilization, however, as will be disussed in the following paper, although this may be more related to germinal vesicle breakdown than to fertilization per se. This paper will discuss the morphology of the vitelline membrane of the surf clam, S. solidissima, in the unfertilized egg, and through the early cleavage stages.
MATERIALS AND METHODS Oocytes of the surf clam, S. solidissima, were obtained, handled, and fertilized following Allen (2). Fixation and embedding procedures for most of this material were described in 17. Briefly, eggs were fixed as suspensions in ice-cold isotonic or hypotonic Dalton's or Palade's fixative and vacuum embedded in prepolymerized methacrylate at about 70°C. In addition, some of the material was embedded in Araldite 502 according to the methods of Finck (9), and some of it was prepared by freeze-substitution. Techniques for the latter procedure are described in 18. Briefly, these consisted of rapid freezing in Freon 12 (dichlorodifluoromethane) at -150°C, dehydration in acetone containing 1% osmium tetroxide (8) at -80°C, embedding in Araldite 502, and subsequent staining of mounted sections with potassium permanganate (12). Eggs were collected by gentle hand centrifugation at various times in the cleavage cycle, from immediately prior to fertilization to after second cleavage, and prepared by one of the above methods. In addition, pieces of ovary were also prepared as above. Sections were cut with a Porter-Blum microtome and observed with an RCA EMU2d microscope.
OBSERVATIONS A . VITELLINE MEMBRANE OF THE UNFERTILIZED EGG
1. Normal structure
The vitelline membrane appears to be a complex, approximately 1/~ thick, consisting of cytoplasmic extensions associated with fibrillar extracellular material. The cytoplasmic extensions (microvilli) have dimension of 1 # (Figs. 1, 2 and 4) in length, and approximately 300-400 A in diameter, tapering to approximately 100 A at the tips (Fig. 3). The latter dimensions are clearly averages since slices perpendicular to the axes of the microvilli show them to be somewhat angular in cross-section (Fig. 3), possibly owing to distortion during preparation. They are arranged in a somewhat irregular hexagonal array, with typical distances between nearest neighbors (center to center) of 500-600 A, and hexagon diameters of 900-1100 A. The bases of
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FIG. 1. Perpendicular section of vitelline membrane of an unfertilized Spisula oocyte. The microvilli (Mi), outer (OF) and inner (IF) fibrillar layers, perivitelline space (P), cortical granules (C), and small cytoplasmic vesicles (Ve) are clearly visible. Note the branching of microvilli at the unlabeled arrows. Isotonic Dalton fixative, embedded in methacrylate, x 29,000. FIG. 2. Vitelline membrane of an unfertilized oocyte. Features similar to those in Fig. 1 can be seen, except that the inner fibrillar layer is not clear and microvilli appear distorted. Fixation by freezesubstitution, Araldite 502 embedding, permanganate staining. Note the double membrane complex at the arrows, x 25,000.
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several microvilli may fuse near the main body of the egg so that branching occurs. The branching always takes place in the perivitelline space (see below), i.e., never more than k # from the egg proper. No normal formed elements of the cytoplasm can be found in the microvilli beyond the branch points, i.e., the microvilli appear be devoid of particles even down to the 150-,~ particles so abundant in free form in the cytoplasm of these eggs (17). The plasmalemma (cell membrane) bounding the microvilli appears single, and about 75-100 & thick in ordinary osmium preparations. In frozen-substituted eggs (Fig. 2) the plasmalemma often appears double, two 25-A layers separated by a 25-A space, similar to the membranes of the ergastoplasm and Golgi bodies of frozen-substituted preparations in these same eggs (18). The tips of the villi in many preparations appear to be somewhat solid rather than tubular. The extracellular fibrillar material associated with the microvilli is arranged in two layers; an outer dense layer about ~-# thick, and an inner, paler layer of the same thickness. The fibrillar material of these layers is probably the same in both, but organized with different density. After fixation with hypotonic fixatives, the fibrillar material appears less dense than after fixation with isotonic fixatives, probably owing to extraction of material. No accurate estimate of the length of these fibrils, or of uniformity of this length, can be obtained from these micrographs because the fibrils, which are oriented parallel to the egg surface, interweave. The fibrils in the inner layer are considerably more randomly oriented. Many of these features can be seen to better advantage in sections which have been stained with KMnO~, since the fibrils avidly bind this substance under the conditions we have employed (see especially the following paper). The final "component" of the surface seen in vertical sections of the vitelline membrane is a space of quite variable thickness, but on the average about k # thick, lying between the egg surface (that which is not extended into microvilli) and the inner border of the inner fibrillar layer. This space appears devoid of electron-dense material in our preparations. We consider it to be homologous to the perivitelline space of other eggs (e.g., Arbacia) and shall so designate it in subsequent disscussion.
2. Association of fibrils and microvilli There are three lines of evidence which indicate that the complex of microvilli and fibrils acts as a unit in many mechanical and chemical processes. One of these stems from the behavior of these elements during cleavage, and will be described below. A second concerns events occurring in the membrane during chemically induced dissolution, and will be described in the following paper. The strongest evidence of a complex comes from the simple observation that the entire vitelline membrane may be removed as a whole, and examination shows what we interpret to be the components described in Section A-1 composing the isolated entity. More specif-
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FIG. 3. Tangential section of a ripe ovarian egg. The section intersects the outer and inner fibrillar layers but appears to be just above the perivitelline space. Note the hexagonal arrangement of microvilli, the angular cross-sections of the individual microvilli, and the beginning of the branching of the microvilli (arrows). The hexagonal array is probably distorted by conditions of fixation and embedding, x 45,000.
ically, we noticed d u r i n g experiments involving particle separations f r o m egg h o m o genates m a d e in isotonic, buffered KC1 m e d i a t h a t the h o m o g e n a t e s a n d the " d e b r i s " layer o b t a i n e d b y centrifugation at 1000 × g for 10 minutes c o n t a i n e d w h a t a p p e a r e d to be t r a n s p a r e n t " e n v e l o p e s , " often with m a n y particles associated, b u t often with no visible a t t a c h e d debris (visible in the phase microscope). Pellets of this m a t e r i a l were fixed in o s m i u m in sea water, a n d r u n up a n d e m b e d d e d in m e t h a c r y l a t e in a
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fashion similar to that of whole eggs. An electron micrograph of a structure which we believe to represent the vitelline membrane appears in Fig. 5. Differences with the vitelline membrane of the intact cell quite clearly exist in that the region of fibrillar material is now occupied by what apears to be considerably denser material. Nevertheless, the microvilli are still identifiable as is the fibrillar material. It is clear then that these light and electron microscope observations indicate that the vitelline membrane may be isolated as a unit, and that this unit is composed of both microvilli and fibrillar material, albeit in somewhat modified form. The microvilli are thus not to be imagined as merely projecting into layers of fibrils, but as intimately associated with those layers.
3. Development of the vitelline membrane Our observations of developing oocytes in the ovary are by no means complete, hut do allow us to make some statements concerning the formation of the microvilli. Specifically, the oocytes appear to develop from columnar-like cells lining tubules of the ovary. Microvilli first appear on the lumenal surface of the cell and such cells, except for the large and distally placed nucleus, appear to resemble absorptive epithel~ ial cells. As development proceeds, these cells enlarge and bulge into the ovarian lumen, and as they do so the microvilli begin to develop over the free surface, finally forming also on surfaces between adjacent cells (see Fig. 6). Our observations are not extensive enough to decide at what stage the fibrillar material forms, but it can certainly be seen after the cells have begun to enlarge and round up.
B. VITELLINE MEMBRANE D U R I N G EARLY CLEAVAGE
1. Precleavage In many eggs (e.g., sea urchins (3, 10)) the vitelline membrane undergoes extensive modification as the result of fertilization, forming a so-called fertilization membrane. In Spisula there are no startling changes which result from fertilization, either as seen with the light or the electron microscope. With the former technique the vitelline membrane appears to increase somewhat in thickness in some eggs, and in some batches of eggs, more than in others. However, in no eggs that we have seen has this increase been more than of the order of ½#, and this in only a rough estimate. In the electron microscope the only difference that we can see after fertilization is an increase in the average width of the perivitelline space, rarely more than about k # in extent, and actually quite difficult to measure accurately because of the irregular shape of the perivitelline space, this irregularity being due to the microvilli projecting into the space. For a comparison of a fertilized and an unfertilized egg in approximately the same orientation see Figs. 1 and 7. That considerable differences exist
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FIG. 4. Vitelline m e m b r a n e of an unfertilized oocyte sectioned somewhat tangentially compared to that in Fig. 1. The granular material (Gr) just external to the tips of the microvilli may represent a precipitated jelly-coat. It appears in many, but n o t all, of our micrographs, x 18,500. FIG. 5. Isolated vitelline membrane. The swollen remnants of microvilli (arrows) can be seen as well as the considerably darkened fibrillar material, x 9500. FIG. 6. Unripe, but nearly fully grown ovarian oocytes. The lower oocyte has not yet completed formation of its vitelline m e m b r a n e (see arrows). Where the m e m b r a n e is complete, the normal components can be seen, except for an (irregularly) decreased inner fibrillar layer. × 18,500. 8
62173313 J. Ultrastrueture Research
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Fit. 7. Vitelline membrane of an egg at approximately first cleavage. The outer and inner fibrillar layers do not appear to have changed. The perivitelline space has increased in width by an irregular amount. Compare with Fig. 1, taken from the same batch of eggs. x 29,500.
in the vitelline membrane proper prior to and subsequent to germinal vesicle breakdown with respect to response to a chemical agent, will be seen in the following paper, However, no structural differences appear to exist with the microscopic techniques so far used (compare Figs. 1-4 and 6 with Figs. 7-11).
2. Vitelline membrane over the cleavage furrow An interesting relationship between the vitelline membrane and the cleavage furrow appears at the time of cytokinesis, which supports the contention (Section A2) that the microvilli and fibrillar material act as a unit. Specifically, as the furrow pinches in, the vitelline membrane separates from the cell surface in the furrow, and remains spanning the deepening cleft. With the light microscope this appears as a layer about 1 # thick, continuous with the vitelline membrane over the rest of the egg, which continues over the furrow, closing it off from the surrounding sea water, but spanning and not dipping in with it (see Fig. 1 of the following paper). In the electron micrographs it can be seen that the spanning membrane is composed of microvilli still embedded in the fibrillar layers, both the dense outer one and the lighter inner one (Figs. 8, 9 and 11). Their bases are now closed over by membranes continuous with
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Fro. 8. First polar body almost completely separated from the egg proper (remaining connection can be seen between arrows). Note that the vitelline membrane (V) remains attached by microvilli to the polar body proper, but that over the furrow the vitelline membrane itself (microvilli, outer and inner fibrillar layers) is detached from the egg surface and spans furrow. Remnants (R) of microvillar connections with the surface can be seen. × 20,000. the walls of the microvilli. In addition, the dimensions a n d spacing of the microvilli d o n o t a p p e a r to change in the s p a n n i n g m e m b r a n e . I n some electron m i c r o g r a p h s , microvilli n e a r the p r o x i m a l b o r d e r s of the f u r r o w a p p e a r to have their bases stretched a l m o s t twice their length (i.e., the t o t a l microvillus is n o w up to 2 # long), as if they were being stretched between the deepening f u r r o w a n d the u n b e n d i n g m e m b r a n e . 8 " - - 62173313
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FIG. 9. The intact vitelline membrane can here be seen spanning the deepening first-cleavage furrow (F). What appear to be remmants of the surface are attached to the membrane (arrows). They would appear to have been pulled off the deepening furrow. Note that the furrow surface itself is intact so that new membrane has probably been formed to compensate for that remaining with vitellline membrane. Note also the subsurface vesicles (Ve). x 13,500. These stretched microvilli m a y c o r r e s p o n d to the " s t r i a t i o n s " which can often be seen with the p h a s e m i c r o s c o p e at these p o i n t s in the cleaving egg. Similar relationships, t h o u g h on a smaller scale, occur over the furrows of the p o l a r bodies (Fig. 8). A g a i n , however, no structural change in terms of the details o f fibrillar a n d microvillar o r g a n i z a t i o n can be seen. C. MISCELLANEOUS OBSERVATIONS
1. Subsurface vesicles I n m a n y eggs small vesicles, 100-500 • in diameter, m a y be seen i m m e d i a t e l y (within 1000 A) b e n e a t h the surface. T h e y resemble, a n d m a y be, m i c r o p i n o c y t o s i s
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Fie. 10. Partially tangential section of fertilized egg over the cleavage furrow (F) where the vitelline membrane is sometimes somewhat puckered. Cross-sections of microvilli can be seen, and the approximately hexagonal arrangement of microvilli seen in unfertilized eggs is seen still to persist (arrow, lower left). × 19,000. vacuoles, such as occur in a b s o r p t i v e epithelia (see Disussion), a l t h o u g h they are by no means as n u m e r o u s in these eggs as in some other cells. T h e y are m o r e easily seen in " s u b t a n g e n t " sections of the surface where the perivitelline space is " s p r e a d o u t " (see Figs. 1, 9 a n d i3). 2. Fertilization cones
W e have on two occasions seen fertilization cones with s p e r m heads e m b e d d e d in them. Fig. 12 shows such a cone, a n d shows it as p r o t r u d i n g a p p r o x i m a t e l y 2 # f r o m
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FIG. 11. An egg entering second cleavage. Note spindle (SP) in late anaphase in the large (CD) cell. Cleavage in the smaller (AB) cell takes place approximately 1-2 minutes after that in the CD cell. The polar bodies (PB) can be seen beneath the vitelline membrane, and the furrow from the previous cleavage with spanning vitelline membrane, can be seen. x 3000.
the outer surface of the vitelline membrane. The cone appears to protrude through a large gap in the vitelline membrane.
3. Jelly-coat A jelly-coat 1-2/z thick surrounds the egg as can be seen if eggs are put into a suspension of Chinese ink in sea water. The ink marks the jelly-coat exterior. The only thing in electron micrographs which may possibly correspond to this coat is the granular layer seen in Fig. 4. This most commonly occurs on ovarian eggs, unfertilized shed eggs, and recently fertilized eggs.
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F~o. 12. A fertilization cone containing a s p e r m h e a d c a n be seen projecting t h r o u g h a n d b e y o n d t h e vitelline m e m b r a n e in this egg at a b o u t 8 m i n u t e s after fertilization, x 25,000.
DISCUSSION The evidence presented above suggests that the vitelline membrane of S. solidissima, which in the light microscope looks like a transparent 1-# layer, is actually an organized complex composed of cytoplasmic projections (microvilli), tightly associated with extracellular fibrillar material. Indeed, in the conditions in which we have observed them, the two components are not separated by mechanical means and further, in experiments to be described in the following paper, the first response of the membrane to chemical dissolution is again unitary. The binding is thus tight enough to cause the microvilli to pinch off at their bases during mechanical isolation, and also during the natural process of cleavage-furrow formation. What the nature of this binding is we do not know. That the fibrillar layer itself is not merely jelly-like, but actually possesses a tension-resisting organization, is indicated by experimental evidence offered by Schechter (21). He showed that the vitelline membrane resists stretching during osmotic swelling. The fibrils in the membrane are in the correct orientation for exerting such a resistance. Microvilli have been described in a number of absorptive epithelia, e.g., proximal
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Fie_,. 13. A tangential view of the egg surface (5 minutes after fertilization) showing the outer and inner fibrillar layers, perivitelline space, etc. The section includes pieces of the egg proper and arrows point to small vesicles in the egg which may possibly represent micropinocytosis vesicles, x 29,500.
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.convoluted tubules of the kidney (19), intestinal epithelial cells (15, 16), and gall bladder (24), to name a few. Their function is generally assumed to be related to increase in surface area involved in absorption processes (11, 15). Relatively constant structures accompanying microvillated borders are tubular and vesicular invaginations extending into the cellular cytoplasm from the bases of some of the microvilli (15, 16, 19, 24). In eggs, microvilli have been reported in a number of forms, e.g., in sea urchins (1), in annelids (5, 7), amphibia (11), and in rodents (4, 22), and would appear to be a constant feature of oocytes, which may, however, disappear at certain times in development (22) (the oocyte of the enteropneust Saccoglossus may be an exception (5)). In addition, jelly-like layers usually accompany these microvilli, e.g., the zona pellucida material in mammals, and the middle and inner border layers in Hydroides (5-7). The existence of pocketings at the bases of microvilli which might indicate micropinocytosis in eggs has been pointed out by Anderson and Beams (4) in guinea pig oocytes, and the results we have presented above do not contradict these suggestions. It should be carefully pointed out, however, that actual pinocytosis has not been shown in any oocyte system, and the participation of microvilli in absorption not demonstrated (see also 16). In our material, however, the oocytes develop as part of an epithelium and such an absorptive process may play a role in early oogenesis. A micropinocytosis mechanism in ripe eggs may be "left over," as it were, from earlier processes. Tubular invaginations which may be related to micropinocytosis have been seen in cleaving sea urchin eggs (13), where their occurrence and distribution suggests a deep involvement with the process of furrow formation. However, no such structures have as yet been seen in Spisula eggs. Tyler (23) has suggested a process of "specific" pinocytosis as being involved in sperm penetration into sea urchin (and presumably other) eggs. The pinocytosis is postulated to be a reaction of possibly expanded tips of microvilli projecting through the vitelline membrane. The block to polyspermy occurs in part through a subsequent withdrawal of microvilli through the vitellinc membrane, and fertilization membrane elevation consequent on cortical granule breakdown. However these hypotheses may fare for sea urchins, they would appear to be inapplicable to Spisula, since (a) it would be difficult to interpret Fig. 12 as an expanded microvillus, and (b) no evidence exists that the microvilli themselves change in any way during feritlization except, possibly, to increase in length owing to perivitelline-space enlargement. It is clear, therefore, that for Spisula the block to polyspermy involves a delicate, rather than grossly obvious change, in the vitelline membrane, and that pinocytosis, if it exists in these eggs, is probably not tied to sperm penetration, as attractive as such a hypothesis might be.
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1. AFZELIUS,B. A., ExptL Cell Research 10, 257 (1956). 2. ALLEN, R. D., Biol. Bull. 105, 213 (1953). 3. - in MCELROY, W. D. and GLASS, B, (Eds.), The Chemical Basis of Development, p. 17. The Johns Hopkins Press, Baltimore, 1958. 4. ANDERSON,E. and BEAMS, H. W., 9". Ultrastructure Research 3, 432 (1960). 5. COLWlN, A. L., COLWlN, L. H. and P~mVOTT, D. E., d. Biophys. Biochem. CytoL 3, 489 (1957). 6. COLWIN,A. L. and COLWlN, L. H., aT.)3io?hys. Biochem. Cytol. 7, 321 (1960). 7. COLWlN, L. H. and COLWlN,A. L., J. Biophys. Biochem. CytoI. 7, 315 (1960). 8. F~DER, N. and SIDMAN,R. L., J. Biophys. Biochem. Cytol. 4, 593 (1958). 9. FINCK, H., Y. Biophys. Biochem. Cytol. 7, 27 (1960). 10. HARVEY, E. B., The American Arbacia and Other Sea Urchins. Princeton University Press, Princeton, 1956. 11. KEMP, N. E., ,1. Biophys. Biochem. Cytol. 2, 281 (1956). 12. LAWN, A. M., J. Biophys. Biochem. Cytol. 7, 197 (1960). 13. MERCER, E. H. and WOLPERT, L., Exptl. Cell Research 14, 629 (1958). 14. METZ, C. B., in TYLER, A., YON BORSTEL,R. C. and METZ, C. B. (Eds.), The Beginnings of Embryonic Development, p. 23. A.A.A.S., Washington, D. C., 1957. 15. PALAY, S. and KARLIN, L. J., J. Biophys. Biochem. Cytol. 5, 363 (1959). 16. - ibid. 3, 373 (1959). 17 REBrIUN, L. I., J. Biophys. Biochem. Cytol. 9, 785 (1961). 18~ - J. Ultrastructure Research 5, 208 (1961). 19.1RHODIN, J., Intern. Rev. Cytol. 7, 485 (1958). 20. ROTHSCHILD,LORD, Fertilization. Methuen & Co., London, 1956. 21. SCHECHTER, V., Exptl. Cell Research 10, 619 (1956). 22. SOTELO,J. R. and PORTER, K. R., J. Biophys. Biochem. Cytol. 5:327 (1959). 23. TYLER, A., Exptl. Cell Research, Suppl. 7, 183 (1959). 24. YAMADA,E., J. Biophys. Biochem. CytoL 1,445 (1955).