- Email: [email protected]
An electron microscope study of the cortex of the sea urchin (Psammechinus miliaris) egg
Experimental
Cell Research
27, I-13
(1962)
1
AN ELECTRON MICROSCOPE STUDY THE SEA URCHIN (PSAMMECHINUS E. H. MERCER Chester
Beatty
Research
Institute
and L. WOLPERT
and Zoology Received
OF THE CORTEX OF MILIARIS) EGG
Department,
February
King’s
College,
London,
England
20, 19611
IT is commonly accepted that both the unfertilized and fertilized eggs of sea urchins have a gel-like region or cortex just beneath the plasma membrane. The evidence for the presence of a cortex comes from only two sources: (a) centrifugation studies-the cortex being that region beneath the surface from which cytoplasmic particles are not dislodged [lo, 13]-and (6) from micromanipulation studies [2, 71. Various estimates, ranging from 1.G ,U to 5 p have been made of its thickness (see [13]); the most detailed measurements are probably Mitchison’s (1.6 ,u) obtained by centrifugation [13], and Hiramoto’s (3 ,LL)which was obtained by micromanipulation [‘il. It has been argued that the cortex is the main structural component of the cell membrane, and that it thus plays an important role in determining its mechanical properties [lo, 121. It has also been suggested that the cortex is the seat of animal-vegetal polarity of the egg since centrifugation of the egg does not alter its polarity and the cortex is the only region of the egg apparently affected by centrifugation j14, 23, 231. In spite of the importance ascribed to the cortex both for the structural and developmental aspects of the egg almost nothing is known about its structure, though Mitchison has, from birefringence data, postulated that it has a looped protein structure [ 121. Though there have been several studies of the surface of the sea urchin egg with the electron microscope [l, 3, 19, 291 none of these have considered specifically the structure of the cortex, though the cortical granules and changes at fertilization have been described [I, 291. We have therefore reinvestigated this problem. Since the cortex has been mainly defined by a centrifugal operation, our main approach has been to examine centrifuged eggs in order to study that region below the surface from which particles are not displaced. Our work with centrifuged eggs was preceded by a detailed study of the uncentrifuged egg in which much of the findings of Afzelius [l] were confirmed [ll, 291. 1 Revised 1 - 621800
version
received
January
5, 1962. Experimental
Cell Research
27
2
E. H. Mercer MATERIALS
and L. Wolpert AND
METHODS
Eggs of Ps. miliaris were washed twice in sea water and sampleswere fixed by adding a suspensionof eggsto cold (O-5%) buffered (pH 7.4) osmium tetroxide (1 per cent) fixative and allowed to fix for 2 hr. They were then gently centrifuged, the fixative poured off, washed and dehydrated in an ethanol sequence. After standing in absolute ethanol for l-2 hr, the eggswere transferred to xylene for 1 hr before being infiltered with the Araldite resin and polymerized at 60°C [4]. For the centrifuged eggsthe procedure was similar. The eggswere centrifuged at 4400 g for 5 min at the interface of an isotonic sucrose-seawater layer as described by Runnstrom and Kriszat [20]. (We are grateful to Dr. Kriszat for centrifuging our unfertilized eggs.) After centrifugation the eggs were transferred to the fixative within 4 min and processedas above. The stratification was clearly visible in the light microscope at all stagesof the preparation. Fertilized eggswere centrifuged for 10 min at 4400 g at about IO min after fertilization. The fertilization membraneswere not removed. In suitably oriented eggs the stratification into three layers (Fig. 1) was clearly visible in the embedded specimen and sections were cut using conventional techniques, parallel to the long axis of the elongated egg. They were examined in a Siemens Elmiskop I and photographed routinely at x 2300, x 8000 or x 40,000. Many sections were stained using lead salts [24]. Owing to the size of the egg of Psammechinus it was impracticable to photograph in its entirety at higher powers a section passing centrally through the cell. Accordingly a successionof photographs was made, running from one end of the egg to the other along the long axis at right angles to the stratification. Low powered survey photographs of the entire section facilitated the building of a strip mosaic, samplesof which are reproduced as Figs. 2, 3, 4, 5, 6. RESULTS
egg.-The major features of the stratification were readily recognized in electron micrographs when these were compared with light microscope images, and are referred to in Fig. 1 (see also [20]). At a moderate enlargement ( x 30,000) the strip mosaic is some 2 m long and therefore for purposes of illustration we are forced to reproduce only four selected areas (Figs. 2, 3, 4) which are, however, adequate to show the composition of the major layers. It is evident that two main displacements have occurred during centrifugation: the mitochondria have moved towards and collected at the heavy pole (Fig. 3), the yolk granules and nucleus have moved and collected at the light pole (Fig. 2). The separation is fairly complete (only very occasional yolk granules or mitochondria are out of place) and leaves centrally within the egg a broad band (clear in the light microscope) which contains a populaCentrifuged
Experimental
unfertilized
Cell
Research
27
Cortex of the sea urchin egg
3
tion of small particulates not greatly influenced by the centrifugation (Fig. 4). These same small particulates (dense particles either free or attached to membranes and small empty vesicles [O.l ,u - 0.2 ,LL]) are to be seen to a lesser degree between the mitochondria and between the more closely packed yolk granules at the light pole. It would seem that they and the soluble proteins etc. remain unmoved to form a continuous “ground substance” more or less diluted by the presence of the larger mobile particulate.
Fig. l.-Location diagram of sections illustrating structure of unfertilized centrifuged egg. The sections is parallel to the direction of centrifugation; the “heavy pole” is at the lower part of the section. IV, nucleus; Y yolk granules; G, cortical granules; m, mitochondria; P, dense ribosomes; T’, small vesicles; R, reticulum. The distribution in the fertilized egg (not shown) is similar but the cortical granules are absent.
It seems reasonable to identify the masses of small dense particles (P), partly free and partly associated with the small vesicles (V) and flattened sacs (II), with RNA containing material, as has been demonstrated in certain mammalian cells [17], and as is rendered probable by both Pasteels et al. [18] and our own investigation on the microsomes of sea urchin eggs (unpublished). The most significant feature at the surface of the unfertilized egg is the layer of cortical granules whose appearance has been described by Afzelius [l]. These granules form an almost continuous layer around the egg about 1 ,D thick and beneath them, in the normal unfertilized egg, the composition of the cytoplasm, in terms of formed elements, appears to be homogeneous. The cortical granules are the only large cytoplasmic inclusions not displaced during centrifugation; the other large inclusions-yolk granules and mitochondria-being freely displaced from all regions including those immediately adjacent to the layer of cortical granules. At the centripetal and centrifugal poles these are driven right against and even between the cortical granules (Figs 2, 3). There was no evidence for a gel layer not permitting free movement of these cytoplasmic particles or preventing them packing hard against the cortical granules. Experimentul
Cell
Research
27
4
E. H. Merccr and L. Wolpert
Fig. 2.--Section at the “light pole” of the centrifuged unfertilized membrane; H, nucleolar “cap”; Y, yolk granules; R, entrapped and M, outer membrane. Note the close packing of yolk granules
egg. N, nucleus; SM, nuclear reticulum; G, cortical granule; right up to the cortical granules.
Centrifuged fertilized egg.-The results from the fertilized egg are substanitally the same as those described for the unfertilized egg with two main differences-the cortical granules have now opened up and a more obvious oil cap forms at the light end. The opening up of the cortical granules, as has been described elsewhere Experimental
Cell Research
27
5
Cortex of the sea urchin egg
Fig. 3.--Section at the heavy P, dense ribosomal material; cortical granules.
end of the centrifuged fertilized egg. m, mitochondria; I’, yolk granule (a rare instance of an undisplaced
V, vesicles; granule); G,
[29], results in highly convoluted surfaces. At both light and heavy poles there again appears to be no barrier to the displacement of the mitochondria and yolk granules. For example, at the light pole the yolk granules are driven to within 0.1 ,u of the plasma membrane (Fig. 5). Again, examination of the lateral surfaces of the elongated egg in the region of the clear equatorial layer shows no sign that a gelled region had prevented free displacement of cytoplasmic particles (Fig. 6). A possible exception to this is the appearance of a greater number of vesicles, about 0.2-0.5 ,u in diameter, adjacent to the surface, which are probably remnants of cortical granules [29]. Experimental
Cell Research
27
E. H. Mercer and L. Wolpert
Fig. 4.--Section within the central hyaline band of the centrifuged as for other figures. L, lamellae of smooth membranes (Golgi type).
unfertilised
egg.
Lettering
A common feature of the fertilized egg w-ere yolk granules (Fig. .5 at 0). apparently in the act of opening. Further, in neither centrifuged nor normal fertilized eggs, even after staining with phosphotungstate or lead salts, have we ever observed structures (tibrils, dense areas, etc.) which could be interpreted as components of a gelled layer. However, at cleavage a new dense layer 0.1 ,U thick, already described by us [ll], does appear just beneath the surface. Experimental
Cell Research
27
7
Cortex of the sea urchin egg
Fig. J.-Light pole of fertilized egg. Note absence of the cortical membrane, P the plasma membrane. Yolk granules (Y) are driven indicating the absence of a cortical layer. Note opening up of yolk
granules. F is the fertilization close to the plasma membrane granule at 0. (Lead stained). Experimental
Cell Research
27
8
E. Ii. Mercer and L. Wolpert
Fig. 6.-The appearance near the plasma membrane (M) in the central part of the centrifuged fertilized egg in the hyaline band. Note that almost all of the formed granules (mitochondria, yolk and larger vesicles) have been moved from the neighbourhood of the membrane thus proving the absence of a coherent “gelled” region. (Lead stained). Experimental
Cell Research
27
Cortex of the sea urchin
9
egg
DISCUSSION
The most striking features of the above observations are the lack of evidence for a gel-like cortical region in Psammechinus and the fact that, while centrifugation at the speeds employed shifts the larger cptoplasmic inclusions, there is a large mass of smaller particulates which are apparently unaffected. Since in the unfertilized egg the cortical granules are not shifted by centrifugation, this might be interpreted as being evidence for their being held in position by a gel. However, closer examination of their relation to the surface shows that their membrane is fused with the plasma membrane of the egg [29]. In fact, we have suggested on the basis of changes occurring at fertilization that their membrane is identical \vith the plasma membrane and that in effect the cortical granules are enclosed within a pocket of this membrane. 1Ve would thus suggest that the fact that they are not shifted by ccntrifugation is due to their direct attachment to the membrane bounding the egg. Relow the cortical granules we could detect no region in lvhich movement of the larger particulates was prevented. The layer of cortical granules, which in Psammechinrzs corresponds to a layer about 1 ,LLthick probably corresponds to the cortex observed by other workers on the unfertilized eggs of other species. However, it seems to us that this is not evidence for a gel layer. Gross et nl. [5] in their study of the centrifuged unfertilized egg of Arbacia also observed that the cortical granules were not dislodged. They bring no further evidence to support the idea of a gel-like layer and do not discuss this question. In the fertilized egg from which the cortical granules have disappeared all the remaining large particles are displaced. Here there is no evidence at all of a gelled layer restricting movement. These observations are supported by time-lapse cinematography studies of the surface of unfertilized and fertilized Psammechinrrs eggs. Such studies show considerable movement of cytoplasmic particulates even in the region immediately beneath the external membrane; only the cortical granules in unfertilized eggs appeared fixed and stationary [ZS]. Recently, Sakai [21] has reported the isolation of the “cortical membrane” from both unfertilized and fertilized sea urchin eggs but gives no details as to the structure of the isolated membrane. In the isolated membrane of Amoeba proteus, to which a cortex is usually ascribed (cf. jl2]) there is no evidence for a cortex when these are examined in the light or electron microscope [16]. Experimental
Cell Research
27
E. H. Alercer and L. Wolpert \Ve must now ask horn our results may he reconciled with those reporting a cortical gel? The possibility of species differences must be mentioned. Different species behave very differently when centrifuged [6, $I’, and there is good light microscopic evidence that in the heavily pigmented Arbrrcirr something does prevent movement of larger cgtoplasmic particles [ 10, 13 1. Also, in the unfertilized egg of Pnrucentrotus there is a band of pigment not alected by centrifugation [II], and in electron micrographs of the uncentrifuged fertilized egg of SfrongyZocenfrofrzs it is clear that there is a zone immediately below the membrane characterized by various vacuoles and from which yolk granules and mitochondria are excluded (Mazia, private communication). In contrast, a cortical gel has not in fact been described in Psanzmechir~zzs milirzris, the species used here. Thus while not denying the evidence brought forhvard to sho\v the presence of a cortical gel in other species, \ve would suggest on the basis of our results that such a zone is not always and invariably present, and that even where detectable it does not prove that the layer is a gel. For example, the micromanipulation studies of cortex thickness of Hiramoto [7] must be interpreted with caution. He used a needle 2 ,U thick at its tip, and observed how close it \vas necessary to bring it to the surface from the inside before a displacement of the surface externally could be observed, and obtained values of 2-3 ,U for the thickness. However, since the egg is packed \vith cytoplasmic inclusions about 1 ,U in diameter, the packing of such particles between needle and membrane could give the impression that such a layer was present. There is also no reason to believe that the presence of a specialized region beneath the surface from lvhich some constituents are excluded, and others cannot be displaced by centrifugation, indicates the presence of a gel. Particles may be held at the surface by quite different mechanisms. In this connection hlazia’s (private communication) comments are of great iniportance. He points out that the classical conception of a intracellular gel is one of a network of linear elements tending to trap particles. However the aster and spindle which are certainly gel-like not only fail to trap particles but actually exclude all but the smallest. In this case the gel is formed from a compact mass of membrane bounded components. Thus we cannot decide a priori whether a cortical gel would trap or exclude particles. In the case of Psammechinus it seems in fact to do neither. The fact that we can detect no cortical gel in this particular species, which has a normal developmental pattern, must raise doubts as to whether a “cortex” can play the role as regards structure and polarity ascribed to it in other species. For example, the birefringence changes of the surface from Experimental
Cell Research
27
Cortex of the sea urchin
11
egg
fertilization through cleavage, which hare been considered by Rlitchison are now very hard to interpret in [la] in terms of changes in the cortex, view of the very different structures revealed at the surface of unfertilized, [22] hare suggested fertilized and cleaving eggs [ 11 , 291. S\vann and hlitchison that mechanically, the sea urchin egg behaves as a thick walled sphere and that this supports the idea that the cortex is the main structural component of the membrane. This conclusion has been criticisrd, and attention drawn to the possibility that the plasma membrane itself may be the main structural component [26, 251. The location of the animal-vegetal gradient in the cortex of the egg comes from the observation that centrifugation does not affect cell polarity LS, 14, 151 and the postulated cortex appeared to be the only region unaffected by centrifugation [l-1, 23, 231. ‘This argument is largely negative, and in fact rests on the supposition that the cytoplasm through \vhich the larger particles are displaced would be so disturbed as to be unable to be the seat of the gradient (cf. [23, 251). If the cortex is not the scat of polarity several other possibilities for the seat may be re-examined, e.g. the cortical granules, the plasma membrane, and the cptoplasmic contents not displaced by centrifugation. The possibility that the polarity could be located in the cortical granules seems to be excluded by the fact that they open up and extrude their contents to form the fertilization membrane and hyaline layer, at fertilisation. ‘That the plasma membrane could be the seat of polarity has been proposed by Elbers [3]. He examined eggs after treatment \vith lithium (which alters the animal-regetal gradient) and could find no diflerence in their structure as shown in electron microscope. He thus concluded that the lithium did not penetrate the egg and that the plasma membrane therefore must be the seat of the gradient, since it alone n-as in contact \vith the lithium. This argument seems rcry unreliable since, among other reasons, there is no reason whatsoever to believe that lithium would effect the egg in a manner that would be detected by the electron microscope. Another possibility is that the polarity is somehow maintained by the small particulate fractions of the cytoplasm which are not shifted at the small centrifugal forces used. For, while the movement of the large particles through the masses of small particulates must certainly disturb their arrangement, we have no idea of their capacity for regulation, nor of how great the differences between various regions must be to provide the basis for polarity. At the moment there is no good evidence which allows us to choose between these various possibilities, and we wish to stress that the problem Experimental
Cell Research
27
E. H. Mercer and L. Wolpert of the cortex in relation to both the mechanical properties of the membrane and to the seat of the egg’s polarity is equivocal, and requires further investigation. SUMMARY A gel-like cortex beneath the surface of the egg has been supposed to be both the main structural component of the membrane and the seat of the animalvegetal gradient of the egg. This layer has been detected by earlier workers in other species by centrifugation and micro-manipulation. ,4n examination in the electron microscope of both normal and centrifuged unfertilized and fertilized eggs has failed to detect any evidence for such a layer. In the unfertilized egg yolk granules and mitochondria are freely displaced to the light and heavy poles of the egg right up to the layer of cortical granules. The cortical granules are not shifted and it is suggested that this is due to their attachment to the cell membrane. Again in centrifuged fertilized eggs yolk granules and mitochondria are displaced right up to the cell surface, there being no layer, now that the cortical granules have opened up, n-hich limits their displacement. The small particulate cytoplasmic fraction appears to 1~ relatively unaffected. The mechanical properties of the membrane and the seat of animal-vegetal polarity in relation to the cortex are discussed in the light of these findings. We are most grateful to Professor I). Mazia for his comments on our manuscript. One of us (L.W.) is indebted to the Royal Society for a grant from the Browne Research Fund and to the Director and Staff of the Marine Zoological Station, Kristineberg, for providing facilities. The photographic enlargements were very kindly made by Mr. Michael Docherty and many of the sections were cut by Mrs. S. Roberts. The investigation was in part supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council, the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund, and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. REFERENCES 1. 2. 3. 4. 5. 6.
AFZELIUS, B., Expfl. Cd Research 10, 257 (1956). CHAMBERS, R., Am. .I. Physiof. 43, 1 (1917). ELBERS, P. F., Thesis. Utrecht, 1959. GLAUERT, M. M., ROGERS, G. I. and GALUERT, GROSS, P. R., PHILPOTT, D. I. and NASS, S., J. HARVEY, E. B., The American Arbacia and other ton, 1956. 7. HIRAMOTO, Y., Embryofogia 3, 361 (1957). 8. H~RSTADIUS, S., Pubbf. Staz. Zool. Napofi 24, 1 Experimental
Cell Research
27
R. H., Nature 178, 803 (1956). Biophys. Biochem. Cytof. 7, 135 (1960). sea urchins. Princeton Univ. Press, Prince(1953).
Cortex of the sea urchin egg 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
13
IMMERS, J., Exptl. Cell Research 19, 499 (1960). MARSLAND, D. A. and LANDAU, J. V., J. Exptt. Zool. 125, 507 (1954). MERGER, E. H. and WOLPERT, L., Eqfl. Cell Research 14,632 (1958). MITCHISON, J. M., Symposia Sot. Expfl. Biol. 6, 105 (1952). __ Quart. J. Microscop. Sci. 97, 109 (1956). MOTOMURA, I. A., Tohuku Sci. Rep. IV, Ser. 10, 211 (1935). MORGAN, T. H. and SPOONER, G. B., Arch. Entwicklungsmech. Organ. 28, 101 (1909). O’NEILL, C. H. and WOLPERT, L., Exptl. Cell. Research 24, 592 (1961). PALADE, G. E. and SIEKEVITZ, P., J. Biophys. Biochem. Cytol. 2, 171 (1956). PASTEELS, .J. J., CASTIAUX, M. D. and VANDERYGERSSCIIE, G., .J. Biophys. Biochem. Cytot. 575 (1958). ROTHSCHILD, LORD, Quart. J. Microscop. Sci. 99, 1 (1958). RUNNSTRBM, J. and KRISZAT, G., Exptl. Celt Research 1, 286 (1950). SAKAI, H., J. Biophys. Biochem. Cytot. 8, 609 (1960). SWANN, M. M. and MITCHBOX, J. M., Biot. Revs. 33, 103 (1958). WADDISGTON, C. H., Principles of Embryology. Allen & Unwin, London, 1956. WATSON, ibf. L., J. Biophys. Biochem. Cytot. 4, 727 (1958). WEISS, P., Principles of Development. Holt, New York, 1939. WOLPERT, L., Roy. Phys. Sot. Edinburgh 28, 107 (1960). WOLPERT, L., Intern. Reu. Cytol. 10, 164 (1960). WOLPERT, L. and GUSTAFSOX, T., In preparation. WOLPERT, L. and MERCER, E. H., Exptt. Cell Research 22, 45 (1961).
Experimental
Cell
Research
4,
27
Cell Research
27, I-13
(1962)
1
AN ELECTRON MICROSCOPE STUDY THE SEA URCHIN (PSAMMECHINUS E. H. MERCER Chester
Beatty
Research
Institute
and L. WOLPERT
and Zoology Received
OF THE CORTEX OF MILIARIS) EGG
Department,
February
King’s
College,
London,
England
20, 19611
IT is commonly accepted that both the unfertilized and fertilized eggs of sea urchins have a gel-like region or cortex just beneath the plasma membrane. The evidence for the presence of a cortex comes from only two sources: (a) centrifugation studies-the cortex being that region beneath the surface from which cytoplasmic particles are not dislodged [lo, 13]-and (6) from micromanipulation studies [2, 71. Various estimates, ranging from 1.G ,U to 5 p have been made of its thickness (see [13]); the most detailed measurements are probably Mitchison’s (1.6 ,u) obtained by centrifugation [13], and Hiramoto’s (3 ,LL)which was obtained by micromanipulation [‘il. It has been argued that the cortex is the main structural component of the cell membrane, and that it thus plays an important role in determining its mechanical properties [lo, 121. It has also been suggested that the cortex is the seat of animal-vegetal polarity of the egg since centrifugation of the egg does not alter its polarity and the cortex is the only region of the egg apparently affected by centrifugation j14, 23, 231. In spite of the importance ascribed to the cortex both for the structural and developmental aspects of the egg almost nothing is known about its structure, though Mitchison has, from birefringence data, postulated that it has a looped protein structure [ 121. Though there have been several studies of the surface of the sea urchin egg with the electron microscope [l, 3, 19, 291 none of these have considered specifically the structure of the cortex, though the cortical granules and changes at fertilization have been described [I, 291. We have therefore reinvestigated this problem. Since the cortex has been mainly defined by a centrifugal operation, our main approach has been to examine centrifuged eggs in order to study that region below the surface from which particles are not displaced. Our work with centrifuged eggs was preceded by a detailed study of the uncentrifuged egg in which much of the findings of Afzelius [l] were confirmed [ll, 291. 1 Revised 1 - 621800
version
received
January
5, 1962. Experimental
Cell Research
27
2
E. H. Mercer MATERIALS
and L. Wolpert AND
METHODS
Eggs of Ps. miliaris were washed twice in sea water and sampleswere fixed by adding a suspensionof eggsto cold (O-5%) buffered (pH 7.4) osmium tetroxide (1 per cent) fixative and allowed to fix for 2 hr. They were then gently centrifuged, the fixative poured off, washed and dehydrated in an ethanol sequence. After standing in absolute ethanol for l-2 hr, the eggswere transferred to xylene for 1 hr before being infiltered with the Araldite resin and polymerized at 60°C [4]. For the centrifuged eggsthe procedure was similar. The eggswere centrifuged at 4400 g for 5 min at the interface of an isotonic sucrose-seawater layer as described by Runnstrom and Kriszat [20]. (We are grateful to Dr. Kriszat for centrifuging our unfertilized eggs.) After centrifugation the eggs were transferred to the fixative within 4 min and processedas above. The stratification was clearly visible in the light microscope at all stagesof the preparation. Fertilized eggswere centrifuged for 10 min at 4400 g at about IO min after fertilization. The fertilization membraneswere not removed. In suitably oriented eggs the stratification into three layers (Fig. 1) was clearly visible in the embedded specimen and sections were cut using conventional techniques, parallel to the long axis of the elongated egg. They were examined in a Siemens Elmiskop I and photographed routinely at x 2300, x 8000 or x 40,000. Many sections were stained using lead salts [24]. Owing to the size of the egg of Psammechinus it was impracticable to photograph in its entirety at higher powers a section passing centrally through the cell. Accordingly a successionof photographs was made, running from one end of the egg to the other along the long axis at right angles to the stratification. Low powered survey photographs of the entire section facilitated the building of a strip mosaic, samplesof which are reproduced as Figs. 2, 3, 4, 5, 6. RESULTS
egg.-The major features of the stratification were readily recognized in electron micrographs when these were compared with light microscope images, and are referred to in Fig. 1 (see also [20]). At a moderate enlargement ( x 30,000) the strip mosaic is some 2 m long and therefore for purposes of illustration we are forced to reproduce only four selected areas (Figs. 2, 3, 4) which are, however, adequate to show the composition of the major layers. It is evident that two main displacements have occurred during centrifugation: the mitochondria have moved towards and collected at the heavy pole (Fig. 3), the yolk granules and nucleus have moved and collected at the light pole (Fig. 2). The separation is fairly complete (only very occasional yolk granules or mitochondria are out of place) and leaves centrally within the egg a broad band (clear in the light microscope) which contains a populaCentrifuged
Experimental
unfertilized
Cell
Research
27
Cortex of the sea urchin egg
3
tion of small particulates not greatly influenced by the centrifugation (Fig. 4). These same small particulates (dense particles either free or attached to membranes and small empty vesicles [O.l ,u - 0.2 ,LL]) are to be seen to a lesser degree between the mitochondria and between the more closely packed yolk granules at the light pole. It would seem that they and the soluble proteins etc. remain unmoved to form a continuous “ground substance” more or less diluted by the presence of the larger mobile particulate.
Fig. l.-Location diagram of sections illustrating structure of unfertilized centrifuged egg. The sections is parallel to the direction of centrifugation; the “heavy pole” is at the lower part of the section. IV, nucleus; Y yolk granules; G, cortical granules; m, mitochondria; P, dense ribosomes; T’, small vesicles; R, reticulum. The distribution in the fertilized egg (not shown) is similar but the cortical granules are absent.
It seems reasonable to identify the masses of small dense particles (P), partly free and partly associated with the small vesicles (V) and flattened sacs (II), with RNA containing material, as has been demonstrated in certain mammalian cells [17], and as is rendered probable by both Pasteels et al. [18] and our own investigation on the microsomes of sea urchin eggs (unpublished). The most significant feature at the surface of the unfertilized egg is the layer of cortical granules whose appearance has been described by Afzelius [l]. These granules form an almost continuous layer around the egg about 1 ,D thick and beneath them, in the normal unfertilized egg, the composition of the cytoplasm, in terms of formed elements, appears to be homogeneous. The cortical granules are the only large cytoplasmic inclusions not displaced during centrifugation; the other large inclusions-yolk granules and mitochondria-being freely displaced from all regions including those immediately adjacent to the layer of cortical granules. At the centripetal and centrifugal poles these are driven right against and even between the cortical granules (Figs 2, 3). There was no evidence for a gel layer not permitting free movement of these cytoplasmic particles or preventing them packing hard against the cortical granules. Experimentul
Cell
Research
27
4
E. H. Merccr and L. Wolpert
Fig. 2.--Section at the “light pole” of the centrifuged unfertilized membrane; H, nucleolar “cap”; Y, yolk granules; R, entrapped and M, outer membrane. Note the close packing of yolk granules
egg. N, nucleus; SM, nuclear reticulum; G, cortical granule; right up to the cortical granules.
Centrifuged fertilized egg.-The results from the fertilized egg are substanitally the same as those described for the unfertilized egg with two main differences-the cortical granules have now opened up and a more obvious oil cap forms at the light end. The opening up of the cortical granules, as has been described elsewhere Experimental
Cell Research
27
5
Cortex of the sea urchin egg
Fig. 3.--Section at the heavy P, dense ribosomal material; cortical granules.
end of the centrifuged fertilized egg. m, mitochondria; I’, yolk granule (a rare instance of an undisplaced
V, vesicles; granule); G,
[29], results in highly convoluted surfaces. At both light and heavy poles there again appears to be no barrier to the displacement of the mitochondria and yolk granules. For example, at the light pole the yolk granules are driven to within 0.1 ,u of the plasma membrane (Fig. 5). Again, examination of the lateral surfaces of the elongated egg in the region of the clear equatorial layer shows no sign that a gelled region had prevented free displacement of cytoplasmic particles (Fig. 6). A possible exception to this is the appearance of a greater number of vesicles, about 0.2-0.5 ,u in diameter, adjacent to the surface, which are probably remnants of cortical granules [29]. Experimental
Cell Research
27
E. H. Mercer and L. Wolpert
Fig. 4.--Section within the central hyaline band of the centrifuged as for other figures. L, lamellae of smooth membranes (Golgi type).
unfertilised
egg.
Lettering
A common feature of the fertilized egg w-ere yolk granules (Fig. .5 at 0). apparently in the act of opening. Further, in neither centrifuged nor normal fertilized eggs, even after staining with phosphotungstate or lead salts, have we ever observed structures (tibrils, dense areas, etc.) which could be interpreted as components of a gelled layer. However, at cleavage a new dense layer 0.1 ,U thick, already described by us [ll], does appear just beneath the surface. Experimental
Cell Research
27
7
Cortex of the sea urchin egg
Fig. J.-Light pole of fertilized egg. Note absence of the cortical membrane, P the plasma membrane. Yolk granules (Y) are driven indicating the absence of a cortical layer. Note opening up of yolk
granules. F is the fertilization close to the plasma membrane granule at 0. (Lead stained). Experimental
Cell Research
27
8
E. Ii. Mercer and L. Wolpert
Fig. 6.-The appearance near the plasma membrane (M) in the central part of the centrifuged fertilized egg in the hyaline band. Note that almost all of the formed granules (mitochondria, yolk and larger vesicles) have been moved from the neighbourhood of the membrane thus proving the absence of a coherent “gelled” region. (Lead stained). Experimental
Cell Research
27
Cortex of the sea urchin
9
egg
DISCUSSION
The most striking features of the above observations are the lack of evidence for a gel-like cortical region in Psammechinus and the fact that, while centrifugation at the speeds employed shifts the larger cptoplasmic inclusions, there is a large mass of smaller particulates which are apparently unaffected. Since in the unfertilized egg the cortical granules are not shifted by centrifugation, this might be interpreted as being evidence for their being held in position by a gel. However, closer examination of their relation to the surface shows that their membrane is fused with the plasma membrane of the egg [29]. In fact, we have suggested on the basis of changes occurring at fertilization that their membrane is identical \vith the plasma membrane and that in effect the cortical granules are enclosed within a pocket of this membrane. 1Ve would thus suggest that the fact that they are not shifted by ccntrifugation is due to their direct attachment to the membrane bounding the egg. Relow the cortical granules we could detect no region in lvhich movement of the larger particulates was prevented. The layer of cortical granules, which in Psammechinrzs corresponds to a layer about 1 ,LLthick probably corresponds to the cortex observed by other workers on the unfertilized eggs of other species. However, it seems to us that this is not evidence for a gel layer. Gross et nl. [5] in their study of the centrifuged unfertilized egg of Arbacia also observed that the cortical granules were not dislodged. They bring no further evidence to support the idea of a gel-like layer and do not discuss this question. In the fertilized egg from which the cortical granules have disappeared all the remaining large particles are displaced. Here there is no evidence at all of a gelled layer restricting movement. These observations are supported by time-lapse cinematography studies of the surface of unfertilized and fertilized Psammechinrrs eggs. Such studies show considerable movement of cytoplasmic particulates even in the region immediately beneath the external membrane; only the cortical granules in unfertilized eggs appeared fixed and stationary [ZS]. Recently, Sakai [21] has reported the isolation of the “cortical membrane” from both unfertilized and fertilized sea urchin eggs but gives no details as to the structure of the isolated membrane. In the isolated membrane of Amoeba proteus, to which a cortex is usually ascribed (cf. jl2]) there is no evidence for a cortex when these are examined in the light or electron microscope [16]. Experimental
Cell Research
27
E. H. Alercer and L. Wolpert \Ve must now ask horn our results may he reconciled with those reporting a cortical gel? The possibility of species differences must be mentioned. Different species behave very differently when centrifuged [6, $I’, and there is good light microscopic evidence that in the heavily pigmented Arbrrcirr something does prevent movement of larger cgtoplasmic particles [ 10, 13 1. Also, in the unfertilized egg of Pnrucentrotus there is a band of pigment not alected by centrifugation [II], and in electron micrographs of the uncentrifuged fertilized egg of SfrongyZocenfrofrzs it is clear that there is a zone immediately below the membrane characterized by various vacuoles and from which yolk granules and mitochondria are excluded (Mazia, private communication). In contrast, a cortical gel has not in fact been described in Psanzmechir~zzs milirzris, the species used here. Thus while not denying the evidence brought forhvard to sho\v the presence of a cortical gel in other species, \ve would suggest on the basis of our results that such a zone is not always and invariably present, and that even where detectable it does not prove that the layer is a gel. For example, the micromanipulation studies of cortex thickness of Hiramoto [7] must be interpreted with caution. He used a needle 2 ,U thick at its tip, and observed how close it \vas necessary to bring it to the surface from the inside before a displacement of the surface externally could be observed, and obtained values of 2-3 ,U for the thickness. However, since the egg is packed \vith cytoplasmic inclusions about 1 ,U in diameter, the packing of such particles between needle and membrane could give the impression that such a layer was present. There is also no reason to believe that the presence of a specialized region beneath the surface from lvhich some constituents are excluded, and others cannot be displaced by centrifugation, indicates the presence of a gel. Particles may be held at the surface by quite different mechanisms. In this connection hlazia’s (private communication) comments are of great iniportance. He points out that the classical conception of a intracellular gel is one of a network of linear elements tending to trap particles. However the aster and spindle which are certainly gel-like not only fail to trap particles but actually exclude all but the smallest. In this case the gel is formed from a compact mass of membrane bounded components. Thus we cannot decide a priori whether a cortical gel would trap or exclude particles. In the case of Psammechinus it seems in fact to do neither. The fact that we can detect no cortical gel in this particular species, which has a normal developmental pattern, must raise doubts as to whether a “cortex” can play the role as regards structure and polarity ascribed to it in other species. For example, the birefringence changes of the surface from Experimental
Cell Research
27
Cortex of the sea urchin
11
egg
fertilization through cleavage, which hare been considered by Rlitchison are now very hard to interpret in [la] in terms of changes in the cortex, view of the very different structures revealed at the surface of unfertilized, [22] hare suggested fertilized and cleaving eggs [ 11 , 291. S\vann and hlitchison that mechanically, the sea urchin egg behaves as a thick walled sphere and that this supports the idea that the cortex is the main structural component of the membrane. This conclusion has been criticisrd, and attention drawn to the possibility that the plasma membrane itself may be the main structural component [26, 251. The location of the animal-vegetal gradient in the cortex of the egg comes from the observation that centrifugation does not affect cell polarity LS, 14, 151 and the postulated cortex appeared to be the only region unaffected by centrifugation [l-1, 23, 231. ‘This argument is largely negative, and in fact rests on the supposition that the cytoplasm through \vhich the larger particles are displaced would be so disturbed as to be unable to be the seat of the gradient (cf. [23, 251). If the cortex is not the scat of polarity several other possibilities for the seat may be re-examined, e.g. the cortical granules, the plasma membrane, and the cptoplasmic contents not displaced by centrifugation. The possibility that the polarity could be located in the cortical granules seems to be excluded by the fact that they open up and extrude their contents to form the fertilization membrane and hyaline layer, at fertilisation. ‘That the plasma membrane could be the seat of polarity has been proposed by Elbers [3]. He examined eggs after treatment \vith lithium (which alters the animal-regetal gradient) and could find no diflerence in their structure as shown in electron microscope. He thus concluded that the lithium did not penetrate the egg and that the plasma membrane therefore must be the seat of the gradient, since it alone n-as in contact \vith the lithium. This argument seems rcry unreliable since, among other reasons, there is no reason whatsoever to believe that lithium would effect the egg in a manner that would be detected by the electron microscope. Another possibility is that the polarity is somehow maintained by the small particulate fractions of the cytoplasm which are not shifted at the small centrifugal forces used. For, while the movement of the large particles through the masses of small particulates must certainly disturb their arrangement, we have no idea of their capacity for regulation, nor of how great the differences between various regions must be to provide the basis for polarity. At the moment there is no good evidence which allows us to choose between these various possibilities, and we wish to stress that the problem Experimental
Cell Research
27
E. H. Mercer and L. Wolpert of the cortex in relation to both the mechanical properties of the membrane and to the seat of the egg’s polarity is equivocal, and requires further investigation. SUMMARY A gel-like cortex beneath the surface of the egg has been supposed to be both the main structural component of the membrane and the seat of the animalvegetal gradient of the egg. This layer has been detected by earlier workers in other species by centrifugation and micro-manipulation. ,4n examination in the electron microscope of both normal and centrifuged unfertilized and fertilized eggs has failed to detect any evidence for such a layer. In the unfertilized egg yolk granules and mitochondria are freely displaced to the light and heavy poles of the egg right up to the layer of cortical granules. The cortical granules are not shifted and it is suggested that this is due to their attachment to the cell membrane. Again in centrifuged fertilized eggs yolk granules and mitochondria are displaced right up to the cell surface, there being no layer, now that the cortical granules have opened up, n-hich limits their displacement. The small particulate cytoplasmic fraction appears to 1~ relatively unaffected. The mechanical properties of the membrane and the seat of animal-vegetal polarity in relation to the cortex are discussed in the light of these findings. We are most grateful to Professor I). Mazia for his comments on our manuscript. One of us (L.W.) is indebted to the Royal Society for a grant from the Browne Research Fund and to the Director and Staff of the Marine Zoological Station, Kristineberg, for providing facilities. The photographic enlargements were very kindly made by Mr. Michael Docherty and many of the sections were cut by Mrs. S. Roberts. The investigation was in part supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council, the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund, and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. REFERENCES 1. 2. 3. 4. 5. 6.
AFZELIUS, B., Expfl. Cd Research 10, 257 (1956). CHAMBERS, R., Am. .I. Physiof. 43, 1 (1917). ELBERS, P. F., Thesis. Utrecht, 1959. GLAUERT, M. M., ROGERS, G. I. and GALUERT, GROSS, P. R., PHILPOTT, D. I. and NASS, S., J. HARVEY, E. B., The American Arbacia and other ton, 1956. 7. HIRAMOTO, Y., Embryofogia 3, 361 (1957). 8. H~RSTADIUS, S., Pubbf. Staz. Zool. Napofi 24, 1 Experimental
Cell Research
27
R. H., Nature 178, 803 (1956). Biophys. Biochem. Cytof. 7, 135 (1960). sea urchins. Princeton Univ. Press, Prince(1953).
Cortex of the sea urchin egg 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
13
IMMERS, J., Exptl. Cell Research 19, 499 (1960). MARSLAND, D. A. and LANDAU, J. V., J. Exptt. Zool. 125, 507 (1954). MERGER, E. H. and WOLPERT, L., Eqfl. Cell Research 14,632 (1958). MITCHISON, J. M., Symposia Sot. Expfl. Biol. 6, 105 (1952). __ Quart. J. Microscop. Sci. 97, 109 (1956). MOTOMURA, I. A., Tohuku Sci. Rep. IV, Ser. 10, 211 (1935). MORGAN, T. H. and SPOONER, G. B., Arch. Entwicklungsmech. Organ. 28, 101 (1909). O’NEILL, C. H. and WOLPERT, L., Exptl. Cell. Research 24, 592 (1961). PALADE, G. E. and SIEKEVITZ, P., J. Biophys. Biochem. Cytol. 2, 171 (1956). PASTEELS, .J. J., CASTIAUX, M. D. and VANDERYGERSSCIIE, G., .J. Biophys. Biochem. Cytot. 575 (1958). ROTHSCHILD, LORD, Quart. J. Microscop. Sci. 99, 1 (1958). RUNNSTRBM, J. and KRISZAT, G., Exptl. Celt Research 1, 286 (1950). SAKAI, H., J. Biophys. Biochem. Cytot. 8, 609 (1960). SWANN, M. M. and MITCHBOX, J. M., Biot. Revs. 33, 103 (1958). WADDISGTON, C. H., Principles of Embryology. Allen & Unwin, London, 1956. WATSON, ibf. L., J. Biophys. Biochem. Cytot. 4, 727 (1958). WEISS, P., Principles of Development. Holt, New York, 1939. WOLPERT, L., Roy. Phys. Sot. Edinburgh 28, 107 (1960). WOLPERT, L., Intern. Reu. Cytol. 10, 164 (1960). WOLPERT, L. and GUSTAFSOX, T., In preparation. WOLPERT, L. and MERCER, E. H., Exptt. Cell Research 22, 45 (1961).
Experimental
Cell
Research
4,
27