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Structural changes following fertilization in the sea urchin egg
Q 1967 by Academic
Experimental
Press Inc.
569
Cell Research 48, 569-581 (1967)
STRUCTURAL
FORMATION
CHANGES FOLLOWING FERTILIZATION IN THE SEA URCHIN EGG AND
DISSOLUTION PATRICIA
OF
HEAVY
BODIES’
HARRIS
Department of Zoology, Oregon State University, Corvallis, Ore. 97331, and Oregon State University Marine Science Center, Newport, Ore., USA Received
February
20, 19672
THE activation of synthetic processes in sea urchin eggs following fertilization, and the means whereby these processes are held in abeyance in the unfertilized egg, have been the subject of a large number of recent biochemical studies. This work has been reviewed in some detail [3, 17, 22, 231. The conclusions which can be drawn from the data at hand can be stated rather succinctly: there is little or no active protein synthesis in the unfertilized egg; protein synthesis is initiated very shortly after the fertilization reaction: no usable messenger RNA, which is necessary for protein synthesis, is produced until much later in the blastula stage; the existing messenger RNA in these cells is of maternal origin, but present in an unusable or “masked” form until fertilization. It is of some importance at this time to examine at the fine structural level the cytoplasmic changes which occur at fertilization and to combine the techniques of biochemistry and electron microscopy in an attempt to answer the following questions: is there really an inactive messenger RNA; if so, how is it packaged, and how is it released? The most likely candidate for such a role in the cytoplasm are the socalled “heavy bodies”, first named and described by Afzelius [l] in unfertilized eggs of several species of European sea urchins. These bodies are compact balls of tightly packed granules of ribosomal size and appearance. They are bounded on several sides, though not completely enclosed, by annulate lamellae having the same structure as the nuclear membrane, and were demonstrated by Afzelius to contain RNA. They are distributed widely throughout the egg cytoplasm, but most interesting of all is their occurrence at the outer surface of the nucleus. It is very suggestive that they are formed 1 Supported by grants from the USPHS National State University General Research Fund. a Revised version received April 24, 1967.
Institutes
of Health
(GM-12963)
Experimental
and Oregon
Cell Research 48
570
Patricia
Harris
here and packaged within annulate lamellae recruited either from the nuclear membrane itself or from existing cytoplasmic vesicle membranes. Their function remains unknown. The present study is a life history of these particles in the eggs of the sea urchin Strongylocentrotus purpuratus from the time of their apparent origin in the unfertilized eggs to the time of their dissolution prior to the first cleavage division. It is hoped that this pictorial presentation will be of some aid in the interpretation of present biochemical data.
MATERIALS
AND
METHODS
Gametes of the sea urchin Strongylocenfrotus purpuratus, collected intertidally from Yaquina Head on the Oregon coast, were obtained by injecting the urchins with 0.5 M KCl. Eggs were fertilized by the addition of dilute sperm, and the fertilization membranes were removed by treatment with mercaptoethylgluconamide and subsequent stripping with bolting silk. The eggs were then washed and allowed to develop at 15°C with constant stirring. Membranes were not removed in samples of unfertilized eggs or those fixed prior to 2 min after fertilization. Samples were fixed at frequent intervals between fertilization and first cleavage for 1 hr in I per cent osmium tetroxide in Sorenson’s phosphate buffer at pH 6.0, dehydrated in ethanol series and embedded in an Epon-Araldite mixture. Sections were cut with glass knives on a Porter-Blum MT-2 microtome, stained with lead hydroxide [15] and observed with an RCA EMUSH electron microscope. Monitoring of living material was carried out with phase microscopy on eggs slightly flattened under coverslips, to establish a timetable of developmental events during the period of observation. The timetable proved to be identical to that obtained in previous studies on California urchins of the same species, thus allowing a large store of unpublished peripheral observations made during these studies to be used as supporting data. The work on the California urchins was carried out by the author while working in the laboratory of Dr Daniel Mazia, University of California, Berkeley, and was supported by grants to Dr Mazia from the USPHS National Institutes of Health.
RESULTS
Timetable
of events determined
by light microscopy
The rate of development of eggs at 15°C varied only slightly between batches from different individuals and was essentially the same regardless of whether the animals were collected on the Oregon or northern California coast. The timetable worked out for Oregon urchins is almost identical to that reported in previous studies on California urchins [14]. Experimental
Cell Research 48
Changes following
fertilization TABLE
Min after fertilization 20 30 45 60 65 70
Electron
Stage Pronuclear fusion. Complete formation of the hyaline layer Clear fusion nucleus Elongated fusion nucleus Streak stage Earliest prophase. Beginning aster formation Prophase. Nuclear membrane breakdown
571
in the sea urchin
I. Min after fertilization 75 85 95 105 110 115
Stage Late prophase. Prometaphase Metaphase Anaphase Telophase. Beginning cleavage Reconstitution of nuclei Division completed
microscopy
(1) Fertilization to pronuclear fusion.-The nucleus of the unfertilized egg is usually somewhat irregular in shape, but becomes extremely irregular after fertilization. At its periphery can be seen varying numbers of dense granular aggregates bounded on one side by the nuclear membrane and elsewhere by fragments of annulate lamellae identical in structure to the nuclear membrane. There is no essential change in this relationship up to as late as 15 min following fertilization, as shown in Fig. 1. These membrane bounded granular aggregates or “heavy bodies”, are found scattered in the cytoplasm with no special distribution or concentration in any particular region of the cell. A rough estimate of the total number of heavy bodies, based on counts of about 100 per median section of several 100 ,u diameter cells, is on the order of 1500 per cell. They may be found close to the nucleus, as in Fig. 1, or scattered as far as the cortical region. The granules themselves are the same size as ribosomes, approximately 150 A, and are equally electron dense, though the size of the aggregates may vary considerably. The amount of annulate membrane associated with these granular aggregates varies from egg to egg. In some, like those shown in Fig. 1, the membranes may extend far beyond the bounds of the granular mass, while others adhering to the nucleus may have very little or no membrane covering (Fig. 2). Unbounded granular masses are never seen free in the cytoplasm, or at least cannot be recognized there. Presumably they either acquire membranes later, or are dispersed immediately. The latest time that the heavy bodies are seen associated with the nucleus is just prior to pronuclear fusion. Fig. 3 shows one of these at 12 min follow37 -
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Fig. I.-Egg nucleus (nu) 15 min after fertilization, showing membrane-bound granular aggregates or “heavy bodies” associated with the nuclear membrane. Other heavy bodies can be seen close by in the cytoplasm. This is essentially the same as the unfertilized egg. x 11,700.
Fig. 2.-Two heavy bodies at the nuclear membrane of an egg 12 min after fertilization. Unlike those shown in the previous figure, these have little or no annulate membrane adhering to them. x 14,500. Fig. 3.-Another egg 12 min after fertilization. The sperm nucleus has already reached the egg nucleus, as can be seen from the oblique section of sperm tail (upper arrow), but has not yet fused with it. Heavy bodies are still seen at the nuclear surface (arrow at left), and persist up to the time of pronuclear fusion. x 18,500. Experimental
Cell Research 48
Changes following
fertilization
in the sea urchin
573
Experimental
Cell Research 48
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Harris
ing fertilization. The proximity of the sperm to the egg nucleus is apparent from the presence of the sperm tail seen in oblique section nearby (arrow). It might be noted here that due to some asynchrony within a single batch of fertilized eggs and between batches from different urchins, there may be a considerable overlap of stages from one time sample to the next. Thus the 12 min sample shown in Fig. 3 may actually represent a slightly later stage than the egg at 15 min after fertilization shown in Fig. 1. Other dense granular aggregates are associated with the inner surface of the nuclear membrane at this time, and may superficially be confused with the heavy bodies. They may even possess annulate membrane within the nucleus, as in Fig. 4, but the granularity is much finer than that of ribosomes and they are easily extracted with osmium fixatives at neutral pH and high salt concentration, similar to the effect on metaphase chromosomes described in another study [7]. (2) Pronuclear fusion to nuclear membrane breakdown.-Following the fusion of the egg and sperm nuclei there is little apparent change in appearance, number or distribution of the heavy bodies in the cytoplasm. There is some suggestion of a decrease in the density of packing of the granules within the aggregates, as shown in Fig. 5, but there is enough variation from egg to egg to make such a conclusion rather questionable. At about 60 min following fertilization the egg enters what is called the “streak stage”, characterized in living material by a clear crescent shaped region, with the horns extending from the opposite ends of a somewhat elongated nucleus. In electron micrographs this region appears as a channel or group of channels through the particulate mass of the cytoplasm, and where heavy bodies happen to be in the path of one of these channels they are aligned parallel to it. With the growth of the asters and just prior to the time when the tubular aster fibers penetrate the nucleus, the nuclear membrane again becomes quite irregular and the outer and inner membranes begin to separate (Fig. 6). At the same time there is a corresponding breakdown of the annulate lamellae of the heavy bodies. The paired membranes separate and begin to break up,
Fig. 4.-A dense body within an egg nucleus 12 min after fertilization. These structures vary considerably in size, some being quite large, and occasionally they have associated annulate membranes within the nucleus, as shown here (arrow). x 50,000. Fig. 5.--Several heavy bodies with typical membrane configurations At this stage the granular aggregates appear somewhat less tightly be seen in the surrounding cytoplasm. Their degree of aggregation conditions of fixation and may not be meaningful. x 36,000. Experimental
Cell Research 48
at 61 min after fertilization. packed. Free ribosomes can varies to some extent with
Changes following
fertilization
in the sea urchin
Experimental
575
Cell Research 48
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Harris
becoming indistinguishable from other cytoplasmic membranes and vesicles. Often the annuli themselves persist and may be seen as if they were floating off quite independently of each other. In Fig. 6 these membranes can be seen in cross section with separating annuli. Corresponding pores of the nuclear membrane can be seen where the membrane has been cut obliquely. Fig. 7 shows another view of a heavy body apparently breaking up. With the breakdown of the nuclear membrane and increasing growth of the asters, many of those heavy bodies caught in the channels of the astral rays become incorporated into the massive membranous portion of the mitotic figure, described in previous work [6]. The annulate lamellae, as part of the aster, often retain their annuli for some time, while the granular mass breaks up and is indistinguishable from the great mass of free ribosomes in the cytoplasm. In none of the material examined were recognizable heavy bodies ever seen in the cytoplasm after the breakdown of the nuclear membrane in preparation for first cleavage.
DISCUSSION
Formation
of the hravy
bodies
While it is difficult to determine with absolute certainty the sequence of events in a cellular process by means of static observations with the electron the probability of doing so is highly increased by adequate microscope, sampling of a well synchronized system such as a population of simultaneously fertilized eggs. Thus the appearance of the heavy bodies at the nuclear surface before and for some time after fertilization, the ending of their association with the nucleus at the time of pronuclear fusion and their final disappearance as discrete entities in the cytoplasm at the time of nuclear membrane breakdown, suggests that this is a process of formation and dissolution. If the heavy bodies are formed by the nucleus, there is some question concerning how this% accomplished. It is doubtful that they are extruded nucleolar fragments. As Afzelius [l] pointed out, the heavy bodies are
Fig. 6.-Another egg fixed at 61 min after fertilization, but in a slightly more advanced stage than that shown in Fig. 5. The nuclear membrane and the annulate lamellae of the heavy body nearby are beginning to breali down, the double membranes are separating and the annuli appear to be drifting away. Annuli of the nuclear membrane can also be seen (arrow), as well as several of the tubular fibers of the forming mitotic figure. x 26,000. Fig. 7.-A membrane Experimental
heavy body apparently in the process of breaking can be seen with persistent annuli. x 37,000. Cell Research 48
down. Fragments
of the annulate
Changes following
fertilization
in the sea urchin
Experimental
577
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Harris
distinctly vitally stained with toluidine blue, while the nucleolus is not, and are not as quickly dissolved in sea water when isolated as is the nucleolus. Likewise, in S. purpurafus the granular masses of the heavy bodies are easily preserved, while the nucleolus-like bodies associated with the inner nuclear membrane are extracted by neutral osmium fixatives containing high salt concentration. There is also no electron microscope evidence that would suggest that ribosomes are being synthesized or assembled in the nucleus at this time and transported across the nuclear membrane. The great bulk of the nucleus is a finely fibrous homogeneous mass, with no visible particles of ribosomal dimensions. The origin of the annulate membrane cannot definitely be shown to be the nuclear membrane itself, though it is suggestive in Fig. 1 and in several of Afzelius’ micrographs. It is equally possible, however, that the membrane is recruited from pre-existing cytoplasmic membranes in the same manner as telophase chromosomes acquire a new nuclear membrane [6]. The time-course of formation of heavy bodies appears to extend from the time of the maturation divisions until pronuclear fusion. Unlike Afzelius’ observations, no heavy bodies were found in immature oocytes, although many were found in the cytoplasm of mature eggs still in the ovaries. No attempt was made in this study to determine the total length of time during which these structures are formed. This presumably would vary, depending on the time at which fertilization took place. The process seems to be a r continuous one with no immediate relation to the fertilization reaction and may possibly be a factor in the “cytoplasmic maturation” described by Harvey [9]. Possible function
of heavy bodies
There is well documented evidence that sea urchin eggs contain all the necessary constituents for protein synthesis [ 10, 12, 13, 18, 211, yet no synthesis occurs in the mature unfertilized egg [4, 5, 16, 191. The primary question is how these components, existing together, remain inactive. Conclusions that have been drawn from the existing literature suggest that either the ribosomes themselves are in some way defective or that the messenger ribonucleic acid (mRNA), which may or may not be bound to ribosomes, is somehow masked and inactive. Several studies have been directed toward localizing the site of early protein synthesis. Hultin [lo, 111 first noted that in homogenates of unfertilized eggs of Paracentrofus there were populations of free ribosomes as well as those bound in what he called “endoplasmic particles”. Either with or Experimental
Cell Research 48
Changes following
fertilization
in the sea urchin
579
without their associated membranes, these endoplasmic particles were shown to be synthetically active in fertilized eggs, but inactive in unfertilized. Spirin and Nemer [24] found in early sea urchin embryos messenger RNA associated with three different structures. Only one of these, the heavy polyribosomes, which apparently contain only preexisting or “maternal” messenger RNA, accounts for the bulk of protein synthesis. One conclusion which might be drawn from the results of these two investigations is that the endoplasmic particles (heavy bodies) are the site of the heavy polyribosome-maternal mRNA complex, since both have been shown to be the primary site of early protein synthesis. To test this hypothesis, Hultin [ 111 used chloramphenicol to inhibit the binding of mRNA to ribosomes, and found a marked decrease in amino acid incorporation in vivo. Hultin’s conclusion was that the appearance of polyribosome-like particles in the fertilized egg was not due to the release of aggregates from larger cell components, such as the endoplasmic particles, but rather a net formation of such aggregates under the influence of messenger RNA. In the light of the data from the present study, showing a continuous formation of the RNA containing heavy bodies by the nucleus from at least as early as the maturation divisions until at least as late as pronuclear fusion, the results of Hultin’s chloramphenicol experiments are not entirely inconsistent with the idea that the heavy bodies are the site of the inactive messenger RNA. Further evidence pointing to this conclusion comes from the work of Monroy et al. [18] showing that the masking is due to a protein removable with trypsin, and Backstrom’s [2] demonstration of RNA and RNA-bound cytoplasmic basic proteins localized in the layer of stratified unfertilized sea urchin eggs shown by Afzelius [l] to contain heavy bodies. The free ribosomes, on the other hand, could account for the synthetic activity induced by the addition of exogenous templates such as polyuridylic acid, shown by Nemer [20], Wilt and Hultin [26], and Tyler 1251. It remains to be demonstrated that the rate of heavy body formation is consistent with the decrease in amino acid incorporation brought about by treatment with chloramphenicol. This work is now in progress. The identification of the heavy bodies as the site of ribosome-bound or inactive messenger RNA will remain only conjecture until these structures can be isolated, characterized biochemically and tested in appropriate protein synthesizing systems. The possibility that this identity does exist, however, demands that some caution be used in the interpretation of the present biochemical literature. Protein synthesizing systems employing whole homogenates of sea urchin eggs cannot be equated with those in which the cellular Experimental
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debris has been removed by centrifugation. Part or all of the heavy bodies may be removed, leaving only free ribosomes or varying mixtures of free ribosomes and heavy bodies. It should also be noted that cellular constituents have different sedimentation characteristics in different species of sea urchin. The heavy bodies in Echinus esculentus shown by Afzelius to occupy the most centrifugal part of the stratified egg, are not-so-heavy bodies in Strongylocentrotus purpuratus and remain in the light halves of eggs centrifugally separated into light and heavy fragments [B]. Unfortunately, there has been little or no attempt in any of the biochemical work reported until now to monitor the preparations with electron microscopy. It is imperative that a closer liaison exist between the biochemical and morphological approaches to the problems of early development in order that all the techniques available be used to greatest advantage and for more meaningful interpretations of existing data. SUMMARY
1. Large aggregates of ribosome-like particles, or “heavy bodies”, bounded by annulate lamellae are formed at the nuclear surface of sea urchin eggs, beginning some time before fertilization and continuing until pronuclear fusion after fertilization. 2. At the time of nuclear membrane breakdown prior to first division the annulate lamellae of the heavy bodies disintegrate in the same manner as the nuclear membrane and the ribosome-like particles become dispersed in the cytoplasm. 3. The possibility that heavy bodies are aggregates of polyribosomes held inactive by protein masking is discussed in relation to current biochemical data. REFERENCES 1. AFZELIUS, B. A., Z. Zellforsch. 45, 660 (1957). S., Exptl Cell Res. 43, 578 (1966). 2. BKCKSTRGM, of Animal Development, Vol. 1, p. 483. 3. GRANT, P., in R. WEBER (ed.), The Biochemistry Academic Press, New York, 1965. 4. GROSS, P. FL, J. Exptl Zoot. 157, 21 (1964). G. H., Exptl Cell Res. 33, 368 (1964). 5. GROSS, P. R. and COUSINEAU, 6. HARRIS, P., J. Biophys. Biochem. Cytot. 11, 419 (1961). J. Cell Biol. 14, 475 (1962). 7. __ Unpublished data. 8. __ E. B., The American Arbacia and Other Sea Urchins, University 9. HARVEY, -p. 75. Princeton Press, Princeton, New Jersey, 1956. T., Exptl Cell Res. 25, 405 (1961). 10. HULTIN, 11. __ Deuelop. Biol. 10, 305 (1964). Experimental
Cell Research 48
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fertilization
in the sea urchin
581
12. MAGGIO, R. and CATALANO,C., Arch. Biochem. Biophys. 103, 164 (1963). 13. MAGGIO, R., VITTORELLI, M. L., RINALDI, A. M. and MONROY, A., Biochem. Biophys. Res. Commun. 15, 436 (1964). 14. MAZIA, D., HARRIS, P. and BIBRING, T., J. Biophys. Biochem. CyfoI. 7, 1 (1960). 15. MILLONIG, G., J. Biophys. Biochem. Cyfol. 11, 736 (1961). 16. MONROY, A., Ezperfenfia 16, 114 (1960). 17. __ Chemistry and Physiology of Fertilization. Holt, Rinehart and Winston, New York, 1965. 18. MONROY, A., MAGGIO, R. and RINALDI, A. M., Proc. Naff Acad. Sci. U.S. 54, 107 (1965). 19. NAKANO, E. and MONROY, A., Expfl Cell Res. 14, 236 (1958). 20. NEMER, M., Ho&em. Biophys. Res. Commun. 8, 511 (1962). 21. NEMER, M. and BARD, S. G., Science 140, 664 (1963). 22. RUNNSTR~M, J., in Advances in Morphogenesis, Vol. 5, p. 221. Academic Press, New York, 1966. 23. SPIRIN, A. S., in A. MONROY and A. A. MOSCONA (eds.), Current Topics in Developmental Biology, Vol. 1, p. 1. Academic Press, New York, 1966. 24. SPIRIN, A. S. and NEMER, M., Science 150, 214 (1965). 25. TYLER, A., Am. Zool. 3, 109 (1963). 26. WILT, F. H. and HULTIN, T., Biochem. Biophys. Res. Commun. 9, 313 (1962).
Experimental
Cell Research 48
Experimental
Press Inc.
569
Cell Research 48, 569-581 (1967)
STRUCTURAL
FORMATION
CHANGES FOLLOWING FERTILIZATION IN THE SEA URCHIN EGG AND
DISSOLUTION PATRICIA
OF
HEAVY
BODIES’
HARRIS
Department of Zoology, Oregon State University, Corvallis, Ore. 97331, and Oregon State University Marine Science Center, Newport, Ore., USA Received
February
20, 19672
THE activation of synthetic processes in sea urchin eggs following fertilization, and the means whereby these processes are held in abeyance in the unfertilized egg, have been the subject of a large number of recent biochemical studies. This work has been reviewed in some detail [3, 17, 22, 231. The conclusions which can be drawn from the data at hand can be stated rather succinctly: there is little or no active protein synthesis in the unfertilized egg; protein synthesis is initiated very shortly after the fertilization reaction: no usable messenger RNA, which is necessary for protein synthesis, is produced until much later in the blastula stage; the existing messenger RNA in these cells is of maternal origin, but present in an unusable or “masked” form until fertilization. It is of some importance at this time to examine at the fine structural level the cytoplasmic changes which occur at fertilization and to combine the techniques of biochemistry and electron microscopy in an attempt to answer the following questions: is there really an inactive messenger RNA; if so, how is it packaged, and how is it released? The most likely candidate for such a role in the cytoplasm are the socalled “heavy bodies”, first named and described by Afzelius [l] in unfertilized eggs of several species of European sea urchins. These bodies are compact balls of tightly packed granules of ribosomal size and appearance. They are bounded on several sides, though not completely enclosed, by annulate lamellae having the same structure as the nuclear membrane, and were demonstrated by Afzelius to contain RNA. They are distributed widely throughout the egg cytoplasm, but most interesting of all is their occurrence at the outer surface of the nucleus. It is very suggestive that they are formed 1 Supported by grants from the USPHS National State University General Research Fund. a Revised version received April 24, 1967.
Institutes
of Health
(GM-12963)
Experimental
and Oregon
Cell Research 48
570
Patricia
Harris
here and packaged within annulate lamellae recruited either from the nuclear membrane itself or from existing cytoplasmic vesicle membranes. Their function remains unknown. The present study is a life history of these particles in the eggs of the sea urchin Strongylocentrotus purpuratus from the time of their apparent origin in the unfertilized eggs to the time of their dissolution prior to the first cleavage division. It is hoped that this pictorial presentation will be of some aid in the interpretation of present biochemical data.
MATERIALS
AND
METHODS
Gametes of the sea urchin Strongylocenfrotus purpuratus, collected intertidally from Yaquina Head on the Oregon coast, were obtained by injecting the urchins with 0.5 M KCl. Eggs were fertilized by the addition of dilute sperm, and the fertilization membranes were removed by treatment with mercaptoethylgluconamide and subsequent stripping with bolting silk. The eggs were then washed and allowed to develop at 15°C with constant stirring. Membranes were not removed in samples of unfertilized eggs or those fixed prior to 2 min after fertilization. Samples were fixed at frequent intervals between fertilization and first cleavage for 1 hr in I per cent osmium tetroxide in Sorenson’s phosphate buffer at pH 6.0, dehydrated in ethanol series and embedded in an Epon-Araldite mixture. Sections were cut with glass knives on a Porter-Blum MT-2 microtome, stained with lead hydroxide [15] and observed with an RCA EMUSH electron microscope. Monitoring of living material was carried out with phase microscopy on eggs slightly flattened under coverslips, to establish a timetable of developmental events during the period of observation. The timetable proved to be identical to that obtained in previous studies on California urchins of the same species, thus allowing a large store of unpublished peripheral observations made during these studies to be used as supporting data. The work on the California urchins was carried out by the author while working in the laboratory of Dr Daniel Mazia, University of California, Berkeley, and was supported by grants to Dr Mazia from the USPHS National Institutes of Health.
RESULTS
Timetable
of events determined
by light microscopy
The rate of development of eggs at 15°C varied only slightly between batches from different individuals and was essentially the same regardless of whether the animals were collected on the Oregon or northern California coast. The timetable worked out for Oregon urchins is almost identical to that reported in previous studies on California urchins [14]. Experimental
Cell Research 48
Changes following
fertilization TABLE
Min after fertilization 20 30 45 60 65 70
Electron
Stage Pronuclear fusion. Complete formation of the hyaline layer Clear fusion nucleus Elongated fusion nucleus Streak stage Earliest prophase. Beginning aster formation Prophase. Nuclear membrane breakdown
571
in the sea urchin
I. Min after fertilization 75 85 95 105 110 115
Stage Late prophase. Prometaphase Metaphase Anaphase Telophase. Beginning cleavage Reconstitution of nuclei Division completed
microscopy
(1) Fertilization to pronuclear fusion.-The nucleus of the unfertilized egg is usually somewhat irregular in shape, but becomes extremely irregular after fertilization. At its periphery can be seen varying numbers of dense granular aggregates bounded on one side by the nuclear membrane and elsewhere by fragments of annulate lamellae identical in structure to the nuclear membrane. There is no essential change in this relationship up to as late as 15 min following fertilization, as shown in Fig. 1. These membrane bounded granular aggregates or “heavy bodies”, are found scattered in the cytoplasm with no special distribution or concentration in any particular region of the cell. A rough estimate of the total number of heavy bodies, based on counts of about 100 per median section of several 100 ,u diameter cells, is on the order of 1500 per cell. They may be found close to the nucleus, as in Fig. 1, or scattered as far as the cortical region. The granules themselves are the same size as ribosomes, approximately 150 A, and are equally electron dense, though the size of the aggregates may vary considerably. The amount of annulate membrane associated with these granular aggregates varies from egg to egg. In some, like those shown in Fig. 1, the membranes may extend far beyond the bounds of the granular mass, while others adhering to the nucleus may have very little or no membrane covering (Fig. 2). Unbounded granular masses are never seen free in the cytoplasm, or at least cannot be recognized there. Presumably they either acquire membranes later, or are dispersed immediately. The latest time that the heavy bodies are seen associated with the nucleus is just prior to pronuclear fusion. Fig. 3 shows one of these at 12 min follow37 -
671802
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Fig. I.-Egg nucleus (nu) 15 min after fertilization, showing membrane-bound granular aggregates or “heavy bodies” associated with the nuclear membrane. Other heavy bodies can be seen close by in the cytoplasm. This is essentially the same as the unfertilized egg. x 11,700.
Fig. 2.-Two heavy bodies at the nuclear membrane of an egg 12 min after fertilization. Unlike those shown in the previous figure, these have little or no annulate membrane adhering to them. x 14,500. Fig. 3.-Another egg 12 min after fertilization. The sperm nucleus has already reached the egg nucleus, as can be seen from the oblique section of sperm tail (upper arrow), but has not yet fused with it. Heavy bodies are still seen at the nuclear surface (arrow at left), and persist up to the time of pronuclear fusion. x 18,500. Experimental
Cell Research 48
Changes following
fertilization
in the sea urchin
573
Experimental
Cell Research 48
574
Patricia
Harris
ing fertilization. The proximity of the sperm to the egg nucleus is apparent from the presence of the sperm tail seen in oblique section nearby (arrow). It might be noted here that due to some asynchrony within a single batch of fertilized eggs and between batches from different urchins, there may be a considerable overlap of stages from one time sample to the next. Thus the 12 min sample shown in Fig. 3 may actually represent a slightly later stage than the egg at 15 min after fertilization shown in Fig. 1. Other dense granular aggregates are associated with the inner surface of the nuclear membrane at this time, and may superficially be confused with the heavy bodies. They may even possess annulate membrane within the nucleus, as in Fig. 4, but the granularity is much finer than that of ribosomes and they are easily extracted with osmium fixatives at neutral pH and high salt concentration, similar to the effect on metaphase chromosomes described in another study [7]. (2) Pronuclear fusion to nuclear membrane breakdown.-Following the fusion of the egg and sperm nuclei there is little apparent change in appearance, number or distribution of the heavy bodies in the cytoplasm. There is some suggestion of a decrease in the density of packing of the granules within the aggregates, as shown in Fig. 5, but there is enough variation from egg to egg to make such a conclusion rather questionable. At about 60 min following fertilization the egg enters what is called the “streak stage”, characterized in living material by a clear crescent shaped region, with the horns extending from the opposite ends of a somewhat elongated nucleus. In electron micrographs this region appears as a channel or group of channels through the particulate mass of the cytoplasm, and where heavy bodies happen to be in the path of one of these channels they are aligned parallel to it. With the growth of the asters and just prior to the time when the tubular aster fibers penetrate the nucleus, the nuclear membrane again becomes quite irregular and the outer and inner membranes begin to separate (Fig. 6). At the same time there is a corresponding breakdown of the annulate lamellae of the heavy bodies. The paired membranes separate and begin to break up,
Fig. 4.-A dense body within an egg nucleus 12 min after fertilization. These structures vary considerably in size, some being quite large, and occasionally they have associated annulate membranes within the nucleus, as shown here (arrow). x 50,000. Fig. 5.--Several heavy bodies with typical membrane configurations At this stage the granular aggregates appear somewhat less tightly be seen in the surrounding cytoplasm. Their degree of aggregation conditions of fixation and may not be meaningful. x 36,000. Experimental
Cell Research 48
at 61 min after fertilization. packed. Free ribosomes can varies to some extent with
Changes following
fertilization
in the sea urchin
Experimental
575
Cell Research 48
576
Patricia
Harris
becoming indistinguishable from other cytoplasmic membranes and vesicles. Often the annuli themselves persist and may be seen as if they were floating off quite independently of each other. In Fig. 6 these membranes can be seen in cross section with separating annuli. Corresponding pores of the nuclear membrane can be seen where the membrane has been cut obliquely. Fig. 7 shows another view of a heavy body apparently breaking up. With the breakdown of the nuclear membrane and increasing growth of the asters, many of those heavy bodies caught in the channels of the astral rays become incorporated into the massive membranous portion of the mitotic figure, described in previous work [6]. The annulate lamellae, as part of the aster, often retain their annuli for some time, while the granular mass breaks up and is indistinguishable from the great mass of free ribosomes in the cytoplasm. In none of the material examined were recognizable heavy bodies ever seen in the cytoplasm after the breakdown of the nuclear membrane in preparation for first cleavage.
DISCUSSION
Formation
of the hravy
bodies
While it is difficult to determine with absolute certainty the sequence of events in a cellular process by means of static observations with the electron the probability of doing so is highly increased by adequate microscope, sampling of a well synchronized system such as a population of simultaneously fertilized eggs. Thus the appearance of the heavy bodies at the nuclear surface before and for some time after fertilization, the ending of their association with the nucleus at the time of pronuclear fusion and their final disappearance as discrete entities in the cytoplasm at the time of nuclear membrane breakdown, suggests that this is a process of formation and dissolution. If the heavy bodies are formed by the nucleus, there is some question concerning how this% accomplished. It is doubtful that they are extruded nucleolar fragments. As Afzelius [l] pointed out, the heavy bodies are
Fig. 6.-Another egg fixed at 61 min after fertilization, but in a slightly more advanced stage than that shown in Fig. 5. The nuclear membrane and the annulate lamellae of the heavy body nearby are beginning to breali down, the double membranes are separating and the annuli appear to be drifting away. Annuli of the nuclear membrane can also be seen (arrow), as well as several of the tubular fibers of the forming mitotic figure. x 26,000. Fig. 7.-A membrane Experimental
heavy body apparently in the process of breaking can be seen with persistent annuli. x 37,000. Cell Research 48
down. Fragments
of the annulate
Changes following
fertilization
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distinctly vitally stained with toluidine blue, while the nucleolus is not, and are not as quickly dissolved in sea water when isolated as is the nucleolus. Likewise, in S. purpurafus the granular masses of the heavy bodies are easily preserved, while the nucleolus-like bodies associated with the inner nuclear membrane are extracted by neutral osmium fixatives containing high salt concentration. There is also no electron microscope evidence that would suggest that ribosomes are being synthesized or assembled in the nucleus at this time and transported across the nuclear membrane. The great bulk of the nucleus is a finely fibrous homogeneous mass, with no visible particles of ribosomal dimensions. The origin of the annulate membrane cannot definitely be shown to be the nuclear membrane itself, though it is suggestive in Fig. 1 and in several of Afzelius’ micrographs. It is equally possible, however, that the membrane is recruited from pre-existing cytoplasmic membranes in the same manner as telophase chromosomes acquire a new nuclear membrane [6]. The time-course of formation of heavy bodies appears to extend from the time of the maturation divisions until pronuclear fusion. Unlike Afzelius’ observations, no heavy bodies were found in immature oocytes, although many were found in the cytoplasm of mature eggs still in the ovaries. No attempt was made in this study to determine the total length of time during which these structures are formed. This presumably would vary, depending on the time at which fertilization took place. The process seems to be a r continuous one with no immediate relation to the fertilization reaction and may possibly be a factor in the “cytoplasmic maturation” described by Harvey [9]. Possible function
of heavy bodies
There is well documented evidence that sea urchin eggs contain all the necessary constituents for protein synthesis [ 10, 12, 13, 18, 211, yet no synthesis occurs in the mature unfertilized egg [4, 5, 16, 191. The primary question is how these components, existing together, remain inactive. Conclusions that have been drawn from the existing literature suggest that either the ribosomes themselves are in some way defective or that the messenger ribonucleic acid (mRNA), which may or may not be bound to ribosomes, is somehow masked and inactive. Several studies have been directed toward localizing the site of early protein synthesis. Hultin [lo, 111 first noted that in homogenates of unfertilized eggs of Paracentrofus there were populations of free ribosomes as well as those bound in what he called “endoplasmic particles”. Either with or Experimental
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without their associated membranes, these endoplasmic particles were shown to be synthetically active in fertilized eggs, but inactive in unfertilized. Spirin and Nemer [24] found in early sea urchin embryos messenger RNA associated with three different structures. Only one of these, the heavy polyribosomes, which apparently contain only preexisting or “maternal” messenger RNA, accounts for the bulk of protein synthesis. One conclusion which might be drawn from the results of these two investigations is that the endoplasmic particles (heavy bodies) are the site of the heavy polyribosome-maternal mRNA complex, since both have been shown to be the primary site of early protein synthesis. To test this hypothesis, Hultin [ 111 used chloramphenicol to inhibit the binding of mRNA to ribosomes, and found a marked decrease in amino acid incorporation in vivo. Hultin’s conclusion was that the appearance of polyribosome-like particles in the fertilized egg was not due to the release of aggregates from larger cell components, such as the endoplasmic particles, but rather a net formation of such aggregates under the influence of messenger RNA. In the light of the data from the present study, showing a continuous formation of the RNA containing heavy bodies by the nucleus from at least as early as the maturation divisions until at least as late as pronuclear fusion, the results of Hultin’s chloramphenicol experiments are not entirely inconsistent with the idea that the heavy bodies are the site of the inactive messenger RNA. Further evidence pointing to this conclusion comes from the work of Monroy et al. [18] showing that the masking is due to a protein removable with trypsin, and Backstrom’s [2] demonstration of RNA and RNA-bound cytoplasmic basic proteins localized in the layer of stratified unfertilized sea urchin eggs shown by Afzelius [l] to contain heavy bodies. The free ribosomes, on the other hand, could account for the synthetic activity induced by the addition of exogenous templates such as polyuridylic acid, shown by Nemer [20], Wilt and Hultin [26], and Tyler 1251. It remains to be demonstrated that the rate of heavy body formation is consistent with the decrease in amino acid incorporation brought about by treatment with chloramphenicol. This work is now in progress. The identification of the heavy bodies as the site of ribosome-bound or inactive messenger RNA will remain only conjecture until these structures can be isolated, characterized biochemically and tested in appropriate protein synthesizing systems. The possibility that this identity does exist, however, demands that some caution be used in the interpretation of the present biochemical literature. Protein synthesizing systems employing whole homogenates of sea urchin eggs cannot be equated with those in which the cellular Experimental
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debris has been removed by centrifugation. Part or all of the heavy bodies may be removed, leaving only free ribosomes or varying mixtures of free ribosomes and heavy bodies. It should also be noted that cellular constituents have different sedimentation characteristics in different species of sea urchin. The heavy bodies in Echinus esculentus shown by Afzelius to occupy the most centrifugal part of the stratified egg, are not-so-heavy bodies in Strongylocentrotus purpuratus and remain in the light halves of eggs centrifugally separated into light and heavy fragments [B]. Unfortunately, there has been little or no attempt in any of the biochemical work reported until now to monitor the preparations with electron microscopy. It is imperative that a closer liaison exist between the biochemical and morphological approaches to the problems of early development in order that all the techniques available be used to greatest advantage and for more meaningful interpretations of existing data. SUMMARY
1. Large aggregates of ribosome-like particles, or “heavy bodies”, bounded by annulate lamellae are formed at the nuclear surface of sea urchin eggs, beginning some time before fertilization and continuing until pronuclear fusion after fertilization. 2. At the time of nuclear membrane breakdown prior to first division the annulate lamellae of the heavy bodies disintegrate in the same manner as the nuclear membrane and the ribosome-like particles become dispersed in the cytoplasm. 3. The possibility that heavy bodies are aggregates of polyribosomes held inactive by protein masking is discussed in relation to current biochemical data. REFERENCES 1. AFZELIUS, B. A., Z. Zellforsch. 45, 660 (1957). S., Exptl Cell Res. 43, 578 (1966). 2. BKCKSTRGM, of Animal Development, Vol. 1, p. 483. 3. GRANT, P., in R. WEBER (ed.), The Biochemistry Academic Press, New York, 1965. 4. GROSS, P. FL, J. Exptl Zoot. 157, 21 (1964). G. H., Exptl Cell Res. 33, 368 (1964). 5. GROSS, P. R. and COUSINEAU, 6. HARRIS, P., J. Biophys. Biochem. Cytot. 11, 419 (1961). J. Cell Biol. 14, 475 (1962). 7. __ Unpublished data. 8. __ E. B., The American Arbacia and Other Sea Urchins, University 9. HARVEY, -p. 75. Princeton Press, Princeton, New Jersey, 1956. T., Exptl Cell Res. 25, 405 (1961). 10. HULTIN, 11. __ Deuelop. Biol. 10, 305 (1964). Experimental
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12. MAGGIO, R. and CATALANO,C., Arch. Biochem. Biophys. 103, 164 (1963). 13. MAGGIO, R., VITTORELLI, M. L., RINALDI, A. M. and MONROY, A., Biochem. Biophys. Res. Commun. 15, 436 (1964). 14. MAZIA, D., HARRIS, P. and BIBRING, T., J. Biophys. Biochem. CyfoI. 7, 1 (1960). 15. MILLONIG, G., J. Biophys. Biochem. Cyfol. 11, 736 (1961). 16. MONROY, A., Ezperfenfia 16, 114 (1960). 17. __ Chemistry and Physiology of Fertilization. Holt, Rinehart and Winston, New York, 1965. 18. MONROY, A., MAGGIO, R. and RINALDI, A. M., Proc. Naff Acad. Sci. U.S. 54, 107 (1965). 19. NAKANO, E. and MONROY, A., Expfl Cell Res. 14, 236 (1958). 20. NEMER, M., Ho&em. Biophys. Res. Commun. 8, 511 (1962). 21. NEMER, M. and BARD, S. G., Science 140, 664 (1963). 22. RUNNSTR~M, J., in Advances in Morphogenesis, Vol. 5, p. 221. Academic Press, New York, 1966. 23. SPIRIN, A. S., in A. MONROY and A. A. MOSCONA (eds.), Current Topics in Developmental Biology, Vol. 1, p. 1. Academic Press, New York, 1966. 24. SPIRIN, A. S. and NEMER, M., Science 150, 214 (1965). 25. TYLER, A., Am. Zool. 3, 109 (1963). 26. WILT, F. H. and HULTIN, T., Biochem. Biophys. Res. Commun. 9, 313 (1962).
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