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Experimental Cell Research 69 (1971) 361-371 A STORAGE FORM OF RIBOSOMES IN MOUSE OOCYTES G. D. BURKHOLDER, D. E. COMINGS and T. A. OKADA Department of Medical Genetics, City of Hope Medical Center, Duarte, Calf{. 91 0/0, USA SUMMARY By utilizing the technique of whole mount electron microscopy, large numbers of very ordered lattice-like structures have been observed in mouse oocyte preparations, and min-section studies have shown that these structures are of cytoplasmic origin. In some preparations, there appeared to be a breakdown of the lattice with the release of particles having the dimensions of ribosomes (225 A). This evidence, together with the results of enzyme digestion studies which show that the lattices are composed of RNA and protein, leads to the conclusion that these structures are highly ordered aggregates of ribosomes. The ribosomes are strung together to form chains which are capable of regular cross-linkLrig with adjacent chains to form the charac teristic lattice. It is suggested that the ribosomes are held together by basic proteins and are stored in an inactive form within the lattice until after fertilization, when they will be released and used in protein synthesis during early cleavage, until the embryo makes its own functional ribosomes. This premise is discussed in relation to the appearance and disappearance of the lattice, the synthesis of ribosomal RNA, the existence of ribosome inactivating proteins, and the possible formation of inactive maternal ribosome-mRNA complexes during oocyte maturation. The nucleus of maturing mammalian oocytes has been examined in extensive detail, how ever few detailed studies have been conducted on the specialized features of the cytoplasm. Thin-section studies [8, 13, 14, 20, 23, 25] have shown that the cytoplasm of mature oocytes from myomorph rodents (mouse, rat, hamster) is packed with highly ordered arrays of parallel chains, or lattice-like structures. These specialized structures are only found in maturing and mature oocytes of this group of animals, and they completely disappear from the cytoplasm during the early cleavage stages after fertilization. They have not been observed in oocytes of other mammals. Relatively little is known concerning the biochemical nature of these structures, and they have been variously referred to as yolk material [20], protein strands [9, 13, 22], or aggregates of ribosomes [14, 25]. The develop ment of a technique for bursting and spread- ing single oocytes on an air-liquid interface (whole mount preparation) has permitted a more detailed study to be made of these peculiar lattices. The results of electron microscopy and enzyme digestion experiments suggest that these arrays are of ribosomes which are held together by proteins and are stored in an inactive form in the lattice until after fertilizai:io1G, when will be released and used in protein synthesis during early cleavage, until the makes its own functional ribosomes. MATERIALS AND METHODS Oocyte preparation The ovaries were removed from mature female mice, cleaned of fat and connective tissue, and placed a depression slide containing oocyte culture medium [6]. All further operations were performed under a dissecting microscope with adjustable magnification (10-40 x ). To release the oocytes, the was held follicles in position with a 25 gauge needle, and Exptl Ce!l Res 69 362 G. D. Burkholder et al. were punctured using a second needle. After all the visible follicles were punctured, repeated jabbing of the ovary released a few more oocytes. Follicular cells adhering to the oocytes were removed by gentle pipetting. Oocytes having a zona pellucida and intact nucleus were collected using a finely drawn Pasteur pipette and were washed twice by transferring them through fresh media. Whole mount preparations In order to burst and spread the oocytes on an air liquid interface, it was both necessary to remove the zona pe!lucida and expose the oocytes to a hypotonic solution. This was accomplished in one step by transferring the occytes to a solution of 0.5 % pronase made in distilled water. The oocytes were left in this solution at room temperature until microscopic observation revealed that all of the zona pellucida had been removed and the oocytes appeared some what swollen. This usually took 10-15 min. Some oocytes were spread directly from the pronase solution, however in most experiments the oocytes were transferred to NMT (0.12 M NaCl, 0.005 M MgCl2, 0.01 M Tris buffer pH 7.0) containing 10 % fetal calf scrum, to avoid prolonged exposure to pronase. For spreading, a depression slide was filled with aqueous 10 % sucrose. In some experiments 0.1 M sucrose was used, but the increased buoyancy of the 10 % solution was preferred. Single oocytes were drawn, with a small amount of NMT + serum, just into the tip of a fine pipette. Focusing on the surface of the sucrose solution with the dissecting microscope, the tip of the pipette, containing the oocyte, was carefully touched to the liquid surface, and the oocyte was gently expelled with a small amount of fluid onto the sucrose. Extreme care must be taken not to create bubbles during this step. The oocyte floats for several seconds and then bursts, thereby releasing its contents onto the surface of the sucrose solution. Immediately after bursting, a Formvar- and carbon coated 75 mesh specimen grid was touched to the surface where the oocyte had been. In this manner each grid picked up only the contents of a sing!� oocyte. The grids were then stained in 2 % uranyl acetate for 10 min, dehydrated through a graded series of ethanol washes, passed through amyl acetate for 10-20 min, and air-dried. The specimens were examined in an Hitachi HS-8-1 electron microscope at 50kV. Some preparations were platinum-carbon shadowed. This same procedure was used for rat and Chinese hamster oocytes. Thin sectioning Whole oocytes were fixed in 2 % glutaraldehyde at 4°C for 1 h, rinsed for 30 min in phosphate buffer pH 7.2, fixed with I % osmic acid for 1-2 h, rinsed for 30 min with Ringer solution, passed through a graded series of · ethanol washes into propylene oxide and embedded in a mixture of Epon-Araldite. After sectioning, specimens were stained with saturated uranyl acetate in 50 % ethanol, and lead citrate. Exptl Cell Res 69 Enzyme and chemical treatments After being picked up from the surface of the sucrose solution, some of the spread oocyte preparations were treated by floating the grids on the surface of the following enzymes or chemicals: (1) DNase (Worthington) 200 µg/ml in 0.12 M NaCl, 5 x 10-3 M MgC12, 0.01 M Tris buffer pH 7.0 (NMT) at 37°C for 5-10 min; (2) RNase-A (Sigma) 200 µg/ml and RNase T, (Calbiochem) 200 µg/ml in 0.12 M NaCl, 0.01 M EDTA, 0.01 M Tris buffer pH 7.0 (NET) at room temperature for 5 min. Both ribonucleases were first heated to 80°C for 10 min to destroy residual DNase; (3) 0.01 % or 0.1 % Trypsin in NMT at 35 ° C for 1-5 min; (4) 0.2 M HCl, room temperature for 1-5 min; (5) 8 M urea, room tem perature for 1�5 min. Following these treatments, the grids were processed as described above. Some of the oocytes were treated with RNase (concentrations as in (2) above) prior to spreading. In these experiments, the oocytes were treated with pronase, washed in NMT + 10 % fetal calf serum for 15 min, and then transferred to enzyme in NET at 30 °C for 4 h. Control oocytes were exposed to NET under the same conditions. Following this treatment, some oocytes were spread on sucrose while others were prepared for sectioning. Blastocysts Mouse blastocysts were kindly provided by Dr John Melnyk, Department of Biology, City of Hope Medical Center. Superovulated mice were mated, and embryos at the 8-cell stage were flushed from the Fallopian tubes on the third day after mating. These embryos were cultured under paraffin oil in the medium described by Mintz [16]. After approximately 3 days, the embryos had progressed to the blastocyst stage. The blastocysts were transferred to 0.5 % pronase to remove the zona pellucida, and were then spread on 10 % sucrose as previously described for oocytes. RESULTS The mouse follicular oocytes used in these experiments had a clearly recognizable zona pellucida and intact nucleus. These oocytes were in the dictyate stage of meiotic prophase and had not yet begun the first maturation division. When the whole mount preparations of these oocytes were examined with the electron microscope, very highly ordered lattice-like structures were commonly observed (fig. 1 (a, b)). These lattices are composed of individual chains each of which resembles a series of beads on a string. The diameter Storage form of ribosomes 363 Fig. 1. Lattice-like configurations in mouse oocytes. (a) Whole mount preparation. x 53 400; (b) whole mount preparation. x 84 000; (c) cytoplasm of mouse oocyte observed in sectioned material. x 53 400. Note the similar appearance of the lattice structure in spread (a, b) and sectioned oocytes (c). 364 G. D. Burkholder et al. pheral region of the cytoplasm tended to be devoid of lattices. In order to determine the biochemical Controia Experimental nature of these arrays, a series of enzyme digestions was performed, the results of which + + DNase + are shown in table 1 . The DNase and trypsin RNase + Trypsin digestions were performed on spread material. Although some RNase digestions were con ( +) indicates structures are preserved; ( - ) indicates ducted on spread material, these enzyme they are destroyed. a Controls were exposed to buffer solutions without digestions were more easily performed on enzyme. intact whole oocytes which were then used for making whole mount preparations or for of the beads averaged 212 A while the con embedding and sectioning. DNase had no nections between beads were approx. 1 25 A effect on the lattices, but they were readily in diameter. destroyed by either trypsin or RNase. In all Sometimes the chains existed singly but cases, control preparations were unaffected. they were often interconnected to one another It may therefore be concluded that these in a highly regular manner. Crosslinks existed structures are composed of protein and RNA. between chains with a periodicity of about Brief exposure to weak trypsin solutions 360 A and were always such that the connec (0.01 %, 1-2 min) resulted in a partial dis tions occurred between two beads on adjacent solution of the chains. Although the charac chains. In this manner, the beads on one teristic beaded appearance was not as obvious chain were always aligned with corresponding after this treatment (fig. 2(a)), the presence beads on adjacent chains thereby giving rise of beads could still be demonstrated by to the lattice configuration. The centre-to platinum-carbon shadowing (fig. 2(b)). The centre distance between neighboring chains interconnections between the beads were not was found to be somewhat variable, ranging obvious however, suggesting that these con from 235-300 A. The number of chains cross nections had been removed by the trypsin linked to form a lattice varied from 2 to 8 and were therefore of a protein nature. or more. Chain length was also highly variable HCl treatment (0.2 M) destroyed the lattice and lattices frequently branched or anasto configuration, but ribosomes were morpho mosed with one another. Sometimes the logically normal and clusters were common. lattices appeared to form layers of intercon Treatment with 8 M urea completely dis rupted both lattices and ribosomes. nected sheets. Further evidence concerning the nature of When oocytes were embedded and section ed, identical structures were found in the the lattices was obtained from some of the cytoplasm (fig. I (c)). Under low magnification, untreated whole mount preparations in which the lattices appear to be arranged in a rather there appeared to be a breakdown of the haphazard manner and impart a whorl-like lattice-work. This was probably caused by appearance on the cytoplasm. Many of the the physical forces occurring at the time of shorter lattices are curved in a semi-circle bursting and spreading of the oocyte. This while the longer ones tend to be straight. apparent breakdown was more commonly In cross-section, they appear as clusters of observed in oocytes spread from NMT + 10 % granules. As found by others [21], the peri- fetal calf serum than in those spread directly Table 1 . Effect of enzymes on the lattice-like conjigurations Exptl Cell Res 69 Storage form of ribosomes 365 Fig. 2. Ribosomal chains exposed to 0.01 % trypsin for 2 min. (a) Whole mount preparation. Stained with uranyl acetate. x 44 500; (b) whole mount preparation. Stained with uranyl acetate and platinum shadowed. x 44 500. The beaded nature of the chains is obvious, but the connections between beads appear to have been partially removed by the treatment. from the pronase solution. In such prepara tions, many of the lattices had fallen apart into single chains. In addition, there was sometimes a breakdown in structure at the ends of individual chains (fig. 3(a-i)), with the apparent release of particles having the dimensions of ribosomes (225 A). This evi dence, taken together with the results of the enzyme digestion studies, suggests that the lattices are composed of chains of ribosomes, held together by some proteinaceous material. Frequently the terminal ribosome appeared to be firmly attached to the end of the chain (fig. 3(a-c)), but occasionally, one or two ribosomes were only connected to the chain by a thin filament (fig. 3(d g - )). In other cases, there appeared to be clusters of ribosomes in the vicinity of the end of the chain, but without any apparent connection to the chain (fig. 3(h, i)). Free ribosomes (fig. 3(h, i)), and sometimes terminal ones (fig. 3(a g - )) were more electron-dense than ribosomes which were still an integral part of the chain. In addition, there was a difference in size bet ween these ribosomes. Those within the chain averaged 2 1 0 A in diameter, terminal ribo somes were 220 A, and free ribosomes were about 225 A in diameter. In small pieces of chain there sometimes appeared to be a simultaneous separation of the component ribosomes from one another (fig. 3(j-l)). It is possible that these ribosomes could be uniting to form a chain but this seems unlikely. In all preparations of mouse oocytes, free ribosomes were never found scattered at random but were aiways observed in clusters (fig. 3(m-p)). Undoubtedly some of these associations are polyribosornes, however most of the clusters are probably derived from broken-down chains. The tendency of the ribosomes within a cluster to have a Exptl Ceil Res 69 366 G. D. Burkholder et al. linear arrangement (fig. 3(n- p)) supports this idea and suggests that there might be some remnant of a physical bond remaining bet ween them. Occasionally a fine filament could be seen connecting two of the ribosomes in such a cluster (fig. 3(m)), but more often, no connections could be demonstrated, even after platinum-carbon shadowing. No lattice-like arrays were observed in whole mount preparations of mouse blasto cysts, however there were a few small single chains which appeared to be in the process of breaking down. The predominant char acteristic of these preparations was a very large number of ribosomes. Some of the ribosomes were attached to membranous material, probably endoplasmic reticulum, while others existed in clusters. The majority however, appeared to be scattered at random over large areas of the grid. This is in direct contrast to the findings in mouse oocytes, where comparatively few ribosomes were always observed in clusters and were never found scattered at random. The existence of lattice-like configurations in rat and Chinese hamster oocytes was verified by thin sectioning. Whole mount preparations of oocytes from these animals did not reveal any obvious chains or lattice, but close linear associations of ribosomes were found in some of these preparations. DISCUSSION The application of whole mount electron microscopy to single mouse oocytes at the dictyate stage of meiosis has provided a new means of examining speeific structural fea tures of these oocytes which heretofore could only be studied in thin sections. Utilizing this technique, large numbers of very highly ordered lattice-like structures have been observed in spread oocyte preparations, and thin-section studies have shown that these structures are of cytoplasmic origin (fig. 1). In favorable preparations, these lattices appear to give rise to particles the size of ribosomes (fig. 3), and this evidence, together with the results of enzyme digestion studies which show that the lattices are composed of RNA and protein (table 1 ), leads to the conclusion that these structures are highly ordered aggregates of ribosomes. The ribo somes are strung together to form chains which are capable of regular crosslinking with adjacent chains to form the characteris tic lattice. It is suggested that the ribosomes are combined with, and held together by, proteins and are stored in an inactive form in the lattice until after fertilization, when they will be released and used in protein syn thesis during the early cleavage stage until the embryo makes its own functional ribo somes. The interconnections between the ribo somes of a chain are largely removed by brief exposure to weak trypsin solutions (0.01 %, 1-2 min), indicating that these connections are proteinaceous (fig. 2). Basic proteins may be responsible for holding the ribosomes together since there was a rapid deterioration of the lattice in 0.2 M HCL The disintegration of both lattice and free ribosomes during treatment with 8 M urea suggests that Fig. 3. Examples of the apparent breakdown of ribosomal chains in untreated whole mount preparations of mouse oocytes. x 53 400. (a-c) Terminal ribosomes (arrows) which are larger and more electron-dense than stored ribosomes within the chain. These terminal ribosomes appear firmly connected to the end of the chain; (d-g) terminal ribosomes which are connected to the chain by thin filaments. Arrows indicate connecting strands; (h-i) clusters of ribosomes in the vicinity of the. end of a chain; (j-l) small chains in which there appears to be a simultaneous separation of the component ribosomes from one another; (m-p) clusters of ribosomes. Arrow in (m) indicates connecting filament between two ribosomes. Exptl Cell Res 69 Storage form of ribosomes 367 Exptl Cell Res 69 368 G. D. Burkholder et al. hydrogen bonds and/or hydrophobic bonds play a significant role in both lattice and ribosome structure. The breakdown of the lattice and concomi tant release of ribosomes in the whole mount preparations is probably caused by the physical forces occurring at the time of bursting and spreading of the oocyte, how ever it could also be a result of the release of intracellular proteases during the prepa ration. In vivo, the release of ribosomes from the lattice after fertilization is probably due to the controlled activity of a specific protease. Free ribosomes and often terminal ribo somes were found to be more electron-dense and somewhat larger than stored ribosomes within a chain (fig. 3(a-i)). This suggests that the association of ribosomes with lattice proteins decreases the electron density of the ribosomes and perhaps alters their configu ration. The ribosomes must become either more compact or else slightly compressed perpendicular to the long axis of the chains to account for their smaller size within a chain or lattice. During release from the lattice, the original configuration and size is restored as indicated by the fact that terminal ribosomes were often intermediate in size between stored and free ribosomes. Large terminal ribosomes had the same electron density as free ribosomes. Lattice-like arrays are not restricted to the mouse. Similar structures have also been found in thin-sectioned oocytes of the rat and Golden hamster f8, 1 3, 14, 20, 23]. They have not been observed in other mammals [23]. Morphological differences in the arrays have been observed between the mouse, hamster, and rat [9, 23]. In the mouse there are many cross-linked chains forming a typical lattice configuration, while in the rat only single chains exist, and in the hamster double chains are found resembling a ladderExptl Cell Res 69 like structure. These single and double chains often form part of large parallel arrays. Whole mount preparations of rat or Chinese hamster oocytes did not reveal any obvious chains or lattices, however close linear asso ciations of ribosomes were found in favour able spreads. The failure to find intact chains in spread preparations of rat or hamster oocytes may reflect a difference in their stability between species. Assuming that the inactivation and storage of ribosomes in oocytes is a characteristic phenomenon of many diverse animal species, a more widespread distribution of lattice like arrays might be expected. It is quite possible however, that ribosomes could be stored in oocyte cytoplasm without the for mation of the elaborate configurations ob served in this study. Rabbit oocytes, which do not exhibit lattice formations, have many free ribosomes arranged in rosette-like clusters in the cytoplasm during maturation [26]. Whether these ribosomes are active or stored is not known. In comparison, free ribosomes or polysomes are not commonly observed in mature oocytes [2 1] or fertilized ova [19] of the rodents with lattice configurations. Pre sumably the bulk of the ribosomes in these oocytes are an integral part of the lattice structure. Obviously more work is required to determine if there is any morphological or biochemical evidence for ribosome storage in other species. It has been suggested that the arrays are primarily proteinaceous in nature [9, 22]. Evidence for this has largely been obtained by testing the effect of various fixatives on these structures. Glutaraldehyde, which is consi dered to be a protein fixative, was found to preserve the lattice, however osmium tetro xide, another protein fixative, was not a good preservative. The solubility of the lattice structure in permanganate fixative led Enders & Schlafke [9] to conclude that it was com- Storage posed of protein strands. They considered permanganate to be a poor protein preserva tive, but according to Glauert [1 1], perman ganate preferentially destroys cytoplasmic components having a high RNA content. Inter pretations of chemical composition based on such studies must be considered tentative at best, and in any case, do not seriously disagree with the findings of the present work in which the lattices are shown to b e composed o f both protein and RNA. It has been suggested [19] that the weak fluorescence in oocyte cytoplasm following acridine orange staining [2], and the weak ultraviolet absorption in this region [ l ] makes it unlikely that large quantities of RNA could be stored in the cytoplasm. Actually, signifi cant fluorescence and UV absorption were associated with granular elements but it was thought that this was due largely to the presence of mononucleotides [2]. These results are not easily reconciled with the present results. One possibility however, is that the association of the lattice protein with the ribosome alters the configuration of the ribosome such that the RNA is masked so that it can n o longer be detected using these techniques. Mazanek [ 1 4} initially suggested that the lattice-like configurations found in rat oocytes, and cells of the early cleavage divi sions, might be aggregates of linearly arranged ribosomes. Zamboni [25] has indi cated that the lattices are formed from poly somes in mouse oocytes. He found that the ribosomes became aligned in a curvilinear pattern and then fused to form chains and he concluded that the resulting structures were "lattices of fibrillar RNA". In rat and Golden hamster oocytes, Weakley [23] found a close association between "lamellae" (lat tices) and large ribosomes. The lattices first appear during oocyte maturation. In a study of oocyte development of ribosomes 369 in the Golden hamster, Weakley found no such structures in follicles sur rounded by a single layer of flattened granulosa ceHs. They first appeared as single chains i n the inner portion of the cytoi,1asm cuboida1 when the oocyte was surrounded granulosa cells. Mature oocytes contained many stacks of double chains . .r r,ee 1rmos,Jm: es. or dusters of ribosomes, were quite abundant in immature oocytes, but were uncomrnon after maturation, although some appe,art::a be e mbedded i n an amorphous matrix. Similar findings have been obtained in the mouse, where the lattices Zamboni increase in number during oocyte maturation and are abundant i n preovulatory and tubal ova. If the lattices are composed ribosomes to be used for protein <svr1th,�<:1 , during the early cleavage stages after ferti lization, the lattices should disappear early development with a concomitant appearance of free ribosomes and po1ysorr1es. No lattice-like arrays were found in blasto cyst preparations, but there were numbers of ribosomes, existing either in clusters, or attached to membranous mate contrast rial. These with those from mouse oocytes, where arrays were in abundance and there were few ribosomes. Corroborating evidence has been m the obtained by Schlafke & Enders rat. They found densely packed arrays of chains in the fertilized ovum, no granular endoplasmic reticulum nor any free ribosomes. A few small clusters of ribo somes appeared at the 2-4 cell stage. 8-cell stage, some ribosomes were attached to endoplasmic reticulum and a few were pn:seiu on the outer nuclear membrane. no noticeable disappearance of the arrays had yet been observed, this would not be clearly apparent at first because of the numbers initially present. In the cells Exptl Cell Res 69 370 G. D. Burkholder et al. blastocyst however, the arrays had started to maternal ribosomes would become available disappear and had become more disorganized. for protein synthesis. The association of protein with ribosomes At this time there was considerable granular endoplasmic reticulum and "clusters of poly in the lattice configuration very likely renders ribosomes filled much of the background the ribosomes inactive in protein synthesis. cytoplasm". The arrays had largely disap The existence of ribosome inactivating pro peared by the time of implantation [10]. The teins has previously been demonstrated i n disappearance of arrays and correlated in sea urchin eggs. Monroy e t al. [181 found crease in number of ribosomes or polysomes that ribosomes from unfertilized eggs are during early development would be expected inactive as sites for protein synthesis in in if, as the present study suggests, the arrays vitro systems, however they could be activated by prior exposure to trypsin. This suggests are a storage form of ribosomes. The present findings also correlate well that the ribosomes of unfertilized eggs are with what is known about the synthesis of i nactivated by a protein coat. Some doubts ribosomal RNA (rRNA) during oogenesis have been expressed (see [4, 1 2] for reviews) and during the early cleavage stages. Unfor concerning these results because untreated tunately , practically nothing is known about ribosomes from unfertilized eggs are actually rRNA synthesis in mammalian oocytes, how capable of synthesizing proteins using arti ever in Xenopus [5], there is a tremendous ficial mRNA, and are therefore not inactive, build up of rRNA during the diplotene lamp at least in systems using the synthetic tem brush stage of meiosis. This RNA is retained plates. It was suggested that the maternal by the oocyte for several months. After mRNAs, rather than the ribosomes, were fertilization, rRNA synthesis first begins inactivated by proteins. Recently however, during the 4-cell stage in the mouse [24), and Metafora et al. [15] have clearly shown that undergoes a tremendous increase after the the ribosomes from unfertilized sea urchin 8-cell stage [7], however these changes may eggs are less efficient than those from ferti also reflect an increase in the uptake of RNA lized eggs in supporting polypeptide synthesis precursors into the cells at this time [3]. Mintz using synthetic mRNA. Furthermore, they [17] has shown that protein synthesis occurs have obtained a protein factor from the in the mouse embryo shortly after fertili ribosomes of unfertilized eggs which is zation, i.e. before rRNA synthesis begins, capable of inhibiting polypeptide synthesis i n and this suggests that maternal ribosomes are a poly(U)-directed cell-free system containing functional during these early stages of devel active ribosomes from fertilized eggs. This opment. Although rRNA is synthesized protein inhibits the binding of mRNA and quite early during embryogenesis, this does aminoacyl tRNA to the ribosomes but whe not, in itself, indicate that functioning ribo ther it affects the binding sites for these somes are being produced. Little is known molecules or alters the configuration of the concerning the synthesis of ribosomal pro ribosome as a whole is not known. It is teins during preimplantation nor how quickly obviously necessary to exercise caution i n new ribosomes are formed or are capable of extrapolating from sea urchins t o mice, but functioning in protein synthesis. Fully func this example does set a precedent for the tional ribosomes may not be produced for existence of ribosome inactivating proteins some time after rRNA synthesis has been in oocytes. initiated and in the meantime the stored One intriguing question raised by the Exptl Cell Res 69 Storage of rib,,so111es present work concerns the possible association lattice structures from the oocytes, biochemical mRNA prior to their assembly into lattices. tion on the RNA studies using diverse animal species (see [4, for review) have shown that stable maternal mRNA is stored in the egg for use after fertilization. These maternal messen� gers are prevented from participating in protein synthesis until after fertilization, i.e. the message is "masked". It is generally believed that this masking is effected by the interaction of protein with mRNA, however there is some debate as to the site of template storage. In this regard, evidence obtained from sea urchin eggs [18] suggests that during oogenesis, a ribosome-mRNA complex is formed which is subsequently inactivated by a protein coat. It was postulated that, after fertilization, proteases were released which removed the protein coat and thereby ren dered the ribosome-mRNA complex active in protein synthesis. Other evidence (reviewed in [4, 12]) suggests that cytoplasmic particles other than ribosomes may be the site of maternal template inactivation. Neither of these two alternatives has been established definitively but it appears reasonable that messenger inactivation might occur at both ribosomal and non-ribosomal sites. Should this prove to be the case, there is a distinct possibility that maternal mRNA might also be associated with the ribosomes stored in the lattice configuration of mouse oocytes. When the individual ribosomes are released from the lattice after fertilization, the asso ciated messenger would be immediately available for translation. The present results, which rely heavily on the interpretation of morphological data, suggest that a biochemical approach might be very informative. If it proves possible to isolate the This work was supported a ship from the Medical Research Council of Canada to G. D. B., and by NIH grant GM-15886. REFERENCES 1 . Austin, C R & Braden, A W H, Aust j biol 6 (1 953) 324. 2. Austin, C R & Bishop, M W H, Exptl cell res 17 (1959) 35. 3. Daentl, D L & Epstein, C J, Dev biol 24 { 1 971) 428. 4. Davidson, E H, Gene activity in ment. Academic Press, New York 5. Davidson, E H, Allfrey, V G & Proc natl acad sci US 52 (1 964) 501 . 6. Do.nahue, R P, J exptl zool 1 69 7. Ellem. K A O & Gwatkin, R (1968) 3 1 1 . 8. Enders, A C , The biology of the blastocyst (ed R J Blandau) p. 71 . University of Chicago Press, Chicago (1971). 9. 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