Cytoplasm Of Unfertilized Eggs Research Articles

Pigments in egg cells and epidermis of sand dollar Scaphechinus mirabilis

The naphthoquinone pigments of epidermal cells and gametes of clypeasteroid sand dollar Scaphechinus mirabilis isolated enzymatically with subsequent alcohol extraction were studied. It was found that naphthoquinone pigments are present in the pigment cells incorporated into the jelly coat of oocytes but not in the cytoplasm of unfertilized eggs. Laser desorption/ionization mass spectra showed that the pigment cells incorporated into the coats of mature egg cells contain spinochromes E and D, and those found in the epidermis of adult urchins contain echinochrome A and spinochrome D. No spinochromes were found in eggs lacking coats. Fertilization of sea urchins is accompanied by oxidative burst associated with the production of hydrogen peroxide from molecular oxygen by quinone- and naphthoquinone-dependent oxidase Udx1. Since there were no quinones in the egg cells of S. mirabilis, it can be assumed that water-soluble spinochrome E, penetrating by diffusion into the egg cytoplasm from the pigment cells of the coat, is used for hydrogen peroxide stimulation of embryogenesis. Echinochrome A, the dominant echinochrome in epidermal cells of the adult urchin, is insoluble in water and, apparently, immobilized by the ethyl group as a hydrophobic anchor.

17β-Estradiol induces supernumerary primordial germ cells in embryos of the polychaete Platynereis dumerilii

In the polychaete Platynereis dumerilii exactly four primordial germ cells (PGCs) arise in early development and are subject to a transient mitotic arrest until the animals enter gametogenesis. In order to unravel the mechanisms controlling the number of PGCs in Platynereis, we tested whether the steroid 17β-estradiol (E2) is able to induce PGC proliferation, as it had been described in other species. Our data provide strong support for such a mechanism, showing that E2 significantly increases the occurrence of larvae with supernumerary PGCs in Platynereis in a dose dependent manner. E2 responsiveness is restricted to early developmental stages, when the PGCs are specified. During these stages, embryos exhibit high expression levels of the estradiol receptor (ER). The ER transcript localizes to the yolk-free cytoplasm of unfertilized eggs and segregates into the micromeres during cleavage stages. Nuclear ER protein is found asymmetrically distributed between daughter cells. Neither transcript nor protein is detectable in PGCs at larval stages. Addition of the specific estradiol receptor inhibitor ICI-182,780 (ICI) abolishes the proliferative effect of E2, suggesting that it is mediated by ER signaling. Our study reports for the first time an ER mediated proliferative effect of E2 on PGCs in an invertebrate organism.

Authors’ response: polarization microscopy and rescue ICSI

1 h We thank Dr Coticchio for his commentary on our article ‘Spindle examination in unfertilized eggs using the polarization microscope can assist rescue ICS’. His view on some key points in our study harmonize with ideas and issues we have discussed and debated over and over. Therefore, we are pleased to be able to highlight answers for issues he has raised. First, we know the issue of whether to perform rescue intracytoplasmic sperm injection (ICSI) or to cancel the current cycle is still under debate. The goal of assisted reproductive technology is to help infertile couples have a healthy baby, not increase the number of transferable embryos of compromised quality. The biological and clinical implications of oocyte ageing are well reported in numerous articles, including cytogenetic abnormalities in the resulting embryo following rescue ICSI (Morton et al., 1997; Pehlivan et al., 2004) as well as congenital abnormalities (Tarin et al., 2002). However, we should also consider that routine ICSI and IVF might also cause an increased risk of major congenital anomalies compared with natural conception (Wen et al., 2012). To our knowledge, there have been reports of successful births following rescue ICSI (Yuzpe et al., 2000; Shalom-PAZ et al., 2011). As previously shown from data in our centre (Shalom-PAZ et al., 2011), out of 20 pregnancies, only one was diagnosed with trisomy 21 and was terminated, while the other children were found to be

Expression and transient nuclear translocation of proprotein convertase 1 (PC1) during mouse preimplantation embryonic development

Preimplantation embryos express a number of hormones, neuropeptides, and membrane receptors known to derive from proteolytic activation of their precursors by the seven-member family of subtilisin-like, calcium-dependent serine proteinases known as proprotein convertases (PCs). The goal of this study was to determine the pattern of PC expression in mouse preimplantation embryos. Transcripts for all PCs, except PC2, were detected by reverse transcription-polymerase chain reaction (RT-PCR) in unfertilized and fertilized eggs. Furin, PACE4, PC1, and PC7 transcripts remained present at subsequent stages of preimplantation embryonic development, whereas the levels of transcripts for PC4 and PC5 gradually disappeared after the 2-cell stage. Proprotein convertase 1 (PC1) expression was further examined at the protein level. Immunoblotting revealed the presence of the zymogen and mature forms of this enzyme in eggs and embryos. Immunofluorescence laser confocal microscopy showed PC1-specific staining throughout the cytoplasm of unfertilized eggs. After fertilization, surprisingly, the staining was concentrated in pronuclei. It relocated to the cytoplasm at postzygotic stages and was particularly strong at junctions between blastomeres. The nuclear translocation of PC1 in fertilized eggs is probably mediated by its prodomain. Indeed, when transduced in human colon carcinoma LoVo cells, a mutant proPC1 incapable of cleaving off its prodomain was shown to accumulate in the nucleus. Furthermore, when N-terminally fused to green fluorescent protein, this domain was able to direct the reporter protein to the nucleus of these cells. Collectively, these data establish that eggs and preimplantation embryos express various PCs necessary for proteolytic activation of precursors of hormones and growth factors. They also raise the possibility of a nuclear function for PC1 during zygote formation.

Disappearance of an epithelial cell surface‐specific glycoprotein (Epith‐1) associated with epithelial–mesenchymal conversion in sea urchin embryogenesis

Cell surface modification during mesenchyme ingression was examined using a monoclonal antibody (mAb), anti-Epith-1 mAb, raised against a protein (Epith-1) that was confined to the lateral surface of the epithelial cells in embryo of the sea urchin, Temnopleurus hardwicki. The mAb epitope was N-glycosylated oligosaccharides of 160 kDa monomeric Epith-1 protein. The glycoprotein was negatively charged, and its isoelectric point (IP) was 4.98. The mAb, however, is not immunologically cross-reactive with other sea urchin embryos including Hemicentrotus pulcherrimus, Strongylocentrotus nudus, and Scaphechinus mirabilis. Epith-1 is present initially in the cytoplasm of unfertilized eggs. Cytoplasmic Epith-1 shifted to the cell surface to be integrated in plasma membrane during the first cleavage, and remained there during early embryogenesis by retaining the same relative molecular mass (Mr). During primary and secondary mesenchyme ingression periods, however, Epith-1 disappears from the presumptive mesenchyme cell surface that was associated with internalization of the protein. In plutei, an additional anti-Epith-1 mAb-positive protein appears at the 142 kDa region, which was not associated with any visible alteration of the histologic localization of the protein in larvae. Anti-Epith-1 mAb IgG did not inhibit the reaggregation of epithelial cells in vitro, which suggests that either the protein is not involved in cell-cell adhesion or that the mAb is not recognizing the active site of the protein.

Cytoplasm of sea urchin unfertilized eggs contains a nucleosome remodeling activity.

After fertilization the sea urchin sperm nucleus transforms into the male pronucleus which later fuses with the female pronucleus re-establishing the diploid genome of the embryo. This process requires remodeling of the sperm chromatin structure including the replacement of the sperm histones by maternally derived cleavage stage histone variants. In recent years, a group of protein complexes that promote chromatin-remodeling in an ATP-dependent manner have been described. To gain understanding into the molecular mechanisms operating during sea urchin male pronuclei formation, we analyzed whether chromatin-remodeling activity was present in unfertilized eggs as well as during early embryogenesis. We report that in the sea urchin Tetrapygus niger, protein extracts from the cytoplasm but not from the nucleus, of unfertilized eggs exhibit ATP-dependent nucleosome remodeling activity. This cytosolic activity was not found at early stages of sea urchin embryogenesis. In addition, by using polyclonal antibodies in Western blot analyses, we found that an ISWI-related protein is primarily localized in the cytoplasm of the sea urchin eggs. Interestingly, SWI2/SNF2-related proteins were not detected neither in the nucleus nor in the cytoplasm of unfertilized eggs. During embryogenesis, as transcriptional activity is increased an ISWI-related protein is found principally in the nuclear fraction. Together, our results indicate that the cytoplasm in sea urchin eggs contains an ATP-dependent chromatin-remodeling activity, which may include ISWI as a catalytic subunit.

Quantity of prelocalized maternal factor is associated with the timing of initiation of an epidermis-specific gene expression of the ascidian embryo.

We produced half-egg-volume ascidian embryos by dividing the unfertilized egg of Halocynthia roretzi at the equatorial plane, and investigated the timing of the initiation of the expression of three tissue-specific genes, a muscle-specific actin gene HrMA4, a notochord-specific gene As-T and an epidermis-specific gene HrEpiC in the half-egg-volume embryos of the animal side and those of the vegetal side. The timing of the onset of HrMA4 and As-T expression in both the animal- and vegetal-half embryos and that of HrEpiC expression in the animal-half embryos were essentially the same as that of normal embryos. In contrast, the timing of HrEpiC expression in the vegetal-half embryos was delayed by one division cycle compared with the normal embryos. This delay was partially recovered by increasing the amount of unfertilized egg cytoplasm of the animal hemisphere, suggesting that the timing of HrEpiC expression is regulated by the amount of a maternal factor which is distributed abundantly in the animal hemisphere of the unfertilized egg.

Expression of a Gene for Major Mitochondrial Protein, ADP/ATP Translocase, during Embryogenesis in the Ascidian Halocynthia roretzi: (Ascidian embryos/ADP/ATP translocase gene/maternal mRNA/mitochondria).

The ADP/ATP translocase is the most abundant integral protein of the inner mitochondrial membrane and it is encoded by the nuclear DNA. Because mitochondria in the ascidian egg appear to be segregated into blastomeres of muscle lineage, we examined the expression of a gene for ADP/ATP translocase during embryogenesis of the ascidian Halocynthia roretzi. Sequence analysis of a cDNA clone for the ascidian ADP/ATP translocase indicated that it contains a single open reading frame that encodes a polypeptide of 304 amino acids. The polypeptide showed extensive similarity to mammalian ADP/ATP translocases, with as much as 74% identity. The genome of H. roretzi contains a single gene, or two genes at most, for the protein. A large amount of maternal mRNA for ADP/ATP translocase was found in unfertilized eggs and early embryos. The amount of this mRNA decreased after the 64-cell stage, and the mRNA became barely detectable in tailbud embryos, although zygotic transcript for the protein was evident in adult tissues. Both in situ hybridization and Northern blot analyses demonstrated that the mRNA is distributed in the entire cytoplasm of unfertilized eggs. The mRNA is segregated during embryogenesis not only into blastomeres of muscle lineage but also into those of non-muscle lineage.

Role of the extracellular matrix protein thrombospondin in the early development of the mouse embryo.

The distribution of the extracellular matrix protein thrombospondin (TSP) in cleavage to egg cylinder staged mouse embryos and its role in trophoblast outgrowth from cultured blastocysts were examined. TSP was present within the cytoplasm of unfertilized eggs; in fertilized one- to four-cell embryos; by the eight-cell stage, TSP was also densely deposited at cell-cell borders. In the blastocyst, although TSP was present in all three cell types; trophectoderm, endoderm, and inner cell mass (ICM), it was enriched in the ICM and at the surface of trophectoderm cells. Hatched blastocysts grown on matrix-coated coverslips formed extensive trophoblast outgrowths on TSP, grew slightly less avidly on laminin, or on a 140-kD fragment of TSP containing its COOH terminus and putative cell binding domains. There was little outgrowth on the NH2 terminus heparin-binding domain. Addition of anti-TSP antibodies (but not GRGDS) to blastocysts growing on TSP strikingly inhibited outgrowth. Consistent with its early appearance and presence in trophoblast cells during implantation, TSP may play an important role in the early events involved in mammalian embryogenesis.

Cytostatic factor (CSF) activity in cytosols extracted from Xenopus laevis eggs

Cytostatic factor (CSF), found in the cytoplasm of unfertilized eggs of amphibians, causes metaphase arrest when microinjected into cleaving blastomeres. Although CSF from Rana pipiens eggs has been extracted and characterized, little is known about CSF extracted from eggs of other species. We investigated the conditions required to preserve CSF activity in cytosols extracted from Xenopus laevis eggs and found that it was necessary to expose the eggs to CO2 prior to extraction and that the extraction buffer must contain sodium beta-glycerophosphate. CSF activity disappeared after 24 h of storage at 2 degrees C. Cytological examination showed that the arrested blastomeres injected with cytosols had been arrested at metaphase and contained a spindle lacking polar asters, in which highly condensed chromosomes were embedded.

Cytostatic factor (CSF) in the eggs of Xenopus laevis

Cytostatic factor (CSF) in unfertilized egg cytoplasm causes metaphase arrest when microinjected into zygotes. This was originally described in Rana pipiens eggs. In Xenopus laevis, CSF has also been demonstrated, but only when the calcium-chelating agent, EGTA, was injected into the egg cytoplasm. In the present study, however, CSF was demonstrated in Xenopus eggs when donor egg activation was prevented by treatment with CO 2 and Mg 2+ instead of by EGTA, and recipient blastomere degeneration was prevented by increasing the KCl in the surrounding medium.

The sea urchin multicatalytic protease: purification, biochemical analysis, subcellular distribution, and relationship to snRNPs.

We have purified and extensively characterized a 19-S particle from sea urchin eggs. This particle is the sea urchin homologue of the "prosome", a particle originally identified in duck erythroblasts. We now show that these sea urchin prosomes contain multiple proteolytic activities. As shown for analogous particles from other cells, these particles hydrolyze synthetic substrates containing neutral hydrophobic or basic amino acids at the carboxy terminus of the synthetic peptides. They contain 16-20 small proteins ranging in molecular weight from 20,000 to 32,000. Peptide mapping shows that most of the polypeptides are unique, however, three exist in two isoelectric forms. We have investigated the possible function of the sea urchin multicatalytic proteases (MCPs) by determining their subcellular distribution, their relationship to egg snRNPs, and their possible role in translational repression. There are almost as many MCPs (2 x 10(8] as ribosomes (6.6 x 10(8] or mRNPs (1.8 x 10(7] per egg. This suggests that like ribosomes, the MCPs are stored in the egg for use during later development. We find that a substantial proportion of egg MCPs move into nuclei by the late blastula stage. Using a specific antibody against one of the sea urchin MCP proteins and antibodies against U1-U6, La, and Ro RNPs, we show that the sea urchin particle is distinct from these RNPs, although the anti-U1-U6 RNP antibody cross-reacts with a single MCP protein. In addition, the sea urchin MCP appears to be associated with a large structure in the cytoplasm of unfertilized eggs and is released under the same conditions that activate egg mRNPs in vitro.

Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo.

Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the CyIIIa cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of CyIIIa gene expression during embryogenesis. This gene is activated in late cleavage, exclusively in aboral ectoderm cell lineages. In this study, we focus on interactions demonstrated in vitro between sequences of the regulatory domain and proteins present in crude extracts derived from sea urchin embryo nuclei and from unfertilized eggs. Quantitative gel-shift measurements are utilized to estimate minimum numbers of factor molecules per embryo at 24 hr postfertilization, when the CyIIIa gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (Kr) of the various factors for their respective sites, relative to their affinity for synthetic DNA competitors. At least 14 different specific interactions occur within the regulatory regions, some of which produce multiple DNA-protein complexes. Values of Kr range from approximately 2 x 10(4) to approximately 2 x 10(6) for these factors under the conditions applied. With one exception, the minimum factor prevalences that we measured in the 400-cell 24-hr embryo nuclear extracts fell within the range of 2 x 10(5) to 2 x 10(6) molecules per embryo, i.e., a few hundred to a few thousand molecules per nucleus. Three developmental patterns were observed with respect to factor prevalence: Factors reacting at one site were found in unfertilized egg cytoplasm at about the same level per egg or embryo as in 24-hr embryo nuclei; factors reacting with five other regions of the regulatory domain are not detectable in egg cytoplasm but in 7-hr mid-cleavage-stage embryo, nuclei are already at or close to their concentrations in the 24-hr embryo nuclei; and factors reacting with five additional regions are not detectable in egg cytoplasm and are low in 7-hr embryo nuclei, i.e., less than or equal to 10% per embryo of the level they attain in 24-hr embryo nuclei. The rise in concentration of factors of the latter class could provide the proximal cause for the temporal activation of the CyIIIa gene at the early blastula stage.

Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA.

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.

Accumulation of fluorescently labeled actin in the cortical layer in sea urchin eggs after fertilization

Actin from sea urchin eggs was fluorescently labeled with fluorescein isothiocyanate (FITC), N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM), or 5-iodoacetamidofluorescein (IAF) and microinjected into sea urchin eggs and oocytes. It distributed evenly in the cytoplasm of unfertilized eggs. Upon fertilization, actin accumulated first around the sperm binding site and, soon afterwards, in the fertilization cone. The accumulation propagated all over the cortex after a latent period of 10-20 sec. In the case of Clypeaster japonicus eggs, propagation of the accumulation coincided with a shape change in the egg, suggesting that the accumulated actin in the cortex generates forces. FITC-actin was incorporated into microvilli and retained in the cortex after cleavage. On the other hand, DACM- or IAF-actin was not incorporated into microvilli and was dispersed from the cortex by cleavage. These differences may be attributable to differences in the properties of the actins labeled at different sites. After photobleaching by laser light irradiation, FITC- or IAF-actin redistributed in the cortex of fertilized egg as quickly as it did before fertilization. When an unfertilized egg was injected with both actin and a calcium buffer (intracellular free Ca2+ concentration 9 microM), the actin accumulation was similar to that during fertilization but without the latent period. This suggests that the accumulation depended on the increase in the intracellular free Ca2+ concentration. When the unfertilized egg was injected with 0.2 M EGTA after injection of labeled actin and then inseminated, it accumulated only in the protrusion of cytoplasm where the sperm had entered, and fertilization was not completed. In immature oocytes, the accumulation was observed in the cortical region, including the huge protrusion of the cytoplasm where the sperm had entered. These results suggest that actin accumulation in the sperm binding site plays an important role in the sperm reception mechanism of the egg.

Regulation of the cell cycle during early Xenopus development

Maturation-promoting factor (MPF) is a partially purified M-phase-specific activity that induces meiosis in frog oocytes and is detectable in mitotic lysates from cells of wide phylogenetic origins. We show here that without protein synthesis, addition and removal of MPF can drive the mitotic cycle in frog eggs, including nuclear membrane breakdown and reformation, chromosome condensation and decondensation, and suppression and initiation of DNA replication on endogenous DNA and on injected plasmid templates. We have also studied M-phase arrest induced by injection of unfertilized egg cytoplasm and show that this arrest blocks an endogenous cytoplasmic cell-cycle oscillator and causes the stabilization of MPF activity. The oscillator can be restarted by injection of Ca ++, which causes chromosome decondensation, reinitiation of DNA replication, and loss of MPF activity. We have looked in more detail at how DNA replication responds to the level of MPF and show that the effects are on the chromatin template and not the replication machinery. These results suggest that in Xenopus embryos cell-cycle events of the nucleus, including DNA replication and mitosis, are controlled by the level of MPF activity, which is driven by or may be part of an autonomous cell-cycle oscillator. The way in which a more complicated somatic cell cycle may arise from the simple embryonic cell cycle is discussed.

A study of hetero-specific sperm-egg interactions in the rat, mouse, and deer mouse using in vitro fertilization and sperm injection.

Hetero-specific fertilization of zone-free eggs is used in these experiments as a tool to analyze the barriers to hybridization and to gain insight into the mechanisms of normal fertilization. When the zonae of rat eggs, which are a barrier to hetero-specific fertilization, are removed with pronase, the eggs can be fertilized by mouse sperm and the zygotes start to develop normally. A rat egg fertilized with mouse sperm completes meiosis and forms both male and female pronuclei. Chromosomes from both parents are found on he spindle at the metaphase stage of the first cleavage division. Under present culture conditions, embryos develop only to the two-cell stage, but this initial development of the hybrid is apparently normal. The question of whether sperm and egg membrane fusion is requisite for normal development is addressed by injecting sperm directly into the cytoplasm of unfertilized eggs. The injection of mouse sperm into rat eggs frequently leads to activation and formation of male and female pronuclei. The first cleavage division is indistinguishable from that following hetero-specific fertilization. Capacitated and uncapacitated sperm react alike when injected into eggs. Egg activation, however, is necessary for male pronucleus formation. Sperm from the deer mouse Peromyscus maniculatus bairdii, which are incapable of fertilizing even zonea-free eggs, respond like mouse sperm when injected into rat eggs. These data indicate that sperm interactions with the egg cytoplasm are less species-specific than interactions at the egg surface. Furthermore, the normal surface interactions of sperm and eggs are not essential for the start of development.

Roles of Divalent Cations in Maturation and Activation of Vertebrate Oocytes

Summary The induction of meiotic maturation in Xenopus laevis oocytes, either by progesterone or ionophore A23187, is suppressed when intracellular free Ca is chelated by EGTA injection. However, the oocytes become resistant to the Ca deprivation after cytoplasmic development of maturation promoting factor (MPF) required for germinal vesicle breakdown (GVBD). MPF is rapidly inactivated by Ca, but requires Mg for its activity. As meiotic maturation proceeds towards metaphase II, the oocytes develop cytostatic factor (CSF) in their cytoplasm, which has the ability to arrest mitosis at metaphase when injected into cleaving blastomeres. Fresh extracts from mature oocytes of Rana pipiens contain a CSF, designated primary CSF (1° CSF), which is rapidly inactivated by Ca, and requires Mg for its activity. However, once inactivated with Ca, the extracts subsequently develop Ca-resistant CSF activity, designated secondary CSF (2° CSF). The Ca-sensitive 1° CSF or native CSF in unfertilized egg cytoplasm is inactivated when injected into zygotes at early times following fertilization, whereas the Ca-resistant 2° CSF is not. These observations lead to the hypothesis that a Ca surge in the early phase of fertilization is responsible for the inactivation of CSF in the oocytes, bringing about the resumption of meiosis, whereas Mg, which enhances CSF activity in vitro, serves to maintain the conditions necessary for metaphase arrest. This hypothesis appears to be supported by the observation in mouse oocytes that resumption of meiosis induced by ionophore A23187 is retarded in the presence of Mg, but is remarkably accelerated by Ca in the absence of Mg.

Maternal mRNA and protein synthesis in the embryo.

ABSTRACT According to the masked maternal messenger RNA hypothesis, a large part if not all the protein synthesis of early development is directed by mRNA already present in the cytoplasm of unfertilized eggs. This mRNA is supposed to be synthesized during oogenesis and stored in some unavailable form until some later time in development, when it is selectively associated with the translational machinery. To the indirect evidence, which is nevertheless very strong, there can now be added a direct proof of the hypothesis for the case of histone mRNA. The five main histones of sea urchin embryos are synthesized on small polyribosomes, directed in part by newly-synthesized messages that sediment as a group at about 9S. Some histone synthesis survives total transcription block, however, suggesting that maternal histone mRNA exists. In competition-hybridization experiments, the egg RNA is shown to contain sequences characteristic of functional, embryonic histone mRNA. The competing RNA is localized in ribonucleoprotein particles of egg homogenates that sediment at 20–40S. These same particles contain RNA that stimulates a cell-free heterologous system to synthesize sea urchin histones. The application of these facts to some problems of translation control and of development generally is discussed.

Cytonucleoproteins in cleaving eggs of Xenopus lœvis

ABSTRACT The cytoplasm of unfertilized eggs of Xenopus laevis induces DNA synthesis in a high proportion of adult and embryonic nuclei introduced into it (Graham, Arms & Gurdon, 1966). This induction of DNA synthesis is not due merely to the fact that the egg cytoplasm supplies precursors of DNA synthesis which might be absent from the nuclei, since adult liver nuclei, at least, of those nuclei which are induced to synthesize DNA after their injection into eggs, do not do so if incubated in vitro with DNA precursors (Arms, 1968). One of the ways in which the cytoplasm of unfertilized eggs might induce DNA synthesis is by supplying other molecules, such as various enzymes, to the introduced nuclei.