Histone synthesis in early amphibian development: histone and DNA syntheses are not co-ordinated

The synthesis of basic proteins has been studied in the oocytes, eggs and embryos of the South African clawed frog, Xenopus laevis. A group of newly synthesized proteins has been identified as histones by the following criteria: solubility properties; incorporation of [³H]lysine and [³H]arginine in the correct proportions, but lack of incorporation of [³H]tryptophan; co-cleotrophoresis with marker histones in various types of polyacrylamide gels, including a type run in two dimensions; peptide analysis of the arginine-rich fraction, F2A1. The four main histone fractions other than F1 were found to be synthesized at all stages of development. F1 histone synthesis was first detected at the late blastula stage.Rates of histone synthesis were estimated for the different stages of development and it was concluded that histone synthesis was not co-ordinated with DNA synthesis either temporally or quantitatively. Histone synthesis was unusual in the following major respects: histones were synthesized in oocytes, and yet in these cells DNA replication had not occurred for several months; histones were synthesized in activated or fertilized eggs at a rate far in excess (about 500 times) of the immediate requirements. We suggest that in order to provide enough histones for the late blastula embryo a store of histone is accumulated during the early cleavage stages and possibly during oogenesis.     

The stability and translation of sea urchin histone messenger RNA molecules injected into Xenopus laevis eggs and developing embryos

Embryonic sea urchin histone mRNA was injected into eggs and developing zygotes of Xenopus. The functional stability of the mRNA was monitored by separating newly synthesized sea urchin histones from those of Xenopus. Just as when injected into Xenopus oocytes, sea urchin H1, H2A, and H2B mRNA molecules have a functional half-life of about 3 hr in the developing embryo. This suggests that the endogenous Xenopus histone mRNA is also unstable and has a number of implications for the amount of histone mRNA that is stored in the oocyte and the time at which histone genes should become active in development. The injected mRNA is translated with little, if any, greater efficiency in the egg than in the oocyte. However, Xenopus histone synthesis increases about 20- to 50-fold during the transition from oocyte to egg. The injection experiments therefore suggest that this increase is brought about primarily by the mobilization of stored mRNA, rather than an increase in the efficiency of histone synthesis.     

The spatial relationship between heterochromatin protein 1 alpha and histone modifications during mouse oocyte meiosis

Heterochromatin protein 1 (HP1) is closely associated with diverse chromatin organization and function in mitosis. However, we almost know nothing about HP1 in mammalian oocyte. Here, we investigated the subcellular distribution of HP1α and its spatial relationship to histone modifications during mouse oocyte maturation. Dynamic migration of HP1α was observed in germinal vesicle with non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) oocytes, which may be essential for the transition of chromatin conformation during the development of antral oocytes. In meiosis, HP1α was clearly detectable at the periphery of chromosomes from pre-metaphase I stage to anaphase-telophase I stage. Spatial correlation between HP1α and histone modifications is highly variable around the time of meiotic resumption. In germinal vesicle oocytes, HP1α almost colocalized with all histone modifications examined in this study except for phosphorylation of serine 28 on histone H3. However, with the breakdown of germinal vesicle, HP1α was detected mostly in the chromosomal domains with strong phosphorylation of serine 10 and 28 on histone H3, and they also partially associated with methylated histones. These results presented the functional implication of histone modifications in the regulation of HP1α during oocyte maturation. In addition, we also showed that blocking the function of HP1α by microinjecting anti-HP1α antibody caused the delay of GVBD, however, this effect may not be achieved through modifying histones.     

Protein phosphatases are involved in the in vivo activation of histone H1 kinase in mouse oocyte

Histone H1 kinase and protein phosphorylation have been studied in mouse oocyte. Histone H1 kinase activity increases when the oocyte enters M-phase at the time of GVBD and is paralleled with a burst of protein phosphorylation. This activity dramatically drops after parthenogenetic activation induced by puromycin. Okadic acid (OA), a potent inhibitor of protein phosphatases, induces GVBD when oocytes are arrested in the first meiotic prophase by dbc-AMP; the continuous presence of the phosphatase inhibitor, however, inhibits the polymerization of metaphase microtubules. Following activation of metaphase II-arrested mouse eggs by puromycin, OA can induce the breakdown of the nuclear envelope and the activation of histone H1 kinase. This indicates that in the absence of protein synthesis, and therefore of cyclin synthesis, inhibition of protein phosphatases may be sufficient to induce the entry into M-phase during the first cell cycle of the mouse parthenogenetic activated oocyte.     

Program of early development in the mammal: Synthesis and intracellular migration of histone H4 during oogenesis in the mouse

Synthesis of histone H4 by mouse oocytes and unfertilized eggs has been examined by using a modified high-resolution two-dimensional gel electrophoresis procedure capable of resolving basic proteins (M. J. LaMarca and P. M. Wassarman, 1979, Develop. Biol.73, 103–119). Histones were separated on such gels and observed rates of incorporation of [³⁵S]methionine into histone H4 were converted into absolute rates of synthesis by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and unfertilized eggs Schultz et al., 1979a, Schultz et al., 1979b. Histone H4 was synthesized at all stages of oogenesis examined, and accounted for 0.07, 0.05, and 0.04% of total protein synthesis in growing oocytes, fully grown oocytes, and unfertilized eggs, respectively. During oocyte maturation the absolute rate of histone H4 synthesis decreased by about 40%, as compared to a 23% decrease in the rate of total protein synthesis during the same period. These measurements indicate that enough histone is synthesized during oogenesis in the mouse to support two to three cell divisions. Examination of the intracellular location of newly synthesized proteins in fully grown oocytes revealed that histone H4 was highly concentrated in the nucleus (germinal vesicle), whereas total protein and tubulin were not. Nearly 50% of the histone H4 synthesized during a 5-hr period was located in the oocyte's germinal vesicle, as compared to 1.9 and 0.9% for total protein and tubulin, respectively. These results are compared with those obtained using oocytes and eggs from nonmammalian animal species.     

Regulation of histone synthesis during early Urechis caupo (Echiura) development

The histones present in mature oocytes and embryos of Urechis caupo and their pattern of synthesis during early development have been characterized. Acid-soluble proteins extracted from mature oocyte germinal vesicles and from embryonic nuclei were analyzed by two-dimensional polyacrylamide gel electrophoresis. Histones are accumulated in the mature oocytes in amounts sufficient to provide for the assembly of chromatin through the 32- to 64-cell stage of embryogenesis. Two H1 histones, which appear to be variants, were found. Germinal vesicles and cleavage-stage nuclei are enriched in H1M (maternal). During late cleavage a faster-migrating H1, H1E (embryonic), appears among the nuclear histones and, as embryogenesis continues, replaces H1M as the predominant H1. No new core histone variants are detected during early development. Examination of [³H]lysine-labeled histones from germinal vesicles and embryonic nuclei reveals stage-specific patterns of histone synthesis. H1M is the major H1 species synthesized in mature oocytes. After fertilization, a switch to the predominant synthesis of H1E occurs. Comparison of the [³H]lysine incorporated into H1E and core histones indicates that H1E synthesis is disproportionately high from midcleavage through the midblastula stage. By the gastrula stage, a balanced synthesis of H1E and each core histone is established. The results indicate that there is noncoordinate regulation of H1 and core histone synthesis during Urechis development.     

Rates of histone synthesis during early development of Rana pipiens

Newly synthesized histones have been extracted from Rana pipiens oocytes or cleaving embryos previously injected with [³H]lysine or [³H]arginine. The radioactive proteins were fractionated by cation-exchange chromatography and electrophoresis on acid/urea or SDS-polyacrylamide gels; histones were identified by coelectrophoresis with authentic markers. From percentage total incorporation in the putative histones, and absolute rates of lysine or arginine incorporation, rates of histone synthesis were estimated. Rates of histone synthesis in two-cell embryos were at least 10-fold higher than in maturing oocytes. Between the two-cell and blastula stages, the rate increased an additional threefold, from about 1200 pg hr^?1 per embryo to about 4500 pg hr^?1 per embryo. While all histone classes are synthesized during cleavage, synthesis of the various classes is not coordinated; histones are not synthesized in the same relative proportions at which they are found in blastula chromatin. The synthesis of histone H4 in particular is barely detectable during cleavage. This, and other observations, suggested the existence of cytoplasmic histone pools. In approaching the possible existence of histone pools, the amount of H4 present in oocytes was determined. Oocytes contain about 74 ng of H4, an amount sufficient to allow development to the blastula stage. These data are compared to those reported by others on histone synthesis during cleavage in Xenopus.     

Histone messenger RNA synthesis and accumulation during early development in the echiuroid worm, Urechis caupo

RNA isolated from Urechis caupo mature oocytes and embryos was analyzed for the presence of histone messenger RNAs (mRNAs) by in vitro translation and by filter blot hybridization to determine the contribution of maternal and newly transcribed histone mRNAs to the pattern of histone synthesis during early development. Histone mRNAs were not detected in mature oocyte RNA which suggests that relatively few if any maternal histone mRNAs are sequestered in the mature oocytes. Histone mRNAs were detected in cleavage-stage RNA and increased in amount from midcleavage through late gastrula stages. The in vitro translation analysis also demonstrated that the amount of H1 histone mRNA in late cleavage- and early blastula-stage embryos exceeds that of the individual core histone mRNAs. The disproportionate accumulation of individual histone mRNAs correlates with the noncoordinate synthesis of H1 and core histones which occurs during early embryogenesis.     

On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes

In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972;Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly(A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70°C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly(A)^? RNA (7S–14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.     

On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis

We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.     

Temporal pattern of synthesis of the mouse cortical granule protein, p75, during oocyte growth and maturation.

We previously demonstrated that a protein of M(r) 75,000 (p75) is localized to cortical granules (CGs) in mouse oocytes and eggs and is released upon activation or fertilization of eggs (K.E. Pierce, M. C. Siebert, G. S. Kopf, R. M. Schultz, and P. G. Calarco, 1990, Dev. Biol. 141, 381-392). To examine the temporal pattern of synthesis of p75 during the early stages of CG formation, growing oocytes, which were isolated from juvenile mice, were incubated for 4 hr in medium containing [35S]methionine, and radiolabeled proteins were immunoprecipitated using an antiserum that detects p75. Synthesis of p75 is detected at low levels in the smallest oocytes examined (less than 20 microns). Synthesis of p75 relative to total protein synthesis increases about 12-fold during oocyte growth from the 20-40 microns size and then remains constant throughout the remaining period of oocyte growth (40-70 microns). In the fully grown, germinal vesicle (GV)-intact oocyte (70-80 microns), immunoprecipitated p75 comprises approximately 1.5% of the total amount of radiolabeled protein. Three hours after the transfer of these oocytes to a medium that supports resumption of meiosis and GV breakdown in vitro, oocytes subjected to a 1-hr labeling pulse display a 35% decrease in the relative level of p75 synthesis. By 15 hr of maturation, p75 synthesis was reduced to 14% of that in the fully grown, GV-intact oocyte and this is similar to the level of p75 synthesis in ovulated eggs. The level of p75 synthesis following in vitro translation of total egg RNA is only 38% lower than that obtained from total oocyte RNA. In addition, synthesis of p75 is observed following in vitro translation of oocyte, but not egg, poly(A)+ RNA. These results are consistent with p75 synthesis during oocyte maturation being under translational control.     

RNA 3' cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis

Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilized eggs of Xenopus laevis and their 3' cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3' cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3' cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilized eggs, the pre-mRNAs underwent 3' cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse beta-globin pre-mRNAs were not detectably 3' cleaved or polyadenylated in either microinjected oocytes or unfertilized eggs.     

Purification and properties of the Xenopus Hat1 acetyltransferase: association with the 14-3-3 proteins in the oocyte nucleus.

We have purified the Xenopus histone acetyltransferase Hat1 holoenzyme from oocytes. The holoenzyme contains the catalytic subunit Hat1, the retinoblastoma associated protein RbAp48, and members of the phosphoserine binding family of 14-3-3 proteins. We have determined that the Hat1 holoenzyme specifically acetylates free histone H4 but not nucleosomal histones. RbAp48 is a phosphoprotein that contains a consensus recognition motif for the 14-3-3 proteins. The 14-3-3 proteins provide a regulatory function for the activity of many phosphoproteins. We find that the hugely abundant Hat1 holoenzyme is present in 10 000-fold excess over somatic cell levels. The holoenzyme is localized in the oocyte nucleus where acetylated histones are stored. The oocyte form of the Xenopus Hat1 holoenzyme may represent a specialized storage form of histone acetyltransferase. Following oocyte maturation and subsequent embryogenesis, the Hat1 enzyme is redistributed to the cytoplasm, where new histones are synthesized.     

The synthesis and storage of histones during the oogenesis of Xenopus laevis

Further data, including two-dimensional gel electrophoresis and peptide mapping of newly synthesized proteins, confirms the view that oocytes make several types of histone. The newly synthesized histone is present in both nucleus and cytoplasm, but at a higher concentration in the oocyte nucleus and in great excess over the DNA binding sites. The unfertilized egg seems to contain a pool of histones detectable on two-dimensional electrophoretograms. The peptide maps of these proteins are consistent with their identification as histones. The egg contains enough histone to support nuclear replication through most of cleavage.     

Meiotic maturation of mouse oocytes in vitro: association of newly synthesized proteins with condensing chromosomes.

The nature, intracellular distribution, and role of proteins synthesized during meiotic maturation of mouse oocytes in vitro have been examined. Proteins synthesized during the initial stages of maturation are concentrated within the nucleus (germinal vesicle) and become intimately associated with the condensing chromosomes. Inhibition of protein synthesis during this period does not prevent germinal vesicle dissolution or chromosome condensation, but meiotic progression is blocked reversibly at the circular bivalent stage. A protein is synthesized during meiotic maturation of the mouse oocyte which exhibits several of the characteristics of the very lysine-rich histone, FI; this and other histones are phosphorylated during the initial stages of maturation. These results are discussed in relation to studies of meiotic maturation of oocytes from non-mammalian species and chromosome condensation in both oocytes and mitotic cells.     

Rate of protein synthesis in oocytes of Paracentrotus lividus

The rate of protein synthesis of Paracentrotus lividus oocytes in comparison with the rate in unfertilized eggs and embryos has been analyzed, both in vivo and after oocyte and egg isolation. It is suggested that oocytes synthesize proteins at the same rate as unfertilized eggs.     

Electrophoretic analysis of the stored histone pool in unfertilized sea urchin eggs: quantification and identification by antibody binding

A maternal store of histones in unfertilized sea urchin eggs is demonstrated by two independent criteria. Stored histones are identified by their ability to assemble into chromatin of male pronuclei of fertilized sea urchin eggs in the absence of protein synthesis, suggesting a minimum of at least 25 haploid equivalents for each histone present and functional in the unfertilized egg. In addition, electrophoretic analysis of proteins from acid extracts of unfertilized whole eggs and enucleated merogons reveals protein spots comigrating with cleavage stage histone standards, though not with other histone variants found in later sea urchin development or in sperm. Quantification of the amount of protein per histone spot yields an estimate of several hundred haploid DNA equivalents per egg of stored histone. The identity of some of the putative histones was verified by a highly sensitive immunological technique, involving electrophoretic transfer of proteins from the two-dimensional polyacrylamide gels to nitrocellulose filters. Proteins in amounts less than 2 x 10(-4) micrograms can be detected by this method.