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.     

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 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.     

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1⁰ protein was predominant in G₀ cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.     

Differential in vivo binding dynamics of somatic and oocyte-specific linker histones in oocytes and during ES cell nuclear transfer

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.     

Mouse oocytes and early embryos express multiple histone H1 subtypes

Oocytes and embryos of many species, including mammals, contain a unique linker (H1) histone, termed H1oo in mammals. It is uncertain, however, whether other H1 histones also contribute to the linker histone complement of these cells. Using immunofluorescence and radiolabeling, we have examined whether histone H10, which frequently accumulates in the chromatin of nondividing cells, and the somatic subtypes of H1 are present in mouse oocytes and early embryos. We report that oocytes and embryos contain mRNA encoding H10. A polymerase chain reaction-based test indicated that the poly(A) tail did not lengthen during meiotic maturation, although it did so beginning at the four-cell stage. Antibodies raised against histone H10 stained the nucleus of wild-type prophase-arrested oocytes but not of mice lacking the H10 gene. Following fertilization, H10 was detected in the nuclei of two-cell embryos and less strongly at the four-cell stage. No signal was detected in H10 -/- embryos. Radiolabeling revealed that species comigrating with the somatic H1 subtypes H1a and H1c were synthesized in maturing oocytes and in one- and two-cell embryos. Beginning at the four-cell stage in both wild-type and H10 -/- embryos, species comigrating with subtypes H1b, H1d, and H1e were additionally synthesized. These results establish that histone H10 constitutes a portion of the linker histone complement in oocytes and early embryos and that changes in the pattern of somatic H1 synthesis occur during early embryonic development. Taken together with previous results, these findings suggest that multiple H1 subtypes are present on oocyte chromatin and that following fertilization changes in the histone H1 complement accompany the establishment of regulated embryonic gene expression.     

Qualitative differences in nuclear proteins correlate with neuronal terminal differentiation

1. Protein composition of neuronal nuclei was studied at two stages of brain maturation, i.e., before (embryonic day 16; E16) and after (postnatal day 10; P10) shortening of the nucleosomal repeat length. Glial nuclei were analyzed in parallel as a control. 2. Total nuclear or HCl- and 5% perchloric acid (PCA)-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1 zero with differentiation, although the H1 class undergoes an overall decrease. 4. The chromatin of mature neurons is also enriched in the ubiquinated form of histone H2A (A24), while the high-mobility group (HMG) proteins 1 and 2 seem to decrease slightly relative to core histones. 5. Both quantitative and qualitative differences in the abundance of nonhistone proteins relative to histones accompany neuronal terminal differentiation.     

Changes in nuclear localization of An3, a RNA helicase,during oogenesis and embryogenesis in Xenopus laevis

The immunolocalization of An3 protein, an ATP-dependent RNA helicase and a member of the DEAD box family, was compared with the localization of fibrillarin, a protein essential for rRNA processing, and snRNPs, which are involved in mRNA splicing reactions, during oogenesis and embryogenesis in Xenopus laevis. Although An3 protein was detected in the cytoplasm of all stages of oocytes, in most stages An3 protein was also present in the nucleus. Prior to stage I An3 protein was uniformly dispersed throughout the entire germinal vesicle; from stages I to V it was in nucleoli. By stage VI nucleolar labeling with anti An3 disappeared and the protein was no longer present within nuclei. An3 reactivity was also present throughout the nuclei of follicle cells surrounding prestage I to stage VI oocytes. Both cytoplasmic and nuclear An3 staining were present in cells of stages 8 to 35 embryos; however, nuclear staining was punctate and uniformly distributed throughout the nucleoplasm. Fibrillarin was diffusely distributed throughout the entire germinal vesicle prior to stage I, localized exclusively to nucleoli of oocytes between stages I and VI and in nucleoli of stages 12 and 35 embryonic cells. Reactivity for snRNPs (anti-Sm) in germinal vesicles of prestage I oocytes was diffuse, and similar to the distribution of An3 and fibrillarin; in later stage oocytes anti-Sm staining was restricted to a population of granules, much fewer in number and more heterogeneous in size than nucleoli. Anti-Sm activity was apparent in nuclei of embryonic cells of stages 8 to 35 embryos. Although colocalization of the Sm epitope and An3 was not observed in developing oocytes and in embryonic cells, Sm reactive material was frequently found in close association with An3-positive nucleoli (oocytes) and nuclear deposits (embryonic cells). In stage IV and V oocytes treated with actinomycin D (4 μg/ml) to inhibit rRNA synthesis, nucleoli, which continued to possess fibrillarin, lacked An3; staining of follicle cell nuclei for An3 was unchanged. Treatment with 200 μg/ml actinomycin D to block mRNA synthesis, inhibited An3 but not fibrillarin staining in nuclei of prestage I oocytes and follicle cells. The changing patterns of An3 reactivity and the differential effects of actinomycin D on such localizations observed here are consistent with a role for An3 in the processing/production of RNA. © 1996 Wiley-Liss, Inc.     

An 83-kDa embryonic-type nuclear antigen is detected within the germinal vesicles of oocytes of the ascidianHalocynthia roretzi

Summary Monoclonal antibodies were raised against germinal vesicles which were isolated from fully grown oocytes of the ascidianHalocynthia roretzi. Immunoblot analyses revealed that one of the antibodies, designated Hgv-2, recognized a single band with a molecular weight of about 83 kDa. The antibody, visualized by indirect immunohistochemistry, reacted only with the germinal vesicles of oocytes and did not react with test cells, follicle cells, and other somatic cells of the gonad. During embryogenesis the antigenicity was found in interphase nuclei of all embryonic cells. The antibody did not react with chromosomes or the mitotic apparatus. The antigenicity was retained by interphase nuclei of larval cells, but it disappeared from nuclei of juveniles about 7 days after metamorphosis.     

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.     

Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse

Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression.     

Correlation of chromatin composition with metabolic changes in nuclei of primitive erythroid cells from chicken embryos

Changes in levels of biosynthesis of DNA, RNA, and histones were compared with relative proportions of each histone class during primitive erythropoiesis in embryonic chicks. We confirmed that erythrocyte-specific histone 5 (H5) was substantial in the earliest accessible, erythroblast-enriched stage and that it doubled in relative amount between polychromatic and orthochromatic stages to about 1 mol per 2 mol of each nucleosomal histone, still considerable less than in adult definitive erythrocytes. No other histones changed during primitive erythropoiesis, but the molar proportion of histone 1 (H1) always exceeded that of H5 in these cells, unlike definitive erythrocytes. The increase in content of H5 was accompanied by continued decline in synthesis of the other histones and DNA. The accumulation of H5 during development appears to occur in steps corresponding to the maturation of the primitive and definitive erythroid cell lines. Lysine-rich histones were more easily extracted from nuclei of the erythrosynthesis in whole cells and in isolated nuclei.     

Developmental Patterns in Histones and Histone Synthesis During Early Embryogenesis of the Sea Urchin Paracentrotus lividus

Abstract. Striking developmental changes in histone and histone synthesis in sea urchin embryos were observed in three histone classes, H1, H2A and H2B. In each case there is a shift in histone synthesis from the early cleavage stage types to other types of histones at the morula stage; Two new forms appear after the blastula stage. In addition, multiple changes in histone types were found during gameto-genesis in the male and female gonads where specific histones, different from the embryonic histones, were observed.     

Nuclear histone deacetylases are not required for global histone deacetylation during meiotic maturation in porcine oocytes

Histone acetylation plays an important role in the regulation of chromatin structure and gene function. In mammalian oocytes, histones H3 and H4 are highly acetylated during the germinal vesicle (GV) stage, and global histone deacetylation takes place via a histone deacetylase (HDAC)-dependent mechanism after GV breakdown (GVBD). The presence of HDACs in the GVs of mammalian oocytes in spite of the high acetylation states of nuclear histones indicates that the HDACs in the nucleus are inactive but become activated after GVBD. However, the fluctuation pattern, the localization of HDAC activity during meiotic maturation and, moreover, the responsibility of nuclear HDACs for global histone deacetylation are still unknown. Here, we demonstrated using porcine oocytes that total HDAC activity was maintained throughout meiotic maturation, and high HDAC activity was observed in both the nucleus and the cytoplasm at the GV stage. The experiments with valproic acid (VPA), a specific class I HDAC inhibitor, revealed that the HDACs in GVs were class I, and those in the cytoplasm were other than class I. Interestingly, VPA had no effect on global histone deacetylation after GVBD, indicating that nuclear HDACs were not required for global histone deacetylation. To confirm this possibility, we removed the nuclei from immature oocytes, injected somatic cell nuclei into the enucleated oocytes, and showed that injected somatic cell nuclei were dramatically deacetylated after nuclear envelope breakdown. These results revealed that nuclear contents, including class I HDACs, are not required for the global histone deacetylation during meiosis, and that cytoplasmic HDACs other than class I are responsible for this process.     

Protein synthesis during meiotic maturation of mouse oocytes in vitro: Synthesis and phosphorylation of a protein localized in the germinal vesicle

Isolated fully grown mouse oocytes, arrested in dictyate of the first meiotic prophase, synthesize a protein with an apparent molecular weight of 28,000 which is localized in the germinal vesicle of the oocyte (germinal vesicle-associated protein; GVAP). Analyses of the distribution of GVAP have been carried out on SDS-polyacrylamide gels using oocytes cultured in vitro in the presence of [³⁵S]methionine or [³H]lysine and germinal vesicles isolated individually from these cultured oocytes. The results of such analyses show that GVAP contains only about 2% of the total radiolabel incorporated into mouse oocyte proteins, but as much as 40% of the total radiolabel incorporated into proteins associated with isolated germinal vesicles. These measurements indicate that GVAP is at least 1000-fold more concentrated in the germinal vesicle than in the cytoplasm of the oocyte. Furthermore, the synthesis and phosphorylation of GVAP are apparently terminated at a time which coincides with germinal vesicle breakdown during spontaneous meiotic maturation of mouse oocytes in vitro. Although the exact nature of GVAP is not known as yet, it appears to be an example of a protein that is selectively sequestered in the germinal vesicle of the oocyte during oogenesis and whose synthesis and modification are dependent upon the presence of an intact germinal vesicle.     

Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab,Eriocheir sinensis

During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis) is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae). The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin.