Histone modifications accompanying the onset of developmental commitment

In the sea urchin, Strongylocentrotus purpuratus, three cell types comprise the 16-cell stage embryo: micromeres, macromeres, and mesomeres. We have analyzed these three cell types for nuclear proteins that were synthesized during the earliest stages of embryonic development. The most striking differences in composition of newly synthesized proteins were found between the micromeres, which are the most committed cell type, and the macromeres and mesomeres. First, the micromeres lacked triply modified forms of histone H3; the levels of doubly modified forms of H3 were also greatly reduced. In contrast, micromeres were enriched in a band which migrated at the position of unmodified, unacetylated, histone H3 protein. Second, the overall distribution of H2A histone variants differed among the three cell types. Compared with macromeres and mesomeres, micromeres had a higher ratio of alpha-stage to cleavage-stage (CS) histone H2A; the micromere nuclei were depleted by 50 and 35%, respectively, in embryonically synthesized histone CS-H2A. Third, micromeres displayed different profiles of H1 histones. (a) They contained a cleavage-stage H1 histone which migrated faster than that of macromeres and mesomeres. This protein displays the electrophoretic behavior expected for a protein with reduced levels of posttranslational covalent modification. (b) Micromeres also had reduced levels of an H1 histone (designated H1 alpha a) band found in the alpha-H1 region of macromeres and mesomeres. These changes in chromatin modification correlate with the degree of commitment of cells in the developing embryo; they may reflect differing activities of the chromatin modifying enzymes in the various cell types at the 16-cell stage. Thus, the newly synthesized chromatin proteins of the individual blastomere types already differ in the developing sea urchin by the 16-cell stage. We suggest that variations in histone subtypes and in the levels of activity of chromatin modifying enzymes, e.g., acetylases and phosphorylases, could be involved in commitment and differentiation of different cell types.     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.     

Nuclear lamin antigens are developmentally regulated during porcine and bovine embryogenesis

The nuclear lamins, proteins that reside on the inner face of the nuclear envelope, are thought to provide attachment sites for anchoring the chromatin to the nuclear envelope, thus facilitating the overall organization of the nucleus. The composition of the nuclear lamin proteins changes during differentiation and development in a variety of mammalian and nonmammalian tissues. Bovine and porcine oocytes and early embryos were prepared for immunocytochemical detection of nuclear lamins using three different antibodies (recognizing lamin B, lamins A/B/C, or lamins A/C). In both species, germinal vesicle nuclei and early cleavage stage nuclei react positively with the antibodies. However, on nuclei of bovine embryos, the A/C epitope was not detectable at the 16-cell stage, compact morula, spherical blastocyst, or the chorionic cell nuclei of a Day 35 conceptus, but was detectable on both amniotic and embryonic ectodermal cell nuclei of a Day 35 conceptus. All three antibodies reacted with nuclei from two bovine tissue culture cell lines (bovine embryonic cells and Madin-Darby bovine kidney cells) and one porcine kidney cell line. Nuclei in porcine embryos followed a similar pattern, except the loss of the A/C epitope occurred at the 8-cell stage and the epitope was absent from compact morula and spherical blastocyst stage nuclei. All interphase nuclei in both species reacted with both anti-lamin A/B/C and anti-lamin B antibodies, whereas metaphase chromosomes did not react with any of the lamin antibodies tested. The change in recognizing the lamin epitope occurred one cell cycle after the expected transition from maternal control to zygotic control of development. Nuclear transplantation showed that 16-cell stage porcine nuclei, which are lamin A/C negative, acquired the A/C epitope after transfer to an enucleated metaphase II oocyte. These results suggest that the A/C epitope is developmentally regulated.     

The Plant Nucleoskeleton: Ultrastructural Organization and Identification of NuMA Homologues in the Nuclear Matrix and Mitotic Spindle of Plant Cells

In the present work we investigate the structural organization of the nucleoskeleton ofAllium cepameristematic root cells. Resinless sections reveal for the first time a residual filamentous network in plant nuclei. This network is composed of branched knobbed filaments with associated globular structures, connected to the lamina and to the dense aggregates of different sizes. Results of immunoblotting show that many components of this network are homologues of intermediate filament-type proteins. NuMA, a coiled-coil protein related to intermediate filaments, found in animal cells, can also be detected in this plant nuclear matrix system. Immunofluorescence reveals a diffuse distribution of the animal NuMA homologues in plant nuclear core filaments in interphase. Resinless immunoelectron microscopy further reveals a distribution along the extended filaments and the dense aggregates. During mitosis, in contrast to the accumulation at the poles in animal cells, NuMA homologues in plant onion cells show a diffuse pattern, which may correspond to the spindle matrix. Our data are the first report of the conservation in plants of NuMA proteins, which may be involved in both nuclear and mitotic spindle organizations.     

Electric field-mediated BrUTP uptake by mouse oocytes, eggs, and embryos

Electropermeabilization was used to introduce 5-bromouridine 5'-triphosphate (BrUTP) into mouse oocytes, zygotes, 2-cell embryos, and parthenogenetic eggs containing nuclei transferred from 3T3 cells. BrUTP incorporated into nascent RNA was detected by indirect immunofluorescence. Two electric pulses of 100 micros duration and of 20 V strength applied at 10 mM concentration of BrUTP loaded most efficiently all cell types tested. Zygotes loaded with BrUTP developed for the next 20 hr in vitro and cleaved to 2-cell stage. The parameters of electric field which promoted BrUTP uptake were also efficient in inducing fusion of blastomeres of 2-cell embryos.     

Characterization of developmental arrest in early bovine embryos cultured in vitro

The susceptibility of early bovine embryos to developmental arrest ("blocking") in vitro was examined. Embryos, obtained from superovulated donors, were cultured in vitro in Ham's F10 culture medium or in vivo in sheep oviducts. Treatments were terminated on Day 7 post-donor estrus (estrus = day 0), and the embryos were evaluated for development. Experiment 1 tested whether the 8- to 16-cell block was reversible. One- to two-cell embryos were cultured in vitro to the 8-cell stage (2 d), then in vivo for 3 d; controls were cultured in vitro or in vivo for 5 d. Forty-two percent (19 45 ) of in vivo controls developed normally; none (0 55 ; 0%) of the in vitro controls cleaved past the 9- to 16-cell stage. Only 4% (2 48 ) of the embryos cultured to eight cells in vitro developed normally after culture in sheep oviducts, indicating that the block was irreversible. Irreversibility was not caused by overt cell death, since 33 33 (100%) of blocked embryos responded positively to fluorescien diacetate vital staining. Experiment 2 tested the effect of in vitro exposure at specific cell stages on subsequent in vivo development. Embryos at the 1- to 2-, 3- to 4-, 5- to 8- and 9- to 16-cell stages were assigned randomly to one of the following treatments: in vivo culture; in vitro culture; or 24 h in vitro culture, followed by in vivo culture. Subsequent in vivo development was affected by 24 h of in vitro culture (P<0.05) only in 3- to 4-cell embryos (11 41 , 27% vs 22 41 , 54% for in vivo controls). We conclude that 1) the block is a manifestation of in vitro exposure during the four- to eight-cell stage, and 2) the block, while irreversible, is not the result of overt embryonic death.     

Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo

Investigations were conducted to quantitate polyadenylic acid and estimate the synthesis of polyadenylated RNA in mouse embryos at several stages of preimplantation development. Poly(A) was assayed by molecular hybridization of total embryonic RNA with [³H]polyuridylic acid. The mean values of poly(A) in the ovulated oocytes and in the one-cell, two-cell, and blastocyst stages of the embryo were 1.9, 1.6, 0.68, and 3.8 pg, respectively. Synthesis of polyadenylated RNA was estimated by affinity chromatography of [³H]uridine-labeled embryo RNA on oligo(dT)-cellulose. The proportions of newly synthesized RNA bound by oligo(dT)-cellulose at the 2-cell, 8- to 16-cell, and blastocyst stages were 6.7, 3.5, and 3.3%, respectively. These results suggest that significant quantities of maternal mRNA are present during early development of the mouse, but that polyadenylation of RNA transcribed from the embryonic genome occurs as early as the two-cell stage.     

植物细胞中NuMA类似蛋白的分布及其在细胞周期中的变化

免疫荧光染色结果说明植物细胞核内含有与抗动物ＮｕＭＡ多抗呈阳性交叉反应的多肽。选择性抽提并结合免疫荧光染色结果说明这种多肽位于核基质纤维蛋白网络上。免疫印迹反应显示胡萝卜（ＤａｕｃｕｓｃａｒｏｔａＬ．）悬浮培养细胞核基质蛋白与抗动物ＮｕＭＡ蛋白多抗的阳性反应条带为７４ｋＤ和７６ｋＤ。有丝分裂各期免疫荧光染色的结果表明植物细胞中的ＮｕＭＡ类似蛋白在有丝分裂过程中呈现有规律的变化。结合选择性抽提的有丝分裂各期的免疫荧光染色的结果表明核基质在此过程中也发生明显变化。应用选择性抽提并结合ＤＧＤ包埋去包埋电镜技术对植物细胞间期及有丝分裂期核基质的形态结构进行了观察。结果显示胡萝卜悬浮培养细胞间期核内存在一个非染色质性的纤维蛋白网络体系,而在正处于分裂的细胞中则未观察到。以上结果说明ＮｕＭＡ类似蛋白是核基质的组分之一并与有丝分裂密切相关。     

Immunofluorescent localization of lysine-rich histones in isolated nuclei from adult and embryonic chicken erythrocytes

Isolated nuclei from adult chicken erythrocytes were stained by indirect immunofluorescence for histones H5 and H1. Nuclei in 0.15 M NaCl stained for H5 showed internuclear variations in intensity of fluorescence from bright to dim. Most individual nuclei were homogeneously stained although some showed a bright rim around a dimmer interior. Treatment of nuclei with Tween 80 in 0.15 or 0.03 M NaCl also gave internuclear variation in intensity. Adult nuclei stained for H1 (in 0.15 or 0.03 M NaCl) showed little internuclear variation; most nuclei stained brightly with a brighter rim. Simultaneous staining of H5 and H1 in the same nuclei confirmed the variable fluorescence of H5 and consistent fluorescence of H1. Most nuclei showed the presence of both histones. Nuclei from embryonic blood cells also showed considerable internuclear variation of H5 fluorescence and less variation with H1 staining. For both histones the proportion of brightly staining nuclei increased with embryonic development. Difficulties in interpreting quantitative variations in immunofluorescence are discussed.     

Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope. Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.