Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.     

Immunolocalization of a nuclear protein bound to the sphere organelle during oogenesis and embryogenesis inPleurodeles waltl

Summary The distribution of a nuclear antigen ofPleurodeles waltl oocytes, recognized by the monoclonal antibody B24/1, has been studied during oogenesis and early embryonic development. In stage I oocytes the antigen was localized in the nucleoplasm and on two atypical structures of lampbrush chromosomes, the spheres (S) and the mass (M). The immunostaining increased as the oocyte developed. In stage VI oocytes, the nucleoplasm and spheres showed intense staining. At this stage, the nucleoplasm often contained free spheres which were also labelled. The staining of M diminished during oogenesis, as did its size. Immunoblots of nuclear proteins of oocytes at different stages confirmed that there was an accumulation of this protein during oogenesis. During embryonic development, the nuclei of all the cells of blastula and gastrula were labelled by this antibody: there was no embryonic regionalization. Starting from the neurula stage, the staining progressively disappeared from the nuclei of ectodermal and mesodermal cells. In the tailbud stage, only the endodermal cell nuclei showed faint staining. Immunoblots of proteins from embryos of different stages showed that the quantity of this protein was constant until the young gastrula stage and then decreased progressively; in the young tailbud stage, this protein was practically absent. B24/1 is the first described protein of the sphere. This protein is accumulated in the oocyte nucleus and behaves like a maternal polypeptide, shifting early in the nuclei during embryonic development. Thus, B24/1 probably has a function required from the early developmental stages, perhaps in relation with small nuclear ribonucleoproteins.     

Dynamics of lamin A/C in porcine embryos produced by nuclear transfer

This study was conducted to investigate the presence of lamin A/C in porcine nuclear transfer embryos and to determine whether lamin A/C can serve as a potential marker for nuclear reprogramming. First, lamin A/C was studied in oocytes and embryos produced by fertilization or parthenogenetic oocyte activation. We found that lamin A/C was present in the nuclear lamina of oocytes at the germinal vesicle stage while it was absent in mature oocytes. Lamin A/C was detected throughout preimplantation development in both in vivo-derived and parthenogenetic embryos. Incubation of the activated oocytes in the presence of alpha-amanitin (an inhibitor of RNA polymerase II), or cycloheximide (a protein synthesis inhibitor) did not perturb lamin A/C assembly, indicating that the assembly resulted from solubilized lamins dispersed in the cytoplasm. In nuclear transfer embryos, the lamin A/C signal that had previously been identified in fibroblast nuclei disappeared soon after fusion. It became detectable again after the formation of the pronucleus-like structure, and all nuclear transfer embryos displayed lamin A/C staining during early development. Olfactory bulb progenitor cells lacked lamin A/C; however, when such cells were fused with enucleated oocytes, the newly formed nuclear envelopes stained positive for lamin A/C. These findings suggest that recipient oocytes remodel the donor nuclei using type A lamins dispersed in the ooplasm. The results also indicate that lamin A/C is present in the nuclear envelope of pig oocytes and early embryos and unlike in some other species, its presence after nuclear transfer is not an indicator of erroneous reprogramming.     

Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer

In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.     

Changes in plasma membrane protein composition during early development of the frog Rana ridibunda.

Plasma membranes of fully-grown oocytes and early developmental stage embryos of Rana ridibunda were isolated by differential and density gradient centrifugation; they were purified 18-fold as indicated by 5'-nucleotidase. The plasma membrane protein pattern of five different developmental stages was studied by two-dimensional polyacrylamide gel electrophoresis. More than 60 protein species could be detected by silver staining. Most of them are largely conserved during development. The rest either show precise stage specificity or exhibit stronger staining intensity at particular stages. The number of proteins specific for each stage is small, neurula being exempted. The changes observed in the plasma membrane profile during development are mostly prominent in a group of proteins with similar molecular weights (40-45 kDa) but with different pI values. The differences observed in the plasma membrane patterns are discussed in relation to the significance of each stage during development.     

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.     

Characterization and localization of tropomyosin proteins in Xenopus embryos with specific antibodies

In the process of monoclonal antibody (mAb) production against the 38kDa protein which is lacking in the gastrula arrested mutant embryos in Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) proteins in Xenopus embryos. The characterization of the corresponding antigens to those mAb was performed by immunoblotting and silver staining for two-dimensional (2-D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescent microscopy.
By 2-D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid-tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole.
Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms.     

Monoclonal antibodies against Drosophila ovaries: their reaction with ovarian and embryonic antigens

A library of monoclonal antibodies (MAbs) against Drosophila ovarian antigens was established. Each of the MAbs was characterized by its immunohistochemical binding pattern to sections from egg chambers at various stages of oogenesis. Sixteen of the 18 MAbs were found to bind to antigens in mature oocytes. Among the 16 antigens, two were also located in cytoplasm of cell types in the egg chamber other than the oocyte, at all stages of oogenesis. Four made their appearance in nurse cell cytoplasm at mid-vitellogenic stages and shifted to oocyte cytoplasm at a later stage, and ten appeared at the vitellogenic stage and confined their distribution to oocyte cytoplasm. All these antigens were distributed evenly in cytoplasm of mature oocytes. However, some of these antigens were noticed to change their distribution during early embryogenesis as to be localized in a specific region of embryos.     

Onset of nucleolus organizer activity in early mouse embryogenesis and evidence for its regulation

Ovulated mouse oocytes and preimplantation embryos were examined for NOR activity by means of selective silver staining. Evidence of the first staining activity appeared in two cell embryos, which was later followed by an increase in nucleolar activity, whereas the ovulated oocytes and pronuclei showed no such activity whatsoever. The staining of chromosomes was restricted to the nucleolus organizing region. Our results agree with earlier observations that genes for ribosomal RNA (rRNA) are transcribed as early as in the 2-cell stage in mouse embryogenesis. In addition to the nuclear staining we also observed some silver staining within the cytoplasm, at least from 4-cell stages onwards. Cytoplasmic staining was resistant to incubation with cycloheximide and actinomycin D. Nuclear staining was depressed, or even totally blocked, after actinomycin D incubation but was not blocked by cycloheximide. The onset of silver staining depends not on a specific embryonic stage but on the time interval following ovulation. This appears to indicate that the initiation of ribosomal cistrons is regulated by molecules which are activated or synthesized within the oocyte soon after ovulation.     

Spindle-pole organization during early mouse development

Spindle-pole organization during early mouse development was examined using a variety of immunological reagents that recognize centrosomal components. Spindle poles of unfertilized eggs and blastocysts were found to react positively with two antisera (centrin and NRS-01), whereas poles of activated eggs and early cleavage-stage embryos were negative when treated with the same sera. In contrast, a third antiserum (5051) showed positive spindle-pole staining throughout the preimplantation stages of development. Two monoclonal antibodies (MPM-1 and MPM-2) that are known to react with mitotic phosphoproteins were also used in this study. Both antibodies stained the cytoplasm of mitotic cells with extremely high intensity. In addition, MPM-2 was found to stain spindle poles. These results suggest that organizational changes in the spindle pole are occurring during early mouse development. Embryos homozygous for a recessive lethal mutation known as oligosyndactyly (Os) were also treated with the reagents described above. This mutation results in a metaphase arrest at the blastocyst stage with intact spindles being present. Spindle poles were observed in Os homozygous mutants stained with centrin, NRS-01, and 5051. However, when Os mutants were stained with the MPM monoclonal antibodies, about half of the mitotic cells completely lacked the dramatic cytoplasmic staining. This observation is in contrast to that observed for wild-type embryos, where greater than 95% of mitotic cells showed positive cytoplasmic staining.     

Development of cellular polarity of hamster embryos during compaction.

Development of cellular polarity is an important event during early mammalian embryo development and differentiation. Blastomeres of hamster embryos at various stages were examined by scanning electron microscopy (SEM) and immunocytochemical staining. SEM observations revealed that 1- to 7-cell-stage embryos showed a uniform distribution of microvilli throughout the cell surface. Microvillous polarization was initially noted in the blastomeres (10-35%) of 8-cell-stage embryos. The polarized microvilli were observed mostly in the basal region of cell-cell contact and occasionally at the apical, outward-facing surface of the blastomere. Fluorescein-isothiocyanate-conjugated concanavalin A failed to reveal any polarity in the blastomeres regardless of the stages of the embryos. Actin staining showed that microfilaments were present beneath the cell surface, and in addition, areas of cell contact were more heavily stained, indicating a thick microfilament domain. Microtubules were located throughout the cytoplasm and were heavily concentrated near the nucleus during interphase, although they became redistributed in the region of the mitotic spindle during karyokinesis. The position of nucleus changed from the cell center to the apical, outward-facing surface of the cell, and it distanced itself from the basal microvillous pole. It is suggested that the changes in the cell surface and nuclear position are the first manifestations of cell polarity in peri-compacted hamster embryos, which appear as early as the 8-cell stage; furthermore, the outward migration of the nuclei may parallel the redistribution of microtubules in the cytoplasm.     

Immunological analysis of chick notochord and cartilage matrix development with antisera to cartilage matrix macromolecules

Transverse frozen sections from the postcephalic region of stage 9-16 chick embryos and from the wing bud region of stage 17-31 embryos were stained with antibodies to the major extracellular matrix components of cartilage. These probes included unfractionated A1 and A2 antisera to the major cartilage proteoglycan, affinity-purified purified antibodies to the proteoglycan core protein and to Type II collagen, and a monoclonal antibody to keratan sulfate. In embryos as early as stage 10, notochord stained specifically with the keratan sulfate monoclonal antibody. At this stage the notochord, as well as surrounding tissues, were negative to cartilage proteoglycan and collagen antibodies. Positive staining with the latter probes was coordinately acquired by notochord cells and their accompanying sheath around stage 15, while surrounding tissues remained negative. At this stage, the ventral region of the perispinal cord sheath exhibited light staining with the proteoglycan and keratan sulfate antibodies though failing to react to Type II collagen antibodies. Positive staining of notochord and ventral spinal cord persisted through later developmental stages. As revealed by immunofluorescence, definitive vertebral chondroblasts first emerged at approximately stage 23 and definitive limb chondroblasts at stage 25. The results are discussed in terms of the possible multiple roles of notochord in early embryogenesis.     

小鼠母源因子对早期胚胎发育的影响

在脊椎动物中发育过程中,卵母细胞要经历MII期停滞、受精、早期胚胎发育的启动、胚胎基因组的转录激活、并指导完成个体的发育过程。同时,核移植过程中,分化的细胞核在去核的卵母细胞中能够重编程到胚胎早期的状态并能完成个体的发育过程。在这些发育过程中母源因子都发挥了极其的重要作用。在小鼠胚胎发育研究中发现,小鼠的基因组激活发生在2细胞期,这一时期标志着合子的发育由卵母细胞控制向胚胎控制的过渡,期间发生一系列复杂的生化过程。体外培养的小鼠的胚胎的发育阻断也易发生的2细胞时期。因此对卵母细胞及早期胚胎母源因子的研究,将有利于了解早期体外培养胚胎和克隆胚胎发育失败的原因,为提高体外培养和克隆胚胎发育的成功率提供理论的基础。     

Remodeling of Centrosomes in Intraspecies and Interspecies Nuclear Transfer Porcine Embryos

Remodeling of donor cell centrosomes and the centrosome-associated cytoskeleton is crucially important for nuclear cloning as centrosomes are the main microtubule organizing centers that play a significant role in cell division and embryo development. Centrosome dysfunctions have been implicated in various diseases including cancer and metabolic disorders and may also play a role in developmental abnormalities that are frequently seen in cloned animals. In the present studies we investigated microtubule organization and the reorganization and fate of the integral centrosome protein γ-tubulin and the centrosome-associated protein centrin in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using antibodies to γ-tubulin or GFP-centrin transfected mouse fibroblasts as donor cells. Microtubules were stained with antibodies to α-tubulin. In-vitro-fertilized oocytes and nuclear transfer (NT) reconstructed oocytes were sequentially analyzed at different developmental stages. Epi-fluorescence results revealed mitotic spindle abnormalities in NT embryos during the first cell cycle (39.4%, 13/33) which were significantly higher than those in IVF embryos (17.0%, 7/41). The abnormalities in IVF embryos are due to polyspermy while the abnormalities in NT embryos are due to donor cell centrosome dysfunctions. In the NT embryos with abnormal microtubule and centrosome organization, γ-tubulin staining revealed multipolar centrosome foci while DAPI staining showed misalignment of chromosomes. In intraspecies and interspecies embryos the GFP-centrin signal was detected until 3 hrs after fusion. GFP-centrin was not detected at 8 hrs after NT which is consistent with previous results using anti-centrin antibody staining in intraspecies NT porcine embryos. These data indicate that 1) abnormalities in microtubule and centrosome organization are associated with nuclear cloning at a higher rate than observed in IVF embryos; 2) centrosome and cytoskeletal abnormalities in IVF embryos are due to polyspermy while centrosome and cytoskeletal abnormalities in NT embryos are due to donor cell centrosome dysfunctions; and 3) GFP-centrin of the donor cell centrosome provides a reliable marker to follow its fate in intraspecies reconstructed embryos.