Immunolocalization of three oocyte nuclear proteins during oogenesis and embryogenesis in Pleurodeles

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 715-728
Author(s):  
C. Abbadie ◽  
D. Boucher ◽  
J. Charlemagne ◽  
J.C. Lacroix
Keyword(s):  
Adult Development ◽  
Nuclear Proteins ◽  
Oocyte Nucleus ◽  
Lampbrush Chromosomes ◽  
Mitotic Chromosomes ◽  
Midblastula Transition ◽  
Adult Age ◽  
Young Embryo ◽  
Stock Pile ◽  
Oocyte Nuclear Proteins

The location of three proteins of the oocyte nucleus of Pleurodeles was studied during oogenesis and embryogenesis using monoclonal antibodies A33/22, C3/1 and C36/1. Immunoblotting of two-dimensional gel electrophoregrams of oocyte nuclear proteins showed that these antibodies recognized proteins whose relative molecular masses and isoelectric points were 80×103 and 6á4, 175×103 and 5 and 270×103 and 7, respectively. In the oocyte, all three proteins were nucleoplasmic; those revealed by antibodies A33/22 and C36/1 were detected on lampbrush chromosomes: the first one on the RNP matrix of the loops, and the second one on both the loops and the chromomeres. Protein A33/22 was observed in most nuclei during embryonic, larval and adult development, except for the young embryo, before the midblastula transition. The distribution of this protein in the oocyte and its behaviour during development suggest that it might be involved in the packaging of RNAs during transcription. Antibody C3/1 recognized an oocyte nucleoplasmic protein with biochemical and biophysical properties similar to those of protein N1-N2. After oocyte maturation, the protein moved into the cytoplasm of the animal hemisphere and, from fertilization to the midblastula stage, it shifted from the cytoplasm into the nuclei as cell division proceeded. Starting from the gastrula stage, this protein became specific to the endoderm nuclei. After hatching, it was no longer detectable. This behaviour seems to correspond to that of a nuclear protein issued from the maternal stock pile. Protein C36/1 behaved similarly during early development, but remained in most nuclei after neurulation until the adult age, with a pattern similar to that of protein A33/22. In addition, it was present on the mitotic chromosomes. Its association with mitotic as well as lampbrush chromosomes connects it with the DNP fibre proteins.

scholarly journals Chromosomes and Nucleoli of the Axolotl, Ambystoma Mexicanum

1966 ◽  
Vol 1 (1) ◽  
pp. 85-108
Author(s):  
H. G. CALLAN
Keyword(s):  
Low Temperature ◽  
Nuclear Periphery ◽  
Great Majority ◽  
Lampbrush Chromosome ◽  
Ambystoma Mexicanum ◽  
Oocyte Nucleus ◽  
Lampbrush Chromosomes ◽  
Mitotic Chromosomes ◽  
Nucleolar Material ◽  
Secondary Constrictions

Amongst the axolotl's haploid complement of fourteen mitotic chromosomes, one of the four largest, with a greater arm asymmetry than the other three, shows a nucleolar constriction subterminally in its shorter arm. Low-temperature treatment causes further secondary constrictions to appear; these constrictions enable most of the mitotic chromosomes to be identified; the constrictions occur at similar sites in the chromosomes of tail-fin epithelial cells, hepatocytes, and brain cells. Homology between the mitotic and oocyte (lampbrush) nucleolar organizers has been established, and thus the several hundred free nucleoli in oocytes are genetically related to the two nucleoli of diploid somatic interphases. During oocyte development the free nucleoli transform from solid structures to rings and back to solid structures again without detectable increase in number. During the contraction and aggregation of the lampbrush chromosomes within the oocyte nucleus as maturity approaches, in most axolotls the free ring-shaped nucleoli become stretched between the nuclear periphery and central chromosome group, and take on a characteristic beaded appearance. These transformations of the free nucleoli are largely paralleled by forms which nucleoli attached subterminally to the shorter arm of lampbrush chromosome III concurrently assume. The question as to whether fully developed nucleoli detach from the organizer loci and add to the population of free nucleoli in oocytes remains undecided. It may well be that virtually all the DNA-generators of free nucleoli detach from the organizer loci before starting to carry out nucleolar functions, and before there is any significant accumulation of protein and RNA around them. If so, the variability in quantity of attached nucleolar material may not reflect different states in a nucleolar synthesis and detachment cycle, but rather variation in the number of nucleolar DNA Anlagen which happen to remain attached to the organizer loci after the synthesis and detachment of the great majority of the Anlagen has ceased. In occasional oocytes the only chromosomal continuity maintained across the organizer locus consists of a nucleolar ‘double bridge’; this indicates that the genetically persistent (i.e. chromosomal) organizer DNA bears the same structural relationship to neighbouring parts of a lampbrush chromosome as any other chromomere with its attendant pair of lateral loops. The lampbrush chromosomes of the axolotl have been provisionally mapped. The centromeres are represented by short portions of chromosome axis without lateral loops, and there are two spheres close to the centromeres of both chromosome VI and chromosome XIII. Other recognition characters are inconspicuous or not very reliable, and features of the lampbrush chromosomes related to the low-temperature induced secondary constrictions of mitotic chromosomes have not been identified.

Differential accumulation of oocyte nuclear proteins by embryonic nuclei of Xenopus

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 829-846 ◽  
Author(s):  
C. Dreyer
Keyword(s):  
De Novo ◽  
Germinal Vesicle ◽  
Intrinsic Property ◽  
Nuclear Proteins ◽  
Cell Nuclei ◽  
Germinal Vesicle Breakdown ◽  
Early Stages ◽  
Nuclear Antigens ◽  
Gradual Process ◽  
Oocyte Nuclear Proteins

Oocyte nuclear proteins of Xenopus are distributed into the cytoplasm of the maturing egg after germinal vesicle breakdown. Later they are found in all cell nuclei of the embryo. At early stages of development, different nuclear proteins behave differently. A class of ‘early shifting’ antigens is accumulated by pronuclei and cleavage nuclei, whereas others appear to be excluded from the nuclei at early stages but are shifted into the nuclei at blastula or during and after gastrulation. Accumulation of ‘late-shifting’ nuclear antigens is a gradual process and occurs during a period characteristic of each protein. Multiple artificial pronuclei can be formed after injection of sperm nuclei, erythrocyte nuclei or pure lambda-DNA into unfertilized eggs. The artificial pronuclei accumulate early- but not late-shifting proteins. Early-migrating proteins rapidly accumulate into the germinal vesicle after de novo synthesis in the oocyte, indicating that the efficiency of translocation into nuclei is an intrinsic property of each protein. Artificial extension of the length of the cell cycle before midblastula transition does not lead to accumulation of the late-shifting nuclear antigens investigated.

scholarly journals Association of nucleoplasmin with transcription products as revealed by immunolocalization in the amphibian oocyte.

1986 ◽  
Vol 103 (3) ◽  
pp. 683-690 ◽  
Author(s):  
N Moreau ◽  
N Angelier ◽  
M L Bonnanfant-Jais ◽  
P Gounon ◽  
P Kubisz
Keyword(s):  
Transcriptional Activity ◽  
Light And Electron Microscopy ◽  
Nuclear Proteins ◽  
Oocyte Nucleus ◽  
Actin Network ◽  
Previously Treated ◽  
Pleurodeles Waltlii ◽  
Chromosome Axis ◽  
Lampbrush Loops ◽  
Nuclear Content

The oocyte nucleus of Pleurodeles waltlii contains a major 32,000-mol-wt acidic protein which is called nucleoplasmin. Rabbit antibodies were raised against total nuclear proteins from Pleurodeles oocytes. Affinity-purified antibodies directed against nucleoplasmin were prepared using antigens bound to nitrocellulose paper. The specificity of the antibody was controlled on two-dimensional electrophoretic gels of nuclear proteins. The intranuclear distribution of nucleoplasmin was analyzed by indirect immunofluorescence and the immunogold technique in light and electron microscopy. The antibody was tested on a spread of the nuclear content prepared in the presence of calcium, on the nuclear content spread in the presence of phalloidin so that an actin network appeared, and on a spread of nuclei from oocytes previously treated by actinomycin D. In all cases, nucleoplasmin appeared to be localized on the lampbrush loops, i.e., on the sites of transcription and, more specifically, on the ribonucleoprotein (RNP) particles; this protein was also associated with the RNP particles of the nuclear sap (free or inserted in the actin network). Nucleoplasmin was localized on large RNP particles that appeared when transcription was blocked. We never found this protein on the chromosome axis. These results suggest that nucleoplasmin may play a role in transcriptional activity.

An introduction to Programmes for Development

Development ◽  
1984 ◽  
Vol 83 (Supplement) ◽  
pp. 1-6
Author(s):  
Herbert C. Macgregor ◽  
Alma P. Swan
Keyword(s):  
Oocyte Nucleus ◽  
Nuclear Chromatin ◽  
Lampbrush Chromosomes ◽  
Unicellular Alga ◽  
Complex Molecules ◽  
Animal Development ◽  
Initial Tendency ◽  
Dna Strand ◽  
Acetabularia Mediterranea ◽  
The University

The symposium of which this book is a record was first suggested in the autumn of 1982. At that time, all members of the BSDB committee were persons who were primarily concerned with studies of animal development, and the initial tendency was to think along these lines. The cytology laboratory in the University of Leicester has a long tradition in studies of the lampbrush chromosomes that are found in the growing oocytes of most animals as well as in certain stages of the life cycle of at least one simple plant, the giant unicellular alga Acetabularia mediterranea (Callan, 1982). At some stage in very early diplotene of oogenesis of an amphibian, the oocyte nucleus begins to enlarge and many regions of the nuclear chromatin begin to transcribe RNA. RNA polymerase molecules attach to hundreds of sites along the chromosomes and move progressively along the DNA strand, synthesizing long and complex molecules of RNA as they go.

The role of Lampbrush Chromosomes in the formation of Nucleoli in Amphibian Oocytes

1965 ◽  
Vol s3-106 (75) ◽  
pp. 215-228
Author(s):  
H. C. MACGREGOR
Keyword(s):  
Phase Contrast ◽  
Nucleolar Organizer ◽  
Plethodon Cinereus ◽  
Oocyte Nucleus ◽  
Lampbrush Chromosomes ◽  
Triturus Cristatus ◽  
Amphibian Oocyte ◽  
Ambystoma Jeffersonianum ◽  
And Function

Theories concerning the mode of origin of peripheral nucleoli in amphibian oocytes have been examined and tested. In Triturus cristatus the giant fusing loops of the 3 shortest lampbrush bivalents resemble nucleoli when viewed in phase contrast and may be considered as possible sites of production of nucleoli. Giant fusing loops, however, differ from peripheral nucleoli in certain important respects, and animals lacking giant fusing loops on their lampbrush chromosomes nevertheless have normal peripheral nucleoli. Therefore, similarity in appearance between objects attached to lampbrush chromosomes and free peripheral nucleoli may not be significant. In oocytes of T. c. carnifex, T. c. karelinii, and T. c. danubialis, peripheral nucleoli do not increase in number during the lampbrush phase of oogenesis, except by division of pre-existing nucleoli towards the end of oogenesis. There are about 1,000 nucleoli per oocyte nucleus in each of these sub-species. In T. c. cristatus there are more nucleoli in large oocytes than in small ones, and it seems likely that in this sub-species the giant fusing loops add to the existing population of nucleoli in an oocyte by successively growing and shedding new nucleoli. A similar situation probably holds in Plethodon cinereus. Hexaploid oocytes from triploid females of Ambystoma jeffersonianum have 3 times as many nucleoli as diploid oocytes from diploid females of the same species. The number of nucleoli in an amphibian oocyte nucleus is therefore related to the number of sets of chromosomes in the cell. In yolky oocytes from hypophysectomized newts most peripheral nucleoli are firmly attached to the inner surface of the nuclear membrane; whereas in similar oocytes from unoperated or gonadotrophin-treated animals none of the nucleoli is so attached. On the basis of these observations 2 mechanisms are suggested for the formation of amphibian oocyte nucleoli. The first of these mechanisms probably operates in T. c. carnifex, where all peripheral nucleoli are formed before or soon after the chromosomes assume the lampbrush form, and no part of a lampbrush chromosome is involved in a process which adds to the existing population of nucleoli. The second mechanism probably operates in T. c. cristatus, where most of the peripheral nucleoli are formed before the lampbrush phase of oogenesis but a nucleolar organizer on the lampbrush chromosomes continues to grow and detach nucleoli throughout oogenesis. Both these mechanisms are discussed in terms of what is known of the chemical composition and function of peripheral nucleoli.

Nonhistone Proteins of the Oocyte Nucleus of the Newt

1974 ◽  
Vol 15 (1) ◽  
pp. 145-161
Author(s):  
R. J. HILL ◽  
K. MAUNDRELL ◽  
H. G. CALLAN
Keyword(s):  
Nuclear Membrane ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Germinal Vesicle ◽  
Oocyte Nucleus ◽  
Lampbrush Chromosomes ◽  
Nonhistone Proteins ◽  
Aerial Oxidation ◽  
A Minor

Evidence has been obtained which indicates that disulphide bond crosslinks contribute to the morphological integrity of isolated lampbrush chromosomes (both chromomeres and lateral loops) and nucleoli. It is suggested that the progressive formation of these bonds in vitro by aerial oxidation may provide the basis for the previously recognized time-dependent hardening or ‘denaturing’ of these structures. Manually isolated germinal vesicle nuclei have been massed and fractionated by low-speed centrifugation into nucleoplasm and chromatin. Phase-contrast microscopy demonstrates the chromatin to consist of nucleoli, lampbrush chromosomes and nuclear membranes. Urea gel electrophoresis has been employed to resolve the reduced and S-carboxymethylated proteins of whole nuclei into some 12 components, negatively charged at pH 8. The nucleoplasm alone gives an essentially similar pattern, but with the distinct depletion of one component and slight depletion of another. Both of these components are much enriched in the chromatin pellet where they predominate over all other proteins. The total chromatin has been subfractionated by microdissection, taking advantage of the differential attachment of nucleoli to the nuclear membrane at different stages of oogenesis. It is concluded that the nuclear membrane per se does not contribute to the major chromatin proteins. The two major polypeptides are components of the nucleoli. Preparations of isolated lampbrush chromosomes have not, to date, provided sufficient material to give a distinctive electropherogram; only one faint band, a major component of whole nuclei, was apparent. Sodium dodecyl sulphate gel electrophoresis has resolved some 25 components in whole nuclei, and again demonstrates the enrichment of the two major species in the total chromatin fraction. The apparent molecular weights of these two species are 43 kilodaltons and 110 kilodaltons. Approximately 20 minor species are also present in the chromatin and are obviously good candidates as components of the nucleolar and chromosomal structures. Histones, at most, make only a minor contribution to the overall chromatin protein population.

Proteins of the newt oocyte nucleus: analysis of the nonhistone proteins from lampbrush chromosomes, nucleoli and nuclear sap

1975 ◽  
Vol 17 (3) ◽  
pp. 579-588
Author(s):  
K. Maundrell
Keyword(s):  
Total Protein ◽  
Electrophoretic Analysis ◽  
Lampbrush Chromosome ◽  
Chromosome Preparation ◽  
Oocyte Nucleus ◽  
Protein Species ◽  
Lampbrush Chromosomes ◽  
Banding Patterns ◽  
Nonhistone Proteins

An electrophoretic analysis has been carried out on the total protein of lampbrush chromosomes, nucleoli and nuclear sap, obtained from newt oocyte nuclei. In each case, distinctive and heterogeneous banding patterns are observed. The absence of detectable quantities of histones in occyte chromatin is noted. In the case of the lampbrush chromosome preparation, it is concluded that all protein species are derived from the ribonucleoprotein matrix of the lateral loops.

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