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.     

DEVELOPMENT OF THE NUCLEAR STRUCTURE AND METABOLISM DURING OOGENESIS OF PERINEREIS CULTRIFERA (ANNELIDA,POLYCHAETA)

An ultrastructural study of the nucleus was carried out, during oogenesis of Perinereis cultrifera, accompanied by an autoradiographic and biochemical study of the syntheses of RNA. The nucleus encloses formations deriving from the dispersal of meiotic chromosomes and a voluminous nucleolus. The latter undergoes morphological development of which each stage is characteristic of a stage of oogenesis. The autoradiographic study shows that the synthesis of RNA of extra-nucleolar origin is highly intense in young oocytes (during the stages of previtellogenesis and vitellogenesis) and that it decreases in older oocytes. The synthesis of RNA of nucleolar origin is very weak during previtellogenesis, increases during vitellogenesis, which is the stage at which it reaches its peak, and then decreases during the stages of the development of cortical alveoli and of maturity. These autoradiographic results are confirmed by a biochemical study which shows that once an oocyte diameter of 80 μm is reached (mid-vitellogenesis), the specific radioactivity of 18 and 28 S rRNA and of 4 and 5 S RNA decreases progressively up to the end of oogenesis.     

Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy

Anti-tubulin immunofluorescence and laser-scanning confocal microscopy were used to examine microtubule organization during Xenopus oogenesis (Dumont stages I-VI). Stage I oocytes contained a poorly ordered microtubule array, characterized by concentrations of microtubule in the cortex, surrounding the germinal vesicle, and associated with the mitochondrial mass. No focus of microtubule organization was detectable by optical sectioning or in microtubule regrowth experiments, suggesting that stage I oocytes lack a functional MTOC. The microtubule array becomes progressively more complex and polarized during oogenesis; an extensive array of microtubules and microtubule bundles was apparent in the animal hemisphere of stage VI oocytes, and a less ordered array was observed in the vegetal hemisphere. A dense network of microtubules surrounded the germinal vesicle, apparently extending from a tubulin- and microtubule-rich region of cytoplasm adjacent to the vegetal surface of the GV. The organization of microtubules in normal oocytes, in oocytes recovering from cold-induced microtubule depolymerization, and in enucleated oocytes, suggested that the germinal vesicle serves as an MTOC in stage VI oocytes. Antibodies to acetylated alpha-tubulin revealed numerous acetylated, presumably stable, microtubules in stage I and stage VI oocytes. The array of oocyte microtubules thus might function as a stable framework for the localization of developmentally important molecules and organelles during oogenesis.     

Expression of DNA ligases I and II during oogenesis and early development of Xenopus laevis.

We have analyzed the expression of DNA ligase I protein during oogenesis and early development of Xenopus laevis. The protein is already present in stage I oocytes and then accumulates throughout oogenesis to reach a steady state level by stage VI. It remains at this level at least until tadpole stage. In stage VI oocytes DNA ligase I protein is almost exclusively localized in the germinal vesicle. We have partially purified a DNA ligase II activity from stage VI oocytes, unfertilized eggs, and stage 8 embryos. An 80-kDa polypeptide can be specifically adenylated in all three purified extracts. It is not recognized by antibodies directed against DNA ligase I and is active on oligo(dT)-poly(rA) substrate. It could therefore represent DNA ligase II protein. The presence of both DNA ligases I and II in oocytes and embryos is inconsistent with the DNA ligase model that had been previously proposed for amphibia.     

Elongation factor 1 alpha (EF-1 alpha) is concentrated in the Balbiani body and accumulates coordinately with the ribosomes during oogenesis of Xenopus laevis

In Xenopus laevis oocytes two distinct systems catalyze the mRNA-dependent binding of aminoacyl tRNA to the A site of ribosomes. These systems are elongation factor 1 alpha (EF-1 alpha) and the 42S nucleoprotein particle. This particle is also implicated in the long-term storage of 5S RNA and aminoacyl tRNA during early oogenesis. We report here that the ribosomes and the storage particles are distributed uniformly in the cytoplasm of previtellogenic (stage I) oocytes. In contrast, EF-1 alpha is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body. When the Balbiani body disperses in early vitellogenic oocytes (stage II), EF-1 alpha becomes evenly distributed in the cytoplasm. The main phase of EF-1 alpha accumulation follows the disappearance of the 42S particles (stage II), but coincides with the main phase of ribosome accumulation (stages III and IV).     

Analysis of the coding activity and stability of messenger RNA in Drosophila oocytes.

The coding activity of the messenger RNA in the ooplasm of late stage 14 (S14) oocytes of Drosophila melanogaster was analyzed by labeling the oocytes in vitro with [³⁵S]methionine and examining the labeled products by two-dimensional gel electrophoresis and fluorography. This analysis was done both with newly formed S14 oocytes from rapidly laying females and with S14 oocytes stored for about 10 days in females that were prevented from laying. Comparison of the fluorographs showed that the proteins labeled in the newly formed oocytes were also labeled in the stored oocytes. Thus, the coding activity of S14 oocyte messenger RNA appears to remain stable during prolonged storage in utero. The oocyte proteins synthesized during oogenesis and incorporated into S14 oocytes were labeled in vivo by injecting [³⁵S]methionine into newly eclosed females, and the S14 oocytes were removed 2 days later for gel electrophoresis and fluorography. Comparison of the fluorographs produced by the in vivo and in vitro labeling procedures showed that most of the oocyte proteins labeled in vivo were also labeled in vitro. The S14 oocytes, therefore, appear to contain messenger RNA for most of the oocyte proteins synthesized during oogenesis. There were also several additional proteins detected only in the fluorographs of the in vivo labeled oocytes; the most prominent of these were identified by immunoprecipitation tests as vitellogenin proteins of yolk granules, which are known to be synthesized outside the oocyte, in fat bodies. The occurrence of stable S14 oocyte messenger RNA for most of the oocyte proteins suggests that the synthesis of those proteins during oogenesis occurs in the developing oocytes, specified by a stable population of oocyte messenger RNA.     

Downregulation of the translation elongation factor 2 kinase in Xenopus laevis oocytes at the final stages of oogenesis

Phosphorylation of translation elongation factor 2(eEF-2) by a specific Ca2+/calmodulin-dependent eEF-2 kinase plays an important role in the regulation of protein synthesis in mammalian cells. We show here that an eEF-2 kinase similar to the mammalian enzyme is present in tissues of the amphibian Xenopus laevis. We investigated changes in the activity of eEF-2 kinase in extracts of Xenopus oocytes at different stages of oogenesis. The eEF-2 kinase activity was constant from stage I to stage IV of oogenesis, but dramatically decreased after stage IV. Extracts of fully grown stage-VI oocytes showed no eEF-2 kinase activity. However, when extracts were analyzed by two-dimensional gel electrophoresis, eEF-2 was found to be present mostly, if not exclusively, in the dephosphorylated form throughout oogenesis. It is suggested that eEF-2 kinase disappears late in oogenesis to make protein synthesis insensitive to changes in intracellular Ca2+ concentration. This may be important for the induction of meiotic maturation.     

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.     

北京油葫芦卵子发生中DNA及RNA动态变化研究

用孚尔根及甲基绿 -派洛宁组织化学染色法了解北京油葫芦Teleogryllusmitratus(Burmeister)卵子发生各时期阶段中卵内DNA及RNA动态变化规律。在卵子发生的最初阶段 ,核中DNA的合成和复制最活跃 ,以后便慢慢减弱 ;而RNA则在第 2阶段合成最旺盛。在卵子发生各个阶段 ,滤泡细胞中DNA ,RNA均为阳性反应 ,并在卵细胞的卵黄形成期活动旺盛 ,为卵母细胞卵黄蛋白形成提供物质基础。卵子发生第 4～ 6阶段 ,滤泡细胞开放时期 ,血淋巴内一些物质可能直接或间接通过滤泡细胞间隙进入卵母细胞内 ,参与卵母细胞的发育和构建。研究表明卵子发生初期卵母细胞的发育和物质构建主要以内源性合成积累为主 ,中后期则有外源性物质的参与。     

Interactions of 40LoVe within the ribonucleoprotein complex that forms on the localization element of Xenopus Vg1 mRNA

Proline rich RNA-binding protein (Prrp), which associates with mRNAs that employ the late pathway for localization in Xenopus oocytes, was used as bait in a yeast two-hybrid screen of an expression library. Several independent clones were recovered that correspond to a paralog of 40LoVe, a factor required for proper localization of Vg1 mRNA to the vegetal cortex. 40LoVe is present in at least three alternatively spliced isoforms; however, only one, corresponding to the variant identified in the two-hybrid screen, can be crosslinked to Vg1 mRNA. In vitro binding assays revealed that 40LoVe has high affinity for RNA, but exhibits little binding specificity on its own. Nonetheless, it was only found associated with localized mRNAs in oocytes. 40LoVe also interacts directly with VgRBP71 and VgRBP60/hnRNP I; it is the latter factor that likely determines the binding specificity of 40LoVe. Initially, 40LoVe binds to Vg1 mRNA in the nucleus and remains with the RNA in the cytoplasm. Immunohistochemical staining of oocytes shows that the protein is distributed between the nucleus and cytoplasm, consistent with nucleocytoplasmic shuttling activity. 40LoVe is excluded from the mitochondrial cloud, which is used by RNAs that localize through the early (METRO) pathway in stage I oocytes; nonetheless, it is associated with at least some early pathway RNAs during later stages of oogenesis. A phylogenetic analysis of 2×RBD hnRNP proteins combined with other experimental evidence suggests that 40LoVe is a distant homolog of Drosophila Squid.     

Lipids during Bufo arenarum oogenesis

The content and composition of phospholipids and triacylglycerols (TAGs) in Bufo arenarum oocytes in stages III and IV of their oogenesis were studied. The total amount of phospholipids in stage IV oocytes is 0.5-fold higher than in stage III oocytes. In both cases, the main phospholipids are phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A striking observation concerns the high level of diphosphatidylglycerol (DPG) in stage III oocytes, which could be indicative of a relatively larger mitochondrial population with respect to other oogenetic stages. A net increase in sphingomyelin content was found during oogenesis. This fact could be related to the role of this phospholipid in the signal transductional pathways. In PC, palmitic (16:0), linoleic (18:2) and oleic (18:1) are the major fatty acids for both types of oocytes, while in PE the main acyl groups are 18:1, 16:0, arachidonic acid (20:4n6) and 18:2. PE is more unsaturated than PC and both phospholipids are more unsaturated in stage III oocytes than in stage IV oocytes. The amount of triacylglycerols is 0.3-fold higher in stage IV oocytes than in stage III oocytes. In both stages, the main fatty acids are 18:2, 18:1 and 16:0. During oogenesis, a significant increase in 18:1 and 18:3n3, and a decrease in 18:2 of TAG were found. The unsaturation index of TAGs from stage IV oocytes is higher than that from stage III oocytes. The TAG increase during oogenesis is consistent with the putative use of these lipids as a source of energy in embryo development.     

Nuclear pore flow rate of ribosomal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis

The number of ribosomal RNA molecules which are transferred through an average nuclear pore complex per minute into the cytoplasm (nuclear pore flow rate, NPFR) during oocyte growth of Xenopus laevis is estimated. The NPFR calculations are based on determinations of the increase of cytoplasmic rRNA content during defined time intervals and of the total number of pore complexes in the respective oogenesis stages. In the mid-lampbrush stage (500–700 μm oocyte diameter) the NPFR is maximal with 2.62 rRNA molecules/pore/minute. Then it decreases to zero at the end of oogenesis. The nucleocytoplasmic RNA flow rates determined are compared with corresponding values of other cell types. The molecular weight of the rRNA precursor transcribed in the extrachromosomal nucleoli of Xenopus lampbrush stage oocytes is determined by acrylamide gel electrophoresis to be 2.5 × 10⁶ daltons. From the temporal increase of cytoplasmic rRNA (3.8 μg per oocyte in 38 days) and the known number of simultaneously growing precursor molecules in the nucleus the chain growth rate of the 40 S precursor RNA is estimated to be 34 nucleotides per second.     

Quantitation of type II topoisomerase in oocytes and eggs of Xenopus laevis

We have generated a monoclonal antibody and a polyclonal antiserum specific for Xenopus laevis topoisomerase II. Using quantitative immunoprecipitation and Western blotting techniques, we have determined the content of topoisomerase II in X. laevis oocytes during oogenesis and in unfertilized eggs. An average stage I oocyte contains 6 pg of topoisomerase II. The content of topoisomerase II per oocyte increases throughout oogenesis to 1.5 ng per stage VI oocyte. The topoisomerase II protein in stage VI oocytes is stored in the germinal vesicles. The cellular content of type II topoisomerase increases significantly when stage VI oocytes are hormonally stimulated to mature into unfertilized eggs.     

Effect of ouabain on the meiotic maturation of stage IV-V Xenopus laevis oocytes

Full-grown stage VI Xenopus laevis oocytes (1,200 to 1,300 micron) respond to progesterone stimulation by undergoing a series of physiological and morphological changes that are referred to as meiotic maturation. Oocytes in earlier stages of oogenesis (I through V) do not undergo these changes and remain in prophase arrest when exposed to this steroid. We have found that oocytes ranging from 850 micron (stage IV) to 1,000 micron (stage V) are capable of responding to progesterone under the appropriate conditions. Oocytes greater than or equal to 850 micron in diameter underwent germinal vesicle breakdown (GVBD) after 10-12 hr of exposure to progesterone when ouabain was added to the medium at a concentration greater than 2.5 X 10(-6) M. Under this culture condition, progesterone was now able to induce a 0.3- to 0.4-unit increase in the intracellular pH of stage IV-V oocytes, a 4- to 5-fold increase in 40s ribosomal protein S-6 phosphorylation, and a 2.3-fold increase in their rate of protein synthesis. All of these physiological changes are characteristic of full-grown stage VI oocytes undergoing meiotic maturation. In addition, we have found that oocytes greater than or equal to 750 micron are capable of amplifying maturation promoting factor (MPF) in their cytoplasm leading to GVBD. Therefore, stage IV-V Xenopus oocytes have the potential for undergoing meiotic maturation, but they are blocked at a point in prophase that appears to be alleviated by the combination of progesterone and ouabain.