Quantitation and subcellular localization of proliferating cell nuclear antigen (PCNA/cyclin) in oocytes and eggs of Xenopus laevis

Proliferating cell nuclear antigen (PCNA/cyclin) is a 36-kDa polypeptide present in the nuclei of mitotically active cells. It is known to be involved in DNA replication through an association with DNA polymerase delta. We examined the total content as well as the subcellular distribution of PCNA in the oocyte and the egg of Xenopus laevis by employing immunocytological staining and immunoblot analysis. While oocytes are not capable of replicating chromosomes, PCNA is abundant in the nucleus (about 65 ng per nucleus). The oocyte cytoplasm, on the other hand, does not contain a significant quantity of this protein. The amount of total PCNA does not change appreciably during oocyte maturation and the subsequent stages of egg cleavage. Thus, PCNA belongs to a class of proteins which are stockpiled during oogenesis in order to be utilized later for early embryogenesis.     

Egg maturation and parthenogenetic recovery of diploidy in the scorpion Liocheles australasiae (Fabricius) (Scorpions, ischnuridae)

In the scorpion Liocheles australasiae, egg maturation and parthenogenetic recoveries of chromosome number and nuclear DNA content were examined by histological, karyological observations and quantitative measurements of DNA. The primary oocyte becomes mature through two successive maturation divisions. The first maturation division takes place in the primary oocyte to produce a secondary oocyte and a first polar body. The second maturation division soon occurs in the secondary oocyte, in which the nucleus is divided into a mature egg nucleus and a second polar body nucleus, not followed by cytoplasmic fission. The first polar body, in one case, was successively divided into two second polar bodies; in the other case it was not divided. In either case, these polar bodies remained attached to the early embryo. The fate of these polar bodies during further embryogenesis were studied. In the karyological analysis, the chromosome number was divided into two groups, one from 27-32, the other was 54-64. The former was presumably the metaphase chromosome number at the meiotic division; the latter was presumably the metaphase chromosome number at the mitotic division. DNA content in the diploid nucleus of the primary oocyte, doubled before the maturation divisions, was reduced through the maturation divisions by one-half in the nuclei of the secondary oocyte and the first polar body and by one-fourth in the nuclei of the egg and the second polar bodies. The first reduction of DNA content corresponded to halving the number of the chromosomes in the first maturation division and the second to the nuclear division in the secondary oocyte. These reductions represent a common process of egg maturation, except the final production of the mature egg with two haploid nuclei, an egg nucleus, and a second polar body nucleus. These two nuclei, which were formed apart in the mature egg, drew near to fuse into a zygote nucleus. The chromosome number and nuclear DNA content were doubled in the zygote and each blastomere in embryos, supporting the hypothesis that the egg nucleus fuses with the second polar body nucleus and this conjugation initiates subsequent embryonic development.     

Cellular localization of DNA polymerase activities in full-grown oocytes and embryos of Xenopus laevis

In full-grown oocytes of Xenopus laevis more than 80 % of the total DNA polymerase activity is found in the germinal vesicle (nucleus) and only about 8% in the cytoplasm. The intracellular distribution of the multiple DNA polymerase forms has been studied in oocytes and in embryonic cells. The oocyte nucleus contains a major DNA polymerase species, sedimenting at about 7S, and a minor species sedimenting at about 5S. These enzymes are comparable, respectively, with the DNA polymerases α and β described in other biological systems. In the oocyte cytoplasm, besides a small amount of the 7S form, an 8–9S DNA polymerase activity is also detectable. In the nuclei of embryonic cells, in addition to the DNA polymerase forms present in the oocyte nucleus, a new major form which seems specific for the eggs and embryos is detectable by DEAE chromatography.     

Automictic parthenogenesis of a geographically parthenogenetic mayfly,Ephoron shigae (Insecta: Ephemeroptera,Polymitarcyidae)

In the geographically parthenogenetic mayfly, Ephoron shigae, egg maturation and counts of chromosome number of unfertilized, parthenogenetic eggs were studied, in comparison with fertilized eggs from a bisexual population. The primary oocyte becomes mature through two successive maturation divisions. The first maturation division (meiotic division) takes place in the primary oocyte to produce a secondary oocyte and a first polar body. The second maturation division soon occurs in the secondary oocyte, in which the nucleus is divided into a mature egg nucleus (female pronucleus) and second polar body nuclei. The first polar body, in some cases, was successively divided into two polar bodies; in other instances, it was not divided. After the successive maturation division, the egg nucleus and the sister second polar body nucleus drew near to fuse into the zygote nucleus. The chromosome number was doubled in the zygote, and this conjugation initiates subsequent embryonic development. This suggests that, in E. shigae, the process of parthenogenetic recovery of diploidy is the automictic type categorized as the ‘terminal fusion’ pattern. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 335–343.     

Mutations in supernova,indicate that this gene is required for the division of germ line cells in Drosophila

Mutations in supernova, previously shown to uncouple chromosome replication from segregation during cleavage in Drosophila embryos, also sanctions extra divisions of cystoblasts and spermatoblasts. This leads either to the formation of egg chambers which contain more than fifteen nurse cells or testes which have an excess of spermatocytes. In maturing egg chambers two potential oocytes may be specified in which case they are often ectopically located and connected with surrounding nurse cells by four ring canals. However, a typical oocyte nucleus is not always present and these chambers usually become necrotic and degenerate. The nurse cells are of variable size, but are still interconnected by a system of ring canals. They all possess a polyploid nucleus. Sequestering of maternal mRNA's from the nurse cells into the potential oocyte(s) takes place but there is no localization of this maternal information within the oocyte probably because of defective microtubule assembly. Many spermatocytes fail to complete meiosis so that bundles of spermatids are reduced in size and the males have reduced fertility. It is proposed that this gene is indirectly involved in regulating the timing of mitotic divisions in both cystoblasts and spermatoblasts through its interference with microtubule assembly which is consistent with its role during embryogenesis.     

Effects of starvation on reproduction of the predacious mite <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> (Acari: Phytoseiidae)

Effects of starvation on gravid females of Neoseiulus californicus were investigated at 20°C and 85% RH. When females that had been reared with abundant prey were swapped, just after laying their first egg, to conditions without any prey and water, they laid 1.8 eggs and survived for 4.3 days. In the body of well-fed females, an egg with eggshell and/or two oocytes were observed in the ventral and dorsal regions, respectively. The larger oocyte had two roundish nuclei and abundant yolk granules, and was enveloped with a vitelline membrane. These two nuclei were not fused but were just close to each other. The smaller oocyte had a nucleus, but had not yet formed yolk granules and vitelline membrane. Females after 12 h starvation had an egg in the ventral region and an oocyte in the dorsal region of the body. After more than 24 h starvation females maintained an oocyte in the dorsal region of the body, but had no egg in the ventral region. The oocyte was filled with abundant yolk granules and contained two irregular nuclei when females were starved for 24 h, but when starved for more than 36 h it contained one irregular nucleus. These findings suggest that (1) gravid females maintained an oocyte in the dorsal region after laying two eggs during starvation, (2) the oocyte was not absorbed during starvation, (3) the oocyte advanced vitellogenesis and the fusion of two nuclei, and (4) the vitellogenic oocyte was not enveloped with an eggshell and had not started embryogenesis.     

Maturation-promoting factor induces nuclear envelope breakdown in cycloheximide-arrested embryos of Xenopus laevis

We have studied the effect of maturation-promoting factor (MPF) on embryonic nuclei during the early cleavage stage of Xenopus laevis development. When protein synthesis is inhibited by cycloheximide during this stage, the embryonic cell cycle arrests in an artificially produced G2 phase-like state, after completion of one additional round of DNA synthesis. Approximately 100 nuclei can be arrested in a common cytoplasm if cytokinesis is first inhibited by cytochalasin B. Within 5 min after injection of MPF into such embryos, the nuclear envelope surrounding each nucleus disperses, as determined histologically or by immunofluorescent staining of the nuclear lamina with antilamin antiserum. The breakdown of the nuclear envelope occurs at levels of MPF comparable to or slightly lower than those required for oocyte maturation. Amplification of MPF activity, however, does not occur in the arrested egg as it does in the oocyte. These results suggest that MPF can act to advance interphase nuclei into the first events of mitosis and show that the nuclear lamina responds rapidly to MPF.     

Deficiency of ovarian ornithine decarboxylase contributes to aging‐related egg aneuploidy in mice

It has been known for more than four decades that during mammalian estrous cycles, luteinizing hormone stimulates a transitory rise in the ovaries of ornithine decarboxylase (ODC) activity and its enzymatic product putrescine, concurrent with oocyte maturation in vivo. Inhibition of this transitory ODC/putrescine rise, however, does not appear to affect oocyte maturation or ovulation. Using several mouse models and combining in vitro and in vivo approaches, we demonstrated that deficiency of ODC during oocyte maturation is correlated with increased levels of egg aneuploidies. These results suggest that the transitory ovarian ODC rise in late proestrus is important for ensuring proper chromosome segregation during oocyte maturation. Older mice (8 months of age) exhibited about 1/3 that of young mice in LH‐stimulated ovarian ODC activity and a corresponding increase in egg aneuploidies. Moreover, a combination of putrescine supplementation in mouse drinking water leading up to oocyte retrieval and in oocyte maturation medium reduced egg aneuploidies of the older mice from 12.7% to 5.3%. Therefore, ovarian ODC deficiency might be an important etiology of maternal aging‐related aneuploidies, and peri‐ovulatory putrescine supplementation might reduce the risk of aneuploid conceptions in older women.     

Human CDC6/Cdc18 Associates with Orc1 and Cyclin-cdk and Is Selectively Eliminated from the Nucleus at the Onset of S Phase

In a two-hybrid screen for proteins that interact with human PCNA, we identified and cloned a human protein (hCdc18) homologous to yeast CDC6/Cdc18 and human Orc1. Unlike yeast, in which the rapid and total destruction of CDC6/Cdc18 protein in S phase is a central feature of DNA replication, the total level of the human protein is unchanged throughout the cell cycle. Epitope-tagged protein is nuclear in G₁ and cytoplasmic in S-phase cells, suggesting that DNA replication may be regulated by either the translocation of this protein between the nucleus and the cytoplasm or the selective degradation of the protein in the nucleus. Mutation of the only nuclear localization signal of this protein does not alter its nuclear localization, implying that the protein is translocated to the nucleus through its association with other nuclear proteins. Rapid elimination of the nuclear pool of this protein after the onset of DNA replication and its association with human Orc1 protein and cyclin-cdks supports its identification as human CDC6/Cdc18 protein.     

Ubiquitination of Cyclin-Dependent Kinase Inhibitor,Xic1, is Mediated by the Xenopus F-box Protein xSkp2

In the frog, Xenopus laevis, the Cip/Kip-type cyclin-dependent kinase (CDK) inhibitor, Xic1, inhibits DNA replication in interphase egg extracts through the binding of CDK2-cyclins and Proliferating Cell Nuclear Antigen (PCNA). During DNA polymerase switching in the replicating Xenopus egg extract, Xic1 is targeted for ubiquitination and degradation when localized to chromatin through its binding to PCNA. To date, the machinery responsible for Xic1 ubiquitination is unknown and although it is predicted that the E3 called SCF may mediate Xic1 ubiquitination, characterization of the SCF in Xenopus is lacking. In this study, we describe the identification and characterization of Xenopus Skp2 (xSkp2) and the role of xSkp2 in the ubiquitination of Xic1. Our results indicate that the expression of xSkp2 appears to be developmentally regulated with low protein levels found in the egg and increased levels found in the developing embryo. We also demonstrate that when ectopically expressed, a xSkp2 F-box deletion mutant inhibits the initiation of DNA replication suggesting a role for the SCF in the onset of S phase in Xenopus egg extracts. We further show that xSkp2 binds to C-terminal residues of Xic1 and when co-expressed with Skp1, promotes the proteolysis of Xic1 in the egg extract. Moreover, the xSkp2 F-box deletion mutant inhibits the DNA-dependent ubiquitination and proteolysis of Xic1 when added to the interphase egg extract. Importantly, our studies demonstrate that SCFxSkp2 supports the ubiquitination of Xic1 in a reconstituted in vitro ubiquitination assay and that this Xic1 ubiquitination does not require either CDK2-cyclins or Cks1. These studies provide the first characterization of the SCF in Xenopus and its role in the ubiquitination of CDK inhibitor, Xic1, during DNA replication initiation.     

Monoubiquitination of proliferating cell nuclear antigen induced by stalled replication requires uncoupling of DNA polymerase and mini-chromosome maintenance helicase activities

Proliferating cell nuclear antigen (PCNA) is a homotrimeric, ring-shaped protein complex that functions as a processivity factor for DNA polymerases. Following genotoxic stress, PCNA is modified at a conserved site by either a single ubiquitin moiety or a polyubiquitin chain. These modifications are required to coordinate DNA damage tolerance processes with ongoing replication. The molecular mechanisms responsible for inducing PCNA ubiquitination are not well understood. Using Xenopus egg extracts, we show that ultraviolet radiation and aphidicolin treatment induce the mono- and diubiquitination of PCNA. PCNA ubiquitination is replication-dependent and coincides with activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent DNA damage checkpoint pathway. However, loss of ATR signaling by depletion of the ATR-interacting protein (ATRIP) or Rad1, a component of the 911 checkpoint clamp, does not impair PCNA ubiquitination. Primed single-stranded DNA generated by uncoupling of mini-chromosome maintenance helicase and DNA polymerase activities has been shown previously to be necessary for ATR activation. Here we show that PCNA ubiquitination also requires uncoupling of helicase and polymerase activities. We further demonstrate that replicating single-stranded DNA, which mimics the structure produced upon uncoupling, is sufficient to induce PCNA monoubiquitination. Our results suggest that PCNA ubiquitination and ATR activation are two independent events that occur in response to a common single-stranded DNA intermediate generated by functional uncoupling of mini-chromosome maintenance (MCM) helicase and DNA polymerase activities.     

The Elusive Acrosome of Chiton Sperm

Summary

In our study of spermiogenesis in the lined chiton Tonicella lineata, we traced the formation and migration of small Golgi vesicles to the apex of the sperm, where they fused to form an apical granule. This apical granule and other Golgi secretions tested positively for acid phosphatase. In preliminary experiments on fertilization, sperm swam inside open hull (chorion) cupules down to the surface of the egg and penetrated it. No micropyle was observed. Serial 1μm sections of eggs fixed during fertilization demonstrated that the sperm nucleus had penetrated not only the hull but also the vitelline and oocyte membranes. Serial thin sections showed that the tip of the anterior filament of the sperm had fused with a single microvillus of the oocyte membrane, creating a membranous tube through which the nucleus had entered the egg cortex. We suggest that the apical granule of chiton sperm is an acrosome that enables the nucleus to penetrate the egg membranes.     

Egg hull formation in Callochiton dentatus (Mollusca,Polyplacophora): the contribution of microapocrine secretion

Abstract. Egg hull formation during oogenesis in the chiton Callochiton dentatus does not follow the typical model of merocrine secretion involving Golgi vesicle exocytosis. Instead, microapocrine secretions are primarily responsible for egg hull formation, although merocrine secretions contribute “areolae” and the vitelline layer. Microapocrine secretion mechanisms are poorly understood, involving a different cellular pathway than is typical. Egg hull formation in C. dentatus involves two types of microapocrine secretions released by the oocyte, one of which is described here for the first time. The plesiomorphic jelly-like egg hull of chitons, as exemplified by the eggs of members of the basal order Lepidopleurida and present also in eggs of C. dentatus (Chitonida: Callichitonidae), may have evolved solely as an oocyte secretion, whereas members of some other families in the order Chitonida form their egg hulls with considerable secretory input from the follicle cells as well.     

Über die Übereinstimmung von Polarität und Bilateralsymmetrie in Embryo,Ei, Oocyte und Oocytenkern beiGryllus domesticus L.

Summary Studied is the establishment during oogenesis of the bilateral symmetry in the banana-shapedGryllus domesticus egg. Follicular length of the panoistic egg follicle is used for classification of oogenetic stages.External rotation symmetry of oocyte and follicle changes to the bilateral symmetric banana form at about 500m of follicular length.Internal bilateral symmetry is already visible at this and younger stages with regard to the eccentric position of the oocyte nucleus. In the banana form of the oocyte the oocyte nucleus is located presumptive dorso-caudally. In the cylindric form it lies in the posterior half of the oocyte.The germinal vesicle itself exhibits a marked polarity: oocyte nucleolus and a crescent of granular material are always eccentrically located in the presumptive dorso-caudal quarter of the nucleus.Germinal vesicle's polarization and its slightly eccentric location become simultaneously visible in follicles 60 to 100m of length. Thus at this time one can anticipate in a very early stage of oogenesis on the oocyte nucleus the future poles and axes of the coming embryo.Oocyte nucleus polarization and location appear to be uniform in all follicles of an ovariole and may presumably be due to external orientating factors.

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Diese Arbeit wurde gefördert durch anregende Diskussionen mit Herrn Priv.-Doz. Dr. F.-W.Schlote.

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Mit Unterstützung durch ein Promotionsstipendium der Stiftung Volkswagenwerk.     

Mitochondrial DNA synthesis in oocytes of Xenopus laevis

Mitochondria isolated from stage 3 (about half-grown) oocytes of Xenopus laevis exhibit a DNA synthetic rate in vitro of 2.35 ± 0.28 pg/oocyte/h. Similarly, stage 6 (full-grown) oocyte mitochondria synthesize DNA (mtDNA) at 0.28 ± 0.02 pg/oocyte/h. By comparison, the rate of mtDNA synthesis by intact stage 6 oocytes following microinjection of [³H]-dTTP was calculated to be 0.43 ± 0.08 pg/oocyte/h, indicating that the observed in vitro rates may represent minimum values. Measurements of DNA polymerase activity associated with mitochondria isolated from stage 3 oocytes are almost three times those recorded with stage 6 oocyte mitochondria. It appears that active replication of complete mtDNA molecules, which accompanies accumulation of mitochondria by the egg, is terminated midway through oogenesis.     

DNA SYNTHESIS AND EVOLUTION,IN PRESENCE OF A SOMATIC NUCLEUS,OF THE FEMALE PRONUCLEUS AFTER EXPERIMENTAL ACTIVATION OF THE EGG OF PLEURODELES WALTLII*

Cytological evolution and DNA synthesis activity of the female pronucleus were analyzed in experimentally activated virgin eggs of Pleurodeles. Pricking with a micropipette is a positive treatment for activation comparable to electric shock. As in normally fertilized eggs, the evolution of a female pronucleus is related to normal DNA synthesis. Thus, the fact that the female gametic nucleus does not achieve the first mitotic cycle, at the end of migration, cannot be related to initial failure of DNA synthesis. Normal mitoses of this nucleus are obtained in an activated egg in which development is insured by grafting a somatic nucleus. These results are discussed in terms of activation factors.     

The Drosophila hus1 gene is required for homologous recombination repair during meiosis

The checkpoint proteins, Rad9, Rad1, and Hus1 (9-1-1), form a complex which plays a central role in the DNA damage-induced checkpoint response. Previously, we demonstrated that Drosophila hus1 is essential for activation of the meiotic checkpoint elicited in double-strand DNA break (DSB) repair enzyme mutants. The hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in these repair enzymes, suggesting that hus1 plays a role independent of its meiotic checkpoint activity. In this study, we further analyzed the function of hus1 during meiosis and discovered that the synaptonemal complex (SC) disassembles abnormally in hus1 mutants. Oocyte nuclear and SC defects of hus1 mutants can be suppressed by blocking the formation of DSBs, implying that the hus1 oocyte nuclear defects depend upon DSBs. Interestingly, eliminating checkpoint activity through mutations in DmChk2 but not mei-41 suppress the oocyte nucleus and SC defects of hus1, suggesting that these processes are dependent upon DmChk2 checkpoint activity. Moreover, we showed that in hus1, DSBs that form during meiosis are not processed efficiently, and that this defect is not suppressed by a mutation in DmChk2. We found a genetic interaction between hus1 and the Drosophila brca2 homologue, which was shown to participate in DNA repair during meiosis. Together, our results imply that hus1 is required for repair of DSBs during meiotic recombination.     

Ribosomal gene amplification does not occur in the oocytes of Locusta migratoria

It has been suggested that Locusta migratoria amplifies its ribosomal RNA genes in the growing oocytes (Kunz (1967) Chromosoma20, 332–370). Cloned ribosomal DNA of L. migratoria was used to analyze rDNA structure and number. The rDNA is localized on three chromosome pairs in six nucleolus organizers. It was found that all structural variants of the rRNA genes which have been described previously are represented in the same relative amounts in DNA from isolated oocytes as in somatic cells. Furthermore, the rRNA gene number is not increased in oocyte DNA, i.e., amplification does not occur. Therefore, the large number of multiple nucleoli seen in the growing oocytes has to be interpreted as the fully extended and fully active set of chromosomal rRNA genes. The total rRNA gene number was determined by dot blot hybridization to be about 3300 genes/haploid genome.     

Immunoelectron microscopy of PCNA as an efficient marker for studying replication times and sites during pollen development

Here we report for the first time the ultrastructural localization of DNA replication sites in the nucleus of plant cells and the timing of replication through the pollen developmental programme by proliferating cell nuclear antigen (PCNA) immunogold labelling. Replication sites were identified by labelling with anti-PCNA antibodies in fibrils of the interchromatin region close to the condensed chromatin, defining a perichromatin subdomain in the interchromatin space where DNA replication takes place. The same nuclear structures are decorated by anti-BrdU (5-bromo-2'-deoxyuridine) immunogold after short pulses of BrdU labelling. Double immunogold labelling for PCNA and DNA show colocalization on these perichromatin structures. PCNA immunoelectron microscopy also allows correlation of replicative activity with the dynamics of chromatin condensation. DNA replication was also monitored at different phases during pollen development by PCNA immunoelectron microscopy, revealing two peaks of DNA synthesis, at the beginning (early tetrad), and the end (late vacuolate), of microspore interphase. High-resolution autoradiography after [3H]thymidine incorporation also showed high replicative activity at the same two periods of microspore interphase. In the bicellular pollen grain, PCNA immunogold labelling revealed that DNA replication in the generative cell starts at an intermediate stage of pollen maturation, whereas the vegetative nucleus does not replicate and is arrested in G1. The use of anti-PCNA antibodies at the ultrastructural level is an easier, faster and more feasible method than the detection of in vivo-incorporated nucleotides, especially in plant systems with long cell cycles. PCNA immunogold labelling is, therefore, proposed as an efficient marker for mapping the sites and timing of replication at the electron microscopy level.     

Oogenesis in a paedogenetic dipteran insect under normal conditions and after experimental elimination of the follicular epithelium

Summary Paedogenetically developing eggs of the gall midgeHeteropeza pygmaea are not deposited, but develop in the hemocoel of the mother larva. The nurse chamber remains present in the cleaving egg, and the follicular epithelium does not form a chorion but envelops the growing egg during embryonic development. It is possible to obtain naked eggs, i.e. eggs lacking the follicular epithelium, which are able to develop up to the blastoderm stage but remain spherical instead of assuming an elongated shape. Oogenesis of normal and naked eggs has been studied at the ultrastructural level with special reference to the nurse chamber. It is shown that the nurse chamber nuclei develop large nucleoli during oogenesis, indicating that the nurse chamber supplies the oocyte with ribosomal RNA (rRNA). The dense bodies in the nurse chamber may represent an intermediate stage in the transport of the rRNA from the nurse chamber to the oocyte; they are probably not related to the polar granules in the oocyte. It is also shown that the intercellular bridge joining the nurse chamber to the oocyte disappears shortly before cleavage initiation. During egg cleavage the follicular epithelium surrounds the nurse chamber, which degenerates and is gradually absorbed by the growing egg plasmodium. Naked cleaving eggs are never attached to a nurse chamber or to relics of it. Naked oocytenurse chamber complexes frequently aggregate, which may indicate a role of the follicular epithelium in follicle separation during normal development.