Biomedical subjects

Koji Sugiura

Publications and source records attributed to Koji Sugiura.

10 recordsLinked to original sources

PubMed2006

Selective degradation of transcripts during meiotic maturation of mouse oocytes.

There is massive destruction of transcripts during the maturation of mouse oocytes. The objective of this project was to identify and characterize the transcripts that are degraded versus those that are stable during the transcriptionally silent germinal vesicle (GV)-stage to metaphase II (MII)-stage transition using a microarray approach. A system for oocyte transcript amplification using both internal and 3'-poly(A) priming was utilized to minimize the impact of complex variations in transcript polyadenylation prevalent during this transition. Transcripts were identified and quantified using the Affymetrix Mouse Genome 430 v2.0 GeneChip. The significantly changed and stable transcripts were analyzed using Ingenuity Pathways Analysis and GenMAPP/MAPPFinder to characterize the biological themes underlying global changes in oocyte transcripts during maturation. It was concluded that the destruction of transcripts during the GV to MII transition is a selective rather than promiscuous process in mouse oocytes. In general, transcripts involved in processes that are associated with meiotic arrest at the GV-stage and the progression of oocyte maturation, such as oxidative phosphorylation, energy production, and protein synthesis and metabolism, were dramatically degraded. In contrast, transcripts encoding participants in signaling pathways essential for maintaining the unique characteristics of the MII-arrested oocyte, such as those involved in protein kinase pathways, were the most prominent among the stable transcripts.

Animals↗

PubMed2006

Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

Animals↗

PubMed2005

Study of germinal vesicle requirement for the normal kinetics of maturation/M-phase-promoting factor activity during porcine oocyte maturation.

Mammalian immature oocytes contain large nuclei referred to as germinal vesicles (GVs). The translocation of maturation/M-phase promoting factor (MPF) into GVs just before the activation of MPF has been reported in several species. To examine whether the GV is required for MPF activation in mammalian oocytes, porcine immature oocytes were enucleated and their MPF activity and CCNB (also known as cyclin B) levels were investigated. The activation of MPF at the start of maturation was detected at normal levels in enucleated oocytes, whereas reactivation to induce the second meiosis was not observed. Although protein synthesis was found to be normal both qualitatively and quantitatively, even in the absence of the nucleus, CCNB1 did not sufficiently accumulate in the enucleated oocytes. The defects in the enucleated oocytes were reversed by the injection of GV material into the enucleated oocytes. Furthermore, the inhibition of CCNB1 degradation revealed drastic accumulation of CCNB1, indicating active synthesis of CCNB1 in enucleated oocytes. The mitogen-activated protein kinase cascade remained unaffected by enucleation. These results indicate that GV is not required for the activation of MPF during the first meiosis, but that it is required for the second meiosis because of its promotion of CCNB1 accumulation.

Animals↗

PubMed2005

Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism.

Intercellular communication between oocytes and granulosa cells is essential for normal follicular differentiation and oocyte development. Subtraction hybridization was used to identify genes more highly expressed in cumulus cells than in mural granulosa cells of mouse antral follicles. This screen identified six genes involved in glycolysis: Eno1, Pkm2, Tpi, Aldoa, Ldh1, and Pfkp. When oocytes were microsurgically removed from cumulus cell-oocyte complexes, the isolated cumulus cells exhibited decreased expression levels of genes encoding glycolytic enzymes, glycolysis and activity of the tricarboxylic acid (TCA) cycle. These decreases were prevented by culturing the cumulus cells with paracrine factors secreted by fully grown oocytes. Paracrine factors from fully grown oocytes exhibited greater ability than those from growing oocytes to promote expression of genes encoding glycolytic enzymes and glycolysis in the granulosa cells of preantral follicles. However, neither fully grown nor growing oocytes secreted paracrine factors affecting activity of the TCA cycle. These results indicate that oocytes regulate glycolysis and the TCA cycle in granulosa cells in a manner specific to the population of granulosa cells and to the stage of growth and development of the oocyte. Oocytes control glycolysis in granulosa cells by regulating expression levels of genes encoding glycolytic enzymes. Therefore, mouse oocytes control the intercellular metabolic cooperativity between cumulus cells and oocytes needed for energy production by granulosa cells and required for oocyte and follicular development.

Animals↗

PubMed2005

Society for Reproductive Biology Founders' Lecture 2005. Control of metabolic cooperativity between oocytes and their companion granulosa cells by mouse oocytes.

Oocytes orchestrate the rate of follicular development and expression of genes in the surrounding granulosa cells. Oocytes are deficient in their ability to carry out some metabolic processes, such as glycolysis and amino acid uptake, and depend on the cooperation of granulosa cells to carry out these processes. In this dependency, the oocyte was previously considered a passive recipient of the nutritional support from granulosa cells. However, recent studies indicate an active role for the oocyte in controlling metabolic activity in granulosa cells. The ability of oocytes to control granulosa cell metabolism is achieved, at least in part, by regulating granulosa cell expression of genes encoding proteins involved in the metabolic processes. This review summarises current knowledge of intercellular communication between oocytes and granulosa cells from the perspective of oocyte control of gene expression in granulosa cells and metabolic cooperativity between the two cell types. The oocyte probably controls metabolism in granulosa cells to provide metabolites for its own development. In addition, we hypothesise that oocytes use their ability to regulate metabolic pathways in granulosa cells to orchestrate the rate of follicular development.

Animals↗

PubMed2005

Cdk2 activity is essential for the first to second meiosis transition in porcine oocytes.

The meiotic progression of Xenopus oocytes has been suggested to depend on the activity of cyclin-dependent kinase 2 (Cdk2). We examined whether Cdk2 is involved in the regulation of mammalian oocyte meiosis by injecting porcine oocytes with anti-Cdk2 antibody. At first, the cross-reactivity of the anti-Cdk2 antibody with Cdc2 kinase was evaluated by immunoprecipitation and immunoblotting experiments using porcine granulosa cell extract, and no cross-reactivity with Cdc2 kinase was observed in the antibody used. In the anti-Cdk2 antibody-injected group, 50.7% of the oocytes were arrested in the second metaphase after 50 h of culture and this rate was significantly lower than those in the non-injected intact oocytes or the oocytes injected with mouse IgG (84.5% and 86.7%, respectively). Most of the other oocytes in the antibody-injected group formed a pronucleus without polar bodies or with only one polar body. The cyclin B1 amount in the antibody-injected and activated oocytes was dramatically decreased compared with that in the intact or mouse IgG-injected oocytes after 50 h of culture. These results suggest that Cdk2 is involved in the meiotic maturation of mammalian oocytes, and that the block of Cdk2 activity results in the failure of cyclin B1 accumulation and second meiosis induction.

Animals↗

PubMed2003

Analysis of the roles of cyclin B1 and cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection.

The function of cyclin B1 (CB1) and cyclin B2 (CB2) during porcine oocyte maturation was investigated by injecting oocytes with their antisense RNAs (asRNAs). At first, protein levels of both cyclin Bs were examined by immunoblotting, revealing that immature oocytes had only CB2, at a level comparable to 1/20 to 1/40 of that detected in first metaphase oocytes. Both cyclin B syntheses were started around germinal vesicle breakdown (GVBD); CB1 and CB2 peaked at the second metaphase and first metaphase, respectively. We obtained a porcine CB2 cDNA fragment, which was 88% homologous with human CB2, by reverse-transcriptase polymerase chain reaction (RT-PCR) using total RNAs of immature porcine oocytes and a primer set of human CB2. Specific asRNAs of CB1 and CB2 were prepared in vitro. Then one, the other, or both were injected into the cytoplasm of immature oocytes. CB1 asRNA inhibited CB1 synthesis specifically; the injected oocytes underwent first meiosis normally but could not arrest at the second meiotic metaphase. CB2 asRNA inhibited CB2 synthesis specifically, but had almost no effect on the maturation of injected oocytes. When both CB1 and CB2 asRNAs were injected, synthesis of both cyclin Bs was inhibited, and GVBD was significantly suppressed but occurred slowly. These results suggest that CB1 is the principal molecule for regulation in mammalian oocyte maturation, whereas CB2 has only an accessory role. They also show that in porcine oocytes, cyclin B synthesis is not necessary for GVBD induction itself, but synthesis of at least one cyclin B, CB1 or CB2, is necessary for GVBD induction in a normal time course.

Animals↗

PubMed2003

Analyses of mitogen-activated protein kinase function in the maturation of porcine oocytes.

The function of mitogen-activated protein kinase (MAPK) during porcine oocyte maturation was examined by injecting oocytes with either mRNA or antisense RNA of porcine c-mos protein, an upstream kinase of MAPK. The RNAs were injected into the cytoplasm of porcine immature oocytes immediately after collection from ovaries, then the oocytes were cultured for maturation up to 48 h. The phosphorylation and activation of MAPK were observed at 6 h after injection of the c-mos mRNA injected-oocytes, whereas in control oocytes, MAPK activation was detected at 24 h of culture. The germinal vesicle breakdown (GVBD) rate at 24 h of culture was significantly higher in c-mos mRNA-injected oocytes than in control oocytes. In contrast, although injection of c-mos antisense RNA completely inhibited phosphorylation and activation of MAPK throughout the maturation period, the GVBD rate and its time course were the same in noninjected oocytes. The degree of maturation-promoting factor (MPF) activation was, however, very low in oocytes in the absence of MAPK activation. Most of those oocytes had both abnormal morphology and decondensed chromosomes at 48 h of culture. These results suggest that MAPK activation is not required for GVBD induction in porcine oocytes and that the major roles of MAPK during porcine oocyte maturation are to promote GVBD by increasing MPF activity and to arrest oocytes at the second metaphase.

Animals↗

PubMed2002

Preimplantation-embryo-specific cell cycle regulation is attributed to the low expression level of retinoblastoma protein.

It is known that a characteristic of the mammalian preimplantation-embryo-specific cell cycle is the substantially shortened G1-phase, although the regulation mechanisms of the unique cell cycle remain unclear. In the present study, we first examined the presence of retinoblastoma (RB) tumor suppressor gene product throughout mouse preimplantation embryo development and found that the RB expression was down-regulated between the four-cell and morula stages. Furthermore, the overexpression of RB protein in the mouse embryos during this phase inhibited their development significantly. These results suggest that the absence of RB protein contributes to the preimplantation-embryo-specific cell cycle.

Animals↗

PubMed2002

Effects of spindle removal on MPF and MAP kinase activities in porcine matured oocytes.

Intracellular localization of maturation/M-phase promoting factor (MPF) and mitogen activated protein (MAP) kinase in mature oocytes has been examined by immunocytochemical methods and the authors of these studies have reported that they are localized on spindles during M-phase. Although these reports showed the relative localization of MPF and MAPK on spindles, it has never been shown whether these kinases are present in the cytoplasm and, if they are present, how many parts of the kinases are localized on the metaphase spindle. In the present study, we made quantitative analyses of MPF and MAP kinase localized on oocyte spindles by kinase assays and immunoblotting after removal of the spindles from porcine mature oocytes. First, we certified their intracellular distribution by immunocytochemical methods and observed sharp signals of cyclin B1 on spindle poles and MAP kinase signals on the microtubule of metaphase spindles. In contrast to these results by immunostaining, the amounts of MPF and MAP kinase localized on spindles examined by immunoblotting and kinase assays were undetectable and less than 20%, respectively. These results indicate that the immunocytochemical technique is a powerful method for showing relative localization, but it is not suitable for quantitative analysis, and that the removal of metaphase spindles from mature oocytes does not have a severe negative impact on the subsequent MPF and MAP kinase activity and on the cell cycle progression in early embryo development.

Animals↗