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Yayoi Obata

Publications and source records attributed to Yayoi Obata.

18 recordsLinked to original sources
PubMed2006

Disruption of parental-specific expression of imprinted genes in uniparental fetuses.

In mammals, imprinted genes show parental origin-dependent expression based on epigenetic modifications called genomic imprinting (GI), which are established independently during spermatogenesis or oogenesis. Due to GI, uniparental fetuses never develop to term. To determine whether such expression of imprinted genes is maintained in uniparental mouse fetuses, we analyzed the expression of 20 paternally and 11 maternally expressed genes in androgenetic and parthenogenetic fetuses. Four genes of each type were expressed in both groups of fetuses. Furthermore, quantitative analysis showed that expression levels deviated from the presumed levels for some imprinted genes. These results suggest that mechanisms acting in trans between paternal and maternal alleles are involved in the appropriate expression of some imprinted genes.

Alleles↗
PubMed2006

Oocyte growth-dependent progression of maternal imprinting in mice.

In mammals, some genes categorized as imprinted genes are exclusively expressed either from maternal or paternal allele. This parental-origin-specific gene expression is regulated by epigenetic modification of DNA methylation in differentially methylated region (DMR), which is independently imposed during oogenesis and spermatogenesis. It is known that methylation of DMR in the female germ line is established during oocyte growth phase. However, the cause of the progression of methylation on DMR, due to either aging of mice or growth-size of oocyte was unclear up to now. Here, we analyzed the methylation of DMR for each eight imprinted genes (Igf2r, Lit1, Zac1, Snrpn, Peg1/Mest, Impact, Meg1/Grb10, and H19) by bisulfite sequencing methylation assay, using oocytes from 10 dpp (days post partum), 15 dpp, 20 dpp, and adult mice. To find whether the size of oocytes is the cause of methylation, above oocytes were classified into seven groups (each oocyte diameter ranging from 40 to 75 microm with intervals of 5 microm). The results from juvenile mice oocytes showed that DMR methylation progressed according to oocyte growth each imprinted gene. More than 85% of DMR methylation was achieved for both Igf2r, Zac1 & Lit1 with oocyte size of reaching 55 microm and Snrpn, Peg1/Mest, Impact, and Meg1/Grb10 with oocyte size of reaching 60 microm. Preferential methylation of maternal allele was observed in Zac1 and Peg1/Mest of juvenile oocytes and in Snrpn of juvenile and adult oocytes. The oocyte size-dependent-methylation progressed equally for all three different-age juvenile mice. The size-dependent-methylation was also recognized in the growing oocytes collected from adult mice, although the progress is slightly slower than that of juvenile mice. From these results, we concluded that DNA methylation is established with oocyte size dependent manner, not with aging of mice.

Alleles↗
PubMed2006

Regulated expression of two sets of paternally imprinted genes is necessary for mouse parthenogenetic development to term.

Mouse parthenogenetic embryos (PEs) are developmentally arrested until embryo day (E) 9.5 because of genomic imprinting. However, we have shown that embryos containing genomes from non-growing (ng) and fully grown (fg) oocytes, i.e. ng(wt)/fg(wt) PE (wt, wild type), developed to E13.5. Moreover, parthenogenetic development could be extended to term by further regulation of Igf2 and H19 expression using mice with deletion of the H19 transcription unit (H19Delta13) together with its differentially unit (DMR). To gain an insight into the extended development of the parthenotes to term, we have here investigated the expression levels of paternally imprinted genes in ng(H19Delta13)/fg(wt) PE throughout their development. In ng(H19Delta13)/fg(wt) Pes that died soon after recovery, the expression of Igf2 and H19 was restored to the appropriate levels except for low Igf2 expression in the liver after E15.5. Further, the paternally expressed Dlk1 and Dio3 were repressed, while the expression levels of the maternal Gtl2 and Mirg were twice those of the controls. However, the above-mentioned four genes showed almost normal expression in the surviving ng(H19Delta13)/fg(wt) PEs. The methylation analysis revealed that the intragenic DMR of the Dlk1-Gtl2 domain was hypermethylated in the ng(H19Delta13)/fg(wt) PEs that survived, but not in the PEs that died soon after recovery. The present study suggests that two sets of co-ordinately regulated but oppositely expressed genes, Igf2-H19 and Dlk1-Gtl2, act as a critical barrier to parthenogenetic development in order to render a paternal contribution obligatory for descendants in mammals.

Animals↗
PubMed2005

Immunologic evaluation of personalized peptide vaccination for patients with advanced malignant glioma.

PURPOSE: The primary goal of this phase I study was to assess the safety and immunologic responses of personalized peptide vaccination for patients with advanced malignant glioma. EXPERIMENTAL DESIGN: Twenty-five patients with advanced malignant glioma (8 grade 3 and 17 grade 4 gliomas) were evaluated in a phase I clinical study of a personalized peptide vaccination. For personalized peptide vaccination, prevaccination peripheral blood mononuclear cells and plasma were provided to examine cellular and humoral responses to 25 or 23 peptides in HLA-A24+ or HLA-A2+ patients, respectively; then, only the reactive peptides (maximum of four) were used for in vivo administration. RESULTS: The protocols were well tolerated with local redness and swelling at the injection site in most cases. Twenty-one patients received more than six vaccinations and were evaluated for both immunologic and clinical responses. Increases in cellular or humoral responses specific to at least one of the vaccinated peptides were observed in the postvaccination (sixth) samples from 14 or 11 of 21 patients, respectively. More importantly, significant levels of peptide-specific IgG were detected in the postvaccination tumor cavity or spinal fluid of all of the tested patients who showed favorable clinical responses. Clinical responses were 5 partial responses, 8 cases of stable disease, and 8 cases of progressive disease. The median overall survival for patients with recurrent glioblastoma multiforme in this study (n = 17) was 622 days. CONCLUSIONS: Personalized peptide vaccinations were recommended for the further clinical study to malignant glioma patients.

Adult↗
PubMed2005

Immunological evaluation of individualized peptide vaccination with a low dose of estramustine for HLA-A24+ HRPC patients.

BACKGROUND: The safety, toxicity, and immunological response of individualized peptide vaccination or human leukocyte antigen (HLA)-A24+ hormone refractory prostate cancer (HRPC) patients in combination with a low dose of estramustine were evaluated. METHODS: Sixteen patients with HLA-A24+ HRPC were enrolled in the phase I/II study. Conducted immune monitorings for those patients were peptide-specific cytotoxic T lymphocyte (CTL) precursor analysis by interferon-gamma production and peptide-reactive immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay. Clinical responses and quality of life (QOL) outcomes using a self-reported patient questionnaire were also evaluated. RESULTS: Vaccinations were well tolerated, but all patients developed grade 1 or 2 local redness and swelling at the injection site. There was no significant immunosuppression in most cases when the peptide and a half dose (280 mg/day) of estramustine were administrated. Augmentation of peptide-specific CTL precursors or peptide-specific IgG was observed in 10 of 14 or 7 of 14 patients at 12 weeks (peptide vaccination alone), and in 6 of 8 or 10 of 12 patients at 24 weeks (during the combination therapy), respectively. All 13 patients treated, with the combination therapy, showed a decrease of serum prostate-specific antigen (PSA) level from the baseline, including six patients with a serum PSA level decrease of >or=50%. QOL outcomes were not deteriorated during the treatment. CONCLUSION: These results might encourage the further evaluation of the combination of peptide vaccination and a low dose of estramustine phosphate for HLA-A24+ HRPC patients.

Aged↗
PubMed2005

Immunological evaluation of vaccination of peptides derived from epithelial cancer-related antigens in two patients with hematological malignancy.

Recent advances in tumor immunology have resulted in identification of many epithelial cancer-related antigens and peptides applicable to specific immunotherapy. We and others have reported that several epithelial cancer-related antigens are also expressed in hematological malignancies. Two patients with hematological malignancy (multiple myeloma and chronic lymphocytic leukemia) were vaccinated with peptides derived from epithelial cancer-related antigens to evaluate the immune responses to peptides under a personalized peptide vaccination regimen. There was no adverse event except for local skin reaction at the injection site. The peptide vaccination augmented both peptide-specific CTLs cytotoxic to hematological malignant cells in post-vaccination peripheral blood mononuclear cells and peptide-specific IgG in post-vaccination sera. A transient but obvious decrease of malignant cells was observed at the early phase of the vaccination in both cases. Vaccines consisting of peptides derived from epithelial cancer antigens safely increased anti-tumor cell activity in patients with hematological malignancies. These results may provide a scientific rationale in use of epithelial cancer-related antigens for specific immunotherapy to patients with hematological malignancies.

Aged↗
PubMed2004

Nuclei of oocytes derived from mouse parthenogenetic embryos are competent to support development to term.

Mouse parthenotes result in embryonic death before 10 days of gestation, but parthenogenetic embryos (ng/fg PE) that contain haploid sets of genomes from nongrowing (ng) oocytes derived from newborn fetuses and fully grown (fg) oocytes derived from adults can develop into 13.5-day-old fetuses. This prolonged development is due to a lack of genomic imprinting in ng oocytes. Here, we show maternal genomes of oocytes derived from ng/fg PE are competent to support normal development. After 28 days of culture, the ovaries from ng/fg PE grew as well as the controls, forming vesicular follicles with follicular antrums. The oocytes collected from the developed follicles were the same size as those of the controls. To determine whether maternal primary imprinting had been established in the oocytes derived from ng/fg PE, we examined the DNA methylation status in differentially methylated regions of three imprinted genes, Igf2r, Lit1, and H19. The results showed that maternal-specific modifications were imposed in the oocytes derived from ng/fg PE. Further, to assess nuclear competence to support development, we constructed matured oocytes containing a haploid genome derived from ng/fg PE oocytes by serial nuclear transfer. After in vitro fertilization and culture and embryo transplantation into recipients, two live pups were obtained. One developed normally to a fertile adult. These results revealed that oocytes derived from ng/fg PE can be normally imprinted during oogenesis and acquire competence to participate in development as female genomes.

Animals↗
PubMed2004

Birth of parthenogenetic mice that can develop to adulthood.

Only mammals have relinquished parthenogenesis, a means of producing descendants solely from maternal germ cells. Mouse parthenogenetic embryos die by day 10 of gestation. Bi-parental reproduction is necessary because of parent-specific epigenetic modification of the genome during gametogenesis. This leads to unequal expression of imprinted genes from the maternal and paternal alleles. However, there is no direct evidence that genomic imprinting is the only barrier to parthenogenetic development. Here we show the development of a viable parthenogenetic mouse individual from a reconstructed oocyte containing two haploid sets of maternal genome, derived from non-growing and fully grown oocytes. This development was made possible by the appropriate expression of the Igf2 and H19 genes with other imprinted genes, using mutant mice with a 13-kilobase deletion in the H19 gene as non-growing oocytes donors. This full-term development is associated with a marked reduction in aberrantly expressed genes. The parthenote developed to adulthood with the ability to reproduce offspring. These results suggest that paternal imprinting prevents parthenogenesis, ensuring that the paternal contribution is obligatory for the descendant.

Animals↗
PubMed2002

Gene silencing: maturation of mouse fetal germ cells in vitro.

Nuclear reprogramming is essential during gametogenesis for the production of totipotent zygotes. Here we show that premeiotic female germ cells derived from mouse fetuses as early as 12.5 days post coitum are able to complete meiosis and genomic imprinting in vitro and that these matured oocytes are highly competent in supporting development to full term after nuclear transfer and in vitro fertilization. To our knowledge, this is the first time that complete oogenesis has been successfully accomplished in vitro.

Aneuploidy↗
PubMed2002

Unregulated expression of the imprinted genes H19 and Igf2r in mouse uniparental fetuses.

The present study shows that the H19 and Igf2r genes, which are imprinted and expressed solely from maternal alleles, are expressed in an unregulatable manner in mouse uniparental, androgenetic, and parthenogenetic fetuses at day 9.5 of gestation. In the androgenetic fetuses, the H19 and Igf2r genes were respectively expressed at 12 and 40% of the levels in biparental fetuses. In addition, the expression of both genes was excessive (1259 and 482%, respectively) in the parthenotes. These expressions of the imprinted genes were not regulated by methylation in the regulatory regions. Moreover, the expression of the antisense Igf2r RNA (Air) was also excessive and was not correlated with Igf2r gene expression in the uniparental fetuses. Taken together, these results indicate that the parental specific expression of imprinted genes is not maintained in particular genes in uniparental embryos, which in turn suggests that both parental genomes are required to establish maternal specific expression of the H19 and Igf2r genes by trans-acting mechanisms.

Alleles↗
PubMed2002

Imprinting of a small nucleolar RNA gene on mouse chromosome 12.

We have identified a novel, maternally expressed imprinted gene encoding a C/D-box small nucleolar RNA (snoRNA) called MBII-343, which may regulate RNA editing or alternative splicing of an as yet unknown target gene. This gene is closely linked to an imprinted gene, Meg3, on mouse distal chromosome 12, which is syntenic to human chromosome 14. The paternal duplication of mouse distal chromosome 12 leads to late embryonal/neonatal lethality, growth promotion, and cardiomyopathy, whereas maternal duplication leads to late embryonal lethality and growth retardation. Human paternal uniparental disomy for chromosome 14 leads to musculoskeletal problems and mental retardation, whereas maternal uniparental disomy leads to intrauterine growth retardation, motor developmental delay, premature puberty, hypotonia, joint laxity, macrocephaly, short statue, neonatal poor sucking, skill with jigsaw puzzles, skin picking, obesity, and maturity onset diabetes of the young.

Animals↗
PubMed2002

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse.

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.

Alleles↗
PubMed2002

A microarray-based method for detecting methylated loci.

CpG island DNA methylation plays an important role in regulating gene expression in development and carcinogenesis. We developed a new microarray-based method called methylation amplification DNA chip (MAD) for detecting differences in methylation. In this method, only methylated CpG islands from the two samples that we wanted to compare were amplified and used for hybridization. The resource material for the microarray was derived from the methylated DNA library of the sample in which we wanted to detect hypermethylation. Choosing the methylated DNA library as the resource material of the microarray increased the percentage of DNA fragments derived from hypermethylated loci on the microarray.

Animals↗
PubMed2002

Nuclear competence for maturation and pronuclear formation in mouse oocytes.

BACKGROUND: In response to gonadotrophins, a fully grown mouse oocyte matures to the metaphase of the second meiotic division and becomes competent for the development of female and male pronuclei after fertilization. The present study was carried out to clarify when during the growth period an oocyte nucleus acquires the ability to promote pronuclei formation after fertilization. METHODS: Fully grown germinal vesicle (GV) oocytes were enucleated and fused with nuclei from growing oocytes from 1-20 day old mice by standard nuclear transfer technique. The reconstructed oocytes were matured and fertilized in vitro, and pronuclear formation was assessed. RESULTS: The oocytes whose nuclei were exchanged for those of the non-growing-stage oocytes matured to the metaphase of the second meiotic stage, but no normal female pronuclei were formed. Female pronuclei first formed in 27% of the oocytes reconstituted with the nuclei of oocytes from 8 day old pups after fertilization. Recondensed sperm chromatin was detected in 27% of the oocytes reconstructed with oocyte nuclei from 8 day old mice, and a male pronucleus was first formed in 6% of the oocytes that had been reconstructed with the nuclei of oocytes from 15 day old mice. The sizes of the female and male pronuclei increased with oocyte donor age, and reached normal size when the oocytes from 15 and 20 day old mice respectively were used. An electron microscopic study using oocytes that had received the oocyte nuclei of 8 day old mice confirmed these results. CONCLUSION: The factors required for pronuclear formation are derived from fully grown GV oocytes, and the transformation from decondensed sperm chromatin to a recondensed male pronucleus is governed by GV-derived factors.

Animals↗
PubMed2002

Domain regulation of imprinting cluster in Kip2/Lit1 subdomain on mouse chromosome 7F4/F5: large-scale DNA methylation analysis reveals that DMR-Lit1 is a putative imprinting control region.

Mouse chromosome 7F4/F5, where the imprinting domain is located, is syntenic to human 11p15.5, the locus for Beckwith-Wiedemann syndrome. The domain is thought to consist of the two subdomains Kip2 (p57(kip2))/Lit1 and Igf2/H19. Because DNA methylation is believed to be a key factor in genomic imprinting, we performed large-scale DNA methylation analysis to identify the cis-element crucial for the regulation of the Kip2/Lit1 subdomain. Ten CpG islands (CGIs) were found, and these were located at the promoter sites, upstream of genes, and within intergenic regions. Bisulphite sequencing revealed that CGIs 4, 5, 8, and 10 were differentially methylated regions (DMRs). CGIs 4, 5, and 10 were methylated paternally in somatic tissues but not in germ cells. CGI8 was methylated in oocyte and maternally in somatic tissues during development. Parental-specific DNase I hypersensitive sites (HSSs) were found near CGI8. These data indicate that CGI8, called DMR-Lit1, is not only the region for gametic methylation but might also be the imprinting control region (ICR) of the subdomain.

Alleles↗
PubMed2001

Maternal primary imprinting is established at a specific time for each gene throughout oocyte growth.

Primary imprinting during gametogenesis governs the monoallelic expression/repression of imprinted genes in embryogenesis. Previously, we showed that maternal primary imprinting is disrupted in neonate-derived non-growing oocytes. Here, to investigate precisely when and in what order maternal primary imprinting progresses, we produced parthenogenetic embryos containing one genome from a non-growing or growth-stage oocyte from 1- to 20-day-old mice and one from a fully grown oocyte of adult mice. We used these embryos to analyze the expression of eight imprinted genes: Peg1/Mest, Peg3, Snrpn, Znf127, Ndn, Impact, Igf2r, and p57(KIP2). The results showed that the imprinting signals for each gene were not all imposed together at a specific time during oocyte growth but rather occurred throughout the period from primary to antral follicle stage oocytes. The developmental ability of the constructed parthenogenetic embryos was gradually reduced as the nuclear donor oocytes grew. These studies provide the first insight into the process of primary imprinting during oocyte growth.

Alleles↗