Hidehiko Ogawa

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.

Deletion of Gtl2 , imprinted non-coding RNA, with its differentially methylated region induces lethal parent-origin-dependent defects in mice

The cluster of imprinted genes located in the Dlk1-Dio3 domain spanning 1 Mb plays an essential role in controlling pre- and postnatal growth and differentiation in mice and humans. The failure of parent-of-origin-dependent gene expression in this domain results in grave disorders, leading to death in some cases. However, little is known about the role of maternally expressed non-coding RNAs (ncRNAs) including many miRNAs and snoRNAs in this domain. In order to further understand the role of these ncRNAs, we created Gtl2-mutant mice harboring a 10 kb deletion in exons 1-5. The mutant mice exhibited a very unique inheritance mode: when the deletion was inherited from the mother (Mat-KO), the pups were born with normal phenotypes; however, all of them died within 4 weeks after birth, probably due to severely hypoplastic pulmonary alveoli and hepatocellular necrosis. Mice carrying the paternal deletion (Pat-KO) showed severe growth retardation and perinatal lethality. Interestingly, the homozygous mutants (Homo-KO) survived and developed into fertile adults. Our results show that these phenotypes occur due to altered expression of the Dlk1-Dio3 cluster genes including miRNAs and snoRNAs via the cis and trans effects.

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.

DNA methylation imprints on the IG-DMR of the Dlk1-Gtl2 domain in mouse male germline.

Mouse genomes show a large cluster of imprinted genes at the Dlk1–Gtl2 domain in the distal region of chromosome 12. An intergenic‐differentially methylated region (IG‐DMR) located between Dlk1 and Gtl2 is specifically methylated in the male germline; IG‐DMR regulates the parental allele‐specific expression of imprinted genes. Here, we show the resetting of IG‐DMR methylation marks during male germ‐cell differentiation. For parental allele‐specific methylation analysis, polymorphisms were detected in a 2.6‐kb IG‐DMR in three mouse strains. Bisulfite methylation analysis showed erasure of the marks by E14 and re‐establishment before birth. The IG‐DMR methylation status was maintained in spermatogonia and spermatocytes of mature testes. The IG‐DMR methylation status established before birth is thus maintained throughout the lifetime in the male germline.

Identification of Genes Aberrantly Expressed in Mouse Embryonic Stem Cell-Cloned Blastocysts

Abstract During development, cloned embryos often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. The long-term effects resulting from embryo cloning procedures would manifest after birth as early death, obesity, various functional disorders, and so forth. Despite extensive studies, the parameters affecting the developmental features of cloned embryos remain unclear. The present study carried out extensive gene expression analysis to screen a cluster of genes aberrantly expressed in embryonic stem cell-cloned blastocysts. Differential screening of cDNA subtraction libraries revealed 224 differentially expressed genes in the cloned blastocysts: eighty-five were identified by the BLAST search as known genes performing a wide range of functions. To confirm their differential expression, quantitative gene expression analyses were performed by real-time PCR using single blastocysts. The genes Skp1a, Canx, Ctsd, Timd2, and Psmc6 were significantly up-regulated, whereas Aqp3, Ak3l1, Rhot1, Sf3b3, Nid1, mt-Rnr2, mt-Nd1, mt-Cytb, and mt-Co2 were significantly down-regulated in the majority of embryonic stem cell-cloned embryos. Our results suggest that an extraordinarily high frequency of multiple functional disorders caused by the aberrant expression of various genes in the blastocyst stage is involved in developmental arrest and various other disorders in cloned embryos.

Epigenetic and transcriptional features of the novel human imprinted lncRNA GPR1AS suggest it is a functional ortholog to mouse Zdbf2linc

Long non-coding RNAs (lncRNAs), transcribed from the intergenic regions of animal genomes, play important roles in key biological processes. In mice, Zdbf2linc was recently identified as an lncRNA isoform of the paternally expressed imprinted Zdbf2 gene. The functional role of Zdbf2linc remains undefined, but it may control parent-of-origin-specific expression of protein-coding neighbors through epigenetic modification in cis, similar to imprinted Nespas, Kcnq1ot1 and Airn lncRNAs. Here, we identified a novel imprinted long-range non-coding RNA, termed GPR1AS, in the human GPR1-ZDBF2 intergenic region. Although GPR1AS contains no human ZDBF2 exons, this lncRNA is transcribed in the antisense orientation from the GPR1 intron to a secondary, differentially methylated region upstream of the ZDBF2 gene (ZDBF2 DMR), similar to mouse Zdbf2linc. Interestingly, GPR1AS/Zdbf2linc is exclusively expressed in human/mouse placenta with paternal-allele-specific expression and maternal-allele-specific promoter methylation (GPR1/Gpr1 DMR). The paternal-allele specific methylation of the secondary ZDBF2 DMR was established in human placentas as well as somatic lineage. Meanwhile, the ZDBF2 gene showed stochastic paternal-allele-specific expression, possibly methylation-independent, in placental tissues. Overall, we demonstrated that epigenetic regulation mechanisms in the imprinted GPR1-GPR1AS-ZDBF2 region were well-conserved between human and mouse genomes without the high sequence conservation of the intergenic lncRNAs. Our findings also suggest that lncRNAs with highly conserved epigenetic and transcriptional regulation across species arose by divergent evolution from a common ancestor, if they do not have identical exon structures.

Nuclei of Oocytes Derived from Mouse Parthenogenetic Embryos Are Competent to Support Development to Term

Abstract 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.

Cell proliferation potency is independent of FGF4 signaling in trophoblast stem cells derived from androgenetic embryos

We previously established trophoblast stem cells from mouse androgenetic embryos (AGTS cells). In this study, to further characterize AGTS cells, we compared cell proliferation activity between trophoblast stem (TS) cells and AGTS cells under fibroblast growth factor 4 (FGF4) signaling. TS cells continued to proliferate and maintained mitotic cell division in the presence of FGF4. After FGF4 deprivation, the cell proliferation stopped, the rate of M-phase cells decreased, and trophoblast giant cells formed. In contrast, some of AGTS cells continued to proliferate, and the rate of M-phase cells did not decrease after FGF4 deprivation, although the other cells differentiated into giant cells. RO3306, an ATP competitor that selectively inhibits CDK1, inhibited the cell proliferation of both TS and AGTS cells. Under RO3306 treatment, cell death was induced in AGTS cells but not in TS cells. These results indicate that RO3306 caused TS cells to shift mitotic cell division to endoreduplication but that some of AGTS cells did not shift to endoreduplication and induced cell death. In conclusion, the paternal genome facilitated the proliferation of trophoblast cells without FGF4 signaling.

Porcine nuclei in early growing stage do not possess meiotic competence in matured oocytes

To determine whether the nuclei of early growing stage porcine oocytes can mature to the MII stage, we examined meiotic competence of nuclei that had been fused with enucleated GV oocytes using the nuclear transfer method. In vitro matured oocytes were enucleated and then fused with early growing oocytes (30-40 μm in diameter) from 5 to 7-wk-old piglets using the hemagglutinating virus of Japan (HVJ). Reconstructed oocytes were cultured for 24 h to the MII stage. Although these oocytes extruded the first polar body, they did not contain normal haploid chromosomes, and the spindles were misaligned or absent at the metaphase II (MII) stage. Furthermore, maturation promoting factor (MPF) activity levels were low in oocytes reconstructed with early growing oocytes at metaphase I (MI) and MII. In contrast, mitogen-activated protein kinase (MAPK) activity was detected between the MI and MII stages, although at slightly lower levels. In conclusion, the nuclei of early growing oocytes did not accomplish normal meiotic division in matured oocytes due to misaligned or absent spindle formation.