[Show abstract][Hide abstract] ABSTRACT: Mammalian tetraploid embryos die in early development because of defects in the epiblast. Experiments with diploid/tetraploid chimeric mice, obtained via the aggregation of embryonic stem cells, clarified that while tetraploid cells are excluded from epiblast derivatives, diploid embryos with tetraploid extraembryonic tissues can develop to term. Today, this method, known as tetraploid complementation, is usually used for rescuing extraembryonic defects or for obtaining completely embryonic stem (ES) cell-derived pups. However, it is still unknown why defects occur in the epiblast during mammalian development. Here, we demonstrated that downregulation of p53, a tumour suppressor protein, rescued tetraploid development in the mammalian epiblast. Tetraploidy in differentiating epiblast cells triggered p53-dependent cell-cycle arrest and apoptosis, suggesting the activation of a tetraploidy checkpoint during early development. Finally, we found that p53 downregulation rescued tetraploid embryos later in gestation.
[Show abstract][Hide abstract] ABSTRACT: We developed a transgenic mouse line with Y chromosome-linked green fluorescent protein expressing transgenes (Y-GFP) by the conventional microinjection into the pronucleus of C57BL/6J fertilized oocytes. Embryonic stem (ES) cells derived from Y-GFP mice enabled not only sexing but also the identification of 39, XO karyotype by the lack of Y chromosome. Actually, when fluorescence activated cell sorting (FACS) was applied to Y-GFP ES cells, non-fluorescent ES cells were conveniently collected and showed the lack of Y chromosome by PCR genotyping and Southern blot analysis. FACS analysis revealed Y chromosome loss occurred at 2.9 % of 40, XY ES cells after five passages. These Y-GFP ES cells are potentially applicable to reduce the time, cost and effort needed to generate the gene-targeted mice by the production of male and female mice derived from the same ES cell clone.
Transgenic Research 07/2014; 23(5). DOI:10.1007/s11248-014-9813-0 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondria play a crucial role in the development and function of germ cells. Mitochondria contain a maternally inherited genome that should be transmitted to offspring without reactive oxygen species-induced damage during germ line development. Germ cells are also involved in the mitochondrial DNA (mtDNA) bottleneck; thus, the appropriate regulation of mtDNA in these cells is very important for this characteristic transmission. In this review, we focused on unique regulation of the mitochondrial genome in animal germ cells; paternal elimination and the mtDNA bottleneck in females. We also summarized the mitochondrial nucleoid factors involved in various mtDNA regulation pathways. Among them, mitochondrial transcription factor A (TFAM), which has pleiotropic and essential roles in mtDNA maintenance, appears to have putative roles in germ cell regulation.
Reproductive Medicine and Biology 01/2014; 13(1). DOI:10.1007/s12522-013-0162-0
[Show abstract][Hide abstract] ABSTRACT: Manipulation of preimplantation embryos in vitro, such as in vitro fertilization (IVF), in vitro culture (IVC), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) and other assisted reproduction technologies (ART), has contributed to the development of infertility treatment and new animal reproduction methods. However, such embryos often exhibit abnormal DNA methylation patterns in imprinted genes and centromeric satellite repeats. These DNA methylation patterns are established and maintained by three DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b. Dnmt3b is responsible for the creation of methylation patterns during the early stage of embryogenesis and consists of many alternative splice variants that affect methylation activity; nevertheless, the roles of these variants have not yet been identified. In this study, we found an alternatively spliced variant of Dnmt3b lacking exon 6 (Dnmt3bΔ6) that is specific to mouse IVC embryos. Dnmt3bΔ6 also showed prominent expression in embryonic stem (ES) cells derived from in vitro manipulated embryos. Interestingly, IVC blastocysts were hypomethylated in centromeric satellite repeat regions that could be susceptible to methylation by Dnmt3b. In vitro methylation activity assays showed that Dnmt3bΔ6 had lower activity than normal Dnmt3b. Our findings suggest that Dnmt3bΔ6 could induce a hypomethylation status especially in in vitro manipulated embryos.
Journal of Reproduction and Development 06/2011; 57(5):579-85. DOI:10.1262/jrd.10-194A · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In mammals, complementary contributions of both the maternal and the paternal genomes are required for normal development because of the parental-allele-specific modification of the genome, called genomic imprinting. Therefore, parthenogenetic embryos (PG) with two maternal genomes cannot develop to term, and PG chimeras show a restricted cell contribution of donor cells and reduced weight, although they can develop to term. On the other hand, parthenogenetic embryonic stem cells (PGES) chimeras are more normal in their tissue contribution of donor cells and body weight compared with PG chimeras. To elucidate the epigenetic mechanisms underlying this, we analyzed the imprint status in donor cells of PGES and PG chimeras. In somatic lineages, genomic imprinting was lost in some PGES chimeras, whereas those in PG chimeras were almost totally maintained. Moreover, loss of imprints correlated to the gene expression pattern of imprinted genes. Therefore, this loss of imprinting in PGES chimeras could improve the tissue contribution and body weight to a normal level. On the other hand, in germ lineages, both PGES and PG in chimeras showed normal erasure of imprints, indicating that the reprogramming in germ lineages is an inevitable event, regardless of the imprint status of primordial germ cells.
[Show abstract][Hide abstract] ABSTRACT: Observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations prompted the creation of the bottleneck theory. A prevalent hypothesis is that a massive reduction in mtDNA content during early oogenesis leads to the bottleneck. To test this, we estimated the mtDNA copy number in single germline cells and in single somatic cells of early embryos in mice. Primordial germ cells (PGCs) show consistent, moderate mtDNA copy numbers across developmental stages, whereas primary oocytes demonstrate substantial mtDNA expansion during early oocyte maturation. Some somatic cells possess a very low mtDNA copy number. We also demonstrated that PGCs have more than 100 mitochondria per cell. We conclude that the mitochondrial bottleneck is not due to a drastic decline in mtDNA copy number in early oogenesis but rather to a small effective number of segregation units for mtDNA in mouse germ cells. These results provide new information for mtDNA segregation models and for understanding the recurrence risks for mtDNA diseases.
[Show abstract][Hide abstract] ABSTRACT: Mammalian parthenotes with the 2 maternal genomes cannot develop to term. By contrast, chimeras produced by parthenogenetic and normal embryos can develop to term. However, parthenogenetic cells contribute to restricted cells and body weights of the chimeras are reduced. These effects are due to aberrant expressions of imprinted genes, with complete methylation of the maternally methylated genes and complete loss of the paternally methylated genes. On the other hand, parthenogenetic ES (PGES) chimeras show more normal tissue contribution of donor cells and body weight compared to parthenogenetic embryo (PG) chimeras. To elucidate the epigenetic mechanisms underlying this, we analyzed the epigenetic status of maternally methylated genes in murine PG and PGES chimeras. To make parthenogenetic chimeras, PG and PGES cells which express green fluorescent protein (GFP) were introduced into normal host embryos. Mouse embryonic fibroblasts (MEFs) from E13.5 chimeric fetuses were sorted by the fluorescence-activated cell sorter (FACS). Methylation status of parthenogenetic cells was analyzed by combined bisulfite restriction analysis (COBRA) and bisulfite sequencing. Methylation of maternally methylated genes, Peg1/Mest, Snrpn, and Igf 2r, was almost totally maintained in PG chimeras. Average methylatation percentages of PG-derived MEFs were 80% in Peg1/Mest, 84% in Snrpn, and 81% in Igf 2r (n = 6). In contrast, methylation in some PGES chimeras was partially reduced to normal level in all 3 genes (10–45%, n = 7). To clarify whether demethylation is correlated with expression of the imprinted genes, gene expression was analyzed by quantitative real-time RT-PCR. Among maternally imprinted genes, Peg1/Mest and Snrpn are expressed from the paternal allele, whereas Igf 2r is expressed from the maternal allele. Therefore, in parthenogenetic cells, loss of imprints is expected to up-regulate Peg1/Mest and Snrpn expression, and down-regulate Igf 2r expression. In fact, PGES-derived MEFs were up-regulated in Peg1/Mest and Snrpn expression, and down-regulated in Igf 2r expression. This study revealed that variations of imprint status were observed frequently in somatic cells of PGES cell origin. Demethylation could have occurred during establishment and/or maintenance of PGES cells. This demethylation that occurred in PGES cells could reprogram the maternally methylated imprinted genes and improve tissue contribution and body weight to normal level. The PGES cells with reprogramming ability might be utilized for fertility treatment and regenerative medicine.
Reproduction Fertility and Development 01/2007; 19(1). DOI:10.1071/RDv19n1Ab222 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neurochondrin is a novel cytoplasmic protein and possibly involved in neurite outgrowth, chondrocyte differentiation, and bone metabolism. Our previous trial in disclosing its role by the loss of function in mice failed because of the lethality in utero. In this study, we eliminated the neurochondrin gene expression preferentially in the nervous system by the conditional knockout strategy. Our results showed that neurochondrin is a negative regulator of Ca(2+)/calmodulin-dependent protein kinase II phosphorylation and essential for the spatial learning process but not for the differentiation or neurite outgrowth of the neuron. In addition, the nervous system-specific homozygous gene disruption resulted in epileptic seizure.
[Show abstract][Hide abstract] ABSTRACT: The developmental competence of domestic pig oocytes that were transferred to somatic cell nuclei of miniature pig was examined. A co-culture system of oocytes with follicle shells was used for the maturation of domestic pig oocytes in vitro. Co-cultured oocytes progressed to the metaphase II stage of meiosis more quickly and more synchronously than non co-cultured oocytes. Oocytes were enucleated and fused with fibroblast cells of Potbelly miniature pig at 48 h of maturation. The blastocyst formation rate of nuclear transfer (NT) embryos using cocultured oocytes (24%) was significantly higher (p < 0.05) than that of non-co-cultured oocytes (13%). Cleaved embryos at 48 h after nuclear transfer using co-cultured oocytes were transferred to the oviducts of 14 Göttingen miniature pigs and four Meishan pigs. Estrus of all Göttingens returned at around 20-31 days of pregnancy. Two of the four Meishans became pregnant. Three and two cloned piglets were born after modest number of embryo transfer (15 and 29 embryos transferred), respectively. These results indicated that oocytes co-cultured with follicle shells have a high developmental competence after nuclear transfer and result in full-term development after embryo transfer.
[Show abstract][Hide abstract] ABSTRACT: We compared four inbred mouse strains in their physical performance, measured as a maximal treadmill running time, characteristics of soleus muscle, anatomic character, and growth. The strains used were Mus musculus domesticus [C57BL/6 (B6) and BALB/c], Mus musculus molossinus (MSM/Ms), and Mus spretus. Maximal running time was significantly different among these four mouse strains. Running time until exhaustion was highest in MSM/Ms and lowest in M. spretus. Maximal times for the laboratory mouse strains were nearly identical. Soleus muscle fiber type and cross-sectional area also differed significantly among the species. In particular, M. spretus was significantly different from the other inbred mouse strains. Growth in the wild-derived inbred mice appeared to be complete earlier than in the laboratory mice, and the body size of the wild strains was about half that of the laboratory strains. From these results, we propose that wild-derived inbred mouse strains are useful models for enhancing phenotypic variation in physical performance and adaptability.
[Show abstract][Hide abstract] ABSTRACT: Fetal calf serum (FCS) has usually been used for culture of embryonic stem (ES) cell as a component of the culture medium. However, FCS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. Recently, a chemically-defined serum replacement, Knockout Serum Replacement (KSR), was developed to maintain ES cells in an undifferentiated state. In this experiment, we examined the effects of KSR on the growth and differentiation of primordial germ cells (PGCs) and embryonic germ (EG) cells. PGCs were collected 8.5 days postcoitum (dpc) from B6D2F1 (C57BL/6JxDBA/2J) female mice mated with B6D2F1 males. Most of the PGCs that were cultured in FCS-supplemented medium (FCS medium) had alkaline phosphatase (AP) activity and acquired a fibroblast cell shape. In contrast, PGCs in KSR-supplemented medium (KSR medium) proliferated, maintaining round and stem cell-like morphology. In addition, EG cells were established more easily from PGCs cultured in KSR medium than from PGCs cultured in FCS medium. The percentage of undifferentiated colonies of EG cells was significantly higher in KSR medium than in FCS medium. The germ line chimera was also produced from EG cells established in KSR medium. These results suggest that KSR can be used for sustaining an undifferentiated state of PGCs and EG cells in vitro.
[Show abstract][Hide abstract] ABSTRACT: The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.
Biology of Reproduction 09/2002; 67(2):643-7. · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Maternal transmission of mitochondrial DNA (mtDNA) allows us to generate mtDNA congenic strain by repeating backcrosses of female mice to male mice of an inbred strain, which carries different mtDNA haplotype from that of the female progenitor. Since genetic backgrounds of inbred strains commonly used (e.g., C57BL/6J [B6] and BALB/c) are mainly derived from an European subspecies of Mus musculus domesticus, congenic strains, in which mtDNA originated from an Asian subspecies M. musculus musculus or an European species M. spretus, give in vivo condition that mismatch occurs between the mitochondrial and the nuclear genome. So far, little has been known how the mismatch condition affects the physiological phenotype of the mice. To address this question, we established two mtDNA congenic strains, C57BL/6J(B6)-mtSPR and BALB/c-mtSHH, which carry M. spretus- and M. m. musculus-derived mtDNAs, representing the conditions of interspecific and intersubspecific mitochondrial-nuclear genome mismatch, respectively. Using these congenic strains, we examined their physical performance by measuring their running time on a treadmill belt until exhaustion. The result clearly showed that the mtDNA congenic strains manifested a significant decrease in the level of physical performance, when compared with their progenitor strains. It also appeared that the congenic mice manifested growth rate. Thus, all results indicated that mismatch between the mitochondrial and the nuclear genome causes phenotypic changes in individuals of mice.
[Show abstract][Hide abstract] ABSTRACT: To examine the effect of mitochondrial DNA (mtDNA) on the developmental ability of mouse embryos, we investigated using the mice from standard and the mtDNA-congenic strain carrying different type of mtDNA under the same nuclear genetic background. When the 2-cell embryos (day 2 after hCG injection) derived from standard strain C57BL/6 (B6) carrying Mus musculus domesticus type mtDNA and from mtDNA-congenic strain C57BL/6.mtSPR (B6-mtSPR) carrying Mus spretus type mtDNA were used to compare the developmental ability in vitro, the percentage of embryos developed to blastocyst of B6-mt SPR strain (34.7%) showed significantly lower than that of B6 strain (93.8%) at day 6 after hCG injection. The results from reciprocal cross mating between B6 and B6-mtSPR to examine the effect of paternal mtDNA indicated that F1 embryos from B6-mtSPR females (38.7%) also exhibited lower developmental ability to blastocysts than those from B6 females (86.7%). When B6 and B6-mtSPR embryos were cultured in the low oxygen (5% O2) condition, development of B6-mtSPR embryos (96.7%) to the blastocyst stage was greatly improved to the level comparable to that of B6 embryos (97.4%). These results suggest that developmental ability of the embryos may be influenced by the type of maternally derived mtDNA through mitochondria respiratory pathway, but a possible involvement of incompativility among nuclear and mitochondrial genome and cytoplasmic factors on preimplantation development still remains to be resolved.
Journal of Reproduction and Development 01/1998; 44(2):129-134. DOI:10.1262/jrd.44.129 · 1.52 Impact Factor