Biomedical subjects

Fumitoshi Ishino

Publications and source records attributed to Fumitoshi Ishino.

At least 19 recordsLinked to original sources

PubMed2006

Peg1/Mest in obese adipose tissue is expressed from the paternal allele in an isoform-specific manner.

Paternally expressed 1 (Peg1)/mesoderm specific transcript (Mest) is an imprinted gene, which is only transcribed from the paternal (father's) allele. In some human cancer tissues, an alternatively spliced variant of PEG1/MEST mRNA using a different promoter of a distinct first exon is expressed from both paternal and maternal alleles. We previously reported that Peg1/Mest expression was markedly up-regulated in obese adipose tissue in mice. Moreover, transgenic overexpression of Peg1/Mest in the adipose tissue resulted in the enlargement of adipocytes in size. Given the potential pathophysiologic relevance in obesity, we examined the nature of increased expression of Peg1/Mest in obese adipose tissue. In obese adipose tissue, expression of Peg1/Mest was increased, but not that of other imprinted genes tested. The transcription rate of Peg1/Mest was increased in obese adipose tissue. We found at least four isoforms of mouse Peg1/Mest generated by use of the alternative first exons. We also demonstrated that the abundantly expressed Peg1/Mest in obese adipose tissue retained monoallelic expression. This is the first report of monoallelic induction of Peg1/Mest in adult tissues.

Adipocytes↗

PubMed2006

Chorioallantoic placenta defects in cloned mice.

Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones.

Animals↗

PubMed2006

Anti-estrogen ICI 182.780 and anti-androgen flutamide induce tyrosine phosphorylation of cortactin in the ectoplasmic specialization between the Sertoli cell and spermatids in the mouse testis.

Our previous study revealed that the ectoplasmic specialization (ES) was deleted by the treatment of anti-estrogen, ICI 182.780 (ICI), and anti-androgen, flutamide (FLUT) in mouse testis. Also, expression of cortactin, an F-actin-binding protein, was decreased by the treatment of FLUT in mouse testis. Cortactin has been suggested to promote actin polymerizer at the ES in the testis, and also actin depolymerization is induced by tyrosine phosphorylation of cortactin. The present study revealed that exogenous treatment of ICI and FLUT caused the deletion of the cortactin in the apical ES and the increase of tyrosine phosphorylated cortactin in mouse testis. These results suggest that the sex hormone antagonists', ICI and FLUT, induced actin depolymerization and tyrosine phosphorylation of cortactin in the mouse testis. Also, the present study may be a key to elucidate the adverse affect exogenous compounds that affect spermiation.

Androgen Antagonists↗

PubMed2006

Flutamide depresses expression of cortactin in the ectoplasmic specialization between the Sertoli cells and spermatids in the mouse testis.

Flutamide (FLUT) has potent anti-androgenic activity and is used in the medical field and in a screening test to detect endocrinologically active compounds. Our previous study demonstrated that FLUT induced histological deformation of spermatids and ultrastructural defects of the apical ectoplasmic specialization (ES) in the mouse testis. The apical ES is an actin-based junctional structure between the Sertoli cells and germ cells. Cortactin, an actin-binding protein, is found in the actin layer of ES. The protein level of cortactin was decreased in FLUT-treated testes as shown by Western blot analysis. The detailed analysis indicated that the protein level was drastically decreased in FLUT-treated seminiferous tubules of stages from VI to IX. Immunohistochemistry and immunoelectron microscopy showed that FLUT depressed cortactin expression in the apical ES. In addition, the effect of FLUT on cortactin localization appeared between 12 h and 8 days (about 180 h) after a one-day treatment. These results suggest that FLUT depressed the expression of cortactin in the apical ES with stage specificity. Therefore, the initial target of FLUT may be the cell-cell interactions between the Sertoli and germ cells. To our knowledge, this study is the first to document the decrease of cortactin expression in the abnormal apical ES following treatment with FLUT.

Androgen Antagonists↗

PubMed2006

Expression of imprinted genes in cloned mice.

Genomic imprinting is a mammalian specific epigenetic modification of the genome. Assessment of the integrity of the imprinting memory in somatic cell cloned animals is important not only for understanding of the "reprogramming" process during cloning by nuclear transfer, but also for the applications of this technique for therapeutic cloning in the future. In this chapter, we summarize the analytical methods for assessment of monoallelic expression of imprinting genes and expression analysis. From a practical point of view, the authors suggest the use of intersubspecific F1 hybrids between the laboratory mouse (Mus musculus musculus) and the JF1 strain (Mus musculus molossinus). We also list the sequence for PCR primers to detect the polymorphism of imprinted genes between musculus and molossinus.

Animals↗

PubMed2006

Estrogen agonists, 17beta-estradiol, bisphenol A, and diethylstilbestrol, decrease cortactin expression in the mouse testis.

Previous reports have revealed that estrogen agonists or anti-androgenic chemicals induce abnormal spermiogenesis in rodents. In the seminiferous epithelium, the apical ectoplasmic specialization (ES) is an actin-based (cell-cell) junctional structure developing between the Sertoli cells and spermatids as is the basal ES also--although it is located between adjoining Sertoli cells. In the apical and basal ES are several adhesion complex proteins that control the spermatid developing process. Cortactin, an actin-binding protein, is one of the ES adhesion proteins, combining with several cell-cell adhesions associating proteins. In the present study, 17beta-estradiol (E2, 1.2 microg/kg), bisphenol A (BPA, 2.4 microg/kg), and diethylstilbestrol (DES, 2.5 microg/kg) were subcutaneously injected in ICR 12-week-old male mice. Mice testes were observed for the expression of cortactin protein after E2, BPA, and DES treatments by Western blot analysis, immunohistochemical analysis, and immunoelectron microscopic analysis. Observations showed that the immunoreactivity of the treated testes was significantly decreased. The immunohistochemical reactivity of cortactin in the apical ES was decreased in the treated testis. In immunoelectron microscopic observations, ultrastructural immunolocalizations of cortactin protein in the apical ES by both E2 and BPA were decreased, and the immuno-gold particles of apical and basal ES by DES were much less than the control. In the toxicological field, cortactin may be considered to be one of the indicator proteins of abnormal spermiogenesis which is affected by exogenous chemicals, such as endocrine disrupting chemicals. In summary, this study helps toward understanding the cortactin protein expression underlying the histological abnormalities of spermatogenesis induced by exogenous hormonal chemical treatment.

Animals↗

PubMed2005

Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality.

By comparing mammalian genomes, we and others have identified actively transcribed Ty3/gypsy retrotransposon-derived genes with highly conserved DNA sequences and insertion sites. To elucidate the functions of evolutionarily conserved retrotransposon-derived genes in mammalian development, we produced mice that lack one of these genes, Peg10 (paternally expressed 10), which is a paternally expressed imprinted gene on mouse proximal chromosome 6. The Peg10 knockout mice showed early embryonic lethality owing to defects in the placenta. This indicates that Peg10 is critical for mouse parthenogenetic development and provides the first direct evidence of an essential role of an evolutionarily conserved retrotransposon-derived gene in mammalian development.

Animals↗

PubMed2005

Segmental and full paternal isodisomy for chromosome 14 in three patients: narrowing the critical region and implication for the clinical features.

We report on segmental and full paternal isodisomy for chromosome 14 in three previously unreported Japanese patients. Patient 1 was a 5(6/12)-year-old girl, Patient 2 was a male neonate, and Patient 3 was a -year-old girl. Physical examination at birth showed various somatic features characteristic of paternal uniparental disomy for chromosome 14 (upd(14)pat) such as hairy forehead, protruding philtrum, micrognathia, small thorax, and abdominal wall defects in Patients 1-3, and the constellation of somatic features was persistently observed in Patients 1 and 3. Radiological studies at birth delineated unique bell-shaped thorax with coat-hanger appearance of the ribs in Patients 1-3, but the thoracic deformity ameliorated in Patients 1 and 3 by mid childhood. Chromosome analysis showed a 46,XX karyotype in Patients 1 and 3 and was not performed in Patient 2. Microsatellite analysis indicated full paternal isodisomy for chromosome 14 in Patients 1 and 2 and segmental paternal isodisomy for chromosome 14 distal to D14S981 at 14q23.3 in Patient 3. Methylation specific PCR assay for the differentially methylated region (DMR) of GTL2 at 14q32 yielded positive products with methylated allele specific primers and no products with unmethylated allele specific primers in Patients 1-3. Since clinical phenotype was similar between Patient 3 with segmental upd(14)pat and Patients 1 and 2 with full upd(14)pat, the results are keeping with the 14q32 localized imprinted genes as the critical components of the phenotype observed in upd(14)pat and help narrow the search for additional genes to the approximately 40 Mb region distal to D14S981. Furthermore, it is likely that the characteristic thoracic deformity ameliorates with age.

Child↗

PubMed2005

Variation in gene expression and aberrantly regulated chromosome regions in cloned mice.

DNA microarray analysis was used to determine the precise genome-wide gene expression profiles of somatic cloned mice derived from Sertoli and cumulus cells. It demonstrated unexpectedly large epigenetic diversity in neonatal cloned mice, despite their normal appearance and genetic identity. In three neonatal tissues of the cloned mice, the expression of 9-40% of the genes examined was more than two times higher or lower in donor cell-dependent or -independent manners compared with normal controls. Relatively few (0.4-4%) of the genes exhibited up- or downregulation in the same manner in both types of clone. A cluster analysis of the variation in gene expression led to the identification of several chromosome regions in which gene expression was aberrantly controlled in the somatic clones. These results provide a more complete understanding of how somatic clones differ from each other and from normal individuals produced by sexual reproduction and indicate the significant difficulties that face the application of somatic cloning in regenerative medicine.

Animals↗

PubMed2005

Genetic and epigenetic properties of mouse male germline stem cells during long-term culture.

Although stem cells are believed to divide infinitely by self-renewal division, there is little evidence that demonstrates their infinite replicative potential. Spermatogonial stem cells are the founder cell population for spermatogenesis. Recently, in vitro culture of spermatogonial stem cells was described. Spermatogonial stem cells can be expanded in vitro in the presence of glial cell line-derived neurotrophic factor (GDNF), maintaining the capacity to produce spermatogenesis after transplantation into testis. Here, we examined the stability and proliferative capacity of spermatogonial stem cells using cultured cells. Spermatogonial stem cells were cultured over 2 years and achieved approximately 10(85)-fold expansion. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, spermatogonial stem cells retained the euploid karyotype and androgenetic imprint during the 2-year experimental period, and produced normal spermatogenesis and fertile offspring. However, the telomeres in spermatogonial stem cells gradually shortened during culture, suggesting that they are not immortal. Nevertheless, the remarkable stability and proliferative potential of spermatogonial stem cells suggest that they have a unique machinery to prevent transmission of genetic and epigenetic damages to the offspring, and these characteristics make them an attractive target for germline modification.

Animals↗

PubMed2005

Meg1/Grb10 overexpression causes postnatal growth retardation and insulin resistance via negative modulation of the IGF1R and IR cascades.

The Meg1/Grb10 protein has been implicated as an adapter protein in the signaling pathways from insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in vitro. To elucidate its in vivo function, four independent Meg1/Grb10 transgenic mouse lines were established, and the effects of excess Meg1/Grb10 on both postnatal growth and glucose metabolism were examined. All of the Meg1/Grb10 transgenic mice showed growth retardation after weaning (3-4 weeks), which indicates that ectopic overexpression of Meg1/Grb10 inhibits postnatal growth that is mediated by IGF1 via IGF1R. In addition, the mice became hyperinsulinemic owing to high levels of insulin resistance, which demonstrates that Meg1/Grb10 also modulates the insulin receptor cascade negatively in vivo. Type II diabetes arose frequently in the two transgenic lines, which also showed impaired glucose tolerance. In these mice, severe atrophy of the pancreatic acinus cells was associated with high-level production of Meg1/Grb10 in the pancreas. These results suggest that Meg1/Grb10 inhibits the function of both insulin and IGF1 receptors in these cells, since a similar phenotype has been reported for Ir and Igf1r double knockout mice. Taken together, these results indicate that Meg1/Grb10 interacts with both insulin and IGF1 receptors in vivo, and negatively regulates the IGF growth pathways via these receptors.

Animals↗

PubMed2005

Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX<-->XY chimeric testes.

We produced XX<-->XY chimeras by using embryos whose X chromosomes were tagged with EGFP (X*), making the fluorescent green female (XX*) germ cells easily distinguishable from their nonfluorescent male (XY) counterparts. Taking advantage of tagging with EGFP, the XX* "prospermatogonia" were isolated from the testes, and the status of their genomic imprinting was examined. It was shown that these XX cells underwent a paternal imprinting, despite their chromosomal constitution. As previously indicated in sex-reversal XXsxr testes, we also found a few green XX* germ cells developed as "eggs" within the seminiferous tubules of XX*<-->XY chimeric testes. These cells were indistinguishable from XX* prospermatogonia at birth but resumed oogenesis in a testicular environment. The biological nature of the "testicular eggs" was examined by recovering the eggs from chimeric testes. The testicular eggs not only formed an egg-specific structure, the zona pellucida, but also were able to fuse with sperm. The collected testicular eggs were indicated to undergo maternal imprinting, despite the testicular environment. The genomic imprinting did not always follow the environmental conditions of where the germ cells resided; rather, it was defined by the sex that was chosen by the germ cells at early embryonic stage.

Animals↗

PubMed2005

Birth of mice produced by germ cell nuclear transfer.

That mammals can be cloned by nuclear transfer indicates that it is possible to reprogram the somatic cell genome to support full development. However, the developmental plasticity of germ cells is difficult to assess because genomic imprinting, which is essential for normal fetal development, is being reset at this stage. The anomalous influence of imprinting is corroborated by the poor development of mouse clones produced from primordial germ cells (PGCs) during imprinting erasure at embryonic day 11.5 or later. However, this can also be interpreted to mean that, unlike somatic cells, the genome of differentiated germ cells cannot be fully reprogrammed. We used younger PGCs (day 10.5) and eventually obtained four full-term fetuses. DNA methylation analyses showed that only embryos exhibiting normal imprinting developed to term. Thus, germ cell differentiation is not an insurmountable barrier to cloning, and imprinting status is more important than the origin of the nucleus donor cell per se as a determinant of developmental plasticity following nuclear transfer.

Animals↗

PubMed2005

Genomic imprinting of IGF2, p57(KIP2) and PEG1/MEST in a marsupial, the tammar wallaby.

Genomic imprinting is widespread amongst mammals, but has not yet been found in birds. To gain a broader understanding of the origin and significance of imprinting, we have characterized three genes, from three separate imprinted clusters in eutherian mammals in the developing fetus and placenta of an Australian marsupial, the tammar wallaby Macropus eugenii. Imprinted gene orthologues of human and mouse p57(KIP2), IGF2 and PEG1/MEST genes were isolated. p57(KIP2) did not show stable monoallelic expression suggesting that it is not imprinted in marsupials. In contrast, there was paternal-specific expression of IGF2 in almost all tissues, but the biased paternal expression of IGF2 in the fetal head and placenta, demonstrates the occurrence of tissue-specific imprinting, as occurs in mice and humans. There was also paternal-biased expression of PEG1/MESTalpha. The differentially methylated region (DMR) of the human and mouse PEG1/MEST promoter is absent in the wallaby. These data confirm the existence of common imprinted regions in eutherians and marsupials during development, but suggest that the regulatory mechanisms that control imprinted gene expression differ between these two groups of mammals.

Alleles↗

PubMed2004

Generation of pluripotent stem cells from neonatal mouse testis.

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.

Animals↗

PubMed2004

Microinsemination with first-wave round spermatids from immature male mice.

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.

Age Factors↗

PubMed2003

The novel dominant mutation Dspd leads to a severe spermiogenesis defect in mice.

Spermiogenesis is a complex process that is regulated by a plethora of genes and interactions between germ and somatic cells. Here we report a novel mutant mouse strain that carries a transgene insertional/translocational mutation and exhibits dominant male sterility. We named the mutation dominant spermiogenesis defect (Dspd). In the testes of Dspd mutant mice, spermatids detached from the seminiferous epithelium at different steps of the differentiation process before the completion of spermiogenesis. Microinsemination using spermatids collected from the mutant testes resulted in the birth of normal offspring. These observations indicate that the major cause of Dspd infertility is (are) a defect(s) in the Sertoli cell-spermatid interaction or communication in the seminiferous tubules. Fluorescent in situ hybridization (FISH) analysis revealed a translocation between chromosomes 7F and 14C at the transgene insertion site. The deletion of a genomic region of chromosome 7F greater than 1 megabase and containing at least six genes (Cttn, Fadd, Fgf3, Fgf4, Fgf15, and Ccnd1) was associated with the translocation. Cttn encodes the actin-binding protein cortactin. Immunohistochemical analysis revealed localization of cortactin beside elongated spermatids in wild-type testes; abnormality of cortactin localization was found in mutant testes. These data suggest an important role of cortactin in Sertoli cell-spermatid interactions and in the Dspd phenotype.

Animals↗

PubMed2003

Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning.

Although it is widely assumed that the cell type and genotype of the donor cell affect the efficiency of somatic cell cloning, little systematic analysis has been done to verify this assumption. The present study was undertaken to examine whether donor cell type, donor genotype, or a combination thereof increased the efficiency of mouse cloning. Initially we assessed the developmental ability of embryos that were cloned from cumulus or immature Sertoli cells with six different genotypes (i.e., 2 x 6 factorial). Significantly better cleavage rates were obtained with cumulus cells than with Sertoli cells (P < 0.005, two-way ANOVA), which probably was due to the superior cell-cycle synchrony of cumulus cells at G0/G1. After embryo transfer, there was a significant effect of cell type on the birth rate, with Sertoli cells giving the better result (P < 0.005). Furthermore, there was a significant interaction (P < 0.05) between the cell type and genotype, which indicates that cloning efficiency is determined by a combination of these two factors. The highest mean birth rate (10.8 +/- 2.1%) was obtained with (B6 x 129)F1 Sertoli cells. In the second series of experiments, we examined whether the developmental ability of clones with the wild-type genotype (JF1) was improved when combined with the 129 genotype. Normal pups were cloned from cumulus and immature Sertoli cells of the (129 x JF1)F1 and (JF1 x 129)F1 genotypes, whereas no pups were born from cells with the (B6 x JF1)F1 genotype. The present study clearly demonstrates that the efficiency of somatic cell cloning, and in particular fetal survival after embryo transfer, may be improved significantly by choosing the appropriate combinations of cell type and genotype.

Animals↗