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Miyashita N.,Japan National Institute of Agrobiological Science | Kubo Y.,National Institute of Livestock and Grassland Science | Yonai M.,Japan National Agricultural Research Center | Kaneyama K.,National Livestock Breeding Center | And 6 more authors.
Journal of Reproduction and Development

Dolly, the first mammal cloned from a somatic cell, had shorter telomeres than age-matched controls and died at an early age because of disease. To investigate longevity and lifetime performance in cloned animals, we produced cloned cows with short telomeres using oviductal epithelial cells as donor cells. At 5 years of age, despite the presence of short telomeres, all cloned cows delivered multiple healthy offspring following artificial insemination with conventionally processed spermatozoa from noncloned bulls, and their milk production was comparable to that of donor cows. Moreover, this study revealed that the offspring had normal-length telomeres in their leukocytes and major organs. Thus, cloned animals have normal functional germ lines, and therefore germ line function can completely restore telomere lengths in clone gametes by telomerase activity, resulting in healthy offspring with normal-length telomeres. © 2011 by the Society for Reproduction and Development. Source

Sugimura S.,Tohoku University | Yokoo M.,Akita Prefectural University | Yamanaka K.-I.,Japan National Agricultural Research Center | Kawahara M.,Saga University | And 5 more authors.
Cellular Reprogramming

Oxygen consumption reflects overall metabolic activity of mammalian embryos. We measured oxygen consumption in individual porcine somatic cell nuclear transfer (SCNT) and in vitro-fertilized (IVF) embryos by modified scanning electrochemical microscopy. Oxygen consumption in IVF embryos rapidly increased at day 5 of the blastocyst stage (D5BL). IVF embryos that consumed >0.81 ×1014/mol sec-1 of oxygen at D5BL exhibited significantly higher hatching and hatched rates at D7BL, whereas D5BL SCNT embryos using porcine fetal fibroblasts did not show an increase in oxygen consumption until D7BL. The numbers of inner cell mass and trophectoderm (TE) cells and incidence of apoptosis did not significantly differ between IVF and SCNT embryos at D5BL. At D7BL, a significant lower number of TE cell and higher incidence of apoptosis were observed in SCNT than in IVF embryos; this significantly correlated with their oxygen consumption at D5BL. Use of cumulus cells as donor cells neutralized the low oxygen consumption in SCNT embryos at D5BL, regardless of the difference between the recipient cytoplasm and donor nucleus. Some of SCNT embryos at D7BL were retrieved the hatching completion and were improved the number of TE cell and apoptosis incidence by using cumulus cells. Thus, anomalous oxygen consumption in porcine SCNT embryos at D5BL could be sign of limited hatchability, which may be responsible for the low TE cell number and high apoptosis incidence. © 2010, Mary Ann Liebert, Inc. Source

Fukuda S.,Hokkaido Animal Research Center | Okada H.,Japanese National Institute of Animal Health | Arai S.,Japanese National Institute of Animal Health | Yokoyama T.,Japanese National Institute of Animal Health | Mohri S.,Japanese National Institute of Animal Health
Journal of Comparative Pathology

Bovine spongiform encephalopathy (BSE) is characterized by the appearance of spongy lesions in the brain, particularly in the brainstem nuclei. This study evaluated the degenerative changes observed in the central auditory brainstem of BSE-challenged cattle. The neuropathological changes in the auditory brainstem nuclei were assessed by determining the severity of vacuolation and the presence of disease-associated prion protein (PrP Sc). Sixteen female Holstein-Friesian calves, 2-4 months of age, were inoculated intracerebrally with BSE agent. BSE-challenged animals developed the characteristic clinical signs of BSE approximately 18 months post inoculation (mpi) and advanced neurological signs after 22mpi. Before the appearance of clinical signs (i.e. at 3, 10, 12 and 16mpi), vacuolar change was absent or mild and PrP Sc deposition was minimal in the auditory brainstem nuclei. The two cattle sacrificed at 18 and 19mpi had no clinical signs and showed mild vacuolar degeneration and moderate amounts of PrP Sc accumulation in the auditory brainstem pathway. In the animals challenged with BSE agent that developed clinical sings (i.e. after 20mpi), spongy changes were more prominent in the nucleus of the inferior colliculus compared with the other nuclei of the auditory brainstem and the medial geniculate body. Neuropathological changes characterized by spongy lesions accompanied by PrP Sc accumulation in the auditory brainstem nuclei of BSE-infected cattle may be associated with hyperacusia. © 2010 Elsevier Ltd. Source

Sawai K.,Iwate University | Takahashi M.,Japan National Agricultural Research Center | Fujii T.,Iwate University | Moriyasu S.,Hokkaido Animal Research Center | And 4 more authors.
Journal of Reproduction and Development

DNA methylation is an important factor for the regulation of gene expression in early embryos. It is well known that the satellite I sequence is more heavily methylated in bovine somatic cell nuclear transfer (NT-SC) embryos than in embryos derived from in vitro fertilization (IVF). However, the methylation status of bovine embryos obtained by other procedures is not well known. To clarify DNA methylation levels of bovine embryos obtained from various procedures, we examined satellite I sequences in bovine blastocyst (BC) embryos derived from NT-SC, NT using embryonic blastomeres (NT-EM), in vivo (Vivo), IVF and parthenogenetic treatment (PA). Furthermore, in order to evaluate the efficacy of DNA demethylation by the NT procedure, we determined the DNA methylation levels in bovine embryos in which NT was recapitulated (Re-NT). Although the DNA methylation levels in the NT-SC embryos were higher than those in the other embryos, the NT-EM embryos exhibited lower DNA methylation levels. The satellite I sequence in the NT-SC embryos was more demethylated than that in the donor cells. Although the DNA methylation level in the individual NT-SC embryos showed variation, the full-term developmental efficacy of these embryos were not different. These findings suggest that the methylation level of the satellite I sequence at the BC stage is not related to the abnormalities of bovine embryos produced by NT-SC. There was no difference in methylation levels between Re- NT and NT-SC embryos. Our results indicated that the DNA methylation status differed among embryos produced by various methods and that at least some of the demethylation of the donor cell genome occurred in the recipient cytoplast after NT-SC, but the demethylation ability of the NT procedure was noted in the first NT but not in the second NT. © 2011 by the Society for Reproduction and Development. Source

Sawai K.,Iwate University | Takahashi M.,Japan National Agricultural Research Center | Moriyasu S.,Hokkaido Animal Research Center | Hirayama H.,Hokkaido Animal Research Center | And 3 more authors.
Cellular Reprogramming

The epigenetic reprogramming of the donor cell nucleus is an important factor in the development of embryos and production of normal offspring derived by somatic cell nuclear transfer (NT-SC). During early development, a dramatic reduction in methylation levels occurs in mouse. In early embryos, this process makes it possible to erase gamete-specific methylation patterns and induce de novo methylation at defined developmental time-points. To clarify changes in DNA methylation in bovine NT-SC embryos, we examined satellite I sequences in bovine embryos derived in vivo (Vivo) and by NT-SC at the blastocyst (BC) and elongated (EL) stages. Because the EL stage embryo consists of the embryo disc (ED) and trophectoderm (TE), the methylation status of each part was analyzed with respect to the progress of differentiation. DNA methylation levels in Vivo embryos were increased during the elongation stage. In contrast, DNA methylation levels in NT-SC embryos remained unchanged in the ED and significantly decreased in the TE. Real-time PCR analysis showed that Dnmt-1 expression in BC embryos derived by NT-SC was significantly lower than that in Vivo embryos; thus, differences in the DNA methylation status may reflect transcript levels of Dnmt-1. Our results suggest that the aberrant methylation level of bovine NT-SC embryos in the satellite I region is corrected as a result of demethylation and retention of methylation as the embryo develops and differentiates. © 2010, Mary Ann Liebert, Inc. Source

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