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TELOMERE RESTORATION IN CLONED CATTLE OCCUR DURING EARLY EMBRYO DEVELOPMENT.
Sendai, Yutaka1, Aoyagi, Kazushige2, Kobayashi, Masato2, Yamashita, Shoko1, Shinkai, Yoichi3, Hoshi, Hiroyoshi1, 1 2 3
ABSTRACT- The ends of mammalian chromosomes are composed of repeated DNA sequence known as telomeres. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that shorten progressively upon each successive cell division. Generally, telomerase activity is present in germ cells but not in somatic cells. Recent studies in bovine have been shown that telomerase activity is present in early embryos fertilized in vitro and in nuclear transferred embryos. Very recently, teromere elongation/restoration has been observed in cloned cattle, suggesting that teromere elongation/restoration after cloning may occurs during embryogenesis. In the present study, we examined whether the restoration of short teromere of nuclear-transferred bovine embryos occurs at early stage of embryogenesis. We performed a telomere restriction fragment (TRF) analysis using genomic DNA to test the telomere shortening in donor fibroblast cells (from 13-years-old Japanese black bull) during in vitro culture. The TRF lengths of donor fibroblast cells at population doubling (PD) 4, 24, 44 and 64 were approximately 18, 16, 14 and 12 kb, respectively. The average telomere shortening was ~100 bp per one PD. Higher rates of blastocyst development were obtained from donor cells of PD 4 and 24, while the rate from PD 64 donor cells was significantly low (37.6 and 42.1% vs. 5.0%). To examine the telomere lengths of nuclear-transferred embryos , we established the blastocyst-derived cells (ES-like cell) from the cloned embryos (PD 4 and PD 64 donor cells). The ES-like cells showed the high telomerase activity detected by polymerase chain reaction-based assay. As early as ~40 days after nuclear transfer, the TRF lengths of the ES-like cells recovered similar levels to those from in vitro-fertilized embryos. These results suggest that the restoration of telomere lengths after cloning occurs during early embryo development. (Supported by Joint/Research Project for Regional Intensive)
KEY WORDS: telomere, telomerase, bovine, nuclear transferred embryo