36 RESTORATION OF TELOMERE LENGTH IN CLONED PIG EMBRYOS DURING EARLY EMBRYOGENESIS IS NOT DEPENDENT ON TELOMERE LENGTH AND TYPE OF DONOR CELLS

Abstract

Analyses on telomere length in cloned animals have revealed diverse results depending on the donor cell types. In mice and cattle, telomere length is reset during morula-blastocyst transition and the restoration is thought to be a telomerase-dependent process. However, it is still unknown whether the pattern of telomere elongation during this transition is dependent on donor cell types. In the present study, we examined the changes of telomere length during morula-blastocyst transition in cloned porcine embryos using different types of donor cell. Embryonic stem-like cells (ES), cumulus cells (C), fibroblasts at passages 7 and 10 (F7 and F10, respectively) were used as donor cells to produce NT embryos (ES, C, F7, and F10 groups, respectively). Telomere lengths of ES (35.8 ± 1.5 kb), C (24.4 ± 0.5 kb), P7 (18.7 ± 0.6 kb), and P10 (17.2 ± 0.1 kb) cells were significantly different. In contrast, cloned morulae in ES, C, F7, and F10 groups did not have any significant differences in telomere length (18.2 ± 0.3, 17.8 ± 0.7, 18.5 ± 0.3, and 18.4 ± 0.4 kb, respectively). Likewise, cloned blastocysts in ES, C, F7, and F10 groups had similar telomere length (22.3 ± 1.5, 23.5 ± 2.6, 20.2 ± 1.0, and 20.9 ± 1.0 kb, respectively). However, the telomere of the blastocyst was significantly longer (P < 0.05) compared with the morula in the respective group. Furthermore, relative telomerase activities of cloned morulae in ES, C, F7, and F10 groups (4.2 ± 0.4, 4.0 ± 0.5, 5.1 ± 0.4, and 4.9 ± 0.4, respectively) were significantly lower (P < 0.01) than those of cloned blastocysts in the same groups (8.2 ± 1.1, 8.6 ± 0.6, 12.5 ± 2.9, and 8.3 ± 1.1, respectively). The proportions of blastocysts in cloned embryos for ES, C, F7, and F10 groups (10.0 ± 5.2, 17.3 ± 2.9, 13.5 ± 2.9, and 13.1 ± 3.6%, respectively) did not significantly differ. Total cell numbers in blastocysts for ES, C, F7, and F10 groups (28.3 ± 2.9, 32.6 ± 3.6, 30.4 ± 3.1, and 27.4 ± 2.2, respectively) were not significantly different as well. In the present study, we found that the telomere elongation in cloned pig embryos occurs during morula-blastocyst transition. This is consistent with the previous findings in in vivo and in vitro fertilization and cloned embryos in cattle and mice. We also revealed that although different types of cells (ES, C, and F) or the same type of cells with different telomere length (F7 and F10) were used for NT, their resultant morulae and blastocysts had similar telomere length. This suggests that the telomere restoration during morula-blastocyst transition is independent of telomere length and type of donor cells. An increase in telomerase activity during morula-blastocyst transition indicates that the elongation of telomere length was likely a telomerase-dependent process. In conclusion, restoration of telomere length in cloned porcine embryos during morula-blastocyst transition was independent of telomere length and type of donor cells, and likely a telomerase-dependent process.