310 NUCLEAR AND MICROTUBULE REMODELING AND IN VITRO DEVELOPMENT OF NUCLEAR TRANSFERRED CAT OOCYTES WITH SKIN FIBROBLASTS OF DOMESTIC CAT AND LEOPARD CAT

Abstract

The leopard cat (Prionailurus bengalensis), a member of the felidae family, is a threatened animal in South Korea. In terms of endangered felids, nuclear transfer is a potentially valuable technique for assuring the continuation of species with dwindling numbers. The protocol for nuclear transfer has been described previously (Yin et al. 2005 Reproduction 129, 245-249). In this experiment we evaluated nuclear and microtubule remodeling and the in vitro developmental potential of enucleated cat oocytes reconstructed with nuclei from either domestic cat fibroblasts (DCF) or leopard cat fibroblasts (LCF). Microtubule aster was allocated to decondensed chromatin at 6 h post-activation following nuclear transfer of fibroblast cells from both DCF and LCF (3/3 in DCF, 2/3 in LCF), suggesting the introduction of a somatic cell centrosome (Kim et al. 1996 Mol. Reprod. Dev. 43, 248-255; Park et al. 2004 Mol. Reprod. Dev. 68, 25-34). At 12 h following nuclear transfer, the nucleus swelled into a large pronucleus-like structure in most reconstructed oocytes (5/9 in DCF and 4/6 in LCF), which showed further enlargement until 18 h after nuclear transfer (4/6 in DCF, 4/6 in LCF). Two microtubule asters were seen near the swollen nucleus. At 18 h following nuclear transfer, the mitotic metaphase (1/6 in DCF) or two cell divisions (1/6 in DCF, 2/6 in LCF) were observed. The percentage of blastocyst formation from nuclear transfer embryos derived from DCF (4/46, 8.6%) was not significantly different from that for nuclear transfer embryos constructed with LCF (4/52, 7.6%). Statistical significance was established at the P < 0.05 level by a X2-test (SAS; SAS Institute, Inc., Cary, NC, USA). These results indicate that nuclear and microtubule remodeling processes and in vitro developmental ability are similar in cat oocytes reconstructed with both domestic cat and leopard cat nuclei.

This work was supported by KOSEF (grant # M10525010001-05N2501-00110).