56 DEVELOPMENT OF INTERSPECIES CLONED EMBRYOS USING SOMATIC CELLS FROM VARIOUS SPECIES AND BOVINE CYTOPLASTS

Abstract

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus–cytoplasm interaction and it provides a possible alternative to cloning animals whose oocytes are limited. In Experiment 1 of the present study, we investigated the developmental potential of iSCNT embryos created from monkey, pig, and goat donor cells and bovine cytoplasts. Bovine ovaries were obtained at a local slaughterhouse and the cumulus-oocyte complexes (COCs) aspirated. COCs were matured in vitro in TCM-199 supplemented with 10 IU mL^–1 pregnant mare serum gonadotropin (PMSG), 10 IU mL^–1 hCG, and 10 ng mL^–1 epidermal growth factor (EGF) at 38.5°C and 5% CO₂ in air for 20–22 h. At the end of IVM, half of the COCs were inseminated using frozen semen (1 × 10⁶ sperm mL^–1) and the remainder were used for iSCNT after the cumulus cells were removed with 0.1% hyaluronidase in TCM-199. The procedure of iSCNT and establishment of donor cells were according to Koo et al. (2002 Biol. Reprod. 67, 487–492). After IVF and iSCNT, presumptive zygotes were cultured in CR1-aa medium supplement with 0.3% BSA. After 3 days, cleaved embryos were transferred to CR1-aa medium supplemented with 10% FBS and cultured for an additional 4 days. In Experiment 2, we investigated the developmental ability of reconstructed embryos produced from monkey cells and bovine cytoplasts using various IVC media, such as IVC-1/2 (InVitroCare, Frederick, MD, USA), G-1/2 (Vitrolife, Inc., Englewood, CO, USA) and complete medium (CM; Irvine Scientific, Santa Clara, CA, USA). All experiments were repeated more than three times and data were analyzed with t-test of one-way ANOVA using the SAS 8.01 program (SAS Institute, Inc., Cary, NC, USA). Cleavage and developmental rate of blastocysts were expressed as mean ± SEM. In Experiment 1, we investigated the development ability among IVF, SCNT (bovine-bovine), and iSCNT (monkey-bovine, pig-bovine, and goatbovine) embryos cultured in CR1-aa medium. Our results showed that the cleavage rate of IVF (73.6 ± 1.8%, 86/117) embryos was not significantly different compared to SCNT (84.6 ± 2.7%, 38/45), and iSCNT (89.3 ± 2.7%, 100/110, monkey; 89.3 ± 3.3%, 45/49, pig; and 86.0 ± 2.3%, 87/95, goat). Although cloned embryos reconstructed with monkey cells did not develop to the blastocyst stage, iSCNT embryos derived from pig and goat cells did (3.3 ± 3.0%, 2/49, and 7.9 ± 1.7%, 7/95, respectively). However, these blastocyst formation rates were significantly lower compared to those of IVF and SCNT bovine embryos (32.5 ± 2.9%, 38/117, and 26.7 ± 2.8%, 12/88, respectively; P < 0.05). The success of iSCNT was confirmed by PCR of mitochondrial DNA, porcine PKA region, and SRY region. In Experiment 2, we investigated the developmental potential of cloned embryos produced by monkey cells using various IVC media (IVC-1/2, G-1/2, and CM). The cleavage rate of iSCNT embryos was not significantly different among these media (86.9 ± 2.7%, 78.1 ± 2.1%, and 82.3 ± 1.8%, respectively). However, we did not observe blastocyst formation using these media. Therefore, we suggest that the cytoplasts of bovine oocytes can support blastocyst development of cloned embryos with pig and goat cells, but they were not suitable for monkey cells. In conclusion, our results suggest that species-specific differences are apparent in the production of iSCNT embryos.