Production, epigenetic patterns and modulation of inter-species cloned cat embryos using the bovine oocytes as recipient cytoplasts

Abstract

Experiment I was purposed to find the appropriate treatment of donor cell modification that gave the highest percentage of G0/G1 phase cells. Skin fibroblast cells from domestic cat were cultured and treated with either serum starvation for 1-5 days, cell confluency-contact inhibition for 5 days or roscovitine at various concentrations (7.5-30 µM) for 24 h. Results show that cells were successfully synchronized to G0/G1 stage using the serum starvation for 3 d, confluency-contact inhibition and roscovitine treatment at concentration 15 µM. However, the serum starvation method also increased the number of apoptotic cells. Therefore, the confluency-contact inhibition or roscovitine treatment at concentration 15 µM may be valuable for preparing cat donor cells for SCNT. Experiment II was conducted to evaluate the effect of modification of donor cell by cell cycle synchronization and modification of cultured procedure modification by treatment of histone deacetylase inhibitor TSA on the developmental ability of cat iSCNT embryos using bovine oocytes matured in vitro. First study was aimed to observe the development of interspecies embryos reconstructed from enucleated bovine oocytes and modified cat donor cells by cell cycle synchronization (confluency-contact inhibition or 15 µM roscovitine) in comparison with intraspecies cat and bovine NT. Results show that the fusion rate of interspecies couplets was significantly greater in the roscovitine group than in the contact inhibition group. In both of treatment groups, most embryos stopped the development at the 2- or 4-cell stage, and none of the iSCNT cat embryos developed to the morula or blastocyst stage. Second study was conducted to compare the effect of TSA at different concentrations on the in vitro development of iSCNT cat embryos. Reconstructed cat–bovine embryos were treated with 0, 25, 50, and 100 nM concentrations of TSA for 24 h following fusion. The results showed that 50 nM TSA treatment contributed significantly higher rates of cleavage and blastocyst formation in iSCNT cat embryos compared with untreated embryos and embryos treated with 100 nM TSA. Experiment III was aimed to determine the differential acetylation on histone H3 lysine 9 (K9), 18 (K18), 23 (K23) and di-methylation on histone H3 lysine 9 (K9) in the cat donor cell and iSCNT cat embryos at the early stage between with and without 50 nM TSA treatment compared to bovine IVF embryos. Results show that the acetylation levels on H3K9, H3K18 and H3K23 of TSA-treated cat cells were significant higher than those of non-TSA treated cells. The acetylation levels of AcH3K9, AcH3K18 and AcH3K23 in TSA-treated embryos and bovine IVF embryos were higher than that of control embryos at all examined stages (2 h PF, PN, 2-cell, 4-cell and 8-cell). Exceptionally, in 6 h PN stage, the levels of AcH3K9 and AcH3K23 in embryos treated with 50 nM of TSA and bovine IVF embryos were significant lower than that of control embryos. At PN stage, the significantly higher intensity levels of Me2H3K9 were found in embryos treated with TSA and bovine IVF than that of control embryos. This suggest that the treatment of 50 nM TSA for 24 h after fusion in iSCNT cat embryos contribute the beneficial effects on the modification of acetylation levels of lysine residues (K9, K18 and K23) on histone H3 and di-methylation levels on histone H3K9 during the early embryogenesis. Experiment IV, The total of 224 TSA-treated iSCNT cat embryos at 2- to 4-cell stages was successfully transferred into five recipients. The pregnancy was assessed at day 30 after the embryo transfer by using real-time, B-mode ultrasonography. However, none of the recipients receiving TSA-treated iSCNT cat became pregnant. In conclusion, cat cells can be reprogrammed in bovine oocytes, which the reconstructed iSCNT embryos could successfully developed to the blastocyst stages when TSA was supplemented. However, the production of offspring has not been achieved.