Submitted to: International Embryo Transfer Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: November 12, 2004
Publication Date: January 7, 2004
Citation: Powell, A.M., Graninger, P.G., Talbot, N.C., Wall, R.J. 2004. Effect of roscovitine of fibroblast's ability to form blastocysts and pregnancies after bovine nuclear transfer. International Embryo Transfer Society Annual Meeting.Reproduction, Fertility and Development. 16:155. Technical Abstract: Cloning efficiency of fibroblast nuclear transfer is dependent on donor cell chromatin status. Chromatin status is commonly regulated by serum starvation or contact inhibition. We have tested a third method of synchronizing chromatin activity, roscovitine exposure (in MEM + 10% serum) for 24 h, with serum starvation (0.5% serum) for 5 days or growth to confluence in 10% serum prior to nuclear transfer. Roscovitine, a specific cyclin-dependent kinase (CDK)2 inhibitor, provides a means of precisely synchronizing bovine fetal fibroblasts (BFF) at G0/G1 cell cycle stage. Fibroblasts were from 100 day Jersey fetuses. Cells, frozen at passage 2, from fetus 10 are known to produce calves. Fetus 13 cells, frozen at passages 1 and 2, were compared for their ability to serve as nuclear donor cells. Oocytes, either purchased from Bomed or harvested from ovaries obtained from a local slaughterhouse and matured in Ham's F10 were enucleated between 18-21 h post-maturation initiation. Couplets were produced and fused by standard techniques. Embryos were activated 2 to 4 hours after fusion by exposure to ionomycin for 4 min, and DMAP for 4 h. Embryos were then held in CR1aa for 12 h before being cultured in G1 media for 3 days and then G2 media for another 3 days (38.5C and 5% O2 + 5% CO2 + 90% N). On day 7, good quality blastocysts were transferred to synchronized recipient heifers. The remaining embryos were evaluated after another day in culture. Blastocyst development [(100) X (total blastocysts/fused couplets)] was not influenced by fetus (BFF10, 31 +/- 3%; BFF13, 26 +/- 2%, p = 0.126). However, a higher proportion of blastocysts were produced when fibroblasts were cultured in 0.5% serum (38 ' 3%) compared to culture in 10% serum (29 +/- 3%) or in roscovitine (23 +/- 2%, p = 0.001). Time in culture, as measured by passage, had a variable effect on the fibroblast's ability to product blastocysts from the three fibroblast culture conditions tested. Passage 1 and 2 fibroblasts responded similarly to the 0.5% and 10% serum treatments (p > 0.80). When cultured in roscovitine, passage 1 fibroblasts performed better then passage 2 fibroblasts (29 +/- 4% vs. 16 +/- 3 % blastocysts, p = 0.010). Embryos have been transferred to 51 recipients to date. Ten recipients have given birth or are still pregnant. The 60 day non-return rate for those animals was 29%, 50%, and 31% for serum starvation, 10% serum and roscovitine treatments, respectively. BFF10 and BFF13 cells have generated similar non-return rates (33% vs. 33%, respectively). In this study, of the 3 methods of synchronizing fibroblast chromatin, serum-starvation had an in vitro advantage. Cells cultured for different lengths of time (passages) responded differently to synchronization treatments. This may reflect a heterogeneous population of cells at early passages. Current non-return rates seem to favor synchronization by contact inhibition, any advantage roscovitine may offer may not be revealed until calving.