68 EFFECT OF ROSCOVITINE ON FIBROBLASTS’ ABILITY TO FORM BLASTOCYSTS AND ESTABLISH PREGNANCIES AFTER BOVINE NUCLEAR TRANSFER

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 3 methods 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-old 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 (38.5°C 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% v. 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 the same non-return rates (33%). 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 offers may not be revealed until calving.