Developmental capacity of bovine cumulus oocyte complexes after transcriptional inhibition of germinal vesicle breakdown

Abstract

Oocytes cultured in the presence of FSH and the transcriptional inhibitor, 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB), remain in meiotic arrest at the germinal vesicle (GV) stage. The objectives of this study were to assess the kinetics of maturation and the developmental capacity of bovine cumulus oocyte complexes (COC) following release from prolonged meiotic arrest by DRB. In Experiment I, COC were cultured for 20 h in Tissue culture medium (TCM)-199 supplemented with 10% estrus cow serum (ECS), 5 μg/ml FSH and 1 μg/ml estradiol in the presence of 120 μM DRB. COC were then released from meiotic arrest and cultured for 20 h in DRB-free medium. Control COC were cultured for 20 h in DRB-free medium, with culture initiated concomitant to the release of DRB-treated COC from meiotic arrest. Nuclear maturation was assessed after 0, 5, 10, 15, and 20 h of culture in DRB-free medium. The proportion of DRB-arrested oocytes reaching metaphase II (MII) following 20 h culture in DRB-free medium was not significantly different from controls (96±4% versus 99±4%). In Experiment II, COC were cultured for 20 h in TCM-199 supplemented with 10% ECS, 10 μg/ml LH, 5 μg/ml FSH, and 1 μg/ml estradiol in the presence or absence of 120 μM DRB. COC in the DRB-treated group were then washed and matured coincident with a second group of control COC for 20 h in DRB-free medium. COC in both groups were fertilized and then randomly assigned to one of two culture systems: TCM-199+10%ECS or mSOF+0.6% fatty acid-free BSA. Development was assessed at 72 h post insemination (hpi), 168 hpi (Day 7) and 216 hpi (Day 9). In this experiment, culture with DRB-arrested oocyte maturation at the GV stage (DRB, 85±3% GV; Control, 2±3% GV; P<0.001). Following release from arrest, maturation and fertilization, the proportion of COC that cleaved by 72 hpi was decreased by treatment with DRB (DRB: 78±3% versus Control: 90±3%; P<0.05). However, no effect of DRB was found on the proportion of cleaved zygotes that reached the blastocyst stage on either Day 7 or Day 9 of culture (Day 7: DRB 16±2% versus Control, 21±2%; Day 9: DRB 23±3% versus Control, 31±3%). More embryos reached the blastocyst stage in the TCM-199/serum culture system compared to the mSOF/BSA system on both Days 7 and 9 (Day 7: TCM-199, 23±2% versus mSOF, 13±2%, P<0.05; Day 9: TCM-199, 32±3% versus mSOF, 22±3%, P<0.05). In summary, bovine COC maintained in meiotic arrest for 20 h by culture in the presence of the transcriptional inhibitor DRB retained their capacity to develop to the blastocyst stage after fertilization in vitro.