46 THREE STAGES ESTABLISHMENT OF IN VITRO MATURATION OF PORCINE OOCYTES USED AS RECIPIENT CYTOPLASTS FOR SOMATIC CELL NUCLEAR TRANSFER

Abstract

Oocytes at either anaphase/telophase of the first meiotic division (AI/TI) or metaphase of the second meiotic division (MII) are potential candidates used as recipient cytoplasts for somatic cell nuclear transfer (SCNT) because they contain active maturation-promoting factor (MPF), which causes nuclear membrane breakdown (NEBD) and premature chromosome condensation (PCC) in the transferred nucleus and may be essential for nuclear reprogramming (Campbell and Alberio 2003 Reprod. Suppl. 61, 477–494). In vitro maturation of porcine oocytes spends longer times (44 to 48 h), progresses asynchronously from immature stage and presents insufficient MPF for NEBD and PCC at AI/TI or MII. Here we have developed 3 stages establishment of in vitro oocyte maturation to select good quality of porcine oocytes used as recipient cytoplasts for SCNT. First, porcine oocytes were assessed by the activity of glucose-6-phosphate dehydrogenase (G6PD) before in vitro oocyte maturation. These oocytes with loss of expression of G6PD failed to enzymatically break down the dye, brilliant cresyl blue (BCB) and thus stain positively (Roca et al. 1998 Reprod. Fertil. Dev. 10, 479–485). Positively BCB stained oocytes reached to MII (67.0% v. 58.4%) and produced more parthenogenetic blastocysts (50.2% v. 29.6%) in comparison with negatively BCB stained oocytes (P < 0.05). Second, positively BCB stained oocytes were treated with 5 μg mL–1 cycloheximide (CHXM, a nonspecific protein-synthesis inhibitor) to block meiotic progression for synchronizing the cell cycle of oocytes for 16 h. All of the oocytes (130/130) were efficiently synchronized at the germinal vesicle (GV) stage by treatment with CHXM, whereas control oocytes (w/o CHXM) were passed through GV stage (80/176). Retrieval from treatment of CHXM could reversibly induce meiotic resumption and progresses synchronously in porcine oocytes. Following incubation with gonadotropin hormones [epidermal growth factor (EGF) 10 ng mL–1, LH 5μg mL–1, FSH 1 μg mL–1], treatment with 5 mM caffeine (a phosphatase inhibitor) for 12 h significantly increased the activity of MPF in porcine oocytes (P < 0.05). However, there was no difference between CHXM treated and nontreated group (21.9% v. 22.3%) in the developmental rate to the blastocyst stage of parthenogenetic embryos. At least 3 replicates were performed. These data may contribute to an improved nuclear reprogramming in SCNT.