5 MPF AND MAP KINASES IN OVINE OOCYTES: EFFECTS OF ENUCLEATION AND CAFFEINE ON ACTIVITY AND DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS

Abstract

In nuclear transfer (NT) embryos, exposure of the donor chromatin to the MII cytoplasm results in premature chromatin condensation (PCC) which may be beneficial for nuclear reprogramming (Campbell KHS and Alberio R 2003 Reprod. Suppl. 61, 477–494). Following enucleation, maturation promoting factor (MPF) activity in murine oocytes is primarily associated with the meiotic spindle. This reduced MPF activity in the cytoplast may result in decreased PCC and reprogramming. Conversely, increasing cytoplast MPF activity may increase reprogramming. The aims of this study were to perform quantitative analysis of MPF and MAPK activities in ovine oocytes: 1. at anaphase/telophase I (A/TI) or MII; 2. following enucleation; 3. following treatment with caffeine (an inhibitor of Myt1/Wee1 activity). The development of ovine NT embryos reconstructed using caffeine-treated oocytes as cytoplast recipients was then determined. Oocytes were matured in TCM 199, 10% FBS, 5 μg mL⁻¹ FSH, 5 μg mL⁻¹ LH,1 μg mL⁻¹ estradiol, 0.3 mM sodium pyruvate and 100 μM cysteamine. 15 h post-onset of maturation (hpm) oocytes were stripped of cumulus cells and enucleated in HSOF containing 5 μg mL⁻¹ Hoechst 33342 and 7.5 μg mL⁻¹ cytochalasin B (CB). Control oocytes were sham-enucleated by removing an equal volume of cytoplasm. Oocytes were cultured in SOF ± 10 mM caffeine. Groups of 10 oocytes were sampled and analyzed for MPF and MAPK activities as previously described (Ye JP et al., 2003 Reproduction 125, 645–656). For NT, primary foetal fibroblasts were quiesced in DMEM containing 0.1% FBS for 2–3 days. Cell fusion was induced with two DC pulses of 25V cM-1 for 80 μs. 3 methods of NT were compared: A. fusion 20 hpm, activation 21 hpm; B. fusion 24 hpm, activation 25 hpm; C. 10 mM caffeine 18–24 hpm, fusion 24 hpm, activation 25 hpm. All oocytes were activated in HSOF containing 5 μg mL⁻¹ calcium ionophore (A23187), cultured in SOF with 10 μg mL⁻¹ of cycloheximide and 7.5 μg mL⁻¹ CB for 5 h, and then transferred to mSOFaaBSA medium, all at 5% CO₂, 5% O₂ and 90% N₂ at 39°C. On Day 2 cleavage was assessed and 10% FBS added to the medium. Development to blastocyst was assessed on Day 7. All data were analyzed by chi-square test. Both MPF and MAP kinase activities were increased at MII compared to A/TI (P < 0.05). There were no differences in activities of both kinases between intact and enucleated oocytes. Following enucleation, both kinase activities were identical in all groups, reaching maximum activities 24 hpm followed by a slow decline. Caffeine increased the activity of both kinases (MPF in particular) in all groups. Following 5 replicates (total oocytes 145, 143, 144 for NT methods A,B,C, respectively), no significant differences were observed between fusion (82.1%, 67.8%, 67.4%), cleavage (90.8%, 88.7%, 89.7%) or development to blastocyst (20.2%, 18.6%, 25.8%). Analysis of total cell numbers on limited numbers of blastocysts (7, 6, 7) were NS (70.9 ± 38.5, 69.3 ± 25.4, 93.3 ± 17.8).