60 EFFECTS OF CAFFEINE ON THE IN VITRO DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS

Abstract

Previous studies have demonstrated that treating ovine oocytes with caffeine increases the activities of both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). When such oocytes are used as cytoplast recipients for nuclear transfer (NT), there is an increase in cell numbers at the blastocyst stage (Lee and Campbell 2004 Rep. Fert. Dev. 16, 125). The objective of this study was to determine the effects of caffeine on MPF and MAPK activities and the development of bovine NT embryos. Oocytes were matured in maturation medium (MM) composed of TCM199, 10% fetal bovine serum (FBS), 5 µg mL^-1 follicle-stimulating hormone FSH, 5 µg mL^-1 lutcinizing hormone (LH) and 1 µg mL^-1 estradiol for 24 h. Subsequently, oocytes were cultured in MM supplemented with 0, 5, 10, and 15 mM caffeine for 6 h. Groups of 10 oocytes were sampled and analyzed for MPF and MAPK activities as previously described (Ye et al. 2003 Reproduction 125, 645-656). Treatment with 15 mM caffeine significantly increased the levels of MPF and MAPK activities in MII oocytes. To study development potential, oocytes at 16 h post-onset of maturation (hpm) were stripped of cumulus cells and enucleated in HSOF containing 5 µg mL^-1 Hoechst 33342 and 7.5 µg mL^-1 cytochalasin B; enucleation was achieved using a blunt (25-µm i.d.) pipette after cutting a hole in the zona pellucida with a XYClone laser (Hamilton Thorne Research, Beverly, MA, USA). Enucleated oocytes were then cultured in MM ±15 mM caffeine for a further 6 h. For NT, quiesced primary bovine foetal fibroblasts were used. Cell fusion was induced with two DC pulses of 35 V for 65 µs at 24 hpm. At 2 h post-fusion, all reconstructed embryos were briefly exposed to ultraviolet light under a fluorescence microscope (Leica Microsystems AG, Wetzler, Germany) in order to assess nuclear morphology, and then activated in HSOF containing 5 µg mL^-1 calcium ionophore (A23187), cultured in SOF with 10 µg mL^-1 cycloheximide and 7.5 µg mL^-1 cytochalasin B for 5 h, and transferred to mSOFaaBSA medium. 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 using the chi-square test. There was a significant increase in the number of reconstructed embryos that underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) when caffeine-treated cytoplast recipients were used (28.6 ± 9.9% and 60.0 ± 11.0% for control and caffeine groups respectively, P < 0.05). Cleavage rates (47.6 ± 10.9% and 50.0 ± 11.1%), development to blastocyst (20.0 ± 4.0% and 30.0 ± 4.6%), and mean cell number (85.0 ± 7.1 and 122.5 ± 3.5) were not statistically different between control and caffeine treated groups, respectively. In summary, treatment of bovine oocytes with 15 mM caffeine increased the activities of two key cell-cycle regulators MPF and MAPK, and statistically increased the occurrence of NEBD and PCC in the donor nuclei. We previously hypothesized that the occurrence and extent of NEBD and PCC may increase nuclear reprogramming in NT embryos (Lee and Campbell 2004 Rep. Fert. Dev. 16, 125; Campbell et al. 2005 Rep. Dom. Anim. 40, 256-268); however, further studies are required to determine the developmental competence of these embryos.