78 BLIND ENUCLEATION OF OOCYTES IS HIGHLY EFFICIENT IN ZONA-FREE BOVINE CLONING

Abstract

In animal cloning, recipient oocytes are commonly enucleated in metaphase II (MII). By removal of the first polar body (PB1) and 20–30% of the adjacent cytoplasm, without chromatin staining (blind enucleation), only 60% of bovine oocytes can be enucleated (Bordignon and Smith 1998 Mol. Reprod. Dev. 49, 29–36). It is possible that PB1 displaces relative to the MII spindle during removal of cumulus cells (Mohamed Nour and Takahashi 1999 Theriogenology 51, 661–666; Dominko et al. 2000 Biol. Reprod. 62, 150–154). The present study examined the efficiency of blind enucleation of oocytes during preparation of ooplasts for zona-free bovine cloning. Abattoir-derived bovine oocytes were maturated in TCM-199. At 16 h post-maturation (hpm), cumulus cells were removed by vortexing. The zona pellucida (ZP) was removed with pronase. Oocytes with PB1 were retained for enucleation. The rest of oocytes were returned to the maturation medium and checked at an interval of 30–60 min for PB1 extrusion. PB1 together with a minimal volume of adjacent cytoplasm was aspirated using a micromanipulator-controlled micropipette (perpendicular broken, 25–30 µm in diameter). After enucleation, presumptive ooplasts and aspirated oocyte fragments with PB1 were stained with Hoechst 33342. The MII spindle location was determined in UV light under a microscope. In 5 experiments, 227 presumptive ooplasts were investigated. By means of blind enucleation, chromatin was eliminated from 220 oocytes. Maternal chromosomes were found in the cytoplasm of only 7 oocytes after enucleation. The success rate of blind enucleation of bovine zona-free oocytes amounted to 96.9% (220/227). Since the PB1 is fixed on the surface of the zona-free oocyte, when chromosomes migrating with the PB1 are still attached to the microtubule of the spindle, it is a clearly discernible and accurate indicator of maternal chromosome location. The volume of the oocyte cytoplasm removed with the MII plate was about 3.0%. Removal of cumulus cells at 15-16 hpm does not decrease the degree of nuclear maturation of oocytes and has no effect on the development of parthenogenetic or cloned embryos (Galli et al. 2002 Cloning Stem Cells 4, 189–196; Li et al. 2006 Mol. Reprod. Dev. 73, 446–451). In our experiments, the ooplasts prepared by the method of blind enucleation of oocytes without ZP ensured the development to the blastocyst stage from 30 to 50% of reconstructed bovine embryos when fetal fibroblasts were used as nuclear donors. The data obtained suggest that blind enucleation of oocytes is a simple and efficient method to prepare high-quality recipient ooplasts for zona-free bovine cloning.