Mammalian oocytes are arrested at the G2-M phase transition of the first meiotic division. In vitro, fully grown oocytes liberated from their follicles spontaneously reinitiate meiosis I, characterized by germinal vesicle breakdown (GVBD), chromatin condensation, spindle assembly, emission of the first polar body and progression to metaphase of the second meiotic division (MII), at which stage they undergo a second arrest until fertilization. After spermatozoa penetrate the oocyte, the second polar body extrudes, male and female pronuclei form and syngamy occurs to start early embryo development. Nuclear changes during oocyte maturation and fertilization are co-ordinated with movements of genetic material and organelles, and with biochemical changes in the cytoplasm to ensure normal embryo development. The normality of early embryogenesis is directly related to the ordered expression of these developmental programmes (Van Blerkom, 1991).Of the numerous cytoplasmic changes that occur, the positioning of mitochondria may be involved in concentrating ATP or calcium to specific regions in oocytes or fertilized eggs to support normal developmental processes. Thus, the distribution of active mitochondria may be indicative of the energy or ion requirement of various key events during oocyte maturation, fertilization and early embryo development. In mice, the perinuclear accumulation of mitochondria between GVBD and metaphase I (MI) (Van Blerkom and Runner, 1984; Van Blerkom, 1991), and the polarized distribution of mitochondria to one half of the oocyte containing the MII spindle (Calarco, 1995) were observed and were regarded as one aspect of the developmental programme of cytoplasmic maturation. Previous observations also revealed that translocation of mitochondria is co-ordinated

Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expression pattern. In order to determine how transgene expression may be regulated in the early embryo, we generated porcine embryos from two distinct genetically modified cell lines by using the nuclear transfer (NT) technique. Both cell lines expressed the enhanced green fluorescent protein (eGFP); the first was a fibroblast cell line derived from the skin of a newborn pig that expressed eGFP, whereas the second was a fetal derived fibroblast cell line into which the eGFP gene was introduced by a retroviral vector. The reconstructed embryos were activated by electrical pulses and cultured in NCSU23. Although the in vitro developmental ability of each group of NT embryos was not different, the eGFP expression pattern was different. All embryos produced from the transduced fetal cell line fluoresced, but only 26% of the embryos generated from the newborn cell line fluoresced, and among those that did express eGFP, more than half had a mosaic expression pattern. This was unexpected because the fetal cell line was not clonally selected, and each cell had potentially different sites of integration. Embryos generated from the newborn cell line were surgically transferred to five surrogate gilts. One gilt delivered four female piglets, all of which expressed eGFP, and all had microsatellites identical to the donor. Here we demonstrate that transgene expression in all the blastomeres of an NT embryo is not uniform. In addition, transgene expression in a genetically manipulated embryo may not be an accurate indicator of expression in the resulting offspring.

Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherinαsubtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherinαsubtypes in porcine oocytes and embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherinα7 (KPNA7), an oocyte and cleavage stage embryo-specific karyopherinαsubtype. We hypothesised that knockdown of KPNA7 would negatively affect porcine cleavage development. To test this hypothesis, in vitro matured and fertilised porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P<0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that embryos have differential requirements for individual karyopherinαsubtypes and that these karyopherinαsubtypes differentially transport intracellular cargo during cleavage development.

Here we report the production of transgenic pigs that express enhanced green fluorescent protein (eGFP). Porcine oocytes were matured in vitro in a serum-free, chemically defined maturation medium, subsequently infected with a replication deficient pseudotyped retrovirus, fertilized and cultured in vitro before being transferred to a recipient female. Two litters were born from these embryo transfers; one pig from each litter was identified as transgenic and both expressed eGFP. From a tool in basic research to direct applications in production agriculture, domestic livestock capable of expressing foreign genes have many scientific applications.

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.