Research articleA simple method for producing tetraploid porcine parthenogenetic embryos
Introduction
Tetraploid embryos have been widely used in animal reproduction. In chimeric mouse embryos, reconstructed with diploid and tetraploid blastomeres, diploid cells contributed to the inner cell mass, whereas most tetraploid cells contributed to trophoectoderm [1], [2], [3]. Furthermore, murine chimeras produced with tetraploid embryos and embryonic stem (ES) cells resulted in mice derived completely from ES cells [4], [5], [6], [7]. Most tetraploid cells contributed to the extraembryonic lineage, whereas diploid cells derived from ES cells contributed to both embryonic and extraembryonic lineages [6], [8], [9], [10], [11], [12]. Therefore, tetraploid cells supported differentiation of diploid cells to form an embryo.
In domestic animals, chimeric calves from ES-like cells aggregated with tetraploid embryos were reported to have been born [13]; however, ES like cells contributed to chimera formation only at a very low level. The authors speculated that putative tetraploid embryos produced by electrically fused diploid blastomeres may not have been completely tetraploid, and furthermore, they indicated the need to identify complete tetraploid embryos before aggregation with ES-like cells. However, porcine chimeras constructed by ES like cells and tetraploid embryos have apparently not been reported. Furthermore, utilization of blastomeres in in vitro produced (IVP) porcine embryos makes it more difficult to produce tetraploid embryos, due to a high frequency of polyspermic fertilization.
Thus, in the present study, the first objective was to produce porcine tetraploid embryos using parthenogenetic embryos, whose ploidy was easy to predict compared with IVP embryos. Porcine immature oocytes underwent maturation culture in the presence of cytochalasin B to inhibit first polar body extrusion, were electrically stimulated, and again treated with cytochalasin B. The second objective was to compare early developmental competence of the resulting tetraploid embryos relative to diploid embryos. Fluorescence in situ hybridization (FISH) was used to detect a porcine chromosome 1-specific sequence in porcine parthenogenetic embryos at various stages, and ploidy transition was examined during early development.
Section snippets
In vitro maturation and development of porcine parthenogenetic embryos
Ovaries from prepubertal gilts were obtained at a local abattoir and transported to the laboratory at 30 to 35 °C. Porcine follicular oocytes were aspirated from antral follicles (3 to 6 mm in diameter) with a 20-gauge needle. Cumulus-oocyte complexes (COCs) were washed three times with HEPES-buffered Tyrode's albumin lactate pyruvate medium [14]; only those with uniform ooplasm and a compact cumulus cell mass were selected for in vitro maturation, which was performed by a two-step method [15],
Effects of maturation culture time on meiotic progression and subsequent pronucleus formation after parthenogenetic activation in porcine oocytes
In the first experiment, meiotic progression of porcine oocytes was observed during in vitro maturation in a two step method described previously (Fig. 1A). Immediately after removal of oocytes from ovarian follicles, almost all oocytes (98.2%; 109/111) were in the resting GV stage. Until 24 h after the beginning of culture, most oocytes remained in the GV stage. However, by 27 h, GVBD oocytes were detected, and by 30 to 33 h, more than 90% of oocytes were GVBD, but MII oocytes had not yet
Discussion
In a preliminary study, meiotic progression and pronucleus formation competence of porcine oocytes were examined during maturation culture, using a culture method described previously. Oocytes that had reached MII appeared 36 h after the beginning of culture and became more abundant as duration of culture increased. At 42 to 48 h, the proportion of MII oocytes peaked at approximately 90%. In contrast, only 6.2% (12/193) of oocytes cultured for 42 h formed pronuclei after electrical stimulation.
Acknowledgments
We are grateful to the pig farm staff (National Institute of Livestock and Grassland Science, Japan) for care and maintenance of pigs and the assistance in handling the animals. We also express our gratitude to Dr. T. Nagai of the National Institute of Livestock and Grassland Science for critical reading of the manuscript.
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