158 LEADING BLASTOMERE OF A 2-CELL-STAGE PORCINE PARTHENOGENETIC EMBRYO CONTRIBUTES TO TROPHECTODERM FIRST

Abstract

Embryonic axis formation is important in normal animal development. Some vertebrate and invertebrate eggs display undeniable polarity along a plane known as the animal–vegetal axis. However, axis formation in mammals, which is studied only in mice, is still a point of issue (Hiiragi and Solter 2004 Nature 430, 360–364; Zernicka-Goetz et al. 2005 Nature 434, 391–395). In the present study, we investigated the embryonic–abembryonic axis formation in porcine species. We used porcine parthenogenetic embryos to prevent a topological change of the 2 apposing pronuclei in the egg center caused by polyspermy and to avoid the influence of the fertilization cone, which indicates the sperm entry position. For lineage tracing, DiI, a lipophilic fluorescence dye, was injected into one blastomere per embryo at the 2-cell stage. After this process, as embryos developed into the 3-cell stage, all the embryos were divided into 2 groups named Leading and Lagging, following the location of the DiI oil drop (Leading: the oil drop was positioned in the first-dividing blastomere; Lagging: the drop was positioned in the late-dividing blastomere). The embryos developed into blastocysts after 6 to 7 days of culture in vitro. Only the inner cell mass (ICM) was labeled in 50% (8/16) of the Lagging group. In some Lagging embryos (2/16), ICMs with a small portion of adjacent trophectodermal cells (TE) were labeled and 62.5% (10/16) of Lagging embryos formed the ICM part in total. On the other hand, 60% (6/10) of the Leading embryos formed only distal TE (opposite side of the ICM). Only one of the Leading embryos formed the ICM. The rest of Lagging embryos (37.5%, 6/16) or the Leading group (40.0%, 4/10) showed an even distribution of blastocysts, regardless of the ICM or TE. Oocytes, zygotes, and 2-cell-stage embryos in porcine species tended to show unequally distributed lipid contents and could be distinguished by their different colors and brightness, although not all of them showed significant differences. We injected DiI into a relatively brighter blastomere of the 2-cell-stage embryos and the results were very similar to those obtained from the Leading group (distal TE: 70.6%, 24/34). High lipid contents may be the cause of delayed development of the blastomere of an embryo. Our findings indicated that the Leading blastomere of 2-cell porcine parthenotes formed distal TE first, and that afterward, the Lagging blastomere not only filled the rest of the TE but also contributed to the ICM.

This study was supported by the Research Project on the Production of Bio-organs (No. 200506030601) and Technology Developmental Program (High-Technology Development Project No. 204117-3) for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea