The Leading Blastomere of the 2-Cell Stage Parthenogenetic Porcine Embryo Contributes to the Abembryonic Part First

Abstract

ABSTRACT. Polarity formation in preimplantation embryos is controversial. To investigate the embryonic-abembryonic axis in the pig, porcine parthenotes were used to prevent the topological change caused by polyspermy as well as to avoid the influences of sperm entry position. For lineage tracing, DiI, a fluorescence dye, was injected into only a blastomere of the 2-cell stage embryos. If the first blastomere to divide was labeled, the embryo was included in the leading group, and while all others were included in the lagging group. In 60.5% of the blastocysts in the lagging group, the progeny of the labeled blastomeres formed the inner cell mass (ICM) and adjacent trophectoderm (TE) hemisphere; 62.1% of the blastocysts in the leading group had progeny of the labeled blastomeres distributed only to the TE (opposite of ICM). The rest of the lagging and leading groups showed random distributions. Unlike murine parthenotes, biased mitochondrial distribution was also found in porcine parthenotes (38.1%). Our findings indicate that the 'leading' blastomere of the 2-cell porcine parthenote forms the distal TE (abembryonic) and that the 'lagging' blastomere forms the remaining portion of the blastocyst, including the ICM (embryonic). Biased distribution of mitochondria in each 2-cell blastomere may contribute partly to this event.

The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals

“…To understand transcript distribution between early embryonic sister blastomeres ( Fig 2B ), ovine 2-cell embryos were collected around 18 h post IVF and used for random labeling of one sister blastomere using the fluorescent dye Dil (1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate) as described by Park et al [ 35 ]. In brief, Dil was dissolved in corn oil at 2.5 mg/ml at 60°C, allowed to cool and then used immediately.…”

“…One blastomere of two-cell stage ovine embryos was randomly labelled with fluorescent Dil. After preparation of injection needle as described above, random labeling of one two-cell blastomere was accomplished by intracytoplasmic injection of the Dil droplet as described by Park et al [ 35 ]. Labeled embryos were subsequently cultured in their respective embryo culture media and checked for the second cleavage every 3–4 h under a stereomicroscope.…”

“…Lineage-tracing of cleavage stage embryos is poorly understood in mammalian species rather than mice, with only two studies assessed embryo patterning in porcine [ 34 , 35 ]. However, choosing the adequate animal model to test biological hypotheses is still an open challenge and increasing evidence suggests that mouse may not be a relevant model for human biological studies.…”

A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

“…Due to a high lipid granule content, porcine oocytes appeared very dark in color [33,34,35]. During the enucleation step of SCNT, fluorescence microscopy with UV exposure often facilitates the removal of the oocyte nucleus stained with Hoechst 33342 fluorescent dye.…”

Transcriptome analysis of in vitro fertilization and parthenogenesis activation during early embryonic development in pigs

“…Among the AS events identified, TSS and TTS were the most abundant types (TSS: 39%-40%; TTS: 37.2%-37.9%), followed by SKIP (nearly 9%), XSKIP and MSKIP (both nearly 2%), IR and XIR (both nearly 1%), and other AS types (all below 1%) (SupplementaryFigure S4). This finding further suggests that mRNA processes related to TSS and TTS of alternative splicing events may occur in the transcription regulation of early embryonic development.6.Comparative analysis of DEGs between IVF and PA Inner cell mass (ICM) is often not obvious and only a few cells are present in porcine early embryos, which cause failure on pig parthenogenesis activation embryo and pose a huge challenge for parthenogenetic embryo stem cell (pESC) culture and breeding parthenogenetic pig(Hall and Hyttel, 2014;Park et al, 2009). Here, we performed differential expression analysis of protein-coding genes in the same stages between IVF and PA. We identified 92 (67 upregulated, 25 downregulated), 3,670(2,731 upregulated, 939 downregulated), and 259 (212 upregulated, 47 downregulated) DEGs in the zygote, morula, and early blastocyst stages, respectively (Supplementary…”