Investigation of transcript assymetry within oocyte and early embryo.

<p>A) Unfertilized ovine MII-oocytes were micro surgically trisected to generate cortical materials containing MII-spindle material (S) and oocyte hemispheres that were either near to (NS) or far from (FS) the MII-spindle. The pools of S, NS, and FS cytoplasmic fragments were prepared and used for comparative RT-qPCR using 21 developmentally important genes. Obtained profiles revealed four patterns of transcript abundances within the three fragments of the oocyte. B) To understand whether ovine early embryos inherit differential transcripts localization observed within MII-oocyte, the transcriptional distribution of transcripts between early embryonic sister blastomeres was investigated using RT-qPCR. For this purpose, one blastomere of ovine early embryo was labeled with Dil and the embryos were followed during the first and second embryonic divisions to generate pools of leading and lagging blastomeres. The leading and lagging pool of blastomeres were used for quantitative analysis of those transcripts that were found to be differentially located with unfertilized MII oocytes.</p

Comparative analysis of total mRNA contents between subcellular structures of MII-oocyte.

<p>^a-c: values with different superscripts are significantly different at p<0.05.</p

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 - Fig 8

<p>A) Comparative analysis of total protein contents within subcellular structures of MII-oocyte. ^a-c: values with different superscripts are significantly different at p<0.05. B) Protein patterns of oocyte substructures revealed by SDS-PAGE electrophoresis. C) Western blot analysis of NANOG and DNMT3A between NS+S and FS oocyte fragments.</p

Developmental competence of corresponding sister blastomeres derived from ovine 2-cell embryos.

<p>The proportion of sister blastomeres that developed to certain developmental stages (y-axis: 2-cell block (2CB), two further cleavage (4-cell block: 4CB), three further cleavage (8-cell block: 8CB), four further cleavage (morula block: MB), and those blastomeres that reached the blastocyst stage (BLS)). The size of circles correspond their relative proportions (numbers within the circles).</p

Contributions of leading and lagging blastomeres to the total cell count of the developed blastocysts.

<p>In majority (75%) of blastocysts, leading blastomeres contributed to significantly higher cells of the blastocyst compared to the lagging counterpart. In the remaining 25%, the contributions of leading and lagging blastomeres to total cell count of the blastocysts were comparable. *: significantly different at P<0.05.</p

Specific primers used for qRT-PCR.

<p>Specific primers used for qRT-PCR.</p

Lineage tracing of the leading and lagging blastomeres.

<p>A cohort group of blastocysts derived from leading-labeled two-cell embryos that are observed using appropriate Hoechst-33342 (A) and Rhodamine (B) filters. C) Criteria for sorting of Dil-labeled embryos. Scoring the orientation of the Em-Ab axis in ovine blastocysts relative to the first cleavage plane of the 2-cell embryo. After drawing imaginary lines of the Em-Ab axis and equatorial plane, the blastocysts were classified to three groups depending on the angular departure of the boundary line of the fluorescent/nonfluorescent cells from the equatorial plane: i) specified: when the angular departure was almost 90°, ii) semi-specified: when the angular departure was ≥45°, iii) un-specified: when the angular departure was <45°. D1-F4) Representative images of specified (D1-D4), semi-specified (E1-E4) and un-specified (F1-F4) observed under bright light (D1, E1, & F1), and H33342 (D2, E2, & F2) and Rhodamine (D3, E3, & F3) filters. D4, E4, & F4 images show merged images of H33342 and Rhodamine. Bar represents 100 μm.</p

The importance of orientation of the embryonic divisions.

<p>Cleavage can occur either equatorially or meridionally, along the animal-vegetal axis, with reference to MII-spindle as the hypothetical animal pole. A result of equatorial first cleavage division is the transcriptome asymmetry between the balstomeres of 2-cell embryo that could persist even after the second cleavage division. By contrast, a result of meridional first cleavage division is the transcriptome symmetry between the balstomeres of 2-cell and 3-cells embryos, despite oocyte transcript polarity. Moreover, indiscriminate separation of 2-cell embryos yields constraints that exclude the possibility that transcriptome asymmetry within MII-oocyte leads to programmed asymmetric transcriptome inheritance between blastomeres of 2-cell embryos.</p

Representative figures and Nile-red staining for investigation of lipid droplet distribution with MII-oocyte (A-A”) and between sister blastomeres of 2-cell embryo (B-C”).

<p>Lipid-droplets are evenly distributed within MII-oocyte (A-A”), and in majority of 2-cell embryos (A-A” & D). However, in some 2-cell embryos, one balstomere had significantly higher lipid droplets (C-C” and E). Bar represents 100 μm. *: significantly different at P<0.05.</p

Topological assessment of sperm entry point (SEP) in ovine eggs.

<p>Fertilzied oocytes were rotated under constant UV-light until MII-spindle was positioned to 3 O’clock. Then, the spatial relationship between SEP and MII-spindle was measured in 4 imaginary oocyte zones. In this scheme, zones I and IV are the closest and the furthest from the MII-spindle, respectively. Table represents the results of topological assessment of SEP.</p