Project summary:
In our project, we aimed to determine whether the velocity of cytoplasmic movement in mural and polar trophectoderm cells of mouse blastocysts could serve as a marker of embryo implantation potential. To this end, we performed time-lapse imaging of E3.5 and E4.5 mouse embryos to record cytoplasmic movement and used Particle Image Velocimetry (PIV) to quantify its velocity.
First, we found that the cytoplasmic movement velocity decreases during late preimplantation embryo development (from E3.5 morulae to E4.5 blastocysts) and is higher in polar than in mural trophectoderm (TE) cells of mouse E3.5 and E4.5 blastocysts (File 1A-E).
Then we checked whether the cytoplasmic movement velocity was affected by changes in the quality of keratin cytoskeleton. To verify this, we prepared blastocysts with Krt8 or Krt18 knockdown by injecting Krt8 or Krt18 siRNA into zygotes. Efficiency of these knockdowns was verified on protein level – by quantifying fluorescence signal in immunostained embryos (Files 2A-F, 3A-F) and mRNA levels (Files 4A-D, 5A-D). Then we compared the cytoplasmic velocity in TE cells of E3.5 and E4.5 blastocysts, which developed from zygotes injected with siRNA specific to Krt8 or Krt18 and scrambled RNA. We found that the depletion of Krt18 altered velocity of cytoplasmic movement in mural, but not in polar TE cells of E3.5 blastocysts. Depletion of Krt8 affected cytoplasmic speed neither in mural nor in polar E3.5 TE cells (Files 6A-F). Depletion of neither Krt18 nor Krt8 affected cytoplasmic velocity in E4.5 TE cells (Files 7A-F).
We also verified whether the cytoplasmic movement velocity was affected by maternal and postovulatory aging. Our data revealed that both maternal and postovulatory aging did not affect the cytoplasmic movement velocity in mouse blastocysts (Files 8A-F).
Next, we investigated whether the velocity of cytoplasmic movement in E3.5 and E4.5 blastocysts was associated with the embryos' ability to implant in vitro. Briefly, we measured the cytoplasmic velocity in E3.5 or E4.5 blastocysts and then, at E4.5 stage, seeded the embryos onto gelatin-coated plates to allow for an outgrowth formation. The outgrowths area was analyzed after a 4-day culture. We examined the association between the velocity of cytoplasmic movement at the blastocyst stage and the area of the resulting outgrowth. We found that the velocity of cytoplasmic movement in mural and polar TE cells of E3.5 blastocysts did not reflect the embryo’s ability to implant in in vitro conditions (Files 9A-B). On the other hand, we showed that the velocity of cytoplasmic movement in polar, but not mural, TE cells in E4.5 blastocysts reflects the embryo’s ability to implant in vitro (Files 10A-B).
Finally, we wished to test whether time-lapse imaging used to record cytoplasmic movement, affected the embryo’s ability to implant in vitro. To this end, we compared the ability to form outgrowths of imaged and not imaged E3.5 (File 11A-B) and E4.5 (File 12A-B) blastocysts and we showed that time-lapse imaging does not affect the embryo’s ability to implant in vitro.
For file descriptions, please consult the readme.txt file.
