P-152 Velocity of cytoplasmic movement in trophectodermal cells depends on the keratin cytoskeleton
Authors: Walewska A , Pilaszek I , Ajduk A 
Published: June 2023

Abstract

Study question
Can dysfunction of the keratin cytoskeleton, known to severely hinder postimplantation development of mammalian embryos, be detected by analysis of cytoplasmic movement in trophectodermal cells?

Summary answer
Depletion of keratins increases cytoplasmic movement in trophectodermal cells in extent depending on the developmental stage and on the location of the trophectodermal cells (mural/polar).

What is known already
Recent literature data indicate that keratins, intermediate filament proteins, are key regulators of trophectoderm function in mouse and human embryos. Keratin knockouts display trophoblast fragility, placental bleeding, and lethality after implantation. However, depletion of keratin 8 (Krt8) and keratin 18 (Krt18), which are most abundant in preimplantation embryos, does not lead to severe phenotype up to the blastocyst stage at least in mice and cattle, so can easily remain unnoticed. However, it affects the biomechanical properties of the trophectoderm. Analysis of cytoplasmic movement allows for biomechanical assessment of the trophectoderm in a non-invasive way.

Study design, size, duration
We analyzed 188 mouse embryos (66 control, 60 with Krt8 depletion, and 62 with Krt18 depletion) to quantitatively assess changes in the velocity of cytoplasmic movement caused by a knockdown of Krt8 and Krt18. Additionally, we correlated the velocity of cytoplasmic movement in trophectodermal cells (n = 70 embryos) with the ability to form outgrowths. Appropriate statistical tests were used to determine the statistical significance of the obtained results. A value of p &lt; 0.05 was considered statistically significant.

Participants/materials, setting, methods
Zygotes were injected with Krt8 and Krt18 siRNAs or with scrambled siRNAs used as a negative control. The efficiency of the knockdown was tested on mRNA and protein levels. The embryos were cultured for 3 or 4 days and the velocity of cytoplasmic movement in polar and mural trophectoderm cells was measured. Additionally, we correlated the cytoplasmic movement velocity in trophectodermal cells in E3.5 embryos with their ability to implant in vitro (i.e., form outgrowths).

Main results and the role of chance
Our research showed that the velocity of cytoplasmic movement of trophectodermal cells varies depending on the embryo's developmental stage. Namely, the velocity of cytoplasmic movement was higher in E3.5 blastocysts than in E4.5 blastocysts. In addition, the velocity of cytoplasmic movement was higher in polar (8.45 ± 2.64 nm/s in E3.5 and 6.79 ± 1.96 nm/s in E4.5 control embryos) than in mural (6.30 ± 2.30 nm/s in E3.5 and 4.98 ± 1.89 nm/s in E4.5 control embryos) trophectodermal cells. Noteworthy, the depletion of Krt8 and Krt18 increased the velocity of cytoplasmic movement both in the mural and polar trophectodermal cells. However, it appears that changes caused by the depletion of these keratins depend on the embryo's developmental stage. For example, the depletion of Krt18 increased the velocity of cytoplasmic movement in mural trophectodermal cells in E3.5 embryos (9.61 ± 3.80 nm/s), while the depletion of Krt8 - in E4.5 embryos (6.65 ± 2.35 nm/s). In addition, data from our preliminary experiments with in vitro implantation suggested that embryos characterized by the higher cytoplasmic movement velocity were unable to form an outgrowth. The above data suggest that the velocity of cytoplasmic movement depends on the keratin cytoskeleton and probably can be used to predict an embryo’s ability to implant.

Limitations, reasons for caution
We used mouse embryos, a well-established model of early mammalian embryo development. However, analogous experiments should be repeated on other mammalian species, including humans, if we wish to pursue the possibility that cytoplasmic velocity in trophectodermal cells is an embryo quality marker.

Wider implications of the findings
Our data indicate that cytoplasmic velocity in trophectodermal cells may be a valuable tool in embryo quality assessment, as it reflects the functionality of the keratin cytoskeleton in embryos as well as correlates with the embryos’ ability to implant.

Trial registration number
not applicable