P-203 The Effect of c-Abl Tyrosine Kinase Inhibition in Cell Fate Specification and Morphogenesis During Preimplantation Mouse Embryo
Authors: Yıldırım E , Onel T , Yaba A 
Published: June 2023

Abstract

Study question
What are the mechanisms governing the regulation of c-Abl tyrosine kinase in preimplantation embryo development?

Summary answer
Inhibition of c-Abl impairs the fate determination of TE and ICM cells during mouse preimplantation embryo development, preventing cell specilization after compaction.

What is known already
The successful completion of polarization, compaction and lineage specification stages is required for the embryo to reach the blastocyst stage from the zygote stage during preimplantation embryonic development. c-Abl is a tyrosine kinase localized in cell nucleus and cytoplasm and also capable of nuclear-cytoplasmic shuttling. c-Abl is activated when DNA damage occurs and plays a role in the DNA repairment mechanism. The small-molecule inhibitor imatinib is classified as type II inhibitors which targets the inactive conformation of the kinase domain and specifically inhibit c-Abl.

Study design, size, duration
C57BL6 female mice at 6-8-wk old were superovulated with 7,5 IU PMSG and after 48 hr 7,5 IU hCG, then mated. 20 hours after hCG enjection, zygotes were collected and cultured for 96h at 5% CO2, 5% O2 at 37oC. We designed 5 experimental groups: control, DMSO, 1 µM, 5 µM and 10 µM (in KSOM+AA) imatinib group. To determine how altering the imatinib might affect embryonic growth and development, morphokinetic parameters were evaluated.

Participants/materials, setting, methods
Immunofluorescence staining for embryos was performed after 4% paraformaldehyde fixation. Then embryos incubated c-Abl, YAP, TEAD4, PARD6, E-cadherin and CDX2. DAPI mounting medium used for nucleus staining. Imaging was performed under a confocal microscope. Fluorescent intensity analysis was performed with ImageJ. We detected c-Abl, Yap, Tead, Cdx2, Oct4 and Nanog mRNA expression levels using the qRT-PCR. For the statistical analysis we used GraphPad Prism (2-Way ANOVA and and One-way ANOVA (used Geisser-Greenhouse correction)).

Main results and the role of chance
Treatment of zygotes with imatinib resulted in embryonic developmental arrest (control, 81%; 1 µM imatinib, 58.5%; 5 µM imatinib, 54%, 10 µM imatinib 0%) and led to a significant decrease in the rate of blastocyst formation. We detected that the expression level of c-Abl was decreased in the imatinib groups compared to the control group. We determined that YAP and TEAD, which should be localized in the nuclei of TE cells, lost their nuclear expression patterns and were expressed in the cytoplasm and their expression levels decreased. We detected the PARD6 pattern, which was evident at the cell borders in the control group, distributed throughout the cytoplasm in the imatinib groups. While cytoplasmic and decreased localization of CDX2, which should be nuclear in TE cells, was determined in imatinib groups, we also defined that E-cadherin distribution was impaired at cell borders compared to the control group. c-Abl mRNA level was decreased in the 1 and 5 µm imatinib groups, Yap mRNA level was decreased in the 5 µm imatinib group. Cdx2 and Nanog mRNA levels were decreased in the 1 and 5 µm imatinib group (*p&lt;0.1, **p &lt;0.01, ***p &lt;0.001, ****p &lt;0.0001).

Limitations, reasons for caution
All animals used in this study were obtained from Yeditepe University Faculty of Medicine Experimental Research Center (YUDETAM) and all the experimental procedures have been approved by Yeditepe University Ethical Committee.

Wider implications of the findings
Imatinib significantly inhibited the development of eight-cell embryos to the blastocyst stage compared to controls a concentration-dependent manner, furthermore, c-Abl inhibition affected linegae specification by changing the localization of YAP/TEAD, markers of lineage differentiation. Therefore, c-Abl can regulate cell specification and cell fate determination during mouse preimplantation embryonic development.

Trial registration number
YAP-AP-SAB-21019