The aim of the present study is to investigate role of FoxO transcription factors in preimplantation embryo development by knocking down FoxO1, FoxO3, and FoxO4 genes and also to assess cell cycle arrest related proteins, p53 and p21, and apoptosis-related proteins, fas ligand (FASL), and cleaved caspase 3.
Knockdown of FoxOs using siRNA was confirmed utilizing RT-PCR and qRT-PCR in gene level and using immunofluorescence in protein level. Following knockdown of FoxO1, FoxO3, and FoxO4 in two-cell mouse embryos with or without resveratrol treatment; developmental competence of embryos and expression patterns of SIRT1, p53, p21, FASL, and CLEAVED CASPASE 3 proteins in embryos by immunofluorescence were assessed after 48 h. ROS levels were measured in knockdown embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine resveratrol dose.
Successful knockdown of FoxO genes in mouse embryos utilizing a non-invasive siRNA method was achieved. Significantly, knockdown of FoxO genes impaired preimplantation embryo development which cannot be prevented by resveratrol treatment. Immunofluorescence results showed that resveratrol could protect embryos from cell cycle arrest and apoptosis. FOXO proteins regulate apoptosis and cell cycle related proteins in mouse preimplantation embryos. Moreover, there might be an autofeedback mechanism where FOXO1, FOXO3, and FOXO4 regulate SIRT1 protein expression.
These results suggest that FOXO transcription factors could contribute to mouse preimplantation embryo development, and it remains to investigate whether they have crucial roles in human preimplantation embryo and infertility.
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This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) with grant number: 114S384.
The experimental protocol was approved by the local ethical committee (number of the ethical approval 2014.04.04)
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Kuscu, N., Gungor-Ordueri, N.E., Sozen, B. et al. FoxO transcription factors 1 regulate mouse preimplantation embryo development. J Assist Reprod Genet 36, 2121–2133 (2019). https://doi.org/10.1007/s10815-019-01555-1
- Preimplantation embryo
- Oxidative stress