FoxO transcription factors 1 regulate mouse preimplantation embryo development

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Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusions

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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Change history

  • 04 December 2019

    The original article unfortunately contained a mistake. There is a misplaced “1” in the article title and the correct title is shown above.

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Funding

This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) with grant number: 114S384.

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Correspondence to Ciler Celik-Ozenci.

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The experimental protocol was approved by the local ethical committee (number of the ethical approval 2014.04.04)

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Electronic supplementary material

Supplement 3.
figure9

In FoxO1 knockdown group two embryos are side by side in the same culture drop (after 48 h). Two cell embryos which were transfected both with FoxO1 siRNA and siGLO were cultured for 48 h. At the end of the experiment, one of them developed to blastocyst stage (lower left) and expressed very high level of FoxO1 protein (green fluorescent). There was no siGLO (red fluorescent) signal in its blastomeres except a nonspecific retention of siGLO by polar body (arrowhead) can be seen. The other embryo which arrested at 2-cell stage (upper central) expressed very low level (almost none) of FoxO1 protein (green fluorescent). Lots of siGLO (red fluorescent) signal in its blastomeres can be seen as little red dots. White arrows indicate the siGLO signals in arrested 2-cell embryo. Transfection with siRNA around cell nucleus (perinuclear) which is indicated by red siGLO signal (circle) can be seen in image with higher magnification (upper right image). (PNG 804 kb)

Supplement 4.
figure10

Resveratrol (RVT) dose was determined utilizing TUNEL method. According to our results, RVT doses higher than 0.5 μM lead to increased cell death in blastocysts. A) Representative images of TUNEL-labeled blastocysts in control, DMSO (vehicle-control), 0.5 μM RVT, 1 μM RVT, and 2 μM RVT treated groups (n = 20). Red signal indicates TUNEL-positive staining and the blue signal (DAPI) indicates the nuclei of the embryonic cells. B) Total number of cells per embryo were counted in the all groups. Embryos treated with 0.5 μM RVT contained more cells (p = 0.01) when compared to controls. Blastocysts cultured in presence of 2 μM RVT displayed a significant increase in the percentage of TUNEL-positive cells when compared to controls (p = 0.044) and to 0.5 μM RVT treated embryos, respectively (p = 0.015). Dose of resveratrol has been determined as 0.5 μM considering TUNEL results. (PNG 2859 kb)

Supplement 1.

List of siRNA duplexes used in the study. (DOCX 176 kb)

Supplement 2.

List of PCR primers used in the study. (PDF 40 kb)

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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

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Keywords

  • FoxO
  • Preimplantation embryo
  • Sirtuin1
  • Resveratrol
  • Oxidative stress
  • Apoptosis