FoxO transcription factors 1 regulate mouse preimplantation embryo development
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EMBRYO BIOLOGY
FoxO transcription factors 1 regulate mouse preimplantation embryo development Nilay Kuscu 1 & Nazli Ece Gungor-Ordueri 2 & Berna Sozen 3 & Dileyra Adiguzel 1 & Ciler Celik-Ozenci 1 Received: 10 April 2019 / Accepted: 1 August 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019
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. Keywords FoxO . Preimplantation embryo . Sirtuin1 . Resveratrol . Oxidative stress . Apoptosis
Introduction The forkhead box (Fox) transcription factor family consists of 19 subclasses of Fox genes, FoxA–FoxS [44, 45]. FOXO family comprises four members as FOXO1, FOXO3,
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10815-019-01555-1) contains supplementary material, which is available to authorized users. * Ciler Celik-Ozenci [email protected] 1
Department of Histology and Embryology, School of Medicine, Akdeniz University, Campus, 07070 Antalya, Turkey
2
Department of Histology and Embryology, School of Medicine, Biruni University, Istanbul, Turkey
3
Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK
FOXO4, and FOXO6 in mammals, and they are characterized by a highly conserved forkhead domain termed the “forkhead box” or a “winged helix” [46]. FoxO transcripti
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