Sirt3 protects in vitro–fertilized mouse preimplantation embryos against oxidative stress–induced p53-mediated developmental arrest
Yumiko Kawamura, Yasunobu Uchijima, Nanao Horike, Kazuo Tonami, Koichi Nishiyama, Tomokazu Amano, Tomoichiro Asano, Yukiko Kurihara, Hiroki Kurihara
Yumiko Kawamura, Yasunobu Uchijima, Nanao Horike, Kazuo Tonami, Koichi Nishiyama, Tomokazu Amano, Tomoichiro Asano, Yukiko Kurihara, Hiroki Kurihara
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Research Article Reproductive biology

Sirt3 protects in vitro–fertilized mouse preimplantation embryos against oxidative stress–induced p53-mediated developmental arrest

Abstract

Sirtuins are a phylogenetically conserved NAD+-dependent protein deacetylase/ADP-ribosyltransferase family implicated in diverse biological processes. Several family members localize to mitochondria, the function of which is thought to determine the developmental potential of preimplantation embryos. We have therefore characterized the role of sirtuins in mouse preimplantation development under in vitro culture conditions. All sirtuin members were expressed in eggs, and their expression gradually decreased until the blastocyst stage. Treatment with sirtuin inhibitors resulted in increased intracellular ROS levels and decreased blastocyst formation. These effects were recapitulated by siRNA-induced knockdown of Sirt3, which is involved in mitochondrial energy metabolism, and in Sirt3–/– embryos. The antioxidant N-acetyl-L-cysteine and low-oxygen conditions rescued these adverse effects. When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.

Authors

Yumiko Kawamura, Yasunobu Uchijima, Nanao Horike, Kazuo Tonami, Koichi Nishiyama, Tomokazu Amano, Tomoichiro Asano, Yukiko Kurihara, Hiroki Kurihara

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Figure 5

Sirt3 knockdown increases intracellular ROS, mainly of mitochondrial origin, in preimplantation embryos.

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Sirt3 knockdown increases intracellular ROS, mainly of mitochondrial ori...
(A and B) Injection of Sirt3 siRNA increased intracellular ROS levels, as estimated by CM-H2DCFDA fluorescence intensity. This increase was abolished by NAC (A) and stigmatellin (B), but was only partially decreased by apocynin (B). Embryos were injected with control or Sirt3 siRNA at the pronuclear stage and were cultured with or without NAC for 72 hours. To identify the major origin of increased intracellular ROS, embryos were treated with apocynin or stigmatellin for 30 minutes before CM-H2DCFDA staining. Quantitative data of fluorescence intensity, obtained using ImageJ, were standardized by dividing each value by the average value of the control group in each experiment. Data are derived from 3 (A) or 4 (B) independent experiments. Statistical assessments were performed by applying Games-Howell test. *P < 0.05; **P < 0.001. (C and D) Representative images of CM-H2DCFDA fluorescence in embryos analyzed in A and B, respectively. Scale bars: 100 μm.