(87) Analysis of Autophagy Degradation Systems in Somatic Cell Nuclear Transfer Embryos

Takaki Tatebe^{1, 2}; Atsuo Ogura^{1, 2}; Kimiko Inoue^{1, 2}

1. Bioresource Engineering Division, RIKEN BioResource Research Center, Tsukuba, Ibaraki, Japan

2. Graduate School of Science and Technology, University of Tsukuba, Tsukuba, Ibaraki, Japan


Abstract Text:

Somatic cell nuclear transfer (SCNT) cloning technology shows promise for applications in regenerative medicine, bioreactor production, and endangered species conservation. However, SCNT embryos exhibit much lower developmental efficiency than normally fertilized embryos; in mice, only 1–5% of transferred embryos develop into offspring. Although recent SCNT studies, especially those involving mice, have focused on epigenomic improvements in SCNT embryos, only a few have examined their cytoplasmic factors. In this study, we investigated autophagy, a cytoplasmic event essential for preimplantation embryogenesis, to identify its possible involvement in reduced SCNT embryo development. We performed immunofluorescence staining for microtubule-associated protein light chain 3 (LC3), an autophagosome marker, to observe autophagy dynamics in SCNT embryos. We also used live fluorescence imaging involving an autophagic flux probe, which can measure autophagic activity, to compare that of fertilization-derived and SCNT embryos. Additionally, we analyzed autophagic activity in SCNT embryos treated with Trichostatin A (TSA), a histone deacetylase inhibitor, or mRNA for Kdm4d, a histone demethylase, which promote embryonic gene activation. Finally, we examined the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, which negatively regulates autophagy, and analyzed mTORC1-related gene expression in fertilization-derived and SCNT embryos by using publicly available RNA-seq data. LC3 immunofluorescence revealed that autophagosome puncta were present in fertilization-derived and SCNT embryos at the late 2-cell and 4-cell stages; however, SCNT embryos exhibited significantly fewer puncta than fertilization-derived embryos (early 2-cell stage, no significant difference; late 2-cell stage, P < 0.001; 4-cell stage, P < 0.001). Consistent with this finding, live fluorescence imaging with an autophagic flux probe showed that the autophagic activity in SCNT embryos was lower than that in fertilization-derived embryos throughout their development to the blastocyst stage. Autophagic activity did not significantly improve in TSA- or Kdm4d-treated SCNT embryos, indicating that autophagy was robustly suppressed in SCNT embryos. However, SCNT embryos showed increased autophagic activity upon treatment with rapamycin, an mTORC1 inhibitor, at the 2-cell stage. Reanalysis of publicly available RNA-seq data showed that mTORC1 activator genes in the mTORC1 signaling pathway were highly expressed in SCNT embryos. These results indicated that the SCNT embryos had lower autophagic activity than the fertilization-derived embryos, and this phenotype was not rescued by enhancing embryonic gene activation. The mTORC1-activating genes were also highly expressed in donor somatic cells, suggesting that somatic epigenomic memory negatively regulates the autophagic-degradation system in SCNT embryos.
This work was supported by RIKEN Junior Research Associate Program and JST SPRING (JPMJSP2124).