Application of auxin-inducible degron technology to mouse oocyte activation with PLCζ

  • MIURA Kento
    RIKEN BioResource Research Center, Ibaraki 305-0074, Japan
  • MATOBA Shogo
    RIKEN BioResource Research Center, Ibaraki 305-0074, Japan
  • OGONUKI Narumi
    RIKEN BioResource Research Center, Ibaraki 305-0074, Japan
  • NAMIKI Takafumi
    Laboratory of Animal Reproduction, Graduate School of Veterinary Science, Azabu University, Kanagawa 252-5201, Japan
  • ITO Junya
    Laboratory of Animal Reproduction, Graduate School of Veterinary Science, Azabu University, Kanagawa 252-5201, Japan
  • KASHIWAZAKI Naomi
    Laboratory of Animal Reproduction, Graduate School of Veterinary Science, Azabu University, Kanagawa 252-5201, Japan
  • OGURA Atsuo
    RIKEN BioResource Research Center, Ibaraki 305-0074, Japan RIKEN Cluster for Pioneering Research, Saitama 351-0198, Japan Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan

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Abstract

<p> In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/μl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ-AID-EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ-AID-EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ-AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.</p>

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