Chimeric analysis with newly established EGFP/DsRed2-tagged ES cells identify HYDIN as essential for spermiogenesis in mice

  • Oura Seiya
    Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
  • Miyata Haruhiko
    Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
  • Noda Taichi
    Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
  • Shimada Keisuke
    Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
  • Matsumura Takafumi
    Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
  • Morohoshi Akane
    Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
  • Isotani Ayako
    Department of Biomedical Science, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
  • Ikawa Masahito
    Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

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Abstract

<p>The CRISPR/Cas9 system can efficiently introduce biallelic mutations in ES cells (ESCs), and its application with fluorescently-tagged ESCs enables phenotype analysis in chimeric mice. We have utilized ESCs that express EGFP in the cytosol and acrosome [EGR-G101 129S2 × (CAG/Acr-EGFP) B6] in previous studies; however, the EGFP signal in the sperm cytosol is weak and the signal in the acrosome is lost after the acrosome reaction, precluding analysis between wild type and ESC derived spermatozoa. In this study, we established an ESC line from RBGS (Red Body Green Sperm) transgenic mice [B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb] whose spermatozoa exhibit green fluorescence in the acrosome and red fluorescence in the mitochondria within the flagellar midpiece that is retained after the acrosome reaction. We utilized these new ESCs to analyze HYDIN, which is reported to function in sperm motility in humans. Analysis of Hydin-disrupted spermatozoa in mice is difficult as Hydin-mutant mice (hy3) die within 3 weeks, before sexual maturation, due to hydrocephaly. To circumvent the early lethality of the whole-body knockout, we disrupted Hydin in RBGS-ESCs and generated chimeric mice, which survived into sexual maturity. Hydin-disrupted spermatozoa obtained from the chimeric mice possessed short tails and were immotile. When we injected Hydin-disrupted spermatozoa into oocytes, heterozygous pups were obtained, which suggests that the genome of Hydin-disrupted spermatozoa can produce viable pups. Consequently, RBGS-ESCs can be a useful tool for screening and analysis of male-fertility related genes in chimeric mice.</p>

Journal

  • Experimental Animals

    Experimental Animals 68 (1), 25-34, 2019

    Japanese Association for Laboratory Animal Science

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