Endoh Mitsuru, Fujimoto Takafumi, Yamaha Etsuro, Arai Katsutoshi
1 Division of Marine Life Science, Laboratory of Aquaculture Genetics and Genomics, Faculty and Graduate School of Fisheries Sciences, Hokkaido University , Hakodate, Japan .
2 Research Fellow of Japan Society for the Promotion of Science, Hokkaido University , Nanae, Japan .
Zebrafish. 2018 Feb;15(1):33-44. doi: 10.1089/zeb.2017.1482. Epub 2017 Dec 20.
Androgenesis is useful for induction of doubled haploids from male genetic resources and contributes to the restoration of individuals from cryopreserved sperm. Here, we determined the suitable conditions for egg in vitro preservation and the suitable dose of UV irradiation for genetic inactivation of the egg nucleus, and established an improved procedure for induction of androgenetic-doubled haploids in zebrafish. The suitable solution for egg preservation was evaluated by the fertilization rate using different types of solutions or conditions. Hank's solution with 0.5% bovine serum albumin (pH8.0) was suitable for the preservation of zebrafish eggs. In addition, we discovered an improvement of fertilization rates by temporal preservation of ovulated eggs in the suitable solution. UV irradiation of eggs at 50-75 mJ/cm induced haploid embryos. Microsatellite genotyping using eight loci revealed the paternity and homozygosity of the putative androgenetic doubled haploids. The yield rate of androgenetic doubled haploids, which were induced by UV irradiation and heat shock, ranged from 0.4% to 10.7%.
雄核发育对于从雄性遗传资源诱导产生双单倍体很有用，并且有助于从冷冻保存的精子中恢复个体。在此，我们确定了卵母细胞体外保存的合适条件以及用于卵细胞核遗传失活的紫外线照射合适剂量，并建立了一种改进的方法来诱导斑马鱼雄核发育双单倍体。通过使用不同类型的溶液或条件下的受精率来评估适合卵母细胞保存的溶液。含有0.5%牛血清白蛋白（pH8.0）的汉克斯溶液适合于斑马鱼卵母细胞的保存。此外，我们发现将排卵后的卵母细胞在合适的溶液中短暂保存可提高受精率。用50-75 mJ/cm的紫外线照射卵母细胞可诱导产生单倍体胚胎。使用八个位点的微卫星基因分型揭示了假定的雄核发育双单倍体的父系来源和纯合性。通过紫外线照射和热休克诱导产生的雄核发育双单倍体的产率在0.4%至10.7%之间。
