Qi-Xin Guo, Lu Xu, Hao Bai, Guo-Hong Chen, Guo-Bin Chang
College of Animal Science and Technology, Yangzhou University, Yangzhou, China.
College of Biology and Food Engineering, Changshu Institute of Technology, Changshu, China.
Anim Biotechnol. 2023 Nov;34(4):1102-1111. doi: 10.1080/10495398.2021.2011742. Epub 2021 Dec 31.
Piwi-like protein 1 (PIWIL1) plays a crucial role in stem cell proliferation, embryogenesis, growth, and development. We aimed to unravel the function of PIWIL1 and its Piwi/Argonaute/Zwille (PAZ) domain in chicken embryogenesis. The expression of PIWI1 at different stages of spermatogenesis was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the PAZ domain was mutated based on its 3D structure model using the clustered regularly interspaced short palindromic repeats Cas9 (CRISPR/Cas9) technology. The results indicated that PIWIL1 mRNA was specifically expressed in spermatogonium cells undergoing meiosis. After targeting the PAZ domain (300-370 amino acid residues), we obtained two mutant DF-1 cell clones with 23-bp and 8-bp deletions. Injection of the pCMV-Cas9-puro-sgRNA-2 construct into 2.5-day embryos resulted in generation of 19 different PAZ mutants (13 males and 6 females), which showed delayed hatching, reduced quality of semen, and decreased expression of PIWIL1 and SOX2 at embryonic days 5 and 18. However, we could not obtain PAZ double knockout (KO) chickens by crossing of the F generation, suggesting that PAZ double KO may halt embryonic development. Our results indicate that PIWIL1 plays an important role in meiosis and that PAZ mutations can lead to decreased sperm quality, whereas its double KO may arrest embryogenesis in chicken.
类Piwi蛋白1（PIWIL1）在干细胞增殖、胚胎发生、生长和发育中起着关键作用。我们旨在揭示PIWIL1及其Piwi/AGO/Zwille（PAZ）结构域在鸡胚胎发生中的功能。通过定量逆转录聚合酶链反应（RT-qPCR）分析PIWI1在精子发生不同阶段的表达，并基于其三维结构模型使用成簇规律间隔短回文重复序列Cas9（CRISPR/Cas9）技术对PAZ结构域进行突变。结果表明，PIWIL1 mRNA在进行减数分裂的精原细胞中特异性表达。靶向PAZ结构域（300 - 370个氨基酸残基）后，我们获得了两个分别缺失23 bp和8 bp的DF-1细胞突变克隆。将pCMV-Cas9-puro-sgRNA-2构建体注射到2.5天龄的胚胎中，产生了19个不同的PAZ突变体（13只雄性和6只雌性），这些突变体表现出孵化延迟、精液质量下降以及在胚胎第5天和第18天时PIWIL1和SOX2的表达降低。然而，通过F代杂交我们未能获得PAZ双敲除（KO）鸡，这表明PAZ双敲除可能会阻止胚胎发育。我们的结果表明，PIWIL1在减数分裂中起重要作用，PAZ突变可导致精子质量下降，而其双敲除可能会使鸡的胚胎发生停滞。
