Complementary Critical Functions of Zfy1 and Zfy2 in Mouse Spermatogenesis and Reproduction.
Complementary Critical Functions of Zfy1 and Zfy2 in Mouse Spermatogenesis and Reproduction.
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DOI：
10.1371/journal.pgen.1006578
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发表时间：
2017-01
期刊：
影响因子：
4.5
通讯作者：
Asahara H
中科院分区：
文献类型：
--
作者：
Nakasuji T;Ogonuki N;Chiba T;Kato T;Shiozawa K;Yamatoya K;Tanaka H;Kondo T;Miyado K;Miyasaka N;Kubota T;Ogura A;Asahara H
哺乳动物的 Y 染色体在精子发生中起着至关重要的作用。然而，Y染色体上每个基因的确切功能尚未完全阐明，部分原因是Y染色体基因靶向分析的困难。 Zfy 最初被认为是性别决定因子，但其在精子发生中的功能最近已被阐明。然而，迄今为止，Zfy基因靶向分析尚未进行。在这里，我们采用高效的 CRISPR/Cas9 系统来产生个体 Zfy1 或 Zfy2 敲除（KO）小鼠以及 Zfy1 和 Zfy2 双敲除（Zfy1/2-DKO）小鼠。虽然个别 Zfy1 或 Zfy2-KO 小鼠在生育能力方面没有表现出任何显着的表型改变，但 Zfy1/2-DKO 小鼠不育，并表现出精子形态异常、受精失败和早期胚胎发育失败。质谱筛选和蛋白质印迹分析证实，与野生型小鼠相比，Zfy1/2-DKO 小鼠精子中 PLCZ1、PLCD4、PRSS21 和 HTT 蛋白表达显着降低。这些结果与双突变小鼠中观察到的表型变化一致。总的来说，我们的策略和研究结果表明，Zfy1 和 Zfy2 在精子发生中具有冗余功能，有助于更好地理解受精失败和早期胚胎发育失败。根据人类遗传证据，已知 Y 染色体在精子发生中发挥着关键作用。然而，负责精子发生的特定Y染色体基因尚未完全阐明，因为Y染色体基因因其独特的结构而难以定向删除。在这项研究中，我们使用高度优化的 CRISPR/Cas9 系统，破坏了双缺失突变小鼠 Y 染色体上的 Zfy1 和 Zfy2 扩增基因。通过对小鼠的分析，我们发现Zfy1和Zfy2具有冗余功能，并且Zfy1和Zfy2同时缺乏会导致男性不育。 Zfy1和Zfy2双缺失突变小鼠的精子表现出严重的异常，包括形态、运动、获能、顶体反应和卵母细胞激活的缺陷，以及染色体畸变，表明Zfy1和Zfy2是精子发生的多个方面所必需的。阐明 Zfy 依赖性精子发生的机制将有助于了解哺乳动物的受精失败和早期胚胎发育。
The mammalian Y chromosome plays a critical role in spermatogenesis. However, the exact functions of each gene in the Y chromosome have not been completely elucidated, partly owing to difficulties in gene targeting analysis of the Y chromosome. Zfy was first proposed to be a sex determination factor, but its function in spermatogenesis has been recently elucidated. Nevertheless, Zfy gene targeting analysis has not been performed thus far. Here, we adopted the highly efficient CRISPR/Cas9 system to generate individual Zfy1 or Zfy2 knockout (KO) mice and Zfy1 and Zfy2 double knockout (Zfy1/2-DKO) mice. While individual Zfy1 or Zfy2-KO mice did not show any significant phenotypic alterations in fertility, Zfy1/2-DKO mice were infertile and displayed abnormal sperm morphology, fertilization failure, and early embryonic development failure. Mass spectrometric screening, followed by confirmation with western blot analysis, showed that PLCZ1, PLCD4, PRSS21, and HTT protein expression were significantly deceased in spermatozoa of Zfy1/2-DKO mice compared with those of wild-type mice. These results are consistent with the phenotypic changes seen in the double-mutant mice. Collectively, our strategy and findings revealed that Zfy1 and Zfy2 have redundant functions in spermatogenesis, facilitating a better understanding of fertilization failure and early embryonic development failure. The Y chromosome is known to play a critical role in spermatogenesis based on human genetic evidence. However, specific Y chromosome genes responsible for spermatogenesis have not been fully elucidated yet, because the targeted deletion of Y chromosome genes is difficult owing to its unique structure. In this study, using our highly optimized CRISPR/Cas9 system, we disrupted Zfy1 and Zfy2 ampliconic genes located on the Y chromosome in double-deletion mutant mice. By analyzing the mice, we revealed that Zfy1 and Zfy2 have redundant functions, and that a lack of both Zfy1 and Zfy2 leads to male infertility. Zfy1 and Zfy2 double-deletion mutant mice showed severe abnormalities in their sperm, including defects in morphology, motility, capacitation, acrosome reaction, and oocyte activation, as well as chromosomal aberrations, indicating that Zfy1 and Zfy2 are required for multiple aspects of spermatogenesis. Elucidating the mechanisms that underlie Zfy-dependent spermatogenesis will shed light on the failure of fertilization and early embryonic development in mammals.