Jung Yena, Bang Hyewon, Kim Young-Hyun, Park Na-Eun, Park Young-Ho, Park Chaeli, Lee Sang-Rae, Lee Jeong-Woong, Song Bong-Seok, Kim Ji-Su, Sim Bo-Woong, Seol Dong-Won, Wee Gabbine, Kim Sunhyung, Kim Sun-Uk, Kim Ekyune
College of Pharmacy, Catholic University of Daegu, Gyeongsan-si 38430, Korea.
Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 28116, Korea.
Animals (Basel). 2021 Apr 7;11(4):1037. doi: 10.3390/ani11041037.
To elucidate the functional role of V-set and immunoglobulin domain-containing 1 (VSIG1) in spermatogenesis and fertilization, we knocked out (KO) VSIG1 in a mouse embryo using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) -mediated genome editing. Reverse transcription PCR was performed using cDNA synthesized from VSIG1 KO testis RNA. Although Western blot analysis using a specific antibody to VSIG1 confirmed VSIG1 protein defects in the KO mice, hematoxylin-eosin staining analysis was similar in the KO and wild-type mice. Additionally, computer-assisted sperm analysis and in vitro fertilization experiments were conducted to confirm the activity and fertilization ability of sperm derived from the KO mouse. Mice lacking VSIG1 were viable and had no serious developmental defects. As they got older, the KO mice showed slightly higher weight loss, male mice lacking VSIG1 had functional testes, including normal sperm number and motility, and both male and female mice lacking VSIG1 were fertile. Our results from VSIG1 KO mice suggest that VSIG1 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. VSIG1 in sperm is dispensable for spermatogenesis and male fertility in mice. As several genes are known to possess slightly different functions depending on the species, the importance and molecular mechanism of VSIG1 in tissues of other species needs further investigation.
为了阐明含V结构域和免疫球蛋白结构域1(VSIG1)在精子发生和受精过程中的功能作用,我们利用CRISPR/Cas9(规律成簇间隔短回文重复序列/CRISPR相关蛋白9)介导的基因组编辑技术在小鼠胚胎中敲除了VSIG1。使用从VSIG1敲除小鼠睾丸RNA合成的cDNA进行逆转录PCR。尽管使用针对VSIG1的特异性抗体进行的蛋白质免疫印迹分析证实了敲除小鼠中VSIG1蛋白存在缺陷,但苏木精-伊红染色分析显示敲除小鼠和野生型小鼠相似。此外,进行了计算机辅助精子分析和体外受精实验,以确认来自敲除小鼠的精子的活性和受精能力。缺乏VSIG1的小鼠存活且无严重发育缺陷。随着年龄增长,敲除小鼠体重减轻略多,缺乏VSIG1的雄性小鼠睾丸功能正常,精子数量和活力正常,缺乏VSIG1的雄性和雌性小鼠均具有生育能力。我们对VSIG1敲除小鼠的研究结果表明,VSIG1可能在精子发生以及正常睾丸发育、功能和维持过程中不发挥关键作用。精子中的VSIG1对小鼠的精子发生和雄性生育能力并非必不可少。由于已知一些基因在不同物种中功能略有差异,VSIG1在其他物种组织中的重要性和分子机制需要进一步研究。