Wu Yangyang, Wang Ting, Zhao Zigao, Liu Siyu, Shen Cong, Li Hong, Liu Mingxi, Zheng Bo, Yu Jun, Huang Xiaoyan
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China.
Center for Reproduction and Genetics, Suzhou Municipal Hospital, the Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China.
PeerJ. 2021 Feb 19;9:e10847. doi: 10.7717/peerj.10847. eCollection 2021.
Retinoic Acid Induced Protein 14 () is an evolutionarily conserved gene that is highly expressed in the testis. Previous experiments have reported that small interfering RNA (siRNA)-mediated gene knockdown (KD) of in rat testis disrupted spermatid polarity and transport. Of note, a gene knockout (KO) model is considered the "gold standard" for in vivo assessment of crucial gene functions. Herein, we used CRISPR/Cas9-based gene editing to investigate the in vivo role of in mouse testis.
Sperm concentration and motility were assayed using a computer-assisted sperm analysis (CASA) system. Histological and immunofluorescence (IF) staining and transmission electron microscopy (TEM) were used to visualize the effects of KO in the testes and epididymides. Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) was used to determine apoptotic cells. Gene transcript levels were calculated by real-time quantitative PCR.
KO in mice depicted normal fertility and complete spermatogenesis, which is in sharp contrast with the results reported previously in a KD rat model. Sperm parameters and cellular apoptosis did not appear to differ between wild-type (WT) and KO group. Mechanistically, in contrast to the well-known role of in modulating the dynamics of F-actin at the ectoplasmic specialization (ES) junction in the testis, morphological changes of ES junction exhibited no differences between KO and WT testes. Moreover, the F-actin surrounded at the ES junction was also comparable between the two groups.
In summary, our study demonstrates that is dispensable for mouse spermatogenesis and fertility. Although the results of this study were negative, the phenotypic information obtained herein provide an enhanced understanding of the role of in the testis, and researchers may refer to these results to avoid conducting redundant experiments.
维甲酸诱导蛋白14（）是一个在进化上保守的基因，在睾丸中高度表达。先前的实验报道，在大鼠睾丸中，小干扰RNA（siRNA）介导的该基因敲低（KD）破坏了精子细胞的极性和运输。值得注意的是，基因敲除（KO）模型被认为是体内评估关键基因功能的“金标准”。在此，我们使用基于CRISPR/Cas9的基因编辑来研究该基因在小鼠睾丸中的体内作用。
使用计算机辅助精子分析（CASA）系统检测精子浓度和活力。组织学、免疫荧光（IF）染色和透射电子显微镜（TEM）用于观察该基因敲除对睾丸和附睾的影响。末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）用于确定凋亡细胞。通过实时定量PCR计算基因转录水平。
小鼠中的该基因敲除表现出正常的生育能力和完整的精子发生，这与先前报道的该基因敲低大鼠模型的结果形成鲜明对比。野生型（WT）和基因敲除组之间的精子参数和细胞凋亡似乎没有差异。从机制上讲，与该基因在调节睾丸外质特化（ES）连接处F-肌动蛋白动力学中的众所周知的作用相反，该基因敲除和野生型睾丸之间ES连接处的形态变化没有差异。此外，两组之间ES连接处周围的F-肌动蛋白也相当。
总之，我们的研究表明该基因对小鼠精子发生和生育能力是可有可无的。尽管本研究结果为阴性，但本文获得的表型信息有助于增强对该基因在睾丸中作用的理解，研究人员可以参考这些结果以避免进行多余的实验。
