Retinoic Acid Induced Protein 14 (Rai14) is dispensable for mouse spermatogenesis

Yangyang Wu; Ting Wang; Zigao Zhao; Siyu Liu; Cong Shen; Hong Li; Mingxi Liu; Bo Zheng; Jun Yu; Xiaoyan Huang

doi:10.7717/peerj.10847

Retinoic Acid Induced Protein 14 (Rai14) is dispensable for mouse spermatogenesis

PeerJ. 2021 Feb 19:9:e10847. doi: 10.7717/peerj.10847. eCollection 2021.

Authors

Yangyang Wu^#¹, Ting Wang^#², Zigao Zhao³, Siyu Liu¹, Cong Shen², Hong Li², Mingxi Liu¹, Bo Zheng², Jun Yu⁴, Xiaoyan Huang¹

Affiliations

¹ State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China.
² Center for Reproduction and Genetics, Suzhou Municipal Hospital, the Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China.
³ Yunnan Institute of Population and Family Planning Science and Technology, Kunming, China.
⁴ Institute of Reproductive Medicine, Medical School, Nantong University, Nantong, China.

^# Contributed equally.

Abstract

Background: Retinoic Acid Induced Protein 14 (Rai14) 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 Rai14 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 Rai14 in mouse testis.

Methods: 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 Rai14 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.

Results: Rai14 KO in mice depicted normal fertility and complete spermatogenesis, which is in sharp contrast with the results reported previously in a Rai14 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 Rai14 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 Rai14 KO and WT testes. Moreover, the F-actin surrounded at the ES junction was also comparable between the two groups.

Conclusion: In summary, our study demonstrates that Rai14 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 Rai14 in the testis, and researchers may refer to these results to avoid conducting redundant experiments.

Keywords: Ectoplasmic specialization; F-actin; Knockout; Rai14; Spermatogenesis.

Grants and funding

This work was supported by the National Natural Science Foundation of China (81901532 and 81901533), the Natural Science Foundation of Jiangsu Province (BK20190188), the Suzhou Introduced Project of Clinical Medical Expert Team (SZYJTD201708) and the Suzhou Key Laboratory of Male Reproduction Research (SZS201718). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.