Background: Spermatozoa become competent for fertilization during transit through the epididymis. As spermatozoa from the proximal caudal epididymis can fertilize eggs, proteins from the caput and corpus epididymis are required for sperm maturation.
Objectives: Microarray analysis identified that more than 17,000 genes are expressed in the epididymis; however, few of these genes demonstrate expression restricted to the epididymis. To analyze epididymis-enriched gene function in vivo, we generated knockout (KO) mutations in nine genes that are abundantly expressed in the caput and corpus region of the epididymis.
Materials and methods: KO mice were generated using the CRISPR/Cas9 system. The histology of the epididymis was observed with hematoxylin and eosin staining. KO males were caged with wild-type females for 3-6 months to check fertility.
Results: We generated individual mutant mouse lines having indel mutations in Pate1, Pate2, or Pate3. We also deleted the coding regions of Clpsl2, Epp13, and Rnase13, independently. Finally, the 150 kb region encoding Gm1110, Glb1l2, and Glb1l3 was deleted to generate a triple KO mouse line. Histology of the epididymis and sperm morphology of all KO lines were comparable to control males. The females mated with these KO males delivered pups at comparable numbers as control males.
Discussion and conclusion: We revealed that nine genes abundantly expressed in the caput and corpus epididymis are dispensable for sperm function and male fecundity. CRISPR/Cas9-mediated KO mice generation accelerates the screening of epididymis-enriched genes for potential functions in reproduction.
Keywords: genetically modified mice; genome editing; sperm maturation.
© 2019 The Authors. Andrology published by John Wiley & Sons Ltd on behalf of American Society of Andrology and European Academy of Andrology.
Conflict of interest statement
The authors declare no conflict of interest.
Effect of interleukin-1 receptor antagonist gene deletion on male mouse fertility.Endocrinology. 2009 Jan;150(1):295-303. doi: 10.1210/en.2008-0848. Epub 2008 Sep 11. Endocrinology. 2009. PMID: 18787019
Region-specific gene expression in the epididymis of Yak.Theriogenology. 2019 Nov;139:132-146. doi: 10.1016/j.theriogenology.2019.08.006. Epub 2019 Aug 5. Theriogenology. 2019. PMID: 31404823
Junctional adhesion molecule A: expression in the murine epididymal tract and accessory organs and acquisition by maturing sperm.Mol Hum Reprod. 2017 Feb 10;23(2):132-140. doi: 10.1093/molehr/gaw082. Mol Hum Reprod. 2017. PMID: 28062807 Free PMC article.
Development and use of surgical procedures to bypass selected regions of the mammalian epididymis: effects on sperm maturation.J Reprod Fertil Suppl. 1998;53:183-95. J Reprod Fertil Suppl. 1998. PMID: 10645277 Review.
Sperm maturation in the domestic cat.Theriogenology. 2006 Jul 1;66(1):14-24. doi: 10.1016/j.theriogenology.2006.03.022. Epub 2006 Apr 18. Theriogenology. 2006. PMID: 16620928 Review.
Cited by 2 articles
Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice.Proc Natl Acad Sci U S A. 2019 Sep 10;116(37):18498-18506. doi: 10.1073/pnas.1908736116. Epub 2019 Aug 27. Proc Natl Acad Sci U S A. 2019. PMID: 31455729 Free PMC article.
Physiological function of seminal vesicle secretions on male fecundity.Reprod Med Biol. 2019 Jun 17;18(3):241-246. doi: 10.1002/rmb2.12282. eCollection 2019 Jul. Reprod Med Biol. 2019. PMID: 31312102 Free PMC article. Review.
- Cho S, Beintema JJ & Zhang J. (2005) The ribonuclease A superfamily of mammals and birds: identifying new members and tracing evolutionary histories. Genomics 85, 208–220. - PubMed
- Johnston DS, Jelinsky SA, Bang HJ, DiCandeloro P, Wilson E, Kopf GS & Turner TT. (2005) The mouse epididymal transcriptome: transcriptional profiling of segmental gene expression in the epididymis. Biol Reprod 73, 404–413. - PubMed
- Levitin F, Weiss M, Hahn Y, Stern O, Papke RL, Matusik R, Nandana SR, Ziv R, Pichinuk E, Salame S, Bera T, Vincent J, Lee B, Pastan I & Wreschner DH. (2008) PATE gene clusters code for multiple, secreted TFP/Ly‐6/uPAR proteins that are expressed in reproductive and neuron‐rich tissues and possess neuromodulatory activity. J Biol Chem 283, 16928–16939. - PMC - PubMed
- P01 HD087157/HD/NICHD NIH HHS/United States
- T32 GM120011/GM/NIGMS NIH HHS/United States
- Takeda Science Foundation/International
- JP17H01394/Japan Society for the Promotion of Science/International
- R01 HD088412/HD/NICHD NIH HHS/United States
- JP18gm5010001/Japan Agency for Medical Research and Development/International
- JP18K14612/Japan Society for the Promotion of Science/International
- JP25112007/Japan Society for the Promotion of Science/International
- OPP1160866/GATES/Bill & Melinda Gates Foundation/United States
- 20170633/Japan Society for the Promotion of Science/International
- Ministry of Education, Culture, Sports, Science and Technology/International