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Original research
Human X chromosome exome sequencing identifies BCORL1 as contributor to spermatogenesis
  1. Chuncheng Lu1,2,
  2. Yan Zhang1,2,
  3. Yufeng Qin3,
  4. Qiaoqiao Xu1,2,
  5. Ran Zhou4,
  6. Yiqiang Cui1,
  7. Yunfei Zhu1,
  8. Xin Zhang1,
  9. Jintao Zhang1,
  10. Xiang Wei1,
  11. Min Wang1,
  12. Bo Hang5,
  13. Jian-Hua Mao5,
  14. Antoine M Snijders5,
  15. Mingxi Liu1,
  16. Zhibin Hu1,6,
  17. Hongbing Shen1,6,
  18. Zuomin Zhou1,
  19. Xuejiang Guo1,
  20. Xin Wu1,
  21. Xinru Wang1,2,
  22. Yankai Xia1,2
  1. 1 State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China
  2. 2 Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China
  3. 3 Epigenetics & Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Durham, North Carolina, USA
  4. 4 Nanjing Maternity and Child Health Care Hospital, Nanjing, Jiangsu, China
  5. 5 Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, California, USA
  6. 6 Department of Epidemiology and Biostatistics and Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China
  1. Correspondence to Professor Yankai Xia, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, China; yankaixia{at}


Background Infertility affects approximately 15% of couples worldwide with male infertility being responsible for approximately 50% of cases. Although accumulating evidence demonstrates the critical role of the X chromosome in spermatogenesis during the last few decades, the expression patterns and potential impact of the X chromosome, together with X linked genes, on male infertility are less well understood.

Methods We performed X chromosome exome sequencing followed by a two-stage independent population validation in 1333 non-obstructive azoospermia cases and 1141 healthy controls to identify variant classes with high likelihood of pathogenicity. To explore the functions of these candidate genes in spermatogenesis, we first knocked down these candidate genes individually in mouse spermatogonial stem cells (SSCs) using short interfering RNA oligonucleotides and then generated candidate genes knockout mice by CRISPR-Cas9 system.

Results Four low-frequency variants were identified in four genes (BCORL1, MAP7D3, ARMCX4 and H2BFWT) associated with male infertility. Functional studies of the mouse SSCs revealed that knocking down Bcorl1 or Mtap7d3 could inhibit SSCs self-renewal and knocking down Armcx4 could repress SSCs differentiation in vitro. Using CRISPR-Cas9 system, Bcorl1 and Mtap7d3 knockout mice were generated. Excitingly, Bcorl1 knockout mice were infertile with impaired spermatogenesis. Moreover, Bcorl1 knockout mice exhibited impaired sperm motility and sperm cells displayed abnormal mitochondrial structure.

Conclusion Our data indicate that the X-linked genes are associated with male infertility and involved in regulating SSCs, which provides a new insight into the role of X-linked genes in spermatogenesis.

  • male infertility
  • non-obstructive azoospermia (NOA)
  • X chromosome exons
  • spermatogonial stem cells (SSCs)

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  • CL, YZ and YQ are joint first authors.

  • CL, YZ and YQ contributed equally.

  • Contributors Yankai Xia and Xinru Wang directed the study, obtained financial support and were responsible for study design. Chuncheng Lu performed overall project management along with Yan Zhang and Yufeng Qin. and drafted the initial manuscript. Qiaoqiao Xu, Yiqiang Cui, Jian-hua Mao and Xuejiang Guo performed statistical analysis. Min Wang and Jintao Zhang were responsible for sample processing and managed the genotyping data. Yunfei Zhu and Xin Zhang were responsible for subject recruitment and sample preparation. Xin Wu, Ran Zhou and Xiang Wei were responsible for functional analysis in SSCs. Mingxi Liu, Bo Hang, Zhibin Hu, Hongbing Shen, Antoine M Snijders and Zuomin Zhou conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

  • Funding This work was supported by the National Key R&D Program of China (2019YFC1005100), the National Natural Science Foundation of China (81471500, 81630085 and 81671461) and the Priority Academic Programme for the Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Ethics approval This study was approved by the institutional review board of Nanjing Medical University, China (FWA00001501), and conducted according to the Declaration of Helsinki.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data are available in a public, open access repository. Sequencing data were deposited in NCBI SRA with accession number SRP072055.