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Integrated Analyses of Phenotype and Quantitative Proteome of CMTM4 Deficient Mice Reveal Its Association with Male Fertility*[S]

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The chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is a gene family that has been implicated in male reproduction. CMTM4 is an evolutionarily conserved member that is highly expressed in the testis. However, its function in male fertility remains unknown. Here, we demonstrate that CMTM4 is associated with spermatogenesis and sperm quality. Using Western blotting and immunohistochemical analyses, we found CMTM4 expression to be decreased in poor-quality human spermatozoa, old human testes, and testicular biopsies with nonobstructive azoospermia. Using CRISPR-Cas9 technology, we knocked out the Cmtm4 gene in mice. These Cmtm4 knockout (KO) mice showed reduced testicular daily sperm production, lower epididymal sperm motility and increased proportion of abnormally backward-curved sperm heads and bent sperm midpieces. These mice also had an evident sub-fertile phenotype, characterized by low pregnancy rates on prolonged breeding with wild type female mice, reduced in vitro fertilization efficiency and a reduced percentage of acrosome reactions. We then performed quantitative proteomic analysis of the testes, where we identified 139 proteins to be downregulated in Cmtm4-KO mice, 100 (71.9%) of which were related to sperm motility and acrosome reaction. The same proteomic analysis was performed on sperm, where we identified 3588 proteins with 409 being differentially regulated in Cmtm4-KO mice. Our enrichment analysis showed that upregulated proteins were enriched with nucleosomal DNA binding functions and the downregulated proteins were enriched with actin binding functions. These findings elucidate the roles of CMTM4 in male fertility and demonstrates its potential as a promising molecular candidate for sperm quality assessment and the diagnosis or treatment of male infertility.

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Highlights

  • CMTM4 is associated with human spermatogenesis and sperm quality.

  • Cmtm4 knockout mouse were generated by CRISPR/Cas9 technology for male fertility research.

  • CMTM4 is required for male fertility but not female fertility.

  • Phenotype and quantitative proteomics of Cmtm4 KO mice reveal an association of CMTM4 with histone-to-protamine exchange, sperm motility and induction of the acrosome reaction.

Cytokines*
iTRAQ
Knockouts*
Mouse models
Absolute quantification
Cas9
CMTM4
Male fertility
Spermatogenesis
Testis

Cited by (0)

The authors declare no conflict of interest.

Author contributions: F.L. and W.H. designed research; F.L., X.-X.L., X.L., P.Z., H.X., and W.W. performed research; F.L., X.-X.L., X.L., T.L., P.Z., Z.L., W.W., X.Y., and J.L. analyzed data; F.L. and W.H. wrote the paper; X.-X.L., X.L., T.L., P.Z., Z.L., H.X., W.W., X.Y., and J.L. contributed new reagents/analytic tools.

*

The current study was supported by the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2018PT31039), National Natural Science Foundation of China (81571490), Shandong Provincial Natural Science Foundation, China (grant no. ZR2014HQ068).

[S]

This article contains supplemental material.