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

Abstract

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.

Keywords: Absolute quantification; CMTM4; Cas9; Cytokines*; Knockouts*; Male fertility; Mouse models; Spermatogenesis; Testis; iTRAQ.

Graphical abstract

Fig. 1.

Expression of CMTM4 in human spermatozoa. Human spermatozoa were collected from normozoospermic, asthenozoospermic, oligoasthenozoospermic, teratozoospermic patients and showed significant differences in progressive sperm motility (A); comparison of CMTM4 expression in spermatozoa in normozoospermia and teratozoospermia was analyzed by retrieving GEO data from GSE6968 (B); Western blot analysis of CMTM4 in human spermatozoa in normozoospermia, asthenozoospermia, oligoasthenozoospermia and teratozoospermia. The quantification of CMTM4 expression was calculated by normalizing band intensities to the respective TUBB expressions by Image Pro software (C–E). Asth, asthenozoospermia, Olig, oligoasthenozoospermia, Tera, teratozoospermia; Lines (A) indicate mean values, whiskers indicate standard deviation; Data were compared by one-way analysis of variance (ANOVA); *, p < 0.05.

Fig. 2.

Expression of CMTM4 in human testes. Johnsen score evaluation of testicular sections from young adults, elderly adults and patients with nonobstructive azoospermia (NOA) (A); quantitative evaluation of CMTM4 expression in testes by Image-Pro (B); representative image of testicular cellular localization of negative control (C) and CMTM4 in young adults (D), elderly adults (E) and NOA patients (F–H); SP, spermatogonia; PS, pachytene spermatocyte; RS, round spermatid; SC, Sertoli cell; Spt, spermatids; Lines (A, B) indicate mean values, whiskers indicate standard deviation; Data were compared by one-way analysis of variance (ANOVA); *, p < 0.05; Each bar represents 20 μm.

Fig. 3.

Construction of Cmtm4 KO mice. Scheme of the genomic target sites in theCmtm4 (A). The untranslated region (UTR) of Cmtm4 is highlighted in green and the coding sequence (CDS) of Cmtm4 is indicated in blue. 179bp sequences in exon 1 were deleted in Cmtm4 KO mice. Polymerase Chain Reaction (PCR) (B), Western blotting (C) and immunohistochemistry analysis (D, E) were performed on mouse testis samples. WT, wild type; HT, heterozygous; KO, knockout; SP, spermatogonia; PS, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids. Each bar represents 20 μm.

Fig. 4.

Characteristics of fertility of Cmtm4 WT and KO male mice. No variance in testicular weight (A), sperm concentration (B) and serum testosterone level (C) between WT and KO mice was observed. Significantly reduced daily sperm production (D) and sperm motility (E, F, G) and increased abnormal sperm morphology (H, K) were observed in Cmtm4 KO mice compared with WT mice. In vitro fertility analyses showed a decreased percentage of induced acrosome reactions (I, M), the numbers in brackets represent the number of mice examined, and the decreased percentage of oocytes developed to two-cell stages. The numbers in brackets represent the number of WT oocytes used (J, L). Values are shown as mean ± S.D. a, curved back sperm head; b, bent sperm midpiece; WT, wild type; KO, knockout; VSL, straight line velocity; VCL, curvilinear velocity; Lines (E, F, G) indicate mean values, whiskers indicate standard deviation; Data were compared by one-way analysis of variance (ANOVA); *, p < 0.05; **, p < 0.01. Each bar represents 20 μm.

Fig. 5.

iTRAQ-based quantitative proteomic analysis of Cmtm4 KO mice testis. Flow chart of iTRAQ analysis of the mouse testicular proteome (A); Venn diagram of the current mouse testis proteome and a previously reported mouse germ cell proteome (B), and the current mouse testicular proteome and a previously reported mouse sperm proteome (C); broad functional analysis of the current mouse testicular proteome in GO terms of molecular functions (D) and biological processes (E).

Fig. 6.

Bioinformatic analysis of differentially expressed proteins in WT and Cmtm4 KO mice testes. Broad functional classification of downregulated proteins (A) and upregulated proteins (B) in KO mice, and biological processes enriched in downregulated proteins (C) and in upregulated proteins (D) in KO mice; interactional network between differential proteins and major biological processes of sperm function (E); tissue expression patterns of downregulated proteins (F) and upregulated proteins (G); testicular germ cell expression patterns of downregulated proteins (H) and upregulated proteins (I) in the normal mouse testis.

Fig. 7.

Western blotting (A, B) and immunohistochemical (C) analysis of PGK2, SORD, HSPA1L and HSPA9 in WT and Cmtm4 KO mice. The integrated optical density (IOD) ratio of target protein to ACTB was used to express the results of the Western blot analysis. Lines (C) indicate mean values, whiskers indicate standard deviation; Data were compared by Student's t test; *, p < 0.05; Each bar represents 50 μm.

Fig. 8.

iTRAQ-based quantitative proteomic analysis of Cmtm4 KO mice sperm. Flow chart of iTRAQ analysis of the mouse sperm proteome (A); Venn diagram of the current mouse sperm proteome and previously reported mouse sperm proteomes (B); broad function of current mouse sperm proteomes (C); enriched molecular functional analysis of downregulated (D) and upregulated proteins (E) in KO sperm.

Fig. 9.

Western blotting and immunofluorescence staining of selected proteins. Western blot analysis of H2A, H2B, H4, PRM1, PRM2. MYH11, ODF2, CFL1, TPM1, α-tubulin, β-tubulin, and ACTB serving as a control (A); quantification of blotting intensity is shown as ratio of normalized IOD of each band (KO/WT) (B); quantitative immunofluorescence of CMTM4 and ODF2 in WT and KO sperm (C), intensity was calculated by using Zeiss LSM 510 Meta software.