CRISPR/Cas9-mediated genome editing reveals 30 testis-enriched genes dispensable for male fertility in mice†

Abstract

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.

Keywords: CRISPR/Cas9; knockout mice; male infertility; spermatogenesis; testis expression.

Figure 1.

Conservation of the testis-enriched genes among species. The presence of orthologs is indicated by solid or shaded squares. Orthologous genes conserved in all species in a taxon are highlighted with solid dark gray. Shaded gray indicates loss of an ortholog in several species within a taxon. Solid light gray indicates that 1700010B08Rik is only found in mice. Question marks indicate potential orthologs in species within a taxon.

Figure 2.

Digital PCR depicting the average TPM value per tissue per gene from 240 published mouse and human RNA-seq datasets. (A) Patterns of gene expression in mouse tissues and organs. (B) Patterns of gene expression in human tissues and organs. White = 0 TPM, black ≥ 30 TPM.

Figure 3.

Phenotypic analysis of Trim17 knockout male mice. (A) Genomic structure and knockout strategy of mouse Trim17. Dual sgRNAs were designed to target the first coding exon and the 3^΄ UTR region downstream the last exon. Fw, forward primer for genotyping; Rv #1/#2, reverse primers for genotyping. (B) Genotype validation of Trim17 knockout mice by genomic PCR and Sanger sequencing. (C) Comparison of testis size and testis to body weight ratios of Trim17 wild-type (WT) and knockout (KO) mice. Scale bar = 2 mm. (D) Histological analyses of testes and epididymides in Trim17 wild-type and knockout mice. Scale bars = 50 μm. (E) Morphology of spermatozoa extracted from the cauda epididymides in Trim17 wild-type and knockout mice. Scale bars = 100 μm. (F) Percentages of motile spermatozoa in Trim17 wild-type and knockout mice. Sperm motility was measured at 10 and 120 min of incubation in TYH media.

Figure 4.

Phenotypic analysis of Mgat4d knockout male mice. (A) Genomic structure and knockout strategy of mouse Mgat4d. Fw, forward primer for genotyping; Rv, reverse primers for genotyping. (B) Genotype validation of Mgat4d knockout mice by MultiNA microchip electrophoresis and Sanger sequencing. (C) Comparison of testis size and testis to body weight ratios of Mgat4d wild-type (WT) and knockout (KO) mice. Scale bar = 2 mm. (D) Histological analyses of testes and epididymides in Mgat4d wild-type and knockout mice. Scale bars = 100 μm. (E) Morphology of spermatozoa extracted from the cauda epididymides in Mgat4d wild-type and knockout mice. Scale bars = 50 μm. (F) Percentages of motile spermatozoa in Mgat4d wild-type and knockout mice. Sperm motility was measured at 10 and 120 min of incubation in TYH media.