Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice

Abstract

Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201–12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract–enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the “gold standard” to determine whether a gene’s function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.

TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa

“…We initially identified Tcte1 as a conserved testis-specific gene using a bioinformatic screen, as recently described (28). To determine the expression profile of Tcte1, we performed multitissue RT-PCR from adult mouse.…”

Widespread transcriptional scanning in the testis modulates gene evolution rates

“…Moreover, recent animal studies have shown that many testis-enriched and evolutionarily-conserved genes are not required for male fertility in mice 17 . A second hypothesis implicates leaky transcription during the massive chromatin remodeling that occurs throughout spermatogenesis 12,18,19 .…”

Revolutionizing male fertility factor research in mice by using the genome editing tool CRISPR/Cas9

“…For example, by utilizing the CRISPR/Cas9 system in addition to other KO methods, >80 KO mice of genes that are predominantly expressed in the testis and conserved in both mice and humans were generated. Surprisingly, 54 of those KO male mouse lines were fertile, meaning that the importance of these genes cannot be based solely on their tissue expression profile . In contrast, there are several genes that were found to be indispensable for male fertility by using the CRISPR/Cas9 system, such as Ppp3r2 , Ccdc63 , and Cabyr .…”

“…There are ~1000 genes that are predominantly expressed in the mouse testis and many of these genes still need to be analyzed with the KO approach. The genes that are essential for male fertility will be revealed more rapidly with the CRISPR/Cas9 system.…”