| 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.Spermatozoa are haploid cells whose role is to convey their genetic information to oocytes (reviewed in refs. 1–4). Because of this special role, spermatozoa possess highly specialized morphology and function. For example, spermatids compact their haploid genome into a small head during spermatogenesis and are equipped with a flagellum for motility. The mitochondrial sheath surrounding the midpiece of the flagellum provides energy for movement. Ejaculated spermatozoa undergo physiological processes called capacitation and the acrosome reaction that confer fertilizing ability. Only acrosome-reacted spermatozoa are capable of fusing with an oocyte.Based on the microarray studies of Schultz et al. (5), many genes are believed to be expressed predominantly in male germ cells (>2,300 genes). In addition to housekeeping genes, many of these testis-enriched genes are thought to play a unique role during spermatogenesis and/or sperm function. However, the functions of most of these genes are still unclear because of the lack of an in vitro system to generate fully functional spermatozoa.In mammals, gene knockout (KO) mice have been pivotal in studying gene function in vivo. Hundreds of genes are essential or important for reproduction, as revealed by various gene manipulation strategies (reviewed in refs. 1–4). There are several advantages to using KO mice in reproductive biology: (i) because reproductive organs are not essential for viability, genes predominantly expressed in these tissues can be studied without conditional KO methodology; (ii) genes essential for reproductive functions can be screened by examining breeding pairs; and (iii) once infertility is confirmed in vivo, there are assisted reproductive technologies (e.g., germ-line stem cells, seminiferous tubule transplantation, in vitro fertilization and embryo transfer, and intracytoplasmic sperm injection) to analyze their defects. Many KO mice are fertile even though the targeted genes were thought essential for fertilization, based on in vitro experiments [e.g., Acr, Zp3r, Zan, B4galt1, Adam1b, and others. (6)]. Alternatively, without a preceding functional assay in vitro, many testis-enriched genes proved to be essential, by KO, for male reproduction. For example, TEX14 is required for intercellular bridge formation and progression through meiosis (7), GASZ functions in the piRNA pathway and is required for suppression of retrotransposons (8), CATSPER1 is a voltage-dependent calcium channel required for sperm hyperactivated motility (9), CLGN is an endoplasmic reticulum chaperone protein essential for sperm migration into the oviduct and fertilization (10), and PPP3R2 is a testis-specific regulatory subunit of calcineurin required for normal motility of the sperm midpiece (11). Taking these facts into account, it would be better to know first whether a gene of interest is essential in vivo before deciding to invest valuable research dollars and experimental time and personnel efforts into the project. In addition, with the advances made in using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to manipulate the mouse genome (12–15), it is likely as inexpensive and quicker to create genetic mutations and test for phenotypes as it is to test by in vitro experimentation.If a KO mouse consistently sires pups, that gene is classified as not essential for male fertility. Whereas research involving KO mice with phenotypes are published, studies of KO mice without phenotypes are usually ignored and rarely published unless the gene is well recognized. The lack of publicity of a fertile mouse KO may mislead other researchers into making the same KO mouse and thereby wasting valuable resources and research staff time. Once these KO mice become available to academia as bioresources, researchers with differing areas of expertise are able to examine their hypothesis using these mice.In this report, we describe our KO of 54 genes that exhibit testis-enriched expression, based on bioinformatic and/or experimental analysis. Using various gene manipulation tools, we discovered these evolutionarily conserved genes (present in at least the mouse and human genomes) are dispensable for male fertility. Thus, our findings indicate that, individually, these testis-enriched genes do not perform critical roles in spermatogenesis or fertilization in mice. |
