Mouse t-complex protein 11 is important for progressive motility in sperm†.

The t-complex is defined as naturally occurring variants of the proximal third of mouse chromosome 17 and has been studied by mouse geneticists for decades. This region contains many genes involved in processes from embryogenesis to sperm function. One such gene, t-complex protein 11 (Tcp11), was identified as a testis-specific gene whose protein is present in elongating spermatids. Later work on Tcp11 localized TCP11 to the sperm surface and acrosome cap and implicated TCP11 as important for sperm capacitation through the cyclic AMP/Protein Kinase A pathway. Here, we show that TCP11 is cytoplasmically localized to elongating spermatids and absent from sperm. In the absence of Tcp11, male mice have severely reduced fertility due to a significant decrease in progressively motile sperm; however, Tcp11-null sperm continues to undergo tyrosine phosphorylation, a hallmark of capacitation. Interestingly, null sperm displays reduced PKA activity, consistent with previous reports. Our work demonstrates that TCP11 functions in elongated spermatids to confer proper motility in mature sperm.

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

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.

Disrupting the male germ line to find infertility and contraception targets.

Genetically-manipulated mouse models have become indispensible for broadening our understanding of genes and pathways related to male germ cell development. Until suitable in vitro systems for studying spermatogenesis are perfected, in vivo models will remain the gold standard for inquiry into testicular function. Here, we discuss exciting advances that are allowing researchers faster, easier, and more customizable access to their mouse models of interest. Specifically, the trans-NIH Knockout Mouse Project (KOMP) is working to generate knockout mouse models of every gene in the mouse genome. The related Knockout Mouse Phenotyping Program (KOMP2) is performing systematic phenotypic analysis of this genome-wide collection of knockout mice, including fertility screening. Together, these programs will not only uncover new genes involved in male germ cell development but also provide the research community with the mouse models necessary for further investigations. In addition to KOMP/KOMP2, another promising development in the field of mouse models is the advent of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas technology. Utilizing 20 nucleotide guide sequences, CRISPR/Cas has the potential to introduce sequence-specific insertions, deletions, and point mutations to produce null, conditional, activated, or reporter-tagged alleles. CRISPR/Cas can also successfully target multiple genes in a single experimental step, forgoing the multiple generations of breeding traditionally required to produce mouse models with deletions, insertions, or mutations in multiple genes. In addition, CRISPR/Cas can be used to create mouse models carrying variants identical to those identified in infertile human patients, providing the opportunity to explore the effects of such mutations in an in vivo system. Both the KOMP/KOMP2 projects and the CRISPR/Cas system provide powerful, accessible genetic approaches to the study of male germ cell development in the mouse. A more complete understanding of male germ cell biology is critical for the identification of novel targets for potential non-hormonal contraceptive intervention.

Loss of smad4 in Sertoli and Leydig cells leads to testicular dysgenesis and hemorrhagic tumor formation in mice.

As the central component of canonical TGFbeta superfamily signaling, SMAD4 is a critical regulator of organ development, patterning, tumorigenesis, and many other biological processes. Because numerous TGFbeta superfamily ligands are expressed in developing testes, there may exist specific requirements for SMAD4 in individual testicular cell types. Previously, we reported that expansion of the fetal testis cords requires expression of SMAD4 by the Sertoli cell lineage. To further uncover the role of Smad4 in murine testes, we produced conditional knockout mice lacking Smad4 in either Leydig cells or in both Sertoli and Leydig cells simultaneously. Loss of Smad4 concomitantly in Sertoli and Leydig cells led to underdevelopment of the testis cords during fetal life and mild testicular dysgenesis in young adulthood (decreased testis size, partially dysgenic seminiferous tubules, and low sperm production). When the Sertoli/Leydig cell Smad4 conditional knockout mice aged (56- to 62-wk old), the testis phenotypes became exacerbated with the appearance of hemorrhagic tumors, Leydig cell adenomas, and a complete loss of spermatogenesis. In contrast, loss of Smad4 in Leydig cells alone did not appreciably alter fetal and adult testis development. Our findings support a cell type-specific requirement of Smad4 in testis development and suppression of testicular tumors.

GASZ promotes germ cell derivation from embryonic stem cells.

Primordial germ cells (PGCs) are the first germ-line population that forms from the proximal epiblast of the developing embryo. Despite their biological importance, the regulatory networks whereby PGCs arise, migrate, and differentiate into gametes during embryonic development remains elusive, largely due to the limited number of germ cells in the early embryo. To elucidate the molecular mechanisms that govern early germ cell development, we utilized an in vitro differentiation model of embryonic stem cells (ESCs) and screened a series of candidate genes with specific expression in the adult reproductive organs. We discovered that gain of function of Gasz, a gene previously reported to participate in meiosis of postnatal spermatocytes, led to the most robust upregulation of PGC formation from both human and murine ESCs. In contrast, Gasz deficiency resulted in pronounced reduction of germ cells during ESC differentiation and decreased expression of MVH and DAZL in genital ridges during early embryonic development. Further analyses demonstrated that GASZ interacted with DAZL, a key germ cell regulator, to synergistically promote germ cell derivation from ESCs. Thus, our data reveal a potential role of GASZ during embryonic germ cell development and provide a powerful in vitro system for dissecting the molecular pathways in early germ cell formation during embryogenesis.

Testicular somatic cells, not gonocytes, are the major source of functional activin A during testis morphogenesis.

Proper development of the seminiferous tubules (or testis cords in embryos) is critical for male fertility. Sertoli cells, somatic components of the seminiferous tubules, serve as nurse cells to the male germline, and thus their numbers decide the quantity of sperm output in adulthood. We previously identified activin A, the protein product of the activin ßA (Inhba) gene, as a key regulator of murine Sertoli cell proliferation and testis cord expansion during embryogenesis. Although our genetic studies implicated fetal Leydig cells as the primary producers of testicular activin A, gonocytes are another potential source. To investigate the relative contribution of gonocyte-derived activin A to testis morphogenesis, we compared testis development in the Inhba global knockout mouse, which lacks activin A production in all cells (including the gonocytes), and a steroidogenic factor 1 (Sf1)-specific conditional knockout model in which activin A expression in testicular somatic cells is disrupted but gonocyte expression of activin A remains intact. Surprisingly, testis development was comparable in these two models of activin A insufficiency, with similar reductions in Sertoli cell proliferation and minor differences in testis histology. Thus, our findings suggest activin A from male gonocytes is insufficient to promote Sertoli cell proliferation and testis cord expansion in the absence of somatic cell-derived activin A. Evaluation of adult male mice with fetal disruption of activin A revealed reduced testis size, lowered sperm production, altered testicular histology, and elevated plasma FSH levels, defects reminiscent of human cases of androgen-sufficient idiopathic oligozoospermia.

Activin A, a product of fetal Leydig cells, is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion.

Formation of tubular structures relies upon complex interactions between adjacent epithelium and mesenchyme. In the embryonic testes, dramatic compartmentalization leads to the formation of testis cords (epithelium) and the surrounding interstitium (mesenchyme). Sertoli cells, the epithelial cell type within testis cords, produce signaling molecules to orchestrate testis cord formation. The interstitial fetal Leydig cells, however, are thought only to masculinize the embryo and are not known to be involved in testis cord morphogenesis. Contrary to this notion, we have identified activin A, a member of the TGF-beta protein superfamily, as a product of the murine fetal Leydig cells that acts directly upon Sertoli cells to promote their proliferation during late embryogenesis. Genetic disruption of activin betaA, the gene encoding activin A, specifically in fetal Leydig cells resulted in a failure of fetal testis cord elongation and expansion due to decreased Sertoli cell proliferation. Conditional inactivation of Smad4, the central component of TGF-beta signaling, in Sertoli cells led to testis cord dysgenesis and proliferative defects similar to those of Leydig cell-specific activin betaA knockout testes. These results indicate that activin A is the major TGF-beta protein that acts directly on Sertoli cells. Testicular dysgenesis in activin betaA and Smad4 conditional knockout embryos persists into adulthood, leading to low sperm production and abnormal testicular histology. Our findings challenge the paradigm that fetal testis development is solely under the control of Sertoli cells, by uncovering an active and essential role of fetal Leydig cells during testis cord morphogenesis.

Epithelial-mesenchymal crosstalk in Wolffian duct and fetal testis cord development.

Interactions between adjacent epithelial and mesenchymal tissues represent a highly conserved mechanism in embryonic organogenesis. In particular, the ability of the mesenchyme to instruct cellular differentiation of the epithelium is a fundamental requirement for the morphogenesis of tubular structures such as those found in the kidneys, lungs, and the developing male reproductive system. Once the tubular structure has formed, it receives signals from the mesenchyme, which can control proliferation, patterning, and differentiation of the epithelium inside the tube. However, the epithelium is not a "silent partner" in this process, and epithelium-derived factors are often required for proper maintenance of the mesenchymal compartment. Although much emphasis has been placed on the characterization of mesenchymally-derived signals required for epithelial differentiation, it is important to note that epithelial-mesenchymal interactions are a two-way street wherein each compartment requires the presence of the other for proper tubule morphogenesis and function. In this review, we discuss epithelial-mesenchymal interactions in the processes of Wolffian duct and fetal testis cord development using the mouse as a model organism and propose inhibin beta A as a conserved mesenchyme-derived regulator in these two male-specific tubular structures.