SPATC1L maintains the integrity of the sperm head-tail junction.

Spermatogenesis is a tightly regulated process involving germ cell-specific and germ cell-predominant genes. Here we investigate a novel germ cell-specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9-mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z-line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L-PKA complex in maintaining the stability of the sperm head-tail junction, thereby revealing a new molecular basis for sperm head-tail integrity.

Characterization of MAGEG2 with testis-specific expression in mice.

Male germ cell development is a well-defined process occurring in numerous seminiferous tubules of the testis. Uncovering testicular novel genes related to intrinsic regulation of spermatogenesis is essential for the understanding of spermatogenesis. In the present study, we investigated mouse Mageg2, which belongs to a group of melanoma-associated antigens (MAGEs). Mageg2 is transcribed in the testis specifically, and its expression level is increased at the pachytene spermatocyte stage, indicating that Mageg2 is expressed predominantly in germ cells. We generated an antibody against mouse MAGEG2 for further characterization at the protein level. Immunoblot analysis suggested that MAGEG2 has specific testicular expression and the expression primarily occurred in pachytene spermatocytes. Proteomic analyses demonstrated that mouse MAGEG2 binded to testicular germ cell-specific serine/threonine-protein kinase 31 (STK31) and heat shock protein 9 (HSPA9). Direct binding with both interaction partners was confirmed by co-immunoprecipitation. We found that STK31 and HSPA9 bind MAGEG2 directly but not with each other. Interestingly, MAGEG2 reduced the kinase activity of STK31. Our study suggests that mouse MAGEG2 has at least two functions, including chaperone activity related to HSPA9 and regulation of pachytene spermatocyte-specific kinase, STK31. Altogether, our results provide the first information about MAGEG2 at the transcript and protein levels and suggest its potential molecular functions.

Reduced Fertility and Altered Epididymal and Sperm Integrity in Mice Lacking ADAM7.

The mammalian epididymis is a highly convoluted tubule that connects the testis to the vas deferens. Its proper functions in sperm transport, storage, and maturation are essential for male reproduction. One of the genes predominantly expressed in the epididymis is ADAM7 (a disintegrin and metalloprotease 7). Previous studies have shown that ADAM7 synthesized in the epididymis is secreted into the epididymal lumen and is then transferred to sperm membranes, where it forms a chaperone complex that is potentially involved in sperm fertility. In this study, we generated and analyzed mice with a targeted disruption in the Adam7 gene. We found that the fertility of male mice was modestly but significantly reduced by knockout of Adam7. Histological analyses revealed that the cell heights of the epithelium were dramatically decreased in the caput of the epididymis of Adam7-null mice, suggesting a requirement for ADAM7 in maintaining the integrity of the epididymal epithelium. We found that sperm from Adam7-null mice exhibit decreased motility, tail deformation, and altered tyrosine phosphorylation, indicating that the absence of ADAM7 leads to abnormal sperm functions and morphology. Western blot analyses revealed reduced levels of integral membrane protein 2B (ITM2B) and ADAM2 in sperm from Adam7-null mice, suggesting a requirement for ADAM7 in normal expression of sperm membrane proteins involved in sperm functions. Collectively, our study demonstrates for the first time that ADAM7 is required for normal fertility and is important for the maintenance of epididymal integrity and for sperm morphology, motility, and membrane proteins.

Identification and characterization of germ cell genes expressed in the F9 testicular teratoma stem cell line.

The F9 cell line, which was derived from a mouse testicular teratoma that originated from pluripotent germ cells, has been used as a model for differentiation. However, it is largely unknown whether F9 cells possess the characteristics of male germ cells. In the present study, we investigated spermatogenic stage- and cell type-specific gene expression in F9 cells. Analysis of previous microarray data showed that a large number of stage-regulated germ cell genes are expressed in F9 cells. Specifically, genes that are prominently expressed in spermatogonia and have transcriptional regulatory functions appear to be enriched in F9 cells. Our in silico and in vitro analyses identified several germ cell-specific or -predominant genes that are expressed in F9 cells. Among them, strong promoter activities were observed in the regions upstream of the spermatogonial genes, Dmrt1 (doublesex and mab-3 related transcription factor 1), Stra8 (stimulated by retinoic acid gene 8) and Tex13 (testis expressed gene 13), in F9 cells. A detailed analysis of the Tex13 promoter allowed us to identify an enhancer and a region that is implicated in germ cell-specificity. We also found that Tex13 expression is regulated by DNA methylation. Finally, analysis of GFP (green fluorescent protein) TEX13 localization revealed that the protein distributes heterogeneously in the cytoplasm and nucleus, suggesting that TEX13 shuttles between these two compartments. Taken together, our results demonstrate that F9 cells express numerous spermatogonial genes and could be used for transcriptional studies focusing on such genes. As an example of this, we use F9 cells to provide comprehensive expressional information about Tex13, and report that this gene appears to encode a germ cell-specific protein that functions in the nucleus during early spermatogenesis.