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.

Profiling of testis-specific long noncoding RNAs in mice.

BACKGROUND: Spermatogenesis, which is the complex and highly regulated process of producing haploid spermatozoa, involves testis-specific transcripts. Recent studies have discovered that long noncoding RNAs (lncRNAs) are novel regulatory molecules that play important roles in various biological processes. However, there has been no report on the comprehensive identification of testis-specific lncRNAs in mice. RESULTS: We performed microarray analysis of transcripts from mouse brain, heart, kidney, liver and testis. We found that testis harbored the highest proportion of tissue-specific lncRNAs (11%; 1607 of 14,256). Testis also harbored the largest number of tissue-specific mRNAs among the examined tissues, but the proportion was lower than that of lncRNAs (7%; 1090 of 16,587). We categorized the testis-specific lncRNAs and found that a large portion corresponded to long intergenic ncRNAs (lincRNAs). Genomic analysis identified 250 protein-coding genes located near (≤ 10 kb) 194 of the loci encoding testis-specific lincRNAs. Gene ontology (GO) analysis showed that these protein-coding genes were enriched for transcriptional regulation-related terms. Analysis of male germ cell-related cell lines (F9, GC-1 and GC-2) revealed that some of the testis-specific lncRNAs were expressed in each of these cell lines. Finally, we arbitrarily selected 26 testis-specific lncRNAs and performed in vitro expression analysis. Our results revealed that all of them were expressed exclusively in the testis, and 23 of the 26 showed germ cell-specific expression. CONCLUSION: This study provides a catalog of testis-specific lncRNAs and a basis for future investigation of the lncRNAs involved in spermatogenesis and testicular 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 promoter and regulatory regions for mouse Adam2 gene expression.

ADAM2, a member of the 'a disintegrin and metalloprotease' (ADAM) family, is a key protein in mammalian fertilization that is specifically expressed in testicular germ cells. Here, we investigated the transcriptional regulation of the mouse Adam2 gene. An in silico analysis identified two conserved non-coding sequences located upstream of the mouse and human ADAM2 genes. The upstream region of the mouse Adam2 gene was found to lack typical TATA and CAAT boxes, and to have a high GC content. Our in vitro transient transfection-reporter analysis identified a promoter in this region of the mouse Adam2 gene, along with regulatory regions that inhibit the activity of this promoter in somatic cells. Site-directed mutagenesis revealed that the caudal-type homeobox 1 and CCTC-binding factor motifs are responsible for the inhibitory activities of the repressor regions. Finally, electrophoretic mobility shift assays showed putative transcription factor-promoter DNA complexes, and DNA-affinity chromatography and proteomic analyses identified myelin gene regulatory factor as a binding partner of the Adam2 promoter. This provides the first identification and characterization of promoter and repressor regions that regulate the transcription of the mouse Adam2 gene, and offers insights into the regulation of this germ-cell-specific gene.

Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes.

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.

Myosin regulatory light chains are required to maintain the stability of myosin II and cellular integrity.

Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC-MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.

Identification of heat shock protein 5, calnexin and integral membrane protein 2B as Adam7-interacting membrane proteins in mouse sperm.

In mammals, sperm acquire their motility and ability to fertilize eggs in the epididymis. This maturation process involves the acquisition of particular proteins from the epididymis. One such secretory protein specifically expressed in the epididymis is Adam7 (a disintegrin and metalloprotease 7). Previous studies have shown that Adam7 that resides in an intracellular compartment of epididymal cells is transferred to sperm membranes, where its levels are dependent on the expression of Adam2 and Adam3, which have critical roles in fertilization. Here, using a proteomics approach based on mass spectrometry, we identified proteins that interact with Adam7 in sperm membranes. This analysis revealed that Adam7 forms complexes with calnexin (Canx), heat shock protein 5 (Hspa5), and integral membrane protein 2B (Itm2b). Canx and Hspa5 are molecular chaperones, and Itm2b is a type II integral membrane protein implicated in neurodegeneration. The interaction of Adam7 with these proteins was confirmed by immunoprecipitation-Western blot analysis. We found that Adam7 and Itm2b are located in detergent-resistant regions known to be highly correlated with membrane lipid rafts. We further found that the association of Adam7 with Itm2b is remarkably promoted during sperm capacitation owing to a conformational change of Adam7 that occurs in concert with the capacitation process. Thus, our results suggest that Adam7 functions in fertilization through the formation of a chaperone complex and enhanced association with Itm2b during capacitation in sperm.

Expression of uncharacterized male germ cell-specific genes and discovery of novel sperm-tail proteins in mice.

The identification and characterization of germ cell-specific genes are essential if we hope to comprehensively understand the mechanisms of spermatogenesis and fertilization. Here, we searched the mouse UniGene databases and identified 13 novel genes as being putatively testis-specific or -predominant. Our in silico and in vitro analyses revealed that the expressions of these genes are testis- and germ cell-specific, and that they are regulated in a stage-specific manner during spermatogenesis. We generated antibodies against the proteins encoded by seven of the genes to facilitate their characterization in male germ cells. Immunoblotting and immunofluorescence analyses revealed that one of these proteins was expressed only in testicular germ cells, three were expressed in both testicular germ cells and testicular sperm, and the remaining three were expressed in sperm of the testicular stages and in mature sperm from the epididymis. Further analysis of the latter three proteins showed that they were all associated with cytoskeletal structures in the sperm flagellum. Among them, MORN5, which is predicted to contain three MORN motifs, is conserved between mouse and human sperm. In conclusion, we herein identify 13 authentic genes with male germ cell-specific expression, and provide comprehensive information about these genes and their encoded products. Our finding will facilitate future investigations into the functional roles of these novel genes in spermatogenesis and sperm functions.

Identification of target genes for spermatogenic cell-specific KRAB transcription factor ZFP819 in a male germ cell line.

BACKGROUND: Zfp819, a member of the Krüppel-associated box (KRAB) family, encodes a spermatogenic cell-specific transcription factor. Zfp819-overexpression induces apoptosis and inhibits proliferation in somatic cell lines. RESULTS: In the present study, we examined the cellular effects of Zfp819 in a male germ cell line (GC-2 cells). Overexpression of Zfp819 demonstrated an increase in the number of apoptotic cells, leading to inhibition of proliferation in GC-2 cells. We further investigated genes regulated by ZFP819 using microarray analysis and chromatin-immunoprecipitation combined with microarray analysis (ChIP-chip) in GC-2 cells. We identified 118 downregulated genes in Zfp819-overexpressing GC-2 cells using microarray analysis. ChIP-chip assay revealed that 1011 promoter sites (corresponding to 262 genes) were specifically enriched in GC-2 cells transfected with Zfp819. Two genes (trinucleotide repeat containing 6b and annexin A11) were commonly found when we compared the data between microarray and ChIP-chip analyses. Consistent with these results, Zfp819 overexpression significantly reduced the transcript levels of the two genes by binding to their promoter regions. Tissue distribution analysis indicated that both genes were predominantly expressed in testis. It has been reported that these two genes function in apoptosis. CONCLUSION: Collectively, our study provides inclusive information on germ cell-specific gene regulation by ZFP819, which is involved in apoptosis, to maintain the integrity of spermatogenesis.

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.

TEX13 is a novel male germ cell-specific nuclear protein potentially involved in transcriptional repression.

The identification and characterization of male germ cell-specific genes is crucial to understanding the mechanisms of male germ cell development. In this study, we investigated the protein encoded by the novel mouse germ cell-specific gene testis-expressed gene 13 (Tex13). We found that TEX13 expression is testis- and germ cell-specific and is regulated in a stage-specific manner via translational repression. Immunostaining of testicular cells and sperm showed that TEX13 is localized in the nuclei of spermatogenic cells and the redundant nuclear envelope of mature sperm. Remarkably, we found that TEX13 possesses transcriptional repressor activity and that its overexpression in GC-2 cells altered the expression levels of 130 genes. Our results suggest that TEX13 has a potential role in transcriptional regulation during spermatogenesis.

Characterization of Mammalian ADAM2 and Its Absence from Human Sperm.

The members of the ADAM (a disintegrin and metalloprotease) family are membrane-anchored multi-domain proteins that play prominent roles in male reproduction. ADAM2, which was one of the first identified ADAMs, is the best studied ADAM in reproduction. In the male germ cells of mice, ADAM2 and other ADAMs form complexes that contribute to sperm-sperm adhesion, sperm-egg interactions, and the migration of sperm in the female reproductive tract. Here, we generated specific antibodies against mouse and human ADAM2, and investigated various features of ADAM2 in mice, monkeys and humans. We found that the cytoplasmic domain of ADAM2 might enable the differential association of this protein with other ADAMs in mice. Western blot analysis with the anti-human ADAM2 antibodies showed that ADAM2 is present in the testis and sperm of monkeys. Monkey ADAM2 was found to associate with chaperone proteins in testis. In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm. This is surprising given the results in mice and monkeys, but it is consistent with the failure of ADAM2 identification in the previous proteomic analyses of human sperm. These findings suggest that the reproductive functions of ADAM2 differ between humans and mice. Our protein analysis showed the presence of potential ADAM2 complexes involving yet-unknown proteins in human testis. Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

Identification and characterization of reproductive KRAB-ZF genes in mice.

The mammalian genome contains numerous genes encoding transcription factors that contain Krüppel-associated box (KRAB) and C2H2-type zinc finger (ZF) motifs (KRAB-ZF). In the present study, we identified KRAB-ZF genes expressed in the mouse testis or ovary, and selected three genes that exhibit gonad-specific or gonad-predominant expression. In vitro analyses showed that these gonadal KRAB-ZF proteins are localized in cell nuclei and are able to repress transcriptional activity. We further analyzed one of the gonad-specific reproductive genes, Zfp819, and found that it is expressed exclusively in spermatogenic cells. Overexpression of Zfp819 suppressed cell proliferation and induced apoptosis. Microarray analysis of Zfp819-overexpressing cells allowed us to identify numerous, potential target genes. A number of the down-regulated genes were found to show gene expression levels inversely correlated with Zfp819 during spermatogenesis. Some of the down-regulated genes were previously reported to play significant roles in spermatogenesis and apoptosis. Collectively, our study provides the first comprehensive information regarding the expression of reproductive KRAB-ZF genes in mice and reveals potential functions of Zfp819.

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.

Expression and function of the testis-predominant protein LYAR in mice.

Mammalian spermatogenesis is a complex process involving an intrinsic genetic program of germ cell-specific and -predominant genes. In the present study, we analyzed the Ly-1 reactive clone (Lyar) gene in the mouse. Lyar, which is known to be expressed abundantly in the testis, encodes a nucleolar protein that contains a LYAR-type C2HC zinc finger motif and three nuclear localization signals. We herein confirmed that Lyar is expressed predominantly in the testis, and further showed that this expression is specific to germ cells. Protein analyses with an anti-LYAR antibody demonstrated that the LYAR protein is present in spermatocytes and spermatids, but not in sperm. To assess the functional role of LYAR in vivo, we used a genetrap mutagenesis approach to establish a LYAR-null mouse model. Lyar mutant mice were born live and developed normally. Male mutant mice lacking LYAR were fully fertile and showed intact spermatogenesis. Taken together, our results demonstrate that LYAR is strongly preferred in male germ cells, but has a dispensable role in spermatogenesis and fertility.

Impaired sperm aggregation in Adam2 and Adam3 null mice.

Previous studies have shown that sperm from Adam2 and Adam3 knockout mice have defective migration in the female reproductive tracts and cannot bind to the egg's zona pellucida (ZP), which leads to infertility. Here, we report that Adam2 and Adam3 knockout sperm have severely impaired sperm aggregation and that this defect is not restored over time during in vitro cultivation, suggesting the requirement of ADAM2 and ADAM3 in normal sperm association.

Expression analysis of the Adam21 gene in mouse testis.

A number of members belonging to a disintegrin and metalloprotease (ADAM) family of cell surface proteins, including ADAM21, are expressed specifically or predominantly in the mammalian testis. Here, we investigated the transcriptional characteristics of the Adam21 gene. We found that Adam21 produces two types of transcripts with different developmental stages and cellular localizations. One type comprises germ cell-specific transcripts with both exons 1 and 2, while the other type corresponds to exon 2 and is expressed in testicular somatic cells. Further, regulatory and promoter regions responsible for the expression of Adam21 in testicular somatic cells were investigated using an in silico sequence analysis and an in vitro transient transfection assay. We identified an essential promoter and mapped regulatory regions that repress the transcription of Adam21. Finally, we confirmed the expression of Adam21 at the protein level in testicular somatic cells in which the promoter of the gene was found to be active. This is the first study to provide information regarding transcriptional regulation of a testicular ADAM family member, which will aid in elucidation of the transcriptional mechanisms of other testicular Adam genes.