Targeted ablation of the histidine-rich Ca(2+)-binding protein (HRC) gene is associated with abnormal SR Ca(2+)-cycling and severe pathology under pressure-overload stress.

The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.

Crystal structure of Sus scrofa quinolinate phosphoribosyltransferase in complex with nicotinate mononucleotide.

We have determined the crystal structure of porcine quinolinate phosphoribosyltransferase (QAPRTase) in complex with nicotinate mononucleotide (NAMN), which is the first crystal structure of a mammalian QAPRTase with its reaction product. The structure was determined from protein obtained from the porcine kidney. Because the full protein sequence of porcine QAPRTase was not available in either protein or nucleotide databases, cDNA was synthesized using reverse transcriptase-polymerase chain reaction to determine the porcine QAPRTase amino acid sequence. The crystal structure revealed that porcine QAPRTases have a hexameric structure that is similar to other eukaryotic QAPRTases, such as the human and yeast enzymes. However, the interaction between NAMN and porcine QAPRTase was different from the interaction found in prokaryotic enzymes, such as those of Helicobacter pylori and Mycobacterium tuberculosis. The crystal structure of porcine QAPRTase in complex with NAMN provides a structural framework for understanding the unique properties of the mammalian QAPRTase active site and designing new antibiotics that are selective for the QAPRTases of pathogenic bacteria, such as H. pylori and M. tuberculosis.

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.

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.

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.

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.

Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.

A Disintegrin And Metalloprotease (ADAM) family members expressed in male reproductive tissues are divided phylogenetically into three major groups. In the present study, we analyzed six ADAMs in one of the groups (ADAMs 4, 6, 24, 26, 29, and 30) of which function is largely unknown. Our results showed that most of the ADAMs undergo unique processing during sperm maturation and are located at the surface of sperm head. We found that the levels of ADAM4 and ADAM6 are dramatically reduced in Adam2 and Adam3 knockout sperm defective in various fertilization processes. We observed premature processing of ADAM4 in the Adam3-null mice. Furthermore, we obtained a result showing complex formation of ADAM6 with ADAM2 and ADAM3 in testis. Taken together, these results disclose involvement of ADAM4 and ADAM6 in a reproductive ADAM system that functions in fertilization.

A novel germ cell-specific protein, SHIP1, forms a complex with chromatin remodeling activity during spermatogenesis.

To determine the mechanisms of spermatogenesis, it is essential to identify and characterize germ cell-specific genes. Here we describe a protein encoded by a novel germ cell-specific gene, Mm.290718/ZFP541, identified from the mouse spermatocyte UniGene library. The protein contains specific motifs and domains potentially involved in DNA binding and chromatin reorganization. An antibody against Mm.290718/ZFP541 revealed the existence of the protein in testicular spermatogenic cells (159 kDa) but not testicular and mature sperm. Immunostaining analysis of cells at various stages of spermatogenesis consistently showed that the protein is present in spermatocytes and round spermatids only. Transfection assays and immunofluorescence studies indicate that the protein is localized specifically in the nucleus. Proteomic analyses performed to explore the functional characteristics of Mm.290718/ZFP541 showed that the protein forms a unique complex. Other major components of the complex included histone deacetylase 1 (HDAC1) and heat-shock protein A2. Disappearance of Mm.290718/ZFP541 was highly correlated with hyperacetylation in spermatids during spermatogenesis, and specific domains of the protein were involved in the regulation of interactions and nuclear localization of HDAC1. Furthermore, we found that premature hyperacetylation, induced by an HDAC inhibitor, is associated with an alteration in the integrity of Mm.290718/ZFP541 in spermatogenic cells. Our results collectively suggest that the Mm.290718/ZFP541 complex is implicated in chromatin remodeling during spermatogenesis, and we provide further information on the previously unknown molecular mechanism. Consequently, we re-designate Mm.290718/ZFP541 as "SHIP1" representing spermatogenic cell HDAC-interacting protein 1.

Characterization of eight novel proteins with male germ cell-specific expression in mouse.

BACKGROUND: Spermatogenesis and fertilization are highly unique processes. Discovery and characterization of germ cell-specific genes are important for the understanding of these reproductive processes. We investigated eight proteins encoded by novel spermatogenic cell-specific genes previously identified from the mouse round spermatid UniGene library. METHODS: Polyclonal antibodies were generated against the novel proteins and western blot analysis was performed with various protein samples. Germ cell specificity was investigated using testes from germ cell-less mutant mice. Developmental expression pattern was examined in testicular germ cells, testicular sperm and mature sperm. Subcellular localization was assessed by cell surface biotin labeling and trypsinization. Protein localization and properties in sperm were investigated by separation of head and tail fractions, and extractabilities by a non-ionic detergent and urea. RESULTS: The authenticity of the eight novel proteins and their specificity to spermatogenic cells were confirmed. In examining the developmental expression patterns, we found the presence of four proteins only in testicular germ cells, a single protein in testicular germ cells and testicular sperm, and three proteins in the testicular stages and mature sperm from the epididymis. Further analysis of the three proteins present in sperm disclosed that one is located at the surface of the acrosomal region and the other two are associated with cytoskeletal structures in the sperm flagellum. We name the genes for these sperm proteins Shsp1 (Sperm head surface protein 1), Sfap1 (Sperm flagellum associated protein 1) and Sfap2 (Sperm flagellum associated protein 2). CONCLUSION: We analyzed eight novel germ cell-specific proteins, providing new and inclusive information about their developmental and cellular characteristics. Our findings will facilitate future investigation into the biological roles of these novel proteins in spermatogenesis and sperm functions.

Identification of mouse heart transcriptomic network sensitive to various heart diseases.

Exploring biological systems from highly complex datasets is an important task for systems biology. The present study examined co-expression dynamics of mouse heart transcriptome by spectral graph clustering (SGC) to identify a heart transcriptomic network. SGC of microarray data produced 17 classified biological conditions (called condition spectrum, CS) and co-expression patterns by generating bi-clusters. The results showed dynamic co-expression patterns with a modular structure enriched in heart-related CS (CS-1 and -13) containing abundant heart-related microarray data. Consequently, a mouse heart transcriptomic network was constructed by clique analysis from the gene clusters exclusively present in the heart-related CS; 31 cliques were used for constructing the network. The participating genes in the network were closely associated with important cardiac functions (e. g., development, lipid and glycogen metabolisms). Online Mendelian Inheritance in Man (OMIM) database indicates that mutations of the genes in the network induced serious heart diseases. Many of the tested genes in the network showed significantly altered gene expression in an animal model of hypertrophy. The results suggest that the present approach is critical for constructing a heart-related transcriptomic network and for deducing important genes involved in the pathogenesis of various heart diseases.

Differentially expressed genes implicated in unexplained recurrent spontaneous abortion.

Recurrent spontaneous abortion occurs in approximately 3% of women with diagnosed pregnancies. The etiology in approximately 40% of recurrent spontaneous abortion is unexplained. To elucidate unexplained recurrent spontaneous abortion at the molecular level, we systemically identified differentially expressed genes during implantation window period in unexplained recurrent spontaneous abortion and characterized their functions in a human endometrial cell line. Expression levels of implantation-related genes selected from previously reported, various microarray data were determined to identify differentially expressed genes between normal fertile and unexplained recurrent spontaneous abortion subjects by real-time quantitative RT-PCR. Of 29 implantation-related genes, the transcript levels of cellular retinoic acid binding protein 2 and olfactomedin 1 were higher, whereas that of complement component 4 binding protein alpha was lower in subjects with unexplained recurrent spontaneous abortion, compared to normal fertile subjects. A correlation was evident between the transcript and protein levels of complement component 4 binding protein alpha and cellular retinoic acid binding protein 2. Expression of cellular retinoic acid binding protein 2 was positively correlated with retinoic acid-related genes in normal fertile subjects, but no significant association was observed in unexplained recurrent spontaneous abortion subjects. In relation to complement component 4 binding protein alpha, C5a receptor protein level was significantly higher in subjects with unexplained recurrent spontaneous abortion. Stable expression of cellular retinoic acid binding protein 2 and olfactomedin 1 in a human endometrial cell line inhibited cell growth and induced cell accumulation in the S and G(2)-M phase fractions, but did not trigger apoptosis. This study represents the first systematic identification of differentially expressed genes in unexplained recurrent spontaneous abortion. Defective cell growth by the differentially expressed genes suggests their implication in implantation failure in women with unexplained recurrent spontaneous abortion.

Integrative characterization of germ cell-specific genes from mouse spermatocyte UniGene library.

BACKGROUND: The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes. RESULTS: We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein. CONCLUSION: We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.

Comprehensive identification and characterization of novel cardiac genes in mouse.

Comprehensive understanding of the molecular and physiological events occurring in cardiac muscle requires identification of unknown genes expressed in this tissue. We analyzed the mouse cardiac muscle UniGene library containing 827 gene-oriented transcript clusters, predicting that 19% of these genes are unknown. We systematically identified 15 authentic novel genes abundantly expressed in cardiac muscle. Northern blot analysis revealed transcriptional characteristics of the genes, such as transcript size and presence of isoforms. Transfection assays performed using various cell lines including mouse cardiac muscle cells provided information on the cellular characteristics of the novel proteins. Using correlation analysis, we identified co-regulated genes from previously reported microarray data sets. Our in silico and in vitro data suggest that a number of the novel genes are implicated in calcium metabolism, mitochondrial functions and gene transcription. In particular, we obtained new and direct evidence that one of the novel proteins is a calcium-binding protein. Taken together, we identified and characterized a number of novel cardiac genes by integrative approach. Our inclusive data establish a firm basis for future investigation into the cardiac gene network and functions of these genes.

Systematic identification and integrative analysis of novel genes expressed specifically or predominantly in mouse epididymis.

BACKGROUND: Maturation of spermatozoa, including development of motility and the ability to fertilize the oocyte, occurs during transit through the microenvironment of the epididymis. Comprehensive understanding of sperm maturation requires identification and characterization of unique genes expressed in the epididymis. RESULTS: We systematically identified 32 novel genes with epididymis-specific or -predominant expression in the mouse epididymis UniGene library, containing 1505 gene-oriented transcript clusters, by in silico and in vitro analyses. The Northern blot analysis revealed various characteristics of the genes at the transcript level, such as expression level, size and the presence of isoform. We found that expression of the half of the genes is regulated by androgens. Further expression analyses demonstrated that the novel genes are region-specific and developmentally regulated. Computational analysis showed that 15 of the genes lack human orthologues, suggesting their implication in male reproduction unique to the mouse. A number of the novel genes are putative epididymal protease inhibitors or beta-defensins. We also found that six of the genes have secretory activity, indicating that they may interact with sperm and have functional roles in sperm maturation. CONCLUSION: We identified and characterized 32 novel epididymis-specific or -predominant genes by an integrative approach. Our study is unique in the aspect of systematic identification of novel epididymal genes and should be a firm basis for future investigation into molecular mechanisms underlying sperm maturation in the epididymis.