Sora Jin

Comprehensive Analysis of Reproductive ADAMs: Relationship of ADAM4 and ADAM6 with an ADAM Complex Required for Fertilization in Mice

Abstract 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.

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.

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.

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. J. Cell. Physiol. 226: 1186–1195, 2011.

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

ABSTRACT 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.

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 eggs 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.

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.

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.