Cecil Han

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.

ADAM7 Is Associated with Epididymosomes and Integrated into Sperm Plasma Membrane

During epididymal transit, mammalian sperm acquire selected proteins secreted by the epididymis. We previously showed that a disintegrin and metalloprotease (ADAM) 7 is expressed specifically in the epididymis and transferred to the sperm surface during epididymal transit. Here, we show that mouse ADAM7 secreted to the epididymal lumen is associated with membranous vesicles known as epididymosomes. Furthermore, we found that ADAM7 can be transferred directly from epididymal vesicles to sperm and that it is an integral plasma membrane protein in sperm. Thus, our study provides new information regarding the unique mode of secretion and interaction of ADAM7 during the epididymis-to-sperm transfer process.

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.

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

BackgroundThe primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes.ResultsWe 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.ConclusionWe 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.

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

BackgroundMaturation 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.ResultsWe 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 β-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.ConclusionWe 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.

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.

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.

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

BackgroundSpermatogenesis 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.MethodsPolyclonal 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.ResultsThe 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).ConclusionWe 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.