Biomedical subjects

Kentaro Yomogida

Publications and source records attributed to Kentaro Yomogida.

At least 19 recordsLinked to original sources

PubMed2006

Transcription factors, cAMP-responsive element modulator (CREM) and Tisp40, act in concert in postmeiotic transcriptional regulation.

We previously isolated 80 TISP (transcript induced in spermiogenesis) genes whose transcription is dramatically induced during spermiogenesis. Our analysis here of the expression of these genes in the testis of the cAMP-responsive element modulator (CREM)-null mouse revealed that 54 TISP genes are under the transcriptional regulation of CREM. One CREM-regulated gene is TISP40, which encodes a basic leucine zipper (bZip)-type transcription factor bearing a transmembrane domain that generates the two proteins Tisp40alpha and Tisp40beta. Both of these proteins function by binding to UPRE (unfolded protein-response element) but do not recognize CRE motifs. We show here that Tisp40alpha mRNA is generated under the direct transcriptional regulation of CREM. CREMtau and Tisp40 form a heterodimer, which functions through CRE but not through UPRE. Furthermore, binding ability of CREM to CRE is dramatically up-regulated by forming a heterodimer with Tisp40alphaDeltaTM, a truncated form of Tisp40alpha that lacks the transmembrane domain. We confirmed that Tisp40 and CREM actually bind to the Tisp40 promoter in vivo by chromatin immunoprecipitation assay. Finally, we demonstrate that the Tisp40DeltaTM-CREMtau heterodimer acts as a recruiter of HIRA, a histone chaperone, to CRE. Taken together, we propose that Tisp40 is an important transcriptional regulator during spermiogenesis.

Animals↗

PubMed2006

The testes-specific bZip type transcription factor Tisp40 plays a role in ER stress responses and chromatin packaging during spermiogenesis.

We previously reported that the spermatid-specific transcription factor Tisp40 functions through UPRE and CRE. To investigate Tisp40 function in vivo, we generated TISP40(-/-) mice. TISP40(-/-) mice were born at expected ratios, were healthy, and mutant males bred normally. However, the ER stress-response protein Grp78/BiP accumulated in the TISP40(-/-) testis and RAMP4 (Ribosome-associated membrane protein 4) mRNA level was up-regulated. Disruption of TISP40 caused ER stress and activation of caspase 12 but not caspase 9, leading to apoptosis of meiotic/postmeiotic germ cells. On the other hand, DAPI staining and electron microscopy revealed that epididymal sperm nuclei were abnormally relaxed in the TISP40(-/-) testis, a phenotype that was independent of the expression and maturation of transition proteins and protamines but due to abnormally retained histones. Histones localized to the cytoplasm as well as to the nucleus and were also retained in epididymal sperm. Histones H2A and H4 were dramatically up-regulated and the acetylation of H2A, H2B and H4 was also enhanced in the TISP40(-/-) testis. Taken together, we conclude that Tisp40 plays an important role in the unfolded protein response of the testis and in regulating the maturation of sperm head nuclei.

Animals↗

PubMed2005

A type I DnaJ homolog, DjA1, regulates androgen receptor signaling and spermatogenesis.

Two type I DnaJ homologs DjA1 (DNAJA1; dj2, HSDJ/hdj-2, rdj1) and DjA2 (DNAJA2; dj3, rdj2) work similarly as a cochaperone of Hsp70s in protein folding and mitochondrial protein import in vitro. To study the in vivo role of DjA1, we generated DjA1-mutant mice. Surprisingly, loss of DjA1 in mice led to severe defects in spermatogenesis that involve aberrant androgen signaling. Transplantation experiments with green fluorescent protein-labeled spermatogonia into DjA1(-/-) mice revealed a primary defect of Sertoli cells in maintaining spermiogenesis at steps 8 and 9. In Sertoli cells of DjA1(-/-) mice, the androgen receptor markedly accumulated with enhanced transcription of several androgen-responsive genes, including Pem and testin. Disruption of Sertoli-germ cell adherens junctions was also evident in DjA1(-/-) mice. Experiments with DjA1(-/-) fibroblasts and primary Sertoli cells indicated aberrant androgen receptor signaling. These results revealed a critical role of DjA1 in spermiogenesis and suggest that DjA1 and DjA2 are not functionally equivalent in vivo.

Adherens Junctions↗

PubMed2005

Tisp40, a spermatid specific bZip transcription factor, functions by binding to the unfolded protein response element via the Rip pathway.

TISP40, a mouse spermatid-specific gene, encodes a CREB/CREM family transcription factor that is predominantly expressed during spermiogenesis. We report here that TISP40 generates two types of proteins, Tisp40alpha and Tisp40beta, both of which contain a transmembrane domain and localize to the endoplasmic reticulum (ER). In contrast, mutant proteins lacking the transmembrane domain (Tisp40alpha/betaDeltaTM) primarily localize to the nucleus. Endoglycosidase H treatment shows that the C-terminus of Tisp40alpha/beta is glycosylated. Protease experiments demonstrate that Tisp40alpha/beta are Type II transmembrane proteins that are released into the nucleus by a two-step cleavage mechanism called 'regulated intramembrane proteolysis' (Rip). Unlike previously published observations, Tisp40alpha does not bind to the NF-kappaB site; instead, it specifically binds to the unfolded protein response element (UPRE). Luciferase assays reveal that Tisp40betaDeltaTM activates transcription through UPRE. Northern blot analysis shows that Tisp40alpha/betaDeltaTM proteins up-regulate EDEM (ER degradation of enhancing alpha-manosidase-like protein) mRNA. These observations unveil a novel event in mouse spermiogenesis and show that the final stage of transcriptional regulation is controlled by the Rip pathway.

Amino Acid Sequence↗

PubMed2005

Regulated expression and dynamic changes in subnuclear localization of mammalian Rad18 under normal and genotoxic conditions.

Rad18 plays a crucial role in postreplication repair in both lower eukaryotes and higher eukaryotes. However, regulation of the Rad18 expression in higher eukaryotes is largely unknown. We found that the RAD18 transcript is expressed ubiquitously in various tissues and very highly in the testis in mammals. Although human RAD18 (hRAD18) transcription levels fluctuate during the cell cycle, being maximal in the late S and minimal in the early G1, the protein levels remain constant throughout the cell cycle. Following UV-irradiation, hRAD18 transcription levels decrease significantly, but Rad18 protein levels change little. The protein levels are maintained at least in part by enhanced translation rates. hRad18 localizes in the nucleus in two forms: a diffused form and a condensed form forming nuclear dots. These nuclear dots disperse rapidly in the nucleoplasm after treatments with various genotoxic agents, resulting in an enhancement of the intranuclear Rad18 concentration of the diffused form. No de novo protein synthesis is required for this process. These results suggest that in higher eukaryotes, the maintenance and dynamic translocation of Rad18 protein is important for postreplication repair.

Animals↗

PubMed2004

Functional characterization of a mouse testicular olfactory receptor and its role in chemosensing and in regulation of sperm motility.

Although a subset of the olfactory receptor (OR) gene family is expressed in testis, neither their developmental profile nor their physiological functions have been fully characterized. Here, we show that MOR23 (a mouse OR expressed in the olfactory epithelium and testis) functions as a chemosensing receptor in mouse germ cells. In situ hybridization showed that MOR23 was expressed in round spermatids during stages VI-VIII of spermatogenesis. Lyral, a cognate ligand of MOR23, caused an increase in intracellular Ca2+ in a fraction of spermatogenic cells and spermatozoa. We also generated transgenic mice that express high levels of MOR23 in the testis and examined the response of their germ cells to lyral. The results provided evidence that lyral-induced Ca2+ increases were indeed mediated by MOR23. In a sperm accumulation assay, spermatozoa migrated towards an increasing gradient of lyral. Tracking and sperm flagellar analyses suggest that Ca2+ increases caused by MOR23 activation lead to modulation of flagellar configuration, resulting in chemotaxis. By contrast, a gradient of a cAMP analog or K8.6 solution, which elicit Ca2+ influx in spermatozoa, did not cause sperm accumulation, indicating that chemosensing and regulation of sperm motility was due to an OR-mediated local Ca2+ increase. The present studies indicate that mouse testicular ORs might play a role in chemoreception during sperm-egg communication and thereby regulate fertilization.

Animals↗

PubMed2003

Dramatic expansion of germinal stem cells by ectopically expressed human glial cell line-derived neurotrophic factor in mouse Sertoli cells.

Although the mammalian germinal stem cell (GSC) provides a good model to investigate the regulation of stem cells, the small number of these cells currently available hampers elucidation of the regulatory mechanism. Here, we show the dramatic amplification of GSCs in mouse testis following transfection of human glial cell line-derived neurotrophic factor cDNA into Sertoli cells using an efficient, in vivo electroporation technique. Transplantation analysis demonstrated not only GSC enrichment but also differentiation from stem cells into sperm. The GSC population, as estimated using a colony-formation assay, was approximately 20-fold greater than in cryptorchid testis, or approximately 500- to 1000-fold greater than in normal adult testis. This system should provide sufficient quantities of GSCs to accelerate our understanding of GSC properties, regulation mechanisms, and behavior control.

Animals↗

PubMed2003

Characterization of histone H2A.X expression in testis and specific labeling of germ cells at the commitment stage of meiosis with histone H2A.X promoter-enhanced green fluorescent protein transgene.

To study the complex molecular mechanisms of mammalian spermatogenesis, it would be useful to be able to isolate cells at each stage of differentiation, especially at the stage in which the cells switch from mitosis to meiosis. Currently, no useful marker proteins or gene promoters specific to this important stage are known. We report here a transgenic mouse line that under the control of the promoter for a histone variant, H2A.X, expressed an enhanced green fluorescent protein (EGFP) in cells at the stage of the mitosis-meiosis switch. Endogenous H2A.X is expressed in type A spermatogonia through meiotic prophase spermatocytes in testis and in some somatic cells. However, despite the fact that its expression was driven by the H2A.X promoter, the EGFP expressed in the transgenic mice specifically labeled only the intermediate spermatogonia stage through the meiotic prophase spermatocyte stage in transgenic mice containing the -600-base pair H2A.X promoter/EGFP construct. Type A spermatogonia and somatic cells of other organs were not labeled. This expression pattern made it possible to isolate living cells from the testis of the transgenic mice at the stage of the mitosis-meiosis switch in spermatogenesis using EGFP fluorescence.

Animals↗

PubMed2003

Novel actin-like proteins T-ACTIN 1 and T-ACTIN 2 are differentially expressed in the cytoplasm and nucleus of mouse haploid germ cells.

We isolated cDNA clones for the novel actin-like proteins T-ACTIN 1 and T-ACTIN 2, which are expressed specifically in the mouse testis. These clones were from a subtracted cDNA library that was enriched for haploid germ cell-specific cDNAs. The mRNA sizes and deduced molecular masses of t-actin 1/mACTl7b and t-actin 2/mACTl7a were 2.2 kilobases (kb) and 1.8 kb, and Mr 43.1 x 10(3) and Mr 47.2 x 10(3), respectively. The two deduced amino acid sequences had 60% homology, and they had approximately 40% homology with other actins. The T-ACTINs contained some of the conserved regions seen in other actins. Although the cellular locations of these two proteins are quite different (T-ACTIN-1 was found in the cytoplasm and T-ACTIN-2 was located in the nucleus), the expression of their proteins and mRNAs is controlled during development and limited during spermiogenesis. In contrast, only T-ACTIN-2 was present in sperm heads and tails. These results suggest that T-ACTINs play important roles in sperm function and in the specific morphogenesis of spermatozoa during spermiogenesis.

Actins↗

PubMed2003

Isolation and characterization of a novel cDNA encoding a DNA-binding protein (Hils1) specifically expressed in testicular haploid germ cells.

A cDNA encoding a protein homologous with histone H1 has been cloned from a haploid germ cell specific cDNA library. Deduced amino acid sequence (170 amino acids) showed 40% identity with histone H1 globular domain. Messenger RNA of the gene was observed exclusively in the testis, and was accumulated after post-natal day 23. Western blotting analysis showed that the protein encoded by this gene is about 19 kDa in molecular weight, and it was exclusively recovered from the nuclei of testicular germ cells. Immunohistochemical analysis showed that the protein was localized to the nuclei of round and elongating spermatids, consistent with the results of immunoblot analysis. Thus, the gene product was named Hils1 (histone H1 like protein in spermatids 1). In vitro DNA-binding experiments using DNA-cellulose mini-columns showed that Hils1 was able to bind to both double and single stranded-DNAs in a non-sequence-specific manner. These findings suggest that Hils1 may play an important role in the structural changes of spermatid nuclei, such as nuclear condensation, and gene regulation of haploid germ cell differentiation.

Amino Acid Sequence↗

PubMed2003

GATA-1 testis activation region is essential for Sertoli cell-specific expression of GATA-1 gene in transgenic mouse.

BACKGROUND: The erythroid transcription factor GATA-1 is also expressed in Sertoli cells of the testis. The testicular expression of GATA-1 is regulated in a developmental and spermatogenic stage-specific manner. To further clarify the regulatory mechanisms of testicular GATA-1 gene expression, we carried out transgenic reporter gene expression analyses. RESULTS: We found that GATA-1 expression in Sertoli cells is markedly decreased concomitant with the emergence of elongated spermatids in the seminiferous tubules. Transgenic reporter mouse analyses revealed that a 15 kb GATA-1 genomic region is sufficient to recapitulate the gene expression profile in Sertoli cells. While the GATA-1 haematopoietic enhancer and the proximal first exon are included within the 15 kb genomic region, these regulatory elements are not essential for GATA-1 expression in Sertoli cells. Further analyses using deletion constructs revealed that a 1.5 kb region 5' to the GATA-1 haematopoietic enhancer is essential for gene expression in Sertoli cells and this region is referred to as the GATA-1 testis activation region. CONCLUSION: These results thus demonstrated that the GATA-1 testis activation region is essential for Sertoli cell-specific expression of GATA-1 gene. The 15 kb genomic region is applicable and useful for the expression vector system specific for adult Sertoli cells in stage VII to IX.

Animals↗

PubMed2003

Expression of Tom34 splicing isoforms in mouse testis and knockout of Tom34 in mice.

The 34-kDa translocase of the outer mitochondrial membrane (Tom34) is a putative mammalian-specific factor involved in protein import into mitochondria. We analyzed the genomic sequence of the mouse Tom34 gene and found it has two alternative initial exons. Using reverse transcription and the polymerase chain reaction (RT-PCR), we found that these two mRNAs differs only in the 5'-proximal sequences corresponding to the two initial exons (exon 1a and 1b). Tom34 mRNA with exon 1a (Tom34a) is expressed ubiquitously, while that with exon 1b (Tom34b) is expressed only in mature testicular germ cells. To explore the in vivo function of Tom34 proteins, we generated Tom34-deficient mice by targeted disruption. The Tom34(-/-) mice were viable and grew normally and had a normal Mendelian inheritance pattern. Male as well as female Tom34(-/-) mice were fertile. In vitro-preprotein import into isolated mitochondria showed no apparent difference between Tom34(-/-) and wild-type mice. These results indicate that Tom34 is dispensable for mouse growth and development under optimal conditions.

Alternative Splicing↗

PubMed2003

Mouse germ cell-less as an essential component for nuclear integrity.

A mouse homologue of the Drosophila melanogaster germ cell-less (mgcl-1) gene is expressed ubiquitously, and its gene product is localized to the nuclear envelope based on its binding to LAP2 beta (lamina-associated polypeptide 2 beta). To elucidate the role of mgcl-1, we analyzed two mutant mouse lines that lacked mgcl-1 gene expression. Abnormal nuclear morphologies that were probably due to impaired nuclear envelope integrity were observed in the liver, exocrine pancreas, and testis. In particular, functional abnormalities were observed in testis in which the highest expression of mgcl-1 was detected. Fertility was significantly impaired in mgcl-1-null male mice, probably as a result of severe morphological abnormalities in the sperm. Electron microscopic observations showed insufficient chromatin condensation and abnormal acrosome structures in mgcl-1-null sperm. In addition, the expression patterns of transition proteins and protamines, both of which are essential for chromatin remodeling during spermatogenesis, were aberrant. Considering that the first abnormality during the process of spermatogenesis was abnormal nuclear envelope structure in spermatocytes, the mgcl-1 gene product appears to be essential for appropriate nuclear-lamina organization, which in turn is essential for normal sperm morphogenesis and chromatin remodeling.

Animals↗

PubMed2003

Conditional loss of PTEN leads to testicular teratoma and enhances embryonic germ cell production.

The tumor suppressor gene PTEN, which is frequently mutated in human cancers, encodes a lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3] and antagonizes phosphatidylinositol 3 kinase. Primordial germ cells (PGCs), which are the embryonic precursors of gametes, are the source of testicular teratoma. To elucidate the intracellular signaling mechanisms that underlie germ cell differentiation and proliferation, we have generated mice with a PGC-specific deletion of the Pten gene. Male mice that lacked PTEN exhibited bilateral testicular teratoma, which resulted from impaired mitotic arrest and outgrowth of cells with immature characters. Experiments with PTEN-null PGCs in culture revealed that these cells had greater proliferative capacity and enhanced pluripotent embryonic germ (EG) cell colony formation. PTEN appears to be essential for germ cell differentiation and an important factor in testicular germ cell tumor formation.

Animals↗

PubMed2002

Type XXVI collagen, a new member of the collagen family, is specifically expressed in the testis and ovary.

HSP47 is a collagen-specific molecular chaperone that specifically recognizes and binds to the triple helical domain of various types of collagens. Here we report the cloning of the entire coding region of a novel collagen-like protein by yeast two-hybrid screening of a 17.5-day whole mouse embryo cDNA library using HSP47 as a bait. The cDNA of this protein and its deduced amino acid sequence are 2,690 bp and 438 amino acids long, respectively. The protein contains two clusters of Gly-X-Y collagenous repeats and three noncollagenous domains. Northern blot analysis showed that its mRNA is specifically expressed in the testis and ovary in adult tissues and that expression in these tissues is highest in the neonate. Biochemical characterization of this protein revealed that its proline residues are hydroxylated, it undergoes N-linked glycosylation, it forms trimers, and it is secreted in vitro. Immunohistochemical studies showed that the myoid cells and the pre-theca cells synthesized it in the testis and ovary, respectively, resulting in the accumulation of this protein in the extracellular spaces of these organs. These observations suggest that this protein is a new member of the collagen protein family. We thus designated this protein as type XXVI collagen.

Aging↗

PubMed2002

Homeostatic regulation of germinal stem cell proliferation by the GDNF/FSH pathway.

Stem cell regulatory mechanisms are difficult to study because self-renewal and production of differentiated progeny, which are both strictly controlled, occur simultaneously in these cells. To focus on the self-renewal mechanism alone, we investigated the behavior of germinal stem cells (GSCs) in progeny-deficient testes with defective GSC differentiation. In these testes, we found that the proliferation of undifferentiated spermatogonia, some of which are GSCs, was accelerated by high concentrations of glial cell line-derived neurotrophic factor (GDNF). Furthermore, we found that follicle-stimulating hormone (FSH) stimulation via homeostatic control was one of the major regulators of GDNF concentration. These results suggest that in mammalian testes, GSC proliferation and population size are regulated homeostatically by the GDNF/FSH pathway.

Animals↗

PubMed2002

Utility of immunohistochemical detection of prostate-specific Ets for the diagnosis of benign and malignant prostatic epithelial lesions.

BACKGROUND: Human prostate-specific Ets (hPSE) belongs to the Ets family. It regulates the proliferation, differentiation, and development of prostate epithelial cells. A recent study showed that hPSE can be detected in normal glands but not in cell lines established from prostate cancer (PCA), suggesting a translational disorder of hPSE from mRNA to protein in PCA. Immunohistochemical detection of hPSE could therefore be another method of differential diagnosis of PCA from other proliferative conditions in the prostate. METHODS: An immunohistochemical detection of hPSE was carried out on the whole mounted prostatectomy specimen obtained from 19 cases with PCA. RESULTS: Basal and secretory luminar cells showed a diffuse cytoplasmic staining for hPSE in normal glands, hyperplastic glands, and prostate intraepithelial neoplasia lesions. Whereas approximately 30% of PCA lesions showed a negative staining for hPSE, the positive rate for hPSE between PCA and benign glands or prostate intraepithelial neoplasia (PIN), was statistically significant (P < 0.05). Staining intensities in normal glands, hyperplastic glands, and PIN lesions were similar, but generally stronger than those in PCA lesions. CONCLUSIONS: Negative immunoreactivity for hPSE strongly suggests malignancy in the prostate glands. Decreased immunoreactivities of glands for hPSE could suggest PCA.

Aged↗

PubMed2002

Electroporated transgene-rescued spermatogenesis in infertile mutant mice with a sertoli cell defect.

The molecular basis of most human male infertility arising from spermatogenesis disruption is poorly understood because of a lack of useful investigation systems. To study the roles of the supporting Sertoli cells in mammalian spermatogenesis, we improved an electroporation technique for seminiferous tubules in vivo. Because Sertoli cells barely proliferate in mature testis, linear transgenes are not incorporated into the genome and quickly degrade. However, circular expression vector is stably expressed in Sertoli cells for a long period. By electrotransformation of a complete cDNA, we rescued defective spermatogenesis in infertile Sl(17H)/Sl(17H) mutant mice with partial dysfunction of stem cell factor in Sertoli cells. Application of this gene transfer system will facilitate both the understanding of spermatogenesis and the development of new gene therapies for human male infertility.

Actins↗