Biomedical subjects

Yoshitake Nishimune

Publications and source records attributed to Yoshitake Nishimune.

At least 37 records · Page 2Linked to original sources

PubMed2004

Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function.

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.

Animals↗

PubMed2003

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development.

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.

Animals↗

PubMed2003

Control of mouse hils1 gene expression during spermatogenesis: identification of regulatory element by transgenic mouse.

Histone H1-like protein in spermatids 1 (Hils1) is a testis- specific histone H1-like protein exclusively expressed in haploid spermatids and should be involved in chromatin remodeling during mouse spermatogenesis. Spatial and temporal regulation of the hils1 gene expression would be critical for the formation of functional sperm, controlled at both transcriptional and translational levels. Previously, we reported that transcripts of the hils1 gene are exclusively expressed in mouse testis from 23 days of age whereas the Hils1 protein is not detected until 28 days of age, suggesting that hils1 is a member of a class of translationally regulated genes. By analyzing transgenic mice, we could demonstrate that 318-base pair (bp) 5'-proximal region corresponding to the first 70-bp proximal TATA-less promoter, and 248 bp of 5'-untranslated region is sufficient to confer testis- and spermatid-specific transcription as well as posttranscriptional control of the mouse hils1 gene in vivo.

Animals↗

PubMed2003

Methylation of CpG dinucleotides in the open reading frame of a testicular germ cell-specific intronless gene, Tact1/Actl7b, represses its expression in somatic cells.

Methylation of CpG islands spanning promoter regions is associated with control of gene expression. However, it is considered that methylation of exonic CpG islands without promoter is not related to gene expression, because such exonic CpG islands are usually distant from the promoter. Whether methylation of exonic CpG islands near the promoter, as in the case of a CpG-rich intronless gene, causes repression of the promoter remains unknown. To gain insight into this issue, we investigated the distribution and methylation status of CpG dinucleotides in the mouse Tact1/Actl7b gene, which is intronless and expressed exclusively in testicular germ cells. The region upstream to the gene was poor in CpG, with CpG dinucleotides absent from the core promoter. However, a CpG island was found inside the open reading frame (ORF). Analysis of the methylation status of the Tact1/Actl7b gene including the 5'-flanking area demonstrated that all CpG sites were methylated in somatic cells, whereas these sites were unmethylated in the Tact1/Actl7b-positive testis. Trans fection experiments with in vitro-methylated constructs indicated that methylation of the ORF but not 5' upstream repressed Tact1/Actl7b promoter activity in somatic cells. Similar effects of ORF methylation on the promoter activity were observed in testicular germ cells. These are the first results indicating that methylation of the CpG island in the ORF represses its promoter in somatic cells and demethylation is necessary for gene expression in spermatogenic cells.

Actins↗

PubMed2003

Haprin, a novel haploid germ cell-specific RING finger protein involved in the acrosome reaction.

The acrosome reaction (i.e. the exocytosis of the sperm vesicle) is a prerequisite for fertilization, but its molecular mechanism is largely unknown. We have identified a cDNA clone for a gene named haprin, which encodes a haploid germ cell-specific RING finger protein. This protein is a novel member of the RBCC (RING finger, B-box type zinc finger, and coiled-coil domain) motif family that has roles in several cellular processes, such as exocytosis. It is transcribed exclusively in testicular germ cells after meiotic division. Western blot and immunohistochemical analyses showed the molecular weight of Haprin protein to be Mr approximately 82,000. It was localized in the acrosomal region of elongated spermatids and mature sperm and was not present in acrosome-reacted sperm. The specific antibody against the RING finger domain of Haprin inhibited the acrosome reaction in permeabilized sperm. These results indicated that the novel RBCC protein Haprin plays a key role in the acrosome reaction and fertilization.

Acrosome↗

PubMed2003

Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis.

Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/Wv mutant mouse testis. In the present study, some of the W/Wv mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/Wv mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/Wv spermatogonia into the seminiferous tubules of normal SCF (W/Wv) or SCF mutant (Sl/Sld) mice demonstrated that the W/Wv spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/Wv testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/Wv testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.

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

A novel protein phosphatase 2C family member (PP2Czeta) is able to associate with ubiquitin conjugating enzyme 9.

In this study we have cloned a novel member of mouse protein phosphatase 2C family, PP2Czeta, which is composed of 507 amino acids and has a unique N-terminal region. The overall similarity of the amino acid sequence between PP2Czeta and PP2Calpha was 22%. On Northern blot analysis PP2Czeta was found to be expressed specifically in the testicular germ cells. PP2Czeta expressed in COS7 cells was able to associate with ubiquitin conjugating enzyme 9 (UBC9) and the association was enhanced by co-expression of small ubiquitin-related modifier-1 (SUMO-1), suggesting that PP2Czeta exhibits its specific role through its SUMO-induced recruitment to UBC9.

Amino Acid Sequence↗

PubMed2003

Functional analysis of the p53 gene in apoptosis induced by heat stress or loss of stem cell factor signaling in mouse male germ cells.

Apoptosis plays an important role in controlling germ cell numbers and restricting abnormal cell proliferation during spermatogenesis. The tumor suppressor protein, p53, is highly expressed in the testis, and is known to be involved in apoptosis, which suggests that it is one of the major causes of germ cell loss in the testis. Mice that are c-kit/SCF mutant (Sl/Sld) and cryptorchid show similar testicular phenotypes; they carry undifferentiated spermatogonia and Sertoli cells in their seminiferous tubules. To investigate the role of p53-dependent apoptosis in infertile testes, we transplanted p53-deficient spermatogonia that were labeled with enhanced green fluorescence protein into cryptorchid and Sl/Sld testes. In cryptorchid testes, transplanted p53-deficient spermatogonia differentiated into spermatocytes, but not into haploid spermatids. In contrast, no differentiated germ cells were observed in Sl/Sld mutant testes. These results indicate that the mechanism of germ cell loss in the c-kit/SCF mutant is not dependent on p53, whereas the apoptotic mechanism in the cryptorchid testis is quite different (i.e., although the early stage of differentiation of spermatogonia and the meiotic prophase is dependent on p53-mediated apoptosis, the later stage of spermatids is not).

Animals↗

PubMed2003

Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes, Tact1 and Tact2.

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.

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

The role of the c-kit receptor in the regenerative differentiation of rat Leydig cells.

To determine the physiological role of the c-kit receptor, which is highly expressed in Leydig cells, the regenerative differentiation of Leydig cells was studied following transient degeneration induced by ethane dimethyl sulphonate (EDS) in c-kit-deficient mutant rats (Ws/Ws). EDS caused the destruction of Leydig cells; their functional recovery was evaluated by the weight change of the target organs of androgens, which occurred at the same rate in Ws/Ws and wild-type rats. These results indicate that the tyrosine kinase activity of the c-kit receptor does not play an essential role in the regenerative differentiation of Leydig cells.

Androgens↗

PubMed2003

Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations.

Although various genetic factors have been implicated in human male infertility, the causative genes for the different types of idiopathic male infertility have not been elucidated. Protamines, which are the major DNA-binding proteins in the sperm nucleus, package the DNA into the sperm head. Analysis of the human protamine-1 (PRM1) and -2 (PRM2) gene sequences in 226 sterile male patients and in 270 proven-fertile male volunteers revealed four single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and one SNP in the PRM2 gene, which produced translation termination. We also observed one SNP in the 3' non-coding region of the PRM1 gene, and two SNPs within the intron of the PRM2 gene. The prevalence of these SNPs was similar in both infertile patients and in proven-fertile volunteers, except that the c248t alteration in the PRM2 gene induced a nonsense codon under conditions of heterozygosity in one infertile patient. Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene that induces translation termination may result in male infertility due to haploinsufficiency of PRM2.

Amino Acid Sequence↗

PubMed2003

Molecular cloning and characterization of the human orthologue of the oppo 1 gene encoding a sperm tail protein.

We report here the molecular cloning and characterization of a human orthologue of oppo 1, a mouse gene encoding a male germ-cell-specific sperm tail protein, and the organization of its genomic structure. The mRNA of the human oppo 1 gene (h-oppo 1) was expressed exclusively in the testis, and the 30 kDa protein encoded by the mRNA was detected in human testis and sperm. Immunohistochemical analyses showed that human OPPO 1 protein was localized in the flagellae of ejaculated sperm. A human genomic DNA database search indicated that the h-oppo 1 gene mapped to chromosome 17. The genomic structure of h-oppo 1 showed differences in exon/intron usage, the sequence of the 5'-flanking region, and the first intron was rich in Alu repeats as compared with the mouse oppo 1 gene. Comparison of the two genomic sequences indicated that human oppo 1 has evolved independently, resulting in substantial differences in the genomic structure after the human-mouse split, whereas the sequence of the basic functional unit of the oppo 1 gene seems to have been relatively well conserved.

Amino Acid Sequence↗

PubMed2003

In vitro and in vivo gene delivery by recombinant baculoviruses.

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy.

Animals↗

PubMed2002

Structure and promoter activity of the gene encoding ornithine decarboxylase antizyme expressed exclusively in haploid germ cells in testis (OAZt/Oaz3).

Ornithine decarboxylase antizyme 1 and 2 (OAZ1 and OAZ2) are expressed ubiquitously, and control the intracellular concentration of polyamines. Their testicular isoform, OAZt/Oaz3, is specifically expressed in differentiated haploid germ cells. We have identified and characterized the gene encoding OAZt in mice. The mouse OAZt gene contains, as does the human ortholog and paralogs, five exons and four introns. Comparison of the mouse OAZt with the human ortholog gene revealed that exon sizes are identical and nucleotide sequences in exons are highly homologous (83% identity). The major transcriptional start site was determined by primer extension assay. Promoter activity was confirmed by transgenic mouse assays, using the upstream region of the mouse OAZt gene fused to a EGFP reporter gene. The OAZt essential promoter located between -133 and +242, has two CREs and an Inr, and lacks a TATA box. These elements are conserved in the human ortholog but not in the paralogs, indicating that such a short upstream region including two CREs and Inr is sufficient to drive endogenous OAZt mRNA expression in the haploid testicular germ cells.

3T3 Cells↗