ESE-1 inhibits the invasion of oral squamous cell carcinoma in conjunction with MMP-9 suppression.

OBJECTIVES: Matrix metalloproteinases (MMPs) regulated by ets transcription factors facilitate carcinoma cell invasion. An ets family member, ESE-1, is expressed specifically in epithelial tissues, but its association with MMPs is obscure. In this study, we investigated whether ESE-1 regulates invasion of oral squamous cell carcinoma (SCC) via transcriptional activity of MMP-9. METHODS: HSC-3 and KB were used as human oral SCC lines. The expression of ESE-1 and MMP-9 was detected by in situ hybridization and immunohistochemistry. Invasion assay, gelatin zymography and Northern blotting were used to detect the invasion activity, the gelatinolytic activity and the expression of MMP-9 in the ESE-1 transfectants. Luciferase assays and mutation analysis were used for the transcriptional analysis of MMP-9 promoter region by ESE-1. RESULTS: ESE-1 was expressed in the intermediate layer but not in the invasive area, in which MMP-9 was expressed, in the oral SCC tissues. ESE-1 suppressed invasion activity and 92 kDa gelatinolytic activity in HSC-3 as a result of transfection. ESE-1 regulates MMP-9 expression in a negative manner and the ets binding site on the MMP-9 promoter contributed to suppression by ESE-1. CONCLUSIONS: These findings indicate that ESE-1 negatively regulates the invasion of oral SCC via transcriptional suppression of MMP-9.

In vivo and in vitro constant expression of GATA-4 in mouse postnatal Sertoli cells.

In the mammalian postnatal testis, the biochemical and structural features of Sertoli cells change, depending on developmental stage and spermatogenic cycle, to support efficient spermatogenesis. Consequently, basic transcription factors that determine fundamental properties should be strictly maintained in postnatal Sertoli cells. We have confirmed that GATA-4 expression is kept at a constant level in mouse Sertoli cells during postnatal development, and is also maintained at a constant level in primary cultures, independent of treatment with hormones or the addition of germ cell fractions. In transient transfection assays with the testicular cell line TM3, established from Leydig cells, GATA-4 induced several Sertoli cell-specific genes. In the Sertoli cell line TM4, and in Sertoli cells in primary culture, GATA-4 slightly up-regulated these genes. These results suggest that GATA-4 plays an important role in the regulation of Sertoli cell function, and is exactly regulated in these cells.

[Efficacy of pilsicainide for the long-term prevention of paroxysmal atrial fibrillation: analysis based on the time of onset].

OBJECTIVES: This study evaluated the efficacy of long-term pilsicainide therapy to maintain sinus rhythm in patients with paroxysmal atrial fibrillation in terms of the time of onset. METHODS: A total of 81 patients (57 men, 24 women, mean age 65 +/- 11 years) were given pilsicainide (150 mg/day) after cardioversion. Paroxysmal atrial fibrillation was divided into three types by the time of onset: the day type (AM 7:00-PM 5:00, n = 13), the night type (PM 5:00-AM 7:00, n = 12) and the mixed type (n = 56). Mean follow-up period was 35.4 +/- 16.1 months. RESULTS: There was a higher incidence of hypertension in the day type (38.5%) and the mixed type (48.2%) than in the night type (8.3%) (p < 0.05). The periods for maintenance of sinus rhythm in the day type, the night type and the mixed type were 15.7 +/- 5.0, 9.9 +/- 2.7 and 5.7 +/- 1.3 months, respectively, with a significant difference between the day type and the mixed type (p < 0.01). Actuarial event free-rates at 1, 3, 6, 9 and 12 months were 76.9%, 69.2%, 61.5%, 53.8% and 53.8% respectively, in the day type, 83.3%, 66.7%, 58.3%, 33.3% and 33.3%, respectively, in the night type and 58.9%, 37.5%, 26.8%, 25.0% and 21.4%, respectively, in the mixed type. There was a significant difference in the rate at 12 months between the day type and the mixed type (p < 0.05). CONCLUSIONS: These results suggest that pilsicainide has a high degree of efficacy for maintaining normal sinus rhythm in patients with the day type onset of paroxysmal atrial fibrillation.

Two mouse piwi-related genes: miwi and mili.

Genes belonging to the piwi family are required for stem cell self-renewal in diverse organisms. We cloned mouse homologues of piwi by RT-PCR using degenerative primers. The deduced amino acid sequences of mouse homologues MIWI and MILI showed that each contains a well-conserved C-terminal PIWI domain and that each shares significant homology with PIWI and their human counterparts HIWI. Both miwi and mili were found in germ cells of adult testis by in situ hybridization, suggesting that these genes may function in spermatogenesis. Furthermore, mili was expressed in primordial germ cells (PGCs) of developing mouse embryos and may therefore play a role during germ cell formation. MIWI may be involved in RNA processing or translational regulation, since MIWI was found to possess RNA binding activity. Our data suggest that miwi and mili regulate spermatogenesis and primordial germ cell production.

Testosterone suppresses spermatogenesis in juvenile spermatogonial depletion (jsd ) mice.

Male juvenile spermatogonial depletion (jsd/jsd) mice are sterile because of a failure of spermatogonial differentiation. We have previously reported the recovery of spermatogonial differentiation by suppressing the levels of gonadotropins and testosterone with Nal-Glu, a GnRH antagonist. To determine whether suppression of testosterone or the gonadotropins was responsible for spermatogenic recovery, we examined the effect of supplementation of LH or FSH along with Nal-Glu treatment. Systemic administration of flutamide, an androgen receptor antagonist, was also examined. LH supplementation elevated both serum and intratesticular testosterone levels and suppressed the recovery of spermatogonial differentiation in a dose-dependent manner. Supplementation with FSH did not affect either testosterone levels or spermatogonial differentiation. Furthermore, the mice treated with flutamide showed some recovery of spermatogonial differentiation. The overall findings revealed that testosterone action mediated by androgen receptors suppressed the spermatogonial differentiation in jsd/jsd mice and suggested that spermatogonial differentiation in the jsd mutant is highly sensitive to testosterone suppression.

Nested genomic structure of haploid germ cell specific haspin gene.

The haspin gene specifically expressed in haploid germ cells encodes a unique Ser/Thr protein kinase. We have cloned a mouse haspin genomic clone using cDNA as a probe. Sequencing data showed that the haspin gene was not interrupted by introns and was bordered by appropriate direct repeat. The transcription start site of the gene was not preceded by a TATA box. The whole transcription unit was located at an intron of integrin alphaM290 gene, and transcription direction of these two genes was different. Southern blotting analysis under stringent condition showed that haspin was a single gene. Phylogenetic analysis suggested that the diversion of haspin gene from other kinase family might be very ancient: the early stage of plant-fungus-animal split.

Defect in germ cells, not in supporting cells, is the cause of male infertility in the jsd mutant mouse: proliferation of spermatogonial stem cells without differentiation.

C57BL/6 (B6)-jsd/jsd male mice are sterile because of lack of spermatogenesis. To find the cause of the deficient spermatogenesis, we have examined whether the mutation phenotype is the result of a defect in germ cells or in supporting cells using germ cell transplantation. In the seminiferous tubules of B6-jsd/jsd mutant mice, donor germ cells derived from the wild type GFP transgenic mouse (B6-+/+GFP) were able to undergo complete spermatogenesis, indicating that the juvenile spermatogonial depletion (jsd/jsd) mouse possesses normal supporting cell functions. In contrast, undifferentiated spermatogonia derived from B6-jsd/jsd mice were unable to differentiate in the seminiferous tubules of W/W v mice, even if the mutant germ cells successfully settled in the tubules. These results demonstrate that the deficiency in spermatogenesis of B6-jsd/jsd mice can be ascribed to a defect in spermatogonia but not in their supporting cell environment. Furthermore, the defect in B6-jsd/jsd spermatogonia is not in their ability to proliferate, but in their differentiation and may result from their hypersensitivity to high concentrations of androgen in the testis.

Transcription factor BACH1 is recruited to the nucleus by its novel alternative spliced isoform.

The transcription factor Bach1 is a member of a novel family of broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) basic region leucine zipper factors. Bach1 forms a heterodimer with MafK, a member of the small Maf protein family (MafF, MafG, and MafK), which recognizes the NF-E2/Maf recognition element, a cis-regulatory motif containing a 12-O-tetradecanoylphorbol-13-acetate-responsive element. Here we describe the gene structure of human BACH1, including a newly identified promoter and an alternatively RNA-spliced truncated form of BACH1, designated BACH1t, abundantly transcribed in human testis. The alternate splicing originated from the usage of a novel exon located 5.6 kilobase pairs downstream of the exon encoding the leucine zipper domain, and produced a protein that contained the conserved BTB/POZ, Cap'n collar, and basic region domains, but lacked the leucine zipper domain essential for NF-E2/Maf recognition element binding. Subcellular localization studies using green fluorescent protein as a reporter showed that full-length BACH1 localized to the cytoplasm, whereas BACH1t accumulated in the nucleus. Interestingly, coexpression of BACH1 and BACH1t demonstrated interaction between the molecules and the induction of nuclear import of BACH1. These results suggested that BACH1t recruits BACH1 to the nucleus through BTB domain-mediated interaction.

Isolation and characterization of a haploid germ cell-specific novel complementary deoxyribonucleic acid; testis-specific homologue of succinyl CoA:3-Oxo acid CoA transferase.

We have isolated a cDNA clone encoding a mouse haploid germ cell-specific protein from a subtracted cDNA library. Sequence analysis of the cDNA revealed high homology with pig and human heart succinyl CoA:3-oxo acid CoA transferase (EC 2.8.3.5), which is a key enzyme for energy metabolism of ketone bodies. The deduced protein consists of 520 amino acid residues, including glutamate 344, known to be the catalytic residue in the active site of pig heart CoA transferase and the expected mitochondrial targeting sequence enriched with Arg, Leu, and Ser in the N-terminal region. Thus, we termed this gene scot-t (testis-specific succinyl CoA:3-oxo acid CoA transferase). Northern blot analysis, in situ hybridization, and Western blot analysis demonstrated a unique expression pattern of the mRNA with rapid translation exclusively in late spermatids. The scot-t protein was detected first in elongated spermatids at step 8 or 9 as faint signals and gradually accumulated during spermiogenesis. It was also detected in the midpiece of spermatozoa by immunohistochemistry. The results suggest that the scot-t protein plays important roles in the energy metabolism of spermatozoa.

Real-time observation of transplanted 'green germ cells': proliferation and differentiation of stem cells.

To elucidate the mechanism of proliferation and differentiation of testicular germ cells, donor testicular germ cells labeled with enhanced green fluorescent protein (eGFP) were transplanted to recipient seminiferous tubules. The kinetics of colonization as well as of differentiation of the donor cells was followed in the same transplanted tubules (alive) under ultraviolet light. One week after transplantation, clusters of fluorescent cells were randomly spread as dots in the recipient seminiferous tubule, whereas non-homed cells flowed out from the testis to the epididymis. By 4 weeks after transplantation, green germ cells were observed with weak and moderate fluorescence along the recipient seminiferous tubule. By 8 weeks, proliferation and differentiation of the germ cells occurred, resulting in strong fluorescence in the middle part of the seminiferous tubule but in weak and moderate fluorescence at both terminals. The length of the fluorescent positive seminiferous tubule became longer. Detailed histological analyses of the recipient tubules indicated that the portions of the seminiferous tubule in weak, moderate, and strong fluorescence contained the spermatogonia, spermatogonia with spermatocytes, and all types of germ cells including spermatids, respectively. Thus, testicular stem cells colonized first as dots within 1 week, and then proliferated along the basement membrane of the seminiferous tubules followed by differentiation.

Identification and characterization of testis specific ornithine decarboxylase antizyme (OAZ-t) gene: expression in haploid germ cells and polyamine-induced frameshifting.

BACKGROUND: Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. However, in the testis, where polyamines such as spermidine and spermine exist in high concentrations, their roles still remains to be elucidated. RESULTS: We have cloned a testis-specific gene encoding an ornithine decarboxylase antizyme known to control intracellular concentrations of polyamines in a feedback manner. The mRNA encoding the protein named ornithine decarboxylase antizyme in testis (OAZ-t) was specifically expressed in haploid germ cells. In contrast, the mRNA level of somatic ornithine decarboxylase antizyme 1 (OAZ1) decreased markedly at the late stages of haploid germ cell differentiation. OAZ-t mRNA was first observed in 23-day-old mice, whereas the OAZ-t protein was detected much later, at 35 days after birth. Further experiments on OAZ-t revealed that polyamines were capable of inducing a frameshifting at the frameshift sequence of OAZ-t mRNA, resulting in the translation of OAZ-t, as was the case with the somatic OAZ1. Transfection of OAZ-t cDNA inactivated the ornithine decarboxylase activity in the HEK293 cells. CONCLUSIONS: Results indicate that the expression of OAZ-t is controlled at both transcriptional and translational levels, and that OAZ-t likely plays a key role in spermatogenesis by regulating the intracellular concentration of polyamines in haploid germ cells.

Molecular cloning and characterization of phosphatidylcholine transfer protein-like protein gene expressed in murine haploid germ cells.

We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF.

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.

Prostate-specific transcription factor hPSE is translated only in normal prostate epithelial cells.

We recently cloned a novel transcription factor gene, hPSE, which belongs to the Ets gene family. hPSE mRNA was expressed specifically in prostate glandular epithelial cells and also in the human prostate carcinoma cell lines PC-3 and LNCaP. On the other hand, on immunoblot analysis with anti-hPSE antiserum, hPSE protein was detected only in human prostate tissue samples and not in PC-3 or LNCaP culture cells. Immunohistochemistry and in situ hybridization analysis revealed that hPSE protein was translated in normal prostate glandular epithelial cells, but not in carcinoma cells with hPSE transcripts. These findings suggest that expression of hPSE is regulated translationally in prostate epithelial cells and that hPSE protein is a candidate for a marker distinguishing normal cells from cancer cells in the prostate. It appeared that the 5'- and 3'-untranslated regions of hPSE transcripts might be necessary for translational control of hPSE, on the basis of results of transfection analysis in non-prostate lineage cells (HEK-293) using some deletion mutants of hPSE cDNA.

Genomic analysis of male germ cell-specific actin capping protein alpha.

The Gsg3 gene which expresses specifically in haploid germ cells is a mouse testicular homolog of somatic cell type actin capping protein alpha (ACP alpha). We have obtained a mouse Gsg3 genomic clone using cDNA as a probe. Sequencing data showed that the Gsg3 gene was not interrupted by introns. The transcription initiation site of the gene was preceded not by a TATA box or GC rich promoter motifs, but by two consensus cAMP-response element (CRE) motifs at the putative position. Southern blotting analysis showed that Gsg3 is a single copy gene in the mouse, and conserved in mammals. Phylogenetic analysis showed that Gsg3 is a novel ACP alpha specific for haploid germ cells.

Molecular cloning and genomic organization of mouse homologue of Drosophila germ cell-less and its expression in germ lineage cells.

Primordial germ cells (PGCs) are founder cells of all gametes. A number of genes which control PGCs development have been identified in invertebrates, whereas such genes are by and large unelucidated in mammals. Here we describe cloning, genomic structure and expression of mouse homologue of germ cell-less (gcl) gene which is required for PGCs formation in Drosophila. The mouse gcl shows 34% identity compared with Drosophila gcl protein and contains BTB/POZ domain. The gcl gene consists of 14 exons and spans more than 50 kb. The CpG islands are found around exon 1 of the gene. Putative promoter region contains potential binding sites for various transcription factors. Northern blot analysis showed that its mRNA is highly expressed in adult testis with lower expression in ovary, ES (embryonic stem) cells, and various other organs. In situ hybridization analysis revealed strong expression of the gcl gene in the pachytene stage spermatocytes. The expression was also observed in post-migratory PGCs, but was not apparent in migratory and pre-migratory PGCs. Further studies including gene disruption analysis would provide an important insight into mammalian germ lineage development.

Identification and characterization of a haploid germ cell-specific nuclear protein kinase (Haspin) in spermatid nuclei and its effects on somatic cells.

We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I-III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (for haploid germ cell-specific nuclear protein kinase). Western blot analysis showed that specific antibodies recognized a protein of Mr 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells.

Sperizin is a murine RING zinc-finger protein specifically expressed in Haploid germ cells.

Through the preparation of a subtracted cDNA library, we have extensively isolated genes whose expression is induced in mouse spermatogenic cells. One of the genes encoded a protein with a RING zinc-finger motif, which we termed sperizin (spermatid-specific ring zinc finger). Transcription of the sperizin gene was not observed in prepubertal testis, but became detectable at day 23. Northern and in situ hybridization analyses indicated that the sperizin gene was exclusively expressed in the round spermatid. The sperizin gene is intronless, and GFP-tagged sperizin was found to be localized in the cytoplasm of ectopically expressed somatic cultured cells. We assigned the chromosomal localization of the sperizin gene to chromosome 17 using an interspecific backcross mapping panel. The data suggest that we have identified a new member of the RING-finger family of proteins that may be involved in spermatogenesis.

Expression of selenoprotein-P messenger ribonucleic acid in the rat testis.

It has been suggested that a plasma protein, selenoprotein P, functions as an antioxidant and that its mRNA is expressed ubiquitously, including in the testis. To determine its physiological function, we have investigated the expression of selenoprotein-P mRNA in the rat testis. Northern blot analysis showed that selenoprotein P was exclusively expressed in the Leydig cell fraction. In situ hybridization experiments further supported this observation. Testes of rats administered ethylene dimethane sulfonate (EDS) were also examined by Northern blot analysis. Selective degeneration of Leydig cells by EDS treatment resulted in disappearance of selenoprotein-P mRNA from the testis. Furthermore, upon recovery, in association with regenerative differentiation of Leydig cells, reappearance of the selenoprotein-P mRNA was observed. These results indicated that selenoprotein-P mRNA was predominantly expressed in the interstitial Leydig cells.