RESUMEN

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.

Asunto(s)

Carcinoma de Células Escamosas/patología , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias de la Boca/patología , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Humanos , Metaloproteinasa 9 de la Matriz/genética , Neoplasias de la Boca/metabolismo , Invasividad Neoplásica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , ARN Mensajero/análisis , Factores de Transcripción/genética , Transcripción Genética/fisiología

RESUMEN

We carried out single nucleotide polymorphism (SNP) and mutation analyses of haploid germ cell-specific genes. An analysis of 13 genes associated with male infertility in approximately 300 infertile male patients and approximately 300 male volunteers with proven fertility revealed two mutations that might produce male infertility, and three SNP/mutations associated with male infertility in 13 germ cell-specific genes. These findings strongly support the hypothesis that dysfunction of germ cell-specific genes causes idiopathic human male infertility.

Asunto(s)

Infertilidad Masculina/genética , Polimorfismo de Nucleótido Simple , Espermatogénesis/genética , Análisis Mutacional de ADN , Frecuencia de los Genes , Humanos , Masculino

RESUMEN

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.

Asunto(s)

Proteínas de Unión al ADN/genética , Células de Sertoli/metabolismo , Factores de Transcripción/genética , Animales , Línea Celular , Células Cultivadas , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/fisiología , Factor de Transcripción GATA4 , Masculino , Ratones , Ratones Endogámicos ICR , ARN Mensajero/análisis , Espermatogénesis , Testículo/citología , Factores de Transcripción/análisis , Factores de Transcripción/fisiología , Transcripción Genética , Transfección , Regulación hacia Arriba

RESUMEN

Kit and its ligand stem cell factor (SCF) play a fundamental role in hematopoiesis, melanogenesis and gametogenesis. Homozygous W(v) mutant mice with a mutation in kit show abnormalities in these cell lineages. Fas is a member of the death receptor family inducing apoptosis. In this study, we generated double-mutant mice (W(v)/W(v):Fas(-/-)) and analyzed histologically their reproductive organs. In testes and ovaries of the double-mutant mice, testicular germ cells and oocytes were detected, respectively, whereas the same-aged W(v)/W(v) mice contained neither cells. In addition, inhibition of Kit signals by administration of anti-Kit mAb, which induces degeneration of testicular germ cells in vivo in wild-type mice, did not cause degeneration in Fas-deficient mice. In testicular germ cells of W(v)/W(v) mutant mice, an increase of Fas expression was observed in spermatogonia. Further, in vitro treatment with SCF was shown to downregulate Fas on fibroblasts expressing exogenous Kit through activation of PI3-kinase/Akt. All the results clearly indicate that Fas-mediated apoptosis is involved in germ cell degeneration accompanied by defects in Kit-mediated signals, and Kit signaling negatively regulates Fas-mediated apoptosis in vivo.

Asunto(s)

Apoptosis/genética , Células Germinativas/crecimiento & desarrollo , Gónadas/crecimiento & desarrollo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor fas/metabolismo , Células 3T3 , Animales , Anticuerpos/farmacología , Cruzamientos Genéticos , Femenino , Células Germinativas/citología , Células Germinativas/metabolismo , Gónadas/citología , Gónadas/metabolismo , Masculino , Ratones , Ratones Noqueados , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Ovario/citología , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-kit/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Espermatocitos/citología , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismo , Factor de Células Madre/metabolismo , Factor de Células Madre/farmacología , Testículo/citología , Testículo/crecimiento & desarrollo , Testículo/metabolismo

RESUMEN

Succinyl CoA:3-oxo acid CoA transferase (SCOT/OXCT; EC 2.8.3.5) is a key mitochondrial enzyme in the metabolism of ketone bodies in various organs (but not in the liver). We identified a cDNA clone of the testicular germ cell-specific succinyl CoA transferase isozyme (SCOT-t). We then isolated a mouse orthologue of the SCOT/OXCT cDNA (SCOT-s) and determined the expression of the two types of SCOT in the testis. The mRNAs of scot-s and scot-t were expressed exclusively in testicular somatic cells (i.e. Leydig and Sertoli cells) and germ cells, respectively. SCOT enzymatic activities were assayed in Leydig cell (SCOT-s) and sperm (SCOT-t) fractions. The SCOT activity in sperm was 2.5-fold higher than that in Leydig cells. We conclude that germ cells and somatic cells differentially express the SCOT enzymes and that the SCOT activity of sperm caused exclusively by SCOT-t should play an important role in sperm activity.

Asunto(s)

Coenzima A Transferasas/genética , Testículo/citología , Testículo/enzimología , Animales , Regulación Enzimológica de la Expresión Génica , Células Intersticiales del Testículo/enzimología , Masculino , Ratones , Mitocondrias/enzimología , Datos de Secuencia Molecular , Células de Sertoli/enzimología , Espermatocitos/enzimología

RESUMEN

In mice, the recessive, non-pleiotropic, juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis, followed by failure of type A spermatogonial stem cells to differentiate, rendering adult males sterile. As part of an effort to identify the gene underlying this mutation, we report here the construction of a high-resolution genetic map involving more than 1000 meioses and 24 polymorphic loci. Our data define a critical jsd interval of approximately 0.4 cM at 49 cM on mouse chromosome 1, between D1Mit215 and 257SP6. We have constructed a physical map spanning the region comprising 24 overlapping BACs. Eighteen of these BACs have been fully sequenced, or are in draft form, allowing us to annotate approximately 2.5 Mb of DNA surrounding the jsd locus. The critical 0.4 cM jsd interval corresponds to a physical distance of approximately 1.5 Mb. Eight genes have been identified in this interval, two of which appear to be possible candidates for the jsd mutation.

Asunto(s)

Cromosomas , Proteínas Cullin , Mutación , Espermatogénesis , Espermatogonias/fisiología , Animales , Proteínas de Ciclo Celular/genética , Mapeo Cromosómico , Prueba de Complementación Genética , Ligamiento Genético , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Mutantes , Análisis de Secuencia de ADN , Testículo/anatomía & histología , Transcripción Genética

RESUMEN

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.

Asunto(s)

Espermatogénesis/efectos de los fármacos , Espermatogonias/citología , Testosterona/farmacología , Antagonistas de Andrógenos/farmacología , Antagonistas de Receptores Androgénicos , Animales , Diferenciación Celular/efectos de los fármacos , Dipéptidos/farmacología , Flutamida/farmacología , Hormona Folículo Estimulante/farmacología , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Hormona Luteinizante/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Androgénicos/fisiología , Testosterona/sangre

RESUMEN

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.

Asunto(s)

Genes/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Secuencia de Bases , Southern Blotting , ADN/química , ADN/genética , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Transcripción Genética

RESUMEN

We report here the molecular cloning and characterization of human haspin cDNA and its genomic DNA construct. The haspin protein is a unique protein kinase, first isolated from mouse testis. Specifically expressed in mouse testicular germ cells, haspin is suggested to play a role in cell cycle arrest in haploid spermatids. Detection of human haspin by Northern blot analysis showed that the major transcript was 2.8 kilobases long and detected exclusively in the testis. The entire coding region of the human cDNA showed 68% identity with mouse haspin. The predicted amino acid sequence showed strong conservation of the kinase catalytic domain, leucine zipper, potential phosphorylation sites, and MEF2B homologous region, but a relatively unique N:-terminal region. Human haspin protein was also demonstrated to have protein kinase activity. The human haspin gene was mapped to chromosome 17p13 by computer database cloning of human genomic DNA. Furthermore, the genomic structure of human haspin was proven to be intronless and the whole transcription unit was found to be located in an intron of the integrin alphaE2 gene.

Asunto(s)

Cromosomas Humanos Par 17 , Leucina Zippers , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico

RESUMEN

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.

Asunto(s)

Infertilidad Masculina/patología , Espermatogonias/patología , Animales , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Túbulos Seminíferos/citología , Espermatogénesis/genética , Espermatogonias/trasplante , Células del Estroma/fisiología , Testículo/citología

RESUMEN

Loss of the endoplasmic reticulum resident chaperone calmegin leads to the production of sterile sperm that do not bind to the egg zona pellucida (M. Ikawa et al., 1997, Nature 387, 607-611). In the present study, we demonstrate that calmegin -/- sperm were defective in migrating into the oviducts and in binding to the egg plasma membrane. Despite the impaired adhesive function, calmegin -/- sperm could fertilize eggs when zonae pellucidae were partially dissected, and eggs fertilized in this manner could develop normally to term. Since these sperm characteristics were similar to those found in fertilin beta -/- sperm, we investigated the interaction of calmegin with fertilin beta. Using immunoprecipitation techniques, calmegin was found to bind to sperm membrane proteins, fertilin alpha and beta, during spermatogenesis. The binding was specific to calmegin: another endoplasmic reticulum chaperone calnexin, a calmegin homologue, was not able to bind to fertilin alpha and beta. In the calmegin -/- mice, a loss of heterodimerization of fertilin alpha and beta was observed and fertilin beta was not detectable in mature sperm. The data not only explain why the calmegin and fertilin beta knockout mouse lines share a common infertile phenotype, but also reveal the importance of the maturation of sperm membrane proteins in the endoplasmic reticulum.

Asunto(s)

Proteínas de Unión al Calcio/fisiología , Calnexina , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/fisiología , Metaloendopeptidasas/metabolismo , Interacciones Espermatozoide-Óvulo , Proteínas ADAM , Animales , Secuencia de Bases , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Cartilla de ADN , Dimerización , Retículo Endoplásmico/metabolismo , Femenino , Fertilinas , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Chaperonas Moleculares

RESUMEN

The Biosafety Committee of the Japanese Association of Laboratory Animal Facilities of National Universities (JALAN) investigated recent episodes of microbiological contamination in genetically modified mice (GMM), and the countermeasures taken when the contaminated GMM were introduced into animal facilities, by questionnaires addressed to 53 animal facilities belonging to JALAN and serological tests. Although almost all of the contaminated GMM were accepted with conditions such as rederivation after or before reception and housing in designated rooms, contamination with a spectrum of microorganisms was demonstrated in GMM transferred domestically and from abroad. In serological tests, Mycoplasma pulmonis, mouse parvovirus, and mouse encephalomylitis virus were detected in GMM transferred from domestic facilities and from abroad. The present results of the questionnaires and serological tests suggest that GMM are highly and widely contaminated with microorganisms compared with mice from commercial breeders. Thus, we propose a microbiological requirement, including microbiological status--excellent, common, and minimum--as a guide for the transfer and procurement of mice and rats in Japan.

Asunto(s)

Transmisión de Enfermedad Infecciosa/prevención & control , Control de Infecciones , Organismos Modificados Genéticamente/microbiología , Animales , Guías como Asunto , Vivienda para Animales , Japón , Virus Maus Elberfeld/aislamiento & purificación , Virus Maus Elberfeld/patogenicidad , Ratones , Mycoplasma/aislamiento & purificación , Mycoplasma/patogenicidad , Parvovirus/aislamiento & purificación , Parvovirus/patogenicidad , Ratas , Medición de Riesgo , Pruebas Serológicas

RESUMEN

The regulatory mechanisms of mammalian hairy and Enhancer of split homologue-1 (HES-1) genes were examined in mouse P19 embryonic carcinoma cells (P19 cells). Undifferentiated P19 stem cells expressed a basal level of the HES-1 gene, whereas the expression of this gene was increased upon induction of the cells to a neural cell lineage using retinoic acid (RA). Reporter co-transfection analysis identified an activating region within the upstream promoter region of HES-1 from nucleotides -201 to -172. This activating region, called activating region X (ARX), shows a high GC content and contains both an AP-2 binding motif and a CCAAT box. An electrophoretic mobility shift assay using nuclear proteins extracted from P19 cells showed that ARX forms a specific DNA-protein complex. Importantly, ARX-dependent transcription, as well as ARX-binding activity, was significantly increased in P19 cells treated with RA. These results indicate that ARX transduces signals that up-regulate HES-1 gene expression in response to RA-treatment. Thus, a novel cis-acting element involved in HES-1 gene regulation that plays a role in RA-induced neural differentiation of P19 cells has been identified.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Tretinoina/farmacología , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleótidos , ARN Mensajero/genética , Factor de Transcripción HES-1 , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas

RESUMEN

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.

Asunto(s)

Coenzima A Transferasas/biosíntesis , ADN Complementario/metabolismo , Células Germinativas/metabolismo , Testículo/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Fraccionamiento Celular , Clonación Molecular , Coenzima A Transferasas/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Biblioteca de Genes , Células Germinativas/enzimología , Células Germinativas/ultraestructura , Haploidia , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN/análisis , ARN/genética , ARN/aislamiento & purificación , Testículo/ultraestructura

RESUMEN

We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3-4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation.

Asunto(s)

Cromatina/genética , Cromatina/metabolismo , Nucleoproteínas , Testículo/fisiología , Cromosoma X , Secuencia de Aminoácidos , Animales , Western Blotting , Cromatina/inmunología , Clonación Molecular , Citoplasma/metabolismo , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica/métodos , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/metabolismo , Secuencias Repetitivas de Aminoácido , Espermátides/citología , Espermátides/fisiología , Testículo/citología

RESUMEN

Spermatogenic cells from a mouse strain expressing enhanced green fluorescent protein (EGFP) under chicken beta-actin promoter were studied under living conditions to analyse stage- and cell-specific expression and hormonal regulation of the transgene. The isolated seminiferous tubules were examined by transillumination and the live cell squashes by phase contrast and fluorescence microscopy. FSH effects were measured in whole seminiferous tubules comparing stages I-VI, VII-VIII and IX-XII of the cycle. Beta-actin was highly expressed in spermatogonia, but almost no expression was found at early meiosis (leptotene spermatocytes). A gradual increase in translation of beta-actin was found during later stages of meiosis and early spermiogenesis, with a maximum in elongating spermatids. FSH increased the translation of beta-actin after 4 h and 24 h of incubation at stages I-VI, after 24 h at stages VII-VIII but not at stages IX-XII of the cycle. The results support the view that beta-actin plays a role in the nuclear elongation of spermatids and that its expression is regulated by FSH in a stage-specific fashion. Techniques used in this study give us new insight to study temporal and hormonal regulation of gene products in living spermatogenic cells.

Asunto(s)

Actinas/genética , Hormona Folículo Estimulante/metabolismo , Proteínas Luminiscentes/genética , Regiones Promotoras Genéticas , Espermatogénesis , Espermatozoides/metabolismo , Animales , Pollos , Fluorescencia , Hormona Folículo Estimulante/farmacología , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes , Técnicas In Vitro , Masculino , Ratones , Ratones Transgénicos , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología

RESUMEN

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.

Asunto(s)

Células Madre/citología , Testículo/citología , Animales , Diferenciación Celular , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Trasplante de Células Madre , Factores de Tiempo

RESUMEN

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.

Asunto(s)

Proteínas/genética , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Células CHO , Línea Celular , Clonación Molecular , Cricetinae , ADN Complementario/química , ADN Complementario/genética , Sistema de Lectura Ribosómico/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes , Haploidia , Humanos , Inmunohistoquímica , Hibridación in Situ , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Ornitina Descarboxilasa/metabolismo , Inhibidores de la Ornitina Descarboxilasa , Poliaminas/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Proteínas/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Espermatozoides/química , Espermatozoides/metabolismo , Testículo/química

RESUMEN

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.

Asunto(s)

Proteína de Unión a Andrógenos , Clonación Molecular , Expresión Génica , Haploidia , Espermatozoides/química , Envejecimiento , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Proteínas Portadoras/química , Proteínas Portadoras/genética , ADN Complementario/química , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas de Transferencia de Fosfolípidos , ARN Mensajero/análisis , Homología de Secuencia , Cola del Espermatozoide/química , Espermátides/química , Testículo/química

RESUMEN

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.

Asunto(s)

Proteínas Proto-Oncogénicas c-kit/fisiología , Espermatogonias/citología , Factor de Células Madre/fisiología , Células Madre/citología , Animales , Membrana Basal/citología , Diferenciación Celular , División Celular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Túbulos Seminíferos/citología , Testículo/citología