RESUMEN
A novel heterozygous mutation A340T leading to the substitution of Phe for the conserved amino acid Ile114 was identified by nucleotide sequencing of the human LH/chorionic gonadotropin receptor (hLHR) of a patient with Leydig cell hypoplasia. This mutation is located in the third leucine-rich repeat in the ectodomain of the hLHR. In vitro expression studies demonstrated that this mutation results in reduced ligand binding and signal transduction of the receptor. Studies of hLHR constructs in which various amino acids were substituted for the conserved Ile114 showed that receptor activity is sensitive to changes in size, shape, and charge of the side chain. A homology model of the wild-type hLHR ectodomain was made, illustrating the packing of conserved hydrophobic side chains in the protein core. Substitution of Ile114 by Phe might disrupt intermolecular contacts between hormone and receptor. This mutation might also affect an LHR-dimer interaction. Thus, the I114F mutation reduces ligand binding and signal transduction by the hLHR, and it is partially responsible for Leydig cell hypoplasia in the patient.
Asunto(s)
Tumor de Células de Leydig/genética , Modelos Moleculares , Mutación Missense/genética , Proteínas/genética , Receptores de HL/genética , Secuencia de Bases , Línea Celular , AMP Cíclico/metabolismo , Citometría de Flujo , Humanos , Proteínas Repetidas Ricas en Leucina , Masculino , Microscopía Fluorescente , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Autism spectrum disorder is a complex neurodevelopmental disorder whose pathophysiology remains elusive as a consequence of the unavailability for study of patient brain neurons; this deficit may potentially be circumvented by neural differentiation of induced pluripotent stem cells. Rare syndromes with single gene mutations and autistic symptoms have significantly advanced the molecular and cellular understanding of autism spectrum disorders; however, in aggregate, they only represent a fraction of all cases of autism. In an effort to define the cellular and molecular phenotypes in human neurons of non-syndromic autism, we generated induced pluripotent stem cells (iPSCs) from three male autism spectrum disorder patients who had no identifiable clinical syndromes, and their unaffected male siblings and subsequently differentiated these patient-specific stem cells into electrophysiologically active neurons. iPSC-derived neurons from these autistic patients displayed decreases in the frequency and kinetics of spontaneous excitatory postsynaptic currents relative to controls, as well as significant decreases in Na+ and inactivating K+ voltage-gated currents. Moreover, whole-genome microarray analysis of gene expression identified 161 unique genes that were significantly differentially expressed in autistic patient iPSC-derived neurons (>twofold, FDR < 0.05). These genes were significantly enriched for processes related to synaptic transmission, such as neuroactive ligand-receptor signaling and extracellular matrix interactions, and were enriched for genes previously associated with autism spectrum disorder. Our data demonstrate aberrant voltage-gated currents and underlying molecular changes related to synaptic function in iPSC-derived neurons from individuals with idiopathic autism as compared to unaffected siblings controls.
Asunto(s)
Trastorno Autístico/genética , Trastorno Autístico/patología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Adolescente , Diferenciación Celular , Línea Celular , Niño , Potenciales Postsinápticos Excitadores , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Activación del Canal Iónico , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Canales de Potasio/metabolismo , Canales de Sodio/metabolismoRESUMEN
CONTEXT: Adrenocortical tumors have been studied at the molecular genetic and cytogenetic levels, but the gene expression profiles of normal and tumor adrenal tissue have not been extensively investigated. OBJECTIVE: The objective of this study was to obtain information about transcriptome differences in hyperplastic adrenal cells. DESIGN AND PATIENTS: We performed serial analysis of gene expression (SAGE) on control adrenal tissue and primary pigmented nodular adrenocortical disease (PPNAD) tissue from two adolescent female patients. MAIN OUTCOME MEASURE: The main outcome measure was to provide quantitative datasets of the vast majority of the transcripts implicated in normal and pathogenic adrenal functioning. RESULTS: The libraries of 28,705 and 31,278 tags represented 14,846 and 16,698 unique mRNAs from the control and PPNAD tissue, respectively. A total of 842 tags from the two libraries did not match any known sequences. We found 127 tags, including 70 no-match tags, to be expressed almost exclusively in control and/or PPNAD adrenals and to be absent or very rare in other human tissues. Examples of well-characterized genes expressed at significantly higher levels in PPNAD included steroidogenic acute regulator, chromogranin A, and those coding for the steroidogenic enzymes P450 cytochromes CYP17A1 and CYP21A2. Pathway analysis revealed Wnt signaling as the most up-regulated in PPNAD. These data were confirmed for selected genes by quantitative RT-PCR and/or immunohistochemistry. CONCLUSIONS: This study was the first of its kind for adrenal tissue and provides important information about the adrenal transcriptome and aberrant signaling in an inherited form of adrenocortical hyperplasia.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Mutación de Línea Germinal , Proteínas/genética , Neoplasias de la Corteza Suprarrenal/metabolismo , Niño , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico , Femenino , Biblioteca de Genes , Humanos , Inmunohistoquímica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Serial analysis of gene expression (SAGE) provides an alternative with additional advantages to microarrays for studying gene expression during spermatogenesis. The digitized transcriptome provided by SAGE of purified mouse germ cells identified 27,504 species of transcripts expressed in type A spermatogonia, pachytene spermatocytes, and round spermatids. Over 2700 of these transcripts were novel. Computational analyses allowed the identification of clusters of co-regulated genes, cell-specific promoter modules, cell-specific biological processes, as well as "preferential" biological networks in different cell types. These analyses provided potential drug targets for interference of specific pathways at different stages of spermatogenesis. Analyses of the transcriptomes revealed the prominent role of cytochrome c oxidase in germ cells and suggest a novel role for this enzyme in cytochrome c-mediated apoptosis in spermatogonia. A number of genes were shown to undergo differential splicing during spermatogenesis giving rise to cell-specific splice variants.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Espermatogénesis/genética , Espermatozoides/metabolismo , Empalme Alternativo/genética , Animales , Biología Computacional , Anticonceptivos Masculinos/farmacología , Complejo IV de Transporte de Electrones/genética , Masculino , Ratones , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Espermatogénesis/efectos de los fármacos , Transcripción GenéticaRESUMEN
The objective of this study was to further understand the genetic mechanisms of vitamin A deficiency (VAD) induced arrest of spermatogonial stem-cell differentiation. Vitamin A and its derivatives (the retinoids) participate in many physiological processes including vision, cellular differentiation and reproduction. VAD affects spermatogenesis, the subject of our present study. Spermatogenesis is a highly regulated process of differentiation and complex morphologic alterations that leads to the formation of sperm in the seminiferous epithelium. VAD causes early cessation of spermatogenesis, characterized by degeneration of meiotic germ cells, leading to seminiferous tubules containing mostly type A spermatogonia and Sertoli cells. These observations led us to the hypothesis that VAD affects not only germ cells but also somatic cells. To investigate the effects of VAD on spermatogenesis in mice we used adult Balb/C mice fed with Control or VAD diet for an extended period of time (6-28 weeks). We first observed the chronology, then the extent of the effects of VAD on the testes. Using microarray analysis of isolated pure populations of spermatogonia, Leydig and Sertoli cells from control and VAD 18- and 25-week mice, we examined the effects of VAD on gene expression and identified target genes involved in the arrest of spermatogonial differentiation and spermatogenesis. Our results provide a more precise definition of the chronology and magnitude of the consequences of VAD on mouse testes than the previously available literature and highlight direct and indirect (via somatic cells) effects of VAD on germ cell differentiation.
Asunto(s)
Expresión Génica , Espermatogénesis/genética , Espermatogonias/patología , Deficiencia de Vitamina A/genética , Animales , Diferenciación Celular , Células Germinativas/metabolismo , Células Germinativas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Epitelio Seminífero/metabolismo , Epitelio Seminífero/patología , Células de Sertoli/metabolismo , Células de Sertoli/patología , Espermatogonias/fisiología , Vitamina A/genética , Vitamina A/metabolismo , Deficiencia de Vitamina A/metabolismo , Deficiencia de Vitamina A/patologíaRESUMEN
Seminiferous tubules of the male testis contain somatic cells (Sertoli and Leydig cells) and germ cells at different stages of spermatogenesis (spermatogonia, spermatocytes and spermatids). Germ cells at different stages of differentiation migrate toward the central lumen via cell junctions formed by Sertoli cells. The protocol described herein consists of the dissection and decapsulation of the testes, disruption of the structure of the seminiferous tubules, and the breaking of cell junctions to release all the cells in suspension. Germ cells are then separated from Sertoli cells by overnight plating of the suspension on plastic to which the germ cells preferentially adhere. And finally, a BSA gradient allows a high-purity separation of the various types of germ cells according to size.
Asunto(s)
Separación Celular/métodos , Espermatozoides/citología , Animales , Recuento de Células , Disección/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Espermatogonias/citología , Testículo/citologíaRESUMEN
To compare methylation profiles and expression levels of Brahma at different stages of spermatogenesis, and to identify the methylation pattern during oogenesis, we analyzed gene expression and methylation patterns in murine germ cells at various developmental stages. The methylation levels of CpG islands within Brahma increased during spermatogenesis and decreased during oogenesis. This change in methylation pattern correlates with the change in expression of Brahma during spermatogenesis. As the degree of methylation increases, the expression decreases. The change in methylation is opposite during oogenesis, which suggests opposite expression levels.
Asunto(s)
Metilación de ADN/fisiología , Oogénesis/fisiología , Espermatogénesis/fisiología , Factores de Transcripción/genética , Cromatina/fisiología , Islas de CpG/fisiología , Femenino , Humanos , Células Intersticiales del Testículo/fisiología , Masculino , Oocitos/fisiología , Células de Sertoli/fisiología , Espermatocitos/fisiologíaRESUMEN
ING2 (inhibitor of growth family, member 2) is a member of the plant homeodomain (PHD)-containing ING family of putative tumor suppressors. As part of mSin3A-HDAC corepressor complexes, ING2 binds to tri-methylated lysine 4 of histone H3 (H3K4me3) to regulate chromatin modification and gene expression. ING2 also functionally interacts with the tumor suppressor protein p53 to regulate cellular senescence, apoptosis and DNA damage response in vitro, and is thus expected to modulate carcinogenesis and aging. Here we investigate the developmental and physiological functions of Ing2 through targeted germline disruption. Consistent with its abundant expression in mouse and human testes, male mice deficient for Ing2 showed abnormal spermatogenesis and were infertile. Numbers of mature sperm and sperm motility were significantly reduced in Ing2(-/-) mice (â¼2% of wild type, P<0.0001 and â¼10% of wild type, P<0.0001, respectively). Their testes showed degeneration of seminiferous tubules, meiotic arrest before pachytene stage with incomplete meiotic recombination, induction of p53, and enhanced apoptosis. This phenotype was only partially abrogated by concomitant loss of p53 in the germline. The arrested spermatocytes in Ing2(-/-) testes were characterized by lack of specific HDAC1 accumulation and deregulated chromatin acetylation. The role of Ing2 in germ cell maturation may extend to human ING2 as well. Using publicly available gene expression datasets, low expression of ING2 was found in teratozoospermic sperm (>3-fold reduction) and in testes from patients with defective spermatogenesis (>7-fold reduction in Sertoli-cell only Syndrome). This study establishes ING2 as a novel regulator of spermatogenesis functioning through both p53- and chromatin-mediated mechanisms, suggests that an HDAC1/ING2/H3K4me3-regulated, stage-specific coordination of chromatin modifications is essential to normal spermatogenesis, and provides an animal model to study idiopathic and iatrogenic infertility in men. In addition, a bona fide tumor suppressive role of Ing2 is demonstrated by increased incidence of soft-tissue sarcomas in Ing2(-/-) mice.
Asunto(s)
Proteínas de Homeodominio/genética , Sarcoma/genética , Espermatogénesis/genética , Proteínas Supresoras de Tumor/genética , Animales , Apoptosis , Atrofia , Western Blotting , Femenino , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Infertilidad Masculina/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma/metabolismo , Sarcoma/patología , Semen/metabolismo , Túbulos Seminíferos/metabolismo , Túbulos Seminíferos/patología , Testículo/metabolismo , Testículo/patología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoAsunto(s)
Factor V/genética , Hipertensión Intracraneal/genética , Trombosis/genética , Adolescente , Adulto , Secuencia de Bases , Niño , ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Femenino , Predisposición Genética a la Enfermedad , Humanos , Hipertensión Intracraneal/complicaciones , Hipertensión Intracraneal/diagnóstico , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Trombosis/etiologíaRESUMEN
Despite the identification of key genes such as Sry integral to embryonic gonadal development, the genomic classification and identification of chromosomal activation of this process is still poorly understood. To better understand the genetic regulation of gonadal development, we performed Serial Analysis of Gene Expression (SAGE) to profile the genes and novel transcripts, and an average of 152,000 tags from male embryonic gonads at E10.5 (embryonic day 10.5), E11.5, E12.5, E13.5, E15.5 and E17.5 were analyzed. A total of 275,583 non-singleton tags that do not map to any annotated sequence were identified in the six gonad libraries, and 47,255 tags were mapped to 24,975 annotated sequences, among which 987 sequences were uncharacterized. Utilizing an unsupervised pattern identification technique, we established molecular staging of male gonadal development. Rather than providing a static descriptive analysis, we developed algorithms to cluster the SAGE data and assign SAGE tags to a corresponding chromosomal position; these data are displayed in chromosome graphic format. A prominent increase in global genomic activity from E10.5 to E17.5 was observed. Important chromosomal regions related to the developmental processes were identified and validated based on established mouse models with developmental disorders. These regions may represent markers for early diagnosis for disorders of male gonad development as well as potential treatment targets.
Asunto(s)
Perfilación de la Expresión Génica , Ratones/embriología , Ratones/genética , Testículo/embriología , Animales , Mapeo Cromosómico , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones/crecimiento & desarrollo , Datos de Secuencia Molecular , Testículo/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
Serial analysis of gene expression (SAGE) provides a global analysis platform for profiling mRNA populations present in cells of interest without the constraint of gene selection and the ambiguous nature of data obtained. However, most of the reports on SAGE and germ cell development are limited to descriptive analyses. Here, we report a series of bioinformatic analyses using recently published SAGE data on the transcriptome of mouse type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd). Tags with a total count of > or =20 in three SAGE libraries were examined. Our aim was to identify and discover potential transcriptional regulators and pathways involved at different stages of spermatogenesis. Unsupervised hierarchical clustering based on tag expression and Gene Ontology analysis were applied to identify genes and biological processes overrepresented at a particular stage of development. The 5' cis-regulatory elements were examined for common regulators in different functional clusters. Potential biological networks were also constructed to reveal the link between the gene candidates. Biological pathways related to the three germ cell stages were constructed. A number of known transcription regulators in spermatogenesis, including NF-kappaB, SP1, AP-1, and EGR, were identified. Novel promoter elements such as the E box in Spga-specific genes, GATA in Spcy-specific genes, and GKLF in Sptd-specific genes were also observed. Taken together, our approach is reliable and provides a foundation for the generation of novel biological hypotheses for studying spermatogenesis.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Espermátides/química , Espermatocitos/química , Espermatogonias/química , Transcripción Genética , Animales , Diferenciación Celular/genética , Biblioteca de Genes , Factor 4 Similar a Kruppel , Masculino , Ratones , Regiones Promotoras GenéticasRESUMEN
Computational analyses have identified the widespread occurrence of antisense transcripts in the human and the mouse genome. However, the structure and the origin of the majority of the antisense transcripts are unknown. The presence of antisense transcripts for 19 of 64 differentially expressed genes during mouse spermatogenesis was demonstrated with orientation-specific RT-PCR. These antisense transcripts were derived from a wide variety of origins, including processed sense transcripts, intronic and exonic sequences of a single gene or multiple genes, intergenic sequences, and pseudogenes. They underwent normal and alternative splicing, 5' capping, and 3' polyadenylation, similar to the sense transcripts. There were also antisense transcripts that were not capped and/or polyadenylated. The testicular levels of the sense transcripts were higher than those of the antisense transcripts in all cases, while the relative expression in nontesticular tissues was variable. Thus antisense transcripts have complex origins and structures and the sense and antisense transcripts can be regulated independently.
Asunto(s)
ARN sin Sentido/genética , Espermatogénesis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Protaminas/genética , Precursores de Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Ubiquitinas/genética , Región del Complejo T del GenomaRESUMEN
The transcriptomes of mouse type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd) were determined by sequencing the respective SAGE (Serial Analysis of Gene Expression) libraries. A total of 444,015 tags derived from one Spga, two Spcy, and one Sptd library were analyzed, and 34,619 different species of transcripts were identified, 5279 of which were novel. Results indicated the germ-cell transcriptome comprises of more than 30,000 transcripts. Virtual subtraction showed that cell-specific transcripts constitute 12-19.5% of the transcriptome. Components of the protein biosynthetic machinery are highly expressed in Spga. In Spcy transcription factors are abundantly expressed while transcripts encoding proteins involved in chromosome remodeling and testis-specific transcripts are prominent in Sptd. The databases generated by this work provide very useful resources for cellular localization of genes in silico. They are also extremely useful as sources for identification of splice variants of genes in germ cells.