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OBJECTIVE: To study the role of estrogen receptor beta in follicle development and maturation and in the response to gonadotropin stimulation aiming at superovulation DESIGN: Experimental study and transcriptomic analysis. ANIMALS: Healthy wild-type and estrogen receptor beta (ERß) knockout female mice undergoing superovulation at 4-weeks, 7-weeks, and 6-months of age. MAIN OUTCOMES: Oocyte yield after superovulation, transcriptomic profiling of cumulus-granulosa cell complexes and oocytes, and immunohistochemical analyses. RESULTS: Superovulation of ERß knockout (Esr2-KO) mice resulted in reduced oocyte yield at 6-months of age compared to wild-type (WT) mice, but younger mice had similar yields. RNA-seq analysis of cumulus cells from superovulated WT and Esr2-KO mice identified genes and pathways associated with among others adhesion, proliferation, Wnt-signaling, and placed ERß in bipotential granulosa cell cluster. Loss of ERß increased expression of the other estrogen receptors Esr1 and Gper1. CONCLUSION: Our results show that ERß has an important role in regulating ovulation in response to exogenous gonadotropins in 6-month-old mice, but not in younger mice. Our transcriptomic and immunohistochemical observations suggest a dysregulation of the granulosa cell communication and lack of tight coordination between granulosa cell replication and antrum expansion. A significant upregulation of other estrogen receptors may support a compensatory mechanism sustaining fertility during younger age in Esr2-KO mice.
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Long terminal repeat (LTR) retrotransposons are widely distributed across the human genome. They have accumulated through retroviral integration into germline DNA and are latent genetic modules. Active LTR promoters are observed in germline cells; however, little is known about the mechanisms underlying their active transcription in somatic tissues. Here, by integrating our previous transcriptome data set with publicly available data sets, we show that the LTR families MLT2A1 and MLT2A2 are primarily expressed in human four-cell and eight-cell embryos and are also activated in some adult somatic tissues, particularly pineal gland. Three MLT2A elements function as the promoters and first exons of the protein-coding genes ABCE1, COL5A1, and GALNT13 specifically in the pineal gland of humans but not in that of macaques, suggesting that the exaptation of these LTRs as promoters occurred during recent primate evolution. This analysis provides insight into the possible transition from germline insertion to somatic expression of LTR retrotransposons.
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Retroelementos , Secuencias Repetidas Terminales , Animales , Primates/genética , Regiones Promotoras Genéticas , Retroelementos/genética , Secuencias Repetidas Terminales/genéticaRESUMEN
BACKGROUND: We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases (GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting. METHODS: Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout-a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams. RESULTS: Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange. CONCLUSIONS: Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.
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Atención a la Salud , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Secuenciación Completa del Genoma , Estudios de Cohortes , Variaciones en el Número de Copia de ADN/genética , Heterogeneidad Genética , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Difusión de la Información , Patrón de Herencia/genética , Repeticiones de Microsatélite/genética , Mutación/genética , Suecia , Disomía Uniparental/genéticaRESUMEN
BACKGROUND: Male estrogen receptor beta (ERß) knockout (BERKO) mice display anxiety and aggression linked to, among others, altered serotonergic signaling in the basolateral amygdala and dorsal raphe, impaired cortical radial glia migration, and reduced GABAergic signaling. The effects on primary motor cortex (M1 cortex) and locomotor activity as a consequence of ERß loss have not been investigated. OBJECTIVE: The aim of this study was to determine whether locomotor activity is altered as a consequence of the changes in the M1 cortex. METHODS: The locomotor activity of male wild-type (WT) and BERKO mice was evaluated using the open-field and rotarod tests. Molecular changes in the M1 cortex were analyzed by RNA sequencing, electron microscopy, electrophysiology, and immunohistological techniques. In addition, we established oligodendrocyte (OL) cultures from WT and BERKO mouse embryonic stem cells to evaluate OL function. RESULTS: Locomotor profiling revealed that BERKO mice were more active than WT mice but had impaired motor coordination. Analysis of the M1 cortex pointed out differences in synapse function and myelination. There was a reduction in GABAergic signaling resulting in imbalanced excitatory and inhibitory neurotransmission as well as a defective OL differentiation accompanied by myelin defects. The effects of ERß loss on OL differentiation were confirmed in vitro. CONCLUSION: ERß is an important regulator of GABAergic interneurons and OL differentiation, which impacts on adult M1 cortex function and may be linked to increased locomotor activity and decreased motor coordination in BERKO mice.
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Receptor beta de Estrógeno/genética , Locomoción/genética , Corteza Motora/fisiopatología , Vaina de Mielina/fisiología , Desempeño Psicomotor , Transmisión Sináptica , Animales , Expresión Génica , Masculino , Ratones , Ratones Noqueados , Corteza Motora/metabolismo , Oligodendroglía/patologíaRESUMEN
As estrogen receptor ß-/- (ERß-/-) mice age, the ventral prostate (VP) develops increased numbers of hyperplastic, fibroplastic lesions and inflammatory cells. To identify genes involved in these changes, we used RNA sequencing and immunohistochemistry to compare gene expression profiles in the VP of young (2-mo-old) and aging (18-mo-old) ERß-/- mice and their WT littermates. We also treated young and old WT mice with an ERß-selective agonist and evaluated protein expression. The most significant findings were that ERß down-regulates androgen receptor (AR) signaling and up-regulates the tumor suppressor phosphatase and tensin homolog (PTEN). ERß agonist increased expression of the AR corepressor dachshund family (DACH1/2), T-cadherin, stromal caveolin-1, and nuclear PTEN and decreased expression of RAR-related orphan receptor c, Bcl2, inducible nitric oxide synthase, and IL-6. In the ERß-/- mouse VP, RNA sequencing revealed that the following genes were up-regulated more than fivefold: Bcl2, clusterin, the cytokines CXCL16 and -17, and a marker of basal/intermediate cells (prostate stem cell antigen) and cytokeratins 4, 5, and 17. The most down-regulated genes were the following: the antioxidant gene glutathione peroxidase 3; protease inhibitors WAP four-disulfide core domain 3 (WFDC3); the tumor-suppressive genes T-cadherin and caveolin-1; the regulator of transforming growth factor ß signaling SMAD7; and the PTEN ubiquitin ligase NEDD4. The role of ERß in opposing AR signaling, proliferation, and inflammation suggests that ERß-selective agonists may be used to prevent progression of prostate cancer, prevent fibrosis and development of benign prostatic hyperplasia, and treat prostatitis.
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Envejecimiento/metabolismo , Regulación hacia Abajo , Receptor beta de Estrógeno/metabolismo , Próstata/metabolismo , Receptores Androgénicos/biosíntesis , Transducción de Señal , Envejecimiento/genética , Envejecimiento/patología , Andrógenos/metabolismo , Animales , Quimiocina CXCL16/biosíntesis , Quimiocina CXCL16/genética , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Clusterina/biosíntesis , Clusterina/genética , Receptor beta de Estrógeno/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Queratinas/biosíntesis , Queratinas/genética , Masculino , Ratones , Ratones Noqueados , Ubiquitina-Proteína Ligasas Nedd4/biosíntesis , Ubiquitina-Proteína Ligasas Nedd4/genética , Fosfohidrolasa PTEN/biosíntesis , Fosfohidrolasa PTEN/genética , Próstata/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores Androgénicos/genética , Proteína smad7/biosíntesis , Proteína smad7/genéticaRESUMEN
Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA.
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Blastocisto/metabolismo , Células Madre Embrionarias/metabolismo , Proteínas de Homeodominio/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Técnica del Anticuerpo Fluorescente Indirecta , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Ratones , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/genéticaRESUMEN
PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development.
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Blastocisto/metabolismo , Proteínas Fetales/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Clonación Molecular , Secuencia de Consenso , ADN Complementario/genética , Proteínas Fetales/biosíntesis , Perfilación de la Expresión Génica , Biblioteca de Genes , Proteínas de Homeodominio/biosíntesis , Células Madre Embrionarias Humanas/metabolismo , Humanos , Familia de Multigenes , Especificidad de Órganos , Células Madre Pluripotentes/metabolismo , Regiones Promotoras Genéticas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/biosíntesisRESUMEN
Estrogen receptor beta (ERß/ESR2) has a central role in mouse ovaries, as ERß knockout (BERKO) mice are subfertile due to an increase in fibrosis around the maturing follicle and a decrease in blood supply. This has a consequence that these follicles rarely rupture to release the mature oocyte. Matrix metalloproteinases (MMPs) are modulators of the extracellular matrix, and the expression of one specific MMP, MMP-19, is normally increased in granulosa cells during their maturation until ovulation. In this study, we demonstrate that MMP-19 levels are downregulated in BERKO mouse ovaries. Using human MCF-7 cells that overexpress ERß, we could identify MMP-19 to be a transcriptional target of ligand-bound activated ERß acting on a specificity protein-1 binding site. These data provide a molecular explanation for the observed follicle rupture defect that contributes to the subfertility of female BERKO mice.
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Receptor beta de Estrógeno/metabolismo , Metaloproteinasas de la Matriz Secretadas/metabolismo , Ovario/metabolismo , Animales , Gonadotropina Coriónica , Regulación hacia Abajo , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Células MCF-7 , Metaloproteinasas de la Matriz Secretadas/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ovulación , Factor de Transcripción Sp1/metabolismoRESUMEN
Transcriptional program that drives human preimplantation development is largely unknown. Here, by using single-cell RNA sequencing of 348 oocytes, zygotes and single blastomeres from 2- to 3-day-old embryos, we provide a detailed analysis of the human preimplantation transcriptome. By quantifying transcript far 5'-ends (TFEs), we include in our analysis transcripts that derive from alternative promoters. We show that 32 and 129 genes are transcribed during the transition from oocyte to four-cell stage and from four- to eight-cell stage, respectively. A number of identified transcripts originates from previously unannotated genes that include the PRD-like homeobox genes ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB and LEUTX. Employing de novo promoter motif extraction on sequences surrounding TFEs, we identify significantly enriched gene regulatory motifs that often overlap with Alu elements. Our high-resolution analysis of the human transcriptome during preimplantation development may have important implications on future studies of human pluripotent stem cells and cell reprograming.
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Blastocisto/metabolismo , Blastómeros/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Oocitos/metabolismo , ARN Mensajero/metabolismo , Retroelementos/genética , Factores de Transcripción/genética , Cigoto/metabolismo , Regiones no Traducidas 5' , Perfilación de la Expresión Génica , Células HEK293 , Proteínas de Homeodominio/metabolismo , Humanos , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismoRESUMEN
Congenital heart defects (CHD) occur in approximately 50% of patients with Down syndrome (DS); the mechanisms for this occurrence however remain unknown. In order to understand how these defects evolve in early development in DS, we focused on the earliest stages of cardiogenesis to ascertain perturbations in development leading to CHD. Using a trisomy 21 (T21) sibling human embryonic stem cell (hESC) model of DS, we show that T21-hESC display many significant differences in expression of genes and cell populations associated with mesodermal, and more notably, secondary heart field (SHF) development, in particular a reduced number of ISL1(+) progenitor cells. Furthermore, we provide evidence for two candidate genes located on chromosome 21, ETS2 and ERG, whose overexpression during cardiac commitment likely account for the disruption of SHF development, as revealed by downregulation or overexpression experiments. Additionally, we uncover an abnormal electrophysiological phenotype in functional T21 cardiomyocytes, a result further supported by mRNA expression data acquired using RNA-Seq. These data, in combination, revealed a cardiomyocyte-specific phenotype in T21 cardiomyocytes, likely due to the overexpression of genes such as RYR2, NCX, and L-type Ca(2+) channel. These results contribute to the understanding of the mechanisms involved in the development of CHD. Stem Cells 2015;33:1434-1446.
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Síndrome de Down/patología , Síndrome de Down/fisiopatología , Corazón/embriología , Corazón/fisiopatología , Células Madre Embrionarias Humanas/metabolismo , Miocitos Cardíacos/patología , Potenciales de Acción , Diferenciación Celular , Línea Celular , Cromosomas Humanos Par 21/genética , Síndrome de Down/genética , Regulación del Desarrollo de la Expresión Génica , Estudios de Asociación Genética , Cardiopatías Congénitas/genética , Humanos , Modelos Biológicos , Miocitos Cardíacos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Transcriptoma/genéticaRESUMEN
Infertility is a worldwide concern that can be treated with in vitro fertilization (IVF). Improvements in IVF and infertility treatment depend largely on better understanding of the molecular mechanisms for human preimplantation development. Several large-scale studies have been conducted to identify gene expression patterns for the first five days of human development, and many functional studies utilize mouse as a model system. We have identified genes of possible importance for this time period by analyzing human microarray data and available data from online databases. We selected 70 candidate genes for human preimplantation development and investigated their expression in the early mouse development from oocyte to the 8-cell stage. Maternally loaded genes expectedly decreased in expression during development both in human and mouse. We discovered that 25 significantly upregulated genes after fertilization in human included 13 genes whose orthologs in mouse behaved differently and mimicked the expression profile of maternally expressed genes. Our findings highlight many significant differences in gene expression patterns during mouse and human preimplantation development. We also describe four cancer-testis antigen families that are also highly expressed in human embryos: PRAME, SSX, GAGE and MAGEA.
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Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Blastocisto/metabolismo , Análisis por Conglomerados , Femenino , Humanos , Masculino , Ratones , Familia de MultigenesRESUMEN
The coxsackievirus and adenovirus receptor (CXADR (CAR)) is a cell adhesion molecule expressed mainly in epithelial cells. Numerous evidence indicate that CXADR has an important role in testis development and function of the blood-testis barrier (BTB) in vitro. The role of CXADR in testis physiology in vivo has, however, not been addressed. We therefore constructed a conditional CXADR knockout (cKO) mouse model in which CXADR can be depleted at any chosen timepoint by the administration of tamoxifen. We report for the first time that testicular depletion of CXADR in adult and pubertal mice does not alter BTB permeability or germ cell migration across the BTB during spermatogenesis. Adult cKO mice display normal junctional ultra-structure and localization of the junctional proteins claudin-3, occludin, junction-associated molecule-A (JAM-A), and ZO1. The BTB was intact with no leakage of biotin and lanthanum tracers into the tubular lumen. Adult CXADR cKO mice were fertile with normal sperm parameters and litter size. Breeding experiments and genotyping of the pups demonstrated that CXADR-negative sperm could fertilize WT eggs. In addition, knocking down CXADR from postnatal day 9 (P9) does not affect testicular development and BTB formation. These cKO mice were analyzed at P49 and P90 and display an intact barrier and uncompromised fertility. We conclude that CXADR possesses no direct role in testicular physiology in vivo.
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Barrera Hematotesticular/metabolismo , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/deficiencia , Espermatogénesis , Espermatozoides/metabolismo , Factores de Edad , Animales , Barrera Hematotesticular/ultraestructura , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/genética , Femenino , Fertilidad , Uniones Intercelulares/metabolismo , Tamaño de la Camada , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Permeabilidad , Embarazo , Maduración Sexual , Proteínas de Uniones Estrechas/metabolismoRESUMEN
MOTIVATION: Recent transcriptome studies have revealed that total transcript numbers vary by cell type and condition; therefore, the statistical assumptions for single-cell transcriptome studies must be revisited. SAMstrt is an extension code for SAMseq, which is a statistical method for differential expression, to enable spike-in normalization and statistical testing based on the estimated absolute number of transcripts per cell for single-cell RNA-seq methods. AVAILABILITY AND IMPLEMENTATION: SAMstrt is implemented on R and available in github (https://github.com/shka/R-SAMstrt). CONTACT: shintaro.katayama@ki.se
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Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Interpretación Estadística de Datos , Humanos , Análisis de la Célula IndividualRESUMEN
The aim of this study was to evaluate mitochondrial alteration and ATP content of germinal vesicle (GV) oocytes isolated from fresh and vitrified ovaries. After superovulation, the ovaries from adult mice were collected and divided into control and vitrified groups. GV oocytes were isolated mechanically from each group. Half were cultured for 24 hours and their maturation was assessed. Metaphase II oocytes were collected and submitted to in vitro fertilization and their fertilization rates and development to the blastocyst stage were evaluated. In the remaining GV oocytes, ATP levels were quantified, and mitochondrial distribution, mitochondrial membrane potential, and intracellular free calcium were detected with rhodamine 123, JC-1 and Flou-4 AM staining, using laser-scanning confocal microscopy. Maturation and fertilization rates of GV oocytes and the developmental rates of subsequent embryos were significantly lower in vitrified samples (P < 0.05). The ATP content and Ca(2+) levels differed significantly in fresh and vitrified GV oocytes (P < 0.05). Most mitochondria were seen as large and homogenous aggregates (66.6%) in fresh GV oocytes compared to vitrified oocytes (50%). No significant differences in mitochondrial membrane potential were found between the groups. The lower maturation and fertilization rates of GV oocytes from vitrified ovaries may be due to changes in their mitochondrial function and distribution.
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Adenosina Trifosfato/metabolismo , Criopreservación , Oocitos/fisiología , Ovario/fisiología , Animales , Blastocisto/citología , Supervivencia Celular/fisiología , Femenino , Fertilización/fisiología , Fertilización In Vitro , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Mitocondrias/metabolismo , Mitocondrias/fisiología , Oocitos/metabolismo , Ovario/citología , VitrificaciónRESUMEN
During development there is a multitude of signaling events governing the assembly of the developing organism. Receptors for signaling molecules such as fibroblast growth factor receptor 2 (FGFR2) enable the embryo to communicate with the surrounding environment and activate downstream pathways. The neural cell adhesion molecule (NCAM) was first characterized as a cell adhesion molecule highly expressed in the nervous system, but recent studies have shown that it is also a signaling receptor. Using a novel single oocyte adaptation of the proximity ligation assay, we here show a close association between NCAM and FGFR2 in mouse oocytes and 2-cell embryos. Real-time PCR analyses revealed the presence of messenger RNA encoding key proteins in downstream signaling pathways in oocytes and early mouse embryos. In summary these findings show a co-localization of NCAM and FGFR2 in early vertebrate development with intracellular signaling pathways present to enable a cellular response.
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Antígeno CD56/metabolismo , Embrión de Mamíferos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Antígeno CD56/genética , Diferenciación Celular , Membrana Celular/metabolismo , Embrión de Mamíferos/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oocitos , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Transducción de SeñalRESUMEN
The cystatin family comprises a group of generally broadly expressed protease inhibitors. The Cres/Testatin subgroup (CTES) genes within the type 2 cystatins differs from the classical type 2 cystatins in having a strikingly reproductive tissue-specific expression, and putative functions in reproduction have therefore been discussed. We have performed evolutionary studies of the CTES genes based on gene searches in genomes from 11 species. Ancestors of the cystatin family can be traced back to plants. We have localized the evolutionary origin of the CTES genes to the split of marsupial and placental mammals. A model for the evolution of these genes illustrates that they constitute a dynamic group of genes, which has undergone several gene expansions and we find indications of a high degree of positive selection, in striking contrast to what is seen for the classical cystatin C. We show with phylogenetic relations that the CTES genes are clustered into three original groups, a testatin, a Cres, and a CstL1 group. We have further characterized the expression patterns of all human members of the subfamily. Of a total of nine identified human genes, four express putative functional transcripts with a predominant expression in the male reproductive system. Our results are compatible with a function of this gene family in reproduction.
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Cistatinas/genética , Evolución Molecular , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Humanos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis.
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Hibridación Genómica Comparativa , Células Madre Embrionarias/patología , Oncogenes/genética , Polimorfismo de Nucleótido Simple , Teratocarcinoma/patología , Aneuploidia , Línea Celular , Aberraciones Cromosómicas , Genoma Humano , HumanosRESUMEN
OBJECTIVE: To study the role of a synthetic insulin-like growth factor-I receptor (IGF-IR) antagonist, picropodophyllin, for mouse preimplantation embryo development in vivo and in vitro. DESIGN: In vitro and in vivo study. SETTING: Hospital-based research unit. ANIMALS: FVB/N mice and mouse embryos. INTERVENTION(S): The effect of picropodophyllin in mouse embryo development in vivo and in vitro, immunohistochemistry, ELISA, polymerase chain reaction. MAIN OUTCOME MEASURE(S): Embryo development, presence of IGF-IR, messenger RNA expression, IGF-I synthesis. RESULT(S): The effect of picropodophyllin on embryo development in vitro and in vivo was not reversible. Mice treated with picropodophyllin 1 to 3 days after mating had a reduced number of blastocysts, 40.5% versus 78.8%, and a higher number of embryos with delayed development, 48.6% versus 11.5%. Insulin-like growth factor-IR protein is present in both phosphorylated and nonphosphorylated form at all stages of embryo development. The relative IGF-IR messenger RNA expression was highest in the oocyte and reduced during development to blastocyst stage. Insulin-like growth factor-I in culture media was reduced after picropodophyllin treatment. CONCLUSION(S): We conclude that IGF-I has an important role in normal mouse embryo development and that its receptor plays an essential role in the embryonic genome activation process.
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Desarrollo Embrionario/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Podofilotoxina/análogos & derivados , Animales , Ratones , Podofilotoxina/farmacología , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/metabolismoRESUMEN
The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), specific to neurons and muscle, supplies aspartate to the cytosol and, as a component of the malate-aspartate shuttle, enables mitochondrial oxidation of cytosolic NADH, thought to be important in providing energy for neurons in the central nervous system. We describe AGC1 deficiency, a novel syndrome characterized by arrested psychomotor development, hypotonia, and seizures in a child with a homozygous missense mutation in the solute carrier family 25, member 12, gene SLC25A12, which encodes the AGC1 protein. Functional analysis of the mutant AGC1 protein showed abolished activity. The child had global hypomyelination in the cerebral hemispheres, suggesting that impaired efflux of aspartate from neuronal mitochondria prevents normal myelin formation.