Basic helix-loop-helix transcription factor Tcf21 is downstream target of male sex-determining gene SRY
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
SUBMITTER: Michael Skinner
PROVIDER: E-GEOD-24666 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:A critical transcription factor required for mammalian male sex determination is SRY (sex determining region on the Y chromosome). The expression of SRY in precursor Sertoli cells is one of the initial events in testis development. The current study was designed to determine the impact of environmentally induced epigenetic transgenerational inheritance on SRY during gonadal sex determination in the male. The agricultural fungicide vinclozolin and vehicle control (DMSO) exposed gestating females (F0 generation) during gonadal sex determination promoted the transgenerational inheritance of differential DNA methylation in sperm of the F3 generation (great grand-offspring). The fetal gonads in F3 generation males were used to identify potential alterations in SRY binding sites in the developing Sertoli cells. Chromatin immunoprecipitation with an SRY antibody followed by genome-wide promoter tiling array (ChIP-Chip) was used to identify alterations in SRY binding. A total of 81 adjacent oligonucleotide sites and 173 single oligo SRY binding sites were identified to be altered transgenerationally in the Sertoli cell vinclozolin lineage F3 generation males. Observations demonstrate the majority of the previously identified normal SRY binding sites were not altered and the altered SRY binding sites were novel and new additional sites. The chromosomal locations, gene associations and potentially modified cellular pathways were investigated. In summary, environmentally induces epigenetic transgenerational inheritance of germline epimutations appears to alter the cellular differentiation and development of the precursor Sertoli cell SRY binding during gonadal sex determination that influence the developmental origins of adult onset testis disease. The experimental design involved the intraperitoneal exposure of gestating female rats to vinclozolin or a vehicle control (dimethyl sulfoxide, DMSO) transiently from embryonic days E8-E14. Sister littermates were divided into control and vinclozolin treatment groups and mated to similar males to minimize the genetic variation between the control and vinclozolin lineages. Sufficient females were used so no inbreeding (sibling or cousin) occurred in any generation. The F1 generation was bred within the lineage to generate the F2 generation and these F2 generation bred to generate the F3 generation. The only exposure was the F0 generation female. The F3 generation control and vinclozolin lineage embryonic day 13 (E13) embryos were collected and the gonads micro-dissected and then sexed with an SRY PCR protocol. The male gonads were pooled from a minimum of three different litters and the pools used to collect DNA. Three different experiments were performed to collect 3 control and vinclozolin E13 F3 generation testis pools, each with different animals (n=25 gonads/pool). The chromatin DNA (not denatured) from each pool was fragmented and used in an SRY chromatin immunoprecipitation (ChIP) procedure for each pool separately. The control and vinclozolin SRY ChIP DNA were paired for a competitive hybridization on a genome-wide promoter tiling array (ChIP-Chip) assay. The hybridization data obtained was used to identify the SRY binding sites that were different in the F3 generation vinclozolin versus control lineage in E13 testis.
2015-08-28 | E-GEOD-72469 | biostudies-arrayexpress
Project description:The current study investigates the direct effects of in utero vinclozolin exposure on the developing rat testis transcriptome. Vinclozolin is a commonly used fungicide in agriculture and is an endocrine disruptor with anti-androgenic activity. Previous studies have demonstrated that exposure to vinclozolin during embryonic gonadal sex determination induces epigenetic modifications of the germ line and transgenerational adult onset disease states that include spermatogenic cell defects, prostate disease, kidney disease, and tumor development. An investigation of the molecular actions of vinclozolin was initiated through an analysis of direct actions on the F1 generation embryonic testis development. Microarray analyses were performed to compare control and vinclozolin treated testis transcriptomes at embryonic day 13, 14 and 16. A total of 576 differentially expressed genes were identified and the major cellular functions and pathways associated with these altered transcripts were examined. The sets of regulated genes at the different development periods were found to be transiently altered and distinct. Interestingly, genes previously shown to be regulated during normal male sex determination were not altered by vinclozolin treatment. Categorization by major known functions of all 576 genes altered by in utero vinclozolin exposure demonstrates transcription, signaling, cytoskeletal and extra cellular matrix associated transcripts are highly represented. Specific cellular process and pathway analyses suggest the involvement of Wnt and calcium signaling, vascular development and epigenetic mechanisms as potential mediators of the direct F1 generation actions of vinclozolin. For Samples 1-12: We used microarrays to determine genes expressed differentially between control and in utero Vinclozolin treated E13, E14, and E16 rat testis. For Samples 13-16: We used microarrays to determine genes expressed differentially between control and in vitro Vinclozolin treated E13 cultured rat testis. For Samples 17-20: We used microarrays to determine genes expressed differentially between control and in vitro Flutamide treated rat E13 cultured testis. For Samples 1-12: RNA samples from two control groups are compared to two Vinclozolin treated groups for each E13, E14, E16 testis. For Samples 13-16: RNA samples from two control groups of E13 cultured testis are compared to two Vinclosolin treated groups of E13 cultured testis. For Samples 17-20: RNA samples from two control groups of E13 cultured testis are compared to two Flutamide treated groups of E13 cultured testis.
2010-02-24 | E-GEOD-20245 | biostudies-arrayexpress
Project description:The cascade of molecular events involved in mammalian sex determination has been shown to involve the SRY gene, but specific downstream events have eluded researchers for decades. The current study identifies one of the first direct downstream targets of the male sex-determining factor SRY as the basic-helix-loop-helix (bHLH) transcription factor TCF21. SRY was found to directly associate with the Tcf21 promoter SRY/SOX9 response element both in vitro and in vivo during male sex determination. TCF21 was found to promote an in vitro sex reversal of embryonic ovarian cells to promote precursor Sertoli cell differentiation. Therefore, SRY acts directly on the Tcf21 promoter to, in part, initiate a cascade of events associated with Sertoli cell differentiation and embryonic testis development.
2011-04-25 | GSE24666 | GEO
Project description:A major event in mammalian male sex determination is the induction of the testis determining factor Sry and its downstream gene Sox9. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. A modified ChIP-Chip analysis using a comparative hybridization was used to identify 71 direct downstream binding targets for SRY and 109 binding targets for SOX9. Interestingly, only 5 gene targets overlapped between SRY and SOX9. In addition to the direct response element binding gene targets, a large number of atypical binding gene targets were identified for both SRY and SOX9. Bioinformatic analysis of the downstream binding targets identified gene networks and cellular pathways potentially involved in the induction of Sertoli cell differentiation and testis development. The specific DNA sequence binding site motifs for both SRY and SOX9 were identified. Observations provide insights into the molecular control of male gonadal sex determination. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. At embryonic day 13 (E13) of pregnancy rats were euthanized and embryonic gonads were collected for chromatin. A modified ChIP-Chip analysis using a comparative hybridization was used to identify direct downstream binding targets for SRY and for SOX9. Then, bioinformatic analysis of the downstream binding targets was done to identify gene networks and cellular pathways that are potentially involved in the induction of Sertoli cell differentiation and testis development.
2014-12-05 | E-GEOD-63855 | biostudies-arrayexpress
Project description:A major event in mammalian male sex determination is the induction of the testis determining factor Sry and its downstream gene Sox9. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. A modified ChIP-Chip analysis using a comparative hybridization was used to identify 71 direct downstream binding targets for SRY and 109 binding targets for SOX9. Interestingly, only 5 gene targets overlapped between SRY and SOX9. In addition to the direct response element binding gene targets, a large number of atypical binding gene targets were identified for both SRY and SOX9. Bioinformatic analysis of the downstream binding targets identified gene networks and cellular pathways potentially involved in the induction of Sertoli cell differentiation and testis development. The specific DNA sequence binding site motifs for both SRY and SOX9 were identified. Observations provide insights into the molecular control of male gonadal sex determination. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. At embryonic day 13 (E13) of pregnancy rats were euthanized and embryonic gonads were collected for chromatin. A modified ChIP-Chip analysis using a comparative hybridization was used to identify direct downstream binding targets for SRY and for SOX9. Then, bioinformatic analysis of the downstream binding targets was done to identify gene networks and cellular pathways that are potentially involved in the induction of Sertoli cell differentiation and testis development.
2014-12-05 | E-GEOD-63854 | biostudies-arrayexpress
Project description:Embryonic day 13 (E13), E14, and E16 rat testes and ovaries were used for microarray analysis, as well as E13 testis organ cultures that undergo testis morphogenesis and develop seminiferous cords in vitro. A list of 109 genes resulted from a selective analysis for genes present in male gonadal development and with a 1.5-fold change in expression between E13 and E16. Characterization of these 109 genes potentially important for testis development revealed that cytoskeletal-associated proteins, extracellular matrix factors, and signaling factors were highly represented. Throughout the developmental period (E13-E16), sex-enriched transcripts were more prevalent in the male with 34 of the 109 genes having testis-enriched expression during sex determination. In ovaries, the total number of transcripts with a 1.5-fold change in expression between E13 and E16 was similar to the testis, but none of those genes were both ovary enriched and regulated during the developmental period. Genes conserved in sex determination were identified by comparing changing transcripts in the rat analysis herein, to transcripts altered in previously published mouse studies of gonadal sex determination. A comparison of changing mouse and rat transcripts identified 43 genes with species conservation in sex determination and testis development. Profiles of gene expression during E13-E16 rat testis and ovary development are presented and candidate genes for involvement in sex determination and testis differentiation are identified. Analysis of cellular pathways did not reveal any specific pathways involving multiple candidate genes. However, the genes and gene network identified influence numerous cellular processes with cellular differentiation, proliferation, focal contact, RNA localization, and development being predominant. Experiment Overall Design: RNA samples from two different groups of 20-40 pooled gonads for each sample. Embrionic testis and ovaries of age E13, E14, or E16, and E13 testis cultured for three days were compared to each other
2008-04-07 | E-GEOD-10558 | biostudies-arrayexpress
Project description:A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided. The F0 generation females were exposed to a vehicle (dimethylsulfoxide DMSO) as control or to vinclozolin, as described in the Methods. The F1 generation offspring were bred to generate the F2 generation and the F2 generation offspring were bred to generate the F3 generation offspring. The timed pregnant F2 generation females were used to isolate the F3 generation control and vinclozolin lineage fetal gonads at the E13 and E16 time points. The F3 generation E13 PGC and E16 prospermatogonia were isolated. DNA was isolated from the freshly isolated cells to examine DNA methylation by methylated DNA immunoprecipitation (MeDIP) followed by analysis on a genome-wide promoter tiling array (Chip) using a comparative hybridization MeDIP-Chip analysis between control and vinclozolin lineage samples as described in Methods. This allowed a comparison of the epigenome alterations in F3 vinclozolin lineage germ cells at E13 and E16. Three separate experimental comparisons of control and vinclozolin-lineage animals involving different germ cell isolations were analyzed with three different MeDIP-Chip analyses at each time point.
2014-07-17 | E-GEOD-59510 | biostudies-arrayexpress
Project description:Gonadal sex determining (GSD) genes that initiate fetal ovarian and testicular development and differentiation are expressed in the cells of the urogenital ridge that differentiate as somatic support cells (SSCs), i.e., granulosa cells of the ovary and Sertoli cells of the testis. To identify potential new mammalian GSD genes, we analyzed the gene expression differences between XX and XY SSCs cells isolated from the gonads of embryonic day (E) 13 mouse fetuses carrying an EGFP reporter transgene expressed specifically in SSCs. In addition, genome wide expression differences between XX and XY E13 whole gonads were examined. Newly identified differentially expressed transcripts are potential GSD genes involved in unexplained human sex reversal cases. Experiment Overall Design: Two seperate RNA samples were obtained from E13 XX and XY sorted EGFP+ cells, and two seperate RNA samples were obtained from E13 XX and XY pooled gonads. Approximately 20ng of total RNA from each sample was used to generate biotin-labelled cDNA. Approximately 2.5ug biotin labelled cDNA of each sample was used for each Mouse Genome 430v2.0 GeneChip array (Affymetrix). Significantly differentially expressed transcripts were identified using R/maanova. Statistical significance was determined at a false discovery rate value of equal to or less than 1%.
2007-11-30 | E-GEOD-4928 | biostudies-arrayexpress
Project description:A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germline is associated with primordial germ cell development and during fetal gonadal sex determination. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation primordial germ cell transcriptome and epigenome (DNA methylation) was altered transgenerationally. Interestingly, the differential DNA methylation regions (DMR) and altered transcriptomes were distinct between the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DMR and transcriptional alterations were observed in the E13 PGC than E16 germ cells. Observations demonstrate an altered transgenerational epigenetic reprogramming and function of the primordial germ cells and subsequent male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided. The combined observations demonstrate ancestral exposure of a gestating female during fetal gonadal sex determination can promote transgenerational alterations in the primordial germ cell and subsequent male germline epigenetic and transcriptional programming. This altered germline programming leads to the epigenetic transgenerational inheritance of disease and phenotypic variation. Observations support the role of the primordial germ cell programming in the molecular mechanism involved and provides insights into the molecular mechanisms that control the epigenetic transgenerational inheritance phenomena. Results suggest a cascade of epigenetic and transcriptional events during germ cell development is needed to obtain the mature germline epigenome that is then transmitted transgenerationally. RNA samples from PGC of 2 F3-control lineage groups were compared to PGC of 2 F3-vinclozolin lineage groups for two embryonic age E13 and E16
2013-01-17 | E-GEOD-43559 | biostudies-arrayexpress
Project description:The neural stem cell decision to self-renew or differentiate is tightly regulated by its microenvironment. Here, we have asked about this microenvironment, focusing on growth factors in the embryonic cortex at a time when it is largely comprised of neural precursor cells (NPCs) and newborn neurons. We show that cortical NPCs secrete factors that promote their maintenance while cortical neurons secrete factors that promote differentiation. To define factors important for these activities, we used transcriptome profiling to identify ligands produced by NPCs and neurons, cell surface mass spectrometry to identify receptors on these cells, and computational modeling to integrate these data. The resultant model predicts a complex growth factor environment with multiple autocrine and paracrine interactions. We tested this communication model, focusing on neurogenesis, and identified IFNγ, Nrtn and glial-derived neurotrophic factor (GDNF) as ligands with unexpected roles in promoting neurogenic differentiation of NPCs in vivo. We prepared E16 cortical neuron, E13 cortical precursor and co-cultured E16 cortical neuron and E13 cortical precursor cultures from three independent biological replicates.
2016-08-23 | E-GEOD-84482 | biostudies-arrayexpress