Germ cell nuclear factor regulates gametogenesis in developing gonads.
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ABSTRACT:
SUBMITTER: Sabour D
PROVIDER: S-EPMC4139263 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
Publications
Germ cell nuclear factor regulates gametogenesis in developing gonads.
Sabour Davood D Xu Xueping X Chung Arthur C K AC Le Menuet Damien D Ko Kinarm K Tapia Natalia N Araúzo-Bravo Marcos J MJ Gentile Luca L Greber Boris B Hübner Karin K Sebastiano Vittorio V Wu Guangming G Schöler Hans R HR Cooney Austin J AJ
PloS one 20140820 8
Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ c ...[more]
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Project description:Expression of germ cell nuclear factor (GCNF, Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads. For RNA isolation, PGCs were sorted by FACS, collected by centrifugation, lysed in RLT buffer (QIAGEN), and processed using the RNeasy micro and mini kits with on-column DNase digestion as per the manufacturer’s instructions. Integrity of RNA samples was quality checked using a 2100 Bioanalyzer (Agilent). When possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA Amplification Kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hr of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality checked on a 2100 Bioanalyser and hybridized as biological or technical duplicates for 18 hr onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader and accompanying software. Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. 8 samples were analyzed: ESC++, ESC control Oct4-GFP, 1 replicate ESC--, ESC Gcnf -/- Oct4-GFP, 1 replicate D15++, Day 15 in vitro derived PGCs from GCNF +/+ Oct4-GFP ESC, 1 replicate D15--, Day 15 in vitro derived PGC from GCNF -/- Oct4-GFP ESC, 1 replicate D12++, Day 12 in vitro derived PGC from GCNF +/+ Oct4-GFP ESC, 1 replicate D12--, Day 12 in vitro derived PGC from GCNF -/- Oct4-GFP ESC, 1 replicate EpiSC, EpiESC, 2 replicates
2015-05-08 | E-GEOD-57506 | biostudies-arrayexpress
Project description:Expression of germ cell nuclear factor (GCNF, Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads. For RNA isolation, PGCs were sorted by FACS, collected by centrifugation, lysed in RLT buffer (QIAGEN), and processed using the RNeasy micro and mini kits with on-column DNase digestion as per the manufacturer’s instructions. Integrity of RNA samples was quality checked using a 2100 Bioanalyzer (Agilent). When possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA Amplification Kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hr of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality checked on a 2100 Bioanalyser and hybridized as biological or technical duplicates for 18 hr onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader and accompanying software. Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab.
2015-05-08 | GSE57506 | GEO
Project description:Gonadotropin-regulated testicular RNA helicase (GRTH) (GRTH/DDX25), is a testis-specific protein essential for completion of spermatogenesis. Transgenic mice carrying 5'-flanking regions of the GRTH gene/green fluorescence protein (GFP) reporter revealed a region (-6.4/-3.6 kb) which directs its expression in germ cells (GCs) via androgen action. This study identifies a functional cis-binding element on the GRTH gene for GC nuclear factor (GCNF) (GCNF/RTR) required to regulate GRTH gene expression in postmeiotic testis GCs and explore the action of androgen on GCNF and GRTH transcription/expression. GCNF expression decreased in mice testis upon flutamide (androgen receptor antagonist) treatment, indicating the presence of an androgen/GCNF network to direct GRTH expression in GC. Binding studies and chromatin immunoprecipitation demonstrated specific association of GCNF to a consensus half-site (-5270/-5252) of the GRTH gene in both round spermatids and spermatocytes, which was abolished by flutamide treatment in round spermatids. Moreover, flutamide treatment of wild-type mice caused selective reduction of GCNF and GRTH in round spermatids. GCNF knock-down in seminiferous tubules from GRTH-transgenic mice (dark zone, round spermatid rich) caused decreased GFP expression. Exposure of tubules to flutamide caused decrease in GCNF and GFP expression, whereas androgen exposure induced significant increase. Our studies provide evidence for actions of androgen on GCNF cell-specific regulation of GRTH expression in GC. GRTH associates with GCNF mRNA, its absence caused increase on GCNF expression and mRNA stability indicative of a negative autocrine regulation of GCNF by GRTH. These in vivo/in vitro models link androgen actions to GC through GCNF, as regulated transfactor that controls transcription/expression of GRTH.
| S-EPMC4664233 | biostudies-literature
Project description:LIM homeobox 8 (Lhx8) is a germ cell-specific transcription factor essential for the development of oocytes during early oogenesis. In mice, Lhx8 deficiency causes postnatal oocyte loss and affects the expression of many oocyte-specific genes. The aims of this study were to characterize the bovine Lhx8 gene, determine its mRNA expression during oocyte development and early embryogenesis, and evaluate its interactions with other oocyte-specific transcription factors. The bovine Lhx8 gene encodes a protein of 377 amino acids. A splice variant of Lhx8 (Lhx8_v1) was also identified. The predicted bovine Lhx8 protein contains two LIM domains and one homeobox domain. However, one of the LIM domains in Lhx8_v1 is incomplete due to deletion of 83 amino acids near the N terminus. Both Lhx8 and Lhx8_v1 transcripts were only detected in the gonads but none of the somatic tissues examined. The expression of Lhx8 and Lhx8_v1 appears to be restricted to oocytes as none of the transcripts was detectable in granulosa or theca cells. The maternal Lhx8 transcript is abundant in GV and MII stage oocytes as well as in early embryos but disappear by morula stage. A nuclear localization signal that is required for the import of Lhx8 into nucleus was identified, and Lhx8 is predominantly localized in the nucleus when ectopically expressed in mammalian cells. Finally, a novel interaction between Lhx8 and Figla, another transcription factor essential for oogenesis, was detected. The results provide new information for studying the mechanisms of action for Lhx8 in oocyte development and early embryogenesis.
| S-EPMC5055334 | biostudies-literature
Project description:BackgroundThe germ cell nuclear factor (GCNF, also known as retinoid acid receptor-related testis-associated receptor, neuronal cell nuclear receptor or NR6A1) is an orphan receptor in the nuclear receptor superfamily found in mammals, amphibians and fish. The mouse Gcnf gene is expressed in the placenta and the developing nervous system and germ cells, and responds to retinoic acid.ResultsWe have defined the intron-exon structure of the mouse Gcnf gene and found that it contains 11 exons. Exons 1-4 encode the 75 amino acid amino-terminal domain and exon 4 also encodes the core DNA-binding domain. The carboxy-terminal extension is encoded by exon 5, exons 6 and 7 encode the hinge region, and exons 7-11 encode the putative ligand-binding domain. Unusually, the two zinc-finger motifs in the DNA-binding domain are encoded by separate exons.ConclusionsThe protein-coding region of GCNF is contained in 11 exons. The genomic structure of this nuclear receptor gene will be useful for further studies.
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Project description:The meiotic cell cycle is required for production of fertilization-competent gametes. Germ cell meiotic commitment requires expression of Stimulated by retinoic acid gene 8 (Stra8), which is transcriptionally activated by retinoic acid (RA). Meiotic suppression in embryonic male germ cells is believed to result from sex-specific differences in CYP26B1-catalyzed RA metabolism in the developing gonads. Here we show in mice that RA-induced Stra8 transcription is epigenetically controlled and requires a co-activator that binds proximal to the RA response elements (RAREs) in the Stra8 promoter. Embryonic male germ cells exposed in utero to the class I/II histone deacetylase (HDAC) inhibitor, trichostatin-A (TSA), show premature Stra8 activation and meiotic entry without altered Cyp26b1 expression. We also show that Stra8 expression is detectable and physiologically regulated in adult mouse ovaries. Further, oogenesis induction in adult females using TSA is associated with Stra8 activation, and these events are absent in mice deficient in the RA precursor vitamin A. Finally, all of the actions of TSA in premeiotic germ cells in vitro and in mouse ovaries in vivo can be reproduced with the small molecule HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA). Thus, the ability of RA to transcriptionally induce expression of the meiosis-commitment gene, Stra8, is epigenetically controlled and this process involves a novel co-activator that functions upstream of the RAREs. These events not only coordinate the sex-specific timing of meiotic entry during embryogenesis, but also contribute to the regulation of oogenesis in adult female mammals.
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Project description:Transitions in cell states are controlled by combinatorial actions of transcription factors. BLIMP1, the key regulator of primordial germ cell (PGC) specification, apparently acts together with PRDM14 and AP2?. To investigate their individual and combinatorial functions, we first sought an in vitro system for transcriptional readouts and chromatin immunoprecipitation sequencing analysis. We then integrated this data with information from single-cell transcriptome analysis of normal and mutant PGCs. Here we show that BLIMP1 binds directly to repress somatic and cell proliferation genes. It also directly induces AP2?, which together with PRDM14 initiates the PGC-specific fate. We determined the occupancy of critical genes by AP2?-which, when computed altogether with those of BLIMP1 and PRDM14 (both individually and cooperatively), reveals a tripartite mutually interdependent transcriptional network for PGCs. We also demonstrate that, in principle, BLIMP1, AP2? and PRDM14 are sufficient for PGC specification, and the unprecedented resetting of the epigenome towards a basal state.
| S-EPMC3796875 | biostudies-literature
Project description:Normal gonad development assures the fertility of the individual. The properly functioning gonads must contain a sufficient number of the viable germ cells, possess a correct architecture and tissue structure, and assure the proper hormonal regulation. This is achieved by the interplay between the germ cells and different types of somatic cells. N-cadherin coded by the Cdh2 gene plays a critical role in this interplay. To gain an insight into the role of N-cadherin in the development of mouse gonads, we used the Cre-loxP system to knock out N-cadherin separately in two cell lines: the SF1+ somatic cells and the OCT4+ germ cells. We observed that N-cadherin plays a key role in the survival of both female and male germ cells. However, the N-cadherin is not necessary for the differentiation of the Sertoli cells or the initiation of the formation of testis cords or ovigerous cords. In the later stages of gonad development, N-cadherin is important for the maintenance of testis cord structure and is required for the formation of steroidogenic cells. In the ovaries, N-cadherin is necessary for the formation of the ovarian follicles. These results indicate that N-cadherin plays a major role in gonad differentiation, structuralization, and function.
| S-EPMC6952792 | biostudies-literature
Project description:Sex determination (SD) is a highly diverse and complex mechanism. In vertebrates, one of the first morphological differences between the sexes is the timing of initiation of the first meiosis, where its initiation occurs first in female and later in male. Thus, SD is intimately related to the responsiveness of the germ cells to undergo meiosis in a sex-specific manner. In some vertebrates, it has been reported that the timing for meiosis entry would be under control of retinoic acid (RA), through activation of Stra8. In this study, we used a fish model species for sex determination and lacking the stra8 gene, the Japanese medaka (Oryzias latipes), to investigate the connection between RA and the sex determination pathway. Exogenous RA treatments act as a stress factor inhibiting germ cell differentiation probably by activation of dmrt1a and amh. Disruption of the RA degrading enzyme gene cyp26a1 induced precocious meiosis and oogenesis in embryos/hatchlings of female and even some males. Transcriptome analyzes of cyp26a1-/-adult gonads revealed upregulation of genes related to germ cell differentiation and meiosis, in both ovaries and testes. Our findings show that germ cells respond to RA in a stra8 independent model species. The responsiveness to RA is conferred by sex-related genes, restricting its action to the sex differentiation period in both sexes.
| S-EPMC7848095 | biostudies-literature
Project description:Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor α1-GFRα1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in mammalian testes. In nonmammalian species, Gdnf and Gfrα1 orthologs have been found but their functions remain poorly investigated in the testes. Considering this background, this study aimed to understand the roles of the Gdnf-Gfrα1 signaling pathway in zebrafish testes by combining in vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibit two paralogs for Gndf (gdnfa and gdnfb) and its receptor, Gfrα1 (gfrα1a and gfrα1b), in accordance with a teleost-specific third round of whole genome duplication. Expression analysis further revealed that both ligands and receptors were expressed in zebrafish adult testes. Subsequently, we demonstrated that gdnfa is expressed in the germ cells, while Gfrα1a/Gfrα1b was detected in early spermatogonia (mainly in types Aund and Adiff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf promoted the creation of new available niches by stimulating the proliferation of both type Aund spermatogonia and their surrounding Sertoli cells but without changing pou5f3 mRNA levels. Strikingly, Gdnf also inhibited late spermatogonial differentiation, as shown by the decrease in type B spermatogonia and down-regulation of dazl in a co-treatment with Fsh. Altogether, our data revealed that a germ cell-derived factor is involved in maintaining germ cell stemness through the creation of new available niches, supporting the development of spermatogonial cysts and inhibiting late spermatogonial differentiation in autocrine- and paracrine-dependent manners.
| S-EPMC9030868 | biostudies-literature