The presence of the Y-chromosome, not the absence of the second X-chromosome, alters the mRNA levels stored in the fully grown XY mouse oocyte
ABSTRACT: The oocytes of B6.Y(TIR) sex-reversed female mouse mature in culture but fail to develop after fertilization because of their cytoplasmic defects. To identify the defective components, we compared the gene expression profiles between the fully-grown oocytes of B6.Y(TIR) (XY) females and those of their XX littermates by cDNA microarray. 173 genes were found to be higher and 485 genes were lower in XY oocytes than in XX oocytes by at least 2-fold. We compared the transcript levels of selected genes by RT-PCR in XY and XX oocytes, as well as in XO oocytes missing paternal X-chromosomes. All genes tested showed comparable transcript levels between XX and XO oocytes, indicating that mRNA accumulation is well adjusted in XO oocytes. By contrast, in addition to Y-encoded genes, many genes showed significantly different transcript levels in XY oocytes. We speculate that the presence of the Y-chromosome, rather than the absence of the second X-chromosome, caused dramatic changes in the gene expression profile in the XY fully-grown oocyte. We compared the gene expression profiles between the fully-grown oocytes of B6.Y(TIR) (XY) females and those of their XX littermates by cDNA microarray Mouse GV oocytes of B6.Y(TIR) were collected for RNA extraction and hybridization to Affymetrix microarray. We sought to extract the differentially expressed genes in the XY oocytes.
Project description:Sex-reversed ‘XYSry-’ female mice that lack Sry due to the 11 kb deletion Srydl1Rlb have very limited fertility, partly due to the effects of posessing only a single X chromosome. However, the fertility deficit is even worse in sex-reversed XY females than in XO females, implicating Y-linked genes in the further loss of fertility. Transgenic addition of Yp-linked genes to XO females and also to normal XX females implicated Zfy2 (but not the related Zfy1) as the cause of this effect. This study examines the transcriptional effects of Zfy2 and Zfy1 in GV oocytes from normal XX females. 18 samples representing 3 biological replicates from each of 6 genotypes. Genotypes are XX (normal control); XX,Zfy2-nf (control with non-functional Zfy2 transgene); XX,Zfy2 (with Zfy2 transgene); XX,Zfy2+Eif2s3y (contaminated sample, XX with Zfy2 transgene and also an Eif2s3y transgene in proportion of the cells), XX,Zfy1-lo (with single-copy Zfy1 transgene); XX-Zfy1-hi (with multi-copy Zfy1 transgene).
Project description:We analyzed the differentiation of the bipotential gonad into a testis or an ovary in 2 strains of mice - 129S1/SvImJ (129S1) and C57BL/6J (B6). Our results provide a high resolution view of the initiation and canalization of the differentiation of the gonad. It also reveals global differences in the transcriptome between 129S1 and B6 mice. Total RNA was obtained from XX and XY embryonic mouse gonads at 6 equally spaced time points between embryonic day (E) 11.0 and E12.0 from 129S1 and B6 mice. Mice were staged using tail somite numbers
Project description:Mice lacking the function of the PcG protein CBX2 (also known as M33) show defects in gonadal, adrenal, and splenic development. In particular, XY knockout mice develop ovaries but not testes, and the gonads are hypoplastic in both sexes. To clarify the molecular basis of the defects, RNA prepared from both wild type and KO gonads were subjected to microarray analyses. Mouse embryonic gonads of Cbx2 knockout (KO) were sectioned and recovered at E11.5 for RNA extraction and hybridization on Affymetrix microarrays. Four different genotypes of embryos at E11.5 (XY WT, XX WT, XY Cbx2 KO, and XX Cbx2 KO) were frozen in OCT compound without fixation. They were sectioned to 30 μm slices and stained with hematoxylin. The developing gonadal cells localized by GATA4 immunostaining and morphology were prepared using a Laser Microdissection System. The specimens prepared from two or three individuals were combined into one group, and three groups were prepared for each genotype.
Project description:This project defines the transcriptomes of XO (male) and XX (female or mutant pseudo-female) Caenorhabditis nematodes. The data allow the overall composition and sexual regulation of the transcriptome within a single species to be determined. In addition, the five related species studied allow meta-comparisons between them. Because two of the five (C. elegans and C. briggsae) produce a self-fertile XX hermaphrodite, while the XX sex in the remaining three (C. japonica, C. remanei, and C. brenneri) are true females, the data are particularly useful for inferring effects of sexual mode on genome-wide gene expression. L4 larvae and adults were pooled for each sex for five species (C. elegans, C. briggsae, C. japonica, C. brenneri, and C. remanei). Each of these 10 species-sex combinations was replicated three times, for a total of 30 samples.
Project description:2 x 2 design, with 2 pooled total RNA samples (5 hemibrains per sample) per genotype (40,XY or 39,XY*O) hybridised to Mouse Gene 1.0 ST array to determine effect of genotype on brain gene expression
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%.
Project description:RNA-Seq was performed on mESCs generated from F1 hybrids from reciprocal crosses of C57BL/6J and CAST/EiJ, denoted B and C hereafter; cell lines are designated as BC or CB, with the maternal line symbolized first Overall design: 3 XX and 3 XY lines were used from each cross as well as two XO lines for a total of 14 samples.
Project description:This experiment depicts RNA-Seq datasets from wild type XY male and XX female, as well as sex chromosomally abnormal XO female (Turner syndrome) and XX male (Klinefelter variant syndrome) mouse germ cells before, during and after germline reprogramming. This range from E6.5 epiblasts, fluorescence activated cell sorted (FACS) highly purified populations of germ cells (EGFP-positive) and gonadal somatic cells (EGFP-negative) from both sexes at E9.5, E11.5, E12.5, E14.5, E15.5, E16.5 and E18.5, as well as purified spermatogonia and leptotene / zygotene spermatocytes from P2 and P11 males, respectively. Non-gonadal somatic cell control datasets were generated from male and female E14.5 liver and tail. Germ cells from individual embryos were processed to make cDNA libraries and served as biological replicates. We generated in total 184 libraries for our analysis from 60 separate conditions.
Project description:Here we exploit the essential process of X-chromosome dosage compensation to elucidate basic mechanisms that control the assembly, genome-wide binding, and function of gene regulatory complexes that act over large chromosomal territories. We demonstrate that a subunit of C. elegans MLL/COMPASS, a gene-activation complex, acts within the dosage compensation complex (DCC), a condensin complex, to target the DCC to both X chromosomes of hermaphrodites and thereby reduce chromosome-wide gene expression. The DCC binds to two categories of sites on X: rex sites that recruit the DCC in an autonomous, sequence- dependent manner, and dox sites that reside primarily in promoters of expressed genes and bind the DCC robustly only when attached to X. We find that DCC mutants that abolish rex-site binding do not eliminate dox-site binding, but instead reduce it to the level observed at autosomal binding sites in wild-type animals. Changes in DCC binding to these non-rex sites occur throughout development and correlate with transcriptional activity of adjacent genes. Moreover, autosomal DCC binding is enhanced by rex-site binding in cis in X-autosome fusion chromosomes. Thus, dox and autosomal sites exhibit similar binding properties. Our data support a model for DCC binding in which low-level DCC binding at dox and autosomal sites is dictated by intrinsic properties correlated with high transcriptional activity. Sex-specific DCC recruitment to rex sites then greatly elevates DCC binding to dox sites in cis, which lack intrinsically high DCC affinity on their own. We also show here that the C. elegans DCC achieves dosage compensation through its effects on transcription. ChIP-chip experiments using antibodies against DPY-27, SDC-3, DPY-30, DPY-26, MIX-1, SMC-4, ASH-2 in wild-type embryos. ChIP-chip experiments using antibodies against SDC-3, DPY-27, DPY-30, ASH-2, and IgG in different DCC mutants. ChIP-chip experiments using antibodies against RNA Pol II (hypophosphorylated and S2 and S5) in wild type and sdc-2 partial loss of function mutants.
Project description:In many species, a dosage compensation complex (DCC) is targeted to X chromosomes of one sex to equalize levels of X gene products between males (1X) and females (2X). Here we identify cis-acting regulatory elements that target the C. elegans X chromosome for repression by the DCC. The DCC binds to discrete, dispersed sites on X of two types. rex sites recruit the DCC in an autonomous, DNA sequence-dependent manner using a 12 bp consensus motif that is enriched on X. This motif is critical for DCC binding, is clustered in rex sites, and confers much of X-chromosome specificity. Motif variants enriched on X by 3.8-fold or more are highly predictive (95%) for rex sites. In contrast, dox sites lack the X-enriched variants and cannot bind the DCC when detached from X. dox sites are more prevalent than rex sites and, unlike rex sites, reside preferentially in promoters of some expressed genes. These findings fulfill predictions for a targeting model in which the DCC binds to recruitment sites on X and disperses to discrete sites lacking autonomous recruitment ability. To relate DCC binding to function, we identified dosage-compensated and non-compensated genes on X. Unexpectedly, many genes of both types have bound DCC, but many do not, suggesting the DCC acts over long distances to repress X gene expression. Remarkably, the DCC binds to autosomes, but at far fewer sites and rarely at consensus motifs. DCC disruption causes opposite effects on expression of X and autosomal genes. The DCC thus acts at a distance to impact expression throughout the genome. Experiment Overall Design: For microarray analysis, the nematode strains and number of experiments was as follows: TY2222, her-1(hv1y101); xol-1(y9) sdc-2(y74) unc-9(e101), XO embryos (8 biological replicas and 6 wild-type XX embryos controls ), dpy-27(y57) XX embryos (3 biological replicas and 3 wild-type XX embryos controls), and sdc-2(y93, RNAi) XX embryos (3 biological replicas and 3 wild-type XX embryos controls).