139 SEX-SPECIFIC GENE EXPRESSION IN PORCINE PRE-IMPLANTATION EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 199
Author(s):  
D. Kradolfer ◽  
J. Knubben ◽  
V. Flöter ◽  
J. Bick ◽  
S. Bauersachs ◽  
...  
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
Current Knowledge ◽  
Critical Time ◽  
Blastocyst Stage ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Specific Gene ◽  
Embryo Survival ◽  
Specific Gene Expression

X-Chromosome inactivation in female mammals starts during early blastocyst stage with expression of the X-inactive specific transcript (XIST), which coats and silences the inactive X chromosome. However, this compensation is not complete in blastocysts, as a large number of X-linked transcripts are more highly expressed in female embryos than in males. Furthermore, the process of X chromosome inactivation is altered in IVF and cloned porcine embryos, possibly explaining problems of embryo survival with these techniques. The aim of this study was to gain more insights into the transcriptional dynamics of the porcine pre-implantation embryo, with a particular focus on sex-specific differences. RNA sequencing (RNA-Seq) was performed for individual blastocysts at 8, 10, and 12 days after ovulation, and the temporal development of sex-specific transcripts was analysed. German Landrace sows were cycle synchronized and inseminated with sperm of the same Pietrain boar. On Days 8, 10, and 12 post-insemination, sows were slaughtered and embryos were removed from the uterus using 10 mL of PBS (pH 7.4) per horn. Single embryos were shock frozen in liquid nitrogen and stored at –80°C until the extraction of RNA and DNA (AllPrep DNA/RNA Micro Kit, Qiagen, Valencia, CA, USA). Using the isolated DNA, the sex of the embryos was determined and 5 female and male embryos, respectively, were analysed per stage. Illumina TruSeq Stranded mRNA libraries (Illumina Inc., San Diego, CA, USA) were sequenced on a HiSEqn 2500 (Illumina Inc.), and 15 to 25 million 100-bp single-end reads were generated per sample. Reads were filtered and processed using Trimmomatic and mapped to the porcine genome assembly Sscrofa10.2 with TopHat2. Mapped reads were counted by the use of QuasR qCount based on the current National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) GFF3 annotation file. Statistical analysis of count data was performed with the BioConductor R (https://www.bioconductor.org/) package DESEqn 2. At all 3 stages, we found 7 Y-linked transcripts that were highly expressed in male embryos (EIF2S3, EIF1AY, LOC100624590, LOC100625207, LOC100624329, LOC102162178, LOC100624937). On the other hand, 47 X-linked transcripts showed increased expression in female blastocysts, most of them at all 3 time points. However, a small number of genes (DDX3X, LAMP2, and RPS6KA3) were more highly expressed in females at Days 8 and 10 but more highly expressed in males at Day 12. Three X-linked genes (OFD1, KAL1, and LOC100525092) were more highly expressed in male embryos, although only at a low fold change of 1.2 to 1.4. Furthermore, expression of 8 transcripts located on autosomes was higher in females. In conclusion, our study expands the current knowledge of sex-specific gene expression in 8- to 12-day-old porcine blastocysts, a critical time period during pre-implantation embryo development.

Allele-specific expression analysis reveals conserved and unique features of preimplantation development in equine ICSI embryos

2021 ◽  
Author(s):  
D E Goszczynski ◽  
P S Tinetti ◽  
Y H Choi ◽  
P J Ross ◽  
K Hinrichs
Keyword(s):  
Gene Expression ◽  
Early Development ◽  
X Chromosome ◽  
Dosage Compensation ◽  
Blastocyst Stage ◽  
X Chromosome Inactivation ◽  
Specific Gene ◽  
Cell Stage ◽  
Specific Gene Expression ◽  
Genome Activation

Abstract Embryonic genome activation and dosage compensation are major genetic events in early development. Combined analysis of single embryo RNA-seq data and parental genome sequencing was used to evaluate parental contributions to early development and investigate X-chromosome dynamics. In addition, we evaluated dimorphism in gene expression between male and female embryos. Evaluation of parent-specific gene expression revealed a minor increase in paternal expression at the 4-cell stage that increased at the 8-cell stage. We also detected eight genes with allelic expression bias that may have an important role in early development, notably NANOGNB. The main actor in X-chromosome inactivation, XIST, was significantly upregulated at the 8-cell, morula, and blastocyst stages in female embryos, with high expression at the latter. Sexual dimorphism in gene expression was identified at all stages, with strong representation of the X-chromosome in females from the 16-cell to the blastocyst stage. Female embryos showed biparental X-chromosome expression at all stages after the 4-cell stage, demonstrating the absence of imprinted X-inactivation at the embryo level. The analysis of gene dosage showed incomplete dosage compensation (0.5 < X:A < 1) in MII oocytes and embryos up to the 4-cell stage, an increase of the X:A ratio at the 16-cell and morula stages after genome activation, and a decrease of the X:A ratio at the blastocyst stage, which might be associated with the beginning of X-chromosome inactivation. This study represents the first critical analysis of parent- and sex-specific gene expression in early equine embryos produced in vitro.

scholarly journals Random X Inactivation and Extensive Mosaicism in Human Placenta Revealed by Analysis of Allele-Specific Gene Expression along the X Chromosome

PLoS ONE ◽  
2010 ◽  
Vol 5 (6) ◽  
pp. e10947 ◽  
Author(s):  
Joana Carvalho Moreira de Mello ◽  
Érica Sara Souza de Araújo ◽  
Raquel Stabellini ◽  
Ana Maria Fraga ◽  
Jorge Estefano Santana de Souza ◽  
...  
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
Human Placenta ◽  
X Inactivation ◽  
Specific Gene ◽  
Specific Gene Expression ◽  
Allele Specific

X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation

Development ◽  
2000 ◽  
Vol 127 (19) ◽  
pp. 4137-4145 ◽  
Author(s):  
I. Okamoto ◽  
S. Tan ◽  
N. Takagi
Keyword(s):  
X Chromosome ◽  
Ectopic Expression ◽  
Blastocyst Stage ◽  
X Chromosome Inactivation ◽  
X Inactivation ◽  
Chromosome Inactivation ◽  
Current View ◽  
Replication Banding ◽  
Xist Rna ◽  
Morula Stage

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6. 5.

scholarly journals Landscape of X chromosome inactivation across human tissues

Nature ◽  
2017 ◽  
Vol 550 (7675) ◽  
pp. 244-248 ◽  
Author(s):  
Taru Tukiainen ◽  
◽  
Alexandra-Chloé Villani ◽  
Angela Yen ◽  
Manuel A. Rivas ◽  
...  
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
Mammalian Cells ◽  
Phenotypic Diversity ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Phenotypic Traits ◽  
Human Tissues ◽  
X Chromosomes ◽  
Chromosomal Genes

Abstract X chromosome inactivation (XCI) silences transcription from one of the two X chromosomes in female mammalian cells to balance expression dosage between XX females and XY males. XCI is, however, incomplete in humans: up to one-third of X-chromosomal genes are expressed from both the active and inactive X chromosomes (Xa and Xi, respectively) in female cells, with the degree of ‘escape’ from inactivation varying between genes and individuals1,2. The extent to which XCI is shared between cells and tissues remains poorly characterized3,4, as does the degree to which incomplete XCI manifests as detectable sex differences in gene expression5 and phenotypic traits6. Here we describe a systematic survey of XCI, integrating over 5,500 transcriptomes from 449 individuals spanning 29 tissues from GTEx (v6p release) and 940 single-cell transcriptomes, combined with genomic sequence data. We show that XCI at 683 X-chromosomal genes is generally uniform across human tissues, but identify examples of heterogeneity between tissues, individuals and cells. We show that incomplete XCI affects at least 23% of X-chromosomal genes, identify seven genes that escape XCI with support from multiple lines of evidence and demonstrate that escape from XCI results in sex biases in gene expression, establishing incomplete XCI as a mechanism that is likely to introduce phenotypic diversity6,7. Overall, this updated catalogue of XCI across human tissues helps to increase our understanding of the extent and impact of the incompleteness in the maintenance of XCI.

Monoallelic Methylation of the Human Androgen Receptor Gene as Detected by the HUMARA Clonality Assay Does Not Consistently Reflect X-Chromosome Inactivation and Is Therefore Unreliable for Assessment of Clonal Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4566-4566
Author(s):  
Sabina Swierczek ◽  
Jaroslav Jelinek ◽  
Neeraj Agarwal ◽  
Andrew Wilson ◽  
Kimberly Hickman ◽  
...  
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
X Chromosome Inactivation ◽  
X Inactivation ◽  
Chromosome Inactivation ◽  
Cag Repeats ◽  
Clonal Hematopoiesis ◽  
Healthy Elderly ◽  
Polymorphic Genes ◽  
Exon 1

Abstract Abstract 4566 Even in the absence of a disease specific chromosomal marker, clonality can be assessed in somatic tissues of female origin by using assays based on the pattern of X-chromosome inactivation. The most widely used technique for quantifying X-chromosome inactivation is the HUMARA method that is based on the putative role of DNA methylation at CpG sites close to trinucleotide repeats in exon 1 in silencing of the AR locus. However, using the HUMARA method, we and others have observed that approximately 30% of healthy elderly volunteers appear to have clonal hematopoiesis. This observation is at odds with the concept that normal hematopoiesis is a polyclonal process, suggesting that the finding of monoclonality or oligoclonality in a high percentage of healthy volunteers by using the HUMARA method, is a technical artifact. To address this issue, we developed a clonality assay based on gene expression of five X-linked polymorphic genes and found no evidence of clonal hematopoiesis in healthy elderly volunteers, although we confirmed extreme skewing of X inactivation (consistent with monoallelic methylation of AR) in 30% of the study subject when analyzed by HUMARA (Swierczek et al., Blood 2008). In the present studies, we have validated the accuracy and reproducibility of our quantitative transcription-based clonality assay (qTCA) using two different methods for quantifying gene expression and compared the results with those obtained using the HUMARA method. DNA and RNA were extracted from peripheral blood samples from 31 healthy female volunteers (age in years as follows: range, 22-55; mean, 35; median, 34). RNA was reverse transcribed (RT) and analyzed by using our qTCA in which expression of three polymorphic genes (MPP1, IDS and FHL1), that are subject to X inactivation, is quantified by allele-specific, real-time RT-PCR. Based on DNA analysis, 25 of the 31 (80%) volunteers were polymorphic for at least one of the test genes. Results are reported as the percentage of each of the two single nucleotide polymorphisms (SNPs) that is present in the sample (e. g., 60% A; 40% G). PCR primers are designed to provide maximum discrimination between SNPs with >13 PCR cycles (i. e., 13 log2) separating true-positive from false-positive amplification. Aliquots of the isolated RNA from the test samples were sent to an independent investigator (JJ), at a separate institution, who was blinded to the results of our qTCA, and the allele ratio was determined by using a different technique (quantitative pyrosequencing). Comparison of the results, confirmed the accuracy and reproducibility of the two methods with coefficients of correlation for each gene as follows: (MPP1, r=0.9385; IDS, r=0.8565; FHL1, r=0.8657). One of the 25 informative females (4%) showed extreme skewing (SNP ratio >75%:25%) of X inactivation by both methods. Based on allelic differences in the number of CAG repeats, 29/31 participants were informative in the HUMARA. Among most of the samples, a good correlation was observed between the pattern of X chromosome inactivation as determined by HUMARA and that determined by both qTCA and quantitative pyrosequencing, however, 8/29 (27%) samples analyzed by the HUMARA showed extreme skewing of allele methylation (ratio >75%:25%). Of the 8 subjects with extreme skewing, 3 were homozygous (i. e., non-informative) for all of the X-chromosome polymorphic genes used in the qTCA. Samples from the 5 informative participants were analyzed by using the qTCA, and, in contrast to the HUMARA results, only one subject showed extreme skewing of the SNP ratio (the same subject as identified in the original qTCA). We also quantified HUMARA gene expression using the difference in the number of exon 1 CAG repeats between the two AR alleles as the polymorphic marker. These experiments showed that, of the 8 volunteers with skewing of X inactivation based on HUMARA, 5 had skewing of AR allele expression and 3 had expression of both AR alleles, indicating that the correlation between DNA methylation at the AR locus and AR mRNA transcription is inconsistent. In conclusion, we found a good correlation between the HUMARA and qTCA in some females; however, this was not the case in many healthy females both elderly and young. These experiments demonstrate the accuracy and reproducibility of the qTCA and confirmed that this technique is not subject to the artifact of aberrant skewing of X-inactivation due to monoallelic methylation of AR that limits the applicability and value of the HUMARA. Disclosures: No relevant conflicts of interest to declare.

Changes in DNA methylation during mouse embryonic development in relation to X-chromosome activity and imprinting

1990 ◽  
Vol 326 (1235) ◽  
pp. 299-312 ◽  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Embryonic Development ◽  
X Chromosome ◽  
De Novo ◽  
Germ Line ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Chromatin Configuration ◽  
Methylation Patterns

Changing DNA methylation patterns during embryonic development are discussed in relation to differential gene expression, changes in X-chromosome activity and genomic imprinting. Sperm DNA is more methylated than oocyte DNA, both overall and for specific sequences. The methylation difference between the gametes could be one of the mechanisms (along with chromatin structure) regulating initial differences in expression of parental alleles in early development. There is a loss of methylation during development from the morula to the blastocyst and a marked decrease in methylase activity. De novo methylation becomes apparent around the time of implantation and occurs to a lesser extent in extra-embryonic tissue DNA. In embryonic DNA, de novo methylation begins at the time of random X-chromosome inactivation but it continues to occur after X-chromosome inactivation and may be a mechanism that irreversibly fixes specific patterns of gene expression and X-chromosome inactivity in the female. The germ line is probably delineated before extensive de novo methylation and hence escapes this process. The marked undermethylation of the germ line DNA may be a prerequisite for X-chromosome reactivation. The process underlying reactivation and removal of parent-specific patterns of gene expression may be changes in chromatin configuration associated with meiosis and a general reprogramming of the germ line to developmental totipotency.

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