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Transcriptome high-throughput sequencing derived from mouse blastocysts with artificial oocyte activation treatment

ABSTRACT: Intracytoplasmic sperm injection (ICSI) has been an effective infertility treatment. Nevertheless, ICSI failures still occurred. One of the factors associated with ICSI failure is oocyte activation deficiency (AOD). The most commonly applied method of artificial oocyte activation (AOA) in humans includes ionophore. Although AOA is performed as a routine process in many assisted reproduction centers, up to date, there is no relevant study to expound whether AOA procedures increase developmental risks by disturbing subsequent gene expression during the embryonic development stages. Our results for the first time provide a profile of the changes in the global patterns of gene expression in AOA treatment versus ICSI generated mouse early embryos. In particular, we focus on the expression changes of imprinted genes. Another key observation in this study is that AOA treatment affects imprinted gene Igf2r expression and mehtylation, which is regulated by the imprinted Airn macro long non-coding (lnc) RNA.

ORGANISM(S): Mus musculus

PROVIDER: GSE155948 | GEO | 2021/11/10

REPOSITORIES: GEO

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Airn transcriptional overlap but not its lncRNA products induce imprinted Igf2r silencing.

Project description:To test if the imprinted long non-coding RNA (lncRNA) Airn transcription or its product silences the protein-coding Igf2r gene, we shortened the endogenous lncRNA to four different lengths by inserting polyadenylation (polyA) cassettes on the paternal chromosome via homologous recombination in ES cells. ES cell differentiation was used to recapitulate the developmental onset of Airn and Igf2r imprinted expression and polyA cassettes inserted before (T3, T16, T27) or after (T31, T51) the Igf2r promoter successfully truncated Airn, with the exception of T27. RNA hybridization to a tiling array (MIRTA) demonstrated loss of Airn upstream of the Igf2r promoter (except T27) and absence of novel spliced variants in all truncation alleles. To further test the transcriptional overlap model we moved the Airn promoter ~700bp before the Igf2r TSS in ES cells that lack an endogenous paternal Airn promoter and called this cell line FAP (Forward-Airn-Promoter). RNA hybridization to a tiling array (MIRTA) demonstrated that the moved Airn promoter expresses Airn in wildtype orientation and terminates at the wildtype 3' end. ES cell lines expressing different Airn variants were differentiated on gelatinized dishes after feeder cell depletion and LIF withdrawal by 0.27M-BM-5M retinoic acid for 5 days and Airn length was assayed.

2012-12-20 | E-GEOD-41444 | biostudies-arrayexpress

Airn transcriptional overlap but not its lncRNA products induce imprinted Igf2r silencing.

2012-12-20 | GSE41444 | GEO

The effect of ionomycin-induced oocyte activation on multiple morphological abnormalities of the sperm flagella

Project description:Artificial oocyte activation (AOA) is considered an effective method to improve clinical outcomes in patients with some forms of male factor infertility and does not increase the risk of birth defects. However, the effects of AOA on patients with multiple morphological abnormalities of the sperm flagella (MMAF) caused by a DNAH1 mutation are still unknown. To explore the effects, our study analyzed a case with MMAF due to DNAH1 homozygous mutation who underwent testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI). The case had 28 MII oocytes. The 28 oocytes were divided randomly and equally into AOA and non-AOA groups. Ionomycin was used for AOA. We compared the clinical outcomes of two groups and selected three blastulation failure embryos from each group for transcriptome analysis. Differentially expressed genes (DEGs) were determined with an adjusted P-value < 0.05 and a |log2-fold change| ≥ 1. The comparison of clinical outcomes showed that the two pronuclei (2PN) rate and grade 1–2 embryo rate at day 3 were not significantly different between the two groups. Transcriptome analyses of blastulation failure embryos showed that the use of AOA had potential risks of chromosome structure defects, transcriptional regulation defects, and epigenetic defects. In conclusion, when the case with MMAF due to DNAH1 mutation underwent TESE-ICSI, ionomycin-induced oocyte activation could not improve the clinical outcomes and introduced the risks of chromosome structure defect, transcriptional regulation defect, and epigenetic defect.

2023-02-11 | GSE216618 | GEO

The Airn RNA does not require any DNA elements within its locus to silence distant imprinted genes

Project description:Long non-coding (lnc) RNAs are numerous and found throughout the mammalian genome, and many are thought to be involved in the regulation of gene expression. However, the majority remain relatively uncharacterised and of uncertain function making the use to model systems to uncover their mode of action valuable. Imprinted lncRNAs target and recruit epigenetic silencing to a cluster of imprinted genes on the same chromosome, making them one of the best characterized lncRNAs to silence distant genes in cis. In this study we examined silencing of the distant imprinted gene Slc22a3 by the lncRNA Airn in the Igf2r imprinted cluster in mouse. Previously we proposed that imprinted lncRNAs may silence distant imprinted genes by disrupting promoter-enhancer interactions by being transcribed through the enhancer, which we called the enhancer interference hypothesis. Here we tested this hypothesis by first using allele-specific chromosome conformation capture (3C) to detect interactions between the Slc22a3 promoter and the locus of the Airn lncRNA that silences it on the paternal chromosome. In agreement with the model we found interactions enriched on the maternal allele, but across the entire Airn gene consistent with multiple enhancer-promoter interactions. Therefore, to further test the enhancer interference hypothesis we devised an approach to delete the entire Airn gene. However, the deletion showed that there are no essential enhancers for Slc22a3 within the Airn gene, strongly indicating that the Airn RNA rather than its transcription is responsible for silencing distant imprinted genes. Therefore we propose an alternative hypothesis whereby the chromosome interactions may initially guide the lncRNA to target imprinted promoters and recruit repressive chromatin, and that these interactions are lost once silencing is established.

2019-07-07 | GSE128513 | GEO

Mus musculus

Project description:Airn transcriptional overlap but not its lncRNA products induce imprinted Igf2r silencing.

| PRJNA177027 | ENA

Mitochondrial supplementation at fertilization enhances early developmental outcome

Project description:Competent oocytes can be discriminated by BCB staining. Positive stained oocytes are considered more competent than BCB negative oocyte, and injection of BCB+ oocyte extracted mitochondria into BCB negative oocytse can increase fertilisation and blastocyst rate. Here we have analysed the impact of mitochondrial supplementation on subsequent blastocyst transcriptome using agilent one color microarray that is specificly design to study the porcine embryo preimplantation period. Blastocysts were produced by intra cytoplasmic sperm injection (ICSI) from BCB positive and BCB negative oocytes as well as BCB negative oocytes supplemented with mitochondrial extract during ICSI (mICSI), and 3-4 single blatocyst transcriptomes were analysed for each group. 3-4 single blastocysts were analysed at the RNA level after whole transcriptome amplification, and level of gene expression was compared between groups, i.e ICSI BCB+ blastocysts (4), ICSI BCB- blastocysts (3) and mICSI BCB- blastocysts (4).

2016-09-01 | E-GEOD-65310 | biostudies-arrayexpress

Macro ncRNAs are abundant in imprinted regions and directly regulated by DNA methylation [tiling array]

Project description:Imprinted macro ncRNAs such as Airn play an important role in silencing protein-coding genes in cis, macro ncRNAs could be a common feature in all imprinted gene clusters. By applying the RNA Expression on Tiling Array (RETA) technique, macro ncRNAs were found to be abundant in 26 known mouse genomic regions containing imprinted genes were detected. All well-known imprinted macro ncRNAs were up-regulated upon depletion of DNA methylation.

2017-02-25 | GSE26718 | GEO

IQCN disruption causes fertilization failure and male infertility due to manchette assembly defect

Project description:Total fertilization failure (TFF) is an important cause of infertility; however, the genetic basis of TFF caused by male factors remain to be clarified. In this study, whole-exome sequencing was firstly used to screen for genetic causes of TFF after intracytoplasmic sperm injection (ICSI), and homozygous variants in the novel gene IQ motif containing N (IQCN) were identified in two affected individuals with abnormal acrosome structure. Then, Iqcn knockout mice were generated by CRISPR-Cas9 technology and showed that the knockout male mice resembled TFF phenotype with oocyte activation failure. Additionally, we found that IQCN regulates the microtubule nucleation during manchette assembly via calmodulin and related calmodulin-binding proteins, which resulted in head deformity with aberrant oocyte activation factor PLCζ. Fortunately, ICSI with assisted oocyte activation can overcome IQCN-associate male infertility. Thus, our study firstly identifies the function of IQCN, highlights the relationship between the manchette assembly and fertilization, and provides a genetic marker and a therapeutic option for male-source TFF.

2022-11-22 | PXD036197 | Pride

Low dose human chorionic gonadotropin hCG

Project description:Influence of ovarian stimulation with 200 IU of hCG, (administered in the late follicular phase among ICSI patients undergoing a GnRH-antagonist protocol), on the endometrium on the day of oocyte pick-up. The purpose of the present study is to assess the influence of the administration of low dose hCG on the endometrium. In addition, by analysing the correlation of the morphological pattern and gene expression profile of human endometrium on the day of oocyte retrieval in patients of both treatment groups, we want to study the implantation potential. Keywords: gene expression analysis

2010-10-02 | GSE18557 | GEO

Transcription profiling of human cumulus cells in vitro isolated from polycystic ovary syndrome lean and obese individuals reveals PCOS in lean and obese individuals is associated with different molecular pathways

Project description:Objective: The etiology of PCOS is mostly unknown. Existing data support both genetic and environmental factors in its pathogenesis. Design: Prospective case - control study. Setting: University Hospital. Patients: 25 patients undergoing IVF-ICSI treatment. Intervention: Genome-wide oligonucleotide microarray technology was used to study differential gene-expression patterns of cultured human cumulus cells from IVF patients divided into 4 groups according to disease state (PCOS vs. Control) and BMI (Obese vs. Lean). Results: Two differential PCOS gene expression profiles were established: Lean-Type was formed by comparing PCOS lean (PL) vs. non-PCOS lean (NL) individuals; Obese-Type was formed by comparing PCOS obese (PO) vs. non-PCOS (NO) obese patients. Conclusions: Different molecular pathways are associated with PCOS in Lean and Obese individuals, as demonstrated by gene expression profiling of cumulus cells. Our findings provide insights into the molecular pathogenesis of PCOS. We used microarrays to study the gene expression of human cultured cumulus cells. We compared the genes expression of lean PCOS, Obese PCOS, lean controls and obese controls. Different molecular pathways are associated with PCOS in Lean and Obese patients. Experiment Overall Design: Cumulus cells obtained from woman undergoing IVF/ICSI. Following oocyte retrieval, cumulus cells were stripped from the oocyte, in preparation for the ICSI process, with a micropipette. After 48h in culture the cumulus cells were collected for RNA extraction and hybridization on Affymetrix microarrays. We compered the expression profile of 4 groups - lean PCOS, obese PCOS, lean controls and obese controls.

2008-06-11 | E-GEOD-10946 | biostudies-arrayexpress

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