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Investigation of microRNA expression and DNA repair gene transcripts in human oocytes and blastocysts.

ABSTRACT: The aim of the study is to investigate the regulation of DNA repair genes by microRNAs (miRNAs). miRNAs are short non-coding RNAs that regulate transcriptional and post-transcriptional gene silencing. Several miRNAs that are expressed during preimplantation embryo development have been shown or are predicted to target genes that regulate cell cycle checkpoints and DNA repair in response to DNA damage.This study compares the expression level of 20 miRNAs and 9 target transcripts involved in DNA repair. The statistical significance of differential miRNA expression between oocytes and blastocysts was determined by t test analysis using the GraphPad Prism v6 software. The possible regulatory roles of miRNAs on their target messenger RNAs (mRNAs) were analysed using a Pearson correlation test.This study shows for the first time that several miRNAs are expressed in human oocytes and blastocysts that target key genes involved in DNA repair and cell cycle checkpoints. Blastocysts exhibited statistically significant lower expression levels for the majority of miRNAs compared to oocytes (p?

SUBMITTER: Tulay P

PROVIDER: S-EPMC4681740 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Investigation of microRNA expression and DNA repair gene transcripts in human oocytes and blastocysts.

Tulay P P Naja R P RP Cascales-Roman O O Doshi A A Serhal P P SenGupta S B SB

Journal of assisted reproduction and genetics 20151005 12

<h4>Purpose</h4>The aim of the study is to investigate the regulation of DNA repair genes by microRNAs (miRNAs). miRNAs are short non-coding RNAs that regulate transcriptional and post-transcriptional gene silencing. Several miRNAs that are expressed during preimplantation embryo development have been shown or are predicted to target genes that regulate cell cycle checkpoints and DNA repair in response to DNA damage.<h4>Methods</h4>This study compares the expression level of 20 miRNAs and 9 targ ...[more]

PMID: 26438643

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Project description:BackgroundMitochondrial DNA (mtDNA) copy number in oocytes correlates with oocyte quality and fertilisation outcome. The introduction of additional copies of mtDNA through mitochondrial supplementation of mtDNA-deficient Sus scrofa oocytes resulted in: (1) improved rates of fertilisation; (2) increased mtDNA copy number in the 2-cell stage embryo; and (3) improved development of the embryo to the blastocyst stage. Furthermore, a subset of genes showed changes in gene expression. However, it is still unknown if mitochondrial supplementation alters global and local DNA methylation patterns during early development.ResultsWe generated a series of embryos in a model animal, Sus scrofa, by intracytoplasmic sperm injection (ICSI) and mitochondrial supplementation in combination with ICSI (mICSI). The DNA methylation status of ICSI- and mICSI-derived blastocysts was analysed by whole genome bisulfite sequencing. At a global level, the additional copies of mtDNA did not affect nuclear DNA methylation profiles of blastocysts, though over 2000 local genomic regions exhibited differential levels of DNA methylation. In terms of the imprinted genes, DNA methylation patterns were conserved in putative imprint control regions; and the gene expression profile of these genes and genes involved in embryonic genome activation were not affected by mitochondrial supplementation. However, 52 genes showed significant differences in expression as demonstrated by RNAseq analysis. The affected gene networks involved haematological system development and function, tissue morphology and cell cycle. Furthermore, seven mtDNA-encoded t-RNAs were downregulated in mICSI-derived blastocysts suggesting that extra copies of mtDNA affected tRNA processing and/or turnover, hence protein synthesis in blastocysts. We also showed a potential association between differentially methylated regions and changes in expression for 55 genes due to mitochondrial supplementation.ConclusionsThe addition of just an extra ~ 800 copies of mtDNA into oocytes can have a significant impact on both gene expression and DNA methylation profiles in Sus scrofa blastocysts by altering the epigenetic programming established during oogenesis. Some of these changes may affect specific tissue-types later in life. Consequently, it is important to determine the longitudinal effect of these molecular changes on growth and development before considering human clinical practice.

Project description:BACKGROUND: Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. METHODOLOGY/PRINCIPAL FINDING: Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. CONCLUSIONS/SIGNIFICANCE: Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer.

Project description:IntroductionMany studies have reported that human-induced pluripotent stem (hiPS)/embryonic stem (hES) cells have an exceptional ability to repair damaged DNA. Moreover, unlike differentiated cells, hES cells have features and mechanisms such as apoptosis-prone mitochondria, which prevent any changes in genetic information caused by DNA damage to be transmitted to their descendants. Type-A (dark) spermatogonia and cancer stem cells are thought to be dormant. However, hiPS/hES cells, the so-called stem cells used in regenerative medicine, generally have a high proliferative capacity. This suggests that in these cells, oxidative DNA damage associated with vigorous proliferation and DNA scission associated with replication occur frequently. Although pluripotency according to change of genomic structure is well studied, the change of DNA repair through reprogramming has not been well studied.MethodsWe analyzed the expression of DNA repair-related genes in hiPS cells using microarray and western blotting analyses and assessed changes in PARP activity through reprogramming.ResultsThrough reprogramming, hiPS cells were found to upregulate poly (ADP-ribose) polymerase (PARP) activity and genes regulating homologous recombination (HR). Simultaneously, the expression level of genes involved in non-homologous end joining (NHEJ) was not high, suggesting that at least at the gene expression level, frequently occurring DNA scission is preferentially dealt with via HR instead of NHEJ. Also, reflecting the high proliferative activity, genes related to mismatch repair (MMR) were upregulated through reprogramming. Conversely, error-prone polymerase was downregulated through reprogramming. These are also likely to be the mechanisms preventing changes in genetic information.ConclusionsHigh PARP activity and HR-related gene expression in hiPS cells were achieved through reprogramming and likely facilitate precise genome editing in these cells in exchange for a high possibility of cell death.

Dataset Information

Investigation of microRNA expression and DNA repair gene transcripts in human oocytes and blastocysts.

Publications

Investigation of microRNA expression and DNA repair gene transcripts in human oocytes and blastocysts.

Similar Datasets

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