18 research outputs found
Deoxyribonucleic acid methylation profiling of single human blastocysts by methylated CpG-island amplification coupled with CpG-island microarray
Objective
To study whether methylated CpG-island (CGI) amplification coupled with microarray (MCAM) can be used to generate DNA (deoxyribonucleic acid) methylation profiles from single human blastocysts.
Design
A pilot microarray study with methylated CpG-island amplification applied to human blastocyst genomic DNA and hybridized on CpG-island microarrays.
Setting
University research laboratory.
Patient(s)
Five cryopreserved sibling 2-pronuclear zygotes that were surplus to requirements for clinical treatment by in vitro fertilization were donated with informed consent from a patient attending Bourn Hall Clinic, Cambridge, United Kingdom.
Intervention(s)
None.
Main Outcome Measure(s)
Successful generation of genome-wide DNA methylation profiles at CpG islands from individual human blastocysts, with common genomic regions of DNA methylation identified between embryos.
Result(s)
Between 472 and 734 CpG islands were methylated in each blastocyst, with 121 CpG islands being commonly methylated in all 5 blastocysts. A further 159 CGIs were commonly methylated in 4 of the 5 tested blastocysts. Methylation was observed at a number of CGIs within imprinted-gene, differentially methylated regions (DMRs), including placental and preimplantation-specific DMRs.
Conclusion(s)
The MCAM method is capable of providing comprehensive DNA methylation data in individual human blastocysts
Clinical and functional investigations of the tissue specific SmN protein
The SmN protein is highly homologous to the constitutively expressed small nuclear ribonucleoprotein particle (snRNP)-associated protein SmB but is expressed in brain and heart only. SmN is demonstrated here to associate with snRNPs when expressed naturally or artificially and interestingly, the nature of this association is different to that exhibited by SmB. SnRNPs catalyse RNA splicing of the primary trancripts within the spliceosome to generate mRNAs encoding for proteins. The primary transcripts of some genes however, are processed differently according to the tissue or cell type to generate distinct mRNAs encoding for different proteins. The tissue specific expression of SmN suggests it plays an important RNA splicing role in the tissues expressing it, and hence is a putative regulator of alternative splicing. The gene encoding SmN in humans, SNRPN has been mapped to the critical region for Prader Willi Syndrome (PWS) in which patients lack a functional paternally inherited copy of the maternally imprinted SNRPN gene. Murine models of PWS that lack expression of the mouse homologue (Snrpn) suffer early postnatal lethality. Data presented here confirms the absence of SmN expression at the protein level in all tissues tested demonstrating that the maternal imprint is not relaxed in these mice. Furthermore, SmB expression was observed to be elevated in the brains of such mice. This suggests that elevated SmB expression cannot compensate for important splicing roles normally performed by SmN in the tissues expressing it. RNA from the brains of these mice was therefore used to study the proposed alternative splicing role of SmN. Genes proposed to be good candidates for regulation by SmN on the basis that their pattern of splicing is different in tissues expressing SmN were analysed by RT-PCR amplification of endogenous transcripts. Analysis of c-src, NCAM the Oct-2 transcription factor, CGRP and the GS alpha and Go alpha subunits of GTP binding proteins argues against a general role of SmN in alternative splicing, but the fate of these mice suggests SmN has unique functions or properties not assigned to SmB. This is consistent with a functional analysis by immunoprecipitation whereby SmN demonstrated different affinities for the U1 and U2 snRNPs, showing preferential association with the U2 snRNP at low levels of expression. In addition, ELISA screening using peptides corresponding to the regions of least sequence homology between SmN and SmB/B' revealed that a subset of Systemic Lupus Erythematosus (SLE) patients generate autoantibodies that can discriminate between and SmN and SmB/B' by binding an SmB/B'-specific epitope (SmB/B' 179- 190). Therefore the differences in primary structure between SmN and SmB/B' are sufficient to alter both protein conformation and the nature of association with snRNPs, implying that SmN- containing snRNPs may be capable of processing RNA differently. In view of these findings, further studies have been performed to determine whether a reported significant elevation in gene transcription of SNRPN in certain SLE patients was reflected at the protein level. Data presented in this study suggests a post-transcriptional control mechanism operates to counter this aberrant SNRPN transcription and that elevated SmN protein expression is not a common occurrence in SLE. This is consistent with both immunoprecipitation data and ELISA data in that SmN does not appear to be the antigen stimulating the immune system in SLE
Variable imprinting of the MEST gene in human preimplantation embryos
There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos
The effects of aging on molecular modulators of human embryo implantation
Advancing age has a negative impact on female fertility. As implantation
rates decline during the normal maternal life course, age-related,
embryonic factors are altered and our inability to monitor these factors
in an unbiased genome-wide manner in vivo has severely limited our
understanding of early human embryo development and implantation. Our
high-throughput methodology uses trophectoderm samples representing the
full spectrum of maternal reproductive ages with embryo implantation
potential examined in relation to trophectoderm transcriptome dynamics
and reproductive maternal age. Potential embryo-endometrial interactions
were tested using trophectoderm sampled from young women, with the
receptive uterine environment representing the most `fertile'
environment for successful embryo implantation. Potential roles for
extracellular exosomes, embryonic metabolism and regulation of apoptosis
were revealed. These biomarkers are consistent with embryo-endometrial
crosstalk/developmental competency, serving as a mediator for successful
implantation. Our data opens the door to developing a diagnostic test
for predicting implantation success in women undergoing fertility
treatment
Potential sperm contributions to the murine zygote predicted by in silico analysis
Paternal contributions to the zygote are thought to extend beyond delivery of the genome and paternal RNAs have been linked to epigenetic transgenerational inheritance in different species. In addition, sperm–egg fusion activates several downstream processes that contribute to zygote formation, including PLC zeta-mediated egg activation and maternal RNA clearance. Since a third of the preimplantation developmental period in the mouse occurs prior to the first cleavage stage, there is ample time for paternal RNAs or their encoded proteins potentially to interact and participate in early zygotic activities. To investigate this possibility, a bespoke next-generation RNA sequencing pipeline was employed for the first time to characterise and compare transcripts obtained from isolated murine sperm, MII eggs and pre-cleavage stage zygotes. Gene network analysis was then employed to identify potential interactions between paternally and maternally derived factors during the murine egg-to-zygote transition involving RNA clearance, protein clearance and post-transcriptional regulation of gene expression. Our in silico approach looked for factors in sperm, eggs and zygotes that could potentially interact co-operatively and synergistically during zygote formation. At least five sperm RNAs (Hdac11, Fbxo2, Map1lc3a, Pcbp4 and Zfp821) met these requirements for a paternal contribution, which with complementary maternal co-factors suggest a wider potential for extra-genomic paternal involvement in the developing zygote
Molecular approaches to the study of gene expression during human preimplantation development
The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro
Mutations Causing Familial Biparental Hydatidiform Mole Implicate C6orf221 as a Possible Regulator of Genomic Imprinting in the Human Oocyte
Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome