[Show abstract][Hide abstract] ABSTRACT: Assisted reproductive technologies (ARTs) represent the best chance for infertile couples to conceive, although increased risks for morbidities exist, including imprinting disorders. This increased risk could arise from ARTs disrupting genomic imprints during gametogenesis or preimplantation. The few studies examining ART effects on genomic imprinting primarily assessed poor quality human embryos. Here, we examined day 3 and blastocyst stage, good to high quality, donated human embryos for imprinted SNRPN, KCNQ1OT1 and H19 methylation. Seventy-six percent day 3 embryos and 50% blastocysts exhibited perturbed imprinted methylation, demonstrating that extended culture did not pose greater risk for imprinting errors than short culture. Comparison of embryos with normal and abnormal methylation didn't reveal any confounding factors. Notably, two embryos from male factor infertility patients using donor sperm harboured aberrant methylation, suggesting errors in these embryos cannot be explained by infertility alone. Overall, these results indicate that ART human preimplantation embryos possess a high frequency of imprinted methylation errors.
[Show abstract][Hide abstract] ABSTRACT: Recently, many advancements in genome-wide chromatin topology and nuclear architecture have unveiled the complex and hidden world of the nucleus, where chromatin is organized into discrete neighbourhoods with coordinated gene expression. This includes the active and inactive X chromosomes. Using X chromosome inactivation as a working model, we utilized publicly available datasets together with a literature review to gain insight into topologically associated domains, lamin-associated domains, nucleolar-associating domains, scaffold/matrix attachment regions, and nucleoporin-associated chromatin and their role in regulating monoallelic expression. Furthermore, we comprehensively review for the first time the role of chromatin topology and nuclear architecture in the regulation of genomic imprinting. We propose that chromatin topology and nuclear architecture are important regulatory mechanisms for directing gene expression within imprinted domains. Furthermore, we predict that dynamic changes in chromatin topology and nuclear architecture play roles in tissue-specific imprint domain regulation during early development and differentiation.
Full-text · Article · Jun 2015 · Biochemistry and Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Many studies have shown that in vitro culture can negatively impact preimplantation development. This necessitates some selection criteria for identifying the best-suited embryos for transfer. That said, embryo selection after in vitro culture remains a subjective process in most mammalian species, including cows, mice and humans. General consensus in the field is that embryos that develop in a timely manner have the highest developmental competence and viability after transfer. Herein lies the key question: what is a timely manner? With emerging data in bovine and mouse supporting increased developmental competency in embryos with moderate rates of development, it is time to question whether the fastest developing embryos are the best embryos for transfer in the human clinic. This is especially relevant to epigenetic gene regulation, including genomic imprinting, where faster developing embryos exhibit loss of imprinted methylation, as well as to sex selection bias, where faster developmental rates of male embryos may lead to biased embryo transfer and, in turn, biased sex ratios. In this review, we explore evidence surrounding the question of developmental timing as it relates to bovine embryo quality, mouse embryo quality and genomic imprint maintenance, and embryo sex.
Full-text · Article · Sep 2014 · Reproduction Fertility and Development
[Show abstract][Hide abstract] ABSTRACT: In this issue of Biology of Reproduction, Kooistra et al. use gene expression studies to show that mouse cumulus-oocyte complexes and oocytes harbor transcripts for folate carrier SLC19A1, an anion exchanger.
Preview · Article · Aug 2013 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: Genomic imprinting is a specialized transcriptional phenomenon that employs epigenetic mechanisms to facilitate parental-specific expression. Perturbations in parental epigenetic asymmetry can lead to the development of imprinting disorders, such as Beckwith-Wiedemann syndrome and Angelman syndrome. DNA methylation is one of the most widely studied epigenetic marks that characterizes imprinted regions. During gametogenesis and early embryogenesis, imprinted methylation undergoes a cycle of erasure, acquisition and maintenance. Gamete and embryo manipulations for the purpose of assisted reproduction are performed during these reprogramming events and may lead to their disruption. Recent studies point to the role of maternal-effect proteins in imprinted gene regulation. Studies are now required to increase understanding of how these factors regulate genomic imprinting as well as how assisted reproduction technologies may alter their function. Assisted reproductive technologies have been linked to the development of Beckwith-Wiedemann syndrome and Angelman syndrome. This may relate to the fact that the use of assisted reproduction technology coincides with crucial events that establish and maintain gene expression during egg and early embryo development. Recent studies have identified several proteins that are found in the egg that may play a role in gene expression in the early embryo. Studies are now required to increase understanding of how these factors work as well as how assisted reproductive technologies may alter their function.
Full-text · Article · Jun 2013 · Reproductive biomedicine online
[Show abstract][Hide abstract] ABSTRACT: Gamete and early embryo development are important stages when genome-scale epigenetic transitions are orchestrated. The apparent lack of remodeling of differential imprinted DNA methylation during preimplantation development has lead to the argument that epigenetic disruption by assisted reproductive technologies (ARTs) is restricted to imprinted genes. We contend that aberrant imprinted methylation arising from assisted reproduction or infertility may be an indicator of more global epigenetic instability. Here, we review the current literature on the effects of ARTs, including ovarian stimulation, in vitro oocyte maturation, oocyte cryopreservation, IVF, ICSI, embryo culture, and infertility on genomic imprinting as a model for evaluating epigenetic stability. Undoubtedly, the relationship between impaired fertility, ARTs, and epigenetic stability is unquestionably complex. What is clear is that future studies need to be directed at determining the molecular and cellular mechanisms giving rise to epigenetic errors.
[Show abstract][Hide abstract] ABSTRACT: During preimplantation development, major epigenetic reprogramming occurs, erasing gametic modifications, and establishing embryonic epigenetic modifications. Given the plasticity of these modifications, they are susceptible to disruption by assisted reproductive technologies, including embryo culture. The current state of evidence is presented for the effects of embryo culture on global DNA methylation and histone modifications, retroviral silencing, X-inactivation, and genomic imprinting. Several salient points emerge from the literature; that culture in the absence of other procedures can lead to epigenetic perturbations; that all media are suboptimal; and that embryo response to in vitro culture is stochastic. We propose that embryos adapt to the suboptimal environment generated by embryo culture, including epigenetic adaptations, and that "quiet" embryos may be the least epigenetically compromised by in vitro culture.
[Show abstract][Hide abstract] ABSTRACT: Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation, and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt de novo methylation acquisition using ERβ and connexin37 genetic models. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20–80 μm control oocytes at three imprinted genes, Snrpn, Peg3, and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition,individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3, or Peg1 in oocytes from Esr2-deficient females, and no perturbation in Snrpn or Peg3
de novo methylation in oocytes from Gja4-null females. However, Gja4 deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine the effects of subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis on imprint maintenance during preimplantation development.
Full-text · Article · Jul 2012 · Frontiers in Genetics
[Show abstract][Hide abstract] ABSTRACT: Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting. One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level. Development of the bisulfite mutagenesis assay has advanced from those originally reported towards ones that are more sensitive and reproducible. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos. Taking clues from agarose embedding of minute DNA amounts including oocytes, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.
[Show abstract][Hide abstract] ABSTRACT: Currently, the stage of embryo development has been proposed as one of many criteria for identifying healthy embryos in infertility clinics with the fastest embryos being highlighted as the healthiest. However the validity of this as an accurate criterion with respect to genomic imprinting is unknown. Given that embryo development in culture generally requires an extra day compared to in vivo development, we hypothesized that loss of imprinting correlates with slower rates of embryonic development. To evaluate this, embryos were recovered at the 2-cell stage, separated into four groups based on morphological stage at two predetermined time points, and cultured to blastocysts. We examined cell number, embryo volume, embryo sex, imprinted Snrpn and H19 methylation, imprinted Snrpn, H19, and Cdkn1c expression, and expression of genes involved in embryo metabolism-Atp1a1, Slc2a1, and Mapk14-all within the same individual embryo. Contrary to our hypothesis, we observed that faster developing embryos exhibited greater cell numbers and embryo volumes as well as greater perturbations in genomic imprinting and metabolic marker expression. Embryos with slower rates of preimplantation development were most similar to in vivo derived embryos, displaying similar cell numbers, embryo volumes, Snrpn and H19 imprinted methylation, H19 imprinted expression, and Atp1a1 and Slc2a1 expression. We conclude that faster development rates in vitro are correlated with loss of genomic imprinting and aberrant metabolic marker expression. Importantly, we identified a subset of in vitro cultured embryos that, according to the parameters evaluated, are very similar to in vivo derived embryos and thus are likely most suitable for embryo transfer.
Full-text · Article · Jan 2012 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: In May 2011, the Canadian Conference on Epigenetics: Epigenetics Eh! was held in London, Canada. The objectives of this conference were to showcase the breadth of epigenetic research on environment and health across Canada and to provide the catalyst to develop collaborative Canadian epigenetic research opportunities, similar to existing international epigenetic initiatives in the US and Europe. With ten platform sessions and two sessions with over 100 poster presentations, this conference featured cutting-edge epigenetic research, presented by Canadian and international principal investigators and their trainees in the field of epigenetics and chromatin dynamics. An EpigenART competition included ten artists, creating a unique opportunity for artists and scientists to interact and explore their individual interpretations of this scientific discipline. The conference provided a unique venue for a significant cross-section of Canadian epigenetic researchers from diverse disciplines to meet, interact, collaborate and strategize at the national level.
Full-text · Article · Oct 2011 · Epigenetics: official journal of the DNA Methylation Society
[Show abstract][Hide abstract] ABSTRACT: To understand the complex regulation of genomic imprinting it is important to determine how early embryos establish imprinted gene expression across large chromosomal domains. Long non-coding RNAs (ncRNAs) have been associated with the regulation of imprinting domains, yet their function remains undefined. Here, we investigated the mouse Kcnq1ot1 ncRNA and its role in imprinted gene regulation during preimplantation development by utilizing mouse embryonic and extra-embryonic stem cell models. Our findings demonstrate that the Kcnq1ot1 ncRNA extends 471 kb from the transcription start site. This is significant as it raises the possibility that transcription through downstream genes might play a role in their silencing, including Th, which we demonstrate possesses maternal-specific expression during early development. To distinguish between a functional role for the transcript and properties inherent to transcription of long ncRNAs, we employed RNA interference-based technology to deplete Kcnq1ot1 transcripts. We hypothesized that post-transcriptional depletion of Kcnq1ot1 ncRNA would lead to activation of normally maternal-specific protein-coding genes on the paternal chromosome. Post-transcriptional short hairpin RNA-mediated depletion in embryonic stem, trophoblast stem and extra-embryonic endoderm stem cells had no observable effect on the imprinted expression of genes within the domain, or on Kcnq1ot1 imprinting center DNA methylation, although a significant decrease in Kcnq1ot1 RNA signal volume in the nucleus was observed. These data support the argument that it is the act of transcription that plays a role in imprint maintenance during early development rather than a post-transcriptional role for the RNA itself.
[Show abstract][Hide abstract] ABSTRACT: To investigate whether superovulation disrupts maternal imprint acquisition in oocytes.
Spontaneously ovulated and superovulated mice.
Low and high hormone dosage treatments were administered to females, and ovulated metaphase II oocytes were collected.
Imprinted DNA methylation was analyzed at Snrpn, Kcnq1ot1, Peg3, and H19 in individual oocytes.
Examination of 125 individual oocytes derived from females subjected to low and high hormone treatments revealed normal imprinted methylation patterns that were comparable to oocytes derived from spontaneously ovulated females.
Maternal imprint acquisition was not affected by superovulation. Given its aberrant effects during preimplantation development, superovulation must instead disrupt maternal-effect gene products that are required after fertilization for imprint maintenance. These results eliminate imprint acquisition per se as the initial stage of imprint loss and point to the importance of analyses on early embryos after procedures involving oocyte manipulation.
Full-text · Article · Jul 2011 · Fertility and sterility
[Show abstract][Hide abstract] ABSTRACT: The two main challenges facing retroviral transgenesis are variable expression and epigenetic silencing. Although modern lentiviral vectors incorporate several elements to increase transgene expression and reduce position effect variegation and silencing, therapeutic research in stem cells, as well as production of transgenic animals, is still hampered by these two key problems. On the basis of recent studies demonstrating the chromatin insulating properties of divergent promoters, we sought to develop a bidirectional lentiviral vector with which to conduct RNA interference (RNAi)-based genetic screens in embryonic and extraembryonic stem cells. To this end, we designed and tested a series of synthetic bidirectional promoters, combining the mouse phosphoglycerate kinase 1 (Pgk1) promoter with other strong mammalian and viral promoters. Here, we demonstrate that a back-to-back configuration of the mouse Pgk1 and human eukaryotic translation elongation factor 1 alpha 1 promoters provided a substantive increase in both transgene expression and RNAi-based transcript depletion as compared with previous designs and other promoter combinations. Using this vector, we were able to achieve stable and robust depletion of a transfected luciferase reporter, as well as an endogenous non-coding RNA. The described constructs are an improved transgene delivery system capable of conducting RNAi screens in stem cells at single copy.
[Show abstract][Hide abstract] ABSTRACT: Assisted reproductive technologies (ARTs) are becoming increasingly prevalent and are generally considered to be safe medical procedures. However, evidence indicates that embryo culture may adversely affect the developmental potential and overall health of the embryo. One of the least studied but most important areas in this regard is the effects of embryo culture on epigenetic phenomena, and on genomic imprinting in particular, because assisted reproduction has been linked to development of the human imprinting disorders Angelman and Beckwith-Wiedemann syndromes. In this study, we performed side-by-side comparisons of five commercial embryo culture systems (KSOMaa, Global, Human Tubal Fluid, Preimplantation 1/Multiblast, and G1v5PLUS/G2v5PLUS) in relation to a best-case (in vivo-derived embryos) and a worst-case (Whitten culture) scenario. Imprinted DNA methylation and expression were examined at three well-studied loci, H19, Peg3, and Snrpn, in mouse embryos cultured from the 2-cell to the blastocyst stage. We show that embryo culture in all commercial media systems resulted in imprinted methylation loss compared to in vivo-derived embryos, although some media systems were able to maintain imprinted methylation levels more similar to those of in vivo-derived embryos in comparison to embryos cultured in Whitten medium. However, all media systems exhibited loss of imprinted H19 expression comparable to that using Whitten medium. Combined treatment of superovulation and embryo culture resulted in increased perturbation of genomic imprinting, above that from culture alone, indicating that multiple ART procedures further disrupt genomic imprinting. These results suggest that time in culture and number of ART procedures should be minimized to ensure fidelity of genomic imprinting during preimplantation development.
Preview · Article · Dec 2010 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: Imprinted genes are expressed in a monoallelic, parent-of-origin-specific manner. Clusters of imprinted genes are regulated
by imprinting control regions (ICRs) characterized by DNA methylation of one allele. This methylation is critical for imprinting;
a reduction in the DNA methyltransferase DNMT1 causes a widespread loss of imprinting. To better understand the role of DNA
methylation in the regulation of imprinting, we characterized the effects of Dnmt1 mutations on the expression of a panel of imprinted genes in the embryo and placenta. We found striking differences among
imprinted domains. The Igf2 and Peg3 domains showed imprinting perturbations with both null and partial loss-of-function mutations, and both domains had pairs
of coordinately regulated genes with opposite responses to loss of DNMT1 function, suggesting these domains employ similar
regulatory mechanisms. Genes in the Kcnq1 domain were less sensitive to the absence of DNMT1. Cdkn1c exhibited imprinting perturbations only in null mutants, while Kcnq1 and Ascl2 were largely unaffected by a loss of DNMT1 function. These results emphasize the critical role for DNA methylation in imprinting
and reveal the different ways it controls gene expression.
Full-text · Article · Aug 2010 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: To prevent insertional mutagenesis arising from retroviral reactivation, cells of embryonic origin possess a unique capacity to silence retroviruses. Given the distinct modes of X chromosome inactivation between embryonic and extraembryonic lineages, we investigated paradigms of viral extinction. We show that trophectoderm stem cells do not silence retroviral transcription, whereas extraembryonic endoderm stem cells aggressively extinguish proviral transcription, even more rapidly than do embryonic stem cells. By using a short hairpin RNA library, we identified epigenetic modifiers of retroviral extinction in extraembryonic endoderm stem cells. Multiple chromatin remodeling and polycomb repressor complex proteins act to modulate integrated, as well as endogenous, retroviral element silencing, with a subset of factors displaying differential effects between stem cell types. Furthermore, our data suggest that small RNAs play a role in this process through interactions with the Argonaute family. Our results further the understanding of mechanisms regulating retroviral transcription in different stem cell lineages.