DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

Abstract

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability to. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

Data availability

The following data sets were generated

Article and author information

Author details

  1. Sebastian Canovas

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  2. Elena Ivanova

    Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Raquel Romar

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  4. Soledad García-Martínez

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  5. Cristina Soriano-Úbeda

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  6. Francisco Alberto A García-Vázquez

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  7. Heba Saadeh

    Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  8. Simon Andrews

    Bioinformatics Group, The Babraham Institute, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Gavin Kelsey

    Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9762-5634
  10. Pilar Coy

    Physiology of Reproduction Group, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain
    For correspondence
    pcoy@um.es
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3943-1890

Funding

Research Councils UK

  • Gavin Kelsey

Ministerio de Economía y Competitividad (AGL2012-40180-C03-01 and AGL2015-66341-R)

  • Pilar Coy

Ministerio de Educación, Cultura y Deporte (PRX14/00348)

  • Pilar Coy

Fundación Séneca. Región de Murcia. Spain (20040/GERM/16)

  • Pilar Coy

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was carried out in strict accordance with the recommendations in the Guiding Principles for the Care and Use of Animals (DHEW Publication, NIH, 80-23). The protocol was approved by the Ethical Committee for Experimentation with Animals of the University of Murcia, Spain (Project Code: 192/2015).

Copyright

© 2017, Canovas et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 4,318
    views
  • 949
    downloads
  • 119
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Sebastian Canovas
  2. Elena Ivanova
  3. Raquel Romar
  4. Soledad García-Martínez
  5. Cristina Soriano-Úbeda
  6. Francisco Alberto A García-Vázquez
  7. Heba Saadeh
  8. Simon Andrews
  9. Gavin Kelsey
  10. Pilar Coy
(2017)
DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids
eLife 6:e23670.
https://doi.org/10.7554/eLife.23670

Share this article

https://doi.org/10.7554/eLife.23670

Further reading

    1. Developmental Biology
    Shannon H Carroll, Sogand Schafer ... Eric C Liao
    Research Article

    Wnt signaling plays crucial roles in embryonic patterning including the regulation of convergent extension (CE) during gastrulation, the establishment of the dorsal axis, and later, craniofacial morphogenesis. Further, Wnt signaling is a crucial regulator of craniofacial morphogenesis. The adapter proteins Dact1 and Dact2 modulate the Wnt signaling pathway through binding to Disheveled. However, the distinct relative functions of Dact1 and Dact2 during embryogenesis remain unclear. We found that dact1 and dact2 genes have dynamic spatiotemporal expression domains that are reciprocal to one another suggesting distinct functions during zebrafish embryogenesis. Both dact1 and dact2 contribute to axis extension, with compound mutants exhibiting a similar CE defect and craniofacial phenotype to the wnt11f2 mutant. Utilizing single-cell RNAseq and an established noncanonical Wnt pathway mutant with a shortened axis (gpc4), we identified dact1/2-specific roles during early development. Comparative whole transcriptome analysis between wildtype and gpc4 and wildtype and dact1/2 compound mutants revealed a novel role for dact1/2 in regulating the mRNA expression of the classical calpain capn8. Overexpression of capn8 phenocopies dact1/2 craniofacial dysmorphology. These results identify a previously unappreciated role of capn8 and calcium-dependent proteolysis during embryogenesis. Taken together, our findings highlight the distinct and overlapping roles of dact1 and dact2 in embryonic craniofacial development, providing new insights into the multifaceted regulation of Wnt signaling.

    1. Developmental Biology
    Sudershana Nair, Nicholas E Baker
    Research Article

    Many cell fate decisions are determined transcriptionally. Accordingly, some fate specification is prevented by Inhibitor of DNA-binding (Id) proteins that interfere with DNA binding by master regulatory transcription factors. We show that the Drosophila Id protein Extra macrochaetae (Emc) also affects developmental decisions by regulating caspase activity. Emc, which prevents proneural bHLH transcription factors from specifying neural cell fate, also prevents homodimerization of another bHLH protein, Daughterless (Da), and thereby maintains expression of the Death-Associated Inhibitor of Apoptosis (diap1) gene. Accordingly, we found that multiple effects of emc mutations on cell growth and on eye development were all caused by activation of caspases. These effects included acceleration of the morphogenetic furrow, failure of R7 photoreceptor cell specification, and delayed differentiation of non-neuronal cone cells. Within emc mutant clones, Notch signaling was elevated in the morphogenetic furrow, increasing morphogenetic furrow speed. This was associated with caspase-dependent increase in levels of Delta protein, the transmembrane ligand for Notch. Posterior to the morphogenetic furrow, elevated Delta cis-inhibited Notch signaling that was required for R7 specification and cone cell differentiation. Growth inhibition of emc mutant clones in wing imaginal discs also depended on caspases. Thus, emc mutations reveal the importance of restraining caspase activity even in non-apoptotic cells to prevent abnormal development, in the Drosophila eye through effects on Notch signaling.