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. 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.DOI:
http://dx.doi.org/10.7554/eLife.23670.001
Preimplantation embryos experience profound resetting of epigenetic information inherited from the gametes. Genome-wide analysis at single-base resolution has shown similarities but also species differences between human and mouse preimplantation embryos in DNA methylation patterns and reprogramming. Here, we have extended such analysis to two key livestock species, the pig and the cow. We generated genome-wide DNA methylation and whole-transcriptome datasets from gametes to blastocysts in both species. In oocytes from both species, a distinctive bimodal methylation landscape is present, with hypermethylated domains prevalent over hypomethylated domains, similar to human, while in the mouse the proportions are reversed. An oocyte-like pattern of methylation persists in the cleavage stages, albeit with some reduction in methylation level, persisting to blastocysts in cow, while pig blastocysts have a highly hypomethylated landscape. In the pig, there was evidence of transient de novo methylation at the 8-16 cell stages of domains unmethylated in oocytes, revealing a complex dynamic of methylation reprogramming. The methylation datasets were used to identify germline differentially methylated regions (gDMRs) of known imprinted genes and for the basis of detection of novel imprinted loci. Strikingly in the pig, we detected a consistent reduction in gDMR methylation at the 8-16 cell stages, followed by recovery to the blastocyst stage, suggesting an active period of imprint stabilization in preimplantation embryos. Transcriptome analysis revealed absence of expression in oocytes of both species of ZFP57, a key factor in the mouse for gDMR methylation maintenance, but presence of the alternative imprint regulator ZNF445. In conclusion, our study reveals species differences in DNA methylation reprogramming and suggests that porcine or bovine models may be closer to human in key aspects than in the mouse model.
This study was funded by Spanish Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER). Grants AGL2012-40180-C03-01 and AGL2015-66341-R. The authors declare no conflict of interest.
AbstractThis work was designed to determine temperature conditions within the reproductive tract of the female pig and study their impact on assisted reproductive technologies. Temperatures were recorded using a laparo-endoscopic single-site surgery assisted approach and a miniaturised probe. Sows and gilts were used to address natural cycle and ovarian stimulation treatments, respectively. According to in-vivo values, invitro fertilisation was performed at three temperature conditions (37.0 °C, 38.5 °C and 39.5 °C) and presumptive zygotes were cultured in these conditions for 20 hours while further embryo culture (21-168 hours post-insemination) was maintained at 38.5 °C. After 20 hours, different fertility parameters were assessed. During embryo culture, cleavage and blastocyst stages were evaluated. Sperm membrane fluidity at the experimental temperatures was studied by using differential scanning calorimetry and fluorescence recovery after photobleaching techniques. An increasing temperature gradient of 1.5 °C was found between the oviduct and uterus of sows (p < 0.05) and when this gradient was transferred to pig in-vitro culture, the number of poly-nuclear zygotes after in-vitro fertilisation was reduced and the percentage of blastocysts was increased. Moreover, the temperature transition phase for the boar sperm membrane (37.0 °C) coincided with the temperature registered in the sow oviduct, and sperm membranes were more fluid at 37.0 °C compared with that of sperm incubated at higher temperatures (38.5 and 39.5 °C). These data suggest that there may be an impact of physiological temperature gradients on human embryo development.
A major limitation of embryo epigenotyping by chromatin immunoprecipitation analysis is the reduced amount of sample available from an embryo biopsy. We developed an in vitro system to expand trophectoderm cells from an embryo biopsy to overcome this limitation. This work analyzes whether expanded trophectoderm (EX) is representative of the trophectoderm (TE) methylation or adaptation to culture has altered its epigenome. We took a small biopsy from the trophectoderm (30-40 cells) of in vitro produced bovine-hatched blastocysts and cultured it on fibronectin-treated plates until we obtained ∼4 × 104 cells. The rest of the embryo was allowed to recover its spherical shape and, subsequently, TE and inner cell mass were separated. We examined whether there were DNA methylation differences between TE and EX of three bovine embryos using whole-genome bisulfite sequencing. As a consequence of adaptation to culture, global methylation, including transposable elements, was higher in EX, with 5.3% of quantified regions showing significant methylation differences between TE and EX. Analysis of individual embryos indicated that TE methylation is more similar to its EX counterpart than to TE from other embryos. Interestingly, these similarly methylated regions are enriched in CpG islands, promoters and transcription units near genes involved in biological processes important for embryo development. Our results indicate that EX is representative of the embryo in terms of DNA methylation, thus providing an informative proxy for embryo epigenotyping.
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