Transcriptional Profiling by Deep Sequencing Identifies Differences in mRNA Transcript Abundance in In Vivo-Derived Versus In Vitro-Cultured Porcine Blastocyst Stage Embryos1

Abstract

In vitro embryo culture systems promote development at rates lower than in vivo systems. The goal of this project was to discover transcripts that may be responsible for a decrease of embryo competency in blastocyst-stage embryos cultured in vitro. Gilts were artificially inseminated on the first day of estrus, and on Day 2, one oviduct and the tip of a uterine horn were flushed and the recovered embryos were cultured in porcine zygote medium 3 for 4 days. On Day 6, the gilts were euthanized and the contralateral horn was flushed to obtain in vivo derived embryos. Total RNA was extracted from three pools of 10 blastocysts from each treatment. First and second strand cDNA was synthesized and sequenced using Illumina sequencing. The reads generated were aligned to a custom-built database designed to represent the known porcine transcriptome. A total of 1170 database members were different between the two groups (P < 0.05), and 588 of those had at least a 2-fold difference. Eleven transcripts were subjected to real-time PCR that validated the sequencing. There was an overall decrease in inner cell mass (ICM) and trophectodermal (TE) cell numbers in embryos cultured in vitro; however, no difference in the ICM:TE ratio was found. Interestingly, the transcript SLC7A1 was higher in the in vitro cultured group. This difference disappeared after addition of arginine to the 4-day culture. Illumina sequencing and alignment to a custom transcriptome identified a large number of genes that yield clues on ways to manipulate the culture media to mimic the in vivo environment.

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

“…These data suggest that in vitro culture significantly alters embryonic gene expression to a lesser extent than previously proposed (Bauer et al, 2010), and a better modulation of the blastocyst transcriptome was achieved by mimicking physiological conditions of fertilization and early embryo development by the addition of reproductive fluids (Natur-IVF).…”

“…In vitro culture systems significantly alter embryonic gene expression as previously observed in pooled pig blastocysts (Bauer et al, 2010). Here, the transcriptomes from three individual day 7.5 blastocysts produced by C-IVF or Natur-IVF were compared with their in vivo counterparts (Figure 3A–B).…”

Dual effect of fetal bovine serum on early development depends on stage-specific reactive oxygen species demands in pigs

“…Indeed, the parameters dictating in vitro developmental competence are frequently in good accordance with TE cell quantity in IVP blastocysts in domestic animals [64, 65]. Moreover, recent reports have shown that the number and proportion of TE cells, unlike ICM, are markedly reduced in IVP porcine blastocysts compared to in vivo blastocysts [66, 67]. Consistent with these demonstrations, the current study showed that modulation of ROS led to changes in the number of TE cells, but not ICM (Figs 2–4, 6 and 7; S3 Fig; S6, S9, S11, S14, S18, and S20 Tables).…”

Effect of peroxiredoxin II on the quality and mitochondrial activity of pre-implantation bovine embryos

“…The expression patterns of OCT4, IFN , and CDX2 during early embryonic development allow assessment of the quality of blastocysts produced (Bauer et al, 2010;Ross et al, 2007). In ruminant, the function of IFN is the recognition of pregnancy (Bazer et al, 2008) and IFN gene expression is higher in hatched blastocysts than in expanded and early embryos (Rekik et al, 2011).…”