The global gene expression outline of the bovine blastocyst: reflector of environmental conditions and predictor of developmental capacity

Abstract

Background: Morphological evaluation of embryos has been used to screen embryos for transfer. However, the repeatability and accuracy of this method remains low. Thus, evaluation of an embryo's gene expression signature with respect to its developmental capacity could provide new opportunities for embryo selection. Since the gene expression outline of an embryo is considered as an aggregate of its intrinsic characteristics and culture conditions, we have compared transcriptome profiles of in vivo and in vitro derived blastocysts in relation to pregnancy outcome to unravel the discrete effects of developmental competence and environmental conditions on bovine embryo gene expression outlines. To understand whether the gene expression patterns could be associated with blastocyst developmental competency, the global transcriptome profile of in vivo (CVO) and in vitro (CVT) derived competent blastocysts that resulted in pregnancy was investigated relative to that of in vivo (NVO) and in vitro (NVT) derived blastocysts which did not establish initial pregnancy, respectively while to unravel the effects of culture condition on the transcriptome profile of embryos, the transcriptional activity of the CVO group was compared to the CVT group and the NVO group was compared to the NVT ones.

Results: A total of 700 differentially expressed genes (DEGs) were identified between CVO and NVO blastocysts. These gene transcripts represent constitutive regions, indel variants, 3'-UTR sequence variants and novel transcript regions. The majority (82%) of these DEGs, including gene clusters like ATP synthases, eukaryotic translation initiation factors, ribosomal proteins, mitochondrial ribosomal proteins, NADH dehydrogenase and cytochrome c oxidase subunits were enriched in the CVO group. These DEGs were involved in pathways associated with glycolysis/glycogenesis, citrate acid cycle, pyruvate metabolism and oxidative phosphorylation. Similarly, a total of 218 genes were differentially expressed between CVT and NVT groups. Of these, 89%, including TPT1, PDIA6, HSP90AA1 and CALM, were downregulated in the CVT group and those DEGs were overrepresented in pathways related to protein processing, endoplasmic reticulum, spliceasome, ubiquitone mediated proteolysis and steroid biosynthesis. On the other hand, although both the CVT and CVO blastocyst groups resulted in pregnancy, a total of 937 genes were differential expressed between the two groups. Compared to CVO embryos, the CVT ones exhibited downregulation of gene clusters including ribosomal proteins, mitochondrial ribosomal protein, eukaryotic translation initiation factors, ATP synthases, NADH dehydrogenase and cytochrome c oxidases. Nonetheless, downregulation of these genes could be associated with pre and postnatal abnormalities observed after transfer of in vitro embryos.

Conclusion: The present study provides a detailed inventory of differentially expressed gene signatures and pathways specifically reflective of the developmental environment and future developmental capacities of bovine embryos suggesting that transcriptome activity observed in blastocysts could be indicative of further pregnancy success but also adaptation to culture environment.

Keywords: Bovine; Embryo; Pregnancy; Transcriptome.

Fig. 1

The experimental design used for comparative gene expression analysis in embryo biopsies. Numbers I, II, III and VI indicate comparisons with regard to the global gene expression profile between competent (CVO) and non-competent (NVO) in vivo derived blastocysts, competent (CVT) and non-competent (NVT) in vitro derived blastocysts, competent in vitro derived blastocysts (CVT) and competent in vivo derived blastocysts (CVO) as well as non-competent in vitro derived blastocysts (NVT) and non-competent in vivo derived blastocysts (NVO), respectively

Fig. 2

Molecular signature associated with the developmental capacity of in vivo derived embryos. Volcano plot demonstrating differentially expressed genes between CVO and NVO blastocysts. Red and green dots indicate up and downregulated genes, respectively in CVO compared to NVO blastocysts. Transcripts highly significant up or downregulated are indicated with arrows

Fig. 3

Gene clusters significantly enriched in in vivo derived competent embryos (CVO) compared to none competent (NVO) ones

Fig. 4

Molecular signature associated with developmental capacity of in vitro derived embryos. The heatmap indicates the expression patterns of the top 58 differentially expressed genes between CVT and NVT. Numbers 1, 2, 3 indicate three biological replicates hybridization whereas 1 μg of labelled Cy-5 labelled CVT samples were hybridized with 1 μg of Cy-3 labelled NVT samples. Numbers 4, 5, 6 indicate dye-swaps in which 1 μg of labelled Cy-3 labelled CVT samples were hybridized with 1 μg of Cy-5 labelled NVT samples

Fig. 5

Molecular functions significantly enriched by differentially expressed genes in competent in vivo derived embryos (CVO vs. NVO). Lists of genes on the right indicate differentially expressed genes involved within these distinct molecular functions

Fig. 6

Molecular pathways significantly enriched by differentially expressed genes in competent in vivo derived embryos (CVO vs. NVO). Lists of genes on the right indicate differentially expressed genes involved within these distinct molecular pathways

Fig. 7

Molecular functions significantly enriched by differentially expressed genes specifically in competent in vitro derived embryos (CVT vs. NVT). Lists of genes on the right indicate differentially expressed genes involved within these distinct molecular functions

Fig. 8

Meta-analysis of genes predictive for developmental capacity of in vivo derived embryos and differential expressed between ICM and TE cells. a Venn diagram depicting genes differentially expressed in CVO vs. NVO as well as in ICM vs. TE cells of in vivo derived embryos. The arrows on the left hand indicate expression trend of genes in CVO compared to NVO blastocysts whereas the arrows on the right side indicate expression trend of these genes in ICM relative to TE cells of in vivo derived embryos. b Venn diagram depicting genes differentially expressed in CVO vs. NVO as well as in ICM vs. TE cells of in vitro derived embryos. The arrows on the left indicate expression trend of genes in CVO compared to NVO blastocysts whereas the arrows on the right side indicate expression trend of these genes in ICM relative to TE cells of in vitro derived embryos. c Molecular pathways enriched by genes differentially expressed both in CVO vs. NVO blastocysts and in ICM vs. TE cells

Fig. 9

Meta-analysis of genes predictive for developmental capacity of in vitro derived embryos and differential expressed between ICM and TE cells. a Venn diagram depicting genes differentially expressed in CVT vs. NVT as well as in ICM vs. TE cells of in vivo derived embryos. The arrows on the left indicate expression trend of genes in CVT compared to NVT blastocysts whereas the arrows on the right side indicate expression trend of these genes in ICM relative to TE cells of in vivo derived embryos. b Venn diagram depicting genes differentially expressed in CVT vs. NVT as well as in ICM vs. TE cells of in vitro derived embryos. The arrows on the left indicate expression trend of genes in CVT compared to NVT blastocysts whereas the arrows on the right side indicate expression trend of these genes in ICM relative to TE cells of in vitro derived embryos. c Molecular pathways enriched by genes differentially expressed both in CVT vs. NVT blastocysts and as in ICM vs. TE cells

Fig. 10

Arrays of gene cluster reflecting environmental conditions in competent embryos (CVT vs. CVO)

Fig. 11

Biological processes (a) and molecular functions (b) significantly enriched by differentially expressed genes specifically modulated by the environmental conditions in competent embryos (CVT vs. CVO). Red, green, black and blue bars in Fig. A indicate functions associated with metabolism, energy production, cell cycle related activities and protein synthesis, respectively whereas yellow and black bars in Fig. B indicate binding and enzymatic activities respectively

Fig. 12

Summary of (a) differential expressed genes modulated by environmental conditions in competent embryos (left, CVT vs. CVO) and non-competent embryos (right, NVT vs. NVO). Venn diagram reports also number of genes differential expressed in common (center). Total numbers of differential expressed probes for each section are presented in italic whereas annotated ones are indicated in bold. b Molecular pathways significantly enriched by genes affected by environmental conditions exclusively in competent embryos (CVT vs. CVO). c Molecular pathways significantly enriched by genes affected by environmental conditions both in competent and non-competent embryos. Black bars indicate numbers of differentially expressed genes and blue dots indicate adjusted p values. NAFLD: Non-alcoholic fatty liver disease

Fig. 13

Meta-analysis of genes reflective for developmental environment and differential expressed between ICM and TE cells. a Venn diagram depicting genes differentially expressed in CVT vs. CVO as well as in ICM vs. TE cells of in vivo derived embryos. The arrows on the left hand indicate expression trend of genes in CVT compared to CVO blastocysts whereas the arrows on the right side indicate expression trend of these genes in ICM relative to TE cells of in vivo derived embryos. b Venn diagram depicting genes differentially expressed in CVT vs. CVO as well as in ICM vs. TE cells of in vitro derived embryos. The arrows on the left hand indicate expression trend of genes in CVT compared to CVO blastocysts whereas the arrows on the right side indicate expression trend of these genes in ICM relative to TE cells of in vitro derived embryos. c Molecular pathways enriched by genes differentially expressed both in CVT vs. CVO blastocysts as well as in ICM vs. TE cells