Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

Abstract

Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.

Results: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, xenobiotic metabolism general signaling pathway, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts further were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.

Conclusions: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

Keywords: Embryo development; In vivo embryo development; Porcine; Transcriptomics; in vitro fertilization.

Fig. 1

Experimental set-up for single embryo RNA-sequencing. The arrows indicate the between-group analyses

Fig. 2

Between-group analyses of the 4-cell stage embryos, morulae and hatched blastocysts of a. In vivo developed embryos, and b. In vitro produced embryos

Fig. 3

Upset plot displaying the differentially expressed genes during embryo development from the 4-cell to the morula stage and the morula to the hatched blastocyst stage in a. In vivo developed embryos, and b. In vitro produced embryos

Fig. 4

Transcriptome dynamics during development displayed by a self-organizing tree algorithm for a. In vivo developed embryos, and b. In vitro produced embryos. The number of genes per cluster and the embryonic sex of the morulae and hatched blastocysts are indicated

Fig. 5

Enriched canonical pathways. Red (−) dots represent canonical pathways of which genes were significantly lower expressed in the 4-cell versus morula and morula versus hatched blastocysts, and blue (+) represent canonical pathways of which genes were significantly higher expressed in the 4-cell versus morula or morula versus hatched blastocysts. The GeneRatio indicates the proportion of DEGs that were identified in an enriched canonical pathway. Shared enriched canonical pathways in both in vivo developed and in vitro produced embryos at the 4-cell versus morula or morula versus hatched blastocyst stage are indicated in purple

Fig. 6

a. Between group analysis of in vivo and in vitro female and male hatched blastocysts. b. Heatmap displaying the 243 DEGs observed by comparing in vivo produced versus in vitro developed females and the in vivo produced versus in vitro developed males. c. Heatmap displaying genes involved in amino acid metabolism, and d. Significantly enriched canonical pathways between the in vivo developed and in vitro produced hatched blastocysts. Red (−) dots represent canonical pathways of which genes were significantly less expressed in the in vivo embryos, while blue (+) represent canonical pathways of which genes were significantly higher expressed in the in vivo embryos. The GeneRatio indicates the proportion of DEGs that were identified in an enriched canonical pathway