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Effects of treatment with bovine oviductal extracellular vesicles on the transcriptome of bovine day 7 in vitro produced embryos

ABSTRACT: Oviductal extracellular vesicles (oEVs) are emerging as key players in gamete/embryo-oviduct interactions contributing to successful pregnancy. Various positive effects of oEVs on gametes and early embryos have been found in vitro. To find out if these effects are associated with changes of embryonic gene expression, the embryonic transcriptome was analyzed after supplementation of bovine embryos with bovine oEVs during in vitro culture (IVC) in comparison to corresponding controls without oEVs. Embryos were cultured during 7 days of IVC with fresh oEVs, oEVs frozen after isolation from oviductal fluid or without oEVs (control), since a previous study showed different effects of fresh and frozen oEVs. Five pools of 10-14 embryos were analyzed for each group by low-input Illumina RNA-sequencing. Analysis of differential gene expression revealed 221 differentially expressed genes (DEGs) for the comparison of frozen oEVs vs control, 67 DEGs for fresh vs frozen oEVs, and only minor differences for the comparison fresh oEVs vs control (28 DEGs). An integrative analysis with the previously studied mRNA and small ncRNA content of bovine oEVs suggested direct effects of oEVs content on the embryonic transcriptome for the supplementation with frozen oEVs, i.e., increase of transcripts found in higher concentrations in oEVs and decrease of transcripts which are potential targets of miRNAs found in oEVs.

ORGANISM(S): Bos taurus

PROVIDER: GSE143596 | GEO | 2020/03/21

REPOSITORIES: GEO

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Project description:Differentiation of totipotent embryonic cells into the first cell lineages (trophectoderm (TE) and inner cell mass (ICM), and further into epiblast (EPI) and primitive endoderm (PE)), although well studied, is only beginning to be understood at the molecular level. An in depth understanding of the regulation of these early differentiation events would enable improvements in the quality of in vitro produced embryos, as well as the study and application of bovine embryonic stem cells. The objective of this work was to perform single cell RNASeq on bovine blastocysts derived in vivo (IVV), in vitro in a conventional in vitro production system (IVC), and in vitro in reduced nutrient media (IVR). All in vitro culture was serum free. In vivo embryonic cells could be assigned based on Leiden clustering analysis, although there was some overlap in expression of marker genes. The two TE groups identified clearly separated in a uniform manifold approximation and projection (UMAP) plot from EPI groups. Putative epiblast cells could be identified. In in vitro embryos produced in a conventional system, considerable overlap in expression of marker genes made Leiden clustering difficult, but a selected count could be used for UMAP to tentatively identify one TE, 2 EPI and 2 PE groups. In assigned cell types we observed higher expression of the identifying marker genes, but some marker genes were expressed in all cell types. In IVC embryos, TE markers clustered while PE and EPI marker genes overlapped. In IVR embryos, again there was considerable overlap between marker gene expression. Like IVC embryos, TE was easily identified by clustering in IVR, but EPI and PE were too similar to distinguish. In summary, the methods used here could partially distinguish putative cell types in bovine blastocysts, although marker gene expression was not always discreet. This may not be surprising given differentiation events are in progress in the blastocyst stage embryo. TE cells were more easily distinguished than EPI and PE cells, which would support this hypothesis.

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Effects of treatment with bovine oviductal extracellular vesicles on the transcriptome of bovine day 7 in vitro produced embryos

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