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- Author: Luc J Peelman x
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Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan, Ghent, Belgium
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Cancer Research Institute Ghent (CRIG), Ghent, Belgium
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In brief
MicroRNAs (miRNAs) carried in extracellular vesicles are one of the essential factors for embryo development. This study demonstrates that miRNA-146 b has negative impacts on the quality and development of bovine embryos and also shows its higher expression in non-blastocyst embryos and related EVs.
Abstract
MicroRNAs (miRNAs), which can be carried inside extracellular vesicles (EVs), play a crucial role in regulating embryo development up to the blastocyst stage. Yet, the molecular mechanisms underlying blastocyst development and quality are largely unknown. Recently, our group identified 69 differentially expressed miRNAs in extracellular vesicles (EVs) isolated from culture medium conditioned by bovine embryos that either developed to the blastocyst stage or did not (non-blastocysts). We found miR-146b to be more abundant in the EVs derived from media conditioned by non-blastocyst embryos. Using RT-qPCR, we here confirmed the upregulation of miR-146b in non-blastocyst (arrested at two- to four-cell and morula stage) embryos compared to blastocysts (P < 0.005), which coincides with the upregulation of miR-146b in EVs derived from the medium of these non-blastocysts. To evaluate a functional effect, bovine embryo culture media were supplemented with miR-146b mimics, resulting in significantly decreased embryo quality, with lower blastocyst rates at day 7 and lower total cell numbers, while the opposite was found after supplementation with miR-146b inhibitors, which resulted in reduced apoptosis rates (P < 0.01). Transcriptomic analysis of embryos treated with miR-146b mimics or inhibitors showed differential expression (P < 0.01) of genes associated with apoptosis, cell differentiation, and the RNA Pol II transcription complex, including WDR36, MBNL2, ERCC6l2, PYGO1, and SNIP1. Overall, miR-146b is overexpressed in non-blastocyst embryos and in EVs secreted by these embryos, and it regulates genes involved in embryo development and apoptosis, resulting in decreased embryo quality.
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The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not a suitable environment for pre-compact morula stage horse embryos. By contrast, exposure to uterocalin during IVP improved capsule formation, although it did not measurably affect the development or expression of a panel of genes known to differ between in vivo and in vitro embryos. Further studies are required to evaluate whether uterocalin serves purely as a carrier protein or more directly promotes improved capsule development.
