Murine Blastocysts Release Mature MicroRNAs Into Culture Media That Reflect Developmental Status.

David Connor Hawke,Andrew John Watson,Danyal Baber Ahmed,Dean Harvey Betts

doi:10.3389/fgene.2021.655882

David Connor Hawke, Andrew John Watson + Show 2 more

Open Access

https://doi.org/10.3389/fgene.2021.655882

Copy DOI

Abstract

Extracellular microRNA (miRNA) sequences derived from the pre-implantation embryo have attracted interest for their possible contributions to the ongoing embryonic–uterine milieu, as well as their potential for use as accessible biomarkers indicative of embryonic health. Spent culture media microdroplets used to culture late-stage E4.0 murine blastocysts were screened for 641 mature miRNA sequences using a reverse transcription–quantitative polymerase chain reaction–based array. We report here 39 miRNAs exclusively detected in the conditioned media, including the implantation-relevant miR-126a-3p, miR-101a, miR-143, and miR-320, in addition to members of the highly expressed embryonic miR-125 and miR-290 families. Based on these results, an miRNA panel was assembled comprising five members of the miR-290 family (miR-291-295) and five conserved sequences with significance to the embryonic secretome (miR-20a, miR-30c, miR-142-3p, miR-191, and miR-320). Panel profiling of developing embryo cohort lysates and accompanying conditioned media microdroplets revealed extensive similarities in relative quantities of miRNAs and, as a biomarker proof of concept, enabled distinction between media conditioned with differently staged embryos (zygote, 4-cell, and blastocyst). When used to assess media conditioned with embryos of varying degrees of degeneration, the panel revealed increases in all extracellular panel sequences, suggesting cell death is an influential and identifiable factor detectable by this assessment. In situ hybridization of three panel sequences (miR-30c, miR-294, and miR-295) in late-stage blastocysts revealed primarily inner cell mass expression with a significant presence of miR-294 throughout the blastocyst cavity. Furthermore, extracellular miR-290 sequences responded significantly to high centrifugal force, suggesting a substantial fraction of these sequences may exist within a vesicle such as an exosome, microvesicle, or apoptotic bleb. Together, these results support the use of extracellular miRNA to assess embryonic health and enable development of a non-invasive viability diagnostic tool for clinical use.

Highlights

MicroRNAs are short ∼19- to 22-nucleotide noncoding RNAs ubiquitously transcribed from animal, plant, and viral genomes (Rana, 2007). miRNAs are potent modulators of widespread biological phenomena, acting to inhibit the transcription of mRNA through a process known as RISCmediated mRNA silencing (Bartel, 2004)
We demonstrate that extracellular media-borne miRNA may be accurately and reproducibly identified within spent microdroplets conditioned with murine pre-implantation embryos and, as a proof of concept, demonstrate their capability in describing the state of the embryos from which they are derived
Spent culture media that was conditioned with healthy murine blastocysts for 17 h was collected along with a cultured blank control, and both were profiled for 641 mature miRNAs using a TaqMan reverse transcription (RT)-quantitative polymerase chain reaction (qPCR)–based array to identify abundant extracellular miRNAs suitable for further study

Summary

Introduction

MicroRNAs (miRNAs) are short ∼19- to 22-nucleotide noncoding RNAs ubiquitously transcribed from animal, plant, and viral genomes (Rana, 2007). miRNAs are potent modulators of widespread biological phenomena, acting to inhibit the transcription of mRNA through a process known as RISCmediated mRNA silencing (Bartel, 2004). MiRNAs bind to complementary sequences in the 3 UTR region of a target mRNA sequence and may inhibit ribosomal translation or induce scission of the mRNA transcript (Rana, 2007). Cellular release of miRNAs occurs either through binding to secreted AGO1 (Cuman et al, 2015) and apolipoprotein proteins (Vickers et al, 2011; Tabet et al, 2014), encapsulation within vesicles such as exosomes (Kosaka et al, 2010; Chevillet et al, 2014; SatoKuwabara et al, 2015), or during apoptosis. Exosomes are known secretory products from nearly every cell type (Simons and Raposo, 2009); most frequently, extracellular miRNA is found in an unencapsulated, protein-bound state (Turchinovich et al, 2011)

Methods

Results

Discussion

Conclusion