Characterization of microRNA in bovine in vitro culture media associated with embryo quality and development

Abstract

Dairy cattle fertility has declined over time due to factors including reduced fertilization and early embryonic loss. To counter fertility problems and better study preimplantation embryonic development, in vitro production systems have been developed. These systems largely assess embryos based on their morphology, which is not a strong indicator of developmental potential. Currently, no biomarkers can be used to noninvasively survey an embryo's potential in terms of its development and ability to establish a pregnancy. Thus, the objective of this study was to characterize and identify microRNA (miRNA) in culture media of embryos of differing developmental competence for future development as noninvasive biomarkers of embryo quality. The MiRNA sequencing of media conditioned by blastocyst and degenerate (those that failed to develop from the morula to blastocyst stage) embryos, revealed 11 differentially expressed miRNA; all were higher in concentration in degenerate conditioned media. Differential expression of mature microRNA (miR)-24, miR-191, and miR-148a was further validated using quantitative real-time PCR. Functional analysis of miR-24 revealed that addition of a mimic miRNA to culture media of morulae embryos resulted in a 27.3% decrease in development to the blastocyst stage. Furthermore, expression of miR-24 was 44.29-fold higher in blastocysts cultured with a miR-24 mimic compared with control blastocysts. Interestingly, the expression of CDKN1b, a target gene of miR-24 was repressed in embryos grown in the presence of the miRNA mimic. Mimic supplementation experiments suggest that miRNA are taken up by the embryo and that extracellular miRNA affect embryonic development. Overall, identification of a rich extracellular milieu in conditioned media sets the framework for future studies to determine the long-term predictive ability of embryo-based miRNA biomarkers on pregnancy outcome.

Sexual Dimorphism of miRNAs Secreted by Bovine In vitro-produced Embryos

“…Embryos and media were procured as described by Kropp and Khatib (2015). Ovaries were obtained from Applied Reproductive Technology, LLC (Monona, WI, USA), and follicles were aspirated to recover oocytes.…”

“…miRNAs can be secreted out of a cell through extracellular vesicles, via apoptotic bodies, or by being bound to AGO proteins (Turchinovich et al, 2013). In cattle, the presence of circulating miRNAs has been reported in bodily fluids such as milk (Sun et al, 2015), blood, and follicular fluid (Noferesti et al, 2015) and in the in vitro culture media of blastocysts (Kropp et al, 2014; Kropp and Khatib, 2015). miRNAs have shown involvement in a variety of biological processes including organismal development, cell proliferation, cell death, hematopoiesis, and immunity (Wahid et al, 2010).…”

Effects of embryo-derived exosomes on the development of bovine cloned embryos

“…Embryos produced in vitro from most mammalian species usually display fewer blastocysts capable of hatching and implantation, altered inner cell mass/trophectoderm (ICM/TE) ratios, higher apoptosis index, and lower total cell numbers. Moreover, these embryos present blastomeres with irregular sizes, increased sensitivity to cryopreservation, and abnormal gene expression and epigenetic modification [3–6]. Suboptimal culture conditions are main factors affecting the developmental competence of embryos produced in vitro [7].…”

Effect of dietary Astragalus Polysaccharide supplements on testicular miRNA expression profiles and enzymatic changes of breeder cocks

“…The differential expression of miRNAs based on normalized counts was analyzed using Student t test according to the experimental design and the significance threshold was set as 0.05 for each test74757677. The fold-change and P-value for each miRNA was calculated, based on the normalized expression using the formula as previously described1273.…”