Characterization of microRNA in bovine in vitro culture media associated with embryo quality and development
Characterization of microRNA in bovine in vitro culture media associated with embryo quality and development
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DOI：
10.3168/jds.2015-9510
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发表时间：
2015-09-01
影响因子：
3.5
通讯作者：
Khatib, Hasan
中科院分区：
文献类型：
--
作者：
Kropp, Jenna;Khatib, Hasan
由于受精减少和早期胚胎丢失等因素，奶牛的生育力随着时间的推移而下降。为了解决生育问题和更好地研究植入前胚胎发育，已经开发了体外生产系统。这些系统主要根据胚胎的形态来评估胚胎，这不是发育潜力的有力指标。目前，没有生物标志物可以用于非侵入性地调查胚胎在发育和建立妊娠能力方面的潜力。因此，本研究的目的是表征和鉴定具有不同发育能力的胚胎培养基中的微小RNA（miRNA），以供未来发育作为胚胎质量的非侵入性生物标志物。对囊胚和退化（未能从桑椹胚发育到囊胚阶段的胚胎）胚胎的条件培养基进行miRNA测序，发现11种差异表达的miRNA;在退化条件培养基中，所有miRNA的浓度都较高。使用定量实时PCR进一步验证成熟microRNA（miR）-24、miR-191和miR-148 a的差异表达。miR-24的功能分析显示，在桑椹胚培养基中添加模拟miRNA导致发育到囊胚阶段的减少27.3%。此外，与对照囊胚相比，用miR-24模拟物培养的囊胚中miR-24的表达高44.29倍。有趣的是，CDKN 1b的表达，miR-24的靶基因在miRNA模拟物存在下生长的胚胎中被抑制。模拟补充实验表明，miRNA被胚胎吸收，细胞外miRNA影响胚胎发育。总的来说，在条件培养基中鉴定丰富的细胞外环境为未来的研究确定基于胚胎的miRNA生物标志物对妊娠结局的长期预测能力奠定了基础。
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.