Understanding developmental competence in preimplantation embryos

牛的生育率下降仍然是一个具有重大经济意义的世界性问题。辅助生殖技术可以为商业养牛业提供克服这些问题的好处，就像过去几十年来为人类所做的那样。体外生产的胚胎在商业工业中的低应用仍然源于早期胚胎死亡率高，最多产生30- 40%的适度囊胚率。这些生殖挑战由于我们缺乏对哪个胚胎被认为是最健康和最有可能发育到足月的了解而加剧。目前的胚胎选择实践，形态学和形态动力学评价，是主观的，有偏见的，不准确的，不足以有效地预测发育潜力的定量。需要一种更加定量的方法，以非侵入性的方式在体外评估胚胎。近年来，代谢组学和胚胎代谢领域已成为人类IVF研究的一个活跃领域，现在在牛IVP中，通过发现生物标志物来帮助胚胎选择。我们的实验室已经确定了16个重要的生物标志物，这些标志物在缓慢生长的胚胎和正常生长的胚胎之间差异表达，其中多胺和磷脂酰胆碱是最显着的。多胺和磷脂酰胆碱的水平是较高的慢胚比在体外培养过程中正常生长的胚胎用过的媒体。这表明发育缓慢的胚胎可能需要额外的努力才能进入下一个发育阶段，或者这些代谢物可能会从胚胎中泄漏出来。因此，为了验证第一个假设，我们计划了一系列实验，使用DFI-LC-MS/MS，RT-qPCR，免疫定位和共聚焦成像，siRNA显微注射技术来检查牛胚胎发育过程中的聚酰胺和磷脂酰胆碱代谢途径。此外，将使用特定化学品或试剂（siRNA显微注射）对代谢途径进行特异性抑制或增强。将使用标准定量程序ELISA和/或DFI-LC-MS/MS进行特定代谢物的定量。为了测试这些代谢物从不健康胚胎中泄漏的第二个假设，将用增加剂量的氧化应激处理胚胎，并观察这些途径的反应，包括组成基因以及用过的培养基中存在的水平。由于在植入前胚胎发育过程中对多胺和磷脂酰胆碱的细胞生物学知之甚少，这些创新性研究将解决这个问题;是什么使牛植入前胚胎存活，以及胚胎发育停滞的分子机制是什么？

Declining fertility rates in cattle continue to be a worldwide problem of significant economical proportions. Assisted reproductive technologies can offer the commercial cattle industry benefits of overcoming these problems in the same manner that they have been doing for humans over the past several decades. The low application of in-vitro produced embryos in the commercial industry continues to stem from the high rate of early embryonic mortality, yielding modest blastocyst rates of 30-40%, at best. These reproductive challenges are augmented by our lack of understanding on which embryo is considered to be the healthiest' and most likely to develop to term. The current practice of embryo selection, a morphological and morphokinetic evaluation, is subjective, biased, inaccurate, and not quantitative enough to be effective at predicting developmental potential. There needs to be a more quantitative method of assessing an embryo in vitro in a non-invasive manner. In recent years, the field of metabolomics and embryo metabolism has become an active area of research in human IVF, and now in bovine IVP, to aid in embryo selection through biomarker discovery. Our laboratory has identified 16 significant biological markers, which are differentially expressed between slow and normal growing embryos with polyamines and phosphotidylcholine being the most notable ones. Levels of both polyamines and phosphotidylcholine are higher in the spent media of slow embryos than the embryos that grow normally during in vitro culture. This suggests that the embryos that are developing slowly might be requiring extra effort to progress to the next stage of development or there could be leakage of these metabolites out of the embryo. Therefore, to test the first hypothesis, we have planned a series of experiments using DFI-LC-MS/MS, RT-qPCR, immunolocalization and confocal imaging, siRNA microinjection techniques to examine the polyamide and phosphatidylcholine metabolic pathways during bovine embryo development. In addition, specific inhibition or augmentation of the metabolic pathways will be undertaken using specific chemicals or reagents (siRNA microinjection). Quantification of specific metabolite will be undertaken using standard quantification procedures, ELISA and/or DFI-LC-MS/MS. To test the second hypothesis that these metabolites are being leaked out of unhealthy embryos, embryos will be treated with increasing doses of oxidative stress and the response of these pathways, both in terms of constituent genes as well as the levels present in the spent media will be observed. Since the cellular biology of polyamines and phosohotidylcholine is poorly understood during preimplantation embryo development, these innovative studies will address the question; what makes a viable bovine preimplantation embryo and what is the molecular mechanism of embryo arrest vis-à-vis development?