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，就像它们在过去几十年里为人类所做的一样。体外培养胚胎在商业工业中的低应用仍然源于早期胚胎死亡率高，最多只能产生30-40%的囊胚率。由于我们不了解哪些胚胎被认为是最健康的，最有可能发育到足月，这些生殖方面的挑战就更大了。目前的胚胎选择实践，即形态和形态动力学评估，是主观的，有偏见的，不准确的，并且不足以定量有效地预测发育潜力。需要一种更定量的方法，以非侵入性的方式评估体外胚胎。近年来，代谢组学和胚胎代谢已成为人类体外受精的一个活跃研究领域，现在在牛体外受精中，通过发现生物标志物来帮助胚胎选择。我们实验室鉴定了16个显著的生物标志物，它们在生长缓慢的胚胎和正常的胚胎之间存在差异表达，其中多胺和磷脂酰胆碱是最显著的。在体外培养过程中，慢胚的废培养基中多胺和磷脂酰胆碱的含量高于正常生长的胚。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显微注射技术来研究牛胚胎发育过程中聚酰胺和磷脂酰胆碱的代谢途径。此外，将使用特定的化学物质或试剂（siRNA显微注射）对代谢途径进行特异性抑制或增强。特定代谢物的定量将使用标准的定量程序，ELISA和/或DFI-LC-MS/MS进行。为了验证第二种假设，即这些代谢物是从不健康的胚胎中泄漏出来的，将对胚胎进行增加剂量的氧化应激处理，并观察这些途径的反应，无论是从组成基因方面还是从废培养基中存在的水平。由于多胺和磷酰胆碱的细胞生物学在胚胎着床前发育过程中知之甚少，这些创新的研究将解决这个问题；是什么造就了一个有活力的牛着床前胚胎？胚胎对-à-vis发育的抑制分子机制是什么？

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?