Understanding developmental competence in preimplantation embryos

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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?