Performing Department
(N/A)
Non Technical Summary
The beef and dairy industry are the primary suppliers of dietary protein sources aiding to meet the growing global demand, which relies heavily on animal reproduction as a central determinant of production efficiency. Unfortunately, dairy cattle fertility is continually declining worldwide, which hampers implemented efforts to advance economic efficiency in the dairy sector. Even though Assisted Reproductive Technologies (ARTs) currently account for a growing portion of livestock and human populations, ART-induced oxidative stress can also contribute to impacts on embryo development with long-term consequences on fetal growth and offspring health. Among the several molecular mechanisms associated with an embryo's response to stress, we have previously found the nuclear factor erythroid 2-related factor 2 - kelch-like ECH-associated protein 1 (NRF2-KEAP1) pathway to act as a first-line defense mechanism, with the ability of the embryo to activate its NRF2-signaling highly associated with its survival and viability under oxidative stress conditions. Therefore, approaches to induce NRF2 protein activity in bovine embryos will contribute to improved survival, viability, and enhanced reproductive efficiencies following ARTs in animals. Moreover, here we aim to implement a dual-targeted approach using CRISPR-cas9 mediated knockdown of KEAP1 gene (NRF2-inhibitor) in Specific Aim 1 and a pharmacological strategy using the antioxidant quercetin in the in vitro culture system in Specific Aim 2, to increase the relative abundance of localized NRF2 protein in bovine preimplantation embryos to promote their survival and viability under oxidative stress conditions. The modulation of this pathway in preimplantation embryos is expected to reduce oxidative damage in the resulting blastocysts to enhance the survival and viability of embryos under suboptimal conditions, particularly with respect to currently implemented ART procedures. Moreover, the activation of NRF2 in the preimplantation period has the potential to mitigate stress-induced abnormal molecular alterations, thereby attenuating long-term negative consequences of stress on fetal growth and offspring health. The planned project will identify genes and genome regions, that can be targets for future intervention against physiological and environmental stress-induced infertility problems in dairy and beef cattle.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The goal of the projectis to develop strategies both by genetic and pharmacological means to tackle the problem of oxidative stress in bovine preimplantation embryos, which results in altered developmental competence and transcriptome and epigenome landscape with a profound effect on fetal development and offspring health. Since oxidative stress in oocytes and embryos is correlated with various pathophysiological conditions, this study will enable us to identify genome regions and genes that are sensitive to oxidative damage providing insight into targets for future fertility treatment strategies. With the follow-up experiments, embryos from different experimental groups in Specific Aims 1 and 2 can be transferred to recipients, and the consequence of inducing NRF2 activity during the preimplantation period on fetal growth and offspring health can be elucidated.
Project Methods
In both specific aims 1 and 2 of the project, the following research methods will be used1. CRISPR-Cas9 based knockdown of the KEAP1 gene (NRF2 inhibitor). For this, the CRISPR complex will be delivered to the zygote stage bovine embryos using an electroporation procedure. The gene editing efficiency will be determined by sequencing of biopsies collected from individual blastocysts.2. In aim 2 of the project, the pharmacological substance quercetin will be supplemented during the culture of bovine embryos.3. Following both CRISPR-based knockdown of the KEAP1 gene and quercetin supplementation, embryos generated will be used for investigation of ROS accumulation, mitochondrial analysis, large-scale transcriptome analysis, and whole genome bisulfite sequencing to identify genes and genome regions that can be targets for future intervention to tackle oxidative stress-induced damages in preimplantation embryos.In addition to a weekly project progress evaluation and discussion during lab meetings, the project progress will be assessed and evaluated by the PD and Co-PDs every three months throughout the year