Performing Department
(N/A)
Non Technical Summary
The utilization of laboratory-derived, or in vitro produced (IVP) embryos, has become vital to the beef and dairy cattle industries. IVP embryos allow for propagation of genetic traits from elite animals and increase the efficiency of cattle production systems, thereby contributing to a more sustainable and secure food source for a continually increasing global population. However, IVP embryos have several roadblocks to successful development and establishment of pregnancy. When the female gamete, or oocyte, is removed from the ovary, it encounters several stressors that can significantly reduce its quality. In fact, less than 40% of oocytes go on to produce a viable embryo for transfer. Therefore, it is critical to address oocyte-specific factors that contribute to poor development of IVP embryos. The overall goal of this research proposal is to understand how stress factors, namely oxidative stress during culture, leads to poor maturation and developmental competence of the oocyte. To accomplish this, we will perform culture of oocytes under different conditions to determine how oxidative stress impacts the way genes are regulated within the oocyte. Certain marks, or methylation, on genes determine their ability to be either degraded or made into protein for a certain function. Specifically, it is our goal to determine how deposition or removal of these methylation marks may be impacted by metabolic changes in the oocyte due to external stressors. We expect that oxidative stress caused by laboratory culture of oocytes alters the proteins that control these methylation marks, thus altering the regulation of genes. Ultimately, our work to uncover these mechanisms may lead to new embryo culture treatments that will allow for a greater number of high-quality embryos produced for beef and dairy cattle production systems.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The overall goal of this proposal is to determine mechanisms that regulate the epitranscriptomic landscape of maternal mRNA in bovine oocytes. This knowledge will be used to develop appropriate treatments to address potential m6A dysfunction due to oxidative stress induced during IVM, thereby improving IVP embryo outcomes. Objective 1 - Determine the impact of oxidative stress on one-carbon metabolism and subsequent METTL3-regulated mRNA methylation.Objective 2 - Examine the effects of redox homeostasis on demethylase activity of FTO during in vitro maturation.
Project Methods
General Oocyte and Embryo Culture:Ovaries will be obtained from slaughterhouse animals by a collaborating scientist at US Meat Animal Research Center in Clay Center, NE. Cumulus oocyte complexes (COCs) will be obtained via follicle puncture and placed into oocyte maturation medium (bOMM). After in vitro maturation (IVM), semen straws from a proven bull will be used to fertilize mature (MII) oocytes, and embryos will be cultured until end of pre-implantation embryo development.Objective 1 - Determine the impact of oxidative stress on one-carbon metabolism and subsequent METTL3-regulated mRNA methylation.Experiment 1A: Determine if METTL3-driven methylation of mRNA in MII oocytes is dependent on SAM availability. COCs will be cultured in bOMM containing the MAT2A inhibitor to deplete endogenous SAM during IVM. COCs will be matured in the presence of increasing concentrations of exogenous SAM. Because the number of oocytes reasonably available is too low to effectively purify METTL3, we will use a two-step approach to determine the activity of METTL3 in SAM-treated oocytes. First, we will perform the MTase-Glo assay to determine SAH concentrations in MII protein extracts. To demonstrate that the change in SAH concentration specifically increases METTL3 activity, we will perform the same assay in oocytes depleted of both SAM and METTL3 (via microinjection of METTL3 siRNA) and then treated with exogenous SAM. SAM-dependent regulation of METTL3 activity will be determined.Experiment 1B: Establish that SIRT1 regulates one-carbon metabolism and mRNA methylation by METTL3 during IVM. COCs will be collected and matured under control conditions (Con), or in the presence of SIRT1 activator (SIRT1+) or SIRT1 inhibitor (SIRT1-). Protein extracts from MII oocytes will be assessed for SAM using the Bridge-It S-adenosyl methionine fluorescence assay. Using a second set of Con, SIRT1+, and SIRT1- MII oocytes, we will assess methylation status of selected maternal mRNAs that we previously demonstrated to be highly methylated in murine in vivo derived MII oocytes. Briefly, after maturation for 24h, cumulus cells will be stripped from oocytes and RNA extracted from pools of 40 MII oocytes. Extracted RNA will then be incubated in the absence (1/2 of each sample, control) or presence (1/2 of each sample) of MazF, which is an enzyme that cuts at unmethylated but not methylated ACA sites. RNA will then be re-extracted, reverse transcribed and subjected to RT-qPCR. Primers will be designed for each maternal gene that span the highly predicted ACA methylation site. Treatment effects on percent methylation will be determined by comparing PCR product from MazF-treated to MazF-untreated samples.Experiment 1C: Determine if supplementation of IVM culture with an antioxidant and/or methyl donor increases SAM availability, global m6A, and embryo development. COCs will be collected and matured in the following treatment groups: Control (Con), Resveratrol (Res), Methionine (Met) and Resveratrol+Methionine (Res+Met). MII oocytes be fertilized and cultured to assess blastocyst development in each treatment group. MII protein extracts will be assessed for relative changes in SAM substrate availability using the assay described in Exp 1B. Treatment-dependent changes in global m6A will be determined via immunofluorescence staining and imaging as described in Exp 1A, using primary antibody against m6A. Resveratrol- and methionine-dependent differences in SAM and global m6A levels will be determined.Objective 2 - Examine the effects of redox homeostasis on demethylase activity of FTO during in vitro maturation.Experiment 2A: Determine if excess oxidative stress during IVM alters NADP/NAPDH and impacts global mRNA methylation in MII oocytes. Bovine COCs will be collected as described and matured at control (21%) or hypoxic (5%) O2 concentrations, which induces oxidative stress via altered NADP production. MII oocytes will be incubated with CellROX Green. These same MII will then be fixed and stained for m6A as described in Exp 1C to determine correlations between increased ROS and global mRNA methylation. Additionally, MII protein extracts will be assayed for redox imbalance using the NADP/NADPH Quantification kit. Lastly, blastocyst development for the two treatment groups will be assessed.Experiment 2B: Determine if NADP increases FTO activity and/or abundance, and global m6A methylation during IVM. During maturation, COCs will be exposed to Control (Con), NADP-increased (+NADP) or NADP-depleted (-NADP) conditions. To deplete NADP, bOMM will be supplemented with glucose-6-phosphate dehydrogenase (G6PD) activator. FTO demethylase activity in MII oocytes will be assessed using the two-step approach involving knock down of FTO as described in Exp 1A. Changes in global m6A of Con, +NADP, and -NADP oocytes will be measured using immunofluorescence. FTO abundance in MII will be measured via immunofluorescence staining using a monoclonal antibody against FTO and further validated in MII protein extracts using the Jess capillary western blot system with the same antibody. NADP-dependent changes in FTO expression will be determined.Experiment 2C: Determine the mechanism by which NADP modulates FTO demethylation of mRNA during IVM. GV oocytes will be co-injected with custom synthetic mRNA that contains a methyladenosine within an ACA motif and FTO siRNA or recombinant FTO protein. Injected GV will then be matured in under control, +NADP, or -NADP conditions. A subset of MII will be stained with anti-FTO to assess effectiveness of knock down and over expression. Following maturation, the MazF enzyme coupled to qPCR assay described in Exp 1B will be performed using primers against the synthetic mRNA. Changes in percent methylation in response to modulated FTO and NADP levels will be determined.