Progress 06/01/19 to 05/31/24
Outputs
Target Audience: Training: 2 Undergraduate students Dispersal of knowledge through abstracts and talks to the scientific community (researchers working in the areas of embryology, nutrition, reproductive physiology, metabolic engineering and metabolism) with the ultimate aim of benefiting the dairy and beef cattle industries Changes/Problems:Restrictions on research and difficulties obtaining resources due to COVID-1 significantly delayed progress. Staffing issues (graduation of a doctoral student and the postdoc leaving for an academic position) slowed progress as there were inadequate funds for new personnel. However, we initiated a collaboration with Dr. Sunny's lab and obtained access to his GC/MS/MS for additional sample processing and analysis (including glycogen analysis). What opportunities for training and professional development has the project provided?Previous trainees were successful in their career advancement: Doctoral student, Dr. Dan Lugar took a computational analysis position in industry (APL) in 2022. The postdoctoral fellow, Dr. Halli Weiner, who led the embryo analysis (protein localization), accepted an academic position (Assistant Clinical Professor) leading an undergraduate First-Year Innovation & Research Experience project (UMD FIRE). Undergraduate trainees: The graduate student responsible for the lipid droplet and re-expansion studies is now working as an embryologist at a fertility clinic. The two undergraduate researchers who were involved in the monitoring of the localization of metabolic enzymes and transcription factors by immunofluorescent staining have since graduated and taken positions at NIH. How have the results been disseminated to communities of interest?A poster was presented on the metabolic capacity of bovine trophectoderm to convert glucose into glycogen at the annual conference of the Society for the Study of Reproduction in Dublin, Ireland, July 16-19, 2024. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Aim 1: 13C Metabolic flux analysis (13C MFA): The manuscript describing our experimental results on isotope labeling of bovine blastocysts and CT1 trophectoderm cell line, Utilization of Glucose and Fructose in Bovine Trophectoderm and Blastocysts Assessed by Metabolic Flux Analysis is being submitted to Molecular Genetics and Metabolism. In this manuscript we report the following findings. 13C MFA was used to measure metabolic flux in individual and small groups of bovine embryos and the trophectoderm cell line, CT-1. The CT-1 cells provided a good metabolic model as flux paralleled that observed in the blastocysts. In both blastocysts and the CT-1 cells glucose and fructose were metabolized primarily to lactate and pyruvate (~70-80%) with a much lower contribution to the TCA cycle and the PPP pathway (10-15%), and glycogen (5-10%). While pyruvate and lactate reach steady state in under 1 h in 1-13C glucose, the M+1 of citrate ramps up gradually to ~30% by 6 h and is still increasing at the end of the 6 h experiment, displaying slower labeling dynamics than pyruvate or lactate. Other TCA metabolites including glutamic and aspartic acids showed slower labeling than citrate (~5-10% labeling at 6 h vs ~30%), corroborating the low flux through the TCA cycle. Fructose flux followed that of glucose with the primary products being lactate and pyruvate, however the relative flux was at a much lower rate than that of glucose. When glucose and fructose were provided in equal amounts (0.75 mM), glucose was primarily used. This glycolytic production of lactate and pyruvate represents a shift in flux as in cleavage stage and morula flux is directed primarily to the PPP with minimal to lactate and pyruvate, representing the need for molecular building blocks at the earlier stages. While the majority (80%) of the heavy isotope (13C) from labeled glucose was identified in products of glycolysis (primarily lactate and pyruvate)and to a lesser extent (10-15%) in TCA metabolites and the pentose phosphate pathway, 5-10% is unaccounted. A portion of the unaccounted metabolites might be stored as glycogen. The presence of glycogen in bovine embryos and CT1 cells was first demonstrated using Periodic Acid-Schiff staining and then confirmed using GC/MS. Studies using U13C-glucose demonstrated that CT1 cells converted glucose and pyruvate derived from the labeled glucose into glycogen in a concentration dependent manner (collaboration with N. Sunny lab). However, the strong staining retained after amylase treatment which removes the glycogen suggested that stained substances were predominantly mucins (i.e., neutral and acid simple non-sulfated and acid complex sulfated types). Further studies are planned to determine if these glycogen stores can be accessed by the blastocyst for subsequent energy use. Aim 2: Altering substrate availability will alter embryo quality as measured by embryonic cell number (ICM/TE ratio), expression of key proteins involved in lineage differentiation (OCT4 and CDX2) and metabolism (GAPDH, PKM2), and lipid density. Embryos were cultured in media containing differing energy substrates (e.g., glucose or fructose), then stained with BODIPY, imaged and analyzed (ImageJ) for lipid accumulation (e.g., amount, droplet size. Provision of glucose or fructose at increasing concentrations (0.75 mM,1.5 mM, 3.0 mM) did not shift droplet size and amount of lipid significantly during short incubations used in the flux studies (24 hr). An energy requiring stress test was used to determine the importance of pyruvate and fatty acid metabolism as energy sources at the blastocyst stage. The effects of inhibitors of long-chain (LC) fatty acids transport (Etomoxir) and the mitochondrial pyruvate transporter (UK5099) on metabolic energy pathways (altering availability of substrates by blocking transport) required for re-expansion were tested. Expanded blastocysts were collapsed using cytochalasin B and ouabain (inhibitor of the sodium-potassium pump). Embryos were then allowed to re-expand in the presence or absence of the inhibitors. Our hypothesis was that as blastocysts primarily use glucose and fructose in aerobic glycolysis other substrates such as fatty acids are utilized by the mitochondria for energy production. Interestingly, inhibition with etomoxir (blocking LC-fatty acids transport into the mitochondria and hence beta-oxidation) did not block re-expansion of collapsed blastocysts. Inhibition of pyruvate transport into the mitochondria had a slight, but significant, effect on re-expansion (67% of UK5099 treated embryos re-expanded vs. 100% of controls), while combination of both inhibitors completely blocked re-expansion. This demonstrates the flexibility of bovine blastocysts in that they can overcome blockage of one pathway by presumably switching to the other but cannot overcome blockage of both pyruvate and LC-fatty acid entry into the mitochondria (contribution to TCA cycle and beta-oxidation, respectively). Some proteins, particularly those involved in metabolic processes have been described as having moonlighting capabilities - that is having a different function when not performing its primary duty. For example, when PKM2 - which functions as a tetrameter when active in glycolysis - is not busy, it can dissociate into dimers and monomers and translocate to the nucleus where it may function in regulation of gene expression. Earlier studies (this grant) suggested that PKM2 could shift between the cytoplasm and nucleus in blastocysts, depending on hexose supplementation (Weiner, H., Tompkins, L., Keefer, C.L. (2022). Glycolytic substrates influence intracellular movement of PKM2 and expression of OCT4 in bovine preimplantation embryos. IETS annual conference, Savannah, GA. January 13, 2022. RFD 34(2) 271). Blastocysts provided with glucose had a more cytoplasmic PKM2 localization, while fructose, which has less flux through glycolysis, resulted in a slight shift to the nucleus. These studies were done using antibodies that were described as specific to PKM1 and PKM2, however, were subsequently found not to be. Therefore, it could not be determined whether or not both of the PKM isoforms were shifting to the nuclear location or just the PKM2 as expected. New validated antibodies were obtained and used in the bovine trophectoderm model to assess whether similar shifts in location were observed. Indeed, the validated antibodies provided confirmation that PKM2 shifts from a predominantly cytoplasmic location to nuclear dependent on hexose supplementation. Nuclear localization of PKM2 was strongest in CT-1 cells cultured with galactose which slows down glycolysis. Fructose, which moves through glycolysis slower than glucose, also shifted to a more nuclear localization. The strength of the shift to nuclear localization was dependent on the hexose used: galactose>fructose>glucose. The CT1 data is being analysis and the embryo study repeated. In addition, samples were collected following incubation in glucose, fructose or galactose and were processed by RNAseq. Analysis of this data may point to changes in gene expression which can be further studied to determine whether shifts in PKM2 nuclear location may directly affect specific gene expression. Additional manuscripts describing the effects of hexose supplementation on morphological characteristics (lipid droplets, mitochondrial potential, and localization of PKM2, PKM1, OCT4, CDX2 and GAPDH) and mRNAseq will be prepared as data analysis is completed.
Publications
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:Training: 1 undergraduate, 1 master's student, 1 doctoral student Dispersal of knowledge through abstracts, publication and talks to the scientific community (researcher working in the areas of embryology, nutrition, reproductive physiology, metabolic engineering and metabolism) with the ultimate aim of benefiting the dairy and beef cattle industries. Changes/Problems:Restrictions on research and difficulties obtaining resources due to COVID-1 significantly delayed progress. Staffing issues (graduation of a doctoral studentand the postdoc leaving for an academic position) slowed progress further, but these issues are being resolved by the addition of a new bioengineering doctoral graduate student (Funke D. Okunrinboye) who is being trained on the Waters LC/MS/MS. In addition, we have obtained access to Dr. Sunny's GC/MS/MS for additional sample processing and analysis (including glycogen analysis). What opportunities for training and professional development has the project provided?Previous trainees were successful in their career advancement: Doctoral student, Dr. Dan Lugar, took a computational analysis position in industry (APL) in 2022. The postdoctoral fellow, Dr. Halli Weiner, who led the embryo analysis (protein localization) portion in the previous year, left to take an academic position leading an undergraduate First-Year Innovation & Research Experience project (UMD FIRE). Laura Tompkins, the MS graduate student, completed her image analysis of lipid droplets. She has taken a position as an embryologist at an IVF clinic. New trainees: An undergraduate (Emma Kellenberger) has taken on culture responsibilities for the CT1 (bovine trophectoderm) cells.She plans on joining the ANSC department as a graduate student next fall.A new doctoral graduate student (Funke D. Okunrinboye) in chemical engineering has startedtraining in Dr. Sriram's laboratoryand will be taking on responsibility for analysis of flux samples on the LC/MS/MS. She is a member of an underrepresented group in STEM (African-American female). How have the results been disseminated to communities of interest?Research progress was presented at the annual Multi-State Research Project (Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock W-4171) which was held in at the University of Florida, March 3, 2023 (both in person and by Zoom). What do you plan to do during the next reporting period to accomplish the goals?We will continue our studies as outlined in the objectives above and described in NIFA grant: A systemic approach to deciphering energy metabolism in preimplantation embryos. More specifically, Stored samples will be processed and analyzed using the LC-MS/MS (Bioengineering) and the GC/MS (Dr. Sunny lab, ANSC) to further develop the flux map. Samples will be collected and analyzed for glycogen (Sunny lab, GC/MS) as studies performed in 2022-2023 suggested glycogen and/or other neutral glycolipids are stored in the embryos and CT1-cells. A manuscript, now in draft form, that describes the flux map produced using labeled glucose and fructose will have been submitted for publication, any requested revisions after review will be made. Additional manuscripts describing the effects of glucose and fructose supplementation on morphological characteristics (lipid droplets, mitochondrial potential, and localization of OCT4 and GAPDH) will be prepared as data analysis is completed.
Impacts
What was accomplished under these goals?
Aim 1: 13C Metabolic flux analysis (13C MFA) was used to investigate carbon source usage and the effects of medium composition on metabolism in preimplantation bovine embryos. Assessing metabolism in individual bovine embryos presents unique challenges owing to the small cell number (< 200 cells). Furthermore, the media are complex and contain multiple carbon sources, thus potentially obfuscating the labeling patterns of the 13C-labeled carbon source. Because of the small sample size, there is only a limited set of metabolites whose labeling patterns can be accurately measured. • We have designed novel labeling experiments and developed a software, eiFlux, to surmount these challenges and obtain a metabolic flux map for this complex system. The software eiFlux performs cumulative isotopomer (cumomer) balancing along with state-of- the-art-optimization to calculate a robust solution to the metabolic flux analysis problem (Lugar & Sriram, 2022). • A manuscript describing our experimental results on isotope labeling of bovine blastocysts and CT1 trophectoderm cell line has been prepared for submission to Molecular Genetics and Metabolism in spring of 2023. Protocols have been developed for the Waters LC-MS/MS instrument and amino acid standards have been run. • Sample collections (spent medium from bovine blastocyst cultures) were completed from experiments designed to probe the relative utilization of glycolysis and the pentose phosphate pathway, as well as the contributions of different carbon sources to metabolites originating at the pyruvate node. These samples are ready for processing. Processing was delayed due to covid and staffing issues, but these have been resolved by obtaining access to another GC/MS/MS in Dr. Sunny's lab whose members can run the samples for us and by the training of a new graduate student who joined Dr. Sriram's lab. Use of these two instruments will allow us to continue our sample analysis, which was halted due to loss of the GC/MS/MS that was used previously on this project. Aim 2: Altering substrate availability will alter embryo quality as measured by embryonic cell number (ICM/TE ratio), expression of key proteins involved in lineage differentiation (OCT4, NANOG, and CDX2) and metabolism (GAPDH, PKM2), and lipid density. • Embryos cultured in media containing differing energy substrates (e.g., glucose or fructose) were imaged for lipid accumulation (e.g., amount, droplet size) using BODIPY stains and Image J analysis. Initial data indicates that the energy substrates shifted droplet size and amount of lipid. The effects of inhibitors (inhibitors of long chain (LC)-fatty acids and pyruvate transport into the mitochondria) on the ability of collapsed blastocysts to re-expand (a stress test todetermine metabolic energy pathways required for re-expansion) were assessed. Interestingly, inhibition with etomoxir (blocking LC-fatty acids transport into the mitochondria and, hence, beta-oxidation) did not block re-expansion of collapsed blastocysts. Inhibition of pyruvate transport into the mitochondria had a slight, but significant, effect on re-expansion (67% of UK5099 treated embryos re-expanded vs. 100% of controls), while a combination of both inhibitors completely blocked re-expansion.This demonstrates the flexibility of bovine blastocysts in that they can overcome blockage of one pathway by presumably switching to the other but cannot overcome blockage of both pyruvate and LC-fatty acid entry into the mitochondria (contribution to TCA cycle and beta-oxidation, respectively). While the majority (80%) of the heavy isotope (13C) from labeled glucose was identified in products of glycolysis (primarily lactate and pyruvate)and to a lesser extent in TCA metabolites, 20% is unaccounted. In order to determine whether there might be significant amounts ofglycogen and/or other neutral glycolipids storage inbovine blastocysts and CT-1 cells, they were stainedusing the Periodic Acid Schiff method. Strong staining was observed which was not susceptible to diastase (amylase) treatment to any great extent which indicated the presence of neutral glycolipids/glycoproteins other than glycogen which is sensitive to diastase digestion.Studies are ongoing to determine the amount of glycogen present (amylase sensitive),the identify of amylase-insensitiveneutral glycolipids orglycoproteins, and if labeled hexose contributes to these stores.
Publications
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Progress 06/01/21 to 05/31/22
Outputs
Target Audience:Training: 1 MS and 1 PhD student; 1 Postdoctoral fellow Dispersal of knowledge through abstracts, publications and talks to the scientific community (researchers working in the areas of embryology, nutrition, reproductive physiology, metabolic engineering and metabolism) with the ultimate aim of benefiting the dairy and beef cattle industries Changes/Problems:Restrictions on research and difficulties obtaining resources (plasticware, etc.) due to COVID-1 significantly delayed progress. The GC/MS has proven unrepairable as the needed part could not be attained. Fortuitously a Waters LC-MS/MS instrument has been added to campus facilities. Additionally, a colleague in ANSC (N. Sunny) has agreed to let us use his GC/MS if the LC/MS/MS doesn't prove satisfactory. What opportunities for training and professional development has the project provided?Doctoral student, Dan Lugar, successfully defended his dissertation and has taken a computational analysis position in industry. He developed a more complex model and software, eiFlux, for interpretation of labeling results in a complex eukaryotic systems (including mammalian systems such as bovine embryos). The software eiFlux for isotope-assisted metabolic flux analysis is ready for release and a manuscript describing eiFlux was accepted and published in PLOS Computational Biology . The postdoctoral fellow, Dr. Halli Weiner, led the embryo analysis (protein localization) portion and mentored the MS student, Laura Tompkins, in her analysis of lipid droplets. As of January 2022, Dr. Weiner left to become a clinical assistant professor leading a FIRE (First-Year Innovation & Research Experience) project at the University of Maryland.A manuscript from her studies should be forthcoming. MS student, Laura Tompkins, has completed her master's research and should defend her thesis in August 2022. A manuscript from her studies should be forthcoming. How have the results been disseminated to communities of interest?Research progress was discussed at the annual Multi-State Research Project (Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock W-4171) which was held inassociationwith the January 2022 IETS annual conference. Moreover, as listed elsewhere, a manuscript on the iFlux software was published. What do you plan to do during the next reporting period to accomplish the goals?We will continue our studies as outlined in the objectives above and described in NIFA grant: A systemic approach to deciphering energy metabolism in preimplantation embryos. More specifically, Stored samples will be processed and analyzed using the LC-MS/MS to further develop the flux map. A manuscript, now in draft form, that describes the flux map produced using labeled glucose and fructose will be submitted for publication. Additional manuscripts describing the effects of glucose and fructose supplementation on morphological characteristics (lipid droplets, mitochondrial potential, and localization of OCT4 and GAPDH) will be prepared.
Impacts
What was accomplished under these goals?
Aim 1: 13C Metabolic flux analysis (13C MFA) was used to investigate carbon source usage and the effects of medium composition on metabolism in preimplantation bovine embryos. Assessing metabolism in individual bovine embryos presents unique challenges owing to the small cell number (< 200 cells). Furthermore, the media are complex and contain multiple carbon sources, thus potentially obfuscating the labeling patterns of the 13C-labeled carbon source. Because of the small sample size, there is only a limited set of metabolites whose labeling patterns can be accurately measured. We have designed novel labeling experiments and developed a software, eiFlux, to surmount these challenges and obtain a metabolic flux map for this complex system. The software eiFlux performs cumulative isotopomer (cumomer) balancing along with state-of- the-art-optimization to calculate a robust solution to the metabolic flux analysis problem (Lugar & Sriram, 2022). A manuscript describing our experimental results on isotope labeling of bovine blastocysts is being prepared for submission to Molecular Genetics and Metabolism in summer/fall 2022. Sample collections (spent medium from bovine blastocyst cultures) were completed from experimentsdesigned to probe the relative utilization of glycolysis and the pentose phosphate pathway, as well as the contributions of different carbon sources to metabolites originating at the pyruvate node. These samples are ready for processing. The addition of a Waters LC-MS/MS instrument to campus facilities will allow us to expand the number of metabolites that we can detect. Training on the new system has been completed and analytical methods have been developed to allow us to continue our sample analysis, which was halted due to loss of theGC-MS previously used on this project. Aim 2: Altering substrate availability will alter embryo quality as measured by embryonic cell number (ICM/TE ratio), expression of key proteins involved in lineage differentiation (OCT4, NANOG, and CDX2) and metabolism (GAPDH, PKM2), and lipid density. Embryos cultured in media containing differing energy substrates (e.g., glucose, fructose) are being assessed for lipid accumulation (e.g., amount, droplet size) using BODIPY stains and Image J analysis. Initial data indicates that the energy substrates may shift droplet size and amount of lipid and affect rate of blastocyst expansion and hatching. Effects of inhibitors (inhibitors of LC-fatty acids and pyruvate transport into the mitochondria) on the ability of collapsed blastocysts to re-expand (determine metabolic energy pathways required for re-expansion)are being assessed. Embryos cultured in media containing differing energy substrates (e.g., glucose, fructose) were assessed for shifts in OCT4, GAPDH, and PKM2 protein localization (cytoplasmic vs. nuclear) using immunocytochemistry and Image J analysis. Results indicate that the energy substrates do not affect OCT4, however shifts in GAPDH and PKM localization (cytoplasmic vs. nuclear) may occur. Analysis is ongoing.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Lugar DJ, Sriram G. Isotope-assisted metabolic flux analysis as an equality-constrained nonlinear program for improved scalability and robustness. PLOS Computational Biology. 2022;18: e1009831. doi:10.1371/journal.pcbi.1009831
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Progress 06/01/20 to 05/31/21
Outputs
Target Audience:Training: 1 MS and 1 PhD student; 1 Postdoctoral fellow Dispersal of knowledge through abstracts, publications and talksto the scientific community (embryologists, nutritionists, reproductive physiologist, and biochemical engineers) with the ultimate aim of benefiting the dairy and beef cattle industries Changes/Problems:Restrictions on research due to COVID-1 significantly delayed progress. Need to replace/repair the GC/MS. A needed part has proven to be unattainable, but fortuitously a Waters LC-MS/MS instrument has been added to campus facilities. What opportunities for training and professional development has the project provided?A doctoral student, Dan Lugar, has developed a more complex model and software, eiFlux, for interpretation of labeling results in a complex eukaryotic systems (including mammalian systems such as bovine embryos). This expands on his previous training with plant cells. He has submitted a manuscript describing this model. The postdoctoral fellow, Dr. Halli Weiner, is leading the embryo analysis (protein localization) and mentoring the MS student, Laura Tompkins, in her analysis of lipid droplets. How have the results been disseminated to communities of interest?Research progress will be discussed at the annual Multi-State Research Project (Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock W-4171) which will likely have a meeting around the time of the January 2022 meeting of the IETS. An invited presentation that was delayed will be given at a satellite symposium during the annual conference of the Society for the Study of Reproduction that is to be held in December 2021. What do you plan to do during the next reporting period to accomplish the goals?We will continue our studies as outlined in the objectives above and described in NIFA grant: A systemic approach to deciphering energy metabolism in preimplantation embryos.
Impacts
What was accomplished under these goals?
Aim 1: 13C Metabolic flux analysis (13C MFA) was used to investigate carbon source usage and the effects of medium composition on metabolism in preimplantation bovine embryos. Assessing metabolism in individual bovine embryos presents unique challenges owing to the small cell number (< 200 cells). Furthermore, the media are complex and contain multiple carbon sources, thus potentially obfuscating the labeling patterns of the 13C-labeled carbon source. Because of the small sample size, there is only a limited set of metabolites whose labeling patterns can be accurately measured. We have designed novel labeling experiments and developed a software, eiFlux, to surmount these challenges and obtain a metabolic flux map for this complex system. The software eiFlux performs cumulative isotopomer (cumomer) balancing along with state-of-the-art-optimization to calculate a robust solution to the metabolic flux analysis problem. Previous experiments revealed that the embryos utilize substantially more glucose than fructose. The development of eiFlux for MFA resulted in a more detailed flux map. As COVID19 restrictions eased in 2021, sample collections were restarted. These experiments have been designed to probe the relative utilization of glycolysis and the pentose phosphate pathway, as well as the contributions of different carbon sources to metabolites originating at the pyruvate node. The recent addition of a Waters LC-MS/MS instrument to campus facilities will allow us to expand the number of metabolites that we can detect. This will substantially improve the MFA. Training on the new system has been completed and sample analysis will be started. Aim 2: Altering substrate availability will alter embryo quality as measured by embryonic cell number (ICM/TE ratio), expression of key proteins involved in lineage differentiation (OCT4, NANOG, and CDX2) and metabolism (GAPDH,PKM2), and lipid density. Embryos cultured in media containing differing energy substrates (e.g., glucose, fructose) are being assessed for lipid accumulation (e.g., amount, droplet size) using BODIPY stains and Image J analysis. Initial data indicates that the energy substrates affect droplet size and amount of lipid. Embryos cultured in media containing differing energy substrates (e.g., glucose, fructose) are being assessed for shifts in OCT4, GAPDH and PKM2 protein localization (cytoplasmic vs. nuclear) using immunocytochemistry and Image J analysis. Initial data indicates that the energy substrates do not affect OCT4, however shifts in GAPDH localization (cytoplasmic vs. nuclear) may occur. Analysis is ongoing.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Lugar DJ, Sriram G. Isotope-Assisted Metabolic Flux Analysis as an Equality-Constrained Nonlinear Program for Improved Scalability and Robustness. PLoS Computational Biology
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Progress 06/01/19 to 05/31/20
Outputs
Target Audience:Training: 1 MS and 1 PhD student; 1 Postdoctoral fellow Dispersal of knowledge to the scientific community with the ultimate aim of benefiting the dairy and beef cattle industries Changes/Problems:Restrictions on research due to COVID-1 Need to replace/repair GS/MS. Dr. Sriram is arranging funding to support the purchase of a new GS/MS. What opportunities for training and professional development has the project provided?A doctoral student is involved and has developed a more complex model for interpretation of labeling results in a mammalian system, which expands on his previous training with plant cells. A postdoctoral fellow has now joined the lab. While she has previous experience in fluxomic analysis, her previous experience was with sperm and she is now expanding her knowledge to embryo development. A graduate student (MS program) is also receiving training. How have the results been disseminated to communities of interest?Research progress will be discussed at annual Multi-State Research Project (Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock W-4171) which will likely have a virtual meeting around the time of the January 2021 meeting of the IETS. An invited presentation was to be given at a satellite symposium during the annual conference of the Society for the Study of Reproduction that was to be held in July 2020, but has been postponed to 2021. What do you plan to do during the next reporting period to accomplish the goals?We will continue our studies as outlined in the objectives above and described in NIFA grant: A systemic approach to deciphering energy metabolism in preimplantation embryos.
Impacts
What was accomplished under these goals?
Aim 1: 13C Metabolic flux analysis (13C MFA) was used to investigate carbon source usage and the effects of medium composition on metabolism in preimplantation bovine embryos. Assessing metabolism in individual bovine embryos presents unique challenges owing to the small cell number (< 200 cells). Furthermore, the media are complex and contain multiple carbon sources, thus potentially obfuscating the labeling patterns of the 13C-labeled carbon source. Because of the small sample size, there is only a limited set of metabolites whose labeling patterns can be accurately measured. We have designed novel labeling experiments to surmount these challenges and obtain a metabolic flux map for this complex system. MFA results comparing flux identifiability resulting from multiple versions of 13C-glucose and 13C-fructose in parallel labeling experiments revealed that the embryos utilize substantially more glucose than fructose. Further experiments have been designed to probe the relative utilization of glycolysis and the pentose phosphate pathway, as well as the contributions of different carbon sources to metabolites originating at the pyruvate node. The methods developed by us demonstrate the extension of the MFA framework to complex mammalian systems. Aim 2: Altering substrate availability will alter embryo quality as measured by embryonic cell number (ICM/TE ratio), expression of key proteins involved in lineage differentiation (OCT4, NANOG, and CDX2) and metabolism (GAPDH,PKM2), and lipid density. Embryos cultured in media containing differing energy substrates (e.g., glucose, fructose) are being assessed for lipid accumulation (e.g., amount, droplet size) using BODIPY stains and Image J analysis. Embryos cultured in media containing differing energy substrates (e.g., glucose, fructose) are being assessed for shifts in GAPDH and PKM2 protein localization (cytoplasmic vs. nuclear) using immunocytochemistry and Image J analysis.
Publications
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