GLUTAMINOLYSIS PROVIDES TCA CYCLE INTERMEDIATES NECESSARY FOR PROLIFERATION IN THE CONCEPTUS TROPHECTODERM OF PIGS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022387
• Grant No.: 2020-67015-31136
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2019-05330
• Project Start Date: Jun 1, 2020
• Project End Date: May 31, 2024
• Grant Year: 2020
• Program Code: [A1211]- Animal Health and Production and Animal Products: Animal Reproduction

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Vet Integrative Biosciences

Non Technical Summary
Twenty to forty percent of embryonic loss in pigs occurs during the first 20 days of pregnancy. At this time the developing embryos are fed by secretions from the uterus, and increased understanding of how these secretions are generated by the uterus and used by the embryos can advance strategies to decrease embryonic loss. Many cells generate energy by converting glutamine into glutamate and then α-ketoglutarate that enters the TCA cycle, a process called glutaminolysis. Our preliminary work suggests that the developing placentae of embryos use glutamine to support their development. The long-term goals of this research is to understand the role of glutaminolysis in the early development of the placentae of porcine embryos. The specific objectives are to: 1) determine if embryos use glutaminolysis to support synthesis of nucleotides and lips; and 2) determine if inactivation of glutaminolysis affects the placental development of early embryos. New knowledge from the proposed research will inform as to the roles of glutamine in rapid growth and development of conceptus trophectoderm of pigs that sets the stage for rapid growth and development of the embryo/fetus. The knowledge gained from the proposed research will provide the framework for mechanism-driven translational research into strategies to enhance reproductive efficiencies in animal agriculture.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3510	1020	100%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1020 - Physiology;

Keywords

conceptus

glutamine

pregnancy

tca cycle anaplerosis

glutaminolysis

reductive carboxylation

Goals / Objectives
Enhanced understanding of the components of histotroph, critical for growth, development and survival of conceptuses, can be used to reduce the 20 to 40 percent embryonic death losses in pigs. Glutaminolysis is the process by which cells convert glutamine into glutamate and then α-ketoglutarate that is a TCA cycle intermediate. Our preliminary results suggest that the trophectoderm (Tr) cells of porcine conceptuses utilize glutamine within the uterine lumen as an alternate carbon source to maintain TCA cycle flux and provide biosynthetic precursors for the synthesis of nucleotides and lipids that is essential to support elongation, migration, and hormone synthesis by porcine conceptuses. The long-term goal of this research is to understand the role of glutaminolysis in the proliferating Tr of pig conceptuses during the peri-implantation period. The Specific Objectives of this proposal are to: 1) determine if glutaminolysis drives TCA cycle anaplerosis through oxidative and reductive pathways to support nucleotide and lipid biosynthesis in pig conceptuses; and 2) determine if inactivation of glutaminolysis and reductive carboxylation affects proliferation and elongation of pig conceptuses during the peri-implantation period. New knowledge from the proposed research will inform as to the roles of glutamine in the rapid growth and development of the conceptus Tr of pigs that sets the stage for rapid growth and development of the embryo/fetus.

Project Methods
For Objective 1, we will determine if glutamine is utilized for nucleotide and lipid synthesis in pig conceptuses in vitro and in vivo by using isotope-labeled glutamine to trace the contribution of glutamine to TCA cycle metabolites, aspartate, nucleotide synthesis, acetyl-CoA and palmitate through oxidative metabolism and reductive carboxylation.Mature pigs who have experienced at least one estrous cycle of normal duration will be mated when detected in estrus (Day 0) and 12 and 24 h later to boars of proven fertility. GLS expression highly increases as conceptuses elongate between Day 13 and Day 15 of pregnancy in Tr cells. Therefore, pigs will be hysterectomized on Day 13 and Day 15 of pregnancy. The lumen of each uterine horn will be flushed with 20 ml physiological saline to recover morphologically normal conceptuses, and the inner cell mass will be removed from the conceptuses to obtain Tr cells. Tr cells will then be cultured for 24 h in medium containing [U-13C5]glutamine (Cambridge isotope). Cells will be washed with PBS and snap-frozen in liquid nitrogen. The 13C-labeled isotopomers of TCA cycle metabolites including citrate, oxaloacetate, and aspartate, and nucleotides will be determined by mass spectrometry and metabolic flux analyses. The Tr cells recovered will also be cultured for 24 h in medium containing [5-13C]glutamine (Cambridge isotope). Cells will be washed with PBS and snap-frozen in liquid nitrogen. The 13C-labelling of acetyl-CoA and palmitate will be determined by mass spectrometry and metabolic flux analyses.Gilts will be mated as described above. On Day 11 of pregnancy, when conceptuses begin to elongate, gilts will be subjected to mid-ventral laparotomy, and both uterine horns ligated proximal to the uterine body. A vinyl catheter will be then inserted about 1 cm distal to the utero-tubal junction into the lumen of the uterine horn. The catheter will be connected to a preloaded and equilibrated Alzet 2ML1 osmotic pump. The Alzet pump will then be affixed to the mesosalpinx supporting the oviduct using cyanoacrylate glue and secured by suturing the mesosalpinx to the perimetrium of the uterine horn using 0 Coated Vicryl suture. The Alzet 2ML1 Osmotic Pump will be calibrated to constantly deliver 10 μl/h (240 μl/day) for 4 days. For each gilt, each ligated uterine horn will be assigned randomly to receive either (1) [U-13C5]glutamine or (2) [5-13C1]glutamine. Gilts will be hysterectomized on Day 15 of gestation when conceptuses are in the early apposition phase and can be flushed from the uterus. The lumen of each uterine horn will be flushed with 20 ml physiological saline to recover conceptuses. Conceptus and endometrial tissues will be immediately snap-frozen for assessment of metabolites. The 13C-labeled isotopomers of TCA cycle metabolites, aspartate, nucleotides, acetyl-CoA, and palmitate will be determined by mass spectrometry and metabolic flux analyses.In Objective 2, we will determine effects of glutaminolysis and reductive carboxylation on proliferation and elongation of pig conceptuses using siRNAs in vitro and CRISPR/CAS9 knockout technology in vivo.Gilts will be mated and will be hysterectomized on Day 15 of pregnancy. The lumen of each uterine horn will be flushed with 20 ml physiological saline to recover morphologically normal conceptuses, and the inner cell mass will be removed from the conceptuses to obtain Tr cells. Tr cells will then be transfected with either control siRNA, GLS siRNA, or IDH1 siRNA. After 12 h, the cells will be cultured in 96-well plates in RPMI-1640. After 12 h, cell proliferation will be assessed using a BrdU cell proliferation ELISA kit according to the manufacturer's protocol. Briefly, the cells will be labeled using 1X BrdU for 12 h. The plates will be spun down in the centrifuge for 5 min at 1,000 rpm, the medium will be aspirated, and cells will be fixed in 3.7% formaldehyde in PBS at room temperature for 30 min. Anti-BrdU monoclonal detector antibody will be added and incubated for 1 h at room temperature. Then, the cells will be washed 3 times using PBS, and peroxidase-conjugated goat anti-mouse IgG antibody will be added and incubated for 30 min at room temperature in the dark. The absorbance of samples of cells will be measured at 450 nm. The average of triplicate samples will be determined for each treatment. Some portions of the cells transfected with siRNA will be cultured for 24 h in medium containing [U-13C5]glutamine or [5-13C1]glutamine. The cells will be washed with PBS and snap frozen in liquid nitrogen. Portions of the cultured cells will be subjected to Western blot analyses for GLS and IDH1 to confirm knockdown of those enzymes. The 13C-labeled isotopomers of TCA cycle metabolites including citrate, oxaloacetate, and aspartate, nucleotides, and acetyl-CoA will be determined by mass spectrometry and metabolic flux analyses.The approach to generate GLS-knockout conceptuses is: (a) design and in vitro validation of gRNAs to build specific CRISPRs for Cas9 nuclease targeted genome editing; (b) fetal fibroblast cell engineering and genotyping; (c) SCNT embryo production; (d) in vitro culture; and (e) embryo transfer and subsequent collection for analysis. Recipient gilts (GLS+/+, GLS-/-) will be hysterectomized on Day 16 of gestation when conceptuses are in the early apposition phase and can be flushed from the uterus. The rationale for obtaining samples on Day 16 is that GLS is highly expressed by Day 15 of pregnancy and SCNT has a slight developmental delay of 1-2 days for conceptuses. The lumen of each uterine horn will be flushed with 20 ml physiological saline to recover conceptuses. The collected conceptuses will be examined under a stereomicroscope, and the morphology (i.e., spherical, ovoid, tubular, or elongated) will be assessed. Conceptuses will be immediately snap-frozen for assessment of protein expression and metabolites. Portions of the conceptuses will be fixed in 4% paraformaldehyde and paraffin-embedded for immunohistochemical analyses. PCNA staining indicates proliferation of the Tr cells during the peri-implantation period of pigs. Western blot analysis and immunofluorescence microscopy for PCNA will be performed to assess effects of glutaminolysis on Tr cell proliferation. Quantification of TCA cycle metabolites in the isolated conceptuses will be measured by HPLC-MS/MS to determine effects on TCA cycle anaplerosis.

Progress 06/01/20 to 12/14/23

Outputs
Target Audience:Our primary target audience has been members of the scientific community who are involved in the study of the molecular aspects of animal reproduction. 2023 July. Invited speaker (Dr. Greg Johnson). Metabolism of Glucose, Fructose, and Glutamine by Elongating & Implanting Porcine Conceptuses. 56th Annual SSR Conference, Peri-Implantation Biology, Ottawa, Canada 2023 June. Invited speaker (Dr. Heewon Seo). Pig trophectoderm utilizes glucose-derived serine for one-carbon metabolism to support conceptus elongation. 11th International Conference on Pig Reproduction (ICPR), Belgium 2023 June. Invited speaker (Dr. Greg Johnson). Metabolic Pathways in the Conceptus, Uterus and Placenta. 11th International Conference on Pig Reproduction (ICPR), Belgium 2022 August. Invited Speaker (Dr. Heewon Seo). New hypotheses for placentation in pigs, sheep and cattle. The Asian- Australasian Association of Animal Production Societies (AAAP), South Korea 2022 August. Plenary Speaker (Dr. Greg Johnson). Metabolic pathways utilized by the porcine conceptus, uterus and placenta. The Asian-Australasian Association of Animal Production Societies (AAAP), South Korea 2022 October. Invited Speaker (Dr. Greg Johnson). Deciphering Pregnancy in Livestock by Focusing on Interactions between the Placenta and Uterus. Uterine Workshop. NIEHS 2022 January, Invited Speaker (Dr. Greg Johnson), Metabolic Pathways Utilized by the Porcine Conceptus, Uterus and Placenta. Veterinary Physiology and Pharmacology (VTPP) seminar 2021 December, Poster presentation (Avery Kramer), SSR 2021 October, Invited Speaker (Dr. Heewon Seo), Comparative Analyses of Placentation in Domestic Species at the International Symposium on Developmental Biotechnology, South Korea. 2020, Invited Speaker, Steroids regulate SLC2A1 and SLC2A3 to deliver glucose and fructose into trophectoderm for metabolism via glycolysis, Trienniel Reproduction Symposium, American Society of Animal Science Annual Meeting. 2021, Invited Speaker, Comparative placentation analysis in domestic livestock, Webinar in Emerging Topics in Reproduction,Texas A&M-FAPESP research program. 2021, Invited Speaker, Porcine Conceptuses Utilize Glucose and Fructose to Support Development during the Peri- Implantation Period of Pregnancy, Interdisciplinary Faculty of Reproductive Biology (IFRB) seminar. 2021, Invited Speaker, Pig conceptuses secrete interferon gamma to recruit T cells which enhance glycolytic metabolism within the hypoxic peri-implantation uterus. Interdisciplinary Faculty of Reproductive Biology (IFRB) seminar. 2021, Invited Speaker, New Insights into the Histologic and Metabolic Basis for Early Placental Development in Pigs, Sheep, Cattle, and Baboons. Interdisciplinary Faculty of Reproductive Biology (IFRB) seminar. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three PhD students gained research experience with animal husbandry, animal treatments and laboratory bench work as well as presented in the local, national, and international scientific meetings. How have the results been disseminated to communities of interest?Results have been disseminated to the scientific community in the form of an invited talk to the International Symposium on Developmental Biotechnology and the Veterinary Physiology and Pharmacology (VTPP) seminar, the American Society of Animal Science Annual Meeting, the Interdisciplinary Faculty of Reproductive Biology (IFRB) seminar, the Society for the Study of Reproduction (SSR), International Conference on Pig Reproduction (ICPR), Asian-Australasian Association of Animal Production Societies (AAAP), and NIEHS as well as poster presentation to the Society for the Study of Reproduction. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the peri-implantation period, pig conceptuses (embryo and placental membranes) rapidly elongate from spherical to tubular to filamentous forms. This elongation requires that trophectoderm (Tr) cells expend significant amounts of energy to undergo timely and extensive proliferation. Therefore, optimal utilization of multiple biosynthetic pathways is likely an essential aspect of early conceptus development and survival. Our preliminary studies indicated that the endometrium of pigs transports glucose into the uterine lumen. Elongating and proliferating conceptuses then potentially act, in a manner similar to cancer cells, to direct the carbon, generated from glucose, away from the tricarboxylic acid (TCA) cycle for utilization in branching pathways of glycolysis, including the pentose phosphate pathway, one-carbon metabolism, and hexosamine biosynthesis. The result is limited availability of pyruvate for maintaining the TCA cycle within mitochondria. Proliferating cells can replenish TCA cycle metabolites via a process known as anaplerosis to convert glutamine into TCA cycle metabolites other than acetyl-CoA. Glutamine can be converted into glutamate and then alpha- ketoglutarate (α-KG), a TCA cycle intermediate, through the enzymes glutaminase (GLS), glutamate dehydrogenase (GLUD), and aminotransferases. Our preliminary results indicated that the conceptus Tr of pigs utilizes the GLS-phosphoserine transaminase 1 (PSAT1) glutaminolysis pathway to metabolically support proliferation for elongation. We collected elongating conceptus tissues from Days 15 and 16 of pregnancy, and incubated these tissues with [1-14C]glucose or [6-14C]glucose, and measured 14CO2 released from the conceptuses to determine whether carbons from glucose enter the PPP. The result showed that carbons derived from glucose actually entered the pentose phosphate pathway with maximum levels of utilization on Day 15 of pregnancy. Next, we examined whether glutaminolysis-derived α-KG is utilized as TCA cycle anaplerotic substrate when deprived of glucose. We isolated pig conceptuses on Day 15 of pregnancy, treated them with 14C-labeled glutamine in the presence or absence of glucose in the culture media, and then measured 14C-labelled CO2 production. Glutamine oxidation by conceptuses significantly increased in the absence of glucose in the media. Further we exposed conceptus tissues to 13C-glutamine to assess movement of glutamine-derived carbons through glutaminolysis in TCA cycle intermediates. The 13C-labeled carbons were accumulated in glutamate, α-ketoglutarate, succinate, malate, citrate, and aspartate in both the presence and absence of glucose in the media, and the accumulation of 13C-labeled carbons significantly increased in the absence of glucose in the media. Collectively, our results indicate that during the peri-implantation period of pregnancy, the proliferating and migrating trophectoderm cells of elongating porcine conceptuses utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux. Overall, we demonstrated that aerobic glycolysis is activated in the elongating porcine conceptuses, and most of the carbon atoms from glucose that has been taken up become diverted into lactate production and branching pathways of glycolysis instead of being routed into the mitochondrial TCA cycle. In order to maintain TCA cycle flux during aerobic glycolysis, porcine conceptuses utilize glutamine through glutaminolysis as an alternate carbon source. In normal cells, after glutamine is converted into α-ketoglutarate (αKG), it is subsequently metabolized to succinate by α-ketoglutarate dehydrogenase through oxidative glutaminolysis. This decarboxylation reaction is catalyzed irreversibly by isocitrate dehydrogenase 3 (IDH3) in the TCA cycle in mitochondria. However, cells undergoing aerobic glycolysis are also able to convert αKG into isocitrate, which is in the reverse direction of the conventional TCA cycle. And this reductive carboxylation of α-KG to isocitrate is catalyzed by either cytosolic IDH1 or mitochondrial IDH2. Nest, we determined if glutaminolysis-derived aKG is metabolized through oxidative or reductive pathways to sustain TCA cycle flux in pig conceptuses within a low oxygen environment. What we found is that 1) IDH3, alpha-ketoglutarate dehydrogenase (OGDH) and succinate dehydrogenase (SDHA), mitochondrial TCA cycle enzymes that convert isocitrate to α-KG and α-KG to succinate and succinate to fumarate, are highly expressed in the trophectoderm during the peri-implantation period. Interestingly, we also found that trophectoderm cells do not express mitochondrial IDH2 but do express cytosolic IDH1, suggesting that glutaminolysis-derived aKG can be exported into the cytosol to be converted into isocitrate through the reverse TCA cycle. Collectively, these results indicate that glutaminolysis-derived aKG is subsequently metabolized through the IDH3-mediated oxidative TCA cycle in mitochondria as well as through the IDH1-mediated reductive TCA cycle in the cytosol.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Johnson GA, Seo H, Bazer FW, Wu G, Kramer AC, McLendon BA, Cain JW. Metabolic pathways utilized by the porcine conceptus, uterus, and placenta. Mol Reprod Dev. 2023 Jul;90(7):673-683. doi: 10.1002/mrd.23570. Epub 2022 Apr 22. PMID: 35460118.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Seo H, Kramer AC, McLendon BA, Cain JW, Burghardt RC, Wu G, Bazer FW, Johnson GA. Elongating porcine conceptuses can utilize glutaminolysis as an anaplerotic pathway to maintain the TCA cycle . Biol Reprod. 2022 Sep 12;107(3):823-833. doi: 10.1093/biolre/ioac097. PMID: 35552608.

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:Our primary target audience has been members of the scientific community who are involved in the study of the molecular aspects of animal reproduction. 2023 July. Invited speaker (Dr. Greg Johnson). Metabolism of Glucose, Fructose, and Glutamine by Elongating & Implanting Porcine Conceptuses. 56th Annual SSR Conference, Peri-Implantation Biology, Ottawa, Canada 2023 June. Invited speaker (Dr. Heewon Seo). Pig trophectoderm utilizes glucose-derived serine for one-carbon metabolism to support conceptus elongation. 11th International Conference on Pig Reproduction (ICPR), Belgium 2023 June. Invited speaker (Dr. Greg Johnson). Metabolic Pathways in the Conceptus, Uterus and Placenta. 11th International Conference on Pig Reproduction (ICPR), Belgium 2022 August. Invited Speaker (Dr. Heewon Seo). New hypotheses for placentation in pigs, sheep and cattle. The Asian-Australasian Association of Animal Production Societies (AAAP), South Korea 2022 August. Plenary Speaker (Dr. Greg Johnson). Metabolic pathways utilized by the porcine conceptus, uterus and placenta. The Asian-Australasian Association of Animal Production Societies (AAAP), South Korea 2022 October. Invited Speaker (Dr. Greg Johnson). Deciphering Pregnancy in Livestock by Focusing on Interactions between the Placenta and Uterus. Uterine Workshop. NIEHS Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A PhD student continues to gain research experience with animal husbandry, animal treatments and laboratory bench work. How have the results been disseminated to communities of interest?Results have been disseminated to the scientific community in the form of an invited talk to the Society for the Study of Reproduction (SSR), International Conference on Pig Reproduction (ICPR), Asian-Australasian Association of Animal Production Societies (AAAP), and NIEHS. What do you plan to do during the next reporting period to accomplish the goals?We will continue to analyze the tissues we have already generated. Further studies will be done to see if the porcine conceptuses actually utilize reductive glutaminolysis by culturing conceptus tissues with 13C-glutamine.

Impacts
What was accomplished under these goals? In our previous studies, we demonstrated that aerobic glycolysis is activated in the elongating porcine conceptuses, and most of the carbon atoms from glucose that has been taken up become diverted into lactate production and branching pathways of glycolysis instead of being routed into the mitochondrial TCA cycle. In order to maintain TCA cycle flux during aerobic glycolysis, porcine conceptuses utilize glutamine through glutaminolysis as an alternate carbon source. In normal cells, after glutamine is converted into α-ketoglutarate (αKG), it is subsequently metabolized to succinate by α-ketoglutarate dehydrogenase through oxidative glutaminolysis. This decarboxylation reaction is catalyzed irreversibly by isocitrate dehydrogenase 3 (IDH3) in the TCA cycle in mitochondria. However, cells undergoing aerobic glycolysis are also able to convert αKG into isocitrate, which is in the reverse direction of the conventional TCA cycle. And this reductive carboxylation of α-KG to isocitrate is catalyzed by either cytosolic IDH1 or mitochondrial IDH2. Therefore, our aim was to determine if glutaminolysis-derived aKG is metabolized through oxidative or reductive pathways to sustain TCA cycle flux in pig conceptuses within a low oxygen environment. What we found is that 1) IDH3, alpha-ketoglutarate dehydrogenase (OGDH) and succinate dehydrogenase (SDHA), mitochondrial TCA cycle enzymes that convert isocitrate to α-KG and α-KG to succinate and succinate to fumarate, are highly expressed in the trophectoderm during the peri-implantation period. Interestingly, we also found that trophectoderm cells do not express mitochondrial IDH2 but do express cytosolic IDH1, suggesting that glutaminolysis-derived aKG can be exported into the cytosol to be converted into isocitrate through the reverse TCA cycle. Collectively, these results indicate that glutaminolysis-derived aKG is subsequently metabolized through the IDH3-mediated oxidative TCA cycle in mitochondria as well as through the IDH1-mediated reductive TCA cycle in the cytosol.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Seo H, McLendon BA, Kramer AC, Cain JW, Burghardt RC, Wu G, Bazer FW, Johnson GA. Elongating Porcine Conceptuses Can Utilize Glutaminolysis as an Anaplerotic Pathway to Maintain the TCA Cycle. Biol Reprod 2022
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Johnson GA, Seo H, Bazer FW, Wu G, Kramer AC, McLendon BA, Cain JW. Metabolic pathways utilized by the porcine conceptus, uterus and placenta. Molecular Reproduction and Development 2022

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:Our primary target audience has been members of the scientific community who are involved in the study of the molecular aspects of animal reproduction. 2022 January, Invited Speaker (Dr. Greg Johnson), Metabolic Pathways Utilized by the Porcine Conceptus, Uterus and Placenta. Veterinary Physiology and Pharmacology (VTPP) seminar 2021 December, Poster presentation (Avery Kramer), SSR 2021 October, Invited Speaker (Dr. Heewon Seo), Comparative Analyses of Placentation in Domestic Species at the International Symposium on Developmental Biotechnology, South Korea. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A master student continue to gain research experience with animal husbandry, animal treatments and laboratory bench work. How have the results been disseminated to communities of interest?Results have been disseminated to the scientific community in the form of an invited talk to the International Symposium on Developmental Biotechnology and the Veterinary Physiology and Pharmacology (VTPP) seminar as well as poster presentation to the Society for the Study of Reproduction. What do you plan to do during the next reporting period to accomplish the goals?We will continue to analyze the tissues we have already generated, and continue with inhibition of glutaminolysis using a glutaminase inhibitor.

Impacts
What was accomplished under these goals? The aims of this project is to determine: 1) if enzymes for glutaminolysis are expressed in the conceptus trophectoderm; and 2) if glutamine-derived carbons are actually incorporated into the TCA cycle intermediates. We demonstrated that: 1) expression of GLS increased in trophectoderm and GLUL increased in endoderm of conceptuses, suggesting that endoderm synthesizes glutamine, and trophectoderm converts glutamine into glutamate; and 2) expression of GLUD decreased while expression of aminotransferases (glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), phophoserine aminotransferase (PSAT1) increased in trophectoderm, suggesting generation of α-KG, serine, alanine, and aspartate which contribute to the biosynthesis of nucleotides. Our results indicated that the conceptus Tr of pigs utilizes the GLS-phosphoserine transaminase 1 (PSAT1) glutaminolysis pathway to metabolically support proliferation for elongation. Next, we isolated pig conceptuses on Day 15 of pregnancy, treated them with 14C-labeled glutamine in the presence or absence of glucose in the culture media, and then measured 14C-labeled CO2 production. Glutamine oxidation by conceptuses significantly increased in the absence of glucose in the media. Further we exposed conceptus tissues to 13C-glutamine to assess movement of glutamine-derived carbons through glutaminolysis in TCA cycle intermediates. The 13C-labeled carbons were accumulated in glutamate, α-ketoglutarate, succinate, malate, citrate, and aspartate in both the presence and absence of glucose in the media, and the accumulation of 13C-labeled carbons significantly increased in the absence of glucose in the media. Collectively, our results indicate that during the peri-implantation period of pregnancy, the proliferating and migrating trophectoderm cells of elongating porcine conceptuses utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Johnson GA, Seo H, Bazer FW, Wu G, Kramer AC, McLendon BA, Cain JW. Metabolic pathways utilized by the porcine conceptus, uterus and placenta. Molecular Reproduction and Development 2022 April (in press)
• Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Seo H, McLendon BA, Kramer AC, Cain JW, Burghardt RC, Wu G, Bazer FW, Johnson GA. Elongating Porcine Conceptuses Can Utilize Glutaminolysis as an Anaplerotic Pathway to Maintain the TCA Cycle. Biol Reprod 2022 (in press)

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Our primary target audience has been members of the scientific community who are involved in the study of the molecular aspects of animal reproduction. 2020, Invited Speaker, Steroids regulate SLC2A1 and SLC2A3 to deliver glucose and fructose into trophectoderm for metabolism via glycolysis, Trienniel Reproduction Symposium, American Society of Animal Science Annual Meeting. 2021, Invited Speaker, Comparative placentation analysis in domestic livestock, Webinar in Emerging Topics in Reproduction,Texas A&M-FAPESP research program. 2021, Invited Speaker, Porcine Conceptuses Utilize Glucose and Fructose to Support Development during the Peri-Implantation Period of Pregnancy, Interdisciplinary Faculty of Reproductive Biology (IFRB) seminar. 2021, Invited Speaker, Pig conceptuses secrete interferon gamma to recruit T cells which enhance glycolytic metabolism within the hypoxic peri-implantation uterus. Interdisciplinary Faculty of Reproductive Biology (IFRB) seminar. 2021, Invited Speaker, New Insights into the Histologic and Metabolic Basis for Early Placental Development in Pigs, Sheep, Cattle, and Baboons. Interdisciplinary Faculty of Reproductive Biology (IFRB) seminar. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two Ph.D. students continue to gain research experience both animal husbandry, animal treatments and laboratory bench work. How have the results been disseminated to communities of interest?Results have been disseminated to the scientific community in the form of an invited talk to the American Society of Animal Science Annual Meeting, the Interdisciplinary Faculty of Reproductive Biology (IFRB) seminar, and the Webinar in Emerging Topics in Reproduction on Texas A&M-FAPESP research program. What do you plan to do during the next reporting period to accomplish the goals?We will continue to analyze the tissues we have already generated, and continue with intrauterine infusion of 13C-labled glutamine.

Impacts
What was accomplished under these goals? During the peri-implantation period, pig conceptuses (embryo and placental membranes) rapidly elongate from spherical to tubular to filamentous forms. This elongation requires that trophectoderm (Tr) cells expend significant amounts of energy to undergo timely and extensive proliferation. Therefore, optimal utilization of multiple biosynthetic pathways is likely an essential aspect of early conceptus development and survival. Our preliminary studies indicated that the endometrium of pigs transports glucose into the uterine lumen. Elongating and proliferating conceptuses then potentially act, in a manner similar to cancer cells, to direct the carbon, generated from glucose, away from the tricarboxylic acid (TCA) cycle for utilization in branching pathways of glycolysis, including the pentose phosphate pathway, one-carbon metabolism, and hexosamine biosynthesis. The result is limited availability of pyruvate for maintaining the TCA cycle within mitochondria. Proliferating cells can replenish TCA cycle metabolites via a process known as anaplerosis to convert glutamine into TCA cycle metabolites other than acetyl-CoA. Glutamine can be converted into glutamate and then alpha- ketoglutarate (α-KG), a TCA cycle intermediate, through the enzymes glutaminase (GLS), glutamate dehydrogenase (GLUD), and aminotransferases. Our preliminary results indicated that the conceptus Tr of pigs utilizes the GLS-phosphoserine transaminase 1 (PSAT1) glutaminolysis pathway to metabolically support proliferation for elongation. We collected elongating conceptus tissues from Days 15 and 16 of pregnancy, and incubated these tissues with [1-14C]glucose or [6-14C]glucose, and measured 14CO2 released from the conceptuses to determine whether carbons from glucose enter the PPP. The result showed that carbons derived from glucose actually entered the pentose phosphate pathway with maximum levels of utilization on Day 15 of pregnancy. Next, we examined whether glutaminolysis-derived α-KG is utilized as TCA cycle anaplerotic substrate when deprived of glucose. We isolated pig conceptuses on Day 15 of pregnancy, treated them with 14C-labeled glutamine in the presence or absence of glucose in the culture media, and then measured 14C-labelled CO2 production. Glutamine oxidation and utilization by conceptuses significantly increased in the absence of glucose in the media, supporting our idea that conceptus utilizes glutamine as a TCA cycle anaplerotic substrate when deprived of glucose. Collectively, our results indicate that in response to a hypoxic environment, the Tr cells of pig conceptuses utilize glucose via the glycolytic biosynthetic pathways for the de novo synthesis of nucleotides, and utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux.

Publications