Source: NORTH CAROLINA STATE UNIV submitted to
TNF-ALPHA PRODUCTION BY CORPUS LUTEUM (CL) MACROPHAGES (MAC) AND ITS CONTROL BY PROGESTERONE IN THE PORCINE CL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022389
Grant No.
2020-67015-31014
Cumulative Award Amt.
$492,000.00
Proposal No.
2019-05296
Multistate No.
(N/A)
Project Start Date
Jun 1, 2020
Project End Date
May 31, 2025
Grant Year
2020
Program Code
[A1211]- Animal Health and Production and Animal Products: Animal Reproduction
Project Director
Poole, D. H.
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Molecular Biomedical Sciences
Non Technical Summary
Currently few effective or cost effective methods exist for the regulation/ synchronization of estrous cycles in female pigs, and thus the rationale for this study is to identify novel drugs and/or approaches to be used for this purpose. The goal for the current project is to improve our understanding of how immune cells control of the corpus luteum (CL; ovarian structure that produces the hormone progesterone, which control estrous cycles), and thus reveal/identify novel targets for estrous cycle regulating drugs. There is an abundant evidence in the literature indicating that immune cells such as macrophages play key roles in the control of the corpus luteum (CL), and our preliminary data suggests that the hormone progesterone plays a critical role in regulating them. Thus the overarching hypothesis for this proposal is that progesterone acts via its receptor (PGR) to inhibit CL macrophages and that this process plays a key role in the control of the corpus luteum during the estrous cycle in the pig. Our objectives for this study are to 1) measure the levels of progesterone receptors in macrophages isolated from the corpus luteum (CL) on different days of the estrous cycle, 2) determine the effect of blocking progesterone synthesis and progesterone receptors (using chemical inhibitors) on CL macrophages maintained in culture, and 3) determine the effect of blocking progesterone synthesis and progesterone receptors using chemical inhibitors given to cycling pigs, on macrophages collected from CL taken from treated animals. We expect that the data generated from this study will confirm our hypothesis and provide irrefutable evidence that progesterone plays a vital role in regulating CL macrophages, and that these immune cells in turn are of critical importance to the control of estrous cyclicity in the female pig.These studies will provide critical new data that will be used to reveal/identify novel approaches and/or drug therapies for regulating and synchronizing estrous cycles in female pigs. The availability and use of novel drugs/approaches to estrous cycle synchronization in female pigs would improve the efficiency of breeding protocols within, and would have significant economic benefits for, the swine industry in the US and other countries worldwide.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30135101020100%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1020 - Physiology;
Goals / Objectives
The porcine corpus luteum (CL) lacks Luteolytic Sensitivity (LS) to prostaglandin F-2alpha (PGF) for ~13 days of the estrous cycle and thus PGF cannot be used to synchronize estrous cycles in pigs; this presents a significant problem for swine producers since few other methods exist. CL macrophages (MAC) play a critical role in regulating LS via Tumor Necrosis Factor-alpha (TNF) production. In preliminary studies it was shown that TNF mRNA expression by MAC was inhibited by progesterone acting via the genomic progesterone receptor (PGR). Additionally, it was shown that PGR mRNA was higher in MAC before D.13 (=before LS) vs. after D.13 (=after LS), while TNF mRNA was higher in MAC after vs. before D.13/LS. The overarching hypothesis for this proposal is that progesterone acting via PGR, inhibits MAC TNF production and plays a key role in regulating LS during the estrous cycle in pigs. The Specific Objectives are:(1)To measure PGR in porcine MAC BEFORE, versus AFTER, D.13 of the estrous cycle.(2)To examine the effects of (A) inhibition of progesterone synthesis, and (B) blockade of PGR,on TNF production by porcine MAC, in vitro(3)To examine the effects of (A) inhibition of progesterone synthesis, and (B) blockade of PGR,on TNF production by porcine MAC, in vivo.It is expected that new data about the regulation of MAC-TNF by progesterone will aid the development of novel drugs to control estrous cycles and improve reproductive efficiency in pigs, for the economic benefit of swine producers.
Project Methods
Specific Aim 1: CL will be collected at the MID (BEFORE D.13) and LATE (AFTER D. 13) stages of the estrous cycle and transported to the laboratory on ice. CL will be dissected out, diced and subjected to collagenase dissociation. The total luteal cell preparation will be subjected to Percoll density centrifugation and CL MAC isolated from the small luteal cell fraction (by immuno-magnetic separation using anti-CD14 coated beads. The purity and viability of macrophage preparations will be confirmed using Flow Cytometry (currently >90%). CL MAC (~ 0.5-1 x 106 cells per replicate; n = 5 replicates per CL stage) will be lysed for total RNA isolation, cDNA synthesis and Q-PCR analysis of PGR) Steady state mRNA levels will be calculated from the real-time Q-PCR curve (DDCt method) for each gene, and expressed relative to the housekeeping gene (GAPDH). Additional aliquots of CL MAC (~0.5-1 x 106 cells per replicate; n = 5 per cell type, per CL stage) will be subjected to cell-lysis and protein extraction, for Western blot analysis of PGR. In brief, 10-20 μg protein will be subjected to PAGE and transferred to PVDF membrane "blots". Blots will be incubated with the primary PGR antibody (~1:200 dilution). Protein bands will be visualized with a Bio-Rad Chemi-Doc imaging system and quantified using the Image Lab software. Quantification of Western blots will be performed relative to b-Actin (housekeeping protein) and data expressed as a ratio. These experiments will be repeated with 5 CL MAC preps. per cycle stage. Specific Aim 2: For SA 2A, we will employ co-cultures of CL MAC with an enriched large luteal cell fraction (LLC) collected from Percoll centrifugation. CL MAC will be isolated as described in SA 1 and plated on 24-well culture plates (~ 0.5 x 106 cells in 1 ml) in RPMI + 10% FBS, for co-culture with LLC as described below. LLC (source of P4; 0.25 x 106 cells per insert) will be plated on cell-culture inserts and placed into CL MAC containing culture wells for 24 h. Trilostane (TRIL; 5 mM) will be used to inhibit P4 synthesis in LLC in co-cultures with CL MAC. These experiments are designed to determine the effects of P4 withdrawal on CL MAC TNF-a production. This experiment will be replicated 5 times with CL MAC and LLC from 5 separate Mid-stage CL (BEFORE D. 13) dissociations. At the end of culture, media will be collected for analysis of P4 by EIA, to confirm the inhibition of progesterone synthesis by TRIL, and for analysis of TNF-a by ELISA. CL MAC will be lysed for mRNA extraction to be used for quantification of TNF-a mRNA expression by Q-PCR (as described in SA 1). For SA 2B, CL MAC will be cultured (~ 0.5 x 106 cells in 1 ml) in 24-well culture plates in RPMI + 10% FBS for 24 h with 0 (control), 0.05, 0.5 or 5 mg/ml (n = 4 per dose) progesterone-agonists (P4 = a non-selective agonist or R5020 = a PGR selective agonist), in the presence of the PGR selective antagonist RU-486 at 0, 1 or 10 mM (n = 4 per dose). At the end of culture, media will be removed for TNF-a analysis by ELISA. In addition, CL MAC will be lysed for mRNA isolation and Q-PCR (see SA#1), for examination of steady state expression of TNF-a mRNA, relative to that of the housekeeping gene GAPDH. The media collected from these cultures will be examined for changes in TNF-a production at the protein level, using commercially available ELISA (porcine) assays. These experiments will be repeated 5 times (with 5 CL MAC pools). Specific Aim 3: SA 3A; We propose to administer TRIL at Mid-cycle (Before D.13), to deplete intra-luteal P4 concentrations in vivo, and allow us to evaluate TNF-a production in CL MAC isolated from the CL of these animals. In addition, some animals will receive a "PGF-2a challenge" given (in vivo) 10h prior to CL removal on day 9, to allow us to confirm the acquisition of LS , in response to TRIL. We expect to observe increased TNF-a production by CL MAC in response to the intra-luteal P4 withdrawal resulting from TRIL treatment. We also expect to observe increased PGF-2a secretion by porcine CL tissue in vitro, as well as increased FOSB and aromatase (CYP19A1) mRNA expression in CL, following the PGF-2a challenge (in vivo), which should confirm the acquisition of LS in response to TRIL. Animals treated with or without TRIL and with or without PGF-2a. On day 7 of the estrous cycle, gilts (n=5 per group) will be randomly assigned to 4 treatment groups in a 2 x 2 design; Group 1: Control (no TRIL, no PGF-2a; Group 2: TRIL (no PGF-2a; Group 3: PGF (no TRIL); Group 4: TRIL & PGF-2a. TRIL will be given orally every 12 hours for 36h beginning on day 7 of the estrous cycle (see Pilot study). PGF-2a or vehicle (no PGF-2a) will be given at 38h. 10 h later (i.e. on day 9), animals will be euthanized, and the ovaries removed and transported on ice to the laboratory (see a-d below). Blood samples will be collected on D.7 and 8 via the ear vein, and on D.9 (at euthanasia) via the jugular vein. CL will be isolated and used as follows: (a) 5 CL will be used for dissociation/isolation of CL MAC (0.5 - 1 x 106 cells; 3 replicates per animal; stored at -70C) for an analysis of TNF-a mRNA as described in SA#2. Additional aliquots of CL MAC (0.5 - 1 x 106 cells; 3 replicates per animal) will be cultured for 24h in RPMI + 10% FBS and media collected for analysis of TNF-a protein by ELISA (see SA#2). CL MAC numbers/viability will be documented per animal by cell counting; (b) 1 CL will be minced and incubated (~ 40 mg tissue per well, in 24 well plate; n = 5 per animal) in M199 with 0.1% BSA at 37C for 2 h to examine progesterone (P4) and PGF-2a secretion in vitro. Media will be collected and assayed for P4 & PGF-2a by EIA (Cayman); (c) 3 CL (n = 3) will be frozen at -70C for future measurement of CYP19A1 (aromatase) and FOSB mRNA analysis by Q-PCR as described previously (SA#1) but using porcine specific primers for FOSB and CYP19A1; (d) 1 CL for extraction and measurement of progesterone concentrations by EIA to confirm the decline in CL P4 concentrations in response to TRIL. Blood (serum) samples will also be assayed for P4 by EIA, to confirm the decline in serum P4 concentrations in response to TRIL administration, and/or the induction of LS. SA 3B: We propose to use the PGR antagonist RU-486 to block P4 actions in CL MAC. We will follow the overall experimental plan described in detail in SA3A for TRIL, and examine TNF-a production by CL MAC, as well as the induction of LS by monitoring luteal PGF-2a synthesis in vitro and FOSB/CYP19A1 mRNA expression. In addition, in this specific aim, CL and serum P4, as well as the P4 secretion levels by minced CL tissue in vitro, should also provide valuable indicators of the induction of LS (i.e. P4 should be decreased in all samples) in PGF-2a "challenged", RU-486-treated animals (vs. all other groups). Thus, the data obtained from this specific aim should demonstrate increased TNF-a production by CL MAC, and provide additional evidence to support a physiological role for increased TNF-a in the induction of LS in response to progesterone withdrawal, in confirmation of the specific hypothesis for this Specific Aim. Animals will be assigned to one of 4 Groups for treatment with or without RU-486 (and with or without PGF-2a. On day (D.) 7 of the estrous cycle, gilts (n=5 per group) will `be randomly assigned to 4 treatment groups in a 2 x 2 design (see above for TRIL; replace RU-486 for TRIL). RU-486 (400 mg i.m./pig/day) will be given 1 x daily on D. 7 and 8. PGF-2a or vehicle (no PGF-2a) will be given on D. ~8.5, and 10 h later (i.e. on D. 9) animals will be euthanized, ovaries removed and transported on ice to the laboratory (see a-d below). Blood samples will be collected on D. 7 and 8 via the ear vein, and on D. 9 (at euthanasia) via the jugular vein assayed for P4 by EIA. CL will be used for the same analyses as described above in SA 3A {(a-d)}.

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

Outputs
Target Audience:Animal Scientists and reproductive biologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Completion of a Master students (Molly Alexander), training of a new Master Student (Mariam Giurgis, August 2023 start date) in the area of research and techniques associated with the studies described above How have the results been disseminated to communities of interest?Results from Specific Aim 3 were published in 2 differnt confernce presentations to expand the audience as well as in a graduate student thesis. Following additional analyses recommended by reviewers Specific Aim 3 will be published in a peer review journal. What do you plan to do during the next reporting period to accomplish the goals?We are currently in the process of completing Specific Aims 2A & B described in the proposal using the prescribed method of ovary & macrophage isolation described above. The tissue collections, cell isolations and in vitro experiments have been conducted to date. Final analysis of gene and protein expression as well and changes in hormone concentrations in currently underway

Impacts
What was accomplished under these goals? Progress has been made on Specific 3 of this proposal, which focused on (A) inhibition of P4 synthesis, and (B) blockade of PGR receptors, on TNF production by porcine CL MAC, in vivo. The specific objective was to determine the effects of decreased P4 production/actions on the acquisition of luteolytic sensitivity, as indicated by the expression of PGR, CYP19A1, FOSB and TNF mRNA (CL tissue), and TNF protein (CL MAC). Cyclic gilts (n= 25) were placed in crates at the Swine Educational Unit for individual feeding and synchronized using commercially available orally active progestin (Matrix; 6.8 mL per gilt once daily for 14 consecutive days) as a top dressing on their daily feed allowance. Following removal of the Matrix, gilts were observed daily to determine the start of the estrous cycle (day 0), which occurred 4 to 9 days after completion of the Matrix. Gilts were be randomly assigned to the following treatments: Control, PGF (25 mg i.m. 10hrs prior to euthanasia), Trilostane, (TRIL, 10 mg/kg per pig/day on days 7, 7.5, 8 and 8.5), RU486 (400 mg i.m./pig/day on days 7 and 8), TRIL+PGF, or RU486+PGF. In order to determine serum P4 concentrations, daily jugular blood samples were taken on days 6 through 9 of the estrous cycle. On day 9 of the estrous cycle, all gilts were euthanized, and the ovaries removed, CL were dissected and ~ 500 mg tissue was immediately frozen in liquid nitrogen for subsequent P4 analysis, via RIA. Additional luteal tissue collected on day 9 was enzyme-dissociated; cultured for 3 hours at 37oC and 5% CO2; and analyzed for P4. These results from Specific Aim 3 were published in 2 differnt confernce presentations to expand the audience as well as in a graduate student thesis. Following additional analyses recommended by reviewers Specific Aim 3 will be published in a peer review journal.Future studies should explore greater P4 reduction sustained for longer period of time prior to analysis. We are currently in the process of completing Specific Aims 2A & B described in the proposal using the prescribed method of ovary & macrophage isolation described above. The tissue collections, cell isolations and in vitro experiments have been conducted to date. Ovaries were collected from a local slaughterhouse and returned to the laboratory where CL were removed, and dissected and ~ 500 mg tissue was immediately frozen in liquid nitrogen for subsequent P4 analysis, via RIA. Additional luteal tissue collected o was enzyme-dissociated; cultured for 3 hours at 37oC and 5% CO2; and analyzed for P4 or further dissoxciation was ultized to isolate small and large luteal cells and luteal macrophages for co-culture experiments with and without progesterone supplementation. Final analysis of gene and protein expression as well and changes in hormone concentrations in currently underway.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Alexander, M.K., Giurgis, M., Wang, X., Gadsby, J.E., Poole, D.H. 2022. Trilostane effectively reduces Progesterone Synthesis prior to Acquisition of Luteolytic Capacity in Cyclic Gilts. Society for the Study of Reproduction, Spokane, WA. JULY 2629, 202


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

Outputs
Target Audience:Reproductive Physiologist, Swine Producers and Extension personnel Changes/Problems:Due to timing and availablity of porcine ovaries and assays, we have requested a No Cost extension for this project. The undergraduate researcher in my lab has been accepted into the graduate program and will complete the remaining experiments of Specific Aim 2A&B as part of her graduate program requirements. What opportunities for training and professional development has the project provided?Completion of a Master students (Molly Alexander), training of a new Master Student (Mariam Giurgis, August 2023 start date) in the area of research and techniques associated with the studies described above. How have the results been disseminated to communities of interest?Results from Specific Aim 3 were published in 2 differnt confernce presentations to expand the audience as well as in a graduate student thesis. Following additional analyses recommended by reviewers Specific Aim 3 will be published in a peer review journal. What do you plan to do during the next reporting period to accomplish the goals?We are currently in the process of completing Specific Aims 2A & B described in the proposal using the prescribed method of ovary & macrophage isolation described above. The tissue collections, cell isolations and in vitro experiments have been conducted to date. Final analysis of gene and protein expression as well and changes in hormone concentrations in currently underway.

Impacts
What was accomplished under these goals? Progress has been made on Specific 3 of this proposal, which focused on (A) inhibition of P4 synthesis, and (B) blockade of PGR receptors, on TNF production by porcine CL MAC, in vivo. The specific objective was to determine the effects of decreased P4 production/actions on the acquisition of luteolytic sensitivity, as indicated by the expression of PGR, CYP19A1, FOSB and TNF mRNA (CL tissue), and TNF protein (CL MAC). Cyclic gilts (n= 25) were placed in crates at the Swine Educational Unit for individual feeding and synchronized using commercially available orally active progestin (Matrix; 6.8 mL per gilt once daily for 14 consecutive days) as a top dressing on their daily feed allowance. Following removal of the Matrix, gilts were observed daily to determine the start of the estrous cycle (day 0), which occurred 4 to 9 days after completion of the Matrix. Gilts were be randomly assigned to the following treatments: Control, PGF (25 mg i.m. 10hrs prior to euthanasia), Trilostane, (TRIL, 10 mg/kg per pig/day on days 7, 7.5, 8 and 8.5), RU486 (400 mg i.m./pig/day on days 7 and 8), TRIL+PGF, or RU486+PGF. In order to determine serum P4 concentrations, daily jugular blood samples were taken on days 6 through 9 of the estrous cycle. On day 9 of the estrous cycle, all gilts were euthanized, and the ovaries removed, CL were dissected and ~ 500 mg tissue was immediately frozen in liquid nitrogen for subsequent P4 analysis, via RIA. Additional luteal tissue collected on day 9 was enzyme-dissociated; cultured for 3 hours at 37oC and 5% CO2; and analyzed for P4. These results from Specific Aim 3 were published in 2 differnt confernce presentations to expand the audience as well as in a graduate student thesis. Following additional analyses recommended by reviewers Specific Aim 3 will be published in a peer review journal.Future studies should explore greater P4 reduction sustained for longer period of time prior to analysis. We are currently in the process of completing Specific Aims 2A & B described in the proposal using the prescribed method of ovary & macrophage isolation described above. The tissue collections, cell isolations and in vitro experiments have been conducted to date. Ovaries were collected from a local slaughterhouse and returned to the laboratory where CL were removed, and dissected and ~ 500 mg tissue was immediately frozen in liquid nitrogen for subsequent P4 analysis, via RIA. Additional luteal tissue collected o was enzyme-dissociated; cultured for 3 hours at 37oC and 5% CO2; and analyzed for P4 or further dissoxciation was ultized to isolate small and large luteal cells and luteal macrophages for co-culture experiments with and without progesterone supplementation. Final analysis of gene and protein expression as well and changes in hormone concentrations in currently underway.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Alexander, M. A., 2022. TNF-alpha Production by Corpus Luteum (CL) Macrophages (MAC) and its Control by Progesterone in the Porcine CL. M.S. Thesis⿿ Animal Science, North Carolina State University.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Alexander, M.K., Wang, X., Gadsby, J.E., Poole, 2022 Trilostane Effectively Reduces CL Progesterone Synthesis Prior to Acquisition of Luteolytic Sensitivity in Cyclic Gilts. In: Journal of Animal Science, Volume 100, Issue Supplement_1, April 2022, Pages 31⿿32, https://doi.org/10.1093/jas/skac028.061
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Alexander, M.K., Giurgis, M., Wang, X., Gadsby, J.E., Poole, D.H. 2022. Trilostane effectively reduces Progesterone Synthesis prior to Acquisition of Luteolytic Capacity in Cyclic Gilts. Society for the Study of Reproduction, Spokane, WA. JULY 26⿿29, 2022


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

Outputs
Target Audience:Reprductive Physiologist, Swine Producers and Extension personnel Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of two Master students (Molly Alexander and Macy Massengill), and an Undergraduate Students (Mariam Giurgis) in the area of research and techniques associated with the studies described above. How have the results been disseminated to communities of interest?Results from Specific Aim 1 were published in Domestic Animal Endocrinology in July 2020 (citation below) whereas results from Specific Aim 3A will be reported at the USDA PI's Annual Meeting in December 14-15, 2021 St. Louis, MO. Gadsby JE, Frandsen S, Chang J, Celestino B, Tucker E, Poole DH. Progesterone inhibits cytokine/TNF-α production by porcine CL macrophages via the genomic progesterone receptor. Domest Anim Endocrinol. 2020 Jul;72:106426. doi: 10.1016/j.domaniend.2019.106426. Epub 2019 Dec 9. PMID: 32244110. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue with Specific Aims 3A & B described in the proposal using the prescribed method of ovary & macrophage isolation described above.

Impacts
What was accomplished under these goals? Progress has been made on Specific aims 1 and 3 of this proposal, which included studies on the genomic progesterone receptor (PGR) in porcine corpora lutea macrophages (CL MAC) collected before (~days 7-12) and after (~days 13-18) onset of luteolytic sensitivity (SA #1), as well as using novel drugs injected into the whole animal during the estrous cycle to block progesterone synthesis (SA #3A - pilot study). Specific aim 1: Using CL MACS isolated from mid-cycle (approx. D 7-12, before LS) and late cycle (approx. D. 13-18, after LS), we confirmed that CL MACs express both genomic progesterone (PGR) and membrane progesterone (mPR) receptors. Genomic progesterone receptor expression was greater in CL MACs collected before (~days 7-12) onset of luteolytic sensitivity when compared to CL MACs collect after (~days 13-18) onset of luteolytic sensitivity. Luteal cells, via progesterone, inhibited cytokine expression (TNF, IL-6, and IL-10 mRNA) in CL MACs. Lastly, TNF expression was greater in CL MACs collected after (~days 13-18) onset of luteolytic sensitivity. Thus contributing to our overall hypothesis that progesterone acting via progesterone receptor (PGR), inhibits corpora lutea macrophages (CL MAC) tumor necrosis factor (TNF) production and plays a key role in regulating luteolytic sensitivity during the estrous cycle in pigs. These data have been published in Domestic Animal Endocrinology. Specific aim 3A: Furthermore, in order to understand progesterone action on macrophage function in the live animal, we need an effective way to reduce P4 concentrations prior to luteolytic sensitivity. Therefore, a pilot study was conducted to confirm the efficacy of Trilostane (TRIL), an inhibitor of 3b-hydroxysteroid dehydrogenase (critical enzyme in progesterone synthesis), as a means to reduce progesterone synthesis in pigs. The goal was to determine the effective dosage of TRIL that reduces progesterone concentrations by 80% compared to control animals, in order to develop our model to investigate P4 role in regulating macrophage function. Cyclic gilts (n= 12) were placed in crates at the Swine Educational Unit for individual feeding and synchronized using commercially available orally active progestin (Matrix; 6.8 mL per gilt once daily for 14 consecutive days) as a top dressing on their daily feed allowance. Following removal of the Matrix, gilts were observed daily to determine the start of the estrous cycle (day 0), which occurred 4 to 9 days after completion of the Matrix. Gilts were be randomly assigned to the following treatments: Control (0 mg/kg of TRIL), 3, 5, 7.5 or 10 mg/kg (n=2 per dose) of TRIL. Treatments were delivered on days 7, 7.5, 8, and 8.5 of the estrous cycle. In order to determine serum progesterone concentrations, daily jugular blood samples were taken on days 6 through 9 of the estrous cycle. On day 9 of the estrous cycle, all gilts were euthanized, and the ovaries removed, CL were dissected and immediately frozen in liquid nitrogen for subsequent progesterone analysis, via RIA. Serum progesterone concentrations were reduced by 50 to 70% within 12 hours of receiving TRIL compared to control animals. The 10 mg/kg dose resulted in 80% reduction after 24 hrs. of treatment, and progesterone concentration remained low throughout the rest of the treatment period. It must be noted that the lower dosages of TRIL eventually reduced serum progesterone concentrations by >77% on day 9 of the estrous cycle. Luteal progesterone concentrations, from CL collected on day 9 of the estrous cycle, revealed a dosage dependent response with 72.4, 82.4, 87.7 and 88.6 % reduction in progesterone concentration with 3, 5, 7.5, and 10 mg/kg of TRIL, respectfully. Based on these preliminary studies, the dose of 10mg/kg of TRIL will effectively suppress serum and luteal progesterone concentrations prior to luteolytic sensitivity and allow us to examine the role of progesterone in regulating macrophage function, as described for the full experiment (Specific Aim #3A.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Gadsby JE, Frandsen S, Chang J, Celestino B, Tucker E, Poole DH. Progesterone inhibits cytokine/TNF-? production by porcine CL macrophages via the genomic progesterone receptor. Domest Anim Endocrinol. 2020 Jul;72:106426. doi: 10.1016/j.domaniend.2019.106426. Epub 2019 Dec 9. PMID: 32244110.