Progress 02/01/16 to 01/31/22
Outputs
Target Audience:The target audience is primarily scientists, with that knowledge eventually being applied in a way that it will be useful to producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Postdoctoral associates, graduate and undergraduate students have developed their skills in development and testing of hypotheses, experimental design and analysis of data. There were taught all of the procedures and assays necessary to perform these experiments. They performed all of the research and were involved in all aspects of the project.Further professional development included the opportunity to present their results at annual meetings of the Society for the Study of Reproduction and the American Society of Reproductive Immunology. How have the results been disseminated to communities of interest?The results have been presented at three different national meetings, sharing the discoveries withthe scientific community. The work shouldresult in 3 full-length peer-reviewed papers. Twoof these papers are in nearlyfinal draft form and will be submitted for publication soon. The third paper is currently in preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Threemanuscripts are currently under preparation for submission to a peer-reviewed journal. These manuscripts report the findings summarized below. 1. The corpus luteum (CL) is comprised of steroidogenic cells and various nonsteroidogenic cells, including T lymphocytes (TC), which support luteolysis and luteal function. It was hypothesized that luteal cells can communicate with TC via secreted extracellular vesicles (EV). Until now, the characteristics of luteal cell EV miRNA cargo and how it may affect nonsteroidogenic cells had not been studied. MicroRNA cargo in luteal EV from midcycle and regressing CL was profiled using Next Generation Sequencing (NGS), which revealed that one miRNA, miR-150, was more abundant in EV from regressing luteal cells (Padj = 0.016) and 10 additional miRNA tended (P < 0.05, Padj > 0.05) to be greater. Ten miRNA tended to be more abundant in EV from midcycle luteal cells (P < 0.05, Padj > 0.05). Pathway analysis of clusters of miRNA identified in luteal EV showed their potential involvement in immune cell functions, fatty acid biosynthesis and metabolism. MiR-1246 was identified as the most abundant miRNA in luteal EV regardless of status of the CL. To identify the actual targets of miR-1246 in bovine TC a pulldown assay was used. Biotinylated miR-1246 mimic was transfected into TC and transcripts were pulled down with streptavidin-labeled beads, followed by NGS. Thirty-eight transcripts were specifically pulled down compared to the negative control pulldown (P < 0.08). Pathway analysis of these transcripts revealed regulation of the Wnt signaling pathway, retinoid X receptor (RXR) activation, amino acid metabolism and activation of TC via nuclear factor of activated T cells (NFAT).To assess miR-1246 effect on TC function, miR-1246 mimic was transfected into cultured peripheral blood bovine TC, followed by the determination of cell cycle progression and the measurement of secreted tumor necrosis factor (TNF). Tumor necrosis factor secretion was decreased in TC under the influence of miR-1246 compared to TNF secreted from cells transfected with mimic negative control (P = 0.056). In summary, miRNA cargo in luteal EV, including the most abundant miR-1246, may affect immune cell functions possibly through targeting pathways associated with Wnt signaling, NFAT and nuclear factor-KB (NF-KB) signaling, which may serve as a mechanism to prevent luteal TC from excessive activation and proinflammatory differentiation. 2.Communication between luteal steroidogenic and nonsteroidogenic cells, including T cells (TC), through extracellular vesicles (EV) has not yet been studied. Extracellular vesicles are membranous structures that enclose various types of cargo, including proteins, that can influence target cells. It was hypothesized that luteal EV protein cargo affects TC functions. Proteins in luteal EV from midcycle and regressing CL were profiled using tandem mass spectrometry (MS/MS). Moreover, to better understand how the luteal microenvironment affects TC, proteomes of luteal resident TC (RTC) isolated from fully functional and regressing CL were profiled using MS/MS. Protein cargo identification in luteal EV revealed that laminin 1 (LAMA1) was more abundant in EV from midcycle CL (Padj<0.0001). Laminin 1 is known as a ligand for integrins, and a regulator of TC migration. Enolase 1 (ENO1), an enzyme involved in glycolysis, was more abundant in luteal EV from regressing CL (P<0.05, Padj=0.26 in MS/MS, P<0.01 in western blotting). Ingenuity Pathway Analysis (IPA) indicated that luteal EV proteins are associated with regulation of metabolic pathways. Analysis of RTC proteomes revealed that ENO1 also tended to be more abundant in RTC in regressing CL (P<0.05, Padj=0.4, P=0.08 in western blotting). Overall, proteins that tended to be different in luteal RTC were associated with metabolic pathways, integrin signaling or actin cytoskeleton signaling, which were common with pathways regulated by luteal EV proteins. To assess which changes in RTC may be driven specifically by luteal EV, peripheral blood TC were treatedin vitrowith luteal EV isolated from cells from functional CL or regressing CL and tumor necrosis factor (TNF), interleukin 4 (IL4) and the percentage of ENO1+TC was measured. Tumor necrosis factor secretion by TC was increased after treatment with EV from either midcycle or regressing luteal cells (P<0.05). Interestingly, luteal EV were not a source of ENO1 for TC during a short incubation, and the percentage of ENO1+TC was decreased after the treatment. In conclusion, luteal EV proteins may be involved in regulation of metabolic pathways in luteal RTC, which results in increased secretion of TNF, a known luteolytic mediator. Perhaps RTC are transiently stimulated to produce the cytokine, but their proteome does not differ greatly depending on CL status.
Publications
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Lupicka M and Pate JL. 2018. Luteal Extracellular Vesicles Carry MicroRNA and Modulate Cytokine Secretion from Immune Cells. Society for the Study of Reproduction, New Orleans, LA
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
Lupicka M and Pate JL. 2019. Cargo proteins in luteal extracellular vesicles have catalytic activity that may regulate activation of luteal resident immune cells. Society for the Study of Reproduction, San Jose, CA
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
M Lupicka, JL Pate. 2020. MiR-1246 is the most abundant miRNA in luteal extracellular vesicles and it regulates T cell transcripts associated with their activation. Society for the Study of Reproduction
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Progress 02/01/20 to 01/31/21
Outputs
Target Audience:Scientists, veterinarians, graduate students, postdocs, producers Changes/Problems:One experiment designed to treat luteal cells with extracellular vesicles from pregnant and nonpregnant cattle proved to be technically challenging, and when cows were sold because of the pandemic, this experiment was halted. Instead, we conducted microRNA and proteomic profiling of luteal EVs, followed by transfection of T cells with one miRNA. This provided new information about a potential role of EV-carried microRNA in the regulation of luteal cell- T cell interactions. What opportunities for training and professional development has the project provided?Graduate and undergraduate students have developed their skills in development and testing of hypotheses, experimental design and analysis of data. They were taught all of the procedures and assays necessary to perform these experiments. Graduate students, undergraduate students and postdoctoral associates performed the research and were involved in all aspects of the project. Further professional development included the opportunity to present their results at annual meetings of the Society for the Study of Reproduction.? How have the results been disseminated to communities of interest?These results were presented at the virtual meeting of the Society for the Study of Reproduction and published as an abstract from that meeting. Three full-length publications are in preparation.? What do you plan to do during the next reporting period to accomplish the goals?The final experiments for this grant were significantly delayed when the laboratory was closed and cows were sold off during the early stages of the pandemic. Experiments are essentially completed, and all data currently being analyzed and prepared for publication. The goal for this final year is to get these papers published in scientific journals.?
Impacts
What was accomplished under these goals?
Extracellular vesicles (EV) are cup-shaped, membranous structures that contain specific cargo used for cell-to-cell communication. Previously, we showed that bovine luteal EV regulate cytokine production in monocytes and T cells. We also characterized miRNA cargo of luteal EV using Next Generation Sequencing (NGS), and it revealed that miR-1246 was the most abundant miRNA regardless of the functional status of the corpus luteum (CL). Ingenuity Pathway Analysis (IPA) of 2377 predicted miR?1246 targets (determined with TargetScan algorithm) showed potential regulation of mRNA associated with NF-KB signaling and proliferation (p<0.05). Therefore, we hypothesized that miR-1246 affects T cell activation. To determine real targets of miR-1246 in bovine T cells, a miRNA pull-down assay was performed. Biotin-labeled miR-1246 mimic was transfected into cultured bovine peripheral blood T cells (n=6). Cells were then treated with PMA (phorbol 12-myristate-13-acetate) and ionomycin to increase expression of genes associated with cell activation. After treatment, miR-1246, together with its bound targets, were isolated using streptavidin-coated beads. Pulled-down targets were sequenced using NGS. Overall, the analysis revealed 37 transcripts that were different (P<0.05) or tended to be different (P<0.08) from negative control pull-down, among which 7 were also predicted miR?1246 targets. Among these identified transcripts, genes associated with cell cycle, such as cAMP regulated phosphoprotein 19 (ARPP19), cell division cycle 27 protein (CDC27) and PWWP domain containing 2A protein (PWWP2A), and genes associated with NFKB signaling, such as nuclear receptor coactivator 3 (NCOA3) were identified. Pathway analysis of pulled-down targets showed involvement of these transcripts in pathways associated with RXR activation, which among other functions also induces cell cycle progression and proliferation. To further test the hypothesis, cell cycle progression was measured in activated T cells transfected with miR?1246 mimics (n=2). After transfection with mimic, 8% of cells progressed to S or G2/M phase, whereas, cell cycle progression occurred in 12% of control cells. In summary, miR-1246 that is shuttled via luteal EV may regulate T cell activation in the CL through inhibition of translation of mRNA associated with cell cycle regulation.In our previous studies, luteal EV induced increased production of proinflammatory cytokines by T cells, but luteal cell-induced proliferation is limited, perhaps, because miR-1246 controls cell cycle progression and resident T cell survival during their activation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Hughes CHK, Bosviel R, Newman JW, Pate JL. 2019. Luteal lipids regulate progesterone production and may modulate immune cell function during the estrous cycle and pregnancy. Front. Endocrinol. 10:662. doi: 10.3389/fendo.2019.00662
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Hughes CHK, Inskeep EK, Pate JL. 2020. Temporal changes in the corpus luteum during early pregnancy reveal regulation of pathways that enhance steroidogenesis and suppress luteolytic mechanisms. Biology of Reproduction, ioaa047, https://doi.org/10.1093/biolre/ioaa047
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Pate JL. 2020. Roadmap to pregnancy during the period of maternal recognition in the cow: Changes within the corpus luteum associated with luteal rescue, Theriogenology, https://doi.org/10.1016/j.theriogenology.2020.01.074
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Vanselow J, Christenson LK and Pate JL. 2020. Editorial: Regulation of Dynamic Changes and Remodeling Events During the Formation, Rescue and Regression of the Corpus Luteum. Front. Endocrinol. 11:244. doi: 10.3389/fendo.2020.00244
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
M Lupicka, JL Pate. 2020. MiR-1246 is the most abundant miRNA in luteal extracellular vesicles and it regulates T cell transcripts associated with their activation. Society for the Study of Reproduction (virtual poster session).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Mezera MA, Hughes CHK, Pate JL, Wiltbank MC. 2020. The impact of pregnancy on the transcriptome of the bovine CL: insights from combining two independent experiments. Society for the Study of Reproduction (virtual poster session).
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Progress 02/01/19 to 01/31/20
Outputs
Target Audience: Scientists, veterinarians, graduate students, postdocs, producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate and undergraduate students have developed their skills in development and testing of hypotheses, experimental design and analysis of data. They were taught all of the procedures and assays necessary to perform these experiments. Graduate students, undergraduate students and postdoctoral associates performed the research and were involved in all aspects of the project. Further professional development included the opportunity to present their results at annual meetings of the Society for the Study of Reproduction How have the results been disseminated to communities of interest?Presentation at scientific meetings. Two journal articles are in preparation. What do you plan to do during the next reporting period to accomplish the goals?We have recently completed pulldown assays to identify the targets of the EV-transported miRNA in T cells. These analyses are ongoing and the papers are in preparation and will be submitted this year.
Impacts
What was accomplished under these goals?
Extracellular vesicles (EV) are cup-shaped, membranous structures that contain specific cargo used for cell-to-cell communication. Previously, we showed that bovine luteal EV regulate cytokine production in monocytes and T cells. We also characterized miRNA cargo of luteal EV using Next Generation Sequencing (NGS), and it revealed that miR-1246 was the most abundant miRNA regardless of the functional status of the corpus luteum (CL). Ingenuity Pathway Analysis (IPA) of 2377 predicted miR?1246 targets (determined with TargetScan algorithm) showed potential regulation of mRNA associated with NF-KB signaling and proliferation (p<0.05). Therefore, we hypothesized that miR-1246 affects T cell activation. To determine real targets of miR-1246 in bovine T cells, a miRNA pull-down assay was performed. Biotin-labeled miR-1246 mimic was transfected into cultured bovine peripheral blood T cells (n=6). Cells were then treated with PMA (phorbol 12-myristate-13-acetate) and ionomycin to increase expression of genes associated with cell activation. After treatment, miR-1246, together with its bound targets, were isolated using streptavidin-coated beads. Pulled-down targets were sequenced using NGS. Overall, the analysis revealed 37 transcripts that were different (P<0.05) or tended to be different (P<0.08) from negative control pull-down, among which 7 were also predicted miR?1246 targets. Among these identified transcripts, genes associated with cell cycle, such as cAMP regulated phosphoprotein 19 (ARPP19), cell division cycle 27 protein (CDC27) and PWWP domain containing 2A protein (PWWP2A), and genes associated with NFKB signaling, such as nuclear receptor coactivator 3 (NCOA3) were identified. Pathway analysis of pulled-down targets showed involvement of these transcripts in pathways associated with RXR activation, which among other functions also induces cell cycle progression and proliferation. To further test the hypothesis, cell cycle progression was measured in activated T cells transfected with miR?1246 mimics (n=2). After transfection with mimic, 8% of cells progressed to S or G2/M phase, whereas, cell cycle progression occurred in 12% of control cells. In summary, miR-1246 that is shuttled via luteal EV may regulate T cell activation in the CL through inhibition of translation of mRNA associated with cell cycle regulation.In our previous studies, luteal EV induced increased production of proinflammatory cytokines by T cells, but luteal cell-induced proliferation is limited, perhaps, because miR-1246 controls cell cycle progression and resident T cell survival during their activation. This project was supported by Agriculture and Food Research Initiative Competitive Grant no.2016-67015-24900 from the USDA National Institute of Food and Agriculture.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Lupicka M and Pate JL. 2019. Cargo proteins in luteal extracellular vesicles have catalytic activity that may regulate activation of luteal resident immune cells. Society for the Study of Reproduction, San Jose, CA
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Hughes CHK, Bosviel R, Newman JW, Pate JL. 2019. Luteal lipids regulate progesterone production and may modulate immune cell function during the estrous cycle and pregnancy. Front. Endocrinol. 10:662. doi: 10.3389/fendo.2019.00662
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Pate JL. 2020. Roadmap to pregnancy during the period of maternal recognition of pregnancy in the cow: Changes within the corpus luteum associated with luteal rescue. Theriogenology. https://doi.org/10.1016/j.theriogenology.2020.01.074
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Progress 02/01/18 to 01/31/19
Outputs
Target Audience:Scientists, veterinarians, graduate students, postdocs, producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate and undergraduate students have developed their skills in development and testing of hypotheses, experimental design and analysis of data. They were taught all of the procedures and assays necessary to perform these experiments. Graduate students, undergraduate students and postdoctoral associates performed the research and were involved in all aspects of the project. Further professional development included the opportunity to present their results at annual meetings of the Society for the Study of Reproduction How have the results been disseminated to communities of interest? Publication in scientific journals and presentations at scientific and extension/stakeholder meetings What do you plan to do during the next reporting period to accomplish the goals? We continue to do the proposed experiments, including profiling of the microRNA and proteins contained in the vesicles as well as proteomic analyses of the target immune cells.
Impacts
What was accomplished under these goals?
Our previous study showed that cells isolated from fully functional corpora lutea induce T cell activation without the need of cell-to-cell contact. These interactions are possibly mediated by extracellular vesicles, and could play a role in the fate of the corpus luteum. Extracellular vesicles are cup-shaped membranous structures secreted by a wide variety of cells. They contain specific cargo, including microRNA, which takes part in cell-to-cell communication. Our hypothesis is that luteal steroidogenic cells communicate with luteal resident immune cells, affecting their functions, via extracellular vesicles, and that their microRNA content differs depending on the estrous cycle stage. In the present study we isolated extracellular vesicles in the size range between 50-450 nm (exosomes and microvesicles), secreted by in vitro cultured luteal cells isolated from day 10 (n=4) and regressing (n=4, 8 hr after PGF2alpha injection) bovine corpus luteum. MicroRNA were profiled in vesicles using Next Generation Sequencing. Only miR-150 was identified as differentially expressed after applying Benjamini-Hochberg false discovery rate correction, and was more prevalent in extracellular vesicles during regression of the corpus luteum compared to vesicles from day 10 of the cycle (logFC= -8.96). Targets for miR?150 were predicted by three different algorithms, and their common targets were subject to pathway analysis. Ingenuity Pathway Analysis revealed that among the top significantly enriched pathways one was associated with T cell proliferation and one with IL7 signaling. Moreover, pathway analysis of predicted targets of the most abundant 15 microRNA in luteal extracellular vesicles showed enrichment in pathways such as fatty acid synthesis and metabolism, TGFbeta receptor signaling, and immune system processes. We also performed an in vitro experiment wherein bovine T lymphocytes or monocytes isolated from peripheral blood were treated for 72 hr with isolated luteal extracellular vesicles. Concentrations of selected cytokines in post-treatment culture media were measured with ELISA. This experiment showed that TNFalpha secretion was greater in monocytes treated with either midcycle or regressing vesicles (n=3), when compared to controls. Transforming growth factor beta concentration was significantly greater in media from monocytes treated with regressing vesicles comparing with the control. Furthermore, both midcycle and regressing vesicles (n=3) induced increased secretion of TNFalpha in T lymphocytes. In summary, our current results suggest that luteal extracellular vesicles' cargo affects immune cell functions. In silico analysis of sequencing experiment results supports the hypothesis that microRNA are regulators of those functions, however further experiments are required to confirm if microRNA that are packaged into luteal vesicles can directly alter immune cell cytokine secretion.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Pate JL, Hughes CK. 2018. Applications of large-scale molecular profiling techniques to the study of the corpus luteum. Anim. Reprod. 15:Suppl. 1, p. 791-804. DOI: 10.21451/1984-3143-AR2018-0038
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Vasudevan S, Kamat MM, Walusimbi SS, Pate JL, Ott TL. 2017. Effects of early pregnancy on uterine lymphocytes and endometrial expression of immune-regulatory molecules in dairy heifers. Biol Reprod 97(1), 104-118.
- Type:
Book Chapters
Status:
Published
Year Published:
2018
Citation:
Pate, JL (2018). Luteolysis. In M. K. Skinner (Ed.), Encyclopedia of Reproduction. vol. 2, pp. 106113. Academic Press: Elsevier. http://dx.doi.org/10.1016/B978-0-12-801238-3.64397-0
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
Hughes CK, Pate JL. 2019. Luteolysis and the Corpus Luteum of Pregnancy. In: The Ovary 3rd Edition, PCK Leung and EY Adashi, eds., Academic Press, Elsevier, pp. 270-295. ISBN 978-0-12-813209-8
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Progress 02/01/17 to 01/31/18
Outputs
Target Audience:Scientists, veterinarians, graduate students, postdocs, producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate and undergraduate students have developed their skills in development and testing of hypotheses, experimental design and analysis of data. They were taught all of the procedures and assays necessary to perform these experiments. Graduate students, undergraduate students and postdoctoral associates performed the research and were involved in all aspects of the project. Further professional development included the opportunity to present their results at annual meetings of the Society for the Study of Reproduction and the American Society of Reproductive Immunology. How have the results been disseminated to communities of interest?Publication in scientific journals and presentations at scientific and extension/stakeholder meetings What do you plan to do during the next reporting period to accomplish the goals?We continue to do the proposed experiments, including profiling of the microRNA and proteins contained in the vesicles.
Impacts
What was accomplished under these goals?
Previous work from this laboratory has provided clear evidence that bovine luteal cells can communicate with immune cells in the absence of cell-to-cell contact. We hypothesized that this communication is accomplished by the release of extracellular vesicles (EV), which carry microRNA and proteins that can alter functions of target cells. Extracellular vesicles were isolated from cultured bovine luteal cells obtained from fully functional or regressing corpora lutea. The EV were used to treat immune cells (monocytes and T lymphocytes) collected from the blood of cows. Expression of cytokine mRNA and proteins in the immune cells following treatment with EV or control liposomes was evaluated using quantitative PCR and ELISA, respectively. Luteal EV stimulated T cells to proliferate, indicating activation of the T cells. Cellular concentration of TNF mRNA was increased by EV from functional luteal cells, but decreased by EV from regressing luteal cells. EV from regressing luteal cells also decreased concentrations of mRNA for IFNG, IL10 and IL4 in T cells. Secretion of TNF by T cells was stimulated by EV from either functional or regressing luteal cells. Secretion of IL4 tended to be greater in the presence of EV from functional luteal cells, but was unaffected by vesicles from regressing luteal cells. In monocytes, EV from both types of luteal cells increased mRNA for IL10, TNF and IL1B. Further, EV stimulated secretion of TNF and TGFB from monocytes. These data provide evidence that luteal cells can communicate with immune cells via release of extracellular vesicles. In the target cells, EV alter the intracellular concentration of cytokine mRNA, either by regulating the balance of mRNA transcription or degradation, or by delivering specific mRNA to the target cells. These changes result in alteration of cytokines that are secreted by the immune cells, suggesting that luteal cells can direct the function of immune cells within the microenvironment of the CL.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Pate, JL and Hughes, CK. Cells and networks that facilitate luteal survival for pregnancy success. Proceedings of the 4th World Congress of Reproductive Biology, Okinawa, Japan.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Walusimbi SS, Wetzel LM, Townson DH, Pate JL. 2017. Isolation of luteal endothelial cells and functional interactions with T lymphocytes. Reproduction 153 (5) 519-533, doi: 10.1530/REP-16-0578
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Progress 02/01/16 to 01/31/17
Outputs
Target Audience:Scientists and producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate and undergraduate students have developed their skills in development and testing of hypotheses, experimental design and analysis of data. They were taught all of the procedures and assays necessary to perform these experiments. Graduate students, undergraduate students and postdoctoral associates performed the research and were involved in all aspects of the project. Further professional development included the opportunity to present their results at annual meetings of the Society for the Study of Reproduction and the American Society of Reproductive Immunology. How have the results been disseminated to communities of interest?Presentations at scientific meetings What do you plan to do during the next reporting period to accomplish the goals?Continue to address the stated objectives with experiments as outlined in the approved project description
Impacts
What was accomplished under these goals?
1. Evidence from coculture of luteal cells (LC) and T cells (TC) suggests that resident TC in the corpus luteum (CL) communicate with luteal cells via paracrine factors and through physical contact, to support luteal function or facilitate luteal regression. The goal of this study was to visualize this physical contact and identify the proteins involved in mediating the interaction between LC and TC. Activated TC from the peripheral blood were cocultured for 4 hours with LC from regressing CL (collected 8 hours after a luteolytic injection of prostaglandin F2 alpha). Cocultures were fixed and visualized using transmission electron microscopy. For flow cytometry experiments, cocultured cells were fixed and labeled for CD3, to identify TC, and with either 3-beta-hydroxysteroid dehydrogenase to identify steroidogenic LC, or lectin from Bandeiraea simplicifolia to identify endothelial LC. Additionally, LC were stained with the cell stain efluor and TC were stained with the membrane stain PKH26 prior to coculture. Following coculture, these conjugates were fixed and stained for CD6 and CD166 or CD28 and CD86. The Amnis FlowSight imaging flow cytometer was used for imaging and analysis of cell populations. Areas of apposition, interdigitation, and junctional complexes were observed between LC and TC. A greater proportion of luteal steroidogenic cells than luteal endothelial cells formed conjugates with TC (n=3; p>0.05). Most conjugates that form between LC and TC coexpress the protein pair CD6 and CD166 and most conjugates coexpress the protein pair CD28 and CD86 (n=3). These protein pairs colocalize at the point of interaction between individual TC and LC in some conjugates. Luteal steroidogenic cells and luteal endothelial cells both form physical interactions with TC. Steroidogenic cells form more conjugates than endothelial cells. CD6-CD166 and CD28-CD86 conjugation may both be important in TC-LC interactions. 2.Macrophages are the most abundant immune cell type within the corpus luteum (CL), however, their phenotypes are unknown. We hypothesized that the functional phenotype of luteal macrophages changes throughout the estrous cycle and during luteal regression. CL were collected at 3 different times in the estrous cycle and during luteal regression. Macrophage protein expression was determined by dual labeling tissues with CD11b and the M1 proteins, inducible nitric oxide synthase (NOS2), indoleamine 2,3-dioxygenase (IDO1) and tumor necrosis factor (TNF), or the M2 proteins, interleukin 10 (IL10) and CD36. CD45 expression was used to determine the proportion of immune cells that are macrophages. The percentage of dual labeled cells per total CD11b cells per tissue was calculated (n=4 CL/stage). The number of macrophages was greatest in early compared to midcycle and late CL. The percentage of CD45+ that are macrophages decreased in late (p=0.014) and tended to decrease in midcycle (p=0.081) compared to early CL. NOS2 decreased in late compared to early CL (p=0.052). No differences in IDO1, TNF, CD36 or IL10 were observed during the estrous cycle. During luteolysis, the percentage of CD45+ cells that are macrophages tended to increase by 1 hour (p=0.085) and to decline at 8 hours (p=0.087) after prostaglandin (PG)F2α. NOS2 increased by 1 hour (p=0.034) and declined by 8 hours (p=0.039) after PGF2α. TNF decreased by 2 hours (p=0.045) after PGF2α. IDO1 tended to increase by 2 (p=0.092) and 8 hours (0.100) after PGF2α. IL10 decreased at 1 hour (p=0.015) but increased (p=0.004), as did CD36 (p=0.020) by 8 hours after PGF2α. Greater NOS2 in early and early regressing CL may be associated with NO-mediated cytotoxicity and restriction of T cell expansion. The increases in NOS2 during early luteolysis and CD36 and IL10 in late luteolysis suggest a shift from M1 macrophages during initiation, to M2 macrophages as luteolysis progresses.
Publications
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