Progress 07/01/23 to 06/30/24
Outputs
Target Audience:Target audiences include: 1) members of reproductive physiology, developmental biology, pig embryology, extracellular vesicles, and tissue engineering research communities, as well as the swine industry; and 2) students in the PI's labs and in courses taught by the PIs, who directly and indirectly benefit from the knowledge gained in this projected. Changes/Problems:Progress on the overall scientific goal was delayed first by the pandemic (award began in summer 2021) and then again as our swine herd at US MARC suffered a PRRS outbreak in Winter/Spring 2022, which limited the availability of gilts and caused a delay in obtaining flushings, cells and embryos for both objectives. Following veterinarian consulting, we began depopulation of all young, weaned pigs including all potential replacement gilts starting March 2022 to ensure proper vaccination protocol of the novel highly virulent PPRS strain within our older breeding females to accrue appropriate immunity before retaining any new pigs in our herd. This eliminated any replacement gilts during the period of June 2022 to March 2023, resulting in no collection of material during this period. After several rounds of negative testing within the herd, we began to retain young gilts in March 2023 and have had no significant issues with PPRS thru June 2024. As a result, we have retained the appropriate number of replacement gilts and reinitiated sample collection in Fall 2023 (~November 2023). In addition, our PhD student on this project, responsible for the bulk of the work, suffered a mental health episode in August 2022, and decided to end her graduate program with us at that time. We recruited a new PhD student who started on the project in May 2023; he is now fully trained in all of our techniques and has been performing all studies outlined above. What opportunities for training and professional development has the project provided?Opportunities include formal graduate coursework, in-lab training in interdisciplinary research methods and techniques, University seminars, and opportunities for learning and networking at local, regional and national scientific meetings. How have the results been disseminated to communities of interest?Research results have been broadly disseminated through presentations at local symposia, and international professional meetings and lectures (NIFA PD meetings in July 2022 and June 2024 and 11th International Conference on Pig Reproduction in 2023), as well as publications. What do you plan to do during the next reporting period to accomplish the goals?For objective 1, complete isolation of EVs from ULF and cultured maternal explant samples via optimized procedures described above, followed by characterization of morphology and content. Size and concentration of particles from the isolated EV samples will be conducted by nano-flow cytometry. TEM will also be used to determine the presence of intact EVs and to verify the average diameter data given by nano-flow cytometry. Transcriptomic analysis on isolated EVs derived from ULF and explant samples will be performed, followed by analysis of the large amount of data generated from these studies. Multiple methods of dissolving alginate beads will be tested to determine if EVs from the encapsulated embryos are trapped within the alginate hydrogel system, and once isolated, those EVs will also be analyzed as described above. Prepare and submit manuscripts reporting the transcriptomic evaluations from ULF, explants and culture embryos. For objective 2, incorporate EVs into the alginate hydrogel system either through addition prior to conjugation, addition to media, or immobilization following conjugation. EVs from ULF and explants will be added to the encapsulated embryos to study the effect of different EVs on the initiation of elongation following day 9 of gestation. Prepare manuscripts reporting the addition of EVs to our culture system.
Impacts
What was accomplished under these goals?
IMPACT: The preimplantation period of porcine pregnancy is marked by dramatic morphological changes as embryos elongate from spherical to filamentous conceptuses. Successful elongation is necessary for maintenance of pregnancy, adequate nutrient exchange, correct spacing of embryos in the uterus, proper placental development, increased litter size, birthweight uniformity, and postnatal piglet survival. Unfortunately, a significant portion of embryos fail to undergo proper elongation, resulting in embryonic loss or non-uniform embryonic development within litters, which impacts pregnancy success and therefore efficiency of swine production. Due in part to the difficulty of replicating this process in vitro, little is known about the specific factors contributing to proper porcine conceptus elongation, including the influence of extracellular vesicles (EVs) present in the preimplantation uterine environment as mediators of maternal-conceptus crosstalk during this crucial developmental period. Therefore, the overall goal of this project is to characterize EVs isolated from maternal and embryonic secretions during the initiation of conceptus elongation, and then investigate the effect of crosstalk mediated by different EV populations on the initiation of porcine conceptus elongation using our unique three-dimensional (3D) in vitro culture system. This project will identify EV factors that are critical for porcine conceptus elongation, which could be manipulated to improve reproductive outcomes in the pig and other domestic agricultural species, improving animal production, specifically benefiting the swine industry, and ensuring a long-term sustainable source of high value protein for a global food security. The overall strategy for Objective 1 is to isolate extracellular vesicles (EVs) produced by the maternal endometrium and embryo at different time points during the initiation of elongation and characterize the EV proteomes and transcriptomes to elucidate potential mechanisms of cell-to-cell communication for promoting successful conceptus elongation. Since initiation of sample collection for Objective 1 and despite the delay over the past years due to the problems highlighted above, we have collected samples from 170 pregnant gilts between days 9 and 11 of gestation. This includes uterine lumen flushings (ULF) from all bred gilts, and also includes 2,400 embryos, of which 420 embryos have been cultured within our 3D alginate culture system and 150 non-encapsulated control embryos to obtain embryonic EVs. In addition, we have isolated and cultured uterine epithelial cells (UECs)/explants from 52 pregnant gilts and collected media to evaluate maternal specific EVs. We have evaluated techniques to isolate EVs from flushings, which contain large amounts of contaminating materials. We have used a new type of analytical technique called nano-flow cytometry, in combination with other techniques such as Western blots and cryogenic electron microscopy (cryo-EM), to characterize EVs (secreted by HEK 293T cells, to ensure a virtual unlimited supply and to preserve our limited porcine EV library) isolated using various methods including differential ultracentrifugation, tangential flow filtration (TFF), and traditional ultrafiltration. Using these techniques we have been able to observe precise quantifying parameters such as total particle and EV-like particle yield, size distributions, as well as their protein and lipid compositions. We have finalized our isolation workflows which include steps of ultracentrifugation, TFF, and ultrafiltration occurring in sequence, isolating size-based subsets of EVs in high purity and yield (that is, we can now routinely isolate small EVs and large EVs and plan to analyze these separately in future analyses). Most recently we have started to test commercial antibodies for their ability to be used for characterizing porcine EVs using both Western blots and nano-flow cytometry. We will use this workflow to isolate and characterize all porcine EVs, as well as to prepare samples for proteomic and transcriptomic analysis. Within Objective 1, we have also investigated methods to isolate EVs from cultured embryos within the culture media using similar methods described above. However, given the hydrogel matrix we use to culture the embryos potentially serves as a trap for EVs, we have had some difficultly isolating EVs, particularly from encapsulated treatment groups. Therefore, we have initiated protocol testing to isolate EVs from within the hydrogel matrix. In addition, we have attempted to isolate EVs from cultured maternal uterine epithelial cells, but due to limitations in that culture system, we have instead begun culturing uterine endometrial explants with success to isolate maternal derived EVs. We have also begun to characterize mRNA expression for glycerophospholipid and sphingolipids from specific cell populations of embryos (trophectoderm or embryonic disc) at the initiation of elongation from the in vivo- produced embryos. These data illustrate substantial up-regulation of sphingolipid metabolism specifically within the trophectoderm, which illustrate a potential involvement of sphingolipids within the conceptus during initiation of elongation. Interestingly, sphingolipids are a significant component of the EV membrane, so perhaps increased sphingolipid within the trophectoderm is playing a role in EV formation. As a result, we plan to further evaluate any potential involvement of sphingolipids on EV formation within various components of the uterine environment during initiation of conceptus elongation. Recently, we have started to investigate methods to extract and quantify miRNA from populations of isolated EV's with moderate success. The total amounts of miRNA extracted from EVs harvested from individual gilts have been nearly sufficient to begin preparing sequencing libraries. However, due to the relatively low quantities, future library preps may need to be derived from pooled miRNA aliquots within each of the days of gestation that are under investigation. The overall strategy for Objective 2 is to investigate the influence of EVs derived from uterine luminal and epithelial cells present in the preimplantation uterine environment on the survival and initiation of conceptus elongation using our 3D culture system. EVs will be isolated from the culture samples with our optimized methods. Isolated EVs will be incorporated into the 3D hydrogel culture system during encapsulation of embryo, where initiation of elongation will be measured after 96 hours of culture. Due to delays from the problems stated above, we not have yet begun studies for Objective 2.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Walsh SC, Miles JR, Broeckling CD, Rempel LA, Wright-Johnson EC, and Pannier AK. 2023. Secreted metabolome of porcine blastocysts encapsulated within in vitro 3D alginate hydrogel culture systems undergoing morphological changes provides insights into specific mechanisms involved in the initiation of porcine conceptus elongation. Reproduction, Fertility and Development. 35(5): 375-394.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Miles JR, Walsh SC, Rempel LA and Pannier AK. 2023. Mechanisms regulating the initiation of porcine conceptus elongation. Mol. Reprod. Develop. 90(7): 646-657(Invited review and presentation at the 11th International Conference on Pig Reproduction, Ghent Belgium)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Pannier AK, Miles JR, Rempel LA, Vyzourek B. Characterizing Maternal and Embryonic Extracellular Vesicles at the Initiation of Porcine Conceptus Elongation. USDA AFRI Animal Reproduction Project Director Meeting, Hybrid, June 25-27, 2024.
|
Progress 07/01/22 to 06/30/23
Outputs
Target Audience:Target audiences include: 1) members of reproductive physiology, developmental biology, pig embryology, extracellular vesicles, and tissue engineering research communities, as well as the swine industry; and 2) students in the PI's labs and in courses taught by the PIs, who directly and indirectly benefit from the knowledge gained in this projected. Changes/Problems:Anticipated progress on the overall scientific goal was delayed as our swine herd at USMARC suffered a PRRS outbreak in Spring 2022, which limited the availability of gilts and caused a delay in obtaining flushings, cells and embryos for both objectives. Following veterinarian consulting, we began depopulation of all young, weaned pigs including all potential replacement gilts starting June 2022 to ensure proper vaccination protocol of the novel highly virulent PPRS strain within our older breeding females to accrue appropriate immunity before retaining any new pigs in our herd. This eliminated any replacement gilts during the period of June 2022 to March 2023, resulting in no collection of material during this period. After several rounds of negative testing within the herd, we began to retain young gilts in March 2023 and have had no issues with PPRS thru June 2023. As a result, we are on schedule to have the appropriate number of replacement gilts and begin sample collection in Fall 2023 (~October 2023). In addition, our PhD student on this project, responsible for the bulk of the work, suffered a mental health episode in August 2022, and decided to end her graduate program with us at that time. We have recruited a new PhD student to start on this project (May 2023), and are looking forward to improved progress and outcomes, once he is trained in all of our techniques and should align nicely with gilt availability in Fall 2023. What opportunities for training and professional development has the project provided?Opportunities include formal graduate coursework, in-lab training in interdisciplinary research methods and techniques, University seminars, and opportunities for learning and networking at local, regional and national scientific meetings. How have the results been disseminated to communities of interest?Research results have been broadly disseminated through presentations at international professional meetings and lectures (NIFA PD meeting and at the 11th International Conference on Pig Reproduction), as well as publications. What do you plan to do during the next reporting period to accomplish the goals?For objective 1, plans for the next year include all isolation of EVs from ULF and cultured UEC samples via optimized procedures described above, followed by characterization of morphology and content. Size and concentration of particles from the isolated EV samples will be conducted by nano-flow cytometry. TEM will also be used to determine the presence of intact EVs and to verify the average diameter data given by nano-flow cytometry. Proteomic and transcriptomic analysis on isolated EVs derived from ULF and UEC samples will be performed as described above, and much of Year 3 will be spent analyzing the large amount of data generated from these analyses. Protocols for pooling samples of isolated EVs derived from UEC will be performed to ensure a yield that can enable the previously described characterization methods. Multiple methods of dissolving alginate beads, including alginate lyase, centrifugation, and EDTA, will be tested to determine if EVs from the encapsulated embryos are trapped within the alginate hydrogel system, and once isolated, those EVs will also be analyzed as described above. For objective 2, plans for next year include some early studies to understand how to incorporate EVs into the alginate hydrogel, but the bulk of objective 2 will be done after year 3 (with planned NCE due to delays). Furthermore, we will continue to evaluate the potential involvement of sphingolipids on EV packaging embryos and uterine samples collected in the previous years. In year 4 (with anticipated NCE), we plan to target components specific to Objective 2 including incorporation of EVs into the alginate hydrogel system either through addition prior to conjugation, addition to media, or immobilization following conjugation. EVs from ULF and UEC will be added to the encapsulated embryos to study the effect of different EVs on the initiation of elongation following day 9 of gestation.
Impacts
What was accomplished under these goals?
IMPACT: The preimplantation period of porcine pregnancy is marked by dramatic morphological changes as embryos elongate from spherical to filamentous conceptuses. Successful elongation is necessary for maintenance of pregnancy, adequate nutrient exchange, correct spacing of embryos in the uterus, proper placental development, increased litter size, birthweight uniformity, and postnatal piglet survival. Unfortunately, a significant portion of embryos fail to undergo proper elongation, resulting in embryonic loss or non-uniform embryonic development within litters, which impacts pregnancy success and therefore efficiency of swine production. Due in part to the difficulty of replicating this process in vitro, little is known about the specific factors contributing to proper porcine conceptus elongation, including the influence of extracellular vesicles (EVs) present in the preimplantation uterine environment as mediators of maternal-conceptus crosstalk during this crucial developmental period. Therefore, the overall goal of this project is to characterize EVs isolated from maternal and embryonic secretions during the initiation of conceptus elongation, and then investigate the effect of crosstalk mediated by different EV populations on the initiation of porcine conceptus elongation using our unique three-dimensional (3D) in vitro culture system. This project will identify EV factors that are critical for porcine conceptus elongation, which could be manipulated to improve reproductive outcomes in the pig and other domestic agricultural species, improving animal production, specifically benefiting the swine industry, and ensuring a long-term sustainable source of high value protein for a global food security. The overall strategy for Objective 1 is to isolate EVs produced by the maternal endometrium and embryo at different time points during the initiation of elongation and characterize the EV proteomes and transcriptomes to elucidate potential mechanisms of cell-to-cell communication for promoting successful conceptus elongation. Since initiation of sample collection for Objective 1 and despite the delay over the past year due to the problems highlighted below, we have collected samples from 114 pregnant gilts between days 9 and 11 of gestation. This includes ULF from all bred gilts, and also includes 1,142 embryos, of which 300 embryos have been cultured with our 3D alginate culture system to obtain embryonic EVs. In addition, we have isolated and cultured UECs from 32 pregnant gilts and collected media to evaluate maternal specific EVs. We have evaluated techniques to isolate EVs from flushings, which contain large amounts of contaminating materials. We reported our findings last year, but given our delays (see below), we have worked this year to continue to refine our isolation and characterization techniques. We used a new type of analytical technique called nano-flow cytometry, in combination with other techniques such as enzyme-linked immunosorbent assay (ELISA), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM), to characterize EVs (secreted by Hek 293T cells, to ensure a virtual unlimited supply and to preserve our limited porcine EV library) isolated using various methods including differential ultracentrifugation, polymer-based precipitation, ultrafiltration, size exclusion chromatography, and immunocapture, and to compare these techniques by quantifying parameters such as EV yield, ELV yield, size distribution, purity, and concentration. Using the nano-flow cytometry assessment, isolation using ultracentrifugation resulted in the highest EV yield and purity, compared to all other isolation techniques. We will use this workflow to isolation and characterization all porcine EVs, as well as to prepare samples for proteomic and transcriptomic analysis. We also investigated methods to isolate EVs from cultured embryos within the culture media using similar methods described above. However, given the hydrogel matrix we use to culture the embryos potentially serves as a trap for EVs, we have had some difficultly isolating EVs, particularly from encapsulated treatment groups. Therefore, we have initiated protocol testing to isolate EVs from within the hydrogel matrix. We have also begun to characterize mRNA expression for glycerophospholipid and sphingolipids from specific cell populations of embryos (trophectoderm or embryonic disc) at the initiation of elongation from the in vivo- produced embryos. These data illustrate substantial up-regulation of sphingolipid metabolism specifically within the trophectoderm, which illustrate a potential involvement of sphingolipids within the conceptus during initiation of elongation. Interestingly, sphingolipids are a significant component of the EV membrane, so perhaps increased sphingolipid within the trophectoderm is playing a role in EV formation. As a result, we plan to further evaluate any potential involvement of sphingolipids on EV formation within various components of the uterine environment during initiation of conceptus elongation. The overall strategy for Objective 2 is to investigate the influence of EVs derived from uterine luminal and epithelial cells present in the preimplantation uterine environment on the survival and initiation of conceptus elongation using our 3D culture system. EVs will be isolated from the ULF and UEC culture samples with our optimized methods. Isolated EVs will be incorporated into the 3D hydrogel culture system during encapsulation of embryo, where initiation of elongation will be measured after 96 hours of culture. Due to delays from the problems stated below, we not have yet begun studies for Objective 2.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Pannier AK, Miles JR, Rempel LA, Walsh S, Biegert M. Influence of Maternal and Embryonic Extracellular Vesicles on the Initiation of Porcine Conceptus Elongation. USDA AFRI Animal Reproduction Project Director Meeting, Spokane, WA (and Hybrid) July 25-26, 2022.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Miles JR, Walsh SC, Rempel LA and Pannier AK. 2022. Mechanisms regulating the initiation of porcine conceptus elongation. (online ahead of print at Mol. Reprod. Develop). doi: 10.1002/mrd.23623.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Miles JR, Rempel LA, Snider AP, and Pannier AK. Characterization of mRNA expression for key glycerophospholipid and sphingolipid enzymes within trophectoderm and embryonic disc during initiation of porcine conceptus elongation. 2023. Abst 43. Final Program Abstract Book for the 11th International Conference on Pig Reproduction, Ghent, Belgium, June 4-7, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Miles JR, Walsh SC, Rempel LA, and Pannier AK. Invited Presentation at the 11th International Conference on Pig Reproduction, Ghent, Belgium, June 4-7, 2023.
|
Progress 07/01/21 to 06/30/22
Outputs
Target Audience:Target audiences include: 1) members of reproductive physiology, developmental biology, pig embryology, extracellular vesicles, and tissue engineering research communities, as well as the swine industry; and 2) students in the PI's labs and in courses taught by the PIs, who directly and indirectly benefit from the knowledge gained in this projected. Changes/Problems:Progress on the overall scientific goal is delayed as our swine herd at USMARC suffered a PRRS outbreak in Spring 2022, which has limited the availability of gilts and has caused a delay in obtaining flushings, cells and embryos for both objectives. Following veterinarian consulting, we have just begun to hold back weaned gilts as of June 2022 to ensure that vaccination protocol allow for proper exposure to young animals. This will extend our limit of gilts through January 2023 at which time we are hopeful gilts will be ready for breeding and we can pick up on sample collection shortly thereafter. What opportunities for training and professional development has the project provided?Opportunities include formal graduate coursework, in-lab training in interdisciplinary research methods and techniques, University seminars, and opportunities for learning and networking at local, regional and national scientific meetings. How have the results been disseminated to communities of interest?Research results have been broadly disseminated through presentations at international professional meetings and lectures (SSR and NIFA PD meeting). What do you plan to do during the next reporting period to accomplish the goals?For objective 1, plans for the next year include more isolation of EVs from ULF and cultured UEC samples via ExoQuick Ultra Isolation Kit followed by characterization of morphology and content. Size and concentration of particles from the isolated EV samples will be conducted by nFCM via NanoAnalyzer. TEM will also be used to determine the presence of intact EVs and to verify the average diameter data given by the nFCM. Proteomic and transcriptomic analysis on isolated EVs derived from ULF and UEC samples will be performed as described above (and we have already submitted samples to the proteomics and transcriptomics cores and thus are awaiting data), and much of Year 2 will be spent analyzing the large amount of data generated from these analyses. Protocols for pooling samples of isolated EVs derived from UEC will be performed to ensure a yield that can enable the previously described characterization methods. Multiple methods of dissolving alginate beads, including alginate lyase, centrifugation, and EDTA, will be tested to determine if EVs from the encapsulated embryos are trapped within the alginate hydrogel system, and once isolated, those EVs will also be analyzed as described above. For objective 2, plans for next year include some early studies to understand how to incorporate EVs into the alginate hydrogel, but the bulk of objective 2 will be done after year 2.
Impacts
What was accomplished under these goals?
IMPACT: The preimplantation period of porcine pregnancy is marked by dramatic morphological changes as embryos elongate from spherical to filamentous conceptuses. Successful elongation is necessary for maintenance of pregnancy, adequate nutrient exchange, correct spacing of embryos in the uterus, proper placental development, increased litter size, birthweight uniformity, and postnatal piglet survival. Unfortunately, a significant portion of embryos fail to undergo proper elongation, resulting in embryonic loss or non-uniform embryonic development within litters, which impacts pregnancy success and therefore efficiency of swine production. Due in part to the difficulty of replicating this process in vitro, little is known about the specific factors contributing to proper porcine conceptus elongation, including the influence of extracellular vesicles (EVs) present in the preimplantation uterine environment as mediators of maternal-conceptus crosstalk during this crucial developmental period. Therefore, the overall goal of this project is to characterize EVs isolated from maternal and embryonic secretions during the initiation of conceptus elongation, and then investigate the effect of crosstalk mediated by different EV populations on the initiation of porcine conceptus elongation using our unique three-dimensional (3D) in vitro culture system. This project will identify EV factors that are critical for porcine conceptus elongation, which could be manipulated to improve reproductive outcomes in the pig and other domestic agricultural species, improving animal production, specifically benefiting the swine industry, and ensuring a long-term sustainable source of high value protein for a global food security. The overall strategy for Objective 1 is to isolate EVs produced by the maternal endometrium and embryo at different time points during the initiation of elongation and characterize the EV proteomes and transcriptomes to elucidate potential mechanisms of cell-to-cell communication for promoting successful conceptus elongation. Since initiation of sample collection for Objective 1, we have collected samples from 114 pregnant gilts between days 9 and 11 of gestation. This includes ULF from all bred gilts, and also includes 1,142 embryos, of which 300 embryos have been cultured with our 3D alginate culture system to obtain embryonic EVs. In addition, we have isolated and cultured UECs from 32 pregnant gilts and collected media to evaluate maternal specific EVs. We have evaluated and determined best methods to store EVs for future analysis, as well as techniques to isolate EVs from flushings, which contain large amounts of contaminating materials. We found the best method for isolation of EVs involves a combination of at least two techniques described by the International Society of Extracellular Vesicles (ISEV). The ExoQuick Ultra Isolation Kit provides a preliminary polymer-based precipitation of EVs from the original sample volume followed by a size exclusion column to obtain a highly concentrated volume of isolated EVs. Nanoparticle tracking analysis (NTA) by the Malvern NanoSight has been performed on isolated EV samples to obtain an average diameter and particle concentration of the EVs. The average diameter of EVs appears to decrease as the day of gestation increases, from 168 nm to 153 nm to 120 nm for gestational days 9, 10, and 11, respectively. The estimated average particle concentration varied between 5.4e11 and 7.8e11 particles/mL across all collection days. Nanoflow Cytometry (nFCM) by the NanoAnalyzer was conducted on EVs derived from ULF on days 8 and 9. The data generated showed a slightly smaller average diameter of EVs compared to the NTA data of around 69 nm and a concentration varying between 3.12e11 and 9.72e10 particles/mL for days 8 and 9, respectively. Transmission electron microscopy (TEM) results provided still images of isolated EVs with diameters closer to those measured by the nFCM, with an average diameter of 108 nm for day 8 ULF EVs and 95 nm for day 9. After running TEM and comparing the results to the nFCM and Nanosight data, we have decided to use the nFCM for all EV analyses in this project. Overall protein analysis via BCA was performed to determine a general concentration of proteins from the isolated EV samples. EVs derived from ULF had an average protein concentration of 200 - 500 µg/mL across days 8, 9, and 10, which is within the range needed to perform proteomic analysis on the isolated EVs. We have begun proteomic analysis on ULF EVs, by first running approximately 9 µg of sample through an SDS-PAGE gel, isolating he gel band and digesting in trypsin followed by peptide extraction and finally analyzing using mass spectroscopy. At this time samples have been sent to the proteomics core for mass spectroscopy and we are awaiting results. We are also currently in the process of preparing samples for transcriptomic analysis by isolating miRNA from the EV samples using the mirVana miRNA Isolation kit. Transcriptomics analysis will be completed by the UNMC Genomics Core. We are also investigating methods to isolate EVs from cultured embryos, given the hydrogel matrix we use to culture the embryos serves as a trap for EVs. The overall strategy for Objective 2 is to investigate the influence of EVs derived from uterine luminal and epithelial cells present in the preimplantation uterine environment on the survival and initiation of conceptus elongation using our 3D culture system. EVs will be isolated from the ULF and UEC culture samples using the ExoQuick Ultra kit. Isolated EVs will be incorporated into the 3D hydrogel culture system during encapsulation of embryo, where initiation of elongation will be measured after 96 hours of culture. We not have yet begun studies for Objective 2.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Pannier AK. In Vitro Culture of Porcine Embryos within an Alginate Hydrogel Matrix. Annual Meeting of the Society for the Study of Reproduction, St. Louis, Missouri, December 18, 2021 (invited).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Pannier AK, Miles JR, Rempel LA, Walsh S, Biegert M. Influence of Maternal and Embryonic Extracellular Vesicles on the Initiation of Porcine Conceptus Elongation. USDA AFRI Animal Reproduction Project Director Meeting, St. Louis, MO Hybrid Meeting, December 14-15, 2021.
|
|