Performing Department
Dean-Veterinary Medicine
Non Technical Summary
Reproductive performance is critical for livestock production systems. The development of a fetus depends on appropriate communication between the placenta and the maternal immune system. Knowledge of how vesicles derived from the placenta modulate the maternal immune system will lead to the identification of potential targets for the prevention of pregnancy losses. The overall objective of this application is to characterize and investigate the effect of placental vesicles collected from healthy pregnancies or abortion-prone pregnancies on fetal development and uterine inflammatory status. The central hypothesis is that the placenta releases vesicles that play an important role in the communication between the fetus and the mother. Additionally, we hypothesize that the maternal uterine environment is modulated differently by vesicles derived from healthy and abortion-prone placentas. We have a unique model that uses pregnancies produced by animal cloning that allows us to study the role of vesicles in pregnancy losses. Our specific aims are to 1) Determine the differences in content of vesicles derived from healthy pregnancies and abortion-prone pregnancies; 2) Determine the effect of uterine infusion of placental vesicles from healthy pregnancies on fetal development and uterine inflammatory status in abortion-prone pregnancies. We expect our results to demonstrate that placental vesicles from healthy pregnancies can regulate the inflammatory response mounted against fetuses produced by animal cloning and promote fetal development. These findings are expected to promote the development of novel approaches to improve reproductive efficiency and lower production costs in livestock.
Animal Health Component
100%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
The overall objective of this application is to characterize extracellular vesicles (EVs)released from the conceptus and investigate the effect of EVs derived from healthy pregnancies or abortion-prone pregnancies on conceptus development and the uterine inflammatory milieu.To address the overall goal, we propose experiments that address two specific aims:1. Determine the differences in the cargo of EVs derived from healthy pregnancies established by artificial insemination (AI)and abortion-prone pregnancies established by somatic cell nuclear transfer (SCNT).2. Determine the effect of intrauterine infusion of trophoblast-derived EVs from healthypregnancies on embryonic development and the endometrial inflammatory milieu inabortion-prone pregnancies.
Project Methods
Specific Aim # 1:Determine the differences in cargo of EVs derived from healthy pregnancies established by AI and abortion-prone pregnancies established by SCNT Rationale and experimental design. We will generate Day 15 and 25 pregnancies established by SCNT and AI (control).Establishment of pregnancies.We plan to do 20 embryo transfers to obtain the necessary number of SCNT pregnancies per collection day (n = 5/group/collection day). Control pregnancies will be established by estrous synchronization followed by AI.Sample collection.On days 15 and 25 of pregnancy, concepti will be flushed from the uteri of cows and chorionic tissue will be separated for tissue digestion and culture.Experiment 1.1. Uterine luminal fluid EV isolation and characterization. Excess medium will be collected into sterile glass jars and used to isolate ULF EVs for experiment 1.1.Experiment 1.2. Trophoblast cell collection, 3D cultures, and EV isolation. A subset of trophoblast tissue will be snap-frozen for later protein analysis. For the 3D culture of Day 15 and 25 trophoblasts, chorionic tissue will be digested and cell aggregates will be cultured in trophoblast organoid conditions. Tissue will cultured, and after 14, 21, and 28 days, culture supernatant will be collected and concentrated, and EVs will be isolated and quantified as described below.Extracellular vesicle isolation. Uterine flush and trophoblast cell culture media will beconcentrated and EVs will be isolated by size exclusion chromatography. The NanoSight NS300 (Malvern Panalytical) will quantify EVs in each fraction.Specific Aim # 2: Determine the effect of intrauterine infusion of trophoblast-derived EVs from healthy pregnancies on embryonic development and the endometrial inflammatory milieu in abortion-prone pregnancies. We hypothesize that trophoblast EVs from healthy pregnancies can restore the normal inflammatory process at the fetal-maternal interface and promote the development of abortion-prone SCNT concepti. We will use trophoblast EVs isolated from 3D trophoblast cell cultures. To determine the effects of intrauterine infusion of trophoblast-derived EVs on embryonic development and the endometrial inflammatory milieu in abortion-prone pregnancies, we will assess conceptus morphology, cell viability and proliferation status, endometrial immune cell transcriptome profiles, and conceptus RNA abundance transcribed from genes related to apoptosis, cell proliferation, trophoblast function, cell growth and metabolism of SCNT pregnancies in response to intrauterine infusions of EVs from AI and SCNT trophoblast cells.Intra-uterine EV infusion. EVs isolated 3D trophoblast cell cultures from SCNT- and AI-derived pregnancies in Specific Aim #1 will be used for intra-uterine infusion. SCNT and AI pregnancies will be established as described above. A pool containing approximately 1 x 10^12 EV particles isolated from the 3D trophoblast culture supernatant of cows at days 15 and 25 of gestation will be infused transcervically into the body of the uterus of SCNT-pregnant cows at gestation days 15, 16, 17, 18, 19, and 20 for the Day 15 group, and at gestation days 25, 26, 27, 28, 29, and 30 for the Day 25 group. These particles will be delivered in 500 μl of PBS and will be derived from culture supernatant from trophoblast 3D cultures from placentas collected from SCNT or AI-established pregnancies at Day 15 (Day 15 group) and 25 (Day 25 group) of gestation. EVs will be loaded into 500 μl semen straws (n = 6/pregnancy day/EV treatment) with the control group receiving sham treatments containing the same volume (500 μl) of PBS present in the EV infusions.Extracellular vesicle uptake by the endometrium. To verify that EVs are being taken up by the endometrium, a subset of extracellular vesicles will be stained with PKH67 (Sigma-Aldrich) according to the manufacturer's instructions and infused into the uterus of two additional cows per treatment group as described above.Sample collection. After EV infusions, pregnant females carrying SCNT-generated concepti will be humanely euthanized on days 22 and 32 of pregnancy. Reproductive tracts will be immediately collected and transported to the laboratory on ice. Concepti and embryonic discs will be measured, and trophoblast and endometrial tissues will be snap frozen for gene expression analyses. Placental and endometrial tissues will also be frozen for later immunohistochemistry analyses. Endometrial tissue will also be collected and digested, and cells will be isolated for single-cell sequencing analysis.Conceptus morphology, cell viability, and cell proliferation. Concepti will be inspected under a stereomicroscope, photographs will be taken, and elongation will be determined by morphological and size changes using ImageJ software. Cell viability will be determined using a Live/Dead staining protocol. The CYQUANT cell proliferation assay will be used to assess cell proliferation as per the manufacturer's instructions.Gene expression profiling of trophoblast cells.To determine how trophoblast-derived EVs change the mRNA expression profile of trophoblast cells in vivo, Eva Green™ high-throughput nanoliter volume microfluidic chip quantitative RT-PCR (48.48 Dynamic Array; Fluidigm) will be used with previously validated primer sets for the following gene categories: growth factor activity, placental development, hypoxia, apoptosis, immune response.Immunohistochemistry. Immunohistochemistry for endometrial leukocytes will be conducted using the following primary monoclonal antibodies: mouse anti-bovine CD4, CD3, CD8, CD25, and γ/δ-TCR. Isotype controls will be used. Stained sections will be analyzed under a microscope, digital images will be acquired and the abundance of endometrial leukocytes will be assessed.Endometrial immune cell isolation. Endometrium from the uterine horn containing the conceptus will be dissected and the tissue will be minced and digested with type I collagenase. Cells in the supernatant suspension will be filtered, and separated, and viable cells will be suspended.Cell preparation and single-cell sequencing. To identify and select the leukocytes in cell preparations, cells will be stained with APC-conjugated CD45 and LIVE/DEAD® Fixable Dead Cell Stain Kit (Invitrogen) and sorted with BD FACSAria cell sorter. The manufacturer's instructions (10x Genomics) will be followed to process samples for single-cell sequencing using a 10x platform at Utah State University. Pairedend RNAseq (125 cycles) will be performed via an Agilent HiSeq next-generation sequencer at the Utah State University genomics core facility. Sequencing reads will be analyzed using 10x Genomics Cell Ranger pipeline and further analysis will be performed with the Seurat R package.