Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Biomedical Sciences
Non Technical Summary
The oviduct provides the optimal microenvironment for gamete maturation, transport, and guidance of spermatozoa towards the oocyte leading to successful fertilization, and supporting early embryo development. Oviductal extracellular vesicles (EVs) have been identified as major components of the oviductal fluid and potential mediators of oocyte/embryo interactions in the course of a successful pregnancy.Organoids recapitulate key physiological aspects of the organ of origin and have become an alternative in vitro model that closely mirrors normal physiology, therefore EVs harvested from organoids should be more similar to those found in vivo.Therefore, in the present project we plan to use the 3D oviductal organoid culture system to 1) study EV mediated response of organoids to thermal stress and 2) investigate the potential of organoid-derived EVs in inducing adaptive responses to bovine preimplantation embryos against recurrent thermal stress in vitro.
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Goals / Objectives
The goalof the present project is to use the 3D oviductal organoid culture system to 1) study the extracellular vesicle-mediated response of organoids to thermal stress, and 2) investigate the potential of organoid-derived extracellular vesicle in inducing adaptive responses to bovine preimplantation embryos against recurrent thermal stress in vitro.
Project Methods
In specific aim 1 of the project, a bovine oviductal organoid culture system will be performed using an established protocol.Briefly, the oviductal lumen will be exposed and scraped with a scalpel blade in Handling Medium (HM; MEM with Earle's salts, 25 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.1 mM pyruvate, 2 mM Glutamax, 5% Bovine Serum Albumin; BSA) to release epithelial glandular tissue.The solution containing the glands will be passed through a 40 mm cell strainer to remove the remaining stromal and inflammatory cells. The glands will be collected in the cell strainer and then backwashed with RPMI-1640. The solution will be centrifuged, the pellet washed and suspended in 20X volume/volume Matrigel. The Matrigel solution will be plated in a 48-well plate and a culture medium will be added. The cultures will be kept in an incubator at 37°C and 5% CO2 for growth prior to elevated temperature exposure.After exposure of half of the plates to an elevated temperature at (42 degrees) for 24 hrs, spent culture media and organoids will be collected. The spent media will be used to isolate extracellular vesicles using ultracentrifugation. The isolated Evs will be morphologically and molecularly characterized using Nano Tracking Analysis, western blotting for marker proteins (ALIX, CD63, and TSG101), and transmission electron microscopy. EVs isolated from spent media of organoids exposed to normal or elevated temperature will be subjected for miRNA isolation and subsequent large-scale miRNA sequencing will be performed using the service provided by Novogen company. After receiving the raw FASTQ data bioinformatic analysis will be performed to identify differentially expressed miRNA and pathways analysis will also be done for candidate miRNAs.Finally, EVs derived from organoids culture cultured either at normal or elevated temperature will be fluorescently labeled and supplemented into the culture media of in IVP embryos at zygote, 8-16 cell, and morula stage embryos. For this bovine oocytes aspirated from ovaries collected from local slaughterhousewill be used in IVM,IVF, and IVC until day 7 blastocyst stage. Following supplementation, the effect on cleavage and blastocyst rate will be documented. Moreover, the resulting blastocysts will be investigated for total cell count, apoptosis level and expression of genes related to heat stress (HSP70 & 90), oxidative stress response (NRF2, SOD1, CAT1), and endoplasmic reticulum stress (GRP78, GRP94). The expression of genes in the blastocysts will be performed using real-time quantitative PCR using GAPDH and B-Actin as endogenous controls.