Location: Livestock Bio-Systems
Project Number: 3040-31000-095-018-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 29, 2021
End Date: May 31, 2024
The overall goal of this proposal is to identify and characterize maternal and embryonic derived extracellular vesicles (EVs) during the initiation of porcine conceptus elongation and then investigate the influences of these maternal and embryonic derived EVs on the regulation of the initiation of conceptus elongation using our unique three-dimensional (3D) in vitro culture system. To determine the influence of maternal and embryonic derived EVs on the initiation of conceptus elongation, we will focus on two complementary, yet independent objectives. Objective 1) isolate and characterize EVs produced and secreted from maternal and embryonic components during the initiation of porcine conceptus elongation. Objective 2) determine the effects of EVs isolated from the uterine environment and cultured maternal epithelial cells on the survival and initiation of conceptus elongation of embryos during in vitro culture within 3D hydrogel culture system.
The overall strategy for Objective 1 is to isolate and characterize maternal- and embryonic-derived EVs during the initiation of conceptus elongation (between gestational day 9-11) including: 1) Uterine luminal fluids (ULFs) at day 9, 10, and 11 of gestation (Subobjective 1a); in vitro culture media of porcine uterine epithelial cells (UECs) isolated from pregnant gilts at day 9, 10, and 11 of gestation (Subobjective 1b); and media following culture of spherical porcine collected at day 9 of gestation and cultured within our 3D hydrogel culture system (Subobjective 1c). EVs form these components will be assessed for morphology, size distribution, concentration, proteome, and transcriptome (mRNA and miRNA). The overall strategy for Objective 2 is to investigate the effects of EV isolated from maternal uterine environment and specific maternal cell types on the survival and initiation of elongation of embryos cultured with in our 3D hydrogel system including 1) EVs isolated from ULF at day 9, 10, and 11 of gestation (Subobjective 2a) and 2) EVs isolated from cultured media of UECs collected at day 9, 10, and 11 of gestation (Subobjective 2b). These EVs will be incorporated into the 3D hydrogel system and embryo development will be assessed by morphological changes, gene expression markers, and molecular secretion of encapsulated embryos following culture.