Identifying Uterine Factors that Drive Development
Between days 9-12 of gestation in the pig, the embryo transforms from spherical morphology (~1-2 mm) to a long, thin filament (> 100 mm) in a process referred to as elongation. Deficiencies in elongation contribute to approximately 20% of embryonic loss, but physiological mechanisms that regulate elongation remain poorly understood. An effective in vitro culture system that can support pig embryo elongation is necessary for evaluating these specific mechanisms of elongation and understanding how these factors are altered during embryonic loss. We hypothesize that previous attempts at achieving embryo elongation in vitro have failed due to, in part, the use of inadequate culture systems that lack the appropriate three-dimensional (3D) structure for maintenance of proper embryo architecture during elongation.
Therefore, we have developed a culture system employing alginate hydrogels as a 3D matrix that can facilitate embryonic morphological changes with corresponding increases in estrogen production and steroidogenic transcript expression, in a similar pattern as in vivo elongation. Our current project objective is to use this system as an in vitro tool for evaluating uterine factors that promote elongation. Knowledge of these mechanisms that regulate elongation can then be used to develop strategies to improve pregnancy outcomes in the pig and potentially other domestic agricultural species.
Transcriptomics and Metabolomics of Developing Embryos
Analysis of global changes in gene expression and metabolic abundance may reveal important mechanisms driving the initiation of porcine embryo elongation. On days 9-11 of gestation, embryos were flushed from pregnant gilts and categorized into stages of elongation based on embryo morphology and length. Uterine flushings of litters containing embryos of the respective elongation stages were submitted for nontargeted profiling by gas chromatography–mass spectrometry (GC–MS) and ultra performance liquid chromatography (UPLC)–MS techniques to compare changes in metabolite content within the uterine milieu as elongation progresses. Embryos of the respective elongation stages were evaluated using RNA-Sequencing (RNA-Seq) to identify changes in global gene expression as the embryo transitions through the initial stages of elongation.
Additionally, spherical embryos harvested from pregnant gilts on day 9 of gestation were encapsulated in our 3D hydrogel system and cultured in vitro for 96 hours. GC-MS and UPLC-MS were utilized to evaluate differences in metabolite content of culture media derived from embryos that did and did not elongate during in vitro culture. RNA-Seq was used to evaluate global gene expression differences between these elongating and non-elongating embryos.
Characterization and Effect of Extracellular Vesicles on Elongation
Another aspect that remains poorly understood during the initiation of porcine embryo elongation is the influence of extracellular vesicles (EVs) present in the preimplantation uterine environment as mediators of maternal-conceptus crosstalk during this crucial developmental period. 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) hydrogel culture system.