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...

Authors: WALSH, SOHPIE - University Of Nebraska; Miles, Jeremy; BROECKLING, COREY - Colorado State University; Rempel, Lea; Wright, Elane; PANNIER, ANGELA - University Of Nebraska

Submitted to: Journal Of Reproduction, Fertility And Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/24/2023
Publication Date: 2/14/2023
Citation: Walsh, S.C., Miles, J.R., Broeckling, C.D., Rempel, L.A., Wright-Johnson, E.C., Pannier, A.K. 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. Journal of Reproduction, Fertility and Development. 35(5):375-394. https://doi.org/10.1071/RD22210.
DOI: https://doi.org/10.1071/RD22210

Interpretive Summary: In the pig, deficiencies in embryo elongation contribute to embryonic loss and reduced pregnancy outcomes. The exact mechanisms regulating elongation are poorly understood due to the complexity of the uterine environment and difficulty to replicate this process in a laboratory environment. We have previously developed a culture system using alginate hydrogels as a three-dimensional matrix that facilitates the initiation of porcine embryo elongation, enabling real-time study of elongation in a controlled laboratory environment. Using an approach to identify unique compounds, the objective of this study was to examine differences within culture media, and corresponding secretions of individual embryos either initiating or not initiating elongation as well as a function of specific alginate hydrogels (unmodified- or modified- hydrogels). This study identified changes in phospholipid, glycosphingolipid, lipid signaling, and amino acid metabolic processes as potential mechanisms of elongation in unmodified hydrogels and identified changes in lysophosphatidylcholine and sphingolipid secretions during elongation in modified hydrogels. The results of this study illustrate the importance of phospholipids and sphingolipids metabolism on regulating the initiation of embryo elongation in the pig, a critical period for embryo survival.

Technical Abstract: Context. The exact mechanisms regulating the initiation of porcine conceptus elongation are not known due to the complexity of the uterine environment. Aims. To identify contributing factors for initiation of conceptus elongation in vitro, this study evaluated differential metabolite abundance within media following culture of blastocysts within unmodified alginate (ALG) or Arg-Gly-Asp (RGD)-modified alginate hydrogel culture systems. Methods. Blastocysts were harvested from pregnant gilts, encapsulated within ALG or RGD or as non-encapsulated control blastocysts (CONT), and cultured. At the termination of 96h culture, media were separated into blastocyst media groups: non-encapsulated control blastocysts (CONT); ALG & RGD blastocysts with no morphological change (ALG- & RGD-); ALG & RGD blastocysts with morphological changes (ALG+ & RGD+) and evaluated for nontargeted metabolomic profiling by liquid chromatography (LC)–mass spectrometry (MS) techniques and gas chromatography–(GC–MS). Key results. Analysis of variance identified 280 (LC–MS) and 1 (GC–MS) compounds that differed (P < 0.05), of which 134 (LC–MS) and 1 (GC–MS) were annotated. Metabolites abundance between ALG+ vs. ALG-, RGD+ vs. RGD-, and RGD+ vs. ALG+ were further investigated to identify potential differences in metabolic processes during the initiation of elongation. Conclusions. This study identified changes in phospholipid, glycosphingolipid, lipid signaling, and amino acid metabolic processes as potential RGD-independent mechanisms of elongation and identified changes in lysophosphatidylcholine and sphingolipid secretions during RGD-mediated elongation. Implications. These results illustrate changes in phospholipid and sphingolipid metabolic processes and secretions may act as mediators of the RGD-integrin adhesion that promotes porcine conceptus elongation.