Utilization of a 3D hydrogel culture system to study reproductive process of conceptus elongation in vitro

Authors: WALSH, SOPHIE - University Of Nebraska; Miles, Jeremy; Wright, Elane; Keel, Brittney; Rempel, Lea; PANNIER, ANGELA - University Of Nebraska

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 8/18/2020
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Introduction: Three-dimensional (3D) in vitro culture systems are a vital tool for studying complex reproductive processes in a controlled, biomimetic environment. During early porcine conceptus development, embryos undergo the complex process of elongation as they rapidly transition from spherical to filamentous morphology. Successful elongation is necessary for the maintenance of pregnancy and proper subsequent conceptus development and survival. Understanding the expression of critical genes by embryos successfully initiating elongation can help identify essential pathways and processes that can be used to improve reproductive outcomes. Previously, our group engineered a 3D alginate hydrogel culture system to study porcine conceptus elongation, which was able to initiate morphological change of encapsulated embryos.1,2 The objective of the current study was to facilitate the initiation of porcine embryo elongation by encapsulation within our 3D hydrogel in vitro culture system, with or without covalent attachment of the GRGDS adhesion peptide (RGD) to the alginate scaffold, determine embryo survival, and isolate embryo RNA. Isolated RNA from elongating and non-elongating embryos can then be used to identify genes and pathways essential to this important developmental process. Materials and Methods: White crossbred gilts (n = 15) were bred at estrus and harvested at day 9 of gestation to recover blastocysts of spherical morphology (n = 287 total embryos), which were randomly assigned to be encapsulated in the 3D culture system within an alginate (ALG) matrix, RGD-alginate (RGD) matrix, or to a control, non-encapsulated group (CONT). Embryos were double encapsulated, as described previously.1,2 Briefly, a single embryo was placed into droplets of 0.7% (w/v) alginate or RGD-alginate solution, and each droplet was pipetted into crosslinking solution (50 mM CaCl2, 140 mM NaCl) for gelation. The resulting encapsulated embryo beads were rinsed, then encapsulated in a second layer of alginate using identical methods as the initial encapsulation. Individual ALG (n = 107), RGD (n = 125), and CONT (n = 55) embryos were cultured for 96 h, at which time images were taken to assess embryo survival and morphological changes. Surviving embryos were categorized as CONT embryos (with no morphological change), ALG and RGD embryos that failed to initiate morphological changes (ALG- and RGD-), and that initiated morphological change (ALG+ and RGD+), and stored at -80°C for transcriptomic evaluation. Survival and morphological data from these embryos were analyzed across treatments using Chi Square Analysis and PROC GLIMMIX. RNA was extracted from a subset of embryos and quality and quantity assessed from individual embryos across all treatments (n = 45 total embryos), which will be subsequently sequenced to identify differentially expressed genes (DEG) and associated enriched pathways. Results and Discussion: Encapsulated embryos had higher survival rates (P = 0.002) in both the ALG (52.3 ± 4.8%) and RGD (41.6 ± 4.4%) treatments than the non-encapsulated CONT (23.6 ± 5.7%). Encapsulation of embryos within the alginate systems resulted in higher rates (P = 0.03) of morphological change for both ALG (37.5 ± 6.5%) and RGD (34.6 ± 6.6%) surviving embryos, as compared to no morphological changes in any non-encapsulated CONT (0%), demonstrating successful initiation of embryo elongation within the 3D culture system. Both the quantity and quality of RNA extracted from cultured embryos were not statistically different across all treatment groups. RNA-Seq will be performed from 45 libraries constructed from single in vivo-produced embryos from the CONT, ALG-, ALG+, RGD-, and RGD+ embryos (n = 9 per treatment), and DEG and pathway analysis will be evaluated. Conclusions: Overall, these data demonstrate the ability of our 3D hydrogel culture system to promo