In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix

Authors: SARGUS-PATINO, CATHERINE - University Of Nebraska; Wright, Elane; PLAUTZ, SARAH - University Of Nebraska; Miles, Jeremy; Vallet, Jeff; PANNIER, ANGELA - University Of Nebraska

Submitted to: Reproduction, Fertility and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/12/2013
Publication Date: 8/1/2014
Publication URL: http://handle.nal.usda.gov/10113/59651
Citation: Sargus-Patino, C.N., Wright, E.C., Plautz, S.A., Miles, J.R., Vallet, J.L., Pannier, A.K. 2014. In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix. Reproduction, Fertility and Development. 26(7):943-953.

Interpretive Summary: Between day 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, which is critical for recognition of pregnancy. Elongation deficiencies contribute to ~20% of embryonic loss, but exact mechanisms of elongation are poorly understood. Knowledge of these mechanisms could lead to the identification of factors that could be targeted to improve pregnancy outcomes. We have established a culture system using alginate hydrogel as a three-dimensional matrix that facilitates transformations in porcine embryo morphology, providing an in vitro model to study mechanisms of elongation. Day 9 embryos were assigned to an alginate hydrogel system or a control, non-encapsulated group and were cultured for five days. Daily monitoring revealed that 32% of the encapsulated embryos that survived throughout culture transformed morphologically, while control embryos had no morphological changes. Furthermore, encapsulated embryos that exhibited morphological changes had greater expression of steroidogenic transcripts compared to non-encapsulated controls. Additionally, a significant time-dependent increase in estradiol in the culture media of encapsulated embryos was identified compared with controls and culture media alone. These results illustrate that porcine embryos cultured in alginate hydrogels can undergo morphological changes with increased estrogen production and differential gene expression, consistent with in vivo elongation.

Technical Abstract: Between day 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in estrogen production for maternal recognition of pregnancy. Elongation deficiencies contribute to ~20% of embryonic loss, but exact mechanisms of elongation are poorly understood due to the lack of an effective in vitro culture system. Our objective was to utilize alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of pre-implantation porcine embryos. White crossbred gilts were bred at estrus (d 0) to Duroc boars and embryos were recovered at d -9, -10, or -11 of gestation. Spherical embryos were randomly assigned to be encapsulated within double-layered 0.7% alginate beads or remain as non-encapsulated controls (ENC and CONT treatment groups, respectively) and were cultured for 96 h. Every 24 h, embryos were imaged to assess morphological changes and cell survival and half of the media was replaced with fresh media. Estradiol-17ß levels were measured in the removed media by radioimmunoassay. Real-time PCR was used to analyze steroidogenic transcript expression at 96 h in ENC and CONT embryos as well as in vivo-developed control embryos (i.e., spherical, ovoid and tubular). Although no differences in cell survival were observed, 32% of the surviving ENC embryos underwent morphological changes characterized by tubal formation with subsequent flattening, while none of the CONT embryos had morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P < 0.07) in ENC embryos with morphological changes (ENC+) compared to CONT embryos and ENC embryos with no morphological changes (ENC-), with more similar expression to later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P < 0.001) in estradiol-17ß was observed in culture media with ENC+ embryos compared to ENC- and CONT groups. These results illustrate that pre-implantation pig embryos encapsulated in alginate hydrogels can undergo morphological changes with increased expression of steroidogenic transcripts and estrogen production consistent with in vivo-developed embryos. This alginate culture system can serve as a tool for evaluating specific mechanisms of embryo elongation that could be targeted to improve pregnancy outcomes.