|LAUGHLIN, TAYLOR - University Of Nebraska|
|PANNIER, ANGELA - University Of Nebraska|
Submitted to: Society for the Study of Reproduction Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 3/20/2015
Publication Date: 6/2/2015
Citation: Miles, J., Laughlin, T., Wright-Johnson, E., Rempel, L., Pannier, A. 2015. Development of pre-implantation porcine embryos cultured within a three-dimensional alginate hydrogel system either conjugated with Arg-Gly-Asp (RGD) peptide or supplemented with secreted phosphoprotein 1 (SPP1) [abstract]. Society for the Study of Reproduction Annual Meeting. p. 172 (Abstract #432). Available: https://www.ssr.org/
Technical Abstract: Many uterine specific factors have been shown to be increased within the uterine milieu as the porcine embryo initiates elongation. Secreted phosphoprotein 1 (SPP1) is increased during this time and contains an Arg-Gly-Asp (RGD) peptide sequence that has been shown to bind to cell surface integrins on the uterine endometrium and trophectoderm promoting cell adhesion and migration. We have previously developed a three-dimensional culture system using alginate hydrogels that provides mechanical support to maintain embryo architecture and facilitate morphological changes of pre-implantation embryos in vitro. The objective of the current study was to evaluate embryo development of pre-implantation porcine embryos cultured within alginate hydrogels either conjugated with RGD or supplemented with SPP1. White crossbred gilts (n = 32) from nine replicate collections were bred at d 0 and 1 of the estrous cycle. At d 9 of gestation, reproductive tracts were collected and flushed with RPMI-1640 media. Embryos were recovered and washed with RPMI-1640 media. Embryos (n = 256) were randomly assigned to be encapsulated using a standard double encapsulation technique (0.7% sodium alginate and 1.5% calcium chloride solution) or used as non-encapsulated controls. Evaluations of embryo development were made for: 1) the standard hydrogel system; 2) the standard hydrogel system conjugated with 2.3 mg/g RGD peptide; 3) the standard hydrogel system supplemented with 0.1 µg/ml SPP1; and 4) non-encapsulated controls. Embryos were cultured individually within 4-well NUNC plates for 96 h in CO2-pretreated RPMI-1640 with 10% FBS media, 5% CO2 in air and 100% humidity. Every 24 h, half of the media was replaced with fresh media and the embryos were imaged to assess cell survival by blastocyst fragmentation and evaluate morphological changes throughout the culture period. All data were analyzed with GLM procedure for ANOVA, reported as least-squares means, and means were separated using a series of orthogonal contrasts. At termination of culture, embryo survival was greater (P < 0.05) for embryos encapsulated in hydrogels conjugated with RGD (42.2 ± 5.9%) and those supplemented with SPP1 (42.2 ± 5.9%) compared to non-encapsulated controls (21.9 ± 5.9%). In addition, survival of embryos encapsulated in the standard hydrogels (35.9 ± 5.9%) tended (P = 0.10) to be greater compared to non-encapsulated controls. Throughout the culture period, all non-encapsulated control embryos remained spherical with no significant morphological change. In contrast, a significant (P < 0.01) proportion of embryos encapsulated in the standard hydrogels (10.9 ± 3.9%), in hydrogels conjugated with RDG (21.9 ± 3.9%), or supplemented with SPP1 (14.1 ± 3.9%) had morphological changes compared to non-encapsulated controls. These changes were characterized by minor to moderate flattening and tubal formation of the embryo through the gel. Furthermore, the proportion of embryos undergoing morphological changes was greater (P = 0.05) for those encapsulated in hydrogels conjugated with RGD compared to the standard hydrogels. These results demonstrate that embryos encapsulated in alginate hydrogels conjugated with RGD or supplemented with SPP1 develop similarly to those in our standard alginate hydrogel system. Furthermore, RGD conjugation within the hydrogel improved embryo development in terms of proportion of embryos undergoing morphological changes.