title: In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix.
abstract: Between Days 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in oestrogen production that triggers maternal recognition of pregnancy. Elongation deficiencies contribute to 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 use alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of preimplantation porcine embryos. White cross-bred gilts were bred at oestrus (Day 0) to Duroc boars and embryos were recovered on Days 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 96h. Every 24h, half the medium was replaced with fresh medium and an image of each embryo was recorded. At the termination of culture, embryo images were used to assess morphological changes and cell survival. 17β-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse steroidogenic transcript expression at 96h 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% (P<0.001) of the surviving ENC embryos underwent morphological changes characterised by tubal formation with subsequent flattening, whereas none of the CONT embryos exhibited morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P<0.07) in ENC embryos with morphological changes (ENC+) compared with CONT embryos and ENC embryos with no morphological changes (ENC-), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P<0.001) in 17β-oestradiol was observed in culture media from ENC+ compared with ENC- and CONT embryos. These results illustrate that preimplantation pig embryos encapsulated in alginate hydrogels can undergo morphological changes with increased expression of steroidogenic transcripts and oestrogen 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.
authors.affiliation: Department of Biological Systems Engineering, University of Nebraska-Lincoln, PO Box 830726, Lincoln, NE 68583, USA.
authors.forename: Catherine N
authors.initials: CN
authors.lastname: Sargus-Patino
authors.affiliation: USDA-ARS US Meat Animal Research Center (USMARC), PO Box 166, Clay Center, NE 68933, USA.
authors.forename: Elane C
authors.initials: EC
authors.lastname: Wright
authors.affiliation: Department of Biological Systems Engineering, University of Nebraska-Lincoln, PO Box 830726, Lincoln, NE 68583, USA.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Plautz
authors.affiliation: USDA-ARS US Meat Animal Research Center (USMARC), PO Box 166, Clay Center, NE 68933, USA.
authors.forename: Jeremy R
authors.initials: JR
authors.lastname: Miles
authors.affiliation: USDA-ARS US Meat Animal Research Center (USMARC), PO Box 166, Clay Center, NE 68933, USA.
authors.forename: Jeff L
authors.initials: JL
authors.lastname: Vallet
authors.affiliation: Department of Biological Systems Engineering, University of Nebraska-Lincoln, PO Box 830726, Lincoln, NE 68583, USA.
authors.forename: Angela K
authors.initials: AK
authors.lastname: Pannier
mesh_terms: D000464:Alginates; D000818:Animals; D001141:Aromatase / Q000235:genetics; D001755:Blastocyst / Q000502:physiology*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics; D003470:Culture Media; D017077:Culture Media, Conditioned / Q000737:chemistry; D046149:Embryo Culture Techniques / Q000379:methods / Q000662:veterinary*; D047108:Embryonic Development / Q000502:physiology*; D004958:Estradiol / Q000032:analysis; D005260:Female; D015870:Gene Expression; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D020100:Hydrogels; D008297:Male; D010750:Phosphoproteins / Q000235:genetics; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D034421:Sus scrofa / Q000196:embryology*; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D000464:Alginates; D003470:Culture Media; D017077:Culture Media, Conditioned; D006603:Hexuronic Acids; D020100:Hydrogels; D010750:Phosphoproteins; D012333:RNA, Messenger; D000098682:Steroidogenic Acute Regulatory Protein; D004958:Estradiol; D020723:Glucuronic Acid; D001141:Aromatase; D002786:Cholesterol Side-Chain Cleavage Enzyme