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 96 h. Every 24 h, 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. 17b-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse 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% (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 (ENCþ) compared with CONT embryos and ENC embryos with no morphological changes (ENCÀ), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P , 0.001) in 17b-oestradiol was observed in culture media from ENCþ compared with ENCÀ 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.
During embryonic and postnatal skeletal development, the shape and length of bones is determined by the activities of the growth plate cartilage. Growth plate cartilage promotes elongation of long bones via regulation of chondrocyte matura tion that is reflected in morphologically and functionally unique cellular domains. 1 Toward the distal (epiphyseal) end resides the resting (or reserve) zone that is composed of the least mature chondrocytes. 2 These resting zone chondrocytes are progres sively recruited into the proliferative zone, where cell cycle ac tivation and changes in cell morphology and cell organization result in expansion of isogenic columns of chondrocytes along the axis of growth. 3 As columns lengthen, chondrocytes at the proximal (meta physeal) end of the column withdraw from the cell cycle and increase in volume (hypertrophy) in two steps that are char acteristic of the prehypertrophic and the hypertrophic chon drocytes. 4,5 Growth is generated from chondrocyte hypertrophy through increased cell mass and deposition of specialized ma trix. 6 Hypertrophic chondrocytes are subsequently replaced by bone through the process of endochondral ossification. 7 Thus, continuous growth over the twodecade span of human devel opment requires tight coordination between cell production in the resting and proliferative zones, and cartilage loss in the hy pertrophic zone. Chondrocyte maturation in growth plate cartilage is coordi nated by a complex paracrine signaling network that is rooted AbstractDefining the final size and geometry of engineered tissues through precise control of the scalar and vector components of tissue growth is a necessary benchmark for regenerative medicine, but it has proved to be a significant challenge for tissue engineers. The growth plate cartilage that promotes elongation of the long bones is a good model system for studying morphogenetic mechanisms because cartilage is composed of a single cell type, the chondrocyte; chondrocytes are readily maintained in culture; and growth trajectory is predominately in a single vector. In this cartilage, growth is generated via a differentiation program that is spatially and temporally regulated by an interconnected network composed of long and shortrange signaling mechanisms that together result in the formation of functionally distinct cellular zones. To facilitate investigation of the mechanisms underlying anisotropic growth, we developed an in vitro model of the growth plate cartilage by using neonatal mouse growth plate chondrocytes encapsulated in alginate hydrogel beads. In bead cultures, encapsulated chondrocytes showed high viability, cartilage matrix deposition, low levels of chondrocyte hypertrophy, and a progressive increase in cell proliferation over 7 days in culture. Exogenous factors were used to test functionality of the parathyroidrelated protein-Indian hedgehog (PTHrPIHH) signaling interaction, which is a crucial feedback loop for regulation of growth. Consistent with in vivo observations, exogenous PTHrP stimulated cell pr...
Appropriate embryonic and fetal development significantly impact pregnancy success and, therefore, the efficiency of swine production. The pre-implantation period of porcine pregnancy is characterized by several developmental hallmarks, which are initiated by the dramatic morphological change that occurs as pig blastocysts elongate from spherical to filamentous blastocysts. Deficiencies in blastocyst elongation contribute to approximately 20% of embryonic loss, and have a direct influence on within-litter birth weight variation.Although factors identified within the uterine environment may play a role in blastocyst elongation, little is known about the exact mechanisms by which porcine (or other species') blastocysts initiate and progress through the elongation process. This is partly due to the difficulty of replicating elongation in vitro, which would allow for its study in a controlled environment and in real-time. We developed a three dimensional (3-D) culture system using alginate hydrogel matrices that can encapsulate pig blastocysts, maintain viability and blastocyst architecture, and facilitate reproducible morphological changes with corresponding expression of steroidogenic enzyme transcripts and estrogen production, consistent with the initiation of elongation in vivo. This review highlights key aspects of the pre-implantation period of porcine pregnancy and the difficulty of studying blastocyst elongation in vivo or by using in vitro systems. This review also provides insights on the utility of 3-D hydrogels to study blastocyst elongation continuously and in real-time as a complementary and confirmatory approach to in vivo analysis.alginate hydrogels, blastocyst elongation, in vitro culture models, porcine, tissue engineering Abbreviations: 2/3D, two/three dimensional; CONT, non-encapsulated control blastocyst; CYP11A1, CytochromeP450 side chain cleavage; CPY19A1, aromatase; ECM, extracellular matrix; ENC[±], blastocysts encapsulated in alginate hydrogels [with or without morphological change]; FGF7, Fibroblast growth factor 7; G, α-L-guluronic acid; IL1B, Interleukin1β; M, β-D-mannuronic acid; RGD, Arg-Gly-Asp; SPP1, Secreted phosphoprotein 1; STAR, Steroidogenic acute regulatory protein; TGFB, Transforming growth factor-β.Mention of trade names is necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the same by USDA implies no approval of the product to the exclusion of others that may also be suitable.The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual's income is derived from any public assistance program.
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