RESUMEN
Insight into the normal and abnormal function of an interphase nucleus can be revealed by using fluorescence in situ hybridization (FISH) to determine chromosome copy number and/or the nuclear position of loci or chromosome territories. FISH has been used extensively in studies of mouse and human early embryos, however, translation of such methods to domestic species have been hindered by the presence of high levels of intracytoplasmic lipid in these embryos which can impede the efficiency of FISH. This chapter describes in detail a FISH protocol for overcoming this problem. Following extensive technical development, the protocol was derived and optimized for IVF porcine embryos to enable investigation of whole chromosome and subchromosomal regions by FISH during these early stages of development. Porcine embryos can be generated in-vitro using semen samples from commercial companies and oocytes retrieved from discarded abattoir material. According to our method, porcine embryos are lyzed and immobilized on slides using Hydrochloric acid and "Tween 20" detergent, prior to pretreatment with RNase A and pepsin before FISH. The method described has been optimized for subsequent analysis of FISH in two dimensions since organic solvents, which are necessary to remove the lipid, have the effect of flattening the nuclear structure. The work in this chapter has focussed on the pig; however, such methods could be applied to bovine, ovine, and canine embryos, all of which are rich in lipid.
Asunto(s)
Embrión de Mamíferos/metabolismo , Hibridación Fluorescente in Situ/métodos , Animales , Sondas de ADN/metabolismo , Embrión de Mamíferos/citología , PorcinosRESUMEN
Skeletal stem and progenitor populations provide a platform for cell-based tissue regeneration strategies. Optimized conditions for ex vivo expansion will be critical and use of serum-free culture may allow enhanced modelling of differentiation potential. Maintenance of human foetal femur-derived cells in a chemically defined medium (CDM) with activin A and fibroblast growth factor-2 generated a unique undifferentiated cell population in comparison to basal cultures, with significantly reduced amino acid depletion, appearance and turnover, reduced alkaline phosphatase (ALP) activity and loss of type I and II collagen expression demonstrated by fluorescence immunocytochemistry. Microarray analysis demonstrated up-regulation of CLU, OSR2, POSTN and RABGAP1 and down-regulation of differentiation-associated genes CRYAB, CSRP1, EPAS1, GREM1, MT1X and SRGN as validated by quantitative real-time polymerase chain reaction. Application of osteogenic conditions to CDM cultures demonstrated partial rescue of ALP activity. In contrast, the addition of bone morphogenetic protein-2 (BMP-2) resulted in reduced ALP levels, increased amino acid metabolism and, strikingly, a marked shift to a cobblestone-like cellular morphology, with expression of SOX-2 and SOX-9 but not STRO-1 as shown by immunocytochemistry, and significantly altered expression of metabolic genes (GFPT2, SC4MOL and SQLE), genes involved in morphogenesis (SOX15 and WIF1) and differentiation potential (C1orf19, CHSY-2,DUSP6, HMGCS1 and PPL). These studies demonstrate the use of an intermediary foetal cellular model for differentiation studies in chemically defined conditions and indicate the in vitro reconstruction of the mesenchymal condensation phenotype in the presence of BMP-2, with implications therein for rescue studies, screening assays and skeletal regeneration research.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Medio de Cultivo Libre de Suero , Activinas/metabolismo , Supervivencia Celular , Medio de Cultivo Libre de Suero/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Edad Gestacional , Humanos , Inmunohistoquímica/métodos , Osteogénesis , Fenotipo , Embarazo , Regeneración , Factores de TiempoRESUMEN
BACKGROUND: Cryopreservation of supernumerary embryos is routinely performed in human-assisted reproduction, providing a source of embryos which can be thawed for use in subsequent treatment cycles. However, the viability of cryopreserved embryos has traditionally relied on morphological assessment, which is a poor predictor of embryo health since freezing leads to a significant overall reduction in implantation potential, and its long-term efficacy is unknown. This study describes how the post-thaw metabolism of human embryos can be used to predict future development to the blastocyst stage. METHODS: HPLC was used to analyse the post-thaw amino acid metabolism of human embryos from day 2 to day 3 of development. RESULTS: It was possible to predict with 87% accuracy which frozen-thawed embryo would develop to the blastocyst stage. Developmentally competent embryos were more metabolically quiescent than their arresting counterparts. Amino acid turnover was also capable of distinguishing between the developmental potential of the best, Grade I embryos P < 0.05. CONCLUSIONS: The data suggests that cryopreservation in IVF is a safe procedure and that amino acid turnover can be used to select which cryopreserved embryo will develop to the blastocyst stage, irrespective of their post-thaw grade.