RESUMEN
A highly accurate 'non-invasive quantitative embryo assessment for pregnancy' (NQEAP) technique that determines embryo quality has been an elusive goal. If developed, NQEAP would transform the selection of embryos from both Multiple Ovulation and Embryo Transfer (MOET), and even more so, in vitro produced (IVP) embryos for livestock breeding. The area where this concept is already having impact is in the field of clinical embryology, where great strides have been taken in the application of morphokinetics and artificial intelligence (AI); while both are already in practice, rigorous and robust evidence of efficacy is still required. Even the translation of advances in the qualitative scoring of human IVF embryos have yet to be translated to the livestock IVP industry, which remains dependent on the MOET-standardised 3-point scoring system. Furthermore, there are new ways to interrogate the biochemistry of individual embryonic cells by using new, light-based methodologies, such as FLIM and hyperspectral microscopy. Combinations of these technologies, in particular combining new imaging systems with AI, will lead to very accurate NQEAP predictive tools, improving embryo selection and recipient pregnancy success.
Asunto(s)
Técnicas de Cultivo de Embriones , Embrión de Mamíferos/fisiología , Desarrollo Embrionario/fisiología , Ganado/embriología , Imagen Óptica/veterinaria , Animales , Humanos , Mamíferos , Imagen Óptica/métodosRESUMEN
Haemoglobin expression is not restricted to erythroid cells. We investigated the gene expression of the haemoglobin subunits haemoglobin, alpha adult chain 1 (Hba-a1) and haemoglobin, beta (Hbb), 2,3-bisphosphoglycerate mutase (Bpgm) and the oxygen-regulated genes BCL2/adenovirus E1B interacting protein 3 (Bnip3), solute carrier family 2 (facilitated glucose transporter), member 1 (Slc2a1) and N-myc downstream regulated gene 1 (Ndrg1) in the murine preimplantation embryo, comparing invivo to invitro gene expression. Relatively high levels of Hba-a1 and Hbb were expressed invivo from the 2-cell to blastocyst stage; in contrast, little or no expression occurred invitro. We hypothesised that the presence of haemoglobin invivo creates a low oxygen environment to induce oxygen-regulated gene expression, supported by high expression of Slc2a1 and Ndrg1 in invivo relative to invitro embryos. In addition, analysis of an invitro-derived human embryo gene expression public dataset revealed low expression of haemoglobin subunit alpha (HBA) and HBB, and high expression of BPGM. To explore whether there was a developmental stage-specific effect of haemoglobin, we added exogenous haemoglobin either up to the 4-cell stage or throughout development to the blastocyst stage, but observed no difference in blastocyst rate or the inner cell mass to trophectoderm cell ratio. We conclude that haemoglobin in the invivo preimplantation embryo raises an interesting premise of potential mechanisms for oxygen regulation, which may influence oxygen-regulated gene expression.
Asunto(s)
Blastocisto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hemoglobinas/metabolismo , Oxígeno/metabolismo , Animales , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Femenino , Hemoglobinas/genética , RatonesRESUMEN
We have constructed reading frames for the HIV-1 YU-2 minor proteins Vpr, Vpu, Vif and Nef that are codon-optimized for high-level expression in mammalian cells. We show that, in the absence of the Rev/Rev-response element system, these codon-optimized reading frames result in greatly increased levels of expression of the corresponding proteins in cell culture systems when compared with the native reading frame. Northern blot analysis shows that the increase in expression found with the codon-optimized reading frames is largely owing to increased steady-state mRNA levels.