Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo

In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2-/- embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7-21% form blastocysts compared to 76.2-83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages

Improving metabolic health in obese male mice via diet and exercise restores embryo development and fetal growth

Paternal obesity is now clearly associated with or causal of impaired embryo and fetal development and reduced pregnancy rates in humans and rodents. This appears to be a result of reduced blastocyst potential. Whether these adverse embryo and fetal outcomes can be ameliorated by interventions to reduce paternal obesity has not been established. Here, male mice fed a high fat diet (HFD) to induce obesity were used, to determine if early embryo and fetal development is improved by interventions of diet (CD) and/or exercise to reduce adiposity and improve metabolism. Exercise and to a lesser extent CD in obese males improved embryo development rates, with increased cell to cell contacts in the compacting embryo measured by E-cadherin in exercise interventions and subsequently, increased blastocyst trophectoderm (TE), inner cell mass (ICM) and epiblast cell numbers. Implantation rates and fetal development from resulting blastocysts were also improved by exercise in obese males. Additionally, all interventions to obese males increased fetal weight, with CD alone and exercise alone, also increasing fetal crown-rump length. Measures of embryo and fetal development correlated with paternal measures of glycaemia, insulin action and serum lipids regardless of paternal adiposity or intervention, suggesting a link between paternal metabolic health and subsequent embryo and fetal development. This is the first study to show that improvements to metabolic health of obese males through diet and exercise can improve embryo and fetal development, suggesting such interventions are likely to improve offspring health.Nicole O. McPherson, Hassan W. Bakos, Julie A. Owens, Brian P. Setchell, Michelle Lan

Multi-objective design under uncertainties of hybrid renewable energy system using NSGA-II and chance constrained programming

The optimum design of Hybrid Renewable Energy Systems (HRES) depends on different economical, environmental and performance related criteria which are often conflicting objectives. The Non-dominated Sorting Genetic Algorithm (NSGA-II) provides a decision support mechanism in solving multi-objective problems and providing a set of non-dominated solutions where finding an absolute optimum solution is not possible. The present study uses NSGA-II algorithm in the design of a standalone HRES comprising wind turbine, PV panel and battery bank with the (economic) objective of minimum system total cost and (performance) objective of maximum reliability. To address the uncertainties in renewable resources (wind speed and solar irradiance), an innovative method is proposed which is based on Chance Constrained Programming (CCP). A case study is used to validate the proposed method, where the results obtained are compared with the conventional method of incorporating uncertainties using Monte Carlo simulation

The clinically active PARP inhibitor AG014699 ameliorates cardiotoxicity but does not enhance the efficacy of doxorubicin, despite improving tumor perfusion and radiation response in mice

AG014699 was the first inhibitor of the DNA-repair enzyme Poly(ADP-ribose) polymerase-1 (PARP-1) to enter clinical trial in cancer patients. In addition to enhancing the cytotoxic effect of DNA-damaging chemotherapies, we have previously shown that AG014699 is vasoactive, thereby having the potential to improve drug biodistribution. The effectiveness of the clinical agent doxorubicin is confounded both by poor tumour penetration and cardiotoxicity elicited via PARP hyperactivation. Here we analysed the impact of AG014699 on doxorubicin tolerance and response in breast (MDA-MB-231) and colorectal (SW620, LoVo) tumour models in vitro and in vivo. As anticipated, AG014699 did not potentiate response to doxorubicin in vitro. In vivo, AG014699 did not influence the pharmacokinetics of doxorubicin, but did ameliorate cardiotoxicity. Both toxicity and extent of amelioration were more pronounced in male versus female mice. AG014699 improved vessel perfusion in both MDA-MB-231 and SW620 tumours yet this did not lead to improved tumour-accumulation of doxorubicin nor enhanced therapeutic response. In contrast, when combined with radiotherapy, AG014699 significantly enhanced response both in vitro and in vivo. Real-time assessment of tumour vessel function and companion histological studies suggest that doxorubicin causes a profound anti-vascular effect that countered the positive effect of AG014699 on perfusion. These data suggest that although AG014699 can enhance response to some chemotherapies via improved delivery, this does not apply to doxorubicin. PARP inhibitors may still be of use to counter doxorubicin toxicity and if the gender effect translates from rodents to humans, this would have greater effect in males

Vasoactivity of AG014699, a clinically active small molecule inhibitor of poly(ADP-ribose) polymerase: a contributory factor to chemopotentiation in vivo?

PURPOSE: Poly(ADP-ribose) polymerase plays an important role in DNA repair and PARP inhibitors can enhance the activity of DNA damaging agents in vitro and in vivo. AG014699 is a potent PARP inhibitor in Phase II clinical development. However the range of therapeutics with which AG014699 could interact via a DNA-repair based mechanism is limited. We aimed to investigate a novel, vascular-based activity of AG014699, underlying in vivo chemosensitisation, that could widen its clinical application. EXPERIMENTAL DESIGN: Temozolomide response was analysed in vitro and in vivo. Vessel dynamics were monitored using “mismatch” following the administration of perfusion markers and real-time analysis of fluorescently-labeled albumin uptake in to tumours established in dorsal window chambers. Further mechanistic investigations employed ex vivo assays of vascular smooth muscle relaxation, gut motility and myosin light chain kinase inhibition. RESULTS: AG014699 failed to sensitise SW620 cells to temozolomide in vitro but induced pronounced enhancement in vivo. AG014699 (1mg/kg) improved tumour perfusion comparably with the control agents nicotinamide (1g/kg) and AG14361 (fore-runner to AG014699; 10mg/kg). AG014699 and AG14361 relaxed pre-constricted vascular smooth muscle more potently than the standard agent, hydralazine, with no impact on gut motility. AG014699 inhibited myosin light chain kinase at concentrations that relaxed isolated arteries, whereas AG14361 had no effect. CONCLUSION: Increased vessel perfusion elicited by AG014699 could increase tumour drug accumulation and therapeutic response. Vasoactive concentrations of AG014699 do not cause detrimental side-effects to gut motility and may increase the range of therapeutics with which AG014699 could be combined with for clinical benefit

Culture medium preferences of pre-implantation cloned mouse embryos

Somatic cloning technology involves the transfer of a somatic cell nucleus into an enucleated oocyte, followed by activation and in vitro culture. Efficiency in terms of live offspring generally remains very low. Little attention has been devoted so far to the impact of culture environment on cloned embryo development. Failure of genomic reprogramming of the donor nucleus in nuclear transfer (NT) experiments could lead to an altered phenotype in these cloned embryos that could be manifested by different medium preferences of the NT embryos. We describe here the application of sequential culture media to support preimplantation development of mouse embryos reconstructed using conventional NT techniques. Embryo-quality analysis was performed on NT blastocysts obtained. Additionally, NT embryos that arrested during development also were analyzed