Search

At refrigeration temperature, mouse embryos can retain their developmental ability for a couple of days. Previous research reports have focused on the effect of cool temperature on the development of 2-cell stage embryos, morulae or blastocysts and determined that the embryo still has the ability to produce offspring after about 48 h storage at refrigeration temperature. Here we examined whether refrigeration temperature affects the development of the eight-cell stage and if the stored eight-cell stage embryo can still be used as a host embryo for ES cell injection. Our results show that eight-cell stage embryos can develop into blastocysts and yield pups after cold storage for 24 and 48 h. After ES cell injection, stored eight-cell stage embryos can support ES cells developing to F0 pups. In summary, cool storage can preserve the developmental ability of eight-cell stage embryos for at least 48 h, allowing transportation of the embryos at refrigeration temperature between different labs and their subsequent use as host embryos for ES cell injection., (Copyright © 2018. Published by Elsevier Inc.)

Reproduction technologies (RTs) for the storage and use of amphibian gametes have rapidly developed since the recognition of the amphibian conservation crisis in the late 20th Century. Of these RTs, the refrigerated storage of oocytes and sperm can help to achieve reliable pair-matching when unexpected deaths could lead to critical gaps in studbook programs, and also to enable gamete transport between facilities or when sampled from field populations. Viable sperm can be reliably stored in vitro in testes, as suspensions in refrigerators for weeks and in situ in refrigerated carcasses for days. However, oocytes have only been reliably stored in vitro and then only for a few hours. We stored mature oocytes of the European common frog Rana temporaria refrigerated at 4 °C: in situ in the oviduct of carcasses for 1-5 days, in vivo in the oviduct of live frogs for 30 days, and in vitro in plastic boxes for 1-5 days. Oocyte viability was measured as the percentage of fertilisation relative to controls and as the percentage hatch of fertilised oocytes. Rana temporaria oocytes in situ or in vitro retained some viability to hatch for up to 5 days. In contrast, when stored in vivo, oocytes showed little loss of viability to hatch after 10 days and moderate viability up to 30 days., (Copyright © 2018. Published by Elsevier Inc.)

During hypothermic preservation of cells (0-4°C), metabolism is diminished and energy-dependent transport processes are arrested. The effect of hypothermic preservation of hepatocytes in endocytic transport following rewarming has not been previously reported. We evaluated the uptake of EGF (Epidermal Growth Factor) ligand conjugated to fluorescent Quantum Dots (QDs) probes in rat hepatocytes after 24 and 72h cold storage in University of Wisconsin (UW) solution at 4°C. QDs uptake was visualized during rewarming to 37°C under air or, in a second approach, at the end of rewarming under 5% CO2. After 24h in UW solution, QDs were internalized under both rewarming conditions similar to non-preserved hepatocytes and cells maintained a normal cytoskeleton distribution. However, in hepatocytes preserved 72h none of the cells internalized QDs, which remained bound to the membranes. After rewarming, this group showed diminished actin staining and 60% reduction in ATP levels, while viability was maintained at ∼70%. Our results present evidence that, hypothermic preservation for 72h in UW solution at 4°C does not prevent EGFR (epidermal growth factor receptor) activation but irreversibly impairs endocytic uptake upon EGF stimulation; presumably due to actin cytoskeleton disassembling besides reduced ATP pool. Our approach can be applied on other membrane receptor systems and with other hypothermic preservation solutions to understand the effect of cooling in endocytic transport and to determine the optimal cold storage period., (Copyright © 2012 Elsevier Inc. All rights reserved.)