Effect of cooling rate and partial removal of yolk on the chilling injury in zebrafish (Danio rerio) embryos

High chilling sensitivity is one of the main obstacles to successful cryopreservation of zebrafish embryos. So far the nature of the chilling injury in fish embryos has not been clear. The aim of this study is to investigate the effect of cooling rate and partial removal of yolk on chilling injury in zebrafish embryos. Zebrafish embryos at 64-cell, 50%-epiboly, 6-somite and prim-6 stages were cooled to either 0 degrees C or -5 degrees C at three different cooling rates: slow (0.3 degrees C/min or 1 degree C/min), moderate (30 degrees C/min), and rapid (approximately 300 degrees C/min). After chilling, embryos were warmed in a 26 degrees C water bath, followed by 3-day culturing in EM at 26 +/- 1 degrees C for survival assessment. When embryos were cooled to 0 degrees C for up to 30 min, 64-cell embryos had higher survival after rapid cooling than when they were cooled at a slower rate. When 64-cell embryos were held at -5 degrees C for 1 min, their survival decreased greatly after both slow and rapid cooling. The effect of cooling rate on the survival of 50%-epiboly and 6-somite embryos was not significant after 1 h exposure at 0 degrees C and 1 min exposure at -5 degrees C. However, rapid cooling resulted in significantly lower embryo survival than a cooling rate of 30 degrees C/min or 1 degree C/min after 1 h exposure to 0 degrees C for prim-6 stage or 1 h exposure to -5 degrees C for all stages. Chilling injury in 64-cell embryos appears to be a consequence of exposure time at low temperatures rather than a consequence of rapid cooling. Results also indicate that chilling injury in later stage embryos (50%-epiboly, 6-somite and prim-6) is a consequence of the combination of rapid cooling and exposure time at low temperatures. Dechorionated prim-6 embryos were punctured and about half of yolk was removed. After 24 h culture at 26 +/- 1 degrees C after removal of yolk, the yolk-reduced embryos showed higher embryo survival than did control embryos after rapid cooling to -5 degrees C for 10 to 60 min. Results suggest that cold shock injury after rapid cooling can be mitigated after partial removal of yolk at the prim-6 stage. These findings help us to understand the nature of chilling sensitivity of fish embryos and to develop protocols for their cryopreservation.     

Flexibility within the Rotor and Stators of the Vacuolar H+-ATPase

The V-ATPase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an ATP-driven 3-stroke rotary motor (V₁) to a multistroke proton pump (V_o). This is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. We have used electron microscopy to reveal distinctive bending along the V-ATPase complex, leading to angular displacement of the V₁ domain relative to the V_o domain to a maximum of ~30°. This has been complemented by elastic network normal mode analysis that shows both flexing and twisting with the compliance being located in the rotor axle, stator filaments, or both. This study provides direct evidence of flexibility within the V-ATPase and by implication in related rotary ATPases, a feature predicted to be important for regulation and their high energetic efficiencies.     

An Investigation of the Structural Diversities of Lithiated HMPA Complexes of o-Mercaptopyridine and Trithiocyanuric Acid: Syntheses, Crystal structures and Model Molecular Orbital Calculations

Reaction of o-mercaptopyridine (o-MPH) and trithiocyanuric acid (TTCyH₃) with one equivalent of BuⁿLi in the presence of HMPA yields the mono-lithiated salts MPLi.HMPA (1) and TTCyH₂Li.2HMPA (2) respectively, which have been characterised by NMR spectroscopy and X-ray crystallography. Reaction of three equivalents of BuⁿLi with anhydrous TTCyH₃ in THF yields the tri-lithiated species TTCyLi₃.4THF (3). In all three compounds the lithium centres have N,S-bridged coordination modes. Whereas 1 is dimeric in the solid state, 2 has an unusual monomeric structure and 3, which is a very rare example of a structurally characterised tri-lithiated compound, has an unprecedented polymeric structure incorporating (NCSLi) _n (n = 1, 2) rings. The structural diversities displayed by 1 and 2 have been probed, and thereby in part rationalised, by ab initio (6-31G*/RHF, 6-31G**/RHF and 6-31G*/MP2 levels) MO calculations on both their thio-keto and thiol isomers and on their uncomplexed and complexed lithiated derivatives. In particular, the optimised structures predict and reproduce the N,S-bridging coordination modes found for lithium and explain why structure 1 is dimeric whereas 2 is monomeric.Electronic Supplementary Material available.     

DNA and RNA Levels in Bundle Sheath and Mesophyll Cells of Pearl Millett (Pennisetum americanum)

The DNA content of bundle sheath cells and mesophyll protoplasts from the C₄ plant pearl millet (Pennisetum americanum, Tift 23DB) was determined by microspectrophotometry to be 1.8 to 2.3 and 3.2 to 4.0 picograms/nucleus, respectively. Measurement of RNA by ultraviolet spectroscopy indicated that bundle sheath cells contain twice as much RNA as mesophyll cells.     

Preliminary results of olfactory testing in rats without deprivation

Although rodents are nocturnal, their behavior is usually tested during the day. The authors present the results of a preliminary study, which suggest that altering the animals' day:night cycle might be the key to eliminating the need for food or water deprivation prior to testing.     

Cytoskeleton proteins F-actin and tubulin distribution and interaction with mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes

The distributional arrangement of mitochondria in the granulosa cells surrounding stage III zebrafish oocyte has been reported as a contiguous aggregation of mitochondria at the margin of the each granulosa cell. The aim of the present study was to further investigate the mitochondrial distribution in the granulosa cell layer in stage III ovarian follicles and the interaction between mitochondria and cytoskeleton elements actin and tubulin. To determine mitochondrial distribution/transport, immunocytochemistry analysis of tubulin and mitochondrial COX-I was carried out along with phalloidin staining of polymerised F-actin. The follicles were also exposed to a range of conditions that are known to affect mitochondria and the cytoskeleton proteins actin and tubulin. The mitochondrial inhibitor FCCP, the anti-mitotic drug nocodazole, and actin polymerisation inhibitor cytochalasin B were used. Levels of ATP, mtDNA copy number, and viability assessed by Trypan blue were also studied after exposure to inhibitors in order to determine the relationship between mitochondrial distribution/activity and ATP production. F-actin showed a hexagonal-polygonal distribution surrounding the mitochondria in granulosa cells, with the F-actin network adjacent to the plasma membrane of each granulosa cell. Tubulin structure presented a less organised distribution than F-actin, it was sparse in the cytosol. Interaction between mitochondria and tubulin was found indicating that mitochondria and tubulin are colocalised in zebrafish ovarian follicles. The exposure of ovarian follicles to inhibitors induced the loss of mitochondrial structural integrity showing that mitochondria distribution in granulosa cells of stage III zebrafish ovarian follicles is determined by the microtubules network.     

Effect of chilling and cryopreservation on expression of Pax genes in zebrafish (Danio rerio) embryos and blastomeres

Cryopreservation is now common practice in the fields of aquaculture, conservation and biomedicine. However, there is a lack of information on the effect of chilling and cryopreservation at the molecular level. In the present study, we used real-time RT-PCR analysis to determine the effect of chilling and cryopreservation on expression of Pax2a, Pax2b, Pax5 and Pax8 which constitute one subgroup of the Pax gene family. As intact embryos of zebrafish have not yet been successfully cryopreserved, we have used two alternatives: chilling of intact embryos and cryopreservation of isolated blastomeres. Cryopreservation was found to affect the normal pattern of gene expression in zebrafish embryonic blastomeres. The trends, profile changes, in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryonic blastomeres which could have a detrimental effect on embryo development.