The use of osmolality as an aid to establishing consistent fixation quality: studies on the kidney of the domestic fowl.

1. Using the proximal convoluted tubule of the fowl kidney as indicator tissue the immersion application of fixatives of differing composition and osmolality was studied. 2. The best results were obtained with Dalton's buffered osmium tetroxide and with sodium cacodylate buffered glutaraldehyde followed by osmium post-fixation. 3. The most satisfactory component concentrations in both cases were those which most reproduced a total osmolality close to that of fowl plasma. With both types of fixation the ultrastructural image of the kidney tubule was sensitive to changes in fixative osmolality and differences were clearly identified over a 30 to 50 mOsm range. 4. With glutaraldehyde/osmium double fixation it was found that it was the total osmolality of the glutaraldehyde buffer combination that was to be considered in a study of osmolotic effects, not the osolality of the buffer vehicle alone. 5. The 30 to 50 mOsm difference in fixative osmolality that was able to be detected by studying the image of the kidney tubule was of the order of magnitude commonly experienced as experimental error in the laboratory as a result of changes in reagent batches or error in making up the solutions. The measurement of osmolality therefore provides an ideal quality control to the laboratory make-up of fixative solutions.     

Mechanistic target of rapamycin is activated in bovine granulosa cells after LH surge but is not essential for ovulation

The LH surge induces functional and morphological changes in granulosa cells. Mechanistic target of rapamycin (mTOR) is an integrator of signalling pathways in multiple cell types. We hypothesized that mTOR kinase activity integrates and modulates molecular pathways induced by LH in granulosa cells during the preovulatory period. Cows were ovariectomized and granulosa cells collected at 0, 3, 6, 12 and 24 hr after GnRH injection. While RHEB mRNA levels increased at 3 and 6 hr, returning to basal levels by 12 hr after GnRH treatment, RHOA mRNA levels increased at 6 hr and remained high thereafter. Western blot analyses revealed increased S6K phosphorylation at 3 and 6 hr after GnRH injection. Similarly, mRNA levels of ERK1/2, STAR and EGR‐1 were higher 3 hr after GnRH treatment. Rapamycin treatment inhibited mTOR activity and increased AKT activity, but did not alter ERK1/2 phosphorylation and EGR1 protein levels in cultured bovine granulosa cells. Rapamycin also inhibited LH‐induced increase in EREG mRNA abundance in granulosa cells in vitro. However, intrafollicular injection of rapamycin did not suppress ovulation. These findings suggest that mTOR is involved in the control of EREG expression in cattle, which may be triggered by LH surge stimulating RHEB and S6K activity.     

Production and characterisation of monoclonal antibodies specific for chicken interleukin-2.

Using genetic immunisation of mice, we produced antibodies against chicken interleukin-2 (ChIL-2), the first produced against a non-mammalian interleukin. After a final injection with a recombinant ChIL-2 protein, two stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of the two MAbs to recombinant ChIL-2 produced by Escherichia coli and COS-7 cells was demonstrated in an indirect ELISA, Western blotting and dot blots. Both of them were able to neutralise the biological activity of the ChIL-2, but neither allowed the detection of ChIL-2 by flow cytometry.