Sex-specific effects of yolk testosterone on survival, begging and growth of zebra finches

Yolk androgens affect offspring hatching, begging, growth and survival in many bird species. If these effects are sex-specific, yolk androgen deposition may constitute a mechanism for differential investment in male and female offspring. We tested this hypothesis in zebra finches. In this species, females increase yolk-testosterone levels and produce male-biased sex ratios when paired to more attractive males. We therefore predicted that especially sons benefit from elevated yolk androgens. Eggs were injected with testosterone or sesame oil (controls) after 2 days of incubation. Testosterone had no clear effect on sex-specific embryonic mortality and changed the pattern of early nestling mortality independent of offspring sex. Testosterone-treated eggs took longer to hatch than control eggs. Control males begged significantly longer than females during the first days after hatching and grew significantly faster. These sex differences were reduced in offspring from testosterone-treated eggs due to prolonged begging durations of daughters, enhanced growth of daughters and reduced growth of sons. The results show that variation in maternal testosterone can play an important role in avian sex allocation due to its sex-specific effects on offspring begging and growth.     

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.     

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

Quantitative metabolomics of microbial cultures requires well-designed sampling and quenching procedures. We successfully developed and applied a differential method to obtain a reliable set of metabolome data for Escherichia coli K12 MG1655 grown in steady-state, aerobic, glucose-limited chemostat cultures. From a rigorous analysis of the commonly applied quenching procedure based on cold aqueous methanol, it was concluded that it was not applicable because of release of a major part of the metabolites from the cells. No positive effect of buffering or increasing the ionic strength of the quenching solution was observed. Application of a differential method in principle requires metabolite measurements in total broth and filtrate for each measurement. Different methods for sampling of culture filtrate were examined, and it was found that direct filtration without cooling of the sample was the most appropriate. Analysis of culture filtrates revealed that most of the central metabolites and amino acids were present in significant amounts outside the cells. Because the turnover time of the pools of extracellular metabolites is much larger than that of the intracellular pools, the differential method should also be applicable to short-term pulse response experiments without requiring measurement of metabolites in the supernatant during the dynamic period.     

In vivo kinetics of primary metabolism in Saccharomyces cerevisiae studied through prolonged chemostat cultivation

In this study, prolonged chemostat cultivation is applied to investigate in vivo enzyme kinetics of Saccharomyces cerevisiae. S. cerevisiae was grown in carbon-limited aerobic chemostats for 70-95 generations, during which multiple steady states were observed, characterized by constant intracellular fluxes but significant changes in intracellular metabolite concentrations and enzyme capacities. We provide evidence for two relevant kinetic mechanisms for sustaining constant fluxes: in vivo near-equilibrium of reversible reactions and tight regulation of irreversible reactions by coordinated changes of metabolic effectors. Using linear-logarithmic kinetics, we illustrate that these multiple steady-state measurements provide linear constraints between elasticity parameters instead of their absolute values. Upon perturbation by a glucose pulse, glucose uptake and ethanol excretion in prolonged cultures were remarkably lower, compared to a reference culture perturbed at 10 generations. Metabolome measurements during the transient indicate that the differences might be due to a reduced ATP regeneration capacity in prolonged cultures.     

Combined molecular and morphological phylogenetic analyses of the New Zealand wolf spider genus Anoteropsis (Araneae: Lycosidae)

Datasets from the mitochondrial gene regions NADH dehydrogenase subunit I (ND1) and cytochrome c oxidase subunit I (COI) of the 20 species in the New Zealand wolf spider (Lycosidae) genus Anoteropsis were generated. Sequence data were phylogenetically analysed using parsimony and maximum likelihood analyses. The phylogenies generated from the ND1 and COI sequence data and a previously generated morphological dataset were significantly congruent (p<0.001). Sequence data were combined with morphological data and phylogenetically analysed using parsimony. The ND1 region sequenced included part of tRNA(Leu(CUN)), which appears to have an unstable amino-acyl arm and no TpsiC arm in lycosids. Analyses supported the existence of five species groups within Anoteropsis and the monophyly of species represented by multiple samples. A radiation of Anoteropsis species within the last five million years is inferred from the ND1 and COI likelihood phylograms, habitat and geological data, which also indicates that Anoteropsis arrived in New Zealand some time after it separated from Gondwana.     

Sex differences in yolk hormones depend on maternal social status in Leghorn chickens (Gallus gallus domesticus)

Maternal hormones are known to be present in avian eggs and can have beneficial effects on chick development. Recently, differences in avian yolk steroid concentrations between the sexes have been demonstrated, and in this context steroids have been proposed to be part of the avian sex-determining mechanism. In our study, we show that it is very unlikely that androgen concentrations alone are the decisive part of the sex-determining mechanism. We found that sex-specific differences in the yolk hormones strongly depend on the social rank of the mother. First, dominant females, but not subdominant females, allocated significantly more testosterone to male eggs than to female eggs. Second, subordinate females increased the testosterone concentrations of female eggs. This pattern of yolk hormone deposition can be functionally explained. In polygynous species such as the chicken, reproductive success is more variable in males than in females. Parental investment in sons or daughters is therefore expected to occur in direct relation to parental rearing capacities. We found that the social status of a hen was indeed negatively correlated with her maternal capacities (for example, body mass, egg mass). Differential androgen deposition might thus provide a mechanism for adaptive maternal investment depending on both the sex of the egg and the social status of the mother.     

Uptake of 13C-glucose by cell suspensions of carrot (Daucus carota) measured by in vivo NMR: Cycling of triose-, pentose- and hexose-phosphates

After a lag phase of 2 days, batch-grown cells of carrot ( Daucus carota L.) cv. Flakkese entered the exponential growth phase and started to accumulate sucrose and hexoses. Short-term feeding ¹³C-glucose in this period resulted in only minor labelling of sucrose or fructose. CO₂ production from [1-¹³C]- and [6-¹³C]-glucose revealed, that at least 40% of the added glucose passed through the oxidative pentose phosphate pathway (OPPP), up to 40% through glycolysis leaving only minor ¹³C-glucose for incorporation in various cell components in the exponential growth phase. After about 11 days of culture, the medium sugars were exhausted, cells entered the stationary growth phase and consumed stored sugar. Both neutral and acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) increased 50% from day 0 to days 11–13; thereafter their levels decreased again. Labelling with ¹³C-glucose resulted in the accumulation of labelled sucrose and fructose during the stationary growth phase. Sucrose labelling was transient, i.e. after 6 h its level started to decrease again. Labelled fructose, however, evolved slower and increased even after 8 h. In sucrose and fructose up to 20% of the ¹³C-label was exchanged from C-1 to C-6 carbons, indicating intensive cycling of at least 40% of the carbon between hexoses and triose phosphates. In the stationary phase only 10% of the labelled glucose passed through the OPPP and about 30% passed through the respiratory pathway; the remaining 60% was incorporated in cell constituents and sugars. Comparing the various cycles revealed that the regulation of the OPPP operated relatively independently from the cytosolic cycling of hexose phosphates through sucrose and from the cycling between hexose phosphates and triose phosphates.     

Vertebrate orthologues of the Drosophila region-specific patterning gene teashirt

In Drosophila the teashirt gene, coding for a zinc finger protein, is active in specific body parts for patterning. For example, Teashirt is required in the trunk (thorax and abdomen) tagmata of the embryo, parts of the intestine and the proximal parts of appendages. Here we report the isolation of vertebrate cDNAs related to teashirt. As in Drosophila, human and murine proteins possess three widely spaced zinc finger motifs. Additionally, we describe the expression patterns of the two murine genes. Both genes show regionalized patterns of expression, in the trunk, in the developing limbs and the gut.