Revealing mechanisms of mating plug function under sexual selection.

Mating plugs are produced by many sexually reproducing animals and are hypothesized to promote male fertilization success under promiscuous mating. However, tests of this hypothesis have been constrained by an inability to discriminate ejaculates of different males in direct competition. Here, we use stable isotope labeling in vivo and proteomics to achieve this in a promiscuous rodent, Myodes glareolus We show that, although the first male's plug is usually dislodged, it can be retained throughout the second male's copulation. Retained plugs did not completely block rival sperm but did significantly limit their numbers. Differences in the number of each male's sperm progressing through the female reproductive tract were also explained by natural variation in the size of mating plugs and reproductive accessory glands from which major plug proteins originate. Relative sperm numbers in turn predicted the relative fertilization success of rival males. Our application of stable isotopes to label ejaculates resolves a longstanding debate by revealing how rodent mating plugs promote fertilization success under competitive conditions. This approach opens new opportunities to reveal cryptic mechanisms of postcopulatory sexual selection among diverse animal taxa.

Proteome Dynamics: Tissue Variation in the Kinetics of Proteostasis in Intact Animals.

Understanding the role of protein turnover in the maintenance of proteostasis requires accurate measurements of the rates of replacement of proteins in complex systems, such as intact animals. Moreover, any investigation of allometric scaling of protein turnover is likely to include species for which fully annotated proteomes are not available. We have used dietary administration of stable isotope labeled lysine to assess protein turnover rates for proteins from four tissues in the bank vole,Myodes glareolus The annotated genome for this species is not available, so protein identification was attained through cross-species matching to the mouse. For proteins for which confident identifications were derived, the pattern of lysine incorporation over 40 days was used to define the rate of synthesis of individual proteins in the four tissues. The data were heavily filtered to retain a very high quality dataset of turnover rates for 1088 proteins. Comparative analysis of the four tissues revealed different median rates of degradation (kidney: 0.099 days(-1); liver 0.136 days(-1); heart, 0.054 days(-1), and skeletal muscle, 0.035 days(-1)). These data were compared with protein degradation rates from other studies on intact animals or from cells in culture and indicate that both cell type and analytical methodology may contribute to variance in turnover data between different studies. These differences were not only due to tissue-specific proteins but were reflected in gene products common to all tissues. All data are available via ProteomeXchange with identifier PXD002054.

Sperm competition risk drives plasticity in seminal fluid composition.

BACKGROUND: Ejaculates contain a diverse mixture of sperm and seminal fluid proteins, the combination of which is crucial to male reproductive success under competitive conditions. Males should therefore tailor the production of different ejaculate components according to their social environment, with particular sensitivity to cues of sperm competition risk (i.e. how likely it is that females will mate promiscuously). Here we test this hypothesis using an established vertebrate model system, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our study tests for the first time how both sperm and seminal fluid components of the ejaculate are tailored to the social environment. RESULTS: Our quantitative proteomics analysis reveals that the relative production of different proteins found in seminal fluid--i.e. seminal fluid proteome composition--differs significantly according to cues of sperm competition risk. Using a conservative analytical approach to identify differential expression of individual seminal fluid components, at least seven of 31 secreted seminal fluid proteins examined showed consistent differences in relative abundance under high versus low sperm competition conditions. Notably three important proteins with potential roles in sperm competition--SVS 6, SVS 5 and CEACAM 10--were more abundant in the high competition treatment groups. Total investment in both sperm and seminal fluid production also increased with cues of heightened sperm competition risk in the social environment. By contrast, relative investment in different ejaculate components was unaffected by cues of mating opportunities. CONCLUSIONS: Our study reveals significant plasticity in different ejaculate components, with the production of both sperm and non-sperm fractions of the ejaculate strongly influenced by the social environment. Sperm competition risk is thus shown to be a key factor in male ejaculate production decisions, including driving plasticity in seminal fluid composition.

An in vivo control map for the eukaryotic mRNA translation machinery.

Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNA(i) to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5'untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5'UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.

Proteome dynamics: revisiting turnover with a global perspective.

Although bulk protein turnover has been measured with the use of stable isotope labeled tracers for over half a century, it is only recently that the same approach has become applicable to the level of the proteome, permitting analysis of the turnover of many proteins instead of single proteins or an aggregated protein pool. The optimal experimental design for turnover studies is dependent on the nature of the biological system under study, which dictates the choice of precursor label, protein pool sampling strategy, and treatment of data. In this review we discuss different approaches and, in particular, explore how complexity in experimental design and data processing increases as we shift from unicellular to multicellular systems, in particular animals.

Heterogenous turnover of sperm and seminal vesicle proteins in the mouse revealed by dynamic metabolic labeling.

Plasticity in ejaculate composition is predicted as an adaptive response to the evolutionary selective pressure of sperm competition. However, to respond rapidly to local competitive conditions requires dynamic modulation in the production of functionally relevant ejaculate proteins. Here we combine metabolic labeling of proteins with proteomics to explore the opportunity for such modulation within mammalian ejaculates. We assessed the rate at which proteins are synthesized and incorporated in the seminal vesicles of male house mice (Mus musculus domesticus), where major seminal fluid proteins with potential roles in sperm competition are produced. We compared rates of protein turnover in the seminal vesicle with those during spermatogenesis, the timing of which is well known in mice. The subjects were fed a diet containing deuterated valine ([(2)H(8)]valine) for up to 35 days, and the incorporation of dietary-labeled amino acid into seminal vesicle- or sperm-specific proteins was assessed by liquid chromatography-mass spectrometry of samples recovered from the seminal vesicle lumen and cauda epididymis, respectively. Analyses of epididymal contents were consistent with the known duration of spermatogenesis and sperm maturation in this species and in addition revealed evidence for a subset of epididymal proteins subject to rapid turnover. For seminal vesicle proteins, incorporation of the stable isotope was evident from day 2 of labeling, reaching a plateau of labeling by day 24. Hence, even in the absence of copulation, the seminal vesicle proteins and certain epididymal proteins demonstrate considerable turnover, a response that is consonant with the capacity to rapidly modulate protein production. These techniques can now be used to assess the extent of phenotypic plasticity in mammalian ejaculate production and allocation according to social and environmental cues of sperm competition.

Diauxic shift-dependent relocalization of decapping activators Dhh1 and Pat1 to polysomal complexes.

Dhh1 and Pat1 in yeast are mRNA decapping activators/translational repressors thought to play key roles in the transition of mRNAs from translation to degradation. However, little is known about the physical and functional relationships between these proteins and the translation machinery. We describe a previously unknown type of diauxic shift-dependent modulation of the intracellular locations of Dhh1 and Pat1. Like the formation of P bodies, this phenomenon changes the spatial relationship between components involved in translation and mRNA degradation. We report significant spatial separation of Dhh1 and Pat1 from ribosomes in exponentially growing cells. Moreover, biochemical analyses reveal that these proteins are excluded from polysomal complexes in exponentially growing cells, indicating that they may not be associated with active states of the translation machinery. In contrast, under diauxic growth shift conditions, Dhh1 and Pat1 are found to co-localize with polysomal complexes. This work suggests that Dhh1 and Pat1 functions are modulated by a re-localization mechanism that involves eIF4A. Pull-down experiments reveal that the intracellular binding partners of Dhh1 and Pat1 change as cells undergo the diauxic growth shift. This reveals a new dimension to the relationship between translation activity and interactions between mRNA, the translation machinery and decapping activator proteins.

Protein turnover: measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids.

The measurement of protein turnover in tissues of intact animals is obtained by whole animal dynamic labelling studies, requiring dietary administration of precursor label. It is difficult to obtain full labelling of precursor amino acids in the diet and if partial labelling is used, calculation of the rate of turnover of each protein requires knowledge of the precursor relative isotope abundance (RIA). We describe an approach to dynamic labelling of proteins in the mouse with a commercial diet supplemented with a pure, deuterated essential amino acid. The pattern of isotopomer labelling can be used to recover the precursor RIA, and sampling of urinary secreted proteins can monitor the development of liver precursor RIA non-invasively. Time-series analysis of the labelling trajectories for individual proteins allows accurate determination of the first order rate constant for degradation. The acquisition of this parameter over multiple proteins permits turnover profiling of cellular proteins and comparisons of different tissues. The median rate of degradation of muscle protein is considerably lower than liver or kidney, with heart occupying an intermediate position.

Social status and ejaculate composition in the house mouse.

Sperm competition theory predicts that males should tailor ejaculates according to their social status. Here, we test this in a model vertebrate, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our analyses reveal that both sperm production and the composition of proteins found in seminal vesicle secretions differ according to social status. Dominant males invested more in ejaculate production overall. Their epididymides contained more sperm than those of subordinate or control males, despite similar testes size between the groups. Dominant males also had larger seminal vesicle glands than subordinate or control males, despite similar body size. However, the seminal vesicle secretions of subordinate males had a significantly higher protein concentration than those of dominant males. Moreover, detailed proteomic analysis revealed subtle but consistent differences in the composition of secreted seminal vesicle proteins according to social status, involving multiple proteins of potential functional significance in sperm competition. These findings have significant implications for understanding the dynamics and outcome of sperm competition, and highlight the importance of social status as a factor influencing both sperm and seminal fluid investment strategies. This article is part of the theme issue 'Fifty years of sperm competition'.

Specificity of the osmotic stress response in Candida albicans highlighted by quantitative proteomics.

Stress adaptation is critical for the survival of microbes in dynamic environments, and in particular, for fungal pathogens to survive in and colonise host niches. Proteomic analyses have the potential to significantly enhance our understanding of these adaptive responses by providing insight into post-transcriptional regulatory mechanisms that contribute to the outputs, as well as testing presumptions about the regulation of protein levels based on transcript profiling. Here, we used label-free, quantitative mass spectrometry to re-examine the response of the major fungal pathogen of humans, Candida albicans, to osmotic stress. Of the 1,262 proteins that were identified, 84 were down-regulated in response to 1M NaCl, reflecting the decrease in ribosome biogenesis and translation that often accompanies stress. The 64 up-regulated proteins included central metabolic enzymes required for glycerol synthesis, a key osmolyte for this yeast, as well as proteins with functions during stress. These data reinforce the view that adaptation to salt stress involves a transient reduction in ribosome biogenesis and translation together with the accumulation of the osmolyte, glycerol. The specificity of the response to salt stress is highlighted by the small proportion of quantified C. albicans proteins (5%) whose relative elevated abundances were statistically significant.

Cross-species proteomics in analysis of mammalian sperm proteins.

UNLABELLED: Many proteomics studies are conducted in model organisms for which fully annotated, detailed, high quality proteomes are available. By contrast, many studies in ecology and evolution are conducted in species which lack high quality proteome data, limiting the perceived value of a proteomic approach for protein discovery and quantification. This is particularly true of rapidly evolving proteins in the reproductive system, such as those that have an immune function or are under sexual selection, and can compromise the potential for cross-species proteomics to yield confident identification. In this investigation we analysed the sperm proteome, from a range of ungulates and rodents, and explored the potential of routine proteomic workflows to yield characterisation and quantification in non-model organisms. We report that database searching is robust to cross-species matching for a mammalian core sperm proteome, comprising 623 proteins that were common to most of the 19 species studied here, suggesting that these proteins are likely to be present and identifiable across many mammalian sperm. Further, label-free quantification reveals a consistent pattern of expression level. Functional analysis of this core proteome suggests consistency with previous studies limited to model organisms and has value as a quantitative reference for analysis of species-specific protein characterisation. SIGNIFICANCE: From analysis of the sperm proteome for diverse species (rodents and ungulates) using LC-MS/MS workflows and standard data processing, we show that it is feasible to obtain cross-species matches for a large number of proteins that can be filtered stringently to yield a highly expressed mammalian sperm core proteome, for which label-free quantitative data are also used to inform protein function and abundance.

Identification of novel peptides for horse meat speciation in highly processed foodstuffs.

There is a need for robust analytical methods to support enforcement of food labelling legislation. Proteomics is emerging as a complementary methodology to existing tools such as DNA and antibody-based techniques. Here we describe the development of a proteomics strategy for the determination of meat species in highly processed foods. A database of specific peptides for nine relevant animal species was used to enable semi-targeted species determination. This principle was tested for horse meat speciation, and a range of horse-specific peptides were identified as heat stable marker peptides for the detection of low levels of horse meat in mixtures with other species.

Protein turnover methods in single-celled organisms: dynamic SILAC.

Early achievements in proteomics were qualitative, typified by the identification of very small quantities of proteins. However, as the subject has developed, there has been a pressure to develop approaches to define the amounts of each protein--whether in a relative or an absolute sense. A further dimension to quantitative proteomics embeds the behavior of each protein in terms of its turnover. Virtually every protein in the cell is in a dynamic state, subject to continuous synthesis and degradation, the relative rates of which control the expansion or the contraction of the protein pool, and the absolute values of which dictate the temporal responsiveness of the protein pool. Strategies must therefore be developed to assess the turnover of individual proteins in the proteome. Because a protein can be turning over rapidly even when the protein pool is in steady state, the only acceptable approach to measure turnover is to use metabolic labels that are incorporated or lost from the protein pool as it is replaced. Using metabolic labeling on a proteome-wide scale in turn requires metabolic labels that contain stable isotopes, the incorporation or loss of which can be assessed by mass spectrometry. A typical turnover experiment is complex. The choice of metabolic label is dictated by several factors, including abundance in the proteome, metabolic redistribution of the label in the precursor pool, and the downstream mass spectrometric analytical protocols. Key issues include the need to control and understand the relative isotope abundance of the precursor, the optimization of label flux into and out of the protein pool, and a sampling strategy that ensures the coverage of the greatest range of turnover rates. Finally, the informatics approaches to data analysis will not be as straightforward as in other areas of proteomics. In this chapter, we will discuss the principles and practice of workflow development for turnover analysis, exemplified by the development of methodologies for turnover analysis in the model eukaryote Saccharomyces cerevisiae.

Elevated glucose represses liver glucokinase and induces its regulatory protein to safeguard hepatic phosphate homeostasis.

OBJECTIVE: The induction of hepatic glucose 6-phosphatase (G6pc) by glucose presents a paradox of glucose-induced glucose intolerance. We tested whether glucose regulation of liver gene expression is geared toward intracellular homeostasis. RESEARCH DESIGN AND METHODS: The effect of glucose-induced accumulation of phosphorylated intermediates on expression of glucokinase (Gck) and its regulator Gckr was determined in hepatocytes. Cell ATP and uric acid production were measured as indices of cell phosphate homeostasis. RESULTS: Accumulation of phosphorylated intermediates in hepatocytes incubated at elevated glucose induced rapid and inverse changes in Gck (repression) and Gckr (induction) mRNA concomitantly with induction of G6pc, but had slower effects on the Gckr-to-Gck protein ratio. Dynamic metabolic labeling in mice and liver proteome analysis confirmed that Gckr and Gck are low-turnover proteins. Involvement of Max-like protein X in glucose-mediated Gck-repression was confirmed by chromatin immunoprecipitation analysis. Elevation of the Gck-to-Gckr ratio in hepatocytes was associated with glucose-dependent ATP depletion and elevated urate production confirming compromised phosphate homeostasis. CONCLUSIONS: The lowering by glucose of the Gck-to-Gckr ratio provides a potential explanation for the impaired hepatic glucose uptake in diabetes. Elevated uric acid production at an elevated Gck-to-Gckr ratio supports a role for glucose regulation of gene expression in hepatic phosphate homeostasis.

Regional variation in parvalbumin isoform expression correlates with muscle performance in common carp (Cyprinus carpio).

The mechanical properties of the axial muscles vary along the length of a fish's body. This variation in performance correlates with the expression of certain muscle proteins. Parvalbumin (PARV) is an important calcium binding protein that helps modulate intracellular calcium levels which set the size and shape of the muscle calcium transient. It therefore has a central role in determining the functional properties of the muscle. Transcript data revealed eight specific isoforms of PARV in common carp (Cyprinus carpio) skeletal muscle which we classified as alpha1 and beta1-7. This study is the first to show expression of all eight skeletal muscle PARV isoforms in carp at the protein level and relate regional differences in expression to performance. All of the PARV isoforms were characterised at the protein level using 2D-PAGE and tandem mass spectrometry. Comparison of carp muscle from different regions of the fish revealed a higher level of expression of PARV isoforms beta4 and beta5 in the anterior region, which was accompanied by an increase in the rate of relaxation. We postulate that changes in specific PARV isoform expression are an important part of the adaptive change in muscle mechanical properties in response to varying functional demands and environmental change.