BACKGROUND: Female animals are often able to store sperm inside their body--in some species even for several decades. The molecular basis of how females keep non-own cells alive is largely unknown, but since sperm cells are reported to be transcriptionally silenced and, therefore, limited in their ability to maintain their own function, it is likely that females actively participate in sperm maintenance. Because female contributions are likely to be of central importance for sperm survival, molecular insights into the process offer opportunities to observe mechanisms through which females manipulate sperm. RESULTS: We used the honeybee, Apis mellifera, in which queens are highly polyandrous and able to maintain sperm viable for several years. We identified over a hundred proteins representing the major constituents of the spermathecal fluid, which females contribute to sperm in storage. We found that the gel profile of proteins from spermathecal fluid is very similar to the secretions of the spermathecal gland and concluded that the spermathecal glands are the main contributors to the spermathecal fluid proteome. A detailed analysis of the spermathecal fluid proteins indicate that they fall into a range of different functional groups, most notably enzymes of energy metabolism and antioxidant defense. A metabolic network analysis comparing the proteins detected in seminal fluid and spermathecal fluid showed a more integrated network is present in the spermathecal fluid that could facilitate long-term storage of sperm. CONCLUSIONS: We present a large-scale identification of proteins in the spermathecal fluid of honeybee queens and provide insights into the molecular regulation of female sperm storage.

STUDY QUESTION: How do the two new fully automated anti-Müllerian hormone (AMH) assays released in September 2014 by two different diagnostic companies perform compared with the clinical standard assay, namely the AMH Gen II enzyme-linked immunosorbent assay (ELISA)? SUMMARY ANSWER: Both fully automated AMH assays perform in a nearly identical fashion compared with the AMH Gen II assay, with a higher analytical sensitivity. WHAT IS KNOWN ALREADY: Owing to the lack of standardization, the results of AMH ELISA assays are sometimes difficult to compare. The BCI AMH Gen II assay became the clinical reference assay over the last few years. Two newly developed fully automated, highly sensitive AMH immunoassays, based on the AMH Gen II antibody composition have become available since September 2014. STUDY DESIGN, SIZE, DURATION: Previously characterized serum samples from 155 women were used to measure AMH with the three immunoassays, focusing on the aspect of predicting ovarian reserve. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples from 94 women with an unfilled desire for a child diagnosed as infertile/subfertile, 29 samples women with polycystic ovary syndrome and 32 women approaching menopause were included to the study. The precision and the linearity in dilutions of the two new AMH assays were determined and the assay results were compared with the clinical reference (the modified version of the BCI AMH Gen II assay) and to the antral follicle counts of the study participants. Cutoff values for the discrimination between each of two predefined groups were calculated using receiver operating characteristic analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The performance evaluation of the fully automated AMH assays resulted in a within-run and intermediate precision of 0.9-1.9% and 2.5-6.5% with the one and 0.9-3.6% or 4.4-10.7% with the other immunoassay, respectively. Pearson's coefficient of correlation was 0.991 for the method comparison between both assays with a bias of 0.003 ng/ml and a slope of 0.97. The discrimination of the new immunoassays between subfertile women and women approaching menopause was significantly better compared with the BCI Gen II assay (87.5 versus 68.8%, P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Owing to the low number of study subjects in each group, the results have to be confirmed in further studies. WIDER IMPLICATIONS OF THE FINDINGS: The findings of the study are in good agreement with studies that used the Ultra Sensitivite AMH and the pico AMH ELISA assays. The application of AMH measurement onto an automated immunoassay platform is a major step forward, allowing health care providers rapid access to the AMH result and facilitating the adoption of AMH measurement into daily clinical practice. STUDY FUNDING/COMPETING INTERESTS: We declare no financial relationships or competing interests.

Given the substantial changes in mitochondrial gene expression, the mitochondrial proteome, and respiratory function during rice (Oryza sativa) germination under anaerobic and aerobic conditions, we have attempted to identify changes in mitochondrial membrane transport capacity during these processes. We have assembled a preliminary rice mitochondrial carrier gene family of 50 members, defined its orthology to carriers of known function, and observed significant changes in microarray expression data for these rice genes during germination under aerobic and anaerobic conditions and across rice development. To determine if these transcript changes reflect alteration of the carrier profile itself and to determine which members of the family encode the major mitochondrial carrier proteins, we analyzed mitochondrial integral membrane protein preparations using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mass spectrometry, identifying seven distinct carrier proteins. We have used mass spectrometry-based quantitative approaches to compare the abundance of these carriers between mitochondria from dry seeds and those from aerobic- or anaerobic-germinated seeds. We highlight an anaerobic-enhanced basic amino acid carrier and show concomitant increases in mitochondrial arginase and the abundance of arginine and ornithine in anaerobic-germinated seeds, consistent with an anaerobic role of this mitochondria carrier. The potential role of this carrier in facilitating mitochondrial involvement in arginine metabolism and the plant urea cycle during the growth of rice coleoptiles and early seed nitrate assimilation under anaerobic conditions are discussed.

Mitochondria are the cellular organelles responsible for respiration, oxidizing organic acids to release carbon dioxide and reducing oxygen to water. In the process, mitochondria synthesize ATP and export it to the cell to drive many energy-utilizing processes in growth and development. To undertake these processes, mitochondria contain many hundreds, and perhaps even thousands, of different proteins. Each protein participates as an enzyme in a complex series of biochemical pathways to complete the task of respiration. In addition, mitochondria in plants are involved in a wide array of other processes like nitrogen metabolism, photorespiration or even making cofactors such as biotin and folate. Mitochondria arose from endosymbiotic bacteria in the ancient eukaryotic cell. Over time, most of the mitochondrial genes were transferred to the nucleus and now the proteins are post-translationally imported into mitochondria from the cytosol. While the transcription, translation, import and processing of these nucleus-encoded mitochondrial proteins has received considerable attention in plants, very little work has been carried out on the array of proteases that are likely to be instrumental in protein stability, turnover and assembly within mitochondria. ‘Arabidopsis Lon1 has been moved from the shadows to the limelight, and the nonredundant role of this class of proteases in plant mitochondrial homeostasis is beginning to be uncovered.’ Proteases from the Lon, FtsH and Clp families are known to be targeted to mitochondria in plants (Adam et al., 2001). Proteolysis plays an important role in post-translational control by the targeted degradation of short-lived proteins and also helps to maintain protein quality control by removing defective, damaged or even damaging proteins. However, how important each of these proteases is to plant mitochondrial structure and function, and what their targets are, has largely remained a mystery. In this issue of New Phytologist, Rigas and colleagues (pp. 588–600) report the discovery of a Lon1 mutant through a forward genetic screen for root growth in Arabidopsis. What follows is a very thorough analysis of this mutant, providing both convincing evidence of the importance of a single mitochondrial protease for root growth, coupled to a detailed molecular analysis of the impact of this protease loss on mitochondrial form and function. As a result, Arabidopsis Lon1 has been moved from the shadows to the limelight, and the nonredundant role of this class of proteases in plant mitochondrial homeostasis is beginning to be uncovered. So what is Lon? Lon is an ATP-dependent protease that was first found in bacteria. In fact, it was the first ATP-dependent protease discovered (Chung & Goldberg, 1981). It is a multidomain polypeptide with a variable N-terminal domain, a central ATPase domain and a C-terminal domain that contains the proteolytic activity (Fig. 1). Its name derives from an Escherichia coli K-12 mutant lon, identified in the 1960s and named for the long form of the mutant cells (Donch & Greenberg, 1968). Lon acts as a heat shock protein in bacteria, being transcriptionally induced by heat. Lon proteases have since been found in virtually all living organisms, from Archaea to Eubacteria, to plants and animals. The protein domains of the Lon protease. (a) The crystal structures of the three domains of the Escherichia coli Lon protease (Botos et al., 2004a,b; Li et al., 2005). Crystal structures were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) database http://www.rcsb.org. (b) A schematic representation of the Lon protease showing the variable N-terminal domain, the AAA+ ATP hydrolysis module adjacent to the sensor and substrate-discrimination module (SSD), in the central domain, and the proteolytic P-domain. The presence of Lon-like proteases in mitochondria was first noted in the 1970s, and an extensive literature on this class of protease in mitochondria from a variety of organisms has traversed the past 30 yr. In the last decade it has become clear that Lon preferentially degrades oxidatively modified proteins in the mitochondrial matrix, notably the oxidized, hydrophobic form of aconitase (Bota & Davies, 2002). As Lon expression and activity are known to decline with age, its inactivation may contribute to the accumulation of the oxidatively modified protein aggregates often observed during aging and in cells from diseased individuals. Beyond its proteolytic role, other functions have been attributed to Lon in mitochondria, including mitochondrial DNA (mtDNA) binding and chaperone activity for the assembly of respiratory chain complexes (Fig. 2; Ngo & Davies, 2007). The functional role of Lon and consequences of loss of Lon function. Substrates for proteolysis by Lon protease can be misfolded, oxidatively damaged, overproduced or aggregated proteins in cells. Lon protease has been shown to be involved in the degradation or refolding of such proteins. Organisms lacking or depleted in Lon protease show a variety of phenotypes, as shown. mtDNA, mitochondrial DNA. In plant mitochondria, little work on Lon proteases has been reported. The four Arabidopsis isomers of the Lon protease are predicted to reside in either chloroplasts or mitochondria (Adam et al., 2001), and proteomic evidence has identified several in mitochondria (Heazlewood et al., 2007). Transcriptionally they are only minimally affected by stresses such as high light, cold and heat, in contrast to other organelle protease classes that are highly induced by such conditions (Sinvany-Villalobo et al., 2004). The only clear documentation of a molecular function that we are aware of in plants is the report of Lon protease being responsible for degradation of a cytoplasmic male sterility-associated protein, ORF239, in bean mitochondria in vegetative, but not in reproductive, tissues (Sarria et al., 1998). In this issue of New Phytologist, Rigas et al. report the story of one particular mutant discovered during the screen of an ethane methyl sulfonate (EMS) mutagenized population of Arabidopsis to identify mutants with impaired root-growth phenotypes. This mutant had not only impaired primary root elongation but also retardation of postgerminative growth that persisted throughout its life cycle. Positional cloning identified the locus responsible for the mutation to be a 40-kb region on the upper arm of chromosome V. Using the power and ease of reverse genetics in Arabidopsis, the eight candidate genes in this region were then tested for complementation of the phenotype, and the stably transformed insertion of At5g26860 was able to rescue the phenotype. This gene encoded a protein with a Lon protease-like amino acid sequence. Sequence analysis further revealed that the mutation was caused by the introduction of a premature termination codon in the At5g26860 locus. To confirm the function of this gene locus, Rigas et al. used a yeast mutant that is missing an ATP-dependent, mitochondrially located protease with 30% identity to the Lon protease from Bacillus brevis (Van Dyck et al., 1994). This mutation leads to disruption of mtDNA and thus to respiratory deficiency in yeast. Complementation of Δpim1 yeast with At-Lon1 allowed growth of the Δpim1 yeast at 30°C on the nonfermentable carbon source, glycerol, that normally prevents Δpim1 growth (van Dyck et al., 1998). However, the authors also showed that At-Lon1 only promoted minimal growth of Δpim1 at 36°C compared with wild-type yeast, suggesting that while there was functional conservation of PIM 1 and At-Lon1, under heat stress At-Lon1 was unable to perform the specific function of PIM1. This difference was further examined by measuring the At-Lon1 transcript abundance when seedlings were grown at elevated temperatures, and the results showed that the Lon-1 transcript is, in fact, mildly downregulated during both acute and prolonged exposure to heat. Interestingly, this result is supported by the observation that maize Lon-1 gene expression also declined in response to thermal stress (Barakat et al., 1998). This is in stark contrast to Lon proteases in many nonplant species where these genes are transcriptionally induced during heat shock (Fig. 2). Interestingly, the authors showed that while Lon is not a heat shock protein in Arabidopsis, loss of Lon in the lon1-1 and lon1-2 lines leads to a temperature-dependent germination phenotype, thus still linking Lon with a heat-induced functional role in plant germination (Fig. 2). Together, these observations suggest that while the plant Lon-1 homologues may perform many similar roles to the yeast PIM1, there are differences in substrate recognition, heat stability and/or transcriptional control that will influence their roles during thermal stress (Fig. 2). The cellular location of At-Lon1 was confirmed by the insertion of the yellow fluorescent protein (YFP) reporter gene into At-Lon1 cDNA. The YFP fluorescence in root cells was seen to overlay directly in transgenic plant seedling stained with MitoTracker Orange, revealing that the Lon-1-YFP is in planta targeted to mitochondria. To dig deeper into the effect of the absence of At-Lon1 on mitochondrial function, the respiratory capacity of isolated mitochondria was tested using oxygen-consumption assays of respiration and direct analysis of specific mitochondrial enzymes by spectrophotometry. These assays showed that while mitochondria of the mutant maintained outer-membrane integrity, respiratory capacity was reduced when oxidizing succinate and cytochrome c, indicating damage or decrease in complexes II and IV. The largest effect was on the activity of complex IV, the cytochrome c terminal oxidase of the respiratory chain, which was only ∼20% of the wild-type level, providing a strong marker for one molecular change in the mutants. By contrast, the activities of Complex I (CI), the external NADH dehydrogenase and the alternative oxidase, were unaffected by the mutation. The absence of Lon1 also led to significant decreases in the activities of five tricarboxylic acid (TCA) cycle enzymes. A morphological examination of hypocotyl tissue using transmission electron microscopy showed that lon1-1 mitochondria were swollen and had a poorly developed internal membrane structure with few discernable cristae. This suggested either a damaged mitochondrial structure or perhaps even an undeveloped structure. Similar-looking organelles, termed protomitochondria, have been observed in dry seeds but are normally modified during germination in a mitochondrial maturation process (Logan et al., 2001; Howell et al., 2006). The lack of transcriptional up-regulation of Lon by heat stress compared with the temperature-induced germination phenotype of the mutants, suggests that Lon may be more of a constitutive protease in plant mitochondria relative to the other proteases present (Sinvany-Villalobo et al., 2004) but this still needs to be verified. While Lon might have important roles during heat stress, a role as a chaperone or assembly factor might better explain its expression pattern that is correlated to tissues with a high growth rate rather than to environmental factors. The dual role of Lon in yeast as chaperone and protease was clearly shown in mutants lacking a functional C-terminal proteolytic domain (Rep et al., 1996). The chaperone role is probably a result of its central AAA+ ATPase domain, a fold often noted in other proteins with chaperone roles (Neuwald et al., 1999). Sophisticated remodeling of Lon1 and other Lon proteases in plants now awaits researchers interested in untangling both their specialization and their multifaceted roles in plant mitochondrial function.

Activation of hepatic stellate cells is considered to be the main step in the development of liver fibrosis, which is characterized by the transition of quiescent vitamin-A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The identification of regulatory genes during early cell activation and transdifferentiation is essential to extend our knowledge of hepatic fibrogenesis. In liver, the gene CSRP2 is exclusively expressed by stellate cells, whereas no transcripts are detectable in hepatocytes, sinusoidal endothelial cells or Kupffer cells. The early activation of stellate cells induced by platelet-derived growth factor is accompanied by an enhanced expression of CSRP2. During later stages of transdifferentiation, the expression of CSRP2 in these cells is suppressed in vitro and in vivo. The CSRP2-encoded cysteine- and glycine-rich double-LIM-domain protein (CRP)2 is proposed to function as a molecular adapter, arranging two or more as yet unidentified protein constituents into a macromolecular complex. To identify these proteins and assign a cellular function to CRP2, a human cDNA library was screened with full-length CRP2 as bait in a yeast two-hybrid screen. The protein inhibitor of activated STAT1 (‘PIAS1’) was shown to associate selectively with the C-terminal LIM domain of CRP2. Physical interaction of both proteins in the cellular environment was confirmed by co-localization experiments with confocal laser scanning microscopy and co-immunoprecipitation analysis. These results establish CRP2 as a potential new factor in the JAK/STAT-signalling pathway and suggest that the suppression of CSRP2 might be a prerequisite for the myofibroblastic transition of hepatic stellate cells.

Liver fibrosis occurs as a consequence of the transdifferentiationof hepatic stellate cells into myofibroblasts and is associated with an increased expression and activation of transforming growth factor (TGF)-beta1. This pluripotent, profibrogenic cytokine stimulates matrix synthesis and decreases matrix degradation, resulting in fibrosis. Thus, blockade of synthesis or sequestering of mature TGF-beta1 is a primary target for the development of antifibrotic approaches. The purpose of this study was to investigate whether the administration of adenoviruses constitutively expressing an antisense mRNA complementary to the 3' coding sequence of TGF-beta1 is able to suppress the synthesis of TGF-beta1 in culture-activated hepatic stellate cells. We demonstrate that the adenoviral vehicle directs high-level expression of the transgene and proved that the transduced antisense is biologically active by immunoprecipitation, Western blot, quantitative TGF-beta1 ELISA, and cell proliferation assays. Additionally, the biological function of the transgene was confirmed by analysis of differential activity of TGF-beta1-responsive genes using cell ELISA, Northern blotting, and by microarray technology, respectively. Furthermore, we examined the effects of that transgene on the expression of TGF-beta2, TGF-beta3, collagen type alpha1(I), latent transforming growth factor binding protein 1, types I and II TGF-beta receptors, and alpha-smooth muscle actin. Our results indicate that the administration of antisense mRNA offers a feasible approach to block autocrine TGF-beta1 signaling in hepatic stellate cells and may be useful and applicable in future to the treatment of fibrosis in chronic liver diseases.

Researchers often want to study the respiratory properties of individual parts of plants in response to a range of treatments. Arabidopsis is an obvious model for this work; however, because of its size, it represents a challenge for gas exchange measurements of respiration. The combination of micro-respiratory technologies with multiplex assays has the potential to bridge this gap, and make measurements possible in this model plant species. We show the adaptation of the commercial technology used for mammalian cell respiration analysis to study three critical tissues of interest: leaf sections, root tips and seeds. The measurement of respiration in single leaf discs has allowed the age dependence of the respiration rate in Arabidopsis leaves across the rosette to be observed. The oxygen consumption of single root tips from plate-grown seedlings shows the enhanced respiration of root tips and their time-dependent susceptibility to salinity. The monitoring of single Arabidopsis seeds shows the kinetics of respiration over 48 h post-imbibition, and the effect of the phytohormones gibberellic acid (GA3 ) and abscisic acid (ABA) on respiration during seed germination. These studies highlight the potential for multiplexed micro-respiratory assays to study oxygen consumption in Arabidopsis tissues, and open up new possibilities to screen and study mutants and to identify differences in ecotypes or populations of different plant species.

Wheat has served as a key species for characterising fundamental aspects of mitochondrial biochemistry and respiratory physiology. Respiratory traits are linked to many important agronomic properties, so identifying the proteins that carry out these molecular processes would define a new set of targets for wheat breeding. To date, systematic proteomic investigations into wheat mitochondria have lagged behind other species, due to the size and complexity of the wheat genome. However this situation is changing with new sequence data increasing the power of proteomics applied to wheat. In this review, we argue that the impact of wheat mitochondrial proteomics on wheat respiratory traits can be improved through integrating data from current proteomics approaches with knowledge from the wheat respiration literature. We present a historical overview of biochemical and physiological studies of mitochondrial respiration in wheat, highlighting respiratory properties linked to agronomically important traits, such as biomass production, stress tolerance and cytoplasmic male sterility. Also, we summarise the current status of the wheat mitochondrial proteome and present a predicted set of 2000 probable mitochondrial proteins from Triticum urartu. Finally, we present a set of strategies outlining how future proteomics experiments can be applied to wheat mitochondria, by targeting studies to build on pre-existing knowledge.

Ejaculates contain sperm but also seminal fluid, which is increasingly recognized to be of central importance for reproductive success. However, a detailed biochemical composition and physiological understanding of seminal fluid is still elusive. We have used MS to identify the 57 most abundant proteins within the ejaculated seminal fluid of the honeybee Apis mellifera. Their amino acid sequences revealed the presence of diverse functional categories of enzymes, regulators and structural proteins. A number have known or predicted roles in maintaining sperm viability, protecting sperm from microbial infections or interacting with the physiology of the female. A range of putative glycoproteins or glycosylation enzymes were detected among the 57, subsequent fluorescent staining of glycolysation revealed several prominent glycoproteins in seminal fluid, while no glycoproteins were detected in sperm samples. Many of the abundant proteins that accumulate in the seminal fluid did not contain predictable tags for secretion for the cell. Comparison of the honeybee seminal fluid proteins with Drosophila seminal fluid proteins (including secreted accessory gland proteins known as ACPs), and with the human seminal fluid proteome revealed the bee protein set contains a range of newly identified seminal fluid proteins and we noted more similarity of the bee protein set with the current human seminal fluid protein set than with the known Drosophila seminal fluid proteins. The honeybee seminal fluid proteome thus represents an important addition to available data for comparative studies of seminal fluid proteomes in insects.

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction.

Wilson's disease is an autosomal recessive disorder in which the liver does not properly release copper into bile, resulting in prominent copper accumulation in various tissues. Affected patients suffer from hepatic disorders and severe neurological defects. Experimental studies in mutant mice in which the copper-transporting ATPase gene (Atp7b) is disrupted revealed a drastic, time-dependent accumulation of hepatic copper that is accompanied by formation of regenerative nodes resembling cirrhosis. Therefore, these mice represent an excellent exploratory model for Wilson's disease. However, the precise time course in hepatic copper accumulation and its impact on other trace metals within the liver is yet poorly understood. We have recently established novel laser ablation inductively coupled plasma mass spectrometry protocols allowing quantitative metal imaging in human and murine liver tissue with high sensitivity, spatial resolution, specificity and quantification ability. By use of these techniques, we here aimed to comparatively analyse hepatic metal content in wild-type and Atp7b deficient mice during ageing. We demonstrate that the age-dependent accumulation of hepatic copper is strictly associated with a simultaneous increase in iron and zinc, while the intrahepatic concentration and distribution of other metals or metalloids is not affected. The same findings were obtained in well-defined human liver samples that were obtained from patients suffering from Wilson's disease. We conclude that in Wilson's disease the imbalances of hepatic copper during ageing are closely correlated with alterations in intrahepatic iron and zinc content.