Aldolase C is a novel molecular marker for folliculo-stellate cells in rodent pituitary.

The anterior pituitary gland is composed of five types of hormone-producing cells and folliculo-stellate cells. Folliculo-stellate cells do not produce anterior pituitary hormones but they are thought to play important roles as stem cells, phagocytes, or supporting cells of hormone-producing cells in the anterior pituitary. S100ß protein has been used as a folliculo-stellate cell marker in some animals, including rats. However, since no reliable molecular marker for folliculo-stellate cells has been reported in mice, genetic approaches for the investigation of folliculo-stellate cells in mice are not yet available. Aldolase C/Zebrin II is a brain-type isozyme and is a fructose-1,6-bisphosphate aldolase. In the present study, we first used immunohistochemistry to verify that aldolase C was produced in the anterior pituitary of rats. Moreover, using transgenic rats expressing green fluorescent protein under the control of the S100ß gene promoter, we identified aldolase C-immunoreactive signals in folliculo-stellate cells and marginal cells located in the parenchyma of the anterior pituitary and around Rathke's cleft, respectively. We also identified aldolase C-expressing cells in the mouse pituitary using immunohistochemistry and in situ hybridization. Aldolase C was not produced in any pituitary hormone-producing cells, while aldolase C-immunopositive signal co-localized with E-cadherin- and SOX2-positive cells. Using post-embedding immunoelectron microscopy, aldolase C-immunoreactive products were observed in the cytoplasm of marginal cells and folliculo-stellate cells of the mouse pituitary. Taken together, aldolase C is a common folliculo-stellate cell marker in the anterior pituitary gland of rodents.

Fructose-1,6-bisphosphate aldolase encoded by a core gene of Mycoplasma hyopneumoniae contributes to host cell adhesion.

Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein-protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong KD of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin.

[Regulation mechanism of HIFs, PPARs and AMPK in hypoxic training-induced reduction of body weight].

Hypoxic exposure activates hypoxia inducible factors (HIFs) to up-regulate the expression of its target genes. These genes encode glucose metabolism related proteins, such as glucose transporters (GLUTs) and glycolysis related enzymes, including lactate dehydrogenase A (LDHA) and aldolase A (ALDA). Therefore, HIFs participate in oxygenolysis of glucose and play an important role in mediating hypoxia response and weight loss. Exercise training influences fatty acid metabolism, insulin sensitivity and body energy balance through activating peroxisome proliferator-activated receptors (PPARs), which plays an active role in losing weight. In addition, hypoxic exposure or exercise training can activate energy sensor 5'-AMP activated protein kinase (AMPK) in cells and promote oxidation of glucose and fatty acid and weight loss. It has been shown that hypoxic training exerts a better effects on controlling weight, compared with either hypoxic exposure or exercise training alone. This paper reviewed synergistic interactions among HIFs, PPARs and AMPK under hypoxic training and proposed possible mechanisms of hypoxic training-induced weight loss via AMPK-HIFs axis or AMPK-PPARs axis, thus providing theoretical guidance for application of hypoxic training in weight control.

Identification of proteins involved in the adhesionof Candida species to different medical devices.

Adhesion is the first step for Candida species to form biofilms on medical devices implanted in the human host. Both the physicochemical nature of the biomaterial and cell wall proteins (CWP) of the pathogen play a determinant role in the process. While it is true that some CWP have been identified in vitro, little is known about the CWP of pathogenic species of Candida involved in adhesion. On this background, we considered it important to investigate the potential role of CWP of C. albicans, C. glabrata, C. krusei and C. parapsilosis in adhesion to different medical devices. Our results indicate that the four species strongly adher to polyvinyl chloride (PVC) devices, followed by polyurethane and finally by silicone. It was interesting to identify fructose-bisphosphate aldolase (Fba1) and enolase 1 (Eno1) as the CWP involved in adhesion of C. albicans, C. glabrata and C. krusei to PVC devices whereas phosphoglycerate kinase (Pgk) and Eno1 allow C. parapsilosis to adher to silicone-made implants. Results presented here suggest that these CWP participate in the initial event of adhesion and are probably followed by other proteins that covalently bind to the biomaterial thus providing conditions for biofilm formation and eventually the onset of infection.

Proteomic analysis of uterine fluid during the pre-implantation period of pregnancy in cattle.

The aims of this study were (i) to characterize the global changes in the composition of the uterine luminal fluid (ULF) from pregnant heifers during pregnancy recognition (day 16) using nano-LC MS/MS; (ii) to describe quantitative changes in selected proteins in the ULF from days 10, 13, 16 and 19 by Isobaric tags for Relative and Absolute Quantification (iTRAQ) analysis; and (iii) to determine whether these proteins are of endometrial or conceptus origin, by examining the expression profiles of the associated transcripts by RNA sequencing. On day 16, 1652 peptides were identified in the ULF by nano-LC MS/MS. Of the most abundant proteins present, iTRAQ analysis revealed that RPB4, TIMP2 and GC had the same expression pattern as IFNT, while the abundance of IDH1, CST6 and GDI2 decreased on either day 16 or 19. ALDOA, CO3, GSN, HSP90A1, SERPINA31 and VCN proteins decreased on day 13 compared with day 10 but subsequently increased on day 16 (P<0.05). Purine nucleoside phosphorylase (PNP) and HSPA8 decreased on day 13, increased on day 16 and decreased and increased on day 19 (P<0.05). The abundance of CATD, CO3, CST6, GDA, GELS, IDHC, PNPH and TIMP2 mRNAs was greater (P<0.001) in the endometrium than in the conceptus. By contrast, the abundance of ACTB, ALDOA, ALDR, CAP1, CATB, CATG, GD1B, HSP7C, HSP90A, RET4 and TERA was greater (P<0.05) in the conceptus than in the endometrium. In conclusion, significant changes in the protein content of the ULF occur during the pre-implantation period of pregnancy reflecting the morphological changes that occur in the conceptus.

Skeletal muscle aldolase an overexpression in endotoxemic rats and inhibited by GSNO via potential role for S-nitrosylation in vitro.

BACKGROUND: Hepatic aldolase (ALD) A mRNA transcription and ALD B S-nitrosylation have been confirmed in endotoxemic rats and mice, respectively. In the present study we investigated whether the skeletal muscle ALD A shared potential for S-nitrosylation to act as a hypoxia-related signaling mechanism in lipopolysaccharide (LPS) challenged rats. MATERIALS AND METHODS: Male Sprague Dawley rats were treated (i.p.) as follows, control group (n = 6) with 0.9% NaCl, tested group (n = 6) with a single dose of 2 mg/kg LPS. Protein S-nitrosylation was determined by biotin switch and dot blotting analysis. ALD A, hypoxia-inducible factor 1α and vascular endothelial growth factor were determined by western blotting. ALD A catalytic activity treated with S-nitrosoglutathione (GSNO), an exogenous NO-donor, was examined in vitro. RESULTS: There were several S-nitrosylated proteins under basal conditions. ALD A was over-expressed in a hypoxia-related way in the skeletal muscle of LPS challenged rats. Importantly, treatment of ALD A with GSNO at concentration 50 µmol/L ∼ 1000 µmol/L that inhibited catalytic activity, increased the number of S-nitrosylated bands and led to hyper-nitrosylation of basally S-nitrosylated proteins of ALD A. Quantization of enzyme S-nitrosothiol showed that a maximal of four cysteines per subunit was modified by S-nitrosylation in the presence of GSNO. CONCLUSIONS: These findings suggested that S-nitrosylation of ALD A might serve as a novel mechanism for controlling ALD A activity at the post-translational level in endotoxemic rats.

The melanoma brain metastatic microenvironment: aldolase C partakes in shaping the malignant phenotype of melanoma cells - a case of inter-tumor heterogeneity.

Previous studies indicated that microglia cells upregulate the expression of aldolase C (ALDOC) in melanoma cells. The present study using brain-metastasizing variants from three human melanomas explores the functional role of ALDOC in the formation and maintenance of melanoma brain metastasis (MBM). ALDOC overexpression impacted differentially the malignant phenotype of these three variants. In the first variant, ALDOC overexpression promoted cell viability, adhesion to and transmigration through a layer of brain endothelial cells, and amplified brain micrometastasis formation. The cross-talk between this MBM variant and microglia cells promoted the proliferation and migration of the latter cells. In sharp contrast, ALDOC overexpression in the second brain-metastasizing melanoma variant reduced or did not affect the same malignancy features. In the third melanoma variant, ALDOC overexpression augmented certain characteristics of malignancy and reduced others. The analysis of biological functions and disease pathways in the ALDOC overexpressing variants clearly indicated that ALDOC induced the expression of tumor progression promoting genes in the first variant and antitumor progression properties in the second variant. Overall, these results accentuate the complex microenvironment interactions between microglia cells and MBM, and the functional impact of intertumor heterogeneity. Since intertumor heterogeneity imposes a challenge in the planning of cancer treatment, we propose to employ the functional response of tumors with an identical histology, to a particular drug or the molecular signature of this response, as a predictive indicator of response/nonresponse to this drug.

Malignant but not naïve hepatocytes of human and rodent origin are killed by TNF after metabolic depletion of ATP by fructose.

BACKGROUND & AIMS: TNF was the first cytokine employed for cancer therapy, but its use was limited due to its insufficient selectivity towards malignant cells. Fructose induces transient hepatic ATP depletion in humans and rodents due to the liver-specific fructose metabolism via fructokinase, while other cells e.g. Muscle cells metabolize fructose via hexokinase. Under ATP depleted conditions hepatocytes are protected against TNF-induced apoptosis. Our aim was to identify metabolic differences between normal and malignant liver cells that can be exploited for selective immunotherapy. METHODS: We analyzed the expression and activities of enzymes involved in fructose metabolism in primary hepatocytes and hepatoma cell lines. Furthermore, we studied the influence of hexokinase II (HKII) on fructose-mediated ATP depletion and cytoprotection in murine hepatocytes. RESULTS: Primary mouse, rat and human hepatocytes depleted of ATP by fructose were fully protected against TNF-induced cytotoxicity. By contrast, hepatic tumor cell lines showed increased HKII expression, lack of fructose-mediated ATP depletion and, therefore, remained susceptible to TNF/ActD-induced apoptosis. Inhibition of hexokinases restored fructose-induced ATP depletion in hepg2 cells. Finally, hypoxia-inducible factor1 (HIF1)-mediated up-regulation of HKII prevented fructose-induced ATP depletion and overexpression of HKII inhibited fructose-mediated cytoprotection against TNF-induced apoptosis in primary murine hepatocytes. CONCLUSION: Increased expression of HKII in malignant cells of hepatic origin shifts the fructose metabolism from liver- to muscle-type, thereby preventing ATP depletion and subsequent cytoprotection of the target cells. Therefore, healthy liver cells are transiently protected from TNF-mediated cell death by fructose-induced ATP depletion, while malignant cells can be selectively eliminated through TNF-induced apoptosis.

A cryptic role of a glycolytic-gluconeogenic enzyme (aldolase) in amino acid transporter turnover in Aspergillus nidulans.

In Aspergillus nidulans the fbaA1013 mutation results in reduced or total loss of growth on glycolytic and gluconeogenic carbon sources, respectively. It also negatively affects growth on several amino acids (including L-proline, L-glutamate or L-aspartate) that the fungus can use as nitrogen source on glycolytic carbon sources. Complementation of the fbaA1013 mutation using an A. nidulans genomic library resulted in cloning of the fbaA gene, which encodes a putative fructose 1,6-biphosphate aldolase (FBA), an enzyme involved in both glycolysis and gluconeogenesis. The fbaA1013 mutation is a chromosome rearrangement in the 5' regulatory region of the fbaA gene resulting in reduced or total loss of transcription in response to glycolytic and gluconeogenic carbon sources respectively. The fbaA gene is essential for growth. A functional FbaA protein is necessary for plasma membrane localization of the AgtA acidic amino acid (L-glutamate/L-aspartate) transporter, as the fbaA1013 mutation results in targeting to and presumably subsequent degradation of AgtA in the vacuole. Our results support a novel role of the FbaA protein that is, involvement in the regulation of amino acids transporters.

Involvement of aldolase A in X-ray resistance of human HeLa and UV(r)-1 cells.

To find novel proteins involved in radio-resistance of human cells, we searched for nuclear proteins, whose expression levels alter after X-ray irradiation in HeLa cells, using agarose fluorescent two-dimensional differential gel electrophoresis following mass spectrometry. We identified 6 proteins, whose levels were increased in nuclei 24h after irradiation at 5Gy, including aldolase A. Nuclear aldolase A levels increased twofold after the irradiation, however, total aldolase A levels did not change. When the expression of aldolase A was suppressed by its specific siRNA, sensitization of the suppressed cells to X-ray-induced cell death was observed. In addition, UV(r)-1 cells with higher aldolase A expression exhibited lower sensitivity to X-ray-induced cell death than the parental RSa cells with lower aldolase A expression. These results suggest that aldolase A may play a role in the radio-response of human cells, probably in nuclei, in addition to its glycolytic role in the cytosol.

Congruence of zebrin II expression and functional zones defined by climbing fiber topography in the flocculus.

The cerebellum is organized into parasagittal zones with respect to the topography of climbing fiber (CF) afferents and the expression of molecular markers such as zebrin II. Zebrin is expressed by a subset of Purkinje cells that are distributed as a parasagittal array of immunopositive and immunonegative stripes. Several studies in rodents suggest that, in general, CFs to the zebrin negative stripes convey somatosensory information, whereas CFs to the zebrin positive stripes convey information from visual and other sensory systems. The pigeon flocculus consists of four pairs of zebrin+/- stripes (P4 +/- through P7 +/-), however the CF input consists entirely of visual inputs. Thus, because the correspondence of zebrin expression and CF information must be different from that proposed for rodents, we investigated this relationship in the pigeon flocculus. Floccular Purkinje cells respond to patterns of optic flow resulting from self-rotation about one of two axes: either the vertical axis (zones 0 and 2), or a horizontal axis (zones 1 and 3). Visual CF afferents projecting to the flocculus arise from the medial column of the inferior olive (mcIO). Zones 0 and 2 receive input from the caudal mcIO, whereas zones 1 and 3 receive input from the rostral mcIO. We injected a fluorescent anterograde tracer into the rostral and/or caudal mcIO and visualized zebrin expression. There was a strict concordance between CF organization and zebrin labeling: caudal mcIO injections resulted in CFs in zebrin bands P4 +/- and P6 +/-, whereas rostral mcIO injections resulted in CFs in zebrin bands P5 +/- and P7 +/-. Thus, zebrin stripes P4 +/- and P6 +/- correspond to the vertical axis zones 0 and 2, whereas P5 +/- and P7 +/- correspond to the horizontal axis zones 1 and 3. This is the first explicit demonstration that a series of zebrin stripes corresponds with functional zones in the cerebellum.

Aldolase B-Mediated Fructose Metabolism Drives Metabolic Reprogramming of Colon Cancer Liver Metastasis.

Cancer metastasis accounts for the majority of cancer-related deaths and remains a clinical challenge. Metastatic cancer cells generally resemble cells of the primary cancer, but they may be influenced by the milieu of the organs they colonize. Here, we show that colorectal cancer cells undergo metabolic reprogramming after they metastasize and colonize the liver, a key metabolic organ. In particular, via GATA6, metastatic cells in the liver upregulate the enzyme aldolase B (ALDOB), which enhances fructose metabolism and provides fuel for major pathways of central carbon metabolism during tumor cell proliferation. Targeting ALDOB or reducing dietary fructose significantly reduces liver metastatic growth but has little effect on the primary tumor. Our findings suggest that metastatic cells can take advantage of reprogrammed metabolism in their new microenvironment, especially in a metabolically active organ such as the liver. Manipulation of involved pathways may affect the course of metastatic growth.

Aldolases a and C are ribonucleolytic components of a neuronal complex that regulates the stability of the light-neurofilament mRNA.

A 68 nucleotide segment of the light neurofilament (NF-L) mRNA, spanning the translation termination signal, participates in regulating the stability of the transcript in vivo. Aldolases A and C, but not B, interact specifically with this segment of the transcript in vitro. Aldolases A and C are glycolytic enzymes expressed in neural cells, and their mRNA binding activity represents a novel function of these isozymes. This unsuspected new activity was first uncovered by Northwestern blotting of a brainstem/spinal cord cDNA library. It was confirmed by two-dimensional fractionation of mouse brain cytosol followed by Northwestern hybridization and protein sequencing. Both neuronal aldolases interact specifically with the NF-L but not the heavy neurofilament mRNA, and their binding to the transcript excludes the poly(A)-binding protein (PABP) from the complex. Constitutive ectopic expression of aldolases A and C accelerates the decay of a neurofilament transgene (NF-L) driven by a tetracycline inducible system. In contrast, mutant transgenes lacking mRNA sequence for aldolase binding are stabilized. Our findings strongly suggest that aldolases A and C are regulatory components of a light neurofilament mRNA complex that modulates the stability of NF-L mRNA. This modulation likely involves endonucleolytic cleavage and a competing interaction with the PABP. Interactions of aldolases A and C in NF-L expression may be linked to regulatory pathways that maintain the highly asymmetrical form and function of large neurons.

Applications of enzymes in synthetic carbohydrate chemistry.

By photosynthesis 200 billion tons of glucose are formed per annum most of which remains in the sugar state. Thus, the well-known properties of saccharides as the basis for structural materials and energy stores is well established. Increasingly, focus is being addressed to their functions as recognition molecule. Therefore, carbohydrate chemistry became en vogue again as documented by classical syntheses of complex hetero-oligosaccharides employing protecting and activating group chemistry. In recent years enzymatic methods for acylation and deacylation in saccharide chemistry came into use. Novel saccharides could be approached following aldolase-catalyzed de novo syntheses. The very complex task of stereospecific glycosylation was addressed by use of enzymes. With glycohydrolases via transglycosylation as well as with glycosyltransferases including cofactor regenerating circles advances were made. Modified acceptor and also donor substrates allowed access to oligosaccharides in preparative quantities. This paper will discuss these modern developments and will focus on some selected examples from others' and our own laboratory.

Modulation of phosphofructokinase action by macromolecular interactions. Quantitative analysis of the phosphofructokinase-aldolase-calmodulin system.

The simultaneous effect of calmodulin and aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) on the concentration-dependent behaviour of muscle phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been analysed by means of a covalently attached fluorescent probe, gel penetration experiments, and using a kinetic approach. We found that calmodulin-induced inactivation of phosphofructokinase is suspended by addition of an equimolar amount of aldolase. This effect was attributed to an apparent competition of calmodulin and aldolase for the dimeric forms of kinase. Moreover, the direct binding of aldolase to calmodulin has also been demonstrated, which resulted in a significant decrease in the kcat value of the enzyme. The quantitative analysis of these interactions in the system phosphofructokinase-calmodulin-aldolase is presented. A possible molecular model for the modulation of phosphofructokinase action by macromolecular interactions is envisaged.

Structural and functional analysis of aldolase B mutants related to hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired function of human liver aldolase (B isoform). 25 enzyme-impairing mutations have been identified in the aldolase B gene. We have studied the HFI-related mutant recombinant proteins W147R, A149P, A174D, L256P, N334K and delta6ex6 in relation to aldolase B function and structure using kinetic assays and molecular graphics analysis. We found that these mutations affect aldolase B function by decreasing substrate affinity, maximal velocity and/or enzyme stability. Finally, the functional and structural analyses of the non-natural mutant Q354E provide insight into the catalytic role of Arg(303), whose natural mutants are associated to HFI.

The first exon of the rat aldolase C gene is essential for restoring the chromatin structure in transgenic mice.

A 13-kb fragment of the rat aldolase C gene contains sufficient information for gene expression. Transgenic mice carrying the 13-kb fragment showed restoration of chromatin structure and tissue-specific, copy number-dependent expression. To localize the regulatory elements responsible for restoring chromatin structure, several mutated constructs were used to produce transgenic mice. Three activities were examined: recreation of DNase hypersensitive sites, restoration of methylation status, and copy number-dependent expression. Deletions of the 3'-flanking region did not affect those activities. Deletion of seven introns affected the mRNA levels but not the restoration of the chromatin structure. The insertion of the LacZ gene into the first exon of the transgene interfered with both the restoration of the chromatin structure and the copy number-dependent expression in transgenic mice. DNase I footprinting assays revealed that brain-specific factors bind to the sequence disrupted by the LacZ insertion. These results suggest that the sequence in the first exon is essential for restoring the chromatin structure of the rat aldolase C gene.

Fructose-bisphophate aldolase exhibits functional roles between carbon metabolism and the hrp system in rice pathogen Xanthomonas oryzae pv. oryzicola.

Fructose-bisphophate aldolase (FbaB), is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique fbaB gene of Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak, led the pathogen not only unable to use pyruvate and malate for growth and delayed its growth when fructose was used as the sole carbon source, but also reduced extracellular polysaccharide (EPS) production and impaired bacterial virulence and growth in rice. Intriguingly, the fbaB promoter contains an imperfect PIP-box (plant-inducible promoter) (TTCGT-N(9)-TTCGT). The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, the expression of fbaB in X. oryzae pv. oryzicola RS105 strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3 were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3 reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in RΔfbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator.

Compartmentalization of a glycolytic enzyme in Diplonema, a non-kinetoplastid euglenozoan.

Glycosomes are peroxisome-related organelles containing glycolytic enzymes that have been found only in kinetoplastids. We show here that a glycolytic enzyme is compartmentalized in diplonemids, the sister group of kinetoplastids. We found that, similar to kinetoplastid aldolases, the fructose 1,6-bisphosphate aldolase of Diplonema papillatum possesses a type 2-peroxisomal targeting signal. Western blotting showed that this aldolase was present predominantly in the membrane/organellar fraction. Immunofluorescence analysis showed that this aldolase had a scattered distribution in the cytosol, suggesting its compartmentalization. In contrast, orotidine-5'-monophosphate decarboxylase, a non-glycolytic glycosomal enzyme in kinetoplastids, was shown to be a cytosolic enzyme in D. papillatum. Since euglenoids, the earliest diverging branch of Euglenozoa, do not possess glycolytic compartments, these findings suggest that the routing of glycolytic enzymes into peroxisomes may have occurred in a common ancestor of diplonemids and kinetoplastids, followed by diversification of these newly established organelles in each of these euglenozoan lineages.

Physiology and endocrinology symposium: a proteome-based model for sperm mobility phenotype.

Sperm mobility is defined as sperm movement against resistance at body temperature. Although all mobile sperm are motile, not all motile sperm are mobile. Sperm mobility is a primary determinant of male fertility in the chicken. Previous work explained phenotypic variation at the level of the sperm cell and the mitochondrion. The present work was conducted to determine if phenotypic variation could be explained at the level of the proteome using semen donors from lines of chickens selected for low or high sperm mobility. We began by testing the hypothesis that premature mitochondrial failure, and hence sperm immobility, arose from Ca(2+) overloading. The hypothesis was rejected because staining with a cell permeant Ca(2+)-specific dye was not enhanced in the case of low mobility sperm. The likelihood that sperm require little energy before ejaculation and the realization that the mitochondrial permeability transition can be induced by oxidative stress arising from inadequate NADH led to the hypothesis that glycolytic enzymes might differ between lines. This possibility was confirmed by 2-dimensional electrophoresis for aldolase and phosphoglycerate kinase 1. This outcome warranted evaluation of the whole cell proteome by differential detergent fractionation and mass spectrometry. Bioinformatics evaluation of proteins with different expression levels confirmed the likelihood that ATP metabolism and glycolysis differ between lines. This experimental outcome corroborated differences observed between lines in previous work, which include mitochondrial ultrastructure, sperm cell oxygen consumption, and straight line velocity. Although glycolytic proteins were more abundant within highly mobile sperm, quantitative PCR of representative testis RNA, which included mRNA for phosphoglycerate kinase 1, found no difference between lines. In summary, we propose a proteome-based model for sperm mobility phenotype in which a genetic predisposition puts sperm cells at risk of premature mitochondrial failure as they pass through the excurrent ducts of the testis. In other words, we attribute mitochondrial failure to sperm cell and reproductive tract attributes that interact to affect sperm in a stochastic manner before ejaculation. In conclusion, our work provides a starting point for understanding chicken semen quality in terms of gene networks.