Fertility of spermatozoa depends on maintenance of the mitochondrial transmembrane potential (⌬ m), which is generated by the electron-transport chain and regulated by an oxidation-reduction equilibrium of reactive oxygen intermediates, pyridine nucleotides, and glutathione (GSH). Here, we report that male mice lacking transaldolase (TAL) ؊/؊ are sterile because of defective forward motility. TAL ؊/؊ spermatozoa show loss of ⌬m and mitochondrial membrane integrity because of diminished NADPH, NADH, and GSH. Mitochondria constitute major Ca 2؉ stores; thus, diminished mitochondrial mass accounts for reduced Ca 2؉ fluxing, defective forward motility, and infertility. Reduced forward progression of TAL-deficient spermatozoa is associated with diminished mitochondrial reactive oxygen intermediate production and Ca 2؉ levels, intracellular acidosis, and compensatory down-regulation of carbonic anhydrase IV and overexpression of CD38 and ␥-glutamyl transferase. Microarray analyses of gene expression in the testis, caput, and cauda epididymidis of TAL ؉/؉ , TAL ؉/؊ , and TAL ؊/؊ littermates confirmed a dominant impact of TAL deficiency on late stages of sperm-cell development, affecting the electrontransport chain and GSH metabolism. Stimulation of de novo GSH synthesis by oral N-acetyl-cysteine normalized the low fertility rate of TAL ؉/؊ males without affecting the sterility of TAL ؊/؊ males. Whereas TAL ؊/؊ sperm failed to fertilize TAL ؉/؉ oocytes in vitro, sterility of TAL ؊/؊ sperm was circumvented by intracytoplasmic sperm injection, indicating that TAL deficiency influenced the structure and function of mitochondria without compromising the nucleus and DNA integrity. Collectively, these data reveal an essential role of TAL in sperm-cell mitochondrial function and, thus, male fertility. F orward motility and fertility of spermatozoa depend on production of reactive oxygen intermediates (ROIs) (1) and maintenance of the mitochondrial transmembrane potential (⌬ m ) (2, 3). ⌬ m is generated by the electron-transport chain and subject to regulation by an oxidation-reduction equilibrium of ROI, pyridine nucleotides (NADH͞NAD ϩ NADPH͞NADP), and reduced glutathione (GSH) (4). In turn, NADPH, a reducing equivalent required for biosynthetic reactions and regeneration of GSH from its oxidized form, is produced by the pentose phosphate pathway (PPP) (5). The PPP was originally formulated based on metabolites and enzymes detected in yeast (6). Thus, PPP comprises two separate oxidative and nonoxidative phases. Enzymes of the oxidative phase, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase, can generate both ribose 5-phosphate (R5P) and NADPH. Although enzymes of the nonoxidative phase, transketolase (TK) and transaldolase (TAL), can convert R5P into glucose 6-phosphate (G6P) for the oxidative phase, and, thus, indirectly, these enzymes can also contribute to the generation of NADPH, the significance of the nonoxidative branch is less well established. Certain organisms (7,8) and mammalian tissu...

TAL (transaldolase) was originally described in the yeast as an enzyme of the PPP (pentose phosphate pathway). However, certain organisms and mammalian tissues lack TAL, and the overall reason for its existence is unclear. Recently, deletion of Ser(171) (TALDeltaS171) was found in five patients causing inactivation, proteasome-mediated degradation and complete deficiency of TAL. In the present study, microarray and follow-up Western-blot, enzyme-activity and metabolic studies of TALDeltaS171 TD (TAL-deficient) lymphoblasts revealed co-ordinated changes in the expression of genes involved in the PPP, mitochondrial biogenesis, oxidative stress, and Ca(2+) fluxing. Sedoheptulose 7-phosphate was accumulated, whereas G6P (glucose 6-phosphate) was depleted, indicating a failure to recycle G6P for the oxidative branch of the PPP. Nucleotide analysis showed depletion of NADPH and NAD(+) and accumulation of ADP-ribose. TD cells have diminished Deltapsi(m) (mitochondrial transmembrane potential) and increased mitochondrial mass associated with increased production of nitric oxide and ATP. TAL deficiency resulted in enhanced spontaneous and H(2)O(2)-induced apoptosis. TD lymphoblasts showed increased expression of CD38, which hydrolyses NAD(+) into ADP-ribose, a trigger of Ca(2+) release from the endoplasmic reticulum that, in turn, facilitated CD20-induced apoptosis. By contrast, TD cells were resistant to CD95/Fas-induced apoptosis, owing to a dependence of caspase activity on redox-sensitive cysteine residues. Normalization of TAL activity by adeno-associated-virus-mediated gene transfer reversed the elevated CD38 expression, ATP and Ca(2+) levels, suppressed H(2)O(2)- and CD20-induced apoptosis and enhanced Fas-induced cell death. The present study identified the TAL deficiency as a modulator of mitochondrial homoeostasis, Ca(2+) fluxing and apoptosis.

Transaldolase (TAL) is a key enzyme of the pentose phosphate pathway (PPP). TAL deficiency is a newly recognized cause of liver cirrhosis. We have developed an ion-pair LC separation combined with negative ion electrospray MS/MS detection method to assess PPP metabolites in urine samples from TAL-deficient mice. Sedoheptulose 7-phosphate (S7P), C5-polyols D-arabitol and D-ribitol, and 6-phosphogluconate (6PG) levels were markedly increased in urine of TALdeficient mice with respect to those of wild-type and heterozygote littermates. The detection limits of S7P, D-arabitol, and 6PG were 0.15 ± 0.015 pmol, 3.5 ± 0.41 pmol, and 0.61 ± 0.055 pmol, respectively. The limit of quantitation was 0.4 ± 0.024 nmol/ml for S7P, 1.6 ± 0.11 nmol/ml for 6PG and 10 ± 0.7 nmol/ml for D-arabitol. Additional metabolites,

The possibility of using firefly luciferase as a substrate for an aspartic proteinase was explored. Several amino acid modifications to the C-terminus of the luciferase were created on the basis of the known substrate of the Arabidopsis thaliana aspartic proteinase, pro-(barley lectin). One luciferase with the sequence Arg-Asp-Gly-Val-Phe-Ala-Ala instead of the native Arg-Glu-Ile-Leu-Ile-Lys-Ala at position -15 to -9 relative to the C-terminus of native luciferase was found to possess 17% of the original luciferase activity. When this modified luciferase was incubated with the aspartic proteinase, a specific loss in activity occurred that was not observed with the original luciferase. However, both enzymes seemed very sensitive to the acidic conditions required for aspartic proteinase activity. The other versions of luciferase with different numbers of pro-(barley lectin) amino acids were not active luciferases. This provided information on the structural requirements of the C-terminal portion of the protein for luciferase activity. The luciferase proteins were also monitored during the digestion by using Western blots and some were shown to be substrates for the aspartic proteinase. Contrary to what had been expected, the modified luciferase that incorporated the pro-(barley lectin) sequences was not simply cleaved at the engineered site but at additional positions in the protein. The Arabidopsis aspartic proteinase cleaved two other standard protein substrates at many sites, suggesting that this proteinase could have a role in the degradation of proteins in addition to processing propeptides in plants.