Male mice express spermatogenic cell-specific triosephosphate isomerase isozymes.

Triosephosphate isomerase 1 (TPI1) is a member of the glycolytic pathway, which is a critical source of energy for motility in mouse sperm. By immunoblotting, we detected two male, germ line-specific TPI1 bands (Mr 33,400 and 30,800) as well as the somatic-type band (Mr 27,700). Although all three bands were observed in spermatogenic cells, somatic-type TPI1 disappeared from sperm during epididymal maturation. In vitro dephosphorylation analysis suggested that the two male, germ line-specific TPI1 bands were not the result of phosphorylation of the 27,700 Mr TPI1 band. The Mr 33,400; 30,800; and 27,700 TPI1 bands corresponded to the respective sizes of the proteins predicted to use the first, second, and third possible initiation codons of the Tpi1 cDNA. We performed immunofluorescence on epididymal sperm and determined that TPI1 specifically localized in the principal piece. The antibody staining was stronger in cauda epididymal sperm than in caput epididymal sperm, a finding consistent with the identification of TPI1 as a cauda epididymal sperm-enriched protein. Immunofluorescence with sodium dodecyl sulfate (SDS)-insoluble flagellar accessory structures showed a strong TPI1 signal only in the principal piece, indicating that TPI1 is a component of the fibrous sheath. Northern blot hybridization detected longer Tpi1 transcripts (1.56 kb) in mouse testis, whereas somatic tissues had shorter transcripts (1.32 kb). As there is only one triosephosphate isomerase gene in the mouse genome, we conclude that the three variants we see in sperm result from the use of alternative translation start codons in spermatogenic cells.

Characterization of a novel tektin member, TEKT5, in mouse sperm.

Tektins are important components of flagella. Alterations in the expression of or mutations in mouse tektins are correlated with defective sperm motility, a cause of male infertility. Our proteomic studies of flagellar accessory structures previously identified a novel tektin, TEKT5, whose function is unknown. To understand the role of TEKT5 in mouse sperm, we characterized the expression of the mouse Tekt5 gene and the presence of TEKT5 in spermatogenic cells and spermatozoa. A complete cDNA encoding the Tekt5 transcript was assembled following reverse transcription-polymerase chain reaction (RT-PCR) and 3'-rapid amplification of cDNA ends and predicted that TEKT5 is a 62 730-dalton protein with an unusual, long C-terminus. Tekt5 mRNA was highly expressed during late stages of spermiogenesis. Among examined tissues, Tekt5 mRNA was present only in testis and brain, and quantitative RT-PCR showed that the expression level of mRNA in testis was 6.8-fold higher than that in brain. At the protein level, TEKT5 was present in sperm and was enriched in the accessory structures of flagella. Immunofluorescence confirmed that TEKT5 was localized throughout the sperm tail in flagellar accessory structures. The expression pattern suggests that TEKT5 plays an important role in flagella formation during spermiogenesis as well as being implicated in sperm motility.