Novel sperm-specific glycolytic isozymes generated by retrotransposition and alternative splicing

Targeted gene disruption of glycolytic enzymes expressed only during spermatogenesis indicates that glycolysis is essential for sperm motility and male fertility. This pathway is compartmentalized in the principal piece of the sperm flagellum, where several spermatogenic cell-specific isozymes are bound to a cytoskeletal structure known as the fibrous sheath. Fructose-1,6-bisphosphate aldolase, or aldolase, is the fourth enzyme in the glycolytic pathway. Genomic and proteomic analyses identified three aldolase A (Aldoa) transcripts generated by retrotransposition and alternative splicing: Aldoa_v2, Aldoart1, and Aldoart2. Unique nucleotide sequence in Aldoart1 provided evidence for an alternatively spliced exon in Aldoa_v2. Expression of all three isozymes was restricted to the male germline and was regulated at transcriptional and translational levels. Both ALDOART1 and ALDOA_V2 are tightly bound to the fibrous sheath and have unique N-terminal extensions that may mediate this binding. ALDOA_V2 is conserved across species and is expressed in rat and human sperm. Preliminary modeling data predicted unique amino acids near functional domains in each isozyme, suggesting distinctive binding and/or catalytic properties. Recombinant sperm ALDOA-related isozymes demonstrated reduced activity when expressed in E. coli, perhaps due to difficulties producing proteins with native conformations. However, analysis of aldolase kinetic parameters in mouse sperm identified a significant portion of the total activity in insoluble fractions, providing initial evidence that the novel ALDOA-related isozymes bound to the fibrous sheath are active.;Glycolytic enzymes with restricted expression during spermatogenesis arose via gene duplication (Gapdhs) and retrotransposition (Pgk2, Aldoart1, Aldoart2). A genomic approach identified all retroposed sequences matching glycolytic enzymes in the human and mouse genomes. Each glycolytic enzyme is encoded by a family of genes, and there is frequent retrotransposition of a single gene in each family. The same orthologous gene is independently retroposed in both species. Results from this study identified an alternative form of Gpi1 transcribed during mouse spermatogenesis. Annotation and expression analysis of all glycolytic enzymes expressed during spermatogenesis will help to understand the regulation of energy metabolism. Since glycolysis is required for sperm motility and male fertility, spermatogenic-cell specific glycolytic enzymes are potential targets for contraceptives. Determining whether defects in the glycolytic pathway are a significant cause of male infertility is also an important clinical need.