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

Frequent and recent retrotransposition of orthologous genes plays a role in the evolution of sperm glycolytic enzymes

<p><b>Abstract</b></p> <p>Background</p> <p>The central metabolic pathway of glycolysis converts glucose to pyruvate, with the net production of 2 ATP and 2 NADH per glucose molecule. Each of the ten reactions in this pathway is typically catalyzed by multiple isozymes encoded by a multigene family. Several isozymes in this pathway are expressed only during spermatogenesis, and gene targeting studies indicate that they are essential for sperm function and male fertility in mouse. At least three of the novel glycolytic isozymes are encoded by retrogenes (<it>Pgk2</it>, <it>Aldoart1</it>, and <it>Aldoart2</it>). Their restricted expression profile suggests that retrotransposition may play a significant role in the evolution of sperm glycolytic enzymes.</p> <p>Results</p> <p>We conducted a comprehensive genomic analysis of glycolytic enzymes in the human and mouse genomes and identified several intronless copies for all enzymes in the pathway, except <it>Pfk</it>. Within each gene family, a single orthologous gene was typically retrotransposed frequently and independently in both species. Several retroposed sequences maintained open reading frames (ORFs) and/or provided evidence of alternatively spliced exons. We analyzed expression of sequences with ORFs and <99% sequence identity in the coding region and obtained evidence for the expression of an alternative <it>Gpi1 </it>transcript in mouse spermatogenic cells.</p> <p>Conclusions</p> <p>Our analysis detected frequent, recent, and lineage-specific retrotransposition of orthologous glycolytic enzymes in the human and mouse genomes. Retrotransposition events are associated with LINE/LTR and genomic integration is random. We found evidence for the alternative splicing of parent genes. Many retroposed sequences have maintained ORFs, suggesting a functional role for these genes.</p

Three male germline-specific aldolase A isozymes are generated by alternative splicing and retrotransposition

Enzymes in the glycolytic pathway of mammalian sperm are modified extensively and are localized in the flagellum, where several are tightly bound to the fibrous sheath. This study provides the first evidence for three novel aldolase isozymes in mouse sperm, two encoded by Aldoart1 and Aldoart2 retrogenes on different chromosomes and another by Aldoa_v2, a splice variant of Aldoa. Phylogenetic analyses and comparative genomics indicate that the retrogenes and splice variant have remained functional and have been under positive selection for millions of years. Their expression is restricted to the male germline and is tightly regulated at both transcriptional and translational levels. All three isozymes are present only in spermatids and sperm and have distinctive features that may be important for localization in the flagellum and/or altered metabolic regulation. Both ALDOART1 and ALDOA_V2 have unusual N-terminal extensions not found in other aldolases. The N-terminal extension of ALDOA_V2 is highly conserved in mammals, suggesting a conserved function in sperm. We hypothesize that the N-terminal extensions are responsible for localizing components of the glycolytic pathway to the fibrous sheath and that this localization is required to provide sufficient ATP along the length of the flagellum to support sperm motility