Abstract
BackgroundCABYR is a polymorphic calcium-binding protein of the sperm fibrous sheath (FS) which gene contains two coding regions (CR-A and CR-B) and is tyrosine as well as serine/threonine phosphorylated during in vitro sperm capacitation. Thus far, the detailed information on CABYR protein expression in mouse spermatogenesis is lacking. Moreover, because of the complexity of this polymorphic protein, there are no data on how CABYR isoforms associate and assemble into the FS.MethodsThe capacity of mouse CABYR isoforms to associate into dimers and oligomers, and the relationships between CABYR and other FS proteins were studied by gel electrophoresis, Western blotting, immunofluorescence, immunoprecipitation and yeast two-hybrid analyses.ResultsThe predominant form of mouse CABYR in the FS is an 80 kDa variant that contains only CABYR-A encoded by coding region A. CABYR isoforms form dimers by combining the 80 kDa CABYR-A-only variant with the 50 kDa variant that contains both CABYR-A and CABYR-B encoded by full length or truncated coding region A and B. It is proposed that this step is followed by the formation of larger oligomers, which then participate in the formation of the supramolecular structure of the FS in mouse sperm. The initial expression of CABYR occurs in the cytoplasm of spermatids at step 11 of spermiogenesis and increases progressively during steps 12-15. CABYR protein gradually migrates into the sperm flagellum and localizes to the FS of the principal piece during steps 15-16. Deletion of the CABYR RII domain abolished the interaction between CABYR and AKAP3/AKAP4 but did not abolish the interaction between CABYR and ropporin suggesting that CABYR binds to AKAP3/AKAP4 by its RII domain but binds to ropporin through another as yet undefined region.ConclusionsCABYR expresses at the late stage of spermiogenesis and its isoforms oligomerize and bind with AKAPs and ropporin. These interactions strongly suggest that CABYR participates in the assembly of complexes in the FS, which may be related to calcium signaling.
Highlights
CABYR is a polymorphic calcium-binding protein of the sperm fibrous sheath (FS) which gene contains two coding regions (CR-A and coding region B (CR-B)) and is tyrosine as well as serine/threonine phosphorylated during in vitro sperm capacitation
Generation of recombinant CABYR-A and CABYR-B proteins and anti-CABYR-A, anti-CABYR-B polyclonal antibodies Mouse CABYR has three alternative splice variants involving two coding regions, coding region A (CR-A) and CR-B, which are separated by dual stop codons [19]
(1) Mouse CABYR forms homodimers, heterdimers and oligomers mainly involving the most abundant 80 kDa variant (CABYR-A only) and the 48 kDa form. (2) Mouse CABYR expression begins in the spermatid cytoplasm at step 11 of spermiogenesis and progressively increases though steps 13-15, and CABYR subsequently localizes on the surface of the FS during steps 15-16
Summary
CABYR is a polymorphic calcium-binding protein of the sperm fibrous sheath (FS) which gene contains two coding regions (CR-A and CR-B) and is tyrosine as well as serine/threonine phosphorylated during in vitro sperm capacitation. Observations indicate that the FS plays important roles in energy metabolism, ATP generation for sperm motility, calcium signaling, and as a scaffold for signaling molecules in addition to its role as a structural girdle surrounding the outer dense fibers and axoneme. A model has been proposed in which the FS represents a highly ordered complex, somewhat analogous to the electron transport chain, in which adjacent enzymes in the glycolytic pathway are assembled to permit efficient flux of energy substrates and products, possibly as a nucleotide shuttle between flagellar glycolysis, protein phosphorylation and mechanisms of motility [17]
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