Rat Spag5 associates in somatic cells with endoplasmic reticulum and microtubules but in spermatozoa with outer dense fibers

Abstract

The leucine zipper motif has been identified as an important and specific interaction motif used by various sperm tail proteins that localize to the outer dense fibers. We had found that rat Odf1, a major integral ODF protein, utilizes its leucine zipper to associate with Odf2, another major ODF protein, Spag4 which localizes to the interface between ODF and axonemal microtubule doublets, and Spag5. The rat Spag5 sequence indicated a close relationship with human Astrin, a microtubule‐binding spindle protein suggesting that Spag5, like Spag4, may associate with the sperm tail axoneme. RT PCR assays indicated expression of Spag5 in various tissues and in somatic cells Spag5 localizes to endoplasmic reticulum and microtubules, as expected for an Astrin orthologue. MT binding was confirmed both in vivo and in in vitro MT‐binding assays: somatic cells contain a 58 kDa MT‐associated Spag5 protein. Western blotting assays of rat somatic cells and male germ cells at different stages of development using anti‐Spag5 antibodies demonstrated that the protein expression pattern changes during spermatogenesis and that sperm tails contain a 58 kDa Spag5 protein. Use of affinity‐purified anti‐Spag5 antibodies in immuno electron microscopy shows that in rat elongated spermatids and epididymal sperm the Spag5 protein associates with ODF, but not with the axonemal MTs. This observation is in contrast to that for the other Odf1‐binding, MT‐binding protein Spag4, which is present between ODF and axoneme. Our data demonstrate that Spag5 has different localization in somatic versus male germ cells suggesting the possibility of different function. Mol. Reprod. Dev.