Expression of tubulin isoforms during the differentiation of mammalian spermatozoa

Using the GT 335 mAb we have previously demonstrated a differential expression of glutamylated tubulin isoforms during spermatogenesis and in spermatooza of the mouse and human. Moreover, the proximodistal decrease of the immunolabeling and its predominance in doublets 1-5-6, corresponding to the plane of the flagellar wave, suggested that the glutamylated tubulin could be involved in a functional heterogeneity of microtubules in peripheral doublets of the sperm flagellum. In order to characterize further the importance of glutamylated tubulin in the sperm model, we analyzed tubulin isoforms by immunoblotting and quantitative immunogold, using antibodies to the C-terminal domain of both subunits including non-glutamylated and glutamylated epitopes. The unique differential immunolabeling of the glutamylated tubulin was confirmed with three mAbs 406-3, 392-2 and B3, in addition to GT 335. This differential labeling was interpreted as a differential accessibility of tubulin epitopes since it was greatly reduced in human spermatozoa lacking dynein arms and after motility inhibition of normal spermatozoa by azide pretreatment. We suggest that the glutamylated tubulin interacts with other axonemal and/or periaxonemal proteins which could be involved in flagellar beating and its regulation.