Composition and organization of tubulin isoforms reveals a variety of axonemal models.

In the flagellum of mammalian spermatozoa, glutamylated and glycylated tubulin isoforms are detected according to longitudinal gradients and preferentially in axonemal doublets 1-5-6 and 3-8, respectively. This suggested a role for these tubulin isoforms in the regulation of flagellar beating. In the present work, using antibodies directed against various tubulin isoforms and quantitative immunogold analysis, we aimed at investigating whether the particular accessibility of tubulin isoforms in the mammalian sperm flagellum is restricted to this model of axoneme surrounded with periaxonemal structures or is also displayed in naked axonemes. In rodent lung ciliated cells, all studied tubulin isoforms are uniformly distributed in all axonemal microtubules with a unique deficiency of glutamylated tubulin in the transitional region. A similar distribution of tubulin isoforms is observed in cilia of Paramecium, except for a decreasing gradient of glutamylated tubulin labeling in the proximal part of axonemal microtubules. In the sea urchin sperm flagellum, predominant labeling of tyrosinated and detyrosinated tubulin in 1-5-6 and 3-8 doublets, respectively, were observed together with decreasing proximo-distal gradients of glutamylated and polyglycylated tubulin labeling and an increasing gradient of monoglycylated tubulin labeling. In flagella of Chlamydomonas, the glutamylated and glycylated tubulin isoforms are detected at low levels. Our results show a specific composition and organization of tubulin isoforms in different models of cilia and flagella, suggesting various models of functional organization and beating regulation of the axoneme.

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.

Glutamylated tubulin as a marker of microtubule heterogeneity in the human sperm flagellum.

Four site-directed monoclonal antibodies (mAbs) to tubulin: DM1A and DM1B general anti-alpha and anti-beta tubulin, 6-11B-1 anti-acetylated alpha tubulin and GT335 anti-glutamylated alpha and beta tubulin were used to study the distribution of tubulin isoforms in the human sperm flagellum. Since indirect immunofluorescence (IIF) did not give reliable results, a quantitative study of the immunogold labelling of the flagellum was performed at five levels: the mid-piece, three successive regions of the principal piece and the terminal piece. A uniform labelling was observed with DM1A, DM1B and 6-11B-1 mAbs. In contrast, the labelling of glutamylated tubulin detected with GT335 mAb decreased from the middle piece to the terminal piece both for peripheral doublets and the central pair. The changes in labelling of peripheral doublets were related to the pattern of outer dense fibre (ODF) changes. Thus doublets 1-5-6, associated with the largest number of ODF, were the most heavily labelled. This predominant labelling corresponded to the plane of flagellar beating suggesting a functional heterogeneity of peripheral doublets.