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

Four site-directed monoclonal antibodies (mAbs) to tubulin: DMA 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 DM(1)A, 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.