Antibody against tubulin from porcine brain was used to examine the distribution of tubulin in developing spermatids of Polytrichum and mature spermatozoids of Sphagnum. Cells were prepared for indirect immunofluorescence microscopy after fixation in buffered paraformaldehyde and brief incubation in cellulase. Pretreatment with cold methanol resulted in considerably enhanced immunofluorescence but exposure to Triton X-100, with or without sonication, had no effect. The antibody showed similar immunological cross-reactivity with the flagella (both basal bodies and axonemes) and the spline microtubules of the multilayered structure. This is the first direct evidence that this rigid array of stable cytoskeletal microtubules consists of tubulin. Particularly intense fluorescence from the lamellar strata of the MLS in developing spermatids provides strong support for the notion that the lamellae comprise a highly structured microtubule organizing centre (MTOC), responsible for the ordered assembly of the overlying spline tubules. The demonstration of immunological cross-reactivity with antitubulin from porcine brain tubulin, within a plant structure other than fully formed microtubules, suggests that immunocytochemistry may have considerable potential for the detection of other MTOCs. By contrast, no detectable fluorescence emanated from the granular matrix cementing the flagellar basal bodies to the spline or the spindle-shaped sheath of fibres present in the spermatozoids of Sphagnum. Disruption of the mature gametes by sonication and treatment with Triton X-100 reveals the presence of particularly strong links between the spline and subjacent nuclear envelope.

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