Differential post‐translational modifications of microtubules in cells of the seminiferous epithelium of the rat: A light and electron microscope immunocytochemical study

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Abstract
The cells of the seminiferous epithelium of the rat testis are a rich source of microtubules and contain distinct microtubular structures such as the meiotic spindle and manchette. Microtubule diversity can be maintained by differential genetic expression of the multiple α- and β-tubulin polypeptides or by tubulin monomer acetylation and detyrosination, post-translational modifications of α-tubulin. In the present analysis, antibodies that specifically recognize acetylated (antiacetylated), tyrosinated (anti-Tyr) and detyrosinated (anti-Glu) α-tubulins were employed to examine the distribution of post-translationally modified microtubules in the cells of the seminiferous epithelium. In the light microscope, a distinct pattern of staining for each antibody was detected using immunoperoxidase techniques on paraffin-embedded testicular sections. In the case of the anti-Glu antibody, a dense immunoperoxidase staining was detected in the cytoplasm of steps 4–7 spermatids. Thereafter, staining was noted over the area corresponding to the manchette of steps 8–15 spermatids, but not over their cytoplasm. The tails of spermatids were also reactive with this antibody. The anti-Tyr antibody was observed to be localized over the cytoplasm of Sertoli cells in their basal, supranuclear, and apical regions. A dense immunoperoxidase staining was also noted in the cytoplasm of pachytene spermatocytes, but it was negligible in the cytoplasm of spermatocytes undergoing their meiotic division; in these cells the centrioles and meiotic spindle were reactive. The spermatid's tails were also reactive. The antiacetylated antibody showed reactivity only over the tails of spermatids. With the electron microscope, a similar pattern of labeling was noted using immunogold labeling on Lowicryl K4M embedded testicular sections. The anti-Glu antibody heavily labeled microtubules of the manchette and the axoneme of tails of spermatids as well as microtubules of the proximal and distal centrioles and centriolar adjunct. The anti-Tyr antibody strongly labeled microtubules of Sertoli cells and the meiotic spindle and midbody of dividing spermatocytes. The anti-Tyr antibody also labeled the microtubules of the axoneme, centrioles, and centriolar adjunct of spermatids, but to a lesser degree than the anti-Glu antibodies; the manchette was faintly labeled. Of the three antibodies, the antiacetylated antibody showed the weakest labeling of microtubules of the centrioles, centriolar adjunct, and midbody, whereas those of the manchette and Sertoli cells were unreactive; the axoneme was moderately labeled. All three antibodies, however, labeled the microtubules of the tall columnar epithelial cells, referred to as transitional cells, lining the terminal part (tubulus rectus) of the seminiferous tubule. These results thus suggest that post-translational modifications of α-tubulin have an essential role in the functional microtubular diversification of the seminiferous epithelium.