Microtubule depolymerization in rat seminiferous epithelium is associated with diminished tyrosination of alpha-tubulin

In the testis, microtubule-disrupting agents cause breakdown of the Sertoli cell cytoskeleton and sloughing of germ cells with associated Sertoli cell fragments, although the mechanism underlying this event is not understood. In this study, we investigated the effects of carbendazim and colchicine o n microtubule polymerization status and posttranslational modifications of tubulin in freshly isolated rat seminiferous tubules. Soluble and polymeriz ed tubulin pools were separated and tubulin was quantified using a competit ive ELISA. Carbendazim and colchicine caused extensive microtubule depolyme rization, shifting the ratio of soluble to polymerized tubulin from 40%:60% to 78%:22%, and to 84%:16%, respectively. Total tubulin levels remained re latively constant after carbendazim treatment but decreased twofold after c olchicine treatment. To determine if modifications to tubulin may be associ ated with polymerization status, tubulin pools were analyzed by immunoblott ing. Acetylated alpha -tubulin and beta III-tubulin distribution in tubulin pools was not affected by treatment. Tyrosinated alpha -tubulin (52 kDa) w as localized in both tubulin pools and had decreased tyrosination in the mi crotubule pool after carbendazim treatment. A 47-kDa protein immunoreactive with both tyrosinated ol-tubulin and general alpha -tubulin antibodies was found only in the microtubule pool. The 47-kDa protein (potentially an alp ha -tubulin isoform) lost tyrosination, yet was still present in the microt ubule pool based on detection with the general alpha -tubulin antibody, aft er carbendazim treatment. Similar effects were seen with colchicine, althou gh loss of total tubulin protein was measured. Thus, decreased tyrosination of the microtubule pool of tubulin appears to be associated with depolymer ization of microtubules.