ISOLATION OF THE RAT SPERMATID MANCHETTE AND ITS PERINUCLEAR RING

The manchette is a transient structure that develops during spermiogen esis. It consists of three components: a perinuclear ring, a microtubu le mantle inserted in the ring, and dense plaques attached at the dist al end of the mantle. A procedure has been developed for the fractiona tion of intact manchettes from rat spermatids. Each fractionation step was monitored by indirect immunofluorescence using an antibody to unm odified alpha-tubulin. Indirect immunofluorescence and electron micros copy demonstrate that fractionated manchettes are relatively intact. A thermocleavage step was used to sever the microtubule mantle from the perinuclear ring. Microtubules of the mantle collected in a stabilizi ng buffer containing Taxol formed long bundles of side-by-side aligned microtubules. The perinuclear ring sample consisted of circular-shape d units of different diameter with truncated microtubules still attach ed to the ring, a property that enabled the initial recognition of the rings by alpha-tubulin antibody staining. Indirect immunofluorescence and immunoblotting experiments using isoform-specific antibodies to a lpha-tubulins show that the manchette contains acetylated, tyrosinated , glutamylated alpha-tubulin and an alpha-3/7 tubulin isoform. The sam e cy-tubulin isoforms were observed in the axoneme of the sperm tail. Two-dimensional polyacrylamide gel electrophoresis fractionation maps of silver-stained proteins of the intact manchette show four predomina nt proteins: alpha- and beta-tubulins, beta-actin, vimentin, and a 62- kDa protein. The latter persisted in thermocleaved perinuclear ring sa mples. Results of this study indicate that the newly developed procedu re for the fractionation of manchettes will facilitate a direct charac terization of posttranslationally modified tubulin variants, microtubu le-associated proteins, and the components of the perinuclear ring of this largely neglected structure of the spermiogenic process. (C) 1998 Academic Press.