Isolation of the rat spermatid manchette and its perinuclear ring.
The manchette is a transient structure that develops during spermiogenesis. It consists of three components: a perinuclear ring, a microtubule mantle inserted in the ring, and dense plaques attached at the distal end of the mantle. A procedure has been developed for the fractionation of intact manchettes from rat spermatids. Each fractionation step was monitored by indirect immunofluorescence using an antibody to unmodified alpha-tubulin. Indirect immunofluorescence and electron microscopy 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 stabilizing buffer containing Taxol formed long bundles of side-by-side aligned microtubules. The perinuclear ring sample consisted of circular-shaped units of different diameter with truncated microtubules still attached 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 alpha-tubulins show that the manchette contains acetylated, tyrosinated, glutamylated alpha-tubulin and an alpha-3/7 tubulin isoform. The same alpha-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 predominant proteins: alpha- and beta-tubulins, beta-actin, vimentin, and a 62-kDa protein. The latter persisted in thermocleaved perinuclear ring samples. Results of this study indicate that the newly developed procedure for the fractionation of manchettes will facilitate a direct characterization of posttranslationally modified tubulin variants, microtubule-associatedproteins, and the components of the perinuclear ring of this largely neglected structure of the spermiogenic process.
Department of Cell Biology and Anatomical Sciences, City University of New York Medical School, New York, New York, 10031, USA.
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Kierszenbaum
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry / Q000648:ultrastructure; D008297:Male; D015388:Organelles / Q000737:chemistry / Q000648:ultrastructure*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013082:Sperm Tail / Q000737:chemistry; D013087:Spermatids / Q000737:chemistry / Q000648:ultrastructure*; D013091:Spermatogenesis; D013347:Subcellular Fractions / Q000737:chemistry / Q000648:ultrastructure; D014404:Tubulin / Q000302:isolation & purification
D014404:Tubulin