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.
Mochida
K
K
Department of Cell Biology and Anatomical Sciences, City University of New York Medical School, New York, New York, 10031, USA.
Tres
L L
LL
Kierszenbaum
A L
AL
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