Isolation of the rat spermatid manchette and its perinuclear ring

被引:47
作者
Mochida, K [1 ]
Tres, LL [1 ]
Kierszenbaum, AL [1 ]
机构
[1] CUNY, Sch Med, Dept Cell Biol & Anat Sci, New York, NY 10031 USA
关键词
sperm nuclear shaping; microtubules; axoneme; sperm tail;
D O I
10.1006/dbio.1998.8942
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
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-associated proteins, and the components of the perinuclear ring of this largely neglected structure of the spermiogenic process. (C) 1998 Academic Press.
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页码:46 / 56
页数:11
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