Isolation and biochemical characterization of the human sperm tail fibrous sheath.
The isolation and biochemical characterization of the human sperm tail fibrous sheath (FS) is described for the first time. Initially, the solubilization properties of the FS were assessed immunocytochemically using GDA-J/F3 and RT97 monoclonal antibodies (MoAbs) and morphologically by electron microscopy. Following extensive investigations to optimize the conditions for the FS isolation, a simple method was developed which involved sequential extraction of the flageller components with Triton-dithiothreitol (DTT) and urea-DTT. The procedure was monitored by phase contrast microscopy and the purity of the FS preparations was confirmed by electron microscopy. SDS-PAGE of the isolated FS revealed seven major protein bands with mol. wt of 97, 76, 62, 55, 33, 28 and 25 kDa. In Western blotting, the reaction of RT97 MoAb with supernatants from the various extraction steps and the isolated FS indicated that its target antigen (AJ-p97) was an integral FS product and that disulphide bonding was probably involved in its stabilization. The reactivity of normal and aprotruded sperm tails with GDA-J/F3 and RT97 MoAbs was not affected by Triton while the GDA-J/F3 staining of the cytoplasmic matrix of other abnormal spermatids was abolished, thus suggesting variation in the biochemical properties of GDA-J/F3 in normal and abnormal germ cells. These and other data indicate that the FS could be a modified form of intermediate filament.
Jassim
A
A
Department of Immunology, London Hospital Medical College, UK.
Gillott
D J
DJ
al-Zuhdi
Y
Y
Gray
A
A
Foxon
R
R
Bottazzo
G F
GF
D000911:Antibodies, Monoclonal; D003598:Cytoskeletal Proteins / Q000276:immunology / Q000302:isolation & purification*; D004220:Disulfides; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D011092:Polyethylene Glycols; D013082:Sperm Tail / Q000737:chemistry* / Q000648:ultrastructure; D013094:Spermatozoa / Q000002:abnormalities; D014508:Urea
D000911:Antibodies, Monoclonal; D003598:Cytoskeletal Proteins; D004220:Disulfides; D011092:Polyethylene Glycols; D014508:Urea; D004229:Dithiothreitol