Isolation and biochemical characterization of the human sperm tail fibrous sheath

Abstract

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.

Immunochemical Characterization of a Human Sperm Fibrous Sheath Protein, Its Developmental Expression Pattern, and Morphogenetic Relationships with Actin

“…Unlike MAb 4F7, AJ-FS1 (Jassim and Chen 1994) and S69 (Beecher et al 1993;Homyk and Herr 1992) bind only to the outer side of the FS. The RT97 anti-neurofilament antibody recognizes a single 97-kD peptide of the human FS, showing that the human FS shares an epitope common to anti-200-kD neurofilament (Jassim et al , 1992Jassim 1991). MAb 4F7, which was produced from human spermatozoa, does not react with various seminiferous components and germ cell nuclei as was observed with the RT97 MAb (Jassim 1991).…”

Proteomic identification of human sperm proteins

“…The general lysis and extraction procedure used has the advantage that it provides a general idea of the types of proteins that constitute the sperm proteome. Therefore, this approach should be complementary to other proteomic approaches focusing on specific compartments or organelles such as the cytoplasm, membrane, cytoskeleton, tail, acrosome or sperm head [8][9][10][11][24][25][26][27][28].…”

“…Many efforts to identify sperm proteins have been directed to the nuclear proteins [1,[5][6][7][8], to identify the proteins required for sperm motility [9][10], and in the understanding of the membrane proteins required for capacitation, egg interaction and fertilization [11]. So far, most of the fundamental knowledge on the sperm protein composition has been gained using conventional protein purification and identification strategies [1-6, 12, 13].…”

A-kinase anchoring protein 4 has a conserved role in mammalian spermatogenesis

“…Besides AKAP4, AKAP3 and GAPDHS, many other proteins are located on the flagellum. At least 17 major proteins have been identified in the fibrous sheath in rats (Kim et al 1995b), 14 in humans (Jassim et al 1992), and 10 in rabbits (Kim et al 1995b(Kim et al , 1997. The amino acid composition of the purified fibrous sheath from human, rabbit, and rat spermatozoa are similar, indicating that mammalian sperm tail fibrous sheaths are composed of similar types of proteins (Kim et al 1997).…”