Antigenic determinants of human sperm tail fibrous sheath proteins.

Mouse anti-fibrous sheath antisera (MAFA) produced by immunizing mice with purified preparations of human sperm tail fibrous sheath (FS) reacted with the principal piece of less than 10% of freshly isolated spermatozoa which were immotile and probably dead. Following their demembranation by detergents or repeated freezing and thawing, all spermatozoa were stained. This was also demonstrated on spermatozoa dried onto slides, but the undiluted xenoantisera showed additional reactivity with the acrosomal zone (AZ). Using immunogold electron microscopy, the target antigens were ultrastructurally localized to the FS, and a few spermatozoa showed some reaction at the AZ subacrosomal perinuclear theca. Following titration of the antibodies, the anti-AZ-reaction became undetectable at a dilution of 1:20 while their reactivity with the principal piece continued to a 1:400-dilution. These results indicated that the xenoantisera probably contained an additional unrelated antibody component which reacted with the AZ. Western blotting and staining of purified FS with MAFA detected seven major protein bands with MW ranging between 25 kDa and 97.4 kDa. In human testes, the 1:50 diluted MAFA reacted with sperm tails only, indicating the late expression of the antigenic determinants during spermatogenesis. MAFA did not react with oesophagus, stomach, duodenum, ileum, nasal lining tissues, uterus, pericardium, pancreas, thyroid gland, or cultured fibroblasts. The xenoantisera did, however stain the skin epidermis and cultured keratinocytes which exhibited filamentous cytoplasmic staining although their target antigens could not be biochemically identified. These results indicate that the FS proteins express antigenic determinants which are not shared with other cytoskeletal elements within the sperm flagellum or a variety of somatic tissues.

AJ-p90: a novel protein of the perinuclear theca in human sperm subacrosome.

A perinuclear theca protein of the human sperm subacrosome was detected using LH43 monoclonal antibody which was originally raised against human keratinocytes. Using indirect immunofluorescence, the antibody stained the acrosomal zone (AZ) of dried ejaculated spermatozoa but did not react with viable cells, thus suggesting that the antigen was intracellular. This was confirmed by immunogold electron microscopy which also revealed the ultrastructural localisation of the antigen to the subacrosomal fibrils. Throughout spermatogenesis the antigen was only detected on the AZ of developed testicular spermatozoa and its expression was continued during their epididymal passage. Biochemically, the protein was insoluble in Triton, and dithiothreitol (DTT) was required for its solubilisation. In Western blotting of sperm and keratinocyte lysates, the antibody detected similar 90-kDa protein doublets (AJ-p90). These biochemical features exclude the identity of AJ-p90 with various cyto- and karyo-skeletal antigens, including the intermediate filaments and microfilaments. AJ-p90 therefore represents a novel product of the subacrosomal perinuclear theca. The significance of these data is discussed together with the importance of the antibody for probing the perinuclear theca in normal and abnormal germ cells, including multinucleated spermatids which also showed reactivity with the antibody.

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.

Human sperm tail fibrous sheath undergoes phosphorylation during its development.

The phosphorylation of the human sperm tail fibrous sheath as a maturational step during its development is reported for the first time. This was demonstrated using GDA-J/F3 and RT97 monoclonal antibodies (MoAb) which recognize the fibrous sheath. In indirect immunofluorescence microscopy of frozen sections of human adult testes, the two antibodies reacted with the assembled fibrous sheath only, but the numbers of sperm tails stained with RT97 were consistently lower than those treated with GDA-J/F3. Furthermore, by using double indirect immunofluorescence, although the majority of spermatozoa were doubly stained with the two MoAbs, some GDA-J/F3-positive sperm tails were negative with RT97. In epididymal and ejaculated spermatozoa, the two antibodies stained all the tails. This indicated that the ontogenic appearance of the GDA-J/F3 epitope precedes that of RT97. In Western blotting and/or indirect immunofluorescence of spermatozoa, treatment of samples with alkaline phosphatase abolished the reactivity of RT97 while that of GDA-J/F3 MoAb was not affected. This finding indicated that the RT97 but not the GDA-J/F3 epitope was phosphorylated. Together, these results therefore reveal that during tail morphogenesis, the fibrous sheath undergoes phosphorylation as part of its structural maturation. Screening of sperm cell precursors recovered from oligozoospermic donors showed reaction of some abnormal germ cells with GDA-J/F3 MoAb but not with RT97, suggesting the possible failure of phosphorylation of the fibrous sheath protein in these cells. The significance of these findings is discussed together with the biological importance of phosphorylation to the fibrous sheath.