| Research Abstract |
Sperm-borne proteins responsible for lysis of the oocyte vitelline coat (VC), that is, VC lysins, were studied in marine mollusks, Haliotis discus (abalone) and Tegula pfelfferi. Biochemical studies have shown that the action of these molluskan VC lysins is stoichiometric rather than enzymatic. To obtain morphological evidence for this uncommon lytic mechanism, the present immunoelectron microscopic study was undertaken using isolated and purified VC lysins and antibodies against these lysins.
Two acrosomal proteins (APs) of molecular weights 20,000 (20K) and 15,500 (15.5K) from abalone spermatozoa are VC lysins. To elucidate the role(s) of each AP in VC lysis, oocytes were examined after treatment with various AP preparations. The intact abalone VC is composed of outer and inner, thin electron-dense layers and a thick main layer of a fine filamentous feltwork. The results suggested that lysis of the outer layer requires both APs but not simultaneously, and that the 15.5K AP caused extr
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eme loosening of the main layer following the action of the 20K AP on the outer layer. Immunogold labeling of dissolved VCs with anti-15.5K lysin antiserum revealed that binding sites of the lysin were densely distributed in loosened VC main layer, indicating non-enzymatic action of the 15.5K lysin. On the other hand, the 20K AP was failed to be localized.
A VC lysin has been isolated from Tegula spermatozoa. The VC of intact oocyte was composed of an innermost basal layer (BL) and an outer layer comprising a number of clusters uniformly distributed along the BL. Higher magnification revealed stellate spicules (SS) underlying cone-shaped deposits of granular structures (GS). Lysin treatment led to elevation of the VC and resultant undulation of the BL, but the composition of the VC appeared unchanged. Immunogold labeling of lysin-treated oocytes with anti-lysin antiserum revealed that binding sites for the lysin were distributed only on the surface of the GS, presenting additional evidence for non-enzymatic action of this lysin. The lysin, however, was adsorbed to the GS and not to the BL, which was changed by the lysin. How is the lysin bound to the GS able to act on the BL across the SS? The answer awaits further study. Less
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