Identification of the sperm motility-initiating substance in the newt, Cynops pyrrhogaster, and its possible relationship with the acrosome reaction during internal fertilization

Motility initiation is a key event during internal fertilization of female-stored sperm, although the underlying mechanisms remain unclear. In internally fertilizing urodeles, quiescent sperm initiate motility on the surface of the egg-jelly, a thick extracellular matrix that accumulates around the egg in oviduct. By immunizing mice with egg-jelly extracts, we successfully generated an alpha34 monoclonal antibody (mAb) which neutralized sperm motility-initiating activity in the egg-jelly of the newt, Cynops pyrrhogaster, in a dose-dependent manner. The alpha34 mAb recognized an unglycosylated 34 kDa protein in the outermost of the six layers that comprise egg-jelly. Under nonreducing conditions, immunoblotting with alpha34 mAb produced many bands in addition to the 34 kDa protein, suggesting that the 34 kDa protein associates not only with the jelly matrix itself, but also with additional substances present in the matrix. Our current results are compatible with the supposed features of sperm motility-initiating substance (SMIS), indicating that the 34 kDa protein itself, or a complex consisting of the 34 kDa protein and some other molecules, is the SMIS in C. pyrrhogaster. Immunofluorescence staining further indicated that SMIS was distributed in a dot-like pattern in the outermost jelly layer and was fully covered with acrosome reaction-inducing substance (ARIS). Immunocytochemical and scanning electron microscopic examinations of the outermost jelly layer strongly suggests that the 34 kDa protein localized in granules (2 microns) and that ARIS was distributed covering the granules and in the sheet-like structure above the granules. These data suggest that the initiation of sperm motility is mediated by the acrosome reaction.