Hyperactivated motility propels mammalian sperm through oviductal matrix. In spite of its significance in internal fertilization, how the hyperactivated sperm motility is established in vertebrate reproduction is unknown. Here, we found that sperm of the newt, Cynops pyrrhogaster showed a specific state of sperm motility for penetration of oviduct-secreted matrix synonymously to hyperactivated motility. It was triggered by sperm motility-initiating substance (SMIS), which was a novel protein critical for acrosome reaction-associated sperm motility initiation in the internal fertilization of the newt, and localized to the surface of oviduct-secreted matrix accumulated on the egg to form jelly layer. The specific motility state was characterized by strong wave of undulating membrane, a specialized structure including an axoneme at sperm tail, with higher wave velocity at the posterior midpiece compared to the anterior. It was induced with a few minute lag time after the initiation of the waving in the jelly extract, and was suggested to raise fertilization rate in insemination assay. Pharmacological study suggested that the specific motility state was maintained by two types of Ca²⁺ channels (L- and T- types) and those of adenylate cyclases (soluble and membrane-associated) acting with different combinations in the anterior and posterior midpiece.

The SMIS enhanced rotary motion of coil-shaped sperm of the tree frog, Rhacophorus arboreus and flagellum beating with high amplitude in sperm of the toad, Bufo japonicus. The unique sperm motion of each species was highly adapted to distinct nature of oviduct-secreted matrix surrounding eggs that was adapted to specific reproductive mode. This suggests an evolutionary history in amphibians that SMIS worked as a bridge between modification of oviduct-secreted matrix to achieve various reproductive modes and that of sperm motility for the success of fertilization, which optimized motion of the sperm for penetration of oviduct-secreted matrix in the internal fertilization.