A novel cysteine knot protein of sperm motility initiating substance revealed a significant process of diversification of reproductive modes to establish internal fertilization in amphibians. (Press release; Oct 18th /2016)

Establishment of internal fertilization in amphibians would have greatly contributed to the transition of vertebrates from water to land, which is suggested by the fact that extant terrestrial vertebrates commonly undergo internal fertilization for their natural reproduction. Whereas, developing reproductive technologies allow reproduction of domestic animals or human by artificial external fertilization. However, little is known about the mechanism involving in the diversification of reproductive modes to establish internal fertilization in vertebrates. Yokoe et al in PLOS ONE 11: e0160445 (2016) first addresses that mechanism by identifying the gene encoding a novel cysteine knot protein of sperm motility initiating substance (SMIS), which is a key protein for the success of internal fertilization of the newt Cynops pyrrhogaster. The SMIS is an amphibian-specific gene since no analogous gene was found in the gene data of any model organisms. Bioassay using an active site peptide of the SMIS demonstrated that SMIS was a common enhancer of sperm motility in anurans and urodeles, and enabled sperm of C. pyrrhogaster and the tree frog Rhacophorus arboreus to penetrate egg coat matrix highly specialized in physicochemical feature for the internal fertilization and for the arboreal fertilization, respectively. Sperm of each species is known to show unique motion based on its specific morphology (long and stiff tail with a undulating membrane in C. pyrrhogaster and spiral shape in R. arboreus), which can provide directional motility only in the viscous matrix of the egg coat. These facts suggest the evolutionary history that sperm motility and morphology has been selectively adapted to the egg coat matrix specialized for the specific reproductive mode in the diversification of reproductive modes of amphibians, and the evolution of SMIS gene might facilitate the selective alteration of sperm motility and morphology to finally establish internal fertilization.


Internal fertilization-specific mechanism of initiation of sperm motility in amphibians: Its molecular basis and evolution

    Reproductive modes of amphibians are highly diversified and, along the evolutional process, internal fertilization is suggested to be established from fundamental external fertilization (Duellman and Trueb. 1986). Actually, 90% of urodele amphibians undergo external fertilization, which occurs in female cloaca. The aim of our project is to understand how the internal fertilization of amphibians is established, which should be a critical event in their adaptation to a variety of conditions on land.


    Amphibian eggs are surrounded by jelly layer, thick extracellular matrices secreted and accumulated in the oviduct. Multiple works show significance of the jelly layer for the success of amphibian fertilization in both external and internal conditions. We have found a new correlation of sperm-egg interaction specific for the urodele mode of internal fertilization in the jelly layer of a red-bellied newt, Cynops pyrrhogaster (Ukita et al., 1999; Watanabe and Onitake, 2003).

     It is based on the specific localizations of acrosome reaction-inducing substance and sperm motility-initiating substance on the jelly surface (Watanabe et al., 2009; Watanabe et al., 2010), which cause a unique pattern of motility initiation among sperm quiescently stored in sperm reservoir for long time. Our recent research focuses on 1) the molecular basis of the novel mechanism of sperm motility initiation and 2) its establishing history on amphibian evolution.


Duellman WE, Trueb L. 1994. Biology of amphibians. Baltimore: Johns Hopkins University Press.
Watanabe A, Onitake K. 2002. Zool. Sci. 19: 1341-1347.
Watanabe A, Fukutomi K, Kubo H, Ohta M, Takayama-Watanabe E, Onitake K. 2009. Mol. Reprod. Dev.79: 399-406.
Watanabe T, Kubo H, Takeshima S, Nakagawa M, Ohta M, Kamimura S, Takayama-Watanabe E, Watanabe A, Onitake K. 2010. Int. J. Dev. Biol. 54: 591-597.