A Novel Cysteine Knot Protein for Enhancing Sperm Motility That Might Facilitate the Evolution of Internal Fertilization in Amphibians

Abstract

Internal fertilization ensures successful reproduction of tetrapod vertebrates on land, although how this mode of reproduction evolved is unknown. Here, we identified a novel gene encoding sperm motility-initiating substance (SMIS), a key protein for the internal fertilization of the urodele Cynops pyrrhogaster by Edman degradation of an isolated protein and subsequent reverse transcription polymerase chain reaction. The SMIS gene encoded a 150 amino-acid sequence including the cysteine knot (CK) motif. No gene with substantial similarity to the SMIS was in the data bank of any model organisms. An active site of the SMIS was in the C-terminal region of the 2nd loop of CK motif. A synthetic peptide including the active site sequence bound to the midpiece and initiated/enhanced the circular motion of C. pyrrhogaster sperm, which allows penetration of the egg jelly specialized for the internal fertilization of this species. The synthetic peptide bound to whole sperm of Rhacophorus arboreus and enhanced the rotary motion, which is adapted to propel the sperm through egg coat matrix specialized for arboreal reproduction, while it bound to the tip of head and tail of Bufo japonicus sperm, and enhanced the vibratory motion, which is suited to sperm penetration through the egg jelly specialized for the reproduction of that species in freshwater. The polyclonal antibody against the active site of the SMIS specifically bound to egg coat matrix of R. arboreus. These findings suggest that diversification of amphibian reproductive modes accompanies the specialization of egg coat and the adaptation of sperm motility to penetrate the specialized egg coat, and SMIS acts as the sperm motility enhancer of anurans and urodeles that might facilitate to adaptively optimize sperm motility for allowing the establishment of internal fertilization.

Sperm motility initiating substance may be insufficient to induce forward motility of Cynops ensicauda sperm

“…Fluorescein isothiocyanate (FITC)‐conjugated P3 peptide, which contains the amino acid sequence of the SMIS active site, bound to the axial rod of the midpiece and principal piece of C. ensicauda sperm (Figure a). In contrast, control FITC‐conjugated P1 peptide, which contains the amino acid sequence in the amino‐terminal portion of Ce SMIS and shows no effect on sperm motility in C. pyrrhogaster (Yokoe et al, ), bound only to the principal piece (Figure b). These results suggest that Ce SMIS affects the midpiece in C. ensicauda sperm as Cp SMIS does in C. pyrrhogaster sperm (Yokoe et al, ).…”

“…Cp SMIS initiates and enhances sperm motility in C. pyrrhogaster (Ukita, Itoh, Watanabe, & Onitake, ; Watanabe et al, ; Yokoe et al, ). To ask if Ce SMIS has a similar effect in C. ensicauda , the sperm of this species were incubated in the Steinberg's salt solution containing P3 peptide.…”

“…Amphibians reproduce by a variety of modes, including external fertilization in freshwater and internal fertilization (Duellman & Trueb, ; Wake & Dickie, ). Sperm motility‐initiating substance (SMIS) is a key protein for internal fertilization in the newt Cynops pyrrhogaster (Watanabe & Onitake, ; Watanabe et al, ; Yokoe et al, ). SMIS is localized to the external layer of egg jelly, and affects sperm that are mechanically discharged from the sperm reservoir of females.…”

“…SMIS initiates and enhances a wave of the undulating membrane, which includes an axoneme that provides these sperm with a thrust force for forward motility. In a number of amphibian species, SMIS‐induced signal transduction is thought to be involved the induction of forward motility to propel sperm through the egg jelly matrix–albeit with adaptive modifications to various reproductive conditions (Yokoe et al, ). Thus, comparative studies on SMIS signaling among amphibian species could reveal how the signal transduction cascade of the sperm‐egg interaction is modified in different species.…”

“…The localization of SMIS in the egg jelly and the binding site of SMIS in sperm were similar between the two species. However, an active site peptide of SMIS, which induces forward motility of C. pyrrhogaster sperm (Yokoe et al, ), strengthened waving of the undulating membrane but did not induce forward motility of C. ensicauda sperm. Forward motility of the sperm was induced in the egg jelly extract containing Ce SMIS, suggesting that although SMIS signaling involves in the initiation and enhancement of sperm motility in the two Cynops species, a species‐specific difference is present to induce forward sperm motility.…”