Motility initiation of <i>C</i>. <i>pyrrhogaster</i> sperm by the P3 peptide in isotonic solution.

(a) Sperm were suspended in modified Steinberg’s salt solution (ST) containing 1 mM P1, P2, or P3. The percentages of sperm showing circular motion were estimated over 5 min. Dose-dependent P3-initiated sperm motility was induced within the range of 1μM-1mM. (b and c) Sperm of R. arboreus (b) or B. japonicus (c) were suspended in ST or 1/10 ST (1 mM for R. arboreus or 10 mM for B. japonicus). (b) Percentages of motile sperm showing stable or unstable rotary motion over 5 min. (c) Percentages of motile sperm whose flagellum beat with a high amplitude or moderate amplitude over 1 min. Asterisks and double asterisks indicate significant differences at p<0.05 and p<0.01, respectively, against 1/10 ST by Student’s t-test.</p

Identification and characterization of SMIS.

(a) Immunoblotting of the SMIS protein in the JE of C. pyrrhogaster. Substances present in the JE were separated through 2D-electrophoresis and transferred to a PVDF membrane. Immunoreaction was performed using an anti-SMIS monoclonal antibody (mAb)[4]. The upper and lower photographs show Coomassie brilliant blue staining of the gel and an immunoblot image, respectively. Arrowheads indicate the SMIS proteins. (b) Amplification of SMIS cDNA through reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was purified from the indicated organs using the TRIzol® reagent (Invitrogen), and 1 μg of each sample was reverse-transcribed with an oligo-(dT) primer. Subsequently, PCR was performed using a primer set specific for the SMIS gene. In the control, primers specific for the ß-tubulin gene were used for PCR. (c) Immunoblotting of recombinant SMIS protein with an anti-SMIS mAb. Recombinant SMIS protein was produced in Escherichia coli by transfection of an expression vector (pCold TF, TaKaRa Bio, Tokyo, Japan) containing the open reading frame of SMIS cDNA. The protein was subjected to 1D-electrophoresis and immunoblotted with the anti-SMIS mAb. The left two columns show Coomassie Brilliant Blue staining of the acrylamide gel. The right shows specific binding of the anti-SMIS mAb. Arrowheads and arrows indicate the proteins produced from expression vectors with (SMIS) and without (Mock) SMIS cDNA, respectively. (d) A CK motif in the deduced amino acid sequence of the SMIS. The CK motif includes 6 cysteine residues at the 53rd, 62nd, 66th, 87th, 117th, 119th from N-terminus. Disulfide bonds are formed between cysteines-53 and -87, -62 and -117, and -87 and -119 to produce one ring and 3 loops (loops 1–3) that are supposed to be exposed on the outside. P1-P3 indicate the corresponding sites for preparation of the peptides for sperm motility analysis.</p

Salt Effect on the Heat-Induced Association Behavior of Gold Nanoparticles Coated with Poly(<i>N</i>-isopropylacrylamide) Prepared via Reversible Addition−Fragmentation Chain Transfer (RAFT) Radical Polymerization

Poly(N-isopropylacrylamide) (PNIPAM) with a narrow molecular weight distribution was prepared by reversible addition−fragmentation chain transfer (RAFT) radical polymerization. A dithioester group at the chain end of PNIPAM thus prepared was cleaved by treating with 2-ethanolamine to provide thiol-terminated PNIPAM with which gold nanoparticles were coated via reactions of the terminal thiol with gold. The thermoresponsive nature of the maximum wavelength of the surface plasmon band and hydrodynamic radius (Rh) for the PNIPAM-coated gold nanoparticles were found to be sensitively affected by added salt. In pure water, Rh for the PNIPAM-coated gold nanoparticles at 40 °C (>lower critical solution temperature (LCST)) was smaller than that at 25 °C (Rh for the PNIPAM-coated gold nanoparticles at 40 °C was significantly larger than that at 25 °C. Heat-induced association and dissociation for the PNIPAM-coated gold nanoparticles were completely reversible in 50 mM NaCl aqueous solutions, which is responsible for the reversible thermoresponsive color change

P3 peptide binding to sperm.

<p>Sperm of <i>C</i>. <i>pyrrhogaster</i> (a, b), <i>R</i>. <i>arboreus</i> (c, d), <i>B</i>. <i>japonicus</i> (e, f), and <i>X</i>. <i>laevis</i> (g, h) were treated with FITC-P3 (1 mM in a, c, g and 10mM in <b>e</b>) or FITC-P1 (1 mM in b, d, h and 10 mM in f). Bar: 10 μm.</p

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

<div><p>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 <i>Cynops pyrrhogaster</i> by Edman degradation of an isolated protein and subsequent reverse transcription polymerase chain reaction. The <i>SMIS</i> gene encoded a 150 amino-acid sequence including the cysteine knot (CK) motif. No gene with substantial similarity to the <i>SMIS</i> 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 <i>C</i>. <i>pyrrhogaster</i> sperm, which allows penetration of the egg jelly specialized for the internal fertilization of this species. The synthetic peptide bound to whole sperm of <i>Rhacophorus arboreus</i> 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 <i>Bufo japonicus</i> 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 <i>R</i>. <i>arboreus</i>. 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.</p></div

Base sequence of <i>SMIS</i> gene and its deduced amino acid sequence.

<p>Bold line indicates the amino acid sequences obtained by Edman degradation of the SMIS. Gray shade indicated the conserved amino acids in the CK motif of the matrix type[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160445#pone.0160445.ref009" target="_blank">9</a>]. Dotted line shows the sites of the P1-P3 peptides. The P3 peptides with different sizes have same effect on motility. Doubled underline shows polyadenylation signal.</p

Immunolocalization of SMIS in the oviduct-secreted matrix of <i>R</i>. <i>arboreus</i>.

<p>Egg coat matrix of <i>R</i>. <i>arboreus</i> was fixed in methanol and immunostained with the anti-P3 antibody or the anti-SMIS antibody. (a-d) Fluorescence staining with the anti-P3 antibody (a-c) and the anti-P3 antibody preabsorbed by the synthetic peptide including P3 site as a control (d). (e-h) Double-staining with the anti-P3 antibody (f) and the anti-SMIS antibody (g). (h) Merged image. Bar: 200 μm (d) and 50 μm (h).</p