Eriko Takayama-Watanabe

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 microm) 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.

Sperm motility initiation by egg jelly of the anuran, Discoglossus pictus may be mediated by sperm motility-initiating substance of the internally-fertilizing newt, Cynops pyrrhogaster

The egg jelly of Discoglossus pictus contains sperm motility-activating activity, the molecular basis of which has not been studied. Discoglossus pictus sperm initiated motility immediately after immersion in egg-jelly extract, as well as after immersion in hyposmotic solution, which initiates sperm motility in the external fertilization of anuran amphibians. Sequential treatment of the D. pictus sperm with these two solutions revealed the predominant effect of hyposmolality in initiation of motility. The motility initiation induced by jelly extract was suppressed by a monoclonal antibody (mAb) that is specific for the 34 kDa sperm motility-initiating substance (SMIS) in the egg jelly of the newt, Cynops pyrrhogaster. Immunoblotting using the anti-SMIS mAb revealed several antigenic proteins that included major ones with sizes of 18- and 34-kDa in D. pictus jelly extract. Scanning electron microscopic observation revealed that granules of jelly matrix, in which SMIS localizes and which have a critical role in the internal fertilization of C. pyrrhogaster, were not observed near the surface of the D. pictus egg jelly. These results suggest that sperm motility-activating activity in egg jelly of D. pictus may be mediated by SMIS homologous proteins that act through a mechanism that is partially different from that of C. pyrrhogaster.

Sperm motility-initiating substance in newt egg-jelly induces differential initiation of sperm motility based on sperm intracellular calcium levels

Sperm motility‐initiating substance (SMIS), a novel motility inducer from newt egg‐jelly, is activated by the release from associated jelly substances at the beginning of internal fertilization and affects female‐stored sperm. We examined motility initiation kinetics of newt sperm in response to SMIS by monitoring the changes of sperm intracellular calcium ([Ca2+]i). In quiescent non‐motile sperm loaded with the Ca2+ indicator Fluo‐4, intracellular free Ca2+ was observed around mitochondria using confocal scanning laser microscopy. A slight increase in [Ca2+]i occurred simultaneously and transiently at motility initiation in sperm treated with either heated jelly extract (hJE) containing activated SMIS, or a low osmotic solution, which naturally initiates motility in externally‐fertilizing amphibians and can initiate motility in urodele sperm. When the increase of [Ca2+]i at motility‐initiation was monitored using spectrofluorometry, large increases in [Ca2+]i occurred immediately in the low osmotic solution and within 1.5 min in the hJE. In the intact jelly extract (no heating), small increases of [Ca2+]i irregularly occurred from around 1 min and for about 4 min, during which motility was differentially initiated among sperm. These results indicate that the SMIS induces differential initiation of sperm motility depending on the activational states of the SMIS and its overall activity. The motility initiation in the jelly extract was delayed in sperm whose intracellular Ca2+ had been chelated with BAPTA‐AM. The relative levels of [Ca2+]i were variable with a mean of 414 ± 256 nmol/L among resting sperm, suggesting that the level of [Ca2+]i in the resting sperm modulates the responsiveness to the SMIS.

Distinct Ca2+ channels maintain a high motility state of the sperm that may be needed for penetration of egg jelly of the newt, Cynops pyrrhogaster

Activation state of sperm motility named “hyperactivation” enables mammalian sperm to progress through the oviductal matrix, although a similar state of sperm motility is unknown in non‐mammalian vertebrates at fertilization. Here, we found a high motility state of the sperm in the newt Cynops pyrrhogaster. It was predominantly caused in egg jelly extract (JE) and characterized by a high wave velocity of the undulating membrane (UM) that was significantly higher at the posterior midpiece. An insemination assay suggested that the high motility state might be needed for sperm to penetrate the egg jelly, which is the accumulated oviductal matrix. Specific characteristics of the high motility state were completely abrogated by a high concentration of verapamil, which blocks the L‐type and T‐type voltage‐dependent Ca2+ channels (VDCCs). Mibefradil, a dominant blocker of T‐type VDCCs, suppressed the wave of the UM at the posterior midpiece with separate wave propagation from both the anterior midpiece and the posterior principal piece. In addition, nitrendipine, a dominant L‐type VDCC blocker, weakened the wave of the UM, especially in the anterior midpiece. Live Ca2+ imaging showed that, compared with the intact sperm in the JE, the relative intracellular Ca2+ level changed especially in the anterior and posterior ends of the midpiece of the blocker‐treated sperm. These suggest that different types of Ca2+ channels mediate the intracellular Ca2+ level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.

Contribution of different Ca 2+ channels to the acrosome reaction-mediated initiation of sperm motility in the newt Cynops pyrrhogaster

Initiation of sperm motility in urodeles, which is induced by a sperm motility-initiating substance (SMIS) in the sequestered granules on the surface of egg jelly, is mediated by the acrosome reaction (AR), which is triggered by an AR-inducing substance (ARIS) on a sheet-like structure. Details of the unique process of the interaction between egg jelly and sperm in these species is still unclear. The current study showed the fine structure of egg jelly in the newt Cynops pyrrhogaster, a urodele species, revealing that its outer surface was covered by a sheet-like structure of approximately 0.29 μm in thickness. Granules of approximately 2 μm in diameter with small particles of approximately 54 nm were attached to its surface and distributed inhomogeneously just beneath the sheet-like structure. Emission spectrometry revealed that the Ca2+ concentration was maintained at a high level compared with that of the blood plasma and the vas deferens fluid, suggesting that egg jelly is a reliable source of Ca2+ for the sperm-egg interaction. Blockers of the T-type voltage-dependent Ca2+ channel (VDCC), but not the L-type VDCC, inhibited both AR and initiation of sperm motility. Conversely, Ni+, which affects the α1 H subunit of T-type VDCC, only inhibited the initiation of sperm motility. These data suggest that, in response to ARIS and SMIS, sequential gating of distinct Ca2+ channels occurs in the AR, followed by the initiation of sperm motility on the surface of the egg jelly in C. pyrrhogaster at fertilization.

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

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.

In silico identification of the genes for sperm-egg interaction in the internal fertilization of the newt Cynops pyrrhogaster

A specific sperm-egg interaction in the oviductal matrix is crucial for internal fertilization of the red-bellied newt, Cynops pyrrhogaster. An understanding of the molecular basis of this interaction is expected to elucidate the evolutionary history of internal fertilization in amphibians. Recently, deep sequencing technology has provided global gene information even in nonmodel animals, allowing us to understand specific features of the molecular mechanisms underlying fertilization in C. pyrrhogaster. In the present study, we screened de novo assembled RNAseq from ovary, testis, and oviduct samples in C. pyrrhogaster and identified the base sequences encoding zona pellucida (ZP) proteins, voltage-dependent Ca(2+) channels, and cysteine-rich secretory proteins (CRISPs), which respectively are sperm receptors for egg envelopes, major mediators of sperm intracellular signaling, and expected extracellular modulators for sperm function in the female reproductive tract. In the ovary, ZP homologues of all six subgroups were found, including a ZP1 homologue that was newly found in amphibians, a ZP4 homologue, and six ZPC homologues. The unique combination of ZP proteins suggests a new mechanism for sperm binding to egg envelopes in the internal fertilization of C. pyrrhogaster. In the testis, CaV1.1, 1.2, and 3.2, which are L- and T-type voltage-dependent Ca(2+) channels, were found as potential mediators for the internal fertilization-specific sperm-egg interaction. We also found CRISP 2 in the oviduct, which is speculated to participate in the sperm-egg interaction. These results indicate that the de novo assembled RNAseq is a powerful tool allowing analysis of the specific sperm-egg interactions in C. pyrrhogaster.

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

Sperm motility‐initiating substance (SMIS) is a key protein for internal fertilization of the newt, Cynops pyrrhogaster, and commonly enhances forward sperm motility in some amphibian species, including external fertilizers. SMIS action varies among different species in correlation with a species‐specific reproductive environment. In the present study, we identified the gene of C. ensicauda SMIS (CeSMIS) and examined the mechanism of SMIS action with reference to that of the closely related Cynops species. The CeSMIS was identified by a 176‐amino acid sequence including seven amino acids critical for the initiation of sperm motility. The amino acid sequence showed 91% homology to the whole sequence of C. pyrrhogaster SMIS (CpSMIS). By immunostaining with an anti‐CpSMIS antibody, CeSMIS was shown to be localized in the outer layer of the egg jelly. A peptide presenting the active site of SMIS was observed to bind to the axial rod of the midpiece in C. ensicauda sperm. The localization and binding patterns of CeSMIS were fundamentally similar to those of CpSMIS. However, the SMIS peptide did not induce forward motility of C. ensicauda sperm, although it induced a fast wave of the undulating membrane. Forward sperm motility was induced in the egg jelly extract containing CeSMIS. These results suggest that the mechanism of initiation of sperm motility is differentiated between C. ensicauda and C. pyrrhogaster.

Acrosome Reaction-Mediated Motility Initiation That Is Critical for the Internal Fertilization of Urodele Amphibians

The reproductive modes of extant amphibians are highly diversified in their adaptation to species-specific reproductive environments. In the external fertilization of most amphibians, fertilization begins from motility initiation of the sperm by the hyposmolality of freshwater at spawning, whereas in internal fertilization, the sperm initiate motility without any change in osmolality. Acrosome reaction (AR)-mediated motility initiation is an initial event of the internal fertilization of the urodele amphibian Cynops pyrrhogaster, also known as the Japanese fire belly newt. It initiates motility of female-stored sperm on the surface of the jelly layer in the cloaca. This unique mechanism of motility initiation is based on the fine structure of the jelly matrix, in which AR-inducing substance (ARIS) and sperm motility-initiating substance (SMIS) are localized in the sheet-like structure covering the outer surface and in the granules beneath it. The ARIS and the SMIS repeatedly increase the sperm intracellular Ca2+ level and result in the sporadic initiation of motility within 4 min. The SMIS activity is also present in the jelly layer of the externally fertilizing anuran amphibian Discoglossus pictus (the Mediterranean painted frog), but a thick layer of matrix simply covers the outer surface of the jelly layer. The AR-mediated mechanism may be established in the reproductive strategies for the internal fertilization of urodeles.

Sperm storage influences the potential for spontaneous acrosome reaction of the sperm in the newt Cynops pyrrhogaster

Sperm storage is supposed to influence sperm quality, although the details remain unclear. In the present study, we found that sperm stored in a sperm storage site, the vas deferens of Cynops pyrrhogaster, spontaneously undergo acrosome reaction following incubation in Steinbergs salt solution (ST). Percentages of acrosome‐reacted sperm increased time‐dependently to about 60% in 24 hr. The concentration of cyclic adenosine monophosphate (cAMP) was elevated after incubating sperm in ST, while dibutylyl cAMP induced an acrosome reaction. Chelating of extracellular Ca2+ suppressed the dibutylyl cAMP‐induced acrosome reaction as well as spontaneous acrosome reaction in ST. These results suggest that cAMP elevation driven by Ca2+ influx can be a cue for spontaneous acrosome reaction. Relatively low Ca2+ concentration and pH in the vas deferens were sufficient to suppress spontaneous acrosome reaction within 1 hr. In addition, the cysteine rich secretory protein 2 gene was expressed in the vas deferens, indicating that it may be involved in the continuous suppression of spontaneous acrosome reaction. Sperm that underwent spontaneous acrosome reaction in ST was significantly increased when stored in the vas deferens for longer periods, or by males experiencing temperatures in excess of 12°C during hibernation conditions. Percentages of the spontaneously acrosome‐reacted sperm were found to differ among males even though they were of identical genetic background. Taken together, C. pyrrhogaster sperm possess the potential for spontaneous acrosome reaction that does not become obvious in the vas deferens, unless promoted in correlation with sperm storage.