A CatSper-Uninvolved Mechanism to Induce Forward Sperm Motility in the Internal Fertilization.

Sperm-specific cation channel (CatSper), sperm-specific Na + /H + exchanger (sNHE), and soluble adenylyl cyclase (sAC) are necessary in the signaling pathways to control sperm motility in many animals, whereas some animals have lost some or all of them. In the present study, we examined CatSper-uninvolved signaling for vigorous undulation of the undulating membrane that is attached to the sperm tail and gives thrust for forward motility in the internally fertilizing newt Cynops pyrrhogaster. Reverse-transcription PCR failed to detect sNHE in the newt sperm. However, the pH of sperm cytoplasm was raised under a high extracellular pH equivalent to that of egg jelly, where sperm motility is initiated by sperm motility-initiating substance (SMIS). Carbonic anhydrase XII/ XVI and SLC4A4/8 were suggested to be present in the sperm, and transported bicarbonates raised the intracellular pH. In egg jelly extract that contained SMIS, the anion transporter inhibitor DIDS weakened the undulation of the undulating membrane, while bicarbonates enhanced it. The cyclic AMP concentration was found to increase in sperm cytoplasm in the egg-jelly extract. An inhibitor of sAC (KH7) weakened the undulation of the undulating membrane, and dibutyryl cyclic AMP blocked the inhibitory effect. Inhibitor of transmembrane AC (DDA) limitedly affected the undulation. The undulation was weakened by an inhibitor of protein kinase A (H89), and by an inhibitor of transient receptor potential (TRP) channels (RN1747). Our results support the conclusions that the high pH of the egg jelly triggers a signaling pathway through sAC, PKA, and TRP channels, and coacts with SMIS to induce forward sperm motility.

Identification and characterization of sperm motility-initiating substance-2 gene in internally fertilizing Cynops species.

Sperm motility-initiating substance (SMIS) is an oviductal protein critical for internal fertilization in urodeles. It contributes to the establishment of various reproductive modes in amphibians and is thus a unique research model for the gene evolution of gamete-recognizing ligands that have diversified among animal species. In this study, a paralogous SMIS gene, smis2, was identified via the RNA sequencing of the oviduct of the newt, Cynops pyrrhogaster. The base sequence of the smis2 gene was homologous (˃90%) to that of the original smis gene (smis1), and deduced amino acid sequences of both genes conserved six cysteine residues essential for the cysteine knot motif. Furthermore, smis2 complementary DNA was identified in the oviduct of Cynops ensicauda, and the base substitution patterns also suggested that the smis gene was duplicated in the Salamandridae. Nonsynonymous/synonymous substitution ratios of smis1 and smis2 genes were 0.79 and 2.6, respectively, suggesting that smis2 gene evolution was independently driven by positive selection. Amino acid substitutions were concentrated in the cysteine knot motif of SMIS2. The smis2 gene was expressed in some organs in addition to the oviduct; in contrast, SMIS1 was only expressed in the oviduct. The SMIS2 protein was suggested to be produced and secreted at least in the oviduct and redundantly act in sperm. These results suggest that smis1 plays the original role in the oviduct, whereas smis2 may undergo neofunctionalization, which rarely occurs in gene evolution.

Functionally integrated waveguide illuminator for common-path digital holographic microscopy through random media.

We designed and fabricated a functionally integrated optical waveguide illuminator specially for common-path digital holographic microscopy through random media. The waveguide illuminator creates two point sources with desired phase shifts, which are located close to one another so that the common-path condition of the object and reference illumination is satisfied. Thereby, the proposed device permits phase-shift digital holographic microscopy free from bulky optical elements such as a beam splitter, an objective lens, and a piezoelectric transducer for phase shifting. Using the proposed device, microscopic 3D imaging through a highly heterogeneous double-composite random medium was experimentally demonstrated by means of common-path phase-shift digital holography.

3D imaging through a highly heterogeneous double-composite random medium by common-path phase-shift digital holography.

A method is proposed for 3D imaging through a highly heterogeneous double-composite random medium made of a thick mildly inhomogeneous medium followed by a thin strongly scattering layer. To realize the immunity to the heterogeneous random medium, a system of common-path phase-shift digital holography is designed in such a manner that the wavefront distortion caused by the first inhomogeneous medium is canceled out by the common-path geometry, and the influence of the random phase introduced by the second scattering layer is removed by the intensity-based recording of the digital hologram on the thin scattering layer. The validity of the method was confirmed by experiments.

Differences of Extracellular Cues and Ca2+ Permeable Channels in the Signaling Pathways for Inducing Amphibian Sperm Motility.

Low osmolality of freshwater and/or sperm motility-initiating substance (SMIS) induce amphibian sperm motility through increases in intracellular Ca2+. In the internally fertilizing newt Cynops pyrrhogaster, the sperm motility-initiating substance engages T type voltage-dependent Ca2 + channels and N-methyl D-aspartate-type glutamate receptors to initiate sperm motility and L type voltage-dependent Ca2+ channels to enhance motility. In the present study, differences in the usages of SMIS and Ca2+ permeable channels for sperm motility regulation were examined in amphibians that undergo different reproductive modes. Proteins of 14-17 kDa were detected by antibody against the active site peptide of SMIS in the oviduct secretion of internal fertilizers (C. pyrrhogaster, Cynops ensicauda, and Ambystoma mexicanum) and arboreal fertilizers (Rhacophorus arboreus and Rhacophorus schlegelii), but not in Buergeria japonica, an external fertilizer in freshwater. In the pharmacological study, a blocker of some transient receptor potential channels (RN1734) additionally suppressed enhancement of sperm motility in C. pyrrhogaster. In R. schlegelii, blockers of four types of channels differently suppressed sperm motility induced by low osmolality with or without the active site peptide of SMIS. Notably, blockers of L type voltage-dependent Ca2+ channels (nifedipine) and N-methyl D-aspartate-type glutamate receptors (MK801) suppressed sperm motility in the presence and the absence of the peptide, respectively. Low osmolality-induced sperm motility was suppressed by RN1734 and MK801 in B. japonica, but not in Xenopus laevis. These results reveal complex differences in the signaling pathways for inducing sperm motility that may be partly related to reproductive modes in amphibians.

Localization of sperm intracellular Ca2+ keeps fertilizability in the newt vas deferens.

Sperm intracellular Ca2+ is crucial for the induction of sperm-egg interaction, but little is known about the significance of Ca2+ maintenance prior to induction. In sperm of the newt Cynops pyrrhogaster, intracellular Ca2+ is localized to the midpiece during storage in the vas deferens, while extracellular Ca2+ is influxed in modified Steinberg's salt solution to promote a spontaneous acrosome reaction related to the decline of sperm quality. In the present study, sperm from the vas deferens were loaded with the Ca2+ indicator Fluo8H, and changes in Ca2+ localization in modified Steinberg's salt solution were examined. Calcium ions expanded from the cytoplasmic area of the midpiece to the entire tail in most sperm during a 1-h incubation and localized to the principal piece in some sperm within 24 h. Similar changes in Ca2+ localization were observed in reconstructed vas deferens solution that included ions and pH at equivalent levels to those in the vas deferens fluid. Sperm with Ca2+ localization in the entire tail or the principal piece weakened or lost responsiveness to sperm motility-initiating substances, which trigger sperm motility for fertilization, but responded to a trigger for acrosome reaction. The change in Ca2+ localization was delayed and transiently reversed by ethylene glycol tetraacetic acid or a mixture of Ca2+ channel blockers including Ni2+ and diltiazem. These results suggest that C. pyrrhogaster sperm localize intracellular Ca2+ to the midpiece through Ca2+ transport in the vas deferens to allow for responses to sperm motility-initiating substances.

NMDA-type glutamate receptors mediate the acrosome reaction and motility initiation in newt sperm.

The N-methyl d-aspartate type glutamate receptor (NMDAR) is a ligand-gated cation channel that causes Ca2+ influx in nerve cells. An NMDAR agonist is effective to the sperm motility in fowls, although the actual role of NMDAR in sperm function is unknown. In the present study, RNA-seq of the spermatogenic testes suggested the presence of NMDAR in the sperm of the newt Cynops pyrrhogaster. Glutamate of at least 0.7 ± 0.5 mM was detected in the egg-jelly substances along with acrosome reaction-inducing substance (ARIS) and sperm motility-initiating substance (SMIS). In the egg-jelly extract (JE) that included the ARIS and SMIS, the acrosome reaction was inhibited by a NMDAR antagonists, memantine and MK801. MK801 also inhibited the spontaneous acrosome reaction in Steinberg's salt solution (ST). Furthermore, memantine and MK801 suppressed the progressive motility of the sperm in JE and spontaneous waving of the undulating membrane, which is the tail structure giving thrust for forward motility, in ST. The spontaneous waving of the undulating membrane was promoted when Mg2+ , which blocks Ca2+ influx through gated NMDARs, was removed from the ST. In addition, the ARIS-induced acrosome reaction was inhibited by a selective antagonist of the transient receptor potential vanilloid 4, whose activation might result in the membrane depolarization to release Mg2+ from the NMDAR. These results suggest that NMDAR acts together with other cation channels in the induction of the acrosome reaction and motility of the sperm during the fertilization process of C. pyrrhogaster.

Combined Theoretical and Experimental Studies of Sodium Battery Materials.

Owing to developments in theoretical chemistry and computer power, the combination of calculations and experiments is now standard practice in understanding and developing new materials for battery systems. Here, we briefly review our recent combined studies based on density functional theory and molecular dynamics calculations for electrode and electrolyte materials for sodium-ion batteries. These findings represent case studies of successful combinations of experimental and theoretical methods.

Three-dimensional microscopic imaging through scattering media based on in-line phase-shift digital holography.

Microscopic three-dimensional imaging and phase quantification for objects hidden behind a scattering medium by using in-line phase-shift digital holography are proposed. A spatial resolution of 1.81 µm and highly accurate quantitative phase imaging are demonstrated for objects behind a scatter plate. Three-dimensional imaging was confirmed using objects with a depth difference of 1.32 mm. Further, imaging was performed using rat skin as a demonstration for imaging through a complex multilayer scattering medium, where a spatial resolution close to the theoretically predicted value was achieved by experiment.

Sodium- and Potassium-Hydrate Melts Containing Asymmetric Imide Anions for High-Voltage Aqueous Batteries.

Aqueous Na- or K-ion batteries could virtually eliminate the safety and cost concerns raised from Li-ion batteries, but their widespread applications have generally suffered from narrow electrochemical potential window (ca. 1.23 V) of aqueous electrolytes that leads to low energy density. Herein, by exploring optimized eutectic systems of Na and K salts with asymmetric imide anions, we discovered, for the first time, room-temperature hydrate melts for Na and K systems, which are the second and third alkali metal hydrate melts reported since the first discovery of Li hydrate melt by our group in 2016. The newly discovered Na- and K- hydrate melts could significantly extend the potential window up to 2.7 and 2.5 V (at Pt electrode), respectively, owing to the merit that almost all water molecules participate in the Na+ or K+ hydration shells. As a proof-of-concept, a prototype Na3 V2 (PO4 )2 F3 |NaTi2 (PO4 )3 aqueous Na-ion full-cell with the Na-hydrate-melt electrolyte delivers an average discharge voltage of 1.75 V, that is among the highest value ever reported for all aqueous Na-ion batteries.

Optical correlation-based cross-domain image retrieval system.

A novel cross-domain image retrieval system that is based on a high-speed optical correlator with a coaxial holographic system is presented. Our newly designed conversion module for the optical correlator allows various kinds of data to be converted to pagedata with a uniform optical intensity by using an autoencoder, which is difficult with other conventional methods. By using our conversion module, an existing model for deep learning could be utilized as a feature extractor. A sketch-based cross-domain image retrieval system with the goal of discovering similar photos by querying freehand human sketches was experimentally demonstrated using our optical correlator. We believe that this proposed optical correlation-based system helps expand the applications of the optical correlator.

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.

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 Steinberg's 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.

Endoplasmic Reticulum (ER) Stress Induces Sirtuin 1 (SIRT1) Expression via the PI3K-Akt-GSK3ß Signaling Pathway and Promotes Hepatocellular Injury.

Sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3ß signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage.

Contribution of different Ca²âº 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.

Sperm proteases that may be involved in the initiation of sperm motility in the newt, Cynops pyrrhogaster.

A protease of sperm in the newt Cynops pyrrhogaster that is released after the acrosome reaction (AR) is proposed to lyse the sheet structure on the outer surface of egg jelly and release sperm motility-initiating substance (SMIS). Here, we found that protease activity in the sperm head was potent to widely digest substrates beneath the sperm. The protease activity measured by fluorescein thiocarbamoyl-casein digestion was detected in the supernatant of the sperm after the AR and the activity was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), an inhibitor for serine or cysteine protease, suggesting the release of serine and/or cysteine proteases by AR. In an in silico analysis of the testes, acrosins and 20S proteasome were identified as possible candidates of the acrosomal proteases. We also detected another AEBSF-sensitive protease activity on the sperm surface. Fluorescence staining with AlexaFluor 488-labeled AEBSF revealed a cysteine protease in the principal piece; it is localized in the joint region between the axial rod and undulating membrane, which includes an axoneme and produces powerful undulation of the membrane for forward sperm motility. These results indicate that AEBSF-sensitive proteases in the acrosome and principal piece may participate in the initiation of sperm motility on the surface of egg jelly.

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 Ca(2+) 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 Ca(2+) imaging showed that, compared with the intact sperm in the JE, the relative intracellular Ca(2+) level changed especially in the anterior and posterior ends of the midpiece of the blocker-treated sperm. These suggest that different types of Ca(2+) channels mediate the intracellular Ca(2+) level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.

High-precision microscopic phase imaging without phase unwrapping for cancer cell identification.

Experiments for cell identification are presented using a high-precision cell phase measurement system that does not require any phase unwrapping. This system is based on a Mach-Zehnder interferometer using a phase-locking technique, and it measures the change in optical path length while the sample is scanned across the optical axis. The spatial resolution is estimated to be less than 1.1 µm. The sensitivity of optical path length difference is estimated to be less than 2 nm. Using experiments, we investigate the potential of this approach for cancer cell identification. In our preliminary experiments, cancer cells were distinguished from normal cells through comparison of optical path length differences.

Mandibular and chin electrodes as a supplemental recording for detection of epileptiform discharges in mesial temporal lobe epilepsy.

Background: We previously demonstrated the usefulness of periorbital electrodes in supplemental recording to detect epileptiform discharges in patients with mesial temporal lobe epilepsy (MTLE). However, eye movement may disturb periorbital electrode recording. To overcome this, we developed mandibular (MA) and chin (CH) electrodes and examined whether these electrodes could detect hippocampal epileptiform discharges. Methods: This study included a patient with MTLE, who underwent insertion of bilateral hippocampal depth electrodes and video-electroencephalographic (EEG) monitoring with simultaneous recordings of extra- and intracranial EEG as part of a presurgical evaluation. We examined 100 consecutive interictal epileptiform discharges (IEDs) recorded from the hippocampus and two ictal discharges. We compared these IEDs from intracranial electrodes with those from extracranial electrodes such as MA and CH electrodes in addition to F7/8 and A1/2 of international EEG 10-20 system, T1/2 of Silverman, and periorbital electrodes. We analyzed the number, rate of laterality concordance, and mean amplitude of IEDs detected in extracranial EEG monitoring and characteristics of IEDs on the MA and CH electrodes. Results: The MA and CH electrodes had nearly the same detection rate of hippocampal IEDs from other extracranial electrodes without contamination by eye movement. Three IEDs, not detected by A1/2 and T1/2, could be detected using the MA and CH electrodes. In two ictal events, the MA and CH electrodes detected the ictal discharges from the hippocampal onset as well as other extracranial electrodes. Conclusion: The MA and CH electrodes could detect hippocampal epileptiform discharges as well as A1/A2, T1/T2, and peri-orbital electrodes. These electrodes could serve as supplementary recording tools for detecting epileptiform discharges in MTLE.

MEK-ERK signaling dictates DNA-repair gene MGMT expression and temozolomide resistance of stem-like glioblastoma cells via the MDM2-p53 axis.

Overcoming the resistance of glioblastoma cells against temozolomide, the first-line chemotherapeutic agent of choice for newly diagnosed glioblastoma, is a major therapeutic challenge in the management of this deadly brain tumor. The gene encoding O(6) -methylguanine DNA methyltransferase (MGMT), which removes the methyl group attached by temozolomide, is often silenced by promoter methylation in glioblastoma but is nevertheless expressed in a significant fraction of cases and is therefore regarded as one of the most clinically relevant mechanisms of resistance against temozolomide. However, to date, signaling pathways regulating MGMT in MGMT-expressing glioblastoma cells have been poorly delineated. Here in this study, we provide lines of evidence that the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK)-murine double minute 2 (MDM2)-p53 pathway plays a critical role in the regulation of MGMT expression, using stem-like glioblastoma cells directly derived from patient tumor samples and maintained in the absence of serum, which not only possess stem-like properties but are also known to phenocopy the characteristics of the original tumors from which they are derived. We show that, in stem-like glioblastoma cells, MEK inhibition reduced MDM2 expression and that inhibition of either MEK or MDM2 resulted in p53 activation accompanied by p53-dependent downregulation of MGMT expression. MEK inhibition rendered otherwise resistant stem-like glioblastoma cells sensitive to temozolomide, and combination of MEK inhibitor and temozolomide treatments effectively deprived stem-like glioblastoma cells of their tumorigenic potential. Our findings suggest that targeting of the MEK-ERK-MDM2-p53 pathway in combination with temozolomide could be a novel and promising therapeutic strategy in the treatment of glioblastoma.