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.

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.

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.

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.

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.

Soft X-ray Emission Studies on Hydrate-Melt Electrolytes.

Highly salt-concentrated aqueous solutions are a new class of electrolytes, which provide a wide potential window exceeding 3 V and, hence, realize possibly inexpensive, safe, and high-energy-density storage devices. Herein, we investigate the evolution of the coordination structure and electronic state depending on the salt concentration through soft X-ray emission spectroscopy and first-principles molecular dynamics calculations. Close to the concentration limit, categorized as a "hydrate melt," a long-range hydrogen-bond network of water molecules disappears with emerging localized electronic states that resemble those in the gas phase. Such localized electronic states are attributed not only to their geometrically isolated nature but also to their dominant electrostatic interaction with Li+ cations. Therefore, the electrical properties of water in the hydrate melt can be more gaslike than liquidlike.

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.

Paroxysmal sympathetic hyperactivity and the later development of epilepsy in a chemotherapy-associated brain damage.

BACKGROUND: Chemotherapy in childhood leukemia potentially induces brain lesions and neurological sequelae. Paroxysmal sympathetic hyperactivity (PSH) is known as a treatment-associated complication; however, the full clinical spectra of PSH remain to be elusive. CASE REPORT: A 5-year-old girl was diagnosed of acute myeloid leukemia (AML) M5. After the intensification therapy, she developed recurrent symptoms of episodic tachycardia, hypertension and perspiration lasting for several hours per day. The low-frequency-high-frequency ratio on Holter electrocardiography was rapidly increased from 0.84 to 2.24 at the onset of the paroxysmal event, whereas the video-monitoring electroencephalography (EEG) never identified ictal patterns of epileptiform discharges during the episodes. Thus, the diagnosis of PSH was given at 7 years of age. Myoclonic and generalized tonic-clonic seizures frequently appeared from 10 years of age, which poorly responded to anticonvulsants. EEG showed diffuse slow-wave bursts with multifocal spikes. Serial head magnetic resonance imaging (MRI) revealed diffuse cerebral and hippocampal atrophy, but not inflammatory lesions in the limbic system. CONCLUSION: We first demonstrate a pediatric case with PSH who developed drug-resistant epilepsy 3 years after the onset of PSH. Our data suggest the pathophysiological link of persistent PSH with chemotherapy-associated brain damage.

Age-related morphological differences in the spike-and-wave complexes of absence epilepsy.

OBJECTIVE: Absence epilepsy shows age-related clinical features, as is observed in childhood and juvenile absence epilepsy. Electroencephalogram (EEG) is characterized by bursts of 3 Hz spike-and-wave complex (SWC). We noticed a morphological variation of the slow-wave component of SWCs between patients. This study investigated whether the waveform of SWC might be associated with the child's age of this epilepsy. METHODS: Digitally-recorded EEGs under medication-free conditions were collected from 25 children who received the diagnosis of childhood or juvenile absence epilepsy. The morphology of slow wave in SWC in the frontal midline region was quantitatively compared between younger and older children using a cluster-based permutation test. RESULTS: At <7 years of age (2.9-6.5 years of age, n = 6), the electrical potential of the descending slope in the slow wave was positively correlated with age whereas this correlation was not observed in patients of ≥7 years of age (7.1-12.9 years, n = 19). A cluster-based permutation test confirmed the results-among the entire slow wave period (0-285 msec), the period of the descending slope (195-260 msec) showed significantly lower potential in patients of <7 years of age in comparison to patients of ≥7 years of age (sum of t-values: 46.57, p-value: 0.011). CONCLUSIONS: The current study demonstrated an age-dependent morphological difference in the slow-wave components of SWCs in EEGs of patients with pediatric absence epilepsy. This finding may provide a clue to understanding the age-related clinical manifestations of this epilepsy.

First-Principles Study on the Cation-Dependent Electrochemical Stabilities in Li/Na/K Hydrate-Melt Electrolytes.

Aqueous alkali-ion batteries, particularly earth-abundant sodium- or potassium-based systems, are potentially safe and low-cost alternatives to nonaqueous Li-ion batteries. Recently, concentrated aqueous electrolytes with Na and K salts as well as Li ones have been extensively studied to increase the voltage of aqueous batteries; however, the potential windows become narrower in the order of Li > Na > K. Here, we study the difference in the potential windows of Li-, Na-, and K-salt concentrated aqueous electrolytes (hydrate melts) by first-principles molecular dynamics. As the Lewis acidity of alkali cations decreases (Li+ > Na+ > K+), the sacrificial reduction of counter anions is less active and water molecules are more aggregated. This situation is unfavorable for achieving stable anion-derived passivation on negative electrodes as well as for being stabilized to oxidation on positive electrodes. Hence, the Lewis acidity of alkali cations is essential to dominate the potential windows of hydrate-melt electrolytes.

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.

Discovery of a new pyrimidine synthesis inhibitor eradicating glioblastoma-initiating cells.

BACKGROUND: Glioblastoma-initiating cells (GICs) comprise a tumorigenic subpopulation of cells that are resistant to radio- and chemotherapies and are responsible for cancer recurrence. The aim of this study was to identify novel compounds that specifically eradicate GICs using a high throughput drug screening approach. METHODS: We performed a cell proliferation/death-based drug screening using 10 560 independent compounds. We identified dihydroorotate dehydrogenase (DHODH) as a target protein of hit compound 10580 using ligand-fishing and mass spectrometry analysis. The medical efficacy of 10580 was investigated by in vitro cell proliferation/death and differentiation and in vivo tumorigenic assays. RESULTS: Among the effective compounds, we identified 10580, which induced cell cycle arrest, decreased the expression of stem cell factors in GICs, and prevented tumorigenesis upon oral administration without any visible side effects. Mechanistic studies revealed that 10580 decreased pyrimidine nucleotide levels and enhanced sex determining region Y-box 2 nuclear export by antagonizing the enzyme activity of DHODH, an essential enzyme for the de novo pyrimidine synthesis. CONCLUSION: In this study, we identified 10580 as a promising new drug against GICs. Given that normal tissue cells, in particular brain cells, tend to use the alternative salvage pathway for pyrimidine synthesis, our findings suggest that 10580 can be used for glioblastoma therapy without side effects.Key Points1. Chemical screening identified 10580 as a novel GIC-eliminating drug that targets DHODH, an essential enzyme for the de novo pyrimidine synthesis pathway. 2. Compound 10580 induced cell cycle arrest, apoptosis, and differentiation in GICs.

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.

First-Principles Study on the Peculiar Water Environment in a Hydrate-Melt Electrolyte.

Aqueous electrolytes have great potential to improve the safety and production costs of Li-ion batteries. Our recent materials exploration led to the discovery of the Li-salt dihydrate melt Li(TFSI)0.7(BETI)0.3·2H2O, which possesses an extremely wide potential window. To clarify the detailed liquid structure and electronic states of this unique aqueous system, a first-principles molecular dynamics study has been conducted. We found that water molecules in the hydrate melt exist as isolated monomers or clusters consisting of only a few (at most five) H2O molecules. Both the monomers and the clusters have electronic structures largely deviating from that in bulk water, where the lowest unoccupied states are higher in energy than that of the Li-salt anions, which preferentially cause anion reduction leading to formation of an anion-derived stable solid-electrolyte interphase. This clearly shows the role of characteristic electronic structure inherent to the peculiar water environment for the extraordinary electrochemical stability of hydrate melts.

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.

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.

Impact of cis- versus trans-Configuration of Butylene Carbonate Electrolyte on Microscopic Solid Electrolyte Interphase Formation Processes in Lithium-Ion Batteries.

The solid electrolyte interphase (SEI) film, which consists of the products of reduction reaction of the electrolyte, has a strong influence on the lifetime and safety of Li-ion batteries. Of particular importance when designing SEI films is its strong dependence on the electrolyte solvent. In this study, we focused on geometric isomers cis- and trans-2,3-butylene carbonates ( c/ t-BC) as model electrolytes. Despite their similar structures and chemical properties, t-BC-based electrolytes have been reported to enable the reversible reaction of graphite anodes [as in ethylene carbonate (EC)], whereas c-BC-based electrolytes cause the exfoliation of graphite [as in propylene carbonate (PC)]. To understand the microscopic origin of the different electrochemical behaviors of t-BC and c-BC, we applied Red Moon simulation to elucidate the microscopic SEI film formation processes. The results revealed that the SEI film formed in c-BC-based electrolytes contains fewer dimerized products, which are primary components of a good SEI film; this lower number of dimerized products can cause reduced film stability. As one of the origins of the decreased dimerization in c-BC, we identified the larger solvation energy of c-BC for the intermediate species and its smaller diffusion constant, which largely diminishes the dimerization. Moreover, the correlation among the Li+ intercalation behavior, nature of the SEI film, and strength of solvation was found to be common for EC/PC and t-BC/ c-BC electrolytes, confirming the importance of solvation of the intermediates in the stability of the SEI film. These results suggest that weakening the solvation of the intermediates is one possible way to stabilize the SEI film for better charge-discharge performance.

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.

Acrosome reaction-inducing substance triggers two different pathways of sperm intracellular signaling in newt fertilization.

The acrosome reaction is induced in the sperm of Cynops pyrrhogaster immediately in response to a ligand protein called acrosome reaction-inducing substance (ARIS) in the egg jelly at fertilization, whereas a spontaneous acrosome reaction occurs time-dependently in correlation with the decline of sperm quality for fertilization. The ARIS-induced acrosome reaction was recently found to be mediated by TRPV4 in association with the NMDA type glutamate receptor, although the intracellular mediators for the acrosome reaction are largely unclear. In the present study, spontaneous acrosome reaction was significantly inhibited by Ni2+, RN1734, and diltiazem, which blocks Cav3.2, TRPV4 or TRPM8, and the cyclic nucleotide-gated channel, respectively. In contrast, expression of Ca2+-activated transmembrane and soluble adenylyl cyclases was detected in the sperm of C. pyrrhogaster by reverse transcription-polymerase chain reaction. Activator of transmembrane or soluble adenylyl cyclases (forskolin or HCO 3-) independently promoted spontaneous acrosome reaction, while an inhibitor of each enzyme (MD12330A or KH7) inhibited it only in the sperm with high potential for spontaneous acrosome reaction. An inhibitor of protein kinase A (H89) inhibited spontaneous acrosome reaction in a manner independent of sperm potential for spontaneous acrosome reaction. Surprisingly, KH7 significantly inhibited ARIS-induced acrosome reaction, but its effect was seen in a small percentage of sperm. H89 had no effect on ARIS-induced acrosome reaction. These results suggest that C. pyrrhogaster sperm possess multiple intracellular pathways for acrosome reaction, involving Ca2+ permeable channels, adenylyl cyclases and PKA, and that two pathways having distinct dependencies on adenylyl cyclases may contribute to ARIS-induced acrosome reaction at fertilization.

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.