Spawning-Induced pH Increase Activates Sperm Attraction and Fertilization Abilities in Eggs of the Ascidian, Phallusia philippinensis and Ciona intestinalis.

In Phlebobranchiata ascidians, oocytes and spermatozoa are stored in the oviduct and spermiduct, respectively, until spawning occurs. Gametes in the gonoducts are mature and fertilizable; however, it was found that the gametes of the ascidians Phallusia philippinensis and Ciona intestinalis could not undergo fertilization in the gonoductal fluids. The body fluids of the ascidians, especially in the gonoducts, were much more acidic (pH 5.5-6.8) than seawater (pH 8.2), and the fertilization rate was low under such acidic conditions. Hence, we examined the effect of pH on gametes. Pre-incubation of gonoductal eggs at pH 8.2 prior to insemination increased fertilization rates, even when insemination was performed under low pH conditions. Furthermore, an increase in ambient pH induced an increase in the intracellular pH of the eggs. It was also found that an increase in ambient pH triggered the release of sperm attractants from the egg and is therefore necessary for sperm chemotaxis. Hence, acidic conditions in the gonoductal fluids keep the gametes, especially eggs, infertile, and the release of eggs into seawater upon spawning induces an increase in ambient pH, which enables egg fertilization.

A novel role for ATP2B in ascidians: Ascidian-specific mutations in ATP2B contribute to sperm chemotaxis.

Sperm chemotaxis, in which sperms are attracted to conspecific eggs via species-specific attractants, plays an important role in fertilization. This phenomenon has been observed in various animals and species-specific sperm attractants have been reported in some species. However, the mechanisms involved in the reception and recognition of the species-specific attractant by the sperms is poorly studied. Previously, we found that the plasma membrane-type Ca2+ /ATPase (PMCA) is the receptor for the sperm-activating and -attracting factor (SAAF) in the ascidian Ciona intestinalis. To determine the role of PMCA in species-specific sperm chemotaxis, we identified the amino acid sequences of PMCAs derived from six Phlebobranchia species. The testis-specific splice variant of PMCA was found to be present in all the species investigated and the ascidian-specific sequence was detected near the 3'-terminus. Moreover, dN/dS analysis revealed that the extracellular loops 1, 2, and 4 in ascidian PMCA underwent a positive selection. These findings suggest that PMCA recognizes the species-specific structure of SAAF at the extracellular loops 1, 2, and 4, and its testis-specific C-terminal region is involved in the activation and chemotaxis of ascidian sperms.

Kulikovia alborostrata and Kulikovia fulva comb. nov. (Nemertea: Heteronemertea) are Sister Species with Prezygotic Isolating Barriers.

The heteronemertean Kulikovia alborostrata (Takakura, 1898) was originally described as Lineus alborostratus based on material from Misaki, Japan. Although this species was regarded as consisting of two color variants, purple and brown-yellow, the identity of these variants has never been examined based on topotypes. In this study, we performed a multi-locus phylogeny reconstruction, species delimitation analyses, and cross-fertilization experiments to examine the species status of Takakura's original taxon concept consisting of these color variants. Our results suggest that the purple type is identical to Lineus alborostratus Takakura, 1898 auct. (currently Kulikovia alborostrata), whereas the brown-yellow type is conspecific with Lineus fulvus Iwata, 1954, originally established from Hokkaido. These two species appear to have a sister-taxon relationship and are reproductively isolated from each other by prezygotic mechanisms involving gamete incompatibility, minimally separated with 2.8% (16S rRNA) and 14.4% (COI) uncorrected p-distances. We propose that the purple type be considered as representing the true identity of the nominal species Lineus alborostratus (currently assigned to the genus Kulikovia) to maintain the common usage of the name. Although Takakura's type material is not extant, we consider that neotypification is unnecessary in this case because no taxonomic/nomenclatural confusion persists. We also propose to transfer Lineus fulvus to yield Kulikovia fulva comb. nov.

DATSURYOKU Sensor-A Capacitive-Sensor-Based Belt for Predicting Muscle Tension: Preliminary Results.

Excessive muscle tension is implicitly caused by inactivity or tension in daily activities, and it results in increased joint stiffness and vibration, and thus, poor performance, failure, and injury in sports. Therefore, the routine measurement of muscle tension is important. However, a co-contraction observed in excessive muscle tension cannot be easily detected because it does not appear in motion owing to the counteracting muscle tension, and it cannot be measured by conventional motion capture systems. Therefore, we focused on the physiological characteristics of muscle, that is, the increase in muscle belly cross-sectional area during activity and softening during relaxation. Furthermore, we measured muscle tension, especially co-contraction and relaxation, using a DATSURYOKU sensor, which measures the circumference of the applied part. The experiments showed high interclass correlation between muscle activities and circumference across maximal voluntary co-contractions of the thigh muscles and squats. Moreover, the circumference sensor can measure passive muscle deformation that does not appear in muscle activities. Therefore, the DATSURYOKU sensor showed the potential to routinely measure muscle tension and relaxation, thus avoiding the risk of failure and injury owing to excessive muscle tension and can contribute to the realization of preemptive medicine by measuring daily changes.

Does the Rainbow Trout Ovarian Fluid Promote the Spermatozoon on Its Way to the Egg?

The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we confirmed here for rainbow trout (Oncorhynchus mykiss). The ovarian fluid affected significantly spermatozoa performance: it supported high velocity for a longer period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout ovarian fluid induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating medium. The interaction of the spermatozoa with the attracting agents was accompanied by the "turn-and-run" behavior involving asymmetric flagellar beating and Ca2+ concentration bursts in the bent flagellum segment, which are characteristic of the chemotactic response. Ovarian fluid created the optimal environment for rainbow trout spermatozoa performance, and the individual peculiarities of the egg (ovarian fluid)-sperm interaction reflect the specific features of the spawning process in this species.

Human Semenogelin 1 Promotes Sperm Survival in the Mouse Female Reproductive Tract.

Semenogelin 1 (SEMG1), a main component of human seminal plasma, is a multi-functional protein involved in the regulation of sperm motility and fertility. SEMG1 is orthologous to mouse seminal vesicle secretion 2 (SVS2), required for sperm survival in the female reproductive tract after copulation; however, its in vivo function remains unclear. In this study, we addressed this issue by examining the effect of recombinant SEMG1 on intrauterine mouse sperm survival. SEMG1 caused a dose-dependent decrease in mouse sperm motility, similar to its effect on human sperm, but SVS2 had no effect on mouse sperm motility. Mouse epididymal sperm in the presence of 100 µM SEMG1, a concentration that does not affect mouse sperm motility, were injected into the mouse uterus (intrauterine insemination, IUI). IUI combined with SEMG1 significantly increased the survival rate of intrauterine mouse sperm. The effect of SEMG1 on intrauterine sperm survival was comparable with that of SVS2. For clinical applications, three potentially sperm-protecting polypeptides that are easy to handle were designed from SEMG1, but their individual use was unable to mimic the ability of SEMG1. Our results indicate that SEMG1 has potential clinical applications for effective IUI and thereby for safe, simple, and effective internal fertilization.

A Population Without Females: Males of Baseodiscus delineatus (Nemertea: Heteronemertea) Reproduce Asexually by Fragmentation.

The valenciniid heteronemertean Baseodiscus delineatus (Delle Chiaje, 1825) was originally described from Naples, Italy, and shows a circumglobal distribution from tropical to temperate seas in both hemispheres. To investigate its reproductive biology, we performed intermittent year-round sampling from 2014 to 2018 in Misaki on the Pacific coast of Honshu, Japan. Of the 40 specimens obtained, 11 were males, while 29 were immature. No female specimen was confirmed during the sampling period. We additionally observed that B. delineatus is fissiparous. A male individual in captivity reproduced asexually by spontaneous posterior fragmentation, an ability that is described here for the first time among Valenciniidae. Autotomy occurred every 2-10 days, with each of the tail fragments having undergone anterior regeneration, which took about 24-36 days before completion of internal organs, such as ocelli, cerebral organs, brain, alimentary canal, rhynchocoel, and proboscis. A review of the literature suggests that the species was absent in the Misaki region 120 years ago. We assume a recent settlement of a male founder, which has putatively reproduced asexually to yield a clonal, unisexual population in Misaki.

High potassium seawater inhibits ascidian sperm chemotaxis, but does not affect the male gamete chemotaxis of a brown alga.

Male gamete chemotaxis towards the female gamete is a general strategy to facilitate the sexual reproduction in many marine eukaryotes. Biochemical studies of chemoattractants for male gametes of brown algae have advanced in the 1970s and 1980s, but the molecular mechanism of male gamete responses to the attractants remains elusive. In sea urchin, a K+ channel called the tetraKCNG channel plays a fundamental role in sperm chemotaxis and inhibition of K+ efflux through this channel by high K+ seawater blocks almost all cell responses to the chemoattractant. This signalling mechanism could be conserved in marine invertebrates as tetraKCNG channels are conserved in the marine invertebrates that exhibit sperm chemotaxis. We confirmed that high K+ seawater also inhibited sperm chemotaxis in ascidian, Ciona intestinalis (robusta), in this study. Conversely, the male gamete chemotaxis towards the female gamete of a brown alga, Mutimo cylindricus, was preserved even in high K+ seawater. This result indicates that none of the K+ channels is essential for male gamete chemotaxis in the brown alga, suggesting that the signalling mechanism for chemotaxis in this brown alga is quite different from that of marine invertebrates. Correlated to this result, we revealed that the channels previously proposed as homologues of tetraKCNG in brown algae have a distinct domain composition from that of the tetraKCNG. Namely, one of them possesses two repeats of the six transmembrane segments (diKCNG) instead of four. The structural analysis suggests that diKCNG is a cyclic nucleotide-modulated and/or voltage-gated K+ channel.

Deletion of a Seminal Gene Cluster Reinforces a Crucial Role of SVS2 in Male Fertility.

Multiple genes, whose functions or expression are overlapping, compensate for the loss of one gene. A gene cluster in the mouse genome encodes five seminal vesicle proteins (SVS2, SVS3, SVS4, SVS5, and SVS6). These proteins are produced by male rodents and function in formation of the copulatory plug following mating. SVS2 plays an essential role in the successful internal fertilization by protecting the sperm membrane against a uterine immune attack. We hypothesized that the four remaining seminal vesicle proteins (SVPs) of this gene cluster may partially/completely compensate for the deficiency of SVS2. For confirming our hypothesis, we generated mice lacking the entire SVP-encoding gene cluster and compared their fecundity with Svs2-deficient (Svs2-/-) mice; that is, mice deficient in Svs2 alone. A single loxP site remained after the deletion of the Svs2 gene. Therefore, we inserted another loxP site by combining the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides (ssODN). Male mice lacking the entire SVP-encoding gene cluster (Svs2-6-/- mice) and thereby all five SVP proteins, generated by the deletion of 100kbp genomic DNA, showed low fecundity. However, the fecundity level was comparable with that from Svs2-/- male mice. Our results demonstrate that SVS3, SVS4, SVS5, and SVS6 do not function in the protection of sperm against a uterine immune attack in the absence of SVS2. Thus, Svs2 is the critical gene in the SVP gene cluster.

Induction of divalent cation permeability by heterologous expression of a voltage sensor domain.

The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K+ channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure.

Synthesis and Complete Structure Determination of a Sperm-Activating and -Attracting Factor Isolated from the Ascidian Ascidia sydneiensis.

For the complete structure elucidation of an endogenous sperm-activating and -attracting factor isolated from eggs of the ascidian Ascidia sydneiensis ( Assydn-SAAF), its two possible diastereomers with respect to C-25 were synthesized. Starting from ergosterol, the characteristic steroid backbone was constructed by using an intramolecular pinacol coupling reaction and stereoselective reduction of a hydroxy ketone as key steps, and the side chain was introduced by Julia-Kocienski olefination. Comparison of the NMR data of the two diastereomers with those of the natural product led to the elucidation of the absolute configuration as 25 S; thus the complete structure was determined and the first synthesis of Assydn-SAAF was achieved.

Seminal vesicle protein SVS2 is required for sperm survival in the uterus.

In mammals, sperm migrate through the female reproductive tract to reach the egg; however, our understanding of this journey is highly limited. To shed light on this process, we focused on defining the functions of seminal vesicle secretion 2 (SVS2). SVS2(-/-) male mice produced sperm but were severely subfertile, and formation of a copulatory plug to cover the female genital opening did not occur. Surprisingly, even when artificial insemination was performed with silicon as a substitute for the plug, sperm fertility in the absence of SVS2 remained severely reduced because the sperm were already dead in the uterus. Thus, our results provide evidence that the uterus induces sperm cell death and that SVS2 protects sperm from uterine attack.

Seminal vesicle proteins SVS3 and SVS4 facilitate SVS2 effect on sperm capacitation.

Mammalian spermatozoa acquire their fertilizing ability in the female reproductive tract (sperm capacitation). On the other hand, seminal vesicle secretion, which is a major component of seminal plasma, inhibits the initiation of sperm capacitation (capacitation inhibition) and reduces the fertility of the capacitated spermatozoa (decapacitation). There are seven major proteins involved in murine seminal vesicle secretion (SVS1-7), and we have previously shown that SVS2 acts as both a capacitation inhibitor and a decapacitation factor, and is indispensable for in vivo fertilization. However, the effects of SVSs other than SVS2 on the sperm have not been elucidated. Since mouse Svs2-Svs6 genes evolved by gene duplication belong to the same gene family, it is possible that SVSs other than SVS2 also have some effects on sperm capacitation. In this study, we examined the effects of SVS3 and SVS4 on sperm capacitation. Our results showed that both SVS3 and SVS4 are able to bind to spermatozoa, but SVS3 alone showed no effects on sperm capacitation. On the other hand, SVS4 acted as a capacitation inhibitor, although it did not show decapacitation abilities. Interestingly, SVS3 showed an affinity for SVS2 and it facilitated the effects of SVS2. Interaction of SVS2 and spermatozoa is mediated by the ganglioside GM1 in the sperm membrane; however, both SVS3 and SVS4 had weaker affinities for GM1 than SVS2. Therefore, we suggest that separate processes may cause capacitation inhibition and decapacitation, and SVS3 and SVS4 act on sperm capacitation cooperatively with SVS2.

Seminal vesicle secretion 2 acts as a protectant of sperm sterols and prevents ectopic sperm capacitation in mice.

Seminal vesicle secretion 2 (SVS2) is a protein secreted by the mouse seminal vesicle. We previously demonstrated that SVS2 regulates fertilization in mice; SVS2 is attached to a ganglioside GM1 on the plasma membrane of the sperm head and inhibits sperm capacitation in in vitro fertilization as a decapacitation factor. Furthermore, male mice lacking SVS2 display prominently reduced fertility in vivo, which indicates that SVS2 protects spermatozoa from some spermicidal attack in the uterus. In this study, we tried to investigate the mechanisms by which SVS2 controls in vivo sperm capacitation. SVS2-deficient males that mated with wild-type partners resulted in decreased cholesterol levels on ejaculated sperm in the uterine cavity. SVS2 prevented cholesterol efflux from the sperm plasma membrane and incorporated liberated cholesterol in the sperm plasma membrane, thereby reversibly preventing the induction of sperm capacitation by bovine serum albumin and methyl-beta-cyclodextrin in vitro. SVS2 enters the uterus and the uterotubal junction, arresting sperm capacitation in this area. Therefore, our results show that SVS2 keeps sterols on the sperm plasma membrane and plays a key role in unlocking sperm capacitation in vivo.

Neurotensin enhances sperm capacitation and acrosome reaction in mice.

Neurotensin (NT) has multiple functions, ranging from acting as a neurotransmitter to regulating intestinal movement. However, its function in reproductive physiology is unknown. Here, we confirmed the expression and localization of NT receptors (NTR1) in mouse epididymal spermatozoa and investigated the effect of NT on sperm function. Sperm protein tyrosine phosphorylation, one of the indices of sperm capacitation, was facilitated dose-dependently by NT administration. In addition, the acrosome reaction was promoted in capacitated spermatozoa, and addition of a selective antagonist of NTR1 and NTR2 blocked the induction. Furthermore, intracellular calcium mobilization by NT addition was observed. This showed that NT was an accelerator of sperm function via its functional receptors. The presence of NT was confirmed by immunohistochemistry and its localization was observed in epithelia of the uterus and oviduct isthmus and ampulla, which correspond to the fertilization route of spermatozoa. The NT mRNA level in ovulated cumulus cell was remarkably increased by treatment with human chorionic gonadotropin (hCG). Using an in vitro maturation model, we analyzed the effects of FSH, epidermal growth factor (EGF), estradiol, and progesterone in NT production in cumulus cells. We found that FSH and EGF upregulated NT release and mRNA expression. Both FSH- and EGF-induced upregulation were inhibited by U0126, an MAPK kinase inhibitor, indicating that FSH and EGF regulate NT expression via a MAPK-dependent pathway. This evidence suggests that NT can act as a promoter of sperm capacitation and the acrosome reaction in the female reproductive tract.

Chronic Encapsulated Intracerebral Hematoma after Carbon Ion Therapy for Chordoma Mimicking Malignant Glioma: A Case Report.

Chronic encapsulated intracerebral hematoma is a rare type of intracerebral hemorrhage. Reportedly, it is associated with vascular malformations, including arteriovenous malformations, cavernous hemangiomas, microaneurysms, and venous malformations. Recently, an association between chronic encapsulated intracerebral hematoma and stereotactic radiosurgery for arteriovenous malformations has been reported. In general, as the hematoma enlarges, symptoms progress slowly. In this report, we present a case of a 50-year-old woman who had undergone clivus chordoma resection and carbon ion therapy for the clivus respectively 27 and 20 years before developing chronic encapsulated intracerebral hematoma with rapidly progressing disturbance of consciousness. She was referred to our hospital because of difficulty walking due to left hemiparesis. Head computed tomography and magnetic resonance imaging showed a cystic lesion in the right temporal lobe with perifocal edema. On the second day of hospitalization, the patient's consciousness worsened. We suspected a malignant glioma and performed an emergency craniotomy; however, the pathological diagnosis was chronic encapsulated intracerebral hematoma. After the rehabilitation therapy, the patient became ambulatory and was discharged. To the date of reporting, the patient remained recurrence-free. Chronic encapsulated intracerebral hematoma may be due to invasive craniotomy or carbon ion therapy. It usually progresses slowly; however, in some cases, such as this one, it may cause rapid deterioration of consciousness.

Role of Mos/MEK/ERK cascade and Cdk1 in Ca2+ oscillations in fertilized ascidian eggs.

Intracellular calcium ion concentration ([Ca(2+)](i)) transients are observed in the fertilized eggs of all species investigated so far, and are critical for initiating several events related to egg activation and cell cycle control. Here, we investigated the role of the Mos/MEK/ERK cascade and Cdk1 on Ca(2+) oscillations in fertilized ascidian eggs. The egg of the ascidian Phallusia nigra shows [Ca(2+)](i) oscillations after fertilization: Ca(2+) waves immediately following fertilization (phase I), and [Ca(2+)](i) oscillations between the first and second polar body extrusions (phase II). Our results show that in P. nigra eggs, ERK activity peaked just before the extrusion of the first polar body, and decreased gradually, eventually disappearing at the extrusion of the second polar body. Cyclin-dependent protein kinase 1(Cdk1) activity decreased to undetectable levels immediately after fertilization, and then periodically increased according to the meiotic and mitotic cell cycle. When the unfertilized eggs were incubated with U0126, an inhibitor of MEK, before insemination, ERK was immediately inactivated, and the phase II [Ca(2+)](i) oscillations disappeared. Alternatively, when the constitutively active Mos protein (GST-Mos) was injected into the unfertilized eggs, ERK activity was preserved for at least 120 min after fertilization, and the phase II [Ca(2+)](i) oscillations lasted for more than 120 min after the second polar body extrusion. These results suggest that ERK activity is necessary for maintaining [Ca(2+)](i) oscillations. GST-ΔN85-cyclin, which maintains Cdk1 activity, caused ERK activity in the eggs to persist for over 120 min after fertilization, and prolonged [Ca(2+)](i) oscillations. Moreover, the effects of GST-ΔN85-cyclin on the egg were abrogated by the application of U0126. Thus, Cdk1-mediated [Ca(2+)](i) oscillations seem to require ERK activity. However, GST-Mos triggered [Ca(2+)](i) oscillations after the second polar body extrusion, whereas GST-ΔN85-cyclin did not, although it prolongs the duration of [Ca(2+)](i) oscillations. Interestingly, GST-ΔN85-cyclin increased the frequency of [Ca(2+)](i) transients in the Mos-induced [Ca(2+)](i) oscillations after the extrusion of the second polar body. Thus, Cdk1 could maintain, but not activate, ERK and [Ca(2+)](i) oscillations. ERK activity and [Ca(2+)](i) oscillations seem to form a negative feedback loop which may be responsible for maintaining the meiotic period.

Alkyl-C60 liquid electrets as deformable mechanoelectric generators.

Special attention is being paid to the potential applicability of various soft electronics in deformable/wearable devices. These devices must be constantly connected to energy sources to ensure their uninterrupted operation. Electrets, which are capable of retaining quasi-permanent electric charges inside or on the surface of materials, are expected to be a battery-less power source. Here, we present a strategy for harvesting the charges in alkyl-C60 liquids. Suitable substitution of bulky yet flexible branched long-alkyl chains generated C60-mono-adducts and regioisomeric bis-adducts as room-temperature solvent-free liquids. These alkyl-C60 liquids were negatively poled by the corona-discharging and soaked in nylon fabric. The liquid of the C60 bis-adduct exhibited better charge retention in comparison to the liquid of the C60 mono-adduct. This suggests that the bulky long-alkyl chains provided proper insulation for the C60 core and charge trapping in the liquid. This charge-trapping behaviour and the inherent fluidity of the alkyl-C60 liquids enabled their fabrication into deformable mechanoelectric generator (MEG) devices. The MEG exhibited applicability as a deformable micropower source or vibration sensor by generating output voltage pulses even under folded/twisted/rolled conditions. The alkylated-liquid-based MEGs worked at frequencies similar to human body motion, showing promising potential for body motion sensors and healthcare applications.

CatSper mediates not only chemotactic behavior but also the motility of ascidian sperm.

Introduction: Sperm motility, including chemotactic behavior, is regulated by changes in the intracellular Ca2+ concentration, and the sperm-specific Ca2+ channel CatSper has been shown to play an important role in the regulation of intracellular Ca2+. In particular, in mammals, CatSper is the only functional Ca2+ channel in the sperm, and mice deficient in the genes comprising the pore region of the Ca2+ channel are infertile due to the inhibition of sperm hyperactivation. CatSper is also thought to be involved in sea urchin chemotaxis. In contrast, in ascidian Ciona intestinalis, SAAF, a sperm attractant, interacts with Ca2+/ATPase, a Ca2+ pump. Although the existence of CatSper genes has been reported, it is not clear whether CatSper is a functional Ca2+ channel in sperm. Results: We showed that CatSper is present in the sperm flagella of C. intestinalis as in mammalian species, although a small level of gene expression was found in other tissues. The spermatozoa of CatSper3 KO animals were significantly less motile, and some motile sperms did not show any chemotactic behavior. These results suggest that CatSper plays an important role in ascidians and mammals, and is involved in spermatogenesis and basic motility mechanisms.

Helicobacter pylori-negative Gastric Adenocarcinoma Mimicking Verrucous Gastritis in the Antrum: A Case Report and Literature Review.

A 46-year-old man was referred to our hospital for the examination of a flat elevated lesion with an erosion-like depression, located on the greater curvature of the antrum. Endoscopic submucosal dissection was performed. Histological findings of the resected specimen demonstrated a well-differentiated tubular adenocarcinoma with a diameter of 12 mm. No atrophy was observed in the tumor-adjacent mucosa. Serum Helicobacter pylori antibody estimation and 13C-urea breath tests yielded negative results. Immunohistochemical staining was positive for both gastric mucin and intestinal mucin. The final diagnosis was well-differentiated tubular adenocarcinoma with a gastrointestinal phenotype that originated in mucosa uninfected by H. pylori.