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.

Metabolic Syndrome Accelerates the Age-Related Increase of Intraductal Papillary Mucinous Neoplasm of the Pancreas.

OBJECTIVES: Aging is associated with a high prevalence of pancreatic cysts and intraductal papillary mucinous neoplasms (IPMNs). Metabolic syndrome (MS) may increase the risk of neoplasms, including those that develop in the pancreas. However, the influence of factors associated with MS on the development of IPMN remains unclear. METHODS: A total of 9363 patients who underwent abdominal ultrasound examinations between April 2012 and May 2013 were included in this study. Multivariate logistic regression analysis was performed to identify factors associated with the presence of IPMN by age. RESULTS: Pancreatic cysts were detected in 198 of 9363 patients, of whom 129 were found to have IPMNs. The presence of IPMN significantly correlated with age (10-year increments; odds ratio, 2.73; 95% CI, 2.28-3.29; P < 0.001). High body mass index, history of smoking, hyperlipidemia, hypertension, and MS were associated with a higher prevalence of IPMN with advancing age. In multivariate analysis, the presence of IPMN was more frequent in elderly patients with MS (odds ratio, 3.14; 95% CI, 3.14-6.72; P = 0.003). CONCLUSIONS: The present study suggests that the incidence of IPMN increases with age and is accelerated in the presence of MS.

Factors Associated with Overlap between Functional Dyspepsia and Nonerosive Reflux Disease in Endoscopy-based Helicobacter pylori-uninfected Japanese Health Checkup Participants: A Prospective, Multicenter Cross-sectional Study.

Objective We assessed the factors associated with overlap between functional dyspepsia (FD) and nonerosive reflux disease (NERD) in endoscopy-based Helicobacter pylori-uninfected Japanese health checkup participants. Methods We utilized baseline data from 3,085 individuals who underwent upper endoscopy for health screening in a prospective, multicenter cohort study. The participants were asked to complete a questionnaire detailing their upper abdominal symptoms and lifestyle. Anxiety was assessed using the State-Trait Anxiety Inventory (STAI) score. FD, postprandial distress syndrome (PDS), and epigastric pain syndrome (EPS) were defined according to the Rome III criteria. NERD was defined as heartburn or regurgitation ≥1 day/week without erosive esophagitis. Results Of the 3,085 participants, 73 (2.4%), 97 (3.1%), and 84 (2.7%) had FD alone, NERD alone, and FD-NERD overlap, respectively. Factors associated with FD-NERD-overlap participants compared with participants with neither FD nor NERD were women [odds ratio (OR): 2.08, 95% confidence interval (CI): 1.24-3.52], body mass index (BMI) <18.5 (OR: 2.87, 95% CI: 1.56-5.07), alcohol consumption ≥20 g/day (OR: 1.85, 95% CI: 1.06-3.15), and a high STAI score (OR: 2.53, 95% CI: 1.62-4.00). Increasing age (OR: 1.06, 95% CI: 1.01-1.11) and EPS symptoms [pure EPS (OR: 3.67, 95% CI: 1.65-8.51) and PDS-EPS overlap (OR: 11.6, 95% CI: 4.09-37.2)] were associated with FD-NERD overlap vs. FD alone. Women (OR: 3.17, 95% CI: 1.47-7.04), BMI <18.5 (OR: 3.03, 95% CI: 1.04-9.90), and acid reflux symptoms ≥2 days a week (OR: 3.57, 95% CI: 1.83-7.14) were associated with FD-NERD overlap vs. NERD alone. Conclusion Understanding the clinical features of overlap between FD and NERD will lead to better management.

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.

[Lymphocytic colitis caused by medicine ingredient modification].

A 49-year-old woman was referred to our hospital for further evaluation and treatment of diarrhea. Colonoscopic findings revealed indistinct vascular patterns and extensive edema in a colon segment, and white granular mucosa and crack-like appearance in the sigmoid colon and rectum. She was diagnosed with lymphocytic colitis (LC) based on lymphocytic infiltration into the epithelium on histopathological examination. Diarrhea symptoms resolved after long-term medication withdrawal. This medicine's composition was changed 4 years ago and this modification possibly triggered LC.

[A case of juvenile gastric polyposis with gastric cancer successfully treated by laparoscopic total gastrectomy -review of 36 reported cases in Japan].

A 43-year-old woman was suffering from epigastric pain. Her gastroscopy revealed polyposis of the stomach, and her biopsy revealed a hyperplastic polyp. During the 18-month follow-up, the polyps proliferated, and the patient was referred to our institute for further investigation and treatment. A juvenile gastric polyposis diagnosis was made. She refused to have the surgery despite the fact that it was necessary due to the anemia and hypoalbuminemia she was experiencing. Endoscopic biopsy results revealed gastric cancer at a follow-up visit 2 years and 3 months later; thus, a laparoscopic total gastrectomy was performed. Pathological examination revealed adenocarcinomas that were scattered and well-differentiated, with hyperplastic polyps in the background. No lymph node metastasis was found. Despite the fact that juvenile gastric polyposis is a pathologically benign disease, there have been numerous case reports of surgery being performed due to anemia, hypoalbuminemia, or gastric cancer associated with the disease. When gastric cancers are discovered in cases of juvenile gastric polyposis, they are usually in an early stage, making them a good candidate for laparoscopic total gastrectomy.

Pembrolizumab-related cholangitis with multiple fatal liver abscesses after endoscopic biliary drainage: a case report and review of the literature.

A 60-year-old male with cStage IVB lung cancer was treated with pembrolizumab. However, after five courses of pembrolizumab, he developed pembrolizumab-related cholangitis. Imaging studies showed enlargement and diffuse wall thickening of the gallbladder and mild dilation of the bile ducts without any obvious obstruction. As the patient experienced severe abdominal pain, we suspected bile stasis and performed biliary drainage. However, his condition did not improve, and he developed multiple liver abscesses and died during immunosuppressive therapy. Our case suggests that in ir-cholangitis, the indication and method of endoscopic retrograde cholangiopancreatography should be carefully judged.

New practical scoring system to predict post-endoscopic retrograde cholangiopancreatography pancreatitis: Development and validation.

BACKGROUND AND AIM: Post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) is the most serious adverse event of ERCP. Therefore, it is important to identify high-risk patients who require prophylactic measures. The aim of this study was to develop a practical prediction model for PEP that identifies high-risk patients. METHODS: Patients who underwent ERCP at three tertiary hospitals between April 2010 and September 2012 were enrolled. The dataset was divided into a training set (two centers) and validation set (one center). Using the training set, univariable and multivariable analyses were performed to identify predictive factors for PEP. We constructed a scoring system and evaluated reproducibility using the validation set. RESULTS: A total of 2719 ERCPs were analyzed. PEP developed in 133 cases (4.9%). Risk factors (adjusted odds ratios [OR]) in the training set were a history of PEP (OR: 4.2), intact papilla (OR: 2.4), difficult cannulation (OR: 1.9), pancreatic guidewire-assisted biliary cannulation (OR: 2.2), pancreatic injection (OR: 2.1), pancreatic intraductal ultrasonography (IDUS)/sampling from the pancreatic duct (OR: 2.2), and biliary IDUS/sampling from the biliary tract (OR: 2.8). A scoring system was constructed using these seven clinical variables. The areas under the receiver-operating characteristic curve of this model were 0.799 in the training set and 0.791 in the validation set. In the high-risk group at a score of 3 or higher, the incidence of PEP was 13.4%, and all severe/fatal cases were in the high-risk group. CONCLUSIONS: This scoring system helps to predict each patient's risk and select preventive measures.

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.

Non-Alcoholic Fatty Liver Disease Is Strongly Associated with Smoking Status and Is Improved by Smoking Cessation in Japanese Males: A Retrospective Study.

BACKGROUND: Cigarette smoking is known to be a significant risk factor associated with non-alcoholic fatty liver disease (NAFLD). We aimed to examine the association between smoking status and the severity of fatty liver with regard to sex and smoking cessation. METHODS: In total, 13,466 subjects (6,642 males and 6,824 females) who had undergone abdominal ultrasonography for health check-up, multivariable logistic regression analysis was retrospectively conducted to assess the association between smoking status and the prevalence of NAFLD stratified by sex after adjusting for other potential confounders. RESULTS: Male sex (odds ratio [OR] 3.27, 95% confidence interval [CI] 3.00-3.57) and smoking history (former smoker: OR 1.23, 95% CI 1.10-1.38, current smoker: OR 1.31, 95% CI 1.17-1.47) were significantly associated with NAFLD. In males with a smoking history, an increased pack-year was strongly associated with the prevalence and severity of NAFLD (prevalence of moderate to severe fatty liver: Pack-year from 0.01 to 9.99, 21.3%; Pack-year from 10.00 to 19.99, 27.2%; Pack-year ≥20.00, 33.7%; P<0.0001), although the prevalence of moderate to severe fatty liver was inversely associated with the duration of smoking cessation (more than 10 years vs. within 5 years, OR 0.71, 95% CI 0.53-0.96). In female subjects, light current smoking was negatively associated with NAFLD (current smoker with a pack-year from 0.01 to 9.99 vs. never smoker, OR 0.41, 95% CI 0.19-0.76). CONCLUSIONS: Smoking status and pack-year were strongly associated with the prevalence and severity of NAFLD, especially in Japanese males. However, smoking cessation improved NAFLD in this population.

[Comparison of Abiraterone Acetate Plus Prednisolone and Combined Androgen Blockade in High-risk Metastatic Hormone-Sensitive Prostate Cancer].

Abiraterone acetate plus prednisone/prednisolone (AAP) and androgen deprivation therapy (ADT) was approved for the treatment of high-risk metastatic hormone-sensitive prostate cancer (mHSPC) in Japan in 2018. We investigated 22 patients with mHSPC showing ≥2 of 3 high-risk factors (Gleason score ≥8, ≥3 bone lesions or measurable visceral metastases) who received AAP plus ADT at our hospital between March 2018 and October 2019. We compared outcomes between the propensity-score matched AAP plus ADT group and the combined androgen blockade (CAB) group (19 matched pairs, 38 patients) to evaluate the efficacy and safety of AAP plus ADT. Prostate-specific antigen progression-free survival (PSA-PFS) was significantly longer in the AAP group than in the CAB group (not reached vs 15.1 months, p=0.014). Time to achievement of serum PSA ≤0.2 ng/ml and ≤0.1 ng/ml was shorter in the AAP group than in the CAB group (6.4 months vs not reached, p=0.001 and 11.0 months vs not reached, p=0.004, respectively). Although no significant intergroup difference was observed in the overall survival rates and PSA-PFS2 (PSAPFS on subsequent anticancer therapy) owing to the shorter follow-up in the AAP group, our data suggest that the clinical efficacy of AAP is betterthan that of CAB in patients with mHSPC.

A pathological complete response by chemotherapy with S-1 and oxaliplatin for a locally advanced duodenal adenocarcinoma in Lynch syndrome: a case report.

BACKGROUND: Although primary duodenal adenocarcinoma (DA) is a rare malignancy representing ~ 0.5% of all gastrointestinal cancers, the incidence of DA is more frequent in Lynch syndrome. Because of its rarity, treatment strategies or optimal chemotherapeutic regimens have not been clearly defined for advanced DA. CASE PRESENTATION: A 72-year-old woman with Lynch syndrome visited our hospital with a right upper abdominal pain. Computed tomography (CT) showed wall thickness with enhancement in the second portion of the duodenum and adjacent abdominal wall, which suggested direct tumor invasion to the abdominal wall. Upper gastrointestinal endoscopy (UGE) showed a large ulcerative tumor in the second portion of the duodenum, and histological analysis revealed a poorly differentiated adenocarcinoma. A cT4N0M0, cStage IIB (Union for International Control Cancer TNM staging) DA was diagnosed. After three courses of chemotherapy with S-1 and oxaliplatin (SOX), follow-up CT and UGE showed shrinkage of the duodenal tumor. Therefore, the patient underwent pancreaticoduodenectomy with lymph node dissection with curative intent. Histological examination showed a pathological complete response to SOX therapy. The postoperative course was uneventful, and the patient was discharged on postoperative day 29. The patient received no adjuvant chemotherapy, and there has been no evidence of recurrence 6 months after the operation. CONCLUSIONS: SOX therapy provided a remarkable response and can be an optimal chemotherapeutic regimen for advanced DA in Lynch syndrome.

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.

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.

[Pancreatic arteriovenous malformation perforated into the common bile and pancreatic ducts:a case report].

A 54-year-old man presented with melena and was conservatively monitored for duodenitis. He developed epigastric pain, and dynamic computed tomography revealed abnormal blood flow in the pancreatic head. The endoscopic retrograde cholangiography revealed that the common bile and pancreatic ducts were simultaneously enhanced, thereby indicating the perforation of an arteriovenous malformation into both ducts. Despite medical treatment, the epigastric pain rapidly worsened and therefore, pancreatoduodenectomy was performed. The present report suggests that if the patient's general condition permits, surgical resection should be actively considered for the treatment of symptomatic pancreatic arteriovenous malformation.

Metachronous skeletal muscle metastasis without any other organ metastases after curative gastrectomy: a case report.

BACKGROUND: Skeletal muscle metastasis from gastric cancer is extremely rare and often accompanied with synchronous metastasis to any other organs. We herein report a case of rapidly developing multiple skeletal metastases from gastric cancer without any other organ metastases. CASE PRESENTATION: A 47-year-old man underwent distal gastrectomy for advanced gastric cancer. Pathological diagnosis was poorly differentiated adenocarcinoma, T2N1M0, Stage IIA. The patient presented with a history of left dorsal tenderness 12 months after the operation. A computed tomography (CT) revealed a solid mass in the left latissimus dorsi muscle. Pathological examination of the ultrasound guided needle biopsy specimen revealed poorly differentiated adenocarcinoma similar to the previously resected gastric cancer, and the tumor was diagnosed as metastasis of gastric cancer. Thereafter, the systemic chemotherapy was administrated. However, the metastases were extended to the paraspinal muscle and quadriceps, and the patient died 7 months after the recurrence. CONCLUSIONS: The prognosis of patients with skeletal muscle metastasis may be extremely poor, even in patients without any other organ metastases. The development of further chemotherapeutic agents and regimens is therefore needed.

Novel erythrocyte clumps revealed by an orphan gene Newtic1 in circulating blood and regenerating limbs of the adult newt.

The newt, a group of urodele amphibians, has outstanding ability to repeatedly regenerate various body parts, even in the terrestrial life-stage. In this animal, when the limb is amputated, a cell mass named the blastema appears on the stump and eventually gives rise to a new functional limb. Erythrocytes (red blood cells) in most non-mammalian vertebrates, including the newt, preserve their nucleus throughout their life-span, although physiological roles of such nucleated erythrocytes, other than oxygen delivery, are not known. Here we report novel behavior of erythrocytes in the newt. We identified an orphan gene Newtic1, whose transcripts significantly increased in the blastema. Newtic1 was expressed in a subset of erythrocytes that formed a novel clump (EryC). EryC formed a complex with monocytes and was circulating throughout the body. When the limb was amputated, EryCs were newly generated in the stump and accumulated into a distal portion of the growing blastema. Our data suggested that the newt erythrocytes carried multiple secretory molecules including growth factors and matrix metalloproteases, and were capable of delivering these molecules into the blastema as a form of EryCs. This study provides insight into regulations and roles of nucleated erythrocytes, that are independent of oxygen delivery.