Fetal Kidney Grafts and Organoids from Microminiature Pigs: Establishing a Protocol for Production and Long-Term Cryopreservation.

Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by patients and researchers, effective methods for producing, retrieving, and cryopreserving pig fetuses are indispensable. Therefore, in this study, to collect fetuses for kidney extraction, we employed cesarean sections to preserve the survival and fertility of the mother pig and a method for storing fetal kidneys by long-term cryopreservation. Subsequently, we evaluated the utility of these two methods. We confirmed that the kidneys of pig fetuses retrieved by cesarean section that were cryopreserved for an extended period could resume renal growth when grafted into mice and were capable of forming renal organoids. These results demonstrate the usefulness of long-term cryopreserved fetal pig organs and strongly suggest the effectiveness of our comprehensive system of pig fetus retrieval and fetal organ preservation, thereby highlighting its potential as an accelerator of xenotransplantation research and clinical innovation.

Multiple ageing effects on testicular/epididymal germ cells lead to decreased male fertility in mice.

In mammals, females undergo reproductive cessation with age, whereas male fertility gradually declines but persists almost throughout life. However, the detailed effects of ageing on germ cells during and after spermatogenesis, in the testis and epididymis, respectively, remain unclear. Here we comprehensively examined the in vivo male fertility and the overall organization of the testis and epididymis with age, focusing on spermatogenesis, and sperm function and fertility, in mice. We first found that in vivo male fertility decreased with age, which is independent of mating behaviors and testosterone levels. Second, overall sperm production in aged testes was decreased; about 20% of seminiferous tubules showed abnormalities such as germ cell depletion, sperm release failure, and perturbed germ cell associations, and the remaining 80% of tubules contained lower number of germ cells because of decreased proliferation of spermatogonia. Further, the spermatozoa in aged epididymides exhibited decreased total cell numbers, abnormal morphology/structure, decreased motility, and DNA damage, resulting in low fertilizing and developmental rates. We conclude that these multiple ageing effects on germ cells lead to decreased in vivo male fertility. Our present findings are useful to better understand the basic mechanism behind the ageing effect on male fertility in mammals including humans.

A small secreted protein NICOL regulates lumicrine-mediated sperm maturation and male fertility.

The mammalian spermatozoa produced in the testis require functional maturation in the epididymis for their full competence. Epididymal sperm maturation is regulated by lumicrine signalling pathways in which testis-derived secreted signals relocate to the epididymis lumen and promote functional differentiation. However, the detailed mechanisms of lumicrine regulation are unclear. Herein, we demonstrate that a small secreted protein, NELL2-interacting cofactor for lumicrine signalling (NICOL), plays a crucial role in lumicrine signalling in mice. NICOL is expressed in male reproductive organs, including the testis, and forms a complex with the testis-secreted protein NELL2, which is transported transluminally from the testis to the epididymis. Males lacking Nicol are sterile due to impaired NELL2-mediated lumicrine signalling, leading to defective epididymal differentiation and deficient sperm maturation but can be restored by NICOL expression in testicular germ cells. Our results demonstrate how lumicrine signalling regulates epididymal function for successful sperm maturation and male fertility.

1700029I15Rik orchestrates the biosynthesis of acrosomal membrane proteins required for sperm-egg interaction.

Sperm acrosomal membrane proteins, such as Izumo sperm-egg fusion 1 (IZUMO1) and sperm acrosome-associated 6 (SPACA6), play essential roles in mammalian gamete binding or fusion. How their biosynthesis is regulated during spermiogenesis has largely remained elusive. Here, we show that 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik is a type-II transmembrane protein expressed in early round spermatids but not in mature spermatozoa. It interacts with proteins involved in N-linked glycosylation, disulfide isomerization, and endoplasmic reticulum (ER)-Golgi trafficking, suggesting a potential role in nascent protein processing. The ablation of 1700029I15Rik destabilizes non-catalytic subunits of the oligosaccharyltransferase (OST) complex that are pivotal for N-glycosylation. The knockout testes exhibit normal expression of sperm plasma membrane proteins, but decreased abundance of multiple acrosomal membrane proteins involved in fertilization. The knockout sperm show upregulated chaperones related to ER-associated degradation (ERAD) and elevated protein ubiquitination; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway underpinning the biosynthesis of acrosomal membrane proteins during spermiogenesis.

CRISPR/Cas9-mediated genome-edited mice reveal 10 testis-enriched genes are dispensable for male fecundity.

As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes.

NELL2-mediated lumicrine signaling through OVCH2 is required for male fertility.

The lumicrine system is a postulated signaling system in which testis-derived (upstream) secreted factors enter the male reproductive tract to regulate epididymal (downstream) pathways required for sperm maturation. Until now, no lumicrine factors have been identified. We demonstrate that a testicular germ-cell-secreted epidermal growth factor-like protein, neural epidermal growth factor-like-like 2 (NELL2), specifically binds to an orphan receptor tyrosine kinase, c-ros oncogene 1 (ROS1), and mediates the differentiation of the initial segment (IS) of the caput epididymis. Male mice in which Nell2 had been knocked out were infertile. The IS-specific secreted proteases, ovochymase 2 (OVCH2) and A disintegrin and metallopeptidase 28 (ADAM28), were expressed upon IS maturation, and OVCH2 was required for processing of the sperm surface protein ADAM3, which is required for sperm fertilizing ability. This work identifies a lumicrine system essential for testis-epididymis-spermatozoa (NELL2-ROS1-OVCH2-ADAM3) signaling and male fertility.

Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells.

It has recently been recognized that inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), upregulate the secretion of matrix metalloproteinase-9 (MMP-9) from cancer cells and thereby promote peritoneal dissemination. In this study, we found that TNF-α also stimulated peritoneal mesothelial cells to secrete MMP-9 as assessed by zymography. MMP-9 gene expression in mesothelial cells induced by TNF-α was confirmed by quantitative RT-PCR analysis. We then utilized the reconstituted artificial mesothelium, which was composed of a monolayer of mesothelial cells cultured on a Matrigel layer in a Boyden chamber system, to examine the effects of TNF-α on carcinoma cell invasion. The transmigration of MKN1 human gastric carcinoma cells through the reconstituted mesothelium was promoted by TNF-α in a dose-dependent manner. The increased MKN1 cell migration was partially inhibited by the anti-α3 integrin antibody, indicating that the invasion process involves an integrin-dependent mechanism. Finally, we observed that the invasion of MMP-9-knockdown MKN1 cells into Matrigel membranes was potentiated by the exogenous addition of purified proMMP-9. These results suggest that TNF-α-induced MMP-9 secretion from mesothelial cells plays an important role in the metastatic dissemination of gastric cancer.

mDia1/3 generate cortical F-actin meshwork in Sertoli cells that is continuous with contractile F-actin bundles and indispensable for spermatogenesis and male fertility.

Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell-germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility.

Predictive Value of Combining the Ankle-Brachial Index and SYNTAX Score for the Prediction of Outcome After Percutaneous Coronary Intervention (from the SHINANO Registry).

The Synergy Between PCI With TAXUS and Cardiac Surgery (SYNTAX) score is effective in predicting clinical outcome after percutaneous coronary intervention (PCI). However, its prediction ability is low because it reflects only the coronary characterization. We assessed the predictive value of combining the ankle-brachial index (ABI) and SYNTAX score to predict clinical outcomes after PCI. The ABI-SYNTAX score was calculated for 1,197 patients recruited from the Shinshu Prospective Multi-center Analysis for Elderly Patients with Coronary Artery Disease Undergoing Percutaneous Coronary Intervention (SHINANO) registry, a prospective, observational, multicenter cohort study in Japan. The primary end points were major adverse cardiovascular and cerebrovascular events (MACE; all-cause death, myocardial infarction, and stroke) in the first year after PCI. The ABI-SYNTAX score was calculated by categorizing and summing up the ABI and SYNTAX scores. ABI ≤ 0.49 was defined as 4, 0.5 to 0.69 as 3, 0.7 to 0.89 as 2, 0.9 to 1.09 as 1, and 1.1 to 1.5 as 0; an SYNTAX score ≤ 22 was defined as 0, 23 to 32 as 1, and ≥ 33 as 2. Patients were divided into low (0), moderate (1 to 2), and high (3 to 6) groups. The MACE rate was significantly higher in the high ABI-SYNTAX score group than in the lower 2 groups (low: 4.6% vs moderate: 7.0% vs high: 13.9%, p = 0.002). Multivariate regression analysis found that ABI-SYNTAX score independently predicted MACE (hazards ratio 1.25, 95% confidence interval 1.02 to 1.52, p = 0.029). The respective C-statistic for the ABI-SYNTAX and SYNTAX score for 1-year MACE was 0.60 and 0.55, respectively. In conclusion, combining the ABI and SYNTAX scores improved the prediction of 1-year adverse ischemic events compared with the SYNTAX score alone.

Risk stratification using the CHA2DS2-VASc score in patients with coronary heart disease undergoing percutaneous coronary intervention; sub-analysis of SHINANO registry.

BACKGROUND: CHADS2 or CHA2DS2-VASc score is used for prediction of stroke in patients with atrial fibrillation (AF). Recently, CHADS2 score is reported to have prognostic value in acute coronary syndrome without AF. However, clinical validation of CHA2DS2-VASc score for prognostic stratification in coronary heart disease (CHD) without AF remains uncertain. In this study, we evaluate whether CHA2DS2-VASc score could predict clinical outcome in CHD without known AF. METHODS: SHINANO registry was a prospective, observational, multicenter cohort study, enrolling 1923 consecutive patients with CHD from August 2012 to July 2013. Two hundred nine patients were excluded because of known AF. We calculated CHA2DS2-VASc score in the remaining 1714 patients (mean age 70 ± 11 years, 23% female) without known AF. To assess the clinical validation of CHA2DS2-VASc score, we divided patients into 3 groups according to the tertiles (score 0-2, 3-4, and ≥ 5). The primary endpoint was MACE including death, nonfatal myocardial infarction, and ischemic stroke at 1 year. RESULTS: One-year follow-up was completed in 1632 patients (95.2%). Cumulative incidence of MACE was 139 cases. In Kaplan-Meier analysis, incidence of MACE was significantly higher in patients with CHA2DS2-VASc score ≥ 5 compared to 3-4 and 0-2 (14.6% vs. 6.8% vs. 5.3%, p < 0.001). In multivariate Cox-regression analysis, CHA2DS2-VASc score was an independent predictor for MACE (hazard ratio 1.26, 95% confidence interval 1.15-1.39p < 0.001). CONCLUSIONS: This study demonstrated that CHA2DS2-VASc score could provide prognostic information in CHD without known AF.

In-hospital clinical outcomes of elderly patients (≥80 years) undergoing percutaneous coronary intervention.

BACKGROUND: The clinical outcomes of elderly patients (≥80 years old) undergoing percutaneous coronary intervention (PCI) has not been well established, despite recent advances in both devices and techniques. METHODS AND RESULTS: We recruited patients from the SHINANO Registry, a prospective, observational, multicenter, cohort study. From August 2012 to July 2013, a total of 1,923 consecutive patients with 2,250 elective/urgent PCIs (2,105 admissions) (mean age, 71±11 years; ≥80 years, 23%; men, 77%) were enrolled. The primary endpoint was procedural success. The secondary endpoints were in-hospital death and in-hospital major adverse cardiovascular events (MACE). The procedural success rate was significantly lower (83.7% vs. 89.1%, P=0.0001), and the rates of in-hospital mortality and MACE were significantly higher in elderly than in non-elderly patients (3.6% vs. 1.5%, P=0.005; 4.4% vs. 2.3%, P=0.016, respectively). For elective PCI, the rates of procedural success and in-hospital MACE were similar between groups (90.3% vs. 91.3%, P=0.65, 2.3% vs. 1.2%, P=0.2, respectively). On multivariate analysis, being elderly was not an independent predictor of procedural failure (OR, 1.15; CI, 0.81-1.61; P=0.43). CONCLUSIONS: In elderly patients, PCI is safe and feasible. The presence of comorbidities is a more important factor than age alone.

Hemodialysis-induced P-wave signal-averaged electrocardiogram alterations are indicative of vulnerability to atrial arrhythmias.

BACKGROUND: Atrial fibrillation (AF) is the most common supraventricular arrhythmia, often occurring during hemodialysis (HD). Prolongation of the total filtered P-wave duration (PWD) and reduction of the root mean square voltages for the last 20 ms of the P wave (RMS20) on a P-wave signal-averaged electrocardiogram (P-SAECG) are predictors of AF. We investigated whether HD induces alterations of P-SAECG, and determined the influential factors. METHODS AND RESULTS: Thirty-three end-stage kidney disease patients (66.7 ± 12.6 years, 23 males) undergoing maintenance HD were enrolled in this study. Digital ambulatory P-SAECG monitoring and laboratory examination of serum proteins and ions were carried out before, during, and after the HD sessions. Data were analyzed by multiple regression analysis. PWD was significantly prolonged, and RMS20 significantly reduced, during HD. These values recovered after completion of HD. Multiple regression analysis showed that prolongation of PWD significantly correlated with HD duration and the rate of removal of body fluid. On the other hand, RMS20 significantly correlated with HD duration and blood urea nitrogen variation. CONCLUSIONS: HD resulted in prolongation of PWD and reduction of RMS20, indicating the vulnerability of HD patients to AF. These P-SAECG changes correlated with HD duration and the rate of removal of the body fluid. These findings underline the importance of the control of dialysis variables in the prevention of atrial arrhythmias following HD.

Catheter ablation of non-inducible atrial tachycardia after surgical repair of heart disease.

We present a patient with non-inducible atrial tachycardia (AT) after atriotomy for surgical repair of heart disease who underwent ablation successfully. Using a 3-D mapping system, we presumed the atriotomy site on the lateral right atrial wall by searching for linear double potentials (DP) during sinus/paced rhythm from the coronary sinus, but it was evaluated incompletely. We could verify the edges of the atriotomy scar precisely by pacing from close to the linear DP lesion and the opposite site. After ablation between the presumed atriotomy scar and the inferior vena cava and cavotricuspid isthmus, no AT recurred without anti-arrhythmic drugs.

Influence of circumferential pulmonary vein isolation for atrial fibrillation on ST elevation in patient with Brugada syndrome.

We present a patient with Brugada syndrome and paroxysmal atrial fibrillation who underwent circumferential pulmonary vein isolation. His electrocardiogram showed normal sinus rhythm and first-degree AV block (P-R 280 ms) with coved-type ST elevation in V1-2 (+2.0 mm) before ablation. During ablation around the left pulmonary vein ostium, atrial fibrillation, progression of ST elevation (+4.5 mm), and T wave alternans occurred. After right pulmonary vein encirclement was complete, ST elevation improved to +1.0 mm. The following day, the ST segments remained lower than baseline levels.

Potentiation of cell invasion and matrix metalloproteinase production by alpha3beta1 integrin-mediated adhesion of gastric carcinoma cells to laminin-5.

We previously reported that the adhesion of gastric carcinoma cells to the peritoneum mediated by the alpha3beta1 integrin-laminin interaction is a key step in the initial process of peritoneal metastatic dissemination. Carcinoma cells subsequently invade through the intercellular gaps of mesothelial linings. In this study, we examined the role of the interaction of carcinoma cells with laminin-5, which is a major component of submesothelial basement membranes and serves as a high-affinity ligand for alpha3beta1 integrin, in carcinoma cell invasion. Human gastric carcinoma cell lines (MKN1, GT3TKB, and NUGC-4) adhered in an alpha3beta1 integrin-dependent manner to the extracellular matrix deposited by peritoneal mesothelial cells. An in vitro invasion assay using the Boyden chamber system revealed that MKN1 cell migration through the membranes increased when the membranes were coated with matrices produced by mesothelial cells or with laminin-5-containing Matrigel as compared to Matrigel alone. The cell migration promoted by laminin-5-containing Matrigel was inhibited by the presence of anti-alpha3 integrin antibody. When MKN1 cells were cultured in a laminin-5-coated plate, these cells were promoted to produce matrix metalloproteinase (MMP)-9, as assessed by gelatin zymography, enzyme-linked immunosorbent assay, and reverse transcription-polymerase chain reaction. These results suggest that the production of MMP-9 by MKN1 cells was potentiated by the alpha3beta1 integrin-laminin-5 interaction, which facilitated their invasion via degradation of the matrix.

[A case of multiple lung abscesses successfully treated with computed tomography guided percutaneous thoracic drainage].

A 48-year-old woman who had abused alcohol admitted to our hospital because of fever and chest pain. Laboratory data presented inflammation and liver dysfunction. Chest X-ray films revealed multiple mass lesions with air fluid levels. We diagnosed multiple lung abscesses and started treatment with antibiotics. However, her fever did not improve and mass lesions increased. On day 6 of hospitalization, three drainage tubes were inserted percutaneously into the mass lesions under computed tomography (CT) guidance, and alpha-hemolytic streptococcus, peptostreptococcus sp. and neutrophils were isolated from the drainage fluid. Abcesses and inflammation were improved by administration of the antibiotics selected according to the result of fluid culture. Because all multiple abcesses were located near the pleura, we successfully treated them with simultaneous percutaneus thoracic drainage.