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Spermatogonial stem cells (SSCs) are the germline stem cells that are essential for the maintenance of spermatogenesis in the testis. However, it has not been sufficiently understood in amphibians, reptiles, and fish because numerous studies have been focused mainly on mammals. The aim of this review is to discuss scientific ways to elucidate SSC models of nonmammals in the context of the evolution of testicular organization since rodent SSC models. To further understand the SSC models in nonmammals, we point out common markers of an SSC pool (undifferentiated spermatogonia) in various types of testes where the kinetics of the SSC pool appears. This review includes the knowledge of (1) common molecular markers of vertebrate type A spermatogonia including putative SSC markers, (2) localization of the markers on the spermatogonia that have been reported in previous studies, (3) highlighting the most common markers in vertebrates, and (4) suggesting ways of finding SSC models in nonmammals. [ABSTRACT FROM AUTHOR]

• Spermatozoa were not detected in cryptorchid pig testes. • Undifferentiated spermatogonia were significantly reduced in cryptorchid pig testes. • Spermatocyte and spermatid were significantly reduced in cryptorchid pig testes. • Vimentin expressed Sertoli cells not changed in cryptorchid pig testes. Male germ cell apoptosis has been described in heat-damaged testes by cryptorchidism. In the present study, wild type pig testes were compared with cryptorchid testes via histological and immunohistological analyses. Spermatozoa were not detected in two cryptorchid testes and the diameters of seminiferous tubules were significantly reduced in cryptorchid pig testes compared with wild type pig testes. Cells expressing marker genes for undifferentiated spermatogonia, such as protein gene product 9.5 was significantly decreased in cryptochid pig testes. In addition, the numbers of cells expressing DEAD-box polypeptide 4 (VASA), synaptonemal complex protein 3, protamine, and acrosin (a biomarker of spermatocyte, spermatid, and spermatozoa) were significantly reduced in cryptochid pig testes. However, the number of vimentin-expressing Sertoli cells was not changed or was significantly increased in cryptorchid pig testes. This result indicates that male germ cells are specifically damaged by heat in cryptorchid pig testes and not Sertoli cells. These findings will facilitate the further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility. [ABSTRACT FROM AUTHOR]

The common essential environmental factors for plant growth are play the regulatory and signaling role in the developmental and metabolic processes of plants. A study aimed at determining the metabolite changes in wild and cultivated Euphorbia supina and their effects on its biological activity was conducted. These two plant groups were clearly discriminated from one another on principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) score plots. More detailed analyses of the extracts by ultra-performance liquid chromatography (UPLC) coupled to high-resolution electrospray ionization mass spectrometry (HRESIMS) on a quadrupole time-of-flight (QTof) analyzer was revealed approximately twenty phytochemicals, including gallotannins, ellagitannins and glycosidic derivatives of flavonoids the secondary phytochemicals in wild E. supina . The accumulation of these metabolites was responsible for the antioxidant content and activity in the group of plants, highlighting their potential utility in understanding the effects of environmental stress conditions on crop productivity and improving the bioactivity of medicinal plants. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance The tuber of Alismataceae Alisma orientale Juzepzuk has been prescribed as a remedy for treating the diseases associated with body fluid dysfunction such as edema and inflammatory lung diseases. Chronic obstructive pulmonary disease (COPD) is a debilitating, inflammatory lung disease without effective treatment. Along with persistent inflammation, autophagy has been recently reported to contribute to COPD. Here, by employing a murine model, we examined whether the tuber of the plant is effective against COPD Materials and methods The ethanol extract of the tuber of A. orientale Juzepzuk (EEAO) was fingerprinted by HPLC. For the establishment of COPD lung, mice received single intratracheal (i.t.) spraying of elastase and LPS per week for 2 weeks. After approximated to the dose prescribed typically to patients, EEAO was administered to the lung 2 h after each LPS treatment. Morphometric analyses, semi-quantitative RT-PCR, and western blot were performed to evaluate the effects of EEAO on emphysema, inflammation, and autophagy in mouse lungs. The effect of EEAO on autophagy was also assessed by western blot at the cellular level with murine macrophages and human lung epithelial cells. Results When receiving i.t. elastase and LPS for 2 weeks, mice developed emphysema and inflammation in the lung. EEAO treatment, however, significantly reduced emphysema and inflammatory cell infiltration to the lung with concomitant decrease of the production of pro-inflammatory cytokines including TNF-α, IL-6, and TGF-β, signature cytokines of COPD. Unlike control mice, the lungs of the COPD mice expressed LC3-II, a biomarker for autophagy formation, which was decreased by EEAO treatment. EEAO also lowered the expression of LC3-II in murine macrophage, RAW 264.7, and human lung epithelial cell, BEAS-2B, which was associated with EEAO activating mTOR. Conclusion EEAO relieved COPD pathologic features in a mouse model, which was associated with suppression of lung inflammation, emphysema, and autophagy. Our results suggest an effectiveness of the tuber of A. orientale in chronic inflammatory lung diseases such as COPD. [ABSTRACT FROM AUTHOR]

Euphorbia supina has been traditionally used for the treatment of furuncle and bloody diarrhea relevant to the inflammatory process. It has been proven to have a variety of pharmacological efficacies including antiarthritic, detoxification, hemostatic, and diuretic activities. RAW 264.7 macrophages and bone marrow-derived mast cells (BMMCs) were used to determine the anti-inflammatory and anti-allergic effects of E. supina (ES). NO production was assayed by measuring the nitrite content of the supernatants of cultured RAW 264.7 cells. β-hexosaminidase, a marker of mast cell degranulation, was quantitated by spectrophotometric analysis. ELISA was used for the analysis of interleukin-6 expression, and Western blotting was used to analyze 5-LOX, iNOS, and MAPK activation. The relevant gene expression upon ES treatment was measured by RT-PCR. ES inhibited inducible nitric oxide synthase (iNOS) in RAW 264.7 cells, and IL-6 and LTC 4 production in PMA- and A23187-induced BMMCs along with the downregulation of 5-LOX gene expression. Furthermore, in the present study, a decrease in p-ERK, p-JNK, and p-P38 expression, as well as the suppression of degranulation, were observed by treatment with ES. Further in vivo study revealed that ES treatment also remarkably inhibited xylene-induced mouse ear edema and MPO levels in mice ears. This study demonstrates that ES has a potential regulatory effect on the expression of inflammatory mediators through the inhibition of both the phosphorylation of MAPK signaling and the activation of degranulation. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Pseudolysimachion rotundum var . subintegrum (Speedwell, Plantaginaceae) is used as a traditional herbal medicine for treating bronchitis, cough and asthma in Korea, China, Russia, and Europe. Aim of the study In this study, we investigated the protective effects of the novel iridoid glycoside, piscroside C (compound 1) isolated from the methanolic extract of P. rotundum var. subintegrum against inflammatory responses using a cigarette smoke induced chronic obstructive pulmonary disease (COPD) and TNF-α-stimulated human airway epithelial NCI-H292 cells. Materials and methods The novel iridoid glycoside piscroside C was isolated from the methanolic extract of P. rotundum var. subintegrum . The chemical structure was established by NMR, HRESIMS, and optical rotation. In in vivo experiment, the mice received 1 h of cigarette smoke for 3 days. Piscroside C was administered to mice by oral gavage 1 h before cigarette smoke exposure for 3 days. In in vitro experiment, we evaluated the effect of piscroside C on proinflammatory mediators in H292 cells stimulated with TNF-α. Results Piscroside C significantly reduced the neutrophil influx, reactive oxygen species production, IL-6, TNF-α, and elastase activity in bronchoalveolar lavage fluid in COPD animals. In addition, piscroside C attenuated NF-κB and IκB phosphorylation, leading to reduced recruitment of inflammatory cells into the lung tissue. Consistent with the results of in vivo experiment, piscroside C significantly inhibited the expression of inflammatory cytokines (IL-6, IL-8 and IL-1β) by inhibiting NF-κB activation, as resulting decrease in the phosphorylation of IKKβ, IκBα and TAK1 in TNF-α-stimulated H292 cells. Conclusion These findings indicate that piscroside C effectively inhibits inflammatory responses, which is an important process in the development of COPD through suppression of IKK/NF-κB activation. Our study suggest that piscroside C might represent a useful therapeutic for the treatment of inflammatory airway disease. [ABSTRACT FROM AUTHOR]

Putative markers have been discovered and are used to identify and separate certain lineage of spermatogonia. The KIT is a marker for differentiating spermatogonial stem cells in several species including mice and goats. The objectives of this study were (1) to investigate reproductive stage-dependent KIT expression patterns in stallions and (2) to identify spermatogonia subpopulations expressing KIT in stallion testes. To achieve these objectives, testicular samples were obtained during routine field castration of stallions. The reproductive stage of the stallions was classified as pre-pubertal (<1 year, n = 3), pubertal (1–1.5 year, n = 4), post-pubertal (2–3 year, n = 6), or adult (4–8 year, n = 6). For immunohistochemistry, KIT was used at a dilution of 1:200. In the pre-pubertal and pubertal stage, most germ cells were immunolabeled with KIT. In the post-pubertal and adult stages, immunolabeling of KIT was evident in the germ cells attached to the basement membrane of the seminiferous tubules with exception of some spermatogonia. Co-immunolabeling with KIT and deleted in azoospermia like (DAZL) showed different co-staining patterns, including KIT only, both KIT and DAZL), or DAZL positive germ cell populations alone. The KIT was not immunolabeled in Sertoli or Leydig cells at any reproductive stages. The result of Western blot analysis verified the cross-activity of the KIT antibody with horse testes tissue. In conclusion, KIT appears to be expressed in differentiating spermatogonia, and may be used to identify and isolate differentiating germ cells from stallions. [ABSTRACT FROM AUTHOR]

Soybean ( Glycine max L.) leaves have unique nutraceutical and pharmacological benefits, and have been widely used as a source of healthy and functional food stuffs in Korea. In this study, we investigated the phytochemical metabolomic changes of soybean leaves depending on growth stages (maturation period) assessed based on UPLC-QTOF-MS analysis. Principal component analysis was carried out to trace the metabolite profiles of the phytochemicals from the vegetable stage (1D) through the seven reproductive stages (R1-R7). On the loading plot, significant changes in the contents of metabolites were found during the growth, and eight flavonoid kaempferol glycosides ( 2, 3, 6, 8, and 10), daidzein ( 14), genistein ( 17), and coumestrol ( 19) were evaluated as growth markers among the 19 isolated metabolites. The kaempferol glycosides were increasingly synthesized from the 1D to the R6 stage but decreased rapidly at stages R7-R8. The extensively synthesized daidzein and genistein were shown during seed growth in the pod (R5-R6), while coumestrol was increased significantly at stages R7-R8 (maturity period). The synthetic pathway of the flavonoids could be elucidated based on the concentration of the individual metabolites. These results demonstrate that the metabolite production changed depending on the growth stage; a possible pathway could be deduced using metabolomic analysis to provide information regarding physiological characterization and optimal harvesting time for crops. [ABSTRACT FROM AUTHOR]

In this study, the anti-inflammatory and anti-allergic effects of the chloroform-soluble extract of Agaricus blazei in mouse bone marrow-derived mast cells (BMMCs) were investigated. The chloroform-soluble extract inhibited IL-6 production in PMA plus A23187-stimulated BMMCs, and down-regulated the phosphorylation of Akt. In addition, this extract demonstrated inhibition of the degranulation of β-hexosaminidase and the production of IL-6, prostaglandin D2 and leukotriene C4 in PMA plus A23187-induced BMMCs. In conclusion, the chloroform-soluble extract of Agaricus blazei exerted anti-inflammatory and anti-allergic activities mediated by influencing IL-6, prostaglandin D2, leukotriene C4, and the phosphorylation of Akt. [ABSTRACT FROM AUTHOR]

Abstract: A new cytotoxic cyclic pentadepsipeptide, neo-N-methylsansalvamide, was produced by Fusarium solani KCCM90040, which was isolated from potato in Korea. The species of Fusarium was identified as F. solani by examining its morphological characteristics and by ITS-5.8rDNA sequence analysis. This compound was analysed for C33H52N4O6 by electrospray ionisation mass spectrometry and combined structural analysis. The one and two-dimensional NMR and the absolute configurations of amino acids spectral data allowed the resolution of five subunits linked in the following order: (S)-2-hydroxy-4-methylpentanoic acid, N-methyl-l-leucine, l-valine, l-leucine, and l-phenylalanine. The in vitro cytotoxic effect of the purified cyclic pentadepsipeptide was evaluated to establish the possibility that it would be a biohazard if present in foods. The concentrations of purified cyclic pentadepsipeptide required to inhibit cell growth in vitro by 50% for A549 (lung cancer), SK-OV-3 (ovarian cancer), SK-MEL-2 (skin melanoma), and MES-SA (uterine sarcoma) cell lines were 10.7±0.15, 11.2±1.23, 10.0±0.53, and 14.0±0.74M, respectively (mean±SE). [Copyright &y& Elsevier]

Abstract: Enniatins (ENs) H, I, and MK1688 and beauvericin (BEA) were purified from concentrated chloroform extracts of Fusarium oxysporum KFCC 11363P submerged cultures using HPLC, and their in vitro cytotoxicities were evaluated against four human carcinoma cell lines (lung, A549; ovarian, SK-OV-3; skin melanoma, SK-MEL-2; and colon, HCT15) using the sulforhodamine B (SRB) method. ENs I and MK1688 inhibited the growth of cancer cell lines most strongly and had similar cytotoxic effects on the tested human cancer cell cultures. The cytotoxicity of ENs I and MK1688 was three- to fourfold higher than that of BEA and EN H. When cultivated in Fusarium-defined medium (FDM), the concentrations of ENs and BEA produced in F. oxysporum KFCC 11363P decreased in the following order: EN MK1688 (0.81g/L) >EN I (0.55g/L) >BEA (0.17g/L) > EN H (0.16g/L). This study has shown that ENs H, I, and MK1688 exhibit cytotoxicity against certain adenocarcinoma cell lines. The results indicate the need for more investigations into the significance of the biological properties of these new ENs. [Copyright &y& Elsevier]

Abstract: Beauvericins and enniatins are cyclohexadepsipeptide mycotoxins that exhibit phytotoxicity and insecticidal activities. In the present study, the production of beauvericin and newly found enniatins (H, I, and MK1688) was characterized in 28 Fusarium strains isolated from potato samples in Korea. The predominant Fusarium species in potato was F. oxysporum (53.6%). Fifteen strains of F. oxysporum and two strains of other Fusarium species produced beauvericin (at concentrations from 3.1 to 743.2 μg/g) in culture on rice. Enniatins H and I were produced by 3 and 11 strains at concentrations from 33.1 to 781.3 μg/g and from 6.5 to 730.3 μg/g, respectively. Five isolates produced enniatin MK1688 at concentrations from 4.6 to 432.6 μg/g. In particular, one isolate (No. 1501) identified as F. oxysporum and two other Fusarium strains (Nos. 804 and 910) produced all of the tested toxins. These results indicate that enniatins H, I, and MK1688 and beauvericin are produced by Fusarium isolates occurring on potato. We do not know if the toxins can accumulate in the environment since it was not demonstrated. [Copyright &y& Elsevier]

Abstract: We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues (244KRKR247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236–252 and 249–267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues (318KKK320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport. [Copyright &y& Elsevier]

Abstract: Busulfan kills spermatogonia with the exception of a few that are attached to the basal membrane of the seminiferous epithelium. In mice, these remaining spermatogonia reacted strongly to a goat anti-mouse IgG antibody. Spermatogonia in untreated testes rarely showed the same reactivity. Testicular IgG levels are normally minimal but increase markedly, 4 weeks after busulfan treatment before peaking at week 6. Laser scanning cytometry analysis of control and busulfan-treated testicular cells showed busulfan treatment increased the frequency of cells that were positive for not only IgG (from 0.67±0.29 to 16.5±3.8%) but also for α6-integrin, β1-integrin, GFR(-1 and/or Ret. Thus, an enrichment in putative male stem cells correlates with appearance of IgG expression. Confocal microscopy revealed busulfan-treated cells contained both IgG and GFRα-1, and that the initial surface IgG became intracellular in the weeks following busulfan treatment. The basement membranes of the seminiferous tubules were compromised by busulfan treatment as the mRNA expression profiles of various adhesion molecules in the basement membranes were altered and electron microscopy revealed severe damage. Serum IgG levels increased in a manner corresponding with the increase in testicular IgG levels. Thus, it appears that in the busulfan-treated testis, small breaches of the blood-testis barrier leak IgG that is then taken up by a significant number of spermatogonia. When the busulfan-resistant germ cells were transferred into recipient germ cell-depleted testes, they settled and repopulated the recipient testes. Thus, the IgG-bearing cells observed after busulfan treatment may be putative spermatogonial stem cells. [Copyright &y& Elsevier]

Extracellular vesicles (EVs) are nanosized membrane-enclosed compartments that serve as messengers in cell-to-cell communication, both in normal physiology and in pathological conditions. EVs can transfer functional proteins and genetic information to alter the phenotype and function of recipient cells, which undergo different changes that positively affect their structural and functional integrity. Biological fluids are enriched with several subpopulations of EVs, including exosomes, microvesicles (MVs), and apoptotic bodies carrying several cargoes, such as lipids, proteins, and nucleic acids. EVs associated with the reproductive system are actively involved in the regulation of different physiological events, including gamete maturation, fertilization, and embryo and fetal development. EVs can influence follicle development, oocyte maturation, embryo production, and endometrial-conceptus communication. EVs loaded with cargoes are used to diagnose various diseases, including pregnancy disorders; however, these are dependent on the type of cell of origin and pathological characteristics. EV-derived microRNAs (miRNAs) and proteins in the placenta regulate inflammatory responses and trophoblast invasion through intercellular delivery in the placental microenvironment. This review presents evidence regarding the types of extracellular vesicles, and general aspects of isolation, purification, and characterization of EVs, particularly from various types of embryos. Further, we discuss EVs as mediators and messengers in reproductive biology, the effects of EVs on placentation and pregnancy disorders, the role of EVs in animal reproduction, in the male reproductive system, and mother and embryo cross-communication. In addition, we emphasize the role of microRNAs in embryo implantation and the role of EVs in reproductive and therapeutic medicine. Finally, we discuss the future perspectives of EVs in reproductive biology. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are nanosized membrane-enclosed compartments that serve as messengers in cell-to-cell communication, both in normal physiology and in pathological conditions. EVs can transfer functional proteins and genetic information to alter the phenotype and function of recipient cells, which undergo different changes that positively affect their structural and functional integrity. Biological fluids are enriched with several subpopulations of EVs, including exosomes, microvesicles (MVs), and apoptotic bodies carrying several cargoes, such as lipids, proteins, and nucleic acids. EVs associated with the reproductive system are actively involved in the regulation of different physiological events, including gamete maturation, fertilization, and embryo and fetal development. EVs can influence follicle development, oocyte maturation, embryo production, and endometrial-conceptus communication. EVs loaded with cargoes are used to diagnose various diseases, including pregnancy disorders; however, these are dependent on the type of cell of origin and pathological characteristics. EV-derived microRNAs (miRNAs) and proteins in the placenta regulate inflammatory responses and trophoblast invasion through intercellular delivery in the placental microenvironment. This review presents evidence regarding the types of extracellular vesicles, and general aspects of isolation, purification, and characterization of EVs, particularly from various types of embryos. Further, we discuss EVs as mediators and messengers in reproductive biology, the effects of EVs on placentation and pregnancy disorders, the role of EVs in animal reproduction, in the male reproductive system, and mother and embryo cross-communication. In addition, we emphasize the role of microRNAs in embryo implantation and the role of EVs in reproductive and therapeutic medicine. Finally, we discuss the future perspectives of EVs in reproductive biology. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are promising alternatives to existing treatments for multidrug-resistant bacteria-infected wounds. Therefore, the effect of protegrin-1 (PG1), a potent porcine AMP with broad-spectrum activity, on wound healing was evaluated. PG1-overexpressing transgenic mice were used as an in vivo model to evaluate its healing efficiency against Staphylococcus aureus-infected (106 colony forming units) wounds. We analyzed the wounds under four specific conditions in the presence or absence of antibiotic treatment. We observed the resolution of bacterial infection and formation of neo-epithelium in S. aureus-infected wounds of the mice, even without antibiotic treatment, whereas all wild-type mice with bacterial infection died within 8 to 10 days due to uncontrolled bacterial proliferation. Interestingly, the wound area on day 7 was smaller (p < 0.01) in PG1 transgenic mice than that in the other groups, including antibiotic-treated mice, suggesting that PG1 exerts biological effects other than bactericidal effect. Additionally, we observed that the treatment of primary epidermal keratinocytes with recombinant PG1 enhanced cell migration in in vitro scratch and cell migration assays. This study contributes to the understanding of broad-spectrum endogenous cathelicidins with potent antimicrobial activities, such as PG1, on wound healing. Furthermore, our findings suggest that PG1 is a potent therapeutic candidate for wound healing. [ABSTRACT FROM AUTHOR]

The extracellular matrix is important in cell growth, proliferation, and differentiation. Gelatin, a support for adhering cells, is used for coating culture plate surfaces of several primary and stem cells. However, gelatin characteristics on culture plates and its cell interactions are not understood. Here, we aimed to identify the effect of gelatin topography on culture plates on the proliferation and colony formation of porcine spermatogonial germ cells (pSGC). To generate different surface topographies, gelatin powder was dissolved in H 2 O at varying melting temperatures (40, 60, 80, and 120 °C) and coated on the surface of the culture plates. At 40 °C, the pores of the gelatin scaffold were regular ellipses 5−6 μm in diameter and 10−30 nm in thickness. However, at 120 °C, irregular pores 20−30 μm in diameter and 10−20 nm in thickness were obtained. Additionally, the number of attached cells and pSGC colonies were significantly more at 40 °C than at 120 °C after a week of culture. Interestingly, the feeder cells did not settle properly at 120 °C but detached easily from the culture dishes. PSGC colonies were 100 μm in diameter at 40 °C, with small and detached colonies observed at 120 °C. Thus, optimal topography of gelatin was obtained at 40 °C, which was sufficient for the proliferation of feeder cells and the formation of pSGC colonies. Thus, gelatin scaffold conditions at 40 °C and 60 °C were optimal for the derivation and culture of pSGC, and gelatin surface morphology is important for the maintenance of supportive feeder cells for pSGC proliferation and colony formation. • The pore size and diameter of pure gelatin were less than 5 μm in the scaffold structure. • The optimal gelatin pore size for pSGC growth is a 4-5 μm scaffold. • Gelatin dissolution temperature is a microenvironmental condition for maintaining the characteristics of pSGC. • The pSGC was observed on the roughness of plates coated with gelatin melted at 40 °C and 60 °C. [ABSTRACT FROM AUTHOR]

The flavonoids mosloflavone, oroxylin A, and norwogonin, which were purified from Scutellaria baicalensis Georgi, significantly protected Vero cells against Coxsackievirus B3 (CVB3)-induced cell death. To investigate the in vivo antiviral activity of oroxylin A, we intraperitoneally inoculated CVB3 into 4-week-old BALB/c mice. Body weights and blood glucose levels of the mice were decreased after CVB3 infection, and these changes were attenuated by the administration of oroxylin A. Importantly, treatment of mice with oroxylin A reduced viral titers in the pancreas and decreased the serum levels of the inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α. Additionally, the administration of oroxylin A mitigated the histological pancreatic lesions and apoptotic cell death induced by CVB3 infection and increased the levels of phospho-eIF2α in infected pancreata. The results suggest that oroxylin A may represent a potent antiviral agent against CVB3 infection. [ABSTRACT FROM AUTHOR]

Precise regulation of the cell cycle of embryonic stem cells (ESCs) is critical for their self-maintenance and differentiation. The cell cycle of ESCs differs from that of somatic cells and is different depending on the cell culture conditions. However, the cell cycle regulation in ESCs via epigenetic mechanisms remains unclear. Here, we showed that the ATP-dependent chromatin remodeler Ino80 regulates the cell cycle genes in ESCs under primed conditions. Ino80 loss led to a significantly extended length of the G1-phase in ESCs grown under primed culture conditions. Ino80 directly bound to the transcription start site and regulated the expression of cell cycle-related genes. Furthermore, Ino80 loss induced cell apoptosis. However, the regulatory mechanism of Ino80 in differentiating ESC cycle slightly differed; an extended S-phase was detected in differentiating inducible Ino80 knockout ESCs. RNA-seq analysis of differentiating ESCs revealed that the expression of genes associated with organ development cell cycle is persistently altered in Ino80 knockout cells, suggesting that cell cycle regulation by Ino80 is not limited to undifferentiated ESCs. Therefore, our study establishes the function of Ino80 in ESC cycle via transcriptional regulation, at least partly. Moreover, this Ino80 function may be universal to other cell types. [ABSTRACT FROM AUTHOR]

A conjugate heat transfer analysis method for the thermal integrity of a reactor vessel under external reactor vessel cooling conditions is developed to resolve light metal layer focusing effect issue for in-vessel retention. The method calculates steady-state three-dimensional temperature distribution of a reactor vessel using coupled conjugate heat transfer between in-vessel three-layered stratified corium (metallic pool, oxide pool and heavy metal and polar-angle dependent boiling heat transfer at the outer surface of a reactor vessel). The three-layer corium heat transfer model is utilizing lumped-parameter thermal-resistance circuit method. For the ex-vessel boiling boundary conditions, nucleate, transition and film boiling are considered. The thermal integrity of a reactor vessel is addressed in terms of heat flux at the outer-most nodes of the vessel and remaining thickness profile. The vessel three-dimensional heat conduction is validated against a commercial code. It is found that even though the internal heat flux from the metal layer goes far beyond critical heat flux (CHF) the heat flux from the outermost nodes of the vessel may be maintained below CHF due to massive vessel heat diffusion. The heat diffusion throughout the vessel is more pronounced for relatively low heat generation rate in an oxide pool. Parametric calculations are performed considering thermal conditions such as peak heat flux from a light metal layer, heat generation in an oxide pool and external boiling conditions. The major finding is that the most crucial factor for success of in-vessel retention is not the mass of the molten light metal above the oxide pool but the heat generation rate inside an oxide pool and the three-dimensional vessel heat transfer provides much larger minimum vessel thickness. [ABSTRACT FROM AUTHOR]

Callicarpa japonica Thunb. (CJT) is traditionally used as an herbal remedy for the treatment of inflammatory diseases. This study aimed to investigate the anti-inflammatory response of CJT in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and LPS-induced acute lung injury (ALI) in mice. The C57BL/6 mice were administered 30 mg/kg of CJT by oral gavage for 3 days. LPS is applied to animals by intranasal administration 1 h after final CJT treatment. LPS is applied to animals by intranasal administration 1 h after final CJT treatment. LPS was delivered intranasally 1 h after the final CJT treatment. In the LPS-stimulated RAW264.7 cells, CJT significantly decreased nitric oxide (NO) and interleukin (IL)-6 in a concentration-dependent manner by reducing inducible NO synthase (iNOS) and IL-6 mRNA levels. In the ALI model, CJT decreased the inflammatory cell count in the bronchoalveolar lavage fluid (BALF) while IL-6 levels were reduced in CJT-treated mice compared with the ALI control mice. CJT also inhibited airway inflammation by reducing iNOS expression in lung tissue. In conclusion, our results indicate that CJT inhibits inflammatory responses in LPS-stimulated RAW264.7 cells and in the LPS-induced ALI model. Therefore, we suggest that CJT has the potential to treat inflammatory diseases such as pneumonia. [ABSTRACT FROM AUTHOR]

The plant Carpesium abrotanoides (CA) is used in Asian herbal medicines as an insecticide and to treat bruises. However, the effect of single compounds from CA blooms and the mechanism of its immunosuppressive effect remain poorly understood. The aim of this study was to investigate the mechanism of the immunosuppressive effect in the three kinds of immune cells, and the immunosuppressive effect of CA bloom extract (CAE) in acute inflammation models (LPS and ConA-induced inflammation). Interleukin-6, IL-4, IL-13, IFNγ, and IL-10—but not TNFα—were significantly reduced in a dose-dependent manner by 4α,5α-epoxy-10α,14-dihydro-inuviscolide (INV). Furthermore, INV inhibited NF-κB transcriptional activation and IL-10 promoter activity in the same manner as for Bay11. Meanwhile, treatment with dexamethasone reduced the levels of IFNγ, but not IL-10, and resulted in no change in NF-κB transcriptional activation or the IL-10 promoter. INV did not affect PMA-induced IκB kinase complex phosphorylation, IκB degradation, or MAPK and the nuclear translocation of p65, as with DEX. The in vivo, CAE has an immunosuppressive effect on the LPS-induced inflammation response model by inhibiting the plasma level of IFNγ and IL-6 levels. CAE treatment also tends to attenuate the plasma level of IFNγ, IL-4, and IL-6 in ConA-induced inflammation. These findings indicate that INV causes the reduction of the cytokine profile by blocking the NF-κB transcription factor activation and the molecular mechanism by which INV operates could provide new insights into the unique mechanisms responsible for NF-κB inhibition, in contrast to established immunosuppressants, as a therapeutic agent for immunopathological treatment. [ABSTRACT FROM AUTHOR]

Chemotherapy is used for childhood cancer but may lead to infertility in patients. Spermatogonia stem cells are present in the testes of prepubertal boys, although they do not produce sperm at this age. Herein, we evaluated the toxicity of cisplatin, a known medicine for cancer treatment, in neonatal mouse testes using in vitro organ culture. Mouse testicular fragments (MTFs) derived from 5.5-d postpartum mouse testes were exposed to 1–10 μg/mL cisplatin. The results showed that cisplatin significantly downregulated the expression of germ cell marker genes, including differentiated and undifferentiated, in a dose-dependent manner. In particular, a high dose of cisplatin (10 μg/mL) led to germ cell depletion. In addition, the expression levels of the Sertoli cell marker gene, the number of SOX9+ Sertoli cells, and the levels of SOX9 protein were markedly decreased in cisplatin-treated MTFs compared to controls. The mRNA expression of steroidogenic enzyme-related genes significantly increased in cisplatin-treated MTFs, except for estrogen receptor 1 (Esr1). Consistently, 3β-hydroxysteroid dehydrogenase protein was also observed in the interstitial regions of cisplatin-treated MTFs. Altogether, our findings showed a significant impairment in germ cell development, Sertoli cell survival, and steroidogenesis in the MTFs of cisplatin-treated mice. [ABSTRACT FROM AUTHOR]

The dynamics of uterine endometrium is important for successful establishment and maintenance of embryonic implantation and development, along with extensive cell differentiation and proliferation. The tissue event is precisely and complicatedly regulated as several signaling pathways are involved including two main hormones, estrogen and progesterone signaling. We previously showed a novel signaling molecule, Serine/threonine protein kinase 3/4 (STK3/4), which is responded to hormone in the mouse uterine epithelium. However, the role and regulation of its target, YES-associated protein (YAP) remains unknown. In this study, we investigated the expression and regulation of YAP in mouse endometrium. We found that YAP was periodically expressed in the endometrium during the estrous cycle. Furthermore, periodic expression of YAP was shown to be related to the pathway under hormone treatment. Interestingly, estrogen was shown to positively modulate YAP via endometrial epithelial receptors. In addition, the knockdown of YAP showed that YAP regulated various target genes in endometrial cells. The knockdown of YAP down-regulated numerous targets including ADAMTS1, AMOT, AMOTL1, ANKRD1, CTNNA1, MCL1. On the other hand, the expressions of AREG and AXL were increased by its knockdown. These findings imply that YAP responds via Hippo signaling under various intrauterine signals and is considered to play a role in the expression of factors important for uterine endometrium dynamic regulation. [ABSTRACT FROM AUTHOR]

The lymphatic system is critical for maintaining the homeostasis of lipids and interstitial fluid and regulating the immune cell development and functions. Developmental anomaly-induced lymphatic dysfunction is associated with various pathological conditions, including lymphedema, inflammation, and cancer. Most lymphatic endothelial cells (LECs) are derived from a subset of endothelial cells in the cardinal vein. However, recent studies have reported that the developmental origin of LECs is heterogeneous. Multiple regulatory mechanisms, including those mediated by signaling pathways, transcription factors, and epigenetic pathways, are involved in lymphatic development and functions. Recent studies have demonstrated that the epigenetic regulation of transcription is critical for embryonic LEC development and functions. In addition to the chromatin structures, epigenetic modifications may modulate transcriptional signatures during the development or differentiation of LECs. Therefore, the understanding of the epigenetic mechanisms involved in the development and function of the lymphatic system can aid in the management of various congenital or acquired lymphatic disorders. Future studies must determine the role of other epigenetic factors and changes in mammalian lymphatic development and function. Here, the recent findings on key factors involved in the development of the lymphatic system and their epigenetic regulation, LEC origins from different organs, and lymphatic diseases are reviewed. [ABSTRACT FROM AUTHOR]

The uterus is essential for embryo implantation and fetal development. During the estrous cycle, the uterine endometrium undergoes dramatic remodeling to prepare for pregnancy. Angiogenesis is an essential biological process in endometrial remodeling. Steroid hormones regulate the series of events that occur during such remodeling. Researchers have investigated the potential factors, including angiofactors, involved in endometrial remodeling. The Hippo signaling pathway discovered in the 21st century, plays important roles in various cellular functions, including cell proliferation and cell death. However, its role in the endometrium remains unclear. In this review, we describe the female reproductive system and its association with the Hippo signaling pathway, as well as novel Hippo pathway genes and potential target genes. [ABSTRACT FROM AUTHOR]

MHC genes are highly polymorphic as antigen presenting molecules for adaptive immune response in vertebrates. In this study, we evaluated the diversity of dog leukocyte antigen (DLA) class II genes among two Korean breeds, the Sapsaree and the Jindo, and three Chinese native breeds, Tibetan Mastiff, Pug, and Pekingese, and determined their genetic relationships. Sequence-based typing of DLA-DRB1 and DLA-DQB1 was performed in 138 dogs. We identified 27 alleles for DRB1 and 24 for DQB1, including five new alleles. DRB1*015:01 was shared among all five breeds and was a major allele for the Pug, Tibetan Mastiff, and Sapsaree. The observed heterozygosity for the five breeds ranged from 0.67 to 1.00 for DRB1 and from 0.80 to 1.00 for DQB1. A total of 40 DRB1-DQB1 haplotypes, including nine new haplotypes, were identified. Interestingly, most haplotypes (33/40, 82%) were specific to a single breed, and only seven were present in multiple breeds. Haplotype sharing analysis together with previously available data from 109 breeds revealed that compared to within region distances, Asian breeds were more distant than breeds from other regions. As a first report on the analysis of DLA genes of dog breeds in Korean peninsula, our results show that these breeds carry unique DLA class II haplotype lineages. Our results indicate that the diversity and breed specificity of DLA class II genes are much higher among East Asian breeds than among breeds from other regions. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) are crucial for maintaining spermatogenesis, studying germ line stem cell biology, and producing transgenic animals. Growth factors, including leukemia inhibitory factor (LIF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and glial cell line-derived neurotrophic factor (GDNF), are essential for in vitro culture of SSCs as well as their self-renewal and maintenance. In this study, we investigated the effects of these growth factors on porcine spermatogonial germ cell (pSGC) colony formation. We determined round alkaline phosphatase (AP)-positive pSGC colonies in the presence and absence of growth factors after 7 days of pSGC culture. EGF was found to be essential to support the formation of AP-positive pSGC colonies. The expression of epidermal growth factor receptor (EGFR) and Erb-B2 receptor tyrosine kinase 2 (ERBB2) was also altered in cultured pSGCs compared to that in feeder cells. We verified the effect of EGF signaling on pSGC colony formation using AG1478 as an EGFR inhibitor and AG879 as an ERBB2 inhibitor. pSGC colonies were observed in low dose AG1478-treated groups with EGF, whereas a high dose of AG1478 suppressed pSGC colony formation. AP-positive colonies were also observed in all AG879-treated groups. Taken together, EGFR and EGF signaling play a critical role in the initiation of colony formation of pSGCs. Our study provides insights into the mechanisms of EGF-mediated colony formation by SGCs derived from porcine testes, and will aid the development of transplantation techniques for the production of transgenic offspring. [ABSTRACT FROM AUTHOR]

The effects of ΔPb-CATH4, a cathelicidin derived from Python bivittatus, were evaluated against Staphylococcus aureus-infected wounds in mice. These effects were comparable to those of classical antibiotics. ΔPb-CATH4 was resistant to bacterial protease but not to porcine trypsin. A reduction in the level of inflammatory cytokines and an increase in the migration of immune cells was observed in vitro. Thus, ΔPb-CATH4 can promote wound healing by controlling infections including those caused by multidrug-resistant bacteria via its immunomodulatory effects. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells can be established from parthenogenetic embryos, which only possess maternal alleles with maternal-specific imprinting patterns. Previously, we and others showed that parthenogenetic embryonic stem cells (pESCs) and parthenogenetic induced pluripotent stem cells (piPSCs) progressively lose the bimaternal imprinting patterns. As ESCs and iPSCs are naïve pluripotent stem cells, parthenogenetic primed pluripotent stem cells have not yet been established, and thus, their imprinting patterns have not been studied. Here, we first established parthenogenetic epiblast stem cells (pEpiSCs) from 7.5 dpc parthenogenetic implantation embryos and compared the expression patterns and DNA methylation status of the representative imprinted genes with biparental EpiSCs. We found that there were no striking differences between pEpiSCs and biparental EpiSCs with respect to morphology, pluripotency gene expression, and differentiation potential, but there were differences in the expression and DNA methylation status of imprinted genes (H19, Igf2, Peg1, and Peg3). Moreover, pEpiSCs displayed a different DNA methylation pattern compared with that of parthenogenetic neural stem cells (pNSCs), which showed a typical bimaternal imprinting pattern. These results suggest that both naïve pluripotent stem cells and primed pluripotent stem cells have an unstable imprinting status. [ABSTRACT FROM AUTHOR]

During embryonic development, cells undergo changes in gene expression, signaling pathway activation/inactivation, metabolism, and intracellular organelle structures, which are mediated by mitochondria. Mitochondria continuously switch their morphology between elongated tubular and fragmented globular via mitochondrial fusion and fission. Mitochondrial fusion is mediated by proteins encoded by Mfn1, Mfn2, and Opa1, whereas mitochondrial fission is mediated by proteins encoded by Fis1 and Dnm1L. Here, we investigated the expression patterns of mitochondria-related genes during the differentiation of mouse embryonic stem cells (ESCs). Pluripotent ESCs maintain stemness in the presence of leukemia inhibitory factor (LIF) via the JAK-STAT3 pathway but lose pluripotency and differentiate in response to the withdrawal of LIF. We analyzed the expression levels of mitochondrial fusion- and fission-related genes during the differentiation of ESCs. We hypothesized that mitochondrial fusion genes would be overexpressed while the fission genes would be downregulated during the differentiation of ESCs. Though the mitochondria exhibited an elongated morphology in ESCs differentiating in response to LIF withdrawal, only the expression of Mfn2 was increased and that of Dnm1L was decreased as expected, the other exceptions being Mfn1, Opa1, and Fis1. Next, by comparing gene expression and mitochondrial morphology, we proposed an index that could precisely represent mitochondrial changes during the differentiation of pluripotent stem cells by analyzing the expression ratios of three fusion- and two fission-related genes. Surprisingly, increased Mfn2/Dnm1L ratio was correlated with elongation of mitochondria during the differentiation of ESCs. Moreover, application of this index to other specialized cell types revealed that neural stems cells (NSCs) and mouse embryonic fibroblasts (MEFs) showed increased Mfn2/Dnm1L ratio compared to ESCs. Thus, we suggest that the Mfn2/Dnm1L ratio could reflect changes in mitochondrial morphology according to the extent of differentiation. [ABSTRACT FROM AUTHOR]

Swine leukocyte antigens play indispensable roles in immune responses by recognizing a large number of foreign antigens and thus, their genetic diversity plays a critical role in their functions. In this study, we developed a new high-resolution typing method for pig SLA-1 and successfully typed 307 individuals from diverse genetic backgrounds including 11 pure breeds, 1 cross bred, and 12 cell lines. We identified a total of 52 alleles including 18 novel alleles and 9 SLA-1 duplication haplotypes, including 4 new haplotypes. We observed significant differences in the distribution of SLA-1 alleles among the different pig breeds, including the breed specific alleles. SLA-1 duplication was observed in 33% of the chromosomes and was especially high in the biomedical model breeds such as SNU (100%) and NIH (76%) miniature pigs. Our analysis showed that SLA-1 duplication is associated with the increased level of SLA-1 mRNA expression in porcine cells compared to that of the single copy haplotype. Therefore, we provide here the results of the most extensive genetic analysis on pig SLA-1. [ABSTRACT FROM AUTHOR]

Resmethrin is a widely used pyrethroid insecticide, which causes low toxicity in mammals. However, its toxicity in testes has not been fully investigated. Therefore, we evaluated the toxicity of resmethrin in mouse testes using an in vitro organ culture. Mouse testicular fragments (MTFs) derived from neonates were cultured in medium containing resmethrin for 30 days. Effects on spermatogenesis in the cultured testes were investigated as functions of both time and dose. Resmethrin significantly downregulated the transcription levels of marker genes for spermatogonia and the number of spermatogenic germ cells relative to those of the controls, according to quantitative PCR and immunostaining. In addition, spermatocyte was observed in the control, but not in 50 μM resmethrin-exposed cultures. Levels of the SYCP3 meiotic marker and phosphorylated H2AX decreased by resmethrin treatment, as observed by Western blotting. Toxic or apoptotic effects of resmethrin in Sertoli and Leydig cells from MTFs were not observed by immunostaining and Tunnel assay. No changes in the expression of steroidogenic enzymes were noted. Apoptosis was only detected in the germ cells of resmethrin-treated MTFs. Thus, the highest dose of resmethrin tested (50 μM) completely inhibited spermatogenesis, because of apoptosis of germ cells and spermatocytes. Although the in vivo toxicity of resmethrin has not yet been studied in detail, significant evidence for cytotoxicity was observed in our organ cultures. This methodological approach is useful for the study of reproductive toxicity before proceeding to animal models, as it greatly reduces the use of laboratory animals. [ABSTRACT FROM AUTHOR]

Human‐induced pluripotent stem cells (hiPSCs) have facilitated studies on organ development and differentiation into specific lineages in in vitro systems. Although numerous studies have focused on cellular differentiation into neural lineage using hPSCs, most studies have initially evaluated embryoid body (EB) formation, eventually yielding terminally differentiated neurons with limited proliferation potential. This study aimed to establish human primitive neural stem cells (pNSCs) from exogene‐free hiPSCs without EB formation. To derive pNSCs, we optimized N2B27 neural differentiation medium through supplementation of two inhibitors, CHIR99021 (GSK‐3 inhibitor) and PD0325901 (MEK inhibitor), and growth factors including basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (hLIF). Consequently, pNSCs were efficiently derived and cultured over a long term. pNSCs displayed differentiation potential into neurons, astrocytes, and oligodendrocytes. These early NSC types potentially promote the clinical application of hiPSCs to cure human neurological disorders. [ABSTRACT FROM AUTHOR]

The amount of concentrated feed supplied to a dairy cow affects milk yield. However, there is no evidence of a relationship between the colostrum proteomic composition and energy intake. We supplied 30 heifers (4–24 months old, two groups of 15 heifers each) with either a normal diet and high-energy diet to investigate the correlation between energy intake and colostrum protein composition. Colostrum milk proteins were analysed on the day of calving and on the third day following calving using two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). Five proteins were identified as differentially expressed between the two feeding groups in the colostrum on the day of calving. The levels of αS2-casein precursor and β-casein was higher in the colostrum from the high-energy diet group (HEG), whereas the levels of IgG3 heavy chain constant region, non-classical MHC class I antigen isoform X2, and β-casein A2 variant were higher in the normal-diet group (NEG) colostrum. Twelve differential proteins were identified on the third day: β-lactoglobulin, αS2-casein, zinc-α2-glycoprotein, lactoferrin, fibrinogen gamma-B chain isoform X1, non-classical MHC class I antigen isoform X2, complement C3, gelsolin isoform A precursor, vitamin D-binding protein isoform X1, immunoglobulin gamma 1 heavy chain constant region, IgG3 heavy chain constant region and polymeric immunoglobulin receptor. All were present at higher levels in the normal-diet group colostrum than in the high-energy diet group colostrum, although the milk yield from mature cows was lower in the normal-diet group. In conclusion, a high-energy diet can enhance milk production; however, the levels of immune-related factors are higher in the colostrum of cows fed a normal diet. Feeding strategies for improving milk production are important in dairy farming in order to maximise profit. The relationship between the colostrum proteomic composition and energy intake was investigated. Adequate energy with a balanced amount of feed intake is a key for better production of the milk component. The results of the present study contributed to the development of feeding strategies and improved milk quality. [ABSTRACT FROM AUTHOR]

The reproductive toxicity of 4-octylphenol (4-OP) has been studied in animals such as mouse and fish. In humans, the exposure of sperm to 4-OP has been shown to decrease motility and viability. In this study, we performed an in vitro assessment of the toxic effects of 4-OP on mouse TM4 Sertoli cells and investigated the underlying molecular mechanisms. TM4 cells were treated with four concentrations (0, 10, 30, and 50 µM) of 4-OP at the following time points: 24, 48, and 72 h. Cell viability and apoptosis assays were conducted following 4-OP exposure. We found that 4-OP significantly decreased cell viability in a concentration- and time-dependent manner, and increased apoptosis. Quantitative PCR analysis showed that the mRNA expression levels of BCL2 Associated X, Apoptosis Regulator (Bax) and BCL2 Antagonist/Killer (Bak) increased while that of BCL2 Apoptosis Regulator (Bcl-2) decreased in 4-OP-exposed cells compared with that in the controls. Western blotting revealed that 4-OP induced caspase-3 activity and Bad phosphorylation in a concentration- and time-dependent manner. Additionally, cytochrome C protein did not colocalize with mitochondrial marker dye by 24 h. Cytochrome c protein expression increased in a time-dependent manner upon exposure to 50 µM 4-OP. These results suggest that 4-OP induces mitochondria-mediated apoptosis through regulation of Bcl-2 family proteins and caspase-3 activation in male Sertoli cells. [ABSTRACT FROM AUTHOR]

Abstract Proline-arginine-rich (PR)-39 is neutrophil antimicrobial peptide that has potent antimicrobial activity against a broad spectrum of microorganisms, including bacteria, fungi, and some enveloped viruses as a part of the innate immune system. We analyzed the nucleotide sequence variations of PR - 39 exon 4, which is the mature peptide region responsible for antimicrobial activity, from 48 pigs of six breeds using sequence-based typing. The analysis identified four alleles including allele PR-35 with a 12-bp deletion near the N-terminus. Interestingly, 16.7% of individuals showed the presence of three alleles per individual, but only in the Berkshire and Duroc breeds. We further analyzed the genetic diversity of PR-39 for the entire genomic region of the gene from PR - 39 exon 1 to the 3′ untranslated region for different alleles by PCR amplification and cloning. The antimicrobial activity of chemically synthesized PR-35 was similar to that of PR-39, but the level of mammalian cell cytotoxicity was lower than the wild type. Better knowledge of the genetic diversity of PR-39 among different individuals and breeds may contribute to improved immune defense of pigs. PR-35, as a natural antimicrobial peptide variant, could be an interesting candidate for the development of peptide antibiotics. Highlights • PR-35, a naturally truncated form of PR-39, was found from genome of Berkshire and Duroc breeds in pigs. • PR-35 exhibited similar bactericidal activity against gram-negative bacteria, like PR-39. • PR-35 showed lower toxicity to Human embryonic kidney cells than PR-39. • PR-39 probably has copy number variations in the pig genome. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are active regulators of numerous biological and physiological processes including most of the events of mammalian reproduction. Understanding the biological functions of miRNAs in the context of mammalian reproduction will allow a better and comparative understanding of fertility and sterility in male and female mammals. Herein, we summarize recent progress in miRNA‐mediated regulation of mammalian reproduction and highlight the significance of miRNAs in different aspects of mammalian reproduction including the biogenesis of germ cells, the functionality of reproductive organs, and the development of early embryos. Furthermore, we focus on the gene expression regulatory feedback loops involving hormones and miRNA expression to increase our understanding of germ cell commitment and the functioning of reproductive organs. Finally, we discuss the influence of miRNAs on male and female reproductive failure, and provide perspectives for future studies on this topic. [ABSTRACT FROM AUTHOR]

Abstract We performed the in silico genome-wide identification of antimicrobial peptides against the available genome sequence of the naked mole rat Heterocephalus glaber (H. glaber). Our results showed the presence of Hg-CATH, the single cathelicidin containing the antimicrobial domain in H. glaber. We chemically synthesized a 25 amino-acid peptide (ΔHg-CATH) corresponding to the predicted antimicrobial-active core region of Hg-CATH, and evaluated its antibacterial activity against seven bacterial strains. The ΔHg-CATH peptide exhibited strong bactericidal activity against gram-negative bacteria, including a multi-drug resistant strain, while showing low toxicity towards mammalian cells, including erythrocytes. Scanning electron microscopy images of bacterial cells treated with ΔHg-CATH showed disruption of their membranes due to the formation of toroidal pores. Identifying novel antimicrobial peptides, such as Hg-CATH, may be important for identifying candidate peptides for the control of multi-drug resistant bacteria. Highlights • Hg-CATH was the single cathelicidin identified in the genome of naked mole rats. • ΔHg-CATH exhibited bactericidal activity against gram-negative bacteria. • ΔHg-CATH showed low toxicity to chicken erythrocyte and mammalian cells. • Peptides like Hg-CATH may protect against multi-drug resistance bacteria. [ABSTRACT FROM AUTHOR]

Abstract: A system for fully automatic contact‐less image‐based measurement of volumetric flow rate in urban drainage structures is presented. The hardware includes two original equipment manufacturer cameras and a single‐board computer on which our custom image processing software is running. The value of water discharge depends on the surface velocity, water level and channel's geometry. The level of the flow is estimated as the difference between distances from the camera to the water surface and from the camera to the channel's bottom. Camera‐to‐water distance is recovered automatically using large‐scale stereo‐matching, whereas the distance to the channel's bottom is measured upon installation. Surface velocity is calculated using cross‐correlation template matching: individual natural particles in the flow are detected and tracked throughout the sequence of images recorded over a fixed time interval. The relative discharge computation error is lower than 1.34% of the theoretical maximal discharge for a given location, which makes our system competitive to commercial components such as ultrasonic flow meters, while using cheaper technologies. [ABSTRACT FROM AUTHOR]

Mammalian testes are maintained at a relatively lesser temperature than the abdominal region so that normal spermatogenesis can occur. Germ cell apoptosis has resulted in heat-damaged testes that occurs as a result of cryptorchidism, but the mechanism is not yet fully understood. To elucidate the cause of germ-cell death by cryptorchidism, cryptorchidism was surgically induced in dog testes and histological and molecular analyses were performed. Histological data indicated that the seminiferous tubules of cryptorchid testes and epididymis contained fewer germ cells. Total RNA sequencing was performed to screen for overexpressed genes in cryptorchid dog testes. Clusterin RNA was in greater abundance (approximately 12.8-fold) in cryptorchid testes than in normal testes. In addition, cleaved caspase-3 and -8 were detected in greater abundance in cryptorchid dog testes. Real time RT-PCR and western blotting analysis indicated there was a greater abundance of clusterin in cryptorchid dog testes. Furthermore, clusterin was detected in extracellular regions of cryptorchid dog testes during the 4 weeks after surgery. Thus, germ-cell specific apoptosis and expression of clusterin genes occur with a resulting presence of this protein in extracellular regions of cryptorchid dog testes. This result will facilitate further study of spermatogenesis and the specific mechanisms by which cryptorchidism results in male infertility. [ABSTRACT FROM AUTHOR]

The stem cell niche is a complex unit comprising key components, such as the extracellular matrix and various paracrine factors, which regulate the differentiation of adult stem cells. In our previous study, we established pig spermatogonial stem cells (pSSCs) in culture and identified the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in pSSCs. The present study investigated not only the expression of IGFBP-3, but also its possible role in pSSCs. In this study, IGFBP-3-expressing cells responded positively to protein gene product 9.5 (PGP9.5), which is a marker for pig spermatogonia. IGFBP-3 expression was significantly increased in 60-dayold pig testes. Additionally, the expression levels of insulinlike growth factor I (IGF-I) and its receptor (IGF-IR) were observed in pSSCs and pig Sertoli cells (pSCs). Furthermore, IGF-I treatment enhanced the proliferation of pSCs and pSSCs when they were co-cultured. Blocking the IGF-I pathway using a specific IGF-IR inhibitor dramatically reduced the proliferation of pSCs. In addition, when heparan sulfate was used to sequester IGFBP-3 from IGF-I binding, a significant increase in the proliferation of pSCs was observed. Exogenous IGF-I treatment also increased the expression level of IGFBP-3 in cultured pSSCs. Furthermore, pSSCs grew well in IGF-I-treated pSC conditioned media. In summary, IGF-I and IGF-IR signaling are important for the proliferation of pSCs, and the germ cell-derived IGFBP-3 had an inhibitory effect on the mitotic activity of IGF-I in pSCs. [ABSTRACT FROM AUTHOR]

The signaling of chemokine stromal cell-derived factor (SDF)-1 and its receptor C-X-C motif chemokine receptor 4 (CXCR4) is involved in the cellular proliferation, survival, and migration of various cell types. Although SDF-1/CXCR4 has been implicated in the maintenance of the spermatogonial population during mouse testis development, their expression patterns and functions in boar testis remain unclear. In the present study, the expression pattern of SDF-1 and CXCR4 was determined during pre-pubertal and post-pubertal stage boar testes and in vitro cultured porcine spermatogonial stem cells (pSSCs). The role of these proteins in colony formation in cultured pSSCs was also investigated. Interestingly, SDF-1 expression was observed in PGP 9.5-positve spermatogonia in all developing stages of boar testis; however, CXCR4 expression was only detected in spermatogonia from 5-day-old boar testis. In addition, SDF-1 and CXCR4 expression was observed in cultured pSSCs from 5-day-old boar testes, and inhibition of the CXCR4 receptor signaling pathway by AMD3100 significantly decreased the colony formation of pSSCs. These results suggest that SDF-1 and CXCR4 are useful markers for detecting stage-specific spermatogonia in boar testis. Our results reveal the role of the SDF-1/CXCR4 axis in pSSC in vitro culture. [ABSTRACT FROM AUTHOR]

Whereas stage-specific markers for spermatogonial cells have been well investigated in mouse, the specific markers of germ cells in the testis of domestic animals have not been well defined. Phosphoglycerate kinase (PGK), an enzyme that converts 1,3-bisphosphoglycerate and adenosine diphosphate to 3-phosphoglycerate and adenosine triphosphate, has two isozymes: PGK1 and PGK2. In mouse, PGK1 exists only during the early stages of spermatogenesis, and PGK2 is then expressed during the pachytene spermatocyte stage. In this study, we investigated the localization of PGK2 in the developing porcine testis, and compared the similarities and differences in its expression with that of the PGK2 in mouse. The PGK2 protein was found to be exclusively expressed in spermatids of the adult mouse testis, whereas PGK2-positive cells were observed in the prepubertal and postpubertal testes of pigs. Based on this result, we examined the expression of PGK2 in in vitro -cultured porcine undifferentiated spermatogonia and found it to be maintained in the cultured cells. To verify this result and identify the spermatogonial stem cell-like potential in recipient testes, PKH26 dye-stained PGK2-positive cells were transplanted into the testes of busulfan-treated immunodeficient mouse that had been depleted of both testicular germ cells and somatic cells. The transplanted cells colonized the recipient testis at 8 weeks post transplantation, and fluorescence microscopy identified the cells in the basement membranes of the seminiferous tubules of the injected mouse. Taken together, our results suggest that PGK2 is expressed differently in the testes of mouse and pigs according to developmental stage. This finding should contribute to the study of spermatogenesis and the production of transgenic domestic animals through in vitro spermatogonial sperm cell culture. [ABSTRACT FROM AUTHOR]

Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis. [ABSTRACT FROM AUTHOR]

The pathogenesis of atopic dermatitis (AD) involves T helper 2 (Th2) cells, and effective therapies remain elusive due to the paucity of animal models. We aimed to develop a mouse model of an immune system aberration caused by allergen. Experiments were conducted in two phases. In experiment 1, BALB/c mice were sensitized with one of four chemical allergens – toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI), trimellitic anhydride (TMA), or 2,4-dinitrochlorobenzene (DNCB) – for 3 weeks. Based on results of experiment 1, immunological features were compared between TMA-sensitized BALB/c mice and NC/Nga mice, after exposure to mite extracts, harmful chemicals and detergents in experiment 2. Sensitization by allergen caused a large number of pathological changes in the skin, and an increase in mast cell number. TMA-sensitized BALB/c mice models showed higher sensitivity to an environmental allergen than NC/Nga mice did. Overall, the initial sensitization with TMA leads to disturbances in Th2-mediated immunity. [ABSTRACT FROM AUTHOR]

The sequence information of the genomic form of the human ABO blood group orthologous gene (erythrocyte antigen A, EAA) is not complete in pigs. Therefore, we cloned and characterized the nucleotide sequence of EAA intron 7, which is critical to understand genetic difference between A and 0 blood groups in pigs, covering complete genomic sequence information of EAA excluding a ~ 560 bp unsequencible gap. We also analyzed genetic polymorphisms within EAA intron 7 and exon 8. We found difference in A0 blood group frequencies among pig breeds. In addition, we designed a new genomic DNA-based A0 blood group typing method and improved the accuracy and simplicity of the typing. [ABSTRACT FROM AUTHOR]

Spermatogenesis begins after puberty and continues throughout a male’s life, and is regulated by spermatogonial stem cells in the seminiferous tubules. Markers of male germ cells, including undifferentiated spermatogonia to fully developed spermatozoa have been identified in rodents, but not in dogs. In this study, to characterize the markers of undifferentiated spermatogonia, histological and immunohistochemical analyses were performed on pre-pubertal (1-month-old), early pubertal (4-month-old), and post-pubertal (7-month-old) dog testes. Expression of chemokine receptor 4 (CXCR4), insulin-like growth factor binding protein 3 (IGFBP3), LIN28, and Sal-like protein 4 (SALL4) genes was confirmed by immunohistochemical analysis. In pre-pubertal and early pubertal dog testes, CXCR4, IGFBP4, and LIN28 genes were expressed in undifferentiated spermatogonia, whereas the SALL4 gene was not expressed in the pre-pubertal stage. In adult dog testes, CXCR4 and IGFBP3 gene expression was detected in undifferentiated spermatogonia and co-localized with protein gene product 9.5 (PGP9.5) near the basement membrane of the seminiferous tubules. The LIN28 and SALL4 genes were expressed in synaptonemal complex protein 3-positive spermatocytes. The CXCR4 and IGFBP3 gene expression is conserved among other species, while LIN28 and SALL4 gene expression varies. Based on results of the present study, it is suggested that undifferentiated spermatogonia markers detected in other species are conserved in dogs. These results may facilitate further studies of the cellular mechanisms of spermatogenesis in dogs. [ABSTRACT FROM AUTHOR]

Spermatogenesis, the complex process of sperm cell development including mitotic cell division and meiosis, relies on spermatogonial stem cells (SSCs). While markers for developing germ cells have been well investigated in mice, developmental stage-specific markers of germ cells in domestic animals have not been identified. Sal-like protein 4 (SALL4) is known as a putative marker for undifferentiated spermatogonia in rodents; however, its expression in domestic animals has not been investigated. The objective of this study was to characterize the expression of SALL4 in the developmental stages of boar testes and SSCs. Interestingly, all SALL4-expressing cells responded positively to PGP9.5, which is known as a spermatogonia marker in boar testes, while some PGP9.5-positive cells did not express SALL4 in pre-pubertal boar testes. At this stage, the expression of SALL4 was observed in GFRα1-positive cells, and its expression was maintained in cultured pSSCs in vitro , suggesting that SALL4 is a marker of early-stage boar spermatogonia that express GFRα1 in pre-pubertal testes. Additionally, SALL4 expression was observed in c-Kit-positive but not in PGP9.5- or SCP3-positive cells in post-pubertal testes. In conclusion, SALL4 is expressed in early undifferentiated spermatogonia in pre-pubertal boar testes and in primary spermatocytes in post-pubertal boar testes. Therefore, SALL4 can be used as a stage-specific marker of developing germ cells in boar testes. [ABSTRACT FROM AUTHOR]