Mast cells (MCs) expressing serine proteases; tryptase and chymase, are associated with fibrosis in various diseases. However, little is known about their involvement in oral submucous fibrosis (OSF). Our goal was to evaluate the role of MC tryptase and chymase in the pathogenesis of OSF and its malignant transformation. Immunohistochemical expression of MC tryptase and chymase was evaluated in 20 cases of OSF, 10 cases of oral squamous cell carcinoma (OSCC) and 10 cases of healthy controls. Subepithelial zone of Stage 1 and 2 while deep zone of Stage 3 and 4 OSF demonstrated increased tryptase positive MCs. OSCC revealed a proportionate increase in tryptase and chymase positive MCs irrespective of areas of distribution. An altered balance in the subepithelial and deep distribution of tryptase and chymase positive MCs play an important role in the pathogenesis of OSF and its malignant transformation. Oral submucous fibrosis (OSF) is a chronic progressive, areca nut chewing habit related, precancerous condition of the oral mucosa predominantly affecting Indians and South Asians. It is clinically characterized by burning sensation of the oral mucosa accompanied by pallor and progressive, irreversible fibrosis leading to difficulty in opening mouth, speech and swallowing In normal wound healing fibroblasts are transiently activated into myofibroblast, a particular type of fibroblast to proliferate and deposit the collagen TC, while mast cell secreting only tryptase are termed MCT. Both MCT and MCTC phenotypes are present in all human tissues. However, the ratio is different in each anatomical site: while MCTC predominates in the skin, heart, synovial and small intestinal submucosa, MCT predominates in the lungs and the small intestinal mucosa Human MCs also contain two types of serine protease, tryptase and chymase. Tryptase is a trypsin-like enzyme, and chymase is a chymotrypsin-like enzyme. According to their protease content, human MCs are divided into two phenotypes: Mast cell secreting both tryptase and chymase are termed MCIt is already known that neoangiogenesis is required for the growth and spread of tumor The present study was undertaken to quantify and characterize the distribution of MC subpopulation in OSF, thereby evaluating the potential role of MC tryptase and chymase in the pathogenesis of OSF & its malignant transformation.The study protocol was approved by the Institutional Review Board and the Local Ethics Committee of Nair Hospital Dental College and was in compliance with ethical standards of the Declaration of Helsinki. Written informed consents were obtained from all the study participants. Twenty (n = 20) previously untreated cases of OSF and ten (n = 10) cases of OSCC with no history of OSF, diagnosed on clinical grounds and confirmed histologically were randomly selected to form the study groups. Ten (n = 10) age and sex matched healthy volunteers without habits were included in the control group. Patients with mouth opening of more than 40 mm were considered as normal for healthy controls The staging of the disease was performed based upon the degree of mouth opening, Stage 1: mouth opening between 35 and 40 mm; Stage 2: mouth opening between 30 and 34 mm; Stage 3: mouth opening between 20 and 29 mm and Stage 4: mouth opening of less than 20 mm .Punch biopsies (5 mm) were performed on OSF patients and control subjects from identical oral site , whereas for OSCC study group the formalin fixed paraffin blocks were retrieved from the department archives and stained with hematoxylin-eosin for histological examination Four-µm-thick formalin fixed paraffin embedded tissue sections were deparafinized in xylene and rehydrated through decreasing graded ethanol solution. Endogenous peroxidase activity was suppressed by incubation for 10 min with 3% hydrogen peroxidase (Dako). The primary monoclonal antibodies used for MC were anti-MC tryptase (Dako) and anti-MC chymase (Abcam). Staining was done at room temperature on automatic staining work station (Dako Autostainer Plus) using the Dako Envision Flex Plus Visualization System (Dako). Counterstaining with hematoxylin was the final step. Normal skin sections were used as positive controls and a negative control was performed in all cases by omitting the primary antibody.Tryptase and chymase positive MCs were counted separately in serial sections of healthy controls, OSF and OSCC using Carl Zeiss, Axiolab microscope. Cytoplasm showing brown colour was considered positive immunoreactivity. MCs were counted in two areas of immunostained sections, namely subepithelial and deep. MC density was assessed in areas showing the highest concentration of tryptase and chymase positive MC (hot spots) as determine by an initial scan at 100X magnification (10X objective and 10X Ocular lens). MCs were then counted from 5 different fields at 400X magnification using an ocular grid of 12.5×12.5 mm size divided into 100 blocks.Statistical analysis was performed using the SPSS software for Windows S version 16.0 . The statistical analysis was carried out using Kruskal-Wallis test to compare mean values  and Mann Whitney U test for individual mean values. For all tests, p-values <0.05 were considered to be statistically significant.T) and those containing both tryptase and chymase (MCTC), only chymase containing MCs are extremely rare, therefore all MCs contain tryptase TC. To determine MC subpopulations  in healthy controls, OSF and OSCC, the formula: Total MCs = MCT+MCTC was used as previously described The present study consisted of 20 OSF, 10 OSCC and 10 healthy controls. There were 5 cases in each stage of OSF (Stage 1 to Stage 4). The patients with OSF were in age group of 18 to 75 years, with the mean age being 33.9 years with a marked male predominance   There arWe found a statistically significant increase in the number of subepithelial as well as deeper distribution of tryptase positive MCs in OSCC group when compared to OSF group (p<0.05). Subepithelial and deeper distribution of tryptase positive MCs in OSF group demonstrated no statistical significance when compared to healthy controls. From our results, it can be comprehended that OSCC group has significantly more number of tryptase positive MCs as compared to OSF group irrespective of the area of distribution .We observed a statistically significant increase in the number of subepithelial distribution of chymase positive MCs in OSCC group when compared to healthy controls and OSF group (p<0.05). Conversely, a statistically significant decrease in the number of subepithelial distribution of chymase positive MCs in OSF group was noted when compared to healthy controls (p<0.05). Hence, it can be concluded that OSF has the least number of chymase positive MCs subepithelially. We recorded a statistically significant decrease in the number of deep distribution of chymase positive MCs in the OSF group when compared to healthy controls and OSCC group (p<0.05) .Stage I OSF showed a statistically significant increase in the number of tryptase positive MCs subepithelially as compared to other stages of OSF, however, no significant difference was noted between stage 2, 3 and 4 OSF .Deeper distribution of tryptase positive MCs was greater in healthy controls, OSCC group and advanced stages of OSF (Stage 3 and Stage 4) as compared to early stages of OSF (Stage 1 and stage 2) .No statistically significant difference for the subepithelial distribution of chymase positive MCs was observed among different stages of OSF. A statistically significant increase in the number of subepithelial distribution of chymase positive MCs was noted in the OSCC group when compared to all the stages of OSF. Conversely, barring Stage 1 OSF, all stages of OSF showed a statistically decrease in the subepithelial distribution of chymase positive MCs when compared to healthy controls .A statistically significant decrease in the deeper distribution of chymase positive MCs was observed in all the stages of OSF when compared to healthy controls & OSCC group. OSCC group showed a statistically significant increase in chymase positive MCs in deeper areas when compared to healthy controls. However no statistically significant correlation was found for chymase positive MCs in deeper areas among different stages of OSF. OSCC group showed maximum number of chymase positive MCs in the deeper region when compared to all the other study groups .OSF is a chronic, inflammatory, premalignant fibrotic condition characterized by excessive deposition of collagen in the submucosa, leading to restricted mouth opening The enzyme profile of MCs in oral tissues resembles that of skin, with most MCs expressing the serine proteases, tryptase and chymase The fibrogenic cytokines influencing the fibrotic process are shown to play an important role in regulating fibroblast function, such as proliferation, migration, matrix synthesis and is likely to play a key role in regulating the initiation and progression of scarring in any fibrotic disease. The present study demonstrated a significantly higher subepithelial distribution of tryptase positive MCs than chymase positive MCs in the early stages of OSF against the increased distribution of tryptase positive MCs than chymase positive MCs in deeper zones of the advanced stages of OSF, which was in accordance with other findings in the literature MC tryptase is a major protease & produces mitogenic effects on various types of cultured cells such as smooth muscles and bronchial epithelial cells. Tryptase has in turn been described to activate TGF-β and collagenase, induce C3a and collagen mRNA, cleave type IV collagen, fibronectin, elastase, and proteoglycans, and induce fibroblast proliferation A statistically significant decrease in subepithelial & deep distribution of chymase positive mast cells in all the stages of OSF  in comparison to healthy controls and OSCC group made us to propose the possible role of MC chymase in the pathogenesis of OSF and its malignant transformation. Chymase can activate collagenase and stromelysin, destruct vitronectin and fibronectin, and induce fibroblast proliferation, suggesting an important role of this mast-cell-specific protease in tissue matrix turnover and renewal. Chymase has been shown to degrade the extra-cellular matrix (ECM) and basement membrane components, digest specific neuropeptides, and convert pro-IL-1B to its active molecule. Furthermore, it can cleave soluble stem cell factor (SCF) from its membrane form MCs are a major source of other pro-fibrotic cytokines like bFGF From the above results it can be inferred that there is an altered balance in the subepithelial and deep distribution of tryptase positive MCs and chymase positive MCs as it progresses from healthy controls to OSF, thereby playing an important role in the pathogenesis of OSF. From the present study it can also be deduced that fibrosis in OSF may be initiating in the subepithelial zone in early stages of OSF (Stage 1 and stage 2) and gradually progressing towards the deeper muscle layer in advanced stages of OSF (Stage 3 and Stage 4). We observed a greater number of MCs in the muscle bundles of advanced stages than early stages of OSF. Based on our findings we assume that process of fibrosis in OSF remains uninterrupted since activated resident MCs continue to secrete fibrogenic & angiogenic cytokines even after the cessation of the areca nut chewing habit, resulting in irrevocable fibrosis.The pathogenesis of OSF and circumstances leading to its proven pre-cancerous outcome have always aroused curiosity but remained enigmatic till date. Fibroblast density and activation are increased in physiological responses, such as wound healing, and in disease states, such as fibrotic disorders and carcinogenesis 3, demand for oxygen supply, a fact that makes necessary the formation of new microvasculature Tumours require a blood supply for their expansive growth. Solid tumours, in order to outgrow the size of 2 mmAs we have observed a significant increase in the tryptase and chymase positive MCs in OSCC group irrespective of the areas of distribution when compared to OSF group, it suggests that upregulation of MCs may play a crucial role in tumour progression during malignant transformation of atrophic epithelium in OSF .. A simiAs part of the process of oral mucosal carcinogenesis in OSF, the connective tissue adjacent to the epithelium gets affected by the topically applied chemical carcinogen and plays a directive role, not yet understood, in the epithelial changes observed. It appears likely that extracellular matrix molecules, growth factors, angiogenic cytokines and proteinases co-operate in influencing epithelium via an autocrine proliferative effect on the atrophic epithelium, while paracrine stimulation of the vascular network may maintain survival and growth In summary, we present data indicating that MC tryptase and chymase contribute to the development of OSF and malignant transformation of the overlying epithelium. Several therapeutic approaches such as the use of MC stabilizer, blockade of stem cell factor or inhibitors of tryptase, chymase and TGF-β have already demonstrated some clinical utility in tissue fibrosis or inflammatory diseases, by inhibiting MC activation. Considering the important role of MC tryptase and chymase in the pathogenesis of OSF, further studies using either administration of a neutralizing antibody against tryptase and chymase or application of a MC tryptase knock-out mouse strategy in experimental models of OSF are required for more definitive determination of the role of tryptase and chymase in the development of OSF.


Mechanisms underlying hypertensive target organ damage (TOD) are not completely understood. The pathophysiological role of mitochondrial oxidative stress, resulting from mitochondrial dysfunction, in development of TOD is unclear. The stroke-prone spontaneously hypertensive rat (SHRSP) is a suitable model of human hypertension and of its vascular consequences. Pathogenesis of TOD in SHRSP is multifactorial, being determined by high blood pressure levels, high salt/low potassium diet, and genetic factors. Accumulating evidence points to a key role of mitochondrial dysfunction in increased susceptibility to TOD development of SHRSP. Mitochondrial abnormalities were described in both heart and brain of SHRSP. Pharmacological compounds able to protect mitochondrial function exerted a significant protective effect on TOD development, independently of blood pressure levels. Through our research efforts, we discovered that two genes encoding mitochondrial proteins, one (Ndufc2) involved in OXPHOS complex I assembly and activity and the second one (UCP2) involved in clearance of mitochondrial ROS, are responsible, when dysregulated, for vascular damage in SHRSP. The suitability of SHRSP as a model of human disease represents a promising background for future translation of the experimental findings to human hypertension. Novel therapeutic strategies toward mitochondrial molecular targets may become a valuable tool for prevention and treatment of TOD in human hypertension. It has been clearly understood that the complexity of the etiopathogenetic basis of human hypertension extends also to the etiopathogenesis of the related TOD . An inteThe SHRSP was established from the SHR 40 years ago based on its higher susceptibility to develop spontaneous cerebrovascular accidents . It deveThe SHRSP can be considered a suitable animal model for studies of human hypertension since it shares several features in common with the human pathological condition. In particular, high BP levels, dietary salt excess, and reduced potassium intake favour cardiovascular damage development, such as cerebrovascular and renal disease, in both SHRSP and in humans .The type of stroke detected in SHRSP is predominantly ischemic (80%) and to a lesser extent hemorrhagic (20%), similarly to humans. Histopathological studies of the brain reveal most of the key signs of small vessel disease: segmental wall damage, depletion of smooth myocytes, accumulation of fibrous tissue, wall thickening, luminal narrowing, blood brain barrier leakage, and perivascular oedema. These alterations can be summarized as a fibrinoid vascular lesion caused by a degenerative-infiltrative-proliferative disease process which affects short segments of penetrating arterioles in multiple, wide scattered foci , 11.The pathophysiological evidence obtained over the years in the SHRSP supports a major role of higher BP levels in producing increased susceptibility to TOD. On the other hand, as previously mentioned, additional causative factors appear to be involved, being triggered by high salt/low potassium dietary feeding. Previous studies exploring the mechanistic aspects of increased vascular damage in high salt fed SHRSP found implications of the RAAS abnormalities , of incrIn fact, therapeutic strategies aimed at counteracting the RAAS activation with ACEI and ARB turned out to be more beneficial than beta-blocker and calcium antagonist drugs despite comparable BP lowering effect –19. InteThus, although hypertension remained a major permissive factor, the above-mentioned evidence indicated that hypertensive TOD had a complex multifactorial pathogenesis in this animal model. In particular, the evidence that SHRSP developed stroke whereas SHRSR did not upon the same high salt dietary feeding, despite similar BP levels, prompted us several years ago to search for BP independent contributing factors. In fact, the existence of hypertension independent etiopathogenetic determinants underlying stroke in this animal model was convincingly demonstrated through the genetic approach .The availability of an inbred genetically homogenous animal model represents a great challenge for the discovery of genes contributing to both hypertension and to the related vascular damage . First oAlthough no specific gene predisposing to either hypertension or its vascular consequences has been precisely identified yet in SHRSP, strong evidence has been collected over the last few years with regard to few candidate genes (mapping within the identified stroke QTLs) and their encoded proteins involved in both stroke and renal damage development.In this regard, an altered sequence and regulation of the atrial natriuretic peptide gene, mapping at the lod score peak of a QTL on rat chromosome 5, STR2, in the SHRSR/SHRSP F2-hybrid cohort , were foIn the present paper, we will discuss the available evidence related to genes encoding mitochondrial proteins, underlying both stroke and renal damage development in SHRSP, that were discovered within STR1. Klk and Mt1pa markers, extending for an overall 80 cM length and showing a 6.7 lod score peak in correspondence with the anonymous marker D1Mit3. Based on our studies, STR1 contributed to 20% of the overall stroke phenotype variance [The major finding of the linkage analysis performed in the SHRSR/SHRSP F2-hybrid cohort was the identification of STR1, a QTL included betweenIn particular, the mitochondrial dysfunction was discovered as a crucial mechanism underlying stroke development in SHRSP.ROS behave as an important intracellular and intercellular second messenger that modulate several downstream signaling molecules leading to vascular smooth muscle cell growth and migration, expression of proinflammatory mediators, and remodeling of extracellular matrix . In addi2) to form H2O. Flow of electrons through the OXPHOS chain is accompanied by pumping of protons across the mitochondrial inner membrane, thus creating a transmembrane electrochemical gradient. Complex I, known as NADH:ubiquinone oxidoreductase, is the largest of the five OXPHOS complexes and it is composed of 45 subunits . Complex I leaks electrons in the intermembrane space to generate mitochondrial anion superoxide (O2−). The reentry of protons into the matrix is used by complex V to synthetize ATP from ADP [2− production [Mitochondria represent a major source of energy for cells and their function is fundamental for life of all organisms . They mefrom ADP . Thus, coduction .2 to O2− [Under physiological condition, ROS are produced as a byproduct of the respiratory chain due to 1-electron reduction of O to O2− . Mitocho to O2− . Accumul to O2− .It is becoming more and more clear that mitochondrial dysfunction causing excessive ROS production may contribute to the pathogenesis of common cardiovascular diseases . NotablyThe mechanisms underlying the increased mitochondrial oxidative stress which has been described in the cardiovascular system of several models of hypertension are multiple . ThroughMitochondrial dysfunction may also contribute to promoting hypertensive TOD. In this regard, an assembly defect in complex I and in complex V of the OXPHOS in SHR brain mitochondria, causing a decreased ATP production and cell energy deficiency, was previously described . EvidencThe SHRSP provides key insights into the contribution of mitochondrial dysfunction in the pathogenesis of vascular damage associated with hypertension. This concept was hypothesized in SHRSP for the first time several years ago, based on evidence of a significant increase of mitochondria in cardiomyocytes of lacidipine treated SHRSP . The preOf note, treatment with S 35171, a compound able to protect mitochondrial function by increasing the inner antioxidant systems, was shown to delay and even to prevent brain damage in high salt fed SHRSP, as a result of protection from systemic processes linked to mitochondria and involved in brain damage . Notablyα agonist action and the consequent stimulation of UCP2 expression levels [Moreover, the significant protective effect described with fenofibrate on both renal and cerebral damage in salt loaded SHRSP  may be mn levels . Ndufc2 silencing in vascular cells led to a significant impairment of complex I assembly, to a reduction of mitochondrial membrane potential and of ATP synthesis, with consequent marked accumulation of ROS, increased inflammation, increased cell necrosis, and reduced cell viability [ Ndufc2-ko rat model, obtained from the parental SHRSR strain, showed a deficient complex I assembly and, more importantly, once fed with the Japanese style diet, developed renal damage followed by stroke occurrence, similarly to what occurs in SHRSP [ Ndufc2-ko model as compared to its strain of origin under the same high salt diet, thus underscoring a direct contributory role of mitochondrial dysfunction in the development of vascular damage. As a consequence of single gene effect, the percentage of stroke events did not go above 40%. The identification of a marker of the human gene, associated with a significant NDUFC2 defective expression in carriers and with early-onset ischemic stroke occurrence [Through a microarray differential expression analysis, including all sequences mapping inside STR1, we discovered the gene encoding the mitochondrial protein Ndufc2  as a sequence significantly downregulated by high salt diet only in the brain of SHRSP as compared to the SHRSR . This eviability . In vivoin SHRSP . No diffcurrence , support Ndufc2 expression is induced under Bendavia treatment, a novel molecule able to counteract mitochondrial dysfunction, in the noninfarcted border zone within the rat heart, underscoring a key role of a regular complex I activity in ischemic heart disease [Function of the Ndufc2 is starting to be understood . The obs disease . NDUFC2  disease , 73. Int disease .2− has been implicated so far in ischemic preconditioning, in aging and, only indirectly, in hypertension [Whereas the above-mentioned evidence highlights the role of NDUFC2 as a novel gene for human diseases, the results obtained in SHRSP bring up to our attention for the first time the importance of an adequate mitochondrial function, through a regular complex I assembly and activity, in order to maintain the vascular health status and to avoid TOD development in hypertension. Of note, complex I mediated production of mitochondrial Ortension . Deficiertension .+) and it dissipates the mitochondrial proton gradient that contributes to anion superoxide formation by reverse electron flow from complex II to complex I [Identification of UCP2 mapping nearby the load score peak of STR1 revealed unexpected interesting implications for the renal injury phenotype of SHRSP. UCP2, belonging to the uncoupling proteins family, is located within the inner mitochondrial membrane where it carries negative ions (Homplex I . Its dysomplex I –80. On tomplex I , 82. It omplex I . Interesomplex I .We discovered a differential regulation of UCP2 expression levels, upon the stroke-permissive high salt diet, in kidneys of SHRSP versus SHRSR with evidence of reduced expression in stroke-prone and increased expression in stroke-resistant rats . We also Brassica oleracea sprouts, was able to counteract the high salt diet induced renal damage of SHRSP, independently of BP levels [ Brassica oleracea sprouts extract was reinforced by evidence that parallel blockade with a selective inhibitor of PPARα, a family of nuclear transcriptional factor receptors that regulate several genes including UCP2 [Interestingly, the stimulation of renal UCP2 expression, by administration of a vegetable extract obtained fromP levels . The keying UCP2 , did noting UCP2 .A similar striking differential modulation, that is suppression of UCP2 gene and protein expression, was found in relation to both hypertension and aging in the brain, heart, and kidneys of SHRSP but not of SHRSR . Thus, b Brassica oleracea sprouts extract, appears a critical step to prevent TOD development in hypertension [Consistent with the critical role of UCP2 in clearance of ROS, whenever UCP2 expression is decreased levels of both oxidative stress and inflammation are remarkably increased, and levels of ATP are decreased with consequent cellular and tissue damage . Restorirtension . Altoget Brassica oleracea, were also reported in humans [Of note, the beneficial cardiovascular effects of vegetable compounds, includingn humans , thus unn humans .Overall, the results of our experimental research in the SHRSP strongly support the hypothesis that mitochondrial dysfunction may be an important contributory pathogenetic mechanism underlying hypertensive vascular damage, independently of BP levels, and that the SHRSP is a suitable animal model of mitochondrial oxidative stress-dependent hypertensive TOD. In fact, through the discovery of two mitochondrial proteins as key determinants of both cerebral and renal damage, a cause-effect relationship between mitochondrial dysfunction and hypertensive vascular damage was revealed in a clear manner in the SHRSP. Of note, apart from the two genes discussed herein, some of those listed in Notably, a gene-environment interaction appears critical to unmask the inherited mitochondrial dysfunction of SHRSP. The precise mechanisms by which salt excess interferes with mitochondrial function still need to be clarified. The role of an epigenetic regulation, as discussed for both Ndufc2 and UCP2, cannot be excluded.Apart from the positive lessons learned from this animal model, some drawbacks should also be outlined. First of all, the complex nature of human hypertensive vascular damage certainly relies on additional genetic factors that remained undiscovered in SHRSP due either to technical limitations of the methodological approaches or to limitations inherent in the model. For instance, no information could be obtained through the use of this model on the role of mitochondrial DNA variants on oxidative vascular damage in hypertension. In this regard, it is known that mitochondrial DNA plays a role in distinct pathological contexts . Its patThe consequences of a dysregulated mitochondrial function obviously apply to each living organism through the cell and tissue damage consequent to the increased oxidative stress and reduced ATP production. In fact, mitochondrial dysfunction has been already established as an important pathogenetic determinant of common neurodegenerative diseases in humans . Ndufc2 looks promising [In contrast, current knowledge on the role of mitochondrial dysfunction in human hypertensive vascular damage is still very scarce. The animal model reveals exciting new results in this regard by highlighting the role of a complex I subunit and of UCP2 into increased renal and cerebrovascular damage. We expect to see a translation of the evidence obtained in SHRSP to the human disease, since several findings reported in this model often anticipated those encountered later on in the human pathological context. For instance, we were able to translate to humans the information provided by SHRSP about the role of the atrial natriuretic peptide gene as a determinant of stroke , 36. Theromising . It suggFurthermore, evidence that UCP2 is involved in human chronic kidney disease , 93, incFuture studies will better define the true pathogenetic relevance of mitochondrial dysfunction in both animal models and human hypertensive vascular disease. It cannot be excluded that an improvement of mitochondrial dysfunction may represent a mechanism by which available antihypertensive drugs can modulate vascular oxidative stress and exert vascular protection beyond their BP lowering effect. In addition, novel targeted strategies, designed toward mitochondria dependent pathogenic mechanisms , may rev


Immunity by Fransen and colleagues aimed to mechanistically decipher the interrelationship between sIgA and microbiota diversity/composition. This commentary will discuss these important new findings, as well as how future therapies can ultimately benefit from such discovery.The intestinal microbiota is a large and diverse microbial community that inhabits the intestine, containing about 100 trillion bacteria of 500-1000 distinct species that, collectively, provide benefits to the host. The human gut microbiota composition is determined by a myriad of factors, among them genetic and environmental, including diet and medication. The microbiota contributes to nutrient absorption and maturation of the immune system. As reciprocity, the host immune system plays a central role in shaping the composition and localization of the intestinal microbiota. Secretory immunoglobulins A (sIgAs), component of the adaptive immune system, are important player in the protection of epithelium, and are known to have an important impact on the regulation of microbiota composition. A recent study published in  The gut host defense system comprises an array of mechanisms to keep the microbiota in check, maintaining an orderly beneficial relationship with the intestinal microbiota . These mA key intestinal strategy to generate immune protection in a non-inflammatory manner is the production of IgA –5, which-/- mice (which have no adaptive immune system owing to the lack of V(D)J recombination-activating protein 1 (RAG1)) revealed profound alterations in their microbiota composition [The main observation showing the importance of immunoglobulin in microbiota composition regulation was made with animals lacking such Ig production. Microbiota analysis of RAG1position . Moreoveposition , 10, 11.position . Mountinposition , 14.Fransen and collaborators recently demonstrated that the abundance and repertoire diversity of innate IgAs  play a central role in regulating the diversity of the intestinal microbiota , 15. TheHowever, it is important to note that low IgA diversity is not the exclusive discrepancy found between these 2 mice strains. Among other differences, C57BL/6 mice have a normal Th1 response while BALB/c mice are deficient in Th1 signaling, which could also have an important impact on the phenotypes described in this study. One important point that still needs to be addressed is whether the decreased microbiota diversity observed in C57BL/6 mice, as a result of low IgA diversity, is associated with an increased pro-inflammatory potential/harmfulness. The investigations of whether the altered microbiota associated with low intestinal IgA predispose mice of subsequent challenges still need to be conducted.via a mechanism involving the ability of bacteria from IgA-low mice to degrade sIgA [It is important to note that, in the study by Fransen and colleagues, co-housing or fecal transplantation had little effect on both IgA production and microbiota composition, highlighting the stability of the intestinal microbiota in an individual, with a central role played by IgA repertoire . The defade sIgA . AltogetCell Host and Microbes in 2013, it was demonstrated that TLR5-/- mice harbor a reduced level of flagellin specific IgA [-/- animals was found to express significantly higher amounts of bioactive flagellin, supporting an impact of intestinal IgA in suppressing levels of flagellin, likely by putting flagellated bacteria at a competitive disadvantage within a complex microbial community.A common feature of colitis-associated microbiota are increased levels of bioactive flagellin and lipopolysaccharide (LPS), which can activate Toll-like receptor 5 (TLR5), NOD-like Receptor 4 (NLRC4) inflammasome, and TLR4 –20. Apprific IgA . ImportaIn addition, recent findings made by flow-cytometric sorting suggest that IgA may mark commensal and pathobionts according to the extent of their individual coating . This stBased on this appealing work by Rescigno and colleagues, we can hypothesis that selected manipulation of the immune system has the potential to alter gut microbiota composition to make it inherently less pro-inflammatory (i.e. more diverse and with a reduced level of innate immune activators), reducing susceptibility to and/or severity of intestinal inflammation development. IgA may be used as a target to shape the intestinal bacterial community in order to maintain a beneficial relationship between the host and the microbiota.


The abnormal elastic behaviors of selected lattice planes of ZnO during phase transition revealed the existence of internal elastic strain, which arise from the lattice misfit between wurtzite and rocksalt phase. Furthermore, the strength decrease of ZnO during phase transition under non-hydrostatic pressure was observed and could be attributed to such internal elastic strain, unveiling the relationship between pressure induced internal strain and mechanical property of material. These findings are of fundamental importance to understanding the mechanism of phase transition and the properties of materials under pressure.Under high pressure, the phase transition mechanism and mechanical property of material are supposed to be largely associated with the transformation induced elastic strain. However, the experimental evidences for such strain are scanty. The elastic and plastic properties of ZnO, a leading material for applications in chemical sensor, catalyst, and optical thin coatings, were determined using  Pressure induced phase transition has been considered as an important and extensively used approach in designing new materials123468101213During a pressure induced first order solid-solid transformation, usually both phases can co-exist over a certain pressure range (transition zone). The crystal structure and lattice parameters of the new phase are different from the original phase, thus, theoretically speaking internal lattice strain should be generated during the nucleation and growth of new phase to accommodate the misfit between nucleus and matrix1417181920in situ high pressure synchrotron x-ray diffraction, we checked the compressibility of ZnO which experiences a wurtzite-rocksalt phase transition under pressure2223In the present work, using d100/d002 for wurtzite phase and the ratio d200/d220 for rocksalt phase. Before phase transition the d100/d002 increases linearly with pressure, and deviates from the linear relationship in the transition zone  direction in wurtzite phase transforms to a square in rocksalt phase (d-spacing of (100) plane of wurtzite phase needs to increase to match the (110) plane of rocksalt phase, as d110 of rocksalt phase is larger than d100 of wurtzite phase. Therefore, d100 of wurtzite phase would be stretched by the larger lattice dimension of (110) plane of rocksalt phase, while accordingly d110 (or d220) of rocksalt phase would be compressed to match the smaller lattice dimension of (100) plane of wurtzite phase. Owing to the symmetry of rocksalt phase, d200 equals to d220 and thus shows more obvious unusual compression behavior than d220 lt phase 26. In thhan d220 , therefoIn the initial stage of transformation, the amount of rocksalt phase is small. The lattice parameter of wurtzite phase does not deviate significantly  due to thkl)>, which refer the arithmetic average of measured differential strains of all lattice planes, increases with pressure. The differential strain of rocksalt ZnO also increases with pressure when the phase transition finished. But during the phase transition, <Q (hkl)> of each phase ZnO decreases steeply with pressure, indicating that plastic flow is achievedTo check the influence of internal strain on the plastic property of ZnO, we derive differential elastic lattice strains for both phases of ZnO from line shifts in radial X-ray diffraction (RXRD) data collected at pressureAs indicated in 19in situ synchrotron XRD measurements under quasi-hydrostatic conditions reveal unusual compression behaviors in ZnO during the wurtzite-rocksalt phase transition, indicating the internal elastic strain on selected lattice planes. Combined with the established phase transition model of ZnO, the observed elastic strain is believed to be caused by the lattice misfit between wurtzite phase and rocksalt phase during phase transition. Additionally, the internal elastic strain is supposed to give rise to strength softening of each phase ZnO during phase transition, which is observed in the present RXRD measurements at non-hydrostatic conditions. These results supply a strong evidence for the existence of phase transition induced elastic lattice strain under pressure and indicate that such strain could have significant affect on the physical property of material.In summary, In situ high pressure AXRD experiments were performed at room temperature, and designed to generate quasi-hydrostatic conditions by using neon as a pressure transmitting medium. Polycrystalline ZnO powder  with a wavelength of 0.6199 Å. The RXRD measurements were carried out at 16-BMD station of the High-Pressure Collaborative Access Team (HPCAT), Advanced Photon Source, Argonne National Laboratory, with a wavelength of 0.3101 Å. Diffraction patterns at various pressures were recorded with a Mar345 imaging plate detector. Collected data were analyzed for lattice parameters and differential elastic lattice strains using the programs FIT2DHow to cite this article: Yan, X. et al. Phase transition induced strain in ZnO under high pressure. Sci. Rep.6, 24958; doi: 10.1038/srep24958 (2016).


The latter involves the expression and activity of a series of ion and water transport proteins. These molecules are grouped together around glial fibrillary acidic protein (GFAP) and water channel protein aquaporin 4 (AQP4) to form functional networks, regulate hydromineral balance across cell membranes and maintain the integrity of the BBB. Intense ischemic challenges can disrupt these capacities of astrocytes and result in their maladaptation. The maladapted astrocytic plasticity in ischemic stroke cannot only disrupt the hydromineral homeostasis across astrocyte membrane and the BBB, but also leads to disorders of the whole neurovascular unit. This review focuses on how the maladapted astrocytic plasticity in ischemic stroke plays the central role in the brain edema formation.Brain edema formation and the ensuing brain damages are the major cause of high mortality and long term disability following the occurrence of ischemic stroke. In this process, oxygen and glucose deprivation and the resulting reperfusion injury play primary roles. In response to the ischemic insult, the neurovascular unit experiences both intracellular and extracellular edemas, associated with maladapted astrocytic plasticity. The astrocytic plasticity includes both morphological and functional plasticity. The former involves a reactive gliosis and the subsequent glial retraction. It relates to the capacity of astrocytes to buffer changes in extracellular chemical levels, particularly K Ischemic stroke is a major cause of death and disability, largely because of brain edema formation  formation and wrap around neurons to modulate neuronal activity  in the infarct zone in parallel with gliosis at the penumbra area or following the initially adaptive gliosis  Dreier,  as well  Dreier, . As a re+, K+, 2Cl− and water cotransporter (NKCC)1  4.1 and sodium pump. Astrocyte NKCC1 is activated by exposure to high levels of extracellular K+ and to the regulatory volume increase in cells shrunken in response to hypertonic challenge (Larsen et al., + levels, known to increase GFAP expression (Wang and Hatton, 2+ influx and intracellular cAMP levels during stroke. The Ca2+ influx induces an increase in intracellular Ca2+ levels and the activation of NKCC1 and Kir4.1 (Song et al., Cytotoxic astrocyte swelling largely depends on osmotic gradients built by changes in ion transport activity, which involves NKCC1, inward rectifier K+ (Strohschein et al., Among many GFAP-associated molecules, AQP4 is distinguished not only by its water permeability but also by its role of a “cotransporter” of osmolytes. AQP4 is colocalized or assembled with the transient receptor potential vanilloid channel 4 (Jo et al., Following the occurrence of stroke, AQP4 expression presents two peaks despite the time variations among individual observations. The first peak occurs at the initial stage of stroke; the second peak appears days after reperfusion and during recovery. As shown in mice (Ribeiro Mde et al., In mice with 1 h transient focal brain ischemia, AQP4 expression is significantly increased at astrocyte endfeet in the lesion core initially and it increases in whole astrocytes at the border of lesion at 48 h (Ribeiro Mde et al., Further studies reveal that the spatial distribution rather than the expression level of AQP4 is closely associated with its functions. The loss of perivascular AQP4 is associated with the disappearance of the perivascular astrocyte membrane (Frydenlund et al., As stated above, astrocytes are not only the major cell type exhibiting cytotoxic edema during ischemia and reperfusion injury, but also a main factor that disrupts BBB integrity. Further analysis reveals that the astrocytic plasticity plays a central role in brain edema formation as detailed below.+ (Strohschein et al., 4+ toxicity and oxidative stress by synthesizing glutamine, antagonizing glutamate toxicity by releasing inhibitory adenosine, taurine and β-alanine (Morán et al., Astrocytes can extensively modulate the activities of other components of the neurovascular units (Theodosis et al., + and glutamate levels through siphoning K+ and glutamate uptake, to re-establish neuronal membrane potential and minimize excitotoxic impact at the cost of least energy. However, uncontrolled gliosis can be harmful because it produces high levels of bioactive compounds that are noxious for neuronal cell functions, such as the bioactive free radical nitric oxide (Ghasemi and Fatemi, In ischemia, the extent of spatiotemporally differentiated reactive gliosis and the associated RVD is highly correlated with the severity of brain swelling, excitotoxicity, oxidative stress, metabolic disorders, and disruption of the BBB. On the one hand, reactive gliosis, indicated by increasing GFAP and AQP4 expression, is a sign of astrocyte swelling at the early stage of mild stroke and in the penumbra of severe stroke. This gliosis is likely an evolutionary wise selection. It allows the brain, in a situation of a substantial energy deficit in ischemic stroke, to rebalance extracellular KOn the other hand, breakdown of this gliosis can lead to degeneration of neural tissues in the lesion core. The reduced volume during the RVD can avoid overload of osmolytes and water in astrocytes, and thus preserve structural integrity of astrocytes. However, the reduced absorption of ion, glutamate and water, decreased volume transmission into the blood in infarct areas as well as the destruction of BBB integrity can seriously disrupt the homeostasis of the whole neurovascular unit, leading to its apoptosis, autophagic cell death and necrosis (Puyal et al., Following the onset of stroke, astrocytes keep changing their structure and function as indicated by the expression of GFAP and AQP4, which determine a conversion of the intracellular edema into extracellular edema in the lesion core while forming a shell of swollen astrocytes at the penumbra. Moreover, overly increased astrocyte volume triggers the RVD wherein the reduced AQP4 could disrupt the adaptive plastic change and dispersion of intracellular volume through the junctional coupling among astrocytes (Nicchia et al., Together with the view presented previously (Verkhratsky et al., All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The dealkylating effect of MGMT is relevant not only in repairing DNA mismatches produced by environmental alkylating agents promoting tumor pathogenesis, but also when alkylating molecules are applied in the chemotherapy of different cancers, including glioma, the most common primary tumor of the central nervous system. Elevated MGMT gene expression is known to confer resistance to the treatment with the alkylating drug temozolomide in patients affected by gliomas and, on the contrary, methylation of MGMT gene promoter, which causes reduction of MGMT protein expression, is known to predict a favourable response to temozolomide. Thus, detecting expression levels of MGMT gene is crucial to indicate the option of alkylating agents or to select patients directly for a second line targeted therapy. Further study is required to gain insights into MGMT expression regulation, that has attracted growing interest recently in MGMT promoter methylation, histone acetylation and microRNAs expression. The review will focus on the epigenetic regulation of MGMT gene, with translational applications to the identification of biomarkers predicting response to therapy and prognosis.O-6-methylguanine-DNA methyltransferase (MGMT) is an abundantly expressed nuclear protein dealkylating O O6-methylguanine-DNA methyltransferase (MGMT) is a ubiquitously expressed nuclear enzyme which removes alkyl groups from the O6-position of O6-MG  has been cloned . So far In silico analysis on the putative consensus sequences for the binding of nuclear transcription factors in MGMT promoter sequence reveals further nuclear factors potentially involved in activating MGMT transcription, for instance AP-2, NF-IL6, and ER-α, besides Sp1, AP-1 and c/EBP, as shown in Further analyses correlated the high expression of MGMT with the presence of a hypovascular central core of the tumor, where activation of the hypoxia inducible factor (HIF)-1α will in turn promote expression of MGMT, particularly in the hypoxic glioma stem cells niches ,15. The Epigenetic modifications of MGMT gene have been found to play a relevant role in MGMT expression in the context of cancer. Histone acetylation and methylation has been extensively investigated in relation to MGMT expression, in different cancer models including gliomas –24. Acetin vitro the effect of the histone deacetylase (HDAC) inhibitor suber-oylalanide hydroxamic acid (SAHA), which increased MGMT expression, thus strengthening resistance to the alkylating agent temozolomide  protein 2 (MeCP2) to methylated CpG islands have been found relevant in silencing MGMT expression . MGMT mewww.mirbase.org) belong to a family of small (19 to 25 nucleotides in length) noncoding RNAs that target specific sequences of mRNAs thereby regulating gene expression   dataset, which includes 424 glioma samples . Comparaex vivo and in experimental glioblastoma cell models in vitro supported the bioinformatics analyses and indicated that downregulation of MGMT expression by miR-181d and miR-767-3p is due to degradation of the MGMT mRNA whereas miR-648 affects MGMT protein translation  which also depletes MGMT by activating its ‘suicidal’ dealkylation mechanism  and peptide nucleic acids (PNAs), are novel tools to upmodulate or downmodulate miRs expression –176, by MGMT expression in patients affected by glioblastoma is a double-edged sword as low levels favour cancer pathogenesis by affecting repair of DNA from environmental alkylating agents whereas high levels are responsible for the resistance to the most effective drug presently utilized, the alkylating molecule temozolomide.Some of the molecular pathways of regulation of expression of MGMT gene have been now cleared, as summarized in Artificial manipulation to silence MGMT expression in patients with unmethylated MGMT promoter to be treated with temozolomide should be explored with both innovative chemical inhibitors of MGMT function and novels molecular biology tools aimed to reduce MGMT expression by targeting MGMT mRNA stability or MGMT mRNA translation.


It has become a current social trend for women to delay childbearing. However, the quality of oocytes from older females is compromised and the pregnancy rate of older women is lower. With the increased rate of delayed childbearing, it is becoming more and more crucial to understand the mechanisms underlying the compromised quality of oocytes from older women, including mitochondrial dysfunctions, aneuploidy and epigenetic changes. Establishing proper epigenetic modifications during oogenesis and early embryo development is an important aspect in reproduction. The reprogramming process may be influenced by external and internal factors that result in improper epigenetic changes in germ cells. Furthermore, germ cell epigenetic changes might be inherited by the next generations. In this review, we briefly summarise the effects of ageing on oocyte quality. We focus on discussing the relationship between ageing and epigenetic modifications, highlighting the epigenetic changes in oocytes from advanced-age females and in post-ovulatory aged oocytes as well as the possible underlying mechanisms. The average age of women bearing children has increased by ∼5 years in the past several decades . HoweverThe risk of childlessness, stillbirth and multiple births for daughters born by aged mothers (≥40 years) is increased when compared with daughters born to young mothers (24–30 years of age) . The BarAltered epigenetic modification may be an important factor for the complications seen in children of older mothers b. In thDamage to the reproductive and other systems increases with ageing in humans and in many other complex organisms, and there is a decrease in the adaptability and energy utilisation of the system. The accumulation of damage is caused by external and internal factors. Genetic factors only explain 20–30% of the variation in the human lifespan for twins and long-lived families, although these factors are crucial for survival to very old ages .Caenorhabditis elegans, Drosophila and mouse model systems and the tissue culture-based replicative senescence model of cell ageing  and Mrgx (Morf412); Previous discussions indicate that the epigenetic modifications of oocytes may be affected by advanced maternal age because the expression of DNMTs and histone acetyltransferases  of Snrpn, Kcnq1ot1, U2af1-rs1 (Zrsr1), Peg1, Igf2r and H19 are not altered. By Restriction Landmark Genome Scanning, the investigators also did not find significant differences in genome-wide DNA methylation in embryos and placentas from aged female mice  activity in mammalian oocytes as revealed by immunostaining . AkiyamaCbx1 and Sirt1) is changed in ageing GV oocytes. Histone 3 lysine 4 methylation in mouse GV oocytes is also changed by ageing  is downregulated in the oocytes, but three genes for the ubiquitin-specific peptidases Dicer, which is crucial for the generation of endo-siRNA and miRNA, in mouse oocytes results in severe spindle and chromosomal segregation defects  injection, respectively, and loss of methylation was observed at 29 h of hCG injection , HDACs, ZFP57, Dicer, and KAP1/TRIM28. If their expression is affected in oocytes, the epigenetic modifications may be altered. DNMT1 and DNMT1o are crucial for maintaining proper methylation and DNMT3a, b and l are key elopment . In MII Hdac2 is downregulated and the expression of histone acetyltransferases (Myst1 and Mrgx) decreases in aged mouse oocytes  as a donor of methyl groups. SAM is biosynthesised using methionine and ATP  supplemented to medium can also decrease ROS levels in post-ovulatory aged oocytes, but only resveratrol increased the fertilisation rate (The above discussions suggest that advanced maternal age and post-ovulatory oocyte ageing are deleterious to oocyte quality, including oocyte maturation, chromosome segregation, epigenetic modifications and mitochondrial function, and the health of the offspring from advanced-age mothers may also be affected by compromised oocyte quality (


Copper is an essential micronutrient and its misbalance in the body is associated with severe neurodegenerative disorders. While the importance of copper in the body is evident, mechanisms by which copper misbalance induces pathologic changes and disease symptoms are poorly understood. In this study, using fibroblasts from Menkes disease patient, as a cellular model of copper accumulation, we examined whether excess copper triggers specific and distinct changes in the redox environment of different cellular compartments.-/-) and his heterozygous mother (ATP7A+/-) were used as an experimental system. Glutathione mediated redox environment and levels of H2O2 were investigated in nuclei, cytosol, and mitochondria of live cells by tagging the respective ratiometric sensors (GRX-roGFP and HyPer) with a compartment-specific localization signal.Skin fibroblasts from Menkes disease patient (YS cells, ATP7AUnder basal conditions, the YS and XS cells show similar glutathione mediated redox environment in the nucleus and cytosol. However, the mitochondria are oxidizing in YS cells. YS cells were observed to accumulate higher level of peroxide in the cytosol and mitochondria. We also found that copper accumulation in cytosol and nuclei of YS cells sensitize cells to glutathione depletion suggesting importance of glutathione in protection of cells against copper overload2O2 accumulation in cytosol in YS cells does not change glutathione balance, whereas mitochondria appear most affected, since both H2O2 levels and glutathione balance are altered. We propose that mitochondria could be a primary site of copper toxicity in Menkes fibroblasts. Molecular mechanisms underlying differential redox responses are presently being investigated.Our experiments revealed differential response of cellular compartments to excess copper in cells. Nuclei, in spite of being a site of copper accumulation, do not show marked redox changes, suggesting presence of robust protective mechanisms operating in this compartment. H


After more than 10 years, where have these researches reached? Which are the major successes achieved and the problems still remaining to be solved? Although during the last years, many reviews have been published about these topics, in the present work, we will focus on an aspect that has been little considered so far, namely a strict comparison between the in vitro and in vivo developmental capabilities of the primordial germ cells (PGCs) isolated from the embryo and the PGC-like cells (PGC-LCs) produced in vitro from different types of stem cells in the mouse, the species in which most investigation has been carried out. Actually, the formation and differentiation of PGCs are crucial for both male and female gametogenesis, and the faithful production of PGCs in vitro represents the basis for obtaining functional germ cells.Stem cells are unique cell types capable to proliferate, some of them indefinitely, while maintaining the ability to differentiate into a few or any cell lineages. In 2003, a group headed by Hans R. Schöler reported that oocyte-like cells could be produced from mouse embryonic stem (ES) cells  Actually, in mammals, germ cells derive from the mortal soma and non-continuous germline do exist. Many researchers devoting to the germ cell biology were, however, surprised by some of these results. In fact, artificial germ cells produced in the Hübner's paper and in others that rapidly followed uncovered in vitro developmental capabilities that were not shown by true germ cells. Oocytes and sperm seemed to magically appear in the culture dish, although scientists of reproductive biology know that gametogenesis is a very complex process of which only some stages can be reconstructed in vitro. At the same time, however, they were fascinated by these results for two main reasons. First, once validated, in vitro model of gametogenesis from stem cells could make it easier to study and elucidate the mechanisms underlying gametogenesis, mainly in the humans, in which experimental approaches are limited. Second, artificial germ cells could greatly improve and make the actual procedures of assisted reproductive technology more efficient and develop alternative infertility treatments. A scenario for radical changes in the reproduction performance of many species, first of all humans, could also be imagined with consequences difficult to foresee. Actually, from Hübner's work, many papers described the production of germ cells from various types of stem cells, even includes humans.8 Particularly important, some authors reported the generation of live offspring from male and female germ cell-like cells obtained in vitro from mouse ES and induced pluripotent stem (iPS) cells.10 As both male and female germline begins from primordial germ cell (PGCs), precise information about the characteristics and developmental capability of the embryo-derived PGCs and their counterpart, PGC-like cells (PGC-LCs) produced in vitro from stem cells, is essential to elucidate the conditions for obtaining functional germ cells. The present review is focused on this topic.From the first work by Hübner in vitro, it is essential to establish the type and the developmental stage of the putative germ cell generated. As most of the studies reviewed here have been performed in the mouse, we summarized a brief outline of murine gametogenesis and key genes involved in germ cell formation and their roles during gametogenesis, as shown in In the attempt to produce germ cells or recreate gametogenesis phases 11 As many mitoses do not end with cytokinesis, germ cells give rise to small cell clusters termed cysts or nests. Subsequently, they either cease mitosis and enter meiosis becoming primary oocytes  in the female or progressively arrest in G0/G1 as prospermatogonia/gonocytes in the male.In the mouse embryo, PGC precursors arise around 6.25 dpc from the proximal epiblast. Subsequently, the precursors move into the extraembryonic allantois mesoderm where they are specified as PGCs. From 8.0 dpc, PGCs migrate into the embryo proper, moving through the hindgut and the dorsal mesentery and around 10.0 dpc begin to colonize the gonadal ridges. Germ cell proliferation occurs throughout the migratory period, and continues for 2–3 days after gonad entering, forming a population of about 25 000 cells at 13.5 dpc when proliferation terminates.13 Subsequently, the surviving oocytes are individually encircled by pregranulosa cells and finally enclosed into a primordial follicle. The primordial follicle pool represents the ovarian reserve from which cohorts of follicles are continuously activated from birth to menopause to undergo extensive growth and development as primary and secondary follicles in a process termed folliculogenesis.In the female, around birth, oocytes arrest at the diplotene stage of the meiotic prophase I, and undergo a loss of more than one-third of their number by apoptosis and/or autophagy.14 The first round of spermatogenesis is completed by puberty and continues up to 1–2 years of age.15 The seminiferous cords of the testis of an adult mouse contain cell types in three principal phases of spermatogenesis lasting as whole for 33–35 days:17 spermatogonial renewal and proliferation, meiosis, and spermiogenesis.In the male, transformation of gonocytes into SSCs occurs between 0 and 6 dpp, with the first appearance of biologically active SSCs occurring at approximately 3–4 dpp.in vitro in any mammalian species. However, some stages of this process occurring in the embryo or foetus have been reproduced and significant progresses in obtaining mature gametes from postnatal gonads in vitro were achieved. At present, the more promising approaches for producing functional gametes from PGCs are based on in vivo transplantation of PGC-containing tissues collected from embryos or after in vitro reaggregation of PGCs with somatic gonadal cells, into the gonads of prepuberal/adult hosts.The process of female or male gametogenesis from the formation of PGCs to functional oocytes or sperm has not been entirely recreated et al.18 devised a robust in vitro culture protocol to induce the differentiation of epiblast cells into PGC-LCs. They added BMPs and WNT3 to the culture dish of isolated floating epiblasts and monitored PGC formation using transgenic fluorescent reporter genes whose expression is controlled by the upstream regulatory elements of the genes encoding Prdm1  or Prdm14, which are expressed in PGC precursors.21 They showed that epiblast cells, in which these reporters were induced, expressed germ cell-specific genes in an appropriate sequence and displayed chromatin modifications characteristic resembling in vivo germ cells. Most importantly, they also demonstrated that male PGC-LCs, like endogenous PGCs, were able to differentiate into spermatozoa when transplanted into testicular tubules of prepuberal mice and even to fertilize oocytes to produce viable mice.In 2005, Ohinata 23 Although the cell monolayers were considered necessary to support PGC survival and proliferation over this time, Farini and her collaborators24 showed that they were not necessary for their entering into meiosis in vitro. Actually, these authors showed that both female and male PGCs with minimal gonadal cell contamination were able to enter and progress into meiotic prophase I up to pachytene/diplotene when providing a cocktail of growth factors. On the other hand, it has been previously shown that mouse PGCs maintained onto embryonic fibroblast monolayers for 7–10 days in the continuous presence of a combination of growth factors, deviated from their normal differentiation pathways and gave rise to colonies of ES-like cells termed EG cells.27 Such behaviour was much more frequent in pregonadal (8.5–10.5 dpc=about 18/100) than in gonadal (11.5–12.5 dpc=about 3/100) PGCs and resembled the transformation of PGCs in embryonic carcinoma cells occurring in the foetal testis of certain mouse strains.28 Similarly, EG cells were derived from human PGCs.29 More recently, it has been shown that mouse PGCs can also give rise to EG cells in the absence of cell monolayers when they were cultured onto fibronectin and in the presence of GSK3 and ERK kinase inhibitors (2i).31Usually mouse PGCs obtained from 11.5 to 12.5-dpc gonadal ridges can be maintained in culture only for 3–4 days before undergoing degeneration through apoptosis  and 2.22in vivo situation.32 In partial disagreement with such results, Farini et al. reported that although isolated 10.5 dpc PGCs were able to engage meiosis and express meiotic genes such as Sycp3, Dmc1 and Rec8, these PGCs appeared unable to progress beyond the leptotene stage after 5 days of culture. PGCs of the same age, however, were able to enter and progress into the meiotic prophase I if cultured within an intact AGM.As far as we know, only one paper has shown that pregonadal male or female PGCs mixed with endogenous somatic cells of the 10.5 dpc AGM region when cultured onto KL-producing cell monolayer were able to enter and progress into meiotic prophase I following a timing comparable with the in vitro from female PGCs to continue their development and be enclosed into follicles was not studied. Indirect data indicate, however, that they could be able to begin the growing phase in vitro also without the companion pregranulosa cells but not to form follicles with ovarian somatic cells. In fact, Klinger and De Felici33 showed that primary oocytes isolated from the foetal ovaries entered the growth phase upon stimulation with KL, while Lei et al.34 observed that 12.5-dpc PGCs after 10–11 days of culture developed into primary oocytes, but were unable to form follicles when aggregated with gonadal somatic cells.The capability of the primary oocytes produced in vitro are able to enter and progress into meiotic prophase I when the right cocktail of growth factors is provided. In any case, the microenvironment of the developing gonadal ridges seems necessary for gonadal PGCs acquiring meiotic competence and capability to enter and progress into the meiotic prophase I.Overall, these experiments indicate that both isolated female and male gonadal PGCs in vitro within their gonadal tissues was investigated following basically three experimental approaches: organotypic culture, culture of pieces of ovarian tissues or PGC-somatic cell aggregates  The formation of EBs and the use of growth factors known to induce PGC differentiation from the epiblast such as BMP4 and WNT3 ; (iii) overexpression of key genes that regulate germ cell formation, such as Dazl and Mvh genes;74 and (iii) conditioned media containing unidentified PGC-LC0inducing factors ; after induction with a cocktail of other growth factors, EpiLCs robustly (about 30%) differentiated into PGC-LCs resembling their in vivo counterparts. Using a similar method, PGC-LCs have been recently obtained from human ES cells.8 Interestingly, the Saitou's group also reported that without growth factors, simultaneous overexpression of three transcription factors, Blimp1, Prdm14 and Tfap2c , directed mouse EpiLCs swiftly and efficiently into a PGC state.77 An apparent improvement of the original Hayashi's method has been recently reported.78 As we will discuss below, Hayashi et al. notably reported that PGC-LCs were capable to differentiate into fertile sperm and oocytes after in vivo transplantation.Between 2011 and 2012, the papers by Hayashi in vitro. In fact, although several authors reported the expression of meiotic genes and proteins by these cells such as SCP3, γH2AX or DMC1, in a few of them, in which such capability was investigated by observing the typical morphological pattern of the meiotic chromosomes, severe abnormalities were reported.81As far as we know, no papers have convincingly shown that PGC-LCs are able to undergo normal meiotic prophase 85 Particularly surprising is the formation of aggregates resembling cumulus-oocyte complexes from either female or male foetal porcine or newborn mouse SDSCs.90 In all cases, however, the OLCs showed morphological and molecular abnormalities and were not proved to be functional. Our interpretation is that under certain in vitro conditions, some types of stem cells can acquire morphological and molecular features of oocytes independently from meiosis and proper follicle assembly.91 A recent paper supports such a possibility. In fact, Dokscin et al.92 showed that mouse oocyte growth and differentiation are dissociable from the chromosomal events of meiosis; they observed that a few surviving Stra8-deficient female germ cells grown and differentiated into OLCs that synthesized zona pellucida, assembled into follicles and were even ovulated and fertilized without meiotic initiation, and noteworthy, these oocytes arrested at the two-cell stage after in vitro fertilization.Despite such results, in the early Hübner's study and in a number of subsequent papers, the formation of follicle-like structures or OLCs from stem cells in the culture dish was observed.in vitro resembling round spermatids or even with a sperm-like morphology from mouse94 and human97 ES, iPS cells and adult stem cells. The efficiency of such processes was, however, very low and when tested, the haploid cells proved to be only partly functional. In fact, in the mouse, two papers showed that haploid cells generated in vitro were able to fertilize oocytes. Geijsen et al.71 detected and isolated haploid cells from mouse EBs treated with RA, and showed that the injection of the cells into oocytes resulted in the formation of blastocyst-like structures at a low rate. Nayernia et al.93 reported the induction of haploid cells with a sperm-like morphology from ES cell monolayers and successful production of offspring by intracytoplasmic injection which had growth abnormalities and died within 5 months after birth.Several papers reported the formation of haploid male germ cells in vitro cultured embryonic ovarian tissues. Under certain culture conditions, such oocytes are able to form follicles and in rare cases to complete the growing phase and be fertilized.42 In all cases, however, the OLCs inside these structures showed morphological and molecular abnormality and were not proved to be functional.As reported above, various experimental approaches indicate that it is relatively simple to induce the development of embryo-derived female PGCs into primary growing oocytes within in vitro within embryo-derived ovarian tissue has been reported by Hayashi and Surani.98 Stella-GFP-positive PGC-LCs obtained from EpiSCs were mixed with gonadal cells from 12.5-dpc ovaries, and after centrifugation, the cell suspension was left to aggregate and seeded through the gaps present between small pieces of 12.5-dpc ovaries on transwell inserts. After 40 days in culture, OLCs with strong expression of Stella-GFP indistinguishable from the endogenous germ cell present in the same co-cultures were observed although with very low frequency around 1 oocyte/5000 PGC-LCs.As far as we know, the only attempt to culture PGC-LCs in vitro cultured within testis tissues apart for their ability to differentiate into prospermatogonia or gonocytes did not account for their capability to undergo spermatogenesis. Likely for this inability and the difficulties in reconstructing spermatogenesis in vitro were discussed in the previous paragraph; no attempts have been performed to maintain male PGC-LCs in vitro for a long time within testis tissues or onto Sertoli cell monolayers. In the works in which male PGC-LCs were identified and maintained in EBs up to the haploid status, we can postulate that such formation might develop into a testis-like structure in which PGC-LCs can differentiate at a certain extent.The available information on male embryo-derived PGCs in vitro spermatogenesis.The new methods allowing spermatogenesis in cultured postnatal or prepuberal gonocytes reported above represent a system to attempt to differentiate XY PGC-LCs to postnatal gonocytes/spermatogonia and even to accomplish complete Because as reported above, a number of papers showed that embryo-derived female PGCs, at least from 13.5 dpc onward, when aggregated with ovarian somatic cells and transplanted under the kidney capsule or ovarian bursa of syngeneic mice were able to give rise to fertilizable oocytes, some authors used this approach to investigate whether PGC-LCs possess such capability.et al.80 co-aggregated GFP-PGC-LCs obtained from mouse ES cells with dissociated newborn mouse ovarian tissues. After transplantation under the kidney capsule of recipient mice, the authors found some GFP oocytes within primary follicles in two of the grafts. Similar results were obtained by Dyce et al. and Chuang et al.99 Hayashi et al. published the most efficient method to obtain oocytes after aggregation of GFP-PGC-LCs obtained from mouse EpiLCs (see above) with 12.5-dpc ovarian cells and in vivo transplantation under the ovarian bursa of host mice. These authors reported an efficiency of about 0.7% fully grown oocyte generated after 4 weeks in a synchronous single wave of oogenesis from both female PGC-LCs and embryo-derived PGCs.Nicholas et al.9 also reported that male PGC-LCs from EpiLCs, like embryo-derived PGCs,63 were capable to differentiate into spermatozoa when transplanted into seminiferous tubules of prepuberal mice. Such spermatozoa gave rise to healthy individuals through in vitro microinsemination. Zhu et al.100 reported that male germ cells produced from mouse iPS cell EBs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids after transplantation into recipient testes. Toyooka et al.61 should be credited for the first successful generation of spermatozoa from male PGC-LCs. The authors using BMP4 to induce germ cells from EBs, reported that male PGC-LCs were able to differentiate into spermatozoa in aggregates with gonadal cells from 12.5-dpc testis when transplanted under a host testis capsule. However, the capability of these cells to fertilize was not investigated.Hayashi in vivo counterparts. Although the reasons of such differences are not clear, these results indicate that PGC-LCs are not identical to embryo-derived PGCs and suggest that the gonadal ridges produce factor(s) essential for PGC survival/proliferation and differentiation. Therefore, it is not surprising that both artificial and real PGCs require to be enclosed inside or re-aggregated with suitable somatic cells to progress through gametogenesis. However, culture conditions suitable to complete oogenesis and spermatogenesis in vitro have not been established yet.The artificial germ cells produced in the mouse proved to be functional because they were capable to differentiate into spermatozoa and oocytes that give rise to healthy individuals. In this regard, the behaviour of the mouse PGC-LCs was quite similar to that of embryo-derived PGCs. However, these still showed some notable differences from their in vitro are the proper entry and progression into the meiosis and the epigenetic changes accompanying both female and male gametogenesis. Determining whether epigenesis and meiosis occur correctly during the process of deriving germ cells from stem cells is obviously crucial for the acquisition of proper gamete functionality. Nevertheless, the apparent failure of stem cell-derived germ cells to undergo meiosis efficiently could provide opportunities to study the molecular mechanisms regulating meiotic entry, progression and further broaden insights into the causes of meiotic failure.Generally, two processes that are the most difficult to be reconstructed in isolated PGC-LCs in vitro, we must first achieve a comprehensive understanding of the mechanisms underlying gametogenesis in vivo. Only the elucidation of the many still unknown aspects of such complex process and the progress in the coculture methods to maintain germ cells for long periods in vitro in 3-D microenvironments, suitable for the different stage-dependent requirements of gametogenesis, will give the necessary tools for rendering the production of germ cells from stem cells a reliable possibility. The first steps for producing germ cells from stem cells in vitro are promising, but there is still a long road ahead.As a prerequisite to the establishment of gametogenesis


There is a resurgent interest in tanycytes, a radial glial-like cell population occupying the floor and ventro-lateral walls of the third ventricle (3V). Tanycytes reside in close proximity to hypothalamic neuronal nuclei that regulate appetite and energy expenditure, with a subset sending projections into these nuclei. Moreover, tanycytes are exposed to 3V cerebrospinal fluid and have privileged access to plasma metabolites and hormones, through fenestrated capillaries. Indeed, some tanycytes act as conduits for trafficking of these molecules into the brain parenchyma. Tanycytes can also act as neural stem/progenitor cells, supplying the postnatal and adult hypothalamus with new neurons. Collectively, these findings suggest that tanycytes regulate and integrate important trophic and metabolic processes and possibly endow functional malleability to neuronal circuits of the hypothalamus. Hence, manipulation of tanycyte biology could provide a valuable tool for modulating hypothalamic functions such as energy uptake and expenditure in order to tackle prevalent eating disorders such as obesity and anorexia. The role and importance of tanycytes in diverse physiological functions—from barrier properties to neuroendocrine, metabolic, and neurogenic processes has been the subject of several excellent reviews  was first coined by Horstmann in 1954 and although some early literature used it to describe Radial glial-like cells found in CVOs, in recent times, it has become synonymous mostly with hypothalamic Radial glia.Hypothalamic tanycytes emerge during late embryonic development, in rats around embryonic day 19 , from similar if not the same neurogenic ventricular zone cells that generated hypothalamic neurons a few days earlier  and beta (β) subtypes and although both have a radial appearance, several characteristics distinguishes them apart  shows that whilst adult β- and α-tanycytes share some markers , there is also a class of β-tanycyte—specific genes/markers. Indeed, generally, the gene/marker profile of α-tanycytes is more resemblant of more dorsal non-tanycytic ependymal cells than β-tanycytes  but this capacity seems to wane with increasing age. For example, Lee et al.  and HaanAlthough the specific roles of tanycytes in the hypothalamus are still under much debate, their strategic proximity to, and relationship with fenestrated capillaries, blood brain barrier, axonal nerve terminals, and hypothalamic nuclei that regulate appetite/energy expenditure has placed them in a privileged position to integrate multiple inputs and regulate homeostasis. Some of their prominent roles are discussed below.Early studies focused on barrier functions of tanycytes, prompted by the finding that when Horse Radish Peroxidase (HRP) is injected into the CSF, for a short period it can diffuse freely into the hypothalamic parenchyma, but not into the Median Eminence (ME). Eventually, HRP reaches ME through the subarachnoid CSF. Conversely, intravenously-injected HRP is locked out of the hypothalamic parenchyma by its blood brain barrier (BBB), but can rapidly diffuse into the ME, and be contained therein , ventromedial (VMN), Lateral (LHN), the arcuate (Arc) nucleus, and the Paraventricular (PVN). At the core of this regulation lies the Arcuate's anorexigenic and orexigenic neurons, marked by their respective neurotransmitters Pro-opiomelanocortin (POMC), and Neuropeptide Y (NPY)/ and Agouti-related Peptide (AgRP), in addition to other minor neurotransmitters. These neurons sense and integrate a diverse array of anorexigenic and orexigenic metabolic signals such as ghrelin, glucose, and Leptin to promote or suppress appetite  are critical regulators of tissue growth and differentiation as well as metabolic homeostasis. Tanycytes also play a critical role in the feedback mechanisms that regulate TH (T4) release, involving the Hypothalamo-Pituitary-Thyroid axis  participate in this regulatory pathway via multiple but complementary mechanisms. First, they can actively uptake T4 from the CSF via two transporter molecules, MCT8 and OATP1, and convert T4 into either a more potent form, T3, or an inactive form by expressing the relevant converting enzymes—Deiodinase 2 (DIO2) and DIO3, respectively. Second, within the median eminence, the axonal terminals of TRH-producing neurons release TRH into the extracellular space, but TRH access to portal blood is restricted by the processes of tanycytes, akin to the GnRH gating scenario (above). Thus, it is thought that circulating TH induces cytoskeletal remodeling and retraction of tanycytic processes to allow TRH access to portal blood. Third, tanycytes also produce important enzymes that regulate TRH degradation, chiefly Pyroglutamyl peptidase  and thus long term control of appetite/energy expenditure Ebling, . Long ph2+) ions, ATP release, activation of P2Y1 purinergic receptors and an initiation of internal Ca2+ release within tanycytes. This effect then induces a propagative wave of Ca2+ signaling across neighboring tanycytes  and occupy a strategic position at the interface of the CSF and the parenchyma where appetite is regulated  or the lateral subventricular zone (SVZ) is insufficient to label tanycytes. Hence, early studies resorted to either multiple intraperitoneal injections or by direct intracerebroventricular infusion of BrdU, with the latter resulting in a greater number of labeled cells . Application of tamoxifen to these mice causes a nuclear translocation of CreERT2 protein to delete the STOP codon and activate the reporter marker, only in cells expressing the gene of interest.Lineage tracing studies have proved instrumental in delineating the fate and potential of diverse neural stem/progenitor cells in the postnatal and adult CNS , our knowledge of tanycytes is still rudimentary. In particular, we know very little about their anterio-posterior regionalization. Given their diverse functions (highlighted above), a set of pertaining questions ensues:If tanycytes constitute a neural stem cell population, what are the endogenous factors and genetic regulators driving their development , or maintenance with their niche? Are these comparable to those that regulate postnatal and adult neural stem cells in SGZ and SVZ?What are the intermediate steps of neurogenesis downstream of tanycytes? Is an intermediate or transient amplifying cell population involved, and how are particular neuronal subtypes produced? Do these properties change with age?How heterogeneous are tanycytes? For example, are specific populations set aside for barrier and transport functions vs. neurogenesis, or does a single tanycyte cell population multi-task? If there is heterogeneity, what factors establish this functional diversification? If not, are the chemosensory and homeostatic functions directly linked to neurogenesis?Tanycytes are found in the human hypothalamus (Flament-Durand and Brion, Fortunately, with the plethora of novel experimental tools and protocols at our disposal—ranging from novel transgenic animal models, gene transducing viruses, and cross-synaptic viral tracers to optogenetics—we are in an exciting era to answer these and other pertinent questions thereby learning more about the biology of tanycytes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.