Recent advances in nanotechnology for programmed death ligand 1-targeted cancer theranostics.

Programmed cell death ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) checkpoint inhibitor-based immunotherapy has provided a unique and potent weapon against cancer in clinical practice. The likelihood of achieving beneficial effects from PD-L1/PD-1 immune checkpoint blockade (ICB) therapy is clinically assessed by detecting PD-L1 expression through invasive tissue biopsies. However, PD-L1 expression is susceptible to tumor heterogeneity and dynamic response to ICB therapy. Moreover, currently, anti-PD-L1 immunotherapy still faces challenges of the low targeting efficiency of antibody drugs and the risk of immune-associated adverse events. To overcome these issues, advanced nanotechnology has been developed for the purpose of quantitative, non-invasive, and dynamic analyses of PD-L1, and to enhance the efficiency of ICB therapy. In this review, we first introduce the nanoprobe-assisted in vitro/in vivo modalities for the selective and sensitive analysis of PD-L1 during the diagnostic and therapeutic process. On the other hand, the feasibility of fabricating diverse functional nanocarriers as smart delivery systems for precisely targeted delivery of PD-L1 immune checkpoint inhibitors and combined therapies is highlighted. Finally, the current challenges are discussed and future perspectives for PD-L1-targeted cancer theranostics in preclinical research and clinical settings are proposed.

Interleukin-22 promotes proliferation and reverses LPS-induced apoptosis and steroidogenesis attenuation in human ovarian granulosa cells: implications for polycystic ovary syndrome pathogenesis.

OBJECTIVE: Interleukin 22 (IL-22) plays a role in inflammatory diseases. However, whether IL-22 affects the function of ovarian granulosa cells (GCs) and its relationship with Polycystic Ovary Syndrome (PCOS)remains unclear. METHODS: We investigated the level of IL-22 in human follicular fluid using ELISA. The expression and localization of the IL-22 receptor 1 (IL-22R1) in GCs were investigated by RT-PCR and immunofluorescence staining, respectively. The proliferation of KGN cells (human GCs line) was assessed by CCK-8 assay and EdU assay after treatment with recombinant human IL-22 (rhIL-22) and lipopolysaccharide (LPS). Apoptosis was assessed using flow cytometry. Apoptotic proteins and steroidogenic genes were detected by western blotting. RESULTS: ELISA's results showed that compared with the control group, PCOS patients showed lower expression of IL-22 in follicular fluid. Immunofluorescence showed that IL-22R1 is expressed and localized in human granulosa cell membranes. IL-22 promoted cell proliferation and reversed LPS-induced inhibition of cell proliferation. IL-22 alone did not affect apoptotic or steroidogenic protein expression, however, it reversed LPS-induced apoptosis via downregulation of Bcl-2, upregulation of Bax and cleaved caspase-3, and restoration of LPS-downregulated StAR, CYP11A1, and CYP19A1 expression. Western blotting confirmed that IL-22 activated the JAK2/STAT3 signaling. CONCLUSION: IL-22 promotes cell proliferation, inhibits apoptosis, and regulates KGN cell steroidogenesis confronted with LPS, and decreased IL-22 may be involved in the development of PCOS.

Acute and Subacute Toxicity Evaluation of Erythrocyte Membrane-Coated Boron Nitride Nanoparticles.

Boron nitride nanoparticles have been reported for boron drug delivery. However, its toxicity has not been systematically elucidated. It is necessary to clarify their potential toxicity profile after administration for clinical application. Here, we prepared erythrocyte membrane-coated boron nitride nanoparticles (BN@RBCM). We expect to use them for boron neutron capture therapy (BNCT) in tumors. In this study, we evaluated the acute toxicity and subacute toxicity of BN@RBCM of about 100 nm and determined the half-lethal dose (LD50) of the particles for mice. The results showed that the LD50 of BN@RBCM was 258.94 mg/kg. No remarkable pathological changes by microscopic observation were observed in the treated animals throughout the study period. These results indicate that BN@RBCM has low toxicity and good biocompatibility, which have great potential for biomedical applications.

TOP2A deficiency leads to human recurrent spontaneous abortion and growth retardation of mouse pre-implantation embryos.

BACKGROUND: Recurrent spontaneous abortion (RSA), is a dangerous pregnancy-related condition and is a subject of debate in the gynaecology and obstetrics communities. The objective of this study was to determine the function of DNA Topoisomerase II Alpha (TOP2A) in RSA and elucidate the underlying molecular mechanisms. METHODS: In vitro models of TOP2A-knockdown and -overexpression were generated by transfecting specific sh-RNA lentivirus and overexpression plasmid, respectively. An in vitro TOP2A inhibition model was established by culturing mouse embryos at the two-cell stage in a medium containing PluriSIn2, a TOP2A inhibitor. Immunohistochemical staining was used to analyse expression of TOP2A in villi tissues of patients with RSA. Western blotting and qRT-PCR were used to analyse the expression of TOP2A and proteins involved in trophoblast functions, the FOXO signalling pathway, and the development of pre-implantation embryos. 5-Ethynyl-2'-deoxyuridine staining, TUNEL assay and flow cytometry were used to further evaluate the effect of TOP2A on cell proliferation and apoptosis. Transwell and wound healing assays were used to evaluate migration and invasion. Moreover, the effect of TOP2A inhibitor on embryos was determined by immunofluorescence and mitochondrial-related dyes. RESULTS: Evaluation of clinical samples revealed that the villi tissues of patients that have experienced RSA had lower TOP2A expression compared with that from women who have experienced normal pregnancy (P < 0.01). In vitro, TOP2A knockdown decreased the proliferation, migration, and invasion of trophoblast cell lines, and increased apoptosis and activation of the FOXO signalling pathway (P < 0.05). Conversely, TOP2A overexpression reversed these effects. Moreover, in vivo experiments confirmed that inhibition of TOP2A impairs trophectoderm differentiation, embryonic mitochondrial function as well as the developmental rate; however, no differences were noted in the expression of zygotic genome activation-related genes. CONCLUSIONS: Collectively, our data suggest that lower TOP2A expression is related to RSA as it inhibits trophoblast cell proliferation, migration, and invasion by activation of the FOXO signalling pathway. Additionally, TOP2A inhibition resulted in impaired development of pre-implantation embryos in mice, which could be attributed to excessive oxidative stress.

Epithelial cell adhesion molecule promotes breast cancer resistance protein-mediated multidrug resistance in breast cancer by inducing partial epithelial-mesenchymal transition.

Overexpression of breast cancer resistance protein (BCRP) plays a crucial role in the acquired multidrug resistance (MDR) in breast cancer. The elucidation of molecular events that confer BCRP-mediated MDR is of major therapeutic importance in breast cancer. Epithelial cell adhesion molecule (EpCAM) has been implicated in tumor progression and drug resistance in various types of cancers, including breast cancer. However, the role of EpCAM in BCRP-mediated MDR in breast cancer remains unknown. In the present study, we revealed that EpCAM expression was upregulated in BCRP-overexpressing breast cancer MCF-7/MX cells, and EpCAM knockdown using siRNA reduced BCRP expression and increased the sensitivity of MCF-7/MX cells to mitoxantrone (MX). The epithelial-mesenchymal transition (EMT) promoted BCRP-mediated MDR in breast cancer cells, and EpCAM knockdown partially suppressed EMT progression in MCF-7/MX cells. In addition, Wnt/ß-catenin signaling was activated in MCF-7/MX cells, and the inhibition of this signaling attenuated EpCAM and BCRP expression and partially reversed EMT. Together, this study illustrates that EpCAM upregulation by Wnt/ß-catenin signaling induces partial EMT to promote BCRP-mediated MDR resistance in breast cancer cells. EpCAM may be a potential therapeutic target for overcoming BCRP-mediated resistance in human breast cancer.

Application of nanomaterials in the treatment of rheumatoid arthritis.

Rheumatoid Arthritis (RA) is a chronic autoimmune disease, which mainly causes inflammation of the synovial joints and destruction of cartilage and bone tissue. At present, a variety of clinical drugs have been applied in the treatment of rheumatoid arthritis. With the development of nanotechnology, more and more nano-drugs have been applied in the treatment of rheumatoid arthritis due to the unique physical and chemical properties of nanomaterials. Treatment of RA with nanomaterials can improve bioavailability and selectively target damaged joint tissue. In this review, we summarized the progress of the application of nanomaterials in the treatment of rheumatoid arthritis and also proposed challenges faced by nanomaterials in the treatment of rheumatoid arthritis.

Downregulation of cytokeratin 18 induces cellular partial EMT and stemness through increasing EpCAM expression in breast cancer.

Induction of epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) characteristics underlie the development of metastasis, chemoresistance, and tumor recurrence in breast cancer. Downregulation of cytokeratin 18 (CK18) is a critical molecular event of EMT; however, its importance in the induction of EMT and CSC features has not been defined to date. This study aimed to investigate the biological significance and underlying molecular mechanisms of CK18 in inducing EMT phenotype and stemness properties of breast cancer cells. Three breast cancer cell lines (i.e., non-metastatic MCF-7, highly metastatic MDA-MB-231, and mitoxantrone (MX)-selected resistant MCF-7/MX cells) and two CK18-knockdown stable cell clones (MCF-7-shCK18-7D and 3C) were used to determine the association between CK18 and EMT and stemness. CK18 expression was extremely low in highly metastatic, resistant, and transforming growth factor (TGF)-ß1/tumor necrosis factor (TNF)-α-treated breast cancer cells with mesenchymal phenotype and increased expression of CSC markers. Depletion of CK18 promoted partial EMT and the acquisition of stemness properties in breast cancer MCF-7 cells. Mechanistically, CK18 interference in MCF-7 cells activated the Wnt/ß-catenin signaling, resulting in the up-regulation of epithelial cell adhesion molecule (EpCAM). Consistently, the stemness properties and metastasis can be attenuated by further knockdown of EpCAM in CK18-depleted cells. In conclusion, downregulation of CK18 promotes partial EMT and enhances breast cancer stemness by increasing EpCAM expression partly via the Wnt/ß-catenin pathway. These findings indicate that CK18 may serve as a potential treatment target for advanced breast cancer.

Derivation and propagation of spermatogonial stem cells from human pluripotent cells.

OBJECTIVES: This study is designed to generate and propagate human spermatogonial stem cells (SSCs) derived from human pluripotent stem cells (hPSCs). METHODS: hPSCs were differentiated into SSC-like cells (SSCLCs) by a three-step strategy. The biological characteristics of SSCLCs were detected by immunostaining with antibodies against SSC markers. The ability of self-renewal was measured by propagating for a long time and still maintaining SSCs morphological property. The differentiation potential of SSCLCs was determined by the generation of spermatocytes and haploid cells, which were identified by immunostaining and flow cytometry. The transcriptome analysis of SSCLCs was performed by RNA sequencing. The biological function of SSCLCs was assessed by xeno-transplantation into busulfan-treated mouse testes. RESULTS: SSCLCs were efficiently generated by a 3-step strategy. The SSCLCs displayed a grape-like morphology and expressed SSC markers. Moreover, SSCLCs could be propagated for approximately 4 months and still maintained their morphological properties. Furthermore, SSCLCs could differentiate into spermatocytes and haploid cells. In addition, SSCLCs displayed a similar gene expression pattern as human GPR125+ spermatogonia derived from human testicular tissues. And more, SSCLCs could survive and home at the base membrane of seminiferous tubules. CONCLUSION: SSCLCs were successfully derived from hPSCs and propagated for a long time. The SSCLCs resembled their counterpart human GPR125+ spermatogonia, as evidenced by the grape-like morphology, transcriptome, homing, and functional characteristics. Therefore, hPSC-derived SSCLCs may provide a reliable cell source for studying human SSCs biological properties, disease modeling, and drug toxicity screening.

Downregulation of cytokeratin 18 enhances BCRP-mediated multidrug resistance through induction of epithelial-mesenchymal transition and predicts poor prognosis in breast cancer.

Multiple drug resistance (MDR) and metastasis have been identified as the two major causes of the poor prognosis of patients with breast cancer. However, the relationship between MDR and metastasis has not been characterized. Epithelial­mesenchymal transition (EMT), a process known to promote metastasis in cancer, has been shown to be associated with the MDR phenotype of many tumor types. Reduced cytokeratin 18 (CK18) expression is thought to be one of the hallmarks of EMT, and the role of CK18 in MDR of metastatic breast cancer remains unknown. In the present study, we revealed that the expression of CK18 was significantly downregulated in breast cancer tissues and in an MDR cell line overexpressing breast cancer resistant protein (BCRP), and the presence of low levels of CK18 was associated with TNM stage, lymph node metastasis, and unfavorable survival in breast cancer patients. Further results demonstrated that CK18 stable knockdown using shRNA increased BCRP expression and induced the EMT process in human breast cancer MCF­7 cells. Moreover, CK18 knockdown was associated with the activation of the NF­κB/Snail signaling pathway, which has been revealed to regulate EMT and BCRP. Based on these findings, we concluded that CK18 knockdown enhanced BCRP­mediated MDR in MCF­7 cells through EMT induction partly via the NF­κB/Snail pathway. These findings provide a valuable insight into the potential role of CK18 in MDR, migration and invasion of breast cancer cells. Reduced expression of CK18 may be a novel biomarker for predicting the poor prognosis of breast cancer patients.

Baicalein attenuates monocrotaline-induced pulmonary arterial hypertension by inhibiting endothelial-to-mesenchymal transition.

AIMS: Endothelial-to-mesenchymal transition (EndoMT) was shown to lead to endothelial cell (EC) dysfunction in pulmonary arterial hypertension (PAH). Baicalein was reported to inhibit epithelial-to-mesenchymal transition (EMT), a biological process that has many regulatory pathways in common with EndoMT. Whether it can attenuate PAH by inhibiting EndoMT remains obscure. MAIN METHODS: PAH was induced by a single subcutaneous injection of MCT (60 mg/kg) in male Sprague Dawley rats. Two weeks after MCT administration, the rats in the treatment groups received baicalein orally (50 or 100 mg/kg/day) for an additional 2 weeks. Hemodynamic changes and right ventricular hypertrophy (RVH) were evaluated on day 28. Cardiopulmonary interstitial fibrosis was detected using Masson's trichrome, Picrosirius-red, and immunohistochemical staining. The reactivity of pulmonary arteries (PAs) was examined ex vivo. The protein expresson of EndoMT molecules, bone morphogenetic protein receptor 2 (BMPR2), and nuclear factor-κB (NF-κB) was examined to explore the mechanism of protective action of baicalein. KEY FINDINGS: Baicalein (50 and 100 mg/kg) significantly alleviated MCT-induced PAH and cardiopulmonary interstitial fibrosis. Furthermore, baicalein treatment enhanced PA responsiveness to acetylcholine (ACh) in PAH rats. The upregulation of EndoMT molecules (N-cadherin, vimentin, Snail, and Slug) strongly suggest that EndoMT participates in MCT-induced PAH, which was reversed by baicalein (50 and 100 mg/kg) treatment. Moreover, baicalein partially reversed MCT-induced reductions in BMPR2 and NF-κB activation in the PAs. SIGNIFICANCE: Baicalein attenuated MCT-induced PAH in rats by inhibiting EndoMT partially via the NF-κB-BMPR2 pathway. Thus, baicalein might be considered as a promising treatment option for PAH.

Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation.

Spermatogonial stem cells (SSCs) have significant applications in both reproductive and regenerative medicine. However, primary human SSCs are very rare, and a human SSC line has not yet been available. In this study, we have for the first time reported a stable human SSC line by stably expressing human SV40 large T antigen. RT-PCR, immunocytochemistry, and Western blots revealed that this cell line was positive for a number of human spermatogonial and SSC hallmarks, including VASA, DAZL, MAGEA4, GFRA1, RET, UCHL1, GPR125, PLZF and THY1, suggesting that these cells are human SSCs phenotypically. Proliferation analysis showed that the cell line could be expanded with significant increases of cells for 1.5 years, and high levels of PCNA, UCHL1 and SV40 were maintained for long-term culture. Transplantation assay indicated that human SSC line was able to colonize and proliferate in vivo in the recipient mice. Neither Y chromosome microdeletions of numerous genes nor tumor formation was observed in human SSC line although there was abnormal karyotype in this cell line. Collectively, we have established a human SSC line with unlimited proliferation potentials and no tumorgenesis, which could provide an abundant source of human SSCs for their mechanistic studies and translational medicine.

Expansion and long-term culture of human spermatogonial stem cells via the activation of SMAD3 and AKT pathways.

Spermatogonial stem cells (SSCs) can differentiate into spermatids, reflecting that they could be used in reproductive medicine for treating male infertility. SSCs are able to become embryonic stem-like cells with the potentials of differentiating into numerous cell types of the three germ layers and they can transdifferentiate to mature and functional cells of other lineages, highlighting significant applications of human SSCs for treating human diseases. However, human SSCs are very rare and a long-term culture system of human SSCs has not yet established. This aim of study was to isolate, identify and culture human SSCs for a long period. We isolated GPR125-positive spermatogonia with high purity and viability from adult human testicular tissues utilizing the two-step enzymatic digestion and magnetic-activated cell sorting with antibody against GPR125. These freshly isolated cells expressed a number of markers for SSCs, including GPR125, PLZF, GFRA1, RET, THY1, UCHL1 and MAGEA4, but not the hallmarks for spermatocytes and spermatozoa, e.g. SYCP1, SYCP3, PRM1, and TNP1. The isolated human SSCs could be cultured for two months with a significant increase of cell number with the defined medium containing growth factors and hydrogel. Notably, the expression of numerous SSC markers was maintained during the cultivation of human SSCs. Furthermore, SMAD3 and AKT phosphorylation was enhanced during the culture of human SSCs. Collectively, these results suggest that human SSCs can be cultivated for a long period and expanded whilst retaining an undifferentiated status via the activation of SMAD3 and AKT pathways. This study could provide sufficient cells of SSCs for their basic research and clinic applications in reproductive and regenerative medicine.

Nodal promotes the self-renewal of human colon cancer stem cells via an autocrine manner through Smad2/3 signaling pathway.

Colorectal cancer is one of the most common and fatal tumors. However, molecular mechanisms underlying carcinogenesis of colorectal cancer remain largely undefined. Here, we explored the expression and function of Nodal in colon cancer stem cells (CCSCs). Nodal and its receptors were present in numerous human colorectal cancer cell lines. NODAL and ALK-4 were coexpressed in human colon cancerous tissues, and NODAL, CD24, and CD44, markers for CCSCs, were expressed at higher levels in human colon cancerous tissues than adjacent noncancerous colon tissues. Human CCSCs were isolated by magnetic activated cell sorting using anti-CD24 and anti-CD44. Nodal transcript and protein were hardly detectable in CD44- or CD24-negative human colorectal cancer cell lines, whereas Nodal and its receptors were present in CCSCs. Notably, Nodal facilitated spheroid formation of human CCSCs, and phosphorylation of Smad2 and Smad3 was activated by Nodal in cells of spheres derived from human CCSCs. Collectively, these results suggest that Nodal promotes the self-renewal of human CCSCs and mediate carcinogenesis of human colorectal cancer via an autocrine manner through Smad2/3 pathway. This study provides a novel insight into molecular mechanisms controlling fate of human CCSCs and offers new targets for gene therapy of human colorectal cancer.

[Imaging analysis of burst injury of the lungs in coal miners workers].

OBJECTIVE: To investigate the imaging features of burst injury of the lung in coal miners and to provide reference for clinical diagnosis and treatment. METHODS: A retrospective analysis was performed on the clinical data and imaging findings of 78 patients with burst injury of the lung. RESULTS: The imaging findings of burst injury of the lung were variable and complex, and they varied over time. Eleven cases showed no abnormalities on X-ray and CT within 24 h, but abnormalities appeared within 3 d; 53 cases showed abnormalities on X-ray and CT and had an exacerbation within 3 d; 8 cases showed improvements when reexamined. Among the 78 patients examined by X-ray and (or) CT, ground glass-like shadows were noted in 15 cases, interstitial changes in 13 cases, segmental consolidation in 24 cases, and diffuse consolidation in 26 cases. The complications included pneumothorax (n = 35), hemothorax (n = 28), costal fracture (n = 24), and pulmonary infection (n = 27). CONCLUSION: X-ray plain film and CT offer a reliable basis for early diagnosis of burst injury of the lung. CT is superior to X-ray plain film in detecting lesions, so chest CT should be performed as early as possible to remedy burst injury of the lung.

Fibroblast growth factor 4 is required but not sufficient for the astrocyte dedifferentiation.

Our recent studies demonstrated that mature astrocytes from spinal cord can be reprogrammed in vitro and in vivo to generate neural stem/progenitor cells (NSPCs) following treatment with conditioned medium collected from mechanically injured astrocytes. However, little is known regarding the molecular mechanisms underlying the reprogramming of astrocytes. Here, we show that fibroblast growth factor 4 (FGF4) exerts a critical role in synergistically converting astrocytes into NSPCs that can express multiple neural stem cell markers (nestin and CD133) and are capable of both self-renewal and differentiation into neurons and glia. Lack of FGF4 signals fails to elicit the dedifferentiation of astrocytes towards NSPCs, displaying a substantially lower efficiency in the reprogramming of astrocytes and a slower transition through fate-determined state. These astrocyte-derived NSPCs displayed relatively poor self-renewal and multipotency. More importantly, further investigation suggested that FGF4 is a key molecule necessary for activating PI3K/Akt/p21 signaling cascades, as well as their downstream effectors responsible for directing cell reprogramming towards NSPCs. Collectively, these findings provide a molecular basis for astrocyte dedifferentiation into NSPCs after central nervous system (CNS) injury and imply that FGF4 may be a clinically applicable molecule for in situ neural repair in the CNS disorders.

Direct transdifferentiation of spermatogonial stem cells to morphological, phenotypic and functional hepatocyte-like cells via the ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E.

BACKGROUND: Severe shortage of liver donors and hepatocytes highlights urgent requirement of extra-liver and stem cell source of hepatocytes for treating liver-related diseases. Here we hypothesized that spermatogonial stem cells (SSCs) can directly transdifferentiate to hepatic stem-like cells capable of differentiating into mature hepatocyte-like cells in vitro without an intervening pluripotent state. RESULTS: SSCs first changed into hepatic stem-like cells since they resembled hepatic oval cells in morphology and expressed Ck8, Ck18, Ck7, Ck19, OV6, and albumin. Importantly, they co-expressed CK8 and CK19 but not ES cell markers. Hepatic stem-like cells derived from SSCs could differentiate into small hepatocytes based upon their morphological features and expression of numerous hepatic cell markers but lacking of bile epithelial cell hallmarks. Small hepatocytes were further coaxed to differentiate into mature hepatocyte-like cells, as identified by their morphological traits and strong expression of Ck8, Ck18, Cyp7a1, Hnf3b, Alb, Tat, Ttr, albumin, and CYP1A2 but not Ck7 or CK19. Notably, these differentiated cells acquired functional attributes of hepatocyte-like cells because they secreted albumin, synthesized urea, and uptake and released indocyanine green. Moreover, phosphorylation of ERK1/2 and Smad2/3 rather than Akt was activated in hepatic stem cells and mature hepatocytes. Additionally, cyclin A, cyclin B and cyclin E transcripts and proteins but not cyclin D1 or CDK1 and CDK2 transcripts or proteins were reduced in mature hepatocyte-like cells or hepatic stem-like cells derived from SSCs compared to SSCs. CONCLUSIONS: SSCs can transdifferentiate to hepatic stem-like cells capable of differentiating into cells with morphological, phenotypic and functional characteristics of mature hepatocytes via the activation of ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E. This study thus provides an invaluable source of mature hepatocytes for treating liver-related diseases and drug toxicity screening and offers novel insights into mechanisms of liver development and cell reprogramming.

Sertoli cells from non-obstructive azoospermia and obstructive azoospermia patients show distinct morphology, Raman spectrum and biochemical phenotype.

STUDY QUESTION: Are there differences in the morphology, spectrum and biochemical phenotype between Sertoli cells from non-obstructive azoospermia (NOA) patients and those from obstructive azoospermia (OA) patients with normal spermatogenesis? SUMMARY ANSWER: Sertoli cells from NOA patients are distinct from those from OA patients in terms of morphological features, Raman spectrum and phenotype including the expression of genes and proteins (e.g. SCF, BMP4 and GDNF). WHAT IS KNOWN ALREADY: NOA affects 10% of infertile men and has been diagnosed in 60% of azoospermic men. In contrast with OA patients who have normal spermatogenesis, NOA patients have an impaired spermatogenesis. STUDY DESIGN, SIZE AND DURATION: This case-control study included 100 NOA patients (as cases) and 100 OA patients with normal spermatogenesis (as controls). The study was performed between January 2012 and January 2013. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Karyotype analysis was performed to check the chromosome content and multiplex PCR was carried out to determine the expression of numerous Y chromosome genes in NOA patients. Human Sertoli cells were then isolated from the testes of NOA and OA patients by two-step enzymatic digestion and differential plating. Transmission electron microscopy was used to determine the ultrastructure of the Sertoli cells and real-time Raman microspectroscopy was used to assess their spectrum. We further compared the two groups of patients for expression of SCF, GDNF and BMP4 in Sertoli cells, using RT-PCR, microarray analysis, immunofluorescence, immunohistochemistry and Western blots. MAIN RESULTS AND THE ROLE OF CHANCE: NOA patients had normal chromosome karyotypes and Y chromosome microdeletions were excluded. In morphology, Sertoli cells isolated from NOA patients had a series of abnormal ultrastructural features compared with the control Sertoli cells: (i) existence of small and spindle-shaped nuclei, (ii) smaller diameter, (iii) deficient nucleolus or endoplasmic reticulum and (iv) more vacuoles. Spectral intensities in Sertoli cells of NOA patients were distinct at four typical Raman peaks compared with the control Sertoli cells. In phenotype, SCF, BMP4 and GDNF transcripts and proteins were significantly lower in Sertoli cells of NOA patients than in the control Sertoli cells. LIMITATIONS AND REASONS FOR CAUTION: The Sertoli cells of NOA patients were not compared with Sertoli cells of normal fertile men due to the fact that it is hard to obtain adult testes from normal donors. WIDER IMPLICATIONS OF THE FINDINGS: This study provides novel insights into understanding the underlying causes for NOA and might offer a basis for developing new therapeutic strategies for patients with NOA.

Characterization of an inositol 1,3,4-trisphosphate 5/6-kinase gene that is essential for drought and salt stress responses in rice.

Drought and salt stresses are major limiting factors for crop production. To identify critical genes for stress resistance in rice (Oryza sativa L.), we screened T-DNA mutants and identified a drought- and salt-hypersensitive mutant dsm3. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative inositol 1,3,4-trisphosphate 5/6-kinase previously named OsITPK2 with unknown function. Under drought stress conditions, the mutant had significantly less accumulation of osmolytes such as proline and soluble sugar and showed significantly reduced root volume, spikelet fertility, biomass, and grain yield; however, malondialdehyde level was increased in the mutant. Interestingly, overexpression of DSM3 (OsITPK2) in rice resulted in drought- and salt-hypersensitive phenotypes and physiological changes similar to those in the mutant. Inositol trisphosphate (IP3) level was decreased in the overexpressors under normal condition and drought stress. A few genes related to osmotic adjustment and reactive oxygen species scavenging were down-regulated in the mutant and overexpression lines. The expression level of DSM3 promoter-driven ß-glucuronidase (GUS) reporter gene in rice was induced by drought, salt and abscisic acid. Protoplast transient expression assay indicated that DSM3 is an endoplasmic reticulum protein. Sequence analysis revealed six putative ITPKs in rice. Transcript level analysis of OsITPK genes revealed that they had different tempo-spatial expression patterns, and the responses of DSM3 to abiotic stresses, including drought, salinity, cold, and high temperature, were distinct from the other five members in rice. These results together suggest that DSM3/OsITPK2 is an important member of the OsITPK family for stress responses, and an optimal expression level is essential for drought and salt tolerance in rice.

E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro.

Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.