Naïve-like conversion of bovine induced pluripotent stem cells from Sertoli cells.

Feeder cells are essential to derive pluripotent stem cells (PSCs). Mouse embryonic fibroblasts (MEF) are widely used as feeder to generate and culture embryonic stem cells (ESCs) and induced PSCs (iPSCs) in many species. However it may not be suitable for livestock ESCs/iPSCs due to interspecies difference. Previously we derived bovine iPSCs from bovine Sertoli cells using MEF feeder. Here we compared the effects of MEF feeder and bovine embryonic fibroblasts (BEF) feeder on the maintenance of bovine iPSC pluripotency and morphology as well their contributions to the naïve-like conversion, based on a naïve medium (NM). The results showed successful conversion of the primed bovine iPSCs to naïve-like state within 3-4 days both on MEF feeder and BEF feeder in NM (termed as MNM and BNM respectively). These naïve-like iPSCs showed normal karyotype. There were more iPSC colonies under BNM condition than MNM condition. Epigenetically, histone modification H3K4 was upregulated, while H3K27 was downregulated in the naïve-like iPSCs. We further analyzed the naïve markers and differentiation potential both in vitro and in vivo of these cells, which were all reserved throughout the maintenance. Together, bovine naïve-like iPSCs can be generated both on MEF and BEF feeder in NM condition. The BNM condition is able to sustain the pluripotency and differentiation potential of the naïve-like bovine iPSCs, and improve the conversion efficiency.

Current Evidence and Future Directions of Berberine Intervention in Depression.

A major type of serious mood disorder, depression is currently a widespread and easily overlooked psychological illness. With the low side effects of natural products in the treatment of diseases becoming the pursuit of new antidepressants, natural Chinese medicine products have been paid more and more attention for their unique efficacy in improving depression. In a view from the current study, the positive antidepressant effects of berberine are encouraging. There is a lot of work that needs to be done to accurately elucidate the efficacy and mechanism of berberine in depression. In this review, the relevant literature reports on the treatment of depression and anxiety by berberine are updated, and the potential pharmacological mechanism of berberine in relieving depression has also been discussed.

Schisandrin A and B affect the proliferation and differentiation of neural stem cells.

Schisandrin A and B (Sch A and B) are the important components of Asian dietary supplement and phytomedicine Schisandra chinensis (S. chinensis). They can enhance adult neurogenesis in vivo; however, these effects still need to be verified. Here NE-4 C neural stem cells (NSCs) were employed as the in vitro model and treated with Sch A and B at 0.1 µg/mL. EdU (5-Ethynyl-2'-deoxyuridine) labeling showed that both Sch A and B treatments enhanced NSC proliferation. Real-time PCR analysis showed the mRNA abundances of telomerase gene Tert and cell cycle gene Cyclin D1 were significantly up-regulated after the treatments. During the neurosphere induction, Sch B enhanced the neurosphere formation and neuronal differentiation, and increased the neurosphere semidiameters. Detection of the neuron differentiation marker Mapt indicates that both Sch A and B, especially Sch B, benefits the induced neuronal differentiation. Sch B treatment also enhanced mRNA expressions of the neurosphere-specific adhesion molecule Cdh2 and Wnt pathway-related genes including Mmp9, Cyclin D1 and ß-catenin. Together, Sch A especially Sch B, promotes the proliferation, affects the survival, differentiation and neurogenesis of NSCs, which is consistent with their in vivo effects. This study provides further clue on the potential neuropharmacological effects of S. chinensis.

Effective isolation of Sertoli cells from New Zealand rabbit testis.

OBJECTIVE: Sertoli cells (SCs) are important sustentacular cells in the seminiferous tubules of the testis. Isolation and identification of SCs are the premise for studying their functions. Since New Zealand rabbit is a stable strain which is widely used for biomedical research and animal farming, this study aimed to develop a simple and effective protocol for SC isolation in New Zealand rabbits. MATERIALS AND METHODS: The SCs of three 30-day-old New Zealand rabbits were isolated by incubation with enzymatic digestion I (Dulbecco's modified Eagle medium supplemented with 1 mg/ml collagenase IV and 50 µg/ml DNase I) and digestion II (digestion I + 1 mg/ml hyaluronidase + 1 mg/ml trypsin), as well as differential plating. The cells were enriched and identified by using immunocytochemical staining and reverse transcription polymerase chain reaction analysis. RESULTS: Homogeneous cells were obtained. They presented the typical large cell body and an irregular pyramidal shape after differential plating and passaging. These cells expressed mRNA of the SC marker sex-determining region Y-box 9 (SOX9) instead of the Leydig cell marker StAR. Immunocytochemically, they are positive of SOX9, GATA binding protein 4, and androgen-binding protein. CONCLUSION: The SCs were enriched from the testicular tissues of prepubertal New Zealand rabbits by a simple and effective protocol, which provides a basis for further theoretical researches and practical applications.

Survivable potential of germ cells after trehalose cryopreservation of bovine testicular tissues.

Germplasm preservation of livestock or endangered animals and expansion of germline stem cells are important. The purpose of this study is to investigate whether supplementation of trehalose to the freezing medium (FM) reduces tissular damage and improves the quality of testicular cells in the cryopreserved bovine testicular tissues. We herein established an optimized protocol for the cryopreservation of bovine testicular tissues, and the isolation as well as culture of bovine germ cells containing spermatogonial stem cells (SSCs) from these tissues. The results showed that FM containing 10% dimethyl sulfoxide (Me2SO/DMSO), 10% knockout serum replacement (KSR) and 20% trehalose (FM5) combined with the uncontrolled slow freezing (USF) procedures has the optimized cryoprotective effect on bovine testicular tissues. The FM5 + USF protocol reduced the cell apoptosis, maintained high cell viability, supported the structural integrity and seminiferous epithelial cohesion similar to that in the fresh tissues. Viable germ cells containing SSCs were effectively isolated from these tissues and they maintained germline marker expressions in the co-testicular cells and co-mouse embryonic fibroblasts (MEF) feeder culture systems respectively, during the short-term culture. Additionally, upregulated transcriptions of spermatogenic differentiation marker C-KIT and meiotic marker SYCP3 were detected in these cells after retinoic acid-induced differentiation. Together, FM5 + USF is suitable for the cryopreservation of bovine testicular tissues, with benefits of reducing the apoptosis, maintaining the cell viability, supporting the testicular structure integrity, and sustaining the survival and differentiation potential of bovine germ cells containing SSCs.

Culture bovine prospermatogonia with 2i medium.

Germplasm cryopreservation and expansion of gonocytes/prospermatogonia or spermatogonial stem cells (SSCs) are important; however, it's difficult in cattle. Since inhibitors of Mek1/2 and Gsk3ß (2i) can enhance pluripotency maintenance, effects of 2i-based medium on the cultivation of bovine prospermatogonia from the cryopreserved tissues were examined. The testicular tissues of newborn bulls were well cryopreserved. High mRNA levels of prospermatogonium/SSC markers (PLZF, GFRα-1) and pluripotency markers (Oct4/Pouf5, Sox2, Nanog) were detected and the PLZF+ /GFRα-1+ prospermatogonia were consistently identified immunohistochemically in the seminiferous cords. Using differential plating and Percoll-based centrifugation, 41.59% prospermatogonia were enriched and they proliferated robustly in 2i medium. The 2i medium boosted mRNA abundances of Pouf5, Sox2, Nanog, GFRα-1, PLZF, anti-apoptosis gene Bcl2, LIF receptor gene LIFR and enhanced PLZF protein expression, but suppressed mRNA expressions of spermatogonial differentiation marker c-kit and pro-apoptotic gene Bax, in the cultured prospermatogonia. It also alleviated H2 O2 -induced apoptosis of the enriched cells and decreased histone H3 lysine (K9) trimethylation (H3K9me3) and its methylase Suv39h1/2 mRNA level in the cultured seminiferous cords. Overall, 2i medium improves the cultivation of bovine prospermatogonia isolated from the cryopreserved testes, by inhibiting Suv39h1/2-mediated H3K9me3 through Mek1/2 and Gsk3ß signalling, evidencing successful cryopreservation and expansion of bovine germplasm.

Decreased abundance of GDNF mRNA transcript in the immature Sertoli cells of cattle in response to protein kinase inhibitor staurosporine.

Sertoli cells (SC) have important functions in spermatogenesis by regulating development of spermatogenic cells. Glial cell line-derived neurotrophic factor (GDNF) are produced by SC. Although the effects of GDNF on spermatogenesis have been well studied, the understanding of how GDNF is synthesized is still limited, especially in food animal producing species. Because protein kinase (PK) has varied functions in multiple cellular processes and the PK pathway modulates SC functions, the objective of the present study was to determine whether PK modulates the abundance of GDNF protein in SC of cattle. To conduct this study, immature SC were enriched from cryopreserved testicular tissues of 1-day-old bulls. These cells had a marked proliferation capacity. Results from immunostaining analysis indicated that there was a sustained abundance of SC mRNA marker protein transcripts and marker proteins: androgen bind protein (ABP), GATA4 and VIMENTIN. There was subsequent characterization of SC treated with the PK inhibitor staurosporine for 0, 1 or 2 h. Results from real-time-PCR and Western blot analyses indicated the treatment (2 h) resulted in a decrease in Gdnf mRNA transcript and GDNF protein. Additionally, the staurosporine treatment resulted in an increase in the abundance of anti-apoptosis Bcl2 and decrease in pro-apoptosis Bax mRNA transcripts. Furthermore, results of the TUNEL assay indicated there was a decrease in apoptosis in the staurosprine-treated SC. Collectively, results indicate the PK signaling is involved in regulation of GDNF protein abundance in the immature SC and the survival of these cells in cattle.

Schisandrin A and B enhance the dentate gyrus neurogenesis in mouse hippocampus.

Schisandrin A and B (Sch A and B) are the main effective components of Schisandra chinensis (S. chinensis), which is traditionally used to enhance mental and intellectual functions in eastern Asia. Previously, we reported Sch A and B remarkably affect adult neurogenesis in the subventricular zone of mouse lateral ventricle. Since the neurogenesis in the hippocampal dentate gyrus (DG) is more important to learning, memory and cognition, here we further examined their effects on the adult DG neurogenesis. Phosphohistone H3 (PHH3) immunostaining showed that Sch B significantly enhanced the cell proliferation in the DG. Glial fibrillary acidic protein (GFAP, mostly labels astrocytes and some stem cells) staining was used to further identify the proliferating cell type. Dramatically, increases of GFAP+ cells in both Sch A and B treated groups were observed. What's more, the total numbers of the mature neurons labeled by neuron-specific nuclear protein (NeuN) were also increased in both Sch A and B treated groups compared with the controls. Together, Sch A and B enhance the adult DG neurogenesis by increasing astrocytes/stem cells and improving the survival and maturation of DG neurons. Our study shed a new light on the neuropharmacological functions of the herbal medicine S. chinensis.

Cryopreservation of calf testicular tissues with knockout serum replacement.

To enrich bovine gonocytes from cryopreserved testicular tissues, the cryoprotection effects of the freezing media containing knockout serum replacement (KSR) were examined. Using Minimum essential medium (MEM) + 10% dimethyl sulfoxide (Me2SO) as the basic medium, calf testicular tissues were cryopreserved in media containing 0, 5, 10, 20, 40, 90% KSR and 5% fetal bovine serum (FBS) respectively. Morphologically, the seminiferous cords and interstitium were well preserved in all groups. The gonocytes were all glial cell line-derived neurotrophic factor (GDNF) family receptor α-1 (GFRα-1) positive. The recovery rates in all KSR groups were higher than that of the 10% Me2SO group, while comparable to the 5% FBS group. The enriched gonocytes expressed gonocyte marker GFRα-1 typically. Collectively, supplementation of 5-10% KSR can achieve comparable cryoprotective effects with using 5% FBS, which is useful in future study due to its defined formulation that is more consistent in quality and stable in supply.

Thymoquinone inhibits proliferation in gastric cancer via the STAT3 pathway in vivo and in vitro.

AIM: To elucidate the mechanism of thymoquinone (TQ)-induced apoptosis in human gastric cancer cells in vitro and in vivo. METHODS: HGC27, BGC823, and SGC7901 cells were cultured in vitro and treated with TQ (0, 10, 25, 50, 75, 100, 125 µmol/L) for 12 h, 24 h, and 36 h, and then the proliferation inhibitory rates were detected by methylthiazole tetrazolium assay. Apoptosis was observed after Hoechst staining. The protein expressions of signal transducer and activator of transcription (STAT)3, p-STAT3, STAT5, p-STAT5, phospho-janus-activated kinase 2 (JAK2), JAK2, p-Src, Src, glyceraldehyde-3-phosphate dehydrogenase, lamin-A, survivin, Cyclin D, Bcl-2, Bax, peroxisome proliferator activated receptor, and caspase-3,7,9 were detected by western blot. Cell cycle and apoptosis were determined with flow cytometry. TQ induced dose-dependent apoptotic cell death in HGC27 cells was measured by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) analysis and Hoechst 33258. RESULTS: TQ inhibited the phosphorylation of STAT3 but not STAT5. TQ-induced downregulation of STAT3 activation was associated with a reduction in JAK2 and c-Src activity. TQ also downregulated the expression of STAT3-regulated genes, such as Bcl-2, cyclin D, survivin, and vascular endothelial growth factor, and activated caspase-3,7,9. Consistent with the in vitro results, TQ was significantly effective as an antitumor agent in a xenograft tumor mouse model. CONCLUSION: This study provides strong evidence that downregulation of the STAT3 signaling pathway mediates TQ-induced apoptosis in gastric cancer.

Kinetic models and pathways of ronidazole degradation by chlorination, UV irradiation and UV/chlorine processes.

Degradation kinetics and pathways of ronidazole (RNZ) by chlorination (Cl2), UV irradiation and combined UV/chlorine processes were investigated in this paper. The degradation kinetics of RNZ chlorination followed a second-order behavior with the rate constants calculated as (2.13 ± 0.15) × 10(2) M(-2) s(-1), (0.82 ± 0.52) × 10(-2) M(-1) s(-1) and (2.06 ± 0.09) × 10(-1) M(-1) s(-1) for the acid-catalyzed reaction, as well as the reactions of RNZ with HOCl and OCl(-), respectively. Although UV irradiation degraded RNZ more effectively than chlorination did, very low quantum yield of RNZ at 254 nm was obtained as 1.02 × 10(-3) mol E(-1). RNZ could be efficiently degraded and mineralized in the UV/chlorine process due to the generation of hydroxyl radicals. The second-order rate constant between RNZ and hydroxyl radical was determined as (2.92 ± 0.05) × 10(9) M(-1) s(-1). The degradation intermediates of RNZ during the three processes were identified with Ultra Performance Liquid Chromatography - Electrospray Ionization - mass spectrometry and the degradation pathways were then proposed. Moreover, the variation of chloropicrin (TCNM) and chloroform (CF) formation after the three processes were further evaluated. Enhanced formation of CF and TCNM precursors during UV/chlorine process deserves extensive attention in drinking water treatment.

[Osteoclasts and early bone remodeling after orthodontic micro-implant placement].

PURPOSE: To observe the incidence of osteoclasts during early bone remodeling after orthodontic micro-implant placement. METHODS: Twenty New Zealand rabbits were randomly allotted into 4 groups. One micro-implant was implanted proximal to the epiphyseal plate of the tibia. Animals were sacrificed on day 3, 7, 14 and 28 (n=5). The sequence of histological changes around the micro-implants were evaluated by hematoxylin and eosin (HE) staining. Osteoclasts were identified by TRAP staining. The differences of the number of the osteoclasts among each time point were analyzed by one way ANOVA with SPSS 19.0 software package. RESULTS: After 3 days of implantation, a large number of erythrocytes, inflammatory cells, mesenchymal cells and bone debris were seen at the implant bone interfaces. Few osteoclasts were observed. On day 7, granular woven bone was formed and some osteoclasts were found in the Howship's lacunae. New bone formation and mineralization were apparent on day 14. Meanwhile, large amounts of osteoclasts were found in the latticed woven bone. On day 28, woven trabeculae with lamellate structures connected to lamellar bone and fewer osteoclasts were identified. Semi-quantitative analysis showed that the number of the osteoclasts was at peak on day 14. There were significant differences among each time point (P<0.01). CONCLUSIONS: Osteoclast activity is closely related to bone formation and remodeling after micro-implant insertion.