Melatonin has been reported to protect neurons from a variety of neurotoxicity. However, the underlying mechanism by which melatonin exerts its neuroprotective property has not yet been clearly understood. We previously demonstrated that melatonin protected kainic acid-induced neuronal cell death in mouse hippocampus, accompanied by sustained activation of Akt, a critical mediator of neuronal survival. To further elucidate the neuroprotective action of melatonin, we examined in the present study the causal mechanism how Akt signaling pathway is regulated by melatonin in a rat primary astrocyte culture model. Melatonin resulted in increased astrocytic Akt phosphorylation, which was significantly decreased with wortmannin, a specific inhibitor of PI3K, suggesting that activation of Akt by melatonin is mediated through the PI3K-Akt signaling pathway. Furthermore, increased Akt activation was also significantly decreased with luzindole, a non-selective melatonin receptor antagonist. As downstream signaling pathway of Akt activation, increased levels of CREB phoshorylation and GDNF expression were observed, which were also attenuated with wortmannin and luzindole. These results strongly suggest that melatonin exerts its neuroprotective property in astrocytes through the activation of plasma membrane receptors and then PI3K-Akt signaling pathway.
Androstadienes ; Animals ; Astrocytes ; Cell Death ; Cell Membrane ; Glial Cell Line-Derived Neurotrophic Factor ; Hippocampus ; Melatonin ; Mice ; Neurons ; Phosphorylation ; Rats ; Receptors, Melatonin ; Tryptamines

OBJECTIVE: To study the expression levels of glial cell line-derived neurotrophic factor family receptor α-1 (GFRα1) and enhancer of zeste homolog 2 (EZH2) in the intestinal tissue of children with Hirschsprung's disease (HSCR), as well as the role of EZH2 in the regulation of GFRα1 gene expression and the pathogenesis of HSCR. METHODS: The samples of colon tissue with spasm from 24 children with HSCR after radical treatment of HSCR were selected as the experimental group, and the samples of necrotized colon tissue from 18 children with neonatal necrotizing enterocolitis after surgical resection were selected as the control group. Real-time PCR and Western blot were used to measure the expression levels of GFRα1 and EZH2 in colon tissue in both groups. Human neuroblastoma SH-SY5Y cells were divided into an EZH2 over-expression group and a negative control group. The cells in the EZH2 over-expression group were transfected with pCMV6-EZH2 plasmid, and those in the negative control group were transfected with pCMV6 plasmid. The expression levels of EZH2 and GFRα1 were measured after transfection. RESULTS: Compared with the control group, the experimental group had significant reductions in the mRNA and protein expression levels of GFRα1 and EZH2 in colon tissue (P<0.05), and the protein expression of EZH2 was positively correlated with that of GFRα1 (r=0.606, P=0.002). Compared with the negative control group, the EZH2 over-expression group had significant increases in the expression levels of EZH2 and GFRα1 after SH-SY5Y cells were transfected with EZH2 over-expression plasmid (P<0.05). CONCLUSIONS Low expression of EZH2 in the colon tissue of children with HSCR may be one of the causes of inadequate expression of GFRα1 and onset of HSCR.
Child ; Colon ; Enhancer of Zeste Homolog 2 Protein ; genetics ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; genetics ; Hirschsprung Disease ; genetics ; Humans ; Infant, Newborn ; RNA, Messenger

OBJECTIVETo establish a simple and highly effective isolation and culture system of mouse spermatogonial stem cells(SSCs)and detect the expression of stem cell-related markers in the isolated cells.

METHODSThe structures of seminiferous tubules of neonatal(6-8 days of age)and adult(26-28 weeks)DBA/2 mice were compared using histochemical examination. Testes of neonatal mice were selected for preparing primary cells. The digestive efficiency of different enzymes was compared. SSCs were isolated according to the different binding abilities of testicle somatic cells and SSCs to gelatin matrix. The effects of different base culture media such as StemPro34 and α-MEM,gelatin,and serum on the SSCs binding activity and growth were studied. The cell morphology was observed during the culture process. Immunofluorescence was used to detect the expression of SSCs and cancer stem cells(CSCs)-related markers in SSCs.

RESULTSThe content of SSCs in the testes of neonatal mice was relatively higher than that in adult mice. Trypsin showed the highest digestive efficiency. In StemPro34 supplemented with 1% fetal bovine serum and on the gelatin matrix,testicular somatic cells could bind with the plate efficiently. Spermatogonial cells grew well when using mitomycin C-treated testicular somatic cells as feeder cells and showed typical characteristic of SSCs. After 13 days of culture,spermatogonial cells formed cell clusters. Immunofluorescence assay showed that SSCs markers glial cell line-derived neurotrophic factor(GDNF)family receptor α1(GFRα1)and VASA protein were highly expressed in the cell clusters. CSCs marker CD44 was expressed in the As,Apr,Aal and the inner cells of the cell clusters,while seldom expressed in the somatic cells.

CONCLUSIONSAn isolation and culture system of SSCs derived from DBA/2 mice was established. CD44 is highly expressed in the early stage of spermatogonial cell development.

Animals ; Biomarkers ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Glial Cell Line-Derived Neurotrophic Factor ; metabolism ; Hyaluronan Receptors ; metabolism ; Male ; Mice ; Mice, Inbred DBA ; Spermatogonia ; cytology ; Stem Cells ; cytology

OBJECTIVETo investigate the association of artemin and GFRalpha3 expressions with perineural invasion and metastasis of pancreatic carcinoma.

METHODSSemi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the expression of artemin and GFRalpha3 in pancreatic carcinoma tissues, adjacent tissues and normal pancreas tissues, and the relevance of artemin and GFRalpha3 expressions to the perineural invasion and metastasis of pancreatic carcinoma were analyzed.

RESULTSThe positivity rates of artemin and GFRalpha3 expressions were 72.09% and 67.44% in pancreatic carcinoma, respectively, significantly higher than those in the adjacent tissue (18.19% and 22.73%). The positivity rates of artemin and GFRalpha3 expressions were significantly higher in patients with perineural invasion than in those without perineural invasion (chi(2)=11.11 and 11.78, respectively, P<0.01). Significantly higher expression of artemin mRNA was noted in pancreatic carcinoma (0.741-/+0.014) than in the normal pancreas tissue (0.101-/+0.031, P<0.05), and patients with perineural invasion showed significantly higher positivity rates of artemin mRNA expression (0.843-/+0.012) than those without perineural invasion (0.512-/+0.017, P<0.05).

CONCLUSIONArtemin and GFRalpha3 expressions may play an important role in perineural invasion of pancreatic carcinoma and can be used a useful indicators for evaluating the biological behavior of pancreatic carcinomas.

Adult ; Aged ; Carcinoma, Pancreatic Ductal ; metabolism ; pathology ; Female ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; genetics ; metabolism ; Humans ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Nerve Tissue Proteins ; genetics ; metabolism ; physiology ; Neurons ; pathology ; Pancreatic Neoplasms ; metabolism ; pathology

OBJECTIVETo discuss the expression and significance of glial cell-derived neurotrophic factor (GDFN) receptor alpha 1 gene (GFR alpha 1) in the recovery spermatogenesis of mice.

METHODSAdult Kunming mice were injected intraperitoneally with 2 doses of busulfan (10 mg/kg) 24 days apart so as to establish the recovery spermatogenesis model. Testes were harvested 1 w, 2 w, 3 w, 4 w, 6 w, 8 w and 10 w after the second injection, and normal testes were used as control. The recovery spermatogenesis was observed by light and electron microscopy, and the GFR alpha 1 mRNA was measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization.

RESULTSThe expression of GFR alpha 1 mRNA increased significantly at 1 w and reached its peak at 2 w after the second injection [(104.72 +/- 24.4)% vs normal control, P < 0.01]; its expression reduced significantly at 3 w and reached its valley at 4 w [(20.77 +/- 4.25)% vs normal control, P < 0.01], and then increased gradually and restored to the normal level at 10 w. GFR alpha 1 mRNA was mainly expressed by undifferentiated spermatogonia.

CONCLUSIONSIn the course of recovery spermatogenesis, the expression of GFR alpha 1 plays a key role in turning the spermatogonial stem cell reactivity to GDNF, which promotes self-renewal at a high level, or results in differentiation at a low level.

Animals ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; In Situ Hybridization ; Male ; Mice ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-ret ; RNA, Messenger ; analysis ; Receptor Protein-Tyrosine Kinases ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatogenesis ; Testis ; pathology

OBJECTIVETo establish the testis transplantation model in rats and to study the mechanism of graft injury.

METHODSThe testis orthotopic transplantation model was established using three-cuff method. The animals were divided into 6 groups. Serum levels of testosterone (T), luteining hormone (LH) and follicle-stimulating hormone (FSH) were determined by radioimmunoassay (RIA). Morphology and ultrastructure were examined by light and electron microscopy. Expression of Glial cell line-derived neurotrophic factor (GDNF) mRNA was studied by reverse-transcription polymerase chain reaction (RT-PCR) technique.

RESULTOn the 7th day postoperatively, the allotransplanted testes showed perivascular massive infiltration of lymphocytes and polymorphonuclear neutrophil (PMN) and reduced number of the sertoli cells under light microscopy. It also showed the broken blood-testis barrier, the atrophy of the sertoli cells and spermatogenic cells arranged in disorder under electron microscopy. The decline of serum T level and the increase of serum LH and FSH levels were similar to those found in bilateral castrates. The levels of GDNFmRNA expression were lower than those in normal controls. On 14th day postoperatively, the spermatogenesis of allotransplanted testes was still not recovered and the expression of GDNFmRNA declined further.

CONCLUSIONThe atrophy and reduced number of the sertoli cells and the breakage of the close connection probably are the main causes of dysfunction of spermatogenesis. The decline of GDNFmRNA expression is in accordance with the dysfunction of the sertoli cells and the spermatogenesis.

Animals ; Follicle Stimulating Hormone ; blood ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; biosynthesis ; genetics ; Luteinizing Hormone ; blood ; Male ; Models, Animal ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Inbred Lew ; Rats, Wistar ; Sertoli Cells ; ultrastructure ; Spermatogenesis ; physiology ; Testis ; transplantation ; ultrastructure ; Testosterone ; blood

OBJECTIVEThe present study aimed to explore the role of P2Y(1) receptor in glial fibrillary acidic protein (GFAP) production and glial cell line-derived neurotrophic factor (GDNF) secretion of astrocytes under ischemic insult and the related signaling pathways.

METHODSUsing transient right middle cerebral artery occlusion (tMCAO) and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, enzyme linked immunosorbent assay (ELISA) were used to investigate location of P2Y(1) receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules.

RESULTSBlockage of P2Y(1) receptor with the selective antagonist N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate diammonium (MRS2179) reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y(1) receptor caused elevation of phosphorylated Akt and cAMP response element binding protein (CREB), and reduction of phosphorylated Janus kinase2 (JAK2) and signal transducer and activator of transcription3 (STAT3, Ser727). After blockage of P2Y(1) receptor and deprivation of oxygen-glucose-serum, AG490 (inhibitor of JAK2) reduced phosphorylation of STAT3 (Ser727) as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase1/2 (MEK1/2) U0126, an important molecule of Ras/extracellular signal-regulated kinase (ERK) signaling pathway, decreased the phosphorylation of JAK2, STAT3 (Ser727), Akt and CREB.

CONCLUSIONThese results suggest that P2Y(1) receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.

Animals ; Astrocytes ; metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Glial Cell Line-Derived Neurotrophic Factor ; biosynthesis ; Glial Fibrillary Acidic Protein ; biosynthesis ; Infarction, Middle Cerebral Artery ; metabolism ; RNA, Messenger ; analysis ; Rats ; Receptors, Purinergic P2 ; metabolism ; Receptors, Purinergic P2Y1 ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; physiology

OBJECTIVETo isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application.

METHODSWe detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro.

RESULTSThe isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity.

CONCLUSIONCD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.

Adult Stem Cells ; cytology ; Biomarkers ; metabolism ; Cell Separation ; methods ; Cell Shape ; Cell Size ; Coculture Techniques ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; metabolism ; Humans ; Immunohistochemistry ; Male ; Receptors, G-Protein-Coupled ; metabolism ; Sertoli Cells ; Spermatogonia ; cytology ; Testis ; metabolism ; Thy-1 Antigens ; isolation & purification ; metabolism ; Ubiquitin Thiolesterase ; metabolism

While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately identify subset-specific SSC marker distributions due to the poor nuclear morphological characteristics associated with fixation in 4% paraformaldehyde. In the present study, testicular cross-sections and whole-mount samples were Bouin fixed to optimize nuclear resolution and visualized by immunohistochemistry (IHC) and immunofluorescence (IF). The results identified an expression pattern of PLZF^highc-KIT^pos in A₁ spermatogonia, while A₂-A₄-differentiating spermatogonia were PLZF^lowc-KIT^pos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric A_pr and false clones were distinguished based on the presence or absence of TEX14, a molecular maker of intercellular bridges, despite having identical nuclear morphology and intercellular distances that were <25 μm. In conclusion, this optimized Bouin fixation procedure facilitates the accurate identification of spermatogonium subsets based on their molecular profiles and is capable of distinguishing asymmetric and false clones. Therefore, the findings presented herein will facilitate further morphological and functional analysis studies and provide further insight into spermatogonium subtypes.
Animals ; Cell Differentiation ; Fluorescent Antibody Technique ; Gene Expression Regulation/genetics* ; Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics* ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred C57BL ; Promyelocytic Leukemia Zinc Finger Protein/genetics* ; Proto-Oncogene Proteins c-kit/genetics* ; Seminiferous Tubules/cytology* ; Spermatogenesis ; Spermatogonia/metabolism* ; Testis/cytology* ; Tissue Fixation ; Transcription Factors/genetics*