Effect of enhancer of zeste homolog 2 on the expression of glial cell line-derived neurotrophic factor family receptor α-1 in the colon tissue of children with Hirschsprung's disease / 中国当代儿科杂志

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.

PLZFposc-KITpos-delineated A1-A4-differentiating spermatogonia by subset and stage detection upon Bouin fixation / 亚洲男科学杂志(英文版)

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 PLZFhighc-KITpos in A1 spermatogonia, while A2-A4-differentiating spermatogonia were PLZFlowc-KITpos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric Apr 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.

Isolation, culture, and identification of human spermatogonial stem cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application.</p><p><b>METHODS</b>We 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>CD90 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.</p>

Artemin and GFRalpha3 expressions and their relevance to perineural invasiveness and metastasis of pancreatic carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the association of artemin and GFRalpha3 expressions with perineural invasion and metastasis of pancreatic carcinoma.</p><p><b>METHODS</b>Semi-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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>Artemin 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.</p>

Establishment of testis transplantation model and study on mechanism of graft injury in rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish the testis transplantation model in rats and to study the mechanism of graft injury.</p><p><b>METHODS</b>The 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.</p><p><b>RESULT</b>On 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.</p><p><b>CONCLUSION</b>The 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.</p>

Expression and significance of GFR alpha 1 gene in the recovery spermatogenesis of mice / 中华男科学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Adult 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>In 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.</p>