Mouse spermatogenesis entails the maintenance and self-renewal of spermatogonial stem cells (SSCs), which require a complex web-like signaling network transduced by various cytokines. Although brain-derived neurotrophic factor (BDNF) is expressed in Sertoli cells in the testis, and its receptor tropomyosin receptor kinase B (TrkB) is expressed in the spermatogonial population containing SSCs, potential functions of BDNF for spermatogenesis have not been uncovered. Here, we generate BDNF conditional knockout mice and find that BDNF is dispensable for in vivo spermatogenesis and fertility. However, in vitro , we reveal that BDNF -deficient germline stem cells (GSCs) exhibit growth potential not only in the absence of glial cell line-derived neurotrophic factor (GDNF), a master regulator for GSC proliferation, but also in the absence of other factors, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin. GSCs grown without these factors are prone to differentiation, yet they maintain expression of promyelocytic leukemia zinc finger ( Plzf ), an undifferentiated spermatogonial marker. Inhibition of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and Src pathways all interfere with the growth of BDNF-deficient GSCs. Thus, our findings suggest a role for BDNF in maintaining the undifferentiated state of spermatogonia, particularly in situations where there is a shortage of growth factors.
Animals ; Male ; Brain-Derived Neurotrophic Factor/genetics* ; Spermatogonia/cytology* ; Mice ; Spermatogenesis/genetics* ; Mice, Knockout ; Cell Differentiation ; Glial Cell Line-Derived Neurotrophic Factor/genetics* ; Promyelocytic Leukemia Zinc Finger Protein/genetics* ; Cell Survival/physiology* ; Signal Transduction/physiology* ; Cell Proliferation/physiology*

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

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*

OBJECTIVETo explore the neuroprotective effects of electroacupuncture (EA) on hypoxic-ischemic encephalopathy (HIE) and to further investigate the role of glial cell line-derived neurotrophic factor (GDNF) family receptor member RET (rearranged during transfection) and its key downstream phosphatidylinositol 3 kinase (PI-3K)/protein kinase B (Akt) pathway in the process.

METHODSA total of 220 seven-day-old SD rats (of either sex, from 22 broods) were randomly divided into two groups, one (30 rats) for sham-surgery group and the other (190 rats) for HIE model group. The HIE model was established using the left common carotid artery ligation method in combination with hypoxic treatment. The successfully established rats were randomly divided into five groups, including control model group, EA group, sham-EA group, antagonist group and antagonist plus electroacupuncture group, with 35 rats in each group. Baihui (GV 20), Dazhui (GV 14), Quchi (LI 11) and Yongquan (KI 1) acupoints were chosen for acupuncture. EA was performed at Baihui and Quchi for 10 min once a day for continuous 1, 3, 7 and 21 days, respectively. The rats were then killed after the operation and injured cerebral cortex was taken for the measurement of neurologic damage by hematoxylin-eosin (HE) staining and the degenerative changes of cortical ultrastructure by transmission electron microscopy. RET mRNA level and Akt protein level were detected by real-time reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively.

RESULTSEA could ameliorate neurologic damage of the first somatic sensory area (S1Tr) and alleviate the degenerative changes of ultrastructure of cortical neurons in rats subjected to HIE. And the longer acupuncture treatment lasted, the better its therapeutic effect would be. This was accompanied by gradually increased expression of GDNF family receptor RET at the mRNA level and its downstream signaling Akt at the protein level in the ischemic cortex.

CONCLUSIONEA has neuroprotective effects on HIE and could be a potential therapeutic strategy for HIE in the neonate. Activation of RET/Akt signaling pathway might be involved in this process.

Animals ; Blotting, Western ; Cerebral Cortex ; pathology ; ultrastructure ; Electroacupuncture ; Female ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Hypoxia-Ischemia, Brain ; genetics ; pathology ; therapy ; Male ; Nerve Degeneration ; pathology ; Neurons ; pathology ; ultrastructure ; Neuroprotective Agents ; therapeutic use ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-ret ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

OBJECTIVETo evaluate the therapeutic effect of transplantation of mesencephalic neural stem cells (mNSCs) genetically modified by glial cell line-derived neurotrophic factor (GDNF) gene in a rat model of Parkinson disease.

METHODSmNSCs isolated from the lateral component of the midbrain of fetal rats at gestational age of 14 or 15 days were cultured for 5 days before genetic modification with GFP or GDNF gene. Rat models of Parkinson disease established by stereotactic injection of 6-hydroxy dopamine in the ventral area of the midbrain and the medial forebrain bundle were randomized into 3 groups to receive PBS injection, GFP gene-modified mNSCs transplantation, or GDNF gene-modified mNSCs transplantation into the right stratum. The behavioral changes of the rats were evaluated by observing rotations induced by intraperitoneal injection of apomorphine after the transplantation, and the survival, migration and differentiation of the transplanted cells were identified by immunohistochemistry.

RESULTSTransplantation with GDNF gene-modified mNSCs significantly improved the behavioral abnormalities of the rat models as compared with PBS injection and GFP gene-modified mNSCs transplantation. At 56 days after the transplantation, a greater number of the transplanted cells survived in the rat brain and more differentiated dopaminergic neurons were detected in GDNF gene-modified mNSCs transplantation group than in GFP gene-modified mNSCs transplantation group.

CONCLUSIONGDNF gene-modified mNSCs transplantation can significantly improve dyskinesia in rat models of Parkinson disease, but the molecular mechanism needs further clarification.

Animals ; Disease Models, Animal ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; therapeutic use ; Mesencephalon ; cytology ; Neural Stem Cells ; transplantation ; Parkinson Disease ; therapy ; Rats ; Stem Cell Transplantation

OBJECTIVETo observe the effects of adipose-derived mesenchymal stem cells (ADSCs) with continous over-expression of glial cell line-derived neurotrophic factor (GDNF) on the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

METHODSFive SD rats were collected to prepare ADSCs with over-expression of GDNF. One hundred and fifty SD rats were divided into normal control group (N), GDNF-ADSCs group (GA), ADSCs group (A), GDNF group (G), and physiological saline group (P) according to the random number table, with 30 rats in each group. Rats in group N were routinely fed without treatment, and rats in the other 4 groups were inflicted with electrical injury on sciatic nerve of thigh of the right hind leg. Rats in groups GA, A, G, and P were respectively injected with 100 µL suspension of ADSCs with over-expression of GDNF (1 x 10(7) cells per mL), 100 [µL ADSCs suspension (1 x 10(7) cells per mL), 100 µL GDNF solution (100 mg/L) , and 100 µL physiological saline to the surface of the injured nerves immediately after injury. Six rats of each group were collected for measuring hind limb stride from post injury week (PIW) 1 to 8, and morphology of the sciatic nerves was observed in PIW 8. In PIW 4, the protein expression of GDNF of sciatic nerves of the rest rats in each group was determined with Western blotting. Data were processed with one-way analysis of variance, analysis of variance of repeated measurement, and SNK test.

RESULTSCompared with that of group N, the hind limb stride values in groups GA, A, G, and P were significantly lower at each time point (with P values below 0.05). Compared with those of group P, the hind limb stride values in group GA from PIW 3 to 8, in group A in PIW 3, 5, and 7, and in group G in PIW 3, 5, 7, and 8 were significantly longer (with P values below 0.05). The hind limb stride values in group GA from PIW 4 to 8 were respectively (10.83 ± 0.97), (13.25 ± 1.40), (12.86 ± 1.42), (14.06 ± 1.50), and (15.09 ± 1.17) cm, which were significantly longer than those in group A [(8.90 ± 0.82), (9.03 ± 0.57), (9.27 ± 0.36), (9.86 ± 0.36), and (9.52 ± 0.58) cm] and group G [(8.87 ± 0.69), (8.51 ± 1.18), (9.34 ± 0.87), (9.76 ± 0.67), and (9.50 ± 1.22) cm], with P values below 0.05. Compared with that of group N, the number of myelinated nerve fibers of sciatic nerves was obviously decreased in group P but obviously increased in groups GA, A, and G; the diameter of axons was obviously shorter, and the myelin thickness was obviously increased in groups GA, A, G, and P in PIW 8 (with P values below 0.05). The number of myelinated nerve fibers in group GA was 31.2 ± 0.8, which was significantly higher than that in group A (23.7 ± 2.7), group G (22.3 ± 2.7), or group P (9.3 ± 2.8), with P values below 0.05. The diameter values of axons among groups P, A, G, and GA were similar (with P values above 0.05). The myelin thickness of rats in group GA was (3.41 ± 0.34) µm, which was significantly thicker than that in group A [(2.64 ± 0.37) µm] or group G [(2.41 ± 0.34) µm], with P values below 0.05. In PIW 4, the protein expression of GDNF of sciatic nerves was significantly higher in groups P, A, G, and GA than in group N (with P values below 0.05), and the protein expression of GDNF in group GA was significantly higher than that in group P, A, or G (with P values below 0.05).

CONCLUSIONSADSCs over-expressing GDNF protein can obviously promote the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

Adipose Tissue ; Animals ; Electrophysiology ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; metabolism ; Nerve Crush ; Nerve Regeneration ; physiology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; pathology ; physiology

OBJECTIVETo investigate the mechanism of high transcription of the glial cell-line derived neurotrophic factor (gdnf) gene induced by hyperacetylation of histone H3 lysine 9 (H3K9) at its promoter region II in rat C6 glioma cells.

METHODSThe acetylation level of H3K9 at Egr-1 binding site in gdnf gene promoter region II and the binding capacity of Egr-1 to its binding site in gdnf promoter were examined by ChIP-PCR in C6 astroglioma cells and normal rat astrocytes, and its changes were investigated in C6 astroglioma cells after treatment with histone acetyltransferase inhibitor curcumin or deacetylase inhibitor trichostatin A.

RESULTSCompared normal astrocytes, C6 astroglioma cells showed significantly increased acetylation level of H3K9 at Egr-1 binding site in gdnf gene promoter region II and Egr-1 binding capacity (P<0.01). Curcumin treatment significantly reduced H3K9 acetylation level at Egr-1 binding site and decreased both the binding of Egr-1 to promoter region II and gdnf mRNA levels in C6 astroglioma cells (P<0.05). Conversely, increased H3K9 acetylation at the Egr-1 binding site induced by trichostatin A significantly increased the binding of Egr-1 to promoter region II and gdnf mRNA expression levels (P<0.05).

CONCLUSIONH3K9 hyperacetylation induces increased Egr-1 binding to gdnf gene promoter II, which might be the reason for the high transcription level of gdnf gene in rat C6 glioma cells.

Acetylation ; Animals ; Astrocytes ; metabolism ; Binding Sites ; Cell Line, Tumor ; Early Growth Response Protein 1 ; metabolism ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; Glioma ; metabolism ; Histones ; chemistry ; Promoter Regions, Genetic ; Protein Processing, Post-Translational ; RNA, Messenger ; Rats ; Transcription, Genetic

OBJECTIVETo observe the effect of Draconis Sanguis-containing serum on the expressions of NGF, BDNF, CNTF, LNG-FR, TrkA, GDNF, GAP-43 and NF-H in Schwann cells, and investigate the possible mechanism of Draconis Sanguis to promote peripheral nerve regeneration.

METHODSD rats were randomly divided into 2 groups: the Draconis Sanguis group (orally administered with Draconis Sanguis-containing balm solution) and the blank group (equivoluminal balm) to prepare Draconis Sanguis-containing serum and blank control serum. Schwann cells were extracted from double sciatic nerves of three-day-old SD rats, divided into 2 groups: the Draconis Sanguis group and the blank control group, and respectively cultured with 10% Draconis Sanguis-containing serum or blank control serum. The mRNA expressions of NGF, BDNF, CNTF and other genes in Schwann cells were measured by RT-PCR analysis 48 hours later.

RESULTMost of the Schwann cells were bipolar spindle and arranged shoulder to shoulder or end to end under the microscope and identified to be positive with the immunocytochemical method. To compare with the blank group, mRNA expressions of NGF, LNGFR, GDNF and GAP-43 significantly increased (P < 0.01). Whereas that of BDNF decreased significantly (P < 0.05), and so did that of TrkA, CNTF (P < 0.01), with no remarkable difference in NF-H-mRNA.

CONCLUSIONTraditional Chinese medicine Draconis Sanguis may show effect in nerve regeneration by up-regulating mRNA expressions of NGF, LNGFR, GDNF and GAP-43 and down-regulating mRNA expressions of TrkA, BDNF and CNTF.

Animals ; Arecaceae ; chemistry ; Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; Cells, Cultured ; Ciliary Neurotrophic Factor ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; GAP-43 Protein ; genetics ; metabolism ; Gene Expression ; drug effects ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Male ; Nerve Growth Factor ; genetics ; metabolism ; Nerve Regeneration ; drug effects ; Neurofilament Proteins ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, trkA ; genetics ; metabolism ; Schwann Cells ; drug effects ; physiology ; Serum ; chemistry

BACKGROUNDSpinal cord injury (SCI) is a serious neurological injury that often leads to permanent disabilities for the victims. The aim of this study was to determine the effects of glial-derived neurotrophic factor (GDNF) mediated by recombinant adeno-associated virus type 2 (rAAV2) alone or in combination with early rehabilitation training on SCI.

METHODSSCI was induced on the T8-9 segments of the spinal cord by laminectomy in adult male Sprague-Dawley rats. Then besides the sham operation group, the SCI rats were randomly divided into four groups: natural healing group, gene therapy group, rehabilitation training group, and combination therapy group (gene therapy in combination with rehabilitation training). Motor dysfunction, protein expression of GDNF, edema formation, and cell injury were examined 7, 14, and 21 days after trauma.

RESULTSThe topical application of rAAV-GDNF-GFP resulted in strong expression of GDNF, especially after the 14th day, and could protect the motor neuron cells. Early rehabilitative treatment resulted in significantly improved motor function, reduced edema formation, and protected the cells from injury, especially after the 7th and 14th days, and increased the GDNF expression in the damaged area, which was most evident after Day 14. The combined application of GDNF and early rehabilitative treatment after SCI resulted in a significant reduction in spinal cord pathology and motor dysfunction after the 7th and 14th days.

CONCLUSIONThese observations suggest that rAAV2 gene therapy in combination with rehabilitation therapy has potential clinical value for the treatment of SCI.

Animals ; Cell Line ; Dependovirus ; genetics ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; physiology ; Humans ; Immunohistochemistry ; Male ; Motor Activity ; genetics ; physiology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism

Vectors used to carry foreign genes play an important role in gene therapy, among which, the adeno-associated virus (AAV) has many advantages, such as nonpathogenicity, low immunogenicity, stable and long-term expression and multiple-tissue-type infection, etc. These advantages have made AAV one of the most potential vectors in gene therapy, and widely used in many clinical researches, for example, Parkinson's disease. This paper introduces the biological characteristics of AAV and the latest research progress of AAV carrying neurotrophic factor, dopamine synthesis related enzymes and glutamic acid decarboxylase gene in the gene therapy of Parkinson's disease.
Animals ; Aromatic-L-Amino-Acid Decarboxylases ; genetics ; Dependovirus ; genetics ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; Glutamate Decarboxylase ; genetics ; Humans ; Nerve Growth Factors ; genetics ; Neurturin ; genetics ; Parkinson Disease ; therapy