|Year : 2014 | Volume
| Issue : 1 | Page : 38-43
Mechanism of glial cell line-derived neurotrophic factor in proliferation of spermatogonial stem cells
Jianxin Hu1, Shujie Xia2, Dalong Song3, Jun Liu3, Zhaolin Sun3
1 Shanghai First People's Hospital, Shanghai Jiao Tong University, Institute of Urology, Post-Doctoral Mobile Stations, Shanghai; Department of Urology, Guizhou Province People's Hospital, Guiyang 550002, Guizhou Province, China
2 Shanghai First People's Hospital, Shanghai Jiao Tong University, Institute of Urology, Post-Doctoral Mobile Stations, Shanghai, China
3 Department of Urology, Guizhou Province People's Hospital, Guiyang 550002, Guizhou Province, China
|Date of Web Publication||25-Jul-2014|
Department of Urology, Guizhou Province People's Hospital, Guiyang 550002
Source of Support: This study was supported by Science and Technology Fund Project, Guizhou, China (Guizhou Branch SY Zi  No. 3045), Conflict of Interest: None
Objectives: The aim was to investigate the mechanism of glial cell line-derived neurotrophic factor (GDNF) in the proliferation of spermatogonial stem cells (SSCs) through detecting the expression of GDNF, receptor tyrosine kinases (RTKs), fyn and focal adhesion kinase (FAK) messenger ribonucleic acids (mRNAs) after gene interference. Materials and Methods: Multiple small interfering RNAs (siRNA) of GDNF were designed and constructed to transfect SSCs. The siRNA of GDNF with the highest efficiency was applied and expressions of GDNF, RTKs, fyn and FAK mRNA and the protein were measured with reverse transcription polymerase chain reaction and western blot, respectively. The proliferation and apoptosis of SSCs were measured with enzyme-labeled meter and flow cytometry, respectively. Results: The expression intensity of GDNF mRNA in SSCs of the experimental group and control group was 12.32 ± 1.22% and 54.25 ± 1.34%, respectively; the apoptosis rate was 25.43 ± 1.91% and 5.61 ± 0.16%, respectively; and there was a significant difference between the two groups (P < 0.01). The expression intensity of RTKs, fyn and FAK in the experimental group was 16.24 ± 1.35%, 18.32 ± 1.34%, and 20.04 ± 1.65%, respectively; the expression intensity of RTKs, fyn and FAK in the control group was 45.35 ± 1.37%, 38.37 ± 1.55% and 43.27 ± 1.28%, respectively; there was a significant difference between the two groups (P < 0.01). Conclusion: These results suggest that the constructed positive vector effectively inhibits the expression of GDNF, RTKs, fyn and FAK mRNAs, and the proliferation of SSCs. GDNF plays an important role in the proliferation of SSCs.
Keywords: Glial cell line-derived neurotrophic factor, ribonucleic acid, small interfering ribonucleic acids, spermatogonial stem cells
|How to cite this article:|
Hu J, Xia S, Song D, Liu J, Sun Z. Mechanism of glial cell line-derived neurotrophic factor in proliferation of spermatogonial stem cells. J Integr Nephrol Androl 2014;1:38-43
|How to cite this URL:|
Hu J, Xia S, Song D, Liu J, Sun Z. Mechanism of glial cell line-derived neurotrophic factor in proliferation of spermatogonial stem cells. J Integr Nephrol Androl [serial online] 2014 [cited 2023 Jan 29];1:38-43. Available from: http://www.journal-ina.com/text.asp?2014/1/1/38/137553
| Introduction|| |
In modern society, the physical and mental health of people is affected by complicated factors such as infertility. More and more males are suffering from the disorder, accounting for 13%~15% of the married men. Regarding the therapy, there is no effective method for azoospermia. Sperms are derived from spermatogonial stem cells (SSCs) which are capable of continuous self-renewal and differentiation. To cultivate sperms by culturing and inducing SSCs to proliferate and differentiate in vitro is the most hopeful therapeutic approach and one hotspot of studies. Our previous study and Spinnler et al. indicated that glial cell line-derived neurotrophic factor (GDNF) increased the proliferation and differentiation of cultured SSCs in vitro, but the mechanism is unclear. ,
Our previous study indicated that GDNF could promote the spermatogenesis.  In order to further investigate its mechanism, the present study designed and synthesized the expression vector of small interfering ribonucleic acid (siRNA) against GDNF gene according to the features of RNA interference (RNAi) in effectively inhibiting the expression of target genes and promoting the proliferation and differentiation of SSCs. Using this vector, we studied the growth and proliferation changes of SSCs after interfering GDNF gene to understand the mechanism of GDNF in spermatogenesis.
| Materials and methods|| |
Healthy male Kunming mice (5-7 days and 12-15 days, n = 40 for each age group, specific pathogen-free grade) were purchased from the experimental animal center of Guiyang Medical College. Other reagents included TRI REAGENT; RNA extraction kit (Molecular Research Center), DEPC (Sigma), chloroform, anhydrous alcohol and isopropanol (Sinopharm Chemical Reagent Co. Limited, Shanghai, China), 10 mmol/L deoxynucleotide mix (Invitrogen), 25 mmol/L MgCl 2 (Invitrogen), 100 bpDNA (Invitrogen), plasmid extraction kit (QIAGEN), reverse transcription polymerase chain reaction (RT-PCR) kit (Invitrogen), agarose (Biowest, Spain), Taq polymerase (TAKARA), T4-DNA ligase (New England), PCR gel extraction kit (QIAGEN), restriction endonuclease (New England), PCR primers (Sangon Biothech Biotech Co Limited, Shanghai, China), ethidium bromide (EB) (Sigma), λDNA/Hind III DNA Marker (Promega), Lipofectamine™ 2000 (Invitrogen), PLKO.1 TRC (Clone TECH), high-glucose Dulbecco's Modified Eagle Medium (GIBCO), fetal bovine serum (FBS) (GIBCO), trypsin (GIBCO), phosphate buffered saline (PBS) (Hycone), horseradish peroxidase (HRP) labeled goat antimouse secondary antibody (Santa Cruz), HRP labeled goat antirabbit secondary antibody (Santa Cruz), GDNF polyclonal antibody, Basement Membrane Matrix BD Pharmingen), and enhanced chemiluminescence (ECL) luminescence kit (Santa Cruz).
Construction and extraction of recombinant plasmid vector
Glial cell line-derived neurotrophic factor messenger RNA (mRNA) was searched in Gene-Bank and designed according to the principle of siRNA. Lentivirus plasmid with GDNF knockdown was constructed with the lentivirus vector of PLKO.1. Sequences of GDNF knockdown was: GGGAC TCTA AGA TGAAG TTAT TTCAAG AGAAT AACT T CATCT TAGAG CCCTT;
siRNA GDNF1 sequence was: GCCATAC ACTT AAA TGTC AC TTT CAAGA GAAG TG AC ATTTA AGTGTA TGGCTT;
siRNA GDNF2 sequence was: GCTAA CAAGTG ACAAA GTAGGT TCAAGA GACCTA CTTTG TCAC TTGT TAGCTT;
siRNA GDNF3 sequence was: GCTAAA CGGTG TGGATG TATCTT CAAGAG ATAC ATCCA CACC GTTT AGCT.
Enzymatic digestion of PLK O.1 TRC vector
The production of enzymatic digestion was collected under ultraviolet and added to preweighted 1.5 mL Eppendorf (EP) tube to measure the weight. After addition of 3-fold volume of QG, the mixture was melt completely in 50°C water bath and added 1-fold volume of isopropanol. The mixture was put on the column for 3-min settlement, centrifuged at 12,000 rpm for 1 min, and settled for 2 min. The flow-through was discarded. The column was added 0.5 ml QG buffer and centrifuged at 12,000 rpm for 1 min and the flow-through was discarded. 0.75 ml PE buffer was added to the column for 5-min settlement. After centrifugation at 10,000 rpm for 1 min, the flow-through was discarded. Another centrifugation was performed at 15,000 rpm for 1 min. Later, the column was put in 1.5 mL EP tube and added 30 μL elution buffer for 5-min settlement and centrifugation at 15,000 rpm for 1 min. A suitable centrifugation production was taken for electrophoresis with 1.5% agarose and identification.
Transformation reaction after ligation reaction
A volume of 20 μL of ligation reaction plasmid DNA was added to melt 100 μL of competent bacteria ( Escherichia More Details coli DH5α) in ice bath for 30 min, followed by 42°C hot shock for 90 s and ice bath for 1-2 min. The reaction system was put in horizontal shaker at 37°C, 200 rpm for 45 min after addition of 600 μL of Luria-Bertani (LB) solution without antibiotics, and then evenly smeared on plate coated with LB and 100 mg/mL ampicillin. The plate was then uprightly put at 37°C for 1 h and then put upside down in 37°C incubator for overnight, and kept at 4°C for future experiments.
293T cells were digested with trypsin at exponential phase with a density of 0.5 × 10 6 /L and then re-inoculated in 25 mL flask (10 cm 2 ) and in 5% CO 2 incubator at 37°C. When the confluence reached 70-80%, the cells were transfected with lentivirus. The medium was removed with sterile PBS and 5 mL serum-free medium was added. Then, the cells were added with 8 μg PLKO. 1-GDDNF, 6 μg psPAX2, 2 μg pMD, then added with opti-minimal essential medium (MEM) to 500 μL, and put at room temperature for 5 min. Another sterile EP tube was added with 450 μL opti-MEM and 50 μL lip 2000, and put at room temperature for 5 min. The liposome was then slowly added to plasmid DNA, mixed evenly and put at room temperature for 20 min. The mixture of DNA and liposome were transferred to medium containing monolayer cells and mixed evenly for 4-6 h culture. Then, the medium containing transfected mixture was discarded. The cells were then rinsed with 15 mL PBS for 3 times and added 15 mL medium containing 10% FBS. When the medium changed to yellow, transfected 294T cells were collected and added with fresh medium containing 10% FBS for 2 times. The collected supernatant was centrifuged at 4°C, 4 000 g for 10 min and the new supernatant was filtered with 0.45 μm filter and split into 2 mL EP tube for future use.
The experiment involved blank control group, negative control group, short hairpin RNA (shRNA)-120 vector group, shRNA-494 vector group, shRNA-634 vector group, and shRNA-743 vector group. The targeting cells were inoculated in 6 cm 2 culture dishes and when the confluence reached 30-45%, added 3 mL viral supernatant, 2 mL medium containing 10% FBS, and polybrene to the final concentration of 4-8 μg/mL, and finally changed to normal medium after 24 h.
Screening of stably transfected cells with drug resistance
At 48 h, the cells were removed of the medium and added Puromycin medium with a final concentration of 3 μg/mL till all cells in the control group died. The normal medium was changed back.
Measurement of glial cell line-derived neurotrophic factor, receptor tyrosine kinases, fyn and focal adhension kinase expression using reverse transcription polymerase chain reaction
The cells were collected and extracted of total RNA, according to the kit manual. The cDNA was transcribed with total mRNA. The reaction system was 20.00 μL, mixed evenly, centrifuged and put in PCR apparatus. The reaction protocol contained the following steps: 30°C 10 min, 42°C 30 min, 99°C 5 min and 4°C 5 min. The reaction system contained GDNF and GAPDH primers. The sequences of GDNF primers were: 5'-CACCGTT CTCCG AACGTGTCA CGTCAAGAG ATTAC GTGACACG TTC GGAGAA TTTTTTG-3' and 5'-GATCCAAAAAATTCTCCGAACG TGTCAC GTAATCTCTTGACG TGACACGTTCGGAGAAC-3'. The sequence of GAPDH primers were: 5'-CACCGCTCACT CAAGATT GTCAGCAATTCAA GAGATTGCTGACAA TCTTGAGTGAGTTTTTTG-3' and 5'-GATCCAAAAAACTCACTCA AGATTGTCAGCAATC TCTTGAATTGCTGACAATCTTGAGTGACC-3'. The reaction system contained 10.00 μL cDNA, 10.00 μL 5 × PCR buffer, 0.25 μL DNA polymerase, 1.00 μL upperstream primer, 1.00 μL downstream primer, 27.75 μL H 2 0, 10.00 μL cDNA, with total volume of 50.00 μL. The reaction condition was predenaturation at 94°C for 2 min, denaturation at 94°C for 40 s, annealing at 57°C for 40 s, extension at 72°C for 1 min, repeated 32 times, followed by extension at 72°C for 10 min. The production was measured with agarose electrophoresis and stained with EB for storage.
Measurement of expression of glial cell line-derived neurotrophic factor, receptor tyrosine kinases, fyn and focal adhesion kinase with western blot
The cells were lysed with radioimmunoprecipitation assay solution (Sigma) and proteins were collected. In order to ensure the same volume of protein samples, the protein concentration was measured by kit from Sangon Biothech Biotech Co Limited and bicinchoninic acid method.
Measurement of protein with sodium dodecyl sulfate polyacrylamide gel electrophoresis
About 12% separation gel was prepared and poured into the gap between two glass plates and covered with 1 mL ddH 2 O on the top. After natural concretion (37°C, 10-15 min), ddH 2 O was poured and absorbed with filter paper. 5% stacking gel was poured above the separation gel, the comb was inserted between two glass plates and the air bubbles were removed. The samples were mixed with 2× buffer at 1:1. The comb was pulled out and the gel was put in electrophoresis chamber. The upper and lower slots were added with 1× Tris-glycin buffer (pH 8.3). The electrophoresis was performed with 8 V/cm for stacking gel and 12 V/cm for separation gel. When EB was moved about 0.5 cm to the bottom, the electrophoresis was stopped.
The polyvinylidene difluoride (PVDF) membrane and filter paper were cut into the same size with the gel. The PVDF membrane was soaked in ethanol solution and then transferred with two filter papers to transferring buffer (2.9 g/L glycin, 5.8 g/L Tris base, sodium dodecyl sulfate 0.37 g/L and 200 ml/L ethanol). The gel was rinsed in transferring buffer for 15 min. The pad, filter paper, gel, PVDF membrane, filter paper, pad were subsequently put into the transfer folder without air bubbles. The transfer folder was put in the electrotransfer slot and the gel was subjected to cathode. The transfer buffer and cold system were added. The transfer was performed at 100 v for 90 min.
Specific antibody measurement
After blotting, the PVDF membrane was added with confining solution (Tris-buffered saline [TBS], 0.1% Tween-20, 2% bovine serum albumin) at room temperature for 1 h, rinsed with TBS for 3 times, 5 min each time. The primary antibody was diluted with confining solution (2 μg/mL osteopontin monoclonal antibody or actin antibody at 1:1000), with total volume of 0.1 ml/cm 2 membrane, was incubated at room temperature for 1.5 h. The PVDF membrane was rinsed with TBS for 3 times, 5 min each time. The ECL luminescence reagents A solution and B solution were mixed and added to the PVDF membrane for reaction at room temperature for 1 min. The excessive liquid was absorbed with filter paper. The PVDF membrane was put between two layers of preservative films and developed in darkness.
The cells were inoculated in 96-well plate (100 μL/well, 5000 cells) and preincubated in 5% CO 2 at 37°C. Then the wells were added 10 μL WST-1 solution/well and were incubated in an incubator for 1-4 day. The absorbance was measured with enzyme-labeled meter at 222 nm.
The cells were digested by trypsin without ethylenediamine-tetraacetic acid, rinsed with PBS for 2 times, centrifuged at 2000 rpm for 5 min, and collected at the density of 1∼5 × 10 5 . Then, 500 μL binding buffer was added to suspend the cells, followed by adding 2 μL annexin V-fluorescein isothiocyanate and 5 μL propidium iodide (PI) and mixed evenly. The cells undertook reaction in darkness at room temperature for 5 min. The apoptosis was measured using flow cytometry (Ex = 488 nm; Em = 530 nm).
The data were expressed as x- ± s and analyzed with SPSS11.0: Statistical Product and Service Solutions 11.0 (SPSS company, Chicago, USA). The intergroup differences were compared with variance analysis. P < 0.05 was set as significant level.
| Results|| |
Construction of plasmid
Sequence measurement of recombinant plasmid with pBSsi-GDNF DNA confirmed the consistence with the designed sequence, suggesting successful construction of siRNA vector against GDNF genes.
Expression of glial cell line-derived neurotrophic factor messenger ribonucleic acid in transfected Sertoli cells More Details
Through the comparison between fluorescent microscope and light microscope, it suggested that the transfection rate of cells reached >80%. There was electrophoresis band of GDNF gene at the location of 222 bp in all groups obviously different in intensity. After RT-PCR, the intensity of electrophoresis band of GDNF mRNA in positive transfection group was obvious weaker than that of the control group while the GAPDH was same in all groups [Figure 1]. ImageMaster TotalLab image analysis system (TotalLab company, England) indicated that the intensity of GDNFa mRNA in the experimental group and control group was 12.32 ± 1.22% and 54.25 ± 1.34%, respectively, and the difference was significant (P < 0.01).
|Figure 1: Expression of glial cell line-derived neurotrophic factor messenger ribonucleic acid in transfected Sertoli cells|
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Apoptosis of spermatogonial stem cells after transfection
The measurement with annexin V and PI double-staining flow cytometry indicated that the apoptosis rate in the experimental group and control group was 25.43 ± 1.91% and 5.61 ± 0.16%, respectively. The number of apoptotic cells in G2 quadrant of the experimental group was obviously larger than that in F2 quadrant of the control group. The difference between the two groups was significant [[Figure 2]; P = 0.035]. These results suggested that the apoptosis significantly increased after effective GDNF RNAi.
Expression of receptor tyrosine kinases, fyn and focal adhesion kinase in spermatogonial stem cells after ribonucleic acid interference
The electrophoresis bands of receptor tyrosine kinases (RTKs), fyn and focal adhesion kinase (FAK) in SSCs after GDNF RNAi were obviously weaker than that of the control group [Figure 3] and [Figure 4]. Analysis with ImageMaster TotalLab indicated that the expression intensity of RTKs, fyn and FAK in the experimental group was 37.34 ± 4.23%, 28.77 ± 3.32% and 39.5 ± 6.02%, respectively; the expression intensity of RTKs, fyn and FAK in the control group was 67.35 ± 5.22%, 66.75 ± 6.31% and 59.1 ± 6.35%, respectively. There was a significant difference between the two groups (P < 0.01).
|Figure 3: Group of glial cell line-derived neurotrophic factor gene interfere|
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|Figure 4: Group of glial cell line-derived neurotrophic factor gene did not interfere|
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| Discussion|| |
As one member of the super-family of transforming growth factor β, GDNF is secreted by the supporting cells of mammalian testicle.  GDNF is an important paracrine regulator of spermiogenesis and is expressed in steps from the growth, differentiation and proliferation of spermatogonium to the spermiongenesis. There are massive GDNF receptors in undifferentiated spermatogonium. GDNF plays an important role in the proliferation and differentiation of SSCs. ,, Multiple studies indicate that GDNF is a key factor in the proliferation and differentiation of SSCs in mammalian testicles.  During in vitro culture, it is found that the addition of suitable GDNF could promote the proliferation of SSCs. Further study indicates that GDNF works through a multiple-component system, which is composed of extramembrane glycosylphosphatidylinositol-coupled receptor bound directly with GDNF (GDNF family receptor α [GFR-α])  and trans-membrane tyrosine kinase Ret. During the proliferation of SSCs, the major mediating signal pathways are the GDNF-mediated two pathways  of which the Ret-dependent pathway is more important and is mainly mediated by the complex of Ret RTK and GFRα-l. Dimer GDNF forms complex with monomer or dimer GFR-α to react with Ret, resulting in formation of Ret dimer and self-phosphorylation of Ret.  Shc is one protein coding the SH domain, and one connexin. The Shc can be phosphorylated by activated Ret at the site of tyrosine and bind to the complex of growth factor receptor-bound protein 2 (Grb2)/son of seven-less (Sos), making Grb2/Sos an activated receptor. Sos accumulates around the cellular membrane and binds to plasmalemma Ras, promoting transformation of Ras guanosine diphosphate to guanosine triphosphate  and activation of Ras, which transiently activates Ras/ERK1/2 pathway and results in phosphorylation and activation of transcription factors, such as Creb-1, Atf-1 and Crem-1.  Finally, GDNF/Ret/Ras up-regulates the transcription of transient response gene c-fos and cellular cycle regulating protein A (Cyclin A) and cyclin-dependent kinase 2 (CDK2). Cyclin A is one key regulating protein modulating the S phase of mammalian cellular cycle and binding to CDK2. In addition, Cyclin A is mainly expressed during spermatogenesis. Cyclin A1 is highly expressed during pachytene stage of spermatogoniums and is necessary for spermatocyte. Cyclin A2 is mainly expressed in A-type spermatogoniums, including SSCs. Therefore, Creb and c-Fos enhance the expressions of Cyclin A and cyclin-dependent kinase 2 (CDK2) in Gfrα-1 positive spermatogoniums, promote the conversion of mitotic G1/S, accelerate the entering of S stage, and promote DNA synthesis and cell proliferation. 
Another signaling pathway is Ret-independent and mainly mediated by neural cell adhesion molecule (NCAM) which plays an important role in nonRet dependent signaling pathway. , NCAM, one kind of adhesion molecules widely expressed on neural membrane, can bind to the GFRα-l of GDNF, activate downstream fyn and FAK, ,, and promote the growth of processes.
The present study applied RNAi to silence GDNF gene, and indicated that the growth of spermatogonium was obviously inhibited, the proliferation was slowed, and the expression of GDNF mRNA and protein was decreased. The fyn and FAK expression in GDNF-mediated Ret-dependent pathway and GDNF-mediated Ret-independent pathway were reduced. These results indicated that the inhibition of GDNF expression inhibited multiple signaling pathways. GDNF plays an important role in the growth and differentiation of spermatogonium, mainly through Ret-dependent and Ret-independent signaling pathways.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]