| Background and objectivesMale sterility, which is a complex clinical syndrome caused by many factors, is one of difficult medical problems to deal with in the world. Infertility rate of married couples as high as10%, for which azoospermia is an important reason. Sertoli cell only syndrome (SCOS) accounts for17-20%azoospermia and30%non-obstructive azoospermia.SCOS, representing a condition of the testes in which only sertoli cells line the seminiferous tubules, was first described in1947It has been suggested that the factors associated with the primary events before or during the proliferation phase of spermatogonia are the candidate regulators of SCOS. And, deletions of the azoospermia factor (AZF) subregions on the Y chromosome, copy number variants of sex-chromosomal or single-nucleotide polymorphisms in the SEPTIN12gene, are associated with the high risk of SCOS. However, the proteins responsible for abnormal spermatogenesis in SCOS remain unclear.Lately, ten genes involved in human spermatogenesis were identified using microarray profile of testicular tissues from individuals with normal spermatogenesis, germinal arrest, and SCOS. Similar to microarray technology, proteomics is also a kind of large-scale approach, which can evaluate complex samples of proteins on a global level rapidly and comprehensively. A typical proteomic technology includes three key components:two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) and bioinformatics. First,2-DE enables separation of highly complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance, and delivers a map of intact proteins reflecting changes in protein expression level, isoforms, or posttranslational modifications. Second, these protein spots of interest can be analyzed and identified after proteolysis by liquid chromatography coupled with tandem mass spectrometry and database search. Then, bioinformatics is usually used to identify proteins that are up-regulated or down-regulated in a disease-specific manner for use as diagnostic markers or therapeutic targets.Proteomic methods can also be effectively used to find novel biomarkers or key proteins in reproductive biology and medicine. Here, we used the clinical samples of testicular tissue from SCOS patients and screened the potential key proteins involved in development and progression of SCOS based on proteomic strategy.Methods:1. Protein Separation and Identification by2-DE and MALDI-TOF MSSpecimens from15SCOS patients and15normal spermatogenesis patients were pooled respectively for2-D analysis. Specimens from each group were mixed and homogenized in lysis buffer, followed by centrifμgation. The supernatant was stored at-80℃for use. About100μg protein was loaded for2-D PAGE. The gels were then underwent silver staining for visualizing proteins. Protein spots of interest were cut from the gels followed by matrix-assisted laser desorption time-of-flight mass spectrometry (ABI4800plus MALDI-TOF/TOF MS).The masses of the tryptic peptides were identified by searching for MASCOT Database (http://www.matrixscience.com/search_form_select.html).The identification results were filtered with peakErazor software (Lighthouse Data, Odense, Denmark)2. Bioinformatics AnalysisFunctional network analysis of the identified proteins by proteomic methods was performed by STRING8.3(http://STRING.embl.de/) and bio-process association analysis of the identified proteins was performed by Pubgene (http://www.pubgene.org/). The association networks retrieved from STRING and Pubgene were visualized in Graphviz software. Graphviz software was also used to map the network of the functional and bio-process annotations of the proteins3. Immunohistochernical evaluation HnRNPL expression in mice model of SCOSSamples paraffin-embedded tissue from mice with injection with busful and DMSO were used in thisstudy. Immunohistochemical analysis was used to detect the expression of HnRNPL in testes of normal spermatogenesis and SCOS mice.4. Effects ofHnRNPLKnockdownon Spermatogenesis in vivo Small interfering RNA (siRNA)(5’-TATGGCTTGGATCAATCTA-3’ GenePharma, Shanghai, China) targeted against HnRNPL transcript were used in vivo and in vitro in our following study.The seminiferous tubules were transfected with20nM siRNA specific for HnRNPL through efferent duct in one testis of adult mouse. Another testis of the same adult mouse was injected with equivalent mock siRNA (5’-TAAGGCTATGAAGAGATA C-3’,GenePharma, Shanghai, China) for control. Approximately20ul of siRNA was injected into the seminiferous tubules of one testis in an8-week-old NIH mouse. Trypan blue (0.8%) was used as an indicator to ensure that the microinjection was successful. Knockdown of HnRNPL was confirmed by western blot assay and immunohistochemistry. After the adult male mice were killed by cervical dislocation, whole testes were removed, free of fat and connection organs. The part marked with trypan blue was cut off for further research. Immunohistochemical analysis, immunofluorescent staining and western blot were used to detect the expression of HnRNPL, growth and apoptosis of germ cells in testes.5. Effects ofHnRNPL Knockdown on Spermatogenesis in vitroTM4and GC-1spg cells were seeded at1×105cells per well in6-well microliter plates. Eight hours later, the cells were transfected with60nM siRNA specific for HnRNPL or negative control with Lipofectamine2000(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Two days after transfection, cells were harvested and used for protein extraction and further analysis.Western blot, MTT and flow cytometry were used to analyze the expression of HnRNPL,cell viability and apoptosis. 6. Proteins Regulated by HnRNPL in GC-1spg and TM4CellsTwo days after transfection, cells were harvested and used for detection proteins regulated by HnRNPL. Samples from GC-1spg and TM4cells were extracted by RNAiso Plus (Takara, Dalian, China) following manufacturer’s instructions. Primescript RT master mix (Takara, Dalian, China) was used to construct the template cDNA for real-time PCR (ABI Prism7500, Perkin Elmer, Foster City, Calif, CA, USA) using SYBR Premix Ex Taq (Takara, Dalian, China). Gene specific primers are retrieved from Primer Bank (http://pga.mgh.harvard.edu/primerbank/) or RTPrimerDB (http://medgen.μgent.be/rtprimerdb/index.php) as follows:GAPDH:5’-ACCCAGAAGACTGTGGATGG-3’(sense) and5’-ACACATTGGG GGTAGGAACA-3’(antisense); HnRNPL:5’-TTGTGGCCCTGTCCAGAGAATT-3’(sense) and5’-GTTCGTGTAGTCCCAAGTATCTTG-3’(antisense);Carcinoembryonic antigen-related cell adhesion molecule1(CEACAM1):5’-ATTTCACGGGGCAAGCATACA-3’(sense) and5’-AAATCGCACAGTCGCCTGAGTACG-3’(antisense);(B-cell lymphoma2) Bcl-2:5’-GTCGCTACCGTCGTGACTTC-3’(sense) and5’-CAGACATGCACCTACCCAGC-3’(antisense);Inducible nitric oxide synthases (iNOS):5’-GGCAGCCTGTGAGACCTTTG-3’(sense) and5’-GCATTGGAAGTAAGCGTTTC-3’(antisense).The initial denaturation phase was30s at95℃followed by an amplification phase of denaturation at95℃for5s, and annealing at60℃for34s for40cycles. Relative quantification of PCR products was calculated after normalization to GAPDH.Results1. Thirteen proteins are identified as potential regulators involved in SCOS by proteomic approachAfter2-DE, sixteen differential spots between normal spermatogenesis group and SCOS group (P<0.05) were selected for subsequent identification by MALDI-TOF/TOF MS. Excluded the three overlapped proteins, thirteen proteins were identified. Eleven proteins were significantly up-regulated and two were down-regulated in SCOS. These up-regulated proteins included Glial fibrillary acidic protein(GFAP), Phosphoglyceratemutase1(PGAM1), Annexin A5(ANXA5), Ribose-phosphate pyrophosphokinase3(PRPS1L1), Ribose-phosphate pyrophosphokinase2(PPRibP/PRPS2), Kappa-actin (FKSG30), Lactoylglutathionelyase(GLO1), Glutathione S-transferase P(GSTP1) Peroxiredoxin-2(PRDX2), Tubulin alpha-3C/D chain (PRDX2) and Transthyretin (TTR). The down-regulated two proteins were ANKRD26-like family C member1B and Heterogeneous nuclear ribonucleoproteinL (HnRNPL).2. HnRNPL was predicted as a key regulator involved in apoptosis, death and growth of spermatogenic cellsPubgene was used to analyze the bio-process of identified proteins. Eight hundred and thirty seven bio-processes were predicted to be associated with these identified thirteen proteins by Pubgene (15bio-processes were showed in table2). Most of these proteins were involved in apoptosis or cell death (ANXA5; GFAP; GLO1; GSTP1; HNRNPL; PRDX2; PRPS2; TTR), and cell growth (ANXA5; GFAP; GLO1; GSTP1; HNRNPL; PGAM1; PRDX2; PRPS2; TTR; TUBA3C).Interestingly, HnRNPL, PGAM1, GSTP1and PRXD2were predicted as functional partners by STRING8.3program. Moreover, single protein analysis indicated that HnRNPL was a key function partner with many hnRNPs family members, such as hnRNPK or hnRNP A2/B1, which are involved in spermiogenesis. HnRNPL was involved in bio-process of apoptosis, death or growth and played as a function partner with many HnRNPs family members, sμggesting a potential key role in spermiogenesis3. Low expression of HnRNPL in testis of SCOS miceIn order to further know the significance of HnRNPL in testis of SCOS mice, we used immunohistochemical analysis to study expression of HnRNPL protein in SCOS mice. Our results indicated that HnRNPL was down regulated in testis of SCOS mice, consistent with the results identified by proteomic methods.4. Knockdown of HnRNPL in vivo leads to impaired growth and apoptosis of spermatogenic cells in testes of NIH mice To investigate the putative role of HnRNPL on spermatogenesis, expression of HnRNPL in the testis of mice was knocked down in vivo by efferent duct administration of siRNA into seminiferous tubules. The HnRNPL protein was suppressed significantly in the testis of mice as early as24h, and consistently suppressed at48h and72h, recovered at96h after injection.Immunohistochemical staining also indicated the expression of HnRNPL was significantly reduced in testis after HnRNPL siRNA injection. Because expression of HnRNPL at72h after in vivo siRNA administration was lowest, we observed the subsequent effects at72h after HnRNPL siRNA injection.The Ki-67index is one of the most commonly used markers of proliferating cells. Immunohistochemical results showed that Ki-67positive cells were spermatogoniums and spermatocytes. The Ki-67negative cells were later spermatids, sperms, and sertoli cells. Compared with that in paired testes in the same mouse, the number of Ki-67positive cells in different stage of spermatids decreased significantly after HnRNPL siRNA were injected with in seminiferous tubules in vivo. Ten round tubules were randomly selected, and the numbers of sertoli cells, Ki-67-positive spermatogonias were counted for quantitative evaluation of spermatogenesis. The mean ratio of spermatogonia to sertoli cells in the HnRNPL siRNA-treated testis was significant lower than that in mock control testes (t=21.880, P=0.000).The caspase family of cysteine proteases plays a key role in apoptosis. Caspase3, a key executioner in the apoptotic pathway, has been extensively studied in caspase family members.The caspase-3positive cells (green) were mainly localized in the bottom and the center of lumen which germ cells were localized. Interestingly, compared with that in paired testes in the same mouse, the number of caspase-3positive cells increased significantly after HnRNPL siRNA injected into seminiferoustubules in NIH mice.5. Knockdown of HnRNPL leads to impaired growth and apoptosis of GC-1spg cells but inhabits apoptosis of TM4cells in vitroIn order to know the potential regulatory effects of HnRNPL on spermatogoniums and sertoli cells, a mouse spermatogonia cell line GC-1spg cells and a mouse sertoli cell line TM4cells were used in our study. We knocked down HnRNPL expressions of GCl spg and TM4cell lines respectively and determined the effects of HnRNPL on cell proliferation and apoptosis by MTT assays and flow cytometry analysis.MTT assay showed that cell proliferation of GC1spg cells decreased significantly after HnRNPL knocked down (F=36.958, P=0.000). Differently, down-regulation of HnRNPL had no significant effects on cell proliferation of TM4cells (F=0.079, P=0.781).The flow cytometry indicated that knockdown of HnRNPL could lead to the apoptosis of GC-1spg cells (t=-11.949, P=0.000). However, knockdown of HnRNPL could not result in increased apoptosis of TM4cells (t=7.924, P=0.001), consistent with regulatory effects of HnRNPL on cell proliferation of TM4cells.Expression of caspase-3increased significantly in GC-1spg cells but decreased in TM4cell after HnRNPL was knocked down, sμggesting the role of activated caspase-3in HnRNPL related apoptotic pathway.6. Knockdown of HnRNPL leads to reduced expression of CEACAM1in GC-1cells and decreased expression of iNOS in TM4cellsHnRNPL is a global regulator of alternative splicing, binding to diverse C/A-rich elements. CEACAM1, bcl-2and iNOS have been reported to be regulated by HnRNPL and be closely related to spermatogenesis. Therefore, we studied the regulatory effects of HnRNPL on expressions of CEACAM1, bcl-2and iNOS. Knockdown of HnRNPL led to reduced expression of CEACAM1(t=4.928, P=0.008)but no change in expression of the iNOS(t=-0.443,P=0.608) in GC-1cells. Interestingly, expression of iNOS decreased significantly (t=3.824, P=0.019) and expression of the CEACAM1did not change after HnRNPL was knocked down in TM4cellsl (t=2.118, P=0.102). Expression of bcl-2had no significant change in both GC-1spg (t=1.524, P=0.202) and TM4cells (t=0.966, P=0.389).Conclusion1. Thirteen proteins are identified as potential regulators involved in SCOS by proteomic approach.2. HnRNPL is a key function partner with many hnRNPs family members and predicted as a key regulator involved in apoptosis, death and growth of spermatogenic cells.3. The expression of HnRNPL is low in the model of SCOS.4. Knockdown of HnRNPL in vivo leads to impaired growth and apoptosis of spermatogenic cells in testes of NIH mice.5. Knockdown of HnRNPL leads to impaired growth and apoptosis of GC-1spg cells but inhabits apoptosis of TM4cells in vitro.6. HnRNPL protein may involve in the pathological spermatogenesis process of SCOS by regulating expressions of CEACAM1in spermatogonia and iNOS in sertoli cells. |
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