The Screening, Identification And Preliminary Function Of The Associated Proteins Of Testis Of The Male Patients With Sertoli Cell-only Syndrome

BACKGROUNDMale infertility is a difficult problem in the word. With development of economics, male fertility has been declining constantly, which we can see from reduction in quality and quantity of the spermatozoa. Infertility rate of married couples is as high as 10%, for which azoospermia is one of common reasons accounts. There are two main types leading to azoospermia:one is that testes have problems of spermatogenic dysfunction, called primary azoospermia or non-obstructive azoospermia; another is that sperm can not be ejaculated from the penis due to vas deferens road congestion, whereas testes has normal ability to produce spermatozoa. This type is called obstructive azoospermia. Primary azoospermia can be divided into Sertoli cell-only syndrome(SCOS), complete spermatogenic arrest, Klinefelter syndrome, hypospermatogenesis and so on. SCOS is an important type in primary azoospermia, which characterizes normal secondary sex characteristics, small testes, azoospermia and high serum follicle-stimulating hormone (FSH),clinically. And its pathological features is spermatogenic cells in testicular seminiferous tubules completely missing and only supporting cells composing seminiferous epithelium, accompanied tissue around the tube fibrosis and interstitial hyperplasia. In the past, we were lack of understanding about it. However, with wide application of testicular biopsy and cytotechnology, the rate of SCOS diagnosis has significantly elevated. Owing to etiology and pathological mechanism unclear, doctors treat patients with SCOS according to their experience and therapeutic effect are not satisfying. As a result, a great many patients have no children. Obviously, the therapeutic breakthrough for SCOS depends on significant progress in fundamental research and understanding the pathological mechanism about it.Testes, one of the most important sexual organs, have the functions of producing sperm, secreting testosterone and so on. So far, most research have stayed on the level of single gene or genome, which revealed testicular fine structure of genome, limited number of genes and relative stability of genic structure. Differing from variability and complexity of the testicular functions and diseases makes us realize that gene is just the carrier of hereditary information. Therefore, it is necessary to strengthen research on protein, the direct executors of life activities, in patients with SCOS.With the accomplishment of genome map, a new era of targeting proteome is dawning. Research on Proteome which is a dynamic whole changing with time and space is aimed at analyzing the composition, expression levels and modification state, interaction and association of the proteins in cells from a holistic point of view, and revealing the functions of proteins as well as laws of cellular activity. The Two-dimensional electrophoresis (2-DE) established by O'Farrell in 1975 is still the first choice for protein separation technology in proteome research.Research on testicular proteome of SCOS is limited both at home and abroad, for that it is relatively difficult to administer testes biopsy, collect samples and persuade the patient to agree with information consent form associate with medical ethics. However, it is meaningful to acquire the proteins associated with SCOS in application of proteome strategy.We conform to the principles of medical ethics and respect patients'right to know the truth. After approval from Nanfang Hospital Ethics Committee, we start to collect testicular tissue samples in patients with azoospermia and win the support of majority. We have established definite testicular biopsy sample collection process and norm, collected the testicular tissue, divided different tissue into different groups according to pathological diagnosis and saved it in the testicular tissue bank. Proteins extracted from testicular tissue in SCOS and normal spermatogenic function groups respectively were analysed by 2-DE combined with mass spectrometry identification technology. Important proteins related SCOS were selected and identified, of which HnRNPL and PRPS2 were studied with immunohistochemical method and Western Blotting on their function. Our study aimed at selecting and identifying the important proteins related to SCOS and exploring the relationships between them.Methods1. Sample collectionThe collection and grouping of the biopsies for the adult male with azoospermia Specimen were cut in each case about 5 mm×3 mm×4 mm. Each specimen was put in freezing tube and frozen in liquid nitrogen. All the specimen were classified according to pathology. Patients signed informed consent before voluntarily donated. Our study was approved by the bioethics committee of Nanfang hospital. At present, we have collected 102 cases specimen, of which spermatogenesis normal or almost normal was 61 cases, mild spermatogenesis dysfunction 7 cases, moderate spermatogenesis dysfunction 4 cases, severe spermatogenesis dysfunction 3 cases, Sertoli cell syndrome 20 cases, spermatogenesis not completely blocked 4 cases, spermatogenesis fully block 1 case, Klinefelter syndrome 2 cases.2. Screening of protein marker2.1 The total protein extraction of specimen:10 cases in every group with normal spermatogenesis and sertoli cell syndrome were randomly selected. The total protein was extracted by liquid nitrogen freeze-thaw cycle:The testicular tissue was removed from freezing tube, dissolved in room temperature and liquid nitrogen freezing. The frozen tissue was grinded into powder and then added 3 to 5 times the volume of lysis buffer (7 mol/L urea,2 mol/L thiourea,65mmol/L dithiothreitol, 40% CHAPS,0.5% IPG buffer, complex protease inhibitors) for homogenate. The suspension was put at room temperature for 1h to make the protein fully dissolved, then followed by centrifugation at 14 000×g for 1 h at 4℃. The supernatant was collected and the density was measured by Modified Bradford assay using BSA as the standard. The supernatant was subpackaged 100μL/tube, stored at-80℃refrigerator spare.2.2 The separation of total protein by two-dimensional gel electrophoresis:The Immobiline Dry strip (pH 3-10, length 17 cm, Bio-Rad) was rehydrated with 120μg protein in 300 ml rehydration buffer containing 7 M urea,2 M thiourea,4% CHAPS, 65 mM DTT,5 mM tributylphosphine,1% IPG buffer [pH 3-10] and 0.002% bromophenol blue for 13 h at room temperature. Isoelectric focusing conditions were 250 V 3h,500 V 1h,1000 V 1h,5000 V 3h,10000 V 60 KVh,500 V 3h. After isoelectric focusing the IPG gel was placed face up on the way to the 2.0% DTT balance solution for 15 min, and then placed in 2.5% balance Iodoacetamide for 15 min, then put on the filter paper with buffer on the surface in order to absorb excess liquid. The balance gel was moved to 0.75 mm thick with concentration of 10.0% SDS-PAGE gel for two dimensional electrophoresis. The condition was 12 mA/gel 30 min,24 mA/gel constant current until the bromophenol blue front reached the bottom of the gel.2.3 Silver-staining:40.0% ethanol,10.0% acetic acid fixed 2 times, each time 15 min.30.0% ethanol,0.2% Na2S2O3,6.8% sodium acetate-sensitized for 30min. Following distilled water washed three times, each time 5 min.2.50‰AgNO3 staining 20min. Following distilled water washed two times, each time 1 min.2.5% Na2CO3,0.04‰formaldehyde to develop an image until protein spots clear. Then we immediately added the 5.0% acetic acid to terminate for 10 min, following distilled water washed three times, each time 5min. At last, we used 30.0% ethanol and 4.6% glycerol to preserve.2.4 Gel image scanning and analysis:the gel was scanned through the UMAX PowerLook 1100 scanner to obtain 2-DE gel image, then using Melanie 4 image analysis software to analyse the image and produce differentially expressed protein spots data.3. The expression of associated protein in clinical testicular tissue of azoospermia patients3.1 The validation of differentially expressed proteins by Western Blot:re-select the Sertoli cell syndrome group and spermatogenesis normal group to extract protein, measure the protein concentration by BCA method. A total of 30μg the protein isolated from the testicular specimens was electrophoresed on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel. The proteins were then transferred from the gel onto Polyvinylidene Xuoride (PVDF) (Immobilon P, Millipore, Bedford, MA, USA) membranes. The membranes were blocked for 1h at room temperature in a solution of phosphate-buffered saline (PBS) containing 5% nonfat milk powder and then incubated overnight at 4℃5% skimmed milk T-TBS at room temperature blocked 1h. The primary antibody was diluted at blocked fluid,4℃overnight shaking, following TTBS rinsed three times. The secondary antibody was diluted in blocked fluid, shaking at room temperature for 1 h, following TTBS rinsed three times. The proteins were detected using an enhanced chemiluminescence (ECL, Pierce, Rockford, IL, USA) detection system. The films were put on the lighting PVDF membrane to image. For gray-scale analysis, calculation the ratio of gray between awaited measure indicators and GAPDH to detect differences proteins in expression of abundance between spermatogenesis normal and spermatogenesis abnormal groups.3.2 The validation of differentially expressed proteins by immunohistochemistry: Apply the paraffin testis biopsy specimens which we have collected from January, 2005 to October,2008. Select the structural integrity, clear specimens. Their corresponding paraffin mass was serial cut into 5μm sections, then mounted on 32-amino-propyl 3 Silicone Alkyl Ethoxy (APES) glass slide. Paraffin sections was dropped the wax then put into the water. The sections were incubated in 3% hydrogen peroxide to quench the endogenous peroxidase activity and washed in PBS for three times. Plus the primary antibody conjugated with biotin (Biotin) at 37℃for 30 minutes, then put in the wet box. Following PBS washed three times, then plus enzyme labeled avidin (Avidin) at 37℃for 30. PBS washed three times, following DAB chromogenic and washed in the running water. The sections was stained, dehydration and mounted at last.Result1. After a series of optimization steps for sample processing, sample volume, choice of gel, we success obtain high-resolution, reproducible human testis two-dimensional electrophoresis maps.2. Compare the electrophoresis maps between sertoli cell syndrome group and the spermatogenesis normal group. We have found 18 different protein spots, of which 16 high expressed and 2 low expressed in sertoli cell syndrome group. According to the results identified by mass spectrometry, we searched the database and identified several changes in protein expression may be related to sertoli cell syndrome or spermatogenesis.3. From the comparison results of western blotting for HnRNPL and PRPS2, we found that HnRNPL expression level in Sertoli cell syndrome group was lower than the spermatogenesis normal group, while PRPS2 expression level was higher. From the immunohistochemistry in the two groups we also found that:HnRNPL expression in nucleus at all levels of spermatogenesis cells and Sertoli cells. HnRNPL staining weaker than the normal group, suggesting that HnRNPL lower expression in Sertoli cell group. The PRPS2 cells expression at all levels of spermatogenesis cytoplasmic. PRPS2 staining stronger than the normal group, suggesting that PRPS2 increase expression in Sertoli cells group. The results of Western blotting and immunohistochemistry are consistent to two-dimensional gel electrophoresis results.Conclusion1. Established a stability and good reproducibility human testis tissue protein group of two-dimensional gel electrophoresis techniques and methods. It laid a foundation for further proteomics research and related diseases study.2. We are the first to compare testicular tissue proteome map of adult male Sertoli cell syndrome group and spermatogenesis normal group;3. We preliminary analysis of 18 different protein spots, of which 16 are high expression in Sertoli cell syndrome group,2 low expression and further identified the different protein by mass spectrometry; 4. The immunohistochemistry and Western Blotting results of HnRNPL and PRPS2 were consistent with the results of two-dimensional gel electrophoresis. It lays a foundation for further study.