Proteome Analysis of Testicular Tissue in Varicocele Rats Using iTRAQ Labeling Technology

Purpose Varicocele (VC) is considered as the main cause of male infertility, clear and denite molecular markers of varicocele disease are helpful for early prevention and timely treatment. This study was in order to examine the effect of varicocele on protein expression in testicular tissue. Methods The testicular tissue samples of normal rats and varicocele rats were used for proteomic analysis and functional bioinformatics analysis. The proteins with a fold change of > 1.2 or < 1.2, and with a P-value < 0.05 were used to identify up- and downregulation proteins between the varicocele and control rats, and two target proteins were selected and veried with western blot. Results It was found that seminiferous epithelium was disordered and spermatogenic cells were injured seriously in varicocele group. A total of 65 differentially expressed proteins were identied compared with normal group by liquid chromatography tandem mass spectrometry (LC-MS/MS) and isobaric tags for relative and absolute quantitation (iTRAQ) analysis, including 31 up-regulated proteins and 34 down-regulated proteins, respectively. Functions of those proteins were mainly related to the following processes: signal transduction, protein cycle, transmembrane transport, protein transport, vesicular mediated transport, cell division, and membrane tissue. Two down regulated proteins of ATPase and Cu 2+ -transporting alpha (ATP7A) and Calcium and integrin-binding protein 1 (CIB1) in varicocele rats were selected and conrmed. different Low expressions of ATP7A and CIB1 may be affect and can used a potential for


Introduction
Varicocele (VC) is a kind of vascular disease, and characterized by varying degrees of dilation and tortuosity of the spermatic veins [1]. VC can lead to male infertility, the incidence of VC in the common male population was 4.4-22.6 %, of which the incidence of primary infertility was 35-40 %, while the incidence of secondary infertility was as high as 80% [2,3]. Now, the main causes of varicocele are included testicular hypoxia, oxidative stress, elevated local temperature, apoptosis of spermatogenic cells, nitric oxide, endocrine system dysfunction [4,5]. However, the exact pathophysiological mechanism of male infertility caused by varicocele is still unclear.
At present, the study of varicocele infertility has been deeply studied in the molecular mechanism. Our previous research found the abberant expression of stem cell factor (SCF) and c-KIT in the testes of VC rats and speculated that VC induced spermatogenesis disorder was related to the abnormal expression of SCF/C-KIT system [6]. We also found the correlation between the apoptosis of spermatogenic cells and overexpression of hypoxia-induciblefactor-1α (HIF-1α) in VC rats. By decreasing the expression of HIF-1α, the apoptosis of spermatogenic cells was reduced, which is bene cial to the recovery of testicular function and the improvement of fertility [7]. Liang et al. observed that increased expression level of reactive oxygen species (ROS) and p53 in VC rats and thought that the increased expression of p53 gene in testicular tissue was caused by the activation of ROS, and the abnormal increase of p53 may inhibit angiogenesis by generating anti-angiogenic factors, which lead to an increase in cell death [8,9]. Hou et al. hold that the deletion of Magea gene could activate the expression of p53 gene, which could increase the apoptosis of spermatogenic cells and lead to male infertility [10].
In order to further explore the molecules involved in VC infertility, this is the rst time to use iTRAQ combined with LC-MS/MS technique to screen the differential proteins in testis between VC rats and normal rats. According to the results of proteome analysis, the molecules that affected the spermatogenesis were selected for veri cation, which laid a foundation for further exploration of its role in VC infertility.

Animals
Twelve male Sprague Dawley rats weighing 200-250g were randomly divided into two groups, named normal group (n=6) and the varicocele group (n=6), respectively. All rats were fed the same diet and raised in a constant environment with a 12-hour light/dark cycle.

Establishment of the VC model
The rats of VC model were referenced from Turner [11]. All experimental procedures were performed in a sterile environment. The rats were anesthetized with an intraperitoneal injection of pentobarbital sodium 40 mg/kg. A midline laparotomy incision was made from xyphoid to pubis to visualize the left renal vein, inferior vena cava, left spermatic vein, left suprarenal vein, and left kidney. Following a careful blunt dissection, a clamp was passed behind the left renal vein just distal to the spermatic vein insertion, a loose ligature of 4-zero silk was placed around the left renal vein at the site, and a rigid hydrophilic 0.8mm-diameter guide wire was placed on the left renal vein. The ligature was tied around the vein over the top of the guide wire, and the wire was withdrawn, allowing vein diameter to be reduced to approximately half of normal. After the injection of penicillin into the abdominal cavity, the midline incision was closed in two layers using 4/0 silk sutures. Successful modeling criteria: the diameter of the spermatic vein was more than 1mm and there was no difference in size between the left and right kidneys [11,12]. And the control group did not do any treatment. All animals were sacri ced after 8 weeks, and the testicular tissue was extracted immediately after the rats were euthanized by intravenous injection of pentobarbital sodium.
Hematoxylin and eosin staining HE staining was conducted according to routine protocols [7]. After xation in 4% paraformaldehyde and dehydrated sequentially with ethanol, the left testicle tissue was embedded in para n and sectioned at 5 um thickness. After routine dewaxing and hydration, sections were stained with hematoxylin for 5 min and eosin for 2 min. The morphology of seminiferous tubules was observed under an optical microscope.
Protein extraction, quanti cation, and digestion These processes were conducted according to routine protocols [13]. Three rats were randomly selected from each group for the experiment. Samples were dissolved in lysis buffer (consisting of 7M urea, 1.4M thiourea, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS)) and fully mixed, and then centrifuged at 14,000 g for 30 min at 4°C. The supernatant was collected and the protein concentration was determined according to the Bradford method. 100μg of each sample was added into 4μl of reducing reagent (60℃, 1h), then 2μl of Cysteine-Blocking reagent was mixed into it (room temperature (RT), 10min). The reductive alkylation protein solution was added into a 10K ultra ltration tube (12,000r, 20min), the solution in the bottom was discarded, and then 100μl of dissolution buffer of the iTRAQ kit was added into it (12,000r, 20min). A new collection tube was used to add trypsin and to react with the acquired solution (37℃, overnight). Next day, the peptide solution digested by enzyme hydrolysis was collected at the bottom of the tube (12,000r, 20min). 50μl of dissolution buffer 5 (12,000r, 20min) was combined with the above products, then 100μl of sample was obtained which is after digestion at the bottom of the collection tube.
The liquid chromatography eluent was directed into an electrospray ionization source for quantitative time-of-ight MS analysis. Electrospray ionization was performed for information-dependent acquisition in positive-ion mode with a spray voltage of 2.1 kV and a selected mass range of 350-1800 m/z. The Thermo Q-Exactive system was operated in data-dependent acquisition mode, as previously described [13,18].
Mass spectrometry data processing and protein quanti cation In our study, the functional annotations of the differentially expressed proteins were acquired by utilizing the Uniport and Rattus norvegicus database. The mass spectrometry analysis of iTRAQ was carried out by Thermo Q-Exactive mass spectrometry. The original mass spectrometry le was processed by proteome discoverer 1.4 (thermo corporation). The analysis and search parameters were as follows: trypsin as the digestion enzyme with allowance for a maximum of two missed cleavage; Carbamidomethyl (C) as a xed modi cation; Oxidation (M), N-terminal, K and iTRAQ 8 plex modi cation as a dynamic modi cation; precursor ion mass tolerance of 15ppm; and fragment ion mass tolerance of 20mmu. The division values of differentially expressed proteins were set as follows: a signi cant difference was considered when the p-value was less than 0.05 and the differentially expressed proteins cutoff value was more than 1.2-fold [14,19].

Bioinformatics analysis
The differentially expressed proteins were analyzed by GO analysis, the cellular components (CC), the molecular function (MF) and the biological process (BP) involved in the differential protein were analyzed, and the metabolic pathway enrichment analysis was carried out using the KEGG database. A p-value ≤ 0.05 was used as the threshold of signi cant enrichment of GO and KEGG pathways.
Western blot to con rm potential proteins Western blot was conducted according to routine protocols [6,7]. The proteins were extracted from the left testicle tissue of rats. 100 ug of protein was subjected to 8% and 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred onto polyvinylidene uoride membranes (EMD Millipore Corporation, Billerica, MA, USA). Membranes were blocked in tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk powder (TBST) for 3-4 hours at room temperature, then was incubated with the primary antibody overnight at 4 ℃. Antibodies used were as follows: anti-ATP7a (1:1000; ab131400; Abcam, Cambridge, UK), anti-CIB1 (1:500; ZP2168BP68; Boster Biotechnology Co., Wuhan, China), and anti-β-actin (1:5000; I102; Bioworld Technology, Co. Ltd. USA). Four washes with TBST for 10 minutes each followed. The membranes were then incubated with HRP-conjugated secondary antibody (1:5000; BA1054; Boster Biotechnology Co., Wuhan, China) for 1 hour at room temperature. After washing, the speci c proteins were visualized by enhanced chemiluminescence and imaged by Chemi DOC XRS + (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative intensity and density of immunoreactive bands was measured using Image J software (National Institute of Health, MD, USA). The results were obtained using at least three separate samples. The relative expression of goal protein was normalized with β-actin as a control.

Statistical analysis
All the statistical analysis and graph preparation were performed using IBM SPSS Statistics version 21.0 (SPSS Inc., Chicago, IL, USA) or GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA).
All data were presented as the mean ± standard deviation. The differences between the two groups were compared by independent sample T test. P<0.05 was considered to indicate a statistically signi cant difference.

Results
Changes in histologic structure of rats testis The layers in the seminiferous tubules of normal group were well organized from external to internal as basal lamina, spermatogonia, spermatocyte, and spermatid. However, the seminiferous epithelium was disorganized and germ cells were seriously reduced in the varicocele group (Fig. 1).

Expression analysis of the candidate protein
In this study, the expression of differential proteins in testicular tissue with varicocele rats were detected by iTRAQ combined with LC-MS/MS. The protein database was searched by protein pilot 4.0 software. In total, 5277 proteins were detected by proteome analysis. Proteins with a relative expression of > 1. 2 or <1.2 and results were regarded as statistically signi cant at p-values less than 0.05. A total of 65 differentially expressed proteins were identi ed in the mixed samples of the two groups, of which 31 were up-regulated (Table 1) and 34 were down-regulated (Table 2) (Fig. 2).
GO and KEGG database analysis GO analysis was conducted to understand the functional basis of the differentially expressed proteins, which were identi ed by iTRAQ. The biological process (BP), cell component (CC) and molecular function (MF) data were the basic information of GO analysis. The metabolic pathway enrichment of differential proteins were analyzed by R language software. GO analysis showed that the identi ed proteins were widely distributed in cells, functionally diverse, and involved in a variety of biological processes. CC analysis showed that the identi ed proteins were mainly distributed in the cytoplasm, nucleus, extracellular region, cytoskeleton, cell membrane, plasma membrane, and mitochondria (Fig. 3a). MF analysis showed that the identi ed proteins were mainly had the following functions: transporter activity, signal transduction activity, metal ion binding, protein binding, receptor binding and so on (Fig. 3b). BP analysis showed that the identi ed proteins were mainly involved in signal transduction, protein cycle, transmembrane transport, protein transport, vesicular mediated transport, cell division, membrane tissue, and other biological processes (Fig. 3c). KEGG pathway analysis was conducted to enrich the potential pathways of the identi ed proteins with differential expression. KEGG analysis showed that the main metabolic pathways involved in the identi ed proteins were antibiotic biosynthesis and protein processing in endoplasmic reticulum (Fig. 3d).

Western-blot validation
The expression levels of ATP7A and CIB1 in the two groups were detected by western-blot. The result showed that the expression levels of ATP7A and CIB1 in the varicocele group were signi cantly decreased compared with that in the normal group (p<0.05), which was the same as the results of proteome analysis (Fig. 4).

Discussion
Varicocele is a common disease in the male genitourinary system, it is one of the most important causes of male infertility [20]. However, the exact molecular mechanism of varicocele inducing male infertility has not yet been clari ed. In this study, the proteome method of iTRAQ combined with LC-MS/MS was used to screen out the new biomarkers that might play an important role in male infertility caused by varicocele.
The function of differential proteins was classi ed by GO analysis, it was found that the main biological functions were binding, signal transduction, protein cycle, transmembrane transport, protein transport and cell division. KEGG results showed that differential proteins were mainly involved in antibiotic biosynthesis and protein processing in endoplasmic reticulum. Among the many screened differential protein molecules, molecules with a more signi cant difference in protein expression between the two groups, and the functional molecules related to spermatogenesis were reported in the literature as our candidate molecules for further study. ATP7A and CIB1 molecules were selected as candidate molecules.
Our study found that the expression of ATP7A and CIB1 in testis of VC rats were signi cantly lower than that of normal rats in the protein level (p<0.05), which is consistent with what proteomic analysis results.
CIB1 was an important calcium-binding regulatory protein of 22 kDa [21]. The expression level of CIB1 was highest in normal testicular tissue, and participated in calcium signaling, cell survival and proliferation, cell migration, cell adhesion and apoptosis [22,23]. CIB1 could regulate the release of intracellular Ca 2+ , the calcium ion signal had special signi cance in the sperm cells, and it was the central regulator factor of many key activities, which included capacitation, superactivation, chemotaxis and acrosome reaction [24,25]. The damage of calcium ion signal in sperm was related to male sterility [26]. CIB1 was necessary for spermatogenesis in mice, the deletion of CIB1 may lead to differentiation and dysfunction of Sertoli cells. Sun et al. found that the expression level of CIB1 in patients with oligospermia and asthenospermia were lower than those in the control group, while the cyclin-dependent kinase 1 (CDK1) were signi cantly increased, and there was a signi cant correlation between CIB1 mRNA level and CDK1 mRNA level. Therefore, they came to conclusion that CIB1 may be involved in the pathogenesis of oligozoospermia through CDK1 signaling pathway [27]. Yuan et al. found that both mRNA and protein expression levels of Cdc2/Cdk1, a regulatory factor of cell cycle, were signi cantly higher in mouse testes with CIB1 deletion than that in the control group, and the proliferation rate of the embryonic broblasts was signi cantly reduced, it is suggested that the expression changes of CDK1 and Cdc2 may destroy the normal interval of Sertoli cell proliferation and lead to the imbalance between the number of Sertoli cells and germ cells, which may lead to the increase of germ cell apoptosis and spermatogenesis defects [21,28]. Therefore, we believe that the decreased expression of CIB1 can be used as an auxiliary biomarker for the diagnosis of varicocele sterility. However, how the decrease of CIB1 leads to male sterility needs further study.
Copper was an essential trace element for normal growth and development of all organisms [29]. It is reported that normal sperm count and sperm motility need to be maintained by physiological copper concentration in seminal plasma, and it was important for spermatogenesis [30,31]. Kowal et al. found that the copper metabolism disorders could affect the sperm motility, sperm count, sperm head morphology, integrity of plasma membrane in tail cells, and the structure of testicular tissue [32]. In human and animal experiments, male gonads were particularly sensitive to the increase of copper concentration, which leads to the decrease of testosterone level, the degradation of spermatogenic tubule epithelium, the decrease of sperm count and motility, the inhibition of acrosome reaction and spermatogenesis , and the increase of cell apoptosis, so that the fertility of the male was reduced [29-35]. ATP7A could protect tissue from excessive copper and played a crucial role in cellular copper homeostasis [36,37]. The results of Ogórek et al. showed that the expression of ATP7A could protect the germ cells from the negative effects of excessive copper, and that ATP7A was mainly active in primary spermatocytes [33]. It is suggested that the concentration of copper in vivo should be strictly regulated. Therefore, we speculate that the downregulation of ATP7A expression in testicular tissue of varicocele infertile rats may result in the copper metabolism disorders, which leads to the decrease of sperm count and sperm motility, the increase of apoptosis, and the decrease of male fertility.

Conclusions
In summary, we speculate that ATP7A and CIB1 may be play an important role in normal spermatogenesis and are essential to maintain normal fertility in rat testicular tissue. they may be very important molecules involving in spermatogenesis of varicocele. The decrease of their expressions will probably cause increased spermatogenic cell apoptosis, further induce increased spermatogenic failure, and nally lead to male infertility.