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Proteomic analysis of seminal plasma in adolescents with and without varicocele

      Objective

      To compare proteomic profiles of seminal plasma from adolescents with varicocele and changes in semen quality with the plasma from adolescents with varicocele without seminal changes and from adolescents without varicocele.

      Design

      Observational study.

      Setting

      Patients in an academic research environment.

      Patient(s)

      Adolescents without varicocele (control group), adolescents with varicocele and normal semen quality (VNS group), adolescents with varicocele and abnormal semen quality (VAS group).

      Intervention(s)

      Two semen collections at 1-week interval. Protein separation by two-dimensional protein electrophoresis, analysis by gel densitometry, and identification by mass spectrometry.

      Main Outcome Measure(s)

      Overexpressed proteins in each group, observed by increased densitometric signal in gels, and exclusively identified proteins in each group.

      Result(s)

      No differences were observed among the three groups regarding clinical parameters. In semen analysis, the VAS group presented lower sperm concentration, motility, and morphology compared with the VNS and control groups. Forty-seven protein spots of interest were submitted to mass spectrometry identification. Apoptosis regulation proteins were overexpressed in the VAS group, whereas spermatogenesis proteins were overexpressed in the VNS group. Controls presented proteins related to homeostasis.

      Conclusion(s)

      Changes in the proteomic profile of adolescents with varicocele and normal semen parameters (VNS group) indicate that normal semen analysis may not reflect alterations in proteins in seminal plasma. Implementation of proteomics will help characterize proteins identified in seminal plasma and will facilitate detection of new proteins associated with spermatogenesis and sperm function.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/zylbersztejnds-proteomics-seminal-plasma-varicocele/
      Varicocele is characterized by dilation of veins of the pampiniform plexus and affects approximately 15%–25% of the male population. It is a time-dependent disease that begins at puberty and is considered the major treatable cause of male factor infertility (
      • Mori M.M.
      • Bertolla R.P.
      • Fraietta R.
      • Ortiz V.
      • Cedenho A.P.
      Does varicocele grade determine extent of alteration to spermatogenesis in adolescents?.
      ,
      • Oster J.
      Varicocele in children and adolescents. An investigation of the incidence among Danish school children.
      ,
      • Steeno O.
      • Knops J.
      • Declerck L.
      • Adimoelja A.
      • van de Voorde H.
      Prevention of fertility disorders by detection and treatment of varicocele at school and college age.
      ). Varicocele accounts for 40% of primary and 80% of secondary causes of male factor infertility (
      • Gorelick J.I.
      • Goldstein M.
      Loss of fertility in men with varicocele.
      ).
      The scrotal temperature required for normal spermatogenesis is 2°–3°C below body temperature. Varicocele impairs the countercurrent heat exchange mechanism, which is the cooling system of the arterial blood supply, resulting in venous stasis and thereby maintaining testicular temperature at body temperature (
      • Dahl E.V.
      • Herrick J.F.
      A vascular mechanism for maintaining testicular temperature by counter-current exchange.
      ,
      • Setchell B.P.
      The Parkes Lecture. Heat and the testis.
      ). This loss of testicular thermoregulation causes an increase in cellular metabolism, which is not accompanied by an increase in testicular blood supply. The resulting lack of adequate cell oxygenation leads to chronic hypoxia, increased stage-specific apoptosis of germ cells at most susceptible stages of spermatogenesis, and excessive production of reactive oxygen species. Excessive reactive oxygen species production that is not counterbalanced by antioxidant systems results in a number of deleterious effects to the sperm, including increased levels of sperm membrane lipid peroxidation (which results in loss of membrane fluidity and changes in membrane permeability), reduced motility, low level of acrosome reaction, decreased mitochondrial activity, increased DNA fragmentation, and apoptosis (
      • Setchell B.P.
      The Parkes Lecture. Heat and the testis.
      ,
      • Halliwell B.
      Reactive oxygen species in living systems: source, biochemistry, and role in human disease.
      ,
      • Yin Y.
      • Hawkins K.L.
      • DeWolf W.C.
      • Morgentaler A.
      Heat stress causes testicular germ cell apoptosis in adult mice.
      ).
      It is widely believed that clinical varicocele in adult men should be surgically corrected when changes in the results of semen analysis or functional tests are confirmed (
      Practice Committee of American Society for Reproductive Medicine. Report on varicocele and infertility.
      ,
      • Blumer C.G.
      • Restelli A.E.
      • Giudice P.T.
      • Soler T.B.
      • Fraietta R.
      • Nichi M.
      • et al.
      Effect of varicocele on sperm function and semen oxidative stress.
      ,
      • Lacerda J.I.
      • Del Giudice P.T.
      • da Silva B.F.
      • Nichi M.
      • Fariello R.M.
      • Fraietta R.
      • et al.
      Adolescent varicocele: improved sperm function after varicocelectomy.
      ). However, this consensus does not apply to adolescents, as the hypothalamic-pituitary-testis axis is still immature during adolescence, thus rendering semen analysis inaccurate. At present, surgical correction is recommended for adolescents with clinical varicocele and reduction in ipsilateral testicular volume of more than 20% compared with the contralateral testis (
      Practice Committee of American Society for Reproductive Medicine. Report on varicocele and infertility.
      ). There is a need for more objective criteria for the indication of surgical correction of varicocele in adolescents. Not all adolescents with varicocele will have reduced fertility potential in the future, but many of them may have severely impaired spermatogenesis. Semen analysis is not reliable for evaluating varicocele in adolescents, and current clinical criteria for the indication of surgical treatment are unsatisfactory, as testicular growth retardation would determine impaired spermatogenesis. Studies of varicocele in adolescents are designed to determine which adolescents would benefit from surgical correction and the best time to perform surgery.
      With the advent of the human genome project and development of bioinformatics, a new science encompassing omics (e.g., transcriptomics, proteomics, and metabolomics) has emerged and is aimed at improving the analysis of biological systems. Proteomics is a novel approach to the study of protein functions and investigation of metabolic processes to better understand the workings of a cell or tissue at the molecular level (
      • Thacker S.
      • Yadav S.P.
      • Sharma R.K.
      • Kashou A.
      • Willard B.
      • Zhang D.
      • et al.
      Evaluation of sperm proteins in infertile men: a proteomic approach.
      ).
      Thus, seminal plasma proteins can serve as potential markers of impaired spermatogenesis and might provide an early indication for varicocelectomy before clinical varicocele is detected by semen analysis or by a reduction in testicular volume. The main objective of the present study was to compare the proteomic profiles of seminal plasma from adolescents with varicocele and changes in semen quality with those from adolescents with varicocele without seminal changes and from adolescents without varicocele.

      Materials and methods

      The study was approved by the Research Ethics Committee of the Federal University of São Paulo, Brazil, and performed in accordance with the ethical standards of the 1964 Declaration of Helsinki and its succeeding revisions. Written informed consent was obtained from all participants or their representatives before their inclusion in the study, and anonymity was assured.
      This was an observational study. A cohort of 156 adolescents from 10–19 years of age was initially evaluated in the National Service of Industrial Learning in São Paulo, Brazil, from August 2009–September 2010. The evaluation included the administration of a standardized questionnaire and physical examination using a Prader orchidometer to measure testicular volume. All adolescents were evaluated by the same physician. Inclusion criteria were age 10–19 years and full sexual maturity (Tanner stage V). The exclusion criteria were the presence of systemic disease, Tanner stages ≤4, endocrine disease, obesity, congenital malformation of the genitalia, genetic syndrome, prior history of inguinoscrotal surgery, orchitis or epididymitis, testicular torsion, testicular dystopia, absence of masturbation, and other conditions that could affect fertility.
      Sixty-seven adolescents entered the study and were allocated to three groups: control group: 21 adolescents without varicocele and normal semen parameters; the VNS group: 28 adolescents with clinical varicocele (grade II or III) and normal semen parameters; and the VAS group: 18 adolescents with clinical varicocele and abnormal semen parameters, as determined by semen analysis.

       Semen Analysis

      Semen samples were obtained by masturbation after 2–5 days of ejaculatory abstinence and analyzed within 1 hour of collection. After liquefaction, the semen was analyzed according to the World Health Organization 1999 criteria and sperm morphology was assessed using Kruger's strict criteria (
      World Health Organization
      WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction.
      ). Semen parameters were considered normal when sperm concentration was ≥20 × 106/mL, motility (% a+b) was ≥50%, and normal sperm forms were >14%. Two samples were collected with a 7-day interval between collections.

       Identification and Quantification of Proteins

      For protein identification, an aliquot of semen was processed immediately after centrifugation. Semen was centrifuged at 4,000 × g, 4°C to separate sperm from seminal plasma. Seminal plasma was submitted to a second centrifugation step, performed at 14,000 × g, 4°C, to remove cellular debris.
      The proteomic profile of seminal plasma was initially determined using two-dimensional polyacrylamide gel electrophoresis. Protein content was quantified using a colorimetric modified Lowry bicinchoninic acid assay (
      • Smith P.K.
      • Krohn R.I.
      • Hermanson G.T.
      • Mallia A.K.
      • Gartner F.H.
      • Provenzano M.D.
      • et al.
      Measurement of protein using bicinchoninic acid.
      ), and pools from each group were formed, normalized to protein content, and analyzed in quadruplicate. In brief, proteins were separated by two-dimensional polyacrylamide gel electrophoresis in 18-cm pH gradient 3–10 strips on the basis of their isoelectric points by isoelectric focusing. The second dimension separation was performed on gradient (10%–17.5% acrylamide) slab gels. The gels were stained with Coomassie brilliant blue, scanned, and compared using ImageMaster 2D Platinum 7.0 software (GE Healthcare). Protein spots of interest (i.e., those that were significantly different between groups) were excised from the gels and the proteins were identified by tandem mass spectrometry (ESI-Quad-TOF spectrometer; Waters Co.,) and using the Mascot search algorithm (Matrix Science) for searching database. The UniProt (SwissProt) webpage (http://www.uniprot.org/) was used for the final identification of proteins.

       Statistical Analysis

      The Statistical Package for the Social Sciences for Windows, version 13.0, was used for the statistical analysis of the seminal variables. Initially, the homoscedastic distribution of the samples was tested and confirmed. Next, analysis of variance (ANOVA) was used for comparison between groups. When a significant difference was observed (P<.05), post-hoc analysis was performed using Fisher's least significant difference test to identify in which groups this difference was present. All statistical tests were performed at a significance level α of 0.05 (P<.05).

      Results

      The clinical results for the three groups of adolescents are shown in Table 1. There were no differences among the three groups of adolescents in age, ejaculatory abstinence, ejaculatory volume, round cells, neutrophils, right or left testicular volume, height, weight, age at first masturbation episode, and ambient temperature during physical examination. The VAS group had a lower concentration of sperm, lower sperm motility, and poorer sperm morphology compared with the VNS and control groups.
      Table 1Clinical characteristics of adolescents without varicocele (control group), adolescents with varicocele but normal semen analysis variables (VNS group), and adolescents with abnormal semen analysis variables (VAS group).
      VariablesControl groupVNS groupVAS groupP value (ANOVA)
      Age (y)
       Mean; SD16.3; 1.316.3; 1.016.4; 1.1.956
       95% CI15.8–16.915.9–16.715.8–17.0
      Abstinence (d)
       Mean; SD3.5; 1.23.8; 1.23.4; 1.0.438
       95% CI2.9–4.33.8–4.32.9–3.9
      Ejaculatory volume (mL)
       Mean; SD2.6; 1.02.5; 1.22.2; 1.1.532
       95% CI2.1–3.02.0–2.91.6–2.7
      Concentration (×106/mL)
       Mean; SD84.1; 49.5a95.9; 87.3a21.7; 39.8b.001
      Statistical significance (ANOVA). Different superscript letters in a same line indicate a significant difference.
       95% CI61.6–106.762.0–129.71.9–41.5
      Progressive motility (%)
       Mean; SD64.0; 10.1a64.2; 10.6a54.7; 14.5b.017
      Statistical significance (ANOVA). Different superscript letters in a same line indicate a significant difference.
       95% CI59.3–68.660.1–68.347.4–61.9
      Morphology (%)
       Mean; SD9.1; 1.7a9.5; 2.8a3.8; 2.3b<.001
      Statistical significance (ANOVA). Different superscript letters in a same line indicate a significant difference.
       95% CI8.4–9.98.4–10.62.7–4.5
      Round cells (×106/mL)
       Mean; SD2.9; 1.73.4; 4.02.1; 3.0.458
       95% CI2.1–3.71.8–4.90.6–3.7
      Neutrophils (106/mL)
       Mean; SD0.3; 0.90.6; 1.20.07; 0.2.1880
       95% CI−0.1–0.70.1–1.0−0–0.2
      Left testicular volume (cm3)
       Mean; SD18.3; 4.118; 2.916.3; 3.9.1730
       95% CI16.5–20.216.8–19.014.3–18.2
      Right testicular volume (cm3)
       Mean; SD18.6; 4.218.8; 3.217.5; 3.7.5104
       95% CI16.7–20.617.5–20.015.7–19.4
      Body height (m)
       Mean; SD1.73; 0.071.72; 0.051.76; 0.08.2221
       95% CI1.7–1.761.7–1.741.72–1.8
      Body weight (kg)
       Mean; SD66.1; 13.066.6; 12.268.6; 11.6.800
       95% CI60.2–72.061.9–71.462.8–74.4
      Age at onset of masturbation (y)
       Mean; SD12.4; 1.112.3; 1.112.2; 1.4.9612
       95% CI11.9–12.911.9–12.811.6–12.9
      Ambient temperature (°C)
       Mean; SD22.8; 1.7722.7; 1.7523.0; 1.9.8872
       95% CI22.0–23.422.0–23.422.0–23.9
      Note: Groups were compared using one-way analysis of variance (ANOVA), followed by a least significant differences post-hoc test. An α of 5% was adopted. 95% CI = 95% confidence interval of the mean.
      c Statistical significance (ANOVA). Different superscript letters in a same line indicate a significant difference.
      Four gels were scanned per group (control, VNS, and VAS groups), for a total of 12 gels. The minimum and maximum numbers of protein spots found in the gels were 181 and 323, respectively. The number of protein spots was greater in the control group than in the VNS and VAS groups. Forty-seven protein spots of interest were excised from the gels and analyzed first by tandem mass spectrometry and then using the Mascot search algorithm. More than one protein was identified per spot; all peptides with a score above the significance threshold were considered to be positively identified. A score was calculated for each peptide based on the number of fragments and length of the peptide in the fixed and variable modifications. Proteins expressed exclusively in the control group are presented in Table 2, in the VNS group in Table 3, and in the VAS group in Table 4. Differentially expressed proteins are presented in Table 5.
      Table 2Protein expression in seminal plasma of adolescents without varicocele (control group), adolescents with varicocele but normal semen analysis variables (VNS group), and adolescents with abnormal semen analysis variables (VAS group).
      Match IDpIMolecular mass (kDa)Significance thresholdScoreProteinControl groupVNS groupVAS group
      1488.57UPP1Exclusive
      14987>2988SEMG1_HUMANExclusive
      1509.86.75>3167SEMG2_HUMANExclusive
      1527.48>2992SEMG1_HUMANExclusive
      1588.49>29153SEMG2_HUMANExclusive
      1636.510UPP2Exclusive
      1645.110.25>29174PIP_HUMANExclusive
      1665.910.75UPP3Exclusive
      1676.611UPP4Exclusive
      1734.811.5>29108CYTS_HUMANExclusive
      1794.912.25UPP5Exclusive
      1834.913UPP6Exclusive
      1845.613.3>2967PIP_HUMANExclusive
      1845.613.3>3059SEMG2_HUMANExclusive
      1895.214.7>30115PIP_HUMANExclusive
      1895.214.7>3078SEMG2_HUMANExclusive
      1906.615>3047NPC2_HUMANExclusive
      1945.818.5>3085NPC2_HUMANExclusive
      1945.818.5>3062KLK3_HUMANExclusive
      1964.218.25UPP7Exclusive
      2004.226.25>3041SHRM3_HUMANExclusive
      2004.226.25>3037QRIC2_HUMANExclusive
      2004.226.25>3032LRMP_HUMANExclusive
      2027.630UPP8Exclusive
      2175.739.5UPP9Exclusive
      2237.544>29192IDHC_HUMANExclusive
      2336.148.5>3035YI001_HUMANExclusive
      2977.313>3050EP3B_HUMANExclusive
      3114.612UPP10Exclusive
      3137.412.25>3051SEMG2_HUMANExclusive
      3208.7519>3039BRE1B_HUMANExclusive
      3435.171>2984SAP_HUMANExclusive
      4365.211.25UPP11Exclusive
      437411.3>3033PPAP_HUMANExclusive
      4405.212.25>29111CYTS_HUMANExclusive
      441512.75>30190CYTS_HUMANExclusive
      441512.75>3045SMG1_HUMANExclusive
      4446.413.7>2932IBP3_HUMANExclusive
      09.89UPP12HighAbsentModerate
      58.39.5>3049SEMG1_HUMANLowLowHigh
      76.59.5UPP13ModerateAbsentHigh
      186.212UPP14LowModerateHigh
      195.913UPP15ModerateAbsentHigh
      225.413UPP16HighAbsentModerate
      326.219.25>29165KLK3_HUMANHighLowLow
      816.143.7>3069PPAP_HUMANHighLowLow
      1244.415.75UPP17AbsentPresentPresent
      Note: BRE1B_HUMAN = E3 ubiquitin-protein ligase BRE1B; CYTS_HUMAN = cystatin-S; EP3B_HUMAN = epididymal secretory protein E3-beta; IBP3_HUMAN = insulin-like growth factor-binding protein 3; IDHC_HUMAN = isocitrate dehydrogenase cytoplasmic protein; KLK3_HUMAN = kallikrein-related peptidase 3; LRMP_HUMAN = lymphoid-restricted membrane protein; NPC2_HUMAN = epididymal secretory protein E1; pI = isoelectric point; PIP_HUMAN = prolactin inducible protein; PPAP_HUMAN = prostatic acid phosphatase; QRIC2_HUMAN = glutamine-rich protein 2; SAP_HUMAN = proactivator polypeptide; SEMG1/2_HUMAN = semenogelins 1 and 2; SHRM3_HUMAN = protein Shroom3; UPP = uncharacterized protein product; YI001_HUMAN = uncharacterized protein FLJ30774.
      Spot intensity was analyzed both qualitatively (presence or absence) and quantitatively (high, moderate, or low protein expression). The UniProt website (a database that contains protein information that has been extracted from the literature and computational analyses) was used for the final identification of proteins.

      Discussion

      Semen analysis is currently the test most commonly used to assess fertility potential in adult men. New tests have been developed to contribute to this assessment process; functional assays are additional tools that can be used for a better diagnosis of infertile men (
      • Aitken R.J.
      Sperm function tests and fertility.
      ). However, neither semen analysis nor functional assays can be reliably used in adolescents given the wide variation in the results (
      • Andrade-Rocha F.T.
      Significance of sperm characteristics in the evaluation of adolescents, adults and older men with varicocele.
      ,
      • Diamond D.A.
      • Gargollo P.C.
      • Caldamone A.A.
      Current management principles for adolescent varicocele.
      ). Our approach was to evaluate varicocele in a group of adolescents, and to group these adolescents according to presence or absence of varicocele, and in the case of a palpable varicocele, to an established alteration in semen quality. In this case, observation of protein expression in seminal plasma patients with altered semen quality would allow suggestions of putative mechanisms and biomarkers for fertile potential. It is also noteworthy that the study used the cutoff values for fertility established in the 1999 World Health Organization guidelines. Reclassification of our study group according to the 2010 guidelines (
      World Health Organization
      WHO laboratory manual for the examination and processing of human semen.
      ) would lead to the exclusion of two adolescents from the VAS group due to semen normality, although they still presented a differential protein expression.
      Seminal plasma contains a number of components, including lipids, carbohydrates, peptides, and proteins, which are secreted from different regions of the male reproductive tract, including the seminal vesicles, vas deferens, periurethral glands, epididymis, and prostate gland (
      • Fung K.Y.
      • Glode L.M.
      • Green S.
      • Duncan M.W.
      A comprehensive characterization of the peptide and protein constituents of human seminal fluid.
      ). The large amount of protein in the seminal plasma (35–55 g/L) makes this an excellent source for protein identification by proteomic analysis.
      Proteins are of fundamental importance in all aspects of cellular life by virtue of their complex molecular structures and ability to interact among themselves and with other compounds (
      • Krogan N.J.
      • Cagney G.
      • Yu H.
      • Zhong G.
      • Guo X.
      • Ignatchenko A.
      • et al.
      Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.
      ). The seminal fluid proteome has been attracting considerable interest from researchers in reproductive physiology and improving our understanding of male factor infertility and prostate and testicular tumors (
      • Pilch B.
      • Mann M.
      Large-scale and high-confidence proteomic analysis of human seminal plasma.
      ).
      Pilch and Mann (
      • Pilch B.
      • Mann M.
      Large-scale and high-confidence proteomic analysis of human seminal plasma.
      ) identified more than 900 proteins in seminal plasma using proteomics analysis. That study made it possible for us to detect 47 proteins well expressed in seminal plasma. These proteins are associated with motility, capacitation, inflammatory and immune responses, spermatogenesis, apoptosis, and transport of molecules of unknown function. Of the 47 proteins, only 17 were not characterized; this is most likely due to an attempt to identify weak spots by tandem mass spectrometry.

       Motility and Capacitation

      We detected the proteins semenogelin I (SgI) and semenogelin II (SgII), which together account for 20%–40% of the total protein in the seminal plasma (
      • Iwamoto T.
      • Gagnon C.
      A human seminal plasma protein blocks the motility of human spermatozoa.
      ,
      • Robert M.
      • Gagnon C.
      Semenogelin I: a coagulum forming, multifunctional seminal vesicle protein.
      ). In addition to inhibiting sperm motility, these proteins prevent premature sperm hyperactivation and capacitation (
      • de Lamirande E.
      • Yoshida K.
      • Yoshiike T.M.
      • Iwamoto T.
      • Gagnon C.
      Semenogelin, the main protein of semen coagulum, inhibits human sperm capacitation by interfering with the superoxide anion generated during this process.
      ). Expression of SgI was low in the control and VNS groups but high in the VAS group. The increased expression of SgI in adolescents with varicocele and abnormal semen parameters may reflect a strategy to counteract the deleterious effects of oxidative stress resulting from high levels of reactive oxygen species and lipid peroxidation. On the other hand, adolescents in the VAS group may exhibit decreased expression of several other proteins, thereby leading to an increase in the relative abundance of semenogelins in the seminal plasma. The increased levels of semenogelins in the VAS group may lead to a reduced fluidity of the plasma membrane, inhibiting premature sperm capacitation. The strong inhibition of sperm motility observed in the VAS group may also be attributed to the high levels of semenogelins in the seminal plasma of these adolescents.
      Kallikrein-related peptidase 3, also known as the prostate-specific antigen (PSA), is the major enzyme responsible for degradation of semenogelin in semen. The expression of PSA was high in the control group, but low in the VNS and VAS groups. This suggests that varicocele may trigger a mechanism that inhibits the expression of PSA. On the other hand, the levels of PSA in the VNS and VAS groups may be depleted due to an increase in PSA activity to prevent a premature sperm capacitation by sperm hyperactivation, which would impair the final process of fertilization.
      In addition to PSA, the prostate contributes to the production of other protein abundant in seminal plasma, the prostatic acid phosphatase. Several investigators have reported that prostatic acid phosphatase can interfere with sperm motility and capacitation and that it becomes inactive at high temperatures (
      • Curi S.M.
      • Ariagno J.I.
      • Chenlo P.H.
      • Mendeluk G.R.
      • Pugliese M.N.
      • Sardi Segovia L.M.
      • et al.
      Asthenozoospermia: analysis of a large population.
      ,
      • Lin M.F.
      • Clinton G.M.
      Human prostatic acid phosphatase has phosphotyrosyl protein phosphatase activity.
      ,
      • Lin M.F.
      • Lee C.L.
      • Clinton G.M.
      Tyrosyl kinase activity is inversely related to prostatic acid phosphatase activity in two human prostate carcinoma cell lines.
      ,
      • Ziyyat A.
      • Barraud-Lange V.
      • Sifer C.
      • Ducot B.
      • Wolf J.P.
      • Soufir J.C.
      Paradoxical increase of sperm motility and seminal carnitine associated with moderate leukocytospermia in infertile patients.
      ). The expression of prostatic acid phosphatase was high in the control group and low in the VNS and VAS groups. Varicocele may cause inactivation of proteins by increasing the gonadal temperature, thereby affecting sperm capacitation and the fertility potential of adolescents. Lymphoid-restricted membrane protein, which was only found in the control group, is also possibly related to sperm capacitation.
      Epididymis is a rich source of proteins secreted in the seminal plasma. Epididymal secretory protein E1, also known as Niemann-Pick disease C2 (NPC2) protein, was only detected in the control group. During epididymal transit, the sperm undergoes changes in cholesterol/phospholipids as the NPC2 protein binds to cholesterol, enhancing the fluidity of the plasma membrane, and consequently promoting maturation and increasing sperm motility (
      • Haidl G.
      • Opper C.
      Changes in lipids and membrane anisotropy in human spermatozoa during epididymal maturation.
      ). The fact that the NPC2 protein was only detected in the control group raises two hypotheses: [1] varicocele can cause a marked reduction in the expression of NPC2 protein, rendering the protein undetectable by two-dimensional polyacrylamide gel electrophoresis; and [2] there is a direct relationship between the reduced expression of NPC2 and reduction of progressive motility in the VAS group. A reduction in plasma membrane fluidity associated with reduced levels of NPC2 may indicate a decrease in sperm capacitation in adolescents with varicocele. The exclusive presence of NPC2 protein in the control group suggests its potential use as a biomarker of impaired spermatogenesis in adolescents with varicocele, as its expression was not even detected in the VNS group.
      The control group exhibited expression of isocitrate dehydrogenase cytoplasmic protein, also known as IDH, which is essential for energy production in aerobic processes (

      Universal Protein Resource (UniProt). Available at: http://www.uniprot.org. Accessed January 2011.

      ). In sperm, IDH is expected to be involved in energy production (in the form of nicotinamide, adenine dinucleotide [NAD]) by the middle-piece mitochondria, and in seminal plasma, IDH may participate in lipid breakdown and energy production for sperm motility.

       Immune Function

      Whether or not varicocele leads to antibody-to-surface antigen (ASA) is controversial and has been the subject of much debate (
      • Djaladat H.
      • Mehrsai A.
      • Rezazade M.
      • Djaladat Y.
      • Pourmand G.
      Varicocele and antisperm antibody: fact or fiction?.
      ,
      • Gilbert B.R.
      • Witkin S.S.
      • Goldstein M.
      Correlation of sperm-bound immunoglobulins with impaired semen analysis in infertile men with varicoceles.
      ). The role of ASAs is to expose the sperm to the immune system, induce sperm agglutination by blocking motility, and block receptors that are essential for fertilization. The prolactin-inducible protein is secreted by the seminal vesicle, binds to a constant fragment of the IgG antibody, thereby blocking its presentation to immune cells and preventing deleterious effects on fertility (
      • Chiu W.W.
      • Chamley L.W.
      Human seminal plasma antibody-binding proteins.
      ). Prolactin-inducible protein was detected only in the control group. Assuming that varicocele leads to ASA, the observed lack of expression of PRL-inducible protein in the VNS and VAS groups may be attributed to a high degradation rate of this protein in an attempt to block its presentation to the immune system.

       Apoptotic Function

      The serine/threonine protein kinase SMG1 and insulin-like growth factor binding protein-3 (IGFBP-3) were exclusively found in the VAS group. These proteins play an important role in the repair of DNA damage and promote apoptosis (
      • Brumbaugh K.M.
      • Otterness D.M.
      • Geisen C.
      • Oliveira V.
      • Brognard J.
      • Li X.
      • et al.
      The mRNA surveillance protein hSMG-1 functions in genotoxic stress response pathways in mammalian cells.
      ,
      • Ikonen M.
      • Liu B.
      • Hashimoto Y.
      • Ma L.
      • Lee K.W.
      • Niikura T.
      • et al.
      Interaction between the Alzheimer's survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis.
      ). The presence of SMG1 and IGFBP-3 in the VAS group may be a consequence of the attempted repair of germ cells undergoing oxidative stress and due to an increased frequency of apoptosis to reduce the number of damaged cells in the ejaculate resulting from abortive apoptosis. The IGFBP-3 regulates insulin-like growth factor (IGF) bioactivity and modulates cell growth and survival (
      • Ikonen M.
      • Liu B.
      • Hashimoto Y.
      • Ma L.
      • Lee K.W.
      • Niikura T.
      • et al.
      Interaction between the Alzheimer's survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis.
      ).

       Function in Spermatogenesis

      The E3 ubiquitin-protein ligase BRE1B was found only in the VNS group. The BRE1B participates in histone ubiquitination, a process responsible for signaling histones to undergo proteolytic degradation. The BRE1B is expressed at high levels in testicular tissue; the presence of BRE1B in seminal plasma suggests that its action arises from testicular processes, without interaction with mature sperm (

      Universal Protein Resource (UniProt). Available at: http://www.uniprot.org. Accessed January 2011.

      ). Therefore, BRE1B may serve as a potential protein marker of spermatogenesis. The fact that BRE1B was observed only in the VNS group may suggest it is up-regulated as a protective mechanism in the presence of varicocele, but further quantitative studies should be carried out to confirm this.

       Inflammatory Function

      In addition to cystatin-S, cystatins A, B, and C are also present in seminal plasma. Jiborn et al. (
      • Jiborn T.
      • Abrahamson M.
      • Wallin H.
      • Malm J.
      • Lundwall A.
      • Gadaleanu V.
      • et al.
      Cystatin C is highly expressed in the human male reproductive system.
      ) reported a high concentration of cystatin-C in the epididymis, which may provide protection for sperm against the proteolytic activity of bacteria. In the present study, cystatin-S was detected in the control and VAS groups. The main function of cystatins is to inhibit endogenous proteases responsible for the catabolism of peptides and proteins. One may infer that the cystatin isoform present in the control group, which comprised all adolescents without varicocele, is the active form of this protein having all functions intact. In the VAS group, the presence of two additional isoforms may indicate that this protein has undergone post-translational modifications that altered its function. This change in protein function can produce an imbalance between the need for proteolytic activity and inhibition of proteases, thereby leading to two different situations: either their activity is reduced or even suppressed due to varicocele, or their activity increases due to extensive damage to spermatogenesis in an attempt to preserve the fertility potential.

       Function in the Transport of Molecules

      Proactivator polypeptide can form a strong link with lipids, thereby acting as a carrier of these molecules into and out of cells (

      Universal Protein Resource (UniProt). Available at: http://www.uniprot.org. Accessed January 2011.

      ). Proactivator polypeptide was detected in the VNS group. Given the importance of the lipid constitution of the plasma membrane, this protein may be expressed in the VNS group in an attempt to preserve the sperm function, as the fluidity of this structure is essential for proper sperm capacitation. In addition, proactivator polypeptide can participate in lipid metabolic processes and be involved in lipid transport, therefore lipid metabolic enzymes can initiate the production of energy (

      Universal Protein Resource (UniProt). Available at: http://www.uniprot.org. Accessed January 2011.

      ).
      Epididymal secretory protein E3-beta presented a likely function in spermatid development, and no functional annotation was found for glutamine-rich protein 2, protein Shroom3, and uncharacterized protein FLJ30774.
      The aim of this study was to better understand the impact of varicocele on the proteomic profile of seminal plasma from adolescents and consequent changes in spermatogenesis and sperm function. Changes in the proteomic profile of adolescents with varicocele and normal semen parameters (VNS group) indicate that a normal semen analysis may not reflect important alterations in protein levels in the seminal plasma.
      The identification of some proteins that were exclusively expressed in each of the three groups provided a better understanding of the damage that oxidative stress causes to sperm function. The IBP-3 and SMG1proteins that were detected only in the VAS group serve to regulate cellular apoptosis. The VNS group exhibited the BRE1B, proactivator polypeptide, and epididymal secretory protein E3-beta proteins, which help maintain spermatogenesis and sperm function during hyperthermia and tissue hypoxia. The control group showed a selective expression of NPC2, IDH, and lymphoid-restricted membrane protein; their presence may be associated with homeostasis and maintenance of sperm function in a nonharmful environment. Therefore, these proteins are potential candidates for new specific biochemical markers for an early diagnosis of a negative impact of varicocele in spermatogenesis, even in the absence of changes in semen analysis.
      The implementation of genomics and proteomics will help further characterize proteins that have been identified in the seminal plasma and will facilitate the detection of new proteins associated with spermatogenesis and sperm function.

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