- Email: [email protected]
Seminal Clusterin Gene Expression Associated with Seminal Variables in Fertile and Infertile Men
Seminal Clusterin Gene Expression Associated with Seminal Variables in Fertile and Infertile Men Adel Zalata, Ayman Z. El-Samanoudy, Dalia Shaalan, Youssef El-Baiomy, Mai Taymour and Taymour Mostafa* From the Medical Biochemistry (AZ, AZES, DS) and Dermatology and Andrology (YEB) Departments, Faculty of Medicine, Mansoura University, Mansoura and Cairo University Hospitals (MT) and Andrology and Sexology Department, Faculty of Medicine (TM), Cairo University, Cairo, Egypt
Abbreviations and Acronyms AT ⫽ asthenoteratozoospermia CLU ⫽ clusterin OAT ⫽ oligoasthenoteratozoospermia OS ⫽ oxidative stress PBS ⫽ phosphate buffered saline PCR ⫽ polymerase chain reaction ROS ⫽ reactive oxygen species RT ⫽ reverse transcriptase Submitted for publication November 25, 2011. * Correspondence: Andrology and Sexology Department, Faculty of Medicine, Cairo University, Cairo, Egypt (telephone: ⫹2 01005150297; e-mail: [email protected]).
Purpose: CLU is a disulfide linked, heterodimeric protein associated with the clearance of cellular debris and apoptosis. We assessed the association of seminal CLU gene expression with seminal variables in fertile and infertile men. Materials and Methods: A total of 124 men were divided into healthy, fertile men with normozoospermia, and men with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia. History was obtained, and clinical examination and semen analysis were done. In semen we assessed sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression. Results: CLU RNA and CLU protein gene expression were significantly increased in semen samples of infertile men with oligoasthenoteratozoospermia ⬎ asthenoteratozoospermia ⬎ asthenozoospermia compared with healthy, fertile controls. CLU gene expression significantly correlated negatively with sperm count, motility, acrosin activity index, linearity index and linear velocity, and significantly correlated positively with the percent of sperm abnormal forms and DNA fragmentation. Conclusion: CLU gene expression was significantly increased in the semen samples of infertile men. It correlated negatively with sperm count, motility, acrosin activity, linearity index and linear velocity, and positively with the percent of sperm abnormal forms and DNA fragmentation. Key Words: testis; infertility, male; DNA fragmentation; spermatozoa; clusterin
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IN humans an association was reported between abnormal semen parameters and an important role of abortive apoptosis in regulating germ cell number and eliminating defective germ cells, thus maintaining normal spermatogenesis.1,2 In addition, the molecular basis of infertile semen samples is not yet fully understood due to the relative lack of knowledge of the proteins involved in normal spermatozoa physiology and the tools available to identify the corresponding proteins.3 CLU is a heterodimeric, disulfide linked glycoprotein that is expressed in
a wide variety of tissues, such as blood plasma, milk, urine, cerebrospinal fluid and semen. It is over expressed in many tissues undergoing stress, such as cancer and neurodegenerative disorders, and in cell survival under cytotoxic conditions.4 Because of its cytoprotective and anti-apoptotic properties, CLU appears to be a potentially pathophysiological gene with multiple functions related to apoptosis, inflammation, proliferation and differentiation.5 Choi6 and O’Bryan7 et al reported that the average CLU concentration in normal seminal plasma is consider-
0022-5347/12/1884-1260/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
http://dx.doi.org/10.1016/j.juro.2012.06.012 Vol. 188, 1260-1264, October 2012 RESEARCH, INC. Printed in U.S.A.
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SEMINAL CLUSTERIN GENE EXPRESSION ASSOCIATED WITH SEMINAL VARIABLES
ably higher than in serum, while it is significantly lower in cases of obstructive or nonobstructive azoospermia than of oligozoospermia or normozoospermia. Martínez-Heredia et al measured the amount of major proteins extracted from sperm samples.8 In men with asthenozoospermia CLU was detected at an increased quantity with CLU precursors. CLU was detected on 10% of spermatozoa, predominantly those that were immature or had abnormal morphology, in which seminal CLU correlated with the fertilization rate in vitro. More recently, Griffiths et al added that CLU facilitates the glycosyl phosphatidylinositol exchange involved in the post-testicular maturation of sperm and it has a role in fertilization, that is between reproductive luminal fluids and human sperm membranes.9 We assessed the association of seminal CLU gene expression with seminal variables in fertile and infertile men.
MATERIALS AND METHODS A total of 124 men from the Andrology Unit at Cairo University Hospital were prospectively included in analysis after institutional review board approval and informed consent. They were divided into 26 healthy, fertile men with normozoospermia, 32 with asthenozoospermia, 31 with AT and 35 with OAT. Semen samples were collected by masturbation after 4 or 5 days of abstinence and subjected to Autosperm (Ferti Pro, Industriepark Noord, Beerneme, Belgium) computer assisted semen analysis according to WHO guidelines.10 Each man provided 2 semen samples 2 weeks apart and the average was considered for categorization and data analysis. Spermatozoa were separated from white blood cells by Sil-Select gradient (Fertipro). Purified spermatozoa were used to assess acrosin activity and sperm DNA fragmentation.
Total RNA and Protein Extraction from Sperm Pellets Total RNA and total proteins isolation were done using the Tri-Fast™ reagent kit. Isolated RNA was determined spectrophotometrically at 260 nm. Each sample (10 l) was added to 990 l diethylpyrocarbonate treated water and quantified by measuring absorbance at 260 nm as the RNA yield in g/ml. RNA purity was determined by formaldehyde agarose gel electrophoresis and ethidium bromide staining, which showed 2 sharp, purified bands of 28S and 18S ribosomal RNA. Semiquantitative RT-PCR was performed using ReadyTo-Go™ RT-PCR Beads for first cDNA synthesis and PCR reaction. Added to the beads were 2 l first strand primer, 3 l 30 pmol PCR gene specific primer (sense), 3 l 30 pmol PCR gene specific primer (anti-sense), 25 l total template RNA containing 1 g and 17 l diethylpyrocarbonate treated water to a volume of 50 l. Dehydrated beads were incubated at 95C for 10 minutes to inactivate Moloney murine leukemia virus RT. Mineral oil (50 l) was added to overlay the reaction. Preparations were transferred to the thermal cycler and incubated at 40C for 30 minutes for cDNA synthesis, followed by incubation at 95C for 5 min-
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utes to inactivate RT and completely denature the template. The sequence of CLU gene oligonucleotide primers, designed using GenBank® sequences, was 5=-CTTGATGCCCTTCTCTCCGTA-3=(sense) and 5=-AACGTCCGAGTCAGAAGTGTG-3=(antisense), located at nucleotides 684 to 704 and 1194 to 1214 of CLU cDNA. The sequence of the oligonucleotide primers of glutaraldehyde-3-phosphate dehydrogenase was sense 5=-CGG AGT CAA CGG ATT TGG TCG TAT-3=and anti-sense 5=-AGC CTT CTC CAT GGT GGT GAA GAC-3=. Thermal cycling reaction was done using 30 cycles of denaturation at 95C for 1 minute, annealing at 55C for 1 minute and extension at 72C for 1 minute with final extension at 72C for 10 minutes. The product was subjected to agarose gel electrophoresis and visualized via an ultraviolet light transilluminator and photographed. To detect CLU protein by immunoblot we used rabbit anti-human CLU polyclonal unconjugated primary antibody against -tubulin as the control. Goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (Alpha Diagnostic International, San Antonio, Texas) was used as the secondary antibody. Colorimetric immunodetection of the protein was done using an enzyme substrate (tetramethylbenzidine) that reacted with horseradish peroxidase and precipitated on the conjugated antibodies. Membrane bands were digitally photographed to detect bands and convert to peaks. The area under each peak was calculated in square pixels for quantification. CLU gene expression and CLU protein levels were determined by calculating the ratio of the square pixel values of target bands and control bands (figs. 1 and 2).
Sperm Acrosin Activity Sperm acrosin activity11 was assessed by gelatinolysis by spreading 20 l 5% gelatin (Merck, Darmstadt, Germany) in distilled water on the slides. The slides were air dried, stored at 4C overnight, fixed and washed in PBS. Purified spermatozoa were diluted 1:10 in PBS containing 15.7 mmol ␣-D-glucose. Semen samples were smeared on the prepared slides and incubated in a moist chamber at 37C for 2 hours. The halo formation rate was calculated per slide as the percent of spermatozoa showing a halo after
Figure 1. RT-PCR product of CLU gene expression. Lane 1, DNA marker. Lane 2, normozoospermia. Lane 3, asthenozoospermia. Lane 4, AT. Lane 5, OAT. Lanes 6 and 7, negative control.
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considered significant. Statistical analysis was done using SPSS®, version 17.
RESULTS
Figure 2. Western blot of CLU protein expression at 40 kDa (A) and tubulin expression internal control at 50 kDa (B). Lane 1, normozoospermia. Lane 2, asthenozoospermia. Lane 3, AT. Lane 4, OAT.
evaluating 100 spermatozoa using the equation, acrosin activity index ⫽ halo diameter ⫻ halo formation rate.
Sperm DNA Fragmentation Analysis Sperm DNA fragmentation analysis12 was assessed by an Enhanced Apoptotic DNA Ladder Detection Kit (BioVision, Mountain View, California). The sperm pellet (5 to 10 ⫻ 105 cells in 1.5 ml tubes) was washed with PBS and centrifuged for 5 minutes at 500 ⫻ gravity. Cells were lysed with 35 l tris-ethylenediaminetetraacetic acid lysis buffer. Enzyme A solution (5 l) was added and cells were incubated at 37C for 10 minutes. Enzyme B solution (5 l) was added and cells were incubated at 50C for 30 minutes. Ammonium acetate solution (5 l) and 50 l isopropanol were added and mixed. The DNA pellet was washed with 0.5 ml 70% ethanol, air dried and dissolved in 20 l DNA suspension buffer. The sample (15 to 30 l) was added to 1.2% agarose gel containing 0.5 g/ml ethidium bromide and run at 88 V for 40 minutes. Ethidium bromide stained DNA was visualized by transillumination with ultraviolet light and photographed.
Statistical Analysis Data are shown as the mean ⫾ SD. Statistical differences were analyzed using the paired Student t test to compare 2 groups. The Spearman rank correlation coefficient was used to study relations between variables with p ⬍0.05
CLU RNA and CLU protein gene expression were significantly increased in the semen samples of infertile men with OAT ⬎ AT ⬎ asthenozoospermia compared with fertile controls (table). There was a significant negative correlation of CLU mRNA and protein expression with sperm count (r ⫽ – 0.754 and – 0.757), sperm motility (r ⫽ – 0.743 and – 0.760), sperm linear velocity (r ⫽ – 0.684 and – 0.730), sperm linearity index (r ⫽ – 0.712 and – 0.659) and sperm acrosin activity (r ⫽ – 0.697 and – 0.744, respectively, each p ⫽ 0.001). There was a significant positive correlation of CLU mRNA and protein expression with the percent of sperm abnormal forms (r ⫽ 0.614 and 0.651) and sperm DNA fragmentation (r ⫽ 0.204 and 0.236, respectively, each p ⫽ 0.001).
DISCUSSION Although it is widely accepted that under normal circumstances spermatozoa are transcriptionally silent, the sperm nucleus is a more dynamic organelle than was originally thought since there is active translation of stored mRNAs.13 In this study increased CLU expression was noted in human semen samples, which was more exaggerated with increased affected parameters. Up-regulation of CLU mRNA and full length protein was increased in infertile semen samples compared with normozoospermic samples. In infertile semen samples it was significantly increased in the OAT ⬎ AT ⬎ asthenozoospermia groups. Generally, CLU is related to the prevention of protein precipitation, agglutination of abnormal spermatozoa and control of complement induced sperm lysis.14 –16 CLU is also part of the cellular response
Semen parameters, DNA fragmentation and clusterin expression
Mean ⫾ SD sperm: Count (106/ml) % Abnormal forms % Motility Linear velocity (m/sec) Linearity index Acrosin activity % Sperm DNA fragmentation (No. pts/total No.) Mean ⫾ SD clusterin expression: RNA Protein
Normozoospermia
Asthenozoospermia
AT
OAT
54.34 ⫾ 5.0 11.42 ⫾ 2.61 60.76 ⫾ 4.49 59.62 ⫾ 8.96 79.03 ⫾ 6.99 13.22 ⫾ 3.34 11.5 (3/26)
38.85 ⫾ 4.04 10.04 ⫾ 3.7 30.90 ⫾ 5.41 44.90 ⫾ 14.68* 64.99 ⫾ 10.22* 10.01 ⫾ 2.36* 18.8 (6/32)*
23.52 ⫾ 8.94 30.80 ⫾ 7.22 23.33 ⫾ 9.43 25.47 ⫾ 11.72† 66.00 ⫾ 11.42* 5.69 ⫾ 3.13† 29.0 (9/31)†
8.00 ⫾ 3.77 39.68 ⫾ 5.6 17.68 ⫾ 10.86 23.82 ⫾ 13.57† 58.49 ⫾ 15.78* 2.54 ⫾ 2.60‡ 40.0 (14/35)‡
0.44 ⫾ 0.11 0.55 ⫾ 0.20
0.81 ⫾ 0.31* 0.82 ⫾ 0.21*
1.22 ⫾ 0.40† 1.95 ⫾ 0.40†
1.76 ⫾ 0.48‡ 3.17 ⫾ 0.65‡
* Significantly different vs normozoospermia. † Significantly different vs normozoospermia and asthenozoospermia. ‡ Significantly different vs all other groups.
SEMINAL CLUSTERIN GENE EXPRESSION ASSOCIATED WITH SEMINAL VARIABLES
to oxidative stress.17 Secretory CLU may act as an extracellular molecular chaperone, scavenging the extracellular misfolded or denatured proteins that can be produced after stress induced injury.18,19 Also, CLU may have antioxidant properties and be capable of protecting cells from apoptosis induced by ROS. In parallel, OS occurs when there is an excess of ROS and/or a decrease in antioxidant levels. ROS has been found in the seminiferous tubules and seminal plasma of most infertile patients, especially those with OAT.20 The concentration of malondialdehyde, a marker of lipid peroxidation, was almost twice as high in the sperm pellet suspensions of men with asthenozoospermia and oligoasthenozoospermia as those with normozoospermia.21 Previously, O’Bryan et al described CLU as present only in human abnormal spermatozoa,14 in accord with the consensus that in defective semen samples high ROS generation is associated with decreased seminal antioxidants.22 Trougakos and Gonos added that the CLU gene can be an extremely sensitive biosensor for ROS,18 emphasizing that CLU gene regulation by oxidative stress is the common link among all pathological conditions in which CLU is implicated. Therefore, seminal CLU may be related to the stress condition associated with abnormal semen patterns. Strocchi et al supported the notion that increased CLU expression may be a physiological defense to decrease cell damage and maintain cell viability during increased OS.23 Viard et al noted that CLU gene expression is induced in response to heat shock and OS, conferring cellular protection.19 Strocchi et al added that CLU mRNA and its over expression are involved in cytoprotection and tissue remodeling due to the cell attempt to protect itself from local stress conditions.24 This is exerted through the ability of CLU to act as a scavenger for the modified molecules induced by altered redox homeostasis in viable cells.
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In bulls CLU positive spermatozoa are abnormal and the presence of CLU inversely correlated with sperm motility, suggesting that sperm CLU in bull semen is associated with morphologically abnormal spermatozoa.15,25 Ibrahim et al concluded that aberrant spermatogenesis induced by scrotal insulation increases the percent of CLU in ram semen, suggesting that this percent could be a useful marker of poor quality ejaculate.26 Increased CLU expression was associated with an increased percent of sperm DNA fragmentation and decreased acrosin activity. The etiology of DNA damage in spermatozoa involves a cascade of changes, progressing from the induction of oxidative stress and oxidized DNA base adduct formation to sperm DNA fragmentation and cell death.27 OS may have a key role in the underlying sperm DNA fragmentation as well as in acrosin activity. Spermatozoa are sensitive to such stress because they have limited endogenous antioxidant protection, while providing abundant substrates for free radical attack in the form of unsaturated fatty acids and DNA.28 When ROS production by sperm mitochondria is excessive, the limited endogenous antioxidant defenses of the gamete are rapidly overwhelmed and oxidative damage is induced, which leads to peroxidation of the sperm acrosomal membrane, decreasing acrosin activity.29,30
CONCLUSIONS CLU gene expression was significantly increased in the semen samples of infertile men. It negatively correlated with sperm count, motility, acrosin activity index, linearity index and linear velocity, and positively correlated with sperm abnormal forms and DNA fragmentation. Seminal CLU expression may be a surrogate marker for seminal oxidative stress.
REFERENCES 1. Hassan A, el-Nashar EM, Mostafa T: Programmed cell death in varicocele-bearing testes. Andrologia 2009; 41: 39. 2. Zedan H, El-Mekhlafi AW, El-Noweihi AM et al: Soluble Fas and gonadal hormones in infertile men with varicocele. Fertil Steril 2009; 91: 420. 3. Rousseaux S, Caron C, Govin J et al: Establishment of male-specific epigenetic information. Gene 2005; 345: 139. 4. Trougakos IP and Gonos ES: Clusterin/apolipoprotein J in human aging and cancer. Int J Biochem Cell Biol 2002; 34: 1430. 5. Devauchelle V, Essabbani A, Pinieux G et al: Characterization and functional consequences of
underexpression of clusterin in rheumatoid arthritis. J Immunol 2006; 177: 6471. 6. Choi NH, Tobe T, Hara K et al: Sandwich ELISA assay for quantitative measurement of SP-40, 40 in seminal plasma and serum. J Immunol Methods 1990; 131: 159.
9. Griffiths GS, Galileo DS, Aravindan RG et al: Clusterin facilitates exchange of glycosyl phosphatidylinositol-linked SPAM1 between reproductive luminal fluids and mouse and human sperm membranes. Biol Reprod 2009; 81: 562.
7. O’Bryan MK, Baker HW, Saunders JR et al: Human seminal clusterin (SP-40,40). Isolation and characterization. J Clin Invest 1990; 85: 1477.
10. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, 4th ed. Cambridge: Cambridge University Press 1999.
8. Martínez-Heredia J, de Mateo S, Vidal-Taboada JM et al: Identification of proteomic differences in asthenozoospermic sperm samples. Hum Reprod 2008; 23: 783.
11. Henkel R, Müller C, Miska W et al: Acrosin activity of human spermatozoa by means of a simple gelatinolytic technique: a method useful for IVF. J Androl 1995; 16: 272.
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12. Daniel PT, Sturm I, Ritschel S et al: Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers. Anal Biochem 1999; 266: 110.
18. Trougakos IP and Gonos ES: Regulation of clusterin/apolipoprotein J, a functional homologue to the small heat shock proteins, by oxidative stress in ageing and age-related diseases. Free Radic Res 2006; 40: 1324.
13. Gur Y and Breitbart H: Mammalian sperm translate nuclear-encoded proteins by mitochondrialtype ribosomes. Genes Dev 2006; 20: 411.
19. Viard I, Wehrli P, Jornot L et al: Clusterin gene expression mediates resistance to apoptotic cell death induced by heat shock and oxidative stress. J Invest Dermatol 1999; 112: 290.
14. O’Bryan MK, Murphy BF, Liu DY et al: The use of anticlusterin monoclonal antibodies for the combined assessment of human sperm morphology and acrosome integrity. Hum Reprod 1994; 9: 1490.
20. Mostafa T, Anis T, El Nashar A et al: Seminal plasma reactive oxygen species-antioxidants relationship with varicocele grade. Andrologia 2012; 44: 66.
15. Ibrahim NM, Gilbert GR, Loseth KL et al: Correlation between clusterin-positive spermatozoa determined by flow cytometry in bull semen and fertility. J Androl 2000; 21: 887. 16. Reyes-Moreno C, Boilard M, Sullivan R et al: Characterization and identification of epididymal factors that protect ejaculated bovine sperm during in vitro storage. Biol Reprod 2002; 66: 159. 17. Vaithinathan S, Saradha B and Mathur PP: Methoxychlor-induced alteration in the levels of HSP70 and clusterin is accompanied with oxidative stress in adult rat testis. J Biochem Mol Toxicol 2009; 23: 29.
21. Suleiman SA, Ali ME, Zaki ZM et al: Lipid peroxidation and human sperm motility: protective role of vitamin E. J Androl 1996; 17: 530. 22. Mostafa T, Anis T, Imam H et al: Seminal reactive oxygen species-antioxidant relationship in fertile males with and without varicocele. Andrologia 2009; 41: 125. 23. Strocchi P, Smith MA, Perry G et al: Clusterin up-regulation following sub-lethal oxidative stress and lipid peroxidation in human neuroblastoma cells. Neurobiol Aging 2006; 27: 1588. 24. Strocchi P, Rauzi F and Cevolani D: Neuronal loss up-regulates clusterin mRNA in living neurons
and glial cells in the rat brain. Neuroreport 1999; 10: 1789. 25. Ibrahim NM, Foster DN and Crabo BG: Localization of clusterin on freeze-preserved bull spermatozoa before and after glass wool-Sephadex filtration. J Androl 2001; 22: 891. 26. Ibrahim NM, Romano JE, Troedsson MH et al: Effect of scrotal insulation on clusterin-positive cells in ram semen and their relationship to semen quality. J Androl 2001; 22: 863. 27. Aitken RJ, De Iuliis GN, Finnie JM et al: Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria. Hum Reprod 2010; 25: 2415. 28. Koppers AJ, Garg ML and Aitken RJ: Stimulation of mitochondrial reactive oxygen species production by unesterified, unsaturated fatty acids in defective human spermatozoa. Free Radic Biol Med 2010; 48: 112. 29. Zalata AA, Ahmed AH, Allamaneni SS et al: Relationship between acrosin activity of human spermatozoa and oxidative stress. Asian J Androl 2004; 6: 313. 30. Sadek A, Almohamdy AS, Zaki A et al: Sperm chromatin condensation in infertile men with varicocele before and after surgical repair. Fertil Steril 2011; 95: 1705.
Abbreviations and Acronyms AT ⫽ asthenoteratozoospermia CLU ⫽ clusterin OAT ⫽ oligoasthenoteratozoospermia OS ⫽ oxidative stress PBS ⫽ phosphate buffered saline PCR ⫽ polymerase chain reaction ROS ⫽ reactive oxygen species RT ⫽ reverse transcriptase Submitted for publication November 25, 2011. * Correspondence: Andrology and Sexology Department, Faculty of Medicine, Cairo University, Cairo, Egypt (telephone: ⫹2 01005150297; e-mail: [email protected]).
Purpose: CLU is a disulfide linked, heterodimeric protein associated with the clearance of cellular debris and apoptosis. We assessed the association of seminal CLU gene expression with seminal variables in fertile and infertile men. Materials and Methods: A total of 124 men were divided into healthy, fertile men with normozoospermia, and men with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia. History was obtained, and clinical examination and semen analysis were done. In semen we assessed sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression. Results: CLU RNA and CLU protein gene expression were significantly increased in semen samples of infertile men with oligoasthenoteratozoospermia ⬎ asthenoteratozoospermia ⬎ asthenozoospermia compared with healthy, fertile controls. CLU gene expression significantly correlated negatively with sperm count, motility, acrosin activity index, linearity index and linear velocity, and significantly correlated positively with the percent of sperm abnormal forms and DNA fragmentation. Conclusion: CLU gene expression was significantly increased in the semen samples of infertile men. It correlated negatively with sperm count, motility, acrosin activity, linearity index and linear velocity, and positively with the percent of sperm abnormal forms and DNA fragmentation. Key Words: testis; infertility, male; DNA fragmentation; spermatozoa; clusterin
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IN humans an association was reported between abnormal semen parameters and an important role of abortive apoptosis in regulating germ cell number and eliminating defective germ cells, thus maintaining normal spermatogenesis.1,2 In addition, the molecular basis of infertile semen samples is not yet fully understood due to the relative lack of knowledge of the proteins involved in normal spermatozoa physiology and the tools available to identify the corresponding proteins.3 CLU is a heterodimeric, disulfide linked glycoprotein that is expressed in
a wide variety of tissues, such as blood plasma, milk, urine, cerebrospinal fluid and semen. It is over expressed in many tissues undergoing stress, such as cancer and neurodegenerative disorders, and in cell survival under cytotoxic conditions.4 Because of its cytoprotective and anti-apoptotic properties, CLU appears to be a potentially pathophysiological gene with multiple functions related to apoptosis, inflammation, proliferation and differentiation.5 Choi6 and O’Bryan7 et al reported that the average CLU concentration in normal seminal plasma is consider-
0022-5347/12/1884-1260/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
http://dx.doi.org/10.1016/j.juro.2012.06.012 Vol. 188, 1260-1264, October 2012 RESEARCH, INC. Printed in U.S.A.
AND
SEMINAL CLUSTERIN GENE EXPRESSION ASSOCIATED WITH SEMINAL VARIABLES
ably higher than in serum, while it is significantly lower in cases of obstructive or nonobstructive azoospermia than of oligozoospermia or normozoospermia. Martínez-Heredia et al measured the amount of major proteins extracted from sperm samples.8 In men with asthenozoospermia CLU was detected at an increased quantity with CLU precursors. CLU was detected on 10% of spermatozoa, predominantly those that were immature or had abnormal morphology, in which seminal CLU correlated with the fertilization rate in vitro. More recently, Griffiths et al added that CLU facilitates the glycosyl phosphatidylinositol exchange involved in the post-testicular maturation of sperm and it has a role in fertilization, that is between reproductive luminal fluids and human sperm membranes.9 We assessed the association of seminal CLU gene expression with seminal variables in fertile and infertile men.
MATERIALS AND METHODS A total of 124 men from the Andrology Unit at Cairo University Hospital were prospectively included in analysis after institutional review board approval and informed consent. They were divided into 26 healthy, fertile men with normozoospermia, 32 with asthenozoospermia, 31 with AT and 35 with OAT. Semen samples were collected by masturbation after 4 or 5 days of abstinence and subjected to Autosperm (Ferti Pro, Industriepark Noord, Beerneme, Belgium) computer assisted semen analysis according to WHO guidelines.10 Each man provided 2 semen samples 2 weeks apart and the average was considered for categorization and data analysis. Spermatozoa were separated from white blood cells by Sil-Select gradient (Fertipro). Purified spermatozoa were used to assess acrosin activity and sperm DNA fragmentation.
Total RNA and Protein Extraction from Sperm Pellets Total RNA and total proteins isolation were done using the Tri-Fast™ reagent kit. Isolated RNA was determined spectrophotometrically at 260 nm. Each sample (10 l) was added to 990 l diethylpyrocarbonate treated water and quantified by measuring absorbance at 260 nm as the RNA yield in g/ml. RNA purity was determined by formaldehyde agarose gel electrophoresis and ethidium bromide staining, which showed 2 sharp, purified bands of 28S and 18S ribosomal RNA. Semiquantitative RT-PCR was performed using ReadyTo-Go™ RT-PCR Beads for first cDNA synthesis and PCR reaction. Added to the beads were 2 l first strand primer, 3 l 30 pmol PCR gene specific primer (sense), 3 l 30 pmol PCR gene specific primer (anti-sense), 25 l total template RNA containing 1 g and 17 l diethylpyrocarbonate treated water to a volume of 50 l. Dehydrated beads were incubated at 95C for 10 minutes to inactivate Moloney murine leukemia virus RT. Mineral oil (50 l) was added to overlay the reaction. Preparations were transferred to the thermal cycler and incubated at 40C for 30 minutes for cDNA synthesis, followed by incubation at 95C for 5 min-
1261
utes to inactivate RT and completely denature the template. The sequence of CLU gene oligonucleotide primers, designed using GenBank® sequences, was 5=-CTTGATGCCCTTCTCTCCGTA-3=(sense) and 5=-AACGTCCGAGTCAGAAGTGTG-3=(antisense), located at nucleotides 684 to 704 and 1194 to 1214 of CLU cDNA. The sequence of the oligonucleotide primers of glutaraldehyde-3-phosphate dehydrogenase was sense 5=-CGG AGT CAA CGG ATT TGG TCG TAT-3=and anti-sense 5=-AGC CTT CTC CAT GGT GGT GAA GAC-3=. Thermal cycling reaction was done using 30 cycles of denaturation at 95C for 1 minute, annealing at 55C for 1 minute and extension at 72C for 1 minute with final extension at 72C for 10 minutes. The product was subjected to agarose gel electrophoresis and visualized via an ultraviolet light transilluminator and photographed. To detect CLU protein by immunoblot we used rabbit anti-human CLU polyclonal unconjugated primary antibody against -tubulin as the control. Goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (Alpha Diagnostic International, San Antonio, Texas) was used as the secondary antibody. Colorimetric immunodetection of the protein was done using an enzyme substrate (tetramethylbenzidine) that reacted with horseradish peroxidase and precipitated on the conjugated antibodies. Membrane bands were digitally photographed to detect bands and convert to peaks. The area under each peak was calculated in square pixels for quantification. CLU gene expression and CLU protein levels were determined by calculating the ratio of the square pixel values of target bands and control bands (figs. 1 and 2).
Sperm Acrosin Activity Sperm acrosin activity11 was assessed by gelatinolysis by spreading 20 l 5% gelatin (Merck, Darmstadt, Germany) in distilled water on the slides. The slides were air dried, stored at 4C overnight, fixed and washed in PBS. Purified spermatozoa were diluted 1:10 in PBS containing 15.7 mmol ␣-D-glucose. Semen samples were smeared on the prepared slides and incubated in a moist chamber at 37C for 2 hours. The halo formation rate was calculated per slide as the percent of spermatozoa showing a halo after
Figure 1. RT-PCR product of CLU gene expression. Lane 1, DNA marker. Lane 2, normozoospermia. Lane 3, asthenozoospermia. Lane 4, AT. Lane 5, OAT. Lanes 6 and 7, negative control.
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considered significant. Statistical analysis was done using SPSS®, version 17.
RESULTS
Figure 2. Western blot of CLU protein expression at 40 kDa (A) and tubulin expression internal control at 50 kDa (B). Lane 1, normozoospermia. Lane 2, asthenozoospermia. Lane 3, AT. Lane 4, OAT.
evaluating 100 spermatozoa using the equation, acrosin activity index ⫽ halo diameter ⫻ halo formation rate.
Sperm DNA Fragmentation Analysis Sperm DNA fragmentation analysis12 was assessed by an Enhanced Apoptotic DNA Ladder Detection Kit (BioVision, Mountain View, California). The sperm pellet (5 to 10 ⫻ 105 cells in 1.5 ml tubes) was washed with PBS and centrifuged for 5 minutes at 500 ⫻ gravity. Cells were lysed with 35 l tris-ethylenediaminetetraacetic acid lysis buffer. Enzyme A solution (5 l) was added and cells were incubated at 37C for 10 minutes. Enzyme B solution (5 l) was added and cells were incubated at 50C for 30 minutes. Ammonium acetate solution (5 l) and 50 l isopropanol were added and mixed. The DNA pellet was washed with 0.5 ml 70% ethanol, air dried and dissolved in 20 l DNA suspension buffer. The sample (15 to 30 l) was added to 1.2% agarose gel containing 0.5 g/ml ethidium bromide and run at 88 V for 40 minutes. Ethidium bromide stained DNA was visualized by transillumination with ultraviolet light and photographed.
Statistical Analysis Data are shown as the mean ⫾ SD. Statistical differences were analyzed using the paired Student t test to compare 2 groups. The Spearman rank correlation coefficient was used to study relations between variables with p ⬍0.05
CLU RNA and CLU protein gene expression were significantly increased in the semen samples of infertile men with OAT ⬎ AT ⬎ asthenozoospermia compared with fertile controls (table). There was a significant negative correlation of CLU mRNA and protein expression with sperm count (r ⫽ – 0.754 and – 0.757), sperm motility (r ⫽ – 0.743 and – 0.760), sperm linear velocity (r ⫽ – 0.684 and – 0.730), sperm linearity index (r ⫽ – 0.712 and – 0.659) and sperm acrosin activity (r ⫽ – 0.697 and – 0.744, respectively, each p ⫽ 0.001). There was a significant positive correlation of CLU mRNA and protein expression with the percent of sperm abnormal forms (r ⫽ 0.614 and 0.651) and sperm DNA fragmentation (r ⫽ 0.204 and 0.236, respectively, each p ⫽ 0.001).
DISCUSSION Although it is widely accepted that under normal circumstances spermatozoa are transcriptionally silent, the sperm nucleus is a more dynamic organelle than was originally thought since there is active translation of stored mRNAs.13 In this study increased CLU expression was noted in human semen samples, which was more exaggerated with increased affected parameters. Up-regulation of CLU mRNA and full length protein was increased in infertile semen samples compared with normozoospermic samples. In infertile semen samples it was significantly increased in the OAT ⬎ AT ⬎ asthenozoospermia groups. Generally, CLU is related to the prevention of protein precipitation, agglutination of abnormal spermatozoa and control of complement induced sperm lysis.14 –16 CLU is also part of the cellular response
Semen parameters, DNA fragmentation and clusterin expression
Mean ⫾ SD sperm: Count (106/ml) % Abnormal forms % Motility Linear velocity (m/sec) Linearity index Acrosin activity % Sperm DNA fragmentation (No. pts/total No.) Mean ⫾ SD clusterin expression: RNA Protein
Normozoospermia
Asthenozoospermia
AT
OAT
54.34 ⫾ 5.0 11.42 ⫾ 2.61 60.76 ⫾ 4.49 59.62 ⫾ 8.96 79.03 ⫾ 6.99 13.22 ⫾ 3.34 11.5 (3/26)
38.85 ⫾ 4.04 10.04 ⫾ 3.7 30.90 ⫾ 5.41 44.90 ⫾ 14.68* 64.99 ⫾ 10.22* 10.01 ⫾ 2.36* 18.8 (6/32)*
23.52 ⫾ 8.94 30.80 ⫾ 7.22 23.33 ⫾ 9.43 25.47 ⫾ 11.72† 66.00 ⫾ 11.42* 5.69 ⫾ 3.13† 29.0 (9/31)†
8.00 ⫾ 3.77 39.68 ⫾ 5.6 17.68 ⫾ 10.86 23.82 ⫾ 13.57† 58.49 ⫾ 15.78* 2.54 ⫾ 2.60‡ 40.0 (14/35)‡
0.44 ⫾ 0.11 0.55 ⫾ 0.20
0.81 ⫾ 0.31* 0.82 ⫾ 0.21*
1.22 ⫾ 0.40† 1.95 ⫾ 0.40†
1.76 ⫾ 0.48‡ 3.17 ⫾ 0.65‡
* Significantly different vs normozoospermia. † Significantly different vs normozoospermia and asthenozoospermia. ‡ Significantly different vs all other groups.
SEMINAL CLUSTERIN GENE EXPRESSION ASSOCIATED WITH SEMINAL VARIABLES
to oxidative stress.17 Secretory CLU may act as an extracellular molecular chaperone, scavenging the extracellular misfolded or denatured proteins that can be produced after stress induced injury.18,19 Also, CLU may have antioxidant properties and be capable of protecting cells from apoptosis induced by ROS. In parallel, OS occurs when there is an excess of ROS and/or a decrease in antioxidant levels. ROS has been found in the seminiferous tubules and seminal plasma of most infertile patients, especially those with OAT.20 The concentration of malondialdehyde, a marker of lipid peroxidation, was almost twice as high in the sperm pellet suspensions of men with asthenozoospermia and oligoasthenozoospermia as those with normozoospermia.21 Previously, O’Bryan et al described CLU as present only in human abnormal spermatozoa,14 in accord with the consensus that in defective semen samples high ROS generation is associated with decreased seminal antioxidants.22 Trougakos and Gonos added that the CLU gene can be an extremely sensitive biosensor for ROS,18 emphasizing that CLU gene regulation by oxidative stress is the common link among all pathological conditions in which CLU is implicated. Therefore, seminal CLU may be related to the stress condition associated with abnormal semen patterns. Strocchi et al supported the notion that increased CLU expression may be a physiological defense to decrease cell damage and maintain cell viability during increased OS.23 Viard et al noted that CLU gene expression is induced in response to heat shock and OS, conferring cellular protection.19 Strocchi et al added that CLU mRNA and its over expression are involved in cytoprotection and tissue remodeling due to the cell attempt to protect itself from local stress conditions.24 This is exerted through the ability of CLU to act as a scavenger for the modified molecules induced by altered redox homeostasis in viable cells.
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In bulls CLU positive spermatozoa are abnormal and the presence of CLU inversely correlated with sperm motility, suggesting that sperm CLU in bull semen is associated with morphologically abnormal spermatozoa.15,25 Ibrahim et al concluded that aberrant spermatogenesis induced by scrotal insulation increases the percent of CLU in ram semen, suggesting that this percent could be a useful marker of poor quality ejaculate.26 Increased CLU expression was associated with an increased percent of sperm DNA fragmentation and decreased acrosin activity. The etiology of DNA damage in spermatozoa involves a cascade of changes, progressing from the induction of oxidative stress and oxidized DNA base adduct formation to sperm DNA fragmentation and cell death.27 OS may have a key role in the underlying sperm DNA fragmentation as well as in acrosin activity. Spermatozoa are sensitive to such stress because they have limited endogenous antioxidant protection, while providing abundant substrates for free radical attack in the form of unsaturated fatty acids and DNA.28 When ROS production by sperm mitochondria is excessive, the limited endogenous antioxidant defenses of the gamete are rapidly overwhelmed and oxidative damage is induced, which leads to peroxidation of the sperm acrosomal membrane, decreasing acrosin activity.29,30
CONCLUSIONS CLU gene expression was significantly increased in the semen samples of infertile men. It negatively correlated with sperm count, motility, acrosin activity index, linearity index and linear velocity, and positively correlated with sperm abnormal forms and DNA fragmentation. Seminal CLU expression may be a surrogate marker for seminal oxidative stress.
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