Heat Shock Protein and Heat Shock Factor Expression in Sperm: Relation to Oligozoospermia and Varicocele

Heat Shock Protein and Heat Shock Factor Expression in Sperm: Relation to Oligozoospermia and Varicocele Alberto Ferlin, Elena Speltra, Cristina Patassini, Mauro A. Pati, Andrea Garolla, Nicola Caretta and Carlo Foresta* From the Section of Clinical Pathology and Centre for Male Gamete Cryopreservation, Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Padova, Italy

Abbreviations and Acronyms AZFb ⫽ azoospermia factor b CN ⫽ control normozoospermia CO ⫽ control oligozoospermia FSH ⫽ follicle-stimulating hormone HSF ⫽ heat shock factor HSP ⫽ heat shock protein PCR ⫽ polymerase chain reaction RT ⫽ reverse transcriptase VN ⫽ varicocele normozoospermia VO ⫽ varicocele oligozoospermia Submitted for publication June 11, 2009. Study received institutional review board approval. Supported by Italian Ministry of University and Research Grant 2007TKYYJR. * Correspondence and requests for reprints: Section of Clinical Pathology and Centre for Male Gamete Cryopreservation, Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Via Gabelli 63, 35121 Padova, Italy (telephone: ⫹39 049 8218517; FAX: ⫹39 049 8218520; e-mail: [email protected]).

Purpose: Varicocele may be associated with normozoospermia or oligozoospermia. Much controversy still exists regarding the diagnosis, management and pathophysiology of spermatogenesis alterations associated with varicocele. The increased temperature induced by varicocele and stress in general may activate heat shock proteins and heat shock factors with a protective function in cells. We analyzed the expression of 5 heat shock proteins and heat shock factors in the sperm of men with normozoospermia and oligozoospermia with or without varicocele. Materials and Methods: We performed a prospective study between June 2008 and February 2009 at an academic clinic in 117 consecutive patients with varicocele and 68 controls without varicocele. Four groups were based on the presence/absence of varicocele and normozoospermia/oligozoospermia. Subjects were studied by history, physical examination, scrotal Doppler ultrasound, semen analysis, reproductive hormone plasma levels and quantitative real-time polymerase chain reaction in RNA extracted from ejaculated sperm to analyze HSP90, HSPA4, HSF1, HSF2 and HSFY expression. Results: Increased HSPA4, HSF1 and HSF2 were observed in the sperm of men with varicocele and in those with oligozoospermia. Levels were maximum when the 2 conditions were present. Increased HSP90 was observed in oligozoospermia cases independent of varicocele. HSFY was up-regulated only in patients with varicocele, especially those with normozoospermia. Conclusions: To our knowledge we describe for the first time the expression of different heat shock proteins and heat shock factors in ejaculated sperm. While some of these proteins are up-regulated in men with oligozoospermia and varicocele, HSFY is up-regulated only in the presence of varicocele and especially in men with normozoospermia. This suggests that it may be a molecular marker of an adequate or inadequate response to the damaging effect of varicocele on spermatogenesis. Key Words: testis, sperm, heat stress disorders, varicocele, proteins

1248

www.jurology.com

VARICOCELE, that is dilatation of veins along the spermatic cord with a backup of blood, is the most common identifiable abnormality in men evaluated for infertility. However, its role in impaired spermatogenesis is still debatable. Varicocele is found in ap-

proximately 35% of men with primary infertility but also in about 15% of the general population and it may be associated with normal fertility.1 In addition to infertility, varicocele may be associated with damaged spermatogenesis and oligoazoospermia as well

0022-5347/10/1833-1248/0 THE JOURNAL OF UROLOGY® © 2010 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION

Vol. 183, 1248-1252, March 2010 Printed in U.S.A. DOI:10.1016/j.juro.2009.11.009

AND

RESEARCH, INC.

HEAT SHOCK PROTEIN AND HEAT SHOCK FACTOR EXPRESSION IN SPERM

as normozoospermia. Despite the long history associated with varicocele there remains much controversy on its diagnosis and management, and knowledge of the pathophysiology of the spermatogenesis alterations associated with it. Many questions regarding varicocele have not yet received an adequate answer. Why does varicocele have a harmful effect on spermatogenesis only in some men? Why is surgical correction for varicocele beneficial in terms of spermatogenesis and fertility in some men but not in others? Are there molecular markers that distinguish men with varicocele and normozoospermia from those with oligoazoospermia and predict the response after varicocelectomy? Several mechanisms may be involved in the pathophysiology of damaged spermatogenesis induced by varicocele, including hypoxia, renal-adrenal reflux, hormonal dysfunction, autoimmunity, oxidative stress and above all hyperthermia.2 Increased temperature and stress in general may activate HSPs, which are in turn regulated by HSFs.3,4 These proteins have an essential role as molecular chaperones by assisting the correct folding of nascent and stress accumulated misfolded proteins, and preventing their aggregation. Thus, HSPs have a protective function, in that they allow cells to survive in otherwise lethal conditions.3–5 The are numerous HSPs and HSFs but only 2 groups have evaluated HSPA2 expression in men with varicocele.6,7 This HSP is down-regulated in the ejaculated sperm of men with oligozoospermia6 and re-expressed at a higher level 6 months after varicocelectomy.7 Although adequate comparison with men with oligozoospermia not associated with varicocele is lacking in these studies, these data suggest that HSPA2 expression may serve as a molecular marker for the acquisition of thermal tolerance in the ejaculated spermatozoa of men with varicocele. We analyzed the expression of 5 HSPs and HSFs (HSP90, HSPA4, HSF1, HSF2 and HSFY) in the ejaculated sperm of men with normozoospermia and oligozoospermia associated or not associated with varicocele. We selected these factors because they are among the best characterized factors involved in the response to stress,5 they are expressed in germ cells and animal models have shown their roles in spermatogenesis.8,9 They also cooperate in the stress response. HSP90 and HSPA4 form a multiproteic complex that corrects misfolded proteins.10 HSF1 and HSF2 stimulate HSPA4 transcription and dissociate from HSP90 after stress, allowing it to activate.11,12 HSFY is not part of this pathway and it has been less studied but we included it in this study because its gene is localized in the AZFb region of the Y chromosome, whose deletion causes a severe alteration in spermatogenesis.13 We previously mapped this gene in the AZFb region, observed its expression in germ cells and proposed it as

1249

a candidate gene for the AZFb phenotype.14 Furthermore, HSFY translocates from cytoplasm to nucleus in a stage dependent manner during spermatogenesis15 and its expression is altered in infertile patients with hypospermatogenesis.16

MATERIALS AND METHODS Subjects Our institutional review board approved this study and each participant provided informed consent. The study groups consisted of 117 patients with grade II or III left varicocele (varicocele group) and 68 without varicocele (control group) recruited consecutively from June 2008 to February 2009 at the Centre for Male Gamete Cryopreservation, University of Padova. All subjects were evaluated by history, physical examination, scrotal Doppler ultrasound, semen analysis and reproductive plasma hormone determinations, including FSH, luteinizing hormone and testosterone. Varicocele group exclusion criteria were grade I varicocele, azoospermia, medication, fever in the previous month, seminal infection, systemic disease and a history of cryptorchidism or orchitis. Varicocele was graded according to international clinical classification guidelines, including grades I—inducible during Valsalva maneuver, II—palpable and III—visible. Scrotal Doppler ultrasound was done to evaluate testicular volume and confirm reflux. According to semen analysis we subgrouped varicocele and control groups into 4 groups, including group 1— 66 men with varicocele and normal sperm concentration with a total sperm count of greater than 40 million (VN group), group 2—51 with varicocele and oligozoospermia with a total sperm count of less than 40 million (VO group), group 3— 42 controls without varicocele and with normozoospermia (CN group), and group 4 —26 controls without varicocele and with oligozoospermia (CO group). Mean ⫾ SD age of men with varicocele and controls was 29.8 ⫾ 4.5 and 30.4 ⫾ 3.8 years, respectively.

Semen Analysis Two semen samples per study subject were collected by masturbation after 2 to 4 days of sexual abstinence. After semen liquefaction at 37C standard semen analysis was performed according to WHO guidelines17 for sperm concentration, motility, morphology and vitality. Samples of the second semen collection were used for statistical analysis and then subjected to a Ficoll density gradient using Ficoll-Paque™ Plus centrifugation to recover sperm for subsequent RNA extraction. Briefly, 1 ml semen was layered on discontinuous gradient formed by 3 layers 50%, 75% and 90% from the top, respectively. Semen was placed on top of the density medium and centrifuged at 800 rpm for 20 minutes. Spermatozoa collected from the bottom layer were washed in phosphate buffered saline and immediately frozen in liquid nitrogen until RNA extraction.

RNA Extraction, cDNA Synthesis and RT-PCR Total RNA was extracted from sperm pellets using the RNA-spin™ Mini kit. First strand cDNA synthesis from total RNA was catalyzed by SuperScript® III RT using

1250

HEAT SHOCK PROTEIN AND HEAT SHOCK FACTOR EXPRESSION IN SPERM

random hexamers, including a deoxyribonuclease treatment according to the manufacturer protocol. All isolated RNA was quantified by spectrophotometry by determining the ratio of optical density at 260/280 nm using a NanoDrop® spectrophotometer. Total cDNA was amplified by PCR using specific primers for each gene to verify sperm cDNA presence and quality. As a negative control, cDNA was omitted. The primers were HSP90 forward 5=-ACAGGTGAGACCAAGGACCA-3= and reverse 5=-CGGTTTGACACAACCACCTT-3= (290 bp, exon spanning), HSP70 forward 5=-ATGAGTATAGCGACCGCTGC-3= and reverse 5=-TCCTTGGACTGTGTTCTTTGC-3= (116 bp, exon spanning), HSF1 forward 5=-GCCTTCCTGACCAAGCTGT-3= and reverse 5=-GTCGAACACGTGGAAGCTGT-3= (96 bp, exon spanning), HSF2 forward 5=-ACGAGTTCATCACCTGGAGC -3= and reverse 5=-GCCTCACAAAGCTTGCCATA-3= (120 bp, exon spanning), and HSFY forward 5=-TTTTGTTCGACAGCTCAACCT-3= and reverse 5=-CCACGCTTGAAATTTGGATT-3= (150bp, exon spanning). To control for intact RNA recovery and the uniform efficiency of each RT reaction ␤-actin was amplified by PCR using after primers, including forward 5=-CACTCTTCCAGCCTTCCTTCC-3= and reverse 5=-CGGACTCGTCATACTCCTGCT-3=. The melting temperature was 60C for all primer pairs. RT-PCR products were electrophoretically analyzed on 2% agarose gel and confirmed by direct sequencing on an ABI® Prism® sequencer.

Quantitative Real-Time PCR HSP90, HSP70, HSF1, HSF2 and HSFY expression in ejaculated sperm was quantified by real-time PCR using the Bio-Rad iQ™5 system according to manufacturer instructions with SYBR® Green PCR Master Mix. Amplification reactions were performed in a 25 ␮l final volume containing 12.5 ␮l Power SYBR Green PCR Master Mix, 1 ␮l primers (10 ␮M) and 4 ␮l (20 ng) cDNA from ejaculated sperm. Amplification was done for 45 cycles. After an initial hot start for 10 minutes each cycle consisted of 15-second denaturation, 30-second annealing at 60C and 30-second extension at 60C. To normalize the amount of expressed mRNA the internal housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (forward 5=-AAGGTGAAGGTCGGAGTCAA-3= and reverse 5=-AATGAAGGGGTCATTGATGG-3=) was used and each cDNA product was tested in triplicate. To calcu-

late data we used the comparative Ct method for relative quantification (⌬⌬Ct), which describes the change in expression of the target gene in the tested sample relative to a calibrator sample from a cDNA library and provides accurate comparison between the initial level of template in each sample. Data were analyzed with iQ5 2.0, version 2.0.148.060623 standard edition optical system software.

Statistical Analysis Comparison of data between the groups was done by the unpaired 2-tailed Student t test after acceptance of normal distribution with the Kolmogorov-Smirnov test. Data are shown as the mean ⫾ SD of the mean. The Bonferroni correction for multiple tests was applied and with p ⬍0.002 considered statistically significant. All statistical analysis was done using R open source statistical software (http://cran.r-project.org).

RESULTS The table lists baseline characteristics of men with varicocele and controls. The CO group had lower left testicular volume (p ⫽ 0.0006), total sperm count (p ⬍0.0001) and percent of A ⫹ B sperm motility (p ⫽ 0.0014) but higher FSH (p ⬍0.0001) than the CN group. The VO group had lower left testicular volume (p ⬍0.0001), total sperm count (p ⬍0.0001) and percent of A ⫹ B sperm motility (p ⫽ 0.0002) but higher FSH (p ⫽ 0.0015) than the VN group. These data confirm that oligozoospermia was due to primary testicular damage. No differences were noted in testicular volume, FSH or semen parameters in VN vs CN or VO vs CO cases. Thus, the groups were homogeneous and had similarly impaired spermatogenesis. Also, the percent of grade II and III varicocele was similar in men with normozoospermia and oligozoospermia. Expression analysis of HSP90, HSPA4, HSF1, HSF2 and HSFY in ejaculated sperm revealed significant differences in men with oligozoospermia vs normozoospermia and in men with vs without varicocele (see figure). Particularly HSPA4, HSF1 and HSF2 seem to have similar behavior while HSP90

Baseline parameters in controls and patients with varicocele

Age Testicular vol (ml): Lt Rt FSH (IU/l) Total sperm count (million) % Sperm motility A⫹B % Normal morphology % Varicocele grade: II III

Mean ⫾ SD CN

Mean ⫾ SD CO

30.3 ⫾ 3.7

30.5 ⫾ 4.3

18.5 ⫾ 4.5 19.7 ⫾ 4.9 3.8 ⫾ 1.9 165.8 ⫾ 57.4 47.8 ⫾ 18.4 35.2 ⫾ 18.4

13.8 ⫾ 6.3 14.7 ⫾ 10.1 7.7 ⫾ 1.6 18.8 ⫾ 9.4 31.2 ⫾ 22.1 36.8 ⫾ 19.7

0.0006 (3.58) 0.0079 ⬍0.0001 (8.72) ⬍0.0001 (12.92) 0.0014 (3.35) 0.7355

— —

— —

— —

p Value (t test)* 0.8394

* Only significant data shown (t test p ⬍0.002), and CN vs VN and CO vs VO not significant.

Mean ⫾ SD VN

Mean ⫾ SD VO

29.4 ⫾ 4.2

30.1 ⫾ 4.6

16.1 ⫾ 4.9 17.2 ⫾ 5.1 4.8 ⫾ 3.7 163.5 ⫾ 38.5 45.5 ⫾ 16.7 37.1 ⫾ 16.3

12.5 ⫾ 4.1 14.6 ⫾ 4.5 7.8 ⫾ 6.2 17.7 ⫾ 8.7 33.5 ⫾ 16.4 33.4 ⫾ 15.8

39 61

35 65

p Value (t test)* 0.3929 ⬍0.0001 (4.23) 0.0048 0.0015 (3.25) ⬍0.0001 (26.50) 0.0002 (3.88) 0.2198 — —

HEAT SHOCK PROTEIN AND HEAT SHOCK FACTOR EXPRESSION IN SPERM

Quantitative real-time PCR of HSP90, HSPA4, HSF1, HSF2 and HSFY mRNA expression in ejaculated sperm of men with normozoospermia and oligozoospermia with vs without varicocele. Open bars indicate CN. Light gray bars indicate CO. Dark gray bars indicate VN. Black bars indicate VO. Brackets indicate p ⬍0.0001.

and HSFY had a different pattern. HSPA4, HSF1 and HSF2 expression were significantly increased in CO vs CN cases and all 3 were significantly increased in VO vs CO cases (each p ⬍0.0001). Levels of these 3 genes were significantly lower in VN vs VO cases but significantly higher vs those in CN cases (each p ⬍0.0001). Thus HSPA4, HSF1 and HSF2 expression in sperm was increased in men with oligozoospermia, especially in those with varicocele. Expression in VN cases was between that in CO and VO cases. These data suggest that increased HSPA4, HSF1 and HSF2 expression is associated with varicocele and with spermatogenic damage, and is maximum when the 2 conditions are present. HSP90 was increased in CO and VO cases but not in VN vs CN cases (p ⬍0.0001). Thus, increased HSP90 expression was noted in men with oligozoospermia independent of the presence of varicocele. In contrast, HSFY expression was not increased in the CO group vs that in the CN group. However, it was increased in men with varicocele, especially in the VN group. Thus, increased HSFY expression seems to be related only to varicocele and VN cases showed higher expression than VO cases.

DISCUSSION To our knowledge we analyzed for the first time the expression of 5 HSPs and HSFs in the sperm of men with normozoospermia and oligozoospermia with or without varicocele. We found 3 expression patterns in relation to oligozoospermia and/or varicocele. A common pattern was observed for HSPA4, HSF1 and HSF2. Increased expression of their mRNA was associated with varicocele and with spermatogenic damage, and was at the highest level when the 2

1251

conditions were present. Thus, expression of these HSPs and HSFs seems to be stimulated by each condition and they represent markers of cellular response to the stress induced by the underlying cause of spermatogenic damage, including varicocele. Oligozoospermia in men with CO was associated with different conditions, including a history of cryptorchidism and previous orchiepididymitis or testicular trauma, or idiopathic causes. The decreased testicular volume and increased FSH corroborate the hypothesis that expression of these HSPs and HSFs is related to primary testicular damage. HSPs are described as chaperonines with protective and antiapoptotic roles in cells in response to thermal and oxidative stress. Thus, expression of this group of HSPs and HSFs in the sperm of men with oligozoospermia that was or was not associated with varicocele may be interpreted as the tendency of the testis to repair cellular damage and spermatogenic impairment. Alternatively one may speculate that high expression of these proteins may damage spermatogenesis. We observed greater expression in the sperm of men with CO vs men with CN and in men with VO vs men with VN. Supporting this hypothesis, transgenic mice that constitutively express HSF1 have arrested spermatogenesis and primary spermatocyte apoptosis while HSF1 knockout mice are fertile and have normal spermatogenesis.8 However, the opposite effect was described for HSF2 and knockout mice for this factor have decreased testicular volume and sperm count.9 Because to our knowledge the role of HSPA4 in spermatogenesis is not known, no conclusion can be drawn. In contrast, our study shows that HSP90 expression increases in oligozoospermia cases independent of varicocele. Its expression is similar in VN cases with respect to CN cases and in VO cases with respect to VN cases, making it a good marker of impaired spermatogenesis. Thus, it distinguishes men with normozoospermia from those with oligozoospermia, suggesting 2 alternative possibilities, including 1) impaired spermatogenesis due to any cause is the stress that induces HSP90 expression or 2) high HSP90 expression is dangerous for spermatogenesis. The latter hypothesis also implies an individual ability to maintain low HSP90, explaining at least in part why varicocele causes spermatogenic damage in some individuals and not in others. However, we have no data to support any of these hypotheses and only prospective studies comparing HSP90 expression. For example, an increased vs a nonincreased sperm count after varicocelectomy could be informative. The third pattern of expression was observed for HSFY, which seems to be related only to varicocele. This is an intriguing result because HSFY expres-

1252

HEAT SHOCK PROTEIN AND HEAT SHOCK FACTOR EXPRESSION IN SPERM

sion, in contrast to that observed of the other HSF and HSP, seems to be completely independent of impaired spermatogenesis and represents a specific varicocele marker. The highest HSFY expression was observed in VN cases, suggesting a protective role of this factor on spermatogenesis. Thus, men with varicocele in whom a defensive mechanism against heat stress does not develop are more likely to experience oligozoospermia. Although the function of HSFY is largely unknown, some facts support this hypothesis. HSFY is expressed in germ cells14,15 but absent in cases of deletion of the AZFb region of the Y chromosome, which causes severe spermatogenic impairment,13,14 and expression is altered in infertile patients with hypospermatogenesis.16 Thus, we hypothesize that HSFY represents a factor whose levels of expression may be important to determine whether a man with varicocele would or would not have spermatogenic damage. Different individual abilities to respond to varicocele in terms

of HSFY expression may contribute to the seminal phenotype in men with VN in those with high levels and to oligozoospermia in those with low levels. Further studies, particularly prospective studies, in men undergoing surgical correction for varicocele are important to support this hypothesis.

CONCLUSIONS To our knowledge we report for the first time that different HSPs and HSFs are expressed at different levels in the ejaculated sperm of men with normozoospermia or oligozoospermia that is or is not associated with varicocele. While some of these proteins are up-regulated in oligozoospermia and varicocele cases, HSFY is up-regulated only in the presence of varicocele. Higher expression in cases of varicocele and normozoospermia suggest that it may represent a molecular marker of adequate or inadequate response to the damaging effect of varicocele on spermatogenesis.

REFERENCES 1. Jarow JP: Effects of varicocele on male fertility. Hum Reprod Update 2001; 7: 59.

infertile men with varicocele. Urology 2005; 66: 610.

2. Fretz PC and Sandlow JI: Varicocele: current concepts in pathophysiology, diagnosis, and treatment. Urol Clin North Am 2002; 29: 921.

8. Nakai A, Suzuki M and Tanabe M: Arrest of spermatogenesis in mice expressing an active heat shock transcription factor 1. EMBO J 2000; 19: 1545.

3. Feder ME and Hofmann GE: Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu Rev Physiol 1999; 61: 243. 4. Katschinski DM: On heat and cells and proteins. News Physiol Sci 2004; 19: 11. 5. Lanneau D, Brunet M, Frisan E et al: Heat shock proteins: essential proteins for apoptosis regulation. J Cell Mol Med 2008; 12: 743.

9. Wang G, Zhang J, Moskophidis D et al: Targeted disruption of the heat shock transcription factor (hsf)-2 gene results in increased embryonic lethality, neuronal defects, and reduced spermatogenesis. Genesis 2003; 36: 48. 10. Zhang H and Burrows F: Targeting multiple signal transduction pathways through inhibition of Hsp90. J Mol Med 2004; 82: 488.

13. Ferlin A, Arredi B, Speltra E et al: Molecular and clinical characterization of Y chromosome microdeletions in infertile men: a 10-year experience in Italy. J Clin Endocrinol Metab 2007; 92: 762. 14. Tessari A, Salata E, Ferlin A et al: Characterization of HSFY, a novel AZFb gene on the Y chromosome with a possible role in human spermatogenesis. Mol Hum Reprod 2004; 10: 253. 15. Shinka T, Sato Y, Chen G et al: Molecular characterization of heat shock-like factor encoded on the human Y chromosome, and implications for male infertility. Biol Reprod 2004; 71: 297.

6. Lima SB, Cenedeze MA, Bertolla RP et al: Expression of the HSPA2 gene in ejaculated spermatozoa from adolescents with and without varicocele. Fertil Steril 2006; 86: 1659.

11. Wang X, Khaleque MA, Zhao MJ et al: Phosphorylation of HSF1 by MAPK-activated protein kinase 2 on serine 121, inhibits transcriptional activity and promotes HSP90 binding. J Biol Chem 2006; 281: 782.

16. Sato Y, Yoshida K, Shinka T et al: Altered expression pattern of heat shock transcription factor, Y chromosome (HSFY) may be related to altered differentiation of spermatogenic cells in testes with deteriorated spermatogenesis. Fertil Steril 2006; 86: 612.

7. Yes¸illi C, Mungan G, Seçkiner I et al: Effect of varicocelectomy on sperm creatine kinase, HspA2 chaperone protein (creatine kinase-M type), LDH, LDH-X, and lipid peroxidation product levels in

12. Ostling P, Björk JK, Roos-Mattjus P et al: Heat shock factor 2 (HSF2) contributes to inducible expression of hsp genes through interplay with HSF1. J Biol Chem 2007; 282: 7077.

17. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. World Health Organization. Cambridge, United Kingdom: Cambridge University Press 1999.