Hum. Reprod. Advance Access originally published online on March 3, 2006
Human Reproduction 2006 21(7):1791-1794; doi:10.1093/humrep/del055
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Oligozoospermia and heat-shock protein expression in ejaculated spermatozoa
1 Department of Surgery, Division of Urology 2 Department of Medicine, Division of Nephrology, São Paulo Federal University, Sao Paulo, Brazil and 3 The Jones Institute for Reproductive Medicine, Norfolk, VA, USA
4 To whom correspondence should be addressed at: Rua Leandro Dupret, 204 cj 43, 04025-010 São Paulo, SP, Brazil. E-mail: samirabl{at}uol.com.br
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Abstract |
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BACKGROUND: Heat-shock protein A2 (HspA2) is correlated with sperm maturity, function and fertility, and a dysfunctional expression of such a gene results in abnormal spermatogenesis. The purpose of this study was to compare HspA2 gene expression in spermatozoa from oligozoospermic men and normozoospermic controls. METHODS: Semen was obtained and analysed according to World Health Organization (World Health Organization, 1999
) guidelines, morphology by Kruger’s strict criteria. Seventeen patients with oligozoospermia and 21 fertile controls were studied. Total RNA was extracted from ejaculated and Percoll density-gradient-separated spermatozoa followed by semiquantitative RT–PCR analysis. The relative expression level of HspA2 was analysed according to the expression level of the housekeeping 
-actin gene. Serum hormonal profiles (FSH, LH and testosterone) and a peripheral karyotype were also performed. RESULTS: All patients possessed normal karyotype, and no significant hormonal differences were found between the two groups. The study group had significantly lower sperm concentration and normal morphology than the controls. Semiquantitative RT–PCR analysis of HspA2 showed significantly lower expression levels in the oligoteratozoospermic men when compared to controls (P = 0.0021). CONCLUSIONS: The HspA2 gene was down-regulated in sperm from infertile men with idiopathic oligoteratozoospermia, suggesting that such anomalies of gene expression might be associated with pathogenesis in some subtypes of male infertility.
Key words: gene expression/HspA2/male infertility/oligozoospermia
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Introduction |
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It is estimated that about 15% of couples present with reduced fertility, and male infertility is responsible for half these cases (World Health Organization, 1999
). A large proportion of infertile patients are only able to conceive through the use of assisted reproduction technologies (ART). Besides the reduction in sperm count, oligozoospermic men generally possess a higher rate of sperm with abnormal morphology and/or low progressive motility, which leads to lower fertility (World Health Organization, 1999). These oligozoospermic men are candidates for ICSI.
Spermatogenesis is a unique process of cellular differentiation in which diploid testicular stem cells differentiate into haploid spermatozoa (Eddy et al., 1991). Much remains to be learned about the regulation of gene expression during this process. Some developmentally regulated genes are expressed exclusively in spermatogenic germ cells, whereas others are expressed in both germ cells and somatic cells. Some of these genes expressed only in germ cells are homologues of genes transcribed in somatic cells and are members of specific gene families (Eddy, 1999), such as the heat-shock proteins (hsps) families, which are highly conserved cellular stress proteins, present in every organism from bacteria to man (Neuer et al., 2000).
The HspA2, a member of the 70-kDa hsp family, is a molecular chaperone that assists in the folding, transport and assembly of proteins in the cytoplasm, mitochondria and endoplasmic reticulum (Georgopoulos and Welch, 1993). hsps have a protective action on the cellular auto-regulation in response to heat (Santoro, 2000) and on the mechanism of homeostasis, providing a balance between protein synthesis and degradation (Shi et al., 1998). The mRNA for one of the hsps, HspA2, has also been shown to be induced by cell-free seminal fluid as well as by isolated motile spermatozoa (Jeremias et al., 1999).
Dysfunctional expression of regulated genes may result in abnormal spermatogenesis (Eddy et al., 1991). In maturation arrest testes, HspA2 protein levels were shown to be lower than those in normal men and are completely absent in testes with Sertoli cell-only syndrome. The authors also demonstrated that HspA2 is expressed during the spermatocyte and spermatid stages of spermatogenesis (Feng et al., 2001). These results agree with Son et al. (2000), who demonstrated repression of HspA2 mRNA in testicular biopsy material from men with abnormal spermatogenesis.
It has also been demonstrated that HspA2 is present in ejaculated human sperm (Huszar et al., 2000) and that a lower expression of HspA2 correlates with a higher percentage of cells with residual cytoplasm, determined by creatine kinase (CK) activity (Huszar and Vigue, 1990). This demonstrates that HspA2 is essential for normal spermatogenesis, which is confirmed by the fact that lower HspA2/CK ratios correlate with lower fertility rates in IVF programmes (Huszar et al., 1992).
Dix et al. (1996) demonstrated that knockout of the HspA2 homologue Hsp70-2 gene in mice leads to maturation arrest of primary spermatocytes at stage 1 of meiosis, and the lack of the HSP-70 gene led to a significant increase in apoptosis. It has been shown in mice that HSP70-2 functions in maintaining the synaptonemal complexes and assisting chromosome crossing-over during meiosis and spermatocyte differentiation, which is demonstrated by the fact that mice with targeted disruption of the hsp70-2 gene present with arrested spermatocyte differentiation and azoospermia (Dix et al., 1997). Spinaci et al. (2005) showed that the HSP70 protein is present in mature sperm and that treatment of the IVF media with anti-HSP70 antibodies led to lower fertilization rates, whereas Kamaruddin et al. (2004) showed that in bovine sperm HSP70 is located in the cytoplasm during the spermatocyte stage, after which it migrates and is located to the acrosome in freshly ejaculated sperm.
In this study, we aimed to investigate the relationship between the HSP70-2 gene in ejaculated human sperm and infertility. For this purpose, we compared the expression levels of the HSP70-2 messenger RNA (mRNA) in ejaculated spermatozoa between oligozoospermic men and normozoospermic controls.
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Materials and methods |
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Patients
This was a prospectively designed, controlled observational study. We investigated 17 oligozoospermic (<20 x 106 sperm/ml) patients with idiopathic infertility diagnosed following completion of infertility work up (study group) and who were candidates for ICSI in our ART programme and 21 normozoospermic men with a history of recent parenthood (control group). The sole inclusion criterion for the study group was oligozoospermia, and the exclusion criterion for both groups was the presence of leukospermia. The age distribution for both groups varied from 24 to 33 years. This study was approved by the Institutional Review Board of São Paulo Federal University and informed consent was obtained from all patients.
Semen samples
Semen samples were collected by masturbation in an area adjacent to the laboratory following 2–4 days of sexual abstinence. After semen liquefaction, standard semen analysis was performed according to the World Health Organization guidelines criteria (World Health Organization, 1999). Sperm morphology was by strict criteria after Diff-Quick staining. All samples tested negative for cultures of micro-organisms and were free of leukocytes (peroxidase staining).
Immediately upon collection and examination, each semen sample was subjected to a separation of the fraction of sperm with high motility. This was accomplished by Percoll density-gradient centrifugation. Briefly, 1 ml of semen was layered on discontinuous two-layer (45 and 90%) Percoll gradients (170891-01/GE Healthcare, Uppsala, Sweden) and centrifuged at 600 x g for 20 min in 15 ml conical tubes. The medium used to dilute the Percoll was human tubal fluid (98270, Irvine, Santa Ana, CA, USA) supplemented with 10% bovine serum albumin (BSA) (A-6003, Sigma, St. Louis, MO, USA).
Spermatozoa collected from the bottom layer (90% layer) were resuspended in HTF medium, and the tube was centrifuged at 600 x g for 10 min. Extreme care was taken to collect the pelleted spermatozoa, avoiding the seminal plasma and interphase material containing any potentially contaminating cells. Furthermore, a small aliquot from the pelleted spermatozoa was again examined to confirm the absence of peroxidase-positive contaminating leukocytes. Immediately thereafter, the sperm pellets were flash frozen in liquid nitrogen. Samples were stored at –80°C until RNA extraction. Post-Percoll sperm motility and concentration were not assessed to avoid mRNA degradation. Thus, HspA2 levels were determined based on the expression of a housekeeping gene (-actin) and not on sperm concentration.
Blood samples
Two blood samples were collected by peripheral venipuncture for (i) karyotype (collected in heparinized syringe, Butantan Genetics Laboratory, São Paulo, Brazil) and (ii) serum testosterone, LH and FSH quantification (collected in silicon vacutainer without anti-clotting, Hormonal Dosage Laboratory of the Human Reproduction Section, São Paulo Federal University). Karyotypes were analysed by a modified Moorhead G banding technique (Moorhead et al., 1960). The hormonal quantification of FSH, LH and testosterone was performed using chemiluminescence (DPC-MedLab®) with an intra-assay coefficient of variation <10%.
Total RNA preparation and RT–PCR analysis
Total RNA was extracted from sperm pellets using the TRIzol reagent according to the manufacturer’s instruction (Gibco BRL®). First-strand cDNA synthesis from total RNA was catalysed by Superscript II RT (Invitrogen, Gaithersburg, MD, USA) using oligo (dT) 12–18 according to manufacturer’s protocol. PCR was performed with 3 µl of cDNA preparation using Open Reading Frame sequence-specific primers made on the basis of gene sequences identified with a BLAST search for -actin: sense, 5'-CGT GAC ATT AAG GAG AAG CTG TGC-3'; antisense, 5'-CTC AGG AGG AGC AAT GAT CTT GAT-3', and the synthetic oligonucleotide primers (343-bp fragment) of HspA2 were sense, 5'-TTG TTG GAA GTC TTT GGT ATA-3' and antisense, 5'-CAT TTG CAT TTA TGC ATT TGT-3' (Son et al., 2000).
Pilot tests were carried out to determine the appropriate amount of RNA to be used, thus optimizing the RT reaction. Five micrograms of RNA was used from each sample for a total of 20 samples. Contaminating DNA was eliminated with the DNAse I enzyme reaction buffer (GIBCO BRL®). After these tests for optimization of the technique, including selection of the best reagent concentrations and for the annealing temperature, the following standard conditions were used: 10 min at 94°C for DNA denaturation followed by 36 cycles (1 min at 94°C, 1 min at 57°C, 1 min at 72°C) and a final extension for 10 min at 72°C using a Master Mix kit (Eppendorf®, Hamburg, Germany).
The cDNA amplification was assessed on 2% agarose gels stained with SYBR Green I (excitation maximum 497 nm and emission maximum 520 nm) nucleic acid gel stain—10 000x in dimethylsulphoxide (DMSO) (Invitrogen, Gaithersburg, MD, USA), which emits blue fluorescence. After this step, the gel was analysed with the optical scanner system STORMTM (Molecular Dynamics®, GE Helathcare, Upsala, Sweden). By fluorescence, it was possible to estimate comparatively the amount of cDNA (semiquantified) referring to the genes under study. The presence of the expected band was established by confirmation of size in base pairs (bp) using as standard the PHIS RFDNA plasmid digested with the restriction enzyme Hae III. The cDNA amplifications of each sample were assessed for the -actin and HspA2 genes. The samples were first analysed for -actin, which characterizes the presence of total RNA in the material under study, and then for HspA2. The HspA2/-actin ratio (absorbance unit at 520 nm) was assessed in both groups through reading with STORMTM and later analysis with the ImageQuant software (Molecular Dynamics®). Maximum inter-assay variation was 10% (mean = 5.61%).
Statistical analysis
Statistical analysis was performed using Student’s t-test. Data are presented as mean ± SD and a value of P < 0.05 was considered to indicate a statistically significant difference.
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Results |
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Patients from each group did not differ in age and all possessed a normal karyotype (46,XY). The hormonal parameters did not show any statistical differences between the groups. No statistical difference was found as to progressive sperm motility. However, significant differences were found in sperm concentration and normal morphology between the oligozoospermic and the normozoospermic men (Table I).
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-Actin levels were similar in both groups and presented a normal distribution. Semiquantitative RT–PCR analysis was performed to examine HspA2 mRNA gene expression and to compare spermatozoa from infertile and fertile groups (Figures 1 and 2). The data demonstrated a significantly lower level of HspA2 mRNA expression in the oligoteratozoospermic men compared to controls (P = 0.0021) (Table I).
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No correlations were observed between sperm HspA2/
-actin levels and sperm morphology (r < 0.30) or motility (r < 0.30). |
Discussion |
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hsps have been identified as a critical component of a very complex and highly conserved cellular defensive mechanism to preserve cell survival under adverse environmental conditions. hsps are preferentially expressed in response to an array of insults, including hyperthermia, oxidative stress, heavy metals, ethanol, amino acid analogues, inflammation and infection (Neuer et al., 2000
). The stress-elicited activation of heat-shock genes is called heat-shock response and is frequently found in clinical situations. The presence of HspA2 is correlated in spermatogenesis with maturity, function and fertility (Huszar et al., 2000). In this study, semiquantitative RT–PCR analysis demonstrated that HspA2 expression was significantly decreased in infertile men with abnormal spermatogenesis leading to oligoteratozoospermia when compared with fertile controls with normozoospermia. Even when considering the 10% inter-assay variation, a significant difference was still observed (P = 0.026), which demonstrates a clear difference between both groups. We speculate that the oligoteratozoospermic group revealed a failure to develop a defensive mechanism, which led to a decreased level of protection. This in turn could have resulted in a decreased sperm production and a compromised morphology.
This observation extends a previous study on the effects of an HSP70 (a homologue for the human HspA2) gene knockout in murine spermatogenesis, which led to failure in meiosis, increased apoptosis and correlated with a male infertility phenotype (Dix et al., 1996). Similar results were demonstrated in infertile men (Moorhead et al., 1960), showing that HspA2 gene expression was down-regulated in human testes with abnormal spermatogenesis, which in turn suggested that the HspA2 gene might play a specific role during meiosis in the human testes. Feng et al. (2001) demonstrated that the hsp, HspA2, was expressed in spermatocytes and spermatids in normal testes and in cases with maturation arrest but that expression was decreased in the latter. These authors thus concluded that lower HspA2 expression might be associated with the pathogenesis of male infertility.
The use of ejaculated spermatozoa as a wholly noninvasive biopsy of the spermatid has been proposed (Miller, 1997), and this may present a very useful tool for the diagnosis of genetic diseases and reproductive anomalies (Miller, 2000). As observed in this study, this technology could be used to assess environmental insults to the male gonad and recovery status from insult because spermatogenesis is a process of continual renewal (Miller, 2000).
In conclusion, this study demonstrated that HspA2 gene expression was significantly down-regulated in ejaculated sperm from infertile men with oligoteratozoospermia. Anomalies in the expression of this gene are associated with spermatogenic and/or spermiogenic dysfunction involved in the pathogenesis of some cases of male infertility, and sperm mRNA analyses may thus be a useful tool in evaluating the infertile man.
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Submitted on August 15, 2005; resubmitted on November 15, 2005; accepted on February 1, 2006.
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