Mol. Hum. Reprod. Advance Access originally published online on January 29, 2004
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Molecular Human Reproduction, Vol. 10, No. 3, pp. 197-202, 2004
© European Society of Human Reproduction and Embryology 2004
Expression of heat shock protein 70 in normal and cryptorchid human excurrent duct
Centre de Recherche en Biologie de la Reproduction and 1Département d’Obstétrique–Gynécologie and 2Chirurgie, Faculté de Médecine, Université Laval, Québec, Canada
3 To whom correspondence should be addressed at: Unité d’Ontogénie–Reproduction, Centre de Recherche, Centre Hospitalier de l’Université Laval, 2705 Blvd Laurier, Ste-Foy, PQ, Canada, G1V 4G2. e-mail: robert.sullivan{at}crchul.ulaval.ca
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ABSTRACT |
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The Hsp70 heat-shock proteins are molecular chaperones that assist other proteins in their folding, transport and assembly into complexes. Most of these proteins are either constitutively expressed or induced by heat shock and other stresses. Heat shock proteins are required for spermatogenesis, and also protect cells from environmental hazards such as heat, radiation, and chemicals. The abdominal position of the cryptorchid testis provokes a temperature elevation which is detrimental to spermatogenesis and causes infertility. The consequences of such a stress on Hsp70 expression were evaluated in normal and cryptorchid epididymides and vas deferens. Hsp70-1 transcript from reproductive organs of normal and cryptorchid men was analysed by real-time quantitative RT–PCR, meanwhile Hsp70 protein was characterized by western blot and immunohistochemical staining analysis. Hsp70-1 mRNA and protein showed equal expression in all segments of normal epididymis and ductus deferens. Hsp70 was specific to basal cells in the epididymal epithelium. For cryptorchid patients, Hsp70-1 mRNA expression in caput epididymidis was unchanged compared with controls. However, in corpus and cauda epididymides as well as in vas deferens, the expression level of Hsp70-1 transcript was higher for the cryptorchid tissues. Changes in mRNA frequency were specifically correlated with the age of the patients. By opposition to the mRNA, western blot analysis revealed that Hsp70 protein levels were not affected by the inguinal location of the epididymis. The data show that Hsp70-1 transcript and protein were constitutively expressed in human excurrent duct and that an inguinal location can stimulate the expression of Hsp70-1 mRNA along the human epididymis and ductus deferens.
Key words: Key words: cryptorchidism/epididymis/gene expression/Hsp70/human
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Introduction |
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The epididymis ensures sperm concentration, maturation, transport and storage (Bedford, Calvin et al., 1973; Brooks, 1983; Cooper et al., 1986). It can be divided into three major sections: the caput, the corpus, and the cauda epididymidis (Robaire and Hermo, 1988; Hermo, 1995). The proximal regions of the epididymis are devoted to sperm maturation (Temple-Smith et al., 1998), while in the distal region, sperm storage takes place (Foldesy and Bedford, 1982). Gene expression in the epididymis is characterized by a particular spatial organization, with specific genes expressed in specific segments of the organ (Cornwall and Hann, 1995; Kirchhoff, 1999). Androgens regulate specific gene expression in the epididymal epithelium (Jones et al., 1980; Moore, 1981; Brooks, 1987) and produce marked changes in the pattern of secreted proteins in every region (Moore, 1981). Moreover, testicular factors are known to influence epididymal function. In this regard, efferent duct ligation in rats has a notable effect on both gene expression (Hinton et al., 1998; Hermo et al., 2000) and cell survival (Turner and Riley, 1999). Temperature is another major factor influencing epididymal physiology (Foldesy and Bedford, 1982). It is known that temperature regulates sperm storage in the cauda epididymidis of the rat and rabbit (Jara et al., 2002).
In humans, failure of testicular descent (cryptorchidism) is one of the most frequent congenital malformations, affecting 1–3% of newborn boys. The abdominal position of the cryptorchid testis provokes a temperature elevation which is detrimental to spermatogenesis and causes infertility. Sperm storage functions of the cauda epididymidis and its ability to support sperm survival are severely affected by an elevation of scrotal temperature.
Cells exposed to elevated temperature and other environmental stress conditions respond by rapidly inducing the expression of heat shock proteins (Hsp) (Morimoto et al., 1990), which function to protect cellular proteins from harmful effects of stress conditions on cellular proteins (Sarge, 1995). Heat shock proteins are members of highly conserved protein families consisting of both constitutive and inducible components. Constitutively synthesized heat shock proteins function as molecular chaperones, aid antigen presentation, and regulate steroid receptor function (Burel et al., 1992; Jeremias et al., 1997). Inducible heat shock proteins prevent protein denaturation and incorrect polypeptide aggregation during exposure to physiochemical insults: elevated temperature, activated oxygen and nitrogen intermediates, inflammatory mediators or infection (Kantengwa et al., 1991). The 70 kDa heat shock protein (Hsp70) is one the most highly conserved members of the Hsp family.
The aim of the present investigation was to explore the role of heat shock protein Hsp70 in the human excurrent duct. The expression pattern of the Hsp70-1 mRNA was investigated using real-time quantitative PCR analysis in normal and pathological situations (cryptorchidism). We also characterized the protein expression in these tissues by western blot analysis and immunohistochemical staining analysis. Here, we report new qualitative and quantitative data concerning the mRNA and protein expression profiles of Hsp70 in human excurrent duct.
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Tissue preparation
Normal human epididymis and vas deferens were obtained as described previously (Boué et al., 1996; Légaré et al., 1999). Briefly, reproductive tissues were recovered from two donors of 48 and 52 years of age registered in our local organ transplantation programme. These donors were victims of accidental death and did not have pathologies that could affect the reproductive function. Tissues were collected while artificial circulation was maintained to preserve organs and tissues assigned for transplantation. For each epididymal section, tissue pieces were frozen in liquid nitrogen and kept at –80°C for RNA and protein extraction. Other pieces were fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C and then included in OCT (Optimal Cutting Temperature; Canemco Supplies, Canada) for immunohistochemistry.
Four men aged between 24 and 57 years referred to urology for unilateral cryptorchidism were included in this study. After physical examination confirming testicle inguinal position, unilateral orchidectomy was performed under general anaesthesia. Within 1 h after surgery, epididymis was dissected in caput, corpus and cauda segments. These tissues as well as scrotal portion of vas deferens were processed as described for normal tissues. Anatomical examination revealed testicular atrophy in all cases and absence of testicular cancer was confirmed by histological examination.
Sexually mature crab-eating macaques (Macaca fascicularis) (5–10 years of age) were also used in this study to ascertain that heat shock protein expression along the normal reproductive tract was not associated with maintenance of artificial circulation procedures applied to normal donors. Before surgical castration, these monkeys were not submitted to experimental procedures. Immediately after castration under general anaesthesia, the epididymides were dissected into three different sections, i.e. the caput, corpus, and cauda epididymidis. For each section, tissue pieces were frozen in liquid nitrogen and kept at –80°C for RNA and protein extraction.
Protocols used in this study were approved by the local animal welfare and ethical committees.
RT–PCR analysis
Total RNA from frozen epididymis sections and vas deferens were isolated using Absolutely RNA RT–PCR Miniprep Kit following the manufacturer’s directions (Stratagene, USA). Residual DNA was removed by treatment with 5 IU of DNAse1 at 37°C for 15 min. The RNA concentrations were estimated by spectrophotometry at 260 nm. A PCR product was generated to evaluate the presence of Hsp70-1 mRNA in human excurrent duct. Five micrograms of total RNA from caput epididymidis was used for first strand cDNA synthesis, using 100 IU of SuperScript (Gibco-BRL, USA), RT buffer (50 mmol/l Tris, pH 8.3, 75 mmol/l KCl, 3 mmol/l MgCl2), 10 mmol/l dithiothreitol (DTT), 200 µmol/l dNTP and 10 µmol/l random primer p(dN)6 (Roche Diagnostics) in a final volume of 20 µl. The cDNA was used as the template in the PCR reaction mixture. The primers 5'-ATAACGGCTAGCCTGAGGA-3' (sense) and 5'-GGTGTTCTGCGGGTTCAG-3' (anti-sense) were derived from nucleotide sequences of human Hsp70-1 cDNA. Reactions were run with 5 µl of RT template or negative control and Taq DNA polymerase (1.5 U) (Pharmacia, Canada) in a final volume of 50 µl. PCR amplifications were achieved following 30 cycles. The PCR cycle consisted of a single incubation at 95°C for 5 min followed by 30 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s with a final single extension step of 72°C for 10 min. An 413 base pair (bp) PCR amplification product was generated and cloned in pGEM-T (Promega, USA). All nucleotide sequences were determined by the dideoxynucleotide termination method (Sanger) using T7 Sequenase v 2.0 kit (Amersham, Canada).
Quantitative real-time RT–PCR
Total RNA of each human epididymidal segment and vas deferens were extracted as described above. Using SuperScript II (Gibco-BRL) and random primer in final volume of 20 µl, cDNA was synthesized from 5 µg of RNA. To amplify the cDNA, 5 µl aliquots of reverse-transcribed cDNA (diluted 1:12.5) were amplified by PCR in 20 µl containing a final concentration of 3 mmol/l MgCl2, 50 ng of each primer (5'-ATAACGGCTAGCCTGAGGA-3'; 5'-GGTGTTCTGCGGGTTCAG-3') and 2 µl of ready-to-use LightCycler DNA master SRBRGreen I (Roche Diagnostics, Canada; x10, containing TaqDNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, SYBRGreen I dye, and 10 mmol/l MgCl2). The plasmid containing the 413 bp fragment was used to validate the LightCycler reaction and to determine the quantification range (calibration curve). The reaction conditions were as follows: initial denaturation at 95°C for 10 min followed by 35 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 5 s, and extension at 72°C for 20 s. The temperature ramp was 20°C/s, except when heating to 72°C, when it was 2°C/s. At the end of the extension step, fluorescence of each sample was measured to allow quantification of the RNA. After amplification, a melting curve was obtained by heating at 20°C/s to 95°C, cooling at 20°C/s to 60°C, and slowly heating at 0.1°C/s to 95°C with fluorescence data collected at 0.1°C intervals. To control for the recovery of intact RNA and for the uniform efficiency of each reverse transcription reaction, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragment was amplified by real-time RT–PCR using these primers: 5'-GAAGACTGTGGATGGCCCCTC-3' and 5'-GTTGAG GGCAATGCCAGCCCC-3'.
Quantitative analysis of the LightCycler data was performed using the LightCycler analysis software. The data analysis was divided into two parts: specificity control of the amplification reaction using the melting curve program of the LightCycler software followed by use of the quantification program. The SYBR Green I signal of each sample was plotted against the number of cycles. Using the LightCycler analysis software, background fluorescence was removed by setting a noise band. This fluorescence threshold was used to determine cycle number that correlated inversely with the log of the initial template concentration. To this end, the log–linear portions of the amplification curves were identified and best-fit lines calculated. The crossing points were the intersections between the best-fit lines of the log–linear region and the noise band. These crossing points correlated inversely with the log of the initial template concentration (LightCycler operator’s Manual, Version II). The crossing points determined for Hsp70-1 mRNA were normalized to those of GAPDH to compensate for variability in RNA amount.
Antibodies
Mouse monoclonal antibody against Hsp70 was purchased from Stressgen Biotechnologies, (Canada) and used at 1 µg/ml for western blot analysis and 5 µg/ml for immunohistochemistry. Biotinylated goat anti-mouse secondary antibody was obtained from Dako Diagnostics (Canada) and used at 1/200 (vol/vol). Goat anti-mouse IgG conjugated to horseradish peroxidase was purchased from BIO/CAN Scientific (Canada) and used at 1/2000 (vol/vol).
Immunohistochemical staining
Cryostat cross-sections (10 µm) were prepared from frozen epididymal tissues. Endogenous peroxidase activity was quenched with 3% H2O2 (v/v) in PBS for 30 min. Non-specific binding sites were then blocked with 10% goat serum in PBS for 1 h. The Hsp70-specific antibodies were diluted in PBS and applied overnight at 4°C. In control sections, the primary antibodies were replaced by the corresponding non-specific IgG and processed in parallel. Sections were subsequently incubated with biotinylated goat anti-mouse antibody for 30 min, and with ABC reagent for 30 min. Immunostaining was revealed using 3-amino-9-ethylcarbazole (AEC). Harris haematoxylin was used for counterstaining, and mounted under cover slip using an aqueous mounting medium (Sigma). Slides were observed under a Zeiss Axioskop2 Plus microscope (Canada) linked to a digital camera from Diagnostics Instruments (USA). Images were captured using the Spot software (Diagnostics Instruments).
Western blot analysis
Samples from normal and cryptorchid epididymal tissue were homogenized in lysis buffer containing 0.01 mol/l Tris, 0.1 mmol/l EDTA, 1% sodium dodecyl sulphate (SDS), and 1 mmol/l phenylmethylsulphonyl fluoride. Total protein concentration in each sample was determined by using the Bio-Rad compatible-detergent protein assay kit and bovine serum albumin (BSA) as standard. Two micrograms of total protein from each sample were separated on a 10% SDS–polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were then blocked in PBS–0.1% Tween–5% milk (wt/vol) solution at room temperature for 1 h. Primary antibody (Hsp70-specific) was incubated overnight at 4°C. Goat anti-mouse IgG conjugated to horseradish peroxidase was used to chemiluminescent detection of proteins (ECL, Amersham).
Statistical analysis
Statistical analysis was performed by analysis of variance using super ANOVA (analysis of variance) software (Abacus Concepts, USA). Results were compared by Student–Newman–Keuls test. Differences were considered to be significant at P <0.05.
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Hsp70 in adult human epididymis and vas deferens
The results presented in Figure 1 correspond to Hsp70 expression in two epididymides and vas deferens from two different men. It appears that in normal tissue, Hsp70-1 mRNA was expressed all along the human excurrent duct (Figure 1A) with a lower level in the vas deferens. Western blot data demonstrated that normal epididymis and vas deferens tissues strongly expressed the Hsp70 protein (Figure 1B). No difference was noticed in the amount of Hsp70 along the human excurrent duct.
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In order to verify that the presence of Hsp70 in normal tissues was not due to stress factors associated with maintenance of artificial circulation in donors, the presence of Hsp70 in the epididymis of cynomolgus monkeys (not subjected to experimental procedures) was investigated. (Figure 2). The same transcript level was found in primate tissues as in normal human epididymis (Figure 2A). The protein was also expressed at high levels all along the monkey epididymis (Figure 2B).
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Cellular distribution Hsp70 protein in human epididymis and vas deferens
The localization of Hsp70 was investigated by immunohistochemistry techniques (Figure 3). The protein was strongly expressed in the cytoplasm of basal cells in the epididymis as well as in vas deferens. Hsp70 was also present in the macrophage cells localized in the interstitium of the caput, corpus and cauda epididymides. The same pattern of distribution was revealed all along the epididymis. No immunohistochemical staining was detected when a preimmune serum was used as a negative control (Figure 3F).
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Modulation of Hsp70 by inguinal cryptorchidism.
To examine the changes in Hsp70-1 mRNA profile in epididymis and vas deferens after cryptorchidism, epididymides and vas deferens of four cryptorchid men were compared to those of normal men (Figure 4). The inguinal location of the epididymis did not influence the expression of Hsp70-1 mRNA in caput epididymidis compared with controls. In corpus and cauda epididymidis as well as in vas deferens, the expression level of Hsp70-1 mRNA was higher for the cryptorchid tissues when compared with normal tissues. A significant increase in Hsp70-1 transcript was found in the corpus epididymidis of the oldest patients (54 and 57 years) as well as in the cauda segment of three of the four cryptorchid men. The expression of GAPDH mRNA used as a housekeeping gene control was not significantly affected in any sample examined (data not shown).
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In contrast to the mRNA, western blot analysis revealed that Hsp70 protein levels were not significantly affected in the inguinally located organs. These results did not vary throughout age distribution (Figure 5). Indeed, for the four cryptorchid men, a similar quantity of Hsp70 was detected all along the excurrent duct. Moreover, the cellular localization of Hsp70 remained the same in the cryptorchid epididymis: as in normal epididymis, Hsp70 was restricted to the basal cells of the epididymal epithelium (data not shown).
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Discussion |
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The heat-induced Hsp plays a cytoprotective role in preventing irreversible damage to cellular proteins by binding to unfolded or partially malfolded peptides to retard thermal denaturation and aggregation of cellular proteins (Morimoto et al., 1994). It is well demonstrated that some Hsp are constitutively expressed in the testis, some are developmentally regulated, some are only induced in response to heat stress and some are specific to testis. No studies have been performed to demonstrate the presence and localization of Hsp70 in human epididymis and vas deferens. In the present investigation we show that Hsp70-1 mRNA is present all along the human excurrent duct and its level of expression correlates with protein expression. Because of the clothed state, scrotal temperature in man is chronically elevated by several degrees. Consequently, the human epididymis is generally maintained at several degrees above its physiological temperature. Thus, the higher scrotal temperature created by clothing could be an explanation for the presence of Hsp70 in human excurrent duct. To eliminate this possibility, we have verified the presence of Hsp70 in the epididymis of another species. In monkey epididymis the transcript level is the same as in normal human epididymis. The protein is also expressed all along the monkey epididymis. It seems that under undisturbed conditions, Hsp70 is constitutively expressed in human and monkey male excurrent duct.
The epididymal epithelium contains several major cell types: principal, basal, narrow, clear and halo cells (Serre and Robaire, 2002). The cellular localization of Hsp70 is selective for basal cells in the human epididymal epithelium. In murine and human epididymis, basal cells express macrophage antigens (Yeung et al., 1994; Seiler et al., 1998). It was proposed that basal cells have a potential immunological function. The hypothetical role of the basal cells in the epididymal local immune defence mechanism could be to phagocytose the antigenic products taken up by the principal cells (Yeung et al., 1994). Moreover, it is known that basal cells play a role in immune defence against sperm autoantigens by avoiding the contact of these antigens with the blood compartment (Poulton et al., 1996). Several studies have raised the possibility that Hsp70 may be involved in various aspects of the immune system (Kaufmann, 1994). Genes encoding two members of the Hsp70 family are found to reside in the MHC complex. It has been reported that Hsp70 act as recognition structure for natural killer (NK) cells (Yenari, 2002). Hsp70, by its expression in basal cells, may be involved in an epididymal protective role. Substantial evidence indicates that Hsp70 is capable of protecting cells, tissues, organs, and animals from subsequent, normally lethal heating, as well as from other types of noxious conditions. Cells transfected with the Hsp70 gene are readily protected from many harmful agents (Kiang and Tsokos, 1998). Therefore, inhibition of Hsp70 expression diminishes cell survival.
Hsp have been observed in many cell types and tissues, under both unstressed and stressed conditions. An accumulation of evidence has shown that environmental and pathological stresses induce Hsp. The degree of induction depends on the level and duration of exposure to stress. The increase is transient, but how long it persists is different in various cell types (Kiang and Tsokos, 1998). Cryptorchidism is a very frequent illness amongst newborn male children. The elevation of scrotal temperature can negatively influence male reproductive function (Setchell, 1998). The effects of temperature on the male tract have been examined in the rat, rabbit and hamster. A small increase in the temperature of the testis does not destroy the germinal epithelium, but it reduces testis weight and sperm production. An increase in temperature augments the risk of oxidative damage to DNA in the developing sperm, and hence also to embryo survival in the later offspring of affected males (Setchell, 1998). Deep body temperature changes at least the ionic and protein composition of cauda epididymal fluid by virtue of effects on the cauda epithelium, and it eliminates the special ability of the cauda epididymidis to store and prolong the life of sperm. In previous studies on the epididymides of cryptorchid animals and on primary epithelial cell cultures, it has been shown that some epididymal gene products are exquisitely sensitive to small temperature changes (Pera et al., 1996; Gebhardt et al., 1999; Kirchhoff et al., 2000).
This study demonstrates that the position of the cryptorchid testis influences the expression of Hsp70-1 mRNA in the distal part of the human excurrent duct. The level of Hsp70-1 mRNA increased notably and seemed to be related to the age of the cryptorchid man: higher expression of Hsp70-1 mRNA being detected in the oldest cryptorchid patient (57 years old). Since the two normal donors were 48 and 52 years old, the age parameter seemed to have no impact on the change of Hsp70-1 mRNA expression in normal human epididymis; at least before the sixties.
Hsp70 appears to be a stable protein. Indeed, in this study, inguinal location did not change the levels of Hsp70. Previous studies using surgical cryptepididymis to evaluate the effects of temperature on cell survival in the mouse cauda epididymidis, also indicated that Hsp70 protein is not altered by temperature increase (Jara et al., 2002). The production of Hsp70 is quantitatively correlated with the degree of stress. The level of synthesis is controlled both transcriptionally and post-transcriptionally through repression of Hsp70 mRNA synthesis and destabilization of Hsp70 transcripts (DiDomenico et al., 1982). It is possible that mRNA degradation is accelerated in a cryptorchid situation, resulting in a reduced half-life of Hsp70-1 mRNA.
Taken together, the results of the present work suggest that Hsp70, specially localized in basal cells, may play a specific role in human epididymis. The inguinal location of the human epididymis influences the expression of the Hsp70-1 transcript without altering the protein level. Further molecular analysis of the gene and protein function is necessary in order to elucidate the mechanism of Hsp70 control level and function in human epididymis.
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Acknowledgements |
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We wish to thank Dr F.Saez for valuable comments and criticisms of the manuscript. This study was supported by a Canadian Institutes for Health Research grant to R.S.
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Submitted on October 1, 2003; resubmitted on November 10, 2003; accepted on November 19, 2003.
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