Hum. Reprod. Advance Access originally published online on June 21, 2007
Human Reproduction 2007 22(8):2169-2177; doi:10.1093/humrep/dem156
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Distribution of the receptor for advanced glycation end products in the human male reproductive tract: prevalence in men with diabetes mellitus
1 Department of Obstetrics and Gynaecology, Institute of Clinical Sciences, School of Medicine, Queen's University of Belfast, Grosvenor Road, Belfast BT12 6BJ UK 2 Division of Basic Medical Sciences/Anatomy, School of Medicine, Queen's University of Belfast, Belfast, UK 3 Centre for Vision Science and Vascular Biology, Queen's University of Belfast, Belfast, UK 4 Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast, UK 5 Department of Urology and Paediatric Urology, Justus Liebig University, Giessen, Germany
6 Correspondence address. Tel: +44 28 90 63 2556; Fax: +44 28 90 32 8247; E-mail: c.mallidis{at}qub.ac.uk
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Abstract |
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BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage.
METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localization on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. Enzyme-linked immunosorbent assay analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalization was employed to evaluate RAGE's precise location on the sperm head.
RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (P < 0.01, P < 0.0001 and P < 0.0001, respectively).
CONCLUSIONS: The presence of RAGE implies that it may play a central role in sperm nDNA damage particularly in diabetic men where the levels are elevated.
Key words: diabetes/epididymis/RAGE/sperm/testis
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Introduction |
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The growing incidence of diabetes mellitus (DM) in the Western world is a major healthcare issue which has gone beyond scientific and medical circles and is now a cause célèbre of the popular press. Of particular concern is the decreasing age of patients at diagnosis, especially in view of the consequent long-term health repercussions for those with early onset diabetes.
Concomitantly, much has been written about falling fertility rates in industrialized countries and various causes have been proposed to explain the phenomenon (Carlsen et al., 1992
; Morgan, 2003; Skakkebaek et al., 2006). Of these, diminished male fertility, in particular decreased sperm counts and quality, has received significant attention (de Kretser, 1996; Jensen et al., 2002).
Clearly, there is an increasing overlap between DM and infertility in the young, but despite this there is little new information available on any possible association between diabetes and male fertility. In the few studies that have examined any possible links between diabetes and semen parameters (i.e. sperm concentration, motility, morphology, etc.) (Handelsman et al., 1985; Vignon et al., 1991; Ali et al., 1993; Niven et al., 1995), the overall findings are at best, confusing and inconclusive. As a consequence, few fertility specialists consider the condition of relevance to their treatment regimen or even note the diabetic status of the male partner in a couple seeking help for infertility (Agbaje et al., 2007).
Recently, our group reported a significant increase in the percentage of fragmented sperm nuclear DNA (nDNA) in men with Type 1 diabetes when compared with sperm from non-diabetic controls (Agbaje et al., 2007s). Increased sperm nDNA damage has been associated with decreased embryo quality, lower implantation rates and possibly the onset of some childhood cancers (Morris et al., 2002; Henkel et al., 2004; Lewis and Aitken, 2005). However, it is rarely included in the routine assessment of the male partner (Trisini et al., 2004).
Currently, the prevailing view is that sperm nDNA damage is the result of oxidative stress encountered during transit through and storage in the epididymis (Fouchecourt et al., 2000; Vernet et al., 2004). Sperm are particularly susceptible to attack by reactive oxygen species (ROS) due to their high unsaturated fatty acid content and the absence of DNA repair mechanisms (Aitken and Sawyer, 2003). Although numerous possible causes have been proposed (Donnelly et al., 2001; Gil-Guzman et al., 2001; Alvarez et al., 2002; Aitken and Baker, 2006), as yet the main factor responsible for the generation of ROS which damage nDNA in the male reproductive tract remains unidentified.
Advanced glycation end products (AGEs) have been implicated in an increasing number of diabetic complications that result from, and in, oxidative damage due to ROS generation (Wautier and Schmidt, 2004; Bohlender et al., 2005; Chekir et al., 2006). Forming on the amino groups of proteins, lipids and DNA through a number of intricate pathways, these complex heterogeneous compounds are part of the normal ageing process and are both produced by, and introduced to, the body (e.g. diet, smoking) (Koschinsky et al., 1997; Vlassara and Palace, 2002).
AGEs are capable of damaging DNA either directly (Vlassara and Palace, 2002) or by the generation of ROS via the activation of the receptor for AGEs (RAGE) (Schmidt et al., 2000; Wautier and Schmidt, 2004; Chekir et al., 2006). RAGE is a member of the IgG superfamily and is a multiligand receptor with a high affinity for, amongst others, several AGE-modified proteins (e.g. 
amyloid, amphoterin and S 100/calgranulins) (Schmidt et al., 2000; Stern et al., 2002). AGE binding to RAGE in many cell types provokes a range of pathophysiological responses linked to the downstream activation of NF
B and other signalling pathways that lead to ROS generation and certain pro-inflammatory responses (Wendt et al., 2003). Suppression of RAGE-ligand binding using soluble RAGE or neutralizing antibodies has been shown to prevent various pathological events in a range of cells (Schmidt et al., 2000; Wautier and Schmidt, 2004).
RAGE normally exists in a basal activation state evoked by nominal RAGE-ligand binding but, in pathological situations such as diabetes, its expanded expression mediates tissue damage and prevents the restoration of homeostasis (Schmidt, 2001).
As we have recently shown the presence of N
-carboxymethyl-lysine (CML), a prominent AGE, in the testis, epididymis and on sperm (Mallidis et al., unpublished data), we hypothesize that RAGE may also be present in the male reproductive tract and that it could play a contributory role in the ROS-initiated damage of sperm nDNA. The aims of our study were, to determine whether RAGE is present in the male reproductive tract, to describe its location and to compare the levels of RAGE protein found in the semen of men with Type 1 diabetes to those in the semen of non-diabetic men.
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Subjects
Written informed consent for participation was obtained and the project was approved by the local research ethics committee and Royal Group of Hospitals Trust Clinical Governance Committee.
Male diabetics (Type-1, n = 14; Type-2, n = 7) aged 18–60 years attending the Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast were invited to participate in this study. The participants were undertaking standard medication regiments commensurate with the requirements of their diabetic condition. Two of the subjects were smokers. Control samples were obtained from men attending the QUB Andrology Laboratory for semen analysis as part of routine infertility investigations (n = 31). No information on the smoking habits of the men providing the control samples was available.
Semen samples were collected after 2–5 days of sexual abstinence. All samples had conventional light microscopic analysis performed according to WHO recommendations (WHO, 1999) for semen volume, sperm concentration and motility. Sperm morphology was assessed according to Tygerberg Strict Criteria (Menkveld and Kruger, 1995). Semen analysis was performed within 1 h of ejaculation after incubation at 37°C to allow liquefaction to occur.
After analysis, the remainder of each sample was centrifuged at 300 g for 10 min, the seminal plasma removed, frozen in liquid nitrogen and stored. The pellet was washed in an equal volume of phosphate-buffered saline (PBS) (Sigma Aldrich, Poole, UK) and centrifuged as before. After discarding the supernatant, the pellet was resuspended in PBS, 10 µl was removed to prepare smears for immunocytochemistry, and the remainder was frozen in liquid nitrogen and stored until needed for protein extraction. Sperm smears were prepared on 3-aminopropyltriethoxysilane (APEs) coated microscope slides, left to air dry, then fixed in absolute ethanol, air dried and stored until use.
Testicular biopsies were obtained from non-diabetic normogonadotrophic men (n = 5) undergoing investigation for obstructive azoospermia at the Department of Urology and Paediatric Urology, Justus Liebig University, Giessen. Tissue was collected by open biopsy procedure, immediately immersed in Bouin's fixative (bovine serum albumin, BSA), then washed, dehydrated through a series of graded ethanol, cleared in xylene and embedded in paraffin using standard techniques. Serial sections of 5 µm for each sample were cut and placed on APEs coated glass microscope slides.
Immunolocalization
Tissue sections were dewaxed, rehydrated using a sequence of xylene, graded ethanol and water, then washed in PBS. The immunodetection procedure employed was a modification to the standard method as recommended by the manufacturers of the antibodies (R&D Systems, Oxon, UK). Briefly, antigen unmasking was performed by incubating the slides with a 50% trypsin:50% vercene solution (Cambrex Bioscience, Berkshire, UK) for 2 min. After being washed in water for 20 min, the tissue was permeabilized in 0.1% Triton X-100 (Sigma Aldrich, UK) for 20 min, rinsed in PBS, and blocked with donkey serum overnight at 4°C. The next day the slides were again washed in PBS and then incubated with either goat anti-human RAGE (1:1000) or normal goat IgG antibody (isotype control), overnight at 4°C. A negative control was carried out by the omission of primary antibody. Finally, samples were washed in PBS, incubated with a donkey anti-goat Alexa 488 fluor antibody (1:200) (Invitrogen, Paisley, UK), washed again, incubated with propidium iodide (1:200) for 20 min at room temperature, washed, mounted and coverslipped. All slides were then examined and evaluated using a Bio-Rad Micro radiance confocal scanning microscope fitted to an Olympus BX690 fluorescent microscope.
Other than the omission of the antigen unmasking step, the procedure used for the immunodetection of RAGE on sperm was identical to that detailed above. For the RAGE/acrosome dual labelling, rhodamine peanut agglutinin (1:500) (Vector Laboratories Ltd, Peterborough, UK) was added for 20 min to sperm smears instead of the propidium iodide at the end of above procedure.
Semi-quantitative sperm assessment
Sperm smears from 10 diabetic and 11 non-diabetic subjects were assessed in this part of the study. Two sets of slides (negative control and RAGE) from each subject were prepared and stained for double labelled fluorescent immunocytochemistry and examined using the above mentioned manner. Images from a minimum of 10 (mean: 13.8; SD: 2.8) randomly selected fields from each slide were photographed, given a non-identifying code and stored electronically. The images were then evaluated by blind assessment. A median of 284 sperm (mean: 324, SD: 177) per patient were examined and the number of immunoreacted and non-reacted sperm counted. Each fluorescing sperm was then further classified based on the specific location of the detected staining (i.e. acrosome, nucleus, tail or any combination thereof).
Protein extraction and quantitation
Stored semen samples were thawed and aliquots of 20 x 106 sperm taken and centrifuged at 16 000 g for 15 min to separate sperm and seminal plasma. The seminal plasma was removed and reserved. A volume of extraction buffer (0.1% Triton X, 1% Tergitol, 0.1% SDS and 0.001% NaN3) in PBS was added and the pellet was sonicated on ice for 30 s using a hand held mini pestle (Sigma Aldrich, UK). The resultant suspension was centrifuged at 10 000 g for 10min and the supernatant collected. Protein content of the sperm extracts and seminal plasmas was determined using a Bichoninic acid kit (Pierce BCATM Protein Assay Kit, Rockford, IL) and microplate reader set at an absorbance of 562 nm.
Enzyme-linked immunosorbent assay
Levels of all forms of RAGE were measured using the DuoSet® ELISA system (R & D Systems, Oxon, UK), according to the manufacturer's protocol, all steps being performed at room temperature and out of direct light. Briefly, 100 µl mouse anti-human RAGE antibody (1.0 µg/ml) was added per well of a 96 well plate (NuncTM Maxisorb, Denmark), covered and incubated at 4°C overnight. Next day the plates were washed with 0.05% Tween in PBS, blocked (1% BSA in PBS for 2 h), washed, 100 µl aliquots of samples and standards (duplicate wells of each) added, covered and incubated with agitation for 2 h. Standards of recombinant human RAGE in protein extraction buffer were serially diluted to give a range from 4000 to 3.9 pg/µl. After washing, 100 µl of biotinylated goat anti-human RAGE antibody (100 ng/ml) was added, the plates covered and incubated for 2 h, before further washing. Streptavidin-horse radish peroxidase (100 µl of a 1:200 dilution) was added and the plates placed out of direct light for 20 min. Following a final washing step, 1:1 hydrogen peroxide-tetramethylbenzidine solution (Sigma Aldrich, UK) was added and the plates returned to the dark with gentle agitation for 20 min. The colour reaction was stopped by the addition of 2 N H2SO4. The optical density of each well was measured using a Tecan Safire microplate reader (Tecan, UK) set to 450 nm and corrected by subtracting the reading obtained at 540 nm. The concentration of RAGE was determined using the appropriate standard curve and standardized according to the total protein initially added to each well.
Statistical analysis
Statistical analysis was performed using SPSS 11 for MAC OS 10 (SPSS INC., Chicago, Illinois, www.spss.com). Semen profiles were compared using Student's t-test. Sperm concentrations were normalized using a square root transformation prior to analysis. To account for the non-Gaussian distribution of the semi-quantitative and ELISA data, the Mann–Whitney U Test was used to compare median values for diabetic and non-diabetic subjects.
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Semen analysis
No statistically significant difference was found in age (37.6 ± 9.5 versus 34.6 ± 5.4 years, mean ± SD) or any aspect of semen quality (Table 1) between the diabetic (regardless of type) and control subjects. Non-significant differences in volume (decreased in diabetics), sperm concentration (decreased in non-diabetics) and total sperm count (decreased in diabetics) were noted. However, all measures obtained were well above the normal limits as recommended by the WHO (1999
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Distribution of RAGE in the testis and epididymis
The immunocytochemical localization of RAGE in the testis displayed a uniform staining pattern throughout the cytoplasm of all cell types present within the seminiferous epithelium (Fig. 1a). The same level of immunoreactivity was also evident in the cytoplasm of the peritubular cells and the constituents of the interstitium. The nuclei of Sertoli cells, germ cells (regardless of developmental stage), myoid peritubular cells and Leydig cells did not display any immunofluorescence. However, in a few regions close to the basal membrane of the seminiferous epithelium where a more intense staining pattern was noted, occasionally a small number of spermatogonial nuclei did show some immunoreactivity (Fig. 1a). Distinct nuclear staining was seen within the blood vessel wall. The luminal space of most tubules contained a diffuse immunofluorescence probably attributable to the sloughing of the spermatogenic cell cytoplasm which may have been a consequence of the biopsy procedure. No specific fluorescence was noted in any of the negative control sections (Fig. 1c).
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Sections of the efferent ducts/caput region of the epididymis displayed various degrees of RAGE immunoreactivity (Fig. 1b). In the epithelium, the entire cytoplasm of the principal cells was clearly stained in those lining the lumen and decreased to a more diffuse fluorescence in those nearer the basal lamina. A distinct band of fluorescence was evident lining the lumen of the duct, suggestive of a greater quantity of RAGE in the ciliated apical domain of the principal cells. The principal cell nuclei and the basal lamina itself were not immunoreactive. However, the nuclei of the basal cells showed distinct and intense fluorescent staining (Fig. 1b). The smooth muscle layer encircling the tubule was strongly immunoreactive and was interspersed with intensely fluorescent nuclei, primarily myoid cell in origin but, possibly, also fibroblasts. The dense connective tissue beyond the smooth muscle ring displayed only small areas of streak-like staining. The negative control sections showed no fluorescent staining (Fig. 1d).
Localization of RAGE on sperm
In samples from both diabetic and non-diabetic men, immunoreactivity was detected in a large number of sperm, primarily in the head region (Fig. 2a). Particularly, prominent was a speckled staining appearance which extended from the mid-section of the head to the very edge of the sperm, approximating the extent of the acrosomal cap (Fig. 2b). An equatorial band just below the acrosome was also very intensely stained and clearly delineated (Fig. 2c) in most immunoreacted sperm regardless of their morphology. Occasionally the tail region was also found to bind the antibody (Fig. 2a and c). However, as this was a relatively rare occurrence, and taking into account the role and function of RAGE in other cell types, this immunoreactivity was taken to be an artefact and consequently disregarded in further assessments.
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The semi-quantitative analysis of the immunostaining of sperm from diabetic and non-diabetic men (Fig. 3a) found that the majority of sperm (>60%) in samples from diabetic men (regardless of type) showed the presence of RAGE, a proportion approximately three times greater than that seen in samples from non-diabetic men (P < 0.01). Evaluation and semi-quantitative analysis of the localization of the immunoreactivity (Fig. 3b) showed that the vast majority of stained sperm, regardless of whether from diabetic (96%) or non-diabetic men (84%), were labelled on the nucleus (i.e. equatorial band and head) region. None of the IgG negative control slides displayed any immunofluoresence (data not shown).
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To determine the specific location of RAGE on the head region, sperm in differing stages of the acrosome reaction were examined. When the acrosomal cap was found to be fully intact, immunoreactivity for RAGE extended from the intensely stained equatorial band throughout the apical region of the sperm head (Fig. 4a). Where the acrosome had partially reacted (Fig. 4b), intense immunostaining was restricted to the equatorial region but a very light staining was evident on the remnants of the acrosome. On sperm that had entirely undergone the acrosome reaction, no evidence of immunoreactivity was noted (Fig. 4c).
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Quantitation of RAGE protein
A significantly higher amount of RAGE protein was detected by ELISA in both the sperm (P < 0.0001) and seminal plasma (P < 0.0001) of diabetic men when compared with that found in samples from non-diabetic men (Fig. 5). The proportion of the difference (~2.2-fold increase) is consistent with the previously mentioned finding (Fig. 3a) that significantly more of the sperm from diabetic men express RAGE when compared with sperm from men without diabetes.
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Discussion |
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The possibility of an association between DM and impaired male fertility has been largely ignored because of the lack of any definitive correlations between DM and alterations in sperm light microscopic profiles. However, with the application of techniques which assess sperm function at a molecular level, suspicions are being aroused that diabetes does indeed have a significant effect on male reproductive function (Agbaje et al., 2007
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In this study, as in our previous investigations (Agbaje et al., 2007; Mallidis et al., unpublished data) and those of others (Paz et al., 1977; Bartak, 1979; Padron et al., 1984), routine light microscopy alone, demonstrated no significant difference in the semen characteristics of diabetics when compared with those of non-diabetic subjects. However, it is now evident that in diabetics (Agbaje et al., 2007), as with infertile men (Saleh et al., 2002), adolescents with varicocele (Bertolla et al., 2006), men over the age of 45 (Moskovtsev et al., 2006) and cigarette smokers (Sepaniak et al., 2006), semen parameters within the WHO normal range (WHO, 1999) do not preclude significant damage to the underlying nDNA. Were it not that the investigation of nDNA integrity is so laborious much greater clinical prominence would be given to it in the investigation of male fertility due to the ever accumulating evidence linking nDNA fragmentation to impaired embryonic development, increased incidence of spontaneous miscarriage and the onset of certain childhood cancers (Lewis and Aitken, 2005).
At present, sperm nDNA fragmentation is considered to be the result of oxidative stress due to excessive ROS generation possibly caused by a variety of factors, both internal and external (Lewis and Aitken, 2005). However, the precise mechanism responsible for the generation of these damaging ROS is, as yet, unknown.
AGEs are influential instigators, mediators and/or contributors to an ever increasing number of diabetic complications (Vlassara and Palace, 2002; Peppa and Vlassara, 2005). Accumulating in the body and the result of the normal ageing process (Chavakis et al., 2004), these complex heterogenous compounds occur at enhanced levels in diabetes and can form very rapidly (Sajithlal et al., 1998; Han et al., 2005). A distinctive feature of AGEs is their ability to interact with a variety of receptors hence resulting in endocytosis, degradation, cellular activation or pro-inflammatory and pro-oxidant events (Peppa et al., 2003).
The receptor for AGEs, RAGE, is a member of the immunoglobulin superfamily of cell surface molecules. It is composed of an extracellular region containing a single ‘V’- type Ig domain and two ‘C’- type Ig domains followed by a hydrophobic transmembrane domain and a short, highly charged cytosolic domain which is essential for the cellular effects mediated by ligand-RAGE binding (Schmidt et al., 2000). The extracellular region confers ligand-binding properties, probably via the ‘V’ domain, and recognizes tertiary structures not specific amino acid sequences. As such it is a pattern recognition receptor able to engage classes rather than individual molecules (Chavakis et al., 2004). Once ligand-RAGE binding occurs, it perturbs cellular properties and sets the stage for the sequelae of AGE generation/accumulation (Ramasamy et al., 2005). RAGE levels are dictated by the accumulation of its ligands. In diabetes, where hyperglycemia triggers accelerated formation and deposition of AGEs, there is enhanced expression of RAGE. However, it is also found in low levels even in normal tissue (Schmidt et al., 2000). Similarly, glucose induced damage is not limited to hyperglycaemic conditions. Glycation accompanies every fundamental process of cellular metabolism and, as such, has been implicated in a variety of seemingly disparate conditions such as the effects of cigarette smoking, ageing and certain neurodegenerative diseases (Kikuchi et al., 2003).
The immunocytochemical localization of RAGE in all cell types within the seminiferous epithelium, the peritubular cells and the constituents of the interstitium is consistent with the pattern of staining seen in various tissues from non-diabetic subjects, including placenta (Chekir et al., 2006), mesothelium and prostate, amongst others (Cheng et al., 2005). The lack of nuclear immunoreactivity together with a diffuse overall cytoplasmic staining, conforms with what Cheng et al. (2005) designated as ‘pattern A’ in their classification of RAGE expression. The only truly distinct nuclear staining was seen in a few cells present in the blood vessels. These were most probably, leukocytes, macrophages or phagocytes, all of which have been shown to express the receptor (Liliensiek et al., 2004). As RAGE expression, even at low levels, is dependent on ligand accumulation, the apparent uniform level of expression of the receptor throughout the testicular compartments suggests that they are all equally bathed in the receptor's ligand(s). This assertion is substantiated by the distribution and localization of CML in the testis as reported in our previous study (Mallidis et al., 2007). This, in turn, implies that the differentiating spermatozoa are continuously influenced by some form of ligand-RAGE instigated/mediated cellular event(s) throughout their development. The type of the ligand(s), the nature of the events, and their effects on the various stages of spermatogenesis are unknown.
The localization of RAGE in the smooth muscle ring surrounding the tubules of the epididymis was not surprising as the observation is consistent with cell culture based studies which have shown that the receptor is not only present, but is also capable of modulating migration, proliferation and the expression of matrix modifying molecules in this cell type (Ramasamy et al., 2005). Similarly, the detection of RAGE on fibroblasts in the epididymis, is consistent with previous studies that have identified RAGE expression on fibroblasts in vitro, where it has been shown to be associated with collagen production (Owen et al., 1998).
The milieu of the epididymis, with its mixture of rete testis fluid, epididymal secretions and the products of the metabolic activity of the spermatozoa themselves (Fouchecourt et al., 2000), constitutes an extremely favorable environment for the formation and accumulation of AGEs. As, such, it is a region where RAGE expression would be expected to be high. This premise is verified by the presence and distribution of RAGE reported in this study and that of CML in our previous report (Mallidis et al., unpublished data). With the localization of RAGE in the cytoplasm of the principal cells of the epididymis, an organ known for its absorptive and phagocytotic capabilities, it is tempting to speculate that the receptors' presence at the site of AGE accumulation bolsters the defences against AGE instigated damage. This is especially likely as the ciliated apical region appears to be expressing higher levels of the receptor. However, unlike other sites to which AGEs bind, and which do participate in their removal and detoxification (e.g. lactoferrin, scavenging receptors I and II, oligosaccharyl transferase-48, 80 K-H phosphoprotein, galectin 3 and CD36) (Chavakis et al., 2004), RAGE has the opposite ability. It neither accelerates nor contributes to AGE clearance or degradation. Rather it activates an array of signal transduction cascades which amplify the damaging effects of AGEs (Ramasamy et al., 2005). Consequently, it is more likely that as in other organs, in the epididymis RAGE contributes to, or mediates, the propagation and/or maintenance of a potentially hostile environment. For example, there is a large body of evidence showing that under a variety of settings, AGE-RAGE binding can trigger the rapid generation of ROS (Ramasamy et al., 2005), via the activation of NADPH oxidase amongst others processes (Wautier et al., 2001), which results in the ongoing generation of oxidative stress and, hence, sustained production of ROS and AGEs (Wendt et al., 2003). This is of particular interest when considering that sperm spend a substantial amount of time in the epididymis where they are particularly vulnerable to oxidative attack and that the epididymis is thought to be the primary site of ROS induced sperm nDNA damage (O'Connell et al., 2002; Suganuma et al., 2005).
The significantly greater number of RAGE expressing sperm found in samples from diabetic men when compared with non-diabetic subjects is probably a result of the heightened rate of AGE accumulation found in hyperglycaemic conditions. However, it is the specificity of RAGE expression on the acrosomal cap and the equatorial region of the sperm head, regardless of the diabetic status of the man, i.e. particularly noteworthy. This precisely mirrors the specificity of distribution of CML on sperm that we previously reported (Mallidis et al., 2007). It is this area, the acrosomal region, that undergoes a number of modifications during epididymal maturation. It constitutes a direct access point to the underlying nDNA and may also be the site of potentially the greatest RAGE induced damage. Beyond their ability to instigate an oxidative cascade, AGEs and RAGE may also facilitate attack from ROS produced by other mechanisms (e.g. leukocytes) by weakening the plasma membrane encapsulating and protecting the genome. The presence of RAGEs in this vulnerable region of the spermatozoa also opens the possibility that the nDNA damage may not only be the result of oxidative attack but could also be due to the destabilization or fragmentation of the DNA by a RAGE mediated process or an AGE modification of nuclear proteins (Gugliucci and Bendayan, 1995) which in turn undermines the integrity of the nDNA packing leading to strand breaks (Vlassara and Palace, 2002).
The presence of RAGE in seminal plasma is somewhat curious considering the receptor's function. However, low levels of RAGE have also been found in other secreted materials of the prostate, thyroid and renal tubule (Cheng et al., 2005).
The role of RAGE and its numerous ligands in diverse processes such as tissue damage, cell death, inflammatory response, oxidative stress and DNA fragmentation in various organs and cells, suggests that the presence of RAGE in the male reproductive tract may be a portent of its involvement in a multitude of similar but, as yet, unexplained conditions which ultimately result in male infertility.
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Acknowledgements |
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The authors thank Professor Andreas Meinhardt for his suggestions and critical reading of the manuscript, Professor Dr Martin Bergmann and Dr. Joachim Woenckhaus for the provision of the biopsy material and Mrs Margaret Kennedy for her technical assistance. We gratefully acknowledge the financial support of the Northern Ireland Research and Development Office, (Recognised Research Group: endocrinology and Diabetes) Belfast, United Kingdom (Grant no. EAT 2539) and the Spear Bell endowment.
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Submitted on February 23, 2007; resubmitted on April 19, 2007; accepted on April 28, 2007.
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