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Keywords:

  • antioxidants;
  • high-density lipoproteins;
  • male subfertility;
  • paraoxonase 1;
  • paraoxonase 2;
  • paraoxonase 3

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Study Type – Aetiology (case series)

Level of Evidence 4

What’s known on the subject? and What does the study add?

Oxidative stress seems to be one of the biochemical causes of defective sperm function. Paraoxonases are antioxidant enzymes that degrade lipid peroxides. There is a paucity of data on the possible role played by these enzymes in the pathophysiology of male sub-fertility.

The present study shows that testicular tissue of sub-fertile patients clearly expresses paraoxonases-1, 2, and 3. These findings suggest a role for these enzymes in the protection against lipid peroxidation inside the cell. However, the concentration and activity of paraoxonase-1 in semen are negligible and are probably the result of cellular catabolism, with no significant biological function.

OBJECTIVE

To characterise the immunohistochemical sites of paraoxonase (PON) 1, PON2 and PON3 in human testicular tissue, and to analyse PON1 levels in semen, aiming to investigate the role played by these enzymes in the pathophysiology of male subfertility.

PATIENTS AND METHODS

The present study was performed in 41 semen samples from normal donors and in 52 semen samples and ten testicle biopsies from patients who were being evaluated for causes of subfertility.

RESULTS

Immunohistochemical analyses showed high levels of PON1 and PON3 expression in testicular tissue. PON2 expression was also detected, albeit at weaker levels. Oxidative stress indicators in biopsies were low and localized in some specific areas of the seminiferous tubules. PON1 was detected in seminal fluid at very low levels but with no significant differences between patients and controls. Receiver-operating characteristic analysis showed a low diagnostic power of semen PON1 levels.

CONCLUSIONS

The present study shows high protein expression levels of PON1, PON2 and PON3 in testicular cells. The concentrations and activities of PON1 in semen are negligible and are probably the result of cellular catabolism, with no significant biological function in the testes.


Abbreviations
HDL

high-density lipoprotein

PON

paraoxonase

TBBL

5-thiobutyl-butyrolactone.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Decreases in male fertility rates are becoming an important problem in industrialized countries. Male subfertility currently affects one in 20 men, and this frequency will increase in the near future. Social as well as biological factors contribute to this phenomenon [1]. Several studies suggest that one of the biochemical causes of defective sperm function is oxidative stress. This can be a result of excessive free radical generation by spermatozoa and leukocytes or the disruption of the antioxidant defence systems in the male reproductive tract [2,3]. The role of exogenous, dietary antioxidants in combating oxidative stress and inflammation and their impact on male fertility has been well documented [2,4]. However, there is a paucity of information available on the putative role of endogenous antioxidants in this process. Paraoxonase (PON) 1 is an enzyme with esterase and lactonase activities found in plasma bound to high-density lipoproteins (HDL). It degrades oxidized phospholipids and thus has an antioxidant function [5]. The PON1 gene belongs to a family comprising three members, PON1, PON2 and PON3, with a common ancestral origin. All three PON enzymes share antioxidant properties. Reduced serum PON1 activities have been reported in several diseases involving increased oxidative stress [6]. PON1 protein expression has been detected in mouse testicular cells [7], and a recent study described low PON1 activity in the semen of subfertile men [8].

Despite the relevance of paraoxonases in the pathophysiology of many diseases, the measurement of these enzymes has been restricted to research laboratories and has not been extensively applied in routine clinical laboratories. The reason for this restriction is a result of methodological difficulties in their measurement [5]. For example, the most extensively used substrate to date for PON1 measurement is paraoxon, a non-physiological, highly toxic xenobiotic. Recently, a new serum test based on the measurement of the lactonase activity of PON1 was proposed and evaluated [9,10]. Lactonase measurement correlates well with the levels of PON1-HDL complex and provides a good indication of the levels and quality of the HDL particles to which the enzyme is bound. Moreover, highly specific antibodies against PON1, PON2 and PON3 have been obtained, and this has facilitated the development of immunological methods aiming to determine the expression of these proteins in tissues. To date, methods for the measurement of paraoxonases concentrations in serum and other biological fluids have only been developed for PON1 [11].

The present study aimed to characterise the immunohistochemical localization of PON1, PON2 and PON3 in human testicular tissue, as well as to analyse PON1 levels in seminal fluid. The measurement employed two assays for the different enzyme activities (esterase and lactonase) and, by also measuring the PON1 protein concentration, the possible role played by this enzyme in the pathophysiology of male subfertility may be elucidated.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

All the procedures described were approved by the Ethics Committees (Institutional Review Boards) of all the Hospitals involved, and written informed consent was obtained from all of the study participants. In total, 52 semen samples from individuals attending the subfertility clinic and Andrology Laboratory of the Centre d’Infertilitat i Reproducció Humana were studied. A further 41 samples were from documented normal semen donors. After abstinence from sexual intercourse for 3 days, semen samples were collected by masturbation into sterile specimen containers. The semen sample was allowed to liquefy over 30 min and a basic analysis was performed in all samples in accordance with the criteria recommended by the World Health Organization [12]. Normospermia was defined as a sperm count of ≥20 × 106/mL, motility a + b ≥ 50%, and >15% normal sperm forms. Oligospermia was defined as a sperm concentration <20 × 106/mL, irrespective of morphology or motility. Asthenospermia was defined as a sperm concentration of ≥20 × 106/mL and motility a + b < 50%, irrespective of morphology. According to these criteria, patients were classified as having oligospermia (n= 5), asthenospermia (n= 4), oligoasthenospermia (n= 41) and aspermia (n= 2). The presence of leukocytes was detected in the samples of five patients. After light microscopy examination, samples were centrifuged at 1500g for 10 min to obtain seminal fluid, which was distributed in aliquots and stored at −80 °C for subsequent batched biochemical analyses.

Biopsies were obtained from ten patients in whom this procedure was clinically indicated as described previously [13]. All were infertile patients undergoing an in vitro fertilization treatment and analyses of meiotic chromosomes were performed to further assess recurrent abortions, poor embryo quality, low rate of fertilization or no pregnancy. A sample of the tissue was set aside for the present investigation with the patient’s agreement. Biopsies were performed according to published methods [14]. The sperm cord was injected with 9 mL of 2% scandicaine (bupivacaine) via a 27-gauge needle just distal to the external inguinal ring. This was followed by an additional 1 mL of 2% local anaesthetic injected into the skin over the anterior scrotum. When anaesthesia had taken effect, a small incision in the testicle’s midportion was performed, cutting through the scrotal skin, tunica vaginalis and the tunica albuginea. A piece of the extruding testicular tissue was cut with fine scissors and placed in a sterile tube containing 4% phosphate-buffered formalin for 24 h at room temperature. The sample was then washed twice with water, dehydrated in 70% ethanol at 4 °C, and embedded in paraffin wax. Microtome paraffin-embedded sections (2 µm thick) were used for all histological analyses.

PON1, PON2 and PON3 protein expressions were assessed in testicular tissue by immunohistochemistry using polyclonal antibodies raised against specific peptides derived from the sequences of the three mature PON proteins [15]. Tubular cell 4-hydroxy-2-nonenal-protein adducts were measured as an index of lipid peroxidation using a monoclonal antibody obtained from the Japan Institute for the Control of Aging (Shizuoka, Japan).

PON1 esterase activity in semen was measured as the rate of hydrolysis of paraoxon at 410 nm and 37 °C in 0.05 mmol/L glycine buffer (pH 10.5) with 1 mmol/L CaCl2[16]. Activity was expressed as U/L (where 1 U = 1 µmol/min of hydrolysed paraoxon). PON1 lactonase activity was measured in undiluted semen samples using an assay reagent containing 1 mmol/L CaCl2, 0.25 mmol/L 5-thiobutyl-butyrolactone (TBBL) and 0.5 mmol/L 5,5′-dithio-bis-2-nitrobenzoic acid in 0.05 mmol/L Tris-HCL buffer (pH 8.0). The increase in absorbance was monitored at 412 nm [9]. Activities were expressed as mU/L (where 1 U = 1 mmol/min of hydrolysed TBBL). PON1 concentration was determined using an ELISA with the polyclonal antibody described above [15]. Total peroxide concentrations in semen samples were analyzed by a colorimetric enzymatic assay (ImmunDiagnostik, AG, Benshein, Germany). Apolipoprotein A-I and HDL-cholesterol concentrations, as estimates of HDL levels in semen, were analysed by standard methods (Beckman-Coulter, Fullerton, CA, USA).

Data are reported as the mean ± SD. Differences between groups were analysed by Student’s t-test. The area under the curve (AUC) of receiver-operating characteristic plots [17] was employed to assess the discriminative ability of PON1 activity and PON1 concentration aiming to distinguish between patients and control subjects. All analyses were performed using SPSS software, version 17.0 (SPSS Inc., Chicago, IL, USA).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Immunohistochemical analyses of testicular tissue showed high levels of PON1 and PON3 expression in the cytoplasm of most spermatogonias. PON2 expression was also detected, albeit at weaker levels. No staining for 4-hydroxy-2-nonenal-protein adducts was observed in the seminiferous tubules of the patients in the present study. A representative example of these findings is provided in Fig. 1.

image

Figure 1. Representative example of the immunohistochemical analysis of paraoxonase (PON) 1 (A), PON2 (B), PON3 (C) and 4-hydroxynonenal (D) in testicular tissue from subfertile patients. Positive staining is indicated by the arrows.

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Very low levels of PON1 were detected in seminal fluid (Table 1). There were no significant differences between patients and controls with respect to either PON1 esterase or lactonase activity or in the PON1 concentration. Receiver-operating characteristic curve analyses indicated a low diagnostic power of PON1 (i.e. the PON1 values were not able to discriminate between patients and controls), with the area under the curve (AUC) being ≈0.60 (Fig. 2 and Table 2). Total peroxides, apolipoprotein A-I and HDL-cholesterol concentration measurements in all donors, as well as patients, were below the detection limits of the respective assays. The likelihood of a prominent role of oxidative stress in this setting is low and scarcely significant values in both PON1 activity and lipid peroxides precluded further correlations. Similarly, there was no correlation with sperm concentration.

Table 1.  PON1 activity and concentration in healthy donors and subfertile patients
ParameterDonors (n= 41)Patients (n= 52)
  1. There were no statistically significant differences in any of the parameters in either of the groups studied. PON, paraoxonase.

PON1 esterase (U/L)21.3 ± 3.819.7 ± 4.7
PON1 lactonase (mU/L) 6.5 ± 5.5 7.8 ± 4.9
PON1 concentration (mg/L) 2.9 ± 3.1 2.7 ± 3.9
image

Figure 2. Receiver-operating characteristic curves for paraoxonase 1 lactonase activity (1), esterase activity (2) and concentration (3).

Download figure to PowerPoint

Table 2.  Area under the curve of receiver-operating characteristic plots for lactonase and esterase activities of PON1 and PON1 concentrations in semen to discriminate between subfertile patients and control subjects
ParameterAUC95% CI
  1. AUC, area under the curve; PON, paraoxonase.

PON1 esterase activity (U/L)0.660.54–0.77
PON1 lactonase activity (mU/L)0.590.47–0.71
PON1 concentration (mg/L)0.610.50–0.73

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

The causes of infertility in a large proportion of subfertile men remain unexplained [18]. Although oxidative stress has been proposed as having a role in the pathophysiology of this derangement [2,3], the available data remain controversial. Several studies reported increased levels of free radical species in only 50% of the subfertile patients studied [19–21], and no relationships were observed between increased lipid peroxidation and sperm function [22,23]. In the present study, 4-hydroxy-2-nonenal was not expressed in testicular cells, and the total peroxide concentrations in seminal fluid were below the detection limit of the assay. These findings indicate that, at least in the specific patients investigated in the present study, alterations in sperm concentration and/or motility may be observed despite there being a low degree of oxidative stress. In this setting, the question arises as to whether the paraoxonases play any significant role in the protection against free radical generation in male infertility.

The role of the paraoxonase family of antioxidant proteins in the protection against free radicals in the testicle has not been sufficiently investigated to date. A previous study by our group reported a strong PON1 and PON3 immunohistochemical staining and a weaker PON2 staining in seminiferous tubules of normal mice [7]. The present study confirms these results in subfertile patients, in whom a similar pattern of staining was observed. The data obtained on paraoxonase expression in the present study also confirm the recent reports of the Swedish Human Protein Atlas (http://proteinatlas.org), with antibodies directed against the complete PON1, PON2 and PON3 protein sequences. Indeed, PON1 and PON2 genetic polymorphisms, which determine the levels of these enzymes in tissues, have recently been associated with semen quality (I. Georgiou, L. Lazaros, N. Xita, A. Kaponis, N. Plachouras, E. Hatzi & K. Zikopoulos, unpublished data). Taken together, these results suggest that the paraoxonase protein family may play a role in the protection against cell oxidative stress in testicular tissue.

However, the results obtained in the present study do not support the concept of PON1 playing a significant role in the protection against free radicals in seminal fluid. Levels of the enzyme were practically negligible. The esterase activity against paraoxon observed in the patients in the present study was very similar to that observed by the spontaneous hydrolysis of this substrate in the reaction media when no sample is added (i.e. blank control in the assay). This was confirmed by the low levels of lactonase activity and PON1 concentration. In addition, PON1 measurement was not able to discriminate between patients and controls with any degree of statistical confidence. These results are not surprising because PON1 is carried in HDL particles in circulation and needs the lypophilic environment inside these lipoproteins to be enzymatically active [24,25]. In the present study, apolipoprotein A-I and HDL-cholesterol concentrations in seminal fluid were lower than the detection limit of the assay in all the individuals studied. This indicates an absence of significant amounts of HDL particles, and can explain why PON1 levels were so low, as well, why the enzyme was mostly inactive. This opinion differs from that expressed in a recent study [8], which, despite also observing a very low PON1 esterase activity, postulated that PON1 in semen plays an important role in the pathogenesis of subfertility, and that its measurement may have a high discriminatory value in the diagnosis of this condition. This concept is difficult to understand from our point of view because PON1 activities and concentrations are more than 20-fold lower in semen than in serum [10,26,27]. It is likely that the traceable amounts of PON1 found in human semen derive from testicular cell catabolism, and do not play any significant role in the protection against oxidative stress. Probably, other antioxidants such as vitamin E [22], superoxide dismutase and catalase [23] play a much more important role than PON1 in the protection of semen against the effects of free radicals.

In summary, the present study shows that testicular tissue of subfertile patients clearly express PON1, PON2 and PON3. These findings suggest a role for these enzymes in the protection against lipid peroxidation inside the cell. However, the concentration and activity of PON1 in semen are negligible and are probably the result of cellular catabolism, with no significant biological function.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

We thank Drs Dan Tawfik, Olga Khersonsky and Leonid Gaidukov, from the Weizmann Institute of Rehovot, Israel, for the generous gift of the TBBL reagent. The present study was supported by grants from the Instituto de Salud Carlos III (FIS 05/1607 and 08/1175), Ministerio de Sanidad, Madrid, Spain. J.M. was the recipient of a grant from the Department of Medicine and Surgery of the Universitat Rovira i Virgili, Reus, Spain. R.B.-D and G.A. were recipients of postgraduate fellowships from the Generalitat de Catalunya (FI08/00064, and FI06/01054, respectively). Editorial assistance was provided by Dr Peter R. Turner from t-SciMed (Reus, Spain).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES