Potential role of reactive oxygen species on testicular pathology associated with infertility

Asian J Androl 2003 Jun; 5: 95-99             

Keywords: reactive oxygen species; testis; infertility; malondialdehyde

Abstract

Aim: To investigate the level of malondialdehyde (MDA), a direct indicator of lipid peroxidation-induced injury by reactive oxygen species (ROS), in testicular biopsy specimens from infertile patients. Methods: Levels of MDA were measured in testicular biopsy specimens from 29 consequent-randomized infertile men, aged 29.584.76 (21~45) years. All patients were evaluated by a complete medical and reproductive history, physical examination, semen analysis (at least two), serum follicle-stimulating hormone and free testosterone levels, testicular biopsy and contact imprint. Scrotal colour Doppler ultrasonography was used to confirm suspected varicocele. The testicular MDA level was measured using the thiobarbituric acid test and the results were expressed per unit tissue weight. Results: As a causal factor in infertility, varicocele was identified in 17 (58.6 %) patients, and idiopathic infertility, testicular failure and obstruction in 4 (13.8 %) patients each. The testicular MDA level was 13.56 (6.01), 49.56 (24.04), 58.53 (48.07), and 32.64 (21.51), 32.72 (13.61), 23.07 (7.82), 42,12 (34.76) pmol/mg tissue in the normal spermatogenesis (control), late maturation arrest, Sertoli cell only (SCO) and hypospermatogenesis (mild, moderete, severe) groups, respectively. The elevation of MDA levels was significant in the testicular tissue from SCO and maturation arrest groups compared with the controls (P<0.05). In addition, the elevation in testicular MDA levels between the SCO and the moderete hypospermatogenesis, and the moderate hypospermatogenesis and the maturation arrest groups was significant (P<0.05). Conclusion: Severe pathologic changes in the testicular tissue are associated with a high level of lipid peroxidation. These findings suggest that overproduction of ROS may play a role in the mechanism of testicular degeneration associated with infertility.

1 Introduction

Defective sperm function is a major cause of human infertility [1] and yet little is known of the aetiology of this condition or the precise nature of the defects responsible for the loss of fertilizing potential. Researh during the last decade implicated oxidative stress is a mediator of sperm dysfunction [2-4]. It has been suggested that this phenomenon was causally related to the ability of germ cells to generate reactive oxygen species (ROS), such as hydrogen peroxide, speroxide anion, and hydroxyl radical. Moreover, action of ROS scavengers in spermatozoa has been described previously [5, 6]. In normal circumstances, there is an equilibrium between the generation of ROS and antioxidant strategies of the male reproductive tract, leaving only a critical amount of ROS reguired for normal sperm functions, as capacitation, acrosome reaction and fusion with the oocyte membrane[7]. Excessive production of ROS, however, results in destruction of the antioxidant capacity of spermatozoa and seminal plasma causing what is called oxidative stress[8]. Due to a high polyunsaturated fatty acid content, spermatozoa plasma membranes are highly sensitive to ROS-induced damage [9, 10]. There are growing evi-dences that oxidative damage to the human sperma-tozoa membrane is an important pathophysiological mechanism in male infertility. Malondialdehyde (MDA) is a stable end product of lipid peroxidation and therefore can be used as an indirect measure of the cumulative lipid peroxidation [11, 12]. Recently, it has been shown that ROS can also induce apoptosis [13-15] and accelerated apoptotic cell death has been observed in the cryptorchid testis [16]. Thus, the aim of the present study was to investigate the level of MDA in testicular biopsy specimens from infertile patients; characterizing the effect of ROS on these specimens may lead to new insight for therapies to protect spermatogenesis.

2 Materials and methods

The study included 29 consequent-randomized infertile men, aged 29.584.76 (range 21~45) years. A complete medical and reproductive history was obtained from all and included age, duration of infertility and history of any previous systemic or genitourinary disorders. All patients underwent a complete physical examination by the same physician (A.K.) in a warm room. A minimum of two semen analyses were performed according to WHO guidelines [17] and serum follicle stimulating hormone (FSH) and free testosterone levels were measured in all patients. Men with a suspected varicocele underwent scrotal colour Doppler ultrasono-graphy. Varicoles were graded as: Grade I, a distinct dilatation of the internal spermatic veins palpable during a Valsalva manoeuvre when upright; Grade II, a-palpable vein when upright with no Valsalva manoeuvre; Grade III, a vein both palpable and visible through the scrotal skin when upright, with no Valsalva manoeuvre. The ultrasono-graphic criteria for identifying a varicocele was an internal spermatic vein which had a luminal diamater of >0.27 cm and/or reflux [18].

All patients underwent testicular biopsy (using the window technique) and a contact imprint with local anaesthesia. Testes were fixed in Bouin's solution and embedded in paraffin. Histological sections were prepared and stained with hematoxylin and eosin. Histological changes were observed by light microscopy. The findings were classified histopathologically as normal spermatogenesis, hypospermatogenesis (mild, moderate, severe), maturation arrest (early, late), germinal cell aplasia (Sertoly cell only syndrome, SCO) and focal spermato-genesis [19].

The testicular MDA level was measured by using the thiobarbituric acid (TBA) test according to the method suggested by Buege and Aust [20] and the results ex-pressed per unit weight of tissue. MDA reacts with thiobarbituric acid to give a red compound absorbing at 535 nm. The stock reagent (2 mL, 12 % w/v trichloroa-catic acid, 0.375 % w/v thiobarbituric acid and 0.25 mol/L hydrochloric acid, warmed if necessary to dissolve the thiobarbituric acid) was mixed with 1 mL of the sample (0.1 mg~2.0 mg of membrane protein or 0.1 mmol/L~ 0.2 mmol/L of lipid phosphate), mixed thoroughly and heated for 15 min in a boiling water bath. After cooling, the flocculent precipitate was removed by centrifugation at 1000 g for 10 min and the absorbance of the super-natant determined at 535 nm against blank containing all the reagents. Standards were generated by acid hydrolysis of 1, 1, 3, 3-tetraethoxypropane in 0.1 mol/L HCI.

Data were expressed in meanSD. Differences be-tween groups were assessed using Mann-Whithey U test, with P<0.05 considered significant.

3 Results

As a causal factor in infertility, varicocele was iden-tified in 17 (58.6 %) patients and idiopathic infertility, testicular failure and obstruction in 4 (13.8 %) each. Fourteen patients (48.2 %) had azoospermia, 9 (31 %) oligozoospermia alone or in combination with astheno-zoospermia, 5 (17.3 %) normozoospermia and 1 (3.5 %) asthenozoospermia. Histopathological exami-nation revealed hypospermatogenesis in 12 (41.4 %, including 4 severe, 4 moderate and 4 mild cases), SCO in 9 (31 %, including SCO and focal spermatogenesis in 2), late maturation arrest in 4 (13.8 %) and normal spermatogenesis in 4 (13.8 %) patients.

The testicular MDA level in the normal spermato-genesis, late maturation arrest, SCO and hyposper-matogenesis (mild, moderete, severe) groups was shown in Table 1. The elevation of testicular MDA levels was significant in the SCO and maturation arrest groups compared with controls (P<0.05). In addition, the elevation in testicular MDA level between SCO and moderate hypospermatogenesis, and moderete hypospermatogenesis and maturation arrest groups was also significant (P<0.05).

Table 1. Testicular MDA levels in subjects with different pathological diagnosis. ^bP<0.05, compared with control; ^eP<0.05, compared with moderate hypospermatogenesis group.

4 Discussion

The production of ROS by spermatozoa is a normal physiological process, which serves as an important mediator in signal transduction mechanisms, regulation of sperm capacitation and facilitation of acrasome reaction and spermatozoon-oocyte attachment [21-25]. ROS may cause defective sperm function as a result of lipid peroxidation of the polyunsaturated fatty acids in the head and mid-pice, alter sperm morphology and lead to decreased motility and ineffective spermatozoon-oocyte fusion. Lipid peroxidation may damage membrane integrity with increased cell membrane permeability, thus leading to enzyme inactivation, structural damage to DNA and cell death [2, 3, 9, 10]. Oxidative stress has been established as one of the major factors leading to an infertile status in men [2, 3, 9, 10, 26]. In addition, a large body of evidence has accumulated to indicate that oxidative stress may play a fundamental role in the regulation of apoptosis [27]. Recently, it has been suggested that degenerative processes of testicular germ cells by heat stress associated with cryptorchidism involve apoptosis [16, 28]. Ikade et al. [15] showed the involvement of oxidative stress in the induction of apoptosis due to heat stress and suggested that oxidative stress itself was a direct inducer of apoptosis in testicular cells. All living cells were claimed to generate ROS constantly [29], and the rate of ROS generation in the testis or spermatozoa appears to be temperature-dependent. Moreover, in experimentally induced cryptorchid rats the activities of several scavenging enzymes were impared, accompained by increased peroxidation of cellular lipids [30]. As compared with the liver, the rat testis expressed equivalent superoxide dismutase (SOD) activity but only 5 % of the glutathione peroxidase activity and 2 % of the catalase activity [31]. Compared with somatic cells, such as the Sertoli cells or peritubular cells, the testicular germ cells presented a different composition characterized by a low glutathione-dependent enzyme activity [32]. Furthermore, Peltola et al. reported that physiological lutuneizing hormone (LH) action in the rat testis caused lipid peroxidation and maintained high activities of peroxide-metabolizing enzymes in the interstitial tissue. The steroidogenic steps regulated by the P450 enzymes were the most likely sites of ROS generation [33]. Combined action of FSH-stimulated Sertoli cells and hCG-stimulated testosterone production may stimulate ROS-producing metabolism or differen-tiation processes in the germ cells [34, 35].

We have previously shown that although there was no significant difference in MDA levels in testicular tissue between men with or withouth varicocele, patients with grade III varicocele had significantly higher levels than those with lower grades, indicating that increasing MDA levels are associated with higher grades of varicocele [36]. In the present study, it was shown that the MDA level was significantly higher in the SCO and maturation arrest groups than that in the controls. However, Yaman et al. reported that testicular tissue MDA and xanthine oxidase concentrations were not statistically different between subjects with normal spermatogenesis and patients with SCO, maturation arrest and hyposper-matogenesis [37]. We also found that the level of MDA was highest in late maturation arrest and SCO groups and suggested that potential sources of ROS production were the morphologically or functionally abnormal spermatozoa and other cells, including the Sertoli cells or Leydig cells.

In view of the physiologic and diagnostic significance of ROS generation, the development of techniques to detect this activity is important. A more specific method of determining ROS measures an oxidized component that can remain in the body fluids such as TBA-reacted MDA, an index of lipid peroxidation damage [38]. The outcome of this assay correlates well with other lipid peroxidation assay methods, including chemilu-minescence, pentane or ethane formation, and colori-metric reactions based on the reduction of phospholipid hydroperoxides with potassium iodide [39]. The onset of lipid peroxidation in susceptible sperm leads to the progressive accumulation of lipid hydroperoxides in the sperm plasma membrane, which then decompose to form MDA. We have previously studied the level of MDA using TBA method in testicular tissue [36, 40].

In conclusion, the present study demonstrates that severe pathologic changes in the testicular tissue are associated with a high level of lipid peroxidation. These findings suggest that overproduction of ROS may play a role in the mechanism of testicular degeneration associated with infertility.

To Kemal Hakan Gulkesen (from Akdeniz University) for performing the statistical analysis of this study and for his interest.

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Correspondence to: Dr. I. Turker Koksal, Department of Urology, Faculty of Medicine, Akdeniz University, Dumlupinar Bulvari, Kampus, 07070, Antalya, Turkey.
Tel. + 90-242-227 4482 Fax: +90-242-227 4480
E-mail: koksal@med.akdeniz.edu.tr
Received 2002-11-06    Accepted 2003-02-14

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