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Effect of endotoxin-induced reactive oxygen species on sperm motility

      Abstract

      Objective: To define the mechanism of infection-induced damage of sperm.
      Design: The effect of lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) on sperm motility and its modification by scavengers were investigated.
      Setting: Research laboratory of a university hospital.
      Patient(s): Normozoospermic semen samples were obtained from 37 healthy volunteers.
      Intervention(s): The sperms were incubated in the presence of LPS with or without scavengers.
      Main Outcome Measure(s): Sperm motility was evaluated by a sperm quality analyzer (SQAIIB). ROS formation in semen samples was measured by a Berthold luminometer (LB953).
      Result(s): Motility of spermatozoa was decreased in the LPS-treated samples compared with that in the control groups. ROS was significantly higher in the LPS-treated groups than in the control groups. The addition of ROS scavengers restored the motility index and suppressed ROS production in the LPS-treated semen samples.
      Conclusion(s): These data suggest that endotoxin-induced excessive production of ROS is responsible for the decrease in sperm motility and that antioxidant therapy may be a therapeutic option for infertile men with bacterial genital tract infection.

      Keywords

      The first suggestion that oxidative stress might be involved in the disruption of normal sperm function came from MacLeod (
      • MacLeod J
      The role of oxygen in the metabolism and motility of human spermatozoa.
      ), who observed that human spermatozoa rapidly lose their motility when incubated under high oxygen tensions. More recently, it has been shown that as many as 25% of semen samples from infertile men produce high levels of reactive oxygen species (ROS) (
      • Iwasaki A
      • Gagnon C
      Formation of reactive oxygen species in spermatozoa of infertile patients.
      ). The higher levels of ROS produced by damaged or deficient spermatozoa have been believed to be associated with a loss of motility and a decreased capacity for sperm–oocyte fusion (
      • Aitken R.J
      A free radical theory of male infertility.
      ).
      The association between bacterial genital tract infection and male infertility has been suggested for several years. Although endotoxins such as lipopolysaccharide (LPS) from gram-negative bacteria have been shown to be toxic for human spermatozoa (
      • Teague N.S
      • Boyarsky S
      • Geen J.F
      Interference of human spermatozoa motility by Escherichia coli.
      ), the mechanism by which a bacterial genital tract infection causes male infertility is still unclear.
      To define the mechanism of infection-induced damage of sperm, we investigated the effect of LPS-stimulated ROS on sperm motility and its modification by scavengers. This is the first report to demonstrate that LPS-induced ROS production causes loss of sperm motility in vitro, which may provide a mechanism underlying male infertility associated with bacterial infection.

      Materials and methods

       Sample collection

      Normozoospermic semen samples were collected from 37 healthy volunteers. Samples were produced by masturbation after 3–5 days of sexual abstinence and allowed to liquefy for 30 minutes at room temperature. Semen analysis using World Health Organization criteria was performed (
      World Health Organization
      ). Normozoospermic samples were defined as possessing the following characteristics: volume ≥2.0 mL, sperm concentration ≥20 × 106 spermatozoa/mL, motility ≥50%, normal morphology ≥30%, and <1 × 106 leukocytes/mL. This study was approved by the Institutional Review Board of Oita Medical University.

       Determination of sperm motility index

      Sperm motility was evaluated by a sperm quality analyzer (SQAIIB, Medical Electronic Systems, Israel), which provided a quantitative estimation of sperm motility. The sperm motility index, a parameter for sperm motility, was determined simultaneously for the same samples evaluated by the SQAIIB. The sperm motility index was determined in three sequential readings by introducing semen into a thin glass capillary tube (internal dimensions: depth 0.3 mm, width 3 mm, length 50 mm), which was housed in a plastic casing with a 2-mm diameter optical aperture. The sample was processed by the Sperm Quality Analyzer according to the manufacturer’s instructions. Using this method, each sperm motility index reading represented the mean of 10-second measurements of the analog signal. Concentration of sperm and sperm motility were also confirmed using a hemocytometer.

       Chemiluminescence

      Determination of ROS activity was carried out by using a Berthold luminometer (LB953, Berthold, Wildbad, Germany). Phorbol 12-myristate acetate (PMA) (Sigma Chemical Co., St. Louis, MO) (0.075 μg/mL) was added to the mixture of sperm and luminol (5-amino-2, 3dihydro-1, 4 phthalazinedione; 30 μg/mL, Sigma Chemical Co.) as a probe in the cuvette, and the luminescence was recorded. ROS formation for 30 minutes was expressed as the number of photons counted per minute (cpm) × 105. Luminol-enhanced chemiluminescence from semen samples was measured in contact with LPS (Escherichia coli, serotype 055:B5, lot 127H4097, Sigma Chemical Co.) and PMA in vitro. Superoxide dismutase (SOD, 100 U/mL, Sigma Chemical Co.), catalase (1 U/mL, Wako Chemical Co., Osaka, Japan), ascorbic acid (1 mM, Wako Chemical Co.), α-tocopherol (10 mM, Wako Chemical Co.), and reduced glutathione (100 nM, Wako Chemical Co.) were simultaneously added with LPS (10 μg/mL).

       Statistical analysis

      Statistical analysis was performed using analysis of variance followed by the Fisher’s exact test. P<.05 was considered to be statisically significant. Values are given as mean ± SD.

      Results

       Effect of LPS on sperm motility and its modification by ROS scavengers

      The values of the sperm motility index demonstrated a significant correlation with the concentration of sperm, the sperm motility, and the motile sperm concentration (data not shown). Motility of spermatozoa was decreased in the LPS-treated samples compared with that in the control groups in a dose-dependent manner. Sperm motility was inhibited by 15%, 21%, and 50% in the presence of 0.1, 1, and 10 μg/mL dose of LPS after 60 minutes of incubation, respectively, compared with that of the control groups after 60 minutes of incubation (P<.05) (Fig. 1). The LPS-induced inhibition of the motility index was significantly reversed by addition of ROS scavengers, such as SOD, glutathione, catalase, α-tocopherol, and ascorbic acid (P<.05) (Fig. 2). These scavengers were without effect on the sperm motility in the absence of LPS (data not shown).
      Figure thumbnail GR1
      Figure 1Effect of LPS on sperm motility. Various concentrations of LPS were added to the semen samples at 37°C. Motility was assessed immediately after 60 minutes and after 120 minutes as described in Materials and Methods. Values depicted are mean ± SD for experiments performed using different LPS and sperm preparations. The value of 100% corresponds to motility of control semen samples observed at 0 minutes of incubation. Motility of spermatozoa decreased in the LPS-treated samples compared with that of controls. Significantly suppressed sperm motility was observed in LPS-treated samples in a dose-dependent manner. Sperm motility was inhibited by 15%, 21%, and 50% in the presence of 0.1, 1, and 10 μg/mL dose of LPS after 60 minutes of incubation, respectively, compared with that of the control groups after 60 minutes of incubation (P<.05).
      Urata. LPS-induced ROS and sperm motility. Fertil Steril 2001.
      Figure thumbnail GR2
      Figure 2Effects of various scavengers on sperm motility. LPS was added to spermatozoa already coincubated with SOD (100 U/mL), catalase (1 U/mL), ascorbic acid (1 mM), α-tocopherol (10 mM), and glutathione (100 nM), and the motility was assessed 60 minutes later. Values are represented as the mean ± SD and are expressed as a percentage of control sperm motility (without LPS), which was 86.2% ± 9.3% after 60 minutes of incubation. The value obtained with LPS (43.4%) was lower than all others (P<.01), and the values obtained with all scavengers were significantly higher than those of LPS only (P<.05). Control (86.2%), LPS 10 μg/mL (43.1%), LPS 10 μg/mL + SOD 100 U/mL (68.7%), LPS 10 μg/mL + glutathione 100 nM (85.0%), LPS 10 μg/mL + catalase 1 U/mL (89.5%), LPS 10 μg/mL + α-tocopherol 10 mM (87.0%), LPS 10 μg/mL + ascorbic acid 1 mM (90.1%).
      Urata. LPS-induced ROS and sperm motility. Fertil Steril 2001.

       Effect of LPS on ROS production and its modification by ROS scavengers

      The relationship between LPS-induced reduction of motility and ROS production in the semen was evaluated. As shown in Figure 3 , ROS production was significantly higher in the LPS-treated groups than in control groups (control: 100%, LPS 0.1 μg/mL: 105%, LPS 1 μg/mL: 130%, LPS 10 μg/mL: 148%) (P<.05). SOD, catalase, ascorbic acid, α-tocopherol, and glutathione significantly reversed the LPS-stimulated ROS increase compared with LPS groups (control: 100%, LPS 10 μg/mL: 136%, LPS 10 μg/mL + SOD 100 U/mL: 114%, LPS 10 μg/mL + glutathione 100 mM: 123%, LPS 10 μg/mL + catalase 1 U/mL: 115%, LPS 10 μg/mL + α-tocopherol 10 mM: 107%, LPS 10 μg/mL + ascorbic acid 1 mM: 102%) (P<.05). In the absence of LPS, the baseline signals generated in the semen samples were unaffected by these scavengers alone (data not shown).
      Figure thumbnail GR3
      Figure 3Time course of ROS production in human semen samples in response to LPS. Chemiluminescence was recorded for 30 minutes. Representative data are presented from one experiment using three concentrations of LPS (0.1, 1, and 10 μg/mL). The values obtained with LPS were significantly higher, in a dose-depended manner, than those of the controls after 14 minutes of incubation (control: 100%, LPS 0.1 μg/mL: 105%, LPS 1 μg/mL: 130%, LPS 10 μg/mL: 148%) (P<.05).
      Urata. LPS-induced ROS and sperm motility. Fertil Steril 2001.

      Discussion

      In the present study, we demonstrated that LPS decreased sperm motility, which was restored by treatment of the semen with ROS scavengers such as SOD, glutathione, catalase, α-tocopherol, and ascorbic acid. The reversibility of the sperm motility by these ROS scavengers suggests that LPS-induced production of ROS may be related to the decreased motility caused by LPS. Our observation that these scavengers inhibited the increase in ROS production in response to LPS is further evidence that there is a relationship between endotoxin-induced ROS production and a decrease in sperm motility. The LPS-induced increase in ROS may cause the lipid peroxidation of spermatozoa (
      • Aitken R.J
      Free radicals, lipid peroxidation and sperm function.
      ,
      • Kobayashi T
      • Miyazaki T
      • Natori M
      • Nozawa S
      Protective role of superoxide dismutase in human sperm motility superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa.
      ) and disrupt its membrane conformation, leading to loss of motility. Endotoxin-induced ROS may also result in oxidative DNA damage (
      • Fraga C.G
      • Motchnik P.A
      • Shigenaga M.K
      • Helbock H.J
      • Jacob R.A
      • Ames B.N
      Ascorbic acid protects against endogenous oxidative DNA damage in human sperm.
      ) that could affect sperm quality and increase the risk of genetic defects.
      Although the generation of ROS has been known to be an essential prerequisite for the normal functioning of many cells, excessive exposure to ROS may be harmful to spermatozoa, as discussed above. Several investigators have shown that ROS levels are significantly higher in idiopathic male infertility patients. D’Agata et al. (

      D’Agata R, Vicari E, Moncada ML, Sidoti G, Calegero AE, Polosa P. Generation of reactive oxygen species in subgroups of infertile men. Int J Andol 1990;344–51.

      ) have reported that an excessive production of ROS by sperm may explain some cases of idiopathic male infertility. Aitken et al. (
      • Aitken R.J
      • Clarkson J.S
      • Fishel S
      Generation of reactive oxygen species, lipid peroxidation, and human sperm function.
      ) have suggested that ROS may play a causative role in lipid peroxidation in the etiology of defective sperm. These observations indicate that the excessive production of ROS, either surrounding spermatozoa or on its own, could be a mechanism underlying male infertility, irrespective of pathogens that induce oxidative stress.
      Bacterial infections in the male genital tract have been implicated in infertility, in which E. coli or Neisseria gonorrhoeae is considered to be the main infectious source (
      • Teague N.S
      • Boyarsky S
      • Geen J.F
      Interference of human spermatozoa motility by Escherichia coli.
      ). LPS, an endotoxin from these gram-negative organisms, potently activates cells in the immune system (
      • Hinshaw L.B
      Pathophysiology of endotoxin action an overview.
      ). The cells in the immune system may include leukocytes in semen that are present physiologically or that increase during bacterial infection (
      • Teague N.S
      • Boyarsky S
      • Geen J.F
      Interference of human spermatozoa motility by Escherichia coli.
      ). Therefore, our observation in this study indicates that LPS stimulates leukocytes, and possibly a portion of spermatozoa in semen, to induce the production of ROS, which affects sperm motility. Wang and Fanning (
      • Wang A
      • Fanning L
      Generation of reactive oxygen species by leukocytes and sperm following exposure to urogenital tract infection.
      ) have reported that LPS-stimulated leukocytes purified from semen produced markedly higher levels of ROS compared with sperm and suggested that the LPS-stimulated production of ROS was mainly derived from leukocytes. In view of the association between leukocytospermia and genital tract bacterial infection (
      World Health Organization
      ), their finding and ours suggest that the increased leukocytes in the infected semen may produce excessive amounts of ROS in response to endotoxins, resulting in the loss of sperm motility.
      Seminal plasma has been reported to contain α-tocopherol (
      • Moilanen J
      • Hovatta O
      Excretion of alpha-tocopherol into human seminal plasma after oral administration.
      ), uric acid, and vitamin C (

      Thiele JJ, Freisleben HJ, Fuchs J, Ochsendorf FR. Ascorbic acid and urate in human seminal plasma: determination and interrelationships with chemiluminescence in washed semen. Hum Reprod 1995;110–5.

      ), as well as enzymatic systems protective against oxidative damage, such as SOD (
      • Nissen H.P
      • Kreysel H.W
      Superoxide dismutase in human semen.
      ), catalase (
      • Jeulin C
      • Soufir J.C
      • Weber P
      • Laval-Martin D
      • Calvayrac R
      Catalase activity in human spermatozoa and seminal plasma.
      ), and a glutathione peroxidase/reductase pair (
      • Alvarez J.G
      • Storey B.T
      Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation.
      ). Human spermatozoa possess the antilipoperoxidative defense enzymes SOD and glutathione peroxidase plus glutathione reductase (
      • Storey B.T
      Biochemistry of the indication and prevention of lipoperoxidative damage in human spermatozoa.
      ,

      Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. J Androl 1987;338–48.

      ). Glutathione, ascorbate, and α-tocopherol play a significant role, in association with antioxidant enzymes, in the antioxidant defenses that preserve the functional competence of spermatozoa exposed to an oxidative attack (
      • Ochsendorf F.R
      • Buhl R
      • Bastlein A
      • Beschmann H
      Glutathione in spermatozoa and seminal plasma of infertile men.
      ,

      Irvine DS. Glutathione as a treatment for male infertility. Rev Reprod 1996;6–12.

      ,
      • Therond P
      • Auger J
      • Legrand A
      • Jouannet P
      α-tocopherol in human spermatozoa and seminal plasma relationships with motility, antioxidant enzymes and leukocytes.
      ,
      • Suleiman S.A
      • Ali M.E
      • Zaki Z.M.S
      • El-Malik E.M.A
      • Nasr M.A
      Lipid peroxidation and human sperm motility protective role of vitamin E.
      ,
      • Lewis S.E.M
      • Sterling E.S.L
      • Young I.S
      • Thompson W
      Comparison of individual antioxidants of sperm and seminal plasma in fertile and infertile man.
      ). These observations indicate that antioxidant defenses may play an important role in the protection from a ROS attack against spermatozoa. Our observation that these ROS scavengers reversed LPS-induced ROS production is also an indication that antioxidant therapy may be useful for infertile men with leukocytospermic or bacterial genital tract infections.

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