Free radical scavenger effect of rebamipide in sperm processing and cryopreservation

Keywords: male infertility; reactive oxygen species; rebamipide

Abstract

Aim: To study the effect of rebamipide added to semen samples and cryoprotectant on reactive oxygen species (ROS) production. Methods: Semen samples from 30 fertile and healthy volunteers were collected by masturbation after 2 days ~ 3 days of abstinence. After liquefaction, the specimens were diluted with sperm wash media to a uniform density of 20?06/mL. Rebamipide was added to semen samples and cryoprotectant to a final concentration of 10 µmol/L, 30 µmol/L, 100 µmol/L or 300 µmol/L. Specimens were incubated at 37 in a 0.5 % CO₂ incubator for 1 h or cryopreserved at -196 LN₂ for 3 days. The sperm motility and viability and the levels of ROS and lipid peroxidation of sperm membrance were assessed before and after incubation and cryopreservation by means of computer assisted semen analyzer, eosin-nigrosin stain, chemiluminescence and thiobarbituric acid assay, respectively. Results: The sperm motility was significantly increased after incubation with 100 µmol/L and 300 µmol/L rebamipide (P<0.05). After cryopreservation, the sperm motility was significantly decreased in all concentrations (P<0.05), but the decrease was less with 100 µmol/L and 300 µmol/L rebamipide than that with other concentrations. The sperm viability showed no significant difference before and after incubation (P>0.05). The levels of ROS and lipid peroxidation in semen were significantly decreased in proportion to the concentrations of rebamipide both after incubation and cryopreservation (P<0.05). Conclusion: Rebamipide is an effective free radical scavenger in semen in vitro.

1 Introduction

Reactive oxygen species (ROS) were detected in 25 % ~ 40 % of the semen of infertile men and in up to 96 % of the semen of patients with spinal cord injuries [1]. Elevated ROS levels in the ejaculate, one of the main causes of sterility [2], reduce sperm motility as well as decrease sperm-oocyte fusion capability in vitro [3].

ROS are the metabolites of oxygen, such as superoxide anions (O₂), hydrogen peroxide (H₂O₂), hydroxyl radicals (OH) and hypochlorite radicals (OHCl) [4]. Excessive ROS in the ejaculate reflects the existence of immature sperm or white blood cells (WBC) due to inflammation in the seminal tract or lower urinary tract. ROS can also be generated during sperm preparation, cryopreservation and other assisted reproductive tech-nologies. For example, sperm become more fragile due to the oxidative damage caused by new ROS formation or removal of endogenous antioxidants in seminal plasma during sperm preparation. The majority of normal cells have various endogenous scavengers, consisting of enzymatic antioxidants, such as superoxide dismutase, glutathione peroxidase and catalase [5, 6, 7], and nonenzymatic antioxidants, such as uric acid, ascorbate and tocopherol [8]. If ROS are produced in levels exceeding the intrinsic scavenger function of the cell, damage of cell membrane lipid, protein and nuclear DNA may ensue [9]. The World Health Organization (WHO) laboratory manual for the examination of human semen considered the evaluation of ROS levels as an important sperm function test [10].

Though experimental research has proven that various antioxidants scavenge ROS within the sperm, the application and utilization of these antioxidants are limited clinically [11, 12]. In the field of male infertility, there is a growing need to develop a safe and effective oral antioxidant for the treatment of elevated ROS levels in semen. Rebamipide is a propionic acid derivative that directly inhibits the production of superoxide (O₂^-) and eliminates ROS in hydroxyl radical (OH) system. It was originally developed as a drug to treat gastritis and gastric ulcer caused by helicobacter pylori infection [13]. It also has an indirect scavenger effect by decreasing the lipid content of the cell membrane and suppressing the activation of neutrophil and the expression of its adhesion molecule. Therefore we assume that rebamipide may also possess the same scavenger effect on sperm (Figure 1).

Figure 1. Endogenous reactive oxygen species formation and direct scavenger effect of rebamipide in sperm.

In the present paper, the sperm motility and viability and the levels of ROS and lipid peroxidation of cell membrane were studied during sperm processing, cryopreser-vation and thawing to evaluate the scavenger effect of various concentrations of rebamipide in vitro.

2 Materials and methods

The sperm motility and viability and the levels of ROS and lipid peroxidation of sperm membrance were analyzed before and after incubation and cryopreservation to estimate the antioxidant effect of rebamipide by means of computer assisted semen analyzer, eosin-nigrosin stain, chemiluminescence and thiobarbituric acid assay, respectively.

The experimental groups were classified in reference to various rebamipide concentrations of 0 (control), 10 µmol/L, 30 µmol/L, 100 µmol/L and 300 µmol/L.

Semen samples were collected by masturbation from 30 normozoospermic fertile men (age: 28 years4.5 years) after 2 days ~3 days of abstinence. Samples with abnormal semen parameters, delayed liquefaction or the presence of white blood cells or erythrocytes were excluded. Routine semen analysis was performed using a Makler Chamber (Fertility-Tech, USA) or a computer assisted semen analyzer (SAIS, Medical Supply Co., Korea) within 1 after liquefaction at 27 room temperature. Normal semen criteria included a volume of at lease 2 mL, a concentration of 2010⁶/mL or more, a motility of 50 % or more and normal morphology sperm of 30 % or more [10]. The semen sample was then diluted with the sperm washing media (Ham's F-10, Life Technologies, USA) to a uniform sperm density of 2010⁶/mL.

The diluted semen was divided into 0.5 mL aliquots for each group and cryoprotectant was added at a ratio of 1:1. They were transferred into a cryovial and slowly frozen for 4 h in a cryomatic cellevator tray (L.A.O. Enterprise, USA). The frozen samples were then cryopreservated at -196 in a LN₂ tank (Harsco Co., Hamilton. USA) for 3 days and then thawed slowly at room temperature.

To induce Fenton reaction, 0.25 mL of 0.2 µmol/L ferrous sulfate and 1 µmol/L sodium ascorbate were added to the specimens [14]. They were then incubated at 37 in a 0.5 % CO₂ incubator (Forma Scientific Co., USA) for 1 h.

The sperm were classified according to their motion capacity: Grade 1: static sperm; Grade 2: slow motion sperm; Grade 3: medium motion sperm and Grade 4: rapid motion sperm. The percentage of motile sperm was the sum of Grades 3 and 4.

Eosin-nigrosin (E-N) stain solution (50 mL) was added to the same volume of diluted semen in 1mL Eppendorf tube and then mixed vortically for a few seconds. About 50 µL of the mixture was applied on a slide, dried overnight and mounted with a mixture of malinol and xylene at a ratio of 2:1. The level of sperm viability was estimated by counting the dead (pink) and live (white) ones in 100 spermatozoa at 1000.

The level of ROS was measured using a luminometer (MicroLumat, LB196T, EG/G, Berthold, Germany). The luminescence level was measured after 25 µL 4 mmol/L luminal (5-amino-2, 3-dihydro-1,4-phthalazinedione, Wako, Japan) was added into 500 µL of double diluted sperm with a Hepes balanced salt solution/bovine serum albumin buffer (130 µmol/L NaCl, 4 µmol/L KCl, 1 µmol/L CaCl₂, 14 µmol/L fructose, 10 µmol/L Hepes, pH 8.0 and 1 mg/mL BSA). The maximum luminescence level was measured after luminol was added for 10 minutes, while the ROS activity was measured using an analog mode with 1000 times susceptibility in 10 seconds.

Malondialdehyde is the end-product of peroxidized fatty acids and a marker of lipid peroxidation and free radical activity. The sperm samples were centrifuged at 2000 rpm for 10 minutes after incubation (at room temperature) or cryopreservation and the supernatant was decanted. The sperm pellet was resuspended in a solution with 750 µL of 1 % phosphoric acid (Kanto Chemicals Co., Tokyo, Japan) and 250 µL of 0.6 % TBA (2-tribarbituric acid, Sigma, St. Louis, USA) using a vortex mixer (Scientific Industries, USA). The mixture was heated at 100 in a water bath (Samma Industrial Co., Korea) for 60 minutes, then vortexed. After being cooled at 4 for 30 minutes in a refrigerator, 1 mL of n-butanol (Junsei Chemical Co., Japan) was added, vortexed and centrifuged at 3000 rpm for 25 minutes. The supernatant was analyzed for absorbance (nmole/mg protein) at 510 nm and 534 nm using a diode array spectrophotometer (Hewlett Packard, USA).

Repeated measurement and analysis of variance using Pearson's correlation coefficient was used for analyzing the data. P<0.05 was considered statistically significant.

3 Results

After incubation, the sperm motility was significantly higher in all the experimental groups than that in the controls. Figure 2 shows the motility of 34.4 %, 36.9 %, 41.8 % and 44.5 % in groups with rebamipide concentrations of 10 µmol/L, 30 µmol/L, 100 µmol/L and 300 µmol/L, res-pectively, compared to 31.9 % in the controls (P<0.05). After cryopreservation and thawing, the sperm motility was also higher in all the experimental groups than that in the controls. Figure 2 shows the motility of 11.6 %8.1 %, 15.8 %8.0 %, 18.3 %8.6 % and 21.8 %9.4 % in groups with rebamipide concentrations of 10 µmol/L, 30 µmol/L, 100 µmol/L and 300 µmol/L, respectively, compared to 9.3 %6.0 % in the controls (P<0.05). The sperm motility was significantly higher with rebamipide concentration of 30 µmol/L or more (P<0.05).

The sperm viability after incubation and cryopreser-vation was not significantly different from each other in the experimental groups (Figure 3) .

After incubation with experimentally induced Fenton reaction, the ROS level was significantly lower in the experimental groups than that in the control groups (P<0.05). Figure 4 shows ROS levels of (13.84.03) AU, (13.13.62) AU, (13.04.1) AU and (12.63.90) AU in groups with rebamipide concentrations of 10 µmol/L, 30 µmol/L, 100 µmol/L and 300 µmol/L, respectively, compared to (14.54.67) AU in the controls (P<0.05). After cryopreservation and thawing, the ROS level was significantly lower in all the experimental groups than that in the control group. Figure 4 shows ROS levels of (12.33.5) AU, (12.43.1) AU, (12.02.9) AU and (11.62.6) AU with rebamipide concentrations of 10 µmol/L, 30 µmol/L, 100 µmol/L and 300 µmol/L, respectively, compared to (12.83.2) AU in the control group (P<0.05).

After incubation, the lipid peroxidation was significantly lower in all the experimental groups than that in the control groups. Figure 5 shows lipid peroxidase level of (38.615.6) nmol/mg, (30.814.1) nmol/mg, (24.013.9) nmol/mg and (15.013.4) nmol/mg protein with rebamipide concentrations of 10 µmol/L, 30 µmol/L, 100 µmol/L and 300 µmol/L, respectively, compared to (51.717.4) nmol/mg protein in the control group (P<0.05). After cryopreservation and thawing, the lipid peroxidation was significantly lower in all the experimental groups than that in the controls. Figure 5 shows lipid peroxidase level of 40.4 nmol/mg, 33.1 nmol/mg, 27.9 nmol/mg and 24.3 nmol/mg protein with rebamipide concentrations of 10 µmol/L, 30 µmol/L, 100 µmol/L and 300 µmol/L, respectively, compared to 57.2 nmol/mg protein in the control group (P<0.05).

In the incubation and cryopreservation groups, the ROS correlated with lipid peroxidation significantly with correlation coefficients of y =1.4168x +11.269, r = 0.23 (P<0.05) and y = 4.5105x -18.287, r = 0.30 (P<0.05), respectively (Figure 6).

Figure 6. Relationship between reactive oxygen species and lipid peroxidase level after incubation and cryopreservation.

In the incubation group, the ROS correlated with lipid peroxidation significantly with a correlation coefficient of y = -0.2225x +22.67, r = 0.54 (P<0.05, Figure 7). In the cryopreservation groups, the ROS and lipid peroxidation had no significant correlation (y = -0.0197x +17.221, r = 0.06; P>0.05, Figure 7). This suggested that the sperm damage was not only caused by ROS but also by other factors, including thermal injury during cryopre-servation and thawing.

Figure 7. Relationship between sperm motility and lipid peroxidase level after incubation and cryopreservation

Though the studies about ROS began more than one century ago, its physiological role in regard to male fertility has only been addressed in the past ten years. Iwasaki and Gagnon [15] reported that in semen, ROS could be detected in 40 % of infertile men and Agarwal et al. [1] reported that ROS levels were significantly higher in at least 25 % of infertile men. Park et al. [16] indicated that ROS levels in the seminal plasma were 60 % higher in the infertile than in the fertile men. Mazzili et al. [17] observed that ROS was detected in the seminal plasma in 87 % of infertile men and 55 % of fertile men, the difference being significant. The sperm in patients with varicocele and spinal injuries continued to produce ROS after washing and the sperm motility decreased proportionally with the levels of the ROS [4]. ROS level in infertile men was higher than that in fertile men, which correlated strongly with oligozoospermia, asthenospermia and abnormal sperm morphology.

Spermatozoa are sensitive to acute stress under aerobic conditions, inducing ROS production from spermatozoa and seminal white blood cells. The peroxidized metabolites of fatty acids originated from the ROS-damaged phosphatide of cell membrane directly damage sperm function and morphology [18, 19]. ROS arise also from assisted reproductive technology, including sperm washing or preparation. The influence of ROS on the fertile capacity in men is increasing with the problems of air pollution, ozone layer destruction, acid rain and endocrine disruptors [20-22]. High levels of ROS in ejaculate influence sperm motility, oval adhesion and fertilizing capacity, but certain levels of ROS are necessary for normal sperm physiology, such as hyperactivation, capacitation and acrosomal reaction [23].

The endogenous ROS produced from sperm are induced by sperm deficit, such as immature or damaged sperm or by external stimulation due to abnormality of the seminal tract, radiation and chemotherapy. In this study, ROS formation and the following lipid peroxidation of the cell membranes were experimentally induced by the Fenton reaction, which entails the addition of ferrous sulfate and sodium ascorbate or through cryopreservation and thawing [15, 24, 25]. Under these conditions, endogenous cellular antioxidation caused by NADPH-oxidase and NADH-dependent oxidoreductase (diaphorase) becomes markedly reduced. Superoxide is produced as primary metabolite from O2 through aerobic metabolism, followed by the formation of hydrogen peroxide and hydroxyl radical by superoxide dismutase and catalase, respectively. In ROS formed in human cells, including sperm, superoxide is produced not only by oxidative metabolism in the mitochondria, but also through enzymatic reactions caused by (-cytochrome enzymes, like xathine oxidase, cytochrome P-450 and other oxidases [12, 25].

Rebamipide is an antioxidant inhibiting the activation of NADPH-oxidase and sperm-diaphorase in the human cell. In the present study the ROS production and lipid peroxidation were significantly inhibited in the experimental group and eventually resulted in the preservation of sperm motility. But rebamipide did not apparently maintain sperm viability, suggesting that ROS only influence sperm function through adequate scavenger action. The lipid peroxidation of sperm membrane was inversely correlated to sperm motility in the incubation group, but not in the cryopreservation groups. During the cryopre-servation and thawing, it appeared that the sperm damage correlated not only with the ROS level but also with thermal injury, chemical reaction with cryoprotectant and other factors. Based on the data, rebamipide is known to be an effective free radical scavenger in semen and may be tried further as an oral antioxidant in patients with male infertility caused by ROS.

The present study confirmed that rebamipide was an effective free radical scavenger to inhibit the endogenous ROS production and the lipid peroxidation of cell membrane in sperm and to enhance the sperm motility during sperm processing and cryopreservation.

The study was sponsored by a grant from the Otsuka Pharmaceutical (OPC12759 in Semen).

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Correspondence to: Nam Cheol Park, M.D., Ph.D, Department of Urology, College of Medicine, Pusan National University, 1-10, Ami-dong, Seo-Ku, Busan 602-739, Korea.
Tel: +82-51-240 7349, Fax: +82-51-247 5443
E-mail: pnc@pusan.ac.kr
Received 2003-06-05   Accepted 2003-07-23