Antioxidative effect of fullerenol on goat epididymal spermatozoa

M. Arul Murugan, Bindu Gangadharan, P.P. Mathur

Asian J Androl  2002 Jun; 4:  149-152             

Keywords: fullerenol; iron; spermatozoa; oxidative stress; antioxidant enzymes; lipid peroxidation; goat

Abstract

Aim: To evaluate the effect of fullerenol on the antioxidant system of goat epididymal sperm. Methods: Fresh epididymides of adult goats were obtained from local slaughter houses and sperm were collected by chopping the epididymis in modified Ringer's phosphate solution (RPS medium). After several washings the sperm samples were equally dispersed in RPS medium and incubated with fullerenol (1, 10 and 100 mmol) and FeSO₄/ascorbate (40/200 mmol) with or without fullerenol (1, 10 and 100 mmol) for 3 h at 32. After incubation, an aliquot of sperm samples were homogenized and centrifuged and the supernatant used for biochemical studies. Results: In FeSO₄/ascorbate-incubated samples, the activities of antioxidant enzymes, superoxide dismutase, glutathione peroxidase and glutathione reductase, were decreased while lipid peroxidation increased as compared to the control sperm samples. In fullerenol-incubated sperm samples, the activities of superoxide dismutase, glutathione peroxidase and glutathione reductase were increased while lipid peroxidation was decreased in a dose-dependent manner. Co-incubation of sperm with fullerenol (1,10 and 100 mmol) and FeSO₄/ascorbate (40/200 mmol) increased the activities of antioxidant enzymes and prevented the iron-induced elevation of lipid peroxidation in a dose-dependent manner. Conclusion: Fullerenol reduces iron-induced oxidative stress in epididymal sperm of goat by increasing the activities of antioxidant enzymes and decreasing lipid peroxidation.

1 Introduction

Fullerene C₆₀, a third allotropic form of carbon, which exhibits high reactivity towards organic radical additions, is soluble in organic solvents. A water-soluble C₆₀ derivative, fullerenol, has been shown to scavenge free radicals in vitro [1] and in vivo [2]. Fullerenol has been shown to block hydrogen peroxide-induced inhibition of population spikes in rat hippocampus [2]. Carboxy-fullerene, another water-soluble derivative of fullerene has been shown to prevent apoptotic injury evoked by N-methyl D-aspartate (NMDA) without interfering with NMDA-induced Ca²⁺ influx in cortical cell cultures [3].

Reactive oxygen species (ROS) refers to all free radicals activated oxygen species, which may cause oxidative injury. At low concentrations, reactive oxidants have biopositive effect and act selectively. There is growing evidence that sperm can be injured by ROS leading to lipid peroxidation thereby preventing fertilization [4]. Sperm plasma membrane is highly rich in polyunsaturated fatty acids and, therefore, susceptible to oxidative stress [4,5]. ROS are generally involved in the regulation of sperm functions such as capacitation and acrosome reaction. However, excessive production of ROS has been observed in some pathological sperm categories [6]. Reactive oxygen metabolites such as superoxide (O^2-), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH^.) have been reported to act as cytotoxic agents and damage unsaturated lipids in membranes [7].

Iron, the most important transition metal, has been shown to produce hydroxyl radicals [8]. Iron (II) and its complexes stimulate membrane lipid peroxidation in mammalian spermatozoa [9]. Iron overload increases oxidative stress in testes and epididymal sperm causing infertility [10]. There are no studies describing the effect of fullerenol on spermatozoa. In the present study we try to clarify if fullerenol prevents iron-induced oxidative stress in goat epididymal sperm in vitro.

2 Materials and methods

2.1 Chemicals

Fullerenol was a generous gift from Dr. Long Y. Chiang, National Taiwan University, Taipei, Taiwan. All other chemicals were of analytical grade and purchased from local commercial sources.

Sperm were collected from the fresh epididymis of adult goats as per the procedure described by Gangadharan et al. [11]. Epididymal sperm samples dispersed in Ringer's phosphate medium were incubated with fullerenol (1, 10 and 100 mmol) alone or with FeSO₄/ascorbate (40/200 mmol) for 3 h at 32. The control samples were maintained without the addition of fullerenol and FeSO₄/ascorbate.

At the end of incubation, sperm cell suspensions, after washing with Ringer's phosphate medium, were homogenized at 4 in a glass teflon homogenizer for ten seconds and centrifuged at 800g for 10 minutes and the supernatant was used for biochemical assays. The activities of antioxidant enzymes, superoxide dismutase [12], glutathione reductase [13] and glutathione peroxidase [14] and the levels of lipid peroxidation [15] and DNA [16] were estimated by the methods indicated in the literature.

The results were expressed as meanSEM. Statistical analysis was performed by one-way ANOVA followed by post-hoc Duncan's multiple range test. P<0.05 was set as significant.

In the sperm incubated with fullerenol (1, 10 and 100 mmol) the specific activities of antioxidant enzymes, superoxide dismutase, glutathione reductase and glutathione peroxidase, were increased significantly (P <0.05) while the levels of lipid peroxidation were significantly (P<0.05) decreased as compared to the controls (Figure 1).

Figure 1. Effect of sperm incubation with fullerenol on antioxidant system in goat epididymal sperm. Specific activity of superoxide dismutase expressed as nmol pyrogallol oxidized/min/10⁹ spermatozoa at 32, activity of glutathione reductase and glutathione peroxidase as nmol NADPH oxidized/min/10⁹ spermatozoa. Lipid peroxidation expressed as mmol malondialdehyde produced/15 min/10⁹ spermatozoa. ^bP<0.05.

In the sperm incubated with iron (II), the specific activities of superoxide dismutase, glutathione reductase and glutathione peroxidase were decreased significantly (P<0.05) while the levels of lipid peroxidation were significantly (P<0.05) increased when compared to the control samples (Figure 2). In the sperm co-incubated with iron (II) and fullerenol (1,10 and 100 mmol) the specific activities of superoxide dismutase, glutathione reductase and glutathione peroxidase were increased significantly (P<0.05) in a dose-dependent manner (Figure 2). On the contrary, the level of lipid peroxidation was significantly (P<0.05) decreased in a dose-dependent manner (Figure 2).

Figure 2. Effect of co-incubation of sperm with fullerenol and FeSO₄/ascorbate on antioxidant system in goat epididymal sperm. Specific activity of superoxide dismutase expressed as nmol pyrogallol oxidized/min/10⁹ spermatozoa 32, activity of glutathione reductase and glutathione peroxidase as nmol NADPH oxidized/min/10⁹ spermatozoa. Lipid peroxidation expressed as mmol malondialdehyde produced/15 min/10⁹ spermatozoa. ^bP<0.05.

Fullerenol, a water soluble C₆₀ derivative of fullerene, consists of C₆₀ cage along with 18-OH moieties susceptible to the attack of highly reactive radicals. Combination of moderate electron affinity and the allylic hydroxyl functional groups of C₆₀ made it an appropriate candidate for applications such as a free radical remover or a water-soluble antioxidant in biological systems. Fullerenol displays properties of scavenging superoxide radicals generated by xanthine/xanthine oxidase in aqueous solution [1] and the free radicals induced by ischemia-reperfusion injury to small intestine [17]. Fullerenol has been shown to decrease the levels of the microsomal enzymes in vivo and the activities of cytochrome P₄₅₀-dependent monooxygenase and mitochondrial oxidative phosphorylation in vitro [18].

However, there are no studies showing the effects of fullerenol on reproductive tissues. The present study is the first one describing the effect of fullerenol on spermatozoa. The cytoplasm of spermatozoa has been shown to be limited in volume and localization, thus the polyunsaturated fatty acids bound in the sperm plasma membrane are very susceptible to ROS attack [19]. Lipid peroxidation has been considered a major consequence of oxidative stress and results from an interaction of ROS with polyunsaturated lipids in cell membrane [20].

When oxidative stress was induced in the spermatozoa with FeSO₄/ascorbate, the activities of superoxide dismutase, glutathione reductase and glutathione peroxidase were decreased whereas the level of lipid peroxidation increased in a dose-dependent manner. Fullerenol was thus able to alleviate iron-induced oxidative stress as evidenced by increased activities of superoxide dismutase, glutathione reductase and glutathione peroxidase and decreased level of lipid peroxidation in dose-dependent manner indicating that fullerenol could scavenge iron-induced free radicals in epididymal spermatozoa. The mechanism is not known. It is proposed that the condensed hexagons and pentagons arranged in spherical form in fullerenol having hydrophilic character may not be able to enter the sperm membrane. Thus it is likely that the antioxidant effect and decreased lipid peroxidation effects of fullerenol are at the membrane level. It is suggested that fullerenol can be used to protect spermatozoa against iron-induced oxidative stress in vitro.

Acknowledgements

The authors thank Dr. Long Y. Chiang, National Taiwan University, Taipei, Taiwan for the generous gift of fullerenol. The authors thank the staff of Bioinfor-matics Center, Pondicherry University, Pondicherry for providing various facilities. PPM acknowledges the receipt of financial support from the Population Council, New York, USA (Grant Nos. B 99.047P-9/ICMC and B99.048R/ICMC).

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Correspondence to: Dr. P.P. Mathur, School of Life Sciences, Pondicherry University, Pondicherry-605 014, India.
Tel: +91-413-65 5212, Fax: +91-413-65 5211
E-mail: ppmathur@hotmail.com; ppmathur@pu.pon.nic.in
Received 2002-01-06      Accepted 2002-04-18