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Research Article |
Institute of Biochemistry,3 Polytechnic University of Marche, 60100 Ancona, Italy
Endocrinology,4 Department of Medical and Surgical Sciences, University of Padova, 35100 Padova, Italy
Endocrinology,5 Department of Internal Medicine, Umberto I Hospital, Polytechnic University of Marche, 60100 Ancona, Italy
Dipartimento di Biologia MCA,6 Università di Camerino, 62032 Camerino (Mc.), Italy
ABSTRACT
N-acylethanolamides are naturally occurring hydrophobic molecules usually present in a very small amount in many mammalian tissues and cells. The presence of N-acylethanolamides has also been demonstrated in human reproductive tracts and fluids, although their biological effects and molecular mechanisms of action are not yet completely elucidated. It is known that some N-acylethanolamides, such as oleoylethanolamide, have antioxidative properties. The aim of this study was to test whether oleoylethanolamide could protect sperm cells from reactive oxygen species-induced oxidative damage in cases of idiopathic infertility, because the excessive generation of these radicals was associated with this pathology. Our results show that 2.5 nM oleoylethanolamide in vitro supplementation significantly reduces DNA strand breaks both in fertile and infertile subjects. Moreover, oleoylethanolamide increases kinematic parameters, such as curvilinear velocity and amplitude of lateral head displacement and hyperactivation, both in the presence and in the absence of oxidative stress. Results of this study support the hypothesis of a possible protective action of oleoylethanolamide against reactive oxygen species, which could explain its beneficial effects on in vitro capacitated spermatozoa.
male reproductive tract, sperm, sperm capacitation, sperm motility and transport, stress
INTRODUCTION
Abnormal sperm function is difficult to evaluate and treat. There is a lack of understanding of the factors contributing to normal and abnormal sperm functions leading to infertility. One of the most important causes of male infertility is associated with an oxidative stress condition, because of either increases reactive oxygen species (ROS) production or insufficient antioxidant capacity [1, 2]. In fact, although ROS play a physiologic role and are required for normal sperm functions [3], including capacitation and acrosome reaction [4], the excessive generation of ROS (superoxide anion, hydroxyl radical, nitric oxide, peroxides, and peroxynitrile) by immature and abnormal spermatozoa and by contaminant leukocytes, together with genitourinary tract inflammation, was associated with idiopathic male infertility [5].
At high concentrations, ROS induce motility loss, lead to sperm dysfunctions [4], and affect sperm integrity at different levels. In particular, the human sperm plasma membrane has a high concentration of polyunsaturated fatty acids, which can undergo lipid peroxidation, initiated by ROS, particularly hydrogen peroxide (H2O2) [4]. Lipid peroxidative damage leads to a loss of plasma membrane integrity. Moreover, recent studies have demonstrated that ROS may affect DNA integrity of human spermatozoa [4] and fragmentation of sperm DNA was shown to be inversely correlated with sperm quality and fertilization rates after in vitro fertilization and intracytoplasmic sperm injection. Thus, it was suggested [5] that a balance between ROS generation and total antioxidant capacity plays a critical role in the pathophysiology of the disease state [5]. Therefore, a supplementation with antioxidants may provide protection to sperm cells.
Several antioxidant defense lines, both enzymatic (superoxide dismutase, catalase, and glutathione peroxidase) and nonenzymatic (
-tocopherol, ß-carotene, ascorbate, urate, etc.) [2], have been reported in seminal plasma [6].
Recently, N-acylethanolamides (NAEs), an important family of lipid-signaling molecules (present in many tissues) have been isolated in seminal plasma and other human reproductive fluids [7]. In particular, the presence of arachidonylethanolamide (AEA; also known as anandamide), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) was shown in seminal plasma, oviductal fluid, and follicular fluids [7], in which the levels of OEA and PEA (32.9 nM and 31.5 nM, respectively) are significantly higher than those of AEA (12.1 nM) [7]. It was shown that the almost ubiquitous AEA and other polyunsaturated NAEs or similar compounds (all identified as endocannabinoids) are endogenous ligands of cannabinoid receptors CNR1 and CNR2 [8]. Moreover, some of the AEA effects are mediated by the ion-channel-type vanilloid receptor [9]. On the other hand, NAEs with saturated acyl chains (such as PEA) and monounsaturated acyl chains (such as OEA) are cannabinoid-receptor inactive [8, 10].
It was suggested that saturated and monounsaturated NAEs, such as OEA and PEA, may act as entourage compounds that enhance the actions of endocannabinoids, likely by preventing their inactivation [11–13].
It was previously demonstrated that PEA and AEA can modulate sperm hyperactivation (HA) [14, 15] and acrosomal reaction [15], which are sperm functions required for fertilization. Recently, it was demonstrated that AEA affects also spermatozoa physiologic functions through cannabinoid receptors [15], in particular CNR1 receptors [16]. On the other hand, the molecular mechanism of action of PEA on spermatozoa was suggested to be mediated by its induced modifications of physicochemical membrane properties [17], which could have an important effect on molecular mechanisms involved in sperm HA. Moreover, a protective action against ROS cannot be excluded, in fact, PEA antioxidative activities were shown in low-density lipoproteins [18] and in rat liver mitochondria [19]. Current results [7, 14, 15] suggest that the NAEs present in the reproductive system could participate in the regulation of the physiologic processes involved in fertilization.
In the present study, we evaluated the effect of OEA (another NAE with antioxidant properties) in human spermatozoa from idiopathic infertile men, incubated under capacitating conditions. Experimental endpoints considered were functional kinematic parameters and HA, both in unchallenged sperms and cells exposed to 100 µM H2O2. Moreover, we studied the effect of OEA on physicochemical properties of the sperm plasma membrane (polarity and microheterogeneity) and on DNA integrity. Standard seminal parameters, membrane physicochemical properties, and DNA damages were evaluated by the computer-assisted semen analysis (CASA) procedure, Laurdan fluorescence measurements, and single-cell gel electrophoresis assay (Comet), respectively.
MATERIALS AND METHODS
Reagents and Culture Media
Fura 2 AM (acetoxymethyl ester derivative of fura 2) was purchased from Molecular Probes (Eugene, OR). Before use, a stock solution of probe was prepared in dimethylsulfoxide (DMSO). OEA was synthesized as previously described [20] and a stock solution was prepared in DMSO. The medium used in this study (Medi-Cult, MA) is based on Earles Balanced Salt Solution (EBSS), buffered with 25 mM HEPES, and it is routinely used to induce sperm capacitation for assisted reproduction. The medium consisted of 116 mM NaCl, 1 mM NaH2PO4, 5.37 mM KCl, 26 mM NaHCO3, 1.8 mM CaCl2, 0.8 mM MgSO4, 5.5 mM D-glucose, 50 µg/ml of both streptomycin and penicillin, phenol red, and a percent concentration of human serum albumin not higher than 0.3% (Sil-Select Stock was acquired from FertilPro NV, Beernem, Belgium). Higher albumin levels sequester cannabinoids and reduce binding of ligands to sperm [21].
Patients and Semen Samples
Patients selection. Twenty-seven patients (aged from 25 to 34 yr) affected by primary idiopathic infertility were enrolled in the study. The patients were selected at the Andrology Unit of the Endocrinology Division, Umberto I Hospital, Polytechnic University of Marche, Ancona, Italy. The criteria of patient selection were chosen to be similar to those used in a previous study regarding the effect of PEA in idiopathic infertility [17]. All subjects underwent medical screening, clinical examination, and history of infertility after at least 2 yr of regular unprotected intercourse. Testicular volume was evaluated in each patient using Prader's orchidometer. To accomplish a complete diagnosis the following investigations were also performed:
The study was approved by the Institutional Review Board of the Polytechnic University of Marche. All patients provided their written consent.
Eligibility criteria. The following criteria were adopted for patient eligibility: 1) sperm count >20 x 106 sperm/ml and normal sperm morphology >30% according to the World Health Organization (WHO) strict criteria [22]; class a + b + c motility was determined according to WHO standards [22] and considered as percent of motility for the purposes of the present study; 2) seminal white blood cells <1 x 106/ml, and negative sperm culture, Chlamydia, and Mycoplasma ureoliticum detection; 3) normal serum levels of gonadotropins, T, E2, and PRL; 4) absence of infectious genital diseases, of anatomic abnormalities of the genital tract (including varicocele), and of anti-spermatozoa antibodies; 5) absence of systemic diseases or treatment with other drugs within 3 mo before enrollment in the present study; 6) absence of smoking, alcohol, or drug addiction and occupational chemical exposure.
Semen analysis. Semen samples were collected as previously described [23], were allowed to liquefy at room temperature, and were processed within 1 h of ejaculation. Semen quality was assessed by the same biologist in terms of sperm concentration and motility, in accordance with the WHO criteria [22]. Sperm count was determined with the Makler chamber. Motile spermatozoa were assessed by phase contrast microscopy (10 µl of semen was delivered onto a glass slide and covered with a 22 x 22-mm coverslip) and graded as follows: class a and b, fast and weak forward motility; class c, nonprogressive motility; class d, immobile spermatozoa. Sperm morphology was evaluated on smears of seminal fluid, stained with the Giemsa method and observed by oil-immersion light microscopy.
The CASA motility assay for sperm cells was additionally performed, as previously reported [23]. One semen aliquot (3 µl) was placed in a 20-µm depth chamber. Two 20-µm depth cell—VU chambers (Conception Technologies, La Jolla, CA) were loaded and six different fields per chamber were randomly examined; at least 200 motile cells for each field of the chamber were scored. Percentages of motile sperm and movement characteristics were analyzed using an automated analyzer at 37°C (CellTrack VP110, Motion Analysis Corporation, Palo Alto, CA). Frame rate and duration of tracking were 60 frames/sec and 
sec, respectively. Sperm velocity and kinetic characteristics were evaluated only for motile sperm and expressed as mean values, considering curvilinear velocity (VCL) and straight progressive velocity (VSL).
Sperm suspension preparation to study OEA effect on functional kinematic parameters. The specimens were allowed to liquefy for 30–60 min at 37°C in a slide warmer. After semen liquefaction, a basic semen analysis was performed. Motile spermatozoa were selected by centrifugation through a two-step density gradient (90% and 47.5% layers), which was prepared from Sil-Select Stock (FertilPro NV). Sil-Select Stock consists of silane-coated colloidal silica particles suspended in the medium (HEPES-buffered EBSS). Stock solutions of OEA (10–3 M in DMSO) were stored at –80°C and diluted to the proper concentration immediately before each experiment. OEA was injected directly into the medium. DMSO was always 0.2% (v/v). The control experiments were performed with the same volume of this solvent. Preliminary experiments showed that this DMSO concentration had no significant effects on the kinematic parameters of sperm cells (data not shown). For each subject, two density gradients were prepared: one containing 2.5 nM OEA and one containing DMSO. The aliquots of semen (up to 2 ml, 40 million cells for each sample) were layered over the upper step of the density gradient and centrifuged for 12 min at 350 x g. The pellet was diluted in the medium and washed two times at 300 x g for 10 min. At the end of centrifugation, the supernatant was removed and the pellet was resuspended in a suitable volume of medium, containing 2.5 nM OEA or DMSO. Oxidation was induced by 100 µM H2O2 at 37°C for 20 h in a subset of semen samples from 11 infertile men. It was demonstrated [24] that this H2O2 concentration results in a significant reduction of important kinematic parameters, such as VCL and amplitude of lateral head displacement (ALH). Furthermore, it was shown that this H2O2 concentration produces DNA fragmentation, as assessed by the Comet assay [4]. The samples were maintained in incubator at 37°C and analyzed to evaluate functional kinematic parameters.
Effects of OEA on HA Sperm Motility
The effects of OEA on sperm HA were evaluated on samples during a 4 h incubation at 37°C in a CO2 incubator (5% CO2 in air at 95% relative humidity). Semen samples were placed in slightly closed tubes. Each sample contained 10 x 106 cells/ml. Aliquots (3 µl) of both sperm samples were withdrawn during the incubation at various times; these aliquots were placed in a MicroCell chamber (Conception Technologies) and assayed using the CASA system computerized semen analyzer (CellTrak VP110, Motion Analysis Corporation).
The kinematic values for at least 200 motile spermatozoa were analyzed in each sample, and cells with VCL 
100 µm/sec; ALH 5 µm; and linearity (LIN) < 60% (VSL/VCL x 100) were considered hyperactivated [25]. HA is linked to the process of capacitation [26] and has been proposed as one of the criteria for evaluating the fertilizing potential of human spermatozoa [27–30]. As a clinical application, human sperm HA has been shown to be directly correlated with the success of in vitro fertilization [31].
Fluorescence Measurements
Laurdan was purchased from Molecular Probes (Eugene, OR). The spectral features of this fluorescent probe are largely sensitive to membrane polarity and can be used as a simple method to evaluate changes of structural and physicochemical features of spermatozoa plasma membranes [23]. A buffer solution of pH 7.4 containing 30 mM HEPES; 1.19 mM MgSO40.7H2O; 5 mM KCl; 120 mM NaCl; and 5.5 mM D-glucose was prepared to wash the cells [23]. Five sperm samples, chosen randomly between the 27 samples analyzed in this study, were washed twice (500 x g; 10 min each) with buffer to eliminate the seminal fluid coagulum. Furthermore, they were equilibrated for 20 h at 37°C in the dark before labeling. From the same specimen, four different samples were prepared; the first was used as control, the others were incubated with 2.5 nM, 25 nM, and 0.25 µM OEA, respectively, for 15 min before fluorescence measurements. Because OEA was dissolved in 2 µl of DMSO, control experiments were performed with the same volume of this solvent: no significant DMSO effect on Laurdan fluorescence was measured (data not shown). Cells (10 x 106 cells/ml) were labeled with Laurdan, as previously described [23]. The sample volume in each cuvette was 2 ml. An LS 55 Perkin-Elmer fluorometer (Perkin-Elmer), equipped with a stirring accessory was used for the steady-state fluorescence measurements. The cuvette temperature was maintained at 37°C in a thermostat-controlled chamber by using a circulating water bath (HAAKE F3). Laurdan fluorescence emission spectra were recorded in the range from 420 to 550, using both 340-nm and 410-nm excitation wavelengths, whereas the fluorescence excitation spectra were obtained in the range from 300 nm to 420 nm, using both 435-nm and 490-nm emission wavelengths. Blank spectra were obtained with unlabeled cells and were subtracted from the spectra of labeled cells. These blanks were always <5% of the samples in each wavelength range considered. From the spectroscopic data, Laurdan emission generalized polarization (GP) spectra were constructed by calculating the GP value for each emission wavelength (emGP) according to Parasassi et al. [32]
where I410 and I340 are the intensities measured at each emission wavelength (from 420 nm to 520 nm).These values are obtained by the fluorescence emission spectra recorded using fixed excitation wavelengths of 410 nm and 340 nm, respectively. The excitation GP (exGP) spectra were constructed using the following formula [32]:
where I435 and I490 are the intensities measured at each excitation wavelength (from 320 nm to 420 nm) on the fluorescence excitation spectra obtained by fixed emission wavelengths of 435 nm and 490 nm, respectively. The choice of 410 nm, 340 nm, 435 nm, and 490 nm for the GP calculations was based on the characteristic excitation and emission wavelengths of pure gel and liquid-crystalline (e.g., fluid) lipid phases, according to Parasassi et al. [32]. Laurdan exGP and emGP spectra were calculated because previous works demonstrated that they show characteristic patterns in the presence of pure or coexisting lipid phases and in the presence of cholesterol [32]. In fact, these spectra can determine whether domains of different composition and phase properties coexist in the plane of the membrane (membrane heterogeneity) [32].These properties are caused by the particular Laurdan spectroscopic features, which are extensively described elsewhere [32–34]. ExGP340 was used as a parameter particularly sensitive to polarity [23, 35], and it can be calculated both by exGP spectra (choosing the value at 340 nm) or by the Laurdan emission spectrum (
ex = 340 nm), used for calculation of the intensities at 435 and 490 nm. These wavelengths correspond to the characteristic emission wavelengths of Laurdan in pure phospholipid gel and liquid-crystalline phases, respectively. ExGP340, which is sensitive to the extent of water polar relaxation processes, gives a mathematically convenient and quantitative method to measure the Laurdan emission spectral shift. On interaction with the membrane, the Laurdan molecule localizes at the water interface of the bilayer and, at steady-state conditions, its lateral and transbilayer partitioning can be considered uniform [33]. No cytotoxicity and decrease of motility is induced by this probe on spermatozoa [36].
Detection of DNA Fragmentation: Comet Assay
The Comet assay, or single-cell electrophoresis, a rapid and sensitive technique to assess DNA damage, was performed according to Singh et al. [37] with slight modification. Semen used for these experiments were derived from three infertile subjects. In summary, microscope slides were cleaned with 100% ethanol, air-dried, dipped in a solution of 0.7% (w/v) normal melting point agarose, and allowed to dry. The protocol modified for sperm cells involved triple-layer minigels composed of two 200-µl layers of 1% low melting point agarose (LMA) containing one layer of 50 µl of 0.7% LMA containing 
2 x 105 cells. All experiments on each sperm sample were prepared in triplicates. The minigels were immersed in cold lysis solution (2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, and 10 mM DTT, pH 10.0) overnight at 4°C. Subsequently, slides were incubated at 37°C in a wet room in the presence of 200 µg/ml of proteinase K. The microscope slides were placed in an electrophoresis tank, and the DNA was allowed to unwind for 20 min in freshly prepared alkaline electrophoresis buffer (1 mM EDTA and 300 mM NaOH, pH > 13). Electrophoresis was performed in the same buffer for 20 min at 31 V and 300 mA in a cold room (4°C). The slides were then neutralized with Tris-HCl buffer (0.4 M, pH 7.5) and stained with YOYO high-resolution DNA dye (Molecular Probes). Fifty cells per slides (150 cells per sample) were observed using the software, Comet Assay II, from Perspective Instruments (Suffolk, UK). Computer-aided image analysis allows the assessment of the DNA damage by means of three parameters: comet tail length quantifies the migration of the fragmented DNA, comet tail intensity is proportional to the percentage of damaged DNA, and comet tail moment integrates the previous parameters.
To compare the response to OEA incubation of idiopathic infertile patients and fertile subjects, we performed the same experiments on sperm from two men of proven fertility (defined as the induction of at least one pregnancy in a female partner and the paternity of at least one living child).
Statistical Significance
Results are expressed as means ± SD. Statistical significance of data was evaluated by paired Student t-test and two-way ANOVA analysis. Differences were considered significant at P < 0.05.
RESULTS
Effects of OEA on Seminal Functional Parameters and HA in the Absence and in the Presence of Oxidation
Sperm kinematic parameters, measured before capacitation in 27 semen samples from infertile patients, are reported as means ± SD of values: cell concentration (54 ± 16 million cells/ml); percent of total motility, a + b + c (53% ± 13%); VSL (22 ± 4 µm/sec); VCL (59 ± 15 µm/sec); LIN (42 ± 10); and ALH (3.3 ± 0.6 µm).
To assess the sensitivity of spermatozoa to incubation with 2.5 nM OEA, we focused our attention on the measure of VCL, ALH, and LIN, because it was demonstrated that these parameters are important indexes for the evaluation of sperm capacitation and HA [25].
Table 1 shows the mean of VCL and ALH values measured during in vitro capacitation. It is evident that OEA significantly increases both VCL and ALH, as evaluated by paired Student t-test. No significant modifications induced by OEA were evident in LIN and motility parameters (data not shown).
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To evaluate the effectiveness of OEA to improve kinematic parameters, we calculated the HA incidence, both in controls and OEA-incubated samples. The results obtained (Table 1) show that the average of HA incidence, calculated during the time of the experiment (4 h), is significantly larger in the presence of OEA. Oxidation was induced by 100 µM H2O2 in a subset of semen samples from 11 infertile men. It is evident that, in oxidized samples, OEA significantly increases semen parameters (in all cases calculated by paired Student t-test): in the absence of OEA, the values of VCL, ALH, and HA were 88 ± 10 µm/sec, 3.68 ± 0.4 µm, and 10% ± 5%, respectively. In the presence of OEA, these parameters were 101 ± 10 µm/sec, 4.24 ± 0.4 µm, and 18% ± 6%, respectively.
Table 2 shows two-way ANOVA factorial analysis of H2O2/OEA treatment on sperm kinematic parameters (VCL and ALH) and HA in the same samples. By performing two-way ANOVA, it has been shown that OEA was able to significantly improve VCL, ALH, and HA, both in the presence and in the absence of oxidation (in all cases, P < 0.05). P interaction was not significant.
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Laurdan Fluorescence
Figure 1 shows the average of Laurdan exGP and emGP spectra measured in spermatozoa resuspended in buffer, as described in Materials and Methods. Sperm cells were from donors affected by idiopathic infertility. In this figure, it is evident that exGP spectra values decrease, whereas emGP spectra values increase with increasing excitation wavelength. The shape of the GP spectra and the GP values obtained in control samples (Fig. 1) indicates that the lipids of the sperm membrane are in a single liquid-crystalline phase with a high conformational order and low polarity, in agreement with previous data obtained on semen from healthy, fertile donors by Palleschi and Silvestroni [36] and on semen from idiopathic infertile men in our previous work [23]. From the figure, it is evident that the control and 2.5 nM OEA-treated samples have similar Laurdan exGP and emGP spectra behavior, indicating no significant changes of plasma membrane heterogeneity. Moreover, the average of Laurdan ExGP340, calculated in the same samples, was 0.364 ± 0.034 and 0.359 ± 0.031 in controls and 2.5 nM OEA-treated samples, respectively. No significant differences were evident between these values. No significant changes of exGP340 values and GP spectra (emGP and exGP) were recorded in sperm cells incubated with higher OEA concentrations (from 25 nM to 250 nM). The insensibility of these parameters to increased OEA membrane concentrations suggests that OEA effects on spermatozoa are not related to the physicochemical and structural bilayer modifications measured by Laurdan.
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Comet Assay
The alkaline comet assay was performed to evaluate the OEA effect on DNA integrity. Aliquots of cells used to evaluate the effect of OEA on seminal standard parameters were studied. The same experiments were also performed on spermatozoa from healthy fertile donors. Comet tail intensity and comet tail moment were evaluated to identify DNA damage. Tables 3 and 4 show the comet tail intensity and the comet tail moment values calculated in three infertile men (Table 3) and two fertile men (Table 4), in the presence and in the absence of OEA. In line with previous studies [38], the DNA damage, measured in the absence of OEA, is increased in spermatozoa from infertile men, as evident by tail moment and tail intensity values. From Tables 3 and 4, it is evident that incubation with 2.5 nM OEA decreases the extent of DNA damage in all samples tested.
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DISCUSSION
Controlled generation of ROS has physiologic roles in important functions involved in fertilization, such as HA, capacitation, and acrosome reaction [39 and references cited therein].
However, increased levels of ROS were demonstrated in semen samples of infertile men [40], such as patients with idiopathic infertility [39], suggesting that ROS-induced damages of spermatozoa, in particular, lipid peroxidation of sperm membrane, could be one of the key mechanisms involved in the pathophysiology of male infertility [39]. Because uncontrolled and excessive ROS concentration has a significant role as one of the major factors leading to the infertile status [39], it is reasonable to expect that it causes alteration of sperm physiologic functions, such as HA.
However, spermatozoa of infertile men have been shown to contain various nuclear alterations, including DNA strand breaks [4]. Therefore, the evaluation of the sperm DNA integrity may be considered a marker of sperm function that serves as a significant prognostic factor for male infertility [41]. Moreover, DNA damage analysis may reveal a hidden abnormality of sperm DNA in idiopathic infertile men with apparently normal standard semen parameters [41].
One of the factors involved in the etiology of DNA damage in spermatozoa is oxidative stress caused by ROS [4, 42]. It is controversial whether the increased levels of ROS are caused by a decreased antioxidant capacity of semen or by an excessive generation of oxidants by leukocytes and spermatozoa [43]. However, it is known that oxidative stress, leading to cellular damage, arises when the equilibrium between the amount of ROS produced and the amount scavenged by antioxidant is disturbed, and this imbalance has been shown to correlate with idiopathic infertility [44]. It was shown that supplementations with antioxidants, such as vitamin E [45] or coenzyme Q [46], both in vitro [45] and in vivo [45, 46] may play a positive role on sperm cells. Moreover, in vitro supplementation with antioxidants can reduce ROS production during Percoll centrifugation, and, consequently, has beneficial effects for sperm DNA integrity [47].
In this study, we first tested the effects of incubation with OEA, usually present in human reproductive tracts and fluids, on standard semen parameters and DNA integrity in cases of idiopathic infertility. Interest in this study is supported by previous papers, which demonstrated that OEA [48] and its analogues, PEA [19] and stearoylethanolamide [19] have an antioxidant effect by inhibiting the radical-induced in vitro oxidation of lipids in liver [19] and cardiac [48] mitochondria. Moreover, our recent results [18] demonstrate that relatively low concentrations of PEA decrease low-density lipoprotein susceptibility to Cu2+-induced oxidation.
Our Comet assay results, although performed on a limited number of subjects, are consistent with a previous study showing that infertile men may exhibit more sperm DNA damage [38], and are in line with some authors [49] who report that the plasma seminal antioxidant capacity is larger in fertile men than in infertile men. In addition, our data show that sperm supplementation with 2.5 nM OEA significantly reduces DNA strand breaks both in samples from infertile and fertile subjects.
Moreover, 2.5 nM OEA in vitro supplementation significantly increases kinematic parameters important for capacitation and HA, such as VCL and ALH. However, these OEA-induced effects on sperm cells are not a direct indication of a protection against ROS. For this reason, the possible OEA beneficial effects against cell oxidative stress were verified by studying H2O2-induced sperm oxidation. Kinematic parameters and HA of oxidized samples treated with OEA are significantly higher with respect to untreated samples in the same condition, indicating the best quality of this semen as a consequence of the OEA effect on sperm oxidation.
The molecular mechanisms at the basis of the OEA protective action against ROS requires further studies, because of the presence of many intracellular and extracellular antioxidants of enzymatic and nonenzymatic systems [50] in the human ejaculate. However, some hypotheses can be advanced.
It was demonstrated that, in the ROS-induced cell oxidative stress, a pivotal role is played by plasma membrane fluidity [51], because it was shown that an increase in bulk membrane fluidity can amplify the ROS-induced oxidative stress. Moreover, the fertilizing ability of spermatozoa is known to be dependent in part on the integrity and fluidity of the sperm plasma membrane [52], therefore, an induced modification of membrane fluidity can be reasonably expected to affect this ability.
The possible OEA-induced changes of plasma membrane features, such as fluidity and microheterogeneity, can be expected, because the localization of this largely hydrophobic molecule within the lipid bilayer was demonstrated [20]. Because our data show that the OEA concentration used in this study does not induce significant changes in membrane fluidity and microheterogeneity measured by Laurdan fluorescence, we suggest that the beneficial effects of this molecule on sperm parameters and on physiologic functions are not directly related to changes of membrane physicochemical characteristics.
However, it is known [47] that DNA can be protected during sperm Percoll centrifugation by the presence of very high antioxidant concentrations (starting from 30, 200, 300 µM) in the media, whereas the OEA concentration used in this work (2.5 nM) is likely too low to be efficient.
Some other hypotheses can be advanced to elucidate the molecular mechanism of the OEA beneficial effects on sperm. For example, it was previously shown that OEA binds with high affinity to serum albumin [53] and protects serum albumin against ROS-induced oxidation (unpublished results). Albumin plays important roles on sperm: it is involved in cholesterol removal during in vitro capacitation [54]; and the albumin sperm-enrichment procedure improves the recovery of higher-quality spermatozoa because of its antioxidant properties [55]. It is suggested that an OEA-human serum albumin complex could be involved, at least in part, in the modulator roles of albumin during sperm in vitro capacitation.
Moreover, other mechanisms of action could occur: for instance, the localization of this largely hydrophobic molecule within the lipid bilayer [20] suggests that OEA could affect the reaction cascade, resulting in production of malondialdehyde or other products of membrane lipid peroxidation. It could be possible that compounds occurring as a consequence of membrane lipid peroxidation may be modulated by OEA, as recently proposed by Foti et al. [56] to explain mechanisms of protective action of low concentrations (ranging between 0.01 and 2.5 µM) of some lipophilic isoflavones.
Data presented in this work clearly show that addition of OEA to in vitro capacitated spermatozoa improves sperm kinematic parameters and HA, both in the presence and in the absence of oxidative stress, conferring. in some measure, protection against oxidative damage. In conclusion, we suggest that patients with idiopathic infertility, who suffer oxidative stress by increased levels of ROS [39] and need assisted reproduction procedures, could benefit from OEA in vitro supplementation during the preparation of their sperm cells for fertilization.
The possible physiologic role of OEA in human reproductive fluids to contribute to the total antioxidant capacity of semen needs to be elucidated by in vivo studies.
ACKNOWLEDGMENTS
The authors thank Prof. Luigi Ferrante for fruitful discussions regarding statistical data.
FOOTNOTES
1 Supported by Ministero dell'Istruzione, Università e Ricerca grant to G.Z. 
2 Correspondence: Annarina Ambrosini, Istituto di Biochimica, Università Politecnica delle Marche, Via Ranieri 65, I-60131 Ancona, Italy. FAX: 39 071 2204398; a.ambrosini{at}univpm.it
Received: 27 July 2005.
First decision: 6 September 2005.
Accepted: 13 December 2005.
REFERENCES
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  S. Batkai, M. Rajesh, P. Mukhopadhyay, G. Hasko, L. Liaudet, B. F. Cravatt, A. Csiszar, Z. Ungvari, and P. Pacher Decreased age-related cardiac dysfunction, myocardial nitrative stress, inflammatory gene expression, and apoptosis in mice lacking fatty acid amide hydrolase Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H909 - H918. [Abstract] [Full Text] [PDF]
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