andrologia 36, 305310 (2004)

Accepted: April 21, 2004

Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa
M. Arabi
Department of Biology, Andrology Unit, Shahrekord University, Shahrekord, Iran Keywords. Comet assaylipoperoxidationnicotinespermatocrit testspermatozoa

Summary. Infertility remains a major problem in society, with recent data suggesting its presence in one of four couples. The objective of the present study was to evaluate the impact of nicotine (0.25, 0.5 and 0.75 mm), as a major component of cigarette smoke, in vitro, on sperm membrane [by spermatocrit and lipoperoxidation (LPO) tests], DNA integrity (by Comet assay), and viability of spermatozoa (by eosin staining) from normozoospermic men. Sperm samples were washed and diluted with phosphate-buffered saline. A drop in spermatocrit values and an increase in thiobarbituric acid-reactive substances/ LPO rate was observed with the addition of nicotine, predominantly at a concentration of 0.75 mm, indicating a deleterious effect of nicotine on sperm membrane intactness. There was also a strong negative correlation between LPO rate and percentage viable sperm cell (r )0.990). Data obtained from Comet assay technique revealed that nicotine could induce double-stranded DNA breaks (11% in 0.75 mm concentration) in the sperm nuclei. The value of r between LPO rate and percentage Comets was found to be +0.976. Taken together, nicotine proved to be a potential oxidant agent in the category of environmental factors to the integrity of sperm plasma membrane and DNA. Introduction During recent years, the human fertility potential has been deteriorated partly due to the effects of increasing toxic factors like cigarette smoke and
Correspondence: Dr Mehran Arabi, Department of Biology, Faculty of Sciences, Shahrekord University, POB 115, Shahrekord - 88186, Iran. Tel.: ++ 98 (381) 4424552; Fax: ++ 98 (381) 4424419; E-mail: mehranarabi@hotmail.com

nicotine in the environment. In man, these effects may be manifested, among other parameters, by abnormalities in the quality of sperm (Wong et al., 2000). Nicotine is one of the major hazardous components of tobacco which mimics most of the deleterious effects of cigarette smoke (Kavitharaj & Vijayammal, 1999). In the present work, the selected concentration of 0.5 mm nicotine is the amount estimated to approximate residual concentration of nicotine in the testes of heavy (chain) smokers (Pekarsky et al., 1995). Approximately, onethird of the worlds population (15 years) smokes regularly (World Health Organization, 1997). Male infertility accounts for 40% of infertility problems (Fleming et al., 1995; Agarwal et al., 2003).In addition to the endocrine aspects, a large number of toxicological substances and pharmacological and physical agents [e.g. radiation, smoking and reactive oxygen species (ROS)] can be detrimental to mans health and fertility potential. The major ROS such as superoxide anion (O 2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH) are capable of adversely modifying cell functions, ultimately endangering the survival of the cell. The main consequence of excessive ROS generation is the peroxidative damage to the sperm plasma membrane elements or lipoperoxidation (LPO), which leads to an impairment of sperm function that is reected in decreased pregnancy rates in vivo as well as in impaired in vitro fertilization (Sukcharoen et al., 1996; Agarwal et al., 2003). Human spermatozoa, however, are particularly sensitive to ROS assault because of existence of high quantities of polyunsaturated fatty acids (PUFA), dominantly 22:6, in their own membranes (Alvarez & Storey, 1995). Infertility may also be linked to the ROS-induced DNA damage, as the sperm DNA of infertile patients is more susceptible to more fragmentations in vitro than from fertile men (Hughes et al., 1996).
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Moreover, both ROS generation and oxidative DNA base damage are elevated in the spermatozoa of infertile men (Barroso et al., 2000). The objective of the present study was to evaluate the anti-fertility effect of nicotine, in vitro, on sperm membrane and DNA integrity, and viability of spermatozoa in normozoospermic individuals.

sodium citrate + 150 m osmol fructose solution), (1:9), was performed no lesser than 30 min at 37 C. Spermatocrit (haematocrit) tubes lled with latter pellets in swelling medium was then measured, using a haematocrit centrifuge set (micro-centrifuge K80h; Wagtech, Birmingham, UK) (15000 g, 3 min). The standardization of percentage spermatocrit values to the statistical analysis was also carried out (Lagares et al., 1999). Lipoperoxidation test

Materials and methods Collection of semen samples A total of 24 ejaculates (semen samples) were obtained from healthy, non-smoking donors (19 and 21-year old) by masturbation and collected in sterile plastic containers. Fourteen men were 19 and the other 10 were 21 years old. After at least 30 min of liquefaction, semen samples were evaluated (World Health Organization, 1999) for percentage of progressive motility at room temperature (under a phase-contrast microscope) and sperm concentration (using an improved Neubauers haemocytometer), multiplying the latter by the volume of the ejaculate. Samples having a nal concentration of >20 106 sperm cells ml)1 and with more than 70% motile cells were selected. Seminal plasma was then discarded by centrifuging the samples at 300 g, for 10 min. The pellet so obtained was suspended in an equal volume of 0.2 m phosphate-buffered saline (PBS) (pH 7.2). Reagents The chemicals including thiobarbituric acid, low melting point agarose (LMPA), normal melting point agarose (NMPA) and acridine orange were obtained from Sigma Chemical Co. (St Louis, MO, USA). Other chemicals were purchased from Fluka Co. (Biochemka, AG CH-9470, Buchs, Switzerland). All solutions were made in tripledistilled water. Spermatocrit test This method was carried out in our laboratory, according to the spermatocrit technique described by Lagares et al. (1999) with some modications which gave rise to another novel and easy technique to evaluate the functional integrity of the sperm. The treated samples with nicotine (0.25, 0.5 and 0.75 mm) as well as the negative control (untreated) groups were incubated at 37 C for 30 min, and then centrifuged at 300 g, for 10 min. The pellet was homogenized by mixing, and dilution with a swelling (hypo-osmotic) solution (Jeyendran et al., 1984) with ionic strength equal to 1.5 (150 m osmol Single cell gel electrophoresis (Comet) assay The Comet assay is a sensitive technique that detects the presence of DNA strand breaks and alkali labile damages in the individual cells. The DNA fragments so produced migrate towards the anode pole at a rate inversely proportional to the size of the fragment during electrophoresis. The alkaline single cell gel electrophoresis (Comet) assay was based on existing methods which was rst described by Singh et al. (1988), modied as indicated below. Pre-cleaned microscope slides were dipped in a solution of 1% NMPA dissolved in 0.2 m PBS (pH 7.2) and air-dried overnight. Again, 200 ll of NMPA was placed on slides and covered with a coverslip and left to solidify at room temperature. Sperm samples of 25 ll (the incubation time for nicotine-treated samples was 30 min at 37 C) plus 75 ll of 1% LMPA was added to the slide. A nal layer of 1% LMPA was added to slides and allowed to solidify at 4 C for at least 1 h. Following removal of the coverslip, slides were then immersed in lysis buffer (2.5 m NaCl, 100 mm EDTA, 10 mm Tris-HCl, 10% DMSO, 1% Triton
ANDROLOGIA 36, 305310 (2004)

The LPO was estimated in terms of thiobarbituric acid reactive substances (TBARS), particularly malondialdehyde (MDA) by the method of Ohkawa et al. (1979). The assay mixture (3 ml) contained 0.1 ml of sperm sample, 0.2 ml of 8.1% sodium dodecyl sulphate, 1.25 ml of 20% glacial acetic acid (pH 3.5), 1.25 ml of 1.2% aqueous solution of TBA and 0.2 ml triple distilled water in the negative control group instead of adding 0.1 ml of nicotine in treated one. The incubation time for nicotinetreated samples was 30 min at 37 C. Finally, after heating, adding 3 ml of n-butanol-pyridine mixture and centrifuging at 2200 g for 10 min, the amount of MDA + TBA complex formed was measured by the absorbance of the upper organic layer at 532 nm (spectrophotometer UV/VIS 3100 pro; Biochrom Ltd, Cambridge, England) which is the kmax of MDA (extinction coeff. of 1.56 105 m)1 cm)1). The LPO rate was nally expressed as mmol MDA mg protein)1 min)1.

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X-100) for 1 h at 4 C. This treatment lyses the nuclear and the cell walls and permits DNA to unfold. Slides were immersed in a horizontal gel tank lled with alkaline buffer (300 mm NaOH/ 1 mm EDTA, pH 12.3) for 20 min to allow DNA to unwind. Electrophoresis was carried out (Horizontal gel set GTH8; Wagtech) for 15 min at 25 V (0.862 V cm)1). The slides were then placed in Coplin jars containing fresh neutralizing solution (0.4 m Tris-HCl, pH 7.4) for 5 min (repeated three times). Acridine orange (AO) as a uorescence dye (20 ll) was used to stain sperm DNA which were imaged by uorescent microscope (Excitation lter 515560 nm; dichroic lter 580 nm; and suppression lter 580 nm). AO bound to single-stranded uoresces red and to double-stranded DNA uoresces green. For each sample, ve replicate slides were prepared and 50 randomly selected microscopic elds (Olympus BH-2RFCA, Tokyo, Japan) were scored on each slide. The percentage of categories of DNA damages undamaged (no migration) and damaged (migrated) were recorded. Eosin staining To determine the percentage of live and dead sperm cells, a vital staining technique with eosin was used (Blom, 1950). A total of 400 spermatozoa on a prepared slide was observed with a light microscope (40). The percentage of live (unstained) and dead (stained) spermatozoa in media at each of treatments as well as control groups were calculated. Statistical analysis All the results are expressed as mean SE. Data were statistically analysed by using the Students t-test to establish the validity of the investigation. Results The observations were categorized into three groups: (1) negative control (intact): only spermatozoa without nicotine (0.25, 0.5 and 0.75 mm) and/ or swelling solution; (2) positive control: without nicotine but spermatozoa + swelling solution; and (3) treated samples: positive control + nicotine concentrations. As shown in Table 1, the swelling reaction of sperm cells as standardized spermatocrits in positive control group had a signicant enhancement (35%, P < 0.05) when compared with negative control group data. In contrast, following 60 min incubation of positive control group at 37 C with different concentrations of nicotine (0.25, 0.5 and
ANDROLOGIA 36, 305310 (2004)

Table 1. Effect of different concentrations of nicotine on the swelling reaction and lipid peroxidation of normal human spermatozoa Study groups Negative control Positive control 0. 25 mm nicotine 0. 5 mm nicotine 0. 75 mm nicotine Spermatocrit 1.0 1.35 1.29 1.21 1.12 LPO rate 0.312 0.309 0.431 0.496 0.537 0.028 0.017 0.019*a 0.021**b 0.030**b

0.081* 0.075 0.083 0.079a

Data are expressed as mean SE. *P < 0.05 and **P < 0.01 compared with negative (no osmotic stress) control. a P < 0.05 and bP < 0.01 compared with positive (+osmotic stress) control.

0.75 mm), a drop in the number of swollen sperm cells as decreased spermatocrits was detected by reading the percentage sedimentation in spermatocrit (haematocrit) tubes, dose-dependently. This decrease was signicant only in the study group supplemented with 0.75 mm nicotine (17.04%, P < 0.05). In order to determine whether nicotine has a deleterious effect on the sperm membrane organization, the LPO test was also carried out. The results show that nicotine at different concentrations (0.25, 0.5 and 0.75 mm) could impose a remarkable peroxidation to spermatozoal membrane elements, dose-dependently by 38, 58 and 72% (P < 0.05, P < 0.01 and P < 0.01, respectively) (Table 1). There was a strong negative correlation between the number of swollen sperm cells and the LPO rate (r )0.981). Data from Table 2 show that the proportion of spermatozoa with intact membrane (viable cells) indicated by eosin staining decreased with an increase in the nicotine concentration (0.25, 0.5 and 0.75 mm) from 85% in negative control to 60%

Table 2. Effect of different concentrations of nicotine on the viability and DNA integrity of normal human spermatozoa Viable sperm by eosin (%) 85 84 75 63 60 5.6 6.1 5.2 4.8*a 4.4*a

Study groups Negative control Positive control 0.25 mm nicotine 0.5 mm nicotine 0.75 mm nicotine

Comet values (%) 2.0 2.0 4.0 8.0 11.0 0.88 0.90 0.83 0.93*a 0.84**b

Data are expressed as mean SE. *P < 0.05 and **P < 0.01 compared with negative (no osmotic stress) control. a P < 0.05 and bP < 0.01 compared with positive (+osmotic stress) control.

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Figure 1. Impact of nicotine on DNA integrity of human sperm cells by Comet assay. Elongated forms are Comets (DNA breaks) diffusely stained tail of damaged DNA to one side of the head, and circular ones are normal nuclei without damage/DNA break.

(P < 0.05) in 0.75 mm nicotine-treated samples. We show an r )0.990 between LPO rate and percentage viable sperm cells detected by eosin staining method. Another attempt was also carried out to show the impact of nicotine on DNA integrity of spermatozoa. Data obtained from Comet assay technique show a linear increase in percentage Comets when nicotine concentrations were supplemented from 2.0% (negative control) to 11.0% (P < 0.05) in 0.75 mm nicotine-treated samples (Table 2 and Fig. 1). Interestingly, almost 95% of Comets were of double-stranded DNA breaks with green uoresces under AO staining method. The value of r between percentage Comets and percentage viable sperm cells was )0.978, and r +0.976 was between percentage Comets and LPO rate values. Discussion The data presented here provide evidence of an important relationship between nicotine concentration effects and intactness of sperm membrane and DNA integrity. Using spermatocrit test as an indirect measurement, we tested whether an intactness of sperm membrane is present after being exposed to different concentrations of nicotine along with a hypo-osmotic condition or not. Jeyendran et al. (1984) developed the HOS test as an easy assay to assess the functional integrity of sperm membrane in which an inux of water results in an expansion of sperm cell volume (swollen cells). High percentage of swellings will be equal to high rate of normal membrane integrity. In the present study, the high spermatocrit values of the positive control group compared with the

negative control group (35%, P < 0.05) could be explained by a large swelling reaction of spermatozoa due to osmotic challenge in spermatocrit tubes, which were indicators of high osmotic resistance of spermatozoa. The higher the nicotine concentration (0.250.75 mm), the lower the spermatocrit values, indicating the deleterious effect of nicotine on sperm membrane intactness towards eliminating the semipermeability of the sperm membrane barrier. A maximal decrease in spermatocrit values was seen in 0.75 mm nicotine-treated sperm samples (17.04%, P < 0.05), probably because of the high percentage of damaged or dead spermatozoa which lose the ability to expand. The remarkable decrease in the number of live cells (eosin-stained) in nicotine-treated samples (Table 2) was indicative of an increase in the number of defective sperm cells under nicotinic stress. Furthermore, a strong positive correlation was seen between decreased spermatocrit values and eosin staining results (r +0.985). Spermatocrit test is used as an indicator of biochemical integrity, whereas the viability staining (eosin) indicates the physical integrity of the sperm membrane (Schrader et al., 1986). The results obtained in this study indicate that nicotine has an oxidant potential when added to sperm samples by increasing the rate of TBARS/ MDA generation, signicantly and dose-dependently. The end point of LPO process is the thiobarbituric acid-reacted MDA as an index of LPO damages (Alvarez et al., 1987). Negatively, the nicotine oxidant potential bore a strong correlation with the number of swollen sperm cells (r )0.981, spermatocrit data). In conclusion, nicotine could alter the organization of sperm membrane lipid elements resulting in defective spermatozoa. LPO also had a deleterious effect (r )0.990) on the aspect of viable sperm cells after staining with eosin. LPO also impairs plasma membrane ion exchanges, which is for maintenance of sperm movements (Rao et al., 1989). Oxidative stress-mediated damage to the sperm membrane may account for defective sperm function observed in a high proportion of infertility cases (Aitken, 1994; Sharma & Agarwal, 1996). An earlier study from our laboratory showed that nicotine addition to human sperm samples leads to a markedly impaired glutathione redox ratio (GSH/GSSG), with an inverse proportion to oxidative stress, and that protection could be provided by supplementing media with antioxidants such as ascorbate and trolox (a water-soluble analogue of vitamin E), indicating a challenge between antioxidants and ROS-mediated LPO process (Arabi et al., 2003).
ANDROLOGIA 36, 305310 (2004)

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Nicotine at 0.8 lm, the very low sub-micromolar level occurring in the tissues of smokers, increases oxidative stress, apoptosis and genotoxic stress in colon preparations (Crowley-Weber et al., 2003). We speculated that nicotine is able to provide free radicals, either by release of accumulated lipid hydroperoxides from sperm membranes or by direct generation of oxygen derivatives, as a powerful fuel for PUFA peroxidation cascade. However, it needs further assessment. Oxidative stress affects the integrity of sperm chromatin and causes high frequencies of single and double-stranded DNA breaks (Aitken & Krausz, 2001; Saleh et al., 2003). Exposing spermatozoa to articially produced ROS signicantly increases DNA damage by modifying all bases and producing base-free sites, defective frameshifts and DNA cross links (Duru et al., 2000). Recent studies have shown a signicant positive correlation between sperm DNA fragmentation and the levels of ROS in the testicular tissue (Rajesh et al., 2002) and in semen (Barroso et al., 2000; Henkel et al., 2003). The extent of DNA or chromatin damage in the spermatozoa depends on the ability of oocyte to repair the damage (Genesca et al., 1992). Zitzmann et al. (2003) demonstrated that male smokers show a decreased success rate of ART procedures, due to alteration in sperm DNA, not only in in vitro fertilization, but also in intracytoplasmic sperm injection. Human sperm samples were also found to exhibit a wide variation in DNA damages as measured by Comet assay due to different contents of enzymatic and non-enzymatic antioxidants (Hughes et al., 1998). In our study, the results obtained from the Comet assay technique revealed that nicotine could establish a linear increase in percentage Comets (DNA breaks) from 2% (baseline in controls) to 11% in 0.75 mm nicotine-treated samples (Table 2 and Fig. 1). However, an excellent positive correlation was found between increased DNA breaks and LPO rate values (r +0.976) indicating that ROS-mediated LPO in samples simultaneously cause DNA breaks in sperm chromatin and oxidation in DNA bases. Meanwhile, ROS modify PUFA structure in sperm membranes to alter viability and movement variables. In the present investigation, r )0.978 was calculated between percentage Comets and sperm viability. The use of AO stain helped us to distinguish single-stranded from double-stranded DNA breaks in sperm nucleus, as AO bound to double-stranded DNA breaks uoresces green and AO bound to single-stranded DNA breaks uoresces red. Almost all the Comet (95%) were of double-stranded DNA breaks, whereas, some single-stranded ones (5%) were also seen. Reports showing that sperm DNA integrity correlates strongly with male fertilizing
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ability in vivo, and infertile men have signicantly poorer sperm DNA integrity and high levels of ROS than fertile controls (Fraga et al., 1996; Zini et al., 2001). Collectively, nicotine proved to be a potential oxidant agent in the category of environmental factors that could induce membrane impairments and DNA fragmentation, signicantly. However, further studies will be necessary to illuminate the other dark sides of nicotinic infertility in the human spermatozoa. Acknowledgements The author is grateful to Dr Behzad Shareghi (PhD), Dean of Faculty of Sciences, Shahrekord University, for providing necessary laboratory facilities and materials, Dr (Mrs) Noha Eftekhari (PhD), Department of Mathematics, Shahrekord University, for the management of statistical analyses, and Mr Rasool Seidaei (MSc), Department of Biology, Shahrekord University, for his endless laboratory assistance. References
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