ARTICLE IN PRESS Reproductive BioMedicine Online (2015) ■■, ■■–■■
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Zinc protects sperm from being damaged by reactive oxygen species in assisted reproduction techniques
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Jinxiang Wu a,*, Shiqiang Wu b, Yuanzhi Xie b, Zhengyao Wang a, Ruiyun Wu c, Junfeng Cai d, Xiangmin Luo c, Suzhen Huang c, Liuxia You d
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Laboratory of Reproduction Medicine, The Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian, China; b Department of Surgery, The Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian, China; c Department of Obstetric and Gynecology, The Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian, China; d Department of Clinical Laboratory, The Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian, China
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Q1 * Corresponding author. E-mail address: [email protected] (J. Wu).
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Jinxiang Wu graduated with a masters degree from Fujian Medical University in 2009. She works in the reproductive medicine laboratory at the Second Affiliated Hospital of Fujian Medical University. She has published two domestic articles and obtained funding for one research project Jinxiang Wu is married and has a 2-year old daughter.
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The aim of this study was to explore the effect of zinc on hydrogen peroxide-induced sperm damage in assisted reproduction techniques. First, sperms were selected from semen samples of 20 healthy men prepared by density gradient centrifugation. Selected sperm were treated with either 0.001% H2O2, 12.5 nM ZnCL2, 0.001% H2O2 + 12.5 nM ZnCL2 or 0.9% NaCl2 (control). After this treatment, the motility, viability, membrane integrity and DNA fragmentation of sperms in each group were analysed by Goodline sperm detection system, optical microscopy and sperm DNA fragmentation assay. Poorer motility, vitality, membrane integrity and more DNA damage were found in sperms treated by H2O2, compared with control. When sperms were treated with both H2O2 and zinc, however, all indicators were improved compared with H2O2 alone. There was a close association between oxidative stimulation and sperm injury; zinc could inhibit hydrogen peroxide-induced damage of sperm in assisted reproductive technology. However, the presence of zinc in culture medium can decrease the sperm quality without addition of peroxide. Abstract
© 2014 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
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Q6 KEYWORDS: assisted reproduction, DNA damage, oxidative stress, reactive oxygen species, sperm
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http://dx.doi.org/10.1016/j.rbmo.2014.12.008 1472-6483/© 2014 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
Please cite this article in press as: Jinxiang Wu, Shiqiang Wu, Yuanzhi Xie, Zhengyao Wang, Ruiyun Wu, Junfeng Cai, Xiangmin Luo, Suzhen Huang, Liuxia You, Zinc protects sperm from being damaged by reactive oxygen species in assisted reproduction techniques, Reproductive BioMedicine Online (2015), doi: 10.1016/ j.rbmo.2014.12.008
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Introduction
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Infertility is a public health problem for many couples of childbearing age. Growing evidence indicates that oxidative stress plays a fundamental role in the cause of male infertility by negatively affecting sperm quality and function, which can be a primary cause of male infertility (Benedetti et al., 2012). Seminal oxidative stress results from an imbalance between reactive oxygen species (ROS) production and ROS scavenging by seminal antioxidants (Agarwal et al., 2014). Thse are are toxic substances that are produced by cells in the normal metabolism. High levels of semen ROS can cause sperm dysfunction, sperm DNA damage and reduced male reproductive potential. Oxidative stress occurs commonly in spermatozoa through lipid peroxidation because the sperm membrane contains high amounts of polyunsaturated fatty acids. The concept of improving fertility potential of infertile patients with oxidative stress by certain antioxidants has gained considerable attention in assisted reproductive technology and infertility practice (Chow, 1991; Donnelly et al., 1999). Zinc is a micronutrient required for the action of more than 200 metallo-enzymes. It plays a vital role in male fertility. Deficiency of zinc can impair spermatogenesis and decrease serum testosterone levels (Wong et al., 2002). Studies have shown that it plays a central role in normal testicular growth, spermatogenesis, and sperm physiology (Elgazar et al., 2005); It conserves genomic integrity in the sperm and stabilizes connection of sperm head to tail (Tuerk and Fazel, 2009). Deficiency of zinc is associated with hypogonadism and insufficient growth of secondary sex characteristics in human beings (Sandstorm and Sandberg, 1992). Low seminal zinc levels were coupled with a decrease in fertilizing ability of sperm (Pandy et al., 1983) and decreased the synthesis of testosterone Q7 (Ebisch et al., 2007; Prasad, 1991). Also, it helps in stabilizing polymeric macromolecules, such as RNA, DNA and protein. Earlier studies have shown that zinc present in the prostatic secretion provides stability to human sperm chromatin (Bjorndahl and Kvist, 2011; Suzuki et al., 1995). Assisted reproduction techniques are the main method of treating infertility. The process of density gradient centrifugation for sperm preparation before treatment, freezing and recovery in vitro will lead to sperm oxidative damage, which Q8 affects semen parameters (De Iuliis et al., 2009; Donnelly et al., 2001a, 2001b). Human seminal plasma is a natural reservoir of antioxidants. Spermatozoa depend on scavenging systems provided by the seminal plasma, with important natural antioxidants such as vitamins C and E, superoxide dismutase, glutathione and thioredoxin that act directly as free radical scavengers (Kobayashi et al., 1991; Niki, 1991). The sperm lose the protective effect of seminal plasma in assisted reproductive technology. The concept of improving fertility potential of infertile patients with oxidative stress by certain antioxidants has gained considerable attention in assisted reproduction techniques and infertility practice (Chow, 1991; Donnelly et al., 1999). The presence of zinc in culture medium has been reported to inhibit capacitation and acrosome reaction (Andrews et al., 1994; Bilaspuri and Babbar, 2007; Liu et al., 2009; Riffo et al., 1992). Zinc, however, has been reported to protect sperm chromosomal stability in vitro (Blazak and Overstreet, 1982; Kotdawala Q9 et al., 2012). Therefore, the present study was undertaken
to assess whether addition of zinc to the culture medium can benefit sperm and protect the human spermatozoa from oxidative damage.
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Materials and methods Preparation of sperm Semen samples were collected by masturbation after the stipulated 2–7 days of abstinence from 20 men with normal semen parameters who attended the infertility clinic. The semen samples were liquefied for 15–30 min at 37°C, following which the sperm parameters were assessed in accordance with the World Health Organization guidelines (World Health Organization, 2010). Samples with high leukocyte concentrations (leukocytes > 1 × 106 cell mL−1) were excluded. The sperm were prepared using a discontinuous PureSperm gradient (Quinn’s, SAGE, USA), which consisted of two layers of PureSperm (1.5 ml): 80% and 40%. Semen sample (2 ml) was deposited on the 40% layer. The gradient was then centrifuged at 300 x g for 15 min. After centrifugation, the sperm precipitate was collected and washed twice with 2 ml of sperm washing medium (Quinn’s, SAGE, USA) at 200× g for 5 min. The pellet was then resuspended in IVF medium (Scandinavian IVF, Gothenburg, Sweden) to sperm concentration of 5 × 106/ml for further study.
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Experimental design
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At first, pre-experiments of gradient dose of H2O2 (0.1%, 0.01% and 0.001%) and ZnCl2 (50, 25, 12.5, 6.2 nmol L-1) were conducted to decide the optimal concentration that can make a significant difference to semen motility and viability. After 5 h of incubation, significant changes were observed only at 0.001% H2O2 and 12.5 nmol/l ZnCl2. Therefore, four groups of experimental samples were prepared with 0.001% H 2 O 2, 12.5 nmol/ml ZnCl2, 0.001% H2O2 and 12.5 nmol/l ZnCl2, as well as the same volumes of saline (control), respectively.
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Sperm motility and viability analysis Sperm motility and viability were determined according to 2010 WHO recommendation (World Health Organization, 2010), after 5 h and 24 h incubation of the samples in the incubator with 5% CO2 at 37°C.
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Sperm hypo-osmotic swelling test
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The sperm membrane integrity was assessed with hypoosmotic swelling test according to 2010 WHO recommendation (World Health Organization, 2010), after 5 h and 24 h incubation of the samples in the incubator with 5% CO2 at 37°C. In brief, the membrane integrity of spermatozoa tail could expand under hypotonic environment. Thus, following 30min treatment with hypotonic solution, the sperm was observed under optical microscopy (×400 times) in randomly
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selected field. After 200 sperms were counted, the percentage of sperm with membrane integrity was calculated according to the following formula:
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the number of sperm tail expanding the percentage of sperm := × 100% with membrane integrity The total number of sperm were observed
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Sperm DNA fragmentation detection
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Sperm DNA fragmentation was detected by sperm chromatin dispersion (SCD) test. This was carried out as described by Fernandez et al., 2003), with minor modifications (Kalthur et al., 2011). Briefly, 60 µl of each group semen sample was mixed with 1% low melting point agarose maintained at 37°C. About 30 µl of this mixture was layered on a slide precoated with 0.65% of normal melting point agarose, after which a cover slip was carefully placed and the gel was allowed to solidify. The slides were then immersed in freshly prepared acid denaturation solution (0.08 M HCl) for 7 min at room temperature in dark. Proteins were removed by incubating the slides in lysing solution (0.4 M Tris, 20 mM DTT, 1 % SDS, 50 mM EDTA, pH 7.5) for 28 min followed by incubation in lysing solution 2 (0.4 M Tris, 2M NaCl, pH 7.5) for 15 min at room temperature. Slides were washed in Tris buffer (0.4 M Tris, pH 7.5) for 2 min serially dehydrated in graded ethanol, and air dried. Cells were stained with Switzerland pigment (0.2 %) and scored under Optical microscope (Olympus-CX31, Japan). Minimum of 500 spermatozoa were scored in each slide. Sperm DNA fragmentation was indicated by no halo or thickness of unilateral halo of less than one-third of the minimum diameter of the sperm head whereas intact sperm DNA was indicated by a large halo (Figure 1). Samples were counted separately under oil immersion objective (1000× magnification). The percentage of sperm with intact DNA was determined from the number of spermatozoa having a large halo out of the total number counted.
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This study was approved by Ethics Committee of The Second Affiliated Hospital of Fujian Medical University (reference number: 20110311 on 11 March 2011.
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Statistical analysis Data were expressed as mean ± SD. The one-way analysis of variance and the LSD post-hoc test were carried out on the data for inter-group comparisons. Database management and statistical analysis were carried out using the Statistical Package for Social Sciences (SPSS) version 20.0 (IBM Corp., USA).
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Results Sperm motility and viability Data on sperm motility and viability are presented in Table 1. We found that, after 5 h of incubation with 0.001% H2O2, sperm motility and viability significantly decreased (P < 0.05) compared with the control. Co-incubation, however, with ZnCl2 and H2O2 improved motility and vitality after 5 h and 24 h compared with H2O2 alone (Table 1). Sperm motility was also significantly decreased after 24 h of incubation with ZnCl2 alone, compared with the control (P < 0.05).
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Sperm membrane integrity Compared with the control, the greatest damage to sperm plasma membrane was observed after the exposure of spermatozoa to H2O2, and the membrane integrity was significantly higher after incubation with ZnCl2 and H2O2 compared with incubation in H2O2 alone (P < 0.05) (Figure 2). No difference was observed between the control and ZnCl2 alone
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Figure 1 Evaluation of the extent of sperm DNA damage. Sperm DNA fragmentation (SDF): with no halo or thickness of unilateral halo less than one-third of the minimum diameter of the sperm head. Sperm DNA integrity (SDI): large halo or thickness of unilateral halo is larger than one-third of the minimum diameter of the sperm head indicating intact DNA.
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Table 1 Sperm motility and viability of the different experimental groups (n = 20) after 5 h and 24 h of incubation.
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Control (mean ± SD)
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ZnCl2 (mean ± SD)
H2O2 (mean ± SD)
ZnCl2 + H2O2 (mean ± SD)
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After 5 h Motility (%) Viability (%) After 24 h Motility (%) Viability (%)
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84.25 ± 5.04 93.86 ± 1.67
82.12 ± 5.21 93.12 ± 2.21
3.43 ± 0.97a,b 34.05 ± 4.56a,b
26.14 ± 3.54a,b 60.30 ± 5.32a,b
78.61 ± 6.45 91.23 ± 2.45
54.62 ± 6.13a 75.78 ± 4.32a
0a,b 4.61 ± 1.32a,b
0.79 ± 0.51a,b 10.11 ± 2.78a,b
P < 0.05, for comparison with control (spermatozoa without H2O2 and ZnCl2). P < 0.05, comparison between group H2O2 (spermatozoa with 0.001% H2O2) and group ZnCl2 + H2O2 (spermatozoa combined successively with 12.5 nmol/l ZnCl2 and 0.001% H2O2).
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Figure 2 Sperm membrane integrity in each experimental group (n = 20) after 5 h and 24 h of incubation. Values are presented as mean ± SD. Experimental groups include spermatozoa without ZnCl2 and H2O2 (control); spermatozoa with 0.001% H2O2 (H2O2); spermatozoa with 12.5 nmol/l ZnCl2 (ZnCl2); and spermatozoa incubated successively with 12.5 nmol/l ZnCl2 and 0.001% H2O2 (ZnCl2 + H2O2). aP < 0.05, compared with control; bP < 0.05, comparison with group H2O2.
Figure 3 Evaluation of sperm DNA damage in different experimental groups (n = 20) after 5 h and 24 h of incubation. Values are presented as mean ± SD. Experimental groups include spermatozoa without ZnCl2 and H2O2 (control); (2) spermatozoa with 0.001% H2O2 (H2O2); (3) spermatozoa with 12.5 nmol/l ZnCl2 (ZnCl2); and (4) spermatozoa incubated successively with 12.5 nmol/l ZnCl2 and 0.001% H2O2 (ZnCl2 + H2O2).aP < 0.05, compared with control. b P < 0.05, comparison with group H2O2.
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after 5 h of incubation, but membrane integrity was also significantly decreased after 24 h of incubation with ZnCl2 (P < 0.05).
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Sperm DNA damage
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Results (Figure 3) revealed that the percentage of spermatozoa with sperm DNA fragmentation was significantly increased after incubation with H2O2 compared with the control; it was significantly decreased after co-incubation with ZnCl2 (both P < 0.05). It was also observed that the percentage of Sperm DNA damage was higher after incubation for 24 h with ZnCl2 alone compared with control (P < 0.05).
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Discussion
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Spermatozoa are particularly susceptible to oxidative injury owing to the abundance of plasma membrane polyunsaturated fatty acids (Aitken and Clarkson, 1987; De Lamirande
and Gagnon, 1992; Zini et al., 2000). Under physiological conditions, sperm produces small amounts of ROS, which are needed for fertilization, acrosome reaction and capacitation. If an increased production of ROS is not associated with a similar increase in scavenging systems, however, peroxidative damage of the sperm plasma membrane and loss of DNA integrity typically occur, which leads to cell death and reduced fertility. Seminal plasma and spermatozoa themselves are well endowed with an array of protective antioxidants to protect spermatozoa from oxidative stress (Agarwal et al., 2004; Saleh and Agarwal, 2002; Tremellen, 2008). In in-vitro fertilization, seminal plasma is removed during semen processing and the toxic oxygen metabolites (generated by immature spermatozoa and leukocytes) are able to attack spermatozoa without being protected by seminal plasma antioxidants. In addition, the detrimental effect of oxidative stress on sperm functional competence can be exaggerated by the in-vitro sperm processing techniques (e.g. centrifugation and prolonged incubation) that usually precede assisted reproduction techniques (Aitken and Baker, 1995; Twigg et al., 1998; Zini et al., 2009). Most in-vitro fertilization (IVF) cases result from male-factor deficiencies. The quality of sperm is one of the factors determining the success rate of IVF.
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ARTICLE IN PRESS Zinc protects sperm from being damaged by ROS in assisted reproduction techniques 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
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It has been reported that zinc levels in the blood are important for spermatogenesis. Henkel et al. (1999) showed that the outer dense fibre incorporates a high amount of zinc during spermatogenesis so that the outer dense fibre can be protected from premature oxidation. Omu et al. (2008) found that the in-vitro induced oxidative stress of this study was caused by a combination of zinc deficiency and high level of sperm DNA fragmentation and apoptosis. Others have observed a high zinc content in seminal plasma to be correlated with good physical characteristics of sperm such as sperm count (Mankad et al., 2006), motility (Chia et al., 2000), and normal morphology (Edorh et al., 2003). Zinc deficiency during spermatogenesis may, therefore, cause defective mature sperm motility. Many reports have shown that zinc therapy in men with asthenozoospermia resulted in significant improvement in sperm quality with increases in sperm concentration, progressive motility, sperm DNA integrity and improved conception and pregnancy rates (Omu et al., 2008; Sorensen et al., 1999; Wong et al., 2002). It has been reported that the presence of zinc in culture medium can inhibit capacitation and acrosome reaction (Andrews et al., 1994; Bilaspuri and Babbar, 2007; Riffo et al., 1992). The addition of 0.5 mmol/l zinc to the culture media had no effect on spermatozoa–zona pellucida binding, but significantly reduced the zona pellucida-induced acrosome reQ10 action in vitro (Liu et al., 2009). On the contrary, high level of sperm DNA fragmentation and apoptosis were associated with zinc deficiency in vitro (Omu et al., 2008). Meanwhile, Blazak and Overstreet (1982) believed that in-vitro zinc was necessary for stabilizing the nuclear chromatin in the spermatozoa of fertile men. Male infertility has been linked with the excessive generation of ROS by defective spermatozoa. When produced in excess, H2O2 activates the lipid peroxidation cascade, which in turn causes loss of membrane fluidity and decreases sperm quality. In this study, an appropriate amount of zinc was found to have a protective effect on sperm motility and viability against in-vitro induced ROS, and can improve sperm membrane and DNA integrity. Our data, however, showed that sperm membrane and DNA integrity did not improve with the addition of zinc only to the culture media. On the contrary, with increasing incubation time, zinc was harmful to sperm. In conclusion, our data clearly suggest that adding zinc to the culture media can significantly protect the spermatozoa from H 2 O 2 damage and maintain the sperm functional characteristics. Without addition of peroxide, the presence of zinc in culture medium for 24 h can decrease the sperm quality.
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Acknowledgements
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This project was supported by research funds with the apQ11 proval of the Ethical Committee of the Faculty of The Second Affiliated Hospital of Fujian Medical University, China. We thank all the scientists in the Andrology Laboratory for collecting sperm samples and all the individuals who volunteered to participate in this study. This study received financial support from the Youth Research Foundation of Fujian Provincial Department of Health, China (Grant No. 2011-1-36).
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Agarwal, A., Nallella, K.P., Allamaneni, S.S., Said, T.M., 2004. Role of antioxidants in treatment of male infertility: an overview of the literature. Reprod. Biomed. Online 8, 616–627. Agarwal, A., Durairajanayagam, D., Halabi, J., Peng, J., VazquezLevin, M., 2014. Proteomics, oxidative stress and male infertility. Reprod. Biomed. Online 29, 32–58. Aitken, R.J., Baker, H.W., 1995. Seminal leukocytes: passengers, terrorists or good samaritans? Hum. Reprod. 10, 1736–1739. Aitken, R.J., Clarkson, J.S., 1987. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertil. 81, 459–469. Andrews, J.C., Nolan, J.P., Hammerstedt, R.H., Bavister, B.D., 1994. Role of zinc during hamster sperm capacitation. Biol. Reprod. 51, 1238–1247. Benedetti, S., Tagliamonte, M.C., Catalani, S., Primiterra, M., Canestrari, F., De Stefani, S., Palini, S., Bulletti, C., 2012. Differences in blood and semen oxidative status in fertile and infertile men, and their relationship with sperm quality. Reprod. Biomed. Online 25, 300–306. Bilaspuri, G.S., Babbar, B.K., 2007. Effect of albumin and zinc on capacitation and acrosome reaction of buffalo spermatozoa. Indian J. Anim. Sci. 77, 688–692. Bjorndahl, L., Kvist, U., 2011. A model for the importance of zinc in the dynamics of human sperm chromatin stabilization after ejaculation in relation to sperm DNA vulnerability. Syst. Biol. Reprod. Med. 57, 86–92. Blazak, W.F., Overstreet, J.W., 1982. Instability of nuclear chromatin in the ejaculated spermatozoa of fertile men. J. Reprod. Fertil. 65, 331–339. Chia, S.E., Ong, C., Chua, L., Ho, L., Tay, S., 2000. Comparison of zinc concentration in blood and seminal plasma and various sperm parameters between fertile and infertile men. J. Androl. 21, 53– 57. Chow, C.K., 1991. Vitamin E and oxidative stress. Free Radic. Biol. Med. 11, 215–222. De Iuliis, G.N., Thomson, L.K., Mitchell, L.A., Finnie, J.M., Koppers, A.J., Hedges, A., Nixon, B., Aitken, R.J., 2009. DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2’deoxyguanosine, a marker of oxidative stress. Biol. Reprod. 81, 517–524. De Lamirande, E., Gagnon, C., 1992. Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J. Androl. 13, 368–378. Donnelly, E.T., McClure, N., Lewis, S.E.M., 1999. The effect of ascorbate and α-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa. Mutagenesis 14, 505–511. Donnelly, E.T., Steele, K.E., McClure, N., Lewis, S.E.M., 2001a. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum. Reprod. 16, 1191–1199. Donnelly, E.T., McClure, N., Lewis, S.E.M., 2001b. Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity. Fertil. Steril. 76, 892–900. Ebisch, I.M.W., Thomas, C.M.G., Peters, W.H.M., Braat, D.D.M., Steegers-Theunissen, R.P.M., 2007. The importance of folate, zinc and antioxidants in the pathogenesis and prevention of subfertility. Hum. Reprod. Update 13, 163–174. Edorh, A.P., Tachev, K., Hadou, T., Gbeassor, M., Sanni, A., Creppy, E.E., Le Faou, A., Rihn, B.H., 2003. Magnesium content in seminal fluid as an indicator of chronic prostitutes. Cell. Mol. Biol. 49, 419– 423. Elgazar, V., Razanov, V., Stoltenberg, M., Hershfinkel, M., Huleihel, M., Nitzan, Y.B., Lunenfeld, E., Sekler, I., Silverman, W.F., 2005. Zinc-regulating proteins, ZnT-1, and metallothionein I/II are
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present in different cell populations in the mouse testis. J. Histochem. Cytochem. 53, 905–912. Fernandez, J.L., Muriel, L., Rivero, M.T., Goyanes, V., Vazquez, R., Alvarez, J.G., 2003. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J. Androl. 24, 59–66. Henkel, R., Bittner, J., Weber, R., Huther, F., Miska, W., 1999. Relevance of zinc in human sperm flagella and its relation to motility. Fertil. Steril. 6, 1138–1143. Kalthur, G., Raj, S., Thiyagarajan, A., Kumar, S., Kumar, P., Adiga, S.K., 2011. Vitamin E supplementation in semen-freezing medium improves the motility and protects sperm from freeze-thawinduced DNA damage. Fertil. Steril. 95, 1149–1151. Kobayashi, T., Miyazaki, T., Natori, M., Nozawa, S., 1991. Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa. Hum. Reprod. 6, 987–991. Kotdawala, A.P., Kumar, S., Salian, S.R., Thankachan, P., Govindraj, K., Kumar, P., Kalthur, G., Adiga, S.K., 2012. Addition of zinc to human ejaculate prior to cryopreservation prevents freeze-thawinduced DNA damage and preserves sperm function. J. Assist. Reprod. Genet. 29, 1447–1453. Liu, D.Y., Sie, B.S., Liu, M.L., Agresta, F., Baker, H.W., 2009. Relationship between seminal plasma zinc concentration and spermatozoa–zona pellucida binding and the ZP-induced acrosome reaction in sub fertile men. Asian J. Androl. 11, 499–507. Mankad, M., Sathawara, N.G., Doshi, H., Saiyed, H.N., Kumar, S., 2006. Seminal plasma zinc concentration and α-glucosidase activity with respect to semen quality. Biol. Trace Elem. Res. 110, 97–106. Niki, E., 1991. Action of ascorbic acid as scavenger of active and stable oxygen radicals. Am. J. Clin. Nutr. 54, 1119S–1124S. Omu, A.E., Al-Azemi, M.K., Kehinde, E.O., 2008. Indications of the Mechanisms Involved in Improved Sperm Parameters by Zinc Therapy. Med. Princ. Pract. 17, 108–116. Pandy, V.K., Parmeshwaran, M., Soman, S.D., Dacosta, J.C., 1983. Concentrations of morphologically normal, motile spermatozoa: Mg+2, Ca+2 and Zn+2 in semen of infertile men. Sci. Total Environ. 27, 49–52. Riffo, M., Leiva, S., Astudillo, J., 1992. Effect of zinc on human sperm motility and the acrosome reaction. Int. J. Androl. 15, 229–237.
Saleh, R.A., Agarwal, A., 2002. Oxidative stress and male infertility: from research bench to clinical practice. J. Androl. 23, 737– 752. Sandstorm, B., Sandberg, A.S., 1992. Inhibitory affects of isolated inositol phosphates on zinc absorption in humans. J. Trace Elem. Electrolytes Health Dis. 6, 99–103. Sorensen, M.B., Stoltenberg, M., Danscher, G., Ernst, E., 1999. Chelation of intracellular zinc ions affects human sperm cell motility. Mol. Hum. Reprod. 5, 338–341. Suzuki, T., Nakajima, K., Yamamoto, A., Yamanaka, H., 1995. Metallothionein binding zinc inhibits nuclear chromatin decondensation of human spermatozoa. Andrologia 27, 161– 164. Tremellen, K., 2008. Oxidative stress and male infertility – a clinical perspective. Hum. Reprod. Update 14, 243–258. Tuerk, M.J., Fazel, N., 2009. Zinc deficiency. Curr. Opin. Gastroenterol. 25, 136–143. Twigg, J., Fulton, N., Gomez, E., Irvine, D.S., Aitken, R.J., 1998. Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum. Reprod. 13, 1429–1436. Wong, W.Y., Merkus, H.M., Thomas, C.M., Menkveld, R., Zielthuis, G.A., Steegers-Theunissen, R.P., 2002. Effect of folic acid and zinc sulphate on male factor sub fertility, a double blind, randomized placed controlled trial. Fertil. Steril. 77, 491–498. World Health Organization, 2010. WHO Laboratory Manual for the Examination and Processing of Human Semen, fifth ed. World Health Organization., Geneva, Switzerland. Zini, A., Garrels, K., Phang, D., 2000. Antioxidant activity in the semen of fertile and infertile men. Urology 55, 922–926. Zini, A., San Gabriel, M., Baazeem, A., 2009. Antioxidants and sperm DNA damage: a clinical perspective. J. Assist. Reprod. Genet. 26, 427–432.
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Declaration: All authors declare no competing financial interests.
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Received 17 September 2014; refereed 8 December 2014; accepted 10 December 2014.
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w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m
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Zinc protects sperm from being damaged by reactive oxygen species in assisted reproduction techniques
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Jinxiang Wu a,*, Shiqiang Wu b, Yuanzhi Xie b, Zhengyao Wang a, Ruiyun Wu c, Junfeng Cai d, Xiangmin Luo c, Suzhen Huang c, Liuxia You d
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Laboratory of Reproduction Medicine, The Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian, China; b Department of Surgery, The Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian, China; c Department of Obstetric and Gynecology, The Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian, China; d Department of Clinical Laboratory, The Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian, China
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Q1 * Corresponding author. E-mail address: [email protected] (J. Wu).
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Jinxiang Wu graduated with a masters degree from Fujian Medical University in 2009. She works in the reproductive medicine laboratory at the Second Affiliated Hospital of Fujian Medical University. She has published two domestic articles and obtained funding for one research project Jinxiang Wu is married and has a 2-year old daughter.
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The aim of this study was to explore the effect of zinc on hydrogen peroxide-induced sperm damage in assisted reproduction techniques. First, sperms were selected from semen samples of 20 healthy men prepared by density gradient centrifugation. Selected sperm were treated with either 0.001% H2O2, 12.5 nM ZnCL2, 0.001% H2O2 + 12.5 nM ZnCL2 or 0.9% NaCl2 (control). After this treatment, the motility, viability, membrane integrity and DNA fragmentation of sperms in each group were analysed by Goodline sperm detection system, optical microscopy and sperm DNA fragmentation assay. Poorer motility, vitality, membrane integrity and more DNA damage were found in sperms treated by H2O2, compared with control. When sperms were treated with both H2O2 and zinc, however, all indicators were improved compared with H2O2 alone. There was a close association between oxidative stimulation and sperm injury; zinc could inhibit hydrogen peroxide-induced damage of sperm in assisted reproductive technology. However, the presence of zinc in culture medium can decrease the sperm quality without addition of peroxide. Abstract
© 2014 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
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Q6 KEYWORDS: assisted reproduction, DNA damage, oxidative stress, reactive oxygen species, sperm
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http://dx.doi.org/10.1016/j.rbmo.2014.12.008 1472-6483/© 2014 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
Please cite this article in press as: Jinxiang Wu, Shiqiang Wu, Yuanzhi Xie, Zhengyao Wang, Ruiyun Wu, Junfeng Cai, Xiangmin Luo, Suzhen Huang, Liuxia You, Zinc protects sperm from being damaged by reactive oxygen species in assisted reproduction techniques, Reproductive BioMedicine Online (2015), doi: 10.1016/ j.rbmo.2014.12.008
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Introduction
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Infertility is a public health problem for many couples of childbearing age. Growing evidence indicates that oxidative stress plays a fundamental role in the cause of male infertility by negatively affecting sperm quality and function, which can be a primary cause of male infertility (Benedetti et al., 2012). Seminal oxidative stress results from an imbalance between reactive oxygen species (ROS) production and ROS scavenging by seminal antioxidants (Agarwal et al., 2014). Thse are are toxic substances that are produced by cells in the normal metabolism. High levels of semen ROS can cause sperm dysfunction, sperm DNA damage and reduced male reproductive potential. Oxidative stress occurs commonly in spermatozoa through lipid peroxidation because the sperm membrane contains high amounts of polyunsaturated fatty acids. The concept of improving fertility potential of infertile patients with oxidative stress by certain antioxidants has gained considerable attention in assisted reproductive technology and infertility practice (Chow, 1991; Donnelly et al., 1999). Zinc is a micronutrient required for the action of more than 200 metallo-enzymes. It plays a vital role in male fertility. Deficiency of zinc can impair spermatogenesis and decrease serum testosterone levels (Wong et al., 2002). Studies have shown that it plays a central role in normal testicular growth, spermatogenesis, and sperm physiology (Elgazar et al., 2005); It conserves genomic integrity in the sperm and stabilizes connection of sperm head to tail (Tuerk and Fazel, 2009). Deficiency of zinc is associated with hypogonadism and insufficient growth of secondary sex characteristics in human beings (Sandstorm and Sandberg, 1992). Low seminal zinc levels were coupled with a decrease in fertilizing ability of sperm (Pandy et al., 1983) and decreased the synthesis of testosterone Q7 (Ebisch et al., 2007; Prasad, 1991). Also, it helps in stabilizing polymeric macromolecules, such as RNA, DNA and protein. Earlier studies have shown that zinc present in the prostatic secretion provides stability to human sperm chromatin (Bjorndahl and Kvist, 2011; Suzuki et al., 1995). Assisted reproduction techniques are the main method of treating infertility. The process of density gradient centrifugation for sperm preparation before treatment, freezing and recovery in vitro will lead to sperm oxidative damage, which Q8 affects semen parameters (De Iuliis et al., 2009; Donnelly et al., 2001a, 2001b). Human seminal plasma is a natural reservoir of antioxidants. Spermatozoa depend on scavenging systems provided by the seminal plasma, with important natural antioxidants such as vitamins C and E, superoxide dismutase, glutathione and thioredoxin that act directly as free radical scavengers (Kobayashi et al., 1991; Niki, 1991). The sperm lose the protective effect of seminal plasma in assisted reproductive technology. The concept of improving fertility potential of infertile patients with oxidative stress by certain antioxidants has gained considerable attention in assisted reproduction techniques and infertility practice (Chow, 1991; Donnelly et al., 1999). The presence of zinc in culture medium has been reported to inhibit capacitation and acrosome reaction (Andrews et al., 1994; Bilaspuri and Babbar, 2007; Liu et al., 2009; Riffo et al., 1992). Zinc, however, has been reported to protect sperm chromosomal stability in vitro (Blazak and Overstreet, 1982; Kotdawala Q9 et al., 2012). Therefore, the present study was undertaken
to assess whether addition of zinc to the culture medium can benefit sperm and protect the human spermatozoa from oxidative damage.
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Materials and methods Preparation of sperm Semen samples were collected by masturbation after the stipulated 2–7 days of abstinence from 20 men with normal semen parameters who attended the infertility clinic. The semen samples were liquefied for 15–30 min at 37°C, following which the sperm parameters were assessed in accordance with the World Health Organization guidelines (World Health Organization, 2010). Samples with high leukocyte concentrations (leukocytes > 1 × 106 cell mL−1) were excluded. The sperm were prepared using a discontinuous PureSperm gradient (Quinn’s, SAGE, USA), which consisted of two layers of PureSperm (1.5 ml): 80% and 40%. Semen sample (2 ml) was deposited on the 40% layer. The gradient was then centrifuged at 300 x g for 15 min. After centrifugation, the sperm precipitate was collected and washed twice with 2 ml of sperm washing medium (Quinn’s, SAGE, USA) at 200× g for 5 min. The pellet was then resuspended in IVF medium (Scandinavian IVF, Gothenburg, Sweden) to sperm concentration of 5 × 106/ml for further study.
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Experimental design
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At first, pre-experiments of gradient dose of H2O2 (0.1%, 0.01% and 0.001%) and ZnCl2 (50, 25, 12.5, 6.2 nmol L-1) were conducted to decide the optimal concentration that can make a significant difference to semen motility and viability. After 5 h of incubation, significant changes were observed only at 0.001% H2O2 and 12.5 nmol/l ZnCl2. Therefore, four groups of experimental samples were prepared with 0.001% H 2 O 2, 12.5 nmol/ml ZnCl2, 0.001% H2O2 and 12.5 nmol/l ZnCl2, as well as the same volumes of saline (control), respectively.
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Sperm motility and viability analysis Sperm motility and viability were determined according to 2010 WHO recommendation (World Health Organization, 2010), after 5 h and 24 h incubation of the samples in the incubator with 5% CO2 at 37°C.
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The sperm membrane integrity was assessed with hypoosmotic swelling test according to 2010 WHO recommendation (World Health Organization, 2010), after 5 h and 24 h incubation of the samples in the incubator with 5% CO2 at 37°C. In brief, the membrane integrity of spermatozoa tail could expand under hypotonic environment. Thus, following 30min treatment with hypotonic solution, the sperm was observed under optical microscopy (×400 times) in randomly
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selected field. After 200 sperms were counted, the percentage of sperm with membrane integrity was calculated according to the following formula:
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the number of sperm tail expanding the percentage of sperm := × 100% with membrane integrity The total number of sperm were observed
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Sperm DNA fragmentation detection
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Sperm DNA fragmentation was detected by sperm chromatin dispersion (SCD) test. This was carried out as described by Fernandez et al., 2003), with minor modifications (Kalthur et al., 2011). Briefly, 60 µl of each group semen sample was mixed with 1% low melting point agarose maintained at 37°C. About 30 µl of this mixture was layered on a slide precoated with 0.65% of normal melting point agarose, after which a cover slip was carefully placed and the gel was allowed to solidify. The slides were then immersed in freshly prepared acid denaturation solution (0.08 M HCl) for 7 min at room temperature in dark. Proteins were removed by incubating the slides in lysing solution (0.4 M Tris, 20 mM DTT, 1 % SDS, 50 mM EDTA, pH 7.5) for 28 min followed by incubation in lysing solution 2 (0.4 M Tris, 2M NaCl, pH 7.5) for 15 min at room temperature. Slides were washed in Tris buffer (0.4 M Tris, pH 7.5) for 2 min serially dehydrated in graded ethanol, and air dried. Cells were stained with Switzerland pigment (0.2 %) and scored under Optical microscope (Olympus-CX31, Japan). Minimum of 500 spermatozoa were scored in each slide. Sperm DNA fragmentation was indicated by no halo or thickness of unilateral halo of less than one-third of the minimum diameter of the sperm head whereas intact sperm DNA was indicated by a large halo (Figure 1). Samples were counted separately under oil immersion objective (1000× magnification). The percentage of sperm with intact DNA was determined from the number of spermatozoa having a large halo out of the total number counted.
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This study was approved by Ethics Committee of The Second Affiliated Hospital of Fujian Medical University (reference number: 20110311 on 11 March 2011.
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Statistical analysis Data were expressed as mean ± SD. The one-way analysis of variance and the LSD post-hoc test were carried out on the data for inter-group comparisons. Database management and statistical analysis were carried out using the Statistical Package for Social Sciences (SPSS) version 20.0 (IBM Corp., USA).
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Results Sperm motility and viability Data on sperm motility and viability are presented in Table 1. We found that, after 5 h of incubation with 0.001% H2O2, sperm motility and viability significantly decreased (P < 0.05) compared with the control. Co-incubation, however, with ZnCl2 and H2O2 improved motility and vitality after 5 h and 24 h compared with H2O2 alone (Table 1). Sperm motility was also significantly decreased after 24 h of incubation with ZnCl2 alone, compared with the control (P < 0.05).
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Sperm membrane integrity Compared with the control, the greatest damage to sperm plasma membrane was observed after the exposure of spermatozoa to H2O2, and the membrane integrity was significantly higher after incubation with ZnCl2 and H2O2 compared with incubation in H2O2 alone (P < 0.05) (Figure 2). No difference was observed between the control and ZnCl2 alone
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Figure 1 Evaluation of the extent of sperm DNA damage. Sperm DNA fragmentation (SDF): with no halo or thickness of unilateral halo less than one-third of the minimum diameter of the sperm head. Sperm DNA integrity (SDI): large halo or thickness of unilateral halo is larger than one-third of the minimum diameter of the sperm head indicating intact DNA.
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Table 1 Sperm motility and viability of the different experimental groups (n = 20) after 5 h and 24 h of incubation.
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Control (mean ± SD)
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ZnCl2 (mean ± SD)
H2O2 (mean ± SD)
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After 5 h Motility (%) Viability (%) After 24 h Motility (%) Viability (%)
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84.25 ± 5.04 93.86 ± 1.67
82.12 ± 5.21 93.12 ± 2.21
3.43 ± 0.97a,b 34.05 ± 4.56a,b
26.14 ± 3.54a,b 60.30 ± 5.32a,b
78.61 ± 6.45 91.23 ± 2.45
54.62 ± 6.13a 75.78 ± 4.32a
0a,b 4.61 ± 1.32a,b
0.79 ± 0.51a,b 10.11 ± 2.78a,b
P < 0.05, for comparison with control (spermatozoa without H2O2 and ZnCl2). P < 0.05, comparison between group H2O2 (spermatozoa with 0.001% H2O2) and group ZnCl2 + H2O2 (spermatozoa combined successively with 12.5 nmol/l ZnCl2 and 0.001% H2O2).
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Figure 2 Sperm membrane integrity in each experimental group (n = 20) after 5 h and 24 h of incubation. Values are presented as mean ± SD. Experimental groups include spermatozoa without ZnCl2 and H2O2 (control); spermatozoa with 0.001% H2O2 (H2O2); spermatozoa with 12.5 nmol/l ZnCl2 (ZnCl2); and spermatozoa incubated successively with 12.5 nmol/l ZnCl2 and 0.001% H2O2 (ZnCl2 + H2O2). aP < 0.05, compared with control; bP < 0.05, comparison with group H2O2.
Figure 3 Evaluation of sperm DNA damage in different experimental groups (n = 20) after 5 h and 24 h of incubation. Values are presented as mean ± SD. Experimental groups include spermatozoa without ZnCl2 and H2O2 (control); (2) spermatozoa with 0.001% H2O2 (H2O2); (3) spermatozoa with 12.5 nmol/l ZnCl2 (ZnCl2); and (4) spermatozoa incubated successively with 12.5 nmol/l ZnCl2 and 0.001% H2O2 (ZnCl2 + H2O2).aP < 0.05, compared with control. b P < 0.05, comparison with group H2O2.
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after 5 h of incubation, but membrane integrity was also significantly decreased after 24 h of incubation with ZnCl2 (P < 0.05).
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Sperm DNA damage
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Results (Figure 3) revealed that the percentage of spermatozoa with sperm DNA fragmentation was significantly increased after incubation with H2O2 compared with the control; it was significantly decreased after co-incubation with ZnCl2 (both P < 0.05). It was also observed that the percentage of Sperm DNA damage was higher after incubation for 24 h with ZnCl2 alone compared with control (P < 0.05).
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Discussion
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Spermatozoa are particularly susceptible to oxidative injury owing to the abundance of plasma membrane polyunsaturated fatty acids (Aitken and Clarkson, 1987; De Lamirande
and Gagnon, 1992; Zini et al., 2000). Under physiological conditions, sperm produces small amounts of ROS, which are needed for fertilization, acrosome reaction and capacitation. If an increased production of ROS is not associated with a similar increase in scavenging systems, however, peroxidative damage of the sperm plasma membrane and loss of DNA integrity typically occur, which leads to cell death and reduced fertility. Seminal plasma and spermatozoa themselves are well endowed with an array of protective antioxidants to protect spermatozoa from oxidative stress (Agarwal et al., 2004; Saleh and Agarwal, 2002; Tremellen, 2008). In in-vitro fertilization, seminal plasma is removed during semen processing and the toxic oxygen metabolites (generated by immature spermatozoa and leukocytes) are able to attack spermatozoa without being protected by seminal plasma antioxidants. In addition, the detrimental effect of oxidative stress on sperm functional competence can be exaggerated by the in-vitro sperm processing techniques (e.g. centrifugation and prolonged incubation) that usually precede assisted reproduction techniques (Aitken and Baker, 1995; Twigg et al., 1998; Zini et al., 2009). Most in-vitro fertilization (IVF) cases result from male-factor deficiencies. The quality of sperm is one of the factors determining the success rate of IVF.
Please cite this article in press as: Jinxiang Wu, Shiqiang Wu, Yuanzhi Xie, Zhengyao Wang, Ruiyun Wu, Junfeng Cai, Xiangmin Luo, Suzhen Huang, Liuxia You, Zinc protects sperm from being damaged by reactive oxygen species in assisted reproduction techniques, Reproductive BioMedicine Online (2015), doi: 10.1016/ j.rbmo.2014.12.008
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It has been reported that zinc levels in the blood are important for spermatogenesis. Henkel et al. (1999) showed that the outer dense fibre incorporates a high amount of zinc during spermatogenesis so that the outer dense fibre can be protected from premature oxidation. Omu et al. (2008) found that the in-vitro induced oxidative stress of this study was caused by a combination of zinc deficiency and high level of sperm DNA fragmentation and apoptosis. Others have observed a high zinc content in seminal plasma to be correlated with good physical characteristics of sperm such as sperm count (Mankad et al., 2006), motility (Chia et al., 2000), and normal morphology (Edorh et al., 2003). Zinc deficiency during spermatogenesis may, therefore, cause defective mature sperm motility. Many reports have shown that zinc therapy in men with asthenozoospermia resulted in significant improvement in sperm quality with increases in sperm concentration, progressive motility, sperm DNA integrity and improved conception and pregnancy rates (Omu et al., 2008; Sorensen et al., 1999; Wong et al., 2002). It has been reported that the presence of zinc in culture medium can inhibit capacitation and acrosome reaction (Andrews et al., 1994; Bilaspuri and Babbar, 2007; Riffo et al., 1992). The addition of 0.5 mmol/l zinc to the culture media had no effect on spermatozoa–zona pellucida binding, but significantly reduced the zona pellucida-induced acrosome reQ10 action in vitro (Liu et al., 2009). On the contrary, high level of sperm DNA fragmentation and apoptosis were associated with zinc deficiency in vitro (Omu et al., 2008). Meanwhile, Blazak and Overstreet (1982) believed that in-vitro zinc was necessary for stabilizing the nuclear chromatin in the spermatozoa of fertile men. Male infertility has been linked with the excessive generation of ROS by defective spermatozoa. When produced in excess, H2O2 activates the lipid peroxidation cascade, which in turn causes loss of membrane fluidity and decreases sperm quality. In this study, an appropriate amount of zinc was found to have a protective effect on sperm motility and viability against in-vitro induced ROS, and can improve sperm membrane and DNA integrity. Our data, however, showed that sperm membrane and DNA integrity did not improve with the addition of zinc only to the culture media. On the contrary, with increasing incubation time, zinc was harmful to sperm. In conclusion, our data clearly suggest that adding zinc to the culture media can significantly protect the spermatozoa from H 2 O 2 damage and maintain the sperm functional characteristics. Without addition of peroxide, the presence of zinc in culture medium for 24 h can decrease the sperm quality.
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Acknowledgements
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This project was supported by research funds with the apQ11 proval of the Ethical Committee of the Faculty of The Second Affiliated Hospital of Fujian Medical University, China. We thank all the scientists in the Andrology Laboratory for collecting sperm samples and all the individuals who volunteered to participate in this study. This study received financial support from the Youth Research Foundation of Fujian Provincial Department of Health, China (Grant No. 2011-1-36).
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Declaration: All authors declare no competing financial interests.
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Received 17 September 2014; refereed 8 December 2014; accepted 10 December 2014.
Please cite this article in press as: Jinxiang Wu, Shiqiang Wu, Yuanzhi Xie, Zhengyao Wang, Ruiyun Wu, Junfeng Cai, Xiangmin Luo, Suzhen Huang, Liuxia You, Zinc protects sperm from being damaged by reactive oxygen species in assisted reproduction techniques, Reproductive BioMedicine Online (2015), doi: 10.1016/ j.rbmo.2014.12.008
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